| Objective: A nanoparticle with tumor cell nuclear targeting was synthesized by coupled with bovine serum protein and nuclear location sequence(NLS)on its surface,the effects and mechanism of Tio2-NLS nanoparticles on radiosensitivity were investigated by human glioma U251 cell and the therapeutic effect was study by exenograft-bearing nude mice.Methods: in vitro : 1.The bovine serum albumin(BSA)were grafted onto the surface of titanium dioxide by the high absorption capacity of titanium dioxide in accordance with certain proportion(1:1)in acidic aqueous solution,then the NLS were modified on it,s surface by free radical polymerization.The structure were characterized by transmission electron microscope(TEM)and dynamic light scattering(DLS),the distribution of nanoparticles were characterized by laser confocal microscopy.2.The effects of nanoparticles combined with X-ray on the viability and proliferation of glioma cells(U251)were detected by MTT and Clonogenic survival assay,the effects of Tio2-BSA and Tio2-NLS with or without IR on u251 cell cycle were analyzed respectively.3.The contents of reactive oxygen(ROS)in the nanoparticle treatment group and the combined treatment group were detected by DPPH.4.The ?-H2 AX foci number in U251 cell in presence of Ti O2-NLS was observed by laser confocal microscopy 12 or 24 hours after X-ray irradiation and the effects of Ti O2-NLS on apoptosis with or without radiation were measured by flow cytometry.in vivo 1.The anti-tumor effect of Ti O2-NLS combined with X-Ray in exenograft-bearing nude mice were evaluated by comparing the volume and weight of tumor tissues after a certain treatment time.2.The retention of Ti O2-NLS and the apoptosis of tumor after combined with X-ay radiation were observed by HE and Tunal fluorescence staining.Result: 1.The TEM results showed that modified with bovine serum can improve the solubility of Tio2 nanoparticles in aqueous solution.The diameter of Ti O2 ?Ti O2-BSA and Ti O2-NLS nanoparticles were 1025±52,43±25 and 45±321respectively.In addition,the Zeta potential values of three nanoparticles were-25.24±2.30,-2.85±1.50 and ±5.32±1.58 respectively.Confocal laster scanning microscope indicated the nanoparticles coupled with NLS accumulated inside the nuclear region and the counterpart is stuck in the cytoplasm..2.MTT assays and Clonogenic survival assay showed that Ti O2-NLS nanoparticles combined with X-ray can reduce the activity and proliferation of U251 cells,Cell cycle experiments show that Ti O2-NLS nanoparticles can block cells in G2/M phase 3.The results of DPPH showed that Ti O2-NLS nanoparticles stimulate the production of reactive oxygen,which is 1.3 times higher than that in the control group without X-ray,and 1.7 times higher than the 8Gy group when combined with X-ray 4.Immunofluorescence experiments showed that Ti O2-NLS nanoparticles can strengthen the X-ray on glioma cell DNA damage and the cell need longer time to complete repair,the data of flow cytometry showed that compared with irradiation group,Ti O2-NLS nanoparticles combined with X-ray could increase the apoptosis rate of U251 cells by 1.55 times.5.The antitumor assay in vivo showed that Ti O2-NLS nanoparticles combined with X-ray radiation can promote the effect of inhibit tumor growth.HE staining observed large area necrosis of tumor cells around the nanoparticles,and Tunel fluorescence staining showed that Ti O2-NLS nanoparticles combined with X-Ray could promote the apoptosis of tumor cells.Conclusion :The Ti O2 nanoparticles coupled with NLS can accumulate inside the nuclear region,inhibit the growth of subcutaneous glioma effectively and enhance the damage of X-ray to glioma cells combined with X-Ray. |
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