基于核酸杂交-酶联桥接分析法在 siRNA 药物定量分析中的应用

摘要

OBJECTIVE To investigate the feasibility and application of enzyme-linked bridging assay(ELBA)method to the pharmacokinetic evaluation of antisense strand siRNA drug. METHODS Antisense strand RNAs were diluted in LNCap cell lysates from 5 to 50 000 pmol·L-1 to construct the quantification curves. We transfected the intact double-strand siRNA at a final concentration 100 nmol·L-1 targeting Polo-like kinase into the LNCap cells and investigated the specificity of ELBA quantitating the siRNA antisense strand in cell supernatant,cell lysates and RNA-induced silencing complex( RlSC). Quantification curves were constructed and validated in biological matrices such as plasma (5-25 000 pmol·L-1 )and multiple tissues(liver,heart,spleen,and kidneys)(3-6250 pmol·L-1 ). The prostate specific membrane antigen aptamer siRNA delivery system with the intact siRNA concentration of 15 nmol·kg-1 was prepared. The siRNAs were delivered into the LNCap xenogrant tumor model in C57 mice by tail vein injection. The concentration of siRNA antisense strand was determined in plasma and tissues 30 min post administration by ELBA. RESULTS The quantitative range of antisense strand siRNA in cell lysates was 5-50 000 pmol·L-1 ,and ELBA method could quantify the siRNA antisense strand concentration from cell lysates and RlSC in LNCap cells transfected with double-strand siRNA. ln addition,ELBA could specifically reflect the single antisense strand concentration instead of intact siRNA double strands in plasma. The quantification range of siRNA antisense strand using ELBA in plasma was 5-25 000 pmol·L-1 and 3-3125 pmol·L-1 in tissues. About 30 min post administration of PSMA aptamer-siRNA,the antisense strand of siRNA was distributed mainly to the tumor,liver,kidneys,blood and spleen in sequence. The distribution profile might be attributed to the target delivery and siRNA pharma-codynamics. CONCLUSION The ELBA method is successfully applied to the siRNA antisense strand pharmacokinetic evaluation,which provides an alternative for pharmacokinetic studies of siRNA-based drugs.%目的：考察基于核酸的酶联桥接分析（ELBA）法定量分析生物基质中双链 siRNA 药物的反义链RNA 的可行性，为研究 siRNA 药物提供可靠的检测方法。方法在 LNCap 细胞空白基质中采用 ELBA 方法建立 siRNA 反义链的标准曲线（5~50000 pmol·L-1）。LNCap 细胞中转染双链 siRNA（终浓度100 nmol·L-1），24 h 后取上清、细胞裂解物及 RNA 诱导沉默复合体（RlSC），根据标准曲线对各组分中反义链 RNA 浓度进行定量。分别配制含双链 siRNA 5~25000 pmol·L-1或反义 siRNA 5~25000 pmol·L-1的小鼠全血浆样品，用小鼠肝、心、肾和脾生物基质配制反义 siRNA 3~6250 pmol·L-1样品，考察 ELBA 方法对血浆和生物基质中 siRNA 反义链进行定量的特异性和定量范围。sc 给予 C57小鼠 LNCap 细胞制备移植瘤模型，iv 给予双链 siRNA 药物15 nmol·kg-1，30 min 后 ELBA 法测定血浆、肿瘤组织、肝、肾、心和脾中反义链siRNA 浓度。结果基于 ELBA 方法对细胞基质中反义链 siRNA 定量范围为5~50000 pmol·L-1，且ELBA可以对转染双链 siRNA 的细胞裂解物和 RlSC 复合物中的反义链 siRNA 进行特异定量。ELBA 对血浆中双链 siRNA 中的反义链无响应，对血浆和组织中 siRNA 反义链的定量分析范围分别为5~25000 pmol·L-1和3~3125 pmol·L-1。双链 siRNA 在前列腺特异膜抗原-适配子递送系统下静脉给药30 min 后，ELBA 检测发现，反义链 RNA 分布丰度依次为肿瘤组织、肝、肾、血和脾。结论 ELBA 方法可用于 siRNA 药物的反义链的定量分析及组织分布研究。