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Effects of gold nanostructures on differentiation of mesenchymal stem cells
Colloids and Surfaces B: Biointerfaces 184 (2019) 110494
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effects of gold nanostructures on differentiation of mesenchymal stem cells Long Yuan
a,1
, Xiaowei Qi
a,1
b
c
a,⁎
, Gaoping Qin , Qi Liu , Fan Zhang , Yong Song
b,⁎
, Jun Deng
d,⁎
T
a
Department of Breast Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China Department of Hepatobiliary Surgery, Provincial People's Hospital, Xian, Shanxi, 710068, China Department of Blood Transfusion, Southwest Hospital, Army Medical University, Chongqing, 400038, China d Institute of Burn Research, Southwest Hospital, State Key Lab of Trauma, Burn and Combined Injury, Army Medical University, Chongqing, 400038, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Gold nanostructures Mesenchymal stem cells Differentiation Cytotoxicity
Nanoparticles are nanocrystals with complex facets and defective structures that do not adopt an idealised shape. Various physicochemical parameters of nanoparticles, such as surface composition, size, and stiffness, can regulate differentiation in mesenchymal stem cells (MSCs), but the influence of shapes with many edges and corner regions has not been investigated. Herein, we investigated the effects of two gold nanostructures modified with 11-mercaptoundecanoic acid, namely gold nanocubes (MUA-AuNCs) and nanooctahedras (MUA-AuNOs), on viability and differentiation in rat bone marrow MSCs (bMSCs). Analysis of cytotoxicity and proliferation demonstrated good biocompatibility, with concentrations < 100 μg·mL−1 not significantly different from untreated controls. Alkaline phosphatase activity and Alizarin Red S staining revealed weaker potential for bMSCs to differentiate into osteoblasts following treatment with both low (5 μg·mL−1) and high (25 μg·mL−1) concentrations of the gold nanostructures. By contrast, Oil Red O staining showed that both nanostructures enhanced adipogenic differentiation, and upregulated peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein-4 (Fabp4) expression at both mRNA and protein levels. The effects on differentiation were both structure- and dose-dependent; MUA-AuNOs were more effective for enhancing adipogenic differentiation and weakening osteogenic differentiation, possibly due to generating higher levels of reactive oxygen species (ROS). These findings lay the foundation for using these nanoparticles as ex vivo labels in MSCbased imaging and therapy.
1. Introduction Stem cell-based therapies that take advantage of the self-renewal and multi-lineage differentiation capabilities of stem cells to treat various diseases are receiving increasing attention [1–3]. Among the various cell types, mesenchymal stem cells (MSCs) benefit from fewer ethical issues and less risk of developing teratoma [4,5], hence they are considered an attractive source for tissue engineering and regenerative medicine. MSCs possess impressive self-renewal capacity and have an inherent ability to differentiate into multiple lineages including chondrocytes, osteocytes, adipocytes, and various ectodermic and endodermic cell types [6–8]. Numerous MSC-based therapies for tissue repair and immune disorders have been tested, but clinical progress is hampered by risks including controlled self-renewal, rapid proliferation, well-defined differentiation, and difficulties in tracking the fate and distribution of differentiated cells [8]. Rapid advances in nanotechnology have led to the development of
nanomaterials for various stem cell therapy applications, including tissue engineering, molecular imaging, and gene/drug delivery [9,10]. Gold nanoparticles (AuNPs) have received much attention due to their simple synthesis, relatively good biocompatibility, and excellent optical properties. Importantly, the shape, size, structure and surface chemistry of AuNPs can be readily modified to meet different demands [11,12]. Physicochemical properties have a strong influence on cytotoxicity, cellular uptake, differentiation, and other cellular properties [13–15]. A range of AuNPs of different sizes (4, 15, 30, 50, 75 and 100 nm) with similar surface chemistry have been synthesised [16,17], and a diameter of 30 nm or 50 nm enhanced osteogenic differentiation most effectively. By contrast, 4 nm AuNPs had a pronounced influence on adipogenic differentiation, and inhibited osteogenic differentiation of MSCs by generating large amounts of reactive oxygen species (ROS) [18]. In another study, bovine serum albumin (BSA)-coated Au nanospheres, nanostars and nanorods with diameters of 40, 70 and 110 nm were fabricated, and their effects on osteogenic differentiation of MSCs
⁎
Corresponding authors. E-mail addresses: [email protected] (F. Zhang), [email protected] (Y. Song), [email protected] (J. Deng). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.colsurfb.2019.110494 Received 5 July 2019; Received in revised form 3 September 2019; Accepted 5 September 2019 Available online 06 September 2019 0927-7765/ © 2019 Published by Elsevier B.V.
Colloids and Surfaces B: Biointerfaces 184 (2019) 110494
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aggregates, and washed five times with ethanol to remove excess PVP. Finally, concentrated AuNCs and AuNOs were centrifuged and collected as stock solutions. A 20 mL sample of stock solution was added to 30 mL of anhydrous ethanol for ultrasonic dispersion for 30 min, and 5 mL of 4 mg mL−1 MUA solution was added and stirred for 12 h at room temperature. The solution was then centrifuged at 10,000 rpm for 30 min at room temperature. A 2 mL sample of fresh MUA solution (4 mg mL−1) was then added and stirred for another 12 h. This process was repeated to remove all PVP.
were investigated [16]. The influence of AuNPs on MSC osteogenic differentiation was dependent on both sphere size and morphology; 40 and 70 nm spheres and 70 nm rods were stimulatory, whereas rods of 40 nm were inhibitory. Interestingly, osteogenic differentiation of MSCs was not influenced by stars of 40, 70 or 110 nm, spheres of 110 nm, or rods of 110 nm. Moreover, AuNPs similar in size but possessing differences in surface chemistry (e.g., AuNPs-NH2, AuNPs−COOH and AuNPs−OH) were also investigated [19], and AuNP−COOH reduced alkaline phosphatase (ALP) activity and matrix mineralisation of MSCs by upregulating expression of TGF-β and FGF-2, while AuNP-NH2 and AuNP−OH had no influence on osteogenic differentiation. More recently, we prepared molecular chiral poly(acryloyl-L(D)-valine)-anchored AuNPs (L(D)-PAV-AuNPs) [20], and found that they activated the p38 mitogen-activated protein kinase (MAPK) pathway and exerted mechanical stress on MSCs, thereby promoting osteogenic differentiation, but no such effects were observed for D-PAV-AuNPs. Most previous studies and applications have focused on the size, shape and surface chemistry of AuNPs. Although NPs are generally categorised based on idealized shape (spheres or rods), they are actually nanocrystals which have complex facets and defective structures [21,22]. The large surface curvature and numerous edges and corner regions can destabilise the NP surface, which is often coated by chemisorbed ligands [23–25], but this is strongly affected by topography, and this has a pronounced influence on interactions between nanoparticles and their surrounding environment. Therefore, the influence of these factors on differentiation of MSCs is a research priority. In the present study, gold nanocubes (AuNCs) and gold nanooctahedras (AuNOs) as two different representative types of AuNPs were synthesized and coated with 11-mercaptoundecanoic acid (MUA) via SH-Au bonds. The effects of structural properties on differentiation were investigated by monitoring cell viability and proliferation, alkaline phosphatase (ALP) activity, the mRNA and protein expression of osteogenic and adipogenic markers, cellular uptake, and (ROS) generation. Interestingly, both promotion of adipogenic differentiation and inhibition of osteogenic differentiation by the NPs were structure-dependent, and MUA-AuNOs were more effective, presumably due to triggering greater ROS generation. The observed structure-dependent regulation of stem cell differentiation reflects the importance of the structure of NPs, showing significance for the design of novel nanoparticles and their applications.
2.3. Characterisation of MUA-AuNCs and MUA-AuNOs The gold mass concentration (μg·L−1) of MUA-AuNCs or MUAAuNOs was measured using inductively-coupled plasma mass spectrometry (ICP-MS) using an instrument from XSENIES (Palo alto, California, USA). Transmission electron microscopy (TEM) (using an H7650 instrument) and scanning electron microscopy (SEM) [using a SIRION-100 field emission instrument (FEI)] were performed to determine the structure and dimensions of MUA-AuNCs or MUA-AuNOs, respectively. The size distribution of MUA-AuNCs and MUA-AuNOs was derived from TEM and SEM micrographs using Image J software (National Institute of Health, Maryland, USA) according to our previous work, with at least 500 particles per sample [26]. The hydrodynamic diameters and zeta potentials of AuNCs and AuNOs were obtained using a Zetasizer Nano instrument (Malvern, Malvern, England). UV–vis spectra were measured using a Shimadzu UV-3600 UV–vis-near-infrared (NIR) spectrophotometer (Shimadzu, Kyoto, Japan). 2.4. Cell isolation and culture Bone marrow mesenchymal stem cells (bMSCs) were isolated from bone marrow from Sprague Dawley rats (6―8 weeks old) as reported previously [20,27], and routinely cultured in low-glucose cell culture medium containing 5% FBS and 1% penicillin-streptomycin (Sigma). All animal experiments were conducted in accordance with the Third Military Medical University (Army Medical University) for the Care and Use of Laboratory Animals. When ˜70―80% confluence was reached, we detached bMSCs, and then sub-cultured them serially. Cells at passage 2 (P2) were employed in the present work.
2. Experimental 2.5. Cell viability and proliferation
2.1. Materials
The viability and proliferation of bMSCs were probed by CCK-8 assays according to the manufacturer’s protocol (Beyotime). Briefly, bMSCs were cultured at a density of 1 × 104 cells/cm2 in a 48-well plate for 24 h, and then co-cultured with various Au concentrations (5―100 μg·mL−1). We replaced the culture medium with fresh NPcontaining medium every 3 days. After treatment for the indicated time, plates were washed three times with phosphate-buffered saline (PBS), CCK-8 reagent was added to each well and incubated for 2 h, and the absorbance/optical density (OD) at 450 nm (OD450nm) was measured using a Varioskan Flash microplate reader (Thermo Scientific, Massachusetts, USA). For cell proliferation assessment, bMSCs were incubated with MUA-AuNCs or MUA-AuNOs at an Au concentration of 5 or 25 μg·mL-1 for 1, 2 or 3 days, and viability was also measured using CCK-8 assays as above described.
Gold(III) chloride hydrate (HAuCl4) and sodium citrate (C6H5Na3O7·2H2O) were obtained from Sinopharm group Co. Ltd (Shanghai, China), and Aladdin (Shanghai, China) supplied 11-mercaptoundecanoic acid (MUA) and 1,5-pentanediol (96%) (PD). Alizarin Red S (ARS), polyvinylpyrrolidone (PVP, Mw ∼55 kDa) and Oil Red O were obtained from Sigma-Aldrich. KeyGEN Co. Ltd. (Nanjing, China) supplied an alkaline phosphatase kit, and HyClone (Logan, Utah, USA) provided a PicoGreen dsDNA kit, fetal bovine serum (FBS), and lowglucose Dulbecco’s modified Eagle’s medium (LDMEM). A cell counting Kit-8 (CCK8) and 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) were bought from Beyotime (Shanghai, China) and DOJINDO Company (Kumamoto, kyushu island, Japan), respectively. Milli-Q water was used throughout all experiments. 2.2. Synthesis of MUA-functionalized gold nanocubes (MUA-AuNCs) and gold nanooctahedras (MUA-AuNOs)
2.6. Cellular uptake of MUA-AuNCs and MUA-AuNOs
AuNCs and AuNOs were synthesised through a high-temperature polyol process according to our previous work [26]. AuNC and AuNO solutions were centrifuged at 5000 rpm for 5 min at 10 °C, the supernatant was transferred to a new tube to remove large particles and
The amount of MUA-AuNCs or MUA-AuNOs internalised by bMSCs was determined by ICP-MS according to our previously reported protocol. The amount of Au per 104 cells based on ICP-MS analysis is presented as the mean ± standard deviation (SD). 2
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2.7. Measurement of reactive oxygen species (ROS)
Table 1 Characterisation of MUA-AuNCs and MUA-AuNOs (mean ± SD, n = 4).
After seeding bMSCs on a 6-well plate at a density of 2 × 105 cells·cm−2 and attaching for 48 h, cells were treated with MUA-AuNCs or MUA-AuNOs (5 μg·mL-1) for 7 or 21 days (the medium was replaced with fresh NP-containing medium every 3 days). Cells were then washed three times with PBS and incubated with 1 mL of 10 μM DCFH-DA for 20 min, washed three times with culture medium without FBS to remove excess extracellular DCFH-DA, and imaged using a DM 6000 B fluorescence microscope (Leica, Wetzlar, Germany).
a
Nanoparticles
Length (nm)
MUA-AuNCs MUA-AuNOs
89 ± 12 50 ± 9
a
Diameter (DLS, Z-average, nm)
Zeta potential (mV)
437.8 ± 36.1 398.9 ± 58.4
−13.3 ± 1.6 −12.4 ± 0.7
Length refers to the edge length of MUA-AuNCs or MUA-AuNOs.
(4 °C), lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA), and finally incubated with specific antibodies overnight; an enhanced chemiluminescence system with ECL Western Blotting Substrate (Pierce, USA) was employed for detection. Image Jsoftware was employed for integral optical density (IOD) determination. Expression profiles of osteocalcin (OCN) and collagen type I (COL I), both related to osteogenic differentiation, were determined by RT-PCR. Sequences of primers used in this study are listed in Table 1. The 18S ribosomal subunit housekeeping gene served as an endogenous reference.
2.8. Measuring alkaline phosphatase (ALP) activity After culturing with MUA-AuNCs or MUA-AuNOs (5 or 25 μg·mL−1) for 7 days and three washes with PBS, bMSCs were treated with 0.5% (v/v) Triton X-100 (Sigma) at 4 °C for 24 h. Samples were then mixed with the appropriate colorimetric kit reagents (KeyGEN Biotech) according to the manufacturer’s instructions, the OD450nm value was recorded using a microplate reader, and a calibration curve was used to calculate ALP activity. A bicinchoninic acid assay kit (KeyGEN Biotech) was employed for determining the total protein content according to the manufacturer’s instructions, and the results presented as ALP activity per g of protein.
2.12. Statistical analysis
2.9. Observation of cell morphology
Results are expressed as mean ± SD, and significant differences between groups were analysed by one-way analysis of variance (ANOVA) for two groups and two-way ANOVA (for more than two groups) using Origin software. Statistical significance was set as p < 0.05 and p < 0.01, respectively.
After co-culturing with MUA-AuNCs or MUA-AuNOs for 7 or 14 days, bMSCs were washed three times with PBS, fixed with 4% formaldehyde solution at room temperature for 30 min, treated with 0.5% Triton X-100/PBS at 4 °C for 10 min, and then washed with PBS three more times. Following treatment with 1% BSA/PBS for 6 h to block nonspecific adsorption, cell nuclei were stained with 100 ng·mL−1 4′,6diamidino-2-phenylindole (DAPI) and the cytoskeleton (F-actin) was stained with 0.2 μM rhodamine-labelled phalloidin solution (Life Technologies) at 37 °C for 1 h. After washing three times with PBS, cells were observed by confocal laser-scanning microscopy (CLSM) using an LSM 510 instrument (Carl Zeiss, Oberkochen, Germany).
3. Results and discussion 3.1. Characterisation of nanoparticles TEM and SEM images showed successful synthesis of MUA-AuNCs and MUA-AuNOs, which had the desired structures (Fig. 1a, b, c, d) and a narrow morphological distribution (Fig. 1e). MUA-AuNCs were 89 ± 12 nm on each side, and MUA-AuNOs edges were 50 ± 9 nm in length (Table 1). The hydrodynamic diameters of MUA-auNCs and MUA-AuNOs were 437.8 ± 36.1 nm and 398.9 ± 58.4 nm, respectively (Table 1). The zeta potential of MUA-AuNCs and MUA-AuNOs was -13.3 ± 1.6 mV and -12.4 ± 0.7 mV, respectively (Table 1). The coherent oscillation of conduction electrons in the gold surface facilitates analysis by surface plasmon resonance (SPR) because of its high sensitivity to the local dielectric environment. The results revealed a red shift in the SPR peak during ligand exchange, at 566 nm for MUAAuNCs and 582 nm for MUA-AuNOs (Fig. 1f). A degree of peak broadening was evident, possibly indicating a slight rounding of NP vertices during the ligand exchange process, because surface plasmon resonance is known to be highly sensitive to the relative sharpness of corners in anisotropic particles [28]. There were significant changes in SPR signals in 10% FBS/LDMEM (Figure S1), indicating good colloidal stability for both MUA-AuNCs and MUA-AuNOs under the cell culture conditions. These results indicate that MUA-AuNCs and MUA-AuNOs essentially shared identical physiochemical properties except for structure, allowing us to correlate cellular behaviour with different nanostructures.
2.10. Analysis of cell differentiation For analysis of cell differentiation, bMSCs were initially at a density of 1.5 × 104 cells/well into 48-well plates attached by incubating overnight. Fresh medium containing 10% FBS/DMEM and MUA-AuNCs or MUA-AuNOs (5 μg·mL−1) was used to replace the overnight medium, and this was changed every 3 days to maintain a stable particle concentration throughout the entire experimental period. After 14 days of culturing and three washes with PBS, bMSCs were fixed in 4% paraformaldehyde for a minimum of 30 min at room temperature, and stained with 1% (w/v) aqueous ARS to detect calcium deposition and evaluate osteogenic differentiation, and 0.6% (w/v) Oil Red O solution (3:2 isopropanol:water) to detect lipid droplets and evaluate adipogenic differentiation, both at room temperature for 15 min. Cells were subsequently washed three times with PBS and observed under an IX81 bright field microscope (Olympus, Tokyo, Japan). ARS dissolved in 10% (w/v) hexadecylpyridinium chloride in PBS (pH 7.4) was used for quantification of calcium deposition by measuring the absorbance at 570 nm using a Model 680 microplate reader (Bio-Rad, California, USA). Data were normalised against particle-free control samples. 2.11. Western blotting (WB) and real-time quantitative reversetranscription PCR (RT-PCR)
3.2. Differentiation of bMSCs Before investigating cell differentiation, the effects of MUA-AuNCs and MUA-AuNOs dosage on cell viability and proliferation were analysed using CCK-8 assays, and no significant cytotoxicity was observed with either NP type, even after treatment at an Au concentration of 100 μg·mL−1 for 24 h (Fig. 2a). Cell differentiation proceeds as a series of
After culturing with MUA-AuNCs or MUA-AuNOs for 21 days, we washed bMSCs with PBS three times and then completely homogenized them in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. After being centrifuged for 15 min at 12,000 rpm 3
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Fig. 1. Characterisation of MUA-AuNCs and MUA-AuNOs. Representative TEM images (a, b) and SEM images (c, d) of (a, c) MUA-AuNCs and (b, d) MUA-AuNOs, respectively. (e) Hydrodynamic diameter of MUA-AuNCs and MUA-AuNOs. (f) UV–vis-NIR spectra of MUA-AuNCs and MUA-AuNOs in PBS (pH 7.4, 10 mM).
osteogenic differentiation. Differentiation of pluripotent progenitor bMSCs into osteoblasts is a crucial step during osteogenesis, and alkaline phosphatase (ALP) activity can serve as an osteogenic marker of the early stages of osteogenesis differentiation in bMSCs (typically up to 7 days). Meanwhile, mineralized nodule formation serves as a phenotypic marker for the latter stages upon formation of mature osteoblasts. Thus, ALP activity was quantified and normalized against total protein content to further explore the influence of NP structure on bMSC differentiation. Compared with controls, ALP activity of bMSCs was significantly decreased following treatment with MUA-AuNCs or MUAAuNOs, and this osteo-inhibitive effect was dose-dependent regardless of NP structure (Fig. 2c). Moreover, inhibition of osteogenic differentiation by the particles was also dependent on structure; ALP activity was significantly lower after treatment of bMSCs with MUA-AuNOs
sequential events that follow initial cell proliferation. Based on these results, two relatively low concentrations of MUA-AuNCs or MUAAuNOs (5 and 25 μg·mL−1) were selected as the appropriate gold NP dose for further studies on the influence of NP structure on differentiation of bMSCs. Cell differentiation is usually achieved by day 21, according to the existing literature and our previous experience, so this timeframe was adopted for culturing in this work. Osteogenic tissues can form extracellular matrix and regulate mineralisation [29]. Therefore, after culturing for 21 days, calcium deposition can serve as an indicator of osteogenic differentiation [30,31]. We therefore measured this parameter using ARS staining [32], and the amount of adsorbed dye (OD) is positively correlated with calcium content. As shown in Fig. 2b, the presence of either MUA-AuNCs or MUA-AuNOs inhibited calcium deposition, indicating suppression of
Fig. 2. (a) Viability of bMSCs treated with MUA-AuNCs or MUA-AuNOs for 24 h. (b) ARS staining (21 days), (c) ALP activity (7 days), and (d) Oil Red O staining (21 days) of bMSCs after culturing with MUA-AuNCs or MUA-AuNOs for the indicated time. 4
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Fig. 3. Western blotting and relative gene expression analysis. (a) Western blotting analysis of OCN, COL I, PPARγ and Fabp4 expression in bMSCs treated with MUA-AuNCs or MUA-AuNOs for 21 days. GAPDH served as a loading control. (b) Relative gene expression of osteogenic markers (OCN and COL I) and adipogenic markers (PPARγ and Fabp4) in bMSCs cultured for 21 days. Error bars represent mean ± standard deviation (SD; n = 4). * and ** denote significant differences at p < 0.05 and p < 0.01, respectively.
cell surface-attached particles present in small quantities). Based on existing literature and our prior experience, cellular loading of gold particles generally reaches equilibrium within 24 h [37,38], hence this timeframe was adopted in the present work. As shown in Fig. 5a and b, cell endocytosis of both MUA-AuNCs or MUA-AuNOs increased significantly (p < 0.01) with an increasing concentration of gold nanoparticles. Even with a fivefold higher concentration of gold NPs, endocytosis of NPs increased by only 30%, which could be attributed to the limited saturation level [39]; a similar trend was observed previously [40]. Additionally, cellular uptake was comparable for MUA-AuNCs and MUA-AuNOs (p > 0.05). These results suggest that the particles may bind to cytoplasmic proteins and/or interact with the cell membrane, resulting in mechanical stress that activates related signalling pathways regulating the expression of genes that induce adipogenic differentiation and suppress osteogenic differentiation. The effects of NPs on the differentiation potential of stem cells have received considerable attention in recent years [13,41,42]. Indeed, NPs are now considered a new class of differentiation activators that act through oxidative stress and various other pathways. ROS production is a major cause of cytotoxicity associated with most NPs characterised to date, and is considered a typical side-effect of NP exposure. NPs stimulate ROS production through various mechanisms, and interactions can damage mitochondrial membranes, disrupt the respiratory chain, and consequently elevate ROS production. Direct interactions between NPs and intracellular enzymes may disturb the cellular redox potential, and NP-integrin interactions may activate intracellular signalling pathways triggering ROS generation. Degradation of NPs may release metal ions and other byproducts that stimulate ROS release. Altering intracellular ROS levels is a general mechanism for regulating differentiation, but lineage-specific regulation of differentiation requires further investigation. Even so, accumulated evidence indicates that high levels of ROS may promote adipogenic differentiation of stem cells, but impede osteogenesis [18,43]. We therefore probed intracellular ROS generation using DCFH-DA, with which the fluorescence intensity of cells increases in the presence of ROS. Compared with controls, ROS levels were clearly elevated in bMSCs treated with MUAAuNCs and MUA-AuNOs. In addition, MUA-AuNOs triggered much greater ROS levels than MUA-AuNCs, indicating that intracellular ROS generation triggered by the gold particles is structure-dependent (Fig. 6). Mesenchymal stem cellular internalization of NPs and the mechanism underlying differentiation induced by NPs are dependent on the physicochemical properties of NPs, including size, shape, hydrophobicity, softness, composition, and surface chemistry [44]. Here, two types of MUA-AuNP had similar cellular internalization, hydrodynamic
than with MUA-AuNCs (p < 0.01; Fig. 2c). Accumulation of lipid-rich vacuoles within cells is an established marker of adipogenic differentiation in bMSCs. Therefore, formation of lipid droplets was characterised using Oil Red O staining after 21 days of culturing, given the positive correlation between the amount of adsorbed dye (OD) and lipid droplet content. As shown in Fig. 2d, the lipid droplet content in bMSCs was significantly higher after treatment with MUA-AuNCs or MUA-AuNOs (p < 0.01), suggesting that both particle types can promote adipogenic differentiation. Moreover, MUA-AuNOs exhibited a stronger capacity to enhance adipogenic differentiation. Furthermore, the gene and protein expression levels of several markers of the latter stages of differentiation were examined. Collagen type I (COL I) and osteocalcin (OCN) play important roles in osteogenic differentiation; COL is abundant in the organic/inorganic composite matrix of bone tissue [33,34], and OCN is a noncollagenous protein rich in the bone matrix essential for bone formation and remodelling [35]. Herein, data were normalized against expression of 18S (PCR) and βactin (WB) as internal standards. As shown in Fig. 3a and b, compared with control groups, COL I and OCN expression were significantly decreased (p < 0.01) at both mRNA and protein levels following treatment with either MUA-AuNCs or MUA-AuNOs, indicating that both particles exhibited osteo-inhibitive effects. Again, MUA-AuNOs displayed stronger osteogenesis inhibition ability. Peroxisome proliferator-activated receptor-gamma (PPARγ) and lipoprotein lipase (LPL), which are also important in adipocyte differentiation, can stimulate lipid uptake and adipogenesis by fat cells [36]. In the presence of MUA-AuNCs or MUA-AuNOs, the mRNA and protein expression of both LPL and PPARγ was significantly increased (p < 0.01) in bMSCs after culturing for 21 days (Fig. 3c and d). Therefore, we can conclude that both MUA-AuNCs and MUA-AuNOs can trigger adipogenic differentiation of bMSCs, and MUA-AuNOs are more potent for promoting adipogenic differentiation. Stem cell differentiation proceeds after initial cell proliferation, and MUA-AuNCs and MUA-AuNOs might act by stimulating osteogenic and/or adipogenic differentiation of bMSCs. As shown in Fig. 4a, the OD value increased almost linearly over 3 days, regardless of the presence of MUA-AuNCs or MUA-AuNOs, consistent with normal cell proliferation. Moreover, absolute values in the presence of MUA-AuNCs or MUA-AuNOs were similar to those of controls after 1, 2 and 3 days (p > 0.05), indicating no significant impairment of metabolic activity or cell proliferation. Additionally, neither MUA-AuNCs nor MUAAuNOs influenced the structure of MSCs after 7 or 14 days (Fig. 4b), indicating cytoskeletal integrity. Since the biological activities of particles may also be associated with differences in cellular uptake, the content of elemental gold was determined by ICP-MS to quantify particle internalisation (including 5
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Fig. 4. The influence of gold nanostructure (MUAAuNCs or MUA-AuNOs) on the proliferation and morphology of bMSCs. (a) Dose-dependent cell proliferation assay of the effects of MUA-AuNCs and MUAAuNOs. (b) CLSM images of bMSCs exposed to MUAAuNCs or MUA-AuNOs. Cells were co-cultured with particles for 7 or 14 days. Nuclei (blue) and the cytoskeleton (F-actin; red) were stained with DAPI and rhodamine phalloidin, respectively. Scale bar = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 5. (a) Inverted microscope images of bMSCs treated with MUA-AuNCs or MUA-AuNOs for 24 h. Red arrows represent gold nanoparticles (MUA-AuNCs or MUA-AuNOs). (b) Cellular uptake of MUA-AuNCs and MUA-AuNOs quantified by ICP-MS. Error bars represent mean ± SD (n = 4). * and ** denote significant differences at p < 0.05 and p < 0.01, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
cytoskeletal integrity of MSCs inhibited osteogenic differentiation. These results correlate well with the structure-dependent stem cell differentiation behaviour described above, and imply that ROS generation triggered by MUA-AuNCs and MUA-AuNOs is a primary factor regulating stem cell differentiation. NPs can provoke ROS generation through multiple interactions. First, the interaction between NPs and mitochondria interaction may induce mitochondrial membrane damage, thus leading to disruption of the respiratory chain and increased ROS production. Second, the direct
diameters, surface chemistry, surface charge, and so on, and only their structures were different. Mechanical stress also plays a vital role in the differentiation potential change of stem cells [13]. The accumulation of NPs inside cells may cause changes in the cell cytoskeleton and cellular mechanical properties [45]. Increasing evidence indicates that high mechanical properties promoted osteogenic differentiation but inhibited adipogenic differentiation of stem cells [46]. Although our results are opposite in that none of our MUA-AuNPs influenced the structure of MSCs, the conclusion seems to be the same in that 6
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Fig. 6. Oxidative stress responses of bMSCs to MUA-AuNCs and MUA-AuNOs. The fluorescence microscope images indicate ROS levels following culturing with MUAAuNCs or MUA-AuNOs for 7 or 14 days. Scale bar = 200 μm.
Fig. 7. Schematic diagram of the effects of gold nanostructures on stem cell differentiation.
4. Conclusion
interactions between NPs and intracellular enzymes could maintain cellular redox potential. Furthermore, NP–integrin interaction could also activate the intracellular signalling, in turn triggering ROS generation. The degradation byproducts of NPs such as metal ions can also induce ROS production [13]. But why was the ROS induced by MUAAuNPs structure-dependent, and MUA-AuNOs elicited more potent effects? We speculate that shapes with more edges and corner regions have more potential for interaction of MUA-AuNPs with MSCs. Li, however, discovered that sphere-40 with fewer edges and corner regions than sphere-70 elicited more promotion of adipogenic differentiation of human mesenchymal stem cells [16]. The inconsistency of conclusions is probably attributable to the different surface chemistry of the NPs, the different sizes of the NPs, the different types of cells, and so on. In addition, the fate of stem cells may be substantially influenced by the surface protein adsorption and the potential cytotoxicity (e.g., lysosomal dysfunction) induced by NPs [13]. Cell differentiation mediated by NPs is a very complicated process involving many possible mechanisms and signalling pathways. Future efforts are needed for a full understanding of the mechanisms for stem cell differentiation induced by NPs.
Two types of nano-sized MUA-AuNPs with similar hydrodynamic diameters (437.8 ± 36.1 nm vs. 398.9 ± 58.4 nm), surface chemistry, and surface charge (-13.3 ± 1.6 mV vs. -12.4 ± 0.7 mV), but different structures were prepared. Both inhibition of osteogenic differentiation and promotion of adipogenic differentiation of bMSCs induced by MUAAuNCs and MUA-AuNOs were structure-dependent, and MUA-AuNOs elicited more potent effects. It is highly probable that accumulation of MUA-AuNOs triggered greater ROS generation, resulting in more extensive adipogenic differentiation of bMSCs (Fig. 7). These findings may support the rational design of new nanomaterials that can be applied to a broad range of biomedical areas.
Author contributions YL and QXW carried out most experiments and wrote the paper, QGP and LQ participated in the analysis of experimental data, and ZF, SY and DJ provided supervision and guidance and participated in manuscript drafting and language correction. All authors read and 7
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approved the final manuscript.
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Availability of data and materials Not applicable. Consent for publication The manuscript is not under consideration for publication elsewhere. Ethics approval and consent to participate Not applicable. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements This study is supported by the National Natural Science Foundation of China (No. 51703243), Prominent Talent Development Program of Southwest Hospital (SWH2018BJKJ-09), and the Natural Science Foundation of Shanxi Provencal Science and Technology Department (Nos. 2017JM8138). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110494. References [1] P.K. Nguyen, J.-W. Rhee, J.C. Wu, Adult stem cell therapy and heart failure, 2000 to 2016: a systematic review, JAMA Cardiol. 1 (2016) 831–841. [2] D. Sakai, G.B. Andersson, Stem cell therapy for intervertebral disc regeneration: obstacles and solutions, Nat. Rev. Rheumatol. 11 (2015) 243. [3] R. Bolli, S. Ghafghazi, Stem cells: cell therapy for cardiac repair: what is needed to move forward? Nat. Rev. Cardiol. 14 (2017) 257. [4] D.G. Phinney, M.F. Pittenger, Concise review: MSC-derived exosomes for cell-free therapy, Stem Cells 35 (2017) 851–858. [5] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [6] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. OrtizGonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418 (2002) 41. [7] A. Uccelli, L. Moretta, V. Pistoia, Mesenchymal stem cells in health and disease, Nat. Rev. Immunol. 8 (2008) 726. [8] A. Trounson, C. McDonald, Stem cell therapies in clinical trials: progress and challenges, Cell Stem Cell 17 (2015) 11–22. [9] M. Srikanth, J.A. Kessler, Nanotechnology-novel therapeutics for CNS disorders, Nat. Rev. Neurol. 8 (2012) 307. [10] L. Ferreira, J.M. Karp, L. Nobre, R. Langer, New opportunities: the use of nanotechnologies to manipulate and track stem cells, Cell Stem Cell 3 (2008) 136–146. [11] K. Saha, S.S. Agasti, C. Kim, X. Li, V.M. Rotello, Gold nanoparticles in chemical and biological sensing, Chem. Rev. 112 (2012) 2739–2779. [12] M.-C. Daniel, D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 104 (2004) 293–346. [13] X. Zhou, L. Yuan, C. Wu, G. Luo, J. Deng, Z. Mao, Recent review of the effect of nanomaterials on stem cells, RSC Adv. 8 (2018) 17656–17676. [14] C. Yi, D. Liu, C.-C. Fong, J. Zhang, M. Yang, Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38 MAPK pathway, ACS Nano 4 (2010) 6439–6448. [15] J. Deng, C. Gao, Recent advances in interactions of designed nanoparticles and cells with respect to cellular uptake, intracellular fate, degradation and cytotoxicity, Nanotechnology 27 (2016) 412002. [16] J. Li, J. Zhang, X. Wang, N. Kawazoe, G. Chen, Gold nanoparticle size and shape influence on osteogenesis of mesenchymal stem cells, Nanoscale 8 (2016) 7992–8007. [17] W.-K. Ko, D.N. Heo, H.-J. Moon, S.J. Lee, M.S. Bae, J.B. Lee, I.-C. Sun, H.B. Jeon, H.K. Park, I.K. Kwon, The effect of gold nanoparticle size on osteogenic differentiation of adipose-derived stem cells, J. Colloid Interface Sci. 438 (2015) 68–76.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Effects of gold nanostructures on differentiation of mesenchymal stem cells Long Yuan
a,1
, Xiaowei Qi
a,1
b
c
a,⁎
, Gaoping Qin , Qi Liu , Fan Zhang , Yong Song
b,⁎
, Jun Deng
d,⁎
T
a
Department of Breast Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, 400038, China Department of Hepatobiliary Surgery, Provincial People's Hospital, Xian, Shanxi, 710068, China Department of Blood Transfusion, Southwest Hospital, Army Medical University, Chongqing, 400038, China d Institute of Burn Research, Southwest Hospital, State Key Lab of Trauma, Burn and Combined Injury, Army Medical University, Chongqing, 400038, China b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Gold nanostructures Mesenchymal stem cells Differentiation Cytotoxicity
Nanoparticles are nanocrystals with complex facets and defective structures that do not adopt an idealised shape. Various physicochemical parameters of nanoparticles, such as surface composition, size, and stiffness, can regulate differentiation in mesenchymal stem cells (MSCs), but the influence of shapes with many edges and corner regions has not been investigated. Herein, we investigated the effects of two gold nanostructures modified with 11-mercaptoundecanoic acid, namely gold nanocubes (MUA-AuNCs) and nanooctahedras (MUA-AuNOs), on viability and differentiation in rat bone marrow MSCs (bMSCs). Analysis of cytotoxicity and proliferation demonstrated good biocompatibility, with concentrations < 100 μg·mL−1 not significantly different from untreated controls. Alkaline phosphatase activity and Alizarin Red S staining revealed weaker potential for bMSCs to differentiate into osteoblasts following treatment with both low (5 μg·mL−1) and high (25 μg·mL−1) concentrations of the gold nanostructures. By contrast, Oil Red O staining showed that both nanostructures enhanced adipogenic differentiation, and upregulated peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein-4 (Fabp4) expression at both mRNA and protein levels. The effects on differentiation were both structure- and dose-dependent; MUA-AuNOs were more effective for enhancing adipogenic differentiation and weakening osteogenic differentiation, possibly due to generating higher levels of reactive oxygen species (ROS). These findings lay the foundation for using these nanoparticles as ex vivo labels in MSCbased imaging and therapy.
1. Introduction Stem cell-based therapies that take advantage of the self-renewal and multi-lineage differentiation capabilities of stem cells to treat various diseases are receiving increasing attention [1–3]. Among the various cell types, mesenchymal stem cells (MSCs) benefit from fewer ethical issues and less risk of developing teratoma [4,5], hence they are considered an attractive source for tissue engineering and regenerative medicine. MSCs possess impressive self-renewal capacity and have an inherent ability to differentiate into multiple lineages including chondrocytes, osteocytes, adipocytes, and various ectodermic and endodermic cell types [6–8]. Numerous MSC-based therapies for tissue repair and immune disorders have been tested, but clinical progress is hampered by risks including controlled self-renewal, rapid proliferation, well-defined differentiation, and difficulties in tracking the fate and distribution of differentiated cells [8]. Rapid advances in nanotechnology have led to the development of
nanomaterials for various stem cell therapy applications, including tissue engineering, molecular imaging, and gene/drug delivery [9,10]. Gold nanoparticles (AuNPs) have received much attention due to their simple synthesis, relatively good biocompatibility, and excellent optical properties. Importantly, the shape, size, structure and surface chemistry of AuNPs can be readily modified to meet different demands [11,12]. Physicochemical properties have a strong influence on cytotoxicity, cellular uptake, differentiation, and other cellular properties [13–15]. A range of AuNPs of different sizes (4, 15, 30, 50, 75 and 100 nm) with similar surface chemistry have been synthesised [16,17], and a diameter of 30 nm or 50 nm enhanced osteogenic differentiation most effectively. By contrast, 4 nm AuNPs had a pronounced influence on adipogenic differentiation, and inhibited osteogenic differentiation of MSCs by generating large amounts of reactive oxygen species (ROS) [18]. In another study, bovine serum albumin (BSA)-coated Au nanospheres, nanostars and nanorods with diameters of 40, 70 and 110 nm were fabricated, and their effects on osteogenic differentiation of MSCs
⁎
Corresponding authors. E-mail addresses: [email protected] (F. Zhang), [email protected] (Y. Song), [email protected] (J. Deng). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.colsurfb.2019.110494 Received 5 July 2019; Received in revised form 3 September 2019; Accepted 5 September 2019 Available online 06 September 2019 0927-7765/ © 2019 Published by Elsevier B.V.
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aggregates, and washed five times with ethanol to remove excess PVP. Finally, concentrated AuNCs and AuNOs were centrifuged and collected as stock solutions. A 20 mL sample of stock solution was added to 30 mL of anhydrous ethanol for ultrasonic dispersion for 30 min, and 5 mL of 4 mg mL−1 MUA solution was added and stirred for 12 h at room temperature. The solution was then centrifuged at 10,000 rpm for 30 min at room temperature. A 2 mL sample of fresh MUA solution (4 mg mL−1) was then added and stirred for another 12 h. This process was repeated to remove all PVP.
were investigated [16]. The influence of AuNPs on MSC osteogenic differentiation was dependent on both sphere size and morphology; 40 and 70 nm spheres and 70 nm rods were stimulatory, whereas rods of 40 nm were inhibitory. Interestingly, osteogenic differentiation of MSCs was not influenced by stars of 40, 70 or 110 nm, spheres of 110 nm, or rods of 110 nm. Moreover, AuNPs similar in size but possessing differences in surface chemistry (e.g., AuNPs-NH2, AuNPs−COOH and AuNPs−OH) were also investigated [19], and AuNP−COOH reduced alkaline phosphatase (ALP) activity and matrix mineralisation of MSCs by upregulating expression of TGF-β and FGF-2, while AuNP-NH2 and AuNP−OH had no influence on osteogenic differentiation. More recently, we prepared molecular chiral poly(acryloyl-L(D)-valine)-anchored AuNPs (L(D)-PAV-AuNPs) [20], and found that they activated the p38 mitogen-activated protein kinase (MAPK) pathway and exerted mechanical stress on MSCs, thereby promoting osteogenic differentiation, but no such effects were observed for D-PAV-AuNPs. Most previous studies and applications have focused on the size, shape and surface chemistry of AuNPs. Although NPs are generally categorised based on idealized shape (spheres or rods), they are actually nanocrystals which have complex facets and defective structures [21,22]. The large surface curvature and numerous edges and corner regions can destabilise the NP surface, which is often coated by chemisorbed ligands [23–25], but this is strongly affected by topography, and this has a pronounced influence on interactions between nanoparticles and their surrounding environment. Therefore, the influence of these factors on differentiation of MSCs is a research priority. In the present study, gold nanocubes (AuNCs) and gold nanooctahedras (AuNOs) as two different representative types of AuNPs were synthesized and coated with 11-mercaptoundecanoic acid (MUA) via SH-Au bonds. The effects of structural properties on differentiation were investigated by monitoring cell viability and proliferation, alkaline phosphatase (ALP) activity, the mRNA and protein expression of osteogenic and adipogenic markers, cellular uptake, and (ROS) generation. Interestingly, both promotion of adipogenic differentiation and inhibition of osteogenic differentiation by the NPs were structure-dependent, and MUA-AuNOs were more effective, presumably due to triggering greater ROS generation. The observed structure-dependent regulation of stem cell differentiation reflects the importance of the structure of NPs, showing significance for the design of novel nanoparticles and their applications.
2.3. Characterisation of MUA-AuNCs and MUA-AuNOs The gold mass concentration (μg·L−1) of MUA-AuNCs or MUAAuNOs was measured using inductively-coupled plasma mass spectrometry (ICP-MS) using an instrument from XSENIES (Palo alto, California, USA). Transmission electron microscopy (TEM) (using an H7650 instrument) and scanning electron microscopy (SEM) [using a SIRION-100 field emission instrument (FEI)] were performed to determine the structure and dimensions of MUA-AuNCs or MUA-AuNOs, respectively. The size distribution of MUA-AuNCs and MUA-AuNOs was derived from TEM and SEM micrographs using Image J software (National Institute of Health, Maryland, USA) according to our previous work, with at least 500 particles per sample [26]. The hydrodynamic diameters and zeta potentials of AuNCs and AuNOs were obtained using a Zetasizer Nano instrument (Malvern, Malvern, England). UV–vis spectra were measured using a Shimadzu UV-3600 UV–vis-near-infrared (NIR) spectrophotometer (Shimadzu, Kyoto, Japan). 2.4. Cell isolation and culture Bone marrow mesenchymal stem cells (bMSCs) were isolated from bone marrow from Sprague Dawley rats (6―8 weeks old) as reported previously [20,27], and routinely cultured in low-glucose cell culture medium containing 5% FBS and 1% penicillin-streptomycin (Sigma). All animal experiments were conducted in accordance with the Third Military Medical University (Army Medical University) for the Care and Use of Laboratory Animals. When ˜70―80% confluence was reached, we detached bMSCs, and then sub-cultured them serially. Cells at passage 2 (P2) were employed in the present work.
2. Experimental 2.5. Cell viability and proliferation
2.1. Materials
The viability and proliferation of bMSCs were probed by CCK-8 assays according to the manufacturer’s protocol (Beyotime). Briefly, bMSCs were cultured at a density of 1 × 104 cells/cm2 in a 48-well plate for 24 h, and then co-cultured with various Au concentrations (5―100 μg·mL−1). We replaced the culture medium with fresh NPcontaining medium every 3 days. After treatment for the indicated time, plates were washed three times with phosphate-buffered saline (PBS), CCK-8 reagent was added to each well and incubated for 2 h, and the absorbance/optical density (OD) at 450 nm (OD450nm) was measured using a Varioskan Flash microplate reader (Thermo Scientific, Massachusetts, USA). For cell proliferation assessment, bMSCs were incubated with MUA-AuNCs or MUA-AuNOs at an Au concentration of 5 or 25 μg·mL-1 for 1, 2 or 3 days, and viability was also measured using CCK-8 assays as above described.
Gold(III) chloride hydrate (HAuCl4) and sodium citrate (C6H5Na3O7·2H2O) were obtained from Sinopharm group Co. Ltd (Shanghai, China), and Aladdin (Shanghai, China) supplied 11-mercaptoundecanoic acid (MUA) and 1,5-pentanediol (96%) (PD). Alizarin Red S (ARS), polyvinylpyrrolidone (PVP, Mw ∼55 kDa) and Oil Red O were obtained from Sigma-Aldrich. KeyGEN Co. Ltd. (Nanjing, China) supplied an alkaline phosphatase kit, and HyClone (Logan, Utah, USA) provided a PicoGreen dsDNA kit, fetal bovine serum (FBS), and lowglucose Dulbecco’s modified Eagle’s medium (LDMEM). A cell counting Kit-8 (CCK8) and 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) were bought from Beyotime (Shanghai, China) and DOJINDO Company (Kumamoto, kyushu island, Japan), respectively. Milli-Q water was used throughout all experiments. 2.2. Synthesis of MUA-functionalized gold nanocubes (MUA-AuNCs) and gold nanooctahedras (MUA-AuNOs)
2.6. Cellular uptake of MUA-AuNCs and MUA-AuNOs
AuNCs and AuNOs were synthesised through a high-temperature polyol process according to our previous work [26]. AuNC and AuNO solutions were centrifuged at 5000 rpm for 5 min at 10 °C, the supernatant was transferred to a new tube to remove large particles and
The amount of MUA-AuNCs or MUA-AuNOs internalised by bMSCs was determined by ICP-MS according to our previously reported protocol. The amount of Au per 104 cells based on ICP-MS analysis is presented as the mean ± standard deviation (SD). 2
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2.7. Measurement of reactive oxygen species (ROS)
Table 1 Characterisation of MUA-AuNCs and MUA-AuNOs (mean ± SD, n = 4).
After seeding bMSCs on a 6-well plate at a density of 2 × 105 cells·cm−2 and attaching for 48 h, cells were treated with MUA-AuNCs or MUA-AuNOs (5 μg·mL-1) for 7 or 21 days (the medium was replaced with fresh NP-containing medium every 3 days). Cells were then washed three times with PBS and incubated with 1 mL of 10 μM DCFH-DA for 20 min, washed three times with culture medium without FBS to remove excess extracellular DCFH-DA, and imaged using a DM 6000 B fluorescence microscope (Leica, Wetzlar, Germany).
a
Nanoparticles
Length (nm)
MUA-AuNCs MUA-AuNOs
89 ± 12 50 ± 9
a
Diameter (DLS, Z-average, nm)
Zeta potential (mV)
437.8 ± 36.1 398.9 ± 58.4
−13.3 ± 1.6 −12.4 ± 0.7
Length refers to the edge length of MUA-AuNCs or MUA-AuNOs.
(4 °C), lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA), and finally incubated with specific antibodies overnight; an enhanced chemiluminescence system with ECL Western Blotting Substrate (Pierce, USA) was employed for detection. Image Jsoftware was employed for integral optical density (IOD) determination. Expression profiles of osteocalcin (OCN) and collagen type I (COL I), both related to osteogenic differentiation, were determined by RT-PCR. Sequences of primers used in this study are listed in Table 1. The 18S ribosomal subunit housekeeping gene served as an endogenous reference.
2.8. Measuring alkaline phosphatase (ALP) activity After culturing with MUA-AuNCs or MUA-AuNOs (5 or 25 μg·mL−1) for 7 days and three washes with PBS, bMSCs were treated with 0.5% (v/v) Triton X-100 (Sigma) at 4 °C for 24 h. Samples were then mixed with the appropriate colorimetric kit reagents (KeyGEN Biotech) according to the manufacturer’s instructions, the OD450nm value was recorded using a microplate reader, and a calibration curve was used to calculate ALP activity. A bicinchoninic acid assay kit (KeyGEN Biotech) was employed for determining the total protein content according to the manufacturer’s instructions, and the results presented as ALP activity per g of protein.
2.12. Statistical analysis
2.9. Observation of cell morphology
Results are expressed as mean ± SD, and significant differences between groups were analysed by one-way analysis of variance (ANOVA) for two groups and two-way ANOVA (for more than two groups) using Origin software. Statistical significance was set as p < 0.05 and p < 0.01, respectively.
After co-culturing with MUA-AuNCs or MUA-AuNOs for 7 or 14 days, bMSCs were washed three times with PBS, fixed with 4% formaldehyde solution at room temperature for 30 min, treated with 0.5% Triton X-100/PBS at 4 °C for 10 min, and then washed with PBS three more times. Following treatment with 1% BSA/PBS for 6 h to block nonspecific adsorption, cell nuclei were stained with 100 ng·mL−1 4′,6diamidino-2-phenylindole (DAPI) and the cytoskeleton (F-actin) was stained with 0.2 μM rhodamine-labelled phalloidin solution (Life Technologies) at 37 °C for 1 h. After washing three times with PBS, cells were observed by confocal laser-scanning microscopy (CLSM) using an LSM 510 instrument (Carl Zeiss, Oberkochen, Germany).
3. Results and discussion 3.1. Characterisation of nanoparticles TEM and SEM images showed successful synthesis of MUA-AuNCs and MUA-AuNOs, which had the desired structures (Fig. 1a, b, c, d) and a narrow morphological distribution (Fig. 1e). MUA-AuNCs were 89 ± 12 nm on each side, and MUA-AuNOs edges were 50 ± 9 nm in length (Table 1). The hydrodynamic diameters of MUA-auNCs and MUA-AuNOs were 437.8 ± 36.1 nm and 398.9 ± 58.4 nm, respectively (Table 1). The zeta potential of MUA-AuNCs and MUA-AuNOs was -13.3 ± 1.6 mV and -12.4 ± 0.7 mV, respectively (Table 1). The coherent oscillation of conduction electrons in the gold surface facilitates analysis by surface plasmon resonance (SPR) because of its high sensitivity to the local dielectric environment. The results revealed a red shift in the SPR peak during ligand exchange, at 566 nm for MUAAuNCs and 582 nm for MUA-AuNOs (Fig. 1f). A degree of peak broadening was evident, possibly indicating a slight rounding of NP vertices during the ligand exchange process, because surface plasmon resonance is known to be highly sensitive to the relative sharpness of corners in anisotropic particles [28]. There were significant changes in SPR signals in 10% FBS/LDMEM (Figure S1), indicating good colloidal stability for both MUA-AuNCs and MUA-AuNOs under the cell culture conditions. These results indicate that MUA-AuNCs and MUA-AuNOs essentially shared identical physiochemical properties except for structure, allowing us to correlate cellular behaviour with different nanostructures.
2.10. Analysis of cell differentiation For analysis of cell differentiation, bMSCs were initially at a density of 1.5 × 104 cells/well into 48-well plates attached by incubating overnight. Fresh medium containing 10% FBS/DMEM and MUA-AuNCs or MUA-AuNOs (5 μg·mL−1) was used to replace the overnight medium, and this was changed every 3 days to maintain a stable particle concentration throughout the entire experimental period. After 14 days of culturing and three washes with PBS, bMSCs were fixed in 4% paraformaldehyde for a minimum of 30 min at room temperature, and stained with 1% (w/v) aqueous ARS to detect calcium deposition and evaluate osteogenic differentiation, and 0.6% (w/v) Oil Red O solution (3:2 isopropanol:water) to detect lipid droplets and evaluate adipogenic differentiation, both at room temperature for 15 min. Cells were subsequently washed three times with PBS and observed under an IX81 bright field microscope (Olympus, Tokyo, Japan). ARS dissolved in 10% (w/v) hexadecylpyridinium chloride in PBS (pH 7.4) was used for quantification of calcium deposition by measuring the absorbance at 570 nm using a Model 680 microplate reader (Bio-Rad, California, USA). Data were normalised against particle-free control samples. 2.11. Western blotting (WB) and real-time quantitative reversetranscription PCR (RT-PCR)
3.2. Differentiation of bMSCs Before investigating cell differentiation, the effects of MUA-AuNCs and MUA-AuNOs dosage on cell viability and proliferation were analysed using CCK-8 assays, and no significant cytotoxicity was observed with either NP type, even after treatment at an Au concentration of 100 μg·mL−1 for 24 h (Fig. 2a). Cell differentiation proceeds as a series of
After culturing with MUA-AuNCs or MUA-AuNOs for 21 days, we washed bMSCs with PBS three times and then completely homogenized them in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. After being centrifuged for 15 min at 12,000 rpm 3
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Fig. 1. Characterisation of MUA-AuNCs and MUA-AuNOs. Representative TEM images (a, b) and SEM images (c, d) of (a, c) MUA-AuNCs and (b, d) MUA-AuNOs, respectively. (e) Hydrodynamic diameter of MUA-AuNCs and MUA-AuNOs. (f) UV–vis-NIR spectra of MUA-AuNCs and MUA-AuNOs in PBS (pH 7.4, 10 mM).
osteogenic differentiation. Differentiation of pluripotent progenitor bMSCs into osteoblasts is a crucial step during osteogenesis, and alkaline phosphatase (ALP) activity can serve as an osteogenic marker of the early stages of osteogenesis differentiation in bMSCs (typically up to 7 days). Meanwhile, mineralized nodule formation serves as a phenotypic marker for the latter stages upon formation of mature osteoblasts. Thus, ALP activity was quantified and normalized against total protein content to further explore the influence of NP structure on bMSC differentiation. Compared with controls, ALP activity of bMSCs was significantly decreased following treatment with MUA-AuNCs or MUAAuNOs, and this osteo-inhibitive effect was dose-dependent regardless of NP structure (Fig. 2c). Moreover, inhibition of osteogenic differentiation by the particles was also dependent on structure; ALP activity was significantly lower after treatment of bMSCs with MUA-AuNOs
sequential events that follow initial cell proliferation. Based on these results, two relatively low concentrations of MUA-AuNCs or MUAAuNOs (5 and 25 μg·mL−1) were selected as the appropriate gold NP dose for further studies on the influence of NP structure on differentiation of bMSCs. Cell differentiation is usually achieved by day 21, according to the existing literature and our previous experience, so this timeframe was adopted for culturing in this work. Osteogenic tissues can form extracellular matrix and regulate mineralisation [29]. Therefore, after culturing for 21 days, calcium deposition can serve as an indicator of osteogenic differentiation [30,31]. We therefore measured this parameter using ARS staining [32], and the amount of adsorbed dye (OD) is positively correlated with calcium content. As shown in Fig. 2b, the presence of either MUA-AuNCs or MUA-AuNOs inhibited calcium deposition, indicating suppression of
Fig. 2. (a) Viability of bMSCs treated with MUA-AuNCs or MUA-AuNOs for 24 h. (b) ARS staining (21 days), (c) ALP activity (7 days), and (d) Oil Red O staining (21 days) of bMSCs after culturing with MUA-AuNCs or MUA-AuNOs for the indicated time. 4
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Fig. 3. Western blotting and relative gene expression analysis. (a) Western blotting analysis of OCN, COL I, PPARγ and Fabp4 expression in bMSCs treated with MUA-AuNCs or MUA-AuNOs for 21 days. GAPDH served as a loading control. (b) Relative gene expression of osteogenic markers (OCN and COL I) and adipogenic markers (PPARγ and Fabp4) in bMSCs cultured for 21 days. Error bars represent mean ± standard deviation (SD; n = 4). * and ** denote significant differences at p < 0.05 and p < 0.01, respectively.
cell surface-attached particles present in small quantities). Based on existing literature and our prior experience, cellular loading of gold particles generally reaches equilibrium within 24 h [37,38], hence this timeframe was adopted in the present work. As shown in Fig. 5a and b, cell endocytosis of both MUA-AuNCs or MUA-AuNOs increased significantly (p < 0.01) with an increasing concentration of gold nanoparticles. Even with a fivefold higher concentration of gold NPs, endocytosis of NPs increased by only 30%, which could be attributed to the limited saturation level [39]; a similar trend was observed previously [40]. Additionally, cellular uptake was comparable for MUA-AuNCs and MUA-AuNOs (p > 0.05). These results suggest that the particles may bind to cytoplasmic proteins and/or interact with the cell membrane, resulting in mechanical stress that activates related signalling pathways regulating the expression of genes that induce adipogenic differentiation and suppress osteogenic differentiation. The effects of NPs on the differentiation potential of stem cells have received considerable attention in recent years [13,41,42]. Indeed, NPs are now considered a new class of differentiation activators that act through oxidative stress and various other pathways. ROS production is a major cause of cytotoxicity associated with most NPs characterised to date, and is considered a typical side-effect of NP exposure. NPs stimulate ROS production through various mechanisms, and interactions can damage mitochondrial membranes, disrupt the respiratory chain, and consequently elevate ROS production. Direct interactions between NPs and intracellular enzymes may disturb the cellular redox potential, and NP-integrin interactions may activate intracellular signalling pathways triggering ROS generation. Degradation of NPs may release metal ions and other byproducts that stimulate ROS release. Altering intracellular ROS levels is a general mechanism for regulating differentiation, but lineage-specific regulation of differentiation requires further investigation. Even so, accumulated evidence indicates that high levels of ROS may promote adipogenic differentiation of stem cells, but impede osteogenesis [18,43]. We therefore probed intracellular ROS generation using DCFH-DA, with which the fluorescence intensity of cells increases in the presence of ROS. Compared with controls, ROS levels were clearly elevated in bMSCs treated with MUAAuNCs and MUA-AuNOs. In addition, MUA-AuNOs triggered much greater ROS levels than MUA-AuNCs, indicating that intracellular ROS generation triggered by the gold particles is structure-dependent (Fig. 6). Mesenchymal stem cellular internalization of NPs and the mechanism underlying differentiation induced by NPs are dependent on the physicochemical properties of NPs, including size, shape, hydrophobicity, softness, composition, and surface chemistry [44]. Here, two types of MUA-AuNP had similar cellular internalization, hydrodynamic
than with MUA-AuNCs (p < 0.01; Fig. 2c). Accumulation of lipid-rich vacuoles within cells is an established marker of adipogenic differentiation in bMSCs. Therefore, formation of lipid droplets was characterised using Oil Red O staining after 21 days of culturing, given the positive correlation between the amount of adsorbed dye (OD) and lipid droplet content. As shown in Fig. 2d, the lipid droplet content in bMSCs was significantly higher after treatment with MUA-AuNCs or MUA-AuNOs (p < 0.01), suggesting that both particle types can promote adipogenic differentiation. Moreover, MUA-AuNOs exhibited a stronger capacity to enhance adipogenic differentiation. Furthermore, the gene and protein expression levels of several markers of the latter stages of differentiation were examined. Collagen type I (COL I) and osteocalcin (OCN) play important roles in osteogenic differentiation; COL is abundant in the organic/inorganic composite matrix of bone tissue [33,34], and OCN is a noncollagenous protein rich in the bone matrix essential for bone formation and remodelling [35]. Herein, data were normalized against expression of 18S (PCR) and βactin (WB) as internal standards. As shown in Fig. 3a and b, compared with control groups, COL I and OCN expression were significantly decreased (p < 0.01) at both mRNA and protein levels following treatment with either MUA-AuNCs or MUA-AuNOs, indicating that both particles exhibited osteo-inhibitive effects. Again, MUA-AuNOs displayed stronger osteogenesis inhibition ability. Peroxisome proliferator-activated receptor-gamma (PPARγ) and lipoprotein lipase (LPL), which are also important in adipocyte differentiation, can stimulate lipid uptake and adipogenesis by fat cells [36]. In the presence of MUA-AuNCs or MUA-AuNOs, the mRNA and protein expression of both LPL and PPARγ was significantly increased (p < 0.01) in bMSCs after culturing for 21 days (Fig. 3c and d). Therefore, we can conclude that both MUA-AuNCs and MUA-AuNOs can trigger adipogenic differentiation of bMSCs, and MUA-AuNOs are more potent for promoting adipogenic differentiation. Stem cell differentiation proceeds after initial cell proliferation, and MUA-AuNCs and MUA-AuNOs might act by stimulating osteogenic and/or adipogenic differentiation of bMSCs. As shown in Fig. 4a, the OD value increased almost linearly over 3 days, regardless of the presence of MUA-AuNCs or MUA-AuNOs, consistent with normal cell proliferation. Moreover, absolute values in the presence of MUA-AuNCs or MUA-AuNOs were similar to those of controls after 1, 2 and 3 days (p > 0.05), indicating no significant impairment of metabolic activity or cell proliferation. Additionally, neither MUA-AuNCs nor MUAAuNOs influenced the structure of MSCs after 7 or 14 days (Fig. 4b), indicating cytoskeletal integrity. Since the biological activities of particles may also be associated with differences in cellular uptake, the content of elemental gold was determined by ICP-MS to quantify particle internalisation (including 5
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Fig. 4. The influence of gold nanostructure (MUAAuNCs or MUA-AuNOs) on the proliferation and morphology of bMSCs. (a) Dose-dependent cell proliferation assay of the effects of MUA-AuNCs and MUAAuNOs. (b) CLSM images of bMSCs exposed to MUAAuNCs or MUA-AuNOs. Cells were co-cultured with particles for 7 or 14 days. Nuclei (blue) and the cytoskeleton (F-actin; red) were stained with DAPI and rhodamine phalloidin, respectively. Scale bar = 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 5. (a) Inverted microscope images of bMSCs treated with MUA-AuNCs or MUA-AuNOs for 24 h. Red arrows represent gold nanoparticles (MUA-AuNCs or MUA-AuNOs). (b) Cellular uptake of MUA-AuNCs and MUA-AuNOs quantified by ICP-MS. Error bars represent mean ± SD (n = 4). * and ** denote significant differences at p < 0.05 and p < 0.01, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
cytoskeletal integrity of MSCs inhibited osteogenic differentiation. These results correlate well with the structure-dependent stem cell differentiation behaviour described above, and imply that ROS generation triggered by MUA-AuNCs and MUA-AuNOs is a primary factor regulating stem cell differentiation. NPs can provoke ROS generation through multiple interactions. First, the interaction between NPs and mitochondria interaction may induce mitochondrial membrane damage, thus leading to disruption of the respiratory chain and increased ROS production. Second, the direct
diameters, surface chemistry, surface charge, and so on, and only their structures were different. Mechanical stress also plays a vital role in the differentiation potential change of stem cells [13]. The accumulation of NPs inside cells may cause changes in the cell cytoskeleton and cellular mechanical properties [45]. Increasing evidence indicates that high mechanical properties promoted osteogenic differentiation but inhibited adipogenic differentiation of stem cells [46]. Although our results are opposite in that none of our MUA-AuNPs influenced the structure of MSCs, the conclusion seems to be the same in that 6
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Fig. 6. Oxidative stress responses of bMSCs to MUA-AuNCs and MUA-AuNOs. The fluorescence microscope images indicate ROS levels following culturing with MUAAuNCs or MUA-AuNOs for 7 or 14 days. Scale bar = 200 μm.
Fig. 7. Schematic diagram of the effects of gold nanostructures on stem cell differentiation.
4. Conclusion
interactions between NPs and intracellular enzymes could maintain cellular redox potential. Furthermore, NP–integrin interaction could also activate the intracellular signalling, in turn triggering ROS generation. The degradation byproducts of NPs such as metal ions can also induce ROS production [13]. But why was the ROS induced by MUAAuNPs structure-dependent, and MUA-AuNOs elicited more potent effects? We speculate that shapes with more edges and corner regions have more potential for interaction of MUA-AuNPs with MSCs. Li, however, discovered that sphere-40 with fewer edges and corner regions than sphere-70 elicited more promotion of adipogenic differentiation of human mesenchymal stem cells [16]. The inconsistency of conclusions is probably attributable to the different surface chemistry of the NPs, the different sizes of the NPs, the different types of cells, and so on. In addition, the fate of stem cells may be substantially influenced by the surface protein adsorption and the potential cytotoxicity (e.g., lysosomal dysfunction) induced by NPs [13]. Cell differentiation mediated by NPs is a very complicated process involving many possible mechanisms and signalling pathways. Future efforts are needed for a full understanding of the mechanisms for stem cell differentiation induced by NPs.
Two types of nano-sized MUA-AuNPs with similar hydrodynamic diameters (437.8 ± 36.1 nm vs. 398.9 ± 58.4 nm), surface chemistry, and surface charge (-13.3 ± 1.6 mV vs. -12.4 ± 0.7 mV), but different structures were prepared. Both inhibition of osteogenic differentiation and promotion of adipogenic differentiation of bMSCs induced by MUAAuNCs and MUA-AuNOs were structure-dependent, and MUA-AuNOs elicited more potent effects. It is highly probable that accumulation of MUA-AuNOs triggered greater ROS generation, resulting in more extensive adipogenic differentiation of bMSCs (Fig. 7). These findings may support the rational design of new nanomaterials that can be applied to a broad range of biomedical areas.
Author contributions YL and QXW carried out most experiments and wrote the paper, QGP and LQ participated in the analysis of experimental data, and ZF, SY and DJ provided supervision and guidance and participated in manuscript drafting and language correction. All authors read and 7
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approved the final manuscript.
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