Photochemistry and Photobiology, 2014, 90: 801–813 Membrane Damage Efficiency of Phenothiazinium Photosensitizers Isabel O. L. Bacellar 1 , Christiane Pavani 1 , Elisa M. Sales 2,3 , Rosangela Itri 2 , Mark Wainwright 4 and Mauricio S. Baptista* 1 1 Departamento de Bioqu  ımica, Instituto de Qu  ımica, Universidade de S~ ao Paulo, S~ ao Paulo, Brasil 2 Departamento de F  ısica Aplicada, Instituto de F  ısica, Universidade de S~ ao Paulo, S~ ao Paulo, Brasil 3 Instituto de Pesquisas Tecnol  ogicas do Estado de S~ ao Paulo, S~ ao Paulo, Brasil 4 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK Received 26 September 2013, accepted 21 February 2014, DOI: 10.1111/php.12264 ABSTRACT Structure–activity relationships have been widely reported for porphyrin and phthalocyanine photosensitizers, but not for phenothiazinium derivatives. Here, four phenothiazinium salts (methylene blue, toluidine blue O, 1,9-dimethyl methy- lene blue and the pentacyclic derivative DO15) were used to investigate how the ability to damage membranes is affected by membrane/solution partition, photophysical properties and tendency to aggregation of the photosensitizer. These two latter aspects were studied both in isotropic solutions and in membranes. Membrane damage was assessed by leak- age of a fluorescent probe entrapped in liposomes and by generation of thiobarbituric acid-reactive species (TBARS), while structural changes at the lipid bilayer were detected by small-angle X-ray scattering. We observed that all com- pounds had similar singlet-oxygen quantum yields in ethanol, but only the photosensitizers that had higher membrane/solu- tion partition (1,9-dimethyl methylene blue and DO15, the latter having the higher value) could permeabilize the lipid bilayer. Moreover, of these two photosensitizers, only DO15 altered membrane structure, a result that was attributed to its destabilization of higher order aggregates, generation of higher amounts of singlet oxygen within the membranes and effective electron-transfer reaction within its dimers. We con- cluded that membrane-based protocols can provide a better insight on the photodynamic efficiency of the photosensitizer. INTRODUCTION Photosensitization is the basis of photodynamic therapy (PDT), a clinical modality available for a variety of cancers and currently under considerable investigation for its application to treat micro- bial infections (1–5). One of the key elements in PDT is the pho- tosensitizer (PS). Absorption of light causes excitation and the production of several reactive species, and subsequent damage to biomolecules and cell death. The excited state of the PS (usually the triplet excited state) can generate the reactive species either by type I or type II pathways, the former comprising electron or hydrogen transfer to or from a substrate and the latter involving energy transfer to molecular oxygen and the generation of singlet oxygen ( 1 O 2 ) (6). These basic action mechanisms seem to occur to different extents in all different classes of PS in use, such as phthalocyanine, porphyrin and phenothiazinium PS (7–10). The search for more efficient PS is commonly performed by improving the efficiency of generation of light-induced reactive species, which is done by maximizing two main characteristics of the PS: absorption in the therapeutic window and quantum yield of 1 O 2 generation (Φ D ), which is considered to be the main species responsible for causing cell death (5,11). Nevertheless, many studies in mammalian cell culture have highlighted that this strategy is not always the best way to proceed, showing the importance of subcellular localization to photodynamic damage (12,13). Crystal violet, for example, localizes in mitochondria without being reduced. Under irradiation, this compound killed HeLa cells more efficiently than methylene blue (MB), a classi- cal 1 O 2 generator (14). Certainly, for prokaryotic cells and viruses the role of PS localization is more restricted, given their simpler internal compartmentalization. Despite PDT being a multitarget strategy and relying on photodamage to several biomolecules and cellular structures (cytoplasmic membrane, organelles, cytoskeleton, etc.), the role of membrane binding of a PS is critical to define the extent of photoinduced membrane damage and consequently the efficiency of cell death (7,8,15–18). This fact is well recognized for por- phyrin and phthalocyanines PS. However, clear structure–activity relationships are still missing for several PS classes such as the phenothiazinium salts (19). Phenothiazinium cations are composed of an oxidized ring system chromophore and attached auxochromic side groups, which contribute significantly to the polarity of the ion. Increased mammalian cell phototoxicity of this class of PS has been observed with more hydrophobic compounds. This enhanced activity was attributed mainly to an increase in Φ D , resistance to reduction to the photodynamically inactive leuco form and higher cell uptake. Among the studied dyes, 1,9-dimethyl methylene blue (DMMB) and DO15 have superior photodynamic activity in many different biological systems (tumor cells, bacteria, virus and fungi) when compared to commercially available PS such as MB and toluidine blue O (TBO). Moreover, these more hydro- phobic compounds usually exhibit larger light/dark cytotoxicity ratio (20–32). The aim of the current work is to clarify parameters that affect the ability of phenothiazinium ions to damage membranes, start- ing from the efficiency of membrane binding and progressing to analyze the properties of the ground and excited states of the PS *Corresponding author email: baptista@iq.usp.br (Mauricio S. Baptista) © 2014 The American Society of Photobiology 801