Nanoparticle based gene therapy using fluorescent upconversion nanoparticles

NANOPARTICLE-BASED GENE THERAPY USING FLUORESCENT UPCONVERSION NANOPARTICLES Muthu kumara gnanasammandhan JAYAKUMAR (B.Tech Biotechnology, Anna University, India) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 I 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 Parts of this thesis had been published or presented in the publications listed in the following section Muthu Kumara Gnanasammandhan 30th May 2012 II List of Publications Peer reviewed articles Dev K Chatterjee, Muthu Kumara Gnanasammandhan, Yong Zhang, Small Upconverting Fluorescent Nanoparticles for Biomedical Applications, Small, 2010, 6:24, 2781-2795 Li Ching Ong, Muthu Kumara Gnanasammandhan, Sounderya Nagarajan, Yong Zhang Upconversion: road to El Dorado of the fluorescence world, Luminescence, 2010, 25:4, July/August 2012, 290-293 Shan Jiang, Muthu Kumara Gnanasammandhan, Yong Zhang, Optical imaging guided cancer therapy with fluorescent nanoparticles, J R Soc Interface, January 2010, Vol no 42, Pp 3-18 Muthu Kumara Gnanasammandhan and Yong Zhang, Simultaneous gene delivery and tracking of cells using fluorescent upconversion nanoparticles for cell therapy MRS Proceedings, 1355, 2011, mrss11-1355-jj07-02 Niagara Muhammad Idris, Muthu Kumara Gnanasammandhan, Ratha Mahendran, Ho-Chi Lui Paul, Yong Zhang, In vivo photodynamic therapy using upconversion nanoparticles as remote controlled nano-transducers, Nature Medicine, 2012, Published Online Muthu Kumara Gnanasammandhan, Niagara Muhammad Idris, Remote activation of biomolecules in deep tissues using NIR-to-UV upconversion nanotransducers, PNAS, 2012, vol 109, issue 22, pp 8483-8488 Peiyan Yuan , Yih Hong Lee , Muthu Kumara Gnanasammandhan , Zhenping Guan , Yong Zhang and Qing-Hua Xu, Plasmon enhanced upconversion luminescence of NaYF4:Yb,Er@SiO2@Ag core–shell nanocomposites for cell imaging, Nanoscale, 2012,4, 5132-5137 III Book Chapter Muthu Kumara Gnanasammandhan, Yong Zhang, Rare earth nanomaterials in fluorescence microscopy, Pan Stanford Publishing, May 14, 2012 ISBN 9789814316309 Conference Presentations Muthu Kumara Gnanasammandhan, Yong Zhang, Simultaneous gene delivery and tracking of cells using upconversion nanoparticles for cell therapy, Tohoku-NUS 3rd East Asian Pacific Student Workshop in Nanobiomedical Engineering, Dec 21-22, 2009, Singapore, Poster Presentation Nagarajan Sounderya , Li Zhengquan , Roullier Victor , Amela Cortes Marian , Muthu Kumara Gnanasammandhan , Grasset Fabien , M-Artzner Valerie and Zhang Yong, “Surface Modification Routes Towards Water Soluble Upconversion Nanoparticles”, Materials Research Society (MRS) 2010 Spring Meeting , Apr 5-9, 2010, San Francisco, USA, Poster Presentation Muthu Kumara Gnanasammandhan, Zhuang Yuhang, Yong Zhang, Light-controlled gene knockdown using fluorescent upconversion nanoparticles for cancer therapy, 2nd NanoToday Conference, December 2011, Hawaii, USA, Oral Presentation Recipient of student travel award for the same Muthu Kumara Gnanasammandhan, Yong Zhang, A Study of the Toxicity of Lanthanide Doped Nanocrystals with Upconversion Fluorescence, International Conference on Materials for Advanced Technologies (ICMAT), Jun 2011, Singapore, Oral Presentation Akshaya Bansal, Muthu Kumara Gnanasammandhan, Yong Zhang, Targeted and Enhanced RNAi using Rare Earth Doped NaYF4 Nanoparticles, The Biomedical Engineering Society 6th Scientific Meeting (BES6SM) , 19th May 2012, Singapore, Oral Presentation IV Acknowledgements I would like to sincerely thank everyone who has contributed to my thesis I would first like to thank Dr Yong Zhang for accepting to be my mentor and his constant guidance throughout the project I’m also grateful for his efforts in shaping me into a wholesome researcher I am also indebted to National University of Singapore and Department of Bioengineering for accepting me into the program and helping me with funding I cannot thank Niagara enough for all her help in acclimatizing me to lab and work culture in the initial stages and her constructive criticisms and guidance throughout the project, especially in interpretations of some tricky data I would like to thank Sounderya, Qingqing, Shashi and Zeming for their part of the effort in my publications I greatly appreciate all the timely help I have received from my lab colleagues and friends from other labs who helped me with reagents and guidance for some experiments I am immensely thankful to my fiancé, Sangeetha for constantly motivating me and being there with me during my ups and downs and also for the insightful discussion and brainstorming sessions during the course of my work I am also grateful to my brother, Selvarajan for his several hours of work in helping me with schematics and animations for publications, presentations and Thesis I’m indebted to my parents for life for all the support, love and guidance they have bestowed onto me and for contributing to what I am today V Table of Contents Declaration I Acknowledgements IV Table of Contents V Summary VII List of Tables IX List of Figures X List of Abbreviations XIV Chapter Introduction Chapter Literature survey 2.1 Nanoparticles for gene delivery and tracking 2.1.1 Polymeric nanoparticles 2.1.2 Metallic Nanoparticles 2.1.3 Quantum dots 10 2.1.4 Rare earth nanoparticles 12 2.2 Control of gene expression 20 2.2.1 Photoactivable gene expression/silencing 21 2.2.2 Photoactivation using nanoparticles 22 2.3 UCNs for gene delivery, tracking and photoactivation 23 2.4 Objectives 24 Chapter Synthesis of UCNs and silica coating 25 3.1 Introduction 25 3.2 Experimental Section 26 3.2.1 Synthesis of UCN core 26 3.2.2 Silica Coating of UCN 27 3.2.3 Mesoporous silica coating 27 3.2.4 Fluorescence spectra measurement 27 3.2.5 Transmission electron Microscopy 28 3.2.6 Hydrodynamic radius and zeta potential 28 3.2.7 Determination of surface area and pore size for mesoporous silica coated UCNs 28 3.2.8 LDH assay 30 3.2.9 MTS assay 30 3.2.10 TUNEL assay 30 3.3 Results & Discussion 31 3.4 Conclusions 40 Chapter Simultaneous gene delivery and tracking of cells using fluorescent upconversion nanoparticles 42 4.1 Introduction 42 4.2 Experimental Section 43 VI 4.2.1 Plasmid Isolation 43 4.2.2 Agarose Gel Electrophoresis 43 4.2.3 Concentration of DNA 44 4.2.4 Nanoparticle-Gene Loading 44 4.2.5 Checking of Gene Conjugation 44 4.2.6 DNA release studies 45 4.2.7 Restriction Digestion Analysis 45 4.2.8 DNAse protection assay 45 4.2.9 Culturing of Myoblasts 46 4.2.10 Nanoparticle Uptake by Cells 46 4.2.11 In-vitro gene delivery 46 4.2.12 In-vivo nanoparticle tracking 47 4.2.13 Hemolytic activity test 47 4.2.14 In-vivo gene delivery 48 4.3 Results & Discussion 48 4.4 Conclusions 58 Chapter Specific Photoactivation of nucleic acids using NIR-to-UV UCNs 59 5.1 Introduction 59 5.2 Experimental Section 60 5.2.1 Phototoxicity studies 60 5.2.2 DNA/siRNA caging with DMNPE 60 5.2.3 Photoactivation of caged siRNA in solution 61 5.2.4 Photoactivation of caged siRNA in cells 61 5.2.5 Photoactivation of caged plasmid DNA in cells 62 5.2.6 Patterned activation of caged plasmids 62 5.2.7 In vivo studies 63 5.2.8 Deep tissue activation of caged plasmid DNA in cells with tissue phantoms 63 5.2.9 In-vivo activation of cells in an implanted device 63 5.3 Results & Discussion 64 5.4 Conclusion 77 Chapter Conclusions and Future directions 78 6.1 Conclusions 78 6.2 Future Directions 79 References 82 VII Summary There has been a tremendous growth in the field of gene therapy and it is being accepted as a conventional mode of treatment for a variety of genetic disorders But there are still a few hurdles to be crossed before it can be used to its full potential There are few major problems which are hindering the progress of gene therapy from the bench to bedside like the safe delivery of genes, tracking the gene delivery process and controlled expression of the delivered genes Delivery of genes remains problematic because nucleic acids are very sensitive and cannot withstand the harsh biological environment and hence need efficient vectors to carry them to the site of interest Also after delivery the expression of them needs to be controlled because over/under expression might cause various complications leading to inefficient therapy and can even cause cancer Finally, tracking the gene delivery process is also troublesome due to the unavailability of efficient fluorescent labels which can be used in deep tissues for extended periods of time In this thesis, fluorescent upconversion nanoparticles (UCNs) with unique optical properties are used to provide a variety of solutions for the problems mentioned above UCNs are unique fluorescent nanoparticles which absorb NIR light and emit in the UV, Visible and NIR region and have various advantageous properties like low cytotoxicity and high photostability The use of NIR light source to excite/activate these nanoparticles offers additional advantages like low tissue autofluorescence and almost nil phototoxicity These nanoparticles have been developed for delivery of genes, tracking them fluorescently and also to specifically activate the genes at the site of interest using their fluorescence Upconversion nanoparticles exhibiting strong fluorescence were synthesized with a mesoporous silica coating for efficient payload delivery They were also highly VIII biocompatible DNA was loaded onto these nanoparticles and transfected into cells This enabled the gene delivery process to be tracked and also allowed for the tracking of these cells when they were injected into animal models, making simultaneous gene delivery and tracking of cells for cell therapies, possible In addition, DNA/siRNA was photocaged to 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