在細胞和亞細胞水平測定膠束包膜紫杉醇的提取方案和液相色譜/串聯質譜法:應用于細胞攝取和分布研究

2018年1月，北京大學腫瘤醫院研究所癌變與轉化研究教育部重點實驗室國家藥物臨床試驗中心；北京大學腫瘤醫院腎癌及黑色素瘤科癌變與轉化研究教育部重點實驗室；中日友好醫院藥劑科；北京大學腫瘤醫院腫瘤轉化研究教育部重點實驗室(Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), National Drug Clinical Trial Center, Peking University Cancer Hospital &Institute, Beijing 100142, China；Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Renal Cancer and Melanoma, Peking University Cancer Hospital & Institute, Beijing 100142, China；Department of Pharmacy, China-Japan Friendship Hospital, Beijing 100029, China；Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Gastrointestinal Surgery, Peking University Cancer Hospital& Institute, Beijing 100142, China) Jiafu Ji老師研究團隊在《Journal of Chromatography B》上發表論文：

“Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study”

“在細胞和亞細胞水平測定膠束包膜紫杉醇的提取方案和液相色譜/串聯質譜法:應用于細胞攝取和分布研究”

Abstract：

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7).

摘要：

紫杉醇負載聚合物膠束(PTX-PM)是臨床上常用的腫瘤靶向納米載體，具有突出的抗腫瘤特性，但其在體外的積累和分布尚缺乏研究。這可能是由于復雜的膠束系統及其在細胞或亞細胞水平上的低濃度。在這項研究中，科研人員開發了一種改進的提取方法，即機械破壞和液液萃取(LLE)相結合，從細胞裂解液和亞細胞區室的膠束中提取總PTX。以多西紫杉醇為內標，優化了超高效液相色譜-串聯質譜(UPLC-MS /MS)同時檢測細胞和亞細胞水平低濃度PTX的方法。結果表明，該方法可完全從膠束中釋放PTX(≥95.93%)，提取回收率≥75.04%。在0.2 ~ 20 ng/mL濃度范圍內線性良好。準確度的相對誤差(RE%)在0.68 ~ 7.56%之間，精密度內和精密度間的相對標準偏差(RSD%)分別小于8.64%和13.14%。該方法得到了充分驗證，并成功應用于人乳腺癌細胞(MCF-7)中ptx負載PLGA-PEG膠束的細胞攝取和分布研究。

該論文中，MCF-7人乳腺癌細胞的體外培養是使用Ausbian特級胎牛血清完成的。欲了解或購買Ausbian特級胎牛血清可以聯系北京締一生物400-166-8600.