- Email: [email protected]
Control of colloidal behavior of polystyrene latex nanoparticles and their cytotoxicity toward yeast cells using water-soluble polymers
Advanced Powder Technology 29 (2018) 2204–2210
Contents lists available at ScienceDirect
Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
Original Research Paper
Control of colloidal behavior of polystyrene latex nanoparticles and their cytotoxicity toward yeast cells using water-soluble polymers Shohei Yumiyama, Eri Fujisawa, Yasuhiro Konishi, Toshiyuki Nomura ⇑ Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
a r t i c l e
i n f o
Article history: Received 15 February 2018 Received in revised form 19 April 2018 Accepted 4 June 2018 Available online 14 June 2018 Keywords: Polystyrene latex nanoparticles Uptake Saccharomyces cerevisiae Single nanoparticle probe Atomic force microscopy
a b s t r a c t Positively charged polystyrene latex (PSL) nanoparticles (NPs) dispersed in physiological saline (154 mM NaCl solution) are taken up by yeast cells. However, in low ionic strength solutions, the yeast cells are covered with the NPs, leading to cell death. The environmental conditions under which NPs are taken up are therefore limited. In this study, we attempted to control the uptake of positively charged PSL NPs by Saccharomyces cerevisiae in 5 mM NaCl solution using a water-soluble polymer. Addition of a small amount of anionic sodium carboxymethylcellulose (CMC), which has a carboxyl group, to 5 mM NaCl solution allowed the uptake of PSL NPs by living yeast cells. In contrast, non-ionic methylcellulose did not affect the NP behavior. This is because the negatively charged CMC adhered to the positively charged PSL NP surfaces and the surface charge changed from positive to negative. Atomic force microscopy using a single-NP probe consisting of one NP immobilized on the flattened end of the silicon nitride tip showed that CMC significantly reduced the interaction force between a negatively charged living yeast cell and a positively charged PSL NP. Ó 2018 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
1. Introduction The interaction forces between engineered nanoparticles (NPs) and biological cells have received considerable attention due to the potential use of NPs in biological applications such as drugand gene-delivery systems [1–6], and their possible adverse impacts on human health and the environment [7,8]. Yeast is widely used as a model unicellular eukaryotic microorganism due to many similarities to animal and plant cells. However, only a few studies have investigated the potential impact of NPs (metal oxides, silver, and fullerene) on yeast cells [9–13]. These studies reported that NPs show little or no toxicity toward yeast. The uptake of positively charged gold NPs by yeast spheroplasts whose cell walls had been almost completely removed was demonstrated [14]. However, although NP uptake by animal cells has been reported, there have been few studies of NP uptake by yeast. This is probably because yeast cells are protected by a robust cell wall [9]. In our previous studies, confocal observation revealed that live yeast cells take up positively charged amine-modified polystyrene latex (PSL) NPs dispersed in physiological saline (154 mM NaCl solution). However, the yeast cells were completely covered with the NPs in a low ionic strength (5 mM NaCl solution), leading to ⇑ Corresponding author. E-mail address: [email protected] (T. Nomura).
cell death [15–17]. The environmental conditions for NP uptake are therefore limited. It is important to know the physicochemical properties of biological cells and NPs that affect colloidal behavior (diffusion, adhesion, uptake). However, theoretical estimation of the biological interactions between cells and NPs are difficult because of their heterogeneous surfaces mediated by various complex surface macromolecules, including proteins, polysaccharides, and lipids. Atomic force microscopy (AFM) can directly measure the interaction forces between surfaces and is a powerful tool for measuring biological surface forces [18,19]. Various methods based on AFM have been used to measure the interaction forces between a solid surface and a biological cell, e.g., colloidal probe [20–22], cell probe [19,23,24], and NP probe methods [25]. However, only a few studies have been performed using a single-NP probe, i.e., only one NP immobilized on the end of the AFM cantilever tip [26,27]. To the best of our knowledge, there are few reports of the biological interaction between a single NP and a biological cell. In this study, we attempted to control the uptake of positively charged PSL NPs by the budding yeast Saccharomyces cerevisiae cells in the low ionic strength dispersion medium (5 mM NaCl solution) using a water-soluble polymer, namely anionic sodium carboxymethylcellulose (CMC) or non-ionic methylcellulose (MC). A single-NP probe consisting of one NP immobilized on the flattened end of the silicon nitride tip of the AFM cantilever was
https://doi.org/10.1016/j.apt.2018.06.004 0921-8831/Ó 2018 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
prepared by a simple method, without surface modification of the tip, and used direct measurement of the force between a living yeast cell and a PSL NP. 2. Materials and methods 2.1. Yeast strain and growth conditions The budding yeast S. cerevisiae JCM 7255 T, which was used as a model unicellular eukaryotic organism, was purchased from the Japan Collection of Microorganisms (Tsukuba, Japan). 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 collected by centrifugation and were washed three times with sterilized 5 mM NaCl solution to remove remaining medium components. A yeast cell suspension was prepared by resuspending the washed cells in the sterilized dispersion medium, and the cell concentration was adjusted using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). 2.2. Polystyrene latex nanoparticles Positively charged amine-modified PSL NPs with a fluorophore (nominal diameter: 100 nm) were purchased from Sigma-Aldrich (L9904, St. Louis, MO). A PSL NP suspension was prepared by suspending the NPs in sterilized dispersion medium. 2.3. Dispersion medium CMC was purchased from Sigma-Aldrich (419273, average molecular weight: 90 000) and 0.5% (w/v) sterilized MC 400 solution was purchased from Wako Pure Chemical Industries, Ltd. (133-14255, Osaka, Japan). The dispersion medium was prepared by dissolving a water-soluble polymer in 5 mM NaCl solution. The final concentration of the polymer varied from 0 ppm (control) to 4 ppm. 2.4. Cytotoxicity test of polystyrene latex nanoparticles toward yeast cells The yeast cell suspension (500 lL) and PSL NP suspension (500 lL) were mixed in a microtube. The microtube was then placed on a tube rotator at 60 rpm for 60 min at room temperature. After exposure, the diluted suspension (100 lL) was spread on YE agar plates and incubated for 2 d at 30 °C. The number of colonyforming units (CFUs) on the plates was counted. Cell viability was estimated by comparing the number of CFUs on the plates with that on a control plate. The suspension spread on the control plate did not include NPs. The location of the PSL NPs was observed using a confocal laser scanning microscopy (CLSM; FV-1000D, Olympus, Tokyo, Japan) with oil-immersion objective lenses of numerical aperture 1.40 (UPLSAPO 100XO, Olympus). After CLSM imaging, the dead cells were stained with trypan blue, which only infiltrates cells with disrupted membranes. The final concentrations of the PSL NPs and yeast cells were 40 µg/mL and 5 105 cells/mL, respectively. 2.5. Immobilization of living yeast cells on glass substrate A non-coated glass slide (NC-glass) (S7224, Matsunami Glass, Kishiwada, Japan) was rinsed with deionized water and then ethanol for 15 min each. After drying with nitrogen gas, 4 mg/mL of dopamine hydrochloride (Sigma-Aldrich) in 10 mM Tris–HCl buffer (pH 8.5) solution (100 lL) was placed on the cleaned NC-glass surface and left for 1 h at room temperature to create a coating consist-
2205
ing of a thin polymer film produced by dopamine polymerization [28,29]. The polydopamine-coated glass slide (PDA-glass) was washed with deionized water and dried under vacuum for 10 min. The yeast cell suspension (100 lL, 1 106 cells/mL) was placed on the prepared PDA-glass, and the sample was left for 1 h to allow cell immobilization on the PDA-glass. The glass was then rinsed with dispersion medium to remove non-immobilized yeast cells in the suspension. The immobilized cells were stained using a LIVE/DEAD Yeast Viability Kit (Molecular Probes, Eugene, OR) and the cell viability was determined using CLSM. 2.6. Preparation of single nanoparticle probe An NP probe was simply prepared as follows. A V-shaped cantilever (OMCL-TR400PSA, Olympus, nominal spring constant = 0.08 N/m) with a silicon nitride pyramidal tip was used to prepare an NP probe. To prepare a flattened tip end for single NP immobilization, the cleaned NC-glass imaging was performed in an airconditioned laboratory (24 ± 2 °C) using AFM (MFP-3D-BIO-J, Oxford Instruments Asylum Research, Santa Barbara, CA) in contact mode (scan range: 20 µm 20 µm, scan rate: 0.5 Hz, set point: 2.0 V, Z voltage: 130 V, scan time: 90 s). After imaging, the cantilever was sequentially rinsed with chloroform, ethanol, and deionized water for 15 min each. The cantilever was cleaned using a plasma cleaner (PDC-32G, Harrick Plasma, Ithaca, NY) operated at the middle level under reduced air pressure for 15 min. Finally, the flattened tip of the cantilever was immersed in a 40 µg/mL PSL NP suspension dispersed in deionized water and dried under vacuum for 10 min. The prepared single-NP probe was coated with a thin layer of osmium using an osmium coater (Neoc-ST, Meiwafosis, Osaka, Japan) and was observed using a field-emission scanning electron microscopy (SEM) (JSM-6700F, JEOL, Tokyo, Japan). 2.7. Direct measurement of interaction force between a nanoparticle and a cell The force between the single NP surface of the NP probe and soft surface of a living yeast cell in dispersion medium was measured using AFM integrated with an inverted optical microscope (Eclipse TE2000, Nikon, Tokyo, Japan). The NP probe was engaged with the yeast cell surface using the inverted optical microscope. Multiple force curves were recorded at various spots (1 µm 1 µm, 10 pixels 10 pixels) using a maximum applied force of 100 pN and contact approach and retraction rates of 200 nm/s. The spring constant of each individual cantilever was calibrated using the thermal-noise method [30] embedded in the AFM software. The cantilever spring constants were 0.095 ± 0.002 N/m. Three yeast cells from independent cultures were probed using independent single-NP probes under each condition. The maximum downward force exerted on the NP probe is referred to as the adhesion force, and was measured relative to a baseline [31]. 2.8. Hydrodynamic diameter and electrophoretic mobility measurements The hydrodynamic diameters and electrophoretic mobilities (EPMs) of yeast cells and PSL NPs dispersed in 5 mM NaCl aqueous solutions containing a water-soluble polymer were measured using a zeta potential and particle size analyzer (ELS-Z, Otsuka Electronics, Hirakata, Japan). 2.9. Contact angle measurements and surface energy estimation The contact angle between the sample and a probe liquid was measured with a contact angle analyzer (FTA125, First Ten
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
Ångstroms, Portsmouth, VA) using the sessile drop technique, following a previously reported procedure [32,33]. Three different types of probe liquid (water, formamide, and abromonaphthalene) were used. The total surface tension cTotal, Lifshitz–van der Waals component cLW, and acid–base component pffiffiffiffiffiffiffiffiffiffiffi cAB, which can be expressed as 2 cþ c , where c and c+ indicate the electron donor and acceptor, respectively. The surface tensions were calculated from at least five independent measured contact angles using the van Oss approach [34]. Yeast cell lawns were prepared by collecting the cells on a 0.45 µm acetate cellulose membrane filter (Advantec No. 2, Tokyo, Japan) [32,33]. The PSL NP lawns were prepared following a previously reported procedure [35]. Briefly, the NP suspension was plated on a cleaned NC-glass and dried overnight at room temperature. The PSL NP layer was heated at 70 °C for 1 h. Data for the NC-glass were taken from our previous study [36].
100
䖃 CMC 䕦 MC
80
Cell viability (%)
2206
60
40
20
0 0
2.10. Calculation of Gibbs free energy
1
2
3
4
5
Polymer concentration (ppm)
The thermodynamic adhesion stability was estimated from the changes in the Gibbs free energy DGTotal, defined as the sum of the free energy changes for the Lifshitz–van der Waals (DGLW) and acid–base (DGAB) interactions [34,37]:
qffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiqffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffi ; DGLW ¼ 2 cLW cLW cLW cLW B L L S
ð1Þ
qffiffiffiffiffiffi qffiffiffiffiffiffi pffiffiffiffiffi ffi pffiffiffiffiffiffi DGAB ¼ 2 cþB cþS cB cS qffiffiffiffiffiffi qffiffiffiffiffiffi ffi pffiffiffiffiffiffi pffiffiffiffiffi cþB cþL cB cL 2 qffiffiffiffiffiffi qffiffiffiffiffiffi ffi pffiffiffiffiffiffi pffiffiffiffiffi cþS cþL cS cL ; 2
ð2Þ
The subscripts B and L refer to the microbial cell and the dispersion liquid, respectively and the subscript S refers to the substrate or the NP. 3. Results and discussion When yeast cells (final concentration: 5 105 cells/mL) were exposed to positively charged PSL NPs in 154 mM NaCl (final concentration: 40 µg/mL), the cells took up the NPs inside the cells and were alive. In contrast, when the concentration of PSLNPs in 5 mM NaCl was over 15 µg/mL, the cells were surrounded by PSL NPs and were dead [15–17]. We also reported similar colloidal behavior and cytotoxicity of the positively charged PSL NPs toward Aspergillus oryzae [38]. To control the colloidal behavior of the NPs toward the cells, a water-soluble polymer (CMC or MC) was added to the 5 mM NaCl dispersion medium and a cytotoxicity test was performed. Fig. 1 shows the viability of the yeast cells after exposure for 1 h to a 40 µg/mL PSL NP suspension as a function of added polymer concentration in the 5 mM NaCl solution. When CMC was added, the cells were all dead at 1 ppm, as in a control experiment (5 mM NaCl solution without polymer), the ratio of living cells increased at 2 ppm, and ca. 80% of the cells were alive above 3 ppm. In contrast, when MC was added, all the cells died regardless of the polymer concentration in the dispersion medium, even at an MC concentration of 20 ppm. These results show that cell death in 5 mM NaCl solution caused by covering with NPs can be controlled by adding an appropriate amount of CMC to the dispersion medium. CLSM was used to determine the location of the PSL NPs. Fig. 2 shows CLSM images of the yeast cells after exposure for 1 h to 40 µg/mL PSL NPs in 5 mM NaCl dispersion medium containing a
Fig. 1. Viability of yeast cells after exposure for 1 h to 40 µg/mL polystyrene latex nanoparticle suspension as a function of added polymer concentration in 5 mM NaCl solution. Mean ± standard error, N = 3.
water-soluble polymer, and transmission images of the cells after trypan blue staining. When CMC was added, the cells were completely covered with green fluorescent PSL NPs at 1 ppm, as in a control (5 mM NaCl without CMC), PSL NPs partly adhered to the cell surfaces at 2 ppm, and the PSL NPs were taken up by the cells and did not adhere to the cell surfaces above 3 ppm. In contrast, when MC was added, all the cells were completely covered with PSL NPs regardless of the concentration of added MC. The cells to which the NPs adhered were stained by trypan blue, indicating that the cells were dead, whereas the cells that took up the NPs were alive. Positively charged PSL NPs were therefore successfully introduced into the living yeast cells by adding a small amount of CMC to a low ionic strength NaCl solution. The EPM and hydrodynamic diameter were measured to investigate the significant changes in the colloidal behavior of aminemodified PSL NPs toward the yeast cells caused by adding CMC to 5 mM NaCl solution. Fig. 3 shows the EPMs and hydrodynamic diameters of the yeast cells and the PSL NPs as a function of added polymer concentration. The EPMs of the yeast cells were almost constant regardless of the polymer concentration. In contrast, the EPMs of the PSL NPs decreased with increasing CMC concentration, and the surface charge changed from positive to negative between 2 and 3 ppm. MC addition hardly changed the EPMs of the PSL NPs. These results indicate that the negatively charged anionic CMC, which contains a carboxyl group, adhere to the positively charged PSL NP surfaces. Oriekhovo and Stoll reported that fulvic acid, which has a carboxyl group, adheres to the positively charged surfaces of cerium oxide particles under acidic conditions [39]. This suggests that adhesion of CMC to the PSL NPs decreased the electrostatic attractive force between the NPs and cells. The hydrodynamic diameter of the PSL NPs gradually increased with increasing added polymer concentration; a considerable increase was observed on addition of 2 ppm CMC. The addition of 2 ppm CMC clearly caused aggregation of the PSL NPs. This is because the absolute value of the EPM was lower than those under other conditions. This could prevent uptake of the PSL NPs by the cells because of a size effect. In addition, the yeast cells did not aggregate as a result of polymer addition. The interaction force between a single NP and a single living cell was measured directly using AFM. One NP was immobilized on the flattened end of the cantilever tip and living cells were immobilized on the PDA-glass surface. Fig. 4A shows typical SEM image
2207
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
1 ppm
2 ppm
3 ppm
4 ppm
A
B
Fig. 2. Location of polystyrene latex nanoparticles (PSL NPs) and viability of yeast cells after exposure for 1 h to 40 µg/mL PSL NP suspension with added (A) carboxymethylcellulose and (B) methylcellulose. Upper images: confocal microscopy images, fluorescent green: PSL NPs; lower images: transmission images, fluorescent blue: dead cells stained with trypan blue.
of a single-NP probe. The image confirm that only one NP was immobilized on the flattened end of the AFM tip using a simple method, without surface modification of the tip. However, we observed ten prepared AFM probes with SEM. As a result, only two probes were successful. Fig. 4B shows a CLSM image of the immobilized cells on the PDA-glass. Most of the cells were fluorescent green, indicating that living yeast cells were successfully immobilized on the PDA-glass substrate. In contrast, few immobilized cells were present on a NC-glass, which had not undergone surface modification after washing. The physicochemical properties of the cells and substrates are important in cell and NP immobilization. The contact angles for various probe liquids with different polarities, along with the surface tensions, are listed in Table 1. The contact angle of water on the PDA-glass was higher than that on the NC-glass. This shows that coating with PDA increased the hydrophobicity of the NC-glass surface. The water contact angle of the amine-modified PSL NP lawn was reported to be 50.5° [40]; the value obtained in this study was similar
(50 ± 2°). Changes in the interfacial free energies when a yeast cell adheres to substrates or a PSL NP are listed in Table 2. When the yeast cells adhered to a glass substrate, DGTotal for the NC-glass was positive, whereas that for the PDA-glass was negative. This means that the PDA-glass was a thermodynamically favorable surface for the immobilization of yeast cells. DGTotal for the PSL NP was positive, meaning that the yeast cells were a thermodynamically unfavorable surface for the adhesion of PSL NPs. Fig. 5A shows the representative approach curves between an NP and a living yeast cell in 5 mM NaCl solutions containing 4 ppm water-soluble polymer. In the 5 mM NaCl solution without a polymer (control), the attractive force was obviously detected due to the electrostatic attractive force. Similarly, the attractive force was detected when adding 4 ppm MC to the 5 mM NaCl solution. As shown in Fig. 3, MC addition did not affect the surface charge on the PSL NPs. The cells were therefore covered with PSL NPs, leading to cell death (Fig. 2). This is presumably because the covering of the yeast cell by NPs causes a decrease in the fluidity
2208
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
Fig. 3. (A) Electrophoretic mobility and (B) hydrodynamic diameter of Saccharomyces cerevisiae and polystyrene latex nanoparticles as a function of added polymer concentration in 5 mM NaCl solution. Mean ± standard error, N = 3.
A
B
Table 2 Interfacial free energies when the yeast adheres to glass substrate or polystyrene latex nanoparticle (mJ/m2).
Tip
100 nm
Object S
DGLW
DGAB
DGTotal
NC-glass PDA-glass PSL NP
6.0 6.0 4.4
44.1 4.2 25.1
38.1 1.8 20.7
NP
Fig. 4. (A) Scanning electron microscopy image of single-nanoparticle probe and (B) confocal microscopy image of immobilized yeast cells on polydopamine-coated glass slide. Fluorescent green: living cells stained with SYTO 9, fluorescent red: dead cells stained with propidium iodide.
of the cell membrane and inhibition of metabolism through the cell membrane [15,41,42]. In contrast, when CMC was added to the 5 mM NaCl solution, the attractive force almost disappeared. As shown in Fig. 3, CMC addition changed the surface charge on the PSL NPs from negative to positive, leading to a reduction in the electrostatic attractive force between the NPs and yeast cells. In addition, CMC adsorbed on the PSL NP surface is presumed to cause steric forces on the polymer present on the cell surface. In our previous study, when the negatively charged PSL NPs were exposed to the yeast cells, the NPs were dispersed in the dispersion medium due to the electrostatic repulsive force [15]. As shown in Fig. 2, addition of 4 ppm CMC to 5 mM NaCl solution allowed the uptake
of PSL NPs by living yeast cells. These results indicate that the repulsive force between the NPs and yeast cells is very weak, allowing NPs to collide with the cells. Fig. 5B shows typical histograms of the adhesion forces between the NP and the living yeast cell, along with the representative retraction curves. The adhesion force was detected in the 5 mM NaCl solution without a polymer (control) at 400 ± 20 pN, and with 4 ppm MC at 260 ± 20 pN, but not with 4 ppm CMC (adhesion force: 20 ± 1 pN). The trend in the adhesion force was similar to that in the attractive force shown in Fig. 5A. These results imply that the adhered NPs on the cell surfaces could not be taken up by cells because of the strong adhesion forces between the NPs and the cell surface. In contrast, it is suggested that in the 5 mM NaCl solution with CMC, the NPs that collided with the cell surfaces as a result of Brownian motion were taken up by endocytosis, resulting that the cells remained alive. Several peaks were detected in the retraction curves for the 5 mM NaCl solutions without a polymer (control) and with 4 ppm MC. These peaks represent multiple polymer chains or binding with multiple sites on the biological surfaces [31,43,44].
Table 1 Contact angles and surface tensions of yeast, glass substrate and polystyrene latex nanoparticle. Mean ± standard error, N 5. Cell/Substrate/NP
h S. cerevisiae NC-glass PDA-glass PSL NP *
Surface tension (mJ/m2)
Contact angle (Degree) Wa*
11 ± 1 17 ± 1 57 ± 1 50 ± 2
Fo*
Br*
h
h
10 ± 1 31 ± 1 21 ± 1 43 ± 2
26 ± 3 21 ± 1 20 ± 1 36 ± 1
Wa, Water; Fo, Formamide; Br, a-Bromonaphthalene.
cLW
c+
c–
cAB
cTotal
40 42 42 36
1.2 0.1 2.7 0.4
54 62 13 32
16 4.1 11 7.1
56 46 53 44
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
2209
Fig. 5. Interaction forces between polystyrene latex nanoparticle and living yeast cell using atomic force microscopy in 5 mM NaCl solution (without polymer, control), 5 mM NaCl solution containing 4 ppm carboxymethylcellulose (CMC), and 5 mM NaCl solution containing 4 ppm methylcellulose (MC): (A) approach curves and (B) histograms of adhesion forces, along with retraction curves. N = 300 curves from three independent experiments.
4. Conclusions The uptake of positively charged PSL NPs by the budding yeast Saccharomyces cerevisiae cells in 5 mM NaCl solution was attempted to control using a water-soluble polymer. CLSM observations demonstrated that the PSL NPs were successfully taken up by the living yeast cells when a small amount of CMC was added to a low ionic strength NaCl solution. In contrast, nonionic MC did not affect the NP behavior. The negatively charged anionic CMC, which has a carboxyl group, adhered to the surfaces of the positively charged PSL NPs and the surface charge changed from positive to negative. These results indicate that the addition of anionic CMC to a PSL NP suspension decreased the electrostatic attractive force between the NPs and cells. AFM measurements using a single-NP probe revealed that CMC significantly reduced the interaction force between a negatively charged cell and a positively charged PSL NP.
[4]
[5] [6]
[7] [8] [9]
[10]
[11]
Acknowledgments [12]
This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Numbers 24310066 and 15H01745. [13]
References [1] B.D. Chithrani, W.C.W. Chan, Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes, Nano Lett. 7 (2007) 1542–1550, https://doi.org/10.1021/nl070363y. [2] O. Faklaris, V. Joshi, T. Irinopoulou, P. Tauc, M. Sennour, H. Girard, C. Gesset, J. Arnault, A. Thorel, J.-P. Boudou, P.A. Curmi, F. Treussart, Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells, ACS Nano 3 (2009) 3955–3962, https://doi.org/10.1021/ nn901014j. [3] K. Kostarelos, L. Lacerda, G. Pastorin, W. Wu, S. Wieckowski, J. Luangsivilay, S. Godefroy, D. Pantarotto, J.-P. Briand, S. Muller, M. Prato, A. Bianco, Cellular uptake of functionalized carbon nanotubes is independent of functional group
[14]
[15]
[16]
and cell type, Nat. Nanotechnol. 2 (2007) 108–113, https://doi.org/10.1038/ nnano.2006.209. J. Lin, H. Zhang, Z. Chen, Y. Zheng, Penetration of lipid membranes by gold nanoparticles: insights into cellular uptake, cytotoxicity, and their relationship, ACS Nano 4 (2010) 5421–5429, https://doi.org/10.1021/ nn1010792. D. Tasis, N. Tagmatarchis, A. Bianco, M. Prato, Chemistry of carbon nanotubes, Chem. Rev. 106 (2006) 1105–1136, https://doi.org/10.1021/cr050569o. T. Wang, J. Bai, X. Jiang, G.U. Nienhaus, Cellular uptake of nanoparticles by membrane penetration: a study combining confocal microscopy with FTIR spectroelectrochemistry, ACS Nano 6 (2012) 1251–1259, https://doi.org/ 10.1021/nn203892h. A. Nel, T. Xia, L. Mädler, N. Li, Toxic potential of materials at the nanolevel, Science 311 (2006) 622–627, https://doi.org/10.1126/science.1114397. A. Kahru, A. Ivask, Mapping the dawn of nanoecotoxicological research, Acc. Chem. Res. 46 (2013) 823–833, https://doi.org/10.1021/ar3000212. K. Kasemets, A. Ivask, H.-C. Dubourguier, A. Kahru, Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae, Toxicol. Vitr. 23 (2009) 1116–1122, https://doi.org/10.1016/j.tiv.2009.05.015. S. Lee, J. Lee, K. Kim, S.-J. Sim, M.B. Gu, J. Yi, J. Lee, Eco-toxicity of commercial silver nanopowders to bacterial and yeast strains, Biotechnol. Bioprocess Eng. 14 (2009) 490–495, https://doi.org/10.1007/s12257-008-0254-6. H. Schwegmann, A.J. Feitz, F.H. Frimmel, Influence of the zeta potential on the sorption and toxicity of iron oxide nanoparticles on S. cerevisiae and E. coli, J. Colloid Interface Sci. 347 (2010) 43–48, https://doi.org/10.1016/j. jcis.2010.02.028. A.N. Hadduck, V. Hindagolla, A.E. Contreras, Q. Li, A.T. Bakalinsky, Does aqueous fullerene inhibit the growth of Saccharomyces cerevisiae or Escherichia coli?, Appl Environ. Microbiol. 76 (2010) 8239–8242, https://doi.org/10.1128/ AEM.01925-10. C. García-Saucedo, J.A. Field, L. Otero-Gonzalez, R. Sierra-Álvarez, Low toxicity of HfO2, SiO2, Al2O3 and CeO2 nanoparticles to the yeast, Saccharomyces cerevisiae, J. Hazard. Mater. 192 (2011) 1572–1579, https://doi.org/10.1016/j. jhazmat.2011.06.081. C. Prescianotto-Baschong, H. Riezman, Morphology of the yeast endocytic pathway, Mol. Biol. Cell. 9 (1998) 173–189, https://doi.org/10.1091/ mbc.9.1.173. T. Nomura, J. Miyazaki, A. Miyamoto, Y. Kuriyama, H. Tokumoto, Y. Konishi, Exposure of the yeast saccharomyces cerevisiae to functionalized polystyrene latex nanoparticles: influence of surface charge on toxicity, Environ. Sci. Technol. 47 (2013) 3417–3423, https://doi.org/10.1021/es400053x. J. Miyazaki, Y. Kuriyama, A. Miyamoto, H. Tokumoto, Y. Konishi, T. Nomura, Adhesion and internalization of functionalized polystyrene latex nanoparticles toward the yeast Saccharomyces cerevisiae, Adv. Powder Technol. 25 (2014) 1394–1397, https://doi.org/10.1016/j.apt.2014.06.014.
2210
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
[17] J. Miyazaki, Y. Kuriyama, H. Tokumoto, Y. Konishi, T. Nomura, Cytotoxicity and behavior of polystyrene latex nanoparticles to budding yeast, Colloids Surf. A Physicochem. Eng. Asp. 469 (2015) 287–293, https://doi.org/10.1016/ j.colsurfa.2015.01.046. [18] Y.F. Dufrene, Atomic force microscopy, a powerful tool in microbiology, J. Bacteriol. 184 (2002) 5205–5213, https://doi.org/10.1128/JB.184.19.5205. [19] Y.F. Dufrene, Sticky microbes: forces in microbial cell adhesion, Trends Microbiol. 23 (2015) 376–382, https://doi.org/10.1016/j.tim.2015.01.011. [20] M. Herzberg, S. Kang, M. Elimelech, Role of extracellular polymeric substances (EPS) in biofouling of reverse osmosis membranes, Environ. Sci. Technol. 43 (2009) 4393–4398, https://doi.org/10.1021/es900087j. [21] H. Shinto, Y. Aso, T. Fukasawa, K. Higashitani, Adhesion of melanoma cells to the surfaces of microspheres studied by atomic force microscopy, Colloids Surf. B Biointerf. 91 (2012) 114–121, https://doi.org/10.1016/ j.colsurfb.2011.10.060. [22] L.-S.Z. Rathje, N. Nordgren, T. Pettersson, D. Rönnlund, J. Widengren, P. Aspenström, A.K.B. Gad, Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness, Proc. Natl. Acad. Sci. USA 111 (2014) 1515–1520, https://doi.org/10.1073/pnas.1300238111. [23] M. Benoit, D. Gabriel, G. Gerisch, H.E. Gaub, Discrete interactions in cell adhesion measured by single-molecule force spectroscopy, Nat. Cell Biol. 2 (2000) 313–317, https://doi.org/10.1038/35014000. [24] S. Kang, M. Elimelech, Bioinspired single bacterial cell force spectroscopy, Langmuir 25 (2009) 9656–9659, https://doi.org/10.1021/la902247w. [25] I.U. Vakarelski, K. Higashitani, Single-nanoparticle-terminated tips for scanning probe microscopy, Langmuir 22 (2006) 2931–2934, https://doi.org/ 10.1021/la0528145. [26] T. Kim, S. Myung, T.H. Kim, S. Hong, Robust single-nanoparticle probe for contact-mode analysis and dip-pen nanolithography, Small 4 (2008) 1072– 1075, https://doi.org/10.1002/smll.200701102. [27] J.K. Vasir, V. Labhasetwar, Quantification of the force of nanoparticle-cell membrane interactions and its influence on intracellular trafficking of nanoparticles, Biomaterials 29 (2008) 4244–4252, https://doi.org/10.1016/j. biomaterials.2008.07.020. [28] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Mussel-inspired surface chemistry for multifunctional coatings, Science 318 (2007) 426–430, https:// doi.org/10.1126/science.1147241. [29] H. Lee, B.P. Lee, P.B. Messersmith, A reversible wet/dry adhesive inspired by mussels and geckos, Nature 448 (2007) 338–341, https://doi.org/ 10.1038/nature05968. [30] J.L. Hutter, J. Bechhoefer, Calibration of atomic-force microscope tips, Rev. Sci. Instrum. 64 (1993) 1868–1873, https://doi.org/10.1063/1.1143970. [31] A. Beaussart, A. Baker, S.L. Kuchma, S. El-Kirat-Chatel, G.A. O’Tolle, Y.F. Dufrene, Nanoscale adhesion forces of Pseudomonas aeruginosa type IV pili, ACS Nano 8 (2014) 10723–10733, https://doi.org/10.1021/nn5044383.
[32] H.J. Busscher, A.H. Weerkamp, H.C. van der Mei, A.W. van Pelt, H.P. de Jong, J. Arends, Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion, Appl. Environ. Microbiol. 48 (1984) 980–983. [33] A. Yoshihara, H. Narahara, Y. Kuriyama, S. Toyoda, H. Tokumoto, Y. Konishi, T. Nomura, Measurement of microbial adhesive forces with a parallel plate flow chamber, J. Colloid Interface Sci. 432 (2014) 77–85, https://doi.org/10.1016/j. jcis.2014.06.052. [34] C.J. Van Oss, M.K. Chaudhury, R.J. Good, Interfacial Lifshitz-van der Waals and polar interactions in macroscopic systems, Chem. Rev. 88 (1988) 927–941, https://doi.org/10.1021/cr00088a006. [35] Y. Zhao, M. Li, Q. Lu, Z. Shi, Superhydrophobic polyimide films with a hierarchical topography: combined replica molding and layer-by-layer assembly, Langmuir 24 (2008) 12651–12657, https://doi.org/10.1021/ la8024364. [36] T. Nomura, E. Fujisawa, S. Itoh, Y. Konishi, Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms, J. Nanopart. Res. 18 (2016) 1–9, https://doi.org/10.1007/s11051-016-3471-5. [37] A. Yoshihara, N. Nobuhira, H. Narahara, S. Toyoda, H. Tokumoto, Y. Konishi, T. Nomura, Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface, Colloids Surf. B Biointerf. 131 (2015) 67–72, https://doi.org/10.1016/j.colsurfb.2015.04.038. [38] T. Nomura, S. Tani, M. Yamamoto, T. Nakagawa, S. Toyoda, E. Fujisawa, A. Yasui, Y. Konishi, Cytotoxicity and colloidal behavior of polystyrene latex nanoparticles toward filamentous fungi in isotonic solutions, Chemosphere 149 (2016) 84–90, https://doi.org/10.1016/j.chemosphere.2016.01.091. [39] O. Oriekhova, S. Stoll, Effects of pH and fulvic acids concentration on the stability of fulvic acids – cerium (IV) oxide nanoparticle complexes, Chemosphere 144 (2016) 131–137, https://doi.org/10.1016/j. chemosphere.2015.08.057. [40] D. Akilbekova, R. Philiph, A. Graham, K.M. Bratlie, Macrophage reprogramming: influence of latex beads with various functional groups on macrophage phenotype and phagocytic uptake in vitro, J. Biomed. Mater. Res. Part A 103 (2015) 262–268, https://doi.org/10.1002/jbm.a.35169. [41] E. Kojro, G. Gimpl, S. Lammich, W. Marz, F. Fahrenholz, Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase ADAM 10, Proc. Natl. Acad. Sci. USA 98 (2001) 5815–5820, https://doi.org/ 10.1073/pnas.081612998. [42] M.K. Shigenaga, T.M. Hagen, B.N. Ames, Oxidative damage and mitochondrial decay in aging, Proc. Natl. Acad. Sci. USA 91 (1994) 10771–10778, https://doi. org/10.1073/pnas.91.23.10771. [43] H.J. Butt, M. Kappl, H. Mueller, R. Raiteri, W. Meyer, J. Rühe, Steric forces measured with the atomic force microscope at various temperatures, Langmuir 15 (1999) 2559–2565, https://doi.org/10.1021/la981503+. [44] N.I. Abu-Lail, T.A. Camesano, Elasticity of pseudomonas putida KT2442 surface polymers probed with single-molecule force microscopy, Langmuir 18 (2002) 4071–4081, https://doi.org/10.1021/la015695b.
Contents lists available at ScienceDirect
Advanced Powder Technology journal homepage: www.elsevier.com/locate/apt
Original Research Paper
Control of colloidal behavior of polystyrene latex nanoparticles and their cytotoxicity toward yeast cells using water-soluble polymers Shohei Yumiyama, Eri Fujisawa, Yasuhiro Konishi, Toshiyuki Nomura ⇑ Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan
a r t i c l e
i n f o
Article history: Received 15 February 2018 Received in revised form 19 April 2018 Accepted 4 June 2018 Available online 14 June 2018 Keywords: Polystyrene latex nanoparticles Uptake Saccharomyces cerevisiae Single nanoparticle probe Atomic force microscopy
a b s t r a c t Positively charged polystyrene latex (PSL) nanoparticles (NPs) dispersed in physiological saline (154 mM NaCl solution) are taken up by yeast cells. However, in low ionic strength solutions, the yeast cells are covered with the NPs, leading to cell death. The environmental conditions under which NPs are taken up are therefore limited. In this study, we attempted to control the uptake of positively charged PSL NPs by Saccharomyces cerevisiae in 5 mM NaCl solution using a water-soluble polymer. Addition of a small amount of anionic sodium carboxymethylcellulose (CMC), which has a carboxyl group, to 5 mM NaCl solution allowed the uptake of PSL NPs by living yeast cells. In contrast, non-ionic methylcellulose did not affect the NP behavior. This is because the negatively charged CMC adhered to the positively charged PSL NP surfaces and the surface charge changed from positive to negative. Atomic force microscopy using a single-NP probe consisting of one NP immobilized on the flattened end of the silicon nitride tip showed that CMC significantly reduced the interaction force between a negatively charged living yeast cell and a positively charged PSL NP. Ó 2018 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
1. Introduction The interaction forces between engineered nanoparticles (NPs) and biological cells have received considerable attention due to the potential use of NPs in biological applications such as drugand gene-delivery systems [1–6], and their possible adverse impacts on human health and the environment [7,8]. Yeast is widely used as a model unicellular eukaryotic microorganism due to many similarities to animal and plant cells. However, only a few studies have investigated the potential impact of NPs (metal oxides, silver, and fullerene) on yeast cells [9–13]. These studies reported that NPs show little or no toxicity toward yeast. The uptake of positively charged gold NPs by yeast spheroplasts whose cell walls had been almost completely removed was demonstrated [14]. However, although NP uptake by animal cells has been reported, there have been few studies of NP uptake by yeast. This is probably because yeast cells are protected by a robust cell wall [9]. In our previous studies, confocal observation revealed that live yeast cells take up positively charged amine-modified polystyrene latex (PSL) NPs dispersed in physiological saline (154 mM NaCl solution). However, the yeast cells were completely covered with the NPs in a low ionic strength (5 mM NaCl solution), leading to ⇑ Corresponding author. E-mail address: [email protected] (T. Nomura).
cell death [15–17]. The environmental conditions for NP uptake are therefore limited. It is important to know the physicochemical properties of biological cells and NPs that affect colloidal behavior (diffusion, adhesion, uptake). However, theoretical estimation of the biological interactions between cells and NPs are difficult because of their heterogeneous surfaces mediated by various complex surface macromolecules, including proteins, polysaccharides, and lipids. Atomic force microscopy (AFM) can directly measure the interaction forces between surfaces and is a powerful tool for measuring biological surface forces [18,19]. Various methods based on AFM have been used to measure the interaction forces between a solid surface and a biological cell, e.g., colloidal probe [20–22], cell probe [19,23,24], and NP probe methods [25]. However, only a few studies have been performed using a single-NP probe, i.e., only one NP immobilized on the end of the AFM cantilever tip [26,27]. To the best of our knowledge, there are few reports of the biological interaction between a single NP and a biological cell. In this study, we attempted to control the uptake of positively charged PSL NPs by the budding yeast Saccharomyces cerevisiae cells in the low ionic strength dispersion medium (5 mM NaCl solution) using a water-soluble polymer, namely anionic sodium carboxymethylcellulose (CMC) or non-ionic methylcellulose (MC). A single-NP probe consisting of one NP immobilized on the flattened end of the silicon nitride tip of the AFM cantilever was
https://doi.org/10.1016/j.apt.2018.06.004 0921-8831/Ó 2018 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
prepared by a simple method, without surface modification of the tip, and used direct measurement of the force between a living yeast cell and a PSL NP. 2. Materials and methods 2.1. Yeast strain and growth conditions The budding yeast S. cerevisiae JCM 7255 T, which was used as a model unicellular eukaryotic organism, was purchased from the Japan Collection of Microorganisms (Tsukuba, Japan). 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 collected by centrifugation and were washed three times with sterilized 5 mM NaCl solution to remove remaining medium components. A yeast cell suspension was prepared by resuspending the washed cells in the sterilized dispersion medium, and the cell concentration was adjusted using a spectrophotometer (UVmini-1240, Shimadzu, Kyoto, Japan). 2.2. Polystyrene latex nanoparticles Positively charged amine-modified PSL NPs with a fluorophore (nominal diameter: 100 nm) were purchased from Sigma-Aldrich (L9904, St. Louis, MO). A PSL NP suspension was prepared by suspending the NPs in sterilized dispersion medium. 2.3. Dispersion medium CMC was purchased from Sigma-Aldrich (419273, average molecular weight: 90 000) and 0.5% (w/v) sterilized MC 400 solution was purchased from Wako Pure Chemical Industries, Ltd. (133-14255, Osaka, Japan). The dispersion medium was prepared by dissolving a water-soluble polymer in 5 mM NaCl solution. The final concentration of the polymer varied from 0 ppm (control) to 4 ppm. 2.4. Cytotoxicity test of polystyrene latex nanoparticles toward yeast cells The yeast cell suspension (500 lL) and PSL NP suspension (500 lL) were mixed in a microtube. The microtube was then placed on a tube rotator at 60 rpm for 60 min at room temperature. After exposure, the diluted suspension (100 lL) was spread on YE agar plates and incubated for 2 d at 30 °C. The number of colonyforming units (CFUs) on the plates was counted. Cell viability was estimated by comparing the number of CFUs on the plates with that on a control plate. The suspension spread on the control plate did not include NPs. The location of the PSL NPs was observed using a confocal laser scanning microscopy (CLSM; FV-1000D, Olympus, Tokyo, Japan) with oil-immersion objective lenses of numerical aperture 1.40 (UPLSAPO 100XO, Olympus). After CLSM imaging, the dead cells were stained with trypan blue, which only infiltrates cells with disrupted membranes. The final concentrations of the PSL NPs and yeast cells were 40 µg/mL and 5 105 cells/mL, respectively. 2.5. Immobilization of living yeast cells on glass substrate A non-coated glass slide (NC-glass) (S7224, Matsunami Glass, Kishiwada, Japan) was rinsed with deionized water and then ethanol for 15 min each. After drying with nitrogen gas, 4 mg/mL of dopamine hydrochloride (Sigma-Aldrich) in 10 mM Tris–HCl buffer (pH 8.5) solution (100 lL) was placed on the cleaned NC-glass surface and left for 1 h at room temperature to create a coating consist-
2205
ing of a thin polymer film produced by dopamine polymerization [28,29]. The polydopamine-coated glass slide (PDA-glass) was washed with deionized water and dried under vacuum for 10 min. The yeast cell suspension (100 lL, 1 106 cells/mL) was placed on the prepared PDA-glass, and the sample was left for 1 h to allow cell immobilization on the PDA-glass. The glass was then rinsed with dispersion medium to remove non-immobilized yeast cells in the suspension. The immobilized cells were stained using a LIVE/DEAD Yeast Viability Kit (Molecular Probes, Eugene, OR) and the cell viability was determined using CLSM. 2.6. Preparation of single nanoparticle probe An NP probe was simply prepared as follows. A V-shaped cantilever (OMCL-TR400PSA, Olympus, nominal spring constant = 0.08 N/m) with a silicon nitride pyramidal tip was used to prepare an NP probe. To prepare a flattened tip end for single NP immobilization, the cleaned NC-glass imaging was performed in an airconditioned laboratory (24 ± 2 °C) using AFM (MFP-3D-BIO-J, Oxford Instruments Asylum Research, Santa Barbara, CA) in contact mode (scan range: 20 µm 20 µm, scan rate: 0.5 Hz, set point: 2.0 V, Z voltage: 130 V, scan time: 90 s). After imaging, the cantilever was sequentially rinsed with chloroform, ethanol, and deionized water for 15 min each. The cantilever was cleaned using a plasma cleaner (PDC-32G, Harrick Plasma, Ithaca, NY) operated at the middle level under reduced air pressure for 15 min. Finally, the flattened tip of the cantilever was immersed in a 40 µg/mL PSL NP suspension dispersed in deionized water and dried under vacuum for 10 min. The prepared single-NP probe was coated with a thin layer of osmium using an osmium coater (Neoc-ST, Meiwafosis, Osaka, Japan) and was observed using a field-emission scanning electron microscopy (SEM) (JSM-6700F, JEOL, Tokyo, Japan). 2.7. Direct measurement of interaction force between a nanoparticle and a cell The force between the single NP surface of the NP probe and soft surface of a living yeast cell in dispersion medium was measured using AFM integrated with an inverted optical microscope (Eclipse TE2000, Nikon, Tokyo, Japan). The NP probe was engaged with the yeast cell surface using the inverted optical microscope. Multiple force curves were recorded at various spots (1 µm 1 µm, 10 pixels 10 pixels) using a maximum applied force of 100 pN and contact approach and retraction rates of 200 nm/s. The spring constant of each individual cantilever was calibrated using the thermal-noise method [30] embedded in the AFM software. The cantilever spring constants were 0.095 ± 0.002 N/m. Three yeast cells from independent cultures were probed using independent single-NP probes under each condition. The maximum downward force exerted on the NP probe is referred to as the adhesion force, and was measured relative to a baseline [31]. 2.8. Hydrodynamic diameter and electrophoretic mobility measurements The hydrodynamic diameters and electrophoretic mobilities (EPMs) of yeast cells and PSL NPs dispersed in 5 mM NaCl aqueous solutions containing a water-soluble polymer were measured using a zeta potential and particle size analyzer (ELS-Z, Otsuka Electronics, Hirakata, Japan). 2.9. Contact angle measurements and surface energy estimation The contact angle between the sample and a probe liquid was measured with a contact angle analyzer (FTA125, First Ten
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
Ångstroms, Portsmouth, VA) using the sessile drop technique, following a previously reported procedure [32,33]. Three different types of probe liquid (water, formamide, and abromonaphthalene) were used. The total surface tension cTotal, Lifshitz–van der Waals component cLW, and acid–base component pffiffiffiffiffiffiffiffiffiffiffi cAB, which can be expressed as 2 cþ c , where c and c+ indicate the electron donor and acceptor, respectively. The surface tensions were calculated from at least five independent measured contact angles using the van Oss approach [34]. Yeast cell lawns were prepared by collecting the cells on a 0.45 µm acetate cellulose membrane filter (Advantec No. 2, Tokyo, Japan) [32,33]. The PSL NP lawns were prepared following a previously reported procedure [35]. Briefly, the NP suspension was plated on a cleaned NC-glass and dried overnight at room temperature. The PSL NP layer was heated at 70 °C for 1 h. Data for the NC-glass were taken from our previous study [36].
100
䖃 CMC 䕦 MC
80
Cell viability (%)
2206
60
40
20
0 0
2.10. Calculation of Gibbs free energy
1
2
3
4
5
Polymer concentration (ppm)
The thermodynamic adhesion stability was estimated from the changes in the Gibbs free energy DGTotal, defined as the sum of the free energy changes for the Lifshitz–van der Waals (DGLW) and acid–base (DGAB) interactions [34,37]:
qffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffiqffiffiffiffiffiffiffiffi qffiffiffiffiffiffiffiffi ; DGLW ¼ 2 cLW cLW cLW cLW B L L S
ð1Þ
qffiffiffiffiffiffi qffiffiffiffiffiffi pffiffiffiffiffi ffi pffiffiffiffiffiffi DGAB ¼ 2 cþB cþS cB cS qffiffiffiffiffiffi qffiffiffiffiffiffi ffi pffiffiffiffiffiffi pffiffiffiffiffi cþB cþL cB cL 2 qffiffiffiffiffiffi qffiffiffiffiffiffi ffi pffiffiffiffiffiffi pffiffiffiffiffi cþS cþL cS cL ; 2
ð2Þ
The subscripts B and L refer to the microbial cell and the dispersion liquid, respectively and the subscript S refers to the substrate or the NP. 3. Results and discussion When yeast cells (final concentration: 5 105 cells/mL) were exposed to positively charged PSL NPs in 154 mM NaCl (final concentration: 40 µg/mL), the cells took up the NPs inside the cells and were alive. In contrast, when the concentration of PSLNPs in 5 mM NaCl was over 15 µg/mL, the cells were surrounded by PSL NPs and were dead [15–17]. We also reported similar colloidal behavior and cytotoxicity of the positively charged PSL NPs toward Aspergillus oryzae [38]. To control the colloidal behavior of the NPs toward the cells, a water-soluble polymer (CMC or MC) was added to the 5 mM NaCl dispersion medium and a cytotoxicity test was performed. Fig. 1 shows the viability of the yeast cells after exposure for 1 h to a 40 µg/mL PSL NP suspension as a function of added polymer concentration in the 5 mM NaCl solution. When CMC was added, the cells were all dead at 1 ppm, as in a control experiment (5 mM NaCl solution without polymer), the ratio of living cells increased at 2 ppm, and ca. 80% of the cells were alive above 3 ppm. In contrast, when MC was added, all the cells died regardless of the polymer concentration in the dispersion medium, even at an MC concentration of 20 ppm. These results show that cell death in 5 mM NaCl solution caused by covering with NPs can be controlled by adding an appropriate amount of CMC to the dispersion medium. CLSM was used to determine the location of the PSL NPs. Fig. 2 shows CLSM images of the yeast cells after exposure for 1 h to 40 µg/mL PSL NPs in 5 mM NaCl dispersion medium containing a
Fig. 1. Viability of yeast cells after exposure for 1 h to 40 µg/mL polystyrene latex nanoparticle suspension as a function of added polymer concentration in 5 mM NaCl solution. Mean ± standard error, N = 3.
water-soluble polymer, and transmission images of the cells after trypan blue staining. When CMC was added, the cells were completely covered with green fluorescent PSL NPs at 1 ppm, as in a control (5 mM NaCl without CMC), PSL NPs partly adhered to the cell surfaces at 2 ppm, and the PSL NPs were taken up by the cells and did not adhere to the cell surfaces above 3 ppm. In contrast, when MC was added, all the cells were completely covered with PSL NPs regardless of the concentration of added MC. The cells to which the NPs adhered were stained by trypan blue, indicating that the cells were dead, whereas the cells that took up the NPs were alive. Positively charged PSL NPs were therefore successfully introduced into the living yeast cells by adding a small amount of CMC to a low ionic strength NaCl solution. The EPM and hydrodynamic diameter were measured to investigate the significant changes in the colloidal behavior of aminemodified PSL NPs toward the yeast cells caused by adding CMC to 5 mM NaCl solution. Fig. 3 shows the EPMs and hydrodynamic diameters of the yeast cells and the PSL NPs as a function of added polymer concentration. The EPMs of the yeast cells were almost constant regardless of the polymer concentration. In contrast, the EPMs of the PSL NPs decreased with increasing CMC concentration, and the surface charge changed from positive to negative between 2 and 3 ppm. MC addition hardly changed the EPMs of the PSL NPs. These results indicate that the negatively charged anionic CMC, which contains a carboxyl group, adhere to the positively charged PSL NP surfaces. Oriekhovo and Stoll reported that fulvic acid, which has a carboxyl group, adheres to the positively charged surfaces of cerium oxide particles under acidic conditions [39]. This suggests that adhesion of CMC to the PSL NPs decreased the electrostatic attractive force between the NPs and cells. The hydrodynamic diameter of the PSL NPs gradually increased with increasing added polymer concentration; a considerable increase was observed on addition of 2 ppm CMC. The addition of 2 ppm CMC clearly caused aggregation of the PSL NPs. This is because the absolute value of the EPM was lower than those under other conditions. This could prevent uptake of the PSL NPs by the cells because of a size effect. In addition, the yeast cells did not aggregate as a result of polymer addition. The interaction force between a single NP and a single living cell was measured directly using AFM. One NP was immobilized on the flattened end of the cantilever tip and living cells were immobilized on the PDA-glass surface. Fig. 4A shows typical SEM image
2207
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
1 ppm
2 ppm
3 ppm
4 ppm
A
B
Fig. 2. Location of polystyrene latex nanoparticles (PSL NPs) and viability of yeast cells after exposure for 1 h to 40 µg/mL PSL NP suspension with added (A) carboxymethylcellulose and (B) methylcellulose. Upper images: confocal microscopy images, fluorescent green: PSL NPs; lower images: transmission images, fluorescent blue: dead cells stained with trypan blue.
of a single-NP probe. The image confirm that only one NP was immobilized on the flattened end of the AFM tip using a simple method, without surface modification of the tip. However, we observed ten prepared AFM probes with SEM. As a result, only two probes were successful. Fig. 4B shows a CLSM image of the immobilized cells on the PDA-glass. Most of the cells were fluorescent green, indicating that living yeast cells were successfully immobilized on the PDA-glass substrate. In contrast, few immobilized cells were present on a NC-glass, which had not undergone surface modification after washing. The physicochemical properties of the cells and substrates are important in cell and NP immobilization. The contact angles for various probe liquids with different polarities, along with the surface tensions, are listed in Table 1. The contact angle of water on the PDA-glass was higher than that on the NC-glass. This shows that coating with PDA increased the hydrophobicity of the NC-glass surface. The water contact angle of the amine-modified PSL NP lawn was reported to be 50.5° [40]; the value obtained in this study was similar
(50 ± 2°). Changes in the interfacial free energies when a yeast cell adheres to substrates or a PSL NP are listed in Table 2. When the yeast cells adhered to a glass substrate, DGTotal for the NC-glass was positive, whereas that for the PDA-glass was negative. This means that the PDA-glass was a thermodynamically favorable surface for the immobilization of yeast cells. DGTotal for the PSL NP was positive, meaning that the yeast cells were a thermodynamically unfavorable surface for the adhesion of PSL NPs. Fig. 5A shows the representative approach curves between an NP and a living yeast cell in 5 mM NaCl solutions containing 4 ppm water-soluble polymer. In the 5 mM NaCl solution without a polymer (control), the attractive force was obviously detected due to the electrostatic attractive force. Similarly, the attractive force was detected when adding 4 ppm MC to the 5 mM NaCl solution. As shown in Fig. 3, MC addition did not affect the surface charge on the PSL NPs. The cells were therefore covered with PSL NPs, leading to cell death (Fig. 2). This is presumably because the covering of the yeast cell by NPs causes a decrease in the fluidity
2208
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
Fig. 3. (A) Electrophoretic mobility and (B) hydrodynamic diameter of Saccharomyces cerevisiae and polystyrene latex nanoparticles as a function of added polymer concentration in 5 mM NaCl solution. Mean ± standard error, N = 3.
A
B
Table 2 Interfacial free energies when the yeast adheres to glass substrate or polystyrene latex nanoparticle (mJ/m2).
Tip
100 nm
Object S
DGLW
DGAB
DGTotal
NC-glass PDA-glass PSL NP
6.0 6.0 4.4
44.1 4.2 25.1
38.1 1.8 20.7
NP
Fig. 4. (A) Scanning electron microscopy image of single-nanoparticle probe and (B) confocal microscopy image of immobilized yeast cells on polydopamine-coated glass slide. Fluorescent green: living cells stained with SYTO 9, fluorescent red: dead cells stained with propidium iodide.
of the cell membrane and inhibition of metabolism through the cell membrane [15,41,42]. In contrast, when CMC was added to the 5 mM NaCl solution, the attractive force almost disappeared. As shown in Fig. 3, CMC addition changed the surface charge on the PSL NPs from negative to positive, leading to a reduction in the electrostatic attractive force between the NPs and yeast cells. In addition, CMC adsorbed on the PSL NP surface is presumed to cause steric forces on the polymer present on the cell surface. In our previous study, when the negatively charged PSL NPs were exposed to the yeast cells, the NPs were dispersed in the dispersion medium due to the electrostatic repulsive force [15]. As shown in Fig. 2, addition of 4 ppm CMC to 5 mM NaCl solution allowed the uptake
of PSL NPs by living yeast cells. These results indicate that the repulsive force between the NPs and yeast cells is very weak, allowing NPs to collide with the cells. Fig. 5B shows typical histograms of the adhesion forces between the NP and the living yeast cell, along with the representative retraction curves. The adhesion force was detected in the 5 mM NaCl solution without a polymer (control) at 400 ± 20 pN, and with 4 ppm MC at 260 ± 20 pN, but not with 4 ppm CMC (adhesion force: 20 ± 1 pN). The trend in the adhesion force was similar to that in the attractive force shown in Fig. 5A. These results imply that the adhered NPs on the cell surfaces could not be taken up by cells because of the strong adhesion forces between the NPs and the cell surface. In contrast, it is suggested that in the 5 mM NaCl solution with CMC, the NPs that collided with the cell surfaces as a result of Brownian motion were taken up by endocytosis, resulting that the cells remained alive. Several peaks were detected in the retraction curves for the 5 mM NaCl solutions without a polymer (control) and with 4 ppm MC. These peaks represent multiple polymer chains or binding with multiple sites on the biological surfaces [31,43,44].
Table 1 Contact angles and surface tensions of yeast, glass substrate and polystyrene latex nanoparticle. Mean ± standard error, N 5. Cell/Substrate/NP
h S. cerevisiae NC-glass PDA-glass PSL NP *
Surface tension (mJ/m2)
Contact angle (Degree) Wa*
11 ± 1 17 ± 1 57 ± 1 50 ± 2
Fo*
Br*
h
h
10 ± 1 31 ± 1 21 ± 1 43 ± 2
26 ± 3 21 ± 1 20 ± 1 36 ± 1
Wa, Water; Fo, Formamide; Br, a-Bromonaphthalene.
cLW
c+
c–
cAB
cTotal
40 42 42 36
1.2 0.1 2.7 0.4
54 62 13 32
16 4.1 11 7.1
56 46 53 44
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
2209
Fig. 5. Interaction forces between polystyrene latex nanoparticle and living yeast cell using atomic force microscopy in 5 mM NaCl solution (without polymer, control), 5 mM NaCl solution containing 4 ppm carboxymethylcellulose (CMC), and 5 mM NaCl solution containing 4 ppm methylcellulose (MC): (A) approach curves and (B) histograms of adhesion forces, along with retraction curves. N = 300 curves from three independent experiments.
4. Conclusions The uptake of positively charged PSL NPs by the budding yeast Saccharomyces cerevisiae cells in 5 mM NaCl solution was attempted to control using a water-soluble polymer. CLSM observations demonstrated that the PSL NPs were successfully taken up by the living yeast cells when a small amount of CMC was added to a low ionic strength NaCl solution. In contrast, nonionic MC did not affect the NP behavior. The negatively charged anionic CMC, which has a carboxyl group, adhered to the surfaces of the positively charged PSL NPs and the surface charge changed from positive to negative. These results indicate that the addition of anionic CMC to a PSL NP suspension decreased the electrostatic attractive force between the NPs and cells. AFM measurements using a single-NP probe revealed that CMC significantly reduced the interaction force between a negatively charged cell and a positively charged PSL NP.
[4]
[5] [6]
[7] [8] [9]
[10]
[11]
Acknowledgments [12]
This work was supported by the Japan Society for the Promotion of Science, KAKENHI Grant Numbers 24310066 and 15H01745. [13]
References [1] B.D. Chithrani, W.C.W. Chan, Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes, Nano Lett. 7 (2007) 1542–1550, https://doi.org/10.1021/nl070363y. [2] O. Faklaris, V. Joshi, T. Irinopoulou, P. Tauc, M. Sennour, H. Girard, C. Gesset, J. Arnault, A. Thorel, J.-P. Boudou, P.A. Curmi, F. Treussart, Photoluminescent diamond nanoparticles for cell labeling: study of the uptake mechanism in mammalian cells, ACS Nano 3 (2009) 3955–3962, https://doi.org/10.1021/ nn901014j. [3] K. Kostarelos, L. Lacerda, G. Pastorin, W. Wu, S. Wieckowski, J. Luangsivilay, S. Godefroy, D. Pantarotto, J.-P. Briand, S. Muller, M. Prato, A. Bianco, Cellular uptake of functionalized carbon nanotubes is independent of functional group
[14]
[15]
[16]
and cell type, Nat. Nanotechnol. 2 (2007) 108–113, https://doi.org/10.1038/ nnano.2006.209. J. Lin, H. Zhang, Z. Chen, Y. Zheng, Penetration of lipid membranes by gold nanoparticles: insights into cellular uptake, cytotoxicity, and their relationship, ACS Nano 4 (2010) 5421–5429, https://doi.org/10.1021/ nn1010792. D. Tasis, N. Tagmatarchis, A. Bianco, M. Prato, Chemistry of carbon nanotubes, Chem. Rev. 106 (2006) 1105–1136, https://doi.org/10.1021/cr050569o. T. Wang, J. Bai, X. Jiang, G.U. Nienhaus, Cellular uptake of nanoparticles by membrane penetration: a study combining confocal microscopy with FTIR spectroelectrochemistry, ACS Nano 6 (2012) 1251–1259, https://doi.org/ 10.1021/nn203892h. A. Nel, T. Xia, L. Mädler, N. Li, Toxic potential of materials at the nanolevel, Science 311 (2006) 622–627, https://doi.org/10.1126/science.1114397. A. Kahru, A. Ivask, Mapping the dawn of nanoecotoxicological research, Acc. Chem. Res. 46 (2013) 823–833, https://doi.org/10.1021/ar3000212. K. Kasemets, A. Ivask, H.-C. Dubourguier, A. Kahru, Toxicity of nanoparticles of ZnO, CuO and TiO2 to yeast Saccharomyces cerevisiae, Toxicol. Vitr. 23 (2009) 1116–1122, https://doi.org/10.1016/j.tiv.2009.05.015. S. Lee, J. Lee, K. Kim, S.-J. Sim, M.B. Gu, J. Yi, J. Lee, Eco-toxicity of commercial silver nanopowders to bacterial and yeast strains, Biotechnol. Bioprocess Eng. 14 (2009) 490–495, https://doi.org/10.1007/s12257-008-0254-6. H. Schwegmann, A.J. Feitz, F.H. Frimmel, Influence of the zeta potential on the sorption and toxicity of iron oxide nanoparticles on S. cerevisiae and E. coli, J. Colloid Interface Sci. 347 (2010) 43–48, https://doi.org/10.1016/j. jcis.2010.02.028. A.N. Hadduck, V. Hindagolla, A.E. Contreras, Q. Li, A.T. Bakalinsky, Does aqueous fullerene inhibit the growth of Saccharomyces cerevisiae or Escherichia coli?, Appl Environ. Microbiol. 76 (2010) 8239–8242, https://doi.org/10.1128/ AEM.01925-10. C. García-Saucedo, J.A. Field, L. Otero-Gonzalez, R. Sierra-Álvarez, Low toxicity of HfO2, SiO2, Al2O3 and CeO2 nanoparticles to the yeast, Saccharomyces cerevisiae, J. Hazard. Mater. 192 (2011) 1572–1579, https://doi.org/10.1016/j. jhazmat.2011.06.081. C. Prescianotto-Baschong, H. Riezman, Morphology of the yeast endocytic pathway, Mol. Biol. Cell. 9 (1998) 173–189, https://doi.org/10.1091/ mbc.9.1.173. T. Nomura, J. Miyazaki, A. Miyamoto, Y. Kuriyama, H. Tokumoto, Y. Konishi, Exposure of the yeast saccharomyces cerevisiae to functionalized polystyrene latex nanoparticles: influence of surface charge on toxicity, Environ. Sci. Technol. 47 (2013) 3417–3423, https://doi.org/10.1021/es400053x. J. Miyazaki, Y. Kuriyama, A. Miyamoto, H. Tokumoto, Y. Konishi, T. Nomura, Adhesion and internalization of functionalized polystyrene latex nanoparticles toward the yeast Saccharomyces cerevisiae, Adv. Powder Technol. 25 (2014) 1394–1397, https://doi.org/10.1016/j.apt.2014.06.014.
2210
S. Yumiyama et al. / Advanced Powder Technology 29 (2018) 2204–2210
[17] J. Miyazaki, Y. Kuriyama, H. Tokumoto, Y. Konishi, T. Nomura, Cytotoxicity and behavior of polystyrene latex nanoparticles to budding yeast, Colloids Surf. A Physicochem. Eng. Asp. 469 (2015) 287–293, https://doi.org/10.1016/ j.colsurfa.2015.01.046. [18] Y.F. Dufrene, Atomic force microscopy, a powerful tool in microbiology, J. Bacteriol. 184 (2002) 5205–5213, https://doi.org/10.1128/JB.184.19.5205. [19] Y.F. Dufrene, Sticky microbes: forces in microbial cell adhesion, Trends Microbiol. 23 (2015) 376–382, https://doi.org/10.1016/j.tim.2015.01.011. [20] M. Herzberg, S. Kang, M. Elimelech, Role of extracellular polymeric substances (EPS) in biofouling of reverse osmosis membranes, Environ. Sci. Technol. 43 (2009) 4393–4398, https://doi.org/10.1021/es900087j. [21] H. Shinto, Y. Aso, T. Fukasawa, K. Higashitani, Adhesion of melanoma cells to the surfaces of microspheres studied by atomic force microscopy, Colloids Surf. B Biointerf. 91 (2012) 114–121, https://doi.org/10.1016/ j.colsurfb.2011.10.060. [22] L.-S.Z. Rathje, N. Nordgren, T. Pettersson, D. Rönnlund, J. Widengren, P. Aspenström, A.K.B. Gad, Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness, Proc. Natl. Acad. Sci. USA 111 (2014) 1515–1520, https://doi.org/10.1073/pnas.1300238111. [23] M. Benoit, D. Gabriel, G. Gerisch, H.E. Gaub, Discrete interactions in cell adhesion measured by single-molecule force spectroscopy, Nat. Cell Biol. 2 (2000) 313–317, https://doi.org/10.1038/35014000. [24] S. Kang, M. Elimelech, Bioinspired single bacterial cell force spectroscopy, Langmuir 25 (2009) 9656–9659, https://doi.org/10.1021/la902247w. [25] I.U. Vakarelski, K. Higashitani, Single-nanoparticle-terminated tips for scanning probe microscopy, Langmuir 22 (2006) 2931–2934, https://doi.org/ 10.1021/la0528145. [26] T. Kim, S. Myung, T.H. Kim, S. Hong, Robust single-nanoparticle probe for contact-mode analysis and dip-pen nanolithography, Small 4 (2008) 1072– 1075, https://doi.org/10.1002/smll.200701102. [27] J.K. Vasir, V. Labhasetwar, Quantification of the force of nanoparticle-cell membrane interactions and its influence on intracellular trafficking of nanoparticles, Biomaterials 29 (2008) 4244–4252, https://doi.org/10.1016/j. biomaterials.2008.07.020. [28] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Mussel-inspired surface chemistry for multifunctional coatings, Science 318 (2007) 426–430, https:// doi.org/10.1126/science.1147241. [29] H. Lee, B.P. Lee, P.B. Messersmith, A reversible wet/dry adhesive inspired by mussels and geckos, Nature 448 (2007) 338–341, https://doi.org/ 10.1038/nature05968. [30] J.L. Hutter, J. Bechhoefer, Calibration of atomic-force microscope tips, Rev. Sci. Instrum. 64 (1993) 1868–1873, https://doi.org/10.1063/1.1143970. [31] A. Beaussart, A. Baker, S.L. Kuchma, S. El-Kirat-Chatel, G.A. O’Tolle, Y.F. Dufrene, Nanoscale adhesion forces of Pseudomonas aeruginosa type IV pili, ACS Nano 8 (2014) 10723–10733, https://doi.org/10.1021/nn5044383.
[32] H.J. Busscher, A.H. Weerkamp, H.C. van der Mei, A.W. van Pelt, H.P. de Jong, J. Arends, Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion, Appl. Environ. Microbiol. 48 (1984) 980–983. [33] A. Yoshihara, H. Narahara, Y. Kuriyama, S. Toyoda, H. Tokumoto, Y. Konishi, T. Nomura, Measurement of microbial adhesive forces with a parallel plate flow chamber, J. Colloid Interface Sci. 432 (2014) 77–85, https://doi.org/10.1016/j. jcis.2014.06.052. [34] C.J. Van Oss, M.K. Chaudhury, R.J. Good, Interfacial Lifshitz-van der Waals and polar interactions in macroscopic systems, Chem. Rev. 88 (1988) 927–941, https://doi.org/10.1021/cr00088a006. [35] Y. Zhao, M. Li, Q. Lu, Z. Shi, Superhydrophobic polyimide films with a hierarchical topography: combined replica molding and layer-by-layer assembly, Langmuir 24 (2008) 12651–12657, https://doi.org/10.1021/ la8024364. [36] T. Nomura, E. Fujisawa, S. Itoh, Y. Konishi, Comparison of the cytotoxic effect of polystyrene latex nanoparticles on planktonic cells and bacterial biofilms, J. Nanopart. Res. 18 (2016) 1–9, https://doi.org/10.1007/s11051-016-3471-5. [37] A. Yoshihara, N. Nobuhira, H. Narahara, S. Toyoda, H. Tokumoto, Y. Konishi, T. Nomura, Estimation of the adhesive force distribution for the flagellar adhesion of Escherichia coli on a glass surface, Colloids Surf. B Biointerf. 131 (2015) 67–72, https://doi.org/10.1016/j.colsurfb.2015.04.038. [38] T. Nomura, S. Tani, M. Yamamoto, T. Nakagawa, S. Toyoda, E. Fujisawa, A. Yasui, Y. Konishi, Cytotoxicity and colloidal behavior of polystyrene latex nanoparticles toward filamentous fungi in isotonic solutions, Chemosphere 149 (2016) 84–90, https://doi.org/10.1016/j.chemosphere.2016.01.091. [39] O. Oriekhova, S. Stoll, Effects of pH and fulvic acids concentration on the stability of fulvic acids – cerium (IV) oxide nanoparticle complexes, Chemosphere 144 (2016) 131–137, https://doi.org/10.1016/j. chemosphere.2015.08.057. [40] D. Akilbekova, R. Philiph, A. Graham, K.M. Bratlie, Macrophage reprogramming: influence of latex beads with various functional groups on macrophage phenotype and phagocytic uptake in vitro, J. Biomed. Mater. Res. Part A 103 (2015) 262–268, https://doi.org/10.1002/jbm.a.35169. [41] E. Kojro, G. Gimpl, S. Lammich, W. Marz, F. Fahrenholz, Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the alpha -secretase ADAM 10, Proc. Natl. Acad. Sci. USA 98 (2001) 5815–5820, https://doi.org/ 10.1073/pnas.081612998. [42] M.K. Shigenaga, T.M. Hagen, B.N. Ames, Oxidative damage and mitochondrial decay in aging, Proc. Natl. Acad. Sci. USA 91 (1994) 10771–10778, https://doi. org/10.1073/pnas.91.23.10771. [43] H.J. Butt, M. Kappl, H. Mueller, R. Raiteri, W. Meyer, J. Rühe, Steric forces measured with the atomic force microscope at various temperatures, Langmuir 15 (1999) 2559–2565, https://doi.org/10.1021/la981503+. [44] N.I. Abu-Lail, T.A. Camesano, Elasticity of pseudomonas putida KT2442 surface polymers probed with single-molecule force microscopy, Langmuir 18 (2002) 4071–4081, https://doi.org/10.1021/la015695b.