Nir Laser Responsive Nanoparticles For Ovarian Cancer Targeted Combination Therapy With Dual-Modal Imaging Guidance

PART ONE:THE PREPARATION AND CHARACTERIZATION OF NIR LASER RESPONSIVE NANOPARTICLESObject1.A targeted nanoparticle（FBPD NPS）containing Bismuth sulfide（Bi₂S₃）and doxorubicin（Dox）was prepared by double emulsification method,and its physical and chemical properties were detected.2.The folic acid targeting performance of FBPD NPS as a drug carrier in vitro was evaluated.Method1.Bi₂S₃,PFP and Dox were encapsulated in FA-PEG-PLGA by ultrasonic double emulsification method to prepare FBPD NPs;The morphology,uniformity,particle size,potential,encapsulation rate and drug loading of Bi₂S₃and Dox of the nanoparticles were also detected;The release of Dox in vitro was detected by UV spectrophotometer;The photothermal conversion efficiency of nanoparticles was monitored by a near-infrared thermal imager（808 nm）;The results of photothermal phase transition of the nanoparticles were observed by inverted fluorescence microscope.2.It is known that folate receptors are highly expressed on the surface of SKOV-3 ovarian cancer cells,so folate-modified FBPD NPs and unmodified BPD NPs were co-incubated with SKOV-3 ovarian cancer cells,and the phagocytosis of nanoparticles was detected by laser confocal method.Result1.FBPD NPs were successfully prepared by ultrasonic double emulsification.The size and the zeta potential of the nanoparticles were measured by Malvern Instruments and determined to be 295.3±40.3 nm and-7.99±6.66 m V,respectively.Scanning electron microscopy（SEM）and transmission electron microscopy（TEM）detected that the nanoparticles were uniform in size and uniformly distributed in a regular spherical shape,and the Bi₂S₃was successfully encapsulated in the shell of the nanoparticle.The measured encapsulation rate of Bi₂S₃is（87.48±0.19）%,and the drug loading is（3.38±0.07）%;the encapsulation rate of Dox is（25.21±3.05）%,and the drug loading is（4.07±0.49）%.Through UV spectrophotometer detection,it was found that after irradiated by 808nm NIR laser,the in vitro release rate of Dox increased significantly.Thermal imager showed that nanoparticles have high photothermal conversion efficiency,and FBPD NPs could quickly undergo liquid-gas phase transition after being irradiated by 808 nm NIR laser.2.Since folate receptors are highly expressed on the surface of SKOV-3 ovarian cancer cells,folic acid-modified FBPD NPs could be efficiently phagocytosed by ovarian cancer cells due to receptor-ligand mediation,while BPD NPs that are not modified by folic acid are significantly less phagocytosed by ovarian cancer cells.Conclusion1.Successfully prepared Bi₂S₃and Dox loaded NIR laser responsive nanoparticles（FBPD NPs）by ultrasonic double emulsification method,and tested the basic properties of the nanoparticles.2.As a drug carrier,FBPD NPs have good biological safety.Under the mediation of receptor-ligand interaction,folic acid-linked modified FBPD NPs could be effectively taken up by SKOV-3 ovarian cancer cells with high receptor expression.PART TWO: EVALUATION OF IN VITRO CT/PA DUAL-MODALITY IMAGING OF NIR LASER RESPONSE NANOPARTICLES AND PHOTOTHERMAL COMBINED CHEMOTHERAPY AGAINSTSKOV-3 OVARIAN CANCER CELLSObject1.Study the imaging ability of FBPD NPs as the CT/PA contrast agents in vitro.2.Evaluate the photothermal/chemotherapy therapeutic effect induce by FBPD nanoparticles to SKOV-3 cells in vitro.Method1.Put different concentrations of FBPD NPs and PBS in test tubes,collect images in GE CT and measure the corresponding CT signal values.Add different concentrations of FBPD NPs and PBS to the agar gel module,and collect PA images.Then the corresponding signal intensity was measured.2.Divide SKOV-3 cells into 8 groups and treat them accordingly（i:FBPD NPs with laser irradiation（FBPD NPs + Laser）;ii: FBPD NPs without laser irradiation（FBPD NPs）;iii: BPD NPs with laser irradiation（BPD NPs + Laser）;iv: BPD NPs without laser irradiation（BPD NPs）;v:FBP NPs with laser irradiation（FBP NPs + Laser）;vi: FBP NPs without laser irradiation（FBP NPs）;vii: Laser only;viii: Control）.After each group of nanoparticles were incubated with SKOV-3 cells for 6 hours,the CCK-8method was used to detect and calculate the survival rate of ovarian cancer cells in each treatment group.Thus evaluated the ability of each treatment to resist SKOV-3 cells.Result1.FBPD NPs have excellent CT and PA imaging effects.And as the concentration of FBPD NPs increased,the CT signal and PA signal also increased linearly.On the contrary,the PBS group did not show obvious CT and PA signals.2.The relative survival rate of ovarian cancer cells in the FBPD NPs +laser group was the lowest（P<0.01）.Conclusion1.FBPD NPs showed excellent enhanced imaging capabilities in in vitro CT/PA dual-modal imaging experiments,proving their potential as CT/PA dual-modal imaging contrast agents.2.The photothermal combined chemotherapy guided by FBPD NPs had the most significant effect against SKOV-3 ovarian cancer cells.PART THREE: THE DUAL-MODALITY CT/PA IMAGING OF THE NIR LASER RESPONSE NANOPARTICLES IN VIVO AND EVALUATION OF THE CHEMO/PHOTOTHERMALTHERAPY TO THE SKOV-3 NUDE MICEObject1.Assess the biological safety of FBPD NPs and their distribution in the body.2.Evaluate the in vivo CT/PA imaging capabilities of FBPD NPs.3.Evaluate the chemo/photothermal therapeutic effect to the SKOV-3tumor-bearing nude mice.Method1.Firstly,the FBPD NPs at a concentration of 5 mg/m L and saline solution were i.v.injected into the mice,and the liver function,kidney function,routine blood and blood biochemical of the mice were measured after 1 d,3 d,7 d,14 d,and 28 d.Fluorescence imaging system was used to study the distribution of FBPD NPs with folate target and BPD NPs without folate target in mice.2.FBPD NPs and BPD NPs were i.v.injected into nude mice,then the tumor area was imaged with spiral CT and PA imager,and the CT/PA images were collected Perform analysis.3.Divide SKOV-3 cells into 8 groups and treat them accordingly（i:FBPD NPs with laser irradiation（FBPD NPs + Laser）;ii: FBPD NPs without laser irradiation（FBPD NPs）;iii: BPD NPs with laser irradiation（BPD NPs + Laser）;iv: BPD NPs without laser irradiation（BPD NPs）;v:FBP NPs with laser irradiation（FBP NPs + Laser）;vi: FBP NPs without laser irradiation（FBP NPs）;vii: Laser only;viii: Control）.Comprehensive evaluation of the effect of each treatment group on tumor proliferation by comparing size of tumor,body weight of mice and pathological sections.Result1.The nude mice injected with FBPD NPs showed no significant difference in liver function,kidney function,blood biochemistry,routine blood and the pathological sections of the nude mice’s internal organs compared with the control group.In vivo fluorescence results showed that after 3 hours of injection,the accumulation of Di R-containing FBPD NPs in the tumor area reached the strongest.And compared with non-targeted nanoparticles,folic acid-modified targeted nanoparticles accumulate more in the tumor area.2.Compared with non-targeted nanoparticles without folic acid modification,targeted nanoparticles have better CT imaging and PA imaging effects in tumors.3.Comprehensive evaluation of tumor growth showed that the FBPD NPs + laser group has the most significant inhibitory effect.Conclusion1.FBPD NPs had excellent biological safety in nude mice.The optimal time for FBPD NPs to accumulate in the tumor area was 3 h,and it accumulated more in the tumor area than BPD NPs.2.FBPD NPs used as CT/PA dual-modal imaging contrast agents,which showing excellent tumor imaging capabilities in nude mice.3.Photothermal combined chemotherapy induced by folic acid-modified FBPD NPs had the best inhibitory effect on tumor growth.