[CANCER RESEARCH 50. 4533-4538, August 1, 1990] Effect of Sulfonation on the Cell and Tissue Distribution of the Photosensitizer Aluminum Phthalocyanine Wai-Shun Chan,1 John F. Marshall, Rüssel Svensen, Joanne Bedwell, and Ian R. Hart Biology of Metastasis Laboratory; Imperial Cancer Research Fund, Lincoln's Inn Fields, London, WC2A 3PX [W-S. C., J. F. M., I. R. HJ; Royal Institution of Great Britain, 21 Albermarie Street, London, WIX 4BS [R. S.J; and The Rayne Institute, Faculty of Clinical Sciences, University College London, London, United Kingdom [J. BJ ABSTRACT Aluminum sulfonated phthalocyanine has potential as a suitable pho- tosensitizer for use in the photodynamic therapy of cancer. In the present study, cellular uptake and retention of the individual mono-, di-, tri-, and tetrasulfonated derivatives ( \IS, jIY) were examined in tissue culture and in normal and neoplastic tissue of tumor-bearing mice. Uptake and retention of the various derivatives by cells in tissue culture correlated inversely with the degree of sulfonation. Accordingly, Colo 26 cells in monola j er culture, 24 h after addition of 10 ;JMof appropriate photosen- sitizer, had accumulated approximately 25-fold more VIS,IV than \IS ,1V- and retained this species longer than more sulfonated derivatives. In contrast to these in vitro results, it was found that Colo 26 growing s.c. in BALB/c mice accumulated photosensitizer to a greater extent when the degree of sulfonation increased, such that AlS«Pc > AlS3Pc > AlS2Pc > AlS,Pc. By 24-48 h after the i.v. injection of 0.1 ml 2.27 mM solution of individual photosensitizer, the relative ratios of tumoradjacent tissue varied from >10:1 to <2:1, showing that selective tumor uptake may be affected profoundly by the composition of the phthalocyanine compound. The livers and spleens of both normal and tumor-bearing mice, unlike other normal tissue, took up the sulfonated derivatives in an order that provided a mirror image of that observed in neoplastic tissue. These complex in vivo distribution and retention characteristics appear to be a consequence of relative hydrophilicity/hydrophobicity properties of the sulfonated species and indicate the extent to which these characteristics may influence photosensitizer distribution and accumulation. INTRODUCTION Metallophthalocyanines are porphyrin-like, photoactivatable dyes with strong red light absorption characteristics, which have been considered as second generation photosensitizers for the PDT2 of cancer (for review, see Refs. 1-4). Because most metallo-Pc are poorly soluble in water, they are not suitable for clinical administration. Problems of insolubility can be over come by the use of specific carrier vehicles, such as entrapment of monomeric form Pc in unilamellar vesicles (5), or by the creation of water-soluble derivatives. This latter change can be achieved by the addition of substituents like amino, carboxylic acid, and sulfonate groups to the outer ring of the isoindole units. To date, the sulfonated derivatives have been the most commonly used, since their synthesis is relatively easy, although subsequent purification may be difficult. One such sulfonated derivative, AlSPc, that we have been investigating is capable of photoinactivating cells in tissue culture (6), exhibits good tu mor-localizing capacity (7, 8), and can be used to achieve marked reduction in tumor burden of various murine tumors of different histológica! origins (7). This dye, therefore, seems to have considerable potential as a photosensitizer for use in PDT. Received 12/18/89; revised 4/6/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed. 2 The abbreviations used are: PDT. photodynamic therapy; AIS.Pc, chloroal- uminum sulfonated phthalocyanine with n sulfonate groups: AlSPc. a mixed sulfonate preparation of chloroaluminum sulfonated phthalocyanine; ex/em, ex citation/emission; HPLC, high pressure liquid chromatography; PBS, phosphate- buffered saline; Pc, phthalocyanine(s). However, the AlSPc used in our studies, and in studies from other groups which also have demonstrated the possible utility of this agent (9-11), consisted of a mixture of mono-, di-, tri-, and tetrasulfonated derivatives. It would seem to be important to know which derivative(s) is (are) responsible for the observed biological activities. Some of the in vitro characteristics of the individual species have been investigated (12-14), although to date there is little information on the in vivo behavior of these various agents (15) and nothing on their distribution patterns. In this paper, we report not only the uptake of individual derivatives of AlSPc by cells in vitro but also their in vivo uptake and retention by normal and neoplastic tissue in tumor-bearing mice. MATERIALS AND METHODS Photosensitizer Preparation. AlSPc was obtained from Ciba-Geigy Dyestuffs and Chemicals (Basel, Switzerland). This material was a complex mixture of mono- to tetrasulfonated derivatives; according to the supplier, it had an average of three sulfonate groups. Individual chloroaluminum mono-, di-, and trisulfonated derivatives (AlSiPc, AlS2Pc, and AlS3Pc, respectively) were purchased from Porphyrin Products (Logan, UT). Chloroaluminum tetrasulfonated Pc (AlS4Pc) was prepared by condensation of aluminum trichloride with four equiv alents of sulfophthalic acid (monosodium salt) using the modified technique of Weber and Busch (16). Stock solutions of all of the dyes were prepared at 2-4 mg/ml in PBS, except the water-insoluble AlSiPc, which was dissolved to the same concentration in either 40% (v/v) ethanohPBS (for in vivo studies) or in dimethylformamide (for in vitro studies). Solutions were sonicated for 3-5 min, heated at 100°C for 5- 10 min, and then stored at 4°Cin darkness. Stock solutions were measured spectrophotometrically (diode array spectrophotometer, model 8452A; Hewlett-Packard, Palo Alto, CA) after 10 ^1 of stock solution had been diluted in 9.990 ml 0.1 M NaOH [in the case of AIS iPc, the diluent was the same volume of a 3:1 (v/v) mixture of absolute ethanol:0.1 M NaOH]. Molarity of the AlS2Pc solution was calculated from absorbance at 666 nm and a molar extinction coefficient (17): E = 1.5x 105M-'cm-' In order to determine the molarity of the solutions of the other derivatives, the area under the curve obtained by measuring absorbance between 500 and 750 nm was compared to that obtained with AlS2Pc. HPLC Analysis. Analysis of the individual Pc species was by re verse-phase HPLC essentially as described elsewhere (18). Briefly, the various sulfonated Pc were dissolved in 10 mM NaOH, buffered with 10 mM phosphate to pH 5, filtered (Waters HV 0.45 microfilters SJHVD43N3), and then injected onto a reverse-phase column (Waters NOVA-PAK C-18 radial compressed unit). Samples were resolved by a gradient mode elution (0-80% methanol in 10 mM phosphate buffer) over an 18-min run period. Eluted Pc were detected by absorption measurements at 365 nm. Tumor Cells. Colo 26 cells from a murine colorectal carcinoma, syngeneic to BALB/c mice, were passaged routinely in E4 growth medium containing 10% fetal calf serum exactly as detailed previously (7). Animals. Specific pathogen-free BALB/c mice were obtained from the Imperial Cancer Research Fund breeding unit. For individual 4533 on July 29, 2015. © 1990 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from