- Email: [email protected]
Uptake of sensitizer by electroporated yeast cells
Bioelectrochemistry and Bioenergetics 47 Ž1998. 175–177
Short communication
Uptake of sensitizer by electroporated yeast cells Xing Wang a , I. Hones ¨ b, Hermann Berg
c,)
a
Biophysics Program, Department of Physics, Nankai UniÕersity, Tianjin 300071, China Department of Molecular Biology, Institute of Molecular Biotechnology, Jena, Germany Laboratory of Bioelectrochemistry, Saxonian Academy of Sciences at Leipzig, Jena, Germany b
c
Received 30 May 1998; revised 16 July 1998; accepted 19 August 1998
Abstract The photodynamic effect on yeast Ž Saccharomyces cereÕisiae . cell viability has been enhanced by electroporation treatment. Exponentially decaying high-voltage pulses caused temporary permeability changes on the cell walls, by which the uptake of the sensitizer thiopyronine ŽTP. has been accelerated. The total killing effect after visible light irradiation, determined by Phloxine B exclusion tests, depended on the pulse strength and the TP concentration as well as the irradiation time. Under optimal condition, 0.01 mM TP and pulse strength 2.35 kVrcm, it was shown that the effect after 5 min irradiation increased to more than 10 times, e.g., 75–80% cells are dead, as much as the control sample, which was treated only by TP and irradiation but without electroporation. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Saccharomyces cereÕisiae; Thiopyronine; Electroporation
1. Introduction
2. Experimental
Photodynamic effects are known since the beginning of our century, and their cytotoxicity mechanisms have been evaluated since 1960s, e.g., Ža. the hydrogen peroxide production w1x; Žb. the photooxidation of guanine and DNA strand-break w2,3x. The mechanism Žb. needs penetration of the sensitizer through the cell envelope for complex formations mainly with nucleic acids for subsequent excitation by visible light irradiation. This transport process is the rate limiting step for the cytotoxic efficiency before fast complex formation with DNA and light treatment. Therefore, we accelerated this process by electropermeation applying single, high exponential pulses w4–6x causing pore formation in the membrane. In this way, a synergistic effect has been found.
2.1. Preparation of yeast cells
) Corresponding author. Laboratory of Bioelectrochemistry, Beutenbergstrasse 11, 077745 Jena, Germany. E-mail: [email protected]
Saccharomyces cereÕisiae H192 ZIMET strain was inoculated to a liquid YEPG medium Ž1% yeast extract, 2% peptone, 2% glucose, pH 6.5. and precultured in a temperature controlled shaking bath at 308C for 18 h. Then the cells were harvested and washed three times in 0.9% NaCl solution by centrifugation Ž2000 rpm, 5 min.. Finally, 1 = 10 7 cellsrml were resuspended in 0.9% NaCl for the experiment. 2.2. Electroporation Preparative electroporation was performed by a BTX Electro Cell Manipulator 600 ŽGenetronix, CA. equipped with a generator of exponentially decaying pulses and disposable cuvette chambers with embedded 0.2 cm gap aluminum electrodes. The experiments on yeast cells were carried out in a higher output voltage mode at ambient temperatures of 20–248C. A total of 200 ml cells suspensions with different concentrations of thiopyronine ŽTP. were subjected to one pulse of different field strengths.
0302-4598r98r$ - see front matter q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 1 6 0 - 3
176
X. Wang et al.r Bioelectrochemistry and Bioenergetics 47 (1998) 175–177
2.3. Irradiation and measurement After the electropermeation, the cell suspension stayed for 5 min for resealing the pores. Then the cuvettes of cell sample and control were positioned on both sides of a 500 W lamp ŽNarva, Berlin. at 37 cm distance in the focus areas of two identical lenses. There are two circulating water volumes, 4 cm in thickness, after the lenses as heat absorbers. We have done the same test on both sides and obtained the same results, which proves that the light beams are identical for this work. Monitoring of the synergistic effect after pulse and irradiation was performed by an inverted light microscope ŽOlympus, Japan. equipped with a CCD-IRIS video camera ŽSony, Japan., using the Phloxine B exclusion test for yeast cells.
3. Results and discussion To investigate concentration dependence of TP on the photodynamic effect, a set of tests were performed under the appropriate pulse strength of 2.35 kVrcm for yeast cell electroporation. The results are shown in the Fig. 1. Along with higher concentrations of TP, the cell killing increased until the optimal wTPx, 1 = 10y5 M. Maintaining wTPx at 1 = 10y5 M, the dependence of the photodynamic effect on the pulse strength has been investigated in detail. For yeast cells, the optimum eletroporation
Fig. 1. Concentration dependence of TP on the photodynamic effect of yeast cells S. cereÕisiae by 5 min visible light irradiation, which followed 5 min after the pulse. The pulse strength is 2.35 kVrcm, while the pulse length is 0.35 ms.
Fig. 2. Pulse strength dependence of the photodynamic effect on yeast cells S. cereÕisiae by 5 min visible light irradiation, which followed 5 min after the pulse. wTPx s1.0=10y5 M.
was obtained by 2.35 kVrcm, which is shown in Fig. 2. The pulse produces several effects on cells, besides pore formation andror the lipid disorders in accelerating molecular transport, cell death occurs if the pores are too big or
Fig. 3. Synergism of electropermeation and photodynamic effect of yeast cells S. cereÕisiae by 5 min visible light irradiation, which followed 5 min after the pulse. Žv . For the test sample, and wTPx is 1.0=10y5 M and the pulse strength is 2.35 kVrcm, while the pulse length is 0.35 ms. ŽB. For the control sample, without pulse. The left part shows the process of resealing after electropermeation.
X. Wang et al.r Bioelectrochemistry and Bioenergetics 47 (1998) 175–177
the number of pores is too high, so that resealing is no more possible. When the pulse increases, both effects are turning stronger, but at different rates. Before reaching 2.35 kVrcm, the former one is the main result, while after that the latter one is predominant. The synergism of photodynamic and electropermeation effects at the optimal condition for yeast cells is shown in Fig. 3. Compared to the control samples, this synergistic effect is ten times higher. One third of cell killing is caused by the pulse and two thirds are coming out by TP radicals according to: TP q hn l TP) q
q
TP) q G Ž DNA . ™ TPH 2 q G Ž DNA . TPH 2 q O 2 ™ TP q H 2 O 2
Ž 1. Ž 2. Ž 3.
Because of the electropermeation, the wTPx in cells reaches quickly the same level as outside. Therefore, the photodynamic damage is accelerated by pore penetration. The efficiency can be increased further by transport of oxygen to the tumor cells since according to Eq. Ž3. the regeneration of TP or another dye will be facilitated.
4. Conclusion Our aim to combine the electropermeation with the photodynamic effect presents results in a synergistic cell killing possibility, which is suitable to increase the cytotoxic efficiency. According to this novel method it is possible to distinguish between pulse killing of original
177
cells and light destruction of mostly resealed cells. The efficiency of both leads to a tenfold higher cytotoxicity than mere photodynamic action as usual.
Acknowledgements Xing Wang thank sincerely the grant from the Boehringer Ingelheim Fonds, Stuttgart, and Dr.Velizarov for invaluable discussion and suggestions.
References w1x H. Berg, H.-E. Jacob, Biologische Wirkungen photochemischer Wasserstoff-peroxydbildung: 2. Mitt., Photochem. Photobiol. 4 Ž1965. 55. w2x F.A. Gollmick, H. Berg, Sensibilisierte Photooxidation durch Meth¨ ylenblau, Thiopyronin und Pyronin: III. Mitt. Uber den Mechanismus der photosensibilisierten Oxydation des Guanosins durch Thiopyronin, Photochem. Photobiol. 16 Ž1972. 447. w3x L. Kittler, G. Lober, F.A. Gollmick, H. Berg, Redox processes during ¨ photodynamic damage of DNA: III. Redox mechanism of photosensitization and radical reaction, Bioelectrochem. Bioenerg. 7 Ž1980. 503. w4x H.-E. Jacob, W. Forster, H. Berg, Microbiological implications of ¨ electric field effects: II. Inactivation of yeast cells and repair of their cell envelope, Z. Allgem. Mikrobiol. 21 Ž1981. 225. w5x M. Muraji, W. Tatebe, T. Konishi, T. Fuyii, H. Berg, Effect of electrical energy on the electropermeabilization of yeast cells, Bioelectrochem. Bioenerg. 31 Ž1993. 77. w6x H.-E. Jacob, W. Forster, M. Hamann, Photodynamic inactivation of ¨ yeast cells by thiopyronine after electric field pulse treatment, Studia Biophysica ŽBerlin. 94 Ž1983. 71–72.
Short communication
Uptake of sensitizer by electroporated yeast cells Xing Wang a , I. Hones ¨ b, Hermann Berg
c,)
a
Biophysics Program, Department of Physics, Nankai UniÕersity, Tianjin 300071, China Department of Molecular Biology, Institute of Molecular Biotechnology, Jena, Germany Laboratory of Bioelectrochemistry, Saxonian Academy of Sciences at Leipzig, Jena, Germany b
c
Received 30 May 1998; revised 16 July 1998; accepted 19 August 1998
Abstract The photodynamic effect on yeast Ž Saccharomyces cereÕisiae . cell viability has been enhanced by electroporation treatment. Exponentially decaying high-voltage pulses caused temporary permeability changes on the cell walls, by which the uptake of the sensitizer thiopyronine ŽTP. has been accelerated. The total killing effect after visible light irradiation, determined by Phloxine B exclusion tests, depended on the pulse strength and the TP concentration as well as the irradiation time. Under optimal condition, 0.01 mM TP and pulse strength 2.35 kVrcm, it was shown that the effect after 5 min irradiation increased to more than 10 times, e.g., 75–80% cells are dead, as much as the control sample, which was treated only by TP and irradiation but without electroporation. q 1998 Elsevier Science S.A. All rights reserved. Keywords: Saccharomyces cereÕisiae; Thiopyronine; Electroporation
1. Introduction
2. Experimental
Photodynamic effects are known since the beginning of our century, and their cytotoxicity mechanisms have been evaluated since 1960s, e.g., Ža. the hydrogen peroxide production w1x; Žb. the photooxidation of guanine and DNA strand-break w2,3x. The mechanism Žb. needs penetration of the sensitizer through the cell envelope for complex formations mainly with nucleic acids for subsequent excitation by visible light irradiation. This transport process is the rate limiting step for the cytotoxic efficiency before fast complex formation with DNA and light treatment. Therefore, we accelerated this process by electropermeation applying single, high exponential pulses w4–6x causing pore formation in the membrane. In this way, a synergistic effect has been found.
2.1. Preparation of yeast cells
) Corresponding author. Laboratory of Bioelectrochemistry, Beutenbergstrasse 11, 077745 Jena, Germany. E-mail: [email protected]
Saccharomyces cereÕisiae H192 ZIMET strain was inoculated to a liquid YEPG medium Ž1% yeast extract, 2% peptone, 2% glucose, pH 6.5. and precultured in a temperature controlled shaking bath at 308C for 18 h. Then the cells were harvested and washed three times in 0.9% NaCl solution by centrifugation Ž2000 rpm, 5 min.. Finally, 1 = 10 7 cellsrml were resuspended in 0.9% NaCl for the experiment. 2.2. Electroporation Preparative electroporation was performed by a BTX Electro Cell Manipulator 600 ŽGenetronix, CA. equipped with a generator of exponentially decaying pulses and disposable cuvette chambers with embedded 0.2 cm gap aluminum electrodes. The experiments on yeast cells were carried out in a higher output voltage mode at ambient temperatures of 20–248C. A total of 200 ml cells suspensions with different concentrations of thiopyronine ŽTP. were subjected to one pulse of different field strengths.
0302-4598r98r$ - see front matter q 1998 Elsevier Science S.A. All rights reserved. PII: S 0 3 0 2 - 4 5 9 8 Ž 9 8 . 0 0 1 6 0 - 3
176
X. Wang et al.r Bioelectrochemistry and Bioenergetics 47 (1998) 175–177
2.3. Irradiation and measurement After the electropermeation, the cell suspension stayed for 5 min for resealing the pores. Then the cuvettes of cell sample and control were positioned on both sides of a 500 W lamp ŽNarva, Berlin. at 37 cm distance in the focus areas of two identical lenses. There are two circulating water volumes, 4 cm in thickness, after the lenses as heat absorbers. We have done the same test on both sides and obtained the same results, which proves that the light beams are identical for this work. Monitoring of the synergistic effect after pulse and irradiation was performed by an inverted light microscope ŽOlympus, Japan. equipped with a CCD-IRIS video camera ŽSony, Japan., using the Phloxine B exclusion test for yeast cells.
3. Results and discussion To investigate concentration dependence of TP on the photodynamic effect, a set of tests were performed under the appropriate pulse strength of 2.35 kVrcm for yeast cell electroporation. The results are shown in the Fig. 1. Along with higher concentrations of TP, the cell killing increased until the optimal wTPx, 1 = 10y5 M. Maintaining wTPx at 1 = 10y5 M, the dependence of the photodynamic effect on the pulse strength has been investigated in detail. For yeast cells, the optimum eletroporation
Fig. 1. Concentration dependence of TP on the photodynamic effect of yeast cells S. cereÕisiae by 5 min visible light irradiation, which followed 5 min after the pulse. The pulse strength is 2.35 kVrcm, while the pulse length is 0.35 ms.
Fig. 2. Pulse strength dependence of the photodynamic effect on yeast cells S. cereÕisiae by 5 min visible light irradiation, which followed 5 min after the pulse. wTPx s1.0=10y5 M.
was obtained by 2.35 kVrcm, which is shown in Fig. 2. The pulse produces several effects on cells, besides pore formation andror the lipid disorders in accelerating molecular transport, cell death occurs if the pores are too big or
Fig. 3. Synergism of electropermeation and photodynamic effect of yeast cells S. cereÕisiae by 5 min visible light irradiation, which followed 5 min after the pulse. Žv . For the test sample, and wTPx is 1.0=10y5 M and the pulse strength is 2.35 kVrcm, while the pulse length is 0.35 ms. ŽB. For the control sample, without pulse. The left part shows the process of resealing after electropermeation.
X. Wang et al.r Bioelectrochemistry and Bioenergetics 47 (1998) 175–177
the number of pores is too high, so that resealing is no more possible. When the pulse increases, both effects are turning stronger, but at different rates. Before reaching 2.35 kVrcm, the former one is the main result, while after that the latter one is predominant. The synergism of photodynamic and electropermeation effects at the optimal condition for yeast cells is shown in Fig. 3. Compared to the control samples, this synergistic effect is ten times higher. One third of cell killing is caused by the pulse and two thirds are coming out by TP radicals according to: TP q hn l TP) q
q
TP) q G Ž DNA . ™ TPH 2 q G Ž DNA . TPH 2 q O 2 ™ TP q H 2 O 2
Ž 1. Ž 2. Ž 3.
Because of the electropermeation, the wTPx in cells reaches quickly the same level as outside. Therefore, the photodynamic damage is accelerated by pore penetration. The efficiency can be increased further by transport of oxygen to the tumor cells since according to Eq. Ž3. the regeneration of TP or another dye will be facilitated.
4. Conclusion Our aim to combine the electropermeation with the photodynamic effect presents results in a synergistic cell killing possibility, which is suitable to increase the cytotoxic efficiency. According to this novel method it is possible to distinguish between pulse killing of original
177
cells and light destruction of mostly resealed cells. The efficiency of both leads to a tenfold higher cytotoxicity than mere photodynamic action as usual.
Acknowledgements Xing Wang thank sincerely the grant from the Boehringer Ingelheim Fonds, Stuttgart, and Dr.Velizarov for invaluable discussion and suggestions.
References w1x H. Berg, H.-E. Jacob, Biologische Wirkungen photochemischer Wasserstoff-peroxydbildung: 2. Mitt., Photochem. Photobiol. 4 Ž1965. 55. w2x F.A. Gollmick, H. Berg, Sensibilisierte Photooxidation durch Meth¨ ylenblau, Thiopyronin und Pyronin: III. Mitt. Uber den Mechanismus der photosensibilisierten Oxydation des Guanosins durch Thiopyronin, Photochem. Photobiol. 16 Ž1972. 447. w3x L. Kittler, G. Lober, F.A. Gollmick, H. Berg, Redox processes during ¨ photodynamic damage of DNA: III. Redox mechanism of photosensitization and radical reaction, Bioelectrochem. Bioenerg. 7 Ž1980. 503. w4x H.-E. Jacob, W. Forster, H. Berg, Microbiological implications of ¨ electric field effects: II. Inactivation of yeast cells and repair of their cell envelope, Z. Allgem. Mikrobiol. 21 Ž1981. 225. w5x M. Muraji, W. Tatebe, T. Konishi, T. Fuyii, H. Berg, Effect of electrical energy on the electropermeabilization of yeast cells, Bioelectrochem. Bioenerg. 31 Ž1993. 77. w6x H.-E. Jacob, W. Forster, M. Hamann, Photodynamic inactivation of ¨ yeast cells by thiopyronine after electric field pulse treatment, Studia Biophysica ŽBerlin. 94 Ž1983. 71–72.