Indian Journal of Experimental Biology Vol. 38, August 2000, pp. 746-752

Review Article

Poly (DL-Iactide-co-glycolide) based delivery systems for vaccines and drugs

Neelam Dhiman, Manisha Dutt &G K KhuIIer*

Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India

Current vaccination and drug delivery strategies emphasize on the development of controlled release techniques for persistent and sustained effects. In the recent years, polymer based systems for the delivery. of bioactive agents have gained

. considerable attention due to their marked adjuvanticity, established biodegradability and biocompatibility, excellent mechanical strength and controlled release profiles. This review deals with the potential applications of synthetic polymers mainly PLG polymers in deli~ery of vaccines and drugs.

During the last two decades, the delivery of bioactive agents from polymeric materials has attracted considerable attention of investigators throughout the scientific community. Among the possible strategies available today, micro and nanoparticles represent excItmg technologies able to improve the pharmacokinetic profile of numerous peptides and drugs to enable a more efficient transport of these molecules across biological barriers. The commonly used biodegradable polymers for systemic delivery of macromolecules include various natural and synthetic polymers and some depot forming formulations which have been listed in Table 1. The use of natural polymers is limited because of their relatively high costs and questionable purity. The most widely investigated and advanced polymers with regard to available toxicological and clinical data are the aliphatic polyesters based on lactic and glycolic acids. Amongst the initial reports on polylactic acid used for controlled release were by the research teams who sought delivery systems for narcotic antagonists, contraceptive hormones, conventional drugs and antibiotics 1-4. Many companies have also exploited polymers for use in medical implants, dental and fracture repairs and surgical dressings5

•

Poly (DL-Iactide-co-glycolide) or PLG polymers They are synthetic, a-hydroxy acids such as poly­

DL-lactide (PLA) and copolymers of lactide and glycolide such as poly DL-lactide-co-glycolide (PLG)

*Correspondent author: Tel: 747585 Ext. 282, 274; Fax: 0172-744401; Email : medinst@pgi .. chd.nic.in

synthesized by the ring opening melt condensation of the cyclic dimer-lactide and glycolide6

. PLG polymers are biodegradable, biocompatible, non­immunogenic with controllable release profiles ranging from several weeks to months. Biodegradation of polymers occurs by bulk erosion7

Table I-Types of biodegradable polymers

or Natural polymers * Proteins and polypeptides

a) Albumin b) Fibrinogen and fibrin c) Collagen d) Gelatin e) Casein

* Polysaccharides a) b) c) d)

* Cells and viruses

Synthetic polymers

Starch, Dextran and Dextrins Alginic acid Hyaluronic acid Chitin and Chitosan.

* Aliphatic polyesters and hydroxy acids a) Poly lactide-co-glycolide

(PLGA)/polylactic acid (PLA) b) Terpolymers ofPLGA c) Poly (P-hydroxybutyric acid) (PHB) d) Poly e-caprolactone (PCL)

* Polyanhydrides (PHA) * Poly (ortho esters) (POE) * Poly alkyIcyanoacrylate (PACA)

or Leuprorelin acetate depot formulation or TRH depot formulation ... TNP-470 microspheres

DHIMAN et at : POLY (DL-LACTIDE-CO-GLYCOLIDE) BASED DELIVERY SYSTEMS 747

or enzymaticalll to monomeric acids, which are eliminated from the body through the Kreb's cycle (Fig. 1).

PLG polymers containing 50:50 ratio of lactic and glycolic acids are hydrolyzed much faster than those containing a higher proportion of either of the two monomers9. PLG has been used for drug delivery in various forms such as capsules, tablets, powder, topical and injectables1

0-13

• Some of the advantageous features offered by this technology are greater drug stability, sustained drug release, protection from unfavourable environment, drug targeting, enhancement of drug uptake and alteration of surface properties ll

•13

. Another advantage of PLG microparticles is their proven adjuvanticity when d . . d II . t II 14,15 a mmlstere ora y or In ranasa y

PLG as a delivery system for drugs Microsphere based drug delivery systems using

various kinds of biodegradable polymers have been extensively studied during the past two decades. Microspheres are microporous matrix systems (size range between 0.1-200~m) where the drug is uniformly dispersed into the polymer. Injectable and biodegradable microspheres appear to be particularly ideal delivery systems due to the avoidance of the insertion of large implants with a local anesthesia and no requirement to remove the device after the drug supply is exhausted. The first application of polylactide as erodible matrices for long acting controlled release of a drug were reported in the early 1970S16.1 8

• Subsequently, Beck et al. 19 proposed a long acting injectable microcapsule system for the controlled release of progesterone. Since then, many drugs have been incorporated into microparticles, and their release rates studied both in vitro and in vivo. Compounds with a short biological half life, instability, degradability in the gastrointestinal tract and high toxicity are good candidates for incorporation into these systems. The microparticles for in vivo drug release are generally prepared by grinding matrix20

, emulsion (O/W, 0/0) - solvent • • 21 d evaporation, solvent evaporatIOn extractton an

22 • h . spray drying method . The mlcrosp ere preparatIOn methods and in vivo drug release rates for water insoluble and water-soluble small drugs have been listed in Tables 2 & 3 respectively. Drugs formulated in such, polymers are released either by diffusion through the polymer barrier or by erosion of the polymer material or by a combination of both.

~CH'+ CH3~O

o DL-Iactide

Poly DL-Iaclide-co·glycolide

1 Hydrolysis o 11

HO-CH2-C-OH

Lactic acid Glycolic acid

Fig. I-Synthesis, structure and biodegradation products ofDL-PLG.

Long circulating carrier systems Though PLA and PLGA microparticles have been

successfully used to deliver drugs for long period of time54, however, because of their large size, it is impossible to direct the drug to the target tissues via systemic circulation or across the mucosal membrane55. After intravenous administration, the uptake of nanoparticles and their subsequent clearance by the reticuloendothelial system (RESi6

impose major hurdles for the targeted delivery of drugs57

•

To overcome these drawbacks, long circulating microparticulate drug carriers have been developed that involve the coating of carriers with certain hydrophilic and flexible polymers primarily with poly (ethylene glycol) i.e. PEG13

• The advantages of using PEG include its excellent solubility in aqueous solutions, high flexibility of its polymer chain, low toxicity, immunogenicity and antigenicity, lack of accumulation in the RES cells and minimum influence on specific biological properties of modified pharmaceuticals58,59 Therefore, PEG modified carriers can help to a<;hieve a better targeting effect for specific ligand modified drugs and

. 6061 drug carners '

lmplantable systems/devices Many groups have reported on the preparation of

drug loaded implants from PLA or PLGA62.

Implantable systems developed so far include devices such as nails tablets, pellets, films, fibres and rods,

, 10,52 circular plates and porous membranes .

748 INDIAN J EXP BIOL, AUGUST 2000

Table 2-Biodegradable microspheres for water-insoluble small drugs

Drug Polymer Preparation Released in vivo

Cyclazocine PLA-4S,ooo Grinding 29%/S3 days (17)

Naltrexone PLGA (90/1 0) Coated beads 90 days (23)

40,000-200,000

WR-lS8122 (Antimalarial) PLGA (2SI7S) Spray dry 14 weeks (3)

Progesterone PLA 2S,OOO OIW SO/I day + 30 days (19)

Norethisterone PLA PLGA (88/12)-40,000 OIW 6 months (24)

Butamben tetra- caine PLA-9,100 -2S,OOO OIW (2S)

Dibucaine PLA-17,ooO OIW 17 days (26)

Bleomycin PLA -2S,OOO - 4S ,OOO OIW 30-4S%/Sdays (27)

Aclarubicin PLA-3S,OOO OIW 94%/I6hr (28)

Lomustine progesterone PLA - 61 ,000 OIW (29)

Hydrocortisone PLA - 33,000 0/0 40 days (30)

Cisplatin PLA - 60,000 OIW 20 days (31)

PLGA - (7S/2S) - 100,000

Prostaglandin E2 PLA - 12,SOO Grinding + heat 68%/60 hr (20)

Quinidine PLA - 120,000 - 2,000 OIW 4-82%/SO hr (32)

NaItrexone PLGA (80/20) - 2S,400 OIW 80%/30 days (33)

Meperidine PLA - 100,000 OIW 60%/3days (33)

Methadone PLA - 100,000 OIW 60%/3days (33)

Promethazine PLA - 100,000 OIW 60%/120 hr (33)

Progesterone PLA - SO,OOO OIW/O 1O-12days (21 )

Clarithromycin PLA - SO,OOO OIW spray dry Sdays (34)

Retinoic acid PLGA (SO/SO) OIW 40days (3S)

Oestrone PLA - SO,OOO OIW S9%l7days (36)

Nifedipine PLGA (7SI2S) - S4,OOO OIW 17-20days (37)

Adapalene PLGA (SO/SO) OIW 80%/Shr (38)

Bupivacaine PLA - 2,000 OIW >Iday (39)

PLGA (7SI2S) - IS ,OOO >Iday

Taxol PLGA (7SI2S) - 10,000 OIW (40)

Ciprofloxacin PLGA (SO/SO) 0/0 90%/6 weeks (41)

PLA - poly lactic acid, PLGA - poly DL (Iactide co-glycoJide) OIW - oil in water, % - Oil in oil.

In addition to these PLG formulations, both injectable devices and implantable systems have been tried as a chemotherapeutic approach towards mycobacterial infections. Various drugs have been entrapped in PLG polymers such as isoniazid52

,

rifampicin5o, clofazirnine51 and have shown promising

results. PLG based microparticles developed in our laboratory exhibited a sustained release of isoniazid and rifampicin in plasma and various organs of mice upto seven weeks when given as single injectable dose (unpublished data).

PLC as delivery system/or vaccines Vaccines offer an excellent opportunity for

controlled release technology because most of the

vaccination schedules require primary immunization followed by repeated booster doses. Wise et al. 63

described the possible usage of polylactide microsphere delivery systems for vaccines.

Beck et al. 64,65 reported significant immune responses with polylactide micro spheres containing vaccines against pneumococcus, herpes simplex viral antigen and bovine chorionic gonadotropin which were administered vaginally to rabbits. The potential use of microparticles for oral immunization has been · reported which exhibited higher IgA and IgG response and induced both proliferative and cytotoxic T cell responses 14. Eldridge et al. 66 reported better immunogenicity of toxoid vaccine using PLG as an

, DHIMAN et al : POLY (DL-LACTlDE-CO-GL YCOLlDE) BASED DELIVERY SYSTEMS 749

Table 3-Biodegradable microspheres for water-soluble small drugs.

Drug Polymer Preparation Release in vitro

Ampicillin PLGA (S2/48) PS 3S%124hr + 2 week (42)

0.S8 dUg (43)

Mitomycin C PLA - 33,000 a/a 96-2S%120hr (44)

Adriamycin PLA - 3,400 a/a 6S-80%IISdays (4S)

Gentamycin sulphate PLA PS 90%1I4days (46)

Chlorpheniramine maleate PLGA (SO/SO) - S7,000 W/O/O/O 70-90% (47)

Timolol maleate PLGA (SO/SO) a/a 40%/40 days (48)

Isoproterenol PLGA (SO/SO) - 3,400 a/a 70%/S min + 12hr (49)

Rifampin PLGA (60/40) (SO/SO) W/O 4-S%l2days (SO)

Clofazimine PLGA (SO/SO) W/O (SI)

Isoniazid PLGA (16/84) (2SI7S) W/O 17.6%/3days (S2)

W/O 11 .6%/3days

Piroxicane Indomethaen PLA (16,000) W/O S-7%/14days (10)

109,000 7 days lag

209,000

330,000

TAK-029 PLGA (7SI2S) 9,000 W/O/w 77%/3 weeks (S3)

PLA - poly lactic acid, PLGA - poly DL (Iactide co-glycolide), PS - particulate suspension, W/O - water in oil, a/a -Oil in oil.

oral delivery system which was due to pulsatile I f · . . 67 re ease 0 antigen In VlVO .

Gilley et at. 68 claimed stronger adjuvanticity of

microspheres less than 10 Jlm as compared to complete freund's adjuvant (CFA). A polypeptide analogue of Pfs 25, a 25 kD surface protein of Plasmodium Jalciparum exhibited much higher and long lasting antibody titre in comparison to commonly used adjuvants69

• Diptheria toxoid loaded polylactide microspheres prepared by the solvent evaporation method exhibited higher immunogenicity after microencapsulation as compared to conventional three injection schedule on alum as an adjuvaneo.

PLG based delivery system has also been evaluated for antituberculous vaccines. The immunodominant 38 kDa antigen from M. tuberculosis when entrapped in PLG particles exhibited ,higher antigen specific IgGJ / IgG2a and IFN-y secretion as compared to those induced in FIA 64. We in our laboratory have demonstrated PLG to be an ideal vaccine delivery system for two highly protective vaccine candidates (30 kDa secretory protein and 71 kDa cell wall protein of M. tuberculosis H37Ra) as compared to other delivery systems such as FlA, liposomes and DDA7I ,n. Further, PLG microparticles based single shot subunit vaccines with these antigen were equally protective to that of three shot schedulen . Moreover, the protection induced by mycobacterial antigens in

PLG was also found to be long tenn as compared to FlAn. Currently, the evaluation of protective efficacy of DNA based vaccines of these mycobacterial antigens in PLG is in progress.

In brief, PLG polymers as delivery systems have generated immense interest due to their excellent biocompatibility and biodegradability. The various PLG based systems are versatile in tenns of the nature of the agent encapsulated, time period of release and diverse routes of their delivery. Therefore, such systems represent novel and exciting technologies for delivery of different biomole.cules.

References 1 Boswell GA & Scribner RM. US Patent, 3, 773, 919 (1973). 2 Yolles S, Eldridge JE, Leafe TO, Woodland JH, Blake DR &

Meyer FJ. Long acting delivery systems for narcotic antagonists. Adv Exp Med Bioi, 47 (1973) 177. .

3 Wise DL, McCormick GJ, Willet GP & Andersen Le. Sustained release of an antimalarial drug using a copolymer of glycolic/lactic acid. Life Sci, 19 (1976) 867.

4 Beck LR, Cowsar DR, Lewis DH, Gibson JW & Flowers CE Jr. New long acting injectable microcapsule contraceptive system. Am J Obstet Gynecol, 13S (1979) 419.

S Holland SJ, Tighe BJ & Gould PLo Polymers for biodegradable medical devices I. The potential of polyesters as controlled macro molecular release system. J Cont Rei, 4 (1986) ISS.

6 Gliding DK & Reed AM. Biodegradable polymers for use in surgery: poly (glycolic)/poly (Iactide acid) homo and copolymers: I. Polymer. 20 (1979) 14S9.

750 INDIAN J EXP BIOL, AUGUST 2000

7 Brady JM, Cutright DE, Miller RA & Battistone GC. Resorbtion rate, route of eliminiation and ultrastructure of the implant site of poly lactic acid in the abdominal wall of the rat. J Biomed Mater Res, 7 (1973) 155.

8 Williams DF & Most E. Enz~me accelerated hydrolysis of poly (glycolic acid). J Bio Eng, I (1987) 231 .

9 DH Lewis, in Biodegradable polymers as drug delivery systems, edited by M Chasin and R Langer, (Marcel Dekker, Inc. , New York), 1990, 1-41.

\0 Jain R, Shah NH, Malick A W & Rhodes CT. Controlled drug delivery by biodegradable poly (ester) devices: different preparative approaches. Drug Develop Indust Pharm, 24 (1998) 703.

11 Deasy PB. Evaluation of drug containing microcapsules. J Microencapsul, II (1994) 487.

12 Okada H & Toguchi H. Biodegradable microspheres in drug delivery. Crit Rev Ther Drug Carr Sys, 12 (1995) I.

13 Torchilin VP. Polymer coated long circulating microparticulate pharmaceuticals. J. Microencapsul, 15 (1998) I .

14 O' Hagan DT, McGee JP, Holmgren J, Mowat AM, Donachie AM, Mills KHC, Galsford N, Rahman P & Challcombe SJ. Biodegradable micropartic\es for oral immunization. Vaccine, 11(1993) 149.

15 Cahill ES, O'Hagan DT, IlIum L & Redhead K. Mice are protected against Bordetella pertussis infection by intra-nasal immunization with filamentous haemagglutinin. FEMS Microbiol Lelt, \07 (1993) 211.

16 Yolles S, Eldridge, JE .& Woodland JHR. New biodegradable delivery systems. Polymers News, I (1970) 9.

17 Woodland JHR, Yolles S, Blake DA, Helrich M & Meyer FJ. Long acting delivery systems for narcotic antagonists. J Med Chem, 16 (8) (1973) 897.

18 Yolles S, Leafe ID, Woodland JHR & Meyer FJ. Long acting delivery systems for narcotic antagonists. 11: Release rate of naItrexone from poly (lactic acid) composites. J Pharm Sci, 64 (1975) 343.

19 Beck LR, Cowsar DR, Lewis DH, Cosgrove RJ, Riddle CT, Lowry SL & Epperly T. A new long acting injectable microcapsule system for the administration of progestrone. Fert Steril, 5 (1979) 545.

20 Zhou MX & Chang TMS. Control release of prostaglandin E2 from poly lactic acid microcapsules, microparticles and modified microparticles. J Microencapsul, 5 (1988) 27.

21 Gupta PK, Mehta RC, Douglas RH & DeLuca PP. In vivo evaluation of biodegradable progestrone microspheres in mares. Pharm Res, 9 (1992) 1502.

22 Pavaneuo F, Genta I, Ginuchedi, P & Conti B. Evaluation of spray drying as a method for polylactide and poly lactide-co­glycolide microsphere preparation. J Microencapsul, 10 (1993) 487.

23 Schwope AD, Wise DL & Howes JF. Lactic/glycolic acid polymers as narcotic antagonis! delivery systems. Life Sci, 17 (1975) 1877.

24 Beck LR, Pope VZ, Flowers CE Jr, Cowsar DR, Tice TR, Lewis DH, Dunn RL, Moore AB & Gilley RM. Poly (DL­lactide-co-glycolide)/norethisterone microcapsules: an injectable biodegradable contraceptive. Bioi Reproduct , 28 (1983) 186.

25 Wakiyama N, Juni K & Nakano M. Influence of physiochemical properties of poly lactic acid on the characteristics and in vitro release patterns of poly lactic acid micro spheres containing local anesthetics. Chem Pharm Bull, 30 (1982) 2621.

26 Wakiyama N, Juni K & Nakano M. Preparation and evaluation in vitro and in vivo of poly lactic acid microspheres containing dibucaine. Chem Pharm Bull, 30 (1982) 3719.

27 Juni K, Ogata J, Matsui N, Kubota M & Nakano M. Control of release rate of bleomycin from poly lactic acid micro spheres by additives. Chem Pltarm Bull, 33 (1985) 1609.

28 Juni K, Ogata J, Matsui N, Kubota M & Nanano M. Modification of the release rate of ac\arubicin from poly lactic acid microspheres by using additives. Chem Pharm Bull, 33 (1985) 1734.

29 Benita S, Benoit JP, Puisieux F & Thies C. Characterization of drug loaded poly (d, I-Iactide) microspheres. J Pharm Sci, 73 (1984) 1721.

30 Leelarasamee N, Howard SA, Malanga Cl, Luzzi LA, Hogan TF, Kandzari SJ & Ma JKH. Kinetics of drug release from poly lactic acid-hydrocortisone microcapsules . J Microencap 3 (1986) 171.

31 Spenlehauer G, Vert M, Bennoit JP & Boddaert A. In vitro and in vivo degradation of poly (DL-Iactide/glycolide) type microspheres made by solvent evaporation method, Biomaterials. 10 (1989) 557.

32 Bodmeier R, Oh KH & Ch en H. The effect of the addition of low molecular weight poly (DL-Iactide) on drug release from biodegradable poly (DL-Iactide) drug delivery systems. Int J Pharmaceut, 51 (1989) I.

33 Cha Y & Piu CG. The acceleration of degradation-controlled drug delivery from polyester microspheres. J COlltr Rei, 8 (1989) 259.

34 Gupta PK, Johnson H & Allexon C. In vitro and in vivo evaluation of c\arithromycin/poly (lactic acid) microspheres for intramuscular drug delivery. J Cont Rei, 26 (1993) 229.

35 Giordaro GG, Refojo MF & Arroyo MH. Sustained delivery of retinoic acid from microspheres of biodegradable polymer in PVR. Invest Ophthalmol Vis Sci, 34 (1993) 2743.

36 Parikh BV, Upadrashta SM, Neau SU & Nuessle NO. Oestrone loaded poly (lactic acid) microspheres: preparation, evaluation and in vitro release kinetics. J Microencapsul, 10 (1993) 141.

37 Sansdrap P & Moes AJ . Influence of manufacturing parameters on the size characteristics and the release profiles of nifedipine from poly (DL-Iactide-co-glycolide) microspheres. Int J Pltarmaceut , 98 (1993) 157.

38 Rolland A, Wagner N, Chatelus A, Shroot B & Schaefer H. Site specific drug delivery to pilosebaceous structures using polymeric microspheres. Pharm Res, 10 (1993) 1738.

39 LeCorre P, LeGuevello P, Gajan V, Chevanne F & LeVerge R. Preparation and characterization of bupivacaine-Ioaded polylactide and poly lactide-co-glycolide microspheres. Int J Pharmaceut, \07 (1994) 41.

40 Sato H, Wang YM, Adachi I & Horikoshi I. Pharmacokinetic study of taxol-Ioaded poly (Iactic-co-glycolic acid) microspheres containing isopropyl Myristate after targeted delivery to the lung in mice. Bioi Pharm Bull, 19 (1996) 1596.

DHIMAN et al : POLY (DL-LACTIDE-CO-GLYCOLIDE) BASED DELIVERY SYSTEMS 751

41 Ramchandani M & Robinson D. In vitro and in vivo release of ciprofloxacin from PLGA 50:50 implants, J Control Rel, 54 (1998) 167.

42 Setterstrom JA, Tice TR & Myers WE. Development of encapsulated antibiotics for topical administration to wounds, in Recent Advances in Drug Delivery Systems, deited by Kim SW, (Plenum Press, New York), 1984, 185.

43 Jacob E, Setterstrom JA, Bach DE, Heath JR Ill, McNiesh LM & Ciemy G Ill. Evaluation of biodegradable ampicillin anhydride microcapsule for local treatment of experimental syaphylococcal osteomyelitis . • Clin Orthopaed Related Res, 267 (1991) 237.

44 Tsai DC, Howard SA, Hogan TF, Malanga 0, Kandzari SJ & Ma JKH. Preparation and in vitro evaluation of poly lactic acid mitomycin C with microcapsules. J Microencapsul, 3 (1986) 181.

45 Wada R, Hyon SH & Ikada Y. Lactic acid oligomer micro spheres containing hydrophilic drugs. J Pharm Sci, 79 (1990) 919.

46 Sampath SS, Garvin K & Robinson DH. Preparation and characterization of biodegradable poly (L-Iactic acid) gentamycin delivery systems. Int J Pharmaceut, 78 (1992) 165.

47 Iwata M & McGinity JW. Dissolution, stability and morphological properties of conventional and multiphase poly (DL-Iactide-co-glycolic acid) microspheres containing water soluble compounds. Pharm Res, 10 (1993) 1219.

48 Sturesson C, Carlfors J, Edsman K & Andersson M. Preparation of biodegradable poly (Iactic-co-glycolic) acid microspheres and their in vitro release of timolol maleate. Int J Pharmaceut, 89 (1993) 235.

49 Lai Y-L, Mehta RC, Thacker AA, Yoo SD, McNamara PJ & Deluca PP. Sustained bronchodilation with isoproterenol poly (glycolide-co-Iactide) microspheres. Pharm Res, 10 (1993) 221.

50 Barrow ELW, Winchester GA, Staas JK, Quenelle DC & Barrow WW. Use of microsphere technology for targeted delivery of rifampin to Mycobacterium tuberculosis infected macrophages. Antimicrob Agents Chemother, 42 (1998) 2682.

51 Kailasam S, Wise DL & Gangadharam PRJ. Bioavailability and chemotherapeutic activity of c10fazimine against Mycobacterium avium complex infections in beige mice following a single implant of a biodegradable polymer. Antimicrob Agents Chemother, 33 (1994) 273.

52 Gangadharam PRJ, Kailasam S, Srinivasan S & Wise DL. Experimental chemotherapy of tuberculosis using single dose treatment with isoniazid in biodegradable polymers. J Antimicrob Chemother, 3 (1994) 265.

53 Takada S, Kurokawa T, Miyazaki K, Iwasa S & Ogawa Y. Sustained release of a water soluble GP II b/Illa antagonist from copoly (DL-Iactide/glycolic) acid microspheres. Int J Pharmaceutics, 146 (1997) 147.

54 Anderson JM & Shivne MS. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Del Rev, 28 (1997) 5.

55 Deshpande AA, Rhodes eT, Shah NH, Malick A W. Controlled release drug delivery systems for prolonged gastric residence: An overview, Drug Dev Indust Pharm. 22(6) (1996) 531.

56 Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V & Langer R. Biodegradable long circulating polymeric nanospheres. Science 263 (1994) 1600.

57 Stolnik S, Dunn SE, Gamett MC, Davis MC, Coombes AGA, Taylor DC, Irving MP, Purkins SC, Tadros TF, Davis SS, IlIum L. Surface modification of poly (Iactide-co­glycolide) nanospheres by novel biodegradable poly (Iactide)- poly (ethylene glycol) copolymers. Pharm Res, 11 (12) (1994) 1800.

58 Yamaoka T, Tabata T, Ikada Y. Distribution and tissue uptake of poly (ethylene glycol) with different molecular weights after intravenous administration in mice. J. Pharm Sci, 83 (1994) 601.

59 Zalipsky S. Chemistry of polyethylene glycol conjugates with biologically active molecules. Adv Drug Deliv Rev, 16 (1995) 215.

60 GrefR, Domb A, Quellec P, Blunk T, Muller RH, Verbavatz JM & Langer R. The controlled intravenous delivery of drugs using PEG coated sterically stabilized nanospheres. Adv Drug Deliv Rev, 16 (1995) 215.

61 Torchilin VP, Trubetskoy VS, Papisov MI, Bogdanov AA, Omelyanenkko VG, Narula J & Khaw BA. Polymer coated immunoliposomes for delivery of pharmaceuticals: targeting and biological stability. In Proc 20th Int Symp Control Release Bioact Mater. (Washington Controlled Release Society), 1993, 194.

62 Sanders LM, Kell BA, McRae GI & Whitehead GW. Prolonged controlled-release of Nafarelin : a lutenizing hormone-releasing hormone analogue, from biodegradable implants: influence of composition and molecular weight of polymer. J Pharm Sci, 75 (4) (1986) 356.

63 Wise DL, Debra IT, Rachel TM & Judith PK. Opportunities and challenge in the design of implanatation biodegradable polymeric systems for the delivery of antimicrobial agents and vaccines. Adv Drug Del Rev, I (1987) 19.

64 Beck LR, Flowers CF, Cowsar DR & Tangary AC, US Patent, 4 (1988) 756.

65 Beck LR, Flowers CF, Coswar DR & Tangary AC, US Patent, 4 (I 988b ) 732.

66 Eldridge JH, Stass JK, Malbrock JA, Tice TR & Gilley RM. Biodegradable and biocompatible poly (DL-Iactide-co­glycolide) microspheres as an adjuvant for Staphylococcal enterotoxin B toxoid which enhances the levels of toxin neutralising antibodies. Infect Immun, 9 (1991) 2978.

67 Stass JK, Eldridge JH, Morgan 10, Finch OB, Tice TR & Gilley RM. Microsphere vaccine: Enhanced immune response through adjuvant effect and mUltiple-pulse release capability. Proc Int Symp Cont Rei Bioact Mater, 18 (1991) 619.

68 Gilley, RM, Staas JK, Tice TR, Morgan 10 & Eldridge JH. Microencapsulation and its application to vaccine developmelt. Proc Int Symp Cont Rei Bioact Mater, 19 (1992) 110.

69 Bathurst IC, Barr PJ, Kashew DC, Lewis DH, Atkinss TJ & Pickey ME. Development of a single injection transmission blocking malaria vaccine using biodegradable microsphere. Proct Int Sym Cont Rei Bioact Mater, 19 (1992) 120.

752 INDIAN J EXP BIOL, AUGUST 2000

70 Singh M, Li XM, Wang H, McGee JP, Zeb BT, Kaff W, Wang CY & O'Hagan DT. Immunogenicity and protection in small animal models with controlled release tetanus toxoid microparticles as a single dose vaccine. Infect Immull, 65 (1997) 1716.

71 Dhiman N & Khuller GK. Protective efficacy of mycobacterial 71 kDa cell wall associated protein using poly

(DL-1actide-co-glycolide) microparticles as carrier vehicles. FEMS Immunol Med Microbiol, 21 (1998) 19.

72 Sharma A, Verma I, Tewari K & Khuller GK. Adjuvant modulation ofT cell reactivity to 30 kDa secretory protein of Mycobacterium tuberculosis H37Rv and its protective efficacy against experimental tuberculosis. J Med Microbiol, 48 (1999) 757.