Biomimetic exosome harnessing exosomal lipidomics and functional proteins for PEDF-pDNA delivery in high altitude pulmonary edema intervention.

In the realm of gene therapy, given the exceptional performance of native exosomes, researchers have redirected their innovative focus towards exosome-mimetic nanovesicles (EMNs); however, the current design of most EMNs relies heavily on native cells or their components, inevitably introducing inter-batch variability issues and posing significant challenges for quality control. To overcome the excessive reliance on native cellular components, this study adopts a unique approach by precisely mimicking the lipid composition of exosomes and innovatively incorporating histone components to recapitulate the gene transfer characteristics of exosomes. We selected sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), and cholesterol as the lipid components, and employed the double emulsion method to prepare biomimetic exosomes carrying histone A and PEDF-DNA plasmids (His-pDNA@EMNs). These vesicles exhibited an ideal particle size (102 ± 2 nm) and Zeta potential (-20 ± 2 mV) with cup-shaped structure, similar to native exosomes. Compared with the commercial gene transfection reagent Lipo6000, His-pDNA@EMNs significantly improved the transfection efficiency of the PEDF gene in HUVEC cells by 18.74 % while significantly reducing cytotoxicity, demonstrating their superior biocompatibility and efficiency. Mechanism exploration revealed that the lipid composition of these EMNs delicately promoted each step of gene delivery: PC facilitated efficient cellular uptake, the synergistic effect of PE and PS significantly enhanced lysosomal escape ability, and the specific combination of PS and SM assisted vesicles in penetrating into the nucleus. Notably, EMNs escaped from lysosomes in their intact form through a local membrane fusion mechanism. Further cellular and animal experiments fully verified that His-pDNA@EMNs could effectively enhance PEDF protein expression both in vitro and in vivo, effectively inhibiting hypoxia-induced vascular remodeling and endothelial injury, providing a novel and effective intervention of high-altitude pulmonary edema (HAPE). In summary, this study not only demonstrates the feasibility of preparing efficient gene delivery vectors by mimicking the functions of native exosomes with synthetic phospholipids and histones, but also opens up a new path for the development of gene therapy vectors. His-pDNA@EMNs also provide a new strategy for the prevention of HAPE.