Cell density-dependent accumulation of low polarity gold nanocluster in cultured vascular endothelial cells

Abstract

We have previously reported the cytotoxicity and various biological responses of organic-inorganic hybrid molecules. However, because all the molecules used were electrophilic, the effect of the hybrid molecule without electrophilicity remains unclear. The glutathione-protected gold nanocluster, Au₂₅(SG)₁₈, is an organic-inorganic hybrid molecule that shows a low intramolecular polarity and high stability. In this study, we examined the cytotoxicity and intracellular accumulation of Au₂₅(SG)₁₈ in cultured vascular endothelial cells and compared these characteristics with those of negatively charged gold nanoparticles (AuNPs). Both Au₂₅(SG)₁₈ and AuNPs accumulated in vascular endothelial cells in a dose-dependent manner without cytotoxicity and more accumulation was observed at low cell densities. However, Au₂₅(SG)₁₈ accumulated significantly less than AuNPs in the cells. These results suggest that the intramolecular polarity of organic-inorganic hybrid molecules could regulate intracellular accumulation.

INTRODUCTION

Nanotechnology has been developing rapidly in recent years and is expected to solve social problems in the fields of materials, energy, environment, and medicine. Under these circumstances, the creation of stable and functional nanomaterials has progressed considerably. Gold compounds have long been used in the medical field as therapeutic agents for rheumatoid arthritis, such as gold thiomalic acid (Goldberg et al., 1981) and auranofin (Finkelstein et al., 1976). In addition, attempts have also been made to apply nanoscale gold particles to biomedical diagnosis (Storhoff and Mirkin, 1999), drug delivery (Voskerician et al., 2003), bioimaging (Shang et al., 2013; Chen et al., 2015), and radiation therapy (Zhang et al., 2014; Riley and Day, 2017). Furthermore, metal nanoclusters with a few or hundreds of metal atoms within 1–2 nm in size are attracting attention as functional nanomaterials with physical properties specific to the size of the cluster, including catalytic activity (Zhu et al., 2010; Yamazoe et al., 2014), optical activity (Knoppe et al., 2012), luminescence (Link et al., 2002; Wu and Jin, 2010), and magnetic properties (Zhu et al., 2009; Antonello et al., 2013).

As the creation of such nanomaterials has become widespread, the toxicity and distribution of the molecules have also been studied (Perrault et al., 2009; Chou and Chan, 2012; Jiang et al., 2015; Foroozandeh and Aziz, 2018). We also reported the cytotoxicity and function of organic-inorganic hybrid molecules —metal complexes and organometallic compounds— as tools for analyzing biological systems of vascular endothelial cells at different cell densities (Fujie et al., 2016, 2019, 2020; Hara et al., 2017, 2018a, 2019, 2020; Takahashi et al., 2018). However, because these hybrid molecules are generally electrophilic, the response of these cells against metal compounds that lack electrophilicity is not well understood. To answer this question, new analytical tools are needed.

Among hybrid molecules, thiolate-protected gold nanoclusters (Au_n(SR)_m) show high stability because the clusters have a structure in which complexes of gold and thiolate (-SR-Au-SR-) are bound to the surface of the gold core. Au₂₅(SG)₁₈, a glutathione-protected gold nanocluster, has a diameter of approximately 1 nm and is particularly stable (Negishi et al., 2005, 2012; Ohta et al., 2013). In addition, as the icosahedral gold core of Au₂₅(SG)₁₈ has a low intramolecular polarity (Wu et al., 2009), this cluster is considered to be a metal compound with no electrophilicity. Therefore, we investigated the cytotoxicity of Au₂₅(SG)₁₈ to endothelial cells.

MATERIALS AND METHODS

Materials

Bovine aortic endothelial cells were obtained from Cell Applications (San Diego, CA, USA). Tissue culture dishes and plates were purchased from AGC Techno Glass (Shizuoka, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and Ca²⁺- and Mg²⁺-free phosphate-buffered saline were obtained from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum, alamarBlue cell viability reagent, and a bicinchoninic acid protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Other reagents of the highest grade available were obtained from Nacalai Tesque (Kyoto, Japan). Standard gold nanoparticles (AuNPs; 5 nm) were purchased from Cytodiagnostics (Burlington,Canada) and the Au₂₅(SG)₁₈ was synthesized as reported previously (Shichibu et al., 2005).

Cell culture and treatment

Vascular endothelial cells were cultured in a humidified atmosphere of 5% CO₂ at 37°C in DMEM supplemented with 10% fetal bovine serum until confluent. Subsequently, the experiments were performed as described below.

Cell viability assay

Vascular endothelial cells were transferred into 24-well culture plates at a density of 1 × 10⁴ cells/cm² and cultured for either 24 hr (“sparse culture”) or until confluence (“dense culture”). The medium was then discarded, and the cells were washed with serum-free DMEM; the medium was then replaced to test media prepared with fresh serum-free DMEM. After a 24-hr incubation, the conditioned media were discarded, the cells were washed with Ca²⁺- and Mg²⁺-free phosphate-buffered saline, and 262.5 µL of DMEM-alamarBlue (20:1) solution was added to each well, and the cells were further incubated for 60 min. After incubation, 200 µL of DMEM-alamarBlue was transferred from each well to a black bottom 96-well plate and fluorescence was measured (Ex = 544 nm, Em = 590 nm) using a multimodal plate reader (PerkinElmer, Waltham, MA, USA).

Intracellular accumulation of gold atoms

Vascular endothelial cells were transferred into 100 mm dishes at a density of 1 × 10⁴ cells/cm² and cultured for 24 hr (“sparse culture”) or transferred into 60 mm dishes until confluence (“dense culture”). In another experiment, the cells were transferred into 60 mm dishes at densities of 1, 2, 4, and 6 × 10⁴ cells/cm² and cultured for 24 hr. The medium was then discarded, and the cells were washed with serum-free DMEM; the medium was then replaced to test media prepared with fresh serum-free DMEM. After a 24-hr incubation, the cell layer was harvested with 80 µL of 50 mM Tris-HCl buffer solution (pH 6.8) containing 2% sodium dodecyl sulfate and 10% glycerol. The cells were then lysed by incubation at 95°C for 15 min, and 50 µL of the lysate was incubated with HNO₃-H₂O₂ solution and dried as described in our previous report (Hara et al., 2018b). The samples were further incubated at 130°C for 48 hr in aqua regia (35% HCl:61% HNO₃ = 3:1) and then dissolved in 5 mL of 0.1 M HNO₃. The gold atom content was then analyzed using inductively coupled plasma mass spectrometry (Nexion 300S, PerkinElmer) with conditions optimized for a plasma output of 1600 W, a plasma gas flow of 18.0 L/min, and a nebulizer gas flow rate of 0.94 L/min. Another portion of the cell lysate was analyzed for protein content using the bicinchoninic acid protein assay kit to express the content of antimony as pmol/mg protein.

Statistical analysis

The data were analyzed for statistical significance using analysis of variance (ANOVA) and the Bonferroni’s multiple t-test, when applicable. P-values of less than 0.05 were considered statistically significant.

RESULTS

We first examined the cytotoxicity of Au₂₅(SG)₁₈ and AuNPs to vascular endothelial cells through morphological observation. Treatment with Au₂₅(SG)₁₈ and AuNPs did not result in marked changes in the morphological appearance of the cells. The cell viability assay also supported the morphological observation and showed that no cytotoxicity was observed after treatment with Au₂₅(SG)₁₈ and AuNPs at concentrations up to 0.78 µg/mL in both dense and sparse cultures (Fig. 1). However, as indicated in Fig. 2, the amount of ¹⁹⁷Au was increased in Au₂₅(SG)₁₈ and AuNPs in a concentration-dependent manner, and both compounds accumulated more in the sparse culture than in the dense culture of vascular endothelial cells. In addition, the amount of ¹⁹⁷Au accumulated in the cells treated with Au₂₅(SG)₁₈ was about 10 times lower than that in the AuNPs-treated cells. Considering the 47.3% occupancy of gold in the molecular weight of Au₂₅(SG)₁₈, this result indicates that Au₂₅(SG)₁₈, which has low polarity, is difficult to accumulate in the cells. Moreover, we examined whether the uptake of Au₂₅(SG)₁₈ is dependent on the density of the cells. Vascular endothelial cells were seeded at different cell densities and treated with Au₂₅(SG)₁₈. As the cell seeding density increased, the accumulation of ¹⁹⁷Au in the cells decreased significantly (Fig. 3).

Fig. 1

Morphological observation and cell viability of bovine aortic endothelial cells. [A and C] Dense and [B and D] sparse culture of the cells were treated with [A and B] glutathione-protected gold nanocluster (Au₂₅(SG)₁₈) and [C and D] gold nanoparticles (AuNPs) at 0.05, 0.10, 0.20, 0.39, and 0.78 µg/mL for 24 hr. The values are mean ± standard error of four samples of the experiment.

Fig. 2

Intracellular accumulation of Au₂₅(SG)₁₈ and AuNPs in vascular endothelial cells. Dense (left panels) and sparse (right panels) cultures of bovine aortic endothelial cells were treated with [A] Au₂₅(SG)₁₈ and [B] AuNPs at 0.05, 0.10, 0.20, 0.39, and 0.78 µg/mL for 24 hr. The values are the mean ± standard error of three samples of the experiment. Significantly different from the corresponding control, *p < 0.05, **p < 0.01.

Fig. 3

Cell density-dependent accumulation of Au₂₅(SG)₁₈ in vascular endothelial cells. Bovine aortic endothelial cells at different cell densities were treated with Au₂₅(SG)₁₈ at 0.78 µg/mL for 24 hr. The values are mean ± standard error of three samples of the experiment. **Significantly different from “1 × 10⁴ cells/cm²”, p < 0.01.

DISCUSSION

What kind of molecular basis can be mediated in this cell density-dependent accumulation of Au₂₅(SG)₁₈? It has been reported that endocytosis is related to the internalization mechanism of lipoic acid-protected gold nanoclusters (Yang et al., 2013; Barbieri et al., 2016) and that clathrin and caveolin are molecules involved in the formation of endocytosis vesicles (Takei and Haucke, 2001; Harris et al., 2002). Endocytosis is classified into the following four types according to structural features: 1) clathrin-dependent endocytosis, 2) caveolae-dependent endocytosis, 3) clathrin/caveolae-independent endocytosis, and 4) macropinocytosis (Lamaze and Schmid, 1995). In particular, clathrin-dependent endocytosis and caveolae-dependent endocytosis behave differently depending on the cell cycle. Clathrin-dependent endocytosis is constant at all stages of mitosis, and internalized clathrin does not return to the membrane surface until the late stage of mitosis (Boucrot and Kirchhausen, 2007). In contrast, caveolae are plasma membrane structures often found in vascular endothelial cells (Lisanti et al., 1994). The caveolae component, caveolin, actively translocates inside the cells during mitosis, localizes to the endosome, and then returns to the cell surface after or during cytokinesis (Boucrot et al., 2011) to modulate transport of nutrients and receptors in growing cells (Hinze and Boucrot, 2018). Namely, caveolae-dependent endocytosis is more likely to occur during mitosis than during clathrin-dependent endocytosis. As the uptake of Au₂₅(SG)₁₈ into the vascular endothelial cells was more in the growing state (Fig. 3), it is speculated that Au₂₅(SG)₁₈ translocated into the cells via caveolae-dependent endocytosis in a dose-dependent manner. This study is an important result of analyzing the cytotoxicity of metal nanomolecules having a small electric charge.

ACKNOWLEDGMENTS

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 15K08047 (to C.Y.), 15K14992 (to T.K.) and 17H05385 (to Y.N.).

Conflict of interest

The authors declare that there is no conflict of interest.

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