Targeted Liposomal Nanocarriers for High-Efficiency Neuronal mRNA Delivery

Purpose Messenger RNA therapeutics enable rapid, transient protein expression without risk of genomic integration, but their delivery to post-mitotic neurons remains challenging. Here, we present a neuron-targeted liposomal platform designed to enhance mRNA delivery efficiency in neurons. Methods We engineered DOTAP: DOPE liposomes to promote nucleic acid loading, stability, and endosomal escape, incorporated PEGylation to improve biocompatibility and circulation, and functionalized the liposome surface with a rabies virus glycoprotein (RVG)-derived peptide for neuronal targeting. We assessed particle size and surface charge, mRNA encapsulation/loading across different lipid: mRNA ratios, cytotoxicity in HEK293T cells as a function of cationic lipid content and PEGylation, and transfection performance in HEK293T cells and primary neurons, benchmarking against a commercial transfection reagent, with functional mRNA delivery further demonstrated in midbrain organoids following microinjection. Results The system enabled efficient mRNA encapsulation, maintained favorable particle size and surface charge characteristics, and supported low cytotoxicity. Across formulations, mRNA loading increased with lipid: mRNA ratio, with only modest reductions following PEGylation. In HEK293T cells, higher cationic lipid content increased its toxicity, whereas PEGylation reduced membrane interactions and improved tolerability. Transfection studies identified the 50:10 DOTAP: DOPE composition as optimal, yielding robust mRNA expression. In primary neurons, RVG-functionalized PEGylated liposomes containing 10 mol% DOPE achieved gene expression levels comparable to those of a commercial transfection reagent at a 10:1 lipid:mRNA ratio. Notably, incorporating 0.1% w/w RVG significantly outperformed the commercial formulation, yielding >30% gene-delivery efficiency in primary neuronal cultures. In midbrain organoids, the optimized mRNA-loaded RVG-functionalized liposomes produced GFP mRNA expression overlapping with NeuN staining, demonstrating functional mRNA delivery and translation within three-dimensional neural tissue. Conclusions Neuron-targeted DOTAP: DOPE liposomes incorporating the RVG peptide represent a promising mRNA delivery system for neurological applications, achieving a favorable balance of potency, safety, and specificity and supporting transient gene modulation in the central nervous system.