| Currently, non-viral gene delivery vectors are widely used to encapsulate plasmid DNA or mRNA into nanoparticles that transport the genetic material to cells of interest. Although non-viral vectors have not received significant attention in the past because of their poor efficiency versus viral vectors, they have been proven bio-safe and not to elicit a severe immune response. The primary objective of this study was to develop and evaluate an injectable biodegradable chitosan-alginate hydrogel that will locally deliver a cationic polymer packaged plasmid DNA vector.;An evenly crosslinked hydrogel was prepared by mixing an oxidized alginate solution and a N-succinyl chitosan solution within a syringe at a volume ratio of 1:2. Hydrogels were characterized for gelation time, morphologies, mechanical properties, in-vitro swelling and degradation behaviors, protein adsorption, and cell toxicity. Nanoparticle "polyplexes" were formed by mixing anionic pDNA (pMAX-GFP) with cationic poly(ethylenimine) (PEI) in a Nitrogen/Phosphorous (N/P) ratio =7. The resultant polyplexes were encapsulated into alginate:chitosan hydrogels and their in-vitro release kinetics were quantified. The in-vitro transfection of fibroblast (BHK) and dendritic cells (DC 2.4 and JAWsII cells) was quantified by transfected cell GFP gene expression at 1 day, 3 days and 5 days.;The chitosan-alginate hydrogels could be injected through state the size of the needle after gelling for around 2 minutes. The hydrogels were proven elastic materials with a compressive modulus between 1.08 to 3.58 kPa, which approximates soft tissues. The hydrophilic nature and high swelling ratios of the hydrogels (from 25.7 to 39.6) directly reflected their high efficiency of substance exchange. In vitro, the hydrogels were found degradable by hydrolysis with the degradation rate varying based on the chitosan-alginate composition. The in-vitro release test demonstrated that between 40-75% of pDNA/PEI polyplexes were continuously released from 10CS/50Alg hydrogel over 14 days mainly by diffusion. Cells (Baby hamster kidney (BHK) cells and two dendritic cell lines) both exhibited GFP gene transfection by day 5 while retaining high cell viability. As expected, the transfection efficiency of BHK cells was about ten times higher than that of either dendritic cell line. |
