Cellular interactions and in vitro DNA transfection studies with poly(ethylene glycol)-modified gelatin nanoparticles

In order to develop a systemically administered safe and effective nonviral gene delivery system, cellular interactions and plasmid DNA transfection with poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticles were examined. The DNA-containing nanoparticles were prepared by a controlled water-ethanol solvent displacement method. The nanoparticles were characterized for particle size, surface charge, and DNA loading, release, and stability. For cellular interaction studies, the control and PEGylated gelatin nanoparticles, complexed either with colloidal gold for transmission electron microscopy or loaded with rhodamine-dextran for fluorescence confocal microscopy, were incubated with NIH-3T3 fibroblast cells. At different time points, the location of the nanoparticles in the cellular environment was investigated. Furthermore, a reporter plasmid expressing the enhanced green fluorescent protein was encapsulated in the control gelatin and PEGylated gelatin nanoparticles for in vitro transfection studies. DNA-containing nanoparticles were prepared in the size range of 100-500 nm, with an average of 200 nm. PEGylated gelatin nanoparticles, with a slight negative surface charge, could stably and efficiently encapsulate plasmid DNA. Both transmission electron microscopy and confocal microscopy images showed that the gelatin and PEGylated gelatin nanoparticles rapidly entered the cell through nonspecific endocytosis followed by vesicular transport in the cytosol. Almost 100% of the administered gelatin and PEGylated gelatin nanoparticles were internalized in NIH-3T3 cells within the first 6 h of incubation. A large fraction of the administered nanoparticles was found to be concentrated in the perinuclear region of the cells after 12 h. Green fluorescent protein expression was observed after 12 h of nanoparticle incubation and remained stable for up to 96 h. Flow cytometry results showed that the DNA transfection efficiency with gelatin and PEGylated gelatin nanoparticles was 43% and 61%, respectively, after 96 h. The results of this study illustrate that PEGylated gelatin nanoparticles were rapidly internalized by the cells through nonspecific endocytosis and remained intact in the cytosol for up to 12 h. In addition, the DNA-encapsulated PEGylated gelatin nanoparticles were found to efficiently transfect cells in culture.