Expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats

Abstract

Aim: To study the expression of human insulin gene wrapped with chitosan nanoparticles in NIH3T3 cells and diabetic rats. Methods: pCMV.Ins, an expression plasmid of the human insulin gene, was constructed. In total, 100 μg pCMV.Ins wrapped with chitosan nanoparticles (chitosan-pCMV.Ins) was transfected to NIH3T3 cells and diabetes rats through lavage and coloclysis, respectively. The transfected cells were grown in Dulbecco's modified Eagle's medium, containing G418, for 72 h after transfection. The clones were selected and continued to grow in G418 medium for 24 d. The expression of human insulin was detected by immunohistochemistry. Human insulin in the culture medium of transfected cells was measured. Fasting blood glucose and plasma human insulin of diabetic rats were measured for 5 d after transfection. RT-PCR and Western blotting were performed to confirm the expression of the human insulin gene in diabetic rats. Results: Approximately 10% of NIH3T3 cells transfected by chitosan-pCMV.Ins expressed human insulin. Human insulin in the culture medium of NIH3T3 cells transfected by chitosan-pCMV.Ins significantly increased compared with that of the control group (P<0.01). Fasting blood glucose levels of the lavage group and the coloclysis group decreased significantly in 5 d (P<0.01) in comparison, while plasma insulin levels were much higher (P<0.01). The human insulin gene mRNA and human insulin were only detected in the lavage and the coloclysis groups. Conclusion: The human insulin gene can be transfected and expressed successfully by chitosanpCMV.Ins in NIH3T3 cells and diabetes rats, which indicates that chitosan is a promising, non-viral vector for gene expression.

In Utero Gene Delivery Using Chitosan-DNA Nanoparticles in Mice

“…4 and 6). The paradoxical observation of in vivo transfection despite quantitatively low levels of transfection in vitro occurred in our study, as well as in the two studies discussed above [37,38]. This phenomenon may be explained, at least in part, by higher levels of bioavailable chitosan nanoparticle in vivo, or due to accumulated, sequential transfection of cells in vivo, resulting from the longer in vivo exposure time.…”

“…Although cell viability was not significantly reduced by acidification, the low transfection efficiency and the dependence on low pH point to the limitations of this specific chitosan polyplex and the model itself, which does not address barriers to target cell delivery in vivo. Another study has looked at in vitro and in vivo chitosan polyplex transfection with the human insulin gene [37]. The transfection efficiency of NIH 3T3 cells (a mouse embryonic fibroblast cell line) with expressed human insulin gene measured by immunohistochemistry was 10%, yet the reported in vivo transfection of rat upper gastrointestinal mucosa and rectum following gastric and colonic lavage respectively was much higher, as measured by RT-PCR and Western blot analysis.…”

Development and Characterization of Nanoparticles for the Delivery of Gemcitabine Hydrochloride

“…Another successful strategy to ameliorate the biopharmaceutical properties of the hydrophilic compound was the use of colloidal drug carriers, that is, nanoparticles. Nanoparticles with a mean diameter of 10–1000 nm are widely used as a carrier in drug delivery system [12, 13]. Tumor cells, hepatic Kupffer cells, and cells of the mononuclear phagocyte system have higher phagocytic rates for the uptake of nanoparticles than cells of other tissues, thus increasing the distribution of GEM in tumors, liver, and spleen [14].…”

Preparation of albumin nanospheres loaded with gemcitabine and their cytotoxicity against BXPC-3 cells in vitro

“…Nanospheres-spherical nanoparticles with a mean diameter of 10-1000 nm-are widely used as carriers in drugdelivery systems in clinical applications [7][8][9] . However, there are no published studies on GEM-loaded serum albumin nanospheres.…”