Biodistribution And Biocompatibility Of Dual-ligand Chitosan Nanoparticles As Novel Targeting Vehicle For Hepatocellular Carcinoma

Obsjective: To elucidate the in vivo biodistribution and targeting efficiency for hepatocellular carcinoma(HCC) cells of galactosylated chitosan nanoparticles modified with glycyrrhetinic acid(GCGA NPs). To further evaluate the cytotoxicity and biocompatibility of the NPs.Methods: The GACS and GCGA NPs were synthetized through two steps of amidation reaction reported in previous article, and fabricated with fluorescein isothiocyanate(FITC)by a chemical reaction. Morphological examination of the NPs was performed using field emission scanning electron microscope(FE-SEM), the size distribution was measured using a Zetasizer 3000 HS. In vitro cellular uptake assay was carried out to compare the targeting capacity of GCGA, GACS, and CS NPs for Hep G2 cells, and the affinity of GCGA NPs between Hep G2 cells and normal liver cells(LO2) under a confocal laser scanning microscopy(CLSM). The cytotoxicity of GCGA NPs at different concentrations to Hep G2 cells was measured by CCK-8 assay. Hemolysis assay in vitro was implemented to assess the hemocompatibility of GCGA NPs. The H22 cell-bearing mouse model in situ was was established by the mothed of liver tunnel embeddedness, and then FITC-labeled NPs solutions were injected to these mice via tail vein for tracking the NPs' biodistribution at different time points, so as to further verify the high targeting property of GCGA NPs for the hepatoma tissues, which over-express the receptors of glycyrrhetinic acid(GA) and lactobionic acid(LA). The biocompatibility assessment of the NPs was performed through testing body weight, hematologic and biochemical parameters, and observing main organs' HE stain slices of the Sprague-Dawley(SD) rats suffering GCGA NPs administration.Results : CS, GACS and GCGA NPs were prepared in compliance with the previously reported method, and then labeled with FITC. FE-SEM images showed that the prepared GCGA, GACS, and CS NPs are all spherical, monodisperse and size-uniform,their average size was about 60 nm. Dynamic light scattering(DLS) revealed the average diameter of them is approximately 93.8 nm, 90.3 nm, and 94.0 nm, respectively. The fluorescence intensity of confocal images of GCGA NPs were much stronger than that of GACS NPs and CS NPs, and a significant Green fluorescence enhancement in Hep G2 cells relative to that in LO2 cells deficient in targeting receptors under the same conditions. The cytotoxicity assay of GCGA NPs exhibited no statistically significant cytotoxicity to Hep G2 at concentration up to 50 ug/m L. The NPs didn't show apparent hemolytic activity to red blood cells(RBCs)even at the highest dose(300 ?g/m L) for 3 h, and Student's t test analysis of optical density(OD) values of the supernatants revealed no significant differences compared to saline(p >0.05). For the group of H22 tumor-bearing mice injected with FITC-GCGA NPs, the relative uptake by the liver tumor was the highest among all the tissues no matter at 4 h or 24 h after injection. The body weight, hematologic and biochemical parameters of SD rats treated with GCGA NPs showed no statistic variance in comparison with those treated with saline at each time point(p > 0.05), and the HE staining slices of major organs also displayed no obvious pathological changes.Conclusion:GCGA NPs represents a safe and effective vehicle for hepatocellular carcinoma,and might play a critical role in the field of HCC targeted chemotherapy in the near future.