Experimental Study On Multimodal Imaging And Phototherapy Combined With Radiotherapy Of IR780/Bi-DTPA-loaded Targeted Nanoparticles For Breast Cancer

PARTⅠSTUDY ON CT IMAGING AND RADIOTHERAPY OF BREAST CANCER WITH BI-DTPAObjective To investigate the feasibility and effect of Bi-DTPA in CT imaging on mouse model of breast cancer,and whether it has the characteristic of sensitizing radiotherapy.Methods Bi-DTPA was prepared by one-pot method,and its synthesis was verified by Fourier transform infrared spectroscopy(FT-IR),~1H-Nuclear Magnetic Resonance(~1H-NMR)and High resolution mass spectrum(HR-MS).In vitro,different concentrations of Bi-DTPA and Iopromide 370 were added to EP tubes for CT imaging,and CT values were recorded;in vivo,tumor-bearing mice were intratumoral injected with Bi-DTPA and Iopromide 370,then CT imaging was performed at different time points and CT values were recorded.After co-incubation of 4T1 cells with different concentrations of Bi-DTPA,CCK-8 was used to detect the cell activity.Radiotherapy was divided into(1)Bi-DTPA+X-ray group,(2)X-ray group,(3)Bi-DTPA group,and(4)control group.After treatments of four groups in vitro,4T1 cells were analyzed for clonal survival in(1)(2)groups,and were tested by CCK-8detection,ROS detection,DNA double-strand break experiment and flow cytometry detection in(1)-(4)groups;after treatments of four groups in vivo,body weight and tumor volume changes of tumor-bearing mice were recorded,and tumor tissues were taken for H&E staining,PCNA and TUNEL detection,and major organs tissues were taken for H&E staining on the second day after treatments.Results The white powder Bi-DTPA was obtained,and its successful synthesis was verified by FT-IR,~1H-NMR and HR-MS.In vitro CT imaging showed that CT values of Bi-DTPA group were higher than those of I 370 group at the same concentration.In both groups,there was a linear relationship between concentrations and CT values,and the slope of Bi-DTPA group was higher than that of I 370 group.In vivo CT imaging showed that tumors were enhanced rapidly after administration,and then CT values decreased gradually with time.CT values of bladders increased first and then decreased gradually,and reached the peak at 1h,which was delayed compared with the peak time of tumors.CT values of tumors and bladders in Bi-DTPA group were higher than those in I 370group during the whole process.When the concentration of Bi-DTPA was≤0.5mg/m L,the cell viability could be maintained above 90%.After in vitro treatments,the results showed that the cell proliferation ability decreased significantly in Bi-DTPA+X-ray group,which had the lowest cell viability,the highest intracellular ROS production,the most DNA damage sites,and the lowest cell survival rate.After in vivo treatments,the effect of inhibiting tumor proliferation was most obvious in Bi-DTPA+X-ray group,the cell nucleuses were significantly shrunken by H&E staining,the cell proliferation ability was the lowest by PCNA detection,and the cell apoptosis was the most by TUNEL detection.There was no obvious damage of main organs by H&E staining,and no difference in the body weight changes of mice among four groups.Conclusion Bi-DTPA was safe and feasible for CT imaging of breast cancer tumor,and it had the characteristic of radiosensitization,which could damage tumor cells and inhibit tumor proliferation by generating ROS.PART Ⅱ PREPARATION AND CHARACTERIZATION OF IR780/BI-DTPA-LOADED TARGETING NANOPARTICLESObjective To prepare IR780/Bi-DTPA-loaded targeted nanoparticles(PLGA-IR780-Bi-DTPA),to detect their characterization and properties,and to evaluate their morphological structure,safety,targeting and in vitro photothermal effect.Methods PLGA-IR780-Bi-DTPA were prepared by double emulsification method.The particle sizes and potentials were detected by Malvern particle size potential analyzer,the surface morphology was observed by scanning electron microscope(SEM),the internal structure and element distribution was observed by transmission electron microscope(TEM),and the encapsulation rate and drug loading of IR780 was detected by UV spectrophotometer.After co-incubation of 4T1 cells with different concentrations of nanoparticles,CCK-8 was used to detect the cell viability.Blood samples were taken from mice for blood routine and blood biochemical index detection,and major organs tissues were H&E stained at different time points before and after the injection of nanoparticles to evaluate the safety of nanoparticles.After co-incubation of Di I-labeled nanoparticles with 4T1 cells,the targeting of nanoparticles was observed under confocal laser scanning microscopy(CLSM).The temperature changes of nanoparticles at different concentrations under the excitation of 1W/cm2 808 nm laser,and at the concentration of 5mg/m L under the irradiation of different powers were recorded to evaluate their photothermal effects in vitro.Results Light green emulsion nanoparticles were successfully prepared.The particle size was(250.7±8.05)nm and the potential was(-4.81±1.94)m V.They were spherical with smooth surface,uniform size and good dispersibility under SEM,and shell-core structure under TEM.The encapsulation rate of IR780 was(83.61±1.61)%,and the drug loading was(3.34±0.06)%.The maximum safe concentration of nanoparticles in vitro was 2.5mg/m L,and the cell viability was maintained at(91.19±1.38)%;blood analysis results in mice were within normal range,and H&E staining of major organs tissues showed no abnomality at different time points before and after nanoparticle injection.Confocal laser scanning microscopy(CLSM)showed that nanoparticles were targeted to around the nucleuses of 4T1 cells and gradually increased over time.The maximum temperature of photothermal in vitro increased with the increase of nanoparticle concentrations,which reached 50 °C at 2.5 mg/m L and 60 °C at 5 mg/m L.The time to reach the maximum temperature was shortened with the increase of irradiation power at the concentration of 5mg/m L,and the temperature could be rapidly heated up and maintained at about 60°C under the power of 1W/cm2.Conclusion The IR780/Bi-DTPA-loaded targeted nanoparticles(PLGA-IR780-Bi-DTPA)were successfully prepared,with suitable size,good dispersibility,high safety,strong targeting and good photothermal property.PART Ⅲ STUDY ON MULTIMODAL IMAGING OF IR780/BI-DTPA-LOADED TARGETED NANOPARTICLESObjective To investigate the ability of IR780/Bi-DTPA-loaded targeted nanoparticles(PLGA-IR780-Bi-DTPA)in photoacoustic(PA),near-infrared fluorescence(NIRF)and CT imaging.Methods(1)PA imaging: In vitro,different concentrations of nanoparticles were added to agarose gel model,then photoacoustic imaging was performed with photoacoustic imager and photoacoustic values were recorded.In vivo,nanoparticles were injected by the tail vein of tumor-bearing mice,then photoacoustic imaging of tumors was performed.Images were taken at different time points and photoacoustic values were recorded.(2)NIRF imaging: In vitro,different concentrations of nanoparticles were added to 96-well plates,then fluorescence imaging was performed with small animal in vivo fluorescence imaging system and fluorescence values were recorded.In vivo,nanoparticles were injected by the tail vein of tumor-bearing mice,then fluorescence imaging of tumors was performed.Images were taken at different time points and fluorescence values were recorded.(3)CT imaging: In vitro,different concentrations of nanoparticles were added to EP tubes,then CT imaging were performed with CT imaging system and CT values were recorded.In vivo,nanoparticles were injected by the tail vein of tumor-bearing mice,then CT imaging of tumors was performed.Images were taken at different time points and CT values were recorded.Results(1)PA imaging: In vitro PA values increased with the increase of nanoparticle concentrations,and there was a linear relationship between them.In vivo PA values of tumors increased with time and peaked at 24 h.(2)NIRF imaging: In vitro fluorescence values increased with the increase of nanoparticle concentrations at first,reached the peak at 6.25 mg/m L,then decreased with the increase of nanoparticle concentrations.In vivo fluorescence values of tumors increased with time and peaked at 24 h.The fluorescence values of tumors were higher than that of main organs after 24 h(P<0.05).(3)CT imaging: In vitro CT values increased with the increase of nanoparticle concentrations,and there was a linear relationship between them.In vivo CT values of tumors increased with time and peaked at 24 h.There was no adverse reaction in the mice during the whole experiment in vivo.Conclusion PLGA-IR780-Bi-DTPA has good targeting and PA/NIRF/CT multimodal imaging ability in vitro and in vivo,and nanoparticles accumulation was highest at tumor areas 24 h after nanoparticle injection.PART Ⅳ STUDY ON PHOTOTHERAPY COMBINED WITH RADIOTHERAPY OF IR780/BI-DTPA-LOADED TARGETED NANOPARTICLESObjective To investigate the therapeutic effect of phototherapy combined with radiotherapy in vitro and in vivo with IR780/Bi-DTPA-loaded targeted nanoparticles(PLGA-IR780-Bi-DTPA).Methods The experimental groups were:(1)PLGA-IR780-Bi-DTPA(P)+X-ray+NIR group;(2)P+X-ray group;(3)P+NIR group;(4)X-ray+NIR group;(5)X-ray group;(6)NIR group;(7)P group;(8)control group.The therapeutic concentration of PLGA-IR780-Bi-DTPA was 2.5 mg/m L in vitro and 5 mg/m L in vivo;X-ray dose was 6 Gy;NIR wavelength was 808 nm and energy was 1 W/cm2.After treatments of 8 groups in vitro,4T1 cells were tested by CCK-8 detection,ROS detection,DNA double-strand break experiment and flow cytometry detection.After treatments of 8 groups in vivo,tumor temperature changes in(3)(6)(8)groups were recorded by near-infrared thermal imaging,blood oxygen changes of tumor area before and after NIR in(3)(6)groups were detected by photoacoustic instrument,and body weight and tumor volume changes of tumor-bearing mice in all groups were recorded.On the second day after treatment,the tumor tissues were taken for H & E staining,PCNA and TUNEL detection,and the main organ tissues were taken for H & E staining.Results After in vitro treatments,the results showed that P+X-ray+NIR group had the lowest cell viability,the highest intracellular ROS production(and higher than the sum of the P+X-ray group and the P+NIR group),the most DNA damage sites,and the lowest cell survival rate.During in vivo treatments,the temperature of the P+NIR group could reach 50°C in about 80 s and remained stable in the later period.The blood oxygen values increased from Pre(29.63±3.17)% to(58±4.43)% after NIR irradiation.After in vivo treatments,there was no significant difference in the growth trend of body weight of mice among 8 groups(P>0.05).In P+X-ray+NIR group,tumors were completely cured and the volume was reduced to 0.Cell nucleuses were completely disappeared by H&E staining,cells showed no proliferative ability by PCNA detection,and cells completely apoptotic by TUNEL detection.There was no damage of main organs in all groups by H&E staining.Conclusion PLGA-IR780-Bi-DTPA phototherapy combined with radiotherapy could not only kill the tumor more effectively,but also improve the efficacy of RT through improving the hypoxic environment in the tumor area by the thermal effect,which achieved a therapeutic effect of 1+1>2.