Biocompatibility Study of Gold Nanoparticles to Human Cells J.H. Fan1, W.I. Hung2, W.T. Li1,3,*, J.M. Yeh2,4 1
Department of Biomedical Engineering, Chung-Yuan Christian University, Taiwan 2 Department of Chemistry, Chung-Yuan Christian University, Taiwan 3 Research and Development Center for Biomedical Microdevice Technologies, Chung-Yuan Christian University, Taiwan 4 Center for Nano-Technology, Chung-Yuan Christian University, Taiwan Abstract — Gold nanoparticle (GNP) is one of the most stable and popular nanoparticles, which receives considerable attention due to their applications in biomedical imaging and diagnostic tests. However, its cytotoxicity has not been fully investigated. Here we report the effects on biocompatibility of water-soluble GNPs with different sizes and concentrations to human bone marrow mesenchymal stem cells (hBMSCs) and human hepatoma carcinoma cells (HuH-7). Cytotoxicity was analyzed at different time points using MTT assay after coculturing with different concentrations of GNPs. Both cells incubated with 71.1 g/mL 15- and 30-nm GNPs exhibited more than 80 % cell survival. However, cell viability decreased to less than 60% after incubated with 31.6 g/mL 5-nm GNPs for 5 days. GNP 15 nm in size was chosen for the following experiments. According to Annexin V and propidium iodide (PI) analysis, the percentage of necrotic cells increased gradually with the concentration of GNPs increased. Approximately 1.5 fold increase in the level of reactive oxygen species (ROS) was found, suggesting that necrosis might be triggered by ROS production after GNPs were endocytosed by cells. Alkaline phosphatase activity and calcium deposition of hBMSCs during osteogenic differentiation were inhibited slightly by coculturing with GNPs. A similar observation was made in adipogenic hBMSCs, in which the accumulation of triacylglycerides was repressed by the addition of GNPs. In conclusion, despite 15- and 30-nm GNPs had little toxicity to hBMSCs and HuH-7 cells, they still had some influence on both osteogenic and adipogenic capabilities of hBMSCs. Keywords — gold nanoparticle, biocompatibility, differentiation, HuH-7, mesenchymal stem cell
I. INTRODUCTION In the past 30 years, nanomaterials have received considerable attention because of their great potential in nanobiotechnology and drug delivery. Most of the research is focused on nanoparticles. For example, quantum dots are colloidal semi-conductor nanocrystals with unique light emitting optical properties that make them can be used as fluorescent probes for long-term cell targeting and imaging [1,2]. Dextran-coated iron oxide nanoparticles have superparamagnetic core which can be used in magnetic resonance imaging and magnetic biosensors [3]. Gold nanoparticles (GNPs) exhibit scattering light when excited due to surface
plasmon resonance [4]. In fact, GNPs are widely used in biomedical imaging and diagnostic tests because they have many advantages over other types of nanoparticles. Besides their characteristics of optical properties, GNPs are chemically inert, easy to prepare and highly stable once synthesized [5]. Conjugation chemistry to biomolecules is simple and well developed in GNPs. Despite the wide application of GNPs, there is a serious lack of information concerning the impact of manufactured GNPs on human health and environment. Because nanoparticles can pass through biological membranes, they can affect the physiology of any cell in human body. This consideration is important for stem cells, where the effect of GNPS on their potential for selfrenewal and differentiation is unknown. Here, we report the effect of GNPs on human cells. In addition, the influence on osteogenesis and adipogenesis of human bone marrow mesenchymal stem cells by GNPs treatment was explored. II. MATERIALS AND METHODS A. Preparation of GNPs GNPs were synthesized as described by Turkevich et al. [6]. A solution of 1 mM HAuCl4 (Alfa Aesar, MA, USA) was heated to boiling while stirring. After boiling was commenced, 38.8 mM sodium citrate (Mallinckrodt Baker, NJ, USA) solution was added slowly to the HAuCl4 boiling solution. The solution changed color from yellow to dark red within 10 min. The solution was slowly cooled down to room temperature (RT). The resulting particles were coated with negatively charged citrate and were well-suspended. The GNPs solution was stored at 4oC for further use. GNPs of 5, 15 and 30 nm in size were prepared using different ratios of HAuCl4 and sodium citrate solutions. B. Cell culture Human hepatoma carcinoma cells (HuH-7, Japanese Collection of Research Bioresources, Japan) were cultured Dulbecco's minimal essential medium (Gibco/BRL, Invitrogen, CA, USA) supplemented with 10 % fetal bovine serum (FBS, Biological Industries, Israel). Human bone marrow
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mesenchymal stem cells (SV40T- and hTERT-immortalized cells, hBMSCs) were grown in Alpha-minimum essential medium (-MEM, Gibco/BRL, Invitrogen, CA, USA) containing 10 % lot selected FBS (Hyclone, Utah, USA). Both cells were maintained at 37oC under atmosphere with 5 % CO2.
was analyzed by an ELISA reader (Fluoroskan Ascent FL, Thermo Fisher Scientific, Inc., MA, USA) (ext: 488 nm). The protein amount in each sample was determined by bicinchoninic acid protein assay (Sigma, MO, USA).
C. Cytotoxicity assay
To observe the effect of GNPs on osteogenic differentiation of hBMSCs, cells (500 cells/well) were grown on a 96well microplate and incubated in the osteogenic medium (MEM containing 2% FBS, 10 M -glycerophosphate, 10-7 M dexamethasone and 50 M ascorbic acid 2-phosphate) containing different concentrations of GNPs 15 nm for 28 days. Fresh medium containing GNPs was replaced every 3 days. Alkaline phosphatase (ALP) activity and calcium deposition of hBMSCs were assessed every week. ALP activity was assayed based on the conversion of pnitrophenyl phosphate (p-NPP) into p-nitrophenol (p-NP) in the presence of ALP. Culture supernant (100 l) was mixed with 100 l of 6.7 mM p-NPP (Sigma, MO, USA), followed by incubation at 37oC for 30 min. After stopping the reaction with 50 l of 3 N NaOH, the absorbance at 405 nm of p-NP was measured with an ELISA reader. Calcium deposition is a late marker for osteogenic differentiation, which can be stained with Alizarin red S. Cells in each well were stained with 50 l of 5% (W/V) Alizarin red S. To quantify the amount of mineralization, 50 l of 5% (W/V) cetylpyridinium chloride (Sigma, MO, USA) was added into each well to dissolve calcium deposition at RT for 3 h. The absorbance at 550 nm was read by an ELISA reader. To observe the effect of GNPs on adipogenic differentiation, hBMSCs (2 x 104 cells/well) were grown on a 24-well plate for 6 days. Then, the medium was changed into adipogenic medium (-MEM containing 2% FBS, 0.5 mM 3isobutyl-1-methylxanthine, 10-6 M dexamethasone, 60 M indomethacin and 5 g/ml insulin) containing different concentrations of GNPs 15 nm and the medium was replaced every 3 to 4 days. Adipogenesis was assessed by Oil Red-O stain. To quantify the amount of triacylglyceride, 50 l of isopropanol was added into each well to dissolve Oil Red-O at RT for 10 min. The absorbance at 510 nm was read by an ELISA reader.
To determine the cytotoxicity of GNPs to HuH-7 cells and hBMSCs, cells (5000 cells/well) were inoculated on a 96-well microplate and allowed to adhere for 24 h. Then, different concentrations of GNPs solution with same volume were added into each well. Cell viability was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay after 24, 72 and 120 h incubation. Medium was removed and cells were rinsed with Dulbecco’s phosphate buffered saline (PBS, Sigma, MO, USA) twice. The cells were then incubated with MTT solution (0.5 mg/ml) at 37 oC for 4 h. The purple formazan formed in each well was dissolved in 0.1 ml of DMSO for 40 min. The absorbance at 570 nm was monitored using an ELISA reader (Multiskan Spectrum, Thermo Fisher Scientific, Inc., MA, USA). D. Assessment of necrosis and apoptosis Annexin V/ Propidium iodide (PI) staining method is commonly used to differentiate necrotic and apoptotic cells. Here, phosphatidylserine externalization and nuclei were stained by the Annexin V-FITC Apoptosis Detection Kit (Sigma, MO, USA). One million HuH-7 cells and hBMSCs were plated on a 60-mm dish. Various concentrations of GNPs15 nm were added to each dish and incubated for 24 h. Cells were trypsinized and resuspended in 1 x binding buffer (0.01 M HEPES, pH 7.4; 140 mM NaCl; 25 mM CaCl2), then incubated with staining solution (Annexin VFITC : PI = 1 : 2) in darkness for 20 min at RT. Immediately after annexin V/PI staining, the sample was analyzed with Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, NJ, USA).
F. Differentiation of hBMSCs
E. Measurement of ROS formation Reactive oxygen species (ROS) formation was detected using 2’,7’-dichlorofluorescin diacetate (DCFDA, Sigma, MO, USA). Cells (106 cells/dish) were seeded a 60-mm dish. Various concentrations of GNPs15 nm were added to each dish. After 24 h incubation, cells were washed with PBS and DCFDA (20 M) was added into each dish. Cells were washed twice with PBS after 1 h, and lysed by sonication. DCF (2',7'-dichlorofluorescein) fluorescence in cell lysate
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III. RESULTS AND DISCUSSION A. Effect of GNPs on cell survival The results of cell survival of HuH-7 cells and hBMSCs treated with various concentrations (0-75.05 g/mL) of GNPs with different sizes are shown in Table 1. Cytotoxicity of GNPs was determined by MTT assay that measures
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the metabolic activity of survival cells. The LD20 value is where the concentration of GNPs that resulted in a 20% reduction in cell survival compared to the untreated control. It is found that cytotoxicity decreased as the size of GNPs increased in HuH-7 cells. GNPs 5 nm in size resulted in less than 80 % cell survival even at low concentrations at day 1. On day 3, the percentages of cell survival for GNPs 15 and 30 nm were higher than 80%, indicating these GNPs had little toxicity to HuH-7 cells. Similar observation was made in hBMSCs, where cell viability increased as the size of GNPs increased. Cytotoxicity of GNPS to HuH-7 cells was higher than that to hBMSCs. Culture media were replaced with fresh media containing GNPs on day 3 (after MTT assay). GNPs endocytosed by cells with GNPs replenished in the fresh medium could lead to increase in total concentration of GNPs, which might be the reason why the LD20 values decreased on day 5. To investigate the mechanism of cell death and the effects on hBMSCs differentiation, GNPs 15 nm was chosen for the following experiments.
with 15-nm GNPs were found that the percentage of live cells decreased as the concentration of GNPs increased. Significant increase in necrotic population was observed after treated with GNPs. In contrast, there was no significant increase in apoptotic cells was seen. Our results indicate that GNPs induce cell death in HuH-7 cells and hBMSCs via necrotic process. C. Effect of GNPs on ROS production ROS production from HuH-7 cells and hBMSCs was observed (Fig. 1). It was found that the fluorescence intensity increased significantly after treated with GNPs. The level of ROS increased 1.5 fold of the non-treated control. The result suggests GNPs may result in cell death elicited from excessive ROS formation.
HuH-7
Table 1 Cell types
LD20 dose of GNPs to human cells
Size of GNPs (nm)
HuH-7
hBMSCs
LD20 (g/mL) Day 1
Day 3
Day 5
5
0.06
0.70
7.90
15
14.10
᧩*
52.21
30
73.30
᧩
0.75
5
16.00
9.31
15.80
15
45.49
57.70
24.44
30
᧩
᧩
37.53
Fold of ROS production
2.0
Percentages of live, apoptotic, and necrotic cells treated with GNPs
Cell types
Cells gated (%)
GNPs (g/mL) 3.56 7.11 17.78
0 35.55 71.1 99.4±0.1 60.4±2.9 71.5±1.0 78.8±1.3 79.2±4.1 75.4±4.2 Live HuH-7 Apoptosis 0±0 0.4±0.2 2.5±0 1.6±0.2 2.2±0.1 3.1±0. 6 Necrosis 0.6±0.1 39.2±3.0 26.0±0.9 19.6±1.5 18.6±4.2 21.5±4.7 98.8±0.1 78.2±2.9 83.7±0.4 81.5±0.6 82.3±0.3 86.0±0.3 Live hBMSCs Apoptosis 0±0 4.9±0.6 3.9±0.4 3.4±0.6 5.3±0.8 3.4±0.9 Necrosis 1.2±0.1 17.0±2.3 12.5±0 15.1±0 12.5±1.1 10.6±1.2
B. Apoptosis versus necrosis caused by GNPs To determine apoptosis and necrosis in HuH-7 cells and hBMSCs treated by GNPs, cells were stained with a combination of Annexin V and PI and subjected to analysis by flow cytometry. As shown in Table 2, both cells treated
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1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.00
7.11
17.78
35.55
71.10
GNPs (ug/mL)
*The percentage of cell survival was higher than 80% within the range of the concentrations of GNPs tested.
Table 2
hBMSCs
Fig. 1 The effect of GNPs on ROS generation D. Effect of GNPs on hBMSCs differentiation Human bone marrow MSCs were subjected to osteogenic induction while treated with GNPs. The osteogenic medium containing GNPs was replaced every 3 days. ALP activity and calcium deposition of MSCs were assessed every week. It was observed that both ALP activity and calcium deposition were significantly inhibited by the addition of GNPs compared with control. ALP activity did not seem to be repressed at early stage of osteogenesis but apparent inhibition was seen at day 14 when GNPs ฺ14.78 g/ml was applied (Fig. 2). Calcium deposition was suppressed at day 28 (Fig. 3). No correlation was found between the decrease of osteogenic differentiation and the concentration of GNPs. Our results suggest that GNPs influence the late stage of hBMSCs osteogenic differentiation.
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ALP (OD at 405 nm / ug protein)
The influence of GNPs on hBMSCs adipogenic differentiation was also studied. It was found that the level of triacylglyceride accumulation decreased as the concentration of GNPs increased during the 14-day period of adipogenesis (Fig. 4). The above results indicate that the addition of GNPs inhibited hBMSCs differentiation despite that hBMSCs showed better tolerance for these nanoparticles.
Day 7 Day 14
10
873
8 6 4
2
IV. CONCLUSIONS
0 0.00
7.11
17.78
35.55
71.10
GNPs (ug/ml)
Fig. 2 The effect of GNPs on ALP activity of hBMSCs
Day 21
4.0
Day 28
Calcium level (OD at 550 nm / ug protein)
3.5 3.0
2.5
Our results showed that low concentration of GNPs exhibited more than 80% cell survival in HuH-7 cells and hBMSCs. Cell survival decreased as the size of GNPs increased. GNPs induce cell death via necrotic process, which might be elicited from excessive ROS formation. The addition of GNPs inhibited hBMSCs differentiation despite that hBMSCs showed better tolerance for these nanoparticles. The molecular mechanisms of GNPs toxicity to hBMSCs remain to be explored further.
2.0
ACKNOWLEDGMENT
1.5 1.0 0.5 0.0 0
7.11
17.78
35.55
71.10
This work was supported in part by the National Science Council, Taiwan, R.O.C. under Grants NSC 96-2218-E033-001, and NSC 97-2627-M-033-005.
GNPs (ug/ml)
REFERENCES
Fig. 3 The effect of GNPs on mineralization of hBMSCs 1. 100 Day 7
Level of triacylglyceride (OD at 510 nm / ug protein)
90
2.
Day 14
80
70
3.
60
50
40 30
4.
20
5.
10 0 0.00
7.11
17.78
35.55
71.10
GNPs (ug/ml)
6.
Fig. 4 The effect of GNPs on adipogenesis of hBMSCs
Chan WH, Shiao NH, Lu PZ (2006) CdSe quantum dots induce apoptosis in human neuroblastoma cells via mitochondrial-dependent pathways and inhibition of survival signals. Toxicol Lett 167:191-200 Maysinger D, Lovric J, Eisenberg A, Savi R (2007) Fate of micelles and quantum dots in cells. Eur J Pharm Biopharm 65:270-281 Schellenberger EA, Reynolds F, Weissleder R, Josephson L (2004) Surface-functionalized nanoparticle library yields probes for apoptotic cells. Chembiochem 5:275-279 El-Sayed IH, Huang X, El-Sayed MA (2005) Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer. Nano Lett 5:829-834 He H, Xie C, Ren J (2008) Nonbleaching fluorescence of gold nanoparticles and its applications in cancer cell imaging. Anal Chem 80:5951-5957 Turkevich J, Stevenson PC, Hillier JA (1951) A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss Faraday Soc 11:55-75 Author: Institute: Street: City: Country: Email:
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Wen-Tyng Li, Ph.D. Chung-Yuan Christian University 200 Chung-Pei Road Chung-Li Taiwan, R.O.C.
[email protected]
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