Development of Janus Nanocomposites as a Multifunctional Nanocarrier for Cancer Therapy A dissertation Submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in the School of Energy, Environmental, Biological & Medical Engineering of the College of Engineering and Applied Science 2013 by Feng Wang B.S., Nanjing University of Technology, China 2005 Committee Chair: Dr. Donglu Shi ABSTRACT With the advancement of nanotechnology, cancer therapy requires that the carrier at nanoscale integrates cell targeting, imaging, drug storage and controlled drug release simultaneously. Extensive efforts have been devoted to isotropic sphere nanoparticle based carrier systems for their uniform surface properties. However, multifunctionality also leads to a challenge issue: different moieties may interfere with each other in bioconjugation process due to the similar conjugation chemistry applied to the same surface. As a result, Janus nanoparticles which are anisotropic in shape, composition or surface chemistry have attracted increasing attention. Asymmetric composition could achieve multifunctionality simultaneously. More importantly, surfaces could be selectively loaded with targeting ligands, imaging probes or drugs, which made the Janus nanoparticles “truly multifunctional entities”. A variety of fabrication methods have been studied to synthesize Janus nanoparticles for applications such as surfactants, magnetic-fluorescent display or imaging, and catalysts, etc. In contrast, exploration in the biomedical application field is rather limited. Based on our previous work on iron oxides@polystyrene matrix multifunctional nanoparticles and yolk-shell nanocomposites, we designed the polystyrene/Fe3O4@SiO2 superparamagnetic Janus nanocomposites (SJNCs). The SJNCs (~300 nm) are composed of a polystyrene (PS) core and a silica half shell embedded with iron oxide nanoparticles. We demonstrated the innovative dual i functionalities on independent surfaces were obtained simultaneously during one-pot facile synthesis, which is much more convenient than the previous report on generating the similar structure through selectively coating. PS surfaces were decorated with carboxyl groups and silanol groups on silica surfaces provided enormous opportunities for further functionalization. To achieve cell targeting and controlled drug release, we conjugated folic acid (FA) to the PS and doxorubicin (DOX), an anti-cancer drug to the silica via a pH-sensitive hydrazone bond (FA-SJNCs-DOX). Drug release behaviors in different buffer solutions (pH 5.0, 6.0 and 7.4) displayed clear pH dependence. To evaluate the in vitro cell targeting and drug release, cell cytotoxicity of FA-SJNCs-DOX against human breast cancer line MDA-MB-231 was tested. Significant difference of the concentrations killing 50 % of the cells (IC50) was observed from targeted and non-targeted group. It is hypothesized that targeted nanocomposites (FA-SJNCs- DOX) could be internalized by cancer cells via folate receptor mediated endocytosis and release the drug at a faster rate in endocytic compartments (pH 4.5~6.5), compared to under physiological condition (pH 7.4) from the non-targeted group (SJNCs-DOX), thus providing highly localized controlled drug delivery. More interestingly, the incorporated iron oxides could provide potential application such as MRI, magnetic targeting and hyperthermia, thus making the Janus nanocomposites a truly versatile platform for wide biomedical application. ii ACKNOWLEDGEMENT I would like to express my sincere gratitude to my academic advisor, Dr. Donglu Shi, for his encouragement, support, patience, and valuable advice in academic research and scientific writing during my study in University of Cincinnati. I would acknowledge my committee members, Drs. Giovanni M. Pauletti, Vesselin Shanov, and Vikram K. Kuppa on for their suggestions on dissertation. This research has been supported by National Natural Science Foundation of China (No.51003077; No.51173135), Shanghai Nano-program (No. 11nm0506100), and the Fundamental Research Funds for the Central Universities. I’m grateful to Drs. Yilong Wang and Giovanni M. Pauletti for their guidance in this highly interdisciplinary study. I have gained a lot of experience on material synthesis and tissue culture and most importantly, confidence to face challenges in unknown fields. I acknowledge Bing Han, Andrew D. Gilpin, Xiaoping Chen and Dr. Bo Hu for helping with characterization and providing valuable suggestions on structure analysis. My gratitude also goes to lab-mates, Ronak Patel, Drs. Sheng Tong, Hoonsung Cho and Chris Huth for their support in every way. I would like to give my sincere thanks to my parents, my uncle and aunt, my cousin and my dear grandma who passed away last month, for their unconditional love, support and patience. I also want to thank my friends in University of Cincinnati and in The Institute for Biomedical Engineering and Nano Science, Tongji University, China, Xuecheng Dong, Dingchuan Xue, Xiang Gao, Linqian Feng, Fei Yu, Hua Li, Peng He, Vibhor Chaswal, Guojun iii Zhang, Yin Chen, Mary Lim, Zhouyang Liu, Mingyu Zhong, Xiaojun Cai, Huiyun Wen and Xuequan Li for their help and encouragement. iv Contents ABSTRACT ..................................................................................................................................... i ACKNOWLEDGEMENT ............................................................................................................. iii Chapter 1. Introduction ............................................................................................................ 1 1.1. Targeting ............................................................................................................................... 3 1.2. Drug Delivery ....................................................................................................................... 5 1.2.1 Micelles ........................................................................................................................... 5 1.2.2. Dendrimers ..................................................................................................................... 7 1.2.3. Quantum Dots ................................................................................................................ 8 1.2.4. Inorganic Nanoparticles ............................................................................................... 10 1.3. Magnetic Resonance Imaging ............................................................................................ 15 1.4. Development of Janus Particles ......................................................................................... 16 1.4.1. Introduction to Janus Particles ..................................................................................... 16 1.4.2. Inorganic Janus Particles .............................................................................................. 18 1.4.3. Polymeric Janus Particles ............................................................................................ 20 1.4.3. Polymeric/Inorganic Janus Composites ....................................................................... 22 1.4.4. Applications of Janus Particles .................................................................................... 25 1.5. Research objectives and strategy: Multifunctional Janus Nanocomposites for Biomedical Application ................................................................................................................................ 26 1.6. References .......................................................................................................................... 27 Chapter 2. Synthesis and Application of Multifunctional Nanocarrier System for Cancer Therapy ....................................................................................................................................................... 36 2.1. Background ........................................................................................................................ 36 2.2. Experimental ...................................................................................................................... 38 2.2.1. QD Conjugation to MNSs ............................................................................................ 38 2.4. Conclusion .......................................................................................................................... 43 2.5. References .......................................................................................................................... 44 Chapter 3. Design and Development of Superparamagnetic Janus Nanoparticles as a Multi- functional Tool for Cancer Therapy .............................................................................................. 45 3.1. Introduction ........................................................................................................................ 45 3.2. Design and Development of Polystyrene/Fe3O4@Silica nanocomposites ......................... 46 3.2.1. Experimental ................................................................................................................ 46 3.2.2. Results and Discussion ................................................................................................ 48 3.3. Design and Development of Dual Functionalized Polystyrene/Fe3O4@Silica