Department of Biomedical Engineering , Washington University in St. Louis , One Brookings Drive , St. Louis, Missouri 63130 , United States.
Department of Radiology , Washington University School of Medicine , St. Louis , Missouri 63110 , United States.
Bioconjug Chem. 2019 May 15;30(5):1451-1458. doi: 10.1021/acs.bioconjchem.9b00192. Epub 2019 Apr 30.
Photodynamic therapy (PDT) is often used in preclinical and clinical treatment regimens. Reactive oxygen species (ROS) generated by photosensitizers (PSs) upon exposure to light induce cell death via diverse mechanisms. PSs can exert therapeutic effects in different cellular organelles, although the efficacy of organelle-specific PDT has yet to be determined as most previous studies use different PSs in different organelles. Here, we explored how a single PS, chlorin e6 (Ce6), targeted to different organelles altered the effectiveness of PDT. Ce6 intrinsically localizes to the ER after 4 h of incubation. Modification of Ce6 via conjugation with an octapeptide (LS765), a monosubstituted triphenylphosphonium (TPP) derivative (LS897), or a disubstituted TPP derivative (LS909) altered the intrinsic localization. We determined that LS765 and LS9897 predominantly accumulated in the lysosomes, but LS909 trafficked equally to both the mitochondria and the lysosomes. Moreover, the conjugation altered the type of ROS produced by Ce6, increasing the ratio of hydrogen peroxide to hydroxyl radicals. Irradiation of identical concentrations of the PSs in solution with 650 nm, 0.84 mW/cm light for 10 min showed that the TPP conjugates nearly doubled the hydrogen peroxide production from ∼0.2 μM for Ce6 and LS765 to ∼0.37 μM for LS897 and LS909. In contrast, Ce6 produced ∼1.5-fold higher hydroxyl radicals than its conjugates. To compare the effect of each PS on cell death, we normalized the intracellular concentration of each PS. Hydrogen peroxide-producing PSs are effective PDT agents in the lysosomes while the hydroxyl-generating PSs are very effective in the ER. Compared to the PSs that accumulated in the lysosomes, only the ER-targeted Ce6 exerted >50% cell death at either low light power or low intracellular concentration. By delineating the contributions of cellular organelles and types of ROS produced, our work suggests that targeting hydroxyl radical-producing PSs to the ER is an exciting strategy to improve the therapeutic outcome of PDT.
光动力疗法（PDT）常用于临床前和临床治疗方案中。光敏剂（PSs）在暴露于光下时产生的活性氧（ROS）通过多种机制诱导细胞死亡。PSs 可以在不同的细胞细胞器中发挥治疗作用，尽管细胞器特异性 PDT 的疗效尚未确定，因为大多数先前的研究都在不同的细胞器中使用不同的 PSs。在这里，我们探索了一种单一 PS，即叶绿素 e6（Ce6），靶向不同细胞器如何改变 PDT 的有效性。Ce6 在孵育 4 小时后内在定位于内质网。通过用八肽（LS765）、单取代三苯基膦（TPP）衍生物（LS897）或双取代 TPP 衍生物（LS909）修饰 Ce6，改变了其内在定位。我们确定 LS765 和 LS9897 主要积聚在溶酶体中，但 LS909 同样转运到线粒体和溶酶体。此外，修饰改变了 Ce6 产生的 ROS 类型，增加了过氧化氢与羟基自由基的比值。用 650nm、0.84mW/cm 的光照射相同浓度的 PSs 在溶液中 10 分钟，结果表明 TPP 缀合物使 Ce6 和 LS765 的过氧化氢产量从约 0.2μM 增加到 LS897 和 LS909 的约 0.37μM，几乎增加了一倍。相比之下，Ce6 产生的羟基自由基比其缀合物高约 1.5 倍。为了比较每种 PS 对细胞死亡的影响，我们对每种 PS 的细胞内浓度进行了归一化。产生过氧化氢的 PSs 是溶酶体中有效的 PDT 试剂，而产生羟基的 PSs 在 ER 中非常有效。与积聚在溶酶体中的 PS 相比，只有靶向 ER 的 Ce6 在低光功率或低细胞内浓度下都能发挥>50%的细胞杀伤作用。通过描绘细胞细胞器和产生的 ROS 类型的贡献，我们的工作表明将产生羟基自由基的 PSs 靶向 ER 是提高 PDT 治疗效果的一种令人兴奋的策略。
