Preparation and Evaluation of Thiol-Modified Gelatin Nanoparticles for Intracellular DNA Delivery in Response to Glutathione

Abstract

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we have developed thiolated gelatin nanoparticles that can release the payload in the highly reducing environment, such as in response to glutathione. Thiolated gelatin was synthesized by covalent modification of the primary amino groups of Type B gelatin using 2-iminothiolane (Traut's reagent). The degree of thiolation of the polymers ranged from 0 to 43.71 mmol of reduced sulfhydryl (SH) groups when the amount of 2-iminothiolane was increased up to 100 mg per gram of the biopolymer. Cytotoxicity evaluations carried out by the formazan (MTS) assay showed that the thiolated gelatin prepared with 20 mg and 40 mg of 2-iminothiolane (SHGel-20 and SHGel-40) per gram of gelatin had comparable cell viability profile to that of the unmodified gelatin. In vitro release studies of fluorescein isothiocyanate (FITC)-labeled dextran (mol wt. 70 000 Da), when encapsulated in gelatin and thiolated gelatin nanoparticles (150-250 nm in diameter), was found to be affected by the presence of glutathione (GSH) in the medium. The presence of GSH was found to enhance the release by about 40% in case of thiolated gelatin and about 20% in gelatin nanoparticles under similar conditions of temperature and GSH concentrations. Qualitative and quantitative analysis of transfection in NIH-3T3 murine fibroblast cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by fluorescence confocal microscopy and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 h. Quantitative results from FACS showed that the thiolated gelatin nanoparticles (SHGel-20) were significantly more effective in transfecting NIH-3T3 cells than other carrier systems examined. The results of this study show that thiolated gelatin nanoparticles would serve as a biocompatible intracellular delivery system that can release the payload in a highly reducing environment.