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Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo
51
J. Photochem. Photobiol. B: Biol., 13 (1992) 51-57
Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo G. P. Gurinovich”, T. E. Zorinab, S. B. Melnov”, N. I. Melnova”, L. A. Grubina”, M. V. Sarzhevskaya” and S. N. Cherenkevichb
I. F. Gurinovich”,
“Institute of Physics, Byelorus Academy of Sciences, Leninsky Prospekt 70, 220602 Minsk (Byelorussia) bByelorussian State University, Leninsky Prospekt 4, 220080 Minsk (Byekwussia) Vnstitute of Genetics and Qtology, Byelow Academy of Sciences, Skoriny 27, 220734 Minsk (Byeloncssia) (Received March 30, 1991; accepted September 17, 1991)
Abstract
Several parameters
of chlorin e6 and its derivative chlorin eb ethylenediamide have been investigated as these compound are potential sensitizers for photodynamic therapy. A study carried out to compare the cellular uptake of the pigments indicates that chlorin eb ethylenediamide possesses an enhanced affinity for tumour cells and cellular membranes. Comparison of the uptake in induced sarcoma shows that chlorin eb ethylenediamide is a much better tumour localizer than chlorin e* The efficiency of phototherapy with chlorin e6 ethylenediamide is higher than that with chlorin e6. These data show the influence of the substitution of the carboxyl groups in chlorin e6 by ester and amide groups on the photosensitizing properties of the pigments.
Keywords: Porphyrins, tumour, cell, tissue.
chlorins, sensitizers,
phototherapy,
photocytotoxicity,
accumulation,
1. Introduction
The selective accumulation of porphyrin pigments in tumours is one of the main factors determining their use in medical practice as sensitizers in photodynamic therapy. The basis of the destructive action of porphyrins is the transfer of excitation energy to oxygen with the generation of singlet oxygen [l]. The properties and therapeutic effects on tumours of haematoporphyrin and its derivatives, phthalocyanines and tetraphenylporphyrins have been studied [l, 21. Each of these sensitizers has its own advantages and disadvantages. However, the problem of seeking low-toxicity photosensitizers with optimal spectral and physicochemical characteristics remains urgent. Chlorin e6 has been investigated as a possible photosensitizer [3-S]. The main absorption band (Q band, A,,,,= 660 nm) of chlorins (chlorophyll derivatives) is located at a wavelength where tissue and blood transparency are high. Moreover, the extinction coefficients at the maxima of the long-wavelength absorption bands of chlorins and haematoporphyrin derivative (HpD) differ by a factor of 10-20. Modification of the lateral groups of the tetrapyrrole bring of the dyes is a possible method of increasing the selectivity of tumour targeting. The interrelationship between
loll-1344/92/$5.00
0 1992 - Elsevier Sequoia. All rights reserved
52 the sensitizer molecular structure, its biodistribution in viva and the efficiency of the photodynamic action on the tumour remains unknown. The establishment of such a relationship is an indispensable stage in a purposeful search for effective sensitizers. In this paper, we report a comparative investigation of the phototoxicity of chlorin e6 and its derivative chlorin e6 ethylenediamide by studying their ability to accumulate selectivity in tumour cells and tumours and to sensitize their photodestruction.
2. Materials and methods Chlorin e6 (Chl e6) was obtained by the modification method of Fisher and Orth [6]. The ethylenediamide of Chl e6 (EDA) was obtained by the method of Pennington et al. [A. The structural formulae of Chl e6 and EDA molecules are given in Fig. 1. The dynamics of accumulation and removal of chlorins from tissues and the efficiency of photodynamic therapy were studied in viva in Af mice (average weight, 20 g) bearing transplantable methylcholantrene-induced mouse sarcoma. Solid tumours were initiated by injection of sarcoma cells (lo6 ml-’ (from a donor mouse) in physiological saline subcutaneously in the right hind leg. On day S-10 after transplantation, when the tumour diameter was approximately 1 cm, the mice were injected intraperitoneally with a volume of pigment solution in phosphate-buffered saline (PBS) corresponding to 10 mg of chlorins per kilogram of body weight. The mice were killed at the required times after administration of the chlorins and normal and tumour tissues were removed and washed with PBS; a weighed amount of tissue was finely minced and homogenized by grinding with quartz sand. An alkaline solution of acetone (10 ml) was added and the dispersion was centrifuged at 3000 rev min-’ for 15 min;
400
600
600
700
Wavelength (run)
Fig. 1. Chemical structures and absorption spectra of Chl e6 (-) and EDA (--) solution (pH 7.2). CM eb: R1 =H, Rz= OH, EDA: R1 = CH,, R,=NH-CH&H&H~
in PBS
53
the supernatant was collected and the chlorin content was analyzed fluorometrically were determined using chlorin (A, = 405 nm, A, = 660 nm). The chlorin concentrations standards. For photodynamic therapy experiments, about 30 mice with approximately equal tumour size were chosen. After 6 h for Chl e6 and 18 h for EDA, when the accumulation of the sensitizers in the tumour reached a maximum, the shaved tumour area was exposed to 600-700 nm light obtained form the emission of a 1000 W xenon lamp by a set of optical filters. The IR radiation was removed by a water filter. The illumination time was 10 min. The light intensity reaching the sample was 600 W mm2 and the total incident fluence was 36 J cm-*. Comparisons of results were made for three groups of animals, 8-10 mice in each group, which were kept under identical conditions. In the first group, pigment was not introduced into the mice and no irradiation was carried out. The second group received the dye preparation but was not subjected to irradiation with light. The third group received pigment and phototreatment. In preliminary experiments, irradiation only did not intluence the tumour growth. The efficiency of phototreatment with Chl e6 and EDA was assessed on the tenth day after irradiation by determining the weight of the tumour and the area of tumour necrosis. The size of the region of tumour necrosis was measured with a calliper in two dimensions, from which the area was calculated using the equation for an ellipse. No necrotic areas were observed in the mice of the control group. For the in vitro experiments, Ehrlich ascite carcinoma (EAC) cells were used, isolated on the seventh day after intraperitoneal tumour transplantation. The cellular suspension was washed with Hanks balanced salt solution (pH 7.4). A total of 30 X lo6 cells were incubated in 6 ml PBS with the chlorins (lo-’ M) at 37 “C. At the required times, 5 X lo6 cells were detached and washed three times with PBS (centrifugation at 1000 rev min- ’ for 10 min) and resuspended in 3 ml PBS containing Triton X100 (0.1%). Determination of chlorin concentration was performed as described for tissues. Membranes integrity was determined by *‘Cr leakage [8]. EAC cells were incubated in 5 ml isotonic NaCl solution containing 150 &i Na2Cr04 for 45 min at 37 “C, washed twice with PBS and resuspended in PBS to a final density of 5 X106 cells ml-‘. Thereafter the cells were incubated in the presence of chlorins (5 X10e6 M) for 30 min and irradiated with light of 600-700 nm wavelength. All solutions were air equilibrated and maintained at 2&22 “C by circulating water. The irradiation fluence reaching the cells was 3.6 W m-*, as measured using a URST-1 (Carl Zeiss) photometer. After irradiation, aliquots of the cell suspension were centrifuged and the “Cr content of the supematant was determined using a Packard liquid scintillation counter. The total ‘lCr content of the cells (100%) was determined by freeze-thawing.
3. Results and discussion A comparative analysis of the efficiency of phototreatment of EAC cells with Chl e6 and EDA shows that the investigated pigments display different photocytotoxicity. Figure 2 shows the dependence of 51Cr release on the irradiation time in the presence of both photosensitizers. EDA is considerably more potent than Chl es. The time required for 50% leakage of ‘lCr photosensitized by Chl e6 exceeds that required in the presence of EDA by 40%-50%. Modification of the lateral groups of the Chl e6 molecule does not lead to a significant change in the photophysical characteristics of the pigment [9]. Therefore the differences in cell photocytotoxicity between Chl e6
54 I 100
.
80
-
s c d 2 d uk Ln
60.
40
-
26
_
20
40
60
Illumination
80 time
100 (mid
Fig. 2. Leakage of “Cr from EAC cells irradiated with visible light in the presence
of Chl e6
(1) and EDA (2). TABLE
1
Accumulation
of chlorins
Pigment
Chl e6 EDA Ci, concentration
in EAC tumour cells (Ci/C, (%)) Time (min) 2
15
30
60
20 48
30 60
34 67
38 70
of pigment
bound by cells; C,, total concentration
of pigment.
and EDA are probably associated with the higher efficiency of EDA accumulation compared with Chl e6 (see Table 1). After 1 h, the EAC cells have accumulated 70% of the EDA introduced into the suspension. Chl e6 is bound less effectively: even after 2 h the amount of Chl e6 accumulated in the cells does not exceed 40% of the total pigment in the solution. In the literature, the relationship between the chemical structure of porphyrins and their ability to sensitize the photodestruction of cells has been extensively studied. The accumulation of different components of HpD is determined by their hydrophobicity and affinity to the cellular structures [lo]. The process of accumulation of sulphonated tetraphenylporphins in cells is greatly influenced by the pigment charge and solubilization in the lipid bilayer [ll]. The critical photodamage of cells occurs at the sites of predominant localization of the sensitizers [2]. In our case, the presence of a methyl group at position 8 and a positively charged amino group at position 9 in EDA increases the number of binding sites for pigment in liposomes and proteins [12]. This
55 provides an explanation for the predominant accumulation of EDA in the plasma membrane of HeLa cells and the larger quantity of EDA (twice as much) taken up by these cells compared with Chl e6 [9]. Therefore by substituting the lateral groups in the Chl e6 molecule, it is possible to change signifkantly the aflinity of the dye for cellular structures and thus increase its photocytotoxicity. An analysis of the concentration of chlorins in different tissues (Fig. 3) shows that, in normal tissues, the maximal Chl e6 accumulation is reached during the first hour after introduction, followed by a sharp decrease. After 24 h, because of excretion, the pigment is no longer detectable in normal tissues. The maximum accumulation in the tumour tissue is registered 6 h after the injection of the pigment corresponding to 1.5 pg g-’ tissue. The content of EDA in almost all tissues, including the tumour, is l&20 times higher than that of Chl e6. However, it should be noted that this pigment is easily removed from the animal and after 2 days is hardly detectable. These properties provide an advantage of EDA over Chl e6 in phototherapy. However, the most important feature of this compound is the difference in the dynamics of its accumulation in tumour and muscular tissues. In a tumour it is accumulated much slower than in muscle. Therefore by the time the EDA concentration in the tumour reaches a maximum (40 pg g-l), a large amount of it has already been removed from the muscle tissue (only 2 lug g-’ remaining). The data show that there is a much higher accumulation of EDA than Chl e6. Likewise, the ratio between tumour and normal tissue is changed: it is about three for Chl e6 and about 20 for EDA. Therefore EDA is superior to Chl e6 in its ability to localize in tumour tissues. This probably explains the higher photodynamic activity of EDA compared with Chl e6. Figure 4 shows the change in tumour mass 10 days after photodynamic therapy. In the group of animals treated with Chl e6, after exposure to light the tumour growth
50
Ai
4.0 30
20 to 50 40 30 20 IO 0 ‘MUM after injection (h) Fig. 3. Dynamics of Cl11e6 (A) and EDA (B) accumulation in tissues of mice: 1, liver; 2, kidney; 3, spleen; 4, induced sarcoma; 5, muscle.
56
A
B
c
B
C
Fig. 4. Influence of photodynamic therapy (PDT) on the tumour mass and degree of tumour necrosis determined 10 days after treatment: (A) group 1, control animals (n=lO); (B) group 2, animals subjected to PDT with Chl e6 (n = 10); (C) group 3, animals subjected to PDT with EDA (n =8). was retarded by 30% compared with the control animals. In animals treated with EDA, after exposure to light the tumour growth was retarded by 65%. Moreover, in the tumours subjected to photoaction in the presence of EDA the area of necrosis was larger than that in mice treated with Chl e6 (Fig. 4). These results confirm that chlorins are powerful photodynamic agents. The substitution of the carboxyl groups in Chl e6 by ester and amide groups enhances the photosensitizing potential and increases tumour pigment accumulation without affecting the photophysical properties. Although EDA penetrates tumour cells in vim much better than Chl e6, it is improbable that this is of decisive importance in the distribution selectivity in tumour-bearing mice. Indeed, the enhanced affinity displayed by EDA for other types of cells only reflects the general interrelationship between porphyrin polarity and the ability to locate in cellular membranes [9, 131. The uptake of the photosensitizer by the tumour strongly depends on the transport mechanism in blood [14]. Related porphyrins with a different charge and hydrophobicity have been shown to be carried by various serum proteins. Kessel [15] has shown that hydrophobic mesochlorin is mainly bound to lipoproteins in the blood plasma, whereas hydrophilic mono-r,-aspartyl chlorin e6 is combined with 70% of serum proteins. According to the data obtained in our laboratory [16], EDA displays a higher affinity for low-density lipoproteins than Chl e6. We suggest that the preferential binding to lipoprotein fractions is responsible for the high distribution selectivity of EDA. References G. Jori, Photodynamic therapy of solid tumours, Radiat. Phys. Chem, 30 (1987) 375-380. T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumours, Phorochem. PhotobioL, 45 (1987) 879889. J. Nelson, W. Roberts and M. Bems, In vivo studies on the utilization of mono-L-aspartyl chlorin (Np eb) for photodynamic therapy, Ccincer Res., 47 (1987) 46814685. W. Roberts, F. Shian, J. S. Nelson, K. Smith and M. Bems, In vitro characterization of monoaspartyl chlorin e6 for photodynamic therapy, J. Natl. Cancer Inst., 80 (1988) 330-336. G. P. Gurinovich, T. E. Zorina, V. P. Z&n, S. B. Melnov, Yu. M. Arcatov, S. N. Cherenkevich and M. V. Sarjevskaya, Localization, accumulation and photodynamic action of chlorin eb and its derivatives on tumor ceils, Abstracts, Znt. Con& on Photodynamic l?aerapv, Sojia, Bulgaria, October 3-5, 1989, Bulgarian Academy of Sciences, Sofia, 1989, p. 6.
57 6 H. Fisher and H. Orth, Die Chemie des Pyrrols, Bd l-2, Akad. Verlaggesselschaft, Leipzig, 1937, p. 63. 7 F. S. Pennington, S. D. Boyd, K. Horston, S. W. Taylor, D. G. Wulf, J. J. Katz and N. N. Strain, Reactions of chlorophylls “a” and “b” with the amines. Isocyclic ring rupture and formation of substitute chlorin-6-amides, J. Am. Chem Sot., 89 (1967) 3871-3876. 8 N. R. Ling, Ly@aocyte Stimulation, North-Holland, Amsterdam, 1968, p. 288. 9 G. P. Gurinovich, T. E. Zorina, Yu. M. Arcatov, M. V. Sajevskaya and S. N. Cherenkevich, Investigation of the distribution of chlorin e6 and its derivatives in HeLa cells by luminescence microscopy, Qtologja, 31 (1989) 1058-1063. 10 M. El-Far, M. Abou-el-Zahal, M. Ghoneim and E. Ibrahim, Tumor localization of newly developed hematoporphyrin using a bladder tumor model: a novel hematoporphyrin derivative, Biochimie, 70 (1988) 251-258. 11 S. G. Bown, C. J. Tralau, P. P. Coleridge Smith, P. Akdemir and N. J. Wieman, Photodynamic therapy with porphyrin and phthalocyanine sensitization. Quantitative studies in normal rat liver, Br. J. Cancer, 54 (1986) 43-52. 12 G. P. Gurinovich, T. E. Zorina, V. P. Zorin, M. V. Sajevskaya and S. N. Cherenkevich, Chlorin- and porphyrin-sensitized structural damage of erythrocytes, Biophyziku, 33 (1988) 314-318. 13 J. Moan, Q. Peng, J. F. Evensen, K Berg, A. Western and C. Rimington, Photosensitizing efficiencies, tumor and cellular uptake of different photosensitizing drugs relevant for photodynamic therapy of cancer, Photochem. PhotobioL, 46 (1987) 713-721. 14 G. Jori, Photosensitizing properties of porphyrins and photodynamic therapy, Photobiochem. Photobiophys. Suppl. (1987) 373-384. 15 D. Kessel, Determinants of photosensitization by mono-L-aspartyl chlorin e6, Photochem. Photobiol., 49 (1989) 447-452. 16 V. P. Zorin, I. I. Khludeyev, T. E. Zorina, M. V. Sarzhevskaya, G. P. Guronovich and S. N. Cherenkevich, Investigation of tumor tissue affinity for chlorin e6 derivative, Eq. Oncol., in the press.
J. Photochem. Photobiol. B: Biol., 13 (1992) 51-57
Photodynamic activity of chlorin e6 and chlorin e6 ethylenediamide in vitro and in vivo G. P. Gurinovich”, T. E. Zorinab, S. B. Melnov”, N. I. Melnova”, L. A. Grubina”, M. V. Sarzhevskaya” and S. N. Cherenkevichb
I. F. Gurinovich”,
“Institute of Physics, Byelorus Academy of Sciences, Leninsky Prospekt 70, 220602 Minsk (Byelorussia) bByelorussian State University, Leninsky Prospekt 4, 220080 Minsk (Byekwussia) Vnstitute of Genetics and Qtology, Byelow Academy of Sciences, Skoriny 27, 220734 Minsk (Byeloncssia) (Received March 30, 1991; accepted September 17, 1991)
Abstract
Several parameters
of chlorin e6 and its derivative chlorin eb ethylenediamide have been investigated as these compound are potential sensitizers for photodynamic therapy. A study carried out to compare the cellular uptake of the pigments indicates that chlorin eb ethylenediamide possesses an enhanced affinity for tumour cells and cellular membranes. Comparison of the uptake in induced sarcoma shows that chlorin eb ethylenediamide is a much better tumour localizer than chlorin e* The efficiency of phototherapy with chlorin e6 ethylenediamide is higher than that with chlorin e6. These data show the influence of the substitution of the carboxyl groups in chlorin e6 by ester and amide groups on the photosensitizing properties of the pigments.
Keywords: Porphyrins, tumour, cell, tissue.
chlorins, sensitizers,
phototherapy,
photocytotoxicity,
accumulation,
1. Introduction
The selective accumulation of porphyrin pigments in tumours is one of the main factors determining their use in medical practice as sensitizers in photodynamic therapy. The basis of the destructive action of porphyrins is the transfer of excitation energy to oxygen with the generation of singlet oxygen [l]. The properties and therapeutic effects on tumours of haematoporphyrin and its derivatives, phthalocyanines and tetraphenylporphyrins have been studied [l, 21. Each of these sensitizers has its own advantages and disadvantages. However, the problem of seeking low-toxicity photosensitizers with optimal spectral and physicochemical characteristics remains urgent. Chlorin e6 has been investigated as a possible photosensitizer [3-S]. The main absorption band (Q band, A,,,,= 660 nm) of chlorins (chlorophyll derivatives) is located at a wavelength where tissue and blood transparency are high. Moreover, the extinction coefficients at the maxima of the long-wavelength absorption bands of chlorins and haematoporphyrin derivative (HpD) differ by a factor of 10-20. Modification of the lateral groups of the tetrapyrrole bring of the dyes is a possible method of increasing the selectivity of tumour targeting. The interrelationship between
loll-1344/92/$5.00
0 1992 - Elsevier Sequoia. All rights reserved
52 the sensitizer molecular structure, its biodistribution in viva and the efficiency of the photodynamic action on the tumour remains unknown. The establishment of such a relationship is an indispensable stage in a purposeful search for effective sensitizers. In this paper, we report a comparative investigation of the phototoxicity of chlorin e6 and its derivative chlorin e6 ethylenediamide by studying their ability to accumulate selectivity in tumour cells and tumours and to sensitize their photodestruction.
2. Materials and methods Chlorin e6 (Chl e6) was obtained by the modification method of Fisher and Orth [6]. The ethylenediamide of Chl e6 (EDA) was obtained by the method of Pennington et al. [A. The structural formulae of Chl e6 and EDA molecules are given in Fig. 1. The dynamics of accumulation and removal of chlorins from tissues and the efficiency of photodynamic therapy were studied in viva in Af mice (average weight, 20 g) bearing transplantable methylcholantrene-induced mouse sarcoma. Solid tumours were initiated by injection of sarcoma cells (lo6 ml-’ (from a donor mouse) in physiological saline subcutaneously in the right hind leg. On day S-10 after transplantation, when the tumour diameter was approximately 1 cm, the mice were injected intraperitoneally with a volume of pigment solution in phosphate-buffered saline (PBS) corresponding to 10 mg of chlorins per kilogram of body weight. The mice were killed at the required times after administration of the chlorins and normal and tumour tissues were removed and washed with PBS; a weighed amount of tissue was finely minced and homogenized by grinding with quartz sand. An alkaline solution of acetone (10 ml) was added and the dispersion was centrifuged at 3000 rev min-’ for 15 min;
400
600
600
700
Wavelength (run)
Fig. 1. Chemical structures and absorption spectra of Chl e6 (-) and EDA (--) solution (pH 7.2). CM eb: R1 =H, Rz= OH, EDA: R1 = CH,, R,=NH-CH&H&H~
in PBS
53
the supernatant was collected and the chlorin content was analyzed fluorometrically were determined using chlorin (A, = 405 nm, A, = 660 nm). The chlorin concentrations standards. For photodynamic therapy experiments, about 30 mice with approximately equal tumour size were chosen. After 6 h for Chl e6 and 18 h for EDA, when the accumulation of the sensitizers in the tumour reached a maximum, the shaved tumour area was exposed to 600-700 nm light obtained form the emission of a 1000 W xenon lamp by a set of optical filters. The IR radiation was removed by a water filter. The illumination time was 10 min. The light intensity reaching the sample was 600 W mm2 and the total incident fluence was 36 J cm-*. Comparisons of results were made for three groups of animals, 8-10 mice in each group, which were kept under identical conditions. In the first group, pigment was not introduced into the mice and no irradiation was carried out. The second group received the dye preparation but was not subjected to irradiation with light. The third group received pigment and phototreatment. In preliminary experiments, irradiation only did not intluence the tumour growth. The efficiency of phototreatment with Chl e6 and EDA was assessed on the tenth day after irradiation by determining the weight of the tumour and the area of tumour necrosis. The size of the region of tumour necrosis was measured with a calliper in two dimensions, from which the area was calculated using the equation for an ellipse. No necrotic areas were observed in the mice of the control group. For the in vitro experiments, Ehrlich ascite carcinoma (EAC) cells were used, isolated on the seventh day after intraperitoneal tumour transplantation. The cellular suspension was washed with Hanks balanced salt solution (pH 7.4). A total of 30 X lo6 cells were incubated in 6 ml PBS with the chlorins (lo-’ M) at 37 “C. At the required times, 5 X lo6 cells were detached and washed three times with PBS (centrifugation at 1000 rev min- ’ for 10 min) and resuspended in 3 ml PBS containing Triton X100 (0.1%). Determination of chlorin concentration was performed as described for tissues. Membranes integrity was determined by *‘Cr leakage [8]. EAC cells were incubated in 5 ml isotonic NaCl solution containing 150 &i Na2Cr04 for 45 min at 37 “C, washed twice with PBS and resuspended in PBS to a final density of 5 X106 cells ml-‘. Thereafter the cells were incubated in the presence of chlorins (5 X10e6 M) for 30 min and irradiated with light of 600-700 nm wavelength. All solutions were air equilibrated and maintained at 2&22 “C by circulating water. The irradiation fluence reaching the cells was 3.6 W m-*, as measured using a URST-1 (Carl Zeiss) photometer. After irradiation, aliquots of the cell suspension were centrifuged and the “Cr content of the supematant was determined using a Packard liquid scintillation counter. The total ‘lCr content of the cells (100%) was determined by freeze-thawing.
3. Results and discussion A comparative analysis of the efficiency of phototreatment of EAC cells with Chl e6 and EDA shows that the investigated pigments display different photocytotoxicity. Figure 2 shows the dependence of 51Cr release on the irradiation time in the presence of both photosensitizers. EDA is considerably more potent than Chl es. The time required for 50% leakage of ‘lCr photosensitized by Chl e6 exceeds that required in the presence of EDA by 40%-50%. Modification of the lateral groups of the Chl e6 molecule does not lead to a significant change in the photophysical characteristics of the pigment [9]. Therefore the differences in cell photocytotoxicity between Chl e6
54 I 100
.
80
-
s c d 2 d uk Ln
60.
40
-
26
_
20
40
60
Illumination
80 time
100 (mid
Fig. 2. Leakage of “Cr from EAC cells irradiated with visible light in the presence
of Chl e6
(1) and EDA (2). TABLE
1
Accumulation
of chlorins
Pigment
Chl e6 EDA Ci, concentration
in EAC tumour cells (Ci/C, (%)) Time (min) 2
15
30
60
20 48
30 60
34 67
38 70
of pigment
bound by cells; C,, total concentration
of pigment.
and EDA are probably associated with the higher efficiency of EDA accumulation compared with Chl e6 (see Table 1). After 1 h, the EAC cells have accumulated 70% of the EDA introduced into the suspension. Chl e6 is bound less effectively: even after 2 h the amount of Chl e6 accumulated in the cells does not exceed 40% of the total pigment in the solution. In the literature, the relationship between the chemical structure of porphyrins and their ability to sensitize the photodestruction of cells has been extensively studied. The accumulation of different components of HpD is determined by their hydrophobicity and affinity to the cellular structures [lo]. The process of accumulation of sulphonated tetraphenylporphins in cells is greatly influenced by the pigment charge and solubilization in the lipid bilayer [ll]. The critical photodamage of cells occurs at the sites of predominant localization of the sensitizers [2]. In our case, the presence of a methyl group at position 8 and a positively charged amino group at position 9 in EDA increases the number of binding sites for pigment in liposomes and proteins [12]. This
55 provides an explanation for the predominant accumulation of EDA in the plasma membrane of HeLa cells and the larger quantity of EDA (twice as much) taken up by these cells compared with Chl e6 [9]. Therefore by substituting the lateral groups in the Chl e6 molecule, it is possible to change signifkantly the aflinity of the dye for cellular structures and thus increase its photocytotoxicity. An analysis of the concentration of chlorins in different tissues (Fig. 3) shows that, in normal tissues, the maximal Chl e6 accumulation is reached during the first hour after introduction, followed by a sharp decrease. After 24 h, because of excretion, the pigment is no longer detectable in normal tissues. The maximum accumulation in the tumour tissue is registered 6 h after the injection of the pigment corresponding to 1.5 pg g-’ tissue. The content of EDA in almost all tissues, including the tumour, is l&20 times higher than that of Chl e6. However, it should be noted that this pigment is easily removed from the animal and after 2 days is hardly detectable. These properties provide an advantage of EDA over Chl e6 in phototherapy. However, the most important feature of this compound is the difference in the dynamics of its accumulation in tumour and muscular tissues. In a tumour it is accumulated much slower than in muscle. Therefore by the time the EDA concentration in the tumour reaches a maximum (40 pg g-l), a large amount of it has already been removed from the muscle tissue (only 2 lug g-’ remaining). The data show that there is a much higher accumulation of EDA than Chl e6. Likewise, the ratio between tumour and normal tissue is changed: it is about three for Chl e6 and about 20 for EDA. Therefore EDA is superior to Chl e6 in its ability to localize in tumour tissues. This probably explains the higher photodynamic activity of EDA compared with Chl e6. Figure 4 shows the change in tumour mass 10 days after photodynamic therapy. In the group of animals treated with Chl e6, after exposure to light the tumour growth
50
Ai
4.0 30
20 to 50 40 30 20 IO 0 ‘MUM after injection (h) Fig. 3. Dynamics of Cl11e6 (A) and EDA (B) accumulation in tissues of mice: 1, liver; 2, kidney; 3, spleen; 4, induced sarcoma; 5, muscle.
56
A
B
c
B
C
Fig. 4. Influence of photodynamic therapy (PDT) on the tumour mass and degree of tumour necrosis determined 10 days after treatment: (A) group 1, control animals (n=lO); (B) group 2, animals subjected to PDT with Chl e6 (n = 10); (C) group 3, animals subjected to PDT with EDA (n =8). was retarded by 30% compared with the control animals. In animals treated with EDA, after exposure to light the tumour growth was retarded by 65%. Moreover, in the tumours subjected to photoaction in the presence of EDA the area of necrosis was larger than that in mice treated with Chl e6 (Fig. 4). These results confirm that chlorins are powerful photodynamic agents. The substitution of the carboxyl groups in Chl e6 by ester and amide groups enhances the photosensitizing potential and increases tumour pigment accumulation without affecting the photophysical properties. Although EDA penetrates tumour cells in vim much better than Chl e6, it is improbable that this is of decisive importance in the distribution selectivity in tumour-bearing mice. Indeed, the enhanced affinity displayed by EDA for other types of cells only reflects the general interrelationship between porphyrin polarity and the ability to locate in cellular membranes [9, 131. The uptake of the photosensitizer by the tumour strongly depends on the transport mechanism in blood [14]. Related porphyrins with a different charge and hydrophobicity have been shown to be carried by various serum proteins. Kessel [15] has shown that hydrophobic mesochlorin is mainly bound to lipoproteins in the blood plasma, whereas hydrophilic mono-r,-aspartyl chlorin e6 is combined with 70% of serum proteins. According to the data obtained in our laboratory [16], EDA displays a higher affinity for low-density lipoproteins than Chl e6. We suggest that the preferential binding to lipoprotein fractions is responsible for the high distribution selectivity of EDA. References G. Jori, Photodynamic therapy of solid tumours, Radiat. Phys. Chem, 30 (1987) 375-380. T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumours, Phorochem. PhotobioL, 45 (1987) 879889. J. Nelson, W. Roberts and M. Bems, In vivo studies on the utilization of mono-L-aspartyl chlorin (Np eb) for photodynamic therapy, Ccincer Res., 47 (1987) 46814685. W. Roberts, F. Shian, J. S. Nelson, K. Smith and M. Bems, In vitro characterization of monoaspartyl chlorin e6 for photodynamic therapy, J. Natl. Cancer Inst., 80 (1988) 330-336. G. P. Gurinovich, T. E. Zorina, V. P. Z&n, S. B. Melnov, Yu. M. Arcatov, S. N. Cherenkevich and M. V. Sarjevskaya, Localization, accumulation and photodynamic action of chlorin eb and its derivatives on tumor ceils, Abstracts, Znt. Con& on Photodynamic l?aerapv, Sojia, Bulgaria, October 3-5, 1989, Bulgarian Academy of Sciences, Sofia, 1989, p. 6.
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