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Two-step laser activation of hematoporphyrin derivative
Volume 88. number
I
CHEMICAL
PHYSICS LETTERS
23 Aprd 1982
TWO-STEP LASER ACTIVATION OF HEMATOPORPHYRIN DERIVATIVE A. ANDREONI, R. CLJBEDDU,S. DE SILVESTRI, P. LAPORTA and 0. SVELTO Centro Eleltronica Quonristim e Srrumenrarione Elemoniar, CNR Istmro di Frsrm dcl Pol~fecnrco.20133 Mhn. Italy Received 9 February 1982; In final form 23 kbruary
1982
A new photoaclrvatron mechamsm of hcmatoporphyrin dcrivatrvc IS presented This process, based on the scqucntral ab. sorption of two photons, leads to the production of cytotoxrc radicals of HpD. The cytocrdal efficnency is dcmonstmted by in vitro experiments.
I. Introduction Dyes such as hematoporphyrin (Hp) or its derrvativc (HpD) [I] are known to exhibit cytocrdal activity on normal and tumor cells [2], when photoactivated by cw hght. This property, together with the fact that they accumulate, wrth a certain degree of specificity, in tissues with a high mitotic index [3], makes them partlcularly attractive for the treatment of tumors. In recent years, the therapeutx ngnificance of photoactivated HpDhas beendemonstratedboth in experimental[4,5] and in human tumors 161. The activationprocessinvolvesthe intermediacy of the cytotoxic agent 1As 02 generated by electronic energy transfer from tnplet HpD (photodynamic action). In this paper we present a new mechanism of photoactivation that results in the production of HpD radicals following the absorption of two near-UV photons. The cytocidal efficrency of this process has been tested in vitro and compared with that of the photodynamic action.
2. Experimental HpD powder, kindly supplied by Dr. T.J. Dougherty from Roswell Park Memorial Hospital (Buffalo, USA), was dissolved in 0.1 M phosphate buffer (pH = 7.4). The value of the HpD concentration was calculated assuming a molecular weight of 598.7. The excitation source for the two-step photoactiva0 009.2614/82/0000-0000/.$02.75
0 1982 North-Holland
tion experunent was a nitrogen laser that provides pulses at 337.1 nrn with 10 ns duratron and peak power up to 250 kW at a repetition rate of 30 HZ. Peak lrradlallon intcnsrties of up to 20 MW/cm2 could be obtamcd. HpD samples of 0.2 cm3 volume were irradiated m a quartz cuvette wrth an optrcal path of 5 mm. The amount of dye decomposed by this process was momtored by measuring the decrease m the fluoresccncc intensity of HpD at X> 500 MI. The fluorescence was cxcrted at 405 nm by a probe pulse of K?OO ps duration, generated by a dye laser[7] pumped by an atmospheric-pressure mtrogen laser and it was obscwcd at 90° through a cut-off filter by an Xp 1210 photo. multiplier tube. The output signal was displayed on a Tektronix 7904 oscilloscope (500 MHz bandwidth). In vitro experiments were performed on an epithehal stram of rat thyroid orrgin (FR-TLS, see ref. [S]). Before irradration the cells were incubated for 2 h with 20 ~.rgHpD per ml of culture medium. Samples of 0.2 cm3 containing =2 X IO5 cells were uniformcly lrradnted in the same quartz cuvette of the prevrous experiment. After irradiation the cell-survival percentage was measured by additron of tripan blue (I% w/v) to the cell suspension and counting thereafter the hving cells in a Burker chamber. cw irradiation experiments on cells were performed in the same conditions, using the 333.6 nm hnc from an argon ion laser.
37
Volume 88. number I
CHE~I~CALPHYSICS LETI’ERS
3. Results and discussion
Under pulsed near-WI excltatlon a decrease in the fluorescence intensity of HpD is observed which mdicates an irreversiblenrodificntlon of the molecule (possable photoionization) leading to the formatIon of photoproducts. A first set of experiments was performed on 1.67 PM HpD solutrons to show the biphotomc character of this process. To evaluate the results, we define the dam-
age probablhty p per laser shot as the quantity p = AM/N,where N = N(tz) is the HpD concentration after the rzth laser pulse and AN is the reductton of this concentration after the next laser shot. Since m our ex~r~lent p 4 1, rt can be shown that [9] ln~~(n~~~v(o)~ =
-np
V#/ v,
(1)
Where V; is the inrerxtlon volume and Y is the total sample volume. Since the ratio N(ft)/N(O) is given by
23 Aprd 1982
the ratio of the fiuorescence signal after the nth laser shot to the in&al one, eq. (f) readily gives the value of p once the number of laser shots n is known. Fig. 1 shows a loganthmic plot ofN(n)/N(O) versus the number of laser shots at 337.1 run. The data were collected for different peak power values of the irradiation pulses. The experimental points at each irradiation intensity could be fitted by a straight line whose slope gives the value of p according to eq. (I), where Vi/V = 0.025. The values of p as a function of the irradiation intensi. ty I are presented in a logarithmic plot in fig. 2. The dependence ofp on the square of the intensrty is found for I values lower than 4 MWjcm2, which IS rndicatrve of a biphotonic process. For higher intensities, the square law is not followed sutce the first transrtio~
begins to saturate. On the basisof the above result whrch demonstrates that the photoproducts
are produced
by a two-step
process, a second set of experiments was performed to compare in vitro the cytocidal efficiency of this photoactivation scheme wrth that of the photodynamic action. The percentages of surviving cells after pulsed (A = 337.1 nm) and cw irradiation (h = 333.6 nm) are shown in fig. 3 (curves (a) and (b) respectively) as a functton of the irradiation time. The experunental
‘O”F’
Fii. 1. Fractton of undamaged HpD molecuIes N ~n)~~(O)as a f~nctton of the number of laser shots n for different N+ser peak intensities.
38
Fig 2. Damagepro~b~~~ per laser shot versus uradiation intennty I. The straight tine has a slope of 2.
Volume 88. number 1
I-
CHEMICAL
PHYSICS LETTERS
1
&
1
23 Aprd I982
teract directly with cells. On the other hand only the first mechanism (photodynamic action) can occur in the case of cw excitation. Thus, the presence of an additlonal cytocidal mechanism beside photodynamic action may account for the higher efficiency obtamed under pulsed Irradlatlon. This photoactivation mechanism of HpD based on the absorption of two photons stems lo be pronusing for possible apphcations in tumor phototherapy.
Acknowledgement We are grateful to Dr. S.F. Ambcsl-lmpiornbato for kindly prowding the ccl1 strain and lo M. Esposito and M. Mastrocinque for their assistance in preparing the ccl1 samples.
References Fig 3. Percentage of su~vlng cells as B function of the undutlon time. (a) pulsed irradiation (h = 337.1 nm); (b) cw uradiat~on(h = 333.6 nm). The avenge laser mtcnsity is 65 mW/cm* in both uses.
points were obtained with an average intensity of 65 mW/cm2 in both casesand could be fitted by stnlght hnes in a logarithmic plot. No decrease in the number of surviving cells was observed when cells incubated with HpD were irradiated in the same experimental conditions. The ratio of the slope of the mterpolating straight line (a) to that of(b) in fig. 3 is ~1.5. This demonstrates that pulsed irradiation has a cytocidal efficiency higher than that of cw. It must be noted that in the case of pulsed excitation the HpD molecules after the absorption of one photon are able to produce either singlet oxygen from their triplet state or, following the absorption of a second photon, radicals which can in-
[ 11T
J Dougherty, J.E. Kaufman. A. Coldhrb. K R. Washaupt, D G. Boyle and A. iMtclman.Canccr Rcs. 38 (1978) 2628. [2] T. Christcnscn and J. Xloan,Canccr Res. 39 (1979) 3735.
[ 31
C.J. Comer and T.J. Dougherty, Cancer Rcs 39 (1979) 146 141 K R. Wcishaupt, C.J. Comer and TJ. Dougherty. Cancer Rcs. 36 (1976) 2326. 151
I Diamond. A.F. McDonagh. C.B. Wdson et al., Lzmcct 2 (1972) 1175.
161 J.E. Kaufman, T.J Dougherty, A. Goldfarb. R J.R Jonhson, hl. D~dolkar, P. Crcavcn, R.R. WcUiaup1 and D.C. Boyle, Proc. Am. Sot. Clmical Oncol. 18 (1977) 275. 171 R. Cubcddu. S. de Sllvcstri and 0. Svclto, Opt Commun. 34 (1980) 460. 181 S.F. Ambcsl-lmplombato, R. Piconc and D. Tnmontano, in. Growth of Cells m Hormonally Defmed Lledls - CSH Conference on Cell Proliferation, Vol. 9. eds. D.A. Subxku. C.H. Sate and A.B. Pnrdee (CSII Press, New York, 1982). to be pubhshcd. 191 A Andrconi. R.Cubcddu, S. de Sdvcstrl. P. Laporta and 0. Svelte. Phys. Rev. Letters 4.5 (1980) 431.
39
I
CHEMICAL
PHYSICS LETTERS
23 Aprd 1982
TWO-STEP LASER ACTIVATION OF HEMATOPORPHYRIN DERIVATIVE A. ANDREONI, R. CLJBEDDU,S. DE SILVESTRI, P. LAPORTA and 0. SVELTO Centro Eleltronica Quonristim e Srrumenrarione Elemoniar, CNR Istmro di Frsrm dcl Pol~fecnrco.20133 Mhn. Italy Received 9 February 1982; In final form 23 kbruary
1982
A new photoaclrvatron mechamsm of hcmatoporphyrin dcrivatrvc IS presented This process, based on the scqucntral ab. sorption of two photons, leads to the production of cytotoxrc radicals of HpD. The cytocrdal efficnency is dcmonstmted by in vitro experiments.
I. Introduction Dyes such as hematoporphyrin (Hp) or its derrvativc (HpD) [I] are known to exhibit cytocrdal activity on normal and tumor cells [2], when photoactivated by cw hght. This property, together with the fact that they accumulate, wrth a certain degree of specificity, in tissues with a high mitotic index [3], makes them partlcularly attractive for the treatment of tumors. In recent years, the therapeutx ngnificance of photoactivated HpDhas beendemonstratedboth in experimental[4,5] and in human tumors 161. The activationprocessinvolvesthe intermediacy of the cytotoxic agent 1As 02 generated by electronic energy transfer from tnplet HpD (photodynamic action). In this paper we present a new mechanism of photoactivation that results in the production of HpD radicals following the absorption of two near-UV photons. The cytocidal efficrency of this process has been tested in vitro and compared with that of the photodynamic action.
2. Experimental HpD powder, kindly supplied by Dr. T.J. Dougherty from Roswell Park Memorial Hospital (Buffalo, USA), was dissolved in 0.1 M phosphate buffer (pH = 7.4). The value of the HpD concentration was calculated assuming a molecular weight of 598.7. The excitation source for the two-step photoactiva0 009.2614/82/0000-0000/.$02.75
0 1982 North-Holland
tion experunent was a nitrogen laser that provides pulses at 337.1 nrn with 10 ns duratron and peak power up to 250 kW at a repetition rate of 30 HZ. Peak lrradlallon intcnsrties of up to 20 MW/cm2 could be obtamcd. HpD samples of 0.2 cm3 volume were irradiated m a quartz cuvette wrth an optrcal path of 5 mm. The amount of dye decomposed by this process was momtored by measuring the decrease m the fluoresccncc intensity of HpD at X> 500 MI. The fluorescence was cxcrted at 405 nm by a probe pulse of K?OO ps duration, generated by a dye laser[7] pumped by an atmospheric-pressure mtrogen laser and it was obscwcd at 90° through a cut-off filter by an Xp 1210 photo. multiplier tube. The output signal was displayed on a Tektronix 7904 oscilloscope (500 MHz bandwidth). In vitro experiments were performed on an epithehal stram of rat thyroid orrgin (FR-TLS, see ref. [S]). Before irradration the cells were incubated for 2 h with 20 ~.rgHpD per ml of culture medium. Samples of 0.2 cm3 containing =2 X IO5 cells were uniformcly lrradnted in the same quartz cuvette of the prevrous experiment. After irradiation the cell-survival percentage was measured by additron of tripan blue (I% w/v) to the cell suspension and counting thereafter the hving cells in a Burker chamber. cw irradiation experiments on cells were performed in the same conditions, using the 333.6 nm hnc from an argon ion laser.
37
Volume 88. number I
CHE~I~CALPHYSICS LETI’ERS
3. Results and discussion
Under pulsed near-WI excltatlon a decrease in the fluorescence intensity of HpD is observed which mdicates an irreversiblenrodificntlon of the molecule (possable photoionization) leading to the formatIon of photoproducts. A first set of experiments was performed on 1.67 PM HpD solutrons to show the biphotomc character of this process. To evaluate the results, we define the dam-
age probablhty p per laser shot as the quantity p = AM/N,where N = N(tz) is the HpD concentration after the rzth laser pulse and AN is the reductton of this concentration after the next laser shot. Since m our ex~r~lent p 4 1, rt can be shown that [9] ln~~(n~~~v(o)~ =
-np
V#/ v,
(1)
Where V; is the inrerxtlon volume and Y is the total sample volume. Since the ratio N(ft)/N(O) is given by
23 Aprd 1982
the ratio of the fiuorescence signal after the nth laser shot to the in&al one, eq. (f) readily gives the value of p once the number of laser shots n is known. Fig. 1 shows a loganthmic plot ofN(n)/N(O) versus the number of laser shots at 337.1 run. The data were collected for different peak power values of the irradiation pulses. The experimental points at each irradiation intensity could be fitted by a straight line whose slope gives the value of p according to eq. (I), where Vi/V = 0.025. The values of p as a function of the irradiation intensi. ty I are presented in a logarithmic plot in fig. 2. The dependence ofp on the square of the intensrty is found for I values lower than 4 MWjcm2, which IS rndicatrve of a biphotonic process. For higher intensities, the square law is not followed sutce the first transrtio~
begins to saturate. On the basisof the above result whrch demonstrates that the photoproducts
are produced
by a two-step
process, a second set of experiments was performed to compare in vitro the cytocidal efficiency of this photoactivation scheme wrth that of the photodynamic action. The percentages of surviving cells after pulsed (A = 337.1 nm) and cw irradiation (h = 333.6 nm) are shown in fig. 3 (curves (a) and (b) respectively) as a functton of the irradiation time. The experunental
‘O”F’
Fii. 1. Fractton of undamaged HpD molecuIes N ~n)~~(O)as a f~nctton of the number of laser shots n for different N+ser peak intensities.
38
Fig 2. Damagepro~b~~~ per laser shot versus uradiation intennty I. The straight tine has a slope of 2.
Volume 88. number 1
I-
CHEMICAL
PHYSICS LETTERS
1
&
1
23 Aprd I982
teract directly with cells. On the other hand only the first mechanism (photodynamic action) can occur in the case of cw excitation. Thus, the presence of an additlonal cytocidal mechanism beside photodynamic action may account for the higher efficiency obtamed under pulsed Irradlatlon. This photoactivation mechanism of HpD based on the absorption of two photons stems lo be pronusing for possible apphcations in tumor phototherapy.
Acknowledgement We are grateful to Dr. S.F. Ambcsl-lmpiornbato for kindly prowding the ccl1 strain and lo M. Esposito and M. Mastrocinque for their assistance in preparing the ccl1 samples.
References Fig 3. Percentage of su~vlng cells as B function of the undutlon time. (a) pulsed irradiation (h = 337.1 nm); (b) cw uradiat~on(h = 333.6 nm). The avenge laser mtcnsity is 65 mW/cm* in both uses.
points were obtained with an average intensity of 65 mW/cm2 in both casesand could be fitted by stnlght hnes in a logarithmic plot. No decrease in the number of surviving cells was observed when cells incubated with HpD were irradiated in the same experimental conditions. The ratio of the slope of the mterpolating straight line (a) to that of(b) in fig. 3 is ~1.5. This demonstrates that pulsed irradiation has a cytocidal efficiency higher than that of cw. It must be noted that in the case of pulsed excitation the HpD molecules after the absorption of one photon are able to produce either singlet oxygen from their triplet state or, following the absorption of a second photon, radicals which can in-
[ 11T
J Dougherty, J.E. Kaufman. A. Coldhrb. K R. Washaupt, D G. Boyle and A. iMtclman.Canccr Rcs. 38 (1978) 2628. [2] T. Christcnscn and J. Xloan,Canccr Res. 39 (1979) 3735.
[ 31
C.J. Comer and T.J. Dougherty, Cancer Rcs 39 (1979) 146 141 K R. Wcishaupt, C.J. Comer and TJ. Dougherty. Cancer Rcs. 36 (1976) 2326. 151
I Diamond. A.F. McDonagh. C.B. Wdson et al., Lzmcct 2 (1972) 1175.
161 J.E. Kaufman, T.J Dougherty, A. Goldfarb. R J.R Jonhson, hl. D~dolkar, P. Crcavcn, R.R. WcUiaup1 and D.C. Boyle, Proc. Am. Sot. Clmical Oncol. 18 (1977) 275. 171 R. Cubcddu. S. de Sllvcstri and 0. Svclto, Opt Commun. 34 (1980) 460. 181 S.F. Ambcsl-lmplombato, R. Piconc and D. Tnmontano, in. Growth of Cells m Hormonally Defmed Lledls - CSH Conference on Cell Proliferation, Vol. 9. eds. D.A. Subxku. C.H. Sate and A.B. Pnrdee (CSII Press, New York, 1982). to be pubhshcd. 191 A Andrconi. R.Cubcddu, S. de Sdvcstrl. P. Laporta and 0. Svelte. Phys. Rev. Letters 4.5 (1980) 431.
39