Mastersizer x

MF59 is Novartis proprietary oil-in-water emulsion consisting of 4.3% (vol/vol) squalene, 0.5% Tween 80, and 0.5% Span 85, in citrate buffer (10 mM). MF59 was prepared by homogenization at 12,000 psi with a Microfluidizer, model 110Y (Microfluidics). The emulsion was sterilized by passage through a polysulfone filter with 0.22 μm pore size (Gelman Sciences) and stored at 4°C. The mean particle size of the emulsion droplets determined with a Mastersizer X (Malvern Instruments) was 194±76 nm. Fluorescently labelled-MF59 was prepared formulating chloroform dissolved DiO fluorescent lipophilic tracer (Molecular Probes; Invitrogen-Life Technologies) in the squalene phase of MF59. The complete evaporation of chloroform was performed before homogenization of the emulsion as described above. The final concentration of DiO was 0.25 μg/ml.
Formulation with MF59 or DiO fluorescently labeled MF59 was prepared diluting the emulsion ½ in the volume dose with physiologic solution. The volume of the immunization dose was 10 μl.
Formulation with ALUM was prepared diluting, ALUM (2 mg/ml) in a solution (prepared with Water For Injection) with 10 mM histidine buffer pH 6.5 and 136 mM NaCl. The volume of the immunization dose was 20 μl.

The size distribution of the microparticles was determined by laser diffraction method using Malvern Mastersizer X (UK) after the formulations had been dispersed in appropriate medium (saturated solution of SS in water) and sonicated for 2 min. The geometrical diameter was expressed as volume median diameter (D50%). Also the Span values of formulations were defined as $\frac{\mathit{D}90\%-\mathit{D}10\%}{\mathit{D}50\%}$ , which represents the breadth of the particle distribution. Each measurement was repeated in triplicate.

Statistical design. PWO-in-water emulsion was performed according to a Box-Behnken experimental design (15 runs, 12 experimental points and three central points). SL content (2–6% of PWO), homogenization time (HT, 1–5 min) and homogenization speed (HS, 11,000–20,000 rpm) were the independent variables. Droplet size (D_[4,3]), and lipid oxidation evaluated as thiobarbituric acid reactive substances (TBARs) were the dependent variables.
Experimental data were fit to a second-order regression model. All the experiments were conducted randomly to avoid systematic bias. Response surface methodology (RSM) was applied at each independent variable and the multiple response optimization was performed using the desirability function, where droplet size and TBARs values were minimized.
Preparation and characterization of PWO emulsion. PWO-in-water emulsions (6 g) were prepared by dispersing SL (0.05–0.16 g) in distilled water (3.17–3.28 g) at 40 °C by stirring at 500 rpm for 20 min. The dispersion was added to PWO (2.67 g) and the mixture was homogenized with a Polytron PT-2100 (Kinematica AG, Luzern, Switzerland). Droplet size of PWO emulsions was determined by laser diffraction, using a particle size analyzer (Mastersizer X, Malvern Instruments, Malvern, UK), and expressed as D_[4,3]. Lipid oxidation was evaluated by TBARs, according to [20 (link)].