Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit

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Imidazolium salts mimicking the structure of natural lipids exploit remarkable properties forming lamellar phases and giant vesicles Patrick Drücker, Andreas Rühling, David Grill, Da Wang, Annette Draeger, Volker Gerke, Frank Glorius, and Hans Joachim Galla Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03182 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Imidazolium salts mimicking the structure of natural lipids exploit remarkable properties forming lamellar phases and giant vesicles Patrick Drücker†‡, Andreas Rühling§, David Grill∥, Da Wang†, Annette Draeger‡, Volker Gerke∥, Frank Glorius§* and Hans-Joachim Galla†* †

Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Str. 2, D-48149 Münster, Germany ‡ Department of Cell Biology, Institute of Anatomy, University of Bern, Baltzerstrasse 2, 3000 Bern 9, Switzerland § Organic Chemistry Institute, University of Münster, Corrensstrasse 40, D-48149 Münster, Germany ∥Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, University of Münster, Von-Esmarch-Str. 56, D-48149 Münster, Germany

* [email protected]; [email protected]

Abstract Tailor-made ionic liquids based on imidazolium salts recently attracted high attention due to their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes was already reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (CnIMe·HI, n = 7, 11, 15) and 1,3-dibenzyl-4,5-dialkylimidazolium (Cn-IBn·HBr, n = 7, 11, 15) salts. Diverse alkyl chain lengths and headgroups differing in their steric demand were employed for membrane interface interaction with bilayer membranes imitating the cellular plasma membrane. Membrane hydration properties and domain fluidization were analyzed by fluorescent bilayer probes in direct comparison to established model membranes in a buffered aqueous environment, which resembles the salt content and pH of the cytosol of living cells. Membrane binding and insertion was analyzed by quartz crystal microbalance and confocal laser scanning microscopy. We show that short chain 4,5-dialkylimidazolium salts with a bulky headgroup were able to disintegrate membranes. Long chain imidazolium salts form bilayer membrane vesicles spontaneously and autonomously without addition of other lipids. These 4,5dialkylimidazolium salts are highly eligible for further biochemical engineering and drug delivery.

Introduction During the last decade the interest in N-heterocyclic carbenes (NHCs) has grown significantly, especially in the field of organometallic complexes, drugs, materials and in catalysis.1-7 However, little attention has been paid to their corresponding imidazolium salts and their potential to mimic lipid properties, which we now investigate to probe their interaction within biological membranes. Recently, SCHMITZER et al. used imidazolium and benzimidazolium salts as transmembrane anion transporters.8-11 In addition, GAYET et al. described vesicles of DPPC that can form in the presence of ionic liquids, which have their alkyl chains attached to the

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imidazolium-nitrogen atoms.12 Further, LIU et al. provided molecular dynamics (MD) simulations, revealing the formation of stable unilamellar vesicles of 1,3-dialkylimidazolium ionic liquids ([Cnmim]Br, n = 10, 12, 14) in aqueous solution.13 Experimentally it was shown for the first time, that single tail 1-alkyl-3-methylimidazolium cations exhibit pH dependent structural changes in an aqueous environment. These ionic liquids (ILs), [Cnmim]+ (n = 12, 14), could transform from micelles to vesicles and thus became valuable tools for drug delivery and biochemical engineering.14 Further, micelles of imidazolium bromides bearing an N-alkyl chain can also aggregate in an aqueous solution.15 The introduction of alkyl chains at the imidazolium backbone in the 4,5-position of N-heterocyclic carbene salts, leads to a high structural similarity to the hydrophobic part of naturally occurring lipids. Thus interactions with cellular membranes in vivo resulted remarkable properties such as enhanced cytotoxicity and anti-tumor activity.16,17 Furthermore, 4,5-dialkylimidazolium salts showed lipid interactions at the surfactant monolayer model in vitro.16, 17 This report expands the knowledge about imidazolium salts and their membrane interactions to bilayer interfaces. Giant unilamellar vesicles and surface tethered liposomes, which can mimic the shape and structure of cellular plasma membranes are employed. We include the consideration of visco-elastic properties and a comparison of binding characteristics to a neutral POPC and a charged POPC/POPS (4:1) vesicle model, which is probed by quartz crystal microbalance measurements in a physiological buffer. The different surface charge of these systems reveals further insights into the interaction with our positively charged imidazolium salts (Figure 1). Further, we characterized the membrane morphology and show that 4,5dialkylimidazolium salts are able to fluidize vesicle membranes. Confocal laser scanning microscopy revealed that C15-IMe·HI can also form bilayer membrane structures, without the addition of other lipids. The structure of our 4,5-alkyl-imidazolium salts has the advantage, that the alkyl chains which mimic the hydrophobic tail of a lipid and the nitrogen substituents which represent the lipid polar headgroup, can be modified independently on demand with respect to the headgroup size and tail length. Therefore, membrane binding in dependence of alkyl chain length and bulkiness of the headgroup could be probed and both were found to affect the membrane integration efficacy.

Figure 1: Lipophilic imidazolium salts used in this study.

Keywords


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Ionic liquid, bilayer membrane, giant unilamellar vesicle, imidazolium salt, N-heterocyclic carbene salt, membrane fluidization.

Abbreviations SM, sphingomyelin; NHC, N-heterocyclic carbene; IL, ionic liquid; MD, molecular dynamics; GUV, giant unilamellar vesicle; HBS, HEPES buffered saline; TBS, Tris-buffered saline; PBS, phosphate bufferet saline; DPH, 1,6-diphenyl-1,3,5-hexatriene; PVA, polyvinyl alcohol; CLSM, confocal laser scanning microscopy.

Materials and Methods Materials Imidazolium salt compounds: 1,3-dimethyl-4,5-diheptylimidazolium iodide (C7-IMe·HI), 1,3dimethyl-4,5-diundecyl-imidazolium iodide (C11-IMe·HI), 1,3-dimethyl-4,5dipentadecylimidazolium iodide (C15-IMe·HI), 1,3-dibenzyl-4,5-diheptylimidazolium bromide (C7-IBn·HBr), 1,3-dibenzyl-4,5-di- undecylimidazolium bromide (C11-IBn·HBr) 1,3-dibenzyl4,5-dipentadecylimidazolium bromide (C15-IBn·HBr) were synthesized as described earlier1, Laurdan, DPH, 16-Mercaptohexadecaneic Alcohol (AOH), 16-Mercaptohexadecaneic Acid (ACOOH), agarose and PVA have been purchased from Sigma Aldrich (Munich, Germany), HEPES from Applichem (Darmstadt, Germany) and 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-Lserine (sodium salt) (POPS), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (PSM), Nstearoyl-D-erythro-sphingosylphosphorylcholine (SSM), 16-Mercapto(8-biotinamido-3,6dioxaoctyl)hexadecaneamide (BBiotin), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-N(cap biotinyl) (DCB) from Avanti Polar Lipids (Alabasta, AL, USA). [1,1' - Dioctadecyl 3,3,3',3' – tetramethylindo-carbocyanine iodide] (DiIC18) from Ana Spec Inc. (Fremont, CA, USA), 2-(4,4-Difluoro-5-methyl-4-bora-3a,4a-diaza-s-indiacene-3-dodecanoyl)-1-hexadecanoyl-sn-gycero-3-phophocholine (BODIPY-PC) from Invitrogen (Carlsbad USA). Streptavidin (Rockland). Solvents (HPLC grade) were supplied by Merck KGaA (Darmstadt, Germany) Buffers: HBS (10 mM HEPES, 150 mM NaCl, pH 7.4), TBS (20 mM Tris, 100 mM NaCl, 1 mM MgCl2, pH 7.4) and PBS, Dulbecco`s PBS, Sigma Aldrich (Munich, Germany). (Ultra-pure water (18.2 MΩ) was generated using a cartridge system from (MilliPore, Billerica, MA, USA). Quartz crystal microbalance (QCM) Gold covered QCM sensors (QSX 301, Q-Sense, Gothenburg, Sweden) were incubated in a 0.1 mM solution of AOH/ACOOH/BBiotin (40:10:1) in CHCl3 over night until a self-assembled monolayer is formed. After washing with CHCl3 and ultra-pure water the surfaces were dried in a stream of nitrogen. The modified sensors were then mounted into the Q-Sense E4 QCM-D (QSense, Gothenburg, Sweden) and equilibrated in TBS buffer at 80.4 µl/min per sensor. Then 0.15 µg/ml streptavidin was incubated, rinsed 5 min by TBS and liposomes, 200 nm, ~ 0.5 mg/ml, doped 1000:1 with DCB were tethered. Surfaces were always rinsed approx. 5 min by TBS buffer before a new solution was applied. Liposomes were prepared from lipid film in a glass test


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tube by extrusion at 50 °C through a 200 nm polycarbonate membrane (Avestin Liposofast, Ottawa, Canada) as described elsewhere.18-20 The setup allowed two parallel measurements, each as duplicate. The data shown is the mean of n = 2, if not otherwise stated and errors are SD. To clarify visual presentation, graphs were time shifted by removing a few data points at equilibrium states, indicated by //. Y-Data remained original. Measurements were performed at 20°C. Laurdan and DPH fluorescence Lipid and imidazolium salt component films containing Laurdan (500:1), or DPH (250:1) were prepared in glass tubes, hydrated in degassed HBS (Laurdan) or PBS (DPH), pH 7.4 buffer at 60 °C and extruded 31 times through a 200 nm polycarbonate membrane (Avestin Liposofast, Ottawa, Canada) at 60 °C. The final concentration was 0.5 mM (Laurdan) or 0.2 mM (DPH) lipid or imidazolium salt. Laurdan fluorescence was excited at 360 nm and emission spectra were recorded at room temperature between 380 nm and 600 nm with a slit of 1 nm.21, 22 DPH fluorescence was excited at 320 nm and spectra were measured at 20 °C between 340 nm and 580 nm with a slit of 5 nm on a Jasco FP-6500 (Jasco, Maryland, USA). DPH fluorescence anisotropy was then converted from measurements at 20°C using the emission intensity with emission polarizer oriented parallel to excitation polarizer and the emission intensity with the emission polarizer being adjusted perpendicular to the excitation polarizer. 23-25 Confocal laser scanning microscopy (CLSM) CLSM employed either a Leica DMI8, equipped with a TCS SP8 scanning unit, using a HC PL APO 63x/1.20 W UVIS CS2 water immersion objective (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) with lasers: OPSL 488 nm (BP 493 nm – 547 nm), OPSL 552 nm (BP 563 nm – 754 nm) and a temperature controlled incubation chamber, or a Zeiss LSM 780, using a Pln APO 63x1.4/ Oil DICII M27 objective (Carl Zeiss Microscopy GmbH, Jena, Germany). Lasers: 488 nm argon (Lasos Lasertechnik GmbH, Jena, Germany) (BP 493 nm – 541 nm) and 561 nm DPSS 561-10 laser (BP 600 nm – 700 nm). Furthermore, a Zeiss LSM 880, using a Pln APO 63x/1.4 Oil DIC M27 (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 488 nm argon laser (Lasos Lasertechnik GmbH, Jena, Germany) (BP 493 nm – 556 nm) as well as a 561 nm DPSS laser (BP 578 nm – 696 nm) was used (MBS 488/561). The LSM 780 and 880 were equipped which a temperature controlled incubation chamber as well. Additionally, a Leica DMRE with a TCS SL scanning unit and a PH HCX PL APO CS 63.0 x 1.32 OIL PH3UV objective (Leica Microsystems Heidelberg GmbH, Mannheim, Germany), with a 488 nm Arlaser (BP 495 nm – 535 nm) and a 543 nm HeNe-laser (BP 620 nm – 750 nm) was used when appropriate. Images are usually CLSM images or wide-field when stated and processed using Leica manufacturers software, Zeiss ZEN 2011 software ore Image J / Fiji. Evaluation of GUV forming efficacy was aided by EVOSTM FL Cell Imaging System, Life Technologies (Carlsbad, CA, USA). Vesicle formation Preparation followed modified protocols as described elsewhere.26-28 Briefly, lipid stocks were made in chloroform/methanol (1:1, v/v) and mixed in molar ratio. Mixtures were doped by 0.4 % BODIPY-PC and DiIC18 where indicated. A film of ultra-low geling agarose or polyvinyl alcohol (PVA) is deposited at 80 °C on a glass slide from a 1% (w/v) agarose or 5% (w/v) PVA solution in ultra-pure water. Subsequently the film was dried, the lipid mixture was deposited and again


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dried under vacuum at 50 °C for 60 min. Vesicles were then formed by equilibration in HBS buffer or 320 mM sucrose for 1h at 60 °C, i.e. above Tm of the lipids / imidazolium salts.

Results and Discussion In previous work, short-chain ionic liquids made of 1-alkyl-3-methylimidazolium salts were reported to disintegrate large unilamellar vesicles (LUV).29 In contrast, we employed imidazolium salts with long alkyl chains, the lipid mimetic 4,5-alkyl-imidazolium salt C15IMe·HI, which has a better structural resemblance to natural lipids16 and probed its membrane interactions. Moreover, we investigated different derivatives with respect to the effect of alkyl chain length on toxicity for cells and the ability to bind, intercalate and lyse membranes.16 We now present further evidence for their membrane interaction considering the viscoelastic properties of bilayer membranes. To obtain membrane binding characteristics an established protocol was used,30, 31 to tether large unilamellar vesicles of either POPC/POPS (4:1) or pure POPC via the strong biotin-streptavidin interaction on a quartz crystal microbalance with dissipation monitoring (QCM-D) setup. POPC is one of the most common natural lipid components in cellular membranes and addition of 20 % POPS incorporates negative charge into the model to analyze the interaction with positively charged imidazolium salts. POPS is also present in the inner leaflet of eukaryotic plasma membranes.32, 33 In a QCM-D measurement, the shift of the sensors resonance frequency (∆F = F(t) – F0) is proportional to the mass adsorbed to the surface and the change in dissipation (∆D = D(t) – D0) is indicative of the viscoelastic properties.34-36 Thus the adsorption of mass, increasing the systems viscoelasticity will shift ∆F towards negative values and increase ∆D. On the streptavidin modified sensor surface, the adsorption of 200 nm POPC/POPS (4:1) vesicles resulted in a relative frequency shift of ∆∆F = 311 ± 16 Hz; n = 7 (Figure 2 arrows 1, WANG et al.16) and immobilization of pure 200 nm POPC liposomes resulted in ∆∆F = 399 ± 17 Hz; n = 7 (Figure 2, arrows 1). Subsequent equilibration of liposomes in TBS buffer, simulating cellular salt and pH conditions, did not considerably change the mass, indicated by a minor change in frequency or viscoelastic properties, represented by the dissipation. Incubation with a 0.1 mM dispersion of C15-IMe·HI on POPC/POPS vesicles lead to an additional frequency shift of ∆∆F = 445 ± 8 Hz demonstrating an intense membrane association (Figure 2A, arrow 2). However, the parallel change in dissipation of ∆∆D = 109·10-6 ± 7·10-6, reveals a significant increase in the systems viscosity, which is promoted by the components alkyl chains intercalating into the membrane bilayer. Although C15-IMe·HI enhances the mass and viscosity of POPC/POPS vesicles, the system can cope with an intense hypo-osmotic stress of ≈ 240 mOsmL-1, induced by washing with ultra-pure water (arrow 3). This results in a relative liposome mass change of 185 ± 2 % after treatment. It is likely that the majority of vesicles remained intact upon treatment. The shorter component, C11-IMe·HI leads to less extended shifts of mass and viscosity on the same membrane, although it is highly lytic under hypo-osmotic conditions (Figure 2A). This lytic action is caused by a hydrophobic mismatch between the C11 alkyl chains of the component and the longer lipid chains, causing a bilayer disturbance. As a control, the incubation with liposomes of similar membrane composition but without any imidazolium salt, did not alter the relative membrane mass or viscoelastic properties of the tethered liposomes. The same class of components, incubated on the neutrally charged, zwitterionic POPC liposomes lead to smaller


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effects in the change of mass and viscosity. Although C15-IMe·HI, resulted in a small lytic activity on hypo-osmotic conditions, membrane binding (i.e. ∆∆F) and the increase in viscosity (∆∆D) are much less pronounced. Here, it resulted in a relative mass change of 73 ± 4 % after hypo-osmotic treatment compared to the original mass of liposomes before incubation (Figure 2A, right, arrow 3). However, C11-IMe·HI was more lytic resulting in a final POPC vesicle mass change of 54 ± 1 % instead of 2 ± 1 % as measured on POPC/POPS vesicles. Therefore, membrane binding and intercalation efficacy of Cn-IMe·HI salts (except the C7 compound, which has only minor effects) strongly depends on membrane charge thus showing, that at least initial binding is facilitated by the positively charged headgroup of these components. A graphical illustration of the observed bilayer interactions and the model of tethered liposomes is discussed later (Figure 7).


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Figure 2: Membrane interaction of IMe- and IBn-salt compounds with LUVs. A biotinylated SAM was incubated by a layer of streptavidin. Then POPC/POPS (4:1) or POPC liposomes (200 nm), doped by DCB, were tethered on the surface (arrows 1). After equilibration, the surface was rinsed with pure TBS buffer for at least 30 min, before 0.1 mM Cn-imidazolium salts were incubated on the membrane interfaces (arrows 2). After the following equilibrium state, the surfaces were again washed for 30 - 60 min by pure TBS, before hypo-osmotic conditions were induced by rinsing with ultra-pure water (arrows 3). A) Cn-IMe·HI, B) CnIBn·HBr. C15- imidazolium salts: red graph, C11- imidazolium salts: blue, C7- imidazolium salts: green. Control: ~ 0.5 mg/ml, 200 nm LUVs of the corresponding lipid mix, presented in black. Each graph represents n = 2.


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To analyze whether membrane binding does not only depend on the alkyl chain length but also on headgroup size, we replaced the methyl groups in 1 and 3 position of our imidazolium salts by bulkier benzyl groups (Figure 1). In contrast to Cn-IMe·HI imidazolium salts, C11-IBn·HBr and C15-IBn·HBr showed no binding effect upon incubation on POPC/POPS (4:1) liposomes. However, incubation of 0.1 mM C7-IBn·HBr on POPC/POPS (4:1) liposomes lead to a reduction of the frequency shift of ∆∆F = 164 ± 3 Hz, which shows that only 46 ± 3 % of liposome mass remained on the surface, even without applying a hypo-osmotic gradient. This is in contrast to C7-IMe·HI, which had a minor effect on the same membrane. The observation is in good agreement with the fact, that C7-IBn·HBr is the most toxic compound (EC50 = 2.5 ± 0.43 mM) on NIH3T3 cells.17, 37 Adsorption on pure POPC liposomes showed only a minor mass effect of C15IBn·HBr, which resulted in a relative liposome mass change to 117 ± 9 % ,without applying hypo-osmotic conditions (Figure 2B, arrow 2). The C7-IBn·HBr salt induced an immediate but small drop in frequency, accompanied by a larger shift in dissipation as observed for the other membrane (Figure 2 B, right). This is an enhancement of the viscoelastic properties of the almost unchanged liposome mass. The other benzyl compound did not induce a relevant change in mass on this membrane. We further probed the unspecific interaction of Cn-IMe·HI and Cn-IBn·HBr imidazolium salts on the streptavidin layer (Figure S1). C15-IMe·HI induced a shift of ∆∆F ≈ 22 Hz which is small in comparison to its effect on liposomes (≈ 5%) and also small in comparison with liposome immobilization (≈ 7%, POPC/POPS or 5%, POPC). The short chain component, C7-IMe·HI, lead to a smaller unspecific adsorption of ∆∆F ≈ 7 Hz. Unfortunately, C11-IMe·HI induced a relative large adsorption on the streptavidin layer of about 125 Hz. However, if POPC/POPS liposomes are immobilized on streptavidin (Figure 1 A arrow 2), this component induced a shift of 200 ± 3 Hz. If the streptavidin layer underneath the tethered liposomes would be exposed to the imidazolium salt in the same manner as in our control, then this could result in a minimal effective shift of about 75 Hz. This shift remains indicative for strong binding. The Cn-IBn·HBr imidazolium salts did not induce any relevant shift (Figure S1 B). The component C15-IMe·HI easily interacts with bilayer membranes without strong indication for a lytic activity, thus we continued to analyze whether it reveals properties of a bilayer membrane using Laurdan and DPH. Laurdan is a fluorescent probe, which has a lauric acid tail that intercalates into bilayers by van der Waals interactions with the hydrocarbon backbone. Its fluorescent, naphthalene moiety resides close to the glycerol backbone of the lipid bilayer.38 The probe’s fluorescence shows a spectral shift in dependence of water molecules present in the membrane caused by a dipolar relaxation of the fluorophore.38 Therefore it has been widely used to characterize bilayer phases and fluidity of membranes. DPH is a well-known probe often used for polarization measurements.23, 24 The fluorescence spectra of Laurdan, embedded in C15-IMe·HI and in the lipid reference membranes, prepared in HBS buffer, are presented in Figure 3 A. Within DPPC vesicles, the probe shows a spectrum with a maximum emission centered at λmax ≈ 445 ± 1 nm, which is typical for a tight packed gel phase.38 BAGATOLLI et al. reported a maximum emission value of λmax ≈ 440 nm for Laurdan in DPPC measured in 20 mM TrisCl·H-50 mM NaCl, pH 7.4.39 The small variation of our value may be explained by a higher ionic strength of our buffer, allowing more water molecules to be in close proximity to the probe and thus causing a minimal shift. However, relative to the maximum emission of Laurdan in DPPC LUVs, a red shifted maximum


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emission in the bilayer models POPC (∆λmax ≈ 20 ± 2 nm) and DOPC (∆λmax ≈ 25 ± 2 nm) is observed together with a relative reduction of fluorescence intensity. These red shifts are typically observed for a liquid-crystalline phase state of the membrane which incorporates a higher amount of water than DPPC.38, 39 The pure C15-IMe·HI incorporates Laurdan, shows a maximum emission centered at λmax ≈ 478 ± 1 nm and induces a strong red-shift of the maximum emission in the Laurdan spectrum (∆λmax ≈ 33 ± 2 nm), which could be indicative of a liquid-crystalline phase (Figure 3 A). In this state, the lipid headgroups are spatially more separated providing additional room for water molecules in close proximity of the probe thus causing dipolar relaxation (quenching) of the fluorophore. Although the exact location of the probe in the C15-IMe·HI membrane is unknown and thus interpretation of any shift is limited, the strong fluorescence signal supports probe incorporation into the membrane. In contrast, the component C15-IBn·HBr did not exhibit incorporation of Laurdan and thus shows much less fluorescence (Figure 3 A).

Figure 3: Fluorescence spectra of Laurdan and DPH incorporated into C15-IMe·HI membranes and standard model membranes. A) Relative fluorescence intensity of Laurdan incorporated into bilayer models and imidazolium salts in HBS buffer. The inset graph shows normalized data to highlight spectral shifts. B) Relative fluorescence intensity of DPH in model membranes and imidazolium salts in PBS buffer. The inset graph shows the anisotropy of DPH in C15-IMe·HI at different temperatures (n = 2). The component C15-IBn·HBr did not display any relevant Laurdan or DPH fluorescence. The probe DPH is also widely used to examine membrane dynamics.23-25 Again this probe is well incorporated into a C15-IMe·HI membrane as it shows a typical spectrum in a bilayer with three maximum intensities and the main being centered at about λmax ≈ 432 ± 1 nm (Figure 3 B). The fluorescence signal is almost as strong as for DPH in a DOPC membrane under similar conditions. For comparison, the maximum intensities of the main peaks of DPH in DPPC, POPC and DOPC bilayers are λmax ≈ 428 ± 1 nm, λmax ≈ 430 ± 1 nm and λmax ≈ 431 ± 1 nm (Figure 3 B). The spectrum of this probe in our C15-IMe·HI membrane appears to be very similar to these reference bilayers, although the exact position of the probe within the membrane was not determined. The imidazolium salt C15-IBn·HBr did not exhibit incorporation of DPH as observed for Laurdan.


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Furthermore, we analyzed the anisotropy of DPH in C15-IMe·HI and our reference model bilayers. When this hydrophobic probe is distributed parallel to the alkyl chain region of the bilayer, the fluorescence anisotropy is a measure for dynamic properties, such as membrane fluidity.23, 40 A lower value reports enhanced rotational freedom of the probe and thus reveals a fluidic membrane, whereas higher values are indicative for reduced dynamics. The fluorescence anisotropy of C15-IMe·HI in PBS, pH 7.4 buffer at 20°C is 0.197 ± 0.005 (n = 3), which is higher as for POPC (0.124 ± 0.003) and DOPC (0.101 ± 0.002) under similar conditions. The anisotropy values of these reference bilayers are in well agreement with previous reports and indicate a liquid membrane state whereas DPPC shows a fluorescence anisotropy of 0.327 ± 0.002 indicating a gel phase.24, 25, 41 Thus, C15-IMe·HI probably forms an intermediate state between a liquid-crystalline or a gel-phase membrane at 20 °C. The temperature dependent anisotropy of DPH in C15-IMe·HI membranes shows a transition between 28 °C and 32 °C (Figure 3B, inset), which is suggestive for a phase transition. This is in good agreement with the lower one of two unidentified endothermic transitions found for pure C15-IMe·HI at Tm1 = 29.3 °C and Tm2 = 35.2 °C, determined by Differential Scanning Calorimetry (WANG et al.42). Similar phase transitions have been deduced from anisotropy transitions as reported for DPPC or the fluorescence polarization of DMPC at different temperatures.23, 24, 41 Additionally, we analyzed by Laser Scanning confocal microscopy whether pure imidazolium salts may form bilayer membranes autonomously. Using a GUV preparation technique according to a modified protocol,26-28 we could generate giant vesicles in sucrose or in a physiological HBS buffer (Figure 4, Suppl. Figure S2).

Figure 4: Giant unilamellar vesicles formed by C15-IMe·HI and C11-IMe·HI.


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A) A film of C15-IMe·HI is hydrated from PVA or agarose in HBS. Scales: 5 µm. B) C11-IMe·HI is swollen in 320 mM sucrose on PVA, diluted 1:4 by HBS buffer and directly observed. Scale 5 µm. C) C11-IMe·HI is swollen in 320 mM sucrose on PVA, diluted 1:4 by HBS buffer and a time series is recorded. Duration: 131 s. BODIPY-PC fluorescence channel, scale 10 µm. Note the prominent shrinkage in the vesicle size. All images represent equatorial slices of a CLSM recording. We incorporated phase selective membrane dyes to distinguish between liquid disordered (ld) and liquid ordered (lo) domains. The fluorophore BODIPY-PC is a membrane label known to incorporate into the ld phase, whereas DiIC18 preferentially marks the lo phase in many membrane compositions.43-45 However, phase selectivity cannot always be easily predicted and can vary for other mixtures or under different conditions.45, 46 C15-IMe·HI forms large vesicles of slightly distorted shape, which consist of multi- and unilamellar membranes (Figure 4 A, Figure S2). They appear homogeneous without any phases observed at room temperature and were stable over longer periods of time (at least 90-120 min), also when they have been diluted 1:4 by HBS buffer after preparation (Figure S2). Their membrane appears to be more rigid, as dynamic fluctuations, like membrane movement of the vesicle surface which is usually observed in a microscopic measurement, seem to be hindered in comparison to other e.g. POPC or DOPC vesicles in a fluid state. For instance, the circumference curvature of the example presented in Figure 4 A, second row, is imperfect as observed for a DPPC GUV in the gel state.47 This is also in good agreement with the observation that DiIC18 shows a prominent photoselection effect48 in C15-IMe·HI due to excitation with linear polarized laser light (Figure 4 A upper row, Figure S2 E). This finding again is in support of a gel-state of the membrane, as the rotational freedom of the probe is restricted. In contrast to C15-IMe·HI, vesicles swollen from C11-IMe·HI form regular shaped, homogeneous bilayer membranes which appear to be unilamellar. However, these vesicles become instable if diluted 1:4 with buffer, which is observed by a continuous shrinkage within a few minutes (Figure 4 C, Figure S2 B, Supplemental Material Video S1). The volume (approximated by a sphere deduced from the membrane diameter at the equatorial cross section) shrinks within 131 s from 417 µm3 to 117 µm3 (~28 %) whereby two attached GUVs are formed. Further examples showed shrinkage from 202 µm3 to 17 µm3 (~8 %) in 133 s or from 552 µm3 to 298 µm3 (~54 %) in 54 s and a total collapse from 674 µm3 in 27s (Figure S2 C). The diversity among these different examples may vary due to different times required to focus on individual vesicles. However, this finding was rather unexpected and was not observed for other model vesicles under similar conditions. The possibility of imidazolium salt bilayer dissolution after dilution is reasonable according to the relative high critical micelle concentration (CMC) of C11-IMe·HI (CMC = 8.5 µM), which easily stabilizes micelles in high ratios.16 Upon swelling, GUVs form at the gel-buffer interface driven by forces that are normal to the lipid layers.26 If C11-IMe·HI vesicles swell from a film deposited on agarose or PVA gel, a concentration gradient of the component may arise in the surrounding media, which is perpendicular to the swelling layer surface. Therefore, this concentration gradient may stabilize structures which thermodynamically collapse upon further dilution. However, vesicle formation by swelling is not entirely quantitative which impedes reliable calculation of the final liposome concentration. Thus it seems likely that C11-IMe·HI can form bilayer vesicles on a kinetically driven basis at elevated temperature and an enhanced concentration. The vesicles then transiently transform into


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micelles after dilution, which cannot be detected by microscopy. This transformation possibly includes the steps of micelle nucleation and elongation at the bilayer surface until detachment of the micelles reduces the observable amount of vesicle membrane. Steps of micellar nucleation and elongation have been reported by YOO et al. using MD calculations for the ionic liquid 1dodecyl-3-methylimidazolium chloride.49 Attempts to form giant vesicles from C7-IMe·HI failed probably because it easily forms relative stable micelles. Therefore, the vesicle formation capability and bilayer stability is in good agreement with observed CMCs of Cn-IMe·HI compounds, where C7-IMe·HI (CMC = 65.9 µM) has the highest and C15-IMe·HI (CMC = 1.4 µM) the lowest CMC.16 Thus, C15-IMe·HI can self-assemble into micelles but also can easily form larger structures like bilayer membranes. Next, we also tried to generate GUVs from C15-IBn·HBr, which forms a droplet-like emulsion instead (Figure 5). These droplets detach from the support, are completely homogeneous as judged by phase selective dyes (BODIPY-PC, DiIC18) and are entirely filled as shown by singleslice confocal images. Additionally, a tubule-like connection between droplets is observed which may indicate the presence of a surrounding bilayer at their surface. These tubule tethers have been already observed for lipid membranes,26 however, here a detailed analysis is beyond the light microscopic resolution.

Figure 5: Droplets formed by C15-IBn·HBr. A dried film of C15-IBn·HBr on agarose is hydrated in HBS. Note the difference of filled structures in comparison to GUVs formed by C15-IMe·HI. The Images represent single slices of a CLSM experiment. Scales = 10 µm. The difference between C15-IMe·HI and C15-IBn·HBr is the structure of the headgroup, with the bulkier benzyl rings potentially inducing stronger intermolecular interactions by π-stacking and thus possibly stabilizing a droplet suspension instead of lamellar membranes. This suspension could be further stabilized through the possible formation of inverse micelles which embed the charged headgroup within these droplets. In order to gain further insights into the miscibility within lipid bilayers we also selected phase separated lipid mixtures and investigated the effect of C15-IMe·HI on the phase behavior. This component was used since it was found to stabilize bilayers and already revealed large bilayer interactions in our QCM survey. We used three different lipid mixtures, all containing cholesterol, which is commonly present in eukaryotic membranes and one mixture additionally containing N-palmitoyl-D-erythro-sphingosylphosphorylcholine (PSM) a sphingomyelin, which


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is a typical lipid-raft associated lipid present in domains important for cellular signaling and trafficking.50, 51 At first we analyzed the influence on the phase behavior of the mixture DOPC/PSM/Chol (33:33:33) prepared in HBS buffer. This mixture was reported by VEATCH et al. to show liquidliquid demixing domains on vesicles, measured below the miscibility transition of Tmiscibility = 3335 °C.52 In our studies we used the probes BODIPY-PC and DiIC18 which showed discernible domains at T = 20-22 °C (Figure S3). However, incorporation of 10 % C15-IMe·HI, which results in the lipid mixture DOPC/PSM/Chol/C15-IMe·HI (33:23:33:10) does not reveal any prominent difference in the same temperature range (not shown). The lipid PSM was chosen to be replaced, since it is the only lipid in the original mixture with only saturated alkyl chains like the imidazolium salt. Under our conditions, small bulging domains and a strong line tension between phases could be observed for the mixture DOPC/PSM/Chol (33:33:33) at elevated temperatures around 26.5-28 °C (Figure 6 A, Figure S4). However, incorporation of 10 % C15-IMe·HI (DOPC/PSM/Chol/C15-IMe·HI; 33:23:33:10) does reduce the line tension between domains and phase boundaries appear to be less noticeable in the same temperature range (Figure 6 B, Figure S4). The relative reduction of the line tension between phase boundaries show that C15-IMe·HI can intercalate into the DOPC/PSM/Chol (33:33:33) mixture, which is in line with our QCM survey for other bilayer models and does fluidize the membrane. Liquid-liquid demixing domains on vesicles do not commonly protrude out of the vesicle plane. However, VEATCH et al. showed that domains can bulge out at temperatures slightly below the miscibility transition temperature,52 which is observed in our case. Furthermore, BAUMGART et al. showed bulging and pinching in brain SM/DOPC/Chol vesicles with lo + ld phase coexistence.53, 54 Domains bulging out of the vesicle surface indicate stronger line tension between phase boundaries in order to minimize the interfacial energy.52, 55 Next, we examined incorporation into the model DOPC/DSPC/Chol (33:33:33), which has a slightly different miscibility transition temperature of Tmiscibility = 35 °C.52 These vesicles present discernible domains observed at T = 22-24 °C (Figure 6 C, Figure S4). Incorporation of 10 % C15-IMe·HI on the cost of saturated DSPC, which results in the mixture DOPC/DSPC/Chol/C15IMe·HI (33:23:33:10), revealed a distinct behavior. Phase boundaries almost fully disappeared and the mixture looks homogeneous when probed with BODIPY-PC and DiIC18 (Figure 6 D, Figure S4). The small red dots on the vesicle surface were unspecific debris and do not represent domains. Note that small, round shaped and darker domains in the lower part of the vesicle in figure 6 C were discernible, which cannot be observed after the incorporation of our imidazolium salt. Together these observations suggest that also in this lipid mixture the imidazolium salt reduces line tension and fluidizes the membrane (Figure 6 D, Figure S4).


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Figure 6: Bilayer domain fluidization in presence of 10 % C15-IMe·HI.


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A) GUVs of DOPC/PSM/Chol (33:33:33) at T = 27.3 °C, scale = 5 µm. B) GUVs of DOPC/PSM/Chol/C15-IMe·HI (33:23:33:10) at T = 27.0 °C, scale = 10 µm. Note the difference in line tension between domains. C) GUVs of DOPC/DSPC/Chol (33:33:33) at T = 22.3 °C, scale = 5 µm. D) GUVs of DOPC/DSPC/Chol/C15-IMe·HI (33:23:33:10) at T = 24.0 °C, scale = 10 µm. Note the difference in domain appearance. E) GUVs of DOPC/SSM/Chol (33:33:33) at T = 38.0 °C, scale = 20 µm. F) GUVs of DOPC/SSM/Chol/C15-IMe·HI (33:23:33:10) at T = 38.0 °C, scale = 20 µm. Note the prominent fluidization of small bulged domains to flat large domains with enhanced dye specificity upon C15-IMe·HI addition. All GUVs were swollen in HBS buffer and images are single CLSM slices, except image D, composite, which is a 2D projection of a 3D reconstruction of several CLSM z-slices. Image C, composite, represents a single slice to highlight small fluid domains on the vesicle´s upper hemisphere and thus appears to be a little smaller that the corresponding equatorial slices. Further, we probed another sphingomyelin containing lipid mixture which has an even higher miscibility transition range. The mixture DOPC/SSM/Chol (33:33:33) also shows liquid-liquid demixing domains on vesicles and has a miscibility transition of Tmiscibility = 42-44 °C.52 Vesicles of this composition were doped with 0.4 mol % BODIPY-PC and 0.4 mol % DiIC18 and analyzed below miscibility transition at 38°C. Discernible but small domains, which protrude out of the membranes surface (Figure 6 E) to minimize the interfacial energy between phase boundaries, were observed. These small protrusions presented contrast in the bright field image in difference to flat domains (Figure 6 E vs. Figure 6 C). In contrast to the examples above, these vesicles are attached to each other, which has no influence on domain morphology. We used the mixture DOPC/SSM/Chol/C15-IMe·HI (33:23:33:10), which has 10 % C15-IMe·HI incorporated on the cost of SSM. When this mixture is also measured under similar conditions, large DiIC18-rich domains appeared which seem to be flat within the vesicle membrane (Figure 6 F). This is supported by the fact, that most of these domains become hardly visible in the bright field image, in comparison to the imidazolium salt free mixture (Figure 6 E and F). Furthermore, these domains vividly move and fuse during the time period required to measure the complete z-stack (Supplemental Material Video S2). Thus, the incorporation of 10 % C15-IMe·HI into this raft-like lipid mixture facilitates the formation of distinct lo domains, which is indicated by the enhanced specificity of DiIC18 dye (Figure 6 E, F, composite),52, 53 whereas the line tension between phases is reduced in comparison to the mixture without imidazolium salt. Therefore, domains become larger and also phase selectivity of the dyes for lo and ld phases is increased. Hydrophobic van der Waals interactions and a reduced mismatch between the stearoyl-SM and the C15 chains of our imidazolium salt as well as the dioctadecyl-hydrocarbon chains of the lo specific DiIC18 possibly promote this pronounced fluidization effect. Furthermore, the temperature of 38 °C at which this fluidizing effect is observed is above our anisotropy transition of pure C15-IMe·HI, which means that this component is possibly in the liquid state and hence facilitates domain miscibility or reduces line tension. The two fluorescent probes used here do not always clearly reveal different specificity for phases under these conditions, as it could be observed by different probes in a different buffer.52 However, they become well suited to provide contrast between domains, when C15-IMe·HI is considered as mixture component, which is important in our study. Moreover, phases on GUVs must be compared with great care because small variations in experimental conditions like temperature or illumination intensity can affect the appearance of


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domains.43, 52 We therefore kept our conditions fairly constant in the three sets of experiments to maintain highest possible comparability. Together these results indicate that the incorporation of C15-IMe·HI reduces the amount of observable bulged domains and fluidizes the bilayer membrane, as observed by a decrease in the line tension between phases.52, 53 This finding is in line with the fluidizing effect observed on the monolayer surfactant model which has been reported by WANG et al.42 Take this into account, a model can be derived for the interaction of (Cn-IMe·HI, n = 7, 11, 15) imidazolium salts with bilayers in order to explain how they form membrane structures under physiological conditions (Figure 7). According to our QCM setup described above, liposomes which are immobilized via biotin-streptavidin binding on the sensor surfaces react different upon incubation with these imidazolium salts. Membrane intercalation (observed for C15-IMe·HI and C11-IMe·HI), disintegration and lysis (C11-IMe·HI) as well as penetration without disruption (C7IMe·HI), strongly depended on alkyl chain length (Figure 7, upper panel, A-C). Based on this relationship it can be further deduced, that the toxicity of this imidazolium salts on cells in vitro17 is mainly determined by the compounds ability to pass a membrane barrier and must not essentially require membrane lysis. WANG and RÜHLING et al. revealed that the short chain imidazolium salts C7-IMe·HI and C7-IBn·HBr showed highest toxic behavior on MDCKII and NIH3T3 cells.17, 37 These compounds did not reveal a membrane disintegration activity on our liposomes. Thus in this case the toxicity is not caused by membrane disruptive effects but by so far unknown intracellular actions that lead to membrane perturbation. The formation of liposomes and / or micelles without aid of other lipids is described in Figure 7 lower panel, D-F. A film of the compounds, i.e. C15-IMe·HI, deposited on substrate glass, a PVA or agarose gel matrix will hydrate until lamellar bilayers are formed, which then further swell to the growth of liposomes. This swelling is a prerequisite state for vesicle formation as it enhances formation efficacy until vesicles eventually bud and detach from the swollen gel. Such process would resemble the mechanism of GUV formation observed for natural lipids.26, 27 Among the tested compounds, C15-IMe·HI showed the highest bilayer mimetic properties. The shorter chain C11-IMe·HI can form, probably kinetically driven, vesicles which transiently transform via micellar nucleation to micelles.49


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Figure 7: Model for membrane interaction and structure formation of imidazolium salts in aqueous solution at physiological conditions. A-C) Bilayer interface interaction of 4,5-dialkylimidazolium salts based on immobilized vesicles in a physiological salt / pH buffered environment as retrieved from Figure 2. Liposomes, (blue) are tethered via biotin linkers (green) and streptavidin (purple) on a self-assembled monolayer


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(brown), which itself is chemisorbed on a gold coated sensor surface (orange). A) Vesicles of C15-IMe·HI can associate, fuse and intercalate into bilayer membranes, which results in an increase of liposome mass. This is possibly accompanied by the formation of mixed micelles in a minor extent. B) Vesicles and micelles of C11-IMe·HI intercalate and lyses bilayer membranes upon application of physical stress, e.g. a hypo-osmolar gradient across the membrane. This leads to membrane rupture and results in a net loss of tethered vesicle mass. Bilayer disintegration is accompanied by the formation of micelles and mixed micelles. C) C7-IMe·HI dissolves to micelles and single molecules and can pass though the membrane without disintegration. D-E) Structures and compartments which are autonomously formed of 4,5-dialkylimidazolium salts in a physiological buffer without addition of other lipids. D) A lipid film of C15-IMe·HI swells in aqueous solution from hydrated lamellae structures (bottom) and thermodynamic stable vesicles bud from the support. E) C11-IMe·HI vesicles and micelles can form from swelling of the lipid lamellae. Vesicles are thermodynamic instable and transiently transform to micelles via micelle nucleation. F) The compound C7-IMe·HI cannot form bilayer vesicles which could be observed under microscopic conditions. It is likely to form micelles directly. The lipid lamellae may contain PVA or agarose gels to improve formation efficacy.

Conclusion We analyzed the interactions of 4,5-dialkylimidazolium salts with bilayer membrane models which can mimic the curvature of cellular membranes due to the size of our liposomes and also considered charge effects of model membranes by use of negative charged lipids. Membrane interactions of 4,5-dialkylimidazolium salts which have a 1,3-dimethyl headgroup strongly depended on the alkyl-chain length. Long chain salts can intercalate with bilayers without disintegration, whereas shorter alkyl components did disintegrate and lyse bilayers. Additionally, we measured association characteristics in dependence of headgroup size and showed that a bulkier, 1,3-bisbenzyl-head reduced binding interactions except for the short chain C7-IBn·HBr component, which reveals the highest lytic activity on our tethered vesicle model membranes. Due to the charge of the imidazolium salts, activity was highest for a negatively charged bilayer system in comparison to neutral POPC liposomes. Thus electrostatic interactions between the imidazolium moiety and the lipid headgroup play an important role for binding interactions prior to membrane insertion. Furthermore, experimental evidence is provided, that C15-IMe·HI itself forms uni- and multilamellar bilayer membranes, which are in a gel- or liquid-crystalline membrane state at room temperature. In previous research, a two tailed cationic surfactant has been reported by LÓPEZ-BARRÓN et al., which forms vesicles in the environment of a protic ionic liquid.56 Here, we present further unique findings, since our C15-IMe·HI autonomously forms compartment-enclosing bilayer structures in the presence of a physiological buffer. This has high potential as vehicle for drug delivery and biochemical engineering. Additionally, its non-lytic intercalation into existing bilayer models may attract future interest in biosensor formation or bio-engineering. Interestingly, the incorporation of C15-IMe·HI into phase separated GUVs, which present distinct domains having a high interfacial energy between boundaries, lead to a remarkable fluidization of bilayer membrane domains. This is in good agreement with the domain fluidization effect shown by incorporation of imidazolium salts into the monolayer Langmuir surfactant model at


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high molar ratios (WANG et al.42). Therefore, application of imidazolium salts as membrane fluidizing agents, which also may improve drug permeability, provides another possible aspect of bio-engineering. Furthermore, carbenes and their imidazolium precursors may also serve as a platform to develop new linkers to either connect fluorophores to bilayer lipophilic imidazolium salt components or to proteins. A new linker approach was recently reported by KOLLMANNSPERGER et al., who investigated a very efficient lock and key approach using a trisNTA modified fluorophores together with the widely present poly-his-tag used in protein purification.57 In a recent report, BLENKE et al, described triene modified lipid headgroups tailored for the functionalization of liposomes via click chemistry.58 Importantly, our imidazolium salts provide the advantage that the group which is susceptible for modification is directly incorporated into the membranes surface and not connected by a spacer. Furthermore, carbenes can efficiently bind Pdnanoparticles2 which therefore can open new routes for catalysis on a membrane associated basis. Hence, our compounds might become potential lead structure candidates for further biotechnological development.

Supporting Information Incubation of imidazolium salts on a streptavidin layer, GUVs prepared from C15-IMe·HI and C7-IMe·HI on polyvinyl alcohol or agarose, additional CLSM images of GUVs presented in figure 4 and 6 to highlight domains, supplemental videos of a shrinking thermodynamic instable C11-IMe·HI GUV and a fluidized DOPC/SM/Chol/C15-IMe·HI (33:23:33:10) GUV.

Acknowledgements Financial support was provided by the Deutsche Forschungsgemeinschaft (SFB 858 and Leibnitz Award) and the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (SNF 31003A_159414).

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Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit

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