German Edition: DOI: 10.1002/ange.201507800 Photodynamic Therapy Very Important Paper International Edition: DOI: 10.1002/anie.201507800 Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome- Localized Photosensitizers for Two-Photon Photodynamic Therapy Huaiyi Huang, Bole Yu, Pingyu Zhang, Juanjuan Huang, Yu Chen, Gilles Gasser,* Liangnian Ji, and Hui Chao* Abstract: Photodynamic therapy (PDT) is a noninvasive medical technique that has received increasing attention over the last years and been applied for the treatment of certain types of cancer. However, the currently clinically used PDT agents have several limitations, such as low water solubility, poor photostability, and limited selectivity towards cancer cells, aside from having very low two-photon cross-sections around 800 nm, which limits their potential use in TP-PDT. To tackle these drawbacks, three highly positively charged ruthenium(II) polypyridyl complexes were synthesized. These complexes selectively localize in the lysosomes, an ideal localization for PDT purposes. One of these complexes showed an impressive phototoxicity index upon irradiation at 800 nm in 3D HeLa multicellular tumor spheroids and thus holds great promise for applications in two-photon photo- dynamic therapy. Photodynamic therapy (PDT) has expanded the range of medical techniques available to treat certain types of cancer, such as lung, bladder, and ophthalmologic cancer, and urinary tumors. [1] PDT treatments rely on the use of a combination of a photosensitizer (PS) and light. Ideally, a PS should be non- toxic in the absence of light but have a high toxicity for cancer cells upon light exposure. [2] Specifically, the excited state of the PS reacts with the ground state of molecular oxygen ( 3 O 2 ) to generate reactive oxygen species and notably singlet oxygen ( 1 O 2 ). In PDT, 1 O 2 is considered as the primary toxic species that can induce cellular organelle or vasculature damage. [3] However, currently available PDT agents are plagued by a number of issues, including a generally poor water solubility and slow clearance from the body, weak photostability, and the necessity for irradiation with a high- energy one-photon (OP) laser beam. [4] To tackle these drawbacks, two-photon PDT (TP-PDT) agents have emerged over recent years as attractive alternatives to the currently approved PSs. [5] Contrary to OP-PDT, TP-PDT uses low- energy near-infrared laser irradiation, which is less damaging to normal cells and allows for reduced photobleaching of the PSs and deeper tissue penetration. [6] In view of TP-PDT applications, viable PSs must have a high efficiency for two-photon absorption (TPA), a property that is quantified by the two-photon cross-section (s 2 ). [7] Readily available PSs (e.g., porphyrin) generally have low s 2 values (< 20 GM, where 1 GM = 10 À50 cm 4 s molecule À1 ). Although these compounds are preferentially taken up by tumors, their two-photon cross-sections are insufficient for adequate clinical efficacy in TP-PDT. [7] Among the different PSs that have been tested for TP-PDT and more generally for PDT, ruthenium(II) polypyridyl complexes were found to be excellent candidates [8] owing to their attractive photophysical properties (i.e., high water solubility, large s 2 , high 3 O 2 production, long luminescence lifetime, and excellent chem- ical and photostability). However, a number of other pertinent factors must be considered for the development of practically useful TP-PDT agents. These include cell uptake and localization, a high phototoxicity index (PI) characterized by a low dark toxicity and high light toxicity, and the biodistribution of the PS. For instance, in cells, the PSs can be localized in the mitochondria, the plasma-membrane endoplasmic reticulum, the lysosomes, the Golgi apparatus, or the nucleus. [9] Owing to the influence of the mitochondrial membrane potential, the mitochondrion is a well-known target for lipophilic, charged metal complexes, such as polypyridyl Ru II complexes, that enter cells mostly by passive diffusion. [10] However, as the mitochondria function as cellular energy houses, it has been pointed out that mitochondrial localization of a PS could result in high dark cytotoxicity. [11] Furthermore, nucleus localization is generally considered to be unfavorable because of the potential for DNA mutation. [12] Therefore, PSs that target cellular organelles other than the mitochondria or the nucleus are in high demand. With all these requirements in mind, we envisaged preparing highly charged Ru II polypyridyl complexes that do not localize either in the mitochondria or in the nucleus. Herein, we present the synthesis, characterization, and in-depth biological investigation of three highly positively charged homoleptic Ru II complexes (Figure 1) as lysosome- targeting TP-PDT agents. This study demonstrates that such complexes have a great potential in TP-PDT. To the best of our knowledge, this is the first report of a Ru II complex suitable as a lysosome-targeting TP-PDT agent. In particular, the [Ru(bpy) 3 ] 2+ core structure was chosen as octupolar tris(bipyridyl) Ru II complexes have been shown to exhibit excellent TPA properties. [13] In this work, tertiary ammonium groups were added to the core structure to allow [*] H. Huang, [+] Dr. B. Yu, [+] Dr. P. Zhang, J. Huang, Dr. Y. Chen, Prof. L. Ji, Prof. Dr. H. Chao MOE Key Laboratory of Bioinorganic and Synthetic Chemistry School of Chemistry and Chemical Engineering Sun Yat-Sen University Guangzhou, 510275 (China) E-mail: ceschh@mail.sysu.edu.cn H. Huang, [+] Prof. Dr. G. Gasser Department of Chemistry, University of Zurich Winterthurerstrasse 190, CH-8057, Zurich (Switzerland) E-mail: gilles.gasser@chem.uzh.ch [ + ] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201507800. Angewandte Chemie 14049 Angew. Chem. Int. Ed. 2015, 54, 14049 –14052 # 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim