- Email: [email protected]
Singlet oxygen lifetime dependence on photosensitizer concentration in lipid films
Journal of Luminescence 131 (2011) 442–444
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Singlet oxygen lifetime dependence on photosensitizer concentration in lipid films Roman Dˇedic, Vojtˇech Vyklicky´, Antonı´n Svoboda, Jan Ha´la Charles University in Prague, Faculty of Mathematics and Physics, Department of Chemical Physics and Optics, Ke Karlovu 3, 121 16 Praha 2, The Czech Republic
a r t i c l e in f o
a b s t r a c t
Available online 18 September 2010
It was shown that lipids substantially influence singlet oxygen lifetime. Question arises whether photosensitizers triplet states and excitation energy transfer to oxygen are also affected by lipids. In this contribution, the influence of lipids on excitation energy transfer from lipophilic photosensitizer tetraphenylporphyrin (TPP) to oxygen was investigated in bulk lipids and dry lipid films. Two components of TPP triplets decays were identified: quenching by oxygen which does not depend on TPP concentration and triplet–triplet annihilation. Rather long lifetimes of the TPP triplets around 1:1 ms are due to low solubility and diffusion coefficient of oxygen in the lipid. They are also reflected in low signal of singlet oxygen luminescence. Kinetics of the singlet oxygen luminescence follow convolutions of two exponential decays: rise-time independent on concentration and well corresponding to the short component of TPP triplet decay and decay time decreasing from 14 to 8 ms with increasing TPP concentration due to quenching of singlet oxygen by TPP. & 2010 Elsevier B.V. All rights reserved.
Keywords: TPP Singlet oxygen Lipids Phosphorescence PDT
1. Introduction Lipids represent basic components of biological membranes. They are also able to form liposomes—vesicles consisting of lipid bilayers. Liposomes are often used as carriers of lipophilic drugs in the bloodstream, for instance in photodynamic therapy [1] where they can be used to even target the drugs to specific cells [2]. Photodynamic therapy (PDT) is a rapidly emerging method to treat wide range of diseases, i. a. tumors, age related wet macular degeneration, rheumatoid arthritis, psoriasis, as well as antibioticresistant infections [3]. The treatment makes use of preferential accumulation of the drug in the target tissue during a delay after administration. The treated site is subsequently irradiated with visible or near infrared light. The light excites photosensitizer molecules to excited states. Excitation energy transfer from triplet states of the photosensitizers generates cytotoxic products, mainly singlet oxygen. These products can cause apoptosis or necrosis of the target tissue leading to the desired therapeutic effect. The relatively high efficiency, low mutagenic potential, and few side-effects compared to radiation therapy or chemotherapy are the key benefits that make for still growing application of the photodynamic therapy in clinical practice. Despite of the growing interest in this promising method of treatment, only little is known about particular mechanisms of the primary processes of excitation energy transfer between photosensitizers, singlet oxygen (as the
Corresponding author.
E-mail address: [email protected] (J. Ha´la). 0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.09.016
main cytotoxic agent in PDT), and biological molecules. Direct time-resolved detection of weak near infrared phosphorescence of both the photosensitizers and singlet oxygen proved to be an invaluable tool to investigate details of these crucial processes [4]. Our previous efforts in examining the interaction of photosensitizers with liposomes [5,6] left question of photosensitization in bulk lipids unanswered. This work addresses the question of lipid influence on photosensitizer triplet lifetimes and excitation energy transfer to oxygen by detection of time- and spectral-resolved emission of both the photosensitizer and singlet oxygen in bulk lipids prepared by solvent evaporation as well as spin-coated dry lipid films.
2. Experimental 2.1. Materials Meso-tetraphenylporphyrin (TPP), chlorin free, was obtained from Frontier Scientific (Logan, USA) and used without further purification. L-a–phosphatidyl-choline (PC, soy, 95%) was provided by Avanti Polar Lipids (Alabaster, USA). Chloroform (Lachema, Neratovice, Czech Republic) was analytical grade. Stock solutions of 350 mM TPP and 200 mM phosphatidylcholine in chloroform were prepared. The stock solutions were mixed to obtain the desired ratio of TPP to PC concentrations. To prepare bulk lipid sample, 0.25 ml of TPP stock solution was mixed with 1 ml of PC stock solution in standard 1 1 cm
spectroscopic fluorescence cell with optically polished bottom, the same type as is usually used for measurements of solutions. The chloroform was then evaporated during 2 h of blowing of the cell by dry nitrogen. Resulting film on the bottom of the cell was rather inhomogeneous with possible traces of the solvent remaining in the film. To improve homogeneity and evaporation of the solvent, the lipid film with different TPP to PC ratios were prepared by spin-coating of the mixture of both the stock solutions of TPP and PC on thin glass substrate. The resulting films contained 0.44, 1.75, 3.5, and 17.5 mmol of TPP per mol of PC.
phosphorescence [a.u.]
R. Dˇedic et al. / Journal of Luminescence 131 (2011) 442–444
10
1
2.2. Methods
3. Results and discussion On contrast to the single-exponential decays of TPP in organic solvents [8], the kinetics of TPP phosphorescence in spin-coated PC films is much more complicated. Typical phosphorescence decay is shown in Fig. 1. The decay does not follow simple linear combination of single exponentials but resembles kinetics obtained before for TPP in DMSO [9,10], where high amount of triplet–triplet quenching was observed due to low solubility and diffusion coefficient of oxygen in the solvent. Anyway, the decays can be rather well approximated by double exponential decays. The lifetimes of both components are shown in Fig. 2 as functions of TPP concentration in the lipid film. No distinctive concentration dependence was obtained for the short component with average value of ð1:6 7 0:2Þ ms. It is substantially longer than ð0:52 70:03Þ ms which we have obtained in TPP solution in chloroform (data not shown). On the other hand, the longer component is decreasing with growing TPP concentration from ð26 7 2Þ ms at 0.44 mmol TPP/mol PC to ð9:9 70:8Þ ms at 17.5 mol TPP/mol PC. This shortening can be attributed to
0
5
10
15 time [µs]
20
25
30
Fig. 1. Phosphorescence kinetics of TPP in lipid film with 17.5 mmol of TPP/mol of PC at 846 nm together with its double exponential fit.
30
lifetime [µs]
25 20 15 10 5 0 0
5 10 15 concentration [mmolTPP: molPC]
20
Fig. 2. Concentration dependence of lifetimes of both components of TPP phosphorescence decays.
120 phosphorescence [a.u.]
Our home-built set-up was used for detection of time- and spectral-resolved luminescence. The details of the device were described elsewhere [7], thus only a brief description of important details is provided here: The bulk lipid sample placed in a standard 1 1 cm spectroscopic fluorescence cell was excited through the optically polished bottom by 6 ns pulses of 10 mJ at 420 nm provided by excimer laser pumped dye laser. Resulting vertical luminescent spot was projected by an optical telescope on entrance slit of high-luminosity monochromator. Two long-pass glass filters RG7 (Schott) were placed between the sample and the first lens to remove scattered excitation light as well as strong fluorescence of the photosensitizer. Another telescope was used to focus the image of the output slit of the monochromator on the cathode of near infrared-sensitive photomultiplier. The signal from the photomultiplier was fed to time-resolved photoncounter/multiscaler with 5 ns time resolution. This set-up was slightly modified to collect luminescence from surface of solid samples, this time of the spin-coated lipid films. Output of detection leg of bifurcated optical fiber with luminescence probe Avantes FCR-7IR400-2-ME was placed in the place, where the luminescent spot in the cell is usually located. The sample was excited using the same pulses as before through the other leg of the fiber. The long-pass filter TECHSPEC 450 nm exhibiting much lower autofluorescence compared to the RG7 filters was put in front of the long-pass filters to block backscattered excitation laser light. However, even this measure is not able to completely dismiss filter luminescence, which occurs at similar wavelengths and timescales as the sample luminescence. Therefore, the background signals measured on films with no photosensitizer have to be subtracted from the data.
443
100 80 60 40 20 0 -20 0
5
10
15
20
25
30
35
40
time [µs] Fig. 3. Singlet oxygen luminescence kinetics lipid film with 1.75 mmol of TPP/mol of PC at 1278 nm together with its fit.
increasing rate of triplet–triplet quenching at higher photosensitizer concentrations. The low solubility and diffusion coefficient of oxygen in lipids result in rather low luminescence of singlet oxygen. Nevertheless the complex TPP triplet decays, the luminescence kinetics of singlet oxygen can be described by simple convolution of two single-exponential functions. It is documented in Fig. 3. Moreover, the rise-times of the kinetics also do not exhibit any concentration dependence with average value of ð1:1 7 0:2Þ ms (see Fig. 4). This value is somewhat shorter than the average TPP triplet lifetime of
444
R. Dˇedic et al. / Journal of Luminescence 131 (2011) 442–444
30
lifetime [µs]
25 20 15 10 5 0 0
5
10
15
20
concentration [mmolTPP: molPC] Fig. 4. Concentration dependence of rise- and decay-times of singlet oxygen at 1278 nm.
phosphorescence [a.u.]
4000 CHCl3 CHCl3+0.2 M PC bulk lipid spin coating
3500 3000 2500 10×
2000
200 mM of the lipid. Finally, it reaches ð1:1 7 0:1Þ ms in both the bulk lipid and the spin-coated samples. On the contrary to the films, the singlet oxygen kinetics in the bulk sample exhibited more complex decay which can be approximated by linear combination of two exponential components with lifetimes of ð5:9 70:6Þ ms and ð357 4Þ ms. These two components are probably due to the presence of two phases, the lipid and the remaining chloroform. Although the amount of the remaining chloroform in the bulk sample is very low, the much higher singlet oxygen luminescence in chloroform compared to the lipid due to higher solubility and diffusion coefficient of oxygen in chloroform makes this component significant (42% of total oxygen luminescence intensity). The lifetime of the shorter decay roughly corresponds to the 8 ms lifetime of the singlet oxygen in the spin-coated film of the highest photosensitizer concentration. The lifetime of the longer component of ð35 74Þ ms is shorter than the singleexponential decay of ð78 7 1Þ ms obtained in 140 mM TPP in chloroform (top curve in Fig. 5). It follows the trend of singlet oxygen lifetimes shortening with increasing TPP concentration due to quenching of singlet oxygen by TPP, similar to that observed in acetone [8]. Taking into account singlet oxygen lifetime of ð156:5 70:9Þ ms in 10 mM TPP in chloroform, the extrapolated concentration of TPP in chloroform phase in the bulk lipid sample would be around 450 mM under assumption of bimolecular quenching mechanism in the solutions.
1500 1000
4. Conclusion
500 0 0.1
1
10
100
time [µs] Fig. 5. Singlet oxygen luminescence kinetics at 1278 nm in chloroform with two different concentrations of PC (0 and 200 mM), in bulk lipid obtained by evaporating the solution in the spectroscopic cell, and in spin-coated glass.
ð1:6 7 0:2Þ ms. The same effect was observed in acetone solutions of TPP [8] as well as in phosphate buffer solutions of TPPS4 (5,10,15,20-tetrakis (4-sulfonatophenyl) porphine) [11] and TMPyP (5,10,15,20-tetrakis (1-methyl-4-pyridinio) porphine) [12]. The differences between lifetimes of photosensitizer triplets and rise-times of singlet oxygen were formely explained in the frame of aggregation and later the role of transient chargetransfer complexes was identified in these publications. The similarity of the current data justifies the identification of the shorter component as being due to oxygen quenching. The longer component exhibits decrease from ð14 7 2Þ ms at the lowest TPP concentration to ð87 3Þ ms but it does not follow bimolecular quenching dependence. The shortening is accompanied also by approximately two-fold decrease of maximal singlet oxygen phosphorescence from the lowest to the highest TPP concentration. Both these phenomena are probably due to quenching of the singlet oxygen by the photosensitizer molecules similarly to the case of acetonic solutions [8]. The very low signal especially at the highest concentration also explains rather big errors of the singlet oxygen lifetimes. The value of 14 ms corresponds to the one published before for PC by Baier [13], but the concentration dependence suggest, that the value extrapolated to the zero photosensitizer concentration should be even higher. Fig. 5 illustrates the trends in singlet oxygen luminescence kinetics in samples with increasing ratio of PC to chloroform. The rise-time of singlet oxygen (roughly corresponding to the lifetime of TPP triplets) increases with lipid concentration from ð0:34 7 0:01Þ ms at pure chloroform to ð0:59 7 0:02Þ ms in the sample containing
On the contrary to organic solvents, two components of TPP triplets decays were identified: relatively slow quenching by oxygen which does not depend on TPP concentration and triplet–triplet annihilation. Low solubility and diffusion coefficient of oxygen in the lipid are also reflected in low signal of singlet oxygen luminescence. Kinetics of the singlet oxygen luminescence follow convolutions of two exponential decays: rise-time independent on concentration and corresponding to the short component of TPP triplet decay and decay with lifetime decreasing from 14 to 8 ms with increasing TPP concentration. This strong dependence points out the importance of quenching of singlet oxygen by photosensitizer in lipid media.
Acknowledgement This work was supported by project number MSM 0021620835 from the Ministry of Education of the Czech Republic. References [1] A.S.L. Derycke, P.A.M. de Witte, Adv. Drug Deliv. Rev. 56 (2004) 17. [2] A. Gijsens, A. Derycke, L. Missiaen, D. De Vos, J. Huwyler, A. Eberle, P. De Witte, Int. J. Cancer 101 (2002) 78. [3] B.C. Wilson, M.S. Patterson, Phys. Med. Biol. 53 (2008) R61. [4] A.A. Krasnovsky Jr., J. Photochem. Photobiol. A-Chem. 196 (2008) 210. [5] A. Molna´r, R. Dˇedic, A. Svoboda, J. Ha´la, J. Mol. Struct. 834 (2007) 488. [6] A. Molna´r, R. Dˇedic, A. Svoboda, J. Ha´la, J. Lumines. 128 (2008) 783. [7] R. Dˇedic, A. Svoboda, J. Pˇsencˇı´k, J. Ha´la, J. Mol. Struct. 651–653 (2003) 301. [8] M. Koˇrı´nek, R. Dˇedic, A. Svoboda, J. Ha´la, J. Fluoresc. 14 (2004) 71. [9] M. Koˇrı´nek, R. Dˇedic, A. Molna´r, A. Svoboda, J. Ha´la, J. Mol. Struct. 744–747 (2005) 727. [10] M. Koˇrı´nek, P. Klinger, R. Dˇedic, J. Pˇsencˇı´k, A. Svoboda, J. Ha´la, J. Lumines. 122 (2007) 247. [11] R. Dˇedic, A. Molna´r, M. Koˇrı´nek, A. Svoboda, J. Ha´la, J. Lumines. 108 (2004) 117. [12] R. Dˇedic, V. Vyklicky´, A. Svoboda, J. Ha´la, J. Mol. Struct. 924–926 (2009) 153. ¨ [13] J. Baier, M. Maier, R. Engl, M. Landthaler, W. Baumler, J. Phys. Chem. B 109 (2005) 3041.
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Singlet oxygen lifetime dependence on photosensitizer concentration in lipid films Roman Dˇedic, Vojtˇech Vyklicky´, Antonı´n Svoboda, Jan Ha´la Charles University in Prague, Faculty of Mathematics and Physics, Department of Chemical Physics and Optics, Ke Karlovu 3, 121 16 Praha 2, The Czech Republic
a r t i c l e in f o
a b s t r a c t
Available online 18 September 2010
It was shown that lipids substantially influence singlet oxygen lifetime. Question arises whether photosensitizers triplet states and excitation energy transfer to oxygen are also affected by lipids. In this contribution, the influence of lipids on excitation energy transfer from lipophilic photosensitizer tetraphenylporphyrin (TPP) to oxygen was investigated in bulk lipids and dry lipid films. Two components of TPP triplets decays were identified: quenching by oxygen which does not depend on TPP concentration and triplet–triplet annihilation. Rather long lifetimes of the TPP triplets around 1:1 ms are due to low solubility and diffusion coefficient of oxygen in the lipid. They are also reflected in low signal of singlet oxygen luminescence. Kinetics of the singlet oxygen luminescence follow convolutions of two exponential decays: rise-time independent on concentration and well corresponding to the short component of TPP triplet decay and decay time decreasing from 14 to 8 ms with increasing TPP concentration due to quenching of singlet oxygen by TPP. & 2010 Elsevier B.V. All rights reserved.
Keywords: TPP Singlet oxygen Lipids Phosphorescence PDT
1. Introduction Lipids represent basic components of biological membranes. They are also able to form liposomes—vesicles consisting of lipid bilayers. Liposomes are often used as carriers of lipophilic drugs in the bloodstream, for instance in photodynamic therapy [1] where they can be used to even target the drugs to specific cells [2]. Photodynamic therapy (PDT) is a rapidly emerging method to treat wide range of diseases, i. a. tumors, age related wet macular degeneration, rheumatoid arthritis, psoriasis, as well as antibioticresistant infections [3]. The treatment makes use of preferential accumulation of the drug in the target tissue during a delay after administration. The treated site is subsequently irradiated with visible or near infrared light. The light excites photosensitizer molecules to excited states. Excitation energy transfer from triplet states of the photosensitizers generates cytotoxic products, mainly singlet oxygen. These products can cause apoptosis or necrosis of the target tissue leading to the desired therapeutic effect. The relatively high efficiency, low mutagenic potential, and few side-effects compared to radiation therapy or chemotherapy are the key benefits that make for still growing application of the photodynamic therapy in clinical practice. Despite of the growing interest in this promising method of treatment, only little is known about particular mechanisms of the primary processes of excitation energy transfer between photosensitizers, singlet oxygen (as the
Corresponding author.
E-mail address: [email protected] (J. Ha´la). 0022-2313/$ - see front matter & 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2010.09.016
main cytotoxic agent in PDT), and biological molecules. Direct time-resolved detection of weak near infrared phosphorescence of both the photosensitizers and singlet oxygen proved to be an invaluable tool to investigate details of these crucial processes [4]. Our previous efforts in examining the interaction of photosensitizers with liposomes [5,6] left question of photosensitization in bulk lipids unanswered. This work addresses the question of lipid influence on photosensitizer triplet lifetimes and excitation energy transfer to oxygen by detection of time- and spectral-resolved emission of both the photosensitizer and singlet oxygen in bulk lipids prepared by solvent evaporation as well as spin-coated dry lipid films.
2. Experimental 2.1. Materials Meso-tetraphenylporphyrin (TPP), chlorin free, was obtained from Frontier Scientific (Logan, USA) and used without further purification. L-a–phosphatidyl-choline (PC, soy, 95%) was provided by Avanti Polar Lipids (Alabaster, USA). Chloroform (Lachema, Neratovice, Czech Republic) was analytical grade. Stock solutions of 350 mM TPP and 200 mM phosphatidylcholine in chloroform were prepared. The stock solutions were mixed to obtain the desired ratio of TPP to PC concentrations. To prepare bulk lipid sample, 0.25 ml of TPP stock solution was mixed with 1 ml of PC stock solution in standard 1 1 cm
spectroscopic fluorescence cell with optically polished bottom, the same type as is usually used for measurements of solutions. The chloroform was then evaporated during 2 h of blowing of the cell by dry nitrogen. Resulting film on the bottom of the cell was rather inhomogeneous with possible traces of the solvent remaining in the film. To improve homogeneity and evaporation of the solvent, the lipid film with different TPP to PC ratios were prepared by spin-coating of the mixture of both the stock solutions of TPP and PC on thin glass substrate. The resulting films contained 0.44, 1.75, 3.5, and 17.5 mmol of TPP per mol of PC.
phosphorescence [a.u.]
R. Dˇedic et al. / Journal of Luminescence 131 (2011) 442–444
10
1
2.2. Methods
3. Results and discussion On contrast to the single-exponential decays of TPP in organic solvents [8], the kinetics of TPP phosphorescence in spin-coated PC films is much more complicated. Typical phosphorescence decay is shown in Fig. 1. The decay does not follow simple linear combination of single exponentials but resembles kinetics obtained before for TPP in DMSO [9,10], where high amount of triplet–triplet quenching was observed due to low solubility and diffusion coefficient of oxygen in the solvent. Anyway, the decays can be rather well approximated by double exponential decays. The lifetimes of both components are shown in Fig. 2 as functions of TPP concentration in the lipid film. No distinctive concentration dependence was obtained for the short component with average value of ð1:6 7 0:2Þ ms. It is substantially longer than ð0:52 70:03Þ ms which we have obtained in TPP solution in chloroform (data not shown). On the other hand, the longer component is decreasing with growing TPP concentration from ð26 7 2Þ ms at 0.44 mmol TPP/mol PC to ð9:9 70:8Þ ms at 17.5 mol TPP/mol PC. This shortening can be attributed to
0
5
10
15 time [µs]
20
25
30
Fig. 1. Phosphorescence kinetics of TPP in lipid film with 17.5 mmol of TPP/mol of PC at 846 nm together with its double exponential fit.
30
lifetime [µs]
25 20 15 10 5 0 0
5 10 15 concentration [mmolTPP: molPC]
20
Fig. 2. Concentration dependence of lifetimes of both components of TPP phosphorescence decays.
120 phosphorescence [a.u.]
Our home-built set-up was used for detection of time- and spectral-resolved luminescence. The details of the device were described elsewhere [7], thus only a brief description of important details is provided here: The bulk lipid sample placed in a standard 1 1 cm spectroscopic fluorescence cell was excited through the optically polished bottom by 6 ns pulses of 10 mJ at 420 nm provided by excimer laser pumped dye laser. Resulting vertical luminescent spot was projected by an optical telescope on entrance slit of high-luminosity monochromator. Two long-pass glass filters RG7 (Schott) were placed between the sample and the first lens to remove scattered excitation light as well as strong fluorescence of the photosensitizer. Another telescope was used to focus the image of the output slit of the monochromator on the cathode of near infrared-sensitive photomultiplier. The signal from the photomultiplier was fed to time-resolved photoncounter/multiscaler with 5 ns time resolution. This set-up was slightly modified to collect luminescence from surface of solid samples, this time of the spin-coated lipid films. Output of detection leg of bifurcated optical fiber with luminescence probe Avantes FCR-7IR400-2-ME was placed in the place, where the luminescent spot in the cell is usually located. The sample was excited using the same pulses as before through the other leg of the fiber. The long-pass filter TECHSPEC 450 nm exhibiting much lower autofluorescence compared to the RG7 filters was put in front of the long-pass filters to block backscattered excitation laser light. However, even this measure is not able to completely dismiss filter luminescence, which occurs at similar wavelengths and timescales as the sample luminescence. Therefore, the background signals measured on films with no photosensitizer have to be subtracted from the data.
443
100 80 60 40 20 0 -20 0
5
10
15
20
25
30
35
40
time [µs] Fig. 3. Singlet oxygen luminescence kinetics lipid film with 1.75 mmol of TPP/mol of PC at 1278 nm together with its fit.
increasing rate of triplet–triplet quenching at higher photosensitizer concentrations. The low solubility and diffusion coefficient of oxygen in lipids result in rather low luminescence of singlet oxygen. Nevertheless the complex TPP triplet decays, the luminescence kinetics of singlet oxygen can be described by simple convolution of two single-exponential functions. It is documented in Fig. 3. Moreover, the rise-times of the kinetics also do not exhibit any concentration dependence with average value of ð1:1 7 0:2Þ ms (see Fig. 4). This value is somewhat shorter than the average TPP triplet lifetime of
444
R. Dˇedic et al. / Journal of Luminescence 131 (2011) 442–444
30
lifetime [µs]
25 20 15 10 5 0 0
5
10
15
20
concentration [mmolTPP: molPC] Fig. 4. Concentration dependence of rise- and decay-times of singlet oxygen at 1278 nm.
phosphorescence [a.u.]
4000 CHCl3 CHCl3+0.2 M PC bulk lipid spin coating
3500 3000 2500 10×
2000
200 mM of the lipid. Finally, it reaches ð1:1 7 0:1Þ ms in both the bulk lipid and the spin-coated samples. On the contrary to the films, the singlet oxygen kinetics in the bulk sample exhibited more complex decay which can be approximated by linear combination of two exponential components with lifetimes of ð5:9 70:6Þ ms and ð357 4Þ ms. These two components are probably due to the presence of two phases, the lipid and the remaining chloroform. Although the amount of the remaining chloroform in the bulk sample is very low, the much higher singlet oxygen luminescence in chloroform compared to the lipid due to higher solubility and diffusion coefficient of oxygen in chloroform makes this component significant (42% of total oxygen luminescence intensity). The lifetime of the shorter decay roughly corresponds to the 8 ms lifetime of the singlet oxygen in the spin-coated film of the highest photosensitizer concentration. The lifetime of the longer component of ð35 74Þ ms is shorter than the singleexponential decay of ð78 7 1Þ ms obtained in 140 mM TPP in chloroform (top curve in Fig. 5). It follows the trend of singlet oxygen lifetimes shortening with increasing TPP concentration due to quenching of singlet oxygen by TPP, similar to that observed in acetone [8]. Taking into account singlet oxygen lifetime of ð156:5 70:9Þ ms in 10 mM TPP in chloroform, the extrapolated concentration of TPP in chloroform phase in the bulk lipid sample would be around 450 mM under assumption of bimolecular quenching mechanism in the solutions.
1500 1000
4. Conclusion
500 0 0.1
1
10
100
time [µs] Fig. 5. Singlet oxygen luminescence kinetics at 1278 nm in chloroform with two different concentrations of PC (0 and 200 mM), in bulk lipid obtained by evaporating the solution in the spectroscopic cell, and in spin-coated glass.
ð1:6 7 0:2Þ ms. The same effect was observed in acetone solutions of TPP [8] as well as in phosphate buffer solutions of TPPS4 (5,10,15,20-tetrakis (4-sulfonatophenyl) porphine) [11] and TMPyP (5,10,15,20-tetrakis (1-methyl-4-pyridinio) porphine) [12]. The differences between lifetimes of photosensitizer triplets and rise-times of singlet oxygen were formely explained in the frame of aggregation and later the role of transient chargetransfer complexes was identified in these publications. The similarity of the current data justifies the identification of the shorter component as being due to oxygen quenching. The longer component exhibits decrease from ð14 7 2Þ ms at the lowest TPP concentration to ð87 3Þ ms but it does not follow bimolecular quenching dependence. The shortening is accompanied also by approximately two-fold decrease of maximal singlet oxygen phosphorescence from the lowest to the highest TPP concentration. Both these phenomena are probably due to quenching of the singlet oxygen by the photosensitizer molecules similarly to the case of acetonic solutions [8]. The very low signal especially at the highest concentration also explains rather big errors of the singlet oxygen lifetimes. The value of 14 ms corresponds to the one published before for PC by Baier [13], but the concentration dependence suggest, that the value extrapolated to the zero photosensitizer concentration should be even higher. Fig. 5 illustrates the trends in singlet oxygen luminescence kinetics in samples with increasing ratio of PC to chloroform. The rise-time of singlet oxygen (roughly corresponding to the lifetime of TPP triplets) increases with lipid concentration from ð0:34 7 0:01Þ ms at pure chloroform to ð0:59 7 0:02Þ ms in the sample containing
On the contrary to organic solvents, two components of TPP triplets decays were identified: relatively slow quenching by oxygen which does not depend on TPP concentration and triplet–triplet annihilation. Low solubility and diffusion coefficient of oxygen in the lipid are also reflected in low signal of singlet oxygen luminescence. Kinetics of the singlet oxygen luminescence follow convolutions of two exponential decays: rise-time independent on concentration and corresponding to the short component of TPP triplet decay and decay with lifetime decreasing from 14 to 8 ms with increasing TPP concentration. This strong dependence points out the importance of quenching of singlet oxygen by photosensitizer in lipid media.
Acknowledgement This work was supported by project number MSM 0021620835 from the Ministry of Education of the Czech Republic. References [1] A.S.L. Derycke, P.A.M. de Witte, Adv. Drug Deliv. Rev. 56 (2004) 17. [2] A. Gijsens, A. Derycke, L. Missiaen, D. De Vos, J. Huwyler, A. Eberle, P. De Witte, Int. J. Cancer 101 (2002) 78. [3] B.C. Wilson, M.S. Patterson, Phys. Med. Biol. 53 (2008) R61. [4] A.A. Krasnovsky Jr., J. Photochem. Photobiol. A-Chem. 196 (2008) 210. [5] A. Molna´r, R. Dˇedic, A. Svoboda, J. Ha´la, J. Mol. Struct. 834 (2007) 488. [6] A. Molna´r, R. Dˇedic, A. Svoboda, J. Ha´la, J. Lumines. 128 (2008) 783. [7] R. Dˇedic, A. Svoboda, J. Pˇsencˇı´k, J. Ha´la, J. Mol. Struct. 651–653 (2003) 301. [8] M. Koˇrı´nek, R. Dˇedic, A. Svoboda, J. Ha´la, J. Fluoresc. 14 (2004) 71. [9] M. Koˇrı´nek, R. Dˇedic, A. Molna´r, A. Svoboda, J. Ha´la, J. Mol. Struct. 744–747 (2005) 727. [10] M. Koˇrı´nek, P. Klinger, R. Dˇedic, J. Pˇsencˇı´k, A. Svoboda, J. Ha´la, J. Lumines. 122 (2007) 247. [11] R. Dˇedic, A. Molna´r, M. Koˇrı´nek, A. Svoboda, J. Ha´la, J. Lumines. 108 (2004) 117. [12] R. Dˇedic, V. Vyklicky´, A. Svoboda, J. Ha´la, J. Mol. Struct. 924–926 (2009) 153. ¨ [13] J. Baier, M. Maier, R. Engl, M. Landthaler, W. Baumler, J. Phys. Chem. B 109 (2005) 3041.