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Photochemistry and Photobiology, 1999, 69(3): 390-396

A Comparative Study of the Cellular Uptake and Photodynamic Efficacy of Three Novel Zinc Phthalocyanines of Differing Charge

D. J. Ball', S. Mayhew', S. R. Wood', J. Griffiths2, D. 1. Vernon1 and S. B. Brown*' 'Centre for Photobiology and Photodynamic Therapy, School of Biochemistry and Molecular Biology, University of Leeds, Leeds, UK and 'Department of Colour Chemistry and Dyeing, University of Leeds, Leeds, UK

Received 22 June 1998; accepted 14 December 1998

ABSTRACT

Three novel substituted zinc (n) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensithers, 5,10,15,2&tetra(m-hydrox- ypheny1)chlorin (m-THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the REF-1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc m phthalocyanine) had improved activity over the other two phthalocyanines and slightly improved activity over PHP and m-THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m-THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low-level light exposure, and this was charge dependent. The anionic phthalocyanine (TGly, tetraglycine zinc [a phthalwyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine CIIxIPc, tetradioctylamine zinc m phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relo- calkation following low-level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.

INTRODUCTION

Photodynamic therapy (PDT)? is a treatment for cancer and other diseases, involving the administration of a photosen-

* To whom correspondence should be addressed at: Centre for Pho- tobiology and Photodynamic Therapy, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT. UK. Fax: 01 13 233 3017; e-mail: [email protected]

trlbbreviations: HEPES. N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid): HSA, 1 -hexane sulfonic acid; LD,,,, extracellular concentration resulting in 50% cell survival; LDSo,,,, intracellular concentration corresponding to the LD,,,; LDL, low-density lipoprotein; m-THPC. 5,10,15,20-tetra(m-hydroxyphen- y1)chlorin; MTT. 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; PDT, pho-

0 1Y99 American Society for Photobiology 0031-8655/99 $5.00+0.00

sitizing compound that, when activated by light, results in tumor destruction via the production of active oxygen species, particularly singlet oxygen ('OJ (1). The main clinical photosensitizer is a porphyrin-based compound referred to as PhotofrinB, consisting of a mixture of porphyrin oligo- mers (2). Other photosensitizers that have been used exper- imentally are phthalocyanines (3) and chlorins (4). In ho- mogenous solution, the factors affecting the efficiency of photosensitization include: the concentration of both oxygen and sensitizer, the reaction rate of the excited sensitizer with oxygen and target molecules and the rate of sensitizer triplet decay (5). It is well established that the efficiency of photosensitizers is reduced upon aggregation (6) and we have recently shown this to be the case for 5,10,15,20-tetra(rn- hydroxyphenyl) chlorin (m-THPC), polyhematoporphyrin (PHP, a compound identical to PhotofrinB) and the three novel zinc (II) phthalocyanines used in the current study (7).

In the more complex environment of a cell, these factors are further complicated by the subcellular localization of the sensitizer and by interactions with cellular material that can affect the aggregation state of photosensitizers (8). In vivo, further levels of complexity exist as damage may occur to tumor cells and/or the tumor vasculature depending on the sensitizer used (9).

The lifetime and diffusion of '02 in a cellular environment is limited by its reactivity with cellular components, and damage is reported to occur close to the site of generation (10,ll). Therefore, many recent reports have concentrated on the subcellular localization of sensitizers in an attempt to identify potential cellular targets. In vitro studies suggest that the more hydrophobic sensitizers target cellular membranes, implicating membrane damage as a primary mechanism of cell death by PDT (8), with internal membranes becoming more sensitive following prolonged incubation (12,13). Cat- ionic compounds appear to have selective affinity for mitochondria (14), lysosomes (15) and membranes (16), whereas lysosomes appear to be the primary target for tetrasubstituted anionic sensitizers (1 4). However, light-induced relocalization of some sensitizers suggests that the initial site of lo-

todynamic therapy; PHP, polyhematoporphyrin; PPC, pyridinium zinc (11) phthalocyanine: M E , receptor-mediated endocytosis; TAB, cetyltrimethylammonium bromide; TDOPc, tetradi- octyiamine zinc (11) phthalocyanine; TGly, tetraglycine zinc (11) phthalocyanine.

390

Photochemistry and Photobiology, 1999, 69(3) 391

calization may not be as important to cell damage andor repair as secondary sites (17,18).

A causal relationship between sensitizer structure, uptake, localization and hence cell death has been postulated (19- 25). In vitro studies have largely been directed toward structure-function relationships between differentially substituted porphyrins and phthalocyanines. In general, it appears that lipophilic compounds are taken up more readily through the plasma membrane than hydrophilic sensitizers, and in most cases this is accompanied by an increased photocytotoxicity. However, additional factors are likely to be involved because the uptake of some phthalocyanines is reported to be greater than porphyrins of similar lipophilicity (26), and the mange- ment of peripheral substituents has been shown to affect cellular uptake (24,27).

We have previously characterized the five photosensitizers with respect to aggregation and photoactivity using amino acid oxidation and red blood cell hemolysis as simple sys- tems (7). Here hydrophobicity and charge were the major determinants of efficacy. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine), but not the anionic (TGly, tetraglycine zinc [II] phthalocyanine) or the neutral (TDOPc, tetradioctylamine zinc [11] phthalocyanine) phthalocyanine, showed slightly improved activity over the clinical photosensitizers, m-THPC and PHP.

In this study, we have used fluorescence microscopy to study the subcellular localization of these novel phthalocyanines, together with that of m-THPC and PHP, in the RIF- 1 cell line. These observations, before and after low-level light exposure, have been related to photodynamic activity in an attempt to determine whether efficiency can be pre- dicted by a particular localization pattern.

MATERIALS AND METHODS fhotosensirizers. The three zinc (II) phthalocyanines, TGIy, TDOPc and PPC, were prepared by Mr. J. Schofield, Department of Colour Chem- istry, Centre for Photobiology and Photodynamic Therapy, University of Leeds. The TGly and TDOPc are tetrasubstituted (J. Schofield, per- sonal communication) while PPC is reported to have an average substitution corresponding to the dication (28). The PHP was supplied by Dr. D. I. Vernon, School of Biochemistry and Molecular Biology, Cen- tre for Photobiology and Photodynamic Therapy, at a concentration of 1 mg mL-' in phosphate-buffered saline (PBS), and stored at -20°C in working aliquots. The m-THPC (Foscana) was kindly donated by Scotia QuantaNova (Guilford, Surrey) and solutions were prepared fresh each day according to the manufacturer's guidelines in polyethylene glycol/ ethanovwater (2:3:5 vol/vol/vol).

Plasma protein binding. Plasma binding of phthalocyanines (0.1 mg mI-I) was determined by density-gradient ultracentrifugation according to the method of Have1 et al. (29) using a gradient of KBr/NaCI prepared according to Redgrave et al. (30). The protein and sensitizer content of each fraction was determined by absor- bance at 280 nm and 680 nm, respectively.

Cell culture. The cell line used was a RIF-1 murine fibrosarcoma (kindly donated by Dr. J. Bremner, MRC Radiobiology Unit, Har- well, Oxford), maintained in a culture medium of RPMI supple- mented with 10% fetal calf serum, 1% glutamine (200 mM) and 0.1% gentamycin (10 mg mI-').

Sensitizer uptake into RIF-I cells. Cells were plated in 35 mm petri dishes at a density of 2 X 104 cells per dish. A period of 18- 24 h was allowed for attachment and growth after which the culture medium was removed and replaced with fresh medium containing sensitizer. The cellular uptake of each sensitizer as a function of extracellular concentration was determined over a 2 h incubation period at 37°C in the dark, following serial dilution in medium from a 1 mg mI- ' stock solution.

Following incubation, the medium containing sensitizer was removed and the monolayers washed three times with cold PBS. The cells were dissolved by the addition of 0.1 M sodium hydroxide. The protein content of each sample was determined by the method of Bradford (31) using the Bio-Rad protein assay. Quantification of sensitizer was achieved as described below, by comparisons to known standards. The amount of cell-associated sensitizer was expressed as nmole per gram cellular protein.

TGly analysis. Sensitizer content was analyzed by fluorescence (Aex = 614 nm, A,, = 684 nm) following dilution in detergent (50 mM N-[2-hydroxyethyl]piperazinc-N'-[2-ethanesulfonic acid] [HE- PES], 10 mM cetyltrimethylammonium bromide [CTAB], pH 7.4).

TDOfc analysis. Cell homogenates were diluted with 50 mM HEPES, 10 mM CTAB, pH 7.4 (1:4). The TDOPc was extracted by the addition of butan-1-01 and centrifugation (2000 g, 5 rnin). The volume of the upper organic layer was measured and the amount of cell-associated sensitizer determined by fluorescence (Aex = 612 nm, A,, = 682 nm).

f P C analysis. Cell homogenates were diluted 1:4 with the ion- pairing reagent, 1-hexane sulfonic acid (HSA) (50 mg mL-'). The PPC was extracted by the addition of butan-1-01 and centrifugation (2000 g, 5 min). The volume of the upper organic layer was measured and the amount of cell-associated sensitizer determined by fluorescence (Aex = 605 nm, A,, = 682 nm).

m-THPC analysis. Cell homogenates were diluted with 50 mM HEPES, 10 mM CTAB, pH 7.4 (1:4). The m-THPC was extracted by the addition of ethyl acetate/acetic acid (4:l vol/vol) and centrifugation (2000 g, 5 min). The volume of the upper organic layer was measured and the amount of cell-associated sensitizer determined by fluorescence (Aex = 418 nm, A,, = 652 nm).

P H f analysis. Cell homogenates were diluted with 50 mM HEPES, 10 mM CTAB, pH 7.4 (1:4) and PHP was extracted by the addition of ethyl acetate/acetic acid (4:l vol/vol) and centrifugation (2000 g, 5 min). The organic layer was removed into a clean centrifuge tube containing an equal volume of 1 M HCI. After mixing, the tubes were recentrifuged (2000 g, 5 min). The upper organic layer was discarded and the lower acid layer was boiled for 30 min to allow hydrolysis of ester and ether linkages, thereby increasing the amount of fluores- cent monomeric material present (32). Following boiling, the samples were allowed to cool, the volume measured and porphyrin content was determined by fluorescence (Aex = 400 nm, A,, = 595 nm).

Subcellular localization and relocalization of photosensitizers. Cells were seeded onto glass coverslips (22 X 22 mm) at a density of 1 X 104 cells per dish. Sensitizer (10 @I phthalocyanine, 10 phf PHP, 1 @I m-THPC) was added in normal growth medium for 2 h at 37°C. Following incubation, the medium containing sensitizer was removed and the monolayers washed three times with PBS (37°C). The cells on coverslips were then inverted onto a specialized chamber slide containing fresh medium (37'C). Sensitizer fluorescence was monitored using a Lietz Dialux 22 upright microscope equipped for epifluorescence illumination. The excitation source for PHP and the phthalocyanines was a HeNe laser (632.8 nm, 4 mW); the excitation light for m-THFT was a filtered compact arc lamp providing light centered on 540 ? 25 nm producing 300 mW at the end of a 5 mm liquid light guide. Emitted light was isolated using interference filters (580-750 nm). Images for PHP and the phthalocyanines were captured, following a 1 min exposure, on a liquid nitrogen-cooled charge-coupled device camera (Asm- med). For m-THPC an exposure of 5 ms was sufficient. The light dose at the cell surface was estimated to be in the region of 0.1-0.5 mW cm-z ( 6 3 0 mJ cm-*). Relocalization of sensitizer following excitation was determined by a series of light exposures, with each exposure corresponding to the capture of one image. Captured images were pro- cessed using Visilog Imagerplus software.

Identijcation of cellular organelles. Organelles were identified using vital stains as described previously (18).

fhotocytotoxicity experiments. Cells were plated in 96 well plates for 18-24 h at a density of 1 X 104 cells per well for attachment and growth. Sensitizer was then added at a range of concentrations for 2 h. Following incubation, the cells were washed three times with PBS and then fresh medium was added. Illumination was performed using a 10 W copper vapor-pumped tunable dye laser (Ox- ford lasers, UK), the light from which was focused into a 600 pm diameter core optical fiber. The distal output was collected and col- limated into a 36 mm diameter beam. The power experienced by

392 D. J. Ball eta/.

Table 1. Hydrophobicity, plasma protein binding and intracellular levels (nmol sensitizedg cell protein) of photosensitizer in RIF-I cells*

Intracellular E concentration

Sensitizer ( M - ' cm I ) Hydrophobicity Major plasma protein binding (nmol g-I)

TGly 1 04 OOO TDOPc 116000 PPC 112000 m -THPC 22 400 PHP 1000

Hydrophilic Hydrophobic Hydrophilic Hydrophobic Amphiphilic

Albumin LDL Albumin Lipoproteins (36) Lipoproteins (37)

56 ? 2 237 f 21

1127 f 35 469 t 12 583 2 19

*Extinction coefficients were calculated in methanol at 680 nm (phthalocyanines). 652 nm (m-THPC) and 630 nm (PHP). Hydrophobicity was determincd by octanol buffer partition experiments as previously described (7). Inuacellular concenuation was determined as described in the text following a 2 h incubation with sensitizer (10 pM). Experiments were performed at least three times with triplicate points per experiment.

the cells (400 niW cm ? ) has been previously reported to be approximately 93% of that which hits the power meter in the absence of the cell dish (33). Following illumination, the medium was as- pirated and replaced with fresh medium. Cell survival was determined 24 h later using the 3-(4,5-dimethylthiazol 2-yl)-2,5-diphenyl tetrazolium bromide (M'IT) assay that measures mitochondria1 dehydrogenase activity (34) and gives comparable results to the clon- ogenic assay as a method of determining cell survival (35). A decrease in dehydrogenase activity has been widely used to indicate the relative PDT effects of different sensitizers.

RESULTS Hydrophobicity

The hydrophobicity of each sensitizer was determined by octanol buffer partition experiments as described previously (7). The hydrophilic sensitizers were found exclusively in the buffer phase while the hydrophobic sensitizers were found in the octanol phase (Table 1). The distribution of PHP was dependent upon pH, but at pH 7 there was an approximately equal amount in the octanol and buffer phase. Extinction coefficients for each sensitizer are also given in Table 1.

Plasma protein binding

The binding of each photosensitizer to the components in human plasma correlated to the hydrophobicity (Table 1). The hydrophilic sensitizers bound to albumin, the hydrophobic neutral phthalocyanine bound exclusively to low-density lipoproteins (LDL), and both m-THPC and PHF' have been reported to distribute among the high-density lipoproteins, LDL and very low-density lipoproteins (36,37).

Cellular uptake studies

The time dependence of uptake was studied for each sensitizer and the results are given in Table 2. The relative

amounts of each sensitizer taken up by the cells showed considerable variation. For this set of data, the extracellular concentrations are expressed in molar terms for comparative purposes and, although this is not consistent for each sensitizer (because experiments were performed with the concentration in pg mI- ' and because of variation in molecular weights), it is nevertheless clear that the intracellular concentration continues to increase at least up to the 24 h incubation time. For further studies, a 2 h incubation was chosen. This proved to give good discrimination between the sensitizers in terms of intracellular concentration, localization and PDT effect. Cellular uptake at 2 h, as a function of extracellular concentration, was dose dependent for each sensitizer, as shown in Fig. 1. In order to determine the relative uptake on an equimolar basis, the sensitizers were each studied at an extracellular concentration of 10 pM. The order of intracellular concentration was in the sequence PPC > PHP > m-THPC > TDOPc > TGly (Table 1).

Cytotoxicity and photocytotoxicity in the RIF-1 cell line

No dark toxicity was observed with any of the photosensitizers at the concentrations used. To correlate cell uptake studies with PDT effect a 2 h incubation was chosen. For each sensitizer cells were incubated with sensitizer at a range of concentrations and illuminated using laser light (Fig. 2). The extracellular concentration required to produce 50% cell kill, as determined by a 50% decrease in MTT reduction (LD,,, value), was thus calculated, and the corresponding intracellular concentrations at each LDSOex (LDSOint values) were determined from separate cell uptake curves construct- ed over the relevant concentration range (Table 3). Gener- ally, an increase in the concentration of extracellular sensitizer was accompanied by an increase in PDT effect at a given light dose. From a plot of the LDSOint versus light dose

Table 2. Intracellular sensitizer levels (nmol g-I) in RIF-I cells following different incubation times*

Sensitizer I*M 2 h 8 h 12 h 24 h

159 t 1 1 TCily 7.2 60 l?r 8 88.5 2 4 119 i- 9 TDOPc 5.6 163 f 8 251 f 23 353 2 39 501 t 80 PPC 10 1127 f 35 1858 i- 62 2918 5 312 3348 2 213 m-THPC 5.87 290 f 24 547 2 16 871 f 183 1766 2 63 PHP 16.66 687 t 45 820 f 49 983 t 69 1364 f 49

*Each value represents the mean of three experiments (t standard deviation).

0 5 10 15 20

(c) 3000,

0 5 10 15 20

0 5 10 15 20

(d)

0 5 10 15 20

0 10 20

Concentration (pM)

Figure 1. The RIF-1 cells were incubated with (a) TGly, (b) TDOPc, ( c ) PPC, (d) m-THPC and (e) PHP for 2 h in the dark at 37°C. Intracellular concentration was determined by extraction pro- cedures as described in the Materials and Methods. Results are expressed as nmol sensitizer per gram cell protein. Points represent the mean of three experiment (+.standard deviation).

(Fig. 3), it can be seen that the intracellular concentration of PHP was much higher than that of the other photosensitizers, whereas relatively less PPC or m-THPC was required to give the same effect.

Subcellular localization and relocalization of sensitizers

The localization of each sensitizer is given in Fig. 4a-e. Cells incubated with the phthalocyanines gave a punctate staining pattern similar to that of the lysomotrophic dye, ac- ridine orange (18). The PHP and m-THPC did not appear to target any particular organelle, with a diffuse fluorescence observed throughout the cell. The similar staining pattern to that of dihexaoxacarbocyanine iodide (18) suggests that these sensitizers have a general affinity for lipophilic membranes. There was also a greater intensity of staining in the perinuclear region suggestive of a more specific localization, possibly the Golgi.

Light exposure of cells at a light dose 1000-fold lower than that used for photocytotoxicity experiments resulted in a relocalization of fluorescence that was sensitizer dependent (Fig. 4a'-e'). The most pronounced effect was observed with


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Figure 2. The RIF-1 cells were incubated with (a) TGly, (b) TDOPc, (c) PPC, (d) m-THPC and (e) PHP for 2 h and then illuminated using laser light (10-40 J cm-2). Loss in mitochondria1 dehydrogenase activity was determined 24 h later by the M T I assay. Light doses were 0 (+). 10 (X), 20 (a) and 40 (0) J cm-'.

Table 3. The extracellular concentration of sensitizer required to give 50% cell kill (LD,,, values) for each sensitizer following 20 J cm-2 laser light illumination*

Sensitizer LD50ex (CLM) LD50~nt (nmol g-'1

TGly 2.1 2 0.05 15.6 t 1.2 TDOPc 2.6 +. 0.04 59 t 2.5 PPC 0.028 2 0.001 2.6 t 0.45

3.6 ? 0.26 m-THPC 141 2 1 1 PHP 2.5 ? 0.1

0.12 +. 0.002

*Intracellular concentrations corresponding to each extracellular concentration (LD50,nt values) were calculated from cell uptake curves prepared over the relevant concentration range. Values are given 2 standard deviation where n = 3-6.


150

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50

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R 10 20 30 40

Light Dose (J/cm2)

Figure 3. The RIF-1 cells were incubated with TGly (A). TDOPc (w), PPC (X), m-THPC ( + ) and PHP (0) for 2 h and then illuminated using laser light (IW J cm ’). The concentration of sensitizer resulting in 50% cell kill was calculated for each light dose and the corresponding intracellular concentration (LD,,,) calculated from cell uptake experiments. Each experiment was performed at least three times. Insert shows the data for PPC and m-THFC on an expanded scale.

TGly; the initial punctate fluorescence disappeared following three 1 min exposures producing a much more diffuse staining pattern that was most intense in the nuclear area. This relocalization of sensitizer was accompanied by a dramatic increase in fluorescence intensity. At this time the cells were still flat in appearance and did not show any morphological changes until exposed to a further 9 min of light, when some blebbing of the plasma membrane started to develop (Fig. 4a‘). Despite the similar initial localization pattern, light exposure of cells incubated with TDOPc was not accompanied by a relocalization of fluorescence until morphological changes occurred (Fig. 4b’). Light exposure of cells incubated with PPC resulted in a more diffuse fluorescence distribution and a slight increase in fluorescence intensity (Fig. 4c’). The PHP and m-THPC gave a diffuse fluorescence throughout the cell with membranes appearing the major target (Fig. 4d, e). Successive light exposure resulted in a decrease in the fluorescence intensity (photobleaching) rather than any relocalization (Fig. 4d‘, e‘).

DISCUSSION Following the initial use of Photofrina and PHP, a number of second-generation photosensitizers have been developed that absorb further in the red (3,4). Many of these new compounds have been peripherally substituted to change the Figure 4. 1,ocalization of (a) TGly, (b) TDOPc, (c) PPC, (d) m- overall charge and hydrophobicity, which in turn affect THPC and (e) PHP in RIF-1 cells. Localization following a further cell uptake and localization (19,21,23-25). Studies of these compounds are important for understanding possible mech-

12 light exposures is also given (a’-e’).


anisms of action and for the development of potentially use- ful PDT agents.

It is generally accepted that non-ionized species can cross the plasma membrane more easily than charged compounds (38,39). Therefore, it would be expected that the charged photosensitizers will be less readily taken up into cells than the neutral and hydrophobic ones. Uptake experiments (Fig. 1) confirmed this to be the case for TGly and are in agreement with that reported for sulfonated zinc (22,25), alumi- num (20,26) and gallium (19) phthalocyanines, in which the tetrasubstituted derivatives had relatively poor uptake compared to the more hydrophobic, lower substituted derivatives. However, PPC had improved uptake over the more hydrophobic sensitizers (Fig. 1 , Tables 1 and 2). Three possible mechanisms of photosensitizer uptake have been postulated: simple diffusion of lipophilic sensitizers, fluid phase endocytosis of water-soluble sensitizers and receptor-mediated endocytosis (RME) of LDL-associated dye (40). The high uptake of PPC may be attributed to the position of the pyridinium groups on the phthalocyanine skeleton (20). The synthesis of PPC gives a mixed product with an average degree of substitution corresponding to the dication (28). Al- though the actual position of the pyridinium groups has not been elucidated, it has been reported that disubstituted sensitizers with adjacent, rather than opposite, groups have improved cellular uptake compared to the tetrasubstituted derivatives (20).

The punctate fluorescence of PPC, TDOPc and TGly is suggestive of lysosomal .localization and is consistent with results that have been previously reported for PPC (18). Plasma protein binding indicated that TDOPc binds to LDL (Table 1) and therefore RME would be a possible mechanism of uptake that would result in a specific localization in the lysosomes. The PPC and TGly did not bind to lipoproteins but delivery to lysosomes would also result from pi- nocytosis. The m-THPC and PHP have also been reported to distribute among the lipoproteins (36,37), suggesting that localization may be lysosomal. However, the diffuse localization of both these compounds suggests that the lysosomes are not the only target (Fig. 4). It is possible that by using different mechanisms of uptake, such as simple diffusion through the lipid bilayer and endocytosis, multiple cellular targets are accessible.

It is generally accepted that photochemical efficiency of metallophthalocyanines is not affected by peripheral substituents as long as the dye is monomeric (6). In solution, a low quantum yield of singlet oxygen produced with lower sulfonated derivatives correlated well with the tendency of these sensitizers to aggregate (6). At the cellular level, as- sociation of sensitizers with cellular proteins and lipids can affect the aggregation state and hence the photodynamic activity (7,13,16,17). Using laser illumination, the efficiency of photoinactivation, based on the extracellular concentration of sensitizer to give 50% MTT reduction (LD5&J, was PPC > m-THPC > PHP > TGly > TDOPc. Comparisons of the intracellular concentrations at these LD,,, values (LDSOint) was in the order PPC < mTHPC < TGly < TDOPc < PHP (Table 3). This suggests that the activity of PHP was improved by the high uptake of this sensitizer, as confirmed by a plot of LDsoint against light dose (Fig. 3). However, the efficiency of PHP is underestimated by these cnfteria alone,

because when comparing photodynamic activity other factors must also be considered such as the extinction coefficient at the treatment wavelength (Table l), the relative efficiency of photoinactivation per quantum of light absorbed, singlet oxygen yields, aggregation state and localization (7). The phthalocyanines all have similar extinction coefficients and their singlet oxygen yields are comparable to methylene blue so these factors do not account for the improved activity of PPC over TGly and TDOPc.

Localization patterns have been shown to be an important determinant in the mode of cell death (38-41). Comparisons of localization suggested that all three phthalocyanines had a similar distribution (Fig. 4). However, when cells were exposed to light, at a much lower level than that used to produce a PDT effect, differences in localization patterns became apparent. The TDOPc fluorescence remained punctate, TGly relocalized to the nuclear area with a concomitant increase in fluorescence intensity and the distribution of PPC became more cytoplasmic (Fig. 4). It appears that relocalization to the nucleus is common to tetrasubstituted, anionic photosensitizers, and it has been reported that this is not accompanied by an increased PDT effect (17). Using NHIK 3025 cells, Berg et al. (42) showed that incubation with differentially substituted meso-tetraphenylporphines resulted in a punctate distribution indicative of lysosomes. Following exposure to light, the tetrasubstituted derivative relocalized to the nucleus, whereas the disubstituted derivative gave a more diffuse cytoplasmic distribution. In agreement with the work presented here, it was shown that the disubstituted derivative was a more efficient photosensitizer after relocalization, whereas the tetrasubstituted derivative was less efficient. The two clinically used photosensitizers, PHP and m- THPC had a diffuse distribution pattern that did not change following light exposure, although some photobleaching occurred.

In conclusion, we have demonstrated that following a 2 h incubation there is a relationship between the uptake and photocytotoxicity of these compounds. The PPC has comparable, if not slightly improved, activity over the two clinically used photosensitizers, m-THPC and PHP and is much better than the anionic (TGly) and neutral (TDOPc) phthalocyanines. This can be explained by the increased extinction coefficient at the treatment wavelength compared to m- THPC and PHP (7) and improved cellular uptake over TGly and TDOPc (Fig. 1, Table 1). However, comparisons of the intracellular levels of PPC required to give a PDT effect were much lower than TGly and TDOPc. The differences in localization of these phthalocyanines following low-level light exposure suggests that the diffuse localization common to PHP, m-THPC and PPC (following relocalization) may be an important determinant of good photodynamic activity.

Acknowledgements-This work was supported by the Yorkshire Cancer Research. We also thank Andy Holroyd for technical assis- tance.

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