The interaction of positively charged phospholipid vesicles with bacteria

Abstract

Streptococcus sanguis strain C104 Proteovesicles (proteoliposomes) have been used for the delivery of bactericides t o several strains of oral and skinassociated bacteria adsorbed on solid surfaces (microtitre plates) I1.21. The preparation and characterisation of proteoliposomes is relatively complex 131 and simpler methods of liposomal targeting and delivery are desirable. One possible approach is t o exploit the negative charge on the surface of bacteria 141 by targeting w i th positively charged liposomes. Here positively charged liposomes were prepared by the extrusion method 151 in phosphate buffered saline (pH 7.4) from dipalmitoylphosphatidylcholine (DPPCI-cholesterol mixtures (weight ratio 1 :0.26) containing variable amounts of stearylamine (SA) in a range up t o 17 w t % of the total liposomal lipid. The liposomes, which had weight-average diameters in the range 100-140nm. were radiolabelled w i th 3H-DPPC. Two methods were used t o study the interaction of the liposomes w i th bacteria; liposome adsorption t o adsorbed biofilms on microtitre plates and adsorption t o bacterial suspensions using a solution-depletion technique. In the former method adsorbed biofilms were prepared by incubation of bacterial suspensions of specific optical absorbance (0.5) in microtitre plate wells for 18 hours. Liposome suspensions were incubated w i th the biofilms for 2 hours at 37°C. after which the liquid phase was removed and the biofilm was dispersed in 1 % w/v sodium ndodecylsulphate and analysed by scintillation counting to determine the extent of liposome adsorption. Liposome adsorption was expressed as the percentage apparent monolayer coverage (% amc) of the biofilm calculated from the projected diameter of the liposomes and the area of the biofilm. In the solution-depletion method the liposomes were incubated w i th bacterial suspensions of known cell concentration (as determined by cell counting) for 2 hours; the suspension was gently centrifuged t o pellet the bacteria (5609 for 10 minutes) and the supernatant analysed for liposomal lipid. From the bacterial cell number and depletion of liposome concentration in the supernatant, the number of liposomes adsorbed per bacterium was determined as a function of liposomal lipid concentration.