This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lin, C.-W. Articles by Foley, J. W. Search for Related Content PubMed PubMed Citation Articles by Lin, C.-W. Articles by Foley, J. W.

Photosensitization, Uptake, and Retention of Phenoxazine Nile Blue Derivatives in Human Bladder Carcinoma Cells¹

Urology Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 [C-W. L., J. R. S., Y-K. W., C. F. S.], and Rowland Institute for Science, Cambridge, Massachusetts 02142 [L. C., J. W. F.]

The overall goal of our research is to develop effective new photosensitizers for tumor-selective photodynamic therapy. Phenoxazine dyes, including several Nile blue analogues, are known to localize selectively in animal tumors. Structural modifications yielded several series of analogues with substantially higher ¹O₂ yields and different photochemical and physicochemical properties. This study examined the photosensitization potency, cellular uptake, and retention of these derivatives in human bladder carcinoma cells (MGH-U1) in culture. Nile blue derivatives containing halogens and/or sulfur substitutes were selected to exhibit different ¹O₂ yields, pKa values, and hydrophobicities.

The effectiveness of these derivatives in mediating photokilling of tumor cells in vitro corresponded well with the ¹O₂ yields of these compounds, indicating that structural modifications which resulted in increased ¹O₂ yields enhanced potency in mediating photocytotoxicity in vitro. Using derivatives (sat-NBS and sat-NBS-6I) with the highest ¹O₂ quantum yield (0.35 and 0.821), over 90% cell kill was achieved at a sensitizer concentration of 5 x 10^-8 M, about 3 orders of magnitude more effective than hematoporphyrin derivative, the only sensitizer currently available clinically. This result suggests that some of the oxazine derivatives could potentially be effective photosensitizers. The correspondence between ¹O₂ yield and photosensitizing potency, together with results showing enhanced photocytotoxicity in the presence of D₂O and reduced photocytotoxicity under hypoxic conditions, strongly suggests that the generation of ¹O₂ is a major mechanism mediating the photocytotoxic effect.

The uptake of Nile blue derivatives by cells in culture exhibited a pattern of rapid initial uptake followed by a gradual increase in cellular dye contents. The uptake does not correlate directly with the individual pKa values or hydrophobicities of the derivatives, indicating that the structural modifications that increased ¹O₂ yields did not significantly alter the uptake and retention of Nile blue derivatives. The highly concentrative uptake by and slow efflux from dye-loaded cells were consistent with an active mechanism for the cellular accumulation of these dyes. On the other hand, the retention of the compounds was directly proportional to dye concentration in the medium over a 1000-fold range of concentrations, and the uptake could proceed at temperatures below 2°C; these observations excluded endocytosis or a carriermediated mechanism for the uptake. The uptake was also unaffected by the presence of serum in the medium. Based on these results, we hypothesize that Nile blue derivatives transport across the cell membrane possibly as deprotonated forms and, upon entering the cell, either partition into lipophilic areas of the cell membranes and/or become sequestered in certain intracellular organelles.

¹ This work was supported by grants from the National Cancer Institute (CA 32259), the Beinecke Foundation, and the Thomas Anthony Pappas Charitable Foundation and by the Rowland Institute of Science.

² To whom requests for reprints should be addressed.

Received 8/10/90. Accepted 11/30/90.

This article has been cited by other articles:

				T. Maisch, C. Bosl, R.-M. Szeimies, N. Lehn, and C. Abels Photodynamic Effects of Novel XF Porphyrin Derivatives on Prokaryotic and Eukaryotic Cells Antimicrob. Agents Chemother., April 1, 2005; 49(4): 1542 - 1552. [Abstract] [Full Text] [PDF]

				A. Gijsens, L. Missiaen, W. Merlevede, and P. de Witte Epidermal Growth Factor-mediated Targeting of Chlorin e6 Selectively Potentiates Its Photodynamic Activity Cancer Res., April 1, 2000; 60(8): 2197 - 2202. [Abstract] [Full Text]