Department of Pharmaceutics, Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, College of Medicine, Jiaxing University, Jiaxing, Zhejiang, PR China; CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal; Department of Medical Biology, Cancer Center Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands.
Department of Medical Biology, Cancer Center Amsterdam, Amsterdam UMC, Amsterdam, The Netherlands; Laboratory of Experimental Oncology and Radiobiology (LEXOR), Cancer Center Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.
J Photochem Photobiol B. 2021 Mar;216:112146. doi: 10.1016/j.jphotobiol.2021.112146. Epub 2021 Jan 29.
Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design.
ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC values were established for each PS at 671 nm and a radiant exposure of 15 J/cm following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed.
In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-μM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 μM and ZnPCS4 at 2.5 μM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC values of 0.13 μM, 0.04 μM, and 0.81 μM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G/G phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT.
Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 μM, i.e., > 37 times the LC value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.
肿瘤光动力疗法（PDT）依赖于光敏剂（PS）来光氧化破坏肿瘤细胞。目前批准的 PS 在治疗浅表和易于接近的肿瘤方面效果令人满意，但在胆管系统和胰腺等内部器官的实体癌方面效果较差。对于这些恶性肿瘤，第二代 PS，如金属化酞菁，更为合适。目前尚不清楚哪种常用的金属化酞菁，即铝酞菁（AlPC）和锌酞菁（ZnPC）以及它们的四磺化衍生物 AlPCS4 和 ZnPCS4，对肿瘤细胞的细胞毒性最大。本研究因此采用损耗性方法，在头对头比较分析和标准化实验设计中确定最适合肿瘤 PDT 的金属化酞菁。
将 ZnPC 和 AlPC 包封在聚乙二醇化脂质体中。在培养的 A431 细胞中进行分析，作为具有功能失调的 P53 肿瘤抑制基因和 EGFR 过表达的肿瘤细胞的模板。首先，使用 WST-1 和磺酰罗丹明 B 测定法评估 PS 浓度作为暗毒性的函数。其次，通过流式细胞术和共聚焦显微镜分别测定细胞内摄取和分布，分别使用 PS 的本征荧光。第三，在 1 小时 PS 暴露后，在 671nm 和 15J/cm2 的辐射暴露下，确定每个 PS 的 LC 值。最后，分析 PDT 后不同时间的细胞死亡方式和 PDT 后 24 小时的细胞周期阻滞。
在没有光照的情况下，AlPC 和 ZnPC 在高达 1.5μM PS 浓度和长达 72 小时的暴露下对细胞没有毒性。在 5μM 的 AlPCS4 和 2.5μM 的 ZnPCS4 中观察到暗毒性。PS 添加到细胞后 1 分钟即可观察到所有 PS 的摄取，并在 2 小时孵育期间增加幅度。60 分钟后，细胞的整个非核空间都被光敏化，PS 积累在多个亚细胞结构中，特别是在 AlPC 和 AlPCS4 的情况下。用 ZnPC、AlPC 和 AlPCS4 敏化的细胞进行 PDT 后 24 小时，LC 值分别为 0.13μM、0.04μM 和 0.81μM，基于磺酰罗丹明 B 测定法。ZnPCS4 没有引起明显的光毒性，这与细胞死亡和细胞周期阻滞数据一致。在 PDT 后 4 小时，细胞死亡方式主要为 ZnPC 和 AlPC 的凋亡，在 AlPC 敏化的细胞中，这种程度在 8 小时期间逐渐加剧。与 4 小时相比，ZnPC 处理的细胞在 PDT 后 8 小时似乎有所恢复，这在另一种细胞系中之前已经观察到。AlPCS4 除了诱导凋亡外还诱导了相当多的坏死，其中大部分细胞死亡在 PDT 后 2 小时已经显现。在 8 小时的过程中，坏死性细胞死亡逐渐转变为主要的晚期凋亡性细胞死亡。细胞死亡信号与 G/G 期（ZnPC、AlPC、AlPCS4）和 S 期（ZnPC、AlPC、AlPCS4）和 G 期（ZnPC 和 AlPC）的细胞周期阻滞一致。AlPC 和 AlPCS4 敏化的细胞中细胞周期阻滞最为明显。
脂质体 AlPC 是最有效的肿瘤 PDT PS，而 ZnPCS4 在 A431 细胞中光动力学惰性。在高达 1.5μM 的 PS 浓度下，即 LC 值的 37 倍以上，AlPC 没有诱导暗毒性，这在临床光毒性问题方面是有利的。AlPC 敏化了多个细胞内位置，这与广泛的、不可逆的细胞死亡信号有关，这有望有益于治疗效果，并可能在体内给予足够的 AlPC 后获得免疫长期肿瘤控制。鉴于不同的药代动力学、细胞内分布和细胞死亡动力学，脂质体 AlPC 可以与 AlPCS4 结合在 PS 鸡尾酒中，以进一步提高 PDT 效果。
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