Journal of Photochemistry and Photobiology, 13." B/ology, 6 (1990) 87-92 87

SITES OF PHOTOSENSITIZATION BY PROTOPORPHYRIN A N D TIN PROTOPORPHYRIN IN LEUKEMIA 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 in vivo [1-3]. Although protoporphyrin (PP) is a poor tumor-localizing agent in 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 in 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.

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2. M a t e r i a l s a n d m e t h o d s

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 anti- biotics (gentamycin). Dextran T-500 was obtained from Pharmacia (Pisca- taway, N J). Poly(ethyleneglycol) was provided by Pierce Chemical Co. (Rock- ford, 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 trime- thylammonium 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

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using the Coulter ZF particle counter. The phases were allowed to separate for 5 min and another 0.5 ml was withdrawn from the center of the top phase. The partition coefficient is expressed as the percentage of total cells which remain in the upper 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 broader Soret band, suggesting some aggregation in this solvent; SnPP has the characteristic two-band spectrum 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 cor- responding 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- -32) occurs at 583.5 nm.

Data relating to the physical and biological propert ies of these dyes are summarized in Table 1. PP is much more hydrophobic (from octanol -water 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 concentrat ion of PP which inhibits t ransport 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 ter

151 /ii 0 / : i, o 1.0 ,

.o ~ 0 . 5 ,,

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)<) over the range 450-650 nm. For SnPP, only the magnified tracing is shown at 450-650 nm.

90

100-

i'i i' :1/ • i l '

8

-'//./ 0 ,, ,.~. ~ ........ ,v ~ ...> ..

'\ o--- - 610 6SO 650 550 605 660

Wavelength (nml Wavelength (nm) Fig. 2. Fluorescence emission spectra of PP. Solvent, from top to bottom: methanol, ethanol, butanol, tetrahydrofuran.

Fig. 3. Fluorescence emission spectra of SnPP. Full line, tetrahydrofuran; broken line, n- butanol; dotted line, methanol.

TABLE 1

Dye uptake and sites of photoaamage

M e a s u r e m e n t P P S n P P

Octanol-water a 4.9 + 0.5 0.21 ± 0.03 IDso (~M) b 0.33±0.06 (5.1) 5.12=0.4 (7.1) Em~ c 635 ( > 4 ) 583 (40) Transport d 0.39 2:0.04 74 ± 3 DNA synthesis e 3.62 ± 0.45 10.9 4- 1.3

L1210 cells were incubated with different extracellular concentrations of dyes for 30 min at 37 °C as described in the text. Data shown represent the mean of 3--4 determinations ± standard deviation (SD). aDistribution of dye between octanol and 50 mM phosphate buffer (pH 7.0). bExtracellular dye level 0zM) needed to reduce viability by 50% on irradiation. The corresponding intracellular dye concentration (/~M) is given in parentheses. ~The wavelength of optimum fluorescence emission from dye-loaded cells. The estimated dielectric constant of the dye-binding site is given in parentheses. dExtracellular dye level (~M) required for 50% inhibition of CL transport on irradiation. eExtracellular dye level 0zM) needed to inhibit incorporation of labeled thymidine into DNA by 50% on irradiation.

c o r r e l a t e d w i th cel l kil l t h a n is the i n h i b i t i o n of CL t r a n s p o r t , b u t m a y n o t r e p r e s e n t the in i t i a l p h o t o t o x i c p h e n o m e n o n .

The t w o - p h a s e p a r t i t i o n i n g r e s u l t s a re s h o w n in T a b l e 2. A n IDso leve l of PP, o n i r r ad i a t i on , h a s a m a r k e d effect o n ce l l - su r f ace h y d r o p h o b i c i t y , as p r o b e d b y the p a r t i t i o n i n g sy s t em, b u t th i s is n o t t he case w i th SnPP . W e ca r r i ed ou t s im i l a r s t u d i e s w i th t he p u r p u r i n s NT2 a n d SnNT2H2, dyes w h i c h h a v e b e e n e x a m i n e d p r e v i o u s l y [10] . Use of IDso leve l s o f t h e s e s e n s i t i z e r s d o e s n o t h a v e a n y d e t e c t a b l e effect o n t he p a r t i t i o n i n g b e h a v i o r of ce l l s ( da t a n o t s h o w n ) . The i m p l i c a t i o n s of t h e s e r e s u l t s a re d i s c u s s e d be low.

TABLE 2

Two-phase partitioning studies

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Trea tmen t Control P P S n P P

Dark 37±4 36±3 39±4 Light 36±4 15±2 37±3

Cells were incubated for 5 min at 37 °C with PP (0.3 /zM) or SnPP (30 p.M), then irradiated as described in the text, or kept in the dark. Cells were then partitioned for 10 min in the two-phase system. Numbers represent the percentage of total cells found in the upper phase. The mean+SD for four experiments is shown.

4. D i s c u s s i o n

The f luo rescence emiss ion s p e c t r a indica te b inding of PP to a si te of low dielectr ic cons tan t , w h e r e a s SnPP b inds to a m u c h m o r e h y d r o p h o b i c locus (Table 1). HPLC (not shown) does no t indica te any conve r s ion of SnPP to PP by L1210 cells. Moreover , incuba t ion of L1210 cells wi th SnPP resu l t s in no m e m b r a n e p h o t o d a m a g e (Table 2), which would be e x p e c t e d if PP was fo rmed . PP and SnPP are equi toxic wi th r ega rd to in t racel lu lar levels r equ i red to p r o d u c e le thal p h o t o d a m a g e , a l though the m o r e effect ive u p t a k e of the f o r m e r lowers the ex t race l lu la r level r equ i red to p r o d u c e a p h o t o t o x i c effect.

The d is t r ibut ion of a cell popu l a t i on b e t w e e n the p h a s e s of the par t i t ion ing s y s t e m ref lects h y d r o p h o b i c in te rac t ions at the cell su r face [11, 12]. In the p r e s e n t invest igat ion, the par t i t ion ing s tudies (Table 2) revea l no ev idence of a l te ra t ion in m e m b r a n e h y d r o p h o b i c i t y w h e n cells a re t r e a t ed wi th an IDso level o f SnPP and i r radiated. However , PP u n d e r s imilar condi t ions m a r k e d l y lowers the par t i t ion ratio. Such a resu l t can der ive f r o m pho to - ox ida t ion of ce l l -surface c o m p o n e n t s .

The resu l t s p r e s e n t e d he re are different f r o m da ta ob t a ined with a pu rpu r in es te r and its t in ana log [10]. The me ta l l opu rpu r in is b e t t e r ac- c u m u l a t e d ( env i ronmen ta l dielectr ic cons tan t , 24) and p r o d u c e s p h o t o d a m a g e at m e m b r a n e loci. In cont ras t , the p a r e n t p u rpu r in is local ized in a cel lular si te of low dielectr ic cons t an t ( less t han 4), and its p r e d o m i n a n t pho tob io log ica l effect is on DNA synthes is . These pu rpur ins are insoluble in water , and were de l ivered via emul s ions in C r e m o p h o r EL. The pa t t e rn s of local izat ion and pho tosens i t i za t ion of the pu rpu r in s reflect, in par t , the use of an a m p h i p a t h i c agen t for the i r delivery. Nei ther purpur in , at the IDso level, af fects the two- p h a s e par t i t ion ing behav i o r of L 1 2 1 0 cells (da ta no t shown) . Pu rpu r in pho to tox i c i t y is thus not a s soc i a t ed wi th a l te ra t ions in the hyd rophob i c i t y of the ou te r cell sur face .

An exp lana t ion cons i s t en t wi th the p o r p h y r i n and pu rpu r in resu l t s is tha t the tin p u r p u r i n (SnNT2H2) a s soc i a t e s wi th a cel lular si te n e a r the inner su r face of the p l a s m a m e m b r a n e , whe re i r radia t ion cause s p h o t o d a m a g e to t r a n s p o r t sys t ems , wi thou t affect ing the par t i t ion ing b e h a v i o r of cells, which

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r e f l e c t s p r o p e r t i e s o f t h e o u t e r ce l l s u r f a c e [11 ]. T h e r e s u l t s w i t h p u r p u r i n (NT2) s u g g e s t a s s o c i a t i o n o f th i s v e r y h y d r o p h o b i c e s t e r ( o c t a n o l : w a t e r p a r t i t i o n r a t i o , 8 0 ) w i t h i n t r a c e l l u l a r l i p id s . T h e u n e x p e c t e d l a c k o f m e m b r a n e p h o t o d a m a g e b y NT2 m a y d e r i v e f r o m t h e m o d e o f d e l i v e r y , w i t h t h e d r u g - c o n t a i n i n g m i c e l l e s f i rs t e x p o s e d to i n t r a c e l l u l a r l i p i d s r a t h e r t h a n to t h e l i p i d - r i c h r e g i o n s o f t h e ce l l m e m b r a n e .

W i t h r e g a r d to t h e p o r p h y r i n s , t h e h y d r o p h o b i c P P f i rs t e n c o u n t e r s t h e ce l l m e m b r a n e w h e r e i t c o n c e n t r a t e s and , o n i r r a d i a t i o n , c a t a l y z e s a l t e r a t i o n s in m e m b r a n e h y d r o p h o b i c i t y a n d t r a n s p o r t . In c o n t r a s t , S n P P is m o r e hy- d r o p h i l i c , l e s s e f f e c t i v e l y a c c u m u l a t e d , a n d c a u s e s p h o t o d a m a g e a t s i t e s c o n s i s t e n t w i th a h y d r o p h i l i c dye , i .e . a t a n i n t r a c e l l u l a r s i te .

Acknowledgment

This w o r k w a s s u p p o r t e d b y g r a n t CA 2 3 3 ? 8 f r o m t h e N a t i o n a l C a n c e r I n s t i t u t e , NIH, DHHS.

References

1 C. Gomer, Photodynamic therapy in the treatment of malignancies, Semin. Oncol., 26 (1989) 27-31.

2 T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumors, Photochem. Photobiol., 45 (1987) 879-890.

3 J. Moan, Porphyrin photosensitization and phototherapy, Photochem. Photobiol., 43 (1986) 681-690.

4 D. Kessel, T. J. Dougherty and T. G. Truscott, Photosensitization by diporphyrins joined via methylene bridges, Photochem. Photobiol., 48 (1988) 741-744.

5 T. Dubbelman, A. De Goeij and J. Van Steveninck, Protoporphyrin-induced photodynamic effects on transport processes across the membrane of human erythrocytes, Biochim. Biophys. Acta, 595 (1980) 133-139.

6 A. Girotti, Protoporphyrin-sensitized photodamage in isolated membranes of human eryth- rocytes, Biochemistry, 18 (1979) 4403-4411.

7 E. J. Land, A. F. McDonagh, D. J. McGarvey and T. G. Truscott, Photophysical studies of tin(IV)-protoporphyrin: potential phototoxicity of a chemotherapeutic agent proposed for the prevention of neonatal jaundice, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5249-5253.

8 A. F. McDonagh and L. A. Palma, Tin-protoporphyrin: a potent photosensitizer of bilirubin destruction, Photochem. Photobiol., 42 (1985) 261-264.

9 V. P. Shanbhag and G. Johansson, Specific extraction of human serum albumin by partition in aqueous biphasic systems containing poly(ethyleneglycol) bound ligand, Biochem. Bio- phys. Res. Commun., 61 (1974) 1141-1146.

10 D. Kessel, Determinants of photosensitization by purpurins, Photochem. Photobiol., 50 (1989) 169-174.

11 H. Walter, E. J. Krob and R. Tung, Hydrophobic affinity partition in aqueous two-phase systems of erythrocytes from different species, Exp. Cell Res., 102 (1976) 14-24.

12 D. Kesscl, Some determinants of partitioning behavior of lymphoblasts in aqueous biphasic systems, Biochim. Biophys. Acta, 578 (1981) 245-249.