Study On Grafted Redox-responsive Nanoprobe In The Theranostic Of Liver Carcinoma

Objective:To discuss the physicochemical property,biosecurity,anti-tumor effect and enhanced magnetic resonance imaging effect of amphipathic,smart and targeting grafted nanoprobe HA-SS-PZLL@SPIO/DOX for diagnosis and treatment of liver carcinoma.Methods:1.Grafted polymeric micelles HA-SS-PZLL with disulfide bond was synthetized by chemical method and formed HA-SS-PZLL@SPIO/DOX nanoprobe for diagnosis and treatment of liver carcinoma with Super paramagnetic Iron Oxide（SPIO）and Doxorubicin（DOX）in aqueous medium by self-assembly method;¹H Nuclear Magnetic Resonance Spectrum（¹HNMR）and Fourier Transform Infrared（FTIR）was adopted to analyze the distinctive proton peak and absorption peak and to confirm the successful synthesis of nano-micelle;Dynamic Light Scattering（DLS）and Transmission Electron Microscope（TEM）was adopted to detect the particle size and morphology of micelle;pyrene fluorescence method was adopted to detect the critical micelle concentration（CMC）of micelle;entrapment efficiency of DOX and loading content of SPIO was determined and evaluated by DOX and SPIO content.Besides,release efficiency and drug delivery（reduction responsiveness determination）of micelle was determined and evaluated by DOX content.T₂ relaxation rate of nanoprobe was calculated by MR scan to reflect the enhanced imaging effect.In the laboratory,HA-PZLL@SPIO/DOX without disulfide bond was prepared for control study.2.Cellular level:Through control experiment,cell viability was calculated by MTT assay toevaluate thebiosecurityof HA-SS-PZLLmicelle,and the lethalityof HA-SS-PZLL@SPIO/DOX nanoprobe on human hepatoma cells;DOX fluorescence observation,DAPI nucleus staining and flow cytometry was adopted to do qualitative and quantitative analysis on the intake and intracellular distribution of human hepatoma cells to nanoprobe and on the anti-tumor ability of nanoprobe;Average T₂ value of cell suspension was measured to do analysis and comparison and to evaluate the enhanced MR imaging effect of nanoprobe in vitro.3.Animal level in vivo:BALB/c nude-mouse with liver cancer in situ model and subcutaneous tumor-bearing model were established and divided into groups for dosing control.Tumorous tissue was confirmed and drug safety on main organs was evaluated by HE staining;blue staining cell distribution was observed by Prussian blue staining;iron distribution of main organs and tumor was given a qualitative detection;tumor growth curve was drawn and tumor inhibitory rate was calculated by measuring the tumor volume and weight in subcutaneous tumor-bearing model to give a statistical analysis and to evaluate the anti-tumor effect of nanoprobe;Average CNR value and T₂ value of tumor was measured by MR inspection of subcutaneous tumor-bearing model to do analysis and comparison and to evaluate the enhanced MR imaging effect of nanoprobe in vivo.Results:1.The distinctive proton peak and absorption peak of HA-SS-PZLL grafted polymeric micelles was confirmed by ¹HNMR and FTIR;the average particle size of HA-SS-PZLL micelle measured by DLS was 128.5±5.7 nm;HA-SS-PZLL micelle and HA-SS-PZLL@SPIO/DOX nanoprobe was spherical with average particle size of 91.5±3.5 nm and 105.5±4.8 nm respectively via TEM measurement;HA-SS-PZLL was measured by pyrene fluorescence method to have relatively low CMC value of 0.0092 mg/mL;entrapment efficiency and loading content of micelle was over 50%by being detected with DOX and SPIO content,and the nanoprobe gained more efficient drug delivery under reducing condition with GSH;higher T ₂ relaxation rate(231.0 Fe m M^-1·s^-1)of nanoprobe was found by MR inspection.2.It was detected by MTT method that HA-SS-PZLL grafted polymeric micelles had high biosecurity with over 80%cell survival rate（48h incubation）,under the condition of high drug concentration（10 ug/m L）,the killing effect of tumor cells in group HA-SS-PZLL@SPIO/DOX and group HA-PZLL@SPIO/DOX was close to that of group Free DOX（incubating 48 h）.Fluorescence observation,DAPI nucleus staining and flow cytometry analysis results showed:fluorescence in Free DOX group was stronger than that in drug-loaded micelle group.Free DOX mainlyenteredintocellnucleus;however,HA-SS-PZLL@SPIO/DOXand HA-PZLL@SPIO/DOX was mainly located in cytoplasm.MRI results in vitro showed that average T₂ value in HA-SS-PZLL@SPIO/DOX group was significantly lower than that in blank control group and SPIO group.3.The average weight of the experimental rats in HA-SS-PZLL@SPIO/DOX group was better growth than that of the control group,and there was no obvious damage to the main organs showed in the HE staining,so it had outstanding biological safety;prussian blue staining showed that more iron aggregated on tumor tissue（it meant drug distribution）in HA-SS-PZLL@SPIO/DOX group;during the treatment cycle,the volume and weight of subcutaneous bearing tumor model in group HA-SS-PZLL@SPIO/DOX was significantly smaller than that in the control group,with a statistically significant difference;by MR inspection of subcutaneous tumor-bearing model,average T₂ value and T₂WI signal of tumor in HA-SS-PZLL@SPIO/DOX group decreased more significantly than that in control group with the difference of statistical significance.Conclusion:HA-SS-PZLL@SPIO/DOX nanoprobe prepared in the laboratory has good physicochemical property,possesses sound biosecurity,intelligence and targeting in vitro and vivo,and has relatively good anti-tumor effect and MR enhanced imaging effect,which is beneficial to the application of diagnosis and treatment of liver carcinoma.