Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are prepared by a modified water-in-oil-in-water emulsion-solvent evaporation (W/O/W) double emulsion-solvent evaporation method.48 (link) Briefly, 100 mg PLGA (MedChem Express) was added to 1 mL dichloromethane by sonication for 30 s. Next, 3 mL of polyvinyl alcohol (PVA) solution (7%, w/v) was added to the W/O single emulsion before being tip-sonicated for 10 min. Then the above solution was dripped into 50 mL of PVA solution (1%, w/v) containing 2% isopropanol and then maintained under mechanical stirring for 1 h at 600 rpm. After centrifugation at 8,000 × g for 40 min, purified PLGA nanoparticles were obtained. The above particles were washed twice, paying attention to avoid agglomeration of particles.
Next, PEI (branched, 25 kDa; Sigma) solution (2 mg/mL) was mixed with PLGA solution (1.5 mg/mL) and then soft vortexing for 15 s. The aforesaid mixture was kept at room temperature (RT) for at least 1 h. After preparation of the PLGA/PEI complex, plasmid DNA solution (1 mg/mL) was mixed with polycation nanoparticles and then soft vortexed for 1 min. The prepared PLGA/PEI/pDNA polyplexes were characterized by their size and zeta-potential with the Zeta sizer Nano ZS (Malvern, Southborough, MA). The nanoparticle vaccines were stabilized for 30 min at RT prior to immunization.
Next, PEI (branched, 25 kDa; Sigma) solution (2 mg/mL) was mixed with PLGA solution (1.5 mg/mL) and then soft vortexing for 15 s. The aforesaid mixture was kept at room temperature (RT) for at least 1 h. After preparation of the PLGA/PEI complex, plasmid DNA solution (1 mg/mL) was mixed with polycation nanoparticles and then soft vortexed for 1 min. The prepared PLGA/PEI/pDNA polyplexes were characterized by their size and zeta-potential with the Zeta sizer Nano ZS (Malvern, Southborough, MA). The nanoparticle vaccines were stabilized for 30 min at RT prior to immunization.