Article pubs.acs.org/journal/abseba
Cite This: ACS Biomater. Sci. Eng. 2019, 5, 3645−3653
The Influence of Water on Choline-Based Ionic Liquids Eden E. L. Tanner,† Kathryn M. Piston,‡ Huilin Ma,‡ Kelly N. Ibsen,† Shikha Nangia,‡ and Samir Mitragotri*,†,§
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†
John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, Massachusetts 02138, United States ‡ Department of Biomedical and Chemical Engineering, Syracuse University, 223 Link Hall, Syracuse, New York 13244, United States § Wyss Institute of Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, Massachusetts 02115, United States S Supporting Information *
ABSTRACT: Choline and geranic acid (CAGE)-based ionic liquids have been recently developed for applications in drug delivery. Understanding the microscopic structures of CAGE in the presence of water is critical for its continued use in biomedical applications as it will undoubtedly come into contact with water in physiological fluids. Water can drastically impact the physicochemical properties of the ionic liquids, including CAGE. Computational and experimental characterization, namely viscosity, conductivity, and self-diffusion coefficient, were employed here to understand the properties of equimolar CAGE (1:1 choline/geranic acid) in the presence of varying amounts of water. It was found that under stored conditions, 1:1 CAGE contained up to 0.20 mole fraction water. Experimental and computational studies indicate that microscopic intraionic interactions within CAGE are not substantially changed until the water content exceeds 0.65 mole fraction. At this point, we theorize that the geranate ions undergo reorganization to minimize contact between the hydrophobic tails and the water molecules. This is evidenced by the plateau in viscosity at this mole fraction, and the increased interactions between the tails of the anions. This suggests that CAGE could be used without predrying in most applications and can be diluted to induce the organization of the anions where desired. KEYWORDS: ionic liquids, deep eutectic solvents, molecular dynamics, simulation, water content
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INTRODUCTION Ionic liquids (ILs) are being increasingly explored for biomedical applications with over 3000 papers published in the last three years alone. Ionic liquids consist of bulky, asymmetric organic cations and anions, and have melting points below 100 °C.1 They have traditionally been employed in a variety of chemical and industrial applications, including catalysis energy applications2−5 and electrochemical sensors,6 where their favorable low volatility,7 large electrochemical window,8 stability,9 and tuneability10 have made them attractive alternatives to traditional molecular solvents. However, many of the traditional ions used, cations such as imidazolium, pyrrolidinium, and pyridinium and anions such as tetrafluoroborate and bistriflimide, exhibit toxicities that limit their biological applications11−13 and so new ILs composed of biologically compatible components have been synthesized to enable their use in a biomedical context.14 Ionic liquids have shown promise in biomedical contexts due to their design modularity and ability to permeate membranes.15−18 This has led to their successful use both as antimicrobial agents19−21 and in drug delivery formulations.22−25 Drugs with poor solubility and bioavailability in © 2019 American Chemical Society
their native forms have also been turned into ILs (termed active pharmaceutical ingredient ionic liquids) in order to overcome these challenges.26,27 In addition to this, the ionic components can themselves be tuned to solvate a range of poorly water-soluble drugs and biologics.28,29 The ability to design specific solvents combined with their ability to transport these drugs across a range of biological barriers make ILs exceptional candidates for drug delivery. Our lab has reported one particular IL, choline and geranate, CAGE (Figure 1), for transdermal24,25 as well as oral30 delivery of insulin by navigating the stratum corneum of the skin, and the tight epithelial junctions of the intestine, respectively. In both cases, insulin was suspended in pure CAGE and placed on the respective epithelia. CAGE-induced permeabilization of the stratum corneum as well as the intestinal epithelium led to high bioavailabilities. While these applications involved the placement of undiluted IL on the epithelia, CAGE eventually undoubtedly comes in contact with water in physiological Received: February 17, 2019 Accepted: May 21, 2019 Published: May 22, 2019 3645
DOI: 10.1021/acsbiomaterials.9b00243 ACS Biomater. Sci. Eng. 2019, 5, 3645−3653
Article
ACS Biomaterials Science & Engineering
left in sealed sample tubes that had not been evacuated or flushed with inert gas but contained atmosphere for >3 months at room temperature and humidity. “Dry” samples were placed on a rotary evaporator at 60 °C and 10 mbar for 3 h prior to testing. “Wet” samples consisted of a 485 μL aliquot of stored CAGE diluted with 15 μL of water.
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DOSY MEASUREMENTS Prior to all measurements, a sample of CAGE was dried at 60 °C on a rotary evaporator under 10 mbar pressure for 3 h. A 0.725g sample of CAGE was placed in a clean NMR tube (KONTES, Kimble-Chase, U.S.A.) alongside a coaxial insert filled with d-DMSO. Measurements were recorded in triplicate on a Varian Unity/Inova 500B spectrometer. The 90° pulse width was set as 11.25. The diffusion gradient length used was 6.0 ms and the diffusion delay was 200 ms across all samples. The temperature was 22.0 ± 0.2 °C.
Figure 1. Molecular line structure of (a) choline and (b) geranate indicating the head and tail conventions adopted in this work. The AA to CG mapping (dashed lines) scheme is indicated along with MARTINI CG bead types for choline.
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media. This is especially significant for applications in oral delivery where the highly hydrated environment in the gastrointestinal tract is expected to lead to hydration of CAGE almost instantaneously. In addition, most ionic liquids, CAGE included, are hygroscopic.31 Thus, CAGE is expected to absorb water under storage conditions, as other hygroscopic ionic liquids have been shown to do.32 Thus, it is critical to understand the impact of water on the structure and properties of CAGE. This understanding, however, is currently absent. A considerable number of experimental and computational studies conducted in other ILs have revealed that water can dramatically alter physical and chemical properties,8,33−35 including electrochemistry,8 viscosity,36 diffusion of molecules,37 and activation parameters.38 All-atom (AA) and coarse grain (CG) molecular dynamics (MD) simulations of IL− water systems have shown a nonhomogenous behavior of the mixtures that segregate into polar and nonpolar nanodomains.39−50 On the basis of the levels of solvation, molecular level changes occur between polar and nonpolar regions. This nanostructural reorganization causes shifts in the diffusion or conductivity; however, these changes vary with the chemical structure of the IL ions. To date CAGE has been studied via MD simulations exclusively in bacterial membrane systems that exclude water.20 Despite the numerous computational and experimental studies on IL and water mixtures, the molecular level interactions between CAGE and water remain unknown. Using a synergistic experimental and computational approach, we elucidate the impact of water on the microscopic interactions and the bulk physical properties of CAGE.
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VISCOSITY MEASUREMENTS Viscosity was measured on an AR-G2 rheometer supplied by TA Instruments using a 20 mm diameter aluminum 2° cone. Ninety microlitres of CAGE was placed on the bottom plate, and the sample was equilibrated at 25 °C for 2 min prior to the experiment being conducted. A steady-state flow method was employed with 5 points per decade being recorded from 1 to 100 Pa. In each case, 3 measurements were recorded with each repeat receiving a fresh aliquot of CAGE.
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CONDUCTIVITY MEASUREMENTS Conductivity was measured using a Horiba-supplied Laqua conductivity meter (model DS-71) using a 3574-10C probe. In each case, 200 μL of CAGE was introduced into the clean probe (rinsed thoroughly with water and nitrogen dried) and a reading was taken after the output was stabilized. This was repeated three times per variant.
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MD SIMULATIONS All-atom (AA) and coarse grain (CG) MD simulations were performed using GROMACS molecular dynamics package (version 2016.4).51,52 In AA systems, OPLS-AA force field53 was used for CAGE and TIP4P for water.54 The PDB files of CAGE components were generated using an Automated Force Field Topology Builder (ATB) and Repository55 and the topology files of CAGE components were obtained using LigParGen web server, an automatic OPLS-AA parameter generator.56−58 Six CAGE-water AA systems in the 0−100 mol percentage range (Table 1) were generated using in-built GROMACS utilities in a cubic simulation box with 10 nm box dimensions. Each AA system was simulated for 1 μs after equilibration. To characterize CAGE−water systems for longer time scales beyond 1 μs, we performed CG simulations for four systems (Table 1). The CG description of choline and geranate was based on MARTINI four-to-one mapping scheme with geranate as three beads (Qa, C1, and C3) and choline as two beads (Q0 and P2). Explicit standard MARTINI water beads were used to represent water. All-Atom System. Energy minimization was performed using the steepest-descent algorithm59 with a 1 fs time-step until the maximum force on any bead was below the tolerance parameter of 100 kJmol−1 nm−1. Periodic boundary conditions were applied in all three dimensions. Equilibration runs were performed in isothermal−isochoric (NVT) and isothermal− isobaric (NPT) ensembles for 0.1 and 0.05 ns, respectively.
MATERIALS AND METHODS
Synthesis and Characterization of ILs. Choline bicarbonate, geranic acid, D2O, and deuterated DMSO were obtained from SigmaAldrich (St. Louis, MO, U.S.A.). CAGE was synthesized as previously reported.22 Choline bicarbonate and geranic acid at a molar ratio of 1:1 were combined at 40 °C for 12 h. The resultant viscous liquid was dried under reduced pressure, first on a rotary evaporator for 3 h, then in a vacuum oven at 60 °C for 36 h. CAGE was characterized via NMR by placing dried, neat CAGE into an NMR tube (KONTES, Kimble-Chase, U.S.A.) containing a coaxial insert filled with DMSO− d6. An Agilent DD2 600 MHz spectrometer was used for all onedimensional (1D) characterizations, which have been reported previously.25 Water Content Measurements. The amount of water in each sample was quantified with the addition of approximately 100 mg (exact mass was calculated using weigh-by-difference) to a Karl Fischer Coulometer (Model 899, Metrohm, U.S.A.). Samples noted as “stored” were dried in a vacuum oven for 3 days for 60 °C but were 3646
DOI: 10.1021/acsbiomaterials.9b00243 ACS Biomater. Sci. Eng. 2019, 5, 3645−3653
Article
ACS Biomaterials Science & Engineering
fraction (99.18% CAGE by mass) indicating that the IL is moderately hygroscopic, and wet CAGE measured at 0.54 mole fraction (94.65% CAGE by mass). In most practical applications, it is the stored condition that is likely to be most relevant, where the amount of water exceeds 0.10 mole fraction, thus necessitating the understanding of the effect of the presence of water in these systems. Effect of Water on CAGE Conductivity, Viscosity, and Diffusion Coefficient. The impact of water content on the CAGE’s physical properties, specifically the conductivity, viscosity, and self-diffusion coefficient, was examined. Conductivity exhibited a strong dependence on water content (Figure 2, blue circles, experimental data; red diamonds,
Table 1. System Details of CAGE−Water Mixtures in AllAtom and CG Representations no. of ions or molecules
resolution
systems
IL mole fraction
AA
1 2 3 4 5 6 7 8 9 10
0.05 0.10 0.30 0.50 0.80 1.00 0.10 0.30 0.80 1.00
CG
choline
geranate
water
system performance (ns/day)
338 408 1220 1328 1450 1321 841 1133 1500 1687
338 408 1220 1328 1450 1321 841 1133 1500 1687
6000 4000 2500 1500 600 0 7576 2644 374 0
88 92 37 36 34 37 2977 3888 4119 3694
The long-range electrostatic interactions were computed using the Particle Mesh Ewald electrostatics.60 The systems were simulated at 1 bar pressure using isotropic Parrinello−Rahman barostat61 with a coupling constant τp = 2.0 ps and compressibility factor of 4.5 × 10−5 bar−1. The temperature was maintained at 296 K by independently coupling the water and CAGE molecules to an external velocity rescaling thermostat62 with τT = 0.1 ps. The neighbor list was updated every 5 steps using 1.0 nm for short-range van der Waals and electrostatic cutoffs. Bonds with H atoms were constrained with the LINCS algorithm.63 The production NPT simulations were performed for 1 μs for all systems and post simulation analyses were performed using in-built GROMACS utilities (gmx rdf; gmx energy; and gmx hbond). Coarse Grain (CG) Simulations. Energy minimization was performed using the steepest-descent algorithm with a 20 fs time-step until the maximum force on any bead was below the tolerance parameter of 10 kJ mol−1 nm−1. Periodic boundary conditions were applied in all three dimensions. The NVT and NPT equilibration runs were performed for 0.2 μs. Isotropic pressure coupling was used, and the systems were maintained at 1 bar using the Berendsen barostat with time constant, τp = 4.0 ps. Temperature was maintained at 296 K by independently coupling the water and CAGE to an external velocity rescaling thermostat with τT = 1.0 ps. The neighbor list was updated every 25 steps using 1.4 and 1.2 nm for shortrange van der Waals and electrostatic cutoffs, respectively. The production NPT simulations were performed for 10 μs for all the systems. Molecular visualization and graphics were generated using visual molecular dynamics (VMD) software.64 Post simulation analyses were performed using in-built GROMACS utilities and in-house python scripts.
Figure 2. Conductivity of 1:1 CAGE. Blue circles are the experimental data (n = 3), and red diamonds are AA MD simulation data. Error bars are encompassed within the area of the marker if not visible.
simulations). Ionic liquids, despite their ionic compositions, generally have lower-than-expected ionic conductivity. This originates from the formation of ion-pairs between the cation and anion,65 leading to charge neutralization, and the difficulty of charge transfer through a network of bulky ions.66 Conductivity of pure CAGE is consistent with this behavior, exhibiting minimal conductivity, partially due to ion pairing between the choline and geranic acid. As water is added, the conductivity increases; however, this increase is modest until CAGE is diluted to nearly 0.3 mole fraction (0.70 water mole fraction), after which the conductivity increased dramatically to a value of ∼1750 mS/m, a value about 200-fold higher than that of pure CAGE. The addition of water disrupts the hydrogen-bonding between choline and geranic acid, allowing charges to migrate more readily through the solution, thus leading to increased conductivity. Vila et al.67 investigated the changes in conductivity of imidazolium and pyridinium ionic liquids with the addition of water and reported a maximum 30fold increase in the conductivity in the diluted solutions. Note that these cations have a higher baseline conductivity because the planar cationic core is less bulky than the ammonium cation discussed here.8 Zhao et al.68 present a modified hole theory to explain the conductivity of ionic liquids, whereby ions can only move through solution if there is an appropriate hole for them to move into. The addition of water widens this hole, improving charge transport and resulting in higher conductivities. There is also discussion in the literature about how the conductivity of pure ionic liquids is governed by momentum conservation of the ion pairs, whereas in wet systems, the presence of water affects the correlation of cations
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RESULTS AND DISCUSSION Water Content Measurements. Water content of CAGE (1:1) was measured under three states of dryness: dry, stored, and wet. These conditions were chosen as representative conditions of relevant to biomedical applications. Dry CAGE corresponds to CAGE dehydrated under controlled conditions (60 °C, 10 mbar for 3 h), stored CAGE represents IL under “storage conditions”, that is, CAGE left in a sealed container for 3 months, and wet CAGE is stored CAGE with the addition of 3% v/v addition of water. Dry 1:1 CAGE measured at 0.99 ionic liquid by mole fraction (or 99.77% CAGE by mass), while stored CAGE measured at 0.89 IL by mole 3647
DOI: 10.1021/acsbiomaterials.9b00243 ACS Biomater. Sci. Eng. 2019, 5, 3645−3653
Article
ACS Biomaterials Science & Engineering
viscosity is measured means that accurately recording an experimental value for mole fractions of IL > 0.7 is very difficult in this ionic liquid because of its hygroscopicity and the exposure to the atmosphere as the measurement is occurring, hence experimental values are only included up to this mole fraction. Unlike diffusivity and conductivity, the viscosity decreased almost linearly, exhibiting a plateau in the range of 0.30−0.50 mole fraction CAGE. The reduction of viscosity of ILs with the addition of water has been widely reported previously in the literature. For example, in imidazolium bistriflimide based ILs the addition of 1% water by mass results in a 30% decrease in viscosity, wherea for a hexafluorophosphate anion, a 0.19% increase in the water content resulted in a 17% decrease in viscosity.36 Seddon, Stark, and Torres72 comprehensively discuss the effect of water on the viscosity of ionic liquids. They report that the addition of water reduces the electrostatic interactions between the ions, lowering the systematic cohesive energy and reducing the viscosity. Note that the plateau in the viscosity at ∼0.30 mole fraction CAGE is peculiar; other ionic liquids report shapes similar to those seen in Figures 2 and 3. This suggests that other interactions (outside of electrostatic interactions and ionpairing) could be potentially contributing to the change in viscosity in CAGE as water is added. This is explored further using the molecular dynamics simulations below. Hydrogen Bonding. The effect of water on hydrogen bonding between various components of CAGE was explored using MD simulations. The calculated number of hydrogen bonds between geranate and water (green), choline and water (red), and geranate and choline (black) changed significantly as a function of water content (Figure 5).
and anions, and the momentum conservation is satisfied by the ions and the water molecules.69 Next, the self-diffusion coefficient of the ionic components was measured using 2D DOSY NMR and simulated using molecular dynamics (Figure 3, blue circles, experimental data;
Figure 3. Diffusion coefficient of 1:1 CAGE (blue circles are the experimental data (n = 3), and red diamonds are AA MD simulations). Error bars are encompassed within the area of the marker if not visible.
red diamonds, simulations). At low water content, the diffusivity of the ions was low at about 3 × 10−12 m2 s−1. However, the diffusivity increased significantly for hydrated CAGE with a water content exceeding 0.70 mole fraction, although the changes in self-diffusion coefficient were more gradual than those observed in the case of conductivity. The change in self-diffusion coefficient upon hydration occurs for similar reasons as that in the case of conductivity, whereby the water disrupts the anion−cation interaction, allowing them to move more freely in solution. Watanabe and colleagues70 report the self-diffusion coefficient for imidazolium-based cations, which possess much faster diffusion coefficients than CAGE. Hanke and Lynden-Bell71 report simulated diffusion coefficients in imidazolium ionic liquids as water is added, with the same trend being seen as in Figure 3. They attribute this to the lower energies of interaction between the ions and water than the ions themselves, resulting in increased diffusion coefficients as water is added. Last, the viscosity of CAGE was measured and compared to simulated data (Figure 4, blue circles, experimental data; red diamonds, simulations). Note that the way in which the
Figure 5. Number of hydrogen bonds between geranate−water (green), choline−water (red) and geranate and choline (black) in 1:1 CAGE as a function of mole fraction of CAGE in water.
The number of hydrogen bonds between choline and geranate exhibits a maximum value of one in pure CAGE, indicative of a stoichiometric match of the ions (black cross, Figure 5), and it decreases monotonically with decreasing CAGE concentration. At 0.05 mole fraction of CAGE, the number of hydrogen bonds is
