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.

Lectin-mediated agglutination of liposomes containing glycophorin. Effect 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molar ratios up to 4.10(-3) have been studied by differential scanning calorimetry and the numbers of lipids withdrawn from participation in the gel-to-lamellar phase transition were found to be 42 +/- 22 (DMPC), 197 +/- 28 (DPPC) and 240 +/- 64 (DSPC).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 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.

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.

Targeting and delivery of bactericide to adsorbed oral bacteria by use of proteoliposomes.

Proteoliposomes having surface-bound succinylated concanavalin A (s-conA) have been prepared from a range of phospholipid mixtures by sonication (SUV) and reverse phase evaporation (REV) covering a range of size (weight-average diameter (dw)) from approx. 35 to 310 nm and weight-average number of protein molecules per liposomes (Pw) from approx. 50 to 3000. The targeting of the proteoliposomes to adsorbed biofilms of the bacteria Streptococcus sanguis and Streptococcus mutans has been assessed from the extent of inhibition of an enzyme-linked immunosorbent assay (ELISA) for bacterial cell surface antigens. The surface-bound lectin enhances targeting relative to 'naked' liposomes of comparable concentration by factors of 2-50 depending on the liposomal lipid composition and Pw. The effect of the bactericide Triclosan on the thermal properties and permeability characteristics of liposomes has been studied. At and above a molar ratio of Triclosan to lipid of 0.6, Triclosan eliminates the gel to liquid-crystalline phase transition in dipalmitoylphosphatidylcholine (DPPC) containing liposomes and increases the bilayer permeability of both liposomes and proteoliposomes to D-glucose. The proteoliposomes have been used to deliver Triclosan to S. sanguis biofilms and the inhibition of growth of the bacteria after treatment with liposomally delivered Triclosan has been determined using a microtitre plate re-growth assay and compared with growth inhibition by 'free' Triclosan. It is shown that for short exposure times (1 to 2 min) proteoliposomally delivered Triclosan is a more effective growth inhibitor than free Triclosan. The results are discussed in terms of the targeting, retention and subsequent release of Triclosan into the bacterial biofilms.

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.

Effect of additives on liposomes: an electric birefringence study.

Under the influence of an electric field, vesicles in suspension partially deform or orientate, rendering the medium optically birefringent. The amplitudes of the birefringence under fields of variable amplitude lead to evaluation of the anisotropy of the electrical delta alpha and optical delta G polarisabilities of the vesicles. By using pulsed fields, rates of establishment and decay of birefringence enable vesicle sizes d to be measured. Samples of 9:1 phosphatidylcholine-phosphatidylserine liposome suspension in water were studied in the presence of various additives, including sucrose, an antibiotic (streptomycin), a steroid (dexamethasone), an anaesthetic (lignocaine) and a fluidising agent (benzyl alcohol). The changes in the three parameters delta alpha, delta G and d were different for each additive and are thought to be indicative of the mode of interaction of each. Electric birefringence also appears to be a rapid means of detecting structure changes.