Y 2 O 3 Nanoparticles Caused Bone Tissue Damage by Breaking the Intracellular Phosphate Balance in Bone Marrow Stromal Cells

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1 Supporting Information Y 2 O 3 Nanoparticles Caused Bone Tissue Damage by Breaking the Intracellular Phosphate Balance in Bone Marrow Stromal Cells Chunyue Gao, Yi Jin*,, Guang Jia, Xiaomin Suo, Huifang Liu, Dandan Liu, Xinjian Yang, Kun Ge, Xing-Jie Liang, Shuxiang Wang*,, and Jinchao Zhang*, College of Chemistry & Environmental Science, Chemical Biology Key Laboratory of Hebei Province, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Hebei University, Baoding , P. R. China College of Medical Science, Hebei University, Baoding , P. R. China College of Pharmacy, Hebei University, Baoding , P. R. China Chinese Academy of Sciences (CAS) Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, and National Center for Nanoscience and Technology, Beijing , P. R. China

2 MATERIALS AND METHODS Synthesis and Characterization of NPs. Firstly, 2.0 mmol of Y(NO 3 ) 3 6H 2 O and 50 mmol of CO(NH 2 ) 2 were added to 30 ml of deionized water. The mixing solution was then heated to 85 C and maintained 3 h with sufficiently stirring. The as-obtained white precipitate was washed by deionized water and ethanol in sequence and dried in air. Finally, the Y 2 O 3 NPs were obtained by an annealing process at 800 C for 2 h. 1 To conjugation of Y 2 O 3 NPs with fluorescein isothiocyanate (FITC), 10 mg Y 2 O 3 NPs were added to 8 ml anhydrous dimethyl formamide (DMF), homogeneous dispersed by a sonicator. Y 2 O 3 NPs suspension reacted with 500 μl aminopropyl triethoxy siloxane (APTES) in the 30 ml DMF, protected with nitrogen, and stirred at 1000 rpm for 12 h. Then FITC reacted with APTES-Y 2 O 3 NPs by adding 2.5 mg FITC slowly in darkness at room temperature and stirring at 1000 rpm for 6 h. Finally, FITC labeled Y 2 O 3 NPs was centrifuged at rpm for 10 min, and the deposit was collected and dried. 2 To synthesis of YPO 4 -Y 2 O 3 NPs, Y 2 O 3 NPs (30 μg/ml) were added into 5 ml LSBF buffer. After incubation for 24 h, the NPs suspensions were centrifuged at rpm for 10 min. The deposit was collected, washed and dried using vacuum drying chamber. 3 The morphology features of NPs were evaluated by scanning electron microscope (SEM), transmission electron microscope (TEM) and dynamic light scattering (DLS). The phase of Y 2 O 3 NPs was identified by X-ray diffraction (XRD) patterns. BMSCs Culture. BMSCs were prepared from ICR mice as described previously. 4 Then, cells were cultured in DMEM medium in a humidified atmosphere of 5% CO 2 at 37 C. Cell Viability Assay. MTT assay was employed to measure the viability of BMSCs upon treatment with Y 2 O 3 NPs or YPO 4 -Y 2 O 3 NPs. Briefly, BMSCs were seeded and treated with Y 2 O 3 NPs for 6, 12, and 24 h, MTT assay was carried out at a wavelength of 570 nm using a microplate reader. 5 Intracellular ATP Level. Intracellular ATP was detected using an ATP assay kit (Nanjing Jiancheng Bioengineering Institute, China). Generally, BMSCs (2 10 5

3 cells/well) were treated with 3.125, 6.25, and 12.5 μg/ml Y 2 O 3 NPs for 3 days. The ATP level was detected according to the manufacturer s instructions. 6 Mitochondrial Membrane Potential. JC-1 staining was used to measure the loss of mitochondrial membrane potential (MMP). 7 After cells were treated with Y 2 O 3 NPs, the cells were washed, stained with JC-1 for 20 min. Afterwards, the fluorescence of green and red JC-1 were observed using confocal laser scanning microscopy. The Biodistribution of Y 2 O 3 NPs in Vivo. For biodistribution, the mice were injected with Y 2 O 3 NPs (50 mg/kg) by tail vein. Y 3+ biodistribution in different tissues (heart, liver, spleen, lung, kidney, and bone) was detected by inductively coupled plasma mass spectrometry (ICP-MS) as previously described. 8 Toxicity Evaluation in Vivo. 4-week-old female ICR mice (19 ± 2 g) were injected 50 mg/kg Y 2 O 3 and YPO 4 -Y 2 O 3 NPs by tail vein in a total volume of 200 μl per mouse. Biochemical analysis was measured after 35 days. The major organs were separated from mice, fixed in formalin and stained with HE as previously described. 9 Supplementary Figures Figure S1. The dynamic light scattering (DLS) data for determining the size distribution of Y 2 O 3 NPs.

4 Figure S2. Mineral deposits were quantitated by elution of alizarin red S. (*p<0.05, n=5) Figure S3. Cell area was measured by image J analysis. (***p<0.001, n=3) Figure S4. XRD pattern of Y 2 O 3 NPs after exposure to LSBF for 24 h. Asterisk indicate the diffraction peaks of tetragonal YPO 4.

5 Figure S5. The change of zeta potential of Y 2 O 3 NPs after exposure to LSBF or water. (***p<0.001, n=3) Figure S6. In vivo distribution of yttrium element in different organs by ICP-MS. (* p<0.05, ** p<0.01, *** p<0.001, n=3) Figure S7. The serum biochemistry analysis of mice treated with Y 2 O 3 and YPO 4 -Y 2 O 3 NPs for 35 days. (** p<0.01, *** p<0.001, n=3)

6 Figure S8. HE-stained tissue sections from mice injected with Y 2 O 3 and YPO 4 -Y 2 O 3 NPs for 35 days. REFERENCES 1. Jia, G.; You, H. P.; Song, Y. H.; Huang, Y. J.; Yang, M.; Zhang, H. J. Facile Synthesis and Luminescence of Uniform Y 2 O 3 Hollow Spheres by a Sacrificial Template Route. Inorg. Chem. 2010, 49, Lord, M. S.; Jung, M.; Teoh, W. Y.; Gunawan, C.; Vassie, J. A.; Amal, R.; Whitelock, J. M. Cellular Uptake and Reactive Oxygen Species Modulation of Cerium Oxide Nanoparticles in Human Monocyte Cell Line U937. Biomaterials 2012, 33, Li, R. B.; Ji, Z. X.; Chang, C.H.; Darren, R. D.; Cai, X. M.; Meng, H.; Zhang, H. Y.; Sun, B. B.; Wang, X.; Dong, J. Y. Surface Interactions with Compartmentalized Cellular Phosphates Explain Rare Earth Oxide Nanoparticle Hazard and Provide Opportunities for Safer Design. ACS Nano. 2014, 8, Liu, D.; Yi, C.; Zhang, D.; Zhang, J.; Yang, M. Inhibition of Proliferation and Differentiation of Mesenchymal Stem Cells by Carboxylated Carbon Nanotubes. ACS Nano. 2010, 4, Jin, Y.; Chen, S.; Duan, J.; Jia, G.; Zhang, J. Europium-doped Gd 2 O 3 Nanotubes Cause the Necrosis of Primary Mouse Bone Marrow Stromal Cells through Lysosome and Mitochondrion Damage. J Inorg. Biochem. 2015, 146, Wang, C.; Liu, D.; Zhang, C.; Sun, J.; Feng, W.; Liang, X.; Wang, S.; Zhang, J. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of

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