Simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems

Robust, sensitive, and versatile analytical methods are essential for quantification of RNA drug cargoes loaded into nanoparticle-based delivery systems. However, simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticles remains a challenge. Here, we developed and validated the use of ion-pair reversed-phase high-performance liquid chromatography combined with UV detection (IP-RP-HPLC-UV) for simultaneous quantification of single- and double-stranded RNA cargoes. Complete extraction of RNA cargo from the nanoparticle carrier was achieved using a phenol:chloroform:isoamyl alcohol mixture. Separations were performed using either a C18 or a PLRP-S column, eluted with 0.1 M triethylammonium acetate solution (TEAA) as ion-pairing reagent (eluent A), and 0.1 M TEAA containing 25% (v/v) CH3CN as eluent B. These methods were applied to quantify mRNA and polyinosinic:polycytidylic acid co-loaded into lipid-polymer hybrid nanoparticles, and single-stranded oligodeoxynucleotide donors and Alt-R CRISPR single guide RNAs co-loaded into lipid nanoparticles. The developed methods were sensitive (limit of RNA quantitation < 60 ng), linear (R2 > 0.997), and accurate (≈ 100% recovery of RNA spiked in nanoparticles). Hence, the present study may facilitate convenient quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems.