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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: J Biomed Mater Res A. 2017 Sep 19;105(12):3350–3359. doi: 10.1002/jbm.a.36200

SPIO-Au core-shell nanoparticles for promoting osteogenic differentiation of MC3T3-E1 cells: concentration-dependence study

Muzhaozi Yuan 1, Ya Wang 2,†, Yi-Xian Qin 3,*
PMCID: PMC5761339  NIHMSID: NIHMS929155  PMID: 28869707

Abstract

This work aims to explore the concentration-dependence of SPIO-Au core-shell NPs (17.3 ± 1.2 nm in diameter) on biocompatibility and osteogenic differentiation of preosteoblast MC3T3-E1 cells. The stability of NPs was first investigated by UV-Vis absorption spectra and zeta potential measurement. Then concentration effects of NPs (1–80 μg/mL) were evaluated on viability, morphology, proliferation, cellular uptake and alkaline phosphate (ALP) activity levels. Results have shown strong stability and no acute toxicity (viability > 93%) or morphological difference at all concentration levels of NPs. The proliferation results indicated that the concentration of NPs below 40 μg/mL does not affect the cell proliferation for 7 days of incubation. Transmission electron microscopy (TEM) images revealed the successful internalization of NPs into MC3T3-E1 cells and the dose-dependent accumulation of NPs inside the cytoplasm. The ALP level of MC3T3-E1 cells was improved by 49% (of control) after treated with NPs at 10 μg/mL for 10 days, indicating their positive effect on early osteogenic differentiation. This study confirmed the excellent biocompatibility of SPIO-Au NPs and their great potential for promoting osteogenic differentiation and promised the future application for these NPs in bone engineering including drug delivery, cell labeling and activity tracking within scaffolds.

Keywords: SPIO-Au core-shell nanoparticles, osteogenic differentiaiton, MC3T3 E1 cells, concentration-dependence study, cell uptake

Introduction

Nanoscale particles (NPs) have attracted considerable attention in tissue engineering, because of their special magnetic, optical and biochemical properties compared with bulk materials.1–3 Recently, an increasing number of in vitro and in vivo works have been done to explore the potential applications of NPs in bone tissue engineering.1,4 For example, nanomaterial scaffolds have been extensively developed to mimic the structure of natural extracellular matrices and to provide a 3-dimentional (3D) network and sufficient support for cell growth.5,6 As a highly sensitive contrast agent,7,8 superparamagnetic iron oxide (SPIO) NPs have been used to label various kinds of cells such as chondrocytes,9 mesenchymal stem cells (MSCs)10 and adipose derived stem cells (ADSCs).11 By the effective labeling with SPIO NPs, the localization of cells inside the scaffolds can be noninvasively visualized using Magnetic Resonance Imaging (MRI).12 SPIO NPs can also be combined with transfection agents like poly-L-lysine and lipofectamine to enhance their cellular uptake into chondrocyte without affecting cells’ phenotype and viability.9 C. Lalande et al.11 have labeled human ADSCs by ultra small SPIO within scaffolds and obtained high contrast T2-weighted images even at low cell density. They were able to detect cells for up to 28 days after implantation. Besides their potential applications as contrast agents, SPIO NPs can also be directed to a specific site by external magnetic fields, which further extends their usage in targeted drug or gene delivery.13

Despite their unique properties, uncoated magnetic NPs have disadvantages including the instability in biological media14 and the cellular toxicity.15 One way to overcome this is to coat them using biocompatible materials, which not only shield the magnetic core from being exposed to surroundings but also make them to be readily functionalized with different groups.3 Among various coating materials, gold (Au) exhibits excellent biocompatibility and low cytotoxicity because of its inertness and stability.16,17 The tunable surface functionalization of Au NPs due to the Au-S chemistry further extends their applications to the fields of gene delivery,18,19 fluorescence imaging,20 cell labeling21 and bio sensing.22 In particular, Au NPs were reported to be alternative osteogenic inductive agents in bone tissue engineering, as they were able to accelerate the osteogenic differentiation of MSCs by stimulating the p38 mitogen activated protein kinase (MAPK) signaling pathway in the cells when interacting with certain proteins inside the cytoplasm.23 This effect of Au NPs was also reported in ADSCs by Dong et al.24 They found the Au NPs in a hydrogel network is capable of promoting the ALP activity level similar to bone morphogenic proteins (BMPs) while overcome the BMPs’ drawbacks such as high cost, local inflammation and unwanted bone formation.25

SPIO-Au core-shell nanoparticle has such a unique composite nanostructure that possesses the magnetic property of SPIO NPs and the surface properties of Au NPs. The magnetic nature of the SPIO core promises the application of this material in MRI,7,26 while the Au shell effectively enhance the biocompatibility besides the aforementioned benefits. The potential application of these NPs in bone tissue engineering is strong. However, their concentration-dependent impact on biocompatibility and osteogenic differentiation has not yet been extensively studied, which will be the focus of this paper. An preosteoblast cell line MC3T3-E1 from mouse27 is chosen as an in-vitro model. The effects of SPIO-Au NPs on cell viability, proliferation, cell uptake and osteogenic differentiation are studied at different concentration levels.

Materials and Methods

Synthesis of SPIO-Au NPs

Briefly, SPIO-Au core-shell NPs (17.3 ± 1.2 nm) were synthesized by the seed growth method28,29 with sodium citrate as a reducing agent to form a Au coating on a SPIO core (10 nm). In this reaction, 0.02 mL of SPIO (EMG-304 (10 nm), Ferrotech, Santa Clara, CA) aqueous solution (0.931 M) was diluted to 3.724 mM by adding 4.98 mL of Deionized (DI) water and then sonicated for 5 minutes. 0.1 mL of diluted SPIO solution (3.724 mM) was then added into 30 mL of DI water and heated to 90°C under vigorous mechanical stirring. Next, 1.1 mL of 1% sodium citrate (Sigma-Aldrich, St. Louis, MO) and 0.5 mL of 1% HAuCl4 (Sigma-Aldrich, St. Louis, MO) were sequentially added to the solution. During the reaction, the color of the solution changed from light brown to deep red in 30 min. Then, the resulting solution was used as a seed solution and the citrate reducing process was repeated again to produce a thicker Au coating. The as-prepared SPIO-Au NPs were directly used in the following experiments without further purification.

Characterization of SPIO-Au NPs

To observe the morphology of the synthesized SPIO-Au NPs, the Transmission electron microscopy (TEM) imaging was performed on a JEOL JEM 1400 Transmission Electron Microscope at an operating voltage of 120 kV. Briefly, 100 μL droplets of each sample were dropped onto a 300-mesh copper grid (Ted Pella Inc., Redding, CA) and then left to dry in the air. To examine the influence of the cell growth medium on the stabilities of NPs, the UV-Vis absorption spectroscopy and the zeta potential characterization were conducted. The solution of SPIO-Au NPs at the concentration of 80 μg/mL was suspended in MC3T3-E1 cell growth medium and in deionized water, respectively. At different time points, the light absorption spectra were recorded at wavelengths between 400 nm and 800 nm at room temperature with a FLAME-S-XR1-ES spectrometer and the SpectraSuite software from Ocean Optics Inc. The zeta potential test was performed with a Malvern Zetasizer Nano ZS (Malvern Instruments Inc.) at 25 °C.

Cell culture

MC3T3-E1 cells at passage-8 were cultured in Petri dishes with cell growth medium, which consisted of 89% alpha Minimum Essential Medium (αMEM) (Gibco, Grand Island, NY), 10% fetal bovine serum (Gibco, Grand Island, NY) and 1% penicillin streptomycin solution (Gibco, Grand Island, NY). The cells were kept in a 5% CO2 atmosphere at 37°C. The cell growth medium was replaced every two days and the cells were passaged at sub-confluency.

Cell viability assessment and cell morphology observation

The cell viability test was performed by using a Cellstain double staining kit (04511, Sigma-Aldrich, St. Louis, MO). Briefly, MC3T3-E1 cells were seeded into 96-well tissue culture plates at a density of 1000 cells per well with growth medium. After 24 h of incubation, the medium was replaced by fresh growth medium containing SPIO-Au NPs at different concentrations (10, 20, 40, and 80 μg/mL). Cells without NPs were seeded as a control group. All the cells were incubated for 1, 3 and 7 day(s) for further testing. At each time period, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) twice and incubated in 100 μL of the assay solution (5 mL of DPBS containing 10 μL of calcein-AM and 5 μL of propidium iodide) in each well for 15 min. The live/dead fluorescence images were captured using an Axiovert 200M Inverted Fluorescence/phase Contrast Microscope (Carl Zeiss Inc.) equipped with an AxioCam CCD camera. The cell morphology was also observed and recorded with this inverted microscope after 3 and 7 days of incubation.

Cell proliferation assay

The proliferation of MC3T3-E1 cells was measured using a CCK-8 cell counting kit (Sigma-Aldrich, St. Louis, MO), following procedures detailed in the previous work.24 MC3T3-E1 cells were seeded into 96-well tissue culture plates at a density of 1000 cells per well with growth medium. After 24 h of incubation, the medium was replaced by fresh medium containing SPIO-Au NPs at different concentrations (1, 5, 10, 20, 40 and 80 μg/mL). Cells without NPs were seeded as a control group. All the cells were incubated for 1, 3 and 7 day(s). At each time period, the cells were washed with DPBS and followed by adding 100 μL of growth medium and 10 μL of CCK-8 solution to each well. Then after 2 h of incubation the intensity was measured at the wavelength of 450 nm by using a microplate reader (SpectraMax i3x, Molecular Devices Inc.).

Transmission electron microscopy (TEM)

To observe the cell uptake of SPIO-Au NPs in MC3T3-E1 cells, cells were seeded on a Aclar® (Electron Microscopy Sciences Inc.) film in a 12-well plate at a density of 1*105 cells per well and incubated with growth medium containing SPIO-Au NPs at concentrations of 40 μg/mL and 80 μg/mL, respectively. After 48 h of incubation, cells were prefixed in 3% Electron Microscopy (EM) grade glutaraldehyde in 0.1 M phosphate buffer saline (PBS), pH 7.4 at room temperature for 1 h. Cells were then fixed in 1% osmium tetroxide in 0.1 M PBS, pH 7.4. Followed by the dehydration in a graded series of ethyl alcohol and embedded in Durcupan resin (Sigma-Aldrich, St. Louis, MO). Ultrathin sections (80 nm) were cut with a Leica EM UC7 Ultramicrotome (Leica Microsystems Inc.) and collected on formvar coated copper slot grids. Then these sections were stained with uranyl acetate and lead citrate and then analyzed by using a FEI Tecnai12 BioTwinG2 transmission electron microscope with an AMT XR-60 CCD Digital Camera system, at an accelerating voltage of 80 kV.

Alkaline phosphatase activity (ALP) assay

To study the performance of SPIO-Au NPs in osteogenic differentiation of MC3T3-E1 cells, the ALP activity level was assessed by using an Abcam® Alkaline phosphatase assay kit (ab83369, Abcam, Cambridge, MA). MC3T3-E1 cells were seeded into a 24-well plate at a density of 5*104 cells per well with growth medium. After 24 h of incubation, the medium was changed to osteogenic induction medium, containing SPIO-Au NPs at different concentrations (0, 1, 5, 10 and 20 μg/mL). The osteogenic induction medium consisted of αMEM (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY), 1% penicillin (Gibco, Grand Island, NY), 10 mM disodium β–glycerophosphate (Sigma-Aldrich, St. Louis, MO), 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO) and 0.28 mM ascorbic acid (Sigma-Aldrich, St. Louis, MO). All the cells were incubated for 7, 10 and 14 days. At each time period, cells were harvested following the manufacturer’s instruction. The intensity was measured at the wavelength of 405 nm on a microplate reader (SpectraMax i3x, Molecular Devices Inc.). The amount of ALP in each well was calculated and normalized by total protein content, which was measured simultaneously using the Pierce™ Coomassie (Bradford) protein assay kit (Thermo Scientific, Rockford, IL).

Statistical analysis

All results were analyzed based on 3 independent experiments and expressed using the standard deviation. Statistical analysis was performed by the one-way analysis of variance (ANOVA) and Tukey post hoc test. Each sample group treated with NPs was compared with the control group. P value less than 0.05 was considered as a significant difference.

Results

Characteristics of SPIO-Au NPs: TEM image, UV-Vis spectra and Zeta Potential

The TEM image (Fig. 1) indicates that the as-synthesized NPs (17.3 ± 1.2 nm) exhibit mono-dispersed uniform quasi-spherical shape. The histogram depicting along with the TEM image shows the narrow size distribution.

Fig. 1.

Fig. 1

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Transmission Electron Microscopy (TEM) image of SPIO-Au NPs used for biocompatibility evaluation. The average particle size is 17.3 ± 1.2 nm calculated from at least 100 NPs shown in TEM images.

To further analyze the stability of SPIO-Au NPs in different medium, the UV-Vis absorption spectroscopy was performed accordingly. A strong absorbance peak of the SPIO-Au NPs in pure DI water was found at 524 nm (Fig. 2a), which is close to the characteristic Surface Plasmon Resonance (SPR) peak of the Au shell.16 This indicates the successful formation of SPIO-Au NPs. The unchanging absorbance spectra for up to 21 days of mixing time imply the high stability of NPs in DI water (Fig. 2a).

Fig. 2.

Fig. 2

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UV-Vis absorption spectra of SPIO-Au NPs at 80 μg/mL in (a) MC3T3-E1 cell growth medium for different time periods with a maximum absorbance at a wavelength of 553 nm and an obvious discrepancy of absorbance at a wavelength of 630 nm. (b) Deionized water for different time periods with a maximum absorbance at a wavelength of 520 nm (c) The spectrum of the cell growth medium only with a maximum absorbance at a wavelength of 560 nm, which explains the shift of the peak of the absorbance curve from (b) to (c).

In the cell growth medium, although a slight broadening of the peak with time is observed, no decrease of the absorbance peak is found even after 21 days (Fig. 2b). Therefore, one can conclude that our SPIO-Au NPs are stable in both the cell growth medium and the DI water for up to 21 days. Interestingly, the absorbance peak shifted to 555 nm for NPs dispersed in the cell growth medium from that of 524 nm for NPs dispersed in the DI water, which is due to the optical effect of the cell growth medium, whose absorbance peak was found at 555 nm (Fig. 2c). The measured zeta potential was changed from −31.7 mV (in DI water) to −11.9 mV (in cell growth medium), indicating the stability of NPs in the cell growth medium. The change of zeta potential can be explained by the surface absorption of serum proteins onto the NPs in the cell growth medium.

Cell viability of SPIO-Au NPs: Fluorescence images and statistics

The treatment of live/dead double staining was performed to assess the viability on cells treated with SPIO-Au NPs at concentrations of 10, 20 40 and 80 μg/mL for 1, 3 and 7 day(s) of incubation. As observed, all groups of cells exhibited high cell viability with scarcely any dead cells (red staining) in comparison to the control group (Fig. 3).

Fig. 3.

Fig. 3

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Fluorescence images for live/dead double staining tests after the cells were cultured with different concentrations of SPIO-Au NPs for 1, 3 and 7 day(s) in growth medium. Live and dead cells were stained by the calcein AM and the propidium iodide respectively. Green color represents viable cells while red color represents dead cells. Scale bar length = 200 μm. Direct observation of live cells and dead cells was provided to guarantee the accuracy.

The number of viable and dead cells in the fluorescence images was counted and plotted (Fig. 4). The percentage of live cells to the total cells showed no significant difference for 1 and 3 day(s) of incubation. Although a slight reduction of viability was observed at the highest concentration of 80 μg/mL for 7 days of incubation, SPIO-Au NPs still had 93% viability of MC3T3-E1 cells. No obvious cytotoxic effect was found with the treatment of SPIO-Au NPs.

Fig. 4.

Fig. 4

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Cell viability determined by fluorescence images of live/dead cells. The number of live cells and dead cells were counted by using Matlab. Cells containing no NPs were used as the control group. Viability higher than 93% was observed for each sample group. Slight reduction of viability happened at the highest concentration after 7 days incubation. ((***) p ≤ 0.001)

Cell morphology: time and concentration dependence study

The morphology of cells treated with the SPIO-Au NPs was observed by an inverted light microscope (Fig. 5). After 3 and 7 days of incubation, MC3T3-E1 cells were formed with spindle shapes. The increase of the number of cells was observed at a longer incubation period (7 days). No significant difference in morphology was observed between the cell groups treated with NPs and the control group at each time period.

Fig. 5.

Fig. 5

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Cell morphology images by using an inverted light microscopy after cells were treated with different concentrations of SPIO-Au NPs for 3 and 7 days of incubation. The increase of cell density was observed at a longer time period. Scale bar length = 100 μm.

Cell proliferation: Curve-fitted modeling and analysis

The proliferation of MC3T3-E1 cells treated with the SPIO-Au NPs at concentrations of 0, 1, 5, 10, 20, 40 and 80 μg/mL was determined at different time points up to 7 days (Fig. 6). The standard curve of Absorbance vs. Cell concentration was generated to reflect the relationship between the absorbance and the cell concentration by using a CCK-8 kit (Fig. 7). Results show that the absorbance was increased at higher concentration, which approximated a linear relationship when the concentration was below the threshold value of 1.5*105 cells/mL. Results of the proliferation assay in Fig. 6 showed a significant reduction of the proliferation rate for 3 days of incubation when the concentration reached 80 μg/mL. However, there was no significant decrease of the proliferation rate at lower concentration levels (≤ 40 μg/mL) for 3 days of incubation and at the whole concentration range (1–80 μg/mL) for 1 day and 7 days of incubation.

Fig. 6.

Fig. 6

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Proliferation of MC3T3-E1 cells treated with different concentrations of SPIO-Au NPs determined by a CCK-8 kit. Cells were cultured for up to 1, 3 and 7 day(s) in cell growth medium. Cells containing no NPs were used as the control group. ((***) p ≤ 0.001)

Fig. 7.

Fig. 7

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Standard curve of absorbance vs. cell concentration. The linear-like region was found at the concentration below 1.5*105 cells/mL. The curve fit was performed by SciDAVis software using Polyfit4 function.

Cellular uptake of SPIO-Au NPs: TEM analysis

To examine the cellular uptake of the SPIO-Au NPs, MC3T3-E1 cells were incubated for 48 h after treated with NPs at concentrations of 40 and 80 μg/mL, respectively. As shown in the TEM images (Fig. 8), the clusters of SPIO-Au NPs were found inside each MC3T3-E1 cell, indicating the successful internalization of NPs at both concentration levels.

Fig. 8.

Fig. 8

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Transmission electron microscopy (TEM) images of MC3T3-E1 cells treated with SPIO-Au NPs at 40 μg/mL (a, b, c) and 80 μg/mL (d, e, f) after 48 h of incubation. Note: (c) and (f) are enlarged images of areas shown inside the dashed circles in (b) and (e). The arrows in (a), (c), (d) and (f) showed the accumulation of SPIO-Au NPs inside the cellular cytoplasm. In particular, (a) and (d) illustrated how NPs penetrated into cells through endocytosis.

Interestingly, the internalized NPs mainly located inside the endosomal vesicles in the cytoplasm, rather than in the nucleus, as preferred. Larger clusters of NPs were found in the vesicles at a higher concentration (80 μg/mL), than that at a lower concentration (40 μg/mL). For both concentrations, some NP clusters were found in the endocytic pathway, suggesting that NPs penetrated into cells mainly through endocytosis.30,31

It can be readily seen how the plasma membrane engulfed the cluster of NPs (the areas pointed by arrows) before their penetration into the cytoplasm (Fig. 8a and 8d), which was the sign of early endocytosis.27 Moreover, examined from all the TEM images, the MC3T3-E1 cells treated with NPs at concentrations of 40 and 80 μg/mL had intact cytoplasmic membranes, indicating the excellent biocompatibility of SPIO-Au NPs.

Promoting the osteogenic differentiation of MC-3T3-E1 cells

The ALP activity assay was conducted to explore the concentration-dependence of SPIO-Au NPs on the osteogenic differentiation (Fig. 9a). Cells were treated with NPs at concentrations of 1, 5, 10, and 20 μg/mL in the osteogenic induction medium for 7, 10 and 14 days. It was found that the ALP level of MC3T3-E1 cells was affected by the treatment of NPs in a time and dose dependent manner. The significant increase of ALP level per gram of protein was displayed at 10 and 20 μg/mL for 14 days of incubation, with the maximum increase of ALP level by 49% at 10 μg/mL.

Fig. 9.

Fig. 9

Fig. 9

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a The Alkaline phosphatase activity level of MC3T3-E1 cells treated with different concentrations of SPIO-Au NPs for 7, 10 and 14 days of incubation (original data without normalization by protein content). Cells containing no NPs were used as the control group. ((**) p ≤ 0.01)

(b) The Alkaline phosphatase activity level of MC3T3-E1 cells treated with different concentrations of SPIO-Au NPs for 7, 10 and 14 days of incubation (Normalize by protein content). Cells containing no NPs were used as the control group. ((*) p ≤ 0.05)

Interestingly, the ALP level of groups treated with NPs was slightly reduced after 7 days of incubation, while promoted after 10 and 14 days of incubation. It was speculated that for a short time incubation (7 days), most NPs might not be able to interfere with the cells yet. The ALP level normalized by protein content exhibited the similar trend (Fig. 9b).

Discussion

Our goal is to evaluate the concentration-dependence of SPIO-Au NPs (17.3 ± 1.2 nm) on the osteogenic differentiation of the MC-3T3-E1 preosteoblast cell line. This work lays foundation for future applications of these NPs in tissue engineering, such as drug or gene delivery, cell labeling and tracking within scaffolds.

To date, many studies have been carried on to evaluate the excellent viability of Au NPs (4 nm, Ellen E et al.32 and 12 nm, Thikra et al.27), and their outstanding biocompatibility (Hela cells, Alexandre et al.33) and surface function (human MSCs, Kawazoe et al.34). However, very few works have been done to evaluate the biological effect of SPIO-Au NPs. The SPIO-Au core-shell NPs (36–56 nm, 1–500 μg/mL) have been proved to be nontoxic to mouse leukemic monocyte macrophage cells (> 90% viability, 3 days of incubation) by Zhang et al.,17 however, no viability studies were reported for incubation periods longer than 3 days and no proliferation evaluation was performed, which is essential to simulate the in-vivo situation for bone tissue engineering.30

Our results showed an excellent cell viability (≥ 93%) and stability for the SPIO-Au core-shell NPs at concentrations of 10, 20, 40 and 80 μg/mL, which were supported by the light absorbance measurement as well (Fig. 2).

Additionally, the cell morphology observation clearly exhibited the unchanged shape and size of cells treated with SPIO-Au NPs at all tested concentrations, and demonstrated that the near-zero influence of cellular internalization of NPs on the cellular morphology. This supports the aforementioned viability result. More morphological work in 3D network, i.e. scaffolds, could be performed in the near future.

The proliferation results proved the optimal concentration range of the NPs (≤ 40 μg/mL) and a saturation could be reached at much faster rate with higher NP concentrations (> 40 μg/mL). The inhibition of proliferation may happen when the concentration of NPs reaches 80 μg/mL, while interestingly the viability of cells still maintained above 93% for concentrations up to 80 μg/mL. This suggests that, longer incubation days (> 1 day) or higher NP concentration levels (> 40 μg/mL) are not necessary for obtaining optimal proliferation benefits. For instance, the treatment of NPs at 10 μg/mL could gain comparable proliferation rate to control group at 7 days of incubation.

TEM analysis provided a direct tool to observe the existence of NPs absorbed by MC3T3-E1 cells. The size and the number of the NP clusters shown in TEM images demonstrated the dose-dependence of cellular NPs uptake. The fact of NPs staying inside the vesicles within the cytoplasm structure implied the possible mechanism of cell internalization by endocytosis at the concentrations of 40 and 80 μg/mL, which was further supported by some TEM images showing the engulfing of NPs by cells (Fig. 8a and 8d). The zeta potential measurement showed that the surface charge of the SPIO-Au NPs is negative (−31.7mV in water) due to the existence of citric acid as the stabilizing ligand, while the surface charge was increased to −11.9 mV (in cell growth medium), which implies the surface modification of SPIO-Au NPs by the nonspecifically absorbed serum proteins. Therefore, we hypothesize that the access of NPs into cells was induced by serum proteins, which were absorbed onto the surface of SPIO-Au NPs, via receptor-mediated endocytosis. This mechanism has been proposed by B. Devika et al.35 and Thikra et al.27 for the study of the internalization of Au NPs by Hela cells and ME3T3-E1 cells, respectively. At the concentration as low as 10 μg/mL, Thikra et al.27 suggested that NPs may penetrate into the cells individually rather than through endocytosis. Moreover, the intact cell membranes indicated the excellent biocompatibility of SPIO-Au NPs, which has been proved before by viability results. We can also conclude that the uptake of NPs into cells did not induce any cellular toxicity (viability > 93%). In addition, the surface modification of SPIO-Au NPs also plays an important role in cell uptake as it determines the surface charge and the hydrophilicity36 of NPs. Eun37 found that the surface conjugation of different groups changed the amount of the NPs internalized by SK-BR-3 cells in the following order: poly(allyamine hydrochloride) > anti-HER2 > antibody > poly(ethylene glycol). To determine the best mechanism for cellular NP uptake and cellular kinetics, longer incubation time, more time points and necessary surface functionalization are needed for future study.

The results from viability, proliferation, morphology and TEM analysis indicated that SPIO-Au NPs did not induce acute toxicity on MC3T3-E1 cell line. Based on the biocompatibility evaluation, The ALP activity measurements were designed and performed to assess the potential of SPIO-Au NPs in osteogenic differentiation. The results exhibited the promoted effect of SPIO-Au NPs on the early osteogenic differentiation of MC3T3-E1 cells. Pure Au NPs have been shown to promote the osteogenic differentiation of ADSCs.24 However the examination of the SPIO-Au NPs core-shell on osteogenesis have not been mentioned in any of previous works. The presented work is the first to confirm the osteogenic effect of SPIO-Au NPs for the application in bone cell regeneration. The ALP level is known to be an early phenotypic marker for osteogenic differentiation.23 In the next stage, matrix mineralization and specific gene expression will be studied to further examine the effect and mechanism of SPIO-Au NPs on osteogenesis. To enhance their promotion effect on bone tissue regeneration, NPs can be embedded into scaffolds in the near future. In particular, composite scaffolds are able to provide enough surface roughness for cellular attachment and improve the mechanical strength of the system.38 Incorporating NPs with osteogenic agents,39 growth factors40 and drugs are some other promising strategies to promote cell differentiation and/or add more functionality to the scaffolds.41 Moreover, to enhance the contrast capability, SPIO-Au NPs can be combined with other type of nanocomposite scaffolds.42

Conclusions

In this work, we synthesized SPIO-Au core-shell NPs with uniform size (17.3 ± 1.2 nm), shape (spherical) and excellent stability in DI water and the MC3T3-E1 cell growth medium. Experimental results suggest that SPIO-Au NPs (10, 20, 40 and 80 μg/mL) didn’t induce acute toxicity for up to 7 days of incubation. The existence of SPIO-Au NPs does not affect the proliferation of MC3T3-E1 cells at the concentration range between 1 and 40 μg/mL. The TEM results revealed the cellular uptake of SPIO-Au NPs through endocytosis and the dose-dependence of internalization of NPs into cells. The enhanced ALP activity level of cells treated by NPs implied their potential in promoting the osteogenic differentiation of MC3T3-E1 cells. Further investigation of SPIO-Au NPs integrated with 3D scaffolds and/or growth factors as well as the in-vivo studies will be performed in the near future to explore their promising applications in bone tissue engineering.

Acknowledgments

The authors would like to thank the support of DOE-AR0000531 and ONR-N000141410230, and the support of NIH (R01 AR 52379 and AR 61821), and National Space Biomedical Research Institute through NASA Contract NCC 9–58. The authors would like to thank the Central Microscopy Imaging Center at Stony Brook University for their assistance of transmission electron microscopy in cell biology.

Contributor Information

Muzhaozi Yuan, Heavy Engineering 133, Department of Mechanical Engineering, State University of New York at Stony Brook, Stony Brook, NY 11794-2300, Tel: 631-891-5208 Fax:(631) 632-8544, Muzhaozi.yuan@stonybrook.edu

Ya Wang, Assistant Professor, LE 153, Department of Mechanical Engineering, State University of New York at Stony Brook, Stony Brook, NY 11794-2300.

Yi-Xian Qin, Professor, 215 Bioengineering Bldg., Dept. of Biomedical Engineering, State University of New York at Stony Brook, Stony Brook, NY 11794-5281.

References

  • 1.Wang Q, Yan J, Yang J, Li B. Nanomaterials promise better bone repair. Materials Today. 2016;19:451–463. [Google Scholar]
  • 2.Chen H, Zeng Y, Liu W, Zhao S, Wu J, Du Y. Multifaceted applications of nanomaterials in cell engineering and therapy. Biotechnology advances. 2013;31:638–653. doi: 10.1016/j.biotechadv.2012.08.002. [DOI] [PubMed] [Google Scholar]
  • 3.Mahmoudi M, Sant S, Wang B, Laurent S, Sen T. Superparamagnetic iron oxide nanoparticles (SPIONs): development, surface modification and applications in chemotherapy. Advanced drug delivery reviews. 2011;63:24–46. doi: 10.1016/j.addr.2010.05.006. [DOI] [PubMed] [Google Scholar]
  • 4.Tautzenberger A, Kovtun A, Ignatius A. Nanoparticles and their potential for application in bone. Int J Nanomedicine. 2012;7:4545–4557. doi: 10.2147/IJN.S34127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Zhao C, Tan A, Pastorin G, Ho HK. Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering. Biotechnology advances. 2013;31:654–668. doi: 10.1016/j.biotechadv.2012.08.001. [DOI] [PubMed] [Google Scholar]
  • 6.Li X, Wang L, Fan Y, Feng Q, Cui FZ, Watari F. Nanostructured scaffolds for bone tissue engineering. Journal of Biomedical Materials Research Part A. 2013;101:2424–2435. doi: 10.1002/jbm.a.34539. [DOI] [PubMed] [Google Scholar]
  • 7.Lee N, Hyeon T. Designed synthesis of uniformly sized iron oxide nanoparticles for efficient magnetic resonance imaging contrast agents. Chemical Society Reviews. 2012;41:2575–2589. doi: 10.1039/c1cs15248c. [DOI] [PubMed] [Google Scholar]
  • 8.Yoffe S, Leshuk T, Everett P, Gu F. Superparamagnetic iron oxide nanoparticles (SPIONs): synthesis and surface modification techniques for use with MRI and other biomedical applications. Current pharmaceutical design. 2013;19:493–509. [PubMed] [Google Scholar]
  • 9.Ramaswamy S, Greco JB, Uluer MC, Zhang Z, Zhang Z, Fishbein KW, Spencer RG. Magnetic resonance imaging of chondrocytes labeled with superparamagnetic iron oxide nanoparticles in tissue-engineered cartilage. Tissue Engineering Part A. 2009;15:3899–3910. doi: 10.1089/ten.tea.2008.0677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Saldanha KJ, Doan RP, Ainslie KM, Desai TA, Majumdar S. Micrometer-sized iron oxide particle labeling of mesenchymal stem cells for magnetic resonance imaging-based monitoring of cartilage tissue engineering. Magnetic resonance imaging. 2011;29:40–49. doi: 10.1016/j.mri.2010.07.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lalande C, Miraux S, Derkaoui SM, Mornet S, Bareille R, Fricain J-C, Franconi J-M, Le Visage C, Letourneur D, Amédée J. Magnetic resonance imaging tracking of human adipose derived stromal cells within three-dimensional scaffolds for bone tissue engineering. Eur Cell Mater. 2011;21:e54. doi: 10.22203/ecm.v021a25. [DOI] [PubMed] [Google Scholar]
  • 12.Nam SY, Ricles LM, Suggs LJ, Emelianov SY. Imaging strategies for tissue engineering applications. Tissue Engineering Part B: Reviews. 2014;21:88–102. doi: 10.1089/ten.teb.2014.0180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Singh N, Jenkins GJS, Asadi R, Doak SH. Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION) Nano Reviews & Experiments. 2010;1:5358. doi: 10.3402/nano.v1i0.5358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Laurent S, Forge D, Port M, Roch A, Robic C, Vander Elst L, Muller RN. Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chemical reviews. 2008;108:2064–2110. doi: 10.1021/cr068445e. [DOI] [PubMed] [Google Scholar]
  • 15.Mahmoudi M, Hofmann H, Rothen-Rutishauser B, Petri-Fink A. Assessing the in vitro and in vivo toxicity of superparamagnetic iron oxide nanoparticles. Chemical reviews. 2011;112:2323–2338. doi: 10.1021/cr2002596. [DOI] [PubMed] [Google Scholar]
  • 16.Xu Z, Hou Y, Sun S. Magnetic Core/Shell Fe3O4/Au and Fe3O4/Au/Ag Nanoparticles with Tunable Plasmonic Properties. Journal of the American Chemical Society. 2007;129:8698–8699. doi: 10.1021/ja073057v. [DOI] [PubMed] [Google Scholar]
  • 17.Zhang S, Qi Y, Yang H, Gong M, Zhang D, Zou L. Optimization of the composition of bimetallic core/shell Fe2O3/Au nanoparticles for MRI/CT dual-mode imaging. Journal of nanoparticle research. 2013;15:1–9. [Google Scholar]
  • 18.Kong L, Alves CS, Hou W, Qiu J, Möhwald H, Tomás H, Shi X. RGD peptide-modified dendrimer-entrapped gold nanoparticles enable highly efficient and specific gene delivery to stem cells. ACS applied materials & interfaces. 2015;7:4833–4843. doi: 10.1021/am508760w. [DOI] [PubMed] [Google Scholar]
  • 19.Muroski ME, Morgan TJ, Jr, Levenson CW, Strouse GF. A gold nanoparticle pentapeptide: gene fusion to induce therapeutic gene expression in mesenchymal stem cells. Journal of the American Chemical Society. 2014;136:14763–14771. doi: 10.1021/ja505190q. [DOI] [PubMed] [Google Scholar]
  • 20.Dreaden EC, Alkilany AM, Huang X, Murphy CJ, El-Sayed MA. The golden age: gold nanoparticles for biomedicine. Chemical Society Reviews. 2012;41:2740–2779. doi: 10.1039/c1cs15237h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ricles LM, Nam SY, Trevino EA, Emelianov SY, Suggs LJ. A dual gold nanoparticle system for mesenchymal stem cell tracking. Journal of Materials Chemistry B. 2014;2:8220–8230. doi: 10.1039/C4TB00975D. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Saha K, Agasti SS, Kim C, Li X, Rotello VM. Gold nanoparticles in chemical and biological sensing. Chemical reviews. 2012;112:2739–2779. doi: 10.1021/cr2001178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Yi C, Liu D, Fong C-C, Zhang J, Yang M. Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38 MAPK pathway. Acs Nano. 2010;4:6439–6448. doi: 10.1021/nn101373r. [DOI] [PubMed] [Google Scholar]
  • 24.Heo DN, Ko W-K, Bae MS, Lee JB, Lee D-W, Byun W, Lee CH, Kim E-C, Jung B-Y, Kwon IK. Enhanced bone regeneration with a gold nanoparticle–hydrogel complex. Journal of Materials Chemistry B. 2014;2:1584–1593. doi: 10.1039/c3tb21246g. [DOI] [PubMed] [Google Scholar]
  • 25.McGovern SC, Fong W, Wang JC. Can bone morphogenetic protein binding peptide increase efficiency of bone formation? Spine. 2010;35:1655–1659. doi: 10.1097/BRS.0b013e3181e83c93. [DOI] [PubMed] [Google Scholar]
  • 26.Li L, Jiang W, Luo K, Song H, Lan F, Wu Y, Gu Z. Superparamagnetic iron oxide nanoparticles as MRI contrast agents for non-invasive stem cell labeling and tracking. Theranostics. 2013;3:595–615. doi: 10.7150/thno.5366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Mustafa T, Watanabe F, Monroe W, Mahmood M, Xu Y, Saeed LM, Karmakar A, Casciano D, All S, Biris AS. Impact of gold nanoparticle concentration on their cellular uptake by MC3T3-E1 mouse osteoblastic cells as analyzed by transmission electron microscopy. J Nanomed Nanotechnol. 2011;2:1000118. [Google Scholar]
  • 28.Lyon JL, Fleming DA, Stone MB, Schiffer P, Williams ME. Synthesis of Fe oxide core/Au shell nanoparticles by iterative hydroxylamine seeding. Nano Letters. 2004;4:719–723. [Google Scholar]
  • 29.Fan Z, Shelton M, Singh AK, Senapati D, Khan SA, Ray PC. Multifunctional Plasmonic Shell–Magnetic Core Nanoparticles for Targeted Diagnostics, Isolation, and Photothermal Destruction of Tumor Cells. ACS Nano. 2012;6:1065–1073. doi: 10.1021/nn2045246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Pauksch L, Hartmann S, Rohnke M, Szalay G, Alt V, Schnettler R, Lips KS. Biocompatibility of silver nanoparticles and silver ions in primary human mesenchymal stem cells and osteoblasts. Acta biomaterialia. 2014;10:439–449. doi: 10.1016/j.actbio.2013.09.037. [DOI] [PubMed] [Google Scholar]
  • 31.Goldman EB, Zak A, Tenne R, Kartvelishvily E, Levin-Zaidman S, Neumann Y, Stiubea-Cohen R, Palmon A, Hovav A-H, Aframian DJ. Biocompatibility of tungsten disulfide inorganic nanotubes and fullerene-like nanoparticles with salivary gland cells. Tissue Engineering Part A. 2014;21:1013–1023. doi: 10.1089/ten.tea.2014.0163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Connor EE, Mwamuka J, Gole A, Murphy CJ, Wyatt MD. Gold Nanoparticles Are Taken Up by Human Cells but Do Not Cause Acute Cytotoxicity. Small. 2005;1:325–327. doi: 10.1002/smll.200400093. [DOI] [PubMed] [Google Scholar]
  • 33.Albanese A, Chan WCW. Effect of Gold Nanoparticle Aggregation on Cell Uptake and Toxicity. ACS Nano. 2011;5:5478–5489. doi: 10.1021/nn2007496. [DOI] [PubMed] [Google Scholar]
  • 34.Kawazoe N, Chen G. Gold nanoparticles with different charge and moiety induce differential cell response on mesenchymal stem cell osteogenesis. Biomaterials. 2015;54:226–236. doi: 10.1016/j.biomaterials.2015.03.001. [DOI] [PubMed] [Google Scholar]
  • 35.Chithrani BD, Ghazani AA, Chan WC. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano letters. 2006;6:662–668. doi: 10.1021/nl052396o. [DOI] [PubMed] [Google Scholar]
  • 36.Albanese A, Tang PS, Chan WCW. The effect of nanoparticle size, shape, and surface chemistry on biological systems. Annual review of biomedical engineering. 2012;14:1–16. doi: 10.1146/annurev-bioeng-071811-150124. [DOI] [PubMed] [Google Scholar]
  • 37.Cho EC, Au L, Zhang Q, Xia Y. The effects of size, shape, and surface functional group of gold nanostructures on their adsorption and internalization by cells. Small. 2010;6:517–522. doi: 10.1002/smll.200901622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Dashnyam K, Perez RA, Singh RK, Lee E-J, Kim H-W. Hybrid magnetic scaffolds of gelatin–siloxane incorporated with magnetite nanoparticles effective for bone tissue engineering. RSC Advances. 2014;4:40841–40851. [Google Scholar]
  • 39.Qiu K, Chen B, Nie W, Zhou X, Feng W, Wang W, Chen L, Mo X, Wei Y, He C. Electrophoretic Deposition of Dexamethasone-loaded Mesoporous Silica Nanoparticles onto Poly (L-lactic acid)/Poly (ε–caprolactone) Composite Scaffold for Bone Tissue Engineering. ACS applied materials & interfaces. 2016;8:4137–4148. doi: 10.1021/acsami.5b11879. [DOI] [PubMed] [Google Scholar]
  • 40.Park JS, Yang HN, Woo DG, Chung H-M, Park K-H. In Vitro and In Vivo Chondrogenesis of Rabbit Bone Marrow–Derived Stromal Cells in Fibrin Matrix Mixed with Growth Factor Loaded in Nanoparticles. Tissue Engineering Part A. 2009;15:2163–2175. doi: 10.1089/ten.tea.2008.0532. [DOI] [PubMed] [Google Scholar]
  • 41.Zheng F, Wang S, Wen S, Shen M, Zhu M, Shi X. Characterization and antibacterial activity of amoxicillin-loaded electrospun nano-hydroxyapatite/poly (lactic-co-glycolic acid) composite nanofibers. Biomaterials. 2013;34:1402–1412. doi: 10.1016/j.biomaterials.2012.10.071. [DOI] [PubMed] [Google Scholar]
  • 42.Ganesh N, Ashokan A, Rajeshkannan R, Chennazhi K, Koyakutty M, Nair SV. Magnetic resonance functional nano-hydroxyapatite incorporated poly (caprolactone) composite scaffolds for in situ monitoring of bone tissue regeneration by MRI. Tissue Engineering Part A. 2014;20:2783–2794. doi: 10.1089/ten.tea.2014.0161. [DOI] [PMC free article] [PubMed] [Google Scholar]

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