Unveiling the Interaction Between Fatty Acid Modified Membrane and

Article pubs.acs.org/JPCB

Unveiling the Interaction between Fatty-Acid-Modified Membrane and Hydrophilic Imidazolium-Based Ionic Liquid: Understanding the Mechanism of Ionic Liquid Cytotoxicity Niloy Kundu,† Shreya Roy,† Devdeep Mukherjee,‡ Tapas Kumar Maiti,‡ and Nilmoni Sarkar*,† †

Department of Chemistry and ‡Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, WB, India W Web-Enhanced Feature * S Supporting Information *

ABSTRACT: Ionic liquids (ILs) are considered as “green solvents” for more than 2 decades. However, recent studies suggest that some ILs exhibit greater toxicity compared to common solvents. As a proactive effort to better understand the molecular origin of the cytotoxicity, the work herein presents the systemic characterization of the interaction between model membrane composed of fatty acids and popular imidazolium-based hydrophilic IL. The fusion kinetics between the vesicles demonstrates the swelling of the vesicle. Further, membrane fluidity is determined using the isomerization kinetics of a lipophilic dye, merocyanine-540, and in the presence of IL, the fluidity of the inner water pool of the vesicle is increased. The results can be directly correlated to the cytotoxicity generated by IL in K562 cell, a human erythroleukemic cell line. High-concentration IL ruptures the cell membrane and causes membrane permeabilization. Thus, the results would help to facilitate the rational design of nontoxic ILs.

1. INTRODUCTION In recent years, room-temperature ionic liquids (RTILs) have been extensively investigated in the field of chemistry, material science, and biology because of their unique physical and chemical properties, such as nonflammability, nonvolatility, high-temperature stability, and so forth.1,2 These properties improve their applicability in a wide range of applications.3−7 From the economic point of view, they can be replacement for various volatile organic solvents in industrial chemical processes.6 However, a popular class of imidazolium-based ionic liquids shows toxic properties toward different aquatic species and organisms and the degree of toxicity is higher than that of conventional solvents.8 Many studies have been aimed to understand the relationship between the IL structure and their toxicity, and it generally depends on (a) the nature of cation and anion, (b) the length of the alkyl side chain in the cation, (c) interactions between cation and anion, and so forth.9 ILs are generally nonbiodegradable in nature and therefore their interaction with the environment is of high importance. It can be assumed that the higher water solubility of ILs corresponds to higher danger to the environment as they can easily penetrate into the ecosystems. Thus, ionic liquids (ILs) have impact on the different stages of life, from single protein to multicellular organism.10,11 As water is crucial for all of the living systems, solubility and interactions between ILs and water are one of the primary factors that determine the biological activities of ILs and it depends on the hydration states of the ILs.12,13 The biological effect of IL−water mixture changes dramatically when one ion pair of the IL is surrounded by seven water molecules, irrespective of the nature of the ions, © 2017 American Chemical Society

and this threshold hydration number is required for several biochemical reactions, such as microbial growth, enzymatic reaction, and so forth.12 To corroborate the dependency of biological activities of IL−water mixture, various simulation studies between aqueous solution of IL and phospholipid bilayer are performed,14−18 which suggest that the behavior of the IL anions depends on their interaction with the water molecules.14 Generally, small hydrophilic anion Cl− stays at the solution, whereas hydrophobic [Tf2N]− moves inside the lipid bilayer along with the imidazolium cation.15 Further, structural analysis by molecular dynamics (MD) simulation study reveals that IL’s cation asymmetrically inserts into the lipid bilayer, which causes the morphological disruption of the lipid bilayer.16,17 However, the insertion of IL, which causes the swelling of the lipid bilayer, strongly depends on the hydrophobicity of the alkyl chain of IL cation as well as the anion.19 Thus, it can be correlated to the cytotoxicity of the IL. However, the mechanism of IL cytotoxicity is yet not well understood. In this context, we present here a comprehensive characterization of fatty-acid-membrane interaction with a popular hydrophilic imidazolium-based ionic liquid, 1-butyl-3methyl imidazolium tetrafluoroborate (Bmim-BF4). Fatty acids spontaneously form bilayer vesicle in aqueous medium when the pH of the system is close to the pKa of the fatty acid.20,21 For this reason, early cell membranes are thought to comprise fatty acids or simple amphiphilic molecules and fatty-acid-based vesicles are considered as one of the important biomimetic Received: June 26, 2017 Published: July 30, 2017 8162

DOI: 10.1021/acs.jpcb.7b06231 J. Phys. Chem. B 2017, 121, 8162−8170

Article

The Journal of Physical Chemistry B

Scheme 1. Structure of the Oleate Ethyl Amine (OEA), Hydrophilic Imidazolium-Based Ionic Liquid, and the Fluorophores (MC-540, DCM)

model membranes.22 The most important feature of fatty-acid vesicles is their self-replication.23 Therefore, they can be utilized to understand the great emergence of cellular life in real biological system.24 Fatty acids form different aggregated structures in solution depending on the pH of the medium.20 Fatty acids have hydrophobic tail part and hydrophilic head group similar to phospholipids, and they can also form the same type of structures, such as bilayer vesicle micelle, depending on the nature of the environment. The structures of these fatty-acid vesicles are much simpler and may have formed more readily in prebiotic environment. However, phospholipid vesicles are unlikely candidates for the protocell membrane as they are less permeable to many nutrients essential for basic cell function.25 Furthermore, they are relatively complex molecules and thus several enzymes are required for their synthesis in modern cells. On the one hand, fatty-acid-based vesicles are better suited as a component of vesicle-based protocells because their bilayer membrane permits the passive diffusion of ions and small molecules in and out of the vesicle compartments.25 On the other hand, oleic acid/oleate vesicle, an important fatty-acid vesicle membrane involves the enzymatic polycondensation of ADP into the homo-polyriboneucleotide, poly(A), which can be considered as a prototype of RNA.26 In case of RNA replication, biophysical consideration suggests that primordial replicating compartments are membranous vesicles composed of fatty-acid vesicles. Szostak et al. showed RNA-copying reaction within fatty-acid vesicles by addition of activated nucleotides to the outside of vesicles, which contain primer template complex.27 Thus, there is a growing consensus that fatty-acid-based vesicles are plausible models of primitive cellular compartments. Oleic acid, an important fatty acid, forms bilayer structure when the pH of the solution is adjusted in between 8 and 9. However, at physiological pH, it forms an oil droplet.28 Thus, the utility of vesicles composed of simple fatty acid is limited because of their poor stability with respect to the pH, and such stability can be enhanced by mixing the fatty acids with different amphiphiles or fatty alcohols and so forth.29 In our recent work, we have synthesized a protic ionic liquid of fatty acid (oleate ethyl amine, OEA) with ethyl amine as a component and it spontaneously formed a vesicle in aqueous solution.29 The formation of the vesicle was confirmed by transmission electron microscopy (TEM) and fluorescence lifetime imaging micros-

copy (FLIM) measurements. This OEA vesicle is considered here as a model membrane system. The chemical structures of the OEA vesicle, IL, and fluorophores, which are used in the FLIM measurements, are shown in Scheme 1. In this work, we have first systematically characterized the interaction between the model membrane and Bmim-BF4 ionic liquid by FLIM and fluorescence correlation spectroscopy (FCS) measurements and further, to correlate the IL cytotoxicity and membrane fluidity of K562 cells, a human erythroleukemic cell line is measured in the presence of IL. Thus, the obtained result will improve our understanding of the mechanism of IL cytotoxicity and it would help to design a new class of ILs exhibiting benign environmental profile.

2. EXPERIMENTAL SECTION 2.1. Materials. Oleic acid and ethyl amine are obtained from Loba Chemie Ltd. (India) and SRL (India), respectively. The synthesis procedure of oleate ethyl amine (OEA) is described in our earlier manuscript.29 Briefly, equimolar amount of oleic acid and ethyl amine mixture is magnetically stirred at low temperature (273−278 K) to obtain a light yellow viscous product. Water is removed by rotary evaporation, followed by lyophilization. Ionic liquid (IL) Bmim-BF4 is purchased from SRL (India), and the water content of the IL is less than 150 ppm. 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)4H-pyran (DCM) obtained from Exciton is used for FLIM measurements. Merocyanine-540 (MC-540) dye purchased from Sigma-Aldrich is used to determine the local viscosity by FCS and FLIM measurements. All of the chemicals are used as received and double-distilled deionized water (Milli-Q) is used in all experiments. The chemical structures of all of the chemicals are shown in Scheme 1 (in the article). 2.2. Maintenance of Cell Lines. Chronic myeloid leukemia cell line (K562) is obtained from National Center for Cell Science, India, and maintained inside a humidified 5% CO2 incubator with regular subculture in Roswell Park Memorial Institute (RPMI-1640) medium, which is supplemented with 10% fetal bovine serum, 100 units of penicillin, and 0.1 mg of streptomycin. 2.3. Cell Viability Assay. K562 cells are seeded in a 96-well plate at a density of 1 × 104 cells/well and incubated with the indicated concentration of Bmim-BF 4 . Then, 3-(4, 58163


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Figure 1. (a) FLIM images and intensity images of formation of different-sized vesicles of 0.1 M OEA in the presence of 0.5 M Bmim-BF4 (scale bars in images (i), (ii), and (iii) are 16, 32, and 18 μm, respectively). (b) DLS intensity distribution profile of 0.1 M probe sonicated OEA vesicles in the presence of different concentrations of Bmim-BF4.

morphological change of vesicles or bilayer disruption is observed in case of OEA vesicles in the presence of Bmim-BF4, and the bilayer of the vesicles is clearly distinguishable in the FLIM images. The images present in Figure 1a are the time domain images of OEA vesicles stained with DCM dye. Figure 1a exhibits the intensity images of the vesicles calculated from the photons in all time channels of the pixels. In the FLIM image, the lifetime obtained for each pixel is enclosed by color (Figure S1a, Supporting Information), and the lifetime is significantly varied in different regions of the vesicles. Thus, the lifetime distribution obtained from the FLIM image is quite broad in nature (Figure S1b, Supporting Information), and it is deconvoluted into two spectra. The short-distribution component contributes to the probe molecules, which are located at the water pool of the vesicle, and the higher lifetime distribution signifies the probe molecules that are at the hydrophobic bilayer of the vesicle. The formation of giant vesicles in the presence of Bmim-BF4 is also established from the field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) images (Figure S2, Supporting Information). In the FE-SEM image, vesicle size of 70 μm is observed. The insertion of Bmim-BF4 causes the swelling of the vesicles, which increases their sizes.19 Now, the increase in the size of the vesicles is also evidenced from the DLS measurements (Figure 1b). For the DLS measurements, larger OEA vesicles are converted into smaller ones by probe sonication for 15 min using an ultrasonicator bath. For the small unilamellar vesicle, the size obtained from DLS measurement is 150 nm. Addition of different concentrations of Bmim-BF4 into the solution leads to increase in the intensity distribution of the OEA vesicles, and it suggests the fusion of the vesicles in the presence of IL. Earlier, we have shown that the fusion of OEA vesicles is triggered by sodium chloride (NaCl),29 and it is suggested that Na+ promotes the close association of the vesicles and due to the osmotic swelling, the rate of fusion is increased. Now, Bmim-BF4 can also behave as

dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay is performed as described elsewhere.30 The absorbance is measured at 570 nm using a microplate reader (Thermo). 2.4. Preparation of Cells for FCS and Lifetime Imaging. K562 cells are seeded on a coverslip and treated with MC-540 for 2 h. Then, the cells are washed thrice with PBS (10 mM, pH 7.4) and finally kept in PBS buffer and analyzed under microscope. For the FCS measurements, the dye to vesicle ratio is kept quite low (1:1 000 000) to ensure that each vesicle has no more than one fluorophore. However, in case of FLIM measurements, the dye concentration is higher (∼0.1 μM). 2.5. Instrumentation. The interaction between IL and OEA vesicles is measured by proton nuclear magnetic resonance (1H NMR) measurements. The change in the size of the vesicles in the presence of IL is measured by dynamic light scattering (DLS) measurements and visualized by electron microscopy and FLIM measurements. The membrane fluidity of the OEA vesicles and K562 cell were measured by FCS measurements using merocyanine-540 (MC-540) as a probe molecule. A detailed description of the instruments is discussed in the Supporting Information.

3. RESULTS AND DISCUSSIONS Figure 1a demonstrates the FLIM images of OEA vesicles in the presence of 0.5 M Bmim-BF4. The size of the vesicles is significantly increased, and a wide variation in the size is observed from the FLIM images. In the images, among the smaller vesicles, very large and spherical vesicles are also observed with diameters up to 20−50 μm. The size of the OEA vesicle in the absence of IL is varied in the range of 0.5−5 μm.29 In case of lipid vesicle, Zhu et al. reported that imidazolium-based IL could disrupt the bilayer of the vesicle, which leads to disintegration of lipid bilayer and formation of IL−lipid mixed micelles in aqueous solution.19 However, no 8164


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Figure 2. FLIM images of OEA vesicle in the presence of 0.1 M Bmim-BF4 collected in different time scales (time zero indicates the time when the image of the sample is started to record; it does not imply the starting time of the reaction after addition of Bmim-BF4 into the medium).

measurements. NMR spectra of OEA vesicles are taken in the presence of different concentrations of Bmim-BF4 (Figure S5, Supporting Information). The protons of the imidazolium ring in Bmim-BF4 are not very acidic, and the hydrogen-bonding interaction between the imidazolium protons and the anions (such as BF4− and Cl−) is reported in the literature.33 In the presence of OEA vesicles, the imidazolium protons (H-a, H-c) are shifted toward lower magnetic field. On the other hand, the protons (H-b) on the side chain of the imidazolium ring and the olefinic protons (H1) of OEA are shifted toward lower magnetic field. Thus, the 1H NMR spectra clearly signify the hydrogen bonding as well as the hydrophobic interaction between the OEA vesicle and Bmim-BF4, and it can be proposed that the alkyl side chain of the Bmim-BF4 inserts into the hydrophobic bilayer of the vesicle, whereas the imidazolium cation interacts with the head group of the vesicle. Membrane fluidity is an important parameter to elucidate the vesicle-fusion kinetics. Now, to determine the local viscosity and membrane fluidity in the presence of different concentrations of Bmim-BF4, we have used merocyanine-540 (MC540) as a probe molecule. MC-540, an anionic lipophilic dye, generally binds to the outer leaflet of the membrane, and its fluorescence is very sensitive to polarity, lipid packing, and so forth.34,35 Extensive photophysical study of MC-540 indicates that the excited-state dynamics of MC-540 can be characterized by the normal (N) state and photoisomer (P) state. The N and P states signify the formation of trans and cis isomers in the excited states, respectively. Due to the large energy barrier between the trans and cis states, the dye molecule exists in trans form in the ground state.35 However, in the first-excited state, the bond order of the central double bond of polymethine chain is significantly reduced, which allows the molecule to twist during its first-excited-state lifetime. The photoisomerization model of MC-540 is described in detail in the Supporting Information. After binding with a loosely packed membrane, MC-50 is oriented parallel to the membrane surface. However, in case of tightly packed membrane, it is oriented perpendicular to the surface and the fluorescence emission spectra are redshifted along with the decrease in its fluorescence quantum yield.34 In our case, with addition of 0.5 M Bmim-BF4 in 0.1 M probe sonicated vesicle, fluorescence intensity is slightly increased although no shift in the emission property is

an electrolyte due to the presence of imidazolium cation and BF4− anion.31 The different events in vesicle fusion are evidenced by performing time scan FLIM measurements (Figure 2). The time duration of the collection of each image is 1 s, and the images are assembled together and converted into a movie frame (Supporting Information, Movie 1). The time scale of the fusion processes is varied from milliseconds to seconds. However, compared to that of the NaCl-induced fusion, the rate of the fusion is much higher in this process. The vesicle fusion can proceed through several distinct steps, such as hemifusion and direct fusion, and the latter consumes much higher energy than the former.32 In case of hemifusion, the outer bilayer leaflets are mixed. After this step, the inner membrane of two vesicles is mixed and forms a nascent fusion pore. At this stage, the water molecules inside the inner pool of the vesicles are exchanged. To understand different events in vesicle fusion, we have measured the lifetime distribution associated with each image in different time. However, to avoid complication, we have focused on the lifetime associated with two vesicles which will be fused (Figure S3a, Supporting Information), and we have observed that the lifetime of DCM is decreased with increasing time (Figure S3b, Supporting Information). As DCM is a hydrophobic dye, it is mainly located at the bilayer region of the vesicles. Thus, during the exchange of water molecules, the lifetime associated with vesicles is gradually decreased. We have measured the ζ potential of the vesicles in the presence of different concentrations of IL (Figure S4, Supporting Information), and the ζ potential value increased with increasing concentration of IL. Thus, it is suggested that the negative charge of the vesicle is neutralized in the presence of Bmim-BF4 and the electrostatic repulsion between the vesicles is decreased. Consequently, addition of IL into the vesicle solution allows the vesicle to come close to each other. Besides the electrostatic interaction between the OEA vesicles and Bmim-BF4, recent MD simulation study suggests that amphiphilic IL easily inserts into the bilayer of vesicles due to the hydrophobic interaction irrespective of the alkyl chain length of the IL.16,17 To elucidate the hydrophobic interaction between OEA membrane and Bmim-BF4, we have performed 1H NMR 8165


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Figure 3. (a) FLIM images of 0.1 M OEA vesicles in the presence of different concentrations of Bmim-BF4 stained with MC-540. (b) Lifetime distribution of MC-540 obtained from (a).

FCS measurements of MC-540 in 0.05 M probe sonicated small OEA vesicles are performed to investigate the extent of the photoisomerization that can be detected in membrane. The FCS traces in vesicle in the absence and presence of Bmim-BF4

observed (Figure S6, Supporting Information). Recently, Chmyrov et al. have found that the isomerization kinetics of MC-540 in lipid vesicles is a much more sensitive technique in the presence of different additives compared to the spectral changes.34 The cis−trans isomerization of MC-540 can be monitored through FCS in a relatively straightforward manner. FCS can be used as a sensitive technique to probe the local viscosity and temperature effect on biomembrane. Using FCS, transition to and from long-lived species (cis form of MC-540), which has a weak fluorescent state, can be detected by analyzing the fluctuations originating from the fluorescence intensity.36 First, FLIM images of 0.1 M OEA vesicles in the presence of different concentrations of Bmim-BF4 are taken, and the images are assembled in Figure 3a. We have observed that with increasing concentration of Bmim-BF4, the sizes of the vesicles are gradually increased, and the lifetime distribution is also plotted in Figure 3b. In the absence of Bmim-BF4, the lifetime distribution of MC-540 is deconvoluted into two spectra. The short distribution contributes to the MC-540 molecules located at the water pool of the vesicle, and the higher lifetime distribution corresponds to the probe molecules at the bilayer of the vesicle. However, in the presence of Bmim-BF4, the lifetime of MC-540 associated with the vesicle sharply decreased (Figure 3b). The higher lifetime distribution (∼2400 ps) arises from the background, and the shorter distribution (∼1000 ps) is assigned to the lifetime of MC-540 located in the vesicle. The decrease in the lifetime of MC-540 is consistent with the disassembly of water pool of the vesicle in the presence of Bmim-BF4. Due to the fusion between vesicles, the fluidity of the water molecules is increased. On the basis of the structure of MC-540, it is presumed that MC-540 is anchored to the hydrocarbon region of the vesicle through the two tertamethylenic tails and benzoxazole end, which is linked to the anionic sulfonate group interacting with the positively charged head group region of the vesicle and points toward the outer surface. Thus, the decrease in the fluorescence lifetime signifies the increase in the water fluidity in the presence of vesicle. To further confirm this, we have performed FCS measurements in OEA vesicles because the photoisomerization of MC-540 is highly sensitive to the microviscosity of the system.

Figure 4. FCS traces of MC-540 in probe sonicated 0.05 M OEA vesicles in the presence of two different concentrations of Bmim-BF4.

are shown in Figure 4, and the traces are fitted with the following equation30 ⎛ ⎞⎜ 1⎛ 1 1 G (τ ) = ⎜ i ⎟⎜ N ⎝ 1 + (τ /τD) ⎠⎜ 1 + 1 ω2 ⎝

( ) τ τDi

⎛ ⎞ Peq ⎜⎜1 + exp( −τ /τiso)⎟⎟ 1 − Peq ⎝ ⎠

⎞1/2 ⎟ ⎟ ⎟ ⎠

(1)

A detailed description of the equation is described in Supporting Information. Considering the isomerization process, the fluorescence fluctuation arises from the translational diffusion and the fluorescence blinking originating from the transition between the trans and cis forms. In pure ethanol, the 8166


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Figure 5. (a) FLIM images of K562 cells stained with MC-540 in the presence of two different concentrations (0.2 and 0.8 M) of Bmim-BF4. (b) Lifetime distribution obtained from the FLIM images.

diffusion time of MC-540 is reported to be 79 μs. In OEA vesicle, the diffusion time is obtained as 180 μs. However, in the presence of 0.1 M Bmim-BF4, the diffusion is further slowed to 5 ms (Table S1, Supporting Information). The slower diffusion in vesicular system contributes to the diffusion of the vesicle as a whole. However, the effect of large viscous drag experienced by MC-540 in the vesicle compared to the bulk solvent may also play a significant role in this case.34 Vesicles have significant polydispersity with respect to the size and dynamics. Due to the inhomogeneous dynamics, the FCS measurements were performed multiple times and the average value is taken. The error associated with each measurement with respect to the average is calculated and the values are tabulated in Table S1, Supporting Information. The isomerization kinetics of MC540 is very sensitive to the laser excitation power. For this reason, the laser intensity is kept same for every measurement. After fitting the FCS traces with eq 1, Peq and τiso values are obtained. By this procedure, the kinetic rate constant, k/iso, can also be obtained and they are tabulated in Table S1, Supporting Information. τiso is related to isomerization of MC-540 and it is obtained as 0.95 μs for OEA vesicles. However, in the presence of Bmim-BF4, no such isomerization kinetics is observed for MC-540 (Figure S7, Supporting Information), and the FCS traces are simply fitted with the equation considering only the translational diffusion of the probe molecule. Earlier, FLIM study suggests that in the presence of Bmim-BF4, microviscosity around the probe surroundings is decreased. For this reason, the rate of isomerization should also be increased and the isomerization kinetics cannot be detected within this time scale. Moreover, it is reported that fluorescence brightness of MC540 depends on the liposome size.34 As the size of the vesicles is increased, the brightness of MC-540 is decreased. Thus, the isomerization process may also be the reason behind the changes in the fluorescence brightness. Overall, from the FCS and FLIM measurements, we can conclude that in the presence of Bmim-BF4, the fluidity in the inner pool of the vesicles is

increased and, due to the swelling, the size of the vesicles is also increased. Ionic liquid shows distinct mechanisms of membrane binding and insertion. Therefore, it can be correlated with their biological activities. We have analyzed the impact of this imidazolium-based hydrophilic IL on K562 cell, a human erythroleukemic cell line. K562 cells are the first-established human immortalized myelogenous leukemia line obtained in blast crisis. Recent studies demonstrate that imidazolium-based IL with long alkyl chain causes plasma-membrane permeabilization.11 However, detailed mechanism of action of these ILs on cell membrane is still unveiled. To demonstrate the interaction between Bmim-BF4 IL and K562 cell membrane, we have used FLIM and FCS techniques using MC-540 as a probe molecule. FLIM study suggests that MC-540 binds to the outer surface of the cell membrane (Figure 5a). The binding affinity of MC-540 to the leukemic cell is found to be much higher compared to that of other cells.37 Thus, leukemic cells and electrically excitable cells can be killed by simultaneous exposure of MC540 and suitable-wavelength light. However, nonexcitable cells and normal hematopoetic stem cells remain unaffected. Therefore, several attempts have been made to exploit this differential photosensitivity in clinical applications.37 In Figure 5a, FLIM images of K562 cells (stained with MC-540) in the absence and presence of Bmim-BF4 are shown, and the lifetime distributions obtained from Figure 5a are shown in Figure 5b. In the presence of 0.8 M Bmim-BF4, some of the cell membranes are disrupted (Figure S8, Supporting Information). Further, lifetime of MC-540 is shifted toward lower value with increasing concentration of Bmim-BF4, and it indicates that the membrane fluidity of K562 cells is increased in the presence of IL and it causes membrane permeabilization. As we have mentioned earlier that the change in the fluidity of cell membrane also affects the photoisomerization of MC-540. Therefore, isomerization kinetics can be determined using FCS measurements. The FCS traces of MC-540 in K562 cells are shown in Figure 6a; they are fitted with eq 1, and the fitted 8167


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Figure 6. (a) Fitted FCS traces of MC-540 in K562 cells treated with different concentrations of Bmim-BF4. (b) K562 cell viability study (MTT assay) after 2 and 6 h exposure to Bmim-BF4 of 0.2, 0.5, and 0.8 M concentrations.

measurements, the alkyl chain of Bmim-BF4 can insert into the bilayer of cell membrane, and similar to OEA vesicles, we have also determined the fluidity of the cell membrane using MC540. The rate of isomerization of MC-540 is gradually decreased with increasing concentration of IL and it suggests that in the presence of IL, the fluidity of the cell membrane is increased similar to that of OEA vesicle and it increases the membrane permeability. Liang et al. have also demonstrated dose-dependent decrease in the viability of different cells, and it is associated with the composition of phosphatidylcholine vesicles, which are incorporated with long-chain imidazoliumbased ILs.38 Thus, investigating the earlier OEA model membrane system and other simulation studies,16,17 the increase in the permeabilization of K562 cells by ILs can be considered as a two-step process. Due to the electrostatic interaction, cationic IL initially covers the membrane surface, and in the second step, the hydrophobic chain of the IL interacts with the hydrophobic inner region of bilayer, which facilitates the membrane permeabilization. Therefore, it can be correlated to the cytotoxicity caused by Bmim-BF4 in K562 cells. In conclusion, the impact of popular hydrophilic imidazolium IL on the morphology of model OEA membrane with the implication of IL cytotoxicity is examined experimentally. Due to the strong electrostatic as well as hydrophobic interaction, the IL cation inserts into the bilayer of vesicle and causes swelling of the vesicle. The fusion kinetics of the vesicles in the presence of ILs is analyzed using time scan FLIM measurements, and the membrane fluidity is also determined by demonstrating the cis−trans isomerization of MC-540. Therefore, this study suggests the underlying mechanism of IL cytotoxicity governed by the IL-induced perturbation on cell membrane of K562 cell. Increase in the membrane fluidity in the presence of IL is mainly responsible for the membrane disruption of K562 cells, and MTT assay further shows the serious cytotoxicity of IL to K562 cells. Thus, this work motivates us to understand IL toxicity, which provides us proper guidance for designing novel biocompatible ILs. We are hopeful that the finding presented here may alter the paradigm of IL research, which has favored the development of cationic IL carrying long alkyl chain.

parameters are enlisted in Table 1. With increasing concentration of IL, rate of isomerization and the diffusion Table 1. Isomerization Parameters and Diffusion Coefficienta of MC-540 in K562 Cells Obtained from the Analysis of FCS Traces in Figure 6a system

τiso (μs)

K/iso (μs−1)

Dt (μm2 s−1)

K562 cell K562 cell + 0.2 M IL K562 cell + 0.8 M IL

4.6 ± 0.5 5.9 ± 0.2 1.83 ± 0.1

0.07 0.09 0.31

4.9 ± 0.1 20.1 ± 0.8 26.9 ± 2.1

a

Measurements are performed multiple times and the mean value is taken. The relative error is calculated with respect to the average.

coefficient of MC-540 are gradually increased. In the presence of low concentration of IL (0.2 M), the isomerization rate does not change appreciably and it is also consistent with the FLIM images. However, when we further increase the concentration of IL, up to 0.8 M, the isomerization rate is sharply increased to 0.31 from 0.07 μs−1 in the absence of IL. The increase in the diffusion coefficient of MC-540 in the presence of IL can also be correlated to the decrease in the local viscosity in cell membrane, and it also increases the isomerization rate of MC-540. Thus, the FCS trace in the presence of 0.8 M IL can also be correlated with the FLIM images. The disruption of K562 cell membranes at this concentration of IL is observed in the FLIM image. To further quantify the toxicity generated by IL, the viability of K562 cells following treatment with different concentrations of IL is evaluated by MTT assay. After 6 h of incubation with increasing concentration of IL up to 0.8 M, less than 20% viability of K562 cells is observed (Figure 6b). Thus, the results demonstrate that Bmim-BF4 IL has serious cytotoxic effect. Cell membranes are mostly composed of different neutral and negatively charged phospholipids. Therefore, negatively charged lipids facilitate the interaction between cationic imidazolium-based IL and cell membrane. Maginn et al. show that one mode of IL toxicity on unicellular organisms is driven by swelling of the cell membrane.18 MD simulation study shows that IL can cause morphological rearrangements of the cell membrane and this may alter the cell functionality (such as small-molecule membrane permeability, lipid lateral mobility, etc.), leading to cell death.18 As evidenced from 1H NMR 8168


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b06231. Instrumentation, FLIM images of vesicle, ζ potential, TEM and FESEM images, NMR measurements, FCS traces in vesicles, and FLIM images of cell (PDF) W Web-Enhanced Features *

Assembly of images collected (Movie 1)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 91-3222-255303. ORCID

Nilmoni Sarkar: 0000-0002-8714-0000 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.S. gratefully acknowledges SERB (Grant No: IR/S1/LU001/2013 dated 24/03/2015), Department of Science and Technology (DST) and Council of Scientific Industrial Research (CSIR), Government of India, for providing generous research grants. N.K. and D.M. acknowledge IIT Kharagpur and CSIR for their research fellowships.

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DEDICATION Dedicated to the memory of late Professor Mihir Chowdhury. REFERENCES

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