Preparation Of A Liposomal Lipopolysaccharide Of Gram-Negative Bacteria Isolated From Clinic And Its Immunoprotective Effects

ObjectTo prepare a liposomal lipopolysaccharide (LPS) of gram-negativebacteria isolated from clinic which were multi-drug resistant. Andto observe the immunoprotective effects of the liposomal LPS on micechallenged by various LPS or bacteria. We are looking forward todevelop a novel vaccine strategy that can be evaluated for surgical,tumor and other high-risk hospitalized patients.MethodsLPSs were extracted from several multi-drug resistant gram-negative bacteria strains which were isolated from clinic by enzymeextraction, and then incorporated into multi-lamellar liposomesconsisting of phosphatidyl choline (PC), phosphatidylserine (PS)and cholesterol in a 4:1:4 molar ratio by the method of reverseevaporating. The basic LPS/lipid ratio was 1:10 (wt/wt). Theendotoxic activities of both the LPS-containing liposomes and thefree LPS were measured by Limulus amoebocyte lysate (LAL) assay andthe entrapment efficiency was presumed. The particle size of theliposomes were measured by laser light scattering instrument. Andthe transmission electron microscopy (TEM) was used to examine themorphology of liposomes. D-galactosamine (D-GalN)-sensitized micemodel were established to investigate the immuno-protective effectsof the liposomes in vivo against various LPS or bacteria. ResultThe multilamella liposomal LPS, consisting of PC, PG andcholesterol in a 4:1:4 molar ratio was prepared according to thereverse evaporating method developed by Dijkstra et. al. with somemodifications, and was opalescent at room tempreture. The basicLPS/lipid ratio was 1:10 (wt/wt). The endotoxic activities of theLPS in liposomes measured by the LAL assay reduced 100 to 1,000-foldcompared with its original value which suggested 99-99.9% of the LPShad been incorporated into the liposomes.During the 90-day stability experiments, there was no change inthe LAL activity of any of these samples at any of the time pointseither under the routine (4℃) or the accelerated (37℃) conditons.The LPS liposomes had a regular morphous under the TEM and well-distributed particle size ranged from 300 to 600 nm measured bylaser light scattering instrument.Active immunization with LPS liposomes to the D-GaiN-sensitizedmice model provided protection against a twice LD₁₀₀ challenge withvarious LPS or gram-negative bacteria. Both the ratio and theduration of survival increased.The liposomes were identified nonpyrogenic and nontoxic.ConclusionThe technique of preparing the LPS liposomes by the reverseevaporating method is feasible, our results of its endotoxicactivities, entrapment efficiency, particle size, morphology,stability and its immuno-protective effects in vivo against variousLPS or bacteria suggested its possibility to become a novel vaccinestrategy to surgical, tumor and other high-risk hospitalizedpatients.