SHAPING ARTIFICIAL RECEPTORS THROUGH NANOPARTICLE SURFACE IMPRINTING OF BIOMOLECULES USING MINIEMULSION POLYMERIZATION NIRANJANI SANKARAKUMAR (B.Tech. ANNA UNIVERSITY, INDIA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Department of Chemical and Biomolecular Engineering NATIONAL UNIVERSITY OF SINGAPORE 2012 ACKNOWLEDGMENTS It gives me immense pleasure in expressing my gratitude to several persons who have immeasurably contributed and helped me throughout my PhD research. I would like to show my deepest gratitude to my supervisor, Prof. Tong Yen Wah for his tremendous support, unreserved guidance, patience and encouragement throughout the years of my PhD study. His scientific and editorial suggestions and comments have helped tremendously in the making of this thesis and working under him, as a graduate student was an enjoyable experience. I’m forever indebted to my parents who have always been the main source of comfort, friendship and encouragement throughout my life and their continuing faith and prayers have brought me this far. This thesis would have been impossible without backing from my husband, and I’m extremely grateful for his understanding, patience and support and for also being a volunteer to proof read this dissertation. I specially thank my senior Dr. Tan Chau Jin (the expert in molecular imprinting!) who had been helpful in clearing all my queries and Mr. Shalom Wangrangsimakul, for taking the time to teach imprinting experimentally during the beginning of my research. My special thanks to my senior friends Dr. Zhu Xinhao and Dr. Shirlaine Koh, my fellow lab mates Anjaneyulu Kodali, Liang Youyun, Lee Tian Jonathan, |i | Acknowledgements Chen Yiren, Dr. Xie Wenyuan, Dr. Luo Jingnan, Sushmitha Sundar, Dr. Chen Wenhui, Wang Honglei, Guo Zhi, Wang Bingfang, He Fang, Dr. Deny Hartono and Dai Mengqiao for their valuable suggestions, stimulating discussions, immense support, the many fun times spent together as a group and for creating a pleasant working atmosphere in the lab. I convey my special acknowledgment to the staff members of the Department of Chemical and Biomolecular Engineering Ms. Li Fengmei and Ms. Li Xiang for their help and assistance regarding lab and safety matters. Finally, I would like to acknowledge funding from the Bill and Melinda Gates foundation and the National University of Singapore. | ii CONTENTS Acknowledgments i Contents iii Summary ix List of Tables xi List of Figures xii List of Symbols xviii List of Abbreviations xx Introduction 1.1 Background 1.2 Hypothesis 1.3 Research objectives 1.4 Scientific and clinical significance Literature review 2.1 Molecular imprinting 10 2.2 Molecular imprinting of biomacromolecules 15 Chapter Chapter | iii | Contents 2.2.1 Bulk imprinting 17 2.2.2 Particle-based imprinting 18 2.2.3 Surface imprinting 20 Molecular imprinting of proteins 21 2.3.1 Template immobilization 23 Molecular imprinting of viruses 28 2.4.1 Bulk imprinting of viruses 33 Materials and methods 36 3.1 Materials 37 3.2 Two-stage core-shell miniemulsion polymerization 38 3.2.1 Preparation of polymeric core nanoparticles 38 3.2.2 Aminolysis 39 3.2.3 Aldehyde functionalization 39 3.2.4 Shell layer synthesis 40 3.2.5 Template removal 41 3.2.6 Non-imprinted polymers 41 Surface imprinting of proteins 42 2.3 2.4 Chapter 3.3 3.3.1 Preparation of protein-immobilized molecuarly 42 imprinted core-shell nanoparticles 3.3.2 Protein concentration assay 42 3.3.3 Protein binding analyses 43 3.3.4 Protein adsorption kinetics 44 | iv | Contents 3.4 3.3.5 Protein desorption kinetics 44 3.3.6 Imprinting Efficiency 45 3.3.7 Selectivity parameters 46 Surface imprinting of viruses 47 3.4.1 Virus and host 47 3.4.2 Host bacteria culture 47 3.4.3 Bacteriophage propagation and purification 48 3.4.4 Bacteriophage enumeration assay 48 3.4.5 Preparation of the virus surface imprinted nanoparticles 49 3.4.6 Preparation of imprinted nanoparticles using two-stage 50 miniemulsion polymerization 3.5 Chapter 3.4.7 Equilibrium virus rebinding analyses 51 3.4.8 Antiviral studies in a host-virus system 51 Analysis and measurement 52 3.5.1 Instrumental methods of analysis 52 3.5.2 Swelling experiments 53 3.5.3 Statistical analysis 54 Protein adsorption behaviour of surface 55 imprinted nanoparticles in batch and competitive conditions 4.1 Introduction 56 |v | Contents 4.2 Poly (MMA-co-EGDMA) imprinted core-shell nanoparticles 61 4.3 Singular protein adsorption kinetics 64 4.4 Equilibrium binding analyses under single protein condition 67 4.4.1 Protein adsorption using Lys 67 4.4.2 Protein adsorption using RNase A 68 4.4.3 Protein adsorption using BSA 70 Competitive protein adsorption 70 4.5.1 Binary adsorption of Lys and RNase A 70 4.5.2 Ternary adsorption of Lys, RNase A and BSA mixture 73 Competitive protein adsorption kinetics 74 4.6.1 Binary protein adsorption kinetics using Lys and RNase 74 4.5 4.6 A mixture 4.6.2 Ternary protein adsorption kinetics 78 4.7 Singular protein desorption kinetics 80 4.8 Conclusions 81 Chapter Treating viral infections using molecularly 83 imprinted polymeric nanoparticles: A novel nanotechnological approach to antiviral therapy 5.1 Introduction 84 5.2 Model host-virus system 86 | vi | Contents 5.3 Virus imprinted nanoparticles using one-stage miniemulsion 88 polymerization 5.4 Virus imprinted nanoparticles using two-stage miniemulsion 93 polymerization 5.5 Equilibrium virus rebinding analyses 96 5.5.1 Equilibrium virus rebinding analysis of vMIPs 97 5.5.2 Equilibrium virus rebinding analysis of viMIPs 100 5.6 Virus adsorption kinetics 101 5.7 Antiviral studies in a model host-virus system 102 5.7.1 Effect of imprinted particles on host cell viability 105 5.7.2 Infectivity of the adsorbed viruses 106 5.8 Treating viral infections using imprinted particles 107 5.9 Conclusions 110 Conclusions and recommendations 112 6.1 Adsorption behaviour of protein imprinted nanoparticles 113 6.2 Antiviral therapy using virus imprinted nanoparticles 115 6.3 Suggestions for future work 117 6.4 Preliminary studies for future work: Incorporation of a virus- 118 Chapter deactivation mechanism 6.4.1 Synthesis of silver encapsulated virus imprinted 118 nanoparticles 6.4.2 Characterization, virus adsorption and antiviral effects 119 | vii | Contents of Ag-viMIPs 6.4.3 Mechanism of viral inactivation by Ag nanoparticles 120 Bibliography 122 Appendix A : List of Publications 138 | viii | Bibliography Dickey, F.H. 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Sankarakumar, N., Tong, Y. W. Protein adsorption behaviour in batch and competitive conditions with nanoparticle surface imprinting, RSC Adv., 3, pp.15191527. 2013 | 138 Appendix A | List of Publications 4. Tan, C. J.; Wangrangsimakul, S.; Sankarakumar, N.; Tong, Y. W. Response to comment on preparation of super paramagnetic ribonuclease A surface-imprinted submicrometre particles for protein recognition in aqueous media, Anal. Chem., 80 (23), pp. 9375-9376. 2008. Conference publications 5. Tong, Y. W., Sankarakumar, N. Inhibition of viral infection by molecularly imprinted nanoparticles – A synthetic antibody approach to antiviral therapy, American Institute of Chemical Engineers (AIChE) Annual Meeting, October 2012, Pittsburgh, Pennsylvania, USA. 6. Sankarakumar, N., Tong, Y. W. Molecularly Imprinted Polymeric Nanoparticles for preventing viral infections. Biomedical Engineering Society 6th Scientific Meeting (BES6SM), May 2012, Singapore. 7. Sankarakumar, N., Tong, Y. W. Core-shell surface imprinted polymeric nanoparticles for selective protein recognition and separation from complex aqueous media. 241st American Chemical Society (ACS) National meeting and Exposition, March 2011, Anaheim, California, USA. | 139 Appendix A | List of Publications 8. Tong, Y. W., Sankarakumar, N. Molecularly imprinted polymeric nanoparticles for treating viral infections – A synthetic antibody approach. AIChE Annual Meeting, October 2011, Minneapolis, Minnesota, USA. 9. Sankarakumar, N., Tong, Y. W. Plastic antibodies: Molecularly imprinted polymeric nanoparticles for recognition of viruses. In Proc. 4th East Asian Pacific Student Workshop on Nano Biomedical Engineering, December 2010, Singapore. 10. Sankarakumar, N.; Tong, Y. W. Ribonuclease A and lysozyme imprinted polymeric nanoparticles as synthetic affinity receptors obtained using a two-stage core-shell miniemulsion polymerization. 5th International Conference on Materials and Advanced Technologies (ICMAT), June 2009, Singapore. | 140 [...]... lower costs of production However, the application of imprinting techniques to large biomolecules like viruses is a challenging task and has not been studied to a great extent In this thesis, molecularly imprinted artificial receptors were developed for the recognition and adsorption of viruses Miniemulsion polymerization was employed as the polymerization method enabling surface imprinting on nanoparticles... shells and surface imprinted memory of viruses was created on nanoparticles A mechanism of imprinting and recognition of viruses through miniemulsion polymerization was presented, and the fabricated nanoparticles displayed significant virus rebinding specificity in aqueous medium Following this, a first-time investigation was conducted on the application of molecularly imprinted polymeric receptors. .. nanoscale particles with large surface area that can specifically target and capture viruses to prevent them from infecting host cells 1.3 Research objectives |5 Chapter 1 | Introduction The objective of this thesis is to develop artificial receptors through nanoparticle surface imprinting via miniemulsion polymerization The receptors can be used for recognition and adsorption of biomacromolecules like... amounts of viruses from contaminated blood or sera Imprinting using miniemulsion polymerization has been reported previously for small molecular templates (Vaihinger et al., 2002) and proteins (Tan and Tong 2007a) Currently, this is the first known report on the application of miniemulsion polymerization to prepare nanoparticles having a specific surface imprinted memory of viruses Molecular imprinting. .. schematic illustration of the traditional process of molecular imprinting (MIP – Molecularly Imprinted Polymer) 13 Figure 2.3 An illustration of surface imprinting using immobilized template proteins and sacrificial supports Reprinted (adapted) with permission from (Li et al., 2006) Copyright © 2006 American Chemical Society 24 Figure 2.4 (a) A schematic depiction of surface imprinting using immobilized... use in the treatment of viral infectious diseases The specific research objectives of this thesis are: 1) To study and understand the critical factors controlling the adsorption behaviour of protein surface imprinted particles in a complex environment 2) To develop, optimize and understand surface imprinting of viruses through miniemulsion polymerization 3) To demonstrate the use of virus imprinted polymers... utilization of molecularly imprinted materials as antivirals for disease treatment With the paucity of research on viral imprinting, there have been only few reports of success Majority of the virus MIP systems have been designed only to detect the presence of viruses in small quantities (Hayden et al., 2006; Tai et al., 2010) However, the nanoparticle surface imprinting technique via miniemulsion polymerization. .. Instead of targeting individual viral protein residues, an artificial receptor for the entire virus was created through the imprinting process A simple bacteriophage was employed as the model virus, and miniemulsion polymerization was employed to fabricate sub-micron scale imprinted particles Miniemulsion polymerization is an effective imprinting polymerization system, which involves the dispersion of monomers... 34 Figure 4.1 A schematic illustration of surface imprinting of proteins via a two-stage miniemulsion polymerization Reprinted (adapted) with permission from (Tan et al., (2008a)) Copyright © 2008 American Chemical Society 60 Figure 4.2 Electron micrographs of particles (a) TEM image of core particles (b) FESEM images of core and (c) imprinted (d) non-imprinted nanoparticles The sample preparation was... of the MIPs Also, such highly cross-linked bulk imprinted polymers pose diffusional limitations reducing the efficiency of template removal and rebinding of macromolecules like proteins Additionally, the sensitive nature of biomolecules necessitates the imprinting process to be performed in aqueous medium In response to these issues, Tan and Tong (2007c) had previously proposed surface imprinting of . SHAPING ARTIFICIAL RECEPTORS THROUGH NANOPARTICLE SURFACE IMPRINTING OF BIOMOLECULES USING MINIEMULSION POLYMERIZATION NIRANJANI SANKARAKUMAR. imprinted artificial receptors were developed for the recognition and adsorption of viruses. Miniemulsion polymerization was employed as the polymerization method enabling surface imprinting on nanoparticles. imprinted memory of viruses was created on nanoparticles. A mechanism of imprinting and recognition of viruses through miniemulsion polymerization was presented, and the fabricated nanoparticles