Photochemistry and Photobiology, 2014, 90: 171–182 Complexes of Chlorin e6 with Pluronics and Polyvinylpyrrolidone: Structure and Photodynamic Activity in Cell Culture Timur M. Zhiyentayev †1 , Umed T. Boltaev ‡1 , Anna B. Solov’eva 2 , Nadezhda A. Aksenova 2 , Nickolay N. Glagolev 2 , Alexander V. Chernjak 3 and Nickolay S. Melik-Nubarov* 1 1 Chemistry Department, M.V. Lomonosov Moscow State University, Moscow, Russia 2 N.N. Semenov Institute of Chemical Physics RAS, Moscow, Russia 3 Institute of Problems of Chemical Physics RAS, prosp. Academika Semenova 1, Chernogolovka, Russia Received 13 July 2013, accepted 24 September 2013, DOI: 10.1111/php.12181 ABSTRACT Polymeric carriers are extensively used in photodynamic therapy (PDT) for increase of efficacy of photosensitizers. Here, we report the influence of nine Pluronic copolymers on phototoxicity of chlorin e6 (Ce6), in particular 5- to 7-fold rise in the phototoxicity caused by hydrophilic Pluronics F127, F108, F68 and F87 and practically no influence on Ce6 of more hydrophobic polymers. The revealed value of 0.2 mg mL 1 of Pluronic F127 concentration sufficient for half-of-maximal increase of Ce6 photodynamic activity proved to be close to 0.16 mg mL 1 inherent in well- documented carrier poly(N-vinylpyrrolidone) (PVP). The dis- sociation constants of Ce6 complexes with Pluronic F127 and PVP that were estimated from UV spectra were 0.252 and 0.036 mg mL 1 , respectively, indicating higher stability of Ce6 complex with PVP. According to the results of 1 H-NMR studies of Ce6 complexes, the porphyrin interacts not only with hydrophobic regions but also with hydrophilic sides of both polymers. INTRODUCTION Photodynamic therapy (PDT) is quickly emerging treatment modality employing the photochemical interaction of three com- ponents, i.e. light, photosensitizer and oxygen. Tremendous pro- gress has been made in the last two decades in new technical development of all components as well as understanding of bio- physical mechanism of PDT (1,2). This technique permanently entered clinical practice for the treatment of superficial tumors and exuding wounds (1,3). In spite of undoubted success of PDT in the treatment of light accessible tumors, yet several limitations of PDT still remain (4). One of them is long-lasting skin photosensitivity caused by some commonly used photosensitizers. In most cases, these side effects are accounted for high affinity of the photosensitizer to cell membranes and thus binding to normal tissues. Either covalent or noncovalent attachment of a photosensitizer to polymeric scaffolds has been proposed as a possible way to overcome this limitation (5). Polymeric carriers were proposed to reduce unspecific binding to normal tissues and hence to increase bioavailability of the photosensitizer and efficacy of PDT. For example, biodistribution of highly hydrophobic silicon-containing phthalocyanine Pc 4 (6) and pheophorbide (7) in tumor-bearing animals was markedly improved upon solubilization in polyethylene glycol-block- polycaprolactone (PEG-PCL) micelles. Similar effect was reported for Zn-phthalocyanine loaded in polylactide-co-glycolide (PLGA) biodegradable nanoparticles (8). Significant reduction in skin sensitivity to photodynamic treatment was achieved for the formulation of meta-tetra(hydroxyphenyl)chlorin in poly(2-ethyl- 2-oxazoline)-block-poly-(D,L-lactide) copolymer micelles (9). Solubilization of a hydrophobic fluorescent tumor marker hypericin in the micelles of block copolymer of methoxy-poly (ethylene glycol) and hexyl-substituted polylactide (mPEG-hex- PLA) resulted in 17-fold increase in tumor localization as com- pared with free hypericin (10). Susceptibility of polycaprolactone hydrophobic block to enzymatic hydrolysis makes such copoly- mers useful for construction of switchable particles capable to release the photosensitizer meta-tetra(hydroxyphenyl)chlorin in presence of lipases (11). Not only amphiphilic micelle-forming polymers but also some hydrophilic homopolymers were found to interact with porphy- rins and increase their contrast for malignant tissues in vivo and phototoxicity both in vitro and in vivo. Extensive studies were focused on water-soluble and biocompatible polymer poly-(N-vi- nylpyrrolidone) (PVP) which forms stable complexes with chlo- rin e6 characterized by effective constant about 10 4 M 1 and approximately 50–60 repeat units that involved in the complex formation (12). This polymer increased contrast of PDT with Ce6 (13). The complexes exhibited substantially lower hydropho- bicity estimated by partitioning in octanol–water system under slightly alkaline conditions and favored Ce6 binding to very low-density lipoproteins (VLDL). Both these factors could be the reasons of the pronounced selectivity of PVP–Ce6 complex as compared with free Ce6 (14). Formation of PVP complexes with hypericin increased the selectivity due to its accumulation in tumors that was used for PDT (15) and diagnostics (16). Biochemical basis for the polymer-induced increase in por- phyrins photodynamic activity has been studied in a number of *Corresponding author email: melik.nubarov@genebee.msu.ru (Nickolay S. Melik- Nubarov) † Current address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA ‡ Current address: Department of Chemistry, Columbia University, New York, NY © 2013 The American Society of Photobiology 171