Use of liposomes to deliver bactericides to bacterial biofilms

Methods are described for the preparation of anionic and cationic liposomes and proteoliposomes with covalently linked lectins or antibodies by the extrusion technique (vesicles by extrusion, VETS). The liposomes are prepared from the phospholipid dipalmitoylphosphatidylcholine (DPPC), together with the anionic lipid phosphatidylinositol (PI) or the cationic amphiphile dioctadecyldimethylammonium bromide (DDAB) together with the reactive lipid DPPE-MBS, the m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) derivative of dipalmitoylphosphatidylethanolamine (DPPE). Proteins (lectin or antibody), after derivatization with N-succinimidyl-S-acetylthioacetate (SATA), can be covalently linked to the surface of the liposomes by reaction with the reactive lipid, DPPE-MBS. The physical and chemical characterization of the liposomes and proteoliposomes by photon correlation spectroscopy (PCS) and protein analysis, to determine the number of chemically linked protein molecules (lectin or antibody) per liposome, are described. The liposomes can be used for carrying oil-soluble bactericides (e.g., Triclosan) or water-soluble antibiotics (e.g., vancomycin or benzylpenicillin) and targeted to immobilized bacterial biofilms of oral or skin-associated bacteria adsorbed on microtiter plates. Techniques for the preparation of immobilized bacterial biofilms, applicable to a wide range of bacterial suspensions, and for the analysis of the adsorption (targeting) of the liposomes to the bacterial biofilms are given. The mode of delivery and assessment of antibacterial activity of liposomes encapsulating bactericides and antibiotics, when targeted to the bacterial biofilms, by use of an automated microtiter plate reader, are illustrated, with specific reference to the delivery of the antibiotic benzylpenicillin encapsulated in anionic liposomes to biofilms of Staphylococcus aureus. The methods have potential application for the delivery of oil-soluble or water-soluble bactericidal compounds to a wide range of adsorbed bacteria responsible for infections in implanted devices such as catheters, heart valves, and artificial joints.