Michael Kaszuba

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.

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.

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.

Polyhydroxy-mediated interactions between liposomes and bacterial biofilms

A theoretical model has been developed for the interaction of the surface polymers of the bacterial glycocalyx with liposomes incorporating lipids with polyhydroxy headgroups such as phosphatidylinositol (PI). The theory is based on a lattice model and equations are derived for the potential energy of interaction between the surfaces of a bacterium and a liposome as a function of their separation. It is shown that a relatively small energy of interaction, less than that of a single hydrogen bond, between the polyhydroxyl headgroup of the liposomal lipid and bacterium surface polymer residues could give rise to a potential energy of interaction in excess of the classical double layer repulsive force and attractive dispersion force interactions. The most important prediction of the theory is that the potential energy of interaction goes through a minimum as a function of the polyhydroxy lipid (PI) concentration in the liposomal surface, thus predicting an optimal liposomal composition for adsorption of liposome to bacterium. This result is in concordance with the adsorption of dipalmitoylphosphatidylcholine-PI liposomes to a range of biofilms of oral and skin-associated bacteria on solid supports, where optimum levels of PI for adsorption have been found. The theory demonstrates that subtle changes in the composition of liposomal and bacterial surfaces involving relatively small interaction energies can markedly influence the nature of their interactions.

The Use of Phospholipid Liposomes for Targeting to Oral and Skin-Associated Bacteria

Phospholipid (dipalmitoylphosphatidylcholine (DPPC) plus phosphatidylinositol (PI)) proteoliposomes with surface bound lectins (succinylated concanavalin A (s con A) and wheat germ agglutinin (WGA)) have been prepared covering a range of size and surface density of lectin. Negatively charged phospholipid liposomes from DPPC-PI mixtures covering a range of PI mole % and positively charged liposomes from DPPC-cholesterol-stearylamine (SA) mixtures covering a range of SA mole % have been prepared. The targeting of the liposomes and proteoliposomes to a range of oral and skin-associated been prepared. The targeting of the liposomes and proteoliposomes to a range of oral and skin-associated bacterial biofilms has been investigated. The oral bacteria Streptococcus mutans and gordonii and the skin-associated bacterium Coryneform hofmanni can be targeted with s con A bearing proteoliposomes while the skin associated bacterium Staphylococcus epidermidis can be targeted with WGA bearing proteoliposomes. Both oral and skin-associated bacteria can be targeted with positively charged liposomes although the extents of adsorption to the biofilm are low except for Staphylococcus epidermidis. In the case of negatively charged liposomes targeting is critically dependent on the PI content of the liposomes and for all the bacteria studied optimum levels PI for targeting have been found. The adsorption of the oral bacterium Streptococcus gordonii to immobilised monolayers having the optimum PI level for adsorption has been studied by total internal reflection microscopy (TIRM). Both the phospholipid and proteoliposomes have been used to deliver the bactericide Triclosan to biofilms. All the systems studied inhibited bacterial growth to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen peroxide production from reactive liposomes encapsulating enzymes.

Reactive cationic and anionic liposomes have been prepared from mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol incorporating dimethyldioctadecylammonium bromide and DMPC incorporating phosphatidylinositol, respectively. The liposomes were prepared by the vesicle extrusion technique and had the enzymes glucose oxidase (GO) encapsulated in combination with horseradish peroxidase (HRP) or lactoperoxidase (LPO). The generation of hydrogen peroxide from the liposomes in response to externally added D-glucose substrate was monitored using a Rank electrode system polarised to +650 mV, relative to a standard silver-silver chloride electrode. The effects of encapsulated enzyme concentration, enzyme combinations (GO+HRP, GO+LPO), substrate concentration, electron donor and temperature on the production of hydrogen peroxide have been investigated. The electrode signal (peroxide production) was found to increase linearly with GO incorporation, was reduced on addition of HRP and an electron donor (o-dianisidine) and showed a maximum at the lipid chain-melting temperature from the anionic liposomes containing no cholesterol. To aid interpretation of the results, the permeability of the non-reactive substrate (methyl glucoside) across the bilayer membranes was measured. It was found that the encapsulation of the enzymes effected the permeability coefficients of methyl glucoside, increasing them in the case of anionic liposomes and decreasing them in the case of cationic liposomes. These observations are discussed in terms of enzyme bilayer interactions.

Antibacterial reactive liposomes encapsulating coupled enzyme systems

Liposomes have been prepared from phospholipid mixtures of dipalmitoylphosphatidylcholine (DPPC) and phosphatidylinositol (PI) encapsulating the enzymes chloroperoxidase (CPO) and lactoperoxidase (LPO) in combination with glucose oxidase (GO) by both extrusion (VET) and/or reverse phase evaporation (REV). The liposomes were characterised in terms of the protein content and activity of the encapsulated enzymes. The antibacterial activity of these reactive liposomes arises from hydrogen peroxide and oxyacids produced in the presence of the substrates glucose and chloride or thiocyanate ions. The liposomes were targeted to biofilms of Steptococcus gordonii, an oral bacterium and their antibacterial activity was measured both as a function of liposome-biofilm incubation time and incubation time with the substrates. Bacterial inhibition increases with both liposome-biofilm and substrate-biofilm incubation time and with the extent of enzyme encapsulation. The reactive liposomes also display antibacterial activity in the presence of saliva. The reactive liposomes have potential value in the context of oral hygiene.

THE VISUALISATION OF THE TARGETING OF PHOSPHOLIPID LIPOSOMES TO BACTERIA

Anionic and cationic phospholipid liposomes have been prepared from dipalmitoylphosphatidylcholine (DPPC) — phosphatidylinositol (PI) and DPPC-cholesterol-stearylamine (SA) mixtures over a range of composition and targeted to biofilms of the skin-associated bacterium Staphylococcus epidermidis to establish the optimum PI and SA content for targeting. The interaction of liposomes of optimum composition with the bacteria were visualized by electron microscopy using negative staining with uranyl acetate and phosphotungstic acid. It has been demonstrated that the liposomes absorb extensively to the bacterial surface. Immunoliposomes have been prepared with a covalently linked monoclonal antibody raised to antigenic determinants on the surface of the oral bacterium Streptococcus oralis. The targeting of the immunoliposomes to this bacterium has been visualized using a second gold labelled anti-antibody. As for the anionic and cationic liposomes the immunoliposomes adsorb to the surface of the bacteria. The results add support to the concept of using liposomes for the delivery of bactericides or therapeutic agents to bacteria.