Preparation and Characterization of Lipid Nanoparticles Co-loaded With DNA and Nitro-Oleic Acid.

Lipid nanoparticles (LNPs) are powerful carriers for nucleic acid delivery, but plasmid DNA-loaded LNPs (pDNA-LNPs) have been limited by inflammation and toxicity. We showed that standard pDNA-LNPs activate the cGAS-STING pathway, leading to severe immune responses and mortality in mice. To overcome this, we co-loaded nitro-oleic acid (NOA), an endogenous STING inhibitor, into pDNA-LNPs. NOA-pDNA-LNPs mitigated inflammation, enabled safe in vivo delivery, and supported sustained gene expression for months. Here, we present a detailed protocol for producing and characterizing NOA-pDNA-LNPs to facilitate safer, long-term gene delivery applications. Key features • Provides a step-by-step protocol to produce plasmid DNA-LNPs co-loaded with nitro-oleic acid (NOA), optimized for both in vitro and in vivo applications. • Includes methods for quantitative assessment of DNA and NOA encapsulation efficiencies, particle size, and quality control metrics.