Structural and Functional Insights into Porphyrin Lipid Nanoparticles for RNA Delivery

Lipid nanoparticles (LNPs) have established themselves as the leading delivery platform for RNA therapeutics, exemplified by the clinical approval of LNP-based siRNA and mRNA treatments. Despite clinical advances, significant challenges and knowledge gaps remain in maximizing their efficacy. These include insufficient RNA endosomal escape, limited understanding of structure-function relationships affecting LNP intracellular behavior, and incomplete characterization of protein corona interactions that impact LNP cellular fate. This thesis addresses these fundamental questions through three interconnected studies.Chapter 1 outlines the motivation, scope and objectives of the thesis. Chapter 2 provides a comprehensive review of the relevant literature. Chapter 3 introduces a novel approach termed Light-Activated siRNA Endosomal Release (LASER) that addresses endosomal escape limitations in LNP delivery systems. By incorporating photoactive porphyrin-lipids into clinically used LNP formulations, this work developed LNP capable of disrupting endosomal membranes upon near-infrared light irradiation, significantly enhancing siRNA delivery and target gene knockdown both in vitro and in vivo. Chapter 4 investigates the relationship between LNP internal structural organization and its intracellular behavior. This research demonstrates that variations in siRNA loading affect the spatial distribution of helper lipids in LNP which influences critical LNP properties including membrane fluidity, cellular uptake mechanisms, and responsiveness to external stimuli. These findings establish important structure-function relationships that can guide rational LNP design. Chapter 5 examines how changes in LNP surface lipid composition alter the arrangement of apolipoprotein E (ApoE) on the LNP surface and subsequently affect cellular delivery outcome. The study reveals that lipid-driven structural changes of ApoE-LNPs significantly alters their receptor-mediated endocytosis, enhances endosomal disruption and cytosolic RNA release. This work provides mechanistic insights on ApoE-LNP interactions and ApoE’s intracellular functionality. Chapter 6 presents the overall discussion and outlook of the thesis. Overall, this dissertation provides a comprehensive understanding of how LNP structure—both internal organization and surface properties—governs biological behavior throughout the intracellular delivery. By utilizing porphyrin-lipids as both functional components and imaging probes across all studies, a unifying framework is established that bridges structural characterization with functional outcomes.