利用可形成鳥糞嘌呤四複合體之核酸適體作為載體進行標靶藥物傳輸之研究

Abstract

鳥糞嘌呤四複合體是一種特化的富有鳥糞嘌呤的DNA結構，它已經被廣泛的研究關於它的物理特性以及在生物上面的影響。這裡我們使用一種全新的方式將鳥糞嘌呤四複合體當作一個藥物載體去標靶癌細胞並且作光動力療法。我們由會形成鳥糞嘌呤四複合體的AS1411核酸適體去攜帶六個TMPyP4(tetra-(N-methyl-4-pyridyl)-porphine，一種紫質衍生物)，可以用來合成apt-TMP複合物。藉由紫外光-可見光光譜儀、螢光光譜儀以及圓二色光譜儀分析此複合物可以發現TMPyP4由卡在核酸適體中間以及外面的形式緊密的接在核酸適體上。因為AS1411的鳥糞嘌呤四複合體結構會標靶有核仁素過量表現的癌細胞，所以在此研究我們評估鳥糞嘌呤四複合體攜帶TMPyP4到癌細胞是否為核仁素攝取的影響。由流式細胞儀以及螢光顯微鏡的結果顯示比起M10正常乳房表皮細胞，apt-TMP複合物能夠使TMPyP更大的累積在MCF7乳癌細胞。在加入過量的核酸適體或是抗核仁素的單株抗體可以有效的抑制TMPyP4被MCF7細胞的攝取。而在給予光刺激180秒之後，apt-TMP複合物對於MCF7細胞的光傷害也比對M10大。這些結果顯示TMPyP4的傳輸是經由apt-TMP複合物和細胞表面的核仁素專一性的交互作用，並且使用會形成鳥糞嘌呤四複合體的AS1411核酸適體當作藥物載體對於癌症治療可以是一個極有潛力的策略。

A specialized G-rich DNA structure, G-quadruplex, has been studied for its special physical characters and biological effect. Here we report a novel strategy of using G-quadruplex as a drug carrier to target cancer cells for photodynamic therapy (PDT). A G-quadruplex forming AS1411 aptamer was physical conjugated with six of porphyrin derivative, tetra-(N-methyl-4-pyridyl)-porphine (TMPyP4), to fabricate the apt-TMP complex. The complex was identified that TMPyP4 molecules was bound tightly to aptamer by intercalation and outside binding by the UV-Vis spectrophotometry, fluorescence spectrophotometer and circular dichroism (CD) spectroscopy. Because the G-quadruplex structure of AS1411 is known to target to nucleolin over-expressed in cancer cells, in this study, the effect of G-quadruplex structure as a carrier for delivery of TMPyP4 into cancer cells by nucleolin-mediated internalization was investigated. Results from flow cytometry and fluorescent microscopy showed that the apt-TMP complex exhibited higher TMPyP4 accumulation in MCF7 breast cancer cells than in M10 normal epithelium cells. The effective inhibition of TMPyP4 uptake from MCF7 cells was further observed by adding an excess of free aptamer or monoclonal anti-nucleolin antibody. After treated with light for 180 sec, the photodamage in MCF7 cells was larger than in M10 cells. These results indicate that TMPyP4 delivery and uptake were mediated by the specific interaction of apt-TMP complex with nucleolin on the cellular surface and the use of G-quadruplex forming AS1411 aptamer as a drug carrier may be a potential tactic for cancer therapy.