Method Optimization and Formulation of Chitosan Nanoparticle for PRPF31 Gene Delivery
Retinitis Pigmentosa (RP), an inherited retinal disease, is characterized by a progressive loss of photoreceptor cells, leading to vision impairment. Current treatments, including retinoids and vitamin A supplements, primarily offer symptomatic relief without addressing the genetic root causes of this disorder. Mutations in the PRPF31 gene are key genetic contributors to the pathology of autosomal dominant RP. Since RP often results from specific genetic anomalies, gene therapy represents an ideal treatment approach. However, despite advancements in gene therapy for RP, effective delivery remains a significant challenge. Chitosan (CS) nanoparticles (NPs) offer a promising solution due to their biocompatibility and potential for gene delivery. This study aims to develop a chitosan NP-based delivery system for the PRPF31 gene in the treatment of RP. The plasmid DNA (pDNA) with PRPF31 gene was cloned and extracted according to the manufacturer's protocol. The CS NPs loaded with pDNA were prepared through a complex coacervation process. The NPs were characterized for particle size, poly dispersity index (PDI), zeta potential, and surface morphology. Additionally, the loading capacity, encapsulation efficiency, protective effect on the pDNA, in vitro transfection efficiency and cytotoxicity were evaluated. The particle size, PDI, and zeta potential of chitosan nanoparticles were 182.28 ± 6.23 nm, 0.281 ± 0.002, and +22.33 ± 8.05 mV, respectively. Scanning Electron Microscopy (SEM) images confirmed the formation of NPs and their spherical shape. The pDNA encapsulation efficiency was 64.46 ± 3.21%, with a loading efficiency of 18.33 ± 1.41%. Gel electrophoresis results indicated that the CS NPs effectively protected the pDNA. Fluorescence microscopy images demonstrated successful cellular transfection and green fluorescent protein (GFP) expression by the CS NPs carrying PRPF31 plasmid. It also confirmed GFP expression in the nucleus. The transfection efficiency for CS-pDNA nanoparticles was 48.30 ± 4.25% which was significantly higher than the naked plasmid. The prepared CS-pDNA nanoparticles were significantly (P < 0.05) less toxic (84.4% cell viability) compared to cells treated with Lipofectamine and pDNA.
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