Assessment of siRNA pharmacokinetics using ELISA-based quantification.
Here, we developed a novel ELISA-based assay for quantifying double-stranded intact siRNAs for in vivo pharmacokinetic analysis. The assay makes use of dual-labeled unmethylated or methylated siRNA, 5'-end-labeled on one strand with biotin (capture marker), and with dinitrophenol (detection marker), on the other end. This ELISA-based assay was linear over the range of 10-100 fmol/ml, with a sensitivity (5.4 fmol/ml) 629-fold higher than fluorometric quantification methods. The coefficient of variation (CV) of the ELISA quantification was 9.4% for intra-assay and 12.1% for inter-assay. The assay was specific for double-stranded siRNAs. The intensity of the detected signal was reduced to background levels in the presence of single-stranded RNA. The ELISA-based assay revealed that the levels of methylated forms of siRNAs after transfection into A549 and HeLa cells were significantly higher than those of unmethylated siRNA forms. Applying this assay to a study of the pharmacokinetic profiles of intravenously administered siRNAs, we found that the higher blood concentrations were achieved using the methylated form of siRNAs than unmethylated form. Moreover, methylated siRNAs complexed to DOTAP-based cationic liposomes showed significantly higher and prolonged blood concentration-time profile, with 2.2-fold lower clearance rate (0.11+/-0.02 ml/min) as compared to the uncomplexed form. These results demonstrate the utility of an ELISA-based assay for evaluating chemically modified siRNAs and cationic delivery systems, particularly from a pharmacokinetic perspective.
Kim
Eun-Joong
EJ
National Research Laboratory for Transporters Targeted Drug Design, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul 151-742, South Korea.
Park
Tae Gwan
TG
Oh
Yu-Kyoung
YK
Shim
Chang-Koo
CK
D000818:Animals; D019921:Biotinylation; D002412:Cations; D004140:Dinitrophenols / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005260:Female; D005470:Fluorometry; D006367:HeLa Cells; D006801:Humans; D007275:Injections, Intravenous; D008081:Liposomes; D008745:Methylation; D051379:Mice; D008807:Mice, Inbred BALB C; D009690:Nucleic Acid Conformation; D015588:Observer Variation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*; D015203:Reproducibility of Results; D014162:Transfection
D002412:Cations; D004140:Dinitrophenols; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D034741:RNA, Small Interfering; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane