(M1330-06-35) Brain-Targeted Delivery of Plasmid VGF through Trifunctionalized Liposomes for the Management of Alzheimer’s Disease

Purpose: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline. Current treatments provide only symptomatic relief, underscoring the need for disease-modifying strategies. VGF, a neurotrophin-inducible protein involved in neurogenesis and synaptogenesis, is significantly reduced in AD and shows therapeutic potential in reducing amyloid-β and phosphorylated tau. However, delivering VGF via viral vectors raises safety concerns, and achieving efficient transport across the blood-brain barrier (BBB) remains a significant challenge. This study aims to develop and characterize mono-, dual-, and trifunctionalized liposomes encapsulating plasmid VGF (pVGF), functionalized with mannose and/or transferrin for BBB targeting and penetratin or TTC to enhance VGF expression in the brain.

Methods: Penetratin, TTC, and transferrin were individually conjugated to DSPE-PEG-NHS. Plain liposomes were prepared via thin-film hydration using DOPE: DOTAP: DSPE-MPEG(2000): cholesterol in a 45:45:8:2 molar ratio, and functionalized with DSPE-PEG-Mannose, DSPE-PEG-Transferrin, DSPE-PEG-Penetratin, or DSPE-PEG-TTC at 4% molar ratios, as applicable. Formulations were characterized for size, PDI, zeta potential, encapsulation efficiency, and stability against nuclease degradation. In vitro assays assessed biocompatibility, cellular uptake of rhodamine-labeled liposomes, and transfection efficiency of pVGF in brain cell lines.

Results: The synthesized liposomes had sizes < 200 nm, PDIs < 0.3, and positive zeta potentials. High plasmid VGF entrapment efficiency and protection against enzymatic degradation were observed. Trifunctionalized liposomes exhibited improved cellular uptake, higher VGF expression levels in brain cell lines compared to controls, and were biocompatible at 100 nM phospholipid concentration.

Conclusion: Our findings indicate that trifunctionalized liposomes show strong promise for efficient BBB penetration, enhanced transfection efficiency, and favorable biocompatibility in brain cell lines at therapeutic concentrations. Future studies will focus on cellular uptake and transfection in primary neurons and BBB permeation assays. In vivo investigations will include pharmacokinetic profiling in wild-type mice and efficacy evaluation in transgenic models of Alzheimer’s disease.

References: 1. Yiannopoulou KG, Papageorgiou SG. Current and Future Treatments in Alzheimer Disease: An Update. J Cent Nerv Syst Dis. 2020 Feb 29;12:1179573520907397. doi: 10.1177/1179573520907397. PMID: 32165850; PMCID: PMC70500253.
2. Quinn JP, Kandigian SE, Trombetta BA, Arnold SE, Carlyle BC. VGF as a biomarker and therapeutic target in neurodegenerative and psychiatric diseases. Brain Commun. 2021 Oct 27;3(4):fcab261. doi: 10.1093/braincomms/fcab261. PMID: 34778762; PMCID: PMC8578498
3. Beckmann, N.D., Lin, WJ., Wang, M. et al. Multiscale causal networks identify VGF as a key regulator of Alzheimer’s disease. Nat Commun 11, 3942 (2020). https://doi.org/10.1038/s41467-020-17405-z
4. Juhairiyah F, de Lange ECM. Understanding Drug Delivery to the Brain Using Liposome-Based Strategies: Studies that Provide Mechanistic Insights Are Essential. AAPS J. 2021 Oct 28;23(6):114. doi: 10.1208/s12248-021-00648-z. PMID: 34713363; PMCID: PMC8553706. 5. Dos Santos Rodrigues B, Oue H, Banerjee A, Kanekiyo T, Singh J. Dual functionalized liposome-mediated gene delivery across triple co-culture blood brain barrier model and specific in vivo neuronal transfection. J Control Release. 2018 Sep 28;286:264-278. doi: 10.1016/j.jconrel.2018.07.043. Epub 2018 Jul 31. PMID: 30071253; PMCID: PMC6138570.

Acknowledgements: This research was supported by the National Institute of Aging (NIA) grants R01 AG083981, RF1 AG068034, and R01 AG051574.


A: Conjugation efficiencies of CPPs/ligand to DSPE-NHS-PEG. Data expressed as mean ± SD, n=4
B: Physicochemical characterization of liposomes encapsulating 1µg chitosan-plasmid VGF complex. Data are expressed as mean ± SD, n=4.
C: Nuclease protection assay of liposomes encapsulating 1µg chitosan-plasmid VGF complex.
D: In vitro biocompatibility of liposomal formulations in brain endothelial cells. Data are expressed as mean ± SD, n=4.
E: In vitro biocompatibility of liposomal formulations in primary astrocytes. Data are expressed as mean ± SD, n=4.
F: In vitro biocompatibility of liposomal formulations in primary neurons. Data are expressed as mean ± SD, n=4.



G: Qualitative cellular uptake of rhodamine-labeled liposomes in brain endothelial (b.End 3) cells (20X magnification).
H: Quantitative cellular uptake of rhodamine-labeled liposomes in brain endothelial (b.End 3) cells at varying time points. Data are expressed as mean ± SD, n=4.
I: Quantitative cellular uptake of rhodamine-labeled liposomes in b.EnD 3 cells at 4hrs. Data are expressed as mean ± SD, n=4 ; P < 0.05 (*), P  < 0.01(**), P  < 0.001 (***), and P  < 0.0001 (****).
J: Qualitative cellular uptake of rhodamine-labeled liposomes in primary astrocytes (20X magnification).
K: Quantitative cellular uptake of rhodamine-labeled liposomes in primary astrocytes at varying time points. Data are expressed as mean ± SD, n=4.
L: Quantitative cellular uptake of rhodamine-labeled liposomes in primary astrocytes at 4hrs. Data are expressed as mean ± SD, n=4 ; P < 0.05 (*), P  < 0.01(**), P  < 0.001 (***), and P  < 0.0001 (****).


M: Qualitative transfection of liposomal formulations encapsulating 1 µg chitosan-pGFP complexes 48 hours post-transfection in b.End 3 cells (20X magnification).
N: Quantitative transfection of liposomal formulations encapsulating 1 µg chitosan-pBgal complexes 48 hours post-transfection in b.End 3 cells. Data are expressed as mean ± SD, n=4 ; P < 0.05 (*), P  < 0.01(**), P  < 0.001 (***), and P  < 0.0001 (****).
O: Quantitative transfection of liposomal formulations encapsulating 1 µg chitosan-pVGF complexes 48 hours post-transfection in b.End 3 cells. Data are expressed as mean ± SD, n=4 ; P < 0.05 (*), P  < 0.01(**), P  < 0.001 (***), and P  < 0.0001 (****).
P: Qualitative transfection of liposomal formulations encapsulating 1 µg chitosan-pGFP complexes 48 hours post-transfection in primary astrocytes (20X magnification).
Q: Quantitative transfection of liposomal formulations encapsulating 1 µg chitosan-pBgal complexes 48 hours post-transfection in primary astrocytes. Data are expressed as mean ± SD, n=4 ; P < 0.05 (*), P  < 0.01(**), P  < 0.001 (***), and P  < 0.0001 (****).
R: Quantitative transfection of liposomal formulations encapsulating 1 µg chitosan-pVGF complexes 48 hours post-transfection in primary astrocytes. Data are expressed as mean ± SD, n=4 ; P < 0.05 (*), P  < 0.01(**), P  < 0.001 (***), and P  < 0.0001 (****).