Use of Dna Aptamers for Targeted Delivery of Chemotherapeutic Agents
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USE OF DNA APTAMERS FOR TARGETED DELIVERY OF CHEMOTHERAPEUTIC AGENTS BY CHRISTOPHER H. STUART A Dissertation to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Molecular Medicine and Translational Science May, 2016 Winston-Salem, North Carolina Approved by: William H. Gmeiner Ph.D., Advisor George J. Christ Ph.D., Chair Akiva Mintz, MD., Ph.D. Timothy D. Pardee, MD., Ph.D. Michael C. Seeds, Ph.D. Acknowledgements I would like to graciously acknowledge the guidance provided by my advisor, Dr. William Gmeiner. He has given me the opportunity to grow into a successful young scientist, the freedom to learn independently and mentorship when I was lost. I will be forever grateful for his guidance. Of course, no Ph.D is earned alone. There have been so many friends that have helped me along the way, and to try to list them all would require a second dissertation in itself. Leah, Hugo, Daniel and Kadie, thank you all for your friendship and good times we shared over the years. I would especially like to thank Ryne and Joanna. They have been my most loyal and sincere friends and the appreciation I have for their support, though the best and worst of times, cannot be understated. Finally, I would like to thank my family. My brothers, Joey and Jacob, and my oldest friend Matt have listened to me even when they have no idea what I am rambling on about, and have given me the encouragement I needed, not only in pursuit of this dissertation but in life as well. My loving parents, Bob and DeeAnn, instilled a curiosity and conviction in me from a young age. They are the reason I am the man I am today. Every success I have had is because of their dedication as parents, confidants, counselors and friends. ii Table of Contents List of Figures……………………………………………………………………………vii List of Tables……………………………………………………………………………...x List of Abbreviations……………………………………………………………………..xi Abstract………………………………………………………………………………….xiv Chapter I General Introduction I.1 Introduction of Chemotherapeutic Concepts: Balancing the Cure and its Cost….........1 I.2 Increasing Drug Efficacy through Targeted Therapy……………...…………………..9 I.3 DNA as a Structure: The use of DNA for Drug Targeting……………………….….17 Chapter II Site-Specific DNA–Doxorubicin Conjugates Display Enhanced Cytotoxicity to Breast Cancer Cells II.1 Abstract…………………………………………………………..….……………...39 II.2 Introduction……………………………………………………..…….…………….40 II.3 Experimental Section………………………………………………………………..47 iii II.4 Discussion…………………………………………………………………………...53 II.5 Conclusions………………………………………………………………………….56 II.6 Supporting Information……………………………………………………………...57 II.7 Acknowledgments…………………………………………………………………..58 Chapter III PSMA-Targeted Liposomes Specifically Deliver the Zn2+ Chelator TPEN Inducing Oxidative Stress in Prostate Cancer Cells III.1 Abstract…………………………………………………………………………….65 III.2 Background…………………………………………………………………….......66 III.3 Methods……………………………………………………………………………68 III.4 Results……………………………………………………………………………...75 III.5 Discussion………………………………………………………………………….82 III.6 Future Directions…………………………………………………………………..98 Chapter IV Selection of a New Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing IV.1 Introduction……………………………………………………………………….107 IV.2 Methods…………………………………………………………………………...110 iv IV.3 Results…………………………………………………………………………….116 IV.4 Discussion………………………………………………………………………...120 Chapter V Discussion and Future Directions V.1 Aptamers as Therapeutics………………………...………………………………..139 V.2 Aptamer Complexes and Multifunctional Aptamers………………………………141 V.3 Improving Aptamer Selection……………………………………………………...146 V.4 Conclusions…………………………………………...…………….……………...148 Appendix Dimeric DNA Aptamer Complexes for High-capacity–targeted Drug Delivery Using pH-sensitive Covalent Linkages A1.1 Abstract…………………………………………………………………………...159 A1.2 Introduction……………………………………………………………………….160 A1.3 Results…………………………………………………………………………….162 A1.4 Discussion………………………………………………………………………...167 A1.5 Materials and Methods……………………………………………………………176 A1.6 Supplementary Material…………………………………………………………..182 A1.7 Acknowledgment…………………………………….…………………………...186 v Curriculum Vitae……………………………………………………………………...190 vi LIST OF FIGURES Chapter I Figure 1: Structure of Doxorubicin………………………………………………………5 Figure 2: Trastuzumab FAB fragment binding to HER2…………………...…………………..12 Chapter II Figure 1: Graphical Abstract…………………………………………………………….40 Figure 2: Reaction of Doxorucin with DNA……………………………………………43 Figure 3: Physical analysis of Dox:DNA hairpins…………….……….……………….44 Figure 4: 3D representation of Dox:DNA hairpin………………………………………45 Figure 5: Fluorescence quenching of Dox in DNA hairpins……………………..……..46 Figure 6: Fluorescence microscopy of cells after DCH treatment……………….….….46 Figure 7: Viability of Cells treated with DCH…………………………………………..47 Figure 8: 2D NMR of Dox:DNA Hairpin molecules...……..…………………………..57 Figure 9: Dox Absorbance after extractions…………...………………………………..57 Figure 10: Internalization of DCH molecules into cells………………………………..58 Chapter III Figure 1: TPEN induces significant oxidative stress in PCa cells …………….……….88 vii Figure 2: Time-dependence of Cu2+ and Zn2+ rescue ……………..……………………89 Figure 3: Characterization of aptamer targeted liposomes…………….…….………….90 Figure 4: Targeted cell binding and delivery of aptamer-targeted liposomes …….……90 Figure 5: Selective cytotoxicity of aptamer-targeted liposomes to C4-2 cells ….……...91 Figure 6: TPEN delivered via aptamer-targeted liposomes induces cell death ………...92 Figure 7: Liposomal loading results in reduced hepatotoxicity ………………….....…..93 Figure 8: Aptamer-targeted liposomes specifically kill targeted tumors …………..….. 94 Figure 9: Mitochondria size quantification……………………………………………...95 Figure 10: Leakage of TPEN from liposomes…………………………………………..96 Figure 11: Scheme of Liposome formation……………………………………………..97 Figure 12: Size distribution of liposomes after dialysis………………………………...97 Chapter IV Figure 1: Selection of VBA-01 via CENGS………………….…. …………………...128 Figure 2: Structure of DVBA-01…………………... …….………..…………………129 Figure 3: Physical characteristics of VBA-01 and its derivatives ….………………...129 Figure 4: Effects of DVBA-01 on BC cells……………………….. ………….………130 Figure 5: Flow cytometry of cells plated on VN and treated with DVBA-01-Dox …..131 Figure 6: Histology of DCIS tissue using VBA-01…………………………… …...…132 viii Figure 7: Sequences of the various aptamers identified via NGS …..………………...132 Figure 8: Phylogenetic tree of aptamer sequences…………….. …………………….. 133 Appendix Figure 1: Secondary structure of the dimeric aptamer complex ……………………...171 Figure 2: Physical characterization of DAC and DAC-D ………………..…...…….…172 Figure 3: Fluorescence of cells treated with DAC……… ….……….………………...173 Figure 4: Endosomal uptake of DAC…….……………………….. ………….………174 Figure 5: Specificity of DAC complexes for PSMA+ cells …………………….……..175 Figure 6: Cytotoxicity of DAC-D on PCa Cells………………………………. …...…176 Figure 7: The sequence of the SZTI01 aptamer …..………………………………......182 Figure 8: Physical Characterization of DAC complexes….. …………….......………. 182 Figure 9: Standard Curve of Dox absorbance………………………………………….183 Figure 10: Flow cytometry of cells treated with DAC………………………………..183 Figure 11: Cytotoxicity on cells treated with DAC for 24hr………………………….184 Figure 12: Relative binding of SZTI01 versus the A10 aptamer………………………185 ix LIST OF TABLES Appendix Table 1: Absorbance values for DAC-D complexes………………………………….185 x LIST OF ABREVIATIONS ATM Ataxia telangiectasia Mutated ATP Adenosine Triphosphate BC Breast Cancer CE Capillary Electrophoresis CHK2 Checkpoint kinase 2 CML Chronic Myelogenous Leukemia Col Collagen CRPC Castration Resistant Prostate Cancer DAC Dimeric Aptamer Complex DCH Doxorubicin Conjugated Hairpin DCIS Ductal Carcinoma In Situ DLS Dynamic Light Scattering DNA Deoxyribonucleic Acid DOX Doxorubicin dsDNA Double Stranded Deoxyribonucleic Acid ECM Extra Cellular Matrix xi EGFR Epidermal growth factor receptor NGS Next Generation Sequencing ER Estrogen Receptor FA Folic Acid FBS Fetal Bovine Serum FN Fibronectin HER2 Human Epidermal Growth Factor Receptor 2 MALA Multiple Aptamers with Low Affinity mRNA Messenger Ribonucleic Acid MUC1 Mucin 1, Cell Surface Associated NHS N-Hydroxysuccinimide NMR Nuclear Magnetic Resonance NTA Nanotracking Analysis PBS Phosphate Buffered Saline PCa Prostate Cancer PCR Polymerase Chain Reaction PSMA Prostate Specific Membrane Antigen RNA Ribonucleic Acid xii ROS Reactive Oxygen Species SELEX Systematic Evolution of Ligands by Exponential Enrichment siRNA Small Interfering Ribonucleic Acid SOD1 Superoxide Dismutase 1 ssDNA Single Stranded Deoxyribonucleic Acid TEM Transmission Electron Microscopy TOPI Topoisomerase I TOPII Topoisomerase II TPEN N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine TS Thymidylate Synthase uPAR Urokinase Receptor VN Vitronectin xiii Abstract The development of safe and efficacious chemotherapeutics has been the goal of cancer researchers for decades. Many different methods have been used to pursue this goal ranging from small molecules that act targets to large biomolecules such as antibodies. Aptamers are a new class of biomolecules similar to antibodies in that they fold into 3D shapes which specifically bind to a target of interest, but differ in that they are composed of single stranded RNA or DNA. Aptamers are selected for using the SELEX method and can be designed to bind nearly any target the researcher desires.