Y2O3 Nanoparticles Caused Bone Tissue Damage by Breaking the

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Y2O3 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 ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b06211 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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Y2O3 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 071002, P. R. China ‡

College of Medical Science, Hebei University, Baoding 071002, P. R. China

£ College

§

of Pharmacy, Hebei University, Baoding 071002, 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 100190, P. R. China

AUTHOR INFORMATION * Corresponding Authors: E-mail: [email protected]; E-mail: [email protected]; E-mail: [email protected]

KEYWORDS: Y2O3 nanoparticles, bone tissue damage, phosphate balance, bone marrow stromal cell, apoptosis

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ABSTRACT: Y2O3 nanoparticles (NPs) have become great promising products for numerous applications in nanoscience especially for biomedical application, therefore increasing the probability of human exposure and gaining wide attention to biosecurity. It is well known that rare earth (RE) materials deposited in the bone and is excreted very slowly. Nevertheless, the effect of Y2O3-based NPs on bone metabolism has not been exactly known yet. In present study, the effects of Y2O3 NPs on the bone marrow stromal cell (BMSCs) and the bone metabolism in mice after intravenous injection were studied. The results demonstrated that Y2O3 NPs could be taken into BMSCs, localized in acidifying intracellular lysosomes, and underwent dissolution and transformation from Y2O3 to YPO4, which could lead to break the intracellular phosphate balance, and induce lysosomal and mitochondrial dependent apoptosis pathways. Furthermore, after being administered to mice, the higher concentration of yttrium occurred in bone, which caused the apoptosis of bone cells and induced the destruction of bone structure. However, the formation of YPO4 coating on the surface of Y2O3 NPs by pretreatment of Y2O3 NPs in lysosomes simulated body fluid (LSBF) could observably decrease the toxicity in vivo and in vitro. This study may be useful for practical application of Y2O3 NPs in the biomedical field.

Rare earth oxide (REO) nanoparticles (NPs) have been gaining largely attention in many fields ranging from industry to agriculture especially in the biomedical field due to their particular optical, electrical and magnetic properties.1 Y2O3 can be doped with a wide variety of rare earth (RE) elements through intramolecular energy transfer, and is considered as an excellent anti-stokes luminescence in vivo imaging field. In particularly, Y2O3 NPs doped with erbium (Er3+) and ytterbium ions (Yb3+) are able to emit higher energy in the near infrared (NIR) range by exciting with a longer wavelength photon at 980 nm, which can penetrate deeper into tissues because of its lower light scattering. This performance may achieve deep imaging of thicker biological samples, and exhibit less damage for biological tissue.2 And furthermore, the peculiar optoelectronic properties of Y2O3 NPs, coupled with biomolecules or targeting molecules, would provide a multifunctional platform by integrating diagnosis and treatment. Setua et al. reported that Y2O3 nanocrystals doped with europium ions (Eu3+) and gadolinium ions (Gd3+) and conjugated with a targeting

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ligand (folic acid), were used for bi-modal fluorescence and magnetic imaging of cancer cells.3 Additionally, Y2O3 NPs can be used as bone repair materials because of its similar mechanical properties to natural bone. Hydroxyapatite (HAP) powder mixed with Y2O3 NPs can reinforce the density and mechanical properties of HAP, which may be used as a promising material for bone regeneration and reconstruction in bone tissue engineering.4 While the rapid development in the production and utilization of Y2O3-based NPs, it has also induced significant concern regarding a question of the hazards and impacts on human health. Therefore, it is essential to understand the Y2O3-based NPs biological interactions and biocompatibility. There is a growing interest in the bioeffect of Y2O3-based NPs in vitro and in vivo. It is well known that NPs characteristics such as particle size, morphology, and surface chemistry can impact on cytotoxic responses even for the same composition. Though numerous literatures on the synthesis of yttrium oxide with a wide variety of morphologies, such as spherical NPs,5 nanorods,6 nanotubes,7 nanosheet,8 have been reported, there are few reports on the Y2O3-based NPs bioeffects and biocompatibility. Tamar et al. synthesized three different morphologies of Y2O3, and found that spherical particles exhibited no cytotoxicity to human foreskin fibroblast (HFF) cells, rod-like particles increased cell proliferation, but platelet particles had marked cytotoxicity.9 The cellular biological effects were different due to the difference of morphologies and surface chemistry. Interestingly, Y2O3 NPs also showed relatively nontoxic to murine hippocampal HT22 cells, and protected nerve cells from oxidative stress. It was also shown that this neuroprotection was independent of NPs size.10 However, recent studies have revealed that Y2O3 NPs caused cytotoxicity due to the generation of reactive oxygen species (ROS).9,11 Y2O3 NPs with spherical shape increased cytotoxicity and genotoxicity in human embryonic kidney cells (HEK293).11 In addition, the biodistribution experiments showed that RE nanomaterials easily accumulated in bone tissue.12 Our previous studies demonstrated that most of [Gd@C82(OH)22]n NPs were accumulated in the bone and had a positive effect on bone formation and prevented osteoporosis in vivo.13 Our another study also showed that europium-doped Gd2O3 nanotubes (Gd2O3:Eu3+ NTs) could enhance the bone mineral density and bone biomechanics in mice by oral gavage.14 However, to the best our knowledge, the effect of Y2O3-based NPs on bone metabolism has not been well reported yet.

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As we known, the balance of calcium and phosphorus metabolism is a highly coordinated process, which is responsible for bone metabolism and necessary to bone formation. In particular, maintaining intracellular phosphate balance has important biological significance for bone metabolism and other essential cellular metabolism processes, such as energy metabolism, cell signaling regulation, and protein synthesis.15,16 Once phosphate metabolism is disordered, chondrocytes will not apoptosis, the normal generation of new bone will be also blocked, rickets and osteomalacia will be developed.17 In addition, REOs are more soluble under acidic conditions, and the released RE ions will transform to REPO4 due to their high binding affinity with phosphate groups.18 While it is not clear whether a similar biological transformation happen to bone tissues when exposed to Y2O3 NPs and this transformation will break the intracellular phosphate balance, resulting in bone tissue damage. Multipotent bone marrow stromal cells (BMSCs) are of paramount physiological importance not only for elucidating their multilineage differentiation, but also developing cell-based therapies for tissue repair and gene therapy.19,20 As a cell model, BMSCs have been widely used in bone tissue engineering fields. In present work, we investigated the effects of Y2O3 NPs on BMSCs in vitro and further studied the effects of Y2O3 NPs on bone metabolism in vivo. The cellular effects were found that Y2O3 NPs induced cell apoptosis via lysosome- and mitochondria-dependent pathways. Combined with our results, we speculated that Y3+ was released from Y2O3 NPs in acidic lysosome, which was rapidly bound with lysosomal phosphate groups to form YPO4, which could break the intracellular phosphate balance simultaneously, and lead to a series of biological responses that trigger BMSCs dysfunction and apoptosis. Then, the in vivo toxicity experiments of Y2O3 NPs indicated that yttrium predominantly aggregated in bone after intravenous injection of Y2O3 NPs in mice, which caused bone cells apoptosis and bone structure destruction. In addition, it can be found that the Y2O3 NPs could transform to YPO4-Y2O3 hybrid materials by treating with lysosomes simulated body fluid (LSBF). The YPO4-Y2O3 could not cause cells apoptosis in vitro and bone damage in vivo, further indicating the intracellular phosphate imbalance was an important mechanism of bone tissue damage caused by Y2O3 NPs. This founding can be beneficial to more rational applications of Y2O3 NPs in the biomedical field. RESULTS AND DISCUSSION

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Y2O3 NPs was synthesized by a homogenous precipitation method. As shown in Figure 1A, the as-obtained Y2O3 NPs are composed of high yield well-dispersed NPs with smooth surface and a narrow size distribution. The TEM image exhibits the perfect spherical morphology, which agrees well with the SEM images. Moreover, the solid core nature of the nanospheres is demonstrated by TEM image (Figure 1B). The mean diameters of Y2O3 NPs are estimated to be 166 nm by Image J analysis (Figure 1C) and the hydrodynamic diameters are determined to be 533.5 nm in water (Figure S1). The phase identification and purity were performed by XRD analysis (Figure 1D). All the diffraction peaks of the sample can be indexed well to the cubic phase of Y2O3 with lattice constants a = 10.58804 nm, and c =10.58804 nm, which is consistent with the literature values of a = 10.601nm, c =10.601 nm [JCPDS No. 65-3178; space group: Ia3(206)]. No impurity peaks can be detected, indicating that the pure phase of Y2O3 can be obtained by this synthesis route. One can also see that the diffraction peaks of the sample are very sharp and strong, indicating the high crystallinity of the Y2O3 NPs. The cytotoxicity assay is a vital step to explain the cellular response to Y2O3 NPs, which can give us some information about cell function, survival, death or metabolic activities. Firstly, the mineralized function of the BMSCs upon treatment with Y2O3 NPs was evaluated by formation of mineralized matrix nodules. The mineralized function of the BMSCs significantly decreased by 1.56 and 3.125 μg/mL Y2O3 NPs treatment for 21 days (Figure 2A). The amount of mineralization was quantitated using elution of alizarin red S from stained mineralized nodules (Figures S2). The results showed that 44.6% and 55.3% decrease in the formation of mineralized nodules upon 1.56 and 3.125 μg/mL Y2O3 NPs treatment, respectively, indicating it is enough to cause BMSCs dysfunction even at a very low concentration (1.56 μg/mL). Next, we have exploited more than one assay to study the viability of BMSCs, including MTT, actin filaments of cytoskeleton staining, and Annexin V/propidium iodide (PI) double-staining assays. Firstly, the morphological changes of BMSCs were observed by cytoskeleton staining after 3 h treatment with 6.25, 12.5, and 25 μg/mL of Y2O3 NPs. Microscopic observations of treated cells revealed the distinct morphological changes. BMSCs could not maintain their fibroblast-like shapes compared with control group. More cells treated with Y2O3 NPs appeared significant shrinkage, restricted extensions and wizened nucleus with the dose increase of Y2O3

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NPs. This result proved definitively that the dysfunction of the cell skeleton as a consequence of Y2O3 NPs treatment (Figure 2B). The similar result could be verified in cell area analysis (Figure S3). Next, the cell viability of the different concentrations of Y2O3 NPs in BMSCs was determined by MTT assay. The cell viability was studied in the concentration range of 3.125-50 μg/mL after 6, 12, and 24 h exposure. Interestingly, it can be found that 3.125 μg/mL Y2O3 NPs did not exhibit any cytotoxicity to BMSCs, while 12.5, 25, and 50 μg/mL Y2O3 NPs caused significant decrease in viability of BMSCs in time- and dose-dependent manners (Figure 2C). Previous report has suggested that Y2O3 NPs could induce apoptosis in HEK293 cells.11 To ascertain whether the treatment of BMSCs with Y2O3 NPs can also cause cell apoptosis, the Annexin V/PI staining assay was further performed. As shown in Figure 2D, after cells were exposed to 12.5 and 25 μg/mL of Y2O3 NPs, the apoptosis percentage was significantly increased. The early apoptotic and late apoptotic cells were 26.72 and 38.61 %, respectively. The physicochemical properties of NPs had the close relation to cellular uptake, bioprocessing, and fate.21 The uptake pathway of NPs into cells is an important factor to determine their intracellular processing and toxicity.22 NPs internalization in a mammalian cell was mainly by pinocytosis or direct penetration pathway. When the size of biomaterials was smaller than the thickness of membrane bilayer (4-10 nm), they could be directly uptaken into the cells by penetration pathway. There were three major modalities to pinocytosis pathway, including micropinocytosis-mediated endocytosis, clathrin-dependent endocytosis, and caveolae-mediated endocytosis.21 As we know, NPs could be uptaken into the cells via different endocytic pathways. In order to understand whether the cytotoxicity was dependent on the mechanism of cellular uptake and the amount of Y2O3 NPs internalized, we first investigated the endocytic pathway using flow cytometry. A series of experiments were performed with specific endocytic inhibitors [wortmannin (400 μmol/L), chlorpromazine (35 μmol/L) and nystatin (10 μmol/L)] and at low temperature (4°C). After BMSCs were treated with 25 μg/mL of Y2O3 NPs at 4°C, we observed the reduced Y2O3 NPs internalization, demonstrating that the internalization process was energy-dependent. And the intensity of side scatter signal decreased in the presence of endocytic inhibitors compared with 37°C group, which suggested that endocytosis was a possible mechanism of internalization in some cases. Here, wortmannin and nystatin

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inhibited endocytosis, showing that micropinocytosis- and caveolae-mediated endocytosis were the possible mechanism for internalization of Y2O3 NPs (Figure 3A). Moreover, Figure 3B and 3C clearly indicated that the uptake of Y2O3 NPs had timeand concentration-dependent manners. Intracellular location and translocation of NPs in cells are also important determinants of cellular responses, which were directly concerned with the cytotoxicity of the internalized NPs. The internalized NPs are normally translocated via either endosomal or lysosomal vesicles, which led to the rapid disintegration and destruction and due to the acidic hydrolases.23 After incubation of BMSCs with FITC labeled Y2O3 NPs for 3 h, green dots could be observed inside the cells and the merge image with yellow fluorescence dots indicated that they were almost in lysosomes (Figure 3D), so lysosome was involved in Y2O3 NPs endocytosis by BMSCs. Previous studies indicated that lysosome had many significant functions in the cell bioprocessing, such as antigen presentation, signal transduction, and cell division, it was also an acidic organelle containing an array of lysosomal hydrolases with the degradative property.24 The integrity of the lysosomal membrane was of great importance for its physiological functions.25 Once the membrane is impaired, large amounts of the hydrolases and exogenous materials are released from the lysosome, which may cause further damage to other organelles. There were many NPs-relevant properties which could trigger lysosome damage including shape, magnetic, and dissolution.26 For example, it was possible that such response led to disruption of phospholipid bilayers of the lysosomal membrane because iron oxide magnetic NPs could generate heat under an alternating magnetic field.27 And metallic oxides (ZnO) were easily dissolved in lysosome and induced pathological changes.28 To verify the integrity of the lysosomal membrane, acridine orange (AO) was used to examine lysosomal membrane permeation (LMP). The intensity of red fluorescence in AOtreated cells reflected the number of intact lysosomes. As shown in Figure 4A, the fluorescence intensity of AO turned to be weaker after internalization of Y2O3 NPs into lysosomes due to the fact that AO was released from impaired lysosomes into the cytosol, especially for 3 h. The result showed that there were significant changes in LMP after internalization of Y2O3 NPs into lysosomes in BMSCs. As we know, several lysosomal acid-dependent proteases have been implicated in the induction of apoptosis as a consequence of LMP, such as cathepsin B and D. These proteases were key factors in regulating acid environment of lysosomes, once released from the

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lysosomes into the cytoplasm, they would cause lysosome alkalinization.29 To further affirm the consequence of lysosomal lesion, we detected the activity of cathepsins B and D in the cytoplasm. The results showed that cathepsins B and D exhibited higher activity after the treatment of Y2O3 NPs (Figure 4B, C), suggesting cathepsins B and D were redistributed from the lysosomes to cytosol as a result of impaired lysosomes. Moreover, confocal images of BMSCs stained with Lyso-Tracker ® Red DND-99, which could accumulate in intracellular acidic organelles, showed that fluorescence intensity gradually decreased with the duration of Y2O3 NPs treatment, indicating that time-dependent alkalinization of lysosomes in Y2O3 NPs-treated cells (Figure 4I). Thus, these data suggested that Y2O3 NPs triggered lysosomal rupture and dysfunction in BMSCs. There was growing evidence that cathepsins released from the lysosome was a prerequisite for their participation in the regulation of apoptosis.29 If cathepsin B, D was translocated from the lysosomes into the cytosol, mitochondrion-dependent apoptosis might be actived. For example, Bid and Bax, two proapoptotic members of the Bcl-2 protein family, could be cleaved by cathepsin. Cathepsin B could cleave Bid to tBid, which was an integrator of different apoptotic pathway. Cathepsin D could activate Bax to induce mitochondrial rupture and cytochrome c release, and then mitochondrion-dependent apoptosis was inevitable.30 Previous studies reported that Bid and Bax could interact with mitochondria outer membrane to cause cytochrome c translocation from the intermembrane space into the cytoplasm, which was a key process for activating caspase 3, a downstream vital step to initiate apoptosis. Once mitochondrial outer membrane permeabilization occurred and the subsequent release of apoptogenic factors represented a point of no return in the mitochondrial-dependent apoptosis pathway. Whether apoptosis induced by Y2O3 NPs through lysosomal- and mitochondrial-dependent pathways remains unclear. So we verified the hypothesis that cathepsins released from the lysosome caused mitochondrial rupture and cytochrome c release, further mediated caspase 3 activation pathway. The data obtained from present study showed the up-regulation of Bid and Bax expression after cells were exposed to Y2O3 NPs (Figure 4D, E). This process might be partly mediated by cathepsins released from the lysosome causing the activation of Bid and Bax. As mitochondrial membrane potential (MMP) was under the control of Bcl-2 family members-Bax, we also accurately quantified MMP changes by measuring JC-1 (Figure 4J). These results showed that MMP decreased within a short time. Hence,

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activation of Bax implied MMP opening and induction of apoptosis through the intrinsic pathway. Next, mitochondrial dependent apoptosis pathway was further examined. The result from ELISA assay showed that cytochrome c release from intact mitochondria after treatment with Y2O3 NPs (Figure 4F). Furthermore, mitochondrial cytochrome c release was one of critical upstream pathways that led to the activation of caspase 3, which was an important indication of apoptosis. To verify whether cytochrome c release causes caspase 3 activation, ELISA assay was used to analysis of caspase 3 activation. As shown in Figure 4G, we observed that Y2O3 NPs also provoked the activation of caspase 3 along with the mitochondrial release of cytochrome c, indicating that mitochondrial outer membranes permeabilization promoted cytochrome c translocation to cytosol, which also facilitated caspase 3 activation. All of these results suggested that apoptosis induced by Y2O3 NPs through lysosomal and mitochondrial cytochrome c-mediated caspase 3 activation pathways. In addition, many studies have shown that induction of ROS could mediate cell apoptosis in various cells by rare-earth based NPs.31−33 ROS generation causes destabilization of lysosomal membrane, the loss of mitochondrial membrane potential and the release of cytochrome c, further leading to the activation of caspase-3, and eventual cell apoptosis. To examine whether ROS was related to the apoptosis induced by Y2O3 NPs, the generation of ROS was measured in BMSCs treated with Y2O3 NPs. As shown in Figure 4H, the ROS levels generated in response to Y2O3 NPs were significantly increased at 12.5 mg/mL for 2 and 3 h, indicating ROS also played a role in the apoptosis caused by Y2O3 NPs. According to previous studies, REOs can be dissolved in the acid solution and the released rare earth ions are ready to form insoluble colloids of phosphate due to their low Ksp values.34 Recent research has shown that REOs, such as La2O3 and Gd2O3, could localize in lysosomes of macrophages and change easily to insoluble phosphates REPO4 due to the high binding affinity to phosphates.18 Moreover, ZnO NPs have similar physicochemical properties to Y2O3, which is very sensitive to dissolution below a pH of 6.7 at physiological temperature. And the dissolution of ZnO NPs could couple with carbonate/phosphate and induce pathological changes.28 Based on above analysis, we speculated whether Y2O3 NPs could dissolve in an acidic lysosomal compartment, lead to the release of Y3+ and subsequently couple with phosphate content of lysosome to form YPO4, which may break the phosphate balance of lysosomes. There were two factors to be concerned with Y2O3 NPs

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transformation. One was component of lysosome, another was the pH value of lysosome. To prove the above speculation, Y2O3 NPs were exposed to different pH of buffer solution with or without lysosomal component: LSBF. As shown in Figure 5A, there was observably increase of Y3+ ions in the buffer at pH 4.5, indicating Y2O3 NPs was easy to dissolve in acid environment. However, there were very small amount of free Y3+ release in the LSBF at pH 4.5 or 7.2. Moreover, the phosphate content significantly decreased in the pH 4.5 of LSBF after exposure to Y2O3 NPs for 24 h (Figure 5B).That is, once taken up into lysosomes, Y2O3 NPs could be dissolved more rapidly in lysosomes (pH ~4.5), Y3+ was released and subsequently reacted with phosphate content, and decreased the phosphate content in lysosomes, which might result in breaking the intracellular phosphate balance. Fourier transform infrared (FTIR) spectra was used to further study the transformation process. The only intense absorption band of pure Y2O3 NPs at 565 cm-1 can be assigned to the Y-O stretching frequencies, confirming the structure of Y2O3. After incubation with lysosomes for 2 and 24 h, the absorption bands of Y2O3 NPs centered at 620 and 1076 cm-1 can be detected besides the characteristic Y-O stretching mode, which are attributed to the bending vibrations of O-P-O group and the asymmetry stretching vibrations of the PO43- groups, respectively. The characteristic bands of the phosphate (PO43-) groups reveals the formation of YPO4 (Figure 5C). The result was consistent with the previous literatures.35−37 In order to further indentify the phase structure of the composite, the XRD of the sample was performed. After exposure to LSBF for 24 h, the diffraction peaks of tetragonal YPO4 [JCPDS 74-2429] can be detected besides the cubic phase of Y2O3 (Figure S4). The result further confirms that the YPO4 have been coated on the surfaces of Y2O3 NPs, which can effectively support the FT-IR result (Figure 5C). The zeta potential in Figure S5 provides powerful evidence for the transformation from Y2O3 to YPO4. Y2O3 NPs incubated in water were positively charged. After being incubated in LSBF, the zeta potential changed to negative charge, which might be attributed to the formation of phosphate. In addition, the morphological changes of the Y2O3 NPs were observed by TEM images (Figure 5D). It can be seen that the shape of Y2O3 NPs was spherical in water, and no obvious morphological change was observed with the increase of reaction time. Noteworthy, the morphology of Y2O3 NPs in LSBF changed to “egg shell” shaped structures at 2 h. By further increasing the reaction time to 24 h, the particle size of Y2O3 NPs decreased and a large amount of disordered fiber-like products formed.

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Such morphological changes could be also a good illustration that Y2O3 NPs in acidifying intracellular lysosome undergo dissolution and transformation from Y2O3 to YPO4. And the crystallization of YPO4 deposits on the Y2O3 NPs surfaces. Moreover, in order to analyze the transformation from Y2O3 to YPO4 in BMSCs, the ultra-thin sections of BMSCs treated with Y2O3 NPs were performed and analyzed by TEM. The results showed that Y2O3 NPs could be taken up into BMSCs and localized in intracellular lysosomes, and the Y2O3 NPs surface formed disordered fiber-like structures in a vesicular compartment of BMSCs (Figure 5E). To further clarify the transformation of Y2O3 to YPO4 in BMSCs, the energy dispersive X-ray (EDX) spectra of the ultra-thin sections of BMSCs with Y2O3 NPs was performed. The result showed the obvious peaks of phosphorus (P) and yttrium (Y) elements after the internalization of Y2O3 NPs in BMSCs (Figure 5F), this provides a direct evidence for the transformation from Y2O3 to YPO4. The result further confirmed that Y2O3 NPs underwent dissolution and formation process of YPO4 on its surface after entering the lysosomes, which directly led to the decrease of phosphate content. Since the dissolution and transformation of Y2O3 NPs from Y2O3 to YPO4 is an important factor in breaking the phosphate balance, we hypothesized whether prior pretreatment of Y2O3 NPs in LSBF to form YPO4 coating would reduce or eliminate the interaction between the materials and lysosomes, and further reduce the toxicity in vitro and in vivo. As-received Y2O3 NPs at 30 μg/mL were suspended in LSBF buffer for 24 h. As shown in Figure 6A, the NPs after YPO4 coating (YPO4-Y2O3) did not apparently reduce the intracellular phosphate. As we known, phosphate was a vital element in life activities, and it could react with ADP to synthesis ATP. As an intracellular energy source, ATP was responsible for all the vital activities of BMSCs, such as osteogenic differentiation, cell proliferation and viability.38 Intracellular ATP levels determine cell death fate, and the decline of ATP synthesis as common early events was critical for the execution of apoptosis.39 As shown in Figure 6B, an extreme decrease of ATP content was detected in BMSCs after the treatment of 3.125, 6.25, and 12.5 μg/mL Y2O3 NPs for 3 days. The viability of BMSCs was obviously increased by treatment with YPO4-Y2O3 than Y2O3 NPs at the same concentration (Figure 6D). And YPO4-Y2O3 could induce significantly less cathepsin B and D release in BMSCs (Figure 6E) and prevent cytochrome c release from inner mitochondrial membrane (Figure 6C). These results confirmed that the decrease of lysosomal phosphate broke the intracellular phosphate

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balance of BMSCs, impaired the mitochondrial ATP synthesis, which might induced dysfunction of BMSCs, even ATP-independent apoptosis. Prior YPO4 coating could alleviate the phosphate imbalance caused by Y2O3 NPs and reduce the cytotoxicity of Y2O3 NPs in vitro. RE compounds tended to deposit in bone and difficult to be excreted due to the similar physicochemical and physiological properties as calcium.40,41 Our previous studies have also confirmed this conclusion.13,14 In present study, we further examined its effect on bone tissue in vivo. We first measured the biodistribution of Y2O3 NPs in vivo after intravenous injection. The biodistribution results showed that the higher concentration of yttrium element accumulated in bone after intravenous injection for 35 days (Figure S6), which is consistent with the previous reports that the rare-earth compounds tend to accumulate in bone. Subsequently, we detected the changes of histopathology, apoptosis and apoptosis-related protein caspase 3 of bone tissue in mice after intravenous injection with Y2O3 and YPO4-Y2O3 NPs. The hematoxylin/eosin (HE) staining result showed that the bone trabecula of the Y2O3 NPs group became less and thinner compared with that of the control group and the YPO4-Y2O3 NPs group (Figure 7A). As shown in Figure 7B, the apoptosis-related proteins expression of caspase 3 was significantly increased in Y2O3 NPs group, while no significant differences between the YPO4-Y2O3 NPs group and control group. Moreover, there was also significant apoptosis of osteocytes in Y2O3 NPs group (Figure 7C). These data indicated that Y2O3 NPs could accumulate in bone after intravenous injection and have the potential toxic effects on bone metabolism, which could induce apoptosis of osteocytes by activation of caspase 3 signaling pathway. While YPO4-Y2O3 NPs have no obvious toxic or adverse effects on bone tissue. To further determine whether the Y2O3 NPs can cause other tissue damage after the exposure, the cardiac, hepatic and renal function indicators were measured by serum biochemistry assay. The data were shown in Figure S7, there were significant differences in liver function (AST, ALT, ALP), renal function (UA), and cardiac function (HBDH) between the Y2O3 NPs treatment groups and the control group, while no significant difference between the YPO4-Y2O3 NPs group and the control group was observed. In other words, Y2O3 NPs have potential detrimental effects on the liver, kidney, and cardiovascular tissues. Furthermore, we harvested major organs (heart, liver, spleen, lung, and kidney) of mice after the treatment at 35 days for HE staining. As shown in Figure S8, the hepatocytes in the liver samples appeared

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necrosis (green arrow), hepatic cord derangement, and alveolar rupture in the lung samples (yellow arrow) in Y2O3 NPs group. While no significant histopathological alteration in heart, liver, spleen, lung and kidney was observed after YPO4-Y2O3 NPs treatment. These results fully demonstrated that phosphate imbalance was an important mechanism of tissue damage caused by Y2O3 NPs. CONCLUSIONS In summary, we combined with physicochemical properties of Y2O3 NPs to systematically study their effects on the bone tissue in vivo and in vitro. Scheme 1 shows the schematic illustration of the mechanism of bone tissue damage caused by Y2O3 NPs. On the one hand, Y2O3 NPs could be taken into BMSCs through macropinocytosis, localized in acidifying intracellular lysosomes, and underwent dissolution and transformation from Y2O3 to YPO4-Y2O3, which could lead to the disruption of intracellular phosphate balance and decrease of the mitochondrial ATP synthesis, resulting in the dysfunction of lysosomal function and induced lysosomal and mitochondrial dependent apoptosis pathways. On the other hand, Y2O3 NPs caused obvious detrimental effects on the liver, kidney, and cardiovascular system, especially, caused the apoptosis of osteocytes and the destruction of bone structure. YPO4-Y2O3 could observably decrease the toxicity in vivo and in vitro. These results confirmed that the breaking of intracellular phosphate balance was an important mechanism of bone tissue damage caused by Y2O3 NPs. These findings may provide the basic data for the toxicological database and valuable information for biological application of these materials. MATERIALS AND METHODS Synthesis of Y2O3 and YPO4-Y2O3 NPs. Y2O3 NPs were synthesized by the hydrothermal method according to our previous work.42 To synthesis of YPO4-Y2O3 NPs, Y2O3 NPs were added into LSBF buffer (142 mg/L Na2HPO4, 6.65 g/L NaCl, 62 mg/L Na2SO4, 29 mg/L CaCl2•3H2O, 250 mg/L glycine, 8.09 g/L potassium phthalate, pH 4.5) at concentration of 30 μg/mL for 24 h, the NPs suspensions were centrifuged.18 The deposit was collected, washed and dried.

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BMSCs Culture. The mouse primary BMSCs were obtained from 4 weeks ICR mice (Charles River Laboratories, China) according to our previous method as described by Liu et al.43 Cell Morphology. The cell morphological changes were evaluated by cytoskeleton staining.25 Cells were seeded in a 24-well plate at a density of 1×105 cells per well and treated with Y2O3 NPs for 3 h, and fixed with 4% paraformaldehyde for 10 min at 37°C. Then, ActinGreen and DAPI were added. Cell morphological changes were observed under a fluorescence microscope. Annexin V/PI Staining. Apoptosis was measured by Annexin V/PI method according to the previous literature.44 After being cultured with Y2O3 NPs for 3 h, the cells were collected. The cells were suspended with 1×Annexin-binding buffer and stained with Annexin V and PI. Then cells were analyzed with a flow cytometry analyzer. Intracellular Localization of Y2O3 NPs. Cells were seeded in 35 mm cell culture cover slips at 4×104 cells per well. Cells were first washed three times, then incubated with FITC-labeled 25 μg/mL of Y2O3 NPs for 3 h and stained with Lyso-Tracker Red DND-99 for 30 min and Hoechst 33342 for 15 min at final concentrations of 50 nM and 10 μg/mL, respectively. The staining cells were washed and observed with a laser confocal scanning microscope using a green channel (488 nm barrier filter), a red channel (577 nm barrier filter) and a blue channel (350 nm barrier filter). Phosphate Assay. 30 μg/mL of Y2O3 NPs were added into 5 mL LSBF buffer for 24 h. Then, the NPs suspensions were centrifuged at 10000 rpm for 10 min. The phosphate was detected by a phosphomolybdenum blue method.45 The phosphate contents were detected at a wavelength of 710 nm using a microplate reader. Evaluation of Lysosomal Stability. Lysosomal stability was assessed using AOuptake method.27 A total of 1×105 cells were cultured in 6 well plates and exposed to 12.5 μg/mL of Y2O3 NPs. The Y2O3 NPs-treated cells were suspended in the 500 μL DMEM containing 6 μg/mL AO for 30 min at 37°C. The fluorescence signals intensity level obtained from flow cytometry demonstrated the damage level of lysosomal integrity. Cathepsin B and D Activity by Immunofluorescence Analysis. Cathepsin B and D activity were detected by immunofluorescence analysis. Briefly, cells were treated with Y2O3 NPs and YPO4-Y2O3 NPs for 3 h.

Then, cells were fixed with 4%

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paraformaldehyde, and permeabilized with 0.5% Tritonx-100. The cells were then incubated in 0.5% BSA for 2 h, and then incubated with cathepsin B and D primary antibodies overnight at 4°C respectively, and amplified by appropriate FITCconjugated secondary antibodies for 1 h. Cellular fluorescence was monitored with a confocal system. Determination of Lysosomal Alkalization. Lysosomal alkalization was used as an assessing assay of lysosome damage.46 BMSCs were collected after Y2O3 NPs treatment. The cells were stained with 50 nM Lyso-Tracker Red DND-99 and 10 μg/mL Hoechst 33342. The staining cells were analyzed by a laser confocal scanning microscope. Detection of Bid, Bax and Cytochrome c. The total 1×106 cells treated by NPs were collected. The ruptured cells were centrifuged. Then, 10 μL testing sample and standard sample were added to the coated ELISA plates, incubated for 30 min at 37°C. Then HRP-conjugate enzyme was added to each well, finally stained with chromogen solution A and B. The expression of Bid, Bax and cytochrome c were detected at 450 nm using microplate reader. Expression of Caspase 3 in Cytoplasm. 5×105 BMSCs cells per mL were seeded in the 6-well plate. After treatment with NPs, the cells were collected, caspase 3 activity was measured by an caspase 3 assay kit. Bone Damage Evaluation In Vivo. 4-week-old female ICR mice (19 ± 2 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co. Ltd. (Beijing, China). The care and treatment of laboratory animals were on the basis of the guidelines of the Institutional Animal Care and Use Committee. The mice were injected 50 mg/kg Y2O3 and YPO4-Y2O3 NPs by tail vein manner in a total volume of 200 μL per mouse. All mice were sacrificed after 35 days. The bone was fixed in formalin and stained with HE. In addition, the apoptosis of osteocytes was detected by terminal transferase-mediated DNA end labelling (TUNEL) assay. The cleaved caspase 3 was detected according to a previously reported method.47 Statistical Analysis. All data were reported as the mean± standard deviation (SD). One-way ANOVA was carried out to analyze the significant difference of the results.

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Scheme 1. Illustration of the mechanism of bone tissue damage caused by Y2O3 NPs.

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Figure 1. The characterization of Y2O3 NPs. SEM (A), TEM (B), particle size distribution (C) and XRD pattern (D) of Y2O3 NPs. The standard data for cubic phase Y2O3 (JCPDS 65-3178) is also presented in the figure for comparison.

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Figure 2. Dysfunction and cytotoxicity of BMSCs caused by Y2O3 NPs. (A) Effect of Y2O3 NPs on the mineralized nodule formation of BMSCs. (B) Effect of Y2O3 NPs on cellular morphology. (C) Cell viability in BMSCs determined by MTT assay after exposure to different concentrations of Y2O3 NPs. (*** p