(T1030-01-03) Chitosan DNA Nanoparticle for PRPF31 Gene Delivery in Retinal Cell Line: Formulation and Characterization

Purpose: 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.

Methods: The pDNA was cloned and extracted according to the manufacturer's protocol. The CS NPs loaded with plasmid DNA (pDNA) were prepared through a complex coacervation process. The NPs were characterized for particle size, zeta potential, and surface morphology. Additionally, the loading capacity, encapsulation efficiency, protective effect on the plasmid DNA, and in vitrotransfection efficiency and cytotoxicity were evaluated.

Results: The average particle size, polydispersity index, and zeta potential were 182.28 ± 6.23 nm, 0.281 ± 0.002, and +22.33 ± 8.05 mV, respectively. Scanning Electron Microscopy images confirmed the formation of nanoparticles and their spherical morphology. The pDNA encapsulation efficiency was 66.80 ± 4.69%, with a loading efficiency of 21.93 ± 1.08%. Gel electrophoresis results indicated that the chitosan nanoparticles effectively protected the pDNA. Fluorescence microscopy images demonstrated successful cellular transfection and green fluorescent protein (GFP) expression by the formulation. It also confirmed protein expression in the nucleus. The chitosan DNA nanoparticle has a significantly higher transfection efficiency than the naked plasmid. The synthesized formulation is significantly less toxic compared to cells treated with Lipofectamine and pDNA.

Conclusion: We have successfully prepared chitosan nanoparticles for PRPF31 gene delivery in nano-size range. Gel electrophoresis affirmed the similarity between the cloned plasmid and the original plasmid. The gel retardation assay helped determine the optimal chitosan-to-plasmid ratio for effective DNA encapsulation. The integrity of encapsulated plasmids within NPs was confirmed by their intact recovery following the enzymatic cleavage of the NPs. The high encapsulation and transfection efficiencies, coupled with low toxicity, highlight the effectiveness of this nanoparticle formulation in delivering genetic material.

References: 1. Corsi et al. Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles. Biomaterials 24 (2003) 1255–1264.
2. Mansouri et al. Characterization of folate-chitosan-DNA nanoparticles for gene therapy. Biomaterials 27 (2006) 2060–2065.
3. Mao et al. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. Journal of Controlled Release 70 (2001) 399–421.