2.2. Adjuvant characterization

With the exception of Alum, GLA-Alum, and covalent liposomes with and without GLA, all other formulations were monitored by HPLC with charged aerosol detection (CAD) to determine GLA concentration and dynamic light scattering (DLS) to determine particle size. Particle size was determined using the Malvern Instruments (Worcestershire, UK) Zetasizer Nano-S or Nano-ZS. For oil-in water emulsions and liposome formulations, samples was prepared at 1:100 dilution by combining 5 μl of each formulation with 500 μl of ultrapure water in a 1.5 ml polystyrene disposable cuvette. For aqueous GLA, a 1:10 dilution was accomplished by combining 50 μl of formulation with 450 μl ultrapure water in a 1.5 ml polystyrene disposable cuvette. In general, three separate cuvettes were prepared. All DLS measurements were then made three times with each of the three separate cuvettes. Zeta potential measurements were also obtained using the Malvern Instruments (Worcestershire, UK) Zetasizer Nano-ZS. Samples were prepared for a 1:20 dilution by combining 50 μl of each formulation with 950 μl of ultrapure water in a 1.5 ml eppitube before being transferred to a disposable capillary cell for analysis.

For HPLC-CAD, samples were prepared for analysis in a 1.5 ml capacity HPLC glass vials obtained from Agilent Technologies (Santa Clara, CA), by combining 50 μl of formulation with 950 μl mobile phase B (1:1 [v:v] methanol:chloroform, 20 mM ammonium acetate, 1% acetic acid) for GLA-emulsion and GLA-liposome samples or mobile phase A (75:15:10 [v:v:v] methanol:chloroform:water, 20 mM ammonium acetate, 1% acetic acid) for aqueous GLA samples. Three separate vials were prepared for each formulation. Samples were then placed on que for injection into a Waters Co. (Milford, MA) Atlantis T3 column attached to an Agilent Model 1100 HPLC (Santa Clara, CA). A gradient consisting of mobile phases A and B was used spanning a 25-minute time period. Detection was performed by a Charged Aerosol Detector obtained from ESA Biosciences (Chelmosford, MA). An 11-point nonlinear quadratic standard curve fit was obtained of GLA standards ranging from 1 to 100 μg/ml GLA.