Date of Award
Chemistry and Biochemistry
David Sabatino, Ph.D.
Wyatt Murphy, Ph.D.
Fr. Gerald J Buonopane, Ph.D.
siRNA, Glucose Regulated Proteins (GRPs), Prostate Cancer, siRNA nanostructures, siRNA bioconjugates (Fatty acids, Gold Nanoparticles, Florescent Probes).
The emerging field of RNA nanotechnology has led to rapid advances in the applications of RNA in chemical biology, medicinal chemistry, and biotechnology. At the forefront of its utility is the ability to self-assemble multiple siRNAs into nanostructure formulations capable of targeting selected oncogenes and potentiating the gene therapy of malignant tumors. Self-assembled siRNA integrates multiple siRNAs within a single molecular platform for silencing multiple oncogenic mRNA targets with high precision and efficacy to potentially induce cancer cell apoptosis through the RNA interference (RNAi) pathway. Furthermore, the conjugation of siRNA self-assemblies with bio-active probes results in multi-functional theranostic (therapy+diagnostic) agents capable of enhancing cancer cell detection and treatment. This includes the introduction of lipids for cell uptake, fluorophores for cell-based detection, and metallic nanoparticle formulations for optimizing siRNA biophysical and biological properties. The latter is a central focus of my thesis research objectives, aimed towards the development of self-assembled siRNA bioconjugates as effective anti-cancer agents.
Glucose-regulated proteins (GRPs) are a class of chaperone proteins of the endoplasmic reticulum that serve as key sensors for misfolded proteins and trigger the unfolded protein response (UPR) under physiological and pathological stress conditions. Moreover, GRPs have been classified as clinically relevant biological markers in cancer detection and treatment and found to be over-expressed and cell surface localized in a wide range of cancer types. In cancer, the GRPs regulate cancer initiation, proliferation, adhesion, and invasion which contributes to metastatic spread and treatment resistance. Over the last decade, our research program has focused on the development of various high-order, self-assembled, multi-functional siRNAs capable of silencing the oncogenic GRPs, and reversing their protective UPR effects related to proliferative, pro-survival, and anti-apoptotic pathways in selected tumors. Chemical tools have been implemented to improve the poor pharmacological properties of siRNAs to maximize their full therapeutic efficacy against tumorigenic disease conditions, which are highlighted in Chapter 1 of this thesis.
Chapter 2 highlights the development of synthetic strategies to improve siRNA metabolic stability, cellular uptake, and delivery, which are still in widespread demand for effective siRNA therapeutic applications. More specifically, this research aims to incorporate a small set of fatty acids into chemically derived siRNAs to improve their metabolic stability and cancer cell permeability. To achieve this goal, we have generated the higher-order siRNAs based on self-assembled branching structures. The self-assembled branch siRNAs were generated from their corresponding RNA template strands, which incorporated a synthetic ribouridine branchpoint synthon. This monomer was developed by an efficient solution-phase synthesis route and can be effectively incorporated within linear, V, and Y-shaped RNA templates by automated solid-phase RNA synthesis. Furthermore, an optimized coupling procedure has been developed for tagging variable saturated and unsaturated fatty acids onto linear, V- and Y-shape RNA templates. The RNA-fatty acid bioconjugates were analyzed, purified, and characterized by LC/MS. They were then hybridized with complementary RNA single strands to afford the amphiphilic self-assembled branch siRNA bioconjugates. The amphiphilic siRNA bioconjugates were detected by native PAGE, CD spectroscopy, and RP IP HPLC, while characterization of their self-assembled nanostructures was determined by DLS and TEM. Furthermore, the self-transfection capabilities of the siRNA-fatty acid bioconjugates and their biological activities within a model prostate cancer (PC-3) cell line revealed partial cell uptake, which contributed to modest RNAi activity when compared to the siRNA controls transfected with a commercially available transfection reagent. Nonetheless, this reported solid-phase RNA bioconjugation approach provides an important entry point for the incorporation of various hydrophobic and amphiphilic functional groups. This strategy may enable further development of new generation RNAi molecules for screening important oncogene targets and for improving cancer gene therapy applications.
Chapter 3 describes the synthesis, characterization, and biological evaluation of a new class of bifunctional gold (Au)-RNA nanoparticles to improve cellular uptake and theranostic utility of the multi-functional siRNA nanostructures in prostate cancer cells. In this study, we have developed a simple, bottom-up approach using alkylamino modified RNAs to produce stable and small Au-RNA nanoparticle formulations bearing either a fluorescent reporter (fluorescein) or a fatty acid group (palmitamide) to track cell uptake in PC-3 prostate cancer cells. The resulting Au-functionalized RNA particles were found to be stable under reducing conditions according to UV-Vis spectroscopy. Sample characterization by DLS and TEM confirmed self-assembly into primarily small (~10-40 nm) spherical shaped nanoparticles anticipated to be applicable to cell biology. The application of Au-functionalized siRNA particles in prostate cancer (PC-3) cells resulted in the knockdown of GRP75, which led to detectable levels of cell death in the absence of a transfection vector. Consequently, this novel Au-RNA theranostic formulation may prove to be a valuable bifunctional probe in the early detection and treatment of prostate cancer and related solid tumors.
Chapter 4 of this thesis summarizes our on-going and future work aimed at incorporating cell-targeting ligands within the Au-siRNA nanoparticle formulations for specific cancer treatment in cell cultures and within tumor-bearing mice models. The study aims to modify the Au-siRNA nanoparticle formulation with cell-targeting peptides (CTPs), which functions to target and bind to a cell surface receptor, in this case, PSMA, found on the surface of select prostate cancer cells. A PSMA-targeting peptide sequence has been selected for targeted delivery of the Au-siRNA formulation directly within PSMA+ prostate cancer cells for the application of cancer-targeting gene therapy. Taken together, this thesis will serve to highlight the synthetic strategies towards the development of higher-order self-assembled siRNA bioconjugates with self-transfection capabilities for silencing multiple oncogenic GRPs in cancer.
Shah, Sunil S., "Theranostic Applications of siRNA Bioconjugates in Cancer Detection and Treatment" (2021). Seton Hall University Dissertations and Theses (ETDs). 2851.
Amino Acids, Peptides, and Proteins Commons, Biostatistics Commons, Lipids Commons, Medicinal-Pharmaceutical Chemistry Commons, Nanomedicine Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons