Exposure of the Yeast Saccharomyces cerevisiae to Functionalized

Article pubs.acs.org/est

Exposure of the Yeast Saccharomyces cerevisiae to Functionalized Polystyrene Latex Nanoparticles: Influence of Surface Charge on Toxicity Toshiyuki Nomura,* Jumpei Miyazaki, Akihisa Miyamoto, Yuta Kuriyama, Hayato Tokumoto, and Yasuhiro Konishi Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan S Supporting Information *

ABSTRACT: Novel nanoparticles with unique physicochemical characteristics are being developed with increasing frequency, leading to higher probability of nanoparticle release and environmental accumulation. Therefore, it is important to assess the potential environmental and biological adverse effects of nanoparticles. In this study, we investigated the toxicity and behavior of surface-functionalized nanoparticles toward yeast (Saccharomyces cerevisiae). The colony count method and confocal microscopy were used to examine the cytotoxicity of manufactured polystyrene latex (PSL) nanoparticles with various functional groups (amine, carboxyl, sulfate, and nonmodified). S. cerevisiae were exposed to PSL nanoparticles (40 mg/L) dispersed in 5−154 mM NaCl solutions for 1 h. Negatively charged nanoparticles had little or no toxic effect. Interestingly, nanoparticles with positively charged amine groups (p-Amine) were not toxic in 154 mM NaCl, but highly toxic in 5 mM NaCl. Confocal microscopy indicated that in 154 mM NaCl, the p-Amine nanoparticles were internalized by endocytosis, whereas in 5 mM NaCl they covered the dead cell surfaces. This demonstrates that nanoparticle-induced cell death might to be related to their adhesion to cells rather than their internalization. Together, these findings identify important factors in determining nanoparticle toxicity that might affect their impact on the environment and human health.

1. INTRODUCTION In coming years, nanotechnology has the potential to produce a variety of new materials with the design of novel nanoparticles (NPs) possessing unique physicochemical characteristics such as high specific surface area, high reactivity, and rapid diffusion, which differ from those of bulk materials of the same composition.1 The increased use of NPs in commercial products is predicted to lead to an accumulation of NPs in the environment. Controlling the release of NPs into the environment at the manufacturing stage is possible. However, controlling NP release at the consumer stage is difficult. Therefore, the potential adverse effects of NPs on the environment and human health must be addressed. Yeast is widely used as a unicellular eukaryotic model microorganism in molecular and cell biology because its cellular structure and functional organization share many similarities with cells in plants and animals; however, the cytotoxicity of NPs toward yeast is still poorly understood.2 A few studies have investigated the potential impact of NPs on yeast.2−6 Kasemets et al.3 showed that the yeast Saccharomyces cerevisiae was relatively resistant to CuO and ZnO NPs when compared with other unicellular microorganisms (bacteria and algae). Lee et al.4 reported that S. cerevisiae showed a higher survival rate than Escherichia coli and Bacillus subtilis after exposure to nanosized silver powder. Schwegmann et al.5 showed that S. cerevisiae was © 2013 American Chemical Society

less affected by the presence of iron oxide NPs compared with Escherichia coli. Hadduck et al.6 mentioned that no reduction in the cell yield of S. cerevisiae was observed in the presence of ́ fullerene. Garcia-Saucedo et al.2 demonstrated that nanoscale HfO, HfO2, SiO2, Al2O3, and CeO2 displayed low or no toxicity toward S. cerevisiae. Overall, the experimental results reported in these studies show that metal oxide NPs and fullerenes exhibit little or no toxicity toward yeast. The uptake of NPs by mammalian cells via endocytosis has been shown for various cell types.7−15 The internalization of NPs into yeast cells has not been studied, but it is supposed that NPs are unable to enter the yeast cell under normal conditions because of its rigid cell wall, whereas dissolved ions and oxidative stress may cause disruption of the cell wall.3 Prescianotto-Baschong and Riezman showed that positively charged nanogold is internalized in the yeast spheroplasts.16 To our knowledge, the internalization of NPs under normal conditions has not been reported. In this study, we evaluated the toxicological effects of surfacefunctionalized NPs on yeast cells. Saccharomyces cerevisiae and Received: Revised: Accepted: Published: 3417

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2.5. Confocal Microscopy. To determine the location of the NPs and the viability of the cells, a confocal laser scanning microscope (FV-1000D, Olympus, Tokyo, Japan) was used to image the fluorescence from stained yeast cells that were exposed to the NP suspension. Live cells have intact membranes and are impermeable to propidium iodide (PI), which only infiltrates cells with disrupted membranes. 4′,6Diamidino-2-phenylindole (DAPI) is a membrane-permeable dye that can enter all cells. The combination of these two dyes provides a rapid and reliable method for discriminating between live and dead yeast cells. 2.6. Time-Lapse Movie. The yeast cell suspension was placed on a glass slide on the confocal microscope. Equal amounts of p-Amine suspension were gently dropped on the cell suspension, and time-lapse images were captured immediately afterward. The yeast cells were not stained by fluorescent dyes. The concentration of the PSL NPs was 40 mg/L. The concentration of the yeast cells was 5 × 105 cells/ mL. The concentrations of NaCl aqueous solution used as a dispersion medium were 5 mM and 154 mM.

manufactured polystyrene latex (PSL) NPs with various functional groups (amine, carboxyl, sulfate, and without modification) were used. A cell viability test after exposure to PSL NPs dispersed in different concentrations of aqueous NaCl solution was carried out using the colony count method. Moreover, the behavior of the PSL NPs, namely internalization, adhesion, and dispersion, was directly observed using confocal laser scanning microscopy (CLSM).

2. MATERIALS AND METHODS 2.1. Yeast Strain. Saccharomyces cerevisiae (strain JCM 7255) was purchased from the Japan Collection of Microorganisms. S. cerevisiae was grown at 30 °C in YE medium (5.0 g/L yeast extract and 30 g/L glucose) with agitation. Cells were harvested in the late exponential growth phase by centrifugation at 8400 × g and 4 °C for 10 min. The yeast cells were washed three times with the sterilized NaCl aqueous solution used in the toxicity test as the dispersion medium, and were resuspended without pH adjustment. The optical density and the number of cells in the cell suspension were measured at 500 nm using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan) and a Petroff-Hausser counting chamber, respectively. 2.2. Polystyrene NPs. Carboxylate- and amine-modified PSL NPs, as well as plain PSL NPs labeled with green fluorophore were purchased from micromod Partikeltechnologie GmbH (29−02−102; 29−01−102; 29−00−102, Rostock, Germany) and are referred to as n-Carboxyl, n-Amine, and nPlain, respectively. Sulfate- and amine-modified PSL NPs labeled with orange fluorophore were purchased from Sigma (L1528; L9904, St. Louis, MO, U.S.) and were named n-Sulfate and p-Amine, respectively. The characters “n” and “p” mean negatively charged and positively charged, respectively. The PSL nanoparticles were suspended in the desired concentration of sterilized NaCl aqueous solution without pH adjustment using a vortex for 10 s just before use. 2.3. Characterization. The electrophoretic mobility (EPM) and the median diameter of the yeast cells were measured using a zeta potential and particle size analyzer (ELSZ, Otsuka Electronics, Osaka, Japan) as a function of ionic concentration. 2.4. Toxicity of PSL NPs toward Yeast Cells. Toxicity tests of the NPs toward the yeast cells were performed with the colony count method. First, the initial concentration of the yeast cell suspension was adjusted to 1 × 106 cells/mL. To investigate the effects of NP concentration on the toxicity toward yeast cells, the initial concentrations of the PSL NP suspensions varied between 0 and 160 mg/L. The concentration of the NaCl aqueous solution used as the dispersion medium varied from 5 to 154 mM (0.029 to 0.9%). Yeast cell suspension (0.5 mL) and PSL nanoparticle suspension (0.5 mL) were added to a microtube, and vortexed for 10 s. Next, the microtube was placed on a Duck rotor at 60 rpm for 1 h at room temperature. After exposure, 0.1 mL of the diluted suspension was spread on YE agar plates and incubated for two days at 30 °C. The YE agar plates were prepared by adding 2.0% (w/v) agar to the YE medium. The number of living cells was determined by counting the number of colony-forming units (CFUs) on the YE agar plates. Toxicity was evaluated by comparing the number of CFUs on the YE agar plates with the number of CFUs on the control plate; the suspension spread on the control plate did not include NPs.

3. RESULTS AND DISCUSSION 3.1. Effect of Surface-Modified PSL Nanoparticles on Cell Viability. Cell viability tests were carried out to investigate the effect of surface-modified PSL NPs on S. cerevisiae. Figure 1

Figure 1. Cell viability after 1 h of exposure to surface-modified PSL NPs dispersed in NaCl aqueous solution. The PSL NP concentration was 40 mg/L. The yeast cell concentration was 5 × 105 cells/mL. The concentrations of NaCl aqueous solution used as a dispersion medium were 5 mM and 154 mM. The pH of the exposure media was between 5 and 6. n-Carboxyl, n-Sulfate, and n-Amine/p-Amine modified PSL NPs and n-Plain PSL NPs were used, where “n” and “p” refer to negatively charged and positively charged surfaces, respectively. (*) P < 0.05 and (**) P < 0.01 versus controls, mean ± SEM, N = 3.

shows the cell viability of S. cerevisiae (5 × 105 cells/mL) after 1 h of exposure to 40 mg/L PSL NP suspensions. When exposed to n-Plain, n-Carboxyl, n-Sulfate, and n-Amine PSL NPs in both 5 mM and 154 mM NaCl aqueous solutions, and to p-Amine PSL NPs in 154 mM NaCl aqueous solution the cell viabilities were >70%. In contrast, when exposed to p-Amine PSL NPs in 5 mM NaCl aqueous solution, the cell viability was 99%) when dispersed in 5 mM NaCl was approximately 15 mg/L. Assuming that the cell is spherical (diameter: Dc) and the 3420

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Figure 5. Time-lapse of confocal laser scanning microscope images of p-Amine NPs’ behavior when yeast cells were exposed to them in (A) 5 mM and (B) 154 mM NaCl aqueous solutions. (C) Fluorescence intensity of the NPs on the surface of the yeast cell and inside the yeast cell at various exposure times. (i): p-Amine labeled with fluorophore, (ii): merged images of (i) and the differential interference contrast image. The cell concentration was 5 × 105 cells/mL; p-Amine concentration was 40 mg/L. The time-lapse movies of parts (A) and (B) are shown in Videos S3 and S4 of the SI.

The yeast cells exposed to the 5 mM NaCl aqueous solution died, and were almost entirely covered with p-Amine PSL NPs. In contrast, nearly all the yeast cells exposed to the 154 mM NaCl aqueous solution were alive. The NPs were transported into the yeast cells and adhered to the surface of the cells. The green fluorescent ring of the p-Amine PSL NPs coating the yeast cells was weaker compared with that in the 5 mM NaCl aqueous solution. The 3D movie images in Videos S1 and S2 of the Supporting Information, SI confirm the covering of the surface of the yeast cell with NPs and the uptake of NPs into the yeast cells. However, the negatively charged NPs, n-Plain, nCarboxyl, n-Amine and n-Sulfate, did not adhere to the surfaces of the yeast cells. These results indicate that the positively charged PSL NPs adhered to the yeast cell surfaces, leading to cell death. To directly examine the behavior of the NPs with exposure time, time-lapse observations were performed. Time-lapse CLSM images of p-Amine NPs’ behavior after exposure of the yeast cells to the p-Amine PSL NP suspension in 5 and 154 mM NaCl aqueous solutions are shown in Figure 5. The timelapse movies are shown in Videos S3 and S4 of the SI. The fluorescence intensities of the p-Amine PSL NPs on and inside the yeast cell with exposure time are also shown in Figure 5. The yeast cell concentration was 5 × 105 cells/mL and the pAmine PSL NP concentration was 40 mg/L. In 5 mM NaCl aqueous solution, the uptake of NPs into the cell was observed within 3 min. The fluorescence intensity inside the cell

surface of the cell is covered with NPs in a close-packed single layer, the total surface area covered by NPs, Sc, is calculated with the following equation: Sc = 0.907(πDc2)Nc, where Nc is the concentration of the cells. The required number of NPs (diameter: Dp), Np, is obtained by dividing Sc by the cross section area of one NP: Np = Sc/(πDp2/4). Finally, the NP concentration necessary to achieve this coverage, Cp, can be estimated by multiplying the NP volume by the NP density, ρp: Cp = Np(πDp3/6) ρp. Thus, the required NP concentration to form a close-packed NP single layer on yeast cells in 5 mM NaCl would be 2.9 mg/L, where the parameters used for the calculation were: the Dc and Dp values listed in Table 1 for 5 mM NaCl, Nc = 5 × 1011 cells/m3, and ρp = 1030 kg/m3. This value agrees well with the NP concentrations that were toxic for yeast. The cell viability of S. cerevisiae decreased with decreasing NaCl concentration, indicating that the electrostatic force between the yeast cell and p-Amine is an important factor in yeast cytotoxicity. Negatively charged PSL NPs do not adhere to the surface of the yeast cells because of electrostatic repulsive forces; therefore, these NPs are not toxic to yeast. 3.4. Confocal Laser Scanning Microscopy of Yeast Cells Exposed to Toxic NPs. To determine the relationship between the location of the PSL NPs and cell viability, confocal laser scanning microscopy (CLSM) was carried out. Typical CLSM images of the yeast suspension taken after 1 h of exposure to 40 mg/L p-Amine PSL NP suspensions in 5 mM and 154 mM NaCl aqueous solutions are shown in Figure 4. 3421

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Time-lapse movies taken after yeast cells were exposed to pAmine NP suspension in 5 mM NaCl aqueous solution. Video S4, Time-lapse movies taken after yeast cells were exposed to pAmine NP suspension in 154 mM NaCl aqueous solution. This material is available free of charge via the Internet at http:// pubs.acs.org.

increased within 3 min, and then saturated. In contrast, the fluorescence intensity on the surface of the cell increased, indicating that the number of NPs adhering to the cell increased with exposure time. In the case of the 154 mM NaCl aqueous solution, the nanoparticles were clearly taken into the cell, as the fluorescence intensity inside the cell gradually increased, while the fluorescence intensity on the cell was barely detectable. In both cases, the uptake of NPs into the cell occurred instantaneously. It has been reported that eukaryotes such as yeast display endocytosis.22,23 In the case of 154 mM NaCl aqueous solution, the p-Amine NPs were internalized in vesicles. The yeast cells with internalized p-Amine NPs (Figure 4B) were not stained by the membrane-impermeable PI, indicating that the cell membrane was not disrupted. This result suggests that the positively charged NPs were internalized via an endocytosis pathway. However, when the yeast cells were exposed to the toxic p-Amine NPs in low concentration NaCl aqueous solution, the p-Amine NPs adhered onto the cell surface. The increased adhesion rate of the NPs onto the yeast cells, because of the increased electrostatic force between the yeast cells and the positively charged NPs, exceeded the uptake rate of the NPs into the yeast cells, resulting in their accumulation on the cell surface. It has been reported that cell activity decreases with decreasing fluidity of the cell membrane.24,25 The covering of the yeast cell by NPs causes a decrease in the fluidity of the cell membrane and inhibition of metabolism through the cell membrane, leading to cell death. The balance between the adhesion and uptake rates of the positively charged NPs on/ into the yeast cell is one of the main factors responsible for toxicity. Further investigation is needed to establish the mechanism of this toxicity. Our understanding of NP toxicity toward S. cerevisiae can be applied to more complex eukaryotic cells and aid in the development of harmless NPs for application in ecological and drug delivery systems, gene therapy, and for use in selective low-dose antimicrobial agents. In conclusion, the toxicity of functionalized PSL NPs toward S. cerevisiae was examined using the colony count method and confocal microscopy. Interestingly, the PSL nanoparticles with amine groups, p-Amine and n-Amine, expressed remarkably different toxicity. The positively charged PSL nanoparticle, pAmine, expressed a remarkable range of toxicity according to the ionic strength of the solution, which is related to the electrostatic interaction between the cell and the NP. Nearly all S. cerevisiae cells exposed to the negatively charged n-Amine PSL NPs survived. Confocal microscopy showed that the surfaces of the dead cells were entirely covered with p-Amine NPs when dispersed in the 5 mM NaCl aqueous solution. In contrast, the internalization of the positively charged PSL NPs in vesicles was clearly observed when using 154 mM NaCl aqueous solution. Neither uptake nor adhesion of the negatively charged NPs to the yeast cells was observed. To our knowledge, this is the first study in which the toxicity of functionalized PSL NPs toward S. cerevisiae has been evaluated using CLSM to directly observe the adhesion and internalization of NPs.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-72-254-9300; fax: +81-72-254-9911; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Number 24310066. The authors thank Ms. K. Okuno for her help with the lab work.

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ASSOCIATED CONTENT

* Supporting Information S

Video S1, 3D images taken after yeast cells were exposed to pAmine NP suspension in 5 mM NaCl aqueous solution. Video S2, 3D images taken after yeast cells were exposed to p-Amine NP suspension in 154 mM NaCl aqueous solution. Video S3, 3422

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