Photophysics in Biomembranes: Computational Insight into the Interaction between Lipid Bilayers and Chromophores.

ConspectusLight is ubiquitously available to probe the structure and dynamics of biomolecules and biological tissues. Generally, this cannot be done directly with visible light, because of the absence of absorption by those biomolecules. This problem can be overcome by incorporating organic molecules (chromophores) that show an optical response in the vicinity of those biomolecules. Since those optical properties are strongly dependent on the chromophore's environment, time-resolved spectroscopic studies can provide a wealth of information on biosystems at the molecular scale in a nondestructive way. In this work, we give an overview on the multiscale computational strategy developed by us in the last eight years and prove that theoretical studies and simulations are needed to explain, guide, and predict observations in fluorescence experiments. As we challenge the accepted views on existing probes, we discover unexplored abilities that can discriminate surrounding lipid bilayers and their temperature-dependent as well as solvent-dependent properties. We focus on three archetypal chromophores: diphenylhexatriene (DPH), Laurdan, and azobenzene. Our method shows that conformational changes should not be neglected for the prototype rod-shaped molecule DPH. They determine its position and orientation in a liquid-ordered (Lo) sphingomyelin/cholesterol (SM/Chol) bilayer and are responsible for a strong differentiation of its absorption spectra and fluorescence decay times in dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) membranes, which are at room temperature in liquid-disordered (Ld) and solid-gel (So) phases, respectively. Thanks to its pronounced first excited state dipole moment, Laurdan has long been known as a solvatochromic probe. Since this molecule has however two conformers, we prove that they exhibit different properties in different lipid membrane phases. We see that the two conformers are only blocked in one phase but not in another. Supported by fluorescence anisotropy decay simulations, Laurdan can therefore be regarded as a molecular rotor. Finally, the conformational versatility of azobenzene in saturated Ld lipid bilayers is simulated, along with its photoisomerization pathways. By means of nonadiabatic QM/MM surface hopping analyses (QM/MM-SH), a dual mechanism is found with a torsional mechanism and a slow conversion for trans-to-cis. For cis-to-trans, simulations show a much higher quantum yield and a so-called "pedal-like" mechanism. The differences are related to the different potential energy surfaces as well as the interactions with the surrounding alkyl chains. When tails of increased length are attached to this probe, cis is pushed toward the polar surface, while trans is pulled toward the center of the membrane.

Oxidation Kinetics of Fluorescent Membrane Lipid Peroxidation Indicators.

The oxidation of the cellular membrane through lipid peroxidation (LPO) is linked to aging and disease. Despite the physiological importance, the chemical mechanisms underlying LPO and oxidative reactions in membranes in general remain incompletely understood, and challenges exist in translating LPO inhibitor efficacies from in vitro to in vivo. The complexity of LPO, including multiple oxidation reactions in complex membrane environments and the difficulty in quantifying reaction kinetics, underlies these difficulties. In this work, we developed a robust and straightforward method for quantifying the oxidation rate kinetics of fluorescent molecules and determined the oxidation kinetics of widely fluorophores used as indicators of membrane LPO, diphenylhexatriene (DPH), BODIPY-C11, and Liperfluo. The measurement is initiated by lipoxygenase, which provides chemical specificity and enables a straightforward interpretation of oxidation kinetics. Our results reveal that the membrane composition significantly impacts the observed kinetics oxidation in DPH and BODIPY-C11 but not Liperfluo. Reaction mechanisms for their lipid peroxide-induced oxidation are proposed. This work provides a foundation for the quantitative analysis of LPO with fluorescence and extricating the complexity of oxidation reactions within membranes.

Fluorescence Polarization (FP) Assay for Measuring Staphylococcus aureus Membrane Fluidity.

Fluorescence polarization is a method to determine membrane fluidity using a hydrophobic fluorescent dye that intercalates into the fatty acid bilayer. A spectrofluorometer is used to polarize UV light as a vertical excitation beam which passes through the dye-labeled membrane where the dye fluoresces. The beams perpendicular and horizontal to the excitation light are then collected and analyzed. Membrane structural properties are largely due to the packing of the fatty acids in the lipid bilayer that determines the membrane biophysical parameters. Staphylococcus aureus contains straight-chain (SCFAs) and branched-chain (BCFAs) fatty acids in the membrane and alters the proportion of membrane fluidizing BCFAs and stabilizing SCFAs as a response to a variety of stresses. Herein, we describe a method for determination of membrane fluidity in S. aureus using diphenylhexatriene, one of the most used fluorescent dyes for this purpose.

Flow Linear Dichroism of Protein-Membrane Systems.

Linear dichroism (LD) is the differential absorbance of light polarized parallel and perpendicular to an orientation direction. Any oriented sample will show a signal in its electronic as well as vibrational transitions. Model membrane small unilamellar vesicles or liposomes provide an oriented system when they are subject to shear flow in a Couette or other type of flow cell. Anything, including peptides and proteins, that is bound to the liposome also gives an LD signal whereas unbound analytes are invisible. Flow LD is the ideal technique for determining the orientation of different chromophores with respect to the membrane normal. To illustrate the power of the method, data for diphenyl hexatriene, fluorene, antimicrobial peptides (aurein 2.5 and gramicidin), are considered as well as another common chromophore, fluorene, often used to increase the hydrophobicity and hence membrane binding of peptides. How LD can be used both for geometry, structure analysis and probing kinetic processes is considered. Kinetic analysis usually involves identifying binding (appearance of an LD signal), insertion (sign change), often followed by loss of signal, if the inserted protein or peptide disrupts the membrane .

Fluorescence Assays to Study Membrane Penetration of Proteins.

Phosphoinositides play important roles in the regulation of protein recruitment at specialized membrane domains, protein activity, and membrane dynamics. Phosphoinositide-protein interplay occurs via multiple mechanisms and proteins associate with membranes through different binding patterns. Determinations of membrane-binding mode and membrane penetration depth of proteins in lipid bilayer are thus important steps in characterizing the molecular mechanisms of membrane-protein interactions. Here, we show two standard in vitro assays using liposomes, diphenylhexatriene (DPH) anisotropy, and fluorescence quenching by brominated lipids to determine membrane penetration of proteins into lipid bilayer. These methods will provide useful tools to study membrane-protein association and uncover molecular details of protein-lipid interplay, which are important for understanding biological functions of membrane-associated proteins and membrane dynamics.

Influence of Membrane Phase on the Optical Properties of DPH.

The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.

Interactions of Ionic Liquids and Spirocyclic Compounds with Liposome Model Membranes. A Steady-State Fluorescence Anisotropy Study.

Understanding the toxicity of ionic liquids (ILs) is crucial in the search of greener chemicals. By comparing in vivo toxicity and in vitro interactions determined between compounds and biomimetic lipid membranes, more detailed toxicity vs. structure relation can be obtained. However, determining the interactions between non-surface-active compounds and liposomes has been a challenging task. Organisational changes induced by ILs and IL-like spirocyclic compounds within 1,6-diphenyl-1,3,5-hexatriene-doped biomimetic liposomes was studied by steady-state fluorescence anisotropy technique. The extent of organisational changes detected within the liposome bilayers were compared to the toxicity of the compounds determined using Vibrio Fischeri bacteria. Four liposome compositions made of pure 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocoline (POPC) and mixtures of POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and cholesterol (Chol) were tested as biomimetic models. Changes observed within the POPC/POPS/Chol 55:20:25 bilayers correlated the best with the toxicity results: ten out of twelve compounds followed the trend of increasing bilayer disorder - increasing toxicity. The study suggests that the toxicity of non-surface-active compounds is dependent on their ability to diffuse into the bilayers. The extent of bilayer's organisational changes correlates rather well with the toxicity of the compounds. Highly sensitive technique, such as fluorescence anisotropy measurements, is needed for detecting subtle changes within the bilayer structures.

Behavior of the DPH fluorescence probe in membranes perturbed by drugs.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is one of the most commonly used fluorescent probes to study dynamical and structural properties of lipid bilayers and cellular membranes via measuring steady-state or time-resolved fluorescence anisotropy. In this study, we present a limitation in the use of DPH to predict the order of lipid acyl chains when the lipid bilayer is doped with itraconazole (ITZ), an antifungal drug. Our steady-state fluorescence anisotropy measurements showed a significant decrease in fluorescence anisotropy of DPH embedded in the ITZ-containing membrane, suggesting a substantial increase in membrane fluidity, which indirectly indicates a decrease in the order of the hydrocarbon chains. This result or its interpretation is in disagreement with the fluorescence recovery after photobleaching measurements and molecular dynamics (MD) simulation data. The results of these experiments and calculations indicate an increase in the hydrocarbon chain order. The MD simulations of the bilayer containing both ITZ and DPH provide explanations for these observations. Apparently, in the presence of the drug, the DPH molecules are pushed deeper into the hydrophobic membrane core below the lipid double bonds, and the probe predominately adopts the orientation of the ITZ molecules that is parallel to the membrane surface, instead of orienting parallel to the lipid acyl chains. For this reason, DPH anisotropy provides information related to the less ordered central region of the membrane rather than reporting the properties of the upper segments of the lipid acyl chains.

Orientational distribution of DPH in lipid membranes: a comparison of molecular dynamics calculations and experimental time-resolved anisotropy experiments.

Characterization of the membrane phases is a crucial task in cell biology. Cells differ in composition of the lipids and consequently in adopted phases. The phases can be discriminated based upon lipid ordering and molecular diffusion and their identification could be used for characterization of cell membranes. Here we used molecular dynamics (MD) simulations to study the behavior of the fluorescent reporter molecule diphenylhexatriene (DPH) in different lipid phases - liquid disordered (Ld), liquid ordered (Lo), and solid ordered (So) composed of phosphatidylcholines (Ld and So) or a sphingomyelin/cholesterol (SM/Chol) mixture (Lo). To the best of our knowledge, this is the first simulation of DPH in Lo SM/Chol and So DPPC membranes. For the considered membrane compositions DPH is mostly oriented parallel to lipid tails. In the Lo phase we observed a significant fraction of DPH positioned in between membrane leaflets, which agrees with experimental findings, but which has not been observed in previous MD simulations of DPH in phosphatidylcholine membranes. Further, we calculated rotational autocorrelation functions (ROTACF) from our MD simulations in order to model the time-resolved fluorescence anisotropy decay. We observed that order parameters P2 and P4 are sufficient to fully describe the orientation distribution of DPH. We analyzed the ROTACFs by a so-called general model for the time-resolved fluorescence anisotropy [W. van der Meer et al., Biophys. J., 1984, 46, 515] and observed an overestimation of P4. We suggest a rescaling of the recovered P4 yielding an orientation distribution of DPH close to the one observed in our MD simulations.

Differential interactions of bacterial lipopolysaccharides with lipid membranes: implications for TRPA1-mediated chemosensation.

Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan's emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms.

De Novo-Designed α-Helical Barrels as Receptors for Small Molecules.

We describe de novo-designed α-helical barrels (αHBs) that bind and discriminate between lipophilic biologically active molecules. αHBs have five or more α-helices arranged around central hydrophobic channels the diameters of which scale with oligomer state. We show that pentameric, hexameric, and heptameric αHBs bind the environmentally sensitive dye 1,6-diphenylhexatriene (DPH) in the micromolar range and fluoresce. Displacement of the dye is used to report the binding of nonfluorescent molecules: palmitic acid and retinol bind to all three αHBs with submicromolar inhibitor constants; farnesol binds the hexamer and heptamer; but ß-carotene binds only the heptamer. A co-crystal structure of the hexamer with farnesol reveals oriented binding in the center of the hydrophobic channel. Charged side chains engineered into the lumen of the heptamer facilitate binding of polar ligands: a glutamate variant binds a cationic variant of DPH, and introducing lysine allows binding of the biosynthetically important farnesol diphosphate.

Metabolite amyloid-like fibrils interact with model membranes.

Amyloid-like structure formation by various metabolites represents a significant extension of the amyloidogenic building block family. Similar to protein amyloids, metabolite amyloids induce apoptotic toxicity, a process that was linked to membrane association. Here, we demonstrate that metabolite amyloids interact with model membranes and study the mechanism by molecular dynamics.

Mesoporous silica for drug delivery: Interactions with model fluorescent lipid vesicles and live cells.

Formulated mesoporous silica nanoparticle (MSN) systems offer the best possible drug delivery system through the release of drug molecules from the accessible pores. In the present investigation, steady state and time resolved fluorescence techniques along with the fluorescence imaging were applied to investigate the interactions of dye loaded MSN with fluorescent unilamellar vesicles and live cells. Here 1,2-dimyristoyl-sn-glycero-3-phospocholine (DMPC) was used to prepare Small Unilamellar Vesicles (SUVs) as the model membrane with fluorescent 1,6-diphenyl-1,3,5-hexatriene (DPH) molecule incorporated inside the lipid bilayer. The interaction of DPH incorporated DMPC membrane with Fluorescein loaded MSN lead to the release of Fluorescein (Fl) dye from the interior pores of MSN systems. The extent of release of Fl and spatial distribution of the DPH molecule has been explored by monitoring steady-state fluorescence intensity and fluorescence lifetime at physiological condition. To investigate the fate of drug molecule released from MSN, fluorescence anisotropy has been used. The drug delivery efficiency of the MSN as a carrier for doxorubicin (DOX), a fluorescent chemotherapeutic drug, has also been investigated at physiological conditions. The study gives a definite confirmation for high uptake and steady release of DOX in primary oral mucosal non-keratinized squamous cells in comparison to naked DOX treatment.

1,6-Diphenyl-1,3,5-hexatriene (DPH) as a Novel Matrix for MALDI MS Imaging of Fatty Acids, Phospholipids, and Sulfatides in Brain Tissues.

1,6-Diphenyl-1,3,5-hexatriene (DPH) is a commonly used fluorescence probe for studying cell membrane-lipids due to its affinity toward the acyl chains in the phospholipid bilayers. In this work, we investigated its use in matrix-assisted laser desorption/ionization (MALDI) as a new matrix for mass spectrometry imaging (MSI) of mouse and rat brain tissue. DPH exhibits very minimal matrix-induced background signals for the analysis of small molecules (below m/z of 1000). In the negative ion mode, DPH permits the highly sensitive detection of small fatty acids (m/z 200-350) as well as a variety of large lipids up to m/z of 1000, including lyso-phospholipid, phosphatidic acid (PA), phosphoethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), and sulfatides (ST). The analytes were mostly detected as the deprotonated ion [M - H]-. Our results also demonstrate that sublimated DPH is stable for at least 24 h under the vacuum of our MALDI mass spectrometer. The ability to apply DPH via sublimation coupled with its low volatility allows us to perform tissue imaging of the above analytes at high spatial resolution. The degree of lipid fragmentation was determined experimentally at varying laser intensities. The results illustrated that the use of relatively low laser energy is important to minimize the artificially generated fatty acid signals. On the other hand, the lipid fragmentation obtained at higher laser energies provided tandem MS information useful for lipid structure elucidation.

Bioactivity In Vitro of Quercetin Glycoside Obtained in Beauveria bassiana Culture and Its Interaction with Liposome Membranes.

Quercetin (Q) was used as substrate for regioselective glycosylation at the C-7 position catalyzed by Beauveria bassiana AM278 strain. As a result the glycoside quercetin 7-O-ß-d-(4″-O-methyl)glucopyranoside (Q 7-MeGlu) was formed. The goal of the studies was to determine the anti-oxidative (liposome membrane protection against free radicals IC50Q 7-MeGlu = 5.47 and IC50Q = 4.49 µM) and anti-inflammatory (COX-1 and COX-2 enzymes activity inhibition) properties of Q 7-MeGlu as compared to Q. Every attempt was made to clarify the antioxidant activity of these molecules, which are able to interact with egg phosphatidylcholine liposomes, using a fluorometric method (by applying the probes MC540, TMA-DPH and DPH). The results indicated that Q 7-MeGlu and Q are responsible for increasing the packing order, mainly in the hydrophilic but also in hydrophobic regions of the membrane (Q > Q 7-MeGlu). These observations, confirmed by a ¹H-NMR method, are key to understanding their antioxidant activity which is probably caused by the stabilizing effect on the lipid membranes. The results showed that Q 7-MeGlu and Q have ability to quench the human serum albumin (HSA) intrinsic fluorescence through a static quenching mechanism. The results of thermodynamic parameters indicated that the process of formation complexes between studied molecules and HSA was spontaneous and caused through Van der Waals interactions and hydrogen bonding.

Membrane fluidity is a driving force for recoverin myristoyl immobilization in zwitterionic lipids.

Recoverin is the only protein for which the phenomenon of calcium-myristoyl switch has been demonstrated without ambiguity. It is located in rod disk membranes where the highest content in polyunsaturated lipid acyl chains can be found. However, although essential to better understand the inactivation of the phototransduction process, the role of membrane fluidity on recoverin recruitment is unclear. We have therefore investigated the immobilization of the recoverin myristoyl moiety in the presence of phosphocholine bilayers using 2H solid-state NMR spectroscopy. Several lipids with different acyl chains were selected to investigate model membranes characterized by different fluidity. Immobilization of the recoverin myristoyl moiety was successfully observed but only in the presence of calcium and in specific lipid disordered states, showing that an optimal fluidity is required for recoverin immobilization.

Interaction of Selected Phenylpropenes with Dipalmitoylphosphatidylcholine Membrane and Their Relevance to Antibacterial Activity.

The effect of structurally closely related phenylpropenes (PPs), estragole, anethole, eugenol, and isoeugenol, on the fluidity of dipalmitoyl phosphatidyl choline (DPPC) liposome membrane was investigated by DSC, Raman, and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Liposomes were prepared by thin-film hydration method at various DPPC:PP molar ratios. The DPH anisotropy measurements of blank and PP-loaded liposomes were performed at 28, 41, and 50 °C, which correspond, respectively, to gel phase, main transition temperature of DPPC, and liquid phase. The Raman images showed the formation of nano- and micrometric spherical multi-lamellar vesicles. All studied PPs exhibited a membrane fluidizing effect which was reinforced by the presence of phenolic hydroxyl group in eugenol and isoeugenol. The PPs interacted with the choline head group and the alkyl chains of DPPC membrane, wherein isoeugenol and anethole possessing the same C7-C8 position of the double bond in the propenyl side chain, incorporated deeply in the bilayer. Additionally, the PPs were analyzed for antibacterial activity against E. coli by macrobroth dilution method. Anethole and estragole were more efficient in inhibiting the bacterial growth than eugenol and isoeugenol. We conclude that the fluidizing effect of PPs on the membrane is a common mechanism that is not related to the hydrophobicity of the PP molecule. Besides, other target sites may be involved in PP antibacterial activity against Gram-negative bacteria. The greater hydrophobicity of these PPs may contribute to their penetrability through the outer bacterial membrane.

Transmembrane Difference in Colloid Osmotic Pressure Affects the Lipid Membrane Fluidity of Liposomes Encapsulating a Concentrated Protein Solution.

A hemoglobin vesicle (Hb-V) is an artificial oxygen carrier encapsulating a highly concentrated hemoglobin solution (40 g/dL) in a liposome. The in vivo safety and efficacy of Hb-V suspension as a transfusion alternative and structural stability during storage have been studied extensively. Because the intraliposomal Hb aqueous solution can possess colloid osmotic pressure (COP, 200-300 Torr) that is much higher than that of blood plasma (20-25 Torr), a question arises as to whether the lipid membrane senses the transmembrane difference in COP. We examined the membrane microviscosity using a fluorescence polarization technique. To avoid the interference of red Hb on the fluorescence measurement, we used human serum albumin (HSA) as a substitute for Hb. Both HSA and Hb solutions show high COP depending on the concentration. Encapsulation of HSA solution (40 g/dL) in the liposome decreased the membrane microviscosity at a lower temperature (949 ± 8 cP → 607 ± 10 cP at 25 °C). The result indicates that the transmembrane osmotic stress induced by HSA encapsulation expands the liposome maximally with increasing spherical surface area, and the membrane fluidity is increased extremely. Even for such a condition, the lowest membrane microviscosity, 377 ± 10 cP at 60 °C, is much higher than that of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine liposome (40 ± 2 cP at 60 °C). Accordingly, Hb-V as well as HSA-V maintains a spherical structure and mechanical stability under transmembrane stress caused by high COP, as described in the literature.

Temperature and pH sensitivity of a stabilized self-nanoemulsion formed using an ionizable lipid-like material via an oil-to-surfactant transition.

Lipids functionalized with tertiary amines (ionizable lipids) for a pH-dependent positive charge have been developed extensively as a carrier material for delivering nucleic acids. We previously developed an SS-cleavable proton-activated lipid-like material (ssPalm) as a component of a functionalized lipid envelope structure of a nanoparticle that encapsulated plasmid DNA and short interfering RNA. In this study, we report on the unique characteristics of such an ionizable lipid: the formation of a nano-sized emulsion (ave. 40nm) via pH-triggered self-emulsification in the absence of a cargo (nucleic acids). The particle has a neutral charge at physiological pH and is stabilized by helper lipids and polyethyleneglycol (PEG)-conjugated lipids. The generalized polarization of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which indicates the surface polarity caused by the invasion of water onto the surface, changes dynamically in response to pH and temperature, while the fluidity of the intra-particle compartment, as measured by the fluorescence anisotropy of 1,6-Diphenyl-1,3,5-hexatriene (DPH), is not affected. Even when the particle contains a high density of PEG on the surface, it shows a high fusogenecity to negatively charged liposomes in response to an acidic pH to a higher degree than a conventional cationic lipid. These characteristics suggest that the ssPalm particle possesses unique properties for delivering lipophilic drugs across the biomembrane.

Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit Remarkable Properties Forming Lamellar Phases and Giant Vesicles.

Tailor-made ionic liquids based on imidazolium salts have recently attracted a large amount of attention because of their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes has already been reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (Cn-IMe·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 the 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 via a 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 the addition of other lipids. These 4,5-dialkylimidazolium salts are highly eligible for further biochemical engineering and drug delivery.