Self-Assembled Terbium-Cysteine Nanoparticles As A Model For Terbium Biosafety And Bone Repair Ability Assessment

Background and ObjectiveDue to the excellent physical and chemical properties,rare earth elements(REEs)have been widely applied in biomedical field.Therefore,the biosafety of REEs has attracted more and more attention.It is noteworthy that bone and liver tissues have been confirmed as the main targets of REEs.Because of the rapid metabolization,REEs usually show the low toxicity to liver tissue,while REEs finally redistribute and concentrate in bone tissue with time.However,the long-term effects of REEs on bone tissue have been rarely reported in detail,which makes people lack of enough confidence in their biosafety during in vivo applications.As is known,doping is the main form in biomedical applications of REEs,which is difficult to simulate the high concentration of REEs enriched in bone tissue.Meanwhile,due to the interference of other components of host material,it is difficult to accurately evaluate the long-term effects of REEs on bone tissue.Therefore,it is particularly important to establish a reasonable model to simulate REEs enrichment state in bone tissue and exclude the interference of other components,so as to accurately evaluate REEs long-term effects on bone and system safety in vivo.As a typical member of REEs,terbium(Tb)has been widely studied in imaging and tracing in vivo due to its promising bioactivity and fluorescence characteristics.In this study,we prepared self-assembled terbium-cysteine nanoparticles(Tb-Cys NPs).Since only Tb3+and Lcysteine(Cys)were included in the composition of Tb-Cys NPs,and Cys has little effect on cell activity,osteogenic-angiogenic differentiation,we evaluated the potential effects of Tb3+on bone tissue and its long-term safety in vivo based on Tb-Cys NPs.In this study,the metalamino acid model also provides a reference for evaluating the biosafety of other REEs.Material and Method1.Synthesis and characterization of Tb-Cys NPs.The Tb(NO3)3·6H2O aqueous solution was added into mixture of NaOH and Cys dropwise.Tb-Cys NPs were synthesized by ion-molecular self-assembly after stirring evenly.The Tb-Cys NPs were characterized by scanning electron microscope(SEM),transmission electron microscope(TEM),Fourier transform infrared spectroscopy(FTIR),X-ray photoelectron spectroscopy(XPS),energy dispersive X-ray spectroscopy(EDS),X-ray diffractometer(XRD)and fluorescence spectrophotometer(FLS).2.Biocompatibility of the Tb-Cys NPs in vitro.Bone mesenchymal stem cells(BMSCs)were cultured in α-MEM medium containing different concentrations of Tb-Cys NPs(0 μg/mL,25 μg/mL,50μg/mL,100 μg/mL,200μg/mL)and 0 μg/mL group was set as the control group.Cell biocompatibility was measured by live/dead cell staining and cell counting kit 8(CCK-8).The fluorescence of Tb-Cys NPs was observed by laser confocal fluorescence microscopy.3.Osteogenic differentiation capacity of BMSCs in Tb-Cys NPs medium in vitro.BMSCs were cultured in α-MEM medium containing different concentrations(0 μg/mL,25μg/mL,50 μg/mL,100μg/mL,200 μg/mL)of Tb-Cys NPs.Alkaline phosphatase(ALP)staining,ALP activity,alizarin red S(ARS)staining,cetylpyridinium chloride(CPC)analysis and quantitative real-time polymerase chain reaction(RT-qPCR)were used to evaluate the effect of Tb-Cys NPs on osteogenic differentiation of BMSCs.4.Angiogenic differentiation capacity of BMSCs in Tb-Cys NPs medium in vitro.BMSCs were cultured in α-MEM medium containing different concentrations(0 μg/mL,25μg/mL,50 μg/mL,100 μg/mL,200 μg/mL)of Tb-Cys NPs.Tube formation assay and RT-qPCR were used to evaluate the effect of Tb-Cys NPs on angiogenic differentiation of BMSCs.5.Effect of Cys on osteogenic-angiogenic differentiation of BMSCs.BMSCs were cultured in α-MEM medium containing different concentrations(0 μg/mL,50 μg/mL)of TbCys NPs for osteogenic and angiogenic induction respectively.The effect of Cys on BMSCs was investigated by RT-qPCR quantitative analysis.6.Mechanism of Tb-Cys NPs promoting osteogenic-angiogenic differentiation of BMSCs.BMSCs in control group and 50 μg/mL Tb-Cys NPs group were cultured by osteogenic induction and angiogenic induction respectively,after which ribonucleic acid(RNA)sequencing was performed.7.Evaluation of long-term safety in vivo.A rat femoral defect model was established.The freeze-dried Tb-Cys NPs powder was implanted into the defect to evaluate the long-term safety and bone repair ability in vivo.The heart,liver,spleen,lung,kidney,muscle(nearby the defect),skin and serum of each group at 1,2,4,and 8 w were harvested to quantify the concentrations of Tb by inductively coupled plasma mass spectrometer(ICP-MS).Meanwhile,paraffin sections were made and hematoxylin and eosin(H&E)staining was conducted to observe the long-term effects of Tb3+in the body.8.Assessment of bone repair in vivo.The model of femoral defect was performed by micro-computed tomography(Micro-CT),reconstruction and quantitative analysis.The femoral specimens were prepared into paraffin sections and stained with H&E and immunohistochemical staining.The new bone and new blood vessels in the defect area were observed under the microscope.Results:1.Tb-Cys NPs were successfully prepared and characterized.The morphology of the prepared Tb-Cys NPs was observed by SEM and TEM.Tb-Cys NPs were relatively uniform.FTIR spectra showed that Cys had the typical peaks at 2554 cm-1(-SH)and 1583 cm-1(COOH).After coordinated with Tb,the peak of the-SH almost disappeared,meaning that-SH participated in the coordination reaction between Cys and Tb.XPS confirmed that the valence state of Tb was+3.EDS results illustrated the presence of Tb,O,C,N and S in Tb-Cys NPs.The XRD pattern confirmed the formation of Tb-Cys NPs through coordination between Tb and Cys.Under excitation light of 488 nm,Tb-Cys NPs could emit characteristic green band fluorescence of Tb3+.2.Tb-Cys NPs possessed good biocompatibility.BMSCs grew well in α-MEM medium containing different concentrations of Tb-Cys NPs.The results of live/dead cell staining and CCK-8 showed that compared with the control group,low concentrations of Tb-Cys NPs promoted the proliferation of BMSCs,while high concentrations slightly inhibited the proliferation of BMSCs.BMSCs could ingest Tb-Cys NPs efficiently characterized by the fluorescence of Tb-Cys NPs under 488 nm excitation by laser confocal fluorescence microscopy.3.Tb-Cys NPs promoted the osteogenic differentiation of BMSCs notably.After osteogenic induction for 7 d,BMSCs treated with 50 μg/mL Tb-Cys NPs showed the highest expression and ALP activity compared with control group.After osteogenic induction for 14 d,the trends of ARS and the relative amount of calcium nodules were similar to ALP.After osteogenic induction for 7 and 14 d,the gene expression trends of ALP,runt-related transcription factor 2(Runx-2),bone sialoprotein(BSP)and osteocalcin(OCN)were almost the same,and the expression was most significantly up-regulated in 50μg/mL Tb-Cys NPs group.4.Tb-Cys NPs promoted the angiogenic differentiation of BMSCs notably.After angiogenic induction for 7 d,the tube formation assay showed that 50 μg/mL Tb-Cys NPs maximized tube formation.After angiogenic induction for 3 and 7 d,the expression of angiogenic related genes angiopoietin-1(ANGPT-1),vascular endothelial growth factor(VEGF),hypoxia inducible factor 1(HIF-1),and hepatocyte growth factor(HGF)enhanced with the increase of Tb-Cys NPs concentration.5.Cys had almost no effect on osteogenic-angiogenic differentiation of BMSCs.After osteogenic induction for 7 and 14 d,there was no significant difference in the expression of osteogenic related genes ALP,Runx-2,BSP and OCN between the two groups.After angiogenic induction for 3 and 7 d,there was no significant difference in the expression of angiogenic related genes ANGPT-1,VEGF,HIF-1 and HGF between the two groups.6.Mechanism of Tb-Cys NPs promoting osteogenic-angiogenic differentiation of BMSCs.Gene ontology(GO)function classification,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis and clustering heat map analysis were performed for the differentiated expression genes between control and Tb-Cys NPs groups.The results showed that Tb-Cys NPs promoted osteogenic differentiation through regulating complement and coagulation cascade signaling pathway,and promoted angiogenic differentiation through regulating cell cycle signaling pathway.7.Tb-Cys NPs possessed good biosafety in vivo.The results of ICP-MS showed that Tb could be metabolized efficiently by the main organs.Further,H&E staining was performed on major organs and tissues at different time points,and the results illustrated that Tb did not cause significant damage to vital organs and tissues.8.Tb-Cys NPs promoted bone repair and angiogenesis in vivo.The results of MicroCT showed that Tb-Cys NPs facilitated bone repair.H&E staining and immunohistochemical staining illustrated that Tb-Cys NPs promoted bone repair and blood vessel formation effectively which provided evidences that Tb-Cys NPs facilitated bone regeneration efficiently.ConclusionIn our study,self-assembled Tb-Cys NPs with high cytocompatibility were designed to reasonably and accurately evaluate the effect of Tb3+ on bone tissue in enrichment state.Cell experiments demonstrated that appropriate concentration of Tb-Cys NPs could promote osteogenesis and angiogenesis of BMSCs,and the interference of Cys on biological effect was also excluded.In vivo experiments confirmed that appropriate concentration of Tb-Cys NPs could effectively promote bone tissue repair.Further,Tb-Cys NPs showed the relatively high long-term biosafety in vivo without obvious damage to major organs.This research not only proves the biosafety and repair ability to bone tissue of Tb,but also provides a suitable model in the form of metal-amino acid assembly for the reasonable assessment of the long-term biosafety and the effects on tissues of other REEs.