title: In vitro bioassay model for screening non-viral neurotrophic factor gene delivery systems for glaucoma treatment.
abstract: The feasibility of a two-layer contact-independent 3D neuronal co-culture model to test the bioactivity of brain-derived neurotrophic factor (BDNF), produced by non-virally transfected A7 astrocytes (trA7), on neurite growth in a second cell population of SH-SY5Y (CRL-2266) neuroblastoma cells with (oxSH-SY5Y) or without oxidative damage (SH-SY5Y) was evaluated. Transfection of A7 astrocytes was carried out with BDNF-encoding plasmid using K2® nanoparticle gene delivery system (K2-NPs). The physicochemical characteristics of K2-NPs, transfection efficiency, and BDNF production were evaluated using dynamic light scattering, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), respectively. Neurite counts and length measurements were performed after anti-neuron-specific β-III tubulin antibody immunostaining using confocal laser scanning microscopy. Transfection efficiency of A7 astrocytes by K2-NPs (diameter 83.9 ± 0.4 nm, zeta potential +57.3 ± 2.8 mV) was 39.5 ± 4.6 % with cell viability of 73 ± 2 %. BDNF levels produced were 3750.8 ± 251.1, 9052.6 ± 1391.2, and 10,367.1 ± 390.8 pg/mL at 24, 48, and 72 h, respectively. The increased number of neurites with higher neurite lengths confirmed the bioactivity of BDNF secreted from the transfected A7 astrocytes over 72 h. Neurite count comparisons showed that both trA7/oxSH-SY5Y and trA7/SH-SY5Y consistently produced higher neurite counts compared to A7/oxSH-SY5Y and oxSH-SY5Y only experimental conditions. The results of this study demonstrate that neurite outgrowth quantitation in astrocyte-SH-SY5Y cell co-culture is a suitable bioassay model for evaluating non-viral gene delivery systems. Furthermore, it also demonstrates a proof-of-concept for nanoparticle-based neurotrophic factor gene delivery to astrocytes and stimulation of neurite outgrowth.
authors.affiliation: School of Pharmacy, Waterloo Institute of Nanotechnology and Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada.
authors.forename: Ding Wen
authors.initials: DW
authors.lastname: Chen
authors.affiliation: School of Pharmacy, Waterloo Institute of Nanotechnology and Center for Bioengineering and Biotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON, N2L 3G1, Canada. foldvari@uwaterloo.ca.
authors.forename: Marianna
authors.identifier: 0000-0001-7437-7917
authors.initials: M
authors.lastname: Foldvari
mesh_terms: D001253:Astrocytes / Q000378:metabolism; D001681:Biological Assay; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D018014:Gene Transfer Techniques; D005901:Glaucoma / Q000628:therapy; D006801:Humans; D016501:Neurites / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism
chemical_list: D019208:Brain-Derived Neurotrophic Factor
keywords: Astrocyte; BDNF; Gene therapy; Glaucoma; Nanoparticle; Neurite outgrowth; Neuroprotection; Non-viral; RGC; Three-dimensional culture