Journal of Photochemistry and Photobiology, 13."B/ology, 6 (1990) 87-92
87
S I T E S OF P H O T O S E N S I T I Z A T I O N BY P R O T O P O R P H Y R I N A N D TIN P R O T O P O R P H Y R I N IN L E U K E M I A L 1 2 1 0 CELLS* DAVID KESSEL and VERONIQUE SCHULZ
Departments of Pharmacology and Medicine, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.) (Received October 5, 1989; accepted December 7, 1989)
Keywords. Fluorescence, porphyrins, photosensitization.
Summary Studies with protoporphyrin (PP) and tin protoporphyrin (SnPP) were carried out to assess the effects of tin insertion on the sites of dye localization. Fluorescence emission spectra and studies on the sites of photodamage were consistent with a concentration of PP at membrane loci. In contrast, SnPP photodamage involved an intracellular site.
1. Introduction Porphyrins are useful agents for the selective photosensitization of neoplastic tissues i n vivo [1-3]. Although protoporphyrin (PP) is a poor tumor-localizing agent i n vivo [4], it is a potent sensitizer in cell culture and in cell-free systems [5, 6]. The metalloporphyrin analog tin protoporphyrin (SnPP) is an equally effective photosensitizer [7], and has other interesting biological properties [8]. In this study, we examined sites of localization of SnPP and PP, using murine leukemia L1210 cells i n vitro. Fluorescence emission spectra were used to characterize the binding loci. The sites of photodamage were also assessed by studying the light-catalyzed loss of viability of tumor cells, together with the loss of membrane transport and the capacity for incorporation of labeled thymidine into DNA. An independent measure of cell-surface photodamage was provided by two-phase partitioning of whole cells.
*Paper presented at the Congress on Photodynamic Therapy of Tumours, Sofia, Bulgaria, October, 1989.
1011-1344/90/$3.50
© Elsevier Sequoia/Printed in The Netherlands
88 2. M a t e r i a l s
and methods
PP and SnPP were purchased from Porphyrin Products (Logan, UT). High performance liquid chromatography (HPLC) indicated 95% purity. Leukemia L1210 cells were grown in Fischer's medium (GIBCO, Grand Island, NY) supplemented with 10% horse serum, 1 ~M mercaptoethanol and antibiotics (gentamycin). Dextran T-500 was obtained from Pharmacia (Piscataway, NJ). Poly(ethyleneglycol) was provided by Pierce Chemical Co. (Rockford, IL) (mean molecular weight 6000). The poly(ethyleneglycol) palmitate (8% esterified) was prepared as described by Shanbhag and Johansson [9]. Dye hydrophobicity was determined by measuring the distribution between 2-octanol and aqueous buffer (10 mM, pH 7). Samples of each phase were diluted with 80% ethanol and the dye concentration was measured by fluorescence. Fluorescence emission spectra were acquired using an SLM 48000 instrument (excitation and emission slit widths, 2 and 1 nm respectively). Drugs were dissolved in specified solvents (1 lzM concentration) for the determination of these spectra, using 400 nm excitation. For biological tests, suspensions of L1210 cells (7 mg m1-1 wet weight) were treated with PP or SnPP (30 min, 37 °C). Fluorescence emission spectra of intracellular dyes were assessed using suspensions of cells (108 m1-1) in 130 mM NaCl and 10 mM HEPES (pH 7.2). Total dye uptake was determined by fluorescence assay, using cell pellets dispersed in 10 mM cetyl trimethylammonium bromide (CTAB). To determine the effects of photodamage, cells were incubated with specified levels of dyes for 30 min at 37 °C, collected by centrifugation, washed once and suspended in fresh growth medium. The cell suspensions were then irradiated using an Oriel model 66170 housing containing a 100 W QH lamp. A 10 cm layer of distilled water removed IR radiation. An interference filter confined the spectrum to 550__+ 10 nm. The light dose rate in the region of dye absorbance (shown in Fig. 1, see Section 3) was adjusted to 1200 mJ cm -2, delivered over a total time of 10 min. During irradiation, cell cultures were maintained at 4 °C to minimize temperature-sensitive repair of photodamage. An aliquot of irradiated cells was suspended in fresh growth medium at 37 °C, and viability was assessed by direct cell counts over 3 days after irradiation [10]. Incorporation of radioactive thymidine and cycloleucine (CL) provided information on photodamage to DNA synthesis and membrane transport [10]. Alterations in cell-surface hydrophobicity were determined by examining the behavior of cells in the two-phase system described previously [11, 12]. Cell suspensions (0.1 ml, 3 × 1 0 8 cells) were added to a 5 ml mixture containing 5% dextran (molecular weight, 500 000) and 4% poly(ethyleneglycol) (molecular weight, 6000) in 130 mM NaC1 and 20 mM phosphate buffer (pH 7) with 0.2 tzg ml -~ poly(ethyleneglycol) palmitate. An initial sample of 0.5 ml was withdrawn and the cell number was determined
89
using the Coulter ZF particle counter. The phases were allowed to separate for 5 min and anot her 0.5 ml was withdrawn from the center of the top phase. The partition coefficient is e x p r e s s e d as the percentage of total cells which remain in the u p p e r phase after a 5 min phase separation.
3. R e s u l t s The absorbance spectra of PP and SnPP (10 /zM, ethanol) are shown in Fig. 1. PP has a b r o a d e r Soret band, suggesting some aggregation in this solvent; SnPP has the characteristic two-band spect rum between 500 and 600 nm. We utilized the absorbance band at approximately 550 nm for photobiological studies with these dyes. The fluorescence emission spectra of PP in several solvents are shown in Fig. 2. The slight shoulder at 620 nm represents the presence of 5% hematoporphyrin. The fluorescence emission optimum from PP-loaded cells is 635 nm, suggesting an environmental dielectric constant of 4. The corresponding value from dye dissolved in tetrahydrofuran (D = 4) is 635 nm (Fig. 2). The fluorescence spectra of SnPP in various solvents are shown in Fig. 3. Dye-loaded cells show a fluorescence emission optimum at 583 nm, which suggests an environmental dielectric constant of approximately 35. The emission optimum in methanol ( D - - 3 2 ) occurs at 583.5 nm. Data relating to the physical and biological properties of these dyes are summarized in Table 1. PP is m uch more hydrophobic (from o c t a n o l - w a t e r partitioning) than SnPP, and is more effectively accumulated by L1210 cells. The extracellular level of PP required, on irradiation, to kill 50% of cells (IDso level) is substantially lower than that of SnPP, but the corresponding intracellular dye levels are similar. The concentration of PP which inhibits transport of CL by 50% is almost identical to the IDso level. Photo-inhibition of DNA synthesis requires a higher dye concentration. In the case of SnPP, inhibition of DNA synthesis is bet t er
151 /ii
0 o 1.0
~
.o 0 . 5
/ : i, ,
,,
So
~0
Fig. 1. Absorbance spectra of PP (full line) and SnPP (broken line). A complete absorbance profile of PP is shown, together with a magnification (4)
87
S I T E S OF P H O T O S E N S I T I Z A T I O N BY P R O T O P O R P H Y R I N A N D TIN P R O T O P O R P H Y R I N IN L E U K E M I A L 1 2 1 0 CELLS* DAVID KESSEL and VERONIQUE SCHULZ
Departments of Pharmacology and Medicine, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.) (Received October 5, 1989; accepted December 7, 1989)
Keywords. Fluorescence, porphyrins, photosensitization.
Summary Studies with protoporphyrin (PP) and tin protoporphyrin (SnPP) were carried out to assess the effects of tin insertion on the sites of dye localization. Fluorescence emission spectra and studies on the sites of photodamage were consistent with a concentration of PP at membrane loci. In contrast, SnPP photodamage involved an intracellular site.
1. Introduction Porphyrins are useful agents for the selective photosensitization of neoplastic tissues i n vivo [1-3]. Although protoporphyrin (PP) is a poor tumor-localizing agent i n vivo [4], it is a potent sensitizer in cell culture and in cell-free systems [5, 6]. The metalloporphyrin analog tin protoporphyrin (SnPP) is an equally effective photosensitizer [7], and has other interesting biological properties [8]. In this study, we examined sites of localization of SnPP and PP, using murine leukemia L1210 cells i n vitro. Fluorescence emission spectra were used to characterize the binding loci. The sites of photodamage were also assessed by studying the light-catalyzed loss of viability of tumor cells, together with the loss of membrane transport and the capacity for incorporation of labeled thymidine into DNA. An independent measure of cell-surface photodamage was provided by two-phase partitioning of whole cells.
*Paper presented at the Congress on Photodynamic Therapy of Tumours, Sofia, Bulgaria, October, 1989.
1011-1344/90/$3.50
© Elsevier Sequoia/Printed in The Netherlands
88 2. M a t e r i a l s
and methods
PP and SnPP were purchased from Porphyrin Products (Logan, UT). High performance liquid chromatography (HPLC) indicated 95% purity. Leukemia L1210 cells were grown in Fischer's medium (GIBCO, Grand Island, NY) supplemented with 10% horse serum, 1 ~M mercaptoethanol and antibiotics (gentamycin). Dextran T-500 was obtained from Pharmacia (Piscataway, NJ). Poly(ethyleneglycol) was provided by Pierce Chemical Co. (Rockford, IL) (mean molecular weight 6000). The poly(ethyleneglycol) palmitate (8% esterified) was prepared as described by Shanbhag and Johansson [9]. Dye hydrophobicity was determined by measuring the distribution between 2-octanol and aqueous buffer (10 mM, pH 7). Samples of each phase were diluted with 80% ethanol and the dye concentration was measured by fluorescence. Fluorescence emission spectra were acquired using an SLM 48000 instrument (excitation and emission slit widths, 2 and 1 nm respectively). Drugs were dissolved in specified solvents (1 lzM concentration) for the determination of these spectra, using 400 nm excitation. For biological tests, suspensions of L1210 cells (7 mg m1-1 wet weight) were treated with PP or SnPP (30 min, 37 °C). Fluorescence emission spectra of intracellular dyes were assessed using suspensions of cells (108 m1-1) in 130 mM NaCl and 10 mM HEPES (pH 7.2). Total dye uptake was determined by fluorescence assay, using cell pellets dispersed in 10 mM cetyl trimethylammonium bromide (CTAB). To determine the effects of photodamage, cells were incubated with specified levels of dyes for 30 min at 37 °C, collected by centrifugation, washed once and suspended in fresh growth medium. The cell suspensions were then irradiated using an Oriel model 66170 housing containing a 100 W QH lamp. A 10 cm layer of distilled water removed IR radiation. An interference filter confined the spectrum to 550__+ 10 nm. The light dose rate in the region of dye absorbance (shown in Fig. 1, see Section 3) was adjusted to 1200 mJ cm -2, delivered over a total time of 10 min. During irradiation, cell cultures were maintained at 4 °C to minimize temperature-sensitive repair of photodamage. An aliquot of irradiated cells was suspended in fresh growth medium at 37 °C, and viability was assessed by direct cell counts over 3 days after irradiation [10]. Incorporation of radioactive thymidine and cycloleucine (CL) provided information on photodamage to DNA synthesis and membrane transport [10]. Alterations in cell-surface hydrophobicity were determined by examining the behavior of cells in the two-phase system described previously [11, 12]. Cell suspensions (0.1 ml, 3 × 1 0 8 cells) were added to a 5 ml mixture containing 5% dextran (molecular weight, 500 000) and 4% poly(ethyleneglycol) (molecular weight, 6000) in 130 mM NaC1 and 20 mM phosphate buffer (pH 7) with 0.2 tzg ml -~ poly(ethyleneglycol) palmitate. An initial sample of 0.5 ml was withdrawn and the cell number was determined
89
using the Coulter ZF particle counter. The phases were allowed to separate for 5 min and anot her 0.5 ml was withdrawn from the center of the top phase. The partition coefficient is e x p r e s s e d as the percentage of total cells which remain in the u p p e r phase after a 5 min phase separation.
3. R e s u l t s The absorbance spectra of PP and SnPP (10 /zM, ethanol) are shown in Fig. 1. PP has a b r o a d e r Soret band, suggesting some aggregation in this solvent; SnPP has the characteristic two-band spect rum between 500 and 600 nm. We utilized the absorbance band at approximately 550 nm for photobiological studies with these dyes. The fluorescence emission spectra of PP in several solvents are shown in Fig. 2. The slight shoulder at 620 nm represents the presence of 5% hematoporphyrin. The fluorescence emission optimum from PP-loaded cells is 635 nm, suggesting an environmental dielectric constant of 4. The corresponding value from dye dissolved in tetrahydrofuran (D = 4) is 635 nm (Fig. 2). The fluorescence spectra of SnPP in various solvents are shown in Fig. 3. Dye-loaded cells show a fluorescence emission optimum at 583 nm, which suggests an environmental dielectric constant of approximately 35. The emission optimum in methanol ( D - - 3 2 ) occurs at 583.5 nm. Data relating to the physical and biological properties of these dyes are summarized in Table 1. PP is m uch more hydrophobic (from o c t a n o l - w a t e r partitioning) than SnPP, and is more effectively accumulated by L1210 cells. The extracellular level of PP required, on irradiation, to kill 50% of cells (IDso level) is substantially lower than that of SnPP, but the corresponding intracellular dye levels are similar. The concentration of PP which inhibits transport of CL by 50% is almost identical to the IDso level. Photo-inhibition of DNA synthesis requires a higher dye concentration. In the case of SnPP, inhibition of DNA synthesis is bet t er
151 /ii
0 o 1.0
~
.o 0 . 5
/ : i, ,
,,
So
~0
Fig. 1. Absorbance spectra of PP (full line) and SnPP (broken line). A complete absorbance profile of PP is shown, together with a magnification (4)
