1.1. Synthetic Hydrogels
Transcript of 1.1. Synthetic Hydrogels
Chapter 1: Introduction
1.1. Synthetic Hydrogels
Hydrogels by definition are three-dimensional swollen networked structures. Certain
materials, when placed in a compatible aqueous medium, are able to swell and retain the
volume of the adsorbed aqueous medium in their three-dimensional swollen network.
Such aqueous gel networks are known as hydrogels or aquagels [1]. Included in this
definition are a wide variety of natural materials of both, plant and animal origins,
chemically modified, naturally occurring materials and synthetic polymeric materials [2].
Synthetic polymeric hydrogels are generally three-dimensional swollen networks of
hydrophilic homopolymers or copolymers covalently or ionically crosslinked [1-8]. The
original polymeric hydrogel network was developed by Wichterle and Lim in
Czechoslovakia in 1954 [9]. It was a copolymer of 2-hydroxyethyl methacrylate (HEMA)
and ethylene dimethacrylate (EDMA) for use as contact lenses. Due to lack of interest
and support from the appropriate authorities, no success was achieved. Wichterle and Lim
however, continued to work on their development and it was not until the 1960s when the
versatility of synthetic polymeric hydrogels was visualised from a commercial point of
view.
Polymeric hydrogel networks may be formed by various techniques, however the most
common synthetic route is the free radical polymerisation of vinyl monomers in the
presence of a bifunctional crosslinking agent and a swelling agent. The resulting polymer
is interesting in the sense that it exhibits both liquid-like and solid like properties [3]. The
liquid-like properties result from the fact that the major constituent (>80%) is water.
However, the polymer also exhibits solid-like properties due to the network formed by the
crosslinking reaction, or more like elastic solids in the sense that there exists a
remembered reference configuration to which the hydrogel returns after being deformed
for a long time [3, 10].
The classification of hydrogels depends on their physical structure and chemical
composition. A common classification, especially useful in biomedical applications
includes neutral hydrogels, ionic hydrogels and swollen interpenetrating networks (IPNs)
[4] is described in detail in Section 1.1.1. The most characteristic property of a hydrogel
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
is that it swells in the presence of an aqueous media and shrinks in its absence [7]. In
general hydrogels swell to an equilibrium value of 10 – 98 % at physiologic temperature,
pH and ionic strength [2,8, 11,]. A dried hydrogel imbibing at least 20 times its own
weight of the aqueous media while retaining its original shape could be referred to as
superabsorbent. The capacity of hydrogels to absorb the aqueous media, however, could
be enormous and can be as much as 1000 times the weight of the polymer [3,12]. Figure 1
depicts a hydrogel network upon placement in water [13].
Figure 1. A schematic representation of a single chain in a hydrogel network upon
placement in water.
Swelling in hydrogels when in aqueous media occurs in a similar manner as that of an
analogous linear polymer dissolving in the media to form an ordinary solution [14].
Mainly the nature, predominantly the hydrophilicity/hydrophobicity of polymer chains
and the crosslinking density determine the extent of swelling. A hydrogel from a linear
polymer upon dissolving in the aqueous media becomes a hydrosol, which is a dispersion
of colloidal particles or simply an aqueous solution from a physical point of view [1,15].
A number of polymer systems may undergo a reversible transformation between hydrogel
and hydrosol but through chemical crosslinking of dispersed particles in hydrosol result in
an irreversible hydrogel [1].
A gel is typically a large macromolecule, which forms network extending from one end to
the other and occupying the whole reaction vessel [16]. Microgels, which are crosslinked
polymer networks as that of macromolecules but composed of small particles with
diameters smaller then 1 µm are water soluble as that of linear polymers due to their
molecular nature [17,18]. The term hydrogel is referred to a material currently in swollen
state but upon drying, the swollen network of the hydrogel collapses due to the high
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Chapter 1: Introduction
surface tension of water rendering a xerogel or a dry gel [1,7]. The overall shape of the
hydrogel is preserved during the swelling and shrinking process.
Synthetic hydrogels have been a field of extensive research for the past four decades and
it still remains a very active area of research today. Hydrogels can be designed with
controllable responses as to shrink or expand with changes in external environmental
conditions [1]. The extent of swelling or de-swelling in response to the changes in the
external environment of the hydrogel could be so drastic that the phenomenon is referred
to as volume collapse or phase transition [19-21].
Hydrogels may respond uniquely to changes in external environmental conditions such as
ionic strength [22], electromagnetic radiation [23], pH [24-28], and temperature
[24,26,29-31]. These conditions could be introduced individually or in combinations and
altered as desired. Other important factors such as the type of salt used for the preparation
of buffer [32,33], solvent used as the medium [34], photoelectric stimulus [35] and
external stress [36,37] are also influential on the hydrogel’s performance. These unique
properties make hydrogels excellent candidates in numerous biomedical, pharmaceutical,
agricultural and consumer-oriented fields [1].
1.1.1. Classification of Hydrogels
Polymeric hydrogels are classified in accordance to their monomeric composition based
on the method of preparation giving some important class of hydrogels namely
homopolymeric hydrogels, copolymeric hydrogels and interpenetrating polymeric
hydrogels. Furthermore, the chemical constituent of monomers used in the preparation of
hydrogels plays an important role in classifying the hydrogels. The hydrogels are classed
as either neutral, anionic, cationic or ampholytic based on the presence of ionic charges
on the monomer. Hydrogels are also classed as amorphous or semi-crystalline materials
based on their physical nature.
1.1.1.1. Homopolymeric Hydrogels
Homopolymers are formally referred to as a polymer network derived from a single
species of the monomer, which is the basic structural unit comprising of any polymer
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
network [38-41]. Homopolymers could have crosslinked or uncrosslinked skeletal
structure depending on the nature of the monomer and polymerisation technique.
Homopolymers, which are generally crosslinked, find important applications such as slow
drug delivery devices and contact lenses. An important category of crosslinked
homopolymeric hydrogels of poly(hydroxyalkyl methacrylates) include poly(3-
hydroxypropyl methacrylate (PHPMA), poly(glyceryl methacrylate), (PGMA) and
poly(2-hydroxyethyl methacrylate) (PHEMA) [2,42]. PHEMA hydrogels is among the
most widely studied and used of all synthetic hydrogel materials [23,42-52].
There are some uncrosslinked homopolymers, which have been of interest to a number of
researchers [53-57]. Poly(N-vinyl-2-pyrrolidinone) (PNVP), poly(acrylamide) (PAM),
poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) are classed as uncrosslinked
water-soluble homopolymers. PNVP has found useful applications in biomedicine due to
its extreme solubility in water and adequate solubility in many other polar and non-polar
solvents [2,53-55]. PVA is another important class of uncrosslinked homopolymeric
material with numerous potential biomedical and agricultural applications when
crosslinked [56,57]. PEG and PAM have been widely used in agricultural applications
[58].
1.1.1.2. Copolymeric Hydrogels
Copolymeric hydrogel networks are comprised of two or more different monomer species
with at least one hydrophilic component, arranged in a random, block or alternating
configuration along the chain of the polymer network [38-41]. The copolymeric hydrogel
networks are generally covalently or ionically crosslinked structures, which are not water
-soluble [1-8].
A wide range of important copolymeric hydrogels with vast combinations of compatible
monomers, some of which include poly(NVP-co-HEMA), poly(HEMA-co-MMA) and
poly(HEMA-co-AA) have been studied by a number of researchers [59-61]. Copolymers
designed to function as hydrogels are confined to the combination of compatible
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Chapter 1: Introduction
monomers, which will give the hydrogels the desirable properties for their intended
potential applications.
1.1.1.3. Interpenetrating Polymer Network (IPN) Hydrogels
IPN, an important class of hydrogel materials, are defined as two independent crosslinked
synthetic and/or natural polymer components contained in a network form as shown in
Figure 2 [62]. A semi-IPN is an IPN where one of the components is a crosslinked
polymer while the other component is a non-crosslinked polymer [1,4,41,63-67].
Figure 2. Structure of an IPN
The two basic synthetic routes to form IPNs are sequential and simultaneous
polymerisation methods [63]. The formation of an IPN increases the compatibility of the
polymer components thus preventing phase separation and allows access to properties
that may be hybrids of those of the component macromolecules [1,66-68]. Park et al [1]
have described the IPN formation between a pH sensitive hydrogel and temperature
sensitive second polymer as an example of such behaviour. The IPN formed will be both,
pH and temperature sensitive. Since there is no chemical bonding between the two
polymeric components, each component may retain its own property while the proportion
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
of each network can be varied independently thus obtaining the desired combinations of
the properties of the two macromolecule components [63,69,70].
The mechanical strength of the hydrogel can be improved by using relatively hydrophobic
second polymer in the IPN. Furthermore, one or both of the macromolecular networks of
the IPN could be made biodegradable [1,71]. A number of IPNs and semi-IPNs based on
polysaccharides such as chitosan and its derivatives, PNVP, PVA, poly(ethylene oxide)
(PEO), poly(N-isopropyl acrylamide) (PNIPAM), PEG and poly (methacrylic acid)
(PMAA) with potential bioapplications as hydrogel materials have been reported by
numerous researchers [66,67,69,72-83].
1.1.1.4. Non-Ionic Hydrogels
Non-ionic hydrogels, often referred to as neutral hydrogels, are homopolymeric or
copolymeric networks, which do not bear any charged groups in their structure. Neutral
hydrogels may be prepared by various polymerisation techniques or by conversion of
existing polymers. Although generalizations can be made about hydrogels, the wide range
of chemical compositions of the monomers used, give them different properties with
regards to biocompatibility, physical and chemical properties of the resultant polymer [2].
Neutral hydrogels swell to equilibrium when the osmotic pressure of the solvent is
balanced with the sub-chain stretching energy. The collapse and swelling of neutral
hydrogel networks occur normally as a result of change in the environmental temperature
[84]. Some neutral monomers commonly utilized to form hydrogels are shown in Figures
3 - 8 [2].
CH2
CH3
O
O
R
H2C C
H2
OH H2C C
HOH
CH3
H2C C
HCH
2
OH
OH
R = ,
Figure 3. Hydroxyalkyl methacrylates
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Chapter 1: Introduction
CH3
CH
CH
R'
R
O R''
CH3
CH3 C
2H
5
CH2CHOHCH
3
R =
R', R'' =
H ,
H , ,
Figure 4. Acrylamide derivatives
NCH
2
CH2 C
H2
CH
CH2
O
CH2
CH
CH
CH
CH
2
OH
CH2
C
H2
C
H2
CH2
CH2
N O
CHCH
2
Figure 5 Figure 6 Figure 7
N-Vinyl pyrrolidinone N-Vinyl caprolactam 2, 4 Pentadiene-1-ol
CH2
R
O
O
R'
CH3
CH3 C
4H
9OMe
CNOCH2CH
2OCH
3
R = H ,
, ,
,
R' =
Figure 8. Hydrophobic acrylics
1.1.1.5. Ionic Hydrogels
Ionic hydrogels also known as polyelectrolytes are prepared from monomer/s
accompanying ionic charges. The charges could be positive or negative thus classing the
hydrogels as cationic or anionic hydrogel respectively and furthermore, a combination of
both positive and negative charges gives an ampholytic macromolecule [2,26,85,86]. The
phase transition phenomenon of the polyelectrolytes was theorized by Dusek and
Patterson (1968) [87]. Inclusion of charged species in the polymer backbone enhances the
stimuli responsive properties, which could be controlled, depending on the nature of the
pendent group thus widening its scope of bioapplications as hydrogels [26,84-89].
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
1.1.1.5.1. Anionic Hydrogels
Anionic hydrogel networks are usually referred to as either homopolymers of negatively
charged acidic or anionic monomers or copolymers of an anionic monomer and a neutral
monomer. However, anionic hydrogels could also be prepared through modification of
existing polymeric non-ionic hydrogels such as by the partial hydrolysis of poly(hydroxy
alkyl methacrylates) or by the addition of excess polyanions in the case of polyelectrolyte
complexes to form anionic hydrogels [2]. Anionic monomers commonly utilized to form
anionic hydrogels are shown in Figures 9-11 [2]. Anionic hydrogels are known to exhibit
a marked increase in the swelling ratio with increase in the environmental pH [88,89].
CH2
OH
R
O
CH3R = H ,
Figure 9. Acrylic acid derivatives
CH
CH
OH
O
CH3
CH
CH
CH
CH
SO3 NaC
H
CH2
Figure 10 Figure 11
Crotonic acid Sodium Styrene sulfonate
1.1.1.5.2. Cationic Hydrogels
Homopolymers of positively charged basic or cationic monomers or copolymers of
cationic and neutral monomers are commonly referred to as cationic hydrogel networks.
Cationic monomers commonly utilized to prepare cationic-based hydrogel networks are
shown in Figures 12-13 [2]. As described in section 1.1.1.5.1, cationic polymeric
networks could also be derived through modifications such as partial hydrolysis of the
existing non-ionic pre-formed polymer networks. It is also possible to synthesize cationic
hydrogels through polyelectrolyte complexation reactions by addition of excess
polycations [2].
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Chapter 1: Introduction
Cationic pendant groups in polymer network in the contrary behaviour to anionic pendant
give rise to hydrogels, which remain collapsed in the basic environment and swollen in
the acidic environment due to the electrostatic repulsion between the positively charged
groups [88,89].
CH2
R
O
O
CH
2
CH
2
HN
R'
R''
CH3 C
4H
9H , ,R, R', R'' =
Figure 12. Aminoethyl methacrylate derivatives
NCH
CH2
Figure 13. 4-Vinyl pyridine
1.1.1.5.3. Polyampholytic Hydrogels
Polyampholytic hydrogel networks are referred to as macromolecules capable of
possessing both positively and negatively charged moieties in the polymer network
[26,90,81]. The presence of ionic species along the polymer chain has a distinct effect on
the solution and solid-state properties of the polyampholytes [90]. The columbic
attractions between the oppositely charged sides afford inter- and intramolecular ionic
interactions that are stronger than Van der Waals forces, yet weaker than covalent bonds
[90].
The net charges on these materials can be changed to achieve the desired functional
property by changing the monomeric composition of the feed mixture [36,91]. Some
common acidic and basic monomer combinations used to prepare polyampholytes are
illustrated in Figures 14 - 16 [90]. Preparation of numerous polyampholytic networks
with a wide range of important biomedical applications including sustained drug delivery
systems have been reported [26, 85,86,91-93].
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
CH2
CH
COOHN
CH
CH2
Acrylic acid 2-Vinylpyridine
Figure 14
OO O
N
CH
CH2
OO
Figure 15
Maleic anhydride N- vinylsuccinimide
CH2
CH
COOH
CH2
ON
CH3
O
CH3
CH3
Acrylic acid
Figure 16
2-(Diethylamino)ethyl 2-methylacrylate
1.1.1.6. Hydrogel Network Structures
Flory (1953) [14] states that the polymeric hydrogel network structure may have several
roles. In an aqueous medium the network may dissociate and take the role of the solute as
in the case of some water-soluble hydrogel networks described in Section 1.1.1.1 or swell
to equilibrium by imbibing the medium in its structure. As the network expands in an
aqueous medium (Figure 1) [13], a force resisting the expansion occurs due to the
elongation of the chain into a lesser entropically desirable conformation. When the
osmotic pressure driving the medium into the hydrogel network is matched by the exerted
expansion resistance force, equilibrium degree of swelling is achieved [8].
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Chapter 1: Introduction
The physical and other properties of the hydrogels depend on the structures of the
polymeric networks [4]. To maintain the three-dimensional structures, polymer chains of
hydrogels are usually crosslinked chemically or physically as described earlier in the sub-
sections of Section 1.1.1. In chemically crosslinked hydrogels, polymer chains are
connected by covalent bonds and thus it is difficult to change the shape of such networks.
Polymer chains of physical gels are connected through non-covalent bonds, such as van
der Waals interactions, ionic interactions, hydrogen bonding, or hydrophobic interactions
[7]. Physical and ionotropic forms represent secondary valence networks while the
covalent form indicates primary valence networks (Figure 17) [3].
Physical CovalentIonotropic
Figure 17. Schematic representation of hydrogel structures
The extent of crosslinking in the hydrogel network is referred to as crosslinking density.
Increased crosslinking density will increase the resistive force to chain elongation
consequently reducing the degree of equilibrium swelling in contrast to hydrogels with
low crosslinking density [8].
The polymeric hydrogel networks can be classified as hydrogen bonded, amorphous, or
semi-crystalline based on the structural analysis of the polymer network using a number
of physiochemical techniques such as small angle X-ray scattering (SAXS), wide angle
X-ray Scattering (WAXS), electron microscopy, differential scanning calorimetry (DSC),
along with electron and neutron diffraction [4,38,39,94].
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
1.1.1.6.1. Amorphous Hydrogel Structures
The term ‘amorphous’ also known as non-crystalline is usually attributed to optically
transparent isotropic polymeric networks that contain randomly arranged macromolecular
chains as suggested by Flory [14]. The amorphous hydrogel network often contains
localized ordered structures or non-homogeneous structures that are not suggested by the
common Flory definition of amorphous polymers. Thus the most acceptable definition of
such networks is a collection of Gaussian chains between crosslinks [4].
The temperature at which the polymeric network undergoes the transformation from a
glassy to a rubbery state is referred to as the glass transition temperature (Tg) [4,38,41].
The characteristic feature of amorphous polymeric networks is that when exposed to
temperature conditions below its Tg value, they pass successfully through the
transformation from a rubbery to glassy state without any clear demarcation between the
two phases [41]. The glassy, transparent nature, an important characteristic of amorphous
hydrogel networks, has widened their scope as bioapplicable materials requiring optical
transmittance [95].
1.1.1.6.2. Semicrystalline Hydrogel Structures
Semicrystalline hydrogel networks are complex mixtures of amorphous and crystalline
phases, which contain dense regions of ordered macromolecular chains (crystallites)
[96,97]. The lack of mechanical strength in some conventional crosslinked hydrogel
network structures for certain biomedical applications has led the development of
anisotropic semicrystalline polymeric networks which are characterized by the presence
of strong covalent bonds along the polymer chain [96,98].
Semicrystalline hydrogel networks are produced by heat treatment of noncrystalline
hydrogels above their Tg [4,98]. Crystallization of polymers in polymer-diluent systems is
the typical method of preparing semicrystalline hydrogel networks [4,99]. In the
crystallization process the short chains that are not able to fold are rejected from the
crystalline phase and thus they participate in the amorphous phase hence the resultant
polymer network contains continuous composition of amorphous and crystalline regions
[4,100]. The tendency of the polymers to crystallize is enhanced by its regularity and
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Chapter 1: Introduction
polarity [4]. Peppas (1977) [101] suggests that when semicrystalline polymer networks
are placed in aqueous medium, only the amorphous regions swell and the crystalline
regions are not affected by the medium thus they play the role of crosslinks in the
polymer network.
1.1.1.6.3. Hydrogen Bonded Hydrogel Structures
Hydrogen bonding is referred to as an electrostatic interaction between electronegative
atoms such as oxygen, nitrogen, fluorine and chlorine and hydrogen atoms that are
covalently bound to similar electronegative atoms [1,102]. The strength of the hydrogen
bonding (< 10 Kcal/mol), however, is far weaker than covalent bonding (> 100 Kcal/mol)
but still stronger than the van der Walls interactions (~ 1 Kcal/mol) [102].
The formation of multiple hydrogen bonds between two water-soluble macromolecules
may result in strong intermolecular structures [1], which are physically crosslinked three-
dimensional polymeric networks such as IPNs and semi-IPNs described in Section
1.1.1.3. The driving force behind the formation of the multiple simultaneous hydrogen
bonds between the macromolecules is the co-operative interaction between the
macromolecules, which is restricted to the chain length of the macromolecule [103].
1.2. Synthesis of Polymeric Hydrogels
Polymerisation reactions based on kinetics can be divided into chain or step
polymerisation reactions [41,64,65]. Step polymerisation reactions generally occur
between functionally substituted monomers and are characterized by a rapid
disappearance of the monomer at an early stage of the reaction and the existence of broad
molecular weight distribution in the later stages of the reaction [41,64].
Chain polymerisation, however, involves a three-step process namely: initiation,
propagation and termination thus allowing the monomer concentration to decrease
steadily with time thus ideally the reaction mixture at any stage of the polymerisation
reaction contains the monomer and the converted high polymer [41,64]. In contrary to
step polymerisation reactions, longer reaction times in chain polymerisation produces
high yield polymer but the molecular weight of the polymer is not affected [64].
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The saturated monomers described in Section 1.1.1 for hydrogel synthesis typically react
through a chain polymerisation process [64]. The characteristic polymerisation of
hydrogel network begins from the reactive centre initiated by the polymerisation source
and terminates upon the loss of the radical reactivity of the radicals as described in
Sections 1.2.1–1.2.3. The reactive centres at the initiation stage could be of free radical
nature or ionic nature [64,104] thus promoting free radical or ionic polymerisation as
described in Section 1.2.4.
1.2.1. Chain Initiation
A trace quantity of an initiator is normally required in the case of photopolymerisation
and thermal polymerisation processes to create free radicals for chain initiation [64]. The
initiators for specific curing processes readily fragment into radicals under the influence
of the applied source, which could be either heat or UV light as described in Scheme 1.
∆R R
/ hv
Initiator Free radical
Scheme 1. Formation of radicals
However, in addition to heat and light some high-energy ionization radiation sources,
which do not require initiators, can also generate radicals through electrochemical means
[64]. The radicals created according to Scheme 1 [104] react with an unsaturated
monomer also referred to as vinyl monomers to create a new species, which is still a
radical as shown in Scheme 2 [104] thus initiating the chain polymerisation process.
R CH
2
CH2
RH2C CH
2
Unsaturated
monomer
Propagating
species
+
Free Radical
Scheme 2. Chain initiation
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Chapter 1: Introduction
The efficiency of the initiator is a measure of the extent to which the number of radicals
formed reflects the number of polymer chains formed [64]. For thermal and photo-curable
systems the percentage of the initiator may vary with the weight of total resin. However,
high amounts of initiator may not necessarily enhance polymerisation, instead it may
have adverse effect since increased free radicals may undergo recombination and inhibit
the polymerisation process.
1.2.2. Chain Propagation
The propagation step involves growth of the polymer chain by rapid sequential addition
of monomer to the active center as shown in Scheme 3 [104]. The reactivity of the
propagating radicals is assumed to be independent of the size or degree of polymerisation
[41].
CH
2
CH2
R CH2
CH2
n
CH
2
CH2
CH
2
CH2
n
Propagating
species
+ R
Chain addition Propagating species
Scheme 3. Chain propagation
1.2.3. Chain Termination
The chain polymerisation does not continue until all the participating monomers are used
up because the free radicals involved are so reactive that they find a variety of ways of
losing their radical activity [64]. Thus the polymer chain terminates by disproportionation
or combination reactions.
CH
2
CH2
CH
2
CH3
n
CH
2
CH2
CH
2
CH2
n
CH
2
CH2
CH
CH2
n
R
R
Propagating species
2
Reaction products
Disproportionation
R
+
Scheme 4. Chain termination through disproportionation reaction
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The disproportionation reaction is characterized by the interaction of two reactive radial
species via hydrogen abstraction process leading to the formation of a saturated and an
unsaturated compound as shown in Scheme 4 [104].
The combination reaction in the termination stage of the polymer chain occurs by the
combination of two reactive radical species to form a single bond and one reaction
compound as shown in Scheme 5 [64,104].
CH
2
CH2
CH
2
CH
2n
CH
2
CH2
CH
2
CH2
n
RR
Propagating species
2
2Single reaction product
Combination
Scheme 5. Chain termination through combination reaction
1.2.4. Nature of the Reactive Radical Species
The nature of the radical species characterizes the type of chain polymerisation. The
categories of chain polymerisation reactions based on the nature of the reactive species
are referred to as free radical for non-ionic radical species, cationic and anionic for
cationic and anionic reactive radical species respectively. The presence of reactive ionic
centres makes ionic chain polymerisations more monomer specific than free radical
polymerisation reactions [64]. Furthermore, the propagating ionic centre is accompanied
by a counter-ion of opposite charge and termination does not occur by the reaction of two
ionic centres since they are of similar charge and thus repel each other [64].
The polarity of the polymerisation solvent and the ability to solvate the counter ion are
significant factors in ionic polymerisation [64]. Cationic active centres are created by the
reaction of an electrophilic monomer in the presence of protonic acids, which serve as
initiators. The propagation mechanism is the same as that of free radical polymerisation
described in Section 1.2.2. The termination step in cationic polymerisation is achieved by
either unimolecular arrangement of the ion pair or through chain transfer [64].
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Chapter 1: Introduction
Anionic chain polymerisation begins with active centres created by the reaction of a
nucleophilic monomer and propagates as described in Section 1.2.2 but there is no
inherent termination process, which is a unique property of such polymers [38,64].
Termination by ion pair arrangement in contrary to cationic polymerisation does not
occur in anionic polymerisation due its unfavourable requirement to eliminate the hydride
ion and furthermore, the alkaline earth metal counter ions used do not have the tendency
to combine with the active carbanion thus rendering the polymer molecule active, also
referred to as living polymers [38,64].
1.2.5. Curing Processes
The polymerisation techniques described in Section 1.2.1 could be carried out using a
variety of curing processes such as thermal, redox and radiation methods. Thermal
polymerisation technique involves the use of heat in the presence of a suitable initiator
while the redox method simply involves a reduction-oxidation reaction between the
participating species. Radiations sources commonly utilized by researchers [105,106] to
synthesize polymeric hydrogels include low energy ultraviolet (UV) radiation technique
and high-energy ionisation techniques such as gamma radiation and electron beam
radiation.
1.2.5.1. Ionizing Radiation Sources
Ionization radiation covers a wide range of different radiations some of which are of
primary source or secondary source. Ionization radiation is a high-energy process
involving electronic radiation of moving particles, which carry enough energy to ionize
simple molecules either in air or water and therefore more penetrative [4]. It involves the
use of either electron beams from an electron accelerator or gamma radiation from a 60
Co
source [4].
1.2.5.1.1. Electron Beam (EB) Radiation Process
Electron beam radiation is a high-energy process, which involves artificially accelerated
electron beams delivered from several systems with energy ranging from 0.5 to 20 MeV
[4]. Electron accelerators, such as commonly used Van de Graaff accelerator, substitute
for isotopes emitting β-rays, which are not utilized in radiation chemistry due to technical
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
problems. The EB process is an efficient process, which does not require initiators in the
reactive mixture, however the penetration of fast electron is lower than that of gamma
radiation [4]. Radical as well as ionic species are capable of participating in the
fundamental process.
1.2.5.1.2. Gamma Radiation Process
Gamma radiation involves emission of γ-rays by radioactive isotopes and they cover a
wide range of energies [4]. The ease of its preparation and fairly long half-life of 5.3
years compared to other present isotopes makes 60
Co, which sources two monochromatic
γ beams with energies of 1.17 and 1.33 MeV, the most widely used isotope for this
purpose [4]. 60
Co is produced by neutron irradiation of normal 59
Co in a nuclear reactor.
Gamma radiation technique does not require the inclusion of chemical initiators of any
sort, however, they can be used to remove any residual initiator that is present after other
conventional polymerisation processes such as thermal or UV curing. Residual initiator
may act as an undesirable contaminant [105-107]. The gamma rays in contrary to electron
beam radiation have very high penetrative power and the dose of radiation could be
varied from 5 to 100 rad/sec [4].
1.2.5.2. Ultra Violet (UV) Radiation Process
UV radiation curing technique involves the use of UV rays from a special light source of
desired intensity normally in the presence of a photosensitive chemical. This chemical
serves as an initiator in the photopolymerisation process to form radicals at a wavelength
of 360nm at which monomers are not affected. The curing process could be achieved by
the use of an electrode type or electrodeless lamp.
A medium pressure mercury lamp is an electrode type quartz tube filled with an inert gas
such as argon or xenon along with small amount of mercury installed with an electrode at
either end. The lamp when connected to an appropriate power source, an electrical arc
passes between the electrodes vaporizing mercury resulting in the energy emission, which
is primarily a white light, infrared and ultraviolet.
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Chapter 1: Introduction
Electrodeless lamp displays a similar spectral emission to that of an electrode type lamp,
however the operation of energization of the lamp is different between the two. The lamp
is energized by microwaves generated by magnetrons rather than an electrical arc. Even
ambient lighting contains UV rays, which could initiate some highly photosensitive
monomers in the presence of a photoinitiator.
The use of UV curing is a field of growing interest. UV curing has unique advantages
over thermal systems, which include cost effectiveness, minimal space requirements,
reduction in energy consumption, and rapid polymeric network formation on heat
sensitive substrates [108,109]. Energy and pollution constraints represent the major
contribution factors to the growth of this technology [109]. However, the only drawback
in this curing method is the use of chemical initiators. The photoinitiators are seldom
consumed fully during the polymerisation process. These materials trapped in the
polymer matrix tend to leach out when the polymer is in contact with an aqueous
medium. The in vivo leaching of additives used during the fabrication of polymers has
been cited as the cause of inflammation and eventual rejection of the implanted
biomaterial [2,110].
1.2.6. Charge-Transfer (CT) Complex Polymerisation
Scott et al [111] and Ellinger [112] were the pioneers in the field who initially reported
the spontaneous polymerisation of N-vinylcarbazole (VCZ) through the formation of
charge-transfer (CT) complexes in the presence of a variety of electron acceptor
monomers in 1963. The CT complex formation phenomenon has been widely recognized
since then, and has been an area of great research interest [113-123]. The complex
formations occur between electron-rich (donor) and electron-poor (acceptor) olefins and
are often spontaneous, that is, no chemical initiator is required in the process [124].
Chemical initiators used in the curing process to speed up the reactions are never fully
consumed in the reaction. Thus the resultant polymer is usually fairly unstable and
susceptible to non-desirable degradation. Chemical initiators besides being one of the
most costly components in the polymerisable compositions are also a significant
contributor to the toxicity of such formulations [122]. CT complex reactions, however,
19
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
may require heat or light depending on the nature of the participating monomers for the
reaction to commence. Thermal and photochemically induced CT complex
polymerisation reactions studied in recent years have been thoroughly revised by Shirota
and Mikawa [115].
1.2.6.1. Charge-Transfer (CT) Interactions
The CT complex system involves two components, an acceptor and a donor. The donor
molecule contains unshared pair of electrons and the acceptor molecule has vacant
molecular orbitals. The interaction between the donor and the acceptor counterparts in CT
polymerisation reactions is based on Mulliken’s charge-transfer theory, which states that
charge-transfer complexes are formed as a result of partial electron transfer from the
donor to the acceptor [125]. The formation of a CT complex depends on the nonbonding
interactions between the pi bonds of a donor and acceptor that create a species lower in
energy [126].
A A D
A A D
A D
A D
A D+ + D**
A D+ + D**
*
A D+*hv
*Scheme 6. Formation of an AD from a ground state CT
Scheme 7. Formation of an exciplex type AD
hv
hv
The degree of charge transfer depends on the compatibility of the donor and acceptor
monomers. For weak electron donor-acceptor pairs, the no-bond structure contributes
greatly to the ground state charge-transfer complex, whereas the electron transferred
dative structure contributes greatly to the excited state complex [121]. Scheme 6 [127]
outlines the formation of an excited state CT complex (AD*) by direct excitation of the
ground state CT complex.
20
Chapter 1: Introduction
The formation of CT complexes not only occurs in the ground state but also in the excited
state of an electron donor or acceptor [121]. Scheme 7 [127] outlines the formation of an
AD* from excited donor or acceptor. The excited donor (D
*) or acceptor (A
*) associates
with the ground state acceptor (A) or donor (D) respectively. Such excited state
complexes are termed exciplexes [121]. Exciplexes are stable in the excited state but
dissociative in the ground state. The enhanced donor and acceptor properties of the
molecules in their excited states contribute to the formation of exciplexes [121]. It has
been suggested in the literature [127,128] that both the mechanisms of AD* formation as
illustrated in Schemes 6 and 7 could lead to the generation of photochemically identical
AD* species.
EHOMO (D) ELUMO (A)
Hückel calculations
Formation of ground state CTC: EHOMO (D) ≈ELUMO (A)
Scheme 8. Relative energy differences between electron donors (D) and acceptors (A)
21
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The Mulliken theory [125] predicts a maximum charge transfer stabilization for the cases
where a maximum overlap exists between the highest occupied molecular orbital
(HOMO) of the donor and the lowest unoccupied molecular orbital (LUMO) of the
acceptor, thus producing coloured species of high ionic character. Scheme 8, adopted
from Jönsson et al [127], indicates the relative energy differences between typical donor
and acceptor monomers. In the case of a strong overlap between the HOMO and LUMO
of the monomers, a thermodynamically favoured separated radical ion pair may be
formed. In such cases the thermal stability and the photo latency are completely lost and a
spontaneous polymerisation occurs [127,120].
1.2.6.1.1. Decay Mechanisms of Excited State CT Complexes
Excited state CT complexes besides decaying directly back to ground state could also go
through a photochemically allowed [2+2] cycloaddition. Such interaction between the
donor and the acceptor olefins involved in the CT complexes is based on Huisgen’s
seminal work on the cycloaddition reactions between electron rich and electron deficient
olefins [129,130]. Huisgen’s work suggests the involvement of tetramethylene
intermediates in the process of cycloaddition.
The tetramethylene intermediates resulting from the decomposition of excited state CT
complexes could be predominantly of 1, 4 biradical in nature or predominantly
zwitterionic in nature [127,124,120]. Influence of ionic/biradical ratio of the short-lived
excited CT complex, depending on the polarity of the olefins involved, is also noticeable
among the intermediates [127,120]. The formation of excited state complex and the decay
mechanism via the formation of possible tetramethylene intermediates is illustrated in
Scheme 9 [120].
Hall and Padias [124,131] suggest that the terminal substituents on the donor and the
acceptor olefins direct the nature of the intermediates formed. The zwitterions are
predominantly in the cis or syn conformation due to the coulombic attraction, while the
biradicals exist in extended trans-conformation [124]. An extensive study on the
formation of these tetramethylene intermediates has been reported by Hall and Padias
[131]. All the possible resultant intermediates from the decomposition of the excited CT
22
Chapter 1: Introduction
complexes proceed to form a thermodynamically stable cyclobutane ring. However, the
relative yield of ring closure via each of these intermediates may vary depending on the
nature of the intermediate [120]. The strength of the CT complex could be controlled to
favour the existence of a free radical process over zwitterionic. Jonssön et al [120] have
also proposed the existence of fused radical ion pair as a possible intermediate upon
acceptor donor interaction. If the fused radical/ion pair dissociates into radical ions, they
will be capable of inducing both, the free radical and the ionic process [120]. The
dissociative mechanism of the fused radical/ion pair is illustrated in Scheme 10.
AD
*
*
D A
D A
D A
AD
AD
D A
- e-
Excited state complex
(AD )
-
1, 4 biradical
Zwitter ion
+
- +
+-
Fused radical/ionic pair
Cyclobutane
Tetramethylene intermediates
+
Scheme 9. Mechanism of [2+2] cycloaddition of the donor (D) and the acceptor (A) via
the possible tetramethylene intermediates.
23
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
AD
AD
AD
AD
- +
+-
Fused radical/ionic pair
- +
+-
Separated radical ionsDissociation
+
+
Scheme 10. Dissociation of the radical/ionic pair
To achieve the formation of 1, 4 biradical intermediate and subsequent free radical
polymerisation, it is crucial to generate an excited state complex with minimum ionic
character as possible [127,120].
1.2.6.2. Inducement of CT Complex Polymerisation
CT complex formulations could be induced by heat or light depending on the sensitivity
of the donor/acceptor monomers involved. Thermal induced reactions normally include
various initiation mechanisms whereas photo-induced reactions generally involve the
intermediacy of ions or radicals [115]. Photo-induced CT complex formations are by far
the predominant area of current research with numerous publications available to date
[120-122,126,127].
The use of ultra-violet light (UV) to cure unsaturated monomers in the absence of
photosensitizers has been known since the introduction of CT complexes. However, it
was restricted to very short wavelength UV lights and thickness of the polymerisable film
[122]. Such systems were not commercially viable. Jönsson et al (1995) [122] developed
UV polymerisable CT complex formulations, which were photoinitiator free and not
restricted to short wavelengths. Furthermore, the process was found to be more efficient
in the sense that the thickness of the polymerisable film was not an interfering factor in
the degree of polymerisation.
Such formulations often referred to as photoinitiator-free UV curable systems involve the
use of ‘charge-transfer complexes’ as a substitute to chemical photoinitiators [132] to
24
Chapter 1: Introduction
accomplish photopolymerisation reactions. Copolymerisation reactions via CT complexes
although being slower in comparison to photoinitiator-initiated systems but are vastly
superior in terms of stability is a field of growing interest [127].
1.2.6.3. Proposed Mechanisms of CT Complex Polymerisation
Polymerisation mechanisms always have to take at least a two-step process into account,
namely initiation and propagation. A number of researchers have proposed various
mechanisms to account for the CT complex polymerisation phenomenon. It has been
suggested in the literature that spontaneous CT polymerisation could be initiated by a
partial electron transfer (ET) from the donor to the acceptor molecule as illustrated in
Scheme 11 [124].
D+
A- e-
D A-
Scheme 11. Radical ions formation in ET
However, this is only true for the CT complex reactions, which involve extremely
electron rich and extremely electron deficient olefins [133]. The initiation of CT complex
polymerisation through the formation of tetramethylene intermediates proposed by Hall
and Padias [124] has been the most conclusive and widely accepted concept to date. In an
ideal system without chain transfer, the polymerisation may start from any of the decayed
species illustrated in Scheme 9 [120]. Depending on the character of the monomers
present in the system, the polymerisation will occur via a free radical or ionic process
[124].
An alternating copolymer has often been the observed as the phenomenal result of CT
complex polymerisation. A number of theories have been proposed in the past to account
for this phenomenon [124]. The CT complex acting as the co-monomer in the reaction as
illustrated in Scheme 12 has been a very attractive theory. This theory by Bartlett and
Nozaki [134] states that the colour observed as the result of CT complex formation fades
as the polymerisation via cycloaddition reaction proceeds.
25
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
D
A
DAD
ADA
D A D A D A
A
D A D A D A
D
AD
+
+
Scheme 12. CT complex acts as the monomer
Scheme 13. Matrix polymerisation
The other theory based on the difference of the polarities between the reacting molecules
was that of the matrix polymerisation, illustrated in Scheme 13. The theory of matrix
polymerisation suggests that the monomers align themselves in an alternating fashion in
the polymerisable mixture. The theories illustrated in Schemes 12 and 13 however, do not
provide sufficient evidence on the CT complex polymerisation [124]. In thermal initiated
CT complex polymerisations, Coote and Davies [135] concluded that the CT complex
was not involved in the propagation step. Hall and Padias suggest that the alternating
propagation is ascribed to the polarity difference of the monomers involved in the free
radical polymer chain.
1.2.6.4. Influential Factors in CT Complex Polymerisation
There are certain factors such as the choice of donor/acceptor monomer pairs involved,
inclusion of catalytic additives such as hydrogen donors or Lewis acids which govern the
efficiency and nature of the CT complex polymerisation. Hall and Padias [124] have shed
light on further advancement on their present theory of CT complex initiation. They
hypothesise that any force that brings the donor or acceptor together will enhance the rate
of tetramethylene intermediate formation and consequently the rate of spontaneous
polymerisation. The duo suggests possible modes of interactions such as protic acid/base,
Coulombic attractions, hydrophobic and hydrophilic interactions, Lewis acid/base, or the
use of templates or tethering beyond CT interaction.
26
Chapter 1: Introduction
1.2.6.4.1. Effect of Monomers
The efficiency and the nature of CT complex polymerisation is heavily dependent on the
choice the acceptor and the donor monomer pairs. The strength of electron donating and
electron withdrawing capabilities of the donor and acceptor respectively is the most
crucial factor, which is taken in consideration for the selection of desirable donor/acceptor
pairs. A strong donor/strong acceptor pair is known to give a CT complex of very ionic
character. Strong donor/weak acceptor or weak donor/strong donor pairs, however, give
CT complexes of absolutely minimum ionic character thus promoting free radical
polymerisation [120,127,136,137]. Table 1, adopted from Jönsson et al [120], summarises
a list of some commonly used acceptor and donor monomers with indicative strength of
electron acceptation and donation.
Table 1. Commonly utilized acceptor/donor monomers
Acceptors (A) Donors (D)
Incr
easi
ng
acc
epto
r st
ren
gth
‡‡
Maleic anhydride
Maleimide
N-methylmaleimide
p-Carbomethoxyphenylmaleimide
N-phenylmaleimide
4, 4- Dimaleimidobisphenol
Dimethyl fumarate
Diallyl maleate
Dimethyl maleate
Tetrahydrofurfuryl vinyl ether
Triethyleneglycol divinyl ether
Paraglycidyloxystyrene
Paramethoxystyrene
4-Propenyloxymethyl-1, 3, 2-dioxolanone
IsoEugenol (IEU)
N-Vinyl pyrrolidinone
††In
crea
sin
g d
on
or
stre
ng
th
Increasing donor or acceptor strength in the direction of the arrows
The donor/acceptor monomer feed ratio also critically affects the efficiency of CT
complex polymerisation. According to Decker et al [138], their study on N-substituted
alkyl maleimide (MI) and vinyl ether (VE) has indicated that the monomer feed
composition plays a decisive role on the polymerisation kinetics. They have reported
similar disappearance rate of both the monomers when VE is in excess yielding an
alternating copolymer. However, when the MI is in excess, the copolymerisation and the
27
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
MI homopolymerisation occur simultaneously to yield a copolymer with isolated VE
units. Based on this observation the authors suggest that the VE radicals act as the main
propagating species.
1.2.6.4.2. Effect of Lewis Acids
Lewis acids enhance the efficiency of spontaneous copolymerisation. The role of Lewis
acids in such reactions is that they make the acceptor olefin more electron deficient [124].
A literature example of alternating copolymerisation of styrene and acrylonitrile in the
presence of Lewis acids is illustrated in Scheme 14 [124].
Ph CN
ZnCl2
+ Alternating copolymer
Scheme 14. Role of Lewis acids in copolymerisation
Inorganic salts such as ZnCl2 or SbCl2 complex with the lone electron pairs on the
electron withdrawing substituent on the acceptor olefin. Cole et al [139], Garnett and
Zilic [140] in their recent studies on CT complexes have reported a significant increment
in the rate of CT complex polymerisation in the presence of SbCl2.
1.2.6.4.2. Effect of Hydrogen Donors
Certain N-substituted maleimides are known to homopolymerise in the absence of
chemical sensitizers [127,138,141] but the process is fairly inefficient. According to
Jönsson et al [127] however, in the presence of strong H-donors, which contain labile
hydrogens, the H-abstraction process will be quite efficient. The hydrogen abstraction
process increases the number of initiating radicals thus increasing the rate of
polymerisation.
Jönsson et al [127] and Clark et al [142] have reported that mixtures of N-
alkylmaleimides and acrylates polymerise rapidly, provided that the mixture contains
easily abstractable hydrogens. Hydrogens could be sourced either by added hydrogen
donors such as tertiary amines, secondary alcohols or by ethyleneglycol/ propyleneglycol
backbones in acrylate monomer/oligomer. The mechanisms of the intramolecular and
28
Chapter 1: Introduction
intermolecular H-abstraction from the excited state N-substituted maleimide, proposed by
Jönsson et al [127] are illustrated in Scheme 15.
N
O
O
N
OH
O
RH
N
O
O
R
R 1
NR
2
R 3
HH
OOR'
H H
R"
HS-R N
OH
O
R
R1 NR
2
R 3
H
OOR'
H
R"
S-R
N
OH
O
R N
OH
O
R N
OH
O
R
RHH
hv
Mechanism of intramolecular H abstraction
+ +
Mechanism of intermolecular H abstraction
hv
Mechanism of radical rearrangement
Scheme 15. Hydrogen abstraction from the excited state N-alkyl maleimide
1.2.6.5. Polymeric Hydrogel Synthesis via CT Complex Formation
Synthesis of polymeric hydrogels through a PI-free method is indeed a novel technique,
which was introduced recently in the course of this study. Although the concept of CT
complex polymerisation has been known for decades and is now well established, to the
29
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
best knowledge of the author, it has never been used to synthesize polymeric hydrogels.
The use of conventional PIs to assist the photopolymerisation of biomedical polymers has
always been a concern due to the possible leach of PIs from the polymer matrix. As
previously mentioned the PIs besides being expensive are fairly toxic materials.
Successful synthesis of polymeric hydrogels formed through the initiation of CT
complexes formed between a series of N-hydroxyalkyl maleimides and NVP, has been
published by the author in the duration of this course [143-148]. The N-hydroxyalkyl
maleimides are strong electron acceptors while NVP is a weak electron donor, thus
making them a compatible donor/acceptor pair for free radical polymerisation. The
hydrogels formed through this novel process has been reported to be very stable, quite
resilient, non-toxic and very promising materials for drug delivery applications.
1.3. Applications of Hydrogels
Polymeric hydrogels, owing to their dynamic structural properties have been commonly
utilized in numerous biomedical and agricultural applications. The biomedical
applications of hydrogels could be classified into three distinct categories namely,
coatings such as catheters, homogeneous materials such as contact lenses and devices
such as sustained drug delivery systems [2,149]. Biomedical applications of hydrogels are
discussed in detail in Section 1.4.
1.3.1. Agricultural Applications
Hydrogels have been commonly utilized in agricultural field mainly as water storage
granules [150,151]. The need for improving the physical properties of soil to increase
productivity in the agricultural sector was visualized in 1950s [152]. This led to the
development of water-soluble polymers such as PVA, PEG and PAM to function as soil
conditioners [58,152] followed by the introduction of water-swellable polymeric
hydrogels in the early 1980s [153,154]. Water-swellable hydrogels from crosslinked
PAM, crosslinked polyacrylates and copolymers of acrylamide and acrylates for such
applications have been reported [153, 154].
30
Chapter 1: Introduction
Soils with moist hydrogels acting as conditioners, increase water-holding capacity of the
potting media by 50 to 100 %. The increased water supply enhances the germination
process by reducing the relative amount of water loss via evaporation and drainage.
Swellable hydrogel delivery systems are also commonly utilized for controlled release of
agrochemicals and nutrients of importance in agricultural applications to enhance plant
growth with reduced environmental pollution. A number of researchers have reported the
versatility of polymeric hydrogels in agricultural applications [150-155].
1.3.2. Other Applications
Hydrogels have been commonly utilized commercially in other important industrial areas
such as cosmetics, food industry, photography and instrumentation. A list of some
important industrial applications of polymeric hydrogel systems are summarised in Table
2 [3].
Table 2. Additional important applications of hydrogels
¬ Hydrogels are used as thickening agents (e.g., starch and gelatin) in foods.
¬ The addition of hydrogel-forming agents to incontinence products increases
the fluid uptake and ensures improved retention capacity.
¬ Technical and electronic instruments can be protected from corrosion and
short-circuit exposure of, or sheathing with highly absorbent hydrogel-forming
agents.
¬ Hydrogels are used in photographic technology because they are light
permeable and can also store light sensitive substances.
¬ In electrophoresis and chromatography, the separation and diffusion
characteristics of the gel structure are exploited. Hydrogels, thus applied,
operate within only a very limited range of swelling.
31
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
1.4. Hydrogels as Biomaterials
The field of biomedical research has advanced rapidly in the past several years, mainly as
a result of attempts to replace body tissues with natural or synthetic biomaterials [1-
8,149]. Success in the application of biomaterials is strictly confined to their
biocompatibility. The term biocompatibility is referred to as the appropriate biological
performance, both local and systematic of a given biomaterial in a specific application
[1]. An appropriate response of the biomaterial for its particular application would be
referred to as an inert or positive interaction with the host [156]. One promising class of
biomaterials in this field of research is that of polymeric hydrogels.
The landmark paper by Wichterle and Lim [9] on the biomedical usage of PHEMA
hydrogel as contact lenses captivated the interest of biomaterial researchers around the
globe. Since then preparation and study of numerous hydrogel systems with various
properties have been reported. Hydrogels resemble in the physical properties of living
tissue more than any other class of synthetic biomaterial [2]. The high water content and
the soft rubbery consistency of hydrogels contribute to their superficial resemblance of
human tissues and may also contribute to their biocompatibility by minimising
mechanical irritation to surrounding tissue [1-8]. The highly hydrated and water-
plasticised polymer network of the hydrogel often has a low mechanical strength, but is
still finding ever-increasing use as biomaterials [157-161].
The wide range of biomedical applications of hydrogels can be attributed to both their
satisfactory performance upon in vivo implantation in either blood contacting or tissue
contacting situations and to their ability to be fabricated into a wide range of
morphologies [1-8,149]. It should be emphasized that a particular hydrogel composition
for one biomedical application may have to be significantly modified for a different
application as described in the earlier sections of this chapter. The various applications of
hydrogels in the biomedical field are summarised in Table 3 [2].
32
Chapter 1: Introduction
Table 3. Biomedical Applications of Synthetic Hydrogels
Coatings
‘Homogeneous”
Materials
Devices
Sutures
Catheters
IUD’s
Blood detoxicants
Sensors
Vascular grafts
Electrophoresis cells
Cell structure substrates
Electrophoresis gels
Contact lenses
Artificial corneas
Vitreous-humour
replacements
Oestrous –Induces
Soft tissue substitutes
Burn dressings
Bone ingrowth sponges
Dentures
Ear drum plugs
Synthetic cartilage’s
Hemodialysis membranes
Particulate carriers of
tumour antibodies
Enzyme therapeutic
systems
Artificial organs
Sustained drug
delivery systems
The area of interest presented in this work is that of controlled drug release. Sustained
delivery of drugs has been one very important application, where hydrogels have been
extensively used [1-8]. Delivery of bioactive agents through sustained release devices has
been a major field of research over the last three decades [60].
1.4.1. Sustained Drug Delivery Devices
The principle of slow release has been utilized since 1950 in the pharmaceutical industry
but it was not until the mid 1960’s that polymers were used for slow release of molecules
[162]. Folkman and Long [163] first reported sustained drug release from polymers in
1964. However, it was not until the 1970’s when polymeric hydrogels were considered as
drug delivery devices [162]. In recent years major emphasis has been put on the studying
of polymeric hydrogels in biomedical research related to drug delivery [1-8,164] due to
their dynamic properties described in Section 1.1 of this chapter.
33
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Until the early 1970’s, drugs were delivered to the human body exclusively via oral and
intravenous means [6]. Though these conventional means of drug administration are still
common to date due to their relative availability and easy excess, there are various
disadvantages associated with them. Firstly, high doses of drug cannot be injected into the
body at one time. Secondly, intravenous delivery leads to high concentration of drug into
the blood stream leading to toxic side effects. Furthermore, only a very small percentage
of the injected drug reaches the affected area in the body and hence multiple injections are
often required for an effective treatment. A typical graphical representation of the drug
dose level in blood when administered through conventional methods is depicted in
Figure 18 [165].
Figure 18. Drug levels in blood with traditional drug dosing
Supplying the appropriate amount of medicine to the body is essential to the success of a
treatment. This concept serves as the foundation for sustained drug release systems [162].
In the early stages of the research on controlled drug delivery devices the major challenge
faced was in obtaining zero-order sustained release of the drug over a prolonged period
[1,164]. The premise of zero-order drug release is to maintain a constant drug
concentration in blood for an extended period of time [1]. Current technology on
sustained drug release devices has improved to such a level that delivery of drugs at a
constant rate could be achieved for a certain period of time ranging from days to years
[1,166,167].
34
Chapter 1: Introduction
A variety of methods have been used to target biologically active molecules to the
specific site and extend their therapeutic lifetimes once inside the body [60,164]. The use
of swellable materials for drug delivery applications has followed experimental and
theoretical investigations of drug transport in polymeric delivery systems [168]. A
graphical representation of drug dosing through a sustained release device is depicted in
Figure 19 [165]. Controlled drug delivery occurs when the polymer, whether natural or
synthetic is judiciously combined with a drug, and the drug is then released over a desired
period into the appropriate biological environment [6,165]. The release of the active drug
may be constant over a long period, cyclic over a long period or triggered by the
environment or other external factors [6,165].
Advantages of controlled drug release devices thus possibly include delivery to the
required site, delivery at required rate, fewer applications, reduced dangers of overdose
and economic advantages by the virtue of more efficient dosage, at the expense of
possibly more complicated fabrication [6,165,169].
Figure 19. Drug levels in blood with controlled drug delivery dosing
Hydrogels have to be biocompatible and biodegradable to be ideal for drug delivery
applications. The degradation products should be non-toxic and should not cause an
inflammatory response. The degradation should also occur within a reasonable period as
required by the application [1-8].
35
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
1.4.2. Mechanisms of Controlled Drug Delivery
A convenient classification of controlled-release systems is based on the mechanism that
triggers the release of the incorporated drug. A drug delivery mechanism is ideal for a
device, which exhibits a zero order drug release [1,6,164].
Table 4. Classification of Controlled Release Systems
Type Controlling step Drug release mechanism
Diffusion-controlled devices
Reservoir (membrane)
devices
Matrix (monolithic)
devices
Concentration difference
Concentration difference
Diffusion
Diffusion
Chemically-controlled devices
Biodegradable
(bioerodible) devices
Pendant chain devices
Degradation
Hydrolysis
Reaction-dependent
diffusion
Reaction and Diffusion
Solvent-activated systems
Osmotically controlled
devices
Swellable systems
Swelling-controlled
systems
Osmosis
Swelling
Swelling front
Osmotic flow
Diffusion
Relaxation-dependent
diffusion
External force-induced release
Magnetically controlled
devices
Magnetic field
Diffusion
36
Chapter 1: Introduction
Depending on the mode of delivery, the mechanisms involved in the controlled release of
the drug from the device may vary. The three primary mechanisms by which the
therapeutic drugs can be released from a delivery system are diffusion, polymeric
degradation and swelling followed by diffusion [6,165,168,170]. Any or all of these
mechanisms may occur for a given drug release system. Table 4, adopted from Peppas
and Korsmeyer [171], summarizes the various types of controlled release systems.
1.4.2.1. Diffusion-Controlled Release
The most common mechanism of release is that of diffusion. In diffusion systems the
therapeutic drug, which may be either encapsulated in the polymer membrane or
suspended within the polymer matrix passes through the polymer that forms the
controlled release device when placed in an aqueous media [6,165]. The medium diffuses
into the matrix, dissolves the incorporated drug, which then diffuses out of its carrier. The
diffusion can occur on a macroscopic scale as through pores in the polymer matrix,
depicted in Figure 20 [165] or on a molecular level by passing between chains as that of
reservoir devices depicted in Figure 21 [165]. In the matrix system the drug release rate
depends upon the amount of drug present at a particular time, thus the rate of release is
time dependent. [6,165,170,172].
Figure 20. Drug delivery from a typical matrix drug delivery system
37
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Figure 21. Drug delivery from typical reservoir devices
(a) transdermal systems (b) implantable or oral systems
In reservoir devices the active ingredient within the polymer matrix forms a core
surrounded by an inert polymeric film or membrane, which acts as the diffusion barrier.
This membrane surrounding the reservoir is the only structure, which effectively limits
the release of the drug molecule in such systems [1,165]. The diffusion of the aqueous
medium through the polymeric membrane surrounding the reservoir device is the rate-
determining step. Furthermore, since the polymeric membrane systems surrounding the
reservoir is essentially uniform and of a non-changing nature, the rate of drug release is
fairly constant and is proportional to the concentration of the incorporated drug initially
present [6,165,170,172].
1.4.2.2. Drug Release Through Biodegradation
Polymer degradation is another interesting phenomenon in which the drug is released
from the carrier device. Biodegradable polymers are designed to degrade into biologically
acceptable and progressively smaller molecules and as the polymer degrades the
imbedded drug is freed into the host [6,165,170,172]. The drug release through polymer
degradation is depicted in Figure 22 [165].
38
Chapter 1: Introduction
Figure 22. Drug delivery through polymer degradation
(a) Bulk erosion (b) Surface erosion
The biodegradation may occur through bulk hydrolysis where the polymer randomly
degrades throughout the matrix. The rate of erosion is dependent on the volume of the
matrix rather than the thickness thus the rate of drug release in this case is unpredictable
and dumping effect of the dose is commonly observed [165,173,174]. This could be
solved by the use of polymer systems that are highly hydrophobic yet contain water labile
linkages [174]. These systems undergo surface erosion with minimum internal
degradation, thus the release rate is proportional to the polymer degradation rate with
proper surface geometry [165,173,174]. The most common formulations of biodegradable
materials are that of microparticles used in oral delivery and injectable delivery systems
[165].
1.4.2.3. Swelling-Controlled Release Systems
The release of the drug could occur from matrix or reservoir devices. The coupling of
diffusion and the macromolecular relaxation of the carrier control the release mechanism
of the incorporated drug providing conditions for zero-order release. A typical polymeric
swelling-controlled release system is depicted in Figure 23 [165]. The dry polymer slab
39
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
when placed in an aqueous medium swells thus increasing its aqueous solvent content
within the formulation as well as the polymer mesh size as a result, allowing the
incorporated drug to diffuse out into the host environment [6,60,165].
Figure 23. Drug delivery from (a) reservoir and (b) matrix
swelling-controlled release systems
1.4.2.3.1. Solute Transport in Swelling-Controlled Release Systems
The incorporated drug is essentially immobile in the glassy region of the polymer but
begins to diffuse out as the polymer swells in the compatible penetrant medium. The
release of drug thus depends on two simultaneous rates processes, medium migration into
the polymer network and the solute diffusion out of the network. The solubility of the
drug for a given medium is also essential.
In swelling-controlled release systems, the polymer has to swell to some extent before the
drug can diffuse out, thus the initial burst effect of the drug is often observed [6,171,175].
The continued swelling of the polymer eases the diffusion of the drug, ameliorating the
40
Chapter 1: Introduction
slow tailing off of the release curve [171,176,177]. The net effect of the swelling process
is to prolong and linearize the release profile of the drug. A schematic representation of
the swelling-controlled release action is illustrated in Figure 24 [171].
P G
M
D
υ
Figure 24. Schematic representation of a swelling controlled release system
The penetrant medium (M) enters the initially glassy polymer (P) with velocity (υ). The
incorporated (D) drug diffuses through the swollen gel layer (G).
1.4.2.3.1.1. Fick’s Laws of Diffusion
Understanding the mechanisms of the penetrant medium diffusion into the swellable
polymer is crucial to define the release profile of the incorporated solute. Fick [178]
developed two differential equations referred to as Fick's First and second laws to
describe the diffusion phenomenon in thin membranes in one dimension. Fick’s first law
is described by Equation 1 where J is the flux, j is the flux per unit area, A is the area
across the diffusional field, D is the diffusional coefficient, c is the concentration of
solute, z is the distance and /∂ is the concentration gradient across the z axis. c∂ z
41
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
c
J Aj ADz
∂= − = − ∂ Equation 1
The law states that the flux of a component of concentration across a membrane of unit
area, in a predefined plane, is proportional to the concentration differential across that
plane. In the case of diffusion without convection and unitary area, Equation 1 could be
re-written as described by Equation 2, which is the starting point for numerous
descriptions of diffusion behaviour in swellable polymers [171,179,180].
c
J Dz
∂= − ∂ Equation 2
Fick’s second law with constant boundary conditions can successfully describe much of
the observed solute transport in polymers. It is frequently successful in describing
transport both above and below Tg [175]. It states that the rate of change of concentration
in a volume element of a membrane, within the diffusional field, is proportional to the
rate of change of concentration gradient at that point in the field, as given by Equation 3
[175,179].
2
2
CD
t x
∂ ∂=∂ ∂C
Equation 3
The boundary conditions are:
t = 0 -1/2 < x < 1/2 C = C1
t > 0 x = + 1/2 C = C0
1.4.2.3.1.2. Fickian and Non-Fickian Diffusion
Diffusion in polymers is known to be associated with the physical properties of the gel
network and the interaction between the polymer and the penetrant medium. Based on
Fick’s law of diffusion, Alfrey et al [181] proposed a classification of diffusional
behaviours namely Fickian (Case I) and non-Fickian (Case II and anomalous) in
swellable polymers. The propositions were made in accordance to the penetrant diffusion
rate and the polymer relaxation rate.
42
Chapter 1: Introduction
In the Fickian (Case I) diffusion the penetrant mobility rate is much lower than the
segmental relaxation rate. The reduced driving concentration gradient slows down the
diffusion rate in the polymer slab geometry. In non-Fickian (Case II) diffusion, however,
the mobility rate of the penetrant is much higher than the segmental relaxation rate. The
sharp boundary between the gel phase formed by the penetrant and the glassy portion of
the polymer becomes the rate-determining step [171,179]. Anomalous diffusion
behaviour is characterized by the intermediate properties between the Fickian and Case II
behaviour [179].
Time dependent swelling behaviour in swellable polymers has been generally described
in the literature [27,171,175,176,182] according to Equation 4, normally termed as the
power-law model, with n being the diffusional exponent.
t nM
KtM ∞
= Equation 4
Mt/M∞ represents the fractional uptake of the penetrant medium or release of the
incorporated solute at time (t) normalized with respect to equilibrium conditions. The k
value is a constant, which incorporates the characteristics of the macromolecular
network/drug system and the dissolution medium [175]. The parameter n determines the
dependence of the medium uptake or release rate on time.
Table 5. Transport mechanisms of penetrant through a polymer slab
Exponent n Type of transport Time dependence
0.5 Fickian diffusion f (t –0.5
)
0.5 < n < 1.0 Non-Fickian diffusion (anomalous) f (t n-1
)
1.0 Case II transport Time-independent
n > 1.0 Super Case II transport f (t n-1
)
Table 5 [171,175] summarizes a list of possible transport mechanisms with their
characteristic n values and time dependence.
43
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Figure 25. The Fickian (A) and Case-II transport (B) penetrant uptake isotherm into a
sphere of radius a, with diffusion coefficient D (for Fickian diffusion) or relaxation
constant ka (for Case-II transport). Mt and M∞ represent mass of the swollen gel at time (t)
and infinity respectively.
A graphical representation of the two extreme solute transport diffusion mechanisms,
Fickian and non-Fickian diffusion behaviour in swellable polymers is shown in Figure 25
[171]. The power-law model despite being widely used in the literature and its importance
in defining Fickian and non-Fickian diffusion pattern has limitations [171,175,176]. The
power-law model generally accommodates only for Mt/M∞ ≤ 0.7. Thus in situations
where it is important to model the entire swelling or release curve, more sophisticated
models must be used [171].
1.4.2.3.1.3. Dimensionless Analytical Parameters
Brazel and Peppas [176] suggest that penetrant uptake and drug delivery from swelling-
controlled release systems can be described sufficiently by the two dimensionless
parameters for most polymer/solvent systems. These parameters are referred to as the
diffusional Deborah number (De), and the swelling interface number (Sw).
Vrentas et al [183] attempted to establish the regions of Fickian and non-Fickian
diffusion in the swellable polymer behaviour by introducing a dimensionless parameter,
the De. The ratio of the characteristic relaxation time to the characteristic diffusion time is
referred to as the De (Equation 5) where λ is the characteristic relaxation time for
polymer when subjected to swelling and θ is the characteristic diffusion time into the
44
Chapter 1: Introduction
swelling sample [176,177,184]. The parameter θ is defined as the square of the
diffusional distance divided by the diffusion coefficient of the penetrant in the polymer.
De = λθ Equation 5
When a glassy polymer is placed in contact with a swelling agent, the swelling process is
accompanied by the transition of glassy to rubbery state as a result of lowering the glass
transition temperature (Tg) of the polymer [171,180]. This is attributed to the increased
mobility of the macromolecular chains in the presence of the swelling agent as shown in
Figure 26 [177].
Figure 26. Effect of penetrant concentration on the glass transition temperature
In Figure 26, the glassy to rubbery state transmission is represented by a dashed curve.
Zone III, defined arbitrarily around this transition, is the region where the De is
approximately equal to one. Non-Fickian (anomalous) transport is observed in this region.
In zone II, which is the rubbery state, the De is much smaller than one and thus Fickian
diffusion is observed. In zone I (glassy state) the De is much larger than one and Fickian
diffusion is again observed.
45
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Fickian diffusion is observed when the time scale of the macromolecular relaxation is
either effectively infinite or zero compared to the time required to establish a
concentration profile in the polymer [175,179,180]. Non-Fickian diffusion behaviour is
normally observed in glassy polymers when the Tg of the polymer is higher than the
environmental temperature [180]. At a specific temperature below the Tg, the polymer
chains are not sufficiently mobile to permit immediate penetration of the medium in the
polymer core [185].
The swelling interface number (Sw) is important in describing the balance between the
solvent penetration and the release of the incorporated drug according to Equation 6
[184].
Sw = s,s
u
D
δ Equation 6
The Sw value (Equation 6), expresses the ratio between the solvent motion and solute
diffusion where u is the velocity of the penetrant front, δ is the thickness of the swollen
region through which the solute diffuses out and Ds,s is the diffusion coefficient of the
incorporated solute [176,177,182,184]. In the case where Sw value smaller or greater than
1 the release pattern has been suggested to be controlled by the solvent penetration or
drug diffusion and the time dependence is Fickian. However, in the case where the Sw
value is in the order of one, anomalous behaviour is observed [176].
1.4.2.3.2. Influential Factors in Swelling-Controlled Release Systems
One of the most remarkable, and useful, features of a polymer's swelling ability manifests
itself when the swelling can be triggered by a change in the environment surrounding the
delivery system [26,165,186]. The external environmental conditions could involve pH,
temperature, magnetic field or ionic strength. The gels may either shrink or swell in
response to such environmental changes as illustrated in Figure 27 [[6,7,165,186-189].
46
Chapter 1: Introduction
Figure 27. Environmental sensitive swelling-controlled release system
The effect of the environmental conditions on the polymer’s performance, however, is
dependent on the nature of the polymer, which could be ionic or neutral. The swelling-
release action in neutral hydrogels is driven by the thermodynamic mixing contribution of
the penetrant medium and the polymer to the overall free energy, which is coupled with
an elastic polymer contribution [175,190]. In ionic hydrogels the driving forces are the
same as that of neutral gels along with some additional contributors such as the ionic
interactions between the charged polymer and the free ions [191]. For most of these
polymers, the structural changes are reversible and repeatable upon additional changes in
the external environment [165].
Hydrogels could be synthesized appropriately to achieve the desired response from a
given environmental condition. Parameters such as the polymer composition, degree of
crosslinking density and the size and nature of the incorporated drug molecule play an
important role in determining the drug release behaviour and thus must be considered
during the design of swelling-controlled release devices [6,8,175].
47
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
1.4.2.3.2.1. Effect of the Polymer Composition
The composition of the polymer defines its nature as a neutral or ionic network and
furthermore, its hydrophilic/hydrophobic characteristics. Neutral and ionic polymeric
hydrogel networks have been discussed in Section 1.1.1. Presence of hydrophilic
components in the polymer network enhances the swelling characteristics of the polymer.
Hydrophobic components on the other hand reduce the swelling efficiency [175,192].
1.4.2.3.2.2. Effect of the Crosslinking Density
Increase in crosslinking density through addition of crosslinking agents such as divinyl
glycol (DVG), divinyl benzene (DVB) or tripropyleneglycol diacrylate (TPGDA) are
known to reduce the equilibrium swelling [6,8]. Reduced swelling is often marked with
reduced diffusion coefficient. Lee et al [193] in their study on the diffusion coefficients in
crosslinked PHEMA hydrogels found a reduction in the diffusion coefficient values with
increased crosslinking density.
1.4.2.3.2.3. Effect of the Environmental pH
Ionic hydrogels, which could be cationic, containing basic functional groups or anionic,
containing acidic functional groups, have been reported to be very sensitive to changes in
the environmental pH [2,26,27,88,175,186]. The swelling properties of the ionic
hydrogels are unique due to the ionization of their pendent functional groups [175]. The
equilibrium swelling behaviour of ionic hydrogels containing acidic and/or basic
functional groups is illustrated in Figure 28 [175].
Ng et al [26] in their studies on cationic rich polyampholytes found increased swelling
activity in acidic conditions and reduced swelling in basic conditions. Brannon-Peppas
and Peppas [27] on the other hand studied the swelling behaviour of pH sensitive anionic
hydrogels based on HEMA, methacrylic acid and maleic anhydride in varied pH
environments. They reported low swelling activity of the hydrogels in acidic medium but
very high swelling activity in basic medium.
48
Chapter 1: Introduction
Figure 27. Equilibrium degree of swelling in response to pH
Most useful pH-sensitive polymers swell at high pH values and collapse at low pH
values, the triggered drug delivery occurs upon an increase in the pH of the environment.
Such materials are ideal for systems such as oral delivery, in which the drug is not
released at low pH values in the stomach but rather at high pH values in the upper small
intestine [26,165].
1.4.2.3.2.4. Effect of the Environmental Temperature
Changes in the environmental temperature may either enhance the swelling ability of the
hydrogel or in contrary could cause the hydrogel to collapse. Physical gels that contain
hydrophilic components exhibit enhanced swelling behaviour at elevated temperatures
and are referred to as thermo-swelling gel [194]. However, gel networks composed of
relatively hydrophobic components shrink at elevated temperatures. These networks are
referred to as thermoshrinking networks [195]. Thermoshrinking gels undergo reversible
swelling and de-swelling in response to changes in environmental temperature [1,195].
Hoffman et al [196] in their studies on thermo-responsive hydrogels based on N-
isopropyl acrylamide and methacrylic acid found that the gels shrunk at elevated
temperatures but swelled to equilibrium at low temperatures. According to their study the
process was reversible and they suggested the existence of a low critical solution
temperature (LCST). The temperature, which induces the polymer to collapse, is referred
to as the LCST [164,175,197]. Tanaka [197] has used the thermodynamic approach to
49
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
explain this behaviour. An increased swelling activity is observed at temperatures lower
than the LCST and the collapse of the hydrogel is observed above the LCST. Thermal
responsive hydrogels and membranes have been extensively evaluated as platforms for
pulsatile delivery of drugs [25,164,188]
1.4.2.3.2.5. Effect of the Ionic Strength
According to the concept of Donnan equilibrium, an increase in the ionic strength of the
swelling agent increases the ionization of a weakly polyelectrolytes system thus leading
to high swelling activity [26,175]. However, once the ionic hydrogel has been fully
ionized, further increase in the ionic content of the swelling agent will cause the hydrogel
to de-swell due to the screening effect of the counterions [187]. Anionic gels are normally
unionized at a pH lower than the gel pKa while cationic gels display the opposite
behaviour and the pH is dependent on the pKb of the gel [175].
Khare and Peppas [187] in their study on anionic hydrogels observed a decrease in the
swelling activity upon a further increase in the ionic strength of the swelling agent at a
constant pH higher than the pKa of the gel. According to a recent study by Ostroha et al
[84], the dependence of the swelling behaviour on the ionic strength is significant when
the operational pH is close to the transitional value of the degree of swelling.
1.4.2.3.2.6. Effect of the Nature and Size of the Drug
The size and the nature of the incorporated drug play a very important role in determining
the efficiency of its release from the carrier. Yasuda et al [198,199] found a linear
dependence of the solute diffusion coefficient in the swollen polymer system on the
molecular size of the solute and the reciprocal of the degree of swelling. An increase in
the molecular size of the drug reduces the drug release rate [6,168].
Ng et al [26] in their recent study on the release rate of the model drugs vitamin B1 and
vitamin B in ionic hydrogels showed that the nature of the drug also affects the release
properties of the carrier. They suggest that the coulombic interactions between the
charges borne by the vitamin and the hydrogel matrices are also influential in the release
pattern of the drugs.
50
Chapter 1: Introduction
1.5. Testing of Biomedical Polymeric Hydrogels
Biomaterials should perform with appropriate host response in a specific application
without toxic, inflammatory, carcinogenic and immunogenic responses [200,201]. An
appropriate response of the biomaterial for its particular application would be referred to
as an inert or positive interaction with the host [156]. Toxicology testing of biomaterials
generally includes examination of the local tissue response, systematic toxicological
response, and allergic, pyrogenic, carcinogenic and teratogenic responses [202].
The local tissue cells are usually tested for toxicity activity of the given biomaterial. A
cell proliferation assay is normally conducted to determine the cell viability, which
indicates whether or not the material for intended biomedical use is biocompatible.
Mosmann [203] developed a quantitative colorimetric assay for mammalian cell survival
and proliferation using a tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide (MTT). The assay has been widely accepted as a better alternative to
the previously used radioactive assays, which made use of hazardous radioactive isotopes.
Furthermore, the technique using the tetrazolium salt reflects metabolic activity rather
than cell division thus providing a new approach for the study of cell function [204].
NN
+
NN
S
N N
N NH
N
S
N
e-
MTT (yellow crystals) Formazan (purple crystals)
Scheme 16. Reductive mechanism in the formazan formation
The MTT assay is based on the conversion of yellow water-soluble MTT salt to water-
insoluble purple formazan crystals in the presence of live cells by the reductive cleavage
51
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
of the tetrazolium ring [203,204] as illustrated in Scheme 16. The exact cellular reactions
involved in the reduction of the salt are not clearly understood to date. However, a
number of reaction models have been proposed. The widely accepted assumption of the
reduction process is based on the study carried out by Slater et al [205]. They suggest that
the mitochondrial succinate dehydrogenase in living cells reduces the MTT to formazan.
The amount of the formazan generated is directly proportional to the live cell numbers
over a wide range, using a homogeneous population. Mosmann [203] also found that the
activated cells produce more formazan than the resting cells, which allowed the
measurement of activation even in the absence of cell proliferation. Gerlier and
Thomasset [204] in their study on cell activation have also reported concurrent findings.
Furthermore, the MTT assay could be used for cell viability assessment of all cell types.
1.6. Research Insight
Polymeric hydrogels, since their discovery and introduction into the biomedical arena in
the early 1960s have been of great research interest [1,2]. Numerous researchers around
the globe have carried out extensive research on intelligent polymeric hydrogels and this
has resulted in some very classical and important developments in such materials.
However, there still seems to be an infinite range of possible applications of these
versatile materials. Thus there remains an everlasting quest to achieve superiority over
present hydrogel systems in terms of biocompatibility, mechanical strength, response to
environment and economy to meet the requirements of such applications. These versatile
materials have found numerous important medical and pharmaceutical applications.
One such area of application of great interest has been that of swelling-controlled drug
release systems. Much of the research on hydrogels has been focussed on application in
controlled drug delivery systems, as these are more effective, more patient friendly and
more cost effective towards a particular treatment in comparison to pills and injections,
which are the conventional drug administration methods [1-8]. However, the need for
cheaper, more responsive and more biocompatible substitute drug delivery systems
continues.
52
Chapter 1: Introduction
1.6.1. Research Direction
The direction of the research in this Ph.D. project will be geared towards obtaining
hydrogels for slow drug delivery applications through an economical and efficient
polymerisation process. These hydrogels would possess enhanced properties such as
mechanical strength, swelling-drug release properties and biocompatibility. Hydrogels
will be prepared from a range of monomers, some with specific functional groups, which
contribute to responses to changes in environmental conditions such as pH, temperature
and ionic strength. The use of UV radiation to achieve polymerisation of hydrogels is a
widely used radiation technique today, which is largely due to its ease of operation,
economy and environmental friendliness.
However, in order to proceed efficiently with the polymerisation process, UV curable
systems normally require the presence of a photoinitiator in the reacting monomer
mixture. A photoinitiator is a photosensitive chemical, which is converted into reactive
radicals upon exposure to the UV light. Photoinitiators besides being costly if not
completely utilized in the polymerisation process can lead to undesirable toxic impurities
trapped in the polymer matrix that may leach out of the matrix in a biomedical
application. This has been a major issue pointed by a number of research publications
[110,122] and this will be addressed in this research project through the formation of
charge-transfer complexes. This process is photoinitiator-free.
This project will involve the study of the swelling-controlled release systems synthesized
via UV radiation in the presence and absence of photoinitiators. NVP and HEMA are the
monomers of interest in this study for the synthesis of hydrogels. PHEMA has been
widely used as controlled release devices [51] due to its high level of biocompatibility. A
good reason for combining NVP, a highly hydrophilic monomer and HEMA is to give
high water absorbing hydrogels [52], which makes them suitable for rapid drug delivery
systems. In this study, photoinitiator-free UV curable systems, which involve electron
donor/acceptor type monomers, will include acrylic acid, HEMA and N-hydroxyalkyl
maleimides as acceptors and NVP as a donor used to form charge-transfer complexes,
which generate initiating radicals essential for the polymerisation process.
53
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
A series of N-hydroxyalkyl maleimides namely, HMMI, HEMI, HPrMI and HPMI will
be used as acceptor type monomers and will be photopolymerised in conjunction with
NVP, a donor monomer in the absence of a photoinitiator. IPNs involving chitosan and its
derivative carboxymethyl chitosan will also be prepared through the PI-free
polymerisation technique. The resultant polymers will be investigated in vitro for their
biocompatibility.
The Differential Photocalorimetric technique will be used to evaluate the suitability of the
donor/acceptor pairs for the formation of CT complexes. The effect of hydrogen donors
such as glucose and glucosamine hydrochloride on the efficiency of CT complex
formation will also be investigated.
Hydrogels synthesized through the conventional photopolymerisation process in presence
of a photoinitiator and through the CT complex polymerisation will be tested for their
water uptake and drug release capabilities. The swelling and drug release tests on these
hydrogels will be conducted in varied pH environments to evaluate the effect of pH
changes on the swelling and drug release behaviour. A number of model drugs with
varying molecular weights will be utilized for the drug release experiments. The kinetics
of drug release from the various hydrogel networks and the effect of the molecular weight
of the incorporated drug on the release kinetics will be investigated.
The hydrogels prepared are intended for biomedical applications as implants or
transdermal controlled drug release devices thus there is a need for these materials to
satisfy the definition of being biocompatible. Hydrogels are known to be biocompatible
owing their dynamic structures. However, these materials are prepared via UV radiation
where the conversion of monomers to polymer networks is never 100 %. Thus the
possibility of some unreacted monomers contained in polymer network is inevitable.
These unreacted components could be highly toxic thus unsuitable for biomedical
applications. The hydrogels prepared will be cleaned thoroughly and subjected to 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay
using human keratinocyte (HaCaT) cells to confirm the inertness or positive response of
the hydrogels to the host cells.
54
Chapter 1: Introduction
1.7. References
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
1.1. Synthetic Hydrogels 1
1.1.1. Classification of Hydrogels 3
1.1.1.1. Homopolymeric Hydrogels 3
1.1.1.2. Copolymeric Hydrogels 4
1.1.1.3. Interpenetrating Polymer Network (IPN) Hydrogels 5
1.1.1.4. Non-Ionic Hydrogels 6
1.1.1.5. Ionic Hydrogels 7
1.1.1.5.1. Anionic Hydrogels 8
1.1.1.5.2. Cationic Hydrogels 8
1.1.1.5.3. Polyampholytic Hydrogels 9
1.1.1.6. Hydrogel Network Structures 10
1.1.1.6.1. Amorphous Hydrogel Structures 12
1.1.1.6.2. Semicrystalline Hydrogel Structures 12
1.1.1.6.3. Hydrogen Bonded Hydrogel Structures 13
1.2. Synthesis of Polymeric Hydrogels 13
1.2.1. Chain Initiation 14
1.2.2. Chain Propagation 15
1.2.3. Chain Termination 15
1.2.4. Nature of the Reactive Radical Species 16
1.2.5. Curing Processes 17
1.2.5.1. Ionizing Radiation Sources 17
1.2.5.1.1. Electron Beam (EB) Radiation Process 17
1.2.5.1.2. Gamma Radiation Process 18
1.2.5.2. Ultra Violet (UV) Radiation Process 18
1.2.6. Charge-Transfer (CT) Complex Polymerisation 19
1.2.6.1. Charge-Transfer (CT) Interactions 20
1.2.6.1.1. Decay Mechanisms of Excited State CT Complexes 22
1.2.6.2. Inducement of CT Complex Polymerisation 24
1.2.6.3. Proposed Mechanisms of CT Complex Polymerisation 25
1.2.6.4. Influential Factors in CT Complex Polymerisation 26
1.2.6.4.1. Effect of Monomers 27
1.2.6.4.2. Effect of Lewis Acids 28
1.2.6.4.2. Effect of Hydrogen Donors 28
1.2.6.5. Polymeric Hydrogel Synthesis via CT Complex Formation 29
1.3. Applications of Hydrogels 30
1.3.1. Agricultural Applications 30
1.3.2. Other Applications 31
1.4. Hydrogels as Biomaterials 32
1.4.1. Sustained Drug Delivery Devices 33
1.4.2. Mechanisms of Controlled Drug Delivery 36
1.4.2.1. Diffusion-Controlled Release 37
1.4.2.2. Drug Release Through Biodegradation 38
1.4.2.3. Swelling-Controlled Release Systems 39
1.4.2.3.1. Solute Transport in Swelling-Controlled Release Systems 40
1.4.2.3.1.1. Fick’s Laws of Diffusion 41
66
Chapter 1: Introduction
1.4.2.3.1.2. Fickian and Non-Fickian Diffusion 42
1.4.2.3.1.3. Dimensionless Analytical Parameters 44
1.4.2.3.2. Influential Factors in Swelling-Controlled Release Systems 46
1.4.2.3.2.1. Effect of the Polymer Composition 48
1.4.2.3.2.2. Effect of the Crosslinking Density 48
1.4.2.3.2.3. Effect of the Environmental pH 48
1.4.2.3.2.4. Effect of the Environmental Temperature 49
1.4.2.3.2.5. Effect of the Ionic Strength 50
1.4.2.3.2.6. Effect of the Nature and Size of the Drug 50
1.5. Testing of Biomedical Polymeric Hydrogels 51
1.6. Research Insight 52
1.6.1. Research Direction 53
1.7. References 55
67
Chapter 2: Experimental
2.1. Materials
In this research work, chemicals of high purity were utilized as received from the
suppliers with the exception of two monomers, which were further purified. These were
N-vinyl-2-pyrrolidinone (NVP) and 2-hydroxyethyl methacrylate (HEMA). HEMA was
purified by passing through an inhibitor remover column supplied by Aldrich to remove
the stabilizer hydroquinone while NVP was distilled off at 95 oC
under vacuum of 7 mm
Hg. The materials used in the experimental work and their suppliers are listed in Table 1.
The chemicals required for the synthesis of a series of N-hydroxyalkyl maleimides,
carboxymethyl chitosan and a model drug, manganese-5, 10, 15, 20-tetrakis(4-
hydroxyphenyl) porphyrin are also included in Table 1.
Table 1. Materials list and respective suppliers
Monomers
N-vinyl caprolactam (98%) Aldrich
N-vinyl-2-pyrrolidinone (99%) Sigma
2-hydroxyethyl methacrylate (98%) Sigma
Acrylic acid (99%) Sigma
Polysaccharide
Chitosan (85 % deacetylation) Sigma
Hydrogen Donors
D(+)-Glucose Sigma
D-Glucosamine hydrochloride Sigma
Photoinitiator
Bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide (Irgacure® 819) Ciba-Geigy
66
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Chemical Reagents- Buffer preparation
Disodium hydrogen orthophosphate (Na2HPO4) (A.R. grade) Ajax Chemicals
Sodium dihydrogen orthophosphate (NaH2PO4) (A.R. grade) BDH
Phosphoric acid (A.R grade) BDH
Chemical Reagents - Synthetic work
Maleic anhydride (95%) Sigma
Maleimide (99%) Aldrich
Formaldehyde 37 wt.% Aldrich
3-Amino-1-propanol (99%) Aldrich
5-Amino-1-pentanol (95%) Aldrich
Ethanolamine (98%) Aldrich
Furan (98%) Sigma
Manganese chloride (A.R grade) Ajax chemicals
Monochloro acetic acid (A.R. grade) BDH
Sodium hydroxide (97%) Aldrich
5, 10, 15, 20 tetrakis(4-hydroxyphenyl)-21H, 23H-porphine (99%) Aldrich
67
Chapter 2: Experimental
Solvents
Dichloromethane (A.R grade) BDH
Chloroform (A.R grade) BDH
Chloroform-d (CDCl3) (A.R grade) Aldrich
Deuterium oxide (D2O) (A.R grade) Aldrich
Acetone (A.R grade) APS Chemicals
Methanol (A.R grade) BDH
Ethanol (A.R grade) BDH
Ethyl acetate (99.5 + %) Aldrich
Levulinic acid (98%) Sigma
Cremophor EL (CRM) Sigma
1,2-propanediol (Prg) Sigma
Toluene (L.R grade) BDH
Petroleum ether (A.R grade) Ajax chemicals
Model Drugs
Theophylline Sigma
Thiamine hydrochloride (vitamin B1) Aldrich
Uranyl Actinometer – Lamp calibration reagents
Uranyl nitrate [UO2 (NO3)2. 6H2O] (A.R grade) Ajax chemicals
Oxalic acid (COOH)2 (99 + %) Aldrich
Potassium permanganate (KMnO4) (A.R grade) BDH
Sulphuric acid (A.R grade) BDH
68
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Biological Reagents- Cytotoxicity test
HaCaT cells Skin Tech, UWS
Fetal calf (bovine) serum JRH Biosciences
Dulbecco's modified Eagle's medium (DMEM) - Ca2+
free Thermo Trace
DMEM – w/o phenol red Thermo Trace
L-Glutamine Sigma
Penicillin/streptomycin (5000U/ml, 5000ug/ml) Thermo Trace
Anhydrous isopropanol Sigma
Hydrochloric acid (HCl) BDH
Ethylenediamine tetraacetic acid Sigma
Dulbecco's phosphate buffered saline Thermo Trace
Trypsin Sigma
D-Glucose Sigma
EDTA Disodium Sigma
Potassium chloride (KCl) Sigma
Sodium bicarbonate (NaHCO3) Sigma
Sodium chloride (NaCl) Sigma
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) Sigma
N-[2-hydroxyethyl] piperazine-N-[2-ethane sulfonic acid] (HEPES) Sigma
69
Chapter 2: Experimental
2.2. Equipment
2.2.1. Radiation Source
A 90 W high-pressure mercury vapour filled lamp, manufactured by Phillips (Holland),
was used as the ultra violet (UV) light source. The lamp was mounted at the centre of an
enclosed drum, which contained a circular rack fitted with a rotor to hold six Pyrex
sample tubes, each with a diameter of 2 cm and a height of 10 cm at a distance of 10 cm
from the lamp. The drum had air vents at the bottom and was also fitted with a fan to
control excessive heat. The energy absorption per sample was determined by an uranyl-
oxalate actinometer.
The calculations were made on the assumption that the energy of all the wavelengths
between 254 nm and 435 nm was absorbed completely. An average wavelength of 350
nm and an average quantum yield value of 0.57 were used in the calculations to determine
the dose rate [1]. The UV lamp calibration procedure is outlined in detail in Section 2.4.4.
The calculated energy flux value from the calibration was 9.65 x 10-2
J s-1
. This dose rate
value was used to determine the radiation dose applied to the monomer mixture samples
to achieve the formation of polymers which function as hydrogels.
2.2.2. Analytical Instruments
2.2.2.1. Ultra Violet - Visible (UV-Vis) Spectrophotometer
A Shimadzu UV-1601 PC spectrophotometer was used as the quantitative analytical tool
in the drug release experiments. The samples were measured in quartz cuvettes with a cell
length of 1 cm. The spectroscopic measurements were carried out in the range of 800 nm
to 200 nm with a slit width value of 2.0 nm. Calibration curves of the standards of each of
the drugs analysed were plotted and used for drug release calculations.
2.2.2.2. Gas Chromatograph Mass Spectrometer (GC-MS)
Low-resolution mass spectra were recorded on a Shimadzu QP5000/GC17A equipped
with an MS ion trap detector. Solid samples were dissolved in CH2Cl2 and subsequently
admitted to the ion source using a direct insertion probe. The probe had at its tip a small
container in which a small amount of sample solution was placed. The probe was inserted
through a vacuum lock to within a few millimetres of the ion beam, where precise and
70
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
controllable internal heating was used to gently evaporate the sample with minimum
decomposition.
2.2.2.3. Nuclear Magnetic Resonance (NMR) Spectrometer
2.2.2.3.1. Carbon-13 (13
C) and Proton (1H) NMR
NMR spectroscopic analysis of liquid samples was performed on Varian unity-plus 300
MHz and Mercury 400 MHz spectrometers. Solid samples were either dissolved in CDCl3
or D2O. Chemical shifts were recorded in ppm. 1H and
13C{
1H} spectra were referenced,
internally to the residual solvent peak. The 13
C and 1H spectra in D2O were referenced
with respect to TSP dissolved in D2O.
2.2.2.3.2. Proton NMR Relaxation (T1 and T2) Measurements
Proton NMR relaxation (T1 and T2) measurements were conducted on Varian unity-plus
300 MHz NMR spectrometer equipped with micro-imaging accessory. A micro-imaging
probe was employed for these measurements. The swollen hydrogel samples were
inserted into the micro-imaging probe with 30 mm insert. T1 measurements were
performed using inversion recovery technique while T2 measurements were performed
employing spin-echo technique with CPMG pulse sequence.
2.2.2.4. Fourier Transform Infrared (FT-IR) Spectrometer
Fourier Transform Infrared spectroscopic analysis was conducted on a BIO-RAD FTS
3000 MX FT-IR spectrometer. Potassium bromide (KBr) discs containing samples to be
analysed were prepared by mixing 0.5 – 1.0 mg of the sample with approximately 100 mg
of powdered KBr. The mixture was ground before being compressed in a special metal
KBr die under pressure of 15 - 30 tonnes to produce transparent KBr discs. Merlin,
Version 1.2 was the analytical software used in conjunction with the FT-IR to process the
data. The sampling range was between 4000 cm-1
and 400 cm-1
with an average of 16
scans per sample.
2.2.2.5. Differential Photocalorimeter (DPC)
A Differential Photocalorimeter/DSC 2910 system by TA Instruments was used for
kinetic studies on CT complex formation of various donor/acceptor systems. Samples
71
Chapter 2: Experimental
were placed in a pre-weighed DSC aluminium pan using a micro-syringe, accurately
weighed to an approximate sample size of 2 mg. The aluminium pans were specially
crimped to maintain uniform sample size. The DSC 2910 system is equipped with a 200
W mercury arc lamp with a variable light intensity of 1 - 56 mW cm-2
. Light intensity was
measured with an IL 1440-A Radiometer supplied by International Light. Samples were
degassed in the DSC for 2 minutes prior to irradiation under a measured light intensity of
55.8 mW cm-2
in the presence of N2. Thermal Solutions software was used to operate the
instrument while Universal Analysis software, Version 2.5 was used to process the
acquired data.
2.2.2.6. Texture Analyser (TA)
A TA.XT2 texture analyser from Stable Micro Systems was used to evaluate the stiffness
and the viscoelasticity of the swollen hydrogel networks. A 1/2" ebonite cylinder was
used as the probe. The hydrated samples were in a cylindrical shape. The height and the
diameter of the samples were recorded prior to placement under the probe. The probe was
set to approach the sample at 1.0 mm s-1
with a trigger force of 0.01 N. Once the probe
was in contact with the sample, the test duration was 30 seconds with increase in
compression distance from 0-1.0 mm at a rate of 0.1000 + 0.0001 mm s-1
in the first 10
seconds and maintained compression distance of 1.0 mm in the later 20 seconds. A
compression stress-strain graph was obtained for each sample.
2.2.2.7. Atomic Absorption Spectrometer (AAS)
A GBC 902 double beam AAS was used to carry out the metal analysis. The samples
were dissolved in de-ionized water and aspirated into the air/acetylene flame. The
measurements were carried out using a hollow cathode lamp with a wavelength and a slit
width of 279.50 nm and 0.2 nm respectively. A range of standard solutions (1 ppm –10
ppm) of the metal was analysed to construct a calibration curve from which the metal
content in the sample was determined.
2.2.2.8. Microplate Reader
A BMG Labtechnologies FLUOstar OPTIMA microplate reader was used for the cell
proliferation assay. The reader was equipped with a high-energy xenon flash lamp as the
72
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
light source and a side window, current type photomultiplier tube as the detector.
Absorbance filters A560 and A650 were used as excitation and background filters
respectively. The absorbance measurements were recorded at 560 nm with background
absorbance of 650 nm in a plate mode over a single cycle with 20 flashes per well. The
FLUOstar OPTIMA v1.30-0 software was used to operate the instrument and process the
acquired raw data.
2.2.2.9. Microscope
A Nikon inverted microscope (TMS) equipped with a Nikon digital sight camera unit
(DS-5M-L1) was used for the image analysis of the cell cultures in the cytotoxicity
experiments. Microscopic examinations were carried out at 200 x magnification and the
images were captured on the digital camera.
2.3. Synthesis of Chemical Compounds
A range of chemical compounds used in this work was synthesized using the chemical
reagents described in Table 1. The syntheses and characterization of these compounds are
described in Sections 2.3.1-2.3.3. Detailed reaction mechanisms of the syntheses of these
compounds are illustrated in Appendix V.
2.3.1. Synthesis of a Model Drug
Manganese-5, 10, 15, 20-tetrakis(4-hydroxyphenyl) porphyrin (Mn-TPP-OH), a cancer
tumour-tracing agent was obtained through insertion of Mn into 5, 10, 15, 20 tetrakis(4-
hydroxyphenyl)-21H, 23H-porphine (TPP-OH). The detailed synthesis of Mn-TPP-OH is
described in Section 2.3.1.1. Mn-TPP-OH was used for the drug release studies.
2.3.1.1. Synthesis of Mn-TPP-OH.
TPP-OH (0.30 g, 0.055 mmol) was added to a 50 ml, three-neck, round-bottomed flask
fitted with a reflux condenser under N2 atmosphere, containing distilled CH2Cl2 (20 ml).
The solution was stirred magnetically for 10 minutes. To this, a solution of a manganese
(II) chloride (0.18 g, 0.053 mmol) in methanol (5 ml) was added and the reaction vessel
was shielded from ambient lighting. The flask was immersed in a water bath and the
solution was refluxed overnight. Insertion of the metal into the porphyrin was monitored
73
Chapter 2: Experimental
by UV-vis spectroscopy for completion. De-ionized water (50 ml) was added to the
precipitated crude product. This product was filtered, dissolved in CH2Cl2 and purified
via open column chromatography by passing through a silica column and eluting the
product fraction with CH2Cl2 : MeOH mixture (100:1) as the eluent. The solvents in the
eluted product fraction were removed under reduced pressure yielding 0.19 g (59 %) of
green crystalline solid Mn-TPP-OH (Figure 1).
NN
N N
OH
OH
OH
OH Mn
Figure 1. Mn-TPP-OH
2.3.1.1.1. UV-Vis Spectroscopic Analysis
UV-vis (CH2Cl2) λmax, ε (L mol-1
cm-1
): 610 nm, 5209; 571 nm, 4768; 520 nm, 4842.
2.3.1.1.2. AAS Analysis
Observed Mn composition: 7.40 % w/w
Calculated Mn composition (based on C44H30MnN4O4.H2O): 7.31 % w/w
2.3.2. Synthesis of N-Hydroxyalkyl Maleimides
A series of N-hydroxyalkyl maleimides, which were used as monomers were synthesized
through reverse Diels-Alder reaction of N-hydroxyalkyl maleimide adducts of furan. The
detailed syntheses of the starting materials and the N-hydroxyalkyl maleimides are
described in Sections 2.3.2.1-2.3.2.8.
74
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.3.2.1. Synthesis of HMMI
The N-hydroxymethyl maleimide (HMMI) (Figure 2) was synthesized according to the
method described by Tawney et al [2]. Maleimide (10 g, 0.103 mol) was added to 10 ml
of a 37 % solution of formaldehyde and heated to approximately 35 oC and 0.31 ml of a 5
% solution of NaOH was added. Within 10 minutes all of the maleimide had dissolved
and an exothermic reaction proceeded. The solution was stirred for 2 hours where white
crystals were observed after cooling to room temperature.
N
O
O
OH
ab c
Figure 2. HMMI
The solution was placed in a freezer overnight and the resulting crystals were filtered and
washed with ice-cold ethanol and diethyl ether. The crude product was purified twice by
sublimation yielding HMMI (9.77 g, 74.6 %) as white crystals, m.p. 104 oC (lit. 104-106
oC). The purity of the product was further evaluated using NMR and mass spectroscopy.
2.3.2.1.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: M+ and (M+1)
+ were not observed.
Calculated (C5H5NO3): 127.10
MS fragmentation, m/z (peak intensity): 99.15 (23.87, (M – 28); 80.15 (10.93, (M – 47));
55.10 (25.11, (M – 66)); 54.10 (100.00, (M – 73)); 53.10 (49.11, (M – 74)).
2.3.2.1.2. NMR Spectroscopic Analysis
1H NMR (D2O): δ ppm 6.79 (s, 2H, Ha); 4.88 (s, 2H, Hc)
13C NMR (D2O): δ ppm 172.26 (Cb); 135.31 (Ca); 59.73 (Cc).
75
Chapter 2: Experimental
2.3.2.2. Synthesis of Furan-A
The 3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalic anhydride (Furan-A) (Figure 3) was
synthesized according to the method described by Narita et al [3]. Maleic anhydride (20
g, 20.4 mmol) and ethyl acetate (25.5 ml) were mixed in a 100 ml round-bottomed flask.
Furan (17.35 g, 25.5 mmol) was added and the reaction mixture was stirred overnight at
room temperature. The precipitate was filtered and washed with ethyl acetate producing
18.15 g (89 %) of a white solid as the final product, m.p. 107 oC. NMR and mass
spectroscopy were used to confirm the purity of the product.
O
O
O
O
ab
c d
Figure 3. Furan-A
2.3.2.2.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: M+ and (M+1)
+ were not observed.
Calculated (C8H6O4): 166.03
MS fragmentation, m/z (peak intensity): 149.10 (0.68, (M – 17); 94.05 (4.14, (M – 72));
69.05 (5.59, (M – 80)), 68.00 (100.00, (M – 98)).
2.3.2.2.2. NMR Spectroscopic Analysis
1H NMR (CDCl3): δ ppm 6.56 (s, 2H, Ha); 5.44 (s, 2H, Hc); 3.15 (s, 2H, (Hb).
13C NMR (CDCl3): δ ppm 172.9 (Cd); 137.0 (Ca); 82.2 (Cb); 48.7 (Cc).
2.3.2.3. Synthesis of HEMI-A
The synthesis of 2-hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide (HEMI-
A) (Figure 4) was carried out in accordance to the method described by Narita et al [3].
Ethanolamine (4.2 g, 6.17 mmol) in ethanol (5 ml) was added drop-wise to a slurry of
Furan-A (10 g, 6 mmol) in ethanol (15 ml) in a 50 ml round-bottomed flask. This mixture
76
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
was then refluxed for 4 hours. After cooling to room temperature overnight, a white solid
had formed. The crude product was filtered and washed with ethanol followed by
petroleum ether yielding 7.27 g (58 %) of a white crystalline solid as the final product,
m.p. 132 oC (lit. 132
oC). NMR and mass spectroscopy were used to further evaluate the
purity of the product.
N
O
O
O
OH
a
bc d
e
f
Figure 4. HEMI-A
2.3.2.3.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: M+ and (M+1)
+ were not observed.
Calculated (C10H11NO4): 209.07
MS fragmentation, m/z (peak intensity): 110.10 (5.56, (M – 99)); 82.10 (3.60, (M – 127)),
68.00 (100.00, (M – 141)).
2.3.2.3.2. NMR Spectroscopic Analysis
1H NMR (CDCl3): δ ppm 6.50 (s, 2H, Hb); 5.26 (s, 2H, Ha); 3.75, 3.68 (m, 2H, Hf, He);
2.87 (s, 2H, Hc); 2.20 (br s, 1H, OH).
13C NMR (CDCl3): δ ppm 176.8 (Cd); 136.5 (Ca); 81.0 (Cb); 60.4 (Cf); 47.5 (Cc); 41.8
(Ce).
2.3.2.4. Synthesis of HEMI
The 2-hydroxy-N-ethyl maleimide (HEMI) (Figure 5) was synthesized according to the
method described by Shigeyoshi [4]. HEMI-A (5.8 g, 2.78 mmol) was refluxed in toluene
(40 ml) under a N2 atmosphere overnight in a 100 ml round-bottomed flask. After cooling
the reaction mixture overnight, the precipitate was filtered and washed with petroleum
ether. The crude product was purified twice by sublimation yielding HEMI (3.25 g, 83 %)
77
Chapter 2: Experimental
as white crystals, m.p. 72 oC (lit. 72
oC). The purity of the product was further evaluated
using NMR and mass spectroscopy.
N
O
O
OH
a bc
d
Figure 5. HEMI
2.3.2.4.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: 141.15 (1.11); 142.15 (0.68).
Calculated (C6H7NO3): 141.04 (1.11); 141.12 (0.81).
MS fragmentation, m/z (peak intensity): 110.10 (85.04, (M – 31)); 82.05 (88.08, (M –
59)), 54.10 (100.00, (M – 87)).
2.3.2.4.2. NMR Spectroscopic Analysis
1H NMR (D2O): δ ppm 6.73 (s, 2H, (Ha)); 3.76, 3.72 (m, 2H, Hc, Hd); 2.13 (t, 1H, J(HH)
5.4 Hz, OH).
13C NMR (D2O): δ ppm 171.1 (Cb); 134.2 (Ca); 60.8 (Cd); 40.6 (Cc).
2.3.2.5. Synthesis of HPrMI-A
Synthesis of 3-hydroxy-N-propyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide (HPrMI-
A) (Figure 6) was carried out in accordance to the method described by Narita et al. [3]
with the exception of using ethanolamine. Instead, 3-amino-1-propanol (7.0 g, 93 mmol)
in ethanol (10 ml) was added drop-wise to a slurry of Furan-A (15.0 g, 90 mmol) in
ethanol (25 ml) in a 50 ml round-bottomed flask. This mixture was then refluxed for 4
hours. After cooling the reaction mixture overnight, a white precipitate had formed. The
crude product was filtered and washed with ethanol followed by petroleum ether yielding
10.27 g (51 %) of a white crystalline solid as the final product, m.p. 117 oC. Mass
spectroscopy and NMR were used to confirm the purity of product.
78
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
N
O
O
O
OH
ab
c de
f
g
Figure 6. HPrMI-A
2.3.2.5.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: M+ and (M+1)
+ were not observed.
Calculated (C11H13NO4): 223.08
MS fragmentation, m/z (peak intensity): 110.10 (85.04, (M – 31)); 125.20 (2.21, (M –
80)), 82.10 (3.08, (M – 123)), 68.05 (100.00, (M – 155)).
2.3.2.5.2. NMR Spectroscopic Analysis
1H NMR (CDCl3): δ ppm 6.51 (s, 2H, Ha); 5.26 (s, 2H, Hb); 3.64 (t, 2H, J(HH) 6.2 Hz, Hg);
3.50 (t, 2H, J(HH) 5.7 Hz, He); 2.86 (s, 2H, (Hc)); 2.48 (br s, 1H, OH); 1.75 (quintet, 2H,
J(HH) 6.2 Hz, Hf).
13C NMR (CDCl3): δ ppm 179.7 (Cd); 136.5 (Ca); 81.1 (Cb); 58.9 (Cg); 47.5 (Cc); 35.9
(Ce); 29.4 (Cf).
2.3.2.6. Synthesis of HPrMI
The 3-hydroxy-N-propyl maleimide (HPrMI) (Figure 7) was synthesized according to the
method described by Shigeyoshi [4]. HPrMI-A (5.0 g, 22.4 mmol) was refluxed in
toluene (40 ml) under a N2 atmosphere for 96 hours in a 100 ml round-bottomed flask.
The reaction mixture was allowed to cool overnight and then the crude product was
filtered and washed with petroleum ether, which gave the final product as a white
crystalline solid with a yield of 70 % (2.43 g). The purity of the product was evaluated
using NMR and mass spectroscopy.
79
Chapter 2: Experimental
N
O
O OH
a bc
de
Figure 7. HPrMI
2.3.2.6.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: 155.10 (0.87); (M+1)+ was not observed.
Calculated (C7H9NO3): 155.06
MS fragmentation, m/z (peak intensity): 137.10 (22.44, (M – 18)); 110.00 (91.35, M –
45)), 82.05 (65.30, (M – 73)), 54.05 (100.00, (M – 101)).
2.3.2.6.2. NMR Spectroscopic Analysis
1H NMR (D2O): δ ppm 6.74 (s, 2H, Ha); 3.48 (m, 4H, Hc, He); 1.69 (quintet, 2H, J(HH) 6.6
Hz, Hd).
13C NMR (D2O): δ ppm 173.4 (Cb); 134.5 (Ca); 59.1 (Ce); 34.7 (Cc); 30.3 (Cd).
2.3.2.7. Synthesis of HPMI-A
Synthesis of 5-hydroxy-N-pentyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide (HPMI-A)
(Figure 8) was carried out in accordance to the method described by Narita et al [3] with
the exception of using ethanolamine. Instead, 5-Amino-1-pentanol (7.6 g, 7.35 mmol) in
ethanol (15 ml) was added drop-wise to a slurry of Furan-A (5 g, 7.13 mmol) in ethanol
(15 ml) in a 50 ml round-bottomed flask. This mixture was then refluxed for 4 hours.
After cooling the reaction mixture overnight, a white precipitate had formed. The crude
product was filtered and washed with ethanol followed by petroleum ether yielding 8.98 g
(50 %) of a white crystalline solid as the final product. The purity of the product was
evaluated using NMR and mass spectroscopy.
80
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
N
O
O
O
OH
ab
c de
f
g
h
i
Figure 8. HPMI-A
2.3.2.7.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: M+ and (M+1)
+ were not observed.
Calculated (C13H17NO4): 251.12
MS fragmentation, m/z (peak intensity): 183.15 (4.30, (M – 68); 165.10 (1.85, (M – 18)),
121.15 (1.17, (M – 62)), 82.05 (68.10, (M – 169)); 68.05 (100.00, (M – 183)).
2.3.2.7.2. NMR Spectroscopic Analysis
1H NMR (CDCl3): δ ppm 6.48 (s, 2H, Ha); 5.24 (s, 2H, Hb); 3.58 (t, 2H, J(HH) 6.4 Hz, Hi);
3.46 (t, 2H, J(HH) 7.2 Hz, He); 2.81 (s, 2H, (Hc); 1.48-1.54 (m, 4H, (Hf, Hh)); 1.31 (m, 2H,
Hg).
13C NMR (CDCl3): δ ppm 176.32 (Cd); 136.48 (Ca); 80.90 (Cb); 62.45 (Ci); 47.33 (Cc);
38.73 (Ce); 32.01 (Ch); 27.18 (Cf); 22.64 (Cg).
2.3.2.8. Synthesis of HPMI
The 5-hydroxy-N-pentyl maleimide (HPMI) (Figure 9) was synthesized according to the
method described by Shigeyoshi [4]. HPMI-A (5.0 g, 1.99 mmol) was refluxed in toluene
(40 ml) under a N2 atmosphere for 48 hours in a 50 ml round-bottomed flask. The
reaction mixture was allowed to cool overnight and then precipitate was filtered and
washed with petroleum ether. The crude product was purified twice by sublimation
yielding HPMI (2.73 g, 75 %) as white crystals, m.p. 51 oC. NMR and mass spectroscopy
were used to confirm the purity of the product.
81
Chapter 2: Experimental
N
O
O
OH
a bc
d
e
f
g
Figure 9. HPMI
2.3.2.8.1. Mass Spectroscopic Analysis
M.S. (molecular ion, m/z (peak intensity)
Observed: 183.15 (3.23); (M+1)+ was not observed.
Calculated (C9H13NO3): 183.09
MS fragmentation, m/z (peak intensity): 153.15 (3.83, (M – 30)), 110.05 (100.00, (M –
73)), 82.05 (4.20, (M – 101)).
2.3.2.8.2. NMR Spectroscopic Analysis
1H NMR (D2O): δ ppm 6.71 (s, 2H, Ha); 3.42 (t, 2H, J(HH) 6.6 Hz, Hg); 3.36 (t, 2H, J(HH)
7.0 Hz, Hc); 1.50-1.30 (m, 4H, Hd, Hf); 1.17 (quintet, 2H, J(HH) 7.1 Hz, He).
13C NMR (D2O): δ ppm 176.3 (Cb); 137.1 (Ca); 64.4 (Cg); 40.4 (Cc); 33.6 (Cd); 30.2 (Cf);
25.2 (Ce).
2.3.3. Water-Soluble Derivative of Chitosan
A water-soluble derivative of chitosan, carboxymethyl (CM) chitosan was prepared
through partial deacetylation of chitosan at a specific temperature. The detailed synthesis
of the CM chitosan is described in Section 2.3.3.1.
2.3.3.1. Synthesis of CM Chitosan
CM chitosan was synthesized according to the method described by Liu et al [5].
Chitosan (10 g) was added to a 500 ml flask containing sodium hydroxide (13.5 g), and
solvent (100 ml, 1/9: water/isopropanol) to swell and alkalise at ~ 10 oC for 1 hour. The
temperature was maintained in a water bath. Monochloroacetic acid (15 g) dissolved in
82
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
isopropanol (20 ml), was then added to the reaction mixture drop-wise for 30 minutes and
allowed to react for 4 hours at the same temperature. The reaction was quenched by
adding 70 % ethyl alcohol (200 ml). The solid was filtered and rinsed with 70-90 % ethyl
alcohol to desalt and dewater followed by vacuum drying at room temperature yielding
sodium salt CM chitosan (6.21 g) as the product.
O
CH2OH
OH
NH2
O
CH2OH
OH
NR2
O
O
CH2OH
OH
NHR
O
CH2OH
OH
NHAc
OO O
(R= CH2COOH)
Figure 10. CM Chitosan
Na salt CM chitosan (1 g) was suspended in 80 % ethyl alcohol aqueous solution (100
ml), and hydrochloric acid (10 ml, 37 %) was added with stirring for 30 minutes. The
solid was filtered, rinsed in 70-90 % ethyl alcohol to neutral pH and dried at room
temperature under vacuum. The final product obtained was the N-form of CM chitosan
(Figure 10) [6] as pale brown flakes. The final product was characterized using FT-IR
spectroscopy.
2.3.3.1.1. FT-IR Analysis
3466 cm-1
(broad -OH stretch), 1747 cm-1
(-COOH peak), 1660 and 1540 cm-1
(-NH3+
peak), and 1070-1136 cm-1
(-C-O- stretch).
The FT-IR spectrum is attached in Section 5.3.2.
2.4. UV Lamp calibration
The UV lamps were calibrated on the basis of the amount of energy, (energy flux), given
out per second. The energy flux given out per second was determined using a chemical
actinometer, which involved the decomposition of oxalic acid in the presence of uranyl
nitrate upon exposure to the UV light. Uranyl nitrate and oxalic acid solution samples
83
Chapter 2: Experimental
were exposed to the lamp for varying time intervals. The excited UO22+
species formed
according to Equation 1 decomposes the oxalic acid as shown in Equation 2. The
irradiated samples were then titrated against standardized KMnO4 to determine the
unreacted oxalate ion concentration according to Equation 3.
UO22+
hv (UO22+
)* Equation 1
(UO22+
)* + (COOH) 2 UO2
2+ + H2O + CO2 + CO Equation 2
2 MnO4- + 5(COOH) 2 + 6H
+10CO2 + 8H2O + 2Mn
2+ Equation 3
2.4.1. Preparation of Solutions
2.4.1.1. Oxalic Acid Solution (COOH) 2
Oxalic acid solution (0.02 M) was prepared in a 100 ml volumetric flask by dissolving
accurately weighed oxalic acid (0.180 g, MW. 90.04 g mol-1
) in milli-Q- water. The
volumetric flask was shaken gently until the solid was entirely dissolved and then the
flask was filled up to the mark with milli-Q-water.
2.4.1.2. Uranyl Nitrate Solution (UO2 (NO3) 2. 6H2O)
Accurately weighed hydrated uranyl nitrate (1.005 g, MW. 502.13 g mol-1
) was dissolved
in a 100 ml volumetric flask with milli-Q-water to give approximately a 0.02 M solution
of uranyl nitrate.
2.4.1.3. Potassium Permanganate Solution (KMnO4)
Potassium permanganate is widely used as a titrant in inorganic and organic analysis.
Potassium permanganate solution (0.001 M) was prepared in a 100 ml volumetric flask by
dissolving an accurately weighed KMnO4 (0.016 g, MW. 158.03 g mol-1
) with milli-Q-
water. The KMnO4 solution was standardized by titration with hot acidified oxalic acid at
60-90 oC until an endpoint was detected upon a colour change from a clear colourless
solution to a pink solution.
84
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.4.1.4. Preparation of Sample Solutions
Sample solutions for UV irradiation were prepared in several test tubes, each containing
20 ml of uranyl nitrate and 20 ml of oxalic acid solution. A reference sample solution was
prepared in the same manner.
2.4.2. Irradiation of Samples
The ultraviolet lamp was turned on and equilibrated for 5 minutes prior to placing the test
tubes containing sample solutions prepared as described in Section 2.4.1.1, in the fitted
rotating circular rack around the lamp. Each sample was irradiated for a designated period
of time. The reference sample solution was not exposed to the ultraviolet light.
2.4.3. Analysis of the Irradiated Samples
Aliquots (5 ml) were removed from the irradiated samples along with the reference
sample (described in Section 2.4.2), heated to 60-90 oC, acidified with 2 M sulphuric acid
and titrated against the standardized solution of KMnO4. Oxalic acid that was not
decomposed by UO22+
ion reacted with KMnO4 according to Equation 3. The titration was
that of a redox reaction. Potassium permanganate and oxalic acid were the oxidising and
reducing agents respectively. The titrations were done in duplicates for each irradiated
sample with UV exposure period ranging from 10-90 minutes.
2.4.4. UV Dose Calculation
The UV lamp calibration was carried over 90 minutes to observe the decomposition rate
of oxalic acid at varying exposure times. A plot illustrating the amount of oxalic acid
decomposed as a function of UV exposure time is attached in Appendix IV. Titration
results are listed below in Table 2.
85
Chapter 2: Experimental
Table 2. Titration results – KMnO4 consumption
UV exposure
time (mins)
Titre 1 values
(KMnO4) (ml)
Titre 2 values
(KMnO4) (ml)
Average titre values
(KMnO4) (ml)
0 14.43 14.45 14.44
10 10.68 10.72 10.70
20 6.98 7.04 7.01
30 3.99 4.03 4.01
60 0.71 0.73 0.72
90 0.23 0.25 0.24
The UV radiation dose rate calculations however, were based on the sample exposed to
the lamp for 30 minutes. The extent of oxalic acid decomposition was obtained by
comparing the moles of acid in the irradiated sample at time (tx = 30 mins) to that in the
non-irradiated (reference) sample. An energy flux or dose rate value of 9.63 x 10-2
J s-1
was calculated as illustrated in Sections 2.4.4.2 - 2.4.4.5.
2.4.4.1. Decomposition of Oxalic Acid
Mol (COOH)2 tx = mol (COOH)2 to x titre vol [(KMnO4 (to) - KMnO4 (tx)) / KMnO4 (to)]
= (0.02 mol L-1
x 0.02 L) x [(14.44 – 4.01 ml) /14.44 ml]
= 2.89 x 10-4
mol of oxalic acid decomposed
Concentration of oxalic acid: 0.02 mol L-1
Volume of oxalic acid in sample: 0.02 L
Where to = non-irradiated sample; tx = irradiated sample for time x.
2.4.4.2. Number of Einstein’s (s-1
) Required
No: Einstein’s = (Moles of decomposed oxalic acid) / [(time of exposure (s)) x Φ] = (2.89 x 10
-4 mol) / [(30 x 60 s) (0.57)]
= 2.82 x 10-7
Einstein’s s-1
Where Φ = 0.57 (Quantum yield of oxalic acid)
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.4.4.3. Energy (J)
E = hc/λ E = [(6.626 x 10
-34 J s
-1) (2.998 x 10
8 m s
-1)] / 3.5 x 10
-7 m
E = 5.68 x 10-19
J
Where λ = 350 nm (average energy quanta between 254-435 nm); h = 6.626 x 10-34
J s
(Planks constant) and c = 2.998 x 108 m s
-1 (speed of light).
2.4.4.4. Dose Rate (J s-1
)
Dose rate = (Einsteins (s-1
)) x (avagadro’s number) x (energy J)
= [(2.82 x 10-7
s-1
) (6.022 x 1023
mol-1
) (5.68 x 10-19
J)
= 9.63 x 10-2
J s-1
2.5. Preparation of Hydrogels
An ultra violet radiation source, described in Section 2.2.1 was used for the curing of the
monomer solutions. A wide range of monomers with exceptional chemical properties was
used to synthesize polymeric hydrogels. The monomers used were HEMA, NVP, acrylic
acid (AA), N-vinyl caprolactam (NVC) and a series of N-hydroxyalkyl maleimides,
synthesized as described in Section 2.3.2. Polysaccharides namely, chitosan and its
derivative CM chitosan were also utilized. In this work, majority of the polymerisation
processes were initiated by the donor/acceptor pairs under the influence of the UV source.
Polymerisation of certain monomer systems was also achieved in the presence of a
photoinitiator under the influence of a UV source.
2.5.1. Hydrogels Synthesis Initiated By Photoinitiator (PI)
The monomers used for the synthesis of copolymers with PI included were HEMA and
NVP. Irgacure 819 was the photoinitiator used. The following ratios by volume: 0:5, 1:4,
2.5:2.5, 4:1 and 5:0 of the respective monomers were added to give a total of 5 ml
solution in each test tube to which was added Irgacure 819 (0.005 g, 0.1 % w/v). Two
formulations (5 ml), each containing 10 % v/v of water were prepared in the following
ratios by volume of HEMA: NVP: H2O; 3.6: 0.9: 0.5 and 0.9: 3.6: 0.5 with added PI
87
Chapter 2: Experimental
(0.005 g, 0.1 % w/v). Approximately 1ml of each monomer mixture was transferred into
non-pigmented polypropylene straws cut to lengths of ~8 cm with a bore diameter of 0.5
cm and were sealed at one end. Straws were used instead of micro test tubes to avoid
problems associated with having to break glass test tubes to retrieve the polymer gels for
testing. The straws containing the monomer samples were placed in test tubes and then
subjected to radiation as described in Section 2.5.3.
2.5.2. Hydrogels Synthesis via Photoinitiator-Free Process
A number of PI free systems were studied in which the formations of hydrogels were
initiated by the monomers, which functioned as electron donor/acceptor pairs. HEMA,
AA and a series of N-hydroxyalkyl maleimides: HMMI, HEMI, HPrMI and HPMI were
used as electron acceptor monomers combined with NVP, which served as an electron
donor monomer. Interpenetrating polymer networks (IPNs) involving chitosan and its
derivative CM chitosan in conjunction with HMMI, NVP and HEMA were also prepared
using the PI- free technique.
2.5.2.1. Preparation of the N-Hydroxyalkyl Maleimide and NVP Systems
The N-hydroxyalkyl maleimides, HMMI, HEMI, HPrMI and HPMI were each combined
with NVP to form donor/acceptor pairs respectively. All these systems were prepared in
the presence of hydrogen donors, glucose and glucosamine HCl. The monomer solutions
each of an approximate total mass of 2 g containing the respective maleimide (MI) were
prepared in accordance to the following composition: NVP (71.45 % w/w); MI (4.68 %
w/w); H2O (23.37 % w/w); H-donor (0.5 % w/w). H-donor and the respective maleimide
were first dissolved in water followed by the addition of NVP. Each solution was briefly
sonicated to ensure homogeneity in the mixture and was then transferred into clear
polypropylene straws, which were then subjected to UV radiation.
2.5.2.2. Preparation of the HPMI- NVP-HEMA System
A stock solution of HEMA (50 % v/v) and NVP (50 % v/v) was prepared. The HPMI-
NVP-HEMA system was prepared in accordance to the procedure described in Section
2.5.2.1 with the exception of substituting NVP (71.45 % w/w) with the HEMA-NVP
stock solution (71.45 % w/w).
88
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.5.2.3. Preparation of the HPMI- NVP-NVC System
Stock solutions of NVP and NVC were prepared according to the following composition
in % v/v, (NVP:NVC; 20:80; 50:50; 80:20). The HPMI-NVP-NVC systems were
prepared in accordance to the procedure described in Section 2.5.2.1 with the exception of
substituting NVP (71.45 % w/w) with the NVP-NVC stock solutions (71.45 % w/w).
2.5.2.4. Preparation of the IPN, HMMI-NVP-Chitosan System
Chitosan (85 % de-acetylated) derived from crab shells was used for this work. A
chitosan stock solution A was prepared by dissolving chitosan (0.4249 g) in levulinic acid
(0.4238 g, 0.09 M) to which was added 40 ml of milli-Q water. The mixture was stirred
overnight to allow chitosan to dissolve fully in the acidic medium. The same method of
preparation was repeated for a stock solution B containing chitosan dissolved in acrylic
acid. The stock solutions were slightly viscous upon full dissolution of chitosan. Both the
stock solutions, A and B were used in separate similar formulations with the exception of
excluding HMMI in formulations containing stock solution B to form IPNs. The
monomer solutions of an approximate total mass of 2 g were prepared with varied
compositions of NVP and chitosan solution. Mixture A contained HMMI (4.0 % w/w);
NVP (62.4 % w/w) and chitosan stock solution (33.6 % w/w). Mixture B contained
HMMI (4.0 % w/w); NVP (33.6 % w/w) and chitosan stock solution (62.4 % w/w).
Mixture C contained HMMI (4.0 % w/w); NVP (48 % w/w) and chitosan stock solution
(48 % w/w). The solutions were sonicated for homogeneity, transferred into clear
polypropylene straws and subjected to UV radiation as described in Section 2.5.3.
2.5.2.5. Preparation of the IPN, HMMI-NVP-CM Chitosan System
CM chitosan synthesized under controlled physical conditions (Section 2.3.3) is a water-
soluble derivative of chitosan. CM chitosan (0.2 g) was accurately weighed in a beaker to
which was added 20 ml of milli-Q-water. The mixture was stirred overnight to allow
complete dissolution of CM chitosan. The CM chitosan solution, which was slightly
viscous, was used as the stock solution in the preparation of the IPN, HMMI-NVP-CM
chitosan system. The monomer solutions of an approximate total mass of 2 g were
prepared with varied compositions of NVP and CM chitosan solution. Mixture A
contained HMMI (4.0 % w/w); NVP (62.4 % w/w) and CM chitosan stock solution (33.6
89
Chapter 2: Experimental
% w/w). Mixture B contained HMMI (4.0 % w/w); NVP (33.6 % w/w) and CM chitosan
stock solution (62.4 % w/w). Mixture C contained HMMI (4.0 % w/w); NVP (48 % w/w)
and CM chitosan stock solution (48 % w/w). The final monomer mixtures were sonicated,
transferred into clear polypropylene straws and subjected to UV radiation.
2.5.2.6. Preparation of the IPN, HEMA-NVP-Chitosan System
Chitosan stock solutions A and B prepared as described in Section 2.5.2.2 were utilized to
prepare the HEMA-NVP-chitosan IPNs. The monomer mixture was prepared in
accordance to the following composition: HEMA (25 % w/w): NVP (25 % w/w):
chitosan solution (50 % w/w) to give a final approximate mass of 2 g. A 2 g mixture
containing HEMA (50 % w/w): chitosan solution (50 % w/w) was also prepared. HMMI
was excluded from these formulations. The mixtures were placed in an ultra sonic bath
for a brief moment, transferred into clear polypropylene straws and subjected to UV
radiation.
2.5.2.7. Preparation of the Hydrogels Based on HEMA-NVP-AA System
The hydrogels based on HEMA-NVP-AA system were prepared in a wide range of
variable compositions of the three monomers. The monomer mixtures, each of a total
volume of 5 ml were prepared in accordance to the following composition in % v/v of
(HEMA:NVP:AA): (50:40:10); (50:10:40); (50:25:25); (50:50:0); (40:50:10); (10:50:40);
(25:50:25); (0:50:50); (50:0:50); (25:25:50); (10:40:50); (10:10:50). Each solution was
mixed thoroughly before being transferred into clear polypropylene straws. The samples
were subjected to UV radiation.
2.5.3. Polymerisation Procedure
The clear polypropylene straws filled with the monomer sample solutions as described in
Sections 2.5.1 and 2.5.2 were placed in test tubes and sealed with a rubber stopper. The
tubes were placed around the mercury lamp in the fitted circular rack drum for exposure
to the UV source. The specifications of the UV drum are described in Section 2.2. The
time taken for each sample solution to polymerise was recorded. The polymeric gel
samples were cut open from the straws and subjected to washing for several days in de-
ionized water to remove uncured monomers present in the gel matrix. The washing media
90
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
was changed regularly with fresh de-ionized water during this period. The washing
process was repeated until the concentration of the leached materials in the wash solution
was less than 1 ppm, estimated using an UV- Vis spectrophotometer. The gel samples
after being washed and dried were subjected to tests described in Sections 2.6, 2.7 and
2.9.
2.6. Equilibrium Water Content (EWC) Evaluation
The polymers synthesized were subjected to swelling test to evaluate the EWC in the
polymeric hydrogels. The swollen hydrogel networks were then subjected to texture
analysis and proton NMR experiments.
2.6.1. Equilibrium Swelling
Equilibrium swelling experiments on the hydrogel samples were conducted at 37 + 0.5 oC
mainly in neutral media with the exception of the use of basic and acidic media for some
hydrogel systems. The experiments were carried out at 37 oC to simulate the body
temperature for the purpose of indicating the gels potential application at such
temperature. Isotonic solution at pH 7.4 was also used as the swelling media for selected
hydrogel systems. Preparations for the buffers at different pH values are described in
Section 2.6.1.1.
The hydrogels prior to EWC evaluation were washed and dried as described in Section
2.5.3. Duplicate hydrogel samples for each system prepared as described in Section 2.5
were accurately weighed prior to immersion into the swelling media. The hydrogel
samples were removed periodically from the swelling media, blotted dry with an
absorbent tissue and weighed. The EWC evaluation experiment was carried out over a
period of 170 hours, which allowed the water content in the hydrogels to reach
equilibrium. A Mettler digital weighing balance was used to carry out the measurements.
The EWC calculations were carried out according to Equation 4 where Wt is the weight of
the swollen hydrogel at time t and Wo is the weight of the dry polymer [7].
EWC (%) = t
t
W W
W
− o x 100 Equation 4
91
Chapter 2: Experimental
2.6.1.1. Preparation of Media
The EWC evaluation experiments were conducted in a range of swelling media. Many
biological reactions in body occur in the pH range of 2 to 8 [8] thus the media at certain
pH values were selected to simulate different pH environments in the body for this work.
Neutral media (milli-Q-water) was commonly used along with phosphate buffers (pH 2
and pH 8) for selected systems. A physiological phosphate-buffered isotonic solution (pH
7.4), which simulates the pH of blood, was also used for selected systems.
2.6.1.1.1. Preparation of Phosphate Buffer (pH 2)
The pH 2 buffer preparation method was adopted from Christian [8]. Monobasic sodium
phosphate, NaH2PO4.2H2O (7.80 g, MW. 156.01 g/mol) was combined with phosphoric
acid (3.4 ml, 85 %, 14.7 M) and prepared to a total volume of 1 L with milli-Q-water to
give a 100 mM solution. The final pH of the solution was adjusted to 2.0 by addition of
diluted phosphoric acid drop-wise to this solution while being monitored by a pH meter.
2.6.1.1.2. Preparation of Phosphate Buffer (pH 8)
The pH 8 buffer preparation method was adopted from Christian [8]. Monobasic sodium
phosphate, NaH2PO4.2H2O (3.12 g, MW. 156.01 g/mol) was added to a 100 ml flask and
made up to the mark with milli-Q-water to give a 0.2 M stock solution A. Stock solution
B (0.2 M) was prepared in a 500 ml flask containing anhydrous dibasic sodium
phosphate, Na2HPO4 (14.20 g, MW. 141.96 g/mol). Stock solution A (21.2 ml) was
combined with stock solution B (378.8 ml) and milli-Q-water (400 ml) to give a pH 8
buffered solution.
2.6.1.1.3. Preparation of Phosphate-Buffered Isotonic Solution (pH 7.4)
The phosphate-buffered isotonic solution preparation method was adopted from Christian
[8]. Two stock solutions, A and B were prepared as described in Section 2.6.1.2. Stock
solution A (76 ml) was combined with stock solution B (324 ml) and milli-Q-water (400
ml) to give a pH 7.4 buffered solution.
92
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.6.2. Texture Analysis
The hydrogels in their swollen state were tested for viscoelasticity and stiffness/elasticity
using a TA.XT2 texture analyser. The specifications of the texture analyser and
experimental conditions are described in Section 2.2.2.6. The samples were subjected to
load for a brief period and let to relax back to their original physical state. A stress-strain
graph was obtained (Appendix III). The samples were checked for any deformation after
the test. The linear portion of the compression-strain curve was used to compute the
Young’s modulus, which defines the flexibility/stiffness of the sample. The Young’s
moduli were calculated according to Equation 5 where stress is the measured compression
force F (N) divided by the contact surface area (m2) of sample and the strain is the ratio of
the deformed length and the undeformed length of the sample.
Young’s Modulus (MPa) = Stress
Strain Equation 5
The relative stress relaxation (SR) values, which are a direct measure of viscoelasticity in
the samples, were calculated according to Equation 6 where F1 is the measured
compression force at t = 10 seconds and F2 is the measured relaxation force exerted by
the sample post the stress period.
Relative1
1
-F F
F= 2
SR Equation 6
2.6.3. Proton NMR Relaxation (T1 and T2) Measurements
The proton NMR relaxations times, T1 and T2 were measured for selected swollen
hydrogel samples to investigate the dynamics of the water molecules in the swollen
networks. The specifications of the instrument and T1 and T2 measurements are described
in Section 2.2.2.3.2.
93
Chapter 2: Experimental
2.7. Equilibrium Drug Release (EDR) Evaluation
Equilibrium drug release experiments were conducted at 37 + 0.5 oC in neutral, acidic and
basic media. Thiamine hydrochloride (HCl), theophylline, and Mn-TPP-OH were the
model drugs used for this work to evaluate the drug release behaviour of the various
hydrogels synthesized.
2.7.1. Preparation of the Model Drug Solutions
The model drugs listed in Section 2.7 are water-soluble with the exception of Mn-TPP-
OH, which is only water soluble upon prior dissolution in 2/3:1/3 cremophor EL: 2-
propan-diol mixture. Concentrated drug solutions (8000 ppm) were prepared to allow
maximum drug loading into the gel matrix. Details of the drug loading technique are
described in Section 2.7.2.
2.7.1.1. Preparation of Theophylline Solution
Theophylline (1,3 dimethylxanthine) is an oral bronchodilator used to treat asthma,
emphysema, and bronchitis [9]. It is a widely used model drug for drug release studies by
a number of researchers [10-20]. The high stability under the experimental conditions
described in Section 2.7, ease of detection under UV-Vis spectroscopy and ready
availability made theophylline (Figure 11) a suitable candidate as one of the model drugs
used for the controlled release studies in this research. An accurately weighed mass of
about 0.8 g of theophylline (MW. 180.16 g/mol) was dissolved in a 100 ml volumetric
flask with milli-Q-water to give a 8000 ppm solution of theophylline. The mixture was
warmed up gently and sonicated to give a uniform solution.
N
NN
NH
O
O
Figure 11. Theophylline
94
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.7.1.2. Preparation of Thiamine HCl Solution
Thiamine HCl (vitamin B1) plays a key role in the body’s metabolic cycle for generating
energy. The molecular weight of the incorporated model drug has been reported to be a
crucial factor in the drug release experiments [10,13]. Thiamine HCl (Figure 12) was
considered as a model drug for this work due its stability at the required experimental
conditions described in Section 2.7 and its relatively higher molecular weight in
comparison to theophylline to allow comparative studies on release rate of the model
drugs based on molecular weight.
Accurately weighed mass of approximately 0.8 g of thiamine HCl (MW. 337.26 g/mol)
was dissolved in a 100 ml volumetric flask with milli-Q-water to give a 8000 ppm
solution. The solution was warmed gently and sonicated briefly to allow complete
dissolution of the drug.
N
SO N
N
NH H
H
Cl+
-
Figure 12. Thiamine HCl
2.7.1.3. Preparation of Mn-TPP-OH Solution
Mn-TPP-OH (Figure 1) is a cancer tumor-tracing agent used in conjunction with the
magnetic resonance imaging (MRI) technique. Interest in this material lies in the fact that
it has a relatively larger molecular weight thus making it a good candidate as a model
drug for observing the effect of large molecular weight on the drug release rate.
Furthermore, the use of Mn-TPP-OH would indicate the possibility of using the hydrogels
under study as carriers of such drugs in cancer therapy.
The Mn-TPP-OH is not readily soluble in water thus had to be pre-dissolved in specially
prepared cremophor EL (CRM) and 1, 2-propanediol mixture. Mn-TPP-OH (0.1531 g,
MW. 733.69 g/mol) was dissolved in CRM (3 ml) and 1, 2-propanediol (1.5 ml) mixture
95
Chapter 2: Experimental
prior to addition of 25 ml of milli-Q-water. The solution was gently warmed and briefly
sonicated to make the solution uniform with a concentration of 8000 ppm.
2.7.2. Drug Loading Technique
The model drug could be loaded into the hydrogel in several ways. In crosslinked
polymer systems it is possible to have the drug incorporated during the formation step but
this could lead to problems associated with other unreacted components leaching out of
the gel matrix along with the incorporated drug. The drug could also be sensitive to the
gelation conditions and may undergo chemical change or possibly interfere with the
polymerisation process. Thus in such instances the drug must be loaded from solution
[21]. In this experiment the drug was loaded from a concentrated drug solution for each
drug prepared as described in Section 2.7.1.
All gel samples were washed and dried as described in Section 2.5.2 prior to drug
incorporation. Duplicate samples of each hydrogel were weighed accurately and each
immersed into 25 ml of concentrated drug solutions. The gel samples were left immersed
in the concentrated drug solution for a period of seven days to allow maximum drug
loading into the hydrogel matrix.
2.7.3. Controlled Drug Release Studies
Controlled drug release experiments were conducted in duplicate samples for each
hydrogel system. The duplicate hydrogel samples were removed from the concentrated
drug solution after seven days of loading and each swollen gel loaded with the model
drug was placed in approximately 100 ml of milli-Q-water for 10 minutes to allow any
excess drug on the surface of the gel to be washed off. The samples were removed from
these wash solutions, blotted dry with a tissue paper and weighed. Each of these samples
was then placed into a test-tube containing accurately measured, 50 ml of release
medium.
Milli-Q-water was mainly used as the release medium with the exceptional use of pH 2
and pH 8 phosphate buffered solutions, prepared as described in Section 2.6.1.1, for
selected hydrogels. The release medium temperature was equilibrated to 37 oC prior to
96
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
placement of the samples. In order to obtain a homogeneous solution, each test-tube was
equipped with a stirrer bar. Aliquots (1 ml) of the release medium were removed
periodically, diluted to 21 ml and analysed.
The periodic removal of the release medium was carried out until the change in the
concentration of the released drug was negligible or had reached equilibrium. At this
stage the release medium was changed to an accurately measured 50 ml of fresh medium
and any further drug released was analysed for several days. This was repeated until
negligible amount of drug released (< 0.1 ppm) into the medium was detected.
The controlled drug release experiments on the polymeric hydrogel samples were
conducted at 37 oC. Calculations on fractional drug release were based on the amount of
drug release analysed at each time interval divided by the total amount of drug released
from each sample. The same procedure was repeated for all the hydrogels synthesized
along with all the model drugs used in conjunction with these gels.
2.7.3.1. The Analytical Technique (Ultraviolet - Visible Spectroscopy)
A Shimadzu UV-1601 PC instrument was used for this work. The specifications of the
instrument are described in Section 2.2.2.1. The working wavelength range for the
analysis of the three model drugs was from 800 nm to 200 nm. Mn-TPP-OH was detected
at 424 nm while theophylline and thiamine HCl were detected at 271 nm and 266 nm
respectively. Five standard solutions of varying known concentrations ranging from 3
ppm to 20 ppm of each model drug was prepared and analysed to construct standard
calibration curve of absorbance against concentration. This curve was used to determine
the concentration of the drug released into the media with the fact that absorbance is
directly proportional to concentration. Fractional drug released (FDR) from each hydrogel
sample was calculated according to Equation 7 where Mt is the mass of the drug released
at time t and M∞ is the total mass of the incorporated drug.
FDR = tM
M ∞ Equation 7
97
Chapter 2: Experimental
The total amount of drug released from each hydrogel system as observed from the UV
analysis was compared with the calculated amount of incorporated drug from weight
measurements made prior and post drug loading to confirm equilibrium drug release.
2.8. Kinetic Studies on Electron Donor/Acceptor Systems
Kinetic studies were conducted on a range of donor/acceptor pairs using a Differential
Photocalorimeter/DSC 2910 system by TA Instruments. These donor/acceptor systems
were utilized to form hydrogels as described previously in Section 2.5.1.2. The
specifications of the DPC, experimental conditions and analytical procedure are described
in Section 2.2.2.5. The electron acceptor monomers, which were tested for this work,
were AA, HEMA and a series of N-hydroxyalkyl maleimides, HMMI, HEMI, HPrMI and
HPMI, synthesized as described in Section 2.3.2. NVP was the electron donor monomer
utilized. Glucosamine HCl and glucose were used as hydrogen donors in the N-
hydroxyalkyl maleimide-NVP systems.
2.8.1. Preparation of NVP-HEMA and AA-NVP Systems
The donor/acceptor monomer combinations in each of the following systems
HEMA:NVP and AA:NVP were prepared in three different mol ratios. For each system,
the monomer pairs were weighed in sample tubes to give (1:2; 1:1; 2:1) mol ratios. The
sample tubes were wrapped in aluminium foil to protect samples from being exposed to
ambient light. Samples were analysed using a DPC unit as described in Section 2.2.2.5.
2.8.2. Preparation of N-Hydroxyalkyl Maleimides-NVP Systems
The four maleimides synthesized as described in Section 2.3 namely HMMI, HEMI,
HPrMI, and HPMI were each combined with NVP in a 1:1 mol ratio and tested for their
relative reactivity in the absence as well as in the presence of hydrogen donors, glucose
and glucosamine HCl. Details of preparation of the systems with and without the
hydrogen donors are described in Sections 2.8.2.1 and 2.8.2.2.
2.8.2.1. Preparation in the Presence of H-Donor
Accurately weighed amounts of NVP, N-hydroxyalkyl maleimide and H2O were
combined to form stock solutions. The final mixture contained NVP (1.42 M); N-
98
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
hydroxyalkyl maleimide (1.42 M) and the H-donor (0.15 M). The mass of the different
maleimides varied in each case due to the difference in the molecular weight and thus the
final mass was adjusted with water. However, the mol ratio of NVP and maleimide was
maintained as 1:1. Two sample mixtures, glucose solution (0.15 g; 0.56 M) and
glucosamine HCl solution (0.15 g; 0.46 M), each combined with stock solution (0.33 g)
were prepared in sample tubes wrapped with aluminium foil to prevent unwanted
polymerisation initiated by light in the laboratory. Each sample was dispensed into an
aluminium DSC pan as described in Section 2.2.2.5 and its exotherm was subsequently
obtained using the DPC technique.
2.8.2.2. Preparation in the Absence of H-Donor
Systems without a hydrogen donor were prepared and analysed in accordance to the
procedure described in Section 2.8.2.1 with the hydrogen donor being excluded. The
concentrations of the acceptor and the donor in the final sample mixture were maintained
by substituting the mass of the hydrogen donor solution with milli-Q-water to maintain
the same total mass of the final mixture as that of the systems with hydrogen donor.
2.9. Cytotoxicity Tests on Mammalian Cells
The study of hydrogels in this work is oriented towards its biomedical application as
controlled drug delivery devices. It is a known fact that the complete conversion of
monomers to polymers may not be achieved in the polymerisation process thus there is
always a certain component of unreacted toxic monomers still present in the polymer
matrix. These monomers have the tendency to leach out of the polymer matrices when the
polymers are in contact with an aqueous medium thus rendering the hydrogel to be non-
biocompatible.
The hydrogel systems prepared as described in Section 2.5 were tested in vitro for their
biocompatibility with human epidermal keratinocyte (HaCaT) cells, provided by the Skin
Technologies Research Centre at the University of Western Sydney. In the panorama of
numerous established cell lines, HaCaT has a very interesting feature, having a close
similarity in the functional competence to that of normal keratinocytes [22]. This cell line
has been used in numerous studies as a paradigm for epidermal cells and furthermore, is
99
Chapter 2: Experimental
readily available, highly sensitive and easily regenerated, making it an ideal candidate as
the cell model for this work.
A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is a
colorimetric based technique for non-radioactive quantification of cellular proliferation,
viability and cytotoxicity was used for this work. The assay is based on the cleavage of
the yellow tetrazolium salt, (MTT) to form purple formazan crystals by dehydrogenase
activity in active mitochondria, which indicate the presence of live cells [23,24].
2.9.1. Sustaining HaCaT Cells
The HaCaT cells were handled with great care to avoid any contaminants thus aseptic
handling techniques were enforced at all times. A specially prepared growth medium was
used to sustain the HaCaT cells during the course of the cytotoxicity experiments.
2.9.1.1. Preparation of HaCaT Growth Medium
The HaCaT medium was prepared in milli-Q-water by adding Dulbecco's modified
Eagle's medium (DMEM)- calcium-free (12.7 g/L) powdered media containing glucose
(4.5 g/L) and sodium pyruvate (0.11 g/L) to which was added sodium bicarbonate (2.2
g/L), penicillin/streptomycin (100 U/ml, 0.1 g/L), N- [2-hydroxyethyl] piperazine-N- [2-
ethane sulfonic acid] (HEPES) (2.38 g/L) and L-glutamine (0.29 g/L).
The mixture was gently stirred and then accurately diluted to a total volume of 900 mL
with milli-Q-water. The medium was filter sterilized through a 0.2 µm membrane filter,
aseptically dispensed into sterile bottles and stored in the dark at 4 oC. For cell growth,
the HaCaT medium was supplemented with sterile heat inactivated fetal calf (bovine)
serum (FCS) (10 % v/v). The media was warmed to 37 oC and filter sterilized each time
prior to feeding cells.
2.9.1.2. Aseptic Techniques
It was absolutely crucial that at no point in time during the cytotoxicity experiments,
contaminants of any kind were introduced to the cells. Aseptic handling procedures were
enforced at all times. The biohazard hood was sterilized with ultra violet radiation for 10
100
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
minutes prior to commencement of every experiment. The working bench under the hood
and surrounding atmosphere was sterilized with 70 % ethanol spray. All biological
reagents including the media and test samples were filter sterilized through a 0.2 µm
membrane filter at all times prior to introduction to the cells.
2.9.1.3. Generation of HaCaT Cells
The supplied HaCaT cell suspension was stored in growth medium supplemented with
FCS (30 % v/v) and DMSO (10 % v/v) in a cryo vial (1 ml) under liquid nitrogen. A
specially prepared growth medium as described in Section 2.9.1.1 was used to culture the
HaCaT cells. The HaCaT medium (5 ml) supplemented with 10 % v/v of fetal calf
(bovine) serum FCS was warmed to 37 oC, filter sterilized and aseptically dispensed into
a centrifuge tube to which was added the thawed HaCaT cell suspension.
The mixture was centrifuged for 5 minutes at 2000 rpm separating the cells from the
suspension. The liquid content of the tube was discarded and the cells were re-suspended
in fresh media (1ml). Two 75 cm2 culture flasks each containing 0.5 ml of the cell
suspension and 10 ml of media supplemented with 10 % v/v FCS were prepared and
incubated at 37 oC in the presence of CO2 (5 %). The culture flasks were replenished with
fresh medium every third day until the cells were fully confluent.
2.9.1.4. Trypsinizing HaCaT Cells
The HaCaT cells after becoming fully confluent were trypsinized using ethylenediamine
tetraacetic acid (EDTA) and trypsin solution. The exhausted media in the culture flask
was discarded and replaced with sterile EDTA solution (10 ml, 0.02 %) prepared in
Dulbecco's phosphate buffered saline (DPBS). The flask was then incubated at 37 oC for
20 minutes. At the end of the incubation period the EDTA solution was discarded and
replaced with trypsin solution (5 ml) containing EDTA (0.02 % w/v), D-Glucose (0.1 %
w/v), KCl (0.04 % w/v), NaCl (0.8 % w/v) and trypsin (0.1 % w/v) followed by a further
5 minutes incubation period at 37 oC. The process involved detachment of the cells from
each other by EDTA treatment, and detachment from the surface of the culture flask by
trypsin treatment.
101
Chapter 2: Experimental
The culture flask was tapped gently on the sides to assist in the separation of the cells and
observed under the microscope to determine whether the cell had dissociated from the
flask and each other. The contents of the flask were emptied into a sterile centrifuge tube
containing fresh filtered media (5 ml). The tube was centrifuged for 5 minutes at 2000
rpm. The supernatant was discarded and the cell pellet re-suspended in fresh media (1ml).
The cell suspension was gently swirled before transferring 20 µL into the counting
chamber of a hemacytometer to determine cell density.
A new culture flask containing fresh media (10 ml) was seeded with an aliquot of the cell
suspension (50 µL) to maintain the cell line. The cells were fed as described in Section
2.9.1.3 and trypsinized upon becoming confluent. The process of subculturing and
sustaining the HaCaT cells was repeated throughout the course of the cytotoxicity
experiments.
2.9.2. Preparation of Test Samples
The hydrogel samples used for this work were cleaned thoroughly as described in Section
2.5.2 to eliminate the leaching of any toxic unreacted monomers from the hydrogel
matrix. The pre-weighed gel samples were further cleaned with milli-Q-water in the
biohazard hood and exposed to ultra violet light for 20 minutes for sterilization. Aseptic
techniques were employed in handling the hydrogel samples post this process to avoid the
growth of micro-organisms on the samples, which could affect the validity of this
experiment.
The gel samples after sterilization were immersed in accurately measured volumes of
milli-Q-water with gel mass to water volume ratio of 20 g/ml and incubated at 37 oC for a
period of 14 days. The water conditioned by the hydrogels (samples) was then filter
sterilized using a 0.2 µm syringe filter and used for cytotoxicity tests described below.
Standard solutions of the monomers, AA, HEMA, NVP and the N-hydroxyalkyl
maleimides were prepared in HaCaT medium without FCS in varying concentrations (125
ppm, 250 ppm, 500 ppm and 1000 ppm) and also tested for cytotoxicity.
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.9.2.1. Preparation of Culture Plates
Greiner 96 well tissue culture plates were utilized for this work. The HaCaT cell
suspension prepared as in Section 2.9.1.4 was used to make cell suspensions of varying
cell concentrations ranging from 1 x 106 – 5 x 10
4 cells/mL. A standard culture plate was
prepared for a standard curve by transferring 100 µL aliquots of each cell suspension per
well in five replicate wells in a 96 well plate. Fresh HaCaT media (100 µL) served as the
blank for background reference.
The experimental culture plate was prepared by adding 100 µL of a cell suspension of 1 x
105 cell/mL into each well in the plate, followed by transferring 20 µL aliquots of each
sample medium prepared as described in Section 2.9.2 making a total volume of 120
µL/well. Fresh HaCaT medium (120 µL) served as the blank. The experimental control,
which was the untreated cell suspension, was prepared by adding 20 µL of FCS-free
medium to 100 µL of 1 x 105 cells/ml cell suspension. The response of the HaCaT cells to
the sample medium at 24 hours and at 48 hours of exposure was evaluated. The
cytotoxicity experiment on each sample medium was repeated in triplicate.
2.9.2.2. MTT Cell Proliferation Assay
Sterile MTT solution (5 mg/ml) was prepared by dissolving MTT (100 mg) in DMEM-
phenol red free medium (20 ml). The solution was filter sterilized and stored in the dark
at minus 20 oC. Anhydrous isopropanol with HCl (0.1 M) was used as the MTT solvent
solution. The standard culture plates prepared as described in Section 2.9.2.1 were
incubated at 37 oC in the CO2 (5 %) atmosphere for 10 minutes to allow the cells to settle.
MTT solution (10 µL) was then added to the wells in the culture plates and incubated at
37 oC in the presence of CO2 (5 %) for 3 hours.
The experimental culture plates were incubated at 37 oC in the CO2 atmosphere for 24
hours and 48 hours prior to the addition of the MTT solution (10 µL). The cell cultures
were examined under a microscope after the designated treatment time. MTT was then
subsequently added to the culture plates followed by a further 3 hour incubation period at
37 oC in the presence of CO2 (5 %). The medium from the wells was replaced with MTT
103
Chapter 2: Experimental
solvent solution (100 µL) after the 3 hour incubation period. Each plate was placed on a
gyratory shaker to dissolve the purple MTT formazan crystals.
A BMG Labtechnologies FLUOstar OPTIMA microplate reader was used to carry out the
absorbance measurements. The readings were carried out at a wavelength of 560 nm with
background absorbance of 650 nm. The FLUOstar OPTIMA v1.30-0 software was used
to analyse the acquired raw data. A standard curve was constructed from the standard
culture plate measurements of known cell densities to estimate the cell density of the
unknown cell cultures treated with the monomers and the hydrogel sample media.
2.9.2.2.1. Statistical Analysis
The MTT assay data were expressed as means + SEM. The MTT assay data obtained for
the hydrogels were compared with that of the monomers and also with the experimental
untreated controls. A one-way analysis of variance (ANOVA) was performed using the
MINITAB 7.2 statistical software. A p value of < 0.05 was regarded as statistically
significant.
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.10. References
1. Leighton, W. G., Forbes, G. S., J. Am. Chem. Soc., 52, 3139-3152, (1930).
2. Tawney, P. O., Snyder, R. H., Conger, R. P., Leibbrand, K. A., Stiteler, C. H.,
Williams, A. R., J. Org. Chem., 26, 15-21, (1961).
3. Narita, M., Teramoto, T., Okawara, M., Bull. Chem. Soc. Jap., 44, 1084-1089,
(1971).
4. Shigeyoshi, H., Chem Abs., 114, 185261z, (1990).
5. Liu, X. F., Guan, Y. L., Yang, D. Z., Li, Z., Yao, K. D., J. Appl. Polymer Sci., 79,
1324-1335, (2001).
6. Muzzarelli, R. A. A., Ilari, P., Petrarulo, M., Int. J. Biol. Macromol., 16, 177-180,
(1994).
7. Aggarwal, S. L., “COMPREHENSIVE POLYMER SCIENCE” – The Synthesis,
Characterisation, Reactions & Applications of Polymers, vol 7, Pergamon Press,
Oxford, pp. 221, (1989).
8. Christian, G. D., “Analytical Chemistry”, 5th
edition, John Wiley & Sons, New
York, pp. 207-209, (1994).
9. Evans, D., Taylor, D., Zetterstrom, O., and Chung, K., New Eng. J. Med., 337,
1412-1418, (1997).
10. Brazel, C. S., Peppas, N. A., Polymer, 40, 3383-3398, (1999).
11. Grassi, M., Colombo, I., Lapasin, R., J. Controlled Release, 76, 93-105, (2001).
12. Ward, J. H., Peppas, N. A., J. Controlled Release, 71, 183-192, (2001).
13. Am Ende, M. T., Peppas, N. A., J. Controlled Release, 48, 47-56, (1997).
14. Inoue, T., Chen, G., Nakamae, K., and Hoffman, A. S., J. Controlled Release, 49,
167-176, (1997).
15. Katime, I., Novoa, R., Díaz de Apodaca, E., Mendizábal, E., Puig, J., Polymer
Testing, 18, 559-566, (1999).
16. Korsmeyer, R. W., Peppas, N. A., J. Controlled Release, 1, 89-98, (1984).
17. Shantha, K. L., Harding, D. R. K., Int. J. Pharm., 207, 65-70, (2000).
18. Shah, S. S., Kulkarni, M. G., Mashelkar. R. A., J. Controlled Release, 15, 121-
131, (1991).
19. Harland, R. S., Peppas, N. A., J. Controlled Release, 26, 157-174, (1993).
105
Chapter 2: Experimental
20. Bettini, R., Colombo, P., Peppas, N. A., J. Controlled Release, 37, 105-111,
(1995).
21. Rosiak, J. M., J. Controlled Release, 31, 9-19, (1994).
22. Pessina, A., Raimondi, A., Cerri, A., Piccirillo, M., Neri, M. G., Croera, C., Foti.
P., Berti, E., Cell Prolif., 34, 243-252, (2001).
23. Mosmann, T., J. Immunol. Methods, 65, 55-63, (1983).
24. Gerlier, D., Thomasset, N., J. Immunol. Methods, 94, 57-63, (1986).
106
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.1. Materials 66
2.2. Equipment 70
2.2.1. Radiation Source 70
2.2.2. Analytical Instruments 70
2.2.2.1. Ultra Violet - Visible (UV-Vis) Spectrophotometer 70
2.2.2.2. Gas Chromatograph Mass Spectrometer (GC-MS) 70
2.2.2.3. Nuclear Magnetic Resonance (NMR) Spectrometer 71
2.2.2.3.1. Carbon-13 (13
C) and Proton (1H) NMR 71
2.2.2.3.2. Proton NMR Relaxation (T1 and T2) Measurements 71
2.2.2.4. Fourier Transform Infrared (FT-IR) Spectrometer 71
2.2.2.5. Differential Photocalorimeter (DPC) 71
2.2.2.6. Texture Analyser (TA) 72
2.2.2.7. Atomic Absorption Spectrometer (AAS) 72
2.2.2.8. Microplate Reader 72
2.2.2.9. Microscope 73
2.3. Synthesis of Chemical Compounds 73
2.3.1. Synthesis of a Model Drug 73
2.3.1.1. Synthesis of Mn-TPP-OH. 73
2.3.1.1.1. UV-Vis Spectroscopic Analysis 74
2.3.1.1.2. AAS Analysis 74
2.3.2. Synthesis of N-Hydroxyalkyl Maleimides 74
2.3.2.1. Synthesis of HMMI 75
2.3.2.1.1. Mass Spectroscopic Analysis 75
2.3.2.1.2. NMR Spectroscopic Analysis 75
2.3.2.2. Synthesis of Furan-A 76
2.3.2.2.1. Mass Spectroscopic Analysis 76
2.3.2.2.2. NMR Spectroscopic Analysis 76
2.3.2.3. Synthesis of HEMI-A 76
2.3.2.3.1. Mass Spectroscopic Analysis 77
2.3.2.3.2. NMR Spectroscopic Analysis 77
2.3.2.4. Synthesis of HEMI 77
2.3.2.4.1. Mass Spectroscopic Analysis 78
2.3.2.4.2. NMR Spectroscopic Analysis 78
2.3.2.5. Synthesis of HPrMI-A 78
2.3.2.5.1. Mass Spectroscopic Analysis 79
2.3.2.5.2. NMR Spectroscopic Analysis 79
2.3.2.6. Synthesis of HPrMI 79
2.3.2.6.1. Mass Spectroscopic Analysis 80
2.3.2.6.2. NMR Spectroscopic Analysis 80
2.3.2.7. Synthesis of HPMI-A 80
2.3.2.7.1. Mass Spectroscopic Analysis 81
2.3.2.7.2. NMR Spectroscopic Analysis 81
2.3.2.8. Synthesis of HPMI 81
2.3.2.8.1. Mass Spectroscopic Analysis 82
2.3.2.8.2. NMR Spectroscopic Analysis 82
2.3.3. Water-Soluble Derivative of Chitosan 82
107
Chapter 2: Experimental
2.3.3.1. Synthesis of CM Chitosan 82
2.3.3.1.1. FT-IR Analysis 83
2.4. UV Lamp calibration 83
2.4.1. Preparation of Solutions 84
2.4.1.1. Oxalic Acid Solution (COOH) 2 84
2.4.1.2. Uranyl Nitrate Solution (UO2 (NO3) 2. 6H2O) 84
2.4.1.3. Potassium Permanganate Solution (KMnO4) 84
2.4.1.4. Preparation of Sample Solutions 85
2.4.2. Irradiation of Samples 85
2.4.3. Analysis of the Irradiated Samples 85
2.4.4. UV Dose Calculation 85
2.4.4.1. Decomposition of Oxalic Acid 86
2.4.4.2. Number of Einstein’s (s-1
) Required 86
2.4.4.3. Energy (J) 87
2.4.4.4. Dose Rate (J s-1
) 87
2.5. Preparation of Hydrogels 87
2.5.1. Hydrogels Synthesis Initiated By Photoinitiator (PI) 87
2.5.2. Hydrogels Synthesis via Photoinitiator-Free Process 88
2.5.2.1. Preparation of the N-Hydroxyalkyl Maleimide and NVP Systems 88
2.5.2.2. Preparation of the HPMI- NVP-HEMA System 88
2.5.2.3. Preparation of the HPMI- NVP-NVC System 89
2.5.2.4. Preparation of the IPN, HMMI-NVP-Chitosan System 89
2.5.2.5. Preparation of the IPN, HMMI-NVP-CM Chitosan System 89
2.5.2.6. Preparation of the IPN, HEMA-NVP-Chitosan System 90
2.5.2.7. Preparation of the Hydrogels Based on HEMA-NVP-AA Systems 90
2.5.3. Polymerisation Procedure 90
2.6. Equilibrium Water Content (EWC) Evaluation 91
2.6.1. Equilibrium Swelling 91
2.6.1.1. Preparation of Media 92
2.6.1.1.1. Preparation of Phosphate Buffer (pH 2) 92
2.6.1.1.2. Preparation of Phosphate Buffer (pH 8) 92
2.6.1.1.3. Preparation of Phosphate-Buffered Isotonic Solution (pH 7.4) 92
2.6.2. Texture Analysis 93
2.6.3. Proton NMR Relaxation (T1 and T2) Measurements 93
2.7. Equilibrium Drug Release (EDR) Evaluation 94
2.7.1. Preparation of the Model Drug Solutions 94
2.7.1.1. Preparation of Theophylline Solution 94
2.7.1.2. Preparation of Thiamine HCl Solution 95
2.7.1.3. Preparation of Mn-TPP-OH Solution 95
2.7.2. Drug Loading Technique 96
2.7.3. Controlled Drug Release Studies 96
2.7.3.1. The Analytical Technique (Ultraviolet - Visible Spectroscopy) 97
2.8. Kinetic Studies on Electron Donor/Acceptor Systems 98
108
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
2.8.1. Preparation of NVP-HEMA and AA-NVP Systems 98
2.8.2. Preparation of N-Hydroxyalkyl Maleimides-NVP Systems 98
2.8.2.1. Preparation in the Presence of H-Donor 98
2.8.2.2. Preparation in the Absence of H-Donor 99
2.9. Cytotoxicity Tests on Mammalian Cells 99
2.9.1. Sustaining HaCaT Cells 100
2.9.1.1. Preparation of HaCaT Growth Medium 100
2.9.1.2. Aseptic Techniques 100
2.9.1.3. Generation of HaCaT Cells 101
2.9.1.4. Trypsinizing HaCaT Cells 101
2.9.2. Preparation of Test Samples 102
2.9.2.1. Preparation of Culture Plates 103
2.9.2.2. MTT Cell Proliferation Assay 103
2.9.2.2.1. Statistical Analysis 104
2.10. References 105
109
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
3.1. Introduction
Wichterle and Lim [1] first developed hydrogels based on 2-hydroxyethyl methacrylate
(HEMA) for their potential application as contact lenses. Polymeric hydrogels have been
heavily utilized in the biomedical arena since then as versatile and commercially viable
materials [2-7]. HEMA is by far the most extensively used monomer for hydrogel
preparation. Numerous publications are available to date on poly(HEMA) (PHEMA)
hydrogels [8-18]. HEMA besides being a proven versatile homopolymeric biomaterial
could be combined with a pronounced hydrophilic co-monomer such as N-vinyl
pyrrolidinone (NVP) to produce copolymers with enhanced structural properties.
Polymers of HEMA and NVP have been investigated in recent years as potential hydrogel
materials for biomedical applications such as sustained drug delivery systems and contact
lenses [19-24].
The attractive bioapplications of HEMA-co-NVP hydrogels is attributed to their
remarkable water absorption ability and durability in harsh environments [9,12,20,22].
The high water content in hydrogels is believed to be intrinsically related to their high
biocompatibility [25,26]. Factors favourable to swelling include high osmotic potential,
high free volume, high chain flexibility, low crosslinking density and strong interaction
with water [27]. The water adsorption ability of a polymer is dependent on the nature and
the composition of the monomers in the polymer [22].
Inclusion of additives such as crosslinkers has been reported to govern the swelling in
polymers. Lai [19], Perera and Shanks [20] have reported a marked reduction in the
swelling of HEMA-co-NVP hydrogels upon inclusion of crosslinking agents, ethylene
glycol dimethacrylate and methylene diacrylamide. The rate of the drug delivery is
controlled by the macromolecular structure of the carrier as defined by the degree of
swelling [28,29].
The swelling and drug release kinetics in hydrogels could range from Fickian diffusion to
high order diffusion such as non-Fickian (anomalous and case II). Fickian diffusion is
characterized by a solute mass uptake that is directly proportional to the square root of
time. High order non-Fickian diffusion however, is characterized by linear mass uptake as
107
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
a function of time [28-30]. Ideal release kinetics of a drug would be a zero-order process
where the diffusion of the solute is time independent as that in the case of case II
diffusion. Bhardwaj et al [31] studied the diffusion behaviour in a range of copolymers of
HEMA and NVP and observed Fickian diffusion kinetics.
In contrary, Korsmeyer and Peppas [21] from their swelling and drug release experiments
on HEMA-co-NVP copolymers observed non-Fickian swelling and drug release kinetics.
However, they also further suggest that the swelling and drug release kinetics is
dependent on the relative composition of HEMA to NVP. Franson and Peppas [32] in an
earlier study on the effect of HEMA and NVP composition on the swelling behaviour of
their copolymers have also suggested the dependence of diffusion kinetics on the
monomeric composition.
Non-fickian hydrogels display a phenomenal abrupt volume change as a result of rapid
swelling in contrary to Fickian gels, which have a gradual swelling process. The diffused
water contained in hydrogel is evidenced to vary in molecular form. Khare and Peppas
[33] suggest the existence of water molecules in the polymer in three states, bound water,
interfacial water and bulk water. The bound water is suggested to be associated with the
polymer chains by means of hydrogen bonding. Hydrophobic interactions between the
functional groups on the polymer chain and water result in interfacial water. Free or bulk
water has the same physical properties as normal water and is not attached to the polymer
matrix [33,34].
Researchers have commonly resorted to Nuclear Magnetic Resonance (NMR)
experiments to investigate the state of water [30,34,35]. The T1 and T2 relaxation times
obtained from NMR experiments have been shown to indicate the relative mobility of
water in the polymer network. Furthermore, it is indicative of the diffusion kinetics of the
macromolecular network.
The stress relaxation phenomenon in polymers is another interesting feature, which
describes the texture of polymers. Polymeric hydrogels are often defined as two phase
systems where one phase is the water insoluble macromolecular network while the other
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
is water [2]. Polymers could behave like solids or liquids depending on the relative water
content [36]. The stress relaxation in polymers could also be termed as the viscoelasticity
of the polymer. Hong et al [37] from their studies on the texture analysis of hydrated
copolymers of HEMA and NVP reported the dependence of viscoelasticity on the
monomer composition.
Hydrogels of HEMA and NVP have been synthesized via a number of techniques such as
thermal and radiation methods [19-24]. Thermal and low energy UV radiation techniques
require additional chemical initiators while high-energy radiation processes such as
gamma and electron beam do not. Furthermore, some researchers have made use of
chemical crosslinkers in the formulations [19,20,24].
Bhardwaj et al [31] reported similar diffusion kinetics in HEMA-co-NVP hydrogels
prepared through chemical initiation and gamma radiation. In a recent study by Malak et
al [22,23], similar observations were made. However, they do suggest that these different
polymerisation protocols could lead to subtly different network structures. They state that
the efficiency of polymerisation would vary in each curing protocol thus the copolymers
could be inhomogeneous with varied crosslinking density.
In the present study HEMA and NVP were photopolymerised in a wide range of varied
compositions in the presence of a photoinitiator, Irgacure 819. Irgacure 819, also
chemically known as bis(2,4,6-trimethylbenzoyl)-phenylphosphineoxide, is an efficient
photoinitiator, which was newly introduced in the market at the time of this research. The
hydrogels formed were evaluated for their equilibrium swelling and drug release ability at
310 K. The effect of water, as an added plasticiser in the polymer formulation, on the
swelling and drug release kinetics of the polymer was investigated.
Hydrogels were also swollen in an isotonic medium to evaluate the effect of ionic
strength on the diffusion characteristic of the hydrogels. Texture analysis experiments
were conducted on the hydrogels to investigate their relative stress relaxation phenomena
after an applied stress. Furthermore, proton NMR experiments were conducted to
109
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
measure the relaxation times, T1 and T2 in the hydrogel samples in their fully hydrated
state.
3.2. Experimental Procedure
HEMA and NVP were combined in varying ratios by volume and subjected to UV
radiation in the presence of an external photoinitiator, Irgacure 819. A fraction of water
was included in some formulations to act as a plasticiser. The detailed synthesis
procedure of HEMA-NVP hydrogels in the presence of a photoinitiator is described in
Section 2.5.1. The hydrogels formed were subjected to swelling drug release experiments
in neutral pH environment at 37 oC. The effect of ionic strength on the swelling ability of
the hydrogels in an isotonic (pH 7.4) environment was also evaluated. Theophylline was
used as the model drug for drug release experiments.
Swelling and drug release experimental specifications and procedure have been described
in detail in Sections 2.6.1 and 2.7. UV-vis spectroscopy was employed as the analytical
tool for quantitative drug release measurements. A TA instrument was used carry out
texture analysis experiments on swollen hydrogel samples to investigate their relative
stiffness and viscoelasticity. NMR spectroscopic technique was used to measure the
relaxation times, T1 and T2 in selected swollen hydrogel samples. Detailed experimental
specifications on NMR and TA analysis are described in Sections 2.2.2.3.2 and 2.2.2.6
respectively.
3.3. Results
3.3.1. Hydrogel Formation
HEMA and NVP formulated in varying ratios by volume in the presence of Irgacure 819
were subjected to UV radiation. Certain formulations were inclusive of 10 % v/v of water,
which served as a plasticiser. The observations on the status of polymerisation upon
applying approximately 174 J of radiation dose are described in Table 1.
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
Table 1. Polymerisation status of HEMA : NVP : H2O formulations in the presence
of Irgacure 819 (0.1 % w/v)
Hydrogel formulation
HEMA: NVP: H2O (% v/v)
Polymerisation status upon application
of curing dose (~ 174 J)
100: 00: 00 Hard, clear pale yellow gel
80: 20: 00 Hard, clear pale yellow gel
50: 50: 00 Hard, clear pale yellow gel
20: 80: 00 Hard, clear pale yellow gel
00: 100: 00 Hard, clear pale yellow gel
15: 75: 10 Hard, clear pale yellow gel
75: 15: 10 Hard, clear pale yellow gel
The radiation dose was calculated according to Equation 1 where t is the total radiation
time in seconds. The monomer formulations were exposed to UV radiation for a period of
approximately 30 mins at a dose rate of 9.6 x 10-2
J s-1
. The dose rate was calculated as
described in Section 2.4.4.4.
Radiation dose (J) = dose rate (J s-1
) x t (s) Equation 1
3.3.2. Experimental Swelling Results
The swelling experimental data of HEMA-NVP hydrogels formed is presented in Tables
2-5. The data reflects the effect polymer composition on the swelling behaviour of the
gels and also the effect on the swelling behaviour upon introduction of a plasticiser,
water. Furthermore, the data also reflects the effect of ions in the swelling medium on the
swelling behaviour of the gels. Poly(NVP) (PNVP) hydrogel disintegrated upon a short
exposure to aqueous swelling medium thus further experiments on this system was not
conducted. Graphical representations of the swelling test data are illustrated in Figures 1-
13.
111
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 2. Swelling test on hydrogels in neutral pH environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C Gel D Gel E Gel F
0.00 0.0 0.0 0.0 0.0 0.0 0.0
0.17 8.2 9.2 15.9 36.1 13.1 44.5
0.33 9.8 11.8 21.8 45.8 15.5 55.4
0.50 10.9 13.8 26.0 52.6 17.4 61.4
0.67 12.1 15.6 28.9 57.1 19.6 65.8
0.83 13.2 17.0 31.5 60.3 21.0 68.9
1.00 14.2 18.5 34.0 62.9 23.2 71.5
2.00 17.5 23.1 42.4 73.4 27.6 79.5
3.00 21.1 26.2 47.6 78.3 30.9 83.3
4.00 22.1 28.4 51.6 81.3 33.7 85.7
5.00 23.9 30.4 55.0 83.3 35.7 87.3
7.00 26.6 33.3 59.5 86.0 39.0 89.4
9.00 28.6 35.4 62.4 87.5 41.3 90.5
12.00 30.4 37.3 65.4 89.1 43.6 91.7
24.00 33.5 40.8 69.7 91.3 47.2 93.5
48.00 35.2 41.8 71.1 92.3 48.6 94.6
72.00 35.8 42.0 71.2 92.3 48.8 94.9
96.00 36.0 41.9 71.1 92.2 48.9 95.0
120.00 36.4 41.9 71.1 92.0 49.1 95.0
144.00 36.7 42.0 70.9 91.8 49.1 95.0
170.00 36.6 41.9 70.9 91.6 49.3 95.0
Hydrogel compositions: (NVP : HEMA : H2O (% v/ v)); 00: 100: 00 (Gel A); 20: 80: 00
(Gel B); 50: 50: 00 (Gel C); 80: 20: 00 (Gel D); 15: 75: 10 (Gel E) and 75: 15: 10 (Gel F).
112
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
The swelling data are expressed as percentage water content at respective time intervals.
The percentage water content values were calculated as described in Section 2.6.1.
Graphical representations of the swelling behaviour observed in the NVP-HEMA
hydrogels in neutral pH environment are illustrated in Figures 1 -6. The comparative plots
illustrate the effect of added water to gel formulation E and F, on the swelling efficiency.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C Gel D
Figure 1. Plot of % water content in Gels A-D at 37 oC in neutral pH environment as a
function of time.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel E Gel B Gel F Gel D
Figure 2. Comparative plot of % water content in Gels B, D, E and F at 37 oC in neutral
pH environment as a function of time.
113
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
ifin
ity
Gel A Gel B Gel C Gel D
Figure 3. Plot of fractional swelling in Gels A-D at 37 oC in neutral pH environment as a
function of the square root of time.
0.0
0.4
0.8
1.2
0 5 10 15t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel B Gel D Gel E Gel F
Figure 4. Comparative plot of fractional swelling in Gels B, D, E and F at 37 oC in
neutral pH environment as a function of the square root of time.
114
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel B Gel E
Figure 5. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of the swelling in Gels A, B and E at 37 oC in neutral pH environment.
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel C Gel D Gel F
Figure 6. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of the swelling in Gels C, D and F at 37 oC in neutral pH environment.
The kinetics of diffusion, namely, Fickian and non-Fickian diffusion mechanisms in
swellable polymers have been discussed in Section 1.4.2.3.1.2. The diffusion mechanisms
in polymers can be observed by plotting the fractional swelling data as a function of the
square root of time. A LOG plot derived from this information gives quantitative
information on the diffusion kinetics.
115
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Equation 2, a mathematical expression where y and x are the values on the y and x-axis
respectively, c is the intercept on y-axis and m is the slope, derived from the LOG graphs
illustrated in Figures 5 and 6.
Equation 2 y mx c= ±
The value of the slope represents the parameter n in the power law expression (Equation
3) where Mt is the swollen mass at time t and M∞ is the swollen mass at equilibrium. The
k value is a constant specific to the polymer/solvent system and the parameter n defines
the diffusion mechanism in operation in the polymer matrix.
Mt/M∞ = k t n Equation 3
The slope (n) values calculated from Figures 5 and 6 for the hydrogels A-F are presented
Table 3.
Table 3. Characteristic exponential n values for diffusion in hydrogels in neutral
medium
Hydrogels n values
Gel A 0.39 + 0.05
Gel B 0.47 + 0.02
Gel C 0.53 + 0.01
Gel D 0.62 + 0.01
Gel E 0.41 + 0.02
Gel F 0.63 + 0.01
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
Table 4. Swelling test on hydrogels in isotonic (pH 7.4) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C Gel D Gel E Gel F
0.00 0.0 0.0 0.0 0.0 0.0 0.0
0.17 6.9 9.0 14.3 28.6 9.5 43.3
0.33 8.6 11.9 18.8 36.7 12.6 49.3
0.50 9.8 13.5 22.2 42.2 15.5 56.4
0.67 11.1 14.9 24.5 46.3 15.8 60.7
0.83 11.9 16.2 27.4 49.6 17.1 64.4
1.00 12.7 17.5 29.2 52.1 18.2 66.1
2.00 16.8 21.7 36.7 63.4 23.6 73.8
3.00 20.2 24.4 41.8 69.3 25.5 78.6
4.00 21.8 26.5 45.5 74.0 27.2 80.6
5.00 23.8 28.2 48.3 75.9 28.8 82.6
7.00 26.7 30.8 52.5 79.7 31.2 84.9
9.00 27.5 32.5 55.8 81.8 33.2 86.4
12.00 31.2 34.2 58.6 83.9 34.5 88.0
24.03 33.8 37.1 62.8 87.1 36.7 90.4
48.03 36.2 38.3 64.0 88.3 37.8 91.7
72.02 35.0 39.0 63.7 88.3 38.1 92.0
96.00 35.2 39.3 63.5 88.1 38.2 91.9
120.00 35.0 39.8 62.7 87.7 38.4 91.8
144.00 34.9 39.7 61.4 86.8 39.1 91.5
170.00 35.0 39.7 61.5 87.1 39.9 91.2
Hydrogel compositions: (NVP : HEMA : H2O (% v/ v)); 00: 100: 00 (Gel A); 20: 80: 00
(Gel B); 50: 50: 00 (Gel C); 80: 20: 00 (Gel D); 15: 75: 10 (Gel E) and 75: 15: 10 (Gel F).
117
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Graphical representations of the swelling behaviour observed in the HEMA-NVP
hydrogels in isotonic (pH 7.4) environment are illustrated in Figures 7 -13.
0
20
40
60
80
100
0 50 100 150 200Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C Gel D
Figure 7. Plot of % water content in Gels A-D at 37 oC in isotonic (pH 7.4) environment
as a function of time.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel B Gel D Gel E Gel F
Figure 8. Comparative plot of % water content in Gels B, D, E and F at 37 oC in isotonic
(pH 7.4) environment as a function of time
118
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
neutral isotonic
Figure 9. Comparative plot of % water content in 50 HEMA: 50 NVP hydrogel at 37 oC
in isotonic (pH 7.4) and neutral environments as a function of time.
0.0
0.4
0.8
1.2
0 5 10 15t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C Gel D
Figure 10. Plot of fractional swelling in Gels A-D at 37 oC in isotonic (pH 7.4)
environment as a function of the square root of time.
119
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel B Gel D Gel E Gel F
Figure 11. Comparative plot of fractional swelling in Gels B, D, E and F at 37 oC in
isotonic (pH 7.4) environment as a function of the square root of time
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel B Gel E
Figure 12. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of the swelling in Gels A, B and E at 37 oC in isotonic (pH 7.4)
environment.
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel C Gel D Gel F
Figure 13. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of the swelling in Gels C, D and F at 37 oC in isotonic (pH 7.4) environment
The slope (n) values for hydrogels A-F calculated from Figures 12 and 13 are presented
in Table 5.
Table 5. Characteristic exponential n values for diffusion in hydrogels in isotonic
(pH 7.4) medium
Hydrogels n values
Gel A 0.39 + 0.01
Gel B 0.41 + 0.01
Gel C 0.50 + 0.01
Gel D 0.57 + 0.01
Gel E 0.42 + 0.01
Gel F 0.56 + 0.01
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
3.3.3. Proton NMR Relaxation (T1 and T2) Measurements
The T1 and T2 relaxation measurements were performed on fully hydrated hydrogel
samples. Table 6 presents the NMR relaxation time data for restricted water in the
hydrated hydrogel samples of varying HEMA and NVP content.
Table 6. 1H NMR relaxation T1 and T2 values
Relaxation times (s)
Hydrogels T1 T2
A 0.45830 + 0.06687 0.00321 + 0.00001
C 0.66080 + 0.03113 0.03678 + 0.00077
D 1.86500 + 0.00917 1.01300 + 0.00385
3.3.4. Texture Analysis
Stress relaxation (SR) evaluation was also conducted on the hydrated hydrogel samples of
varying HEMA and NVP content. The hydrogel samples were subjected to a load of
certain magnitude for a period of 30 seconds and then allowed to relax. The stress
relaxation phenomenon is related to the viscoelasticity of the polymer. The Young’s
modulus (E) values of the samples were computed from the linear portion of the stress-
strain curves. The relative SR and E values were calculated as described in Section 2.6.2.
The SR and E values are summarised below in Table 7.
Table 7. Relative SR and E values of the hydrogel samples
Hydrogel samples Relative SR values E (MPa)
Gel A 0.1302 + 0.0291 0.3572 + 0.0218
Gel B 0.1231 + 0.0182 0.0837 + 0.0030
Gel C 0.1020 + 0.0070 0.0468 + 0.0071
Gel D 0.0347 + 0.0109 0.0135 + 0.0003
Gel E 0.1447 + 0.0095 0.0844 + 0.0026
Gel F 0.0424 + 0.0085 0.0096 + 0.0001
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
3.3.5. Experimental Drug Release Results
The experimental data expressed as fractional drug release at specific time intervals are
presented in Table 8. The fractional drug release values were calculated as described in
Section 2.7. Graphical representations of the drug release behaviour in the HEMA-NVP
hydrogels are illustrated in Figures 14 – 19.
Table 8. Theophylline release from HEMA - NVP hydrogels at 37 oC in neutral pH
environment
Average fractional theophylline release values at time (t)
Time (h) Gel A Gel B Gel C Gel D Gel E Gel F
0.00 0.00 0.00 0.00 0.00 0.00 0.00
0.17 0.07 0.09 0.17 0.20 0.10 0.22
0.33 0.10 0.15 0.25 0.31 0.15 0.32
0.50 0.12 0.18 0.33 0.39 0.19 0.41
0.67 0.14 0.21 0.38 0.44 0.22 0.47
0.83 0.16 0.24 0.44 0.50 0.25 0.52
1.00 0.18 0.25 0.48 0.55 0.28 0.56
2.00 0.24 0.35 0.65 0.76 0.40 0.74
3.00 0.29 0.44 0.76 0.83 0.48 0.84
4.00 0.35 0.50 0.83 0.88 0.55 0.89
5.00 0.40 0.54 0.87 0.88 0.61 0.92
7.00 0.44 0.62 0.88 0.89 0.71 0.94
9.00 0.49 0.69 0.90 0.90 0.77 0.94
12.00 0.56 0.74 0.92 0.92 0.83 0.96
24.00 0.73 0.83 0.94 0.93 0.95 0.94
48.00 0.83 0.88 0.93 0.94 0.96 0.95
Hydrogel compositions: (NVP : HEMA : H2O (% v/ v)); 00: 100: 00 (Gel A); 20: 80: 00
(Gel B); 50: 50: 00 (Gel C); 80: 20: 00 (Gel D); 15: 75: 10 (Gel E) and 75: 15: 10 (Gel F).
123
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 10 20 30 40 50 6
Time (h)
Fra
ctio
nal
Dru
g R
elea
se
0
Gel A Gel B Gel C Gel D
Figure 14. Plot of fractional release of theophylline from Gels A-D at 37 oC in neutral pH
environment as a function of time.
0.0
0.4
0.8
1.2
0 10 20 30 40 50 6
Time (h)
Fra
ctio
nal
Dru
g R
elea
se
0
Gel B Gel D Gel E Gel F
Figure 15. Comparative plot of fractional release of theophylline from Gels B, D, E and F
at 37 oC in neutral pH environment as a function of time.
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
0.0
0.4
0.8
1.2
0 2 4 6 8
t1/2
(h1/2
)
Fra
ctio
nal
dru
g r
elea
se
Gel A Gel B Gel C Gel D
Figure 16. Plot of fractional release of theophylline from Gels A-D at 37 oC in neutral pH
environment as a function of the square root of time.
0.0
0.4
0.8
1.2
0 2 4 6 8
t1/2
(h1/2
)
Fra
ctio
nal
Dru
g R
elea
se
Gel B Gel D Gel E Gel F
Figure 17. Comparative plot of fractional release of theophylline from Gels B, D, E and F
at 37 oC in neutral pH environment as a function of the square root of time
125
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel A Gel B Gel E
Figure 18. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time, in the initial stages of the theophylline release from Gels A, B and E at 37 oC in
neutral pH environment
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel C Gel D Gel F
Figure 19. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time, in the initial stages of the theophylline release from Gels C, D and F at 37 oC in
neutral pH environment
126
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
Table 9 presents the slope (n) values calculated from Figures 18 and 19.
Table 9. Characteristic exponential n values for theophylline release from hydrogels
in neutral medium
Hydrogels n values
Gel A 0.51 + 0.03
Gel B 0.52 + 0.02
Gel C 0.61 + 0.01
Gel D 0.56 + 0.02
Gel E 0.55 + 0.01
Gel F 0.55 + 0.02
127
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
3.4. Discussion
3.4.1. Hydrogel Formation
HEMA and NVP based hydrogels of varying monomer compositions were prepared using
a photoinitiator, Irgacure 819, which was found to be a very efficient photoinitiator. The
photolytic decomposition of Irgacure 819 into subsequent free radicals is illustrated in
Scheme 1. Hydrogel formulations upon subjection to UV radiation were successfully
polymerised in ~ 30 minutes at a low dose rate of 9.65 x 10-2
J s-1
. The monomer
formulations containing a higher percentage of HEMA polymerised faster than those
containing high percentage of NVP.
OO
P
O
O
P
O
O
O
P
O+
+
hv
hv
Irgacure 819
Scheme 1. Photolysis of Irgacure 819 under the influence of UV light
128
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
The results on the rate of polymerisation of the hydrogels shed light on the relative
reactivity of the monomers. The free radical formations of the monomers under the
influence of UV light are shown below in Schemes 2 and 3.
N
CH2
C
H2
CH2
OCH
CH2 CHC
H2
R N
CH2
C
H2
CH2
O
R+hv
NVP 2o Free Radical
Scheme 2. Free radical formation of NVP
O
CH3
CH2
O
C
H2
CH
2
OHR O
CH3
O
C
H2
CH
2
OHCH2
R
+hv
HEMA 3o Free Radical
Scheme 3. Free radical formation of HEMA
Lai [19,38], Perera and Shanks [20] have reported the efficient reactivity of HEMA over
NVP. They further suggest that NVP and HEMA do not polymerise well based on
difference of reactivity of the two monomers. According to them, HEMA being a more
reactive monomer tends to enter the polymer more quickly by homopolymerising then
NVP. As a result, NVP is not fully consumed by HEMA and thus it may remain in
partially homopolymerised or unreacted state where it is susceptible to leaching. The
secondary free radical of NVP is an electron donor while the tertiary free radical of
HEMA is electron deficient, thus an acceptor (Schemes 2 and 3). HEMA and NVP can
possibly form a donor/acceptor pair. Scheme 4 illustrates a possible donor/acceptor
interaction between HEMA and NVP.
129
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
N
O
Donor (D) Acceptor (A)
hv
DA interaction
(NVP) (HEMA)
O
OOH
O
OOH
N
O+
Scheme 4. Interaction of HEMA and NVP
However, the copolymerisation reaction between of NVP and HEMA tends to favour the
homopolymerisation of HEMA over the copolymerisation reaction. The relative reactivity
of HEMA could be related to the number of α-hydrogens available for abstraction in
comparison to NVP.
O
CH3
O
OHCH2
R H
HH
H
CH
2
R N
CH2
C
H2
O
H
H
H
HEMA NVP
Figure 20. Structures of HEMA and NVP radicals with available abstractable hydrogens
Figure 20 illustrates the number of α-hydrogens available on the structure on HEMA
radical and NVP radical. HEMA has four abstractable hydrogens over NVP, which has
only three abstractable hydrogens. Thus HEMA tends to preferably undergo
intramolecular hydrogen abstraction leading to favourable homopolymerisation reaction.
However, the presence of an efficient photoinitiator such as Irgacure 819 enhances the
rate of polymerisation providing effective co-polymerisation of HEMA and NVP. All the
hydrogel samples formed from the photopolymerisation process were clear, transparent
and yellowish in colour. The yellow colour is a characteristic of the photoinitiator.
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
3.4.2. Swelling and Drug Release Investigations
Fickian or non-Fickian diffusion kinetics was used to characterize the swelling and drug
release phenomena in the HEMA-NVP hydrogels. Fickian and non-Fickian diffusion
behaviour have been discussed in detail in Section 1.4.2.3.1.2. The swelling action in
polymers is generally time dependent, however, a time independent diffusion is desirable
in drug release applications which gives a zero order release. Diffusion kinetics could be
determined according to a power law expression (Equation 3).
As described in Section 1.4.2.3.1.2, a n value of 0.5 is indicative of Fickian diffusion
while a value of n higher than 0.5 represents non-Fickian diffusion behaviour which could
be further described as anomalous (0.5 < n < 1), case II (n = 1) and super case II (n > 1).
The process of the medium diffusing into the polymer matrix and the incorporated solute
diffusing out of the polymer is simultaneous. The drug release in swellable
macromolecular networks is dependent on the degree of swelling of the network.
Influential factors such as the nature and composition of monomers used in the synthesis
govern the degree of swelling in the hydrogels. Furthermore, the nature of swelling agent
is also an influential contributing factor to swelling efficiency of the hydrogel.
3.4.2.1. Hydrogel Swelling Behaviour
3.4.2.1.1. Effect of the Monomeric Composition on Swelling
Most of these polymer samples when immersed in water produced clear colourless
hydrogels. PNVP hydrogel was found to be water soluble due to insufficient crosslinking
of the macromolecular network. Uncrosslinked PNVP has been reported in the literature
to be water-soluble [2,6,7]. NVP is known to be an extremely hydrophilic monomer. For
polymers, which are highly hydrophilic, and of relatively low molecular weight, the
equilibrium state is an aqueous solution of the polymer.
A gradual increase in water uptake was observed in PHEMA (Gel A), 80 HEMA-20 NVP
(Gel B) and 50 HEMA-50 NVP (Gel C) hydrogels in neutral pH environment at 37 oC
yielding equilibrium water content (EWC) values of 36.6 %, 41.9 % and 70.9 %
respectively (Table 2, Figure 1). The hydrogels showed a linear mass increase upon
131
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
swelling as a function of the square root of time (Figure 3) indicating typical Fickian
diffusion kinetics. This was confirmed by the LOG plots (Figure 5 and 6, Table 3) from
which average n values (Equation 2, 3) of 0.39 for hydrogel A, 0.47 for hydrogel B and
0.53 for hydrogel C were calculated. In the later stages of the swelling process, Case II
diffusion behaviour prevailed, which was indicated by a gradual decrease in the rate of
water uptake by the hydrogels. At this stage the swelling process becomes time
independent where the parameter, n = 1. The rate of polymer chain relaxation at this point
is equal to the rate of solute transport [29].
The 80 NVP-20 HEMA (Gel D) hydrogels however, showed an exponential increase in
water uptake in the first hour followed by a gradual decrease in the swelling around 48
hours yielding an EWC value of 91.6 % (Table 2, Figure 1). The diffusion kinetics in this
hydrogel system was a characteristic of a high order non-Fickian (anomalous) diffusion in
the initial stages followed by case II diffusion in the later stages (Figure 3). Non-Fickian
diffusion behaviour was confirmed by a LOG plot (Figure 6, Table 3), which yielded an
average n value of 0.62. The high water content of the hydrogels containing high
percentage of NVP is due the high hydrophilicity/polarity nature of NVP. Korsmeyer and
Peppas [21] have reported non-Fickian swelling kinetics in copolymers of HEMA and
NVP whilst Bhardwaj et al [31] reported Fickian diffusion kinetics in copolymers of
HEMA and NVP. These researchers have made use of copolymers of varying ratios of
HEMA and NVP.
Bhardwaj et al [31] used high HEMA containing copolymers while Korsmeyer and
Peppas used high NVP containing copolymers. Franson and Peppas [32] have suggested
the dependence of diffusion kinetics on the copolymer composition of HEMA and NVP.
Thus the present results obtained for diffusion kinetics of the hydrogels under study
ranging from Fickian to non-Fickian with increasing NVP content is in agreement with
these researchers.
Water was added to selected formulations in 10 % v/v to enhance the porosity of the
network. The 80 HEMA-20 NVP hydrogel and 20 HEMA-80 NVP hydrogel were the
chosen systems. Thus upon addition of 10 % v/v of H2O, the final formulations of 75
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
HEMA-15 NVP-10 H2O (Gel E) and 15 HEMA-75 NVP-10 H2O (Gel F) were obtained.
Hydrogels E and F upon swelling displayed Fickian and non-Fickian (anomalous)
behaviour with EWC values of 49.3 % and 94.3 % respectively (Tables 2 and 3, Figures
2, 4 and 6). A gradual water uptake was observed in Gel E, however, the efficiency of
swelling was higher than that of 80 HEMA-20 NVP hydrogel. Gel F displayed
exponential swelling in the initial stages with enhanced swelling efficiency in comparison
to 80 NVP-20 HEMA hydrogel. Water acts as a plasticiser for many hydrophilic
polymers and if they can exhibit hydrogel character, the backbone and the side chains
exhibit relatively unrestricted rotational mobility [39].
3.4.2.1.2. Effect of the Ionic Strength on Swelling
PHEMA, copolymers of HEMA-co-NVP and copolymers of HEMA-co-NVP with 10 %
v/v H2O were subjected to swelling in isotonic, pH 7.4 environment at 37 oC. The
hydrogels displayed reduced swelling behaviour in the isotonic environment. Hydrogels
A-F displayed EWC values of 35.0 %, 39.7 %, 61.5 %, 87.1 %, 39.9 % and 91.2 %
respectively (Table 4, Figures 7 and 9). The diffusion kinetics was also influenced by the
presence of ions in the environment. A typical Fickian diffusions kinetics was observed to
be in operation with the exception of hydrogels D and F, which adhered to slight non-
Fickian (anomalous) behaviour with n values of 0.57 and 0.56 respectively (Table 5,
Figures 10-13]. Reduction in the swelling efficiency of these non-ionic hydrogels in
isotonic environment could be attributed to the increase in the ionic strength in the
environment, which effectively reduces the polymer mesh thus reducing the swelling
ratio. Another notable behaviour observed particularly in high swelling hydrogels was
that upon reaching EWC value they began to de-swell upon continual swelling
experimentation.
This observation could be attributed to the concentration gradient established between the
restricted ions in the hydrogels and the free ions in the medium. Hence once the
concentration of the restricted ions is higher than the free ions the diffusion direction is
reversed. The absorbed medium with the ions starts diffusing out of the polymer matrix.
This phenomenal diffusion process is repeatedly reversed in order to maintain an
equilibrium between the ionic concentration of restricted and free ions while the hydrogel
133
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
maintains its maximum swelling capacity for the given environment. This behaviour was
not apparent in low swelling hydrogels due to low swelling activity as that in the case of
high HEMA content hydrogels.
3.4.2.2. Drug Release Studies
A significant variation in the drug release behaviour was observed with varying
monomeric compositions. Hydrogels A-F were tested for theophylline release in neutral
medium at 37 oC. Hydrogels A-F yielded equilibrium drug release (EDR) values of 0.83,
0.88, 0.93, 0.94, 0.96 and 0.95 respectively (Table 8, Figures 14 and 15). A characteristic
rapid release of theophylline was observed in the initial 20 minutes of the experiment.
This rapid release could be described as the burst effect release [29].
PHEMA hydrogel baring a slight initial burst displayed the slowest drug release. The
release kinetics in Gels A and B was that of typical Fickian diffusion behaviour with n
values of 0.51 and 0.52 (Table 9, (16-19). The release profile in the initial stages of
experiment was characterized by a linear increase in the release rate of theophylline as a
function of the square root of time. Hydrogels C-F on the other hand displayed non-
Fickian (anomalous) diffusions kinetics with n value of 0.61, 0.56, 0.55 and 0.55
respectively (Table 9).
The diffusivity of theophylline was observed to increase with increasing water content in
the hydrogel. Hydrogels with high NVP content displayed high theophylline diffusivity.
Thus it could be suggested that the theophylline release was dependent on the swelling
ratio of the polymer. NVP being the more hydrophilic component in the copolymer
contributed to the increased swelling efficiency of the hydrogel leading to an increase in
the polymer mesh size thus allowing rapid diffusion of the incorporated theophylline from
the network. The dependence of solute diffusion on the polymer mesh is well documented
[40-43].
Peppas and Korsmeyer [21] in their study on the swelling-drug release kinetics in
poly(HEMA-co-NVP) hydrogels observed increased diffusivity of theophylline from
networks with increasing NVP content. Teijón et al [13] and Trigo et al [18] from their
134
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
study on the release kinetics in PHEMA hydrogels observed Fickian release mechanism
characterized by a slow linear release of the drug as a function of the square root of time.
The release kinetics observed for PHEMA and poly(HEMA-co-NVP) hydrogels in the
present study is in agreement with these researchers.
3.4.3. Proton NMR Relaxation (T1 and T2) Measurements
The relaxation times T1 and T2 reflect the dynamics of water molecules in the polymer
matrix. The copolymers of HEMA and NVP displayed longer relaxation times with
longer T1 and T2 values with increasing NVP content in the copolymer (Table 6).
PHEMA on the other hand displayed significantly shorter relaxation times. The
experimental data on the relaxation time measurements indicated that the T2 values were
more sensitive to the variation in the polymer composition and their relative water content
than the T1 values.
A short T2 indicates high mobility of water in the polymer matrix, thus indicating that the
water present is interfacial water, which is repelled by the relative hydrophobicity of the
polymer and is effectively mobile. The increased mobility of water in PHEMA sample
could also be attributed to a relatively short T1 value. Shorter T1 values result from rapid
tumbling of free water molecules in the polymer matrix. Due to minimum hydrogen
bonding or hydrophilic attraction between polymer and the water molecules, a faster
rotational motion (tumbling) of the water molecules is observed in the matrix of PHEMA.
The HEMA-co-NVP copolymers have a relatively longer T2 times. This is indicative of
the fact that these copolymers have increased hydrophilicity in comparison to PHEMA.
Furthermore, longer T2 values in the copolymers with higher NVP content could be
attributed to the highly hydrophilic nature of NVP. The study suggested that the
proportion of restricted water, which is due to high hydrogen bonding, is significantly
higher in HEMA-co-NVP hydrogels in comparison to PHEMA hydrogel.
The T1 and T2 relaxation data also correlate to the swelling data and are in agreement with
the deduced diffusion kinetics of the hydrogels under study. Shorter relaxation times are
indicative of Fickian diffusion kinetics while longer relaxation times are indicative of
135
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
non-Fickian diffusion kinetics. Barbieri et al [34], Ghi and Hill [30] in their study on the
diffusion kinetics of HEMA based hydrogels have reported similar results. They also
suggest a direct relevance of relaxation times to the swelling kinetics in polymers.
3.4.4. Texture Analysis (Stress Relaxation)
Texture analysis experiments revealed that the hydrogels under study were viscoelastic.
However, the hydrogels displayed varying degrees of viscoelasticity. The SR values
(Table 7) of the hydrogels decreased with increasing NVP content of the hydrogels.
PHEMA showed a SR value of 0.1302. In comparison, hydrogels D and F displayed
relatively low SR values of 0.0347 and 0.0424 respectively. The viscoelasticity of a
polymer can be described in terms of local frictional forces encountered by a short
segment of a moving chain, together with additional entanglement coupling to other
chains. The entanglements profoundly inhibit long-range conformational rearrangements
and separation of chains from each other.
The SR data suggested that the stress relaxation process in high HEMA content hydrogels
(Gels A, B, C and E) was relatively slower in comparison to high NVP content hydrogels
(Gels E and F). The longer stress relaxation time in high HEMA content hydrogels could
be attributed to the compact nature of the networks. The compactness of the networks
gives rise to increased local frictional force restricting the conformational rearrangement
and separation of the chains.
Hong et al [37], Chirila and Hong [44] recently reported stress relaxation behaviour in
HEMA and NVP based hydrogels. They observed a decrease in the SR value with
increasing NVP content in the hydrogels. The SR data from the present study is in
agreement these researchers. The study suggests that high HEMA content hydrogel
networks (Gels A, B, C and E) are more elastic whilst the hydrogels (Gel D and F) with
high NVP content hydrogel networks have characteristics similar to that of a viscous
fluid.
PHEMA displayed the highest Young’s modulus value of 0.3572 MPa followed by
hydrogels B (0.0837 MPa), E (0.0844 MPa), C (0.0468 MPa), D (0.0135 MPa) and F
136
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
(0.0096 MPa). The Young’s modulus values decreased in the hydrogels with increasing
NVP content suggesting a decrease in the stiffness of the hydrogels. Davis and Huglin
[45] studied the mechanical properties of copolymers of HEMA and NVP of varying
compositions and found increasing stiffness with increasing HEMA content of the
copolymer. The trend of stiffness in relation to HEMA-NVP composition observed in the
present study is in agreement with Davis and Huglin. Furthermore, it is indicative from
this study that there exists a trend between Fickian and non-Fickian diffusion to the SR
values. Hydrogels, which displayed non-Fickian diffusion kinetics, were observed to
behave more like viscous fluid whilst the opposite was observed for hydrogels displaying
Fickian diffusion kinetics. However, the data obtained in the present study does not
sufficiently confirm this trend.
3.5. Conclusions
Irgacure 819 was found to efficiently polymerise NVP and HEMA. High HEMA content
polymers exhibited shorter time of polymerisation in comparison to high NVP content
polymers. Experimental swelling data revealed that the diffusion kinetics in HEMA-NVP
hydrogels range from Fickian to high order non-Fickian (anomalous) diffusion behaviour
in the earlier stages of the experiment followed by case II diffusion in the later stages.
Inclusion of water into the hydrogel formulation resulted in an increased swelling activity
of hydrogels. Presence of ionic environment was found to reduce the swelling efficiency
of the hydrogels.
The proton NMR relaxation times, T1 and T2 were found to correlate with the diffusion
kinetics in polymers. Longer relaxation times were observed in hydrogels, which
displayed non-Fickian diffusion behaviour while hydrogels, which adhered to Fickian
diffusion kinetics displayed shorter relaxation times. Longer relaxations times resulted
from large proportion of restricted water due to enhanced hydrophilic nature of the
hydrogels. Hydrophobic interactions between the polymer and water molecules resulted
in interfacial water, which was increasingly mobile leading to shorter relaxation times.
The texture analysis indicated that hydrogels understudy were viscoelastic with longer
stress relaxation time in high HEMA content hydrogels. The Young’s modulus values
137
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
indicated that hydrogels with high HEMA content were more rigid. The present study
suggests a possible trend between the SR data and the diffusion kinetics exhibited by the
hydrogels, however the present study does not sufficiently confirm this trend.
The drug release experiments revealed that only PHEMA hydrogel adhered to Fickian
transport mechanism in releasing theophylline into the neutral pH environment at 37 oC.
The copolymers of HEMA-co-NVP and copolymers with added plasticiser, water
displayed non-Fickian diffusion behaviour. The burst effect release of the drug was
observed in the initial stages of the release experiment followed by linear release profile.
The rate of theophylline release gradually increased with increase in the concentration of
the hydrophilic monomer, NVP suggesting direct dependence of release kinetics on the
swelling ratio of the polymers.
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Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
3.6. References
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Fundamentals, Peppas, N. A., ed., vol I, CRC Press, Inc., Florida, pp. 1-25,
(1986).
4. Mack, E. J., Okano, T., Kim, S. W., “Hydrogels in Medicine and Pharmacy”-
Polymers, Peppas, N. A., ed., vol II, CRC Press, Inc., Florida, pp. 65-93, (1987).
5. Park, H., Park, K., “Hydrogels and Biodegradable Polymers for Bioapplications”,
Ottenbrite, R. M., Huang, S. J., Park, K., eds., American Chemical Society,
Washington, D.C., pp. 2-10, (1996).
6. Ratner, B. D., Hoffman, A. S., “Hydrogels for Medical and Related Applications”,
Andrade, J. D., ed., American Chemical Society, Washington, D.C., pp. 1-36,
(1976).
7. Park, K., Shalaby, W. S. W., Park, H., “Biodegradable Hydrogels for Drug
Delivery”, Technomic Publishing Company, Inc., Basel, pp. 1-12, (1993).
8. Brannon-Peppas, L., Peppas, N. A., Biomaterials, 11, 635-644, (1990).
9. Hsieh, K.-H., Young, T.-H., ”Polymer Materials Encyclopedia”, Salamone, J. C.,
ed., vol 5, CRC Press, New York, pp. 3087-3091, (1996).
10. Clayton. A. B., Chirila, T. V., Dalton, P. D., Polymer. Int., 42, 45-56, (1997).
11. Hill, D. J. T., Lim, M. C. H., Whittaker, A. K., Polymer Int., 48, 1046-1052,
(1999).
12. Ferreira, L., Vidal, M. M., Gil, M. H., Int. J. Pharm., 194, 169-180, (2000).
13. Teijón, J. M., Trigo, R. M., García, O., Blanco, M. D., Biomaterials, 18, 383-388,
(1997).
14. Hoch, G., Chauhan, A., Radke, C. J., J. Membrane Sci., 214, 199-209, (2003).
15. Jeyanthi, R., Rao, K.P., J. Controlled Release, 13, 91-98, (1990).
16. Yıldırmaz, G., Akgöl, S., Arıca, M. Y., Sönmez, H., Denizli, A., React. Func.
Polym., 56, 103-110, (2003).
17. Duncan, A. C., Boughner, D., Campbell, G., Wan, W. K., Eur. Polym. J., 37,
1821-1826, (2001).
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
18. Trigo, R. M., Blanco, M. D., Teijón, J. M., Sastre, R., Biomaterials, 15, 1181-
1186, (1994).
19. Lai, Y.-C., J. Appl. Polym. Sci., 66, 1475-1484, (1997).
20. Perera, D. I., Shanks, R. A., Polymer Int., 39, 121-127, (1996).
21. Korsmeyer, R. W., Peppas, N. A., J. Controlled Release, 1, 89-98, (1984).
22. Malak, M., Hill, D. J. T., Whittaker, A. K., Polymer. Int., 52, 1740-1748, (2003).
23. Malak, M., Hill, D. J. T., Whittaker, A. K., Polymer. Int., 53, 235, (2004).
24. El-Din, H. M. M. N., Maziad, N. A., El-Naggar, A. W. M., J. Appl. Polym. Sci.,
91, 3274-3280, (2004).
25. Corkhill, P. H., Jolly, A. M., Ng, C. O., Tighe, B., Polymer, 28, 1758-1766,
(1987).
26. Bruck, S. D., J. Biomed. Mat. Res., 7, 387-404, (1973).
27. Ratner, B. D., “Comprehensive Polymer Science” – The Synthesis,
Characterisation, Reactions & Applications of Polymers, Aggarwal, S. K., ed., vol
7, Pergamon Press, Oxford, pp. 201-247, (1989).
28. Peppas, N. A., Khare, A. R., Adv. Drug Deliv. Rev., 11, 1-35, (1993).
29. Peppas, N. A., Korsmeyer, R. W., “Hydrogels in Medicine and Pharmacy”-
Properties and Applications, Peppas, N. A., ed., vol III, CRC Press, Inc., Florida,
pp. 109-135, (1987).
30. Ghi, P. Y., Hill, D. J. T., Maillet, D., Whittaker, A. K., Polymer Commun., 38,
3985-3989, (1997).
31. Bhardwaj, Y. K., Sabharwal, A., Majali, A. B., J. Polym. Mater., 11, 29-34,
(1994).
32. Franson, N. M., Peppas, N. A., J. Appl. Polym. Sci., 28, 1299-1310, (1983).
33. Khare, A., Peppas, N. A., Polymer, 34, 4736-4739, (1993).
34. Barbieri, R., Quagila, M., Delfini, M., Brosio, E., Polymer, 39, 1059-1066,
(1998).
35. Yung, K.-T., Magnetic Resonance Imaging, 21, 135-144, (2003).
36. Kulicke, W. M., Nottelmann, H., “Polymers in Aqueous Media” – Performance
Through Association, Glass, J. E., ed., American Chemical Society, Washington,
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140
Chapter 3: Photo-cured Hydrogels Containing 2-Hydroxyethyl Methacrylate and N-Vinyl-2-Pyrrolidinone
37. Hong, Y., Chirila, T. V., Cuypers M. J. H., Constable, I. J., J. Biomater. Appl., 11,
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38. Lai, Y.-C., J. Polym. Sci. Part A: Polym. Chem., 35, 1039-1046, (1997).
39. Ratner, B. D., “Hydrogels in Medicine and Pharmacy”- Fundamentals, Peppas, N.
A., ed., vol I, CRC Press, Inc., Florida, pp. 85-94, (1986).
40. Yasuda, H., Ikenberry, L. D., Lamaze, C. E., Makromol. Chem., 125, 108-118,
(1969).
41. Wood, J. M., Attwood, D., Collet, J. H., J. Pharm. Pharmacol., 34, 1-4, (1982).
42. Am Ende, M. T., Hariharan, D., Peppas, N. A., React. Polym., 25, 127-137,
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141
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
3.1. Introduction 107
3.2. Experimental Procedure 110
3.3. Results 110
3.3.1. Hydrogel Formation 110
3.3.2. Experimental Swelling Results 111
3.3.3. Proton NMR Relaxation (T1 and T2) Measurements 122
3.3.4. Texture Analysis 122
3.3.5. Experimental Drug Release Results 123
3.4. Discussion 128
3.4.1. Hydrogel Formation 128
3.4.2. Swelling and Drug Release Investigations 131
3.4.2.1. Hydrogel Swelling Behaviour 131
3.4.2.1.1. Effect of the Monomeric Composition on Swelling 131
3.4.2.1.2. Effect of the Ionic Strength on Swelling 133
3.4.2.2. Drug Release Studies 134
3.4.3. Proton NMR Relaxation (T1 and T2) Measurements 135
3.4.4. Texture Analysis (Stress Relaxation) 136
3.5. Conclusions 137
3.6. References 139
142
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
4.1. Introduction
Rapidly polymerisable systems, which respond to light without the necessity of adding a
photosensitiser has been one of the most extensively explored frontiers in photocurable
free radical polymerisation process in recent years [1-17]. The widespread popularity of
such systems is attributed the disadvantages associated with photoinitiators (PI).
The most critical concern in the use of PI is that they are only partially consumed in the
polymerisation process since relatively high concentrations are utilized to ensure adequate
light absorption. This unused PI leads to considerable leachable undesirable small
molecule toxic contaminants in the polymer matrix [1,7]. The PI in the polymer matrix
may cause undesirable degradation leading to an early mechanical failure of the polymer
network thus limiting its possible applications. Generation of coloured and harmful side
products in addition to the primary radical photoproducts of the initiation of the
polymerisation is also a drawback factor in the use of PI [1,7].
The development of these photoinitiator-free photocurable systems has led to the
development of more versatile polymers with enhanced stability and superior
performance. A range of olefins, which function as acceptors and donors could be utilized
to achieve polymerisation in the absence of added photoinitiator. In recent years efficient
photoinduced free radical polymerisation by excited state acceptor monomers, such as N-
substituted maleimides has been shown in numerous donor/acceptor pair combinations.
Jönsson et al [2,8,11,14], Morel et al [13], and Decker et al [5,16] in their respective
study on donor/acceptor pair systems have reported successful use of a range of vinyl
ethers and N-vinyl pyrrolidinone (NVP) as donors combined with a range of N-
substituted maleimides as acceptors. Yamada et al [18] initially reported that maleimides
(MI) and their N-substituted derivatives could homopolymerise in the absence of added
initiator when photo-radiated.
Maleimides undergo a transition from ground to an excited triplet state when subjected to
ultraviolet radiation [17,19]. The excited N-substituted maleimides, which have molar
absorptivity of ~700 M-1
cm-1
have been reported to readily abstract available hydrogens
142
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
from either a vinyl ether backbone or undergo an intra-molecular hydrogen abstraction
process [1,11,12,16]. The resulting free radicals initiate a rapid alternating
copolymerisation [1,2,5,9,10-17]. The excitation and hydrogen abstraction processes of
the maleimides have been described in detail in Section 1.2.6.
MI*
DH
MI
MI*
VE
MI
D
P
P
P
P DH PH D
P
DH
+ +
- Growing polymer chain
- Hydrogen donor
Scheme 1. Interaction of a typical MI with a vinyl ether (VE)
The excited state maleimides besides abstracting available hydrogens from the present co-
monomer such as vinyl ether could also abstract hydrogens from an external additive.
Introducing additives such as hydrogen donors to the donor/acceptor formulation could
enhance the process of donor/acceptor polymerisation initiation [1,2,4,5,9-17,20-22].
Hydrogen donors contain abstractable labile hydrogens that are located adjacent to
heteroatoms such as oxygen, nitrogen and sulfur. An excited state maleimide interacting
with a vinyl ether in the presence of a hydrogen donor is illustrated in Scheme 1 [5].
The unique feature of such systems is their ability to function as the initiator and as well
as the co-monomer in the formulation [9,21,22]. Although the photoinitiator-free
polymerisation process is not as rapid as formulations with added external photoinitiator,
143
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
they are rapid enough to be potentially useful in various applications. Formulations
containing N-substituted maleimides are transparent at wavelengths above 300 nm [7].
This is due to the replacement of N-substituted maleimides by succinimide
chromophores, which have virtually no ultraviolet absorbance at wavelengths greater than
300 nm [1,7]. This ability contributes to a greater stability of such systems in prolonged
ultraviolet exposed environments.
Despite the availability of this photoinitiator- free technology for quite some time, it has
never been utilized to synthesize potential biopolymers. Photoinitiators are also known to
be significant contributors towards the toxicity of finished polymers, thus make
photoinitiator-free formulations ideal candidate for biomedical applications [7]. In this
present work the author has made use of donor/acceptor pairs involving a range of water-
soluble N-hydroxyalkyl maleimides as moderately strong acceptors and NVP as a strong
donor monomer to synthesize polymers, which could function as hydrogels for drug
delivery.
This part of the study firstly involved the evaluation of the efficiency of copolymerisation
of NVP with a series of N-hydroxyalkyl maleimides namely, N-hydroxymethyl
maleimide (HMMI), 2-hydroxy-N-ethyl maleimide (HEMI), 3-hydroxy-N-propyl
maleimide (HPrMI) and 5-hydroxy-N-pentyl maleimide (HPMI). A Differential
Photocalorimetric (DPC) technique was used to investigate the relative kinetics of these
donor/acceptor pairs. Glucose and glucosamine hydrochloride (HCl) were utilized as
added hydrogen donors to enhance the rate of polymerisation.
Suitable donor/acceptor pairs for the initiation of polymerisation were evaluated based on
kinetics data obtained from the DPC measurements. In the synthesis of hydrogels,
comparatively less hydrophilic monomers such as 2-hydroxyethyl methacrylate (HEMA)
and N-vinyl caprolactam (NVC) were also utilized as additional monomers to vary the
swelling capacity of the polymers. Results on photopolymerisation of hydrogels for
sustained drug delivery applications via photoinitiator-free process involving
donor/acceptor interaction have been published by the author in the duration of this
course [20-25].
144
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
4.2. Experimental Procedure
The DPC technique was used to evaluate the efficiency of complex formation between
the donor/acceptor pairs. Experimental specifications and detailed procedure of DPC
measurements are outlined in Sections 2.2.2.5 and 2.8. The hydrogels formed through this
photoinitiator-free process were subjected to swelling drug release experiments in neutral
pH environment at 37 oC. The effect of ionic strength on the swelling ability of hydrogels
in an isotonic (pH 7.4) environment was also evaluated. Three model drugs, theophylline,
thiamine hydrochloride (vitamin B1) and Mn-tetrahydroxyphenyl porphyrin (Mn-TPP-
OH) were utilized for drug release investigations on the hydrogels.
Experimental procedures for the synthesis of photoinitiator-free hydrogels based on N-
hydroxyalkyl maleimides, NVP, HEMA and NVC are outlined in detail in Sections
2.5.2.1-2.5.2.2. Experimental specifications and procedure of swelling-drug release
experiments have been described in Sections 2.6.1 and 2.7. UV-vis spectroscopy was
utilized for the quantitative drug release measurements.
4.3. Results
4.3.1. DPC Measurements
The DPC measurements were performed in N2 atmosphere at an isothermally controlled
temperature of 15 oC under a light intensity of 55 mW cm
-2. The reaction temperature of
15 oC was chosen to eliminate undesirable evaporation of volatile components in the
formulation, which would contribute to inconsistent results. The presence of nitrogen
eliminates atmospheric oxygen, which is known to inhibit the polymerisation reaction.
The maleimides, HMMI, HEMI, HPrMI were each formulated with NVP. The
formulations were exposed to the UV light in the DPC equipment with and without the
presence of hydrogen donors, glucose and glucosamine HCl. The DPC results are
expressed in Figures 1- 4. The calculations on the rate of charge-transfer (CT) complex
polymerisation of the hydroxyalkyl maleimides and NVP are presented in Table 1.
145
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
HMMI-NVP
HPrMI-NVP
HPMI-NVP
-2
8
18H
eat
Flo
w (
W/g
)
0 200 400 600Time (sec)Exo Up
HEMI-NVP
Figure 1. Photo-exotherms of the N-hydroxyalkyl maleimides with NVP in the absence
of an external H-donor
146
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
HPrMI-NVP-Glucose
HPMI-NVP-Glucose
HMMI-NVP-Glucose
-2
8
18H
eat
Flo
w (
W/g
)
0 200 400 600Time (sec)Exo Up
HEMI-NVP-Glucose
Figure 2. Photo-exotherms of the N-hydroxyalkyl maleimides with NVP in the presence
of glucose as the external H-donor
147
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
HPrMI-NVP-Glucosamine HCl
HPMI-NVP-Glucosamine HCl HMMI-NVP-Glucosamine HCl
-2
8
18H
eat
Flo
w (
W/g
)
0 200 400 600Time (sec)Exo Up
HEMI-NVP-Glucosamine HCl
Figure 3. Photo-exotherms of the N-hydroxyalkyl maleimides with NVP in the presence
of glucosamine HCl as the external H-donor
148
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
No H-donor
Glucosamine HCl
-2
8
18H
eat
Flo
w (
W/g
)
0 200 400 600Time (sec)Exo Up
Glucose
Figure 4. Photo-exotherms illustrating the effect of H-donors on the rate of acceptor/
donor interaction between NVP and the N-hydroxyalkyl maleimides.
Table 1. Comparison of the rate of polymerisation of the N-hydroxyalkyl maleimides
and NVP with and without the presence of external H-donors
Rate (J g-1
s-1
) at 15 oC, N2 Systems
1:1 mol No H-donor Glucose Glucosamine HCl
HPMI:NVP 1.18 1.57 2.01
HMMI:NVP 0.73 1.15 1.63
HPrMI:NVP 0.42 0.61 1.39
HEMI:NVP 0.31 0.54 1.08
149
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
The polymerisation rates between the donor/acceptor pairs were calculated according to
Equation 1 where t is the time taken to reach peak max.
Rate of polymerisation (J g-1
s-1
) = -1Peak max (J g )
t (s) Equation 1
4.3.2. Hydrogel Formation
The N-hydroxyalkyl maleimides were formulated with NVP in the presence of glucose
and glucosamine HCl and subjected to UV-radiation. Other monomers, HEMA and NVC
were also included in selected formulations. The observations on the extent of
polymerisation upon applying approximately 9 KJ of radiation dose are described in
Tables 2- 4.
Table 2. Polymerisation status of formulations in the presence of glucosamine HCl
Hydrogel formulation Polymerisation status upon application
of curing dose (~9 KJ)
NVP-HMMI DNP
NVP-HEMI DNP
NVP-HPrMI DNP
NVP-HPMI DNP
Table 3. Polymerisation status of formulations in the presence of glucose
Hydrogel formulation Polymerisation status upon application
of curing dose (~9 KJ)
NVP-HMMI Hard, clear gel
NVP-HEMI Hard, clear gel
NVP-HPrMI Hard, clear gel
NVP-HPMI Hard, clear gel
150
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 4. Polymerisation status of HPMI-NVP formulations with additional
monomers, HEMA and NVC in the presence of glucose
Hydrogel formulation Polymerisation status upon application
of curing dose (~9 KJ)
NVP-HPMI-HEMA Hard, clear gel
NVP-HPMI-NVC Hard, clear pale yellow gel
The details of the monomer compositions are described in Section 2.5.2. DNP indicates
that the formulation did not polymerise. The radiation dose was calculated according to
Equation 2 where t is the total radiation time in seconds. The samples were exposed to
UV radiation for approximately 25 hours at a dose rate of 9.6 x 10-2
J s-1
. The dose rate
was calculated as described in Section 2.4.4.4.
Radiation dose (J) = dose rate (J s-1
) x t (s) Equation 2
4.3.3. Experimental Swelling Results
The swelling experimental data of the N-hydroxyalkyl maleimide-NVP hydrogels formed
are presented in Tables 5-10. The data reflect the effect on the swelling behaviour of the
hydrogels upon introduction of additional monomers and changes in the nature of the
swelling environment such as presence of an ionic environment. HEMI-NVP hydrogels
disintegrated upon a short exposure to aqueous swelling medium thus further experiments
on this system were not conducted.
151
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
Table 5. Swelling test on N-hydroxyalkyl maleimide -NVP hydrogels at 37 oC in
neutral pH environment
Average % water content values at time (t)
Time (hr) Gel A Gel B Gel C Gel D Gel E
0.00 0.0 0.0 0.0 0.0 0.0
0.17 44.0 44.1 47.4 25.9 16.0
0.33 54.2 56.0 58.2 36.0 20.0
0.50 60.6 63.1 64.2 44.4 24.4
0.67 64.9 67.3 68.6 48.8 27.2
0.83 68.1 70.3 71.7 53.3 30.2
1.00 70.6 73.6 73.8 56.3 32.5
2.00 79.5 82.0 81.4 67.4 42.4
3.00 83.6 85.8 85.1 73.0 48.4
4.00 85.9 88.1 87.1 76.8 52.8
5.00 87.4 89.3 88.5 79.3 56.2
7.00 89.3 91.2 90.2 82.3 60.9
9.00 90.5 92.3 91.2 84.4 63.9
12.00 91.6 93.4 92.2 86.3 66.8
24.00 93.5 95.2 93.8 89.9 71.6
48.00 94.4 96.1 94.5 92.1 73.4
72.00 94.7 96.4 94.7 92.5 73.8
96.00 94.8 96.4 94.7 92.7 73.6
120.00 94.8 96.4 94.7 92.8 73.3
144.00 94.8 96.4 94.7 92.8 73.1
170.00 94.8 96.3 94.7 92.7 73.0
Hydrogel compositions: HPMI-NVP (Gel A), HPrMI-NVP (Gel B), HMMI-NVP (Gel
C), HPMI-NVP-NVC (Gel D), HPMI-NVP-HEMA (Gel E)
152
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The swelling data are expressed as percentage water content at respective time intervals.
The percentage water content values were calculated as described in Section 2.6.1.
Graphical representations of the swelling behaviour observed in the NVP-maleimide
hydrogels in neutral pH environment are illustrated in Figures 5 -10.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C
Figure 5. Plot of % water content in hydrogels A - C at 37 oC in neutral pH environment
as a function of time.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel D Gel E
Figure 6. Plot of % water content in hydrogels A, D and E at 37 oC in neutral pH
environment as a function of time.
153
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C
Figure 7. Plot of fractional swelling in hydrogels A - C at 37 oC in neutral pH
environment as a function of the square root of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel D Gel E
Figure 8. Plot of fractional swelling in hydrogels A, D and E at 37 oC in neutral pH
environment as a function of the square root of time.
154
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-2.0
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel B Gel C
Figure 9. Plot of the LOG of fractional swelling in hydrogels A - C in the initial stages of
the swelling experiment at 37 oC in neutral pH environment as a function of the LOG of
time.
-2.0
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel D Gel E
Figure 10. Plot of the LOG of fractional swelling in the hydrogels A, D and E in the
initial stages of the swelling experiment at 37 oC in neutral pH environment as a function
of the LOG of time
155
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
As previously stated the value of the slope represents the parameter n in the power-law
expression (Equation 3) where Mt is the swollen mass at time t and M∞ is the swollen
mass at equilibrium. The k value is a constant specific to the polymer/solvent system and
the parameter n determines the dependence of the medium uptake or release rate on time.
Mt/M∞ = k t n Equation 3
Table 6 presents the slope (n) values for hydrogel A - E calculated from Figure 9 and 10.
Table 6. Characteristic exponential n values for diffusion in hydrogels A - E in
neutral medium
Hydrogels n values
Gel A 0.60 + 0.01
Gel B 0.64 + 0.02
Gel C 0.60 + 0.01
Gel D 0.65 + 0.04
Gel E 0.60 + 0.03
156
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 7. Swelling test on HPMI -NVP, HPMI-NVP-HEMA and HPMI-NVP-NVC
hydrogels at 37 oC in pH 7.4, isotonic environment
Average % water content values at time (t)
Time (h) Gel A Gel D Gel E
0.00 0.0 0.0 0.0
0.17 54.1 23.7 10.8
0.33 63.2 34.8 14.8
0.50 68.1 42.7 18.2
0.67 71.3 48.9 20.8
0.83 73.9 53.7 23.2
1.00 76.1 57.7 25.4
2.00 82.3 70.5 33.9
3.00 85.1 76.0 39.2
4.00 86.8 79.2 43.4
5.00 88.0 81.4 46.3
7.00 89.4 84.3 50.8
9.00 90.3 86.1 53.7
12.00 91.1 87.9 56.6
24.00 92.3 91.0 61.3
48.00 92.6 92.7 63.5
72.00 92.4 93.3 63.7
97.00 92.2 93.5 64.1
120.00 91.9 93.6 63.5
144.00 91.6 93.6 62.5
170.00 91.2 93.6 61.9
157
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
Graphical representations of the effect of isotonic environments on the swelling
behaviour of the NVP-HPMI, NVP-HPMI-HEMA and NVP-HPMI-NVC hydrogels are
illustrated in Figures 11 - 14.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel D Gel E
Figure 11. Plot of % water content in hydrogels A, D and E at 37 oC in isotonic, pH 7.4
environment as a function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel D Gel E
Figure 12. Plot of fractional swelling in the hydrogels A, D and E at 37 oC in isotonic, pH
7.4 environment as a function of the square root of time.
158
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 5 10 15t1/2
(h1/2
)
Mt/
Min
fin
ity
Neutral pH 7.4
Figure 13. Comparative plot of fractional swelling in hydrogel E in isotonic and neutral
pH environments at 37 oC as a function of the square root of time
-2.0
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/M
infi
nit
y
Gel A Gel D Gel E
Figure 14. Plot of the LOG of fractional swelling in gels A, D and E in the initial stages
of the swelling experiment at 37 oC in pH 7.4 environment as a function of the LOG of
time.
159
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
Table 8 presents the slope (n) values for hydrogels A, D and E calculated from Figure 14.
Table 8. Characteristic exponential n values for diffusion in hydrogels A, D and E in
isotonic medium at 37 oC
Hydrogels n values
Gel A 0.57 + 0.02
Gel D 0.83 + 0.07
Gel E 0.57 + 0.02
160
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
4.3.4. Experimental Drug Release Results
The drug release experiments on the N-hydroxyalkyl maleimide–NVP hydrogels were
conducted at 37 oC in neutral pH environment. Theophylline, thiamine HCl and Mn-TPP-
OH were used as the model drugs. The experimental data expressed as fractional drug
release at specific time intervals are presented in Tables 9-12.
Table 9. Theophylline release from N-hydroxyalkyl maleimide -NVP hydrogels at 37
oC in neutral pH environment
Average fractional theophylline release values at
time (t)
Time (h) Gel A Gel B Gel C Gel D Gel E
0.00 0.00 0.00 0.00 0.00 0.00
0.17 0.22 0.24 0.24 0.26 0.20
0.33 0.35 0.37 0.34 0.37 0.30
0.50 0.43 0.45 0.43 0.46 0.38
0.67 0.49 0.52 0.49 0.52 0.46
0.83 0.55 0.58 0.52 0.58 0.53
1.00 0.60 0.63 0.57 0.64 0.55
2.00 0.76 0.80 0.74 0.80 0.72
3.00 0.85 0.88 0.82 0.87 0.85
4.00 0.92 0.93 0.88 0.93 0.88
5.00 0.92 0.95 0.91 0.92 0.93
7.00 0.94 0.97 0.92 0.96 0.98
9.00 0.94 0.97 0.92 0.94 0.99
12.00 0.95 0.97 0.91 0.97 0.96
24.00 0.96 0.97 0.95 0.96 0.95
48.00 0.95 0.97 0.95 0.95 0.97
Hydrogel compositions: HPMI-NVP (Gel A), HPrMI-NVP (Gel B), HMMI-NVP (Gel
C), HPMI-NVP-NVC (Gel D), HPMI-NVP-HEMA (Gel E)
161
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
The fractional drug release values were calculated as described in Section 2.7. Graphical
representations of the drug release behaviour in the N-hydroxyalkyl maleimide-NVP
hydrogels are illustrated in Figures 15 – 20.
0.0
0.4
0.8
1.2
0 10 20 30 40 50 6
Time (h)
Fra
ctio
nal
Dru
g R
elea
se
0
Gel A Gel B Gel C
Figure 15. Plot of the fractional release of theophylline from hydrogels A-C at 37 oC in
neutral pH environment as a function of time.
0.0
0.4
0.8
1.2
0 20 40
Time (h)
Fra
ctio
nal
Dru
g R
elea
se
60
Gel A Gel D Gel E
Figure 16. Plot of the fractional release of theophylline from hydrogels A, D and E at 37
oC in neutral pH environment as a function of time.
162
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 2 4 6 8
t1/2
(h1/2
)
Fra
ctio
nal
Dru
g R
elea
sed
Gel A Gel B Gel C
Figure 17. Plot of the fractional release of theophylline from hydrogels A-C at 37 oC in
neutral pH environment as a function of the square root of time.
0.0
0.4
0.8
1.2
0 2 4 6 8
t1/2
(h1/2
)
Fra
ctio
nal
Dru
g R
elea
sed
Gel A Gel D Gel E
Figure 18. Plot of the fractional release of theophylline hydrogels A, D and E at 37 oC in
neutral pH environment as a function of the square root of time.
163
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel A Gel C Gel B
Figure 19. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time in the initial stages of theophylline release from hydrogels A-C in neutral
environment at 37 oC.
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel A Gel D Gel E
Figure 20. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time in the initial stages of theophylline release from hydrogels A, D and E in neutral
environment at 37 oC.
164
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 10 presents the slope (n) values for theophylline release from NVP-HPMI, NVP-
HPrMI and NVP-HMMI hydrogel systems calculated from Figures 19 and 20.
Table 10. Characteristic exponential n values for theophylline release kinetics of
NVP - N-hydroxyalkyl maleimide hydrogels in neutral medium at 37 oC
Hydrogels n values
Gel A 0.53 + 0.01
Gel B 0.52 + 0.02
Gel C 0.51 + 0.02
Gel D 0.50 + 0.04
Gel E 0.60 + 0.02
165
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
Table 11. Theophylline, thiamine HCl and Mn-TPP-OH release from HMMI -NVP
hydrogels at 37 oC in neutral pH environment
Average fractional release values for the
model drugs at time (t)
Time (h) Mn-TPP-OH Thiamine HCl Theophylline
0.00 0.00 0.00 0.00
0.17 0.08 0.28 0.24
0.33 0.16 0.42 0.34
0.50 0.20 0.52 0.43
0.67 0.28 0.59 0.49
0.83 0.28 0.67 0.52
1.00 0.32 0.69 0.57
2.00 0.46 0.85 0.74
3.00 0.57 0.90 0.82
4.00 0.60 0.91 0.88
5.00 0.60 0.91 0.91
7.00 0.62 0.93 0.92
9.00 0.63 0.92 0.92
12.00 0.70 0.93 0.91
24.00 0.82 0.95 0.95
48.00 0.85 0.95 0.93
Graphical representations of the effect of varying molecular weight and nature of the
model drugs on the release behaviour in NVP-HMMI hydrogels are illustrated in Figures
21 – 23.
166
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 20 40Time (h)
Fra
ctio
nal
Dru
g R
elea
sed
60
Mn-TPP-OH Thiamine HCl Theophylline
Figure 21. Plot of the fractional release of theophylline, thiamine HCl and Mn-TPP-OH
from NVP-HPMI hydrogels at 37 oC in neutral pH environment as a function of time.
0.0
0.4
0.8
1.2
0 2 4 6
t1/2
(h1/2
)
Fra
ctio
nal
Dru
g R
elea
sed
8
Mn-TPP-OH Thiamine HCl Theophylline
Figure 22. Plot of the fractional release of theophylline, thiamine HCl and Mn-TPP-OH
from NVP-HPMI hydrogels at 37 oC in neutral pH environment as a function of the
square root of time.
167
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Mn-TPP-OH Thiamine HCl Theophylline
Figure 23. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time in the initial stages of Mn-TPP-OH, thiamine HCl and theophylline release from
NVP-HMMI hydrogels in neutral pH environment at 37 oC.
Table 12 presents the slope (n) values calculated from Figure 23 for release kinetics of
varying molecular weight drugs from the NVP-HMMI hydrogel systems.
Table 12. Characteristic exponential n values for release kinetics of theophylline,
thiamine HCl and Mn-TPP-OH from HMMI-NVP hydrogel in neutral medium
Model drugs n values
Mn-TPP-OH 0.74 + 0.08
Thiamine HCl 0.52 + 0.01
Theophylline 0.52 + 0.02
168
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
4.4. Discussion
4.4.1. DPC Measurements
The DPC measurements (Table 1) revealed that the HPMI-NVP system was the most
efficient donor/acceptor pair system followed by HMMI-NVP and then HPrMI-NVP. The
rate calculations were carried out according to Equation 1. Efficient systems were
characterized by a sharp peak in the photo-exotherms (Figures 1-4). The HEMI-NVP
system was found to be the least efficient donor/acceptor pair combination. Furthermore,
the rates of polymerisation of these systems were observed to have enhanced upon
addition of hydrogen donors, including glucose and glucosamine HCl. Glucosamine HCl
was found to be a more superior hydrogen donor over glucose as suggested by the
polymerisation rate calculations in Table 1.
The polymerisation reaction of the N-hydroxyalkyl maleimides and NVP in the absence
of external hydrogen donors could be explained as an electron/proton transfer process
between NVP and the N-hydroxyalkyl maleimides. Scheme 2 [9] illustrates a typical
electron/proton transfer between a N-hydroxyalkyl maleimide and NVP leading to the
formation of radical initiators. The relative efficiency of the polymerisation of N-
hydroxyalkyl maleimides with NVP could be related to the strength of the CT complex
formed between the donor/acceptor pair. The strength of the CT complex relies on the
nature of its donor/acceptor pair in terms of the chemical structure, which governs their
compatibility and reactivity.
4.4.1.1. Effect of the Monomer Structure on Polymerisation
The chemical structures of the acceptor and the donor monomers play a very crucial role
in determining the rate of polymerisation. Ng et al [26] in their study on CT complex
polymerisation reactions have emphasised the importance of donor/acceptor monomer
structures on the strength of CT complex formed. The only difference between the four
maleimides under study is the chain length of the hydroxyalkyl N-substituent. The
presence of α hydrogens, which are self abstracted by the maleimides is a very influential
structural characteristic in determining their relative efficiencies. HPMI, HPrMI and
HEMI have four readily abstractable α-hydrogens in comparison to HMMI, which has
only two α abstractable hydrogens as illustrated in Figure 24.
169
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
N OH
O
O
H
H
H
HOH
H
HN
O
O
H
H
N OH
O
O
H
H
H
H
N
O
O
H
H
OH
(1)
(2)
(3)
(4)
(1)
(2)
(3)
(4)
HPMI
HMMI
HPrMI
HEMI
(1)
(2)
(1)
(2)
(3)
(4)
Figure 24. Structures of the N-substituted maleimides illustrating the number of available
abstractable hydrogens located adjacent to heteroatoms, N and O
Based on the fact on the number of abstractable hydrogens contained by the maleimides,
the order of expected reactivity would be as such: HPMI = HEMI = HPrMI > HMMI.
However this was only true for HPMI, which showed the highest reactivity (1.18 J g-1
s-1
).
HMMI despite containing only two abstractable hydrogens was found to be more reactive
(0.73 J g-1
s-1
) than HEMI (0.31 J g-1
s-1
) and HPrMI (0.42 J g-1
s-1
). The present study
thus suggests the involvement of further influential factors such as the chain length of the
N-substituent hydroxyalkyl group. The relative inertness of HEMI paired with NVP in
comparison to HMMI, HPrMI and HPMI paired with NVP could be explained along this
line.
Jönsson et al [9] from their studies on the role of N-hydroxyalkyl maleimides as initiators
and acceptors in donor/acceptor formulations have suggested a predominant intra-
molecular H-abstraction of HEMI over the inter-molecular abstraction from the donor.
They further suggest a phenomenal thermodynamically favoured seven membered ring
formation of HEMI upon excitation as illustrated in Scheme 2.
170
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
N R
O
O
N R
O
O
N R
O
O
N R
O
O
N R
O
O
N+
O
HH
N R
O
O
N R
O
O
N+
OH
H
N
O
H
N R
OH
O
N R
O
O
e- H+
Transfer/
-CH2CH
2CH
2CH
2CH
2OH
-CH2CH
2CH
2OH
-CH2CH
2OH
-CH2OH
hv
*
(RMI)
NVP
+
+
R:
Scheme 2. Direct excitation of a typical N-substituted maleimide by electron/proton
transfer from NVP
171
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
N
O
OH
OH
H
N
O
OH
OH
H
N
O
OHH
OHN
O
OH H
OH
N
O
OH
H
OH
N
O
OH
OHH
NH
O
O
OHH
H+ - Transfer +
hv
Scheme 3. Intramolecular H-abstraction for HEMI leading to a thermodynamically stable
ring formation
172
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
However, the ring formation inevitably reduces the number of initiating radicals, thus
reducing the rate of polymerisation. The extent of the predominance of intra-molecular H-
abstraction over inter-molecular H-abstraction in HEMI however has not been established
to date. The ring formation of the hydroxyalkyl group with the carbonyl centre as
suggested in Scheme 3 would not be thermodynamically favourable at all for HMMI and
HPMI. The hydroxymethyl N-substituent of HMMI is very short in chain length thus the
possibility of ring formation would be a three membered ring, which is highly strained
and thermodynamically unstable.
The hydroxypentyl N-substituent of HPMI has a fairly long chain length, which would
inhibit the ring formation. Firstly, being a longer chain there will be a considerable
amount of steric hindrance for it to form a ring with the carbonyl centre. Secondly, a
possible the ring formation would be a 10 membered ring, which would also be
thermodynamically unstable due to a relatively high angle strain.
The slow reactivity of HPrMI compared to HMMI and HPMI could also be attributed to
some extent to the ring formation phenomenon displayed by HEMI. However, a faster
reactivity of HPrMI as opposed to HEMI suggests that the ring formation in HPrMI is not
as thermodynamically favourable as that in HEMI. The availability of four readily
abstractable α-hydrogens on HPMI in comparison to two readily abstractable α-
hydrogens on HMMI explains the polymerisation efficiency of HPMI over HMMI.
4.4.1.2. Hydrogen Abstraction
NVP has three readily abstractable α hydrogens, which are abstracted by the excited state
maleimide via an inter-molecular process. However, the introduction of glucose and
glucosamine HCl, which have several abstractable α hydrogens, caused a dramatic
increase in the rate of polymerisation reaction by increasing the number of initiating free
radicals. Hydrogens available for inter-molecular hydrogen abstractions are illustrated in
Figure 25.
173
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
O
OH OH
OH
OH
H
HH
H
OH
H
HH
O
OH NH3
+
OH
OH
H
HH
H
OH
H
HH
N
O
H
HH Cl
(1)
(2)(3)
(4)
(5)
(6)(7)
(1)
(2)(3)
(4)
(5)
(6)(7)
Glucose Glucosamine HClNVP
(1)
(2)(3)
Figure 25. Structures of the NVP, glucose and glucosamine HCl illustrating the number
of available hydrogens located adjacent to heteroatoms, N and O for inter-molecular
hydrogen abstraction.
Scheme 4 [21] illustrates the inter-molecular H-abstraction from a glucose molecule by an
excited state N-substituted maleimide. Glucose and glucosamine HCl have the same
number of abstractable α-hydrogens, however, glucosamine HCl was found to be a more
superior H-donor and reaction rate enhancer. This observation could be attributed to the
fact that glucosamine HCl (pKa = 7.75) is more acidic than glucose (pKa = 12.34) due to
the presence of a positive charge on the nitrogen making it a superior hydrogen donor.
Hence a rapid CT complex polymerisation reaction was observed from the DPC
measurements
4.4.2. Hydrogel Formation
Hydrogel formations via ultraviolet radiation were attempted using the donor/acceptor
formulations of N-hydroxyalkyl maleimides and NVP (Tables 2-4). Formulations
containing glucosamine HCl did not polymerise despite its enhancement of reaction
activity shown in the DPC measurements. A clear colourless formulation containing
glucosamine HCl turned into an intense orange coloured solution after being exposed to
UV light. UV light could not penetrate through the intense coloured formulation hence a
complete polymerisation was not achieved.
174
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
N
O
O
R
N
O
O
R
T
N
OH
O
R
O
OH OH
OH
OH
H
HH
H
OH
H
H
O
OH OH
OH
OH
H
HH
H
OH
H
HH
O
OH OH
OH
OH
HH
H
OH
H
H
OH OH
OH
OH
HH
H
OH
O
H
N
O
O
R
H
H
H
(1)
(2)(3)
(4)
(5)
*
1
hv
H- abstraction
Rearrangement
+
+
+
+
(1)
(2)(3)
(4)
(5)
(6)
(1)
(2)(3)
(4)
(5)
(2)(3)
(4)(5)
(7)
(7) (6)
(7) (6)
(7) (6)
Scheme 4. Direct intermolecular H-abstraction of an excited state N-substituted
maleimide from a glucose molecule
175
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
However, the colouration of the formulation can be due to several factors. Glucosamine
HCl could also react with the carbonyl group on N-hydroxyalkyl maleimide via a
nucleophilic attack on the carbonyl followed a proton transfer from nitrogen to oxygen
leading to the formation of an imine. The colour could also be due to the formation of
short polymer chains. The formation of the coloured compound was not investigated in
sufficient detail thus no definite conclusions could drawn on nature of the coloured
compound from this experimental observations. The photocurable sample size should also
be taken into consideration with respect to the light intensity. The sample sizes for DPC
measurements were much smaller than the sample formulation for hydrogel formation.
Furthermore, the UV light source used to cure the hydrogel samples has a relatively lower
intensity thus requiring longer polymerisation time. Thus it could be suggested from this
observation that a more intense light source over a short span of time would be required
to achieve complete polymerisation of such systems.
Hydrogel formulations containing glucose as the hydrogen donor on the other hand were
successfully synthesized. HEMA and NVC, monomers less hydrophilic in nature
compared to NVP, were also included in some formulations, and resulted in successfully
synthesized polymers. Glucose was also observed to enhance the mechanical strength of
the polymeric hydrogels, allowing them to be more resilient upon swelling.
4.4.3. Swelling and Drug Release Investigations
The swelling and drug release behaviour in the hydroxyalkyl maleimide-NVP hydrogels
were characterized on the basis of Fickian or non-Fickian diffusion behaviour. Fickian
and non-Fickian diffusion have been discussed in detail in Section 1.4.2.3.1.2. As
previously described, the swelling action in polymers is generally time dependent and
could be described according Equation 3. The parameter n in the equation characterizes
the diffusion kinetics in the polymer. The characteristic n values signifying Fickian and
non-Fickian diffusion have been mentioned in Section 1.4.2.3.1.2.
176
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
4.4.3.1. Hydrogel Swelling Behaviour
4.4.3.1.1. Effect of the Monomeric Composition on Swelling
The HEMI-NVP hydrogel network disintegrated after a short exposure to the aqueous
swelling medium. This was indicative of the fact that the hydrogel network was not
suitably crosslinked. The bulk of the composition of the hydrogel formulation is NVP
(71.45% w/w), which gives a water-soluble polymer upon homopolymerisation. The
water solubility of the HEMI-NVP hydrogel network thus suggests that HEMI did not
actively crosslink with NVP. The final polymer was simply a homopolymer of NVP,
which disintegrated upon placement in the swelling medium. This can be explained by the
ring formation mechanism (Scheme 3) of HEMI, which suggests that upon preferable ring
formation, HEMI became inert and did not react with NVP.
An exponential increase in the water uptake by the HMMI-NVP, HPrMI-NVP and
HPMI-NVP hydrogels in neutral pH environment at 37 oC was observed in the first hour
(Figure 7) indicating non-Fickian (anomalous) transport behaviour. This was confirmed
by a LOG plot (Figure 9, Table 6) from which average n values (Equation 3) of 0.64 for
HPrMI-NVP and 0.60 for HMMI-NVP and HPMI-NVP were calculated. In the later
stages of the swelling process, Case II diffusion behaviour prevailed which was indicated
by a gradual decrease in the rate of water uptake by the hydrogels.
The gradual decrease in the rate of water uptake was observed around 7 hours of swelling
which became constant upon 48 hours of constant swelling, thus indicating equilibrium
water content was achieved (Table 5, Figure 5). At this stage the swelling process became
time independent where the parameter n = 1. The rate of polymer chain relaxation at this
point is equal to the rate of diffusion [27]. Equilibrium water content (EWC) values of
94.8 %, 96.3 % and 94.7 % were observed for the HPMI-NVP, HPrMI-NVP and HMMI-
NVP hydrogels respectively.
The HPMI-NVP-HEMA and HPMI-NVP-NVC hydrogels also adhered to non-Fickian
(anomalous) diffusion in the initial stages of the swelling experiment followed by case II
transport in the later stages (Figures 8 and 10). However, a reduced swelling activity was
observed from these systems in comparison to HPMI-NVP system. Equilibrium swelling
177
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
was achieved after 48 hours of constant experimental swelling which yielded EWC values
of 73.4 % and 92.1 % for HPMI-NVP-HEMA and HPMI-NVP-NVC hydrogels
respectively (Table 5, Figure 6). HPMI-NVP-HEMA hydrogel displayed significant
suppressed swelling in comparison to HPMI-NVP hydrogel throughout the swelling
process with the percentage water content values ranging from 16.0 % at 10 minutes to an
EWC value of 73.4 % at 48 hours.
HPMI-NVP-NVC gel showed suppressed swelling in the initial stages of the swelling
experiment with % water content values ranging from 25.9 % at 10 minutes to an EWC
value of 92.1 % at 48 hours. Inclusion of relatively hydrophobic monomers such as
HEMA and NVC has led to a reduction in the equilibrium water content uptake with
HEMA proving to be a more effective monomer in reducing water uptake. HEMA is
slightly acidic and also a weak acceptor monomer while NVC is a slightly basic and
could function as a donor. Thus it could be stated that HEMA and NVC were effectively
consumed in the formulation upon UV curing, as their presence was clearly demonstrated
by the swelling process.
4.4.3.1.2. Effect of the Ionic Strength on Swelling
The HPMI-NVP, HPMI-NVP-NVC and HPMI-NVP-HEMA hydrogels were subjected to
swelling in isotonic, pH 7.4 environment at 37 oC. The hydrogels were observed to de-
swell in the isotonic environment (Table 7, Figures 11 - 13). The isotonic environment
had a more pronounced de-swelling effect on HPMI-NVP-HEMA hydrogels in
comparison to HPMI-NVP and HPMI-NVP-NVC hydrogels. The diffusion order was
however influenced by the presence of ions in the environment. A slight reduction in the
swelling efficiency of the hydrogels was observed.
HPMI-NVP, HPMI-NVP-NVC and HPMI-NVP-HEMA hydrogels displayed non-Fickian
(anomalous) behaviour in the initial stages of the swelling experiment in isotonic
environment. This was confirmed by a LOG plot (Figure 14, Table 8) from which average
n values of 0.57, 0.83 and 0.57 were obtained for hydrogels A, D and E respectively.
EWC values of 93.6 %, 91.3 % and 61.9 % for HPMI-NVP-NVC, HPMI-NVP and
HPMI-NVP-HEMA respectively were observed after 48 hours of constant swelling.
178
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Another notable behaviour observed particularly in HPMI-NVP and HPMI-NVP-HEMA
was that upon reaching EWC value these two hydrogels began to de-swell upon continual
swelling process. This observation could be attributed to the concentration gradient
established between the restricted ions in the hydrogels and the free ions in the medium.
Hence once the concentration of the restricted ions is higher than the free ions, the
diffusion direction is reversed. The absorbed medium with the ions starts diffusing out of
the polymer matrix. This phenomenal diffusion process is repeatedly reversed in order to
maintain an equilibrium between the ionic concentration of restricted and free ions while
the hydrogel maintains its maximum swelling capacity for the given environment.
4.4.3.2. Drug Release Studies
4.4.3.2.1. Effect of the Monomeric Composition on Drug Release
The drug release experiments conducted on HPMI-NVP, HPrMI-NVP and HMMI-NVP
hydrogels (Table 9, Figures 15 and 17) in neutral pH environment at 37 oC using
theophylline as the model drug yielded equilibrium drug release (EDR) values of 0.95,
0.97 and 0.93 respectively in 48 hours. The drug release rate increased rapidly in the first
2 hours of the experiment after which the release rate gradually slowed down and
eventually became constant around 7 hours. The quick release of the drug from the carrier
in the early stages of the experiment is attributed to the burst effect release [27]. As
described previously the polymer containing the incorporated solute has to swell to a
certain extent before it can release its contents. The resultant effect is the fast release of
the solute in a short span of time. The LOG plot (Figure 19, Table 10) indicated Fickian
diffusion kinetics of theophylline release with an average n value of 0.53, 0.52 and 0.51
obtained for hydrogels A, B and C respectively.
The drug release experiment on HPMI-NVP-NVC and HPMI-NVP-HEMA using
theophylline at 37 oC in neutral pH environment yielded EDR values of 0.95 and 0.97
respectively in 48 hours (Table 9, Figures 16, 18 and 20). The theophylline release trends
in HPMI-NVP-NVC and HPMI-NVP-HEMA were similar to the HPMI-NVP hydrogel
with a burst effect characterized by a rapid release of theophylline in the first 2 hours. The
release rate in the hydrogels gradually slowed down post this period and became constant
around 7 hours.
179
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
The drug release rate however in HPMI-NVP-HEMA hydrogel was slightly suppressed in
the initial stages of the experiment confining to non-Fickian solute transport behaviour
(Figure 18). This was confirmed by a LOG plot (Figure 20, Table 10) which yielded an
average n value of 0.6. The reduction in release of theophylline in HPMI-NVP-HEMA
hydrogel could be explained with relation to the reduced hydrophilic nature of the gel.
HPMI-NVP-NVC hydrogel adhered to Fickian transport with very similar drug release
rate to that of HPMI-NVP hydrogel despite the fact that HPMI-NVP-NVC had a
relatively lower swelling activity. This observation could be attributed to the relatively
low molecular weight of theophylline, which diffused with ease through the pores of
these two hydrogel networks at a similar rate.
4.4.3.2.2. Effect of the Nature and the Molecule Size of the Drug
The release experiment conducted on HMMI-NVP hydrogel using three model drugs,
theophylline, thiamine HCl and Mn-TPP-OH yielded EDR values of 0.93, 0.95 and 0.85
respectively in 48 hours (Table 11, Figures 21-23). A rapid release of theophylline and
thiamine HCl was observed in the first 2 hours followed by a gradual reduction in the
drug release activity post this period. The release rate became constant around 7 hours.
Mn-TPP-OH was observed to release with an initial burst followed by a gradual increase
in the release rate in the first two hours of the experiment and then a gradual reduction in
the release rate post this period.
The release of Mn-TPP-OH (MW. 733.69 g mol-1
) was the slowest among the model
drugs. The release profile adhered to typical non-Fickian solute transport behaviour
(Figure 22). The slow release could be attributed to its relatively large molecular mass.
Brazel and Peppas [28] have reported similar observation from their study on release rates
of drugs with varying molecular weights. However, theophylline (MW. 180.16 g mol-1
)
release was observed to be slightly slower than thiamine HCl (MW. 337.26 g mol-1
) in the
initial stages of the drug release experiment but followed by similar release rates in the
later stages of the release experiment. An average n value of 0.52 obtained from the LOG
graph (Figure 23, Table 12) for thiamine HCl and theophylline suggested that Fickian
transport mechanism was in operation. Furthermore, the study suggested that the release
rate of the drug was not only affected by the molecular mass but also the nature of the
180
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
drug in terms of solubility. Based on the molecular weights of the drugs, the order of
expected release rate would be: theophylline > thiamine HCl > Mn-TPP-OH. This only
holds true for Mn-TPP-OH. The faster release of thiamine HCl over theophylline despite
the fact that thiamine HCl has a larger molecular mass could be explained in terms of the
relative solubility of the drugs. Colombo et al [29] in their drug release studies observed
an increased release rate with increased solubility of the drug. Theophylline has a limited
solubility of 8.33 x 10-3
g ml-1
in water at 25 oC compared to thiamine HCl, which is a salt
that readily dissolves in water. Thus, despite thiamine HCl having a larger molecular
weight, its ease of dissolutions allows it to be released more efficiently than theophylline.
4.5. Conclusions
The kinetic studies on the N-hydroxyalkyl maleimides and NVP as donor/acceptor pairs
revealed that HPMI was the most efficient acceptor monomer followed by HMMI and
then HPrMI. HEMI-NVP was found to be the least efficient donor/acceptor pair.
Inclusion of hydrogen donors, glucose and glucosamine HCl were found to enhance the
rate of polymerisation with glucosamine HCl being a more efficient donor based on DPC
measurements.
Polymeric hydrogels based on N-hydroxyalkyl maleimides and NVP were successfully
synthesized via photoinitiator-free UV curing technique. Inclusion of more hydrophobic
monomers, HEMA and NVC resulted in successfully synthesized hydrogels. The
hydrogels were found to be resilient and competent drug delivery devices.
Experimental swelling data revealed that the HPMI-NVP, HPrMI-NVP and HMMI-NVP
hydrogels adhered to non-Fickian (anomalous) diffusion behaviour in the early stages of
the experiment followed by case II diffusion in the later stages. Inclusion of HEMA and
NVC into the hydrogel network resulted in a reduced swelling activity of hydrogels.
Presence of ionic environment was found to reduce the swelling efficiency of the
hydrogels with a more pronounced effect on HPMI-HEMA-NVP hydrogels. A time
independent, case II swelling was observed in HPMI-NVP and HPMI-NVP-HEMA
hydrogels while HPMI-NVP-NVC hydrogel displayed a super case II swelling behaviour
in isotonic environment
181
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
The drug release experiments revealed that HPMI-NVP, HPrMI-NVP and HMMI-NVP
hydrogels adhered to Fickian transport mechanism in releasing theophylline into the
neutral pH environment at 37 oC. The burst effect on the release of drugs was observed in
the initial stages of the release experiment followed by a linear release profile. The rate of
theophylline release from HPMI-NVP, HPrMI-NVP and HMMI-NVP hydrogels were
found to be similar. Inclusion of NVC, despite showing reduced swelling, had a very
similar release rate to that observed in the HPMI-NVP hydrogel without NVC indicating
that theophylline being a low molecular weight drug could diffuse through the hydrogels
at a similar rate.
The drug release experiments involving the various model drugs in neutral pH
environment indicated a faster release of thiamine HCl followed by theophylline and then
Mn-TPP-OH. The study suggested that increase in the molecular weight of the drug
reduces the release rate. Additionally the nature of the drug such as the solubility
parameter is also crucial and could predominantly govern the kinetics of release as
indicated by the experimental data on a faster release of thiamine HCl over theophylline.
182
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
4.6. References
1. Hoyle, C. E., Clark, S. C., Jönsson, S., Shimose, M., Polymer, 38, 5695-5697,
(1997).
2. Jönsson, S., Sundell, P.-E., Hultgren, J., Sheng, D., Hoyle, C. E., Prog. Org.
Coat., 27, 107-122, (1996).
3. Lee, C.-W., Kim, J.-M., Han, D. K., Ahn, K.-D., J.M.S.-Pure Appl. Chem., A36,
1387-1399, (1999).
4. Jönsson, S., Viswanathan, K., Hoyle, C. E., Clark, S. C., Miller, C., Morel, F.,
Decker, C., Nuclear Instruments Methods Phys. Res. B, 151, 268-278, (1999).
5. Decker, C., Morel, F., Jönsson, S., Clark, S., Hoyle, C., Macromol. Chem. Phys.,
200, 1005-1013, (1999).
6. Clark, S. C., Jönsson, S., Hoyle, C. E., Polym. Prep., 37, 348-349, (1996).
7. Jönsson, S., Sundell, P.-E. G., Schaeffer, W. R., United States Patent., 5446073,
(1995).
8. Jönsson, S., Ericsson, J. E., Sundell, P.-E., Shimose, M., Clark, S. C., Miller, C.,
Owens, J., Hoyle, C., Proc. RadTech North America’96, Nashville, USA, pp. 377-
385, (1996).
9. Jönsson, S., Yang, D., Viswanathan, K., Nguyen, C. K., Miller, C., Lindgren, K.,
Hoyle, C. E, Proc. RadTech Asia’01, Kunming, China, pp. 182-195, (2001).
10. Jönsson, S., Hultgren, J., Sundell, P.-E., Shimose, M., Owens, J., Vaughn, K.,
Hoyle, C. E., Proc. RadTech Asia’95, Bangkok, Thailand, pp.118-125, (1995).
11. Jönsson, S., Sundell, P.-E., Shimose, M., Owens, J., Miller, C., Clark, S. C.,
Hoyle, C. E., ACS Polymeric Mater. Sci. Eng., 74, 319-320, (1996).
12. Miller, C. W., Hoyle, C. E., Howard, C., Polym. Prep., 37, 346-347, (1996).
13. Morel, F., Decker, C., Jönsson, S., Clark, S. C., Hoyle, C. E., Polymer, 40, 2447-
2454, (1999).
14. Jönsson, S., Sundell, P.-E., Shimose, M., Clark, S., Miller, C., Morel, F., Decker,
C., Hoyle, C. E., Nuclear Instruments Methods Phys. Res. B, 131, 276-290,
(1997).
15. Clark, S. C., Hoyle, C. E., Jönsson, S., Morel, F., Decker, C., Polymer, 40, 5063-
5072, (1999).
183
Chapter 4: Photoinitiator-Free Photopolymerisation of N-Substituted Maleimides and N-Vinylpyrrolidinone Hydrogels
16. Decker, C., Morel, F., Jönsson, S., Clark, S. C., Hoyle, C. E., ACS Polymeric
Mater. Sci. Eng., 75, 198-199, (1996).
17. Viswanathan, K., Clark, S., Miller, C., Hoyle, C. E., Jönsson, S., Shao, L., Polym.
Prep., 39, 644-645, (1998).
18. Yamada, M., Takase, I., Koutou, N., Polym. Lett., 6, 883-888, (1968).
19. Aida, H., Takase, I., Nozi, T., Makromol. Chem., 190, 2821-2831, (1989).
20. Ng, L.-T., Jönsson, S., Lindgren. K., Swami, S., Hoyle, C. E., Clark, S., Proc.
RadTech Europe’01, Basel, Switzerland, 609-613, (2001).
21. Ng, L.-T., Swami, S., Jönsson, S., Radiation Phys. Chem., 69, 321-328, (2004).
22. Swami, S., Ng, L.-T., Jönsson, S., Proc. RadTech Asia ’03, Yokohama, Japan, pp.
677-680, (2003).
23. Ng, L.-T., Jönsson, S., Swami, S., Lindgren, K., Polym. Int., 51, 1398-1403,
(2002).
24. Jönsson, S., Viswanathan, K., Lindgren, K., Swami, S., Ng, L.-T., Polym. Prep.,
44, 7-8, (2003).
25. Garnett, J. L., Ng, L.-T., Nguyen, D., Swami, S., Zilic, E., Radiation Phys. Chem.,
63, 459-463, (2002).
26. Ng, L.-T., Garnett, J. L., Zilic, E., Ngyuen, D., Radiation Phys. Chem., 62, 89-98,
(2001).
27. Peppas, N. A., Korsmeyer, R. W., “Hydrogels in Medicine and Pharmacy”-
Properties and Applications, Peppas, N. A., ed., vol III, CRC Press, Inc., Florida,
pp. 109-135, (1987).
28. Brazel, C. S., Peppas, N. A., S.T.P. Pharm. Sci., 9, 473-485, (1999).
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Release, 39, 231-237, (1996).
184
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
4.1. Introduction 142
4.2. Experimental Procedure 145
4.3. Results 145
4.3.1. DPC Measurements 145
4.3.2. Hydrogel Formation 150
4.3.3. Experimental Swelling Results 151
4.3.4. Experimental Drug Release Results 161
4.4. Discussion 169
4.4.1. DPC Measurements 169
4.4.1.1. Effect of the Monomer Structure on Polymerisation 169
4.4.1.2. Hydrogen Abstraction 173
4.4.2. Hydrogel Formation 174
4.4.3. Swelling and Drug Release Investigations 176
4.4.3.1. Hydrogel Swelling Behaviour 177
4.4.3.1.1. Effect of the Monomeric Composition on Swelling 177
4.4.3.1.2. Effect of the Ionic Strength on Swelling 178
4.4.3.2. Drug Release Studies 179
4.4.3.2.1. Effect of the Monomeric Composition on Drug Release 179
4.4.3.2.2. Effect of the Nature and the Molecule Size of the Drug 180
4.5. Conclusions 181
4.6. References 183
185
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
5.1. Introduction
Interpenetrating network (IPN) hydrogels are an important class of materials, which are
defined as two independent synthetic and/or natural polymer components contained in a
network form [1-6]. Figure 1 illustrates an IPN where at least one component is
crosslinked in the immediate presence of another [1,2].
Figure 1. Interpenetrating polymer network.
Two separate polymer networks interpenetrating each other.
IPN formation is an excellent way to enhance the compatibility of the polymeric
components. An IPN displays a more superior performance for its particular application
in comparison to its component macromolecules [1]. Various synthetic routes of
obtaining such materials have been discussed in detail in Section 1.1.1.3. Numerous
researchers have reported the use of a variety of combination of synthetic and natural
polymers to form IPNs [4-11). The use of polysaccharides such as chitosan and its
derivatives in conjunction with other compatible synthetic or natural polymers to form
IPNs has been very common in recent years as indicated by the publications [4-11].
Chitosan also chemically known as (1-4)-[2-amino-2-deoxy-β-D-glucan] is a natural
derivative of chitin, obtained through its partial deacetylation [11,12]. Chitin, extracted
from crustacean shells, is the most abundant mucopolysaccharide after cellulose and is
known to consist of β-[(1-4)-2-acetamido-2-deoxy-D-glucose] units [11-15]. Amine
185
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
functional groups on the chitosan structure contribute to its cationic character and it is
well known to have the ability to form intermolecular complexes with carboxylic and
polycarboxylic acids [11]. Furthermore, chitosan is biodegradable and highly
biocompatible, which contributes to its versatility as an extremely useful material in
biomedical applications [16-20]. Figure 2 [15] illustrates chemical structures of chitin and
its derivatives.
H
NHCOCH3
H
OH
H
CH2OH
HOH H
H OO H
O
H
CH2OH
H
H NHCOCH3
n
H
NH2
H
OH
H
CH2OH
HOH H
H OO H
O
H
CH2OH
H
H NHCOCH3
n
H
NHCOCH3
H
OH
H
CH2OH
H
OH
OH
NHCH2COOHH
OH
H
CH2OH
HOH H
H OO H
O
H
CH2OH
H
H NH2
n
Chitin
Chitosan
Carboxymethyl chitosanN-
Figure 2. Structures of chitin, chitosan and CM chitosan
186
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
Chitosan, however, has certain limitations to its reactivity and processability [21,22]. In
recent years several researchers have attempted to obtain functional derivatives of
chitosan with enhanced properties such as biocompatibility and water-solubility through
partial N-acetylation or chemical modification [23-27]. It has been suggested in the
literature that the degree of deacetylation of the starting chitosan governs the solubility of
the derivatives [15,24,27]. Experimental conditions such as reaction temperature and
solvent used for deacetylation have also been suggested to affect the nature of the
derivative [26].
Derivatives of chitosan with enhanced reactivity and processability have been utilized to
form IPN hydrogels for bioapplications. Chen et al [7] have reported successful synthesis
of IPN based on a water-soluble chitosan derivative, carboxymethyl (CM) chitosan for
drug delivery application. Chen et al [10] in their study of IPN based on CM chitosan
obtained through varying degree of deacetylation of chitosan found phenomenal
sensitivity of the gels to changes in environmental pH. They further suggest the
dependence of the nature of the derivative on the degree of deacetylation.
In this present work the author has made use of chitosan and its water-soluble derivative,
CM chitosan in conjunction with NVP and its copolymer with HEMA to synthesize IPNs
for swelling-drug release applications. The IPNs were synthesized by allowing NVP to
polymerise within the matrix of the polysaccharides through the formation of charge-
transfer (CT) complexes with HEMA and HMMI as the acceptors. The CT complex
formation reactions have been discussed in Sections 1.2.6 and 4.4.1.
5.2. Experimental Procedure
The IPNs formed through this photoinitiator-free process were subjected to swelling drug
release experiments at 37 oC in neutral pH environment. The effect of acidic, basic and
isotonic environments on the swelling behaviour of the IPNs was also evaluated. A model
drug, theophylline was used for drug release investigations. Detailed experimental
procedure of IPN formation is outlined in Section 2.5.2.3-2.5.2.5. Spectroscopic
techniques such as FT-IR and UV-vis were employed in this work for IPN
characterization and the quantitative measurements of drug release respectively.
187
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Experimental specifications and procedure of swelling-drug release experiments have
been described in Sections 2.6.1 and 2.7.
5.3. Results
5.3.1. Polymerisation of IPNs
The IPNs of chitosan and its derivative CM chitosan were prepared using the NVP and
HMMI via UV curing in the absence of a photoinitiator. Due to restricted solubility of
chitosan, it had to be pre-dissolved in a carboxylic acid prior to usage in the IPN
formulation. CM chitosan on the other hand is water-soluble thus it did not require any
additive for assistance in dissolving.
5.3.1.1. Chitosan Based IPNs
Two carboxylic acids namely, acrylic and levulinic acid were used to pre-dissolve
chitosan prior to use. The viscous mixtures of the chitosan in acid were combined with
NVP, HEMA and HMMI in varied proportions. The observations on the extent of
polymerisation upon applying approximately 9 KJ of radiation dose are described in
Tables 1 and 2.
Table 1. Polymerisation status of formulations with chitosan pre-dissolved in acrylic
acid
IPN formulation (% w/w) Polymerisation status upon application
of curing dose (~9 KJ)
NVP (65 %)-chitosan (35 %) DNP
NVP (50 %)-chitosan (50 %) DNP
NVP (35 %)-chitosan (65 %) DNP
HEMA (50 %)-chitosan (50 %) DNP
NVP (25 %)-HEMA (25 %)-chitosan (50 %) DNP
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
Table 2. Polymerisation status of formulations with chitosan pre-dissolved in
levulinic acid
IPN formulation (% w/w) Polymerisation status upon
application of curing dose (~9 KJ)
NVP (62.4 %)-chitosan (33.6 %)-HMMI (4.0 %) Soft, rubbery and partially opaque gel
NVP (48 %)-chitosan (48 %)-HMMI (4.0 %) Soft, rubbery and partially opaque gel
NVP (33.6 %)-chitosan (62.4 %)-HMMI (4.0 %) DNP
HEMA (50 %)-chitosan (50 %) DNP
NVP (25 %)-HEMA (25 %)-chitosan (50 %) Hard, clear and pale yellow gel
The monomer compositions are expressed as percentage w/w. DNP indicates that the
formulation did not polymerise. The radiation dose was calculated according to Equation
1 where t is the total radiation time in seconds. The samples were exposed to UV
radiation for approximately 25 hours at a dose rate of 9.6 x 10-2
J s-1
. The dose rate was
calculated as described in Section 2.4.4.4.
Radiation dose (J) = dose rate (J s-1
) x t (s) Equation 1
5.3.1.2. CM Chitosan Based IPNs
CM chitosan, a water-soluble derivative of chitosan was dissolved in milli-Q-water. The
viscous mixture of the CM chitosan was then added to the mixture of NVP and HMMI.
The observations on the extent of polymerisation upon applying approximately 9 KJ of
radiation dose are described in Table 3.
Table 3. Polymerisation status of formulations with CM chitosan
IPN formulation (% w/w)
(Presence of HMMI (4.0 %)
Polymerisation status upon application
of curing dose (~9 KJ)
NVP (62.4 %)-CM chitosan (33.6 %) Soft, rubbery and partially opaque gel
NVP (48 %)-CM chitosan (48 %) Soft, rubbery and partially opaque gel
NVP (33.6 %)-CM chitosan (62.4 %) DNP
189
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The monomer compositions are expressed as percentage w/w of the components. DNP
indicates that the formulation did not polymerise. The radiation dose was calculated
according to Equation 1.
5.3.2. Characterization of the IPNs
The IPNs were thoroughly cleaned in milli-Q-water to remove any unreacted component
and then characterized using the FT-IR spectroscopic technique. The 48 % chitosan- 48 %
NVP- 4 % HMMI and 48 % CM chitosan- 48 % NVP- 4 % HMMI IPNs, though
successfully synthesized, were found to be water-soluble. These IPNs disintegrated
during the washing process. The presence of characteristic bands of the monomers on the
FT-IR spectra of IPN gel samples confirmed the IPN formations. The spectra of chitosan,
CM chitosan and the IPNs are illustrated in Figures 3 and 4.
5.3.2.1. FT-IR Data Analysis
The characteristics of the FT-IR spectra corresponding to the chemical compositions of
the polysaccharides and the successfully synthesized water swellable IPNs are described
in Sections 5.3.2.1.1 – 5.3.2.1.5. Figures 3 and 4 illustrate the characteristic peaks of
chitosan, CM chitosan and the IPNs: 33.6 % CM chitosan- 62.4 % NVP- 4 % HMMI (Gel
A), 33.6 % chitosan- 62.4 % NVP- 4 % HMMI (Gel B) and 50 % chitosan- 25 % NVP-
25 % HEMA (Gel C) observed in the FT-IR spectra.
5.3.2.1.1. Chitosan
Figure 4 shows the characteristic peaks of chitosan observed at 3479 cm-1
(-O-H stretch),
2956 cm-1
(-CH stretch), 1720 cm-1
(-NH2 deformation), 1581 cm-1
(-NH bend), 1170 cm-1
(bridge-O-stretch) and 1099 cm-1
(-C-O- stretch).
5.3.2.2.2. CM Chitosan
Figure 3 shows the characteristic peaks of CM chitosan observed at 3466 cm-1
(broad -OH
stretch), 1747 cm-1
(-COOH peak), 1660 and 1540 cm-1
(-NH3+
peaks), and 1070-1136
cm-1
(-C-O- stretch).
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
5.3.2.2.3. IPN Gel A
Figure 3 shows the characteristic peaks of 33.6 % CM Chitosan- 62.4 % NVP- 4 %
HMMI IPN observed at 3479 cm-1
(broad -OH stretch), 1732 cm-1
(-COO stretch (acid)),
1662 cm-1
(-COO stretch (amide)), 1384-1467 cm-1
(-CH bend (saturated)) and 1298 cm-1
(-C-O stretch).
5.3.2.1.4. IPN Gel B
Figure 4 shows the characteristic peaks of 33.6 % Chitosan- 62.4 % NVP- 4 % HMMI
IPN observed at 3500 cm-1
(broad -OH stretch), 2375 cm-1
(-NR2 peak), 1683 cm-1
(-COO
peak (amide)), 1392-1487 cm-1
(-CH bend (saturated)) and 1296 cm-1
(-C-O -stretch).
5.3.2.1.5. IPN Gel C
Figure 4 shows the characteristic peaks of 50 % Chitosan- 25 % NVP- 25 % HEMA IPN
observed at 3500 cm-1
(broad -OH stretch), 2375 cm-1
(-NR2 peak), 1740 cm-1
(-COO
stretch (ester)), 1683 cm-1
(-COO stretch (amide)), 1392-1487 cm-1
(-CH bend
(saturated)), 1296 cm-1
(-C-O stretch) and 1188 cm-1
(-COOR peak).
191
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
34
54
400140024003400
Wavenumber (cm-1
)
% T
ran
smit
tan
ce
CM Chitosan Gel A
Figure 3. FTIR spectrum illustrating the characteristics of CM chitosan and the IPN
formed of CM chitosan-NVP-HMMI (Gel A).
40
70
100
400140024003400
Wavenumber (cm-1
)
% T
ran
smit
tan
ce
Gel B Gel C Chitosan
Figure 4. FTIR spectrum illustrating the characteristics of chitosan and the IPNs formed
of chitosan-NVP-HMMI (Gel B) and chitosan-NVP-HEMA (Gel C).
192
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
5.3.3. Experimental Swelling Results
The IPNs formed were subjected to swelling test in varying pH environments at 37 oC.
The IPNs were found to be resilient with high swelling efficiency and also slightly
sensitive to pH variations. The data on the degree of swelling of the IPNs: Gel A, Gel B
and Gel C in varying pH conditions are presented in Tables 4-11.
Table 4. Swelling test on IPN hydrogels at 37 oC in neutral pH environment
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C
0.00 0.0 0.0 0.0
0.17 44.3 48.0 20.5
0.33 54.3 60.1 26.7
0.50 60.1 66.9 31.6
0.67 64.9 70.3 35.1
0.83 67.5 73.6 37.4
1.00 70.0 75.9 40.0
2.00 78.5 83.1 49.2
3.00 82.2 86.1 54.7
4.00 84.3 88.3 59.9
5.00 85.6 89.1 61.2
7.00 87.4 90.3 65.1
9.00 88.4 91.1 67.8
12.00 89.4 91.9 70.4
24.00 90.9 93.0 75.0
48.00 91.3 93.3 77.4
72.00 91.3 93.4 78.4
96.00 91.5 93.5 79.2
120.00 91.5 93.6 79.8
144.00 91.5 93.6 79.9
170.00 91.4 93.6 79.6
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The swelling data is expressed as percentage water content at the respective time
intervals. The percentage water content values were calculated as described in Section
2.6.1. The chitosan-NVP-HMMI formulation containing 48 % w/w of NVP and the CM
chitosan-NVP-HMMI formulation containing 48 % w/w of NVP disintegrated after a
short exposure to milli-Q-water during the washing process thus no further experiments
were conducted on these samples. Graphical representations of the swelling behaviour
observed in the IPN hydrogels in neutral pH environment at 37 oC are illustrated in
Figures 5 - 7.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C
Figure 5. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in neutral
pH environment as a function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C
Figure 6. Plot of fractional swelling in the IPNs: Gel A, Gel B and Gel C at 37 oC in
neutral pH environment as a function of the square root of time.
194
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel B Gel C
Figure 7. Plot of the LOG of fractional swelling in the IPNs: Gel A, Gel B and Gel C in
the initial stages of the swelling experiment at 37 oC in neutral pH environment as a
function of the LOG of time.
As previously discussed, the value of n in the power-law equation defines the kinetics of
the diffusion in the polymer, which in turn governs the solute release. The value of the
slope from the LOG graph fractional swelling against time represents the n value.
Table 5 presents the slope (n) values for the IPNs: Gel A, Gel B and Gel C calculated
from Figure 7.
Table 5. Characteristic exponential n values for diffusion in IPN hydrogels in
neutral medium
IPN hydrogels n values
Gel A 0.61 + 0.01
Gel B 0.66 + 0.02
Gel C 0.52 + 0.02
195
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
5.3.3.1. Swelling Test in Varying pH Environments
The data on the swelling behaviour of the IPN hydrogels observed at 37 oC in pH 2,
isotonic (pH 7.4) and pH 8 environments is presented in Tables 6-10.
Table 6. Swelling test on IPN hydrogels in acidic (pH 2) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C
0.00 0.0 0.0 0.0
0.17 47.2 47.9 20.6
0.33 56.1 57.6 26.1
0.50 63.9 64.3 31.0
0.67 66.4 69.1 34.6
0.83 69.5 71.3 36.3
1.00 72.6 74.4 38.9
2.00 79.7 82.2 46.7
3.00 83.1 85.1 52.0
4.00 85.0 86.7 55.6
5.00 86.2 88.0 58.5
7.00 87.8 89.7 62.2
9.00 87.8 90.4 65.3
12.00 89.0 91.3 67.7
24.00 90.1 92.7 70.3
48.00 90.8 93.3 71.3
72.00 90.9 93.6 71.4
96.00 91.0 93.6 71.5
120.00 91.2 93.5 71.6
144.00 91.3 93.4 71.9
170.00 91.3 93.3 71.8
Graphical representations of the swelling behaviour observed in the IPN hydrogels in
acidic (pH 2) environment at 37 oC are illustrated in Figures 8 - 10.
196
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C
Figure 8. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in acidic
(pH 2) environment as a function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C
Figure 9. Plot of fractional swelling in the IPNs: Gel A, Gel B and Gel C at 37 oC in
acidic (pH 2) environment as a function of the square root of time.
197
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
inif
init
y
Gel A Gel B Gel C
Figure 10. Plot of the LOG of fractional swelling in the IPNs: Gel A, Gel B and Gel C in
the initial stages of the swelling experiment at 37 oC in acidic (pH 2) environment as a
function of the LOG of time.
Table 7 presents the slope (n) values for IPNs: Gel A, Gel B and Gel C calculated from
Figure 10.
Table 7. Characteristic exponential n values for diffusion in IPN hydrogels in acidic
medium
IPN hydrogels n values
Gel A 0.68 + 0.01
Gel B 0.74 + 0.04
Gel C 0.48 + 0.03
198
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
Table 8. Swelling test on IPN hydrogels in isotonic (pH 7.4) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C
0.00 0.0 0.0 0.0
0.17 51.3 49.0 20.0
0.33 61.5 58.1 27.4
0.50 69.6 67.2 30.9
0.67 73.7 70.7 33.5
0.83 76.2 74.0 36.8
1.00 78.2 76.8 39.0
2.00 83.9 83.0 47.8
3.00 86.4 85.4 53.2
4.00 88.0 86.8 57.1
5.00 88.8 87.5 60.0
7.00 90.1 88.7 63.5
9.00 91.1 89.5 65.6
12.00 91.6 89.6 68.5
24.00 92.2 90.8 71.2
48.00 92.8 91.6 72.3
72.00 92.8 91.7 72.1
96.00 92.7 91.7 71.7
120.00 92.7 91.8 71.7
144.00 92.8 91.9 70.9
170.00 92.8 91.9 70.8
Graphical representations of the swelling behaviour observed in the IPN hydrogels in
isotonic (pH 7.4) environment at 37 oC are illustrated in Figures 11 - 13.
199
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C
Figure 11. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in
isotonic (pH 7.4) environment as a function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C
Figure 12. Plot of fractional swelling in the IPNs: Gel A, Gel B and Gel C at 37 oC in
isotonic (pH 7.4) environment as a function of the square root of time.
200
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel B Gel C
Figure 13. Plot of the LOG of fractional swelling in the IPNs: Gel A, Gel B and Gel C in
the initial stages of the swelling experiment at 37 oC in isotonic (pH 7.4) environment as a
function of the LOG of time.
Table 9 presents the slope (n) values for the IPNs: Gel A, Gel B and Gel C calculated
from Figure 13.
Table 9. Characteristic exponential n values for diffusion in IPN hydrogels in
isotonic medium
IPN hydrogels n values
Gel A 0.70 + 0.03
Gel B 0.70 + 0.04
Gel C 0.51 + 0.02
201
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 10. Swelling test on IPN hydrogels in basic (pH 8) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C
0.00 0.0 0.0 0.0
0.17 42.6 40.2 19.1
0.33 52.8 53.5 25.4
0.50 62.9 63.0 28.3
0.67 64.8 66.3 31.4
0.83 68.5 68.8 34.0
1.00 71.1 71.7 36.5
2.00 78.7 79.7 45.2
3.00 82.4 83.6 49.7
4.00 84.7 85.5 53.3
5.00 86.1 87.0 56.2
7.00 87.5 88.8 59.1
9.00 88.6 90.0 61.7
12.00 89.6 90.8 64.3
24.00 90.3 92.4 67.1
48.00 91.0 92.9 68.5
72.00 91.0 93.0 68.5
96.00 91.1 93.0 68.5
120.00 91.3 93.0 68.2
144.00 91.4 93.0 67.9
170.00 91.4 92.8 67.5
Graphical representations of the swelling behaviour observed in the IPN hydrogels in
basic (pH 8) environment at 37 oC are illustrated in Figures 14 -16.
202
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C
Figure 14. Plot of % water content in the IPNs: Gel A, Gel B and Gel C at 37 oC in basic
(pH 8) environment as a function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/ M
infi
nit
y
Gel A Gel B Gel C
Figure 15. Plot of fractional swelling in the IPNs: Gel A, Gel B and Gel C at 37 oC in
basic (pH 8) environment as a function of the square root of time.
203
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
inif
inty
Gel A Gel B Gel C
Figure 16. Plot of the LOG of fractional swelling in the IPNs: Gel A, Gel B and Gel C in
the initial stages of the swelling experiment at 37 oC in basic (pH 8) environment as a
function of the LOG of time.
Table 11 presents the slope (n) values for the IPNs: Gel A, Gel B and Gel C calculated
from Figure 16.
Table 11. Characteristic exponential n values for diffusion in IPN hydrogels in basic
medium
IPN hydrogels n values
Gel A 0.60 + 0.02
Gel B 0.64 + 0.02
Gel C 0.50 + 0.02
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
Figures 17-19 illustrate the comparative swelling behaviour observed for the IPNs: Gel A,
Gel B and Gel C in varied pH environments as a function of time.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
pH 2 neutral isotonic (pH 7.4) pH 8
Figure 17. Comparative plot of % water content in Gel A in varied pH environments at
37 oC as a function of time.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
pH 2 neutral isotonic (pH 7.4) pH 8
Figure 18. Comparative plot of % water content in Gel B in varied pH environments at
37 oC as a function of time.
205
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
20
40
60
80
100
0 50 100 150 200Time (h)
% W
ate
r C
on
ten
t
pH 2 neutral isotonic (pH 7.4) pH 8
Figure 19. Comparative plot of % water content in Gel C in varied pH environments at
37 oC as a function of time.
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
5.3.4. Experimental Drug Release Results
The drug release experiments on the IPNs were conducted at 37 oC in neutral pH
environment using theophylline as the model drug. The experimental data expressed as
fractional drug release at specific time intervals is presented in Table 12.
Table 12. Drug release test on IPN hydrogels at 37 oC in neutral pH condition
Average fractional theophylline released
values at time (t)
Time (h) Gel A Gel B Gel C
0.00 0.00 0.00 0.00
0.17 0.28 0.28 0.27
0.33 0.40 0.44 0.41
0.50 0.49 0.54 0.49
0.67 0.55 0.63 0.58
0.83 0.61 0.69 0.63
1.00 0.65 0.73 0.67
2.00 0.82 0.89 0.86
3.00 0.89 0.93 0.93
4.00 0.91 0.95 0.96
5.00 0.93 0.96 0.98
7.00 0.94 0.97 0.99
9.00 0.94 0.96 0.97
12.00 0.95 0.97 0.99
24.00 0.95 0.96 0.98
48.00 0.97 0.96 0.99
The fractional drug release values were calculated as described in Section 2.7. Graphical
representations of the drug release behaviour in the IPN hydrogels are illustrated in
Figures 20 - 22.
207
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 20 40
Time (h)
Fra
ctio
nal
Dru
g R
elea
sed
60
Gel A Gel B Gel C
Figure 20. Plot of the fractional release of theophylline from the IPNs: Gel A, Gel B and
Gel C at 37 oC in neutral pH environment as a function of time.
0.0
0.4
0.8
1.2
0 2 4 6 8
t1/2
(h1/2
)
Fra
ctio
nal
dru
g r
elea
se
Gel A Gel B Gel C
Figure 21. Plot of the fractional release of theophylline from the IPNs: Gel A, Gel B and
Gel C at 37 oC in neutral pH environment as a function of the square root of time.
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel A Gel B Gel C
Figure 22. Plot of the LOG of fractional drug released (FDR) from the IPNs: Gel A, Gel
B and Gel C in the initial stages of release experiment at 37 oC in neutral environment as
a function of the LOG of time.
Table 13 presents the slope (n) values for IPNs: Gel A, Gel B and Gel C calculated from
Figure 22.
Table 13. Characteristic exponential n values for theophylline release from IPN
hydrogels in neutral medium
IPN hydrogels n values
Gel A 0.50 + 0.01
Gel B 0.50 + 0.02
Gel C 0.50 + 0.01
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
5.4. Discussion
Attempts to synthesize several IPN formulations via photoinitiator-free UV curing
technique resulted in a number of successfully synthesized IPNs (Tables 1-3). These
successfully synthesized IPNs were characterized using FT-IR (Figures 3 and 4) to
confirm their chemical composition. They were then subjected to a number of
experiments to evaluate their ability to function as biocompatible slow drug release
devices. The FT-IR characterizations of these IPN hydrogels are specified in Section
5.3.2.1.
The first part of this discussion is focussed on the formation of the IPNs using various
formulations, some of which were unsuccessful. A number of factors, which are
influential towards the fate of the polymerisation, have been proposed. The composition
of the formulation could be regarded as the utmost important factor. The relative
reactivity of the components and secondly the compatibility of the components are the
key issues outlined by this factor. Furthermore, reaction conditions such as the solvent
type and radiation dose are also influential towards the fate of the polymerisation. These
factors may either lower the reaction kinetics as to minimise the rate of polymerisation or
hinder the polymerisation path by initiating undesirable side reactions.
5.4.1. Influential Factors on Polymerisation
The chemical composition and the type of solvent used for the preparation of IPNs were
found to be critically influential factors towards the efficiency and the nature of the
polymerisation process. These factors are discussed in Sections 5.4.1.1 - 5.4.1.2.
5.4.1.1. Effect of Solvent on Polymerisation
Chitosan is insoluble in neutral and basic medium due to its cationic nature, however it is
soluble in acidic medium. The solubility of chitosan is essentially governed by the
possibility of formation of inter and intramolecular hydrogen bonds [20]. The acidic
solvents used to achieve this were also evaluated for their role in the polymerisation
process. IPN formulations, which involved chitosan pre-dissolved in acrylic acid and
NVP resulted in a dark reddish coloured complex, which did not solidify upon the applied
radiation dose of approximately 9 KJ. However, formulations containing chitosan pre-
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
dissolved in levulinic acid resulted in three successfully polymerised IPN out of the four
formulations.
The solvents, which are carboxylic acids in this instance, are known to form an
intermolecular complex with chitosan, seem to play an important role in the nature of the
polymerisation reaction. Acrylic acid (AA) was the first choice of solvent for this work
due to the fact that it is a moderately strong electron withdrawing monomer. Since this
work was based on CT complex polymerisation, it was proposed that AA would make an
excellent acceptor monomer in the donor/acceptor pair with NVP as the donor and at the
same time, AA would also serve as a solvent. Thus, inclusion of another solvent or an
acceptor monomer could be avoided.
The formulation, which contained pre-dissolved chitosan in acrylic acid and NVP and
NVP/HEMA formed a strong coloured complex observed after a short exposure to the
UV light. This indicated the formation of a CT complex of very high ionic character [28].
Chitosan has a number of labile abstractable hydrogens, which would effectively enhance
the rate of CT complex formation between AA and NVP. Ng et al [29,30] in their studies
on CT complex systems have described a significant positive effect of hydrogen donors
on the efficiency of CT complex formation.
Due to the formation of a strong coloured complex, the UV light could not penetrate
through the sample thus a complete polymerisation reaction was not achieved. In the case
with levulinic acid as the solvent, it can be concluded that the acid did not affect the
nature of polymerisation. However, its participation in the reaction must be accounted for
as an acid base reaction with the chitosan, which is a weak base with a pKa value between
6.2-7 [31]. Scheme 1 illustrates the formation of an intermolecular complex via an
interaction between weakly basic chitosan and carboxylic acids [32,33].
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
O
OH N
OH
OO
H
H
O
OH N+
OH
OO
H
H H
R O
O
HRO
O
H+
+
Chitosan Carboxylic acid Intermolecular complex
+
Scheme 1. Acid base interaction between a carboxylic acid and chitosan
.
5.4.1.2. Effect of Monomer Composition on Polymerisation
IPN formulations containing pre-dissolved chitosan in levulinic acid showed positive
signs of forming IPN with NVP. However, formulations containing high amount of
chitosan/acid mixture showed very low reactivity of IPN formation upon exposure to the
UV light. This could be attributed to the fact that sufficient amount of the NVP and
HMMI complex was not present to initiate the polymerisation of NVP leading to the
formation of an IPN with chitosan. A similar observation was made in formulations
containing CM chitosan. It was experimentally shown that the formulations with low
NVP-HMMI mixture or the formulation containing 50 % HEMA and 50 % chitosan/acid
solution did not successfully yield an IPN when exposed to UV radiation.
HEMA is an acceptor monomer, which has been previously described in Sections 3 and 4
to effectively homopolymerise through the intramolecular hydrogen abstraction process.
However, the cause of unsuccessful polymerisation in the case of 50 % HEMA and 50 %
chitosan/acid solution formulation could be attributed to the fact that a higher
composition of HEMA was required to initiate the polymerisation process. The 25 %
HEMA -25 % NVP -50 % chitosan/levulinic acid solution formulation however resulted
in a successfully synthesized IPN. The successful IPN formation between HEMA-NVP
and chitosan could be attributed to the donor/acceptor interaction between HEMA and
NVP, which initiated the polymerisation leading to the IPN formation.
The IPNs formed from chitosan and CM chitosan were opaque upon curing with the
exception of chitosan-HEMA-NVP IPN, which was clear. It could be stated from this
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
observation that the IPNs containing just the polysaccharides and NVP in the presence of
HMMI were crystalline in nature. The optical clarity in the chitosan-HEMA-NVP is
indicative of an amorphous network.
5.4.2. Swelling and Drug Release Evaluation
The IPN hydrogels formed were subjected to the swelling and drug release experiments.
The 48 % chitosan- 48 % NVP- 4 % HMMI and 48 % CM chitosan- 48 % NVP- 4 %
HMMI, though successfully synthesized were found to be water-soluble. The solubility of
these IPNs could be attributed to the lack of sufficient crosslinking in the structure. The
swelling and drug release behaviour in the IPN hydrogels were characterized on the basis
of Fickian or non-Fickian diffusion behaviour. The swelling action in polymers is
generally time dependent and could be described according to the power-law equation
described in previous sections. The value of n in the power-law equation indicates the
diffusion kinetics. The characteristic n values for Fickian and non-Fickian diffusion
kinetics have been previously mentioned in Section 1.4.2.3.1.2.
5.4.2.1. IPN Swelling Behaviour
5.4.2.1.1. Effect of the Monomeric Composition on Swelling
An exponential increase in the water uptake by Gel A and Gel B in neutral pH
environment at 37 oC was observed in the first hour (Figure 6) indicating non-Fickian
anomalous transport behaviour. This was confirmed by a LOG plot (Figure 7, Table 5)
from which average n values of 0.61 and 0.66 were calculated for gels A and B
respectively. In the later stages of the swelling process, Case II diffusion behaviour
prevailed which was indicated by a gradual decrease in the rate of water uptake by the
IPNs.
The gradual decrease in the rate of water uptake was observed around 7 hours of swelling
which became constant upon 24 hours of constant swelling thus indicating that
equilibrium water content uptake was achieved (Table 4, Figure 5). At this stage the
swelling process becomes time independent where the parameter n = 1. Equilibrium water
content (EWC) values of 91.4 % and 93.6 % were observed for the Gel A and Gel B
respectively. The slight reduction in the swelling efficiency of Gel A could be attributed
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
to the formulation composition. A slight reduction in NVP content of the IPN
composition could have been the cause of slight variation in the swelling efficiencies
between the IPNs.
Gel C however adhered to Fickian diffusion behaviour in the initial stages of the swelling
experiment followed by an anomalous transport in the later stages (Figures 6 and 7, Table
5). Equilibrium swelling was achieved after 48 hours of constant experimental swelling,
which yielded an EWC value of 79.6 % (Table 4, Figure 5). Inclusion of a relatively
hydrophobic monomer such as HEMA has led to a reduction in the equilibrium water
content. As previously described, HEMA will form a donor/acceptor pair with NVP. Thus
it could be stated that HEMA was effectively consumed in formulation upon UV curing,
as its presence was effective on the swelling process. Presence of HEMA in the IPN was
also evidenced by the FT-IR spectroscopic data as described Section 5.3.2.1.
5.4.2.1.2. Effect of the Environmental pH on Swelling
Risbud et al [34] have reported highly pH sensitive IPNs based on chitosan and NVP.
Chitosan is polycationic thus it will respond to pH changes below its pKa value. Gupta
and Kumar [35] in their study on the diffusion behaviour in chitosan beads over a wide
pH range have also reported similar observations. Chen et al [10] have reported the
amphoteric nature of CM chitosan based IPNs. This could be attributed to the fact that
CM chitosan has basic amine groups as well as acidic acetyl groups. CM chitosan based
IPNs have a certain isoelectric point (IEP) at which these IPNs shrink the most. The IEP
of polyampholytic hydrogels is described as a point where equal amount of anionic and
cationic units exist on their backbones and thus at near the IEP, the Coulombic attraction
between the oppositely charged units within the hydrogel matrix causes the collapse of
the network. However, upon deviating the environmental pH from the IEP these gels
display an increased swelling behaviour.
The results of the swelling experiments on the IPNs at varied pH environments showed
slight variations in the swelling efficiency. The chitosan based IPNs, Gel B and Gel C
showed a slight reduction in the swelling efficiency in pH 8 environment in comparison
to neutral, isotonic and pH 2 environments (Tables 6,8 and 10, Figures 8-10, 11-13,14-
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Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
16). Gel C displayed EWC values of 71.8 %, 70.8 % and 67.5 % in pH 2, pH 7.4 and pH
8 environments respectively. EWC values of 93.3 %, 91.9 % and 92.8 % were observed
in pH 2, pH 7.4 and pH 8 environments respectively for Gel B. These results on chitosan
based IPNs are in agreement with other researchers [34,35]. However, a drastic variation
in the swelling behaviour was not observed as reported by Risbud et al [34], Gupta and
Kumar [35]. The low pH sensitivity of the chitosan based IPNs could be attributed to the
fact that a low amount of chitosan was used in the formulation thus the pH sensitivity of
the polysaccharide was suppressed.
The results on Gel B in different pH environments indicated non-Fickian diffusion
kinetics in the early stages of swelling, with n values (Tables 7,9, 11) of 0.74, 0.70 and
0.64 in pH 2, pH 7.4 and pH 8 environments respectively. The diffusion mechanism in
Gel C is reflective of Fickian diffusion kinetics in the early stages of swelling, with n
values of 0.48, 0.51 and 0.50 in pH 2, pH 7.4 and pH 8 environments. The chitosan based
IPNs showed a rapid swelling up to 7 hours after which a gradual decrease in the swelling
efficiency was observed with equilibrium swelling achieved around 48 hours.
The swelling experiments on Gel A in different pH environments are indicative of
ampholytic behaviour. However, as in the case of chitosan based IPNs, the sensitivity to
varied pH environments were not pronounced. The IPNs yielded EWC values of 91.3 %,
91.8 % and 91.4 % in pH 2, pH 7.4 and pH 8 environments respectively. Despite similar
EWC values in varied pH environments, a slight reduction in the swelling efficiency was
observed in pH 8 environment in the initial stages of swelling in comparison to pH 2,
neutral and pH 7.4 environments. The swelling efficiency was observed to be highest in
pH 7.4 environment followed by pH 2, neutral and pH 8 environment. The % water
content values of 44.3 %, 47.2 %, 51.3 % and 42.6 % in neutral, pH 2, pH 7.4 and pH 8
environments respectively were observed at the initial 10 minutes of swelling.
The sharp increase in swelling activity from neutral to pH 7.4 environment could be
attributed to the increase in the ionic strength of the swelling agent. Ng et al [36] in their
studies on polyampholytic hydrogels have described a similar observation in relation to
the concept of Donnan equilibrium. The pH 7.4 buffer has an ionic strength of 0.2 in
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
comparison to neutral medium which is just milli-Q-water with an assumed ionic strength
of zero.
According to the concept of Donnan equilibrium, more electrolytes will penetrate into the
hydrogel matrices with increase in ionic strength of the swelling agent and the mobile
counterions surrounding the charged groups in the hydrogel matrices will affect the net
osmotic pressure within the hydrogel network. Chen et al [7] have also observed
increased swelling behaviour in CM chitosan based IPN in pH 7.4 environment. They
suggest that the carboxylic acid groups become progressively ionized at pH 7.4, thus the
significant electrostatic repulsion between the ionized acid groups cause an increase in
swelling.
However, the experimental swelling data indicated reduced swelling activity in pH 8
environment. The data thus suggest the presence of IEP in the vicinity of pH 8, which has
caused the slight reduction in the swelling activity of the IPN. Gel A displayed non-
Fickian swelling kinetics in the early stages of swelling, with n values of 0.68, 0.70 and
0.60 in pH 2, pH 7.4 and pH 8 environments respectively (Tables 7, 9 and 11).
5.4.2.2. Drug Release Studies
The drug release experiments conducted on the chitosan-NVP-HEMA, chitosan-NVP and
CM chitosan-NVP IPNs (Table 12, Figures 20-22) in neutral pH environment using
theophylline as the model drug yielded equilibrium drug release (EDR) values of 0.99,
0.96 and 0.97 respectively in 48 hours. The drug release rate increased rapidly in the first
2 hours of the experiment after which the release rate gradually slowed down and
eventually became constant around 7 hours. The quick release of the drug from the carrier
in the early stages of the experiment is attributed to the burst effect release [37]. As
previously described, the polymer containing the incorporated solute has to swell to a
certain extent before it can release its contents. The resultant effect is the fast release of
the solute in a short span of time.
The drug release profile observed in the IPNs could be described as Fickian transport
behaviour as illustrated in Figure 21 where the fractional drug released at time t is directly
216
Chapter 5: IPN Hydrogels Synthesized Through Photoinitiator-Free Polymerisation Technique
proportional to the square root of time. The LOG plot (Figure 22, Table 13) of the
fractional drug released from the IPNs yielded an average n value of 0.50, which
indicated that Fickian release mechanism was in operation. A very similar release rate
was observed in all the IPNs even though the chitosan-NVP-HEMA had a relatively low
swelling activity. This could be attributed to the relatively low molecular weight of
theophylline, which diffuses with ease through the pores of all the IPNs at a similar rate.
5.5. Conclusions
IPN hydrogels based on the polysaccharides, chitosan and CM chitosan in conjunction
with NVP and HEMA were successfully synthesized through a photoinitiator-free curing
technique. Levulinic acid was found to be a better solvent over AA in the formulation
resulting in successfully synthesized IPNs. However, it was necessary to have NVP-
HMMI and NVP-HEMA in sufficient quantity for efficient curing and adequate
crosslinkage in the IPN matrix. The hydrogels were found to be resilient and competent
drug delivery devices.
Experimental swelling data revealed that the CM chitosan-NVP and chitosan-NVP IPNs
adhere to non-Fickian anomalous diffusion behaviour in the earlier stages of the
experiment followed by case II diffusion in the later stages. Inclusion of HEMA into the
IPN resulted in reducing the swelling activity in the IPN, which adhered to a typical
Fickian behaviour. The IPNs under study were found to be sensitive to variations in the
environmental pH with chitosan based IPNs displaying cationic behaviour while CM
chitosan based IPN displayed ampholytic behaviour. However, a pronounce pH
sensitivity of the IPNs based on polysaccharides under study as reported by other
researchers was not observed.
The drug release experiments revealed that all the IPNs understudy adhered to Fickian
transport mechanism in releasing theophylline into the neutral pH environment at 37 oC.
The burst effect release of the drug was observed in the initial stages of the release
experiment followed by linear release profile. The rate of release of theophylline in all the
IPNs under study were found to be similar indicating that theophylline being a low
molecular weight drug could diffuse through the IPN membranes with ease.
217
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
5.6. References
1. Sperling, L. H., “Interpenetrating Polymer Networks and Related Materials”,
Plenum Press, New York, pp. 1-10, (1981).
2. Park, K., Shalaby, W. S. W., Park, H., “Biodegradable Hydrogels for Drug
Delivery”, Technomic Publishing Company, Inc., Basel, pp. 37-39, (1993).
3. Sperling, L. H., Mishra, V., Polym. Adv. Technol., 7, 197-208, (1996).
4. Maolin, Z., Jun, L., Min, Y., Hongfei, H., Radiation Phys. Chem., 58, 397-400,
(2000).
5. Xuequan, L., Maolin, Z., Jiuqiang, L., Hongfei, H., Radiation Phys. Chem., 57,
477-480, (2000).
6. Bartolotta, A., Di Marco, G., Lanza, M., Carini, G., D’Angelo, G., Tripodo, G.,
Fainleib, A., Danilenko, I., Grytsenko, V., Sergeeva, L., Materials Sci. Eng. A,
370, 288-292, (2003).
7. Chen, S.-C., Wu, Y.-C., Mi, F.-L., Lin, Y.-H., Yu, L.-C., Sung, H.-W., J.
Controlled Release, 96, 285-300, (2004).
8. Lee, J. W., Kim, S. Y., Kim, S. S., Lee, Y. M., Lee, K. H., Kim, S. J., J. Appl.
Polym. Sci., 73, 113-120, (1999).
9. Risbud, M. V., Bhat, S. V., J. Mater. Sci. Mater. Med., 12, 75-79, (2001).
10. Chen, L., Tian, Z., Du, Y., Biomaterials, 25, 3725-3732, (2004).
11. Peniche, C., Argüelles-Monal, W., Davidenko, N., Sastre, R., Gallardo, A.,
Román, J. S., Biomaterials, 20, 1869-1878, (1999).
12. Yao, K.-D., Liu, J., Cheng, G.-X., Zhao, R.-Z., Wen, H. W., Wei, L., Polym. Int.,
45, 191-194, (1998).
13. Furlan, L., De Fàvere, V. T., Laranjeira, M. C. M., Polymer, 37, 843-846, (1996).
14. Smirnova, L, A., Semchikov, Y. D., Tikhobaeva, Y. G., Pastukhova, N. V.,
Polym. Sci. Series B, 43, 33-36, (2001).
15. Kumar, M. N. V. R., React. Func. Polym., 46, 1-27, (2000).
16. Gupta, K. C., Kumar, M. N. V. R., J. M. S. Rev. Macromol. Chem. Phys, 40, 273-
308, (2000).
17. Mi, F.-L., Tan, Y.-C., Liang, H.-F., Sung, H.-W., Biomaterials, 23, 181-191,
(2002).
18. Chandy, T., Sharma, P., Biomater. Artif. Cells Artif. Organs, 18, 1-24, (1990).
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19. Singh, D. K., Ray, A. R., J. Appl. Polym. Sci., 53, 1115-1121, (1994).
20. Domard, A., Domard, M., “Polymeric Biomaterials”, Dumitriu, S, ed., 2nd
ed.,
Marcel & Decker, Inc., New York, pp.187-212, (2002).
21. Amass, W., Amass, A., Tighe, B., Polym. Int., 47, 89-144, (1998).
22. Illum, L., Pharm. Res., 15, 1326-1331, (1998).
23. Baumann, H., Faust, V., Carbohydr. Res., 331, 43-57, (2001).
24. Muzzarelli, R. A., A., Ilari, P., Petrarulo, M., Int. J. Biol. Macromol., 16, 177-180,
(1994).
25. Le Dung, P., Milas, M., Rinaudo, M., Desbrières, J., Carbohydr. Polym., 24, 209-
214, (1994).
26. Chen, X.-G., Park, H.-J., Carbohydr. Polym., 53, 355-359, (2003).
27. Chen, L., Du, Y., Zeng, X., Carbohydr. Res., 338, 333-340, (2003).
28. Jönsson, S., Hultgren, J., Sundell, P.-E., Shimose, M., Owens, J., Vaughn, K.,
Hoyle, C. E., Proc. Radtech Asia’95, Bangkok, Thailand, pp. 118-125, (1995).
29. Ng. L.-T., Jönsson, S., Swami, S., Lindgren, K., Polym. Int., 51, 1398-1403,
(2002).
30. Ng, L.-T., Jönsson, S., Lindgren. K., Swami, S., Hoyle, C., Clark, S., Proc.
Radtech Europe’01, Basel, Switzerland, pp. 609-613, (2001).
31. Hejazi, R., Amiji, M., “Polymeric Biomaterials”, Dumitriu, S, ed., 2nd
ed., Marcel
& Decker, Inc., New York, pp. 213-237, (2002).
32. Shamov, M. V., Bratskaya, S. Y., Avramenko, V. A., J. Colloid Interf. Sci., 249,
316-321, (2002).
33. Chavasit, V., Kienzle-Sterzer, C., Torres, J. A., Polym. Bull., 19, 223-230, (1988).
34. Risbud, M. V, Hardikar, A. A., Bhat, S. V., Bhonde, R. R., J. Controlled Release,
68, 23-30, (2000).
35. Gupta, K. C., Kumar, M. N. V. R., Polymer Int., 49, 141-146, (2000).
36. Ng, L-T., Arsenin, A., Nguyen, D., Proc. RadTech Asia’03, Yokohama, Japan, pp.
669-672, (2003).
37. Peppas, N. A., Korsmeyer, R. W., “Hydrogels in Medicine and Pharmacy”-
Properties and Applications, Peppas, N. A., ed., vol III, CRC Press, Inc., Florida,
pp. 109-135, (1987).
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
5.1. Introduction 185
5.2. Experimental Procedure 187
5.3. Results 188
5.3.1. Polymerisation of IPNs 188
5.3.1.1. Chitosan Based IPNs 188
5.3.1.2. CM Chitosan Based IPNs 189
5.3.2. Characterization of the IPNs 190
5.3.2.1. FT-IR Data Analysis 190
5.3.2.1.1. Chitosan 190
5.3.2.2.2. CM Chitosan 190
5.3.2.2.3. IPN Gel A 191
5.3.2.1.4. IPN Gel B 191
5.3.2.1.5. IPN Gel C 191
5.3.3. Experimental Swelling Results 193
5.3.3.1. Swelling Test in Varying pH Environments 196
5.3.4. Experimental Drug Release Results 207
5.4. Discussion 210
5.4.1. Influential Factors on Polymerisation 210
5.4.1.1. Effect of Solvent on Polymerisation 210
5.4.1.2. Effect of Monomer Composition on Polymerisation 212
5.4.2. Swelling and Drug Release Evaluation 213
5.4.2.1. IPN Swelling Behaviour 213
5.4.2.1.1. Effect of the Monomeric Composition on Swelling 213
5.4.2.1.2. Effect of the Environmental pH on Swelling 214
5.4.2.2. Drug Release Studies 216
5.5. Conclusions 217
5.6. References 218
220
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
6.1. Introduction
Variation in pH is the most important signal in the human body, which occurs naturally in
different parts of the body. The pH of the gastric condition is much lower than that of the
enteric condition [1]. A pH sensitive hydrogel is an excellent candidate for an intelligent
delivery device, which will respond to the variation in the environmental pH [1-6].
Numerous pH sensitive hydrogel based drug delivery systems for varied pH domains of
the human body such as periodontal, oral, gastric and intestinal applications have been
developed in recent years [3,7-11].
Polymeric hydrogels, which exhibit pH sensitivity contain either acidic or basic pendent
groups on it structure. In appropriate pH environment with adequate ionic strength, these
pendent groups ionize and develop fixed charges on the hydrogel [4]. The degree of the
ionization determines the swelling efficiency of the ionic hydrogel network [12-14].
These polymers with large number of ionizable groups are also referred to as
polyelectrolytes [2,5,15-18].
Polyelectrolytes could be anionic, cationic or ampholytic in nature depending on the
nature of pendent groups. Anionic hydrogels containing acidic pendent groups exhibit a
marked increase in the degree of swelling at high pH whilst the opposite response is
observed for cationic hydrogels containing basic pendent groups [15-19]. Ampholytic
behaviour in hydrogels is referred to as having both acidic and basic pendent groups on
the polymer structure [20].
Figure 1. Swelling of an ionic hydrogel network due to ionization of pendent groups at
specific pH values.
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Figure 1 illustrates a typical swelling and de-swelling behaviour of an ionic hydrogel in
response to environmental pH variations [4]. Ionic hydrogels have been described in
detail in Section 1.1.1.5.
A wide range of acidic, neutral and basic monomers has been used by the researchers in
numerous varied compositions to achieve desirable environmental sensitivity in specific
applications [11-27]. Use of AA, HEMA and NVP has been commonly reported in the
literature. Kaczmarek et al [26], Devine and Higginbotham [11] reported the use of NVP
and AA where the monomers were individually polymerised and then let to form an inter-
macromolecular complex. Khare et al [14], Am Ende et al [19,24], Khare and Peppas
[12] made use of AA and HEMA to synthesize anionic hydrogels. Sahoo et al [25]
copolymerised NVP and AA in the presence of a crosslinking agent, NN’ methylene bis-
acrylamide to function as hydrogel nanoparticles.
Chapiro and Trung [28] studied interactions between AA derivatives and NVP, and they
described the formation of a complex due to an donor/acceptor interaction. Garnet and
Zilic [29] in a recent study on charge transfer (CT) complexes have also commented on
the suitability of AA as an electron acceptor monomer. However, to date, the concept of
CT complex formation has not been applied to the synthesis of ionic hydrogels.
Researchers [11-26] have either used high-energy source such as gamma radiation or
conventional photo and thermal curing methods in the presence of initiators to prepare
this particular class of hydrogels.
In the present work the author has made use of HEMA, NVP and AA to form negatively
charged anionic polymeric networks via a photoinitiator-free process. These networks
were intended to function as pH sensitive hydrogels for slow drug delivery applications.
The hydrogel formation is based on the concept of donor/acceptor pair interaction
discussed previously in Section 4. In this study HEMA and AA were utilized as electron
acceptor monomers combined with an electron donor monomer, NVP.
The first part of the work involved kinetic studies using the Differential Photocalorimetric
(DPC) technique on the donor/acceptor pair interactions with AA and HEMA as
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Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
acceptors and NVP as the donor. The donor/acceptor pair were formulated in varying
mole ratios of the acceptor and donor and subjected to DPC measurements. Following the
kinetics study, which indicated the suitability of the donor/acceptor pairs, the anionic
hydrogels were synthesized through the photoinitiator-free process. They were
subsequently tested for their swelling and drug release behaviours in acidic, neutral and
basic pH environments.
6.2. Experimental Procedure
DPC measurements were carried out to evaluate the efficiency of complex formation
between the donor/ acceptor pairs. Experimental specifications and detailed procedure of
DPC measurements have been outlined in Sections 2.2.2.5 and 2.8. The NVP-HEMA-AA
hydrogels formed through this photoinitiator-free process were subjected to swelling drug
release experiments at 37 oC. The effect of acidic and basic environment on the swelling
and drug release behaviours of NVP-HEMA-AA hydrogels was also investigated. A
model drug, theophylline was used for drug release investigations. Detailed experimental
procedure for the synthesis of NVP-HEMA-AA hydrogels is outlined in Section 2.5.2.7.
Specifications and procedure of swelling-drug release experiments including the
preparation of the swelling and drug release media have been described in Sections 2.6.1
and 2.7. Quantitative drug release measurements were carried out on a UV-vis
spectrophotometer.
222
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
6.3. Results
6.3.1. DPC Measurements on AA/NVP and HEMA/NVP Systems
AA and HEMA were considered as potential acceptors, which were combined with NVP,
a donor monomer in varying mole ratios. The photo-exotherms (Figures 2 and 3) and
rates of polymerisation (Tables 1 and 2) indicate the relative efficiency of the
donor/acceptor pair.
AA : NVP 1mol : 1mol
AA : NVP 2mol : 1mol
-2
8
18
Hea
t F
low
(W
/g)
0 100 200 300 400 500Time (sec)Exo Up
AA : NVP 1mol : 2mol
Figure 2. Photo-exotherms of AA: NVP in varying mole ratios
Table 1. Rate of polymerisation of AA: NVP in varying ratios
Molar composition
AA: NVP
Polymerisation rate (J g-1
s-1
)
at 15 oC, N2
1 mol: 1 mol 0.53
1 mol: 2 mol 0.23
2 mol: 1 mol 0.37
223
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
1 mol HEMA : 2 mol NVP
1 mol HEMA : 1 mol NVP
-1
0
1
Hea
t F
low
(W
/g)
0 200 400 600Time (sec)Exo Up
2 mol HEMA : 1 mol NVP
Table 2. Rate of polymerisation of HEMA: NVP in varying ratios
Figure 3. Photo-exotherms of HEMA: NVP in varying mole ratios
Molar composition
HEMA: NVP
Polymerisation rate (J g-1
s-1
)
at 15 oC, N2
1 mol: 1 mol DNP
1 mol: 2 mol DNP
2 mol: 1 mol DNP
DNP indicates that the sample did not polymerise within the duration of DPC
measurement. The polymerisation rates between the donor/acceptor pairs were calculated
according to equation 1 where t, is the time taken to reach peak max.
Rate of polymerisation (J g-1
s-1
) = -1Peak max (J g )
t (s) Equation 1
224
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
6.3.2. Photopolymerisation of Hydrogels Containing NVP, AA and HEMA
NVP, AA and HEMA were combined in varying ratios by volume and subjected to UV
radiation in the absence of photoinitiator. The observations on the extent of
polymerisation upon applying approximately 9 KJ of radiation dose are described in
Table 3.
Table 3. Polymerisation status of formulations of NVP, HEMA and AA in varying
% v/v ratios
HEMA-NVP-AA hydrogel formulation
NVP : HEMA : AA (% v/v)
Polymerisation status upon application
of curing dose (~9 KJ)
50 : 00: 50 Hard, clear, pale yellow gel
50 : 50 : 00 Hard, clear, colourless gel
50 : 40 : 10 Hard, clear, colourless gel
50 : 10 : 40 Hard, clear, colourless gel
50 : 25 : 25 Hard, clear, colourless gel
00 : 50 : 50 Hard, clear, pale yellow gel
10 : 50: 40 Hard, clear, colourless gel
40 : 50 : 10 Hard, clear, colourless gel
25 : 50 : 25 Hard, clear, colourless gel
10 : 40 : 50 Hard, clear, colourless gel
40 : 10 : 50 Hard, clear, colourless gel
25 : 25 : 50 Hard, clear, colourless gel
The monomer compositions are expressed as percentage v/v. The radiation dose was
calculated according to Equation 2 where t is the total radiation time in seconds. The
samples were exposed to UV radiation for approximately 25 hours at a dose rate of 9.6 x
10-2
J s-1
. The dose rate was calculated as described in Section 2.4.4.4.
Radiation dose (J) = dose rate (J s-1
) x t (s) Equation 2
225
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
6.3.3. Experimental Swelling Results
The anionic hydrogels based on HEMA, AA and NVP in varying compositions were
subjected to swelling test in varying pH environments at 37 oC. The hydrogels were in
general found to respond to the pH variations with varying extent of swelling with
varying hydrogel compositions and the environmental pH. The swelling test results on the
hydrogels at varied pH environments expressed as % water content values at designated
time intervals are presented in Tables 4-21. The 50 NVP: 50 AA hydrogel though
successfully polymerised was found to be water-soluble, thus no further tests were
conducted on this hydrogel. Graphical representations of the swelling data are illustrated
in Figures 4-32.
226
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 4. Swelling test on the formulations of HEMA (50 % v/v) with varied ratios (
% v/v) of NVP and AA in neutral pH environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C Gel D
0.00 0.0 0.0 0.0 0.0
0.17 10.6 9.7 9.2 7.8
0.33 14.1 13.5 12.4 11.0
0.50 16.5 16.5 15.4 13.2
0.67 18.2 18.2 16.4 18.2
0.83 19.7 20.2 17.7 17.4
1.00 20.5 21.4 19.1 18.3
2.00 27.9 28.3 25.3 24.3
3.00 30.5 31.6 27.0 26.2
4.00 33.3 33.4 29.9 29.3
5.00 35.6 35.8 32.4 30.8
7.00 39.0 39.0 36.0 34.8
9.00 41.1 41.3 37.5 36.7
12.00 43.3 43.4 40.2 38.8
24.00 47.5 46.8 45.2 42.0
48.00 49.2 48.1 45.9 43.4
72.00 49.5 48.2 46.2 42.7
96.00 48.9 47.9 46.2 42.3
120.00 49.2 47.5 46.2 42.4
144.00 48.8 47.8 47.2 42.5
170.00 49.1 48.4 46.6 42.6
Hydrogel compositions: (NVP : HEMA : AA) (% v/ v)); 00 : 50 : 50 (Gel A); 10 : 50 : 40
(Gel B); 40 : 50 : 10 (Gel C) and 25 : 50 : 25 (Gel D)
227
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
The swelling data are expressed as percentage water content at respective time intervals.
The percentage water content values were calculated as described in Section 2.6.1.
Graphical representations of the swelling behaviour observed in the NVP-HEMA-AA
hydrogels in neutral pH environment at 37 oC are illustrated in Figures 4 - 6.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C Gel D
Figure 4. Plot of % water content in Gels A - D at 37 oC in neutral pH environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C Gel D
Figure 5. Plot of fractional swelling in Gels A - D at 37 oC in neutral pH environment as
a function of the square root of time.
228
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
inif
inty
Gel A Gel B Gel C Gel D
Figure 6. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels A - D at 37 oC in neutral pH environment.
As previously described in Sections 1 and 3, the parameter n is the kinetic exponential
value in the power law equation (equation 3), which indicates the time dependence
swelling and solute release kinetics in a swellable polymeric material.
Mt/M∞ = k t n Equation 3
The n values were calculated as the slope of the LOG graph. Table 5 presents the slope
(n) values calculated from Figure 6.
Table 5. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in neutral medium
Hydrogels n values
Gel A 0.44 + 0.03
Gel B 0.52 + 0.01
Gel C 0.47 + 0.02
Gel D 0.53 + 0.01
229
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Table 6. Swelling test on the formulations of AA (50 % v/v) with varied ratios ( %
v/v) of NVP and HEMA in neutral pH environment at 37 oC
Average % water content values at time (t)
Time (h) Gel E Gel F Gel G
0.00 0.0 0.0 0.0
0.17 34.5 12.0 15.4
0.33 42.7 16.1 20.8
0.50 48.7 19.7 24.3
0.67 53.7 21.3 28.0
0.83 57.8 23.4 30.3
1.00 60.0 25.1 32.4
2.00 69.3 32.6 41.1
3.00 72.4 36.7 45.7
4.00 75.0 40.1 50.0
5.00 76.9 43.3 52.0
7.00 79.0 46.3 55.7
9.00 80.0 48.5 57.4
12.00 81.3 50.7 59.5
24.00 82.9 54.5 62.1
48.00 84.1 55.0 63.0
72.00 84.6 54.6 63.0
96.00 84.7 54.3 63.3
120.00 85.0 54.4 63.1
144.00 85.1 54.4 63.4
170.00 85.6 55.1 63.5
Hydrogel compositions: (NVP: HEMA: AA (% v/ v)); 40 : 10 : 50 (Gel E); 10 : 40 : 50
(Gel F) and 25 : 25 : 50 (Gel G)
230
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel E Gel F Gel G
Figure 7. Plot of % water content in Gels E - G at 37 oC in neutral pH environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/ M
infi
nit
y
Gel E Gel F Gel G
Figure 8. Plot of fractional swelling in Gels E - G at 37 oC in neutral pH environment as a
function of the square root of time.
231
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel E Gel F Gel G
Figure 9. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels E - G at 37 oC in neutral pH environment.
Table 7 presents the slope (n) values calculated from Figure 9.
Table 7. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in neutral medium
Hydrogels n values
Gel E 0.60 + 0.02
Gel F 0.50 + 0.02
Gel G 0.52 + 0.01
232
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 8. Swelling test on the formulations of NVP (50 % v/v) with varied ratios (%
v/v) of AA and HEMA in neutral pH environment at 37 oC
Average % water content values at time (t)
Time (h) Gel H Gel I Gel J Gel K
0.00 0.0 0.0 0.0 0.0
0.17 13.8 11.6 18.3 8.7
0.33 18.3 15.9 22.0 11.8
0.50 21.6 19.1 24.9 14.4
0.67 24.8 21.8 28.3 15.6
0.83 27.0 24.4 30.8 17.4
1.00 29.4 25.9 32.5 19.0
2.00 38.0 34.4 39.8 25.2
3.00 42.3 36.7 43.8 26.8
4.00 46.5 39.8 47.5 29.0
5.00 49.4 42.6 49.7 31.5
7.00 53.6 46.6 52.6 34.4
9.00 55.8 49.2 53.4 36.8
12.00 58.2 52.3 55.0 39.1
24.00 62.5 57.8 56.5 42.9
48.00 64.4 60.1 57.4 44.2
72.00 64.5 60.8 57.8 43.7
96.00 64.5 60.9 57.4 43.9
120.00 64.6 61.5 57.2 43.0
144.00 64.8 61.8 57.1 43.4
170.00 65.2 62.1 59.2 43.2
Hydrogel compositions: (NVP : HEMA : AA (% v/ v)); 50 : 50 : 00 (Gel H); 50 : 40 : 10
(Gel I); 50 : 10 : 40 (Gel J) and 50 : 25 : 25 (Gel K)
233
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel H Gel I Gel J Gel K
Figure 10. Plot of % water content in Gels H - K at 37 oC in neutral pH environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel H Gel I Gel J Gel K
Figure 11. Plot of fractional swelling in Gels H - K at 37 oC in neutral pH environment as
a function of the square root of time.
234
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
inif
init
y
Gel H Gel I Gel J Gel K
Figure 12. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels H - K at 37 oC in neutral pH environment.
Table 9 presents the slope (n) values calculated from Figure 12.
Table 9. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in neutral medium
Hydrogels n values
Gel H 0.53 + 0.01
Gel I 0.52 + 0.01
Gel J 0.44 + 0.04
Gel K 0.49 + 0.03
235
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Table 10. Swelling test on the formulations of HEMA (50 % v/v) with varied ratios
(% v/v) of NVP and AA in acidic (pH 2) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C Gel D
0.00 0.0 0.0 0.0 0.0
0.17 10.7 12.1 8.3 8.3
0.33 12.7 13.1 10.7 9.8
0.50 15.0 16.0 12.6 12.7
0.67 17.4 18.4 14.4 13.3
0.83 19.7 21.0 15.1 15.7
1.00 20.3 21.3 16.6 17.6
2.00 26.4 29.2 22.6 23.1
3.00 29.3 31.3 24.5 26.3
4.00 31.6 33.4 26.9 27.8
5.00 32.8 35.8 28.8 29.3
7.00 35.9 40.0 31.9 32.0
9.00 37.6 40.4 34.1 34.4
12.00 40.2 42.8 36.9 36.9
24.00 43.3 45.3 41.5 39.9
48.00 45.4 45.6 43.2 40.7
72.00 44.6 46.5 43.1 40.5
96.00 44.5 45.9 43.1 40.5
120.00 43.9 45.5 42.8 40.3
144.00 44.2 45.3 42.7 39.8
170.00 44.7 45.4 42.8 39.7
Hydrogel compositions: (NVP : HEMA : AA) (% v/ v)); 00 : 50 : 50 (Gel A); 10 : 50 : 40
(Gel B); 40 : 50 : 10 (Gel C) and 25 : 50 : 25 (Gel D)
236
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C Gel D
Figure 13. Plot of % water content in Gels A - D at 37 oC in pH 2 environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C Gel D
Figure 14. Plot of fractional swelling in Gels A - D at 37 oC in pH 2 environment at 37
oC in pH 2 environment as a function of the square root of time.
237
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
-1.5
-1.0
-0.5
0.0
-1.0 -0.5 0.0
LOG time
LO
G M
t/ M
inif
inty
Gel A Gel B Gel C Gel D
Figure 15. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels A - D at 37 oC in pH 2 environment.
Table 11 presents the slope (n) values calculated from Figure 15.
Table 11. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in acidic medium
Hydrogels n values
Gel A 0.45 + 0.04
Gel B 0.42 + 0.05
Gel C 0.44 + 0.03
Gel D 0.47 + 0.02
238
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 12. Swelling test on the formulations of AA (50 % v/v) with varied ratios ( %
v/v) of NVP and HEMA in acidic (pH 2) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel E Gel F Gel G
0.00 0.0 0.0 0.0
0.17 28.7 14.5 15.3
0.33 37.4 17.5 20.1
0.50 43.0 20.9 23.4
0.67 46.7 22.6 26.8
0.83 49.6 25.0 28.6
1.00 52.3 26.5 30.2
2.00 58.7 32.3 38.9
3.00 60.8 36.9 43.8
4.00 62.7 38.9 46.9
5.00 63.0 41.3 49.6
7.00 63.4 44.7 52.6
9.00 63.8 46.2 54.5
12.00 64.6 48.2 56.2
24.00 64.0 50.7 58.1
48.00 64.1 51.5 59.3
72.00 64.2 51.5 58.9
96.00 64.2 51.3 58.8
120.00 64.1 51.1 58.8
144.00 64.4 51.1 58.7
170.00 63.6 50.8 59.0
Hydrogel compositions: (NVP: HEMA: AA (% v/ v)); 40 : 10 : 50 (Gel E); 10 : 40 : 50
(Gel F) and 25 : 25 : 50 (Gel G)
239
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel E Gel F Fel G
Figure 16. Plot of % water content in Gels E - G at 37 oC in pH 2 environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel E Gel F Gel G
Figure 17. Plot of fractional swelling in Gels E - G at 37 oC in pH 2 environment as a
function of the square root of time.
240
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel E Gel F Gel G
Figure 18. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels E - G at 37 oC in pH 2 environment
Table 13 presents the slope (n) values calculated from Figure 18.
Table 13. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in acidic medium
Hydrogels n values
Gel E 0.53 + 0.01
Gel F 0.43 + 0.03
Gel G 0.50 + 0.02
241
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Table 14. Swelling test on the formulations of NVP (50 % v/v) with varied ratios ( %
v/v) of AA and HEMA in acidic (pH 2) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel H Gel I Gel J Gel K
0.00 0.0 0.0 0.0 0.0
0.17 13.5 11.8 18.3 7.4
0.33 17.2 15.0 22.8 11.0
0.50 20.1 16.8 26.7 14.4
0.67 22.4 19.1 29.5 15.5
0.83 24.2 20.7 30.4 16.0
1.00 27.0 22.0 32.6 17.1
2.00 34.2 30.2 39.4 22.8
3.00 38.8 32.1 42.1 25.1
4.00 43.2 35.3 44.1 27.3
5.00 45.2 37.2 46.3 29.1
7.00 49.1 40.9 48.2 32.1
9.00 51.9 43.3 49.9 34.2
12.00 54.0 46.0 49.8 36.8
24.00 58.0 50.5 50.8 40.1
48.00 59.8 51.8 51.1 41.3
72.00 60.6 52.2 51.5 41.2
96.00 60.5 52.2 51.1 40.5
120.00 60.3 52.3 50.9 40.3
144.00 59.7 52.4 50.7 40.4
170.00 59.9 52.7 50.2 40.5
Hydrogel compositions: (NVP : HEMA : AA (% v/ v)); 50 : 50 : 00 (Gel H); 50 : 40 : 10
(Gel I); 50 : 10 : 40 (Gel J) and 50 : 25 : 25 (Gel K)
242
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel H Gel I Gel J Gel K
Figure 19. Plot of % water content in Gels H - K at 37 oC in pH 2 environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel H Gel I Gel J Gel K
Figure 20. Plot of fractional swelling in Gels H - K at 37 oC in pH 2 environment at 37
oC in pH 2 environment as a function of the square root of time.
243
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
inif
init
y
Gel H Gel I Gel J Gel K
Figure 21. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels H - K at 37 oC in pH 2 environment.
Table 15 presents the slope (n) values calculated from Figure 21.
Table 15. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in acidic medium
Hydrogels n values
Gel H 0.47 + 0.02
Gel I 0.42 + 0.03
Gel J 0.43 + 0.04
Gel K 0.53 + 0.01
244
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 16. Swelling test on the formulations of HEMA (50 % v/v) with varied ratios (
% v/v) of AA and NVP in basic (pH 8) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel A Gel B Gel C Gel D
0.00 0.0 0.0 0.0 0.0
0.17 25.2 25.6 24.2 25.8
0.33 36.7 34.5 31.5 35.1
0.50 41.8 40.9 36.6 41.8
0.67 42.5 46.6 42.6 46.6
0.83 44.9 51.3 47.0 51.1
1.00 46.5 55.4 50.0 54.4
2.00 60.7 68.7 65.1 69.5
3.00 64.9 72.6 69.6 73.4
4.00 69.7 74.9 72.9 77.7
5.00 72.3 77.8 76.1 80.7
7.00 76.9 81.1 80.8 84.6
9.00 79.9 82.7 83.9 86.8
12.00 82.7 86.3 88.0 89.5
24.00 87.4 89.4 91.1 93.1
48.00 88.8 90.1 91.6 93.2
72.00 89.3 90.4 91.6 93.0
96.00 89.2 89.8 91.6 92.8
120.00 89.0 89.6 91.6 92.8
144.00 89.1 89.6 91.7 92.9
170.00 89.1 89.8 91.6 93.0
Hydrogel compositions: (NVP : HEMA : AA) (% v/ v)); 00 : 50 : 50 (Gel A); 10 : 50 : 40
(Gel B); 40 : 50 : 10 (Gel C) and 25 : 50 : 25 (Gel D)
245
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel A Gel B Gel C Gel D
Figure 22. Plot of % water content in Gels A - D at 37 oC in pH 8 environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel A Gel B Gel C Gel D
Figure 23. Plot of fractional swelling in Gels A - D at 37 oC in pH 8 environment at 37
oC in pH 8 environment as a function of the square root of time.
246
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-2.0
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel A Gel B Gel C Gel D
Figure 24. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels A - D at 37 oC in pH 8 environment.
Table 17 presents the slope (n) values calculated from Figure 24.
Table 17. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in basic medium
NVP : HEMA : AA n values
Gel A 0.51 + 0.03
Gel B 0.71 + 0.04
Gel C 0.65 + 0.02
Gel D 0.69 + 0.02
247
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Table 18. Swelling test on the formulations of AA (50 % v/v) with varied ratios ( %
v/v) of NVP and HEMA in basic (pH 8) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel E Gel F Gel G
0.00 0.0 0.0 0.0
0.17 92.8 29.0 34.6
0.33 94.1 38.6 46.2
0.50 95.3 45.3 53.6
0.67 95.3 50.1 58.7
0.83 95.5 53.5 62.6
1.00 95.7 56.3 66.0
2.00 96.0 68.3 76.9
3.00 96.0 73.1 81.5
4.00 96.0 77.5 84.8
5.00 96.0 79.6 86.9
7.00 96.1 81.9 89.6
9.00 96.0 84.6 91.2
12.00 96.0 84.9 92.4
24.00 95.9 87.7 94.1
48.00 95.9 88.6 94.4
72.00 96.0 89.3 94.3
96.00 96.1 89.3 94.4
120.00 96.2 89.2 94.4
144.00 96.1 89.0 94.4
170.00 96.0 89.0 94.4
Hydrogel compositions: (NVP: HEMA: AA (% v/ v)); 40 : 10 : 50 (Gel E); 10 : 40 : 50
(Gel F) and 25 : 25 : 50 (Gel G)
248
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel E Gel F Gel G
Figure 25. Plot of % water content in Gels E - G at 37 oC in pH 8 environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel E Gel F Gel G
Figure 26. Plot of fractional swelling in Gels E - G at 37 oC in pH 8 environment as a
function of the square root of time.
249
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
-2.0
-1.5
-1.0
-0.5
0.0
-2.0 -1.5 -1.0 -0.5 0.0
LOG time
LO
G M
t/ M
inif
init
y
Gel E Gel F Gel G
Figure 27. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels E - G at 37 oC in pH 8 environment
Table 19 presents the slope (n) values calculated from Figure 27.
Table 19. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in basic medium
Hydrogels n values
Gel E 2.13 + 0.10
Gel F 0.65 + 0.04
Gel G 0.72 + 0.02
250
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 20. Swelling test on the formulations of NVP (50 % v/v) with varied ratios ( %
v/v) of AA and HEMA in basic (pH 8) environment at 37 oC
Average % water content values at time (t)
Time (h) Gel H Gel I Gel J Gel K
0.00 0.0 0.0 0.0 0.0
0.17 15.0 31.2 51.0 37.4
0.33 20.5 38.3 62.8 47.4
0.50 23.5 44.4 68.7 54.8
0.67 27.6 48.7 73.3 59.6
0.83 28.9 53.1 76.0 63.5
1.00 30.8 55.3 78.1 66.3
2.00 37.5 69.3 86.0 78.8
3.00 44.0 73.1 90.1 81.7
4.00 47.9 79.9 92.1 84.8
5.00 50.9 83.2 93.4 87.4
7.00 54.6 86.4 95.0 90.6
9.00 57.5 88.6 95.9 92.3
12.00 59.9 90.2 96.6 93.9
24.00 65.7 92.2 97.3 96.3
48.00 67.3 92.3 97.3 96.5
72.00 67.3 92.6 97.3 96.6
96.00 67.1 92.3 97.3 96.5
120.00 67.5 92.2 97.3 96.5
144.00 67.1 92.3 97.4 96.5
170.00 67.1 92.4 97.4 96.5
Hydrogel compositions: (NVP : HEMA : AA (% v/ v)); 50 : 50 : 00 (Gel H); 50 : 40 : 10
(Gel I); 50 : 10 : 40 (Gel J) and 50 : 25 : 25 (Gel K)
251
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel H Gel I Gel J Gel K
Figure 28. Plot of % water content in Gels H - K at 37 oC in pH 8 environment as a
function of time.
0.0
0.4
0.8
1.2
0 5 10 15
t1/2
(h1/2
)
Mt/
Min
fin
ity
Gel H Gel I Gel J Gel K
Figure 29. Plot of fractional swelling in Gels H - K at 37 oC in pH 8 environment as a
function of the square root of time.
252
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-2.0
-1.5
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G M
t/ M
infi
nit
y
Gel H Gel I Gel J Gel K
Figure 30. Plot of the LOG of fractional swelling as a function of the LOG of time, in the
initial stages of swelling in Gels H - K at 37 oC in pH 8 environment.
Table 21 presents the slope (n) values calculated from Figure 30.
Table 21. Characteristic exponential n values for diffusion in NVP-HEMA-AA
hydrogels in basic medium
Hydrogels n values
Gel H 0.52 + 0.02
Gel I 0.57 + 0.02
Gel J 0.69 + 0.03
Gel K 0.72 + 0.02
253
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Figure 31 illustrates comparative effect of varying pH environments on the swelling
behaviour of 40 NVP: 10 HEMA: 50 AA hydrogel. Figure 32 illustrates the comparative
effect of the variations in the environmental pH in relation to the hydrogel composition on
the swelling behaviour of the anionic hydrogels.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
pH 8 neutral pH 2
Figure 31. Comparative plot of % water content in the 40 NVP: 10 HEMA: 50 AA
hydrogel in varying pH environments at 37 oC as a function of time.
0
20
40
60
80
100
0 50 100 150 200
Time (h)
% W
ate
r C
on
ten
t
Gel H (pH 2) Gel H (pH 8) Gel H (neutral)Gel K (pH 2) Gel K (pH 8) Gel K (neutral)
Figure 32. Comparative plot of % water content in Gel H and Gel K in varying pH
environments at 37 oC as a function of time.
254
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
6.3.4. Experimental Drug Release Results
The drug release experiments on NVP-AA-HEMA hydrogels H - J were conducted at 37
oC in varied pH environments using theophylline as the model drug. The experimental
data expressed as fractional drug release at specific time intervals are presented in Tables
22, 24 and 26. The hydrogels tested were of the following compositions: (NVP : HEMA
: AA (% v/ v)); 50 : 50 : 00 (Gel H); 50 : 10 : 40 (Gel J) and 50 : 40 : 10 (Gel I)
Table 22. Drug release test on formulations of NVP, AA and HEMA in varied
ratios (% v/v) in neutral environment at 37 oC
Fractional theophylline released
values at time t
Time (h) Gel H Gel I Gel J
0.00 0.00 0.00 0.00
0.17 0.24 0.19 0.28
0.33 0.33 0.28 0.39
0.50 0.41 0.34 0.54
0.67 0.46 0.39 0.64
0.83 0.51 0.43 0.70
1.00 0.55 0.46 0.75
2.00 0.73 0.63 0.92
3.00 0.82 0.72 0.97
4.00 0.88 0.79 0.98
5.00 0.92 0.84 0.97
7.00 0.96 0.90 0.99
9.00 0.95 0.94 0.99
12.00 0.97 0.95 0.98
24.00 0.98 0.97 0.99
48.00 0.98 0.98 0.99
255
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
The fractional drug release values were calculated as described in Section 2.7. Graphical
representations of the drug release behaviour in the IPN hydrogels are illustrated in
Figures 33 - 35.
0.0
0.4
0.8
1.2
0 10 20 30 40 50 6
Time (h)
Fra
ctio
na
l D
rug
Rel
ease
d
0
Gel H Gel I Gel J
Figure 33. Plot of the fractional release of theophylline from Gels H - J at 37 oC in
neutral pH environment as a function of time.
0.00
0.40
0.80
1.20
0 2 4 6
t1/2
(h1/2
)
Fra
ctio
na
l D
rug
Rel
ease
d
8
Gel H Gel I Gel J
Figure 34. Plot of the fractional release of theophylline from Gels H - J at 37 oC in
neutral pH environment as a function of the square root of time.
256
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel H Gel I Gel J
Figure 35. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time, in the initial stages of theophylline release from Gels H - J at 37 oC in neutral pH
environment.
Table 23 presents the slope (n) values calculated from Figure 35.
Table 23. Characteristic exponential n values for drug release from NVP-HEMA-
AA hydrogels in neutral medium
Hydrogels n values
Gel H 0.49 + 0.01
Gel I 0.47 + 0.04
Gel J 0.58 + 0.03
257
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Table 24. Drug release test on formulations of NVP, AA and HEMA in varied
ratios (% v/v) in acidic (pH 2) environment at 37 oC
Fractional theophylline released
values at time t
Time (h) Gel H Gel I Gel J
0.00 0.00 0.00 0.00
0.17 0.20 0.15 0.17
0.33 0.30 0.22 0.27
0.50 0.39 0.28 0.33
0.67 0.44 0.32 0.40
0.83 0.49 0.36 0.44
1.00 0.53 0.39 0.47
2.00 0.70 0.53 0.64
3.00 0.80 0.63 0.76
4.00 0.87 0.70 0.82
5.00 0.91 0.76 0.87
7.00 0.95 0.83 0.94
9.00 0.97 0.88 0.95
12.00 0.97 0.93 0.96
24.00 0.99 0.98 0.99
48.00 0.97 0.98 0.98
258
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0.0
0.4
0.8
1.2
0 10 20 30 40 50 6
Time (h)
Fra
ctio
na
l D
rug
Rel
ease
d
0
Gel H Gel I Gel J
Figure 36. Plot of the fractional release of theophylline from Gels H - J at 37 oC in pH 2
environment as a function of time.
0.0
0.4
0.8
1.2
0.0 2.0 4.0 6.0 8.0
t1/2
(h1/2
)
Fra
ctio
na
l D
rug
Rel
ease
d
Gel H Gel I Gel J
Figure 37. Plot of the fractional release of theophylline from Gels H - J at 37 oC in pH 2
environment as a function of the square root of time.
259
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel H Gel I Gel J
Figure 38. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time, in the initial stages of theophylline release from Gels H - J at 37 oC in pH 2
environment.
Table 25 presents the slope (n) values calculated from Figure 38.
Table 25. Characteristic exponential n values for drug release from NVP-HEMA-
AA hydrogels in acidic medium
Hydrogels n values
Gel H 0.53 + 0.01
Gel I 0.52 + 0.01
Gel J 0.56 + 0.01
260
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 26. Drug release test on formulations of NVP, AA and HEMA in varied
ratios (% v/v) in basic (pH 8) environment at 37 oC
Fractional theophylline released
values at time t
Time (h) Gel H Gel I Gel J
0.00 0.00 0.00 0.00
0.17 0.31 0.35 0.32
0.33 0.41 0.47 0.43
0.50 0.49 0.56 0.51
0.67 0.54 0.62 0.57
0.83 0.59 0.69 0.63
1.00 0.63 0.72 0.67
2.00 0.79 0.87 0.82
3.00 0.87 0.93 0.88
4.00 0.91 0.95 0.91
5.00 0.93 0.97 0.93
7.00 0.95 0.97 0.94
9.00 0.96 0.97 0.93
12.00 0.96 0.96 0.93
24.00 0.97 0.97 0.95
48.00 0.98 0.99 0.95
261
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
0.0
0.4
0.8
1.2
0 10 20 30 40 50 6
Time (h)
Fra
ctio
na
l D
rug
Rel
ease
d
0
Gel H Gel I Gel J
Figure 39. Plot of the fractional release of theophylline from Gels H - J at 37 oC in pH 8
environment as a function of time.
0.0
0.4
0.8
1.2
0 2 4 6
t1/2
(h1/2
)
Fra
ctio
na
l D
rug
Rel
ease
d
8
Gel H Gel I Gel J
Figure 40. Plot of the fractional release of theophylline from Gels H - J at 37 oC in pH 8
environment as a function of the square root of time.
262
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
-1.0
-0.5
0.0
-1.0 -0.8 -0.6 -0.4 -0.2 0.0
LOG time
LO
G F
DR
Gel H Gel I Gel J
Figure 41. Plot of the LOG of fractional drug released (FDR) as a function of the LOG of
time, in the initial stages of theophylline release from Gels H - J at 37 oC in pH 8
environment.
Table 27 presents the slope (n) values calculated from Figure 41.
Table 27. Characteristic exponential n values for drug release from NVP-HEMA-
AA hydrogels in basic medium
NVP : HEMA : AA n values
Gel H 0.40 + 0.02
Gel I 0.42 + 0.02
Gel J 0.41 + 0.03
263
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
6.4. Discussion
6.4.1. DPC Measurements
The DPC measurements on AA: NVP systems at varied mol ratios revealed that the 1:1
mol ratio of AA:NVP was the most optimum donor/acceptor pair yielding the highest
polymerisation rate of 0.53 J g-1
s-1
(Table 1). The AA: NVP formulations with either
excess of AA or excess of NVP showed lower reactivity. The formulation containing
excess NVP displayed the lowest reactivity of 0.23 J g-1
s-1
indicating its relative
inertness. The rate calculations were carried out according to Equation 1. Efficient
systems were characterized by an intense peak in the photo-exotherms (Figure 2). The
optimum ratio of 1mol:1mol of AA:NVP could be described in terms of the number of
double bonds present in the monomers, which effectively take part in the complex
formation. Scheme 1 illustrates the 1:1mol interaction between AA and NVP.
N
O
OH
O
N
O
OH
O
NVP AA
Donor Acceptor
+ hv
NVP-AA complex
Scheme 1. 1:1 mol of NVP:AA interaction
AA and NVP each have 1 pair of double bonds, which form the ring closure (cyclobutane
ring) as illustrated in Scheme 1. Thus when excess NVP or AA is present, they are not
consumed due to insufficient quantity of the other. The resultant possibly is the
homopolymerisation of the excess monomer, which is relatively less efficient than the
copolymerisation reaction. The formulation containing excess AA showed a slightly
higher rate over the formulation containing excess NVP.
Chapiro and Trung [28] studied the reactivity of AA and NVP under the influence of
gamma radiation. They observed a relatively low reactivity with increasing NVP content
264
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
in the polymer. Khodzhaev and Mushraipov [30] made use of NMR spectroscopy to study
the interaction between AA and NVP. The also suggest low reactivity of high NVP
content formulation. This observation could be attributed to the fact that AA being an
acrylate has a relatively higher reactivity than NVP, thus AA homopolymerises more
efficiently than NVP.
The kinetic studies using the DPC technique on HEMA-NVP system revealed that it was
not an efficient donor/acceptor system as compared to AA-NVP as it did not completely
polymerise when exposed to the UV light (Table 2). The non-existence of sharp
exotherms (Figure 3) as in the case of AA-NVP illustrates the incomplete polymerisation
of the HEMA-NVP system. This could be attributed to the fact HEMA did not efficiently
react with NVP probably due to insufficient exposure to the UV light under the DPC
measurement condition. The relative reactivity of HEMA and NVP has been discussed in
Section 3.
6.4.2. Hydrogel Formation
All the AA-NVP-HEMA formulations in varied compositions resulted in successful
hydrogel synthesis (Table 3). The formulations were cured upon an applied UV dose of 9
KJ. HEMA-NVP system despite showing low reactivity in DPC measurements resulted in
successful synthesis of a hydrogel in the absence of photoinitiator. This observation could
be related to the total dose of UV radiation applied to the monomer mixture. The radiation
time in DPC was only several minutes in comparison to curing time of approximately 25
hours for hydrogel synthesis. Thus the study suggests that HEMA and NVP could be
polymerised in the absence of a photoinitiator provided a longer radiation time is allowed.
HEMA and AA when exposed to the UV source also resulted in a successfully
polymerised hydrogel in the absence of a photoinitiator. Synthesis of anionic hydrogels
based on HEMA and AA has been reported by Khare and Peppas [12] who utilized redox
polymerisation technique in the presence of initiators to achieve copolymerisation of
these two monomers. Am Ende et al [19,24] have utilized thermal polymerisation
technique in the presence of initiators to obtain HEMA-co-AA hydrogel networks. The
successful polymerisation of AA-HEMA system as observed in the present study could be
265
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
attributed to the possible role of HEMA as a donor to AA. HEMA contains a slightly
electron rich methyl group adjacent to the carbon-carbon double bond on its structure.
Thus AA being a relatively strong electron acceptor could form a complex with HEMA,
which changes its role from a weak electron acceptor to an electron donor monomer as
illustrated in Scheme 2.
CH3
O
OOH
O
OH
O
OH
CH3
O
OOH
HEMA AA
Possible donor Acceptor
+hv
HEMA-AA complex
Scheme 2. Possible donor/acceptor interaction between HEMA and AA
6.4.3. Swelling and Drug Release Evaluation
The AA-NVP-HEMA hydrogels were subjected to swelling and drug release experiments
in pH 2, neutral and pH 8 environments. The phenomena of Fickian and non-Fickian
diffusion kinetics were used to characterize the swelling and drug release behaviours in
these anionic hydrogel networks. Equation 3 describes time dependent swelling action of
hydrogel networks. The parameter n in the power-law equation as described previously,
effectively indicates the type of diffusion mechanism in the hydrogel network. As
previously mentioned, Fickian diffusion is characterized by a n value of 0.5. High order
non-Fickian diffusion could be described in various degrees of non-Fickian diffusion
behaviour. A non-Fickian (anomalous) behaviour is characterized by 0.5 < n < 1. Case II
diffusion is characterized by a n value of 1 while super case II behaviour is characterized
by n > 1.
266
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
6.4.3.1. Swelling Behaviour of AA-NVP-HEMA Hydrogels
The hydrogels were thoroughly washed in milli-Q-water to remove any unreacted
component in order to avoid any variations in the degree of swelling. The uncrosslinked
copolymer of AA-co-NVP disintegrated during the washing process. The early
disintegration of AA-co-NVP network could be the cause of insufficient crosslinkage in
the polymer network. The hydrogels with HEMA however were reasonably resilient. The
swelling experiments revealed that the degree of swelling in the anionic AA-NVP-HEMA
hydrogels was significantly dependent on pH.
6.4.3.1.1. pH Dependent Swelling Behaviour
The anionic AA-NVP-HEMA hydrogels were found to be significantly dependent on the
environmental pH. Hydrogels swelled in varied pH environments showed an increase in
the degree of swelling with increase in the environmental pH. The extent of pH
sensitivity, however, varied with the monomeric composition.
The 50 % HEMA hydrogels with varied ratios of NVP and AA hydrogels displayed very
low degree of swelling in pH 2 buffer solution with hydrogels A-D yielding average
EWC values of 44.7 %, 45.4 %, 42.8 % and 39.7 % respectively (Table 10, Figure13).
The hydrogels remained collapsed in this acidic environment with a very gradual water
uptake adhering to Fickian diffusion kinetics (Table 11, Figures 14 and 15). The swelling
curve (Figure 13) showed a gradual reduction in the water uptake rate around 12 hours
reaching equilibrium swelling in 48 hours.
In neutral pH environment a very slight increase in the swelling behaviour was observed
with hydrogels A-D yielding EWC values of 49.1 %, 48.4 %, 46.6 % and 42.6 %
respectively (Table 4, Figure 4). Hydrogels A-D displayed a gradual increase in water
uptake in the initial stages of swelling followed by a gradual reduction in the water
absorption rate leading to equilibrium swelling around 48 hours. The swelling data of
hydrogels A-D in neutral pH environment indicate Fickian diffusion mechanism was in
operation (Figures 5 and 6, Table 5).
267
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
However, in pH 8 buffer solution, a significant increase in the swelling behaviour was
observed with hydrogels A-D yielding average EWC values of 89.1 %, 89.8 %, 91.6 %
and 93.0 % respectively (Table 16, Figure 22). A rapid increase in water content was
observed in the initial stages with gradual reduction in swelling efficiency around 9 hours
and equilibrium swelling was reached in 48 hours. The swelling experimental data on the
hydrogel samples suggested that non-Fickian diffusion kinetics was in operation with the
exception of hydrogel A, which adhered to Fickian diffusion kinetics yielding a n value of
0.51 (Table 17, Figures 23 and 24).
Hydrogels composed of 50 % AA with varying ratios of NVP and HEMA behaved in a
similar fashion in swelling to that of hydrogels A-D. The degree of swelling observed in
pH 2 environment was slightly lower than that in neutral and basic environments.
Hydrogels E-G displayed EWC values of 63.6 %, 50.8 % and 59.0 % respectively (Table
12, Figure 16). A gradual reduction in the swelling efficiency was observed around 7
hours with equilibrium swelling achieved in 48 hours. Hydrogels E-G adhered to Fickian
diffusion kinetics in the initial stages of swelling (Table 13, Figures17 and 18). An
increase in EWC was observed in neutral environment with hydrogels E-G yielding EWC
values of 85.6 %, 55.1 % and 63.5 % (Table 6, Figure 7). A rapid diffusion was observed
in Gel E, which adhered to anomalous diffusion kinetics (Table 7, Figures 8 and 9)
reaching equilibrium swelling in 48 hours. The diffusion process in hydrogels F and G
were indicative of Fickian diffusion kinetics characterized by gradual swelling leading to
equilibrium swelling in 48 hours.
Hydrogels E-G showed rapid water uptake behaviour in pH 8 environment yielding EWC
values of 96.0 %, 89.0 % and 94.4 % respectively (Table 18, Figure 25). Hydrogel E
showed a phenomenal increase in water uptake reaching near equilibrium swelling within
the initial 10 minutes of swelling yielding a water content value of 92.8 %. Hydrogels F
and G displayed a gradual reduction in the swelling efficiency around 7 hours reaching
equilibrium swelling in 48 hours. Hydrogels adhered to non-Fickian (anomalous)
diffusion kinetics in the initial stages of swelling experiment with the exception of
Hydrogel E, which displayed extreme non-Fickian diffusion behaviour described as super
case II diffusion kinetics with a n value of 2.13 (Table 19, Figures 26 and 27).
268
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Formulations containing 50 % NVP with varied ratios of HEMA and NVP also showed
significant pH dependent swelling behaviour. In pH 2 environment hydrogels H-K
displayed a low degree of swelling yielding EWC values of 59.9 %, 52.7 %, 50.2 % and
40.5 % respectively (Table 14, Figure19). A gradual increase in water uptake was
observed in the initial stages of the swelling experiment followed by a reduction in the
water uptake rate around 9 hours, reaching equilibrium swelling in 48 hours. The
diffusion kinetics observed in the hydrogels is typical of Fickian diffusion behaviour
(Table 15, Figures 20 and 21). An increase in the EWC was observed in neutral pH
environment with hydrogels H-K yielding EWC values of 65.2 %, 62.1 %, 59.2 % and
43.2 % respectively (Table 8, Figure 10). However, a gradual water uptake rate in
hydrogels H-K was observed indicating that the Fickian diffusion mechanism was in
operation (Table 9, Figures 11 and 12). Equilibrium swelling in hydrogels H-K was
observed around 48 hours of constant swelling.
In pH 8 buffer solution, a rapid increase in the swelling efficiency was observed in
hydrogels I-K, which yielded EWC values of 92.4 %, 97.4 % and 96.5 % respectively
(Table 20, Figure 28). Hydrogel H showed a slight increase in swelling kinetics with an
EWC value of 67.1 %. Hydrogels showed increasing water uptake rate in the initial stages
of the experiment followed by a gradual decrease in the water uptake rate around 9 hours
reaching equilibrium swelling in 48 hours. Hydrogels adhered to non-Fickian diffusion
(anomalous) kinetics in the initial stages of the experiment with the exception of hydrogel
H, which displayed Fickian diffusion kinetics with a n value of 0.52 (Table 21, Figures 29
and 30).
The experimental swelling data could be explained in terms of the monomer composition
of the hydrogels and their relative responses to variations in the environmental pH. The
swelling behaviour of the hydrogels in neutral pH revealed that the formulations
containing high amounts of AA and NVP showed high water absorption ability. The
hydrogel formulations containing 50 % AA (hydrogels E-G) and 50 % NVP (hydrogels
H-K) displayed higher degree of swelling in neutral pH environments in comparison to
hydrogel formulations with 50 % HEMA (hydrogels A-D).
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Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
Hydrogels E-K showed a reduction in the degree of swelling with increasing HEMA
content. This effect was more significant in hydrogels E-G, which displayed the highest
equilibrium swelling in neutral environment. The high swelling behaviour in hydrogels
with high NVP and AA contents could be attributed to their higher hydrophilic nature as
compared to HEMA. Poor water uptake ability of high HEMA content hydrogels has
been discussed in Section 3. The relatively higher swelling activity of hydrogels E-G in
neutral medium in comparison to H-K suggests superior hydrophilicity of AA over NVP.
Am Ende et al [19] in their studies on the swelling activity in HEMA-co-AA hydrogels
observed reduced swelling with increasing HEMA content. They suggested that increase
in AA content in the copolymer led to a significant reduction in the molecular weight
between crosslinks, which led to high degree of swelling.
However, deviation in the environmental pH from neutral to acidic and basic conditions
resulted in variations in the degree of swelling of the AA-NVP-HEMA hydrogels. The
extent of the variations in the swelling behaviour varied with sample compositions. A
swelling and de-swelling phenomenon was observed in basic and acidic environments
respectively. Furthermore, swelling experiments in varying pH environments indicated
increasing sensitivity of hydrogels with high AA content to variations in the
environmental pH. Hydrogels displayed higher EWC values with increase in the
environmental pH with a more pronounced increase with high AA content hydrogels. .
Significant increase in EWC values with increasing in environmental pH could be
attributed to the significantly acidic nature of AA, which has a pKa value of 4.25. As the
environmental pH is increased from acidic to basic condition, the H+ ions from the
carboxyl groups in AA react with the OH- in alkali leading to an increase in the
dissociation of AA to form carboxyl anions. The degree of dissociation of AA in varying
pH environments was calculated according to Equation 4 [20,22] and the results are
summarised in Table 28.
( )
1
10 1apK pHα −= + Equation 4
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Table 28. Degree of dissociation of AA with respect to pH variation
pH Degree of dissociation ( ) α2 0.0056
7 0.9982
8 0.9998
The enhanced swelling action in the hydrogels with increase in the environmental pH
could be described as an increase in the electrostatic repulsion within the polymer matrix,
which led to significant swelling. The anionic pendent groups became increasingly
ionized with increase in pH leading to enhanced electrostatic repulsion within the
polymer matrix. This resulted in a disruption of hydrogen bondings between the acid
groups, leading to an increase in the mesh size, thus increasing the swelling ratio. In
acidic condition (pH 2 buffer solution) the anionic hydrogels are non-ionized where they
are in a compact conformation with relatively smaller mesh size thus causing a reduction
in the swelling fraction. The high EWC values observed for the hydrogels under study
suggested high ionization activity of AA in pH 8 buffer solution. However, in milli-Q-
water (neutral pH) environment only a slight increase in swelling was observed despite a
high degree of dissociation of AA as described in Table 28.
This could be attributed to the fact that the nature of the swelling agent, which is milli-Q-
water in this instance, has an ionic strength of zero. Thus the sharp increase in swelling
efficiency in the pH 8 buffer solution in comparison to milli-Q-water (neutral pH) could
also be explained in terms of the ionic strength of the immediate environment. Khare and
Peppas [12] reported an increase in the swelling fraction of HEMA-co-AA in buffered
solutions in comparison to unbuffered milli-Q-water and they described this behaviour
according to the Donnan equilibrium. The Donnan equilibrium, previously discussed in
Section 5.4.2, suggests that an increase in the ionic strength of the swelling medium leads
to a higher equilibrium water uptake by the ionic hydrogels. Thus in milli-Q-water, due to
limited ionization of the hydrogel, the swelling fraction was lower than that in the pH 8
buffered solution where the degree of ionization was relatively higher.
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Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
The 50 HEMA-50 NVP hydrogel was found to be slightly sensitive to the variations in
the environmental pH. Copolymers of HEMA and NVP have always been reported as
neutral networks. In a recent study, Firreira et al [31] evaluated PHEMA hydrogels as
drug delivery devices in varying pH environments and observed a slight increase in the
degree of swelling with increase in pH. This is in agreement with Svecik et al [32] who
reported partial hydrolysis of PHEMA in basic environment to be the cause of increase in
the degree of swelling. Brannon-Peppas and Peppas [33] also observed a slight increase in
the swelling ratio of PHEMA between pH 6 and pH 8. They explained the slight pH
dependent swelling to be due to the delocalisation of the electron density on the OH
group of HEMA to the electron attracting carbonyl group in basic environment. This
phenomenon explains the slight variations observed in the swelling ratio of the 50
HEMA-50 NVP hydrogel in varying pH environments. The slight pH dependant swelling
behaviour of this network thus could be attributed to the presence of HEMA in the
network.
6.4.3.2. Drug Release Studies
Hydrogels tested were 50 NVP-50 HEMA (Gel H), 50 NVP-40 AA-10 HEMA (Gel J)
and 50 NVP-10 AA-40 HEMA (Gel I). In neutral pH, hydrogels H and I displayed an
initial burst effect release followed by gradual increase in the release rate of theophylline
in the initial stages of the experiment adhering to Fickian diffusion kinetics with n values
of 0.49 and 0.47 respectively (Tables 22 and 23, Figures 33-35). The rate of release
gradually decreased around 7 hours leading to equilibrium drug release around 9 hours.
Hydrogel J displayed a burst effect release followed by a rapid release of the theophylline
in the initial stages adhering to non-Fickian diffusion (anomalous) kinetics with a n value
of 0.58. A gradual reduction in the release rate was observed in hydrogel J around 3 hours
leading to case II, time independent release around 5 hours.
In acidic environment, lower drug release rates were observed for hydrogels H-J. A
gradual increase in release rate of theophylline was observed in initial stages of the
experiments baring an initial burst followed by a gradual decrease in the release rate
around 7 hours (Tables 24 and 25, Figures 36-38). Hydrogels H and I adhered to Fickian
release kinetics with n values of 0.53 and 0.52 respectively while hydrogel J showed a
272
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
slight non-Fickian anomalous behaviour with a n value of 0.56. Equilibrium drug release
was observed around 12 hours, which was characterized by case II time independent
diffusion indicated by a constant release rate as function of time.
A relatively high diffusivity of theophylline from hydrogel H-J was observed in pH 8
environment with slightly higher diffusivity of theophylline observed in hydrogel I. An
initial burst effect release of theophylline was observed in the initial stages followed by a
constant linear increase leading to time independent release around 7 hours (Table 26,
Figure 39). The release kinetics in the hydrogels was characteristic of Fickian release
behaviour as illustrated by a linear increase in the release rate as a function of the square
root of time with characteristic n values of 0.40, 0.41 and 0.42 for hydrogels H, J and I
respectively (Table 27, Figures 40 and 41).
In general an increased amount of theophylline release was observed with increase in the
environmental pH. As described previously, anionic hydrogels become increasingly
ionized with increase in environmental pH thus allowing a rapid release of theophylline
from the hydrogels. In neutral environment the hydrogel J displayed the highest
diffusivity of theophylline followed by hydrogels H and I. This behaviour could be
explained in terms the relative hydrophilicity/hydrophobicity of the hydrogel network and
their degree of ionization at the particular pH. The high diffusivity of theophylline from
hydrogel J in neutral environment indicates that the network is highly hydrophilic in
nature, which, as described previously, could be attributed to high AA and NVP content.
Thus a higher swelling activity in hydrogel J led to higher theophylline diffusivity.
Hydrogels H and I on the other hand contained higher amount of HEMA in the
formulation with respect to hydrogel J. As previously described, HEMA is relatively less
hydrophilic than AA and NVP. Thus the hydrogel networks H and I were less favourable
to swelling in neutral environment, which subsequently retarded the release rate of
theophylline.
In pH 2 environment lower release rates were observed but in contrast to neutral
environment, hydrogel H displayed slightly higher release rate in comparison to
hydrogels I and J. This behaviour could be explained in terms of high pH sensitivity of
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Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
AA containing hydrogels J and I, which remain collapsed in acidic environment due to
nonionization of the carboxyl groups. Thus mesh size of the polymer is significantly
smaller, which restricts the diffusion of theophylline. In pH 8 environment, higher
theophylline diffusivity was observed. This could be explained in terms of increase in the
ionization of carboxyl groups, which lead to an increase in the mesh size thus allowing
high diffusivity of theophylline.
It has been well established in the literature that the diffusion of an entrapped solute from
a swollen polymer increases with the degree of swelling [13,19,27,34-36]. Bettini et al
[13] studied the release kinetics of theophylline from pH sensitive anionic HEMA-co-AA
hydrogels in isotonic environment and reported that the release rate of theophylline was
dependent on the swelling ratio of the hydrogels. Am Ende et al [19] also reported a
similar trend of theophylline release from HEMA-co-AA hydrogels. Shah et al [36]
studied theophylline release behaviour in pH sensitive HEMA-co-4-carboxy styrene
hydrogels and have reported similar observations. Theophylline is a non-ionizable drug
hence it does not interact with the anionic polymer chains. Thus as reported by other
researchers, the rate of solute release is typically dependent on the swelling ratio of the
hydrogels.
The high diffusivity of theophylline from hydrogels H-J in basic medium (Figures 37 and
38) is in agreement with other researchers. A slight increase in the diffusivity of
theophylline was observed in hydrogel I in comparison to hydrogel J despite a higher
EWC value was observed for hydrogel J in pH 8 environment. This observation could be
explained in terms of the extent of ionization achieved by the network. As previously
described, according to the Donnan equilibrium, upon full ionization of the polymer
chain, a further increase in the ionic strength would cause the anionic polymer network to
de-swell thus reducing its mesh size. Thus the slightly lower diffusivity of theophylline
from hydrogel J in comparison to hydrogel I could be attributed to the fact that hydrogel
J, which contained high amounts of AA reached maximum ionization in the presence of a
basic drug, theophylline (pKb = 13.5) within its structure in pH 8 environment. A slightly
lower diffusivity could be due to a reduction of the polymer mesh size as a result of the
shrinking of the network. However, this behaviour was not highly significant.
274
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
6.5. Conclusions
The DPC measurements revealed that the donor/acceptor pair of AA and NVP was
significantly more efficient than that of HEMA and NVP in CT complex formation. The
kinetics data of AA-NVP system indicate an optimum reactive ratio at 1:1 mol of AA:
NVP. HEMA and NVP on the hand were found to be an inefficient donor/acceptor pair,
which did not completely polymerise when exposed to the UV light in the differential
photocalorimeter.
Anionic hydrogels were successfully synthesized via the photoinitiator-free process.
Hydrogels were found to be reasonably resilient and competent drug delivery devices.
Hydrogels with high AA and NVP content were found to be highly water-swellable in
comparison to high HEMA content networks, which exhibited relatively low swelling
behaviour. The swelling experiments at varied pH environments revealed that the pH
sensitivity in the hydrogel networks was contributed by the presence of AA and HEMA.
However, the pH sensitivity was more pronounced in high AA containing networks.
The drug release experiments were also indicative of pH dependence of the hydrogel
networks in releasing theophylline. The release of theophylline from the anionic networks
increased with increase in the environmental pH, suggesting that the release rate was
controlled by the swelling ratio of the hydrogels. Increase in the mesh size of the anionic
hydrogels with increase in pH led to high amount of drug released. The study on these pH
sensitive photoinitiator-free anionic hydrogels indicate that these promising materials
would be ideal as drug delivery systems for pH sensitive bioapplications such as delivery
devices for the intestinal tract where varied pH environments exist.
275
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
6.6. References
1. Miyata, T., Uragami, T., “Polymeric Biomaterials”, Dumitriu, S, ed., 2nd
ed.,
Marcel & Decker, Inc., New York, pp.960-966, (2002).
2. Qiu, Y., Park, K., Adv. Drug Deliv. Rev., 53, 321-339, (2001).
3. Risbud, M. V, Hardikar, A. A., Bhat, S. V., Bhonde, R. R., J. Controlled Release,
68, 23-30, (2000).
4. Peppas, N. A., Curr. Opin. Colloid Interf. Sci., 2, 531-537, (1997).
5. Ostroha, J., Pong, M., Lowman, A., Dan, N., Biomaterials, 25, 4345-4353, (2004).
6. Kost, J., Langer, R., Adv. Drug. Deliv. Rev., 46, 125-148, (2001).
7. Needleman, I. G., Smales, F. C., Martin, G. P., J. Clin. Periodontol., 24, 394-400,
(1997).
8. Dong, L.-C., Hoffman, A. S., J. Controlled release, 15, 141-152, (1991.
9. Ravichandran, P., Shanta, K. L., Rao, K. P., Int. J. Pharm., 154, 89-94, (1997).
10. Park, K., Robinson, J R., Int. J. Pharm., 19, 107-127, (1984).
11. Devine, D. M., Higginbotham, C. L., Polymer, 44, 7851-7860, (2003).
12. Khare, A. R., Peppas, N. A., Biomaterials, 16, 559-567, (1995).
13. Bettini, R., Colombo, P., Peppas, N. A., J. Controlled Release, 37, 105-111,
(1995).
14. Khare, A. R., Peppas, N. A., Massimo, G., Colombo, P., J. Controlled Release,
22, 239-244, (1992).
15. Schwarte, L. M., Peppas, N. A., Polymer Prep., 38, 596-597, (1997).
16. Inoue, T., Chen, G., Nakamae, K., Hoffman, A. S., J. Controlled Release, 49, 167-
176, (1997).
17. Şen, M., Güven, O., Radiation Phys. Chem., 55, 113-120, (1999).
18. Rosso, F., Barbarisi, A., Barbarisi, M., Petillo, O., Margarucci, S., Calarco, A.,
Peluso, G., Mater. Sci., Eng., 23, 371-376, (2003).
19. Am Ende, M. T., Hariharan, D., Peppas, N. A., React. Polym., 25, 127-137,
(1995).
20. Ng, L-T., Arsenin, A., Nguyen, D., Proc. RadTech Asia’03, Yokohama, Japan,
669-672, (2003).
21. Alvarez-Lorenzo, C., Concheiro, A., J. Controlled Release, 80, 247-257, (2002).
276
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
22. Sutani, K., Kaetsu, I., Uchida, K., Matsubara, Y., Radiation Phys. Chem., 64, 331-
336, (2002).
23. Kusonwiriyawong, C., Van de Wetering, P., Hubbell, J. A., Merkle, H. P., Walter,
E., Eur. J. Pharm. Biopharm., 56, 237-246, (2003).
24. Am Ende, M. T., Peppas, N. A., J. Controlled Release, 48, 47-56, (1997).
25. Sahoo, S. K., De, T. K., Gosh, P. K., Maitra, A., J. Colloid Interf. Sci., 206, 361-
368, (1998).
26. Kaczmarek, H., Szalla, A., Kamińska, A., Polymer, 42, 6057-6069, (2001).
27. Vyavahare, N. R., Kulkarni, M. G., Mashelkar, R. A., J. Membrane Sci., 54, 221-
228, (1990).
28. Chapiro, A., Trung, L. D., Eur. Polym. J., 10, 1103-1106, (1974).
29. Garnett, J. L., Zilic, E., Proc. RadTech Europe’01, Basel, Switzerland, pp.233-
238, (2001).
30. Khodzhaev, S. G., Mushraipov, R., Polym. Sci. U.S.S.R., 32, 1254-1263, (1990).
31. Ferreira, L., Vidal, M. M., Gil, M. H., Int. J. Pharm., 194, 169-180, (2000).
32. Sveick, S., Vacik, J., Chmelikova, D., Smetana, Jr, K., J. Mater. Sci. Mater. Med.,
4, 505-509, (1995).
33. Brannon-Peppas, L., Peppas, N. A., J. Controlled Release, 16, 319-330, (1991).
34. Yasuda, H., Ikenberry, L. D., Lamaze, C. E., Makromol. Chem., 125, 108-118,
(1969).
35. Wood, J. M., Attwood, D., Collet, J. H., J. Pharm. Pharmacol., 34, 1-4, (1982).
36. Shah, S. S., Kulkarni, M. G., Mashelkar, R. A., J. Controlled Release, 15, 121-
132, (1991).
277
Chapter 6: pH Sensitive AA-NVP-HEMA Hydrogels Synthesized via Photoinitiator –Free UV Curing Technique
6.1. Introduction 220
6.2. Experimental Procedure 222
6.3. Results 223
6.3.1. DPC Measurements on AA/NVP and HEMA/NVP Systems 223
6.3.2. Photopolymerisation of Hydrogels Containing NVP, AA and HEMA 225
6.3.3. Experimental Swelling Results 226
6.3.4. Experimental Drug Release Results 255
6.4. Discussion 264
6.4.1. DPC Measurements 264
6.4.2. Hydrogel Formation 265
6.4.3. Swelling and Drug Release Evaluation 266
6.4.3.1. Swelling Behaviour of AA-NVP-HEMA Hydrogels 267
6.4.3.1.1. pH Dependent Swelling Behaviour 267
6.4.3.2. Drug Release Studies 272
6.5. Conclusions 275
6.6. References 276
278
Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells
7.1. Introduction
Polymeric hydrogels as sustained drug delivery devices have been discussed in detail in
Sections 1-6. However, hydrogels must be biocompatible in order to be considered for
biomedical applications. Biocompatibility is the ability of a material to perform with an
appropriate host response in a specific application without toxic, inflammatory,
carcinogenic and immunogenic responses [1-7]. An appropriate response of the
biomaterial for its particular application would be referred to as an inert or positive
interaction with the host [8]. The biocompatibility of a biomaterial is directly related to its
chemical and biochemical characteristics [1,6].
Cytotoxicity of a biomaterial can be evaluated in vitro by incubating the biomaterial
samples for prolonged periods in the direct presence of suitable host environmental cells
or indirectly through biomaterial leachates [6,7,9-14]. However, for eventual regulatory
approval of a biomaterial, in vitro and in vivo tests are necessary [15]. In vitro testing is
generally less costly, and a non-invasive means of primary cytotoxicity testing, preferably
used by researchers to avoid extensive testing on animals [9,15].
In vitro investigations into possible cytotoxicity of biomedical devices, their component
materials and leachates evaluate lysis, growth inhibition and other impacts on cell
viability using morphological, biochemical and metabolic criteria. [7]. Determination of
cell viability and proliferation are common assays in cytocompatibility testing of
biomaterials in vitro. The ability of the cells to survive and proliferate is used as the
measure of functional status of the cells [10,16].
A tetrazolium-based colorimetric assay is often used for quantitative measurements of
mammalian cell survival and proliferation using the tetrazolium salt, 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) [7]. The assay developed
by Mosmann [17] is based on the reduction of a yellow tetrazolium salt, MTT, by the
mitochondrial dehydrogenase in living cells to insoluble purple formazan crystals. The
MTT assay has been widely accepted as a better alternative to the previously used
radioactive assays, which made use of hazardous radioactive isotopes [7,18-20]. The
purple formazan crystals upon dissolution in an appropriate solvent can be measured
278
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
spectrophotometrically [20]. The amount of the formazan generated is directly
proportional to the live cell numbers over a wide range, using a homogeneous population
[18,21].
In the present study, the hydrogel networks described in Sections 3-6 were evaluated for
their cytocompatibility in vitro using an indirect contact methodology. A complete
conversion of monomers to polymers may not be achieved in the polymerisation process,
thus there is always a certain component of unreacted monomers, which may be toxic,
still present in the polymer matrix. These monomers have the tendency to leach out of the
polymer matrices when the polymer is in contact with an aqueous environment causing
discomfort to the host environment. However, through thorough cleaning of the finished
polymer, the unreacted toxic components present in the polymer matrix could be
eliminated [7,11-13].
In the study, hydrogel leachates were introduced to human keratinocyte (HaCaT) cells
and incubated for 48 hours. HaCaT cells are epidermal cells, which closely resemble
normal keratinocytes [22]. Furthermore, HaCaT cells are readily available, highly
sensitive and easily regenerated making them an ideal candidate as the human cell model
for in vitro studies. The effect of the hydrogel leachates on the HaCaT cells after 48 hours
of incubation was evaluated using the MTT cell proliferation assay.
7.2. Experimental Procedure
Sterile hydrogel pieces (see Section 2.9.2) were immersed in milli-Q-water, which served
as the sample media, at 37 oC for a period of 14 days. These sample media were subjected
to in vitro cytotoxicity experimentation on HaCaT cells using the MTT cell proliferation
assay. The monomeric components in the hydrogels were also tested individually in their
free form. N-vinyl-2-pyrrolidinone (NVP), acrylic acid (AA), 2-hydroxethyl methacrylate
(HEMA), N-hydroxymethyl maleimide (HMMI), 2-hydroxy-N-propyl maleimide
(HPrMI) and 5-hydroxy-N-pentyl maleimide (HPMI) were the monomers tested. Detailed
experimental specifications and procedure of the cytotoxicity experiments have been
described in Section 2.9. A microplate reader was used to carry out the absorbance
measurements. The MTT assay data obtained for the hydrogels were compared with the
279
Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells
monomers and also with the untreated experimental control. The MTT assay data were
expressed as means + SEM of the replicate percentage cell numbers with respect to the
experimental control. A one-way analysis of variance (ANOVA) was performed using
MINITAB 7.2 statistical software. A p value of < 0.05 was regarded as statistically
significant.
7.3. Results
7.3.1. HaCaT Cell Proliferation in the Presence of Monomers
The monomers utilized to synthesize the hydrogels were tested for their effect on HaCaT
cell growth using the MTT cell proliferation assay. The HaCaT cells were treated with the
N-hydroxyalkyl maleimides, AA, NVP or HEMA at varying concentrations for 24 and 48
hours. However, results for the 24 hr treatment did not indicate significant growth or
inhibitory activity, thus suggesting that the treatment time was not sufficient for the cells
to grow and show any inhibitory effect on growth imposed by the samples. The results
after 48 hr treatment on the other hand clearly showed the effect of the samples on HaCaT
cell growth and the treatment time was considered adequate as the untreated experimental
control showed that the cells were fully confluent.
The MTT assay data revealed that HPrMI and HPMI did not have any adverse effects on
the HaCaT cells. The assay data indicated that the cells had proliferated in the presence of
HPrMI or HPMI. HMMI on the other hand, had a negative effect on cell growth. All the
cells died when treated with HMMI at 250 ppm and 500 ppm. A very small percentage (~
15 %) of cells were viable in the presence of HMMI at 125 ppm. Microscopic
examinations on the cells cultures treated with 500 ppm of HMMI, HPrMI or HPMI
revealed that cells in HPMI and HPrMI environment had become confluent and were still
attached (Figure 2 C & D). The cells treated with HMMI for 48 hours, died and had
detached from the surface of the culture dish (Figure 2 B).
The MTT assay data on HaCaT cell growth observed in the presence of the N-
hydroxyalkyl maleimides after 48 hours of treatment is presented in Table 1. The cell
numbers observed in the treated cultures are expressed as a percentage of the cell density
280
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
measured in the untreated control culture. A graphical representation of the percentage
number of HaCaT cells after the treatment is illustrated in Figure 1.
Table 1. Percentage HaCaT cell numbers after treatment with the N-hydroxyalkyl
maleimides at varying concentrations for 48 hours
N-hydroxyalkyl
maleimides
Concentration
(ppm) % Live cell numbers + SEM
125 14.76 + 7.42
250 5.35 + 5.35 HMMI
500 5.05 + 5.05
125 124.47 + 7.48
250 121.54 + 6.26 HPrMI
500 116.08 + 6.32
125 106.61 + 6.77
250 114.66 + 6.84 HPMI
500 102.06 + 9.21
0
50
100
150
200
125 250 500Monomer Concentration (ppm)
% L
ive
Cel
l N
um
ber
s
HMMI HPrMI HPMI
******* ***
*** ******
****
Figure 1. Percentage number (density) of HaCaT cells after 48 hr treatment with HMMI,
HPrMI or HPMI at varying concentrations. Significant cell growth in comparison to
untreated control is represented by ∗ where p < 0.05. Highly significant growth compared
to the control is represented by ∗ ∗ ∗ where p < 0.001.
281
Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells
Untreated cells
(A)
Cells treated with HMMI (500 ppm)
(B)
Cells treated with HPrMI (500 ppm)
(C)
Cells treated with HPMI (500 ppm)
(D)
Figure 2. HaCaT cells cultured for 48 hours in the presence of HMMI (B) (500 ppm),
HPrMI (C) (500 ppm) or HPMI (D) (500 ppm). Micrograph A is a representation of the
untreated control. The cell cultures were observed at a magnification of 200 x. The
micrographs illustrate live, attached cells in the HPrMI and HPMI environments. Where
as the HMMI environment caused the cells to round up and die, and detach from the
culture dish surface.
282
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
The MTT assay data revealed that the monomers AA and HEMA did not have adverse
effect on the HaCaT cells at 250 ppm. However, at 500 and 1000 ppm, a slight growth
inhibitory effect imposed by the presence of HEMA and AA was observed. NVP on the
other hand did not have any adverse effect on the cell growth. The MTT assay data
indicated that cells proliferated in the presence of NVP at 250 ppm, 500 ppm and 1000
ppm. The MTT assay data on AA, HEMA and NVP are presented in Table 2 and
illustrated in Figure 3. Microscopic examinations were carried out on the cell cultures
treated with 1000 ppm of HEMA, AA or NVP for 48 hours. The cells were found to be to
be fully confluent and attached to the culture dish surface in NVP environment after 48
hours of treatment. HaCaT cell cultures in HEMA and AA showed slow cell growth. The
number of attached cells in AA and HEMA environment after 48 hours of treatment was
relatively lower in comparison to that in the NVP environment. The micrographs of the
cell cultures in AA, HEMA or NVP environment are illustrated in Figure 4 (A-D).
Table 2. Percentage HaCaT cell numbers after treatment with HEMA, NVP and AA
at varying concentrations for 48 hours
Monomers Concentration
(ppm) % Live cell numbers + SEM
250 105.00 + 7.86
500 84.07 + 6.77 HEMA
1000 64.17 + 4.41
250 98.71 + 9.13
500 101.17 + 8.52 NVP
1000 105.25 + 7.91
250 97.82 + 8.99
500 91.30 + 7.70 AA
1000 63.97 + 7.29
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Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells
0
50
100
150
200
250 500 1000Monomer Concentration (ppm)
% L
ive
Cel
l N
um
ber
s
HEMA NVP AA
*** ***
*
Figure 3. Percentage number of HaCaT cells after 48 hr treatment with HEMA, NVP or
AA at varying concentrations. Significant cell growth in comparison to untreated control
is represented by where p < 0.05 while a highly significant growth compared to the
control is represented by ∗ ∗ ∗ where p < 0.001. Absence of ∗ indicates insignificant
cellular proliferation in comparison to the untreated control where p > 0.05.
∗
284
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Untreated cells
(A)
Cells treated with AA (1000 ppm)
(B)
Cells treated with NVP (1000 ppm)
(D)
Cells treated with HEMA (1000 ppm)
(C)
Figure 4. HaCaT cells cultured for 48 hours in the presence of AA (B) (1000 ppm),
HEMA (C) (1000 ppm) or NVP (D) (1000 ppm). The untreated experimental control is
illustrated in micrograph A. The cell cultures were observed at a magnification of 200 x.
The cells were attached to the culture dish and had become confluent in the presence of
NVP environment after 48 hours of treatment. The presence of HEMA and AA seemed to
have inhibited cell growth to some extent as the HaCaT cells were not fully confluent
after 48 hours of treatment.
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7.3.2. HaCaT Cell Proliferation in the Presence of Hydrogels
The polymeric hydrogels described in Sections 3-5 were tested for cytotoxicity on HaCaT
cells. The cells were treated with the hydrogel leachates for 48 hours at 37 oC. The effect
of the hydrogel leachates on the HaCaT cell growth was observed and compared with the
effects imposed by the monomeric components of the hydrogels on the cells. The results
of the MTT assay on the effect of hydrogel leachates samples on the cells are described in
Sections 7.3.2.1-7.3.2.4. The cell numbers observed in the treated cultures are expressed
as a percentage of the cell density measured in the untreated control cell culture.
7.3.2.1. Effect of N-Hydroxyalkyl Maleimide-NVP Hydrogels
The MTT assay data on the HaCaT cells treated with HMMI-NVP, HPrMI-NVP, HPMI-
NVP, HPMI-NVP-HEMA or HPMI-NVP-NVC (Table 3 and Figure 5) revealed that the
hydrogel leachates did not contain cytotoxins. The assay indicated that the cells had
proliferated after 48 hours of treatment. A significant (p < 0.05) increase in cell growth
was observed in the presence of the N-hydroxyalkyl maleimide-NVP hydrogel leachates.
The percentage cell numbers observed after the 48 hr treatment are tabulated in Tables 3
and illustrated graphically in Figure 5. Microscopic examinations revealed that the cells
were fully confluent and were still attached to the culture dish after treatment. A
micrograph representing the status of the HaCaT cells after 48 hours of treatment with
hydrogels based on N-hydroxyalkyl maleimides is illustrated in Figure 6.
Table 3. Percentage HaCaT cell numbers after 48 hr treatment with HMMI-NVP,
HPrMI-NVP, HPMI-NVP, HPMI-NVP-HEMA and HPMI-NVP-NVC hydrogels
Hydrogel samples % Live cell numbers + SEM
Gel A HMMI-NVP 141.59 + 13.57
Gel B HPrMI-NVP 165.02 + 16.80
Gel C HPMI-NVP 170.27 + 15.43
Gel D HPMI-NVP-HEMA 143.36 + 9.05
Gel E HPMI-NVP-NVC 151.48 + 24.44
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0
50
100
150
200
Gel A Gel B Gel C Gel D Gel E
Hydrogel Samples
% L
ive
Cel
l N
um
ber
s
***
*** ***
******
Figure 5. Percentage number of HaCaT cells after 48 hr treatment with leachates of
HMMI-NVP (Gel A), HPrMI-NVP (Gel B), HPMI-NVP (Gel C), HPMI-NVP-HEMA
(Gel D) or HPMI-NVP-NVC (Gel E). The ∗ ∗ ∗ represent highly significant cell growth
in comparison to the untreated control where p < 0.001.
Figure 6. HaCaT cells treated with the N-hydroxyalkyl maleimides–NVP hydrogel
sample leachates after 48 hours of treatment. The cells were mostly attached to the culture
dish and were confluent. The micrograph (magnification 200 x) is a representation of the
cell cultures observed in all the hydrogel samples in this category.
7.3.2.2. Effect of IPN Hydrogels
The MTT assay data (Table 4 and Figure 7) revealed that the IPN hydrogel leachates did
not have any adverse effects on the HaCaT cell growth. The cells were observed to
proliferate after 48 hours of treatment with the IPN hydrogel leachates, thus indicating
that the leachates were free of any cytotoxins. Furthermore, the leaches appeared to
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significantly (p < 0.05) stimulate cell growth. Microscopic examinations also indicated
that the cells were fully confluent and were still attached after 48 hours of treatment with
the leachates. A micrograph representing the HaCaT cell culture treated with the IPN
hydrogel leachate is illustrated in Figure 8.
Table 4. Percentage HaCaT cell numbers after 48 hr treatment with IPN leachates
IPN hydrogel samples % Live cell numbers + SEM
Gel A CM chitosan-NVP-HMMI 154.37 + 27.72
Gel B HEMA-NVP-chitosan 168.24 + 26.56
Gel C Chitosan-NVP-HMMI 136.52 + 27.92
0
50
100
150
200
Gel A Gel B Gel C
IPN Hydrogel Samples
% L
ive
Cel
l N
um
ber
s
******
***
Figure 7. Percentage number of HaCaT cells after 48 hr treatment with leachates of CM
chitosan-NVP (Gel A), chitosan-HEMA-NVP (Gel B) or chitosan-NVP (gel C) IPN
hydrogel. The ∗ represent highly significant cell growth in comparison to the
untreated control where p < 0.001.
∗ ∗
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
Figure 8. HaCaT cells treated with the IPN hydrogel sample leachates for 48 hours.
HaCaT cells were observed to have become confluent with the majority of the cells
attached to the culture plate. The micrograph (magnification 200 x) is a representation of
the cell cultures treated with all the hydrogel samples in this category.
7.3.2.3. Effect of HEMA-NVP-AA Anionic Hydrogels
The MTT assay data (Tables 3-5 and Figures 9-11) indicated that the HEMA-NVP-AA
ionic hydrogels did not have any significant adverse effect on the HaCaT cell viability.
However, the percentage cell numbers indicated by the assay were relatively lower than
that observed for the cells treated with N-hydroxyalkyl maleimide-NVP and IPN
hydrogel leachates. The treated cultures observed under microscope revealed that the cells
were attached to the culture dish thus indicating that the cells were alive after the 48 hr
treatment. A micrograph illustrating the status of the cell culture treated with the HEMA-
AA-NVP anionic hydrogels is illustrated in Figure12.
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Table 5. Percentage HaCaT cell numbers after 48 hr treatment with HEMA-NVP-
AA anionic hydrogel leachates
Hydrogel samples
HEMA:NVP:AA % Live cell numbers + SEM
Gel A 50:00:50 122.70 + 13.64
Gel B 50:40:10 111.45 + 7.60
Gel C 50:25:25 124.55 + 15.45
Gel D 50:10:40 111.68 + 11.76
Gel E 10:40:50 98.06 + 9.03
Gel F 25:25:50 113.54 + 9.13
Gel G 40:10:50 98.89 + 15.43
Gel H 10:50:40 94.96 + 14.93
Gel I 25:50:25 113.93 + 11.73
Gel J 40:50:10 124.29 + 15.15
0
50
100
150
200
Gel A Gel B Gel C Gel D
Hydrogel Samples
% L
ive
Cel
l N
um
ber
s
*** **** *
Figure 9. Percentage number of HaCaT cells after 48 hr treatment with leachates of 50
HEMA-50 AA (Gel A), 50 HEMA-40 NVP-10 AA (Gel B), 50 HEMA-25 NVP-25 AA
(Gel C) or 50 HEMA-10 NVP-40 AA (Gel D). Significant cell growth in comparison to
the untreated control is represented by where p < 0.05 while a highly significant growth
compared to the control is represented by where p < 0.001.
∗∗ ∗ ∗
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
50
100
150
200
Gel E Gel F Gel G
Hydrogel Samples
Per
cen
tag
e C
ell
Nu
mb
ers
*
Figure 10. Percentage number of HaCaT cells after 48 hr treatment with leachates of 50
AA- 40 NVP-10 HEMA (Gel E), 50 AA-25 NVP-25 HEMA (Gel F) or 50 AA-10 NVP-
40 HEMA (Gel G). The ∗ represents significant cell growth in comparison to the
untreated control where p < 0.001. The absence of ∗ indicates insignificant cell growth in
comparison to the untreated control where p > 0.05.
0
50
100
150
200
Gel H Gel I Gel J
Hydrogel Samples
Per
cen
tag
e C
ell
Nu
mb
ers
***
Figure 11. Percentage number of HaCaT cells after 48 hr treatment with leachates of 50
NVP- 40 AA-10 HEMA (Gel H), 50 NVP-25 AA-25 HEMA (Gel I) or 50 NVP-10 AA-
40 HEMA (Gel J). The ∗ ∗ ∗ represent highly significant cell growth in comparison to
the untreated control where p < 0.001. Insignificant cell growth in comparison to the
untreated control is indicated by the absence of ∗ where p > 0.05.
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Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells
Figure 12. HaCaT cells treated with the AA-NVP-HEMA hydrogel sample leachates for
48 hours. HaCaT cells were attached to the culture dish indicating cellular viability. The
micrograph (magnification 200 x) is a representation of the cell cultures observed in all
the hydrogel samples in this category.
7.3.2.4. Effect of HEMA-NVP Hydrogels
The MTT assay data (Table 6 and Figure 13) revealed that the hydrogel leachates from
HEMA-NVP hydrogels were free of any cytotoxins. The assay data indicated that the
HaCaT cells were viable after 48 hours of treatment. Furthermore, a significant (p < 0.05)
cellular proliferation was observed. Microscopic examination revealed that the cells were
fully confluent and attached to the culture plate indicating cellular viability (Figure 14).
Table 6. Percentage HaCaT cell numbers after 48 hr treatment with HEMA-NVP
hydrogel leachates
Hydrogel samples % Live cell numbers + SEM
Gel A PHEMA 150.50 + 42.76
Gel B 80 HEMA-20 NVP 148.76 + 25.61
Gel C 50 HEMA-50 NVP 142.41 + 15.33
Gel D 20 HEMA-80 NVP 116.37 + 5.79
Gel E 75 HEMA-15NVP-10H2O 111.17 + 19.59
Gel F 15 HEMA-75NVP-10H2O 156.57 + 40.90
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
0
50
100
150
200
Gel A Gel B Gel C Gel D Gel E Gel F
Hydrogel Samples
% L
ive
Cel
l N
um
ber
s
******
******
******
Figure 13. Percentage number of HaCaT cells after 48 hr treatment with the leachates of
PHEMA (Gel A), 80 HEMA-20 NVP (Gel B), 50 HEMA-50 NVP (Gel C), 20 HEMA-80
NVP (Gel D), 75 HEMA-15NVP-10H2O (Gel E) or 15 HEMA-75 NVP-10H2O (Gel F).
The represent highly significant cell growth in comparison to the untreated control
where p < 0.001.
∗ ∗ ∗
Figure 14. HaCaT cells treated with the poly(HEMA-co-NVP) hydrogel leachates for 48
hours. The cells were attached to the culture dish indicating cellular viability. The
micrograph (magnification 200 x) is a representation of the cell cultures observed in all
the hydrogel samples in this category.
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7.4. Discussion
7.4.1. Effect of the Monomers on HaCaT Cells
The monomers HMMI, HPrMI, HPMI, NVP, HEMA and NVP were utilized to make
hydrogels in various combinations. HEMA, NVP and AA are widely used monomers for
polymeric hydrogel synthesis. Numerous researchers have made use of HEMA and NVP
in varying compositions for specific biomedical applications [2,12,13,23-29]. AA has
been commonly used to form pH sensitive hydrogels for stimuli response drug delivery
applications [2,30-32]. The N-hydroxyalkyl maleimides, HMMI, HPrMI and HPMI have
been used as suitable acceptor monomers in charge-transfer complex polymerisation [33].
However, the use of photoinitiator-free polymers composed of N-hydroxyalkyl
maleimides as bioapplicable hydrogels has been recently reported by the author [34,35].
In vitro cytotoxicity investigations using the MTT assay revealed that these monomers
bring about a characteristic response in the HaCaT cells. The monomers were therefore
classified in accordance to the level of cytotoxicity exhibited towards the HaCaT cells.
The N-hydroxyalkyl maleimides were found to be fairly inert with the exception of
HMMI. The HaCaT cells were exposed to the maleimides at varying concentrations (125
ppm, 250 ppm and 500 ppm). The MTT assay data revealed that HPrMI and HPMI did
not have a negative impact on cellular viability and proliferation after 48 hours of
exposure at both, high and low concentrations (Table 1, Figure 1). Microscopic
examinations revealed that the cells were attached to the culture dish and the cell cultures
had become confluent indicating cellular growth (Figure 2 (C & D)).
HMMI on the other hand had adverse effects on the HaCaT cells. The cells treated with
HMMI at 125 ppm showed low survival rate (~15%) (Table 1, Figure 1). At higher
concentrations all the cells died. Microscopic observation indicated that all the cells had
detached or had died after 48 hours of exposure thus suggesting that HMMI is toxic to
HaCaT cells (Figure 2 (B)).
The relative toxicity of HMMI in comparison to HPrMI and HPMI could be attributed to
the structural features of these N-hydroxyalkyl maleimides. HMMI, owing to it low
molecular weight and enhanced solubility due to the presence of hydroxyl (-OH) group,
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
will easily diffuse across the cell membrane in comparison to HPrMI and HPMI. Cooney
et al [36] from their studies on biochemical and toxicological properties on N-substituted
maleimides suggested that the addition of alkyl substitutents diminishes the cytotoxicity
of maleimides. The cytotoxicity results of the N-hydroxyalkyl maleimides in this study
are in agreement with Cooney et al. HPMI and HPrMI being larger molecules with longer
N-alkyl substitutents did not have apparent toxic effect at a high concentration of 500
ppm.
NVP did not have an adverse effect on cell viability. The cells were viable as indicated by
the MTT assay data after 48 hours of treatment with NVP at 250 ppm, 500 ppm and 1000
ppm (Table 2, Figure 3). Microscopic examination on the HaCaT cells cultured in NVP
(1000 ppm) revealed that the cells were attached to the culture dish (Figure 4 (H)).
Furthermore, the cultures were almost confluent after 48 hours of treatment, indicating
cellular growth and the inertness of the monomer. NVP is an extremely hydrophilic
neutral monomer. The absence of charged groups on its structure makes it relatively inert.
Van de Wetering et al [37,38] observed a marked reduction in the cytotoxicity of
polymers with increasing NVP content. They described this phenomenon as a result of the
reduction in the charge density on the polymer due to increasing content of a non-charged
monomer, NVP.
HEMA and AA did not show cytotoxic effect on the cell cultures at 250 ppm after the 48
hr treatment (Table 2, Figure 3). The cells appeared viable and appeared to have
proliferated in this environment. However, when the concentration of AA and HEMA
was increased to 500 ppm and 1000 ppm, a toxic effect was observed. A slight growth
inhibitory effect was observed in the 500 ppm environment followed by a pronounced
effect in the 1000 ppm environment. Microscopic examinations revealed that the number
of viable cells had increased with low concentration of AA and HEMA. The cells
remained attached to the culture dish after 48 hours of treatment. However, at 1000 ppm,
the live cell numbers were reduced as indicated by a lower number of attached cells
(Figure 4 (B & C)).
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The study suggests that HEMA and AA at high concentrations inhibit cell proliferation
and have a toxic effect. The relative cytotoxicity of HEMA and AA in comparison to
NVP at high concentration could be attributed to their slightly acidic nature in
comparison to NVP. HEMA and AA are both of low molecular weight. Furthermore, they
are water-soluble owing to the -OH group on their structure, which would dissociate in
the culture medium. The dissociation of the -OH group will alter the pH of the immediate
cellular environment. As suggested by the experimental data, at low concentrations of the
monomer, change in the pH of the environment was negligible thus the effect on the
cellular growth was not pronounced. However, at high concentration when the pH change
was significant, the cell growth was inhibited in the acidic environment, thus a lower
number of attached cells were observed (Figure10).
Yoshii [39] studied the cytotoxic effect of a range of acrylates and methacrylates and
found the dependence of the cytotoxicity of the monomers on their chemical structures.
Yoshii observed enhanced cytotoxicity in acrylates in comparison to methacrylates.
Furthermore, a more pronounced cytotoxicity was observed in the monomers containing
OH groups. The enhanced cytotoxicity of acrylates in comparison to the methacrylates
could be attributed to the presence of the methyl group on the methacrylates, which
stabilize the -OH bonds thus reducing the degree of dissociation. Bouillaguet et al [40,41]
studied the cytotoxic effect of HEMA, and suggested that HEMA is cytotoxic at high
concentrations.
The results obtained on the cytotoxicity of AA and HEMA in the present work are in
agreement with other researchers. However, as suggested by Yoshii, AA being an
acrylate should be more cytotoxic due to a higher dissociation. In the present study, a
marked cytotoxic effect of AA in comparison to HEMA was not observed. This could be
due to the fact the monomer concentrations used for this work were not sufficiently high
for AA to show a more pronounced effect.
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7.4.2. Cytocompatibility of the Polymeric Hydrogels
7.4.2.1. Hydrogels Based on N-Hydroxyalkyl Maleimides and NVP
The data from the MTT assay (Table 3, Figure 5) revealed that the N-hydroxyalkyl
maleimide-NVP hydrogels synthesized via a photoinitiator-free technique were free of
any toxins. The leachates from the hydrogels had a significant positive effect on HaCaT
cell growth. Cells were observed to proliferate at a higher growth rate than the untreated
cell cultures. Microscopic observation showed the cells were still attached to the culture
dish and the cultures were almost confluent after 48 hours of exposure thus indicating that
the hydrogels did not have any adverse effect on HaCaT cell growth (Figure 6).
Despite a significant cytotoxicity effect of HMMI in its free form on HaCaT cells, the
HMMI-NVP hydrogels not only sustained the HaCaT cells but also stimulated positive
growth. The concentration of HMMI in the hydrogel was approximately 1000 ppm,
determined from the formulation described in Section 2.5.2 and the sample preparation
for cytotoxicity (Section 2.9.2). The study suggests that the traces of the unreacted
monomers that may have been present in the gel matrix were successfully washed off as
no apparent cytotoxic effect was observed. Furthermore, the inertness of the polymeric
hydrogels to the cells also suggests that the networks are very stable under the
experimental conditions similar to the physiological environment of pH 7.4 and a
temperature of 37 oC. Thus the hydrogels based on the N-hydroxyalkyl maleimides and
NVP could be successfully applied in these specified conditions without any adverse
effects. Hydrogels for sustained drug delivery applications based on N-hydroxyalkyl
maleimides and NVP have been discussed in Section 4.
7.4.2.2. IPN Hydrogels Based on Polysaccharides and NVP
The IPN hydrogels discussed in Section 5 also revealed cytocompatibility with the
HaCaT cells. The MTT assay data (Table 4, Figure 7) showed significant cell
proliferation activity (> 100 %) in the presence of the IPN hydrogel leachates. The IPNs
were synthesized via the photoinitiator-free technique using HMMI in the presence of
NVP and two polysaccharides, chitosan and CM chitosan. As observed previously in the
case of the HMMI-NVP hydrogels, despite apparent cytotoxicity of HMMI, the IPNs
sustained and significantly stimulated HaCaT cell growth.
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Microscopic investigation revealed that the cells were still attached to the culture dish and
the cultures were almost confluent after 48 hours of exposure thus indicating that the
hydrogels did not have any adverse effect on HaCaT cell growth (Figure 8). The
enhanced cell proliferation (> 100 %) in the presence IPNs could be attributed to the
presence of the polysaccharides in the structure.
Domard and Domard [42] have described polysaccharides to be highly cytocompatible
towards a number of cell types including keratinocytes. Chitosan, although considered
non-toxic, has been suggested to be a strong elicitor of biological activity. They have
further suggested enhanced cell proliferation of keratinocytes in the presence of chitosan
and its derivatives. Risbud et al [11] from their studies on indirect in vitro cytotoxicity
evaluation of chitosan and poly(N-vinyl-2-pyrrolidinone) IPNs on epithelial cells have
reported similar results of enhanced cellular proliferation. The results on the
cytocompatibility of the IPNs based on chitosan and its water-soluble derivative are in
agreement with other researchers. Furthermore, CM chitosan has been reported as an
extremely useful derivative of chitosan owing to its solubility in water and high
biocompatibility [43,44]. Thus the enhanced growth effect by the CM-chitosan based
IPN, as that of chitosan based IPNs could be explained in terms of its biocompatibility.
7.4.2.3. Hydrogels Composed of AA, NVP and HEMA
AA-NVP-HEMA hydrogels discussed in detail in Section 6 are polyanionic in nature.
Results of the MTT assay using these gels indicated that the AA-NVP-HEMA hydrogel
leachates did not have an adverse effect on the HaCaT cells (Table 5, Figures 9-11).
However, the cell proliferation in some of these gels was considerably lower than the
previously discussed samples in Sections 7.4.2.1 and 7.4.2.2. Thus it could be suggested
that these hydrogel networks do not significantly stimulate HaCaT cell growth. Cell
numbers expressed as a percentage of the control were between ~ 90-120% for most of
these hydrogels.
Microscopic investigations revealed that the cells were attached to the culture dish after
48 hours of treatment indicating that the cells were still viable (Figure 12). The hydrogels
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Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
containing large amounts of AA were found to display slightly more growth inhibitory
behaviour than those systems containing lesser amounts of AA.
As previously described, AA is acidic in nature with a pKa value of 4.25, which readily
dissociates in the culture medium altering the pH of the surrounding environment. The
hydrogels were thoroughly washed prior to testing which eliminates the presence of any
unreacted monomers such as free AA. It could be suggested that the relative growth
inhibitory effect of the hydrogels with high AA content on the HaCaT cell growth is an
intrinsic property of the hydrogel and was not caused by the free monomers. Furthermore,
the data from the MTT assay and microscopic observations also revealed that the
hydrogels tested did not have an adverse effect on the cells. The cells were still alive after
48 hours of exposure to the hydrogel leachates. Therefore the hydrogels can be
considered to be cytocompatible.
Recently Foss and Peppas [45] studied cytotoxic effects of AA based copolymers
containing varying ratios of AA and observed a reduction in the cellular growth with
increase in AA content of the copolymer. They suggested that an increase in AA made the
copolymer increasingly ionizable. Thus increasing ionization of the AA units would
reduce the pH of the immediate environment making it acidic. As a result, the cellular
growth will be reduced in the acidic environment. Hydrogels responsive to pH variations
such as anionic networks based on AA are useful in stimuli responsive release
applications.
7.4.2.4. Poly(HEMA-co-NVP) Hydrogels
Investigations on the cytocompatibility of the hydrogel networks based on HEMA and
NVP revealed that the systems were cytocompatible with the HaCaT cells. Furthermore,
the hydrogels seemed to stimulate cell proliferation. Leachates from the hydrogels were
observed to significantly enhance HaCaT cell growth (Table 6, Figure 13). Cells were
observed to proliferate (>100%) after 48 hours of treatment. Microscopic observations
revealed that the cells were still attached to the culture dish and the cultures were almost
confluent after 48 hours of exposure indicating that the hydrogels did not have any
adverse effect on HaCaT cell proliferation and viability (Figure 14).
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The biocompatibility of HEMA and NVP based hydrogels has been well established in
recent years [2,12,13,23-29,46-50]. The cytotoxicity evaluation of HEMA-NVP
hydrogels in the present study confirms the biocompatibility of these gels as reported in
the literature. HEMA despite showing some cytotoxicity in its free monomeric form at
high concentration does not show any adverse effect on the HaCaT cells, once
polymerised. Polymeric hydrogel networks based on HEMA and NVP for sustained drug
delivery applications have been discussed in detail in Section 3.
7.5. Conclusions
The monomers were found to have no effect on HaCaT cell growth with the exception of
HEMA, AA and HMMI, which inhibited cell growth at high concentrations with HMMI
being the most toxic monomer. The relative cytotoxicity of the monomers has been
suggested to be dependent on the chemical structure of the monomers. However,
structure-cytotoxicity relationships were not clearly established in this work.
HaCaT cells were observed to be viable and to proliferate in the presence of all the
hydrogel samples tested. The positive cell growth is indicative of the fact that the cells
were not affected by the presence of the hydrogel leachates suggesting that the hydrogels
were free of cytotoxins. This study provides in vitro evidence of the biocompatibility of
the polymeric hydrogels synthesized via UV radiation. These hydrogels may therefore be
suitable for biomedical applications.
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303
Chapter 7: Biocompatibility of Hydrogels – In Vitro Cytotoxicity Investigations on Mammalian (HaCaT) Cells
7.1. Introduction 278
7.2. Experimental Procedure 279
7.3. Results 280
7.3.1. HaCaT Cell Proliferation in the Presence of Monomers 280
7.3.2. HaCaT Cell Proliferation in the Presence of Hydrogels 286
7.3.2.1. Effect of N-Hydroxyalkyl Maleimide-NVP Hydrogels 286
7.3.2.2. Effect of IPN Hydrogels 287
7.3.2.3. Effect of HEMA-NVP-AA Anionic Hydrogels 289
7.3.2.4. Effect of HEMA-NVP Hydrogels 292
7.4. Discussion 294
7.4.1. Effect of the Monomers on HaCaT Cells 294
7.4.2. Cytocompatibility of the Polymeric Hydrogels 297
7.4.2.1. Hydrogels Based on N-Hydroxyalkyl Maleimides and NVP 297
7.4.2.2. IPN Hydrogels Based on Polysaccharides and NVP 297
7.4.2.3. Hydrogels Composed of AA, NVP and HEMA 298
7.4.2.4. Poly(HEMA-co-NVP) Hydrogels 299
7.5. Conclusions 300
7.6. References 301
304
Chapter 8: Research Outcomes and Future Recommendations
8.1. Research Outcomes
The research work carried out on polymeric hydrogels resulted in a number of versatile
materials with potential applications on sustained drug delivery. A range of experiments
including swelling, drug release and cytotoxicity tests were conducted on these materials
to evaluate their potentials as controlled drug delivery biomedical devices.
A novel photoinitiator-free curing method was proposed to synthesize hydrogels using
monomers, which functioned as donor/acceptor pairs. Kinetics studies were conducted on
these donor/acceptor monomers to evaluate their suitability as donor/acceptor pairs. A
range of N-hydroxyalkyl maleimides and acrylic acid were found to be competent
acceptor monomers in combination with N-vinyl-2-pyrrolidinone (NVP), which served as
an excellent donor monomer. Hydrogels based on these donor/acceptor pairs were
successfully synthesized. Interpenetrating polymer networks were also successfully
formed using chitosan and its water-soluble derivative, carboxymethyl chitosan with NVP
via the photoinitiator free method.
Swelling and drug release experiments conducted on the hydrogel systems prepared
revealed the versatility of these materials as potential drug delivery systems. The swelling
and drug release behaviour observed in the hydrogel networks was described in terms of
Fickian and non-Fickian diffusion kinetics. A wide range of swelling-drug release
kinetics was observed in the hydrogels, which varied from Fickian diffusion to non-
Fickian diffusion kinetics. Furthermore, the polyelectrolyte hydrogel networks displayed
pH dependent swelling and drug release behaviour suggesting their potential application
as stimuli response delivery systems. Finally it could be added that these hydrogel
systems studied could serve a wide range drug delivery applications ranging from slow to
fast releasing systems.
In vitro cytotoxicity experiments on human keratinocyte (HaCaT) cells revealed that
hydrogels did not have any adverse effect on the HaCaT cells suggesting that they were
satisfactorily biocompatible with the host HaCaT cells. A biomaterial must be
satisfactorily biocompatible to be considered for bioapplications. Thus the in vitro
304
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
biocompatibility studies further indicate the suitability of these hydrogel networks as
biomaterials.
8.2. Future Recommendations
Hydrogels as more effective, more patient friendly and more cost effective substitutes to
the conventional pills and injections for delivery of bioactive agents has been investigated
for several decades. However, the need for cheaper, more stimuli responsive and more
biocompatible substitute drug delivery systems continues. In the present work, hydrogels
were cured via a novel photoinitiator-free curing technique, which were found to be cost
effective, free of toxins and competent drug delivery systems.
Future work could involve exploration of a wider range of donor/acceptor pairs, which
would lead to more efficient polymerisable systems. Furthermore, these monomers may
possess characteristics such as ionic, hydrophilic or hydrophobic nature, which could be
effectively used to design hydrogels with desirable swelling-drug release properties for
specific applications.
As emphasized previously, the ability of the hydrogel to be biocompatible governs its
possible use in biomedical applications. Hydrogels synthesized in the present work were
tested in vitro, which provides sufficient preliminary evidence on the biocompatibility of
the hydrogels. However, in vivo tests are more important as they provide a better image of
the biocompatibility nature of a biomaterial. Thus as a suggestion for future work, in vivo
studies involving clinical trials on the hydrogels prepared in the present work would
provide a better understanding of their biocompatibility. Furthermore, in vivo studies will
confirm the findings from this work on the suitability of these materials for biomedical
use.
305
Appendix I: NMR Spectra
1H NMR spectrum of HMMI in D2O
306
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
13
C NMR spectrum of HMMI in D2O
307
Appendix I: NMR Spectra
1H NMR spectrum of HEMI in D2O
308
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
13
C NMR spectrum of HEMI in D2O
309
Appendix I: NMR Spectra
1H NMR spectrum of HPrMI in D2O
310
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
13
C NMR spectrum of HPrMI in D2O
311
Appendix I: NMR Spectra
1H NMR spectrum of HPMI in D2O
312
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
13
C NMR spectrum of HPMI in D2O
313
Appendix I: NMR Spectra
Inversion recovery pulse response of a hydrated gel showing typical T1 relaxation profile
314
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
CPMG pulse sequence of a hydrated gel displaying typical T2 relaxation profile
315
Appendix II: UV-visible Spectra
316
UV-vis spectrum of theophylline
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
317
UV-vis spectrum of thiamine hydrochloride
Appendix II: UV-visible Spectra
318
UV-vis spectrum of Mn-TPP-OH
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
319
UV-vis spectra of TPP-OH and Mn-TPP-OH
Appendix III: Texture Analysis
Appendix III. Texture Analysis
The swollen hydrogels were compressed for 30 seconds with increasing compression
distance (0.1 mm/s) in the first 10 seconds and maintained compression distance in the
later 20 seconds of the experiment. The load was withdrawn from the sample after 30
seconds and the sample was let to relax back to its original physical state. The
compression stress-strain graph (Figure AIII.1) bellow illustrates the behaviour of a
swollen hydrogel under load where F is the compression force measured in the linear
portion of the stress-strain curve, used to calculate the Young’s modulus with the
corresponding strain parameter. F1 is the maximum compression force measured at 10
seconds with a compression distance of 1.0 mm. F2 is the measured maximum relaxation
force exerted by the sample once the load was removed.
Figure AIII.1. A typical compression stress-strain graph of a swollen hydrogel under
load
320
Appendix IV: UV Lamp Calibration
Appendix IV. UV Lamp Calibration
The UV lamp calibration was done using a uranyl-oxalate actinometer. As described in
Section 2.4, 0.02 L of 0.02 M oxalic acid was mixed with 0.02 L of 0.02 M uranyl nitrate
solution and subjected ultra-violet radiation. The radiation dose rate calculations were
carried out according to the decomposition rate of oxalic acid at varying exposure time
intervals. The rate of decomposition of oxalic acid as a function of exposure time is
illustrated in Figure AIV.1.
0.0
1.0
2.0
3.0
4.0
5.0
0 20 40 60 80 100
Time of exposure (mins)
Oxali
c aci
d d
ecom
pose
d
(mol
x 1
0-4
)
Figure AIV.1. Plot of the amount of oxalic acid decomposed as a function of exposure
time to the UV light
A linear decomposition rate of oxalic acid was observed in first 30 minutes of UV
exposure with ~ 70 % oxalic acid decomposed followed by a gradual reduction in
decomposition rate until complete decomposition of the acid. The UV radiation dose rate
calculations as described in Section 2.4.4 were carried out for the uranyl nitrate –oxalic
acid mixture exposed to the UV lamp for 30 minutes on the basis of maximum exposure
time with a linear decomposition rate.
321
Appendix V: Organic Syntheses - Reaction Mechanisms
Appendix V. Organic Syntheses - Reaction Mechanisms
A cancer tumour-tracing agent, Mn-TPP-OH, a series of N-hydroxyalkyl maleimides and
a water-soluble derivative of chitosan were synthesized in accordance to methods
published in the literature. The detailed syntheses and characterization of the compounds
are described in Section 2.3. The reaction mechanisms of the compounds are illustrated in
Schemes 1-10.
NN
N N
R
R
MnR R
NNH
N NH
R
R R
R
OHR
Mn2+
H+
=
TPP-OH Mn-TPP-OH
-2
Scheme 1. Insertion of Mn into the TPP-OH cavity
N
O
O
OH
NH
O
O
H
H
OOH-
Maleimide HMMI
+
Formaldehyde
Scheme 2. Reaction of maleimide with formaldehyde to yield HMMI
322
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
O
O
O
OO
O
O
O +
Furan Maleic anhydride Furan-A
Scheme 3. Preparation of the maleic anhydride adduct of furan (Furan-A)
N
O
O
O
OH
O
O
O
O
NH2
OH
Furan-A HEMI-A
+
Ethanolamine
Scheme 4. Preparation of the HEMI adduct of furan (HEMI-A)
N
O
O
OH
N
O
O
O
OH
O
HEMI-A HEMI
+
Furan
Scheme 5. Decomposition of the HEMI adduct of furan to yield HEMI
N
O
O
O
OH
O
O
O
O
NH2
OH
Furan-A HPrMI-A3-Amino-1-propanol
+
Scheme 6. Preparation of the HPrMI adduct of furan (HPrMI-A)
323
Appendix V: Organic Syntheses - Reaction Mechanisms
N
O
O OH
N
O
O
O
OH
O
HPrMIHPrMI-A
+
Furan
Scheme 7. Decomposition of the HPrMI adduct of furan to yield HPrMI
N
O
O
O
OH
O
O
O
O
NH2
OH
Furan-A HPMI-A5-Amino-1-pentanol
+
Scheme 8. Preparation of the HPMI adduct of furan (HPMI-A)
N
O
O
OH
N
O
O
O
OH
O
HPMI-A HPMI
+
Furan
Scheme 9. Decomposition of the HPMI adduct of furan to yield HPMI
324
Radiation Synthesis of Polymeric Hydrogels for Swelling-Controlled Drug Release Studies
O
CH2OH
OH
O
NH2
O
CH2OH
OH
O
NHAc
O
CH2OH
OH
O
NHR
O
CH2OH
OH
O
NH2
O
CH2OH
OH
O
NR2
O
CH2OH
OH
O
NHAc
ClOH
O
NaOH
O
CH2OH
OH
O
NHR
O
CH2OH
OH
O
NH2
O
CH2OH
OH
O
NR2
O
CH2OH
OH
O
NHAc
H+
O
O
Na+
OH
O
Chitosan
Na Salt CM Chitosan
R =
CM Chitosan
R =
Scheme 10. Partial deacetylation of chitosan to yield carboxymethyl chitosan
325
STATEMENT OF AUTHENTICATION
I, Salesh Narayan Swami, hereby declare that the content of this thesis submitted under
the Doctor of Philosophy program is solely my own work carried out at the School of
Science, Food and Horticulture research laboratory at the University of Western Sydney.
No part of this work has been previously submitted to an educational institution for an
award or postgraduate degree. Information obtained from published or unpublished work
of other researchers has been appropriately acknowledged.
Salesh N. Swami
Doctor of Philosophy Candidate
25 / 07 / 2004
ACKNOWLEDGEMENTS
The author wishes to express his gratitude to several people whose help and suggestions
have been so valuable towards the completion of his doctoral research. Foremost is his
principal supervisor, Dr. Loo-Teck Ng, who has tirelessly supported and guided him
throughout the course of this work with great patience. This work would not have
completed without her invaluable assistance. He wishes to acknowledge with many
thanks, Dr. Clare-Gordon Thompson and Prof. Phil Moore for their help and guidance in
the cytotoxicity studies, Dr. Narshima Reddy for being so helpful in processing the NMR
data, Dr. Michael G. Stevens for his helpful suggestions, advice and support and Dr. Janet
Patterson for her assistance and advice on the texture analysis. The author is grateful to
the technical staff, Mr. Paul Roddy, Mr. Bert Aarts and Ms. Sylvia DeNetto, who have
been very helpful in taking care of the technical needs of the project. He also thanks his
fellow students for their helpful suggestions and encouragement. The author would like to
extend his appreciation to the APA scholarship for the financial support and also the
scholarships selection committee at UWS for such an opportunity. Above all, the author
is very much indebted to his beloved mum and uncle, Mr. Laurie Reilly for their
unconditional love, support and guidance at every step of the way.
i
AWARD
Recipient of the Australian Postgraduate Award (APA) 2001
PUBLICATIONS
Loo-Teck Ng, Salesh Swami and Sonny Jönsson, 2004. “Kinetics study of the
photopolymerisation of donor/acceptor pairs using the differential photocalorimetric
technique and the relation of the kinetics data to hydrogels formation,” Radiation Physics
and Chemistry, 69: pp. 321-328.
Salesh Swami, Loo-Teck Ng and Sonny Jönsson, 2003. “Kinetics studies of
photopolymerisation initiated by donor/acceptor pair systems based on NVP with a series
of N-hydroxyalkyl maleimides, and hydrogel formations via these systems,” Conference
Proceedings of RadTech Asia’03, Yokohama, Japan, pp. 677-680.
Sonny Jönsson, Viswanathan Kalyanaraman, Karin Lindgren, Salesh Swami and Loo-
Teck Ng, 2003. Photopolymerisation of maleimide/N-vinylpyrrolidinone hydrogels,”
Polymer Preprints, 44 (1): pp. 7-8.
Loo-Teck Ng, Sonny Jönsson, Salesh Swami and Karin Lindgren, 2002. “Synthesis of a
hydrogel for drug delivery studies utilising photoinitiator-free photopolymerisation
process based on donor/acceptor pair, N-vinylpyrrolidinone and hydroxypentyl
maleimide,” Polymer International, 51: pp. 1398-1403.
John L. Garnett, Loo-Teck Ng, Duc Nguyen, Salesh Swami and Elvis Zilic, 2002. “CT
complexes in radiation polymerisation processes including grafting, curing and hydrogel
formation,’’ Radiation Physics and Chemistry, 63: pp. 459-463.
Loo-Teck Ng, Sonny Jönsson, Karin Lindgren, Salesh Swami, Charles Hoyle and Shan
Clark, 2001. “Efficiency of hydrogen donors in photo-induced copolymerisation of NVP
and water soluble N-alkyl maleimide,” Conference Proceedings of RadTech Europe’01,
Basel, Switzerland, pp. 609-613.
ii
CONFERENCE PRESENTATIONS
Salesh Swami, Loo-Teck Ng and Clare-Gordon Thompson (poster presentation),
“Hydrogels prepared by photoinitiator-free UV curing technique and their effect on
human keratinocyte (HaCat) cell viability,” 7th
World Biomaterials Congress, 17th
-21st
May 2004, Sydney, Australia.
Loo-Teck Ng and Salesh Swami (poster presentation), “Chitosan-NVP IPN hydrogels
synthesized through photoinitiator-free photopolymerisation technique for drug delivery,”
31st Annual Meeting & Exposition of the Controlled Release Society, 12
th-16
th June 2004,
Hawaii, USA.
Salesh Swami, Loo-Teck Ng and Michael G. Stevens (oral presentation), “Radiation
synthesis of polymeric hydrogels for swelling-controlled drug release studies,” 1st Annual
CSTE Innovation Conference, 8th
-10th
June 2004, UWS, Sydney, Australia.
Salesh Swami, Loo-Teck Ng and Sonny Jönsson (oral presentation), “Kinetics studies of
photopolymerisation initiated by donor/acceptor pair systems based on NVP with a series
of N-hydroxyalkyl maleimides, and hydrogel formations via these systems,” RadTech
Asia’03 Conference, 9th
-12th
December 2003, Yokohama, Japan.
Salesh Swami, Andjelka Arsenin, Vera El-Khoury, Loo-Teck Ng and Sonny Jönsson
(poster presentation), “Hydrogels for controlled-release of drugs prepared through
photoinitiator-free photopolymerisation,” RACI QLD Polymer Group Symposium-
Polymers in Dentistry, Medicine and Surgery, 6th
-8th
February 2002, Brisbane, Australia.
Loo-Teck Ng, Sonny Jönsson, Karin Lindgren, Salesh Swami and Charles Hoyle (oral
presentation), “ Efficiency of hydrogen donors in photo-induced copolymerisation of
NVP and water soluble N-alkyl maleimide,” RadTech Europe’01 Conference, 8th
-10th
October 2001, Basel, Switzerland.
iii
ABSTRACT
Hydrogels are three-dimensional networks of hydrophilic homopolymers or copolymers
generally covalently or ionically crosslinked. They interact with aqueous media by
swelling to some equilibrium value by retaining the aqueous media in their structures.
This study concerns the investigation of the swelling and the controlled drug release
behaviour of hydrogels synthesized via the photopolymerisation process.
Copolymers of varying compositions of 2-hydroxyethyl methacrylate (HEMA) and N-
vinyl-2-pyrrolidinone (NVP) were prepared by allowing the monomers to be exposed to
ultra violet (UV) radiation in the presence of a photoinitiator (PI), Irgacure 819.
However, the use of photoinitiators is a growing concern with regards to their incomplete
usage in the polymerisation process leading to undesirable residual toxic impurities in the
polymer matrix. Hence NVP and HEMA based polymers were synthesized via the
“photoinitiator-free” (PI-free) photopolymerisation process, which was initiated by
charge transfer (CT) complexes formed when electron donor/acceptor pairs were under
the influence of the UV source.
A series of N-hydroxyalkyl maleimides, namely N-hydroxymethyl maleimide (HMMI),
2-hydroxy-N-ethyl maleimide (HEMI), 3-hydroxy-N-propyl maleimide (HPrMI) and 5-
hydroxy-N-pentyl maleimide (HPMI) were synthesized and used as acceptor monomers.
The acceptor monomers were combined with NVP, which was the electron donor
monomer to form the donor/acceptor pairs. Kinetic studies were conducted on these
donor/acceptor pairs in the CT complex formation by using the Differential
Photocalorimeter (DPC) technique. This technique evaluated their efficiency in
polymerisation with respect to the heat released. The kinetics data revealed HPMI-NVP
system as the most efficient followed by HMMI-NVP system, then HPrMI-NVP and
HEMI-NVP being the least efficient. Glucosamine hydrochloride (HCl) and glucose were
use as hydrogen donors. Glucosamine HCl was found to be a more superior hydrogen
donor than glucose with its relative efficiency in enhancing the polymerisation rate.
iv
Neutral hydrogels were prepared based on these N-hydroxyalkyl maleimides as the
acceptors and NVP as the donor.
Prior to the synthesis of anionic hydrogels using the PI-free polymerisation technique
using acrylic acid (AA) as the acceptor and NVP as the donor, kinetics studies using
various mole ratios of these monomers were performed using the DPC technique. The
kinetics data reflected that the 1:1 mole ratio of AA:NVP was the most efficient system
with the highest exotherm. Hydrogels based on AA and NVP were subsequently
synthesized.
Interpenetrating polymer networks (IPNs) involving chitosan and its derivative
carboxymethyl chitosan were synthesized by allowing NVP and NVP/HEMA to
polymerise within the matrices of these polysaccharides via the PI-free technique. The
IPNs were characterized using the Fourier Transform Infrared (FT-IR) spectroscopic
technique.
The polymeric hydrogels synthesized were evaluated for their potential applications as
hydrogels through swelling and drug release experiments conducted at 37 oC in aqueous
media. Polyelectrolyte hydrogels were evaluated for their swelling and drug release
behaviour in varying pH environments. The effect of polymer composition on the drug
release and swelling behaviour of the hydrogels were evaluated.
The drug release studies were conducted using Mn-tetrahydroxyphenyl porphyrin (Mn-
TPP-OH), thiamine hydrochloride and theophylline as the model drugs. The effect of
varying molecular weights of the model drugs on the equilibrium drug release was also
evaluated. The equilibrium water content (EWC) and the equilibrium drug release (EDR)
values from swelling and drug release experiments respectively were reflective of varying
polymer compositions. The differences in these properties of the hydrogels were
explained in terms of heterogeneous crosslinkage and the hydrophilicity/hydrophobicity
of the components. Hydrogels showed a decrease drug release rate with increase in the
molecular weight of the incorporated drug.
v
The study of hydrogels in this work was oriented towards their biomedical applications
as controlled drug delivery devices. It is a known fact that the complete conversion of
monomers to polymers may not be achieved in the polymerisation process thus there is
always a certain component of unreacted toxic monomers still remained in the polymer
matrix. These monomers have the tendency to leach out of the polymer matrices when the
polymers are in contact with an aqueous medium thus rendering the hydrogel to be non-
biocompatible. The polymers synthesized in this work were washed thoroughly in milli-
Q-water and then evaluated in vitro for any possible toxic effect on human keratinocyte
(HaCaT) cells using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
(MTT) cell proliferation assay. The cytotoxicity results indicated that the hydrogels
understudy sustained and allowed a positive growth of the HaCaT cells in the duration of
the cytotoxicity experiment, thus proving to be satisfactorily biocompatible.
vi
LIST OF ABBREVIATIONS
AA Acrylic acid
AAS Atomic Absorption Spectrometer
ANOVA Analysis of variance
CM Carboxymethyl
CPMG Carr-Purcell-Meiboom-Gill
CT Charge transfer
DMEM Dulbecco's modified Eagle's medium
DMSO Dimethyl sulfoxide
DNP Did not polymerise
DPBS Dulbecco's phosphate buffered saline
DPC Differential Photocalorimeter
DSC Differential Scanning Calorimeter
EDMA Ethylene dimethacrylate
EDR Equilibrium drug release
EDTA Ethylenediamine tetraacetic acid
ER Electron transfer
EWC Equilibrium water content
FCS Fetal calf (bovine) serum
FDR Fractional drug released
FT-IR Fourier transform infrared
Furan-A 3,6-Endoxo-1, 2,3,6-tetrahydrophthalic anhydride
GC-MS Gas Chromatograph Mass Spectrometer
HaCaT Human keratinocyte
HEMA 2-Hydroxyethyl methacrylate
HEMI 2-Hydroxy-N-ethyl maleimide
HEMI-A 2-Hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide
HEPES N-2-Hydroxyethyl piperazine-N-2-ethane sulfonic acid
HMMI N-Hydroxypropyl maleimide
vii
HOMO Highest occupied molecular orbital
HPMI 5-Hydroxy-N-pentyl maleimide
HPMI-A 5-Hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide
HPrMI 3-Hydroxy-N-propyl maleimide
HPrMI-A 3-Hydroxy-N-ethyl-3, 6-endoxo-1, 2, 3, 6-tetrahydrophthalimide
IPNs Interpenetrating polymer networks
LCST Low critical solution temperature
LUMO Lowest unoccupied molecular orbital
MA Maleic anhydride
Mn-TPP-OH Manganese-5, 10, 15, 20-tetrakis(4-hydroxyphenyl) porphyrin
MRI Magnetic Resonance Imaging
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
MW Molecular weight
NMR Nuclear Magnetic Resonance
NVC N-Vinyl caprolactam
NVP N-Vinyl-2-pyrrolidinone
PI Photoinitiator
PAM Poly(acrylamide)
PEG Poly(ethylene glycol)
PEO Poly(ethylene oxide)
PGMA Poly(glyceryl methacrylate)
PHEMA Poly(2-hydroxyethyl methacrylate)
PHPMA Poly(3-hydroxypropyl methacrylate)
PMMA Poly(methacrylic acid)
PNIPAM Poly(N-isopropyl acrylamide)
PNVP Poly(N-vinyl-2-pyrrolidinone)
PVA Poly(vinyl alcohol)
SAXS Small angle X-ray scattering
SEM Standard error mean
SR Stress relaxation
viii
TA Texture analysis
TPGDA Tripropylene glycol diacrylate
TPP-OH 5, 10, 15, 20-Tetrakis(4-hydroxyphenyl)-21H, 23H-porphine
TSP 2, 2, 3, 3-d(4)-3-(Trimethylsilyl)propionic acid sodium salt
UV Ultra violet
VCZ N-Vinylcarbazole
VE Vinyl ether
WAXS Wide angle X-ray scattering
ix