NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR CONTROLLED AND TARGETED DELIVERY OF PROTEIN DRUGS SMALL AND MOLECULE DRUGS LEE SIE HUEY NATIONAL UNIVERSITY OF SINGAPORE 2007 NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR CONTROLLED AND TARGETED DELIVERY OF PROTEIN DRUGS SMALL AND MOLECULE DRUGS BY LEE SIE HUEY (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER NANOENGINEERING NUS NANOSCIENCE & NANOTECHNOLOGY INITIATIVE (NUSNNI) NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS Firstly, I would like to express my most sincere gratitude to my respected supervisor A/P Feng Si-Shen for his constant encouragement, invaluable advice and patient guidance throughout the course of my Master candidature I am very proud to have A/P Feng as my mentor I am profoundly grateful to Mr Zhang Zhiping for providing me helpful discussion and constructive comments for my research studies I would like to thank all members of the group and my colleagues for their kind help and good suggestion: Dr Dong Yuancai, Dr Zhao Lingyun, Dr Gajadhar Bahkta, Miss Tan Mei Yee Dinah, Miss Chen Shilin, Miss Wang Yan, Miss Ng Yee Woon, Miss Wang Junping, Miss Sun Bingfeng and Mr Pan Jie I feel very lucky to be a member of this group and very happy to enjoy their friendship I would also like to extend my special thanks to many others laboratory and administrative staff for their technical and administrative supports I am greatly grateful to my parents, my brother, my sister and my boyfriend for their love, continuous spiritual support and consideration Their unfailing encouragements and unselfish supports through my life have helped me pull through this difficult period Last but not least, I sincerely appreciated the research scholarship provided by the Nanoscience and Nanotechnology Initiative, National University of Singapore (NUSNNI) and Economy Development Board (EDB), Singapore i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY vii NOMENCLATURE ix LIST OF FIGURES xi LIST OF TABLES xiii LIST OF PUBLICATIONS xiv CHAPTER INTRODUCTION………….…………………………….………… 1.1 General background 1.2 Objective and thesis organization CHAPTER LITERATURE REVIEW………………………………………… … 2.1 Nanoparticles of biodegradable polymers for drug delivery……………… 2.1.1 Nanoparticles drug delivery systems 2.1.2 Biodegradable polymers .8 2.1.3 Nanoparticles fabrication methods .9 2.1.3.1 Solvent extraction/evaporation method .9 2.1.3.2 Nanoprecipitation method 12 2.1.3.3 Dialysis method 13 2.1.3.4 Supercritical fluid method 14 2.1.3.5 Polymerization method 14 2.2 Peptide/protein drug delivery 15 2.2.1 Structural aspect of protein 15 2.2.2 Challenges in peptide/protein drug delivery 17 2.2.3 Approaches for delivery of peptide/protein drugs 18 ii 2.2.3.1 Parenteral delivery .18 2.2.3.2 Oral delivery 18 2.2.3.3 Other non-parenteral delivery .19 2.2.3.4 Biodegradable particle as delivery system 19 2.3 Anticancer drug delivery .19 2.3.1 Cancer, cancer causes and cancer treatment … 19 2.3.2 Cancer chemotherapy 21 2.3.3 New-concept of chemotherapy 21 2.3.4 Targeted therapeutics in anticancer therapy ………………………… 22 2.3.5 Doxorubicin and its anticancer mechanism 24 2.4 Vitamin E TPGS 25 2.4.1 Chemistry of TPGS 25 2.4.2 Application in drug delivery .26 2.4.2.1 Bioavailabilty enhancer .26 2.4.2.2 Anticancer property 27 2.4.2.3 Excellent elmusifier/additive .27 CHAPTER MATERIALS AND METHODS……………………………………… 29 3.1 Materials 29 3.2 Methods 31 3.2.1 Synthesis of copolymers and conjugates 31 3.2.1.1 PLA-TPGS copolymer 31 3.2.1.2 DOX-PLGA-TPGS conjugate 32 3.2.1.3 TPGS-FOL conjugate 34 3.2.2 Characterization of copolymers and conjugates 35 3.2.2.1 FT-IR and 1H NMR 35 iii 3.2.2.2 GPC .36 3.2.3 Preparation of nanoparticles .36 3.2.3.1 Preparation of BSA-loaded nanoparticles 36 3.2.3.2 Preparation of DOX-loaded nanoparticles……………………… .37 3.2.4 Characterization of nanoparticles 37 3.2.4.1 Size and size distribution 37 3.2.4.2 Surface charge .38 3.2.4.3 Surface morphology 38 3.2.4.4 Surface chemistry 38 3.2.5 Drug encapsulation efficiency 39 3.2.5.1 Drug encapsulation efficiency of BSA-loaded nanoparticles……… 39 3.2.5.2 Drug encapsulation efficiency of DOX-loaded nanoparticles 39 3.2.6 In Vitro release and degradation of nanoparticles 39 3.2.6.1 In vitro BSA release and degradation of nanoparticles .39 3.2.6.2 In vitro DOX release 40 3.2.7 Stability of protein 41 3.2.7.1 SDS-PAGE 41 3.2.7.2 Circular dichroism spectroscopy 41 3.2.8 Cell line experiment .41 3.2.8.1 Cell culture 41 3.2.8.2 In vitro cell viability 42 3.2.8.3 In vitro cell uptake .42 iv CHAPTER NANOPARTICLES OF POLY(LACTIDE)-TOCOPHERYL POLYETHYLENE GLYCOL (PLA-TPGS) COPOLYMERS FOR PROTEIN DRUG DELIVERY .44 4.1 Introduction 44 4.2 Results and discussion 46 4.2.1 Characterization of PLA-TPGS copolymer 46 4.2.2 Effects of Formulation variables on nanoparticles characteristics .48 4.2.2.1 Effects of BSA Loading .48 4.2.2.2 Effects of TPGS Content 49 4.2.3 Surface chemistry of BSA-loaded PLA-TPGS nanoparticles 51 4.2.4 Degradation of BSA-loaded PLA-TPGS nanoparticles .53 4.2.5 In vitro BSA release .58 4.2.6 Stability of BSA released from nanoparticles 61 4.2.7 In vitro cellular uptake of PLA-TPGS nanoparticles loaded with FITCBSA .64 4.3 Conclusions 65 CHAPTER FOLATE-DECORATED POLY(LACTIDE-CO-GLYCOLIDE)- VITAMIN E TPGS NANOPARTICLES FOR TARGETED DRUG DELIVERY 67 5.1 Introduction 67 5.2 Results and discussion 69 5.2.1 Characterization of the synthesized conjugates 69 5.2.2 Characterization of DOX-loaded nanoparticles 71 5.2.3 Surface chemistry 74 5.2.4 In vitro drug release 75 5.2.5 In vitro cytotoxicity .76 v 5.2.6 In vitro cellular uptake of nanoparticles .79 5.3 Conclusion 82 CHAPTER CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK …… 83 6.1 Conclusions 83 6.2 Suggestions for future work 84 REFERENCES 86 vi SUMMARY Owing to the development of nanotechnology and biotechnology, nanoparticles of biodegradable polymers as effective drug delivery systems have received significant attention They have the ability to carry various therapeutic agents including anticancer drugs, DNA, peptides and proteins Among various FDA-approved biodegradable polymers, poly(lactide acid) (PLA), poly(lactide-co-glycolide) (PLGA) and poly(ε-caprolactone) (PCL) are most oftenly used in these areas However, most of them have not been able to meet these demands due to their hydrophobic nature They are not biocompatible with hydrophilic drugs Biodegradable block copolymers with better hydrophobic and hydrophilic balance thus are desired and this can be done by inserting hydrophilic elements into the hydrophobic chains of the polymers In the thesis, d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply TPGS), which is actually a PEGylated vitamin E was introduced into the hydrophobic polymer backbone of PLA and PLGA to form PLA-TPGS and PLGA-TPGS block copolymers These block copolymers are an example of amphiphiles Their uses as different carriers for delivery of protein and anticancer drug and as targeted agents for target specific delivery were addressed in this thesis Poly(lactide) – tocopheryl polyethylene glycol (PLA-TPGS) copolymers with various PLA:TPGS ratios were synthesized Nanoparticles of PLA-TPGS were prepared by double emulsion method for protein drug formulation with bovine serum albumin (BSA) as a model protein Influence of the PLA:TPGS component ratio and the BSA loading level on the drug encapsulation efficiency (EE) and in vitro drug release behavior were investigated The proteins released from the PLA-TPGS nanoparticles vii retained good structural integrity for at least 35 days at 37 oC as indicated by SDSPAGE and circular dichroism (CD) spectroscopy Confocal laser scanning microscopy (CLSM) observation demonstrated the intracellular uptake of the PLA-TPGS nanoparticles by NIH-3T3 fibroblast cell and Caco-2 cancer cell This research suggests that PLA-TPGS nanoparticles could be of great potential for clinical formulation of proteins and peptides Vitamin E TPGS-folate (TPGS-FOL) conjugate and doxorubicin-poly(lactide-coglycolide)-vitamin E TPGS (DOX-PLGA-TPGS) conjugate were synthesized DOXloaded nanoparticles composed of TPGS-FOL and DOX-PLGA-TPGS conjugates with various blend ratios were prepared by solvent extraction/evaporation method for targeted chemotherapy of folate-receptor rich tumors X-ray photoelectron spectroscopy (XPS) demonstrated that folate was distributed on the nanoparticle surface while the drug molecules were entrapped in the core of the nanoparticles The nanoparticles were found to be ~350 nm size and exhibited a biphasic pattern of in vitro drug release over weeks The cellular uptake and cell viability of the two types of DOX-loaded nanoparticles were investigated by using MCF-7 breast cancer cell line and C6 glioma cell line, which were found to be dependent on the content of targeting TPGS-FOL These results suggest that our novel TPGS-FOL decorated PLGA-TPGS nanoparticles can be applied for targeted chemotherapy viii Chansiri, G., Lyons, R T., Patel, M V and Hem, S L Effect of surface charge on the stability of oil/water emulsions during steam sterilization, Journal of Pharmaceutical Sciences, 88, pp.454-458 1999 Chauhan, A S., Jain, N K., Diwan, P V and Khopade, A J Solubility enhancement of indomethacin with poly(amidoamine) dendrimers and targeting to inflammatory regions of arthritic rats, Journal of Drug Targeting, 12, pp.575-583 2004 Cho, K Y., Choi, S H., Kim, C H., Nam, Y S., Park, T G and Park, J K Protein release microparticles based on the blend of poly(D,L-lactic-co-glycolic acid) and oligo-ethylene glycol grafted poly(L-lactide), Journal of Controlled Release, 76, pp.275-284 2001 Chorny, M., Fishbein I., Danenberg H D and Golomb G Lipophilic drug loaded nanospheres prepared by nanoprecipitation: effect of formulation variables on size, drug recovery and release kinetics Journal of Controlled Release, 83, pp.389-400, 2002 Cohen, S., Yoshioka, T., Lucarelli, M., Hwang, L H and Langer, R Controlled delivery systems for proteins based on poly(lactic glycolic acid) microspheres, Pharmaceutical Research, 8, pp.713-720 1991 Coombes, A G A., Yeh, M K., Lavelle, E C and Davis, S S The control of protein release from poly(DL-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method, Journal of Controlled Release, 52, pp.311-320 1998 Courvreur P., Couarraze G., Devissaguet J.-P., Puisieux F Nanoparticles: preparation and characterization In Microencapsulation: Methods and Industrial Application, ed by S., Benita pp.183-211 New York: Marcel Dekker 1996 Couvreur, P and Puisieux, F Nanoparticles and microparticles for the delivery of polypeptides and proteins, Advanced Drug Delivery Reviews, 10, pp.141-162 1993 88 Crotts, G and Park, T G Stability and release of bovine serum albumin encapsulated within poly(,-lactide-co-glycolide) microparticles, Journal of Controlled Release, 44, pp.123-134 1997 De Rosa, G., Iommelli, R., La Rotonda, M I., Miro, A and Quaglia, F Influence of the co-encapsulation of different non-ionic surfactants on the properties of PLGA insulin-loaded microspheres, Journal of Controlled Release, 69, pp.283-295 2000 Deng, X M., Xiong, C D., Cheng, L M., Huang, H H and Xu, R P Studies on the block copolymerization of D,L-lactide and poly(ethylene glycol) with aluminum complex catalyst, Journal of Applied Polymer Science, 55, pp.1193-1196 1995 Desai, M P., Labhasetwar, V., Walter, E., Levy, R J and Amidon, G L The mechanism of uptake of biodegradable microparticles in Caco-2 cells is size dependent, Pharmaceutical Research, 14, pp.1568-1573 1997 Dintaman, J M and Silverman, J A Inhibition of P-glycoprotein by D-alphatocopheryl polyethylene glycol 1000 succinate (TPGS), Pharmaceutical Research, 16, pp.1550-1556 1999 Dong, Y C and Feng, S S Methoxy poly(ethylene glycol)-poly(lactide) (MPEGPLA) nanoparticles for controlled delivery of anticancer drugs, Biomaterials, 25, pp.2843-2849 2004 Drotleff, S., Lungwitz, U., Breunig, M., Dennis, A., Blunk, T., Tessmar, J., et al Biomimetic polymers in pharmaceutical and biomedical sciences, European Journal of Pharmaceutics and Biopharmaceutics, 58, pp.385-407 2004 Dunn, R L Polymeric matrices In Polymeric drugs and drug delivery systems, ed by R L.Dunn, R M Ottenbrite pp.11-23 Washington: American Chemical Society 1990 Ehrlich, P Chemotherapeutics: scientific principles, methods and results, Lancet II, pp.445-451 1913 89 Faraasen, S., Voros, J., Csucs, G., Textor, M., Merkle, H P and Walter, E Ligandspecific targeting of microspheres to phagocytes by surface modification with poly(Llysine)-grafted poly(ethylene glycol) conjugate, Pharmaceutical Research, 20, pp.237246 2003 Feng, S S and Chien, S Chemotherapeutic engineering: Application and further development of chemical engineering principles for chemotherapy of cancer and other diseases, Chemical Engineering Science, 58, pp.4087-4114 2003 Feng, S S., Mu, L., Win, K Y and Huang, G F Nanoparticles of biodegradable polymers for clinical administration of paclitaxel, Current Medicinal Chemistry, 11, pp.413-424 2004 Feng, S S Nanoparticles of biodegradable polymers for new-concept chemotherapy, Expert Review of Medical Devices, 1, pp.115-125 2004 Feng, S.S New-concept chemotherapy by nanoparticles of biodegradable polymers: where are we now?, Nanomedicine, 1, pp.297-309 2006 Fessi, H., Puisieux, F., Devissaguet, J P., Ammoury, N and Benita, S Nanocapsule formation by interfacial polymer deposition following solvent displacement, International Journal of Pharmaceutics, 55, pp.R1-R4 1989 Fischer, J R., Harkin, K R and Freeman, L C Concurrent administration of watersoluble vitamin E can increase the oral bioavailability of cyclosporine a in healthy dogs, Veterinary therapeutics : research in applied veterinary medicine, 3, pp.465-473 2002 Florence, A T The oral absorption of micro- and nanoparticulates: Neither exceptional nor unusual, Pharmaceutical Research, 14, pp.259-266 1997 Fu, K., Pack, D W., Klibanov, A M and Langer, R Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres, Pharmaceutical Research, 17, pp.100-106 2000 90 Gao, H., Wang, Y N., Fan, Y G and Ma, J B Synthesis of a biodegradable tadpoleshaped polymer via the coupling reaction of polylactide onto mono(6-(2aminoethyl)amino-6-deoxy)-beta-cyclodextrin and its properties as the new carrier of protein delivery system, Journal of Controlled Release, 107, pp.158-173 2005 Gao, X H., Cui, Y Y., Levenson, R M., Chung, L W K and Nie, S M In vivo cancer targeting and imaging with semiconductor quantum dots, Nature Biotechnology, 22, pp.969-976 2004 Gref, R., Luck, M., Quellec, P., Marchand, M., Dellacherie, E., Harnisch, S., et al 'Stealth' corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption, Colloids and Surfaces B-Biointerfaces, 18, pp.301-313 2000 Hans, M L and Lowman A M Biodegradable nanoparticles for drug delivery and targeting, Current Opinion in Solid State & Materials Science, 6, pp.319-327 2002 Jani PU, Halbert GW, Langridge J, Florence AT The uptake and translocation of latex nanospheres and microspheres after oral-administration to rats, Journal of Pharmarcy Pharmacology, 41, pp.809-812 1989 Jani PU, McCarthy DE, Florence AT Nanosphere and microsphere uptake via Peyer patches - Observation of the rate of uptake in the rat after a single oral dose, International Journal of Pharmaceutics, 86, pp.239-246 1992 Jeong, Y I., Nah, J W., Lee, H C., Kim, S H and Cho, C S Adriamycin release from flower-type polymeric micelle based on star-block copolymer composed of poly(gamma-benzyl L-glutamate) as the hydrophobic part and poly(ethylene oxide) as the hydrophilic part, International Journal of Pharmaceutics, 188, pp.49-58 1999 Jeong, Y I., Cho, C S., Kim, S H., Ko, K S., Kim, S I., Shim, Y H., et al Preparation of poly(DL-lactide-co-glycolide) nanoparticles without surfactant, Journal of Applied Polymer Science, 80, pp.2228-2236 2001 91 Jeong, Y I., Shim, Y H., Choi, C Y., Jang, M K., Shin, G M and Nah, J W Surfactant-free nanoparticles of poly(DL-lactide-co-glycolide) prepared with poly(Llactide)/poly(ethylene glycol), Journal of Applied Polymer Science, 89, pp.1116-1123 2003 Jiang, W L and Schwendeman, S P Stabilization and controlled release of bovine serum albumin encapsulated in poly(D, L-lactide) and poly(ethylene glycol) microsphere blends, Pharmaceutical Research, 18, pp.878-885 2001 Kataoka, K., Matsumoto, T., Yokoyama, M., Okano, T., Sakurai, Y., Fukushima, S., et al Doxorubicin-loaded poly(ethylene glycol)-poly(beta-benzyl-l-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance, Journal of Controlled Release, 64, pp.143-153 2000 Ke, W T., Lin, S Y., Ho, H O and Sheu, M T Physical characterizations of microemulsion systems using tocopheryl polyethylene glycol 1000 succinate (TPGS) as a surfactant for the oral delivery of protein drugs, Journal of Controlled Release, 102, pp.489-507 2005 Khin, Y W and Feng, S S Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs, Biomaterials, 26, pp.2713-2722 2005 Khin, Y W and Feng, S S In vitro and in vivo studies on vitamin E TPGSemulsified poly(D,L-lactic-co-glycolic acid) nanoparticles for paclitaxel formulation, Biomaterials, 27, pp.2285-2291 2006 Kim, H K and Park, T G Microencapsulation of human growth hormone within biodegradable polyester microspheres: Protein aggregation stability and incomplete release mechanism, Biotechnology and Bioengineering, 65, pp.659-667 1999 92 Kim, S Y and Lee Y M Taxol-loaded block copolymer nanospheres composed of methoxy poly(ethylene glycol) and poly(epsilon-caprolactone) as novel anticancer drug carriers Biomaterials, 22, pp.1697-1704, 2001 Kimura, Y., Matsuzaki, Y., Yamane, H and Kitao, T Preparation of block copoly(ester ether) comprising poly(L-lactide) and poly(Ooxypropylene) and degradation of its fiber in vitro and in vivo, Polymer, 30, pp.1342-1349 1989 Kukowska-Latallo, J F., Candido, K A., Cao, Z Y., Nigavekar, S S., Majoros, I J., Thomas, T P., et al Nanoparticle targeting of anticancer drug improves therapeutic response in animal model of human epithelial cancer, Cancer Research, 65, pp.53175324 2005 Kumar, N., Ravikumar, M N V and Domb, A J Biodegradable block copolymers, Advanced Drug Delivery Reviews, 53, pp.23-44 2001 Lamprecht, A., Ubrich, N., Perez, M H., Lehr, C M., Hoffman, M and Maincent, P Biodegradable monodispersed nanoparticles prepared by pressure homogenizationemulsification, International Journal of Pharmaceutics, 184, pp.97-105 1999 Langer, R and Folkman, J Polymers for the sustained release of proteins and other macromolecules, Nature, 263, pp.797-800 1976 Lappi, D.A Tumor targeting in cancer therapy pp 449-457, Totowa, NJ: The Human Press Inc 2002 Leamon, C P and Reddy, J A Folate-targeted chemotherapy, Advanced Drug Delivery Reviews, 56, pp.1127-1141 2004 Lee, E S., Na, K and Bae, Y H Polymeric micelle for tumor pH and folate-mediated targeting, Journal of Controlled Release, 91, pp.103-113 2003 93 Lee, J W., Lu, J Y., Low, P S and Fuchs, P L Synthesis and evaluation of taxolfolic acid conjugates as targeted antineoplastics, Bioorganic & Medicinal Chemistry, 10, pp.2397-2414 2002 Lee, S C., Kim C., Kwon I C., Chung H and Jeong S Y Polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(epsilon-caprolactone) copolymer as a carrier for paclitaxel Journal of Controlled Release, 89, pp.437-446, 2003 Lee, Y K Preparation and characterization of folic acid linked poly(L-glutamate) nanoparticles for cancer targeting, Macromolecular Research, 14, pp.387-393 2006 Malafa, M P and Neitzel, L T Vitamin E succinate promotes breast cancer tumor dormancy, Journal of Surgical Research, 93, pp.163-170 2000 Manning, M C., Patel, K and Borchardt, R T Stability of protein pharmaceuticals, Pharmaceutical Research, 6, pp.903-918 1989 Marchais, H., Boury, F., Damge, C., Proust, J E and Benoit, J P Formulation of bovine serum albumin loaded PLGA microspheres - Influence of the process variables on the loading and in vitro release, STP Pharma Sciences, 6, pp.417-423 1996 Mawson, S., Johnston, K P., Combes, J R and Desimone, J M Formation of poly(1,1,2,2-tetrahydroperfluorodecyl acrylate) submicron fibers and particles from supercritical carbon-dioxide solutions, Macromolecules, 28, pp.3182-3191 1995 McGee, J P., Davis, S S and Ohagan, D T The immunogenicity of a model protein entrapped in poly(lactide-co-glycolide) microparticles prepared by a novel phaseseparation technique, Journal of Controlled Release, 31, pp.55-60 1994 Minko, T., Kopeckova, P., Pozharov, V and Kopecek, J HPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line, Journal of Controlled Release, 54, pp.223-233 1998 94 Mo, Y and Lim, L Y Paclitaxel-loaded PLGA nanoparticles: Potential of anticancer activity by surface conjugation with wheat germ agglutinin, Journal of Controlled Release, 108, pp.244-262 2005 Moghimi, S M., Porter, C J H., Muir, I S., Illum, L and Davis, S S Nonphagocytic uptake of intravenously injected microspheres in rat spleen - Influence of particle-size and hydrophilic coating, Biochemical and Biophysical Research Communications, 177, pp.861-866 1991 Mu, L and Feng, S S Vitamin E TPGS used as emulsifier in the solvent evaporation/extraction technique for fabrication of polymeric nanospheres for controlled release of paclitaxel (Taxol (R)), Journal of Controlled Release, 80, pp.129144 2002 Mu, L and Feng, S S A novel controlled release formulation for the anticancer drug paclitaxel (Taxol (R)): PLGA nanoparticles containing vitamin E TPGS, Journal of Controlled Release, 86, pp.33-48 2003 Mu, L., Teo, M M., Ning, H Z., Tan, C S and Feng, S S Novel powder formulations for controlled delivery of poorly soluble anticancer drug: Application and investigation of TPGS and PEG in spray-dried particulate system, Journal of Controlled Release, 103, pp.565-575 2005 Neuzil, J., Weber, T., Gellert, N and Weber, C Selective cancer cell killing by alphatocopheryl succinate, British Journal of Cancer, 84, pp.87-89 2001 Neuzil, J Vitamin E succinate and cancer treatment: a vitamin E prototype for selective antitumour activity, British Journal of Cancer, 89, pp.1822-1826 2003 Ogawa, Y., Yamamoto, M., Okada, H., Yashiki, T and Shimamoto, T A new technique to efficiently entrap leuprolide acetate into microcapsules of polylactic acid or copoly(lactic glycolic) acid, Chemical & Pharmaceutical Bulletin, 36, pp.10951103 1988 95 Ohagan, D T., Jeffery, H and Davis, S S The preparation and characterization of poly(lactide-co-glycolide) microparticles Microparticle/polymer degradation rates and the in-vitro release of a model protein, International Journal of Pharmaceutics, 103, pp.37-45 1994 Panyam, J., Dali, M A., Sahoo, S K., Ma, W X., Chakravarthi, S S., Amidon, G L., et al Polymer degradation and in vitro release of a model protein from poly(D,Llactide-co-glycolide) nano- and microparticles, Journal of Controlled Release, 92, pp.173-187 2003 Park, E K., Lee, S B and Lee, Y M Preparation and characterization of methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric nanospheres for tumor-specific folate-mediated targeting of anticancer drugs, Biomaterials, 26, pp.1053-1061 2005a Park, E K., Kim, S Y., Lee, S B and Lee, Y M Folate-conjugated methoxy poly(ethylene glycol)/poly(epsilon-caprolactone) amphiphilic block copolymeric micelles for tumor-targeted drug delivery, Journal of Controlled Release, 109, pp.158168 2005b Pawar, R., Ben-Ari, A and Domb, A J Protein and peptide parental controlled delivery, Expert Opinion on Biological Therapy, 4, pp.1203-1212 2004 Peracchia, M T., Gref, R., Minamitake, Y., Domb, A., Lotan, N and Langer, R PEGcoated nanospheres from amphiphilic diblock and multiblock copolymers: Investigation of their drug encapsulation and release characteristics, Journal of Controlled Release, 46, pp.223-231 1997 Potineni, A., Lynn, D M., Langer, R and Amiji, M M Poly(ethylene oxide)modified poly(beta-amino ester) nanoparticles as a pH-sensitive biodegradable system for paclitaxel delivery, Journal of Controlled Release, 86, pp.223-234 2003 96 Prokop, A., Kozlov, E., Newman, G W and Newman, M J Water-based nanoparticulate polymeric system for protein delivery: Permeability control and vaccine application, Biotechnology and Bioengineering, 78, pp.459-466 2002 Quellec, P., Gref, R., Perrin, L., Dellacherie, E., Sommer, F., Verbavatz, J M., et al Protein encapsulation within polyethylene glycol-coated nanospheres I Physicochemical characterization, Journal of Biomedical Materials Research, 42, pp.45-54 1998 Rege, B D., Kao, J P Y and Polli, J E Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers, European Journal of Pharmaceutical Sciences, 16, pp.237-246 2002 Roy, S., Pal, M and Gupta, B K Indomethacin-Loaded Microspheres - Design and preparation by a multiple-emulsification technique and their in vitro evaluation, Pharmaceutical Research, 9, pp.1132-1136 1992 Ruan, G., Feng, S S and Li, Q T Effects of material hydrophobicity on physical properties of polymeric microspheres formed by double emulsion process, Journal of Controlled Release, 84, pp.151-160 2002 Ruan, G and Feng, S S Preparation and characterization of poly(lactic acid)poly(ethylene glycol)-poly(lactic acid) (PLA-PEG-PLA) microspheres for controlled release of paclitaxel, Biomaterials, 24, pp.5037-5044 2003 Ryu, J G., Jeong, Y I., Kim, I S., Lee, J H., Nah, J W and Kim, S H Clonazepam release from core-shell type nanoparticles of poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) triblock copolymers, International Journal of Pharmaceutics, 200, pp.231-242 2000 Saltzman, W M, Sanchez-Santos, A, Shen, H and Tan, J Nanotechnology: opportunities in drug delivery, 2003 controlled release society 30th annual meeting proceedings, pp.59 2003 97 Saul, J M., Annapragada, A., Natarajan, J V and Bellamkonda, R V Controlled targeting of liposomal doxorubicin via the folate receptor in vitro, Journal of Controlled Release, 92, pp.49-67 2003 Schiffelers, R M., Storm, G., ten Kate, M T and Bakker-Woudenberg, I A J M Therapeutic efficacy of liposome-encapsulated gentamicin in rat Klebsiella pneumoniae pneumonia in relation to impaired host defense and low bacterial susceptibility to gentamicin, Antimicrobial Agents and Chemotherapy, 45, pp.464470 2001 Seidman, A D., Tiersten, A., Hudis, C., Gollub, M., Barrett, S., Yao, T J., et al Phase-II trial of paclitaxel by 3-hour infusion as initial and salvage chemotherapy for metastatic breast-cancer, Journal of Clinical Oncology, 13, pp.2575-2581 1995 Shi, M., Yang, Y Y., Chaw, C S., Goh, S H., Moochhala, S M., Ng, S., et al Double walled POE/PLGA microspheres: encapsulation of water-soluble and waterinsoluble proteins and their release properties, Journal of Controlled Release, 89, pp.167-177 2003 Shin, I L G., Kim, S Y., Lee, Y M., Cho, C S and Sung, Y K Methoxy poly(ethylene glycol) epsilon-caprolactone amphiphilic block copolymeric micelle containing indomethacin I Preparation and characterization Journal of Controlled Release, 51, pp.1-11, 1998 Shmeeda, H., Mak, L., Tzemach, D., Astrahan, P., Tarshish, M and Gabizon, A Intracellular uptake and intracavitary targeting of folate-conjugated liposomes in a mouse lymphoma model with up-regulated folate receptors, Molecular Cancer Therapeutics, 5, pp.818-824 2006 Sinha, V R and Trehan, A Biodegradable microspheres for protein delivery, Journal of Controlled Release, 90, pp.261-280 2003 Somavarapul, S., Pandit, S., Gradassi, G., Bandera, A., Ravichandran, E and Alpar, O H Effect of vitamin E TPGS on immune response to nasally delivered diphtheria 98 toxoid loaded poly(caprolactone) microparticles, International Journal of Pharmaceutics, 298, pp.344-347 2005 Soppimath, K S., Aminabhavi, T M., Kulkarni, A R and Rudzinski, W E Biodegradable polymeric nanoparticles as drug delivery devices, Journal of Controlled Release, 70, pp.1-20 2001 Sudimack, J and Lee, R J Targeted drug delivery via the folate receptor, Advanced Drug Delivery Reviews, 41, pp.147-162 2000 Talmadge, J E The pharmaceutics and delivery of therapeutic polypeptides and proteins, Advanced Drug Delivery Reviews, 10, pp.247-299 1993 Torchilin, V P Drug targeting, European Journal of Pharmaceutical Sciences, 11, pp.S81-S91 2000 Traber, M G., Schiano, T D., Steephen, A C., Kayden, H J and Shike, M Efficacy of water-soluble vitamin-E in the treatment of vitamin-E malabsorption in short-bowel syndrome, American Journal of Clinical Nutrition, 59, pp.1270-1274 1994 Uchida, T., Yoshida, K., Ninomiya, A., Goto, S., Optimization of preparative conditions for polylactide (PLA) microspheres containing ovalbumin, Chemical and Pharmaceutical Bulletin, 43, pp.1569-1573 1995 Vila, A., Sanchez, A., Tobio, M., Calvo, P and Alonso, M J Design of biodegradable particles for protein delivery, Journal of Controlled Release, 78, pp.15-24 2002 Vinogradov, S V., Bronich, T K and Kabanov, A V Nanosized cationic hydrogels for drug delivery: preparation, properties and interactions with cells, Advanced Drug Delivery Reviews, 54, pp.135-147 2002 Wang, S., Lee, R J., Mathias, C J., Green, M A and Low, P S Synthesis, purification, and tumor cell uptake of Ga-67-deferoxamine-folate, a potential radiopharmaceutical for tumor imaging, Bioconjugate Chemistry, 7, pp.56-62 1996 99 Wang, S., Luo, J., Lantrip, D A., Waters, D J., Mathias, C J., Green, M A., et al Design and synthesis of [In-111]DTPA-folate for use as a tumor-targeted radiopharmaceutical, Bioconjugate Chemistry, 8, pp.673-679 1997 Witt, C., Mader, K and Kissel, T The degradation, swelling and erosion properties of biodegradable implants prepared by extrusion or compression moulding of poly(lactide-co-glycolide) and ABA triblock copolymers, Biomaterials, 21, pp.931938 2000 Wu, J., Liu, Q and Lee, R J A folate receptor-targeted liposomal formulation for paclitaxel, International Journal of Pharmaceutics, 316, pp.148-153 2006 Xiong, C D., Cheng, L M., Xu, R P and Deng, X M Synthesis and characterization of block-copolymers from D,L-lactide and poly(tetramethylene ether glycol), Journal of Applied Polymer Science, 55, pp.865-869 1995 Yang, Y Y., Chung, T S and Ng, N P Morphology, drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method, Biomaterials, 22, pp.231-241 2001a Yang, Y Y., Wan, J P., Chung, T S., Pallathadka, P K., Ng, S and Heller, J POEPEG-POE triblock copolymeric microspheres containing protein - I Preparation and characterization, Journal of Controlled Release, 75, pp.115-128 2001b Yoo, H S and Park, T G In vitro and in vivo antitumor activities of nanoparticles based on doxorubicin PLGA conjugate, Abstracts of Papers of the American Chemical Society, 219, pp.U442-U442 2000 Yoo, H S and Park, T G Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer, Journal of Controlled Release, 70, pp.63-70 2001 100 Yoo, H S and Park, T G Folate receptor targeted biodegradable polymeric doxorubicin micelles, Journal of Controlled Release, 96, pp.273-283 2004a Yoo, H S and Park, T G Folate-receptor-targeted delivery of doxorubicin nanoaggregates stabilized by doxorubicin-PEG-folate conjugate, Journal of Controlled Release, 100, pp.247-256 2004b Youk, H J., Lee, E., Choi, M K., Lee, Y J., Chung, J H., Kim, S H., et al Enhanced anticancer efficacy of alpha-tocopheryl succinate by conjugation with polyethylene glycol, Journal of Controlled Release, 107, pp.43-52 2005 Zambaux, M F., Bonneaux, F., Gref, R., Maincent, P., Dellacherie, E., Alonso, M J., et al Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method, Journal of Controlled Release, 50, pp.31-40 1998 Zhang, Z P and Feng, S S Nanoparticles of poly(lactide)/vitamin E TPGS copolymer for cancer chemotherapy: Synthesis, formulation, characterization and in vitro drug release, Biomaterials, 27, pp.262-270 2006a Zhang, Z P and Feng, S S The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles, Biomaterials, 27, pp.4025-4033 2006b Zhang, Z P and Feng, S S Self-assembled nanoparticles of poly(lactide) - Vitamin E TPGS copolymers for oral chemotherapy, International Journal of Pharmaceutics, 324, pp.191-198 2006c Zhou, S B., Liao, X Y., Li, X H., Deng, X M and Li, H Poly-D,L-lactide-copoly(ethylene glycol) microspheres as potential vaccine delivery systems, Journal of Controlled Release, 86, pp.195-205 2003 101 Zhou, X H Overcoming enzymatic and absorption barriers to non-parenterally administered protein and peptide drugs, Journal of Controlled Release, 29, pp.239252 1994 Zhu, G Z., Mallery, S R and Schwendeman, S P Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide), Nature Biotechnology, 18, pp.52-57 2000 Zubay, G Biochemistry pp 3-69, MA: Addison-Wesley Publishing Co Inc 1983 102 .. .NANOPARTICLES OF BIODEGRADABLE POLYMERS FOR CONTROLLED AND TARGETED DELIVERY OF PROTEIN DRUGS SMALL AND MOLECULE DRUGS BY LEE SIE HUEY (B.Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF. .. backbone of PLA and PLGA to form PLA-TPGS and PLGA-TPGS block copolymers These block copolymers are an example of amphiphiles Their uses as different carriers for delivery of protein and anticancer... Peptide /protein drug delivery 15 2.2.1 Structural aspect of protein 15 2.2.2 Challenges in peptide /protein drug delivery 17 2.2.3 Approaches for delivery of peptide /protein drugs