Date of Completion

8-4-2020

Embargo Period

8-4-2022

Keywords

nucleic acids, nanoparticles, gene knockdown, targeted delivery

Major Advisor

Jessica Rouge

Associate Advisor

Challa Kumar

Associate Advisor

Ashis Basu

Field of Study

Chemistry

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

The first chapter of this work describes a novel, enzyme-responsive nanocapsule for the intracellular delivery of therapeutic nucleic acids and small molecule drugs. The stepwise assembly of this new construct, referred to as a nucleic acid nanocapsule (NAN), is presented. The cellular uptake properties of the NAN are investigated through confocal microscopy. Additionally, the functionalization of the NAN with a therapeutic nucleic acid is described. For these experiments, the NAN was functionalized with a DNAzyme, a catalytically active DNA molecule that can specifically bind and cleave a target mRNA sequence. The DNAzyme utilized in these studies is specific for the mRNA of GATA-3, a protein involved in the inflammatory pathway of asthma. An initial investigation into the activity of the DNAzyme on the NAN surface is described. The second chapter expands on the characterization of the DNAzyme-NAN, including an investigation into the stability of the chemically unmodified nucleic acid presented on the NAN surface and the effect of the surfactant on the endosomal escape of the DNAzyme. Additionally, the cellular uptake properties of the DNAzyme-NAN are investigated through confocal microscopy, and the catalytic activity of the DNAzyme on the NAN surface is investigated through a series of mRNA and protein knockdown studies. In the third chapter, in vitro studies of the DNAzyme-NANs are expanded to primary human immune cells, showing the versatility of the NAN construct. DNAzyme-NANs were also tested in in vivo studies in an associated allergic airway disease mouse model. Chapter four investigates the ability of the NAN to co-deliver a therapeutic oligonucleotide with a small molecule drug. The encapsulation of a novel gold(III) metallodrug in the hydrophobic core of the NAN, and the functionalization of the NAN surface with siRNA targeting Bcl-2, a gene involved in cellular apoptosis, are described. The enhanced cytotoxicity of the drug when co-delivered with the therapeutic nucleic acid using the NAN platform is presented. The last chapter describes the functionalization of NANs with an aptamer targeting Annexin A2, a cell-surface protein, for targeted delivery and cell-specific gene knockdown. In closing, future outlook and potential applications of the NAN platform are discussed.

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