Abstract
This protocol assesses proinflammatory properties of nucleic acid nanoparticles (NANPs) using a validated preclinical model, peripheral blood mononuclear cells (PBMCs), that is highly predictive of cytokine responses. The experimental procedure details the preparation of pyrogen-free NANPs, isolation of PBMCs from freshly collected human blood, and analysis of characteristic biomarkers (type I and III interferons) produced by PBMCs transfected with NANPs. Although representative NANPs with high and low immunostimulatory potential are used as standards throughout the procedure, this protocol can be adapted to any NANPs or therapeutic nucleic acids, irrespective of whether they are carrier based or carrier free; additional cytokine biomarkers can also be included. We test several commercial platforms and controls broadly accessible to the research community to quantify all biomarkers in either single- or multiplex format. The continuous execution of this protocol takes <48 h; when immediate analysis is not feasible, single-use aliquots of the supernatants can be frozen and stored (-20 °C; 12 months).