Stability of protamine-RNA particles. Protamine 5000 diluted 14 times with water or with Ringer lactate solution was mixed 1-to-1 with RNA18 diluted to 1 mg/mL with water or Ringer lactate solution to generate nano- or microparticles, respectively. The formulation was repeated at several time intervals. (A) Particles were diluted with 9 volumes of PBS and incubated at room temperature. Particles of 220 nm (B) or 1200 nm (C) were diluted with 9 volumes of fresh human serum and incubated at 37°C. Fresh human PBMCs were added to the particles after various incubation times as indicated. After overnight incubation, the interferon-α (for stimulation with nanoparticles) or TNF-α (for stimulation with microparticles) concentrations in the culture supernatants were measured by ELISA. The data shown are representative of 5 independent experiments.