Effects Of MSNs-IL-4 On The Biological Properties Of Macrophages

Obejective:Immune system plays pivotal roles in biomaterials mediated functional tissue regeneration.Plasity and diversity is known to be the hallmark of macrophage.The macrophage polarization strongly affects inflammation and tissue regeneration after biomaterials implanted.IL-4 is the key mediator of macrophage polarization.Biotherapy development based on recombinant IL-4 has been hampered by their short half lives in vivo.Due to the highly specific surface area,large pore volume,controllable pore size and vast surface functionalization capabilities,mesoporous silica nanoparticles(MSNs)can load large amount of cytokines or other biological high weight molecules and deliver to cells and tissues to maintain and prolong the bioactivity of the loaded drugs.Therefore,we synthesize and characterize MSNs-IL-4 and evaluate their effect on the proliferation,morphology and gene expression of Raw 264.7cells.Materials and methods:1.We synthesized MSNs by a modified sol-gel method,and loaded MSNs with IL-4 by physical absorbtion.2.The element analysis was performed by the SEM accessary energy dispersive spectrometer system.The small angle X-ray diffraction(SAXRD)pattern of the samples was recorded using X-ray powder diffractometer.The Brunauer-Emmett-Teller(BET)specific surface area and pore volume analysis were used to calculate the specific surface area,pore volume and pore size of the material.3.Transmission electron microscope(TEM)was used to acquire TEM images of the samples.Scanning electron microscopy(SEM)was used to obtain SEM images of the samples.4.After the stimulation of Raw 264.7 cell with MSNs and MSNs-IL-4,the live and dead staining and CCK8 were used to evaluate cell viability,immunofluorescence was used to study the morphology of Raw264.7 cells,q RT-PCR was used to study the gene expression of inflammatory factors of Raw 264.7 cells.After the stimulation of human umbilical vein endothelial cells(HUVECs)with MSNs/Raw 264.7 and MSNs-IL-4/ Raw 264.7 condition medium,the gene expression of angiogenic factor and VEGF pathway-related factors from HUVECs was assessed by q RT-PCR.Results:1.According to Energy-dispersive X-ray spectroscopy analysis,Si was calculated to account for 55.14% and O accounted for 44.86% of the material's weight which proved the purity of the synthesized mesoporous silica nanoparticles.2.The SAXRD pattern of MSNs showed an intense peak and two weaker peaks,which further proved that the synthesized MSNs have hexagonal type of mesoporous structure.3.The typical type IV nitrogen sorption isotherms suggested a mesoporous structure in the MSNs.Meanwhile,the specific surface area,pore volume and pore size of the material were calculated to be1,037.9497 m~2/g,1.626754cm~3/g and 6.2691 nm which suggested that the prepared MSNs had excellent drug loading capacity.4.TEM and SEM images displayed the typical hexagonal array of mesoporous channels and that the nanoparticles were well dispersed.After grafted with IL-4 protein,the channels of MSNs were filled and became inordered which proved that IL-4 was successfully loaded.5.CCK8 analysis indicated that MSNs-IL-4 can stimulate the proliferation of Raw 264.7 cells.6.Immunofluorescence staining showed that MSNs-IL-4 promoted the rearrangement of the cytoskeleton microfilament structure of Raw264.7 cells.7.The q PCR results showed that MSNs-IL-4 increased the expression of M2-related genes transforming growth factor,beta 1(Tgfb1),interleukin 1 receptor antagonist(IL-1ra)and arginase,liver(Arg-1)and decreased the expression of M1-related gene interleukin 6(IL-6).Furthermore,MCM-41/Raw 264.7 condition medium improved the gene expression of matrix metalloproteinase-9(MMP9)and platelet derived growth factor receptor alpha(PDGFR?)of HUVECs,and MSNs-IL-4/Raw 264.7 condition medium stimulated upregulation of the HUVECs of MMP9 expression.Conclusion:MSNs with a large average pore diameter(6.3 nm)were successfully synthesized by a modified sol-gel method,then high weight molecular IL-4 was loaded.MSNs-IL-4 stimulated the proliferation of Raw 264.7cells,promoted the rearrangement of the cytoskeleton microfilament structure.In addition,MSNs-IL-4 increased the expression of M1-related genes,decreased the expression of M2-related gene and induced the transformation of M2 macrophage.Moreover,MSNs-IL-4/Raw 264.7condition medium improved the angiogenic gene expression of the HUVECs which suggest MSNs-IL-4 may be utlized as an immunomodulatory agent for bone tissue engineering applications.