Open Access
Add to BibSonomy
Abstract
Since the discovery of photodynamic therapy, scientists have constantly been searching for more effective and ideal photosensitizers (PSs). As part of our ongoing interest in the development of more potent photosensitizers, quinoline-8-yloxy-substituted zinc(II) phthalocyanine (ZnPc-Q1) has been identified as a promising photosensitizers in tumor cells. This study aims to explore the photodynamic mechanism and in vivo photodynamic efficacy of ZnPc-Q1, and further evaluate its potential in clinical photodynamic therapy application. The single crystal structure of ZnPc-Q1 enables the easy control of clinical quality standards. In comparison with Photofrin, ZnPc-Q1 exhibits considerably higher in vitro anticancer activity by dual dose-related mechanisms (antiproliferative and apoptosis). In addition, the in vivo results demonstrate that ZnPc-Q1 exhibits significant tumor regression with less skin photosensitivity by both direct killing and apoptosis anticancer mechanisms. In conclusion, ZnPc-Q1 can be considered to be a promising ideal PS for clinical application owing to its defined chemical structure without phthalocyanine isomerization, good absorption of tissue-penetrating red light, improved photodynamic therapy efficacy, and reduced skin phototoxicity.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Photodynamic therapy (PDT) has emerged as a non-invasive efficacious therapeutic modality for a range of tumors [1–3]. During PDT, two non-cytotoxic component photosensitizers (PSs) and light are administrated in the presence of tissue molecular oxygen, producing cytotoxic reactive oxygen species (ROS), which are an efficacious remedy for tumor eradication [1,4–7]. Therefore, the application of PDT has been greatly facilitated by the recent progress in light delivery and more importantly the development of more potent PSs [8–12]. Since the use of haematoporphyrin (HpD) derivatives to destroy tumor tissue in the 1960s, scientists have been constantly working toward identifying better PSs [10–13]. Photofrin, a purified version of HpD, was the first approved PS for clinical treatment of bladder cancer, early non-small cell lung cancer, and esophageal cancer in the late 1990s [14,15]. However, although Photofrin is the most widely used PS for clinical application, it has a number of adverse side effects, thereby significantly limiting its clinical application [15,16]. First, it is a complex mixture of monomeric and aggregated porphyrins, and has a complicated composition, which makes it difficult to control its clinical quality standards [15–17]. Additionally, Photofrin is characterized by an extremely small decadic molar absorption coefficient at its absorption wavelength (λabs = 630 nm, ɛ = 1.17 × 103 L/mol·cm), and this wavelength has low percentage of light transmission through tissue; therefore, Photofrin cannot be activated effectively [15–17]. Finally, patients must stay in the dark for a long time after treatment because of the severe skin photosensitivity of Photfrin, which is induced by its accumulation in the skin tissue during PDT [18–19]. Therefore, second- and third- generation PSs have been developed.
Zinc phthalocyanines (ZnPcs) are promising second generation PSs in PDT application owing to their less skin photosensitivity, strong absorption in the therapeutic window (650–800 nm) and strong anticancer effect [20–24]. In the past few years, we have synthesized a number of ZnPcs and evaluated in vitro PDT activity to expand the scope of ZnPcs and search for novel ZnPcs with greater PDT efficacy [25–32]. Among them, mono (quinolin-8-yloxy) zinc(II) phthalocyanine (ZnPc-Q1), which combines the PS ZnPc and a derivative of the drug quinoline, shows a high potency with low IC50 values (20 nM) of cell count under a low light dose of 1.5 J/cm2 in HepG2 and BGC-823 cells. More importantly, as reported, most ZnPc PSs contain several isomers, which may lead to difficulty in clinical approval and quality standard control, thereby increasing the cost of drug research and development [33,34]. ZnPc-Q1 possesses a simple and definite structure without any isomers and is characterized by simple synthetic procedures, making its development as a clinically approved PS easier and more eco-friendly in the future. Based on the criteria of an ideal PS [16,35], these advantages of ZnPc-Q1 demonstrated its potential as an ideal PS for clinical PDT application. However, to further evaluate its potential and lay an experimental foundation for clinical photodynamic anticancer application, more detailed studies on its crystal structure, in vitro and in vivo PDT mechanism and anticancer effect on animal models need to be conducted.
In this study, X-ray crystallography of ZnPc-Q1 was performed and its in vitro photocytotoxicity toward a variety of tumor cells was determined through comparison with Photofrin. Then, Hoechst 33258 staining, apoptosis detection, and cell cycle analysis were performed to study its in vitro anticancer mechanisms. Finally, its in vivo PDT efficacy and mechanisms were evaluated in H460 tumor-bearing nude mice.
2. Materials and methods
2.1 General
Dulbecco's modified eagle medium (DMEM), penicillin, streptomycin, and Annexin V/PI apoptosis detection kit were obtained from Invitrogen (Taastrup, Denmark). Photofrin was obtained from Axcan Pharma Inc. (Mont-Saint-Hilaire, QC, Canada). RPMI 1640 medium was obtained from Gibco Life Science (Grand Island, NY, USA). Fetal bovine serum was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (Zhejiang, Hanzhou, China). Cremophor EL was obtained from BASF (Luwigshafen, Rhineland-Palatinate, Germany). All cells used in this study were obtained from Institue of Materia Medica, Chinese Academy of Medical Science (Beijing, China). Other chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2 Preparation of ZnPc-Q1 and Photofrin
ZnPc-Q1 was synthesized according to the literature [30]. To prepare ZnPc-Q1 solution for photodynamic activity assays, it was first dissolved in N,N-dimethylacetamide (DMAC) containing 3.8% polyoxyethylene-35-castor oil (Cremphor EL) to produce a 5 mg/mL (6.9 mM) stock solution. The ZnPc-Q1 solution was diluted to the desired concentration with phosphate buffered saline (PBS) or cell medium. Photofrin was obtained from Axcan Pharma Inc. To prepare Photofrin for photodynamic activity assays, it was first dissolved in PBS to give 2 mg/mL (3.3 mM) stock solution.
2.3 X-ray crystallography
The X-ray diffraction study of ZnPc-Q1 was conducted on a Rigaku Saturn 724+ MicroMax 007 CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). Absorption corrections were performed using a multi-scan method. The structure of ZnPc-Q1 was solved by direct methods using SHELXS-97 and refined by full-matrix least-squares fitting on F2 using the SHELX software package. All non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms of the ligand were calculated in ideal positions with isotropic displacement parameters. As the lattice solvent molecules in the structure were disordered, the residual intensities were removed by PLATON/SQUEEZE routine, leading to better refinement.
2.4 Light source
The light exposure was acquired using PDT-670 semiconductor laser PDT unit for ZnPc-Q1 and a PDT-630II semiconductor laser PDT unit with less than ± 10% laser output instability and high homogeneity, which were equipped with built-in optical fiber output detection system and optical fibers (both were obtained from Guilin Xingda Photoelectric Medical Equipment Co., LTD. Guangxi, China). For in vitro PDT, the output power and irradiation time were set to 1,500 mW and 180 s, respectively, and the spot diameter was set to 8 cm, resulting in a 50 cm2 circular spot size. Thus, the power density was determined to be 30 mW/cm2, and the light fluence was 5.4 J/ cm2. There was no reflection from the bottom of the cell containers. For in vivo PDT: the output power and irradiation time were set to 100 mW and 600 s, respectively, and the spot diameter was set to 1 cm, resulting in a 0.785 cm2 circular spot size. Thus, the power density was determined to be 127.3 mW/cm2, and the light fluence was 76.2 J/ cm2.
2.5 Methyl thiazolyl tetrazolium detection
Tumor cells were seeded onto 96-well plates at 1500-3000 cells per well and incubated overnight. ZnPc-Q1 and Photofrin were separately diluted to the required concentration (DMAC = 0.06%) and added to six-plicate wells (5 wells for each concentration). A blank control group (no drug and no DMAC; referred to as Blank) and solvent control group (DMAC = 0.06%; referred to as Vehicle) were also set up. After 4 h of incubation, the medium containing drugs was replaced with fresh medium and the cells were illuminated. The cells after irradiation were incubated again for 24 h, and then a methyl thiazolyl tetrazolium (MTT) solution in PBS (10 µL, 5 mg/mL) was added to each well followed by incubation for 4 h. Then, 180 µL of DMSO was added to each well. The plate was incubated at room temperature (about 25 °C) for 30 min. The absorbance at 545 nm at each well was recorded by a microplate reader. The survival curves plotted as a function of concentration of PSs and cell viability, IC50 values were then calculated. The experiment was repeated thrice.
2.6 Hoechst 33258 staining
The cells in the logarithmic growth phase were seeded onto a 6-well plate at 400,000 cells/well. After 80% cell confluence was reached, ZnPc-Q1 or Photofrin in its corresponding concentrations (DMAC = 0.01%) was added and incubated for 4 h. The supernatant was discarded and fresh medium were added. The cells were illuminated and incubated again for 4 h. The supernatant was discarded and 0.5 mL of 10% formalin fixative was added. After 10 min of incubation, the fixative was removed and the cells were washed twice with PBS. Then, 10 g/mL Hoeschst 33258 was added, followed by incubation for 10 min; the dyes were discarded, and cells were washed again with PBS and observed under an inverted fluorescence microscope followed by photograph.
2.7 Apoptosis detection
After PDT treatment, the cells were incubated again for 12 h, washed twice with PBS and detached with 0.25% trysin/ethylene diamine tetraacetic acid (EDTA). The cell suspension was followed by centrifugation (200 g, 5 min), washing, re-suspension, and counting. The cell number was more than 200,000. Based on the manufacturer’s instructions, the cells were resuspended in 100 µL annexin-binding buffer and incubated with 5 µL Alexa Fluor 488 annexin V and 1 µL PI for 15 min. Then, the resulting samples was analyzed using a flow cytometer.
2.8 Propidium iodide staining
After PDT treatment, the cells in the 6-well plates were incubated again for 12 h. Then, the cells were harvested by trypsinization, washed twice with PBS, resuspended and fixed with 70% ice-cold ethanol at 4 °C for 24 h. The fixed cells were harvested, washed twice with PBS, and incubated with PI solution (50 µg/mL PI and 20 µg/mL RNase A) at 37 °C for 30 min in the dark. Finally, the cells were analyzed by flow cytometry.
2.9 Nude mice xenograft tumor model
Nu/Nu female mice (18–20 g) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals were housed in cages at constant temperature and humidity under a 12 h light/dark cycle. The animals had free access to food and water. All experiments on the tumor-bearing mice were approved by the University Animal Experimentation Ethics Committee. All operations were performed under the guidance of veterinarians in accordance with international guidelines concerning the care and treatment of experimental animals. To develop the tumor model, H460 cells (50,000,000/mL, 0.2 mL) in the logarithmic phase were harvested and injected subcutaneously into the axilla of nude mice. The tumor size and mouse body weight were measured. Two parameters of the tumor (length and width) were measured using a micrometer digital caliper for the calculation of the tumor volume (TV). TV was calculated as follows: V = a×b2/2, where a is tumor length and b is the tumor width). Two weeks later, subcutaneous xenograft tumors (V = 0.5–1 cm3) were developed and removed under sterile conditions. The tumor tissues were chopped into small pieces of approximately 2–7 mm3 and then transplanted subcutaneously into the axilla of Nu/Nu mice. At the end of every experiment, all mice were sacrificed by cervical dislocation.
2.10 In vivo photodynamic activity assay
The H460 tumor-bearing mice were divided into four groups (Blank group, Vehicle group, ZnPc-Q1 group, and Photofrin group) randomly. Each group contained seven mice. The mice were injected with 200 uL/20 g mouse body weight of their corresponding samples (solvent, 1.2 mg/kg ZnPc-Q1, 20 mg/kg Photofrin). The tumor sites were exposed to 630 nm laser light at 76.2 J/cm2 24 h post-injection (i.v). After 24 h, the mice received 10 min of light irradiation (630 nm for Photofrin and 670 nm for ZnPc-Q1, 127.3 mW/cm2, 76 J/cm2). After PDT treatment, the tumor size and mouse weight were measured.
2.11 Histology examination
The mice from different groups were sacrificed 48 h post PDT treatment, and the tumor tissues were excised. Every specimen was divided into two parts. One part was fixed in 2% glutaraldehyde, and then 1% osmium acid, dehydrated, and soaked with acetone step by step, and then embedded in epoxy resin 812; the sliced tissue sections were observed using transmission electron microscopy. The other part was fixed in 10% formalin and then embedded in paraffin. The sliced tissue sections were stained with hematoxylin and eosin (H&E) staining and observed using light microscopy.
2.12 Statistical analysis
Quantitative data are reported as mean ± SD/SEM from at least three independent experiments. Comparisons between the groups were performed using the one-way analysis of variance (ANOVA) test, and p values of < 0.05 were considered statistically significant.
3. Results
3.1 Crystal X-ray structure analyses
Single crystals of ZnPc-Q1 suitable for X-ray crystal structure analysis was obtained by slow interface diffusion of CH2Cl2 into the tetrahydrofuran (THF) solution of ZnPc-Q1, which crystallized in a C2/c space group with one monosubstituted phthalocyanine molecule sitting in the asymmetric unit [Figs. 1(a) and (b)]. X-Ray crystallography reveals that the central zinc ion is penta-coordinated in a square-pyramidal geometry by four isoindole nitrogen atoms of the phthalocyanine molecule and one oxygen atom from the axial ligand of water. The zinc ion slightly protrudes from the basal plane formed by the four nitrogen atoms with a deviation of 0.40 Å. The average Zn-N bond length is approximately 2.04 Å and the Zn-O1W bond length is 2.51 Å. The crystal packing diagram in one-unit cell of ZnPc-Q1 is illustrated in Fig. 1(c). Face-to-face dimers are formed through hydrogen bonding interaction with an N9…O1W distance of 2.747 Å and an N5…O1W distance of 2.885 Å (see Fig. 1(d)). To form intermolecular hydrogen bonds, the plane defined by a quinolinol group has to deviate from the plane of the attached isoindole group, with the dihedral angle between these two planes of 61.76°. And correspondingly C6-O1-C33 angle is 120.2°. The single crystals of ZnPc-Q1 make it could be purified by crystallization with reduced cost and green chemistry for further pharmaceutical development. The results indicated that ZnPc-Q1 is chemically well defined without isomers, which makes it easy to approve and control clinical quality standards in PDT application.
Fig. 1. (a) Chemical structure of ZnPc-Q1. (b) X-ray crystal structure of ZnPc-Q1 (C: grey, N: blue, O: red, H: white, Zn: green). (c) The molecular packing of ZnPc-Q1. For clarity, all H atoms are omitted and strong hydrogen bonds are shown by green dash lines (C: grey, N: blue, O: red, Zn: green). (d) Close packing of a face-to-face dimmer formed by hydrogen bond.
3.2 In vitro photocytotoxicity study
ZnPc-Q1 has been proved to exhibit significantly high photocytotoxicity toward HepG2 cells in comparison with other ZnPc derivates under a low light dose of 1.5 J/cm2 [30]. To expand the scope of ZnPc-Q1 PDT indications in clinical application, the photocytotoxicity of ZnPc-Q1 toward a spectrum of other cancer cells was determined by MTT assay and a colony formation assay. ZnPc-Q1 or Photofrin was incubated with cells for 4 h before irradiation. As depicted in Figs. 2(a)–2(b) and Table 1, ZnPc-Q1 exerts a cytotoxic effect on eight different cell lines derived from cancers of the lung, liver, pancreas, kidney, breast, oral cavity, and nervous system. Furthermore, the anticancer activity of ZnPc-Q1 is notably 2- to 6-fold greater than that of Photofrin (IC50 0.235–0.945 µg/mL (0.326–1.310 µM) vs. 1.206–1.826 µg/mL (1.833–3.015 µM), p < 0.0001). Photofrin is approved for non-small lung cancer; thus, H460 cells are chosen in the following experiments to compare the photodynamic activities of ZnPc-Q1 and Photofrin. To further evaluate the long-term proliferative potential of cancer cells following PDT, a colony formation assay was performed [Fig. 2(c) and Table 2]. The colony formation rate of the H460 cells was 15.97–2.83% when using a 2.5 µg/mL (4.1 µM) concentration of Photofrin. Only 0.5 µg/mL (0.7 µM) ZnPc-Q1 resulted in a remarkable decrease in the colony formation ratio (1.01% to 0.25%) with a statistically significant difference in comparison with Photofrin (p < 0.01). These results indicate that ZnPc-Q1 is more effective than Photofrin which is the most widely used PS in clinical PDT.
Fig. 2. Cytotoxic effects of Photofrin and ZnPc-Q1 toward (a) H460 cells and (b) Bel7042 cells (incubation time: 4 h; data expressed as mean ± SD from three experiments, each performed in quadruplicate). (c) Colony formation assay of Photofrin and ZnPc-Q1 toward H460 cells (incubation time: 4 h).
Table 1. In vitro anticancer activities (IC50) of Photofrin and ZnPc-Q1 in different cancer cells (mean ± SEM).
Table 2. Colony formation rates of Photofrin and ZnPc-Q1.
3.3 In vitro photodynamic mechanism
To investigate the cell death (necrosis or apoptosis) pathway that was induced by ZnPc-Q1, Hoechst 33258 staining, Annexin V-FITC/PI staining, and cell cycle analysis were conducted to study the photodynamic mechanism of ZnPc-Q1 on nuclear condensation, DNA fragmentation, externalization of phosphatidylserine, and cell cycle.
The H460 cells were first stained with Hoechst 33258 to detect the cell and nuclei morphology. As depicted in Fig. 3(a), the control group does not display visible nuclear changes with low fluorescence intensity. H460 cells treated with an extremely low dose (0.5 µg/mL, 0.7 µM) of ZnPc-Q1 mediated PDT demonstrated an increased chromatin density, as indicated by a brighter color, and DNA fragmentation, which are the typical characteristics of apoptosis. However, in the higher-dose (5 µg/mL, 8.3 µM) Photofrin PDT treatment group, fewer apoptotic cells were observed. These results are consistent with the MTT data and indicate that ZnPc-Q1 induces an apoptotic response during PDT.
Fig. 3. (a) Fluorescence microscopy images of H460 cells and Bel7402 cells, which were stained with Hoechst33258 immediately after PDT (scale bar: 50 µM). (b) Annexin V-FITC/PI dual staining assay of H460 cells after PDT treatment with light and ZnPc-Q1 (*p < 0.05).
To understand the mechanism behind the PDT activity and cellular damage induced by ZnPc-Q1, we investigated the extent of post-PDT cell apoptosis by Annexin V-FITC/PI double staining, and the results were analyzed by flow cytometric analysis. This assay provided estimates of the populations of viable cells (Q3), early apoptotic cells (Q4), late-stage apoptotic and necrotic cells (Q2), and only necrotic cells (Q1) in four quadrants of the dot plots. As depicted in Fig. 3(b), upon 3 µg/mL of ZnPc-Q1 PDT, the percentages of the early and late apoptotic cells significantly elevated in comparison with those of the vehicle group (7.82% vs 0.79%, 60.55% vs 3.23%, p < 0.05). These results confirm that ZnPc-Q1 is capable of inducing apoptosis upon light irradiation.
Cell cycle analysis can reveal DNA fragmentation and cell cycle arrest resulting from exposure to anticancer drugs. To investigate the anticancer effects of ZnPc-Q1 on DNA and cell cycle progression, the H460 cells were treated with different concentrations of ZnPc-Q1 or with the reference drug Photofrin and stained with PI. The profiles in Figs. 4(a) and 4(b) illustrate two different cell cycle arrests with low concentrations (0.03–0.30 µg/mL, 0.04–0.42 µM) and high concentrations (1.00–3.00 µg/mL, 1.39–4.16 µM) of ZnPc-Q1 treatment. They indicate that low concentrations of ZnPc-Q1 can increase the relative number of cells in the G0/G1 phase and decrease that in the S and G2/M phases. Therefore, its proliferation index and S phase fraction (SPF) were significantly reduced (p < 0.05), indicating that cell proliferation was inhibited by a low concentration ZnPc-Q1 treatment. When the concentrations of ZnPc-Q1 increased to 1.00–3.00 µg/mL (1.39–4.16 µM), the number of cells accumulated in the S phase increased with a significant decline of that in G2/M and G0/G1, suggesting that the cells were blocked in the S phase. Furthermore, there was a significant elevation in the cell population at the sub-G1 phase, indicating an increase in the occurrence of apoptosis. This result was confirmed by the profiles depicted in Fig. 4(c). These results suggest high concentrations of ZnPc-Q1 can induce cell apoptosis. Therefore, ZnPc-Q1 demonstrates dual dose-related in vitro anticancer mechanisms.
Fig. 4. Cell cycle analysis determined by flow cytometry with propidium iodide (PI) staining. (a) Effects of Photofrin (2.00–5.00 µg/mL, 3.30–8.25 µM) and ZnPc-Q1 (0.03-3.00 µg/mL, 0.04–4.16 µM) PDT on cell cycle distribution of H460 cells. (b) Cell cycle phase (sub-G1, G1, S and G2/M) distributions of H460 cells after PDT. (c) Percentage of apoptosis cell population. Each value is the mean ± SD from three experiments, each performed in triplicate with *p < 0.05.
3.4 In vivo photodynamic anticancer activity
To evaluate the PDT anticancer efficacy of ZnPc-Q1, we performed in vivo experiments with the H460 tumor-bearing nude mice. The mice were randomized into four groups (seven mice per group): blank group (no drug, no light, negative control), vehicle group (only saline and light, solvent and light control), ZnPc-Q1 group (1.2 mg/kg ZnPc-Q1 upon light irradiation), and Photofrin group (20 mg/kg Photofrin upon light irradiation, positive control). The tumor sites were exposed to 630 nm laser light at 76.2 J/cm2 24 h post-injection. The change in the tumor volume and mouse body weight as a function of time after treatment was monitored, and the excised tumors were weighed after 15 days. As illustrated in Fig. 5, a significant reduction (p < 0.05, versus the control group) in the tumor growth of mice in the ZnPc-Q1 group was observed, indicating good tumor growth inhibition efficacy of ZnPc-Q1 at a considerably low dose. However, saline and light (vehicle group) alone did not notably delay the tumor growth, suggesting that the treatment efficacy of ZnPc-Q1 was not due to the solvent and light exposure alone. The same tumor growth inhibition could only be achieved with Photofrin when the dose was up to 15 times higher than that of ZnPc-Q1, suggesting improved efficacy of ZnPc-Q1. The photographs of excised tumors and mice from the representative mice [Fig. 5(d)] visually display the same results. The biosafety of ZnPc-Q1 was studied by examining the body weights of mice during PDT. As depicted in Fig. 5(c), no loss of body weight or other signs of toxicity were observed, indicating a high biosafety index of ZnPc-Q1.
Fig. 5. (a) H460 tumor volume curves of mice treated with only light (Blank), light and solvent (Vehicle), 20 mg/kg Photofrin, and 1.2 mg/kg ZnPc-Q1 (light fluence 76.2 J/ cm2 and irradiation time 600 s). (b) H460 tumor inhibition rate in each group after treatment. (c) Body weight curves of mice in each group after treatment. (d) Images of excised H460 tumors and mice in each group after treatment on the 15th day. (*p < 0.05).
Skin phototoxicity is the most common undesired side effect of Photofrin in clinical PDT [36–38]. To further highlight the advantages of ZnPc-Q1 in clinical PDT, we observed its potential skin phototoxicity in comparison with Photofrin. As depicted in Fig. 5(d), all of the local tumor skin in the Photofrin group, which was exposed to irradiation, exhibited obvious edemas and ulcers and then showed great crusting. However, similar to the control and vehicle groups, no or slight edema and crusting were observed on the local skin illuminated by laser irradiation upon ZnPc-Q1 treatment. In summary, these results demonstrate the great potential of ZnPc-Q1 as a PS in PDT, which not only exhibits higher anticancer effect, but also reduced skin phototoxicity.
3.5 In vivo photodynamic anticancer mechanism
We studied the mechanism of tumor destruction following ZnPc-Q1-mediated PDT by investigating the morphological changes of the tumors. Tumor sections of the mice from different groups were collected, fixed and stained two days after treatment and then observed using a transmission electron microscope and light microscope. As depicted in Fig. 6, the osmium acid-fixed tumor sections in the control group exhibit typical ultrastructural characteristics of malignant tumor cells; conspicuous and double nucleoli are present together with scanty mitosis, and the structures of the cells and subcellular organelles, such as mitochondria, are well maintained and intact. After ZnPc-Q1-mediated PDT, the characteristics of apoptosis and necrosis were observed: vacuolation of cellular plasma and nucleus, loss of cellular and subcellular organelle structure, distention and membrane damage of subcellular organelles, and decrease and breaking of mitochondrial cristae. H&E staining of the tumor sections in ZnPc-Q1 group revealed more severe tumor damages but no significant differences in the number of blood vessels in comparison with those of the control group. Thus, these results suggest that ZnPc-Q1 acts in two anticancer mechanisms: direct killing with cell necrosis and inducing apoptosis of tumor cells.
Fig. 6. Micrographs of osmium acid-fixed and H&E-stained H460 tumor sections obtained using transmission electron microscopy and light microscopy, respectively, collected from the different groups two days after treatment. The scale bars are 2 µm for osmium acid fixing and 5 mm for H&E staining, respectively.
4. Discussion
PS is one of the key determinants in PDT clinical application [1–4]. An ideal PS should fulfil the following criteria: easy synthesis, chemically well defined, no isomerization, good absorption of tissue-penetrating red light, high quantum yield of ROS, low dark toxicity, and high photodynamic anticancer activity [1–4]. Based on these criteria, ZnPc-Q1 is considered as a promising PS for the PDT clinical application.
Single crystals of ZnPc-Q1 without phthalocyanine isomerization can be easily obtained, resulting in a number of potential clinical advantages, such as a reduction in complex drug interactions and development cost, simplified pharmacokinetics and production process, and easy clinical quality standard control in PDT clinical application [39]. Additionally, a previous study has demonstrated that ZnPc-Q1 exhibits good absorption at long wavelength (λabs = 672 nm, ɛ = 1.7 × 105 L/mol·cm) and high singlet oxygen quantum (ΦΔ = 0.60 in DMF) [30]. ZnPc-Q1 fulfils the chemical criteria of an ideal PS. Furthermore, its simple synthetic and purification procedures enable its development as a clinically approved PS in an easier and eco-friendly manner in the future.
ZnPc-Q1 exhibits enhanced photodynamic anticancer activity toward different cancer cell lines derived from cancers of the lung, liver, pancreas, kidney, breast, oral cavity, and nervous system (H460, Bel7402, HepG2, MDA-MB-232, KB, ACHN, SH-SY5Y, and SW1990). In comparison with the well-known PS Photofrin, ZnPc-Q1 exhibits stronger photodynamic activity in all these cell lines. This result demonstrates the in vitro enhanced photodynamic anticancer activity of ZnPc-Q1 with a wide scope of PDT indications in clinical application. To study the mechanism of ZnPc-Q1-mediated cell death, Hoechst 33258 staining, Annexin V-FITC/PI staining and cell cycle analysis were conducted. Hoechst 33258 staining revealed an increased chromatin density and DNA fragmentation. Additionally, Annexin V-FITC/PI double staining demonstrated that the percentages of early and late apoptotic cells were significantly elevated. Cell cycle analysis further demonstrates that ZnPc-Q1 at high concentrations could induce cell apoptosis. It is clear that ZnPc-Q1 induces an apoptotic response during PDT, which is preferably the most potent defense mechanism against cancer.
An ideal PS should possess enhanced anticancer efficacy not only in cancer cell lines but also in animal models. Significant tumor regression was demonstrated in the H460 tumor-bearing nude mice compared with the FAD-approved PS Photofrin. Additionally, reduced skin phototoxicity was observed, which can be attributed to the red shift of the main absorption peak and smaller overlap of the absorption spectrum with the sunlight emission spectrum in comparison of Photofrin. More detailed and suitable light and skin models will be adopted to further demonstrate its reduced skin phototoxicity and study the mechanism in the future. Morphological changes of the tumors indicate that ZnPc-Q1 acts in two anticancer mechanisms: direct killing and induced apoptosis of tumor cells. No obvious blood vessel destruction was observed in our ZnPc-Q1 mediated PDT protocol perhaps because of a long drug-light interval (24 h) we used. On the one hand, the long drug-light interval results in lower concentration of PS in the vessel lumen [40], thereby there is no significant damage of blood vessels in our study. On the other hand, the long drug-light interval would reduce skin reaction of PS, which meets the safety requirement of newly approved therapeutic agents by the Food and Drug Administration (FDA) in most countries [40]. Therefore, we choose the long drug-light interval protocol, which ZnPc-Q1 can accumulate in the tumor cells, results in direct killing and induced apoptosis of tumor cells. Additionally, as reported, one important anticancer PDT mechanism is induced anticancer immunity [1–4]. Therefore, to further comprehensively support its clinical anticancer application, more detailed studies on its PDT mechanisms and immune effect experiments, and preclinical evaluation are in progress.
5. Conclusions
In summary, based on the criteria of an ideal PS and the above reported results, we successfully demonstrated the PDT clinical potential of ZnPc-Q1. It is chemically well-defined without phthalocyanine isomerization, and exhibits good absorption of tissue-penetrating red light and improved in vitro and in vivo PDT efficacy on different cancer cells and animal models with reduced skin phototoxicity. These results lay an experimental foundation for future clinical anticancer therapy.
Funding
National Health and Family Planning Commission Jointly Established Scientific Fund (WKJ2016-2-14); National Natural Science Foundation of China (81703345).
Disclosures
The authors declare that there are no conflicts of interest related to this article.
References
1. W. M. Sharman, C. M. Allen, and J. E. van Lier, “Photodynamic therapeutics: basic principles and clinical applications,” Drug Discovery Today 4(11), 507–517 (1999). [CrossRef]
2. D. E. Dolmans, D. Fukumura, and R. K. Jain, “Photodynamic therapy for cancer,” Nat. Rev. Cancer 3(5), 380–387 (2003). [CrossRef]
3. A. P. Castano, P. Mroz, and M. R. Hamblin, “Photodynamic therapy and anti-tumour immunity,” Nat. Rev. Cancer 6(7), 535–545 (2006). [CrossRef]
4. J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans, K. S. Samkoe, S. Verma, B. W. Pogue, and T. Hasan, “Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization,” Chem. Rev. 110(5), 2795–2838 (2010). [CrossRef]
5. Z. J. Zhou, J. B. Song, L. M. Nie, and X. Y. Chen, “Reactive oxygen species generating systems meeting challenges of photodynamic cancer therapy,” Chem. Soc. Rev. 45(23), 6597–6626 (2016). [CrossRef]
6. X. Li, N. Kwon, T. Guo, Z. Liu, and J. Yoon, “Innovative Strategies for Hypoxic-Tumor Photodynamic Therapy,” Angew. Chem., Int. Ed. 57(36), 11522–11531 (2018). [CrossRef]
7. J. F. Lovell, T. W. B. Liu, J. Chen, and G. Zheng, “Activatable Photosensitizers for Imaging and Therapy,” Chem. Rev. 110(5), 2839–2857 (2010). [CrossRef]
8. M. Yang, T. Yang, and C. Mao, “Enhancement of Photodynamic Cancer Therapy by Physical and Chemical Factors,” Photodiagn. Photodyn. Ther. 58(40), 14066–14080 (2019). [CrossRef]
9. T. S. Mang, “Lasers and light sources for PDT: past, present and future,” Photodiagnosis Photodyn. Ther. 1(1), 43–48 (2004). [CrossRef]
10. X. Li, S. Lee, and J. Yoon, “Supramolecular photosensitizers rejuvenate photodynamic therapy,” Chem. Soc. Rev. 47(4), 1174–1188 (2018). [CrossRef]
11. X. Li, B. D. Zheng, X. H. Peng, S. Z. Li, J. W. Ying, Y. Y. Zhao, J. D. Huang, and J. Yoon, “Phthalocyanines as medicinal photosensitizers: Developments in the last five years,” Coord. Chem. Rev. 379, 147–160 (2019). [CrossRef]
12. B. M. Luby, C. D. Walsh, and G. Zheng, “Advanced Photosensitizer Activation Strategies for Smarter Photodynamic Therapy Beacons,” Photodiagn. Photodyn. Ther. 58(9), 2558–2569 (2019). [CrossRef]
13. T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori, D. Kessel, M. Korbelik, J. Moan, and Q. Peng, “Photodynamic Therapy,” J. Natl. Cancer Inst. 90(12), 889–905 (1998). [CrossRef]
14. D. A. Bellnier, W. R. Greco, G. M. Loewen, H. Nava, A. R. Oseroff, and T. J. Dougherty, “Clinical Pharmacokinetics of the PDT Photosensitizers Porfimer Sodium (Photofrin), 2-[1-Hexyloxyethyl]-2-Devinyl Pyropheophorbide-a (Photochlor) and 5-ALA-Induced Protoporphyrin IX,” Lasers Surg. Med. 38(5), 439–444 (2006). [CrossRef]
15. A. E. O’Connor, W. M. Gallagher, and A. T. Byrne, “Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy,” Photochem. Photobiol. 85(5), 1053–1074 (2009). [CrossRef]
16. M. Ethirajan, Y. Chen, P. Joshi, and R. K. Pandey, “The role of porphyrin chemistry in tumor imaging and photodynamic therapy,” Chem. Soc. Rev. 40(1), 340–362 (2011). [CrossRef]
17. D. Van Straten, V. Mashayekhi, H. S. De Bruijn, S. Oliveira, and D. J. Robinson, “Oncologic Photodynamic Therapy: Basic Principles, Current Clinical Status and Future Directions,” Cancers 9(12), 19–72 (2017). [CrossRef]
18. Y. Zhang and J. F. Lovell, “Porphyrins as Theranostic Agents from Prehistoric to Modern Times,” Theranostics 2(9), 905–915 (2012). [CrossRef]
19. J. Zhang, C. Jiang, J. P. Figueiró Longo, R. B. Azevedo, H. Zhang, and L. A. Muehlmann, “An updated overview on the development of new photosensitizers for anticancer photodynamic therapy,” Acta Pharm. Sin. B 8(2), 137–146 (2018). [CrossRef]
20. T. Patrice, N. Rousset, L. Bourré, and S. Thibaud, Sensitizers in Photodynamic Therapy (The Royal Society of Chemistry, 2003), Vol. 2, pp. 59–66.
21. N. Brasseur, Sensitizers for Photodynamic Therapy: Phthalocyanines (The Royal Society of Chemistry, 2003), Vol. 2, pp. 105–118. [CrossRef]
22. I. Gurol, M. Durmus, V. Ahsen, and T. Nyokong, “Synthesis, photophysical and photochemical properties of substituted zinc phthalocyanines,” Dalton Trans. 34, 3782–3791 (2007). [CrossRef]
23. N. Sekkat, H. v. d. Bergh, T. Nyokong, and N. Lange, “Like a Bolt from the Blue: Phthalocyanines in Biomedical Optics,” Molecules 17(1), 98–144 (2011). [CrossRef]
24. S. Singh, A. Aggarwal, N. V. S. D. K. Bhupathiraju, G. Arianna, K. Tiwari, and C. M. Drain, “Glycosylated Porphyrins, Phthalocyanines, and Other Porphyrinoids for Diagnostics and Therapeutics,” Chem. Rev. 115(18), 10261–10306 (2015). [CrossRef]
25. X. Jia, F.-F. Yang, J. Li, J.-Y. Liu, and J.-P. Xue, “Synthesis and in Vitro Photodynamic Activity of Oligomeric Ethylene Glycol–Quinoline Substituted Zinc(II) Phthalocyanine Derivatives,” J. Med. Chem. 56(14), 5797–5805 (2013). [CrossRef]
26. F.-L. Zhang, Q. Huang, K. Zheng, J. Li, J.-Y. Liu, and J.-P. Xue, “A novel strategy for targeting photodynamic therapy. Molecular combo of photodynamic agent zinc(ii) phthalocyanine and small molecule target-based anticancer drug erlotinib,” Chem. Commun. 49(83), 9570–9572 (2013). [CrossRef]
27. F.-L. Zhang, Q. Huang, J.-Y. Liu, M.-D. Huang, and J.-P. Xue, “Molecular-target-based anticancer photosensitizer: synthesis and in vitro photodynamic activity of erlotinib–zinc(II) phthalocyanine conjugates,” ChemMedChem 10(2), 312–320 (2015). [CrossRef]
28. J. Chen, H. Ye, M. Zhang, J. Li, J. Liu, and J. Xue, “Erlotinib analogue-substituted zinc(II) phthalocyanines for small molecular target-based photodynamic cancer therapy,” Chin. J. Chem. 34(10), 983–988 (2016). [CrossRef]
29. F.-L. Zhang, M.-R. Song, G.-K. Yuan, H.-N. Ye, Y. Tian, M.-D. Huang, J.-P. Xue, Z.-H. Zhang, and J.-Y. Liu, “A Molecular Combination of Zinc(II) Phthalocyanine and Tamoxifen Derivative for Dual Targeting Photodynamic Therapy and Hormone Therapy,” J. Med. Chem. 60(15), 6693–6703 (2017). [CrossRef]
30. J. Chen, Y. Fang, H. Liu, N. Chen, S. Chen, and J. Xue, “Quinolin-8-yloxy-substituted zinc(II) phthalocyanines for enhanced in vitro photodynamic therapy,” J. Porphyrins Phthalocyanines 22(09n10), 807–813 (2018). [CrossRef]
31. X. Zhao, Y. Huang, G. Yuan, K. Zuo, Y. Huang, J. Chen, J. Li, and J. Xue, “A novel tumor and mitochondria dual-targeted photosensitizer showing ultra-efficient photodynamic anticancer activities,” Chem. Commun. 55(6), 866–869 (2019). [CrossRef]
32. X. Zhao, H. Ma, J. Chen, F. Zhang, X. Jia, and J. Xue, “An epidermal growth factor receptor-targeted and endoplasmic reticulum-localized organic photosensitizer toward photodynamic anticancer therapy,” Eur. J. Med. Chem. 182, 111625 (2019). [CrossRef]
33. K. Berg, S. Nordstrand, P. K. Selbo, D. T. T. Tran, E. Angell-Petersen, and A. Høgset, “Disulfonated tetraphenyl chlorin (TPCS2a), a novel photosensitizer developed for clinical utilization of photochemical internalization,” Photochem. Photobiol. Sci. 10(10), 1637–1651 (2011). [CrossRef]
34. M. Hanack, G. Schmid, and M. Sommerauer, “Chromatographic Separation of the Four Possible Structural Isomers of a Tetrasubstituted Phthalocyanine: Tetrakis(2-ethylhexyloxy)phthalocyaninatonickel(II),” Photodiagn. Photodyn. Ther. 32(10), 1422–1424 (1993). [CrossRef]
35. A. Kamkaew, S. H. Lim, H. B. Lee, L. V. Kiew, L. Y. Chung, and K. Burgess, “BODIPY dyes in photodynamic therapy,” Chem. Soc. Rev. 42(1), 77–88 (2013). [CrossRef]
36. W. Gregory Roberts, K. M. Smith, J. L. Mcculiough, and M. W. Berns, “Skin photosensitivity and photodestruction of several potential photodynamic sensitizers,” Photochem. Photobiol. 49(4), 431–438 (1989). [CrossRef]
37. K. Haedicke, S. Graefe, U. Teichgraeber, and I. Hilger, “Lowering photosensitizer doses and increasing fluences induce apoptosis in tumor bearing mice,” Biomed. Opt. Express 7(7), 2641–2649 (2016). [CrossRef]
38. Z. Dong, L. Feng, Y. Hao, M. Chen, M. Gao, Y. Chao, H. Zhao, W. Zhu, J. Liu, C. Liang, Q. Zhang, and Z. Liu, “Synthesis of Hollow Biomineralized CaCO3–Polydopamine Nanoparticles for Multimodal Imaging-Guided Cancer Photodynamic Therapy with Reduced Skin Photosensitivity,” J. Am. Chem. Soc. 140(6), 2165–2178 (2018). [CrossRef]
39. A. J. Hutt and S. C. Tan, “Drug chirality and its clinical significance,” Drugs 52 Supplement 5, 1–12 (1996). [CrossRef]
40. H. Y. Yeung, P. C. Lo, D. K. Ng, and W. P. Fong, “Anti-tumor immunity of BAM-SiPc-mediated vascular photodynamic therapy in a BALB/c mouse model,” Cell. Mol. Immunol. 14(2), 223–234 (2017). [CrossRef]
