Impact of gaussian curvature lipids on lipid nanoparticle mediated RNA delivery
Zheng, Lining
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Permalink
https://hdl.handle.net/2142/127383
Description
Title
Impact of gaussian curvature lipids on lipid nanoparticle mediated RNA delivery
Author(s)
Zheng, Lining
Issue Date
2024-12-04
Director of Research (if dissertation) or Advisor (if thesis)
Leal, Cecilia
Doctoral Committee Chair(s)
Leal, Cecilia
Committee Member(s)
Schroeder, Charles M
Wang, Hua
Smith, Andrew
Department of Study
Materials Science & Engineerng
Discipline
Materials Science & Engr
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Lipids
Nanomedicine
Rna Delivery
Language
eng
Abstract
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.
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