Tumor Biol. DOI 10.1007/s13277-015-3395-1
RESEARCH ARTICLE
Antitumor activity of photodynamic therapy with a chlorin derivative in vitro and in vivo Lai-Xing Wang 1 & Jian-Wei Li 2 & Jian-Yue Huang 2 & Jian-Hong Li 2 & Li-Jun Zhang 3 & Donal O’Shea 4 & Zhi-Long Chen 3
Received: 18 December 2014 / Accepted: 25 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Chlorin derivatives are promising photosensitive agents for photodynamic therapy (PDT) of tumors. The aim of the current study is to investigate the PDT therapeutic effects of a novel chlorin-based photosensitizer, mesotetra[3-(N,N-diethyl)aminomethyl-4-methoxy]phenyl chlorin (TMPC) for gliomas in vitro and in vivo. Physicochemical characteristics of TMPC were recorded by ultraviolet visible spectrophotometer and fluorescence spectrometer. The rate of singlet oxygen generation of TMPC upon photo-excitation was detected by using 1,3-diphenylisobenzofuran (DPBF). The accumulation of TMPC in gliomas U87 MG cells was measured by fluorescence spectrometer. The efficiency of TMPC-PDT in vitro was analyzed by MTT assay and clonogenic assay. The biodistribution and clearance of TMPC were determined by fluorescence measuring. Human gliomas U87 MG tumor-bearing mice model was used to evaluate the antitumor effects of TMPC-PDT. TMPC shows a singlet oxygen generation rate of 0.05 and displays a characteristic long wavelength absorption peak at 653 nm (ε=15, 400). The accumulation of TMPC increased with the increase of incubation time. In vitro, PDT using TMPC and laser showed laser dose- and concentration-dependent cytotoxicity
* Zhi-Long Chen [email protected] 1
Changhai Hospital, Shanghai 201620, People’s Republic of China
2
Yiwu City Central Hospital, Zhejiang 322000, People’s Republic of China
3
Department of Pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University, Shanghai 201620, People’s Republic of China
4
Center for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin, Ireland
to U87 MG cells. In U87 MG tumor-bearing mice, TMPCPDT significantly reduced the growth of the tumors. Both in vitro and in vivo, TMPC showed little dark toxicity. In vitro and in vivo studies, it found that TMPC has excellent antitumor activities. It suggests that TMPC is a potential photosensitizer of photodynamic therapy for cancer. Keywords Photodynamic therapy . Photosensitizer . Chlorin derivative . Antitumor
Introduction Glioblastomas are highly aggressive tumors characterized by a high recurrence and high mortality. The median survival time for patients with glioblastoma is less than 15 months [1]. Recently, photodynamic therapy (PDT) has been regarded as an effective treatment approach for some tumors, such as skin cancers, breast cancers, early lung cancers, and tumors of the head and neck [2]. PDT has three elements: a photosensitizer (PS), light, and oxygen. PDT is based on the light of a specific wavelength excitation of a photosensitizer which results in the formation of reactive oxygen species (ROS) including superoxide, hydroxyl radical, singlet oxygen, and hydrogen peroxide [3]. The produced ROS can directly kill tumor cells by the induction of necrosis and/or apoptosis, can cause destruction of tumor vasculature, and can produce an acute inflammatory response that attracts leukocytes like dendritic cells and neutrophils [4]. Many advances in PDT suggest that a class of compounds that meet most of the requirements for ideal PDT agents are chlorin derivatives [5–7]. For example, mTHPC (Foscan, Biolitec Pharma, Scotland, UK) has been approved in Europe for use against head and neck cancer, and additional indications have been filed for prostate and pancreatic tumors
Tumor Biol.
[5]. This class of molecules have Q band absorption maxima at wavelengths (λmax=650–670 nm) with a molar absorption coefficient in the range of 105/M/cm, and they often have high singlet oxygen quantum yield [8]. Some of them show limited skin photosensitivity and rapid clearance from tissues [9]. In the present paper, we report that TMPC, a chlorin derivative, has a strong absorption band at relatively long wavelength (λmax=653 nm) and shows low dark toxicity, significant cytotoxicity in the presence of light in vitro and in vivo. Its biodistribution and clearance also have been evaluated in U87 MG tumor-bearing mice model and revealed that TMPC can be cleared rapidly from tissues.
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 ultraviolet visible 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 path length of 1 cm. TMPC was dissolved in DMSO to get 5 μM of solution. Singlet oxygen generation detection The singlet oxygen ability of TMPC was monitored by chemical oxidation of DPBF in the DMF solution. TMPC (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 2 min at an absorbance maximum of 417 nm of DPBF. Cell culture U87 MG human glioma cells (U87 MG) 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.
Cellular uptake of TMPC in U87 MG cells The U87 MG cells (1×105cells/well) were seeded in 24-well cell culture plates. After 24 h, the time-dependent drug accumulation in the cells was investigated by exposing the cells to 8 μM TMPC in culture medium for various time intervals, from 1 up to 24 h. At the end of the incubation time, the cells were washed three times with PBS and solubilized in 300 μL/ well DMSO. The retention of cell-associated TMPC was measured by fluorescence using excitation/emission wavelengths of 426 and 656 nm. The calibration curve (fluorescence vs. concentration) was performed and the concentration of TMPC per cell (pM) was calculated. MTT cell viability assay U87 MG cells were plated at a density of 1×104 cells/well in 96-well microplates and incubated overnight at 37 °C until 70–80 % of confluence. Media containing TMPC in different concentrations were administered to cells and allowed to uptake for 24 h. Media containing TMPC were then removed, a complete fresh culture medium was added, and cells were exposed to 1 to 16 J/cm2 light doses with an Nd: YAG laser at 650 nm. In parallel, non-irradiated cells were used to study dark cytotoxicity. Following treatment, cells were incubated for an additional 24 h period and cell viability was determined by means of MTT reduction assay [10]. Clonogenic assay U87 MG cells were treated with TMPC in different concentrations for 24 h before irradiation with 8 J/cm2. Following PDT, cells were seeded in 6-well plates at densities of 1000 and 200 cells/well. Cells were allowed to form colonies for approximately 14 days, and then the cells were fixed in 75 % ethanol for 15 min at 4 °C. Fixing agent was removed and plates were allowed to air dry, before cells were stained with a 0.25 % solution of crystal violet. Animal and tumor models Five-week-old male BALB/c nude mice were anesthetized and 5×106 U87 MG cells were injected subcutaneously in 200 μL PBS into the 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 10 mm and 5–7 mm in diameter after implantation (14–21 days), the male BALB/c nude mice were used for studies of biodistribution and PDT efficacy of TMPC.
Tumor Biol.
In vivo biodistribution and clearance of TMPC
Statistical analysis
The male BALB/c nude mice bearing U87 MG tumors were injected at a dose of 20 mg/kg in 0.2 mL solution via the lateral tail vein. Eight tissues and serum samples from tumor-bearing BALB/c nude mice were analyzed to investigate the biodistribution and clearance of TMPC at the indicated times after injection. These samples included the tumor, brain, liver, spleen, lung, kidney, muscle, skin, and serum samples. Mice were sacrificed under ether anesthesia, and tissue and serum samples were collected at 1, 3, 6, 12, and 24 h (five mice per time). Serum was separated from the whole blood by centrifugation at 2000g for 15 min after standing for 2 h at 37 °C. The samples were stored at −80 °C until analysis. Concentrations of TMPC in the tissue and serum samples were determined using fluorescence measurements. The wet weight of each tissue was recorded, and the tissue and serum samples were mixed with ultrapure water (100 mg wet tissue or 100 μL serum/500 μL ultrapure water). The tissue samples were homogenized, then all of the samples including serum samples were centrifuged at 12,000g for 5 min at 4 °C, and the supernatants were collected. Fluorescence in the supernatants was measured with a fluorescence spectrometer at 656 nm, and the excitation wavelength used for the measurement was 426 nm. Concentrations of TMPC in the tissue and fluid samples were calculated from standard concentration curves constructed by measuring the fluorescence of several different known concentrations of TMPC in ultrapure water solution. The mean concentration±standard error of the mean (SE) was calculated for each time point (n=5).
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.
In vivo PDT efficacy The male BALB/c nude mice bearing U87 MG tumors were injected at a dose of 5 mg/kg in 0.2 mL solution via the lateral tail vein. PDT was performed at 3 h following injection with laser light (650 nm, 80 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. There are numerous in vivo studies assessing effectiveness of new PSs. In this regard, research carried out by Hongyou Zhao et al. [11] In bearing mouse osteosarcoma S180 BALB/c nude mice with the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB) provides a valuable reference for benchmarking purposes. Experimental conditions of present work are quite similar to those of the photosensitizer mentioned above. This includes the drug dose and light dose.
Results UV-vis and fluorescence spectra The UV-vis absorption spectrum for TMPC was gathered in Fig. 1b. TMPC displayed an intense Soret band at 426 nm and Q band at 528, 553, 600, 653, and 743 nm, respectively. Molar absorption coefficients of TMPC are shown in Table 1. Fluorencese emission spectra of TMPC was observed and collected in Fig. 1c, d. In DMSO system, when excited at 426 nm, TMPC showed strong emission peak at 656 nm and weak fluorescence at 720 nm. Singlet oxygen generation ability The photosensitizer triplet transfers energy to groundstate triplet oxygen, which produces singlet oxygen. The generation ability of singlet oxygen gives a sign of the potential of the compound as photosensitizers in application. For TMPC, the absorption intensity of DPBF (λ=417 nm) continuously decreased as the irradiation time increasing (Fig. 2a). The rate of singlet oxygen generation is calculated by the following equation described by Wei Tang et al. [12]. ln ½DPBFt =½DPBF0 ¼ ‐kt Where [DPBF]t and [DPBF]0 are the concentrations of DPBF after and prior irradiation, respectively. Values of k
Table 1 Molar absorption coefficients of TMPC
Wavelength (nm)
Molar absorption coefficient ε (M−1 cm−1)
528 553 600 653 743
10,400 8200 5300 15,400 6500
Tumor Biol.
Fig. 1 Chemical structure and spectrum properties of TMPC in DMSO. a Chemical structure. b UV-vis absorption spectrum in DMSO. c Fluorescence emission spectrum in DMSO. Excitation wavelengths: 426 nm. d The matrix of excitation and emission spectra (Ex: 300–550 nm, Em: 600–780 nm)
Fig. 2 The singlet oxygen generation rate of TMPC. a UV-vis spectra for the determination of singlet oxygen generation rate of TMPC in DMF use DPBF as quencher. b First-order plot of DPBF absorbance versus time of TMPC
Tumor Biol.
are the rate of singlet oxygen generation and t is the time of irradiation. In Fig. 2b, high singlet oxygen generation rate is observed for TMPC (k=0.05).
significant uptake of TMPC was observed over 1–12 h of incubation then stabilizes.
MTT cell viability assay Cellular uptake of TMPC in U87 MG cells To investigate in vitro cell uptake behavior of TMPC in a tumor-like environment, we employed U87 MG Cells. The amount of TMPC that was taken up was determined by fluorescence spectrometry after cell lysis and expressed relative to the cellular protein amount. As shown in Fig. 3a, a rapid
Fig. 3 Cellular uptake and phototoxicity of TMPC in vitro. a Cellular uptake of TMPC by human glioma U87 MG cells. b Light dose- and concentration-effect curves for TMPC. U87 MG cells were treated at the indicated conditions. Cell viability was examined using MTT assay. c
The phototoxicity of TMPC toward U87 MG cells was determined after 24 h of incubation and subsequent illumination with laser (Fig. 3b). No noticeable reduction was observed up to the highest concentration used (16 μM) in the percentage of viable U87 MG cells in the dark. The viability of U87 cells that received the same concentration of TMPC was sharply decreased
Loss of clonogenicity in U87 MG cells treated with varying TMPC doses and 8 J/cm2 light dose. The error bars denote standard deviation from three replicates
Tumor Biol.
with the irradiation light dose increasing. And when exposed to the same radiation dose, the higher concentration TMPC significantly decreased the percentage of viable U87 MG cells. A light dose- and concentrationdependent response was observed in the percent viability of cells. The IC50 values, which represent the concentrations of TMPC that are required to kill 50 % of cells with different light doses, were calculated for each curve, and they are summarized in Table 2. Clonogenic assay Clonogenic assays were used to determine the long-term survival of U87 MG cells post-PDT using various TMPC doses with 8 J/cm2 light dose or alone. No noticeable reduction was observed up to the TMPC highest concentration (16 μM) used alone on the number of colonies (data not given). A >80 % reduction in clonogenic ability was observed in samples treated with 16 μM and 8 J/cm2 light when compared with light alone and untreated control samples (Fig. 3c), confirming the findings in the MTT assay experiment. In vivo biodistribution and clearance of TMPC The in vivo biodistribution and clearance of TMPC was evaluated. Figure 4 shows the distribution of TMPC in BALB/c nude mice tissues and serum for periods ranging from 1 to 24 h after intravenous injection of 20 mg/kg. The accumulation of TMPC in tumor tissue was less than that in the liver, skin, and serum at 1 h after injection, and then TMPC showed great tumor accumulation, which was more than in all other tissues except for liver and serum at 3 h after administration. In tumor tissue, the accumulation of TMPC was three times of that in skin at 3 h after injection, and the clearance of TMPC in other tissues and serum was superior to that in tumor tissue. At 12 h after administration, TMPC was slightly detected in all tissues. TMPC had been almost completely discharged from all tissues at 24 h after administration.
Fig. 4 In vivo biodistribution of TMPC in U87 MG tumor-bearing BALB/c mice. TMPC (20 mg/kg) were injected intravenously into U87 MG tumor-bearing BALB/c mice. Eight tissues and serum samples from tumor-bearing mice were analyzed at the indicated times after injection. Concentrations of TMPC in the tissue and serum samples were determined using fluorescence measurements. All data represent means± SE (n=5)
tumor xenografts model after irradiation at 650 nm for PDT. Figure 5 presented the representative digital photos of tumors at times of the 1st, 3rd, 5th, 7th, and 14th days after treatment. It showed that the control group and only laser irradiation group had no significant differences. The only laser irradiation had little influence on tumor growth. The PDT treatment group with an injection dose of 5 mg/kg TMPC and 80 J/cm 2 showed a very effective therapeutic effect. In this group, the tumor growth was significantly suppressed. One day after irradiation, there was a clear injury in the tumor sites, a scar formed, and then the scar of tumors fell off and normal healthy skin reconstructed 14 days post-irradiation. The volume growth curves of tumors were provided in Fig. 6.
Discussion PDT efficacy of TMPC in vivo The in vivo therapeutic efficacy of PDT using TMPC was evaluated by measuring tumor growth rates in a U87 MG Table 2 IC50 values of TMPC in U87 MG cells treated for 24 h and then exposed to increasing doses of red light (650 nm)
Malignant gliomas is the most common primary brain tumors and associated with a dismal prognosis and poor quality of life [1]. Chlorin derivatives are currently being developed as 2nd
IC50 (μM) Light dose (J/cm2) U87 MG cell line
1 8.39±0.13*
2 6.47±0.11*
Results represent mean±SD of three independent experiments *P
RESEARCH ARTICLE
Antitumor activity of photodynamic therapy with a chlorin derivative in vitro and in vivo Lai-Xing Wang 1 & Jian-Wei Li 2 & Jian-Yue Huang 2 & Jian-Hong Li 2 & Li-Jun Zhang 3 & Donal O’Shea 4 & Zhi-Long Chen 3
Received: 18 December 2014 / Accepted: 25 March 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Chlorin derivatives are promising photosensitive agents for photodynamic therapy (PDT) of tumors. The aim of the current study is to investigate the PDT therapeutic effects of a novel chlorin-based photosensitizer, mesotetra[3-(N,N-diethyl)aminomethyl-4-methoxy]phenyl chlorin (TMPC) for gliomas in vitro and in vivo. Physicochemical characteristics of TMPC were recorded by ultraviolet visible spectrophotometer and fluorescence spectrometer. The rate of singlet oxygen generation of TMPC upon photo-excitation was detected by using 1,3-diphenylisobenzofuran (DPBF). The accumulation of TMPC in gliomas U87 MG cells was measured by fluorescence spectrometer. The efficiency of TMPC-PDT in vitro was analyzed by MTT assay and clonogenic assay. The biodistribution and clearance of TMPC were determined by fluorescence measuring. Human gliomas U87 MG tumor-bearing mice model was used to evaluate the antitumor effects of TMPC-PDT. TMPC shows a singlet oxygen generation rate of 0.05 and displays a characteristic long wavelength absorption peak at 653 nm (ε=15, 400). The accumulation of TMPC increased with the increase of incubation time. In vitro, PDT using TMPC and laser showed laser dose- and concentration-dependent cytotoxicity
* Zhi-Long Chen [email protected] 1
Changhai Hospital, Shanghai 201620, People’s Republic of China
2
Yiwu City Central Hospital, Zhejiang 322000, People’s Republic of China
3
Department of Pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University, Shanghai 201620, People’s Republic of China
4
Center for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin, Ireland
to U87 MG cells. In U87 MG tumor-bearing mice, TMPCPDT significantly reduced the growth of the tumors. Both in vitro and in vivo, TMPC showed little dark toxicity. In vitro and in vivo studies, it found that TMPC has excellent antitumor activities. It suggests that TMPC is a potential photosensitizer of photodynamic therapy for cancer. Keywords Photodynamic therapy . Photosensitizer . Chlorin derivative . Antitumor
Introduction Glioblastomas are highly aggressive tumors characterized by a high recurrence and high mortality. The median survival time for patients with glioblastoma is less than 15 months [1]. Recently, photodynamic therapy (PDT) has been regarded as an effective treatment approach for some tumors, such as skin cancers, breast cancers, early lung cancers, and tumors of the head and neck [2]. PDT has three elements: a photosensitizer (PS), light, and oxygen. PDT is based on the light of a specific wavelength excitation of a photosensitizer which results in the formation of reactive oxygen species (ROS) including superoxide, hydroxyl radical, singlet oxygen, and hydrogen peroxide [3]. The produced ROS can directly kill tumor cells by the induction of necrosis and/or apoptosis, can cause destruction of tumor vasculature, and can produce an acute inflammatory response that attracts leukocytes like dendritic cells and neutrophils [4]. Many advances in PDT suggest that a class of compounds that meet most of the requirements for ideal PDT agents are chlorin derivatives [5–7]. For example, mTHPC (Foscan, Biolitec Pharma, Scotland, UK) has been approved in Europe for use against head and neck cancer, and additional indications have been filed for prostate and pancreatic tumors
Tumor Biol.
[5]. This class of molecules have Q band absorption maxima at wavelengths (λmax=650–670 nm) with a molar absorption coefficient in the range of 105/M/cm, and they often have high singlet oxygen quantum yield [8]. Some of them show limited skin photosensitivity and rapid clearance from tissues [9]. In the present paper, we report that TMPC, a chlorin derivative, has a strong absorption band at relatively long wavelength (λmax=653 nm) and shows low dark toxicity, significant cytotoxicity in the presence of light in vitro and in vivo. Its biodistribution and clearance also have been evaluated in U87 MG tumor-bearing mice model and revealed that TMPC can be cleared rapidly from tissues.
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 ultraviolet visible 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 path length of 1 cm. TMPC was dissolved in DMSO to get 5 μM of solution. Singlet oxygen generation detection The singlet oxygen ability of TMPC was monitored by chemical oxidation of DPBF in the DMF solution. TMPC (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 2 min at an absorbance maximum of 417 nm of DPBF. Cell culture U87 MG human glioma cells (U87 MG) 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.
Cellular uptake of TMPC in U87 MG cells The U87 MG cells (1×105cells/well) were seeded in 24-well cell culture plates. After 24 h, the time-dependent drug accumulation in the cells was investigated by exposing the cells to 8 μM TMPC in culture medium for various time intervals, from 1 up to 24 h. At the end of the incubation time, the cells were washed three times with PBS and solubilized in 300 μL/ well DMSO. The retention of cell-associated TMPC was measured by fluorescence using excitation/emission wavelengths of 426 and 656 nm. The calibration curve (fluorescence vs. concentration) was performed and the concentration of TMPC per cell (pM) was calculated. MTT cell viability assay U87 MG cells were plated at a density of 1×104 cells/well in 96-well microplates and incubated overnight at 37 °C until 70–80 % of confluence. Media containing TMPC in different concentrations were administered to cells and allowed to uptake for 24 h. Media containing TMPC were then removed, a complete fresh culture medium was added, and cells were exposed to 1 to 16 J/cm2 light doses with an Nd: YAG laser at 650 nm. In parallel, non-irradiated cells were used to study dark cytotoxicity. Following treatment, cells were incubated for an additional 24 h period and cell viability was determined by means of MTT reduction assay [10]. Clonogenic assay U87 MG cells were treated with TMPC in different concentrations for 24 h before irradiation with 8 J/cm2. Following PDT, cells were seeded in 6-well plates at densities of 1000 and 200 cells/well. Cells were allowed to form colonies for approximately 14 days, and then the cells were fixed in 75 % ethanol for 15 min at 4 °C. Fixing agent was removed and plates were allowed to air dry, before cells were stained with a 0.25 % solution of crystal violet. Animal and tumor models Five-week-old male BALB/c nude mice were anesthetized and 5×106 U87 MG cells were injected subcutaneously in 200 μL PBS into the 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 10 mm and 5–7 mm in diameter after implantation (14–21 days), the male BALB/c nude mice were used for studies of biodistribution and PDT efficacy of TMPC.
Tumor Biol.
In vivo biodistribution and clearance of TMPC
Statistical analysis
The male BALB/c nude mice bearing U87 MG tumors were injected at a dose of 20 mg/kg in 0.2 mL solution via the lateral tail vein. Eight tissues and serum samples from tumor-bearing BALB/c nude mice were analyzed to investigate the biodistribution and clearance of TMPC at the indicated times after injection. These samples included the tumor, brain, liver, spleen, lung, kidney, muscle, skin, and serum samples. Mice were sacrificed under ether anesthesia, and tissue and serum samples were collected at 1, 3, 6, 12, and 24 h (five mice per time). Serum was separated from the whole blood by centrifugation at 2000g for 15 min after standing for 2 h at 37 °C. The samples were stored at −80 °C until analysis. Concentrations of TMPC in the tissue and serum samples were determined using fluorescence measurements. The wet weight of each tissue was recorded, and the tissue and serum samples were mixed with ultrapure water (100 mg wet tissue or 100 μL serum/500 μL ultrapure water). The tissue samples were homogenized, then all of the samples including serum samples were centrifuged at 12,000g for 5 min at 4 °C, and the supernatants were collected. Fluorescence in the supernatants was measured with a fluorescence spectrometer at 656 nm, and the excitation wavelength used for the measurement was 426 nm. Concentrations of TMPC in the tissue and fluid samples were calculated from standard concentration curves constructed by measuring the fluorescence of several different known concentrations of TMPC in ultrapure water solution. The mean concentration±standard error of the mean (SE) was calculated for each time point (n=5).
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.
In vivo PDT efficacy The male BALB/c nude mice bearing U87 MG tumors were injected at a dose of 5 mg/kg in 0.2 mL solution via the lateral tail vein. PDT was performed at 3 h following injection with laser light (650 nm, 80 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. There are numerous in vivo studies assessing effectiveness of new PSs. In this regard, research carried out by Hongyou Zhao et al. [11] In bearing mouse osteosarcoma S180 BALB/c nude mice with the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB) provides a valuable reference for benchmarking purposes. Experimental conditions of present work are quite similar to those of the photosensitizer mentioned above. This includes the drug dose and light dose.
Results UV-vis and fluorescence spectra The UV-vis absorption spectrum for TMPC was gathered in Fig. 1b. TMPC displayed an intense Soret band at 426 nm and Q band at 528, 553, 600, 653, and 743 nm, respectively. Molar absorption coefficients of TMPC are shown in Table 1. Fluorencese emission spectra of TMPC was observed and collected in Fig. 1c, d. In DMSO system, when excited at 426 nm, TMPC showed strong emission peak at 656 nm and weak fluorescence at 720 nm. Singlet oxygen generation ability The photosensitizer triplet transfers energy to groundstate triplet oxygen, which produces singlet oxygen. The generation ability of singlet oxygen gives a sign of the potential of the compound as photosensitizers in application. For TMPC, the absorption intensity of DPBF (λ=417 nm) continuously decreased as the irradiation time increasing (Fig. 2a). The rate of singlet oxygen generation is calculated by the following equation described by Wei Tang et al. [12]. ln ½DPBFt =½DPBF0 ¼ ‐kt Where [DPBF]t and [DPBF]0 are the concentrations of DPBF after and prior irradiation, respectively. Values of k
Table 1 Molar absorption coefficients of TMPC
Wavelength (nm)
Molar absorption coefficient ε (M−1 cm−1)
528 553 600 653 743
10,400 8200 5300 15,400 6500
Tumor Biol.
Fig. 1 Chemical structure and spectrum properties of TMPC in DMSO. a Chemical structure. b UV-vis absorption spectrum in DMSO. c Fluorescence emission spectrum in DMSO. Excitation wavelengths: 426 nm. d The matrix of excitation and emission spectra (Ex: 300–550 nm, Em: 600–780 nm)
Fig. 2 The singlet oxygen generation rate of TMPC. a UV-vis spectra for the determination of singlet oxygen generation rate of TMPC in DMF use DPBF as quencher. b First-order plot of DPBF absorbance versus time of TMPC
Tumor Biol.
are the rate of singlet oxygen generation and t is the time of irradiation. In Fig. 2b, high singlet oxygen generation rate is observed for TMPC (k=0.05).
significant uptake of TMPC was observed over 1–12 h of incubation then stabilizes.
MTT cell viability assay Cellular uptake of TMPC in U87 MG cells To investigate in vitro cell uptake behavior of TMPC in a tumor-like environment, we employed U87 MG Cells. The amount of TMPC that was taken up was determined by fluorescence spectrometry after cell lysis and expressed relative to the cellular protein amount. As shown in Fig. 3a, a rapid
Fig. 3 Cellular uptake and phototoxicity of TMPC in vitro. a Cellular uptake of TMPC by human glioma U87 MG cells. b Light dose- and concentration-effect curves for TMPC. U87 MG cells were treated at the indicated conditions. Cell viability was examined using MTT assay. c
The phototoxicity of TMPC toward U87 MG cells was determined after 24 h of incubation and subsequent illumination with laser (Fig. 3b). No noticeable reduction was observed up to the highest concentration used (16 μM) in the percentage of viable U87 MG cells in the dark. The viability of U87 cells that received the same concentration of TMPC was sharply decreased
Loss of clonogenicity in U87 MG cells treated with varying TMPC doses and 8 J/cm2 light dose. The error bars denote standard deviation from three replicates
Tumor Biol.
with the irradiation light dose increasing. And when exposed to the same radiation dose, the higher concentration TMPC significantly decreased the percentage of viable U87 MG cells. A light dose- and concentrationdependent response was observed in the percent viability of cells. The IC50 values, which represent the concentrations of TMPC that are required to kill 50 % of cells with different light doses, were calculated for each curve, and they are summarized in Table 2. Clonogenic assay Clonogenic assays were used to determine the long-term survival of U87 MG cells post-PDT using various TMPC doses with 8 J/cm2 light dose or alone. No noticeable reduction was observed up to the TMPC highest concentration (16 μM) used alone on the number of colonies (data not given). A >80 % reduction in clonogenic ability was observed in samples treated with 16 μM and 8 J/cm2 light when compared with light alone and untreated control samples (Fig. 3c), confirming the findings in the MTT assay experiment. In vivo biodistribution and clearance of TMPC The in vivo biodistribution and clearance of TMPC was evaluated. Figure 4 shows the distribution of TMPC in BALB/c nude mice tissues and serum for periods ranging from 1 to 24 h after intravenous injection of 20 mg/kg. The accumulation of TMPC in tumor tissue was less than that in the liver, skin, and serum at 1 h after injection, and then TMPC showed great tumor accumulation, which was more than in all other tissues except for liver and serum at 3 h after administration. In tumor tissue, the accumulation of TMPC was three times of that in skin at 3 h after injection, and the clearance of TMPC in other tissues and serum was superior to that in tumor tissue. At 12 h after administration, TMPC was slightly detected in all tissues. TMPC had been almost completely discharged from all tissues at 24 h after administration.
Fig. 4 In vivo biodistribution of TMPC in U87 MG tumor-bearing BALB/c mice. TMPC (20 mg/kg) were injected intravenously into U87 MG tumor-bearing BALB/c mice. Eight tissues and serum samples from tumor-bearing mice were analyzed at the indicated times after injection. Concentrations of TMPC in the tissue and serum samples were determined using fluorescence measurements. All data represent means± SE (n=5)
tumor xenografts model after irradiation at 650 nm for PDT. Figure 5 presented the representative digital photos of tumors at times of the 1st, 3rd, 5th, 7th, and 14th days after treatment. It showed that the control group and only laser irradiation group had no significant differences. The only laser irradiation had little influence on tumor growth. The PDT treatment group with an injection dose of 5 mg/kg TMPC and 80 J/cm 2 showed a very effective therapeutic effect. In this group, the tumor growth was significantly suppressed. One day after irradiation, there was a clear injury in the tumor sites, a scar formed, and then the scar of tumors fell off and normal healthy skin reconstructed 14 days post-irradiation. The volume growth curves of tumors were provided in Fig. 6.
Discussion PDT efficacy of TMPC in vivo The in vivo therapeutic efficacy of PDT using TMPC was evaluated by measuring tumor growth rates in a U87 MG Table 2 IC50 values of TMPC in U87 MG cells treated for 24 h and then exposed to increasing doses of red light (650 nm)
Malignant gliomas is the most common primary brain tumors and associated with a dismal prognosis and poor quality of life [1]. Chlorin derivatives are currently being developed as 2nd
IC50 (μM) Light dose (J/cm2) U87 MG cell line
1 8.39±0.13*
2 6.47±0.11*
Results represent mean±SD of three independent experiments *P
