Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for treatment of human hepatocellular carcinoma

Abstract

The therapeutic efficiency of combined chemotherapy and gene therapy on human hepatocellular carcinoma HepG2 cells was investigated using double-walled microspheres that consisted of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(l-lactic acid) (PLLA) shell layer and fabricated via the precision particle fabrication (PPF) technique. Here, double-walled microspheres were used to deliver doxorubicin (Dox) and/or chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53), loaded in the core and shell phases, respectively. Preliminary studies on chi-DNA nanoparticles were performed to optimize gene transfer to HepG2 cells. The transfection efficiency of chi-DNA nanoparticles was optimal at an N/P ratio of 7. In comparison to the 25-kDa branched polyethylenimine (PEI), chitosan showed no inherent toxicity towards the cells. Next, the therapeutic efficiencies of Dox and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor suppressor p53 and apoptotic caspase 3 proteins. Overall, the combined Dox and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either Dox or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. High p53 expression was maintained during a five-day period, and was largely due to the controlled and sustained release of the microspheres. Moreover, increased activation of caspase 3 was observed, and was likely to have been facilitated by high levels of p53 expression. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy.