Bioconjugated Carbon Dots For Delivery Of SiTnfα To Enhance Chondrogenesis Of Mesenchymal Stem Cells By Suppression Of Inflammation

OBJECTIVE:Bone marrow mesenchymal stem cell（BMSC）is a promising method for the treatment of cartilage defects.In this process,inflammatory cytokine tumor necrosis factorα（TNFα）plays an important role and may be a potential intervention target.We studied the transfection of mesenchymal stem cells by using fluorescent carbon dot nanocarriers,and through TNFαgene transfection to inhibit inflammation and promote the differentiation of stem cells towards cartilage to accomplish cartilage regeneration,so as to achieve perfect repair of cartilage defects.METHODS:In our study,carbon dots（CD）were bound to siRNA by4-（n-maleimide methyl）cyclohexane-1-carboxylic acid sulfonate sodium salt（SMCC）as biological coupling agent,and transfected with TNFαgenes into bone marrow mesenchymal stem cells.The common commercial transfection reagent Polyetherimide（PEI）was used as a control.The experimental study was divided into four groups:pure bone marrow mesenchymal stem cells group,CD+SMCC group,PEI+siRNA group and CD+SMCC+siRNA group.Zeta potential was used to detect the Zeta potential of pure carbon and carbon dot nanocarriers and transfected gene complexes.DLS and fluorescence microscopy were used to detect the changes of grain size and fluorescence characteristics before and after carbon dot coupled transfection.The molecular structure and chemical composition of carbon dot and carbon dot nanocarrier and transfection gene complex were analyzed by infrared spectrometer.The toxicity of carbon dot nanocarriers was detected by MTT assay.Transmission electron microscope was used to observe the mechanism of the entry of the CD-SMCC-si Tnfαcomplex into cells before and after the carbon dots coupling.The confocal laser microscope was operated to observe the entry of CD-SMCC-siTnfαcomplexes into cells.Flow cytometry was applied to explore the optimal transfection concentration and time.qRT-PCR was used to detect the inhibition of TNFαexpression after siRNA transfection into bone marrow mesenchymal stem cells.DNA content of bone marrow mesenchymal stem cells after siRNA transfection was found to reflect cell proliferation.Staining was used to find cell morphology and GAG secretion.The expression of chondrospecific genes（Sox9,Acan,Col2a1）in chondrogenic differentiation of stem cells was detected by qRT-PCR.Four different groups of cells and materials were implanted into the chondral defects of the femoral condyles of the knee joint in rats.General observation and pathological sections were performed at 4 and 8 weeks after surgery.Immunohistochemistry was performed on the sections to detect the contents of Col2a1 and Tnfαin cartilage tissue,and the repair effect of cartilage defect in vivo was determined by the above methods.RESULTS:CD and CD-SMCC-si Tnfαshowed a monodispersed spherical structure,and the average particle size of CD coupled with SMCC increased from 4-10nm to 13-22nm.Under different excitation light of fluorescence microscope,CD-SMCC fluorescence can be observed in blue,green and red.After the coupling of CD and SMCC,the UV absorption spectrum changed.The excitation wavelength was from 220 nm to 400 nm,and the intensity peak was360 nm.Infrared spectrum detection showed that the spectral peak changed after CD modified by SMCC,located at 2939 cm^-1,1809 cm^-1 and 1780 cm^-1.The best coupling ratio between CD-SMCC and siRNA was 20:1,and the zata potential at the carbon dots after coupling was changed from 17.06±2.3 mv to 21.33±1.4 mv.Compared with PEI,the cytotoxicity of CD-SMCC was significantly lower.Transmission electron microscopy and laser confocal microscopy found that CD-SMCC,as the carrier,could successfully transport si Tnfαto cells.Flow cytometry detection showed that the transfection efficiency was the highest when the mass weight ratio of CD-SMCC and siTnfαcomplex was 20:1,while the optimal transfection time was 12 hours,and the transfection efficiency of CD-SMCC was significantly higher than PEI.The results of qRT-PCR showed that after TNFαsiRNA was transported into the cells,it could exert interference effect on the bone marrow mesenchymal stem cells（BMSCs）,and the fluorescence of carbon dot played a dynamic role in the transfection process.Tnfαwas down-regulated in the CD-SMCC-siTnfαgroup 7 days after transfection,and the expression level of Tnfαwas gradually increased after 14days and 21 days,indicating that the carbon dot transfection was in the early stage and could achieve higher intervention effect within a week.When TNFαgenes were inhibited in BMSCs cells,the expression levels of chondrogenic specific genes such as Sox9,Acan and Col2a1 were significantly increased.The increased expression of chondrospecific genes in the process of chondrogenic differentiation of BMSCs indicates that the rate of chondrogenic differentiation of stem cells can be significantly accelerated after gene modification.Finally,we tested the genetically modified seed cells and found no significant difference in the amount of DNA in the CD-SMCC-siTnfαcytoplasm group and the pure stem cell group,and the amount of GAG in the CD-SMCC-si Tnfαcytoplasm group was significantly higher than that in the PEI group,indicating that the carbon dot nanocarrier material has good biocompatibility with BMSCs.In vivo experiment,from the general view,gross score,histological staining,and histological grading of comprehensive evaluation can be found that the CD-SMCC-siTnfαgroup joint repair effect is better than PEI and CD-SMCC group,blank group repair effect is the worst,and articular cartilage defect repair was achieved by promoting bone marrow mesenchymal stem cells into cartilage differentiationthroughinhibitinginflammation.Accordingto immunohistochemical staining and qRT-PCR detection,the GAG and Col2a1expression in CD-SMCC-siTnfαgroup were higher than those in the other three groups,revealing that the fluorescent carbon dot nanocarriers can effectively mediate the transfection of TNFαsiRNA into BMSCs,and the BMSCs modified by TNFαsiRNA were able to promote the repair of articular cartilage defects.CONCLUSION:The results of this study confirmed that,after being modified by SMCC as a nanocarrier,fluorescent carbon points can successfully transfect BMSCs with TNFαsiRNA,the transfection efficiency is higher than PEI,and the dynamic tracer of gene transfection is achieved.Expression of inhibited Tnfαincreased the expression level of cartilage specific genes（Sox9,Acan,Col2a1）,promoted the chondrogenic differentiation of stem cells,and enhanced their ability to repair cartilage defects.At last,the proliferation and chondrogenic differentiation of the genetically modified mesenchymal stem cells were stronger,and the new cartilage presented hyaline cartilage morphology.