| Part I:The synthesis and surface-modification of Fe3O4nanoparticlesObjective:(1)We increased the stability,dispersion and biological compatibility of Fe3O4nanoparticles in the electrolyte solution through optimalizing the synthesis and surface modification methods of the Fe3O4nanoparticles.(2) Detect the cell uptake of Fe3O4nanoparticles in tumor cells and their cell toxicity, leading to their further applications in tumor treatment in the future.Methods:(1) We synthesized Fe3O4nanoparticles by thermal decomposition method, and modified them by packaging TiO2, DSPE-mpeg, PAA to the surfaces of them, respectively.(2)Detected their characteristics:particle size analyzer to detect the aqueous size and dispersion, transmission electron microscopy (TEM) to detect the particle size and distribution, XRD to measure elements and their configuration, VSM to determine the magnetic characteristics;(3) To confirm the distribution of nanoparticles in cells, we used confocal fluorescent microscope to detect fluorescent linked nanoparticles distribution, use the light microscope and transmission electron microscope to detect the nanoparticles distribution.(4) Detected the temperature rising and cell uptake ratio of different concentration of Fe3O4@PAA nanoparticles under alternating magnetic field by infrared thermometer.(5)Detected the uptake quality of nanoparticles of tumor cells after incubated with different concentration of Fe3O4@PAA for24hrs.(6) HE staining to observe the nanoparticles distribution in major organs by tail vein injection of H22transplantation tumor model mice.(7)MTT assay to detect the toxicity of nanoparticles to the tumor cells.Results:(1) XRD showed that the characteristic peaks of Fe3O4nanoparticles prepared by thermal decomposition method fitted to the JCPDS card database. The size of Fe3O4nanoparticles was8-10nm, saturation magnetization was18emu/g. The sizes of Fe3O4@TiO2, Fe3O4@DSPE-mpeg, Fe3O4@PAA were260nm,120nm,68nm,respectively; The saturation magnetization of them were12emu/g,42emu/g,12emu/g,respectively; According to the results of the modifications, Fe3O4@PAA nanoparticles were chose as the material for the further experiments.(2) Fe3O4@PAA nanoparticles were observed in the cytoplasm of tumor cells by light microscopy, confocal microscope and transmission electron microscopy (TEM),respectively.(3) The temperature of nanoparticles suspension increased with the extension of time under500kHZ alternating magnetic field, and the rising of temperature was positive correlation with concentration. The nanoparticles in the cells were all at20ug without parallel with concentration.(4) We successfully established the mice model of H22xenograft, and we found that nanoparticles were mainly distributed in the tumor tissue after which were injected into the rat tail vein.(5)MTT assay results showed that the Fe3O4@PAA nanoparticles had no obvious inhibitory effect to H460,CNE-2and HNE-1cells in50-800ug/ml concentration.Conclusions:(1)We successfully synthesized Fe3O4nanoparticles by thermal decomposition method, and successfully took the surface modifications:Fe3O4@TiO2, Fe3O4@DSPE-mpeg, Fe3O4@PAA. According to characterization we proved Fe3O4@PAA nano materials were more fitted for the further biomedical experiments in grain size, dispersion,stability,super-paramagnetism.(2)Cell uptake experiments showed that nanoparticles were mainly distributed in the cytoplasm.(3) Fe3O4@PAA nanoparticles suspension in20ug/ml concentration under500KHZ alternating magnetic field did not have a distinct temperature increasing, but the temperature rose as the concentration increased;(4)Animal experiments showed that the Fe3O4@PAA nanoparticles had EPR effect, which mainly distributed in the transplantation tumor tissue;(5) MTT assay showed that the Fe3O4@PAA nanoparticles had no obvious toxicity to H460, CNE-2,and HNE-1cells. PartⅡ:Fe3O4nanoparticles intracellular hyperthermia enhanced tumor radiosensitivity under the alternating magnetic fieldObjective:(1) Fe3O4@PAA nanoparticles was taken into tumor cells and carried on intracellular hyperthermia under the alternating magnetic field, combined with radiotherapy, testing their killing effect on tumor cells, discussing tumor radiotherapy sensitization of nano intracellular hyperthermia and its mechanism.(2)Fe3O4@PAA nanoparticles were injected into the transplantation tumor of nasopharyngeal carcinoma cell in nude mice, carried on thermotherapy under the alternating magnetic field, and combined with radiotherapy, monitoring the volume of tumors, discussing the radiosensitivity of nano intracellular hyperthermia to the transplantation tumor of nasopharyngeal carcinoma cells in nude mice.Methods:(1) MTT assay to detect the inhibition of nanoparticles intracellular hyperthermia combined with radiotherapy on H460, CNE-2and HNE-1cells.(2) Formating colony assay to detect the radiosensitivity of nanoparticles intracellular hyperthermia under alternating magnetic field to three tumor cell lines.(3)Western-blot analysis to detect the HSP70and caspase-3protein expression of three tumor cell lines after treatment.(4)Flow cytometry to detect the apoptosis of three tumor cell lines after treatment by AnnexinV-FITC/PI double staining;(5) Flow cytometry to detect the cell cycle changes of three tumor cell lines after treatment by single PI staining.(6)Gamma H2AX foci assay to detect the number of foci changes with time after treatment in three tumor cell lines.(7)Draw tumors growth curve to detect the radiosensitivity of nanoparticles intracellular hyperthermia by treating the CNE-2transplantation tumor nude mice.Results:(1) MTT assay showed:H460cells:cell survival of radiotherapy group was61.7±4%, which of combination group was42±5%; CNE-2cells:cell survival of radiotherapy group was60.1±1.9%, which of combination group was40.6±1.4%after heat treatment; HNE-1cells:cell survival of radiotherapy group was56.2±2.6%, combined nanometer granulocyte cell survival was36.7±1.1%after heat treatment.(2) Formation colony assay results:the SER of H460cells=1.3847; the SER of CNE-2cells=1.2755; the SER of HNE-1cells=1.4421.(3) Western blot analysis results showed that protein expression in three tumor cell lines had the same trend:HSP70expression in control group was the same with nanoparticles group,which was lower than the nanoparticles intracellular hyperthermia group(P<0.01); Compared with radiation therapy group, which was significantly lower than that of combined with nanoparticles intracellular hyperthermia group (P<0.01). The caspase-3expression had the same trend with HSP70protein.(4)FCM-Apoptosis results:H460cells: apoptosis rate of radiotherapy group was24.57±0.6545%, which of combination group was34.92±0.6983%; CNE-2cells:apoptosis rate of radiotherapy group was25.77±0.3332%, which of combination group was60.53±0.8386%; HNE-1cell: apoptosis rate of radiotherapy group was18.09±0.1170%, which of combination group was48.35±0.3837%.(5)FCM-cell cycles results:H460cells:G2phase of control group=5.22±0.028%, G2phase of nano intracellular hyperthermia group=30.82±3.32%; CNE-2cells:G2phase of control group=6.72±2.16%, G2phase of nano intracellular hyperthermia group=24.24±1.47%; HNE-1:G2phase of control group=6.65±0.33%, G2phase of nanoparticles intracellular hyperthermia group=37.19±1.25%.(6) Gamma H2AX foci results:the number of foci in three tumor cell lines had the same trend:the foci reduced with time, the foci of radiotherapy combined intracellular group were much than the which of radiotherapy alone group obviously (P<0.01).(7)We successfully established CNE-2cells transplantation tumor model in nude mice, from the tumors growth curve we found that tumor growth of intracellular hyperthermia group was significantly suppressed compared with the control group (P<0.01); And tumor volume of intracellular hyperthermia combined radiotherapy group were significantly smaller than that of radiotherapy group (P<0.01).Conclusions:(1) Fe3O4@PAA nanoparticles under the alternating magnetic field heat the cells, which could enhance the radiosensitivity of tumor cells.(2) We investigated the mechanisms of radiotherapy sensitivity enhanced by Fe3O4@PAA nanoparticles under the alternating magnetic field:nanoparticles intracellular hyperthermia increased the expression of HSP70and caspase-3,which inceased the apoptosis of tumor cells.The intracellular hyperthermia of nanoparticles blocked tumor cell in G2phase, which is sensitive to irradiation; The nanoparticles intracellular hyperthermia could block the DSB repairs in tumor cells, and the DSB was caused by irradiation.(3)In nude mice experiments,we also found that the Fe3O4@PAA nano intracellular hyperthermia enhanced the tumor radiosensitivity. |
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