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(SNA) Constructs for Cancer Therapy and Imaging Colin Calabrese Washington University in St

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(SNA) Constructs for Cancer Therapy and Imaging Colin Calabrese Washington University in St Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Summer 8-15-2017 Synthesis, characterization, and biological activity of spherical nucleic acid (SNA) constructs for cancer therapy and imaging Colin Calabrese Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Chemistry Commons, and the Nanoscience and Nanotechnology Commons Recommended Citation Calabrese, Colin, "Synthesis, characterization, and biological activity of spherical nucleic acid (SNA) constructs for cancer therapy and imaging" (2017). Arts & Sciences Electronic Theses and Dissertations. 1236. https://openscholarship.wustl.edu/art_sci_etds/1236 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Department of Chemistry Dissertation Examination Committee: Prof. John-Stephen Taylor, Chair Prof. Jonathan Barnes Prof. Yongjian Liu Prof. Liviu Mirica Prof. Chad A. Mirkin Synthesis, Characterization, and Biological Activity of Spherical Nucleic Acid (SNA) Constructs for Cancer Therapy and Imaging by Colin Calabrese A dissertation presented to The Graduate School of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2017 St. Louis, Missouri © 2017, Colin Calabrese Table of Contents List of Figures…….…………….……………….……….……….……….……….……………..iii List of Schemes….……….……….……….……….……….……….……………….……..….....vi List of Tables.…….……….……….……….……….……….……….……….……...…….........vii Acknowledgements….……….……….……..….……….……….……….……….……...….....viii Dedication….……….……….……….……….………….….……….………………..………......x Abstract…………………………………………………………………………………………...xi Chapter 1 Introduction 1 Chapter 2 Development of a biocompatible Fe3+ coordination strategy for the 13 synthesis of nucleic acid complexes, nanoparticles, and metallogels Chapter 3 Biocompatible infinite-coordination-polymer nanoparticle-nucleic-acid 33 conjugates for antisense gene regulation Chapter 4 Biodegradable DNA-brush block copolymer spherical nucleic acids 56 enable transfection agent-free intracellular gene regulation Chapter 5 CCR5 pre-targeted nanosystem for breast cancer imaging and therapy 80 based on in vivo nucleic acid hybridization of spherical nucleic acid nanoclusters Chapter 6 Synthesis, characterization, and biological properties of neutral 105 phosphorodiamidate morpholino (PMO) spherical nucleic acids Chapter 7 Conclusions 125 Appendix 132 Bibliography 151 ii List of Figures Chapter 1 Figure 1.1 Chemical structures of nucleic acids. 1 Figure 1.2 Comparison of natural and synthetic assembly of functional 3D nucleic acid nanostructures. 2 Figure 1.3 Structures of selected synthetic nucleic acid analogues. 3 Chapter 2 Figure 2.1 Speciation of deferiprone-Fe3+ complexes. 22 Figure 2.2 Crystal structure of a Δ-fac-FeL3 complex 23 Figure 2.3 UV-Vis spectra of modified (AL) oligonucleotide with and without Fe3+ 25 Figure 2.4 UV-Vis spectra of unmodified (A) oligonucleotide with and without Fe3+ 25 Figure 2.5 Titration of a 100 µM solution of (AL) with ferric nitrate 26 Figure 2.6 UV-Vis spectra of (AL) titration with ferric nitrate 26 Figure 2.7 Gelation of duplex DNA in the presence of Fe3+ at room temperature 27 Figure 2.8 Temperature-dependent gelation of DNA metallogels 28 Figure 2.9 DLS size distribution of particles purified by GFC 29 Figure 2.10 AFM image of particles deposited on mica 29 Figure 2.11 Synthesis of dye-labelled particles and fractions obtained from GFC showing settling of heavy particles. 30 Figure 2.12 Uptake of dye-labelled particles in C166 cells 30 Figure 2.13 Survivin expression in skmel-28 melanoma cells, 30 nM total DNA, quantitated by RT-qPCR. 31 iii Chapter 3 Figure 3.1 Bare colloidal ICP-N3 nanoparticles stored overnight at varying concentrations of NaCl 48 Figure 3.2 Characterization of DNA-ICP particles 49 Figure 3.3 Agarose gel analysis of ICP degradation after 48 hours 51 Figure 3.4 UV-Vis analysis of DNA-ICP particles 51 Figure 3.5 Cellular uptake and gene knockdown of DNA-ICP particles 53 Chapter 4 Figure 4.1 Characterization of as-synthesized polycaprolactone-based micelle- SNAs. 69 Figure 4.2 Cellular uptake of micelle-SNAs 72 Figure 4.3 Confocal microscopy of fluorescein-labelled DBBC-SNA and immunofluorescence staining of organelle markers 74 Figure 4.4 Gene regulation by DBBC-based micelle-SNAs 75 Figure 4.5 The pH-dependent degradation of DBBC-based micelle-SNAs 76 Figure 4.6 Cellular toxicity of DBBC-based micelle-SNAs analyzed by a standard MTT assay 77 Chapter 5 Figure 5.1 Blood circulation of AuNC SNAs 1 hour post-injection in female C57 mice 91 Figure 5.2 Core size, hydrodynamic diameter, and zeta potential characterization of AuNC PMO SNAs. 92 Figure 5.3 Biodistribution of 64Cu-OMe-RNA radiotracer and PMO SNA nanocluster in female C57 mice 1 hour post-injection. 93 Figure 5.4 Binding kinetics measurements 94 Figure 5.5 Representative capture-and-release curves of PMO AuNC SNAs. 95 iv Figure 5.6 Uptake of PMO and DNA AuNCs with varying surface ligand coating in 4T1 breast cancer cells. 96 Figure 5.7 4T1 cell surface association of DNA and PMO AuNCs with varying surface ligand coating after NaCN treatment of cell pellet. 97 Figure 5.8 Representative PET images of tumor-bearing mice at different pretargeting intervals 99 Figure 5.9 Accumulation of 64Cu-OMe-RNA tracer in tumor tissue at different pretargeting intervals. 99 Figure 5.10 Tumor uptake of active vs. scrambled 64Cu-OMe-RNA tracer in 4T1 tumor mice. 100 Figure 5.11 Post-PET biodistribution quantification of 64Cu-OMe-RNA tumor uptake. 101 Figure 5.12 Representative images of tumor sections and their activity visualized by autoradiography. 102 Chapter 6 Figure 6.1 Hydrodynamic radius of PMO SNAs vs NaCl concentration. 113 Figure 6.2 TEM image of PEG5K/PMO SNAs stained with uranyl acetate. 113 Figure 6.3 1:1 PEG/PMO SNAs after centrifugation and resuspension in water 114 Figure 6.4 Binding kinetics measurements 115 Figure 6.5 Melting behavior of DNA and PMO SNAs bearing complementary or non-complementary sequences. 119 Figure 6.6 Melting behavior of single-stranded DNA-DNA and DNA-PMO pairs 120 Figure 6.7 Uptake of PMO SNAs in SKOV-3 ovarian cancer cells after 24 h compared to single-stranded PMO oligonucleotides. 122 Figure 6.8 Confocal microscope images of cellular uptake in KB cells. 122 Figure 6.9 Comparison of uptake of PEG5K/PMO and PEG1K/PMO SNAs. 123 Figure 6.10 Analysis of HER2 expression in SKOV-3 cells by Western Blot 124 v List of Schemes Chapter 1 Scheme 1.1 Functionalization of gold nanoparticles to afford spherical nucleic acids (SNAs). 4 Scheme 1.2 Displacement of nano-flares by target messenger RNA restores fluorescence of attached fluorophores. 7 Chapter 2 Scheme 2.1 Synthesis of ligand phosphoramidite. 23 Scheme 2.2 Synthesis of extened DNA-Fe3+ coordination networks by a two-step assembly process. 26 Chapter 3 Scheme 3.1 Synthesis and assembly of ICP particles and their cellular uptake 47 Chapter 4 Scheme 4.1 Schematic showing the synthesis of DNA grafted block copolymer-based micelle SNAs 58 Chapter 5 Scheme 5.1 Proposed pre-targeting nanosystem based on in vivo SNA hybridization. 90 vi List of Tables Chapter 2 Table 2.1 Stability constants for Fe3+/deferiprone 22 Table 2.2 Table of oligonucleotides 24 Table 2.3 Screening of conditions required for gelation of DNA/ Fe3+ solutions 27 Table 2.4 Temperature-dependent gelation of DNA/Fe3+ solutions 27 Chapter 3 Table 3.1 Table of oligonucleotides 50 Chapter 4 Table 4.1 Table of oligonucleotides 68 Chapter 5 Table 5.1 Table of oligonucleotides 89 Chapter 6 Table 6.1 Table of oligonucleotides 112 Table 6.2 Rate constants, dissociation constants, and surface coverage of SNAs discussed in Chapters 5 & 6. 116 vii Acknowledgments First, I would like to thank my advisor and mentor, Prof. Chad Mirkin, for his invaluable support and guidance during my graduate school career. I feel I have grown immensely as a chemist and critical thinker under his tutelage, and I consider myself extremely privileged to have worked with such an outstanding scientist. I also owe a deep debt of gratitude to Chancellor Mark Wrighton, Prof. William Buhro, and Prof. Richard Loomis for giving me the opportunity to continue my graduate work at Washington University in St. Louis. I am honored that you believed in me to take such a great leap into a new environment and explore new research challenges. I deeply appreciate the assistance from Rachel Dunn in helping me navigate the requirements of the Chemistry Department and Barbara Tessmer for assisting me in the dissertation defense process. I thank Prof. John-Stephen Taylor and Prof. Yongjian Liu for advising me during my tenure at Washington University in St. Louis. Both of you have proven extremely accessible and helpful any time I needed guidance or a critical set of eyes and ears on my work. I also thank Prof. Liviu Mirica and Prof. Jonathan Barnes for serving on my defense committee and taking the time to learn about my research and offer their insights and critical suggestions. I also want to acknowledge all the incredible colleagues and friends I have worked with throughout my graduate school career who helped me with various collaborations and contributed to the skill set I am so grateful to be equipped with today. I particularly acknowledge Dr. Alexander Scott and Dr. Todd Hovey for not only their material support but also their emotional support and friendship which I believe will be lifelong. I also acknowledge my friends, collaborators and co-authors Dr. Jonathan Choi, Dr. Resham Singh Banga, Nikunjkumar Savalia, Dr. William Briley, Dr.
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