| Section one:Synthesize PIO_NPs and detect its basic characteristicsObjective:To synthesize Oxygen-carried and PTX/ICG-loaded nanoparticle(PIO_NPs)and study its basic characteristics.Methods:The PIO_NPs was synthesized by a double emulsification method.The morphology,size and zeta potential before and after near-infrared irradiation were measured by optical microscope and Malvern Zetasizer.The drug loading efficiency(LE)and encapsulation efficiency(EE)of PTX were detected by HPLC analysis.The LE and EE of ICG were measured by UV spectrophotometry.Controlled release of PTX and optical stability obtained by the above methods.Moreover,the capability to generate singlet oxygen of PIO_NPs after laser and ultrasound exposure were detected by using a SOSG kit.Results:The PIO_NPs were prepared successfully with 186.4±1.9 nm diameter and-19.43±0.55 mV zeta potential.After phase-shifted by irradiation,the size was larger than before,but zeta potential increased no significantly.The LE of PTX and ICG were 3.46±0.21%and 1.73±0.15%,respectively.The absorption spectra and fluorescence spectra of PIO_NPs were basically consistent with that of free ICG,but more stable than ICG.Within 15 days,the absorption intensity of PIO_NPs decreased by almost20%,the fluorescence intensity decreased by nearly 25%,but ICG decreased by over 80%.PTX spontaneously released 41.65±1.69%within 48h and released 86.65±3.43%after laser and ultrasound exposure.The reactive oxygen species(ROS)produced by PIO_NPs was detected by SOSG.After exposed to laser and ultrasound,the fluorescence intensity of SOSG increased by 319.76±12.54%in PIO_NPs group,compared with the control group,P<0.001.Conclusion:The PIO_NPs can be successfully prepared which has drug-loading and phase change ability.And it is more stability than free ICG.Moreover,it can be controlled release PTX and produce ROS.Section two:Dual-mode imaging of PIO_NPs in vitro and in vivoObjective:To explore the imaging ability of PIO_NPs in ultrasound and photoacoustic imaging in vitro and in vivo.Methods:An agar gel phantom with holes was used as container in study in vitro.The NPs or free ICG solution was deposited into holes.During each agent exposed to laser-irradiation(1.5W/cm~2,2 min),B-mode and contrast mode images were captured by ultrasonic diagnostic instrument and were measured the Echo Intensity(EI).PA-mode images of the samples were obtained and analyzed using the VEVO LASR PA imaging system by method described above.Some similar Tumor-bearing nude mice were anesthetized with 1%sodium pentobarbital.Then,B-mode and PA-mode imaging were performed.The mice were injected with PIO_NPs through tail vein.Images were obtained at 2,4,6,12,14,48 and 72h respectively.In additional,after injection of free ICG and PIO_NPs,the tumors were subjected to NIR irradiation(1.5W/cm~2,5min)and obtain images.Analyzed the Echo intensity(EI)in B-Mode and the PA average value by the same method as imaging in vitro.Results:In B-mode and CEUS,there were obvious enhanced after irradiation in O_NPs group and PIO_NPs group.The EI of PIO_NPs group increased more significantly,increasing from 34.21±2.09 to 90.80±3.77 for B-mode and from 3.82±0.76 to 50.24±2.59 for CEUS.There were no significant differences in PA imaging for PBS and O_NPs group.The photoacoustic average value(a.u.)of ICG group was decreased from0.40±0.03 to 0.12±0.04 after irradiation,but the value of PIO_NPs groups increased after irradiation from 0.41±0.02 to 1.25±0.07.In the in vivo experiment,4-12 h after injection of PIO_NPs,the PA signal was significantly enhanced at the tumor and peaked at 6 h.The average PA value of PIO_NPs was 0.26±0.02,increased by 39.73%after irradiation.Conclusion:PIO_NPs have the effect of enhancing ultrasound and photoacoustic imaging before and after laser irradiation,and it is a potential dual-mode imaging contrast agent.Section three:Therapeutic effect of PIO_NPs in vitro and in vivoObjective:To investigate the therapeutic effect and mechanism of PIO_NPs on SKOV3 cells and tumor-bearing nude mice.Methods:The uptake of PIO_NPs by SKOV3 cells were observed by laser confocal microscopy and analyzed by flow cytometry.The in vitro experiments were as follows:(1)control group;(2)LU group(only laser/ultrasound);(3)free PTX group;(4)IO_NPs+LU group;(5)PI_NPs+LU group;(6)PIO_NPs+LU group.The cell survival rate was measured by MTT assay,apoptotic rate was obtained by cytometry and reactive oxygen species were detected using ROS kit.In vivo experiments,the bearing-tumor mice were divided into five 5groups:(1)control group;(2)PTX group;(3)PI_NPs+LU group;(4)IO_NPs+LU group(5)PIO_NPs+LU group.Body weight and tumor size were measured during treatment.Eight nude mice were randomly killed in each group 24h after treatment and collect tumor tissues.Five of them were detected tumor cell apoptotic index(AI)by TUNEL,intratumoral microvessel generation were detected by immune-histochemical test of VEGF and CD34.The other tumors were determined the expression of HIF-1a and MDR-1 by western blotting.In additional,the remaining nude mice in each group were observed for a total of 60 days and the survival curves were plotted.Results:PIO_NPs could be taken by SKOV3 cells and the uptake rate was 96.45±3.96%,significant higher than free ICG,P<0.001.The cell survival rate in the PIO_NPs+L.U group was 38.17±4.41%,and the apoptotic rate was 74.09±7.64%.There was a statistically significant difference from other groups,P<0.005.Compared with control,the ROS generation rate was increased by 160.04±9.20%in PIO_NPs+L.U group,P<0.05.In vivo experiments,the weight of the tumor-bearing mice was20.20±0.35g after PIO_NPs+L.U treatment,while control group was down to 18.73±0.15g.And the tumor growth inhibition value of PIO_NPs+L.U group was 89.42%,it was significant compared with other groups,P<0.05.After PIO_NPs+LU therapy,the apoptotic index(AI)of tumor cells was61.36±5.08%,which was higher than that in other groups(P<0.005).And the microvessel density was 16.33±2.34/HP,lower than others,P<0.005.Western blot results showed that HIF-1a/GAPDH was 0.54±0.06,MDR-1/GAPDH was 0.40±0.07,were significantly lower than other groups,P<0.05.In addition,the median survival time for the control group was 35days,while the PIO_NPs+L.U group was extended to 55 days.Conclusion:PIO_NPs have significant therapeutic effects on SKOV3cells and tumor-bearing mice after laser and ultrasound exposure.In addition to the effect of PTX chemotherapy,there is PSDT effect,which produces intracellular reactive oxygen species to enhanced anti-tumor efficacy.Moreover,PIO_NPs can also significantly improve the tumor hypoxia and drug resistance,which is beneficial to promote the therapeutic effect. |
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