Imidazolium-Based Ionic Liquids Affect Morphology and Rigidity of

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Imidazolium-Based Ionic Liquids Affect Morphology and Rigidity of Living Cells: an Atomic Force Microscopy Study Massimiliano Galluzzi, Carsten Schulte, Paolo Milani, and Alessandro Podestà Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01554 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Imidazolium-Based Ionic Liquids Affect Morphology and Rigidity of Living Cells: an Atomic Force Microscopy Study Massimiliano Galluzzi1,2, Carsten Schulte2, Paolo Milani2, Alessandro Podestà2*

1

Shenzhen Key Laboratory of Nanobiomechanics, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China.

2

C.I.Ma.I.Na and Dipartimento di Fisica “Aldo Pontremoli”, Università degli Studi di Milano, via Celoria 16,

20133 - Milano, Italy. * Corresponding author: Prof. Alessandro Podestà, e-mail: [email protected]

Keywords. Ionic liquids; biomembranes; living cells; mechanisms of toxicity; Atomic Force Microscopy (AFM); cell mechanics; nanoindentation.

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Abstract The study of the toxicity, biocompatibility, and environmental sustainability of room-temperature Ionic Liquids (ILs) is still in its infancy. Understanding the impact of ILs on living organisms, especially from the aquatic ecosystem, is urgent, since large amounts of these substances are starting to be employed as solvents in industrial chemical processes, and on the other side evidences of toxic effects of ILs on microorganisms and single cells have been observed. To date, the toxicity of ILs have been investigated by means of macroscopic assays aimed at characterizing the effective concentrations (like the EC50) that cause the dead of a significant fraction of the population of microorganisms and cells. These studies allowed to identify the cell membrane as the first target of the IL interaction, whose effectiveness was correlated to the lipophilicity of the cation, i.e. to the length of the lateral alkyl chain. Our study aimed at investigating the molecular mechanisms underpinning the interaction of ILs with living cells. To this purpose, we carried out a combined topographic and mechanical analysis by Atomic Force Microscopy of living breast metastatic cancer cells (MDA-MB-231) upon interaction with imidazolium-based ILs. We showed that ILs are able to induce modifications of the overall rigidity (effective Young modulus) and morphology of the cells. Our results demonstrate that ILs act on the physical properties of the outer cell layer (the membrane linked to the actin cytoskeleton), already at concentrations below the EC50. These potentially toxic effects are stronger at higher IL concentrations, as well as with longer lateral chains in the cation.

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Introduction Room-Temperature Ionic Liquids (RTILs, or simply ILs) are used as effective and tunable new solvents in several applications.

1-2

ILs represent an interesting alternative to the standard aqueous electrolytes in

applications aimed at the conversion and storage of energy, such as electrochemical supercapacitors,3-4 Grätzel solar cells,5 and Li-ion batteries,6-7 as well as good lubricants in micro-electro-mechanical devices.8 ILs are also increasingly used as solvents in several industrial chemical processes, for example, a recent commercialization of RTIL technology from gram to ton scale was performed by Petronas in Malaysia in order to remove hazardous Hg contaminants from natural gas.9 Other industrial large-scale applications of ILs are reported elsewhere.10-11 The increasingly important massive use of ILs in industrial processes raise concerns due to the possibility that upon leakage or spill these substances contaminate the (aqueous) ecosystem.12-13 Despite the fact that a green label is often attributed to ILs by virtue of their controlled flammability, miscibility, and negligible vapor pressure,2 which are expected to mitigate environmental contamination in the event of leakages, a potential hazard of ILs for living organisms in the aqueous medium, or upon direct contact with humans, cannot be excluded.14 In this framework, the study of IL toxicity is challenging due not only to the huge variety of these substances, but also because of the diverse nature of the biological systems and environments that represent the potential target of the IL toxicity. Nevertheless, a rigorous investigation on the interaction of ILs with living organisms is important to design environmentally sustainable ILs, and fully exploit their potential in different fields. Alongside with the toxicological characterization, the investigation of ILs interacting with biological matter can be important for several applications exploiting their tunable chemical and physical properties. For example, in pharmacology,15-16 ILs are tested as anti-cancer,17-19 antimicrobial treatment20-22 or as media for extraction and purification of proteins.23 ILs can be used as thin conductive films covering delicate cells/tissues specimens for scanning electron microscopy (SEM) investigation.24-25 These treatments could decrease time and efforts for preparation of biological samples prior SEM, although ingress of ions and water exclusion could produce dehydration of cells/tissues. A progressive uptake of IL in the cytoplasmic region (especially at high IL concentrations) could be exploited for other diagnostic purposes, for example using ILs as preserving agents. Majewski et al.26-27 evaluated the potential of ILs as formaldehyde substituents, for embalming and preservation of biological tissues. At odds with formaldehyde, ILs do not bind proteins, therefore excluding stiffening effects induced by the cross-linking of the cytoskeletal filaments. Moreover, ILs restrain access of water by internal substitution, and they can kill bacteria and fungi, therefore inhibiting degradation.27

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Cellular toxicity is frequently assessed by characterizing the effective concentration EC50 of the substance, which induces the maximum effect (death in this case) in 50% of the cell population under test, after a standard period (usually 24h). The general toxic action of ILs on living organisms seems to be connected mainly to the nonspecific lipophilic interaction between the lipid membrane of the cells and the alkyl chain of the cations. In particular, EC50 values were found to be in linear correlation with lipophilicity parameters (i.e. water/octanol partition coefficient) highlighting the primary interaction of ILs on lipid-rich membrane.28-29 In a previous study,30 a series of imidazolium based ILs interacting with dioleoyl phosphatidylcholine (DOPC) monolayer supported on a mercury electrode were investigated by means of electrochemical techniques. The system was acting as an efficient sensor to screen the role of anions and lateral side chain of cations. As expected, the strength of interaction was related to the length of carbon chain of the cation, although, not systematically related to the nature of the anion. The detection limits (LOD) of electrochemical techniques for imidazolium ILs, (along with octanol/water partitioning coefficients) were quantitatively correlated with toxicity data, highlighting the role of the biomembrane in the toxicity mechanisms. Cations with long hydrocarbon chains show an increased lipophilic and toxic character in comparison with shorter chain analogues. The toxic effect of the anions was shown to be secondary compared to the cationic effect, although fluorinated anions (such as BF4 or NTf2) may demonstrate an increased cytotoxicity.21 A variety of experimental conditions and biological systems were investigated (reviewed in recent refs.13, 16, 31-32), confirming the direct link between the IL cation lipophilicity and toxicity, in particular related to the adsorption/intercalation of cations into biomembranes resulting in membrane perturbation/disruption and cellular uptake.28-29, 32-34 Due to the importance of the interaction with the cell membrane, model biomembranes (such as phospholipid bilayers) can be artificially reconstructed in order to use surface techniques for a deep investigation, for example atomic force microscopy (AFM),34-36 neutron scattering,37 X-ray,34, 38-39 quartz crystal balance,40 nanoplasmonics,41 electrochemical impedance measurements,30 and molecular dynamic simulations.42-44 ILs were shown to be capable of strongly interacting with supported phospholipid structures, creating defects and perturbations, including structural rearrangement and fluidification, formation of holes, and complete disintegration of the assemblies.28 Morphological and structural rearrangement of phospholipid bilayers on mica upon interaction with ILs was recently observed by Rotella et al.,36 by means of AFM-based imaging and indentation analysis. This results are relevant, since they demonstrate that ILs are able to markedly interact with lipid system, and in particular with simplified models of the cell membrane. Despite the study of the interaction of ILs with biomembranes is still in its infancy, the field is attracting increasing attention, due to the importance of unravelling the basic molecular mechanisms of the IL toxicity, as highlighted in two recent reviews and perspectives on this topic.28, 45

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However, the use of artificial membranes neglects important aspects of the more complex situation in cells, where the interaction of the membrane with the underlying cortical actin cytoskeleton is essential for the cellular physiology and functioning. Most evidently, this membrane/cytoskeleton linkage regulates structural aspects by defining the cell shape and biomechanical properties (e.g. tension), but it contributes also to cell signaling, and is therefore strongly involved in many cellular processes, such as cell adhesion, migration, division and differentiation.46-50 In this study, we evaluated the morphological and mechanical modifications induced by three different imidazolium-based ILs (C4MIM][Cl], [C4MIM][BF4], and [C8MIM][Cl]) on human breast adenocarcinoma (MDA-MB-231) living cells. We used the breast adenocarcinoma cell line MDA-MB-231, a long-known and well-established cellular model,51 in particular widely used for the study of cytoskeletal organization- and cell migration-related processes, because these cells are characterized by large lamellipodia, and a highly dynamic cytoskeletal remodeling.50, 52-53 Therefore, MDA-MB-231 is a suitable cellular model to study the effects of ILs on the membrane/cytoskeleton interface. To this purpose, we carried out a systematic analysis on several populations of living cells in IL-containing aqueous cell culture media by AFM. AFM allows studying the mechanical properties of biologically-relevant interfaces in physiological environments.54-55 Recently, AFM was shown to be able to detect alterations of single cell rigidity correlated to pathophysiological conditions,56-58 substrate stiffness,59-61 nanotopography,62 as well as interaction with drugs.63-64 The potential of AFM for the investigation of the IL-biomembrane (together with the cytoskeleton) interactions was recently highlighted by Benedetto et al. in a review and perspective article.28, 45 Here, we applied an AFM-based protocol to perform a combined topographic and mechanical imaging of living cells,65-66 at increasing IL concentrations, using custom colloidal probes.67 This protocol has been recently employed in our group to specifically detect modulations in the membrane/cytoskeleton interface during cell adhesion- and migration-related processes.62, 68-69 We focused our attention on the elastic properties of the cell outer envelope, composed by the cell membrane linked to the actin cytoskeleton, since this is the cell region where the first action of ILs is expected to take place.28, 45 We have tested different anions for the same cation, and also different length of the lateral alkyl chain, in order to verify whether the IL action is correlated to its lipophilicity.

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Materials and Methods Ionic Liquids The ILs used in the experiments are: 1-butyl -3-methyl imidazolium chloride ([C4MIM][Cl]), 1-butyl- 3methyl imidazolium tetrafluoroborate ([C4MIM][BF4]), and 1- methyl- 3-octyl imidazolium chloride ([C8MIM][Cl]) (Sigma Aldrich, purity grade > 98.0%). Because of their hydrophilicity, ILs have been stored in a desiccator under mild vacuum condition (≈ 5 mbar) before preparation of water solutions. Considering the high critical micelles concentration (CMC) for these ILs (not measurable for [C4MIM] + and 223 mM for [C8MIM] +),70 we prepared mother solutions at 1 M and 0.1 M for [C4MIM]+ and [C8MIM] + respectively. Experiments were performed exploring different range of concentrations: [C4MIM][Cl] and [C4MIM][BF4] was in the range 1-100 mM, while that of [C8MIM][Cl] was in the range 1-100 µM. ILs solutions at high concentration were injected in the cell specimen, far from measurement location, in order to minimize fluid volume perturbation. The cells-IL systems were typically allowed to equilibrate for 20 minutes after every IL injection in the same specimen. Considering equilibration time and measurement time (≈ 1 h for 3 cells) a total measurement time Ttot = 5 h was used for each sample influenced by increasing concentration of ILs. Within the intrinsic resolution of AFM mechanical measurements and biological variation, we did not notice any evident time-dependent variations in Young’s modulus during the measurement time.

Preparation of Living Cells Specimens for AFM Analysis The MDA-MB-231 cell line were cultured in Dulbecco’s modified eagle medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), L-glutamine 5 mM, 100 units/ml penicillin and 100 units/ml streptomycin in 5% CO2, 98% air-humidified incubator (Galaxy S, RS Biotech, Irvine, California, USA) at 37°C. Cells were detached from culture dishes using a 0,25% Trypsin-EDTA in HBSS (Sigma-Aldrich), centrifuged at 1000 rpm for 5 minutes, and resuspended in the culture medium. Subcultures or culture medium exchanges were routinely established every 2nd to 3rd day into Petri dishes (diameter 10 cm). In order to thermalize the culture plates at a temperature close to 37 °C, a custom fluid transparent cell for AFM measurements was used.65 The presence of the heated fluid cell does not affect significantly the transparency of the optical path, and allows to acquire measurements on the same sample for several hours (typically up to 6h) before the cells manifest signs of distress.

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Measuring the Effective Young’s Modulus of Living Cells The general procedure used for the nanomechanical measurements on living cells and the complete analysis is exhaustively discussed in our recent methodological work.65 Briefly, the measurements were performed with custom spherical colloidal probes.67 Borosilicate glass microspheres were strongly attached to commercial AFM tipless cantilever. The radius of the sphere was characterized by means of AFM reverse imaging of the colloidal probe on a spiked grating (TGT1 from NT-MDT).67 Compared to standard sharp tips, spherical tips are particularly suitable for nanomechanical experiments on living cells because their geometry is well-defined, and the pressure applied to the soft sample is reduced thanks to the increased surface area. Using a larger micrometer-sized probe in nanoindentation experiments also allows a robust statistical averaging over a mesoscopic interaction area (volume), for the sake of better characterizing the effect of ILs on the cell membrane, yet providing a satisfactory lateral resolution compared to the typical cell dimensions. The typical probe radius was about 5 µm, and the cantilever force constant was about 0.2 N/m. The topographic and mechanical imaging was performed in Force Volume (FV) mode, a force spectroscopy technique in which a force-distance curve (FC) is recorded for each point in a grid spanning a finite area across a cell.71 Standard parameters for the acquisition of FCs were: ramp size 5 µm, force setpoint FMAX ≈ 10nN, approaching velocity va = 43.4 µm/s, retracting velocity vr = 200 µm/s, ramp rate 7.1 Hz. A total of 64X64 = 4096 force curves were typically acquired in each FV, with 2048 points per curve, corresponding to a resolution of about 2.5 nm/point. The z-piezo was operated in close-loop mode in order to get rid of nonlinearities and hysteresis, and record accurate vertical distance axes. The raw (compressed) topographic maps of the cells were built using the local z-position corresponding to the maximum setpoint force. The real uncompressed topographic map is obtained by adding the local elastic indentation. All the experiments were performed in the cell culture medium, using the thermostatic fluid cell. Figure S1 in the Supporting Information shows that repeated imaging of the same cell with the colloidal probe for about two hours produced only minor damage and disruption to the cell, and almost no changes in the Young’s modulus maps and histograms. The contact part of each FC is analyzed using the Hertz model (Equation 1):

= √ /

(1)

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Here F is the applied force, δ the indentation, ν the Poisson’s ratio (assumed to be 0.5, as for incompressible materials), E the effective Young’s modulus of the cell, and R the radius of the spherical probe. As shown in Figure S2a-b, adhesive interactions are negligible, ensuring the validity of the nonadhesive Hertz model. Despite the fact that the ramp frequency is relatively high, we did notice only very minor hysteresis between the approaching and the retracting curves (Figure S2c), suggesting that viscoelastic effects are not relevant in our system. We have applied the finite thickness correction proposed by Dimitriadis et al.

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, since this effect is stronger with colloidal, whose radius is comparable to the

thickness of the cells (see Equation S1 in the Supporting Information, and the description therein). Noticeably, the application of the finite thickness correction and the corresponding renormalization of the Young’s modulus values, allowed us to exploit the mechanical data from the entire surface of the cell, and not only from the thicker region (the nuclear region). In particular, regions like lamellipodia, or the cytoplasmic protrusions, can be very informative, since they are rich(er) of actin cytoskeleton elements. To minimize artifacts, we typically applied a mask based on topography to filter off the extreme cell-substrate boundary region, where obvious artifacts are present. The Hertz model requires homogenous and isotropic samples, while living cells are definitely heterogeneous and anisotropic. The cells can be considered as effective multi-layered systems, in order to take into account the complex cytoskeletal structure,73 and in particular the contribution of the external membrane-actin layer, and of the internal portions of the cytoskeleton. In order to consider the most external layers of the cell, where ILs should primarily interact, for the MDA-MB-231 cell line we have fitted the Hertz model between 0% and 20% of the total indentation range (corresponding to Fmax ≈ 2 nN). This range was calibrated and selected based on previous works from the authors, where different Hertzian regimes were identified in the force curves, likely due to the contributions of effective elastic layers located at different depths inside the cell.65 We point out that the mechanical contribution of the external layers (cell membrane + outer actin network) is always convoluted with the inner layers (tubulin, intermediate filaments, nucleus and organelles). Recent efforts of the authors, focusing on the development of a deconvolution procedure based on coupling AFM analysis with finite element simulations, support our methodological approach.74-75 For each cell condition and cell region (body or periphery), N cells were measured (see Table 1). The Young’s modulus values from the different regions were selected using masks built from the topographic maps. The distributions of the Young’s modulus values for the different regions were typically log-normal, and were fitted in semi-logarithmic scale (base 10) with single Gaussian curves. The final mean value of the Young’s modulus E for each cell condition and cell region, and its associated effective error σ, were

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calculated following the procedure described in details in Ref.65, 76 Briefly, for a single FV measurement, representing a single cell out of a total of N cells studied at a given condition, the error σFV associated to the mean Young’s modulus of the cell is calculated taking into account the propagated uncertainty in the probe calibration, the variability of Young’s modulus within the cell, and the number of force curves (typically several hundreds). Then, the average Young’s modulus E of cells at a given condition for a specific region is calculated as the mean of N single cell mean values (the population mean), with its standard deviation of the mean σE. Eventually, the error σ associated to E (an effective standard deviation of the mean) is calculated as the sum in quadrature of σE and the average σFV. The statistical significance of the differences of Young’s modulus values for different conditions was calculated using the two-tails t-test.

Results and Discussion The morphological and nanomechanical AFM investigation was aimed at studying the interactions of different ILs ([C4MIM][Cl], [C4MIM][BF4], and [C8MIM][Cl] respectively) at increasing concentrations with the cell membrane and, likely, the outermost actin filaments of living human breast adenocarcinoma cells from the MDA-MB-231 line. We selected the concentration range for the different ionic species following the 24h EC50 concentrations measured for Photobacterium phosphoreum77 and IPC-81,78 A549,79 HeLa cell lines (numerical values tabulated in Table S1). Moreover, we confirmed experimentally the concentration range limits: [C4MIM]+ did not produce any modifications at 0.1 mM concentration (data not shown), while [C4MIM]+ above 100mM caused a complete detachment of cells from the substrate after 20 min (a condition not suitable for the AFM analysis). Analogue toxic effect was experienced using [C8MIM]+ at 1mM, therefore concentrations in µM range were used. The complete collection of AFM data and results is available in the Supporting Information (Data S1, Data S2, and Data S3 for [C4MIM][Cl], [C4MIM][BF4]; and [C8MIM][Cl], respectively). On average, we have characterized 6-8 single cells for each condition, for a total of nearly 100 cells measured. MDA-MB-231 cells in vitro are known to display a variety of morphological forms80 due to their frequent mesenchymal migration phenotype with single or multiple extended lamellipodia (Figure 1a and Figure 2a), other cells were more rounded (Figure 3a). Cells grown in bigger colonies (not shown) were avoided during the AFM analysis. Indeed, in the Supporting Information, it is documented that we found and analyzed a variety of morphological shapes for all used ILs.

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We also noticed a variation in the average Young’s modulus for the control cells (before interaction with ILs, see Table 1). This variability reflects the nature of the MDA-MB-231 cell line that is characterized by a high morphological diversity, and by the presence of various cell subpopulations.81 Differences in the baseline do not affect the conclusions of this work, since the effects of the ILs were always assessed relatively to the specific control samples (see the Supporting Information for the complete data for the different experimental conditions).

Effect of the Short Lateral Chain

Figure 1. Representative combined topographical and nanomechanical AFM investigation of MDA-MB-231 cells interacting with [C4MIM][Cl] at different concentrations. Along a row, the uncompressed topographic map, the Young’s modulus map, and the Young’s modulus values histograms (cell body and periphery) are shown. Panels a),

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b) and c) represent the control, while the concentration of IL is 1 mM for d), e) and f); 10 mM for g), h) and i); 100 mM for j), k) and l), respectively.

Young's Modulus (E ± σ / Pa)

C4MIM Cl Conc. [mM]

N

Cell Body

Periphery

p value Body

p value Periphery

None 1 10 100

8 8 8 11

442 ± 79 369 ± 46 588 ± 83 2622 ± 510

851 ± 119 702 ± 78 924 ± 164 2060 ± 456

---n.s. 0.037 0.0002

---n.s. n.s. 0.017

Conc. [mM]

N

Cell Body

Periphery

p value Body

p value Periphery

none 1 10 100

13 11 7 5

419 ± 27 481 ± 30 420 ± 31 6084 ± 1032

1054 ± 82 1022 ± 142 683 ± 117 2504 ± 487

---n.s. n.s. 0.001

---n.s. n.s. (0.076) 0.012

Conc. [mM]

N

Cell Body

Periphery

p value Body

p value Periphery

none 10-3 10-2 10-1

7 5 8 6

768 ± 81 530 ± 47 427 ± 12 390 ± 18

1804 ± 200 1008 ± 84 821 ±75 682 ± 59

---0.046 0.024 n.s.

---0.01 n.s. n.s.

C4MIM BF4

C8MIM Cl

Table 1. Summary of the measured average Young’s modulus values of number N of MDA-MB-231 cells interacting with [C4MIM][Cl], [C4MIM][BF4], and [C8MIM][Cl] at increasing concentrations, in the low indentation regime. Results are presented as E ± σ (population mean ± effective standard deviation of the mean). The p values for assessing the statistical significance of the difference of Young’s modulus values between neighboring conditions (obtained applying the two-tails t-test) are shown (n.s. means not significant, i.e. p > 0.05). Data are divided per cell region: cell body and peripheral region.

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Figure 1 shows a series of representative AFM topographic and mechanical maps of MDA-MB-231 cells upon interaction with [C4MIM][Cl], with the corresponding histograms of Young’s modulus values for the different regions analyzed. As shown in Figures 1a,d, the cell morphology starts to be affected at 1mM concentration of [C4MIM][Cl]. At this concentration, we observed a small decrease of the Young’s modulus, more evident for the external protrusions, although the differences are not statistically significant (Table 1). Increasing the concentration of [C4MIM][Cl] in MDA-MB-231 cell culture (up to 10 mM concentration) a somewhat unexpected phenomenon takes place. The cells start stiffening at 10 mM (+33%) the Young’s modulus reaching the maximum value at a concentration of 100 mM (+600%). Moreover, the topographic maps (Figure 1a, d, g, j) highlight the change of the cell morphology (in size and shape) upon increasing the IL concentration: less protrusions are visible, and cells assume a more compact/rounded configuration. The overall evolution of MDA-MB-231 cells exposed to increasing concentration of [C4MIM][BF4], which differs from [C4MIM][Cl] only for the anion, is similar (Figure 2). However, the cells interacting with [C4MIM][BF4] are still significantly soft at 10 mM concentration; at the highest IL concentration, the stiffening for the BF4 anion is twice as large than that for the Cl anion. The trend of the Young’s modulus measured for short-chain ILs with the [Cl]- and [BF4]- anions is presented in Figure 4a,b. At 100 mM it is not unlikely to observe cells in advanced apoptosis status, which significantly limits the number of cells suitable for the AFM analysis, compared to the lower concentrations. Although experiments were performed after only 20 min of IL-cell interaction, the samples were heavily affected at concentrations likely beyond the EC50 (24h) values (Table S1). The exact threshold however could not be predicted precisely, since at present, to the best of our knowledge, toxicological EC50 values for MDA-MB-231 cells, relative to the ILs investigated in this study, are still missing. Nevertheless, based on the behavior of other cells and bacteria with the same ILs, we speculate that the selected concentration ranges include the EC50 values, meaning that through the nanomechanical channel, and in particular through the mechanical response of the cell membrane-actin cytoskeleton, we were able to detect cellular responses to the IL action at concentration well below the critical threshold. Previous works30, 82-83 report a negligible effect of the anion in the interaction of ILs with lipid membranes and organisms. Here we observe a similar qualitative behavior of C4MIM based ILs, although the stiffening of the cell is anticipated at lower concentrations in the case of the Cl- anion, and the absolute stiffening is higher in the case of the BF4- anion. Concerning the role of the cation, we observe that the short-chain [C4MIM]+ cation, in agreement with the idea that shorter alkyl chains are easily attracted to the membrane surface, partially modify the

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structure of the cell membrane and destabilize the compact structure of the phospholipid bilayer. This perturbation of the phospholipid network at low IL concentration (1 mM) could decrease the membrane tension (the area stretching module), determining a decrease of the apparent Young’s modulus measured at shallow indentation.49 The perturbation at the membrane followed by destabilization is reported by recent molecular dynamics (MD) simulations describing a phospholipid bilayer interacting with several ionic liquids.42-43, 84 We cannot exclude that the Young’s modulus decrease can also be due to cations crowding at the surface, causing osmotic unbalance, and triggering a moderate hyperosmotic shrinkage of the cell volume (evidence of shrinkage is reported in Table S2). This effect would lead to a relaxation of the membrane tension and therefore of the apparent Young’s modulus of the cell (a dramatic reduction of the cell volume can lead in turn to internal crowding and overall stiffening of the cell, see discussion below). It was also recently measured the effect of ILs on gramicidin ion channels influencing ion passage regulation.85 Eventually, based on our data, we notice that the cations of short-chain ILs could act similarly to cholesterol, which is known to increase at low concentration the fluidity of phospholipid bilayers, while at higher concentrations is able to induce ordering of the membrane, a reduction of the area per lipid, and an increase of its rigidity, although at higher concentrations other effects become dominant, as we discuss below.86-87 Therefore, destabilization and structural relaxation of membrane can cooperate to decrease the rigidity of the cell membrane- actin cytoskeleton interface.

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Figure 2. Representative combined topographical and nanomechanical AFM investigation of MDA-MB-231 cells interacting with [C4MIM][BF4] at different concentrations. Along a row, the uncompressed topographic map, the Young’s modulus map, and the Young’s modulus values histograms (cell body and periphery) are shown. Panels a), b) and c) represent the control, while the concentration of IL is 1 mM for d), e) and f); 10 mM for g), h) and i); 100 mM for j), k) and l), respectively.

A possible explanation of the observed cell stiffening at higher IL concentrations is the following. While at moderate IL concentrations the structural destabilization of the membrane-actin cytoskeleton interface prevails, at higher concentrations membrane damage and/or permeabilization,88 with the consequent IL ingress into the cytoplasm, deeply influence cell metabolism and survival. This is confirmed by the observed compaction of the cell morphology, the cell membrane roughening (blebbing) and the formation of globular structures (see Figure 2j and Data S1, S2 in the Supporting Information), and by the eventual detachment of the cell from the substrate. All these symptoms are representative of cell in advanced state of apoptosis. The contraction of actomyosin during the early stage of apoptosis is known to cause blebs formation, with consequent increase of membrane tension and actin polymerization,89-91 which

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is compatible with the increase of the Young’s modulus. A similar behavior was reported by Wang et al.92, where HeLa cells treated with imidazolium-based ILs underwent apoptosis, showing vesicles in the cytoplasm, as well as surface blebs (confirmed by SEM analysis). Moreover, the increase of stiffness correlated with late apoptosis is well documented, especially in anti-cancer studies about drugs inducing apoptosis.93-95 At high IL concentration (100 mM) a hyperosmotic shock could contribute to the cell shrinkage, in particular considering that the lipophilic nature of cations could further enhance the local crowding of ions near the membrane, and therefore increase the ionic concentration. This osmotic imbalance at extreme levels could produce cytoplasmic crowding due to cell volume reduction, inhibiting the mobility of internal organelles, increasing the cytoplasmic viscosity, and eventually increasing the Young’s modulus. We analyzed cellular volumes from AFM morphologies shown in Figures 1-2, and found a 40% decrease at 100 mM in comparison with control (data in Supporting Information, Table S2), such shrinking could be an effect of apoptosis, as well as of hyperosmotic shock, or a combination of both. This behavior is in agreement with the results reported by Zhou et al.96 and Guo et al.97, where cells that were compressed by hyperosmotic stress show a progressive stiffening induced by cytoplasmic crowding, which is reminiscent of the glass transition for colloidal suspensions.

Effect of the long lateral chain The effectiveness of the interaction of the long-chain [C8MIM][Cl] with the cell membrane compared to ILs with shorter lateral chain cations is expected to be higher, because of the higher lipophilicity. The maximum concentration of [C8MIM]+ used in this study, coherently with the reported EC50 values, was three orders of magnitude lower than those used for [C4MIM]+. The results of the AFM investigation are shown in Figure 3. The trend of the Young’s modulus across the complete concentration range is shown in Figure 4c. We observed a progressive decrease of the Young’s modulus from the cell membrane-actin cytoskeleton region, similarly to the case of the short-chain [C4MIM]+ cation at low concentrations, but in the case of the long-chain IL the decreasing trend continues up to the maximum concentration of 100µM. No stiffening of cells was observed.

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Figure 3. Representative combined topographical and nanomechanical AFM investigation of MDA-MB-231 cells interacting with [C8MIM][Cl] at different concentrations. Along a row, the uncompressed topographic map, the Young’s modulus map, and the Young’s modulus values histograms (cell body and periphery) are shown. Panels a), b) and c) represent the control, while the concentration of IL is 1 µM for d), e) and f); 10 µM for g), h) and i); 100 µM for j), k) and l), respectively.

Gradually increasing the concentration of [C8MIM][Cl], cells retract the protrusions and assume a more rounded morphology. Using a similar long-chain IL ([C8MIM][Br]), Li et al. 98 observed that PC12 cells upon interaction with the IL lost adhesion capability, and became spherical. [C8MIM][Br] was supposed to attack the biomembrane and easily enter the cytoplasm, inducing the production of excessive reactive oxygen species in mitochondria (a key generator of apoptosis).98-99 Also DNA fragmentation, induced by [C8MIM][Br] in nucleus, could be an additional factor inducing apoptosis. Similar results and conclusions were presented by Jing et al. treating human hepatocarcinoma QGY-7701 cells with [C8MIM][Cl]. Radosevic et al. 100 observed that treatment with various imidazolium ILs decreased CCO cell viability; they reported a similar dependency on the alkyl chain length. The enhanced membrane permeability and evident

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membrane disruption were more pronounced for the longer alkyl chain, increasing the proportion of necrotic and apoptotic cells. Although the investigation of the cell morphology upon interaction with [C8MIM][Cl] (see also the collected data in the Supporting Information, Table S3) showed no remarkable signs of apoptosis, increasing the concentration of IL at 1 mM caused detachment, or very poor adhesion, of all the cells. If we interpret the observed retraction of protrusions and the cell rounding as pre-apoptotic symptoms, we can conclude that the transition to apoptosis of MDA-MB-231 cells induced by the long-tail [C8MIM][Cl] is faster than for the shorter-tail ILs.

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Figure 4. Graphical representation of Young’s modulus mean values ± standard deviations (data also reported in Table 1) in the small indentation range for MDA-MB-231 cells interacting with (a) [C4MIM][Cl], (b) [C4MIM][BF4], and (c) [C8MIM][Cl], at different concentrations.

The absence of the cell stiffening at higher concentration of the long-chain IL could be another manifestation of the quicker transition to apoptosis induced by the interaction of cells with long-tail ILs, which in turn could be attributed to the higher affinity of the long alkyl chains for the apolar tails of the phospholipids in the cell membrane. The lateral chains of the cations of the long-tail ILs more easily penetrate into the cell membrane and, consequently, induce a more pronounced perturbation and weakening of the cell membrane. The transition from a progressive softening of the membrane-actin cytoskeleton interface and cell apoptosis is therefore faster and takes place within less than one decade of IL concentration, at odds with the case of short-tail ILs. We cannot exclude that at higher concentrations of [C8MIM][Cl] the increased affinity of the cations with the lipid membrane could also determines a higher hyperosmotic stress, although the related cell stiffening would be probably overshadowed by the apoptotic response of the cells. In perspective, in the case of [C8MIM][Cl] it would be interesting to study with greater resolution the concentration range 100 µM - 1 mM. In general, the availability of EC50 data for MDA-MB231 cells will allow a finer tuning of the concentration range for the ILs used in this study. As a closing remark, we notice that we have based our discussion on a simplified model of the cell outer layer, composed by a phospholipid bilayer coupled to the actin cortex; the real cell membrane however is a far more complex system, with a variety of different proteins and protein complexes located in, an through, the lipid bilayer. Therefore, it would be very important to consider the specific contribution to the toxicity of ILs of IL-protein interactions101-102 at the cell membrane level. In particular, it could be very insightful to perform experiments similar to ours using both protein-friendly ILs, like those containing amino-acidic residues,102-104 or ILs with opposite character.

Conclusions We have carried out a systematic investigation of the effects of short- and long-chain imidazoliumbased ionic liquids on living MDA-MB-231 cells by means of atomic force microscopy. AFM allowed to investigate morphological and mechanical changes of the cells upon interaction with the ILs at different concentrations. The AFM analysis was focused on the external membrane-actin cytoskeleton interface, where the ILs are expected to exert their first action.

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A softening of the cell was detected already at IL concentrations below the expected EC50, accompanied by a change of the cell morphology, characterized by a compaction and retraction of the cell protrusions, a global rounding and loss of cell volume, and eventually, at the highest concentrations, by cell apoptosis and detachment. An intermediate behavior was observed for short-tail ILs, characterized by a marked stiffening of the cells. Our results suggest that at lower concentrations a progressive inclusion of the alkyl chains of the cations within the phospholipid bilayer structure takes place, with a greater efficiency of the process for the longer lateral chains. The inclusion of the alkyl chains into the membrane-actin cytoskeleton wall weakens the cell rigidity. At higher concentrations different phenomena were observed, depending on the length of the alkyl chain of the cation. In the case of short-chain [C4MIM][Cl] and [C4MIM][BF4], the observed increase of the effective Young’s modulus, up to a concentration of 100mM, could be interpreted as the combination of two effects: i) actomyosin contraction during blebs formation in apoptosis process; ii) hyperosmotic shock induced by the local concentration of ions at the cell membrane, with consequent crowding and reduced mobility of cytoplasmic organelles, increased viscosity of the cytoplasm. In the case of long-chain [C8MIM][Cl], the cell softening was observed up to the maximum concentration investigated (100 µM), with no trace of stiffening, and a quicker transition to advanced apoptotic and necrotic conditions was observed, within less than a decade of IL concentration. This result is probably related to the greater affinity of the longer alkyl chains with the apolar tails of the phospholipids in the cell membrane, which also determine a more effective and anticipated disruption of the membrane. Our results provide a systematic and comprehensive view of the direct effects of imidazolium-based ionic liquids on the cell morphological and mechanical properties, and highlight changes in the physical properties of cells that could have a strong toxic potential, already at concentrations below the macroscopically determined EC50 thresholds. Besides, our results also demonstrate the efficacy, for the assessment of the mechanisms of toxicity of ILs, of an analysis strategy based on atomic force microscopy and, possibly, other tools, with peculiar interfacial sensitivity and mapping capability.

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Associated Content Supporting Information. Details of the analysis of force curves; EC50 values of the selected ILs; Volume analysis of MDA-MB-231 cells; AFM data collection; Bibliography.

Acknowledgements The authors thank L. Puricelli, R. Simonetta, and F. Fanalista for support in the development and test of the nanomechanical protocol, and C. Lenardi for support in the cell-biology laboratory of CIMaINa. A.P. thanks the University of Milano for financial support under the projects ‘‘Piano di Sviluppo dell’Ateneo per la Ricerca 2014. Linea B: Supporto per i Giovani Ricercatori’’ and “Transition Grant 2015/2017 – Horizon 2020”. A.P. also thanks the COST Action TD1002 for providing a stimulating environment for the discussion of AFM-based nanomechanics of cells and soft matter. M.G. acknowledges the Shenzhen Science and Technology Innovation Committee (JCYJ20170818160503855) for support.

Conflict of Interest The authors declare no conflict of interest.

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Figure 1. Representative combined topographical and nanomechanical AFM investigation of MDA-MB-231 cells interacting with [C4MIM][Cl] at different concentrations. Along a row, the uncompressed topographic map, the Young’s modulus map, and the Young’s modulus values histograms (cell body and periphery) are shown. Panels a), b) and c) represent the control, while the concentration of IL is 1 mM for d), e) and f); 10 mM for g), h) and i); 100 mM for j), k) and l), respectively. 177x198mm (300 x 300 DPI)


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Figure 2. Representative combined topographical and nanomechanical AFM investigation of MDA-MB-231 cells interacting with [C4MIM][BF4] at different concentrations. Along a row, the uncompressed topographic map, the Young’s modulus map, and the Young’s modulus values histograms (cell body and periphery) are shown. Panels a), b) and c) represent the control, while the concentration of IL is 1 mM for d), e) and f); 10 mM for g), h) and i); 100 mM for j), k) and l), respectively. 177x199mm (300 x 300 DPI)


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Figure 3. Representative combined topographical and nanomechanical AFM investigation of MDA-MB-231 cells interacting with [C8MIM][Cl] at different concentrations. Along a row, the uncompressed topographic map, the Young’s modulus map, and the Young’s modulus values histograms (cell body and periphery) are shown. Panels a), b) and c) represent the control, while the concentration of IL is 1 µM for d), e) and f); 10 µM for g), h) and i); 100 µM for j), k) and l), respectively. 177x196mm (300 x 300 DPI)


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Figure 4. Graphical representation of Young’s modulus mean values ± standard deviations (data also reported in Table 1) in the small indentation range for MDA-MB-231 cells interacting with (a) [C4MIM][Cl], (b) [C4MIM][BF4], and (c) [C8MIM][Cl], at different concentrations. 82x161mm (300 x 300 DPI)


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Graphical abstract 43x23mm (300 x 300 DPI)


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Imidazolium-Based Ionic Liquids Affect Morphology and Rigidity of

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