Lokras Abhijeet, Chakravarty Akash, Rades Thomas, Christensen Dennis, Franzyk Henrik, Thakur Aneesh, Foged Camilla
Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark.
Department of Infectious Disease Immunology, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark.
Int J Pharm. 2022 Oct 15;626:122171. doi: 10.1016/j.ijpharm.2022.122171. Epub 2022 Sep 5.
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 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) CHCN 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 > 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.
强大、灵敏且通用的分析方法对于定量装载到基于纳米颗粒的递送系统中的RNA药物货物至关重要。然而，同时定量共装载到纳米颗粒中的多种RNA货物仍然是一项挑战。在此，我们开发并验证了使用离子对反相高效液相色谱结合紫外检测（IP-RP-HPLC-UV）来同时定量单链和双链RNA货物。使用苯酚：氯仿：异戊醇混合物实现了从纳米颗粒载体中完全提取RNA货物。使用C柱或PLRP-S柱进行分离，用0.1 M三乙铵乙酸盐（TEAA）溶液作为离子对试剂（洗脱液A）和含有25%（v/v）CHCN的0.1 M TEAA作为洗脱液B进行洗脱。这些方法被应用于定量共装载到脂质-聚合物杂化纳米颗粒中的mRNA和聚肌苷酸：聚胞苷酸，以及共装载到脂质纳米颗粒中的单链寡脱氧核苷酸供体和Alt-R CRISPR单向导RNA。所开发的方法灵敏（RNA定量限<60 ng）、线性（R>0.997）且准确（纳米颗粒中加标的RNA回收率≈100%）。因此，本研究可能有助于方便地定量共装载到基于纳米颗粒的递送系统中的多种RNA货物。
