Polyethylene Glycol Modified Gelatin Nanoparticles

To develop safe and effective systemically administered non-viral gene therapy vectors for solid tumors, PEGylated gelatin was synthesized and fabricated into nanoparticles. The PEG-modified gelatin was synthesized by reacting Type-B gelatin with PEG-epoxide that was further confirmed by electron spectroscopy for chemical analysis (ESCA), the results of which had shown an increase in the presence of an ether carbon (-C-O-) peak in the PEGylated gelatin nanoparticles. The experiments performed using these polymeric nanoparticulate carriers proved their ability to encapsulate DNA, improved transfection efficiency in vitro, and enhanced the biodistribution profile when the carriers were injected into mice bearing tumors.56-58

The in vitro cytotoxicity assays indicate that both gelatin and PEGylated gelatin are non-toxic to the cells. The cell uptake and trafficking studies of the control (gelatin) and PEGylated gelatin nanoparticles encapsulated with electron-dense gold nanoparticles confirmed that the particles were internalized by an endocytic pathway and remained stable during the vesicular transport. Further, the transfection studies carried out using the DNA (pEGFP-N1) encapsulated nanoparticles were internalized in NIH-3T3 fibroblast cells within the first six hours of incubation. Green fluorescent protein expression was observed after 12 h of nanoparticle incubation and remained stable for up to 96 hours. Flow cytometry results showed that the DNA transfection efficiency of PEGylated gelatin nanoparticles was better than that of gelatin.57

In order to determine the biodistribution profile, nanoparticles were radiolabeled with iodine-125 [125I] and intravenously injected into mice bearing Lewis lung carcinoma (LLC) tumors. From the radioactivity of the plasma and other organs collected, it was evident that the majority of the PEGylated gelatin nanoparticles remained in the blood pool or were taken up by the tumor mass and liver. A two-fold increase in concentration of the PEGylated nanoparticles was observed in plasma even at three-hour time points, and about 4-5% of the recovered dose of PEGylated gelatin nanoparticles was found to be present in the tumor mass for up to 12 h. Upon non-compartmental pharmacokinetic analysis of the plasma and tumor concentration-time profiles, it was observed that PEGylated gelatin nanoparticles had greater mean residence time in plasma and a more than six-fold increase in half-life in the tumor. The results of this study showed the stealth nature of the PEGylated gelatin nanoparticles in avoiding uptake by RES, allowing the nanoparticles to circulate longer in plasma.59

The in vivo transfection efficiency of plasmid DNA-encapsulated (pCMV-b encoding b-galactosidase) PEGylated gelatin nanoparticles was evaluated by injecting these nanoparticles intravenously into LLC tumor-bearing female C57BL/6J mice. Following systemic administration, the animals were sacrificed, and the transgene expression in different organs was quantitatively determined by using o-nitrophenyl-b-d-galactopyranosidase (ONPG), a clear substrate of b-galactosidase that is converted to a yellow-colored product with an absorbance maximum at 420 nm, and qualitatively by X-galĀ® tissue staining. When compared to the gelatin nanoparticles, the PEGylated gelatin nanoparticles were found to efficiently transfect the tumor cells with the b-galactosidase expression increasing at time points up to 96 h post-transfection with the absor-bance value at 420 nm increasing from 0.6 to 0.85 starting from 12 h post-transfection. The results of these studies clearly indicate that the long circulating, biocompatible, and biodegradable nano-particles of PEGylated gelatin would be ideal for gene delivery applications to solid tumors.60

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