Innovative DNA Delivery Systems Based on Natural Polymers Grafted to Poly(2-Oxazolines)

In this investigation we manufactured and characterized novel polymer derivatives based on poly(2-oxazoline) (POx) covalently linked to partially quaternized chitosan or dextran. These materials are explored for their potential to form stable polymer-DNA polyplexes for ocular gene delivery. The polymers were characterized using proton nuclear magnetic resonance (¹HNMR) spectroscopy and size exclusion chromatography (SEC) to confirm their structure and molecular weight. The grafted polysaccharides were employed to complex plasmid DNA at varying nitrogen-to-phosphate (N/P) ratios. Dynamic (DLS) and electrophoretic (ELS) light scattering analyses demonstrated the formation of stable electrostatic complexes with hydrodynamic radius ranging from 40 and 100 nm and a zeta potential of approximately +40 mV, suggesting the formation of stable supramolecular structures. Agarose gel electrophoresis and fluorescence spectroscopy confirmed efficient DNA condensation at N/P ~ 2 for chitosan and N/P ~ 5 for dextran. Biological assays were conducted using retinal pigment epithelial (ARPE-19) and HeLa cells highlighting the versatility of the developed polyplexes for ocular and general gene delivery applications. The MTT assays showed that the polyplexes exhibited low cytotoxicity at biologically relevant concentrations. However, the electrostatic polyplexes alone were inefficient in gene transfection, leading to the incorporation of small aliquots Polyethylenimine (PEI). The addition of a small quantity of PEI resulted in a significant enhancement of transfection efficiency, surpassing the performance of PEI itself at equivalent concentrations. These findings demonstrate the potential of hybrid gene delivery systems that combine natural polymers and PEI to improve the efficacy of polymeric vectors for gene therapy. This research provides critical insights into the development of advanced polymer-based systems for gene therapy, with promising implications in ocular applications.