J. Photo&em.
Photobiol.
B: Biol., 12 (1992) 29-36
29
A long-lived transient resulting from flash photolysis of hematoporphyrin in aqueous solution Gerald J. Smith Physics and Engineering Laboratory, Department of Scient@ic Research, P.O. Box 31313, Lower Hutt (New Zealand]
and Industrial
(Received October 5, 1990; accepted March 20, 1991)
Abstract A long-lived transient with a lifetime of several hundred microseconds was observed following the flash photolysis of aqueous solutions of hematoporphyrin buffered at pH 7.5. The transient-ground state difference absorption spectrum was determined 500 c~s after flash photolysis. The yield of this species was found to increase with increasing hematoporphyrin concentration and it was also found to depend on the excitation wavelength. The lifetime of the species is not significantly affected by the presence of oxygen. Because the triplet state of hematoporphyrin is not the only long-lived species produced by flash photolysis of aqueous hematoporphyrin solutions, the observed triplet state extinction coefficients will be lower than the true value and hence the triplet state yields of hematoporphyrin determined by the flash photolysis, “complete conversion” technique, are only upper limits. The formation of the long-lived species is discussed in terms of electron transfer between the monomer partners in hematoporphyrin dimer and aggregates which are present in aqueous solutions of hematoporphyrin, particularly in concentrated solutions.
Keywords:
Hematoporphyrin, aggregation, phototherapy, radical ions.
1. Introduction As a result of the use of derivatives of hematoporphyrin (HP) in the phototherapy of tumours [ 1 ] the photophysics of the parent compound, HP, has been the subject of considerable investigation in recent years [ 21. The photophysics of HP in aqueous solution is complex, largely because it exists in such solutions as a mixture of monomer and various molecular aggregates. The composition of the mixture depends on the concentration of HP and the nature of the solvent [3]. Estimates of the composition vary but it appears that in aqueous solutions at concentrations less than or equal to 1 X lo-’ mol dmW3 the monomeric form predominates, while above approximately 10 X lo-’ mol drn3 HP exists primarily as molecular aggregates [4, 51. The monomer and its aggregates have been found to have significantly different fluorescence and triplet state yields [6, 71, singlet state lifetimes [8, 91 and, apparently, different triplet state extinction coefficients [lo]. Because the Elsevier Sequoia
30
HP monomer and aggregates also have different absorption spectra [5], the photophysical properties of HP aqueous solutions will depend on the excitation wavelength as well as concentration. For example, in an 8 X 10V6 mol dmm3 solution, the triplet state formed following excitation at 347 run has an observed triplet-ground state difference extinction coefficient at 450 nm of 2.8 x lo3 dme3 mol-’ cm-i [ 111, whereas following excitation of a similar solution at 527 nm, the observed triplet-ground state extinction coefficient at 450 run is 5.3 X lo3 dm3 mol- ’ cm-’ [ 121. These differences in the value determined for the triplet state extinction coefficient are going to affect the value obtained for the triplet state yield [ 13 1.This is an important determinant of the efficacy of HP in turnour phototherapy because the triplet state is believed to be responsible for the photolethal action of HP on biological material [ 14, 151. The object of the work reported here was to examine the cause of the variation in the observed triplet state extinction coefficient with HP concentration and excitation wavelength. In particular, this study focused on the observation of a transient absorption following flash photolysis of aqueous solutions of HP which was very much longer lived than that of the triplet state of the HP monomer or its aggregates [ 7, 11, 161. Because the determination of the HP triplet state extinction coefficients using flash photolysis requires making the assumption that the ground state is totally converted to the triplet state by a sufliciently intense pulse of radiation [ 131, the presence of any additional long-lived species will introduce a systematic error in the value obtained for the triplet state extinction coefficient. 2. Materials
and methods
2.1. Materials Hematopdrphyrin IX dihydrochloride obtained from Porphyrin Products was used as received. The water was distilled and further purified by passage through a Millipore Super-Q filter. Ajax spectroscopic grade methanol and BDH spectroscopic grade cyclohexane were used in the preparation of actinometer solutions. The anthracene was purilied by recrystallization and the methylene blue was purified by the method described by Bergmann and O’Konski [ 171. Aqueous solutions of HP were prepared under minimum lighting just prior to experimentation by dissolving 3.0 mg of HP in a few drops of 0.1 mol drne3 sodium hydroxide solution and diluting with phosphate buffer solution to the desired concentration. The pH of the resulting solution was 7.5. The HP concentration was determined directly for the most dilute solution used in this work (2 X lo-’ mol drnb3) from its absorbance at 390 run, taking the extinction coefficient as 1.4 x lo5 dm3 mol-’ cm-’ for dilute solutions [ 181. When required, the solution were deoxygenated by gentle bubbling with oxygen-free nitrogen gas. 2.2. Flash photo&&s Solutions contained in a 10 mm square fluorometric cell were exposed to single, 30 ns pulses of 527 m-n or 351 run radiation which were the
31
doubled or tripled output of a J K Lasers neodymium laser. The laser flash photolysis system has been described in detail previously and has a response time of approximately 300 ns [ 191. Transient absorbance measurements reported here are the average of at least five independent determinations and the estimated errors are the extreme deviations of the measurements from the average. The transient-ground state difference absorption spectrum from 480 nm to 640 run, 500 ps after the laser flash was determined for a 1 x 10e4 mol dm- 3 solution. The decays of transient absorptions at 605 run were recorded following flash photolysis of aqueous HP solutions at concentrations ranging from 2 x lop6 mol drC3 to 1 X 10m4mol dme3. The excitation wavelength was either 351 run or 527 run. Low laser powers were used to avoid errors associated with ground state depletion [ 13 ]. In determinations of the relative yield of the long-lived transient the laser power was adjusted so that the excitation energy absorbed by the solution was the same irrespective of HP concentration or excitation wavelength. This was achieved by monitoring the quantity of triplet states produced by the laser pulse in degassed actinometer solutions with known triplet state yields. For 530 run radiation methylene blue in methanol was used as the actinometer. In such solutions methylene blue exists only in its monomeric form and possible complications related to the presence of dimers are avoided. For 531 nm excitation, anthracene in cyclohexane was used as the actinometer. Similar actinometric or quantum counting procedures have been described elsewhere [ 13 ]. The concentrations of the actinometer solutions were such that the absorbances at 530 nm and at 351 nm were equal to those of the HP solutions studied at these excitation wavelengths. The absorption spectra of these solutions, together with those of the component HP monomer and dimer or aggregate, have been published previously [5]. Using the procedure described above, the relative quantum efficiencies for the production of the long-lived transient (greater than 500 ps) at different HP concentrations resulting from excitation at different wavelengths were determined.
3. R.esults 3.1. i’%-an.sW decay kinetics The decay of the transient observed at 605 nm in a 1 X 10m4mol dmW3 deoxygenated, aqueous HP solution is shown in Fig. 1. It displays a relatively rapidly decaying component, and a second component which persists for several hundred microseconds. Indeed, the lifetime of the second component is too long for the laser flash photolysis system to determine accurately. Over the initial 500 ps the decay is well described by first-order kinetics, i.e. a function of the form, exp( - kt) + c. The rate constant for the decay is 1 X lo4 s- ‘. The lifetime and yield of this relatively rapidly decaying component is substantially reduced by the presence of oxygen. The rate
32
0
5b
I
100
I
150
I
200
Time,
I
250
I
300
I
350
I
400
1
450
ps
Fig. 1. The decay of the transient absorption at 605 nm observed following 351 nm flash photolysis of a 1 X 10m4 mol dmm3 deoxygenated, aqueous solution of HP.
constant for the reaction of this species with oxygen is approximately 1 X 10’ dm3 mol- ’ s-l. However, the lifetime of the longer-lived component is not significantly affected by oxygen. In air-saturated solutions, the short-lived component only makes a small contribution to the transient. Under these conditions, the long-lived species displays no “grow-in” phase and it is estimated that it is formed in a period less than 1 ps after the flash. 3.2. Transient absorptiun spectrum By comparison with the shorter lived species, the contribution that the long-lived species makes to the transient at 445 run is negligible. However, the long-lived species makes significant contributions to the transient absorption at wavelengths greater than or equal to 480 nm. Its transient-ground state difference absorption spectrum at wavelengths greater than or equal to 480 nm measured 500 ps after excitation of a 1 X 10e4 mol dmm3 HP solution is shown in Fig. 2. 3.3. Transient quantum yields The relative quantum yield of the long-lived species is dependent on excitation wavelength. Following excitation of a 1 X 10d4 mol dmm3 HP solution at 351 run, the yield of the long-lived species is 1.8 times greater than that observed from the same solution following excitation at 527 nm. The relative quantum yield of the long-lived species also depends on the concentration of HP as set out in Table 1 where the quantum yields resulting ,from excitation at 35 1 nm have been normalized relative to the value obtained at a concentration of 1 X 10e4 mol drC3.
33
JI,,,,,,,,,,,,,,,,,,,, 500 450
550
600
Wavelength,
650
nm
Fig. 2. The difference absorption spectrum of the transient observed 500 @ after excitation of a deoxygenated, 1 x lo-*
mol dme3 HP solution.
TABLE 1 The relative quantum yields of the long-lived transient absorbing at 605 nm following 351 nm flash photolysis of aqueous solutions of HP at different concentrations HP concentration (mol dme3) 5x
10-B
20x 10-p 100x 1o-e
Relative quantum yield 0.58 * 0.05 0.66 f 0.05 1.0*0.05
4. Discussion The initial, relatively short-lived component observed in the transient absorption at 605 nm appears to be due to the triplet state of HP. Its decay rate constant is similar to the 1.5 x 1O4 s- ’ rate constant determined for a transient absorbing at 450 run in flash photolysed, 1 X 10m4mol dmm3HP solution [6] and within the range of lifetimes determined for the triplet state of HP by other workers [ 7, 11, 201. In addition, the transient is quenched by oxygen with a rate constant of approximately 1 X lo9 dm3 mol- ’ s- ’ which is similar to the rate constant measured previously for the reaction of the HP triplet state with oxygen [7, 201. The main interest in the results from the present work relates to the long-lived component of the transient. Previously published work suggests it is due to HP radical ions. At times greater than 500 ps, the transien+-ground state difference absorption spectrum at wavelengths longer than 550 run shown in Pig. 2 agrees closely with that obtained immediately following flash photolysis of degassed, aqueous HP solution and ascribed to HP’+ [16].
34
The spectrum is also similar to those observed following pulse radiolysis of HP in NzO saturated,aqueous solution containing azide ions or alcohol where HP radical cations and anions respectively are generated [ 2 11.Similar spectra were also observed at relatively long times after pulse radiolysis of aqueous HP solutions under reducing conditions by Hare1 and Mayerstein [ 221. A radical anion would be expected to react with oxygen. Because the species responsible for the long-lived transient is not quenched by the presence of oxygen, it is concluded that it is not a radical anion. In homogeneous aqueous solution it was found that the yield of the long-lived radical species increaseswith concentration of HP, i.e. HP aggregate. This implies that the HP dimer or aggregate can be a source of HP radicals. Photoinduced charge transfer is believed to occur between the molecular pairs of some porphyrin dimer or aggregates in water [23], provided the thermodynamic determinant of such a process, i.e. the change in free energy AGcr, is sufliciently small or negative. AGCT has been shown to be related to the energy of the excited electronic state of the electron donor or acceptor E,, the reduction and oxidation potentialsEFd and Egx of the electron acceptor and donor respectively, and a coulombic stabilization term C [24] The dependence of yield of the long-lived transient on excitation wavelength can also be explained in terms of its generation from the HP dimer or aggregate. Previous investigation of the ground state absorption spectra of HP in aqueous solution has established that the absorption spectra of the monomer differs from that of the aggregates [ 51. Further, both fluorescence lifetime and yield measurements indicate that there are at least two types of HP aggregate present in aqueous solution which have different absorption spectra [6, 91. Thus, excitations at 351 nm and at 527 nm are expected to excite different relative amounts of aggregates and monomer, and thereby produce different relative yields of long-lived HP radicals and HP triplet state. Irrespective of the exact nature of the long-lived species observed in this work, its slow rate of reaction with oxygen suggests that it is not a triplet state (and not a radical anion). The formation of such a species in addition to the triplet state is a significant fact in relation to the determination of the triplet state extinction coefficient. Such determinations rely on the ground state of HP present in solution being completely converted to the triplet state within the duration of the laser pulse by sticiently intense photolysis. When this occurs, the initial concentration of the triplet state is equal to the ground state concentration prior to excitation [ 131 and from the observed optical density of the transient, the extinction coefficient can be obtained. Hence, it is clear [ 131 that if another species is produced by photolysis in addition to the triplet state, the concentration of the triplet state will be less than that of the ground state prior to photolysis and the value of triplet state extinction coefficient determined will therefore be less than the true value. Conversion of the HP ground state to both monomer
35
and aggregate triplet states is believed to contribute to the observed dependence of the triplet state extinction coefficient on HP concentration aggregation [ 10, 111. The present work emphasizes the additional impact that free radical formation resulting from charge transfer in aqueous solutions of HP will have on the determination of triplet state extinction coefficients and the effect of HP concentration on observed triplet extinction coefficients and yields. References 1 T. J. Dougherty, J. E. Kaufman, A. Goldfar, K. R. Weishaupt, D. Boyle and A. Mittleman, Photoradiation therapy for the treatment of malignant tumors, Cancer Res., 38 (1978) 2628-2635. 2 R. Pottier and T. G. Truscott, The photochemistry of hematoporphyrin and related systems, Int. J. Radiat. Biol., 50 (1986) 421-452. 3 W. I. White, in D. Dolphin (ed.), The Porphyrins, Academic Press, New York, p. 303. 4 W. B. Brown, M. Shillock and P. Jones, Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution, Biochem. J., 153 (1976) 279-285. 5 G. J. Smith, K. P. Ghiggino, L. E. Bennett and T. Nero, The Q-band absorption spectra of the hematoporphyrin monomer and aggregate in aqueous solution, Photo&em. Photobiol., 49 (1989) 49-53. 6 G. J. Smith, The effects of aggregation on the fluorescence and the triplet state yield of hematoporphyrin, Photo&em. Photobiol., 41 (1985) 123-126. 7 E. Reddi, G. Jori, M. A. J. Rodgers and J. D. Spikes, Flash photolysis studies of hemato and coproporphyrins in homogeneous and microheterogeneous aqueous dispersions, Photo&em. Photobiol, 38 (1983) 639-645. 8 A. Andreoni, R. Cubeddu, S. De Silvrestri, G. Jori, P. La Porta and E. Reddi, Tune resolved fluorescence of hematoporphyrin, Z. Natudich., 8 (1983) 83-89. 9 A. R. Andreoni and R. Cubeddu, Photophysical properties of photofrin II, Chem Phgs. I.&t., 108 (1984) 141-144. 10 T. G. Truscott, The photochemistry of hematoporphyrin and some related species, J. Cha. Sot., Faraday l+-ans. 2, 82 (1986) 2177-2181. 11 M. Craw, R. Redmond and T. G. Truscott, Laser flash photolysis of hematoporphyrin in some homegeneous and heterogeneous environments, J. Chem. Sot., Faraday !lkans. 1, 80 (1984) 2293-2299. 12 G. J. Smith, unpublished results. 13 R. Be nsasson, C. R. Goldschmidt, E. J. Land and T. G. Truscott, Laser intensity and the comparative method for determination of triplet quantum yields, Photo&em. PhotobioL, 28 (1978) 277-281. 14 T. J. Dougherty, C. J. Gomer and K. R. Weishaupt, Energetics and efficiency of photoactivation of murine tumor cells containing hematoporphyrin, Cancer Res., 36 (1976) 2330-2333. 15 S. Okuda, S. Mimura, M. I&ii and M. Tatsuta, Experimental studies on HPD-photoradiation therapy for upper gastrointestinal cancer, in A. Andreoni and R. Cubeddu (eds.), Porphyrins in Tumor Phototherapy, Plenum, New York, 1984, pp. 413-421. 16 S. M. Murgia, A. Poletti and A. Pasqua, Laser flash photolysis studies on hematoporphyrin IX in aqueous and micellar systems, Med. BioL En&-on., 10 (1982) 279-283. 17 D. Bergman and C. T. O’Konski, A spectroscopic study of methylene blue monomer, dimer and complexes with montmorillonite, J. Phys. Chem., 67 (1963) 2169-2177. 18 L. I. Grossweiner, A. S. Pate1 and J. B. Grossweiner, Type I and type II mechanisms in the photosensitized lysis of liposomes by hematoporphyrin, Photo&em. PhotobioL, 36 (1982) 159-167. 19 G. J. Smith, Enhanced intersystem crossing in the oxygen quenching of aromatic hydrocarbon triplet states, J. Chem. Sot., Faraday Trans. 2, 78 (1982) 769-773.
36 20 S. M. Murgia, A. Pasqua and A. Poletti, Laser flash photolysis of hematoporphyrin in solvent mixtures, Chem. Phys Z&t., 98 (1983) 179-183. 21 R. Bonnet& C. Lambert, E. J. Land, P. A. Scourides, R. S. Sinclair and T. G. Truscott, The triplet and radical species of hematoporphyrin and its derivatives, Photo&em. Photobiol., 38 (1983) 1-8. 22 Y. Hare1 and D. Meyerstein, The mechanism of reduction of porphyrins: a pulse radiolytic study, J. Am. Chem. Sot., 96 (1974) 2720-2727. 23 U. Hofstra, R. B. M. Koehorst and T. J. Schaafsma, Excited-state properties of porphyrin dimers, Chem. Phys. L&t., 130 (1986) 555-559. 24 H. Knibbe, D. Rehm and A. Weller, Intermediates and kinetics of fluorescence quenching by electron transfer, Ber. Bunsenges Phys. Chem., 2 (1968) 257-262.
Photobiol.
B: Biol., 12 (1992) 29-36
29
A long-lived transient resulting from flash photolysis of hematoporphyrin in aqueous solution Gerald J. Smith Physics and Engineering Laboratory, Department of Scient@ic Research, P.O. Box 31313, Lower Hutt (New Zealand]
and Industrial
(Received October 5, 1990; accepted March 20, 1991)
Abstract A long-lived transient with a lifetime of several hundred microseconds was observed following the flash photolysis of aqueous solutions of hematoporphyrin buffered at pH 7.5. The transient-ground state difference absorption spectrum was determined 500 c~s after flash photolysis. The yield of this species was found to increase with increasing hematoporphyrin concentration and it was also found to depend on the excitation wavelength. The lifetime of the species is not significantly affected by the presence of oxygen. Because the triplet state of hematoporphyrin is not the only long-lived species produced by flash photolysis of aqueous hematoporphyrin solutions, the observed triplet state extinction coefficients will be lower than the true value and hence the triplet state yields of hematoporphyrin determined by the flash photolysis, “complete conversion” technique, are only upper limits. The formation of the long-lived species is discussed in terms of electron transfer between the monomer partners in hematoporphyrin dimer and aggregates which are present in aqueous solutions of hematoporphyrin, particularly in concentrated solutions.
Keywords:
Hematoporphyrin, aggregation, phototherapy, radical ions.
1. Introduction As a result of the use of derivatives of hematoporphyrin (HP) in the phototherapy of tumours [ 1 ] the photophysics of the parent compound, HP, has been the subject of considerable investigation in recent years [ 21. The photophysics of HP in aqueous solution is complex, largely because it exists in such solutions as a mixture of monomer and various molecular aggregates. The composition of the mixture depends on the concentration of HP and the nature of the solvent [3]. Estimates of the composition vary but it appears that in aqueous solutions at concentrations less than or equal to 1 X lo-’ mol dmW3 the monomeric form predominates, while above approximately 10 X lo-’ mol drn3 HP exists primarily as molecular aggregates [4, 51. The monomer and its aggregates have been found to have significantly different fluorescence and triplet state yields [6, 71, singlet state lifetimes [8, 91 and, apparently, different triplet state extinction coefficients [lo]. Because the Elsevier Sequoia
30
HP monomer and aggregates also have different absorption spectra [5], the photophysical properties of HP aqueous solutions will depend on the excitation wavelength as well as concentration. For example, in an 8 X 10V6 mol dmm3 solution, the triplet state formed following excitation at 347 run has an observed triplet-ground state difference extinction coefficient at 450 nm of 2.8 x lo3 dme3 mol-’ cm-i [ 111, whereas following excitation of a similar solution at 527 nm, the observed triplet-ground state extinction coefficient at 450 run is 5.3 X lo3 dm3 mol- ’ cm-’ [ 121. These differences in the value determined for the triplet state extinction coefficient are going to affect the value obtained for the triplet state yield [ 13 1.This is an important determinant of the efficacy of HP in turnour phototherapy because the triplet state is believed to be responsible for the photolethal action of HP on biological material [ 14, 151. The object of the work reported here was to examine the cause of the variation in the observed triplet state extinction coefficient with HP concentration and excitation wavelength. In particular, this study focused on the observation of a transient absorption following flash photolysis of aqueous solutions of HP which was very much longer lived than that of the triplet state of the HP monomer or its aggregates [ 7, 11, 161. Because the determination of the HP triplet state extinction coefficients using flash photolysis requires making the assumption that the ground state is totally converted to the triplet state by a sufliciently intense pulse of radiation [ 131, the presence of any additional long-lived species will introduce a systematic error in the value obtained for the triplet state extinction coefficient. 2. Materials
and methods
2.1. Materials Hematopdrphyrin IX dihydrochloride obtained from Porphyrin Products was used as received. The water was distilled and further purified by passage through a Millipore Super-Q filter. Ajax spectroscopic grade methanol and BDH spectroscopic grade cyclohexane were used in the preparation of actinometer solutions. The anthracene was purilied by recrystallization and the methylene blue was purified by the method described by Bergmann and O’Konski [ 171. Aqueous solutions of HP were prepared under minimum lighting just prior to experimentation by dissolving 3.0 mg of HP in a few drops of 0.1 mol drne3 sodium hydroxide solution and diluting with phosphate buffer solution to the desired concentration. The pH of the resulting solution was 7.5. The HP concentration was determined directly for the most dilute solution used in this work (2 X lo-’ mol drnb3) from its absorbance at 390 run, taking the extinction coefficient as 1.4 x lo5 dm3 mol-’ cm-’ for dilute solutions [ 181. When required, the solution were deoxygenated by gentle bubbling with oxygen-free nitrogen gas. 2.2. Flash photo&&s Solutions contained in a 10 mm square fluorometric cell were exposed to single, 30 ns pulses of 527 m-n or 351 run radiation which were the
31
doubled or tripled output of a J K Lasers neodymium laser. The laser flash photolysis system has been described in detail previously and has a response time of approximately 300 ns [ 191. Transient absorbance measurements reported here are the average of at least five independent determinations and the estimated errors are the extreme deviations of the measurements from the average. The transient-ground state difference absorption spectrum from 480 nm to 640 run, 500 ps after the laser flash was determined for a 1 x 10e4 mol dm- 3 solution. The decays of transient absorptions at 605 run were recorded following flash photolysis of aqueous HP solutions at concentrations ranging from 2 x lop6 mol drC3 to 1 X 10m4mol dme3. The excitation wavelength was either 351 run or 527 run. Low laser powers were used to avoid errors associated with ground state depletion [ 13 ]. In determinations of the relative yield of the long-lived transient the laser power was adjusted so that the excitation energy absorbed by the solution was the same irrespective of HP concentration or excitation wavelength. This was achieved by monitoring the quantity of triplet states produced by the laser pulse in degassed actinometer solutions with known triplet state yields. For 530 run radiation methylene blue in methanol was used as the actinometer. In such solutions methylene blue exists only in its monomeric form and possible complications related to the presence of dimers are avoided. For 531 nm excitation, anthracene in cyclohexane was used as the actinometer. Similar actinometric or quantum counting procedures have been described elsewhere [ 13 ]. The concentrations of the actinometer solutions were such that the absorbances at 530 nm and at 351 nm were equal to those of the HP solutions studied at these excitation wavelengths. The absorption spectra of these solutions, together with those of the component HP monomer and dimer or aggregate, have been published previously [5]. Using the procedure described above, the relative quantum efficiencies for the production of the long-lived transient (greater than 500 ps) at different HP concentrations resulting from excitation at different wavelengths were determined.
3. R.esults 3.1. i’%-an.sW decay kinetics The decay of the transient observed at 605 nm in a 1 X 10m4mol dmW3 deoxygenated, aqueous HP solution is shown in Fig. 1. It displays a relatively rapidly decaying component, and a second component which persists for several hundred microseconds. Indeed, the lifetime of the second component is too long for the laser flash photolysis system to determine accurately. Over the initial 500 ps the decay is well described by first-order kinetics, i.e. a function of the form, exp( - kt) + c. The rate constant for the decay is 1 X lo4 s- ‘. The lifetime and yield of this relatively rapidly decaying component is substantially reduced by the presence of oxygen. The rate
32
0
5b
I
100
I
150
I
200
Time,
I
250
I
300
I
350
I
400
1
450
ps
Fig. 1. The decay of the transient absorption at 605 nm observed following 351 nm flash photolysis of a 1 X 10m4 mol dmm3 deoxygenated, aqueous solution of HP.
constant for the reaction of this species with oxygen is approximately 1 X 10’ dm3 mol- ’ s-l. However, the lifetime of the longer-lived component is not significantly affected by oxygen. In air-saturated solutions, the short-lived component only makes a small contribution to the transient. Under these conditions, the long-lived species displays no “grow-in” phase and it is estimated that it is formed in a period less than 1 ps after the flash. 3.2. Transient absorptiun spectrum By comparison with the shorter lived species, the contribution that the long-lived species makes to the transient at 445 run is negligible. However, the long-lived species makes significant contributions to the transient absorption at wavelengths greater than or equal to 480 nm. Its transient-ground state difference absorption spectrum at wavelengths greater than or equal to 480 nm measured 500 ps after excitation of a 1 X 10e4 mol dmm3 HP solution is shown in Fig. 2. 3.3. Transient quantum yields The relative quantum yield of the long-lived species is dependent on excitation wavelength. Following excitation of a 1 X 10d4 mol dmm3 HP solution at 351 run, the yield of the long-lived species is 1.8 times greater than that observed from the same solution following excitation at 527 nm. The relative quantum yield of the long-lived species also depends on the concentration of HP as set out in Table 1 where the quantum yields resulting ,from excitation at 35 1 nm have been normalized relative to the value obtained at a concentration of 1 X 10e4 mol drC3.
33
JI,,,,,,,,,,,,,,,,,,,, 500 450
550
600
Wavelength,
650
nm
Fig. 2. The difference absorption spectrum of the transient observed 500 @ after excitation of a deoxygenated, 1 x lo-*
mol dme3 HP solution.
TABLE 1 The relative quantum yields of the long-lived transient absorbing at 605 nm following 351 nm flash photolysis of aqueous solutions of HP at different concentrations HP concentration (mol dme3) 5x
10-B
20x 10-p 100x 1o-e
Relative quantum yield 0.58 * 0.05 0.66 f 0.05 1.0*0.05
4. Discussion The initial, relatively short-lived component observed in the transient absorption at 605 nm appears to be due to the triplet state of HP. Its decay rate constant is similar to the 1.5 x 1O4 s- ’ rate constant determined for a transient absorbing at 450 run in flash photolysed, 1 X 10m4mol dmm3HP solution [6] and within the range of lifetimes determined for the triplet state of HP by other workers [ 7, 11, 201. In addition, the transient is quenched by oxygen with a rate constant of approximately 1 X lo9 dm3 mol- ’ s- ’ which is similar to the rate constant measured previously for the reaction of the HP triplet state with oxygen [7, 201. The main interest in the results from the present work relates to the long-lived component of the transient. Previously published work suggests it is due to HP radical ions. At times greater than 500 ps, the transien+-ground state difference absorption spectrum at wavelengths longer than 550 run shown in Pig. 2 agrees closely with that obtained immediately following flash photolysis of degassed, aqueous HP solution and ascribed to HP’+ [16].
34
The spectrum is also similar to those observed following pulse radiolysis of HP in NzO saturated,aqueous solution containing azide ions or alcohol where HP radical cations and anions respectively are generated [ 2 11.Similar spectra were also observed at relatively long times after pulse radiolysis of aqueous HP solutions under reducing conditions by Hare1 and Mayerstein [ 221. A radical anion would be expected to react with oxygen. Because the species responsible for the long-lived transient is not quenched by the presence of oxygen, it is concluded that it is not a radical anion. In homogeneous aqueous solution it was found that the yield of the long-lived radical species increaseswith concentration of HP, i.e. HP aggregate. This implies that the HP dimer or aggregate can be a source of HP radicals. Photoinduced charge transfer is believed to occur between the molecular pairs of some porphyrin dimer or aggregates in water [23], provided the thermodynamic determinant of such a process, i.e. the change in free energy AGcr, is sufliciently small or negative. AGCT has been shown to be related to the energy of the excited electronic state of the electron donor or acceptor E,, the reduction and oxidation potentialsEFd and Egx of the electron acceptor and donor respectively, and a coulombic stabilization term C [24] The dependence of yield of the long-lived transient on excitation wavelength can also be explained in terms of its generation from the HP dimer or aggregate. Previous investigation of the ground state absorption spectra of HP in aqueous solution has established that the absorption spectra of the monomer differs from that of the aggregates [ 51. Further, both fluorescence lifetime and yield measurements indicate that there are at least two types of HP aggregate present in aqueous solution which have different absorption spectra [6, 91. Thus, excitations at 351 nm and at 527 nm are expected to excite different relative amounts of aggregates and monomer, and thereby produce different relative yields of long-lived HP radicals and HP triplet state. Irrespective of the exact nature of the long-lived species observed in this work, its slow rate of reaction with oxygen suggests that it is not a triplet state (and not a radical anion). The formation of such a species in addition to the triplet state is a significant fact in relation to the determination of the triplet state extinction coefficient. Such determinations rely on the ground state of HP present in solution being completely converted to the triplet state within the duration of the laser pulse by sticiently intense photolysis. When this occurs, the initial concentration of the triplet state is equal to the ground state concentration prior to excitation [ 131 and from the observed optical density of the transient, the extinction coefficient can be obtained. Hence, it is clear [ 131 that if another species is produced by photolysis in addition to the triplet state, the concentration of the triplet state will be less than that of the ground state prior to photolysis and the value of triplet state extinction coefficient determined will therefore be less than the true value. Conversion of the HP ground state to both monomer
35
and aggregate triplet states is believed to contribute to the observed dependence of the triplet state extinction coefficient on HP concentration aggregation [ 10, 111. The present work emphasizes the additional impact that free radical formation resulting from charge transfer in aqueous solutions of HP will have on the determination of triplet state extinction coefficients and the effect of HP concentration on observed triplet extinction coefficients and yields. References 1 T. J. Dougherty, J. E. Kaufman, A. Goldfar, K. R. Weishaupt, D. Boyle and A. Mittleman, Photoradiation therapy for the treatment of malignant tumors, Cancer Res., 38 (1978) 2628-2635. 2 R. Pottier and T. G. Truscott, The photochemistry of hematoporphyrin and related systems, Int. J. Radiat. Biol., 50 (1986) 421-452. 3 W. I. White, in D. Dolphin (ed.), The Porphyrins, Academic Press, New York, p. 303. 4 W. B. Brown, M. Shillock and P. Jones, Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution, Biochem. J., 153 (1976) 279-285. 5 G. J. Smith, K. P. Ghiggino, L. E. Bennett and T. Nero, The Q-band absorption spectra of the hematoporphyrin monomer and aggregate in aqueous solution, Photo&em. Photobiol., 49 (1989) 49-53. 6 G. J. Smith, The effects of aggregation on the fluorescence and the triplet state yield of hematoporphyrin, Photo&em. Photobiol., 41 (1985) 123-126. 7 E. Reddi, G. Jori, M. A. J. Rodgers and J. D. Spikes, Flash photolysis studies of hemato and coproporphyrins in homogeneous and microheterogeneous aqueous dispersions, Photo&em. Photobiol, 38 (1983) 639-645. 8 A. Andreoni, R. Cubeddu, S. De Silvrestri, G. Jori, P. La Porta and E. Reddi, Tune resolved fluorescence of hematoporphyrin, Z. Natudich., 8 (1983) 83-89. 9 A. R. Andreoni and R. Cubeddu, Photophysical properties of photofrin II, Chem Phgs. I.&t., 108 (1984) 141-144. 10 T. G. Truscott, The photochemistry of hematoporphyrin and some related species, J. Cha. Sot., Faraday l+-ans. 2, 82 (1986) 2177-2181. 11 M. Craw, R. Redmond and T. G. Truscott, Laser flash photolysis of hematoporphyrin in some homegeneous and heterogeneous environments, J. Chem. Sot., Faraday !lkans. 1, 80 (1984) 2293-2299. 12 G. J. Smith, unpublished results. 13 R. Be nsasson, C. R. Goldschmidt, E. J. Land and T. G. Truscott, Laser intensity and the comparative method for determination of triplet quantum yields, Photo&em. PhotobioL, 28 (1978) 277-281. 14 T. J. Dougherty, C. J. Gomer and K. R. Weishaupt, Energetics and efficiency of photoactivation of murine tumor cells containing hematoporphyrin, Cancer Res., 36 (1976) 2330-2333. 15 S. Okuda, S. Mimura, M. I&ii and M. Tatsuta, Experimental studies on HPD-photoradiation therapy for upper gastrointestinal cancer, in A. Andreoni and R. Cubeddu (eds.), Porphyrins in Tumor Phototherapy, Plenum, New York, 1984, pp. 413-421. 16 S. M. Murgia, A. Poletti and A. Pasqua, Laser flash photolysis studies on hematoporphyrin IX in aqueous and micellar systems, Med. BioL En&-on., 10 (1982) 279-283. 17 D. Bergman and C. T. O’Konski, A spectroscopic study of methylene blue monomer, dimer and complexes with montmorillonite, J. Phys. Chem., 67 (1963) 2169-2177. 18 L. I. Grossweiner, A. S. Pate1 and J. B. Grossweiner, Type I and type II mechanisms in the photosensitized lysis of liposomes by hematoporphyrin, Photo&em. PhotobioL, 36 (1982) 159-167. 19 G. J. Smith, Enhanced intersystem crossing in the oxygen quenching of aromatic hydrocarbon triplet states, J. Chem. Sot., Faraday Trans. 2, 78 (1982) 769-773.
36 20 S. M. Murgia, A. Pasqua and A. Poletti, Laser flash photolysis of hematoporphyrin in solvent mixtures, Chem. Phys Z&t., 98 (1983) 179-183. 21 R. Bonnet& C. Lambert, E. J. Land, P. A. Scourides, R. S. Sinclair and T. G. Truscott, The triplet and radical species of hematoporphyrin and its derivatives, Photo&em. Photobiol., 38 (1983) 1-8. 22 Y. Hare1 and D. Meyerstein, The mechanism of reduction of porphyrins: a pulse radiolytic study, J. Am. Chem. Sot., 96 (1974) 2720-2727. 23 U. Hofstra, R. B. M. Koehorst and T. J. Schaafsma, Excited-state properties of porphyrin dimers, Chem. Phys. L&t., 130 (1986) 555-559. 24 H. Knibbe, D. Rehm and A. Weller, Intermediates and kinetics of fluorescence quenching by electron transfer, Ber. Bunsenges Phys. Chem., 2 (1968) 257-262.
