Development of amino acid-modified drug delivery systems for RNA delivery in cancer therapeutics
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Abstract
The primary objective of my research is to develop polymer and lipid-based nanoparticles to effectively deliver therapeutic RNAs for life-threatening diseases. Small interfering RNAs (siRNAs) and messenger RNAs (mRNAs) are rapidly expanding as a new class of therapeutics. Particularly, the use of RNA therapeutics to treat cancer is a very promising approach. However, delivery of RNAs to tumor cells still remains as a challenge. The recent success of siRNA delivery to the liver has incentivized the development of novel biomaterials for siRNA delivery into tumors. Our laboratory has discovered a cholesteryl peptide-based siRNA delivery system and demonstrated its effectiveness in delivering the IKBKE siRNA to breast cancer cells. Herein, we hypothesize that amino acid-modified lipid or dendrimer could deliver siRNAs to cancer cells. In chapter 1, we briefly introduced the background about cancer therapeutics, the intracellular mechanism of RNAs, the challenges of RNA delivery to tumors, the NF-κB pathway in cancer development, and the importance of the IKBKE and IKKα silencing in cancer treatment. We also presented the Statement of the Problems and Objectives that includes the limitations and applications of delivery vehicles in RNA delivery. In chapter 2, we reviewed the delivery systems for RNA-based therapeutics and discussed the challenges and biological barriers to RNA delivery. We discussed the potential applications of optimizing physiochemical characterization of nanoparticles in the development of effective therapeutics. We highlighted the importance of optimizing pKa in the development of effective nanoparticles for RNA delivery and the significant impact of the apparent pKa of nanoparticles on their stability, efficacy, and toxicity. In chapter 3, we developed a novel class of amino acid-modified lipids for siRNA delivery to cancer cells. A series of lipids were developed by headgroup modification using histidine and lysine. The lipids were screened for luciferase silencing and cellular cytotoxicity study in PC3-luciferase stable cells. The best LHHK lipid nanoparticle (LNP) protects siRNA from serum and exhibits higher endosomal release and cellular uptake compared to other lysine-modified lipids in PC3 cells. Moreover, The LHHK LNP exhibits significant silencing activity of IKKα and IKBKE in prostate cancer cells and pancreatic cancer cells, respectively. LHHK LNPs encapsulating the IKBKE siRNA suppresses the proliferation of pancreatic cancer cells in vitro and tumor growth in vivo. These results suggest that amino acid-modified lipids possess great potential for siRNA delivery in cancer therapy. In chapter 4, we synthesized the amphiphilic Cholesteryl-dendrimer using the amino acids and fabricated nanoparticles to entrap siRNAs and mRNAs. Amphiphilic dendrimer has the potential to entrap RNAs to deliver them safely into targeted organ. Dendrimer-based nanoparticles were characterized for size and PDI. The efficacy of the developed formulations was evaluated for gene silencing and protein expression of siRNAs and mRNAs, respectively. In chapter 5, we developed another series of lipids using the tail modification from the highly active lipid LHHK to further improve its efficacy in RNA delivery. The LNPs were evaluated for passive targeting and ligand-based active targeting. The lipids were characterized and screened using luciferase silencing assay in PANC-1/LUC cells. 12AHHK lipid was used to develop RGD-labeled LNPs, which showed improved cellular uptake in integrin-overexpressing BX-PC3 cells compared to unmodified LNPs. The results of the biodistribution study suggest that the 18=AHHKK LNPs showed the highest tumor accumulation among all the active LNPs.
Table of Contents
Introduction -- Literature review -- Development of amino acid-modified biodegradable lipid nanoparticles for siRNA delivery -- Development of amphiphilic dendrimer for RNA delivery -- Development of tumor-specific lipid nanoparticles for RNA delivery -- Summary and conclusion
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Ph.D. (Doctor of Philosophy)