Study On Treating CIA Model Arthritis Of Rats By Ultrasound Combined With Microbubbles Mediated Targeted Cell-mimetic Mesoporous Silica Nanoparticles Loaded With Methotrexate

Background and Purpose:As a common type of inflammatory arthropathy,rheumatoid arthritis(RA)often involves multiple joints of the whole body.It may cause bone erosion and damage other important tissues or organs including the heart,blood vessels,lungs and so on.Inflammatory intra-articular hyperplasia synovium is the pathological basis of RA joint damage.Inflammatory factors produced by macrophages in the synovial membrane are an important cause of disease progression.Therefore,inflammatory joints are the target of treatment.Since RA is often unhealed after treatment,it is recognized as a refractory disease which easily leading to disability.Antirheumatic drugs(DMARDs),such as methotrexate(MTX),are most widely used drugs in firstline to treat RA due to their significant efficacy and wide indications.MTX can not only inhibit joint inflammation,but also reduce joint damage and inhibit disease progression.However,MTX also shows disadvantages including low bioavailability,poor treatment targeting,and high systemic toxicity or side effects.Therefore,it is necessary to search for a durable,highly effective,targeted and low side effect therapy with MTX loaded.Recently,different types of drug carriers have been designed,including liposomes,micelles and nanoparticles.Particularly,mesoporous silica nanoparticles(MSN)indicate good biocompatibility,stability and high drug loading rate.However,these MSN nano drug carriers are often recognized as foreign objects by our immune system and quickly removed,so it is difficult to achieve long circulation and efficient drug release in vivo.Inspired by bionics,polydopamine(PDA)can be firmly coated on the surface of MSN nanoparticles loaded with drug and it may release drugs.In addition,the cell-mimetic nanoparticles coated with cell membrane can evade the recognition and clearance of the immune system because of their immune escape function.It’s reported that red cell membrane(RBCM)has good biocompatibility,biodegradability and long circulation ability.Synovial macrophages in RA are important inflammatory cells,which highly express folate receptor β(FRβ)on the surface,and can specifically bind with folate acid(FA)ligand.Therefore,FRβ of inflammatory synovial macrophages can be used as an ideal target for RA therapy.We aimed to design and construct folate acid targeted mesoporous silica nanoparticles loaded with MTX for targeted therapy of RA.In this chapter,MSNMTX@PDA@RBCM-FA NPs were prepared and characterized.The drug release in vitro mediated by different p H,temperature and ultrasound combined with microbubble(US + MB)was evaluated,providing experimental basis for the safety,targeting and therapeutic efficacy of MSN-MTX@PDA@RBCM-FA NPs in vitro.Materials and Methods:1.MSN-MTX and MSN-MTX@PDA nanoparticles were prepared by dopamine self-polymerization method.Folate targeted mesoporous silica nanoparticles with MTX loaded(MSN-MTX@PDA@RBCM-FA NPs)were prepared by Michael addition reaction and erythrocyte membrane coating methods.2.The particle size and zeta potential were measured by Brookhaven particle size potential analyzer.3.Fourier transform infrared spectrum(FTIR)and thermogravimetric analysis(TGA)were used to analyze the thermal properties of the MSNMTX@PDA@RBCM-FA NPs.4.The morphology of nanoparticles was observed by transmission electron microscope(TEM),and whether RBCM exists on the surface of MSNMTX@PDA@RBCM-FA NPs was observed by TEM and fluorescence microscope.5.Flow cytometry(FCM)was used for quantitatively evaluating the fluorescence carrying capacity of MSN-MTX@PDA@RBCM-FA.6.Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)was employed to verify the presence of RBCM membrane protein on the surface of MSNMTX@PDA@RBCM-FA NPs.7.US + MB was used to trigger and control drug release.The drug loading and release experiments of nanoparticles were detected by UV spectrum analyzer and calculated using the standard curve.Dialysis bag method was applied to evaluate the drug release of MSN-MTX@PDA@RBCM-FA NPs at different time points.Results:1.Loading rate and encapsulation efficiency of MTX: the MTX loading rate and encapsulation efficiency of MSN-MTX@PDA@RBCM-FA NPs were 92.25 ± 4.11%and 24.19 ± 0.48%,respectively.2.Morphology and particle size distribution: TEM showed that the MSNMTX@PDA@RBCM-FA NPs were spherical in shape,and the surface of the NPs was successfully coated with RBCM.Fluorescence microscopy showed that the surface of nanoparticles was successfully coated with RBCM.The particle size of MSN-MTX@PDA@RBCM-FA NPs measured by Brookhaven particle size analyzer was about 143.01 ± 1.36 nm,and remained unchanged after 30 days storage at 4 °C.3.The FTIR and TGA analysis showed that MSN-MTX@PDA@RBCM-FA NPs contained MSN,MTX,PDA and FA.4.Fluorescence microscopy and FCM assay verified the presence of RBCM on the surface of MSN-MTX@PDA@RBCM-FA NPs.5.SDS-PAGE results showed that the membrane protein expression of RBCM coated MSN-MTX@PDA@RBCM-FA NPs was consistent with that of pure RBCM.6.The release of MTX mediated by US + MB in simulated normal and inflammatory humoral environments: under the simulated normal humoral environment(p H 7.4,temperature 37 °C)and inflammatory humoral environment(p H5.3,temperature 43 °C),the release rate of MTX from MSN-MTX@PDA and MSNMTX@PDA@RBCM-FA NPs decreased when compared with MSN-MTX NPs.Moreover,the release rate of MTX from MSN-MTX@PDA@RBCM-FA NPs was the slowest.However,the release rate of MTX mediated by US + MB was only slower than MSN-MTX NPs.Conclusion:In this study,we successfully prepared mesoporous silica nanoparticles loaded with MTX and coated with RBCM and FA with high stability,good,dispersion and high drug loading rate.MSN-MTX@PDA@RBCM-FA NPs showed dual drug release effect,and can promote the release of MTX mediated by US + MB.Background and Purpose: In order to solve the problems of low bioavailability,poor targeting and high toxicity of MTX in the treatment of RA,we have successfully prepared MSNMTX@PDA@RBCM-FA NPs in the first chapter.Furthermore,US+MB can promote the release of MTX.However,whether MSN-MTX@PDA@RBCM-FA NPs have toxicity to normal cells,and whether it can deliver MTX to the inflammatory cells,as well as the effectiveness of in vitro treatment have not been investigated.Human umbilical vein endothelial cells(HUVECs)are often used to study cell toxicity because of the convenience to obtain and culture.In the synovial membrane of RA inflammatory joint hyperplasia,there are a large number of new microvessels and macrophages gathered,which play a crucial role in the occurrence and development of joint inflammation.Moreover,RAW264.7 cells derived from mouse macrophages have showed a strong response when respond to inflammatory stimuli such as lipopolysaccharide(LPS)which is the most widely used inflammatory stimuli.In this experiment,we will firstly investigate the cytotoxicity of MSNMTX@PDA@RBCM-FA NPs on HUVECs.In addition,the targeting and activity inhibition of LPS stimulated RAW264.7 cells mediated by US + MB MSNMTX@PDA@RBCM-FA NPs were observed.The results may provide a feasible reference for the further study of CIA model of rats in vivo.Materials and Methods: 1.HUVECs and RAW264.7 cells were cultured in vitro.2.After HUVECs were co-cultured with different nanoparticles loaded with MTX for 24 hours,the viability of HUVECs was quantitatively detected by methyl thiazolyl tetrazolium(MTT).The survival of HUVECs was observed by living / dead cell fluorescence staining.3.The activated RAW264.7 cells were incubated with Di O fluorescent dye,and the RAW264.7 cells were stained with Rhodamine B and 4 ’,6-diamino-2-phenylindole(DAPI).The uptake of different nanoparticles by RAW264.7 cells was observed by confocal laser scanning microscopy(CLSM),and the overall fluorescence intensity of Di O was quantitatively analyzed by image J software.4.Fluorescence microscopy and flow cytometry(FCM)were used to observe the uptake rate and fluorescence intensity of MSN-MTX@PDA@RBCM-FA NPs when handled with activated RAW264.7 cells.5.The relative activity of RAW264.7 cells in groups with different concentration of LPS was detected by MTT assay.The survival of RAW264.7 cells was observed by living/dead cell staining.6.The apoptosis of RAW264.7 cells under different ultrasound conditions and various concentrations of nanoparticles was quantitatively detected by FCM.At the same time,the apoptosis of RAW264.7 cells under different ultrasound conditions and different concentrations of nanoparticles was observed by fluorescence microscope.7.The diluted red blood cell suspension was extracted and incubated with MSNMTX@PDA@RBCM-FA NPs,normal saline and pure water at 37 °C.The hemolysis was observed and hemolysis rate of NPs was calculated.Results: 1.In vitro safety: MTT results showed the lowest cytotoxicity of MSNMTX@PDA@RBCM-FA NPs to HUVECs,and the cell activity of HUVECs was 98.2% ± 5.0%.However,the activity of HUVECs was 78.2% ± 2.9% in MSN group,81.5% ± 5.6% in MSN-MTX@PDA group and 65.0% ± 1.5% in MSN-MTX group.Living / dead cell staining showed the similar number of living cells and few dead cells in MSN-MTX@PDA@RBCM-FA group as compared to the control group.2.In vitro targeting: all of the fluorescence microscopy,CLSM and FCM quantitative analysis showed the most amount and the strongest green fluorescence of activated RAW264.7 cells labeled with Di O MSN-MTX@PDA@RBCM-FA NPs,which was significantly higher than that of MSN-MTX@PDA NPs and the control group(P < 0.05).3.In vitro therapeutic effectiveness:(1)MTT results showed that,the activity of RAW264.7 cells in MSNMTX@PDA@RBCM-FA+US+MB group was the lowest compared with MTX group and US + MB group(P<0.01).As compared to MSN-MTX@PDA@RBCM-FA group,the activity of RAW264.7 cells in MSN-MTX@PDA@RBCM-FA+US+MB group was significantly lower(P < 0.05).(2)FCM showed that higher concentration of NPs,stronger ultrasonic intensity,longer ultrasonic irradiation time and larger ultrasonic duty cycle leaded to the increased apoptosis rate of activated RAW264.7 cells.Moreover,the differences among each group were statistically significant(P < 0.01).(3)Fluorescence microscope observation of apoptosis showed that with the increase of ultrasonic intensity,ultrasonic irradiation time,ultrasonic duty cycle and the increase of MSN-MTX@PDA@RBCM-FA NPs concentration,the total number of apoptotic cells in activated RAW264.7 cells increased.4.Blood compatibility: the hemolysis test showed the hemolysis rate of MSNMTX@PDA@RBCM-FA NPs was 2.32%.Conclusion: MSN-MTX@PDA@RBCM-FA NPs show good blood compatibility,free toxicity to normal cells,and have obvious targeting to activated RAW264.7 cells.Moreover,it can significantly inhibit the activity of activated RAW264.7 cells,effectively induce the apoptosis of activated RAW264.7 cells,and the efficiency is more significant with US + MB mediation.The results provided experimental basis in vitro for further study of MSN-MTX@PDA@RBCM-FA NPs’ application in targeted therapy of rats CIA model in vivo.Background and Purpose: The formation of synovial pannus in inflammatory joints causing the erosion of articular cartilage and bone is the pathological basis of RA.Inflammatory synovial tissue can produce a large number of inflammatory cytokines,such as TNF-α,IL-1 β,IL-6 and so on,which further damage articular cartilage and bone tissue.These inflammatory cytokines are mainly secreted by the activated synovial macrophages,so the macrophages are the targets for treatment.In the Chapter 1,we successfully prepared the MSN-MTX@PDA@RBCM-FA NPs.In Chapter 2,the safety of NPs on normal cells and its targeting and effective inhibitory effect on activated RAW264.7 cells were confirmed by cell experiments in vitro.Moreover,NPs showed a better inhibitory effect on activated RAW264.7 cells mediated by US + MB.However,whether MSN-MTX@PDA@RBCM-FA NPs can inhibit the activity of activated synovial macrophages,and whether they can inhibit the development of RA inflammation,still need to be further verified in vivo.The combination of US and MB can increase the permeability of blood vessel and cell membrane,promote the localized targeted and timed drug release,and enter the cells to enhance the therapeutic effect.Therefore,this study was aimed evaluate the safety,targeting and effectiveness of MSN-MTX@PDA@RBCM-FA NPs used for the treatment of CIA model,and to explore the application prospect and value of MSNMTX@PDA@RBCM-FA NPs for the treatment of RAMaterials and Methods: 1.The CIA model of SD rats was successfully established with bovine type II collagen and incomplete Freund’s adjuvant.The rats were randomly divided into five groups:(1)control group,(2)MTX group,(3)US + MB group,(4)MSNMTX@PDA@RBCM-FA Group(5)MSN-MTX@PDA@RBCM-FA+US+MB group.There were 6 rats in each group.2.Arthritis score and body weight of each group were measured.3.On day 16,18 and 20 after the initial immunization,the corresponding drug loaded nanoparticles(MTX dose equivalent was about 2 mg / kg,PBS solution of the same volume was injected into the control group)were injected into the tail vein of each group,respectively.The ultrasound irradiation group should be given ultrasound irradiation at the same location and timing on both ankle joints.4.Three rats in each group were randomly selected for Micro-CT examination of ankle joint.5.Verifying targeting in vivo(Di R labeled nanoparticles were injected into the tail vein):(1)The fluorescence signal intensity of nanoparticles in ankle joint was observed by fluorescence imaging in vivo.(2)After injecting MSN-MTX@PDA@RBCM-FA NPs from tail vein,frozen sections were made in the synovial tissue of the ankle joint,and the fluorescence expression was observed by fluorescence microscope.6.For verifying the safety and efficiency of MSN-MTX@PDA@RBCM-FA NPs in vivo,the rats were sacrificed on day 28 and analyzed by pathology.Specifically,HE staining was used to examine the heart,liver,spleen,lung and kidney.HE staining,safranin O-fast green staining,qualitative observation and bone surface/bone volume quantitative analysis of Micro CT,were used to examine the ankle joint.The serum level of inflammatory cytokines including TNF-α and IL-1β were detected by ELISA.Results: 1.Gross observation showed that:(1)After treatment,there was no obvious swelling of ankle joint in both of the MSN-MTX@PDA@RBCM-FA group and the MSN-MTX@PDA@RBCMFA+US+MB group,but significant swelling of ankle joint existed in US + MB group,MTX group and control group.(2)Except for the control group,there was no significant weight loss in the other groups after treatment.(3)After treatment,the arthritis scores of the control group and US + MB group was maintained as 8-11,which was higher than that of other treatment groups at the same time.The arthritis score was significant declined in MSNMTX@PDA@RBCM-FA group and MSN-MTX@PDA@RBCM-FA+US+MB group,and the arthritis score of MSN-MTX@PDA@RBCM-FA+US+MB group was the lowest.2.Safety of nanoparticles in vivo: HE staining of the heart,liver,spleen,lung and kidney showed that there were no obvious pathomorphological changes in all experimental groups when compared with normal rats.3.Targeting of nanoparticles in vivo:(1)The results of in vivo fluorescence imaging showed that after injection of Di R labeled MSN-MTX@PDA@RBCM-FA NPs through the tail vein,the fluorescence signal could be detected in the ankle joint of rats since 2nd hour.In the presence of US + MB,the fluorescence signal increased rapidly at 4th hour,and reached the peak at 8th hour,then decreased slowly and continued to be detected with strong fluorescence signal at 24 th hour.Without US + MB mediated MSN-MTX@PDA@RBCM-FA NPs,the fluorescence signal increased continuously within 24 hours,but the fluorescence intensity was significantly lower than that of MSN-MTX@PDA@RBCMFA+US+MB Group.(2)The frozen section of ankle joint showed that after the tail vein injection of Di R labeled MSN-MTX@PDA@RBCM-FA NPs,the red fluorescence signal was detected in the inflammatory ankle synovial cells.Furthermore,the fluorescence signal was more uniform and extensive in the synovial macrophages with the mediation of US + MB.Meanwhile,the MSN-MTX@PDA@RBCM-FA NPs had stronger red fluorescence signal in synovial macrophages when treating more severe arthritis.4.Micro CT of ankle joint: three-dimensional reconstruction showed that the bone cortex of ankle joint in both MSN-MTX@PDA@RBCM-FA and MSNMTX@PDA@RBCM-FA+US+MB group was smooth and continuous.The quantitative analysis results showed that BS/BV values in the MSNMTX@PDA@RBCM-FA+US+MB group and MSN-MTX@PDA@RBCM-FA group were significantly higher than that of control group(all P < 0.01).5.Serum ELISA: compared with the control group,the serum levels of TNF-α and IL-1β in MSN-MTX@PDA@RBCM-FA group and MSNMTX@PDA@RBCM-FA+US+MB group were significantly decreased(P < 0.01).And the levels of TNF-α and IL-1 β in MSN-MTX@PDA@RBCM-FA+US+MB group decreased more significantly than that in MSN-MTX@PDA@RBCM-FA group(P < 0.05). 6.HE staining of ankle joint:(1)Histological observation: there were a lot of synovial hyperplasia and inflammatory cell infiltration in the control group.The inflammation of ankle joint in MTX group and US + MB group was slightly alleviated,but synovial hyperplasia was still significant.In the MSN-MTX@PDA@RBCM-FA group and MSNMTX@PDA@RBCM-FA+US+MB group,there was no obvious synovial hyperplasia and inflammatory cell infiltration.(2)Pathological score: the pathological scores of ankle joint in the control group,the MSN-MTX@PDA@RBCM-FA group,and the MSN-MTX@PDA@RBCMFA+US+MB group were 9.0 ± 0.9,4.6 ± 0.6 and 1.4 ± 0.8,respectively.Among them,the MSN-MTX@PDA@RBCM-FA+US+MB group showed the lowest pathological score,and it’s significantly lower than the other two groups(P < 0.001,P < 0.01,respectively).7.Ankle safranin O-fast green staining: The surface of articular cartilage in control group and US + MB group was lightly red stained,and no red stained cartilage was found in some areas,suggesting that the articular cartilage tissue was seriously degraded and destroyed.MSN-MTX@PDA@RBCM-FA and MSNMTX@PDA@RBCM-FA+US+MB groups showed the basically intact cartilage with extremely deep red staining and no continuous interruption.Conclusion:(1)MSN-MTX@PDA@RBCM-FA NPs has no damage to the important tissues and organs of CIA model arthritis in rats,suggesting the good biological safety in vivo.(2)MSN-MTX@PDA@RBCM-FA NPs can be targeted uptake by synovial macrophages,and the targeting ability is better when mediated by US + MB.At the same,the MSN-MTX@PDA@RBCM-FA NPs had a better targeting when treating more severe arthritis.(3)MSN-MTX@PDA@RBCM-FA NPs has a good therapeutic effect on CIA model arthritis in rats,especially under the US + MB mediation.It can significantly reduce the degree of joint redness and swelling in CIA model rats,down-regulate the level of inflammatory cytokines in blood,inhibit the synovial hyperplasia and inflammatory cell infiltration of inflammatory joints,and effectively protect the cartilage and bone tissue of inflammatory joints.US + MB mediated MSN-MTX@PDA@RBCM-FA NPs shows safety,targeting and efficacy in the treatment of CIA model in rats,which provides valuable experimental basis for the future application in targeted treatment of RA using MSNMTX@PDA@RBCM-FA NPs with US+MB mediation.