(T1130-07-42) Triple Functionalized Liposomes for Improved Penetration and Targeting of VGF Gene Across BBB in Alzheimer's disease Management

Purpose: VGF, a neuroprotective-neuropeptide precursor, is a potential therapeutic target for neurodegenerative disorders like Alzheimer’s disease (AD). In AD patients, VGF mRNA and protein levels are markedly reduced in the hippocampus and prefrontal cortex compared to healthy controls, correlating with cognitive decline and amyloid-beta plaque burden. This suggests VGF deficiency contributes to AD progression. However, delivering VGF across the blood-brain-barrier (BBB) poses a significant challenge, limiting its therapeutic use. To overcome this, we developed multi-functionalized liposomes for VGF gene delivery, incorporating cell-penetrating peptides (CPPs): Rabies Virus Glycoprotein (RVG) and Rabies Virus- derived Peptide (RDP) and brain-targeting ligands: Mannose (Man) and Transferrin (Tf) to enhance BBB-permeation and brain targeting.

Methods: 2000-NHS (4%) for functionalization. VGF gene-chitosan polyplexes were post-inserted and characterized for size, zeta potential, PDI, and gene entrapment efficiency. Their biocompatibility and uptake in brain cells were assessed in vitro. Transfection efficiency was evaluated using reporter genes and VGF protein expression was quantified with ELISA.

Results: Conjugates achieved >80% coupling efficiency. VGF gene-loaded liposomes had a particle size < 200 nm, PDI < 0.3, positive zeta potential, and >80% gene encapsulation efficiency. At 100 nmoles, formulations showed 80 - 95% cell viability, with peak cellular uptake at 4 hours. Tri-functionalized liposomes (RVGManTf and RDPManTf) exhibited significantly higher transfection efficiency than mono- or dual-functionalized ones, with VGF protein expression ~8-fold higher in treated brain cells versus untreated controls.

Conclusion: Tri-functionalized liposomes demonstrate efficient gene targeting and expression in-vitro, suggesting potential to cross the BBB and elevate brain VGF levels, offering a promising approach for AD treatment.

References: dos Santos Rodrigues, B., Oue, H., Banerjee, A., Kanekiyo, T. and Singh, J., 2018. Dual functionalized liposome-mediated gene delivery across triple co-culture blood brain barrier model and specific in vivo neuronal transfection. Journal of controlled release, 286, pp.264-278.
Arora, S. and Singh, J., 2021. In vitro and in vivo optimization of liposomal nanoparticles-based brain targeted vgf gene therapy. International journal of pharmaceutics, 608, p.121095.
Muolokwu, C.E., Gothwal, A., Kanekiyo, T. and Singh, J., 2024. Synthesis and Characterization of Transferrin and Cell-Penetrating Peptide-Functionalized Liposomal Nanoparticles to Deliver Plasmid ApoE2 In Vitro and In Vivo in Mice. Molecular Pharmaceutics, 22(1), pp.229-241.

Acknowledgements: This research was supported by the National Institute of Aging (NIA) grants R01 AG083981, R01 AG068034, and R01 AG051574


Figure 1: A. Characterization of liposomes (size, PDI, Zeta Potential, Gene Entrapment Efficiency); In-vitro Cytotoxicity of liposomal formulations at different concentrations in B. brain endothelial (bEnd.3) cells, C. Astrocytes



Figure 2: A. Fluorescence microscopy images of Qualitative Cellular uptake of Rhodamine labelled Liposomes (100 nmoles/ml) at 4 hours in bEnd.3 cells, scale bar 100 µm; B. Quantitative cellular uptake of Rhodamine labelled liposomes in Astrocytes, (n=4)



Figure 3: A. Fluorescence microscopy images of GFP expression in primary astrocytes treated with Liposomes encapsulating 1µg chitosan-pGFP complexes 48h post transfection; β-galactosidase expression levels 48 h after transfection in B. bEnd.3 cell, C. Astrocytes with liposomal formulations, statistically significant differences (p < 0.005) as ***, (n=4); VGF expression levels 48 h after transfection in D. bEnd.3 cell, E. Astrocytes with liposomal formulations. Statistically significant differences (p < 0.0001) as **** (n=4)