Use of upconversion fluorescent nanoparticles for simultaneous imaging, detection and delivery of sirna

USE OF UPCONVERSION FLUORESCENT NANOPARTICLES FOR SIMULTANEOUS IMAGING, DETECTION AND DELIVERY OF SIRNA JIANG SHAN (B.Sc., Harbin Institute of Technology) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISION OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 PREFACE This thesis is hereby submitted for the degree of Doctor of Philosophy in the Division of Bioengineering at the Faculty of Engineering, National University of Singapore. This thesis, either in part or whole, has never been submitted for any other degree or equivalent to another university or institution. This thesis contains all original work, unless specifically mentioned and referenced to other works. Parts of this thesis had been published or presented in the following: Peer Reviewed Journal Publications: 1. Shan Jiang, Yong Zhang, Kian Meng Lim, Eugene K W Sim and Lei Ye. NIR-to-visible upconversion nanoparticles for fluorescent labeling and targeted delivery of siRNA. 2009. Nanotechnology 20(15):9. 2. Shan Jiang, Muthu Kumara Gnanasammandhan, Yong Zhang. Optical Imaging Guided Cancer Therapy with Fluorescent Nanoparticles. 2010. Journal of the Royal Society Interface 7(42): 3-18. (Review paper) 3. Shan Jiang, Yong Zhang. Upconversion nanoparticle based FRET system for study of siRNA in live cells. 2010. Langmuir. In press ii 4. Wee Beng Tan, Shan Jiang, Yong Zhang. Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference. 2007. Biomaterials 28: 1565–1571. 5. Zhengquan Li, Yong Zhang, Shan Jiang. Multicolor Core/Shell-Structured Upconversion fluorescent Nanoparticles. 2008. Advanced materials 20: 4765 – 4769. International Conferences Presentations: 1. Shan Jiang, Yong Zhang, Kian Meng Lim. Fluorescence resonance Energy transfer (FRET) of oppositely charged upconversion nanoparticles and quantum dots. 4th World Congress on Bioengineering (WACBE), 26-29 Jul, 2009, Hong Kong Polytechnic University, Hong Kong, China. Poster Presentation 2. Shan Jiang, Yong Zhang. IR-to-visible Upconversion Nanoparticles for in Vitro Fluorescent Imaging. 4th Kuala Lumpur International Conference on Biomedical Engineering, 25-28 June 2008, Malaysia, Kuala Lumpur. IFMBE Proceedings 21, 330-332. Oral Presentation. 3. Shan Jiang, Yong Zhang. Use of IR-to-Visible Upconversion Fluorescent Nanoparticles for Tracking of siRNA Delivery. The Sixth IASTED International Conference on Biomedical Engineering, 13-15 Feb 2008, Innsbruck, Austria. iii Proceeding 368-371. Oral Presentation. 4. Shan Jiang, Yong Zhang. Effective Delivery of Small Interference RNA to Cancer Cells by Using Up-converting Nanoparticles. The 3rd WACBE World Congress on Bioengineering, 9-11 July 2007, Bangkok, Thailand. Proceeding. Oral Presentation. 5. Shan Jiang, Wee Beng Tan, Yong Zhang. Imaging Assisted siRNA Delivery Using Multifunctional Nanoparticles. Materials Processing for Properties and Performance. 11-15 Dec 2006, Singapore. Proceeding 46-48. Oral Presentation. 6. Shan Jiang, Wee Beng Tan and Yong Zhang. Multifunctional Nanoparticles-mediated siRNA Delivery for Breast Cancer Therapy. The 2nd Tohoku-NUS Joint Symposium on the Future Nano-medicine and Bioengineering in the East Asian Region, 4-5 Dec 2006, National University of Singapore, Singapore. Proceeding 15-16. Oral Presentation. iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to each and everyone who has contributed towards the completion of my thesis. First and foremost, I would like to acknowledge the contributions of my supervisor A/P Zhang Yong for his constant encouragement, support and patience throughout the entire course of work. Especially, he offered me immense guidance and advice on the research design and article writing. I am also grateful to my co-supervisors, A/P Lim Kian Meng and A/P Eugene KW Sim for their support and assistance. I am thankful to my colleagues in Cellular and Molecular Bioenigneering lab for their help. Mr. Tan Wee Beng taught me the basic research skills and how to research when I just begin my project. Dr. Li Zhengquan and Dr. Qian Haisheng supplied the nanoparticles and discussed some chemistry questions with me. Dr. Dev Kumar Chatterjee, Miss Muhammad Idris Niagara, Mr. Muthu Kumara Gnanasammandhan and Mr. Shashi Ranjan helped me revise my writing. Dr. Guo Huichen discussed some biological questions with me. I would also like to extend my thanks to the undergraduates including Mr. Ng Weiguang, Ms Sandra Ho Pei Rong and Ms Ho Lay Hoon who have put in a long time and challenged me with their questions. Finally, I express my deep thanks to my parents, Mr. Jiang Yongcheng and Ms. Sun Hongyun, for their constant love and support to help me through the toughest time. A v special acknowledgment goes to my lover, Mr. Dong Hongliang who brought me many happiness and joy. Jiang Shan September, 2009 vi TABLE OF CONTENTS PREFACE ii ACKNOWLEDGEMENTS v TABLE OF CONTENTS vii SUMMARY x LIST OF TABLES .xi LIST OF FIGURES xii ABBREVIATIONS . xvii CHAPTER LITERATURE REVIEW 1.1 Fluorescent nanoparticles . 1.1.1 Organic dye doped nanoparticles . 1.1.2 Quantum dots . 1.1.3 Upconversion nanoparticles . 1.2 Molecular cancer diagnosis 16 1.2.1 In vitro imaging of cancer 16 1.2.2 In vivo detection of cancer . 19 1.3 Multifunctional nanoparticles 27 1.3.1 Integration of imaging and therapy 28 1.3.2 siRNA imaging and delivery 32 1.3.3 FRET based biosensing 36 1.4 Thesis overview . 41 CHAPTER CHITOSAN/QDS NANOPARTICLES FOR SIRNA DELIVERY46 2.1 Introduction 47 2.2 Materials and Methods . 49 2.2.1 Materials 49 2.2.2 Cell Culture 49 2.2.3 Targeted siRNA conjugated Chitosan/QDs nanoparticles . 50 2.2.4 Determination of conjugation efficiency and release profile of siRNA from chitosan/QDs NPs 51 2.2.5 Cell viability . 52 2.2.6 Flow cytometry analysis 53 2.2.7 Imaging 53 2.2.8 siRNA-mediated luciferase gene silencing 54 vii 2.3 Results and Discussion 55 2.3.1 Properties of targeted siRNA-conjugated chitosan/QDs nanoparticles 55 2.3.2 Ligand mediated cellular uptake 59 2.3.3 siRNA-mediated inhibition of gene expression . 62 2.4 Conclusion . 63 CHAPTER PROPERTIES OF UPCONVERSION NANOPARTICLES .64 3.1 Introduction 65 3.2 Materials and Methods . 68 3.2.1 Synthesis of silica coated NaYF4 nanoparticles . 68 3.2.2 Physical characterization of UCNs 69 3.2.3 Optical characterization of UCNs 69 3.2.4 Cell viability . 69 3.2.5 Imaging 70 3.3 Results and Discussion 71 3.3.1 Physical properties of UCNs 71 3.3.2 Optical properties of UCNs . 72 3.3.3 Cytotoxicity of UCNs 75 3.3.4 Cellular uptake of UCNs 76 3.4 Conclusion . 78 CHAPTER UPCONVERSION NANOPARTICLES FOR FLUORESCENT IMAGING .80 4.1 Introduction 81 4.2 Materials and Methods . 83 4.2.1 Materials 83 4.2.2 Amino/Carboxyl group modification of UCNs 83 4.2.3 Anti-HER2 antibody/Folic acid/Streptavidin conjugation to UCNs 84 4.2.4 Imaging 86 4.3 Results and Discussion 87 4.3.1 Detection of HER2 receptors with UCNs 87 4.3.2 Detection of folate receptor with UCNs 90 4.3.3 Detection of Actin Filaments of 3T3 cells . 93 4.4 Conclusion . 96 CHAPTER UPCONVERSION NANOPARTICLES FOR DETECTION OF SIRNA .98 5.1 Introduction 99 5.2 Materials and Methods . 102 5.2.1 Materials 102 5.2.2 Complexing of BOBO-3 stained siRNA with UCNs (UCN/siRNA-BOBO3) . 102 viii 5.2.3 Release and biostability of siRNA attached on UCNs . 103 5.2.4 Intracellular release of siRNA . 104 5.2.5 Imaging 104 5.3 Results and discussion . 105 5.3.1 Synthesis of UCN/siRNA-BOBO3 complex . 105 5.3.2 Characterization of UCN/siRNA-BOBO3 complex 109 5.3.3 Release of siRNA from UCNs 111 5.3.4 Biostability of siRNA attached on UCNs .113 5.3.5 Intracellular release of siRNA 116 5.4 Conclusion 118 CHAPTER UPCONVERSION NANOPARTICLES FOR TARGETED SIRNA DELIVERY .120 6.1 Introduction 121 6.2 Materials and Methods . 124 6.2.1 Materials 124 6.2.2 Anti-HER2 antibody conjugated UCNs with attachment of siRNA 124 6.2.3 Imaging 125 6.2.4 Inductively coupled plasma (ICP) analysis 126 6.2.5 Luciferase assay . 126 6.3 Results and Discussion 127 6.3.1 Anti-HER2 antibody conjugated UCNs with attachment of siRNA 127 6.3.2 Ligand mediated cellular uptake 130 6.3.3 Long-term tracking of siRNA delivery 132 6.3.4 siRNA-mediated inhibition of gene expression . 136 6.4 Conclusion . 138 CHAPTER CONCLUSION AND FUTURE WORK 139 7.1 Conclusion . 140 7.2 Future work 142 REFERENCES .145 ix SUMMARY Recent advancements in the synthesis of fluorescent nanoparticles have made them a promising material for cancer detection. Furthermore, combining different modalities on one particle has sparked great research interest and efforts have been made to develop multifunctional nanoparticles. In this thesis, near-infrared (NIR)-to-visible upconversion fluorescent nanoparticles with unique properties such as low autofluorescence, high tissue penetration depth, low cytotoxicity, good photostability, and minimum photodamage to biological tissues are developed and used for imaging, detection and delivery of siRNA. Silica coated NaYF4 upconversion nanoparticles co-doped with lanthanide ions (Yb/Er) are synthesized with strong NIR-to-visible upconversion fluorescence. These nanoparticles were conjugated to ligands which can specifically bind to cell membrane receptors and cytoskeleton for a high sensitivity of detection with strong signal-to-background ratio for imaging of cells. In addition, the nanoparticles were also used for targeted delivery of siRNA into cells. 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Characterization of UCN /siRNA- BOBO3 complex (A) FRET efficiency in UCN /siRNA- BOBO3 complex at siRNA/ UCN ratios of 0, 31.25, 62.5, 125 and 250 (B) FRET efficiency in UCN /siRNA- BOBO3 complex at dye/bp ratios of 0, 0.025, 0.05, 0.1 and 0.2 111 Figure 5.5 Release of siRNA from nanoparticles (A) Photoluminescence spectra of free UCN nanoparticles, UCN /siRNA- BOBO3 complex and UCN /siRNA- BOBO3 complex with siRNA. .. realized In this chapter, we review the types and characteristics of fluorescent nanoparticles, in vitro and in vivo imaging of cancer using fluorescent nanoparticles, and 2 multifunctional nanoparticles for simultaneous tumor imaging and treatment 1.1 Fluorescent nanoparticles Optical imaging is the latest trend in imaging guided therapy which involves the detection of light photons transmitted through tissues... image of siRNA Lane1: DNA control; lane2: siRNA control; lane3: free siRNA in UCN /siRNA- BOBO3 complex solution; lane4: free siRNA in the solution of UCN /siRNA- BOBO3 added with NaOH 113 Figure 5.6 Biostability of siRNA attached on nanoparticles (A) Photoluminescence spectra of free UCNs, UCN /siRNA- BOBO3 complex solution, UCN /siRNA- BOBO3 complex digested with RNase A and UCN bound with digested siRNA- BOBO3... applications Upconversion nanoparticles have gained popularity in recent years and have been used for various biological applications summarized in Table 1.3 Upconversion nanoparticles, also known as up-converting phosphors (UCP), upconversion nanophosphors or upconversion nanocrystals, were initially used for the fluorescent detection of biological molecules in the buffer For example, Corstjens and his... of siRNA Lane 1: DNA ladder; lane 2: free siRNA control; lane 3: free siRNA digested with RNase A; lane 4: free siRNA in UCN /siRNA- BOBO3 complex solution; lane 5: siRNA relased from UCN /siRNA- BOBO3 complex; lane 6: siRNA relased from UCN /siRNA- BOBO3 complex digested with RNase A; lane 7: free siRNA in the solution of UCN bound with digested siRNA- BOBO3 115 Figure 5.7 Intracellular release of siRNA. .. feasibility, and meanwhile it would not cause photodamage to the tissues NIR quantum dots have many comparable properties of upconversion nanoparticles like high penetration depth, low autofluroescence and low photodamage, but they are comparatively costlier than upconversion nanoparticles and more toxic The upconversion fluorescence output of upconversion nanoparticles is also higher than that of quantum... green and red colors is shown by yellow arrows, and red color emitted from BOBO-3 is shown by red arrows The bar scale is 20 µm 118 Figure 6.1 Gel electrophoresis image of siRNA Lane 1: UCN-Ab -siRNA nanoparticles, lane 2: first supernatant (S1) of UCN-Ab -siRNA nanoparticles, lane 3: second supernatant (S2) of UCN-Ab -siRNA nanoparticles, lane 4: third supernatant (S3) of UCN-Ab -siRNA nanoparticles, ... 2h 132 Figure 6.5 Images of SK-BR-3 cells incubated with UCN-Ab -siRNA for 1, 3, 6, 12 and 24 h The confocal fluorescent image of UCNs (left) and superimposed xv image of UCNs and DAPi (for nucleus) are shown 135 Figure 6.6 Yttrium concentration as measured by ICP revealed the cellular uptake of UCN-Ab -siRNA nanoparticles in SK-BR-3 cells harvested 1, 3, 6, 12, 18, and 24h incubation ... efficiency of siRNA to chitosan/QDs NPs 57 Figure 2.4 Release profile of siRNA from chitosan/QDs NPs in PBS over a period of 6 days 58 Figure 2.5 Cell viability of HT-29 cells treated with chitosan/QDs nanoparticles and non-encapsulated QDs at different concentration of QDs 59 Figure 2.6 Specific uptake of chitosan/QDs nanoparticles by MCF-7 and SK-BR-3 cells Targeted NPs = siRNA- conjugated... monitor the progression of disease and therapy Conventional fluorophores such as fluorescent dyes, bioluminescent proteins, and fluorescent proteins were used initially But the recent advancements in the development of fluorescent nanoparticles have made them potential candidates for imaging guided therapy and they have a lot of advantages over their predecessors 1.1.1 Organic dye doped nanoparticles Recently . USE OF UPCONVERSION FLUORESCENT NANOPARTICLES FOR SIMULTANEOUS IMAGING, DETECTION AND DELIVERY OF SIRNA JIANG SHAN (B.Sc., Harbin Institute of Technology) . good photostability, and minimum photodamage to biological tissues are developed and used for imaging, detection and delivery of siRNA. Silica coated NaYF 4 upconversion nanoparticles co-doped. 4.3.2 Detection of folate receptor with UCNs 90 4.3.3 Detection of Actin Filaments of 3T3 cells 93 4.4 Conclusion 96 CHAPTER 5 UPCONVERSION NANOPARTICLES FOR DETECTION OF SIRNA 98 5.1 Introduction