Graduation Date

Fall 12-19-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Dr. David Oupicky

Abstract

The development of non-viral delivery systems for pulmonary delivery of nucleic acid therapeutics is driven by the need for safer and more accessible approaches to treat lung diseases, while avoiding the systemic side effects and immune responses associated with viral vectors. Such systems encompass both non-invasive inhalation-based platforms, and systemically administered platforms designed to achieve localized lung delivery. These strategies aim to enable high drug concentrations at the site of action, rapid onset of therapeutic effect, and greater therapeutic efficacy than existing treatments. Designing effective pulmonary delivery systems requires precise optimization of particle properties—including size, morphology, surface characteristics, cell-specific targeting, and evasion of host defenses. Rigorous formulation development and characterization are essential to ensure the safety, stability, and therapeutic efficacy of these complex systems.

This dissertation focuses on the development of delivery platforms that enable lung-selective delivery of nucleic acid therapeutics through two distinct routes: (1) perfluorocarbon (PFC)-based RNA nanocapsules for inhaled administration of microRNA (miRNA) therapeutics, and (2) macrocyclic ionizable lipid–based lipid nanoparticles (LNPs) for intravenous administration of mRNA and gene-editing therapeutics.

The first part of the dissertation focuses on the development and characterization of inhalable PFC-based RNA nanocapsules for the selective delivery of miRNAs to metastatic lung tumors. These nanocapsules are stabilized by PAMD-C, a cholesterol-modified polymeric analog of the FDA-approved CXCR4 antagonist AMD3100 (plerixafor), which confers tumor-targeting capability by recognizing CXCR4-overexpressing cancer cells. The PFC nanocapsules demonstrated remarkable ability to evade immune cell-mediated clearance while achieving passive targeting to type II alveolar and bronchial epithelial cells. In an orthotopic lung metastasis model, a single aerosolized dose resulted in more than 60% of cancer cells internalizing the nanocapsules, with pulmonary retention lasting over 48 hours. The platform induced negligible cytokine release, thus enabling repeated dosing. Treatment with therapeutic miR-34a delivered via this platform suppressed metastatic outgrowth, enhanced anti-tumor immunity, and doubled median survival relative to control paclitaxel chemotherapy. Collectively, this platform addresses key biological barriers to inhalable RNA medicines - including mucus entrapment, immune clearance, navigating through heterogeneous lung tissue architecture - and provides a promising and translatable path for treating a broad range of lung diseases.

The second part of the dissertation focuses on the synthesis and pharmacological evaluation of a LNP library derived from ten cyclam-based ionizable lipids, incorporating three distinct linker chemotypes and varied hydrophobic tails. Unlike conventional LNPs that predominantly accumulate in the liver, all cyclam-based formulations demonstrated remarkable lung tropism following intravenous administration, with lead candidates exhibiting lung-to-liver delivery ratios exceeding 100-fold. The cyclam-based LNPs distinguish themselves from standard DLin-MC3-DMA formulations by exhibiting improved safety profile, as they achieve lung-selective delivery without the need for cationic lipids like DOTAP, thereby circumventing lung thrombosis and other toxicities. Lipids incorporating benzylmethyl carbonate (BMC) linkers produced LNPs with compact sizes, higher RNA encapsulation efficiency, greater RNA recovery, and superior lung transfection potency compared to other linker types. In Ai9 reporter mice, two intravenous doses of Cre mRNA formulated with the lead cyclam lipid achieved ~30% lung-specific gene editing with minimal off-organ activity. Structure-activity analyses revealed that increased lipophilicity and aromatic content were inversely correlated with particle size and positively correlated with encapsulation efficiency. These findings establish design rules for macrocycle-guided delivery and provide a rational framework for engineering next-generation LNPs optimized for pulmonary gene therapy.

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2025 Copyright, the authors

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