488  2002 American Society for Photobiology 0031-8655/01 $5.00+0.00 Photochemistry and Photobiology, 2002, 75(5): 488–494 The Effect of pH and Surfactant on the Aggregation Behavior of Chlorin p 6 : A Fluorescence Spectroscopic Study ¶ Anindya Datta*, Alok Dube, Beena Jain, Arjun Tiwari and Pradeep Kumar Gupta Biomedical Applications Section, Centre for Advanced Technology, Indore, India Received 23 October 2001; accepted 6 February 2002 ABSTRACT Steady state and time-resolved fluorescence properties of chlorin p 6 , a potential drug for photodynamic therapy, have been investigated as functions of pH. A decrease in pH of the medium has been shown to cause protonation of the ionizable carboxylic acid side chain, leading to an increase in hydrophobicity and consequent aggregation. The aggregates dissociate on further protonation. The dissociation is explained in terms of formation of cations and their mutual repulsion. A synchronous fluorescence spectroscopic study revealed the presence of two anionic forms in equilibrium at physiological pH, with a shift in the equilibrium on slight decrease in the pH. The anionic nature of chlorin p 6 in aqueous solutions at physiological pH has been confirmed by complexation with surfactants. The nature of the charge on the headgroups of the sur- factants has been found to govern the formation of chlor- in–surfactant complexes. INTRODUCTION Photodynamic therapy (PDT) for treatment of cancer has been a growing field of interest over the last 25 years (1–3). The technique is based on selective localization of photo- sensitizers in tumor cells and subsequent destruction of the tumor cells by photoexcitation of the photosensitizers (2). The desirable features of a photosensitizer for use in PDT are strong absorption in the so-called therapeutic window of 650–900 nm, where penetration in the tissues is the maxi- mum (4), tumor selectivity and high quantum efficiency for singlet oxygen generation. The last is affected by its state of aggregation and primary photophysics. The intracellular dis- tribution of the drug is mainly determined by the charge on it (5). The increase in lipophilicity, induced by protonation, has been reported to result in a higher cellular uptake for ¶Posted on the web site on 22 February 2002. *To whom correspondence should be addressed at: Biomedical Ap- plications Section, Laser R&D Block D, Centre for Advanced Technology, Indore 452 013, India. Fax: 91-731-488430; e-mail: anindya@cat.ernet.in Abbreviations: AR, analytical reagent; CMC, critical micellar con- centration; CTAB, cetyl trimethyl ammonium bromide; HPLC, high-performance liquid chromatography; PBS, phosphate buff- ered saline; PDT, photodynamic therapy; SDS, sodium dodecyl sulfate; TLC, thin-layer chromatography. several photosensitizers (6,7). The interplay of photophysical properties, structure and intracellular distribution has been examined in several recent studies (7–10). Chlorin derivatives are attractive photosensitizers for PDT not only because they have strong absorption in the thera- peutic window but also because of the fact that the singlet oxygen yield has been shown to be considerably high for this class of compounds (11). This has led to the synthesis and study of various chlorin derivatives by several groups (7,12–16). Chlorin p 6 and purpurin 18 (Fig. 1) are two such closely related compounds. On hydrolysis in alcoholic NaOH, the anhydride ring in purpurin 18 opens to form chlorin p 6 , which has the two carboxylic acid groups directly bound to the aromatic skeleton (13). In phosphate buffered saline (PBS), at physiological pH, purpurin 18 exists as ag- gregates whereas chlorin p 6 exists as monomers. This dif- ference in hydrophilicity is ascribed to the presence of polar, ionizable –COOH groups attached directly to the ring system of chlorin p 6 and the lack of such groups in purpurin 18. It is important to understand the nature of the species present in aqueous solutions of these compounds at physiological pH, because it governs hydrophilicity, lipophilicity, aggre- gation and cellular uptake of the drugs (7,17). With this mo- tive, we have studied the effect of variation of pH on the absorption and fluorescence properties of chlorin p 6 , using steady state and time-resolved fluorescence spectroscopy. The presence of more than one anionic species has been observed around physiological pH. The ionic nature of chlor- in p 6 has been explored further by studying its interaction with surfactants. MATERIALS AND METHODS Synthesis of photosensitizers and preparation of solutions. Purpurin 18 was prepared from dry spinach leaves following the procedure of Hoober et al. (13). Chlorin p 6 was prepared by alkaline hydrolysis of purpurin 18 (13), which was purified by silica column and pre- parative thin-layer chromatography (TLC) (R f = 0.56) before hy- drolysis. The purity of chlorin p 6 was tested by TLC, high-perfor- mance liquid chromatography (HPLC) and superimposability of the absorption and fluorescence excitation spectra. Approximately 20 mM solutions of chlorin p 6 were prepared in UV spectroscopic grade methanol. Aliquots of 50 L of these solutions were added to 10 mL of PBS solutions of different pH. HCl and NaOH used to vary the pH were from E-Merck, Mumbai, India. The ionic strength of these solutions was maintained at 0.2 mM by addition of analytical reagent (AR) grade NaCl from E-Merck. The AR grade surfactants cetyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS), obtained from SD Fine Chemicals, Mumbai, India,