Synthesis, characterization and cytotoxic evaluation of chitosan nanoparticles: In Vitro Liver Cancer Model (preliminary report)

Aim . To evaluate cytotoxic effect of chitosan nanoparticles (CSNPs) in vitro human liver cancer cell model (HepG-2) and their possible application as drug delivery system. We synthesized water soluble chitosan nanoparticles, investigated their properties and extensively evluated their cytotoxic activities on a cellular and molecular levels. Methods. Human liver cancer cell line (HepG-2) was used as a model of human liver cancer. Chitosan nanoparticles were characterized using transmission electron microscopy (TEM), Fourier-Transform Infrared spectroscopy(FT-IR), Zeta analysis. Cytotoxic effects of CSNPs on HepG-2 cells were followed by colorimetric SRB cell viability assays and flow cytometric analysis. Molecular investigations included DNA fragmentation and expression of some apoptotic genes on a transcriptional RNA level were performed. Results. Treatment of HepG-2 with different concentrations of 150 nm diameter CSNPs did not show alteration of cell morphology after 24 hrs of cell exposure. Also, when cells treated with 100µg/ml of CSNPs, they killed 12% of cells and IC50 reached at 239µg/ml after 48h of cell exposure. Flow cytometry evaluation of CSNPs revealed mild accumulation in the G2/M phase followed by cellular DNA fragmentation after 48h of cell exposure. Extensive evaluation of cytotoxic effect of CSNPs showed mRNA apoptotic genes expression (P53, Bak, Caspase3 ) after 24h of cell exposure with no expression of mRNA of caspase 3 gene after 48h of cell exposur, suggesting involvement of intrinsic apoptotic caspase independent pathway by increasing exposure time of 100µg/ml of chitosan nanoparticles. Conclusion.  Our engineered CSNPs nanoparticles was controlled to 150nm size and charges of 40mV and a concentration of 100µg/ml revealed genotoxic effect on HepG-2 after 48h of cell exposure through intrinsic apoptotic caspase independent mechanisms. Further quantitative analysis on the molecular and protein levels are still required to confirm impact of chitosan size and time on genotoxic effect before reaching a final conclusion and starting its biomedical application.