(M1130-11-72) Effect of Tetanus Toxoid Fragment C (TT_c) and Transferrin (T_f) Functionalized Liposomal Nanoparticle Encapsulating Plasmid APOE2 for the Treatment of Alzheimer’s Disease

Purpose: Alzheimer’s disease is a progressive neurodegenerative disorder that disrupts cognitive function, eventually leading to loss of memory, loss of language function, and a deranged perception of space. This disease is caused by misfolded proteins (amyloid beta) and phosphorylated TAU accumulation in the brain, blocking electrical signals to neuronal cells, hence brain cells begin to die. The Apo lipoprotein E is a major lipid transporter possessing three (3) isoforms; APOE2- the protective, APOE3- the neutral, and APOE4- the primary genetic risk factor of Alzheimer’s disease. It has been accepted that the elevated level of ApoE2 reduces the risk of AD progression. The purpose of this study is to deliver plasmid ApoE2 to the brain using dual-functionalized liposomes for an effective treatment strategy for AD. The Liposomes were modified with brain-specific cell-penetrating peptide -Tetanus Toxoid Fragment C and targeting ligand – Transferrin.

Methods: The TT_c-T_f functionalized liposomes were prepared using thin film hydration method and characterized for their particle size, zeta potential, and polydispersity index. The encapsulation efficiency of plasmid ApoE2 complexed with chitosan was further determined. The cytotoxicity and blood compatibility studies were performed in b.End3 cells and erythrocytes, respectively. Cell uptake studies were performed using Lissamine-rhodamine labeled TT_c-T_f liposome. While, transfection potential was analyzed using green fluorescent protein (GFP) in brain endothelial cells, primary astrocytes, and neurons. In-vitro blood-brain barrier model was developed using primary astrocytes and brain endothelial cells. The model’s barrier integrity was enhanced using hydrocortisone.

Results: The characterized TT_c-T_f dual functionalized liposomes particles size values obtained were 156.0 ± 8.6, poly-diversity index 0.18 ± 0.01, zeta potential 16.4 ± 1.0, and encapsulation efficiency 84.4 ± 0.7. Cytotoxicity assay(MTT) and hemolysis assay showed a concentration of 100 nmoles of the liposomal formulation to be non-toxic and non-hemolytic respectively. Quantitative analysis of cellular uptake of lissamine-rhodamine labeled liposome showed a statistically significant difference p  < 0.05 between the single functionalized liposomes and dual functionalized liposomes with the highest uptake at 4 h. TT_c-T_f liposomes have exerted significantly higher transfection in brain endothelial cells, primary astrocytes, and neurons. Trans-endothelial electrical resistance and Permeability coefficient of different in-vitro blood-brain barrier models constructed using bEnd3 as a monolayer, bEnd3/Astrocytes as co-culture showed the TEER of bEnd3/Astrocytes treated with hydrocortisone to be statistically significantly higher (p  < 0.05) than bEnd3/Astrocytes without HC treatment and bEnd3 cells only. The permeability coefficient showed a statistically significant difference (p  < 0.05) between the groups.

Conclusion: TT_c-T_f functionalized liposomes were successfully prepared, with cationic zeta potential and uniform size distribution. The developed liposome have shown higher in-vitro transfection and cellular uptake. From the ongoing research, we expect plasmid ApoE2 encapsulated with dual functionalized TT_c-T_f liposomes to cross the blood-brain barrier and increase ApoE2 production in neurons for the treatment of Alzheimer’s disease. 

Acknowledgements: This research was supported by the National Institutes of Health (NIH) Grants R01 AG051574, RF1 AG068034, and 3RFI AG068034-01A1S1

Qualitative Analysis Of Cell Uptake in Brain Endothelial Cells at Different Time Points using TTc-Tf Dual Functionalized Liposome

Qualitative Analysis of Green Florescent Protein (GFP) Transfection in Primary Neurons using Fluorescence Microscope (20x)

Trans-endothelial electrical resistance and permeability coefficient of developed in-vitro Blood brain barrier model using Hydrocortisone