| Hepatocellular Carcinoma(HCC)is the most common primary malignant liver tumor.HCC is occult,mostly advanced to middle and advanced stage at the time of diagnosis,with high tumor heterogeneity,limited treatment and low sensitivity.It has been reported that nearly 70%of HCC patients have recurrence within 5 years after operation.With the development of tumor treatment methods,some new tumor therapy have been discovered.For example,Sonodynamic Therapy(SDT)has good targeting and non-invasive advantages in solid tumor treatment.Immune Checkpoint inhibitors(ICIs),which can inhibit tumor growth by regulating host immune response and tumor immune microenvironment,have their unique advantages in the treatment of advanced liver cancer.However,both SDT and ICIs have certain limitations in the clinical application of anti-solid tumor treatment.Therefore,this study attempts to prepare a carrier for the sound sensitizer and ICIs through specific modification of nanobubbles,and deliver the sound sensitizer and ICIs through ultrasound targeted microbubble destruction(UTMD)mediated nano-drug delivery system.So as to improve the concentration of drug aggregation and release in tumor area,and to combine sonodynamic therapy with immunotherapy.Therefore,this project aims to prepare sound sensitizer and ICIs nanobubbles(PD-L1 Ab/Ce6-NBs)with stable physical and chemical properties and strong targeting,and verify its inhibitory effect on tumor growth and explore its mechanism through anti-liver cancer experiment in mice.This topic is based on the combination of molecular targeted therapy,sonodynamic therapy and immunotherapy,in order to provide a new concept for the treatment of liver cancer.The main contents are as follows:Part Ⅰ:Preparation and Characterization of PD-L1 Ab/Ce6 NanobubblesObjective:To design and prepare lipid microbubbles carrying Ce6 and PD-L1 Ab(PD-L1 Ab/Ce6-NBs),and detect the general physical,chemical properties and stability of PD-L1 Ab/Ce6-NBs.Methods:PD-L1 Ab/Ce6-NBs were prepared by thin film hydration method.Optical microscope,laser confocal microscope and scanning electron microscope were used to observe the morphological characteristics and distribution of PD-L1 Ab/Ce6-NBs.The particle size range and ZETA potential of PD-L1 Ab/Ce6-NBs were measured by ZETA potential analyzer.The encapsulation rate of Ce6 was determined by membrane dialysis.The stability of PD-L1 Ab/Ce6-NBs was detected at 0-96 h.Results:The average particle size and Zeta potential of the PD-L1 Ab/Ce6-NB s were 460.0±80.7 nm and 9.9±4.4 mV,respectively.The microbubbles were uniform in size,with good dispersion,spherical shape and smooth surface were observed by optical microscope,laser confocal microscope and scanning electron microscope.The encapsulation rate of Ce6 was 71.22±4.26%.PD-L1 Ab/Ce6-NBs has no significant change in particle size,dispersion,and without obvious aggregation,agglomeration and other changes within 24h of the refrigerator at 4℃,and owned good encapsulation rate of Ce6.Conclusion:PD-L1 Ab/Ce6-NBs owned uniform particle size,good dispersion,high Ce6 entrapment efficiency,meets the requirements of molecular targeted ultrasound contrast agent,and its stability can meet the requirements of later in vivo and vitro.Part Ⅱ:The Reserch of Targeting and Pharmacokinetic on PD-L1 Ab/Ce6 NanobubblesObjective:To detect the connection rate of Biotin-PD-L1 Ab and Ce6 in PD-L1 Ab/Ce6-NBs,and the expression of PD-L1 on the surface of hepatocellular carcinoma cells so as to lay a theoretical foundation for targeting experiments in vivo and vitro.Then,the targeting ability of PD-L1 Ab/Ce6-NBs was detected in vivo and vitro.At the same time,the aggregation and metabolism of PD-L1 Ab/Ce6-NBs in vivo and tumor sites were fluorescence imaged by imaging of small animals in vivo.Methods:Firstly,the connection of Ce6 and Biotin-PD-L1 Ab to NBs were detected by confocal laser microscopy and flow cytometry,respectively.Then,the expression level of PD-L1 protein on the surface of mouse hepatocellular carcinoma H22 cells was detected by immunofluorescence and flow cytometry Finally,the ability of PD-L1 Ab/Ce6-NBs to target PD-L1 in vitro and vivo was verified by in vivo contrast-enhanced ultrasound and in vitro target seeking experiments.At the same time,Ce6 uptake test and reactive oxygen species generation were used to detect the sonodynamic therapeutic efficacy of different groups of Ce6,and the pharmacokinetic characteristics of Ce6 in mice and the accumulation of Ce6 in tumor sites were detected by in vivo imaging of small animals and frozen section of tumor tissue.Results:Confocal laser microscopy showed that Ce6 and Biotin-PD-L1 Ab were well connected to NBs in PD-L1 Ab/Ce6-NBs.The double positive rate of Ce6 and Biotin-PD-L1 Ab in PD-L1 Ab/Ce6-NBs detected by flow cytometry was about 63.37±5.53%.Immunofluorescence results showed that the expression of PD-L1 on the membrane surface of hepatocellular carcinoma cells was identified by FITC-PD-L1 Ab,and the positive rate of PD-L1 protein in hepatocellular carcinoma cells was 40.56±3.82%detected by flow cytometry.In vitro targeting experiments showed that PD-L1 Ab/Ce6-NBs could specifically adhere to the surface of hepatoma cells.The results of contrast-enhanced ultrasonography in mouse liver cancer transplanted tumor model showed that AUC and Imax in PD-L1 Ab/Ce6-NBs group were significantly higher than those in NBS group(P<0.05,P<0.01).The results of small animal imaging system and frozen sections of tumor tissue showed that fluorescence signal reached the peak at 6h after injection,and the concentration of Ce6 in tumor tissue in PD-L1 Ab/Ce6-NBs group was significantly higher than that in free-Ce6 group at 3h,6h,9h and 12h after injection(P<0.01).The aggregation time of Ce6 drugs in the tumor was longer,and the fluorescence of Free-Ce6 and Ce6-NBs disappeared at the tumor site after 36 hrs,while the red fluorescence signal of PD-L1 Ab/Ce6-NBs group can last for 48 hrs.The concentration of Ce6 in tumor tissues in PD-L1 Ab/Ce6-NBs group was significantly higher than that in free-Ce6 group(P<0.05).Conclusion:Ce6 and Biotin-PD-L1 Ab had a high connection rate to NBs in PD-L1 Ab/Ce6-NBs,meanwhile,PD-L1 was highly expressed on the surface of hepatocellular carcinoma cells;related experiments proved that PD-L1 Ab/Ce6-NBs had good activity in vitro and vivo targeting hepatocellular carcinoma cells.Meanwhile,the detection of Ce6 metabolism in vivo showed that PD-L1 Ab/Ce6-NBs could significantly enhance the local enrichment of Ce6 in tumor tissues and significantly prolong the metabolic time of Ce6 in tumor tissues.Part Ⅲ:The Effect and Mechanism of PD-L1 Ab/Ce6 Nanobubbles on the Anti-liver Cancer in vivoObjective:The subcutaneous transplanted tumor model of mouse liver cancer was constructed to observe the anti-liver cancer effect of PD-L1 Ab/Ce6-NBs in vivo and explore the main mechanism of anti-liver cancer effect of PD-L1 Ab/Ce6-NB s.Methods:A total of 42 SPF Balb/c mice were randomly divided into 6 groups with 7 mice in each group.Grouping:Control group(Control),nanobubbles group(NBs),Free Ce6 group(Free-Ce6),nanobubbles loding Ce6 group(Ce6-NBs),nanobubbles carrying PD-L1 antibody group(PD-L1 Ab-NBs),and combined treatment group(PD-L1 Ab/Ce6-NBs).Mice were injected with 200 μl drug through tail vein every other day.Control group was injected with the same amount of normal saline.The tumor was irradiated by ultrasound for 120s after injection with the irradiation conditions were as follows:power 1.82W/cm2,frequency 1MHz,duty-factor is 50%.Repeat 4 times.Tumor growth curves of each time for each group were plotted,and tumor volume and mass inhibition rates were calculated.Using HE staining to observe the tumor treatment group organization structure change,immunofluorescence test between groups within the tumor tissue apoptosis corpuscle,immunohistochemical method and RT-PCR to detect each group of tumor tissue in the amount of the expression of Bax and Bcl-2 in the cells,the detection of each treatment group of spleen lymphocyte proliferation index,CD8+cells and NK cells expression,percentage of lactate dehydrogenase experimental tests.The lethality of CTL and NK cells was detected by lactate dehydrogenase test,and the related immune factors(INF-γ,CD80,CD86,IL-2,TGF-β)were detected by immunofluorescence and real-time fluorescence quantitative.The expressions of PD-L1 and CRT in tumor tissues were detected by immunofluorescence and immunohistochemistry.Finally,the toxicty and side effects of drugs in each treatment group were evaluated by continuous monitoring of body weight,liver index and pathological damage of important organs in mice during treatment.Results:Using mouse liver cancer transplanted tumor model the volume and mass tumor inhibition rates in PD-L1 Ab/Ce6-NBs group were 63.14%and 67.80%,respectively(P<0.05),the necrosis of tumor tissue was obvious and apoptotic cells were increased significantly.Compared with the control group,the protein and mRNA of Bax in PD-L1 Ab/Ce6-NBs group were significantly increased,while the protein and mRNA of Bcl-2 were significantly inhibited(P<0.01).The proliferation and anti-tumor activity of spleen lymphocytes in PD-L1 Ab-NBs group and PD-L1 Ab/Ce6-NB s group were significantly higher than those in the control group(P<0.01),the proliferation rate of spleen lymphocytes,the positive rate of CD8+cells and NK cells in PD-L1 Ab/Ce6-NBs group were significantly higher than those in the control group(P<0.01),and the killing activities of CTL and NK cells were significantly higher than those in the control group(P<0.01).Through gene detection of tumor tissue related immune factors,the results showed that:The mRNA expression levels of INF-γ,CD80,CD86 and IL-2 in PD-L1 Ab/Ce6-NBs group were significantly increased(P<0.01),while the expression levels of PD-L1 and TGF-β were significantly decreased(P<0.01).Immunohistochemistry and RT-PCR was used to detect the expression of(mRNA)PD-L1 in each treatment group.The results showed that the expression of PD-L1 were significantly down-regulated in PD-L1 Ab-NBs group and PD-L1 Ab/Ce6-NBs groups.By detecting the expression of immunogenic death marker CRT,it was found that the expression of CRT protein in Ce6-carrying group was significantly up-regulated compared with the control group(P<0.01).In addition,under the control of drug dose and curative time,there were no obvious toxic and side effects in important organs during drug treatment.Conclusion:In vivo using mouse transplanted hepatocellular carcinoma model PD-L1 Ab/Ce6-NBs could exert synergistic anti-hepatocellular carcinoma effect through combined UTMD,targeted molecular therapy,sonodynamic therapy and immunotherapy,providing a new insight to the clinical treatment of liver cancer. |
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