No 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.

The prospects for mRNA vaccines are very promising. Like other types of nucleic acid vaccines, mRNA vaccines have the potential to combine the positive attributes of live attenuated vaccines while obviating many potential safety limitations. Although data from initial clinical trials appear encouraging, mRNA vaccines are far from a commercial product. These initial approaches have spurred innovations in vector design, non-viral delivery, large-scale production and purification of mRNA to quickly move the technology forward. Some improvements have already been tested in preclinical models for both prophylactic and therapeutic vaccine targets and have demonstrated their ability to elicit potent and broad immune responses, including functional antibodies, type 1 T helper cells-type T cell responses and cytotoxic T cells. Though the initial barriers for this nucleic acid vaccine approach seem to be overcome, in our opinion, the future and continued success of this approach lies in a more extensive evaluation of the many non-viral delivery systems described in the literature and gaining a better understanding of the mechanism of action to allow rational design of next generation technologies.

Poly(ethylene glycol) (PEG) modified thiolated gelatin nanoparticles (PEG-SHGel) were developed as a long-circulating passively-targeted delivery system that respond to intracellular glutathione concentrations to enhance DNA delivery and transfection. Reporter plasmid expressing enhanced green fluorescent protein (EGFP-N1) was encapsulated in the nanoparticles. DNA-containing gelatin (Gel) and thiolated gelatin (SHGel) nanoparticles were found to have a size range of 220-250 nm, whereas surface modification with PEG resulted in particles with slightly larger size range of 310-350 nm. PEG modification was confirmed by electron spectroscopy for chemical analysis (ESCA), where an increase in the ether peak intensities of the C1s spectra corresponds to the surface presence of ethylene oxide residues. In addition, the PEG-SHGel nanoparticles released encapsulate plasmid DNA in response to varying concentrations of glutathione (0 -5.0 mM GSH in phosphate buffered saline). The stability of the encapsulated DNA was confirmed by agarose gel electrophoresis. Lastly, from the qualitative and quantitative results of in vitro transfection studies in murine fibroblast cells (NIH-3T3), PEG-Gel and PEG-SHGel nanoparticles afforded the highest transfection efficiency of the reporter plasmid. The results of these studies show that PEG-modified thiolated gelatin nanoparticles could serve as a very efficient nanoparticulate vector for systemic DNA delivery to solid tumors where the cells are known to have significantly higher intracellular glutathione concentrations.