DOI: 10.1002/cmdc.201300501 Towards Matched Pairs of Porphyrin–Re I / 99m Tc I Conjugates that Combine Photodynamic Activity with Fluorescence and Radio Imaging Teresa Gianferrara, [a] Cinzia Spagnul, [a] Roger Alberto,* [b] Gilles Gasser, [b] Stefano Ferrari, [c] Vanessa Pierroz, [b, c] Alberta Bergamo, [d] and Enzo Alessio* [a] Introduction The development of new multifunctional agents that combine different diagnostic imaging modalities in a single molecule [1, 2] or both diagnostic and therapeutic functionalities (theranostic agents), [3] is one of the major research goals in medicinal inor- ganic chemistry. As each imaging technique has its own dis- tinctive features (particularly in terms of resolution and sensi- tivity), a clinical or biological problem is best investigated with a multi-modality imaging probe. Affording exact co-localization greatly simplifies and improves image interpretation. Different strategies are being developed for the combined diagnosis and treatment of several diseases, with an emphasis on cancer. [4] In this respect, photodynamic therapy (PDT), a clinical- ly approved medical technique involving visible light-induced generation of cytotoxic singlet oxygen ( 1 O 2 ) from endogenous 3 O 2 , mediated by a photosensitizer (PS), [5–7] has great potential for combination with other modalities. [8, 9] Porphyrins—either natural or synthetic—are particularly at- tractive intrinsic bimodal photosensitizers, as they can be used for both PDT activity and fluorescence imaging (and therefore, image-guided phototherapy) by changing the irradiation con- ditions. [5, 6] Porphyrins have distinctive features that make them unique for the development of imaging and therapy probes: 1) they are very chemically robust molecules with intense elec- tronic absorptions in the visible region and relatively long fluo- rescence decay time with large Stokes shifts; 2) their chemistry is well developed, both for synthesis and functionalization, making them extremely versatile chemical scaffolds; 3) their optical and redox properties can be fine-tuned by appropriate peripheral modification and/or core metallation; and 4) they have tumor-localizing properties, as they typically show prefer- ential uptake and retention by tumor tissues. [5, 7] Indeed, most of the clinical applications of PDT already involve porphyrins and related chromophores (e.g. chlorins). [7] Fluorescence imag- ing, exploiting the strong emission of porphyrins, has excellent resolution (down to the nanometer scale). This allows for local- ization of the compounds on the cellular or subcellular level, and can provide real-time imaging. However, it has quite limit- ed depth penetration (millimeters for visible light, centimeters for near infrared), and is thus unsuitable for whole body imag- ing. Implementing an additional imaging modality in the same molecule, capable of observing structures deep inside tissue, enables us to overcome this limitation. In this context, porphyrin–metal conjugates offer exciting perspectives. The concept of attaching chelators for metal co- ordination to the periphery, rather than into the porphyrin core, gives the possibility of site-specific and highly stable la- beling without interference of the tetrapyrrolic system. [10, 11] We recently prepared two novel water soluble porphyrins bearing a single peripheral chelator, either diethylenetriamine (1) or bipyridyl (2), tethered to one meso position. The prepara- tion of their conjugates with a fac-{ 99m Tc(CO) 3 } + fragment and the potential of these resulting conjugates as fluorescence and radio imaging tools were also described. In this work, we fo- cused on the corresponding non-radioactive analogues that bear the fac-{Re(CO) 3 } + fragment (diethylenetriamine 3 and bi- pyridyl 4). We report on the uptake, in vitro PDT activity, and cellular localization of Re I conjugates 3 and 4 in comparison to the parent porphyrins 1 and 2. Compounds 1–4 have modest or negligible cytotoxicity in the dark against HeLa human cer- vical cancer cells but become remarkably cytotoxic after expo- sure to moderate doses of red visible light (590–700 nm). This phototoxicity was found to be directly proportional to the total light dose. Although the four compounds show distinct uptake patterns, they have comparable PDT activity. Confocal fluorescence measurements showed that porphyrin 1 and its Re I conjugate 3 have different cellular localization patterns in HeLa cells. [a] Dr. T. Gianferrara, Dr. C. Spagnul, Prof. Dr. E. Alessio Department of Chemical & Pharmaceutical Sciences, University of Trieste Via L. Giorgieri 1, 34127 Trieste (Italy) E-mail : alessi@units.it [b] Prof. Dr. R. Alberto, Prof. Dr. G. Gasser, V. Pierroz Department of Chemistry, University of Zurich Winterthurerstr. 190, 8057 Zurich (Switzerland) E-mail : ariel@chem.uzh.ch [c] S. Ferrari, V. Pierroz Institute of Molecular Cancer Research, University of Zurich Winterthurerstr. 190, 8057 Zurich (Switzerland) [d] Dr. A. Bergamo Callerio Foundation Onlus Via A. Fleming 22-31, 34127 Trieste (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201300501.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2014, 9, 1231 – 1237 1231 CHEMMEDCHEM FULL PAPERS