Development and characterization of fusogenic ApoE-liposome complexes for targeted drug delivery to brain endothelial cells

Decay of the cerebral vasculature is a major contributor to age-related cognitive decline, highlighting the need for efficient drug delivery strategies that enable the targeted transport of vasoprotective compounds such as resveratrol (RSV) to the brain endothelium. Against this background, the present study aimed to investigate whether the natural affinity of apolipoprotein E (ApoE) for cationic liposomes can be exploited for the development of brain-specific drug delivery systems. Owing to its central role in cerebral lipid homeostasis, this protein unites pronounced membrane affinity with selective binding to endothelial surface receptors and is thus a promising mediator for the targeted translocation of liposomes into the brain. Structurally, the system was based on fusogenic liposomes (FL). Unlike conventional liposome formulations, these fuse directly with the cellular plasma membrane and do not rely on endocytic pathways. This property enables rapid and efficient drug delivery while avoiding endosomal degradation. Moreover, the fluid membrane structure of FL is particularly well suited for the transport of hydrophobic compounds such as RSV, as it promotes their intercalation within the lipid bilayer. This results in high partition coefficients and permits the incorporation of significant RSV quantities without compromising liposomal stability and functionality. Quick and reproducible production of homogeneous FL/RSV was achieved via ethanol injection. The inherent affinity of ApoE for FL was validated by analyzing the composition of the protein corona in blood plasma and investigated further through physicochemical binding studies. Due to extensive and versatile interactions with the lipid membrane, ApoE exhibits exceptionally strong binding to FL, rendering it a persistent element of the protein corona. This creates optimal conditions for protein-mediated targeting, as it facilitates the functionalization of FL/RSV and enhances the stability of the artificial protein corona in the presence of competing protein species. Comprehensive physicochemical characterization and optimization of the ApoE amount used for functionalization resulted in the formation of stable and fully functional protein-liposome complexes. The potential of these complexes for cerebral drug delivery was confirmed by in vitro and in vivo experiments. Studies on hCMEC/D3 indicated cellular uptake of ApoE-FL/RSV via receptor-mediated docking and subsequent membrane fusion. Moreover, intravenous administration of the complexes in mice led to their accumulation in the cerebral microvasculature, providing additional evidence for the effectiveness of the targeting approach. Collectively, these findings identify ApoE-functionalized FL as a promising platform for the targeted delivery of RSV to the brain, opening new therapeutic avenues for the treatment of age-related cerebral dysfunctions. Furthermore, the study demonstrates how profound knowledge about the protein corona can guide the rational design of biomimetic targeting strategies that harness the natural affinity of plasma proteins and preserve their functionality by avoiding covalent attachment to the nanomaterial.