Tumor Biol. DOI 10.1007/s13277-015-3745-z
RESEARCH ARTICLE
Antitumor effects evaluation of a novel porphyrin derivative in photodynamic therapy Jian-Wei Li 1 & Zhong-Ming Wu 1 & Davor Magetic 2 & Li-Jun Zhang 3 & Zhi-Long Chen 3
Received: 11 May 2015 / Accepted: 30 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract In this paper, the antitumor activity of a novel porphyrin-based photosensitizer 5,10,15,20-tetrakis[(5diethylamino)pentyl] porphyrin (TDPP) was reported in vitro and in vivo. The photophysical and cellular properties of TDPP were investigated. The singlet oxygen generation quantum yield of TDPP was detected; it showed a high singlet oxygen quantum yield of 0.52. The intracellular distribution of photosensitizer was detected with laser scanning confocal microscopy. The efficiency of TDPP-photodynamic therapy (PDT) in vitro was analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay and in situ trypan blue exclusion test. Treated with a 630-nm laser, TDPP can kill cultured human esophageal cancer cell line (Eca-109) cells and reduce the growth of Eca109 xenograft tumors significantly in BABL/c nude mice. And histopathological study was also used to confirm the antitumor effect. It has the perspective to be developed as a new antitumor drug in photodynamic therapy and deserves further investigation.
* Zhi-Long Chen [email protected] 1
Yiwu City Central Hospital, Zhejiang 322000, People’s Republic of China
2
Division of organic chemistry and biochemistr, Rudjer Boskovic Institute, Zagreb, Croatia
3
Department of Pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University, Shanghai 201620, People’s Republic of China
Keywords TDPP . Porphyrin . Tumor . Photosensitizer . Photodynamic therapy
Introduction Photodynamic therapy (PDT) is an approved therapeutic procedure to treat cancers using combinations of chemical photosensitizers and light [1, 2]. Administration of a photosensitizer followed by illumination by an appropriate wavelength, the excited photosensitizer transfers its energy to neighboring molecular oxygen species, producing reactive oxygen species (ROS) to destroy cancer cells [3, 4]. Antitumor effects of PDT are derived from three inter-related mechanisms: direct cytotoxic effects on tumor cells, damage to the tumor vasculature, and induction of a robust inflammatory reaction that may lead to the development of systemic immunity [5]. Photosensitizer, oxygen, and light are the three most important elements of photodynamic therapy [6, 7]. The development of photosensitizers has become popular in recent years. An ideal photosensitizer should be pure, should have high absorption coefficient in the long wavelength region (600–800 nm), should have no dark toxicity, and should be able to produce singlet oxygen efficiently [8]. Many novel photosensitizers have been researched in preclinical and in clinical settings [9–11]. Porphyrin derivatives have attracted considerable attention. Such as the porphyrinbased photosensitizer, Photofrin is administered systemically for the clinical treatment of esophageal and bronchial carcinomas as well as for treating Barrett’s esophagus. Porphyrins are naturally occurring organic compounds which are involved in a variety of important biological processes [12]. Herein, the antitumor activity of 5,10,15,20tetrakis[(5-diethylamino)pentyl] porphyrin (TDPP) (Fig. 1), a porphyrin-based photosensitizer, was
Tumor Biol.
Cell culture Human esophageal cancer cell line (Eca-109) was obtained from the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in normal RPMI-1640 culture medium with 10 % fetal bovine serum (FBS), 50 units/mL penicillin, and 50 μg/mL streptomycin in 5 % CO2 at 37 °C. All cell culture-related reagents were purchased from Shanghai Ming Rong Bio-Science Technology Co., Ltd. MTT cell viability assay Fig. 1 Chemical structure of TDPP
investigated. TDPP shows high singlet oxygen quantum yield of 0.52, low dark toxicity, and significant cytotoxicity in the presence of light in vitro and in vivo. This study suggests that TDPP may become the promising candidate for non-invasive treatment of cancer in PDT.
Materials and methods Chemicals 1,3-diphenylisobenzofuran (DPBF), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) were obtained from Sinopharm Chemical Reagent Co., Ltd. All the chemicals and reagents were of analytical grade and used without any purification.
Absorption and emission spectra UV–vis absorption spectrum was recorded on an ultravioletvisible spectrophotometer (Model V-530, Japan). Fluorescence spectra were carried out using a fluorescence spectrometer (FluoroMax-4, France). Slits were kept narrow to 1 nm in excitation and 2 nm in emission. Right angle detection was used. All the measurements were carried out at room temperature in quartz cuvettes with a path length of 1 cm. TDPP was dissolved in DMSO to get 5 μM of solution.
Singlet oxygen generation detection The singlet oxygen ability of TDPP was monitored by chemical oxidation of DPBF in the DMF solution. TDPP (5× 10−6 M) and DPBF (2×10−5 M) were mixed and irradiated. The reaction was monitored spectrophotometrically by measuring the decrease in optical density every 1 min at an absorbance maximum of 410 nm of DPBF.
Eca-109 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % (v/v) FBS, collected with 0.25 % (w/v) trypsin, and seeded in 96-well plates at 6×103 cells per well. The cells were allowed to attach to the bottom of the wells for 24 h prior to start of the experiment. DMEM containing MPP in different concentrations (range from 0 to 16 μM) was administered to cells and allowed to uptake for 24 h. DMEM containing TDPP was removed, and cells were washed with PBS before irradiation with different light doses (range from 4 to 16 J/cm2) using an Nd:YAG laser at 630 nm. The cell viability was evaluated by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay 24 h after treatment. In parallel, nonirradiated cells were used to investigate the dark cytotoxicity. In situ trypan blue exclusion test Trypan blue test was performed directly on 12-well plates (without trypsinization) to evaluate the effectiveness of cell photosensitization. To this end, samples were analyzed 30 min after treatment with 8 μM TDPP for 12 h followed by red light irradiation (630 nm, 8 J/cm2). The culture medium of control and treated cells was removed and replaced by PBS and then trypan blue was added (0.4 % in PBS) directly to the wells for 5 min. Subsequently, wells were washed three times with PBS and cells were observed and photographed directly using an inverted microscope. Intracellular localization Eca-109 cells grown on cover slips were incubated with the solution (8 μM) of TDPP for 4 h in the dark. Then, after removing the solution with PBS, cells were stained with 1 μg/mL Hoechst 33342 to label the nucleus. After washing with PBS, cover slips were fixed for 10 min at 4 °C with 4 % paraformaldehyde and cells were then examined by fluorescence with a confocal microscopy (Carl Zeiss LSM 700, Jena, Germany). TDPP was excited at 500 nm and its emitted light was monitored through a 600–650-nm band-pass filter, and Hoechst 33342 was excited at 350 nm and blue fluorescence was detected through a 450–500-nm band-pass filter.
Tumor Biol.
Animal and tumor models Five-week-old male BALB/c nude mice (5 weeks old) were anesthetized and 5×106 Eca-109 cells were injected subcutaneously with 200 μL PBS into right forelimb. When implanted tumor sizes were more than 10 mm in diameter, tumors were excised and small pieces of the tumor (approximately 2-mm square pieces) were implanted subcutaneously into the right dorsal area of male BALB/c nude mice (5 weeks old). When tumor sizes had reached 5–7 mm in diameter after implantation (14–21 days), the male BALB/c nude mice were used for studies of PDT efficacy of TDPP. In vivo PDT efficacy The male BALB/c nude mice bearing Eca-109 tumors were injected at a dose of 5 mg/kg in 0.2 mL solution via the lateral tail vein. PDT was performed following injection with laser light (630 nm, 100 J/cm2, 180 mW/cm2). Visible tumors were measured using two orthogonal measurements L and W (perpendicular to L); the volumes were calculated using the formula V=LW2 /2 and recorded. Histology examination To confirm the PDT efficacy of TDPP for tumor therapy, histology analysis of tumor tissues were performed after treatment. Tumor tissues of every group were separated and fixed with 10 % neutral buffered formalin and embedded in paraffin. The sliced organs were stained with hematoxylin–eosin (H&E) and examined under a microscope. Statistical analysis All experiments were performed in triplicate and the data were expressed as mean plus and minus the standard error of the mean. Analysis of variance (ANOVA) and Student’s t test were used to determine the statistically significant difference among different groups when appropriate. The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Donghua University.
Results UV–vis absorption and fluorescence spectra As shown in Fig. 2a, TDPP had the characteristic Soret and Q band absorptions at 401 nm (Soret), 500, 532, 569, and 622 nm (Q band) in DMSO solution, respectively. When excited at 569 nm, TDPP showed strong emission peaks at 623 and 689 nm (Fig. 2b).
Fig. 2 Spectrum properties of TDPP. a UV–vis absorption spectrum of TDPP in DMSO. TDPP had absorptions at 401 nm (Soret), 500 nm, 532 nm, 569 nm, and 622 nm (Q band), respectively. b Emission spectrum of TDPP, which was excited at 569 nm, and its emission peaks were at 623 and 689 nm. c UV–vis spectra for the determination of singlet oxygen generation quantum yield of TMPC in DMF use DPBF as quencher
Tumor Biol.
Singlet oxygen quantum yield The singlet oxygen quantum yield (ΦΔ) value was determined in DMF solution using DPBF as a quencher. The disappearance of DPBF spectra were monitored at 410 nm using UV– vis spectrophotometer; the absorption intensity of DPBF (λ= 410 nm) continuously decreased with the irradiation time increasing (Fig. 2c). The singlet oxygen (1O2) quantum yield ΦΔ of TDPP in DMF was 0.52. TDPP cytotoxicity on human esophageal cancer cells The effect of TDPP on the viability of cultured Eca-109 cells was evaluated by MTT assay. As shown in Fig. 3a, there was almost no dark cytotoxicity observed when exposed up to 8 μM TDPP, and the Eca-109 cells’ viability was more than 90 % when incubation with the highest concentration (16 μM) TDPP in the dark. After irradiation, a significant cytotoxicity
Fig. 3 Cytotoxicity of TDPP in vitro. a Light dose- and concentration-effect curves for TDPP. Eca-109 cells were treated at the indicated conditions. Cell viability was examined using MTT assay. The error bars denote standard deviation from three replicates. b Light microscopy of Eca-109 cells stained in situ with trypan blue exclusion test
was detected. Low concentration (1 μM) caused moderate damage at the largest light dose (16 J/cm2). Raising the concentration from 1 to 16 μM, the cell viability was decreased sharply at the same light dose. The cell viability below 10 % was observed when treated with 16 μM and 16 J/cm2. Incubation with the same concentration TDPP, the cell viability was decreased with the increase of light dose. The data in Fig. 3a indicate that the efficacy of PDT depends on the irradiation energy and the concentration of TDPP. The cell viability of Eca-109 cells that were exposed to light without TDPP pre-incubation was similar to those cells without any treatment (data not shown). A trypan blue cell-death assay was also performed directly on plastic wells 30 min after photodynamic treatments, in order to independently evaluate the effectiveness of cell photosensitization. As shown in Fig. 3b, Eca-109 cells incubated with 8 μM for 12 h and irradiated with 8 J/cm2 were nonviable (blue stained) 30 min after treatment.
Tumor Biol.
Intracellular localization The localization of TDPP in cells were evaluated by confocal microscopy after incubating Eca-109 cells for 4 h in the dark and staining them with fluorescent dye (Hoechst 33342) for the nucleus. TDPP was mainly found in the cytoplasm and nuclear membrane corresponding to the red fluorescence and blue fluorescence represents the signal for Hoechst 33342 in Fig. 4.
had a difference; the tumor growth in the TDPP-PDT group was suppressed more effectively than that in the HpD-PDT group. The volume growth curves of tumors were provided in Fig. 5b; the tumor volume increased about 10-fold for 12 days in the control group and the laser irradiation group. TDPPmediated PDT decreased the tumor volume at the 3rd day post treatment, and the tumor growth was the slowest of all groups.
Histopathological study with H&E staining In vivo photodynamic therapy In order to examine the effects of TDPP-mediated PDT on Eca-109 tumors in vivo, an Eca-109 xenograft tumor model was used. TDPP and hematoporphyrin derivative (HpD) were injected into tumor bearing BALB/c nude mice via the tail vein at a dose of 5 mg/kg followed by irradiation of red light. Figure 5a presented the digital photos of tumors at time of the 14th day with different treatments. There was no significant difference between the control group (without any treatment) and the laser irradiation group (irradiated without TDPP administration). And the tumors in the control group and the laser irradiation group were larger than the tumors in the HpD-PDT group and the TDPP-PDT group. However, the tumors in the HpD-PDT group and the TDPP-PDT group
Fig. 4 Intracellular localization of TDPP in Eca-109 cells. Eca109 cells were incubated with 8 μM TDPP for 4 h in the dark and then stained with Hoechst 33342. Red fluorescence corresponds to TDPP and blue fluorescence represents the signal for Hoechst 33342
The antitumor effect of PDT using TDPP was histologically evaluated in Eca-109 tumor bearing BALB/c nude mice. The nude mice were sacrificed immediately after treatment, and coronal sections were stained with H&E for light microscopic examination. As shown in Fig. 6, the tumor cells in the HpDPDT group and the TDPP-PDT group were shrunken and necrotic (the white arrow indicated). In the destructed area, polymorphonuclear cells were also observed, indicating inflammatory responses. And the necrotic cells in the TDPPPDT group were much more than that in the HpD-PDT group. However, the tumor cells from nude mice in the control group and the laser irradiation group were round in shape and exhibited well-delineated margins with very limited signs of local infiltrations.
Tumor Biol.
Fig. 5 Efficacy of TDPP against esophageal cancer in nude mice bearing Eca-109 tumors. a Digital photos of tumors in every group 14 days post treatment. b Tumor volume at different time points post-PDT. The tumors in the control group and the laser irradiation group continued to grow and
were significantly larger than that in the TDPP-PDT group after 3 days post treatment. The data shown is the means±SD of three independent experiments
Discussion
short-lived excited singlet state [7]. The excited electron changes its spin and produces a longer lived triplet state by intersystem crossing. The PS triplet transfers energy to ground-state triplet oxygen, which produces 1O2 [5, 19]. TDPP shows a high singlet oxygen quantum yield of 0.52. In vitro, there was no significant difference in the cell viability compared to the control group treated with TDPP only, without light irradiation. When irradiated with 630 nm light, TDPP showed a substantial cytotoxic effect on the cultured Eca-109 cells. The results of the present study suggested that TDPP had low dark and high phototoxicity on Eca-109 cells. And the phototherapeutic effect of TDPP-PDT is related to the drug concentration and dose of light irradiation. In situ trypan blue exclusion test showed that the cells treated with TDPP and light can be dyed by trypan blue; however, the control
PDT has been regarded as a promising method for the treatment of various cancer and vascular proliferative diseases; developments of suitable photosensitizers (PSs) are highly desired to improve the prospect for the use of PDT [13]. While there is considerable attention in the synthesis of new PSs with improved PDT characteristics, porphyrin derivatives are particularly strong under investigations in preclinical and clinical PDT of tumors [14–16]. In the present research, the antitumor activity of TDPP, a porphyrin derivative, was evaluated in vitro and in vivo. In PDT, 1O2 is a key cytotoxic agent that is responsible for the destruction of tumor [3, 17, 18]. Especially in PDT type II reaction, the PS absorbs light and an electron moves to the first Fig. 6 Histological examination of the Eca-109 xenograft tumors with H&E. Excised tumors were fixed, sectioned, and stained with hematoxylin–eosin. The sections were examined under a light microscope and photographed. H&E ×200
Tumor Biol.
cells without any treatment cannot be dyed. These illustrate that the membranes of cells, which were treated with TDPPPDT, had been broken. The subcellular localization of PS is crucial for its activity. Some PSs show a broad distribution, while some may localize more specifically. A recent review indicated that several porphyrin derivatives are mainly distributed in lysosomes, mitochondria, ER, Golgi apparatus, and other organelles [20]. We found that TDPP was mainly distributed in the cytoplasm and nuclear membranes, to a less degree in the cell nuclei, which may represent the preferential target of the photosensitized process. The in vivo TDPP-mediated PDT was performed using an Eca-109 tumor xenografts model. The tumors in the control group and the laser irradiation group were larger than the tumors in the HpD-PDT group and the TDPP-PDT group. The tumor in the TDPP-PDT group was smaller than that in the HpD-PDT group. One day post irradiation, tumors were excised and sectioned, and a significant tissue loss was observed in the TDPP-PDT group. H&E staining clearly indicated severe apoptosis. In contrast, control and light-treated tumors showed no signs of apoptosis. Three main mechanisms of PDT-mediated tumor destruction involve direct cellular damage, vascular damage, and immune reaction [6, 21–23]. The relative contribution of each mechanism is not clear in the present study. Any of these routes could be responsible for the effectiveness of TDPP-PDT in vivo.
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Conclusion In summary, this study demonstrates that TDPP-mediated PDT is able to restrain the growth of Eca-109 cells with high efficiency in vitro and destroy the Eca-109 tumors in BABL/c nude mice. Based on the potential and characteristics of TDPP displayed in this paper, we believe that it is worthwhile pursuing TDPP as a PS for antitumor PDT. Acknowledgments This work was supported by the Chinese National Natural Science Foundation (No. 21372042, 21402236, 81101298, 81301878), Foundation of Shanghai government (No. 14431906200, 14140903500, 13431900700, 13430722300, 13ZR1441000, 13ZR1440900, 14ZR1439800, 14ZR1439900, 15ZR1439900, 15XD1523400, 14SJGGYY08, 201370), International Cooperation Foundation of China and Croatia (6-11), and Foundation of Yiwu Science and Technology Bureau (No. 2012-G3-02, 2013-G3-03). Conflicts of interest None
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RESEARCH ARTICLE
Antitumor effects evaluation of a novel porphyrin derivative in photodynamic therapy Jian-Wei Li 1 & Zhong-Ming Wu 1 & Davor Magetic 2 & Li-Jun Zhang 3 & Zhi-Long Chen 3
Received: 11 May 2015 / Accepted: 30 June 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract In this paper, the antitumor activity of a novel porphyrin-based photosensitizer 5,10,15,20-tetrakis[(5diethylamino)pentyl] porphyrin (TDPP) was reported in vitro and in vivo. The photophysical and cellular properties of TDPP were investigated. The singlet oxygen generation quantum yield of TDPP was detected; it showed a high singlet oxygen quantum yield of 0.52. The intracellular distribution of photosensitizer was detected with laser scanning confocal microscopy. The efficiency of TDPP-photodynamic therapy (PDT) in vitro was analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) assay and in situ trypan blue exclusion test. Treated with a 630-nm laser, TDPP can kill cultured human esophageal cancer cell line (Eca-109) cells and reduce the growth of Eca109 xenograft tumors significantly in BABL/c nude mice. And histopathological study was also used to confirm the antitumor effect. It has the perspective to be developed as a new antitumor drug in photodynamic therapy and deserves further investigation.
* Zhi-Long Chen [email protected] 1
Yiwu City Central Hospital, Zhejiang 322000, People’s Republic of China
2
Division of organic chemistry and biochemistr, Rudjer Boskovic Institute, Zagreb, Croatia
3
Department of Pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University, Shanghai 201620, People’s Republic of China
Keywords TDPP . Porphyrin . Tumor . Photosensitizer . Photodynamic therapy
Introduction Photodynamic therapy (PDT) is an approved therapeutic procedure to treat cancers using combinations of chemical photosensitizers and light [1, 2]. Administration of a photosensitizer followed by illumination by an appropriate wavelength, the excited photosensitizer transfers its energy to neighboring molecular oxygen species, producing reactive oxygen species (ROS) to destroy cancer cells [3, 4]. Antitumor effects of PDT are derived from three inter-related mechanisms: direct cytotoxic effects on tumor cells, damage to the tumor vasculature, and induction of a robust inflammatory reaction that may lead to the development of systemic immunity [5]. Photosensitizer, oxygen, and light are the three most important elements of photodynamic therapy [6, 7]. The development of photosensitizers has become popular in recent years. An ideal photosensitizer should be pure, should have high absorption coefficient in the long wavelength region (600–800 nm), should have no dark toxicity, and should be able to produce singlet oxygen efficiently [8]. Many novel photosensitizers have been researched in preclinical and in clinical settings [9–11]. Porphyrin derivatives have attracted considerable attention. Such as the porphyrinbased photosensitizer, Photofrin is administered systemically for the clinical treatment of esophageal and bronchial carcinomas as well as for treating Barrett’s esophagus. Porphyrins are naturally occurring organic compounds which are involved in a variety of important biological processes [12]. Herein, the antitumor activity of 5,10,15,20tetrakis[(5-diethylamino)pentyl] porphyrin (TDPP) (Fig. 1), a porphyrin-based photosensitizer, was
Tumor Biol.
Cell culture Human esophageal cancer cell line (Eca-109) was obtained from the Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in normal RPMI-1640 culture medium with 10 % fetal bovine serum (FBS), 50 units/mL penicillin, and 50 μg/mL streptomycin in 5 % CO2 at 37 °C. All cell culture-related reagents were purchased from Shanghai Ming Rong Bio-Science Technology Co., Ltd. MTT cell viability assay Fig. 1 Chemical structure of TDPP
investigated. TDPP shows high singlet oxygen quantum yield of 0.52, low dark toxicity, and significant cytotoxicity in the presence of light in vitro and in vivo. This study suggests that TDPP may become the promising candidate for non-invasive treatment of cancer in PDT.
Materials and methods Chemicals 1,3-diphenylisobenzofuran (DPBF), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) were obtained from Sinopharm Chemical Reagent Co., Ltd. All the chemicals and reagents were of analytical grade and used without any purification.
Absorption and emission spectra UV–vis absorption spectrum was recorded on an ultravioletvisible spectrophotometer (Model V-530, Japan). Fluorescence spectra were carried out using a fluorescence spectrometer (FluoroMax-4, France). Slits were kept narrow to 1 nm in excitation and 2 nm in emission. Right angle detection was used. All the measurements were carried out at room temperature in quartz cuvettes with a path length of 1 cm. TDPP was dissolved in DMSO to get 5 μM of solution.
Singlet oxygen generation detection The singlet oxygen ability of TDPP was monitored by chemical oxidation of DPBF in the DMF solution. TDPP (5× 10−6 M) and DPBF (2×10−5 M) were mixed and irradiated. The reaction was monitored spectrophotometrically by measuring the decrease in optical density every 1 min at an absorbance maximum of 410 nm of DPBF.
Eca-109 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10 % (v/v) FBS, collected with 0.25 % (w/v) trypsin, and seeded in 96-well plates at 6×103 cells per well. The cells were allowed to attach to the bottom of the wells for 24 h prior to start of the experiment. DMEM containing MPP in different concentrations (range from 0 to 16 μM) was administered to cells and allowed to uptake for 24 h. DMEM containing TDPP was removed, and cells were washed with PBS before irradiation with different light doses (range from 4 to 16 J/cm2) using an Nd:YAG laser at 630 nm. The cell viability was evaluated by 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay 24 h after treatment. In parallel, nonirradiated cells were used to investigate the dark cytotoxicity. In situ trypan blue exclusion test Trypan blue test was performed directly on 12-well plates (without trypsinization) to evaluate the effectiveness of cell photosensitization. To this end, samples were analyzed 30 min after treatment with 8 μM TDPP for 12 h followed by red light irradiation (630 nm, 8 J/cm2). The culture medium of control and treated cells was removed and replaced by PBS and then trypan blue was added (0.4 % in PBS) directly to the wells for 5 min. Subsequently, wells were washed three times with PBS and cells were observed and photographed directly using an inverted microscope. Intracellular localization Eca-109 cells grown on cover slips were incubated with the solution (8 μM) of TDPP for 4 h in the dark. Then, after removing the solution with PBS, cells were stained with 1 μg/mL Hoechst 33342 to label the nucleus. After washing with PBS, cover slips were fixed for 10 min at 4 °C with 4 % paraformaldehyde and cells were then examined by fluorescence with a confocal microscopy (Carl Zeiss LSM 700, Jena, Germany). TDPP was excited at 500 nm and its emitted light was monitored through a 600–650-nm band-pass filter, and Hoechst 33342 was excited at 350 nm and blue fluorescence was detected through a 450–500-nm band-pass filter.
Tumor Biol.
Animal and tumor models Five-week-old male BALB/c nude mice (5 weeks old) were anesthetized and 5×106 Eca-109 cells were injected subcutaneously with 200 μL PBS into right forelimb. When implanted tumor sizes were more than 10 mm in diameter, tumors were excised and small pieces of the tumor (approximately 2-mm square pieces) were implanted subcutaneously into the right dorsal area of male BALB/c nude mice (5 weeks old). When tumor sizes had reached 5–7 mm in diameter after implantation (14–21 days), the male BALB/c nude mice were used for studies of PDT efficacy of TDPP. In vivo PDT efficacy The male BALB/c nude mice bearing Eca-109 tumors were injected at a dose of 5 mg/kg in 0.2 mL solution via the lateral tail vein. PDT was performed following injection with laser light (630 nm, 100 J/cm2, 180 mW/cm2). Visible tumors were measured using two orthogonal measurements L and W (perpendicular to L); the volumes were calculated using the formula V=LW2 /2 and recorded. Histology examination To confirm the PDT efficacy of TDPP for tumor therapy, histology analysis of tumor tissues were performed after treatment. Tumor tissues of every group were separated and fixed with 10 % neutral buffered formalin and embedded in paraffin. The sliced organs were stained with hematoxylin–eosin (H&E) and examined under a microscope. Statistical analysis All experiments were performed in triplicate and the data were expressed as mean plus and minus the standard error of the mean. Analysis of variance (ANOVA) and Student’s t test were used to determine the statistically significant difference among different groups when appropriate. The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Donghua University.
Results UV–vis absorption and fluorescence spectra As shown in Fig. 2a, TDPP had the characteristic Soret and Q band absorptions at 401 nm (Soret), 500, 532, 569, and 622 nm (Q band) in DMSO solution, respectively. When excited at 569 nm, TDPP showed strong emission peaks at 623 and 689 nm (Fig. 2b).
Fig. 2 Spectrum properties of TDPP. a UV–vis absorption spectrum of TDPP in DMSO. TDPP had absorptions at 401 nm (Soret), 500 nm, 532 nm, 569 nm, and 622 nm (Q band), respectively. b Emission spectrum of TDPP, which was excited at 569 nm, and its emission peaks were at 623 and 689 nm. c UV–vis spectra for the determination of singlet oxygen generation quantum yield of TMPC in DMF use DPBF as quencher
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Singlet oxygen quantum yield The singlet oxygen quantum yield (ΦΔ) value was determined in DMF solution using DPBF as a quencher. The disappearance of DPBF spectra were monitored at 410 nm using UV– vis spectrophotometer; the absorption intensity of DPBF (λ= 410 nm) continuously decreased with the irradiation time increasing (Fig. 2c). The singlet oxygen (1O2) quantum yield ΦΔ of TDPP in DMF was 0.52. TDPP cytotoxicity on human esophageal cancer cells The effect of TDPP on the viability of cultured Eca-109 cells was evaluated by MTT assay. As shown in Fig. 3a, there was almost no dark cytotoxicity observed when exposed up to 8 μM TDPP, and the Eca-109 cells’ viability was more than 90 % when incubation with the highest concentration (16 μM) TDPP in the dark. After irradiation, a significant cytotoxicity
Fig. 3 Cytotoxicity of TDPP in vitro. a Light dose- and concentration-effect curves for TDPP. Eca-109 cells were treated at the indicated conditions. Cell viability was examined using MTT assay. The error bars denote standard deviation from three replicates. b Light microscopy of Eca-109 cells stained in situ with trypan blue exclusion test
was detected. Low concentration (1 μM) caused moderate damage at the largest light dose (16 J/cm2). Raising the concentration from 1 to 16 μM, the cell viability was decreased sharply at the same light dose. The cell viability below 10 % was observed when treated with 16 μM and 16 J/cm2. Incubation with the same concentration TDPP, the cell viability was decreased with the increase of light dose. The data in Fig. 3a indicate that the efficacy of PDT depends on the irradiation energy and the concentration of TDPP. The cell viability of Eca-109 cells that were exposed to light without TDPP pre-incubation was similar to those cells without any treatment (data not shown). A trypan blue cell-death assay was also performed directly on plastic wells 30 min after photodynamic treatments, in order to independently evaluate the effectiveness of cell photosensitization. As shown in Fig. 3b, Eca-109 cells incubated with 8 μM for 12 h and irradiated with 8 J/cm2 were nonviable (blue stained) 30 min after treatment.
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Intracellular localization The localization of TDPP in cells were evaluated by confocal microscopy after incubating Eca-109 cells for 4 h in the dark and staining them with fluorescent dye (Hoechst 33342) for the nucleus. TDPP was mainly found in the cytoplasm and nuclear membrane corresponding to the red fluorescence and blue fluorescence represents the signal for Hoechst 33342 in Fig. 4.
had a difference; the tumor growth in the TDPP-PDT group was suppressed more effectively than that in the HpD-PDT group. The volume growth curves of tumors were provided in Fig. 5b; the tumor volume increased about 10-fold for 12 days in the control group and the laser irradiation group. TDPPmediated PDT decreased the tumor volume at the 3rd day post treatment, and the tumor growth was the slowest of all groups.
Histopathological study with H&E staining In vivo photodynamic therapy In order to examine the effects of TDPP-mediated PDT on Eca-109 tumors in vivo, an Eca-109 xenograft tumor model was used. TDPP and hematoporphyrin derivative (HpD) were injected into tumor bearing BALB/c nude mice via the tail vein at a dose of 5 mg/kg followed by irradiation of red light. Figure 5a presented the digital photos of tumors at time of the 14th day with different treatments. There was no significant difference between the control group (without any treatment) and the laser irradiation group (irradiated without TDPP administration). And the tumors in the control group and the laser irradiation group were larger than the tumors in the HpD-PDT group and the TDPP-PDT group. However, the tumors in the HpD-PDT group and the TDPP-PDT group
Fig. 4 Intracellular localization of TDPP in Eca-109 cells. Eca109 cells were incubated with 8 μM TDPP for 4 h in the dark and then stained with Hoechst 33342. Red fluorescence corresponds to TDPP and blue fluorescence represents the signal for Hoechst 33342
The antitumor effect of PDT using TDPP was histologically evaluated in Eca-109 tumor bearing BALB/c nude mice. The nude mice were sacrificed immediately after treatment, and coronal sections were stained with H&E for light microscopic examination. As shown in Fig. 6, the tumor cells in the HpDPDT group and the TDPP-PDT group were shrunken and necrotic (the white arrow indicated). In the destructed area, polymorphonuclear cells were also observed, indicating inflammatory responses. And the necrotic cells in the TDPPPDT group were much more than that in the HpD-PDT group. However, the tumor cells from nude mice in the control group and the laser irradiation group were round in shape and exhibited well-delineated margins with very limited signs of local infiltrations.
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Fig. 5 Efficacy of TDPP against esophageal cancer in nude mice bearing Eca-109 tumors. a Digital photos of tumors in every group 14 days post treatment. b Tumor volume at different time points post-PDT. The tumors in the control group and the laser irradiation group continued to grow and
were significantly larger than that in the TDPP-PDT group after 3 days post treatment. The data shown is the means±SD of three independent experiments
Discussion
short-lived excited singlet state [7]. The excited electron changes its spin and produces a longer lived triplet state by intersystem crossing. The PS triplet transfers energy to ground-state triplet oxygen, which produces 1O2 [5, 19]. TDPP shows a high singlet oxygen quantum yield of 0.52. In vitro, there was no significant difference in the cell viability compared to the control group treated with TDPP only, without light irradiation. When irradiated with 630 nm light, TDPP showed a substantial cytotoxic effect on the cultured Eca-109 cells. The results of the present study suggested that TDPP had low dark and high phototoxicity on Eca-109 cells. And the phototherapeutic effect of TDPP-PDT is related to the drug concentration and dose of light irradiation. In situ trypan blue exclusion test showed that the cells treated with TDPP and light can be dyed by trypan blue; however, the control
PDT has been regarded as a promising method for the treatment of various cancer and vascular proliferative diseases; developments of suitable photosensitizers (PSs) are highly desired to improve the prospect for the use of PDT [13]. While there is considerable attention in the synthesis of new PSs with improved PDT characteristics, porphyrin derivatives are particularly strong under investigations in preclinical and clinical PDT of tumors [14–16]. In the present research, the antitumor activity of TDPP, a porphyrin derivative, was evaluated in vitro and in vivo. In PDT, 1O2 is a key cytotoxic agent that is responsible for the destruction of tumor [3, 17, 18]. Especially in PDT type II reaction, the PS absorbs light and an electron moves to the first Fig. 6 Histological examination of the Eca-109 xenograft tumors with H&E. Excised tumors were fixed, sectioned, and stained with hematoxylin–eosin. The sections were examined under a light microscope and photographed. H&E ×200
Tumor Biol.
cells without any treatment cannot be dyed. These illustrate that the membranes of cells, which were treated with TDPPPDT, had been broken. The subcellular localization of PS is crucial for its activity. Some PSs show a broad distribution, while some may localize more specifically. A recent review indicated that several porphyrin derivatives are mainly distributed in lysosomes, mitochondria, ER, Golgi apparatus, and other organelles [20]. We found that TDPP was mainly distributed in the cytoplasm and nuclear membranes, to a less degree in the cell nuclei, which may represent the preferential target of the photosensitized process. The in vivo TDPP-mediated PDT was performed using an Eca-109 tumor xenografts model. The tumors in the control group and the laser irradiation group were larger than the tumors in the HpD-PDT group and the TDPP-PDT group. The tumor in the TDPP-PDT group was smaller than that in the HpD-PDT group. One day post irradiation, tumors were excised and sectioned, and a significant tissue loss was observed in the TDPP-PDT group. H&E staining clearly indicated severe apoptosis. In contrast, control and light-treated tumors showed no signs of apoptosis. Three main mechanisms of PDT-mediated tumor destruction involve direct cellular damage, vascular damage, and immune reaction [6, 21–23]. The relative contribution of each mechanism is not clear in the present study. Any of these routes could be responsible for the effectiveness of TDPP-PDT in vivo.
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Conclusion In summary, this study demonstrates that TDPP-mediated PDT is able to restrain the growth of Eca-109 cells with high efficiency in vitro and destroy the Eca-109 tumors in BABL/c nude mice. Based on the potential and characteristics of TDPP displayed in this paper, we believe that it is worthwhile pursuing TDPP as a PS for antitumor PDT. Acknowledgments This work was supported by the Chinese National Natural Science Foundation (No. 21372042, 21402236, 81101298, 81301878), Foundation of Shanghai government (No. 14431906200, 14140903500, 13431900700, 13430722300, 13ZR1441000, 13ZR1440900, 14ZR1439800, 14ZR1439900, 15ZR1439900, 15XD1523400, 14SJGGYY08, 201370), International Cooperation Foundation of China and Croatia (6-11), and Foundation of Yiwu Science and Technology Bureau (No. 2012-G3-02, 2013-G3-03). Conflicts of interest None
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