Influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine/cholesterol bilayers

Effects of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers containing cholesterol as well as on 1H NMR spectra were studied. Two series of experiments were performed. In the first one the surfactants were added to the water phase while in the other directly to the lipid phase (a mixed film from cholesterol, surfactant and phosphatidylcholine was formed). The effects of particular surfactants on the main phase transition temperature, Tm, were more pronounced when added to the lipid phase (2nd method) than to the water phase (1st method); the opposite happened when cholesterol was absent (Rózycka-Roszak and Pruchnik 2000, Z. Naturforsch. 55c, 240-244). Furthermore, in the case of the first method the transitions were asymmetrical while in the second method nearly symmetrical. It is suggested that surfactant poor and surfactant rich domains are formed when surfactants are added to the water phase.     

Interaction of surfactants with bilayer of negatively charged lipid: effect on gel-to-liquid-crystalline phase transition of dilauroylphosphatidic acid vesicle membrane

The interaction of surfactants with the vesicle membrane of the negatively charged lipid, dilauroylphosphatidic acid, was investigated through their effect on the gel-to-liquid-crystalline phase transition of the lipid bilayer. Three types of surfactants (anionic, cationic and non-ionic) with different hydrocarbon chain length were examined. (i) Anionic sodium alkylsulfates affected the phase transition temperature, Tm, only weakly. (ii) Non-ionic alkanoyl-N-methylglucamides decreased Tm monotonously with increasing concentration. The depression of Tm induced by these surfactants was analyzed by applying the van't Hoff model for the freezing-point depression, and the partition coefficients of the surfactants between bulk water and lipid membrane were estimated. (iii) Cationic alkyltrimethylammonium bromides affected Tm in a complex manner depending on the hydrocarbon chain length of the surfactants. Octyl-/tetradecyl-trimethylammonium bromide depressed/elevated Tm monotonously with increasing concentration, whereas the change in Tm induced by decyl- and dodecyltrimethylammonium bromides was not monotonous but biphasic. This complex behavior of the phase transition temperature was well explained, based on the statistical mechanical theory presented by Suezaki et al. (Biochim. Biophys. Acta, 818 (1985) 31-37), which takes into account the interaction between surfactant molecules incorporated in the lipid membrane.     

Influence of dodecyltrimethylammonium halides on interaction of phenyltin compounds with model membranes

The effects were studied of dodecyltrimethylammonium chloride (DTAC), dodecyltrimethylammonium bromide (DTAB) and dodecyltrimethylammonium iodide (DTAI) on thermotropic phase behaviour of phosphatidylcholine bilayers, as well as on 1H NMR and 31P NMR spectra, in the presence of diphenyltin dichloride (DPhT) and triphenyltin chloride (TPhT). The obtained results indicate that in the presence of the surfactant studied the interaction of phenyltin compounds with model membranes was changed and the changes depended on the kind of the counterion. The surfactants studied (especially DTAC) decrease the ability of phenyltin compounds to induce structural changes in the bilayer. It is suggested that DTAB, and especially DTAC, prevent DPhT induced interdigitated phase formation as well as formation of an inverted hexagonal phase (H(II)) in the case of TPhT/DPPC liposomes.     

Interaction between N-dodecyl-N,N-dimethyl-N-benzylammonium halides and phosphatidylcholine bilayers-the effect of counterions

The interaction of N-dodecyl-N,N-dimethyl-N-benzylammonium halides (DBeAX) with two types of phospholipid vesicles (MLV and SUV) was investigated using DSC and 1H NMR. It was suggested that the benzyl group like the micellisation process (J. Colloid Interface Sci. 218 (1999) 529) changes its position when interacting with phosphatidylcholine bilayers and incorporates into the bilayer. In order to enhance counterion-water interactions, the surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). It follows from the obtained results that for both types of liposomes and both manners in which the surfactant was added, the interaction of DBeAX with liposomes and consequent changes in the phospholipid bilayer organisation depend on the kind of counterion. Results are discussed in terms of counterion ability to modify water structure.     

The influence of cholesterol on the interaction between N-dodecyl-N,N-dimethyl-N-benzylammonium halides and phosphatidylcholine bilayers

Effects of N-dodecyl-N,N-dimethyl-N-benzylammonium halides (DBeAX) on thermotropic phase behavior of phosphatidylcholine/cholesterol bilayers as well as on 1H NMR spectra were studied. The surfactants were added either to the water phase or directly to the lipid phase (a mixed film was formed). The benzyl group, opposite to liposomes without cholesterol, is not incorporated into the bilayer in the gel state but only in the liquid state. All the halides DBeAX (particularly the chloride DBeAC) showed greater ability to destabilize the membrane structure in the presence than in the absence of cholesterol. The interaction of DBeAX with DPPC/cholesterol bilayers and subsequent changes in the phospholipid bilayer organization depended on the kind of counterion. The strongest effects were observed for chloride (most electronegative ion) and for iodide (largest ion). The effects of chloride and bromide on phase transition and 1H NMR spectra in the presence and absence of cholesterol were opposite. This is discussed in terms of the influence of counterions on the pair cholesterol-DPPC interactions.     

Synergistic permeability enhancing effect of lysophospholipids and fatty acids on lipid membranes

The permeability-enhancing effects of the two surfactants, 1-palmitoyl-2-lyso-sn-gycero-3-phosphocholine (lysoPPC) and palmitic acid (PA), on lipid membranes that at physiological temperatures are in the gel, fluid, and liquid-ordered phases were determined using the concentration-dependent self-quenching properties of the hydrophilic marker, calcein. Adding lysoPPC to lipid membranes in the gel-phase induced a time-dependent calcein release curve that can be described by the sum of two exponentials, whereas PA induces a considerably more complex release curve. However, when lysoPPC and PA were added simultaneously in equimolar concentrations, a dramatic synergistic permeability-enhancing effect was observed. In contrast, when both lysoPPC and PA are added to liposomal membranes that are in the fluid or liquid-ordered phases, no effect on the transmembrane permeation of calcein was observed.     

Synergistic permeability enhancing effect of lysophospholipids and fatty acids on lipid membranes

The permeability-enhancing effects of the two surfactants, 1-palmitoyl-2-lyso-sn-gycero-3-phosphocholine (lysoPPC) and palmitic acid (PA), on lipid membranes that at physiological temperatures are in the gel, fluid, and liquid-ordered phases were determined using the concentration-dependent self-quenching properties of the hydrophilic marker, calcein. Adding lysoPPC to lipid membranes in the gel-phase induced a time-dependent calcein release curve that can be described by the sum of two exponentials, whereas PA induces a considerably more complex release curve. However, when lysoPPC and PA were added simultaneously in equimolar concentrations, a dramatic synergistic permeability-enhancing effect was observed. In contrast, when both lysoPPC and PA are added to liposomal membranes that are in the fluid or liquid-ordered phases, no effect on the transmembrane permeation of calcein was observed.     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

Structures of pulmonary surfactant films adsorbed to an air-liquid interface in vitro

Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air-fluid interface.     

Engineering lipobeads: properties of the hydrogel core and the lipid bilayer shell

We previously reported on a novel system termed Lipobead that consists of hydrogel beads encased within an anchored lipid bilayer. The hydrogel particles are formed by inverse suspension polymerization of dimethylacrylamide with N,N'-ethylenebis(acrylamide). During the polymerization stage, the water in oil emulsion is interfacially stabilized by small molecule surfactants as well as a small percentage of lipid functionalized with a vinyl group. The functionalized lipid becomes tethered to the bead surface and promotes the assembly of a lipid bilayer on the surface of the hydrogel beads. The presence of the functionalized lipid during polymerization dramatically alters the yield, average size, and size distribution of beads produced. This paper examines the effect of various chemical and physical processing parameters on the average size and size distribution of beads produced when lipid is a component of the surfactant mixture. Relationships between the processing parameters, average bead size, and size distribution were established. Macroscopic properties of the lipid bilayers of Lipobeads were also evaluated including phase transition temperature as well as permeability to the small polar molecule, adenosine triphosphate. It was established that the presence of functionalized lipid improves the organization of the bilayer on the Lipobead surface.     

Surfactant-mediated gene transfer for animal cells

A commercially available cationic surfactant, dimethyl-dioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. DDAB easily dissolved in water at 60 °C and formed lipid vesicles at room temperature. The lipid vesicles showed very low cytotoxicity compared with other cationic surfactants. After the lipid vesicles were mixed with plasmid DNA solution, the solution was added to mammalian cells. The addition of a nonionic surfactant (Tween 80) to the cationic lipid vesicles at the weight ratio of 1:1 enhanced transfection efficiency. Adding more or less than the optimal amounts of DNA and lipid vesicles resulted in decreased transfection efficiency. With the optimal amounts of DNA (pCMVβ) and lipid vesicles, about 90–95% of CHO-K1 and BHK-21C13 cells transiently expressed β-galactosidase activity 24 h after transfection. By this procedure, stable transformants around 10⁵ cells corresponding to 10% efficiency could be obtained by one batch transfection. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Studies of Phase Equilibria and Efficiency Assessment for Self-Emulsifying Lipid-Based Formulations

The study was designed to build up a database for the evaluation of the self-emulsifying lipid formulations performance. A standard assessment method was constructed to evaluate the self-emulsifying efficiency of the formulations based on five parameters including excipients miscibility, spontaneity, dispersibility, homogeneity, and physical appearance. Equilibrium phase studies were conducted to investigate the phase changes of the anhydrous formulation in response to aqueous dilution. Droplet size studies were carried out to assess the influence of lipid and surfactant portions on the resulted droplet size upon aqueous dilution. Formulations containing mixed glycerides showed enhanced self-emulsification with both lipophilic and hydrophilic surfactants. Increasing the polarity of the lipid portion in the formulation leaded to progressive water solubilization capacity. In addition, formulations containing medium chain mixed glycerides and hydrophilic surfactants showed lower droplet size compared with their long chain and lipophilic counterparts. The inclusion of mixed glycerides in the lipid formulations enormously enhances the formulation efficiency.     

Excretion of glutamate from Corynebacterium glutamicum triggered by amine surfactants.

Corynebacterium glutamicum is used for the industrial production of glutamate. Excretion of the amino acid may be induced by various means. We have analyzed the characteristics of glutamate excretion induced by two amine surfactants, dodecylammonium acetate (DA) and dodecyltrimethylammonium bromide (DTA). Addition of these surfactants induced an immediate efflux of internal glutamate. It also induced a perturbation of the energetic parameters of the cell (decrease of delta mu H, decrease of the internal ATP concentration). The efflux was not the result of these perturbations: glutamate is taken up by the cells via an ATP-dependent unidirectional active transport system and no efflux took place as a consequence of an artificial decrease of the energetic parameters. In addition, amine surfactants also induced an excretion of other species, in particular potassium. We have tested the possibility that the effluxes result from a permeabilization of the lipid bilayer by analyzing the interactions between the surfactants and liposomes.     

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.     

Formation of 2-D paracrystals of F-actin on phospholipid layers mixed with quaternary ammonium surfactants.

Two-dimensional paracrystalline arrays of F-actin have been formed on positively charged lipid layers composed of phosphatidylcholine (PC) and quaternary ammonium surfactants. These quaternary ammonium surfactants were found to be better promoters of two-dimensional order than PC lipid layers mixed with stearylamine. In addition, the length of the hydrocarbon chain was found to influence the achievement of 2-D order. Lipid layers composed of dilauryl-PC and didodecyldimethylammonium bromide, which are saturated C12 lipids, promoted 2-D crystallization better than mixtures of dipalmitoyl-PC, a saturated C16 lipid, and dioctadecyldimethylammonium bromide, a saturated C18 lipid. Thus, the hydrocarbon chain length, which influences lipid layer fluidity, had a significant effect on paracrystal formation. We suggest that quaternary ammonium surfactants may have advantages in some cases for forming ordered arrays on lipid layers. In addition to investigating the effect of lipid layer composition on paracrystal formation, we found that the injection of G-actin rather than F-actin under a fluid lipid layer into a polymerizing solution produced better ordered paracrystals.     

Study of water vapor and surfactant absorption by lipid model systems using the quartz crystal microbalance

Skin is constantly exposed to surfactants which compromise the essential barrier function of normal healthy skin. To model the interactions of surfactants with the barrier lipids of the stratum corneum (SC), it is essential to develop in vitro and in vivo quantitative measurement methods to predict, evaluate, and demonstrate the effect of the different surfactant chemistries and technologies on skin. In the current work, in vitro water vapor uptake and surfactant absorption onto skin lipid model films were quantitatively studied using a technique based on the piezoelectric effect, the quartz crystal microbalance (QCM). This approach is straightforward and reliable in providing subtle surface/interface related mass change information with high resolution and sensitivity. The results show that barrier properties of the lipid model system may be damaged by surfactant absorption, as well as by long-term water exposure due to alterations to the lipid film structure. Surfactant absorption is found to be concentration dependent even beyond its critical micelle concentration (CMC). QCM results for different surfactant systems are consistent with reported clinical data in showing that clinically milder surfactants (SLES) do not perturb the film as much as clinically harsh surfactants (SDS).     

The effect of chain length on the melting temperature and size of dialkyldimethylammonium bromide vesicles

Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) were used to obtain the gel to liquid-crystalline phase transition temperature (Tm) and the apparent hydrodynamic radius (Rh) of spontaneously formed cationic vesicles of dialkyldimethylammonium bromide salts (CnH2n+1)2(CH3)2N+.Br-, with varying chain lengths. The preparation of cationic vesicles from aqueous solution of these surfactants, for n=12, 14, 16 and 18 (DDAB, DTDAB, DHDAB and DODAB, respectively), requires the knowledge of the surfactant gel to liquid-crystalline phase transition temperature, or melting temperature (Tm) since below this temperature these surfactants are poorly or not soluble in water. That series of cationic surfactants has been widely investigated as vesicle-forming surfactants, although C12 and C18, DDAB and DODAB are by far the most investigated from this series. The dependence of Tm of these surfactants on the number n of carbons in the surfactant tails is reported. The Tm obtained by DSC increases non-linearly with n, and the vesicle apparent radius Rh is about the same for DHDAB and DODAB, but much smaller for DDAB.     

Effect of synthetic surfactants and soapwort (Saponaria officinalis L.) extract on skin-mimetic model lipid monolayers

The effect of a saponin-rich extract from rhizomes of Soapwort (Saponaria officinalis L) and four synthetic surfactants: sodium lauryl sulphate (SLS), sodium laureth sulphate (SLES), ammonium lauryl sulphate (ALS) and cocamidopropyl betaine (CAPB) on two model lipid monolayers is analyzed using surface pressure, surface dilatational rheology and fluorescence microscopy. The following monolayers were employed: dipalmitoylphosphatidylcholine/cholesterol mixture in a molar ratio of 7:3 (DPPC/CHOL) and Ceramide [AP]/stearic acid/cholesterol in a molar ratio of 14:14:10 (CER/SA/CHOL). They mimicked a general bilayer structure and an intercellular lipid mixture, respectively. Both lipid mixtures on Milli-Q water were first compressed to the initial surface pressure, Π₀ = 30 mN/m and then the subphase was exchanged with the respective (bio)surfactant solution at 1% (w/w). All four synthetic surfactants behaved in a similar way: they increased surface pressure to about 40 mN/m and reduced the storage modulus of surface dilational surface rheology, E′, to the values close to zero. The corresponding fluorescence microscopy pictures confirmed that the lipids mimicking the stratum corneum components were almost completely removed by the synthetic surfactants under the present experimental conditions. The components of the Soapwort extract (SAP) increased surface pressure to significantly higher values than the synthetic surfactants, but even more spectacular increase was observed for the storage modulus of the SAP-penetrated lipid monolayers (up to E′= 715 mN/m).     

Effects of different surfactants on motility and DNA integrity of brown trout (Salmo trutta fario) and common carp (Cyprinus carpio) spermatozoa

In this study we evaluated effects of surfactants on motility parameters and DNA integrity of spermatozoa of freshwater teleost fish. Common carp (Cyprinus carpio) and brown trout (Salmo trutta fario) spermatozoa were exposed to either sodium dodecyl sulphate (SDS, anionic surfactant) or octoxynol 9 ( Triton X-100, nonionic surfactant). Both surfactants added at activation caused a decrease in sperm motility characteristics measured by computer-assisted sperm analysis (CASA). Intraspecific differences in speed and trajectory of movement were detected. Triton X-100 and SDS when added to non activated sperm were also effective in the decrease of sperm motility and caused an increase of DNA fragmentation. Our results suggest that not only sperm motility apparatus but also DNA are targets for surfactant action. Therefore any exposure of spermatozoa to surfactants, in aquaculture conditions or natural environment, would have a negative impact on fish reproduction.     

Lipid radicals: properties and detection by spin trapping

Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe2+/LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were similar to those of alkoxyl radical adducts, which were independently synthesized in model experiments using Fe3+-catalyzed nucleophilic addition of methanol or t-butanol to the respective spin trap.