Study On Treatment Of Ovarian Cancer And Ultrasound Imaging In Vitro Using PTX-loaded Anti-LHRHR Targeted Phase Change Nanoparticles Combined With Low Intensity Focused Ultrasound

Objective1.To prepare PTX-loaded targeted phase-change lipid nanoparticles.The basic characteristics of its physical and chemical properties,drug loading rate,entrapment efficiency,in vitro release and so on.2.To investigate the cell viability of phase-change lipid nanoparticles in A2780 cells and to evalulate the cellular uptake of PTX-LHRHR-PCNPs by the A2780 tumor cells.3.To observe the conditions of liquid-gas phase transition and effects on ultrasound imaging in vitro.Methods1.PTX-targeted phase-change lipid nanoparticles were prepared by film-hydration method and single emulsion were carried out by accurately weighing DSPE-PEG2000-Biotin,DPPC and DPPA,respectively.The size and surface potential of PTX-LHRHR-PCNPs were measured by Malvern size zeta analyzer.The morphology of PTX-LHRHR-PCNPs were observed by transmission electron microscopy(TEM).The PTX phase transition lipid with different quality of paclitaxel was detected by high performance liquid chromatography The PTX encapsulation efficiency,PTX loading efficiency and drug release behavior were investigated by high performance liquid chromatography(HPLC)with absorption peak at 227 nm.The immunofluorescence assay was used to detect the connectivity LHRHR antibody and PCNPs.2.To evaluate the cellular targeting of LHRHR-targeted phase change lipid nanoparticles in vitro by the confocal fluorescence microscopy;the cell viability of PCNPs in A2780 tumor cell was measured using CCK8 assay.3.Thermotropic transformation process: The phase change lipid nanoparticles liquid to gas phase transition process on heating-plate was observed by optical microscope instantaneously.Acoustic droplet vaporization(ADV):After using low intensity focused ultrasound(LIFU)irradiation,the effects on ultrasound imaging were observed in vitro.Results1.The appearance of PTX-loaded targeted phase change lipid nanoparticles was well dispersed,uniform and spherical,The size of the particle size measured by Malvern particle size analyzer is about(508 ± 11.26)nm and the potential is about(-30.53 ± 6.34)mv.The images of transmission electron microscopy(TEM)show that the PTX-LHRHR-PCNPs are round and uniform in size.The results showed that the entrapment efficiency and drug loading rating were highest when the quality of PTX was 3mg,respectively.The drug release behaviors of PTX-PCNPs in 48 hours was observed that results showed that LIFU irradiation group of PTX release amount can reach 50%after 1h,while the control group of the release of just about 30%after 1h.48 h The cumulative release of LIFU irradiation group reached 95%after 48 h,and the cumulative release of the control group was about 80% after 48 h,which indicated that LIFU irradiation could accelerate the release of PTX.The connectivity rate of LHRHR antibody and PCNPs were showed by the specifically binding of FITC-labeled rabbit anti-human antibody that was(85.58 ± 2.01)% using flow cytometry.CCK8 was used to detect the cell viability.When the concentration of lipid phase change nanoparticles was reached to 1.5mg/ml,the viability of the cells was still no less than 80%,which indicated that the prepared lipid nanoparticles had low toxicity and biocompatibility;2.The results of flow cytometry showed that the binding rate of LHRH receptor-positive ovarian cancer A2780 cells to targeted phase change nanoparticles was 86.40 ± 2.85%.Confocal microscopy showed a large number of LHRHR-PCNPs combined with ovarian cancer A2780 cell membrane.3.When the temperature of heating-plate is reaching to 38.5℃,the phase change of the lipid nanoparticles starts to change phase,and the droplets gradually become microbubbles,and as the heating temperature becomes higher and higher,and the results showed that there was a good in vitro imaging at 37℃water bath with LIFU irradiation(5w,30min)in vitro,and the ultrasound imaging effect of PCNPs was still strong after 1h.Conclusion1.The PTX-LHRHR-PCNPs with high entrapment efficiency and drug loading rate were successfully prepared and had good ability of in vitro targeting to improve the local drug concentration and reduce systemic side effects with good treatment effect.2.Phase change lipid nanoparticles can undergo liquid-gas phase transition under specific conditions,which is stable and lasting in vitro development,and is expected to be a new type of contrast agent.