Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

This dissertation focuses on the development of biocompatible oligonucleotide-based nanomaterials, known as spherical nucleic acids (SNAs), as therapeutic and diagnostic agents for intracellular gene regulation and in vivo cancer imaging by positron emission tomography (PET). SNAs consist of a nanoparticle core functionalized with a dense shell of oligonucleotides such as deoxyribonucleic acid (DNA). Detailed synthetic procedures and characterization of novel SNAs with purpose-built biocompatible core materials are described. The SNAs exhibit comparable chemical, physical and biological properties regardless of core composition. The functionalities of the SNAs were further tailored by deliberate design and chemical modification of their oligonucleotide sequences.

The SNA nanostructures studied in this dissertation include those with cores made from infinite coordination polymers (ICPs), self-assembled block copolymer micelles, 64Cu-alloyed gold nanoclusters, and commercially-available monodisperse gold nanoparticles. In vitro and in vivo investigations of each SNA type are described. The first two examples, functionalized on their surface with DNA, entered a variety of cell lines without the need for transfection agents, and inhibited the translation of disease-related messenger RNA (mRNA). The 64Cu-alloyed gold nanoclusters, functionalized on their surface with either DNA or phosphorodiamidate morpholino (PMO) oligonucleotides, were evaluated in vivo to determine their pharmacokinetics and biodistribution in mice. The blood circulation time of PMO-modified nanoclusters was far superior to DNA-modified nanoclusters, enabling their use in tumor pre-targeting studies wherein a non-radioactive nanocluster was first injected, allowed to accumulate at the tumor site, and then probed by injection of a complementary oligonucleotide labeled with 64Cu. Studies in mouse tumor models demonstrated the feasibility of in vivo hybridization-based pre-targeting using SNA nanomaterials. Furthermore, the basic chemical and biophysical properties of PMO-modified commercial gold nanoparticles were studied. It was determined that PMO-based SNAs display very similar cellular interactions as compared to their DNA analogues, including the capability of entering cells in a transfection-free manner and effecting gene knockdown. The neutral charge of the SNAs based upon PMO oligonucleotides exhibit more rapid capture kinetics of complementary oligonucleotides compared to DNA SNAs, as well as higher melting temperatures of hybridized duplexes. Taken together, the chemical versatility of SNAs has led to significant advances in understanding their basic properties and improving their viability for in vivo translation for biomedical applications.

Specifically, we uncovered further evidence for the central hypothesis that the unique properties of SNAs, specifically active cellular uptake and their sharp thermal denaturation curves, are core-independent and rely on the spherical orientation of DNA around a nanoparticle core. We also discovered that the cellular interactions of SNAs, mainly active uptake, are likewise independent of the charge of the nucleic acid shell. However, the chemical composition of the SNA-bound oligonucleotides strongly influences their in vivo pharmacokinetics, with neutral oligonucleotides conferring much longer blood circulation times compared to negatively-charged SNAs. We also found that the kinetics and thermodynamics of hybridization of neutral SNAs to complementary oligonucleotides are superior to those of negatively-charged SNAs, expanding their potential uses not only as biomedical agents but also as in vivo “click chemistry” tools capable of the bioorthogonal connection of multiple components under in vivo physiological conditions. Collectively, the studies presented herein demonstrate that the rational design of the core material and oligonucleotide shell impart novel properties to SNAs that expand their capabilities as therapeutic, diagnostic, and bioorthogonal assembly agents.

Language

English (en)

Chair and Committee

John-Stephen Taylor

Committee Members

Chad A. Mirkin, Liviu Mirica, Jonathan C. Barnes, Yongjian Liu,

Comments

Permanent URL: https://doi.org/10.7936/K7XP74CR

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