DELIVERY OF NEUROACTIVE MOLECULES FROM BIODEGRADABLE MlCROSPHERES

Xudong Cao

A thesis submitted in confomity with the requirements for the Degree of Master of Applied Science.

Department of Chernical Engineering and Applied Chemistry. University of Toronto

O Copyright by Xudong Cao

1997

Nationai Library 1*1 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et Bibliographic Services services bibliographiques

395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A O N 4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence dowing the exclusive permettant ii la National Library of Canada to Bïbiiothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nIm, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantid extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otheMrise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

Delivery of Neuraactive molecules from biodegradable microspheres M.A.Sc. Degree thesis, 1997 By Xudong Cao Department of Chemicaf Engineering and Applied Chemistry, University of Toronto

ABSTRACT The defining characteristic of traumatic central newous system injury is the inability

of damaged axons to repair themselves or regenerate. Delivery of neurotrophic factors for

exarnple, may enhance axonal newe regeneration afier spinal card injury. The aim of this

project is to encapsulate and deliver neuroactive molecules, such as nerve growth factor

(NGF), from biodegrada ble polymeric microsp heres.

Biodegradable polymeric microspheres were prepared from poly(ladde-CO-glycolide)

50150 (PLGA50/50), PLGA85115, poly(~-caprolactone) (PCL) and a blend of PLGA50150. the

latter of which enabled the degradation rate to be tailored. Proteins (i.e. NGF and ovalburnin)

were successfully encapsulated into biodegradable polymeric rnicrospheres by the solvent

evaporation technique. The release profile ovalbumin was followed by ultraviolet-visible

spectrophotometry (UV-vis) and that of NGF by the NGF-enzyrne linked immunosorbent

assay (ELISA). The degradation profiles of polyrner microspheres were followed by changes

in molecular weight, morphology and m a s loss. PLGA50150 showed the fastest degradation

followed by PLGA85175 and the blend , with PCL showing the slowest degradation.

Ovalbumin. having the similar molecular weight to that of NGF, was encapsulated as a

model dnig for nerve growth factor in release study. The data obtained from both release

and degradation studies suggested that protein release from PLGA50150 was mainly due to

degradation of polyrner matrix whereas protein release from PCL was mainly due to the

interconneded pore structures. The bioactivity of released NGF was assessed by adrenal rat

pheochromacytoma (PCi2) cell culture for up to 90 d. Results showed that after 90 d,

released NGF maintained its bioactivii to induce neurite outgrowth.

The author would like to take this opportunity to express his greatest

gratitude and appreciation to those who have influenœd and helpcd him

throughout the last two yean.

Professor Molly Shoichet for her guidance, advice and encouragement

throughout the project The helpful comments, discussions and critical review of

the thesis provided by her are also gratefully acknowledged.

Dr. Xue-Ying Shen for her valuable technical advice and discussions on

ce11 culture. which contributed to the successful completion of this work.

Professor J .E. Davis for generously providing cell culture facilities, Yu-Ling

Cheng for generously providing the access to ultraviolet-visible

spectrophotometer and Professor M.A. Winnik for generously providing the

access to gel permeation chromatography.

The author also feels in deep debt to the colleagues in the lab, especially

Yen Wah Tong, Sabeshan Kanagalingam, Chantal E. Holy and Samar

Saneinejad for their friendship and seffless support without which the completion

of this work would not be possible.

Special gratitude is extended to my parents and my brother who gave their

love and moral support throughout the coune of the work.

iii

TABLE OF CONTANTS

1. INTRODUCTION

Nerve regeneration Nerve growth factor Drug delivery to the CNS Historical perspective of dmg delivery systems Microsp heres and microcapsules Literature sunrey Poly (lactidelg lycolide) polymers Biocompatibility Methodology of microencapsulation Project goals H ypothesis Objectives

Materials Methods Microencapsulation C haracterization of microsp heres Particle size and size distribution Scanning electron microscopy Distribution of protein within the microsphere matrix Degradation study Mass loss Scanning electron microscopy Gel penneation chromatography (GPC) Release study of OVA from biodegradable microspheres The detemination of efficiency of encapsulation PC12 cell culture Assessment of bioactivity of released NGF Preliminary 5 d assessment Assessment of bioactivity of released NGF up to 90 d Release study of NGF: NGF-ELISA Test principle of NGF-ELISA Assay procedures Statistics

3 RESULTS

C haracterization of microsp heres ~ o u l t e r counter resuk SEM Protein distribution within the rnicrosphere matrix Degradation study Mass loss SEM results GPC results Efficiency of encapsulation Release study Release of OVA as a model dnig Release of NGF Assessment of bioactivity of encapsulated NGF Preliminary assessrnent for 5 d Assessment of bioadivity of released NGF up to 90 d

4 DISCUSSION

4.1 Morphology of microspheres 4.2 Degradation 4.3 Efficiency of encapsulation 4.4 Protein stability 4.5 Release mechanism

5 CONCLUSIONS

6 FUTUREWORK

7 APPENDICES A? Preparations of solutions for NGF-ELISA A2 Construction of UV-vis calibration curve

8 REFERENCES

LIST OF TABLES

Table 1.1 Microspheres prepared by the solvent evaporation technique 13

Table 3.1 Average size of microspheres 31

Table 3.2 Average neurite outgrowth extending frorn each cell 43

Table 4.1 Polydispersity index 55

Figure 1.1 Representative schemes of microsphere and microcapsules

Figure 1.2 Chemical structures of a) PM. b) PGA, c) PLGA and d) PCL

Figure 1.3 Schematic configuration of a standard spray dryer

Figure 1.4 Scheme of spray wating

Figure 2.1 Schematic diagram of solvent evaporation technique used to prepare microspheres

Figure 2.2 Schematic illustration of the principle of NGF-ELISA

Figure 3.1 a) diameter distnbution of PLGA50150 microspheres without protein powder encapsulated

Figure 3.1 b) diameter distribution of PLGA85115 microspheres without protein powder encapsulated

Figure 3.1 c) diameter distribution of PCL microspheres without protein powder encapsulated

Figure 3.1 d) diameter distribution of blend of PLGA50150 and PCL microspheres without protein powder

Figure 3.1 e) diameter distribution of PLGA50150 microspheres with protein powder encapsulated

Figure 3.1 f ) diameter distribution of PLGA85115 microspheres with protein powder encapsulated

Figure 3.1 g) diameter distribution of PCL microspheres with protein powder encapsulated

Figure 3.1 h) diameter distribution of blend PLGA50150 and PCL microspheres with protein powder encapsulated

Figure 3.2 SEM micrographs of microspheres a) without and b) with protein powder encapsulated

Figure 3.3 ?CL microsp heres without protein encapsulated

Figure 3.4 Protein distribution within PLGA85115 microsphere matrix

vii

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.1 O

Figure 3.1 1

Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 3.16

Figure 3.16

Figure 3.17

Figure 3.17

Figure 3.1 8

Degradation of microspheres determined by mass loss 34

SEM micrographs of microspheres after 35 d of degradation a) PLGA50/50; b) PLGABW15; c) PCL and d) blend of PLGA50/50 and PCL (1 : 1, w h ) 35

SEM micrographs of microspheres after 70 d of degradation (a) PLGA50/50; (b) PLGA85115; (c) PCL and (d) blend of PLGA50/50 and ?CL (1 :1, w/w) 36

Change in weight average molecular weight as detemined by GPC for PLGA50150 microspheres 37

Change in weight average molecular weight as detemined by GPC for PLGA85115 microspheres 38

Change in weight average molecular weight as detemined by GPC for PCL microspheres 38

Change in weight average molecular weig ht as determined by GPC for blend microspheres 39

Efficiency of microencapsulation 40

Cumulative release profile of OVA from biodegradable polymeric microspheres as a percentage of the initial OVA encapsulated

Cumulative release profile of OVA from biodegradable polyrneric microspheres expressed as mass of protein released over time

NGF release determined by ELISA

a) Neurite outgrowth after 4 d of release and 24 h of incubation

b) Neurite outgrowth after 4 d of release and 48 h of incubation

a) Neurïte outgrowth after 8 d of release and 24 h of incubation

b) Neurite outgrowth after 8 d of release and 48 h of incubation

a) Neurite outgrowth after 12 d of release and 24 h of incubation

Figure 3.18 b) Neurite outgrowth after 12 d of release and 48 h of incubation

Figure 3.1 9 a) Neurite outgrowth after 20 d of release and 24 h of incubation

Figure 3.1 9 b) Neurite outgrowth after 20 d of release and 48 h of incubation

Figure 3.20 a) Neurite outgrowth after 44 d of release and 24 h of incubation

Figure 3.20 b) Neurite outgrowth after 44 d of release and 48 h of incubation

Figure 3.21 a) Neurite outgrowth after 91 d of release and 24 h of incubation

Figure 3.21 b) Neurite outgrowth after 91d of release and 48 h of incubation

Figure 3.22 Bioactivy of NGF was assessed after 24 h of incubatingreleased NGF with PC12 cells by assessing the average number of neurites per cell body 51

Figure 3.23 Bioactivty of NGF over time was assessed after 24 h of incubating released NGF with PCi2 cells by calculating the percentage of cells bearing one or more neurites that exceeded the cell body length 51

Figure 4.1 GPC chromatographs of PCL at (a) t=O, (b) t=14 d, and (c) t=21 d

Figure 4.2 Schematic illustration of degradation controlled mechanism 60

Figure 4.3 Schematic illustration of diffusion controlled mechanisrn 60

Figure 4.4 Plot of cumulative release vs. t'" 62

LIST OF APPENDICES

A l Preparations of solutions for NGF-ELISA A2 Construction of UV-vis calibration curve

1. INTRODUCTION

1.1 Nerve regeneration

Traumatic injury to the central nervous system (CNS) is a significant clinical

problem which is under intense research. The injury to the mature CNS is

characterized by the inability of the axons to repair themselves or regenerate.

Functional recovery from such injuries is therefore nonexistent, particularly for

those patients with spinal cord injuries. Although palliative measures are

currently available to improve the quality of a patient's life, there is still no

accepted treatment to restore impaired sensory or motor function. leaving

patients penanently disabled.

While some injured CNS neurons can send out axonal processes, the

regenerative activity after traumaüc lesions is Yaint and sluggishn (11. In contrast,

lesions to peripheral nerves evoke rigorous axonal sprouting. Implantation

experiments have revealed that CNS neurons can extend axonal processes into

peripheral nervous system (PNS) grafts but not into CNS tissue [Il. This was

attributed to a number of tissue elements which are available in the PNS but are

lacking in the CNS. For example, the production of nerve growth factor (NGF)

and NGF receptor (NGFr) in Schwann cells of axotomized nerves rnay have a

trophic and guiding action on regrowing PNS axons [2,3]. The absence of

extracellular matrix components, such as the basal lamina-associated

glycoprotein larninin. rnay impede CNS axon regrowth [3]. Researchers have

shown promising results in attaining nerve cell regeneration in the CNS. Knoops

et al. [4] studied the axonal regeneration of septal cholinergic neurons after

lesioning the septohippocampal pathway of adult rats and implanting tubes

containing sections of predegenerated sciatic nerves. After six weeks of

implantation, regenerated axons were observed mainfy longitudinally oriented

along the axis of the sciatic newe whereas no regenerated structures were found

in the empty conduit. Marchand et al. [5] also reported that spinal axons grew

into a collagen matrix implanted between the stumps of a transected spinal cord.

Regeneration over 2-3 mm optic nerve lesion was obsetved by Aebischer et al.

using a semipermeable acrylic copolymer channel lined with peripheral Schwann

cells. It was wncluded that the incorporation of growth factors or tissue

transplants within the guidance channel lumen may have promoted more

extensive axonal elongation and myelination into and within the regenerated

structure [6]. Tuszynski m used grafts of favorable axonal growth substrates and

transient NGF infusions to promote rnorphological and functional recovery in the

adult rat brain after lesion of the septohippocarnpal projection. It was observed

that after 9 weeks of implantation, both long-terni septal cholinergie neuronal

rescue and partial hippocampal reinnervation were achieved, resulting in partial

functional rewvery on a simple task assessing habituation. It can be concluded

that (i) adult CNS tissue is unfavorable for regeneration; (ii) aduit CNS neurons

still possess the intrinsic mechanisms for axonal growth over long distances if

provided with a permissive environment [Il; and (iii) the administration of

neuroactive molecules into the CNS, such as neurotrophic factors, may help to

promote axonal nerve regeneration in the CNS.

1.2 Nerve growth factor

Growth factors are generally defined as polypeptides that, at very low

concentrations and through specific receptor mediated mechanisms, inliate and

sustain complex cellular processes. These processes include cell proliferation in

the case of cells capable of mitosis, and cell survïval, neurite outgrowth, and

biochemical differentiation in the case of post-mitotic cells such as neurons [8].

An increasing nurnber of growHi factors have been identified over the past few

decades, most of which are now grouped into several 'families'. At present, there

are at least 130 factors that have been identified and grouped into 20 different

growth factor families based on their structural homology [9]. NGF, a

neurotrophic factor, is found to be essential for normal neuronal developrnent; in

the adult animal, it en hances axonal regeneration [ I O . 1 1,121.

1.3 Drug delivery to the CNS

It may be beneficial to administer NGF into the CNS to enhanœ CNS nerve

recovery. Delivery by the conventional forms of dtug administration is, however,

inadequate because the BBB limits transport of molecules into the CNS. The

BBB contains tight endothelial cell junctions and acts like a selectively permeable

membrane by rigorously maintaining a well-defineci, homeostatic environment

[13, 141. It restricts the transport of molecules, except small neutral arnino acids

and lipophilic molecules. preventing the entry of potentially damaging

substances. Systernic delivery of molecules aimed at the CNS by conventional

methods also encounter with problems that rnay inhibl the successful clinical

application due to (i) adverse systemic effects, (ii) peripheral metabolism, (iii)

erratic absorption and (iv) poor patient cornpliance [15]. For example, when L-

dopa, the precursor for dopamine was administered orally in a standard tablet

formulation, the response fiuctuated and dopamine was widely distributed

throughout the body [15]. Due to poor transport across the BBB, only about 1 %

of the administered dose was available to the brain. When administered by an

implantable drug delivery systern (DDS), L-dopa can be delivered directly to the

CNS. Moreover, the DDS maintained the drug dosage within a desired

therapeutic range for a predetemined tirne via a controlled release mechanism.

1.4 Historical perspective of drug delivery systems

The concept of polymeric DDS was first introduced in the early 1960s by

Folkman and Long [16]. They fabricated heart valves using biocompatible

silicone elastomer that absorbed certain dyes from solution and subsequently

released these dyes reversibly. This observation triggered extensive studies on

the feasibility of using silicone polymer capsules as carriers for prolonged,

continuous and subcutaneous administration of cardioregulatory drugs. These

investigations dernonstrated the concept of controlled drug delivery by confining

a depot of active ingredient within a polymeric vehicle. Only microdoses of the

ingredients diffused through the capsule wall at a controlled release rate, thereby

achieving a pre-determined dose level for a prolonged period of time. The

potential biomediml application of controlled drug delivery systems generated

widespread interest in silicone elastorner and other polymers, such as

polyethylene, polyamide, polystyrene copolymen and acetyl cellulose.

In the 1970s. the long-ten controlled release of contraceptive steroids,

narwtic antagonists, local anesthetics, antirnaladal and anticancer agents were

mainly investigated to attain potenüation of the pharmacological activities and

elimination of the inconvenienœ of repeated injections. For example, Dzuik [16]

introduced a steroid releasing silicone capsule for long-term fertility control.

Successive work led to the approval of Progestasert IUDs, a one-year

intrautedne birai control device, in 1976 by the FDA.

In the 1970s and 1980s, twa developments in the pharmaceutical industry

led to considerable interest in polymeric delivery systems for macromolecules

[17]. First, advances in genetic engineering allowed companies to produce a

variety of useful macromolecular drugs, such as hormones. Second, the isolation

of potent new macromolecules normally produced by the body. including

endorphins and enkephalines provided the basis for new types of

macromolecular drugs. Thereafter, in the late 1980s, biodegradable polymers

were investigated intensively for those peptides and proteins to both achieve

satisfactory efficiency and increase patient cornpliance.

Currently, DDS is being investigated for various applications. Brem et al.

[18] reported the delivery of nitrosourea carmusthe (BCNU) from biodegradable

polyanhydride disks. The disks were implanted into monkey brains to investigate

the feasibility of employing DDS to treat brain tumors. Other applications under

intensive investigation include transdermal drug delivery [19, 141, controlled

release of vaccines [20, 211, drug delivery through lungs (221 and drug delivery to

the CNS [15, 231. The DDS can be either biodegradable by using degradable

polymers such as poly(1actide-CO-glycolide) (PLGA) [24,25, 26, 27,281, fibnn

[29], and bovine serum albumin [30, 31, 32, 331; or biostable by using non-

biodegradable polyrners such as poly(ethy1ene oxide) (PEO) [34] and ethylene-

vinyl acetate copolymer (EVAc) [35]. Injectable and biodegradable microspheres

appear to be a particulariy ideal delivery systems because (i) they are small and

(ii) they degrade, obviating the necessity to remove the deviœ after the dnig

supply is exhausted.

1.5 Microspheres and microcapsules

As shown in Figures 1 .l a and 1.1 b, microspheres are fine spherical

particles that contain drugs that can be divided into two categorîes: (i)

homogeneous or monolithic microspheres in which the drug is dissolved or

dispersed throughout the polymer matrix and (ii) reservoir-type microspheres in

which the drug is surrounded by the polyrner matrix in a mononuclear state.

Particles belonging to (i) and (ii) are referred to as microspheres and

microcapsules, respectively. There is. however. no clear-cut between these two,

because the morphological structures are sometimes mixed. For exarnple, in

some systems, most of the drug core is surrounded by the polymer but sorne

drug molecules are dispersed separately or adsorbed on the surface of the

polymer. In this work, micros~here(s) is used to emphasize the monolithic type

morphological structures and microca~sule(s) is ernployed to emphasize the

mononuclear intemal morphology unless othenivise indicated.

a. microsphere b. microcapsule

Figure 1.1 Representative schemes of microsphere and microcapsule (a) a microsphere has

drug dispersed throughout white (b) a microcapsule has a core of drug within.

1.6 Liierature survey

1 .S. 1 porV (lactid&'gIycolide) polymers

Linear polyesters of lactide (PM) and glycolide (PGA) have been used for

more than three decades for a vanety of medical applications [36]. They were

among the first synthetic degradable polymers to find application as surgical

suture materials, controlled drug release deviœs, as well as orthopedic and

reconstrucüve implants [23]. Low molecular weight PLA and PGA are prepared

by the direct condensation of lactic acid and glycolic acid, respectively, whereas

poly(1actide-CO-glycolide) (PLGA) is obtained by the condensation of both lactic

and glycolic acids with or without catalysts. High molecular weigh polymers are

produced by the ring opening method with a catalyst such as dialkyl zinc [37].

Having an asymmetric carbon atom, lactic acid has two optical isorners.

Therefore, its polymer consists of L-, D- and Dl L- lactic acid in which the L- or O-

polymers have a crystalline f o m and Dl L- poiymers are amorphous and more

rapidly degraded [36].

Poly(~-caprolactone) (PCL), another biodegradable polyester has been

synthesized from the anionic, cationic or coordination polyrnerization of E-

caprolactone. It has a melting temperature between 59 OC and 64 OC and a glass

transition temperature (Tg) of -60 O C [38]. Figures 1.2 show the chemical

structures of PM, PGA, PLGA and PCL polymers. In PLGA, the molar ratio of

lactide and glycolide is denoted as m and n.

Figure 1.2 Chernical structure of a) PLA , b) PGA , c) PLGA and d) PCL * indicates the

asymmetric carbon atom.

PLA and its copolymers with less than 50% glycolic acid content are

soluble in common organic solvents such as acetone, chlorinated hydrocarbons,

tetrahydrofuran (THF) and ethyl acetate. PGA is insoluble in these common

organic solvents but is soluble in hexafluoroisopropanol (HFIP). The Tg of PLA,

PGA and PLGA is between 30 OC and 60 OC depending on the composition of

the polymers and the molecular weight.

Degradation of PLA, PGA, PLGA copolymers and PCL has been studied

extensively both in vitro and in vivo [39,40,41,42,43]. Degradation can be

followed by changes in mass [39, 38, 44,451, molecular weight [40,46, 47, 45,

481, morphology [39, 42,49,48], mechanical strength [42, 501 as well as in Tg

and crystallinity [40, 511. It has been shown that the degradation of PLA, PGA,

PLGA and PCL is due to the hydrolysis of the ester bonds along the backbone of

the polymer chains [38-411, and the degradation products are carboxylic acids

and alcohols.

Once a polyrner device is immersed into water, the first event that

happens is water uptake. This results in the bulk water penetration throughout

the polymer's amorphous domains whereas crystalline domains remain intact

because they are less accessible to water. Due to the hydrolysis of the ester

bonds along the polymer chah, the molecular weight of the degrading polymer

decreases. As the degradation tirne increases, some of the degraded polymers

are cleaved so small that they eventually become water soluble and leach out of

the polymer matrix, resulting in the mass loss of the degrading device. Random

scission of the ester bonds also resuits in a mechanical strength decay [40] and

decrease in Tg. The water impermeable domains, however, degrade much more

slowly, giving rise to a muitimodal molecular weight distribution as observed by

gel permeation chromatography (GPC) for a degrading semicrystalline polyrner

1401-

In general, factors that affect water uptake and hydrolysis reaction of the

ester bond will affect the degradation of polymers, Le. chernical structures,

degree of crystallinity, molecular weight, degradation conditions (e.g. pH and

temperature) and the size of degrading device [36].

Furthemore, two phenomena are of critical importance in considering the

degradation of PLGA. First, degradation causes an increase in the number of

carboxylic acid chain ends that are known to autocatalyse the ester bond

hydrolysis [39, 461. Second, only oligomen which are water soluble in the

surrounding aqueous medium can escape from the matrix. It therefore can be

predicted that during degradation, soluble oligomers that are close to the surface

can leach out once they are produced, whereas those which are located well

inside the matrix may remain entrapped and contribute to the autocatalytic effect.

The autocatalytic effect. in tum, results from the production of the carboxylic acid

groups, resulting in faster degradation. This ultimately results in a steep mass

loss observed in the PLGA degradation profile [39.46]. A subsequent sudden

release of degradation products in vivo, can render a sudden local environment

acidic and induce an inflamrnatory reaction or even tissue necrosis. Recum et al.

[44] successfully employed a blend of different molecular weight PLAs to

address this problem. In this work, we used a blend of two polymers - PLGA50150 and poly(~-caprolactone) (PCL) -No polymers with different

degradation rates, to circumvent this problem.

1.6.2 Biocompatibility

PLA. PGA, PLGA and PCL have good biocompatibility and now are being

used clinically in human therapy, such as sutures and bone fracture devices [52.

531. Visscher et al. [54] studied the tissue reaction to PLGA50/50 microspheres

by injecüng the rnicrospheres intrarnusculariy. There was no inflammatory

reaction to the microspheres after 56 days of injection when microspheres were

completely absorbed, although a few foreign body cells and the encapsulation of

microspheres by immature fibrous connective tissue were observed in the first

several days after injection. It was concluded that PLGA50/50 is a biocompatible

material. Another experiment was conducted by Menei et al. [55] in an attempt

to extend the application of degradable PLGA polymers into neurosurgery. The

brain tissue's reaction to the injected P LGA50150 microsp heres was investigated .

PLGA50150 microspheres were stereotactically injected into the rat brain. An

astrocytic proliferation, which is typically found following damage to the CNS was

observed, and sorne foreign-body giant cells were also found. However the

infiammatory and macrophagous reaction decreased drarnatically after 1 month

and almost disappeared after 2 months when the microspheres were totally

degraded. They concluded that PLGA50150 is a biocompatible material for

implantation in the brain.

1.6.3 Mefhodology of microencapsulation

Microencapsulation embodies a series of techniques for the entrapment of

solids or liquids within polymer coatings or matrices. The principle has been

utilized in a wide variety of industrial applications, including phamaceuticals

where, for exarnple, it has been used to improve drug stability. to rnask taste, to

provide sustained release, and to produce targeted dnig delivery [56]. Since

PLGA and PCL are used in this work, the techniques of encapsulation reviewed

will be focused on the techniques suitable for PLGA and PCL as matrix

materials.

The oldest and perhaps the most widely used microencapsulation

technique is coacervation or phase separation. This method was initially

employed in the 1950s. The principle of 'coacervation' was introduœd to

describe macromolecular aggregation by partial desolvation of fully solvated

macromolecules. This involves the deposition of a polymer from the solution

around the material (te. drug) to be coated, by temperature change. the addition

of salt, non-solvents or incompatible polymers [56]. Simple coacervation involves

the deposlion of a single coating polymer whereas complex coacervation

involves the deposition of several polymers that interact to fom the

encapsulating coating.

Microencapsulation by spray drying [57l involves the dispersion of the

active agent in a polymer solution, followed by spraying of the mixture into a

heated chamber. This leads to evaporation of the solvent and the formation of

matrix type microspheres. Figure 1.3 shows a schematic configuration of a

standard spray dryer used for microencapsulation in industry.

exhaust air q-, feed suspension

- rnicrospheres

Figure 1.3 Schematic configuration of a standard spray dryer (adapted fmm reference [5a)

The sprayer feed is prepared by mixing the core rnaterial with the polymer

solution in the presence of a surfactant. Atomization and spraying of the feed

into the hot chamber produces a shower (i.e. microdroplets) of the polymer

solution containing the dispersed core material. The microdroplets lose their

solvent to the hot air, which is blown concurrently with the microdroplets. The

resuiting microspheres are then collected in the cyclone separator. Many water-

soluble coatings have been used in the spray drying microencapsulation

technique at elevated temperatures. For example, various aromatic oils that are

used as flavorings in the formulation of pharmaceuticals are microencapsulated

in this manner and produce a free-fiowing powder with reduced volatility. Spray

drying has the advantage over many other rnicroencapsulation procedures by

being a rapid, single-stage operation that is suitable for batch or continuous

production of large quantities of productç. The drawbacks are that the spray

drying produces less uniform rnicrospheres as compared with other

microencapsulation methods. Also the capital cost of running a spray dryer is

high which makes the procedure expensive unless large production runs are

required. The extensive porous surface morphology of the spray dried

microsp heres makes this method unsuitable for drug delivery.

Another comrnonly used encapsulation method is the coating process

[57], including air suspension coating, pan coating and fluidized bed coating.

They are routinely employed in the phamaceutical industry. Air suspension

coating is an attractive method in that the process is rapid and fully automated.

Figure 1.4 shows the details of its coating chamber. The core particles are

placed at the bottom of the central chamber (c), and blown upwards through the

central column by hot air blown in the same direction. The coating polymer

solution is also sprayed upwards through the central column (c). In this way the

core particles pass through a simultaneous coatingldrying process in the first

flight upwards. After reaching the top of column (c), the partially coated particles

move downwards through the annular column (d) and undergo further drying. At

the end of column (d) they are re-directed back up through column (c), and the

coatingldrying operation is repeated until the desired thickness is reached. The

overall quality of microspheres obtained is controlled by a number of process

variables, including the spraying rate of both the coating solution and hot air and

the temperatures of the inlet and exhaust air. Release behavior of the resulting

microspheres can be controlled by the nature and concentration of the coating

polymer.

Figure 1.4. Scheme of spray coating. A: air distribution plate; 6: coating spray; C: central coatingldrying chamber; D: outer drying chamber. (adapted from reference [571)

Solvent evaporation is the most commonly used technique to prepare

microspheres/capsules. Microencapsulation by solvent evaporation is

conceptually a simple oil-in-water ( o h ) procedure. It involves, first, the

emulsification of a polymer solution containing drug which is either dissolved or

suspended in an immiscible liquid phase containing a surfactant to fonn a

dispersion of drug-polymer-solvent droplet. Second, the solvent is removed from

the dispersed droplet by application of heat, vacuum or by allowing evaporation

at room temperature to leave a suspension of drug-containing polymenc

microspheres that can then be separated by centrifugation. Finally. the

microspheres are washed and dried, foming free flowing microspheres. Solvent

evaporation is extensively employed in the preparation of encapsulation of active

ingredients. Table 1. l sumrnarizes some of the microsphere systems prepared

by this technique.

Table 1.1 Microspheres prepared by solvent evaporation

1 Polymer 1 D W 1 reference 1 P LGA50150

PLA

PLGA50/50

Solvent evaporation is a versatile technique in that the final properties of

the microspheres, such as size, size distribution, drug loading, surface

morphology and release profile cm al1 be tailored by the preparative variables.

There are several main factors that should be taken into consideration

when developing a procedure for a desired rnicroencapsulation of a

pharrnaceutical using solvent evaporation. Central to the process of

microencapsulation by solvent evaporation is the selection of the two liquid

phases. one to contain drug and polymer (the dispersed phase) and one to

cantain the surfactant (the continuous phase). Some factors that should be

considered when making the selection are listed below [56]:

A. Important criteria for dispersed phase solvent:

1. Ability to dissolve the poiymer of choie

2. lmmiscibility with the continuous phase

3. Lower boiling point than the continuous phase solvent

4. Low toxicity

PLGA5015O

PLGA75125

PLGA70/30

BSA

Leuprorelin

BSA

58

59

60

Leuprorelin

BSA

NGF

61

62

63

B. Important critena for wntinuous phase solvent:

1. lrnrniscibility with the dispersed phase solvent

2. lnability to dissolve polymer

3. Higher boiling point than that of dispersed phase solvent

4. Low toxicity

5. Ability to allow easy clean-up of microspheres

Dichloromethane and chlorofomi are the most widely used solvents for

producing PLGA microspheres by the solvent evaporation method. These

solvents are highly volatile thereby that facilitating easy removal by evaporation.

One of the problems with the solvent evaporation technique is the high affinity

between the solvent and the polymer resulting in residual organic solvent [5%-

6% (w/w)] within the resulting dried microspheres [39]. Since there are very few

reports on the detemination of residual organic solvents, the problems that

associated with it might be more serious than have been realized.

Another problem wlh this technique is the poor encapsulation efficiency of

rnoderately water-soluble and water-soluble drugs. It has been shown that water-

soluble drugs such as caffeine and salicylic acid can not be entrapped effectively

within the poly(D,L-lactide) microspheres using o h emulsion system whereas

drugs of low water solubility were successfully entrapped within the microspheres

[55]. One remedy reported by Jalil et al. [a] was the water-inail (wlo) system.

They used phenobarbitone (PB) as a water-soluble drug and poly(L4actide) (L-

PLA) as a coating polymer to make microspheres. In the w/o system, both PB

and 1-PLA were dissolved in acetonitrile with the aid of sonication and heating.

Light liquid paraffin was used as the oil phase with span-40 as the surfactant.

This dramatically increased the encapsulation eficiency, but the removal of oil

phase after encapsulation was problematic.

This problem was circumvented by the use of a novel water-in-oil-in-water

(wloh) system, which was originally proposed by Ogawa [37,65]. In the wloiw

system, a water-soluble drug, leuprorelin acetate was dissolved into water and

emulsified with concentrated P L , in dichloromethane solution, forming a primary

emulsion. The prïmary emulsion was then poured into an aqueous solution of

poly(viny1 alcohol) (PVA) onder stirring. This wfoh emulsion was stirred gently

for 3 hr to evaporate the organic solvent. An average of 90% efficiency

enmpsulation was achieved with a theoretical dmg loading of 12%. For drugs

with pH sensitive solubilities, it is also possible to increase the entrapment by

adjusting the pH of aqueous phase. Bodmerier et al. [66] achieved a high

entrapment of quinidine at a pH above 10, where the solubility was low and a low

entrapment at a pH below 7, where the solubility was high. P L , was used in both

cases as the coating material. Poor entrapment results when the protein is

partitioned from the inner organic phase to the outer aqueous phase. In this

work. we tried to tailor the preparative parameters to promote the precipitation of

polymer at the organic-aqueous interface, thus forming a bamer to prevent the

water-soluble protein from leaching.

The role of the surfactant in the preparation of microspheres by solvent

evaporation is to stabilize the suspended polyrner droplets for a short time period

before hardening. Once adequate solvent evaporation has taken place to

produce some hardening of the drug-polymer droplets, coalescence and

aggregates will not occur. Most commonly used o/w techniques utilize polymeric

surfactants such as gelatin. PVA, methylcellulose (MC) and sodium dodecyl

sulfate (SDS); o h system uses Span 80 and sodium oleate. For a concentration

of 1% surfactant, microspheres are produced in the following rank order of

decreasing particle size: gelatin > MC > SDS s PVA [67].

The principal parameter controlling particle size is the mixing condition,

Le. rotation speed. A study was conducted by Chien et al. [68] to determine the

influence of shear rate on the resuiting microspheres. The wlohiv solvent

evaporation technique was employed to encapsulate bovine serurn alburnin

(BSA) in PLGA 75/25. The shear rate was varied by changing the rotation speed

during ernulsification. This study revealed pronounced effects of shear rate on

the size and size distribution of resulting the microspheres. It was found that the

higher the shear rate, the smaller the size and the narrower the distribution. It

was also shown that a low shear rate produced microspheres wlh a high initial

burst release of BSA. whereas microspheres using hig h shear rate exhibited a

release profile without initial burst release.

In surnmary, it is crucial to take into account al1 the considerations above

in order to develop a desirable drug delivery system using the solvent

evaporation technique.

1.7 Project goals

The aim of this project is to study the encapsulation and delivery of

neuroactive molecules, such as NGF, from biodegradable polymeric

microsp heres.

1 -8 Hypothesis

1. Proteins can be released out of degradable polymeric microspheres by

either degradation or percolation mechanism.

2. The released NGF maintains its bioactivity for a prolonged perïod of time.

1.9 Objectives

1. To study the release profile of OVA from biodegradable microspheres

2. To determine the effect of polymer degradation on the protein release

profile.

3. To study the release profile and bioactivity of neuroactive molecules (e.g.

NGF)

2. EXPERIMENTAL

2.1 Materials

All chernicals were purchased from Sigma (St. Louis, MO) and used as

received unless othennn'se indicated. Biodegradable polyrners were obtained from

Birmingham Polymers, Inc. (Birmingham, AL). The polymers are described as

follows: (1) poly D,L-(lacticco-glycolic acid) with lactic to glycolic acid repeat unit

ratio of 50:50 (PLGA 50/50), with an intrinsic viscosity of 0.53 dVg in CHCl3 at 30

OC; (2) PLGA 85/15 with an intrinsic viscosity of 1.1 1 dUg in CHCl3 at 30 OC;

(3) poly(~-caprolactone) (PCL) with an intrinsic viscosity of 1.21 dVg in CHCIS at

30 OC. Poly(viny1 alcohol) (PVA) was purchased from Polysciences Inc.

(Warrington, PA) with a molecular weight of 6,000g/mol and hydrolyzed to 80%.

Mouse nerve growth factor-p (NGF) was purchased from Cedarlane Lab. Ltd.

(Homby, ON) and the adrenal rat pheochromacytoma (PC-12) cell line was

purchased from American Type Culture Collection (ATCC) (Rockville, MD). Anti-

B (2.5SI 7s) nerve growth factor (clone 27/21), anti-6 (2.5S, 7s) nerve growth

factor-p-gal, and mouse nerve growth factor -P standard for NGF-enzyrne linked

immunosorbent assay (NGF-ELISA) were purchased from Boehringer

Mannheim, Germany. HEPES. chlorophenol red-P-D-galactopyranoside (CPRG),

Tris-HCL, rit on" X-100 were also obtained from Boehringer Mannheim,

Gerrnany. Analytical reagent grade sodium chloride, calcium chloride, sodium

carbonate, sodium bicarbonate were purchased from BDH and magnesium

chloride was from APC chernical Inc. (Montreal, Que.). Deioinized distilled water

was obtained from Milli-RO 10 Plus and Mi11i-Q UF Plus (Bedford, MA) and used

at 18 Mt2 tesistance.

2.2. Methods

2.2.1 . Microencapsulation

Microspheres were fabn'cated by a modifieci solvent evaporation technique

[37,69, 73-75]. 0.2 g of ovalbumin (OVA) powder (or a total of 0.2 g OVA plus

NGF) was added to a solution of 1 g PLGA 85/15 dissolved in 4 ml of chloroform

and the solution was homogenized (Polytron PT3000, Brinkman, Westbury, NY)

for 90 seconds at 7500 rpm at ambient temperature. This mixture was then

poured into 20 ml of an aqueous solution of 1 % PVA and homogenized for an

additional 90 seconds at 7500 rpm, thereby fonning an emulsion. The latter was

added to 300 ml of 0.1% PVA which was wntinuously stirred for 3 h at room

temperature to evaporate the organic solvent. The microspheres were

centrifuged, washed repeatedly with distilled water and then freezeslried

(Labconco freezedryer with a vacuum higher than 10 Fm Hg, Kansas City, MO)

for 48 h to obtain free flowing microspheres. Vsing the same methodology,

microspheres were prepared from PLGA 50150, PCL and a blend of PCUPLGA

50/50 (1 :Il wh) . Microspheres were kept at -20 O C and desiccated until used.

Figure 2.1. describes the solvent evaporation technique used in

microencapsulation. Protein powder was ground thoroughly using a marble

pestle and mortar for 5 min in order to obtain fine protein powder prior to

encapsulation. The microspheres used for degradation studies were prepared

identically without the protein powder.

Concentrated Polvmer I

I Mixture I

1 Emulsion 1 ' I 1 Suspension 1

1 Evaporate Organic Solvent 1

Centrifuge, Wash w Freeze Drv w

I Microspheres I Figure 2.1. Schematic diagram of solvent evaporation technique used to prepare rnicrospheres

In an attempt to increase the efficiency of microencapsulation of the

protein powder into the microspheres, the emulsion was prepared at a lower

temperature of 10 O C using a cold circulating water bath, and by increasing the

volume of surfactant solution to 40 ml. Unless othenMse explicitly described, this

modified procedure was not used in this work.

2.2.2. Characterization of microspheres

2.2.2.1 Particle size and size distribution [37, 69, 701

The size distribution of microspheres was detetmined by a ~oui te$

counter (Model ZM, Coulter Electronics Limited, England). Approximately 0.1 g of

microspheres were suspended in a 20 ml lsoton' II (~oulter@" balanced

electrolyte solution, Couiter corporation, Miami, FL), mildly vortexed and

transferred to a counting chamber. The number of rnicrospheres within a specific

size range was counted. Size range was set to start from 0.2 to 5 pm and up to

100 pm with an increment of 5 Fm. The diameter distribution of the microspheres

was represented as the fraction occurring within every size range relative to the

entire size range (O - 100 pm).

2.2.2.2 Scanning electron rnicroscopy (SEM) [37,69, 70, 71, 651

Field emission H ITACH l Model S-4500 scanning electron microscope was

used to examine the morphology of microspheres. Samples were mounted on

metal studs with double adhesive tape and were sputter coated with gold for 15

seconds wlh a current of 20 mA using a SEM coaüng system (Polaron

Equipment Ltd., WatTord, England). An acceleration voltage of 1.0 kV was

applied to the samples to avoid any sample degradation due to heating.

2.2.2.3 Distribution of protein within the microsphere matrix

.Microspheres made of PLGA85115 were embedded in Tissue-Tek 4538

(Miles, IN) and frozen (Tissue-Tek 4538: 10.24% whnr polyvinyl alcohol, 4.26%

w/w polyethylene glycol and 85.5% wEw non-reactive ingredients). The frozen

blocks were sectioned at -20 OC in a cryostat to a thickness of 6 pm. The sections

were dried at room temperature and the proteins exposed on the cross-section

were stained with Ladd's multiple stain (Ladd Industries. Burlington, VT). The

stained cross-sections of rnicrospheres were mounteâ in Mounting Media (EM

Lab, Oshawa, ON) and photographed under the light microscope.

2.2.3. Degradation study [39,40, 41, 42,49, 721

After weighing by Sartorius MC5 microbalance (Germany) with the

precision of 0.001 mg. 10 mg of rnicrospheres were sealed in a nylon mesh bag

(8 Fm mesh size, Spectrum, Houston, TX) and plaœd into identical flasks

containing phosphate-buffered saline solution (PBS) (pH 7.4) with 1 % penicillin-

streptomycin and incubated at 37 OC. A constant microsphere mass to PBS

volume ratio of 1:2,000 was rnaintained for al1 samples. At one week intervals (for

up to 10 weeks), 3 samples from each polymer were collected, washed

repeatedly with deionized water and freezedried for 48 h before being analyzed.

Degradation was followed by rnass loss, gel permeation chromatography (GPC)

and SEM.

2.2.3.i Mass loss [38, 39,44, 451

Mass loss was evaluated by weighing dry samples. The percentage of

mass loss was calculated from:

rnass loss % = [(WO - Wt) / Wo] x 100 %,

where Wo and Wt are the initial mass and the mass ai time t, respectively, for the

same dried sample. Measurements were done in tnplicate. and the results were

presented as the mean f standard deviation.

2.2.3.2 SEM [38,42,48,49]

The morphology of samples both before and after degradation were

observed by SEM after sputter coating with gold for 15 s to maintain the detailed

structure on the surface of interest.

2.2.3.3 GPC [40,44, 48, 50.721

Polymer molecular weight and distribution were determined using GPC

(Varian 5000) equipped with a differential refractive index detector (Waters R-

400). Microspheres were dissolved in tetrahydrofuran (THF) at a concentration of

0.5% (wlv). A sample size of 75 pl was injected and eluted through a series

configuration of columns (UMrastyragel500 and 100,000) at a flow rate of 0.8

mUmin. THF was used as mobile phase and narrow distribution polystyrene

standards (Mp = 4,000, 20,000,63,000, and 200,000 respectively, polydispersity

index =1.04, Polysciences, Warrington, PA) were used to construct a calibration

curve. The molecular weights of the sample polymers were then calculated from

the calibration curve. Toluene was added to the polymer solution to serve as an

interna1 standard for each sam ple to ensure constant column conditions.

2.2.4. Release study of O VA from biodegadable microspheres [37, 55, 65,

71, 73-77]

For each polymer, 0.5 g of microspheres loaded with OVA were added to

10 ml PBS (pH 7.4) with 1 % antibiotics (penicillin-streptomycin) and incubated at

37 OC. At a given time, the microspheres were centrifuged for 10 minutes at 1000

rpm and 5 ml of the supernatant was collected for analysis by ultraviolet-visible

spectrophotometry (UV-vis) (Hewlett Packard 8452A Diode array

spectrophotometer). The supernatant's absorbance at 280 nm was compared to

that of a calibration curve (range of calibration was from 10 ppm to 1,000 ppm). 5

ml of fresh PBS was added to the diffusion medium to replenish the volume to 10

ml. Spheres were re-suspended by using a vortex for 5 min. This experiment was

carried out in duplicate.

2.2.5. The detenninathn of emciency of microencapsulation [S. i l ]

Approximately 0.2 g of microspheres loaded with OVA were dissolved in

100 ml of chlorofom. 150 ml of deionized water was added into the organic

phase to extract OVA into the aqueous phase. The extraction was camed out in

a 500 ml separatory funnel. The solution consisted of three layen: (1) an

aqueous phase as the top layer; (2) an organic chlorofom as the bottom layer;

and (3) a milky layer in the middle. The middle layer was collected and the

chloroform in the mixture was rernoved by rotary evaporator (Bnnkman,

Westbury, NY) at 35 O C with a rotating speed of 120 rpm. The rernaining

aqueous solution was combined with the upper layer in the separatory funnel and

the amount of protein in the solution was detemiined by UV-vis. The efficiency of

encapsulation is given by:

efficiency of encapsulation = (W -,,=& 1 W lheOretical)~ 100% ,

where W m,,ed is the amount of protein recovered and W aeomtica~ is the

theoretical arnount of protein entrapped. The efficiency of encapsulation was also

calculated for microspheres prepared by the modified solvent evaporation

technique. All measurements were taken in triplicate and the results were

presented as the mean f standard deviation.

This method was validated by adding a 0.2 g of OVA powder to 4 ml of a

25% polymer solution in chloroform in a ~eflon@ mold. The organic solution was

removed under vacuum, resulting in a polymeric disc. The OVA was recovered

by dissolving the polymeric disc in chloroform and extracting the protein into the

aqueous phase as described above. This was done in triplicate for PLGA 85/15

and PCL.

2.2.6. PC f2 cell culture

PC12 single cell clonal line which responds reversibly to NGF [78, 791 by

induction of the neuronal phenotype was used to assess the bioactivity of

released NGF over time. Cells were seeded in T-25 cell culture flasks (Falcon,

Becton Dickinson Labware, Franklin Lakes, NJ) with a concentration of 1 x 1 o5 cellslml in medium. The ceIl culture medium consisted of 84% RPMl 1460, 10%

heat-inactivateci horse senim, 5% fetal bovine serum and 1 % penicillin-

streptomycin. Cells were incubated at 37 OC in a 5% CO2 in air atrnosphere [81].

Cell medium was changed every other day and cells were subcultured once per

week.

Cells used in the bioactivity assessrnents were within 3 passages.

2.2.7. Assessment of bioactivity of released NGF

2.2.7.1 Preliminary 5 d assessrnent

1. Preparation of poly(L-lysine) coated surfaces

A stock solution of poly(L4ysine) (PLL) (Sigma, MW = 36,600 glmol) was

prepared at a concentration of 50 pg1100 ml (wlv) and the solution was

steAe-filtered through a MiIlipore cellulose aœtate filter (0.22 pm). 2 ml of the

sterile PLL solution was added aseptically to each well of a 6-well cell culture

plate (flat bottom, 35 mm, Coming, NY) for 1 h in ambient temperature. The

plates were then washed twice with 3 ml of Hank's balanced salt solution

(HBSS) (Gibco BRL, Burlington ON) [81]

2. Sterilization of NGF loaded PLGA 8511 5 microspheres

Microspheres were prepared by the solvent evaporation technique as

previously described. 10 mg of microspheres (NGF:OVA = 1 : 8,000) were

sterilized by the exposure to UV light for 3 h.

3. Assessment of bioactivity of released NGF [82]

PC12 cells were seeded on the PLL pre-coated surfaces at a

concentration of 1 XI o5 cellslml and incubated with sterile microspheres at 37

O C in an atmosphere of 95% air and 5% COz for a period of 5 d. Cells

incubated with NGF supplemented cell culture medium at a NGF

concentration of 25 ngfml were used as positive controls and those incubated

with only ceIl culture medium were used as blank controls.

2.2.7.2 Assessrnent of bioactivity of released NGF up to 90 d

PLGA 85/15 microspheres loaded with OVA and NGF were prepared by

solvent evaporation technique as descnbed above. Four batches of PLGA 85/15

microspheres were fabricated with four different NGF to OVA mass ratios: (a)

1 :2,000, (b) 1 : 10,000, (c) 1 :100,000 and (d) OVA without NGF. which were

designated as NGF-1, NGF-2 and NGF-3 and OVA respectively. Each of 0.5 g

microspheres from NGF-1, NGF-2, NGF-3 and OVA were added to 10 ml PBS

(pH 7.4) with 1% penicillin-streptomycin and incubated at 37 OC. At each time

point, the suspension of microspheres was centrifuged for 10 min at 1000 rpm

and 5 ml of the supenatant was collected. 1.5 ml of this supematant, collected

from each sample, was sterile-filtered through a M~II~X@-GV filter unit (0.22 Pm,

Millipore) and incubated with PC12 cells that had been plated on the PLL pre-

coated 6-well plates. The number of neurites extending from each of 50 cell

bodies were cuunted for every sample after 24 h and 48 h of incubation. Cells

incubated with the NGF (25 nglml) supplemented cell culture medium served as

positive controls whereas those incubated with cell culture medium or cell culture

with only OVA released medium were used as controls.

The supematant that was collected from every sample was stored at -80

O C before being assayed by the NGF-ELISA to detemine the concentration of

released NGF in the supematant.

2.2.8. Release study of NGF

NGF-ELISA

2.2.8.1 Test principle (Sandwich ELISA)

In the first step, the anti-NGF antibody which is specific for NGF was fixed

adsorptively on the wells of the 96-well microtiter plate. Subsequently, non-

specific binding sites on the well were saturated with the blocking solution.

Standards with known amounts of NGF and unknown samples were then

incubated in the plate's wells and were bound by the immobilized antibody. In

the third step, anti-NGF-p-gal. an enzyme-linked antibody conjugate that is

also specific for NGF, was added and bound ta NGF. Unbound anti-NGF-p-

gal was then washed away and a substrate chlorophenol red-p-D-

galactopyranoside (CPRG) was added. The enzyme-NGF antibody conjugate

would catalyze the hydrolysis of CPRG which allowed a color to develop:

chlorophenol red-P-Pgalactopyranoside (CPRG) + H20

1 P- galadosidase -anti-NGF

galactopyranose + chlorophenol red (red color in basic solution)

The intensity of color is proportional to the amount of bound NGF and can be

determineci by a plate reader. The principle is illustrateci in Figure 2.2.

Figure 2.2 The principle of NGF-ELISA. 1. plate well; II anti-NGF antibody; III. NGF; IV. anti-

NGF-P-gal. enzyme-linked antibody conjugate; V. substrate (CPRG). (Ref 1831)

2.2.8.2 Assay procedures (Refer to Appendix A about the preparation of

solutions) [96]

1. Coating the microtiter plate with antibody

150 pl of coating solution (II) was pipetted into the wells of the 96-well

microtiter plate (Nunc-lmmuno Plate. Maxisorp Nunc. Denmark). The plate

was tightly covered and incubated for 2 h at 37 OC.

2. Blocking

The coating solution was removed thoroughly. 200 pl of blocking solution (III)

was added to each well of the microtiter plate. The plate was tightly covered,

and was incubated for 30 min at 37 OC.

3. Washing

The blocking solution was removed thoroughly from the wells and the wells

were washed 3 times with the washing buffer (VI) to ensure complete removal

of the blocking buffer.

4. Incubation with samples and standard solutions

100 pl of sample or standard solution (X) was added to the well and the plate

was tightly covered and incubated overnight at 4 OC.

s. Washing

As described under 3.

6. Incubation with anti-NGF-pqal

100 pl of anti-NGF-P-gal solution (lx) was added to each well. The plate was

tightly covered, and was incubated for 4 h at 37 OC.

7. Washing

As described under 3.

8. Substrate reaction

200 pl of substrate solution (VIII) was added to each well of microtiter plate

and incubateci at 37 OC until the development of the color was sufficient.

9. Measurement

The absorbance of the substrate solution was measured against substrate

solution (Ml) as a blank value at 640 nrn using Thermo Spectro III plate

reader (Labinstruments, Australia)

Measurements were carried out in triplicate for both standards and unknowns.

One series of calibrations was perforrned on every rnicrotiter plate to minimize

plate specific variances.

2.2.9. Statistics

Data obtained from the bioactivity of NGF assessment were analyzed by

SAS (SAS lnstitute Inc., Caiy, NC). One way ANOVA with 6 different levels

(NGF-1, NGF-2, NGF-3, Blank, OVA, and NGF) was employed assuming a

constant standard deviation in every level. Statistical analysis was carried out

with 95% confidence. Results were presented as groups within which levels were

not significantly different.

3. RESULTS

3.1 Characterization of microspheres

3.1 .1 cou~feP counter tesuit:

As shown in Figures 3.1, microspheres prepared by the sohrent evaporation

technique have a wide size distribution. There are no significant differences

between microspheres made of different polymen or rnicrospheres encapsulated

with and without protein incorporated.

3 13 23 33 43 53 63 73 83 93

diameter (p)

Figure 3.1. a) diameter distribution of PLGA50150 microspheres without protein powder

3 13 23 33 43 53 63 73 83 93

diarneter (p)

Figure 3.1. b) diameter distribution of PLGA85/15 microspheres without protein powder

3 13 23 33 43 53 63 73 83 93

diamater (p)

Figure 3.1. c) diameter distribution of PCL rnicmspheres without protein powder

3 13 23 33 43 53 63 73 83 93

diarneter (p)

Figure 3.1. d) diarneter distribution of blend of PLGA50150 and PCL microspheres without protein

powder

diameter (pm)

Figure 3.1. e) diameter distribution of PLGA50/50 rnicmspheres with protein powder

encapsu lated

diameter (pm)

Figure 3.1. f ) diameter distribution of PLGABSII 5 microspheres with protein powder encapsulated

3 13 23 33 43 53 63 73 83 93

diameter (pm)

Figure 3.1. g) diarneter distribution of PCL microspheres with protein powder encapsulated

diameter (pm)

Figure 3.1. h) diameter distribution of blend PLGASOISO and PCL microspheres with protein

powder encapsulated

More than 70% of the microspheres have diameter of less than 40 Pm. Table 3.1

summarizes the ~oulte$ counter results.

Table 3.1. Average Size (rt standard deviation) of microspheres (n > 1,500 )

Microsp here Microsphere diameter (pm ) PLGA50150 17.0k4.3 PLGA 8511 5 PCL Blend: PCUPLGA 50150

PLGA 50150 + OVA 22.3k5.2 PLGA 8511 5 + OVA 22.7e.4 PCL + OVA 18.26.3 Blend + OVA 19.4s-6

The scanning electron micrographs in Figure 3.2 show that empty

microspheres have a very smooth surface while those encapsulated with protein

powder have a rough surface morphology and seemingly porous structures. The

porous structures likely provide pathways for the protein to dinuse out

independent of degradation.

Figure 3.2 a

Figure 3.2 SEM micrographs of microspheres a) without and b) with protein powder encapsulated

The SEM results also show similar size distribution of microspheres to that

obtained from the ~oulter@ counter as shown in Figure 3.3.

Figure 3.3

Figure 3.3. PCL microspheres without protein encapsulated

3.1.3 Pmtein distribution within the microsphere maoix

As shown in Figures 3.4, the encapsulateci proteins were distributed

throughout the microspheres. The relative volume percent of protein to polymer

in the microspheres was estimateâ, from the two dimensional cross-sectional

areas, to be 17.5%.

Figure 3.4a

Figure 3.4. Protein distribution within polymer mi

Figure 3.4b

3.2 Degradation study

3.2.1 M a s loss

Figure 3.5 shows the mass loss of rnicrospheres over 84 d of observation as

the result of polymer degradation. PLGA50f50 microspheres completely

degraded after 84 d of incubation in PBS whereas PCL microspheres degraded

by 30 % over the same period of time. As expected, mass loss of the blend of

PLGA50/50 and PCL was between that of PLGA50f50 and PCL; PLGA85/15

degraded slower than PLGA50150, which is also as anticipated.

It is of interest to notice the sudden mass loss of PLGA50150 between 28 and

42 d, represented by the steep slope in the mass loss profile. This phenornena is

characteristic for al1 biodegradable poly(a-hydroxy ester)s and can be attributed

to the autocatalytic hydrolytic degradation mechanisrn in which released

carboxylic acids act as catalysts for an increased rate of degradation. An

accelerated mass loss was a result of the accelerated degradation which was

Time (days) + plga50t50 -A- plga85115 + pcl + blend

Figure 3.5 Degradation of microspheres determined by mass loss

catalyzed by the accumulated carboxylic acids. The carboxylic acids, in turn,

were produced by degradation itseif. PLGA50/50 lost 50% of its mass in a period

of 14 d (from 28 d to 42 d) while the blend, a mixture of two polymers of different

degradation rates lost only 25% of its initial mass during the same period of time.

By blending materials with different degradation rates, for example, the blend of

PLGA50i50 and PCL, the amount of acid released to the surrounding aqueous

media can be moderated.

3.2.2 SEM

Initially, the empty micmspheres that were used for the degradation study had

a smooth outer surface (cf. Figure 3.2a). Figures 3.6 show SEM micrographs of

microspheres after 35 d of degradation. After 35 d of degradation, al1

microspheres were still spherical except those of the polymer blend which

showed a dramatically different morphology (cf Figure 3.6d). The morphology

changes of the blend sample can mainly be attributed to the degradation of the

PLGA50150, a wmponent of the blend. PLGA50150 alone lost about 50% of its initial weight as indicated in the mass loss profile (cf. Figure 3.5). Composed of

50% PLGA50f50 by mass. the blend microspheres lost their integnty and thus a

remarkable morphology change occurred even though these rnicrospheres did

not have the highest mass loss.

Several pores formed after 35 d of incubation on the originally smooth

surfaces of both PLGA50/50 and PLGA85/15. (cf Figures 3.6a and 3.6b,

respectiveiy). The pores were likely formed by the leaching out of water soluble

degradation products.

Figure 3.6. SEM micrographs of microspheres a Zr 35 d of degradation a) PLGASOISO; b)

PLGA85115; c) PCL; d) blend of PLGA50t50 and PCL (1 :1, wiw)

Vert et al. [40,51,72] reported the heterogeneous degradation of PLGA with

the fastest degradation rate being inside the device, resulting in a hollow

structure within the device as the degradation products leached out. Our

observations agree with their conclusions. As shown in Figure 3.7, the SEM

micrographs which were taken after 70 d of incubation in PBS showed that PLGA

50/50 microspheres degraded and had a hollow structure with a highly porous

skin structure (cf. Figure 3.7a). The formation of a porous skin contradicts Vert's

prediction that polymer spheres of less than 200 pn will degrade by a

homogeneous manner (401. The morphology of PCL did not change substantially

after both 35 d and 70 d of incubation. This is in a good agreement with the mass loss data which suggested that only 30% mass loss was observed over 84 d. The

porous structure that forrned as a result of degradation facilitates the release of

proteins incorporated into PLGA 50/50.

rr 70 d of degradation (a) PLGASOISO; (b)

PLGA85f 15; (c) PCL; (d) blend of PLGA50150 and PCL (1 : 1, wEw)

3.2.3 GPC riesuIts

Figure 3.8 shows the changes of weight average molecular weight (MW)

of PLGA 50150 detemined by GPC over time during the degradation study. It can be seen that the sudden mass loss was accompanied by a sharp decrease of

molecular weight which was due to the accelerated hydrolysis rate. The

accelerated hydrolysis rate in tum was catalyzed by the carboxylic acids

produced by the hydrolytic degradation. Due to the complete degradation of

PLGA 50150 after 84 d, there was no sample available for GPC analysis. It is

very interesting to see a remarkable increase in MW for PLGA 85/15, PCL and

the blend as shown in Figures 3.9, 3.10, and 3.1 1, respecüvely. The first three

GPC chromatograms of PCL from t=O to t=21 d have similar retention times at

the beginning of the peaks, but the tails of peaks shift gradually towards less

retention time (Le. higher molecular weight). This irnplies that the apparent

increase in the average MW resulted from the loss of lower molecular weight

oligorners and not from crosslinking. The same is also true for both PLGA85115

and blend samples.

Figure 3.8. Change in weight average rnolecular weight as detemined by GPC for PLGA 50/50

microsp heres

Figure 3.9. Change in weight average rnolecular weight as detennined by GPC for PLGA 85/15

microspheres

O 7 14 21 28 35 42 49 56 63 70 n s4 l ime (days)

0 MW + Mass l o s

Figure 3.10. Change in weight average rnolecular weight as detennined by GPC for PCL

microsp heres

Figure 3.11. Change in weight average molecular weight as determined by GPC for blend

rnicrospheres

3.3 Efficiency of encapsulation

The amount of protein encapsulated in each of the polyrneric

microspheres was determined by calculating the amount of protein that could be

recovered after dissolving microspheres in chloroform and extracting the proteins

into water. The validation method indicated that 95.7k 4.6% of the total protein

could be recovered. As shown in Figure 3.12, the efficiency of encapsulation

differed for each polymer investigated, with PCL having the highest eficiency of

encapsulation and PLGA 50150 having the lowest The efficiency of

encapsulation is dependent on the material. In order to obtain a high

encapsulation efïtciency of water-soluble proteins into the organic polymer phase

by solvent evaporation, the polymer must precipitate quickly at the organic-

aqueous interface to form a bamer against protein dissolution into the aqueous

phase. PCL had the highest efficiency of microencapsulation among the

polymers investigated, because it precipitated the fastest. In an attempt to

improve the microencapsulation emciency, a lower temperature and a higher

volume ratio of aqueous phase to organic phase during emulsification were used

to promote polymer precipitation. Figure 3.1 2 shows that a significant

improvement in efficiency of microencapsulation was obtain by the modified

technique.

PLGASOi50 PLGA85/15 PCL BLEND

I NORMAL MODlFlED

Figure 3.1 2. efficiency of microencapsulation

3.4 Release study

3.4.1 Release of OVA as a mode/ drug

Figure 3.13 shows the cumulative protein release profile relative to the

initial amount encapsulated in the polymeric microspheres. As expected, the

highest percentage of OVA was released over 76 d from PLGA 50150

microspheres which had the hig hest degradation rate. Although PLGA 85/15 has

the second highest degradation rate, a lower percentage of OVA was released

from 1 than was released from ?CL. Combining the release profile (cf. Figure

3.13) and mass loss profile (cf Figure 3.5), 75% of protein was released from

PCL during which only 30% of the mass was lost. This implies that the

rnechanism for protein release from PCL microspheres is not due to degradation

alone. The same is also tme for al1 the microspheres at the very beginning of

their release profile. Furthemore, for PLGA50150, there is an accelerated protein

release in conjunction with an accelerated mass loss (Le. between 28 d and 42

d). In Figure 3.14, the sarne data set is plotted in a different way of absolute

amount of protein released vs. time. PCL had the highest amount of protein

released whereas PLGASO/SO had the second least amount released. The

protein release profile from al1 polymeric microspheres was influenced by both

polyrner degradation and protein percolation. Figure 3.14 together with Figure

3.1 2 (efficiency of encapsulation) indicate that PCL microsp heres, having the

greatest amount of protein encapsulated, dominantly released protein by

percolation, whereas PLGA 50150, with the highest degradation rate, released

protein mainly by degradation.

+ plga50150 -t- plga85115 + pcl -a- blend

Figure 3.1 3. Cumulative reiease profile of OVA from biodegradable polymeric microspheres as a percentage of the initial OVA encapsulated.

time (days) + plga50150 + plga85115 -rt pcl + blend

Figure 3.14. cumulative release profile of OVA from biodegradable polymeric microspheres expresseci as mass of protein released over time

3.4.2 Release of NGF

Figure 3.15 shows the results of the amount of NGF released determined by

ELISA. NGF-7 showed a 'burst release profile for the first 12 d followed by a

constant release; NGF-2 samples showed a slight burst at 8 d followed hy a

constant release; and NGF-3 samples showed a constant release over time. The

burst release of NGF-1 is consistent with the higher concentration of NGF CO-

encapsulated with OVA whereas NGF-3 had too little amount of NGF

encapsulated to show any burst release.

Since ELISA employs NGF antibody which is specific to NGF, the amount of

NGF 'visible' to ELISA should be NGF that is still stable.

tirne (days) + NGF-1 + NGF-2 -t- NGF-3

Figure 3.15. NGF release determined by ELISA

3.5 Assessment of bioactivity of encapsulated NGF

3.5.1 Preliminary assessment for 5 d

As shown in Table 3.2, it is evident that the encapsulated NGF maintained its

bioactivity after being released out of the microspheres. It is of interest to note

that the microsphere group is inferior to the NGF control group in the inducement

of neurite outgrowth but superior to the Blank control in the first two days as

indicated in Table 3.2. (p<0.0001). The increasing number of neurites over time

in the microsphere group implies that the sustained release of NGF has a

prolonged effect. At day 3, the effect of NGF released frorn microspheres was the

same as that from the NGF-supplemented media (pc0.0001). In cornparison wÏth

the increasing number of neurites over time from the microsphere group. the

decreasing number of neurite outgrowth observed from NGF-supplemented

media may suggest the depletion of NGF in the NGF-supplemented media. This

is even clear at 5 d when the microsphere group showed significantly more

neurîte outgrowth (pe0.0001) whereas the NGF group had the same effect as the

blank which served as a baseline in this assessment.

Table 3.2. Average rrumber of neurites extending per ce11 average over 50 cells

Time (d) Blank NGF PLGA85/15

medium microsp heres

2 d O.iS(A*) 3.76(C) 2.98 (B)

3 d 1.12 (A) 2.94 (B) 3.00 (B)

4 d 0.88 (A) 1.98 (B) 3.06 (C)

5 d 1.56 (A) 1.70 (A) 3.24 (B)

*Means the with same letters are not significantly different (n=50, az0.05)

3.5.2 Assessrnent of bioactivity of mleased NGF up to 90 d

This experiment was designed to assess the bioactivity of NGF after being

encapsulated into and released out of the biodegradable microspheres over time.

Figures 3.16 show that the released NGF from group NGF-1 (NGF:OVA =

1:2000) had the same effect as NGF-supplemented group in stimulating neurite

outgrowth for the first 44 days. The significantly more neurite outgrowth from

NGF-2 and NGF-3 than those from blank suggests that NGF maintains its activity

in the process of encapsulation and is still active in promoting neunte outgrowth

after being released. As the release time increased, NGF-1 started to promote

less neurite outgrowth than NGF group after 44 d of release as indicated in

Figure 3.20, whereas NGF-3 did not induce any significant more neurite

outgrowth when compared with blank after 8 d of release. This may be an

indication of loss of bioactivity over time or the presence of less NGF. After 91 d

of release, though NGF-1 still induced significant neurite outgrowth (compared

with blank), its effect was less than that of the NGF group; neither NGF-2 nor

NGF-3 stimulated any significant neurite outgrowth. Relative to the blank control,

OVA alone did not induce any significant neunte outgrowth in comparison with

the Blank (p<0.0001), demonstrating that OVA coencapsulated with NGF did not

have any inherent neurite-promoting ability.

As shown in Figure 3.22, at 52 d and beyond, the NGF released from

NGF-1 samples promoted less neurite outgrowth than the NGF-supplemented

group indicating that significantly less NGF was released by the microspheres

than was supplemented into the medium by the positive control (i.e. 25 ng/ml)

(Pc 0.0001). PC12 cell cultures were supplemented with NGF at a concentration

similar to that observed by the NGF-ELISA at 52 d (Le. 0.95 ng/ml) to detemine

whether the released NGF was at too low a concentration to show the saine

activity or simply inactive. The neurite response observed for NGF-1 samples

was similar to that observed for the 0.95 nglml NGF supplernented cultures.

confirming the continued bioactivity of the released NGF.

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.16. a) neurite outgrowth after 4 d of release and 24 h of incubation

OVA BMNK NGF

Figure 3.16. b) neurite outgrowth after 4 d of release and 48 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.17. a) neunte outgrowth after 8 d of release and 24 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.17. b) neurite outgrowth after 8 d of release and 48 h of incubation

A

B 6

O -- -. ~ i i .

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.18. a) neurite outgrowth after 12 d of release and 24 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.18. b) neurite outgrowth after 12 d of release and 48 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.19. a) neurite outgrowth after 20 d of release and 24 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.19. b) neurite outgrowth after 20 d of release and 48 h of incubation

A

NGF NGF-3 OVA BLANK

Figure 3.20. a) neurite outgrowth after 44 d of release and 24 h of incubation

NGF NGF-3 OVA BLANK

Figure 3.20. b) neurite outgrowth after 44 d of release and 48 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.21. a) neunte outgrowth after 91 d of release and 24 h of incubation

NGF-1 NGF-2 NGF-3 OVA BLANK NGF

Figure 3.21. b) neurite outgrowttr after 91d of release and 48 h of incubation

Figure 3.22 Bioactivy of NGF was assessed after 24 h of incubating released NGF with PC12

cells by assessing the average number of neurites per cell body

t i m e ( d )

Figure 3.23 Bioactivty of NGF over tirne was assessed after 24 h of incubating released NGF

with PC12 cells by calculating the percentage of cells bearing one or more neurites that exceeded

the cell body length

NGF activity was followed over time by calculating the perœntage of cells

bearing one or more neurites longer than the cell body length for the three

samples and three controls as shown in Figure 3.23. The NGF released from

NGF-1 samples showed decreased activity after 66 d, but maintained greater

activity than the Blank and OVA controls up to 90 d. NGF-2 samples

demonstrated bioactivity for the 90 d period. but had a significantly lower

response than the NGF 25 nglml supplemented wntrol. NGF-3 samples

demonstrated a PC12 cell response only slightly higher than that of the Blank

and OVA controls.

4. DISCUSSION

4.1 Morphology of microspheres

The encapsulation of protein powder gives the resulüng microspheres a

seemingly porous outer surface morphology. The pores on the surface likely

provide a pathway for entrapped protein to be released from the polymer matrix

even before significant degradation takes place. The pore lself is likely created

by the dissolution of protein which is either on the surface or interconnected to

the surfaœ during washing. Microencapsulation using conventional solvent

evaporation techniques (e-g. encapsulating a protein solution) produces a

smooth rnorphology on the surfaœ [84]. In addition, a 'burst' release is often

reported [59, 85, 861 due to the dissolution of protein adsorbed on or near the

outer surface. The use of protein powder, as opposed to the protein solution. for

encapsulation presents several advantages. The protein molecules experienœ

reduced shear stress, and the amount of protein encapsulated is not limited by

the solubility of protein in the aqueous phase. One drawback. however, is a

decrease in the eficiency of encapsulation due to the incomplete encapsulation

of large or irregular protein particles.

4.2 Degradation

PLGA and PCL degrade heterogeneously, a result which has been

previously reported by Vert et al. [40, 51, 721. Once microspheres are immerçed

in an aqueous medium in vitro, water uptake occurs immediately. The rate of

water penetration is very fast when cornpared with the degradation rate for a

number of reasons. including the small size of the water molecule. It can,

therefore, be considered that the hydrolysis of the ester bond starts

homogeneously from the beginning throughout the amorphous domain while the

crystalline domain remains intact because it is less accessible to water. There

are two facts that are well known to occur during degradation [39, 51, 521: first,

degradation causes an increase in the number of carboxylic acid end groups

which are known to autocatalyse the ester bond hydrolysis; and second, only the

degradation products, which are -ter soluble in the surrounding aqueous

medium, can escape frorn the microsphere matrix. As the degradation time

increases, the soluble degradation products which are close to the surface leach

out easily whereas those located inside the mat& leach out slowly and

contribute to the autocatalytic effect. Consequently. there will be a faster

degradation rate inside the microspheres, which eventually results in the hollow

sphere structure once the water soluble oligomers leach out, as shown in Figure

3.7.a. As more degradation products leach out from the inside matrix to the

surrounding aqueous solution, the outer surface morphology becomes more

porous and there are an increasing number of interconnected channels

throughout the rnatrix (cf. Figures 3.6.a, and 3.7.a). The porous structure,

therefore, likely provides another pathway for protein release.

Generally, the degradation products can be proœssed through normal

metabolic pathways and are ultirnately elirninated from the body through the

respiratory system [44]. A sudden rise in carboxylic acid in vivo, however, can

render the local environment acidic and induce an inflarnmatory response.

Consequently, for tissue engineering applications, a modulated release of

carboxylic acid at levels that can be accommodated by the adjacent tissue is

desired. PLGA 50150 lost 50% of its rnass in a period of 14 d (cf. Figure 3.5)

while the blend of PLGA50150 and PCL, lost only 25% of its initial mass during

the same period of time. Since the sudden release of degradation products is

characteristic of al1 poly(a-hydroxy ester)s due to their autocatalytic hydrolysis

degradation mechanism, each polymer has its own degradation rate and 'sudden

release' profile. The approach of combining materials with difFerent degradation

rates into one microsphere allows the degradation and sudden release profile to

be tailored such that the degradation products can be released over a longer

time period than what would be observed with only one polymer. It has been

shown in this work that blending PLGA 50/50 and PCL can be used to control the

rate of degradation. Consequently the amount of acid released should be

moderated over tirne as well.

Vert [39] predicted that the degradation products inside will be easily

released if the device size is smaller than 200 pm and hence the device

degrades homogeneously; however, a hollow structure was observed by SEM for

PLGA 50150 (cf. Figure 3.7.a) after 70 d of degradation. This contradicts Vert's

prediction because the microspheres had diameters less than 50 pm and yet

they still showed heterogeneouç degradation. This observation contradicts the

predicted 200 pm cnücal size threshold.

It has been reported in several papers that there is no significant mass

loss at the beginning of degradation [39,40,41,44] until the degraded oligorners

are small enough to be soluble in the aqueous medium. Yet, al1 the polyrners

investigated herein showed instant mass loss upon degradation. This

discrepancy may be attributed to the existence of water-soluble oligorners in the

matenals. The leaching out of the pre-existed oligomers is reflected by the

weight average molecular weight and the molecular weight distribution

detemined by GPC. Table 4.1 shows that for al1 the polyrners. an inlially broad

molecular weight distribution became narrower for the first several days and then

broadened again as would be expected from a random cleavage of the ester

bond along the polymer backbone. The narrowed rnolecular weight distribution

detected at the beginning of the degradation may be attributed to the leaching

out of small water-soluble molecules frorn the polymer matrices.

Table 4.1. Polydispersity index

Time(d) O 7 14 21 28 35 42 49 56 63 84 PLGA 2.96 2.56 1.87 1.85 2.14 2.65 3.29 4.10 4.78 3.80 n/a

PLGA 3.00 1.95 2.02 1.99 1.69 1.75 1.88 1.51 1.67 1.91 1.44

PCL 2.09 2.16 2.10 1.86 2.40 2.16 2.12 1.80 1.65 1.79 1.67

BLEND 2.72 3.23 4.60 4.61 6.45 6.72 12.1 9.68 11.1 28.5 1.82

Further evidence is show in Figure 4.1 where GPC chromatograms of

PCL degradation sample are over laid. On the GPC chromatogram, the high

molecular weight region is represented by the point where the peak begins.

Figure 4.1 GPC chromatographs of PCL at (a) t=O, (b) t=14 d, and (c) t=21 d (please notice the

shift of the peak tails)

As shown in Figure 4.1, for the first several days the polymers had sirnilar high

molecular weight regions represented by the similar retention times where peaks

began. The tails where the peaks ended. however, slightly shifted to the left (high

molecular weight region) as degradation time increased. This implies that the

apparent increase in the weight average molecular weight (as shown in Figures

3.8 to 3.1 1) was due to the leaching out of the water-soluble oligomers. (The low

molecular weight portion is also visible from chromatogram (a), where there is a

small peak following the main peak.) The high content of water-soluble oligomers

rnay be attributed to the microencapsulation process du ring which polymer

chains were cleaved by the high shear stress employed (871.

4.3 Efficiency of encapsulation

Efficiency of encapsulation is one of the most important issues in

microencapsulation of water-soluble proteins by the solvent evaporation

technique. This is especially tnie when the protein availability is very limiteci.

Althoug h solvent evaporation has been used successfully to encapsulate

hydrophobic materials into PLGA microspheres (881, poor entrapment has been

observed with hydrophilic agents. Poor entrapment results from partlioning of

protein from the inner organic phase to the outer aqueous phase. Bodmeier et ai.

[66] succeeded in achieving a high entrapment of quinidine in PLA microspheres.

They adjusted the pH of the aqueous phase to greater than pH1 O in order to

achieve minimal drug solubility and to saturate the aqueous phase with the drug

thereby minimizing diffusion and dnig loss. Ogawa et al. [37] reported an

increase in the entrapment efficiency of the hydrophilic peptide leuprolide

acetate after adding gelatin to the peptide solution prior to ernulsification. Jeffery

[73] observed the same entrapment increase with a different water-soluble

protein - albumin. Other attempts found that the effîciency of encapsulation

depends on the concentration of the polymer solution [89] and the molecular

weight of the polymer [84].

As shown in this work. the efficiency of encapsulation also depends on

the chernical structure of the polymer. In order to improve the encapsulation

efficiency of water-soluble proteins into microspheres. the ultimate goal was to

forrn a barrier to prevent contact between the protein and the aqueous medium.

Precipitation of the polymer by phase inversion results in a barrier that prevents

proteins from leaching out. In this work, both a lower temperature and a higher

ratio of aqueous phase to organic phase were used during the emulsification

step to promote faster polymer precipitation and consequently improve the

efficiency of encapsulation. This shows that it is the polymer precipitation rate,

which is influenced by the polymer chernical structure and molecular weight, that

mainly affects the efficiency of encapsulation of water soluble dmgs, such as

water soluble proteins. It can then be predicted that the higher the concentration,

the faster the polymer to precipitation and the hig her the efficiency of

encapsulation (Irregular shapes were reported when the polymer concentration

was too high). Similarly, the higher the molecular weight, the faster the

precipitation, and the hig her efficiency of encapsulation. These predicted results

were confirrned in previous works [84, 891. Furaiemore, the rnodified

encapsulation technique that promotes polyrner precipitation at the interface

eventually improves the efficiency of microencapsufation dramatically (cf. Figure

3.12).

4.4 Protein stabilw

During rnicroencapsulation, the protein has often been exposed to

various unfavorable stresses, both mechanically and chemically. In practice,

exposure to an organic solvent is problematic in most of the cornmon solvent

evaporation processes, because the presence of the organic soivents will disrupt

the hydrophobic interactions responsible for the stable core of globular proteins

[go]. In addition, high shear stress may result in molecular deterioration of the

protein such as denaturation or aggregation. The acidic environment within the

m icrosp heres during the polyrner degradation rnay also adversely affect the

stability of encapsulated proteins in the microspheres. By using sodium dodecyl

sulfate - polyacrylate gel electrophoresis (SDS - page), Yan et al [QI] reported

the structural integrity of BSA molecules in aqueous solution when encapsulated

into PLGA50150. Conversely. Park et al. observed BSA aggregation and

precipitation in a 2 month release period using SDS - page [92]. In a similar

study also conducted by Park et a/. [93], the encapsulated eristostatin, a low

molecular weight polypeptide (MW 5,725) was found to aggregate and fragment

soon after being incorporated into PLGA50150.

An elegant piece of work in assessing the bioactivity of encapsulated

protein has been done by Agrawal et al. [94] who incorporated osteoinductive

bone morphogenetic protein (BMP) into PLA. The activity of BMP in the implant

was verified as the ability of the implant to induce bone formation when placed in

the thigh muscle of mice.

The bioactivity assessrnent made in this work was based on cell culture.

The advantage of this method is that the bioactivity released into the incubation

medium can be monitored for up to 90 d. This is not usually feasible because of

the problems involved in cell culture, such as refeeding and subculture

techniques required for PC12 cells. Since PC12 cells respond reversibly to NGF

by induction of a neuronal phenotype, the reduced amount of NGF in the

medium will reduce the inducement of neurite outgrowth. In the preliminary

study. the decreasing trend of neurite outgrowth from NGF supplemented

cultures showed the depletion NGF in the medium whereas substantially

increasing neurite outgrowth from microsphere cultures over time suggested a

substantial release of NGF from microspheres. Furthemore, it can be concluded

that the processing from which microsphere were made did not totally disrupt the

acüvity of NGF. The assessrnent of bioactivity for up to 91 d demonstrates the

stabilization effed of OVA on NGF. Knowing that NGF is stable in an aqueous

solution for one month at 4 OC and for about 4 d at 37 O C (information provided

by the manufacturer). the bioactivity displayed by NGF-1 over 91 d indicates that

OVA stabilized NGF. This method, however, did not quantify how much, if any,

bioactivity was lost.

It should be emphasized that the bioactivity we are investigating is the

activity of NGF which has been released into the aqueous medium. It would be

of interest to study the protein bioactivity when it is still within microspheres. This

is especially important in designing drug delivery systerns to irnprove the stability

of active ingredients. One technical problem of studying protein stability within

microspheres is the unavailability of an appropriate noninvasive method to

analyze the protein within the solid matrix. Unfortunately. the extraction of protein

molecules from microspheres by first dissolving the polymer matrix into an

organic solvent, with subsequent extraction into an aqueous phase. was found to

cause aggregation and denaturation [92]. So far, there is no satisfactory method

available to investigate the stabil0Ry of proteins within the polymer matrix.

4.5 Release mechanism

The mechanism for protein release from a biodegradable polymer matrix

is controvenial. There are two main proposed mechanisms:

1. Degradation-controlled mechanism ri51

The degradation of biodegradable polymers is a critical factor in determining

the release of proteins from these matrices. As shown in Figure 4.2, proteins are

released either by diffusion through polyrner mata or diHusion through water

filfed pores that were created by the degraded polymer matrix.

T=O

Figure 4.2. Degradation-controlled rnechanisrn

2. Diffusion controlled mechanism (percolation theory):

Altemativeiy, protein release can be attributed to the diffusion of protein

molecules through water filled preexisting pores and interconnected channels

within the polymer matrix as shown in Figure 4.3, that were created after protein

dissolution.

- - - - . - - - -

T=O

Figure 4.3 Difision controlled mechanism

OVA was released from the microspheres from the beginning of the

release. There was an 18% to 30% protein release from ail the polymeric

microspheres during the first 10 d of release; however, only less than 5% mass

loss was observed. Sinœ OVA's release from these polyrner matrices by

partition diffusion is minimal. the mechanism of protein release during this period

of time is attributed to the diffusion of protein from water filled pores which may

be a result of dissolution of protein powder on the surface during washing.

Interestingly. the release profile of PLGA 5W50 showed a significant increase

from day 30 to day 42. This is accompanied by a drarnatic decrease in molecular

weight as well as mass loss, indicating a rapid breakdown of polymer due to

autocatalytic hydrolysis. Similar increases in protein release in conjunction with

accelerated polyrner degradation have been reported by Agrawal[94] and Chiu

[45]. The release profile of PCL, on the other hand, sees a rather constant

release. SEM micrographs show that there is no pore formation in PCL

microspheres due to degradation. Within the time that 75% of the encapsulated

protein was released, PCL only experienced a 30% rnass loss as a result of

degradation. This indicates that unlike PLGA 50150, PCL released protein mainly

via the percolation mechanism. The results of the effciency of encapsulation

showed that PCL, having the fastest precipitation at the interface, had the

highest entrapment of protein. Wiih the most protein encapsulated, PCL had the

greatest Iikelihood for protein release via percolation. Therefore, it is evident that

PLGA 50/50, having the fastest degradation rate, released protein mainly in

concert with degradation while PCL. having the slowest degradation rate but the

most protein encapsulated, released protein via percolation.

If diffusion controlled mechanism and perfect sink conditions were

assumed . release behavior from rnatrix type microsp heres should obey Fick's

second law [95]. The equation can be simplified to:

where M* is the total dtwg incorporated, Mt is the amount of drug released at time

t . Deff is the diffusivity of protein through polymer matrices and 1 is the diameter

of microspheres in this case. By plotting Mt/ M* against t '" insight can be

gained into the protein release mechanism from the biodegradable polymeric

microspheres.

0 - - - -. - - -- - - - - ---- y- ---

O 2 4 6 8 10

ümeA0.5 (dayA0.5)

+ PLGA50150 A PLGA85115 i PCL blend L i n e a r (PCL)

Figure 4.4 plot of cumulative release vs. t ln

As shown in Figure 4.4, the cumulative release from PCL microspheres is

proportional to t'n, which suggests that the protein release mechanism from PCL

is primarily diffusion whereas data from PLGA50150. PLGA85/15 as well as

blend do not yield statistically significant linear relationship from regression. ( R ~

are 0.84, 0.95 and 0.91 for PLGA50/50, PLGA85115 and blend respectively)

The NGF release detenined by ELlSA showed an 'burst' for NGF-1

release followed by a constant release profile. Given that the amount of NGF

released after 20d was constant, yet the extent of neurite outgrowth was

reduced, implies that the released NGF bas either biologically inactive or at a

lower concentration than the positive control. Experiments of PC12 cell cultures

supplemented with NGF at a concentration similar to that observed by the NGF-

ELISA was employed to detenine whether the released NGF was at too low a

concentration to show the same activity or simply inactive and the result showed

that the released NGF was still biologically active.

The release profile in this study shows a substantial release over 90 d.

The desired pattern of release required to maximize the therapeutic effects,

however, can only be detemined after the mechanism of action,

phamacokinetics. pharmacodynamies and receptor dynarnics are defined. Then

a rational dosage and design of the delivery system c m be established.

5. CONCLUSIONS

Microspheres. having an average size of 19.3k5 .O, were prepared from

biodegradable polyesters - PLGA50f50, PLGA85115, PCL and a blend of

PLGABOI5OIPCL (1 : 1, whv). The release profile of ovalbumin (OVA), a model

protein for nerve growth factor (NGF), was found to depend upon protein loading

(Le. percolation) and polymer degradation. The released profile of NGF. CO-

encapsulated with OVA was followed by the NGF-ELISA while bioactivity was

detemined by neurite outgrowth from PC12 cells. NGF release was followed

over 90 d and found to be bioactive for 90 d.

1 OVA was released from microspheres in a sustained manner over a period of

84 d.

2 By comparing the degradation and release profile, degradation was the

predominant pathway for protein release from PLGA50150 whereas the

interconnected pore structure was the main pathway to release from PCL.

3 The encapsulated NGF maintained bioactivity after release for 90 d. The ratio

of protein NGF to OVA provided a means to tailor the concentration of NGF

released.

6. FUTURE WORK

In this work, it was shown that protein release profiles were affected both by the

degradation rate of the biodegradable polymer matrix and by the protein loading

which leads to interconnected pathway for protein to diffuse. Although al1 the

other preparative parameters were kept identical when microsp heres were

prepared from different biodegradable polymers (PLGA50i50, PLGA85115, PCL

and blend of PLGA50/50 and PCL 111 (wiw)), difFerent protein loadings were

obtained due to different precipitation rates of polymers at the polymer solution-

surfactant aqueous interface. By tailoring the parameters to control the rate of

polymer precipitation, protein loading could be manipulated. (cf Figure 3.12) In

order to better understand the roles of degradation and loading on protein

delivery from biodegradable microspheres, it would be interesting to conduct the

following experiments:

To investigate the effect of degradation on the release profile. Study the

protein release profiles from different polymen (wlh different degradation

rates) with the same protein loading.

To investigate the effect of the percolation on the release profile. Study the

release profile from the sarne polymer with different protein loading.

To incorporate microspheres into 3dimentional hydrogels and investigate the

release profile in cornpanson with that from microspheres alone.

7 APPENDIX

PREPARATlONS OF SOLUTIONS FOR NGF-ELISA

Solutions

1. Coating buffer

Na2COJNaHC03, 50 mmoM; sodium azide, 0.1 %

II Coating solution

Shortly before use, dilute the reconstituted anübody solution wÏth coating

bufier (1). The concentration depends on the plates used and range between

0.1-1 pg aantibodylml.

I I I Blocking solution

Bovine serum albumin, 0.5% (wlv) in coating buffer (1).

IV Sampleistandard and conjugate buffer

Tris-HCI, 50 mmoiA; NaCI, 200 mmoVI; CaCb, 10 mmoU1; bovine serum

albumin, 1 % (wfv); Triton X-100, 0.1 % (wlv); sodium azide, 0.05% (wlv).

V Extraction buffer

VI Washing buffer

Sample buffer (IV) without bovine serum albumin.

VI1 Substrate buffer

Hepes, 100 mmol/l; NaCI, 150 mmoIl1; MgCi* 2 rnrnoVi; bovine serum albumin,

1% (wlv) ; sodium azide, 0.1% (wiv)

IIX Substrate solution

40 mg Chlorophenolred-B-D-galactopyranoside with 20 ml substrate buffer

(W). Stir solution for at lest 20 min at room temperature.

IX Antibody-p-gal-conjugate solution

Mix 1 volume of the reconstituted anti-NGF-P-gal conjugate with 9 volumes of

conjugate bMer (IV)

X NGF-B standard solution

Construction of UV-Vis calibration curve

Preparation of standard solutions

0.0503 g of OVA was weighed and caref'ully transferred to a 50 ml volumetric

fiask. The OVA was dissolved in PBS (pH 7.4) solution in the fiask. The

volumetric fiask was filled to the line by adding PBS (pH 7.4) foming the most

concentrated standard solution for the calibration (approximately 1000 ppm).

Other standards were prepared in serial dilutions with a concentration of 115,

111 0, 1/50, 111 00, 1/200 of the most concentrated standard. Figure

Appendix. 1 is the calibration curved constructed and used for OVA

concentration determination.

O 500 1 O00 1500

concentration (ppm)

Figure Appendix.? OVA UV-vis calibration curve

Determination of h ,, h ,, a wavelength at which protein has the most absorbance was

determined by the general smn over the entire wavelength range from 190

nm to 820 nm. Figure Appendix.2, is the result of the scan from the most

concentrated standard. The two wavelengths represent the wavelengths at

which OVA has the rnost absorbanœ. Sinœ 280 nm is the well documented

wavelength to detemine the concentraüon of protein solution. this wavelength

was chosen to determine the OVA concentration in this study.

M a r ked WaveLengths Reg A: L 232 = 2.3901 Reg A : L 280 = 0.61264

8. REFERENCES 7. John Nicholls and Norman Saunders, Regeneration of immature mamrnalian spinal cord

after injury, Tiends Neumsci.. 19, 229-234,1996.

2. M. Risling, K. Fried, H. Linda, T. Carlsted, S. Cullheim, Regrowth of motor axons following

spinal cord lesions: distribution of laminin and collagen in the CNS scar tissue, Brain Res.

Bull., 30,405-414, 1993.

3. F. Xavier Santos, Gonzalo Bilbao, Jose R~drigo, Jorge Fernandez, David Martinez, Edgard

Mayoral, Julian Rodriguez, Experimental mode1 for local administration of nenre growth

factors in microsurgical nerve recunnections, Micmurgery, 16.71 -76, '1 995

4. Bernard Knwps, Cecile Ponsar, Isabelle Hubert, Philipe Van Den Bosch De Aguilar,

Regeneration of lesioned cholingeric septal neurons of the adult rat can be promoted by

peripheral newe grafts and a fibrin-fibroneticin-containing mat* of peripheral regeneration

cham bers, Brah Res. Bull., 30,433437, 1 993

5. R. Marchand, S. Woerly, L, Bertrand, N. Valdes, Evatuation of two cross-linked cullagen gels

implanted in the transected spinal cord, Brain Res. Bull. 30,415-422, 1993

6. P-Aebischer, R.F. Valentini, S.R. Winn and P.M. Gatletti, The use of a semi-penneaable tube

as a guidance channel for a transected rabbit optic nerve, Prog in Brain Res,, 78, 599-603,

1988

7. Mark H. Tuszynski and Fred H. Gage, Bridging grafts and transient newe growth factor

infusion promote long-term central newous system neuronal rescue and partial functional

recovery, Proc. NaU. Acad. Sci. USA, 92,4621 4625,1995.

8. David J. Berlove, Seth P. Finklestein, 'Groth factors and brain injury' in Growth factors,

peptides and receptors (Teny W. Moody Ed.), Plenum Press, New York, 1993.

9, William H. Burgess, 'Structure-function analysis of fibroblast growth factor-1' in Growth

factors, pepüdes and receptors (Terry W. Moody Ed.), Plenum Press, New York, 1993.

10, Ann Logan, James J. Oliver, Martin Berry, Growth factors in CNS repair and regeneration,

Progres in gmwth factor research, 5, 379405, 1994

1 1. David M. Fim, Julie K. Andersen, James M. Schumacher, M. Priscilla Short, Ole Isacson,

Xandra Breakefield, 'Gene transfer into central newous system: Neutrophic factors' in in in

Growth facfors, peptides and receptors (Terry W. Moody Ed.), Plenum Press, New York,

1993.

12. R. Ishikawa, K Nishikori and S. Funikawa, Appearance of nerve growth hctor and acidic

fibroblast growth factor with different time courses in the cavity-lesion cortex of the rat brain,

Neurosci. Le@. , 127, 70-74,1991.

13. Molly S. Shoichet, Frank T. Gentile, Shelly R. Winn, The use of polymers in the treatrnent of

neurological disorders, Trend in Polymer, 3, 374-380, 1995.

14. Robert Langer, New methods of Drug delivery, Science, 29, 1527-1533,1990.

15. J. Fiiipovic-Grcic, O. Mayainger, 1. Jalsenjak, Microparticels with neuroactive agents, J.

Micmencapsulation, 12, 343-362, 1995

16. R.L Juliano, Dmg delivery systems: characteristics and biomedical applications, Oxford

University Press, 11980

17. Robert Langer, 1994 Whitaker lecture: Pofymers for drug delivery and tissue engineering,

Annal. Biomed. Eng., 23, 101-1 1 1, 1995

18. Henry Brem, Rafanel J. Tamargo, Alessandro Olivi, Michael Pinn, Jon D. Weingart, Moody

Whararn Jonathan 1. Epstein, Biodegradable polymers for controlled delivery of

chemotherapy with and without radiation therapy in the rnonkey brain, J. Neurosurg., 80,

283-290, 1994

19. M. T. Aguado. P.-H. Lambert, Controlled release vaccine- biodegradable polylacb'de/

polyglycolide (PUPG) microspheres as antigen vehicies, Irnmunobid, 184, 1 13-1 25, 1992

20. Hongkee Sah, Rohinton Toddywala, Yie W. Chie, Continuous release af protein from

biodegradable microcapsules in vivo evaluation of their potential as a vaccine adjuvant, J.

Contmlled Release, 35, 1 37-1 44, 1995.

21. Ming-Kung Yeh, A.G.A. Coambes, P.G. Jenkins, S.S. Davis, A novel emulsification-solvent

extraction technique for production of protein loaded biodegradable microparücles for

vaccine and drug delivery, J. Controlled Release, 33,43745, 1995

22. Davis A Edwards, Justin Hanes, Giovanni Caponetti, Jeffrey Hrkach, Abdelaziz Ben-Jebria,

Mary Lou Eskew, Jeffrey Mintzes, Daniel Deaver, Noah Lotan, Robert Langer, Large porous

particles for pulmonary drug delivery, Science, 276, 1868-1 871, 11 997

23. Philippe Menei, Jean-Pierre benoit, Michelle Boisdron-Celle, Dominiqe Fournier, Philippe

Mercier, Gilles Guy, Dnig targeting into the central nenrous system by stereotactic

implantation of biodegradable microspheres, Neurosurgery, 34, 1058-1 064, 1994

24. Hiroaki Okada, Yayoi Doken, Yasuaki Ogawa, Hajime Toguchi, Sustained suppression of the

pituitary- gonadal axis by Leuprorelin three month depot microspheres in rats and dogs,

Pharmaceutical Res., 1 1, 1 1 99-1 1 O3,l 994

25. Ichiro Yamakawa, Tuki Tsushima, Ryochi Machida, Sumio Watanabe, In vitro and in vivo

release of poly ( DL-lactic acid) microspheres containing neurotensin analogue prepared by

novel oil-in-water solvent evaporation rnethod, J. Pham. Sci., 81, 808-81 1, 1992

26. P.K Gupta, H. Johnson, C. Allexon, In vitro and in vivo evaluation of clarithromycin/

poly(1actic acid) microspheres for intramuscular drug delivery, J. Controleû Release, 26,

229-238, 1993

27. Reza Arshady, Preparation of biodegradable microspheres and microcapsules: 2.

Polyactides and relateû polyesters, J. Controlled Release, 17, 1-22, 1991

28. Reza Arshady, Biodegrada ble microcapsular dnig del ivery systems: manufacturing

methodology, release control and targeting prospects, J. Bioactive Compatible Polymers, 5,

315342,4990

29. Hsiu-O Ho, Chih-Chuan Hsiao, Theodore D. Sokoloski, Chau-Yang Chen, Ming-Thau Sheu,

Fibrin-based drug delivery systems Ill: the evaluation of the release of macromolecules from

microbeads, J. Controlled Release, 34.65-70,1995

30. Takafurni Ishizaka, Michiyo Motojirna, Shigeni Kounosu, Masumi Koishi, In vitro release of

poly (ethylene oxide) h m serum albumin microspheres, Chem. Pham. Bull., 34, 3341-

3347,1986

31. Wu Lin, Martin C. Gamett, Martyn C. Davies, Fabio Bignotti, Paulo Femiti, Stanley S. Davis,

Lisbeth Illurn, Preaparation of surface-modified albumin nanospheres, Biomatenals, 18, 559-

565,1997

32. Reza Arshady, Albumin microspheres and microcapsules: methodology of manufacturing

thechniques, J. ControIled Release, 14, 1 1 1 -1 3 1,1991

33. P.K Gupta, C.T. Hung, Albumin micnspheres II: applications in dnig delivery, J.

Microencapsulation, 6,463-472, 1989

34. A. Apicella, B. Cappella, M.A. Del Nobile, M.I. La Rotonda, G. Mensitoeri, L- Nicolais, Poly

(ethylene oxide) (PEO) and different molecular weight PEO blends monolithic devices for

dnig release, Biomatenals, 1 4, 83-90, 1 993

35. Elizabeth M. Powell, M. Rovena Sobarzo, W. Mark Saltzman, Controlted release of nerve

growth factor from polymeric implant, &in Res., 515, 309-31 1, 1990

36. Abraham J. Domb, Implantable biodegradable polymers for site-specific drug delivery in

"polymeric site-specific pharmacotherapy" (A.J. Domb, Ed.), John Wley & Sons Ltd., 1994

37. Yasuaki Ogawa, Masaki Yamamoto, Hiroaki Okada, Takatsuka Yashiki, and Tsugio

Shimamoto, A New Technique to Efficientiy Entrap Leuprolide Acetate into Microcapsules of

Potyactic Acid or Copoly (Lactic/Glycolic) Acid, Chem. Pham. Bull., Vol. 36, No. 3, 1 O S -

llO3,1988

38. C.G. Pitt, M.M. Gratzl, G.L. Kimmel, J. Surles, A. Schindler, Aiiphatic polyesters II. The

degradation of ploy (DL-lactide), poly (E-caprolactone), and their coploymers in vivo,

Biomaterials, 2, 2 1 5-220, 1981

39. 1. Grizzi, H. Garreau, S. Li, M. Vert, Hydrolic degradation of devices based on poly (DL-lactic

acid) size-de pendence, Biomaterials, ? 6, 305-3 1 1 , 1 995

40. Hans Pistner, Dieter R. Bendk, Joachim Muhling, Jurgen F. Reuther, Poly(L4actide): a long-

term degradation study in vivo, Part III. Analyücal characterization, Biomaterials, 14, 291 - 298,1992

41. KW. Leong, B.C. Brott, R. Langer, Bioerodible polyanhydrides as drug-carrier matrices. 1:

characterization, degradation, and release characteristics, J. Biomed. Mater. Res., 19, 949 - 955,1985

42. Hans Pisten, Harald Stallforth, Ralf Gutwald, Joachim Muhling, Jurgen Reuther, Christian

Michel, Poly (L-lactide): a long-tenn degradation study in vivo. Part II: physico-mechanical

behaviour of implants, Biomatenals, 1 5, 439-450, 1994

43. P. Robert, J. Mauduit, R.M. Frank, M. Vert, Biocompatibility and resorbability of a polylactic

acid membrane for periodonal guided tissue regeneration, Biomatenals, 15, 353-358, 1993

44. Horst A. von Recum, Robert L. Cleek, Suzanne, G. Eskin, Antonios G. Mikos, Degradation

of polydispersed poly (L-lactic acid) to modulate lactic acid release, Biomatenals, 16,441-

447,1995

45. G. Spenlehauer, M. Vert, J.P. Benoit, A Boddaert, In vitro and in vivo degradation of poly

(QL-lactide/glycoIide) type microspheres made by solvent evaporation method, Biomaterials,

1 O, 557-563,1989

46. Jacques Mauduit, Eric Perouse, Michel Vert, Hydrolytic degradation of films prepared from

belnds of high and low molecular weight poly (DL-lactic acid)s, J. Biomed. Mater. Res., 30,

201 -207,1996

47. Masaharu Asano, Masaru Toshida, Hideki Omichi, Tooru Mashimo, Kazuhiko Okabe, Hisako

Yuasa, Hidetoshi yamanaka, Seiki Monmoto, Hido Sakakibara, Degradable poly (DL-lactic

acid) fomulations in a calcitonin delivery system, Biomatenals, 14, 797-799, 1993

48. L.K. Chiu, W.J. Chiu, Y.-L. Cheng, Effects of polymer degradation on drug release - a

mechanistic study of morphology and transport properües in 50:50 poly(D1-lactide-m-

glycolide), International J. Pham., 126, 169-1 78, 1995

49. Giovanni G. Giordano, Patricia Chevez-Bamos, Miguel F. Refojo, Charles A. Garcia,

Biodegaradtion and tissue reaction to inbavitreous biodegradable poly (DL-lactic-coglycolic)

acid microspheres, Cun. Eye Res., 14, 761-768,1995

50. Yukihiko Kinoshita, Mitsuhiro Kirigakubo, Masaru Kobayashi, Takatoshi Tabata, Kaito

Shimura, Yashito Ikada, Study on the efftcacy of biodegradable poly (L-lactide) mesh for

supporting transplanted particulate cancellous bone and marrow experiment involving

subcutaneous implantation in dogs, Biomaterials, 14, 729-736, 1993

51. Tae Gwan Park, Degradation of poly (lactic-CO-glywlic acid) rnicrospheres: effect of

copolymer composition, Biomaterials, 16,1123-1 130, 1995

52. P. Mainil-Varlet, B. Rahn, S. Gogolewski, Long-ten in vivo degradation and bone reaction to

various polylactides, Biomaterials, 8, 257-266,1997

53. M. van der Elst, A..A Dijkema, C.P.AT. Klein, P. Patka, H. J.Th.M. Haarman, Tissue reaction

on PLLA versus stainless steel linterlocking nails for fracture fixation: an animal study,

Biomaterials, 1 6, 1 03-1 06, 1 995

54. G.E. Visscher, R.L. Robison, H.V. Maulding, J.W. Fong, J.E. Pearson, and G.J. Argentieri,

Note: Biodegradation of and Tissue Reaction to poly(DL-Lactide) Microcapsules, J. Biomed.

Mater. Res., 20,667-676,1986

55. P. Menei, V. Daniel, C- MontemMenei, M. Brouillard, A Pouplard-Barthelaix, and J.P.

Benoit, Biodegradation and Brain Tissue Reaction to Poly(D, L-lactide-coGlycolide)

Micros pheres, BiomatenaIs, 1 4, 470-478, 1 995

56. P.J. Watts, M.C. Davies, C.D. Melia, Microencapsulation using emulsification/solvent

evaporation: an overview of technique and applications, Crit-cal Reviews in 77?erapeutic Dmg

Camer Systems, 7, 235-259,1990

57. Reza Arshady, Microcapsules for food, J. of Micmencapsulation, lO,4l 3435,1993

58. H. Jeffery, S.S. Davis, D.T. OHagan, Preparation and characterisation of poIy (laetide-co-

glycolide) microparticles. t: Oil-in-water emulsion solvent evaporation, Inter. J. Pham., 77,

169-1 75,1991

59. Hiroaki Okada, Masaki Yamamoto, Tosshiro Heya, Yayoi Inoue, Shigeru Kamei, Yasuaki

Ogawa, Hajime Toguchi, Drug delivery using biodegradable microspheres, J. Contmlled

Release, 28, 121 -1 29, 1994.

60. Tae Gwan Park, Weiqi Lu, George Crotts, Importance of in vitro experimental conditions on

protein release kinetics, stability and polymer degradation in protein encapsulated poly (D,L-

lactic acid-co-glycolic acid) microspheres, J. ControlIed Release, 33.21 1-222, 1995

61. Yashuaki, Ogawa, Hiroaki Okada, Masaki Yamamoto, Tsugio Shimamoto, ln vivo profile of

Leuprolide acetate from rnic~ocapsules prepared with polylactic acid or copoly

(lacüc/glycolic) acids and in vivo degradation of these polyrners, Chem. Pham. Bull, 36,

2576-2581,1988

62. Smadar Cohen, Toshio Yoshioka, Melissa Lucarelli, Lena H. tiwang, Robert Langer,

Controlled delivery systems for protein based on poly(lactic/glycolic acid) rnicrospheres,

Phami. R ~ s . , 8,7 1 3-720, 1991

63. Paul 3. Carnarata, Raj Suryanarayanan, Dennis A. Turner, Richard G. Parker, Timothy J.

Ebner, Sustained release of newe growth factor from biodegradable polymer microspheres,

Neumsurg., 30, 31 3-319, 1992.

64. R. JaM, J.R. Nixon, Microencapsulation using poly(L-lactic acid) 1: microcapsule properties

affected by the preparative tech niques, J. Micmencapsulation, 6,473-484, 1 989

65. Hiroaki Okada, Yayoi Doken, Yasuaki Ogawa, and Hajime Toguchi, Preparation of Three-

Month Depot Injectable Microspheres of Leuprorelin Acetate Using Bidegradable Polymers,

Pham. Res., 11,1143-1 147,4994

66. R. Bodmerier, McGinÏty LW, Polylactic acid microspheres containing quinidine base and

quinidine sulphate prepared by the solvent evaporation techniques, J. Microcapsules, 4, 279-

286,9987.

67. Hiroaki Okada, Hajime Toguchi, Biodegradable microspheres in drug delivery, Critical

Reviews in Thenpeutic Dmg Camer Systems, 12,l-99,1995

68. H.K. Sah, R. Tddywala, Y-W. Chien, Biadegradable microcapsules prepared by a w/o/w

technique: effects of shear force to make a pnmary wlo emulsion on their rnorphology and

protein release, J. Microencapsuiation, 1 2,59-69, 1995

69. L. S. C. Wan, P.W.S. Heng and LW. Chan, Drug Encapsulation in Alginae Microspheres by

Ernulsification, J. Micmncapsulation, 9, 309-316, 1992

70. Maria Grazia Cascone, Bushra Sim, and Sandra Downes, Blends of Synthetic and Natural

Polymers as Dnig Delivery Systerns for Growth Hormone, Biomafenals, 16, 569-574, i995

71. S.S. Shah, Y. Cha and CG. Pitt, Poly (Glycolic Acid-Co-DL-Lactic Acid): Diffusion or

Degradation ControBed Dnig Delivery?, J. Controlled Release, 1 8, 26 l-ZïO1l992

72. Michel Thenn, Pascal Christel, Suming Li, Henri Garreau, Michel Vert, In vivo degradation of

massive poly (a-hydroxy acids): validation of in vitro findings, Biomaterials, 13, 594-560,

1995

73. Hayley Jeffery, Stanley S. Davis, and Derek T. OIHagan, The Preparation and

C haracterization of Poly(1actide-CO-g lycolide) micro particles. II. The Entrapment of a Model

Protein Using a (Water-in-0il)-in-Water Emulsion Solvent Evaporation Technique, Pham

Res, 10, 362-368,1993

74. Paul J. Carnarata, M.D., Raj Suryanarayanan, Ph-D., Dennis A. Turner, M.D., Richard G.

Parker, M.D., and Timothy J. Ebner, M.D., Ph-O., Sustained Release of Nerve Growth Factor

from Biodegradable Polymer Microspheres, Experimental and Clinic Studies, 30, 31 3-31 9,

1992

75. Samdar Cohen, Toshio Yoshioka, Melissa Lucarelli, Lena H. Hwang, and Robert Langer,

Controlled Delivery Systems for Proteins Based on Poly(LacticlGlycolic Acid) Microspheres,

Pham Res, 8,713-720.1991

76. Alain Rolland, Nathafie Wagner, Alain Chatelus, Braharn Shroot, and Hans Schaefer, Site-

Specific Drug Delivery to Pilosebaceous Structures Using Polymeric Microspheres, Pham.

Res, 1 O, 1738-1 1744,1993

77. Valer J. Csemus, Bela Szende, and Andrew V. Schally, Release of Peptides from Sustained

Delivery S ystems (microcapsules and microparücles) in vivo, /nt J. Peptide Protein, 35, 557-

565,1990

78. Lloyd A Greene, Arthur S. Tischler, Establishment of a noradrenergic clonal line of rat

adrenal pheochromocytoma cells which respond to nerve growth factor, Pro. Nat/. Acad. Sci.

USA, 73,2424-2428,1976

79. Arthur S. Tischler, Lloyd A Greene, Paul W. Kwan, Val W. Slayton, Ultrastructure effects of

nerve growth factor on PC 12 pheochromocytoma cells in spinner culture, Ce11 i k u e Res.,

228,641-648,1983

80. R. lan Freshney, Culture of animal cells (2m edition), Alan R. Liss Inc., New York, 1987

81. Ephraim Yavin, Ziva Yavin, Attachment and cutture of dissociated cells frorn rat embryo

cerebral hernisp heres on polylysinecoated surface, J. Ce11 Biology, 62, 540-546, 1974

82- Lloyd A Greene, A quantitative bioassôy for nerve growth factor (NGF) activity employing a

clonal pheochrornocytoma ce11 line, Brain Res., 133, 350-353, 1977

83. lan MaKay, lrene Leig hl Growth Bctors: a pmcücal appmach Oxford University Press, New

York, 1993

M. F. Boury, H. Marchais, J.E. Proust, J.P. Benoit, Bovine serum albumin release from poly (a-

hydroxy acid) microspheres: effects of polymer molecular weight and surface properties, J.

Controlled Release, 45, 75-86, 1997

85. Balaji Narasimhan, Robert Langer, Zero-order release of micro- and macromolecules from

polymeric devices: the role of the burst effect, J, ControIIed Release, 47, 13-20, 1997

86. Yasuhiko Tabata, Yoshihiro Takebayashi, Torataro Ueda, and Yoshito Ikada, A Fomlation

Method Using D,L-lacüc Acid Oligomer for Protein Release with Reduced Iniüal Burst, J.

Controlled Release, 23,5564, 1993

87. Tae Gwan Park, degradation of ploy (D, L-lactic acid) microspheres: effect of molecular

weight, J. Confrolled Release, 30, 161-173, 1994

88. lchiro Yamakawa, Yuki Tsushima, Ryoichi Machida, and Sumio Watanabe, Preparation of

Neurotensin Analogue-containing Poly(d1-lactic acid) Microsphere Formed by Oil-in-Water

Solvent Evaporation, J. Pham Sci., 81, 899-903, 1992

89. H.T. Wang, E. Schmitt, D.R. Flanagan, R.J. Linhardt, Influence of formulation rnethods on

the in vitro controlled release of protein from poly (ester) microspheres, J. Controlled

Release, 17, 23-32, 1991

90. Albert L. Lehninger, David L. Nelson, Michael M. Cox. Princples of biochemistry (2*

Edition), Worth Publishers, Inc., New York, 1982

91. Changhong Yan, James H. Resau, John Hewetson, Michael West, Wayne L. Rill, Meir

Kende, Characterization and morphological analysis of protein-loaded poly (lactide-co-

glycolide) microparticels prepared by water-in-oil-in-water emulsion technique, J Controlled

Release, 32,231-241, 1994

92. Weiqi Lu, and Tae Gwan Park, Protein Release from Poly(1actic-CO-glycolic acid)

Microspheres: Protein Stabil'i Prolems, PDA J.Phann. Sci. and Tech., 49, 13-19, 1995

93. Weiqi Lu, and Tae Gwan Park, In Vitro Release Profiles of En'stostaün from Biodegradable

Poly meric Microsp heres: Protein Aggregation Problerns, Biotechnol. Prog, 1 1 , 224-227,

1995

94. C.M. Agrawal, J. Best, J.D. Heckman and B.D. Boyan, Protein Release Kinetics of a

Biodegradable Implant for Fracture Non-Unions, Biomatenals, 16, 1255-1 260, 1995

95. Ronald k Siegel, 'Modeling of drug release from porous polyrners' in Conhlled release of

dmgs (Morton Rasoff Eds), 1-51. VCH Publishers Inc., New York, 1988

96. Product specification for NGF-ELISA by Boehringer Hannheim, Gemany, 1997

l MAGE NALUATION TEST TARGET (QA-3)

APPLIED IMAGE. lnc fi 1653 Eaçt Main Street - -* - Rochester. NY 14609 USA -- --= Phone: 71 6/482-0300 =-= Fax 71 ô/2û8-5989

O 1993. Applmd image. lm., AU Rqhts bsemd