COAXIAL DOUBLE WALLED MICROSPHERES FOR DRUG AND GENE DELIVERY APPLICATIONS

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. 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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