Synthesis and Characterization of Transferrin and\nCell-Penetrating Peptide-Functionalized Liposomal Nanoparticles to\nDeliver Plasmid ApoE2 <i>In Vitro</i> and <i>In Vivo</i> in Mice

Alzheimer’s disease (AD) is\na prevalent neurodegenerative\ncondition characterized by the aggregation of amyloid-β plaques\nand neurofibrillary tangles in the brain, leading to synaptic dysfunction\nand neuronal degeneration. Recently, new treatment approaches involving\ndrugs such as donanemab and lecanemab have been introduced for AD.\nHowever, these drug regimens have been associated with adverse effects,\nleading to the exploration of gene therapy as a potential treatment\noption. The apolipoprotein E (ApoE) isoforms (ApoE2, ApoE3, and ApoE4)\nplay pivotal roles in AD pathology, with ApoE2 known for its protective\neffects against AD, making it a promising candidate for gene therapy\ninterventions. However, delivering therapeutics across the blood–brain\nbarrier (BBB) remains a crucial challenge in treating neurological\ndisorders. Liposomes, lipid-based vesicles, are effective nanocarriers\ndue to their ability to shield therapeutics from degradation, though\nthey often lack specificity for brain delivery. To address this issue,\nliposomes were functionalized with cell-penetrating peptides such\nas penetratin (Pen), cingulin (Cgn), and a targeting ligand transferrin\n(T<sub>f</sub>). This modification strategy aimed to enhance the delivery\nof therapeutic ApoE2 plasmids across the BBB to neurons, thereby increasing\nthe level of ApoE2 protein expression. Experimental findings demonstrated\nthat dual-functionalized liposomes (CgnT<sub>f</sub> and PenT<sub>f</sub>) exhibited higher cellular uptake, biodistribution, and transfection\nefficiency than single-functionalized (Pen, Cgn, or T<sub>f</sub>)\nand nonfunctionalized liposomes. <i>In vitro</i> studies\nusing primary neuronal cells, bEnd.3 cells, and primary astrocytes\nconsistently supported these findings. Following a single dose treatment\nvia tail vein administration in C57BL6/J mice, <i>in vivo</i> biodistribution results showed significantly higher biodistribution\nlevels in the brain (∼12% ID/gram of tissue) for dual-functionalized\nliposomes. Notably, treatment with dual-functionalized liposomes resulted\nin a 2-fold increase in ApoE2 expression levels compared to baseline\nlevels. These findings highlight the potential of dual-functionalized\nliposomes as an efficacious delivery system for ApoE2 gene therapy\nin AD, highlighting a promising strategy to address the disease’s\nunderlying mechanisms.