The Impact of Lipid Nanoparticle Chemistry on RNA Delivery and Therapeutic Outcomes

This dissertation aims to understand how two individual components of the traditional four-component lipid nanoparticle system, the PEG lipid component and the ionizable cationic lipid component, impact RNA delivery. To systematically investigate how PEG lipid chemistry impacted LNP formulation and RNA delivery, a series of linear-dendritic poly(ethylene glycol) (PEG) lipids were synthesized with modulated hydrophobic domains. The chemical structure of the hydrophobic domain did not impact the formulation of 5A2-SC8 LNPs, including nanoparticle size, RNA encapsulation, and stability. However, the chemical structure did affect RNA delivery efficacy both in vitro and in vivo. The chemical structure of the hydrophobic domain of the PEG lipids impacted the escape of 5A2-SC8 LNPs from endosomes at early cell incubation time points. Overall, the results indicated that PEG lipid anchoring and chemical structure modulated RNA delivery. Although most LNPs accumulate in the liver after intravenous administration (suggesting that liver delivery is straightforward), it was observed that two similar LNP formulations (5A2-SC8 and 3A5-SC14 LNPs) resulted in distinct RNA delivery within the liver organ. Despite both LNPs possessing similar physical properties, the ability to silence RNA in vitro, strong accumulation within the liver, and sharing a pKa of 6.5, only 5A2-SC8 LNPs were able to functionally deliver RNA to hepatocytes. Protein corona analysis indicated that 5A2-SC8 LNPs bind Apolipoprotein E (ApoE), which can drive LDL-R receptor mediated endocytosis in hepatocytes. In contrast, the surface of 3A5-SC14 LNPs was enriched in Albumin but depleted in ApoE, which likely led to Kupffer cell delivery and detargeting of hepatocytes. In an aggressive MYC-driven liver cancer model, 5A2-SC8 LNPs carrying let-7g miRNA were able to significantly extend survival compared the non-treatment group. Since disease targets exist in an organ- and cell-type specific manner, the clinical development of RNA LNP therapeutics will require an improved understanding of LNP cellular tropism within organs. Overall, the results from this work illustrates the importance of understanding the cellular localization of RNA delivery and incorporating further checkpoints when choosing nanoparticles beyond biochemical and physical characterization, as small changes in the chemical composition of LNPs can have an impact on both the biofate of LNPs and therapeutic outcomes.