Delivering the Undeliverable: Rational Engineering of Membrane-Based and Synthetic Nanoparticle Platforms

Abstract

Delivering therapeutic macromolecules to defined cell populations in vivo remains a central unsolved problem in biomedicine. Proteins, nucleic acids and viral vectors are cleared by phagocytes, degraded by enzymes, blocked by endothelial and interstitial barriers, and trapped in acidifying endolysosomes. Pre-existing immunity against viral capsids excludes many patients from gene therapy, while intravenously administered nanoparticles accumulate predominantly in the liver. This thesis asks whether delivery platforms can be designed rationally, by exploiting endogenous biological and physical mechanisms, rather than by empirical formulation screening. Four carriers were developed. NEO-TOP-EVs integrate PI(4,5)P₂-mediated membrane targeting, ESCRT-dependent vesicle scission and self-assembly-driven cargo clustering; stepwise addition of these modules progressively improved loading and enabled delivery of adenine base editor ribonucleoproteins, achieving over 90% editing in reporter cells and approximately 65% PCSK9 knockdown in hepatocytes, with preliminary evidence of functional protein delivery in vivo. NEO-AAV applies the same envelopment logic to assembled AAV capsids, yielding vesicle-enveloped particles that resist neutralising antibodies and, through a CD7/CD3/CD28 tri-chimera, mediate single-step targeting, activation and transduction of primary human T cells, including CAR-T generation. Lipid nanoparticles exploit the transient vascular permeability created by ischaemia-reperfusion injury to deliver modRNA to the infarcted myocardium, predominantly to cardiac fibroblasts. Magnetically responsive lipid nanoparticles, guided by an external field, reproducibly redirect mRNA expression away from the liver towards the heart and lungs. Together, these studies show that mechanistic exploitation of vesicle biogenesis, disease-induced permeability and physical field guidance provides a principled basis for carrier design, and argue for a modular toolkit rather than a single optimal delivery modality.