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Wavelength dependence of the fluorescence and singlet oxygen quantum yields of new photosensitizers
23 December 1994
ELSEVlER
CHEMlCiL PHYSICS LETTERS
Chemical Physics Letters 231 (1994) 144-150
Wavelength dependence of the fluorescence and singlet oxygen quantum yields of new photosensitizers Adina Lavi a, Fred R/f,Johnson b, Benjamin Ehrenberg a aDepartment ofPhysics, Bar Ilan University, Ramar Can 32-900, Israel b Department ofPhysics, California State University, Fullerton, CA 92634, USA Received 22 February 1994; in final form 6 October 1994
Abstract
The photophysi~al properties of Mg and Zn tetra~nzopo~hy~ns and Cd-texaphyrin are presented. These sensitizers have strong absorption bands in the red and near-IR regions that make them good candidates for biological photosensitization. Singlet oxygen quantum yields which were determined in an absolute manner, in several solvents, are reported. We show an unusual behavior regarding adherence to Kasha’s and Vavilov’s rules: upon excitation to different electronic states, different values of singlet oxygen quantum yields were obtained. We also show an unusual wavelength dependence of singlet oxygen and fluorescence yields upon excitation to different vibrational levels within the same electronic state.
1. Introduction Fhot~sensitization which employs porphyrins and porphyrin-like chromophores has become an active field in recent years. It is the basis of photodynamic therapy (PDT), an experimental method for treatment of malignant tumors [ 1,2]. The utilization of some porphyrins as biological sensitizers relies on their preferential uptake and retention by cancerous tissues and on their ability to cause lethal damage to cells upon illumination. The photosensitized damage is mediated mainly by the formation of singlet oxygen O2 f ‘A,) (type II mechanism ) [ 31. Long-wavelength light is preferred for clinical uses, because of its deeper penetration and lesser absorption by other chromophores which exist in a living tissue. The photosensitization action spectra of regular porphyrins, such as hematoporphyrin derivative (HPD) and photofrin-II (PF-II), which are presently used for clinical applications, exhibit only marginal absorption bands in the red, peaking around 630
nm [ 41, The effective penetration depth is only l-3 mm, and as a result, high doses have to be administered. Consequently, ~onside~ble effort has been devoted in the last few years to the development of effective sensitizers with strong absorption bands in the biologically convenient spectral region (600-800 nm) and which have high quantum yields of singlet oxygen generation [ 5,6]. In this Letter we report on the spectroscopic and photophysical properties of three sensitizers: Mg and Zn tetrabenzoporphyrins (MgTBP and ZnTBP) [ 71 and Cd-texaphyrin (CdTX ) [ 8 1. These sensitizers have strong absorption bands in the red and near-IR regions. We thus studied the photophysical properties of these sensitizers. We employed 9,10-dimethylanthracene (DMA) as a spectroscopically monitored molecular target for singlet oxygen, to obtain the absolute value of the quantum yield of singlet oxygen generation of the sensitizers in several organic solvents. We found that this quantum yield, as well as the fluorescence quantum yield exhibited an unusual
OOOP-2614/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDIOOOP-2614(94)01262-S
A. Lavi et al. /Chemical Physics Letters 231 (1994) 144-150
dependence on the excitation wavelength, comparing two different electronic absorption and even within the same electronic band.
when bands,
2. Materials and methods 2.1. Chemicals Mg2+-tetrabenzoporphyrin ( MgTBP) was prepared according to Barrett et al. and Linstead [ 9,10 1. To ensure purity the final step in the organic synthesis was sublimation of the compound at 450-500°C. The chemical formula MgC36N4H20, was confirmed via high resolution mass spectrometry. ZnTBP was obtained from Porphyrin Products, Logan, UT. Cdtexaphyrin was a gift from Dr. Jonathan L. Sessler from the University of Texas at Austin. DMA and rhodamine-6G were from Sigma. Concentrated stock solutions of MgTBP, ZnTBP and CdTX were prepared in DMF. 2.2. Irradiations A HeNe laser (Uniphase, Stevenage, Hertfordshire, UK) or a HeCd laser was used as the irradiation source for the tetrabenzoporphyrins. A diode laser (783 nm) or a HeCd laser was used as the irradiation source for texaphyrin. For measurements of the dependence of singlet oxygen quantum yield on the exciting wavelength the irradiation source was a dye laser (Coherent, model CR-599) using rhodamine B, pumped by an Ar+ laser (Coherent, Palo Alto, CA, model Innova 200). The laser light, tunable in the range 605-635 nm, was transferred to the top of the sample cuvette with an optical fiber (Fiberguide Industries, Stirling, NJ ), core diameter 400 pm. The beam power on the sample’s surface ( 12.5 mW for HeNe, 7.5 mW for HeCd, 12.5 mW for diode laser and lo- 15 mW for wavelengths obtained by the dye laser) was measured with a laser power meter (Ophir, Israel, model PD2-A) prior and after the measurements, which lasted less than 10 min. The dye laser exhibited fast power ripples of less than 5% peak to peak and no long term drift. The sample was airsaturated and stirred magnetically to obtain uniform irradiation of the whole sample.
2.3. Spectroscopic
145
measurements
Absorption spectra were measured on a Varian (Mulgrave, Australia) UV-visible spectrometer (model DMS-200). The fluorescence time-drive measurements were performed on a digital fluorimeter (Perkin-Elmer, Norwalk, CT, model LS-50), which is controlled by a PC. In the DMA photosensitization experiments, its fluorescence was excited at 360 nm (for DMA concentrations of 5 pm ) or at 420 nm (for concentrations of 1 mM and 2 mM). The excitation wavelength was chosen so that the optical density at this wavelength was always lower than 0.05, to maintain a linear correspondence between concentration afid fluorescence intensity. The fl:lorescence was monitored at 457 nm when excited with the HeNe or the diode laser and at 468 nm upon excitation with the HeCd. Laser irradiation of the sample was carried out in situ in the fluorimeter. A broad band (20 nm fwhm) interference filter (457 or 468 nm) was placed in the emission beam to isolate the fluorescence of DMA from stray light. The fluorescence traces were transferred to an IBM-PC or IBM 3270 mainframe computer, and fitted to analytical functions by least squares fitting programs. The self-sensitization rates of the photosensitizers were measured, in duplicate samples and under the same irradiation procedures as for DMA photosensitizations, as follows: for MgTBP and ZnTBP a fluorescence time drive was run. The excitation wavelength was 6 15 nm and the emission wavelength was 700 nm. A 640 nm cut-on filter was placed at the emission beam to filter out the HeNe laser light. The fluorescence excited by the HeNe laser did not interfere in the measurements, since the fluorimeter detects only light that is in phase with its chopped light source. Because of the diode laser stray light interference, the self-sensitization of CdTX was monitored by measuring the fluorescence intensity after short periods (3-5 s each interval) of irradiation with the diode laser. For the measurements of fluorescence quantum yield the excitation spectra of the sensitizers were taken within the Soret band and within the Q band (slit width 2.5 nm), with the emission wavelength set at 638 nm (slit width: 5 nm). The concentration was 0.25-0.3 MM for measurements in the Soret band and
A. Lavi et al. /Chemical PhysicsLetters 231 (1994) 144-150
146
0.6-0.7 uM for the Q band. The concentrations were chosen so that the optical density at maximum absorption did not exceed 0.1 to avoid a geometrical problem in light collection from the cuvette. For each excitation wavelength the integrated area under the fluorescence spectra was calculated from the fluorescence intensity at 638 nm. The excitation spectra were corrected for the absorbancies, by dividing by l10-O.d.. Absolute values of Q+ were determined by comparison with rhodamine 6G in methanol (0.5 pM) whose fluorescence quantum yield is 0.83 [ 111.
3. Results and discussion
tern crossing of the sensitizer to the triplet level and the yield of formation of singlet oxygen from the triplet. Usually, @* values are determined relative to each other or to a sensitizer with a known yield. We use here a modified method for the determination of the absolute yield of singlet oxygen [ 13 1. This method employs a molecule which acts as a target for singlet oxygen attack, and which exhibits a spectral change upon the oxidation. The quantum yield of the disappearance of the target A is dA k[Al ‘Ao=-dtll=@AkAIA]+k,,+(k,+k,)[Q] =@
k[Al ‘k,[A]+k’
Fig. 1 shows the absorption spectra of MgTBP, ZnTBP, CdTX and photofrin-II. One should notice the substantially stronger absorption of the TBPs at 633 nm (the HeNe line which is used for clinical applications with porphyrins) as compared to the absorption of hematoporphyrin. The CdTX has its maximum absorption at 760 nm. At this wavelength, penetration into the tissue is much deeper than in the visible range. Molecular singlet oxygen is produced by a collisional energy transfer and spin exchange with the photosensitizer in its excited triplet state [ 121. The quantitative parameter which defines the sensitizer’s efficiency is the quantum yield of singlet oxygen production per photon absorbed in the sensitizer, @*. This parameter is the product of the yield of intersys-
(1)
’
where I is the rate of photons’ absorption by the sensitizer (computed from the power of the laser, the photon’s energy at the exciting wavelength and the sensitizer’s optical density), the kA, k,, kQ and k, are the rate constants of the photooxidation of A, of singlet oxygen decay, of physical or chemical quenching of singlet oxygen by any possible quencher, respectively. The fluorescence was excited at a wavelength at which the optical density of the target was less than 0.05 so that the fluorescence could be taken as linearly dependent on the concentration [A] =/IF. Substituting in Eq. ( 1), we get F i=@AjIF+k’/kA
= @Aj3F:a’
(2)
It follows from Eq. (2 ) that when (Y> j3F the rate of the fluorescence decay of the target is of first order regarding F, as shown in
d[Fl dt
@AIF
=(Y
(3)
Under such conditions, @Acannot be determined. However, at higher target concentrations, solving Eq. (2) yields the following analytical expression, from which (Y,or the quantum yield can be extracted, lnF+{F=lnF,++$F,-?t. Fig. 1. Absorption spectra CdTX (--.,--) and PF-II absorption of MgTBP and of the longest wavelength
of 5 FM MgTBP (-). ZnTBP ( ...), (---) in ethanol. The inset shows the ZnTBP, compared to PF-II, in the range Q band, from 600 to 650 nm.
(4)
We chose DMA as our target because of its high reaction rate with singlet oxygen and because it can be followed easily by its fluorescence, which is not
141
A. Lavi et al. / Chemical Physics Letters 231 (1994) 144-150
affected by the spectral properties
of the sensitizers
[141. As seen in Eq. ( 3), in order to calculate the value of @* from the first-order kinetic trace, one has to know the value of (Y, which is an intrinsic chemical parameter, and is also solvent dependent. We thus evaluated (Yby fitting the kinetic traces of photosensitization of DMA at high concentration, by a nonlinear fitting program, to Eq. (4). We employed DMA at initial concentrations of 1 or 2 mM. In Fig. 2 we show, as an example, the kinetic trace of photosensitization of DMA by CdTX in pyridine and its fit to Eq. (4 ). The excitation wavelength for these experiments was 420 nm, where the absorbancies were low enough for the relation [A] = j?F to hold. Table 1 shows the values of a!, in four solvents, compared to literature values [ 15 1. We now employed the values of a! obtained above to analyze the kinetics at low DMA concentration. In Fig. 3 we show three representative cases of the kinetic curves for DMA photosensitization by MgTBP in various solvents. The initial concentration of the target was 5 pm which agrees with LY> j3F (since a typical value of LYis lop3 M); the concentration of the sensitizer was 10 uM. The kinetic traces were thus fitted to a simple exponent taking into consideration an asymptotical background fluorescence. T, the time constant of the decay was obtained from the fit. In the case of ZnTBP in methanol the kinetic curve did not lit a simple exponent.
Table 1 The parameter 01of DMA in several solvents. (Ywas obtained by fitting kinetic traces ofphotosensitization of DMA at higher concentration to Eq. (4) a and is compared to literature values Solvent
OIb (x10-‘M)
crc (x~O-~M)
acetonitrile benzene methanol pyridine
0.34 1.31 * 0.962 0.61
0.2TO.04 0.3, 2+0.5, 1.2 c 8f0.5, 3TO.6’ 1.4, 1.0 d
a All values are within accuracy of 5%. b Values of o from tit to Eq. (4). E Values from Ref. [ 151. dAverage of two measurements. ’ Different values appear in literature.
“,,A ‘.
0
100
200
300
400
500
600
time (set) Fig. 3. Fluorescence kinetic traces of DMA (5 pM) sensitized by MgTBP ( 10 FM) in three different solvents and their tits to Eq. (3). Data (---), fit (---); (A) benzene, (B) pyridine, (C) acctonitrile. 1,,,=360 nm, A,,=457 nm. The irradiation source was a 10 mW HeNe laser.
0
100
200 time (set)
300
400
Fig. 2. Fluorescence time drive of DMA at high concentration (00o) and the tit to Eq. (4) (p). DMA ( 1 mM in pyridine) was sensitized by 10 pM CdTX, which was irradiated by 12.5 mW of a diode laser at 783 nm. The fluorescence of DMA was measured at ,I,,, = 420 nm, A,,,,=468 nm.
Table 2 shows the calculated values of 0. for the three sensitizers, in various solvents: It can be seen that the singlet oxygen formation is very efficient, and in some solvents is as high as that of the best photosensitizers. The high values of @* together with the strong absorption at long wavelengths make these sensitizers good candidates for biological sensitization. In all the reported cases in this study the self-sensitization of the sensitizer was much slower than the sensitization of the DMA. Therefore the solutions (the exponent and Eq. (4) ) are not complicated by a decrease in the sensitizers’ concentration. A wavelength dependence is generally not ex-
148
A. Luvi et al. / Chemical Physics Letters 231 (1994) 144-150
Table 2
Quantum yields of singlet oxygen production Solvent
by MgTBP, ZnTBP and CdTX in various
MgTBP
solvents
ZnTBP
CdTX
@*
@A
0.75 1 a, 0.998 b 0.497 a e 0.3 a
d 0.727 =, 0.855 b 0.244 ’ 0.33 c
@*a acetonitrile benzene methanol pyridine
0.601 0.34 0.126 0.317
0.481 0.565 0.159 0.367
’ Irradiation at 632.8 nm. ’ Irradiation at 441.6 nm. ‘Irradiation d Values of @* obtained were higher than 1. ’ Fluorescence time drive did not fit a single exponent function. ‘All values are within 6% accuracy except for MgTBP in acetonitrile
petted in photochemical reactions because of efficient internal conversion from an upper to the lowest excited singlet state (Kasha’s rule). However, since the Soret band of the tetrabenzoporphyrins is much stronger than the long wavelength band, we decided to investigate the wavelength dependence of the photosensitization reaction. We compared the values of @A of our sensitizers upon excitation to different electronic states, using the HeCd 44 1.6 nm line and HeNe 632.8 nm (for the TBPs) or a diode laser line at 783 nm (for CdTX). Table 2 shows the values of @,, determined from the photosensitization process at two different irradiation wavelengths. As can be seen, in the case of MgTBP in benzene, @Afor excitation at 632.8 nm was 0.34 as opposed to 0.56 when excited at 441.6. This trend of a lower quantum yield when excited at the longer wavelength can be observed in the table in additional solvents and with the other sensitizers as well. A possible explanation for a greater efficiency of singlet oxygen formation upon excitation with shorter wavelengths is that intersystern-crossing from Sz to the triplet states competes successfully with the internal conversion of Sz to S, and results in a more efficient formation of O2 ( ‘4). Intersystem-crossing from higher excited singlet levels to the triplet ladder has been observed previously with some other sensitizers as well as with other molecules [ 16-201. Wavelength dependence of photochemical reactions or of fluorescence/phosphorescence intensity ratio was ascribed to this effect. An interesting case is that of CdTX in acetonitrile where, again, the @* is higher upon excitation at 441.6 nm than at 632.8, however both numbers are higher than 1, 1.47 and 1.23, respectively. Singlet oxygen
at 783 nm.
where error margin is 10%.
production yields which are higher than 1 can be explained by the formation of one molecule of singlet oxygen by energy transfer from the sensitizer’s excited singlet and another molecule produced by the sensitizer’s excited triplet [ 2 11. We also checked the wavelength dependence of @* upon excitation at different wavelengths within the Q band. By tuning the dye laser, we excited MgTBP in benzene, pyridine and acetonitrile at several wavelengths. As can be seen in Fig. 4 the trend of a lower quantum yield when excited at the longer wavelength can be observed in this case as well. This behavior of wavelength dependence is even more exceptional since it occurs upon excitation to different vibrational levels within the same electronic state, and in-
. y
.
.
.
XX,
x x x
I
I
615
620
wavelength
625
630
(nm)
Fig. 4. Dependence of singlet oxygen quantum yields of MgTBP on the exciting wavelength. Singlet oxygen quantum yields of MgTBP ( 10 FM) in acetonitrile ( n ), benzene ( X ) and pyridine (0). Irradiation source was a dye laser using rhodamine B, pumped by an Ar+ laser. DMA: 5 FM, I,,,=360 nm, A,,=457 nm.
A. Lavi et al. / Chemical Physics Letters231 (1994) 144-150
dicates that intersystem-crossing competes even with vibrational relaxation within the excited electronic state. This effect has been reported before only in a very limited number of cases [ 22,231. Since the quantum yieldof singlet oxygen formation does not obey l&ha’s rule we decided to check whether the fluorescence quantum yield has the same behavior and also exhibits an equivalent dependence on the excitation wavelength. Our results show that upon exciting the sensitizer with different wavelengths within the same electronic state, different values of @r are obtained. In the case of MgTBP in benzene (Fig. 5) the fluorescence yield decreases upon excitation to higher vibrational states. A clear behavior of opposite trends of the wavelength dependencies in @, and @., is observed, although the range of overlap of the two phenomena is limited due to experimental limitations (weak fluorescence excitation at low wavelengths and the dye laser’s range). For MgTBP in acetonitrile @r increased upon excitation with longer wavelengths and this observation remains without an explanation. Fluorescence yields measured for rhodamine 6G showed no wavelength dependence (less than 5%) should be noted that in all cases the shape of the fluorescence spectrum was independent of the excitation wavelength. This indi-
>:
d 0"
I ‘I
0.2 0.16.
k
0.12~
s 6
0.08.
z
0.04.
, ‘\
__~~_
410
Acknowledgement
References
-2
0
cates that the emission occurs from the lowest relaxed, vibrational state, and in addition, it excludes the possibility of fluorescent contamination in the samples or of excited-state heterogeneity due to solvation or other similar effects. In conclusion, we have shown here that MgTBP, ZnTBP and CdTX which have strong absorption bands at long wavelengths, also exhibit efficient intersystem-crossing to the excited triplet level, and they photosensitize the production of singlet oxygen. We have demonstrated a simple method for absolute determination of 0*. We have also demonstrated that these sensitizers do not obey the classical Vavilov rule, and exhibit wavelength dependence of the fluorescence and singlet oxygen quantum yields. This behavior is observed not only when changing from one electronic band to another but also within the Q band, indicating that intersystem-crossing is possible from higher vibrational levels in the excited singlet state. The wavelength dependence of the singlet oxygen production efficiency is of practical relevance for biological photosensitization for a proper selection of the wavelength of the exciting light source.
This work was supported by a grant from the Israel Science Foundation administered by the Israel Academy of Sciences and Humanities. We thank Dr. Jonathan L. Sessler and Greg Hemmi from the University of Texas, Austin for the gift of CdTX.
-------~70.12
n
0.24~
149
-.-A __
---
430
450
wavelength
600
\_,
620
640
-0
(nm)
Fig. 5. Dependence of fluorescence quantum yields of MgTBP in benzene on the exciting wavelength. Fluorescence quantum yields (---) of MgTBP in the Soret (left ordinate) and Q (right ordinate) bands are shown together with the absorption spectrum (ordinate is not shown). The concentration was 0.25-0.3 pM for measurements in the Soret band and 0.6-0.7 nM for the Q band. Absolute values of @were determined by comparison with rhodamine 6G in methanol (0.5 uM) whose fluorescence quantum yield is 0.83.
[ 1] J. Moan, Photochem. Photobiol. 43 (1986) 681. [ 21 B.W. Henderson and T.J. Dougherty, Photochem. Photobiol. 55 (1992)
145.
[ 31 L.I. Grossweiner,
AS. Pate1 and J.B. Grossweiner, Photochem. Photobiol. 36 (1982) 159. [ 4 ] C.J. Gomer, D.R. Doiron, N. Rucker, N.J. Razum and SW. Fountain, Photochem. Photobiol. 39 (1984) 365. [ 51 M. Kreimer-Birnbaum, Seminars Hematol. 26 ( 1989) 157. [ 61 J. Moan, J. Photochem. Photobiol. B 5 ( 1990) 52 1. [ 71 B. Ehrenberg and F.M. Johnson, Spectrochimica Acta A 46 (1990) 1521. [ 81 A. Harriman, B.C. Maiya, T. Murai, G. Hemmi, J.L. Sessler and T.E. Mallouk, J. Chem. Sot. Chem. Commun. (1989) 314.
150
A. Lavi et al. / Chemical Physics Letters 231 (1994) 144-150
[9] R.P. Barrett, P. Linstead, F.G. Rundall and G.A.D. Tuey, J. Chem. Sot. (1940) 1079. [lo] P. Linstead, J. Chem. Sot. (1953) 2873. [ 111 J.B. Birks, Photophysics of aromatic molecules, Vol. 1 (Wiley, New York, 1969). [ 121 H.H. Wasserman and R.W. Murray, eds., Singlet oxygen (Academic Press, New York, 1979). [ 131 E. Gross, B. Ehrenberg and F.M. Johnson, Photochem. Photobiol. 57 (1993) 808. [ 141 M.A. Rubio, E. Lemp, M.V. Encinas and E.A. Lissi, Bol. Sot. Chil. Quim. 37 (1992) 33. [ IS] F. Wilkinson and J. Brummer, J. Phys. Chem. Ref. Data 10 (1981) 809. [ 161 M.F. O’dwyer, J. Chem. Phys. 36 (1962) 1395. [ 171 W. Star, J. Versteeg, W. Van Putten and H. Marijnissen, Photochem. Photobiol. 52 ( 1990) 547.
[ 181 A. Seret, M. Hoebeke and A. Van de Vorst, Photochem. Photobiol. 52 ( 1990) 60 1. [ 191 J.W. Arbogast, A.P. Darmanyan, C.S. Foote, Y. Rubin, F.N. Diederich, M.M. Alvarez, S.J. Anz and RI. Whetten, J. Phys. Chem.95 (1991) 11. [20] M.T. Allen, M. Lynch, A. Lagos, R.W. Redmond and I.E. Kochevar, Biochim. Biophys. Acta 1075 (1991) 42. [ 2 1 ] R.C. Kanner and C.S. Foote, J. Am. Chem. Sot. 114 ( 1992) 678. [22] G. Jones and W.G. Becker, J. Am. Chem. Sot. 103 (1981) 4630. [23] H. Morrison, C. Bernasconi and G. Pandey, Photochem. Photobiol. 40 (1984) 549.
ELSEVlER
CHEMlCiL PHYSICS LETTERS
Chemical Physics Letters 231 (1994) 144-150
Wavelength dependence of the fluorescence and singlet oxygen quantum yields of new photosensitizers Adina Lavi a, Fred R/f,Johnson b, Benjamin Ehrenberg a aDepartment ofPhysics, Bar Ilan University, Ramar Can 32-900, Israel b Department ofPhysics, California State University, Fullerton, CA 92634, USA Received 22 February 1994; in final form 6 October 1994
Abstract
The photophysi~al properties of Mg and Zn tetra~nzopo~hy~ns and Cd-texaphyrin are presented. These sensitizers have strong absorption bands in the red and near-IR regions that make them good candidates for biological photosensitization. Singlet oxygen quantum yields which were determined in an absolute manner, in several solvents, are reported. We show an unusual behavior regarding adherence to Kasha’s and Vavilov’s rules: upon excitation to different electronic states, different values of singlet oxygen quantum yields were obtained. We also show an unusual wavelength dependence of singlet oxygen and fluorescence yields upon excitation to different vibrational levels within the same electronic state.
1. Introduction Fhot~sensitization which employs porphyrins and porphyrin-like chromophores has become an active field in recent years. It is the basis of photodynamic therapy (PDT), an experimental method for treatment of malignant tumors [ 1,2]. The utilization of some porphyrins as biological sensitizers relies on their preferential uptake and retention by cancerous tissues and on their ability to cause lethal damage to cells upon illumination. The photosensitized damage is mediated mainly by the formation of singlet oxygen O2 f ‘A,) (type II mechanism ) [ 31. Long-wavelength light is preferred for clinical uses, because of its deeper penetration and lesser absorption by other chromophores which exist in a living tissue. The photosensitization action spectra of regular porphyrins, such as hematoporphyrin derivative (HPD) and photofrin-II (PF-II), which are presently used for clinical applications, exhibit only marginal absorption bands in the red, peaking around 630
nm [ 41, The effective penetration depth is only l-3 mm, and as a result, high doses have to be administered. Consequently, ~onside~ble effort has been devoted in the last few years to the development of effective sensitizers with strong absorption bands in the biologically convenient spectral region (600-800 nm) and which have high quantum yields of singlet oxygen generation [ 5,6]. In this Letter we report on the spectroscopic and photophysical properties of three sensitizers: Mg and Zn tetrabenzoporphyrins (MgTBP and ZnTBP) [ 71 and Cd-texaphyrin (CdTX ) [ 8 1. These sensitizers have strong absorption bands in the red and near-IR regions. We thus studied the photophysical properties of these sensitizers. We employed 9,10-dimethylanthracene (DMA) as a spectroscopically monitored molecular target for singlet oxygen, to obtain the absolute value of the quantum yield of singlet oxygen generation of the sensitizers in several organic solvents. We found that this quantum yield, as well as the fluorescence quantum yield exhibited an unusual
OOOP-2614/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDIOOOP-2614(94)01262-S
A. Lavi et al. /Chemical Physics Letters 231 (1994) 144-150
dependence on the excitation wavelength, comparing two different electronic absorption and even within the same electronic band.
when bands,
2. Materials and methods 2.1. Chemicals Mg2+-tetrabenzoporphyrin ( MgTBP) was prepared according to Barrett et al. and Linstead [ 9,10 1. To ensure purity the final step in the organic synthesis was sublimation of the compound at 450-500°C. The chemical formula MgC36N4H20, was confirmed via high resolution mass spectrometry. ZnTBP was obtained from Porphyrin Products, Logan, UT. Cdtexaphyrin was a gift from Dr. Jonathan L. Sessler from the University of Texas at Austin. DMA and rhodamine-6G were from Sigma. Concentrated stock solutions of MgTBP, ZnTBP and CdTX were prepared in DMF. 2.2. Irradiations A HeNe laser (Uniphase, Stevenage, Hertfordshire, UK) or a HeCd laser was used as the irradiation source for the tetrabenzoporphyrins. A diode laser (783 nm) or a HeCd laser was used as the irradiation source for texaphyrin. For measurements of the dependence of singlet oxygen quantum yield on the exciting wavelength the irradiation source was a dye laser (Coherent, model CR-599) using rhodamine B, pumped by an Ar+ laser (Coherent, Palo Alto, CA, model Innova 200). The laser light, tunable in the range 605-635 nm, was transferred to the top of the sample cuvette with an optical fiber (Fiberguide Industries, Stirling, NJ ), core diameter 400 pm. The beam power on the sample’s surface ( 12.5 mW for HeNe, 7.5 mW for HeCd, 12.5 mW for diode laser and lo- 15 mW for wavelengths obtained by the dye laser) was measured with a laser power meter (Ophir, Israel, model PD2-A) prior and after the measurements, which lasted less than 10 min. The dye laser exhibited fast power ripples of less than 5% peak to peak and no long term drift. The sample was airsaturated and stirred magnetically to obtain uniform irradiation of the whole sample.
2.3. Spectroscopic
145
measurements
Absorption spectra were measured on a Varian (Mulgrave, Australia) UV-visible spectrometer (model DMS-200). The fluorescence time-drive measurements were performed on a digital fluorimeter (Perkin-Elmer, Norwalk, CT, model LS-50), which is controlled by a PC. In the DMA photosensitization experiments, its fluorescence was excited at 360 nm (for DMA concentrations of 5 pm ) or at 420 nm (for concentrations of 1 mM and 2 mM). The excitation wavelength was chosen so that the optical density at this wavelength was always lower than 0.05, to maintain a linear correspondence between concentration afid fluorescence intensity. The fl:lorescence was monitored at 457 nm when excited with the HeNe or the diode laser and at 468 nm upon excitation with the HeCd. Laser irradiation of the sample was carried out in situ in the fluorimeter. A broad band (20 nm fwhm) interference filter (457 or 468 nm) was placed in the emission beam to isolate the fluorescence of DMA from stray light. The fluorescence traces were transferred to an IBM-PC or IBM 3270 mainframe computer, and fitted to analytical functions by least squares fitting programs. The self-sensitization rates of the photosensitizers were measured, in duplicate samples and under the same irradiation procedures as for DMA photosensitizations, as follows: for MgTBP and ZnTBP a fluorescence time drive was run. The excitation wavelength was 6 15 nm and the emission wavelength was 700 nm. A 640 nm cut-on filter was placed at the emission beam to filter out the HeNe laser light. The fluorescence excited by the HeNe laser did not interfere in the measurements, since the fluorimeter detects only light that is in phase with its chopped light source. Because of the diode laser stray light interference, the self-sensitization of CdTX was monitored by measuring the fluorescence intensity after short periods (3-5 s each interval) of irradiation with the diode laser. For the measurements of fluorescence quantum yield the excitation spectra of the sensitizers were taken within the Soret band and within the Q band (slit width 2.5 nm), with the emission wavelength set at 638 nm (slit width: 5 nm). The concentration was 0.25-0.3 MM for measurements in the Soret band and
A. Lavi et al. /Chemical PhysicsLetters 231 (1994) 144-150
146
0.6-0.7 uM for the Q band. The concentrations were chosen so that the optical density at maximum absorption did not exceed 0.1 to avoid a geometrical problem in light collection from the cuvette. For each excitation wavelength the integrated area under the fluorescence spectra was calculated from the fluorescence intensity at 638 nm. The excitation spectra were corrected for the absorbancies, by dividing by l10-O.d.. Absolute values of Q+ were determined by comparison with rhodamine 6G in methanol (0.5 pM) whose fluorescence quantum yield is 0.83 [ 111.
3. Results and discussion
tern crossing of the sensitizer to the triplet level and the yield of formation of singlet oxygen from the triplet. Usually, @* values are determined relative to each other or to a sensitizer with a known yield. We use here a modified method for the determination of the absolute yield of singlet oxygen [ 13 1. This method employs a molecule which acts as a target for singlet oxygen attack, and which exhibits a spectral change upon the oxidation. The quantum yield of the disappearance of the target A is dA k[Al ‘Ao=-dtll=@AkAIA]+k,,+(k,+k,)[Q] =@
k[Al ‘k,[A]+k’
Fig. 1 shows the absorption spectra of MgTBP, ZnTBP, CdTX and photofrin-II. One should notice the substantially stronger absorption of the TBPs at 633 nm (the HeNe line which is used for clinical applications with porphyrins) as compared to the absorption of hematoporphyrin. The CdTX has its maximum absorption at 760 nm. At this wavelength, penetration into the tissue is much deeper than in the visible range. Molecular singlet oxygen is produced by a collisional energy transfer and spin exchange with the photosensitizer in its excited triplet state [ 121. The quantitative parameter which defines the sensitizer’s efficiency is the quantum yield of singlet oxygen production per photon absorbed in the sensitizer, @*. This parameter is the product of the yield of intersys-
(1)
’
where I is the rate of photons’ absorption by the sensitizer (computed from the power of the laser, the photon’s energy at the exciting wavelength and the sensitizer’s optical density), the kA, k,, kQ and k, are the rate constants of the photooxidation of A, of singlet oxygen decay, of physical or chemical quenching of singlet oxygen by any possible quencher, respectively. The fluorescence was excited at a wavelength at which the optical density of the target was less than 0.05 so that the fluorescence could be taken as linearly dependent on the concentration [A] =/IF. Substituting in Eq. ( 1), we get F i=@AjIF+k’/kA
= @Aj3F:a’
(2)
It follows from Eq. (2 ) that when (Y> j3F the rate of the fluorescence decay of the target is of first order regarding F, as shown in
d[Fl dt
@AIF
=(Y
(3)
Under such conditions, @Acannot be determined. However, at higher target concentrations, solving Eq. (2) yields the following analytical expression, from which (Y,or the quantum yield can be extracted, lnF+{F=lnF,++$F,-?t. Fig. 1. Absorption spectra CdTX (--.,--) and PF-II absorption of MgTBP and of the longest wavelength
of 5 FM MgTBP (-). ZnTBP ( ...), (---) in ethanol. The inset shows the ZnTBP, compared to PF-II, in the range Q band, from 600 to 650 nm.
(4)
We chose DMA as our target because of its high reaction rate with singlet oxygen and because it can be followed easily by its fluorescence, which is not
141
A. Lavi et al. / Chemical Physics Letters 231 (1994) 144-150
affected by the spectral properties
of the sensitizers
[141. As seen in Eq. ( 3), in order to calculate the value of @* from the first-order kinetic trace, one has to know the value of (Y, which is an intrinsic chemical parameter, and is also solvent dependent. We thus evaluated (Yby fitting the kinetic traces of photosensitization of DMA at high concentration, by a nonlinear fitting program, to Eq. (4). We employed DMA at initial concentrations of 1 or 2 mM. In Fig. 2 we show, as an example, the kinetic trace of photosensitization of DMA by CdTX in pyridine and its fit to Eq. (4 ). The excitation wavelength for these experiments was 420 nm, where the absorbancies were low enough for the relation [A] = j?F to hold. Table 1 shows the values of a!, in four solvents, compared to literature values [ 15 1. We now employed the values of a! obtained above to analyze the kinetics at low DMA concentration. In Fig. 3 we show three representative cases of the kinetic curves for DMA photosensitization by MgTBP in various solvents. The initial concentration of the target was 5 pm which agrees with LY> j3F (since a typical value of LYis lop3 M); the concentration of the sensitizer was 10 uM. The kinetic traces were thus fitted to a simple exponent taking into consideration an asymptotical background fluorescence. T, the time constant of the decay was obtained from the fit. In the case of ZnTBP in methanol the kinetic curve did not lit a simple exponent.
Table 1 The parameter 01of DMA in several solvents. (Ywas obtained by fitting kinetic traces ofphotosensitization of DMA at higher concentration to Eq. (4) a and is compared to literature values Solvent
OIb (x10-‘M)
crc (x~O-~M)
acetonitrile benzene methanol pyridine
0.34 1.31 * 0.962 0.61
0.2TO.04 0.3, 2+0.5, 1.2 c 8f0.5, 3TO.6’ 1.4, 1.0 d
a All values are within accuracy of 5%. b Values of o from tit to Eq. (4). E Values from Ref. [ 151. dAverage of two measurements. ’ Different values appear in literature.
“,,A ‘.
0
100
200
300
400
500
600
time (set) Fig. 3. Fluorescence kinetic traces of DMA (5 pM) sensitized by MgTBP ( 10 FM) in three different solvents and their tits to Eq. (3). Data (---), fit (---); (A) benzene, (B) pyridine, (C) acctonitrile. 1,,,=360 nm, A,,=457 nm. The irradiation source was a 10 mW HeNe laser.
0
100
200 time (set)
300
400
Fig. 2. Fluorescence time drive of DMA at high concentration (00o) and the tit to Eq. (4) (p). DMA ( 1 mM in pyridine) was sensitized by 10 pM CdTX, which was irradiated by 12.5 mW of a diode laser at 783 nm. The fluorescence of DMA was measured at ,I,,, = 420 nm, A,,,,=468 nm.
Table 2 shows the calculated values of 0. for the three sensitizers, in various solvents: It can be seen that the singlet oxygen formation is very efficient, and in some solvents is as high as that of the best photosensitizers. The high values of @* together with the strong absorption at long wavelengths make these sensitizers good candidates for biological sensitization. In all the reported cases in this study the self-sensitization of the sensitizer was much slower than the sensitization of the DMA. Therefore the solutions (the exponent and Eq. (4) ) are not complicated by a decrease in the sensitizers’ concentration. A wavelength dependence is generally not ex-
148
A. Luvi et al. / Chemical Physics Letters 231 (1994) 144-150
Table 2
Quantum yields of singlet oxygen production Solvent
by MgTBP, ZnTBP and CdTX in various
MgTBP
solvents
ZnTBP
CdTX
@*
@A
0.75 1 a, 0.998 b 0.497 a e 0.3 a
d 0.727 =, 0.855 b 0.244 ’ 0.33 c
@*a acetonitrile benzene methanol pyridine
0.601 0.34 0.126 0.317
0.481 0.565 0.159 0.367
’ Irradiation at 632.8 nm. ’ Irradiation at 441.6 nm. ‘Irradiation d Values of @* obtained were higher than 1. ’ Fluorescence time drive did not fit a single exponent function. ‘All values are within 6% accuracy except for MgTBP in acetonitrile
petted in photochemical reactions because of efficient internal conversion from an upper to the lowest excited singlet state (Kasha’s rule). However, since the Soret band of the tetrabenzoporphyrins is much stronger than the long wavelength band, we decided to investigate the wavelength dependence of the photosensitization reaction. We compared the values of @A of our sensitizers upon excitation to different electronic states, using the HeCd 44 1.6 nm line and HeNe 632.8 nm (for the TBPs) or a diode laser line at 783 nm (for CdTX). Table 2 shows the values of @,, determined from the photosensitization process at two different irradiation wavelengths. As can be seen, in the case of MgTBP in benzene, @Afor excitation at 632.8 nm was 0.34 as opposed to 0.56 when excited at 441.6. This trend of a lower quantum yield when excited at the longer wavelength can be observed in the table in additional solvents and with the other sensitizers as well. A possible explanation for a greater efficiency of singlet oxygen formation upon excitation with shorter wavelengths is that intersystern-crossing from Sz to the triplet states competes successfully with the internal conversion of Sz to S, and results in a more efficient formation of O2 ( ‘4). Intersystem-crossing from higher excited singlet levels to the triplet ladder has been observed previously with some other sensitizers as well as with other molecules [ 16-201. Wavelength dependence of photochemical reactions or of fluorescence/phosphorescence intensity ratio was ascribed to this effect. An interesting case is that of CdTX in acetonitrile where, again, the @* is higher upon excitation at 441.6 nm than at 632.8, however both numbers are higher than 1, 1.47 and 1.23, respectively. Singlet oxygen
at 783 nm.
where error margin is 10%.
production yields which are higher than 1 can be explained by the formation of one molecule of singlet oxygen by energy transfer from the sensitizer’s excited singlet and another molecule produced by the sensitizer’s excited triplet [ 2 11. We also checked the wavelength dependence of @* upon excitation at different wavelengths within the Q band. By tuning the dye laser, we excited MgTBP in benzene, pyridine and acetonitrile at several wavelengths. As can be seen in Fig. 4 the trend of a lower quantum yield when excited at the longer wavelength can be observed in this case as well. This behavior of wavelength dependence is even more exceptional since it occurs upon excitation to different vibrational levels within the same electronic state, and in-
. y
.
.
.
XX,
x x x
I
I
615
620
wavelength
625
630
(nm)
Fig. 4. Dependence of singlet oxygen quantum yields of MgTBP on the exciting wavelength. Singlet oxygen quantum yields of MgTBP ( 10 FM) in acetonitrile ( n ), benzene ( X ) and pyridine (0). Irradiation source was a dye laser using rhodamine B, pumped by an Ar+ laser. DMA: 5 FM, I,,,=360 nm, A,,=457 nm.
A. Lavi et al. / Chemical Physics Letters231 (1994) 144-150
dicates that intersystem-crossing competes even with vibrational relaxation within the excited electronic state. This effect has been reported before only in a very limited number of cases [ 22,231. Since the quantum yieldof singlet oxygen formation does not obey l&ha’s rule we decided to check whether the fluorescence quantum yield has the same behavior and also exhibits an equivalent dependence on the excitation wavelength. Our results show that upon exciting the sensitizer with different wavelengths within the same electronic state, different values of @r are obtained. In the case of MgTBP in benzene (Fig. 5) the fluorescence yield decreases upon excitation to higher vibrational states. A clear behavior of opposite trends of the wavelength dependencies in @, and @., is observed, although the range of overlap of the two phenomena is limited due to experimental limitations (weak fluorescence excitation at low wavelengths and the dye laser’s range). For MgTBP in acetonitrile @r increased upon excitation with longer wavelengths and this observation remains without an explanation. Fluorescence yields measured for rhodamine 6G showed no wavelength dependence (less than 5%) should be noted that in all cases the shape of the fluorescence spectrum was independent of the excitation wavelength. This indi-
>:
d 0"
I ‘I
0.2 0.16.
k
0.12~
s 6
0.08.
z
0.04.
, ‘\
__~~_
410
Acknowledgement
References
-2
0
cates that the emission occurs from the lowest relaxed, vibrational state, and in addition, it excludes the possibility of fluorescent contamination in the samples or of excited-state heterogeneity due to solvation or other similar effects. In conclusion, we have shown here that MgTBP, ZnTBP and CdTX which have strong absorption bands at long wavelengths, also exhibit efficient intersystem-crossing to the excited triplet level, and they photosensitize the production of singlet oxygen. We have demonstrated a simple method for absolute determination of 0*. We have also demonstrated that these sensitizers do not obey the classical Vavilov rule, and exhibit wavelength dependence of the fluorescence and singlet oxygen quantum yields. This behavior is observed not only when changing from one electronic band to another but also within the Q band, indicating that intersystem-crossing is possible from higher vibrational levels in the excited singlet state. The wavelength dependence of the singlet oxygen production efficiency is of practical relevance for biological photosensitization for a proper selection of the wavelength of the exciting light source.
This work was supported by a grant from the Israel Science Foundation administered by the Israel Academy of Sciences and Humanities. We thank Dr. Jonathan L. Sessler and Greg Hemmi from the University of Texas, Austin for the gift of CdTX.
-------~70.12
n
0.24~
149
-.-A __
---
430
450
wavelength
600
\_,
620
640
-0
(nm)
Fig. 5. Dependence of fluorescence quantum yields of MgTBP in benzene on the exciting wavelength. Fluorescence quantum yields (---) of MgTBP in the Soret (left ordinate) and Q (right ordinate) bands are shown together with the absorption spectrum (ordinate is not shown). The concentration was 0.25-0.3 pM for measurements in the Soret band and 0.6-0.7 nM for the Q band. Absolute values of @were determined by comparison with rhodamine 6G in methanol (0.5 uM) whose fluorescence quantum yield is 0.83.
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A. Lavi et al. / Chemical Physics Letters 231 (1994) 144-150
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