siRNA pharmacokinetic studies

Sprague-Dawley rats were purchased from Beijing Vital River Laboratory Animal Technology. All rats were group-housed in a specific pathogen-free facility under a 12:12-h light/dark cycle at 22°C–24°C. All rats were males aged 8–10 weeks, weighing 401 ± 17 g. Six rats were randomly assigned to two groups to evaluate the pharmacokinetic (PK) feature of anti-SMO siRNAs: the intravenous and intra-articular injection groups. Each group contained three rats. The rats were administered a single dose of si-S1A3-Chol intravenously or intra-articularly at a 25 nmol/kg dose of body weight. The blood samples were harvested using the retro-orbital eye bleed procedure at 0, 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 h after administration. Heparin served as an anticoagulant, and blood samples were centrifuged to harvest plasma. The concentrations of the sense and antisense strand of si-S1A3-Chol in plasma were quantified by stem-loop qPCR as described previously.⁵⁷ In brief, plasma samples were heated in 0.25% Triton X-100 for 10 min. The siRNA-containing supernatants then underwent stem-loop reversed transcription followed by qPCR using the Bulge-Loop miRNA qRT-PCR Starter Kit (Ribobio, {"type":"entrez-nucleotide","attrs":{"text":"C10211","term_id":"1535282","term_text":"C10211"}}C10211). The sequences of the primers are listed in Table S6. The siRNA concentrations were determined using a standard curve. Mean siRNA plasma concentration-time profiles were used to estimate PK parameters, including C_max, t_max, and AUC.