| Part Ⅰ Preparation,characterization and in vitro drug release of IL-10@exosomes Background and Objective:Rheumatoid arthritis(RA)is a systemic autoimmune disease,which is an important cause of joint destruction and deformity in young and middle-aged people.The proliferative synovial membrane of inflammatory joints is the basis of RA and also the therapeutic target.A variety of existing anti-rheumatic drugs need long-term systemic medication,which leads to high-dose medication and has the risks of inducing infection and tumor for patients.Targeted therapy for inflammatory joints is of great significance.There are a large number of new microvessels and macrophages in the hyperplastic synovial membrane of inflammatory joint,which play a vital role in the occurrence and development of joint inflammation.Macrophages have polarization,which can be divided into M1pro-inflammatory type and M2 anti-inflammatory type.Effective regulating M1 macrophages in inflammatory synovium polarizate to M2 macrophages is a promising new treatment for RA.Interleukin-10(IL-10),as an immunosuppressive cytokine,can reduce the secretion of proinflammatory cytokines and regulate macrophage polarizate to M2 type.However,it has immunogenicity,which may lead to the failure of the body to produce anti-drug antibodies,and long-term systematic use may also produce a variety of side effects.Targeting drug delivery system directly into inflammatory synovial tissue is safer and more effective than systemic drug delivery.Exosomes are multivesicular bodies secreted by living cells and are natural endogenous targeting vectors in organisms.Tumor susceptibility gene 101(TSG101)and CD63 are commonly used as markers of exosomes,which are are positive in exosomes.And calnexin is the product of cell lysis,which is negative in exosomes.Taking exosomes as carriers for drug loading has become a new drug loading method.Ultrasound can noninvasively enter the body,which can realize the fixed-point timing release of drugs,and becomes an important way for drug targeted controlled release.In this study,the exosomes in the supernatant of mouse RAW264.7 macrophages were extracted,and IL-10@exosomes were constructed.The morphology and size of IL-10@exosomes were observed,and their physical properties were also detected.A variety of methods were used to verificate the drug loading,and the drug entrapmentefficiency was measured.Finally,the effect of ultrasound on the drug releasing was explored.This study laid a theoretical and experimental foundation for subsequent in vivo and in vitro experiments.Materials and Methods:The mouse RAW264.7 macrophages were resuscitated,frozen and passaged.The exosomes in the cell supernatant were extracted by precipitation method.The exogenous IL-10 was loaded into the exosomes by electroporation to prepare IL-10@exosomes.Transmission electron microscopy(TEM)was used to observe the morphology of exosomes and IL-10@exosomes.And the particle sizes and potentials of Exosomes and IL-10@exosomes were measured by nanoparticle tracking analyzer when they were prepared and stored at-80 ℃ for 90 days.Western blotting(WB)was used to detect the expression of exosomes marker protein TSG101,CD63 and cell lysate calnexin of Exosomes and IL-10@exosomes;WB method,laser confocal microscopy(CLSM,Exosomes membrane was labeled with red fluorescence,and IL-10 was labeled with green fluorescence)and ultraviolet spectrophotometry were used to determine whether IL-10 was successfully loaded into the Exosomes.Enzyme-linked immunosorbent assay(ELISA)was used to detected the weight of IL-10 in IL-10@exosomes.The drug entrapmentefficiency(EE)of IL-10@exosomes was calculated according to the calculation formula.Finally,dialysis bag method was use to explore the effect of ultrasound on the in vitro drug release on IL-10@exosomes.Results:1.Morphological observation of mouse RAW264.7macrophages: mouse RAW264.7macrophages were round or oval,no obvious protrusions,good refraction,adherent or semi-adherent growth observed by optical microscope.2.Morphological observation of Exosomes and IL-10@exosomes: Exosomes and IL-10@exosomes have complete morphology,relatively uniform size,concave disc vesicles and scattered distribution observed by TEM detection.3.Particle size and potential of Exosomes and IL-10@exosomes: when they were prepared,the particle size of IL-10@exosomes was larger than that of Exosomes,and the particle sizes of Exosomes and IL-10@exosomes were 146.60±55.40 nm and 170.00±68.90 nm,respectively.After 90 days of storage at-80 ℃,the particle sizes of both did not change significantly.After 90 days of storage at-80 ℃,the particle sizes of both did not change significantly.When they were prepared,there was no significant difference in the potentials of Exosomes and IL-10@exosomes,both of which had negative charges.The potentials of Exosomes and IL-10@exosomes were-29.50±1.11 m V and-30.93±0.67 m V,respectively.After 90 days of storage at-80 ℃,the potential of exosomes did not change significantly,and the absolute value of the potential of IL-10@exosomes increased compared with that of the preparation.4.Detection of protein expression of Exosomes and IL-10@exosomes: WB detection showed that exosome marker proteins TSG101 and CD63 of Exosomes and IL-10@exosomes were both positive,while cell lysate calnexin was both negative,suggesting that both of them conformed to exosomes performance,and there was no cell component in them.IL-10 in Exosomes was negative and IL-10@exosomes was positive.5.Detection of drug loading of Exosomes and IL-10@exosomes: Exosomes showed only red fluorescence,IL-10@exosomes showed red and green fluorescence by CLSM detection,and co-localization of IL-10@exosomes showed orange fluorescence.Image J image processing software was used for co-localization analysis of red and green fluorescence.It can be seen that the co-localization correlation coefficient r of the two was 0.836,indicating that the coincidence degree of the two was very high.Absorbance of IL-10@exosomes and IL-10 both reached the peak at the wavelength of 244 nm detected by UV spectrophotometry indicating that the peaks of the two were at the same wavelength.These results indicated that IL-10 was successfully loaded into exosomes.6.Entrapmentefficiency of IL-10@exosomes: according to the calculation formula,the drug EE of IL-10@exosomes was calculated as 30.06 ± 9.15 %.7.Effect of ultrasound on drug release of IL-10@exosomes in vitro: the cumulative drug release in each group gradually increased with time.After 24 h,the cumulative drug release(%)of IL-10 group,IL-10@exosomes group,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound +microbubbles group were 95.67±2.08,79.42±2.50,82.33±2.08 and 85.00±5.00,respectively.Compared with IL-10@exosomes group and IL-10@exosomes+ ultrasound group,the drug release of IL-10@exosomes + ultrasound +microbubbles group was higher,and the drug release of IL-10@exosomes +ultrasound group was higher than that of IL-10@exosomes group.Conclusion:In this part,IL-10@exosomes were successfully constructed.The morphology,size and potential of the drug-carrying exosomes were stable,and the drug entrapmentefficiency was high.Ultrasound could promote the drug release of the IL-10@exosomes in vitro,and the effect was more obvious when combined with microvesicles,which laid important experimental foundation for the following in vivo and in vitro experiments.Part Ⅱ Safety,targeting and effectiveness of ultrasound combined with IL-10@exosomes in vitroBackground and Objective: Joint synovitis is the pathogenesis basis of rheumatoid arthritis(RA).There are a large number of new microvessels and a large number of macrophages in the proliferative synovial membrane.These macrophages have functions such as phagocytosis,chemotaxis and immune regulation,which play a vital role in the occurrence and development of arthritis.Under different stimulation factors,Macrophages can be differentiated into two subtypes with different phenotypes and functions,which is named macrophage polarization.It can be divided into M1 pro-inflammatory type and M2 anti-inflammatory type.M1 macrophages,characterized by secreting a variety of pro-inflammatory cytokines,have strong pro-inflammatory effects.By releasing tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and other proinflammatory factors and chemokines,M1 macrophages mediate tissue damage and induce inflammatory reactions.CD86 and CD32 are highly expressed on the cell surface.M2 macrophages are characterized by the secretion of arginase1 1(Arg1),transforming growth factor(TGF-β),and other anti-inflammatory or immunosuppressive molecules,which are involved in tissue repair and healing.CD206 and CD163 are highly expressed on the cell surface.The polarization state of M1 and M2 macrophages is not stable,and can be induced by different stimuli.Many studies have confirmed that,IL-10 can regulate macrophage polarize to M2 type.Exosomes are lipid bilayer vesicles secreted by living cells,carrying specific proteins,lipids,RNA and DNA,which are natural endogenous targeted carriers in organisms.Exosomes have good biocompatibility,low toxicity and low immunogenicity,and can stably and efficiently deliver content to target cells.Ultrasound can produce physical effects such as mechanical effect,thermal effect and cavitation effect,and increase the permeability of cell membrane and vascular wall.Combined with ultrasonic microbubbles,cavitation effect can be enhanced,and its permeability can be further increased,so as to promote the targeted entry of drugs,genes and nanocarriers into tissues and cells.In the first part of this study,IL-10@exosomes were successfully constructed.The morphology,size and potential of IL-10@exosomes were stable,and the encapsulation efficiency was high.Ultrasound could promote the release of IL-10@exosomes in vitro.We hope that through the role of IL-10@exosomes,macrophages of joints in CIA mice can be polarized from M1 pro-inflammatory to M2 anti-inflammatory,which can inhibit inflammation and promote tissue repair,so as to produce therapeutic effect on arthritis.Before IL-10@exosomes were applied to the joints of collagen-induced arthritis(CIA)mice,their safety,targeting and effect of regulating macrophage polarization need to be verified in vitro.In this part of the experiment,we will verify the in vitro safety of IL-10@exosomes,the in vitro targeting and effectiveness of IL-10@exosomes combined ultrasound and microbubbles,and provide a theoretical basis and experimental basis for further in vivo experiments on mouse CIA model arthritis.Materials and Methods: 1.Experimental groups:(1)PBS control group;(2)Exosomes group;(3)IL-10@exosomes group;(4)IL-10@exosomes + microbubbles group.The cytotoxicity of IL-10@exosomes on macrophages and human umbilical vein endothelial cells(HUVECs)was verified by cell proliferation and toxicity test(CCK-8)and FDA/PI staining.2.Experimental groups:(1)phosphate buffer saline(PBS)control group;(2)Exosomes group;(3)IL-10@exosomes group;(4)IL-10@exosomes + ultrasound group;(5)IL-10@exosomes + ultrasound + microbubbles group.Mouse RAW264.7 macrophages will differentiate into M1 type when stimulated by inflammatory stimuli lipopolysaccharide(LPS).Laser confocal microscopy(CLSM)and flow cytometry(FCM)were used to verify the targeting of ultrasound combined with IL-10@exosomes on M1 type differentiated macrophages.3.Experimental groups:(1)PBS control group;(2)IL-10 group;(3)Exosomes group;(4)IL-10@exosomes;(5)IL-10@exosomes + ultrasound group;(6)IL-10@exosomes + ultrasound + microbubbles group.RAW264.7 macrophages were activated by LPS and grouped accordingly.The surface markers of macrophages,the concentration of cytokines in the supernatant and the expression of intracellular messenger ribonucleic acid(m RNA)and were detected by FCM,enzyme-linked immunosorbent assay(ELISA)and polymerase chain reaction(PCR)respectively to determine whether ultrasound combined with IL-10@exosomes could effectively regulate the polarization of macrophages from M1 pro-inflammatory type to M2 anti-inflammatory type in vitro.Results: 1.Safety test in vitro Cytotoxicity test: after 24 h treatment with Exosomes,IL-10@exosomes,and the mixture of IL-10@exosomes and microbubbles on RAW264.7 macrophages and HUVECs,the average survival rates of cells detected by CCK-8 were more than 95 %,and the differences were not statistically significant(P > 0.05).Cell activity detection: Exosomes,IL-10@exosomes,and the mixture of IL-10@exosomes and microbubbles were incubated with macrophages and HUVECs for 24 h,and the cells were stainned by FDA/PI.Fluorescence microscope showed that the majority of living cells with green fluorescence were in both of them,and only a few cells emitted red fluorescence.2.Targeting detection in vitro CLSM detection: in addition to the absence of red fluorescence in the PBS control group,red fluorescence was observed in the cells of the other groups.After co-localization,the red exosomes overlapped with the green cytoskeleton and showed orange color.In addition,it can be seen that the red fluorescence of IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubble group was brighter than that of the other two groups.FCM detection: except that there was no obvious fluorescence in the cells of PBS control group,the fluorescence carrying rates of other groups were all greater than 97 %.The fluorescence carrying rates of IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were all greater than 99 %.Average fluorescence intensity(MFI)analysis showed that IL-10@exosomes + ultrasound + microbubbles group was higher than the other groups,and IL-10@exosomes + ultrasound group was higher than PBS control group,Exosomes group and IL-10@exosomes group,Exosomes group and IL-10@exosomes group were higher than PBS control group,the differences were statistically significant(P < 0.05).There was no significant difference in MFI between Exosomes group and IL-10@exosomes group(P > 0.05).3.Effectiveness test in vitro Detection of cell surface markers: by FCM detection,M1 macrophage surface marker CD86+ cell ratio,IL-10 group,IL-10@exosomes,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were lower than PBS control group and Exosomes group,and the differences were statistically significant(P < 0.05).There was no significant difference in the ratio of CD86+ cells among other groups(P > 0.05).M2 macrophage surface marker CD206+ cell ratio,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were higher than PBS control group and Exosomes group,the differences were statistically significant(P < 0.05).IL-10 group and IL-10@exosomes group were also higher than PBS control group and Exosomes group,but the differences were not statistically significant(P > 0.05).There was no significant difference in the ratio of CD206+ cells among other groups(P > 0.05).Cytokine detection in cell supernatant: by ELISA detection,the concentration of M1 macrophage factor TNF-α in the supernatant,IL-10 group was lower than the other groups;IL-10@exosomes + ultrasound + microbubbles group was lower than the PBS control group,Exosomes group,IL-10@exosomes group and IL-10@exosomes + ultrasound group;IL-10@exosomes group and IL-10@exosomes + ultrasound group were lower than the PBS control group and Exosomes group;Exosomes group was lower than PBS control group.And the differences were statistically significant(P < 0.05).The concentration of M2 macrophage factor Arg1 in the supernatant,IL-10 group and IL-10@exosomes + ultrasound + microbubbles group were higher than PBS control group,Exosomes group and IL-10@exosomes group;IL-10@exosomes + ultrasound group was higher than PBS control group and Exosomes group;Exosomes group and IL-10@exosomes group were higher than PBS control group,the differences were statistically significant(P < 0.05),and there was no significant difference among other groups(P > 0.05).Detection the m RNA of cell factor: by PCR detection,the relative expression levels of CD86 m RNA in IL-10 group,IL-10@exosomes group,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were all lower than those in PBS control group and Exosomes group,and the differences were statistically significant(P < 0.05).And there was no significant difference among other groups(P > 0.05).The relative expression levels of TNF-α m RNA in IL-10 group,Exosomes group,IL-10@exosomes group,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were lower than those in PBS control group,and the difference was statistically significant(P < 0.05).There was no significant difference among other groups(P > 0.05).The relative expression levels of CD206 m RNA in IL-10 group,IL-10@exosomes group,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were higher than those in PBS control group and Exosomes group,and the differences were statistically significant(P < 0.05).And the differences among other groups were not statistically significant(P > 0.05).The relative expression levels of Arg1 m RNA in the Exosomes group,IL-10 group,IL-10@exosomes group,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were higher than those in the PBS control group,and the difference was statistically significant(P < 0.05).And there was no significant difference among other groups(P > 0.05).Conclusion: This part of the experiments proved that the IL-10@exosomes constructed by us had good safety and high targeting in vitro,and could regulate the polarization of macrophages from M1 to M2.Moreover,ultrasound combined with microbubbles could further improve the in vitro targeting and the effect of regulating macrophage polarization,which laid an in vitro experimental foundation for ultrasound combined with the drug-carrying exosomes to regulate macrophage polarization in the treatment of CIA model arthritis in mice.Part Ⅲ Ultrasound combined with IL-10@exosomes to regulate macrophage polarization in the treatment of CIA model arthritis in mice in vivoBackground and Objective: Rheumatoid arthritis(RA)is a kind of systemic autoimmune disease,which is characterized by chronic joint synovitis.It is persistent and recurrent,causing the irreversible destruction of articular cartilage and bone,and joint deformity and dysfunction in the late stage,which seriously reduces the working capacity and the living standards of patients,brings heavy social and economic burden.The treatment of RA has always been a clinical difficulty.Glucocorticoids,disease modifying anti-rheumatic drugs,non-steroidal anti-inflammatory drugs,biological agents,and so on are used to treat RA.However,there are problems of high dosage and bad side effects for systemic administration.Therefore,there is an urgent need for targeted and effective treatment for inflammatory joints.The proliferative synovial tissue in RA inflammatory joints can produce inflammatory factors,which is the pathological basis and therapeutic target of the disease.Effectively regulating the polarization of M1 pro-inflammatory macrophages in inflammatory synovium to M2 anti-inflammatory macrophages is conducive to promoting inflammation regression and tissue repair,which is a promising new treatment method.As an immunosuppressive cytokine,IL-10 has been proved to be able to effectively regulate macrophages polarize to M2 type,thereby reducing the secretion of pro-inflammatory factors.Exosomes have good biocompatibility,low toxicity and low immunogenicity,and can stably and efficiently deliver the contents to target cells,which can be used as a good drug carrier for IL-10.However,exosomes can be non-specific uptake by other tissues,resulting in endogenous off-target effect.Therefore,it is of great significance to improve the targeting of drug-loaded exosomes to inflammatory joints.Studies have shown that ultrasound-targeted destruction of microbubbles can promote the targeted aggregation of exosomes in vivo ultrasound irradiation area,improve the therapeutic effect and further reduce the side effects of drugs.The clinical manifestations,pathological and immunological characteristics,laboratory indexes and joint radiological manifestations of mouse collagen-induced arthritis(CIA)model are very similar to that of human RA,and it has become a classic animal model for RA experimental research.In the first part of this study,IL-10@exosomes were successfully constructed.The morphology,size and potential of the drug-carrying exosomes were stable,and the drug entrapmentefficiency was high.Ultrasound can promote the drug release of the drug-carrying exosomes in vitro.The second part of the experiment confirmed that IL-10@exosomes had good safety and high targeting in vitro,which could regulate the polarization of macrophages from M1 type to M2 type in vitro.Moreover,ultrasound combined with microbubbles could further improve the in vitro targeting and the effect of regulating macrophage polarization.In this part of the experiment,we will evaluate the in vivo targeting,safety,feasibility and therapeutic effect of ultrasound combined with IL-10@exosomes in the treatment of mice CIA model arthritis.Materials and Methods: The mice model of CIA arthritis was established and randomly divided into six groups:(1)PBS control group;(2)IL-10 group;(3)Exosomes group;(4)IL-10@exosomes group;(5)IL-10@exosomes + ultrasound group;(6)IL-10@exosomes + ultrasound + microbubbles group.After intravenous injection of the above drugs,the ankle joints in groups(5)and(6)were subjected to ultrasonic irradiation.The in vivo targeting of ultrasound-assisted IL-10@exosomes was evaluated by in vivo fluorescence imaging detection immediately,2 h,4 h and 24 h after drug injection or ultrasonic irradiation;Four treatments were given at 31,34,37 and 40 days after the initial immunization,and the mice were sacrificed on the 43 rd day.The in vivo targeting of ultrasound combined with IL-10@exosomes was further evaluated by intra-articular IL-10 concentration detection.The in vivo safety of IL-10@exosomes and the feasibility and therapeutic effect of ultrasound combined with IL-10@exosomes in regulating macrophage polarization in the treatment of mice CIA arthritis were evaluated by arthritis score,high-frequency ultrasound and micro-computed tomography(Micro-CT)examination,serum inflammatory factor detection,intra-articular cytokine detection and histopathological detection.Results: 1.In vivo fluorescence imaging of mice: no fluorescence in the lower limb joint areas could be seen in the PBS control group and IL-10 group.Exosomes group,IL-10@exosomes group,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound+ microbubbles group showed fluorescence in bilateral lower limb joint areas at each time point.With the prolongation of time,the fluorescence intensity in the lower limb joint areas of the IL-10@exosomes + ultrasound group and the IL-10@exosomes + ultrasound + microbubbles group was higher than that of the Exosomes group and the IL-10@exosomes group,and the IL-10@exosomes + ultrasound + microbubbles group was higher than that of the IL-10@exosomes + ultrasound group.2.Arthritis score: the arthritis score of the PBS control group showed an upward trend,while that of the other groups showed a decreasing trend over time,and the decrease in the IL-10@exosomes + ultrasound + microbubbles group was the most obvious.3.High frequency ultrasound examination: synovial hyperplasia in PBS control group was the most obvious,followed by IL-10 group,Exosomes group,IL-10@exosomes group and IL-10@exosomes + ultrasound group,and IL-10@exosomes + ultrasound + microbubbles group.4.Micro-CT examination: the ankle bone injury in PBS control group was the most serious,and obvious bone erosion and osteophyte formation were observed on the surface of bone cortex;The degree of bone erosion and osteophyte formation on cortical bone surface in IL-10 group,Exosomes group,IL-10@exosomes group and IL-10@exosomes + ultrasound group was followed,and the degree of ankle bone injury in IL-10@exosomes + ultrasound + microbubble group was the hightest.5.Detection of serum inflammatory factors: the concentration of TNF-α in serum,IL-10@exosomes + ultrasound + microbubbles group was lower than PBS control group,IL-10 group and Exosomes group;IL-10 group,Exosomes group,IL-10@exosomes group and IL-10@exosomes + ultrasound group were lower than PBS control group,and the differences were statistically significant(P < 0.05).There was no significant difference among other groups(P > 0.05).The concentration of IL-1 β in serum,IL-10 group,Exosomes group,and IL-10@exosomes group were lower than PBS control group;IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubble group were lower than PBS control group,IL-10 group,Exosomes group and IL-10@exosomes group,the differences were statistically significant(P<0.05).There was no significant difference among other groups(P > 0.05).6.Detection of IL-10 concentration in ankle joints: IL-10@exosomes + ultrasound group was higher than PBS control group,IL-10 group and Exosomes group;IL-10@exosomes + ultrasound + microbubbles group was higher than PBS control group,IL-10 group,Exosomes group and IL-10@exosomes group.And the differences were statistically significant(P < 0.05).And there was no significant difference among other groups(P > 0.05).7.Detection of cytokines in the ankle joints: the concentration of M1 macrophage surface marker CD86,the IL-10@exosomes + ultrasound group and the IL-10@exosomes + ultrasound + microbubbles group were lower than the other groups;the IL-10@exosomes group was lower than PBS control group,the IL-10 group and the Exosomes group;the IL-10 group was lower than PBS control group and the Exosomes group.And the differences were statistically significant(P < 0.05).There was no significant difference among other groups(P > 0.05).The concentration of M1 macrophage cell factor TNF-α,IL-10@exosomes + ultrasound + microbubbles group was lower than PBS control group and IL-10 group;IL-10 group,Exosomes group,IL-10@exosomes group and IL-10@exosomes + ultrasound group were lower than PBS control group.And the differences were statistically significant(P < 0.05).There was no significant difference among other groups(P > 0.05).The concentration M2 macrophage surface marker CD206,IL-10@exosomes + ultrasound + microbubbles group were higher than control group,IL-10 group and Exosomes group;IL-10@exosomes + ultrasound group were higher than control group and Exosomes group;IL-10 group and IL-10@exosomes were higher than PBS control group.And the differences were statistically significant(P < 0.05).There was no significant difference among other groups(P > 0.05).The concentration of M2 macrophage cell factor Arg1,IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubbles group were higher than other groups;IL-10@exosomes group was higher than PBS control group and Exosomes group;IL-10 group was higher than PBS control group.And the differences were statistically significant(P < 0.05).There was no significant difference among the other groups(P > 0.05).8.HE staining and pathological tissue score: the heart,liver,spleen,lung,kidney and soft tissue around joints of mice showed no damage in each group.Histopathological score of joints,IL-10 group was lower than PBS control group;IL-10@exosomes group was lower than PBS control group and Exosomes group;IL-10@exosomes + ultrasound group and IL-10@exosomes + ultrasound + microbubble group were lower than control group,IL-10 group and Exosomes group.And the differences were statistically significant(P < 0.05).There was no significant difference among other groups(P > 0.05).9.Saffron O-fixation green staining: there were only a few light red cartilage components in the ankle joints of mice in the PBS control group,and the continuity of cartilage was interrupted and most of them disappeared.The cartilage of ankle joint in IL-10 group and Exosomes group was light red,thicker than PBS control group,and some continuity was interrupted or even disappeared.Compared with the PBS control group,IL-10 group and Exosomes group,the cartilage tissue in the ankle joint of IL-10@exosomes group increased,with darker color,uneven surface and obvious thinning of some areas.The cartilage tissue of ankle joint in IL-10@exosomes + ultrasound group was dark red,which was increased compared with PBS control group,IL-10 group,Exosomes group and IL-10@exosomes group,and the cartilage surface was not smooth or some areas were thinner.The cartilage of IL-10@exosomes + ultrasound + microbubble group was deep red,without obvious thinning,and some regions were mildly not smooth.10.Tartrate-resistant acid phosphatase(Trap)staining: A large number of red osteoclasts appeared in the ankle of mice in PBS control group.Osteoclasts in the ankle joints of mice in IL-10 and Exosomes groups were decreased compared with PBS control group;Intra-articular osteoclasts in IL-10@exosomes group were significantly reduced compared with the PBS control group,IL-10 group and Exosomes group.IL-10@exosomes + ultrasound group showed scattered osteoclasts;IL-10@exosomes + ultrasound + microbubbles group had no obvious or a little osteoclasts in the ankle joint.Conclusion: Ultrasound combined with IL-10@exosomes in the treatment of mice CIA model arthritis has targeting,good safety and therapeutic effect in vivo and microbubbles can further improve its in vivo targeting and therapeutic effect on mice CIA model arthritis,which provides a new strategy for the treatment of RA. |
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