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Journal of Photochemistry and Photobiology B..mmtm'r B: Biology 28 (1995) 219-221 ELSEVIER

Modes of photodynamic vs. sonodynamic cytotoxicity

David Kessel ~'*, Julie Lo t,, Russel l Jeffers c, j. Brian Fowlkes d, Charles Cain c "Department of Pharmacology, Wayne State UniversiOJ School of Medicine, Detroit, M1 48201, USA

b Department of Medical Physics, Wayne State University School of Medicine, Detroit, MI 48201, USA Department of Electrical Engineenng and Computer Science, University of Michigan, Ann Arbor, M1 49109, USA

d Department of Radiology, University of Michigan, Ann Arbor, MI 49109, USA

Received 20 October 1994; accepted 4 January 1995

Abstract

We compared effects of ultrasound-induced vs. photodynamic cytotoxicity in cell culture. The photodynamic effects mediated ~y mesoporphyrin caused a delayed toxic reaction, the presence of a "shoulder" on the dose-response curve, indicating the capacity for limited repair of photodamage. In contrast, ultrasound-induced loss of viability resulted from rapid cell destruction and was proportional to the time of sonication. Photodynamic damage to cells before exposure to ultrasound potentiated cell breakage but did not affect the clonogenicity of the surviving cell population. Photodamage after exposure to ultrasound decreased the viability of cells which had survived ultrasonic treatment.

l~eywords: Photosensitization; Ultrasound; Porphyrins

1. Introduction

The term "photodynamic therapy" (PDT) relates to the cytotoxic process initiated by irradiation of pho- tosensitized tissues and involves production of oxygen aad/or oxygen radicals [1-3]. "Sonodynamic therapy" (SDT) is a newer concept which relates to the ability of ultrasound to produce cytotoxic effects on tissues. Cytotoxicity of SDT can be enhanced by the presence of porphyrins and porphyrin analogs, both in cell culture and in experimental animal tumour models [4--8]. The structure-activity relationships are not related to the PDT efficacy of these agents [9]. In this study we have examined the kinetics of photo- and sonodamage, along ~vith interactions between the two modalities.

2. Methods

Mesoporphyrin (MP) was provided by Porphyrin Products, Logan, UT. To avoid effects related to the et~hanced sonodamage caused by very dilute solutions ~f organic solvents [10], MP was dissolved in 10 mM NaOH for these studies. Murine leukemia L1210 cells were grown in Fischer's medium (GIBCO, Grand Island, N i') supplemented with glutamine, 10% horse serum

': Corresponding author.

10~ 1-1344/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved ,vS.91 1011-1344(94)07111-3

and gentamicin. All operations were carried out at 37 °C. Exponentially growing cells were collected by cen- trifugation, resuspended (5 × 106 cells ml-~) in serum- free Fischer's medium buffered to pH 7.0 with 20 mM HEPES and incubated (if specified) with 5 /zM MP for 30 min. Subsequent irradiation was carried out using a 600 W quartz-halogen lamp. The wavelength of irradiation was limited to 630 + 5 nm by an interference filter. IR irradiation was further attenuated by a heat- absorbing filter (transmission 400-850 nm) and a 10 cm layer of water. The light dose was determined with a calibrated EGG 450-1 radiometer. The resulting light flux was 5 mW cm -2 and the light dose was varied from 0 (dark controls) to 0.36 J cm-2.

Exposure of cell suspensions to ultrasound was carried out in 2.5 cm diameter Petri dishes in a bath of degassed water as described previously [9]. A 2.54 cm diameter transducer (Valpey-Fisher) was driven by a power amplifier (ENI 240L) with a continuous wave, 1.94 MHz sinusoidal signal source (Wavetek Model 23). The acoustic intensity, expressed in terms of the spatial peak temporal average (SPTA), was 7.5 W cm- 2. Further details are provided in Ref. [9].

In some experiments, cells were exposed to light, then ultrasound, or this was done in the reverse order. The light dose was 0.27 J cm -2, sufficient to cause an approximately 25% decrease in viability. Ultrasound was delivered for a period of 15 s so as to afford an

220 D. Kessel et al. / J. Photochem. Photobiol. B: Biol. 28 (1995) 219-221

immediate 50% decrease in the cell number. Cell counts before and after experimental procedures were deter- mined with a Coulter Electronics ZM Particle Analyzer and Model 256 Channelyzer. Viability testing involved dilution into fresh medium such that the initial density was less than 6000 m1-1. Cell counts were again de- termined after incubation for sufficient time to allow the number of control (untreated) cells to reach 200 000 m1-1, approximately five doublings. In parallel studies we established that use of a soft agar assay system yielded viability data which were not significantly dif- ferent from the growth curve information.

3. Results and discussion

The photodynamic inhibition of cell growth exhibited a shoulder, with no loss of viability observed until the light dose exceeded 0.2 J cm -2 (Fig. 1). Light or MP alone had no effect on viability (not shown). The SDT dose-response curve (power 7.5 W cm -2) had no de- tectable shoulder, indicating an "all or none" effect (Fig. 2).

Effects of SDT vs. PDT are shown in Fig. 3. The solid bars indicate the number of cells which remain unbroken, expressed as per cent control. Cross-hatched bars indicate the viability of these unbroken cells, also as per cent control. Irradiation of MP-loaded cells (0.27 J cm -2) caused no detectable cell breakage, but there was a 25% decrease in viability (Fig. 3B). Exposure of control cells to ultrasound for 8 s resulted in breakage of 35% of the cell population; the viability of the remaining cells was not significantly diminished (Fig. 3C). Exposure of cells to ultrasound in the presence of MP enhanced cell breakage, but the viability of the remaining cell population was unaffected (Fig. 3D). If different conditions are chosen, a substantial promotion of ultrasound-induced damage in the presence of MP can be demonstrated [9].

M 5000

• ~ 1 0 0 0 '

T l o o ~ 50 i t L t I L __

0 10 20 30 40 50 60 70

Time (sec) Fig. 2. Sonodynamic cell destruction as a function of the time of exposure of cells to 7.5 W cm -2 at 1.94 MHz, applied for the specified interval.

11111

8O

o t _

60 t -

O

40

20

A B C D E F

T r e a t m e n t

Fig. 3. Effects of experimental procedures on immediate cell de- struction (solid bars) vs. viability of surviving cells (cross-hatched bars): A, untreated controls; B, cells exposed to 5 /s.M MP and light (0.27 J cm-2); C, ceils treated with 7.5 W cm -2 for 8 s; D, cells exposed to ultrasound after incubation with MP; E, cells loaded with MP and exposed to ultrasound, then light; F, cells loaded with MP and e ~ d to light, then ultrasound.

200

100 i - -- . z

E ,-i =,-

If) o

10

1

0 .5 0 .00 0 .10 0 .20 0 .30 0 .40

l ight d o s e in J l c m 2

Fig. 1. Photodynamic effects of mesoporphyrin on cell viability as a function of the light dose. D a t a a r e m e a n s + S D (standard deviation) of three replicate experiments.

When cells are loaded with MP, exposed to light and then ultrasound, cell breakage was significantly enhanced, although the viability of the surviving cells was nearly 100% (Fig. 3F). This result suggests that membrane photodamage sensitizes cells to breakage by ultrasound but has no "delayed" effect on cell viability. If MP-loaded cells were first exposed to ultrasound and then to light, the extent of cell breakage was unaffected but there was a marked decrease in the viability of surviving cells (Fig. 3E). This appears to be more than an additive effect and could derive from PDT-induced inhibition of an otherwise highly effective process for repair of sublethal effects of ultrasound. Alternatively, ultrasound could promote relocalization of MP to intracellular loci such that photodynamic cytotoxicity is enhanced.

D. Kessel et al. / J. Photochem. Photobiol. B: Biol. 28 (1995) 219-221 221

The mechanism of promotion of SDT by porphyrins and porphyrin analogs remains to be established. We have reported that cavitation is an important factor in production of sonodamage [9]. Formation of singlet oxygen during porphyrin sonication has been suggested as a possible mechanism [11], but this is difficult to reconcile with the finding [9] that SDT can be enhanced by copper protoporphyrin [9].

The studies reported here suggest a potential role for ultrasound in the enhancement of a photodynamic response. In considering potential applications, it must be remembered, however, that effects of PDT in vivo generally involve the tumor vasculature [1], so that therapeutic predictions based solely on in vitro results may be incorrect. An additional factor to be considered in the design of in vivo experiments is the selectivity :gf the effect. While photodynamic therapy has been demonstrated to target neoplastic tissues [1-3], effects 3f ultrasound may involve general tissue damage. Use :)f tumor-localizing agents to potentiate SDT may in- :rease selective cytotoxicity.

~,cknowledgements

This work was supported by grants CA 55357, CA 23378 and CA 52997 from the National Cancer Institute, ~IIH, DHHS.

References

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[3] D. Kessel, Photodynamic therapy and neoplastic disease, OncoL Res., 4 (1992) 219-225.

[4] S. Umemura, S. Yurnita, R. Nishigaki and K. Umemura, Mech- anism of cell damage by ultrasound in combination with he- matoporphyrin, Jpn. Z Cancer Res., 81 (1990) 962-966.

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[8] IC Tachibana, N. Kimura, M. Okumura, H. Egushi and S. Tachibana, Enhancement of cell killing of HL-60 cells by ultrasound in the presence of the photosensitizing drug Photofrin II, Cancer Lett., 72 (1993) 195-199.

[9] D. Kessel, R. Jeffers, J.B. Fowlkes and C.A. Cain, Porphyrin- induced enhancement of ultrasound cytotoxicity, Int. J. Radiat. Biol., 66 (1994) 221-228.

[10] R.J. Jeffers, R.Q. Feng, J.B. Fowikes, J.W. Hunt and C.A. Cain, Enhanced cytotoxicity of dimethylformamide by ultrasound in vitro, Proc. IEEE Ultrason. Syrup., 9 (1992) 1241-1244.

[11] N. Yumita, R. Nishigaki, K. Umemura, P.D. Worse, H.M. Swartz, C.A. Cain and S. Umemura, Sonochemical activation of hematoporphyrin: an ESR study, Radiat. Res., 138 (1994) 171-176.