Nano-Embedded Bioplatform for Ocular Delivery of Genetic Material

Abstract

This thesis explores gene therapy as a promising strategy for treating retinal degenerative diseases, with a particular focus on the development and future of gene delivery vectors. Effective gene delivery systems must be biocompatible and versatile, capable of encapsulating and delivering various nucleic acid payloads. However, current vectors often face limitations such as immunogenicity, low payload capacity, non-specific tropism, and suboptimal transfection efficiency. Numerous studies over the past decade highlight that the limited success of gene therapy is largely due to the inefficiencies of these delivery systems. To address this, the thesis proposes a biocompatible, cationic gene delivery nanosystem targeting CD44 receptors on retinal pigment epithelium (RPE) cells. This targeted approach enhances tissue specificity and supports nucleic acid encapsulation and delivery. A dual delivery system was developed comprising a thermoresponsive hydrogel designed to encapsulate the nanosystem and support ocular cell proliferation for tissue regeneration. The gene delivery vehicle referred to as the nano-polyplex was constructed using hyaluronic acid (HA) conjugated to 2-aminobenzimidazole and oleylamine, forming an amphiphilic, cationic structure. Upon loading with the plasmid pcDNA6.2-EmGFP (pEmGFP), the nano-polyplex exhibited spherical morphology, with particle sizes ranging from 75 nm (unloaded) to 150 nm–3.6 μm (loaded), and zeta potentials ranging from 2.5 mV to 27 mV, depending on the nitrogen-to-phosphate (N:P) ratio. Encapsulation efficiencies exceeded 90% for most N:P ratios, except for N:P 5, which achieved 19.5%. The formulations effectively protected pEmGFP from nuclease degradation and enabled sustained release over 96 hours. Cell viability remained above 90% across all tested formulations. Transfection efficiency, evaluated via GFP expression and fluorescence microscopy, was highest for the N:P 15 formulation. In Phase II, the nano-polyplex was embedded in a pluronic-chitosan hydrogel to form the Gen-I system, a "Trojan horse" platform enabling sustained gene delivery and promoting cell viability. The Gen-I system supported cell survival and proliferation over 36 hours in vitro. A seven-day pilot in vivo study demonstrated the system’s safety and the sustained presence of pEmGFP following intravitreal administration, with only moderate inflammation observed. Collectively, this work presents a novel, biocompatible gene delivery platform with targeted tropism and regenerative potential for retinal gene therapy applications.