Europium-Doped Gd2O3 Nanotubes Increase Bone Mineral Density in

Jan 25, 2017 - †College of Chemistry & Environmental Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education...
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Europium-Doped Gd2O3 Nanotubes Increase Bone Mineral Density in Vivo and Promote Mineralization in Vitro Huifang Liu,†,‡,& Yi Jin,†,§,& Kun Ge,†,# Guang Jia,† Zhenhua Li,† Xinjian Yang,† Shizhu Chen,† Min Ge,† Wentong Sun,† Dandan Liu,*,† and Jinchao Zhang*,† †

College of Chemistry & Environmental Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Chemical Biology Key Laboratory of Hebei Province, and ‡College of Pharmaceutical Science, Key Laboratory of Pharmaceutical Quality Control of Hebei Province, Hebei University, Baoding, 071002, China § College of Basic Medical Science, Hebei University, Baoding 071000, China # Affiliated Hospital of Hebei University, Baoding 071000, China S Supporting Information *

ABSTRACT: Europium-doped Gd2O3 nanotubes (Gd2O3:Eu3+ NTs) have been extensively applied in the field of bioscience for their photostability and magnetic properties. Nevertheless, the distribution and interaction between Gd2O3:Eu3+ NTs and metabolism of bone are not yet sufficiently understood. In this study, a systematic study of the toxicity and distribution of Gd2O3:Eu3+ NTs in mice after oral administration was carried out. The results showed that a small number of the Gd2O3:Eu3+ NTs could pass through biological barriers into the lung, liver, and spleen, but a high concentration was observed in bone. Furthermore, the effects of Gd2O3:Eu3+ NTs on bone metabolism were systematically studied in vitro and in vivo when accumulating in bone. After being administered to mice, the Gd2O3:Eu3+ NTs extremely enhanced the bone mineral density and bone biomechanics. In vitro the Gd2O3:Eu3+ NTs increased the alkaline phosphatase (ALP) activity and mineralization and promoted the expression of osteogenesis genes in preosteoblasts MC3T3-E1 through activation of the BMP signaling pathway. This study will be significant for appropriate application of Gd2O3:Eu3+ NTs in the biomedical field and expounding the molecular mechanism of bone metabolism. KEYWORDS: europium-doped Gd2O3 nanotube, bone mineral density, BMP signaling pathway, mineralization, phosphorylated-Smad1/5 showed minimal toxicity to RAW264.7 cells and S18 cells.13 Moreover, there was no cytotoxicity from micelle-coated Gd2O3 NPs to human embryonic kidney 293 cells.14 However, Gd2O3:Tb3+ NPs had cytotoxicity in high doses to human neonatal foreskin fibroblast cells.15 Briefly, the cytotoxicity of Gd2O3 nanostructures was dependent upon the dosage, size, and morphologies of nanomaterials. However, the toxicity of Gd2O3:Eu3+ NTs and the behaviors of Gd2O3:Eu3+ NTs in the body are still not fully understood, and clinical applications of Gd2O3:Eu3+ NTs will be limited until the potential toxicity to humans is fully investigated. Therefore, the toxicity and biodistribution of Gd2O3:Eu3+ NTs in biomedical applications should be intensively illuminated. Many mineral materials affect bone metabolism. Hydroxyapatite (HAP) is the main inorganic component in bone and has good biological compatibility and biological activity. So HAP has been applied widely in the field of bone tissue repair.

1. INTRODUCTION Europium-doped Gd2O3 nanoparticles (Gd2O3:Eu3+ NPs) have been extensively applied in the fields of bioscience and medicine due to their photostability and magnetic properties,1 including use in contrast agents of magnetic resonance imaging (MRI), ultrasound scanners, X-ray computed tomography (CT),2 and neutron capture therapy (NCT) to treat cancers.3,4 Moreover, europium-doped Gd2O3 nanotubes (Gd2O3:Eu3+ NTs) with inner cavities can be loaded with drugs, and the outer surface can be modified for better biocompatibility and target ability,5 which have been potentially applied in the field of biomedicine for targeted-cancer therapy.6−9 For example, Gd2O3:Eu3+ hollow spheres were potentially applied in the field of biomedicine for fluorescence labeling and drug targeting due to their luminescent and magnetic properties.10 Zhou and coworkers synthesized single-phase mesoporous Gd2O3:Eu3+ nanorods, which had high loading capacity and multimodal imaging properties.11 Toxicity of nanomaterials was a critical factor for the biomedical application. It was reported that Gd2O3:Yb3+,Er3+ nanostructures had no cytotoxicity to B-cell hybridomas,12 and ligand-free monoclinic Gd2O3 nanocrystals © XXXX American Chemical Society

Received: November 15, 2016 Accepted: January 25, 2017 Published: January 25, 2017 A

DOI: 10.1021/acsami.6b14682 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

previous work.28 To observe the luminescent properties, the samples were configured to 1.0 and 10.0 mg/mL suspensions with deionized water, the suspensions were irradiated using ultraviolet light in the darkness, and deionized water was used as control. Moreover, zeta potentials in H2O, PBS, and MEM are shown in Figure S2, indicating the stabilization of the Gd2O3:Eu3+ NTs suspension. 2.3. Animals and Treatments. Male ICR mice (23−25 g) and female ICR mice (18−20 g) were fed in the animal facility (20 ± 2 °C; 60 ± 10% relative humidity; 12 h light/dark cycle) with free access to food and water. For animal experiments, guidelines of the Institutional Animal Care and Use Committee were followed. After 7 d acclimation, the mice were randomized into four groups by sex (n = 5): control group and three medication groups. The mice of three medication groups were administered by oral gavage at different doses of Gd2O3:Eu3+ NTs suspension: low-dose group (20.0 mg/kg), middledose group (100.0 mg/kg), and high-dose group (500.0 mg/kg). Meanwhile, the mice of the control group were administered 0.1% (hydroxylpropyl)methyl cellulose (HPMC) at the same volume, and all mice were treated every day for 35 consecutive days. 2.4. Detection of Gadolinium Biodistribution. Gadolinium biodistribution in different tissues was analyzed with inductively coupled plasma mass spectrometry (ICP-MS) (Elan9000/DRC-e, PerkinElmer) .28 2.5. Biochemical Analysis and Blood-Element Test. Liver function (AST, ALT, TBIL, and ALP), renal function (Cr, UA, and BUN), and myocardial enzyme (LDH, CK, and HBDH) were determined. The blood-element test: red blood cell (RBC), hemoglobin (HGB), red cell distribution width corpuscular volume (RDW-CV), white blood cell (WBC), hematocrit (HCT), blood platelet (PLT), mean cell hemoglobin concentration (MCHC), and mean corpuscular volume (MCV) were assayed using assay kits.28,29 2.6. Histopathological Examination. For pathological studies, the vital organs (heart, kidney, liver, lung, and spleen) were examined with hematoxylin−eosin (H&E).29 The left femur was decalcified in disodium ethylenediamine tetraacetic acid (EDTA-2Na) supersaturated solution. EDTA-2Na supersaturated solution was exchanged every other day until the bone changed into a soft doughy state. After that, the specimens were examined according to our previous work.29 2.7. BMD Measurement. After being treated by oral gavage for 35 d, the mice were anaesthetized with ether, and BMD values of the mice were measured as grams per square centimeter by dual-energy X-ray absorptiometry (DXA, NORLANDXR-36, France).30 2.8. Biomechanics Detection. Muscle and connective tissue were cleanly removed from the mice femurs. Right femurs were used for the three-point bending test.31 The right femurs were packaged with gauze dipped in physiological saline and preserved in a refrigerator (−80 °C). The femurs were put in the physiological saline and wetted about 3 h at room temperature before measurement. Then a Material Mechanics Testing Machine (WD-1, Changchun, China) was used for the three-point bending test. The displacement accuracy was about 0.001 mm, the loading rate was about 2 mm/min, and the span was about 8 mm. Then the load−deformation curve was recorded. 2.9. MC3T3-E1 Cell Culture. MC3T3-El cells were cultured according to the literature.32 2.10. Assay for Proliferation. MC3T3-E1 cells were treated with Gd2O3:Eu3+ NTs at concentrations of 0.01 and 0.1 1.0 μg/mL. MTT assay was used to determine the proliferation of MC3T3-E1 cells, and cells without Gd2O3:Eu3+ NTs treatment were set as the control group. The detailed procedure was reported in our previous study.33 2.11. Assay for ALP Activity. Briefly, MC3T3-E1 cells were treated with osteogenic supplements (OS) and 0.01, 0.1, and 1 μg/mL Gd2O3:Eu3+ NTs suspension. The control group was only treated with OS, and the positive control group was treated with OS and 1 μM NaF. After incubation for 7 and 14 d, ALP activity was measured according to the routine method illustrated in the ALP assay kit and microprotein assay kit. 2.12. Assay for Mineralized Matrix Formation. MC3T3-E1 cells were treated with osteogenic supplements (OS) and 0.01, 0.1, and 1 μg/mL Gd2O3:Eu3+ NTs suspension for 21 d. The medium containing only OS was used as the control group, and 1 μM NaF was

Many excellent bone tissue engineering materials contain HAP, and they can promote the growth of osteoblasts (MG63), protein adsorption, cell adhesion, and bone regeneration.16−19 They also improve the activity of ALP and the expression of OCN. 20 In addition to HAP, nano gold, 21 silicon, 22 lanthanum,23 cerium,24 and other minerals also promote MC3T3-E1 cell or mesenchymal stem cell proliferation and mineralization. Gadolinium (Gd) is one member of the lanthanide series and has physicochemical and physiological properties similar to those of calcium (Ca), such as “boneseeking”.25 For example, [Gd@C82(OH)22]n NPs were intraperitoneally injected to rats, and the gadolinium concentration was the highest in bone.26 However, the effects of Gd2O3:Eu3+ NTs on bone formation and the corresponding cells are not fully known yet. Therefore, the in vivo toxicity and biodistribution of Gd2O3:Eu3+ NTs in organs were first evaluated through gavage administration in mice. In particular, the effects of Gd2O3:Eu3+ NTs on bone metabolism were systematically studied both in vivo and in vitro when accumulating in bone. In this work, the toxicity of Gd2O3:Eu3+ NTs in vivo to liver, kidney, spleen, lung, blood, and heart was tested through gavage administration in mice. Furthermore, the potential effects on bone metabolism of Gd2O3:Eu3+ NTs were studied by testing bone mineral density (BMD), biomechanics, and histopathology of the femur. The cellular effects of Gd2O3:Eu3+ NTs on the proliferation, osteogenesis, and mineralization of murine calvarial preosteoblasts (MC3T3-E1) cells were assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, and alizarin red staining (ARS) assays, respectively. Moreover, the signal transduction pathways underlying these events were examined by Western blotting and real-time polymerase chain reaction (real-time PCR). More importantly, the response of the bone morphogenesis protein (BMP) signaling pathway to Gd2O3:Eu3+ NTs treatment was evaluated by phosphorylation of Smad1/5/8. All in all, these results should be significant for rational application of Gd2O3:Eu3+ NTs in the biomedical field and expounding the molecular mechanism of bone metabolism.

2. EXPERIMENTAL SECTION 2.1. Animals and Materials. ICR mice were provided by Vital River Experimental Animal Co. Ltd. (SCXK (Beijing)) 2002-0003 (Beijing, China). MC3T3-E1 cells were provided by Beijing Union Cell Resource Center (Beijing, China). Eu(NO3)3 and Gd(NO3)3 were obtained from Xinuo Chemical Industry Co. Ltd. (Nanjing, China). The assay kits of aspartate aminotransferase (AST), total bilirubin levels (TBIL), alanine aminotransferase (ALT), ALP, bloodelement, creatinine (Cr), blood urea nitrogen (BUN), and uric acid (UA) were obtained from Randox Laboratories Ltd. UK (Northern Island, UK). The kits of α-hydroxybutyrate dehydrogenase (HBDH), creatine kinase (CK), and lactate dehydrogenase (LDH) were purchased from Merit Choice Bioengineering Co. Ltd. (Beijing, China). MTT, dimethyl sulfoxide (DMSO), β-glycerophosphate, ascorbic acid, dexamethasone, ARS, and NaF were purchased from Sigma-Aldrich (St. Louis, MO). DEPC and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA). RNeasy Mini Kit was purchased from Qiagen (Valencia, CA). Ex Taq DNA polymerase was from TaKaRa (Dalian, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and primary antibodies (Smad 1, phospho-Smad 1/ 5, and noggin) were purchased from Bioss Biological Technology Co., Ltd. (Beijing, China). 2.2. Synthesis and Characterizations of Gd2O3:Eu3+ NTs. Gd2O3:Eu3+ NTs were synthesized on the basis of previous work with a modification.27 The characterizations of the sample are based on our B

DOI: 10.1021/acsami.6b14682 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces the positive control group. Ddetailed procedures were reported in our previous work.26 2.13. Quantitative Real-Time PCR. Cells were treated with 0.01, 0.1, and 1 μg/mL Gd2O3:Eu3+ NTs, and 0.01, 0.1, and 1 μg/mL Gd2O3:Eu3+ NTs were treated with noggin for 7 d. The TRIzol reagent was used to extract the total RNA from MC3T3-E1 cells. Then cDNA was synthesized according to the TaKaRa protocol. The levels of mRNA for osteogenesis-related genes (BMP-2, Runx-2, and OCN) in 100 μg/mL DHAP-treated MC3T3-E1 cells were measured by realtime PCR using StepOnePlus Real-Time PCR System (StepOnePlus, Applied Biosystems, Foster City, CA). According to the manufacturer’s intructions, 2.5 μg of RNA was used for the reverse transcriptase reaction in a 20 μL mixture. Real-time PCR was conducted in a total volume of 25 μL containing 4 μL of cDNA, l μL of primer pair stock (7.5 μM), 12.5 μL of MltraSYBR mixture, and 7.5 μL of H2O. The amplification cycles and reaction conditions were operated according to our previous work.34 Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used to analyze the gene quantitatively. The primer sequences are listed in Table S2. 2.14. Western Blotting Analysis. MC3T3-E1 cells (2 × 104 cells/well) were treated with 0.01 and 1 μg/mL Gd2O3:Eu3+ NTs for 7 d. Meanwhile, noggin, as an antagonist of the BMP signaling pathway, was added to the medium, and NaF was the positive control group. Subsequently, Western blot analysis was performed as in our previous work.26 2.15. Statistical Analysis. Results were expresed as mean ± standard deviation (SD). Data were analyzed through one-way analysis of variance (p < 0.05 was considered to be statistically significant).

ratio (Figure S1A). The characteristic red emission of Eu3+ ions (611 nm, 5D0−7F2) was dominant to other transition lines (Figure 1D, Figure S1B), which agrees well other Gd2O3:Eu3+ materials.27 3.2. Biodistribution of Gadolinium. According to extensive application of Gd2O3:Eu3+ NTs in biomedical fields, it will enter the body through some administration routes such as oral, intramuscular, or intravenous injection.28 So it is important to understand the biodistribution, toxicity, and effects of Gd 2O 3:Eu3+ NTs in vivo.29 The results of biodistribution in mice indicated that the highest concentration of gadolinium occurred in bone. Moreover, gadolinium content in spleen, liver, and lung was higher (Figure 2).

3. RESULTS AND DISCUSSION 3.1. Characterizations of Gd2O3:Eu3+ NTs. As shown in Figure 1A, the SEM image indicated that the Gd2O3:Eu3+

Figure 2. Biodistribution of elemental gadolinium in mice measured by ICP-MS. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control.

3.3. Biochemical Parameters in Serum and BloodElements. As shown in Table 1, no significant differences in liver function (AST, ALT, ALP, AST/ALT, and TBIL), renal function (Cr, UA, and BUN), and myocardial enzyme (LDH, CK, and HBDH) between the medication groups and the control group (p > 0.05) were observed. This suggested that Gd2O3:Eu3+ NTs had no obvious detrimental effects on the cardiovascular system, liver, and kidney. Furthermore, the results for examination of blood-elements are shown in Table 2, and there were no significant differences between the medication groups and the control group. In other words, Gd2O3:Eu3+ NTs did not cause hematological toxicity even at the highest dose. In addition, the body weight and mean food intakes of mice had no significant difference after Gd2O3:Eu3+ NTs treatment when compared with the control group (Figure S5). Furthermore, Gd2O3:Eu3+ NTs possessed good biocompatibility by hemolytic analysis (Figure S6). 3.4. Histopathology of Vital Organs and Bone Tissue. The histopathology was directed to observing the toxicity of Gd2O3:Eu3+ NTs in the vital organs (Figure 3). The data indicated that there was no significant clinical histopathological alteration in spleen, kidney, lung, liver, and heart after Gd2O3:Eu3+ NTs treatment. Our previous work has shown that the obvious lesions of kidney, heart, and liver were caused by high-dose (400.0 mg/kg) Gd2O3:Eu3+ NTs, but no significant pathological lesions were observed in middle-dose (40.0 mg/kg) and low-dose (4.0 mg/kg) groups in mice after intraperitoneal injection.29 In this work, we systematically evaluated the toxicity, biodistribution, and effects of the Gd2O3:Eu3+ NTs by gavage administration. Contrary to intraperitoneal injection, Gd2O3:Eu3+ NTs showed no obvious toxicity to liver, kidney, spleen, lung, blood, and heart (Figure 3) by gavage administration. Similar to conventional drug molecules, nanomaterials can also be involved in pharmacoki-

Figure 1. Characterizations of Gd2O3:Eu3+ NTs. (A) SEM images, (B) TEM images, (C) XRD pattern, and (D) PL excitation and emission spectra.

sample consisted of uniform nanotubes, whose lengths and diameters were around 300 and 50 nm, respectively. The hollow nature of the nanotubes were directly verified by TEM (Figure 1B). The XRD peaks of the sample were well in accordance with the cubic phase of Gd2O3 (JCPDS No. 862477), and no other impurity phases were found (Figure 1C). The excitation spectrum of the samples was a broad band, whose center was at about 257 nm (Figure 1D). Moreover, the ratio of Eu:Gd is 0.043%, which was in accord with the original C

DOI: 10.1021/acsami.6b14682 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

± ± ± ± ± ± ± ± ± ± ±

20 mg/kg 0.72 ± 0.30 34.76 ± 4.81 168.62 ± 38.81 4.85 ± 0.86 73.46 ± 14.82 153.80 ± 52.86 17.40 ± 1.88 7.29 ± 0.87 1255.78 ± 471.25 827.46 ± 112.58 462.30 ± 93.72

100 mg/kg 0.69 ± 0.36 40.48 ± 11.52 152.88 ± 36.80 3.93 ± 1.25 70.80 ± 19.84 131.32 ± 20.72 17.25 ± 2.70 7.73 ± 1.52 1094.82 ± 295.44 796.02 ± 90.36 426.74 ± 123.08

500 mg/kg 0.70 ± 0.40 35.06 ± 3.64 157.44 ± 40.15 4.53 ± 1.22 72.88 ± 11.47 118.70 ± 17.59 17.50 ± 2.44 8.77 ± 3.41 1267.78 ± 412.90 798.60 ± 104.44 392.46 ± 117.56

control 0.68 ± 0.16 39.36 ± 5.56 116.64 ± 18.96 2.99 ± 0.56 53.88 ± 5.78 157.29 ± 24.77 16.46 ± 2.37 9.94 ± 2.08 1032.12 ± 198.89 729.76 ± 169.18 315.38 ± 94.03

20 mg/kg 0.72 ± 0.29 42.84 ± 4.89 120.10 ± 15.79 2.82 ± 0.41 46.38 ± 8.03 130.30 ± 27.12 15.62 ± 1.11 10.54 ± 1.59 1120.78 ± 168.22 666.54 ± 110.91 273.50 ± 59.81

D

± ± ± ± ± ± ± ± 1.87 1.39 44.77 2.95 2.22 76.54 1.32 99.32

20 mg/kg 6.16 5.08 121.28 38.54 50.66 402.80 16.62 605.92

± ± ± ± ± ± ± ±

2.22 2.60 43.91 4.36 1.42 227.15 0.45 136.78

control

5.44 6.33 133.38 36.62 51.98 426.60 15.48 650.04

parameters

WBC (109/L) RBC (109/L) HGB (g/L) HCT (L/L) MCV (10−15/L) MCHC (g/L) RDW (%) PLT (109/L)

female ± ± ± ± ± ± ± ± 1.47 1.33 18.84 6.33 1.44 116.24 0.62 108.88

100 mg/kg 5.10 6.22 130.16 37.46 51.78 359.52 16.32 722.42

± ± ± ± ± ± ± ± 2.38 1.61 20.08 4.02 0.80 74.01 1.40 227.84

500 mg/kg 6.06 6.30 135.54 38.56 52.10 357.38 16.85 676.88

± ± ± ± ± ± ± ±

1.93 2.67 25.07 10.95 2.83 57.46 1.57 496.33

control 6.46 6.97 134.84 39.48 51,54 374.70 15.84 732.38

± ± ± ± ± ± ± ±

1.46 2.71 40.79 14.12 1.13 10.14 1.92 250.30

20 mg/kg 5.20 7.25 138.60 36.50 50.18 322.60 17.02 819.92

100 mg/kg

male 5.06 8.38 141.78 41.90 51.70 348.00 17.65 883.06

± ± ± ± ± ± ± ±

1.92 2.47 24.68 11.46 1.35 55.79 1.59 276.87

100 mg/kg

0.69 ± 0.38 37.56 ± 7.39 125.36 ± 18.39 3.40 ± 0.52 53.28 ± 7.67 119.84 ± 9.37 15.38 ± 2.36 9.57 ± 1.34 1293.70 ± 399.16 706.68 ± 121.87 281.80 ± 50.91

male

Table 2. Blood-Element Examination of Male and Female Mice after Oral Exposure to Eu:Gd2O3 NTs for 35 d (n = 8, mean ± SD)

0.24 14.32 24.48 0.68 15.18 49.42 2.26 1.26 414.37 99.51 217.68

control

0.66 48.36 173.64 3.72 60.32 167.98 18.18 7.35 1357.56 751.92 552.70

parameters

TBIL (μmol/L) ALT (U/L) AST (U/L) AST/ALT ALP (U/L) UA (μmol/L) Cr (μmol/L) BUN (mmol/L) CK (U/L) LDH (U/L) HBDH (U/L)

female

Table 1. Biochemical Values in the Serum of Male and Female Mice after Oral Exposure to Eu:Gd2O3 NTs for 35 d (n = 8, mean ± SD) 500 mg/kg

5.46 7.45 128.16 37.42 50.16 345.40 17.12 870.88

± ± ± ± ± ± ± ±

1.63 1.79 27.19 9.62 1.51 39.98 1.43 235.10

500 mg/kg

0.70 ± 0.32 36.50 ± 4.03 129.66 ± 22.26 3.60 ± 0.79 50.10 ± 8.82 117.44 ± 32.13 16.10 ± 3.38 10.28 ± 2.00 1438.88 ± 522.72 576.74 ± 95.64 243.76 ± 47.59

ACS Applied Materials & Interfaces Research Article

DOI: 10.1021/acsami.6b14682 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

physicochemical and physiological properties similar to those of calcium.38−41 Previously, we detected Gd2O3:Eu3+ NTs distribution in mice after intraperitoneal injection (ip). The results showed that the Gd2O3:Eu3+ NTs were distributed in all measured tissues and organs, such as bone, lung, the spleen, liver, and kidney, and that their distribution in bone was higher.28,29 In addition, it has been reported that Gd2O3:Eu3+ nanoparticles were distributed into all tissues and organs, such as kidney, lung, spleen, liver, heart, bone, large intestine, and blood through endotracheal instillation.42 Yang et al. studied the biodistribution and effect of [Gd@C82(OH)22]n nanoparticles on bone formation. The results showed that the [Gd@ C82(OH)22]n nanoparticles were mainly deposited in the bone and promoted bone formation, but no damage was induced by [Gd@C82(OH)22]n nanoparticles.26 Therefore, on the basis of this research, we proposed a hypothesis that Gd2O3:Eu3+ NTs could increase the bone mineral density in vivo and promote mineralization in vitro due to physicochemical and physiological properties similar to those of Ca. In this study, we systemically studied the changes in BMD, biomechanics, and histopathology of mice influenced by Gd2O3:Eu3+ NTs after gavage administration. BMD is a common indicator to assess sclerotin that decreases only when bone mass is reduced significantly.43 Bone biomechanics is based on engineering mechanics theory and is the science of researching the mechanical properties of bone and biological effects.44 As shown in Figure 4E and Figure 4F, the BMD of mice (both female and male) was significantly increased after Gd2O3:Eu3+ NTs treatments (p < 0.001). The results of bone mechanics (Figure 4G and Figure 4H) indicated that the maximal load of the female mice femurs in all three medication groups increased (p < 0.05) and that the maximal load of the male mice femurs in middle-dose and low-dose groups extremely increased (p < 0.01) when compared with the control group. Particularly, the maximal load of the male mice femurs in the high-dose group increased statistical significance (p < 0.001). Moreover, the histopathological analysis showed significant changes in femurs caused by Gd2O3:Eu3+ NTs, such as more hairchested bone trabecular, more obvious bone deposition line, and more abundant osteoids than in the control group. 3.6. Gd2O3:Eu3+ NTs Increased ALP Activity and Promoted Proliferation and Mineralization of MC3T3E1 Cells. To understand how the Gd2O3:Eu3+ NTs promoted BMD, in vitro we studied the proliferation and ALP activatity of MC3T3-E1 cells. The murine preosteoblast MC3T3-E1 cell line has great potential application for evaluating osteogenesis because of excellent differentiation capability and a fully differentiated phenotype. After 4 h incubation, Gd2O3:Eu3+ NTs were observed in lysosomes by TEM (Figure S3). Furthermore, Gd2O3:Eu3+ NTs almost did not dissolve into Gd3+ in lysosomes (pH 5.0) and cytoplasm (pH 7.4) (Figure S4). As shown in Figure 5A, 0.01, 0.1, and 1.0 μg/mL Gd2O3:Eu3+ NTs all promoted the proliferation of MC3T3-E1 in a dose-dependent manner for 24, 48, and 72 h incubation. ALP activity as a marker of early osteogenic differentiation was significantly increased at day 7 and 14 when treated with OS and 0.01, 0.1, and 1.0 μg/mL Gd2O3:Eu3+ NTs in a timedependent manner (Figure 5B). Cells treated with OS and NaF (1 μM) were used as control and positive control, respectively. Mineralized nodules as the mature stage of osteogenic differentiation represent the phenotype of osteogenic differentiation.24 As shown in Figure 5C−H, a significant increase in

Figure 3. Histopathology of heart, spleen, liver, kidney, and lung of mice (400×).

netics in vivo, including absorption, distribution, metabolism, and excretion.35 Owing to the existence of physiological barriers, the absorption of nanomaterials is affected. For example, the intestine is one of the important physiological barriers, and it can effectively prevent entry of poisonous substances into the body.36 Therefore, no obvious toxicity to liver, kidney, spleen, lung, blood, and heart was observed. However, it is generally accepted that a small amount of nanoparticles can be taken up by the gastrointestinal epithelium.37 Therefore, the Gd2O3:Eu3+ NTs could distribute into all measured tissue and organs, including bone, lung, the spleen, liver, and kidney. In particular, the content in bone was the highest. The histopathological results of mice femurs are presented in Figure 4A−D. The results showed that the bone trabeculas of

Figure 4. Histological images of bone tissue in mice stained with H&E: (A) control group, (B) low-dose group, (C) middle-dose group, (D) high-dose group. (E and F) BMD. (G and H) Maximal load.

mice in all groups were arranged closely and in order and that the bone trabeculas presented net-like character and had good continuity. However, in the medication groups the bone trabeculas of mice were thicker, the bone deposition line was more obvious, and the osteoid was abundant compared with that of the control group. Moreover, they exhibited obvious dose dependence. 3.5. BMD and Bone Biomechanics Detection in Mice. It has been reported that rare-earth compounds were apt to accumulate in bone and difficult to be excreted because of E

DOI: 10.1021/acsami.6b14682 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 5. Effects of Gd2O3:Eu3+ NTs on the viability and osteogenic differentiation of MC3T3-E1 cells. (A) Viability of MC3T3-E1 cells in the presence of Gd2O3:Eu3+ NTs. (B) Effects of Gd2O3:Eu3+ NTs on ALP activity. (C−H) Effects of Gd2O3:Eu3+ NTs on the mineralized matrix formation: (C) control group (cells treated with OS only), (D) 0.01 μg/mL Gd2O3:Eu3+ NTs + OS, (E) 0.1 μg/mL Gd2O3:Eu3+ NTs + OS, (F) 1 μg/mL Gd2O3:Eu3+ NTs + OS, (G) positive group (NaF + OS). (H) The mineralization promotion rate was determined by quantification of ARS staining. *p < 0.05, **p < 0.01, ***p < 0.001 versus the control.

Figure 6. (A) Effects of osteogenesis-related gene expression in cells treated with Gd2O3:Eu3+ NTs. (B) The expression levels of key proteins involved in the BMP signaling pathway.

ment.48−50 BMPs can combine with BMP receptors (type I and type II) and signal by activating phosphorylating Smad 1 and 5, which form heterodimeric complexes with Smad 4. Subsequently, Smad heterodimers enter the nucleus and regulate relevant gene transcription.51−53 Liu et al. have reported that the osteogenesis of MC3T3-E1 cells was promoted by gold nanoparticles through the BMP signaling pathway.33 Furthermore, La3+ also possessed the same effect and mechanism on bone marrow stromal cells (MSCs). 23 Therefore, we hypothesized that Gd2O3:Eu3+ NTs might preferentially induce osteogenic differentiation of MC3T3-E1 cells through activation of the BMP signaling pathway. In addition, a large number of antagonists (the Spemann organizer, noggin, follistatin, chordin, etc.) can combine with BMPs and prevent their combination with signaling receptors. Among these antagonists that block the BMP receptor, noggin is the most studied.54−57 Western blotting analyses were carried out in this work to evaluate the key signal protein of the BMP signaling pathway using noggin. As shown in Figure 6B, the phosphorylation of Smad 1/5 was highly elevated by the Gd2O3:Eu3+ NTs. Total Smad 1 and GAPDH were used as internal references. Moreover, noggin decreased the p-Smad1/5 level, resulting in the suppression of osteogenesis-related gene expression (Figure 6B), which was enhanced by Gd2O3:Eu3+ NTs (Figure 6A). Figure 7 illustrates how the Gd2O3:Eu3+ NTs induces osteogenic differentiation of MC3T3-E1 cells by activating the BMP signaling pathway through phosphorylation of Smad1/5. The heterodimeric complexes of p-Smad1/5 and Smad 4 enter the nucleus and enhance transcriptional activity, including osteogenesis-related genes.

mineralized nodules was observed by Gd2O3:Eu3+ NTs treatment in a dose-dependent manner after 21 d incubation. 3.7. Gd2O3:Eu3+ NTs Enhanced Osteogenesis-Related Gene Expression in MC3T3-E1 Cells. To confirm the osteogenesis of MC3T3-E1 cells induced by Gd2O3:Eu3+ NTs, the expression of bone formation-related genes (BMP-2, OCN, and Runx-2) was examined by real-time PCR. Runx-2 belongs to the Runx family and is regarded as the most specific gene for the osteogenic differentiation of MC3T3-E1 cells. It has been demonstrated that the absence of Runx-2 could lead to the failure of osteogenesis.45 Correspondingly, BMP-2 belongs to the TGF-β family and can enhance osteoblastic differentiation, stimulate the generation of bone structural proteins (Col-I and OCN), and promote the mineralization of bone matrix.46 Moreover, OCN plays an important role in osteoblast differentiation and mineralization.47 At the molecular level, the expression of BMP-2, Runx-2, and OCN was examined by real-time PCR after Gd2O3:Eu3+ NTs treatment. As shown in Figure 6A, the expression of BMP-2 (p < 0.05) and OCN (p < 0.01) was significantly increased after 7 d Gd2O3:Eu3+ NTs treatment at a concentration of 0.1 μg/mL, and the expression of BMP-2 (p < 0.05), OCN (p < 0.05), and Runx-2 (p < 0.01) was also increased at a concentration of 1 μg/mL. Throughout all the experiments, Gd2O3:Eu3+ NTs promoted osteogenic differentiation of MC3T3-E1 cells both on the cellular level and on the molecular level. 3.8. Gd2O3:Eu3+ NTs Promoted Bone Mineralization through Activation of BMP Signaling Pathway. Increasing evidence proves that the Smad-dependent BMP signaling pathway is involved in the osteogenic differentiation of MC3T3-E1 cells, and BMPs are regarded as one of the most specific proteins in bone formation and skeletal develop-

4. CONCLUSION In summary, we researched the biodistribution of Gd2O3:Eu3+ NTs in mice in vivo as well as induced toxicity. The results showed that Gd2O3:Eu3+ NTs were nontoxic and that Gd content in bone was the highest after gavage administration F

DOI: 10.1021/acsami.6b14682 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Professionals (CG2015003009), Natural Science Foundation of Hebei Province (B2015201097).

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Figure 7. Gd2O3:Eu3+ NTs increase bone mineral density in vivo and promote mineralization in vitro.

with Gd2O3:Eu3+ NTs. Moreover, BMD of mice was increased and the bone trabeculas of mice were promoted by Gd2O3:Eu3+ NTs. In vitro we systematically demonstrated the effects of Gd2O3:Eu3+ NTs on osteogenic differentiation and found that Gd2O3:Eu3+ NTs increased the ALP activity and mineralization of MC3T3-E1 cells. In addition, the levels of osteogenesisrelated proteins and genes were up-regulated in MC3T3-E1 cells by activation of the BMP signaling pathway. These findings may be meaningful for more rational application of Gd2O3:Eu3+ NTs in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b14682. The zeta potential, luminosity, and subcellular location of europium-doped Gd2O3 NTs, body weight and mean, food intakes, coefficients of organs, hemolysis of mice, and PCR primer sequences (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jinchao Zhang: 0000-0002-5279-0468 Author Contributions &

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Natural Science Foundation project (21271059, 31470961, 31500812, 21471044), Key Basic Research Special Foundation of Science Technology Ministry of Hebei Province (14961302D), Hebei Province “Hundred Talents Program” (BR2-202), Hebei Province “Three Three Three Talents Program” (A201401002), Science and Technology Research Project of Higher Education Institutions in Hebei Province (QN2015230), Fund Program for the Scientific Activities of Selected Returned Overseas G

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