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  

Application of enzyme-linked bridging assay method to siRNA pharmacokinetic evaluation

作　　者：付洁[1] 刘潜 欧伦[1] 孙云娟[1] 李梦怡[1] 张晶[1] 宋海峰[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所药理毒理学研究室,北京100850 [2]湘乡市人民医院临床药学科,湖南湘乡411400

出　　处：《中国药理学与毒理学杂志》2014年第5期743-747,共5页Chinese Journal of Pharmacology and Toxicology

基　　金：国家科技重大专项(2012ZX09301003-001-007)~~

摘　　要：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（5-50000pmol·L-1）。LNCap细胞中转染双链siRNA（终浓度100nmol·L-1），24h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siRNA5-25000pmol·L-1或反义siRNA5-25000pmol·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siRNA3-6250pmol·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15nmol·kg-1，30min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5-50000pmol·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5-25000pmol·L-1和3-3125pmol·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30min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。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 al

关 键 词：核酸杂交-酶联桥接分析法 SIRNA 药代动力学 组织分布 

分 类 号：R927[医药卫生—药学] O657[理学—分析化学]