Phthalocyanine−Peptide Conjugates: Receptor-Targeting Bifunctional Agents for Imaging and Photodynamic Therapy Elena Ranyuk, † Nicole Cauchon, † Klaus Klarskov, ‡ Brigitte Gue ́ rin,* ,† and Johan E. van Lier* ,† † Department of Nuclear Medicine and Radiobiology and ‡ Department of Pharmacology Faculty of Medicine and Health Sciences, Universite ́ de Sherbrooke, Sherbrooke, Qué bec, Canada * S Supporting Information ABSTRACT: The synthesis of a series of new zinc phthalocyanine−peptide conjugates targeting the gastrin- releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine−glycine−aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor- mediated cell uptake. In the case of the phthalocyanine−bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor. ■ INTRODUCTION Multifunctional agents for simultaneous imaging and targeted therapy of cancer, known as theranostics, have become of increasing interest over the past decade. Various noninvasive imaging techniques can be used in tandem with therapeutic modalities for image-guided therapy, and particularly the combination of photodynamic therapy (PDT) and fluorescence imaging has gained growing attention over the last decades. 1,2 PDT combines a photosensitizer (PS) and light of an appropriate wavelength to impart cytotoxicity via the generation of reactive molecular species. 3 The light-induced electronic excitation of a PS can result not only in a cytotoxic effect but also in the emission of fluorescence due to relaxation of the excited-singlet-state PS back to the ground state. 4 The phenomenon that the same entity (PS) can act both as therapeutic and imaging agent due to ability of the PS to fluoresce is a unique advantage of PDT. The PS’s fluorescence can be used in diagnostics, therapy guidance, monitoring, treatment assessment, and mechanistic studies. 1 Phthalocyanines (Pc) are of particular interest as PS for PDT due to their suitable physical and chemical properties. They can be synthesized in a straightforward manner and modified to alter hydrophilicity, absorption, and emission wavelengths for different applications. 5,6 Pc exhibit high photo- and chemical stability, which is desirable for chemical modifications as well as in vitro and in vivo applications. They show long-wavelength absorption with high extinction coefficients (ε > 10 5 M −1 cm −1 ), red fluorescence emission approaching NIR wavelengths for deeper tissue imaging, and high singlet oxygen quantum yields. 7 Recent evidence that Raman imaging of Pc distribution can identify malignant areas of a tumor may further contribute to improve Pc-PDT effectiveness. 8 Sulfonated Pc (PcS) are anionic water-soluble phthalocyanines that are effective PS to kill tumor cells in vitro 9 and cause tumor regression in vivo. 10 Because of the presence of negatively charged sulfonate groups, PcS show higher water-solubility and reduced aggregation as compared to nonsubstituted Pc. Combined these properties make PcS attractive PS for PDT as well as molecular probes for fluorescence imaging. The successful outcome of PDT depends to a large extent on the tissue and intracellular localization of the PS. Nonspecific localization often leads to suboptimal treatment outcome and toxicity to healthy tissues. Therefore, the development of PS with improved specificity, selectivity, and efficacy is highly desirable for the successful PDT of tumors while preserving healthy adjacent tissues. To achieve efficient and reliable delivery of chemotherapeutics and diagnostics to cancer cells, a number of delivery agents have been investigated in recent years. 11 Among them, tumor cell-targeting peptides have emerged as the most valuable nonimmunogenic tools. In contrast to larger molecules such as monoclonal antibodies (mAbs), short synthetic peptides have excellent tumor penetration properties, which in combination with their selective binding and rapid internalization, make them ideal carriers of therapeutics to both primary and metastatic tumor sites. Unlike viral delivery vectors and mAbs, peptides are nearly invisible to the immune system and are expected to Received: September 11, 2012 Published: January 28, 2013 Article pubs.acs.org/jmc Published 2013 by the American Chemical Society 1520 dx.doi.org/10.1021/jm301311c | J. Med. Chem. 2013, 56, 1520−1534