A NOVEL μ-FLUIDIC CHANNEL ASSISTED ENCAPSULATION TECHNIQUE FOR LAYER-BY-LAYER POLYMER NANO- AND MICROCARRIER FABRICATION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Jingyu Li August, 2015 A NOVEL μ-FLUIDIC CHANNEL ASSISTED ENCAPSULATION TECHNIQUE FOR LAYER-BY-LAYER POLYMER NANO- AND MICROCARRIER FABRICATION Jingyu Li Thesis Approved: Accepted: Advisor Department Chair Dr. Younjin Min Dr. Sadhan C. Jana Committee Member Dean of the College Dr. Mukerrem Cakmak Dr. Eric J. Amis Committee Member Interim Dean of the Graduate School Dr. Hossein Tavana Dr. Rex D. Ramsier Date ii ABSTRACT Layer-by-layer (LbL) assembly is a popular technique for fabricating thin multilayer films on templates by depositing alternating layers of oppositely charged polyelectrolytes with rinsing steps in between. The LbL technique fabricated capsules can be used for biomedical applications such as drug and vaccine delivery, biosensors and bioreactors. LbL assembly enables us to use various types, sizes, and shapes of particles as templates, like silica particles, calcium carbonate particles and metal particles. In addition, a suite of water-soluble polymers with different properties can be applied to LbL assembly to meet researchers` requirements. Due to accurate control on size and shape, LbL technique is capable of fabricating small particles below 200 nm, which is in the optimal size for drug carriers and small enough to avoid clogging blood capillaries. The versatility of nano- and microcapsules has captured the attention of researchers to develop fabrication methods of LbL particles. The traditional LbL assembly method has simple process, but which is inefficient and has limitations due to repeated centrifugation steps. The long preparation time could lead to premature drug release before use. The capsules containing drugs, dyes and targeting molecules is too fragile to be centrifuged. The optimal capsule size is typically around 200 nm for many drug delivery systems. Nanoparticles under 200 nm are hard to separate from solution by centrifugation. The classical method has many drawbacks; researchers attempt to improve it for a long time. Some advanced iii methods, like atomization techniques or electrophoretic polymer assembly, have been created to better tailor properties of multilayer capsules, but influence the uniformity of films and limit the choice of polyelectrolytes and particles. In this study, our group aims to design a novel μ-fluidic device for layer-by-layer particle fabrication, which overcomes some of shortcomings of other methods. The device takes one seventh of time to assemble the same number of layers as the traditional method (μ-fluidic device takes 20 min per layer, the traditional method takes more than 2 h per layer). The device can easily prepare twenty layers of silica particles in 8 h, which formerly requires 2 days. Dynamic light scattering, ζ-potential analyzer and transmission electron microscopy were used to demonstrate the particle size, film thickness, surface charge and morphology. Drug-loaded poly(D,L-lactide-co-glycolide) (PLGA) particles were prepared for in vitro experiments with breast cancer cells. Imaging of treated cells and a biochemical analysis were used to determine the cell population and morphology. Our observation indicates that the positively charged particles have an advantage on cell uptake and drug delivery for MDA-MB-231 cancer cells. The LbL paclitaxel-loaded PLGA particles show a better anti-cancer effect than the free paclitaxel. iv ACKNOWLEDGEMENTS I am grateful to all those people who helped me to accomplish the project and master thesis. First and foremost, let me express gratitude to my advisors, Dr. Younjin Min for her continuous guidance, support and encouragement for my research work and master study. Dr. Min not only provided me training in experimental and research skills, but also built my scientific qualities, curiosity, skepticism and writing skills. Next I would like to thank Prof. Mukerrem Cakmak and Prof. Hossein Tavana for being my thesis committee member. Also a special thanks to Dr. Rong Bai for Atomic Force Microscopy (AFM) training, Dr. Bojie Wang for Transmission Electron Microscopy (TEM) training, Prof. Nicole Zacharia for providing Malvern Instruments ZEN 3690, Prof. Hossein Tavana and his student Stephanie Lemmo for providing materials for in vitro experiment. And I would like to thank all the group members: Mr. Yuanzhong Zhang, Mr. Yupeng Hu, Mr. Tianxin Zhao, Miss Xiaoxu Lu, Mr. Haoran Wang, Mr. Shihao Wen, Miss Junyan Wang and friends who once helped me in experiments. Last, my gratitude also goes out to my parents and my boyfriend who helped me a lot in daily life. They always supported and encouraged me in all areas. I can achieve nothing without them. v TABLE OF CONTENTS Page LIST OF FIGURES ...................................................................................................... viii LIST OF TABLES...........................................................................................................xi CHAPTER I. INTRODUCTION ...................................................................................................... 1 1.1 Layer-by-layer Assembly ...................................................................................... 1 1.2 Fabrication methods of LbL particles .................................................................... 3 1.2.1 Spray-assisted LbL deposition............................................................................ 3 1.2.2 Membrane filtration.......................................................................................... 6 1.2.3 Electrophoretic polymer assembly (EPA) ........................................................... 7 1.2.4 μ-fluidic systems ............................................................................................... 9 1.3 Biomedical Applications of LbL Nano- and Microcapsules .................................. 11 1.4 Mechanism of Cell Internalizing Particles ........................................................... 12 1.5 Mechanism of Drug Release From Drug-loaded Particles in Vitro ....................... 13 1.6 Physicochemical Characterizations ..................................................................... 14 1.6.1 Transmission Electron Microscopy (TEM) ........................................................ 14 1.6.2 Dynamic Light Scattering (DLS) ........................................................................ 15 1.6.3 ζ-potential Analyzer ........................................................................................ 16 1.6.4 Atomic Force Microscope (AFM) ..................................................................... 17 1.6.5 High Performance Liquid Chromatography (HPLC) ........................................... 19 1.6.6 Fluorescence Microscope ................................................................................ 20 vi 1.6.7 Microplate Reader .......................................................................................... 21 II. MATERIAL SPECIFICATION AND EXPERIMENTAL TECHNIQUES.............................. 23 2.1 Introduction ....................................................................................................... 23 2.2 Experiment Section ............................................................................................ 26 2.2.1 Material………………………………………………………………………………………………….….. .. 26 2.2.2 Layer-by-layer Assembly on Silica Particle. ...................................................... 28 2.2.3 Preparation of Nile Red PLGA Nanoparticles and Drug-Loaded PLGA Nanoparticles……………………………………………………………………………………………….…………28 2.2.4 Layer-by-layer Assembly on Drug Loaded and Nile Red PLGA Nanoparticles. ... 29 2.2.5 In Vitro Experimentation. ................................................................................ 29 2.2.6 Characterization of Particles. ........................................................................... 30 2.3 Configuration and Work Principle of μ-Fluidic Device ......................................... 27 III. RESULT AND DISCUSSION..................................................................................... 31 3.1 The Optimum Experimental Condition of μ-fluidic Device .................................. 31 3.2 LbL Silica Particles .............................................................................................. 37 3.3 In Vitro Experiment ............................................................................................ 40 3.3.1 Fabrication of LbL Blank and LbL Drug-loaded PLGA Particles .......................... 41 3.3.2 Cytotoxicity of LbL Blank PLGA Particles .......................................................... 42 3.3.3 MDA-MD-231 Cell Co-culturing With Free Drug .............................................. 45 3.3.4 Effect of Drug-loaded LbL Particles .................................................................. 48 IV. CONCLUSION AND FUTURE WORK ...................................................................... 56 REFERENCES ............................................................................................................ 58 vii LIST OF FIGURES Figure Page 1.1. The schematic of traditional film-deposition process ................................... 3 1.2. Illustration of the PRINT process................................................................... 4 1.3. SEM images of particles prepared