Impact of Amphiphilic Biomass-Dissolving Ionic Liquids on Biological

Article pubs.acs.org/est

Impact of Amphiphilic Biomass-Dissolving Ionic Liquids on Biological Cells and Liposomes Suvi-Katriina Mikkola,† Alexandra Robciuc,‡,§ Jana Lokajová,† Ashley J. Holding,∥ Michael Lam ̈ merhofer,⊥ ∥ ‡ ∥ ,† Ilkka Kilpelaï nen, Juha M. Holopainen, Alistair W. T. King, and Susanne K. Wiedmer* †

Department of Chemistry and ∥Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, A. I. Virtasen Aukio 1, Post Office Box 55, FIN-00014 University of Helsinki, Finland ‡ Ophthalmology, Helsinki Eye Lab, University of Helsinki and Helsinki University Hospital, Haartmaninkatu 4 C, Post Office Box 220, FIN-00029 Helsinki University Hospital, Finland § Public Health Genomic Unit, National Institute for Health and Welfare, Biomedicum Helsinki 1, Haartmaninkatu 8, FI-00290 Helsinki, Finland ⊥ Institute of Pharmaceutical Sciences, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany S Supporting Information *

ABSTRACT: The toxicity of some promising biomass-dissolving amidinium-, imidazolium-, and phosphonium-based ionic liquids (ILs), toward two different cell lines, human corneal epithelial cells and Escherichia coli bacterial cells, was investigated. In addition, dynamic light scattering (DLS) and ζ potential measurements were used to study the effect of the ILs on the size and surface charge of some model liposomes. Capillary electrophoresis (CE) was used for determination of the electrophoretic mobilities of the liposomes and for determination of the critical micelle concentration (cmc) of the ILs. The toxicity of the phosphonium ILs was highly dependent on the longest linear chain of the IL, due to increasing hydrophobicity, with the long-chain phosphonium ILs being toxic while the shorter-chain versions were significantly less toxic or not toxic at all. Amidinium and imidazolium ILs showed no significant effect on the cells, within the concentration range used. Moreover, the more hydrophobic ILs were found to have a major effect on the surface charges and size distributions of the model liposomes, which can lead to disruption of the lipid bilayer. This indicates that the cytotoxicity is at least to some extent dependent on direct interactions between ILs and the biomembrane.

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INTRODUCTION Their unique properties and nearly unlimited structural combinations give ionic liquids (ILs) great potential for application in a variety of fields, such as lubricants, electrolytes, solvents/catalysts for organic reactions, bioprocessing, and, importantly, dissolution or extraction of biologically relevant compounds and materials,1−6 for example, cellulose dissolution or wood extraction. The most commonly used ILs for this purpose contain 1,3-dialkylimidazolium moieties. These ILs have received much attention in the literature and their application is much more widespread than other classes. Recently amidinium and guanidinium structures have become of interest as more recyclable structures for cellulose and hemicellulose processing.7−9 Likewise, phosphonium-based ILs have also shown potential for biomass processing.2,10,11 In addition, they have also shown great importance in a vast number of industrial and pharmaceutical applications, such as lubricants, electrolytes, or solvents/catalysts for organic reactions, to mention a few.1−4 Phosphonium-based ILs have several advantages over archetypical ILs, such as drastically © 2015 American Chemical Society

improved chemical and thermal stabilities. In addition, the common commercial cations have long aliphatic chains, which typically make them more “hydrophobic” than other IL classes, which should have an effect on the cellulose-dissolving capability of the ionic liquid.12,13 This also makes them miscible with more hydrophobic solvents and they are ideal for being tuned for liquid−liquid phase separation or as media for biphasic reactions.14 In some cases, depending on the chain lengths, the ILs are immiscible with water. Currently, some phosphonium-based ILs are commercially available on a larger scale.15 The increasing interest in the use of ILs for industrial, pharmaceutical, or chemical applications in general forces society to pay attention to their possible environmental impact. Importantly, the risk of air pollution is minimized due to their Received: Revised: Accepted: Published: 1870

October 7, 2014 January 9, 2015 January 12, 2015 January 12, 2015 DOI: 10.1021/es505725g Environ. Sci. Technol. 2015, 49, 1870−1878

Article

Environmental Science & Technology

cells were from Stratagene. 1-Palmitoyl-2-oleyl-sn-glycero-3[phospho-rac-(1-glycerol)] (sodium salt) (POPG) was purchased from Genzyme Pharmaceuticals (Liestal, Switzerland), L-α-phosphatidylcholine (egg, chicken) (eggPC) and cholesterol were from Avanti Lipids (Alabaster, AL). Polybrene (hexadimethrine bromide) was purchased from Fluka (Buchs, Switzerland). Sodium hydrogen phosphate was purchased from Sigma (Darmstadt, Germany), and sodium dihydrogen phosphate monohydrate and HPLC-grade methanol were from Mallinckrodt Baker (Deventer, The Netherlands). The pH solutions (7 and 10) used for calibrating the pH meter were purchased from Merck (Darmstadt, Germany). Sodium hydroxide (1.0 M) was from FF-Chemicals (Haukipudas, Finland), and chloroform was from Rathburn (Walkerburn, U.K.). Distilled water was further purified with a Millipore water-purification system (Millipore, Molsheim, France). For the ionic liquids preparation: acetic acid AcOH (M = 60.1 g/ mol, purity >99.85%, CAS no. 64-19-7) was purchased from Sigma−Aldrich (Helsinki, Finland). 1,5-Diazabicyclo(4.3.0)non-5-ene, DBN (M = 124.2 g/mol, purity 99.9%, CAS no. 3001-72-7), was purchased from Fluorochem Ltd. (Hadfield, U.K.). 1-Ethyl-3-methylimidazolium chloride [emim]Cl (M = 146.6 g/mol, purity >98%, CAS no. 65039-09-0) and 1-ethyl-3methylimidazolium acetate [emim][OAc] (M = 170.2 g/mol, purity 95%, CAS no. 146614-17-4) were purchased from Iolitec GmbH (Heilbronn, Germany). Octyltributylphosphonium chloride [P8444]Cl (M = 351.0 g/mol, purity 96.4%, CAS no. 56315-19-6), tributyl(tetradecyl)phosphonium chloride [P14444] Cl (M = 435.2 g/mol, 50 wt % aq, CAS no. 81741-28-8), and tetrabutylphoshonium chloride [P4444]Cl (M = 288.8 g/mol, 80 wt % aq, CAS no. 2304-30-5) were provided by Cytec Industries (Woodland Park, NJ). 1-Ethyl-3-methylimidazolium dimethylphosphate [emim][Me2PO4] (M = 233.2 g/mol), 1-ethyl-3-methylimidazolium methylhydrogenphosphonate [emim][MeHPO3] (M = 206.2 g/mol), and 1,5-diazabicyclo[4.3.0]non-5-enium acetate [DBNH][OAc] (M = 184.2 g/mol) were synthesized according to previous articles9,50 Synthesis and characterization of ionic liquids tetrabutylphosphonium acetate [P4444][OAc] (M = 312.4 g/mol), octyltributylphosphonium acetate [P8444][OAc] (M = 374.6 g/mol), tributyl(tetradecyl)phosphonium acetate [P14444][OAc] (M = 458.7 g/mol), and trihexyl(tetradecyl)phosphonium acetate [P14666][OAc] (M = 542.9 g/mol) are shown in Supporting Information (Figures S1−S6). The structures and a solubility table for all ILs are shown in Supporting Information (Figure S7 and Table S1). Liposome and Buffer Preparation. Lipid vesicles were prepared from stock solutions of eggPC (20 mM) and POPG (13 mM). Cholesterol was added in certain cases to observe the effect of cholesterol rafts on the interaction of liposomes with ILs. The 100 nm liposomes and sodium phosphate buffer, which was used as solvent for the liposomes and ILs and as background electrolyte (BGE) solution for CE (pH 7.4, ionic strength 10 mM) were prepared as previously described.51 Cell Culture Preparation. SV-40 immortalized human HCE cells were cultured in D-MEM/F12 medium, supplemented with 15% fetal bovine serum, 5 μg/mL insulin, 10 ng/ mL human EGF, 1 μg/mL glutamine, and 40 μg/mL gentamycin.52 XL1-blue E. coli cells were cultured in LB medium (Luria/Miller) containing 100 μg/mL ampicillin. See Supporting Information for details. Cytotoxicity Assay. To reliably measure the extent of cell death induced by ILs, we used the alamarBlue assay

negligible vapor pressure. ILs are thus often considered as “green” replacements for industrial volatile organic compounds (VOCs). The reputation of these solvents as “environmentally friendly” chemicals is justified by their nonvolatility. However, this does not consider contamination of humans, soil, or aquatic ecosystems through accidental spills or industrial drains. Given the excellent stability of ILs, their reduced water solubilities, and the fact that they have generally slow biodegradabilities, even minute amounts can have a large impact on organisms. In addition, the compounds may accumulate in the environment, causing an even greater impact on the ecosystem. Considering this and the fact that most ILs are poorly studied for their ecotoxicity, ILs can hardly be automatically assumed to be “green solvents”. Therefore, fundamental understanding of the ecotoxicity of ILs is necessary for their large-scale application. In addition, before production and use of specific ILs occurs, they should be assessed for their impact on environment and human health.16−19 It has been suggested that the main factors affecting toxicity are (1) length of the alkyl chain in the cation, (2) degree of functionalization in the side chain of the cation, (3) anion nature, (4) cation nature, and (5) mutual influence of anion and cation.20 There are some reports on the health aspects and toxicities of imidazolium-based ILs to soil invertebrates,21−23 various cell lines,23−26 bacteria,23,26−29 enzymes,23 terrestrial plants,23 yeast,27,30 and aquatic systems17,31 such as algae,23,32−35 fish,36 marine invertebrates,28,37−40 and marine plants.23,41 Despite the increasing number of studies assessing aquatic toxicity of ILs, only limited information is available and uncertainties arise for the majority of untested ILs. Up to now, there are only a few phosphonium-based IL toxicity studies concerning lipid biomembranes. No systematic studies have been performed. Phosphonium-based ILs have shown toxicity toward aquatic organisms, having been tested against algae33,42 and bacterial-cell tissues.29,42,43 The toxicity tests are generally based on determining median lethal or effective concentrations (LC50 and EC50, respectively) in different types of model organisms, such as various human cell lines.44−46 These are extensively revised in a review discussing the environmental fate and toxicity of ILs.17,47 In this study the toxicities of some phosphonium-, imidazolium-, and amidinium-based ILs toward a human corneal epithelial cell line (HCE) and E. coli bacterial cells were investigated. In addition, a liposome model system was used to study the effect of the ILs on the size and surface charge of liposomes. Furthermore, capillary electrophoresis (CE) was used to determine the electrophoretic mobilities of the liposomes and the critical micelle concentrations (cmcs) of the ILs. Phosphonium-, amidinium-, and imidazolium-based ILs, commonly used for cellulose or biomass processing, were chosen for this study, due to the potential scale of related biomass-processing applications and to aid in our IL choice for further bioprocessing applications.2,9,48,49

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EXPERIMENTAL SECTION Materials. Dulbecco’s modified Eagle’s medium (D-MEM)/ F12 medium, fetal bovine serum, insulin, human epidermal growth factor (EGF), glutamine, and gentamycin were purchased from Invitrogen Corp. (Carlsbad, CA). Lysogeny broth (LB) medium was purchased from Carl Roth (Karlsruhe, Germany), and ampicillin sodium salt was purchased from AppliChem (Darmstadt, Germany). E. coli XL-1 blue bacterial 1871

DOI: 10.1021/es505725g Environ. Sci. Technol. 2015, 49, 1870−1878

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Figure 1. Cellular toxicity of ILs on HCE cells. The cells were incubated for 18 h with ILs at various concentrations and the cytotoxicity was assessed by an alamarBlue assay. (A) Long-chain phosphonium ILs; (B) shorter-chain phosphonium ILs; (C) imidazolium ILs; (D) [DBNH][OAc] and its unconjugated species. Cell viability is presented as control normalized fluorescence units, proportional to the number of viable cells.

and ζ potential (surface charge) measurements, respectively. The samples were degassed with a vacuum degasser.

(Invitrogen). This assay incorporates a fluorometric/colorimetric indicator and is based on the detection of the oxidation−reduction (redox) capacity of viable cells. Specifically, the assay measures the level of chemical reduction of the reagent in metabolically active cells, by measuring the change from the oxidized (nonfluorescent, blue) form to the reduced (fluorescent, red) form. See Supporting Information for details. Interleukin 8 Secretion. Interleukin 8 (IL-8) secretion was measured from cell-conditioned medium, using a DuoSet enzyme-linked immunosorbent assay (ELISA) for human CXCL8/IL-8 (R&D Systems, Minneapolis, MN) and a protocol recommended by the manufacturer. See Supporting Information for details. Capillary Electrophoresis and Critical Micelle Concentration Determinations. A Hewlett-Packard 3DCE system (Agilent, Waldbronn, Germany), equipped with a diode array detector (wavelength 200 nm), was used for the CE studies (electroosmotic flow and cmc experiments). Experimental details are described in Supporting Information. Size and Surface Charge Determinations of Liposomes. Dynamic light scattering (DLS) measurements for determining the sizes of the liposomes were made on a Malvern Zetasizer Nano ZS (Malvern Instruments, Malvern, U.K.), employing a 20 mW helium/neon laser (633 nm), at a constant temperature of 25 °C. Liposome dispersions were diluted to a concentration equal to 0.05 mM to yield an optimal scattering intensity. The different ILs were added to the liposomes at various concentrations, in the range from 2.2 × 10−4 − 16.7 mM (10−5 to 10−1 %, w/v). The solvent for both liposomes and ILs was phosphate buffer at pH 7.4 (ionic strength 10 mM). Laser Doppler microelectrophoresis, using the same instrumentation, was used to measure the ζ potentials (surface charges) of the liposomes. Measurements were conducted in disposable cuvettes and disposable folded capillary cells for size

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RESULTS AND DISCUSSION Cellular Toxicity of Ionic Liquids on Human Corneal Epithelial Cells. Using two complementary methods, we tested the toxicity of a selection of cellulose-dissolving ILs on a HCE cell line. The HCE cells are adherent cells that grow in monolayers in cell culture. The HCE cells were considered a good model as they retain many of the characteristics of the tissue of origin (corneal epithelium). To test the extent of cell death induced by the ILs, the cellular monolayers were incubated with IL solutions (2.2 × 10−4 to 16.7 mM, 10−5 to 10−1 %, w/v), in order to determine the concentration range of the median effective concentration for the aforementioned compounds. Results obtained with a metabolic activity indicator are presented in Figure 1, where the fluorescence measured for the reagent is directly proportional to the number of viable cells. With this method, the EC50 for the long-chain phosphonium ILs (Figure 1A) is below ([P14444]+) or in the 0.02 mM concentration range and shows an inverse relationship with the length of the side chains (below 0.3 mM for [P8444]+ and above 3 mM for [P4444]+) (Figure 1B). The amidinium- and imidazolium-based ILs showed no significant toxicity toward the HCE cells at these low concentrations, and the differences between the anions was minimal, with only [emim][MeHPO3] affecting the cell viability slightly (Figure 1C). [DBNH][OAc] and the unconjugated DBN and HOAc showed no toxicity toward HCE cells (Figure 1D). Similar to skin epithelium, the corneal epithelium (HCE source tissue) is a tissue in continuous contact with the outside environment and hence participates in the immune response to environmental aggression. One example is the secretion of interleukin 8 from the HCE cells as a response to stress. Interleukin 8 is an acute-phase cytokine, one of the first to be 1872


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Figure 2. Effects of ILs on HCE cells. (A) HCE secretion of interleukin 8 as a result of extracellular stress induced by ILs. The dotted line represents basal (noninduced) interleukin 8 secretion. (B) Cell survival as determined from total protein concentration measurement after incubation with ILs, normalized to the control (considered here 1.00). The dotted line marks a 50% cell loss.

Figure 3. Cellular toxicity of ILs on E. coli bacterial cells. The cells were incubated for 2 h with selected ILs at various concentrations, and the cytotoxicity was assessed by alamarBlue assay. (A) Long-chain phosphonium ILs; (B) shorter-chain phosphonium ILs; (C) imidazolium ILs; (D) [DBNH][OAc] and its unconjugated species. Cell viability is presented as absorption units, which are normalized to negative and positive controls. Viabilities are proportional to the number of viable cells.

of injury. Specifically, we measured the amount of interleukin 8 secreted by HCE cells as a response to ILs in the medium (Figure 2A). The increased secretion of interleukin 8 induced

secreted after stimulation. Cells secrete cytokines as a response to extracellular stress, and in the tissue of origin, interleukin 8 is an important stress signal that recruits immune cells to the site 1873


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cellular models. In addition, there was no clear difference between Cl− and [OAc]− anions but with short alkyl chain phosphonium cations a slight lethal effect of Cl− anion was noticed. Four ILs based on [emim]+ were included in the study, and the results proved that there is a distinction between phosphonium and short-chain imidazolium cations with regard to their toxicity toward the tested cells. For the imidazolium ILs only [emim][MeHPO3] slightly affected the cells’ viability, most likely due to the phosphonate anion. Toxicity measured for the E. coli culture was remarkably similar to the results from the human epithelial cell line, which supports the idea that the decreased fluorescence intensity is due to cell death. Although no significant insight into the mechanism of toxicity is provided with these fast screening tests, some additional information is offered by the measurement of cytokine secretion induced by the ILs. Secretion is a timedependent event; hence, the cells have to survive long enough to activate the stress-response pathway and interleukin 8 secretion pathway. Significant levels of cytokine in the medium are reached after 2 h. Moreover, even if the cytotoxic level is similar, the ILs might interact differently with the cells. For example, [P4444]Cl induced a dose-dependent secretion of interleukin 8, while [P4444][OAc] did not. Both [P8444]+ ILs were toxic to HCE cells at 2.8 mM, but interleukin 8 secretion was increased even when there were fewer than 40% of the cells on the plate. This observation suggests that the loss of viability was a lengthy process with the cells surviving for several hours before cell death. The mechanism behind IL toxicities is poorly understood, but some hypotheses for molecular interactions between imidazolium-based ILs and lipid membranes exist.57,58 In these models the long alkyl chains of the ILs are assumed to be inserted in the lipid membrane, due to lipophilic effects. Due to the rigid structure of imidazolium-based ILs, it is very possible that the ILs will remain more or less close to the outer polar surface of the lipid membrane, having only part of the molecule embedded in the lipid environment. In addition, it is shown that cationic ILs can exert their toxic effect through narcosis, due to their ability to penetrate into the lipid bilayer and furthermore causing membrane-bound protein disruption.39,59−61 Our studies show that the n-alkyl chain length of the IL is directly correlated with toxicity, such that longer chain lengths showed higher toxicity values. Determination of Critical Micelle Concentrations of Ionic Liquids. The cmc values of all relevant ILs were determined in order to see whether the ILs are occurring as monomers or as aggregates in the EC50 concentration range. Even though there is not much information about the interactions between ILs and lipid bilayers or vesicles, some computer simulation studies have been performed. They have suggested that the alkyl chains of the tested ILs are deeply inserted into the hydrophobic region of the lipid bilayer and the penetration is dependent on the alkyl chain length. With increasing IL concentrations the bilayer surface become rough, which can eventually lead to bilayer disruption.62 Furthermore, some charged molecule aggregates, especially cations, were shown to form pores or channels into the lipid bilayer, causing structural damage.63−65 In addition, bioanalytical analyses have shown that the IL exhibiting the strongest micellization effect has the most pronounced binding and disruption effect on membrane bilayers.57 The electric current in a fused silica capillary, at a constant voltage, was determined by use of CE instrumentation (see

by some phosphonium ILs suggests that the cells are surviving the encounter with the ILs long enough to engage the stressresponse pathway. When the stimulus (i.e., the IL) is too damaging, interleukin secretion is either not initiated or there are too few viable cells for measurable results ([P14444]Cl and [P14444][OAc], Figure 2A). Interestingly, cells responded to most phosphonium ILs and the cytokine secretion showed concentration dependence. [P4444][OAc] was neither cytotoxic at the tested concentrations nor an inducer of cytokine secretion. In addition, imidazolium ILs did not induce cytokine secretion. The fact that [P 4444][OAc] does not show cytotoxicity suggests that there is no significant toxicity attributable to the phosphonium functionality. However, [P4444]Cl does induce a significant stress response at higher concentrations, not present for [P4444][OAc], which indicates an effect of the counterion on the cytotoxicity. Figure 2B presents total protein concentration from attached cells after incubation with ILs (dead or damaged cells detach from the cell culture substrate and are excluded from this measurement). Results from the total protein assay were in good agreement with the data from Figure 1 and also allow a percent estimation of cell death. Escherichia coli Cells. E. coli is the most widely studied prokaryotic model organism in the field of biotechnology and microbiology, mainly because the bacterium can be grown easily and inexpensively in a laboratory setting. It has been intensively studied for over 60 years. The results of E. coli cell death induced by different concentrations of the ILs in the present study are presented in Figure 3. The EC50 for [P14444]+ is below 0.02 mM, which is consistent with the results obtained for HCE cells. The [P8444]+ ILs showed lower toxicity toward E. coli cells than toward HCE cells (Figure 3B) and were slightly dependent on the anion. Effective concentrations were below 0.29 and 2.67 mM for [P8444]Cl and [P8444][OAc], respectively. Because of phase separation of [P14666]+ ILs in the LB medium, their effect on cell death could not be determined. Amidinium, imidazolium, and [P4444]+ ILs were not toxic toward E. coli cells in the applied concentration range. However, [P4444]Cl was slightly cytotoxic at a concentration of 3.5 mM, which is in accordance with the suggestion that the counterion has an effect on the cytotoxicity. Even though [DBNH][OAc] was not toxic at the measured concentration range, the 8.05 mM concentration of DBN slightly affected the viability of the cells. As DBN is a known superbase with extremely high affinity for protons, this effect may be caused simply by the increased pH, which may also affect cell viability and lead to toxicity. In addition, 16.65 mM HOAc was lethal to E. coli cells, which is in agreement with previous reports on the effect of acetic acid on biological systems.53,54 However, the toxicity of acetic acid is pH-dependent, because it is the undissociated form that diffuses through the cell membrane.55 As previously shown,18,20 the toxicity of phosphonium ILs is influenced by the length of the alkyl side chains, with the longer chains being more toxic than short-chain phosphonium ILs, as can be seen from Figures 1 and 3. With increasing alkyl chain length of the ILs, the hydrophobicity increases; in fact, Ranke et al.56 demonstrated a correlation between lipophilicity and toxicity and concluded that cation lipophilicity was the dominating factor for IL cytotoxicity. [P14444]+ and [P14666]+ were toxic for more than 50% of the cells at the 0.02 mM concentration and [P8444]+ was toxic in the 0.3−2.7 mM concentration range, while the EC50 for [P4444]+ could not be determined at the tested concentrations (>3.2 mM) on both 1874


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Figure 4. ζ Potentials of 0.05 mM eggPC/POPG 75/25 (mol %) and eggPC/POPG/cholesterol 50/25/25 (mol %) liposomes, with the addition of ILs at various concentrations: (A) [P14444]Cl, (B) [P14444][OAc], (C) [P8444]Cl, and (D) [emim][OAc] ILs.

Determination of Liposome Size and Surface Charge. The toxicity of the IL analogues and homologues suggests that the toxicity arises from interaction with lipophilic cell membranes. Even the adsorption of the compound on the biomembrane is expected to affect the toxicity of the compound, as this might hinder membrane transportation. Therefore, we applied dynamic light scattering to observe the effect of ILs on model liposomes, composed of phosphatidylcholine and phosphatidylglycerol. We also added cholesterol to the liposomes to observe the effect of rigid rafts on the interaction with ILs. We followed the changes in sizes and surface charges of the liposomes in the presence of different ILs, at various concentrations in the range 2.18−5.88 mM. For the size experiments by DLS we selected the ILs that showed strong toxicity even at low concentrations, namely, [P14444]Cl and [P14444][OAc]. [P8444]Cl, which possesses shorter alkyl chains and exhibited toxicity only at slightly higher concentration (see Figures 1−3), was also chosen. As a control we used [emim][OAc], which showed no toxicity. At certain concentrations of the ILs in solution, we observed that the liposomes were shrinking (Figure S8, Supporting Information). The shrinkage of liposomes in the presence of [P8444]Cl was not that strong compared to when [P14444]Cl or [P14444][OAc] were added to the sample. Moreover, the liposomes started to be disrupted in the presence of 0.2 mM or higher concentrations of [P14444]Cl or [P14444][OAc] (Figure S9, Supporting Information). The profiles of the size distribution of the liposomes were different and some particle aggregation was also observed. This disruption of the model liposomes indicate that instead of suppression of biological activity, the mechanism of IL cytotoxicity is at least partially affected by the direct interaction between ILs and the biomembrane.

Supporting Information). This modification of a conventional conductivity method is based on measurements of the difference in mobility of the surfactant, occurring either as single monomers or in an aggregated form, that is, in the form of micelles. [emim]+ cations are not able to form stable spherical aggregates due to their short alkyl chains; however, some anions (e.g., tosylate) have been shown to create clusters, stabilized by a network of cations.66 In addition, [Cnmim]+ cations, where n > 4, have been shown to form regularly sized and close to spherical charged micelles. Other types of aggregates, like disks, have also occurred in solution.67 At this stage we are not able to comment on the type of aggregates formed by the ILs used in this work. The electric current was plotted against an increasing concentration of IL. A bilinear dependency was observed, and the intersection point derived by a smooth connection of the two linear trend lines gave the cmc. The cmcs of [P14444]Cl were 1.0 mM in water and 0.3 mM in sodium phosphate buffer. The corresponding values for [P14444][OAc] were 3.2 and 0.3 mM, respectively. The cmc values of [P14666][OAc] were 0.8 mM in water and 0.1−0.2 mM in sodium phosphate buffer. These values correspond well with the general observation that longer-chain surfactants aggregate at lower concentrations. Some preaggregation of [P8444]Cl occurred in water at an IL concentration of 0.3 mM. However, preliminary data obtained by other techniques suggest the actual cmc of [P8444]Cl to be much higher. This is currently under investigation in our group. The cmc values for all amidinium- and imidazolium-based ILs used in this work could not be determined, due to lack of stable micelles in the solution. In addition, the cmc values of studied ILs were lower in water than in sodium phosphate buffer due to the lack of stabilizing ions. Our data show that the cmcs of the phosphonium-based ILs were in the same concentration range, or higher, as there was an increase in the toxicity of the ILs. 1875


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Environmental Science & Technology ζ Potentials at various concentrations of the tested ILs are shown in Figure 4. A clear decrease in the surface charge of the liposome was observed, with an increase in IL concentration for all toxic compounds, while there was no change in ζ potential for the nontoxic [emim][OAc]. Addition of [P8444]Cl to the liposome only diminished the negative surface charge on the liposome. However, both ILs with longer alkyl chains, [P14444]Cl, and [P14444][OAc], even reversed the surface charge of the liposome at concentrations between 10−2 and 10−3 %. This can be explained by a rather thick layer of [P14444]Cl or [P14444][OAc] adsorbed on the surface of the liposome membrane. From the size and ζ potential determinations, we can assume that the presence of possible cholesterol rafts in the liposome did not have any significant effect on the interaction of ILs with liposomes (see Figure 4 and Figures S8 and S9 in Supporting Information). Capillary Electrophoresis Determinations. The liposomes were injected into the silica capillary, filled with various concentration of [P14444]Cl, [P14444][OAc], and [P8444]Cl, to see if we can use CE as a method for observing a change in the surface charge of the liposome due to interactions with ILs. We tested two liposomes composed of eggPC/POPG 75/25 (mol %) and eggPC/POPG/cholesterol 50/25/25 (mol %), and we calculated the effective electrophoretic mobility of the liposomes. The results in Figure S10 (Supporting Information) show the same profiles for almost all liposome mobilities as the ζ potential data, observed by the Malvern Zetasizer. However, we could not get any result for a 2.3 mM concentration of [P14444]Cl, and the effective mobilities of the liposomes at the 0.02 mM concentration differed from the data obtained from the Malvern Zetasizer measurements, probably due to the presence of Polybrene on the surface of the capillary. The profiles observed by CE when [P14444][OAc] was added support the results obtained by the Malvern Zetasizer. The effective electrophoretic mobilities of the liposomes could not be determined when the [P14444][OAc] concentration was 0.22 mM because of reduction of the mobilities to close-to-zero. Liposome surface charges were reversed to positive when the [P14444][OAc] concentration was increased to 0.69 mM. In addition, the effect of [P8444]Cl on the liposome surface charges was minimal. The results show that the studied cells were strongly influenced by the type and concentration of IL used. The shorter-chain amidinium and imidazolium ILs showed no significant effect on the cells within the concentration range used, while most of the longer-chain phosphonium ILs demonstrated a significant toxicity effect, especially at higher concentrations. However, shorter-chain phosphonium ILs (e.g., [P4444]+) did not exhibit any significant toxicity effect. This indicates that the toxicity of phosphonium ILs depends highly on the hydrophobicity of IL, which itself is dependent on the alkyl chain length. The DLS, ζ potential determinations, and CE methods were found to be suitable for studying changes in the sizes and surface charges of phosphatidylcholine and phosphatidylglycerol liposomes, with or without cholesterol, when various ILs were added. The more hydrophobic ILs had a major effect on the surface charges and size distributions of the model liposomes. Moreover, the model liposomes started to be disrupted in the presence of [P14444]+ cations, suggesting that the cytotoxicity is not only dependent on the suppression of bioactivity but also dependent on the direct interaction of ILs and the biomembrane. The proposed methods also offer a

convenient way to obtain data on IL toxicity, which is of importance for numerous applications in industry as well as for obtaining knowledge to develop new “green” environmentally friendly solvents. In regard to the design of ILs for biomass processing, amphiphilicity is obviously important for cellulose dissolution. While the smaller phosphonium cations do not demonstrate any overt toxicity toward these systems, too large a hydrophobic content can drastically increase the toxicity of potential structures. Overall, an optimum balance between cellulose dissolution capability, recyclability, and environmental harmfulness must therefore be found by varying the cationic or anionic chain lengths.

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ASSOCIATED CONTENT

* Supporting Information S

Additional text describing IL preparation, cell studies, CE determination, determination of cmcs, and determination of sizes by DLS; 10 figures showing ATR-IR and 1H and 13C NMR spectra of [P8444][OAc], 1 H NMR spectra of [P4444][OAc], [P14444][OAc], and [P14666][OAc], IL structures and van der Waals volumes of IL cations, size (number distribution) of liposomes with the addition of ILs, change of the size profile (volume distribution) of liposomes in the presence of IL, and effective electrophoretic mobilities of liposomes in the presence of various ILs; one table listing solubility of ionic liquids in different solvents. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +358 2941 50183; e-mail: susanne.wiedmer@helsinki. fi. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Stefan Polnick from the University of Tübingen is acknowledged for assistance with the E. coli toxicity determintations. Financial support from the Academy of Finland, Project 266342 (S.K.W.), and the Magnus Ehrnrooth Foundation (S.K.W.) is acknowledged. Jeff Dyck and Al Robertson from Cytec Industries are gratefully acknowledged for the provision of their off-the-shelf phosphonium chloride starting salts. Financial support was also provided through the Future Biorefinery (FuBio) Project, from the Finnish Bioeconomy Cluster (FIBIC). S.K.W. and M.L. are grateful for financial support by the Academy of Finland (Project 276075) and German Academic Exchange Service (Project 54655966), respectively.

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NOTE ADDED AFTER ASAP PUBLICATION This article published January 21, 2015 with a mistake in Figure S7 of the Supporting Information. The corrected SI file published January 23, 2015.

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