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Preparation and detection of oxygen and indocyanine green loaded microparticlesObjective: To prepare a novel oxygen and indocyanine green(ICG)loaded microparticles(OI-MPs)and to evaluate its characteristics.Methods: OI-MPs were prepared by gas-driven coaxial flow focusing(CFF)method.The particle size and color were observed using microscope and confocal microscopy.The diameter of synthesized OI-MPs was measured by Image-Pro Plus 6.0.The optical property and stability of OI-MPs were evaluated by ultraviolet–visible U-2810 spectrophotometer and Xenogen IVIS Imaging System.The dissolved oxygen level was tested by a dissolved oxygen instrument in the presence and absence of US.The entrapment efficiency(EE)and loading efficiency(LE)of ICG was monitored by ultraviolet–visible U-2810.Results: The fluorescence image indicated that the ICG was encapsulated within the microparticles.The OI-MPs were dispersed individually with a well-defined spherical shape and homogeneously distribution.The diameter of synthesized OI-MPs measured by Image-Pro Plus 6.0 was(37.5 ± 4.3)?m,which was quite stable without the size change for several months.The EE of ICG encapsulated in OI-MPs was(98.39 ± 1.18)%,while the LE was(0.02 ± 0.005)%.OI-MPs were able to release oxygen after sonication.The fluorescence property of OI-MPs was significantly more stable than that of free ICG(P < 0.05).Conclusion: OI-MPs were successly synthesized by a gas-driven CFF process.This OI-MPs may become a novel type of multiple imaging and therapy in ovarian cancer.PART? The study of ultrasound mediated OI-MPs destruction in multiple imagingObjective: To identify the multiple imaging effect of OI-MPs before and after ultrasound irritationMethod: The OI-MPs was activated by an ultrasound(1 W/cm2,the ultrasound frequency was 1 MHz,the duty cycle was 100%,and duration was 30 s).The Ultrasound images of the phantom were acquired by an M5 multifrequency linear transducer before and after irradiation.Xenogen IVIS Imaging System was used to collect the fluorescence signals before and after excitation while superposing pork from 0 to 1.5 cm.The protocol was also repeated for free ICG alone in PBS solution.Then using post-processing software analysed the intensity of fluorescence image.The results were normalized by the initial fluorescence intensities.Result: The mean diameter of the OI-MPs before stimulation was(37.56 ± 4.33)?m;and reached to(178.37 ± 13.78)?m after stimulation.The individual microparticles increased approximately 6 times the diameter of the original microparticles.The backscatter signals in the ultrasonography was significantly enhanced after irritation.Compared to free ICG and OI-MPs after stimulation.the fluorescence intensity of OI-MPs before stimulation was strongest in all depths.And the detectable NIR penetration depth was observed to 1.5 cm.However,After stimulation by ultrasound,The fluorescent intensity was decreased but still stronger than free ICG in PBS.And the maximum detectable depth of free ICG was 1 cm.Conclusion: The acoustic droplet vaporization(ADV)process of OI-MPs could be induced by low intensity ultrasound.At the same time,significantly enhanced the ultrasound imaging and fluorescence imaging.The OI-MPs could be used as a dual-mode imaging.PART? The effection and mechanism of ultrasound mediated oxygen and indocyanine green loaded microparticles on the apoptosis and proliferation in SKOV-3 ovarian cancer cellsObjective: To investigate the effection and mechanism of ultrasound mediated OI-MPs on the proliferation and apoptosis in SKOV-3 ovarian cancer cells.Method: We use Singlet Oxygen Sensor Green kit to investigate the production of ROS in the group of Control,Ultrasound+OI-MPs,Ultrasound+OI-MPs+Na N3 in cell free system.Then,the 96-well plate was randomly divided into the following groups:(1)Control group(Control),(2)Ultrasound only(US),(3)OI-MPs followed by ultrasound destruction(OI-MPs + US).The concentration of OI-MPs was 0.36 mg/m L(5.8x104 of microparticles).The cells were treated with OI-MPs followed by ultrasound immediately.After various treatments,cells were incubated at 37 ? in a humidified 5% CO2 atmosphere for 6 h or 24 h before further operation.At 6 h after treatment,the apoptosis of cells was determined by Hoechst 33342/PI and Annexin V-FITC/PI staining with flow cytometry.The cell viability was detected by MTT at 24 h after treatment.Result: The fluorescence intensity was significantly increased in OI-MPs(107.39 ± 8.01)% compared with the control(16.66 ± 2.89)% and OI-MPs + Na N3(21.30 ± 5.04)%,respectively.Control group stained with light blue fluorescent and almost no PI red fluorescence.Compared with control group,cells treated with ultrasound alone had no significant changes.Whereas,typical cellular shrinkage,floating round shapes and bright blue as well as red staining were observed in the groups of US + OI-MPs.The apoptosis efficacy induced by singlet oxygen after ultrasound mediated OI-MPs explosion was evaluated quantitatively by flow cytometry.The apoptosis efficiencies of Control,US and US+OI-MPs were(3.10 ± 0.52)%,(5.93 ± 0.94)% and(34.11 ± 3.57)%,respectively.It showed that ultrasound mediated OI-MPs explosion induced significantly cell apoptosis as compared with control group and US(p < 0.05).Compared with the control group(96.73 ± 3.14% of cell viability),the cytotoxic effect of US + OI-MPs(49.97 ± 6.29% of cell viability)was significant increased(p < 0.05).The result was consistent with the apoptosis experiment.Conclusion: These results indicated in the presence of ultrasound,the ROS could be incuced by Ol-MPs effectively.Ultrasound mediated OI-MPs destruction significantly enhanced the apoptosis and decreased the cell viability of SKOV-3 cells.The mechanism may be related to the singlet oxygen and cavitation induced by ultrasound mediated OI-MPs destruction. |
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