具葉酸標的功能之多醣體作為抗癌藥物載體之製備及分析

以葉酸作為標的之藥物載送系統已被使用於各種癌症療法及腫瘤診斷照影上。本研究中，我們利用PEG1000作為spacer經由碳二醯胺脫水縮合劑將葉酸 (Folic acid；FA)接上硫酸化軟骨素 (Chondroitin Sulfate；ChS)。從MALDI-MASS及1H-NMR圖譜證明FA-PEG及FA-PEG-ChS conjugates之化學結構。依據1H-NMR圖譜計算，約有24 mol %之葉酸取代於硫酸化軟骨素上。在體外細胞毒性測試方面，我們將有標的分子之FA-PEG-ChS/Chi、 FA-PEG-ChS/Chi-FITC與沒有標的分子之Naproxen-PEG-ChS/Chi、Naproxen-PEG-ChS/Chi-FITC利用大量表現葉酸受器 (Folate Receptor, FR)之鼻咽癌細胞株 (KB cell line)和不表現葉酸受器肺癌細胞株 (A549 cell line)以MTT assay來進行細胞毒性測試。利用FA-PEG-ChS與帶有螢光之幾丁聚醣 (Chi-FITC)在水相中形成之聚電解質錯合物可進一步從流式細胞儀和共軛聚焦顯微鏡證明FA-PEG-ChS之cell uptake 效果。從螢光光譜儀和共軛聚焦顯微鏡均可證明FA-PEG-ChS/Chi-FITC及Naproxen-PEG-ChS /Chi-FITC對於KB cell之不同傳輸功效。在包覆抗癌藥物方面，經由UV光譜分析可計算出doxorubicin在FA-PEG-ChS/Chi-FITC錯合物中之包覆率。利用流式細胞儀和共軛聚焦顯微鏡皆可證明擁有葉酸片段之錯合物的確可以有效載送抗癌藥物doxorubicin進入鼻咽癌細胞內。

關鍵字

葉酸；硫酸化軟骨素；幾丁聚醣

並列摘要

Folate targeted drug delivery has emerged as an alternative therapy for the treatment and imaging of many cancers. In this study, folic acid was linked to a natural biocompatible polysaccharide, chondroitin sulfate (ChS), using PEG1000 as a spacer through carbodiimide chemistry. MALDI-MASS and 1H-NMR spectrometers were used to confirm the chemical structures of the folic acid-PEG adduct (FA-PEG) and the FA-PEG-ChS conjugate. The molar percent of FA substituent on ChS was determined by 1H-NMR. The FA substitution which calculated by 1H-NMR spectrometer was ~ 24 mol%. The in vitro cytotoxicity of FA-PEG-ChS/Chi and FA-PEG-ChS /Chi-FITC, compared to a conjugate without targeting moiety (Naproxen-PEG-ChS/Chi and Naproxen-PEG- ChS/Chi-FITC), was tested in FR-positive KB cancer cell lines as well as in FR-deficient A549 cancer cell lines by MTT assay (thiazolyl blue tetrazolium bromide). The evidence of cell-uptake of the FA-PEG-ChS conjugate was done by forming a polyelectrolyte complex with chitosan, which is labeled with fluorescein isothiocyanate (Chi-FITC) in an aqueous phase. The folate-mediated efficacy between FA-PEG-ChS/ Chi-FITC (FA-complex) and Naproxen-PEG-ChS/Chi-FITC (Nap- complex) was evidenced by fluorescence plate reader as well as by confocal microscopy. The loading efficiency and encapsulation efficiency of doxorubicin into complexes were calculated by UV spectrometer. The cellular uptake efficiency of the loaded anticancer agent, doxorubicin, to FA-complex in various time periods and doses was investigated using flow cytometry as well as confocal microscopy.