Direct measurements of colloidal behavior of polystyrene nanoparticles into budding yeast cells using atomic force microscopy and confocal microscopy

doi:10.1016/j.colsurfa.2018.07.057

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Volume 555, 20 October 2018, Pages 653-659

https://doi.org/10.1016/j.colsurfa.2018.07.057 Get rights and content

Abstract

The colloidal behavior of positively charged polystyrene latex (PSL) nanoparticles (NPs) toward yeast cells (Saccharomyces cerevisiae) was investigated using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging revealed that the conically-shaped pits connected with endocytosis were observed in 154 mM NaCl, and the surface was completely covered with NPs within 60 s in 5 mM NaCl. AFM force measurements revealed that the adhesion rate of NPs in 5 mM NaCl was faster than that in 154 mM NaCl. This strongly suggested that in 5 mM NaCl, the NPs accumulated on the cell surface over a short time followed by cell death. Furthermore, the accumulation of NPs on the cell surface in 5 mM NaCl could be suppressed by adding polyethylene glycol to the medium, resulting in an increase in the number of living cells. This suggests that the adhesion rate of NPs primarily depended on the interaction forces between the surfaces, and the viscosity of the medium. Thus, the colloidal behavior of the positively charged PSL NPs toward yeast cells was controlled by the balance between the adhesion rate of NPs on the cells and the uptake rate of NPs into the cells. Additionally, the uptake of the PSL NPs and an endocytosis marker into the cells was inhibited by Latrunculin B and NaN₃. However, their locations without inhibitor treatment were obviously different, indicating that NPs were not transported to the vacuole and accumulated in the vesicles.

Graphical abstract

Introduction

Understanding the interactions between engineered nanoparticles (NPs) and biological cells has received considerable attention because of the potential use of NPs as drug and gene delivery systems among other applications [[1], [2], [3], [4], [5], [6]] and their adverse impacts on human health and the environment [7,8]. Yeast has many similarities with animal cells and plant cells. Therefore, it is widely used as a eukaryotic model cell. However, reports on the risk assessment of NPs such as metal oxides, silver, fullerenes, and polymers using yeast cells are limited. This is because various types of NPs have been reported to show little or no cytotoxicity toward yeast [[9], [10], [11], [12], [13]]. In addition, the delivery of NPs to mammalian cells has been extensively studied in relation to drug and gene delivery systems [[1], [2], [3], [4], [5], [6]]. However, uptake of NPs into eukaryotic cells using yeast cells has not been studied. This is because yeast cells are surrounded by robust cell walls [9]. Here, it has been reported that yeast spheroplasts with cell walls almost completely removed take up positively charged nanogold [14]. We demonstrated that in physiological saline (154 mM NaCl solution) positively charged amine modified polystyrene (PS) NPs with particle size less than 100 nm were taken up by living yeast cells [[15], [16], [17]]. In contrast, in 5 mM NaCl solution, the surface of yeast cells was completely covered with the NPs and caused cell death.

Confocal laser scanning microscopy (CLSM) is one technique used to directly observe dynamic NP behavior at the micro-level, but it is not suitable for distinguishing the location of the NPs at the nano-level. Instead, atomic force microscopy (AFM) is a powerful tool for nanoscale imaging of biological surface topography. In this study, we used AFM to visualize yeast cell topography at the nano-level after exposure to PS NPs in the presence of different concentrations of NaCl, as a way to better understand the colloidal behavior of the NPs toward the yeast cells, along with the direct measurement of interaction forces between the NP and the cell. Additionally, an attempt was made to control the NP behavior by adding a thickener to the dispersion medium, and the mechanism of NP uptake was investigated using an inhibitor and an endocytosis marker. The location of PSL NPs and the cell viability after NP exposure were observed directly using CLSM at the macro-level. Saccharomyces cerevisiae and positively charged amine-modified PSL NPs were used as a model eukaryotic microorganism and model NPs, respectively.

Section snippets

Yeast strain and growth condition

The budding yeast Saccharomyces cerevisiae JCM 7255^™ was purchased from the Japan Collection of Microorganisms. S. cerevisiae was grown in YE medium (5 g/l yeast extract and 30 g/l glucose) at 30 °C with agitation at 120 rpm. The yeast cells were harvested in the late exponential growth phase by centrifugation at 8400 × g and 4 °C for 10 min. To remove the leftover medium components, the harvested cells were washed three times with a sterilized NaCl solution used as a dispersion medium in the

Results and discussion

Dynamic behaviors of positively charged PS-NH₂ toward yeast cells in different concentrations of NaCl solutions (5 mM and 154 mM) were previously observed using CLSM [15]. CLSM can directly observe the dynamic NP behavior at the micro-level. However, it is impossible to analyze the location of NPs at the nano-level. AFM is a powerful tool for nanoscale imaging of biological surface topography [22,23]. Fig. 1 shows the AFM images of the yeast cell surface topography after exposure to positively

Conclusions

In conclusion, we investigated the course of NP uptake into yeast cells using AFM and CLSM. Time-lapse imaging of the yeast cell surface topography using AFM revealed that the conically-shaped pits connected with endocytosis were observed in a 154 mM NaCl solution, and the surface was completely covered with NPs within 60 s in a 5 mM NaCl solution. AFM force measurements confirmed that the electrostatic attractive force in the 5 mM NaCl solution was obviously stronger than that in the 154 mM

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Number 24310066, 26550065 and 15H01745. The authors thank Ms. A. Yasui for her help with the laboratory work.

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Direct measurements of colloidal behavior of polystyrene nanoparticles into budding yeast cells using atomic force microscopy and confocal microscopy

Abstract

Graphical abstract

Introduction

Section snippets

Yeast strain and growth condition

Results and discussion

Conclusions

Acknowledgments

Toxicol. In Vitro

J. Colloid Interface Sci.

J. Hazard. Mater.

Adv. Powder Technol.

Colloids Surfaces A Physicochem. Eng. Asp.

Mechatronics

Chem. Eng. Sci.

Biomaterials

Colloids Surf. B Biointerfaces

Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes

Nano Lett.

Photoluminescent Diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells

ACS Nano

Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type

Nat. Nanotechnol.

Penetration of lipid membranes by gold nanoparticles: insights into cellular uptake, cytotoxicity, and their relationship

ACS Nano

Chemistry of carbon nanotubes

Chem. Rev.

Cellular uptake of nanoparticles by membrane penetration: a study combining confocal microscopy with FTIR spectroelectrochemistry

ACS Nano

Toxic potential of materials at the nanolevel

Science.