(M1430-11-71) Development and Characterization of RVG9R-Tansferrin Liposomal Nanoparticles Encapsulating Plasmid GFP for Targeted Delivery to the Brain

Purpose: Gene therapy has been identified to possess immense potential for the treatment of numerous neurological diseases. Liposome has been used as a vehicle for drug and gene delivery because of their biocompatibility and the possibility to incorporate both hydrophilic and hydrophobic drugs. The purpose of this work is focused on delivering plasmid green fluorescent protein (pGFP) encapsulated with RVG9R and Transferrin (T_f) functionalized liposomes across the blood-brain barrier for protein expression in brain cells.

Methods: Liposomes were prepared using a thin film hydration technique. Briefly, the DOTAP 45mol%, DOPE 45mol%, DSPE-PEG 8mol%, and Cholesterol 2% were dissolved in chloroform (2): methanol (1) followed by evaporation using a Rota-vapor to form a thin film. The thin film was rehydrated using HEPES buffer PH 7.4 and sonicated for 15 minutes. The cell-penetrating peptide-RVG9R and Transferrin were conjugated to DSPE-PEG-NHS by reacting the N-hydroxysuccinimide ester of the PEGylated lipid with the primary amines of the cell-penetrating peptides under alkaline conditions to form stable amide bonds. The characteristics of the liposomal formulations were determined by dynamic light scattering using a zeta sizer for their particle size, zeta potential, and poly-dispersity index. The encapsulation efficiency of plasmid GFP complexed with chitosan was further determined. Cytotoxicity and blood compatibility studies were performed in b.End3 cells and erythrocytes respectively. Lissamine-rhodamine labeled liposome cell uptake studies at varying time points and qualitative analysis of transfection using green fluorescent protein (GFP) were determined in brain endothelial cells, primary astrocytes, and neurons.

Results: The size of the developed liposomes was 184.6 ± 6.3, poly-diversity index 0.21 ± 0.03, zeta potential 19.9 ± 1.4, and encapsulation efficiency 86.4 ± 2. 5% considering mean ± SD (n =4) with single modified liposomes having lesser encapsulation efficiency. Cytotoxicity assay (MTT) showed a concentration of 100nmoles to be non-toxic. Quantitative analysis of cellular uptake of lissamine-rhodamine labeled liposome showed a statistically significant difference p  < 0.05 between single functionalized and dual functionalized liposomes with the highest uptake at 4 h. The functionalized liposomes exerted higher transfection in brain endothelial cells, primary astrocytes, and neurons.

Conclusion: RVG9R-T_f dual functionalized liposomes with uniformly distributed size and cationic zeta potential was successfully prepared. The dual functionalized liposome- RVG9R-T_f have shown higher in-vitro transfection potential and cellular uptake. From the ongoing research, we expect plasmid GFP encapsulated with dual functionalized liposomes-RVG9RTf to cross the blood-brain barrier and show higher protein expression when compared to single modified liposomes in brain cells.

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

Characterization of Different Liposomal Formulations

Quantitative Analysis of Cellular Uptake of Lissamine-Rhodamine Labelled Liposomal Formulations at Different Time points in Brain Endothelial Cells

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