Sensitivity and Resilience of Phosphatidylcholine and

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B: Biomaterials and Membranes

Sensitivity and Resilience of Phosphatidylcholine and Phosphatidylethanolamine Lipid Membranes Against Cholinium Glycinate Bio-Compatible Ionic Liquid Pratibha Kumari, and Hemant K. Kashyap J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b02800 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 13, 2019

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The Journal of Physical Chemistry

Sensitivity and Resilience of Phosphatidylcholine and Phosphatidylethanolamine Lipid Membranes Against Cholinium Glycinate Bio-compatible Ionic Liquid Pratibha Kumari and Hemant K. Kashyap∗ Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India E-mail: [email protected] Phone: +91-(0)11-26591518. Fax: +91-(0)11-26581102

Abstract Choline amino acid ([Ch][AA]) based ionic liquids (ILs) are considered to be highly biodegradable and bio-compatible solvents. The toxicological scrutiny and environmental fate analysis of these ILs are fundamental requisites to employ these ILs on a large scale applications. In the present work, we investigate how the presence of the simplest form of [Ch][AA] ILs, cholinium glycinate ([Ch][Gly]), affects the structure and stability of homogeneous 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (POPE) lipid bilayers by using atomistic molecular dynamics simulation. The study reveals a considerable compression of POPC bilayer along with an enhanced ordering of hydrocarbon lipid tails on increasing the concentration of [Ch][Gly] IL. On the other hand, the stability and structure of the POPE bilayer is hardly affected at lower concentration of [Ch][Gly], however at higher concentration (20 mol%) the structure of the bilayer is slightly changed. The H-bond analysis reveals that [Ch]+ cations have greater propensity to H-bond with phosphate and ester group oxygens of POPC than POPE lipid molecules. The structural properties of POPE bilayer are influenced by [Ch][Gly] IL to lesser extent compared to POPC bilayer, which appears to be a direct consequence of the ability of POPE lipids to form strong inter- as well as intra-molecular H-bonding network among themselves and adapting a more compact packed bilayer structure. Enhanced accumulation of [Gly]− anions at water-membrane interfacial regions is unambiguously observed for both POPC and POPE bilayers.

1

Introduction

treme low vapor pressure and non-flammability, these liquids were emerged as a greener substitute for conventional organic solvents. 8 However, toxicity and non-biocompatibility have been one of the most undesirable properties of conventional room-temperature ILs. Various experimental investigations and toxicity assessments performed on different biological test systems, such as enzymes acetyl-

Ionic liquids (ILs), broadly defined as liquid salts with melting point below 100 ◦ C, are versatile solvents due to their unique physicochemical characteristics, such as low boiling point, low flammability, thermal and chemical stability, negligible vapor pressure, and a wide electrochemical window. 1–9 Initially, owing to ex-

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cholinestrase, 10 antioxidant enzyme system, 11 human cell line HeLa, 12 algae, 13 aquatic eukaryotes Daphnia magna, 14 Zebrafish (Danio Rerio), 15 model lipid membranes, 16 and many others 17–19 indicate that some of the ILs show substantial toxicity or antimicrobial activity which could have extremely negative impact on the environment. It has been found that ILs comprising of aromatic ring in the cation part, such as imidazolium and pyridinium have significant toxic propensity which increases with increasing alkyl chain length of cation. However, incorporation of polar functional groups like amide and hydroxide seems to enhance the biodegradability of these ILs. Though the understanding of toxicity mechanism of ILs on biological system is still scarce, it has been proposed that cations of IL penetrate the cell line which subsequently disrupt the biological membranes. 16,17 Such major concerns of toxicity and bio-compatibility of ILs have instigated the scientific community to design neoteric solvents on the basis of green chemistry principles. However, the opportunity of fine-tuning the physicochemical properties as well as biocompatibility of ILs by the appropriate choice of cations and anions has motivated much of the excitement regarding the applicability of these molten salts. 20 Recently, cholinium cation-based ILs have been recognized as a promising candidate due to their low cost, high biodegradability, and environmentally benign nature. Also, choline is an essential micronutrient as it belongs to a vital class of vitamin B and is considered to play an essential role in our daily life. 21,22 In fact, a number of ILs and deep eutectic solvents (DESs) based on cholinium cation have been modeled and used in various applications including catalysis, organic synthesis, preparation of novel polymorphous material, carbon dioxide capture, and separation of bio-molecules. 23–32 Recently, Moriel and coworkers 33 have introduced a new class of biocompatible ILs based on cholinium cation and amino acids as anions, collectively known as cholinium amino acid ILs ([Ch][AA] ILs). These are obtained from natural and renewable feedstocks, choline and amino acids, and hence are

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highly biodegradable and biocompatible in nature. Amino acids are one of the most abundant organic compounds and can be obtained in large quantity at economical cost with high purity. Liu et al. 34 have prepared a series of [Ch][AA] ILs using simple and green chemical route and performed their physical and chemical characterizations. [Ch][AA] ILs are found to be excellent catalyst for organic synthesis and possess highly suitable solvent properties for biomass pretreatment. 35–37 In addition to this, these liquids are also used as lubricants, 38–41 CO2 capturing agent, 42–44 in lignite and thermal coal pre-treatment. 45 Due to their solubility in both aqueous and organic media coupled with their non-toxic nature, they have been considered as potential candidate for various biotechnological applications including the formulation of bio-pharmaceuticals and media for bio-catalysis. 46–48 This widespread applicability of [Ch][AA] ILs in industry and technology have raised many concerns related to the behavior of these synthetic materials in human body as well as in the living eco-system. Recently, Hou and coworkers 49 have investigated the toxicity and biodegradability of a range of [Ch][AA] ILs towards various bacteria (Escherichia coli, Staphylococcus aureus, Salmonella enteritidis, and Listeria monocytogenes) and enzyme (Acetylcholinesterase) and confirmed their lower toxicity and higher biodegradability in comparison to conventional ILs. Yazdani et al. 50 assessed microbial incompatibility and biodegradability of these ILs in the industrial sewage water and observed the EC50 values in the range of 160-1120 mg/ml, which is considered as practically harmless amount according to hazardous rankings. Other experimental toxicological investigations performed on various aquatic organisms reveal that these ILs are not harmful for marine organisms and are also highly bio-degradable and nontoxic. 41,51–53 Bisht and co-workers 54 have studied the effect of cholinium glycinate and cholinium bromide on the stability and activity of stem bromelain and observed that cholinium glycinate is weaker stabilizer of stem bromelain as compared to cholinium bromide. Tarannum et al. 55 found that in the presence


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The Journal of Physical Chemistry Oleoyl tail

Oleoyl tail

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Palmitoyl tail

Palmitoyl tail

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(b) O H2N

N

O

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Figure 1: Chemical structures of (a) POPC, (b) POPE, (c) cholinium ([Ch]+ ) cation, and (d) glycinate ([Gly]− ) anion.

of [Ch][AA] ILs the rheological properties, dielectric behavior and conformation of collagen protein are altered. These experimental studies of relative toxicity of [Ch][AA] ILs towards specific biological systems and cell types provide useful information on how these ILs affect toxicity patterns, but can not provide a molecular description of the interaction between ILs and the cell membrane. Over the past few decades, molecular simulations have become an important tools for finding molecular level information of complex multi-scale biological phenomena. 56–62 As expected, atomistic molecular dynamics (MD) simulations can provide a great deal of insight in understanding of impact of ILs on various biological systems. 16,63 In one of the only few studies, Yoo et al. 16 performed MD simulation to investigate the molecular mechanism of interaction of a range of different imidazolium-based ILs with a POPC lipid bilayer. The study revealed that the IL cations could spontaneously enter into the hydrophobic part of lipid bilayer regardless of the length of alkyl chain of cation. Further, Lim and co-workers 63,64 have explored the mechanism of translocation of conventional IL cations in the hydrophobic regions of bacterial lipid membranes and thereby changes in their structural properties. In the present MD simulation study, we have indirectly tested the cytotoxicity of cholinium glycinate ([Ch][Gly]) IL and explored its im-

pact on the structure and stability of homogeneous POPC (1-Palmitoyl-2-oleoyl-sn-glycero3-phosphocholine) and POPE (1-Palmitoyl-2oleoyl-sn-glycero-3-phosphoethanolamine) lipid bilayers. The two lipids are chosen here because of their good proportion present in several biological membranes. 65 Our results on the microscopic properties studied here, such as lipid tail order parameters and H-bonding, reveal that while POPE shows significant resilience, the structure of POPC bilayer is considerably affected by increase in [Ch][Gly] concentration. These results were also found to be consistent with the other microscopic properties of the bilayers investigated herein.

2

Simulation Details

Bilayers of POPC and POPE lipids comprising 256 lipid molecules, 128 in each leaflet, and 40 water molecules per lipid were constructed using CHARMM GUI. 66–68 All-atom CHARMM36 lipid force-field parameters for the lipids and CHARMM general force field (CGenFF) 69,70 parameters for cholinium glycinate bio-IL were used, along with TIP3P 68 model for water molecules. In order to circumvent charge transfer and polarization effects we have used scaled down charges for cholinium cation (+0.85e) and glycinate anion (-0.85e) as suggested and utilized in the past studies. 71,72 The chemical structures of POPC


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and POPE lipid molecules along with cholinium ([Ch]+ ) and glycinate ([Gly]− ) ions are shown in Fig. 1 and a summary of the systems investigated herein is provided in Table 1. All the MD simulations were performed using NAMD program. 73 For equilibration of pure fully hydrated homogeneous POPC and POPE bilayers, standard equilibrium procedure, provided in CHARMM-GUI, that slowly removes restraints on the lipid molecules was used. Pure hydrated bilayers equilibrated for 50 ns were subsequently used to prepare systems with cholinium glycinate and water at desired concentrations as mentioned in Table 1. PACKMOL 74 package was used to incorporate a layer of desired numbers of ion couples of IL on the top of water layer of equilibrated bilayer. For mixing of the top and bottom layers of cholinium glycinate IL with water, first only water and cholinium glycinate were equilibrated for 1 ns by restraining lipid molecules. After mixing of cholinium glycinate IL with water, the restraining forces on the lipid molecules were released for full-fledged further equilibration. A cutoff of 12 ˚ A was used to calculate non-bonded and electrostatic interactions at every time step. Particle mesh Ewald (PME) 75 summation method with an interpolation order of 6 was used to compute long-range Coulomb interactions. The hydrogen involving bonds were constrained to their equilibrium bond length using SHAKE algorithm. All simulations were performed in isothermal-isobaric condition. The temperature and pressure of each system were kept at 310 K and 1 bar, respectively, by using Langevin dynamics and Nos´e-Hoover Langevin piston. 76,77 Integration time-step of 2 fs was used. The semi-isotropic cell was used with x-direction constrained to be equal to y-direction, but allowed to vary independently with respect to z-direction, which is along the bilayer normal, for all the systems. All bilayer-[Ch][Gly] systems were run for 300 ns and the last 100 ns part of each trajectory was used for analyzing various properties of the systems. These properties include area per lipid, bilayer thickness, deuterium order parameter of lipids palmitoyl tail, P-N vector angle and hydrogen-bonding interactions.

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The partial number density profiles for the Table 1: Summary of the simulated bilayer-[Ch][Gly] systems. [Ch][Gly] Lipid Water (mol%) 2.5 256 15360 5.0 256 15360 10.0 256 15360 20.0 256 15360

[Ch][Gly] ionic couples 394 810 1708 3840

*Here [Ch][Gly] mol% is defined by considering the number of water molecules and [Ch][Gly] ionic couples present in the system.

species of interest were obtained by dividing the equilibrated simulation cell into 103 bins of equal volume along z-direction. The number of species of interest present in each bin was added and averaged over number of frames to obtain the corresponding number density profile. The area per lipid (APL) was calculated from the product of the x- and y-dimensions of the simulation cell divided by the number of lipids per leaflet. Lipid order parameter (SCD) is a measure of orientational preference of the C-D bond in the lipid tails and equivalently in MD simulations SCD is defined by the following equation 78–80

SCD = 1.5 cos2 θ − 0.5 , (1) where θ represents the angle between the bilayer normal and C-D (in fact C-H in the present simulation) vector. Here, < · · · > indicates ensemble average. The magnitude of SCD quantifies the degree of average orientation of lipid molecules with respect to the bilayer normal. The geometrical criteria to define a H-bond, a cutoff of 30◦ for hydrogen-donor-acceptor angle and 2.5 ˚ A for hydrogen-acceptor distance was kept. Thorough equilibration of each system was tested by monitoring the time dependence of area per lipid (APL) of POPC and POPE bilayers at different concentrations of [Ch][Gly] IL (see Figs. S1(a) and S1(b)). We observe that the APLs are converged beyond 150 ns for all the systems. The ensemble averages of APL and thickness (DPP ) of POPC and POPE bilayers as a function of [Ch][Gly] IL concentra-


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Table 2: [Ch][Gly] mol% dependence of average APL and bilayer thickness, DPP , in POPC and POPE bilayer systems. DPP is defined as the distance between the peaks in the phosphate electron density profile along bilayer normal. Bilayer [Ch][Gly] APL DPP (mol%) (˚ A2 ) (˚ A) POPC 2.5 64.3 38.6 5.0 62.1 39.3 10.0 60.4 40.5 20.0 57.2 43.3 POPE 2.5 57.1 42.7 5.0 56.5 42.5 10.0 56.7 43.2 20.0 55.1 42.1

also be seen. These observations indicate that the distance between the opposing leaflets of POPC bilayer increases with increasing concentration of [Ch][Gly] IL, which is also evident from the variation of the bilayer thickness as exhibited in Fig. S2(b) of the Supporting Information. In contrast, for POPE-[Ch][Gly] systems, partial NDPs of POPE lipid molecules do not deviate much from that of pure POPE bilayer, suggesting that the overall structure of POPE bilayer remains intact upon addition of [Ch][Gly] IL. Though, at higher concentrations of [Ch][Gly] IL, a slight increase in the density in the middle of the bilayer can be seen (Fig. 2(b)). Number distribution of water, [Ch]+ and [Gly]− species along bilayer normal for POPC[Ch][Gly] and POPE-[Ch][Gly] systems are shown in Figs. 3(a) and 3(b), respectively. It can be gleaned through Fig. 3(a) (upper panel) that the interfacial region formed between lipid-water contracts with increasing amount of [Ch][Gly] in POPC-[Ch][Gly] systems. Viewing this in conjunction with NDPs of [Ch]+ cations (middle panel) and [Gly]− anions (bottom panel) reveal that the water molecules present at the interface are replaced by [Ch]+ and [Gly]− ions to some extent. Moreover, the NDPs of [Gly]− anions shown in Fig. 3(a) (bottom panel) demonstrate an exceptional tendency of these negative ions to accumulate near the lipid-water interface. The intensity of corresponding peak for [Gly]− anions increases with increasing concentration of [Ch][Gly] IL. The examination NDPs of water molecules, [Ch]+ cations and [Gly]− anions for POPE-[Ch][Gly] systems shows that though the width of the lipid-water interface do not change, there is enhanced accumulation of [Gly]− anions near this interface at higher concentration of [Ch][Gly] IL (Fig. 3(b)). However, as shown in Figs. 4-5, a visual inspection of the equilibrium snapshots of both POPC-[Ch][Gly] and POPE-[Ch][Gly] systems indicates that in membrane lateral direction [Ch]+ and [Gly]− ions are evenly distributed within the membrane-water interface at all [Ch][Gly] IL concentrations studied.

P-N angle (◦ ) 73.2 73.8 73.9 75.0 81.3 81.0 79.4 76.2

tion are shown in Figs. S2(a) and S2(b) and the corresponding data are provided in Table 2. It is clear that the presence of [Ch][Gly] significantly decreases the APL of POPC bilayer with increasing concentration of [Ch][Gly] IL. Apart from this, a concurrent increase in the POPC bilayer thickness can be observed from Fig. S2(b). On the other hand, the presence of [Ch][Gly] IL causes minute changes in the APL and thickness of POPE bilayer.

3 3.1

Results and Discussion Bilayer Structure and Partitioning of water, [Ch]+ and [Gly]−

In order to appreciate how the distribution of lipid molecules varies by addition of [Ch][Gly] IL, the simulated partial number density profiles (NDPs) of POPC and POPE lipids as a function of position along the bilayer normal (z-axis) are depicted in Figs. 2(a) and 2(b), respectively. From Fig. 2(a), we can observe that with increasing concentration of [Ch][Gly] IL, the NDPs of POPC lipid molecules shift away from the center of bilayer. In addition, a decrease in the intensity at the center (at z=0) of the bilayer can


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The Journal of Physical Chemistry 0.12

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0.02 0

-20

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Figure 2: Number density profiles of lipid molecules along bilayer normal (z-axis) for (a) POPC[Ch][Gly]and (d) POPE-[Ch][Gly] systems containing 0, 2.5, 5, 10, and 20 mol% of cholinium glycinate biocompatible liquid.

3.2

Distribution of [Ch]+ and [Gly]− Atoms Along Bilayer Normal

density peaks for phosphorous, nitrogen and ester groups corresponding to two leaflets become distant along with the change in their intensities, which is more prominent for the system containing 20 mol% of [Ch][Gly]. In addition to this, these peaks becomes sharper indicating that POPC lipid head-groups becomes more ordered than that of neat bilayer. However, despite the change in the positions and densities of peaks of phosphorus, nitrogen and ester groups of lipid tails, the order of the peaks are maintained such that peak of nitrogen is located slightly outside the peaks of the phosphorous and ester-groups densities. For POPE-[Ch][Gly] systems, Figs. 6(c) and 6(d) clearly show that [Ch]+ atoms have slightly more probability of finding inside the bilayer than [Gly]− atoms which tend to accumulate near water-bilayer interface, especially close to locations of phosphorus and ester groups. The oxygen atoms of [Ch]+ are found to be slightly more closer to the peaks of phosphorus atom and ester groups densities than that of other atomic species, suggesting that hydroxyl group of [Ch]+ ions prefers to interact with Hbond acceptor groups of POPE lipids. Furthermore, the peaks corresponding to POPE headgroups become slight wider and show reduction

In order to get an in-depth understanding of structural changes in the bilayer due to of [Ch][Gly] IL, we have separately monitored the atomic number density profiles for the polar lipid head-groups, specifically phosphorus, nitrogen and ester groups of lipid as a function of position along the bilayer normal (z-axis) for the pure bilayers and bilayer-[Ch][Gly] systems consisting lowest (2.5%) and highest (20%) concentrations of [Ch][Gly] IL. Fig. 6 shows number density distribution of the polar groups together with that of N and O atoms of [Ch]+ cations and [Gly]− anions in the respective systems. Owing to lower APL of POPE bilayer, the densities of the peaks of phosphorus, nitrogen and ester groups of pure POPE lipid molecules is higher than that of pure POPC. From Figs. 6(a) and 6(b) one can observe that N and O atoms of [Ch]+ cations can penetrate POPC bilayer deeper than [Gly]− ions. As shall also be shown in section 3.5 the [Ch]+ ions prefer to associate more with H-bonding accepting groups of POPC lipid molecules. It can be seen that in the presence of [Ch][Gly] IL, the number


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Water 0.08

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Figure 3: Number density profiles of water, [Ch]+ cations, and [Gly]− anions along z-direction for (a) POPC and (b) POPE bilayer consisting 2.5, 5, 10, and 20 mol% of [Ch][Gly] bio-ionic liquid. Number density profiles of water for respective pure bilayer is also shown for comparison.

P-N angle are provided in Table 2. In case of POPC-[Ch][Gly] systems, P-N vector angle distribution does not depend much on the presence of [Ch][Gly] IL as demonstrated by only a meager increase in the average P-N angle. Along with this, inspection of corresponding distributions of the angle between the PN vector with respect to the bilayer normal shows a slight expansion towards the region of higher angle values, suggesting an increase in the fraction of lipid molecules whose head groups point inside the bilayer interior indicating the expansion of POPC lipid profile along the bilayer normal. These results are consistent with observed increase in the bilayer thickness upon addition of [Ch][Gly] IL. Figure 8(b) depicts that for POPE[Ch][Gly] systems consisting upto 10 mol% of [Ch][Gly] IL, P-N vector angle distribution are similar to neat POPE bilayer. However, at 20 mol% an overall shift of the distribution of PN angle towards lower angles is quite evident. This shift of P-N vector angle in the vicinity of lower angle values stipulate that the P-N vector protrude towards the aqueous region which could cause the decrease of lipid bilayer thickness. This found to be in agreement with slight decrease in the bilayer thickness and slight in-

in the densities in the presence of [Ch][Gly]IL.

3.3

Orientational Ordering of Lipid Tails and P-N Vector in Lipid Head Group

The calculated orientational order parameters for carbon atoms of palmitoyl tails of POPC and POPE lipids for different [Ch][Gly] containing systems are shown in Figs. 7(a)-7(b). For POPC-[Ch][Gly] systems, from Fig. 7(a) it is clear that hydrocarbon lipid tails become more ordered with the addition of [Ch][Gly] IL. In contrast, Fig. 7(b) depicts that for POPE[Ch][Gly] system containing up to 10 mol% of [Ch][Gly], SCDs for C3-C15 show minute changes. However, at 20 mol% of [Ch][Gly] IL, a notable increase in the ordering of POPE lipid tail occurs. In addition to the deuterium order parameter of the lipid tails, it is possible to investigate ordering of lipid head-groups in the similar manner. To do that, we have computed the probability distributions of the angle of phosphorus-nitrogen (P-N) vector of lipid headgroup with respect to the bilayer normal at different amount of [Ch][Gly] IL and are presented in Figs. 8(a)-8(b). The average values of the


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(a)

(b)

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(d)

Figure 4: Equilibrium shapshots showing the distribution (seen from top) of [Ch]+ and [Gly]− within lipid-water interfacial region in POPC-[Ch][Gly] system containing (a) 2.5 (b) 5 (c) 10 and (d) 20 mol% of [Ch][Gly] IL. In the snapshots, the [Ch]+ cations and [Gly]− anions are shown in red and green colors, respectively. The lipid molecules are shaded in white along with molecular representation of phosphate groups. Water molecules are not shown for clarity.

(a)

(b)

(c)

(d)

Figure 5: Equilibrium shapshots showing the distribution (seen from top) of [Ch]+ and − [Gly] within lipid-water interfacial region in POPE-[Ch][Gly] system consisting (a) 2.5 (b) 5 (c) 10 and (d) 20 mol% of [Ch][Gly] IL. In the snapshots, the [Ch]+ cations and [Gly]− anions are shown in red and green colors, respectively. The lipid molecules are shaded in white along with molecular representation of phosphate groups. Water molecules are not shown for clarity. ˚) emerges and lower length scale (around 6 A becomes a major peak for bilayer containing 20 mol% of [Ch][Gly], suggesting that the addition of [Ch][Gly] decreases the spacing between nitrogen atoms of POPC head groups. Further, Fig. 9(b) also exhibits that the correlation between P-P pairs increases in the presence of [Ch][Gly] IL as evident from the increased height of the first solvation peak of P-P RDF. This implies that at shorter distance the lateral correlations between the lipid head group atoms significantly increase at higher concentration of [Ch][Gly]. Consequently, the average spacing between the lipid molecules decreases and hence APL also decreases at higher IL concentrations. The presence of pronounced peaks beyond 10 ˚ A for N-N and P-P correlations indicate an in-

crease in the density in the middle of bilayer at this concentration.

3.4

Lateral Membrane Structure

In order to get further insight, how [Ch][Gly] IL affects the lateral structure of lipid bilayer twodimensional radial distribution functions (2D RDFs) for self-correlations between N-N and PP atoms of POPC and POPE lipids are shown in Figs. 9(a)-9(d). From Fig. 9(a) one can observe that N-N RDF for the neat POPC bilayer consists of a well defined peak around 8 ˚ A. In the presence of [Ch][Gly] IL, this peak shift towards lower length scale along with gradual decrease in its intensity. Moreover, it can be seen through Fig. 9(a) that a new peak at


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N (pure POPC) P (pure POPC) COO-oleoyl tail (pure POPC) COO-palmitoyl tail (pure POPC) N (POPC+[Ch][Gly]) P (POPC+[Ch][Gly]) COO-oleoyl tail (POPC+[Ch][Gly]) COO-palmitoyl tail (POPC+[Ch][Gly]) N[Ch]+

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Figure 6: Atomic number density profiles of nitrogen (N), phosphorous (P), ester groups of lipid tails along with oxygen and nitrogen atoms of [Ch]+ cations and [Gly]− anions for POPC bilayer (a,b) and POPE bilayer (c,d) containing lowest (2.5%) and highest (20%) concentration of [Ch][Gly] IL. Atomic number density profiles of respective neat lipid bilayer are also plotted for comparison.


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Figure 7: Orientational order parameter (SCD) of palmitoyl tail of lipid molecules for POPC[Ch][Gly] and POPE-[Ch][Gly] systems. The order parameter profiles of hydrocarbon lipid tail of respective neat bilayer are also shown for comparison.

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0.015 P(θ)

0.015 P(θ)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 21

0.01

0.005

0 0

0.01

0.005

30

60

90 120 θ [degrees]

150

180

0 0

30

(a)

60

90 120 θ [degrees]

150

180

(b)

Figure 8: The variation of probability distribution function P(θ)(in arbitrary units) of the angle θ between the lipid headgroup P-N vector and the bilayer normal in (a) POPC and (b) POPE lipid bilayer for different concentration of [Ch][Gly] IL.


10

Page 11 of 21

2

POPC

N- N

2

g(r)

g(r)

POPC

P-P


1.5

1.5 1

1

0.5

0.5 0 0

10

5

0 0

15

5

(a)

2

2

1.5

10

15

POPE

P-P

g(r)

1.5

1 0.5 0 0

15

(b)

POPE

N-N

10

r (Å)

r (Å)

g(r)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

0.5

10

5

15

0 0

r (Å)

5

r (Å) (d)

(c)

Figure 9: Two-dimensional RDFs for (a) N-N and (b) P-P atoms of lipids in POPC-[Ch][Gly] systems. RDFs for (c) N-N and (d) P-P atoms of lipids in POPE-[Ch][Gly] systems.


11


Table 3: Ensemble average of number of H-bond per lipid molecule computed for POPC-[Ch]+ , POPC-[Gly]− , POPC-water pairs and per solvent molecules for water[Ch]+ , and water-[Gly]− pairs in POPC-[Ch][Gly] systems.

0.5

+

POPC-[Ch]


0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1 0

20

40 60 80 Time (ns)

0

0.05 0.1 0.15 Occurance

0

0.15

POPC-[Gly]

6.593 6.101 5.184 3.671 0.15

0.1

0.1

0.05

0

0.05

0

20

40

60

80

0.1

0.2

0

Occurance

(a) No. of H-bonds / solvent molecule

0

Time (ns)

water-[Gly]−

0.031 0.057 0.091 0.119

-

10

0.167 0.314 0.549 0.837 10

POPC-water

8

8

6

6

4

4

2

2

0

0

20

40 60 80 Time (ns)

(b) 0.2

+

water-[Ch]

0.2

POPC

0.15

0.15

0.1

0.1

0.05

0.05

0

0

20

40 60 80 Time (ns)

0

0.01 0.02 Occurance

0

No. of H-bonds / solvent molecule

0

POPC-water water-[Ch]+

0.005 0.011 0.028 0.056 No. of H-Bonds / POPC molecule

0.5

POPC-[Gly]−

No. of H-Bonds / POPC molecule

[Ch][Gly] POPC-[Ch]+ mol% 2.5 0.038 5 0.075 10 0.159 20 0.274 No. of H-bonds / POPC molecule

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 21

0 0.005 0.01 0.015 Occurance

0

(c) 1

water-[Gly]

-

1

POPC

0.75

0.75

0.5

0.5

0.25

0.25

0 0

20

40 60 80 Time (ns)

(d)

0

0.008 0.016 Occurance

0

(e)

Figure 10: [Ch][Gly] concentration dependence of number of (a) POPC-[Ch]+ , (b) POPC-[Gly]− , (c) POPC-water, (d) water-[Ch]+ , and (e) water-[Gly]− H-bonds for POPC-[Ch][Gly] systems.

crease in the long range correlations and local structural ordering of headgroup atoms for [Ch][Gly] IL containing bilayer. These observations are also aligned with the increase order parameters of carbon atoms of lipid tail of POPC lipid molecules with inclusion of [Ch][Gly] IL as exhibited in Figs. 7(a)-7(b). Fig. 9(c) for POPE-[Ch][Gly] systems exhibits that N-N correlation show a large first solvation peak at ∼6.6 ˚ A along with two nonnegligible peaks at larger distances for pure POPE lipid bilayer. The intensity of first solvation peak of N-N correlation increases along with shift in its position towards larger length scales with rise of [Ch][Gly] content. For POPE

systems containing equal to or more than 10 mol% [Ch][Gly] IL, this peak bifurcates into two peaks positioned at ∼4.5 ˚ A and ∼6.0 ˚ A, stipulating a change in the arrangement of lateral structural order and decrease in the the spacing between nitrogen atoms of POPE lipids. Concomitantly, Fig. 9(d) also depicts an increase in the nearest neighbor correlation between PP with increasing [Ch][Gly] IL concentration.


12

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molecules. The probability of H-bonding increases with increasing of [Ch][Gly] IL amount. Moreover, [Ch]+ cations show higher susceptibility towards H-bonding interaction with lipid molecules than [Gly]− anions. Further analysis of H-bonding clearly indicates that hydroxyl hydrogen of [Ch]+ cations have propensity to H-bond with all possible H-bond acceptor groups of POPC lipids (phosphoryl oxygens and C=O group), thereby causing an increase in the number of H-bonds with increasing amount of [Ch][Gly] IL. In contrast, hydrogen atoms of NH2 group of [Gly]− anions have lesser probability to H-bond with any H-bonding acceptor groups of POPC lipid molecules, justifying the fact that these hydrogen are more basic due to the presence of negative charge. These observations corroborate well with the previously mentioned number distribution of [Ch]+ and [Gly]− ions at the lipid-water interface in Section 3.2. Representative configurations showing association of [Ch]+ and [Gly]− ions with POPC lipid molecule via H-bonding and electrostatic interactions are shown in Fig. 13. Furthermore, it can be gleaned through Figs. 10(c), 10(d) and 10(e) that the number of POPCwater H-bonds decreases whereas [Ch]+ -water and [Gly]− -water H-bonds increases substantially for all POPC-[Ch][Gly] systems, suggesting that water molecules at membrane surface are being displaced by [Ch][Gly] IL which results in the receding of partial NDPs of water molecules from the center of the bilayer with increase in the concentration of [Ch][Gly] IL as observed from Fig. 3(a) (upper panel).

3.8

1.9

3.6

1.7

(a)

(b)

Figure 11: Frequently observed configurations ((a) and (b)) showing association of POPC lipid molecule with [Ch]+ and [Gly]− ions for system containing 20 mol% [Ch][Gly] IL. In the figures, nitrogen and oxygen atoms of IL ions are shown in blue and red colors, respectively. Representations shown here clearly indicate that [Gly]− ions stay close to the choline group of POPC lipid due to electrostatic interaction whereas [Ch]+ cations can penetrate in the interfacial region and stay close to the phosphoryl or ester group oxygens of the lipids. The distances shown by dashed lines are in ˚ A unit.

3.5

Hydrogen Bonding

3.5.1

Hydrogen Bonding [Ch][Gly] Systems

in

POPC-

The chemical structure of [Ch][Gly] IL is allowing it to establish H-bonding interactions with water and different polar groups of POPC and POPE lipid membrane. The change in the number of POPC-[Ch]+ , POPC-[Gly]− , POPCwater, water-[Ch]+ , and water-[Gly]− H-bonds with addition of [Ch][Gly] concentration are manifested in Figs. 10(a)- 10(e), respectively. The corresponding average number of H-bonds are also provided in Table 3. For comparison, the number of POPC-water H-bonds for pure bilayer is also shown in Fig. 10(c). From Figs. 10(a) and 10(b), one can appreciate that [Ch]+ cations and [Gly]− anions render H-bonding interaction with POPC lipid

3.5.2

Hydrogen Bonding [Ch][Gly] Systems

in

POPE-

For POPE-[Ch][Gly] systems, the change in the number of POPE-[Ch]+ , POPE-[Gly]− , POPEwater, water-[Ch]+ , and water-[Gly]− H-bonds with increasing concentration of [Ch][Gly] are shown in Figs. 12(a)-12(e), respectively, and their corresponding average number of H-bonds are provided in Table 4. Figs. 12(a) and 12(b) depict that the number of POPE-[Ch]+ and POPE-[Gly]− H-bonds increase with addition of [Ch][Gly]. It is observed that the dif-


13


Table 4: Ensemble average of number of H-bond per lipid molecule computed for POPE-[Ch]+ , POPE-[Gly]− , POPE-water pairs and per solvent molecules for water[Ch]+ and water-[Gly]− pairs in POPE-[Ch][Gly] systems.

0.5

+

POPE-[Ch]

0.4

0.4

0.3

0.3

0.2

0.2

0.1 0

20

40 60 80 Time (ns)

0

0.05 0.1 0.15 Occurance

0

POPE-[Gly]

7.056 6.364 5.075 3.067 2

1.5

1.5

1

1

0.5 0

0.5

0

20

40 60 80 Time (ns)

(a) No. of H-bonds / solvent molecule

0.032 0.057 0.092 0.121

-

0

0.02 0.04 Occurance

0

10

0.2

+

water-[Ch]

0.2

POPE

0.15

0.15

0.1

0.1

0.05

0.05

0

0

20

40 60 80 Time (ns)

0

0.01 0.02 Occurance

0

0.165 0.310 0.543 0.834 10

POPE-water

8

8

6

6

4

4

2

2

0

0

20

40 60 80 Time (ns)

(b) No. of H-bonds / solvent molecule

0

0.1

2

water-[Gly]−

POPE-water water-[Ch]+

0.179 0.366 0.747 1.246 No. of H-Bonds / POPE molecule

0.5

POPE-[Gly]−

No. of H-Bonds / POPE molecule

[Ch][Gly] POPE-[Ch]+ mol% 2.5 0.042 5 0.084 10 0.178 20 0.317 No. of H-Bonds / POPE molecule

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 21

0 0.005 0.01 0.015 Occurance

0

(c) 1

water-[Gly]

-

1

POPE

0.75

0.75

0.5

0.5

0.25

0.25

0

0

20

40 60 80 Time (ns)

(d)

0

0.008 0.016 Occurance

0

(e)

Figure 12: [Ch][Gly] concentration dependence of number of (a) POPE-[Ch]+ , (b) POPE-[Gly]− , (c) POPE-water, (d) water-[Ch]+ , and (e) water-[Gly]− H-bonds for POPE-[Ch][Gly] systems.

[Gly]− ions also suggested the accumulation of these ions below lipid-water interface as depicted in Figs. 6(c) and 6(d). Similar to POPC[Ch][Gly] systems, a decrease in the H-bonding interaction for POPE-water and an increase in the H-bonding interaction for [Ch]+ -water and [Gly]− -water for all POPE-[Ch][Gly] systems can be observed from Figs. 12(c)-12(e). The decreased H-bonding interaction between POPE-water molecules leads to shifting of water molecules away from lipid-water interface.

ference in the headgroup structure of POPC and POPE lipid molecules play a vital role in rendering the H-bonding interaction with [Ch][Gly] IL. Here, the headgroup of POPE lipid molecule consists of H-bond donor (NH3 + ) as well as H-bond acceptor groups (phosphoryl oxygens and C=O group at lipid tails) whereas POPC contains only H-bond acceptor groups. The presence of H-bond donor (NH3 + ) group in POPE lipid molecule employs it to have strong H-bonding interactions with carboxylate group of [Gly]− ions. Typical conformations of POPE lipid molecule involved in the H-bond with [Ch]+ and [Gly]− ions are shown in Fig. 13. The atomic number density profiles of oxygen and nitrogen atoms of [Ch]+ and


14

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1.9 1.8

4.2

(a)

showed a decrease in the spacing between headgroup atoms of the lipids causing a considerable decrease in APL of bilayer in the presence of [Ch][Gly] IL. For POPE-[Ch][Gly] systems, while [Ch]+ cations show lesser propensity to H-bond with phosphoryl and ester group oxygens of POPE lipids, the [Gly]− anions remained near the water-membrane interfacial region. However, for POPE bilayer containing [Ch][Gly] the structural properties including APL, NDPs of lipid molecules and bilayer thickness are minutely affected because of strong intra- and intermolecular H-bonding between POPE lipids. Furthermore, perhaps expected from a bio-IL, within the stipulated run time, we did not observe any [Ch][Gly] ions in the hydrophobic part of POPC and POPE bilayers for all the concentrations of [Ch][Gly] investigated. On the other hand, MD simulation studies performed on imidazolium-based IL demonstrate deep insertion of imidazolium cation with long alkyl chains into the hydrophobic part of a lipid bilayer. Yoo et al. 16 explored the interaction of 1-n-alkyl-3-methylimidazolium with a POPC bilayer and reported that imidazolium cation with long alkyl chains deeply inserted into the hydrophobic part of the bilayer and oriented in a manner that imidazolium ring and alkyl chain strongly interact with the lipid head and tail groups, respectively. This results in the disruption of the bilayer and is a potential source of ionic liquid toxicity. Lim and co-workers 63,64 also observed a higher affinity of imidazoliumbased cation to insert into the model bacterial plasma membrane and thereby causing an increase in the disordering of lipid tails and hence inducing destabilization of the membrane.

1.9

4.2

(b)

Figure 13: Representative configurations ((a) and (b)) showing association of POPE lipid molecule with [Ch]+ and [Gly]− ions for system containing 20 mol% [Ch][Gly] IL. Representations in (a) and (b) indicate that [Gly]− ions prefer to stay close to the ammonium group of POPE lipid because of H-bonding or electrostatic interaction (shown as dashed lines). [Ch]+ ions are observed to be close to the phosphoryl or ester group oxygens of lipids. The distances shown by dashed lines are in ˚ A unit.

4

Conclusions

We have reported an all atom molecular dynamics study on the concentration-dependent effects of [Ch][Gly] bio-compatible ionic liquid on the structure and stability of POPC and POPE lipid bilayers, perhaps for the first time. It is shown that the main effect of [Ch][Gly] biocompatible ionic liquid on POPC lipid is determined by the [Ch]+ ions binding to the phosphoryl and ester group oxygens in the deeper region of the membrane. The [Gly]− anions mostly remain accumulated near the watermembrane interfacial region. Our H-bonding analysis revealed that hydroxyl hydrogens of [Ch]+ ions bind to phosphoryl and ester group oxygens of lipid. Partial NDPs also indicated that most of [Ch][Gly] IL sits in the bulk phase and only few ions of [Ch][Gly] IL are able to enter in the interfacial region. The examination of 2D RDFs of POPC headgroup atoms

Supporting Information Figures for variation of area per lipid (APL) with simulation time and figures for average APL and bilayer thickness as a function of [Ch][Gly] IL mol%. Notes: The authors declare no competing financial interest.


15


Acknowledgments

Page 16 of 21

(9) Castner, Jr., E. W.; Wishart, J. F. Spotlight on Ionic Liquids. J. Chem. Phys. 2010, 132, 120901.

P.K. thanks IIT Delhi for fellowship. This work is financially supported by the Department of Science and Technology (DST), India, through FIST grant awarded to Department of Chemistry, IIT Delhi for augmentation of HPC facility. Partial financial support from DSTNanomission (SR/NM/NT-1049/2016) is also acknowledged.

(10) Stock, F.; Hoffmann, J.; Ranke, J.; Stormann, R.; Ondruschka, B.; Jastorff, B. Effects of Ionic Liquids on the Acetylcholinesterase - A Structure-Activity Relationship Consideration. Green Chem. 2004, 6, 286–290. (11) Yu, M.; Li, S.-M.; Li, X.-Y.; Zhang, B.J.; Wang, J.-J. Acute Effects of 1-Octyl3-methylimidazolium Bromide Ionic Liquid on the Antioxidant Enzyme System of Mouse Liver. Ecotoxicol. Environ. Saf. 2008, 71, 903–908.

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