In Vitro Demonstration of Apoptosis Mediated Photodynamic Activity and NIR Nucleus Imaging through a Novel Porphyrin Suneesh C. Karunakaran, † P. S. Saneesh Babu, ‡ Bollapalli Madhuri, § Betsy Marydasan, † Albish K. Paul, † Asha S. Nair, ‡ K. Sridhar Rao, § Alagar Srinivasan, ∥ Tavarekere K. Chandrashekar,* ,∥ Ch. Mohan Rao,* ,§ Radhakrishna Pillai,* ,‡ and Danaboyina Ramaiah* ,† † Chemical Sciences and Technology Division, Photosciences and Photonics, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Trivandrum-695019, Kerala, India ‡ Rajiv Gandhi Centre for Biotechnology, Trivandrum-695014, Kerala, India § CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad-500 007, Andhra Pradesh, India ∥ National Institute of Science Education and Research (NISER), Bhubaneswar-751005, Odisha, India * S Supporting Information ABSTRACT: We synthesized a novel water-soluble porphyrin THPP and its metalated derivative Zn-THPP having excellent triplet excited state quantum yields and singlet oxygen generation efficiency. When compared to U.S. Food and Drug Administration approved and clinically used sensitizer Photofrin, THPP showed ca. 2−3-fold higher in vitro photodynamic activity in different cell lines under identical conditions. The mechanism of the biological activity of these porphyrin systems has been evaluated through a variety of techniques: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide (MTT) assay, comet assay, poly(ADP-ribose)- polymerase (PARP) cleavage, CM-H 2 DCFDA assay, DNA fragmentation, flow cytometric analysis, fluorescence, and confocal microscopy, which confirm the apoptotic cell death through predominantly reactive oxygen species (ROS). Moreover, THPP showed rapid cellular uptake and are localized in the nucleus of the cells as compared to Hoechst dye and Photofrin, thereby demonstrating its use as an efficient sensitizer in photodynamic therapy and live cell NIR nucleus imaging applications. P hotodynamic therapy (PDT) is an emerging treatment for different types of cancers. 1−4 It involves the inactivation of living cells by the combined action of light, a photoactive molecule called sensitizer and molecular oxygen. 5−7 A variety of sensitizers have been examined for their use in PDT. These include porphyrins, chlorins, bacteriochlorins, phthalocyanines, metallophthalocyanines, porphycenes, squaraines, cyanines, rose bengal, methylene blue, aminolevulinic acid, and their derivatives. 8−11 Among these, porphyrins have attracted much attention as sensitizers due to their higher cellular affinity and very low dark toxicity. 12−14 For example, the hematoporphyrin derivative (HpD) and its commercial variant, i.e., Photofrin, is being currently used in the treatment of various types of cancers. 15−17 However, HpD is a mixture of at least nine components, causes cutaneous photosensitivity and immuno- suppression, and, more importantly, has only a weak absorption in the red region of the spectrum. 18,19 The drawbacks associated with the first generation photosensitizer HpD have provided the great impetus to search for new photosensitizers with greater specificity and efficacy. 20,21 In this context, we designed a novel porphyrin derivative THPP and have investigated its photophysical and in vitro photobiological properties when compared to its zinc complex, Zn-THPP, and U.S. Food and Drug Administration approved and clinically used sensitizer, Photofrin. Interestingly, THPP showed high solubility in buffer and significant quantum yields of triplet excited state and singlet oxygen generation. Uniquely, THPP exhibited ca. 2−3-fold high photocytotoxicity and localized specifically in the nucleus when compared to Photofrin, indicating thereby its potential as an efficient sensitizer for PDT and NIR imaging applications. Synthesis of the new porphyrin derivatives THPP and Zn- THPP was achieved through the modi fied Lindsey’ s method 22,23 (Scheme 1) and were characterized on the basis of elemental analysis and spectral evidence (Supplementary Figures 1 and 2). These derivatives showed high solubility in alcoholic solvents and aqueous medium. The free base THPP showed the characteristic absorption of the porphyrin chromophore having a Soret band at 416 nm and four Q- bands at 514, 548, 592, and 648 nm, while its fluorescence Received: August 31, 2012 Accepted: October 23, 2012 Published: October 23, 2012 Letters pubs.acs.org/acschemicalbiology © 2012 American Chemical Society 127 dx.doi.org/10.1021/cb3004622 | ACS Chem. Biol. 2013, 8, 127−132