Room-Temperature Ionic Liquids and Biomembranes: Setting the

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Room-Temperature Ionic Liquids and Bio-membranes: Setting the Stage for Applications in Pharmacology, Bio-Medicine and Bio-nano Technology Antonio Benedetto, and Pietro Ballone Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04361 • Publication Date (Web): 06 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Room-Temperature Ionic Liquids and Bio-membranes: Setting the Stage for Applications in Pharmacology, Bio-medicine and Bio-nano Technology Antonio Benedetto(1,2,3)† , and Pietro Ballone(4) (1) School of Physics, University College Dublin, Dublin 4, Ireland (2) School of Chemistry, University College Dublin, Dublin 4, Ireland (3) Laboratory for Neutron Scattering, Paul Scherrer Institute, Villigen PSI, Villigen 5232, Switzerland and (4) Italian Institute of Technology, Via Morego 30, 16163 Genova, Italy

Abstract Empirical evidence and conceptual elaboration reveal and rationalise the remarkable affinity of organic ionic liquids for biomembranes. Cations of the so-called room-temperature ionic liquids (RTILs), in particular, are readily absorbed into the lipid fraction of biomembranes, causing a variety of observable biological e↵ects, including generic cytotoxicity, broad anti-bacterial potential, and anti-cancer activity. Chemical-physics analysis of model systems made of phospholipid bilayers, RTIL ions and water confirm and partly explain this evidence, quantifying the mild destabilising e↵ect of RTILs on structural, dynamical and thermodynamic properties of lipids in biomembranes. Our Feature Article presents a brief introduction to these systems and to their role in biophysics and in biotechnology, summarising recent experimental and computational results on their properties. More importantly, it highlights the many developments in pharmacology, biomedicine and bio-nano technology expected from the current research e↵ort on this topic. To anticipate future developments we speculate on: (i) potential applications of (magnetic) RTILs to a↵ect and control the rheology of cells and biological tissues, of great relevance for diagnostics; and on: (ii) the usage of RTILs to improve the durability, reliability and output of bio-mimetic photovoltaic devices.

† Corresponding Author: [email protected]

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INTRODUCTION

The combination of lipid bilayers, enzymes and oligo-saccharides that we call biomembranes plays a major role in the life of cells, contributing to their feeding, replication, respiration, energy harvesting from light, and to the excretion of waste.1 Moreover, membrane receptors mediate communications among cells, while specialised polymeric structures sprouting out of biomembranes tether cells to external solid supports, and allow their congregation into multicellular communities such as bio-films made of and by bacteria, or into functional tissues of higher-level organisms. This variety of functions and roles makes biomembranes a primary target of biochemical, pharmacological and bio-medical approaches to control the functioning and evolution of living cells. This research e↵ort, in turn, could provide the basis for a massive new advance in bio-nanotechnologies. A most promising counterpart to this rapidly evolving picture might be represented by the vast class of organic ionic compounds of low melting temperature that are being synthesised and characterised at a sustained high rate because of their potential application as novel solvents for industrial processing. Ions from this family could represent a new choice of versatile ligands, able to interact with lipids and proteins, a↵ecting the structure, dynamics and activity of bio-membranes. We will focus our discussion on the so called room-temperature ionic liquids2 (RTILs) which are organic ionic compounds whose melting temperature fall below 100 C. RTILs can be divided into two broad classes, corresponding to protic and aprotic ionic liquids. Protic ionic liquids3,4 result from the reaction of a Brønsted acid with a Brønsted base. Aprotic ionic liquids represent the majority of RTILs, and, formally, they might be seen as the result of substituting the acidic proton in a protic IL with a broad choice of more extended and less labile cationic moiety, such as an alkyl group. Structural formulae of representative RTIL cations and anions are reported in Fig. 1. An essential structural feature of RTILs consists of the interplay of ionic, polar and apolar groups within the cation, less often within the anions. This characteristic organisation of their structure endows many RTIL ions with a marked amphiphilic character, that naturally matches the overall properties of lipids in solution. These simple and intuitive considerations already raise the prospect of innumerable applications in biochemistry and biotechnology, medicine, pharmacology and bio-nanotechnology. Applications, in turn, drive the surge of interest and research e↵ort devoted to the mutual interaction of RTILs and biomembranes, which is precisely the subject of this Feature Article. The remarkable affinity of RTILs for biomolecules, and for lipids in particular, arises first of all from the similarity of their structure and inter-molecular interactions.5–7 Both classes of compounds are predominantly organic, with a strong electrostatic signature, whose aggregation at ambient tem-

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FIG. 1: Structural formula of cations and anions from selected room-temperature ionic liquids (RTILs). The MILs (magnetic ionic liquids) sub-category consists of magnetic ionic liquid anions. AAILs (amino acid ionic liquids) represents anions made of deprotonated amino acids. perature into a solid structure of long range ordering is prevented by the large size and geometrical asymmetry of their natural building blocks. The first flag raised by the RTIL affinity for biomolecules of course concerns toxicity and environmental safety issues,8,9 since the eventual introduction of RTILs into industrial processes will certainly cause their dispersion into the environment. Early biological assays revealed toxic e↵ects of RTILs at high concentration, marked by the death of a variety of organisms from bacteria10 to fish.11 These results fuelled controversy, but did not stop further interest in RTILs. After all, even NaCl precludes life at high concentration, as shown by its usage to preserve food, or as displayed on the grand scale by the Dead Sea. Cyto- and environmental-toxicity, of course, are just the dark side of the general biological relevance of RTILs, whose bright side is represented by a large number of opportunities for beneficial applications, ranging from their usage as active pharmaceutical ingredient (API)12 to their potential role in manufacturing and manipulating bio-nano systems. The results of early studies along these lines motivated an increasing number of investigations of the e↵ect of RTILs on bio-systems, using a variety of empirical methods, borrowed primarily from biology. The results fuelled the first wave of exploration of biophysical, bio-medical and nano-bio applications, generating in turn an increasing level of expectation on future new formulations of RTILs as drugs, food additives, solvent for enzymatic reactions, or ingredients in the pre-processing of biomass for renewable energy harvesting. The progression of RTIL-biomembrane investigations into a subjects in itself, in particular, has

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been marked by the first measurements of microscopic structural and dynamical properties, focusing on somewhat simplified models of membrane consisting primarily of phospholipid bilayers, sometimes with membrane proteins embedded into them, often deposited on a metal electrode. Studies based on neutron-13 and X-ray scattering,14 di↵erential scanning calorimetry (DSC), luminescence,15 NMR,16 vibrational and optical spectroscopy, electrochemistry and, last but not least, computer simulation,17 provided a fresh look into the chemical physics e↵ects of combining RTILs with bio-membranes. At this stage, experimental and computational studies of structural and dynamical properties of RTILs interacting with simplified model biomembranes arguably represent the mainstay in the biophysical investigation of such systems. Studies of this kind are still critically needed to clarify the microscopic mechanisms underlying many phenomena and properties whose understanding is required to progress with applications. This background activity is what we consider the present state of the art, continuing research lines that already provided a wide range of results, and are still expected to produce a wealth of new knowledge. Hence, our first aim is to briefly summarise this state of the art, to some extent starting from our own activity on RTILs and phospholipid bilayers. Moreover and more importantly, we aim to provide an educated prediction of the most likely and most beneficial developments expected in this field over the next five to ten years. In this exercise we proceed at first by extrapolation based on continuity, but eventually we point to new directions of research that to our eyes appear to be particularly promising. Thus, a major expected development that is already taking place concerns the rapid growth of investigations of pharmaceutical aspects of RTILs, with a strong focus on the e↵ect of RTILs on biomembranes. The close relation of pharmaceutical potential and organic ionic character is emphasised by the fact that, on the one hand, RTILs are organic salts typically in the ⇠ 500 Dalton

range, that corresponds well to the size and complexity of popular APIs.18 On the other hand, many molecules of pharmacological interest, such as antibiotic peptides, are ionic species in physiological conditions, sometimes carrying more than a single charge and often having a complex ionic and polar pattern. This shifting boundary between RTILs and drugs is exemplified by the fact that RTILs are known to display bactericidal properties, an activity that often is mediated by their interaction with the cell membrane. This research field is already expanding rapidly. A recent review reports more than 860 references on pharmacology aspects alone.19 In its last sections, our Feature Article takes a bolder view at the future. In a first speculation, we discuss how the addition of RTILs could modify and control the properties, evolution and behaviour of biological systems larger (thicker) than just natural and artificial biomembranes, progressing to the level of whole cells and of biological tissue. From the biophysical point of view, the aspects of 4


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interest concern mechanical, optical, rheological and adhesion properties of cells. The timeliness of this development is supported by the availability of biophysical techniques such as AFM to accurately measure these properties. The rationale for our interest is the expected role of membranes (together with the cytoskeleton) in the determination of the mechanical properties of whole cells, and the already apparent strong coupling of biomembranes and RTILs. Applications of this research line concern medical diagnostics and imaging, the control of biofilms and their removal from medical equipment, the tuning of sample and environment properties during the growth of new biological tissue and of replacement organs. In a second speculation, we explore the complementary direction of lower time and size scales, considering bio-nano applications of RTILs. As an example, we outline the possible role of RTILs in preparing, stabilising and improving bio-mimetic photovoltaic devices. These consist primarily of enzymes (photosystems) embedded into natural or artificial membranes, that might represent the ultimate bio-nano factory. This conceptual exercise illustrates the variety of ways RTILs could be employed in the bio-nano domain, and highlights the role of two complementary approaches to exploit the vast number and variety of RTIL compounds, consisting of their usage of high throughput screening, and in the enhancement of basic and microscopic knowledge on the e↵ect of RTILs on bio-molecules.

EXPERIMENTAL AND COMPUTATIONAL APPROACHES

The investigation of the e↵ects of RTILs on phospholipid bilayers and on biomembranes is a broadly interdisciplinary subject. By choice, our contribution emphasises its chemical physics content, which here is highlighted by the methods currently used to investigate thermodynamic, structural and dynamical properties of these systems. A cartoon illustration of approaches, biased towards the methods we use, is shown in Fig. 2 Because of its sensitivity to light elements and to hydrogen in particular, neutron scattering (NS) is a major player in our discussion, being currently used in its reflectometry (NR), small angle (SANS), elastic (ENS) and quasi-elastic (QENS) flavours to provide quantitative data on the structure and dynamics of systems made by water, lipids and RTILs. In general, NR is the method of choice to determine the structure of layered systems, probed by a beam of neutrons aimed towards the surface of a planar sample (see Fig. 2 left and Fig. 3). Provided the in-plane distribution of molecules is uniform, the knowledge of the coefficient of specular reflection (i.e., at elastic conditions)) over the widest range of accessible incidence angles allows one to reconstruct the distribution of scattering density along the direction z perpendicular to the solid

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substrate. Since RTIL, water, polar and apolar moieties of phospholipids have di↵erent scattering densities, it is possible to decompose the total scattering into the sum of individual contributions along z (see Panel (b) in Fig. 3). This decomposition is a difficult inverse problem, solved by fitting the measured total scattering density using trial densities for the individual species. The power of the method is greatly enhanced by the unique capability of NS to exploit isotopic substitution, that is particularly e↵ective for systems rich in hydrogen atoms, because of their relatively easy replacement with deuterium. The measurement and the interpretation of results remain challenging, especially because the scattering cross section of a molecularly thin layer is tiny, but NR still represents the most powerful tool to measure structural parameters of solid supported phospholipid bilayers in clean water and in RTIL water solutions. The same role of planar probe could be filled by a beam of X-rays, whose sensitivity to light elements, however, is poor, and, in general, lack the analogue of isotopic substitution. Nevertheless, vast numbers of photons are available up to X-ray energies at synchrotron radiation sources allowing much faster measurements than with neutrons. This, in turn, opens the way to time-dependent measurements of high time resolution, reaching into the ps-ns range. A similar comparison of neutrons and X-rays concerns small angle scattering, i.e., SANS and SAXS, respectively. The technique requires globally homogeneous samples, which can be obtained by vesicles and micelles floating in water. The method is close in spirit to general di↵raction techniques. The low-momentum transfer condition implies that the method is particularly suitable to measure features on the mesoscopic (100 nm) scale, and is currently used to investigate the size and shape of liposomes. A careful analysis of scattering intensities, however, allows the determination also of more microscopic parameters, such as the leaflet separation of phospholipid bilayers in water and in electrolyte solutions.20 To summarise, the comparison of neutron versus X-ray scattering is largely influenced by the di↵erent availability of radiation sources. Neutron approaches always require the complexity and cost of a large scale facility. In the case of X-rays, synchrotron radiation is strictly required for studies like X-ray reflectometry, or any other measurement carried out on single, solid-supported bilayers, for which the total number of scattering atoms is tiny. Structural investigations by X-ray di↵raction, especially on 3D stacks of bilayers, can use simpler and cheaper traditional X-ray sources available in several medium-scale laboratories. Despite the widely repeated statement that the function of biomolecules can be understood on the basis of structure, knowledge of the dynamics of atoms and molecules often provides valuable insight into the system properties. Inelastic neutron scattering, for instance, characterises the spectrum of 6


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FIG. 2: Cartoon representation of neutron reflectometry (NR) and atomic force microscopy (AFM) measurements on solid-supported lipid bilayers. Molecular dynamics represents an essential tool to complement and explain the experimental results. The cartoon selection reflects the methods we use. long lived excitations in condensed matter systems by counting the neutrons that change momentum and energy through their scattering. The quasi-elastic QENS is a particular form of inelastic neutron scattering focusing on a limited interval of energy transfer. QENS is used to investigate di↵usive modes over time scales of the order of ps to ns, limited by the energy resolution of the neutron detector. Contributions from QENS applied to the RTILs/lipids/water problem are starting to appear in the literature.21 Valuable information on the system dynamics is provided also by neutron spin echo (NSE),22 measuring the intermediate scattering function F (Q, t) using a beam of spin polarised neutrons. To a large extent, the equilibrium size and shape of liposomes can be determined also by a table-top apparatus based on dynamic light scattering (DLS),23 or by simple varieties of a drift tube, in which the driving force might be provided by mild centrifugation. Further valuable information is available on thermodynamic properties, also in this case made available by table-top equipment. Di↵erential scanning calorimetry, in particular, represents a very sensitive tool to identify phase changes, even in sub-systems such as micelles in solution, accounting for only a tiny fraction of the sample mass and thermal capacity.24 The method allows the investigation of the mechanism and kinetics of liposomes’ fusion, with implications in drug delivery and cyto7


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toxycity studies.25 Quartz crystal microbalances with dissipation monitoring provide information on relaxation processes.26 The well known Langmuir-Blodgett technique represents an auxiliary approach to prepare samples and an intriguing tool to analyse an extended set of thermodynamic properties of lipid monolayers floating on water, whose properties can also be a↵ected by the addition of RTILs. Representative results from this low complexity apparatus are reported in Ref. 27. A technique posed to acquire a larger role is certainly atomic force microscopy (AFM), that, after a few pioneering applications,15 has been used sparingly to characterise the e↵ect of RTIL on biomembranes, or, more in general, on biomolecules. In the lipid bilayer and biomembrane context, AFM usually requires planar samples deposited on a solid support. AFM has the two-fold ability to measure the topography, and to characterise a variety of mechanical properties such as the bulk modulus of bilayers, or the force needed to punch a hole through them. The versatility of AFM is enhanced by the di↵erent modes of operation, that include contact and tapping mode. Both modes can be used to determine the surface topography. The latter mode provides information on elasticity and relaxation processes within the bilayer through the measurement of the dynamical response of the tip driven at frequency !0 by a piezoelectric oscillator joined to the cantilever. Resolution is not atomistic but very detailed information can be obtained by sophisticated deconvolution of a time dependent signal, or by functionalisation of the AFM tip, of the lipids molecules, or a combination of these. Functionalisation with anti-bodies, in particular, can add sensitivity to a variety of chemical groups. The broadly interdisciplinary character of the subject makes it relevant a panoply of other methods often borrowed from biology, which in several cases provide structural information, and sometimes measure dynamical properties. Examples are fluorescence correlation spectroscopy (FCS),28,29 fluorescence resonance energy transfer (FRET),23 , surface plasmon resonance,30 confocal scanning microscopy, scanning electron microscopy and NMR. This section on methods could not be complete without a short outline of computational approaches, represented primarily by molecular dynamics (MD) simulations. Applications to the systems of interest for our discussion are an outgrowth of the sprawling activity on simulation of bio-systems, whose impact is difficult to overestimate. MD consists of the integration of Newton’s equations of motion, based on an approximate description of the system potential energy surface. The efficient application of the method relies on standard simulation packages, highly optimised for large, parallel computers, often with an hybrid architecture involving traditional CPU’s as well as graphics processing units (GPU). The latter typically provide a significant speed increase at an acceptable cost. At present the great value of MD consists in its ability to follow the real-time evolution of systems, 8


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typically up to a few hundred ns. Perhaps more importantly, MD allows one to investigate the microscopic mechanisms underlying the experimental observations, that, in general, do not reach the length and time resolution of MD. Last but not least, the largest length scale accessible by MD starts to overlap with the range covered by AFM and by other mesoscopic imaging techniques. The most basic model underlying MD simulations represents the system as a collection of particles (atoms, in most cases) organised into molecules of fixed topology. The validity of the model relies on the wide transferability of bonding parameters across organic systems, that apparently also covers ionic species. General force fields, able to describe wide classes of organic and biological molecules are available, and models specialised to RTILs have been tuned and validated.31 The matching of di↵erent force fields, required to simulate heterogeneous systems made of RTILs, biomolecules and water, is a critical ingredient, whose reliability and accuracy is not sufficiently discussed. Often MD is carried out under equilibrium conditions. In such a case, it provides information on time averaged quantities such as thermodynamic properties and static structural correlations. MD trajectories, however, provide information also on the di↵usion coefficient for all species, from water to lipids and ions, on linear transport coefficients such as, for instance electrical (ionic) and thermal conductivity. Fluctuations of the bilayer around planarity allow one to compute the bilayer/water interfacial tension (which should vanish) and the bending rigidity. Accurate estimations however require long production runs (usually reached) and large bilayer cross-sections, and this is often not the case. Less often, MD is used to investigate genuine time-dependent properties, in a non-equilibrium setting. Given the oversimplification of the force field description of the potential energy surface, the success of MD is remarkable. Broadly speaking, results of MD simulations using standard force fields and simulation packages are qualitatively correct, and error bounds on the computed properties can be estimated fairly reliably. A fully quantitative description requires better approximations for the potential energy surface. Ab-initio methods might soon provide some help, but their extensive usage to simulate large, complex systems over ns time scales might take a few years to materialise. A special mention is deserved for advanced statistical mechanics methods devised to accelerate the exhaustive sampling of the system configuration space, which will acquire a rapidly growing role in the investigation of activated and thus rare events in complex systems such as RTILs and biomolecules.32

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EARLY STUDIES AND STATE OF THE ART

Because of their charge and amphiphilic character, RTIL compounds share properties with most biomolecules, but the similarity is perhaps more apparent with lipids. This fact, together with the idea that biomembranes are the first biological structure making contact with a foreign species, has directed the early interest towards the interaction of RTILs with phospholipid bilayers, seen as the simplest yet relevant model of biomembrane. In their liquid phase RTILs generally display2 high viscosity, non negligible ionic conductivity, good thermal conductivity, low flammability and, because of their high cohesion, remarkably low vapour pressure. From the biochemical point of view, a relevant aspect of RTILs might be nanostructuring,33 that produces an alternation of polar and apolar regions for the solvation of third species, with a characteristic length scale that might induce high selectivity in the interaction with macromolecules.34 As for other organic systems, RTILs may form a variety of phases, including several liquid crystal ones. Protic ionic liquids made of not overly asymmetric ions are known to form solid plastic phases,35 in which ions are bound to regularly spaced lattice sites, but can freely rotate. RTILs form both monoand bi-phasic mixtures with water, depending on the relative weight of the charged, polar and apolar portions of the ionic species. Even in the mono-phasic case, a variety of inhomogeneous conditions may occur, with RTILs giving origin to a variety of structures characteristic of amphiphilic species in water,20 including vesicles, micelles, extended or folded bilayers. Another phase of RTILs in water very relevant for biophysics is represented by microemulsions.36 On the other hand, lipids form vesicles and bilayers in pure ionic liquids.37 At first this observation might seem a mere curiosity, but it changes our understanding of the mechanisms underlying the stability of lipid vesicles, micelles and bilayers in water. The solvophobic and solvophilic e↵ect needed to stabilise the same structures in RTILs might arise from the hydrogen bonding network like in water. However, according to Ref. 38, it might also arise from the interplay of polar and non-polar moieties in the RTIL ions, representing a new stabilisation mechanism that might play a major role in the RTIL-biomolecule context. Many of those properties suggest the possibility of combining RTIL ions and phospholipids into a wide range of possible structures. More than anything else, however, the amphiphilic character of RTILs, shared with surfactants and detergents, points to a potential de-stabilising, or, at least, liquifying e↵ect of RTILs on self-assembled layered phospholipid structures. Most phospholipids found in biomembranes present a gel phase at relatively low T and a liquid phase at higher T , separated by the so-called main transition temperature, that often falls around

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FIG. 3: Results from neutron reflectometry (NR) on RTIL/phospholipid bilayers in water.13 ). (a): raw data from the NR measurement; (b) Density distribution of all distinct components (RTIL, lipid head, lipid tail, etc.) as a function of coordinate z perpendicular to the lipid bilayer; (c) Cartoon representation of the position and orientation of RTIL cations, phospholipid molecules and water. Such a detailed view is accessible only through MD simulations. Adapted from Ref. 13, and reproduced with permission from the publisher. room temperature. With only a few exceptions, studies of RTIL-phospholipid-water systems have been carried out in the liquid range of the lipid fraction, above the main transition temperature. A first characterisation of structural damage on 1,2-dielaidoylphosphocholine (DOPC) bilayers was obtained by monitoring the leakage of luminescent molecules from liposomes floating in water upon addition of RTIL based on 1-alkyl-3 methylimidazolium cations ([Cn C1 Im]+ ) of alkyl group Cn from C4 to C8 , in combination with a few di↵erent anions.15 All RTILs caused at least some leakage at all concentrations. The damage increases rapidly with increasing length n of the alkyl chain. For a given n, damage tends to increase with increasing concentration, but the critical concentration (CMC) at which RTILs cations form micelles by themselves, greatly a↵ects this dependence. On the one hand, the formation of micelles limits the number of free cations in solution, and thus represents a upper cut-o↵ concentration. On the other hand, micelles made of cations display an enhanced ability to peel o↵ patches of lipids from the bilayer, forming new micelles of mixed composition and greater stability. In this respect, the CMC represents a lower cut-o↵ concentration for extended damage to the bilayer. Which of these two mechanisms prevails is strongly system dependent. Anion hydrophobicity is a secondary parameter needed to assess the RTIL e↵ect on the bilayers, and the most hydrophobic among them, such as [Tf2 N] contribute at least slightly to the overall damage of the bilayer. In general, however, anions do not seem to contribute much to the picture, although they are required to enforce the system neutrality.

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FIG. 4: A microscopic view of [C4 C1 Im]+ cations in a POPC bilayer from molecular dynamics and force field modelling. [Cl] anions floating in the water interlayer are represented by the green particles. Taken from Ref. 17, and reproduced with permission from the publisher. It is important to remark that the e↵ect of adding alkali halide salts (LiCl, NaCl, KCl) to the solution, that causes only minimal leakage of luminescent molecules from liposomes, represents the baseline to assess the results in Ref. 15. To summarise, up to high concentration [C4 C1 Im]+ cations cause localised holes into DOPC bilayer, while [C8 C1 Im]+ causes the collapse of liposomes already at moderate concentrations. The trend as a function of n suggests that the observed disruption follows the incorporation of cations into the phospholipid bilayer, that becomes stronger with increasing size of the apolar moiety. It is tempting to relate this observation directly to the biological e↵ects of RTILs on cells, that also tend to increase with increasing size of the neutral portion of the cation. Further early studies39,40 introduced the usage of AFM into this subject. Topographical imaging of the damage on a 1,2-dielaidoylphosphatidylcholine (DEPC) bilayer supported on a solid substrate revealed that [C8 C1 Im]+ removes lipids from the bilayer, increasing the interfacial roughness to 2.8 nm (about the width of a lipid monolayer) compared to the 0.2 nm of the same bilayer in clean water. [Tf2 N] anions were shown to create pore-like defects, sufficient to cause permeability in the bilayer. These observation are again potentially very significant in the biophysical context, and for applications in drug delivery. The direct view of the incorporation and eventual location of RTIL cations in phospholipid bilayers had to wait for experimental measurements based on neutron reflectometry.13 This study paired two di↵erent phospholipid systems, i.e., 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC)

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and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), with two typical RTILs represented by 1-butyl-3-methyl-imidazolium chloride ([C4 C1 Im][Cl]) and choline chloride ([Chol][Cl]) in water solution at fairly high concentration. The choice of a highly soluble anion was meant to isolate the e↵ect of the cations, representing the only species able to interact strongly with the lipid fraction of the sample. The system being probed consists of a single bilayer deposited on a solid substrate, in contact with a reservoir of water in which electrolytes might be dissolved. The results (see Fig. 3) provide the confirmation of qualitative results from previous studies, adding much needed quantitative details to the picture. First of all, reflectometry confirms that cations such as [C4 C1 Im]+ and [Chol]+ are readily absorbed into the bilayers, but up to fairly high concentrations (0.5M) cause only limited disruption. The width of the bilayers shrinks slightly (⇠ 1 ˚ A) upon the cation’s absorption, despite the RTIL fraction representing ⇠ 5

10 % of the bilayer volume, implying a corresponding expansion of the

area per lipid. The distribution of molecules in real space, provided by the analysis of reflectivity data, shows that the peak of the RTIL density overlaps the neutral portion of the lipid bilayer, while the superposition of the RTIL distribution with the phospholipid heads is limited. Moreover, in the experiment of Ref. 13 only one of the two leaflets is directly exposed to the RTIL solution, and, not surprisingly, the distribution of absorbed RTIL reflects this asymmetry. In the POPC case, in particular, cations are confined to the exposed leaflet. In the DMPC case, instead, both leaflets are populated, although still with an asymmetric concentration. Absorption is irreversible, and a few % of cations (in volume) are still mixed in the lipid phase after rinsing the bilayer with pure water. The planar average of densities provided by reflectometry turns into a picture of atoms, ions and molecules (see Fig. 3 (c)) through molecular dynamics which, in addition to purely geometrical information, provided an approximate yet relevant quantification of time scales (see Fig. 4).17 In an early study devoted to POPC bilayer floating into water solutions of [C4 C1 Im][Cl], [C4 C1 Im][PF6 ] and [C4 C1 Im][Tf2 N] at 0.5M concentration, MD has captured [Cn C1 Im]+ cations entering the POPC bilayer. Absorption occurs spontaneously, it takes place within a relatively short time of the order of a ns, and cations enter tail first into the lipid range. In this way they are able to simultaneously solvate their hydrophobic tail into the non-polar range of the bilayer, while screening the cation charge by the carbonyl oxygen negative charge. Although the Coulomb energy is usually high, and greatly a↵ects the absorption process, the non-polar solvation contribution should not be neglected, as emphasised by the results of simulations for RTILs absorption in bilayers made of cholesterol,41 whose Coulombic signature is less pronounced than in the phospholipid case. A concurrent MD study42 considered the di↵erent membrane composition of healthy and cancerous 13


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cells, the latter having a sizeable concentration of anionic phospholipids such as phosphatidylserine in their outer leaflet, and also a 50% lower cholesterol concentration than healthy cells. The overall negative charge causes an increased affinity of [C8 C1 Im]+ for the membrane of cancerous cells, with the potential for enhanced disruption. This di↵erence from healthy cells provides the basis for di↵erential e↵ects, and thus the result is potentially of interest. To the best of our knowledge, this paper has been the first to report quantitative data for free energies of RTIL absorption into lipids. A third MD study from the pioneering stage43 also considered the mixing properties of POPC and RTILs based on [Cn C1 Im]+ cations, in combination with anions ([Cl] , [BF4 ] , [PF6 ] , [NTf2 ] ) of increasing hydrophobicity. The results confirm the incorporation of cations into the bilayer, and their de-stabilising e↵ect on the lipid structure, revealed through the increased lateral di↵usion coefficient of POPC, and especially through an increase of the roughness of the POPC/water interface. Umbrella sampling simulations estimate a free energy gain of ⇠ 30 kJ/mol for the absorption of [Cn C1 Im]+ into POPC at normal conditions. The results of these early studies have opened the way to a stream of ongoing research, which, as written in the introduction, we consider representative of the state of the art. A major recent contribution has been provided by X-ray reflectivity measurements14 on DPPC (1,2dipalmitoyl-sn-glycero-3-phosphocholine) bilayers supported by an anionic polymer cushion deposited on silicon and covered by a water solution of [C4 C1 Im][BF4 ]. Like other popular phospholipid varieties, DPPC presents a main transition around room temperature, separating a gel phase at low T from a genuine (2D) liquid phase at high T . The main transition temperature of DPPC is Tm = 42 C. Measurements were carried out for bilayers in the gel phase at T = 35 C and in the liquid phase at T = 48 C. The results confirm the disordering e↵ect of the RTIL on the lipid structure, and also confirm that it has to be attributed to the penetration of cations into the lipid range. There is also evidence of permeability of the bilayers to the RTIL cations, that in that way reach the negative charge of the polymer cushion. Geometrical parameters from X-ray reflectivity qualitatively agree with those of neutron reflectometry.13 In particular, the width of the bilayer decreases upon RTIL addition but the change from 47.4 ˚ A for clean water to 32.5 ˚ A for a bilayer in [C4 C1 Im][BF4 ] at 0.15 M concentration measured by X-rays is much higher than that measured by neutron reflectometry. A dependence of the results on the lipid chain configuration (ordered-disordered) was revealed by the measurements and pointed out in the paper. A popular interpretation of the bilayer shrinking upon incorporation of RTIL cations is based on the increasing tilt of phospholipid molecules due to the shape mismatch between lipids and RTIL cations. This e↵ect is highly lipid and cation dependent, thus agreement and disagreement 14


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FIG. 5: (a) Newly synthesised lipid-like cations; (b), (c), (d) Dependence of their interaction mechanism with phospholipid bilayers on the length of the cation tails. Adapted from Ref. 44 and reproduced with permission from the publisher. of shrinkage measured on di↵erent systems might not be very significant. SAXS on mono- and multi-lamellar eggPC vesicles in water solution also corroborates the shrinking inter-layer separation with increasing concentration of RTIL, represented by phosphonium cations and [Cl]

or acetate anions.45 The loss of ordering in the lipid layers with RTIL concentration is

apparent from the progressive loss of di↵raction peaks, until the transition of the system to low ordering structures. These results on phosphonium-based RTILs are particularly relevant since these compounds display higher toxicity than imidazolium-based RTILs. A quantitative comparison (not yet available) of toxicity and structural e↵ect on lipid bilayers could be highly illuminating. Remarkably, the quantitative change of inter-layer spacing (by 4 ˚ A )measured by SAXS is intermediate between those measured by neutron reflectometry (1 ˚ A ) and by X-ray reflectometry (10 ˚ A ). These di↵erences are likely to be due to di↵erences in composition and geometry in the systems considered by the three studies, but they might also partly reflect di↵erences in the reference surfaces that are measured by the di↵erent probes. Similar results are obtained again by SAXS on vesicles and confocal scanning microscopy on planar lipid bilayers made of ↵-PC and a few [Cn mim][Cl] ionic liquids.46 Also in this case, tilting and shrinking is high for short [Cn mim]+ cations, negligible for longer cations, while very short cations cross the lipid range without leaving behind any visible damage. These results anticipate those of Ref. 44 on di↵erent but related ions. Comparison of results from di↵erent measurements and simulations obviously should account for the density of RTIL ions on or into the (2D) lipid bilayer, distinct from the overall RTIL concentration over the entire (3D) system. The precise control of this 2D interfacial property, however, is uncertain, 15


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hampering a fully quantitative characterisation of these systems. QENS has the ability to estimate the displacement of lipids and water molecules on the ps to ns time scales. First results on the e↵ect of adding [C4 C1 Im][BF4 ] and [C12 C1 Im][BF4 ] to the water environment of DMPC bilayers show that the RTIL causes the enhancement of the lateral displacement and of the local ri-orientation of lipid molecules.21 It would be interesting to match this information with data on longer time scales reaching into the genuine di↵usion regime, accessible, for instance by NMR measurements (ms), or by neutron spin echo (NSE, µs). To the best of our knowledge, however, di↵usion data for ions, water and lipids on the µs to ms time scale are not available yet. The interplay of RTILs and lipids reaches a new hight in a recent study44,47 introducing newly synthesised cations based on the imidazolium ring, decorated by two alkane chains (4,5dialkylimidazolium) matching the basic structure of phospholipid molecules (See Fig. 5). Morphology and structure determination by DLS, confocal laser scanning microscopy, and fluorescence measurements show that long tail cations are incorporated into liposomes without major disruption of the lipid structure. Medium tail cations destabilise liposomes, possibly because of geometrical mismatch between the lipid and RTIL head size and length. Short tail cations are able to cross the bilayer without leaving behind major damage. The observed behaviour, thus, confirms the results of Ref. 46, but di↵ers somewhat from the traditional view of the e↵ect of 1-alkyl-3-methyl-imidazolium cations, whose disruptive power increases monotonically with increasing length of the alkyl-residue, at least up to about 16 carbon atoms. More importantly, according to these studies, the power to disrupt bilayers and the biological activity in this case do not evolve in parallel,48–50 since the shortest cations are by far the most cytotoxic compared to both medium and long-tail cations. These observations disconnect the disruption of bilayers by RTILs and their biological e↵ects. Up to now, this is the clearest indication that membrane disruption might not be the most general mechanism for cytotoxicity. The simulation activity continues at a steady rate, and recent studies51–53 contributed a wealth of new microscopic information on lipid bilayers and RTILs in water. Simulations of [C4 C1 Im][PF6 ] absorption in and on POPC in solution focused on dynamical properties such as water, RTIL and lipid di↵usion, and on mechanical properties such as interfacial tension between the bilayer and the electrolyte solution, and the bending rigidity of the bilayer. The interfacial tension vanishes at the conditions of simulation. The bending rigidity decreases upon RTIL addition, supporting the idea that the absorption of [C4 C1 Im]+ destabilises the bilayer. Bending rigidity, however, is a tiny quantity above the main transition, and its determination remains challenging. The peculiar ability of MD to focus on microscopic aspects is highlighted by the analysis of the density distribution of cations and anions across the the lipid / water interface in samples doped by [C4 C1 Im][Cl] and [C4 C1 Im][PF6 ] 16


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FIG. 6: Pictures from recent MD simulations. (a) Simulation snapshot showing [C4 C1 Im][PF6 ] ions on POPC in water; (b) Comparison of the density distribution of cations and anions in [C4 C1 Im][Cl] and in [C4 C1 Im][PF6 ]. The light shaded area corresponds to the density profile of lipid atoms. (c) Representative configuration of a cation in close contact with a POPC molecule. Adapted from Ref. 17, and reproduced with permission from the publisher. (See Fig. 6). A second molecular dynamics study54 analysed the absorption of [C8 C1 Im]+ into a lipid bilayer whose composition (64 POPE and 16 POPG) was devised to mimic the phospholipid fraction of a bacterial cell membrane. It had a sizable concentration of negatively charged lipids in the outer leaflet, which in the eukaryotic membrane is made predominantly by zwitterionic lipids. The study quantifies the partition of [C8 C1 Im]+ between water and the lipid fraction, finding that the concentration is 47 times higher in the lipid that in water. Such a high partition ratio also depends on the presence of anionic lipids. According to this study, the thickness of the bilayer is no longer 2D-homogeneous in the presence of the RTIL, being significantly thinner (by 6 ˚ A ) in proximity of absorbed [C8 C1 Im]+ ions. The [C8 C1 Im]+ ions within the bilayer are found to attract each other, provided they are bridged by an anionic phospholipid screening their charge. The free energy profile for cation pairs as a function of distance has a minimum of 5.9 kcal/mol with respect to dissociation, that is reached after overcoming an activation barrier of 10.1 kcal/mol. A third major simulation study51 was part of a further comprehensive and multi-approach investi17


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FIG. 7: Results from molecular dynamics simulations based on coarse grained modelling of [Cn C1 Im][Cl] on POPC bilayers. Adapted from Ref. 53, and reproduced with permission from the publisher. gation of the molecular mechanisms underlying the cytotoxicity of RTILs. MD simulations show that the asymmetric absorption of cations by the two leaflets of a bilayer is a major cause of disruption, since it generates strain which above a critical value is released by shedding micelles of mixed RTILlipid composition. This study is also one of the few (the other being Ref. 46) that investigates the role of the edge of the bilayer, that might help equalising the composition of the two leaflets, releasing strain. The description of large scale, long time features in water, lipid and RTIL systems requires sizeable increases in the reach of MD simulation, that, eventually will need to rely on new computational advances. One active research line concerns the development of coarse grained force fields, representing chemical groups by single particles. These, in turn, interact with ad-hoc force fields intended to reproduce the large scale properties of the system. To the best of our knowledge, the first application of coarse graining to water, lipids and RTIL interfaces has been reported in Ref. 53 (See Fig. 7), achieving a significant extension of the length scale covered by simulation. An additional advantage of coarse graining is an apparent speed up of the system dynamics, that contributes to extending also the time scale. In this way, phenomena such as large amplitude, long wavelength fluctuations were observed, reaching up to the regime of transformations of the bilayer morphology. These included the budding of lipid-RTIL micelles, that might be an important step in the collapse of bilayers. Seen in reverse, this process could also be a step in the formation of bilayers on a solid support out of micelles floating in solution. A note of caution concerns the dynamical speed up o↵ered by coarse graining, that might not be the same for di↵erent types of the representing coarse grained particles. Thus, applying coarse graining to systems made of molecules of di↵erent size, complexity and rather di↵erent chemical properties such as water, RTIL and lipids, still represents a significant challenge, especially in the interpretation of dynamical properties. 18


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A new class of solvents closely related to RTILs is represented by the so-called deep eutectic solvents (DES).55 The analysis of their interaction with POPC lipid bilayers has been investigated by molecular dynamics simulations.56 The results show some similarity with the RTIL case, but reveal also important di↵erences, due to the neutral species that complete the formulation of DES. These neutral molecules act as donors in strong hydrogen bonds, and compete with cations for suitable absorption sites within the lipid layer. The disruption of the phospholipid bilayer structure is less severe than in the pure RTIL case, with a clear dependence on the hydrophobicity of the neutral hydrogen bond donor species in the DES. A di↵erent research line only indirectly connected to biomembranes concerns the characterisation of lipid monolayer at the air/water interface, following the well known Langmuir-Blodgett approach. RTILs can be added to the water side of the interface, and change the monolayer equation of state represented by the system isotherms, measuring the monolayer area versus the in-plane (2D) pressure.27 Results on monolayers reflect and extend those from bilayers, and enjoy the refreshing properties of being accessible using relatively simple table-top equipment.

APPLICATION ASPECTS OF RTILS / BIOMEMBRANES SYSTEMS

The systematic investigation of chemical physics properties of water, lipids and RTIL systems, eventually extending to real life biomembranes, provides the basis and the support for the ongoing development of RTIL applications in pharmacology and bio-medicine. Since these two aspects represent a growing portion of research on the topic of our Feature Article, we discuss them in some more detail in the following two subsections.

Pharmacology of RTILs

Our first expectation for the future is that pharmacology will grow increasingly significant in RTIL research. The progression of this sub-field to full maturity is documented in comprehensive reviews such as Ref. 19. For the sake of definiteness, our discussion is limited to those aspects of the RTIL pharmacology that are directly related to their interaction with biomembranes. The basis of this pharmacology is the di↵erence in the membrane structure and properties of different cells, such as bacteria and eukaryotic cells, or healthy and diseased cells, such as, for instance, cancerous cells. An obvious example that might illustrate the point is the di↵erent phospholipid composition of these membranes. The outer leaflet of the biomembrane surrounding healthy eukary-

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otic cells has a high fraction of POPC, and is predominantly neutral. The same membrane leaflet has a clear anionic character in bacteria and in cancer cells, due to the presence, for instance, of phosphatidylglycerol and phosphatidic acid in bacteria,57 or phosphatidylserine in cancer cells. These di↵erences, coupled to the inherent versatility of RTILs, confer selectivity to the RTIL-biomembrane interactions, opening the way to a↵ect the behaviour and eventual fate of cells. Last, but not least, one could also consider that phospholipid-based biomembranes surround a variety of cellular organelles (nucleus, mitochondria, chloroplast, ribosomes, vacuoles, etc.). Each of these membranes has unique structure and composition, that provides a basis for the selective interaction of RTILs with sub-cellular organelles. The factual counterpart of these conceptual remarks is that several RTILs display a marked biological activity, including, in particular, generic cytotoxicity and a broad antibacterial activity, covering both Gram-positive and Gram-negative bacteria, but also mycobacteria and fungi. Such properties are shared by several RTIL families, including those based on imidazolium,58,59 pyridinium,60 ammonium and phosphonium. Empirical evidence shows that the mechanism of action is not unique, since in some cases cytotoxicity and bactericidal activity manifest themselves through the collapse of cells, following the lysis of their outer membrane. In other cases the RTIL ions interfere with basic cellular processes, or trigger the apoptosis stage. It might be useful to remark that the bactericidal activity is not the same as generic toxicity. Pyridinium salts, in particular, kill bacteria without damaging mammalian cells.60 Needless to say, this is an essential property for any pharmaceutical application. At present, prediction of cytotoxicity and bactericidal ability is carried out almost exclusively by quantitative structureactivity relationship (QSAR) modelling.8 To the best of our knowledge, no comprehensive data base of either properties is currently maintained. Cytotoxicity and bactericidal activity of RTILs are not unexpected. As noted in Ref. 12, many known antibiotics are cationic, and are formulated as salts, although not classified as RTILs because of medium-high or unknown melting point. The activity of antimicrobial peptides depends strongly on their electrostatic signature,61,62 which drives their first interaction with the outer membrane of bacteria. On the other hand, quaternary ammonium cations have been known for some time for their anti-microbial properties.63 Similar properties are displayed by quaternary phosphonium cations,64 with a special mention for a phosphonium di-cation compound,65 whose activity depends on the length of the alkane chain joining the two cationic terminations (see Fig. 8). This compound is particularly remarkable because it is active against Gram-negative bacteria, whose external wall includes a layer made of a peptidoglycan gel. The first puzzle, therefore, is how the phosphonium di-cation crosses this first protective barrier. The second remarkable property of the phosphonium di-cation is that it 20


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FIG. 8: Structural formula of the phosphonium di-cation. is non-toxic towards epithelial cells, while mono-phosphonium varieties are toxic to both bacteria and epithelial cells. The bactericidal activity of pure RTILs usually is retained in water solution where molecules are nominally dissociated, implying that the bactericidal ability is a property of one or both ions. In practice, the prevalent role of cations is known, and at least partly explained by the results of the previous sections. The most active RTILs share a common structural motif, being made of a sizeable cationic core (imidazolium, pyridinium, quaternary ammonium) and one or more apolar tails. Because of their relevance in this context, let us focus for a moment on the case of imidazolium-based RTILs. For these compounds, the most clear indication of a membrane mechanism for cell toxicity is the correlation (up to about 16 carbon groups) of antibacterial activity and size of the lipo-philic domain of the cation. This observation, however, does not represent an unambiguous conclusion to the story. Di↵erent or concurrent mechanisms could be at play, concerning the coagulation of the cytoplasm, or the inhibition of enzymes (such as acetylcholinesterase),66,67 interfering with crucial energy or selfrepair processes. The correlation with lipophilicity, in fact might point to a preliminary, necessary but not unique step, represented by the crossing of the membrane by cations, which is favored by their affinity for phospholipids. Only those cations that cross the membrane could progress to the decisive step such as forming stable complexes with nucleic acids, whose electrostatic signature is predominantly anionic. If interpretations based on electrostatic charge are correct, then geminal cations65,68 should show higher activity. This has been confirmed, and extended to trigeminal ionic liquids.69 Additional relevant information is that the anions of popular RTILs of known antibacterial activity do not seem to be involved, but new dual-activity compounds have been developed, in which both cations and anions do have an e↵ect. To this aim, anions incorporate intrinsically antimicrobial species such as silver and copper ions.70 Double activity is a possible approach against drug resistance. An interesting point being debated concerns the relation between cytotoxicity and anti-electrostatic properties.71 . Anti-electrostatic agents are defined as species able to prevent or dissolve the accumulation of charge at surfaces, and this property is characterised by measuring surface electrical resistance, 21


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as well as the maximum voltage and the time to neutrality upon charging by corona discharge. In principle, RTIL ions are strong anti-electrostatic agents, since they turn the water environment into an electrical conductor. In practice their anti-electrostatic capability is moderated by their interaction with the surface of the phospholipid bilayer. Somewhat surprisingly, and certainly unexplained, this property shows positive correlation with antibacterial activity, at least in the case of cholinium-based RTILs.71 A similar correlation, however, is not so clear for other families of RTILs, such as those based on phosphonium. Because of our chemical-physics bias, our discussion focused on the RTIL interaction with the lipid fraction of biomembranes. Their interaction with the protein fraction, however, might be even more significant for pharmaceutical applications, and already now it might play a role in the acquired resistance of bacteria to RTIL compounds, similar to what is known for general antibiotics resistance. Although largely representative, antibacterial activity is not the only relevant biological property of RTILs. In perspective, their anti-cancer role might become even more important, as suggested by the rapid growth of this subfield.19,49,50 Up to now, the knowledge of the underlying microscopic mechanisms is even more rudimentary in the case of anti-cancer than in the case of the antibacterial activity. Since this knowledge could boost the development of new approaches to cancer, the investigation of these mechanisms by the methods discussed in the previous sections is a new wide field open for research. Deep eutectic solvents might be the next important players also in the pharmacology context, exploiting the interplay of ionic and hydrogen bonding. Up to now the investigation of their biological activity has been devoted primarily to phosphonium-base DES, but the scope of these investigations will certainly expand to a wider variety of compounds.

Biomedical applications

The direct API significance of selected RTIL compounds is complemented by two important side topics, concerning the potential of RTILs as adjuvants in the formulation of drugs, and the many possible applications of RTILs in biomedicine. As already pointed out in Ref. 12, for instance, the basic chemical-physics properties of RTILs provide improvements in terms of solubility, and thus of bio-availability, or slow release, when the RTIL forms micelles or vesicles in the physiological solution. These considerations connect the biochemical aspects of the RTIL pharmacology to the broad context of system biology, which analyses, among many other things, how a metabolite (the RTIL ions, in this case) becomes available to an organism.

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FIG. 9: Scanning electron microscopy micrographs of biofilm in the endotracheal tubes. Taken from Ref. 72, and reproduced with permission from the publisher. Perhaps more importantly, the liquid phase of RTILs might prevent problems with the polymorphism of drugs, that represents a constant challenge to drug development.73 Notice that DES enjoy similar properties and might find similar applications. RTIL nanostructuring together with the virtually unlimited choice of functional additions to the basic structure, o↵er many opportunities in drug delivery. Even a moderate permeabilisation of biomembranes could favour the penetration of drug molecules through cellular boundaries.74 Functionalisation of RTILs could result in new families of pro-drugs, releasing the actual API only after crossing otherwise impassable membranes. Besides strict pharmacology, the applications of biomembranes and RTILs in bio-medicine are so many that here we can mention only two examples, concerning the control of biofilms, and the visualisation of cells by scanning electron microscopy (SEM). Biofilms76 are a topic of great medical significance, since most bacteria exist as free-floating (planktonic form) organisms, or as biofilms, consisting of sessile bacterial congregations embedded into a polymeric extra-cellular matrix produced by the bacteria themselves (see Fig. 9). Bacterial biofilms play a major role in di↵using a host of chronic infections, from pneumonia to endocarditis. Their eradication is hampered by the matrix, that also provides a medium favourable for the transmission of genetic resistance. Furthermore, bacterial biofilms tend to colonise medical devices from catheters to implanted tubes, posing an additional threat. RTILs of known anti-bacterial activity have been tested with relatively positive results against biofilms. Representative studies are reported in Ref. 77–79 Removing biofilms from medical devices and producing low-fouling surfaces is required, for instance, 23


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FIG. 10: SEM images of pollen pre-treated with RTIL. A) Primula juliae, B) Anemone coronaria, C) Leucoglossum paludosum, and D) Lathyrus odoratus. Taken from Ref. 75, and reproduced with permission from the publisher.

FIG. 11: Identification of cancer cells by AFM measurement of their sti↵ness. RTILs a↵ects the rigidity of cell boundaries, and could be used as contrasting agent in dagnostic applications. Taken from Ref. 80, and reproduced with permission from the publisher. for advanced diagnostic techniques. Cancer screening is often based on detecting markers in bio-fluids, using, for instance, surface plasmon resonance (SPR). This, however, is hampered by the fouling of all surfaces exposed to biofluids. The problem is particularly relevant when the test is made by lysing cells, with the release of a wide variety of macromolecules, including lipids and nucleic acids. Spreading ionic liquids on the surface of SPR greatly improves the ability of the method to detect markers binding anti-bodies at the surface.81 ( see Fig. 1 in Ref. 81).

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Biofilms in skin infections protect themselves by hiding below the so-called stratum corneum (SC), that represents the outer layer of the skin and acts as a barrier against drug penetration. Screening of a broad set of RTILs has identified choline-geranate as a most promising compound for transdermal drug delivery based on its verified ability to penetrate the stratum corneum and check the growth of the bio-film without causing adverse skin reactions.74,82 Scanning electron microscopy (SEM) is a fundamental technique to visualise cells and cellular organelles. Imaging, however, requires high vacuum, and samples need to be dry. Moreover, to avoid the accumulation of charge from the electron beam, samples need to be electrically conductive. For all these reasons, SEM imaging requires first the replacement of water by some suitable solvent, and the deposition by sputtering of a thin metal film on the sample surface. This long and complex preparation could be replaced by covering the sample with a thin RTIL film, that, at the same time prevents evaporation from wet samples, and provides sufficient surface conductivity83 for SEM imaging (See Fig. 10).75 The simplified preparation is suitable, for instance, for imaging biological samples as delicate as neural cells with their dendrites and meter long axons.84 The list of RTIL applications to SEM is growing rapidly and includes, in particular, hydrous biological samples from bacteria85 to crustacean,86 or the imaging of biofilms,87 whose structure, made predominantly by water, might be modified by dehydration. Remarkably, images of RTIL-treated biofilms look di↵erent from those obtained from traditional (SEM) protocols. These results are compatible with the SEM imaging at ambient temperature of hydrated RTIL structures with up to 20% water (volume) without prior preparation of the sample surface.88 Thus, pre-treatment of samples by RTILs could drastically reduce the preparation time, and expand the application of SEM in research and especially in diagnostics. Electron conductivity by ionic conductors such as RTILs will play a role also in the discussion of bio-mimetic photo-voltaic devices that is the topic of the last sub-section.

PLOTTING THE COURSE OF NOVEL APPLICATIONS

The investigation of systems made of water solutions of organic electrolytes solvating lipid bilayers or biomembranes has established itself as a sizeable research area, based and supported by a solid background of chemical physics studies, and pursuing a broad range of research objectives especially in the context of pharmacology and of biomedicine. Research, however, is also about the unplanned and the unforeseen. By definition these topics cannot be listed, therefore we proceed by presenting a few examples, selected from the new activitis

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FIG. 12: Graphical summary of RTIL applications in controlling cells’ rheology (upper panels) and in bio-mimetic photo-voltaic devices (lower panel). Exploratory stages on these topics are under way. in the authors’ group. In the first of the two examples the view of the previous sections is expanded from membranes to whole cells and even tissues. The second example concerns the application of water, RTIL and biomembrane interfaces to energy devices, with potential applications in in-vivo power generation. The two research lines are summarised in Fig. 12.

RTILs and the rheology of cells

The cell boundary is known to operate a host of processes, from the adhesion to a substrate and the joining of cells into tissues, to intercellular communications through transduction. Adhesion, for instance, is due to a variety of proteins protruding from cell’s membranes, reaching out into a substrate, into another cell, or into the gel-like environment (extra-cellular matrix) among cells. RTILs could a↵ect the properties of each of these components, possibly enhancing the adhesion of cells, or preventing their sticking to a surface or to each other. This last task could use the well known lubrication properties of many RTILs.2 Thus, adhesion or the lack thereof a↵ects cells’ crawling on surfaces, and their rheology in tissues. All these aspects have diagnostic and medical relevance,89 and could provide open fields of applications for RTILs. Biomembranes and the cytoskeleton interact with each other to give the cell its shape, determining in this way the elastic properties (Young’s modulus, for instance) of whole cells. The interest in these

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FIG. 13: AFM topography scans showing the e↵ect of adding [C4 C1 Im][Cl] at 0.1M concentration on osteoblast cells. Panel (a): before the addition of [C4 C1 Im][Cl]; panel (b): after the addition of [C4 C1 Im][Cl]. properties is growing because of the availability of approaches to measure them, including first of all AFM, but also magnetic and optical tweezers, cell stretchers, microplates, cell poking.90 The results show that cells behave as highly viscoelastic systems, i.e., their mechanical properties are strongly non linear, being dependent on the time and size scale of the perturbation. Moreover, cells turn out to be very soft materials, despite being made of components such as the cytoskeleton and the membrane whose softness is much less marked. Remarkably, cancerous cells are even much softer (up to 70%, Ref. 91) than healthy cells. Softness is important to enhance the mobility of cancer cells into tissue, favouring the spread of cancer through metastasis. Also in this case, the di↵erence between healthy and diseased cells is the basis for exploiting mechanical properties in diagnostics and in treatment.92 Moreover, and more importantly for our discussion, properties of this kind can be modified through the addition of RTILs, especially because of their privileged interaction with the cell membrane. Screening the endless variety of RTILs with di↵erent functionalisation is bound to produce compounds able to a↵ect this di↵erence, (i) reducing it and thus negating the advantage of cancer cells with respect to mobility, or (ii) amplifying it, using RTIL ions as a contrasting agent with possible applications in diagnostics (See Fig. 11). To support the feasibility of these approaches, recent data on the e↵ect of [C4 C1 Im][Cl] on the morphology of osteoblast cells are reported in Fig. 13, while the results of an AFM scan of the local elastic modulus measured again before and after the addition of [C4 C1 Im][Cl] on osteoblast cells are shown in Fig. 14. These figures have been obtained within our group, and are representative of the new directions that we intend to pursue in our activity.

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FIG. 14: AFM scan of the local elastic modulus of osteoblast cells before (panel (a)) and after the addition of [C4 C1 Im][Cl] at 0.1M concentration, panel (b). This intriguing picture, admittedly rather speculative, becomes even more so by considering new classes of RTILs, such as the new family of magnetic ionic liquids (MILs), consisting of organic ionic compounds whose (usually) anion carries a net magnetic moment, since it consists of a complex of a metal ion of sizeable spin.93 Examples of MIL anions include [FeCl4 ] , [MnBr3 ] , [CoCl3 ] . Only a few studies have been published so far on these systems, and none of them concerned biological aspects. Up to now, even the precise nature of the magnetic state is still unclear, and, since anisotropy forces are tiny, it is not known how the orientation of the magnetic moment a↵ects the structure and dynamics of ions in the liquid phase at room temperature, although it has been conjectured that low molecular symmetry and itinerant exchange interactions might play a role.94 To be precise, none of these compounds shows long range magnetic ordering (ferro-, anti-ferro-, ferric-, etc) above temperatures of a few K. However, their paramagnetic response to fields in the ⇠ 5 T range apparently modifies their properties,95 and a↵ects their structure and dynamics. This, in turn, is bound to a↵ect their interactions with bio-systems. To amplify this e↵ect, one needs to enhance the affinity of magnetic MILs anions with biomembranes, or to transfer the magnetic functionality to cations, whose affinity for biomembranes is already apparent. Both options should be available, given the versatility of RTILs. Once this tuning is achieved, the application of a magnetic field to a responsive fluid such as MILs could change the interaction of cells with the environment, through forces emanating from their boundaries. The control of the cells’ rheology achieved in this way could have great medical and especially diagnostic value, with little chances of adverse e↵ects, since, apart from metal ions, biological matter is diamagnetic, displaying little interaction with magnetic fields. The feasibility of this approach is supported by the outcome of many studies aiming at similar results using nanoparticles driven into cells by magnetic forces.96 In the MILs approach the magnetic

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FIG. 15: Drift of DMPC liposomes of 100 nm diameter floating in a water solution of magnetic ionic liquid ([bmim][FeCl4 ] at 0.1M) under the e↵ect of an external magnetic field of ⇠ 1 Tesla. agent is spread throughout the system, or, more precisely, it permeates the biomembrane of cells. In this way, magnetic forces might be locally less intense since they are far more pervasive. Preliminary experiments carried out in our group confirm the possibility of manipulating liposomes in [bmim]FeCl4 ] at 0.1 M concentration using magnetic fields of about 1 Tesla (see Fig. 15).

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Bio-mimetic membranes in energy technology

Eventually the most significant developments will arise from considering biomembranes in their full complexity, consisting of an impermeable lipid bilayer with enzymes embedded in it, carrying out a variety of specialised tasks from pumping species across the bilayer to catalysing chemical reactions. In this view, biomembranes might represent the ultimate bio-nano factory, with RTILs representing the technical fluids operating them. RTILs, in particular, could tune the fluidity and permeability of the lipid bilayer, a↵ect the conformation and solvation of membrane proteins into the lipid phase, and possibly optimise their enzymatic throughput. A proof of principles for all these e↵ects is provided by recent works97–99 on gramicidine A, embedded into a model membrane deposited onto a solid support (See Fig. 16). As an example of research plan along these lines, let us consider the case of a bio-mimetic photovoltaic device. The plan is based on the fact that the all-important photosynthetic process, of which photovoltaic is a first essential stage, is in fact a membrane process, in which both the lipid and the protein components of the bio-membrane play an essential role. The basic conversion of a photon into an electron excited above the conduction band occurs at photosystems made of (usually) transmembrane proteins and antenna species. The lipid bilayer, however, is required to orient the trans-membrane proteins, and to force the direction for the propagation of the photo-electron. The high efficiency of photosynthesis and of its photovoltaic stage has been perfected by billions of years of evolution, motivating the search for bio-mimetic and hybrid analogues. Functional photovoltaic interfaces are routinely made by depositing natural photosystems, i.e., PSI absorbing at 700 nm, or PSII, absorbing at 840 nm on a solid electrode. Artificial photosystems are also extensively used, based on a wider variety of photo-chromic proteins such as GFP or cytochrome c, or even synthetic or modified proteins. The correct orientation of the proteins and a suitable level of conformational flexibility can be obtained by mimicking nature, i.e., by embedding proteins into a lipid bilayer deposited onto a solid conducting substrate. Up to now, however, the composite nature of artificial devices, the matching of a soft layer with a metal electrode, and especially the inclusion of water confer to bio-mimetic devices an intrinsic low stability and poor reliability. The available knowledge on the photovoltaic processes and the accumulated experience on photovoltaic bio-mimetic systems combine with the experimental evidence of RTIL-lipids and RTIL-protein affinity pointing to new strategies to optimise the efficiency and durability of bio-mimetic devices. The full exploitation of their potential however, requires a detailed knowledge of the distribution and interactions of RTILs and enzymes in a membrane-like environment. In this context, the results of

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FIG. 16: Cartoon representation of the stabilisation e↵ect of RTIL ions on the structure and ionic conductivity of gramicidin-A embedded into a supported phospholipid bilayer. Adapted from Ref. 97, and reproduced with permission from the publisher. Refs. 97,98 & 99 detailing the changes of conformation and dynamics in the gramicidine ion channel upon addition of [C10 C1 Im][Cl] (Fig. 16) become highly relevant. Because of the similarity of systems and aims, these references provide a clear blueprint for several promising approaches to the fabrication of photovoltaic devices. Given the mild surfactant activity of RTILs, improved reliability is expected more from increasing the fluidity ad thus the self-repair ability of the lipid fraction, than from the stabilisation of a static configuration. A major aim of research is to identify the RTIL and the conditions such that the device can operate without water. Since water-less activity of enzymes in RTILs is rather widespread,100 replacing water with RTILs is mainly a matter of choosing the right compound. Lacking a predictive approach, high throughput screening is now the only available option. The replacement of water with an RTIL would already drastically enhance durability and reliability, since it changes a volatile solvent such as water with RTILs of very low vapour pressure. The RTIL choice could also ease the (major) problem of electrically connecting the interface to the external circuit. First of all, the choice of a semiconducting RTIL able to act as a transporter of energised electrons could endow the system with an intrinsic conductivity of electrons83 , opening the way to the direct electrical connection to an external circuit. This possibility, together with the ability of operating the device in anhydrous conditions would give rise to a whole new generation of bio-mimetic hybrid devices. A di↵erent route to electrical connectivity could be represented by depositing a nanometer-scale layer of metal on the (now) anhydrous surface, cushioned by a thin RTIL layer This possibility is demonstrated in Ref. 101. 31


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Devices of this kind are unlikely to become major players in power generation. Their soft and potentially bio-compatible character, however, could open a niche opportunity for in-vivo power generation devices.102

CONCLUSIVE REMARKS

At first sight, the interaction of biomembranes and RTILs might be perceived as a niche subject, of interest for a specialised community at the crossing point of the biochemistry of cells and the chemicalphysics of organic ionic liquids of low melting temperature. A closer look reveals a robust research topic, bustling with activity, standing on a solid foundation of recent experimental and computational results, and permeated by the expectation of incipient new discoveries and innovative applications. The present Feature Article aims at providing at first a faithful view of this field, summarising past studies, taking a snapshot of current activities, and making an attempt to predict the short term evolution of research. The variety of results summarised in our paper are currently giving origin to a number of further lines of investigation, that are progressively being explored and expanded by the community. A development that in many ways is already with us is the growth of pharmaceutical research on RTILs, based on their interaction with biomembranes. Because of the obvious interest in API for new drugs, and because of the urgent need for new antibiotics, this field is growing very rapidly, and is being expanded to cover the anti-cancer potential of RTILs. This last topic will undoubtedly require consideration of the protein fraction of biomembranes and of the related enzymatic activity, opening a new front of biochemical and chemical physics research. Researchers in this field are aware that in vitro activity of a compound is only the first uncertain step in the development of a successful drug, but even a statistical modest success rate in this e↵ort could provide sizeable advantages for health treatment and for society. Besides these strictly pharmacology considerations, RTILs hold the promise for a vast number of applications in bio-medicine. In our discussion we could only illustrate these concepts by a few examples, concerning primarily the all-important control of biofilms, and the usage of RTILs in SEM imaging. The RTIL ability to control biofilms, in particular, is already validated, and might soon find practical applications. First but already impressive results from the usage of RTILs in the preparation of biological samples for SEM imaging have been reported in the literature. Developments of this kind, expanding the reach of SEM and improving its quality have great application potential in diagnostics, once coupled to automatic high-throughput equipment.

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Looking back at our discussion we realise that the focus has steadily shifted from early concerns on safety to the heightened expectation for new discoveries and promising developments in health care and in nano-bio technologies. This shift is partly due to the inherent optimism of research, but it also reflects the reality of systems such as RTILs whose future role in health care and nano technology could surpass our current imagination, driven by their privileged interactions with biological structures and biomembranes in particular.

OUTLOOK

The applications listed in the previous section are likely to promote further extensive research on biomembranes and RTILs. An equally important role, however, will be played by basic investigations, addressing a few key questions whose answer could greatly ease the parallel development of applications. The first crucial question concerns the mechanism by which RTILs kill cells. While it is apparent that the collapse of the biomembrane is the major mechanism at high salt concentration, it is equally clear that other mechanisms are at play at lower concentrations, or with species able to cross the lipid bilayer barrier without permanently disrupting it. Likely possibilities include the blocking or over-activation of enzymes, as well as a direct interaction with the DNA/RNA machinery that repairs or updates the enzyme library of cells, driven by the large negative charge of nucleic acids. The rapid increase of cytotoxicity with increase charge of the RTIL cation points directly to the last of these possibilities. Two related issues concern (1) the search and characterisation of multi-valent RTIL cations and (2) the detailed investigation of the interaction of RTIL ions with peripheral and integral membrane proteins. A few studies have already been published on the latter issues, an the rate of new publication on this topic is on the rise. The task of exploring all relevant cases, however, is so large to keep the still small RTIL-biochemistry community busy for years to come. Less attention has been devoted to the first issue, and most RTIL compounds characterised and described in the literature are made of mono-valent ions. On the one hand, increasing the ionic charge is likely to increase the melting temperature of the pure compound above the conventional limit for RTILs of 100 C. On the other hand, most applications in the biological context concern systems in a water solution, that gives fluid-like conditions also to compounds of relatively high melting point. A key issue of interest for bio-nano technology is the integration of biomembranes and of the en-

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zymes they carry with inorganic components, giving origin to hybrid structure such as those required for electrochemical applications. In this respect, RTILs have a great potential, because of the already proven ability of selected RTILs to combine ionic as well as electronic conductivity, displaying properties of organic semiconductors. A last major issue concerns extending the scope of RTIL applications to their theoretical limit, exploiting the vast number and chemical variety characteristic of this class of compounds. Until now, the exploration of new applications has often been limited to the most popular compounds, somewhat limiting the range of opportunities provided by RTILs. These issues, and closely related ones such as endowing RTILs with luminescence properties, or exploiting field responsive magnetic ionic liquids, point to a vast new field of fundamental research holding the promise of equally exciting developments in applications.

ACKNOWLEDGMENTS: A.B. acknowledges support from (i) The European Commission under the Marie-Curie Fellowship Grants HYDRA (No. 301463) and PSI-FELLOW (No. 290605), and (ii) Science Foundation Ireland (SFI) under the Start Investigator Research Grant 15-SIRG-3538. A.B. acknowledges the additional support provided by the School of Physics and the School of Chemistry, University College Dublin, Ireland, and the Laboratory for Neutron Scattering, Paul Scherrer Institute, Switzerland. A.B. thanks Prof. Brian Rodriguez for the access to the UCD AFM labs and for fruitful discussions, and Mrs. Pallavi Kumari for providing Fig. 12 and 13. We thank Prof. Padraig Dunne at the School of Physics, University College Dublin, for a careful reading of the manuscript.

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TOC:


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Biography Antonio Benedetto:

Antonio Benedetto was born in Padua, Italy, in August 1984. He is a Science Foundation Ireland PI based at the School of Physics, University College Dublin (Ireland), and an Associate at the Laboratory for Neutron Scattering, Paul Scherrer Institut in Switzerland. As an invited scientists, he regularly visits several of the largest neutron facilities worldwide, i.e. Institute Laue-Langevin (FR); Rutherford Appleton Laboratory (UK); National Institute of Standards and Technology (USA); Oak Ridge National Laboratory (USA); and Heinz-Maier-Leibnitz Zentrum (DE). In 2011, he obtained his PhD in Physics with the mention of Doctor Europaeus (University of Messina, Italy) defending a thesis on proteins’ dynamics with the experimental part carried out at the Institute Lau-Langevin in Grenoble, France. His PhD thesis has been awarded two national prizes by the Italian Chemical Society and by the Italian Biophysical Society, respectively. In March 2012, supported by an individual Endeavour Research Fellowship of the Australian Government, he moved “Down Under” joining the Bragg Institute, and the University of Sydney. In September 2012, supported by a Marie Curie Individual Fellowship of the European Commission, he joined the School of Physics, University College Dublin (UCD) in Ireland. At this time, he started to work on the interaction between ionic liquids and phospholipid bilayers. In January 2015, supported by another Marie Curie Fellowship, he moved to the Paul Scherrer Institute (PSI) in Switzerland. At PSI, he extended his interest in ionic liquids towards their effect on biomolecules at large. In January 2017, he came back to UCD School of Physics supported by a Start Investigator Research Grant awarded by Science Foundation Ireland. More info available at www.antoniobenedetto.eu


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Pietro Ballone received his Physics Degree from Scuola Normale Superiore, Pisa, in 1981 and his PhD from the International School of Advanced Studies (SISSA), Trieste, Italy, in 1986. Over the years, he has been Associate Professor at the Department of Physics, University of Messina from 1998 to 2005, and Full Professor (Chair in Atomistic Simulation) at Queen’s University of Belfast from 2005 to 2012. At present, he is a temporary researcher at the Italian Institute of Technology in Genova. His research interests include a variety of subjects in the chemical physics of soft condensed matter, that he investigates by computer simulation.

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Pietro Ballone:


Room-Temperature Ionic Liquids and Biomembranes: Setting the

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