Lipid Bilayer Phase Research Articles

Permeability of Dermatological Solutes through the Short Periodicity Phase of Human Stratum Corneum Lipid Bilayers.

Determining the permeability of drug-like solutes through the densely packed and heterogeneous stratum corneum lipid layer presents a significant challenge. In this study, we employed umbrella sampling with a periodic weighing function applied to the center of mass of the lipid bilayers. Precise umbrella sampling was conducted with an interframe distance of 0.5 Å, spanning from the bilayer center to the water phase, and each frame was simulated for at least 20 ns. Autocorrelation functions, potential of mean force (PMF), and diffusivity profiles were analyzed for six solutes (testosterone, benzene, caffeine, ethanol, mannitol, and histidine). The results revealed that autocorrelations were dependent solely on the medium, whether water or lipid phase. Diffusivity and PMF profiles along the reaction coordinate were influenced by the hydrophilicity of the solute rather than its size. For hydrophobic solutes, the PMF curves exhibited a minimum at the bilayer center, while for hydrophilic solutes, the PMFs peaked at the bilayer center and lipid tails (where the lipid tails are not interacting with the cholesterol). Diffusivity curves were low at the bilayer center and water phase, with peaks observed at the headgroup or the boundary between fatty acid and cholesterol (1 nm from the bilayer center). The quantitative findings presented in this work hold significance for pharmacists and drug designers.

Disorder within disordered regions of protein-free lipid bilayers.

Optical microscopy-based reports on protein-free lipid bilayers with heterogeneous compositions generally consider observed phase separations as thermodynamic endpoints; while MD simulations indicate kinetic characteristics of domain formations. Here, we study GUVs of POPC:DOPE (9:1) and POPC:DPPC (9:1), labeled with disordered-phase probe Liss-RhodPE (1/1000 w/w), using confocal imaging. In addition to confirming previously well-reported disorder and/or phase-separations in vesicular membranes of above compositions, we report the discovery of non-uniform probe distributions within disordered phases, indicating varying degrees of disorder (VDD) for the same vesicular membrane. We further examine dynamics of probe distributions in VDD regions of vesicular membranes. Regression analysis of time-dependent changes in average pixel intensities of probe molecules (avPIs) in different regions of interest (ROIs) allowed quantitative characterization of local (within ROIs) and global (overall vesicular membrane) motion of probe molecules within disordered phases. Optics-related artifacts (e.g., photobleaching, excitation and/or emission fluctuations) were ruled out by noting negligible variations in whole-vesicle avPIs with time. However, avPIs within certain ROIs showed considerable changes with time. In fact, in the same vesicular membrane, avPIs in different ROIs within disordered phases show (a) linear dependence on time (indicating diffusive motion), (b) dependence on square of time (indicating convective motion), and (c) other dependencies on time (e.g., logarithmic, exponential, power, 1/time etc.) - all clubbed together as non-diffusive-non-convective motions. Thus, we rigorously establish existence of further (at least three) levels of heterogeneity, based on dynamics of probe molecules, within disordered phases of protein-free lipid bilayers. Such heterogeneity has previously been ascribed only to biologically active membranes due to presence of membrane proteins. Interestingly, while diffusive motion may be linked to overall membrane viscosity, convective motion may be linked to concepts such as line-tension resulting from heterogeneous compositions.

Breaking sensitivity limits of QCM-D: An integrated nano-honeycomb sensor for characterization molecular interactions with lipid bilayers.

In the last few years Quartz Crystal Microbalance with Dissipation (QCM-D) emerged as an indispensable label-free method for monitoring formation of lipid bilayers on surfaces and assessing quality of the bilayers formed. QCM-D provides time-resolved data on mass and viscoelastic properties of molecular layers at the sensor surface and therefore is suitable for detecting vesicle adhesion, rupture, and spreading to form planar surface-supported bilayers. However, the small surface areas of QCM sensors impose severe limitation on mass sensitivity of the method. This is, perhaps, the main reason why QCM-D is rarely employed for studying lipid bilayer phase transitions and membrane binding events. To overcome these limitations, we have integrated the QCM sensor with an array of rigid honeycomb-packed anodic aluminum oxide (AAO) nanopores of ca. 25-80 nm in diameter and 2-10 μm in length by anodizing aluminum film deposited on the electrode surface. The resulting nanostructured QCM sensor increased surface area and improved sensitivity of the method by >100-fold. AAO-QCM electrode also provided a nanoscale template for self-assembly of lipid bilayers into nanotubular structures without any chemical attachment. We then employed such nanopore-confined bilayers to observe the main and more difficult to characterize pre-transition of membranes prepared from either pure 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC) or 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) lipids. Observed changes in QCM frequency reflected variations in lipid density upon phase transitions and occurred at temperatures in agreement with literature data. The signal-to-noise ratio of the phase transition peaks increased by 300-fold when compared to conventional QCM-D sensors. To further explore potential applications of AAO-QCM-D, ethanol-induced biphasic effects of phospholipid bilayers were studied. High sensitivity of AAO-QCM electrodes makes the method highly attractive for developing biosensing applications based on detecting changes in lipid packing at the nanoscale.

Photothermal Properties of IR-780-Based Nanoparticles Depend on Nanocarrier Design: A Comparative Study on Synthetic Liposomes and Cell Membrane and Hybrid Biomimetic Vesicles

Biomimetic nanoparticles hold great promise for photonic-mediated nanomedicine due to the association of the biological functionality of the membrane with the physical/chemical goals of organic/inorganic structures, but studies involving fluorescent biomimetic vesicles are still scarce. The purpose of this article is to determine how photothermal therapy (PTT) with theranostic IR-780-based nanoparticles depends on the dye content, cholesterol content, lipid bilayer phase and cell membrane type. The photophysical responses of synthetic liposomes, cell membrane vesicles and hybrid nanoparticles are compared. The samples were characterized by nanoparticle tracking analysis, photoluminescence, electron spin resonance, and photothermal- and heat-mediated drug release experiments, among other techniques. The photothermal conversion efficiency (PCE) was determined using Roper's method. All samples excited at 804 nm showed three fluorescence bands, two of them independent of the IR-780 content. Samples with a fluorescence band at around 850 nm showed photobleaching (PBL). Quenching was higher in cell membrane vesicles, while cholesterol inhibited quenching in synthetic liposomes with low dye content. PTT depended on the cell membrane and was more efficient for melanoma than erythrocyte vesicles. Synthetic liposomes containing cholesterol and a high amount of IR-780 presented superior performance in PTT experiments, with a 2.4-fold PCE increase in comparison with free IR-780, no PBL and the ability to heat-trigger doxorubicin release.

Preparation and Characterization of Trastuzumab Fab-Conjugated Liposomes (Immunoliposomes).

Immunoliposomes are made by conjugating antibodies or antibody fragments on liposome surfaces. Antibody fragments Fab', single-chain Fv fragments (scFv), or new constructs such as nanobodies are commonly used instead of whole IgGs for reduced risk immunogenicity. Here we described the preparation and characterization of immunoliposome-containing trastuzumab Fabs on the surface. The targeting ligand Fab-PEG-DSPE was synthesized by site-specific coupling between the C-terminal cysteine of the Fab and the maleimide group at the distal end of a DSPE-PEG. It was incorporated into preformed liposomes at 60°C above the lipid bilayer phase transition temperature. The binding avidity of the immunoliposomes containing different Fab valencies was characterized using biolayer interferometry.

Elucidating lipid conformations in the ripple phase: Machine learning reveals four lipid populations

A new mixed radial-angular, three-particle correlation function method in combination with unsupervised machine learning was applied to examine the emergence of the ripple phase in dipalmitoylphosphatidylcholine (DPPC) lipid bilayers using data from atomistic molecular dynamics simulations of system sizes ranging from 128 to 4096 lipids. Based on the acyl tail conformations, the analysis revealed the presence of four distinct conformational populations of lipids in the ripple phases of the DPPC lipid bilayers. The expected gel-like (ordered; Lo) and fluid-like (disordered; Ld) lipids are found along with their splayed tail equivalents (Lo,s and Ld,s). These lipids differ, based on their gauche distribution and tail packing. The disordered (Ld) and disordered-splayed (Ld,s) lipids spatially cluster in the ripple in the groove side, that is, in an asymmetric manner across the bilayer leaflets. The ripple phase does not contain large numbers of Ld lipids; instead they only exist on the interface of the groove side of the undulation. The bulk of the groove side is a complex coexistence of Lo,Lo,s, and Ld,s lipids. The convex side of the undulation contains predominantly Lo lipids. Thus, the structure of the ripple phase is neither a simple coexistence of ordered and disordered lipids nor a coexistence of ordered interdigitating gel-like (Lo) and ordered-splayed (Lo,s) lipids, but instead a coexistence of an ordered phase and a complex mixed phase. Principal component analysis further confirmed the existence of the four lipid groups.

Liquid-crystalline, liquid-ordered, rippled and gel lipid bilayer phases as observed with nile red fluorescence

The basic matrix of cellular membranes consists of a double layer (bilayer) of phospholipids. Semisynthetic lipid bilayers are commonly used in biophysical studies of membranes. According to temperature and composition, lipid bilayers can exist in liquid-crystalline (or liquid-disordered), liquid-ordered, rippled, and gel phases. In the present study, the hydrophobic, solvatochromic molecule Nile red has been used as a fluorescent probe to examine the physical state of bilayers of different compositions in the 15–60 °C range. Phospholipids with saturated or unsaturated acyl chains, in the presence or absence of cholesterol have been studied. Nile red shows absorption maxima at 520–550 nm and emission maxima at 580–640 nm, single photon excitation not being damaging to the system. A red/orange intensity ratio (ROIR) index has been used to normalize the results. ROIR varies clearly and reproducibly with the lipid phase, increasing in the order: liquid-ordered < gel < rippled < liquid-crystalline. It increases with temperature and decreases with cholesterol contents in the bilayers. Nile red allows an unusually clear observation of the rippled-to-liquid crystalline phase transition in saturated phospholipids. FLIM studies with Nile red also show differences between lamellar phases. Rotational relaxation times have been determined for Nile red in liquid-disordered (0.72 ± 0.010 ns), gel (1.16 ± 0.070 ns), and liquid-ordered (1.79 ± 0.14 ns) phases, the large value of the liquid-ordered phase being an indication of the sterol hindering probe tumbling in the hydrophobic matrix.

Quantifying Acidity in Heterogeneous Systems: Biphasic pKa Values.

Acidities of lipophilic compounds, such as various ligands or catalysts, in systems consisting of an aqueous phase at equilibrium with a water-immiscible phase (lipid bilayers, phase transfer catalysis, sensor membranes, to name just few) are typically approximated by the aqueous pKa values. Our research shows that such approximations can lead to seriously biased estimations of the acidities as the bulk of solvated H+ ions reside in the aqueous phase, while the lipophilic species─both neutral acid and anion─predominantly reside in the organic phase. Therefore, the use of aqueous pKa in such situations is not justified. In this work, we provide a more accurate description of the acidities of acids in such systems by applying the biphasic pKa concept. Biphasic pKa values (pKaow values) of 35 acids of various structures and chemical properties were determined in a 1-octanol:water system. We provide detailed descriptions of the UV-vis and NMR measurement methods. The directly obtained (apparent) pKaow values depend on concentration. Concentration-independent values were obtained by extrapolating the apparent values to zero concentration using a Debye-Hückel model.

Solid-like Dynamic Nuclear Polarization Observed in the Fluid Phase of Lipid Bilayers at 9.4 T.

Dynamic nuclear polarization (DNP) is a powerful method to enhance NMR sensitivity. Much progress has been achieved recently to optimize DNP performance at high magnetic fields in solid-state samples, mostly by utilizing the solid or the cross effect. In liquids, only the Overhauser mechanism is active, which exhibits a DNP field profile matching the EPR line shape of the radical, distinguishable from other DNP mechanisms. Here, we observe DNP enhancements with a field profile indicative of the solid effect and thermal mixing at ∼320 K and a magnetic field of 9.4 T in the fluid phase of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers doped with the radical BDPA (1,3-bis(diphenylene)-2-phenylallyl). This interesting observation might open up new perspectives for DNP applications in macromolecular systems at ambient temperatures.

Ripple-like instability in the simulated gel phase of finite size phosphocholine bilayers.

Atomistic molecular dynamics simulations have reached a degree of maturity that makes it possible to investigate the lipid polymorphism of model bilayers over a wide range of temperatures. However if both the fluid Lα and tilted gel [Formula: see text] states are routinely obtained, the [Formula: see text] ripple phase of phosphatidylcholine lipid bilayers is still unsatifactorily described. Performing simulations of lipid bilayers made of different numbers of DPPC (1,2-dipalmitoylphosphatidylcholine) molecules ranging from 32 to 512, we demonstrate that the tilted gel phase [Formula: see text] expected below the pretransition cannot be obtained for large systems (equal or larger than 94 DPPC molecules) through common simulations settings or temperature treatments. Large systems are instead found in a disordered gel phase which display configurations, topography and energies reminiscent from the ripple phase [Formula: see text] observed between the pretransition and the main melting transition. We show how the state of the bilayers below the melting transition can be controlled and depends on thermal history and conditions of preparations. A mechanism for the observed topographic instability is suggested.

Factors determining barrier properties to oxygen transport across model and cell plasma membranes based on EPR spin-label oximetry.

This review is motivated by the exciting new area of radiation therapy using a phenomenon termed FLASH in which oxygen is thought to have a central role. Well-established principles of radiation biology and physics suggest that if oxygen has a strong role, it should be the level at the DNA. The key aspect discussed is the rate of oxygen diffusion. If oxygen freely diffuses into cells and rapidly equilibrates, then measurements in the extracellular compartment would enable FLASH to be investigated using existing methodologies that can readily measure oxygen in the extracellular compartment. EPR spin-label oximetry allows evaluation of the oxygen permeability coefficient across lipid bilayer membranes. It is established that simple fluid phase lipid bilayers are not barriers to oxygen transport. However, further investigations indicate that many physical and chemical (compositional) factor can significantly decrease this permeation. In biological cell plasma membranes, the lipid bilayer forms the matrix in which integral membrane proteins are immersed, changing organization and properties of the lipid matrix. To evaluate oxygen permeability coefficients across these complex membranes, oxygen permeation across all membrane domains and components must be considered. In this review, we consider many of the factors that affect (decrease) oxygen permeation across cell plasma membranes. Finally, we address the question, can the plasma membrane of the cell form a barrier to the free diffusion of oxygen into the cell interior? If there is a barrier then this must be considered in the investigations of the role of oxygen in FLASH.

Biomimetic light-activatable graphene-based nanoarchitecture for synergistic chemophotothermal therapy

• Lipid coated biocompatible rGO-mesoporous-silica nanocomposites with was prepared with enhanced hydrophilicity and stability. • The high loading capacity of the nanodrug carriers was proved. • The in vivo antitumor evaluations confirmed the efficacy of nanodrug carriers. The development of biomimetic technology offers excellent opportunities for the construction of therapeutic platforms with enhanced biocompatibility and antitumor performance. In this study, lipid-bilayer-coated reduced graphene oxide (rGO) modified with mesoporous-silica nanosheets was constructed via facile lipid self-assembly for effective synergistic chemophotothermal therapy. These nanodrug carriers were suitable for a high loading rate (53.3%) of doxorubicin (DOX). In vitro , after cellular internalization of these nanosheets, upon near infrared light (NIR) irradiation, the rGO in the nanoarchitecture generated heat not only for hyperthermia treatment but also for triggering lipid bilayer phase transition and on-demand controlled DOX release for chemotherapy. As expected, systematic in vivo antitumor evaluations confirmed the efficient solid tumor removal without recurrence. Our results revealed the excellent integration of the mesoporous-silica-coated rGO and lipid bilayer, which enabled the construction of a platform with the merits of both materials within a single unit. Thus, our results also indicate the great potential for the applications of graphene-based nanomaterials in biomedicine.

Effect of alcohol on the phase separation in model membranes

The discovery of coexisting liquid-ordered and liquid-disordered phases in multicomponent lipid bilayers has received widespread attention due to its potential relevance for biological systems. One of the many open questions is how the presence of additional components affects the nature of the coexisting phases. Of particular interest is the addition of alcohols because their anesthetic properties may arise from modulating bilayer behavior. We use coarse-grained Molecular Dynamics simulations to gain insight into the partitioning preferences of linear n-alcohols into ordered and disordered bilayers alongside their effects on local membrane structure. We find that alcohols cause only small changes to membrane composition alongside a lack of significant effects on membrane thickness and lipid tail order. Cholesterol and n-alcohol trans-bilayer motion is measured and found to be near or within the range of previous atomistic results. The cholesterol flip-flop rates increase with both n-alcohol length and concentration for octanol, dodecanol, and hexadecanol, indicating a decrease in lipid order. Umbrella sampling simulations of removing cholesterol from tertiary membranes find no significant difference with or without n-alcohols at various concentrations. Simulations of a phase-separated bilayer show that octanol preferentially partitions into the liquid-disordered phase in a ratio of approximately 3:1 over the liquid-ordered phase. Furthermore, partition coefficients of alcohol in single-phase membranes show a preference for longer alcohols (dodecanol and hexadecanol) to partition preferentially into the liquid-ordered phase, while decreasing the length of the alcohol reverses this trend. Our work tests experimental results while also investigating the ability for coarse-grained MARTINI simulations to capture minute differences in model membrane spatial arrangements on the nanoscale level.

Control of Lipid Bilayer Phases of Cell-Sized Liposomes by Surface-Engineered Plasmonic Nanoparticles.

Liquid-ordered (Lo)-phase domains, a cholesterol-rich area on lipid bilayers, have attracted significant attention recently because of their relevance to lipid rafts, the formation/collapse of which is associated with various kinds of information exchange through the plasma membrane. Here, we demonstrate that the formation/collapse of Lo-phase domains in cell-sized liposomes, that is, giant unilamellar vesicles (GUVs), can be controlled with bioactive plasmonic nanoparticles and light. The nanoparticles were prepared by surface modification of gold nanorods (AuNRs) using a cationized mutant of high-density lipoprotein (HDL), which is a natural cholesterol transporter. Upon the addition of surface-engineered AuNRs to GUVs with the mixed domains of Lo and liquid-disorder (Ld) phases, the Lo domains collapsed and solid-ordered (So)-phase domains were formed. The reverse phase transition was achieved photothermally, with the AuNRs loaded with cholesterol. During these transitions, the AuNRs appeared to be selectively localized on the less fluidic domain (Lo or So) in the phase-mixed GUVs. These results indicate that the phase transitions occur through the membrane binding of the AuNRs followed by spontaneous/photothermal transfer of cholesterol between the AuNRs and GUVs. Our strategy to develop bioactive AuNRs potentially enables spatiotemporal control of the formation/collapse of lipid rafts in living cells.

Mechanical Properties Determination of DMPC, DPPC, DSPC, and HSPC Solid-Ordered Bilayers

Lipid bilayers are active participants in many crucial biological processes. They can be observed in different phases, liquid and solid, respectively. The liquid phase is predominant in biological systems. The solid phase, both crystalline and gel phases, is under investigation due to its resilience to mechanical stress and tight packing of lipids. The mechanical properties of lipids affect their dynamics, therefore influencing the transformation of cell plasma and the endomembrane. Mechanical properties of lipid bilayers are also an important parameter in the design and production of supramolecular lipid-based drug delivery systems. To this end, in this work, we focused on investigating the effect of solid phases of lipid bilayers on their structural parameters and mechanical properties using theoretical molecular dynamics studies on atomistic models of whole vesicles. Those include area per lipid, membrane thickness, density vesicle profiles, bending rigidity coefficient, and area compressibility. Additionally, the bending rigidity coefficient was measured using the flicker noise spectroscopy. The two approaches produced very similar and consistent results. We showed that, contrary to our expectations, bending rigidity coefficients of solid-ordered bilayers for vesicles decreased with an increase in lipid transition temperature. This tendency was reverse in planar systems. Additionally, we have observed an increase of membrane thickness and area compressibility and a decrease of area per lipid. We hope these results will provide valuable mechanical insight for the behavior in solid phases and differences between spherical and planar confirmations.

Effect of Dipole Moment on Amphiphile Solubility and Partition into Liquid Ordered and Liquid Disordered Phases in Lipid Bilayers

Effect of Dipole Moment on Amphiphile Solubility and Partition into Liquid Ordered and Liquid Disordered Phases in Lipid Bilayers

Fluid bilayer phase in aqueous mixtures of fatty alcohol and cationic surfactant

The $L_\alpha$ phase of lipid bilayers is a fluid self-assembled state, key to the formulation of cosmetics, detergents and pharmaceutics. Despite having been extensively scrutinized in self-assembled phospholipid or surfactant bilayers, the formation of a fluid $L_\alpha$ state has defied understanding in mixtures of fatty alcohols, surfactants and water, where is viewed as the essential step for the preparation of creamy dispersions. Here, atomistic molecular dynamics simulations show the existence of a fluid bilayer in aqueous mixtures of cetyl (C$_{16}$OH) and stearyl (C$_{18}$OH) alcohols, and cetyl-trimethylammonium chloride (CTAC). These simulated bilayer systems display not only a rich temperature phase diagram with many of the features seen in experiments but carry also the unambigous signature of fluid bilayer behavior.

A machine learning study of the two states model for lipid bilayer phase transitions.

We have adapted a set of classification algorithms, also known as machine learning, to the identification of fluid and gel domains close to the main transition of dipalmitoyl-phosphatidylcholine (DPPC) bilayers. Using atomistic molecular dynamics conformations in the low and high temperature phases as learning sets, the algorithm was trained to categorise individual lipid configurations as fluid or gel, in relation with the usual two-states phenomenological description of the lipid melting transition. We demonstrate that our machine can learn and sort lipids according to their most likely state without prior assumption regarding the nature of the order parameter of the transition. Results from our machine learning study provide strong support in favour of a two-states model approach of membrane fluidity.

A REVIEW ON PHYSICO-CHEMICAL PARAMETERS OF LIPOSOMAL DOXORUBICIN

The development of a generic liposomal doxorubicin product requires the study of critical physicochemical properties of the formulation. Food and Drug Administration (FDA) draft guideline has suggested few parameters to be tested for in vitro bioequivalence study which include liposomal composition, state of encapsulated drug, internal environment, liposomal morphology and number of lamellae, lipid bilayer phase transition, liposomal size distribution, grafted Polyethylene Glycol (PEG) at liposomal surface, electric surface potential or charge and in vitro leakage under multiple conditions. Characteristic features of components of liposomal doxorubicin formulation and detail of parameters to be studied have been discussed. This review compile specific, current and historical research outcomes on in vitro analysis of liposomal doxorubicin and highlights the important features that have a critical impact on properties of liposomal doxorubicin formulation. It will provide a better insight to the generic manufacturers and will help them to identify the critical quality attributes during the formulation development phase.

Effect of dipole moment on amphiphile solubility and partition into liquid ordered and liquid disordered phases in lipid bilayers

Association of amphiphiles with biomembranes is important for their availability at specific locations in organisms and cells, being critical for their biological function. A prominent role is usually attributed to the hydrophobic effect, and to electrostatic interactions between charged amphiphiles and lipids. This work explores a closely related and complementary aspect, namely the contribution made by dipole moments to the strength of the interactions established. Two xanthene amphiphiles with opposite relative orientations of their dipole and amphiphilic moments have been selected (Rhodamine-C14 and Carboxyfluorescein-C14). The membranes studied have distinct lipid compositions, representing typical cell membrane pools, ranging from internal membranes to the outer and inner leaflet of the plasma membrane. A comprehensive study is reported, including the affinity of the amphiphiles for the different membranes, the stability of the amphiphiles as monomers and their tendency to form small clusters, as well as their transverse location in the membrane. The orientation of the amphiphile dipole moment, which determines whether its interaction with the membrane dipole potential is repulsive or attractive, is found to exert a large influence on the association of the amphiphile with ordered lipid membranes. These interactions are also responsible for the formation of small clusters or stabilization of amphiphile monomers in the membrane. The results obtained allow understanding the prevalence of protein lipidation at the N-terminal for efficient targeting to the plasma membrane, as well as the tendency of GPI-anchored proteins (usually lipidated at the C-terminal) to form small clusters in the membrane ordered domains.