Immobilization of cationic rifampicin-loaded liposomes on polystyrene for drug-delivery applications

Polymer-associated infections are a major problem in implanted or intravascular devices. Among others, microorganisms of the staphylococcal family have been identified as the most important culprit. Prevention of bacterial adhesion and colonization of polymeric surfaces by release of antimicrobial agents incorporated into the polymers itself are currently under study. We have developed a novel method for the functionalization of a polymeric surface which is based on the deposition of covalently coupled lipid structures from antibiotic loaded vesicles. We have found that such process significantly reduces the bacterial growth on polystyrene material. In this work, lipid coverage obtained from multilamellar (MLVs) and extruded unilamellar (LUVs) vesicles were analyzed with respect to their adhesion efficiency on three types of polystyrene (PS) well-plates. Two methods of lipid deposition were characterized and compared in terms of surface lipid density and time stability: deposition of cationic vesicles on negatively charged surfaces and formation of covalent linkages between functionalized lipids and amines enriched surfaces. In order to study the antibiotic encapsulation efficiency we measured how the rifampicin (RIF) loading was affected by changes of liposome charge upon introduction of various amounts of stearylamine (SA), distearoyl-trimethylammonium propane (DSTAP) or dioleoyloxypropyl-trimethylammonium chloride (DOTAP) into the liposomal formulation. RIF-coated polymeric surfaces were also tested against a Staphylococcus epidermidis strain to evaluate their efficacy in vitro, showing that only approximately 2% of such bacteria inoculated on MLV-treated PS substrate were able to proliferate. Covalently immobilized lipid films showed about a tenfold increase in time stability compared to electrostatically bonded lipid films. Furthermore, substrates covalently modified with RIF-loaded MLVs retained an antibacterial activity for up to 12 days when aged in buffer at 37 degrees C. Such antimicrobial polymer coatings show promise for their use as antibacterial barrier for the prevention of catheter-related infections.     

Self-Assembly of a Peptide Amphiphile Containing l-Carnosine and Its Mixtures with a Multilamellar Vesicle Forming Lipid

The self-assembly of the peptide amphiphile (PA) hexadecyl-(β-alanine-histidine) is examined in aqueous solution, along with its mixtures with multilamellar vesicles formed by DPPC (dipalmitoyl phosphatidylcholine). This PA, denoted C(16)-βAH, contains a dipeptide headgroup corresponding to the bioactive molecule l-carnosine. It is found to self-assemble into nanotapes based on stacked layers of molecules. Bilayers are found to coexist with monolayers in which the PA molecules pack with alternating up-down arrangement so that the headgroups decorate both surfaces. The bilayers become dehydrated as PA concentration increases and the number of layers in the stack decreases to produce ultrathin nanotapes comprised of 2-3 bilayers. Addition of the PA to DPPC multilamellar vesicles leads to a transition to well-defined unilamellar vesicles. The unique ability to modulate the stacking of this PA as a function of concentration, combined with its ability to induce a multilamellar to unilamellar thinning of DPPC vesicles, may be useful in biomaterials applications where the presentation of the peptide function at the surface of self-assembled nanostructures is crucial.     

Formation of tubules and giant vesicles from large multilamellar vesicles

This study reports an observation of submicrometer multilamellar vesicles (MLVs) prepared by simply freeze-thawing a phospholipid dispersion at full hydration that transformed into giant vesicles (GVs) and tubules (TUs) when confined between microscope glass slides. Cover slide cleaning and surface treatment did not hamper the formation of GVs or TUs. However, when small unilamellar vesicles (SUV) were prepared or when MLVs were not confined but rather freely moved between the glass slides or when the phospholipid was in its gel phase, neither GVs nor TUs were observed. Altogether, our results suggested that MLVs would play a role as a lipid reservoir and that the liquid flow between the glass slides induces the peeling of the external bilayers, yielding the formation of tubules and giant unilamellar vesicles.     

Supported lipid bilayer formation by the giant vesicle fusion induced by vesicle–surface electrostatic attractive interaction

The effect of the electrostatic attractive force between giant unilamellar vesicles (GUVs) and the SiO₂ surface on the formation of a Ca²⁺-free supported lipid bilayer (SLB) was investigated by atomic force microscopy and fluorescence microscopy. When negatively charged GUVs were incubated for 1 h without Ca²⁺, the surface coverage of lipid bilayer was <1% on the SiO₂ surface. In contrast, a high coverage was obtained without addition of Ca²⁺ on the positively charged surface modified by aminopropyldimethylethoxysilane, and the coverage of SLBs decreased with increasing KCl concentrations. The thickness of the water layer under SLB was reduced by modification of APS.     

Influence of nanotopography on phospholipid bilayer formation on silicon dioxide

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.     

Effective form of sulfoquinovosyldiacyglycerol (SQDG) vesicles for DNA polymerase inhibition

Sulfoquinovosyldiacyglycerol (SQDG) has a wide range of biological activities that make it an attractive compound for the development of new drugs. Chemically synthesized beta-SQDG-C(18:0) (1,2-di-O-stearoyl-3-O-(6-deoxy-6-sulfo-beta-d-glucopyranosyl)-sn-glycerol), for example, has a potent inhibitory effect on DNA polymerases. We investigated the properties of the vesicle form of beta-SQDG-C(18:0) as the monomer has low solubility in water. The structure of the beta-SQDG-C(18:0) vesicles are highly influenced by NaCl concentration in preparation process. At low NaCl concentrations, the beta-SQDG-C(18:0) vesicles have high surface curvature and form small unilamellar vesicles. Increases in NaCl concentration, resulted in decreased surface curvature and a tendency for beta-SQDG-C(18:0) to form large multilamellar vesicles. The small unilamellar vesicles showed a potent inhibitory effect on DNA polymerase beta, whereas the large multilamellar vesicles had no such effect. We investigated further the relationship between vesicle size and activity by preparing smaller vesicles (262, 99 and 43 nm in diameter) using an extrusion technique. These smaller vesicles had a greater inhibitory effect on DNA polymerase beta activity than non-extruded vesicles. beta-SQDG-C(18:0) vesicles, especially those of small size, were effective in DNA polymerase inhibition and are expected to have high applicability in DNA polymerase study.     

Thin wetting films from aqueous solutions of surfactants and phospholipid dispersions

The microscopic thin wetting film method was used to study the stability of wetting films from aqueous solution of surfactants and phospholipid dispersions on a solid surface. In the case of tetradecyltrimethylammonium bromide (C(14)TAB) films the experimental data for the receding contact angle, film lifetime, surface potential at the vapor/solution and solution/silica interface were used to analyze the stability of the studied films. It is shown that with increasing C(14)TAB concentration charge reversal occurs at both (vapor/solution and solution/silica) interfaces, which affects the thin-film stability. The spontaneous rupture of the thin aqueous film was interpreted in terms of the earlier proposed heterocoagulation mechanism. The presence of the mixed cationic/anionic surfactants was found to lower contact angles and suppresses the thin aqueous film rupture, thus inducing longer film lifetime, as compared to the pure amine system. In the case of mixed surfactants hetero-coagulation could arise through the formation of ionic surfactant complexes. The influence of the melting phase-transition temperature T(c) of the dimyristoylphosphatiddylcholine (DMPC) on the stability of thin films from dispersions of DMPC small unilamellar vesicles on a silica surface was studied by measuring the film lifetime and the TPC expansion rate. The stability of thin wetting films formed from dispersions of DMPC small unilamellar vesicles was investigated by the microinterferometric method. The formation of wetting films from diluted dispersions of DMPC multilamellar vesicles was studied in the temperature range 25-32 degrees C. The stability of thin film of lipid vesicles was explained on the basis of hydrophobic interactions. The results obtained show that the stability of wetting films from aqueous solutions of single cationic and mixed cationic-anionic surfactants has electrostatic origin, whereas the stability of the phospholipid film is due to hydrophobic interaction.     

Effect of a PEG lipid (DSPE-PEG2000) and freeze-thawing process on phospholipid vesicle size and lamellarity

Liposomes containing distearoylphosphatidylethanolamine with covalently linked polyethylene glycol of molecular weight 2,000 (DSPE-PEG2000) covering a range of 0–30 mol% were prepared by a mechanical dispersion or detergent-removal method. The effects of DSPE-PEG2000 on particle sizes and lamellarity of liposomes were investigated. The average diameters of vesicles prepared from both methods decreased when the concentration of DSPE-PEG2000 was increased. The decrease in vesicle size with increase in DSPE-PEG2000 was ascribed to the steric hindrance of strongly hydrated PEG. The significant decrease in the sizes of DSPE-PEG2000-containing EggPC vesicles prepared by the detergent-removal method could be explained by the postvesiculation size growth in the process of micelle–vesicle transition. For DMPC vesicles prepared by the detergent-removal method, electron micrographs showed that inclusion of DSPE-PEG2000 promoted vesicle formation. Based on the results of investigation of calcein entrapment efficiency, we concluded that the lamellarity of liposomes is reduced as PEG lipid concentration is increased. Fragmentation of multilamellar vesicles into smaller unilamellar vesicles occurred more readily when the liposome suspension was subjected to repetitive freeze-thawing. After five cycles of freezing and thawing, vesicles containing more than 0.5 mol% DSPE-PEG2000 were fragmented into unilamellar vesicles with diameters smaller than 300 nm.     

How the stereochemistry of a central cyclopentyl ring influences the self-assembling properties of archaeal lipid analogues: synthesis and cryoTEM observations

The relative stereochemistry (cis or trans) of a 1,3-disubstituted cyclopentane unit placed in the middle of tetraether archaeal bipolar lipid analogues was found to have a dramatic influence on their supramolecular self-assembling properties. The synthesis of two diastereomers varying only by the stereochemistry of the cyclopentyl unit was achieved following a multistep diastereoselective route. The corresponding lipid films were hydrated and were observed by cryoTEM. The micrographs showed several types of unilamellar nano-objects such as lamellas or irregular vesicles for the cis-isomer, whereas the trans-isomer exhibited exclusively multilamellar vesicles with a regular spherical shape. Even if the cyclopentyl ring takes part of a long alkyl chain (32 carbon atoms), the pseudorotation of the carbocycle would influence the global conformation of the bipolar lipid and consequently would modify the orientation of the lactosyl polar headgroups.     

Kinetics of formation of single phospholipid bilayers on self-assembled monolayer supports,as monitored by surface plasmon resonance

The unrolling of small unilamellar vesicles onto a variety of mixed and single-component self-assembled monolayer surfaces has been studied via surface plasmon resonance. In particular, the effect of varying the vesicle concentration has been explored in order to assess the effects of varying the rate of diffusion of vesicles to the surface on the initial rate of lipid layer formation.     

Spontaneous formation of vesicles of diblock copolymer EO6BO11 in water: a SANS study

Small angle neutron scattering (SANS) is used to study the structures formed in water by a diblock copolymer EO6BO11 (having 6 ethylene oxide, EO, and 11 butylene oxide, BO, units). The data show that polymer solutions over a broad concentration range (0.05-20 wt %) contain vesicular structures at room temperature. Interestingly, these vesicles could be formed without any external energy input, such as extrusion, which is commonly required for the formation of other block copolymer or lipid vesicles. The EO6BO11 vesicles are predominantly unilamellar at low polymer concentrations, whereas at higher polymer concentrations or temperatures there is a coexisting population of unilamellar and multilamellar vesicles. At a critical concentration and temperature, the vesicular structures fuse into lyotropic arrays of planar lamellar sheets. The findings from this study are in broad agreement with the work of Harris et al. (Langmuir, 2002, 18, 5337), who used electron microscopy to identify the vesicle phase in the same system.     

Properties of mixed lipid monolayers assembled on hydrophobic surfaces through vesicle adsorption

Supported lipid films are becoming increasingly important tools for the study of membrane protein function because of the availability of high-sensitivity surface analytical and patterning techniques. In this study, we have characterized the physical chemical properties of lipid films assembled on hydrophobic surfaces through the spontaneous adsorption of large unilamellar lipid vesicles composed of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC). The density of the lipid films was measured with surface plasmon resonance spectroscopy as the lipid composition of the vesicles and ionic concentration were varied. As expected, monolayer films were formed, but the density of the monolayers was found to be weakly dependent on the lipid composition of the vesicles and strongly dependent on the ionic concentration of the solution in contact with the monolayer. Atomic force microscopy (AFM) images of the lipid films indicate that they are composed of a homogeneous monolayer. Surface force measurements were used to determine the surface charge and DOPG density of the monolayers. The DOPG content of the films was found to be weakly dependent on the DOPG composition of the vesicles and strongly dependent on the salt concentration of the environment. A model has been developed to describe the behavior of the lipid composition of the films in terms of the hydrophobic, electrostatic, and steric forces acting on the lipid monolayer on the hydrophobic surface.     

Phospholipids as an alternative to direct covalent coupling: surface functionalization of nanoporous alumina for protein recognition and purification

Anodic aluminum oxide (AAO) substrates with aligned, cylindrical, non-intersecting pores with diameters of 75 nm and depths of 3.5 or 10 μm were functionalized with lipid monolayers harboring different receptor lipids. AAO was first functionalized with dodecyl-trichlorosilane, followed by fusion of small unilamellar vesicles (SUVs) forming a lipid monolayer. The SUVs' lipid composition was transferred onto the AAO surface, allowing us to control the surface receptor density. Owing to the optical transparency of the AAO, the overall vesicle spreading process and subsequent protein binding to the receptor-doped lipid monolayers could be investigated in situ by optical waveguide spectroscopy (OWS). SUV spreading occurred at the pore-rim interface, followed by lateral diffusion of lipids within the pore-interior surface until homogeneous coverage was achieved with a lipid monolayer. The functionality of the system was demonstrated through streptavidin binding onto a biotin-DOPE containing POPC membrane, showing maximum protein coverage at 10 mol% of biotin-DOPE. The system enabled us to monitor in real-time the selective extraction of two histidine-tagged proteins, PIGEA14 (14 kDa) and ezrin (70 kDa), directly from cell lysate solutions using a DOGS-NTA(Ni)/DOPC (1:9) membrane. The purification process including protein binding and elution was monitored by OWS and confirmed by SDS-PAGE.     

Tethered bilayer lipid membranes studied by simultaneous attenuated total reflectance infrared spectroscopy and electrochemical impedance spectroscopy

The formation of tethered lipid bilayer membranes (tBLMs) from unilamelar vesicles of egg yolk phosphatidylcholine (EggPC) on mixed self-assembled monolayers (SAMs) from varying ratios of 6-mercaptohexanol and EO(3)Cholesteryl on gold has been monitored by simultaneous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS). The influence of the lipid orientation (and hence the anisotropy) of lipids on a gold film on the dichroic ratio was studied by simulations of spectra with a matrix method for anisotropic layers. It is shown that for certain tilt angles of the dielectric tensor of the adsorbed anisotropic layer dispersive and negative absorption bands are possible. The experimental data indicate that the structure of the assemblies obtained varies with varying SAM composition. On SAMs with a high content of EO(3)Cholesteryl, tBLMs with reduced fluidity are formed. For SAMs with a high content of 6-mercaptohexanol, the results are consistent with the adsorption of flattened vesicles, and spherical vesicles have been found in a small range of surface compositions. The kinetics of the adsorption process is consistent with the assumption of spherical vesicles as long-living intermediates for surfaces of a high 6-mercaptohexanol content. No long-living spherical vesicles have been detected for surfaces with a large fraction of EO(3)Cholesteryl tethers. The observed differences between the surfaces suggest that for the formation of tBLMs (unlike supported BLMs) no critical surface coverage of vesicles is needed prior to lipid bilayer formation.     

Supramolecular self-assembling properties of membrane-spanning archaeal tetraether glycolipid analogues

The self-assembling properties of a new series of archaeal tetraether glycolipid analogues 1-6 that are characterized by a bipolar architecture with two similar or different glycosidic and/or phosphate polar heads and a lipid core possessing a cyclopentane unit and/or branched chains were studied by means of differential scanning calorimetry, optical microscopy, X-ray scattering, freeze-fracture electron microscopy and dynamic light scattering. Unsymmetrical phosphate derivatives 1 and 2 spontaneously formed thermostable multilamellar and unilamellar vesicles in which most of the bipolar lipids adopted a trans-membrane conformation, as revealed by freeze-fracture electron microscopy. Supramolecular aggregates of neutral glycolipids 3-6 were found to depend on both the saccharidic polar heads and the chain composition. The presence of one glycosidic residue with rather marked hydrophilic properties, such as the lactosyl moiety, was required to allow the formation of multilamellar vesicles. Surprisingly, the introduction of a cyclopentane unit in the bridging chain was able to induce an apparent two-by-two membrane association: this unusual behaviour might be the result of unsymmetrical interfacial properties of the lipid layer caused by the presence of the cyclopentane unit.     

The effects of coexisting lipids on the action of Bacillus thuringiensis phosphatidylinositol-specific phospholipase C toward liposomal substrate.

The hydrolytic activity of phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) from Bacillus thuringiensis was studied in detail toward mixed liposomes consisting of PI and one of other phospholipids and cholesterol. Among PI-liposomes, small unilamellar vesicles (SUV) were the most sensitive to PI-PLC; the enzymatic hydrolysis of PI in SUV was not less than 10-fold that in large unilamellar vesicles (LUV) or in multilamellar vesicles (MLV). Thus, in a survey of the effects of coexisting lipids on PI-PLC activity, PI-SUV was used. Phosphatidylcholine (PC) was stimulative for the enzyme activity toward PI-SUV at any molar ratio of PC to PI. Also, the effects of the addition of sphingomyelin (SM), phosphatidylethanolamine (PE) and cholesterol on the enzymatic hydrolysis of PI were studied in detail on the basis of concentration of total lipids or PI.     

Freeze-fracture transmission electron microscopy studies on the self-assemblies of amphiphilic solutions

Self-assemblies of amphiphiles in solutions were investigated by using freeze-fracture transmission electron microscopy (FF-TEM). Especially, vesicles were characterized by FF-TEM and the transition of self-assemblies was determined. The stacked lamellar La-phase was prepared without shear forces by a chemical reaction. The stacked lamellar La-phase can be transformed into multilamellar vesicles by the shearing forces that occur when the stacked lamellar La-phase sample is turned upside down a few times. The multilamellar vesicles can also be transformed into unilamellar vesicles by high shearing forces. These transitions were demonstrated by FF-TEM measurements. 2n2+-induced vesicle formation in the single-chain surfactant solutions was first achieved.     

Enhancement of gemcitabine affinity for biomembranes by conjugation with squalene: differential scanning calorimetry and Langmuir-Blodgett studies using biomembrane models

Molecular interactions between gemcitabine, alone or conjugated with squalene to form the gem-squalene prodrug, with dimyristoylphosphatidylcholine have been investigated by differential scanning calorimetry and Langmuir film balance techniques to gain information about the interaction of gemcitabine and its prodrug with mammalian cell membranes and to evaluate the potential of liposomes as a delivery system for gemcitabine prodrugs. Phospholipids assembled as multilamellar vesicles or monolayers (at the air water interface) have been used as biomembrane models. Different interactions of gemcitabine, its prodrug, and squalene with the lipid were detected by dispersing the compounds in the MLV and were compared with kinetic experiments carried out to consider the ability of the examined compounds to dissolve in an aqueous medium, to migrate through it, and to be captured by multilamellar vesicles. Their ability to be released from drug-loaded liposomes and be taken up by empty vesicles mimicking biomembranes was also considered. Analysis of the differential scanning calorimetry curves reveals that gemcitabine has very little interaction with multilamellar vesicles whereas the gem-squalene prodrug strongly interacts with multilamellar vesicles. The kinetic experiments suggest that an aqueous medium does not permit the prodrug uptake by the biomembrane models, whereas it is allowed when gem-squalene is gradually released by the liposomes. The molecular area/surface pressure isotherms of the gemcitabine/lipid, gem-squalene/lipid, and pure compound monolayers, in agreement with the calorimetric results, indicate that gem-squalene interacts with the phospholipid monolayer with the squalene moiety in contact with the phospholipid chains and gemcitabine protruding in the aqueous medium.     

Dissipation-enhanced quartz crystal microbalance studies on the experimental parameters controlling the formation of supported lipid bilayers

We report on the investigations of the transformation of spherically closed lipid bilayers to supported lipid bilayers in aqueous media in contact with SiO(2) surfaces. The adsorption kinetics of small unilamellar vesicles composed of dimyristoyl- (DMPC) and dipalmitoylphosphatidylcholine (DPPC) mixtures on SiO(2) surfaces were investigated using a dissipation-enhanced quartz crystal microbalance (QCM-D) as a function of buffer (composition and pH), lipid concentration (0.01-1.0 mg/mL), temperature (15-37 degrees C), and lipid composition (DMPC and DMPC/DPPC mixtures). The lipid mixtures used here possess a phase transition temperature (T(m)) of 24-33 degrees C, which is close to the ambient temperature or above and thus considerably higher than most other systems studied by QCM-D. With HEPES or Tris.HCl containing sodium chloride (150 mM) and/or calcium chloride (2 mM), intact vesicles adsorb on the surface until a critical density ((c)) is reached. At close vesicle contact the transformation from vesicles to supported phospholipid bilayers (SPBs) occurs. In absence of CaCl(2), the kinetics of the SPB formation process are slowed, but the passage through (c) is still observed. The latter disappears when buffers with low ionic strength were used. SPB formation was studied in a pH range of 3-10, yet the passage through (c) is obtained only for pH values above to the physiological pH (7.4-10). With an increasing vesicle concentration, (c) is reached after shorter exposure times. At a vesicle concentration of 0.01-1 mg/mL, vesicle fusion on SiO(2) proceeds with the same pathway and accelerates roughly proportionally. In contrast, the pathway of vesicle fusion is strongly influenced by the temperature in the vicinity of T(m). Above and around the T(m), transformation of vesicles to SPB proceeds smoothly, while below, a large number of nonruptured vesicles coexist with SPB. As expected, the physical state of the membrane controls the interaction with both surface and neighboring vesicles.     

The behaviour of water molecules associated with structural changes in negatively charged phosphatidyl-glycerol assemblies as studied by DSC

Variation of the thermotropic behaviour of both lipid assemblies and associated water molecules with an increase in water content was investigated for negatively charged phosphatidyl-glycerol (PG)-water system up to 90 wt.% water by DSC. The number of water molecules existing in interbilayer regions of the present gel phase was estimated from a deconvolution analysis of ice-melting DSC curves. On the basis of a result of the calorimetric analysis, a water-distribution diagram was constructed over the water content range from 0 to 90 wt.%. The diagram presented a continuous incorporation of interlamellar water up to 90 wt.% water, related to unilamellar-vesicle forming properties of charged lipids. Furthermore, similarly to a result for neutral lipid systems previously reported by us, the present diagram also showed the existence of a specific water content region (i.e., pre-region) where a structural change of planar to curved bilayers for multilamellar structures proceeds with the aid of bulk-like water before finally reaching unilamellar vesicles.