Stable oil-in-water emulsions: preparation and use as vaccine vehicles for lipophilic adjuvants.

Many of the most potent immunoadjuvants for inclusion in vaccines are extremely hydrophobic surfactants. Lipophilic vehicles are needed as carriers for these water-insoluble adjuvants and to provide the hydrophilic-hydrophobic interfacial surface at which they act. We used emulsifiers comprised of fatty acid esters of polyoxyethylene sorbitan (Tweens) or sorbitan (Spans) to prepare oil-in-water (O/W) emulsions of hexadecane. Emulsion stability could be predicted from a ratio of spectrophotometric absorbance readings at 800 and 400 nm but not from published hydrophilelipophile balance (HLB) values. Emulsions that were stable even after heating or freezing resulted when equal volumes of hexadecane and a 70:30 blend of Tween 80/Span 80(T80/S80) were mixed and then diluted with normal saline solution to the desired hexadecane concentration. This blend of monooleate esters has an HLB value of 11.8. Other Tween-Span formulations were mixed to yield emulsifiers with the same HLB value, but only those that contained either T80 or S80 were effective stabilizers. Instability resulted when both esters were derived from saturated fatty acids. Addition of bovine serum albumin (BSA) antigen to the oil phase of 5% hexadecane emulsions tended to destabilize the emulsions, especially at higher protein concentrations. The surface active adjuvant, hexadecylamine, increased emulsion stability. Highest antibody responses in mice were seen when BSA was added to the internal phase of emulsions, i.e., the oil phase of O/W emulsions and the aqueous phase of W/O (Freund's) emulsions. Addition of the hydrophilic T80 to the aqueous phase of O/W emulsions was detrimental to antibody production. In general, stability, oil concentration, and T80/S80 concentration of emulsions had little effect on IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)