Abstract
Abstract: :
Purpose: To study the ability of bovine (BRPE) and human Retinal Pigment Epithelium cells (HRPE) to internalize PLGA Nanoparticles (NP) loaded with antisense oligonucleotide and evaluate the therapeutic potentials of this approach. Methods: HRPE and BRPE cells were subcultured and incubated with various concentrations of Nile red labeled PLGA NPs loaded with 6 FAM labeled anti VEGF–R2 ODN. Added NPs were washed away from the RPE cultures after contact times of 1, 6 or 24 hours. NPs internalization by the cultured RPE cells and intracellular ODN kinetics were assessed by phase and fluorescent microscopy. Cell viability was assessed using trypan blue and the MTT test. Cell morphology was examined using inverted and phase contrast microscopy. The integrity of internalized oligonucleotides was assessed by cellular DNA extraction and gel electrophoresis. Results: No significant differences between BRPE and HRPE cells were detected in any of the evaluated parameters. As early as one hour of incubation, NP are observed within the cells; after 6 hours, the number of ingested particles remains constant. The number of ingested particles per cell within the cultures increases in parallel with higher NP concentrations, reaching a plateau at 2 mg/ml. Concentrations of NP as high as 4 mg/ml induced no significant RPE cell toxicity nor they interfered with their proliferation. Free fluorescent ODN was initially observed in the cytoplasm 2 days after ingestion of the loaded NPs, and fluorescence within the nucleus could be observed starting 2 days. At all tested time points, the number of cells per culture and the MTT values during the first 5 days in culture were similar when cultures with loaded NPs were compared to the control cultures incubated with blank NPs, free ODN or culture media. Despite their observation on microscopy, the internalized ODN could not be recovered from the cultured cells in quantity large enough to allow their identification by electrophoresis. Probably, the encapsulation rate within the NPs was too low to allow for their recovery after DNA extraction. The functional assessment of internalized anti–NOS2 loaded NP is being performed. Conclusions: This non–viral approach of gene material vectorization is appealing. Improvement of the encapsulation rate of antisense ODN is needed and is under investigation.
Keywords: retinal pigment epithelium • gene transfer/gene therapy • pharmacology