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Novel unsymmetrical silicon(IV) phthalocyanines as highly potent anticancer photosensitizers. Synthesis, characterization, and in vitro photodynamic activities
Dyes and Pigments 177 (2020) 108286
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Novel unsymmetrical silicon(IV) phthalocyanines as highly potent anticancer photosensitizers. Synthesis, characterization, and in vitro photodynamic activities Bi-Yuan Zheng a, 1, Lei Wang a, b, 1, Qing-Yan Hu a, Jie Shi a, Mei-Rong Ke a, Jian-Dong Huang a, * a
College of Chemistry, State Key Laboratory of Photocatalysis on Energy and Environment, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou, 350116, China Institute of Pharmacology, Shandong First Medical University, Shandong Academy of Medical Sciences, Taian, 271016, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Silicon(IV) phthalocyanine Unsymmetrical Tyramine Oligomeric ethylene glycol Photodynamic therapy
A series of novel unsymmetrical silicon (IV) phthalocyanines (SiPcs) di-substituted axially with tyramine and different oligomeric ethylene glycol moieties have been synthesized by alkoxy exchange reactions, and their singlet oxygen quantum yields (ΦΔ) and in vitro photodynamic activities have also been evaluated. The un symmetrical SiPcs 2–5 show slightly higher photosensitizing efficiency (ΦΔ ¼ 0.08–0.12) than the corresponding symmetrical SiPc 1 (ΦΔ ¼ 0.05), but are less efficient compared to the symmetrical SiPc 6 (ΦΔ ¼ 0.33). All the compounds exhibit high photocytotoxicities against BGC-823 cancer cells with IC50 values ranging from 3 nM to 20 nM. Particularly for the unsymmetrical SiPc 5, the IC50 value toward BGC-823 cells is significantly lower than its corresponding symmetrical SiPcs 1 and 6, which could be attributed to its higher cellular uptake. In addition, SiPc 5 can essentially bind to mitochondria and induces cell death of BGC-823 cells mainly through apoptosis. So SiPc 5 is a highly promising photosensitizer for photodynamic therapy.
1. Introduction Photodynamic therapy (PDT) is a novel and minimally invasive treatment for cancers [1–3]. It includes three critical factors: molecular oxygen, photosensitizer, and light. Under the excitation of appropriate wavelength of light, the tumor-targeted photosensitizer produces reac tive oxygen species (ROS), particularly singlet oxygen, which can induce cell impairment and cause destruction of tumor [2–4]. Phthalocyanines as representative of the second-generation photosensitizers, are prom ising candidates for PDT, due to their intense absorption in the near-infrared region (600–800 nm), low dark toxicity, and ease of chemical modification [4–7]. However, phthalocyanine-based phtho sensitizers also have some drawbacks, such as the poor hydrophilicity and aggregation tendency as a result of the macrocyclic skeleton, which remain to be further improved. Modification with various hydrophilic ligands at axial positions or on the periphery of phthalocyanines could alleviate the hydrophobic nature of the macrocyclic skeleton, lessen aggregation behavior, and improve photodynamic activities [7–13]. Apart from the desirable properties of common phthalocyanines, silicon
(IV) phthalocyanines (SiPcs) show less aggregation due to the axial steric effect. Moreover, it is worth noting that although the axially modified SiPcs have been widely reported, the unsymmetrical SiPcs as well as their structure-activity relationships have been rarely reported [14–19]. Unsymmetrical silicon phthalocyanines show some special advan tages. Compared to symmetrical SiPcs, introducing two different func tional groups at axial positions of SiPcs can afford more functions in one molecule. In addition, unlike unsymmetrically multi-substituted zinc(II) phthalocyanines, they has no structural isomers. The unsymmetrical SiPc Pc4, which has been used in clinical trial due to its high photody namic anti-tumor activity, needs a tedious preparation procedure [14, 19]. Therefore, more efficient and easy ways to synthesize unsymmet rical SiPcs and their structure-activity relationships are need to be further explored. Very recently, we have reported a series of interesting and efficient symmetrical SiPc-based photosensitizers [20–26], including an amino-substituted phthalocyanine 1 and an oligomeric ethylene glycol-substituted phthalocyanine 6 (Scheme 1 and Fig. S1). It has been
* Corresponding author. E-mail address: [email protected] (J.-D. Huang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.dyepig.2020.108286 Received 27 June 2019; Received in revised form 4 December 2019; Accepted 14 February 2020 Available online 16 February 2020 0143-7208/© 2020 Elsevier Ltd. All rights reserved.
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reported that the symmetrical SiPc 1 showed poor solubility in water and moderate photocytotoxicity [23]. In order to improve its solubility in water and photocytotoxicity, one substituent on compound 1 was designed to be replaced by different oligomeric ethylene glycol moieties, which have good amphiphilicity. Thus, four novel unsymmetrical SiPcs axially substituted with tyramine and different oligomeric ethylene glycol moieties were synthesized with moderate yields of 23–31%. Their photophysical and photochemical properties, singlet oxygen generation efficiencies, cellular uptake, and in vitro photodynamic activities were investigated. Meanwhile, the effects of length of oligomeric ethylene glycol moieties on photochemical properties, cellular uptake, and in vitro photodynamic activities were also discussed. It is interesting to find that their cellular uptake and photocytotoxicities generally enhanced with the increase of ethylene glycol chains. In addition, it is surprising to find that the IC50 value toward BGC-823 cells for the unsymmetrical SiPc 5 is c.a. 2–7 times lower than its corresponding symmetrical SiPcs 1 and 6. Thus this work can provide an important reference for further devel opment of unsymmetrical phthalocyanine-based photosensitizers that have high photodynamic activities.
summarized in Table 1. For comparison, the data of symmetrical SiPcs 1 and 6 are also added in Table 1. They all show very similar UV–vis spectra in DMF, displaying an intense and sharp Q-band at 673–681 nm, which are typical UV–vis spectra for non-aggregated phthalocyanines (Fig. S2). The UV–vis spectra of 5 in DMF at various concentrations given as an example are shown in Fig. S2e. The spectrum at tested concentrations depicts an intense and sharp Q-band at 676 nm, and with the increase of the concentration, the intensity of the Q-band peak raises linearly following the Beer-Lambert law, indicating that the SiPc 5 is free from aggregation in DMF [27]. Similar trend was observed for the other compounds 1–4 and 6. Upon excitation at 610 nm, all symmetrical SiPcs 2–5 show a fluorescence emission at 681–682 nm, with a fluorescence quantum yield (ΦF) of 0.02–0.08, relative to unsubstituted zinc(II) phthalocyanine (ZnPc) (ΦF ¼ 0.28) [28], which is slightly higher than their synthetic precursor 1 (ΦF ¼ 0.01), but much lower than the control 6 (ΦF ¼ 0.42) (see Table 1). The singlet oxygen quantum yields (ΦΔ) of the all compounds 1–6 were also measured in DMF using 1,3-diphenyli sobenzofuran (DPBF) as the scavenger. The values of ΦΔ, which were determined by the method described previously [20], are shown in Table 1. All the synthesized compounds 2–4 were found to be having slightly higher singlet oxygen generation efficiency with ΦΔ values of 0.08–0.12 than that of precursor 1 (ΦΔ ¼ 0.05) relative to ZnPc (ΦΔ ¼ 0.56) [29], but much lower than that of control 6 (ΦΔ ¼ 0.33). The
2. Results and discussion 2.1. Synthesis Scheme 1 shows the synthetic route of these unsymmetrical silicon (IV) phthalocyanines. Treatment of silicon (IV) phthalocyanine 1 with various lengths of oligomeric ethylene glycol in the presence of sodium hydride (NaH) in toluene led to ligand-exchange reaction, giving the compounds 2–5 in moderate yields (23–31%). Such ligand-exchange reactions remain extremely rare [14]. All the phthalocyanine de rivatives could be purified by silica gel column chromatography and characterized with various analysis methods including HRMS, 1H NMR, FTIR and so on [23,24].
Table 1 Photophysical and photochemical data for SiPcs 1–6 in DMF.
2.2. Photophysical and photochemical properties
SiPcs
λmax (nm)
λem (nm)a
Stokes Shift (nm)
ε � 105
ΦFb
ΦΔc
1 2 3 4 5 6
681 677 676 677 676 673
689 682 682 681 682 680
8 5 6 4 6 7
1.50 1.25 1.96 1.90 1.27 2.16
0.01 0.04 0.02 0.08 0.05 0.42
0.05 0.10 0.10 0.12 0.08 0.33
a b
The spectroscopic properties of the synthesized unsymmetrical SiPcs 2–5 were measured in N,N-dimethylformamide (DMF), and the data are
c
1
cm 1)
Excited at 610 nm. Using ZnPc in DMF as the reference (ΦF ¼ 0.28). Using ZnPc in DMF as the reference (ΦΔ ¼ 0.56).
Scheme 1. Preparation of SiPcs 2–5 and SiPc 6 [24]. 2
(M
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reasons for these observations can be ascribed to the strong photoin duced electron transfer (PET) effect of tyramine group(s) on SiPcs 1–5, which reduces their photosensitizing efficiency [22,29]. In addition, it also reveals that introduction of oligomeric ethylene glycol moiety on the axial position of phthalocyanine had significant effect on their fluorescence and singlet oxygen quantum yields. In order to better simulate the phenomenon that SiPcs 1–6 applied in a biological environment, the electronic absorption spectra of com pounds 1–6 (6 μM) in the Roswell Park Memorial Institute (RPMI) 1640 medium (formulated with 0.05% Cremophor EL, w.t.) were also measured. As shown in Fig. 1, they all exhibit a similar electronic ab sorption spectrum with a sharp and strong Q-band at 676–683 nm, which was slightly red-shifted (2–3 nm) compared to that in DMF. This phenomenon indicates that these compounds mainly exist as a mono meric form in the RPMI 1640 medium with 0.05% Cremophor EL. And it is what we expected, because non-aggregated form is very important in the process of photodynamic therapy. Since aggregation provides an efficient nonradiative energy relaxation pathway, thereby greatly shortening the triplet lifetime of the phthalocyanine molecule and then drastically reducing the overall photosensitizing efficient [21,30].
Fig. 2. Comparison of the cytotoxic effects of SiPcs 4 and 5 on BGC-823 cells in the absence and presence of light (L, λ > 610 nm) at a dose of 27 J cm 2. Values represent mean � standard deviation of three separate experiments (Student’s ttest, *p < 0.05, compared to the control group). Table 2 The IC50 values upon irradiation of SiPcs 1–6 against BGC-823 cells.
2.3. In vitro studies 2.3.1. Photocytotoxicities against BGC-823 cells The photodynamic activities of the six silicon phthalocyanines 1–6 against malignant gastric cancer cells BGC-823 were evaluated by the 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously [21]. As shown in Fig. 2, all these com pounds are essentially non-cytotoxic in the absence of light, but become cytotoxic upon illumination with red light. The IC50 values, defined as the photosensitizer concentration demanded to kill 50% of the cells, can be obtained from the dose-dependent survival curves in the presence of light. Their IC50 values are summarized in Table 2. From Table 2, it could be found that various axial ligands result in different photody namic activities on BGC-823 cancer cells. The phototoxicity towards BGC-823 cancer cells follows the trend 5 > 6 > 2 � 4 > 3 > 1. Com pound 5 conjugated with tyramine and tetraethylene glycol exhibits the highest phototoxicity with an IC50 value as low as 3 nM, which is higher than that of corresponding symmetrical SiPcs 1 (IC50 ¼ 20 nM) and 6 (IC50 ¼ 6 nM). Compared with the symmetrical SiPc 1, the phototoxic ities of the synthesized unsymmetrical SiPcs 2–5 are all improved with an IC50 value down to 3–10 nM. These results suggest that the oligomeric ethylene glycol substituents not only improve their singlet oxygen generation efficiency, but also improve their phototoxicities. By the way, compound 5 shows higher phototoxicity than that of compound 3. This result suggests that their photocytotoxicities generally enhanced slightly with the increase of ethylene glycol chains, which is consistent
SiPcs
1
2
3
4
5
6
IC50 (nM)
20 � 2
9.1 � 2.1
10 � 1
7.9 � 0.9
3.0 � 0.5
6.0 � 0.5
with our previously reported observations [24]. 2.3.2. Cellular uptake and partition coefficient To further account for the photocytotoxicities, the cellular uptake of these SiPcs 1–6 by BGC-823 cells was examined. Taking the different fluorescence generation efficiency of the SiPcs 1–6 into account, the cellular uptake of these SiPcs 1–6 was evaluated by an extraction method. After incubation with these photosensitizers for 2 h, dime thylsulfoxide (DMSO) was used to lyze the cells and extract the dyes. The dye concentrations inside the cells were quantified by measuring their fluorescence intensity. As shown in Fig. 3, the uptake of 5 was much higher than that of any other compounds, which should be one of the main reasons for its highest photocytotoxicity. It also can be seen that the uptake of these compounds follows the trend: 5 > 4 > 1 � 2 � 3 � 6. Compared to symmetrical SiPcs 1 and 6, the unsymmetrical SiPcs 2–3 substituted with triethylene glycol moiety showed comparable cellular uptake, but the unsymmetrical SiPcs 4–5 substituted with tetraethylene glycol moiety showed much higher cellular uptake. Compared to SiPc 3, SiPc 5 showed much higher photocytotoxicity, which can be attributed to its much higher cellular uptake. These results indicated that their cellular uptake and photocytotoxicity could be generally enhanced with the increase of ethylene glycol chains. In addition, SiPc 5 also showed higher cellular uptake than that of SiPc 4, which suggested that the
Fig. 1. UV–vis absorption spectra of SiPcs 1–6 (6 μM), formulated with 0.05% Cremophor EL, in RPMI 1640 cellular culture medium. The inset plots the Qband absorbance versus the concentration of 5.
Fig. 3. Percentage cellular uptake for SiPcs 1–6 determined by an extraction method. Data are expressed as mean value � standard deviation of three in dependent experiments. 3
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cellular uptake could be enhanced when the terminal methyl group was replaced by hydroxyl group, and this trend is consistent with their photocytotoxicity. Besides, the symmetrical SiPc 6 showed low cellular uptake, but it still exhibit a high photocytotoxicity, which may be due to its much higher singlet oxygen generation efficiency. Thus their differ ence in photocytotoxicities can be rationalized by their difference in cellular uptake and singlet oxygen generation efficiency. Besides, it is believed that the hydrophilic-hydrophobic character istics of the photosensitizers have an important effect on their cellular uptake [25], so their octanol/water partition coefficients (PO/W) were measured. As shown in Table S1, The Log PO/W values follow the order: 1 > 2 > 3 > 4 > 5 > 6. The symmetrical tyramine-substituted phtha locyanine 1 shows the highest Log PO/W value (Log P ¼ 1.59), meaning that it is the most lipophilic. As expected, when one tyramine group on SiPc 1 was replaced by different oligomeric ethylene glycol moieties, SiPcs 2–5 show more hydrophilic with lower Log PO/W value (Log P ¼ 0.84–1.27) than SiPc 1. In addition, the SiPcs 4 and 5 substituted with longer ethoxy chains have lower Log PO/W value relative to their analogs 2 and 3, respectively, which indicates that the elongation of ethoxy chain can enhance the hydrophilicity. The relationships between partition coefficient and cellular uptake are shown in Fig. S10. It can be found that the cellular uptake increases gradually with the decrease of Log PO/W values from 1.59 to 0.84. The cellular uptake of 5 with a Log PO/W value of 0.84 reached a maximum among these SiPcs 1–6. Although the symmetrical SiPc 6 substituted with tetraethylene glycol groups shows the most hydrophility, its cellular uptake is lower than that of SiPc 5. These results indicate that the amphiphilic photosensitizer with an appropriate Log PO/W value is more favorable for cellular uptake by BGC-823 cells than both highly hydrophobic and low hydrophobic photosensitizers.
2.3.3. Subcellular localization Since mitochondria and lysosome are important target of the initia tion of apoptosis induced by PDT, it would be interesting to reveal whether they have affinity to the subcellular component [31,32]. The subcellular localization of SiPcs 1–6 were investigated by CLSM. So, we stained the cells with SiPcs 1–6 and two fluorescent probes, Mito Tracker Green and Lyso Tracker Red, which were specific fluorescence dye for mitochondria and lysosome, respectively. As shown in Fig. 4, the fluorescence of compound 5 in BGC-823 cells is essentially overlapped with that of MitoTracker Green and partially superimposed with Lyso Tracker Red, demonstrating that 5 accumulates mainly into mitochon dria of BGC-823 cells. Similar results could be observed according to the fluorescence images of 1–4, 6 and two fluorescent probes (Fig. S5-9 in the Supporting Information). 2.3.4. Apoptosis Due to the excellent antitumor activity of SiPc 5, the mode of cell death induced by SiPc 5 was also investigated using double staining with annexin V-FITC and propidium iodide (PI) by fluorescence confocal microscopy [33,34]. As shown in Fig. 5, after PDT treatment with compound 5 for 24 h, the fluorescence of annexin V-FITC could be observed in most of BGC-823 cells, and the fluorescence of PI could be detected in more than half of the cells. Meanwhile, the cells in bright fields showed serious morphological alterations with cells shrinkage and cells membrane damage. These observations indicate that the cells after PDT treatment with 5 for 24 h were almost at early apoptotic or late apoptotic stage. However, it could be found that most of BGC-823 cells are negative for annexin V-FITC and PI after the treatment with 5 in the absence of light, and the cells in bright fields showed no morphological alterations with intact cell membrane and shuttle shape. The above phenomenon indicated that compound 5 was essentially noncytotoxic Fig. 4. Visualization of the intracellular fluorescence of BGC-823 cells by using filter sets specific for (a) SiPc 5 (in red), (b) Mito Tracker Green (in green), (c) Lyso Tracker Red (in blue), (d) Bright field image, and the corresponding superimposed image of (e) SiPcMito Tracker Green and (f) SiPc- Lyso Tracker Red. Scale bar: 25 μm. (g) The fluorescence intensity pro files of 5 and the trackers traced along the purple lines in Figure (e) and (f). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
4
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Fig. 5. The intracellular fluorescence images of Annexin V-FITC (in green) and PI (in red) in BGC-823 cells after different treatment: 5 with light (5 þ L), 5 (both at 4 nM) and control (C). The bright field and merged images are given in column 1 and 4, respectively. Scale bar: 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
toward BGC-823 cells in dark. Therefore, it can be concluded that BGC-823 cells underwent an apoptotic process when PDT-treated with 5.
highest photocytotoxities with an IC50 value as low as to 3 nM, which may be due its highest cellular uptake. In addition, SiPc 5 can essentially bind to mitochondria and induces cell death of BGC-823 cells mainly through apoptosis. Moreover, SiPc 5 shows a significant in vivo PDT effect resulting in 65% tumor growth inhibition. Thus, the compound 5 can serve as a promising photosensitizer for PDT.
2.4. Preliminary in vivo studies Finally, the in vivo tumor suppression experiment was carried out to confirm the PDT effect of SiPc 5. As shown in Fig. S11, approximately 65% of the tumor growth was inhibited after 12 days in mice treated with SiPc 5 (~2 nmol/g) and irradiation with a laser (685 � 4 nm, 0.1 W cm 2) for 5 min. This antitumor effect is comparable to a tumor tar geting photosensitizer (SiPc-RGD) under similar condition [24]. It is worth to further investigate in vivo antitumor effect of SiPc 5, in order to evaluate its clinical potency as anticancer photosensitizer. Besides, it is just our preliminary in vivo study, the antitumor effect of SiPc 5 can be enhanced by optimizing the drug dose, light dose, irradiation time after i.v. injection, and number of treatments. In contrast, mice treated with SiPc 5 without light showed a signif icant level of tumor growth, which was comparable to the growth in the mice treated with saline both with and without irradiation.
4. Experimental 4.1. General All the reactions were performed under an atmosphere of nitrogen. Dimethyl formamide (DMF) and CHCl3 was distilled over calcium hy dride. Toluene was distilled by sodium. Triethylene glycol monomethyl ether, triethylene glycol, tetraethylene glycol monomethyl ether, tetra ethylene glycol, and NaH were purchased from Acros organics (Belgium). Cremophor EL, silicon (IV) phthalocyanine dichloride (SiPcCl2), and unsubstituted zinc(II) phthalocyanine (ZnPc) were ob tained from Sigma-Aldrich. All other solvents and reagents were of re agent grade and used as received. 1 H NMR spectra were recorded on Bruker AVANCE III 400 spec trometer in DMSO‑d6. Chemical shifts were relative to internal SiMe4 (δ ¼ 0 ppm). High resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Exactive Plus Orbitrap LC-MS spectrometer. Electronic absorption spectra were measured on a Shimadzu UV-2450 UV–vis spectrophotometer. Fluorescence spectra were taken on an Edinburgh FL900/FS900 spectrofluorometer. ΦF and ΦΔ were deter mined as described in our previous manuscripts [20,21]. The oil/water partition coefficients (Log PO/W) were determined as described in our previous manuscript [35].
3. Conclusions A series of novel unsymmetrical SiPcs 2–5 di-substituted axially with tyramine and different oligomeric ethylene glycol moieties were pre pared and characterized. And they all show higher singlet oxygen gen eration efficiency than their synthetic precursor 1. All the compounds 1–6 are essentially non-aggregated and exhibit similar spectroscopic properties in RPMI 1640 medium with 0.05% Cremophor EL. For the in vitro photodynamic activities, all the compounds present high photo cytotoxicities against BGC-823 cancer cells with IC50 values ranging from 3 nM to 20 nM. Interestingly, the unsymmetrical SiPc 5 showed the 5
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4.2. Synthesis
ml) at 37 � C in a humidified 5% CO2 atmosphere. Stock solutions of the studied SiPcs 1–6 (all at 1 mM) were prepared in DMF and stored at 4 � C in the dark. The solution was then diluted to 80 μM with an aqueous solution of Cremophor EL (1%, 1 g in 100 ml of water), and further diluted with the cellular culture medium to appro priate concentrations.
Silicon phthalocyanine 2. A mixture of SiPc 1 (60 mg, 0.074 mmol), triethylene glycol monomethyl ether (2a) (485 mg, 2.95 mmol), and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) was refluxed for 7 h. After evaporating the solvent in vacuo, the residue was treated with water (100 mL) and then filtered to obtain a blue crude, which was washed thoroughly with water to remove residual NaH and compound 2a. The crude product was further purified by column chromatography (silica gel) using N,N-dimethylformamide (DMF) as eluent to give a blue solid 2 (17 mg, 27%). IR (KBr, cm 1): 3416.0, 3064.0, 2928.0, 1608.6, 1502.7, 1335.0, 1123.3, 1081.3, 909.6, 735.8. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.63–9.72 (m, 8H, Pc-Hα); 8.49–8.58 (m, 8H, Pc-Hβ); 5.46 (d, J ¼ 7.6 Hz, 2H, Ar–H); 3.01 (s, 3H, CH3); 2.84 (t, J ¼ 4.8 Hz, 2H, OCH2); 2.74 (t, J ¼ 5.6 Hz, 2H, OCH2); 2.31 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.02 (d, J ¼ 8 Hz, 2H, Ar–H); 2.08 (t, J ¼ 7.2 Hz, 2H, NCH2); 1.87 (t, J ¼ 8.4 Hz, 2H, Ar-CH2); 1.60 (t, J ¼ 4.4 Hz, 2H, CH2); 0.37 (t, J ¼ 4.4 Hz, 2H, CH2); 1.95 (t, J ¼ 4.8 Hz, 2H, CH2); HRMS (ESI): m/z calcd for C47H41N9NaO5Si [MþNa]þ, 862.2898; found, 862.2863. Silicon phthalocyanine 3. According to the above procedure, SiPc 1 (60 mg, 0.074 mmol) was treated with triethylene glycol (3a) (499 mg, 3.32 mmol) and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) obtained a blue solid 3 (19 mg, 31%). IR (KBr,cm 1): 3400.0, 3064.0, 2928.0, 1612.6, 1518.7, 1338.9, 1123.3, 1079.3, 909.6, 739.8. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.57–9.78 (m, 8H, Pc~ Hα); 8.41–8.63 (m, 8H, Pc~ Hβ); 5.38 (d, J ¼ 8.4 Hz, 2H, Ar–H); 4.31 (s, 1H, OH); 3.11 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.80 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.33 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.17 (d, J ¼ 8.4 Hz, 2H, Ar–H); 1.95 (t, J ¼ 6.8 Hz, 2H, N–CH2); 1.66 (t, J ¼ 6.8 Hz, 2H, Ar-CH2); 1.60 (t, J ¼ 5.2 Hz, 2H, OCH2); 0.38 (t, J ¼ 4.8 Hz, 2H, OCH2); 1.94 (t, J ¼ 4.8 Hz, 2H, OCH2); HRMS (ESI): m/z calcd for C46H39N9NaO5Si [MþNa]þ, 848.2741; found, 848.2703. Silicon phthalocyanine 4. According to the above procedure, SiPc 1 (60 mg, 0.074 mmol) was treated with tetraethylene glycol monomethyl ether (4a) (615 mg, 2.95 mmol) and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) obtained a blue solid 4 (17 mg, 26%). IR (KBr,cm 1): 3336.0, 3272.0, 3028.0, 2932.0, 1594.6, 1516.7, 1472.8, 1123.3, 1079.3, 909.6, 735.8. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.65–9.70 (m, 8H, Pc~ Hα); 8.50–8.56 (m, 8H, Pc~ Hβ); 5.46 (d, J ¼ 8.4 Hz, 2H, Ar–H); 3.24 (t, J ¼ 4.8 Hz, 2H, OCH2); 3.12 (s, 3H, OCH3); 3.08 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.83 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.32 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.24 (t, J ¼ 4.0 Hz, 2H, OCH2); 2.21 (d, J ¼ 8.4 Hz, 2H, Ar–H); 2.08 (t, J ¼ 7.6 Hz, 2H, N–CH2); 1.86 (t, J ¼ 7.6 Hz, 2H, Ar-CH2); 1.61 (t, J ¼ 5.2 Hz, 2H, OCH2); 0.38 (t, J ¼ 5.2 Hz, 2H, OCH2); 1.94 (t, J ¼ 5.2 Hz, 2H, OCH2); HRMS (ESI): m/z calcd for C49H45N9NaO6Si [MþNa]þ, 906.3160; found, 906.3117. Silicon phthalocyanine 5. According to the above procedure, SiPc 1 (60 mg, 0.074 mmol) was treated with tetraethylene glycol (5a) (645 mg, 3.32 mmol) and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) ob tained a blue solid 5 (15 mg, 23%). IR (KBr,cm 1): 3392.6, 3214.8, 3054.2, 2924.1, 1613.0, 1336.3, 1126.5, 1081.1, 912.4, 739.5. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.64–9.76 (m, 8H, Pc~ Hα); 8.49–8.63 (m, 8H, Pc~ Hβ); 5.45 (d, J ¼ 7.6 Hz, 2H, Ar–H); 4.47 (s, 1H, OH); 3.20 (t, J ¼ 4.8 Hz, 2H, OCH2); 3.11 (t, J ¼ 4.8 Hz, 2H, OCH2); 2.85 (t, J ¼ 4.8 Hz, 2H, OCH2); 2.32 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.24 (t, J ¼ 4.0 Hz, 2H, OCH2); 2.21 (d, J ¼ 8.4 Hz, 2H, Ar–H); 2.09 (t, J ¼ 7.6 Hz, 2H, N–CH2); 1.85 (t, J ¼ 7.6 Hz, 2H, Ar-CH2); 1.60 (t, J ¼ 5.2 Hz, 2H, OCH2); 0.37 (t, J ¼ 4.8 Hz, 2H, OCH2); 1.94 (t, J ¼ 4.8 Hz, 2H, OCH2); HRMS (ESI): m/ z calcd for C48H43N9NaO6Si [MþNa]þ, 892.3003; found, 892.3021.
4.3.2. In vitro photocytotoxicity The photocytotoxicity assay is the same as the procedure described previously [21]. Briefly, About 2 � 104 BGC-823 cells per well were seeded in 96-well plate and incubated overnight for cell adherence and growth. The cells were then incubated with various concentrations of SiPcs 1–6 for 2 h under the same conditions. After that, the cells were rinsed with PBS and re-fed with 100 μl of the culture medium before irradiated with 27 J/cm2 of a red light (λ > 610 nm, 15 mW/cm2 for 30 min). The light source consisted of a 500 W halogen lamp (Philips), a water tank for cooling and a color glass filter cut-on 610 nm. After illumination, the cells were incubated at 37 � C under 5% CO2 overnight. Then cell viability was determined by the colorimetric MTT assay as described previously [21]. 4.3.3. Cellular uptake About 2 � 104 BGC-823 cells in RPMI 1640 culture medium were seeded in 96-well plate and incubated overnight. The cells were then incubated with SiPcs 1–6 (all at 2 μM) for 2 h under the same conditions. After that, the cells were washed with PBS twice. After removing the PBS, the cells were lyzed with DMSO and sonicated for 5 min. The extracted solution were measured in a fluorescence plate reader (TECAN Infinite M200, Switzerland), which excited at 610 nm and monitored at 680 � 10 nm. The cellular uptake was calculated by comparing with the respective calibration curves to give the uptake concentrations. 4.3.4. Subcellular localization studies About 6 � 104 BGC-823 cells in the culture medium were seeded on a confocal dish. After removing the medium, the cells were incubated with the solutions of 1–6 in the medium (both at 2 μM, 0.5 ml) for 0.5 h under the same condition, and then Lyso Tracker Red (5 μM, 20 μl) was added for further 60 min co-incubation, followed by Mito Tracker Green (5 μM, 20 μl) for further 30 min co-incubation, leading to a total incubation time of 2 h for the photosensitizer, 1.5 h for Lyso Tracker Red, and 0.5 h for Mito Tracker Green. Subsequently, the cells were then rinsed with PBS and viewed with a LEICA TCS SPE confocal microscope equipped with 488 nm, 532 nm, and 635 nm solid-state lasers. Mito Tracker Green and Lyso Tracker Red were excited at 488 nm and 532 nm monitored at 499–529 nm and 552–617 nm, respectively, while compounds 1–6 were excited at 635 nm and monitored at 645–750 nm. The subcellular lo calizations of 1–6 were revealed by comparing the intracellular fluo rescence images caused by Mito Tracker Green, Lyso Tracker Red, and these phthalocyanines. 4.3.5. Apoptosis About 1 � 105 BGC-823 cells in the culture medium (0.5 ml) were seeded on a confocal dish (three dishes for each compound) and incu bated overnight at 37 � C under 5% CO2. After removing the medium, the cells were incubated with the solution of 5 in the medium (4 nM, 0.5 ml) for 2 h under the same conditions. The cells were then rinsed with PBS and refilled with 0.5 ml of the culture medium. Subsequently, the cells were illuminated using a red light for 30 min (λ > 610 nm, 15 mW/cm2, 27 J/cm2) or kept in the dark. After that, the cells were further incubated in the dark for 24 h. At the set time, the cells were rinsed with PBS, followed by addition of 0.5 ml of binding buffer containing Annexin VFITC (5 μl) and propidium iodium (PI) (5 μl), and further incubation at 37 � C for 10 min in the dark. The cells were then viewed using a LEICA TCS SPE fluorescence confocal microscope. Annexin V-FITC was excited at 488 nm and monitored at 499–529 nm, while PI was excited at 532 nm and monitored at 545–617 nm. The cell populations at different
4.3. In vitro studies 4.3.1. Cell culture The BGC-823 human gastric cancer cells (from ATCC) were main tained in RPMI 1640 medium (with phenol-red) supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 μg/ 6
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phase of cell death, namely viable (annexin V- FITC /PI ), early apoptotic (annexin V-FITCþ/PI ), and necrotic or late-stage apoptotic (annexin V-FITCþ/PIþ), were determined by digitally merging fluores cence images and phase contrast images.
[8] Schmitt F, Juillerat-Jeanneret L. Drug targeting strategies for photodynamic therapy. Anticancer Agents Med. Chem. 2012;12:500–25. [9] Shirasu N, Nam SO, Kuroki M. Tumor-targeted photodynamic therapy. Anticancer Res 2013;33:2823–31. [10] Taquet J-P, Frochot C, Manneville V, Barberi-Heyob M. Phthalocyanines covalently bound to biomolecules for a targeted photodynamic therapy. Curr Med Chem 2007;14:1673–87. [11] Sharman WM, van Lier JE, Allen CM. Targeted photodynamic therapy via receptor mediated delivery systems. Adv Drug Deliv Rev 2004;56:53–76. [12] van Dongen GAMS, Visser GWM, Vrouenraets MB. Photosensitizer-antibody conjugates for detection and therapy of cancer. Adv Drug Deliv Rev 2004;56: 31–52. [13] Li F, Zhao Y, Mao C, Kong YQ, Ming X. RGD-modified albumin nanoconjugates for targeted delivery of a porphyrin photosensitizer. Mol Pharm 2017;14:2793–804. [14] Lo P-C, Huang J-D, Cheng DY, Chan EY, Fong WP, Ko WH, Ng DK. 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Synthesis, spectroscopic and fibroblast activation protein (FAP)-responsive properties of phthalocyanine-doxorubicin conjugates. Chemistry 2018;3:5405–11. [19] Oleinick NL, Antunez AR, Clay ME, Rihter BD, Kenney ME. New phthalocyanine photosensitizers for photodynamic therapy. Photochem Photobiol 1993;57:242–7. [20] Shen X-M, Jiang X-J, Huang C-C, Zhang H-H, Huang J-D. Highly photostable silicon (IV) phthalocyanines containing adamantane moieties: synthesis, structure, and properties. Tetrahedron 2010;66:9041–8. [21] Shen XM, Zheng BY, Huang XR, Wang L, Huang JD. The first silicon(IV) phthalocyanine-nucleoside conjugates with high photodynamic activity. Dalton Trans 2013;42:10398–403. [22] Zheng B-Y, Jiang X-J, Lin T, Ke M-R, Huang J-D. Novel silicon(IV) phthalocyanines containing piperidinyl moieties: synthesis and in vitro antifungal photodynamic activities. Dyes Pigments 2015;112:311–6. [23] Zheng Y-W, Chen S-F, Zheng B-Y, Ke M-R, Huang J-D. A silicon(IV) phthalocyanine-folate conjugate as an efficient photosensitizer. Chem Lett 2014; 43:1701–3. [24] Zheng B-Y, Yang X-Q, Zhao Y, Zheng Q-F, Ke M-R, Lin T, Chen R-X, Ho KKK, Kumar N, Huang J-D. Synthesis and photodynamic activities of integrin-targeting silicon(IV) phthalocyanine-cRGD conjugates. Eur J Med Chem 2018;155:24–33. [25] Li X, Peng X-H, Zheng B-D, Tang J, Zhao Y, Zheng B-Y, Ke M-R, Huang J-D. New application of phthalocyanine molecules: from photodynamic therapy to photothermal therapy by means of structural regulation rather than formation of aggregates. Chem Sci 2018;9:2098–104. [26] Zheng B-Y, Lin T, Yang H-H, Huang J-D. Photodynamic inactivation of Candida albicans sensitized by a series of novel axially di-substituted silicon (IV) phthalocyanines. Dyes Pigments 2013;96:547–53. [27] Sun Q, Zheng B-Y, Zhang Y-H, Zhuang J-J, Ke M-R, Huang J-D. Highly photocytotoxic silicon(IV) phthalocyanines axially modified with L-tyrosine derivatives: effects of mode of axial substituent connection and of formulation on photodynamic activity. Dyes Pigments 2017;141:521–9. [28] Scalise I, Durantini EN. Synthesis, properties, and photodynamic inactivation of Escherichia coli using a cationic and a noncharged Zn (II) pyridyloxyphthalocyanine derivatives. Bioorg Med Chem 2005;13:3037–45. [29] Maree MD, Kuznetsova N, Nyokong T. Silicon octaphenoxyphthalocyanines: photostability and singlet oxygen quantum yields. J Photochem Photobiol Chem 2001;140:117–25. [30] Li X-Y, He X, Ng ACH, Wu C, Ng DKP. Influence of surfactants on the aggregation behavior of water-soluble dendritic phthalocyanines. Macromolecules 2000;33: 2119–23. [31] Morgan J, Oseroff AR. Mitochondria-based photodynamic anti-cancer therapy. Adv Drug Deliv Rev 2001;49:71–86. [32] Mroz P, Yaroslavsky A, Kharkwal GB, Hamblin MR. Cell death pathways in photodynamic therapy of cancer. Cancers 2011;3:2516–39. [33] Zheng B-Y, Shen X-M, Zhao D-M, Cai Y-B, Ke M-R, Huang J-D. Silicon(IV) phthalocyanines substituted axially with different nucleoside moieties. Effects of nucleoside type on the photosensitizing efficiencies and in vitro photodynamic activities. J Photochem Photobiol B 2016;159:196–204. [34] Vermes I, Haanena C, Steffens-Nakkena H, Reutellingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184:39–51. [35] Zheng BY, Zhang HP, Ke MR, Huang JD. Synthesis and antifungal photodynamic activities of a series of novel zinc(II) phthalocyanines substituted with piperazinyl moieties. Dyes Pigments 2013;99:185–91.
4.4. In vivo studies Hepatoma H22 cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China); KM mice were purchased from Wushi Animal Co. Ltd. (Fuzhou, China). All animal studies were performed in compliance with guidelines of the Animal Care Committee of Fuzhou University, and also approved by the committee. To build a subcutaneous tumor model, H22 cells (~1 � 107 cells in 200 μl) were inoculated subcutaneously on the axilla of the KM mice (20–22 g). When the tumors had grown to about100 mm3, the tumor bearing mice were assigned to four groups and treated with PBS, laser (L), SiPc 5, and PDT (SiPc 5 þ L). For PDT treatment, a SiPc 5 aqueous solution containing 1% Cremophor EL (200 μM, 200 μl) was intravenously injected into the tail vein of the tumor-bearing mice (5 mice each group). After 24 h postinjection, the mice were irradiated with a laser (685 � 4 nm, 0.1 W cm 2) for 5 min. The tumor size was determined every other day by using a caliper for a duration of 12 days and calculated using the following formula: volume ¼ (tumor length) � (tumor width)2 � 0.5. Declaration of competing interest There are no interests to declare. CRediT authorship contribution statement Bi-Yuan Zheng: Formal analysis, Writing - original draft. Lei Wang: Formal analysis, Writing - original draft. Qing-Yan Hu: Formal analysis. Jie Shi: Formal analysis. Mei-Rong Ke: Formal analysis, Writing original draft. Jian-Dong Huang: Supervision. Acknowledgements This work was supported by Scientific Research Fund of the National Board of Health and Family Planning (the Joint Research Project of Health and Education of Fujian), China (Grant No. WKJ-FJ-32), Marine High-Tech Programs of Fujian Province, China (Grant No. 2016–14), the Natural Science Foundation of Fujian, China (Grant No. 2018J05016), and the Foundation of Education Department of Fujian, China (No. JAT170106). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2020.108286. References [1] Robertson C, Evans DH, Abrahamse H. Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications for PDT. J Photochem PhotobiolB: Biol 2009;96:1–8. [2] Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Canc 2003;3:380–7. [3] Brown SB, Brown EA, Walker I. The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol 2004;5:497–508. [4] Detty MR, Gibson SL, Wagner SJ. Current clinical and preclinical photosensitizers for use in photodynamic therapy. J Med Chem 2004;47:3897–915. [5] Triesscheijn M, Baas P, Schellens JHM, Stewart FA. Photodynamic therapy in oncology. Oncol 2006;11:1034–44. [6] Bugaj AM. Targeted photodynamic therapy–a promising strategy of tumor treatment. Photochem Photobiol Sci 2011;10:1097–109. [7] Li X, Zheng B-D, Peng X-H, Li S-Z, Ying J-W, Zhao Y, Huang J-D, Yoon J. Phthalocyanines as medicinal photosensitizers: developments in the last five years. Coord Chem Rev 2019;379:147–60.
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Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
Novel unsymmetrical silicon(IV) phthalocyanines as highly potent anticancer photosensitizers. Synthesis, characterization, and in vitro photodynamic activities Bi-Yuan Zheng a, 1, Lei Wang a, b, 1, Qing-Yan Hu a, Jie Shi a, Mei-Rong Ke a, Jian-Dong Huang a, * a
College of Chemistry, State Key Laboratory of Photocatalysis on Energy and Environment, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou, 350116, China Institute of Pharmacology, Shandong First Medical University, Shandong Academy of Medical Sciences, Taian, 271016, China
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Silicon(IV) phthalocyanine Unsymmetrical Tyramine Oligomeric ethylene glycol Photodynamic therapy
A series of novel unsymmetrical silicon (IV) phthalocyanines (SiPcs) di-substituted axially with tyramine and different oligomeric ethylene glycol moieties have been synthesized by alkoxy exchange reactions, and their singlet oxygen quantum yields (ΦΔ) and in vitro photodynamic activities have also been evaluated. The un symmetrical SiPcs 2–5 show slightly higher photosensitizing efficiency (ΦΔ ¼ 0.08–0.12) than the corresponding symmetrical SiPc 1 (ΦΔ ¼ 0.05), but are less efficient compared to the symmetrical SiPc 6 (ΦΔ ¼ 0.33). All the compounds exhibit high photocytotoxicities against BGC-823 cancer cells with IC50 values ranging from 3 nM to 20 nM. Particularly for the unsymmetrical SiPc 5, the IC50 value toward BGC-823 cells is significantly lower than its corresponding symmetrical SiPcs 1 and 6, which could be attributed to its higher cellular uptake. In addition, SiPc 5 can essentially bind to mitochondria and induces cell death of BGC-823 cells mainly through apoptosis. So SiPc 5 is a highly promising photosensitizer for photodynamic therapy.
1. Introduction Photodynamic therapy (PDT) is a novel and minimally invasive treatment for cancers [1–3]. It includes three critical factors: molecular oxygen, photosensitizer, and light. Under the excitation of appropriate wavelength of light, the tumor-targeted photosensitizer produces reac tive oxygen species (ROS), particularly singlet oxygen, which can induce cell impairment and cause destruction of tumor [2–4]. Phthalocyanines as representative of the second-generation photosensitizers, are prom ising candidates for PDT, due to their intense absorption in the near-infrared region (600–800 nm), low dark toxicity, and ease of chemical modification [4–7]. However, phthalocyanine-based phtho sensitizers also have some drawbacks, such as the poor hydrophilicity and aggregation tendency as a result of the macrocyclic skeleton, which remain to be further improved. Modification with various hydrophilic ligands at axial positions or on the periphery of phthalocyanines could alleviate the hydrophobic nature of the macrocyclic skeleton, lessen aggregation behavior, and improve photodynamic activities [7–13]. Apart from the desirable properties of common phthalocyanines, silicon
(IV) phthalocyanines (SiPcs) show less aggregation due to the axial steric effect. Moreover, it is worth noting that although the axially modified SiPcs have been widely reported, the unsymmetrical SiPcs as well as their structure-activity relationships have been rarely reported [14–19]. Unsymmetrical silicon phthalocyanines show some special advan tages. Compared to symmetrical SiPcs, introducing two different func tional groups at axial positions of SiPcs can afford more functions in one molecule. In addition, unlike unsymmetrically multi-substituted zinc(II) phthalocyanines, they has no structural isomers. The unsymmetrical SiPc Pc4, which has been used in clinical trial due to its high photody namic anti-tumor activity, needs a tedious preparation procedure [14, 19]. Therefore, more efficient and easy ways to synthesize unsymmet rical SiPcs and their structure-activity relationships are need to be further explored. Very recently, we have reported a series of interesting and efficient symmetrical SiPc-based photosensitizers [20–26], including an amino-substituted phthalocyanine 1 and an oligomeric ethylene glycol-substituted phthalocyanine 6 (Scheme 1 and Fig. S1). It has been
* Corresponding author. E-mail address: [email protected] (J.-D. Huang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.dyepig.2020.108286 Received 27 June 2019; Received in revised form 4 December 2019; Accepted 14 February 2020 Available online 16 February 2020 0143-7208/© 2020 Elsevier Ltd. All rights reserved.
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reported that the symmetrical SiPc 1 showed poor solubility in water and moderate photocytotoxicity [23]. In order to improve its solubility in water and photocytotoxicity, one substituent on compound 1 was designed to be replaced by different oligomeric ethylene glycol moieties, which have good amphiphilicity. Thus, four novel unsymmetrical SiPcs axially substituted with tyramine and different oligomeric ethylene glycol moieties were synthesized with moderate yields of 23–31%. Their photophysical and photochemical properties, singlet oxygen generation efficiencies, cellular uptake, and in vitro photodynamic activities were investigated. Meanwhile, the effects of length of oligomeric ethylene glycol moieties on photochemical properties, cellular uptake, and in vitro photodynamic activities were also discussed. It is interesting to find that their cellular uptake and photocytotoxicities generally enhanced with the increase of ethylene glycol chains. In addition, it is surprising to find that the IC50 value toward BGC-823 cells for the unsymmetrical SiPc 5 is c.a. 2–7 times lower than its corresponding symmetrical SiPcs 1 and 6. Thus this work can provide an important reference for further devel opment of unsymmetrical phthalocyanine-based photosensitizers that have high photodynamic activities.
summarized in Table 1. For comparison, the data of symmetrical SiPcs 1 and 6 are also added in Table 1. They all show very similar UV–vis spectra in DMF, displaying an intense and sharp Q-band at 673–681 nm, which are typical UV–vis spectra for non-aggregated phthalocyanines (Fig. S2). The UV–vis spectra of 5 in DMF at various concentrations given as an example are shown in Fig. S2e. The spectrum at tested concentrations depicts an intense and sharp Q-band at 676 nm, and with the increase of the concentration, the intensity of the Q-band peak raises linearly following the Beer-Lambert law, indicating that the SiPc 5 is free from aggregation in DMF [27]. Similar trend was observed for the other compounds 1–4 and 6. Upon excitation at 610 nm, all symmetrical SiPcs 2–5 show a fluorescence emission at 681–682 nm, with a fluorescence quantum yield (ΦF) of 0.02–0.08, relative to unsubstituted zinc(II) phthalocyanine (ZnPc) (ΦF ¼ 0.28) [28], which is slightly higher than their synthetic precursor 1 (ΦF ¼ 0.01), but much lower than the control 6 (ΦF ¼ 0.42) (see Table 1). The singlet oxygen quantum yields (ΦΔ) of the all compounds 1–6 were also measured in DMF using 1,3-diphenyli sobenzofuran (DPBF) as the scavenger. The values of ΦΔ, which were determined by the method described previously [20], are shown in Table 1. All the synthesized compounds 2–4 were found to be having slightly higher singlet oxygen generation efficiency with ΦΔ values of 0.08–0.12 than that of precursor 1 (ΦΔ ¼ 0.05) relative to ZnPc (ΦΔ ¼ 0.56) [29], but much lower than that of control 6 (ΦΔ ¼ 0.33). The
2. Results and discussion 2.1. Synthesis Scheme 1 shows the synthetic route of these unsymmetrical silicon (IV) phthalocyanines. Treatment of silicon (IV) phthalocyanine 1 with various lengths of oligomeric ethylene glycol in the presence of sodium hydride (NaH) in toluene led to ligand-exchange reaction, giving the compounds 2–5 in moderate yields (23–31%). Such ligand-exchange reactions remain extremely rare [14]. All the phthalocyanine de rivatives could be purified by silica gel column chromatography and characterized with various analysis methods including HRMS, 1H NMR, FTIR and so on [23,24].
Table 1 Photophysical and photochemical data for SiPcs 1–6 in DMF.
2.2. Photophysical and photochemical properties
SiPcs
λmax (nm)
λem (nm)a
Stokes Shift (nm)
ε � 105
ΦFb
ΦΔc
1 2 3 4 5 6
681 677 676 677 676 673
689 682 682 681 682 680
8 5 6 4 6 7
1.50 1.25 1.96 1.90 1.27 2.16
0.01 0.04 0.02 0.08 0.05 0.42
0.05 0.10 0.10 0.12 0.08 0.33
a b
The spectroscopic properties of the synthesized unsymmetrical SiPcs 2–5 were measured in N,N-dimethylformamide (DMF), and the data are
c
1
cm 1)
Excited at 610 nm. Using ZnPc in DMF as the reference (ΦF ¼ 0.28). Using ZnPc in DMF as the reference (ΦΔ ¼ 0.56).
Scheme 1. Preparation of SiPcs 2–5 and SiPc 6 [24]. 2
(M
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reasons for these observations can be ascribed to the strong photoin duced electron transfer (PET) effect of tyramine group(s) on SiPcs 1–5, which reduces their photosensitizing efficiency [22,29]. In addition, it also reveals that introduction of oligomeric ethylene glycol moiety on the axial position of phthalocyanine had significant effect on their fluorescence and singlet oxygen quantum yields. In order to better simulate the phenomenon that SiPcs 1–6 applied in a biological environment, the electronic absorption spectra of com pounds 1–6 (6 μM) in the Roswell Park Memorial Institute (RPMI) 1640 medium (formulated with 0.05% Cremophor EL, w.t.) were also measured. As shown in Fig. 1, they all exhibit a similar electronic ab sorption spectrum with a sharp and strong Q-band at 676–683 nm, which was slightly red-shifted (2–3 nm) compared to that in DMF. This phenomenon indicates that these compounds mainly exist as a mono meric form in the RPMI 1640 medium with 0.05% Cremophor EL. And it is what we expected, because non-aggregated form is very important in the process of photodynamic therapy. Since aggregation provides an efficient nonradiative energy relaxation pathway, thereby greatly shortening the triplet lifetime of the phthalocyanine molecule and then drastically reducing the overall photosensitizing efficient [21,30].
Fig. 2. Comparison of the cytotoxic effects of SiPcs 4 and 5 on BGC-823 cells in the absence and presence of light (L, λ > 610 nm) at a dose of 27 J cm 2. Values represent mean � standard deviation of three separate experiments (Student’s ttest, *p < 0.05, compared to the control group). Table 2 The IC50 values upon irradiation of SiPcs 1–6 against BGC-823 cells.
2.3. In vitro studies 2.3.1. Photocytotoxicities against BGC-823 cells The photodynamic activities of the six silicon phthalocyanines 1–6 against malignant gastric cancer cells BGC-823 were evaluated by the 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously [21]. As shown in Fig. 2, all these com pounds are essentially non-cytotoxic in the absence of light, but become cytotoxic upon illumination with red light. The IC50 values, defined as the photosensitizer concentration demanded to kill 50% of the cells, can be obtained from the dose-dependent survival curves in the presence of light. Their IC50 values are summarized in Table 2. From Table 2, it could be found that various axial ligands result in different photody namic activities on BGC-823 cancer cells. The phototoxicity towards BGC-823 cancer cells follows the trend 5 > 6 > 2 � 4 > 3 > 1. Com pound 5 conjugated with tyramine and tetraethylene glycol exhibits the highest phototoxicity with an IC50 value as low as 3 nM, which is higher than that of corresponding symmetrical SiPcs 1 (IC50 ¼ 20 nM) and 6 (IC50 ¼ 6 nM). Compared with the symmetrical SiPc 1, the phototoxic ities of the synthesized unsymmetrical SiPcs 2–5 are all improved with an IC50 value down to 3–10 nM. These results suggest that the oligomeric ethylene glycol substituents not only improve their singlet oxygen generation efficiency, but also improve their phototoxicities. By the way, compound 5 shows higher phototoxicity than that of compound 3. This result suggests that their photocytotoxicities generally enhanced slightly with the increase of ethylene glycol chains, which is consistent
SiPcs
1
2
3
4
5
6
IC50 (nM)
20 � 2
9.1 � 2.1
10 � 1
7.9 � 0.9
3.0 � 0.5
6.0 � 0.5
with our previously reported observations [24]. 2.3.2. Cellular uptake and partition coefficient To further account for the photocytotoxicities, the cellular uptake of these SiPcs 1–6 by BGC-823 cells was examined. Taking the different fluorescence generation efficiency of the SiPcs 1–6 into account, the cellular uptake of these SiPcs 1–6 was evaluated by an extraction method. After incubation with these photosensitizers for 2 h, dime thylsulfoxide (DMSO) was used to lyze the cells and extract the dyes. The dye concentrations inside the cells were quantified by measuring their fluorescence intensity. As shown in Fig. 3, the uptake of 5 was much higher than that of any other compounds, which should be one of the main reasons for its highest photocytotoxicity. It also can be seen that the uptake of these compounds follows the trend: 5 > 4 > 1 � 2 � 3 � 6. Compared to symmetrical SiPcs 1 and 6, the unsymmetrical SiPcs 2–3 substituted with triethylene glycol moiety showed comparable cellular uptake, but the unsymmetrical SiPcs 4–5 substituted with tetraethylene glycol moiety showed much higher cellular uptake. Compared to SiPc 3, SiPc 5 showed much higher photocytotoxicity, which can be attributed to its much higher cellular uptake. These results indicated that their cellular uptake and photocytotoxicity could be generally enhanced with the increase of ethylene glycol chains. In addition, SiPc 5 also showed higher cellular uptake than that of SiPc 4, which suggested that the
Fig. 1. UV–vis absorption spectra of SiPcs 1–6 (6 μM), formulated with 0.05% Cremophor EL, in RPMI 1640 cellular culture medium. The inset plots the Qband absorbance versus the concentration of 5.
Fig. 3. Percentage cellular uptake for SiPcs 1–6 determined by an extraction method. Data are expressed as mean value � standard deviation of three in dependent experiments. 3
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cellular uptake could be enhanced when the terminal methyl group was replaced by hydroxyl group, and this trend is consistent with their photocytotoxicity. Besides, the symmetrical SiPc 6 showed low cellular uptake, but it still exhibit a high photocytotoxicity, which may be due to its much higher singlet oxygen generation efficiency. Thus their differ ence in photocytotoxicities can be rationalized by their difference in cellular uptake and singlet oxygen generation efficiency. Besides, it is believed that the hydrophilic-hydrophobic character istics of the photosensitizers have an important effect on their cellular uptake [25], so their octanol/water partition coefficients (PO/W) were measured. As shown in Table S1, The Log PO/W values follow the order: 1 > 2 > 3 > 4 > 5 > 6. The symmetrical tyramine-substituted phtha locyanine 1 shows the highest Log PO/W value (Log P ¼ 1.59), meaning that it is the most lipophilic. As expected, when one tyramine group on SiPc 1 was replaced by different oligomeric ethylene glycol moieties, SiPcs 2–5 show more hydrophilic with lower Log PO/W value (Log P ¼ 0.84–1.27) than SiPc 1. In addition, the SiPcs 4 and 5 substituted with longer ethoxy chains have lower Log PO/W value relative to their analogs 2 and 3, respectively, which indicates that the elongation of ethoxy chain can enhance the hydrophilicity. The relationships between partition coefficient and cellular uptake are shown in Fig. S10. It can be found that the cellular uptake increases gradually with the decrease of Log PO/W values from 1.59 to 0.84. The cellular uptake of 5 with a Log PO/W value of 0.84 reached a maximum among these SiPcs 1–6. Although the symmetrical SiPc 6 substituted with tetraethylene glycol groups shows the most hydrophility, its cellular uptake is lower than that of SiPc 5. These results indicate that the amphiphilic photosensitizer with an appropriate Log PO/W value is more favorable for cellular uptake by BGC-823 cells than both highly hydrophobic and low hydrophobic photosensitizers.
2.3.3. Subcellular localization Since mitochondria and lysosome are important target of the initia tion of apoptosis induced by PDT, it would be interesting to reveal whether they have affinity to the subcellular component [31,32]. The subcellular localization of SiPcs 1–6 were investigated by CLSM. So, we stained the cells with SiPcs 1–6 and two fluorescent probes, Mito Tracker Green and Lyso Tracker Red, which were specific fluorescence dye for mitochondria and lysosome, respectively. As shown in Fig. 4, the fluorescence of compound 5 in BGC-823 cells is essentially overlapped with that of MitoTracker Green and partially superimposed with Lyso Tracker Red, demonstrating that 5 accumulates mainly into mitochon dria of BGC-823 cells. Similar results could be observed according to the fluorescence images of 1–4, 6 and two fluorescent probes (Fig. S5-9 in the Supporting Information). 2.3.4. Apoptosis Due to the excellent antitumor activity of SiPc 5, the mode of cell death induced by SiPc 5 was also investigated using double staining with annexin V-FITC and propidium iodide (PI) by fluorescence confocal microscopy [33,34]. As shown in Fig. 5, after PDT treatment with compound 5 for 24 h, the fluorescence of annexin V-FITC could be observed in most of BGC-823 cells, and the fluorescence of PI could be detected in more than half of the cells. Meanwhile, the cells in bright fields showed serious morphological alterations with cells shrinkage and cells membrane damage. These observations indicate that the cells after PDT treatment with 5 for 24 h were almost at early apoptotic or late apoptotic stage. However, it could be found that most of BGC-823 cells are negative for annexin V-FITC and PI after the treatment with 5 in the absence of light, and the cells in bright fields showed no morphological alterations with intact cell membrane and shuttle shape. The above phenomenon indicated that compound 5 was essentially noncytotoxic Fig. 4. Visualization of the intracellular fluorescence of BGC-823 cells by using filter sets specific for (a) SiPc 5 (in red), (b) Mito Tracker Green (in green), (c) Lyso Tracker Red (in blue), (d) Bright field image, and the corresponding superimposed image of (e) SiPcMito Tracker Green and (f) SiPc- Lyso Tracker Red. Scale bar: 25 μm. (g) The fluorescence intensity pro files of 5 and the trackers traced along the purple lines in Figure (e) and (f). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 5. The intracellular fluorescence images of Annexin V-FITC (in green) and PI (in red) in BGC-823 cells after different treatment: 5 with light (5 þ L), 5 (both at 4 nM) and control (C). The bright field and merged images are given in column 1 and 4, respectively. Scale bar: 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
toward BGC-823 cells in dark. Therefore, it can be concluded that BGC-823 cells underwent an apoptotic process when PDT-treated with 5.
highest photocytotoxities with an IC50 value as low as to 3 nM, which may be due its highest cellular uptake. In addition, SiPc 5 can essentially bind to mitochondria and induces cell death of BGC-823 cells mainly through apoptosis. Moreover, SiPc 5 shows a significant in vivo PDT effect resulting in 65% tumor growth inhibition. Thus, the compound 5 can serve as a promising photosensitizer for PDT.
2.4. Preliminary in vivo studies Finally, the in vivo tumor suppression experiment was carried out to confirm the PDT effect of SiPc 5. As shown in Fig. S11, approximately 65% of the tumor growth was inhibited after 12 days in mice treated with SiPc 5 (~2 nmol/g) and irradiation with a laser (685 � 4 nm, 0.1 W cm 2) for 5 min. This antitumor effect is comparable to a tumor tar geting photosensitizer (SiPc-RGD) under similar condition [24]. It is worth to further investigate in vivo antitumor effect of SiPc 5, in order to evaluate its clinical potency as anticancer photosensitizer. Besides, it is just our preliminary in vivo study, the antitumor effect of SiPc 5 can be enhanced by optimizing the drug dose, light dose, irradiation time after i.v. injection, and number of treatments. In contrast, mice treated with SiPc 5 without light showed a signif icant level of tumor growth, which was comparable to the growth in the mice treated with saline both with and without irradiation.
4. Experimental 4.1. General All the reactions were performed under an atmosphere of nitrogen. Dimethyl formamide (DMF) and CHCl3 was distilled over calcium hy dride. Toluene was distilled by sodium. Triethylene glycol monomethyl ether, triethylene glycol, tetraethylene glycol monomethyl ether, tetra ethylene glycol, and NaH were purchased from Acros organics (Belgium). Cremophor EL, silicon (IV) phthalocyanine dichloride (SiPcCl2), and unsubstituted zinc(II) phthalocyanine (ZnPc) were ob tained from Sigma-Aldrich. All other solvents and reagents were of re agent grade and used as received. 1 H NMR spectra were recorded on Bruker AVANCE III 400 spec trometer in DMSO‑d6. Chemical shifts were relative to internal SiMe4 (δ ¼ 0 ppm). High resolution mass spectra (HRMS) were recorded on a Thermo Fisher Scientific Exactive Plus Orbitrap LC-MS spectrometer. Electronic absorption spectra were measured on a Shimadzu UV-2450 UV–vis spectrophotometer. Fluorescence spectra were taken on an Edinburgh FL900/FS900 spectrofluorometer. ΦF and ΦΔ were deter mined as described in our previous manuscripts [20,21]. The oil/water partition coefficients (Log PO/W) were determined as described in our previous manuscript [35].
3. Conclusions A series of novel unsymmetrical SiPcs 2–5 di-substituted axially with tyramine and different oligomeric ethylene glycol moieties were pre pared and characterized. And they all show higher singlet oxygen gen eration efficiency than their synthetic precursor 1. All the compounds 1–6 are essentially non-aggregated and exhibit similar spectroscopic properties in RPMI 1640 medium with 0.05% Cremophor EL. For the in vitro photodynamic activities, all the compounds present high photo cytotoxicities against BGC-823 cancer cells with IC50 values ranging from 3 nM to 20 nM. Interestingly, the unsymmetrical SiPc 5 showed the 5
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4.2. Synthesis
ml) at 37 � C in a humidified 5% CO2 atmosphere. Stock solutions of the studied SiPcs 1–6 (all at 1 mM) were prepared in DMF and stored at 4 � C in the dark. The solution was then diluted to 80 μM with an aqueous solution of Cremophor EL (1%, 1 g in 100 ml of water), and further diluted with the cellular culture medium to appro priate concentrations.
Silicon phthalocyanine 2. A mixture of SiPc 1 (60 mg, 0.074 mmol), triethylene glycol monomethyl ether (2a) (485 mg, 2.95 mmol), and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) was refluxed for 7 h. After evaporating the solvent in vacuo, the residue was treated with water (100 mL) and then filtered to obtain a blue crude, which was washed thoroughly with water to remove residual NaH and compound 2a. The crude product was further purified by column chromatography (silica gel) using N,N-dimethylformamide (DMF) as eluent to give a blue solid 2 (17 mg, 27%). IR (KBr, cm 1): 3416.0, 3064.0, 2928.0, 1608.6, 1502.7, 1335.0, 1123.3, 1081.3, 909.6, 735.8. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.63–9.72 (m, 8H, Pc-Hα); 8.49–8.58 (m, 8H, Pc-Hβ); 5.46 (d, J ¼ 7.6 Hz, 2H, Ar–H); 3.01 (s, 3H, CH3); 2.84 (t, J ¼ 4.8 Hz, 2H, OCH2); 2.74 (t, J ¼ 5.6 Hz, 2H, OCH2); 2.31 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.02 (d, J ¼ 8 Hz, 2H, Ar–H); 2.08 (t, J ¼ 7.2 Hz, 2H, NCH2); 1.87 (t, J ¼ 8.4 Hz, 2H, Ar-CH2); 1.60 (t, J ¼ 4.4 Hz, 2H, CH2); 0.37 (t, J ¼ 4.4 Hz, 2H, CH2); 1.95 (t, J ¼ 4.8 Hz, 2H, CH2); HRMS (ESI): m/z calcd for C47H41N9NaO5Si [MþNa]þ, 862.2898; found, 862.2863. Silicon phthalocyanine 3. According to the above procedure, SiPc 1 (60 mg, 0.074 mmol) was treated with triethylene glycol (3a) (499 mg, 3.32 mmol) and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) obtained a blue solid 3 (19 mg, 31%). IR (KBr,cm 1): 3400.0, 3064.0, 2928.0, 1612.6, 1518.7, 1338.9, 1123.3, 1079.3, 909.6, 739.8. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.57–9.78 (m, 8H, Pc~ Hα); 8.41–8.63 (m, 8H, Pc~ Hβ); 5.38 (d, J ¼ 8.4 Hz, 2H, Ar–H); 4.31 (s, 1H, OH); 3.11 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.80 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.33 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.17 (d, J ¼ 8.4 Hz, 2H, Ar–H); 1.95 (t, J ¼ 6.8 Hz, 2H, N–CH2); 1.66 (t, J ¼ 6.8 Hz, 2H, Ar-CH2); 1.60 (t, J ¼ 5.2 Hz, 2H, OCH2); 0.38 (t, J ¼ 4.8 Hz, 2H, OCH2); 1.94 (t, J ¼ 4.8 Hz, 2H, OCH2); HRMS (ESI): m/z calcd for C46H39N9NaO5Si [MþNa]þ, 848.2741; found, 848.2703. Silicon phthalocyanine 4. According to the above procedure, SiPc 1 (60 mg, 0.074 mmol) was treated with tetraethylene glycol monomethyl ether (4a) (615 mg, 2.95 mmol) and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) obtained a blue solid 4 (17 mg, 26%). IR (KBr,cm 1): 3336.0, 3272.0, 3028.0, 2932.0, 1594.6, 1516.7, 1472.8, 1123.3, 1079.3, 909.6, 735.8. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.65–9.70 (m, 8H, Pc~ Hα); 8.50–8.56 (m, 8H, Pc~ Hβ); 5.46 (d, J ¼ 8.4 Hz, 2H, Ar–H); 3.24 (t, J ¼ 4.8 Hz, 2H, OCH2); 3.12 (s, 3H, OCH3); 3.08 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.83 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.32 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.24 (t, J ¼ 4.0 Hz, 2H, OCH2); 2.21 (d, J ¼ 8.4 Hz, 2H, Ar–H); 2.08 (t, J ¼ 7.6 Hz, 2H, N–CH2); 1.86 (t, J ¼ 7.6 Hz, 2H, Ar-CH2); 1.61 (t, J ¼ 5.2 Hz, 2H, OCH2); 0.38 (t, J ¼ 5.2 Hz, 2H, OCH2); 1.94 (t, J ¼ 5.2 Hz, 2H, OCH2); HRMS (ESI): m/z calcd for C49H45N9NaO6Si [MþNa]þ, 906.3160; found, 906.3117. Silicon phthalocyanine 5. According to the above procedure, SiPc 1 (60 mg, 0.074 mmol) was treated with tetraethylene glycol (5a) (645 mg, 3.32 mmol) and NaH (8.9 mg, 0.37 mmol) in toluene (30 mL) ob tained a blue solid 5 (15 mg, 23%). IR (KBr,cm 1): 3392.6, 3214.8, 3054.2, 2924.1, 1613.0, 1336.3, 1126.5, 1081.1, 912.4, 739.5. 1HNMR (400 MHz, DMSO‑d6, ppm): δ9.64–9.76 (m, 8H, Pc~ Hα); 8.49–8.63 (m, 8H, Pc~ Hβ); 5.45 (d, J ¼ 7.6 Hz, 2H, Ar–H); 4.47 (s, 1H, OH); 3.20 (t, J ¼ 4.8 Hz, 2H, OCH2); 3.11 (t, J ¼ 4.8 Hz, 2H, OCH2); 2.85 (t, J ¼ 4.8 Hz, 2H, OCH2); 2.32 (t, J ¼ 5.2 Hz, 2H, OCH2); 2.24 (t, J ¼ 4.0 Hz, 2H, OCH2); 2.21 (d, J ¼ 8.4 Hz, 2H, Ar–H); 2.09 (t, J ¼ 7.6 Hz, 2H, N–CH2); 1.85 (t, J ¼ 7.6 Hz, 2H, Ar-CH2); 1.60 (t, J ¼ 5.2 Hz, 2H, OCH2); 0.37 (t, J ¼ 4.8 Hz, 2H, OCH2); 1.94 (t, J ¼ 4.8 Hz, 2H, OCH2); HRMS (ESI): m/ z calcd for C48H43N9NaO6Si [MþNa]þ, 892.3003; found, 892.3021.
4.3.2. In vitro photocytotoxicity The photocytotoxicity assay is the same as the procedure described previously [21]. Briefly, About 2 � 104 BGC-823 cells per well were seeded in 96-well plate and incubated overnight for cell adherence and growth. The cells were then incubated with various concentrations of SiPcs 1–6 for 2 h under the same conditions. After that, the cells were rinsed with PBS and re-fed with 100 μl of the culture medium before irradiated with 27 J/cm2 of a red light (λ > 610 nm, 15 mW/cm2 for 30 min). The light source consisted of a 500 W halogen lamp (Philips), a water tank for cooling and a color glass filter cut-on 610 nm. After illumination, the cells were incubated at 37 � C under 5% CO2 overnight. Then cell viability was determined by the colorimetric MTT assay as described previously [21]. 4.3.3. Cellular uptake About 2 � 104 BGC-823 cells in RPMI 1640 culture medium were seeded in 96-well plate and incubated overnight. The cells were then incubated with SiPcs 1–6 (all at 2 μM) for 2 h under the same conditions. After that, the cells were washed with PBS twice. After removing the PBS, the cells were lyzed with DMSO and sonicated for 5 min. The extracted solution were measured in a fluorescence plate reader (TECAN Infinite M200, Switzerland), which excited at 610 nm and monitored at 680 � 10 nm. The cellular uptake was calculated by comparing with the respective calibration curves to give the uptake concentrations. 4.3.4. Subcellular localization studies About 6 � 104 BGC-823 cells in the culture medium were seeded on a confocal dish. After removing the medium, the cells were incubated with the solutions of 1–6 in the medium (both at 2 μM, 0.5 ml) for 0.5 h under the same condition, and then Lyso Tracker Red (5 μM, 20 μl) was added for further 60 min co-incubation, followed by Mito Tracker Green (5 μM, 20 μl) for further 30 min co-incubation, leading to a total incubation time of 2 h for the photosensitizer, 1.5 h for Lyso Tracker Red, and 0.5 h for Mito Tracker Green. Subsequently, the cells were then rinsed with PBS and viewed with a LEICA TCS SPE confocal microscope equipped with 488 nm, 532 nm, and 635 nm solid-state lasers. Mito Tracker Green and Lyso Tracker Red were excited at 488 nm and 532 nm monitored at 499–529 nm and 552–617 nm, respectively, while compounds 1–6 were excited at 635 nm and monitored at 645–750 nm. The subcellular lo calizations of 1–6 were revealed by comparing the intracellular fluo rescence images caused by Mito Tracker Green, Lyso Tracker Red, and these phthalocyanines. 4.3.5. Apoptosis About 1 � 105 BGC-823 cells in the culture medium (0.5 ml) were seeded on a confocal dish (three dishes for each compound) and incu bated overnight at 37 � C under 5% CO2. After removing the medium, the cells were incubated with the solution of 5 in the medium (4 nM, 0.5 ml) for 2 h under the same conditions. The cells were then rinsed with PBS and refilled with 0.5 ml of the culture medium. Subsequently, the cells were illuminated using a red light for 30 min (λ > 610 nm, 15 mW/cm2, 27 J/cm2) or kept in the dark. After that, the cells were further incubated in the dark for 24 h. At the set time, the cells were rinsed with PBS, followed by addition of 0.5 ml of binding buffer containing Annexin VFITC (5 μl) and propidium iodium (PI) (5 μl), and further incubation at 37 � C for 10 min in the dark. The cells were then viewed using a LEICA TCS SPE fluorescence confocal microscope. Annexin V-FITC was excited at 488 nm and monitored at 499–529 nm, while PI was excited at 532 nm and monitored at 545–617 nm. The cell populations at different
4.3. In vitro studies 4.3.1. Cell culture The BGC-823 human gastric cancer cells (from ATCC) were main tained in RPMI 1640 medium (with phenol-red) supplemented with 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (50 μg/ 6
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phase of cell death, namely viable (annexin V- FITC /PI ), early apoptotic (annexin V-FITCþ/PI ), and necrotic or late-stage apoptotic (annexin V-FITCþ/PIþ), were determined by digitally merging fluores cence images and phase contrast images.
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4.4. In vivo studies Hepatoma H22 cells were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China); KM mice were purchased from Wushi Animal Co. Ltd. (Fuzhou, China). All animal studies were performed in compliance with guidelines of the Animal Care Committee of Fuzhou University, and also approved by the committee. To build a subcutaneous tumor model, H22 cells (~1 � 107 cells in 200 μl) were inoculated subcutaneously on the axilla of the KM mice (20–22 g). When the tumors had grown to about100 mm3, the tumor bearing mice were assigned to four groups and treated with PBS, laser (L), SiPc 5, and PDT (SiPc 5 þ L). For PDT treatment, a SiPc 5 aqueous solution containing 1% Cremophor EL (200 μM, 200 μl) was intravenously injected into the tail vein of the tumor-bearing mice (5 mice each group). After 24 h postinjection, the mice were irradiated with a laser (685 � 4 nm, 0.1 W cm 2) for 5 min. The tumor size was determined every other day by using a caliper for a duration of 12 days and calculated using the following formula: volume ¼ (tumor length) � (tumor width)2 � 0.5. Declaration of competing interest There are no interests to declare. CRediT authorship contribution statement Bi-Yuan Zheng: Formal analysis, Writing - original draft. Lei Wang: Formal analysis, Writing - original draft. Qing-Yan Hu: Formal analysis. Jie Shi: Formal analysis. Mei-Rong Ke: Formal analysis, Writing original draft. Jian-Dong Huang: Supervision. Acknowledgements This work was supported by Scientific Research Fund of the National Board of Health and Family Planning (the Joint Research Project of Health and Education of Fujian), China (Grant No. WKJ-FJ-32), Marine High-Tech Programs of Fujian Province, China (Grant No. 2016–14), the Natural Science Foundation of Fujian, China (Grant No. 2018J05016), and the Foundation of Education Department of Fujian, China (No. JAT170106). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2020.108286. References [1] Robertson C, Evans DH, Abrahamse H. Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications for PDT. J Photochem PhotobiolB: Biol 2009;96:1–8. [2] Dolmans DE, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Canc 2003;3:380–7. [3] Brown SB, Brown EA, Walker I. The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol 2004;5:497–508. [4] Detty MR, Gibson SL, Wagner SJ. Current clinical and preclinical photosensitizers for use in photodynamic therapy. J Med Chem 2004;47:3897–915. [5] Triesscheijn M, Baas P, Schellens JHM, Stewart FA. Photodynamic therapy in oncology. Oncol 2006;11:1034–44. [6] Bugaj AM. Targeted photodynamic therapy–a promising strategy of tumor treatment. Photochem Photobiol Sci 2011;10:1097–109. [7] Li X, Zheng B-D, Peng X-H, Li S-Z, Ying J-W, Zhao Y, Huang J-D, Yoon J. Phthalocyanines as medicinal photosensitizers: developments in the last five years. Coord Chem Rev 2019;379:147–60.
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