[CANCER RESEARCH 51, 2710-2719, May 15, 1991] Lysosomal Localization and Mechanism of Uptake of Nile Blue Photosensitizers in Tumor Cells1 Chi-Wei Lin,2 Janine R. Shulok, Sandra D. Kirley, Louis Cincotta, and James W. Foley Urology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 [C-W. L., J. R. S., S. D. KJ, and Rowland Institute for Science, Cambridge, Massachusetts 02142 ¡L,C., J. W. F.] ABSTRACT tosensitizers with high tumor selectivity will enable effective treatment of multiple, infiltratili!*, and invisible tumors, thus Nile blue derivatives have been shown to be potentially effective expanding the utility of PDT as a useful tool in cancer therapy photosensitizers for photodynamic therapy of malignant tumors. Results with intent to cure. Active research is under way to search for of a previous study suggested that the high accumulation of these dyes more tumor-selective sensitizers (4-6) and to improve the sen- in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, sitizer delivery system for better tumor targeting (7-10). results of studies are presented from an investigation of the subcellular Several early studies with animal tumor models have shown localization and mechanism of accumulation of these dyes in cells in that benzophenoxazines, including several Nile blue analogues, vitro. A video-enhanced fluorescence microscopy was used, and a punctate constitute a special class of dyes that are selectively localized in pattern of fluorescence was seen, most of which was localized in the tumors (11-15). Results of recent work have demonstrated that perinuclear region with extracellular dye concentrations between 1 to 100 Nile blue A can be converted to derivatives with substantially UM.These particles resembled characteristic particles identified by stand increased photoactivity (16-18). Furthermore, structural mod ard lysosomal dyes. At higher dye concentrations (1 pM or above), ifications of the parent dye can result in analogues having fluorescence in the perinuclear region was too intense to resolve into substantially altered pKa values and hydrophobicities, proper discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher ties which may be significant in dye localization in tumors. In dye concentrations (10-100 MM).Nile blue derivatives were seen with a a previous study (19, 20), we showed that Nile blue derivatives having high "O^ yields are effective in mediating photocytotox- light microscope as blue particles, the size and location of which resem icity in vitro. Derivatives with "O2 quantum yields of 35-80% bled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) can mediate a 90% in vitro photocytotoxicity with extracellular histochemical staining of dye-loaded cells with the lysosomal marker dye concentrations as low as 5 x 10~8M. This is about 3 orders enzyme acid phosphatase, which showed similar localization of the lower than with hematoporphyrin derivative. The finding thus enzyme-staining and dye-containing particles, (b) phototreatment of dye- suggests that these compounds are potentially effective photo loaded cells which obliterated the majority of the acid phosphatase- sensitizers for PDT. The cellular uptake of Nile blue derivatives stained particles, and (c) treatments with agents affecting the membrane is rapid, highly concentrative, and directly proportional to the pH gradient reduced the uptake and enhanced the efflux of dyes, while extracellular dye concentration. The uptake can proceed at agents that alter cellular membrane potentials had no effect on dye temperatures below 2°C,thus excluding endocytosis or a car accumulation. The uptake of the dyes was partially inhibited by inhibitors rier-mediated mechanism for the uptake. The overall results of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results show suggest that high cellular accumulation of these dyes may result ing that the cellular uptake of these dyes is highly concentrative and from dye aggregation, partition in membrane lipids, and/or proportional to the extracellular dye concentration over a wide range, are sequestration in certain intracellular organelles (20). consistent with the hypothesis that the dyes are mainly localized in the In the present study, the intracellular localization of Nile lysosomes via an ion-trapping mechanism. Results of the present study blue derivatives and the mechanism of their accumulation in also suggest that the lysosomes may be an intracellular target for pho human bladder carcinoma cells were examined. Findings from todynamic killing of tumor cells mediated by Nile blue photosensitizers this study suggest that the lysosome is the main site of localiza and that lysosomotropic photosensitization may be a strategy for effective tion and ion trapping is likely the process by which Nile blue and selective destruction of tumor cells. dyes are accumulated in cells. INTRODUCTION MATERIALS AND METHODS PDT' is an investigational treatment procedure for malignant Nile Blue Derivatives. Previous reports (16-18) indicated that struc tumors (1-3). The effectiveness of the treatment relies, to a tural modifications of Nile blue A yielded derivatives with enhanced great extent, on the tumor selectivity of the photosensitizer photoactivity as well as different photochemical properties. The six Nile blue derivatives used in this study and their designations are shown which, upon photoactivation, imparts a photodynamic action in Table 1. The photochemical properties of these derivatives have been for cytotoxicity and tumor destruction. The availability of pho- described previously (16-20). All six derivatives were examined in subcellular localization studies using fluorescence and light microscopy. Reccived 12/19/90; accepted 3/6/91. The costs of publication of this article were defrayed in part by the payment In studies involving uptake and sequestering mechanisms, derivatives of page charges. This article must therefore be hereby marked advertisement in NBA and NBA-6I were used to represent derivatives with different pK. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. values and hydrophobicities. In studies involving photodynamic treat 1This work was supported by grants from the National Cancer Institute (CA ment of the cell, derivatives with moderate photoactivity, NBA-6I and 32259), the Beinecke Foundation, the Thomas Anthony Pappas Charitable Foun NBS-6I, were used. dation, and the Rowland Institute for Science. 2To whom requests for reprints should be addressed, at Urology Research Tumor Cells. The cell line used for this study, MGH-U1, is a Laboratory, Massachusetts General Hospital, Boston, MA 02114. subculture of I 24. a well established human bladder carcinoma cell 3The abbreviations used are: PDT, photodynamic therapy; PBS. phosphate- line (21). The cells were grown routinely in McCoy's 5A medium buffered saline at pH 7.4: DPBS. Dulbecco's phosphate-buffered saline at pH 7.4; 'Oi. singlet oxygen; SIT, silicon-intensified-target; TPP, tetraphenyl-phos- supplemented with 5% fetal calf serum. phonium: FCCP.p-trifluoromethoxyphenyl hydrazone; 2,4-DNP, 2,4-dinitrophe- Microscopic Observations of Nile Blue Derivatives in Cells. Both nol. The designations of the Nile blue derivatives are listed in Table 1. fluorescence and light microscopies were used to examine the subcel- 2710 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1991 American Association for Cancer Research. LYSOSOMAL LOCALIZATION OF NILE BLUE PHOTOSENS1TIZERS Table 1 Structures and designation of Nile blue derivatives used in this study examining dye localizations in cells (22), subconfluent cells grown on with their absorption maxima, pK,, and partition coefficients" coverslips were incubated for 30 min at 37°Cwith 10 ml of dye Designation Structure , (nm) pK* P/ solutions; dye concentrations ranging from 10 MMto 0.1 nM were used. At the end of the incubation, the cells were removed from the dye NBA (C2H5)2N 623 10.0 173 solution, washed with DPBS, mounted on Lab-Tek chambers, and observed under the fluorescence microscope with the aid of a Hama- matsu C2400 SIT camera connected to a high resolution color monitor and a Sony UP-5000 video printer. At dye concentrations >10 MM,Nile blue dyes in ceils can be directly NBA-6I (C2H5)2N 642 6.6 5625 seen under the light microscope. This was performed by incubating subconfluent cells, grown on glass slides, in 100 mm culture dishes with 10 ml of various dye solutions at 20 MM,at 37°Cfor 10-30 min to permit dye uptake. Cells were rinsed to remove residual dye solution, mounted with dye-free medium, and observed immediately under the NBS 645 10.0 356 microscope. In experiments designed to examine the intracellular trans- location of the dyes under conditions of short uptake times and rela tively high dye concentration, cells grown on 22-mm coverslips were incubated with 100 /il of 10-100 MMdye solutions at 37°Cfor 5 min. Cells were rinsed twice to remove residual dye, placed in dye-free NBS-6I 660 6.5 5027 medium, and returned to the incubator for specified times before microscopic observation. Acid Phosphatase Staining. To verify that the blue stained