Simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems
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Simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems. / Lokras, Abhijeet Girish; Chakravarty, Akash; Rades, Thomas; Christensen, Dennis; Franzyk, Henrik; Thakur, Aneesh; Foged, Camilla.
In: International Journal of Pharmaceutics, Vol. 626, 122171, 2022.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Simultaneous quantification of multiple RNA cargoes co-loaded into nanoparticle-based delivery systems
AU - Lokras, Abhijeet Girish
AU - Chakravarty, Akash
AU - Rades, Thomas
AU - Christensen, Dennis
AU - Franzyk, Henrik
AU - Thakur, Aneesh
AU - Foged, Camilla
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
U2 - 10.1016/j.ijpharm.2022.122171
DO - 10.1016/j.ijpharm.2022.122171
M3 - Journal article
C2 - 36070841
VL - 626
JO - International Journal of Pharmaceutics
JF - International Journal of Pharmaceutics
SN - 0378-5173
M1 - 122171
ER -
ID: 318522599