Nanoparticles of defined size can be easily obtained by simply mixing Protamine, a pharmaceutical drug that is used to neutralize heparin after surgery, and RNA in the form of oligonucleotides or messenger RNA. Depending on the concentrations of the two reagents and their salt contents, homogenous nanoparticles with a mean diameter of 50 to more than 1000 nm can spontaneously be generated. RNA is a danger signal because it is an agonist of for example TLR-3, -7, and -8; therefore, Protamine-RNA nanoparticles are immunostimulating. We and others have shown in vitro that nanoparticle size and interferon-alpha production by human peripheral blood mononuclear cells (PBMCs) are inversely correlated. Conversely, nanoparticle size and TNF-alpha production by PBMCs are positively correlated (Rettig et al., Blood 115:4533-4541, 2010). Particles of less than 450 nm are most frequently used for research and clinical applications because they are very stable, remain polydispersed and induce interferon-alpha proteins, which are a natural antiviral and anticancer protein family with 12 members in humans. Herein, we describe a method to generate 130 nm nanoparticles as well as some of their physical and biological characteristics.
