Interactions between model membranes and lignin-related compounds studied by immobilized liposome chromatography

In order to elucidate the modes of interaction between lignin precursors and membranes, we have studied the influence of temperature, lipid composition and buffer composition on the partitioning of monolignol and dilignol model substances into phospholipid bilayers. The partitioning was determined by immobilized liposome chromatography, which is an established method for studies of pharmaceutical drugs but a new approach in studies of lignin synthesis. The temperature dependence of the retention and the effect of a high ammonium sulfate concentration in the mobile phase demonstrated that the interaction involved both hydrophobic effects and polar interactions. There was also a good correlation between the partitioning and the estimated hydrophobicity, in terms of octanol/water partitioning. The partitioning behavior of the model substances suggests that passive diffusion over the cell membrane is a possible transport route for lignin precursors. This conclusion is strengthened by comparison of the present results with the partitioning of pharmaceutical drugs that are known to pass cell membranes by diffusion.     

Biopartitioning micellar chromatography: an in vitro technique for predicting human drug absorption

The main oral drug absorption barriers are fluid cell membranes and generally drugs are absorbed by a passive diffusion mechanism. Biopartitioning micellar chromatography (BMC) is a mode of micellar liquid chromatography that uses micellar mobile phases of Brij35 under adequate experimental conditions and can be useful to mimic the drug partitioning process in biological systems. In this paper the usefulness of BMC for predicting oral drug absorption in humans is demonstrated. A hyperbolic model has been obtained using the retention data of a heterogeneous set of 74 compounds, which shows predictive ability for drugs absorbed by passive diffusion. The model obtained in BMC is compared with those obtained using the well-known systems (Caco-2 and TC-7) that use intestinal epithelium cell lines. The use of BMC is simple, reproducible and can provide key information about the transport properties of new compounds during the drug discovery process.     

Biophysical characterization of mycobacterial model membranes and their interaction with rifabutin: Towards lipid-guided drug screening in tuberculosis

Lipid structure critically dictates the molecular interactions of drugs with membranes influencing passive diffusion, drug partitioning and accumulation, thereby underpinning a lipid-composition specific interplay. Spurring selective passive drug diffusion and uptake through membranes is an obvious solution to combat growing antibiotic resistance with minimized toxicities. However, the spectrum of complex mycobacterial lipids and lack thereof of suitable membrane platforms limits the understanding of mechanisms underlying drug-membrane interactions in tuberculosis. Herein, we developed membrane scaffolds specific to mycobacterial outer membrane and demonstrate them as improvised research platforms for investigating anti-tubercular drug interactions. Combined spectroscopy and microscopy results reveal an enhanced partitioning of model drug Rifabutin in trehalose dimycolate-containing mycobacterial membrane systems. These effects are apportioned to specific changes in membrane structure, order and fluidity leading to enhanced drug interaction. These findings on the membrane biophysical consequences of drug interactions will offer valuable insights for guiding the design of more effective antibiotic drugs coupled with tuned toxicity profiles.     

The interactions of bilirubin with model and biological membranes

The partitioning of bilirubin between albumin and model and biological membranes and the differential partitioning of bilirubin between membranes with different lipid and protein compositions were measured. Partition coefficients were independent of the concentration of bilirubin in membranes up to at least 7 mol of bilirubin/mol of phospholipid. The avidity of albumin for bilirubin was greater than that of membranes, but the avidity of the latter for bilirubin depended on the composition of the membrane. Bilirubin partitioned preferentially into model membranes comprised of microsomal lipids greater than dioleoylphosphatidylcholine = plasma membrane lipids much greater than egg phosphatidylcholine = dimyristoylphosphatidylcholine. Partitioning into membranes was increased if these contained proteins, but the effect of proteins could not be attributed to specific binding to sites on proteins, as reflected by the temperature independence of partition coefficients. Differential partitioning of bilirubin into different membranes of pure lipids also was independent of temperature. Differences in the bulk phase fluidity of membranes does not appear to account for the preferential partitioning of bilirubin into some membranes. It appears that bilirubin partitions into elements of free volume of differing sizes in membranes with variable lipid compositions and that the size of these elements can be increased by adding proteins to membranes.     

Effect of sodium bicarbonate as a pharmaceutical formulation excipient on the interaction of fluvastatin with membrane phospholipids

Excipients in the pharmaceutical formulation of oral drugs are notably employed to improve drug stability. However, they can affect drug absorption and bioavailability. Passive transport through intestinal cell walls is the main absorption mechanism of drugs and, thus, involves an interaction with the membrane lipids. Therefore in this work, the effect of the excipient NaHCO₃ on the interaction of the anticholesterolemic drug fluvastatin sodium (FS) with membrane phospholipids was investigated by ¹H NMR and FTIR spectroscopy. Sodium bicarbonate is often combined with fluvastatin for oral delivery to prevent its degradation. We have used model DMPC/DMPS membranes to mimic the phospholipid content of gut cell membranes. The results presented in this work show a 100% affinity of FS for the membrane phospholipids that is not modified by the presence of the excipient. However, NaHCO₃ is shown to change the interaction mechanism of the drug. According to our data, FS enters the DMPC/DMPS bilayer interface by interacting with the lipids’ polar headgroups and burying its aromatic moieties into the apolar core. Moreover, lipid segregation takes place between the anionic and zwitterionic lipids in the membranes due to a preferential interaction of FS with phosphatidylserines. The excipient counteracts this favored interaction without affecting the drug affinity and location in the bilayer. This work illustrates that preferential interactions with lipids can be involved in passive drug permeation mechanisms and gives evidence of a possible nonpassive role of certain excipients in the interaction of drugs with membrane lipids.     

Association of ethanol with lipid membranes containing cholesterol, sphingomyelin and ganglioside: a titration calorimetry study.

The association of ethanol at physiologically relevant concentrations with lipid bilayers of different lipid composition has been investigated by use of isothermal titration calorimetry (ITC). The liposomes examined were composed of combinations of lipids commonly found in neural cell membranes: dimyristoyl phosphatidylcholine (DMPC), ganglioside (GM(1)), sphingomyelin and cholesterol. The calorimetric results show that the interaction of ethanol with fluid lipid bilayers is endothermic and strongly dependent on the lipid composition of the liposomes. The data have been used to estimate partitioning coefficients for ethanol into the fluid lipid bilayer phase and the results are discussed in terms of the thermodynamics of partitioning. The presence of 10 mol% sphingomyelin or ganglioside in DMPC liposomes enhances the partitioning coefficient by a factor of 3. Correspondingly, cholesterol (30 mol%) reduces the partitioning coefficient by a factor of 3. This connection between lipid composition and partitioning coefficient correlates with in vivo observations. Comparison of the data with the molecular structure of the lipid molecules suggests that ethanol partitioning is highly sensitive to changes in the lipid backbone (glycerol or ceramide) while it appears much less sensitive to the nature of the head group.     

Importance of the cell membrane on the mechanism of action of cyclotides

Their distinctive structures, diverse range of bioactivities, and potential for pharmaceutical or agricultural applications make cyclotides an intriguing family of cyclic peptides. Together with the physiological role in plant host defense, cyclotides possess antimicrobial, anticancer, and anti-HIV activities. In all of the reported activities, cell membranes seem to be the primary target for cyclotide binding. This article examines recent literature on cyclotide-membrane studies and highlights the hypothesis that the activity of cyclotides is dependent on their affinity for lipid bilayers and enhanced by the presence of specific lipids, i.e., phospholipids containing phosphatidylethanolamine headgroups. There is growing evidence that the lipid composition of target cell membranes dictates the amount of cyclotides bound to the cell and the extent of their activity. After membrane targeting and insertion in the bilayer core, cyclotides induce disruption of membranes by a pore formation mechanism. This proposed mechanism of action is supported by biophysical studies with model membranes and by studies on natural biological membranes of known lipid compositions.     

Endocytosis of liposomes by Entamoeba histolytica and transport of encapsulated molecules toward the cytoplasm

We have examined the interaction of a highly phagocytosing cell: Entamoeba histolytica with liposomes of different lipid compositions, and followed, by a semi-quantitative method, the intracellular fate of the entrapped molecules. Liposomes containing a small molecule, 6-carboxyfluorescein, are first phagocytosed. Then the encapsulated compound migrates from the vacuoles to the cytoplasm. Liposomes containing macromolecular substances, such as fluorescent albumin or ferritin, are also phagocytosed, but the encapsulated molecules remain within the vacuoles. We conclude that the transfer of carboxyfluorescein does not involve a fusion between liposomes and vacuoles, but more likely occurs via diffusion through membranes. The lipid composition of the liposomes does not affect phagocytosis of liposomes. In contrast, oleic acid greatly increases the transfer of carboxyfluorescein from vacuole to cytoplasm.     

Sphingolipids and the formation of sterol-enriched ordered membrane domains

This review is focused on the formation of lateral domains in model bilayer membranes, with an emphasis on sphingolipids and their interaction with cholesterol. Sphingolipids in general show a preference for partitioning into ordered domains. One of the roles of cholesterol is apparently to modulate the fluidity of the sphingolipid domains and also to help segregate the domains for functional purposes. Cholesterol shows a preference for sphingomyelin over phosphatidylcholine with corresponding acyl chains. The interaction of cholesterol with different sphingolipids is largely dependent on the molecular properties of the particular sphingolipid in question. Small head group size clearly has a destabilizing effect on sphingolipid/cholesterol interaction, as exemplified by studies with ceramide and ceramide phosphoethanolamine. Ceramides actually displace sterol from ordered domains formed with saturated phosphatidylcholine or sphingomyelin. The N-linked acyl chain is known to be an important stabilizer of the sphingolipid/cholesterol interaction. However, N-acyl phosphatidylethanolamines failed to interact favorably with cholesterol and to form cholesterol-enriched lateral domains in bilayer membranes. Glycosphingolipids also form ordered domains in membranes but do not show a strong preference for interacting with cholesterol. It is clear from the studies reviewed here that small changes in the structure of sphingolipids alter their partitioning between lateral domains substantially.     

β-Cyclodextrin hydrogels as potential drug delivery systems

β-cyclodextrins (βCD) are cyclic oligosaccharides which have been widely employed for pharmaceutical applications. Discs of insoluble polymers were synthesized by crosslinking β-cyclodextrins with the reagent epichlorohydrin. In this work, the possibility of employing a polymer containing 60 ± 3% βCD for drug delivery of two antiinflammatory (naproxen and nabumetone) and two antifungal drugs (naftifine and terbinafine) has been investigated. The interaction of Naproxen with the polymers was evidenced by X-ray diffractometry, FTIR spectroscopy and differential thermal analysis. Drug release kinetics were carried out at physiological conditions of pH and temperature, and kinetic and diffusion constants were calculated by fitting 60% of the release profile according to the Korsmeyer-Peppas equation. Also, diffusion coefficients were calculated according to the simplified Higuchi model. The drug release followed a simple Fickian diffusion mechanism for all the model drugs. This study suggests that these hydrogel matrices are potentially suitable as sustained release systems.     

Sphingolipids and the formation of sterol-enriched ordered membrane domains

This review is focused on the formation of lateral domains in model bilayer membranes, with an emphasis on sphingolipids and their interaction with cholesterol. Sphingolipids in general show a preference for partitioning into ordered domains. One of the roles of cholesterol is apparently to modulate the fluidity of the sphingolipid domains and also to help segregate the domains for functional purposes. Cholesterol shows a preference for sphingomyelin over phosphatidylcholine with corresponding acyl chains. The interaction of cholesterol with different sphingolipids is largely dependent on the molecular properties of the particular sphingolipid in question. Small head group size clearly has a destabilizing effect on sphingolipid/cholesterol interaction, as exemplified by studies with ceramide and ceramide phosphoethanolamine. Ceramides actually displace sterol from ordered domains formed with saturated phosphatidylcholine or sphingomyelin. The N-linked acyl chain is known to be an important stabilizer of the sphingolipid/cholesterol interaction. However, N-acyl phosphatidylethanolamines failed to interact favorably with cholesterol and to form cholesterol-enriched lateral domains in bilayer membranes. Glycosphingolipids also form ordered domains in membranes but do not show a strong preference for interacting with cholesterol. It is clear from the studies reviewed here that small changes in the structure of sphingolipids alter their partitioning between lateral domains substantially.     

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

Organized assembly or aggregation of sphingolipid-binding ligands, such as certain toxins and pathogens, has been suggested to increase binding affinity of the ligand to the cell membrane and cause membrane reorganization or distortion. Here we show that the diffusion behavior of the fluorescently tagged sphingolipid-interacting peptide probe SBD (Sphingolipid Binding Domain) is altered by modifications in the construction of the peptide sequence that both result in a reduction in binding to ganglioside-containing supported lipid membranes, and at the same time increase aggregation on the cell plasma membrane, but that do not change relative amounts of secondary structural features. We tested the effects of modifying the overall charge and construction of the SBD probe on its binding and diffusion behavior, by Surface Plasmon Resonance (SPR; Biacore) analysis on lipid surfaces, and by Fluorescence Correlation Spectroscopy (FCS) on live cells, respectively. SBD binds preferentially to membranes containing the highly sialylated gangliosides GT1b and GD1a. However, simple charge interactions of the peptide with the negative ganglioside do not appear to be a critical determinant of binding. Rather, an aggregation-suppressing amino acid composition and linker between the fluorophore and the peptide are required for optimum binding of the SBD to ganglioside-containing supported lipid bilayer surfaces, as well as for interaction with the membrane. Interestingly, the strength of interactions with ganglioside-containing artificial membranes is mirrored in the diffusion behavior by FCS on cell membranes, with stronger binders displaying similar characteristic diffusion profiles. Our findings indicate that for aggregation-prone peptides, aggregation occurs upon contact with the cell membrane, and rather than giving a stronger interaction with the membrane, aggregation is accompanied by weaker binding and complex diffusion profiles indicative of heterogeneous diffusion behavior in the probe population.     

Dynamic Partitioning of a Glycosyl-Phosphatidylinositol-Anchored Protein in Glycosphingolipid-Rich Microdomains Imaged by Single-Quantum Dot Tracking

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.     

A differential vapor-pressure equipment for investigations of biopolymer interactions

The design and performance of an equipment for the measurement of vapor pressures over liquid or solid samples is presented. The equilibrium pressure difference, ΔP, between a sample and a reference of known vapor pressure is recorded as a function of composition and/or temperature. Through the use of high-accuracy capacitance manometers and a leak-tight system of stainless steel pipes, below-sealed valves and metal-gasket fittings, ΔP can be measured with a resolution of about 0.5 μbar (0.05 Pa) in some applications. This sensitivity level, along with other features of the equipment, particularly a “gas-phase titration” routine for changing the cell composition, makes it effective for the investigations of several types of biopolymer interactions. These include isothermal studies of net affinities such as the adsorption of water to proteins or membranes, the preferential interaction of biopolymers with the components of a mixed solvent, the partitioning of solutes between a membrane and the aqueous bulk and the weak, specific binding of ligands to macromolecules. Furthermore, a temperature-scanning mode allows real-time elucidation of such interactions at thermally induced conformational changes in biopolymers. Selected examples of these applications are presented and discussed.     

Lignification in wheat roots parasitised by Gaeuntannomyces graminis and Phialophora radicicola

Preliminary results suggest a possible relationship between lignin synthesis in wheat roots and the observed interaction between Gaeumannomyces graminis (Sacc.) Arx & Olivier and Phialophora radicicola Cain var. graminicola Deacon when they parasitise wheat roots. It was found that colonisation of wheat roots by P. radicicola resulted in a qualitative change in the lignin of the root, such that the content of the p-hydroxy type of aromatic nucleus was reduced almost to zero. It was also found that some of the metabolic precursors of lignin were inhibitory to the growth of G. graminis in Petri-dish culture. Most inhibitory of these precursors was caffeic acid, which reduced the growth rate of G. graminis by half at a concentration of 37 ppm. It is tentatively suggested that colonisation by P. radicicola results in an increased activity of polyphenol oxidase in the root tissues. This would lead to a more rapid synthesis of caffeic acid, with a depletion of the level of p-coumaric acid, and probably an increase in the levels of ferulic acid and sinapic acid. As well as bringing about a change in the composition of the lignin of the root, as the results show, the possible accumulation of caffeic acid in the root tissues might explain the greater resistance to infection by G. graminis observed in roots colonised first by P. radicicola.     

Glycolipid transfer protein interaction with bilayer vesicles: modulation by changing lipid composition

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.     

A Fickian diffusion transport process with features of transport catalysis. Doxorubicin transport in human red blood cells

The transport of the antineoplastic drug doxorubicin (Adriamycin) in human red blood cells was investigated by measuring the net efflux from loaded cells. Previous data indicated that doxorubicin transport was a Fickian diffusion transport process of the electrically neutral molecule through the lipid domain of the cell membrane (Dalmark, 1981 [In press]). However, doxorubicin transport showed saturation kinetics and a concentration-dependent temperature dependence with nonlinear Arrhenius plots. The two phenomena were related to the doxorubicin partition coefficient between 1-octanol and a water phase. This relationship indicated that the two phenomena were caused by changes in the physiochemical properties of doxorubicin in the aqueous phase and were not caused by interaction of doxorubicin with cell membrane components. The physicochemical properties of doxorubicin varied with concentration and temperature because of the ability of doxorubicin to form polymers by self-association in aqueous solution like other planar aromatic molecules through pi-electron orbital interaction. The hypothesis is proposed that doxorubicin transport across cell membranes takes place by simple Fickian diffusion.     

Partitioning of membrane molecules between raft and non-raft domains: insights from model-membrane studies

The special physical and functional properties ascribed to lipid rafts in biological membranes reflect their distinctive organization and composition, properties that are hypothesized to rest in part on the differential partitioning of various membrane components between liquid-ordered and liquid-disordered lipid environments. This review describes the principles and findings of recently developed methods to monitor the partitioning of membrane proteins and lipids between liquid-ordered and liquid-disordered domains in model membranes, and how these approaches can aid in elucidating the properties of rafts in biological membranes.     

Poly(N-isopropylacrylamide) surface-grafted chitosan membranes as a new substrate for cell sheet engineering and manipulation

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosan membranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance ((1)H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously described methodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.