Folic Acid Conjugated Reduced Graphe Ne Oxide Loaded With Hematoporphyrin Monomethyl: Synthesis,characterization And Efficacy In Vitro And Vivo

In this study,a novel folic acid conjugated reduced graphene oxide was designed and prepared as targeting drug delivery system,into which a second generation photosensiter named hematoporphyrin monomethyl ether(HMME)was loaded.The mainly purpose is to explore the optical properties of this nanocarrier,and text its anti-tumor activity,in vitro and vivo.Firstly,the reaction system of DCC/NHS is used to activate folic acid to produce active ester(FA-NHS),which can synthetic folic acid-amino functionalized graphene oxide(FA-NH-GO)further.Then through physical stirring the HMM E adsorbed on folate amino functionalized graphene oxide.Then a series of characterization of the product had beendone.Also,some other pharmaceutical and optical properties was tested respectively.In vitro,CNE-2 cell was treated with the nanocarrier on a way of photodynamic therapy,and tested by MTT colorimetry and flow cytometry.In vivo,CNE-2 tumor-bearing mice model is established before being treated with the photosensitizer nanocarrier.In order to detect tissue distribution of the drug in diffe rent points of time after administration.The tissue distribution in vivo was also visually graphical by means of vivo imaing.Further more,pathological section of tumor,liver and kidney was made.Results of the characterizations show that HMME successfully loaded in FA-NH-GO,the drug loading rate was 69.8%.In-vitro release rate ofFA-NH-GO/HMME is over 90%within 24 h.Fluorescence quantum yields of HMME and FA-NH-GO/HMME were 0.71 and 0.55,respectively.Singlet oxygen quantum yield of FA-NH-GO/HMME was markedly higher than that of HMME.In in-vitro experiments,MTT assay showed that the concentrations of HMME and FA-NH-GO/HMME were both positively correlated with the cytotoxicity of CNE-2 nasopharyngeal carcinoma cells while independent of the laser energy density at drug concentration of less than 100?M and laser energy density of over 18 J/cm~2.Besides,the cytotoxicity was in marked positive correlation with both the concentration and laser energy density when the laser ener gy density did not exceed 18 J/cm~2.FCM indicated that the early apoptosis rates induced by HMME and FA-NH-GO/HMME were both over 80%.Meanwhile,a number early apoptotic nuclei was observed.After tail vein injection and in-vivo metabolism of the two drugs in tumor-bearing mice for 30 min,the tumor concentrations of these two drugs both peaked,which remained at fairly high levels in 180 min.Compared with HMME,FA-NH-GO/HMME exhibited lower and more stable accumulation concentrations in liver and kidney tissues within 3 h.After intratumor injection,the accumulation concentrations in liver and kidney were all lower than the tail vein administration group at the same time points,and HMME was eliminated faster than FA-NH-GO/HMME in the tumor tissues.The results of in-vivo imaging showed that FA-NH-GO/HMME was intensively distributed in the tumor tissues 2 h afteradministration.24 h later,the photosensitizer was almost eliminated in the tumor tissues.The novel drug carrier FA-NH-GO/HMME has similar optical properties to HMME,and its singlet quantum yield is quite high.Combined with the cytotoxicity assay,its PDT efficacy can be considered superior to HMME.The novel carrier has low accumulation concentration in organs like liver and kidney in vivo,whereas in the meantime,its metabolic duration is not shortened.