Inflammation-mediated Targeted Microbubble Destruction Technology Inhibits HIF Pathway On Plaque Stability

The First PartPreparation of an improved model of rabbit abdominal aortic atherosclerotic plaqueObjective:Using the high-fat feeding-inflammation-immune injury combined method,a better animal model was prepared within a certain experimental period to simulate the occurrence and development of human plaques,which laid the foundation for the follow-up imaging and targeted therapy research of plaques.Methods:1.Inflammatory damage factorsBone marrow was extracted from the femur of 2-week-old suckling rabbits and put into a culture system containing Stem cell factor(SCF),Interleukin-3(IL-3)andβ-mercaptoethanol to induce mast cells.Platelets were isolated,both serving as inflammatory injury factors in the modeling process.2.Experimental animals and groupingOne-month-old healthy New Zealand male rabbits were randomly divided into 4 groups with 10 rabbits in each group.If the rabbit died during the experiment,the random supplement method was used to ensure that the number of each group remained unchanged.Normal feeding(Normal diet,ND)group:normal feeding-normal saline;Normal feeding+intervention(Normal intervene,NI)group:normal feeding inflammatory immune combined injury;High-fat diet(HFD)group:high-fat feeding-saline;High-fat+intervene(HFI)group:high-fat feeding inflammatory immune combined injury;Among them,inflammatory injury:injection of mast cells+platelets;immune injury:injection of calf serum albumin.3.Observation of the modeling situationThe blood lipid related indexes triglyceride(TG),total cholesterol(TC)and low density lipoprotein-c(LDL-C)of all experimental rabbits were detected at 0W,at the end of 4W,at the end of 8W and at the end of 12W respectively.The modeling was observed by ultrasound,and the pathological examination was performed at the end of 12W.Results:1.Induction of mast cellsAfter 6 weeks of culture,when the concentrations of SCF and IL-3 were both l0ng/ml and β-mercaptoethanol was added:A.Toluidine blue staining showed more mast cells containing metachromatic granules;B.Flow cytometry results The ratio of CD 117+and FcεRIα+double-positive cells was 96.2%,which was statistically significant compared with that without β-mercaptoethanol(P<0.05).2.Model preparationOf the 47 experimental rabbits,40 survived(the survival rate was 85.1%).The survival rates of the ND group,NI group,HFD group,and HFI group were 90.1%,90.1%,76.9%,and 83.3%.3.Blood lipidsWith the prolongation of feeding time,the blood lipid-related indexes(TC,TG,LDL-C)of rabbits in high-fat feeding group(HFI group and HFD group)gradually increased(all P<0.05),which were higher than those in ordinary feeding group(NI group and ND group)(all P<0.05).4.Evaluation of Rabbit Abdominal Aortic PlaqueUltrasound observation of plaque formation:10 plaques in HFI group;1 plaque in HFD group;3 plaques in NI group;no plaque formation in ND group.With the prolongation of feeding time,the abdominal aorta intima-media thickness(IMT)of the rabbits in the high-fat feeding group(HFI group and HFD group)gradually increased(all P<0.05),and both were thicker than the abdominal aorta.Ordinary feeding group(NI group and ND group)(all P<0.05).The pathological results showed that plaques were formed in the other three groups except the ND group.Among them,the plaques formed in the HFI group were the largest,densest and the most widely distributed,and the prepared plaques contained a large number of lipid cells,collagen fibers and smooth muscle components.The total coincidence rate between ultrasonography and pathological findings was 92.9%.The coincidence rate of ND group,HFD group and HFI group was 100%;the coincidence rate of NI group was 75%.Conclusions:1.Using SCF,IL-3 β-The culture system of mercaptoethanol can successfully induce mast cells.2.High-fat feeding and injection of mast cells,platelets,and calf serum albumin(high-fat feeding-inflammatory-immune damage)combined method is a non-invasive,more efficient,and more controllable plaque model Preparation.3.The total coincidence rate of ultrasound observation and pathological results is more than 90%.Ultrasound can be used as an early monitoring method for AS.The second part Inflammatory factor-traced targeted microvesicles inhibit the HIF pathway to assess Plaque stabilityObjective:Preparation of inflammatory factors Interleukin-8(IL-8)Intercellular cell adhesion molecule-1(ICAM-1)antibody as the tracer,hypoxia-inducible factor-1α(HIF-1α)antibody as the main therapeutic factor carrying monoclonal antibody and bifunctional ligand-carrying targeted ultrasound microbubbles,The tracer drives the microbubbles to enrich and stay in the plaque.After the microbubbles are blasted locally in the plaque with the ultrasonic destruction technology,the HIF-1α antibodies and inflammatory factor antibodies carried by the microbubbles are localized in the plaque.Release,by inhibiting the hypoxia pathway and reducing inflammation,to achieve the effect of targeted therapy.Methods:1.Preparation and observation of targeted microbubblesMonoclonal antibody-carrying and bifunctional ligand targeting microvesicles were prepared by bridging "biotin-avidin".2.Experimental animals and groupingSeventy-five rabbit models of abdominal aortic atherosclerotic plaques(the modeling method was the same as that of the HFI group in the first part)were randomly divided into 5 groups according to different treatment methods,with 15 rats in each group:(1)Naked microbubble group:A.Control group(Control group):simple injection of LS naked microbubbles;B.Blank microbubble group(NMB group):LS bare microbubbles combined with microbubble destruction operation.(2)Simple targeting group(carrying single antibody group):HIF-1α intervention group(MB group):HIF-1α monoclonal antibody contrast agent combined with targeted destruction of microbubbles.(3)Tracer targeting group(carrying bifunctional ligand group):A.IL-8 tracer intervention group(MBD1 group):IL-8+HIF-1α dual functional ligand contrast agent combined with targeted destruction of microbubbles;B.ICAM tracer intervention group(MBD2 group):ICAM-1+HIF-1α dual function ligand contrast agent combined with targeted destruction of microbubbles.3.Evaluation of treatment effectSerological parameters,ultrasound-related parameters,and pathological parameters were detected in each group at each treatment stage,and abdominal active ring budding experiment was performed after treatment.Results:1.Preparation of Targeted MicrobubblesThe "biotin-avidin" bridging method was used to successfully construct antibody-targeted ultrasound microbubbles,and the microbubbles in each group were uniformly dispersed.The binding rate of antibodies to microvesicles detected by flow cytometry was above 90%.2.Ultrasound Evaluation of Rabbit Abdominal AortaWith the prolongation of treatment time,the Intimal-media thickness(IMT)and maximum plaque Thickness of the dual-functional ligand group(MBD1 group and MBD2 group)after treatment were smaller than those of the monoclonal antibody group(MB group)and naked microbubble group(Control group and NMB group)(all P<0.05).3.Contrast-enhanced ultrasonography to evaluate the stability of abdominal aortic plaquesThe plaque Maximumintensity(IMAX)in the dual-functional ligand group(MBD1 group and MBD2 group)was lower than that in the monoclonal antibody group(MB group)and the naked microvesicle group(Control group and NMB group)(all P<0.05);the TTP was higher than that in the monoclonal antibody group(MB group)and the naked microvesicle group(Control group and NMB group)(all P<0.05).4.Serum related indicators(1)Blood lipid indexes(TC,TG,LDL-C):With the prolongation of treatment time,the blood lipid related indexes in each experimental group(MB group,MBD1 group and MBD2 group)gradually decreased(Control group and NMB group)(all P<0.05).(2)The contents of HIF-1α and Matrix metalloprotein-9(MMP-9):with the prolongation of treatment time,the antibody group(MB group、MBD1 group and MBD2 group)gradually decreased(all P<0.05),which were lower than those in the naked microvesicle group(Control group and NMB group)(all P<0.05).5.Abdominal Aortic Sprouting ExperimentThe number and length of sprouting vessels in the bifunctional ligand group(MBD1 group and MBD2 group)were smaller than those in the monoclonal antibody group(MB group)and the naked microvesicle group(Control group and NMB group)(all P<0.05).6.Pathological examinationPre-treatment immunofluorescence observed highest levels of antibody in plaques.With the prolongation of treatment time,the MVD,HIH-1α,and MMP-9 in the plaques in the dual-functional ligand group(MBD1 group and MBD2 group)were lower than those in the monoclonal antibody group(MB group)and the naked microbubble group(Control group and NMB group)after treatment(all P<0.05).7.Correlation between ultrasound and pathologyThe IMAX of the plaques in each group was significantly positively correlated with the protein expressions of MVD,HIH-1α and MMP-9 in the plaques(all P<0.05).Conclusions:1.Ultrasonic microbubbles carrying monoclonal antibodies and bifunctional ligands were successfully constructed using the "biotin-avidin" bridging method.2.The evaluation of plaque stability by CEUS is better than that of 2D ultrasound.The smaller the CEUS parameter IMAX and the larger the Time to peak(TTP),the more stable the plaque.3.Targeted ultrasound microbubbles carrying dual functional ligands combined with low-power ultrasound to destroy microbubbles can not only actively seek targets and reduce inflammatory responses,but also inhibit the HIF pathway to achieve "dual targeting" of targeted targeting and targeted therapy" effect,thereby increasing the stability of the plaque.The therapeutic effect is better than that of the monoclonal antibody-targeted ultrasound microbubbles.