The Construction Of CS-PDC-M Based Nano Systems And Anti-hepatoma Effects In Vitro And In Vivo

In this study,chitosan cross-linked pectin-doxorubicin conjugates macromolecular pro-drug(CS-PDC-M)was prepared to enhance the therapeutic effects on liver cancer.Preparation and characterization of CS-PDC-M: CS-PDC-M was synthesized and prepared through incorporating the positively charged chitosan onto the PDC-M surface with negatively charged pectin.Scanning electron microscopic was used to observe morphology of nano-micelles.The micells size,distribution and stability were analyzed by nano ZS90.Besides,drug loading efficiency was measured by UV spectrophotometer.The CS-PDC-M were observed to have an unregularly spherical shape and were uniform in size and the average particle size was about 140 nm.The encapsulation efficiency and drug loading were 57.82% ± 3.7%(n = 3)and 23.852% ±2.3%(n = 3),respectively.The CS-PDC-M achieved prolonged releasing ability was demonstrated by the in vitro drug release and in vitro cellular uptake assay.The biocompatibility study of CS-PDC-M in vitro: cell viability of CS-PDC-M and DOX in endothelial cells and L02 cells was evaluated by MTT;Hemolysis activity was investigated and protein adsorption of CS-PDC-M and DOX was tested by bovine serum albumin.The results revealed that the resulting CS-PDC-M possesses excellent cell compatibilities and low cytotoxicities in comparison with that of the free DOX.The in vitro study on anti-tumor effect of CS-PDC-M: Anti proliferative effect of CS-PDC-M and DOX in Hep G2 cells,MCF-7 cells and A549 cells was determined by MTT.According to the IC50 values,CSPDC-M against Hep G2 cells were more effective than CS-PDC-M against MCF-7 cells and A549 cells.The enhanced in vitro cytotoxicity of CSPDC-M against Hep G2 cells may be attributed to galactose acid of pectin that can combine with asialoglycoprotein receptor(ASGP-R)of mammalian hepatoma cells.To investigate the important role of galactose acid in the cellular uptake of CS-PDC-M,we conducted a receptor competition assay by selecting galactose acid as the competitive reagent.Consistent with our hypothesis,when PDC-M and galactose were added to the wells simultaneously,the fluorescence intensity of Hep G2 cells significantly decreased.It suggested that the cellular uptake of galactosylated nanomedicine was mediated by the ASGP-R.The in vivo study on anti-tumor effect of CS-PDC-M: The antitumor efficacy of PDC-M and CS-PDC-M was studied in athymic BALB/c nude mice bearing Hep G2 cell xenografts.Antitumor activity was evaluated in terms of tumor volume,body weight,tumor growth and terminal tumor weight.Compared to DOX,CS-PDC-M had higher in vivo safety and therapeutic activity in animal models.Organ damage assays of CS-PDC-M: The organ damage assays were performed by evaluating corresponding functional enzymes with fully automatic clinical chemistry and immunoturbidimetric analyzer.SD rates treated with free DOX showed increased ALT values(mean value 146.0 ± 20.59 U/L)and CK-MB values(mean value 2644.4 ± 731.997 U/L)in plasma,which is indicative of early signs of liver and heart damage.Besides,when the DOX doses dropped from 5 mg/kg to 2.4 mg/kg,mice still died before the end of the experiment in free DOX group owing to the toxicities of DOX.On the contrary,the parameters of CS-PDC-M group substantially maintained at normal levels,indicating that CS-PDC-M did not lead to serious damage in liver,kidney and heart of mice in the treatment duration.