Dc cholesterol

A certain mass of lipid [5 mg DPPC, 2 mg DSPE-PEG (2000)-Amine and 2 mg DC-cholesterol(Avanti Polar Lipids, Inc, Alabaster, AL, USA)] were mixed together and dissolved into 10 mL trichloromethane(CHCl3) (Fisher Scientific, Waltham, MA, USA). The solution was moved to the rotary evaporator (Yarong Inc, Shanghai, China) at 50 °C to remove the organic solvent and form a thin lipid film. One hour later, the resulting thin lipid films was hydrated in 2 mL Double steaming water forming a translucent opalescent suspension. Then 100 µl of Perfluorohexane (PFH) was dropwise into suspension after which the suspension was emulsified in an ice bath using a sonicator (Sonics &Materials Inc., Newtown, CT, USA) with power of 130 W for 5 min (5 s on and 5 s off), then the prepared cationic liquid nanoparticles (CL-NPs) were harvested. The nanoemulsions were centrifuged at 6000 rpm for 5 min and then washed in Double steaming water to wipe off disasociated lipids and PFH. The centrifugation and washing process were repeated three times. Finally, the prepared CL-NPs were stored at 4 °C for later use. To prepare fluorescent nanodroplets, DiI (1 mg) or DiR (1 mg) fluorescent dye were added to the lipid solution and aluminum foil papers was used to prevent light exposure.

PRINT technology was used to manufacture 80×320 nm poly (lactic co-glycolic acid) (PLGA, 50:50, 35 kDa, Lakeshore Biomaterials) particles as previously described [34 (link),35 (link)]. In short, PLGA and DC-cholesterol (Avanti Polar Lipids) were dissolved in chloroform (9:1 w/w ratio) and casted into a thin film on a PET-sheet (KRS Plastics). The film was oriented in order to contact the molds and carefully heated. Next, the film was split and the mold content was transferred to a second PET-sheet by passing through a laminator. Then water with 0.1% polyvinyl alcohol (PVOH) was added to the PET-sheet to release the nanoparticles (NPs). The harvested particles were sterilized and concentrated by sterile filtration and tangential flow filtration.
To adsorb the rE proteins to NP surfaces, rE was incubated with PLGA NPs in a 1% rE/NP (w/w%) ratio for 15 mins at room temperature in 0.1% PVOH in water with 9.25% sucrose to establish 100% adsorption efficiency for all serotypes.