Photochemistry and Photobiology Vol. 42, No. 2, pp. 129 - 133, 1985 Printed in Great Britain. All rights reserved

0031-8655/85 $03.00+0.00 Copyright @ 1985 Pergamon Press Ltd

PHOTOSENSITIZED INACTIVATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

E. BEN-HUR'I and I. ROSENTHAL~' 'Department of Radiobiology, Nuclear Research Center-Negev. P.O. Box 9001, Beer-Sheva, Israel

and 'A.R.O., Department of Food Science. The Volcani Center, P.O. Box 6, Bet-Dagan, Israel

(Received 28 November 1984; accepted 19 February 1985)

Abstract-Chloroaluminum phthalocyanine was found to sensitize cultured Chinese hamster cells upon exposure to white fluorescent light. Elimination of wavelengths below 370 nm did not reduce the effect significantly, indicating that the effective wavelengths were those absorbed by the Q band (600-700 nm) of phthalocyanine. The magnitude of the photosensitizing effect increased with the dye concentration and the time of its contact with the cells prior to light exposure. Although photosensitization was drastically reduced in the absence of oxygen, the lack of effect of glycerol and D 2 0 during exposure suggests that neither hydroxyl radicals nor '02 are responsible for the cytotoxic response. The efficiency of the photosensitized induced cell killing did not vary with the position of the cells in the cell cycle, in contrast to exposure to X-rays. The improved spectral properties, the reported low toxicity and the selective retention by neoplasms, make phthalocyanines promising candidates for use in photodynamic therapy of cancer.

INTRODUCTION

Photodynamic inactivation of biological systems by exposure to certain dyes and visible light has been studied since the beginning of this century (Raab, 1900; Spikes, 1982). Oxygen is required for these photochemical reactions, which proceed either by direct interaction of the electronically excited dye molecule with a cellular target, followed by reaction of the transients formed with molecular oxygen (Type I), or via singlet oxygen produced by interac- tion of O2 with the dye in its excited triplet state (Type 11). Clinical application of such a process was prompted by the observation that malignant tumors can retain porphyrin derivatives for longer time than adjacent normal tissues. Subsequent exposure to light of proper wavelength, can cause eradication of the tumor. This modality is now termed PDT and uses primarily haematoporphyrin derivative (HPD)? (Kessel and Dougherty, 1982). Haematoporphyrin derivative is a complex mixture of porphyrins of a somewhat variable composition derived from hema- toporphyrin by chemical reactions (Clezy etal . , 1980; Bonnett et al., 1981). The most active component was recently suggested to be dihematoporphyrin ether (Dougherty and Weishaupt, 1983). Although the main absorption band of porphyrins is around 400 nm, for therapy the dye is activated by red light (A = 630 nm) where a minor absorption peak exists,

*To whom correspondence should be addresseil. tA bbreviations: CAPC, chloroaluminum phthalocy-

anine; DMEM, Dulbecco's modified Eagle's medium; HPD, hematoporphyrin derivative; HU, hydroxyurea; HVL, half value layer; OER, oxygen enhancement ratio; PBS, phosphate buffered saline; PDT, photodynamic therapy.

because of the increased transparency of tissues in the red (Anderson and Parrish, 1982). This leads to a poor efficiency of the overall photoprocess and the need for very accurate tuning when using a dye laser. A major side effect of PDT is a lingering skin photosensitivity due to the slow clearance of HPD from the dermal tissues.

In search for alternative sensitizers for PDT, we reported the photobiological activity of CAPC (Ben- Hur and Rosenthal, 1985). Metallophthalocyanines are porphyrin-like compounds (Fig. 1) that absorb strongly in the red (the Q band, 600-700 nm, E - lo5 1 mol-' cm-'1. Phthalocyanines are used as hetero- genous catalysts in dehydrogenations and oxidations and as dyes and pigments (Moser and Thomas, 1984). There has also been a considerable interest in the photophysical properties of phthalocyanines since

PHTH ALOCYAN I NE Figure 1. The molecular structure of metallophthalo-

cyanine.

129

130 E . BEN-HUR and I. ROSENTHAL

the first description of their use as passive Q switches with ruby lasers (Hercher et al., 1968), and the photoconduction and semiconductor properties of thin films and powders (Moser and Thomas, 1984). The mechanistic photochemistry of several phtha- locyanines has been investigated in recent years (Day et al., 1968; McVie et al., 1978; Dolphin et al., 1980; Harriman and Richoux, 1980; Lever et al., 1981; Muralidharan and Ferrandi, 1983).

Following our initial observation that CAPC is a potent photosensitizer in mammalian cells (Ben-Hur and Rosenthal, 1985), we now report the characteris- tics of the response of Chinese hamster cells exposed to CAPC and visible light. It is noteworthy that CAPC photosensitization, although requiring 0 2 , probably does not involve lo2 suggesting a Type I photodynamic reaction.

MATERIALS AND METHODS

Cell culture. Chinese hamster fibroblasts, line V79- B310H, were grown as a monolayer in 50 mm plastic petri dishes using DMEM containing 10% fetal calf serum. The cells doubled in number every 9 h at 37°C in a humidified atmosphere containing 5% CO,. Cell survival was deter- mined using colony formation as an end-point (Ben-Hur et al., 1978). Growth curves were obtained by trypsinizing duplicate plates at intervals and quantitating the number of cells using a hemocytometer. To obtain cells synchronized at the GI/S border, HU was used as previously described (Elkind et al., 1968). Briefly, log-phase cells were incubated for 3.5 h in DMEM containing 2 mM HU. Under these conditions cells in S phase are killed and the remainder of the population is accumulated at the G,/S border.

Chemicals. CAPC was purchased from Eastman Kodak Co (Laser Grade) and further purified before use (Rosen- thal, 1978). Cells were treated with CAPC by adding the required volume of 0.1 mM solution of CAPC in ethanol into the culture medium. HU (Sigma Chemical Co) was added from a 0.1 M stock solution in PBS. Light exposure. Prior to light exposure the growth

medium was removed, the cell monolayer was rinsed with PBS and the plates containing 3 me PBS and with the lids on were then exposed to visible light at room temperature. The light source was a bank of three 40 W fluorescent tubular lamps (Sylvania, Daylight) held in a reflector. The fluence rate was 64 W m-’ at the level of the cell monolayer. In some experiments, A < 370 nm were removed by filtering the light through a Tedlar PVF film No. 100BG30UT (DuPont Co., Wilmington, DE 19898).

X-Ray-irradiation. A 50-kV X-ray machine was used as the radiation source. With a beam current of 20 mA and a filter of 0.18 Al (HVL = 0.16 mm), the dose rate was 15 Gyimin. Cells were exposed while attached in dishes with the growth medium removed. Immediately after exposure the growth medium was added back.

Oxygen removal. The exposure of cells in the absence of oxygen was performed by the “full medium” technique of Ling et al. (1981). Briefly, cells grown in flat-bottom prescription glass bottles were flushed with humidified N2 (- 5 ppm 0,) for 45 min at room temperature. OER for exposure to ionizing radiation under these conditions was 3.06 indicating that complete hypoxia was achieved.

RESULTS

Addition of CAPC to cells in log-phase for 16 h, with a subsequent exposure to white fluorescent light resulted in a growth delay (Fig. 2). The magnitude of

W

p‘

m

W U

20 40

GROWTH TIME ( h r )

Figure 2. Photosensitized inhibition of Chinese hamster cells growth. Cells in log-phase were grown with 0.4 pM CAPC for 16 h and then exposed to various intensities of

fluorescent light, as indicated.

the delay increased with the fluence of the light. After exposure to more than 10 kJ m-’ the cells did not resume growth for the entire duration of the experi- ment (not shown). Neither CAPC nor light affected the rate of cell growth when applied separately.

Figure 3 shows that the combined treatment of CAPC and light not only delays cell growth but is also

LIGHT INTENSITY ( kJ /m2)

Figure 3. Survival of Chinese hamster cells exposed for 16 h to 0.4 pM or 1 pM CAPC, as indicated, followed by light exposure. Squares denote exposure to light in PkS contain- ing 1 Mglycerol, triangles are for PBS inD,O and circles are

for PBS in HzO.

Photosensitization by phthalocyanines 131

lethal to the cells. The survival curve has a pro- nounced shoulder region followed by an exponential cell killing. The shoulder on the survival curve was reduced and the final slope became steeper when the concentration of CAPC in the growth medium prior to light exposure was increased from 0.4 p M to 1 p M . It is noted that after 16 h incubation with 0.4 p M CAPC, ca. 10' dye molecules were bound per cell, as estimated using [3H]CAPC (E. Ben-Hur and I. Rosenthal, unpublished). From Fig. 3 it can also be seen that when PBS during exposure to light con- tained either 99% D20 or 1 M glycerol the response of the cells was not affected, confirming our earlier report (Ben-Hur and Rosenthal, 1985).

The kinetics of CAPC photosensitization are shown in Fig. 4. Evidently, photosensitivity develops over a 3 h period during contact of the cells with CAPC. Further contact with CAPC confers only little additional photosensitivity. Disappearance of photosensitivity is quite rapid upon removal of medium containing the dye and incubation of the cells in dye-free DMEM. After 1 h the cells were no longer sensitive to light. The presence of serum is essential for the loss of photosensitivity. Full sensitiv- ity was retained when the cells were incubated in PBS or serum-free DMEM (data not shown). This is most probably due to the very low solubility of CAPC in water. Chloroaluminium phthalocyanine binds

strongly to bovine serum albumin (our unpublished results) and thereby becomes solubilized in the growth medium.

Photosensitization by CAPC depends to a large extent on the presence of oxygen (Fig. 5) . In the

N2 \

0.4 pM CAPC, I6 h

I 1 I I I

0 2 4 6 8 1 0

LIGHT INTENSITY ( kJ/m2)

Figure 5. Survival of Chinese hamster cells exposed for 16 h to 0.4 FLM CAPC followed by light exposure. Triangles indicate exposure with a UV cutoff filter. Filled circles are

for cells exposed in the absence of oxygen.

I I

0 1 2 ~ 3 l

INCUBATION TIME ( h r )

Figure 4. Kinetics of CAPC photosensitization. Solid circles denote the development of hotosensitivity

show the disappearance of photosensitivity when cells incubated for 16 h with 0.4 FLM CAPC are rinsed and exposed to 6 kJ m-' after various incubation times in dye-free DMEM.

as a function of time in the presence of 0.4 pLM CAPC followed by exposure to 8 kJ m- P . Empty circles

PAP 42:2-C

132 E. BEN-HUR and I. ROSENTHAL

I

5 KJ m-2

' ' 0 f- \o-

A

6 KJ mm2 + -

Z m M H U ( 3 . 5 hr)- TIME- X

l o o 1

I 04pM CAPC , I 6 hr I

1 8 0 - 2 6 8

TIME AFTER H U REMOVAL ( h r )

Figure 6. Age-response of Chinese hamster cells exposed to X-ray-irradiation or CAPC-plus-light . Cells were syn- chronized at the G,/S border by HU after 16 h in 0.4 pM CAPC. The cells were exposed to 8 Gy X-rays or fluorescent light, as indicated, at various times after removal of HU. Incubation after HU removal was in DMEM containing 0.4

pM CAPC to avoid loss of photosensitivity.

absence of O2 the cytotoxic effect is drastically inhibited. A control experiment revealed that oxygen removal resulted in on OER of 3.06 for ionizing radiation. This indicates an oxygen level of less than 10 ppm. Also shown in Fig. 5 are results using a UV cutoff filter. The fact that this has only a minimal effect on the response, indicates that photosensitiza- tion is mediated under our conditions by the visible light absorbed by CAPC.

The response of cells to cytotoxic agents can vary as a function of their position in the cell cycle. For example, Fig. 6 shows that for X-irradiation Chinese hamster cells are maximally sensitive at the GliS border and are most resistant at late S , 4 h after removal of HU. There is no change in the response to CAPC photosensitization throughout the cell cycle (Fig. 6), and the age response curve is virtually flat.

DISCUSSION

Our results show that CAPC is an efficient photo- sensitizer of mammalian cells. Photo-induced cell killing was observed at a very low concentration of the dye and moderate light intensities. The CAPC exhibits absorption bands in the visible range at 670, 638 (s), and 600 nm. The observed effect is due to the excitation of these bands, as deduced from a UV cutoff experiment (Fig. 5) and further verified using a

dye-laser. Thus CAPC-induced photoinactivation was also obtained using a DCM dye laser in the tuning range of 654-663 nm, pumped by a copper- vapor laser. It was noted in these preliminary laser experiments that the shape of the survival curve was different from the one obtained with fluorescent light. The various aspects of the laser-induced photo- inactivation are presently under investigation.

Photoexcited non-transition metallophthalocy- anines may reach, with a very low quantum yield, the triplet state which lies at an energy level of ca. 1.1 eV (Vincett et al., 1971). The quenching of this state by oxygen was reported (Darwent etal . , 1982). Howev- er, '02 is apparently not produced to a significant extent, judging from lack of enhancement of the effect in the presence of D 2 0 (Fig. 3). A note of caution regarding the use of D 2 0 as a test for '02 involvement should be added. We observed that cells incubated in D 2 0 growth medium after CAPC and light treatment become more sensitive due to inhibi- tion by D20 of repair processes. As a result, expo- sure to light in D 2 0 can show an enhanced response when the fluence rate is sufficiently low and exposure time is longer than 30 min.

We found that the exposure to light (A = 620 k 10 nm) of a dimethyl sulfoxide solution of CAPC in the presence of 5,5-dirnethyl-l-pyrroline-l-oxide (DMPO) as a spin trap, in the cavity of an ESR spectrophotometer, resulted in the formation of 0; - spin adduct (aN = 12.7 G, a r = 10.3 G). Whether 0, is responsible for the large oxygen effect (Fig. 5) can not be decided at this stage. Furthermore, hydroxyl radicals do not appear to be involved since glycerol, an effective scavenger of OH radicals, had no protective effect against photosensitization by CAPC. It is noted that O H radicals could not be detected upon exposure to light of phthalocyanine dispersions in aqueous medium (Horburn and Hair, 1978). It is possible that the photoexcited CAPC reacts directly with a vital intracellular target. In- deed, nanosecond laser spectroscopy has shown that the photoexcited triplet state of CAPC is oxidized by electron acceptors and reduced by electron donors (Ohno et al. , 1983). An electron donor in the cell that is semioxidized by CAPC could then further react with 0 2 in a typical Type I photodynamic reaction. Preliminary results using the alkaline elution techni- que, indicate that some of the damage is localized in DNA but whether this damage is responsible for the observed cell killing remains to be determined.

Unlike most DNA-damaging agents (e.g. X-rays and UVC), the response of the cells to CAPC-plus- light does not change throughout the cell-cycle (Fig. 6). However, this is not an indication that CAPC- induced damage in DNA is not biologically impor- tant. A flat age response structure can be seen even when cellular damage is strictly localized in DNA, as when 5-bromodeoxyuridine incorporated into DNA is photolysed (Ben-Hur and Elkind, 1972).

Phthalocyanine derivatives are reported to be non-toxic. Thus copper phthalocyanine trisulfonic

Photosensitization by phthalocyanines 133

acid, barium and sodium salts are practically non toxic to various species of protozoa, crustaceaus, nematodes, fish, rabbits, rats and guinea pigs when given for long periods (Moser and Thomas, 1984). Tetrasulfonated copper phthalocyanine was given in doses up to 100 mg/kg to a large number of rabbits, mice, guinea pigs, cats and dogs with apparent impunity. Furthermore, this latter dye was reported to be retained preferentially in experimentally pro- duced intracranial neoplasms in mice (Wrenn et al., 1951). Similarly, uranyl tetrasulfonate phthalocy- anine was shown to accumulate in brain tumors (Frigerio, 1962). Although the blood-brain barrier is often disturbed in such tumors, and thereby drug uptake is generally increased, a recent report also indicates a preferential uptake of Tc-tetrasulfo- phthalocyanine into mammary adenocarcinoma in rats (Rousseau et al., 1983). These observations suggest that phthalocyanine derivatives could be promising photosensitizers for use in PDT. Their potential for this purpose is currently being explored in our laboratories.

Acknowledgements-Some experiments were carried out in Dr. M. M. Elkind’s laboratory, to whom we are grateful. We are also indebted to Dr. J . S. Bedford for providing facilities and advising with the experiments involving ox- ygen removal.

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