Development of Lipid Nanoparticles for Effective Endosomal Escape

Development of Lipid Nanoparticles for Effective Endosomal Escape PROJECT SUMMARY Nucleic acid-lipid nanoparticles (NA-LNPs) are internalized by cells primarily through endocytosis. Upon binding to the cell surface, the plasma membrane invaginates to trap the LNPs within a cellular vesicle known as the endosomes. Research indicates that only 2–4% of nucleic acid cargo escapes the endosome during NA- LNP treatment, underscoring the necessity of enhancing cytoplasmic release to fully utilize LNPs’ potential. Another significant challenge is the reactogenicity of LNPs, which refers to the inflammatory response triggered by vaccination. While vectors can enhance the host’s antigen-specific immune response and reduce dosage requirements, they often increase reactogenicity. This suggests that these components, independent of nucleic acid cargo, possess structural components that induce such responses. Even FDA-approved LNP vectors, like BNT162b2, exhibit reactogenicity, as evidenced by phase 2/3 trial safety results. Therefore, addressing both these issues, including optimizing both endosomal escape and reactogenicity, is crucial for balancing efficacy of NA-LNP therapy with acceptable levels of pain and potential risks. Ionizable lipids (ILs) play a pivotal role in determining both the efficiency of LNP endosomal escape and reactogenicity. Herein, we propose three universal strategies: (a) enhancing electrostatic interactions between LNPs and endosomal membranes using pH-switchable pyridine carboxybetaine ILs; (b) integrating lipophobicity into LNPs through fluorinated zwitterionic ILs; and (c) improving hydrophobic interactions between LNPs and membranes by utilizing virus protein-mimicking ILs. Over the past 20 years, the PI has demonstrated the excellent biocompatibility of zwitterionic materials in various biomedical applications. Incorporating zwitterionic groups into the chemical design of ILs offers a promising approach to reduce reactogenicity. For each approach, we will synthesize unique lipid headgroups to establish the desired properties and understand their structure- property relationships. Once these criteria are met, we will attach the most effective headgroups to a library of lipid tails. LNPs will then be formulated, optimized, and subjected to thorough physical characterization to assess how the structure-property relationships of small molecule headgroups translate to the final LNP performance. This research aims to uncover new and universal structure-function relationships to guide the design of unique lipids with innovative chemical structures, ultimately enhancing the technology of nucleic acid therapeutics, particularly for mRNA therapy. The successful execution of this work will address the critical knowledge gaps needed to advance this field.