Development of a dispersal procedure for the lipid A analog E5531 using a `pH-jump method'

Abstract

We have developed a `pH-jump method' in order to facilitate the dispersion of E5531, a synthetic analog of lipid A, at neutral pH for use in pharmaceutical preparations. The `pH-jump method' procedure involves dispersing E5531 at pH 11.0 (above p K 2) at 50°C (higher than the phase transition temperature) followed by mixing with phosphate buffer in order to adjust the solution to a neutral pH. The size of the aggregates prepared by the pH-jump method was approximately 15 nm. However, when E5531 was dispersed in neutral pH (7.3), directly (normal dilution method), the size of aggregates was approximately 120 nm and when sonication was used (sonication method), the size of the aggregates was approximately 45 nm and larger than that prepared by pH-jump method. E5531 aggregates form vesicle structures. The membrane fluidity and micropolarity of the aggregates prepared by the pH-jump method was higher than that of those prepared by the normal dilution and sonication methods. This study therefore shows that the pH-jump method will provide smaller, fully hydrated aggregates of E5531 than either the normal dilution or sonication methods.