Headgroup Region Research Articles

Abstract 16947: Sotagliflozin, a Dual SGLT 1 and 2 Inhibitor, Has High Membrane Affinity and Hydrocarbon Core Location Compared With Empagliflozin

Background: The dual SGLT-1 and 2 inhibitor sotagliflozin (sota) reduced atherothrombotic events and hospitalization for worsening heart failure in patients with diabetes in clinical trials (SCORED, SOLOIST). Unlike selective SGLT2 inhibitors such as empagliflozin (empa), sota binds with higher affinity to SGLT1, which is located in the intestine, kidney, heart, brain, and vascular endothelial cells. To elucidate the physico-chemical basis for differences in tissue distribution and receptor affinity, we compared the effects of these drugs on membrane binding and molecular location. Methods: Membranes reconstituted from cardiac phospholipid and cholesterol were used to measure drug partitioning across a range of cholesterol concentrations. Multilamellar vesicles were prepared as mixtures of cardiac phospholipid (CPL), cholesterol, and each agent at 30 μM or vehicle. Drug-membrane partitioning was determined by rapid centrifugation and UV/Vis spectrophotometry. In parallel experiments, we used small angle x-ray scattering (SAXS) to identify drug location in model membranes containing phosphatidylcholine and cholesterol. Results: Sota had a membrane partition coefficient (8590 ± 2078) that was 16-fold higher than empa (519 ± 195, p<0.001) in CPL membranes. This high membrane affinity was preserved over a range of cholesterol levels compared to empa (p<0.001). Consistent with high membrane affinity, SAXS analysis showed that sota had a location evidenced by a broad increase in electron density in the hydrocarbon core extending to approximately 20 Å from the membrane center. By contrast, empa had a location limited to the polar headgroup region, consistent with its lower lipophilicity and tissue penetration. Conclusions: Sota had high membrane affinity and distinct location in the hydrocarbon core compared to empa. This may contribute to the effect of its dual SGLT-1 and 2 inhibition in vivo across various tissues including the heart. These distinct membrane interactions may also partially explain the reduced atherothrombotic risk as demonstrated in outcome trials with sota but not selective SGLT-2 inhibitors.

Capric Acid and Myristic Acid Permeability Enhancers in Curved Liposome Membranes.

Liposomes are considered as advanced drug delivery systems for cancer treatment. A generation of pH-sensitive liposomes is being developed that use fatty acids (FAs) as a trigger for drug release in tumor tissues. However, FAs are also known to enhance permeability, and it is unclear whether FAs in liposomes may cause drug leakage or premature drug release. The passive permeability of the drug through the membrane of the liposome is thus a crucial factor for timely drug delivery. To investigate how the curvature and lipid composition of liposomes affect their passive permeability, coarse-grained molecular dynamics were performed. The permeability was determined with a counting method. Flat bilayers and three liposomes with varying diameters were studied, which had varying lipid compositions of dipalmitoylphosphatidylcholine, cholesterol, and deprotonated or neutral saturated FAs. The investigated permeants were water and two other small permeants, which have different free energy profiles (solubility) across the membrane. First, for the curvature effect, our results showed that curvature increases the water permeability by reducing the membrane thickness. The permeability increase for water is about a factor of 1.7 for the most curved membranes. However, a high curvature decreases permeability for permeants with free energy profiles that are a mix of wells and barriers in the headgroup region of the membrane. Importantly, the type of experimental setup is expected to play a dominant role in the permeability value, i.e., whether permeants are escaping or entering the liposomes. Second, for the composition effect, FAs decrease both the area per lipid (APL) and the membrane thickness, resulting in permeability increases of up to 55%. Cholesterol has a similar effect on the APL but has the opposite impact on membrane thickness and permeability. Therefore, FAs and cholesterol have opposing effects on permeability, with cholesterol's effect being slightly stronger in our simulated bilayers. As all permeability values were well within a factor of 2, and with liposomes usually being larger and less curved in experimental applications, it can be concluded that the passive drug release from a pH-sensitive liposome does not seem to be significantly affected by the presence of FAs.

Effect of Host Cholesterol on the Membrane Dynamics of Outer Membrane Lipids of Mycobacteria.

The ability of Mycobacterium tuberculosis to remain dormant after primary infection represents the prime cause of new TB cases throughout the world. Hence, diagnosis and treatment of individuals hosting dormant mycobacterium is one of the crucial strategies to be adopted for the prevention of Tuberculosis. Among many strategies unleashed by the latent bacterium, one of them is scavenging host cholesterol for carbon source. Cholesterol modifies lipid membranes over many scales and here, its effect on mycobacterial membrane biophysics and the subsequent effect on partitioning of antibiotics into cholesterol- enriched mycobacterial membranes was investigated. Our research showed that cholesterol alters the phase state behavior of mycobacterial outer membrane lipids by enhancing the overall membrane order at the headgroup and acyl chain region and is integrated into both ordered and disordered domains/phases, with a preference for the latter. Exogenous cholesterol further alters the drug partitioning behavior of structurally different drugs, pointing to a larger clinical potential of using more hydrophobic medications to target dormant bacteria.

Investigation of the Membrane Localization and Interaction of Selected Flavonoids by NMR and FTIR Spectroscopy.

In this report, we discuss the effects of undescribed flavone derivatives, HZ4 and SP9, newly isolated from the aerial parts of Hottonia palustris L. and Scleranthus perennis L. on membranes. Interaction of flavonoids with lipid bilayers is important for medicinal applications. The experiments were performed with FTIR and NMR techniques on liposomes prepared from DPPC (dipalmitoylphosphatidylcholine) and EYPC (egg yolk phosphatidylcholine). The data showed that the examined polyphenols incorporate into the polar head group region of DPPC phospholipids at both 25 °C and 45 °C. At the lower temperature, a slight effect in the spectral region of the ester carbonyl group is observed. In contrast, at 45 °C, both compounds bring about the changes in the spectral regions attributed to antisymmetric and symmetric stretching vibrations of CH2 and CH3 moieties. Similarly, as in DPPC lipids, the tested compounds interact with the fingerprint region of the polar head groups of the EYPC lipids and cause its reorganization. The outcomes obtained by NMR analyses confirmed the localization of both flavonoids in the polar heads zone. Unraveled effects of HZ4 and SP9 in respect to lipid bilayers can partly determine their biological activities and are crucial for their usability in medicine as disease-preventing phytochemicals.

Hydrophobic Interfacing of Fluorescent Membrane Probes

AbstractFluorescent flippers have been introduced as small‐molecule probes to image membrane tension in living systems. While the hydrophilic headgroup region has been modified extensively for intracellular targeting, little is known about the hydrophobic interfacing with the surrounding membrane. To tackle this challenge, the design, synthesis and evaluation of a glutamine‐derived flipper collection is reported. Considering the importance of tension‐induced phase separation for tension imaging, this study is focused on how to modulate the distribution of functional flippers between ordered and disordered microdomains. Also of interest was control over intermembrane transfer without loss of function for the specific labeling of plasma and intracellular membranes. Evidence is presented for a two‐step insertion mechanism through more accessible disordered domains into better matching ordered domains. This process also explains differences between partition coefficients and bioimaging. It is further demonstrated that interdomain and intermembrane distribution can be regulated by hydrophobic interfacing to control brightness in fluorescence lifetime imaging microscopy and responsiveness to membrane tension. Irreversible partitioning inhibits intermembrane transfer and coincides with internalization into cells. These results demonstrate that hydrophobic interfacing can improve probe performance and provide guidelines on how to proceed.

Effect of aromatic ring on surface and self‐assembly properties of synthesized heterocyclic‐based surfactants

AbstractHeterocyclic‐based cationic surfactants with dodecyl tail group bridged to the head group either via aromatic ether [3‐(4‐dodecyloxybenzyl)‐1‐methyl‐imidazolium bromide (Ar‐I) and 4‐(4‐dodecyloxybenzyl)‐4‐methyl‐morpholinium bromide (Ar‐M)] or via aliphatic ether [3‐(2‐dodecyloxyethyl)‐1‐methyl imidazolium bromide (Al‐I) and 4‐(2‐dodecyloxyethyl)‐4‐methyl morpholinium bromide (Al‐M)] were synthesized and characterized using different spectral techniques. For the synthesis of Ar‐I and Ar‐M, 4‐hydroxybenzoic acid was esterified initially, followed by O‐alkylation, reduction, bromination and finally quaternization either with 1‐methylimidazole or with 4‐methylmorpholine. For the synthesis of aliphatic analogues (Al‐I and Al‐M), ethylene glycol was initially alkylated, followed by tosylation, bromination and quaternization either with 1‐methylimidazole or with 4‐methylmorpholine. Synthesized surfactants were evaluated for surface and self‐assembly properties. Impact of the presence or absence of phenyl ring bridging the polar head and nonpolar tail of the surfactant and also type of heterocyclic moiety in the head group region on micellization and surface properties such as wetting, foaming and emulsification was studied. Surfactants with the bridged aromatic ether moiety were observed to display lower critical micelle concentration than those with the bridged aliphatic ether moiety irrespective of type of heterocyclic molecule in the head group region. Foam volume and stability of the aromatic ether‐bridged surfactant were observed to be superior to their corresponding aliphatic analogs. Similar trend was observed in stabilizing the emulsion. However, aromatic ether‐bridged surfactant depicted poor wetting ability compared to their aliphatic analog. Aliphatic ether bridged surfactant showed higher aggregation number compared to their aromatic counterparts, but depicted similar micellar core polarity.

Binding of βL-Crystallin with Models of Animal and Human Eye Lens-Lipid Membrane.

Several discoveries show that with age and cataract formation, β-crystallin binds with the lens membrane or associates with other lens proteins, which bind with the fiber cell plasma membrane, accompanied by light scattering and cataract formation. However, how lipids (phospholipids and sphingolipids) and cholesterol (Chol) influence β-crystallin binding to the membrane is unclear. This research aims to elucidate the role of lipids and Chol in the binding of β-crystallin to the membrane and the membrane's physical properties (mobility, order, and hydrophobicity) with β-crystallin binding. We used electron paramagnetic resonance (EPR) spin-labeling methods to investigate the binding of βL-crystallin with a model of porcine lens-lipid (MPLL), model of mouse lens-lipid (MMLL), and model of human lens-lipid (MHLL) membrane with and without Chol. Our results show that βL-crystallin binds with all of the investigated membranes in a saturation manner, and the maximum parentage of the membrane surface occupied (MMSO) by βL-crystallin and the binding affinity (Ka) of βL-crystallin to the membranes followed trends: MMSO (MPLL) > MMSO (MMLL) > MMSO (MHLL) and Ka (MHLL) > Ka (MMLL) ≈ Ka (MPLL), respectively, in which the presence of Chol reduces the MMSO and Ka for all membranes. The mobility near the headgroup regions of the membranes decreases with an increase in the binding of βL-crystallin; however, the decrease is more pronounced in the MPLL and MMLL membranes than the MHLL membrane. In the MPLL and MMLL membranes, the membranes become slightly ordered near the headgroup with an increase in βL-crystallin binding compared to the MHLL membrane. The hydrophobicity near the headgroup region of the membrane increases with βL-crystallin binding; however, the increase is more pronounced in the MPLL and MMLL membranes than the MHLL membrane, indicating that βL-crystallin binding creates a hydrophobic barrier for the passage of polar molecules, which supports the barrier hypothesis in cataract formation. However, in the presence of Chol, there is no significant increase in hydrophobicity with βL-crystallin binding, suggesting that Chol prevents the formation of a hydrophobic barrier, possibly protecting against cataract formation.

Nucleoside Analog Reverse-Transcriptase Inhibitors in Membrane Environment: Molecular Dynamics Simulations.

The behavior of four drugs from the family of nucleoside analog reverse-transcriptase inhibitors (zalcitabine, stavudine, didanosine, and apricitabine) in a membrane environment was traced using molecular dynamics simulations. The simulation models included bilayers and monolayers composed of POPC and POPG phospholipids. It was demonstrated that the drugs have a higher affinity towards POPG membranes than POPC membranes due to attractive long-range electrostatic interactions. The results obtained for monolayers were consistent with those obtained for bilayers. The drugs accumulated in the phospholipid polar headgroup region. Two adsorption modes were distinguished. They differed in the degree of penetration of the hydrophilic headgroup region. Hydrogen bonds between drug molecules and phospholipid heads were responsible for adsorption. It was shown that apricitabine penetrated the hydrophilic part of the POPC and POPG membranes more effectively than the other drugs. Van der Waals interactions between S atoms and lipids were responsible for this.

Statin Action Targets Lipid Rafts of Cell Membranes: GIXD/PM-IRRAS Investigation of Langmuir Monolayers.

Lipid rafts are condensed regions of cell membranes rich in cholesterol and sphingomyelin, which constitute the target for anticholesterolemic drugs - statins. In this work, we use for the first time a combined grazing-incidence X-ray diffraction (GIXD)/polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS)/Brewster angle microscopy (BAM) approach to show the statin effect on model lipid rafts and its components assembled in Langmuir monolayers at the air-water interface. Two representatives of these drugs, fluvastatin (FLU) and cerivastatin (CER), of different hydrophobicity were chosen, while cholesterol (Chol) and sphingomyelin (SM), and their 1:1 mixture were selected to form condensed monolayers of lipid rafts. The effect of statins on the single components of lipid rafts indicated that both the hydrophobicity of the drugs and the organization of the layer determined the drug-lipid interaction. For cholesterol monolayers, only the most hydrophobic CER was effectively changing the film structure, while for the less organized sphingomyelin, the biggest effect was observed for FLU. This drug affected both the polar headgroup region as shown by PM-IRRAS results and the 2D crystalline structure of the SM monolayer as evidenced by GIXD. Measurements performed for Chol/SM 1:1 models proved also that the statin effect depends on the presence of Chol-SM complexes. In this case, the less hydrophobic FLU was not able to penetrate the binary layer at all, while exposure to the hydrophobic CER resulted in the phase separation and formation of ordered assemblies. The changes in the membrane properties were visualized by BAM images and GIXD patterns and confirmed by thermodynamic parameters of hysteresis in the Langmuir monolayer compression-decompression experiments.

Transport of Heptane Molecules across Water-Vapor Interfaces Laden with Surfactants.

Molecular transport across liquid-vapor interfaces covered by surfactant monolayers plays a key role in applications such as fire suppression by foams. The molecular understanding of such transport, however, remains incomplete. This work uses molecular dynamics simulations to investigate the heptane transport across water-vapor interfaces populated with sodium dodecyl sulfate (SDS) surfactants. Heptane molecules' potential of mean force (PMF) and local diffusivity profiles across SDS monolayers with different SDS densities are calculated to obtain heptane's transport resistance. We show that a heptane molecule experiences a finite resistance as it crosses water-vapor interfaces covered by SDS. Such interfacial transport resistance is contributed significantly by heptane molecules' high PMF in the SDS headgroup region and their slow diffusion there. This resistance increases linearly as the SDS density rises from zero but jumps as the density approaches saturation when its value is equivalent to that afforded by a 5 nm thick layer of bulk water. These results are understood by analyzing the micro-environment experienced by a heptane molecule crossing SDS monolayers and the local perturbation it brings to the monolayers. The implications of these findings for the design of surfactants to suppress heptane transport through water-vapor interfaces are discussed.

Comparative analysis of a POPC bilayer and a DPC micelle comprising an interfacial anchored peptide using all-atom MD simulations

Abstract Biological membranes are complex systems due to their composition and dynamics. Therefore, membrane mimetics are widely used to investigate lipid properties and interactions between molecules and membrane lipids. Using all-atom molecular dynamics simulations, within this study two systems composed of different membrane mimetics are compared: a 1-palmitoyl-2-oleoyl-3-glycero-phosphatidylcholine (POPC) bilayer or a dodecylphosphocholine (DPC) micelle and a nonapeptide (V94-T-K-Y-W-F-Y-R-L102). Previous 1H-NMR experiments have demonstrated that, in the presence of DPC micelles, this peptide folds as a stable amphipathic helix located in the polar head group region with the tryptophan residue pointing toward the inside of the micelle. The present comparison reveals a hydrophobic surface twice as large for the micelle as for the bilayer and a different arrangement of the acyl chains. The peptide secondary structure is not strongly affected by the membrane mimetics whereas the peptide is more deeply inserted in the bilayer than in the micelle. The contacts between the peptide and the DPC or POPC molecules are analysed and although the distances and lifetimes of these contacts are very different in the micelle and the bilayer, similar specific interactions were found that mainly involved the side chains of the residues R101 and L102.

Phase transfer mechanisms of fluorophore-labeled cell-penetrating peptide ε-poly-l-α-lysine at liquid|liquid interfaces

The transfer reaction of a novel cell-penetrating peptide, ε-poly-l-α-lysine (εPL) as a bacterial polycationic isopeptide, was investigated at the water|1,2-dichloroethane (DCE) interface. Spectroelectrochemical analysis elucidated the interfacial reaction mechanism of covalently carboxyfluorescein (FAM)-labeled εPL (FAM-εPL) in detail. FAM-εPL showed the concentration-dependent interfacial mechanisms in the positive potential region, i.e., the adsorption from the aqueous side of the interface was occurred at the low concentration of FAM-εPL, while the ion transfer across the interface accompanied by the adsorption process at the organic side proceeded further as the concentration increased. The facilitated ion transfer of FAM-εPL was observed at the biomimetic interface with a phospholipid layer. The transfer process of FAM-εPL was accelerated by the specific interaction at the aqueous side of the biomimetic interface, that is, the polar headgroup region of the phospholipid layer. The interfacial and membrane permeation mechanisms of εPL were elucidated through the spectroelectrochemical measurements by the fluorescent FAM modification and the phase transfer of a membrane impermeable FAM was also achieved by forming the conjugate with εPL.

L-Phenylalanine Partitioning Mechanisms in Model Biological Membranes.

Time-resolved fluorescence spectroscopy in combination with differential scanning calorimetry (DSC) was used to study the chemical interactions that occur when l-phenylalanine is introduced to solutions containing phosphatidylcholine vesicles. Studies reported in this work address open questions about l-Phe's affinity for lipid vesicle bilayers, the effects of l-Phe partitioning on bilayer properties, l-Phe's solvation within a lipid bilayer, and the amount of l-Phe within that local solvation environment. DSC data show that l-Phe reduces the amount of heat necessary to melt saturated phosphatidylcholine bilayers from their gel to liquid-crystalline state but does not change the transition temperature (Tgel-lc). Time-resolved emission shows only a single l-Phe lifetime at low temperatures corresponding to l-Phe remaining solvated in aqueous solution. At temperatures close to Tgel-lc, a second, shorter lifetime appears that is assigned to l-Phe already embedded within the membrane that becomes hydrated as water starts to permeate the lipid bilayer. This new lifetime is attributed to a conformationally restricted rotamer in the bilayer's polar headgroup region and accounts for up to 30% of the emission amplitude. Results reported for dipalmitoylphosphatidylcholine (DPPC, 16:0) lipid vesicles prove to be general, with similar effects observed for dimyristoylphosphatidylcholine (DMPC, 14:0) and distearoylphosphatidylcholine (DSPC, 18:0) vesicles. Taken together, these results create a complete and compelling picture of how l-Phe associates with model biological membranes. Furthermore, this approach to examining amino acid partitioning into membranes and the resulting solvation forces points to new strategies for studying the structure and chemistry of membrane-soluble peptides and selected membrane proteins.

Effects of antimicrobial peptides on membrane dynamics: A comparison of fluorescence and NMR experiments

Antimicrobial peptides (AMPs) represent a promising class of compounds to fight resistant infections. They are commonly thought to kill bacteria by perturbing the permeability of their cell membranes. However, bacterial killing requires a high coverage of the cell surface by bound peptides, at least in the case of cationic and amphipathic AMPs. Therefore, it is conceivable that peptide accumulation on the bacterial membranes might interfere with vital cellular functions also by perturbing bilayer dynamics, a hypothesis that has been termed “sand in the gearbox”. Here we performed a systematic study of such possible effects, for two representative peptides (the cationic cathelicidin PMAP-23 and the peptaibol alamethicin), employing fluorescence and NMR spectroscopies. These approaches are commonly applied to characterize lipid order and dynamics, but sample different time-scales and could thus report on different membrane properties. In our case, fluorescence anisotropy measurements on liposomes labelled with probes localized at different depths in the bilayer showed that both peptides perturb membrane fluidity and order. Pyrene excimer-formation experiments showed a peptide-induced reduction in lipid lateral mobility. Finally, laurdan fluorescence indicated that peptide binding reduces water penetration below the headgroups region. Comparable effects were observed also in fluorescence experiments performed directly on live bacterial cells. By contrast, the fatty acyl chain order parameters detected by deuterium NMR spectroscopy remained virtually unaffected by addition of the peptides. The apparent discrepancy between the two techniques confirms previous sporadic observations and is discussed in terms of the different characteristic times of the two approaches. The perturbation of membrane dynamics in the ns timescale, indicated by the multiple fluorescence approaches reported here, could contribute to the antimicrobial activity of AMPs, by affecting the function of membrane proteins, which is strongly dependent on the physicochemical properties of the bilayer.

Interaction of Uperin Peptides with Model Membranes: Molecular Dynamics Study

The interaction of antimicrobial and amyloid peptides with cell membranes is a critical step in their activities. Peptides of the uperin family obtained from the skin secretion of Australian amphibians demonstrate antimicrobial and amyloidogenic properties. All-atomic molecular dynamics and an umbrella sampling approach were used to study the interaction of uperins with model bacterial membrane. Two stable configurations of peptides were found. In the bound state, the peptides in helical form were located right under the head group region in parallel orientation with respect to the bilayer surface. Stable transmembrane configuration was observed for wild-type uperin and its alanine mutant in both alpha-helical and extended unstructured forms. The potential of mean force characterized the process of peptide binding from water to the lipid bilayer and its insertion into the membrane, and revealed that the transition of uperins from the bound state to the transmembrane position was accompanied by the rotation of peptides and passes through the energy barrier of 4-5 kcal/mol. Uperins have a weak effect on membrane properties.

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.

Location of dopamine in lipid bilayers and its relevance to neuromodulator function

Dopamine (DA) is a neurotransmitter that also acts as a neuromodulator, with both functions being essential to brain function. Here, we present the first experimental measurement of DA location in lipid bilayers using x-ray diffuse scattering, solid-state deuterium NMR, and electron paramagnetic resonance. We find that the association of DA with lipid headgroups as seen in electron density profiles leads to an increase of intermembrane repulsion most likely due to electrostatic charging. DA location in the lipid headgroup region also leads to an increase of the cross-sectional area per lipid without affecting the bending rigidity significantly. The order parameters measured by solid-state deuterium NMR decrease in the presence of DA for the acyl chains of PC and PS lipids, consistent with an increase in the area per lipid due to DA. Most importantly, these results support the hypothesis that three-dimensional diffusion of DA to target membranes could be followed by relatively more efficient two-dimensional diffusion to receptors within those membranes.

Interactions of beta-crystallin with a model of human eye lens-lipid membranes.

β-Crystallin helps maintain the eye lens's structure and refractivity. With age and cataract formation, α-, β-, and γ-crystallin may undergo various mutations and post-translational modifications, and consequently form higher molecular weight complexes, some of which bind to lens membranes. However, the binding of β-crystallin alone to the lens membrane is still unclear. This study aims to investigate the binding of β-crystallin to the model of the human lens-lipid (MHLL) membrane and the physical properties of the MHLL membrane after β-crystallin binding. Small unilamellar vesicles of MHLL membranes, resembling the human lens-lipid composition, were prepared using the rapid solvent exchange method and probe-tip sonication. The 1 mol% cholesterol analogue spin label (CSL) integrated into the membrane, which resides near the lens-lipid headgroup region, probes β-crystallin binding to the membrane using the electron paramagnetic resonance (EPR) spin-labeling method. Our results showed that β-crystallin binds to the MHLL membrane with a binding affinity (Ka) of 0.082 ± 0.006 μM−1 and saturates around 5% of the membrane surface occupied. Furthermore, β-crystallin binding to the MHLL membrane decreased the mobility parameter, making the headgroups of the membrane less mobile. However, β-crystallin binding to the MHLL membrane showed no significant change in maximum splitting, meaning it does not affect the order near the headgroup regions of the MHLL membrane. Moreover, the hydrophobicity of the MHLL membrane also did not change significantly with the binding of β-crystallin, meaning hydrophobicity near the headgroup regions does not significantly change with β-crystallin binding. Our results showed that β-crystallin binding with MHLL membranes changes the physical property of membranes, implying that β-crystallin binding to the lens membrane may change the integrity of the membrane and has the potential to promote cataract formation.

Association of adenosine triphosphate with lipid headgroups measured by NMR spectroscopy.

Adenosine triphosphate (ATP) is the energy currency of biological processes because of its central role in energy conversion in biological cells. Given the complexity and the molecular disorder of the cellular environment, the mechanism of energy conversion into useful work still poses challenging questions in molecular biophysics. Using nuclear magnetic resonance spectroscopy and dynamic light scattering, we show that ATP molecules have a measurable affinity to phosphatidylcholine lipid headgroups. The adenosine and ribose groups of the ATP molecule insert into the headgroup region of the membrane as seen by 2D NOESY spectra. Based on 31P NMR measurements, the most likely orientation for the phosphate chain is away from the membrane surface into the water space and the interaction of ATP with lipid headgroups leads to electrostatic charging of membrane surfaces. The interaction of adenosine triphosphate with lipid membranes has relevance to biophysical and biochemical studies of energy conversion in biological cells. In particular, the affinity of ATP to lipid headgroups can affect the interaction with specialized receptors present in biological cells. The observed association of ATP with membrane surfaces primarily through the adenosine group allows phosphate chains to participate in hydrolysis reactions consistent with biochemical data.

Ethosome as a potential transdermal drug delivery system

Ethosomes are elastic nanovesicles with phospholipid bases that are noninvasive delivery vehicles and have a high ethanol concentration (20–45%). As transdermal drug delivery confers poor penetration, the major obstacle is the low diffusion rate of drugs across the stratum corneum. The sophisticated ethosomal delivery systems enable drugs to reach the deep skin layers and/or the systemic circulation. The development of these new carriers involves the employment of several preparatory processes. Ethosomal dispersions are added to gels, patches, and creams for ease of use and stability. Ethanol is known as an efficient permeation enhancer and has been added in the vesicular systems to prepare elastic nanovesicles. It has the potential to interact with the polar head group region of lipid molecules, lowering the melting point of the stratum corneum lipid and raising lipid fluidity and cell membrane permeability as a result. Ethosomes' special structure allows them to enclose and transmit through the skin highly lipophilic substances like propranolol and trihexyphenidil as well as cationic medicines like testosterone and minoxidil. This article provides a detailed review of the ethosomal structure, mechanism of penetration along with various methods of preparation. Also, the article focuses on the applications of ethosomal carriers and opportunities for the research and future development of novel improved therapies.