Up200h system

Slight modifications to our earlier reported double-emulsion approach followed by solvent evaporation [17 (link),18 (link)] were employed to encapsulate whole cell lysate (Tag) in immune-activating NPs generating CpG-NP-Tag NPs (nasal nano-vaccine) and control formulations (NP-Tag and NP) capable of intranasal immunization. First, NP-Tag NPs were prepared using primary emulsion (w/o) obtained from vortexing 200 μL of Tag solution (25 μg/μL) in 1 mL of the organic phase (PLGA (70 mg) in ethyl acetate (1 mL)) for 1 min. Next, the primary emulsion (w/o) was added to 3 mL aqueous (w) phase (BS3 (0.5 mg/mL) in 2.5% polyvinyl alcohol). The mixture was sonicated on ice using the ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany) for 1 min to form activated nanoparticles (NPs) (Figure 1). After three washes with distilled water, NPs were resuspended in 5% w/v sucrose solution. Finally, NPs were freeze-dried and lyophilized (under 200 µm vacuum) on the ATR FD 3.0 system and stored at −20 °C until further use. For optimal ligation of CpG, CpG ligand, and resuspended NPs (1:240 w/w ratio) were incubated on an orbital shaker for 45 min at room temperature, and excess ligand was removed with PBS washes to obtain intranasal CpG-NP-Tag.