Model Lipid Membranes Research Articles

Is lipophilicity the main factor determining the effective drug penetration through lipid membranes? Combined GIXD and PM-IRRAS studies on the example of anthracyclines

Cell membranes constitute the first barrier for the drugs on their way to the cell. Therefore, their penetration determines the effectivity of the treatment and is defined by the drug-lipid interactions. A systematic, comprehensive study to assess the impact of different aspects influencing the effective penetration of model lipid membranes by the drugs including lipophilicity, charge, ability to form dimers as well as stearic hindrance was performed. Four anthracyclines differing in the structure and with lipophilicity increasing in the following order: doxorubicin (DOx) < pirarubicin (THP) < daunorubicin (DNR) < idarubicin (IDA) were chosen to assess their impact on 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) monolayers mimicking cancer cell membranes. The investigation performed at the air–water interface involved the application of multiple surface-sensitive techniques to assess the drug interactions with both polar heads of the phospholipid and the penetration of the acyl chain region. The obtained results allowed us to state that although electrostatic interactions and lipophilicity of the drug are one of the key factors, the ability to form dimers as well as stearic hinderance may strongly prevent from the effective penetration of the phospholipid monolayers even for drugs with higher lipophilicity and therefore have to be also taken into account in the development of new therapeutic agents.

Analysis of the Polyphenolic Composition of Vaccinium L. Extracts and Their Protective Effect on Red Blood Cell Membranes

The blueberry fruit of the genus Vaccinium, including high blueberry, low blueberry, and wild bilberry, is consumed for its flavor and medicinal properties. The purpose of the experiments was to investigate the protective effect and mechanism of the interaction of blueberry fruit polyphenol extracts with the erythrocytes and their membranes. The content of polyphenolic compounds in the extracts was determined using the chromatographic UPLC–ESI–MS method. The effects of the extracts on red blood cell shape changes, hemolysis and osmotic resistance were examined. Changes in the order of packing and fluidity of the erythrocyte membrane and the lipid membrane model caused by the extracts were identified using fluorimetric methods. Erythrocyte membrane oxidation was induced by two agents: AAPH compound and UVC radiation. The results show that the tested extracts are a rich source of low molecular weight polyphenols that bind to the polar groups of the erythrocyte membrane, changing the properties of its hydrophilic area. However, they practically do not penetrate the hydrophobic part of the membrane and do not damage its structure. Research results suggest that the components of the extracts can defend the organism against oxidative stress if they are delivered to the organism in the form of dietary supplements.

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C4TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C4TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C4TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C4TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C4TPP-X series, with the exception of C4TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.

Long-Time Dynamics of Selected Molecular-Motor Components Using a Physics-Based Coarse-Grained Approach.

Molecular motors are essential for the movement and transportation of macromolecules in living organisms. Among them, rotatory motors are particularly efficient. In this study, we investigated the long-term dynamics of the designed left-handed alpha/alpha toroid (PDB: 4YY2), the RBM2 flagellum protein ring from Salmonella (PDB: 6SD5), and the V-type Na+-ATPase rotor in Enterococcus hirae (PDB: 2BL2) using microcanonical and canonical molecular dynamics simulations with the coarse-grained UNRES force field, including a lipid-membrane model, on a millisecond laboratory time scale. Our results demonstrate that rotational motion can occur with zero total angular momentum in the microcanonical regime and that thermal motions can be converted into net rotation in the canonical regime, as previously observed in simulations of smaller cyclic molecules. For 6SD5 and 2BL2, net rotation (with a ratcheting pattern) occurring only about the pivot of the respective system was observed in canonical simulations. The extent and direction of the rotation depended on the initial conditions. This result suggests that rotatory molecular motors can convert thermal oscillations into net rotational motion. The energy from ATP hydrolysis is required probably to set the direction and extent of rotation. Our findings highlight the importance of molecular-motor structures in facilitating movement and transportation within living organisms.

Enzymatically Polymerized Organic Conductors on Model Lipid Membranes.

Seamless integration between biological systems and electrical components is essential for enabling a twinned biochemical-electrical recording and therapy approach to understand and combat neurological disorders. Employing bioelectronic systems made up of conjugated polymers, which have an innate ability to transport both electronic and ionic charges, provides the possibility of such integration. In particular, translating enzymatically polymerized conductive wires, recently demonstrated in plants and simple organism systems, into mammalian models, is of particular interest for the development of next-generation devices that can monitor and modulate neural signals. As a first step toward achieving this goal, enzyme-mediated polymerization of two thiophene-based monomers is demonstrated on a synthetic lipid bilayer supported on a Au surface. Microgravimetric studies of conducting films polymerized in situ provide insights into their interactions with a lipid bilayer model that mimics the cell membrane. Moreover, the resulting electrical and viscoelastic properties of these self-organizing conducting polymers suggest their potential as materials to form the basis for novel approaches to in vivo neural therapeutics.

Lung SPLUNC1 Peptide Derivatives in the Lipid Membrane Headgroup Kill Gram-Negative Planktonic and Biofilm Bacteria.

SPLUNC1 (short palate lung and nasal epithelial clone 1) is a multifunctional host defense protein found in human respiratory tract with antimicrobial properties. In this work, we compare the biological activities of four SPLUNC1 antimicrobial peptide (AMP) derivatives using paired clinical isolates of the Gram-negative (G(-)) bacteria Klebsiella pneumoniae, obtained from 11 patients with/without colistin resistance. Secondary structural studies were carried out to study interactions between the AMPs and lipid model membranes (LMMs) utilizing circular dichroism (CD). Two peptides were further characterized using X-ray diffuse scattering (XDS) and neutron reflectivity (NR). A4-153 displayed superior antibacterial activity in both G(-) planktonic cultures and biofilms. NR and XDS revealed that A4-153 (highest activity) is located primarily in membrane headgroups, while A4-198 (lowest activity) is located in hydrophobic interior. CD revealed that A4-153 is helical, while A4-198 has little helical character, demonstrating that helicity and efficacy are correlated in these SPLUNC1 AMPs.

Metal Complexes of Omadine (N-Hydroxypyridine-2-thione): Differences of Antioxidant and Pro-Oxidant Behavior in Light and Dark Conditions with Possible Toxicity Implications.

Omadine or N-hydroxypyridine-2-thione and its metal complexes are widely used in medicine and show bactericidal, fungicidal, anticancer, and photochemical activity. The redox activity of omadine complexes with iron, copper, and zinc on lipid peroxidation under light and dark conditions has been investigated. The monitoring of the oxidation of linoleic acid micelles, resembling a model of lipid membrane, was carried out using nuclear magnetic resonance (1H-NMR). It has been shown that the omadine-zinc complex can induce the oxidation of linoleic acid under light irradiation, whereas the complexes with iron and copper are photochemically stable. All the chelating complexes of omadine appear to be redox-inactive in the presence of hydrogen peroxide under dark conditions. These findings suggest that omadine can demonstrate antioxidant behavior in processes involving reactive oxygen species generation induced by transition metals (Fenton and photo-Fenton reactions). However, the omadine complex with zinc, which is widely used in shampoos and ointments, is photochemically active and may cause oxidative cell membrane damage when exposed to light, with possible implications to health.

Interactions between Layered Double Hydroxide Nanoparticles and Egg Yolk Lecithin Liposome Membranes.

The burgeoning need to study the applications of nanoparticles (NPs) in biomedical and pharmaceutical fields requires an understanding of their interactions with lipid membranes for further in vivo studies. In this paper, negatively charged egg yolk lecithin liposome (EYL) has been prepared and used as model lipid membranes. Positively charged Mg3Al-layered double hydroxides (LDHs) are viewed as models of clay particles. The ability of the LDH NPs, a two-dimensional nanostructure with an average diameter of 100 nm (LDHs-100) or 500 nm (LDHs-500) to cross the membranes, has been thoroughly investigated via (high-resolution) transmission electron microscopy (TEM), optical microscopy (OM), scanning electron microscopy (SEM), confocal fluorescence microscopy (CLSM), and dynamic light scattering (DLS). The liposomes with an average diameter of 1.5 μm were prepared by the thin-film rehydration method followed by an extrusion technique. A calcein leakage assay and steady-state fluorescence measurement displayed the variation of membrane integrity and polarity of the pyrene-located microenvironment during the interaction between EYL and calcein-interacted LDH NPs (CE-LDHs) or LDH NPs, respectively. These results imply that not only spherical particles but also even more sophisticated nanostructured materials are able to effectively cross the lipid bilayers, thereby engineering new compounds that may be encapsulated for safe and potential use in biomedical applications.

Effect of Desmosterol, Lathosterol and Coprostanol on the phase behaviour of phospholipid membranes

Sterols have played an important role in membrane evolution. The effect of these molecules, particularly the naturally occurring ones, such as cholesterol, ergosterol, lanosterol, phytosterols and oxysterols on the properties of lipid model membranes have been extensively investigated. Unfortunately, other naturally occurring sterols such as desmosterol and lathosterol have received very little attention. Using small angle X-ray scattering (SAXS), we have carried out detailed investigations on the influence of desmosterol and lathosterol along with coprostanol on the phase behaviour of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes. SAXS results indicate both that these sterols modulate the properties of the DPPC bilayer in a manner similar to cholesterol. However, we have found desmosterol to be more efficient than cholesterol in suppressing the main transition of DPPC, with the fluid phase stabilized at around 15 mol% (compared to around 20 mol% in cholesterol). Coprostanol on the other hand was found to be less efficient in stabilizing the fluid phase than the other two. The results obtained from our study in combination with those existing in the literature will help in establishing a complete picture of how different sterols influence the properties of lipid membranes.

Modulation of the Dipole Potential of Model Lipid Membranes with Phytochemicals: Molecular Mechanisms, Structure-Activity Relationships, and Implications in Reconstituted Ion Channels.

Phytochemicals, such as flavonoids, stilbenoids, alkaloids, terpenoids, and related compounds, have a wide range of useful pharmacological properties which cannot be ascribed to binding to a single peptide or protein target alone. Due to the relatively high lipophilicity of phytochemicals, the lipid membrane is thought to mediate their effects via changes in the properties of the lipid matrix, in particular, by modulating the transmembrane distribution of the electrical potential and, consequently, the formation and functioning of the ion channels reconstituted in the lipid bilayers. Therefore, biophysical studies on the interactions between plant metabolites and model lipid membranes are still of interest. This review represents an attempt to provide a critical analysis of a variety of studies on altering membranes and ion channels with phytochemicals via disturbing the potential drop at the membrane-aqueous solution interface. Critical structural motifs and functioning groups in the molecules of plant polyphenols (alkaloids and saponins are identified) and the possible mechanisms of dipole potential modulation with phytochemicals are discussed.

Novel Helical Trp- and Arg-Rich Antimicrobial Peptides Locate Near Membrane Surfaces and Rigidify Lipid Model Membranes.

Antibiotics are losing effectiveness as bacteria become resistant to conventional drugs. To find new alternatives, antimicrobial peptides (AMPs) are rationally designed with different lengths, charges, hydrophobicities (), and hydrophobic moments (), containing only three types of amino acids: arginine, tryptophan, and valine. Six AMPs with low minimum inhibitory concentrations (MICs) and <25% toxicity to mammalian cells are selected for biophysical studies. Their secondary structures are determined using circular dichroism (CD), which finds that the % -helicity of AMPs depends on composition of the lipid model membranes (LMMs): gram-negative () inner membrane (IM) >gram-positive () > Euk33 (eukaryotic with 33 mol% cholesterol). The two most effective peptides, E2-35 (16 amino acid [AA] residues) and E2-05 (22 AAs), are predominantly helical in IM and LMMs. AMP/membrane interactions such as membrane elasticity, chain order parameter, and location of the peptides in the membrane are investigated by low-angle and wide-angle X-ray diffuse scattering (XDS). It is found that headgroup location correlates with efficacy and toxicity. The membrane bending modulus displays nonmonotonic changes due to increasing concentrations of E2-35 and E2-05 in and LMMs, suggesting a bacterial killing mechanism where domain formation causes ion and water leakage.

Interfacial adsorption and activity of pancreatic lipase-related protein 2 onto heterogeneous plant lipid model membranes

Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depend greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and the physical state of its substrate. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipids (GL), and/or phospholipids (PL), and/or phytosterols (pS), presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their gPLRP2 catalyzed digestion kinetics were investigated in presence or in absence of bile salts (NaTDC) during static in vitro, so called “bulk”, digestion.The enzymatic activity of gPLRP2 onto the galactolipid-based monolayers was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids. However, the compositional complexity in the heterogeneous uncharged model systems induced a lag phase before the initiation of lipolysis. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes.

Cholesterol and Melatonin Exert Opposite Effects on the Interaction of Model DMPC Membranes with the SARS‐CoV‐2 S‐Protein: A SANS Study

AbstractAn effect of receptor‐binding domain (RBD) of SARS‐CoV‐2 S‐protein on structural parameters of model lipid membranes presented by dimyristoylphosphatidylcholine (DMPC) systems with cholesterol and melatonin impurities is studied by small angle neutron scattering (SANS). It is shown that an increase in melatonin concentration in the lipid membrane leads to a decrease in the thickness of the lipid bilayer, while an increase in the concentration of cholesterol leads to an increase in its thickness. It is suggested that increasing the concentration of melatonin in a membrane prevents the interaction of coronaviral S‐protein with a lipid membrane of a cell. In the presence of cholesterol in the system, the interaction of a lipid membrane with an active part of S‐protein occurs depending on a phase state of the lipid: in the case of a gel phase, there is no changes in structural parameters, but at higher temperatures in the case of a liquid crystal phase, an addition of RBD SARS‐CoV‐2 to the system causes a reduce in the membrane thickness.

Activation energy for pore opening in lipid membranes under an electric field

The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-μm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.

A Biophysical Research on the Determination of Morphological and Structural Properties of Coumarin-Loaded Liposomes

The major goal of this research was to examine how coumarin affects lipid model membranes. For this reason, liposome membranes were formed using dimyristoyl phosphatidylcholine (DMPC) as zwitterionic lipid. The influence of coumarin on the morphology, packing order, fluidity, and hydration state of lipid membranes was specifically investigated by means of microscopic (field emission scanning electron microscopy (FE-SEM)) and spectroscopic (attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy) techniques. Taken into account the results obtained with FE-SEM images and analysis, liposomes without and with coumarin have uniform structures and spherical shapes in appearance. However, coumarin-loaded liposomes are observed with an increase in size when compared to a mean diameter of unloaded-liposomes. Considering ATR-FTIR analysis, the investigation of the vibrational bands which belong to the hydrophobic and hydrophilic parts of DMPC lipid reveals that coumarin alters the physical features of the DMPC liposomes by decreasing the order and increasing the fluidity of the system and making hydrogen bonding with the interfacial and headgroup regions of zwitterionic lipid DMPC. Finally, performing more biophysical studies on the interactions of biologically active compounds with model membranes plays an important role in determining the molecular action mechanisms of these compounds in drug discovery and formulations.

Polar localization of new flavonoids from aerial parts of Scleranthus perennis and Hottonia palustris and their modulatory action on lipid membranes properties

The aim of this study was to characterize, for the first time, the interactions, location, and influence of flavonoids isolated from aerial parts of Scleranthus perennis (Caryophyllaceae) and Hottonia palustris (Primulaceae) on the properties of model lipid membranes prepared from dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine (EYPC). The tested compounds incorporated into liposomes into the region of the polar heads or at the water/membrane interface of DPPC phospholipids. Spectral effects accompanying the presence of polyphenols revealed their effect on ester carbonyl groups apart from SP8. All polyphenols brought about reorganization of the polar zone of liposomes as it was observed by FTIR technique. Additionally, fluidization effect was noted in the region of symmetric and antisymmetric stretching vibrations of the CH2 and CH3 groups with exception to HZ2 and HZ3. Similarly, in EYPC liposomes, they interacted mainly with the regions of the choline heads of the lipids and had various effects on the carbonyl ester groups with exception to SP8. The region of polar head groups is restructured due to the presence of the additives in liposomes. The outcomes obtained using the NMR technique confirmed the locations of all of the tested compounds in the polar zone and indicated a flavonoid-dependent modifying effect towards lipid membranes. HZ1 and SP8 raised motional freedom in this region whereas opposite effect was revealed for HZ2 and HZ3. In the hydrophobic region restricted mobility was noted. In this report we discuss the mechanism of previously undescribed flavonoids in terms of their actions on membranes.

The role of hydrophobic patches of de novo designed MSI-78 and VG16KRKP antimicrobial peptides on fragmenting model bilayer membranes

Antimicrobial peptides (AMPs) with cell membrane lysing capability are considered potential candidates for the development of the next generation of antibiotics. Designing novel AMPs requires an in-depth understanding of the mechanism of action of the peptides. In this work, we used various biophysical techniques including 31P solid-state NMR to examine the interaction of model membranes with amphipathic de novo-designed peptides. Two such peptides, MSI-78 and VG16KRKP, were designed with different hydrophobicity and positive charges. The model lipid membranes were constituted by mixing lipids of varying degrees of ‘area per lipid’ (APL), which directly affected the packing properties of the membrane. The observed emergence of the isotropic peak in 31P NMR spectra as a function of time is a consequence of the fragmentation of the membrane mediated by the peptide interaction. The factors such as the charges, overall hydrophilicity of the AMPs, as well as lipid membrane packing, contributed to the kinetics of membrane fragmentation. Furthermore, we anticipate the designed AMPs follow the carpet and toroidal pore mechanisms when lysing the cell membrane. This study highlights the significance of the effect of the overall charges and the hydrophobicity of the novel AMPs designed for antimicrobial activity.

Phospholipases and Membrane Curvature: What Is Happening at the Surface?

In this revision work, we emphasize the close relationship between the action of phospholipases and the modulation of membrane curvature and curvature stress resulting from this activity. The alteration of the tridimensional structure of membranes upon the action of phospholipases is analyzed based on studies on model lipid membranes. The transient unbalance of both compositional and physical membrane properties between the hemilayers upon phospholipase activity lead to curvature tension and the catalysis of several membrane-related processes. Several proteins' membrane-bound and soluble forms are susceptible to regulation by the curvature stress induced by phospholipase action, which has important consequences in cell signaling. Additionally, the modulation of membrane fusion by phospholipase products regulates membrane dynamics in several cellular scenarios. We commented on vesicle fusion in the Golgi-endoplasmic system, synaptic vesicle fusion to the plasma membrane, viral membrane fusion to host cell plasma membrane and gametes membrane fusion upon acrosomal reaction. Furthermore, we explored the modulation of membrane fusion by the asymmetric adsorption of amphiphilic drugs. A deep understanding of the relevance of lipid membrane structure, particularly membrane curvature and curvature stress, on different cellular events leads to the challenge of its regulation, which may become a powerful tool for pharmacological therapy.

Use of scattering techniques to probe molecular interactions between novel Trp- and Arg-rich antimicrobial peptides (AMPs) in lipid model membranes (LMMs).

Antibiotics, one of the most important medical developments of the twentieth century, are losing effectiveness as diverse bacteria become resistant to standard and last resort commercial drugs. To address this challenge and provide alternatives to traditional antibiotics, we have rationally designed AMPs with different lengths, charges, hydrophobicities (H) and hydrophobic moments(µH). Five AMPs (out of 86) with the lowest minimum inhibitory concentrations (MICs) and <25% toxicity to mammalian cells were selected for biophysical studies. Their secondary structure was determined using circular dichroism (CD), which found that % α-helicity of AMPs depends on lipid composition. There was decreasing helicity with these LMMs: G(-) inner membrane (IM)> G(+) > Euk33 (eukaryotic with 33 mol% cholesterol). E2-35 (16-mer) and E2-05 (22-mer) are largely helical in G(-) IM LMM, (PE:PG:CL,7:2:1) and G(+) LMM (PG:DOTAP:PE:CL,6:1.5:1.5:1). LE-53 (12-mer) and LE-55 (16-mer) are mostly random coil when interacting with both G(-) IM and G(+) LMMs. The higher α-helical content of E2-35 and E2-05 correlated with a lower MIC and higher toxicity, suggesting that helicity is important for designing new AMPs. Other aspects of AMP/membrane interactions such as membrane elasticity, chain order parameter and location of the peptides in the membrane were investigated by low angle and wide angle x-ray diffuse scattering (XDS). A headgroup or interfacial location correlated with toxicity. The membrane bending modulus Kc displayed a non-monotonic change due to E2-35 and E2-05 perturbation in LMMs. The chain order parameter (Sxray) paralleled Kc in most cases. We suggest that juxtaposition of domains with different bending moduli could lead to leakage of ions and water along domain walls between softer and more rigid domains, thus killing the bacteria.

Exploring the self-quenching of fluorescent probes incorporated into model lipid membranes using electrophoresis and fluorescence lifetime imaging microscopy.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility and interactions of biomolecules. However, fluorophores can undergo “self-quenching” of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artefacts in fluorescence images and is relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores within supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of Texas Red (TR) fluorophores were generated within 100 × 100 µm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively-charged TR molecules towards the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mole/mole), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mole/mole), leading to maximal quenching of 30% up to 70%. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The shape of the calculated concentration profiles revealed evidence of subtle lipid-lipid interactions at high packing densities. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.