Impact of gaussian curvature lipids on lipid nanoparticle mediated RNA delivery

RNA therapeutics have garnered significant attention for their potential to address a wide range of diseases, with the COVID-19 mRNA vaccine being a notable success in combating the global pandemic. Among RNA delivery systems, lipid nanoparticles (LNPs) have emerged as some of the most effective. However, expanding their application to treat various conditions depends on the improvement of their delivery efficiency. A major challenge in this regard is the release of cargo from endocytosed LNPs, with endosomal escape remaining a key bottleneck. This is due to the difficulty LNPs face in disrupting or fusing with the endosomal membrane in a timely manner. My thesis research focuses on how Gaussian curvature lipids—those capable of forming saddle splay or negative Gaussian curvature membranes—influence the endosomal escape of LNPs. This thesis explores the interplay between nanostructures, mechanical properties of LNP formulations, and their impact on RNA delivery, particularly focusing on endosomal escape and fusogenicity. In the first section, the study reveals how LNP nanostructures—specifically bicontinuous cubic and inverse hexagonal phases—synergize with lipid composition to enhance the escape from endosomes. By promoting topological transitions during LNP-endosome fusion-pore formation, cuboplex structures that contain negative Gaussian curvature membranes demonstrate superior fusion abilities, as confirmed by fusion assays and live-cell imaging, compared to lipoplex counterparts. This finding highlights nanostructure as a crucial factor for engineering more efficient LNPs. The second part of the thesis delves into the mechanical properties of endosomal membrane models, with a focus on the role of Gaussian curvature lipids like GMO. The bending moduli of DOPC giant unilamellar vesicles (GUVs) with varying GMO content were measured, showing that the addition of GMO decreases membrane stiffness. Furthermore, active endosomal models were developed by reconstituting V-ATPase, the key proton pump responsible for endosomal acidification, into GUV membranes. Although these V-ATPase-containing systems are still being analyzed for their mechanical properties, this work sheds light on how the inclusion of active membrane components influences the physical behavior of LNPs during endosomal escape. The third section investigates the fusogenicity of lipid formulations by quantifying how nanostructures impact membrane fusion. As demonstrated in the first part, nanostructures like cubic and inverse hexagonal phases play a significant role in endosomal escape. However, LNPs often exhibit multiple nanostructures simultaneously, making it difficult to quantify fusogenicity based solely on structure. To address this, the study measures lattice parameter changes of lipid formulations at different temperatures, allowing for the extraction of the monolayer Gaussian modulus to bending modulus ratio. Results show that increasing GMO content enhances the fusogenicity of LNPs, and this method offers a robust tool for evaluating the fusogenic potential of other lipid formulations. Overall, this research provides key insights into how LNP nanostructures, mechanical properties, and fusogenicity contribute to more efficient RNA delivery, and offers practical guidelines for optimizing LNP design in therapeutic applications.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Materials Science & Engr

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Zheng, Lining

Contributors dc:contributor

• Leal, Cecilia
• Schroeder, Charles M
• Wang, Hua
• Smith, Andrew

• Lipids
• Nanomedicine
• Rna Delivery

Statement dc:rights

• Copyright 2024 Lining Zheng

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/127383

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F127383