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Pharmacokinetic and tumour-photosensitizing properties of methoxyphenyl porphyrin derivative
Biomedicine & Pharmacotherapy 57 (2003) 163–168 www.elsevier.com/locate/biopha
Original article
Pharmacokinetic and tumour-photosensitizing properties of methoxyphenyl porphyrin derivative M. Gabriela Alvarez a, Flavia Morán a, E. Inés Yslas a, N. Belén Rumie Vittar a, Mabel Bertuzzi a, Edgardo N. Durantini b, Viviana Rivarola a,* a
Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal No 3, 5800 Río Cuarto, Córdoba, Argentina b Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal No 3, 5800 Río Cuarto, Córdoba, Argentina Received 13 August 2002; accepted 21 November 2002
Abstract The photokilling activity of 5-[4-N-(2’,6’-dinitro-4’-trifluoromethylphenyl) aminophenyl]-10,15,20-tris(2,4,6-trimethoxy phenyl) porphyrin (CF3) was evaluated on a Hep-2 human larynx-carcinoma cell line. An apoptotic mechanism of cell death was found at low irradiation doses. Pharmacokinetic and tumour-photosensitizing properties were studied in mice. The results show that a different mechanism of cell death can be induced depending on the irradiation doses in the photodynamic treatments with CF3, which makes this agent an interesting sensitizer for potential tumour photosensitizer. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: CF3; Photodynamic therapy; Photosensitizer; Cancer; Porphyrin
1. Introduction Photodynamic therapy (PDT) is a treatment that is used for the destruction of certain types of tumours [1]. This treatment is performed with photosensitizers that generate reactive oxygen species in the presence of light and oxygen [2,3]. The combination of the drug uptake in neoplasic tissues and the delivery of selective visible light, provides an effective tumour therapy with an effective cytotoxicity and a limited damage to the contiguous normal tissue [1]. Under treatment conditions, reactive oxygen species may cause both cell death and damage to blood vessels, thus contributing to tumour regression [4]. Photodynamic studies, in model systems and biological media carried out with a series of 5,10,15,20-tetrakis (methoxyphenyl) porphyrins, have shown that these synthetic porphyrins are effective photosensitizers that can be used as model compounds to investigate the effects of photosensitizers used in PDT [5–8].
* Corresponding author. E-mail address: [email protected] (V. Rivarola). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0753-3322(03)00030-1
Recently, several porphyrin derivatives covalently linked to active molecules have been tested for a potential use in the treatment of tumours [9]. In a previous work, we had synthesized 5-[4-N-(2’,6’-dinitro-4’-trifluoromethylphenyl) aminophenyl]-10,15,20-tris(2,4,6-trimethoxy phenyl) porphyrin (CF3, Fig. 1) [10]. In this structure, the extra methoxy groups were included in the porphyrin moiety and the lipophilic trifluoromethyl group. The influence of the trifluoromethyl group, in biologically active molecules, is often associated with the increased lipophilicity that is imparted by this substituent [11]. In the present study, we have investigated the photodynamic effects of CF3 on Hep-2 cell line, in vitro and in female Balb/c mice, in vivo. First, the cytotoxicity of CF3 was evaluated, in vitro, under dark conditions and in relation to the illumination from visible light. Then, the new porphyrin was studied in mice for its distribution and possible toxic effects. The photokilling mechanism was studied on mice tumours which were injected with CF3 and then irradiated. These tumours were either kept under dark or illuminated conditions. The results can be used to design photodynamic treatments using this porphyrin as a sensitizer.
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1% (v/v) 0.2 M L-glutamine. The cells were incubated at 37 °C in a humidified 5% CO2 atmosphere and the medium was changed everyday. The Hep-2 cell line was routinely grown either in 35-mm culture dishes (Costar, Cambridge, MA) or on 22-mm square glass coverslips placed in the dishes. 2.5. In vitro studies of PDT
Fig. 1. Chemical structure of CF3.
2. Materials and methods 2.1. Drugs All of the solvents were of HPLC quality from Merck and were used without further purification. Dipalmitoyl phosphatidylethanolamine purchased from Sigma was used in liposome preparation. Thiazolyl blue (MTT) was purchased from Sigma.
2.5.1. Treatments and cell survival Treatments were performed with 1–10 µM of liposomes in DMEM containing 1% FCS. Hep-2 cells were incubated at 37 °C under dark conditions with CF3, at different points of time. After washing with the complete DMEM, the cells were either allowed to grow in this medium or subjected to light irradiation. Cell survival was assessed for untreated (control) and CF3-treated cultures. The surviving cells were evaluated 24 h after the treatment using the MTT method [14]. 2.5.2. Irradiation The light source used was that of a Kodak slide projector equipped with a 150 W lamp. The light was filtered through a 3 cm water layer to absorb the heat. A wavelength of the range of 350–800 nm was selected with the aid of optical filters [15], and the light intensity at the treatment site was 100 mW/cm2.
The photosensitizer CF3 was synthesized from the coupling reaction between 5-(4-aminophenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl) porphyrin and 1-chloro-2,6dinitro-4-trifluorometilbenzene as described previously in Fig. 1 [10].
2.5.3. Morphology and cell counting Changes in cell morphology were analysed using fluorescence microscopy. After fixing in methanol at –20 °C for 10 min, the cells were stained with H-33258 (10 µg/ml in distilled water, 5 min) for the visualization of chromatin DNA. After washing and air drying, the preparations were mounted in DPX and observed under UV excitation [16].
2.3. Liposome preparation
2.6. In vivo studies of PDT
The incorporation of CF3 into the phospholipid bilayer of the D,L-dipalmitoyl phosphatidylethanolamine was achieved by a modification of the ethanol injection method [12]. Typically, 2 ml of a solution composed of: 9.60 mM of phospholipid, 1.91 mM of cholesterol and 0.27 mM of CF3 in ethanol-tetrahydrofuran (1:1 v/v) was injected into 10 ml of phosphate-buffered saline (PBS, 10 ml) at 80 °C. The injection was performed at a speed of 50 µl/min with magnetic stirring [13]. The final volume was reduced to 10 ml by evaporation of organic solvents.
2.6.1. Animal and tumour model Female Balb/c 6–8 weeks old, were obtained from the Fundación Balseiro (Buenos Aires) and maintained in standard cages with free access to tap water and normal dietary chow. When required, the animals were anaesthetized with Ketalar (150 mg/kg, by i.p. injection). The experimental tumours LM-2, originally obtained from Hospital Roffo (Buenos Aires. Argentine) were s.c. implanted by a sterile injection of ca. 106 cells in phosphate-buffered saline into the right foreleg. The pharmacokinetic and/or phototherapeutic experiments were performed on the sixth day after implantation, when the tumour was of 0.6–0.7 cm in the outer diameter. Spontaneous tumour necrosis was minimal or absent for these tumour sizes.
2.2. Photosensitizer
2.4. Cell culture A human larynx-carcinoma cell line (Hep-2) (purchased from Asociación Banco Argentino de Células ABAC, Instituto Nacional de Enfermedades Virales Humanas, Pergamino, Argentina) kept in liquid nitrogen was used for the investigation. The cells were grown as a monolayer employing Dulbecco’s modified Eagle’s medium (DMEM) containing 7% foetal calf serum (FCS) and 50 µg gentamycin/ml and
2.6.2. Pharmacokinetic studies CF3 (10 mg/kg) was administered by i.p. injection. The animals were sacrificed during different points of time (six mice at 24 h and three mice at other points of time), varying between 1 h and 1 week, after administration. The brain,
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spleen, kidney, duodenum, and the liver were removed. About 200 mg of tissues were homogenized in THF. The homogenates were centrifuged at 3000 rev/min for 15 min, and the fluorescence of the supernatants was measured, setting the excitation wavelength at 420 nm and recording the emission spectrum from 500 and 800 nm. Serum samples, isolated from the blood by centrifugation, were diluted with suitable volumes of 500 µl THF and the CF3 content was measured by fluorescence. In all the cases, CF3 amounts were determined by the interpolation of emission intensity and CF3 concentration plotted on a standard curve. 2.6.3. Phototherapeutic studies The effect of PDT on tumour growth was assessed using the in vivo/in vitro model previously described by Fukuda et al. [16] with modifications [17]. Briefly, 24 h after i.p. injection of CF3 at a dose of 10 mg/kg, the animals were sacrificed and the tumours were removed. Uniform tumour tissue fragments were obtained using a biopsy punch and incubated for 2 h at 37 °C in 5 ml of serum-free MEM. Irradiated explants were exposed for 15 min to light at a light dose of 90 J/cm2. Appropriate controls, of non-irradiated explants incubated with CF3 and irradiated and non-irradiated explants incubated without CF3, were included. Irradiated and nonirradiated tumour samples were fixed in 10% formaldehyde. The samples were then dehydrated in a series of graded ethanol, cleared in xylene and embedded in paraffin. Sections, 5 µm thick, were cut and stained with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) to show the typical morphology of apoptotic cells.
3. Results 3.1. In vitro studies of PDT Direct cytotoxic effects of CF3 were studied, for different concentrations, in the absence of light. As evaluated by the MTT method, 24 h after treatment, CF3 does not show any dark toxicity (Table 1), which is evidenced by the absence of lethal effects caused by this porphyrin under dark conditions. On the contrary, 24 h of incubation with CF3 treatments, followed by light irradiation, induced a significant decrease of cell viability, which was related to both drug concentration and irradiation time (Fig. 2) The strongest photodynamic effect (10 µM and 15 min of irradiation) resulted in 13% of cell viability.
Fig. 2. Inactivation of Hep-2 cells incubated for 24 h with different concentrations of CF3 and then exposed to several irradiation periods with visible light, 5 µM (n), 8 µM (•), 10 µM (m) and irradiation control (.). Concentration: 0.2 mg of MTT/ml medium. Mean values ± S.D. from at least three different experiments.
Cell death was not found to occur when untreated cell cultures were irradiated with visible light; hence the cell death caused after irradiation, following the abovementioned treatment, was due to the effect of visible light and photosensitizer. When stained with H-33258, characteristic morphological changes were found in the photosensitized cells on incubation with 10 µM CF3 for 24 h; revealing different cell death mechanisms depending on the irradiation time. When analysed 24 h after photodynamic treatments with 15 min irradiation (Fig. 3), a great amount of apoptotic cells were obtained. On the contrary, necrotic cells were observed using the same drug concentration and incubation conditions, but after 15 min irradiation, 1 h of incubation at 37 °C and another 15 min of irradiation. Fig. 4 shows a characteristic photography of normal apoptotic and necrotic cells obtained from the control cells or those treated with CF3.
Table 1 Cytotoxicity of Hep-2 cells treated with several CF3 concentrations for 24 h in the dark CF3 (µM) 0 0.1 1 8 10
Survival cells (%) 100 99 98 99 98
Fig. 3. Effect of cell death of 10 µM CF3 in Hep-2 cells after 24 h of incubation at 37 °C. Hep-2 cells were incubated with a photosensitizer for (A) 24 h in the dark (control), (B) cells irradiated for 15 min (C) cells irradiated for 15 min + 1 h heat incubation + 15 min irradiation. An evaluation of cell types was made using H-33258 staining.
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A
B
C
Fig. 5. Biodistribution of CF3 in mice without tumours: Recovery of CF3 from Balb/c mice injected with 10 mg/kg of drug. Values represent the average of the three experiments (A: liver, B: spleen, C: kidney, D: brain, E: muscle, F: skin, G: lung, H: duodenum).
Fig. 5. In mice without tumour, high levels of photosensitizer in the liver was observed to diminish in the fourth week, while in the spleen, no variation was observed. In the kidney, an intermediate value was found and it remained stable. However, a high percentage of CF3 was found in the duodenum on the seventh day post-injection time, perhaps due to its elimination from the organism via bile-gut pathway [18]. The brain, skin, muscle and the lung presented low values with no significant variations with regard to post-injection time. The low quantity detected in the brain would show that the photosensitizer was unable to cause some type of alteration to the central nervous system; while in the skin, it could be favourable, as it would cause photosensitization. The results obtained in tumour-bearing mice are shown in Fig. 6. The CF3 concentration increase in the spleen, liver and intestine of mice when treated with the photosensitizer. This result is characteristic when lipidic drugs are given as they are eliminated via the bile-gut pathway [19]. The insignificant accumulation of the drug in the brain confirms that the porphyrinic components are unable to go through the haemato-encephalic barrier; consequently any risk of toxic effects of CF3 in the central nervous system could be rejected. The highest concentrations were observed in the lung, the duodenum and the liver at 3 h post-injection treatment; these results declined slowly over the course of the post-injection time. On the contrary, the results obtained from the brain, skin, muscle and tumour maintained a low accumulation of the CF3, reaching the highest values at the shortest post-
Fig. 4. Fluorescence photomicrographs of Hep-2 cells stained with H-33258. (A) Untreated cells. (B) Apoptotic cells induced by treatment with CF3; 15 min of illumination. (C) Necrotic cells induced by treatment with CF3.
3.2. In vivo studies 3.2.1. Pharmacokinetics studies: biodistribution in tumour-free and tumour-bearing mice The distribution of CF3 was examined in different selected tissues of female mice Balb/c, between 1 d and 4 weeks after i.p. administration of 10 mg/kg corporal weight. CF3 distribution was studied in tumour bearing mice at 3, 24 h and 7 d post-injection periods. The results are shown in
Fig. 6. Biodistribution of tumour bearing mice: Recoveries of CF3 from tumour-bearing Balb/c mice injected with 10 mg/kg of drug. Values represent the average of the three experiments (A: liver, B: spleen, C: kidney, D: tumour, E: brain, F: muscle, G: skin, H: lung, I: duoden).
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A
B Fig. 7. Fluorescence images of fluorescein-stained apoptotic cells (bright green) in tumour sections of animals treated with CF3 10 mg/kg (100×). (A) Tumour illuminated, (B) tumour without illumination (see Section 2).
irradiation time (3 h). Histological cuts were performed in organs (liver, kidney, duodenum), where high levels of CF3 were detected and no irreparable damages were found (manuscript in preparation). A low detectable amount of CF3 was found in the tumour, however, PDT treatment could be performed when the neoplastic tissues are identified previously. 3.2.2. Phototherapeutic studies Mice were injected with CF3 10 mg/kg after tumours were taken out and irradiated in vitro. Histological cuts and TUNEL were performed to determine apoptotic cells. The results are shown in Fig. 7. The amount of dead cells, by apoptosis, in irradiated tumours (Fig. 7A) is higher than that in non-irradiated ones (Fig. 7B). These results show that under these conditions, the combination of CF3 and light produces cell death mainly by apoptosis.
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tion (24 h) period with the highest drug concentration used (10 µM). The efficiency of photokilling increased with CF3 concentration and light doses. It is worth noting that by using the same CF3 concentration (1 × 10–6 M) and incubation time (24 h), different photokilling mechanisms were observed, depending on the irradiation time. According to many authors, the occurrence of apoptosis or necrosis after PDT treatments could depend on (i) characteristics of the photosensitizer [20], (ii) kind of cell line [21] and cellular density [22], and (iii) experimental protocol, which include: drug concentration, incubation time and light doses [23,24]. The results show that the mechanism of cell death (necrosis or apoptosis) induced by CF3 is clearly dependent on the treatment used in the irradiation conditions. On the other hand, in vivo results indicate that CF3 can be a candidate for PDT. Although the efficiency of CF3 incorporation in tumoural tissue is not high in relation to other tissues, a high antitumour activity is observed when excited by visible light, since a high percentage of cells in apoptosis appeared in tumours that were obtained from mice already treated with CF3 and illuminated. The liver, the spleen and, to a minor extent, the duodenum, retained detectable amounts of CF3 for prolonged periods. Several reports indicated that many photosensitizers are accumulated in high concentrations by the components of the reticuloendothelial system [25]. Small amounts of CF3 accumulated by kidneys suggest that this porphyrin is mostly eliminated from the organism via the bile-gut pathway. Likewise, only negligible amounts of CF3 accumulate in the skin, making low skin photosensitive. In summary, the present results show that CF3 is a potential tumour photosensitizer. A different mechanism of cell death occurs in Hep-2 cell cultures depending on the irradiation doses in the photodynamic treatments with CF3. Also, the irradiation of tumour tissues produces cell inactivation mainly by the apoptotic way. The development of experimental protocols using a given sensitizer in vitro and in vivo can contribute to the understanding of the photokilling effect in basic oncological research and to assess the potential for clinical applications in the PDT of cancer.
Acknowledgements The authors are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina, Agencia Nacional de Promoción Científica y Tecnológica and SECYT of Universidad Nacional de Río Cuarto for financial support. V.R. and E.N.D. hold the position of researchers at CONICET. M.G.A. thanks CONICET for a research fellowship.
4. Discussion A new photosensitizer was tested on a human carcinoma cell line and in vivo. The studies on Hep-2 cells show that the dark toxicity of CF3 was not found even after a long incuba-
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Original article
Pharmacokinetic and tumour-photosensitizing properties of methoxyphenyl porphyrin derivative M. Gabriela Alvarez a, Flavia Morán a, E. Inés Yslas a, N. Belén Rumie Vittar a, Mabel Bertuzzi a, Edgardo N. Durantini b, Viviana Rivarola a,* a
Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Agencia Postal No 3, 5800 Río Cuarto, Córdoba, Argentina b Departamento de Química, Universidad Nacional de Río Cuarto, Agencia Postal No 3, 5800 Río Cuarto, Córdoba, Argentina Received 13 August 2002; accepted 21 November 2002
Abstract The photokilling activity of 5-[4-N-(2’,6’-dinitro-4’-trifluoromethylphenyl) aminophenyl]-10,15,20-tris(2,4,6-trimethoxy phenyl) porphyrin (CF3) was evaluated on a Hep-2 human larynx-carcinoma cell line. An apoptotic mechanism of cell death was found at low irradiation doses. Pharmacokinetic and tumour-photosensitizing properties were studied in mice. The results show that a different mechanism of cell death can be induced depending on the irradiation doses in the photodynamic treatments with CF3, which makes this agent an interesting sensitizer for potential tumour photosensitizer. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: CF3; Photodynamic therapy; Photosensitizer; Cancer; Porphyrin
1. Introduction Photodynamic therapy (PDT) is a treatment that is used for the destruction of certain types of tumours [1]. This treatment is performed with photosensitizers that generate reactive oxygen species in the presence of light and oxygen [2,3]. The combination of the drug uptake in neoplasic tissues and the delivery of selective visible light, provides an effective tumour therapy with an effective cytotoxicity and a limited damage to the contiguous normal tissue [1]. Under treatment conditions, reactive oxygen species may cause both cell death and damage to blood vessels, thus contributing to tumour regression [4]. Photodynamic studies, in model systems and biological media carried out with a series of 5,10,15,20-tetrakis (methoxyphenyl) porphyrins, have shown that these synthetic porphyrins are effective photosensitizers that can be used as model compounds to investigate the effects of photosensitizers used in PDT [5–8].
* Corresponding author. E-mail address: [email protected] (V. Rivarola). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 10.1016/S0753-3322(03)00030-1
Recently, several porphyrin derivatives covalently linked to active molecules have been tested for a potential use in the treatment of tumours [9]. In a previous work, we had synthesized 5-[4-N-(2’,6’-dinitro-4’-trifluoromethylphenyl) aminophenyl]-10,15,20-tris(2,4,6-trimethoxy phenyl) porphyrin (CF3, Fig. 1) [10]. In this structure, the extra methoxy groups were included in the porphyrin moiety and the lipophilic trifluoromethyl group. The influence of the trifluoromethyl group, in biologically active molecules, is often associated with the increased lipophilicity that is imparted by this substituent [11]. In the present study, we have investigated the photodynamic effects of CF3 on Hep-2 cell line, in vitro and in female Balb/c mice, in vivo. First, the cytotoxicity of CF3 was evaluated, in vitro, under dark conditions and in relation to the illumination from visible light. Then, the new porphyrin was studied in mice for its distribution and possible toxic effects. The photokilling mechanism was studied on mice tumours which were injected with CF3 and then irradiated. These tumours were either kept under dark or illuminated conditions. The results can be used to design photodynamic treatments using this porphyrin as a sensitizer.
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1% (v/v) 0.2 M L-glutamine. The cells were incubated at 37 °C in a humidified 5% CO2 atmosphere and the medium was changed everyday. The Hep-2 cell line was routinely grown either in 35-mm culture dishes (Costar, Cambridge, MA) or on 22-mm square glass coverslips placed in the dishes. 2.5. In vitro studies of PDT
Fig. 1. Chemical structure of CF3.
2. Materials and methods 2.1. Drugs All of the solvents were of HPLC quality from Merck and were used without further purification. Dipalmitoyl phosphatidylethanolamine purchased from Sigma was used in liposome preparation. Thiazolyl blue (MTT) was purchased from Sigma.
2.5.1. Treatments and cell survival Treatments were performed with 1–10 µM of liposomes in DMEM containing 1% FCS. Hep-2 cells were incubated at 37 °C under dark conditions with CF3, at different points of time. After washing with the complete DMEM, the cells were either allowed to grow in this medium or subjected to light irradiation. Cell survival was assessed for untreated (control) and CF3-treated cultures. The surviving cells were evaluated 24 h after the treatment using the MTT method [14]. 2.5.2. Irradiation The light source used was that of a Kodak slide projector equipped with a 150 W lamp. The light was filtered through a 3 cm water layer to absorb the heat. A wavelength of the range of 350–800 nm was selected with the aid of optical filters [15], and the light intensity at the treatment site was 100 mW/cm2.
The photosensitizer CF3 was synthesized from the coupling reaction between 5-(4-aminophenyl)-10,15,20-tris(2,4,6-trimethoxyphenyl) porphyrin and 1-chloro-2,6dinitro-4-trifluorometilbenzene as described previously in Fig. 1 [10].
2.5.3. Morphology and cell counting Changes in cell morphology were analysed using fluorescence microscopy. After fixing in methanol at –20 °C for 10 min, the cells were stained with H-33258 (10 µg/ml in distilled water, 5 min) for the visualization of chromatin DNA. After washing and air drying, the preparations were mounted in DPX and observed under UV excitation [16].
2.3. Liposome preparation
2.6. In vivo studies of PDT
The incorporation of CF3 into the phospholipid bilayer of the D,L-dipalmitoyl phosphatidylethanolamine was achieved by a modification of the ethanol injection method [12]. Typically, 2 ml of a solution composed of: 9.60 mM of phospholipid, 1.91 mM of cholesterol and 0.27 mM of CF3 in ethanol-tetrahydrofuran (1:1 v/v) was injected into 10 ml of phosphate-buffered saline (PBS, 10 ml) at 80 °C. The injection was performed at a speed of 50 µl/min with magnetic stirring [13]. The final volume was reduced to 10 ml by evaporation of organic solvents.
2.6.1. Animal and tumour model Female Balb/c 6–8 weeks old, were obtained from the Fundación Balseiro (Buenos Aires) and maintained in standard cages with free access to tap water and normal dietary chow. When required, the animals were anaesthetized with Ketalar (150 mg/kg, by i.p. injection). The experimental tumours LM-2, originally obtained from Hospital Roffo (Buenos Aires. Argentine) were s.c. implanted by a sterile injection of ca. 106 cells in phosphate-buffered saline into the right foreleg. The pharmacokinetic and/or phototherapeutic experiments were performed on the sixth day after implantation, when the tumour was of 0.6–0.7 cm in the outer diameter. Spontaneous tumour necrosis was minimal or absent for these tumour sizes.
2.2. Photosensitizer
2.4. Cell culture A human larynx-carcinoma cell line (Hep-2) (purchased from Asociación Banco Argentino de Células ABAC, Instituto Nacional de Enfermedades Virales Humanas, Pergamino, Argentina) kept in liquid nitrogen was used for the investigation. The cells were grown as a monolayer employing Dulbecco’s modified Eagle’s medium (DMEM) containing 7% foetal calf serum (FCS) and 50 µg gentamycin/ml and
2.6.2. Pharmacokinetic studies CF3 (10 mg/kg) was administered by i.p. injection. The animals were sacrificed during different points of time (six mice at 24 h and three mice at other points of time), varying between 1 h and 1 week, after administration. The brain,
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spleen, kidney, duodenum, and the liver were removed. About 200 mg of tissues were homogenized in THF. The homogenates were centrifuged at 3000 rev/min for 15 min, and the fluorescence of the supernatants was measured, setting the excitation wavelength at 420 nm and recording the emission spectrum from 500 and 800 nm. Serum samples, isolated from the blood by centrifugation, were diluted with suitable volumes of 500 µl THF and the CF3 content was measured by fluorescence. In all the cases, CF3 amounts were determined by the interpolation of emission intensity and CF3 concentration plotted on a standard curve. 2.6.3. Phototherapeutic studies The effect of PDT on tumour growth was assessed using the in vivo/in vitro model previously described by Fukuda et al. [16] with modifications [17]. Briefly, 24 h after i.p. injection of CF3 at a dose of 10 mg/kg, the animals were sacrificed and the tumours were removed. Uniform tumour tissue fragments were obtained using a biopsy punch and incubated for 2 h at 37 °C in 5 ml of serum-free MEM. Irradiated explants were exposed for 15 min to light at a light dose of 90 J/cm2. Appropriate controls, of non-irradiated explants incubated with CF3 and irradiated and non-irradiated explants incubated without CF3, were included. Irradiated and nonirradiated tumour samples were fixed in 10% formaldehyde. The samples were then dehydrated in a series of graded ethanol, cleared in xylene and embedded in paraffin. Sections, 5 µm thick, were cut and stained with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabelling) to show the typical morphology of apoptotic cells.
3. Results 3.1. In vitro studies of PDT Direct cytotoxic effects of CF3 were studied, for different concentrations, in the absence of light. As evaluated by the MTT method, 24 h after treatment, CF3 does not show any dark toxicity (Table 1), which is evidenced by the absence of lethal effects caused by this porphyrin under dark conditions. On the contrary, 24 h of incubation with CF3 treatments, followed by light irradiation, induced a significant decrease of cell viability, which was related to both drug concentration and irradiation time (Fig. 2) The strongest photodynamic effect (10 µM and 15 min of irradiation) resulted in 13% of cell viability.
Fig. 2. Inactivation of Hep-2 cells incubated for 24 h with different concentrations of CF3 and then exposed to several irradiation periods with visible light, 5 µM (n), 8 µM (•), 10 µM (m) and irradiation control (.). Concentration: 0.2 mg of MTT/ml medium. Mean values ± S.D. from at least three different experiments.
Cell death was not found to occur when untreated cell cultures were irradiated with visible light; hence the cell death caused after irradiation, following the abovementioned treatment, was due to the effect of visible light and photosensitizer. When stained with H-33258, characteristic morphological changes were found in the photosensitized cells on incubation with 10 µM CF3 for 24 h; revealing different cell death mechanisms depending on the irradiation time. When analysed 24 h after photodynamic treatments with 15 min irradiation (Fig. 3), a great amount of apoptotic cells were obtained. On the contrary, necrotic cells were observed using the same drug concentration and incubation conditions, but after 15 min irradiation, 1 h of incubation at 37 °C and another 15 min of irradiation. Fig. 4 shows a characteristic photography of normal apoptotic and necrotic cells obtained from the control cells or those treated with CF3.
Table 1 Cytotoxicity of Hep-2 cells treated with several CF3 concentrations for 24 h in the dark CF3 (µM) 0 0.1 1 8 10
Survival cells (%) 100 99 98 99 98
Fig. 3. Effect of cell death of 10 µM CF3 in Hep-2 cells after 24 h of incubation at 37 °C. Hep-2 cells were incubated with a photosensitizer for (A) 24 h in the dark (control), (B) cells irradiated for 15 min (C) cells irradiated for 15 min + 1 h heat incubation + 15 min irradiation. An evaluation of cell types was made using H-33258 staining.
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A
B
C
Fig. 5. Biodistribution of CF3 in mice without tumours: Recovery of CF3 from Balb/c mice injected with 10 mg/kg of drug. Values represent the average of the three experiments (A: liver, B: spleen, C: kidney, D: brain, E: muscle, F: skin, G: lung, H: duodenum).
Fig. 5. In mice without tumour, high levels of photosensitizer in the liver was observed to diminish in the fourth week, while in the spleen, no variation was observed. In the kidney, an intermediate value was found and it remained stable. However, a high percentage of CF3 was found in the duodenum on the seventh day post-injection time, perhaps due to its elimination from the organism via bile-gut pathway [18]. The brain, skin, muscle and the lung presented low values with no significant variations with regard to post-injection time. The low quantity detected in the brain would show that the photosensitizer was unable to cause some type of alteration to the central nervous system; while in the skin, it could be favourable, as it would cause photosensitization. The results obtained in tumour-bearing mice are shown in Fig. 6. The CF3 concentration increase in the spleen, liver and intestine of mice when treated with the photosensitizer. This result is characteristic when lipidic drugs are given as they are eliminated via the bile-gut pathway [19]. The insignificant accumulation of the drug in the brain confirms that the porphyrinic components are unable to go through the haemato-encephalic barrier; consequently any risk of toxic effects of CF3 in the central nervous system could be rejected. The highest concentrations were observed in the lung, the duodenum and the liver at 3 h post-injection treatment; these results declined slowly over the course of the post-injection time. On the contrary, the results obtained from the brain, skin, muscle and tumour maintained a low accumulation of the CF3, reaching the highest values at the shortest post-
Fig. 4. Fluorescence photomicrographs of Hep-2 cells stained with H-33258. (A) Untreated cells. (B) Apoptotic cells induced by treatment with CF3; 15 min of illumination. (C) Necrotic cells induced by treatment with CF3.
3.2. In vivo studies 3.2.1. Pharmacokinetics studies: biodistribution in tumour-free and tumour-bearing mice The distribution of CF3 was examined in different selected tissues of female mice Balb/c, between 1 d and 4 weeks after i.p. administration of 10 mg/kg corporal weight. CF3 distribution was studied in tumour bearing mice at 3, 24 h and 7 d post-injection periods. The results are shown in
Fig. 6. Biodistribution of tumour bearing mice: Recoveries of CF3 from tumour-bearing Balb/c mice injected with 10 mg/kg of drug. Values represent the average of the three experiments (A: liver, B: spleen, C: kidney, D: tumour, E: brain, F: muscle, G: skin, H: lung, I: duoden).
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A
B Fig. 7. Fluorescence images of fluorescein-stained apoptotic cells (bright green) in tumour sections of animals treated with CF3 10 mg/kg (100×). (A) Tumour illuminated, (B) tumour without illumination (see Section 2).
irradiation time (3 h). Histological cuts were performed in organs (liver, kidney, duodenum), where high levels of CF3 were detected and no irreparable damages were found (manuscript in preparation). A low detectable amount of CF3 was found in the tumour, however, PDT treatment could be performed when the neoplastic tissues are identified previously. 3.2.2. Phototherapeutic studies Mice were injected with CF3 10 mg/kg after tumours were taken out and irradiated in vitro. Histological cuts and TUNEL were performed to determine apoptotic cells. The results are shown in Fig. 7. The amount of dead cells, by apoptosis, in irradiated tumours (Fig. 7A) is higher than that in non-irradiated ones (Fig. 7B). These results show that under these conditions, the combination of CF3 and light produces cell death mainly by apoptosis.
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tion (24 h) period with the highest drug concentration used (10 µM). The efficiency of photokilling increased with CF3 concentration and light doses. It is worth noting that by using the same CF3 concentration (1 × 10–6 M) and incubation time (24 h), different photokilling mechanisms were observed, depending on the irradiation time. According to many authors, the occurrence of apoptosis or necrosis after PDT treatments could depend on (i) characteristics of the photosensitizer [20], (ii) kind of cell line [21] and cellular density [22], and (iii) experimental protocol, which include: drug concentration, incubation time and light doses [23,24]. The results show that the mechanism of cell death (necrosis or apoptosis) induced by CF3 is clearly dependent on the treatment used in the irradiation conditions. On the other hand, in vivo results indicate that CF3 can be a candidate for PDT. Although the efficiency of CF3 incorporation in tumoural tissue is not high in relation to other tissues, a high antitumour activity is observed when excited by visible light, since a high percentage of cells in apoptosis appeared in tumours that were obtained from mice already treated with CF3 and illuminated. The liver, the spleen and, to a minor extent, the duodenum, retained detectable amounts of CF3 for prolonged periods. Several reports indicated that many photosensitizers are accumulated in high concentrations by the components of the reticuloendothelial system [25]. Small amounts of CF3 accumulated by kidneys suggest that this porphyrin is mostly eliminated from the organism via the bile-gut pathway. Likewise, only negligible amounts of CF3 accumulate in the skin, making low skin photosensitive. In summary, the present results show that CF3 is a potential tumour photosensitizer. A different mechanism of cell death occurs in Hep-2 cell cultures depending on the irradiation doses in the photodynamic treatments with CF3. Also, the irradiation of tumour tissues produces cell inactivation mainly by the apoptotic way. The development of experimental protocols using a given sensitizer in vitro and in vivo can contribute to the understanding of the photokilling effect in basic oncological research and to assess the potential for clinical applications in the PDT of cancer.
Acknowledgements The authors are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina, Agencia Nacional de Promoción Científica y Tecnológica and SECYT of Universidad Nacional de Río Cuarto for financial support. V.R. and E.N.D. hold the position of researchers at CONICET. M.G.A. thanks CONICET for a research fellowship.
4. Discussion A new photosensitizer was tested on a human carcinoma cell line and in vivo. The studies on Hep-2 cells show that the dark toxicity of CF3 was not found even after a long incuba-
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