Construction Of Synergistic Microbubbles And Application In The Treatment Of Non-small Cell Lung Cancer

Background: Lung cancer is the type of cancer with the highest morbidity and mortality worldwide,and NSCLC is the main type of lung cancer.For the past few years,the most exciting antitumour drug class has been PD1/PD-L1 checkpoint inhibitors,which can counteract the abilities of tumour cells to suppress the immune system and promote self-tolerance.Based on extensive clinical practice,immunotherapy has become as effective as chemotherapy in the treatment of advanced non-small cell lung cancer,and Immune checkpoint inhibitors(ICIs)have been listed in the NCCN guidelines as a first-line treatment for advanced NSCLC.Clinical evidence has shown that the combination of a PD1/PD-L1 checkpoint inhibitor with a chemotherapeutic drug can achieve better therapeutic effects than monotherapy.However,in addition to the cardiotoxicity of PD1/PD-L1 checkpoint inhibitors,this combination of free drugs also aggravates haematotoxicity,hepatotoxicity,and neurotoxicity.Therefore,an appropriate drug delivery system is needed to reduce the adverse events of these two drugs.In this project,we synthesized the phospholipid microbubbles in DTX and connected the surface of the microbubbles with anti-PD-L1 antibodies.With the active and passive targeting effect,chemotherapy drugs can be enriched in large quantities in tumor cells,promoting the apoptosis of tumor cells and inhibiting the tumor cell cycle.Futhermore,we established the mouse lung tumor model in situ and verified the therapeutic effect of the new multifunctional microbubbles on the mouse lung tumor in situ under the effect of low-frequency ultrasound.We established a model of subcutaneous tumor with incomplete ablation of lung cancer,and applied microbubbles to the treatment of residual lesions after incomplete microwave ablation,verifying the control effect and mechanism of this synergistic function of ultrasonic microbubbles on residual lesions after incomplete microwave ablation.Part I Construction synergistic microbubbles and effect on lung cancerObjective: To construct synergistic phospholipid microbubbles with docetaxel(DTX)and antiPD-L1 antibodies co-loaded and to study the killing effect on the lung cancer cells.Methods: The synergistic phospholipid microbubbles loaded with docetaxel and anti-PD-L1 mAb were prepared by the thin film-hydration method.The morphology of microbubbles was detected by electron microscope and Laser confocal microscope.Particle size and Zeta potential were detected by Marvin laser analyzer.The encapsulation rate and the drug oading rate of docetaxel in microbubbles were measured by high performance liquid chromatography.The release curve of Docetaxel was measured,and the ultrasound imaging ability of microbubbles in vitro and in vivo was detected.The biosafety of microbubbles was evaluated by hemolysis test and animal biochemical indexes.The effect of anti-PD-L1 mAb microbubbles on the uptake of drugs in lung cancer cells was examined by laser confocal microscopy and flow cytometry.The expression of PD-L1 on the surface of mouse lung cancer cells(LLC cells)and human lung cancer cells(NCI-H460,NCI-1299 and A549 cells)was detected by flow cytometry,and the relationship between the killing effect of microbubbles on different kinds of cell lung cancer cells and the expression of PD-L1 was detected by CCK-8 method.The ability of microbubbles to promote apoptosis and cell cycle inhibition of LLC tumor cells was detected by flow cytometry.Results: The synergistic phospholipid microbubbles with docetaxel and anti-PD-L1 antibodies co-loaded had a good morphology.The particle size was 666.4±35.9 nm,encapsulation rate was 57.34±2.61%,and drug loading rate was 4.45±0.91%.Under the effect of ultrasound,the release rate of docetaxel in the microbubbles was significantly increased.The attachment of anti-PD-L1 antibody on the surface of microvbubbles had no effect on the drug release of docetaxel.Laser confocal microscopy demonstrates that fluorescence-labeled anti-PD-L1 mAb was binded on the surface of microbubbles.Ultrasound imaging showed that the microbubbles was not affected by DTX and antibodies.In vitro experiments showed that microbubbles did not produce hemolytic effect,and in vivo experiments showed that synergistic microbubbles had no obvious effect on biochemical indexes of mice,and had no obvious damage to vital organs.Laser confocal microscopy and flow cytometry showed that microbubbles loaded anti-PD-L1 antibodies could promote the absorption of drugs by lung cancer cells,and low-frequency ultrasound could further increase the absorption of drugs by lung cancer cells.CCk-8 experiments showed that the inhibitory effect of microbubbles loaded with anti-PD-L1 mAb on tumor cell proliferation was positively correlated with the expression of PD-L1 on tumor cell surface,while low-frequency ultraosound irradiation enhanced the inhibitory effect on tumor cells.The effect of synergistic microbubles on tumor cell apoptosis was detected by flow cytometry,the results showed that ultrasound irradiation combined with anti-PD-L1 mAb resulted in the highest proportion of total apoptosis of lung cancer cells.At the same time,the targeting effect of anti-PD-L1 mAb and lowfrequency ultrasound irradiation could enhance the inhibition effect of docitaxel on tumor cell cycle.Conclusions: The synergistic phospholipid microbubbles with docetaxel and anti-PD-L1 antibodies synthesized by thin film hydration has good imaging ability,and can release drugs rapidly under the action of low-frequency ultrasound,the synergistic phospholipid microbubbles has good biological safety.These microbubbles can promote the absorption of drugs by lung cancer cells.Phospholipid microbubbles loaded with docetaxel and anti-PD-L1 mAb have a stronger inhibitory effect on the proliferation of tumor cells.Under the effect of low-frequency ultrasound,the synergistic phospholipid microbubbles can promote the apoptosis of lung cancer cells and better inhibit cell cycle.Part II Evaluation of synergistic function in the treatment of lung cancer modelObjective: To establish mouse lung cancer subcutaneous tumor model and orthotopic tumor model,and to evaluate the therapeutic effect of synergistic functional microbubbles on the lung cancer models.Methods: We established the subcutaneous lung cancer model in mice.The microbubbles carried on lipotropy fluorescent probe DiR,docetaxel microbubbles carried on DiR(DMs),synergistic phospholipid microbubbles with docetaxel and anti-PD-L1 antibodies(PDMs).We compared concentration in the tumor site under different treatment methods(free DiR,DiR-DMs,DiR–PDMs,PDMs combined with low-frequency ultrasoud).We evaluated the coordinated function of phospholipids microbubble ultrasound break after the enrichment ability of the drug in the tumor site.In vivo imaging of vital organs was performed to observe tissue distribution.With the control group(PBS),Free DTX(Free DTX solution),Free combo(injection Free DTX and anti-PD-L1 mAb),DMs,PDMs,collaborative treatment group(PDMs + US)for the treatment of subcutaneous tumor model in mice,we observed the mice weight,tumor size and survival differences,TUNEL and immunohistochemical staining of tumor samples(CD31 and Ki67).We also detected the change of CD4 +,CD8 + T cells in the tumor samples.We used Western blot to detect Cleaved caspase 3,Cleaved caspase 8,and Cleaved caspase 9 in tumor tissues.TNF-?,TGF-and VEGF were detected by ELISA.The orthotopic lung tumor model of mice was established under the guidance of DSA.The above different treatment schemes were adopted,and the changes of tumor volume,body weight and survival time of mice were observed through CT follow-up.Results: Compared with free DiR-DMS and DiR-DMS,DiR-PDMs enhanced signals in in vivo in tumor tissues,which were further enhanced under US irradiation.The maximum drug concentration in the tumor was reached on the 1st hour by the combination of DiR-PDMs and low-frequency ultrasound,and the maximum drug concentration was reached on the 6th hour by the injection of DIR-PDMS alone.The enrichment degree of free DiR and DiR-DMs in the tumor was always low.In the subcutaneous tumor model,the PDMs combined with US irradiation group had the highest survival rate and the best inhibition effect.The treatment effect of PDMs alone was superior to the free drug combination.Synergistic treatment was more effective than chemotherapy,DMs had slightly improved therapeutic effect on tumor volume inhibition and survival rate.There was no significant difference in body weight between groups.In the immunohistochemical staining of tumor tissues.TUNEL apoptosis rate was the highest in the combined treatment group,while CD31 and Ki67 were the lowest.In the PDMs combined with US group,the proportion and number of infiltrated CD4+ and CD8+ T cells were the highest.The expressions of apoptotic channel proteins cleaved caspase-3,cleaved caspase-8 and cleaved caspase-9 were also the highest.At the same time,PDMs combined with US irradiation group had the highest TNF-and the lowest TGF-,VEGF.CT evaluation showed that the combined treatment group had the lowest tumor growth rate,the longest survival period,and the best survival.Conclusion: The model of subcutaneous tumor shows that the synergistic function of phospholipid microbubbles combined with ultrasound can promote the drug concentration in the tumor and obtain the highest drug concentration.PDMS combined US produced the best therapeutic effect.This treatment method has the same effect in the in situ tumor model.Part ? Evaluation of synergistic function microbubbles(PDBMs)in the treatment of lung cancer with incomplete ablation modelObjective: To establish a mouse model of incomplete lung cancer ablation and evaluate the therapeutic effect of synergistic microbubbles on the model.Methods: The synergistic phospholipid microbubbles loaded with docetaxel,bindarit and antiPD-L1 mAb were prepared by the thin film-hydration method.The morphology and spectrum of microbubbles were detected by electron microscope and UV spectrometer,and the encapsulation rate and drug loading rate of docetaxel and Bindarit in microbubbles were measured by HPLC.The biosafety of microbubbles was evaluated by animal biochemical indexes and HE staining sections of important organs.Incomplete microwave ablation group(iMWA)was used at 45°C for 15 min,and complete ablation group was used at 65°C for 15 min in vitro.The effect of microwave ablation plus PDBMs on apoptosis of lung cancer cells was detected by flow cytometry.The effects of microwave ablation combined with PDBMs on the expression of monocyte chemoattractant protein-1(CCL2,Mc P-1),calreticulin(CRT)and PD-L1 in lung cancer were detected by laser confocal microscopy and flow cytometry.The lung cancer cells treated by microwave ablation combined with PDBMs.The lung cancer cells and DC cells were incubated in Transwell chamber,and the effect of microwave ablation combined with PDBMs on the activation of mouse bone marrow derived DC cells(BMDCs)was detected by flow cytometry.The lung cancer cells treated with iMWA + PDBMs,BMDCs and T lymphocytes were incubated together.The proliferation and killing ability of T lymphocytes were evaluated by flow cytometry detection of Ki67 and Granzyme B.Microwave parameters for constructing incomplete ablation were explored through specimen and in vivo.An incomplete ablation model of lung cancer in mice was established and randomly divided into five groups: Control,iMWA,iMWA + DTX,iMWA + Bindarit,iMWA +PDBMs.We followed up tumor volume,calculated survival time,and measured the number of lung metastases.We used TUNEL staining to evaluate the effects of different treatment methods on tumor apoptosis,Ki67 staining to evaluate the proliferation capacity of tumor tissue.We detected the percentages of CD4+ and CD8+T cells and Treg cells in tumor tissues to evaluate the infiltration of immune cells in tumor tissues.Besides,the levels of TGF-,VEGF and IL-10 cytokines in tumor tissues were detected.Results: We successfully synthesized phosphatic functional microbubbles loaded with docetaxel,bindarit and anti-PD-L1 mAb.The microbubbles showed regular round shape under electron microscope,and the waveform of PDBMs was displayed by ultraviolet spectrum.The HPLC detection results showed that the encapsulation rate of DTX and drug loading rate in PDBMs were 35.56 ± 5.86% and 4.25 ± 0.67%,respectively,and those of Bindarit was 37.85 ± 6.75% and 4.67 ± 0.53% respectively.PDBMs was injected through tail vein once per week,in total of five times.All the biochemical indexes were followed up within the normal range,and no abnormalities were found in HE staining of heart,liver,spleen,lung and kidney.The completed ablation group(c MWA)had the strongest apoptotic effect on lung cancer cells.The total apoptosis rate of lung cancer cells in iMWA,iMWA + DTX,iMWA + Bindarit and iMWA + PDBMs ranged from 40% to 50%.CCL2 expression was detected by confocal and flow cytometry in the all groups.The results showed that c MWA group was the weakest,iMWA group had higher CCL2 expression than the control group,iMWA + DTX group had no statistically significant difference from the control group,and the CCL2 expression of iMWA + Bindarit group and iMWA + PDBMs decreased under bindarit.Confocal and flow cytometry showed that the expression of CRT in lung cancer cells increased after incomplete ablation.DTX,Bindarit and anti-PD-L1 mAb had no significant effect on the expression of CRT.DTX and bindarit had no significant effect on the expression of PD-L1 in lung cancer cells.In terms of BMDCs activation detection,the proportion of DCs activation in iMWA +PDBMs group increased,and the secretion of IL-12p70 from DC increased correspondingly.In terms of T lymphocyte activation,iMWA +PDBMs can increase the proliferation and killing ability of CD8+T cells compared with other groups.We established an incompleted ablation model.iMWA + PDBMs could significantly reduce the tumor growth after ablation,prolong the survival time,and reduce the number of metastatic nodules.The results of TUNEL staining and Ki67 immunohistochemistry after ablation showed that the iMWA + PDBMs group had the highest proportion of tumors and the least proliferation ability.Flow cytometry detection results of tumor tissues showed that the proportion of CD4+ and CD8+T cells increased in the iMWA + PDBMs group,in which the proportion of CD8+T cells increased more significantly and the proportion of Treg cells decreased.Intracellular cytokine detection showed that TGF-and VEGF increased after microwave ablation,and TGF-,VEGF and IL-10 decreased after microbubble treatment.Conclusion: Synergistic microvesicle PDBMs has good biological security.Synergistic microbubbles can inhibit the expression of CCL2 in lung cancer cells,improve the activation ratio of DC cells and T lymphocytes.It can also reduce the growth rate and the number of lung metastasis after incomplete ablation of lung cancer,and prolong the survival period of mice.The synergistic function microbubble PDBMs has high potential application value in the treatment of lung cancer after microwave ablation.