Effect Of Pluronic F127 Modified Graphene Oxide Loading Doxorubicin On Treating Human Glioma: In Vitro Experiment

Objective:Study the toxicology of graphene oxide(GO) and pluronic F127 modified graphene oxide(PF127-GO) on human astrocytes cell and human glioma cell. Perform PF127 modified GO loading doxorubicin(PF127-GO-DOX) and evaluate its effect on treating human glioma. In order to provide new methods for exploring the technology of diagnosis and treatment of glioma. Methods:The experiment is divided into three parts:In the first part, the toxicity of GO and PF127-GO were studied on human astrocytes(AS) cell and human glioma(U251) cell. Using Cell Counting Kit(CCK-8) to detect cell viability. Experimental groups:(1) GO: 5μg/ml, 10μg/ml, 20μg/ml, 50μg/ml, 100μg/ml, 200μg/ml;(2) PF127-GO: 5μg/ml, 10μg/ml, 20μg/ml, 50μg/ml, 100μg/ml, 200μg/ml;(3) Control. Different concentrations of GO and PF127-GO were cultured with AS cell and U251 cell for 24 h, 48 h, 72 h, in respectively. The cell viability was detected by Cell Counting Kit(CCK-8). Analysis the toxicity of GO and PF127-GO on human astrocytes cell and human glioma cell. Determine the safe concentration rang of GO and PF127-GO. In this part, astrocytes cell is primary isolated human fetal brain astrocytes, identified by GFAP immunofluorescence.The second part, developed PF127 modified GO loading doxorubicin(PF127-GO-DOX) and detect it. Loaded doxorubicin(DOX) onto PF127-GO via simple π-stacking. Use UV-visible spectrophotometer to detect if weather the drug were loaded successfully, draw the DOX standard curve, using the data to calculate the drug loading rate. Preparation of FITC-PF127-GO and FITC-PF127-GO-DOX, observe the distribution of nanoparticles by fluorescence microscopy.The third part, evaluate the effect of PF127-GO-DOX on treating human glioma. A series different concentrations of PF127-GO-DOX and DOX were cultured with U251 cell for 24 h, 48 h, 72 h, in respectively. Using Cell Counting Kit(CCK-8) to detect cell viability. Analysis the toxicity of PF127-GO-DOX and DOX on human glioma cell(U251), compare the difference of PF127-GO-DOX and DOX, select the appropriate concentration in further experiments. Different concentrations of PF127-GO, PF127-GO-DOX and DOX were cultured with U251 cell for 24 h, 48 h, 72 h, in respectively. Using Cell Counting Kit(CCK-8) to detect cell viability. Using invert microscope to observe cell morphologic change. Evaluate the effect of PF127-GO-DOX on human glioma. Results:(1) There are no obvious toxicity of PF127-GO and GO to human astrocytes cells. under 5μg/ml.(2) PF127-GO and GO under 5μg/ml were not observed obvious toxicity to human glioma cells.(3) PF127 modified GO loading Dox was designed, Dox loading ratio was 0.83mg/mg.(4) At the same dose of DOX, the cell survival rate decreased with the increase of the concentration of DOX and PF127-GO-DOX, and there was no significant difference between the two groups. Conclusion:(1) Small concentration of GO and PF127-GO have no obvious toxic effect on AS and U251 cells.(2) Doxorubicin was loaded on to PF127-GO(PF127-GO-DOX), with efficiency 0.83 mg doxorubicin per gram PF127-GO via noncovalent interactions.(3) PF127-GO-DOX exhibited significantly impoved therapeutic efficacy for human glioma cell in vitor.