M.D. Reboiras

The Interaction of Phospholipid Liposomes with Bacteria and Their Use in the Delivery of Bactericides

Liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) incorporating the cationic lipids stearylamine (SA), dimethyldioctadecylammonium bromide (DDAB) and dimethylaminoethane carbamoyl cholesterol (DCchol) and the anionic lipids dipalmitoylphosphatidylglycerol (DPPG) and phosphatidylinositol (PI). Their adsorption to biofilms of skin-associated bacteria (Staphylococcus epidermidis and Proteus vulgaris) and oral bacteria (Streptococcus mutans and sanguis) has been investigated as a function of mole % cationic and anionic lipid. Targeting (adsorption) was most effective for the systems DPPC-chol-SA, DPPC-DPPG and DPPC-PI liposomes to S. epidermidis. The effect of extracellular mucopolysaccharide on targeting was investigated for S. epidermidis biofilms. It was found that targeting increased with the level of extracellular mucopolysaccharide for all liposome compositions studied. The delivery of the oil-soluble bactericide Triclosan and the water soluble bactericide chlorhexidine was studied for a number of liposomal compositions. Superior delivery of both bactericides relative to the free bactericide occurred for DPPC-chol-SA liposomes and for Triclosan delivery by DPPC-DPPG and DPPC-PI liposomes targeted to S. epidermidis at low bactericide concentrations. DPPC-chol-SA liposomes were also effective for delivery of Triclosan to S. sanguis biofilms. Double labelling experiments using [14C]-chlorhexidine and [3H]-DPPC suggested that there was exchange between adsorbed liposomes which had delivered bactericide to the biofilm and those in the bulk solution implying a diffusion mechanism for bactericide delivery.

Characterisation of phosphatidylcholine/phosphatidylinositol sonicated vesicles. Effects of phospholipid composition on vesicle size

Phosphatidylinositol (PI), dipalmitoylphosphatidylcholine (DPPC) and mixed lipid (DPPC plus PI) sonicated vesicles have been prepared covering a range of composition. The vesicles were characterised by gel filtration, electron microscopy and photon correlation spectroscopy. The dimensions of the vesicles as measured by electron microscopy were in good accord with those obtained from photon correlation spectroscopy measurements. The number average diameters of the vesicles increase on increasing the PI content and range from approx. 30-80 nm as the weight % of PI is increased from 0 to 100. Gel filtration on Sepharose 4B columns gave anomalous results indicating that PI-containing vesicles were retarded on the gel possibly due to an interaction between the inositol headgroup and the gel matrix. Electrophoretic measurements on multilamellar vesicles show that the surface charge density increases with the PI content of the vesicles upto 50 weight % PI and remains constant thereafter. The radii of sonicated vesicles also increase with PI content which reflects a decreasing liposome curvature with increasing surface charge density.

The mechanism of phospholipid adsorption from vesicle dispersions onto glass surfaces

Abstract The adsorption of phospholipid onto glass beads from aqueous dispersions of phospholipid vesicles formed from L-α-dipalmitoylphosphatidylcholine (DPPC) and its mixtures with phosphatidylinositol (PI) has been investigated. The temperature dependence of adsorption of DPPC from vesicles is characterised by marked changes in both the adsorption constant and limiting adsorption in the region of the gel to liquid-crystalline phase transition. Below the phase transition temperature the area per molecule of adsorbed lipid corresponds to more than monolayer adsorption while above the phase transition temperature the area per molecule exceeds that in a close-packed monolayer. For vesicles consisting of 50 wt% DPPC and PI the adsorption constant is independent of temperature. The time course for the adsorption of lipid shows a dependence on the surface roughness of the glass beads consistent with initial adsorption of vesicles, followed by vesicle disruption resulting in an increase in particle size in the dispersed phase. Contact angle measurements were made on lipid monolayers on glass made by the Langmuir-Blodgett technique and by deposition from vesicles. The contact angles were independent of the method of lipid deposition and decreased from 44° for DPPC to 8° for PI. It is suggested that a proportion of the lipid molecules in the adsorbed layers are oriented with their head groups into the aqueous phase; this orientation being increasingly favoured for PI rich layers.

Interaction of n-octyl-β-d-glucopyranoside with globular proteins in aqueous solution

The binding of n-octyl glucoside to a range of ten globular proteins in aqueous solution (pH 6.4, ionic strength 0.132 m) at 25°C has been measured by equilibrium dialysis. The binding isotherms which rise steeply at free n-octyl glucoside concentrations below the critical micelle concentration have been fitted to the Hill equation. For all the proteins the Hill coefficients are greater than unity indicating that binding is a positively cooperative process and the Gibbs energies of binding lie in a range from −9.7 kJ mol−1 for bovine serum albumin to −11.4 kJ mol−1 for fibrinogen and Aspergillus niger catalase. The enthalpies of interaction have been measured directly by microcalorimetry and are found to be very small relative to the Gibbs energies of binding consistent with a hydrophobic interaction. No evidence was found for the denaturation of the proteins by octyl glucoside. Binding has been analysed and discussed in terms of a model in which the proteins are incorporated into an octyl glucoside micelle. On this basis saturation binding to ribonuclease, lysozyme and α-chymotrypsin would be satisfactorily described in terms of a prolate ellipsoidal micelle whereas for bovine and Aspergillus niger catalase on oblate ellipsoid would describe the data more satisfactorily.

Adsorption of phospholipid vesicles on solid surfaces

Sonicated vesicles have been prepared from dipalmitoylphosphatidylcholine (DPPC) and phosphatidylinositol (PI) and their mixtures covering a range of composition. The adsorption of lipid from the vesicle dispersions onto the surfaces of glass beads has been measured by a batch procedure using a radiochemical assay. Lipid deposition onto the glass surface occurs via vesicle adsorption rather than monomeric lipid adsorption and a fluorescent assay has been used to demonstrate that the vesicles disrupt on contact with the glass. The adsorption isotherms are of the Langmuir type and for pure DPPC the limiting areas at the glass-aqueous interface are 0.39 nm2 molecule–1 and 0.64 nm2 molecule–1 at 25 and 50 °C, respectively. These figures suggest that a monolayer of DPPC is formed at the interface. Limiting adsorption could not be correlated with the electrokinetic properties of the vesicles and was not markedly dependent on the acyl chain length for a series of diacylphosphatidylcholines.

The electrophoretic properties and aggregation behaviour of dipalmitoylphosphatidylcholine—phosphatidylinositol vesicles in aqueous media

Abstract Mixed lipid vesicles have been prepared from dipalmitoylphosphatidylcholine (DPPC)—phosphatidylinositol (PI) mixtures. The rate of aggregation of sonicated vesicles by Ca 2+ and Mg 2+ ions is dependent on the DPPC: PI ratio. For vesicles of composition DPPC: PI, 75: 25 wt% the rate goes through a maximum as a function of divalent ion concentration in the range 0–25 m M . Microelectrophoresis measurements have been made on multilamellar vesicles of composition DPPC:PI, 75: 25 wt% as a function of Ca 2+ and Mg 2+ ion concentration. The multilamellar vesicles are negatively charged in the presence of divalent ions at low concentrations but have zero points of charge for Ca 2+ and Mg 2+ ions of approximately 2 and 15 m M , respectively. The electrophoretic data has been used to derive a Gibbs energy of binding of Ca 2+ ions to the vesicle surface of −27.9 kJ mol −1 at 25°C and a specific Gibbs energy of interaction between Ca 2+ ions and PI of −64.7 kJ mol −1 .

The electrophoretic properties of dipalmitoylphosphatidylcholine—phosphatidylinositol vesicles in the presence of lanthanum ions

Abstract Large multilamellar vesicles have been prepared from dipalmitoylphosphatidylcholine (DPPC)—phosphatidylinositol (PI) mixtures over the complete range of composition. The electrophoretic mobilities of the vesicles have been measured in a buffer at pH 6.6 and as a function of lanthanum ion concentration over a range covering the zero point of charge. The zeta potentials of the vesicles increase with PI content and the degree of ionisation of the PI molecules lies in the range 0.16 to 0.23. The zeta potentials have been interpreted in terms of La 3+ ion binding using a Gouy—Chapman—Stern model as proposed by Ottewill and co-workers. Apparent and intrinsic binding constants and the number of binding sites have been determined. The Gibbs energy of ion adsorption (Δ G 0 ads ) has been found to obey the linear relationship: Δ G 0 ads (kJ mol −1 ) = −(66.9 (± 1.7) x PI + 41.2 (± 0.8) x DPPC ) which demonstrates that each phospholipid molecule binds La 3+ ions independently.

Liposome adsorption to Candida albicans

Abstract The adsorption of anionic and cationic liposomes to the yeast form of the fungal cell Candida albicans in the stationary phase has been studied as a function of liposome composition and concentration in phosphate-buffered saline (PBS) at pH 7.4. For liposomes of composition dipalmitoylphosphatidylcholinephosphatidylinositol (DPPC-PI) it has been found that there is an optimum composition for adsorption for liposomes containing 18 mol% PI. Adsorption studies as a function of liposome concentration give Langmuir-type isotherms for liposomes of composition DPPC-PI, DPPC-cholesterol-stearylamine and DPPC-cholesterol-dimethyldioctadecylammonium bromide from which saturation values of liposomes adsorbed per cell and the association constants could be derived. The saturation values are consistent with the adsorption of a monolayer, though not necessarily close-packed, on the cell surface. The Gibbs energies of adsorption lie in a range −12 to −19 kJ mol−1. The interaction between the cells of DPPC-PI liposomes is probably dominated by H-bonding between the polyhydroxy head group of PI and the oligosaccharides in the cell surface whereas for the cationic liposomes the interactions are predominantly ionic.

The interaction of lanthanum ions with dipalmitoylphosphatidylcholine—phosphatidylinositol vesicles

Abstract Sonicated and multilamellar dipalmitoylphosphatidylcholine (DPPC)—phosphatidylinositol (PI) vesicles have been prepared with different DPPC/PI weight ratios. The interaction of the sonicated vesicles with lanthanum (La 3+ ) ions has been studied by absorbance measurements at 25°C. Lanthanum ions do not fuse DPPC—PI vesicles (or phosphatidylserine vesicles) so that the absorbance changes are interpreted in terms of vesicle aggregation. The rates of aggregation pass through maxima as a function of La 3+ ion concentration. The concentration of La 3+ ions needed to reach the maxima increases with the PI content of the vesicles. The results are consistent with the formation of multivesicular aggregates. Microelectrophoresis measurements on multilamellar vesicles as a function of La 3+ ion concentration show that the vesicles adsorb La 3+ ions. Upon addition of La 3+ , the zero point of charge is located at higher La 3+ concentrations as the vesicle PI content is increased. The electrophoretic data have been analysed on the basis of the Stern model of the electrical double layer. In water (pH 6.6) at 25°C the intrinsic Gibbs energy for La 3+ ion binding to vesicles containing 25 and 50 wt% PI (Δ G 0 ads ) is found to be −61 kJ mol −1 . Differential scanning calorimetry has been used to determine the effect of La 3+ ions on the enthalpy (Δ H ) and the gel-to-liquid crystalline transition temperature ( T c ) in dispersions of multilamellar vesicles. At low molar ratios of La 3+ to PI, minima were observed in both Δ H and T c . The results are interpreted in terms of the withdrawal of DPPC molecules from participation in chain-melting and suggest a disruption of the cooperative behaviour of DPPC in the immediate environment of PI molecules on binding La 3+ ions.

Preparation and characterisation of polymerised proteovesicles

Abstract The cationic double-chain surfactant, hexadecyl[11-(methacryloyloxy)undecyl] ammonium bromide in combination with dipalmitoylphosphatidylethanolamine (DPPE) derivatised with maleimidobenzyl- N -hydroxysuccinimide (MBS) has been used to make polymerisable vesicles which can be conjugated with a protein (lysozyme) to form polymerised proteovesicles. Polymerisation was carried out at room temperature using Fentons reagent (hydrogen peroxide and ferrous ions). It was found that unpolymerised proteovesicles can be prepared by this procedure but such pre-formed proteovesicles cannot be polymerised without initiating precipitation; however if polymerisation precedes protein conjugation, proteovesicles can be prepared in the dispersed state. In contrast to unpolymerised proteovesicles, polymerised proteovesicles form small aggregates in solution together with physically adsorbed lysozyme, suggesting that the rigidity of the underlying vesicle bilayers significantly influences the physical adsorption process.