COAXIAL DOUBLE-WALLED MICROSPHERES FOR DRUG AND GENE DELIVERY APPLICATIONS XU QINGXING NOEL (B.Eng.(Hons.), NUS) A THESIS SUBMITTED FOR THE NUS-UIUC JOINT DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ----------------------------------------Xu Qingxing Noel May 2014 i Acknowledgements ACKNOWLEDGEMENTS It would not have been possible to complete this doctoral thesis without the assistance and support of the kind people around me, some of whom I would like to give particular mention here. I would like to express my sincere gratitude to my thesis advisors, Prof. ChiHwa Wang at National University of Singapore (NUS, Singapore) and Prof. Daniel W. Pack at University of Illinois at Urbana-Champaign (UIUC, USA). The good advice, support and patience from Prof. Wang have been invaluable on both an academic and a personal level, for which I am extremely grateful. He has provided a simulating and challenging environment for learning and thinking, and opportunities in preparing grant proposals and making seminar presentations, all of which have groomed me into becoming a researcher. The enthusiastic and creative ideas, and valuable suggestions from Prof. Pack have been invaluable in the improvement of my research work and manuscript preparation. I would like to thank the scholarship support from Agency for Science, Technology and Research (A*STAR, Singapore) for NUS-UIUC Joint Ph.D. Program. I would like to also thank the funding support from the National Institute of Health (NIH, USA) and National Medical Research Council (NMRC, Singapore). Acknowledgements ii It has been a rewarding experience to be working with my colleagues in Prof. Pack’s laboratory, Dr. Kalena Stovall, Dr. Kara Smith, Yujie Xia, Dr. Rahul Keswani, Dr. Mark Hwang, Victor Shum and Mihael Lazebnik, and colleagues in Prof. Wang’s laboratory, Dr. Yongpan Cheng, Dr. Hemin Nie, Dr. Alireza Rezvanpour, Chenlu Lei, Jian Qiao, Pooya Davoodi, Yanna Cui and Hao Qin. The undergraduate students that I have worked with, Bei Shi Wong, Kang Chi Neo, Kenneth Teow, Shi En Chin, Kar Kay Chin, Yitong Sun, Jun Quan Yeo, Jiayu Leong, Qi Yi Chua, Yu Tse Chi, Zhenyuan Yin and others, were cooperative and helpful in my research work. Special thanks go to the staff at Materials Research Laboratory, UIUC, particularly James Mabon and Wacek Swiech, the staff in Imaging Technology Group at Beckman Institute, UIUC, particularly Charles Bee and Leilei Yin, and the staff in the Department of Chemical and Biomolecular Engineering, NUS, particularly Phai Ann Chia, Fengmei Li, Xiang Li, Evan Tan, Joey Lim and Wee Siong Ang, for all the useful technical support. Last but not least, I am thankful to my parents, my sister and my girlfriend who have been understanding and caring during my Ph.D. studies. They always stood by me when I needed them most. It has been a wonderful and rewarding experience over at NUS and UIUC. May this work mark the beginning of new and better things to come. iii Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS iii SUMMARY vi LIST OF TABLES ix LIST OF FIGURES xi LIST OF SYMBOLS xxii Chapter 1: Introduction 1.1. Background and motivation 1.2. Studies and objectives 1.3. Structure of the thesis Chapter 2: Literature Review 2.1. Drug delivery 2.2. Techniques in microsphere fabrication 26 2.3. Double-walled microspheres 34 2.4. Doxorubicin 34 2.5. p53 gene therapy 36 iv Table of Contents Chapter 3: Coaxial Electrohydrodynamic Atomization 45 Process for Production of Polymeric Double-Walled Microspheres Chapter 4: 3.1. Introduction 45 3.2. Experiments 49 3.3. Numerical simulation 53 3.4. Results and discussion 65 3.5. Conclusions 93 Mechanism of Drug Release from 95 Double-Walled PDLLA(PLGA) Microspheres Chapter 5: 4.1. Introduction 95 4.2. Materials and methods 99 4.3. Results and discussion 106 4.4. Conclusions 123 Combined Modality Doxorubicin-Based 124 Chemotherapy and Chitosan-Mediated p53 Gene Therapy Using Double-Walled Microspheres for Treatment of Human Hepatocellular Carcinoma 5.1. Introduction 124 5.2. Materials and methods 127 5.3. Results 141 5.4. Discussion 173 5.5. Conclusions 176 v Table of Contents Chapter 6: Conclusions and Recommendations 178 REFERENCES 185 LIST OF PUBLICATIONS 220 LIST OF CONFERENCE PRESENTATIONS 221 vi Summary SUMMARY Polymeric double-walled microspheres were developed by coaxial electrohydrodynamic atomization (CEHDA) and precision particle fabrication (PPF) techniques. Here, we focus on double-walled microspheres consisting of a poly(D,L-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(D,Llactic acid) (PDLLA) or poly(L-lactic acid) (PLLA) shell layer. The first study involves bridging the experimental work on the fabrication of double-walled microspheres from CEHDA and the simulation work on the generation of compound droplets from the same process. Process conditions and solution parameters were investigated to ensure the formation of doublewalled microspheres with a doxorubicin-loaded PLGA core surrounded by a relatively drug-free PDLLA shell layer. Numerical simulation of CEHDA process was performed based on a computational fluid dynamics (CFD) model in Fluent. The simulation results were compared with the experimental work to illustrate the capability of the CFD model to predict the production of consistent double-walled microspheres. The second study involves drug release and degradation behavior of two double-walled microsphere formulations consisting of a doxorubicin-loaded PLGA core surrounded by a PDLLA shell layer. It was postulated that Summary vii different molecular weights of the shell layer could modulate the erosion of the outer coating and limit the occurrence of water penetration into the inner drug-loaded core on various time scales, and therefore control the drug release from the microspheres. For both microsphere formulations, the drug release profiles were observed to be similar. Interestingly, both microsphere formulations exhibited occurrence of bulk erosion of PDLLA on a similar time scale despite different PDLLA molecular weights forming the shell layer. The shell layer of the double-walled microspheres served as an effective diffusion barrier during the initial lag phase period and controlled the release rate of the hydrophilic drug independent of the molecular weight of the shell layer. The third study involves designing and evaluating double-walled microspheres loaded with chitosan-p53 nanoparticles (chi-p53, gene encoding p53 tumor suppressor protein) and/or doxorubicin in the shell and core phases, respectively, for combined gene therapy and chemotherapy. The microspheres were monodisperse with a mean diameter of 65 to 75 μm and uniform shell thickness of to 17 μm. The encapsulation efficiency of doxorubicin was significantly higher when it was encapsulated alone compared to coencapsulation with chi-p53. However, the encapsulation efficiency of chi-p53 was not affected by the presence of doxorubicin. As desired, chi-p53 was released first, followed by simultaneous release of chi-p53 and doxorubicin at a near zero-order rate. Next, the therapeutic efficiencies of doxorubicin and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor Summary viii suppressor p53 and apoptotic caspase proteins in human hepatocellular carcinoma HepG2 cells. Overall, the combined doxorubicin and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either doxorubicin or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy. References 207 Millau JF, Bastien N, Drouin R. P53 transcriptional activities: a general overview and some thoughts. Mutat Res 2009;681:118-33. Miller RA, Brady JM, Cutright DE. Degradation rates of oral resorbable implants (polylactates and polyglycolates): rate modification with changes in PLA/PGA copolymer ratios. J Biomed Mater Res 1977;11:711-9. Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53mediated transactivation. Cell 1992;69:1237-45. Momparler RL, Karon M, Siegel SE, Avila F. Effect of adriamycin on DNA, RNA, and protein synthesis in cell-free systems and intact cells. Cancer Res 1976;36:2891-5. Muggia F, Hamilton A. Phase III data on Caelyx in ovarian cancer. Eur J Cancer 2001;37:S15-8. Muvaffak A, Gurhan I, Hasirci N. Prolonged cytotoxic effect of colchicine released from biodegradable microspheres. J Biomed Mater Res B Appl Biomater 2004;71:295-304. Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 2001;7:683-94. Naraharisetti PK, Ong BYS, Xie JW, Lee TKY, Wang CH, Sahinidis NV. In vivo performance of implantable biodegradable preparations delivering Paclitaxel and Etanidazole for the treatment of glioma. Biomaterials 2007;28:886-94. References 208 Ng IO, Lai EC, Chan AS, So MK. Overexpression of p53 in hepatocellular carcinomas: a clinicopathological and prognostic correlation. J Gastroenterol Hepatol 1995;10:250-5. Nie H, Dong Z, Arifin DY, Hu Y, Wang CH. Core/shell microspheres via coaxial electrohydrodynamic atomization for sequential and parallel release of drugs. J Biomed Mater Res A 2010a;95:709-16. Nie H, Fu Y, Wang CH. Paclitaxel and suramin-loaded core/shell microspheres in the treatment of brain tumors. Biomaterials 2010b;31:8732-40. Noda A, Ning Y, Venable SF, Pereira-Smith OM, Smith JR. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp Cell Res 1994;211:90-8. Northfelt DW, Martin FJ, Working P, Volberding PA, Russell J, Newman M, et al. Doxorubicin encapsulated in liposomes containing surface-bound polyethylene glycol: pharmacokinetics, tumor localization, and safety in patients with AIDS-related Kaposi's sarcoma. J Clin Pharmacol 1996;36:55-63. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 2000;288:1053-8. Ogris M, Brunner S, Schüller S, Kircheis R, Wagner E. PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 1999;6:595-605. References 209 Olsen E, Duvic M, Frankel A, Kim Y, Martin A, Vonderheid E, et al. Pivotal phase III trial of two dose levels of denileukin diftitox for the treatment of cutaneous T-cell lymphoma. J Clin Oncol 2001;19:376-88. Paine MD, Alexander MS, Stark JP. Nozzle and liquid effects on the spray modes in nanoelectrospray. J Colloid Interface Sci 2007;305:111-23. Pareta R, Edirisinghe MJ. A novel method for the preparation of biodegradable microspheres for protein drug delivery. J R Soc Interface 2006;3:573-82. Park TG. Degradation of poly(D,L-lactic acid) microspheres: effect of molecular weight. J Control Release 1994;30:161-73. Park TG. Degradation of poly(lactic-co-glycolic acid) microspheres: effect of copolymer composition. Biomaterials 1995;16:1123-30. Passerini N, Craig DQ. An investigation into the effects of residual water on the glass transition temperature of polylactide microspheres using modulated temperature DSC. J Control Release 2001;73:111-5. Patankar SV, Numerical Heat Transfer and Fluid Flow. Hemisphere, NewYork; 1980. Pekarek KJ, Dyrud MJ, Ferrer K, Jong YS, Mathiowitz E. In vitro and in vivo degradation of double-walled polymer microspheres. J Control Release 1996;40:169-78. Pekarek KJ, Jacob JS, Mathiowitz E. Double-walled polymer microspheres for controlled drug release. Nature 1994a;367:258-60. References 210 Pekarek KJ, Jacob JS, Mathiowitz E. One-step preparation of double-walled microspheres. Adv Mater 1994b;6:684-7. Poeta ML, Manola J, Goldwasser MA, Forastiere A, Benoit N, Califano JA, et al. TP53 mutations and survival in squamous-cell carcinoma of the head and neck. N Engl J Med 2007;357:2552-61. Pollauf EJ, Berkland C, Kim KK, Pack DW. In vitro degradation of polyanhydride/polyester core-shell double-wall microspheres. Int J Pharm 2005b;301:294-303. Pollauf EJ, Kim KK, Pack DW. Small-molecule release from poly(D,Llactide)/poly(D,L-lactide-co-glycolide) composite microparticles. J Pharm Sci 2005a;94:2013-22. Prabha S, Labhasetwar V. Nanoparticle-mediated wild-type p53 gene delivery results in sustained antiproliferative activity in breast cancer cells. Mol Pharm 2004;1:211-9. Prives C, Hall PA. The p53 pathway. J Pathol 1999;187:112-26. Qin L, Pahud DR, Ding Y, Bielinska AU, Kukowska-Latallo JF, Baker JR Jr, et al. Efficient transfer of genes into murine cardiac grafts by Starburst polyamidoamine dendrimers. Hum Gene Ther 1998;9:553-60. Rahman NA, Mathiowitz E. Localization of bovine serum albumin in doublewalled microspheres. J Control Release 2004;94:163-75. Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. J Biomed Mater Res 1997;34:21-8. References 211 Regele JD, Papac MJ, Rickard MJA, Dunn-Rankin D. Effects of capillary spacing on EHD spraying from an array of cone jets. J Aerosol Sci 2002;33:1471-9. Regnström K, Ragnarsson EG, Köping-Höggård M, Torstensson E, Nyblom H, Artursson P. PEI - a potent, but not harmless, mucosal immuno-stimulator of mixed T-helper cell response and FasL-mediated cell death in mice. Gene Ther 2003;10:1575-83. Roberts JC, Bhalgat MK, Zera RT. Preliminary biological evaluation of polyamidoamine (PAMAM) Starburst dendrimers. J Biomed Mater Res 1996;30:53-65. Rosell-Llompart J, Fernández de la Mora J. Generation of monodisperse droplets 0.3 to μm in diameter from electrified cone-jets of highly conducting and viscous liquids. J Aerosol Sci 1994;25:1093-119. Rulison AJ, Flagan RC. Scale-up of electrospray atomization using linear arrays of Taylor cones. Rev Sci Instrum 1993;64:683-6. Rutledge GC, Fridrikh SV. Formation of fibers by electrospinning. Adv Drug Deliv Rev 2007;59:1384-91. Sahara S, Aoto M, Eguchi Y, Imamoto N, Yoneda Y, Tsujimoto Y. Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation. Nature 1999;401:168-73. Samaranayake H, Määttä AM, Pikkarainen J, Ylä-Herttuala S. Future prospects and challenges of antiangiogenic cancer gene therapy. Hum Gene Ther 2010;21:381-96. References 212 Sancar A, Lindsey-Boltz LA, Unsal-Kaçmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004;73:39-85. Sato T, Ishii T, Okahata Y. In vitro gene delivery mediated by chitosan. effect of pH, serum, and molecular mass of chitosan on the transfection efficiency. Biomaterials 2001;22:2075-80. Saville DA. Electrohydrodynamics: the Taylor-Melcher leaky dielectric model. Annu Rev Fluid Mech 1997;29:27-64. Sax JK, Fei P, Murphy ME, Bernhard E, Korsmeyer SJ, El-Deiry WS. BID regulation by p53 contributes to chemosensitivity. Nat Cell Biol 2002;4:842-9. Seymour LW, Olliff SP, Poole CJ, De Takats PG, Orme R, Ferry DR, et al. A novel dosage approach for evaluation of SMANCS [poly-(styrene-co-maleylhalf-n-butylate) - neocarzinostatin] in the treatment of primary hepatocellular carcinoma. Int J Oncol 1998;12:1217-23. Shen Y, White E. p53-dependent apoptosis pathways. Adv Cancer Res 2001;82:55-84. Shenderova A, Burke TG, Schwendeman SP. The acidic microclimate in poly(lactide-co-glycolide) microspheres stabilizes camptothecins. Pharm Res 1999;16:241-8. Shi M, Yang YY, Chaw CS, Goh SH, Moochhala SM, Ng S, et al. Double walled POE/PLGA microspheres: encapsulation of water-soluble and waterinsoluble proteins and their release properties. J Control Release 2003;89:16777. References 213 Shieh L, Tamada J, Chen I, Pang J, Domb A, Langer R. Erosion of a new family of biodegradable polyanhydrides. J Biomed Mater Res 1994;28:146575. Slee EA, O'Connor DJ, Lu X. To die or not to die: how does p53 decide? Oncogene 2004;23:2809-18. Soengas MS, Alarcón RM, Yoshida H, Giaccia AJ, Hakem R, Mak TW, et al. Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition. Science 1999;284:156-9. Srikar R, Yarin AL, Megaridis CM, Bazilevsky AV, Kelley E. Desorptionlimited mechanism of release from polymer nanofibers. Langmuir 2008;24:965-74. Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell 1998;1:949-57. Stavridi ES, Huyen Y, Sheston EA, Halazonetis TD. The three-dimensional structure of p53. In The p53 Tumor Suppressor Pathway and Cancer. Edited by Zambetti GP. New York: Springer Science Business Media; 2005: 25-52. Stommel JM, Marchenko ND, Jimenez GS, Moll UM, Hope TJ, Wahl GM. A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J 1999;18:1660-72. Sudimack J, Lee RJ. Targeted drug delivery via the folate receptor. Adv Drug Deliv Rev 2000;41:147-62. 214 References Takahashi S, Moriya T, Ishida T, Shibata H, Sasano H, Ohuchi N, et al. Prediction of breast cancer prognosis by gene expression profile of TP53 status. Cancer Sci 2008;99:324-32. Takemoto K, Nagai T, Miyawaki A, Miura M. Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects. J Cell Biol 2003;160:235-43. Tamada JA, Langer R. Erosion kinetics of hydrolytically degradable polymers. Proc Natl Acad Sci USA 1993;90:552-6. Tan EC, Lin R, Wang CH. Fabrication of double-walled microspheres for the sustained release of doxorubicin. J Colloid Interface Sci 2005;291:135-43. Tang Y, Zhao W, Chen Y, Zhao Y, Gu W. Acetylation is indispensable for p53 activation. Cell 2008;133:612-26. Toledo F, Wahl GM. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat Rev Cancer 2006;6:909-23. Tomihata K, Ikada Y. In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials 1997;18:567-75. Tsang WP, Chau SP, Kong SK, Fung KP, Kwok TT. Reactive oxygen species mediate doxorubicin 2003;73:2047-58. induced p53-independent apoptosis. Life Sci References 215 Tsuji H, Mizuno A, Ikada Y. Properties and morphology of poly(L-lactide). III. Effects of initial crystallinity on long-term in vitro hydrolysis of high molecular weight poly(L-lactide) film in phosphate-buffered solution. J Appl Polym Sci 2000;77:1452-64. Turunen MP, Hiltunen MO, Ruponen M, Virkamäki L, Szoka FC Jr, Urtti A, et al. Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer-plasmid complexes. Gene Ther 1999;6:6-11. Uchida T, Yoshida K, Goto S. Preparation and characterization of polylactic acid microspheres containing water-soluble dyes using a novel w/o/w emulsion solvent evaporation method. J Microencapsul 1996;13:219-28. Vey E, Rodger C, Booth J, Claybourn M, Miller AF, Saiani A. Degradation kinetics of poly(lactic-co-glycolic) acid block copolymer cast films in phosphate buffer solution as revealed by infrared and Raman spectroscopies. Polym Degrad Stab 2011;96:1882-9. Vidaurreta M, Maestro ML, Sanz-Casla MT, Rafael S, Veganzones S, de la Orden V, et al. Colorectal carcinoma prognosis can be predicted by alterations in gene p53 exons and 8. Int J Colorectal Dis 2008;23:581-6. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004;10:789-99. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307-10. References 216 von Harpe A, Petersen H, Li Y, Kissel T. Characterization of commercially available and synthesized polyethylenimines for gene delivery. J Control Release 2000;69:309-22. Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol 2007;8:275-83. Weinberg BD, Blanco E, Gao J. Polymer implants for intratumoral drug delivery and cancer therapy. J Pharm Sci 2008;97:1681-702. Whibley C, Pharoah PD, Hollstein M. p53 polymorphisms: cancer implications. Nat Rev Cancer 2009;9:95-107. Wightman L, Kircheis R, Rössler V, Carotta S, Ruzicka R, Kursa M, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J Gene Med 2001;3:362-72. Wiradharma N, Tong YW, Yang YY. Self-assembled oligopeptide nanostructures for co-delivery of drug and gene with synergistic therapeutic effect. Biomaterials 2009;30:3100-9. Wiseman JW, Goddard CA, McLelland D, Colledge WH. A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo. Gene Ther 2003;10:1654-62. Wiwattanapatapee R, Carreño-Gómez B, Malik N, Duncan R. Anionic PAMAM dendrimers rapidly cross adult rat intestine in vitro: a potential oral delivery system? Pharm Res 2000;17:991-8. 217 References Wu GS, Burns TF, McDonald III ER, Jiang W, Meng R, Krantz ID, et al. KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat Genet 1997;17:141-3. Wu Y, Yu B, Jackson A, Zha W, Lee LJ, Wyslouzil BE. Coaxial electrohydrodynamic spraying: a novel one-step technique to prepare oligodeoxynucleotide encapsulated lipoplex nanoparticles. Mol Pharm 2009;6:1371-9. Xie J, Marijnissen JC, Wang CH. Microparticles developed by electrohydrodynamic atomization for the local delivery of anticancer drug to treat C6 glioma in vitro. Biomaterials 2006;27:3321-32. Xie J, Ng WJ, Lee LY, Wang CH. Encapsulation of protein drugs in biodegradable microparticles by co-axial electrospray. J Colloid Interface Sci 2008;317:469-76. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993;366:701-4. Xu L, Pirollo KF, Chang EH. Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy. J Control Release 2001;74:11528. Xu Q, Wang CH, Pack DW. Polymeric carriers for gene delivery: chitosan and poly(amidoamine) dendrimers. Curr Pharm Des 2010;16:2350-68. Yamaguchi Y, Takenaga M, Kitagawa A, Ogawa Y, Mizushima Y, Igarashi R. Insulin-loaded biodegradable PLGA microcapsules: initial burst release controlled by hydrophilic additives. J Control Release 2002;81:235-49. References 218 Yan F, Farouk B, Ko F. Numerical modeling of an electrostatically driven liquid meniscus in the cone-jet mode. J Aerosol Sci 2003;34:99-116. Yang YY, Chung TS, Bai XL, Chan WK. Effect of preparation conditions on morphology and release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion method. Chem Eng Sci 2000;55:2223-36. Yang YY, Shi M, Goh SH, Moochhala SM, Ng S, Heller J. POE/PLGA composite microspheres: formation and in vitro behavior of double walled microspheres. J Control Release 2003;88:201-13. Yoon H, Woo JH, Ra YM, Yoon SS, Kim HY, Ahn S, et al. Electrostatic spray deposition of copper-indium thin films. Aerosol Sci Technol 2011;45:1448-55. Young KH, Weisenburger DD, Dave BJ, Smith L, Sanger W, Iqbal J, et al. Mutations in the DNA-binding codons of TP53, which are associated with decreased expression of TRAILreceptor-2, predict for poor survival in diffuse large B-cell lymphoma. Blood 2007;110:4396-405. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMA induces the rapid apoptosis of colorectal cancer cells. Mol Cell 2001;7:673-82. Zhang S, Kawakami K, Yamamoto M, Masaoka Y, Kataoka M, Yamashita S, et al. Coaxial electrospray formulations for improving oral absorption of a poorly water-soluble drug. Mol Pharm 2011;8:807-13. References 219 Zhao X, Yu SB, Wu FL, Mao ZB, Yu CL. Transfection of primary chondrocytes using chitosan-pEGFP nanoparticles. J Control Release 2006;112:223-8. Zheng W. A water-in-oil-in-oil-in-water (W/O/O/W) method for producing drug-releasing, double-walled microspheres. Int J Pharm 2009;374:90-5. Zolnik BS, Burgess DJ. Effect of acidic pH on PLGA microsphere degradation and release. J Control Release 2007;122:338-44. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997;90:405-13. 220 List of Publications LIST OF PUBLICATIONS Xu Q, Qin H, Yin Z, Hua J, Pack DW, Wang CH. Coaxial electrohydrodynamic atomization process for production of polymeric composite microspheres. Chemical Engineering Science 2013;104:330-46. Xu Q, Leong J, Chua QY, Chi YT, Chow PKH, Pack DW, Wang CH. Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for treatment of human hepatocellular carcinoma. Biomaterials 2013;34:5149-62. Xia Y, Xu Q, Wang CH, Pack DW. Protein encapsulation in and release from monodisperse double-wall polymer microspheres. J Pharm Sci 2013;102:1601-9. Xu Q, Chin SE, Wang CH, Pack DW. Mechanism of drug release from double-walled PDLLA(PLGA) microspheres. Biomaterials 2013;34:3902-11. Xu Q, Xia Y, Wang CH, Pack DW. Monodisperse double-walled microspheres loaded with chitosan-p53 nanoparticles and doxorubicin for combined gene therapy and chemotherapy. Journal of Controlled Release 2012;163:130-5. Xu Q, Wang CH, Pack DW. Polymeric carriers for gene delivery: chitosan and poly(amidoamine) dendrimers. Current Pharmaceutical Design 2010;16:235068. List of Conference Presentations 221 LIST OF CONFERENCE PRESENTATIONS Xu Q, Qin H, Yin Z, Pack DW, Wang CH. Simulation based coaxial electrohydrodynamic atomization process for production of polymeric doublewalled microspheres. Controlled Release Society Annual Meeting. Honolulu, Hawaii, USA. July 21 to 24, 2013. Xu Q, Wang CH, Pack DW. Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy enhances inhibition of hepatocellular carcinoma HepG2 cell growth. Controlled Release Society Annual Meeting. Québec City, Canada. July 15 to 18, 2012. Xu Q, Chin SE, Wang CH, Pack DW. In vitro degradation of double-walled PLA(PLGA) microspheres. Controlled Release Society Annual Meeting. Québec City, Canada. July 15 to 18, 2012. Xu Q, Neo KC, Pack DW, Wang CH. Fabrication, characterization and longterm in vitro release of hydrophilic drug using double-walled microspheres from coaxial electrospraying. 9th World Biomaterials Congress. Chengdu, China. June to 5, 2012. Xu Q, Wang CH, Pack DW. Encapsulation of doxorubicin and chitosan-p53 nanoparticles in monodispersed double-walled microspheres. The 14th Asia Pacific Confederation of Chemical Engineering Congress. Singapore. February 21 to 24, 2012. 222 List of Conference Presentations Xu Q, Wang CH, Pack DW. Encapsulation of doxorubicin and chitosan-p53 nanoparticles in monodispersed double-walled microspheres. Controlled Release Society Annual Meeting. National Harbor, Maryland, USA. July 30 to August 3, 2011. Xu Q, Wong BS, Pack DW, Wang CH. Controlling morphology and size of double-walled microspheres from coaxial electrospraying. Controlled Release Society Annual Meeting. National Harbor, Maryland, USA. July 30 to August 3, 2011. Xu Q, Wang CH, Pack DW. Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for cancer treatment. American Institute of Chemical Engineers Annual Meeting. Salt Lake City, Utah, USA. November to 12, 2010. Xu Q, Pack DW, Wang CH. Effect of fabrication conditions on the formation of double-walled microspheres and microfibers by coaxial electrospraying/electrospinning technique. American Institute of Chemical Engineers Annual Meeting. Salt Lake City, Utah, USA. November to 12, 2010. [...]... significance level The mean and standard deviation for the fitted distribution is indicated above For (a), the p values for the Gaussian and Poisson distribution fits are 0.154 and 0.027, respectively For (b), the p values for the Gaussian and Poisson distribution fits are 0.628 and 0.016, respectively For (c), the p values for the Gaussian and Poisson distribution fits are 0.962 and 0.037, respectively... (CEHDA) and the simulation work on the generation of compound droplets from the same process, ii) to examine the drug release and degradation behavior of two double- walled microsphere formulations consisting of a drug- loaded core surrounded by a shell layer with different molecular weights, and lastly, iii) to explore the therapeutic potential of double- walled microspheres for combined gene therapy and. .. Figures Figure 5.10: In vitro doxorubicin and chi-p53 release from doublewalled PLA(PLGA) microspheres: (a) doxorubicin from formulation B microspheres, (b) chi-p53 nanoparticles from formulation C microspheres, (c) doxorubicin from formulation D microspheres, and (d) chi-p53 nanoparticles from formulation D microspheres 157 Figure 5.11: Comparison of combined Dox and chi-p53 FD treatment with Dox FD or... oral, pulmonary and parenteral injection, and they do not need surgical removal after release of the drug is completed Since an important goal of drug delivery systems is to attain well-controlled drug release rates, double- walled microspheres with a particle core surrounded by a shell layer are fabricated The ability to form double- walled microspheres exhibiting a predefined core diameter and shell thickness... depicting the surface morphology of double- walled PLLA(PLGA) microspheres for various microsphere samples listed in Table 4.1 Partial encapsulation was observed for samples A1, A2, B1, B2, and C1 to C3 Fully formed double- walled microspheres were observed in samples A3 and B3 Scale bar = 50 μm 109 Figure 4.3: SEM images depicting the surface morphology of doublewalled PDLLA(PLGA) microspheres with a low PDLLA... drug- loaded core on various time scales, and therefore control the drug release from the microspheres, and finally, iii) the doublewalled microspheres could deliver drug and gene simultaneously for improved treatment of human hepatocellular carcinoma Specific studies and their corresponding objectives are listed as follows: a) This study aims to bridge the experimental and simulation work of the CEHDA process... approach and many drug formulations have already been approved by the US Food and 8 Chapter 2 Drug Administration (FDA) Some representative drug delivery systems which have received regulatory approval have been summarized in Table 2.1 2.1.1 Drug delivery systems Two key aims most drug delivery systems attempt to achieve are i) to minimize drug entering the normal cells, and ii) to maintain drug concentration... molecular weight shell layer (formulation A) and a high PDLLA molecular weight shell layer (formulation B) at different stages of the degradation process (a) and (b) are images of initial microspheres before degradation, (c) and (d) 26 days, (e) and (f) 33 days, (g) and (h) 40 days, and (i) and (j) 47 days after degradation The inserts show microspheres with pore or cavity formation Scale bar = 50 µm... after 33 and 40 days of degradation respectively (b) and (d) are the confocal images of formulation B microspheres after 33 and 40 days of degradation respectively Scale bar = 50 μm 118 Figure 4.7: Molecular weight profiles as a function of incubation time for double- walled PDLLA(PLGA) microspheres during degradation (a) Weight-averaged molecular weight (M w ) profiles for formulations A and B microspheres. .. doublewalled PLA(PLGA) microspheres The distribution of doxorubicin in formulations B and D microspheres is indicated in green The distribution of chi-p53 nanoparticles in formulations C and D microspheres is indicated in red and yellow (colocalization of red and green), respectively Scale bar = 50 μm 150 Figure 5.6: FTIR spectra of blank double- walled PLA(PLGA) microspheres (formulation A) in comparison . COAXIAL DOUBLE- WALLED MICROSPHERES FOR DRUG AND GENE DELIVERY APPLICATIONS XU QINGXING NOEL (B.Eng.(Hons.), NUS) A THESIS SUBMITTED FOR THE NUS-UIUC. treated with microspheres than those treated with free drugs. Overall, double- walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy and (b) are images of initial microspheres before degradation, (c) and (d) 26 days, (e) and (f) 33 days, (g) and (h) 40 days, and (i) and (j) 47 days after degradation. The inserts show microspheres