Date of Award

Fall 12-2025

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Hamidreza Montazeri Aliabadi

Second Advisor

Jennifer E. Totonchy

Third Advisor

Kamaljit Kaur

Fourth Advisor

Hasan Uludağ

Abstract

RNA interference (RNAi) is a powerful tool that can selectively downregulate the expression of any protein without the need for expensive and time-consuming drug development processes. Despite the initial excitement and extensive efforts, the potential impact of RNAi in clinical settings has been limited due to challenges in delivering RNA molecules effectively and safely. In parallel, the CRISPR/Cas9 system has revolutionized genome editing by enabling targeted, permanent genetic modifications; however, its success also depends on the development of reliable delivery systems for single-guide RNA (sgRNA)-Cas9 complexes. Lipid nanoparticles (LNPs) have emerged as one of the most successful non-viral carriers for nucleic acids, highlighted by the approval of the first small interfering RNA (siRNA) therapy and the rapid deployment of LNP-based mRNA vaccines against COVID-19. However, achieving efficient and targeted delivery of nucleic acids to solid tumors remains a significant barrier. Polyethyleneimine (PEI), in particular, was once considered the gold standard in polymer-based nucleic acid delivery due to its high transfection efficiency, but its clinical application has been limited by toxicity. In this study, we investigate a novel lipid–polymer nanoparticle (LPNP) platform for the targeted delivery of sgRNA- Cas9 complexes to triple-negative breast cancer (TNBC) cells (MDA-MB-231) and siRNA against Respiratory Syncytial Virus (RSV) viral proteins in human lung cancer A549 cells. We hypothesize that incorporating hydrophobically modified polyethyleneimines (PEIs) into optimized LNP formulations will enhance the delivery of nucleic acids. We first optimized LNP formulations as a benchmark for cellular uptake, cytotoxicity, and silencing efficiency, guided by advanced experimental designs using Design-Expert software. We then systematically replaced the ionizable lipid with specifically engineered cationic polymers, either partially or fully, to generate hybrid LPNPs. Our findings demonstrate that these LPNPs significantly improve nucleic acid delivery to MDA-MB-231 cells. To further confirm the platform’s effectiveness, we tested the best siRNA formulations in A549 cells. Overall, LPNPs showed strong cellular internalization, which translated to silencing efficiency, suggesting that the LPNP systems could be useful in breast cancer and beyond.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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