In Vitro Evaluation Of Lysozyme-protected Ultra-small Gold Nanoclusters For The Treatment Of Osteoporosis Through The Dual Roles Of Induction Of Osteogenic Differentiation And Inhibition Of Osteoclast Formation

Objective Exploring the effects of lysozyme-protected gold nanoclusters on osteoblasts and osteoclasts and their potential mechanisms of action.Methods1.Constructing lysozyme-protected gold nanoclusters(Lys-Au NCs)and observing the material dimensions by high-resolution transmission electron microscopy(TEM).2.The proliferation rate of Lys-Au NCs on MC3T3E-1 cells(mouse embryonic osteoblast precursor cells),RAW 264.7 cells(mouse mononuclear macrophage leukaemia cells)and NIH/3T3 cells(mouse embryonic cells)was detected by Cell Counting Kit-8(CCK8).3.The effects of Lys-Au NCs on collagen 1(Col-1),alkaline phosphatase(ALP)and PI3K/Akt signalling pathways were analysed by immunoprotein blotting and confocal fluorescence imaging.4.The effect of Lys-Au NCs on the bone mineralisation level during osteogenesis induction was analysed by alizarin red staining and alkaline phosphatase staining.5.RAW 264.7 cells were continuously induced with nuclear factor κB ligand(RANKL)and macrophage colony-stimulating factor(M-CSF)to become osteoblasts,and Lys-Au NCs were analysed by immunoprotein blotting and confocal fluorescence imaging against tartrate acid phosphatase(TRAP)and NFATc-1/c-Fos/V-ATPase-d2/ CTSK signaling pathway.Results The research results show that Lys-Au NCs have an ultra-small size and in in vitro cellular assays,Lys-Au NCs were found to be insignificantly toxic and to promote osteoblast proliferation.In the osteogenic differentiation assay,Lys-Au NCs were more effective in promoting osteoblast differentiation than lysozyme,gold nanoparticles(Au NPs)or a mixture of lysozyme and Au NPs,by promoting the expression of Col-1,ALP and upregulating the PI3K/Akt signalling pathway;while in the osteoclast induction assay,Lys-Au NCs Lys-Au NCs inhibited TRAP,NFATc-1 expression and down-regulated NFATc-1/c-Fos/V-ATPase-d2/CTSK signaling pathway,thereby inhibiting osteoclast formation.ConclusionsWe have designed an ultra-small biocompatible nanomaterial for the treatment of osteoporosis,an ultra-small gold nanocluster supported by lysozyme protection,and found that this nanocluster can dual regulate osteogenesis and osteolysis by up-regulating the PI3K/Akt signalling pathway and down-regulating the NFATc-1/c-Fos/V-ATPase-d2/CTSK signalling pathway,which has the potentially therapeutic effect on osteoporosis and is expected to be one of the nanomedicines for the treatment of osteoporosis.