The size of protamine-RNA complexes depends on the ionic conditions of formulation. An 18-residue RNA (RNA18) oligonucleotide (A,C-F) or mRNA coding enhanced green fluorescent protein (B) were diluted to 1 mg/mL using water (A-B,D) or Ringer lactate solution (C,E) and mixed at indicated mass ratios (A,C) or at 1:1 mass ratios (B,D-F) with Protamine 5000 diluted to 1 mg/mL using water (A-B,D) or Ringer lactate solution (C,E). The size of the particles (A, left, B-C) was assessed by dynamic light scattering (the number shown in each graphic indicates the average hydrodynamic diameter) and electron microscopy (transmission in panel D and scanning in panel E). Data shown are representative of 10 independent experiments. The zetapotentials of the nanoparticles (A right) are measured using a Zetasizer (Malvern). (F) A summary of the results from dynamic light scattering measurements of particles produced with protamine and RNA diluted to 1 mg/mL using different solutions of varying ionic strength. Each formulation was repeated 3 times and measured. Error bars indicate the standard variation within those triplicate experiments.