Lipid Bilayer Interactions Research Articles

Exploring the Interaction of Lipid Bilayers with Curcumin-Laponite Nanoparticles: Implications for Drug Delivery and Therapeutic Applications.

Curcumin, the active compound in turmeric, is renowned for its anti-inflammatory, antioxidant, and antimicrobial properties, making it beneficial for treating conditions like arthritis, neurodegenerative diseases, and various cancers. Despite its promising therapeutic potential, curcumin's poor bioavailability-due to its rapid metabolism and low solubility-limits its clinical efficacy. To address this, recent research has focused on enhancing curcumin delivery using nanoparticles, liposomes, and novel nanomaterials. Among these, laponite, a synthetic nanoclay, has shown promise in improving curcumin delivery due to its unique properties, including large surface area, dual charge, and stability in solution. This study explores the use of curcumin-laponite nanoparticles as carrier vehicles for controlled delivery to in vitro model membranes. Utilizing advanced techniques such as neutron reflectometry, atomic force microscopy, quartz crystal microbalance with dissipation, and infrared spectroscopy, the interaction between curcumin-laponite nanoparticles and solid-supported lipid bilayers is monitored, revealing enhanced stability and controlled release of curcumin across the membrane. These findings pave the way for the development of curcumin-based therapies targeting cardiovascular, neurological, and oncological diseases, leveraging the synergistic effects of curcumin's biological activity and laponite's delivery capabilities.

Influence of Simvastatin and Pravastatin on the Biophysical Properties of Model Lipid Bilayers and Plasma Membranes of Live Cells.

Statins are among the most widely used drugs for the inhibition of cholesterol biosynthesis, prevention of cardiovascular diseases, and treatment of hypercholesterolemia. Additionally, statins also exhibit cholesterol-independent benefits in various diseases, including neuroprotective properties in Alzheimer's disease, anti-inflammatory effects in coronary artery disease, and antiproliferative activities in cancer, which likely result from the statins' interaction and alteration of lipid bilayers. However, the membrane-modulatory effects of statins and the mechanisms by which statins alter lipid bilayers remain poorly understood. In this work, we explore the membrane-modulating effects of statins on model lipid bilayers and live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM) combined with viscosity-sensitive environmental probes, we demonstrate that hydrophobic, but not hydrophilic, statins are capable of changing the microviscosity and lipid order in model and live cell membranes. Furthermore, we show that hydrophobic simvastatin is capable of forming nanoscale cholesterol-rich domains and homogenizing the cholesterol concentrations in lipid bilayers. Our results provide a mechanistic framework for understanding the bimodal effects of simvastatin on the lipid order and the lateral organization of cholesterol in lipid bilayers. Finally, we demonstrate that simvastatin temporarily decreases the microviscosity of live cell plasma membranes, making them more permeable and increasing the level of intracellular chemotherapeutic drug accumulation.

Palmitoylation enhances short polar peptide permeation across stratum corneum lipid bilayer: A molecular dynamics study

Designing chemical molecules that target the skin for non-invasive transdermal drug delivery is of significant interest for both wound healing and skincare applications. These skin-targeting molecules must permeate the outermost protective layer of the skin, the stratum corneum (SC), which consists of dead corneocytes embedded in a lipid matrix, to fulfill their biological functions. Adsorption onto and diffusion through the lipid matrix in the SC represent two key steps for the successful permeation of a skin-targeting molecule across the SC into the underlying skin layers. Here we compare the effects of cyclization and palmitoylation on the adsorption and diffusion of a short polar peptide across a model SC lipid bilayer using molecular dynamics simulations. The cyclized peptide showed slightly better binding to the SC lipid bilayer and similar interaction energies with SC lipids compared to the unmodified peptide. In contrast, the palmitoylated peptide exhibited much stronger interaction with SC lipids via insertion of its attached fatty acid tail into the SC lipid bilayer. The average diffusivity of the cyclized peptide across the SC lipid bilayer was approximately twice that of the unmodified peptide, whereas the palmitoylated peptide’s diffusivity was about 2.7 times higher. Thus, palmitoylation appears to be a promising strategy for enhancing the binding and permeability of short polar peptides across the SC lipid matrix.

Fingolimod phosphoramidate prodrugs: Synthesis, photophysical characterisation and lipid bilayer interaction of fluorescent tagged Prodrug

FTY720 (Fingolimod, Gilenya, 1), a structural analog of sphingosine (2) was the first orally administered drug approved for the treatment of multiple sclerosis (MS). However, the phosphate derivative of Fingolimod, namely Fingolimod-1-phosphate (FTY720-1P, 3) is the biologically active molecule in MS as well as in various lysosomal storage diseases such as mucolipidosis IV and Nieman-Pick. In all cases, Fingolimod requires to be phosphorylated, resembling the natural sphingosine phosphate (4), to accomplish its therapeutic/biological effects. Here, we report the synthesis of a small library of prodrugs that could provide the efficient delivery of 3, the bioactive species. Moreover, to gain insight about the biological behaviour of these novel candidates within human body such as cell and brain blood barrier penetration we have prepared and spectroscopically characterised a fluorescent tagged version of one of the new prodrugs. The behavior of our prodrugs with respect to biological membranes was evaluated by studying the interaction between the fluorescent prodrug 18 and DOPC/DPPC liposome models.

Do Nanodisc Assembly Conditions Affect Natural Lipid Uptake?

Lipids play critical roles in modulating membrane protein structure, interactions, and activity. Nanodiscs provide a tunable membrane mimetic that can model these endogenous protein-lipid interactions in a nanoscale lipid bilayer. However, most studies of membrane proteins with nanodiscs use simple synthetic lipids that lack the headgroup and fatty acyl diversity of natural extracts. Prior research has successfully used natural lipid extracts in nanodiscs that more accurately mimic natural environments, but it is not clear how nanodisc assembly may bias the incorporated lipid profiles. Here, we applied lipidomics to investigate how nanodisc assembly conditions affect the profile of natural lipids in nanodiscs. Specifically, we tested the effects of assembly temperature, nanodisc size, and lipidome extract complexity. Globally, our analysis demonstrates that the lipids profiles are largely unaffected by nanodisc assembly conditions. However, a few notable changes emerged within individual lipids and lipid classes, such as a differential incorporation of cardiolipin and phosphatidylglycerol lipids from the E. coli polar lipid extract at different temperatures. Conversely, some classes of brain lipids were affected by nanodisc size at higher temperatures. Collectively, these data enable the application of nanodiscs to study protein-lipid interactions in complex lipid environments.

Effect of Glycone Diversity on the Interaction of Triterpenoid Saponins and Lipid Bilayers.

Triterpenoid saponins are organic compounds widely available in the plant kingdom. These molecules have received extensive attention due to their antibacterial activity against both Gram-negative and Gram-positive bacteria. Recent studies identified the antibacterial activity of saponins closely relates to their interaction with bacterial membrane lipids; however, molecular details of this interaction remain unclear. Increased understanding of the mechanisms to disrupt bacterial lipid bilayers can help to mitigate development of antibiotic resistance. Here, we examined the effect of chemical structure and deprotonation states of saponin on its interaction with a bacterial membrane model using molecular dynamics simulations. We run multiple simulations with a ternary lipid mixture of POPE/POPG/DPPG (80/15/5 mol %) and different saponin molecules. While all saponin structures can permanently bind the membrane, their location and orientation inside the bilayer depend on the sugar chains attached to their backbone. Similarly, cluster formation and stability also depend on the chemical structure of the saponin molecule. Deprotonation site affects interactions with the bilayer by modulating hydrophilicity of the molecules. At the low concentrations simulated in this work, there is no statistically significant change in the membrane properties upon saponin(s) binding, but the molecules do preferentially partition to POPE lipid environment.

Recombination between 13C and 2H to Form Acetylide (13C22H-) Probes Nanoscale Interactions in Lipid Bilayers via Dynamic Secondary Ion Mass Spectrometry: Cholesterol and GM1 Clustering.

Although it is thought that there is lateral heterogeneity of lipid and protein components within biological membranes, probing this heterogeneity has proven challenging. The difficulty in such experiments is due to both the small length scale over which such heterogeneity can occur, and the significant perturbation resulting from fluorescent or spin labeling on the delicate interactions within bilayers. Atomic recombination during dynamic nanoscale secondary ion imaging mass spectrometry (NanoSIMS) is a non-perturbative method for examining nanoscale bilayer interactions. Atomic recombination is a variation on conventional NanoSIMS imaging, whereby an isotope on one molecule combines with a different isotope on another molecule during the ionization process, forming an isotopically enriched polyatomic ion in a distance-dependent manner. We show that the recombinant ion, 13C22H-, is formed in high yield from 13C- and 2H-labeled lipids. The low natural abundance of triply labeled acetylide also makes it an ideal ion to probe GM1 clusters in model membranes and the effects of cholesterol on lipid-lipid interactions. We find evidence supporting the cholesterol condensation effect as well as the presence of nanoscale GM1 clusters in model membranes.

Investigating the Lipid Selectivity of Membrane Proteins in Heterogeneous Nanodiscs.

The structure and function of membrane proteins can be significantly impacted by the surrounding lipid environment, but membrane protein-lipid interactions in lipid bilayers are often difficult to study due to their transient and polydisperse nature. Here, we used two native mass spectrometry (MS) approaches to investigate how the Escherichia coli ammonium transporter trimer (AmtB) and aquaporin Z (AqpZ) selectively remodel their local lipid environment in heterogeneous lipoprotein nanodiscs. First, we used gas-phase ejection to isolate the membrane protein with bound lipids from heterogeneous nanodiscs with different combinations of lipids. Second, we used solution-phase detergent extraction as an orthogonal approach to study membrane protein remodeling of lipids in the nanodisc with native MS. Our results showed that Triton X-100 and lauryldimethylamine oxide retain lipid selectivity that agrees with gas-phase ejection, but C8E4 distorts some preferential lipid interactions. Both approaches reveal that AmtB has a few selective binding sites for phosphatidylcholine (PC) lipids, is selective for binding phosphatidylglycerols (PG) overall, and is nonselective for phosphatidylethanolamines (PE). In contrast, AqpZ prefers either PC or PG over PE and prefers PC over PG. Overall, these experiments provide a picture of how membrane proteins bind different lipid head groups in the context of mixed lipid bilayers.

Lipid membrane-mediated assembly of the functional amyloid-forming peptide Somatostatin-14

Membrane-mediated assembly has been well characterised for toxic amyloid species such as the amyloid-β peptide implicated in Alzheimer's disease. However, little is known about the membrane-mediated assembly of functional-amyloid forming peptides, recently identified as a natural storage state for neuropeptide hormones in vivo. Here, we study the aggregation of somatostatin-14 (SST-14) co-incubated with model lipid membranes. Atomic force microscopy (AFM) studies confirmed that nanofibrils formed in the presence of various lipid membranes display reduced fibrillogenesis and promote the formation of non-fibrillar oligomers. Both circular dichroism (CD) and intrinsic tryptophan fluorescence studies confirmed interaction between the peptide and the lipid bilayer; this interaction appears to drive changes in membrane-mediated aggregation kinetics. We show that both the surface charge of the membrane and chain packing drive changes in the electrostatic and hydrophobic interactions between the peptide and the membrane, and hence the rate of assembly. The similarities in the effect of the lipid membrane on aggregation of functional amyloids and the more well studied toxic amyloids suggest strong aggregation modifying lipid bilayer interactions are a ubiquitous feature of all amyloid fibrils and highlight the need for further investigation as to why this leads to toxicity in some systems and not others.

Interaction of Cyanine-D112 with Binary Lipid Mixtures: Molecular Dynamics Simulation and Differential Scanning Calorimetry Study.

This comprehensive molecular dynamics (MD) simulation and experimental study investigates the lipid bilayer interactions of dye D112 for potential photodynamic therapy (PDT) applications. PDT involves formation of a reactive oxidant species in the presence of a light sensitive molecule and light, interrupting cellular functions. D112 was developed as a photographic emulsifier, and we hypothesized that its combined cationic and lipophilic nature can render a superior photosensitizing property–crucial in various light therapies. The focus of this study is to elucidate the binding and insertion mechanisms of D112 with mixed lipid bilayers of anionic dipalmitoyl-phosphatidylserine (DPPS) and zwitterionic dipalmitoyl-phosphatidylcholine (DPPC) lipids to resemble cancer cell membranes. Our studies confirm initial electrostatic binding between the positively charged moieties of D112 and negatively charged lipid headgroups. Additionally, MD simulations combined with differential scanning calorimetry (DSC) studies confirm that D112-lipid interactions are governed by enthalpy-driven nonclassical hydrophobic effects in the membrane interior. It was further noted that despite the electrostatic preference of D112 toward the anionic lipids, D112 molecules colocalized on DPPC-rich domains after insertion. Atomistic level MD studies point toward two possible insertion mechanisms for D112: harpoon and flip. Further insights from the simulation showcase the interactions of low and high aggregates of D112 with the bilayer as the concentration of D112 increases in solution. The size of aggregates modulates the orientation and degree of insertion, providing important information for future studies on membrane permeation mechanisms.

Recombination between 13C and 2H to form acetylide (13C22H-) probes nanoscale interactions in lipid bilayers: cholesterol and GM1 clustering

While it is widely believed that lipid and protein components in membranes exhibit lateral organization on a length scale comparable to molecular dimensions, this has been very difficult to characterize. Fluorescent or spin label labels have been used, however, especially for lipids, these probes may perturb the delicate interactions responsible for lateral heterogeneity. We have developed imaging mass spectrometry using the Cameca NanoSIMS 50L to probe composition in model membranes using only non-perturbing isotopic labels.

ComplexMixtures.jl: Investigating the structure of solutions of complex-shaped molecules from a solvent-shell perspective

Distribution functions are used to investigate the interactions between the components of condensed-phase systems, while allowing the computation of thermodynamic properties that can be probed experimentally. Radial distribution functions are the most fundamental and easily understood of these distributions, but fail to provide a molecular picture of the interactions when one or all species have complex shapes. On the other hand, regardless of the complexity of the molecular structures involved, minimum-distance distribution functions (MDDFs) can provide a molecular viewpoint on solute–solvent contacts. Here, we describe the ComplexMixtures.jl package, which provides a practical implementation of MDDFs and corresponding Kirkwood-Buff integrals to analyze Molecular Dynamics and Monte-Carlo simulations. Examples are provided for the study of macromolecules in solutions of multiple cosolvents, homogeneous systems, polymer solvation by organic solvents and lipid bilayer interactions with disruptive agents. The distribution functions can be examined using tools to assess the contributions of each atom, group of atoms, and amino acid residues, for example. ComplexMixtures.jl is free software and is compatible with the most common molecular simulation trajectory formats. The software is available as a Julia package with a comprehensive documentation at: http://m3g.iqm.unicamp.br/ComplexMixtures.

Hydration Forces Dominate Surface Charge Dependent Lipid Bilayer Interactions under Physiological Conditions.

Lipid bilayer interactions are essential to a vast range of biological functions, such as intracellular transport mechanisms. Surface charging mediated by concentration dependent ion adsorption and desorption on lipid headgroups alters electric double layers as well as van der Waals and steric hydration forces of interacting bilayers. Here, we directly measure bilayer interactions during charge modulation in a symmetrically polarized electrochemical three-mirror interferometer surface forces apparatus. We quantify polarization and concentration dependent hydration and electric double layer forces due to cation adsorption/desorption. Our results demonstrate that exponential hydration layer interactions effectively describe surface potential dependent surface forces due to cation adsorption at high salt concentrations. Hence, electric double layers of lipid bilayers are exclusively dominated by inner Helmholtz charge regulation under physiological conditions. These results are important for rationalizing bilayer behavior under physiological conditions, where charge and concentration modulation may act as biological triggers for function and signaling.

FRET detects lateral interaction between transmembrane domain of EGF receptor and ganglioside GM3 in lipid bilayers

Ganglioside GM3 in the plasma membranes suppresses cell growth by preventing the autophosphorylation of the epidermal growth factor receptor (EGFR). Biological studies have suggested that GM3 interacts with the transmembrane segment of EGFR. Further biophysical experiments are particularly important for quantitative evaluation of the peptide-glycolipid interplay in bilayer membranes using a simple reconstituted system. To examine these interactions in this way, we synthesized the transmembrane segment of EGFR bearing a nitrobenzoxadiazole fluorophore (NBD-TM) at the N-terminus. The affinity between EGFR and GM3 was evaluated based on Förster resonance energy transfer (FRET) between NBD-TM and ATTO594-labeled GM3 in bilayers where their non-specific interaction due to lateral proximity was subtracted by using NBD-labeled phospholipid. This method for selectively detecting the specific lipid-peptide interactions in model lipid bilayers disclosed that the lateral interaction between GM3 and the transmembrane segment of EGFR plays a certain role in disturbing the formation of active EGFR dimers.

Assembly of Model Membrane Nanodiscs for Native Mass Spectrometry.

Native mass spectrometry (MS) with nanodiscs is a promising technique for characterizing membrane protein and peptide interactions in lipid bilayers. However, prior studies have used nanodiscs made of only one or two lipids, which lack the complexity of a natural lipid bilayer. To better model specific biological membranes, we developed model mammalian, bacterial, and mitochondrial nanodiscs with up to four different phospholipids. Careful selection of lipids with similar masses that balance the fluidity and curvature enabled these complex nanodiscs to be assembled and resolved with native MS. We then applied this approach to characterize the specificity and incorporation of LL-37, a human antimicrobial peptide, in single-lipid nanodiscs versus model bacterial nanodiscs. Overall, development of these model membrane nanodiscs reveals new insights into the assembly of complex nanodiscs and provides a useful toolkit for studying membrane protein, peptide, and lipid interactions in model biological membranes.

Structure and Interaction of Lipid Bilayers in the Presence of ATP

Even in the absence of membrane receptors, adenosine triphosphate (ATP) has been shown to interact with lipid membranes and to affect their interactions. Measurements of zeta potentials by dynamic light scattering (DLS) show that ATP creates a positive zeta potential of phosphatidylcholine (PC) lipid vesicles. A similar effect is seen on phosphatidylserine (PS) bilayers in that the negative zeta potential of PS diminishes in magnitude in the presence of ATP. We further investigate the effect of ATP on lipid bilayers by 2H NMR spectroscopy which gives us an order parameter profile for the carbon-deuterium bonds along lipid acyl chains.

Nanoprobes to investigate nonspecific interactions in lipid bilayers: from defect-mediated adhesion to membrane disruption.

When a lipid membrane approaches a material/nanomaterial, nonspecific adhesion may occur. The interactions responsible for nonspecific adhesion can either preserve the membrane integrity or lead to its disruption. Despite the importance of the phenomenon, there is still a lack of clear understanding of how and why nonspecific adhesion may originate different resulting scenarios and how these interaction scenarios can be investigated. This work aims at bridging this gap by investigating the role of the interplay between cationic electrostatic and hydrophobic interactions in modulating the membrane stability during nonspecific adhesion phenomena. Here, the stability of the membrane has been studied employing anisotropic nanoprobes in zwitterionic lipid membranes with the support of coarse-grained molecular dynamics simulations to interpret the experimental observations. Lipid membrane electrical measurements and nanoscale visualization in combination with molecular dynamics simulations revealed the phenomena driving nonspecific adhesion. Any interaction with the lipidic bilayer is defect-mediated involving cationic electrostatically driven lipid extraction and hydrophobically-driven chain protrusion, whose interplay determines the existence of a thermodynamic optimum for the membrane structural integrity. These findings unlock unexplored routes to exploit nonspecific adhesion in lipid membranes. The proposed platform can act as a straightforward probing tool to locally investigate interactions between synthetic materials and lipid membranes for the design of antibacterials, antivirals, and scaffolds for tissue engineering.

Mechanistic Insights into Pore Formation by an α-Pore Forming Toxin: Protein and Lipid Bilayer Interactions of Cytolysin A.

Pore forming toxins (PFTs) are the largest class of bacterial toxins playing a central role in bacterial pathogenesis. They are proteins specifically designed to form nanochannels in the membranes of target cells, ultimately resulting in cell death and establishing infection. PFTs are broadly classified as α- and β-PFTs, depending on secondary structures that form the transmembrane channel. A unique feature about this class of proteins is the drastic conformational changes and complex oligomerization pathways that occur upon exposure to the plasma membrane. A molecular understanding of pore formation has implications in designing novel intervention strategies to combat rising antimicrobial resistance, targeted-cancer therapy, as well as designing nanopores for specialized technologies. Central to unraveling the pore formation pathway is the availability of high resolution crystal structures. In this regard, β-toxins are better understood, when compared with α-toxins whose pore forming mechanisms are complicated by an incomplete knowledge of the driving forces for amphiphatic membrane-inserted helices to organize into functional pores. With the publication of the first crystal structure for an α-toxin, cytolysin A (ClyA), in 2009 we embarked on an extensive multiscale study to unravel its pore forming mechanism. This Account represents the collective mechanistic knowledge gained in our laboratories using a variety of experimental and theoretical techniques which include large scale molecular dynamics (MD) simulations, kinetic modeling studies, single-molecule fluorescence imaging, and super-resolution spectroscopy. We reported MD simulations of the ClyA protomer, oligomeric intermediates, and full pore complex in a lipid bilayer and mapped the conformational transitions that accompany membrane binding. Using single-molecule fluorescence imaging, the conformational transition was experimentally verified by analysis of various diffusion states of membrane bound ClyA. Importantly, we have uncovered a hitherto unknown putative cholesterol binding motif in the membrane-inserted helix of ClyA. Distinct binding pockets for cholesterol formed by adjacent membrane-inserted helices are revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation. We posit that this mechanistic understanding of the complex action of α-PFTs has implications in unraveling pore assembly across the wider family of bacterial toxins. With emerging antimicrobial resistance, alternate therapies may rely on disrupting pore functionality or oligomerization of these pathogenic determinants utilized by bacteria, and our study includes assessing the potential for dendrimers as pore blockers.

Stability of Lipid Coatings on Nanoparticle-Decorated Surfaces.

Lipid membranes supported on solid surfaces and nanoparticles find multiple applications in industrial and biomedical technologies. Here, we explore in silico the mechanisms of the interactions of lipid membranes with nanostructured surfaces with deposited nanoparticles and explain the characteristic particle size dependence of the uniformity and stability of lipid coatings observed in vitro. Simulations are performed to demonstrate the specifics of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid membrane adhesion to hydrophilic and hydrophobic nanoparticles ranging in size from 1.5 to 40 nm using an original coarse-grained molecular dynamics model with implicit solvent and large simulation boxes (scales up to 280 × 154 × 69 nm3). We find that one of the major factors that affects the uniformity and stability of lipid coatings is the disjoining pressure in the water hydration layer formed between the lipid membrane and hydrophilic solid surface. This effect is accounted for by introducing a special long-range lipid-solid interaction potential that mimics the effects of the disjoining pressure in thin water layers. Our simulations reveal the physical mechanisms of interactions of lipid bilayers with solid surfaces that are responsible for the experimentally observed nonmonotonic particle size dependence of the uniformity and stability of lipid coatings: particles smaller than the hydration layer thickness (<2-3 nm) or larger than ∼20 nm are partially or fully enfolded by a lipid bilayer, whereas particles of the intermediate size (5-20 nm) cause membrane perforation and pore formation. In contrast, hydrophobic nanoparticles, which repel the hydration layer, tend to be encapsulated within the hydrophobic interior of the membrane and coated by the lipid monolayer. The proposed model can be further extended and applied to a wide class of systems comprising nanoparticles and nanostructured substrates interacting with lipid and surfactant bilayers and monolayers.

Towards a Quantitative Understanding of Protein-Lipid Bilayer Interactions at the Single Molecule Level: Opportunities and Challenges.

Protein-lipid interfaces are among the most fundamental in biology. Yet applying conventional techniques to study the biophysical attributes of these systems is challenging and has left many unknowns. For example, what is the kinetic pathway and energy landscape experienced by a polypeptide chain when in close proximity to a fluid lipid bilayer? Here we review the experimental and theoretical progress we have made in addressing this question from a single molecule perspective. Some remaining impediments are also discussed.