亞甲基藍-金奈米複合物於光動力治療之應用

Abstract

光動力治療主要是經由讓目標細胞將光敏劑攝入後，以特定波長之雷射光激發產生自由基來達到殺死目標細胞之效果。光動力治療通常會遭遇到兩個問題:光敏劑的暗毒性以及光敏劑在目標細胞與正常體細胞之間的低選擇性。本研究中選擇海拉細胞(Hela cell)來做為光動力治療之目標細胞，光敏劑則選用亞甲基藍。為降低亞甲基藍之暗毒性，本研究中將亞甲基藍與經由水熱反應合成之奈米金粒子藉由亞甲基藍與奈米金粒子表面poly styrene-alt-maleic acid (PSMA) 高分子層之分子間作用力來做結合以獲得一具有光敏劑性質的奈米複合物。 本研究中中利用UV-visible光譜儀、電子穿隧式顯微鏡、X光散射儀與界面電位分析儀來分析亞甲基藍-金奈米複合物之物理及光學性質，此外還應用9, 10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA)來檢測此複合物在波長660奈米雷射激發下產生自由基之能力。結果顯示亞甲基藍-金奈米複合物可以成功被合成而且亞甲基藍的光學性質與產生自由基之能力在與奈米金粒子結合後依然被保留。本研究接著選用海拉細胞(Hela cell)來做複合物之毒性測試，在與奈米金粒子接合後，亞甲基藍的暗毒性有效地被降低，並且在功率100 mW波長660奈米雷射照射4分鐘之下可以保有與純亞甲基藍相近之光動力治療效率。另外，為了提升複合物的癌細胞標靶能力，本研究中將運鐵蛋白藉由EDC/NHS反應與複合物接合以提升複合物在目標細胞(海拉細胞)與非癌正常細胞(3T3纖維母細胞)之間的選擇性，之後使用原子吸收光譜儀來做細胞內複合物胞吞量之定量。此外，本研究中還使用2′, 7′-Dichlorofluorescin diacetate (DCFH-DA)與螢光顯微鏡來偵測細胞內部之自由基產生。結果顯示運鐵蛋白可以有效提升癌細胞標靶能力，並且在細胞實驗中證實可以顯著提升光動力治療效果。最後，藉由Annexin V-FITC/PI螢光染色分析，我們發現大多數細胞是死於細胞凋亡。 本研究成功合成一能搭載光敏劑且同時具備高生物相容性和癌細胞標靶能力之多功能奈米複合物。我們希望能應用此複合物在施行癌症之光動力治療上，以期能有效減少治療之副作用，並且能夠以更少劑量去達到更大的治療效果。

Photodynamic therapy (PDT) mainly involves cellular uptake of photosensitizer (PS) and excitation of light at specific wavelength to induce generation of reactive oxygen species (ROS) inside the targeted cells. This approach usually suffers from two main deficiencies: dark toxicity of PS and poor selectivity of cellular uptake between targeted cells and normal tissues. In this work, a known effective PS, methylene blue (MB) which can be excited by 660 nm red light source was chosen. Hela cells were used as targeted cells for PDT. To address obstacles in PDT, the MB conjugated Au nanocomposites were prepared via a hydrothermal synthesis method following by an intermolecular interaction between a poly styrene-alt-maleic acid (PSMA) layer on the Au nanoparticles (AuNPs) and MB. The structure and optical property of MB-AuNP conjugate were characterized by UV-visible spectrometer, transmission electron microscopy (TEM), X-ray diffraction pattern, and zeta-potential analysis. Furthermore, generation of ROS from MB-AuNP conjugate after excitation of 100 mW hand-held laser at 660 nm wavelength was detected by using 9, 10-Anthracenediyl-bis(methylene) dimalonic acid (ABDA). Results indicated that the MB-AuNP conjugate was successfully prepared by wet-chemistry reaction since the optical property of MB was remained after conjugation. Then toxic effect of MB-AuNP conjugate on Hela cell was examined. With a single 4 min hand-held 100 mW laser treatment, cell works proved that AuNPs as goodness carrier could effectively reduce the cell toxicity of MB and still remain the PDT efficiency. Moreover, transferrin (Tf) was grafted on the particles via EDC/NHS reaction and followed by MB attachment as Tf-MB-AuNP conjugate to enhance the selectivity of cellular uptake between cancer cells (Hela cell) and non-malignant cells (3T3 cell, fibroblast). Atomic absorption spectroscopy (AA) was used for cellular uptake quantification. In vitro ROS generation was also monitored by 2′, 7′-Dichlorofluorescin diacetate (DCFH-DA). It was shown that Tf could effectively promote cancer cell targeting. In addition, Tf-MB-AuNP was proved that could enhance PDT efficiency significantly in cell works. Finally, cell death pathway was found to be mainly apoptosis by Annexin V-FITC/PI staining. We proposed that by applying this biocompatible and cancer cell targeting Tf-MB-AuNP conjugate for PDT treatment could simultaneously reduce dark toxicity of MB and enhance the photodynamic therapy efficiency. This multi-functional AuNP could provide a less harmful alternative for PDT in cancer treatments.