Department of Experimental Surgery Academic Medical Center, University of Amsterdam Meibergdreef 9 1105 AZ Amsterdam, The Netherlands.
Curr Med Chem. 2014;21(3):377-91. doi: 10.2174/09298673113209990211.
Photodynamic therapy (PDT) has been successfully used to treat various solid tumors. However, some cancer types respond poorly to PDT, including urothelial carcinomas, nasopharyngeal carcinomas, and extrahepatic cholangiocarcinomas. The therapeutic recalcitrance is in part due to the use of photosensitizers with suboptimal optical/ photochemical properties and unfavorable pharmacokinetics.
To circumvent these drawbacks, a second-generation photosensitizer with improved optical/photochemical properties, zinc phthalocyanine (ZnPC), was encapsulated in interstitially targeted, polyethylene glycol-coated liposomes (ITLs) intended for systemic administration. The ZnPC-ITLs were examined for reactive oxygen species (ROS) generation and oxidation capacity and validated for tumoricidal efficacy in human extrahepatic cholangiocarcinoma (Sk-Cha1) cells. ZnPC-ITL uptake and the mechanism and mode of PDT-induced cell death were studied.
The ITL formulation was optimized on the basis of fluorescence spectroscopy and photon correlation spectroscopy. The extent of ROS generation, protein oxidation, and membrane oxidation were determined by the 2',7'-dichlorodihydrofluorescein, tryptophan oxidation, and calcein leakage assays, respectively. PDT efficacy was evaluated by measuring mitochondrial activity and apoptosis-/necrosis-specific staining in combination with flow cytometry. The uptake of fluorescently labeled ITLs was assayed by confocal microscopy, flow cytometry, and fluorescence spectroscopy.
ZnPC-ITLs exhibited maximum ROS-generating and oxidation potential at a ZnPC:lipid molar ratio of 0.003. PDT of Sk-Cha1 cells incubated with ZnPC-ITLs induced cell death in a lipid concentration- dependent manner. The mode of PDT-induced cell death comprised both apoptosis and necrosis, with necrotic cell death predominating. Post-PDT cell death was attributable to pre-PDT ZnPC-ITL uptake by cancer cells, which was more efficient at smaller ITL diameters and a more positive surface charge.
ZnPC-ITLs constitute a nanoparticulate photosensitizer delivery system capable of inducing apoptosis and necrosis in cultured extrahepatic cholangiocarcinoma cells by PDT-mediated oxidative processes. PDT-induced cell death is dependent on the extent of ITL uptake, which in turn relies on ITL size and zeta potential.
光动力疗法(PDT)已成功用于治疗各种实体瘤。然而,一些癌症类型对 PDT 的反应较差,包括膀胱癌、鼻咽癌和肝外胆管癌。治疗的顽固性部分归因于使用光学/光化学性质不佳和药代动力学不利的光敏剂。
为了规避这些缺点,一种具有改进的光学/光化学性质的第二代光敏剂,锌酞菁(ZnPC),被包裹在间隙靶向、聚乙二醇包被的脂质体(ITL)中,用于系统给药。研究了 ZnPC-ITL 的活性氧(ROS)生成和氧化能力,并在人肝外胆管癌细胞(Sk-Cha1)中验证了其杀肿瘤功效。研究了 ZnPC-ITL 的摄取以及 PDT 诱导细胞死亡的机制和模式。
根据荧光光谱和光子相关光谱对 ITL 配方进行了优化。通过 2',7'-二氯二氢荧光素、色氨酸氧化和钙黄绿素渗漏测定分别确定 ROS 生成、蛋白质氧化和膜氧化的程度。通过测量线粒体活性和凋亡/坏死特异性染色并结合流式细胞术评估 PDT 疗效。通过共聚焦显微镜、流式细胞术和荧光光谱法测定荧光标记的 ITL 的摄取。
ZnPC-ITL 在 ZnPC:脂质摩尔比为 0.003 时表现出最大的 ROS 生成和氧化潜力。用 ZnPC-ITL 孵育的 Sk-Cha1 细胞的 PDT 以脂质浓度依赖的方式诱导细胞死亡。PDT 诱导的细胞死亡模式包括凋亡和坏死,其中坏死细胞死亡为主。PDT 后细胞死亡归因于癌症细胞对预 PDT ZnPC-ITL 的摄取,这在 ITL 直径较小和更正表面电荷时更为有效。
ZnPC-ITL 构成纳米颗粒光敏剂递送系统,能够通过 PDT 介导的氧化过程诱导培养的肝外胆管癌细胞凋亡和坏死。PDT 诱导的细胞死亡依赖于 ITL 摄取的程度,而 ITL 摄取的程度又取决于 ITL 的大小和 ζ 电位。