Synthesis and in vitro cellular evaluation of novel anti-tumor norcantharidin-conjugated chitosan derivatives

Norcantharidin-conjugated chitosan conjugates (NCTD-CSs) with different degrees of substitution (DS, 60.2% and 97.9%) were synthesized and characterized by 1H NMR, FT-IR, and XRD. In comparison with CS, the NCTD-CSs had lower crystallinity and better water solubility. Less than 6% NCTD was released from the NCTD-CSs through the hydrolysis of ester bonds in phosphate-buffered solution (PBS, pH 5.0 and 7.4) within 16 days, showing sustained drug release characteristic. The result of cytoxicity study showed that the NCTD-CSs were cytotoxic to MGC80-3 cells and their cytotoxicity was lower than that of NCTD. Fluorescence microscope and flow cytometry analysis demonstrated that the amount of the NCTD-CSs uptaken by MGC80-3 cells increased as incubation time. Confocal microscopy study showed that the NCTD-CSs were internalized into cells by endocytosis and mainly localized in lysosomes within 24h. It was found that the NCTD-CSs arrested MGC80-3 cell cycle at G2/M phase and induced cell death via cell apoptosis similarly to NCTD. These results indicated that the NCTD-CSs might be an efficient NCTD delivery system for cancer therapy.