ABSTRACT
This protocol assesses pro-inflammatory properties of nucleic acid nanoparticles (NANPs) using a validated preclinical model, peripheral blood mononuclear cells (PBMCs), which are 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. While representative NANPs with high and low immunostimulatory potential are used as standards throughout the procedure, this protocol can be adapted to any other NANPs or therapeutic nucleic acids that are either carrier-based and carrier-free; additional cytokine biomarkers can also be included. Several commercial platforms and controls broadly accessible to the research community are tested to quantify all biomarkers either in single- or multi-plex format. The continuous execution of this protocol does not exceed 48 hours and when the immediate analysis is not feasible, single-use aliquots of supernatants can be frozen and stored.
