| Background:High intensity focused ultrasound(HIFU),as a model in the field of ultrasound treatment,has been widely used in clinical applications.HIFU is mainly used in the treatment of various benign and malignant tumors,such as uterine fibroids,prostate cancer,liver cancer and so on.The biological mechanism of high intensity focused ultrasound on tumor therapy mainly includes thermal effect and mechanical effect: when the temperature of the ablated tumor tissue rises to about 55 to 60 °C for a duration of several seconds,coagulative necrosis will occur;tiny bubbles in the acoustic field will produce inertial and non-inertial cavitation.Shear stress,micro jet and shock wave facilitate HIFU to mechanically disintegrate the tumor tissue.During the HIFU treatment,imaging guidance is indispensable,and the main guidance technology for HIFU therapy is ultrasound imaging and magnetic resonance imaging.At present,scholars have found that after HIFU irradiation in some patients with abdominal or pelvic mass,apparent peritoneal adhesions are found in local organs such as bowel,normal uterus and appendages when the patients receive surgery.Some of the HIFU experts in clinical practice have repeatedly encountered reducing preset power or interruption of treatment in some patients with uterine fibroids,pancreatic cancer or thyroid tumor,due to severe pain in areas beyond the focal region.In view of the complications in the treatment of HIFU,to improve the accuracy of image guidance,enhance the ablation efficacy of HIFU,and reduce the intensity threshold of HIFU treatment is of great importance.Therefore,this study intends to the encapsulate mesoporous silica nanocapsules(MSN)with liposomes to prepare a novel theranostic nanoparticle-mesoporous silica nanocapsules encapsulated liposomes(MSN-LIPO).The hollow interior cavity of MSN contains a small amount of isoamyl acetate with high boiling point and easy volatilization characteristics,which serves as the cavitation agent for enhancing HIFU therapy;the MSN shell contains Fe3O4,which serves as MRI T2 weighted imaging agent.And we verified the functions of MSN-LIPO on improving HIFU image guidance and therapy efficacy both in vitro and in vivo.Part? The Preparation and Characterization of Mesoporous Silica Nanocapsules Encapsulated LisosomesObjective To prepare mesoporous silica nanocapsules encapsulated liposomes(MSN-LIPO)and blank liposomes(B-LIPO),and to detect its particle size,structure characteristics,stability,cell uptake,biocompatibility and other characteristics.Materials and Methods Thin film hydration method was used to prepare MSN-LIPO and B-LIPO.Particle sizes and Zeta potentials of MSN-LIPO and B-LIPO was tested by nanoparticle size and zeta potential analyzer.The structure characteristics of MSN-LIPO was defined by transmission electron microscopy(TEM).The particle size of MSN-LIPO was continuously measured for 4 weeks to verify its stability.Confocal scanning laser microscopy(CSLM)and flow cytometry were adopted to confirm the cell uptake of MSN-LIPO by U87 MG human malignant glioma cells.And the biocompatibility of MSN-LIPO was test by cytotoxicity assay.Results The prepared MSN-LIPO was rust colored,and B-LIPO was light milky white.The particle sizes of MSN-LIPO and B-LIPO was(128.6±2.11)nm and(86.9±0.59)nm,respectively.And the Zeta potential was(-22.9±0.77)m V and(-22.7±0.20)m V respectively.Transmission electron microscopy showed that the MSN was successfully encapsulated by liposomes,and the particle size was similar to that measured by the particle size and Zeta potential analyzer.The particle sizes of MSN-LIPO continuously measured for 4 weeks had no statistical significance(F=0.378,P>0.05),which indicated the good stability of MSN-LIPO.CSLM confirmed that U87 MG glioma cells could successfully uptake MSN-LIPO with good time dependence.The fluorescence intensity of different time groups had statistical significance(F=675.81,P < 0.05).SNK-q multiple comparisons showed that the fluorescence intensity of 30 min group had statistical significance with 0min group with increased cellular uptake of MSN-LIPO(q=8.90,P < 0.05);the fluorescence intensity of 1h group had statistical significance with 30 min group with increased cellular uptake of MSN-LIPO(q=2.43,P < 0.05);the fluorescence intensity of 2h group had statistical significance with 1h group with increased cellular uptake of MSN-LIPO(q=19.77,P < 0.05);the fluorescence intensity of 6h group had statistical significance with 2h group with increased cellular uptake of MSN-LIPO(q=14.05,P < 0.05).Cytotoxicity assay showed that different concentrations of MSN-LIPO had no significant cytotoxicity on U87 MG glioma cells,MCF-7 human breast cancer cells and SK-BR-3 breast cancer cells.The residual cell viability were all above 85%,which indicated the good biocompatibility of MSN-LIPO.Conclusion MSN-LIPO was successfully prepared with small particle size,good stability,favorable cellular uptake and good biocompatibility.Part? Mesoporous Silica Nanocapsules Encapsulated Liposomes Enhance Magnetic Resonance T2 Weighted ImagingObjective To detect the magnetic property of MSN-LIPO,verify the enhanced T2 weighted magnetic resonance imaging function,and investigate the application value of MSN-LIPO as a HIFU contrast agent.Materials and Methods In vitro MRI phantom was prepared to carry different concentrations of MSN-LIPO.Siemens MAGNETOM Trio a Tim 3T MRI was used to verify the magnetic property of MSN-LIPO.BALB / C nude mice malignant glioma model was conducted.MRI T2 image of the liver parenchyma was captured before and 5min after intravenous injection of MSN-LIPO.MRI T2 image of the tumor was captured before and 5min after intra-tumor injection of MSN-LIPO.And the MRI T2 image of the tumor was captured before and 30 min after intravenous injection of MSN-LIPO.Results In vitro MRI indicated that MSN-LIPO had MRI T2 weighted negative enhancement function.The MRI signal intensity decreased gradually with the increase of MSN-LIPO concentration.Based on the relaxation rate curve,MSN-LIPO had a r2 value of 413 m M-1s-1.In vivo MRI showed that the signal intensity of the liver parenchyma before and 5min after intravenous injection of MSN-LIPO was 91.84 ± 14.41 and 30.34 ± 2.61 respectively with statistical significance(t=7.27,P<0.05);the signal intensity of the tumor before and 5min after intra-tumor injection of MSN-LIPO was 211.44 ± 5.34 and 15.34 ± 1.24 respectively with statistical significance(t=36.38,P<0.05);the signal intensity of the tumor before and 30 min after intravenous injection of MSN-LIPO was235.99 ± 5.17 and 179 ± 4.35 respectively with statistical significance(t=14.34,P<0.05).Conclusion The prepared MSN-LIPO had a good MRI T2 weighted contrast enhancement function.Part ? Mesoporous Silica Nanocapsules Encapsulated Liposomes Enhance the Tumor Ablation Efficacy of HIFUObjective In vitro,to verify the synergistic effect of MSN-LIPO on HIFU ablation on tumor cell culture;in vivo,to verify the synergistic effect of MSN-LIPO on HIFU ablation on BALB/c nude mice tumor model.And to investigate the application value of MSN-LIPO as a HIFU sensitizer.Materials and Methods The samples of MSN-LIPO and B-LIPO after HIFU radiation were observed under microscope to inspect the formation of microbubbles.In vitro,CCK-8 test was conducted to define the residual viability of U87 MG cells after HIFU radiation with different intensities.Accordingly,optimized HIFU parameter was adopted to test the HIFU ablation effect of different groups:(1)Control,(2)HIFU,(3)HIFU + B-LIPO,(4)HIFU + MSN-LIPO.After HIFU radiation of different groups,CCK-8 test was used to detect the cell proliferation activity,and flow cytometry was used to determine the percentage of apoptosis and necrosis cells.In vivo,35 tumor-bearing nude mice was included.5 mice were set as Control group,and the other 30 mice were divided into 6 groups as therapuetic groups,5 for each: 180 V,5s,(1)HIFU,(2)HIFU + B-LIPO,(3)HIFU + MSN-LIPO;220V,5s,(1)HIFU,(2)HIFU + B-LIPO,(3)HIFU + MSN-LIPO.After treatment,nude mice from Control group and 180 V,5s radiation groups received contrast enhanced ultrasound examination to detect the blood perfusion of the tumors.Finally,all the tumor were taken out from the nude mice,and TTC staining method was used to inspect the HIFU ablation efficacy of different groups.And the coagulative necrosis volumes were calculated.Results Microbubble formation was observed in the samples of MSN-LIPO after HIFU ablation under microscope,while it was negative for B-LIPO samples.In vitro,the residual cell viability was 70% after HIFU ablation at the parameter of 40 V,1min.And this parameter was selected for further experiment.For Control group,the residual cell viability was 100%;for HIFU group,the residual cell viability was 70%;for HIFU + B-LIPO group,the residual cell viability was 72%;and for HIFU + MSN-LIPO group,the residual cell viability was 52%.Statistical significance was detected between groups by One-way ANOVA test(F=193.7,P<0.05).SNK test showed that the residual cell viability of HIFU + MSN-LIPO group has statistical significance compared with HIFU group(P<0.05).Flow cytometry showed that cell apoptosis and necrosis rate was 1.61% for Control group,20.37% for HIFU group,23.45% for HIFU + B-LIPO and 42.86% for HIFU + MSN-LIPO.Statistical significance was detected between groups by One-way ANOVA test(F=332.37,P<0.05).SNK test showed that cell apoptosis and necrosis rate of HIFU + MSN-LIPO group has statistical significance compared with HIFU group(P<0.05).In vivo,contrast enhanced ultrasound examination showed that tumors of the Control group had good blood perfusion;tumor blood perfusion of the HIFU group and HIFU + B-LIPO group decreased;and tumor blood perfusion of the HIFU + MSN-LIPO decreased significantly.TTC staining showed that,at the HIFU parameter of 180 V,5s,the damaged volume was(232.73±19.77)mm3 for HIFU group;(238.46±20)mm3 for HIFU + B-LIPO group;(771.75±19.33)mm3 for HIFU + MSN-LIPO group.Statistical significance was detected between groups by One-way ANOVA test(F=1234.09,P<0.05).SNK test showed that the damaged volume of HIFU + MSN-LIPO group has statistical significance compared with HIFU and HIFU + B-LIPO group(P<0.05).At the HIFU parameter of 220 V,5s,the damaged volume was(325.13±29.44)mm3 for HIFU group;(343.91±29.91)mm3 for HIFU + B-LIPO group;(1474.88±59.8)mm3 for HIFU + MSN-LIPO group.Statistical significance was detected between groups by One-way ANOVA test(F=1218.12,P<0.05).SNK test showed that the damaged volume of HIFU + MSN-LIPO group has statistical significance compared with HIFU and HIFU + B-LIPO group(P<0.05).More importantly,the damaged volume of 180 V,5s HIFU + MSN-LIPO group had statistical significance compared with 220 V,5s HIFU group(t=28.349,P<0.05).Conclusion MSN-LIPO showed good synergistic effect for HIFU tumor ablation both in vitro and in vivo. |
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