Objective: This research was carried out to optimize luteolin-loaded transfersome formula with independent variables such as lipid-surfactant (total lipid) concentration and luteolin concentration.Methods: Luteolin-loaded transfersome was optimized by response surface methodology based on four parameters, namely, particle size (Z-average), polydispersity index, zeta potential, and entrapment efficiency. The transfersome formula was prepared using central composite design, and the selected independent variables were the total lipid (mixture of phospholipid and Tween 80) and luteolin concentrations. 14 formulas of luteolinloaded transfersome were prepared by thin film hydration, followed by the sonication method. Results:The total lipid and luteolin concentration significantly affected the entrapment efficiency only. The other parameters were not affected by a change in these variables. The optimum formula of 4.88% total lipid and 0.5% luteolin with desirability value of 0.609 conformed with the prediction parameters. Vesicle imaging using transmission electron microscopy revealed spherical particles and the occurrence of particle aggregation. The optimum formula of luteolin-loaded transfersome possessed the following characteristics: Particle size of 286.03±8.46 nm, polydispersity index of 0.480±0.013, zeta potential of -18.67±0.379 mV, and entrapment efficiency of 94.97±0.28 %. However, these values did not correspond to the predicted values and were confirmed by the low adjusted and predicted R-squared values. Conclusion:This method can be applied to optimize the entrapment efficiency, and in the future, it can be used for further optimizing formula of transfersome by including more variables.

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Aluminum hydroxide (AH) is one of the aluminum-based adjuvants that forms needle-like crystalline structure with the average particle size of 1-20 µm. Due to their favorable safety profile, AH adjuvant has been used widely in various human vaccine in diphtheria, tetanus, pertussis, hepatitis, and papilloma virus vaccines. The antigen-adjuvant interaction is strongly governed by the adjuvant's physicochemical characters, such as shape, dimension and surface charge. Based on this concept, tremendous efforts to amplify the performance of the adjuvant have been made by modifying its particle size into nano-sized adjuvant. Also, during vaccine formulation, several physical processes may take place and potentially affect the antigen-adjuvant interaction, such as dilution, salt and aging exposure, pH change, heat, pressure and shear forces stress. Therefore, in this study we have examined the effect of sterilization and pH of AH nanoparticle (AHNp) with the average size of ~160 nm on its physical characterization and adjuvant-antigen interaction. The obtained results were compared with those from AH microparticle (AHMp) and commercial AH adjuvant (AHC).AHMp and AHNp were prepared by using common precipitation method through the addition of alkali to the solution of aluminum chloride. The aluminum content of both AH suspension is 12.1 mg/mL. The average particle size of AHMp and AHNp by static light scattering are 4.21 µm and 0.16 µm, respectively. For evaluating the effect of autoclave on AH properties, all AH samples on glass container were autoclaved at 121 °C for 30 dan 60 min. The result shows that all samples did not give significant physical character change after autoclave. All measured parameters including the solid content of AH suspension, the acquired mass after freezing dry, pH value, and visual appearance remained the same as those before autoclave. The X-ray diffractogram of AHMp and AHNp revealed that the diffraction bands were sharper after autoclaving.Following autoclaving, all fraction of (bovine serum albumin) BSA added into the adjuvant could be completely adsorbed by AHNp, AHMp, and AHC samples, as high as its pre-autoclaved condition, achieving maximum adsorption capacity. Such phenomena could be obtained even when volume ratio of adjuvant/BSA multiplied 20-times. It indicates that the binding efficiency BSA to all adjuvant