| The unique cage structure of fullerene endows them with novel physical and chemical properties,such as excellent biocompatibility,biosafety,ROS scavenging capability,and anti-inflammation and so on.Thus,fullerenes are widely applied in biomedical fields including immunoregulation and cancer therapeutics.Curdlan is a polysaccharide derivative with superior biocompatibility,susceptibility to phagocytosis by macrophages,and immunoregulative ability.The design of the nanoparticles with fullerene and curdlan could not only improve the water solubility and biocompatibility of fullerenes,but also increase the phagocytosis of fullerenes by macrophages and strengthen the immunomodulation.Therefore,we synthesized a kind of curdlan functionalized fullerene derivative,and systematically explored the therapeutic effects of it on autoimmune hepatitis in vitro and in vivo.Firstly,the curdlan and fullerenes were chemically modified,respectively,the curdlan was modified by azidation,and the fullerene was modified by amino acid.Secondly,the hydrophilic modification of the branched chain of curdlan was introduced with,a small molecule with terminal amino and alkynyl group.The end of the alkynyl group was connected with the azide group of the curdlan through click reaction,while the amino terminal was connected with glycine on the fullerene through amide reaction.Finally,Cy7 was used as a fluorescent marker to quantitatively evaluate the biodistribution of nanoparticles in mice.The structure and grafting rate of the product were confirmed by NMR,MS,IR and elemental analysis.The grafting of fluorescent markers was clarified by UV spectra and fluorescence spectra.The particle size and zeta potential of the product were determined by DLS.And the effect of the product on scavenging hydroxyl radical was evaluated by ESR test.We investigated the cytotoxicity,ROS-scavenging ability,anti-oxidative damage and endocytosis by RAW264.7 cells of Cur-NH2,C60-gly and Cur-C60 in vitro.The results indicated that there was no observable cytotoxicity at a certain concentration（0-200μM）of Cur-NH2,C60-gly and Cur-C60.And the ROS levels in H2O2-treated RAW264.7 cells were reduced by C60-gly and Cur-C60,analyzing by confocal microscopy.In particular,Cur-C60 exhibited superior ROS-scavenging ability than C60-gly.Moreover,we found that C60-gly and Cur-C60at higher concentrations（200μM）could protect against H2O2-induced cellular death.Inspiringly,the endocytosis of Cur-C60by RAW264.7 cells was more than that of C60-gly,indicating that curdlan promoted the accumulating of in Cur-C60mcrophage.In vivo,the biodistribution of Cur-C60 was evaluated by fluorescence imaging,revealing that Cur-C60 mainly accumulated in the liver and kidney,instead of other organs after intravenous injection.This provided the possibility of Cur-C60 for liver-disease treatment.Then,the mouse model of autoimmune hepatitis was constructed by tail intravenous injecting Con A.The photos and HE staining analysis demonstrated that Cur-C60 effectively reduced the damage of liver,spleen and lymph nodes.The Elisa analysis suggested that Cur-C60 inhibited the secretion of inflammatory factors and alleviated autoimmune hepatitis.Notably,the histopathological analysis and blood routine tests indicated that,Cur-C60 possessed no significant toxicity with good biosafety and biocompatibility.Altogether,considering the excellent biological effects of fullerene and curdlan in the field of biomedicine,we synthesized a kind of macrophage-targeted curdlan functionalized fullerene derivative with excellent biocompatibility and biosafety,and systematically studied its therapeutic effect on autoimmune hepatitis.This study would provide a novel direction for the development of the drug delivery system for autoimmune hepatitis in the future. |
