Soriano Jorge, García-Díaz María, Mora Margarita, Sagristá Maria Lluïsa, Nonell Santi, Villanueva Angeles, Stockert Juan Carlos, Cañete Magdalena
Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain.
Biochim Biophys Acta. 2013 Oct;1830(10):4611-20. doi: 10.1016/j.bbagen.2013.05.021. Epub 2013 May 28.
The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.
Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.
m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.
Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.
These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.
光动力疗法（PDT）后激活的细胞死亡途径受多种参数控制，包括光敏剂的化学结构、其亚细胞定位以及诱导的光动力损伤。本研究旨在表征一种合适的间-四羟基苯基卟啉（m-THPPo）脂质体制剂，确定其在HeLa细胞中的亚细胞定位，并建立光动力治疗后激活的细胞死亡机制。
由二棕榈酰磷脂酰胆碱（DPPC）和二肉豆蔻酰磷脂酰甘油（DMPG）以9:1的摩尔比混合制备含m-THPPo的脂质体。为获得最佳包封效率，考虑了m-THPPo/脂质摩尔比。将HeLa细胞与脂质体m-THPPo孵育，并研究m-THPPo的亚细胞定位。光动力治疗后进行了几种检测，如TUNEL、膜联蛋白V/碘化丙啶和Hoechst-33258染色。通过细胞色素c和半胱天冬酶-2免疫荧光评估凋亡起始。
与溶解在四氢呋喃中的m-THPPo相比，包封在脂质体中的m-THPPo的荧光和单线态氧量子产率降低。脂质体m-THPPo与溶酶体示踪剂（LysoTracker®）共定位，并通过凋亡机制诱导HeLa细胞光失活。在凋亡细胞中未检测到细胞色素c的重新定位，但在光敏治疗后立即半胱天冬酶-2呈阳性。
脂质体m-THPPo光动力治疗导致HeLa细胞出现显著比例的凋亡形态。半胱天冬酶-2的激活且无细胞色素c的重新定位表明是一种不依赖线粒体的凋亡机制。
这些结果有助于更好地理解脂质体m-THPPo光动力治疗后诱导的细胞死亡机制。
