Ian G. Lyle

The characterisation of liposomes with covalently attached proteins

The problem of characterising liposomes with covalently attached proteins has been analysed theoretically in terms of a normal weight distribution of liposome diameters. The polydispersity of protein conjugation is considered in terms of the width (standard deviation) of the liposome size distribution. It is shown that the weight-average number of proteins per liposome is a convenient parameter to use to define the protein content of proteoliposomes. Two types of proteoliposome have been prepared (small unilamellar vesicles and reverse phase evaporation vesicles) in which wheat germ agglutinin is covalently coupled to the liposomal surface. The liposomes cover a range of weight average diameter from 65 to 240 nm and of polydispersity (weight to number average diameter (dw/dn) from 2.6 to 11.4. The liposomes have been characterised by chemical analysis and photon correlation spectroscopy and the results are discussed in terms of the theoretical consequences of an equivalent normal weight distribution of diameters.

The targeting of phospholipid liposomes to bacteria

Phospholipid liposomes have been prepared from phospholipid mixtures including dipalmitoylphosphatidylcholine/phosphatidylinositol (DPPC/PI) and DPPC/dipalmitoylphosphatidylglycerol (DPPC/DPPG) mixtures and targeted to adsorbed biofilms of the skin-associated bacteria Staphylococcus epidermidis and Proteus vulgaris and the oral bacterium Streptococcus sanguis. The effects of time, liposome concentration and density of bacteria in the biofilm have been studied in detail for Staphylococcus epidermidis. The targeting (as assessed by the apparent monolayer coverage of the biofilms by liposomes) to the biofilms was found to be sensitive to the mol% of PI and DPPG in the liposomes and optimum levels of PI were found for targeting to each bacterium. The use of PI and DPPG-containing liposomes for the delivery of the bactericide, Triclosan, to biofilms of Staphylococcus epidermidis was studied as a function of the amount of Triclosan carried by the liposomes. All the liposome systems tested inhibited the growth of bacteria from the biofilms after brief (2 min) exposure to Triclosan-carrying liposomes. At low Triclosan levels bacterial growth inhibition by Triclosan-carrying liposomes exceeded that by an equivalent level of free Triclosan. After short periods (min) of exposure of biofilms to Triclosan-carrying liposomes the bactericide was shown to preferentially concentrate in the biofilms relative to its liposomal lipid carrier. The results suggest that phospholipid liposomes with appropriately chosen lipid composition have potential for the targeting and delivery of bactericide to bacteria.

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 targeting of lectin-bearing liposomes to skin-associated bacteria

Abstract Liposomes have been prepared by the vesicle extrusion technique (VETs) from mixtures of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylinositol (PI) incorporating a reactive phospholipid (the m -maleimidobenzoyl- N -hydroxysuccinimide ester (MBS) derivative of dipalmitoylphosphatidylethanolamine (DPPE) and conjugated with the N -succinimidyl- S -acetylthioacetate (SATA) derivatives of succinyl concanavalin A (sConA) or wheat germ agglutinin (WGA). The liposomes had diameters in the range 75–110 nm and weight-average numbers of lectin molecules per liposome up to 135 (sConA) and 40 (WGA). The targeting of the liposomes to biofilms on microtitre plates of bacteria including Streptococcus sanguis from the oral cavity and the skin-associated bacteria Staphylococcus epidermidis , Proteus vulgaris and Corynebacterium hofmanni was assessed using a radiochemical method which detects strong associations between the lectin-bearing liposomes and the bacterial biofilm. It was found that sConA-bearing liposomes targeted effectively to S. sanguis and C. hofmanni but not to S. epidermidis or P. vulgaris. S. epidermidis could be targeted using WGA-bearing liposomes. Targeting increased with liposomal lipid concentration and with the number of surface-bound lectin molecules per liposome. The results suggest that a considerable degree of selectivity in targeting to skin-associated bacteria can be achieved using lectins.

Lectin-mediated agglutination of liposomes containing glycophorin: Effects of acyl chain length

Glycophorin from human erythrocytes has been incorporated into liposomes of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC). The thermal properties of unsonicated liposomes with glycophorin/lipid molar ratios up to 4.10(-3) have been studied by differential scanning calorimetry and the numbers of lipids withdrawn from participation in the gel-to-lamellar phase transition were found to be 42 +/- 22 (DMPC), 197 +/- 28 (DPPC) and 240 +/- 64 (DSPC). The initial rates of agglutination of sonicated liposomes with glycophorin/lipid molar ratios up to 4.10(-3) by wheat germ agglutinin in the concentration range 0-7 microM have been measured over a range of temperature. Below the gel-to-lamellar phase transition (Tc) the rates of agglutination increase with acyl chain length in the sequence DMPC less than DPPC less than DSPC. Agglutination is found to be second order in liposome concentration and is completely reversed on saturation of the wheat germ agglutinin-binding sites by N-acetylglucosamine. Agglutination rates decrease with increasing temperature below Tc and are largely independent of temperature above Tc. The results are discussed in relation to the clustering of glycophorin in the phospholipid bilayers and its effect on binding and subsequent interliposomal bridge formation by wheat germ agglutinin.

The effect of surface-bound protein on the permeability of proteoliposomes.

Proteoliposomes have been prepared from mixtures of dipalmitoylphosphatidylcholine and phosphatidylinositol by sonication (SUV) and reverse phase evaporation (REV) and conjugated with succinyl concanavalin A (sConA). The proteoliposomes were characterised in terms of size and composition and covered a range of size (weight-average diameter) from approx. 80 to 300 nm and surface-bound sConA (weight-average number of protein molecules per liposome) from approx. 200 to 1800. The permeabilities of the proteoliposomes to encapsulated D-glucose have been measured and found to increase linearly with protein conjugation. The D-glucose permeability also increases with temperature and passes through a maximum in the region of the gel to liquid-crystalline phase transition temperature. Conjugation has no effect on the chain-melting temperature but slightly decreases the enthalpy of the transition consistent with the withdrawal of some phospholipid participation in chain-melting. The D-glucose permeabilities and thermotropic properties of the proteoliposomes are discussed in terms of the dislocation of the bilayer by the possible off-axis motion of the lipid which anchors the protein to the liposomal surface.

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.

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 control of protein surface concentration on proteoliposomes

Abstract Proteoliposomes having surface-bound succinylated concanavalin A (sConA) have been prepared by sonication of small unilamellar vesicles (SUV) and reverse-phase evaporation (REV) from lipid mixtures or dipalmitoylphosphatidylcholine, phosphatidylinositol and the reactive lipid phosphatidylethanolamine derivatised with maleimidobenzoyl- N -hydroxysuccinimide. The liposomes were conjugated with protein by reaction with the N -succinimidyl- S -thioacetate (SATA) derivative of sConA and had weight-average diameters ( d w ) in the range 45–260 nm, as determined by photon correlation spectroscopy, and weight-average numbers or protein molecules per proteoliposome ( P w ) up to approximately 2000. The factors controlling the extent of conjugation, including activation or the SATA derivative of sConA, the molar ratio of SATA to sConA and the reactive lipid content of the liposomes, have been studied. The surface concentration of sConA is directly controllable by manipulation of the reactive lipid content of the proteoliposomes. The results are compared with previous studies on wheatgerm agglutinin-conjugated proteoliposomes, and it is shown that for both types of proteoliposome the area per protein in the liposomal surfaces reaches limiting values of approximately 100 nm 2 for REV and 40 nm 2 for SUV. This observation is discussed in terms of the projected excluded area of protein on the liposomal surface.