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1.0 BRIEF INTRODUCTION OF DENDRIMER
A dendrimer is generally described as a macromolecule, which is characterized by its
highly branched 3D structure that provides a high degree of surface functionality and
versatility. Dendrimers are a unique class of polymers which play an important role in
emerging nanotechnology. Targeted drug delivery is one of the most attractive
potential applications of Dendrimers. The present review briefly describes about
dendrimer synthesis strategies, dendrimer functionlisation properties which having
crucial importance and their potential applications such as carrie molecule,
multivalent diagnostics for MRI, Sensors, multivalent bioconjugate, Light harvesting,
catalysis, artificial enzymes, coatings, inks and other application. Due to their
multivalent and monodisperse character, dendrimers have stimulated wide interest in
the field of chemistry and biology, especially in applications like drug delivery, gene
therapy and chemotherapy.
1.1 MOLECULAR ARCHITECHTURE[[
Dendrimers are an interesting class of macromolecules that have a highly
symmetrical, hyper-branched and spherical structure. Dendrimers possess an
architecture consisting of (1) a core; (2) an interior of shells (generation); and (3) an
exterior (outermost layer), which often has terminal functional groups [Buhleier, et al,
1978] . The core determines the size and shape of the dendrimer, the interior
determines the amount of void space that can be enclosed by the dendrimer, and the
exterior allows growth of the dendrimer or other chemical modification. This unique
architecture makes dendrimers monodispersed macromolecules compared to classical
linear polymers[Richardson, et al, 2000] . In dendritic structures, the number of
terminal groups increases exponentially with a linear increase in the generation of the
dendrimer. This relationship limits the ultimate size of the dendrimer due to steric
crowding of the terminal groups. Several new branching points are available at each
repeating unit in their structure for hyperbranched growth [Tomalia, et al,
1985] .Polyamidoamine (PAMAM) dendrimers are a newer class of polymer that
possess a number of interesting and useful pharmaceutical applications. PAMAM
dendrimers have capability of enhancing the solubility of low solubility drugs. They
also have potential application for the delivery of DNA and oligonucleotide and as
platforms for the development of cancer therapeutics. Dendrimers have been shown to
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cross cell barriers at sufficient rates to act as potential carrier/delivery systems.
Surface engineering has been found to reduce cytotoxicity and enhance transport
studies on the mechanism of dendrimer transport across epithelial cells [Newkome, et
al, 1985]. The core typically consists of a polyfunctional molecule, where the
functionality governs the number of branches that extend from it. The tetrahedral core
gives a spherical polymer whereas linear polymeric cores give rod-shaped
dendrimers[Johansson, et al, 1992].
Figure: 1.1.1 structure of dendrimer
.1.2 TYPES OF DENDRIMER
Types of dendrimers
1. Liquid crystalline dendrimers
They consist of mesogenic (liq. crystalline)monomerse.g. mesogen
functionalized carbosilane dendrimers. Functionalization of end group of carbosilane
dendrimers with 36 mesogenic units, attached through a C-5 spacer, leads to liquid
crystalline dendrimers that form broad smetic A phase in the temperature range of 17–
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130 ◦C[Liu, et al, 2007] . claims that they have synthesized first photosensitive liquid
crystalline dendrimer with terminal cinnamoyl groups[Tomalia, et al, 1990]. They
have confirmed the structure and purity of this LC dendrimer by 1H NMR and GPC
methods. It was shown that such a dendrimer, under UV irradiation, can undergo E-Z
isomerization of the cinnamoyl groups and [2 + 2] photocycloaddition leading to the
formation of a three-dimensional network[Emrick, et al, 2000].
2. Tecto dendrimers
Tecto-dendrimers are composed of a core dendrimer, which may or may not
contain the therapeutic agent, surrounded by dendrimers. The surrounding dendrimers
are of several types, each type designed to perform a function necessary to a smart
therapeutic nanodevice the PAMAM core–shell tecto dendrimer[Boas, et al, 2004].
The Michigan Nanotechnology Institute for Medicine and Biological Sciences (M-
NIMBS) are developing a tecto dendrimers which perform the following functions:
diseased cell recognition, diagnosis of disease state, drug delivery, reporting location
and reporting outcome of therapy[Sunder,et al, 2000,] They have already made and
tested functional dendrimers which perform each of these functions. Even after a
many questions on the way they are planning to produce a smart therapeutic
nanodevice for the diseased cell like a cancer cell or a cell infected with a virus[Gong,
et al, 1999].
Fig:Schematic representation of PAMAM core–shell tecto dendrimer.
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3. Chiral dendrimers
The chirality in these dendrimers is based upon the construction of
constitutionally different but chemically similar branches to chiral core[Agarwal, et
al, 2008]. Chiral, nonracemic dendrimer with well-defined stereochemistry is
particularly interesting subclass, with potential applications in asymmetric catalysis
and chiral molecular recognition[Peterson, et al, 2003,].
4. PAMAMOS dendrimersRadially layered poly(amidoamine-organosilicon) dendrimers (PAMAMOS)
are inverted unimolecular micelles that consist of hydrophilic, nucleophilic
polyamidoamine (PAMAM) interiors and hydrophobic organosilicon (OS) exteriors.
These dendrimers areexceptionally useful precursors for the preparation of
honeycomblike networks with nanoscopic PAMAM and OS domains[Agarwal, et al,
2008].
5. Hybrid dendrimers
Hybrid dendrimers are combination of dendritic and linear polymers in hybrid
block or graft copolymer forms. The small den- drimer segment coupled to multiple
reactive chain ends provides an opportunity to use them as surface active agents,
compatibilizers or adhesives, e.g. hybrid dendritic linear polymers .
6. Peptide dendrimers
Denrimers having peptides on the surface of the traditional dendrimer
framework and dendrimers incorporating amino acids as branching or core units are
both defined as ‘peptide dendrimers’[Esfand, at al, 2000]. Also peptide dendrimers
can be defined as macromolecules that contain peptide bonds in their structure.
Because of biological and therapeutical relevance of peptide molecules, peptide
dendrimers play an important role in diverse areas including cancer, antimicrobials,
antiviral, central nervous system, analgesia, asthma, allergy and Ca+2 metabolism. On
the basis of their ability to be taken up by cells, making peptides very useful for drug
delivery[Hawker,et al, 1991]. One more interesting application of peptide dendrimers
is that can be used as contrast agents for magnetic resonance imaging (MRI),
magnetic resonance angiography (MRA), fluorogenic imaging and serodiagnosis .
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7. Glycodendrimers
The term ‘glycodendrimers’ is used to describe dendrimers that incorporate
carbohydrates into their structures . Glycodendrimers can be classified as: (i)
carbohydrate-coated; (ii) carbohydrate centered; and (iii) fully carbohydrate-based.
Glycodendrimers have been used for a variety of biologically relevant applications
such as, glycodendrimers with surface carbohydrate units have been used to study the
protein–carbohydrate interactions that are in many intercellular recognition
events[Grubbs, et al, 1997]. The accessibility of the sugars is an important
consideration for glycodendrimers used effectively to evaluate protein–carbohydrate
interactions. Like this, study of protein–carbohydrate interactions, incorporation into
analytical devices, formulation of gels, targeting of MRI contrast agents, drugs and
gene delivery systems are some of the areas where glycodendrimers are likely to be
beneficial[Guan,et al, 1999].
8. PAMAM dendrimerPerhaps the family of dendrimers most investigated for drug delivery is the
polyamidoamine (PAMAM) dendrimer[Cheng, et al, 2008] . Manysurface
modifiedPAMAMdendrimers are non-immunogenic, water-soluble and possess
terminal-modifiable amine functional groups for binding various targeting or guest
molecules[Yin, et al, 1998]. PAMAM dendrimers generally display concentration-
dependent toxicity and haemolysis.PAMAMdendrimers are hydrolytically degradable
only under harsh conditions because of their amide backbones, and hydrolysis
proceeds slowly at physiological temperatures . The internal cavities of PAMAM
dendrimers can host metals or guest molecules because of the unique
functionalarchitecture, which contains tertiary amines and amide linkages. PAMAM
dendrimers are generally prepared by divergent method and product up to generation
10 (G10) have been obtained.PAMAM dendrimers are the most extensively reported
moiety for almost all existing applications of dendrimers [Pushkar, et al, 2006]. .
1.3 SYNTHETIC APPROACH OF DENDRIMER
Dendritic polymers are synthesized using a stepwise repetitive reaction
sequence that leads to monodispersed, with a nearly perfect hyper branched topology
radiating from a central core and grown generation by generation. The synthetic
procedures developed for dendrimer preparation by controlling the critical molecular
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design parameters, such as size, shape, surface/interior chemistry, flexibility, and
topology[Sakthivel , et al, 2003]. .
1.3.1 Divergent Synthesis
In the divergent approach, dendrimers are prepared from the core as the starting point
and built up generation by generation. In the divergent way, problems occur from an
incomplete reaction of the end groups, since these structural defects accumulate with
the buildup of further generation. As the side products possess similar physical
properties, chromatographic separation is not always possible[Balogh,et al, 1998].
The higher generations of divergently constructed dendrimers always contain certain
structural defects. To prevent side reactions and to force reactions to completion large
excess of reagents is required. This may cause some difficulties in the purification of
the final product[Yiyun, et al, 2008].
Divergent synthesis of dendrimer
1.3.2.Convergent synthesis
The convergent approach starts from the surface and ends at the core, where
the dendrimer segments (dendron’s) are coupled. In this way, only a small number of
reactive sites are functionalized in each step, giving a small number of possible side-
reactions per step[Rajca, et al, 1993].In divergent method, purification of the high-
generation dendrimer becomes more difficult because of increasing similarity
between reactants and formed product. In convergent method, with proper purification
after each step, gives dendrimers without defects. On the other side, it does not allow
the formation of high generations because steric problems occur in the reactions of the
dendron’s and the core molecule [Hawker,et al, 1993].
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Convergent synthesis of dendrimer
1.3.3 Double exponential and mixed growth [
In this approach two products (monomers for both convergent and divergent
growth) are reacted together to give an orthogonally protected trimer ,which may be
used to repeat the growth process again[Liu, et al,1999] .strength of double
exponential growth is more stuble than the ability to build large dendrimers in
relatively few steps.
1.3.4 Hypercores and branched monomers growth
This method involved the pre-assembly of oligomeric species which can be
linked to give dendrimers in fewer steps or higher yields [Frechet and Tomalia,
2001].
1.4 PROPERTIES OF DENDRIMER
Nanoscale sizes that have similar dimensions to important bio-building
blocks, e.g., proteins, DNA.
Numbers of terminal surface groups (Z) suitable for bio-conjugation of drugs,
signaling groups, targeting moieties or biocompatibility groups.
Surfaces that may be designed with functional groups to augment or resist
trans-cellular, epithelial or vascular biopermeability[Rajca, et al, 1998].
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An interior void space may be used to encapsulate small molecule drugs,
metals, or imaging moieties. Encapsulating in that void space reduces the
drug toxicity and facilitates controlled release. [[
Positive biocompatibility patterns that are associated with lower generation
anionic or neutral polar terminal surface groups as compared to higher
generation neutral apolar and cationic surface groups[Svenson, et al, 2005].
Non- or low-immunogenicity associated with most dendrime surfaces
modified with small functional groups or polyethylene glycol (PEG)[ Miller,
et al, 1997].
Surface groups that can be modified to optimize biodis-tribution; receptor
mediated targeting, therapy dosage or controlled release of drug from the
interior space.
Ability to arrange excretion mode from body, as a function of nanoscale
diameter.
Dendrimers are monodisperse macromolecules, unlike linear polymers. The
classical polymerization process which results in linear polymers is usually
random in nature and produces molecules of different size, whereas size and
molecular mass of dendrimers can be specifically controlled during
synthesis[Liu, et al, 2000].
Because of their molecular architecture, dendrimers show some significantly
improved physical and chemical properties when compared to traditional
linear polymers.
In solution, linear chains exist as flexible coils; in contrast, dendrimers form
a tightly packed ball. This has a great impact on their rheological properties.
Dendrimer solution has significantly lower viscosity than linear
polymers[Svenson, et al, 2009] . When the molecular mass of dendrimers
increases, their intrinsic viscosity goes through a maximum at the fourth
generation and then begins to decline. Such behavior is unlike that of linear
polymers. For classical polymers the intrinsic viscosity increases continuously
with molecular mass[Hong, et al, 2007].
The presence of many chain-ends is responsible for high solubility and
miscibility and for high reactivity. Dendrimers solubility is strongly
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influenced by the nature of surface groups. Dendrimers terminated in
hydrophilic groups are soluble in polar solvents, while dendrimers having
hydrophobic end groups are soluble in nonpolar solvents[Zhuo, et al, 1999]. In a solubility test with tetrahydrofuran as the solvent, the solubility of
dendritic polyester was found remarkably higher than that of analogous linear
polyester. A marked difference was also observed in chemical reactivity.
Dendritic polyesters was debenzylated by catalytic hydrogenolusis whereas
linear polyester was unreactive[Esfand, et al, 2001].
Dendrimers have some unique properties because of their globular shape and
the presence of internal cavities. The most important one is the possibility to
encapsulate guest molecules in the macromolecule interior[ Sonke and
Tomalia, 2005].
1.4.1 Encapsulation of drugs within the dendritic architecture .
Dendritic artitecture (open nature ) has led several groups to investigated
the possibility of encapsulation drug molecules within the branches of a
dendrimer .This offers the potential of dendrimers to interact with labile or poorly
soluble drugs ,enhances drug stability ,bioavailability and controlling its release ,the
nature of drug encapsulation within a dendrimer may be simple physical
entrapment ,or can involve non- bonding interactions with a dendrimer may be simple
physical entrapment ,or can involve non-bonding interactions with specific structure
within the dendrimer[Christine, et al, 2005].
1.4.2 Surface interactions between drugs and dendrimers
The external surface of dendrimers have been investigated as potential sites of
interaction with drugs .although the number of guest molecules incorporated into a
dendrimer may be dependent to a limited extent on the architecture of a
dendrimer ,the loading capacity may be dramatically increased by the formation of a
complex with the large number of groups on the dendrimer surface. The dendrimer
with each increasing generation of dendrimer[Freeman and Frechet, et al, 2005].
1.4.3 Electrostatic interaction between drug and dendrimer
The presences of large number of ionisable groups on the surface of
dendrimers provides an interesting opportunity for electrostatic attachment of
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numerous ionizable drugs ,providing the resultant complex retains sufficient water
solubility. for example electrostatic interaction can occur between the carboxyl group
of the dendrimers .it has been estimated that approximately 40 ibuprofen molecule
interact with G4 PAMAM dendrimers at pH 10.5 causing a considerable enhancement
of drug solubility[Hawker, et al, 1990].
1.4.4 Conjugation of drug to dendrimer
The covalent attachment of drugs to the surface groups of dendrimers through
hydrolysable or biodegradable linkages offers the opportunity for a greater control
over drug released by ester hydrolysis of the PEG-PAMAM (G3) conjugate was
approximately the same (within 3% ) as that of non modified penicillin [Barbara and
Maria,et al, 2001].
1.5 DENDRIMERS IN DRUG DELIVERY
The development of an efficient drug delivery system is very important to
improve the pharmacological activity of drug molecules. Dendrimers have emerged as
new alternatives and efficient tools for delivery of drug molecule[Hong, et al,. 2004].
As compare to linear polymeric carriers, the multivalent functionalities of dendrimers
can be linked to drug molecules or ligands in a well-defined manner and can be used
to increase the binding efficiency and affinity of therapeutic molecules to receptors
via synergistic interaction[Patel, et al, 2007].
1.5.1 Oral drug delivery
Oral drug delivery systems are very important to the field of medicine, since
most of the common illnesses are treated via oral rout of medication. Dendrimers
those are able to hold medication with good durability but also biodegradable within a
biological system[. By combining the ideas of drug carriers and degradability,
research has recently focused on controlled degradation of dendrimers and release of
compounds via oral route. Encapsulation and conjugation of drug with dendrimers
have shown immense employment for delivery of hydrophobic and labile
drugs[Tomalia, et al, 1985] . Transport of dendrimers throughout epithelial part of
gastrointestinal tract depends upon its characteristics. Packaging a drug in a dendrimer
host not only makes it soluble but also allows it to bypass the transporter protein that
would normally stop it from being absorbed in the intestines after it has been taken
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orally. Anticancer and antihypertensive drugs are most promising candidate for oral
rout using dendrimer as carrier[Soulie, et al, 1999]
1.5.2 Ocular drug delivery
The topical application of active drugs to the eye is the most prescribed route
of administration for the treatment of various ocular disorders. It is generally agreed
that the intraocular bioavailability of topically applied drugs is extremely poor. These
results mainly due to drainage of the excess fluid via nasolacrimal duct and
elimination of the solution by tear turnover. Several research advances have been
made in ocular drug delivery systems by using specialized delivery systems such as
polymers, liposomes, or dendrimers to overcome some of these disadvantages.
1.5.3 Transdermal drug delivery
In recent era, dendrimers have found applications in transdermal drug delivery
systems. Generally, bioactive drugs have hydrophobic moieties in their structure,
resulting in low water-solubility that inhibits efficient delivery into cells[Morgan, et
al, 2003]. Dendrimers designed to be highly water-soluble and biocompatible have
been shown to be able to improve drug properties such as solubility and plasma
circulation time via transdermal formulations and to deliver drugs efficiently. The
viscosity imparting property of a dendrimers solution allows for ease of handling of
highly concentrated dendrimer formulations for these applications. Dendrimers have
been shown to be useful as transdermal drug delivery systems for nonsteroidal anti-
inflammatory drugs (NSAIDs), antiviral, antimicrobial, anticancer, or
antihypertensive drugs[Caminati, et al, 1990].
1.5.4 Targeted gene delivery
Dendrimers can act as carriers, called vectors, in gene therapy. Vectors
transfer genes through the cell membrane into the nucleus. Currently liposomes and
genetically engineered viruses have been mainly used for this[Thomas, et al, 2004].
PAMAM dendrimers have also been tested as genetic material carriers. Cationic
dendrimers (Polypropylenimine (PPI) dendrimer) deliver genetic materials into cells
by forming complexes with negatively charged genetic materials through electrostatic
interaction. Cationic dendrimers lend themselves as non-viral vectors for gene
delivery because of their ability to form compact complexes with DNA[Newkome, et
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al, 2001]. Another potential application could be the use of dendrimers coated with
sialic acid for the influenza virus to attach to the cell surface. In addition, dendrimers
are non-immunogenic and are thus uniquely suited as carrier structures for drugs or
bioactive molecules without degradation in immune system[Adronov, et al, 2000].
1.5.5 Dendrimers in pulmonary drug deliveryDendrimers have been reported for pulmonary drug delivery also. During one
study, efficacy of PAMAM dendrimers in enhancing pulmonary absorption of
Enoxaparin was studied by measuring plasma anti-factor Xa activity, and by
observing prevention efficacy of deep vein thrombosis in a rodent model[Jain, et al,
2001] . G2 and G3 generation positively charged PAMAM dendrimers increased the
relative bioavailability of Enoxaparin by 40%, while G2.5 PAMAM, a half generation
dendrimers, containing negatively charged carboxylic groups had no effect.
Formulations did not adversely affect mucociliary transport rate or produce extensive
damage to the lungs. respectively, as compared to free drugs[Boris and Rubinstein, et
al, 1996].
1.6 LIPOSOME DRUG DELIVERY SYSTEM
Liposomes are the leading drug delivery systems for the systemic
(intravenous) administration of drugs. There are now liposomal formulations of
conventional drugs that have received clinical approval and many others in clinical
trials that bring benefits of reduced toxicity and enhanced efficacy for the treatment of
cancer and other life-threatening diseases[Louvet, et al, 2000]. The mechanisms
giving rise to the therapeutic advantages of liposomes, such as the ability of long-
circulating liposomes to preferentially accumulate at disease sites such as tumours,
sites of infection and sites of inflammation are increasingly well understood. Further,
liposome-based formulations of genetic drugs such as antisense oligonucleotides and
plasmids for gene therapy that have clear potential for systemic utility are increasingly
available[Thomas, et al, 2005] . This work reviews the liposomal drug delivery field,
summarises the success of liposomes for the delivery of small molecules and indicates
how this success is being built on to design effective carriers for genetic drugs[Cottu,
et al, 2000
1.6.1 liposome with dendrimer
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This is the first study demonstrating the successful encapsulation of a 'guest-
host' oxaliplatin-dendrimer complex (i.e. oxaliplatin [guest] contained in a
poly(amidoamine) (PAMAM) dendrimer (G=4) [host]), which is then in turn
encapsulated within a liposome to produce a 'modulatory liposomal controlled release
system' (MLCRS)[ Malik, et al 2000]. These new constructs have exhibited unique,
modulatory drug release profiles compared to traditional drug-liposome (guest-host
complexes). Furthermore, they have manifested enhanced oxaliplatin activity against
lung cancer cell lines DMS114 and NCI-H460 with a (10x) one order of magnitude
higher growth inhibition activity against the NCI-H460 cancer cell line[Tomalia,
2005].
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1.7 DRUG PROFILE
1.7.1 Oxaliplatin
Oxaliplatin is a platinum containing antineoplastic agent[Jansen, et al, 1995] . It is thought to exert its cytotoxic action in a similar manner to alkylating agents by
causing inter- and intrastrand cross links in DNA, inhibiting DNA synthesis and
inducing apoptotic cell death. l Oxaliplatin is licensed for the first line treatment of
metastatic colorectal cancer in combination with 5-fluorouracil and folinic
acid[Jevprasesphant, et al, 2003] . l The addition of oxaliplatin to 5-fluorouracil and
folinic acid significantly improves the overall response rate in patients with
previously untreated colorectal cancer[Wiseman, et al, 1999].
1.7.2 Identification
Molecular weight 395.296 gm/mol
Chemical formula C8H12N2O4Pt
Structure
Synonyms Oxaliplatin [Usan:Inn:Ban] Oxaliplatino [Spanish] Oxaliplatinum [Latin] Oxaloplatine [French] Oxaloplatino [Spanish][ Bleiber, et al,
1996]IUPAC name (3aR,7aR)-octahydro-2',5'-
dioxaspiro[cyclohexa[d]1,3-diaza-platinacyclopentane-2,1'-cyclopentane]-3',4'-dione
Specific optical rotation + 74.5 to + 78.0 (dried substance
Solubility Water: slightly
Methanol: very slightly
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Ethenol: practically soluble
Acetone: practically soluble
Melting point 198ºc and 292ºc[Giaccheti, et al, 2000]
PH 3 to 9[De Gramont, et al, 2000]
Common trade name Eloxatin
Classification Alkylating agent
Additional Names [(1R,2R)-1,2-cyclohexanediamine-N,N¢][oxalato (2-)-O,O¢]platinum; oxalato(1R,2Rcyclohexanediamine)platinum(II); Pt(oxalato)(trans-l-dach); oxalato (trans-l-1,2-diaminocyclohexane)platinum (II); oxalatoplatin; oxalatoplatinum; l-OH[13,14,15]
1.7.3 Pharmacology
1.7.3.1 Mechanism of action
Oxaliplatin belongs to a new class of platinum agent. It contains a platinum
atom complexed with oxalate and diaminocyclohexane (DACH). The bulky DACH is
thought to contribute greater cytotoxicity than cisplatin and carboplatin[Misset, et al,
2000]. The exact mechanism of action of oxaliplatin is not known. Oxaliplatin forms
reactive platinum complexes which are believed to inhibit DNA synthesis by forming
interstrand and intrastrand cross-linking of DNA molecules. Oxaliplatin is not
generally cross-resistant to cisplatin or carboplatin, possibly due to the DACH group
and resistance to DNA mismatch repair[Freyer,et al, 2000]. Preclinical studies have
shown oxaliplatin to be synergistic with fluorouracil and SN-38, the active metabolite
of irinotecan[Carraro,at al 2000] Oxaliplatin is a radiation-sensitizing agent[Culy, at
al, 2000].It is cell-cycle-phase nonspecific[Graham,et al, 200
1.7.4 Pharmacokinetics
Distribution Minimal in plasma accumulation ;in
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erythrocytes does not diffuse into plasma
or act as drug reservoir
Volume of distribution Ultrafilterable platinum;581+261 L6
Plasma protein binding 70-95%[Souglakos, et al, 2002]
Metabolism Rapid nonengymatic biotransformation to
reactive platinum complexes
Excretion Platinum is mainly by renal excretion and
tissue distribution while platinum
metabolites are mainly by renal excretion
[Scheithauer, et al,2003].
Terminal half life Platinum elimination from
erythrocytes :48 days
1.7.5 Pharmacodynamics
Oxaliplatin exhibits a wide spectrum of both in vitro cytotoxicity and in vivo
antitumour activity in a variety of tumour model systems, including human colorectal
cancer models. Oxaliplatin also demonstrates in vitro and in vivo activity in various
cisplatin resistant models. A synergistic cytotoxic action has been observed in
combination with fluorouracil both in vitro and in vivo[Andre,at al, 1999].
1.7.6 Uses
Primary uses : colorectal cancer
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Other uses : breast cancer ,gastric canser ,germ cell cancer head and neck
cancer,lung cancer ,non small cell,ovarian cancer ,pancreatic cancer,prostate cancer
[Giacchetti, et al, 2000]
1.7.7 Side effects
Organ site Side effects
Allergy/immunology
blod /bone marrow
Febrile neutropenia
Anaphylaxis(0.5-2%)
Anemia(64-83%,severe 4-5%)
Immune hemolytic anemia(rare)
Neutropenia:single agent(15%,severe 3%)with fluorouracil
and leucovorin(76%,sever 4%)[Goldberg, et al, 2004]
Constitutional
symptoms
demetology /skin
Fever 36%
Extravasation hazard:irritant
Alopecia 2%
Gastrointestinal Emetogenic potential:heigh moderate
Diarrhea:single agent(41%,severe 5%),with fluorouracil and
leucovorin(58%,severe 10%)
Nausea,vomiting
Hepatic Liver function abnormalities
Infection Infection 23%
Neurology Central neuro toxicity(rare)
1.8 LITERATURE REVIEW
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M P Toraskar et al (2011) has reported that the dendrimers, a nanoparticles based
drug-delivery system have numerous applications in pharmaceuticals such as
enhancing the solubility of poorly soluble drugs, enhancing the delivery of DNA and
oligonucleotides, targeting drug at specific receptor site and ability to act as carriers
for the development of drug delivery systems.
V. Rajesh Babu et al (2010) has reported that the dendrimers are a new class of
synthetic polymers which have the structure like a tree or star shape, with a central
core, interior branches and terminal groups which decorate the surface.
Barbara Klajnert et al (2001) has reported that the dendrimers are a new class of
polymeric materials. They are highly branched, monodisperse macromolecules. The
structure of these materials has a great impact on their physical and chemical
properties. As a result of their unique behaviour dendrimers are suitable for a wide
range of biomedical and industrial applications. The paper gives a concise review of
dendrimers’ physico-chemical properties and their possible use in various areas of
research, technology and treatment.
G.srinubabu et al (2005) has reported that the a simple and reproducible
spectrophotometric method has been developed for the estimation of Oxaliplatin in
pure and dosage forms. The method is based on the reaction of the drug with 1, 10
Phenanthroline in presence of ferric chloride, which forms a blood red colored
complex exhibiting maximum absorption at 520 nm.
Claudia Burzlet et al (2000) has reported that the to evaluate the therapeutic
efficacy of oxaliplatin and to analyze the pharmacokinetics of both ultrafiltrable (free)
and protein–bound platinum in patients with metastatic colon cancer.
Norbert Maurer et al (2001) has reported that the liposomes are the leading drug
delivery systems for the systemic (intravenous) administration of drugs. There are
now liposomal formulations of conventional drugs that have received clinical
approval and many others in clinical trials that bring benefits of reduced toxicity and
enhanced efficacy for the treatment of cancer and other life-threatening diseases.
18
Nahid latif et al (1984) has reported that the liposomes, the artificial phospholipid
vesicles, have the capacity of entrapping water soluble substances in their aqueous
compartments. Of the many possible potentials of liposomes their application in
immunology is most significant. Recent studies have shown an adjuvant and a carrier
effect of liposomes to a number of antigens.
AndrewE et al (2006) has reported that the Pegylated liposomal oaliplatin is a
formulation of oxaliplatin in which the molecule itself is packaged in a liposome
made of various lipids with an outer coating of polyethylene glycol. Liposomal
technology is being used in increasing amounts in the therapy of a variety of cancers,
including ovarian cancers. This article reviews the mechanistic actions of this
formulation, the Phase II and Phase III data that helped define the role of pegylated
liposomal oxaliplatin in recurrent ovarian cancer, as well as a discussion of some of
the side-effects and their managemen.
Buleier, E et al, (2009) has reported that the dendritic polymers are belonging to a
special class of macromolecules. They are called "Dendrimers." Similar to linear
polymers, they composed of a large number of monomer units that were chemically
linked together. Due to their unique physical and chemical properties, dendrimers
have wide ranges of potential applications. These include adhesives and coatings,
chemical sensors, medical diagnostics, drug-delivery systems, high-performance
polymers, catalysts, building blocks of supermolecules, separation agents and many
more(116).
Aristarchos Papagiannaros et al ( 2007), has reported that the liposomes
incorporating oxaliplatin-PAMAM complex exhibited release properties which were
advantageous compared to the conventional type of liposomal oxaliplatin in terms of
oxaliplatin toxicity and its availability to the tumor site. This liposomal formulation
may show improved therapeutic properties in vivo.
Andreas Wagne et al, (2010) has reported that the liposomes, spherical vesicles
consisting of one or more phospholipid bilayers, were first described by Bangham
and coworkers. Today, numerous lab scale but only a few large-scale techniques are
available. However, a lot of these methods have serious limitations in terms of
19
entrapment of sensitive molecules due to their exposure to mechanical and/or
chemical stress.
Rok Podlipec et al, (2010) has reported that the Liposome-cell interaction which is
exchange of lipids, absorbtion of liposomes on cell membranes and specially fusion or
endocytosis, can be described with proper equations of mass action.
Buleier, E et al, (2009) has reported that the dendritic polymers are belonging to a
special class of macromolecules. They are called "Dendrimers." Similar to linear
polymers, they composed of a large number of monomer units that were chemically
linked together. Due to their unique physical and chemical properties, dendrimers
have wide ranges of potential applications. These include adhesives and coatings,
chemical sensors, medical diagnostics, drug-delivery systems, high-performance
polymers, catalysts, building blocks of supermolecules, separation agents and many
more.
Gardikis K, et al, (2010) has reported that the liposomal locked-in dendrimers
(LLDs), the anticancer drug oxaliplatin was loaded into pure liposomes or LLDs and
the final products were subjected to lyophilization. The loading of oxaliplatin as well
as its in vitro release rate from all systems was determined and the interaction of
liposomes with dendrimers was assessed by thermal analysis and fluorescence
spectroscopy. The results were very promising in terms of drug encapsulation and
release rate, factors that can alter the therapeutic profile of a drug with low therapeutic
index such as oxaliplatin. Physicochemical methods revealed a strong, generation
dependent, interaction between liposomes and dendrimers that probably is the basis
for the higher loading and slower drug release from the LLDs comp to pure
liposomes.
20
1.9 AIM OF OBJECT
Cancer is a large heterogeneous class of disease in which a group of cells
display uncontrolled growth ,invasion that intrudes upon and destroys adjacent tissues
and often metasizes ,wherein the tumour cells spread to other location in the body via
the lymphatic system or through the blood stream[Augustin, et al, 1998] .these three
malignated properties of cancer differentiate malignant tumors from benign
tumors ,which do not grow uncntollably ,directly invade locally ,or metastazie to
regional lymph nodes or distant body sites like brain ,bone ,liver or orther organs
[Sorbye, et al, 2004].
Platinum-based complexes are important drugs for the treatment of cancer
diseases. Compared to the commonly used Pt(II) compounds oxaliplatin, the recently
reported complexes containing Pt(IV) seem to have several advantages; they are safer,
can be used orally, have a higher scope of anticancer effect, and do not show cross-
resistance to cisplatin[Bennett, et al, 1999]
Liposomes incorporating the oxaliplatin-PAMAM complex exhibited release
properties which were advantageous compared to the conventional type of liposomal
oxaliplatin in terms of oxaliplatin toxicity and its availability to the tumor site. This
liposomal formulation may show improved therapeutic properties in vivo[ Bangham,
et al, 1964].
In the present investigation include to prepare liposomal formulation of
oxaliplatin-pamam complex and their characterization .
The present work is based on following report :
1.Stathopoulos GP et al (2006) has reported that the lipoxal is a liposomal
oxaliplatin, which reduces the cytotoxic agent's adverse reactions without reducing
effectiveness. Our objectives were to determine the adverse reactions, dose-limiting
toxicity (DLT) and the maximum tolerated dose (MTD) of lipoxal.
2.Moraes ML et al (2008) has reported that the cationic pamam dendrimers
were incorporated in the aqueous interior of liposomes order to increase the
encapsulation efficiency for the anticancers drugs.
21
3.Moraes ML et al (2008) has reported that the loading of drug indeed
increased proportionally to dendrimer generation while the system decreased.
4.Papagiannaros A et al (2005) has reported that the first study
demonstrating the successful encapsulation of a 'guest-host' oxaliplatin-dendrimer
complex (i.e. oxaliplatin [guest] contained in a poly(amidoamine) (PAMAM)
dendrimer (G=4) [host]), which is then in turn encapsulated within a liposome to
produce a 'modulatory liposomal controlled release system' (MLCRS).
22
1.10 PLAN OF WORK
1.Identification of oxaliplatin
a. Melting point
b. U.v spectroscopy
c. I .R spectroscopy
d. Solubility profile
2. Analytical method development
a. Preparation of calibration curve of oxaliplatin in phosphate buffer solution using
uv method (PH-7.4,λmax 207).
b.Preparation of calibration curve of oxaliplatin in water using uv method at λmax
207nm
c.Preparation of calibration curve of oxaliplatin in water using hplc method
d.Preparation of calibration curve of oxaliplatin in plasma using hplc method
3.Preparation of formulation
Preparation of liposome of oxaliplatin with and without dendrimer
Characterization of liposome
1.Particle size determination
a.Optical microscopic method
b.Particle size and size distribution
2.Estimation of dendrimer in external phase
3. Effect of Tris dendrimer and NH2 surface on encapsulation efficiency
4. Invitro release study
5. Stability studies
a. Effect of aging
23
b. Effect of temperature
4. Determination of pharmacokinetics parameters
a. AUC
b. tmax
c. Cmax
24
This chapeter presnts preparation and characterization of dendrimer
phospholipid composition. An attempet was made to understand the formulation
behavior during hydration by various microscopic technique and to estabilish the
presence of dendrimer in the liposomal structure. we have also proved that the
presence of dendrimer modulates the encapsulation, release and pharmacodynamics
properties of the encapsulated drug. The details of experimental methods are as
follows .
2.0 MATERIALS
1. Instruments: Magneticstir with hot plate (simco microscope), Ultrasonic bath
sonicater (relex), Systronics double beam spectrophotometer, Shimadzu hplc,
Electronic balance (Vibra &Essae),whatman filter paper, 2.4nm semipermeable
membrane .
2. Drugs and chemicals: PAMAM dendrimer (Nanosynthons,USA), oxaliplatin(sun
pharma) cholesterol(sigma chemical), lecithine (himedia), HPLC water(rankem),
potassium dihydrogen phosphate(merkindia limited), HPLC grade
methanol(qualigens), choloroform(qualigens).
3. Animal and housing: In the present study male albino rats (150-200g) were used.
All the animals were procured from the disease-free animal house of Central Drug
Research Institute, Lucknow, India. The animals had free access to food and drinking
water as per CPCSEA dietary norms. They were subjected to natural light-dark cycle
(12 hours each). The animals were acclimatized for at least 5 days to the laboratory
conditions prior to experimentation. The experimental protocol was approved by the
Institutional Animal Ethics Committee dated 13-04-2012. The care of the animals
was taken as per the guidelines of CPCSEA, Ministry of Forests & Environment, and
Government of India(Approval no- IAEC/ceu/10).
2.1 METHODS
2.1.1 Preformulation study: Preformulation may be described as a phase of the
research and development process where we characterizes the physical, chemical and
mechanical properties of a new drug substance, in order to develop stable, safe and
25
effective dosage form.Thus we performed this study for authentication and
identification of the drug.
2.1.2 Identification of Oxaliplatin
Drug was identified as per B.P (2010)
2.1.3 Appearance
Physical appearance of the drug was noted by visual observation
2.1.4 Chemical test
Chemical test of the drug sample were performed according to B.P (2010)
Method: Dissolve 0.01 gm in water and dilute to 50 ml with the same solvent.
2.1.5 Melting point
Melting point of drug sample was determined using melting point apparatus.
2.1.6 Solubility profile
The solubility of drug at room temperature was determine in different
solvents.
2.1.7 Scanning of drug
Accurately weight quantity of drug (10mg) was taken in10 ml volumetric
flask, dissolved in 1 ml of water and volume was made upto 100 ml with HPLC grade
water.
Drug solution (0.001%) in water and scanned in range 200-400 nm by
Systronics double beam spectrophotometer and absorption maxima were noted.
2.1.8 Infrared spectroscopy
The IR spectrum of drug sample was obtained by KBr disc method by shimadzu IR
Spectrophotometer CDRI Lucknow (sample code-oxs).
26
2.2 ANALYTICAL METHODS DEVELOPMENT
A calibration curve we were used to figure out the concentration of drug in a sample,
by comparing the sample to a set of predetermined data.
2.2.1 Preparation of calibration curve of oxaliplatin in phosphate buffer solution
using U.V method (PH-7.4, λmax 207)
The wavelength of maximum absorbtion (λmax) was determined by scanning
the drug. The media used for oxaliplatin was phosphate buffer (PH-7.4). The buffer
was prepared according to I.P (2007).
Preparation of stock solution and standard curve: Accurately weight 10 mg of
oxaliplatin was dissolved in sufficient quantity of water and volume was made up to
100 ml with hplc grade water .This gave a stock solution of 0.1 mg/ml. Aliquots were
prepared by transferring 0.2,0.4,0.6,0.8 and 1.0 in 10 ml of volumetric flask then
volume was made upto 10 ml with phosphate buffer solution this gave a
concentration of 2,4,6,8 and 10 µg/ml .The absorbance was measured at 207 nm
against phosphate buffer solution as a blank .
2.2.2 Preparation of calibration curve of oxaliplatin in water using U.V method
at λmax 207nm
The oxaliplatin was estimated spectrophotometrically in water by systronics
double beam spectrophotometer. The calibration curve was prepared as follows .
Preparation of stock solution and standard curve: Accurately weighed 10
mg of oxaliplatin was dissolved in sufficient amount of water and volume was made
upto 10 ml with hplc grade water to produce stock solution of concentration 1 mg/ml.
Aliquots were prepared by transferring 0.2,0.4,0.6,0.8 and 1.0 ml in 10 ml of
volumetric flask, then volume was made upto 10 ml with HPLC water this gave a
concentration of 2,4,6,8 and 10 µg/ml . The absorbance was measured at 207 nm
against water as a blank.
27
2.2.3 Preparation of calibration curve of oxaliplatin in water using HPL method
Equipment: The HPLC was performed with a modular system consisting of a
variable wavelength UV visible detector and auto sampler.
Mobile phase: Mobile phase consisted of a mixture of methanol: water (25:75).
Preparation of stock solution and standard curve: Accurately weight quantity of
drug 10 mg was taken in 10 ml volumetric flask ,dissolved in sufficient quantity of
water and volume was made upto 100 ml with HPLC grade water. This gave a stock
solution of .1 mg/ml.
Aliquots were prepared by transferring 0.1,0.2,0.3 and 0.4 ml in 10 ml
volumetric flasks and volume was made up to 10 ml using mobile phase (methanol
and water in the ratio of 25:75), then all aliquots were filtered by whattman filter
paper this gave a concentration of 1,2,3 and 4 µg/ml. The solution was then injected
in the 200 µl loop attached to the pump, the mobile phase was run at the rate of 1
ml/min. Detection were done at 207 nm sample concentration were calculated by
measuring covered area and plotting against standard concentration.
2.2.4 Preparation of calibration curve of oxaliplatin in plasma using HPLC
method.
The HPLC method reported by Srinubabuin et al. (2006) was followed for
estimation of oxaliplatin in biological sample.
Equipment: The hplc was performed with a modular system consisting of a variabe
wavelength U.V. visible detector and auto sampler.
Mobile phase: Mobile phase consisted of a mixure of methanol: water (25:75).
Preparation of stock solution and standard curve: Accurately weight quantity of
drug 10 mg was taken in 100 ml volumetric flask ,dissolved in sufficient quantity of
water and volume was made upto 10 ml with HPLC grade water. This gave a stock
solution of .1mg/ml.
28
Aliquots were prepared by transferring 0.1,0.2,0.3, 0.4 and 0.5 ml in 10 ml
volumetric flasks this gave a concentration of 1,2,3,4 and 5 µg/ml mixed with 0.2
ml of rat plasma homogenated and the volume was made upto 10 ml then all aliquots
were filtered by whatman filter paper. The solution was then injected in the 200 µl
loop attached to the pump. The mobile phase was run at the rate of 1ml/min.
Detection were done at 207 nm sample concentration were calculated by measuring
peak height and plotting against standard concentration.
2.3 PREPARATION OF LIPOSOMAL FORMULATION WITH AND
WITHOUT PAMAM DENDRIMER
Liposomal preparation having different lipid composition, aqueous phase
composition or quantity of oxaliplatin in feed were prepared by film hydration method
encoded and summarized in (Table 4.2.1) The lipid (2.59×10ˉ5m)consisting of soya
lecithine ,cholesterol in 1.5:1:1 molar ratio, with and without PAMAM
dendrimer(1.4x10ˉ7m ) form a film that was hydrated with 10 ml aqueous phase. The
liposomes were sonicated at 60 w for 2min with a bath sonicator,. Oxaliplatin
solution (1mg/ml) was then added to the dispersion and allowed to stand in 60ᵒ bath
for 6 hrs with mild stirring with PH adjustment to 7.4.
2.3.1 Characterization
2.3.2 Particle size determination
2.3.3 Optical microscopic method
Particle size of denrimeric liposomal formulations was determined by optical
microscopy in which the eye piece micrometer were calibrated by stage micrometer.
A 50µl of dispersion was placed on a microscopy glass slide below the objective of
the microscope and was spread evenly over the slide and a cover slip was placed over
the sample .They were left to equilibrate until the particles slow down their visible
movements. Then particle counts of 100 numbers for its size were determined for
each formulation under 10 X magnification. Values are expressed as average particle
size( Robinson, et al, 1994).
29
2.3.3 particle size and size distribution
It was determined by Malvern Particle Size analyzer from CDRI Lucknow (sample code were: TD101, SD104, ND104).
2.3.4 Estimation of dendrimer in external phase
Presence of dendrimer in external phase in blank experiment was observed by
shift in absorbtion maximum of copper sulfate. 1% copper sulfate solution was added
to the 1ml of formulation and uv absorbtion spectra was recorded against the
formulation blank. The shift in absorbtion maximum was considered as a qualitative
measured for the presence of the dendrimer(Diallo et al.,1999).
2.3.5 Effect of Tris dendrimer and NH2 suface on encapsulation efficiency
It was dertermined by taking .1ml of formulation in 10 ml of volumetric flask
and volume was made upto 10ml with hplc water this gave a concentration of
0.01mg/ml then the solution was analysed by Spectrophotometrically at 207nm.
2.3.6 In -vitro release study
The release of oxaliplatin was studied by placing the formulation equivalent
to 1mg of drug in pretreated dialysis tube with dialysis membrane. The tube was
immersed into vessel containing 100ml of phosphate buffer on hot plate .The
release studies were carried out at temperature 37ᵒc under mechanical stirrer at 50
rpm. At fixed interval of 1hr ,2hr ,3hr upto 24hr samples were withdrawn from the
solution and oxaliplatin content were determined by Spectrophotometrically.
2.3.7 Stability studies
All the selected formulations were subjected to a stability testing for 0,1,2 and
3 days at a room temperature 37ºc .Then all the selected formulations were visually
analyzed for the change in appearance ,pH or drug content (Table :4.1.10).
2.3.8 In-vivo studies
30
Determination of Pharmacokinetic oxaliplatin
The albino rats (average wt.250 gm) were divided into three groups each
containing six rats. First group was administered drug solution equivalent to 15
mg/kg of oxaliplatin through i.v route. Second group was administered NH2D(G4)L2
liposomal formulation and third group was administered OHD(G4)L3 liposomal
formulation. The blood sample was collected at 5,10,15,20,25 hr time intervals from
the tail vein in a heparinized syringe. The sample were centrifuged and plasma were
collected. From each sample 200 µl of plasma were taken in different volumetric
31
3.0 PREFORMULATION
3.1 Identification of Oxaliplatin
The identification test were perform according to B.P 2010. All identification
test were positive. Chemical test was positive that is found similar to reported as per
B.P (2010). The melting point was found 198-292ºC which is similar to reported by
[Overington, et al, 2006], all the data are shown in. (Table 3.1).
Table 3.1 Physicochemical parameter of oxaliplatin
Parameter Observation Appearance
Melting point
White crystalline powder
198ºc and 292ºc
3.2 Scanning of drug
The drug sample was scan in spectrophotometer in the range λmax 200-400nm
.The absorption maxima for oxaliplatin were found at 216,273,254 and 264nm, which
is fully complied with pharmacopoeia specification. The drug sample was almost 99%
pure as analyze by official method (Figure 3.2).
Fig 3.2 U.V spectra of oxaliplatin (Reported )
32
33
3.2 Fig U.V spectra of oxaliplatin (sample)
34
3.3 Infrared spectroscopy
The infrared spectra of the drug was performed and compared with the
standard spectra of drug, which confirm the identity of drug is shown in (Figure 3.3).
3.3 Fig IR spectra of oxaliplatin (Reported )
3.3 Fig I R spectra of oxaliplatin (sample)
35
The powder drug was studied for solubility in various solvents. It was slightly
soluble in water , very slightly in methanol and practically soluble in ethanol and
acetone (Table 3.4).
Table 3.4 Solubility of oxaliplatin in different solvent
Solvent Solubility
Water Slightly
Methanol Very slightly
Ethanol Practically soluble
Acetone Practically soluble
3.5 Analytical method development
After identification on assessment of purity of sample, calibration curves were
prepared in phosphate buffer (PH-7.4) solution, HPLC grade water and in biological
36
sample for oxaliplatin using spectrophotometric method, for in vitro performance
evaluation during formulation development (Table 3.5,3.6,3.7,3.8 and figure 3.5, 3.6,
3.7, 3.8). The spectrophotometric method was selected to its sensitivity and
simplicity. The absorption maxima (λmax) selected was 207 nm for oxaliplatin shows
excellent linearity and obeys bears-lamberts law in the concentration used (20-100
µg/ml) for oxaliplatin . The correlation coefficient values were greater than 0.99 for
the drug.
Table 3.5.1 Standard curve data of oxaliplaatin in Phosphate buffer at λmax
. 207nm
S.no. Conc. (µg/ml) Absorbance (nm)
1.
2.
3.
4.
5.
2
4
6
8
10
0.540
0.772
0.831
0.842
0.972
Fig 3.5.1 Standard curve of oxaliplatin in phosphate buffer at λmax
37
207nm
1 2 3 4 5 6 7 8 9 10 110
0.2
0.4
0.6
0.8
1
1.2
f(x) = 0.1016 xR² = 0.999939764871648
Conc(ug/ml)
Abso
rban
ce(n
m)
Table 3.5.2 Standard curve data of oxaliplatin in Water by UV method at
λmax 207nm
S.NO Conc.(µg/ml) Absorbance(nm)
1.
2.
3.
4.
5.
2
4
6
8
10
0.13
0.15
0.23
0.37
0.63
38
Fig 3.5.2 Standard curve of oxaliplatin in Water by UV Method at λmax
207nm
1 2 3 4 5 6 7 8 9 10 110
0.1
0.2
0.3
0.4
0.5
0.6
0.7
f(x) = 0.0610508816062502 xR² = 0.999760628769013
Conc(ug/ml)
Abso
rban
ce n
m
Table 3.5.3 Standard curve data of oxaliplatin in water by HPLC method at
λmax 207nm
S.NO Conc(µg/ml) Area Height Area% Height%
1. 1 82162 218799 46.595 23.737
2. 2 156419 16258 21.219 9.811
3. 3 295231 17327 29.896 18.629
4. 4 337860 13573 23.023 18.711
Fig 3.5.3 Standard curve of oxaliplatin in water by HPLC method at
39
λmax 207nm
0.5 1 1.5 2 2.5 3 3.5 4 4.50
500000
1000000
1500000
2000000
2500000
3000000
f(x) = 609666.666666667 xR² = 0.999695480924973
Conc (ug/ml)
Area
Table 3.5.4 Standard curve data of oxaliplatin in Biological sample by
HPLC method at λmax 210 nm
S.NO. Conc.
(µg/ml)
Area Height Area% Height%
1 1 294374 13573 23.023 18.711
2 2 534940 17327 29.896 18.629
3 3 954425 23204 24.516 14.557
4 4 959599 16258 21.219 9.811
5 5 21205005 218799 46.595 23.757
40
Figure 3.5.4 Standard curve of oxaliplatin in biological Sample By HPLC
method at λmax 210n
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.50
5000000
10000000
15000000
20000000
25000000
f(x) = 4096363.63636364 xR² = 0.999870781310171
Conc(ug/ml)
Area
3.6 LIPOSOMAL FORMULATION WITH AND WITHOUT DENDRIMER
Liposomal formulation ,differing in lipid composition aqueous phase
composition or quantity of oxaliplatin in feed were prepared by film hydration method
encoded in (Table: 4.1). To summarize three type of liposomal formulations were
prepared neutral and negatively charged soyalecithine and cholesterol basic liposomal
formulations. PAMAM dendrimer G4 generation were included in these formulation
in which NH2 surface PAMAM dendrimer was used in lipid film formation and Tris
dendrimer was used in hydration step.
3.6 Table Coding of liposomal formulation with or without PAMAM
dendrimer .
Formulation
code
LIPID FILM COMPOSITION Aqueous phase
(m)Lecithine (m) Cholestrol (m) Dendrimer (m)
41
SI L1 2.59×10-5 2.21×10-5 dr(2.5×10-5).wa
NH2 D(G4) L2 9.4×10-5 5.4×10-5 1.4×10-7 dr(2.5×10-5).wa
OH D(G4) L3 9.7×10-4 2.1×10-4 dr(2.5×105).OH
(5.5×10-7)
SI L1:- Simple liposome 1
NH2 D(G4) L2:- NH2 dendrimer (G4) liposome 2
OH D(G4) L3:- OH dendrimer liposome 3
Molecular weight of NH2 dendrimer : 14215
Stock solution : 2 mg/ml
Molecular weight of OH dendrimer : 18128
Stock solution : .1 mg/ml
Molecular weight of oxaliplatin : 397.29gm/mol
Stock solution of drug : 1mg/m
3.6.1 Particle size determination
Liposomal formulation containing dendrimer were sonicated for reduction in
size and . The size of freshly filtered liposome were given in( Table 4.2.2,4.2.3) . The
particle size SI L1 of liposomal formulation without dendrimer could be more than
NH2 D(G4) L2 and OH D(G4) L3.The particle sizes reported have were determined
by instrumentally using optical microscope and malveren particle size analyzer
3.6.1 Table Average particle size by Optical Microscope
FORMULATION CODE AVERAGE PARTICLE
SIZE(µm)
SI L1 0.76 ± 0.034
NH2 D(G4) L2 0.16 ± 0.02
42
OH D(G4) L3 0.74 ± 0.02
3.6.1 Fig Average particle size by Optical Microscope
SL1 NH2(4G)L2 OHDL30
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
formulation code
size(
µm)
[
3.6.1 Table Average particle size by Malvern Particle Sizer
FORMULATION CODE AVERAGE PARTICLE SIZE (µm)
SI L1 0.78 ±0.012
NH2 D(G4) L2 0.18±0.014
OH D(G4) L3 0.67±0.011
3.6.1 Fig Average particle size by Malveren Particle Sizer [
43
SI LI NH2 D(G4) L2 OH D(G4)L30
0.10.20.30.40.50.60.70.80.9
formulation code
size(
µm)
3.6.2 Estimation of dendrimer in external phase
The copper sulfate solution have absorbtion maximum at 209nm on addition
of dendrimer the maximum is shifted to 216nm indicating red shift. This can visually
be observed by change in color of copper sulfate from dark blue to light blue. This
reaction is very tentative and could detect small amount of dendrimer in solution by
shift in absorbtion maxima of copper sulfate solution .
44
3.6.2 Fig UV spectra of copper sulfate
45
3.6.2 Fig Estimation of dendrimer in external phase
46
3.6.3 Invitro release study
The release was lowered in dendrimeric liposomal formulation than simple
liposome (Table:). The charge of liposomal lipid was not found to affect the release of
drug with different type of PAMAM dendrimer. The study shows that it is possible to
modulate the release of drug from the dendritic liposomal formulations by changing
the composition of lipid and dendrimer.
3.6.3 Table Drug release of oxaliplatin from dendritic liposomal formulation
TIME (hr) % DRUG RELEASE SI L1 OH D(G4) L3 NH2 D(G4) L2
1 19.6 15.7 9.2 2 37.7 25.9 19.6 3 49.4 38.6 28.3 4 69.3 47.9 36.8 5 79.7 57.34 47.8 6 79.15 63.60 58.1 7 79 .39 63.19 58.11 8 79.67 63.47 58.27 9 63.57 58.47 10 63.67 58.67 24 63.87 58.79
3.6.3 Fig Drug release of oxaliplatin from dendritic liposomal formulation
0 1 2 3 4 5 6 7 8 9 10 240
10
20
30
40
50
60
70
80
90
NH2 D(G4)L2OHD(G4)L3SI L1
Time(hr)
% D
rug
rele
ase
47
3.6.4 Effect of Tris dendrimer and NH2 surface on drug entrapment
The encapsulation of drug was prapotional to the Tris and NH2 surface
dendrimer (Table:3.4.4) .The liposome containing G4 NH2 surface dendrimer were
able to encapsulate drug approximately two or four times greater than OH D(G4) L3
and SI L1 another possibility may be that the proportion of the dendrimers attached
with the surface phosphate group was unable to encapsulate drug due to their open
structure.
3.6.4 Table Effect of Tris dendrimer and NH2 suface on drug entrapment
FORMULATION CODE ENCAPSULATION EFFICIENCYSI L1 68%NH2 D(G4)L2 92.9%OH D(G4)L3 87.9%
3.6.5 Stability studies
The stability of 4th generation dendrimer containing formulation (NH2 G4D
L2) was slightly greater than( OH G4D L3) and (SI L1) formulation. Perhaps because
of the protective effect of higher generation dendrimer on the encapsulation of drug.
The formulation kept at 37ºc were clear. There was drop in pH values from adjusted
7.2 to 5.2 suggesting that there was hydrolysis of phospholipids and pH drop was
produced despite that use of fully hydrogenated phospholipid .Overall the stability of
the formulation was sufficiently promising for its successfully applicability.
3.6.5 Table Stability Studies
Serial no Formulation Days Appearance pH Drug content(%)
1. SI L1 0 Clear 6.7 681 Clear 6.7 67.12 Clear 6.3 66.43 Clear 6.3 65.8
2. NH2 D(G4)L2 0 Clear 7.2 92.91 Clear 7.2 91.52 Clear 7.1 91.33 Clear 7.1 89.2
3. OH D(G4)L3 0 Clear 5.4 87.91 Clear 5.4 86.12 Clear 5.3 84.63 Clear 5.2 83.1
48
3.6.6 In –vivo studies
From the plasma clearance data of oxaliplatin from formulation ,the pharmacokinetics
parameters were calculated by open one compartment model indicated that AUC of
formulation or oxaliplatin increased by 3.74, 3.96, 4.30 for OH D(G4)L3, NH2
D(G4)L2 respectively than free drug .The AUC also increased with different
concentration and types of PAMAM dendrimer .This may be due to open to globular
structure of dendrimer .
3.6.6 Table Determination of pharmacokinetics parameters
Time Drug FormulationOH D(G4)
Formulation NH2D(G4)
0 11 ± 0.1 10 ± 0.21 8 ± 0.145 15.1 ± 0.14 17.2 ± 0.36 20.1 ± 0.4610 12.5 ± 0.22 20.9 ± 0.14 23.8 ± 0.2815 10.9 ± 0.24 20.11 ± 0.16 23.15 ± 0.2520 20.8 ± 2.7 23.14 ± 1.0525 20.5 ± 2.5 23.11 ± 1.05
3.6.6 Fig Determination of pharmacokinetics parameter
0 5 10 15 20 25 300
5
10
15
20
25
Drug OHD(G4)L3NH2D(G4)L2
Time(hr)
Plas
ma
drug
conc
entr
ation
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3.6.6 Table Pharmacokinetic parameter
Pharmacokinetics parameters
Drug (oxaliplatin) Formulation OH D(G4)l2
Formulation NH2 D(G4)l3
Cmax( µg/ml) 18.7 19.8 21.5
tmax (hr) 5 5 5
Auc (µg h/ml) 3.74 3.96 4.30
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4.0 SUMMARY AND CONCLUSION
Dendrimers are a new class of synthetic polymers which have the structure
like a tree or star shape, with a central core, interior branches and terminal groups
which decorate the surface. Cavities inside the core and the interior branches can be
modified to carry hydrophobic and hydrophilic drugs. Recently, dendrimers have
successfully proved themselves as promising nanocarriers for drug delivery because
they can render drug molecules a greater water-solubility, bioavailability, and
biocompatibility. The terminal groups on the surface can also be adapted to carry
drugs. Thanks to the unique molecular architecture it is possible to design the
hyperbranched polyester in numerous ways to reach desired properties in different
application.
Liposomal drugdelivery systems have come of age in recent years, with
several liposomal drugs currently in advanced clinical trials or already on the market.
It is clear from numerous pre-clinical and clinical studies that drugs, such as antitumor
drugs, packaged in liposomes exhibit reduced toxicities, while retaining, or gaining
enhanced, efficacyons. Liposomal drugs exhibit reduced toxicities and retain, or gain
enhanced, efficacy compared with their free counterparts. Liposomes that allow
enhanced drug delivery to disease sites, by virtue of long circulation residence times,
are now achieving clinical.
In the present investigation include to prepare liposomal formulation of
oxaliplatin-pamam complex with different concentration of PAMAM dendrimer and
their characterization.
Two types such as NH2 surface and Tris dendrimer of different concentration
of pamam dendrimer was used for encapsulation of oxaliplatin into liposome through
film hydration method . Liposomal preparation having different lipid composition,
aqueous phase composition or quantity of oxaliplatin, The lipid consisting of soya
lecithine ,cholesterol in 1.5:1:1 molar ratio, with and without PAMAM dendrimer
form a film that was hydrated with 10 ml aqueous phase with. The liposomes were
sonicated at 60 w for 2min with a bath sonicator and Oxaliplatin solution (1mg/ml)
was then added to the dispersion and allowed to stand in 60ᵒ bath for 6 hrs with mild
stirring with PH adjustment to 7.4. Then chraeteriation had done after preparation .
51
Particle size of denrimeric liposomal formulations was determined by optical
microscopy in which the eye piece micrometer were calibrated by stage micrometer.
Then dispersion was placed on a microscopy glass slide below the objective of the
microscope and was spread evenly over the slide and a cover slip was placed over the
sample. Then particle counts of 100 numbers for its size were determined for each
formulation. Particle size and size distribution was determined by Malvern Particle
Size analyzer from CDRI Lucknow.
Presence of dendrimer was observed by shift in absorbtion maximum of
copper sulfate. 1% copper sulfate solution was added to the 1ml of formulation and uv
absorbtion spectra was recorded against the formulation blank..
Effect of Tris dendrimer and NH2 suface on encapsulation efficiency was
dertermined by taking .1ml of formulation in 10 ml of volumetric flask and volume
was made upto 10ml with then the solution was analysed by Spectrophotometrically
at 207nm.
Invitro release study of oxaliplatin was studied by placing the formulation
drug in pretreated dialysis tube with dialysis membrane. The tube was immersed into
vessel containing phosphate buffer on hot plate .The release studies were carried out
at temperature 37ᵒc at 50 rpm. At fixed interval upto 24hr samples were withdrawn
from the solution and oxaliplatin content were determined by Spectrophotometrically.
Stability studies was found by placing the formulation under room temperature
for 3 days and where the formulation were relatively stable .
Invivo studies were performed in male albino rats (150-200g) . All the
animals were procured from the disease-free animal house of Central Drug Research
Institute, Lucknow .Then pharmacokinetics parameters such as AUC Cmax and
tmax was found for drug and different formulation of PAMAM dendrimeric
liposomal system and bioavilability were determined from the plasma profile of
oxaliplatin .,
Conclusion : Thus liposomal drug delivery system incorporating a complex of
oxaliplatin-PAMAM G4 dendrimers was prepared and compared to simple liposomal
formulation encapsulating oxaliplatin with the same lipid composition regarding
release properties, encapsulation efficiency, stability studies, particle size and
52
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