Polymeric Nanoparticles for Gene Delivery to Human Astrocytes

Purpose: Currently available therapies for the treatment of neurodegenerative disorders (NDD) are inadequate. Challenges include low blood-brain barrier (BBB) permeability, brain structure complexity. Nanoparticles (NPs) and gene therapy are the two suggested approaches to overcome these problems. Small diameter of NPs (100-200 nm) may allow them to cross the BBB and gene therapy can target specific type of cells to alter gene regulation and cellular function. In this study, we combined both approaches and tested gene delivery to astrocytes, the principal type of glial cells in the brain, via two NPs formulations. Methods: A5P50, an Arginine-based polyethylenimine (PEI) polymer and poly-lactic-co-glycolic-acid (PLGA) NPs were tested for their gene delivery potential in primary human neural cells and cell lines. AFM imaging was carried out to determine A5P50 and PLGA NP dimensions. CMV- or GFAP-promoter-driven luciferase reporter plasmids (pGL3) served as test genes. Cytotoxicity was measured using MTT, LDH, and DNA fragmentation assays. Luciferase assay and Yoyo-dye labeling was used to evaluate the efficiency of gene delivery. Results: FDA-approved, biodegradable PLGA NPs were able to deliver pGL3 across the cell membrane in astrocytes. However, pGL3 expression was negligible or absent. In parallel, A5P50 successfully delivered and expressed the pGL3 in all tested cell types including astrocytes. But, it was not optimally biocompatible in human neurons at higher treatment concentrations. Then, both NPs were used in combination and a significant change in delivery and expression was seen in all types of cells including astrocytes. AFM imaging showed that the size of NPs remained similar when combined indicating absence of direct interaction. Live imaging with Yoyo-labeled pGL3 indicated that presence of A5P50 facilitated PLGA-mediated pGL3 delivery across the nuclear membrane by an unknown mechanism. Conclusions: A5P50-PLGA-combination system was successfully used for gene delivery to astrocytes as well as other cell types. Low A5P50 concentration in the combination eliminated biocompatibility issues in human neurons. Further in vivo testing is necessary to establish this system for future therapeutic use. Presented in vitro results are promising to progress in that direction.