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TEMPO Conjugated Gold Nanoparticles for Reactive Oxygen Species Scavenging and Regulation of Stem Cell Differentiation Jingchao Li, Jing Zhang, Ying Chen, Naoki Kawazoe, and Guoping Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12486 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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ACS Applied Materials & Interfaces

TEMPO Conjugated Gold Nanoparticles for Reactive Oxygen Species Scavenging and Regulation of Stem Cell Differentiation

Jingchao Li1,2, Jing Zhang1,2, Ying Chen1,2, Naoki Kawazoe1 and Guoping Chen*1,2

1

Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki,

Tsukuba, Ibaraki 305-0044, Japan 2

Department of Materials Science and Engineering, Graduate School of Pure and Applied Sciences,

University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan

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ABSTRACT Controlling the differentiation of human mesenchymal stem cells (hMSCs) shows a great potential in regenerative medicine. Because overproduced reactive oxygen species (ROS) have obvious inhibitory effect on the differentiation and functions of hMSCs, it is highly desirable to develop effective strategy for ROS scavenging and stem cell differentiation controlling. In this study, gold nanoparticles

(Au

NPs)

with

an

average

size

of

40

nm

were

conjugated

with

2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) to endow them with ROS scavenging capacity while holding the beneficial effect of Au NPs. The TEMPO-conjugated Au NPs (Au-PEG-TEMPO NPs) were used for culture of hMSCs to investigate their effect on ROS scavenging, proliferation, and osteogenic and adipogenic differentiation of hMSCs. The Au-PEG-TEMPO NPs had negligible influence on cell viability and proliferation of hMSCs and could effectively reduce the ROS level of hMSCs under H2O2-exposed condition because of their excellent cellular uptake. Similar to the counterparts without surface TEMPO modification (Au-mPEG NPs), the Au-PEG-TEMPO NPs could promote osteogenic differentiation while inhibit adipogenic differentiation of hMSCs. The results indicated that the TEMPO-conjugated Au NPs had high scavenging capacity for overproduced ROS and maintained the promotive effect of Au NPs on osteogenic differentiation of hMSCs without the inhibitory effect of free TEMPO. This study should offer a promising strategy for ROS scavenging to control stem cell differentiation in stem cell transplantation and regenerative medicine.

KEYWORDS: Gold nanoparticles, ROS scavenging, hMSCs, osteogenic differentiation, adipogenic differentiation


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INTRODUCTION Mesenchymal stem cells (MSCs) have shown great prospect for stem cell transplantation and regenerative medicine owing to their high potential of self-renewal and capacity of differentiation into different cell lineages.1-3 Multifarious biological, physical and chemical stimulations have shown prominent effects on the differentiation and functions of MSCs.4-7 The reactive oxygen species (ROS) are considered as a common stimulation for MSCs, which may be induced by age and inflammation.8-10 It has been reported that high ROS levels inhibit osteogenic differentiation while promote adipogenic differentiation of MSCs.11 Furthermore, overproduced ROS have the potential to result in membrane and DNA damage, protein denaturation and cell apoptosis.12-15 Therefore, protection of the MSCs from ROS-enriched environment to maintain their functions and survivability is in urgent need. To address the adverse influence from oxidative stress, a great deal of efforts have been made to reduce the ROS overproduction inside cells, such as the conventional administration of antioxidants and ROS scavengers.16-17 However, these approaches in general fail to achieve desired efficiency due to the limited cellular uptake of these low-molecular-weight drugs and their side effect.18 Overmuch accumulation of drugs may induce inessential redox reactions in cells, leading to cytotoxicity and mitochondrial dysfunctions.19 It is highly desirable to deliver ROS scavengers into ROS-enriched stem cells with high efficient and minimal side effects. Furthermore, the negative influence of ROS scavengers on stem cell differentiation should be avoided. Recently, gold nanoparticles (Au NPs) have received more and more attentions owing to their wide applications in biomedical fields.20-24 They are considered as one of the most attractive nanomaterials for biomedical applications due to their tunable shape and size, facile synthesis and surface modification, excellent optical property and good biocompatibility. The shape, size and ACS Paragon Plus Environment


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surface property of Au NPs have been controlled to modulate their interaction with cells.25-27 Currently, Au NPs have been widely used for molecular imaging, photothermal cancer therapy, biosensing and drug/gene delivery.28-34 Moreover, Au NPs are considered as bioactive in regenerative medicine because they can regulate the differentiation of stem cells.35 Influence of their physicochemical property on stem cell differentiation has also been reported.1, 6 More recently, Au NPs with optimized surface chemistry have been used as nanocarriers for delivering gene into mesenchymal stromal cells.28 Therefore, the versatility and evidences of Au NPs suggest they may be an useful carrier for delivery of ROS scavengers. The 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and its derivatives are potentially powerful candidates for ROS scavenging.36 Redox micelles loaded with TEMPO have been used to suppress inflammation and treat drug-resistant cancers.17, 37 However, the influence of free TEMPO on stem cell differentiation has been barely reported. Developing an effective strategy for ROS scavenging in stem cells without negative influence on cell differentiation is highly desirable. Compared with free TEMPO, clustering of TEMPO molecules on nano-sized carriers may have a different performance on stem cell functions. Therefore, in this study Au NPs were used as carriers for preparation of TEMPO clusters and delivery of TEMPO clusters into human bone marrow-derived mesenchymal stem cells (hMSCs). ROS scavenging effect of TEMPO-conjugated Au NPs and their influence on osteogenic and adipogenic differentiation of hMSCs were studied.

EXPERIMENTAL SECTION Materials. 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Trypsin/EDTA, perchloric acid, Dulbecco’s modified Eagle’s Medium (DMEM), fast blue RR salt, methyl-isobutylxanthine, thiol-poly(ethylene glycol)-carboxylic acid (SH-PEG-COOH, Mw = 2100), ascorbic acid, naphthol AS-MX phosphate, penicillin, proline, streptomycin, dexamethasone, sodium pyruvate, glutamine, nonessential amino ACS Paragon Plus Environment

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acids, β-glycerophosphate, Oil red O, insulin, 2-amino-2-methyl-1,3-propanediol, indomethacin, and Alizarin Red S were purchased from Sigma-Aldrich (St. Louis, MO, USA). Thiol-poly(ethylene glycol)-methoxy (SH-mPEG, Mw = 2000) was from Yuka Sangyo Co., Ltd. (Tokyo, Japan). Trisodium citrate, 4% paraformaldehyde, 2-propanol and hydrogen tetrachloroaurate tetrahydrate (HAuCl4· 4H2O, 99.9%) were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Synthesis and Characterization of Au-PEG-TEMPO NPs. A typical EDC cross-linking reaction was used to synthesize SH-PEG-TEMPO conjugate. EDC (0.20 mmol, 38.34 mg) and NHS (0.20 mmol, 23.02 mg) were successively added into 20.0 mL DMSO solution of SH-PEG-COOH (0.02 mmol, 42.00 mg) and the mixture solution was stirred at 25 ℃ for 3 hours. TEMPO (0.20 mmol, 34.13 mg) dissolved in 5.0 mL DMSO was mixed with the above-mentioned solution. After being kept stirring at 25 ℃ for 3 days, the mixture solution was purified by dialysis and the obtained SH-PEG-TEMPO conjugate was lyophilized for further use. A seed-mediated method was used to synthesize Au NPs with surface TEMPO modification (Au-PEG-TEMPO NPs). Au seed solution was prepared by the citrate reduction of HAuCl4 solution in oil bath.6 To synthesize the Au-PEG-TEMPO NPs, HAuCl4 solution (1.1 mL, 30.0 mg/mL), Au seed solution (44.0 mL), ascorbic acid solution (1.0 mL, 29.3 mg/mL) and SH-PEG-TEMPO solution (1.0 mL, 4.0 mg/mL) were successively added into 20.0 mL aqueous solution. After 24 hours of stirring at 25 ℃, the obtained Au-PEG-TEMPO NPs were purified by centrifugation. The Au-mPEG NPs without TEMPO modification were synthesized according to our previous study and used as a control.38 The SH-PEG-TEMPO conjugate was characterized by using an JEOL FT-NMR system (300 MHz, Tokyo, Japan). The UV-Vis absorption spectra of the NPs solutions were recorded by using a V-660 UV-Vis spectrophotometer (Jasco Corp., Tokyo, Japan). Particle size and morphology were observed under a JEOL 2100F transmission electron microscope (TEM, JEOL, Tokyo, Japan). ACS Paragon Plus Environment


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Thermal gravimetric analysis (TGA) was carried out on a Seiko Exstar 6000 TG/DTA 6200 thermal gravimetric analyzer (SII Nanotechnology Inc., Tokyo, Japan). A ELSZ-2000 zeta-potential & particle size analyzer (Otsuka Electronics Co., Ltd, Tokyo, Japan) was used to measure the hydrodynamic size and zeta potential. Au element concentration analysis was performed on a SPS3520UV-DD Leeman Prodigy inductively coupled plasma-optical emission spectroscopy (ICP-OES) system (SII Nano technology Inc., Tokyo, Japan). Cell Viability and Proliferation Assay. The hMSCs at passage 4 (P4) were used in the following experiments. For cell viability assay, hMSCs were seeded into 96-well cell culture plates (0.5 × 104 cells/cm2). After culture overnight, the medium was removed and fresh growth medium containing the Au-mPEG NPs or Au-PEG-TEMPO NPs at different Au concentrations was added. The cells were cultured with the NPs for 1 day and then the medium was changed to growth medium containing 10% WST-1 reagent. The cells were cultured with the WST-1 regent for another 3 hours. A plate reader (Bio-Rad, Hercules, CA, USA) was employed to measure the absorbance at 440 nm. For cell proliferation assay, hMSCs were incubated with the Au-mPEG NPs or Au-PEG-TEMPO NPs in cell culture medium at an Au concentration of 0.05 mM for 1, 3 and 7 days and then cell viability was measured using WST-1 assay as above described. Then hMSCs after incubation were also stained with calcein-AM and propidium iodide (PI) and an inverted fluorescence microscope (Olympus, Tokyo, Japan) was used to capture the live/dead images. Cellular Uptake Assay. To investigate cellular uptake efficiency of the Au-mPEG NPs and Au-PEG-TEMPO NPs, hMSCs were seeded into 6-well cell culture plates (1 × 104 cells/cm2) and cultured overnight. Then the medium was changed to growth medium containing the Au-mPEG NPs or Au-PEG-TEMPO NPs at different Au concentrations (0.05, 0.10 and 0.20 mM) and the cells were cultured for 3 days. After the culture, the cells were washed to remove the free NPs, collected and counted with a hemocytometer. Aqua regia solution was used to lyse the cells and dissolve the ACS Paragon Plus Environment

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internalized Au NPs for 2 days. The lysis solution was diluted with pure water and used for ICP-OES measurement. ROS Scavenging Experiment. To evaluate ROS scavenging capacity of the Au-PEG-TEMPO NPs, hydrogen peroxide (H2O2) was used to induce ROS generation inside cells.39 In this experiment, the growth medium without supplement of ascorbic acid was used. The hMSCs seeded in 48-well cell culture plates (1 × 104 cells/cm2) were cultured overnight for cell attachment. The cells treated with H2O2 (500 µM) for 1 hour were used as ROS-enriched hMSCs. The cells with or without H2O2 treatment were cultured in 0.25 mL growth medium containing free TEMPO (0.2 and 100.0 µg/mL), Au-mPEG NPs or Au-PEG-TEMPO NPs at an Au concentration of 0.50 mM. After 1 day of culture, the hMSCs were washed with PBS and stained with DCFH-DA.36 To quantify ROS level, Triton X-100 solution was used to lyse the stained cells. The cell debris in each well were centrifuged (2500× g, 10 minutes) to obtain the supernatant. After transferring the supernatant into a 96-well plate, the absorbance in each well at 525 nm was measured by using a plate reader.40 The ROS level in each group was expressed as a relative value to the control group without H2O2 treatment. Osteogenic Differentiation of hMSCs. The hMSCs seeded in 24-well cell culture plates (0.5 × 104 cells/cm2) were cultured in growth medium for 1 day. Then the cell culture medium was changed to osteogenic induction medium containing free TEMPO (10.0 µg/mL), Au-mPEG NPs or Au-PEG-TEMPO NPs at an Au concentration of 0.05 mM. The cell culture medium in each well was changed every 3 days. The osteogenic induction medium was growth medium supplemented with 100 nM dexamethasone and 10 mM β-glycerophosphate. After osteogenic induction culture for 14 days, the cells were washed and fixed with paraformaldehyde (4%). The fixed cells were incubated with 0.1% naphthol AS-MX phosphate and 0.1% fast blue RR salt in 56 mM 2-amino-2-methyl-1,3-propanediol working solution for 10 minutes. The unbound dye was removed by washing and the stained images were captured by using ACS Paragon Plus Environment


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an optical microscope. Sensolyte® pNPP alkaline phosphatase assay kit was used to quantify ALP activity according to our previous work.1 The ALP activity was expressed as the ALP amount per cell. After osteogenic induction culture for 21 days, Alizarin Red S (ARS) staining was performed to confirm the formation of calcium deposition. The cells in different groups were washed, fixed and then immersed in 0.1% ARS solution for 30 minutes. After the cells were washed with PBS again, the stained images were captured by using an optical microscope. For calcium deposition quantification, PBS solution in each well was carefully discarded and the stained cells were air-dried overnight. Perchloric acid (5%) was used to elute the ARS dye in each well. The dissolved solution in each well was placed into 96-well plates and their absorbance at 405 nm was measured by a plate reader. The calcium deposition level was expressed as a relative value to the control group. Expression of osteogenic marker genes was analyzed by real-time polymerase chain reaction (PCR) after 14 days of osteogenic induction culture. The cells with or without treatments of NPs were collected for RNA extraction by using RNAeasy Mini kit (Qiagen, Netherlands). A Thermo Scientific NanoDrop ND-1000 spectrophotometer (Wilmington, DE) was used to quantify the RNA concentration. After RNA was converted to cDNA, a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) was used to assess the gene expression. The primers and probes of osteogenic markers are listed in Table S1 in supporting information. Adipogenic Differentiation of hMSCs. For adipogenic differentiation, the hMSCs seeded in 24-well cell culture plates (2 × 104 cells/cm2) were cultured in growth medium for 1 day to allow attachment. The cell culture medium was then replaced with adipogenic induction medium containing free TEMPO (10.0 µg/mL), Au-mPEG NPs or Au-PEG-TEMPO NPs at an Au concentration of 0.05 mM. The medium in each well was changed every 3 days and the cells were cultured for 14 days. The adipogenic induction medium was growth medium supplemented with 0.5 ACS Paragon Plus Environment

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mM methyl-isobutylxanthine, 200 µM indomethacin, 1 µM dexamethasone and 10 µg/mL insulin. Oil red O staining was performed to confirm the formation of oil droplets in hMSCs. The cells were at first fixed, washed and soaked in 2-propanol solution (60%) for 5 minutes. Subsequently, the cells were soaked in Oil red O working solution for 5 minutes and washed with pure water. An optical microscope was used to observe the stained cells. For quantification of oil droplet formation, water in each well was carefully discarded and the cells were air-dried. The stained cells in each well were soaked in 2-propanol for 30 minutes to extract the Oil red O dye. The lysis solution was transferred into 96-well plates to measure their absorbance at 540 nm using a plate reader. The levels of oil droplet formation was expressed as a relative value to the control group. Expression of adipogenic marker genes was assessed by using real-time PCR. The procedures were the same as the above-mentioned PCR experiment except the primers and probes. The primers and probes of adipogenic markers are listed in Table S1 in supporting information. Statistical Analysis. All experiments were repeated in triplicate (n = 3) and the results were expressed as mean ± SD. Statistical calculation of experimental data was performed using One-way ANOVA statistical analysis. A p value of 0.05 was selected as the level of significance, and the data were classified according to their p values and denoted by (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001. RESULTS AND DISCUSSION Synthesis and Characterization of Au-PEG-TEMPO NPs. Au NPs were used as a carrier to prepare TEMPO clusters owing to their great potential in biomedical applications, which include stem cell differentiation, cancer treatment and molecular imaging.41-42 The preparation scheme of Au-PEG-TEMPO NPs and their interaction with stem cells are shown in Figure 1. TEMPO molecules were conjugated to Au NPs by using biocompatible polyethylene glycol (PEG) as a spacer. In addition, PEG plays an important role in improving the colloidal stability of Au NPs.32 TEMPO ACS Paragon Plus Environment


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clusters on Au NP surface were compared with free TEMPO to elucidate their different effects on scavenging overproduced ROS and controlling stem cell functions. To conjugate TEMPO onto the surface of Au NPs, SH-PEG-TEMPO conjugate was synthesized through the reaction of carboxylic group in SH-PEG-COOH and amino group in TEMPO. 1H NMR spectroscopy was used to characterize the synthesis of SH-PEG-TEMPO conjugate. The characteristic peaks of TEMPO at 1-2 ppm (methyl proton peaks) could be clearly observed in the spectrum of SH-PEG-TEMPO conjugate (Figure S1, supporting information). The average number of TEMPO conjugated to each PEG was estimated to be 0.628 based on the NMR integration. Au NPs were synthesized and then conjugated with SH-mPEG or SH-PEG-TEMPO conjugate for biomedical applications. In the UV-Vis spectra, the absorbance peaks at 529 and 530 nm were observed for Au-mPEG NPs and Au-PEG-TEMPO NPs, respectively (Figure S2, supporting information), confirming the formation of Au NPs in the solution. These results indicated the similar size of Au-mPEG NPs and Au-PEG-TEMPO NPs because the absorption of spherical Au NPs was predominantly dependent on their particle size.1 The absorption peak of Au-PEG-TEMPO NPs displayed a slight red-shift as compared to that of Au-mPEG NPs, which should be due to the surface modification of TEMPO.23 The morphologies and sizes of NPs were observed by using TEM and it was clear that both Au-mPEG NPs and Au-PEG-TEMPO NPs were spherical with a homogeneous distribution (Figure 2a and b). The size of Au-mPEG NPs and Au-PEG-TEMPO NPs was measured to be 39.6 ± 7.9 nm and 40.3 ± 4.8 nm, respectively (Figure 2c). The shape, size and surface property of Au NPs have been reported to play very vital roles in regulating osteogenesis of hMSCs.1, 6 Spherical Au NPs with a size of 40 nm have been found to be the most effective in enhancing osteogenic differentiation of stem cells.1,

43

In addition, the promotive effect of 40-nm Au

nanospheres on osteogenic differentiation is stronger than Au nanorods and Au nanostars at the same size.1 The 40-nm spherical Au NPs promote osteogenic differentiation while inhibit adipogenic ACS Paragon Plus Environment

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differentiation of hMSCs predominantly because they enhance the cytoskeleton tension and cellular mechanical property.38 Therefore, 40-nm spherical Au NPs were chosen in this study. The weight of SH-mPEG or SH-PEG-TEMPO conjugate layer on the particle surface was quantitatively analyzed by TGA (Figure 2d). The naked Au NPs without conjugation had a slight weight loss of 0.35% during heating. On the other hand, the weight loss was 5.31% and 4.32% for the Au-mPEG NPs and Au-PEG-TEMPO NPs, respectively. Therefore, total number of SH-mPEG or SH-PEG-TEMPO molecules on the surface of each Au NP was calculated to be 9152 and 8085, respectively. Based on the result that the TEMPO conjugation efficiency to PEG was 62.8%, the density of TEMPO on the surface of Au-PEG-TEMPO NPs was estimated to be 1.0 molecule/nm2. Dynamic light scattering (DLS) measurement showed that the hydrodynamic size of Au-mPEG NPs and Au-PEG-TEMPO NPs dispersed in water was 62.0 ± 2.2 nm and 65.2 ± 5.3 nm, respectively (Figure 2e). After 7 days of storage, no obvious changes in the hydrodynamic size of the Au-mPEG NPs and Au-PEG-TEMPO NPs were observed, suggesting their good colloidal stability (Figure S3, supporting information). The zeta potential was -26.5 ± 1.4 mV for Au-mPEG NPs and -29.7 ± 2.6 mV for Au-PEG-TEMPO NPs (Figure 2f). The Au-mPEG NPs and Au-PEG-TEMPO NPs had almost the same surface charge. Cell Viability and Proliferation Assay. The hMSCs were cultured with the Au-mPEG NPs or Au-PEG-TEMPO NPs at different Au concentrations for 1 day to check their influence on cell viability. After the incubation, cell viability of hMSCs had no obvious changes after treatments with either the Au-mPEG NPs or Au-PEG-TEMPO NPs at the studied Au concentration range (0.05-1.00 mM) as compared with the control (Figure 3a). These results indicated the Au-mPEG NPs and Au-PEG-TEMPO NPs had no obvious cytotoxicity even at an Au concentration up to 1.00 mM. Influence of the Au-mPEG NPs and Au-PEG-TEMPO NPs on proliferation of hMSCs was investigated after the cells were cultured with these NPs at the Au concentration of 0.05 mM for ACS Paragon Plus Environment


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different time. Live/dead staining showed that scarcely any dead cells (red color) were observed in the NP-treated groups, which was similar to the control (Figure S4, supporting information). The number of live cells (green color) increased gradually with extending the culture time in all the three groups. Quantitative WST-1 assay showed that cell viability in the three groups was at the same level for each time point (Figure 3b). The increasing tendency of cell viability in the NP-treated groups was almost the same as that in the control group. These results suggested the negligible influence of Au-mPEG NPs and Au-PEG-TEMPO NPs on the viability and proliferation of hMSCs. Cellular Uptake Assay. As carriers for drug and gene delivery, excellent cellular uptake of Au NPs is very important. Obvious particle clusters were observed inside the cells after culture with the Au NPs as compared to the control cells, indicating the cellular uptake of Au NPs (Figure 3c-e). Uptake amount of the Au-mPEG NPs and Au-PEG-TEMPO NPs was quantified by using ICP-OES. Compared with the control cells, the NP-treated cells had obvious cellular uptake of Au and the uptaken Au amount per cell increased with the Au concentration for both Au NPs (Figure 3f). Uptake efficiency of the Au-mPEG NPs and Au-PEG-TEMPO NPs was almost at the same level when the same Au concentration was used for cell culture. Surface modification with TEMPO had not significantly effect on cellular uptake of the Au NPs. Highly efficient delivery of TEMPO clusters into cells could be achieved because of the excellent cellular uptake of Au-PEG-TEMPO NPs. ROS Scavenging Experiment. To investigate the ROS scavenging activity of the Au-PEG-TEMPO NPs, the ROS in hMSCs after different treatments were stained with DCFH-DA. For the cells without H2O2 treatment, almost no fluorescence was observed in the studied five groups (Figure 4a). However, for the cells pre-treated with H2O2, the control cells and the cells cultured with free TEMPO (0.2 µg/mL) and Au-mPEG NPs showed very strong green fluorescence signal. The fluorescence in these three groups was much stronger than that in the cells cultured with free TEMPO (100.0 µg/mL) and Au-PEG-TEMPO NPs. Intracellular ROS level was also quantified by ACS Paragon Plus Environment

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measuring the absorbance in different groups. Without H2O2 pre-treatment, the ROS in the control cells and the cells cultured with free TEMPO (0.2 and 100.0 µg/mL), the Au-mPEG NPs and Au-PEG-TEMPO NPs was almost at the same level, suggesting that culture with the Au-mPEG NPs did not induce ROS generation in normal hMSCs. The H2O2 pre-treatment significantly increased the ROS level in the hMSCs, indicating the overproduction of ROS (Figure 4b). The hMSCs with overproduced ROS were cultured with free TEMPO, the Au-mPEG NPs and Au-PEG-TEMPO NPs to compare their scavenging effect on the overproduced ROS. After the ROS overproduced hMSCs were cultured with free TEMPO at a low concentration (0.2 µg/mL) and Au-mPEG NPs, the intracellular ROS levels were still high. However, culture with free TEMPO at a high concentration (100.0 µg/mL) and the Au-PEG-TEMPO NPs obviously reduced the ROS levels in the ROS overproduced hMSCs. These results indicated that the Au-PEG-TEMPO NPs could effectively scavenge the overproduced ROS. According to the TGA results, the weight percentage of TEMPO and Au element in the Au-PEG-TEMPO NPs was 0.21% and 95.33%, respectively. The volume of the cell culture medium in this experiment was 0.25 mL, therefore the TEMPO and Au element amount in the Au-PEG-TEMPO NP group was estimated to be 0.055 µg and 24.625 µg, respectively. It should be noted that the TEMPO amount in the Au-PEG-TEMPO NP group was comparable to that in the free TEMPO-0.2 (0.2 µg/mL) group (0.050 µg TEMPO in total). The total TEMPO amount in the free TEMPO-100 (100 µg/mL) group was calculated to be 25.0 µg. Free TEMPO at a low concentration (0.2 µg/mL) failed to scavenge the overproduced ROS inside cells and a much high dosage (100.0 µg/mL) was required. The high ROS scavenging capacity of Au-PEG-TEMPO NPs should be due to the high cellular uptake efficiency of Au NPs that led to high accumulation of TEMPO clusters inside the cells. Osteogenic Differentiation of hMSCs. When the Au-PEG-TEMPO NPs are applied for ROS scavenging in stem cells, it is necessary to investigate their influence on differentiation of stem cells. ACS Paragon Plus Environment


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Detailed screening experiments were performed in our previous works to optimize the Au concentration for stem cell differentiation.1,

38

It was demonstrated that Au NPs at an Au

concentration of 0.05 mM were effective in inducing osteogenic differentiation of stem cells without influence on cell viability and proliferation.1, 38 Therefore, the concentration of Au NPs was set at 0.05 mM for stem cell differentiation in this study. At first, the osteogenic differentiation of hMSCs was investigated by adding Au-PEG-TEMPO NPs into osteogenic induction medium. The ALP expression is an early phenotypic marker for osteogenesis and formation of calcium deposition is a later marker for mature osteoblasts.44-46 Therefore, osteogenic differentiation level of hMSCs after different treatments was investigated by quantifying the ALP activity and calcium deposition. The cells cultured with the Au-mPEG NPs and Au-PEG-TEMPO NPs showed more intense ALP staining than did the control cells (Figure S5, supporting information). Quantitative analysis showed the ALP activity in hMSCs cultured with the Au-mPEG NPs and Au-PEG-TEMPO NPs increased significantly as compared to the control cells (Figure 5a). There was no significant difference of ALP activity between the cells cultured with the Au-mPEG NPs and Au-PEG-TEMPO NPs. The mineralized levels of hMSCs after culture with the NPs for 21 days were confirmed by ARS staining. The cells cultured with the Au-mPEG NPs and Au-PEG-TEMPO NPs had much stronger staining as compared to the control cells (Figure 5b). The ARS staining was then eluted to quantify the formation of calcium deposition. The results showed that the Au-mPEG NPs and Au-PEG-TEMPO NPs significantly enhanced the calcium deposition in hMSCs (Figure 5c). The amount of calcium deposition in these two groups was almost the same. On the other hand, free TEMPO was found to inhibit osteogenic differentiation of hMSCs. The cells cultured with free TEMPO (10.0 µg/mL) showed obvious decrease in ALP activity and calcium deposition (Figure S6, supporting information). These results indicated that conjugation with TEMPO clusters did not significantly interfere with the promotive effect of Au NPs on ALP activity and calcium deposition. ACS Paragon Plus Environment

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The Au-PEG-TEMPO NPs were still effective in increasing the ALP activity and calcium deposition of hMSCs. Expression of three important osteogenic marker genes was analyzed to further confirm the influence of Au-PEG-TEMPO NPs on osteogenic differentiation of hMSCs at gene expression level. ALP and runt-related transcription factor 2 (Runx2) are two important marker genes in the early-stage osteogenesis.1 The expression of ALP and Runx2 in the cells cultured with the Au-mPEG NPs and Au-PEG-TEMPO NPs was upregulated as compared to that in the control cells (Figure 6a and b). Secreted phosphoprotein I (SPP1) is a late-stage marker gene and its expression is important for osteogenesis of stem cells.47 The Au-mPEG NPs and Au-PEG-TEMPO NPs significantly up-regulated the expression of SPP1 in hMSCs (Figure 6c). The expression of ALP, Runx2 and SPP1 was similar for the cells cultured with the Au-PEG-TEMPO NPs and Au-mPEG NPs. Gene expression results were consistent with the results of ALP activity and calcium deposition, indicating that the Au-PEG-TEMPO NPs after TEMPO conjugation maintained their advantage in promoting osteogenic differentiation of stem cells. Au NPs have shown great promise in accelerating osteogenic differentiation of hMSCs and their physicochemical property is important for regulating osteogenic differentiation.25, 35 The 40-nm spherical Au NPs are the most potent in enhancing ALP expression and mineralization of hMSCs.1 It has also been reported that Au NPs with different surface chemistries have different influences on stem cell osteogenic differentiation.6, 44 Au NPs modified with amine and hydroxyl groups do not inhibit osteogenic differentiation, but Au NPs modified with carboxyl groups can reduce ALP activity and matrix mineralization of hMSCs.6 In another study, poly(acryloy-L-valine) anchored Au NPs are more potent in inducing the osteogenic differentiation of MSCs than do the poly(acryloy-D-valine) anchored counterparts.44 In the present study, the Au-mPEG NPs and Au-PEG-TEMPO NPs showed no obvious difference in promoting the osteogenic differentiation of ACS Paragon Plus Environment


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hMSCs, suggesting conjugation of Au NPs with TEMPO did not significantly influence osteogenic differentiation of hMSCs. Adipogenic Differentiation of hMSCs. Effect of the Au-PEG-TEMPO NPs on adipogenic differentiation of hMSCs was investigated by checking the oil droplets formation and gene expression of adipogenic marker genes. Intracellular lipid accumulation is a distinctive marker for adipogenesis and Oil red O staining is generally performed to confirm the oil droplets formation.48 The results of Oil red O staining showed that a much lower staining intensity in the NP-treated groups was observed (Figure 7a). Oil droplets formation was further quantified by measuring the absorbance of the extracted Oil red O dye. The control cells showed a significantly higher level of oil droplets formation than the cells cultured with the Au-mPEG NPs and Au-PEG-TEMPO NPs (Figure 7b). Culture with free TEMPO (10.0 µg/mL) slightly inhibited the oil droplet formation in hMSCs (Figure S7, supporting information). Expression of adipogenic marker genes including lipoprotein lipase (LPL), CCAAT/enhancer binding protein (CEBPA), and fatty acid synthase (FASN) was analyzed by real-time PCR. Both the Au-mPEG NPs and Au-PEG-TEMPO NPs down-regulated the expression of LPL, FASN and CEBPA (Figure 8). Expression of LPL and CEBPA in the Au-PEG-TEMPO NP-treated cells was slightly lower than that in the Au-mPEG NP-treated cells. The results of Oil red O staining and gene expression suggested that the Au-mPEG NPs and Au-PEG-TEMPO NPs had inhibitory effect on adipogenic differentiation of hMSCs. Conjugation of TEMPO on the surface of Au NPs slightly increased the inhibitory effect of Au NPs on adipogenic differentiation of hMSCs. It has been reported that the ROS generation increases during adipogenic differentiation of stem cells.49-51 Intracellular ROS facilitates differentiation of stem cells into adipocytes.52 On the other hand, use of antioxidants or ROS scavengers is a potential way to inhibit adipogenesis of stem cells.53 Internalization of Au NPs suppressed adipogenic differentiation of hMSCs in this present ACS Paragon Plus Environment

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study is in a good agreement with a previous work.35 The Au-PEG-TEMPO NPs were slightly more effective than the Au-mPEG NPs in suppressing the MSC adipogenesis, which may be because the internalized Au-PEG-TEMPO NPs reduced the ROS generation during the process of differentiation. Treatment with free TEMPO at a concentration of 10.0 µg/mL inhibited osteogenic and adipogenic differentiation of hMSCs. It should be due to the same reason as previously reported that high concentration of TEMPO disrupted the normal redox reactions and induced mitochondrial dysfunctions of stem cells.19, 54 However, TEMPO clusters on the NP surface weakened the negative influence of TEMPO on stem cell differentiation. In addition, the Au NPs with different surface TEMPO density may result in different influence on osteogenic and adipogenic differentiation of hMSCs compared to the Au-mPEG NPs because the accumulation amount of TEMPO should be different after the cellular uptake of the different NPs. The intracellular ROS have obtained increasing attentions because of their obvious influence on cell fate and cell functions.55-58 Profited from the generated ROS, various nanomaterials have been developed for cancer therapy and bacteria killing.59-60 On another hand, some redox nanomaterials have been used for protection of stem cells from ROS-enriched environment.61 It remains a great challenge to prepare functional NPs for ROS scavenging with a high efficiency and minimal side effect, in particular for stem cell transplantation. The influence of these NPs on differentiation of stem cells should be considered. Both the ROS scavenging ability of TEMPO and the role of Au NPs in stem cell differentiation are already known, while using Au NPs for TEMPO delivery has not been reported and their combinational effect on stem cell functions is unclear. The Au-PEG-TEMPO NPs in the present study showed a higher capacity in scavenging overproduced ROS than did the free TEMPO due to the accumulation of TEMPO clusters in cells. Meanwhile, TEMPO conjugated Au NPs showed a positive influence on osteogenic differentiation while a negative influence on adipogenic differentiation of hMSCs. Such effects should be extremely useful for stem cell ACS Paragon Plus Environment


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transplantation of osteoporosis and other diseases. Conjugation of TEMPO molecules on Au NPs facilitated the TEMPO delivery into cells, enabling their good scavenging effect on ROS. Furthermore, the negative effect of TEMPO on stem cell differentiation was weakened when they were conjugated to Au NPs. The conjugated TEMPO clusters had a much larger size than the free TEMPO, which was considered to be the reason of different effects on stem cell functions. The exact reason why the conjugated TEMPO on Au NPs had different cellular effects in comparison with free TEMPO needs to be further explored. Clustering of TEMPO molecules on the surface of Au NPs should provide a useful tool for stem cell transplantation and regenerative medicine. CONCLUSION In summary, Au NPs with an average size of 40 nm were prepared as carriers for clustering and delivery of ROS scavengers into hMSCs. The Au-PEG-TEMPO NPs had desired colloidal stability and negligible cytotoxicity at the studied concentration range. The conjugated TEMPO was efficiently delivered into ROS-enriched cells because of the excellent cellular uptake of Au NPs. Hence, compared to free TEMPO, the Au-PEG-TEMPO NPs were potent in reducing the overproduced ROS in hMSCs even at a low TEMPO dosage. In contrast to the suppressive effect of free TEMPO on stem cell differentiation, the Au-PEG-TEMPO NPs showed a promotive influence on osteogenic differentiation while a suppressive influence on adipogenic differentiation of hMSCs. The Au-PEG-TEMPO NPs had great potential for the treatments of ROS-induced dysfunctions while maintaining the beneficial property of Au NPs to regulate stem cell differentiation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Primers and probes for real-time PCR (Table S1); characterization of the product (Figure S1-S3); ACS Paragon Plus Environment

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cell viability assay (Figure S4); assessment of stem cell differentiation (Figure S5-S7) (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (G.C.). ORCID Guoping Chen: 0000-0001-6753-3678. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the World Premier International Research Center Initiative (WPI) on Materials Nanoarchitectonics from the Ministry of Education, Culture, Sports, Science and Technology, Japan and JSPS KAKENHI Grant Number 15H03027. REFERENCES 1. Li, J.; Li, J. E. J.; Zhang, J.; Wang, X.; Kawazoe, N.; Chen, G., Gold Nanoparticle Size and Shape Influence on Osteogenesis of Mesenchymal Stem Cells. Nanoscale 2016, 8 (15), 7992-8007. 2. Lü, L.-X.; Zhang, X.-F.; Wang, Y.-Y.; Ortiz, L.; Mao, X.; Jiang, Z.-L.; Xiao, Z.-D.; Huang, N.-P., Effects of Hydroxyapatite-Containing Composite Nanofibers on Osteogenesis of Mesenchymal Stem Cells In Vitro and Bone Regeneration In Vivo. ACS Appl. Mater. Interfaces 2013, 5 (2), 319-330. ACS Paragon Plus Environment


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Figure Captions Figure 1. Schematic illustration of synthesis of the Au-PEG-TEMPO NPs and their application for ROS scavenging and stem cell differentiation controlling. Figure 2. TEM images of the Au-mPEG NPs (a) and Au-PEG-TEMPO NPs (b); the size distribution histograms (c), TGA analysis (d), hydrodynamic size (e) and zeta potential (f) of the Au-mPEG NPs and Au-PEG-TEMPO NPs. Figure 3. Cell viability (a) and cell proliferation assay (b); optical micrographs of hMSCs without culture (c) and with culture of the Au-mPEG NPs (d) or Au-PEG-TEMPO NPs (e); quantitative assay of cellular uptake of the NPs (f). Figure 4. Fluorescence images (a) and quantitative ROS level (b) of hMSCs or H2O2-treated hMSCs after culture with free TEMPO, Au-mPEG NPs or Au-PEG-TEMPO NPs for 1 day. Figure 5. ALP activity (a), ARS staining (b) and calcium deposition assay (c) of hMSCs after culture with the Au-mPEG NPs or Au-PEG-TEMPO NPs for 14 or 21 days. Figure 6. Osteogenic marker gene expression of ALP (a), Runx2 (b) and SPP1 (c) of hMSCs after culture with the Au-mPEG NPs or Au-PEG-TEMPO NPs for 14 days. Figure 7. Oil red O staining (a) and oil droplets formation (b) of hMSCs after culture with the Au-mPEG NPs or Au-PEG-TEMPO NPs for 14 days. Figure 8. Adipogenic marker gene expression of LPL (a), FASN (b) and CEBPA (c) of hMSCs after culture with the Au-mPEG NPs or Au-PEG-TEMPO NPs for 14 days.


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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Figure 7



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Figure 8


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TOC graphic
