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In vitro and in vivo antitumor activity of a novel hypocrellin B derivative for photodynamic therapy Hongyou Zhao a,1, Rong Yin b,1, Defu Chen c, Jie Ren a, Yucheng Wang d, Jiaying Zhanga a, Hong Deng e, Ying Wang a, Haixia Qiu a, Naiyan Huang a, Qianli Zou f, Jingquan Zhao e, Ying Gu MD a,∗ a
Department of Laser Medicine, Chinese PLA General Hospital, Beijing 100853, PR China Department of Dermatology, The Second Hospital, Shanxi Medical University, Taiyuan 030001, PR China c School of Information and Electronics, Beijing Institute of Technology, Beijing 100081, PR China d College of Medicine, Nankai University, Tianjin 300071, PR China e Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, PR China f Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China b
Received 17 December 2013; received in revised form 29 January 2014; accepted 31 January 2014
KEYWORDS PDT; Hypocrellin B derivative; Mitochondria; Apoptosis; In vivo PDT activity
∗ 1
Summary Background: Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by irradiating photosensitizers with light exposure to produce reactive oxygen species (ROS). An ideal photosensitizer is a crucial element to PDT. In the current study, we evaluated the photodynamic activity of a novel photosensitizer, the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB), both in vitro and in vivo. Methods: Physicochemical characteristics of the novel photosensitizer were compared with that of its parent HB. The intracellular distribution of photosensitizers and mitochondrial membrane potential were detected with laser scanning confocal microscopy. The pathway of cell death was analyzed by flow cytometry. The release of proapoptotic proteins was evaluated by Western blot. S180 tumor model was used to evaluate the antitumor effects of PENHB-mediated PDT. Results: Compared with its parent HB, water solubility of the derivative was improved enormously (6.6 mg/ml vs. 4.6 g/ml), rendering its intravenous injection feasible without auxiliary solvent. The derivative had better PDT effect than HB in vitro under similar dark cytotoxicity.
Corresponding author. Tel.: +86 010 6693 9394; fax: +86 010 6822 2584. E-mail address: [email protected] (Y. Gu). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.pdpdt.2014.01.003 1572-1000/© 2014 Elsevier B.V. All rights reserved.
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H. Zhao et al. Moreover, PENSHB-mediated PDT was able to induce mitochondrial inner membrane permeabilisation, cytochrome c release, caspase-3 activation and subsequent apoptotic death. In vivo study showed that more than half of tumor bearing mice were cured by PENSHB-mediated PDT. Conclusions: In vitro and in vivo studies suggest that PENSHB is an effective photosensitizer for PDT to tumors. Therefore, PENSHB as a novel photosensitizer has a good prospect of clinical application. © 2014 Elsevier B.V. All rights reserved.
Introduction Photodynamic therapy (PDT) is now well established as a clinical treatment modality for various diseases, particularly for tumors [1,2]. Cell death is induced by photoexcitation of a sensitizer, generally through production of reactive oxygen species (ROS). The first photosensitizer clinically employed for cancer therapy is a water-soluble mixture of porphyrins called hematoporphyrin derivative (HPD), of which there is a purified form, porfimer sodium, later known as photofrin. Currently, most of the photosensitizers used in cancer therapy are based on a tetrapyrrole structure, similar to that of the protoporphyrin contained in hemoglobin [3,4]. Unfortunately, many properties of the currently used photosensitizers such as inefficient delivery, poor water solubility, low selective accumulation and low absorbance in the optical window still hamper the efficacy of cancer treatments [5—7]. Besides tetrapyrrole structures, a lot of naturally occurring and synthetic dyes that are non-porphyrin have also been evaluated for their photosensitizing ability against cancer, such as methylene blue, nileblue, nile red analogs and the chalcogenopyrylium class of photosensitizers. These compounds, however, suffer from a major drawback due to their inherent dark cytotoxicity [8,9]. One alternative class of non-porphyrin photosensitizers being studied is the hypocrellins, including hypocrellin A (HA) and hypocrellin B (HB), which are extracted originally from wild Hypocrellabambusae. In the past decade, many hypocrellin derivatives were synthesized and analyzed [10—13]. However, low water solubility and low absorbance in the optical window are also their shortcomings for PDT of solid tumors [14,15]. It was found that any improvement in the aqueous solubility would unexceptionally lead to decrease in PDT activity [15,16]. In addition to the physicochemical properties of photosensitizers, the location of photosensitizers in cells is another key factor for PDT effect. Many reports have shown mitochondria as important targets of PDT. Photosensitizers on mitochondria are reported to be more efficient in killing cells than those at other cellular sites [17,18]. In the current study, we evaluated the photodynamic activity of a novel photosensitizer, the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB), both in vitro and in vivo (Fig. 1A). The preparation procedure for the derivative has been reported in our previous study (Compound 4) [19]. The current work showed that the PENSHB not only got a better water solubility but also an advanced PDT effect. We further investigated the mechanism involved in cell death induced
by PENSHB-PDT. The derivative can selectively locate on the mitochondria in cytoplasm, the phototoxicity mediated by the novel photosensitizer capable of inducing mitochondrial depolarization resulting in cell apoptosis. On the basis of the relative PDT activity, aqueous solubility, and clinically required concentration, it was estimated that PENSHB could be directly deliverable via intravenous injection without assistance of any drug-delivery vehicle. Irradiation of a 630 nm laser after intravenous administration of PENSHB cured more than half of tumor bearing mice. To the best of our knowledge, this is the first time the derivative of HB has been used through systemic administration to achieve an efficient in vivo PDT effect for cancer treatment.
Materials and methods Cell culture Human Gastric Adenocarcinoma Cells (BGC-823) were obtained from the Department of Tumor, Chinese PLA General Hospital. Cells were cultured in DMEM supplemented with 15% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) in 5% CO2 at 37 ◦ C in a humidified incubator.
Reagents The procedure for the preparation of hypocrellin B (HB) and PENSHB was reported in our previous study [19]. The following fluorophore probes were used: Mito-Tracker Green (200 nM, Invitrogen Life Technologies, Inc.) to label mitochondria, tetramethylrhodamine methyl esters (TMRM, 200 nM, Invitrogen Life Technologies, Inc.) to indicate mitochondrial transmembrane potential (m). 5Aminolevulinic acid (Sigma—Aldrich) to compare the PDT efficacy of PENSHB with that of current clinically used PDT agents.
The biophysical properties of HB and PENSHB The detection of biophysical properties of photosensitizers was performed as previously described [19]. The solutions were measured for the absorption spectra on a Shimadzu UV1601 spectrophotometer. Steady-state absorption spectra were recorded on a Shimadzu UV-1601 spectrophotometer. Lipid/water partition coefficient was calculated by the ratio of photosensitizer concentration in L-octanol to that in PBS
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intensities were processed with physiology software (Zeiss Rel3.2, Jena, Germany).
Assessment of cell survival The cells in logarithmic phase were detached by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA)-PBS and diluted to a density of 5 × 104 cells/ml in the presence of DMEM supplemented with 5% FBS. The cell suspension was seeded in wells (100 l each) of 96-well microplates and incubated under 5% CO2 at 37 ◦ C for 24 h. One column of wells did not receive cells to serve as the blank. Then the cells were transferred to DMEM medium containing photosensitizers of various concentrations and incubated at 37 ◦ C in dark for 4 h. After removal of the above medium containing photosensitizers and addition of fresh DMEM medium, the cells were irradiated with a 630 nm He—Ne laser of 10 mW/cm2 for 600 s and then incubated in dark for 24 h before survival assessment. Meanwhile, dark toxicity was measured under the same conditions without irradiation in parallel. The cell survival was estimated by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 24 h after irradiation [21]. Briefly, after 20 l of MTT (5 mg/ml) was added to each well, the cells were further incubated for 120 min. MTT solution was removed, and 200 l of DMSO was added to dissolve the formazan crystals. Then the plates were shaken at room temperature for 10 min, and the optical density at 570 nm was read on a microplate reader, from which cell survivals were derived. Figure 1 Physicochemical characteristics of the novel photosensitizer. (A) Structures of hypocrellin B (HB) and 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB). (B) The comparison of two photosensitizers in physicochemical properties. (C) Absorption spectra of HB and PENSHB at the concentration of 50 M in dimethyl sulfoxide (DMSO).
18 h after PDT treatment, the percentage of apoptotic and necrotic cells was analyzed by a FACScanto flow cytometer (Becton Dickinson, Mountain View, CA). Fluorescent emissions of FITC and DNA-PI complexes were measured at 515—545 nm and at 564—606 nm, respectively.
solution. The DPA-bleaching method was used to determine the quantum yields of 1 O2 according to Ref. [20].
Mitochondrial isolation and Western blotting analysis
Imaging analysis of living cells
For mitochondria isolation, cells were harvested and then fractionated using Cytosol/Mitochondria Fractionation Kit (Merch, Germany) according to the supplier’s recommendations. The cytosol extraction was subjected to Western blotting analysis of cytochrome c. The purity of fractions was tested by immunoblotting with antibodies specific for the cytosolic proteins -actin. The antibodies used for Western blotting include antibodies against -actin and cytochrome c (Cell Signaling Technology, Danvers, MA).
Fluorescent emission from Mito-Tracker Green was monitored by a commercial laser scanning microscope (LSM 510 Meta) combination system (Carl Zeiss, Jena, Germany) equipped with a Plan-Neofluar 40×/1.3 numerical aperture (NA) oil differential interference contrast (DIC) objective. Excitation wavelength and detection filter settings for each of the fluorescent indicators were as follows: photosensitizer’s fluorescence was excited at 488 nm with an Ar-ion laser, and emitted light was recorded through a 600—650 nm band-pass filter. Mito-Tracker Green fluorescence was excited at 488 nm with a He—Ne laser, and the emitted signal was recorded through a 500—550 nm band pass filter. TMRM was excited using the He—Ne laser. The excitation wavelength was 543 nm, and the emission detection filter was band pass 565—615 IR. For intracellular measurements, a desired measurement position was chosen in the LSM image. To quantify the results, the emission
Annexin flow cytometry
In vivo photodynamic therapy Mouse osteosarcoma S180 cells, purchased from Fourth Military Medical University Experimental Centre were maintained in RPMI-1640 culture media under 5% CO2 at 37 ◦ C. Cells were detached from the substrate using 0.5% trypsin in PBS, centrifuged, and then resuspended in PBS at a concentration of 5 × 106 cells/ml. Female BALB/c mice (Center of Experimental Animal, PLA General Hospital, Beijing, China)
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were housed in an environmentally controlled animal facility with regular 12/12 cycle. Hair on the upper backs of the mice was removed with a chemical depilatory agent (Na2 S 8% aqua-solution), then each mouse was injected with a 100 l cell suspension solution in the sub-dermal dorsal area [22]. The mice were treated with PDT when the tumor diameter was 8—9 mm. Photosensitizer (5 mg/kg) was administered via tail vein respectively at 1, 3 and 6 h prior to the laser irradiation (630 nm, 100 mW/cm2 × 1200 s). The irradiation light source was a semiconductor laser (Shenzhen Laser Medical Tech Co., Ltd., China). The laser output was coupled into an optical fiber (Medlight, S.A. Switzerland) with a lens for an even 3 cm2 irradiation area that covered the tumors. Mice were randomized into three groups: control, laser only and PDT. After the treatment, the tumor dimensions were measured with a caliper every 2 days. The tumor volume was determined using the formula for the volume of an ellipsoid (tumor volume = diameter × width2 × 3.14/6) [23].
Statistics analysis All assays were performed a minimum of three times. Data were presented as mean ± standard errors of the mean (SEM). Statistical analysis was performed with Student’s paired t-test. Differences were considered statistically significant at p < 0.05.
Results Physicochemical characteristics Singlet oxygen quantum yield (˚ ) is one of the most important parameters indicating efficacy of the potential photosensitizers for PDT [24]. As shown in Fig. 1B, ˚ of PENSHB was higher than that of HB, suggesting the derivative had the primary property to be a photosensitizer. Moreover, the solubility of the novel derivative in PBS was much higher than that of HB (6.6 mg/ml vs. 4.6 g/ml). Compared with HB, PENSHB with required lipid/water partition coefficient (PC) value was appropriate candidates for intravenous administration and cellular uptake. The absorption spectra of HB and PENSHB are shown in Fig. 1C. PENSHB had greatly strengthened absorption over a range of 600—700 nm as compared with HB.
The damage of mitochondria induced by PENSHB-PDT To validate the intracellular distribution of the novel photosensitizer in tumor cells, BGC-823 cells were loaded with HB or PENSHB in complete medium for 4 h. Photosensitizers’ fluorescence was imaged using a laser scanning confocal microscopy. The results showed that the photosensitizers were distributed in cytoplasm, but not in cell nuclei (Fig. 2A and B). To assess whether the new photosensitizer bound to the mitochondria, cells were co-loaded with Mito-Tracker, a mitochondria-specific dye. Our results revealed that the derivative was mainly located at mitochondria in cells (Fig. 2B). So the derivative could be used as a mitochondriatargeting photosensitizer. To investigate the direct impact
on mitochondria, the change of mitochondrial membrane potential (m) was detected in living cells by confocal microscopy after PDT treatment. The bright spheres disappeared, indicating mitochondrial depolarization (Fig. 2C). The results suggested that the rate of m descent in the cells treated with PENSHB-PDT was faster than that in cells treated with HB-PDT (Fig. 2D).
In vitro PDT activity In order to evaluate the PDT activity of the two photosensitizers, their dark toxicity on BGC-823 cells were previously tested. Fig. 3A shows the survival rates of cells under various concentrations of HB, and PENSHB. The results implicated that the dark toxicity of two photosensitizers was not statistically significantly different. Subsequently, BGC-823 cells were incubated with HB and PENSHB in varying concentrations for 4 h, and then irradiated by 20 J/cm2 of 630 nm laser. Fig. 3A shows that the concentrations previously used (0—60 nM) had no dark toxicity to the cells. The result suggested that the PDT activity of the novel photosensitizer was much higher compared with parent HB. This is in concordance with the rate of m descent (Fig. 2D) after PDT treatment with the same dose. It can be explained by two reasons, first, PENSHB presents a stronger red-light absorption in the phototherapeutic window (Fig. 1C); second, mitochondria are the most sensitive targets for PDT and PENSHB preferentially binding to them (Fig. 2B). In order to compare the PDT efficacy of PENSHB with that of other current clinically used PDT agents, the PDT activity of the 5-aminolevulinic acid (5-ALA) was tested (Fig. 3B). Fig. 3 shows the survival curve of BGC-823 cells under a series of concentrations of PENSHB or 5-ALA irradiated by 20 J/cm2 of 630 nm laser, from which IC50 values derived are 29.6 M and 2910 M for PENSHB and 5-ALA, respectively. This result suggests that the PDT efficacy of PENSHB is much greater than that of 5-ALA.
Cell death pathway induced by PENSHB-PDT PDT can stimulate three main cell death pathways: apoptosis, necrosis, and autophagy-associated cell death. To detect the pathway of cell death involved in the PENSHBPDT process, the percentage of apoptotic and necrotic cells after PDT treatment was analyzed by FACS (Fig. 4A). The result suggested that apoptosis was a generally major cell death modality in cells responding to PDT with the derivate (Fig. 4B). Cytochrome c is a pivotal effector during apoptosis, the release of cytochrome c is an important process in PDT-induced apoptotic death. Our result showed that the quantity of released cytochrome c and the activation of the caspase-3 in cells treated by PENSHB-PDT were higher than in cells treated by HB-PDT. However, there was a significant reduce of cyto-c release and caspase-3 activation after adding ROS scavenger NAC to the culture medium of cells treated by PENSHB-PDT (Fig. 4C). This result proved that PENSHB-mediated PDT could activate the mitochondrial pathway of apoptosis by inducing intracellular ROS.
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Figure 2 The damage of mitochondria induced by PENSHB-PDT. The intracellular distribution of (A) HB and (B) PENSHB. BGC-823 cells were loaded with 10 M photosensitizers and 100 nM Mito-Tracker Green. Mito-Tracker Green fluorescence (left panel), photosensitizer fluorescence (middle panel) and overlay fluorescence (right panel) were visualized by confocal microscopy. Bar = 10 m. (C) Time sequence of m disappearance under PDT treatment. TMRM (red emission) was localized in mitochondria in cells in response to the m. Cells with no treatment were set as control. Bar = 20 m. (D) The fluorescence intensity of TMRM (n = 5) with the standard deviations as the error bars in BGC-823 cells.
Figure 3 In vitro PDT activity. Viability of BGC-823 cells with or without irradiation (630 nm laser, 10 mW/cm2 , 600 s) in the presence of photosensitizers ((A) PENSHB or HB and (B) 5-ALA). The error bars denote standard deviation from three replicates.
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Figure 4 Cell death pathway induced by PENSHB-PDT. (A) Cell death 18 h after PDT treatment. BGC-823 cells were stained with FITC-conjugated Annexin V and propidium iodide (PI). (B) Quantified analysis of apoptotic and necrotic cell percentage by FACS (photosensitizers concentration, 30 nM, irradiation, 630 nm laser, 10 mW/cm2 , 600 s). All data are representative of three independent experiments. (C) Immunoblot analysis of proapoptotic proteins (cytochrome c and cleaved caspase-3).
In vivo PDT activity
Discussion
Based on the in vitro results, it was expected that the derivative possessed in vivo PDT activity. To evaluate the therapeutic potential in vivo, a S180 tumor model was used to evaluate the antitumor effects of PENSHB-mediated PDT. PENSHB was injected into tumor bearing mice via tail vein. Thereafter, tumor sites of all mice were irradiated by the 630 nm laser for 20 min. Two days after irradiation, there was a clear injury in the tumor sites of mice treated with PDT, implying effective antitumor therapy (Fig. 5A). From the only laser treated mice, we found the tumor was not damaged after irradiation. Fig. 5B shows the tumor growth rate curve of different treatment groups. For survival studies, 7 mice were used per treatment group and the mice were monitored for 100 days after tumor inoculation. Although the tumors of 3 mice relapsed at 6 days after treatment, 4 of the 7 mice were alive with complete tumor regression in the PDT group. There were no long-term survivors in the laser-only group (Fig. 5C).
In PDT process, photosensitizers are administered via vein and transported to tumor tissues through blood circulating system, and enter tumor cells and intercellular regions through the lipid membranes of blood vessels and cells, which requires water- and lipid-soluble photosensitizers. Thus a moderate lipid/water PC is important for photosensitizer [25]. Considering commonly usable volume of physiological saline for intravenous injection is no more than 20—30 ml, drug concentration should be higher than 2 mg/ml [26]. Our results showed that PENSHB not only had a high solubility but also a moderate lipid/water PC (Fig. 1B). Therefore, PENSHB is easy to be prepared as a clinical acceptable aqueous solution. One disadvantage of the currently approved PDT photosensitizers is their low absorbance in the optical window for photosensitizer excitation. In the visible spectral regions below 600 nm, light penetration into the skin is only a few millimeters which reduce the efficiency of PDT [27]. The novel photosensitizer
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Figure 5 Antitumor effects of PENSHB mediated PDT. (A) Images of tumor-bearing mice treated with PENSHB-PDT or laser irradiation, Mice with no treatment were set as control. (B) Volumetric change in tumor sizes of different treatment groups. n = 7 per group. *indicates difference at the p < 0.001 significance level. Error bars represent standard errors of the mean (SEM). (C) Survival rates of tumor-bearing mice of three groups. n = 7 per group.
with >600 nm excitation wavelengths allows access into deeper biological tissues for effective treatment of abnormal tissues of larger volume (Fig. 1C). The intracellular distribution of photosensitizers is a very important feature, which is used to evaluate their safety and more importantly has a great effect on their PDT cytotoxicity. Fig. 2A and B shows that the photosensitizers were distributed in cytoplasm, but not in cell nuclei, suggesting the novel photosensitizer did not affect reproduction of DNA or proliferation of cell. Many reports have implicated mitochondria as important targets of PDT. Photosensitizers at mitochondria are reported to be more efficient in killing cells than those at other cellular sites [17]. Our results showed that PENSHB was largely located at mitochondria in cytoplasm (Fig. 2B). Mitochondrial m is an essential parameter for mitochondrial. The descent of m is a consequence of the opening of permeability transition (PT) pores in the mitochondrial [28]. As a consequence of
PT pore opening, mitochondria uncouple and undergo large amplitude swelling. The outer membrane ruptures during swelling to induce the release of mitochondrial inter membrane proteins, including cytochrome c and other apoptosis inducing factors [29]. In the present study, a change of mitochondrial membrane potential (m) was detected in living cells after PDT treatment (Fig. 2D). The result indicated that the novel photosensitizer-mediated PDT was able to destroy the function of mitochondria and result in cell apoptosis consequently. PDT can evoke the three main cell death pathways: apoptosis, necrosis, and autophagy-associated cell death [1]. Apoptosis is a form of programmed cell death characterized by distinct morphological changes including cell rounding, membrane blebbing, cytoskeletal disassembly and DNA fragmentation [29]. The apoptotic activity limits leakage of intracellular material to the immediate environment, and thereby prevents tissue inflammation [30]. In contrast,
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necrosis is morphologically characterized by vacuolization of the cytoplasm and swelling, and breakdown of the plasma membrane, resulting in an inflammatory reaction due to the release of cellular contents and proinflammatory molecules [31]. Autophagy is characterized by a massive vacuolization of the cytoplasm and the formation of autophagosome. This is a lysosomal pathway for the degradation and recycling of intracellular proteins and organelles. Recent studies suggest that autophagy is a mechanism to preserve cell viability after PDT [32]. Fig. 4C and D shows that apoptosis is the major death pathway after PENSH-PDT treatment. S180 is type of very malignant tumor cell line. Even though a small number of viable cells remain after treatment, they are always expanding and proliferating, which makes it difficult to cure. In previous studies [27,33], no complete tumor cure was obtained due to the shorter absorption wavelength of HB, thus facilitating regrowth of tumors (>5 mm) treated with PDT. In the current study, a high cure percentage was obtained after PDT at 1 h druglight interval to the tumors of 8—9 mm (Fig. 5C). It has been reported that direct antitumor effects of PDT derive from two aspects: cytotoxic effects on tumor cells and damage to the tumor vasculature [1]. Previous study suggested that extending the drug-light interval increases the contribution of direct tumor toxicity and reduce the contribution of damage to the tumor vasculature [34]. In this study, we found PDT six hours after drug administration led to tumor regrowth delay rather than cure (data not shown). This result suggested that vascular damage seems to contribute significantly to the overall PDT effect. In this work, the biological properties of the novel derivative of HB, PENSHB, were studied. Our results showed that it was readily dissolved in an aqueous solvent (water, PBS and physiological saline) in a clinically acceptable concentration. Furthermore, apoptosis is generally a major cell death modality in cells responding to the novel derivative-mediated PDT, because its preferential location at mitochondria in cytoplasm. More importantly, PENSHB can be directly used for intravenous injection without further drug preparation. These characteristics suggested PENSHB was suitable for PDT applied in vascular diseases and solid tumors. Our in vivo study showed that more than half of the tumor bearing mice were cured by PENSHB-mediated PDT. Taken altogether, based on the evidence of our in vitro and in vivo studies, we therefore propose that PENSHB is an effective photosensitizer for PDT in tumor therapy and has a promising prospect of clinical application.
Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 60878055; 61036014; 31170963), Beijing Municipal Natural Science Foundation (No. 4122090) and the National 863 Research Project of China (No. 2008AA030117).
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[29] Liu X, Kim CN, Yang J, et al. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 1996;86:147—57. [30] Evan G, Littlewood T. A matter of life and cell death. Science 1998;281:1317—22. [31] Kroemer G, Reed JC. Mitochondrial control of cell death. Natural Medicines 2000;6:513—9. [32] Reiners Jr JJ, Agostinis P, Berg K, Oleinick NL, Kessel D. Assessing autophagy in the context of photodynamic therapy. Autophagy 2010;6:7—18. [33] Chen B, Xu Y, Roskams T. Efficacy of antitumoral photodynamic therapy with hypericin relationship between biodistribution and photodynamic effects in the RIF-1 mouse tumor model. International Journal of Oncology 2001;93:275—82. [34] Stewart F, Baas P, Star W. What does photodynamic therapy have to offer radiation oncologists (or their cancer patients)? Radiotherapy and Oncology 1998;48:233—48.
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Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx
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In vitro and in vivo antitumor activity of a novel hypocrellin B derivative for photodynamic therapy Hongyou Zhao a,1, Rong Yin b,1, Defu Chen c, Jie Ren a, Yucheng Wang d, Jiaying Zhanga a, Hong Deng e, Ying Wang a, Haixia Qiu a, Naiyan Huang a, Qianli Zou f, Jingquan Zhao e, Ying Gu MD a,∗ a
Department of Laser Medicine, Chinese PLA General Hospital, Beijing 100853, PR China Department of Dermatology, The Second Hospital, Shanxi Medical University, Taiyuan 030001, PR China c School of Information and Electronics, Beijing Institute of Technology, Beijing 100081, PR China d College of Medicine, Nankai University, Tianjin 300071, PR China e Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, PR China f Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China b
Received 17 December 2013; received in revised form 29 January 2014; accepted 31 January 2014
KEYWORDS PDT; Hypocrellin B derivative; Mitochondria; Apoptosis; In vivo PDT activity
∗ 1
Summary Background: Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by irradiating photosensitizers with light exposure to produce reactive oxygen species (ROS). An ideal photosensitizer is a crucial element to PDT. In the current study, we evaluated the photodynamic activity of a novel photosensitizer, the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB), both in vitro and in vivo. Methods: Physicochemical characteristics of the novel photosensitizer were compared with that of its parent HB. The intracellular distribution of photosensitizers and mitochondrial membrane potential were detected with laser scanning confocal microscopy. The pathway of cell death was analyzed by flow cytometry. The release of proapoptotic proteins was evaluated by Western blot. S180 tumor model was used to evaluate the antitumor effects of PENHB-mediated PDT. Results: Compared with its parent HB, water solubility of the derivative was improved enormously (6.6 mg/ml vs. 4.6 g/ml), rendering its intravenous injection feasible without auxiliary solvent. The derivative had better PDT effect than HB in vitro under similar dark cytotoxicity.
Corresponding author. Tel.: +86 010 6693 9394; fax: +86 010 6822 2584. E-mail address: [email protected] (Y. Gu). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.pdpdt.2014.01.003 1572-1000/© 2014 Elsevier B.V. All rights reserved.
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H. Zhao et al. Moreover, PENSHB-mediated PDT was able to induce mitochondrial inner membrane permeabilisation, cytochrome c release, caspase-3 activation and subsequent apoptotic death. In vivo study showed that more than half of tumor bearing mice were cured by PENSHB-mediated PDT. Conclusions: In vitro and in vivo studies suggest that PENSHB is an effective photosensitizer for PDT to tumors. Therefore, PENSHB as a novel photosensitizer has a good prospect of clinical application. © 2014 Elsevier B.V. All rights reserved.
Introduction Photodynamic therapy (PDT) is now well established as a clinical treatment modality for various diseases, particularly for tumors [1,2]. Cell death is induced by photoexcitation of a sensitizer, generally through production of reactive oxygen species (ROS). The first photosensitizer clinically employed for cancer therapy is a water-soluble mixture of porphyrins called hematoporphyrin derivative (HPD), of which there is a purified form, porfimer sodium, later known as photofrin. Currently, most of the photosensitizers used in cancer therapy are based on a tetrapyrrole structure, similar to that of the protoporphyrin contained in hemoglobin [3,4]. Unfortunately, many properties of the currently used photosensitizers such as inefficient delivery, poor water solubility, low selective accumulation and low absorbance in the optical window still hamper the efficacy of cancer treatments [5—7]. Besides tetrapyrrole structures, a lot of naturally occurring and synthetic dyes that are non-porphyrin have also been evaluated for their photosensitizing ability against cancer, such as methylene blue, nileblue, nile red analogs and the chalcogenopyrylium class of photosensitizers. These compounds, however, suffer from a major drawback due to their inherent dark cytotoxicity [8,9]. One alternative class of non-porphyrin photosensitizers being studied is the hypocrellins, including hypocrellin A (HA) and hypocrellin B (HB), which are extracted originally from wild Hypocrellabambusae. In the past decade, many hypocrellin derivatives were synthesized and analyzed [10—13]. However, low water solubility and low absorbance in the optical window are also their shortcomings for PDT of solid tumors [14,15]. It was found that any improvement in the aqueous solubility would unexceptionally lead to decrease in PDT activity [15,16]. In addition to the physicochemical properties of photosensitizers, the location of photosensitizers in cells is another key factor for PDT effect. Many reports have shown mitochondria as important targets of PDT. Photosensitizers on mitochondria are reported to be more efficient in killing cells than those at other cellular sites [17,18]. In the current study, we evaluated the photodynamic activity of a novel photosensitizer, the derivative of hypocrellin B (HB), 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB), both in vitro and in vivo (Fig. 1A). The preparation procedure for the derivative has been reported in our previous study (Compound 4) [19]. The current work showed that the PENSHB not only got a better water solubility but also an advanced PDT effect. We further investigated the mechanism involved in cell death induced
by PENSHB-PDT. The derivative can selectively locate on the mitochondria in cytoplasm, the phototoxicity mediated by the novel photosensitizer capable of inducing mitochondrial depolarization resulting in cell apoptosis. On the basis of the relative PDT activity, aqueous solubility, and clinically required concentration, it was estimated that PENSHB could be directly deliverable via intravenous injection without assistance of any drug-delivery vehicle. Irradiation of a 630 nm laser after intravenous administration of PENSHB cured more than half of tumor bearing mice. To the best of our knowledge, this is the first time the derivative of HB has been used through systemic administration to achieve an efficient in vivo PDT effect for cancer treatment.
Materials and methods Cell culture Human Gastric Adenocarcinoma Cells (BGC-823) were obtained from the Department of Tumor, Chinese PLA General Hospital. Cells were cultured in DMEM supplemented with 15% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) in 5% CO2 at 37 ◦ C in a humidified incubator.
Reagents The procedure for the preparation of hypocrellin B (HB) and PENSHB was reported in our previous study [19]. The following fluorophore probes were used: Mito-Tracker Green (200 nM, Invitrogen Life Technologies, Inc.) to label mitochondria, tetramethylrhodamine methyl esters (TMRM, 200 nM, Invitrogen Life Technologies, Inc.) to indicate mitochondrial transmembrane potential (m). 5Aminolevulinic acid (Sigma—Aldrich) to compare the PDT efficacy of PENSHB with that of current clinically used PDT agents.
The biophysical properties of HB and PENSHB The detection of biophysical properties of photosensitizers was performed as previously described [19]. The solutions were measured for the absorption spectra on a Shimadzu UV1601 spectrophotometer. Steady-state absorption spectra were recorded on a Shimadzu UV-1601 spectrophotometer. Lipid/water partition coefficient was calculated by the ratio of photosensitizer concentration in L-octanol to that in PBS
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intensities were processed with physiology software (Zeiss Rel3.2, Jena, Germany).
Assessment of cell survival The cells in logarithmic phase were detached by treatment with trypsin-ethylenediaminetetraacetic acid (EDTA)-PBS and diluted to a density of 5 × 104 cells/ml in the presence of DMEM supplemented with 5% FBS. The cell suspension was seeded in wells (100 l each) of 96-well microplates and incubated under 5% CO2 at 37 ◦ C for 24 h. One column of wells did not receive cells to serve as the blank. Then the cells were transferred to DMEM medium containing photosensitizers of various concentrations and incubated at 37 ◦ C in dark for 4 h. After removal of the above medium containing photosensitizers and addition of fresh DMEM medium, the cells were irradiated with a 630 nm He—Ne laser of 10 mW/cm2 for 600 s and then incubated in dark for 24 h before survival assessment. Meanwhile, dark toxicity was measured under the same conditions without irradiation in parallel. The cell survival was estimated by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 24 h after irradiation [21]. Briefly, after 20 l of MTT (5 mg/ml) was added to each well, the cells were further incubated for 120 min. MTT solution was removed, and 200 l of DMSO was added to dissolve the formazan crystals. Then the plates were shaken at room temperature for 10 min, and the optical density at 570 nm was read on a microplate reader, from which cell survivals were derived. Figure 1 Physicochemical characteristics of the novel photosensitizer. (A) Structures of hypocrellin B (HB) and 17-(3-amino-1-pentanesulfonic acid)-substituted hypocrellin B Schiff-base (PENSHB). (B) The comparison of two photosensitizers in physicochemical properties. (C) Absorption spectra of HB and PENSHB at the concentration of 50 M in dimethyl sulfoxide (DMSO).
18 h after PDT treatment, the percentage of apoptotic and necrotic cells was analyzed by a FACScanto flow cytometer (Becton Dickinson, Mountain View, CA). Fluorescent emissions of FITC and DNA-PI complexes were measured at 515—545 nm and at 564—606 nm, respectively.
solution. The DPA-bleaching method was used to determine the quantum yields of 1 O2 according to Ref. [20].
Mitochondrial isolation and Western blotting analysis
Imaging analysis of living cells
For mitochondria isolation, cells were harvested and then fractionated using Cytosol/Mitochondria Fractionation Kit (Merch, Germany) according to the supplier’s recommendations. The cytosol extraction was subjected to Western blotting analysis of cytochrome c. The purity of fractions was tested by immunoblotting with antibodies specific for the cytosolic proteins -actin. The antibodies used for Western blotting include antibodies against -actin and cytochrome c (Cell Signaling Technology, Danvers, MA).
Fluorescent emission from Mito-Tracker Green was monitored by a commercial laser scanning microscope (LSM 510 Meta) combination system (Carl Zeiss, Jena, Germany) equipped with a Plan-Neofluar 40×/1.3 numerical aperture (NA) oil differential interference contrast (DIC) objective. Excitation wavelength and detection filter settings for each of the fluorescent indicators were as follows: photosensitizer’s fluorescence was excited at 488 nm with an Ar-ion laser, and emitted light was recorded through a 600—650 nm band-pass filter. Mito-Tracker Green fluorescence was excited at 488 nm with a He—Ne laser, and the emitted signal was recorded through a 500—550 nm band pass filter. TMRM was excited using the He—Ne laser. The excitation wavelength was 543 nm, and the emission detection filter was band pass 565—615 IR. For intracellular measurements, a desired measurement position was chosen in the LSM image. To quantify the results, the emission
Annexin flow cytometry
In vivo photodynamic therapy Mouse osteosarcoma S180 cells, purchased from Fourth Military Medical University Experimental Centre were maintained in RPMI-1640 culture media under 5% CO2 at 37 ◦ C. Cells were detached from the substrate using 0.5% trypsin in PBS, centrifuged, and then resuspended in PBS at a concentration of 5 × 106 cells/ml. Female BALB/c mice (Center of Experimental Animal, PLA General Hospital, Beijing, China)
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were housed in an environmentally controlled animal facility with regular 12/12 cycle. Hair on the upper backs of the mice was removed with a chemical depilatory agent (Na2 S 8% aqua-solution), then each mouse was injected with a 100 l cell suspension solution in the sub-dermal dorsal area [22]. The mice were treated with PDT when the tumor diameter was 8—9 mm. Photosensitizer (5 mg/kg) was administered via tail vein respectively at 1, 3 and 6 h prior to the laser irradiation (630 nm, 100 mW/cm2 × 1200 s). The irradiation light source was a semiconductor laser (Shenzhen Laser Medical Tech Co., Ltd., China). The laser output was coupled into an optical fiber (Medlight, S.A. Switzerland) with a lens for an even 3 cm2 irradiation area that covered the tumors. Mice were randomized into three groups: control, laser only and PDT. After the treatment, the tumor dimensions were measured with a caliper every 2 days. The tumor volume was determined using the formula for the volume of an ellipsoid (tumor volume = diameter × width2 × 3.14/6) [23].
Statistics analysis All assays were performed a minimum of three times. Data were presented as mean ± standard errors of the mean (SEM). Statistical analysis was performed with Student’s paired t-test. Differences were considered statistically significant at p < 0.05.
Results Physicochemical characteristics Singlet oxygen quantum yield (˚ ) is one of the most important parameters indicating efficacy of the potential photosensitizers for PDT [24]. As shown in Fig. 1B, ˚ of PENSHB was higher than that of HB, suggesting the derivative had the primary property to be a photosensitizer. Moreover, the solubility of the novel derivative in PBS was much higher than that of HB (6.6 mg/ml vs. 4.6 g/ml). Compared with HB, PENSHB with required lipid/water partition coefficient (PC) value was appropriate candidates for intravenous administration and cellular uptake. The absorption spectra of HB and PENSHB are shown in Fig. 1C. PENSHB had greatly strengthened absorption over a range of 600—700 nm as compared with HB.
The damage of mitochondria induced by PENSHB-PDT To validate the intracellular distribution of the novel photosensitizer in tumor cells, BGC-823 cells were loaded with HB or PENSHB in complete medium for 4 h. Photosensitizers’ fluorescence was imaged using a laser scanning confocal microscopy. The results showed that the photosensitizers were distributed in cytoplasm, but not in cell nuclei (Fig. 2A and B). To assess whether the new photosensitizer bound to the mitochondria, cells were co-loaded with Mito-Tracker, a mitochondria-specific dye. Our results revealed that the derivative was mainly located at mitochondria in cells (Fig. 2B). So the derivative could be used as a mitochondriatargeting photosensitizer. To investigate the direct impact
on mitochondria, the change of mitochondrial membrane potential (m) was detected in living cells by confocal microscopy after PDT treatment. The bright spheres disappeared, indicating mitochondrial depolarization (Fig. 2C). The results suggested that the rate of m descent in the cells treated with PENSHB-PDT was faster than that in cells treated with HB-PDT (Fig. 2D).
In vitro PDT activity In order to evaluate the PDT activity of the two photosensitizers, their dark toxicity on BGC-823 cells were previously tested. Fig. 3A shows the survival rates of cells under various concentrations of HB, and PENSHB. The results implicated that the dark toxicity of two photosensitizers was not statistically significantly different. Subsequently, BGC-823 cells were incubated with HB and PENSHB in varying concentrations for 4 h, and then irradiated by 20 J/cm2 of 630 nm laser. Fig. 3A shows that the concentrations previously used (0—60 nM) had no dark toxicity to the cells. The result suggested that the PDT activity of the novel photosensitizer was much higher compared with parent HB. This is in concordance with the rate of m descent (Fig. 2D) after PDT treatment with the same dose. It can be explained by two reasons, first, PENSHB presents a stronger red-light absorption in the phototherapeutic window (Fig. 1C); second, mitochondria are the most sensitive targets for PDT and PENSHB preferentially binding to them (Fig. 2B). In order to compare the PDT efficacy of PENSHB with that of other current clinically used PDT agents, the PDT activity of the 5-aminolevulinic acid (5-ALA) was tested (Fig. 3B). Fig. 3 shows the survival curve of BGC-823 cells under a series of concentrations of PENSHB or 5-ALA irradiated by 20 J/cm2 of 630 nm laser, from which IC50 values derived are 29.6 M and 2910 M for PENSHB and 5-ALA, respectively. This result suggests that the PDT efficacy of PENSHB is much greater than that of 5-ALA.
Cell death pathway induced by PENSHB-PDT PDT can stimulate three main cell death pathways: apoptosis, necrosis, and autophagy-associated cell death. To detect the pathway of cell death involved in the PENSHBPDT process, the percentage of apoptotic and necrotic cells after PDT treatment was analyzed by FACS (Fig. 4A). The result suggested that apoptosis was a generally major cell death modality in cells responding to PDT with the derivate (Fig. 4B). Cytochrome c is a pivotal effector during apoptosis, the release of cytochrome c is an important process in PDT-induced apoptotic death. Our result showed that the quantity of released cytochrome c and the activation of the caspase-3 in cells treated by PENSHB-PDT were higher than in cells treated by HB-PDT. However, there was a significant reduce of cyto-c release and caspase-3 activation after adding ROS scavenger NAC to the culture medium of cells treated by PENSHB-PDT (Fig. 4C). This result proved that PENSHB-mediated PDT could activate the mitochondrial pathway of apoptosis by inducing intracellular ROS.
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Figure 2 The damage of mitochondria induced by PENSHB-PDT. The intracellular distribution of (A) HB and (B) PENSHB. BGC-823 cells were loaded with 10 M photosensitizers and 100 nM Mito-Tracker Green. Mito-Tracker Green fluorescence (left panel), photosensitizer fluorescence (middle panel) and overlay fluorescence (right panel) were visualized by confocal microscopy. Bar = 10 m. (C) Time sequence of m disappearance under PDT treatment. TMRM (red emission) was localized in mitochondria in cells in response to the m. Cells with no treatment were set as control. Bar = 20 m. (D) The fluorescence intensity of TMRM (n = 5) with the standard deviations as the error bars in BGC-823 cells.
Figure 3 In vitro PDT activity. Viability of BGC-823 cells with or without irradiation (630 nm laser, 10 mW/cm2 , 600 s) in the presence of photosensitizers ((A) PENSHB or HB and (B) 5-ALA). The error bars denote standard deviation from three replicates.
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Figure 4 Cell death pathway induced by PENSHB-PDT. (A) Cell death 18 h after PDT treatment. BGC-823 cells were stained with FITC-conjugated Annexin V and propidium iodide (PI). (B) Quantified analysis of apoptotic and necrotic cell percentage by FACS (photosensitizers concentration, 30 nM, irradiation, 630 nm laser, 10 mW/cm2 , 600 s). All data are representative of three independent experiments. (C) Immunoblot analysis of proapoptotic proteins (cytochrome c and cleaved caspase-3).
In vivo PDT activity
Discussion
Based on the in vitro results, it was expected that the derivative possessed in vivo PDT activity. To evaluate the therapeutic potential in vivo, a S180 tumor model was used to evaluate the antitumor effects of PENSHB-mediated PDT. PENSHB was injected into tumor bearing mice via tail vein. Thereafter, tumor sites of all mice were irradiated by the 630 nm laser for 20 min. Two days after irradiation, there was a clear injury in the tumor sites of mice treated with PDT, implying effective antitumor therapy (Fig. 5A). From the only laser treated mice, we found the tumor was not damaged after irradiation. Fig. 5B shows the tumor growth rate curve of different treatment groups. For survival studies, 7 mice were used per treatment group and the mice were monitored for 100 days after tumor inoculation. Although the tumors of 3 mice relapsed at 6 days after treatment, 4 of the 7 mice were alive with complete tumor regression in the PDT group. There were no long-term survivors in the laser-only group (Fig. 5C).
In PDT process, photosensitizers are administered via vein and transported to tumor tissues through blood circulating system, and enter tumor cells and intercellular regions through the lipid membranes of blood vessels and cells, which requires water- and lipid-soluble photosensitizers. Thus a moderate lipid/water PC is important for photosensitizer [25]. Considering commonly usable volume of physiological saline for intravenous injection is no more than 20—30 ml, drug concentration should be higher than 2 mg/ml [26]. Our results showed that PENSHB not only had a high solubility but also a moderate lipid/water PC (Fig. 1B). Therefore, PENSHB is easy to be prepared as a clinical acceptable aqueous solution. One disadvantage of the currently approved PDT photosensitizers is their low absorbance in the optical window for photosensitizer excitation. In the visible spectral regions below 600 nm, light penetration into the skin is only a few millimeters which reduce the efficiency of PDT [27]. The novel photosensitizer
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Figure 5 Antitumor effects of PENSHB mediated PDT. (A) Images of tumor-bearing mice treated with PENSHB-PDT or laser irradiation, Mice with no treatment were set as control. (B) Volumetric change in tumor sizes of different treatment groups. n = 7 per group. *indicates difference at the p < 0.001 significance level. Error bars represent standard errors of the mean (SEM). (C) Survival rates of tumor-bearing mice of three groups. n = 7 per group.
with >600 nm excitation wavelengths allows access into deeper biological tissues for effective treatment of abnormal tissues of larger volume (Fig. 1C). The intracellular distribution of photosensitizers is a very important feature, which is used to evaluate their safety and more importantly has a great effect on their PDT cytotoxicity. Fig. 2A and B shows that the photosensitizers were distributed in cytoplasm, but not in cell nuclei, suggesting the novel photosensitizer did not affect reproduction of DNA or proliferation of cell. Many reports have implicated mitochondria as important targets of PDT. Photosensitizers at mitochondria are reported to be more efficient in killing cells than those at other cellular sites [17]. Our results showed that PENSHB was largely located at mitochondria in cytoplasm (Fig. 2B). Mitochondrial m is an essential parameter for mitochondrial. The descent of m is a consequence of the opening of permeability transition (PT) pores in the mitochondrial [28]. As a consequence of
PT pore opening, mitochondria uncouple and undergo large amplitude swelling. The outer membrane ruptures during swelling to induce the release of mitochondrial inter membrane proteins, including cytochrome c and other apoptosis inducing factors [29]. In the present study, a change of mitochondrial membrane potential (m) was detected in living cells after PDT treatment (Fig. 2D). The result indicated that the novel photosensitizer-mediated PDT was able to destroy the function of mitochondria and result in cell apoptosis consequently. PDT can evoke the three main cell death pathways: apoptosis, necrosis, and autophagy-associated cell death [1]. Apoptosis is a form of programmed cell death characterized by distinct morphological changes including cell rounding, membrane blebbing, cytoskeletal disassembly and DNA fragmentation [29]. The apoptotic activity limits leakage of intracellular material to the immediate environment, and thereby prevents tissue inflammation [30]. In contrast,
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necrosis is morphologically characterized by vacuolization of the cytoplasm and swelling, and breakdown of the plasma membrane, resulting in an inflammatory reaction due to the release of cellular contents and proinflammatory molecules [31]. Autophagy is characterized by a massive vacuolization of the cytoplasm and the formation of autophagosome. This is a lysosomal pathway for the degradation and recycling of intracellular proteins and organelles. Recent studies suggest that autophagy is a mechanism to preserve cell viability after PDT [32]. Fig. 4C and D shows that apoptosis is the major death pathway after PENSH-PDT treatment. S180 is type of very malignant tumor cell line. Even though a small number of viable cells remain after treatment, they are always expanding and proliferating, which makes it difficult to cure. In previous studies [27,33], no complete tumor cure was obtained due to the shorter absorption wavelength of HB, thus facilitating regrowth of tumors (>5 mm) treated with PDT. In the current study, a high cure percentage was obtained after PDT at 1 h druglight interval to the tumors of 8—9 mm (Fig. 5C). It has been reported that direct antitumor effects of PDT derive from two aspects: cytotoxic effects on tumor cells and damage to the tumor vasculature [1]. Previous study suggested that extending the drug-light interval increases the contribution of direct tumor toxicity and reduce the contribution of damage to the tumor vasculature [34]. In this study, we found PDT six hours after drug administration led to tumor regrowth delay rather than cure (data not shown). This result suggested that vascular damage seems to contribute significantly to the overall PDT effect. In this work, the biological properties of the novel derivative of HB, PENSHB, were studied. Our results showed that it was readily dissolved in an aqueous solvent (water, PBS and physiological saline) in a clinically acceptable concentration. Furthermore, apoptosis is generally a major cell death modality in cells responding to the novel derivative-mediated PDT, because its preferential location at mitochondria in cytoplasm. More importantly, PENSHB can be directly used for intravenous injection without further drug preparation. These characteristics suggested PENSHB was suitable for PDT applied in vascular diseases and solid tumors. Our in vivo study showed that more than half of the tumor bearing mice were cured by PENSHB-mediated PDT. Taken altogether, based on the evidence of our in vitro and in vivo studies, we therefore propose that PENSHB is an effective photosensitizer for PDT in tumor therapy and has a promising prospect of clinical application.
Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 60878055; 61036014; 31170963), Beijing Municipal Natural Science Foundation (No. 4122090) and the National 863 Research Project of China (No. 2008AA030117).
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