Synthesis and Characterization of Transferrin and Cell-Penetrating Peptide-Functionalized Liposomal Nanoparticles to Deliver Plasmid ApoE2 In Vitro and In Vivo in Mice.

Alzheimer's disease (AD) is a prevalent neurodegenerative condition characterized by the aggregation of amyloid-β plaques and neurofibrillary tangles in the brain, leading to synaptic dysfunction and neuronal degeneration. Recently, new treatment approaches involving drugs such as donanemab and lecanemab have been introduced for AD. However, these drug regimens have been associated with adverse effects, leading to the exploration of gene therapy as a potential treatment option. The apolipoprotein E (ApoE) isoforms (ApoE2, ApoE3, and ApoE4) play pivotal roles in AD pathology, with ApoE2 known for its protective effects against AD, making it a promising candidate for gene therapy interventions. However, delivering therapeutics across the blood-brain barrier (BBB) remains a crucial challenge in treating neurological disorders. Liposomes, lipid-based vesicles, are effective nanocarriers due to their ability to shield therapeutics from degradation, though they often lack specificity for brain delivery. To address this issue, liposomes were functionalized with cell-penetrating peptides such as penetratin (Pen), cingulin (Cgn), and a targeting ligand transferrin (T_f). This modification strategy aimed to enhance the delivery of therapeutic ApoE2 plasmids across the BBB to neurons, thereby increasing the level of ApoE2 protein expression. Experimental findings demonstrated that dual-functionalized liposomes (CgnT_f and PenT_f) exhibited higher cellular uptake, biodistribution, and transfection efficiency than single-functionalized (Pen, Cgn, or T_f) and nonfunctionalized liposomes. In vitro studies using primary neuronal cells, bEnd.3 cells, and primary astrocytes consistently supported these findings. Following a single dose treatment via tail vein administration in C57BL6/J mice, in vivo biodistribution results showed significantly higher biodistribution levels in the brain (∼12% ID/gram of tissue) for dual-functionalized liposomes. Notably, treatment with dual-functionalized liposomes resulted in a 2-fold increase in ApoE2 expression levels compared to baseline levels. These findings highlight the potential of dual-functionalized liposomes as an efficacious delivery system for ApoE2 gene therapy in AD, highlighting a promising strategy to address the disease's underlying mechanisms.