DESIGNING SPRAY DRIED NANOEMULSION AS VACCINE ADJUVANT

Abstract

In this study, oil in water nanoemulsion was prepared as an adjuvant by using high pressure homogenization technique. Various compositions of wheat germ oil alone or with Vitamin E acetate were used as lipid. Tween80 and Span85 were used as surfactants and concentrations were varied in order to obtain the optimized nanoemulsion with the smallest particle size. Optimized nanoemulsion with wheat germ oil alone as FA (formula A) having the particle size of 111 nm, zeta potential of -29 and wheat germ oil with vitamin E acetate (0.9:0.1) as FB (formula B) having the particle size of 100 nm, zeta potential of -24 mV were chosen for spray drying. Maltodextrin (MD) was used as carrier loaded with FA and FB to investigate the effect of parameters during spray drying. FB5-BL was then chosen, which had the particle size of 152 nm and zeta potential of -22 mV after reconstitutions. BSA as a model antigen was loaded into FB5-BL investigation was performed. Characterization of nanoemulsion and spray dried powder was done by TEM and SEM, respectively. The morphology of nanoemulsion was spherical in shape and the spray dried particles had a porous like surface with spherical shape. Spray dried particle size was also analyzed by mastersizer and the particle size of FB5-BL, FB5-BSA1 and FB5-BSA3 was around 3-7 µm. Thus, spray dried powder could be administered by inhalation while reconstituted spray dried powder could be administered by parenteral administration. Thermographic behavior as well as crystallinity of spray dried powder was also examined and all the powder formulation were amorphous in state .There was no interaction between the compositions MD, BSA and nanoemulsion in spray dried powder confirmed by FTIR. BSA integrity was analyzed by circular dichroism and SDS-PAGE. It could be concluded that the integrity of BSA was conserved even after spray drying but a slight shift of secondary structure was note due to the protein extraction by small amount of organic solvents. Moreover, stability evaluation of both nanoemulsion and spray dried powder as well as its reconstitutions were performed at 4°C and at ambient temperature and the particle size of formulations at each time point was analyzed for 3 months. The stability of both formulation at two condition had good results except the spray dried powder at ambient temperature showed increased in particle size. It could be due to slight stickiness and low concentrations of maltodextrin to stabilize the droplets. Two types of cell lines; human monocyte macrophage cell line (CRL-9855) and human nasal cell line (CCL-30) was cultured and cell viability was tested for all optimized formulations. Cell viability testing with monocyte cells gave nearly 100% viability in both FB5-BL and FB5-BSA1. For cell viability in human nasal cells, the concentration of 35µg/ml gave 100% viability. Furthermore, uptake study was also carried out by using macrophage cells. The confocal laser scanning microscopy has confirmed that FB5-BSA1 as well as FB5-BL without BSA was uptake by the cells. It can be concluded that spray dried nanoemulsion adjuvant was successfully developed using BSA as a model antigen with retained integrity of BSA Hence, it could be used as potential powder adjuvant.