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

Robust, sensitive, and versatile analytical methods are essential for quantification of RNA drug cargos loaded into nanoparticle-based delivery systems. However, simultaneous quantification of multiple RNA cargos 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 cargos. Complete extraction of RNA cargo from the nanoparticle carrier was achieved using a phenol:chloroform:isoamyl alcohol mixture. Separations were performed using either a C ₁₈ or a PLRP-S column, eluted with 0.1 M triethylammonium acetate (TEAA) solution as ion-pairing reagent (eluent A), and 0.1 M TEAA containing 25 % (v/v) CH ₃ CN 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 quantification < 60 ng), linear (R ²  > 0.997), and accurate (≈ 100 % recovery of RNA spiked in nanoparticles). Hence, the present study may facilitate convenient quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Camilla Foged reports financial support was provided by Independent Research Fund Denmark. Camilla Foged reports financial support was provided by Innovative Medicines Initiative. Camilla Foged reports financial support was provided by European Commission Seventh Framework Programme for Research and Technological Development.].
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