Intracellular uptake, absorption spectrum and stability of the bacteriochlorin photosensitizer 5,10,15, 20-tetrakis (m-hydroxyphenyl) bacteriochlorin (mTHPBC). Comparison with 5,10,15,20-tetrakis (m-hydroxyphenyl) chlorin (mTHPC)

Jourludof AND

tI-IJII2~t3IIX~ B:BiOLOGY

ELSEVIER

Journal of Photochemistry and Photobiology B: Biology 37 (1997) 261-266

Intracellular uptake, absorption spectrum and stability of the bacteriochlorin photosensitizer 5,10,15,20-tetrakis (mhydroxyphenyl) bacteriochlorin (mTHPBC). Comparison with 5,10,15,20-tetrakis (m-hydroxyphenyl) chlorin ( mTHPC ) Michael F. Grahn a,., Anita McGuinness a,b, Robin Benzie a, Rachael Boyle b, Martin L. de Jode "~,Michael G. Dilkes c, Babar Abbas b, Norman S. Williams a a Academic Surgical Unit, St. Bartholomew's and The Royal London Hospital School of Medicine and Dentistry, Whitechapel, London, E1 1BB, UK b School of Life Sciences, Kingston University, Kingston-upon-Thames, Surrey, KT1 2EE, UK Department of ENT Surgery, Northwick Park Hospital, Harrow, Middlesex HA1 3UJ, UK Received 23 May 1996; accepted 5 July 1996

Abstract

The bacteriochlorin photosensitizer 5,10,15,20-tetrakis (m-hydroxyphenyl) bacteriochlorin ( mTHPB C ) i s a member of a series of related compounds which includes the well-known compound 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (mTHPC) (temoporfin). Although this bacteriochlorin has near-ideal spectral characteristics in pure solvents, little is known of its stability or other characteristics within tumour cells. This study compares mTHPBC with mTHPC in both solvents in vitro and monolayers of the mouse colon tumour cell line Colo26. In aqueous protein-containing solvents, mTHPBC shows signs of aggregation and is oxidized to mTHPC at a rate of 2% h- i. Both drugs are taken up by the cells at similar rates and to the same extent, with plateau levels being reached between 9 and 30 h of incubation. Between 25% and 33% of the bacteriochlorin within the cells is oxidized to chlorin in 24 h, after which no further net oxidation is observed. The intracellular absorption spectra suggest that mTHPBC exists in more than one form within the cells. Measurements of photodynamic therapy (PDT) activity confrm that mTHPBC is active within these cells, but with between 0.6 and 0.7 of the potency of mTHPC. Although aggregation and oxidation of the bacteriochlorin will reduce its overall effectiveness, this must be balanced against the potential effect of the greater red light penetration in vivo and the presence of a green light peak which may be employed to treat thin lesions where there is a risk of perforation of a hollow organ. © 1997 Elsevier Science S.A. All rights reserved. Keywords: Cultured cells; Mesoporphyrins; Photochemotherapy; Spectrophotometry

1, I n t r o d u c t i o n Photodynamic therapy (PDT) depends on the activation of a tumour-localizing sensitizer by light to catalyse the formation of reactive oxygen species within the target tissue ( see Refs. [ 1,2 ] for reviews). The ideal PDT photosensitizer must be capable of being taken up by cells and of efficiently capturing light at those wavelengths in the red portion of the spectrum which best penetrate tissue. Improvements in both the magnitude and wavelength of the lowest energy absorption band of porphyrin-based molecules can be achieved by reduction of the porphyrin ring, first to a chlorin and secondly to a bacteriochlorin. Because of these potential advantages, * Corresponding author. Tel.: +44 171 377 7000 (ext. 3156); fax: +44 171 377 7283; e-mail: [email protected] 1011-1344/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved PHS1011-1344(96)07421-0

several families of bacteriochlorin compounds have undergone investigation as potential photosensitizers for PDT [3-61. The bacteriochlorin photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin ( m T H P B C ) is a member of a family of related tetrahydroxyphenyl compounds, which includes the well-known compound 5,10,15,20tetrakis(m-hydroxyphenyl)chlorin ( m T H P C ) [ 7 - 9 ] . In these closely related compounds, both the magnitude and wavelength of the red light absorbance and the bioactivity in a mouse-implanted tumour model increase from porphyrin t o chlorin to bacteriochlorin. Although it shows the greatest bioactivity, the bacteriochlorin has not been chosen for further evaluation at this time because of concerns about its relative instability [9]. Instead, the chlorin mTHPC has been

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developed and is now showing very encouraging results in experimental models and clinical trials [ 10,11 ]. Because mTHPC is showing such success, and in view of the potential advantages of using a bacteriochlorin, a detailed examination of the stability and cellular uptake of mTHPBC was undertaken and is reported here.

2. Experimental details

showed spectral characteristics closest to those in the cells and using the assumption that the effective path length was the estimated depth of the cell monolayer. The absorption spectra of the drugs in solution were recorded in standard cuvettes in a U3000 spectrophotometer using a bandwidth of 1 nm and recording the absorbance at 0.5 nm intervals. Data files were transferred to a PC-compatible format for subsequent analysis. The solutions were held at 37 °C in an incubator between measurements.

2.1. Preparation of photosensitizer solutions

2.4. Measurement of PDT cytotoxicity

The photosensitizers mTHPC (temoporfin) and mTHPBC were obtained from Scotia Pharmaceuticals Ltd., Guildford, UK. Stock solutions and subdilutions for addition to cell cultures were made using the recommended solvent (1 g ethanol and 1.5 g poly(ethylene glycol) (PEG) 400 made up to 5 ml with water). Reference spectra in water, phosphatebuffered saline, RPM11640 glutimax medium, with and without the addition of 10% heat-inactivated foetal calf serum (Gibco, Paisley, UK), and ethanol were prepared by a single 1 : 500 dilution of a 5 mg ml-~ stock solution into each solvent.

The photodestruction of cells was measured using a colorimetric (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) assay [12] which has been well characterized for use as a measure of the PDT effect [ 13]. Equal numbers (3 X 104) of Colo26 cells were placed into the wells of a 96-well microtitre plate in 0.2 ml of medium and different amounts of each photosensitizer were added in 50 ~1 medium to give final concentrations in the range 0-1 I~g ml-1 mTHPC and mTHPBC. The cells were incubated in the presence of photosensitizer at 37 °C in the dark for 24 h, after which they were irradiated (except the dark controls) with 4 J cm -2 of white (simulated daylight) light. Incubation was then continued in the dark for a further 24 h, after which MTI" reagent was added and the colour, which is proportional to the number of surviving cells, was developed over a 2 h period. The results are expressed as the percentage cell survival relative to the controls of groups of eight incubations.

2.2. Tumour cell cultures Cells of the mouse tumour cell line Colo26 were maintained using standard tissue culture techniques. In order to obtain absorption spectra from cell monolayers, cells were grown on a plastic support slide. This was specially made from tissue-culture-grade plastic and was designed to fit within a standard 1 cm spectrophotometer cuvette. The support slides were placed into small ( 100 mm x 15 mm) Petri dishes (Falcon type 1029; growth area, 55 cm z) to which 20 mi of a suspension of (80-160) X 103 cells ml -~ in growth medium was added. The growth medium consisted of RPMI 1640 with glutimax (Gibco, Paisley, UK) containing 10% foetal calf serum. The dishes were held in an incubator at 37 °C and 5% CO2 for 48 h during which a monolayer of cells adhered to the support slides. The slides were then, with care, removed from the Petri dishes without disturbing the adherent monolayer. This was facilitated by the use of Petri dishes made of non-tissue-culture-grade plastic so that the cells would not strongly adhere to the dish. 2.3. Spectrophotometry and data analysis After varying lengths of exposure to 14.7 I~M mTHPBC or mTHPC, the support slide was removed from the culture medium, washed by immersion in fresh medium and placed within a 3 ml cuvette thermostatically controlled at 37 °C in a Hitachi U3000 spectrophotometer. The absorption spectrum due to the photosensitizer in the cells was obtained by subtracting the spectra of corresponding control incubations. Intracellular drug concentrations were estimated using molar extinction coefficients obtained in those solvents which

3. Results 3.1. Spectra and stability in solvents in vitro Initial determinations of the spectral properties of the bacteriochlorin were made in ethanol and water (containing sufficient ethanol and PEG 400 to maintain solubility). The wavelength (Am~), half-height bandwidth (BW) and molar extinction coefficient (E) of the absorption peak used for PDT of the two photosensitizers in these solvents are shown in Table 1 and selected spectra are given in Figs. 1 and 2. The bacteriochlorin is moderately stable in ethanol solution. Over a period of 60 h of incubation (at 4 °C in the dark), the mTHPC content increases by 0.11% h - ~to 10% from the 3.4% initially present. This contrasts with the results obtained on incubation in water, where chlorin is formed almost 30 times more rapidly at 3% h - 1 over 5 h of incubation. The replacement of water with phosphate-buffered saline (pH 7.4) results in a marked precipitation of the bacteriochlorin with time, but formation of chlorin at a similar rate to that observed in water alone is also still apparent. The addition of 5% ( w / v ) bovine serum albumin to an aqueous solution in RPMI 1640 medium abolishes the precipitation of mTHPBC and lowers the rate of formation of chlorin to 2% h - i over 5

M.F. Grahn et al./ Journal of Photochemistryand Photobiology B: Biology 37 (1997) 261-266

263

Table 1 Spectral characteristics of the photosensitizersin water and ethanol mTHPC

Water Ethanol

mTHPBC

)tmax (rim)

Bandwidth

652.0 650.0

20.0 12.5

(nm)

¢

Arnax ( n m )

Bandwidth

23 000 39 000

740.0 734.5

40.0 12.5

~200

(nm)

45 000 139 000

0.14

i u 0.100 0.12

l ~ 100

'~ 0,00 O

:; 0.06

360

i 400

450

F 600

i 550

r 600

i " i 660 700

I"' 760

i 800

~

0.04

,~' 0.02

Wawl©tt~h Ore)

Fig. 1. Molar absorption spectra of mTHPC in water and ethanol. The absorption of a stock solution of mTHPC was measured in ethanol ) and water (. • • ) as described in the text. 200

i.~ 150 •~e. 1oo

i< 0 350

I 400

I 450

I 500

P 550

I 600

I 6,50

I 700

, 750

i S00

Wavel©nllth (am)

Fig. 2. Molar absorption spectra of mTHPBC in water and ethanol. The absorption of a stock solution of mTHPBC was measured in ethanol ( ) and water ( • • • ) as described in the text. h. Since the compound appears to be somewhat stabilized by the presence o f protein, it seems to be justified to continue to measure its uptake and stability in an in vitro cell culture system. 3.2. lntracellular uptake, spectra and stability The intracellular absorption spectra of both mTHPC and mTHPBC were clearly distinguishable within a few hours of incubation in medium containing photosensitizer. The increase in absorption of the intracellular 652 nm and 741 nm peaks is shown in Fig. 3. The uptake of both sensitizers is approximately linear over the first 4 h of incubation. Over longer incubation times, there is evidence that the intracellular concentrations of both drugs reach a plateau between 9 and 30 h following initial exposure to the drug. The intracellular oxidation of the bacteriochlorin to form the chlorin was monitored by observing the formation of a peak at 652 nm in monolayers of cells incubated with mTHPBC. At the early time points, it is difficult to quantify

O

.........

0

'10

20

30 Time

I ......

40 (Hours)

I .........

60

I .........

00

I

70

Fig. 3. Time course of intracellularaccumulation of mTHPC and mTHPBC. The graph shows the intracellularabsorption of mTHPC at 652 nm (O) and mTHPBC at 740 nm ( • ) after 0.5~o0 h of incubation at 37 °C. the relative proportion of mTHPC because o f the small size of the peak. After 24 h of incubation, mTHPC comprises between one-quarter and one-third o f the total cellular photosensitizer content (range results from the application of water and ethanol extinction coefficients). At 60 h of incubation, this proportion remains unchanged, indicating that oxidation is limited to the earlier part of the time period before the intracellular drug content has reached its plateau. 3.3. P D T cytotoxicity The PDT activity of mTHPC and m T H P B C in the Colo26 cell system was confirmed using the M T r test, following irradiation of the cells after 24 h of incubation with each drug. The dose-response curves for Colo26 cell survival, following 24 h incubation with a range of concentrations ( 0 - 1 I~g m l - ]; 0-1.46 ~ M ) of mTHPC and mTHPBC and irradiation with 4 J cm - 2 of white light, are shown in Fig. 4. Both compounds induce photonecrosis, with 50% cell kill obtained with approximately 50 pg m l - 1 (73 p M ) of each drug. There is no " d a r k toxicity" evident in similar incubations of these drugs without light irradiation (data not shown).

4.

Discussion

It is apparent that the mTHPBC absorption spectrum is considerably more sensitive to changes in the polarity of the solvent than is that o f m T H P C (Table 1 ). In both compounds, the absorbance of the band I peak is less in water than in ethanol, and this hypsochromic effect is considerably more marked with the bacteriochlorin than with the chlorin (32%

264

M.F. Grahn et al. / Journal of Photochemistry and Photobiology B: Biology 37 (1997) 261-266 120

--

100

--

00

--

~ el

80

--

~

40

-

o

o

60,0 (a)

610

620

(130

640

650

$S0

670

680

690

700

Wavcl©nsth(nm)

= ID

0

2O --

0

,

.001

~ ~,,

,

.01

.1

1

, ,,,r,,

I

10

Photosensitiser (pM)

Fig. 4. Photodestruction of Colo26 cells by mTHPC and mTHPBC. The graph shows the percentage survival relative to drug-free control incubations of Colo26 cells incubated for 24 h in the presence of 0.0073-1.48 IxM mTHPC ([]) and mTHPBC (11) before irradiation with 4 J cm -2 of simulated daylight. The points show the mean _+standard error of the mean of eight incubations.

and 59% respectively). However, the molar absorptivity of the bacteriochlorin in water is still greater than that of the chlorin in ethanol. A bathochromic shift in the wavelength of the band I absorption peak is also observed. This shift is similar to that previously reported for these compounds between methanol and aqueous medium [ 8 ]. The changes in the spectral characteristics are accompanied by a large reduction in the fluorescence yield (data not shown), and may be a consequence of the aggregation of the molecules as has been suggested for mTHPC [14]. Because of the relative instability of mTHPBC, observed in aqueous media, such solutions cannot be stored. However, this may not be a problem in practice, since mTHPC is presently prepared for clinical use by dissolving the photosensitizer in its vehicle immediately before injection. Intracellular absorption spectra were obtained using a cell monolayer in a large-aperture, narrow-beam spectrophotometer. This avoids the problem of light scattering, which is encountered when a cell suspension is used, and results in a greater sensitivity than when an integrating-sphere spectrophotometer is used. Measurements of photosensitizer fluorescence were not used in the present study, although they are considerably more sensitive, because of the marked changes in fluorescence yield which can occur, e.g. on aggregation, which makes a correlation between the fluorescence intensity and photosensitizer concentration problematic. Although it is technically more difficult to measure in vitro, the photosensitizer absorption is much less affected by such changes and is directly proportional to the concentration. The uptake of both photosensitizers appears to have a broadly linear initial phase and reaches a plateau by 30 h. The Beer-Lambert law can be used in combination with the molar extinction coefficients of each photosensitizer in water and ethanol to obtain upper and lower estimates of the intracellular photosensitizer concentration. If it is assumed that the cell monolayer is between 1 and 5 Ixm thick, the estimated intracellular steady state concentration of mTHPC will be

I ........ I ........ ; .......... i .......... I ......... I .......... 1.......... i .......... I .......... I .......... I 700 710 720 730 740 750 780 7TO 780 790 800 (1~) Wsv©l®nsth

(rim)

Fig. 5. mTHPCand mTHPBCabsorptionpeaksin cells and solvents.Comparison of the shape of the main absorption peak of mTHPC (a) and mTHPBC (b) in cells ( ), water (. • • ) and ethanol ( - - - ) . All spectra were normalizedto the maximumabsorbancevalue.

between 3 and 26 mM and that of mTHPBC between 5 and 27 mM. Thus it appears that both compounds are taken up by Colo26 cells to a similar extent and reach comparable steady state levels. An examination of the intracellular absorption spectra under equilibrium conditions (at 60 h) shows that the spectrum of mTHPC most closely resembles that of the sensitizer in ethanol solution (Fig. 5 ( a ) ) , with an absorption peak at 651 nm and a bandwidth of 15 nm. However, the intracellular absorption spectrum of mTHPBC (Fig. 5 ( b ) ) shows differences from both the ethanol and aqueous reference spectra. There appear to be two maxima, at 740 and 725 nm, with peaks extending over a 50 nm range. It is possible that the smaller peak represents the photosensitizer in a different state or location within the cell. Further investigations, using a combination of both absorption and fluorescence spectroscopy, are required. The relatively extended bandwidth of the far-red peak of the bacteriochlorin suggests that the photodynamic action will also be produced over a range of irradiation wavelengths. This may be of advantage when using filtered arc lamps or the newer solid state light sources [ 15]. In the case of lightemitting diodes, the entire bandwidth of the light output may be used, whilst in the case of diode lasers, a less stringent wavelength matching and temperature control of the individual laser diodes may be necessary. The bacteriochlorin also has an absorption peak in the green portion of the spectrum at 517 nm in ethanol and 525 nm in water (Fig. 2), which is also observed in the intracellular spectrum (at 520 nm). This peak may be of practical use, since green light has been proposed for the treatment of shallow lesions of relatively thin organs in which perforation is unacceptable, such as the oesophagus [ 16,17 ].

M.F. Grahn et al. / Journal of Photochemistry and Photobiology B: Biology 37 (1997) 261-266

265

light penetration and, possibly, tumour selectivity in vivo more than compensates for the bacteriochlorin's lower potency in vitro. Further work using monochromatic activation in a number of model systems is required.

5. Conclusions 4) LU 0

3

._> 4-*

4) rY 2

300

4DO

500

SOD

7111)

81)0

This preliminary study suggests that, although it is subject to significant oxidation within tumour cells, the bacteriochlorin has sufficient stability, both in potential delivery vehicles and within cells, to be used as a photosensitizer for PDT. Where oxidation does occur, the product is another, wellcharacterized, photosensitizer. The photodynamic potency of mTHPBC may be less than that of mTHPC. However, it is quite possible that these disadvantages will be outweighed by an increased effect due to the improved light penetration at the bacteriochlorin red peak. In addition, the presence of two distinct photosensitizers which can be activated at different wavelengths may enable novel treatment protocols to be developed. In situations in which the perforation of hollow organs is a danger, additional advantage may be obtained from the activation of the photosensitizer using green light.

Wavelength (nm) Fig. 6. Spectral distribution of the light source. The relative spectral energy distribution of the simulated daylight source is shown between 300 and 800 nm.

In the original work by Bonnett et al. [8], mTHPBC appeared to be about twice as potent a photosensitizer as mTHPC. In that study, similar tumour necrosis in mice was obtained using approximately half as much mTHPBC as mTHPC at the same light dose. The present spectroscopic studies suggest that mTHPBC may be more sensitive to alterations in the solvent state, and so may lose some of its potential PDT potency through aggregation within tumour cells. A comparison of the PDT activity with that of mTHPC was therefore carried out in the Colo26 cell line. Although 50% cell kill was observed at a similar drug concentration for both photosensitizers, a direct comparison of PDT potency is not possible because a white light source was used. The spectral energy distribution of the light source is shown in Fig. 6. The relative activation efficiency, in terms of the number of photons absorbed by each photosensitizer, was estimated by numerically integrating the product of the power emitted by the light source per unit wavelength and the specific absorbance with respect to wavelength over the range 300-800 nm. On this basis, mTHPBC is activated between 1.5 and 1.7 times as much as mTHPC by the "daylight" lamps. That both drugs killed half the cells at a similar concentration therefore suggests that mTHPBC is less potent than mTHPC in this system, by a factor of between 0.6 and 0.7, depending on whether the conversion to mTHPC is taken into account. The discrepancy between these results and those obtained earlier in mice could be merely due to differences in the model systems used, or may suggest that a combination of greater

Acknowledgements A. McGuinness was supported by a research studentship from Scotia Pharmaceuticals Ltd. We are grateful to Professor R. Bonnett of Queen Mary and Westfield College for advice and comments.

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