Palmitoyloleoyl Phosphatidylcholine Research Articles

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration, and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner one yield less stretchable membranes with higher compressibility modulus compared with their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. In addition, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol %), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such domain formation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques.

Synergistic Anti-Inflammatory Activity of Apolipoprotein A-I and CIGB-258 in Reconstituted High-Density Lipoproteins (rHDL) against Acute Toxicity of Carboxymethyllysine in Zebrafish and Its Embryo.

CIGB-258 is a 3 kDa altered peptide ligand from heat shock protein (HSP) 60 that exhibits anti-inflammatory activity against the acute toxicity of carboxymethyllysine (CML) with antioxidant and anti-glycation activities via protection of high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I). It is necessary to test a synergistic interaction between apoA-I and CIGB-258 in reconstituted high-density lipoproteins (rHDL). Several rHDLs were synthesized containing palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apoA-I, and CIGB-258 at molar ratios of 95:5:1:0, 95:5:1:0.1, 95:5:1:0.5, and 95:5:1:1 for rHDL-(1:0), rHDL-(1:0.1), rHDL-(1:0.5), and rHDL-(1:1), respectively. As the CIGB-258 content in rHDL was increased, the particle size of rHDL was 1.4-times higher than rHDL-(1:0) to rHDL-(1:1), from 60 nm to 83 nm, respectively. As the CIGB-258 content was increased, the rHDL showed the most resistance to isothermal denaturation by a urea treatment, and rHDL-(1:1) exhibited the highest structural stability and the strongest antioxidant ability against LDL oxidation. Co-treatment of rHDL-(1:0), rHDL-(1:0.5), and rHDL-(1:1) resulted in up to 10%, 24%, and 34% inhibition of HDL glycation, inhibition of HDL glycation, which was caused by the CML, with protection of apoA-I. Microinjection of each rHDL into zebrafish embryos in the presence of CML showed that a higher CIGB-258 content in rHDL was associated with higher survivability with the least inflammation and apoptosis. Furthermore, an intraperitoneal injection of rHDL and CML showed that a higher CIGB-258 content in rHDL was also associated with higher survivability of zebrafish and faster recovery of swimming ability. The rHDL-(1:1) group showed the lowest triglyceride, AST, and ALT serum levels with the least production of interleukin-6, oxidized product, and neutrophil infiltration in hepatic tissue. In conclusion, CIGB-258 could bind well to phospholipids and cholesterol to stabilize apoA-I in the rHDL structure against denaturation stress and larger particle sizes. The rHDL containing CIGB-258 enhanced the in vitro antioxidant ability against LDL oxidation, the anti-glycation activity to protect HDL, and the in vivo anti-inflammatory activity against CML toxicity in zebrafish adults and embryos. Overall, incorporating apoA-I and CIGB-258 in rHDL resulted in a synergistic interaction to enhance the structural and functional correlations in a dose-dependent manner of CIGB-258.

Structural Changes Induced by Resveratrol in Monounsaturated and Polyunsaturated Phosphatidylcholine-Enriched Model Membranes.

Resveratrol (Resv) is considered to exert a beneficial impact due to its radical scavenger, anti-microbial and anti-inflammatory properties through several mechanisms that could include its interaction with the cell plasma membrane. To address this issue, we investigated the influence of Resv on membrane lipid order and organization in large unilamellar vesicles composed of different lipids and ratios. The studied lipid membrane models were composed of phosphatidylcholine (PC) species (either palmitoyl-docosahexaenoyl phosphatidylcholine (PDPC) or palmitoyl-oleoyl phosphatidylcholine (POPC)), sphingomyelin (SM) and cholesterol (Chol). This study found that the addition of Resv resulted in complex membrane reorganization depending on the degree of fatty acid unsaturation at the sn-2 position, and the Lipid/Resv and SM/Chol ratios. Resv rigidified POPC-containing membranes and increased liquid-ordered (Lo) domain formation in 40/40/20 POPC/SM/Chol mixtures as this increase was lower at a 33/33/34 ratio. In contrast, Resv interacted with PDPC/SM/Chol mixtures in a bimodal manner by fluidizing/rigidifying the membranes in a dose-dependent way. Lo domain formation upon Resv addition occurred via the following bimodal mode of action: Lo domain size increased at low Resv concentrations; then, Lo domain size decreased at higher ones. To account for the variable effect of Resv, we suggest that it may act as a "spacer" at low doses, with a transition to a more "filler" position in the lipid bulk. We hypothesize that one of the roles of Resv is to tune the lipid order and organization of cell plasma membranes, which is closely linked to important cell functions such as membrane sorting and trafficking.

Enhancement of Antioxidant and Anti-Glycation Properties of Beeswax Alcohol in Reconstituted High-Density Lipoprotein: Safeguarding against Carboxymethyllysine Toxicity in Zebrafish.

The antioxidant and anti-inflammatory abilities of beeswax alcohol (BWA) are well reported in animal and human clinical studies, with a significant decrease in malondialdehyde (MDA) in the blood, reduced liver steatosis, and decreased insulin. However, there has been insufficient information to explain BWAs in vitro antioxidant and anti-inflammatory activity owing to its limited solubility in an aqueous buffer system. Herein, three distinct reconstituted high-density lipoproteins (rHDL) were prepared with palmitoyloleoyl phosphatidylcholine (POPC), cholesterol, apolipoprotein A-I (apoA-I), and BWA at molar ratios of 95:5:1:0 (rHDL-0), 95:5:1:0.5 (rHDL-0.5), and 95:5:1:1 (rHDL-1) and examined for antioxidant and anti-glycation effects. A rHDL containing BWA, precisely rHDL-1, displayed a remarkable anti-glycation effect against fructose (final 250 mM), induced glycation of HDL, and prevented proteolytic degradation of apoA-I. Also, BWA incorporated rHDL-0.5, and rHDL-1 displayed substantial antioxidant activity by inhibiting cupric ion-mediated low-density lipoprotein (LDL) oxidation. In contrast to rHDL-0, a 20 and 22% enhancement in ferric ion reduction ability (FRA) and paraoxonase (PON) activity was observed in HDL treated with rHDL-1, signifying the effect of BWA on the antioxidant activity enhancement of HDL. rHDL-1 efficiently inhibits Nε-carboxylmethyllysine (CML)-induced reactive oxygen species (ROS) generation and apoptosis in zebrafish embryos, consequently improving embryo survivability and developmental deformities impaired by the CML. The dermal application of rHDL-1 to the CML-impaired cutaneous wound of the adult zebrafish inhibited ROS production and displayed potent wound-healing activity. Conclusively, incorporating BWA in rHDL significantly enhanced the anti-glycation and antioxidant activities in rHDL via more stabilization of apoA-I with a larger particle size. The rHDL containing BWA facilitated the inherent antioxidant ability of HDL to suppress the CML-induced toxicities in zebrafish embryos and ameliorate CML-aggravated chronic wounds in adult zebrafish.

Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches.

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.

Solubilization of biomimetic lipid mixtures by some commonly used non-ionic detergents

Detergents are amphiphilic molecules often used to solubilize biological membranes and separate their components. Here we investigate the solubilization of lipid vesicles by the commonly used non-ionic detergents polyoxyethylene (20) oleyl ether (Brij 98), n-octyl-β-D-glucoside (OG), and n-dodecyl β-D maltoside (DDM) and compare the results with the standard detergent Triton X-100 (TX-100). The vesicles were composed of palmitoyl oleoyl phosphatidylcholine (POPC) or of a biomimetic ternary mixture of POPC, egg sphingomyelin (SM) and cholesterol (2:1:2 molar ratio). To follow the solubilization profile of large unilamellar vesicles (LUVs), 90° light scattering measurements were done along the titration of LUVs with the detergents. Then, giant unilamellar vesicles (GUVs) were observed with optical microscopy during exposure to the detergents, to allow direct visualization of the solubilization process. Isothermal titration calorimetry (ITC) was used to assess the binding constant of the detergents in POPC bilayers. The results show that the incorporation of TX-100, Brij 98 and, to a lesser extent, OG in the pure POPC liposomes leads to an increase in the vesicle area, which indicates their ability to redistribute between the two leaflets of the membrane in a short scale of time. On the other hand, DDM incorporates mainly in the external leaflet causing an increase in vesicle curvature/tension leading ultimately to vesicle burst. Only TX-100 and OG were able to completely solubilize the POPC vesicles, whereas the biomimetic ternary mixture was partially insoluble in all detergents tested. TX-100 and OG were able to incorporate in the bilayer of the ternary mixture and induce macroscopic phase separation of liquid-ordered (Lo) and liquid-disordered (Ld) domains, with selective solubilization of the latter. Combination of ITC data with turbidity results showed that TX-100 and OG can be incorporated up to almost 0.3 detergent/lipid, significantly more than Brij 98 and DDM. This fact seems to be directly related to their higher capacity to solubilize POPC membranes and their ability to induce macroscopic phase separation in the biomimetic lipid mixture.

Cuban Sugar Cane Wax Alcohol Exhibited Enhanced Antioxidant, Anti-Glycation and Anti-Inflammatory Activity in Reconstituted High-Density Lipoprotein (rHDL) with Improved Structural and Functional Correlations: Comparison of Various Policosanols

Policosanols from various sources, such as sugar cane, rice bran, and insects, have been marketed to prevent dyslipidemia, diabetes, and hypertension by increasing the blood high-density lipoproteins cholesterol (HDL-C) levels. On the other hand, there has been no study on how each policosanol influences the quality of HDL particles and their functionality. Reconstituted high-density lipoproteins (rHDLs) with apolipoprotein (apo) A-I and each policosanol were synthesized using the sodium cholate dialysis method to compare the policosanols in lipoprotein metabolism. Each rHDL was compared regarding the particle size and shape, antioxidant activity, and anti-inflammatory activity in vitro and in zebrafish embryos. This study compared four policosanols including one policosanol from Cuba (Raydel® policosanol) and three policosanols from China (Xi'an Natural sugar cane, Xi'an Realin sugar cane, and Shaanxi rice bran). The synthesis of rHDLs with various policosanols (PCO) from Cuba or China using a molar ratio of 95:5:1:1 with palmitoyloleoyl phosphatidylcholine (POPC): free cholesterol (FC): apoA-I:PCO (wt:wt) showed that rHDL containing Cuban policosanol (rHDL-1) showed the largest particle size and the most distinct particle shape. The rHDL-1 showed a 23% larger particle diameter and increased apoA-I molecular weight with a 1.9 nm blue shift of the maximum wavelength fluorescence than rHDL alone (rHDL-0). Other rHDLs containing Chinese policosanols (rHDL-2, rHDL-3, and rHDL-4) showed similar particle sizes with an rHDL-0 and 1.1-1.3 nm blue shift of wavelength maximum fluorescence (WMF). Among all rHDLs, the rHDL-1 showed the strongest antioxidant ability to inhibit cupric ion-mediated LDL oxidation. The rHDL-1-treated LDL showed the most distinct band intensity and particle morphology compared with the other rHDLs. The rHDL-1 also exerted the highest anti-glycation activity to inhibit the fructose-mediated glycation of human HDL2 with the protection of apoA-I from proteolytic degradation. At the same time, other rHDLs showed a loss of anti-glycation activity with severe degradation. A microinjection of each rHDL alone showed that rHDL-1 had the highest survivability of approximately 85 ± 3%, with the fastest developmental speed and morphology. In contrast, rHDL-3 showed the lowest survivability, around 71 ± 5%, with the slowest developmental speed. A microinjection of carboxymethyllysine (CML), a pro-inflammatory advanced glycated end product, into zebrafish embryos resulted in severe embryo death of approximately 30 ± 3% and developmental defects with the slowest developmental speed. On the other hand, the phosphate buffered saline (PBS)-injected embryo showed 83 ± 3% survivability. A co-injection of CML and each rHDL into adult zebrafish showed that rHDL-1 (Cuban policosanol) induced the highest survivability, around 85 ± 3%, while rHDL-0 showed 67 ± 7% survivability. In addition, rHDL-2, rHDL-3, and rHDL-4 showed 67 ± 5%, 62 ± 37, and 71 ± 6% survivability, respectively, with a slower developmental speed and morphology. In conclusion, Cuban policosanol showed the strongest ability to form rHDLs with the most distinct morphology and the largest size. The rHDL-containing Cuban policosanol (rHDL-1) showed the strongest antioxidant ability against LDL oxidation, anti-glycation activity to protect apoA-I from degradation, and the highest anti-inflammatory activity to protect embryo death under the presence of CML.

Asymmetric distribution of aminophospholipids stiffens fluid membranes.

Cellular envelopes contain a large number of lipid species that distribute asymmetrically between the two leaflets of the bilayer. For example, mammalian plasma membranes are composed of an outer leaflet enriched in cholinephospholipids, while the majority of the aminophospholipids are confined to the inner leaflet. Membrane asymmetry can theoretically lead to distinct effects regarding its elastic behavior, either due to lipid specific properties (e.g. size, shape), or simply lipid over/under-crowding of a given leaflet. Corresponding experimental data are scarce. This prompted us to determine the structure and bending rigidity of minimal mammalian plasma membrane mimics composed of milk sphingomyelin (MSM), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and palmitoyl oleoyl phosphatidylserine (POPS) using small-angle neutron/X-ray scattering and neutron spin echo spectroscopy. Most strikingly, we observed an anomalous stiffening of freely-floating fluid vesicles (size ∼ 100 nm) in case of inner leaflets composed of POPE/POPS mixtures and outer leaflets enriched in POPC. That is, the bending rigidities of POPCout/(POPE/POPS)in vesicles did not only exceed those of their scrambled (symmetric) analogs, but also those of symmetric vesicles mimicking either their inner and outer leaflets. Addition MSM in the outer leaflet reduced but not abolished this effect, possibly due to hydrocarbon chain interdigitation. To reconcile our findings, we speculate that lipid shape and charge asymmetry may increase the weight of short-wavelength undulatory modes in membranes by inducing a coupling at distances smaller than the membrane thickness.

Phosphatidylcholine Liposomes Reprogram Macrophages toward an Inflammatory Phenotype

Phospholipids are the major components of cellular membranes and cell-derived vesicles such as exosomes. They are also key components of artificial lipid nanoparticles, allowing the encapsulation and transport of various biological or chemical cargos. Both artificial and natural vesicles could be captured by cells delivering important information that could modulate cellular functions. However, the potential contribution of phospholipids within vesicles altering cellular physiology has been largely underestimated. Here, we showed that macrophages exposed to liposomes made exclusively with palmitoyl oleoyl phosphatidylcholine (POPC) in vivo resulted in a dramatic alteration of the transcriptome profile. Differential gene expression analysis indicated that the exposure to POPC liposomes resulted in a change in the expression of 1598 genes. Moreover, 146 genes were upregulated, and 69 genes were downregulated by incubation with POPC liposomes in contrast to palmitoyl oleoyl phosphatidylserine (POPS) exposure. Signaling pathway impact analysis revealed that 24 signaling pathways were significantly modulated after exposure to POPC liposomes, including the activation of the NF-κB pathway. Indeed, the expression of several cytokines (TNF-α, IL-6, and IL-10) and chemokines (Cxcl1 and Cxcl2) were increased. These observations were validated by the exposure of macrophages to POPC liposomes in culture conditions. In addition, the proteomic analysis of peritoneal cells exposed to POPC liposomes performed by mass spectrometry revealed that the expression of 107 proteins was downregulated after POPC exposure, whereas the expression of 12 proteins was significantly upregulated by this treatment, including seven proteins involved in the neutrophil degranulation pathway. This observation was confirmed by flow cytometry analysis showing the rapid recruitment of neutrophils into the peritoneal cavity after POPC exposure. Overall, these findings demonstrate that the presence of phospholipids within artificial and natural vesicles could be responsible for changes in the function of target cells.

Characterization of phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers using laurdan fluorescence with log-normal multipeak analysis

Phase separation phenomena in hybrid lipid/block copolymer/cholesterol bilayers combining polybutadiene-block-polyethylene oxide (PBdPEO), egg sphingomyelin (egg SM), and cholesterol were studied with fluorescence spectroscopy and microscopy for comparison to lipid bilayers composed of palmitoyl oleoyl phosphatidylcholine (POPC), egg SM, and cholesterol. Laurdan emission spectra were decomposed into three lognormal curves. The temperature dependence of the ratios of the areas of the middle and lowest energy peaks revealed temperature break-point (Tbreak) values that were in better agreement, compared to generalized polarization inflection temperatures, with phase transition temperatures in giant unilamellar vesicles (GUVs). Agreement between GUV and spectroscopy results was further improved for hybrid vesicles by using the ratio of the area of the middle peak to the sum of the areas all three peaks to find the Tbreak values. For the hybrid vesicles, trends at Tbreak are hypothesized to be correlated with the mechanisms by which the phase transition takes place, supported by the compositional range as well as the morphologies of domains observed in GUVs. Low miscibility of PBdPEO and egg SM is suggested by the finding of relatively high Tbreak values at cholesterol contents greater than 30 mol%. Further, GUV phase behavior suggests stronger partitioning of cholesterol into PBdPEO than into POPC, and less miscibility of PBdPEO than POPC with egg SM. These results, summarized using a heat-map, contribute to the limited body of knowledge regarding the effect of cholesterol on hybrid membranes, with potential application toward the development of such materials for drug delivery or membrane protein reconstitution.

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Interfacial behavior of phospholipid monolayers revealed by mesoscopic simulation

A mesoscopic model with molecular resolution is presented for dipalmitoyl phosphatidylcholine (DPPC) and palmitoyl oleoyl phosphatidylcholine (POPC) monolayer simulations at the air-water interface using many-body dissipative particle dynamics (MDPD). The parameterization scheme is rigorously based on reproducing the physical properties of water and alkane and the interfacial property of the phospholipid monolayer by comparison with experimental results. Using much less computing cost, these MDPD simulations yield a similar surface pressure-area isotherm as well as similar pressure-related morphologies as all-atom simulations and experiments. Moreover, the compressibility modulus, order parameter of lipid tails, and thickness of the phospholipid monolayer are quantitatively in line with the all-atom simulations and experiments. This model also captures the sensitive changes in the pressure-area isotherms of mixed DPPC/POPC monolayers with altered mixing ratios, indicating that the model is promising for applications with complex natural phospholipid monolayers. These results demonstrate a significant improvement of quantitative phospholipid monolayer simulations over previous coarse-grained models.

Estimation of the Phospholipase A2 Selectivity on POPC/POPG Membranes Using the Interaction Map

The regulation of the activity and selectivity of phospholipase A2 (PLA2), which is capable of cleaving fatty acid in the second position (sn-2) of the phospholipid, is carried out through the membrane-binding and catalytic sites of the enzyme. For hydrolytic activity, PLA2 must first bind to the phospholipid membrane, and the binding efficiency depends on the composition of the membrane. The membrane-binding site of PLA2 is formed by several tens of amino acids and its composition differs from enzyme to enzyme; hydrophobic and positively charged amino acids play a key role in the interaction. In this work, we investigated the interaction of PLA2 from bee venom with phospholipid bilayers of palmitoyl oleoylphosphatidylcholine (POPC) containing different amounts of palmitoyloleoylphosphatidylglycerol (POPG). On the basis of the measurements of the protein intrinsic fluorescence and the anisotropy of the fluorescence of the lipid probe we propose the construction of lipid–protein interaction maps, which reflect both the efficiency of protein binding and changes in the structure of the membrane. These changes cause alterations in the fluorescence anisotropy of the label, which in turn is a measure of the mobility of the lipid environment of the fluorescent probe. Analysis of interaction maps showed that there is a relationship between lipid mobility and enzyme binding efficiency: the optimum interaction of PLA2 with membranes from a POPC/POPG mixture lies in the region of the highest lipid mobility, and not in the region of the highest negative charge. This dependence complements the existing understanding of the process of recognition of the membrane surface by the enzyme and the selection of lipids by the enzyme already bound to the membrane. The proposed mapping method can be extended to other membrane-active proteins.

The Role of Hyaluronic Acid in Cartilage Boundary Lubrication.

Hydration lubrication has emerged as a new paradigm for lubrication in aqueous and biological media, accounting especially for the extremely low friction (friction coefficients down to 0.001) of articular cartilage lubrication in joints. Among the ensemble of molecules acting in the joint, phosphatidylcholine (PC) lipids have been proposed as the key molecules forming, in a complex with other molecules including hyaluronic acid (HA), a robust layer on the outer surface of the cartilage. HA, ubiquitous in synovial joints, is not in itself a good boundary lubricant, but binds the PC lipids at the cartilage surface; these, in turn, massively reduce the friction via hydration lubrication at their exposed, highly hydrated phosphocholine headgroups. An important unresolved issue in this scenario is why the free HA molecules in the synovial fluid do not suppress the lubricity by adsorbing simultaneously to the opposing lipid layers, i.e., forming an adhesive, dissipative bridge between them, as they slide past each other during joint articulation. To address this question, we directly examined the friction between two hydrogenated soy PC (HSPC) lipid layers (in the form of liposomes) immersed in HA solution or two palmitoyl–oleoyl PC (POPC) lipid layers across HA–POPC solution using a surface force balance (SFB). The results show, clearly and surprisingly, that HA addition does not affect the outstanding lubrication provided by the PC lipid layers. A possible mechanism indicated by our data that may account for this is that multiple lipid layers form on each cartilage surface, so that the slip plane may move from the midplane between the opposing surfaces, which is bridged by the HA, to an HA-free interface within a multilayer, where hydration lubrication is freely active. Another possibility suggested by our model experiments is that lipids in synovial fluid may complex with HA, thereby inhibiting the HA molecules from adhering to the lipids on the cartilage surfaces.

Elasticity and phase behaviour of biomimetic membrane systems containing tetraether archaeal lipids

Archaeal single-cell microorganisms are characterised by particularly strong resistance to harsh environments at extreme temperature ranges, pH values, high pressure and salinity. The recognition of molecules rendering the archaeal membranes stable in such conditions represents an important step towards understanding the molecular mechanisms, which underlie the cellular survival, in order to effectively exploit them in biomedicine and bionanotechnology. Here we report on the bending elasticity and phase behaviour of model lipid membranes containing tetraether archaeal lipids. The bending elasticity modulus of membranes composed of palmitoyl-oleoyl phosphatidylcholine and glycerol dialkyl glycerol tetraether extract from the archaeon Thermoplasma acidophilum’s plasma membrane is measured by analysis of the thermal shape fluctuations of nearly spherical giant unilamellar vesicles (tens of micrometers in size). The bending rigidity is reported to non-monotonically depend on the archaeal lipids’ concentration in the bilayer. At 50 wt% of tetraether lipids in the membrane we measure around 20% decrease of the bending modulus compared to the studied single-component phosphocholine bilayer. The membrane resistance to bending increases at archaeal lipids’ content higher than 70 wt%. These findings support the hypothesis about a probable looping conformation at very low amounts of bipolar lipids in the membrane and prevailing spanning tetraether molecules at higher concentrations. Fluorescence microscopy reveals structural phase coexistence at low temperatures (6 °C) for 75 wt% of bolalipids in the bilayer. Laurdan spectroscopy measurements of large unilamellar vesicles (hundreds of nanometers in size) provide evidences for increased lipid ordering at the glycerol level induced by the presence of up to 90 wt% of bolalipid in the bilayer. At this archaeal lipid content we measure increased bending rigidity of the membrane. The fatty acyl chain mobility probed by DPH fluorescence spectroscopy is significantly reduced in the presence of bolalipids. The lipid ordering decreases with increasing the temperature to an extent depending on the bolalipid content in the membrane. The presence of bipolar lipids in the bilayer affects the lipid packing more strongly at the glycerol level compared to the hydrophobic core of the membrane. By elucidating the effect of tetraether lipids on the structural and mechanical properties of the bilayer, the reported results are expected to help in advancing on liposome-based pharmaceuticals and biomedical applications as well as for developments in sensorics and nanotechnology.

Lipid ratios as a marker for red blood cell storage quality and as a possible explanation for donor gender differences in storage quality.

Red blood cells that are stored for transfusions as red cell concentrates (RCCs) undergo changes during the storage period, culminating in the lysis of the cells. The goal of this work is to find markers that are linked to high haemolysis, in order to explain the inter-donor variability that is known to occur in storage quality, and also the known differences between RCCs from male and female donors. The relative amounts of lipids at the end of the storage period were compared for one group of low haemolysis samples (24 units, all ≤0·15% haemolysis), and one group of high haemolysis samples (26 units, all ≥0·5% haemolysis). Representative lipids were analysed from different lipid classes, including cholesterol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and ceramide. Whole membrane preparations were analysed with one mass spectrometry technique, and lipid extracts were analysed with a second mass spectrometry technique. The ratio of palmitoyl-oleoyl phosphatidylcholine (POPC) to sphingomyelin was different for the high and low haemolysis groups (P=0·0001) and for the RCCs from male and female donors (P=0·0009). The ratio of cholesterol to phospholipids showed only minimal links to haemolysis. Higher relative amounts of sphingomyelin were associated with lower haemolysis, and higher relative amounts of ceramides were associated with increased haemolysis. The level of sphingomyelinase activity and the resulting ratio of sphingomyelin to POPC is proposed as a possible marker for RCC storage quality.

Revealing the Mode of Action of Halictine Antimicrobial Peptides: A Comprehensive Study with Model Membranes.

Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, ζ-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an α-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 103-104 M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.

The small heat shock proteins, HSPB1 and HSPB5, interact differently with lipid membranes.

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu. Several mechanisms have been proposed, including direct interaction with the plasma membrane or their release within extracellular vesicles (ECV). HSPB1 (Hsp27), which belongs to the small hsp family, was detected within the membrane of ECV released from stressed HepG2 cells. To further investigate this finding, we studied the interaction of HSPB1 with lipid membranes using liposomes. We found that HSPB1 interacted with liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG), with different characteristics. Another member of the small hsp family, HSPB5 (αB-crystallin), has also been detected within ECV released from HeLa cells transfected with this gene. This protein was found to interact with liposomes as well, but differently than HSPB1. To address the regions interacting with the membrane, proteoliposomes were digested with proteinase K and the protected domains within the liposomes were identified by mass spectroscopy. We observed that large parts of HSPB1 and HSPB5 were embedded within the liposomes, particularly the alpha-crystallin domain. These observations suggest that the interaction with lipid membranes may be part of the mechanisms of export of these proteins.

Behavior of Doxorubicin Lipophilic Conjugates in Liposomal Lipid Bilayers

Preparation of liposomal formulations containing water-soluble drugs in the form of lipophilic prodrugs in their lipid bilayer is of considerable interest. Previously, we synthesized doxorubicin dioleoyl glyceride and oleoyl conjugates intended for incorporation into fluid-phase liposomal bilayers. In this work, we studied the behavior of lipid conjugates in bilayers prepared from palmitoyl oleoyl phosphatidylcholine and dimyristoyl phosphatidylcholine using methods of fluorescence spectroscopy and molecular modeling. The conjugates were shown to have limited mobility in lipid bilayers, which can be explained by the formation of hydrogen bonds between the doxorubicin aglycone and the lipid phosphate groups. In the liposome membrane, lipophilic conjugates also tend to form clusters through interaction of doxorubicin moieties. Oleoyl chains stretch in parallel to the acyl residues of phospholipids. Due to the formation of a larger number of hydrogen bonds, the oleoyl conjugates interacted with the bilayer more effectively than the dioleoyl glyceride counterparts. These properties of doxorubicin conjugates can affect both the possibility of their incorporation into the lipid bilayer (from the therapeutic effect point of view) and intracellular release of the antibiotic drug by means of enzymolysis.

Sucrose solutions alter the electric capacitance and dielectric permittivity of lipid bilayers

Understanding sugar-membrane interactions is of fundamental and technological relevance considering the role of sugars in drought-protection mechanisms of plants as well as the cryo- and bio-preserving effect of carbohydrates in many industrial and medical applications. In this work, we investigated the effect of sucrose on the electrical properties of membranes. In particular, we measured the specific capacitance of palmitoyl-oleoyl phosphatidylcholine membranes in aqueous solutions of sodium chloride. Different concentrations of sucrose were examined. The capacitance was assessed from the frequency-dependent deformation of giant unilamellar lipid vesicles in alternating electric field. Our measurements on giant vesicles in sugar-free aqueous solutions yield lower specific capacitance compared to values obtained for suspended and supported bilayers. This might be a result of the higher membrane tension in the latter systems, which is coupled to smaller thickness of the bilayer. We also report an increase of the bilayer capacitance upon increasing the sugar content in water. This finding is consistent with the sugar-induced thinning of membranes reported in the literature. However, the thinning is not sufficient to explain the observed capacitance increase with rising sugar concentration. We interpret the trend as resulting from an increase in the membrane dielectric permittivity.