Article pubs.acs.org/jmc

A Dual Activatable Photosensitizer toward Targeted Photodynamic Therapy Janet T. F. Lau, Pui-Chi Lo,* Xiong-Jie Jiang, Qiong Wang, and Dennis K. P. Ng* Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China S Supporting Information *

ABSTRACT: An unsymmetrical bisferrocenyl silicon(IV) phthalocyanine has been prepared in which the disulfide and hydrazone linkers can be cleaved by dithiothreitol and acid, respectively. The separation of the ferrocenyl quenchers and the phthalocyanine core greatly enhances the fluorescence emission, singlet oxygen production, intracellular fluorescence intensity, and in vitro photocytotoxicity. The results have been compared with those for the two symmetrical analogues which contain either the disulfide or hydrazone linker and therefore can only be activated by one of these stimuli. For the dual activatable agent, the greatest enhancement can be attained under a slightly acidic environment (pH = 4.5−6.8) and in the presence of dithiothreitol (in millimolar range), which can roughly mimic the acidic and reducing environment of tumor tissues. This compound can also be activated in tumor-bearing nude mice. It exhibits an increase in fluorescence intensity in the tumor over the first 10 h after intratumoral injection and can effectively inhibit the growth of tumor upon illumination.



called photodynamic molecular beacons,24−30 in which the photosensitizing unit is linked to a quencher via a cleavable peptide or a nucleic acid. In virtually all of these systems, their photoactivity is triggered by a single stimulus. It is logical to extend the study to systems that can be activated by more than one tumor-associated stimulus. It is envisaged that these novel systems can exhibit further enhanced tumor selectivity. This strategy has been employed for intracellular delivery of anticancer drugs using pH- and redox-responsive carriers.31−33 However, to our knowledge, dual activatable photosensitizers remain extremely rare. Ozlem and Akkaya briefly reported a boron dipyrromethene based photosensitizer of which the singlet oxygen generation efficiency increased by about 6-fold at low pH and high concentration of sodium ion in acetonitrile, but the study was not extended to biological systems.34 As part of our continuing interest in pH- and redoxresponsive photosensitizers,35−38 we report herein a novel phthalocyanine-based photosensitizer that exhibits dual acid and thiol activation. Both stimuli are associated with the characteristics of tumors39,40 and therefore are desirable targets for activation.

INTRODUCTION Photodynamic therapy (PDT) has emerged as a promising treatment modality for some localized and superficial cancers, as well as certain noncancerous conditions.1,2 It involves the combined action of a photosensitizer, light, and oxygen to generate cytotoxic reactive oxygen species, in which the photosensitizer plays an extremely vital role in determining the therapeutic outcome. As a result, a large number of functional dyes3−6 and carriers7−10 have been studied with a view to identifying desirable photosensitizing systems and optimizing their photodynamic efficacy. To address the issue of selectivity toward malignant tissues, various approaches have been explored.11−14 These include bioconjugation to tumorspecific vehicles such as the epidermal growth factor and monoclonal antibodies, and encapsulation in colloidal nanocarriers such as polymeric micelles and silica-based nanoparticles. Recently, “smart” photosensitizing systems that can be activated selectively by tumor-associated stimuli have received considerable attention.15,16 In these systems, the photosensitizers are either self-quenched due to aggregation or deactivated by the neighboring quenchers. Upon interactions with the acidic and reducing environment of tumors, cancerrelated proteases, or mRNAs that have high tumor specificity, the photosensitizers are disaggregated or detached from the quenchers, resulting in restoration of their fluorescence and photosensitizing properties. This approach can therefore overcome the drawback of nonspecific activation of phototoxicity in PDT. Among the activatable photosensitizers reported so far, most of them are self-quenched polymeric systems17−23 and the so© 2014 American Chemical Society



RESULTS AND DISCUSSION Synthesis and Characterization. Scheme 1 shows the synthetic route for this dual activatable photosensitizer. We employed ferrocenyl moieties as the quenchers,41 which were linked to the phthalocyanine core via cleavable hydrazone and Received: December 27, 2013 Published: May 3, 2014 4088

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Scheme 1. Synthesis of Dual Activatable Photosensitizer 4

Figure 1. Structures of phthalocyanines 5 and 6.

Compounds 4 and 5 were characterized with various spectroscopic methods. The 1H NMR spectrum of 4 in CDCl3 (Figure S1 in Supporting Information) clearly showed all the expected signals. With reference to the spectra of 5 and 6 and with the aid of 1H−1H COSY spectroscopy (Figure S2 in Supporting Information), all the signals could be assigned unambiguously. It is worth mentioning that the protons that are close to the silicon center are strongly shielded by the phthalocyanine ring, resulting in a large upfield shift of their signals (δ −1.77 and −0.38 for the oxyethylene protons of the disulfide-linked ferrocene and δ −0.70 for the benzylic protons of the hydrazone-linked ferrocene). In addition, as the two ferrocenyl units of 4 are at different environment, they resonate

disulfide linkers. Silicon(IV) phthalocyanine dichloride (1) was first treated with the less reactive alcohol 238 before the more reactive benzylic alcohol 3 (Scheme S1 in Supporting Information) was added. The triethylene glycol chain in 3 was introduced to enhance the solubility and reduce the aggregation of the resulting phthalocyanine, both before and after the cleavage. Under these conditions, the target unsymmetrical phthalocyanine 4 was obtained in 15% yield, while the symmetrical product 5 was isolated as the major product (43% yield) and the other symmetrical phthalocyanine 638 was not obtained (Figure 1). The yield of 5 could be increased to 79% by treating 1 with 3 only. 4089

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the extent reached 62% after 6 h, 85% after 10 h, and 97% after 24 h. Compound 5, which does not have the disulfide linker, showed negligible changes in fluorescence intensity up to 40 mM DTT. The effect of pH on the fluorescence emission of 4−6 was also examined. Figure S6 (Supporting Information) shows the time-dependent fluorescence intensities of these compounds in PBS at pH 4.5, 6.0, and 7.4, which were used to mimic the tumor lysosomal compartment, tumor interstitial environment, and extracellular environment of normal tissues, respectively. For 5, there was no significant change in fluorescence intensity over 12 h at pH 7.4, while the intensity increased significantly at pH 6.0 and 4.5, particularly the latter. This observation could be attributed to the hydrolytic cleavage of the hydrazone bonds under an acidic environment, thus detaching the ferrocenebased quenchers. The fluorescence intensity of 5 increased by about 6-fold when the pH decreased from 7.4 to 4.5. Compound 4 showed a moderate increase in fluorescence intensity at pH 4.5 and 6.0 due to the release of only one ferrocenyl moiety. For 6, which does not possess the acid-labile hydrazone linkage, no significant fluorescence enhancement was observed at pH 4.5−7.4. The combined DTT and pH effect on the fluorescence intensity of 4 is shown in Figure 2a. Higher intensity was

as two sets of signals, each showing a singlet and two slightly broadened signals with an integration ratio of 5:2:2, which are typical for monosubstituted ferrocenes. Electronic Absorption and Photophysical Properties. These properties of 4−6 measured in N,N-dimethylformamide (DMF) are listed in Table 1. The UV−vis spectrum of 4 Table 1. Electronic Absorption and Photophysical Data for 4−6 in DMF compd 4 5 638

λmax (nm) (log ε)

λem (nm)a

ΦF b

ΦΔ c

330 (4.84), 354 (4.86), 496 (3.47), 608 (4.53), 646 (4.49), 677 (5.29) 354 (4.79), 611 (4.48), 647 (4.43), 678 (5.25) 332 (4.90), 353 (4.86), 494 (3.67), 608 (4.51), 646 (4.44), 676 (5.29)

687

0.05

0.10

688

0.03

0.07

688

0.04

0.17

a

Excited at 610 nm. bUsing ZnPc in DMF as the reference (ΦF = 0.28). cUsing ZnPc as the reference (ΦΔ = 0.56 in DMF).

(Figure S3 in Supporting Information) showed typical absorptions of metallophthalocyanines, together with two additional bands at 330 and 496 nm assignable to the πconjugated ferrocenyl moieties.38 Its fluorescence quantum yield (ΦF = 0.05) was much weaker than that of zinc(II) phthalocyanine (ZnPc) (ΦF = 0.28) as a result of quenching by the two ferrocenyl moieties. The singlet oxygen quantum yield (ΦΔ) of 4 was also determined in DMF using 1,3diphenylisobenzofuran (DPBF) as the scavenger. The value (ΦΔ = 0.10) was also significantly lower than that of ZnPc (ΦΔ = 0.56). Among the three compounds, compound 5 showed the lowest ΦF (0.03) and ΦΔ (0.07) values. This suggested that the hydrazone-linked ferrocenyl moiety is a slightly stronger quencher than the disulfide-linked counterpart, which may be a result of its shorter separation from the phthalocyanine core. The UV−vis spectrum of 4 in RPMI 1640 culture medium with 0.5% Cremophor EL also showed a sharp and intense Qband, indicating that the compound remained nonaggregated even in aqueous media. The fluorescence was also significantly weaker than that of the reference compound containing two axial -OCH2CH2S-SCH2CH2OH chains linked to the silicon(IV) phthalocyanine core. As expected, the fluorescence of 5 and 6 was also very weak because of the presence of the ferrocenyl quenchers (Figure S4 in Supporting Information). Redox- and pH-Responsive Properties. The redoxresponsive properties of 4 and 5 were first examined by monitoring their changes in fluorescence intensity with time in the presence of different concentrations of dithiothreitol (DTT) in phosphate buffered saline (PBS). The results are summarized in Figure S5 (Supporting Information), which also includes the response of 6 reported earlier for comparison.38 It can be seen that 6 shows a large increase in fluorescence intensity in the presence of 10 or 40 mM DTT, while the increase is small in the absence or presence of 2 μM DTT. With reference that the intracellular glutathione concentration (1−10 mM) is significantly higher than the extracellular levels (2 μM in plasma),42 the above conditions were used to mimic the intracellular and extracellular reducing environments, respectively. Fluorescence enhancement was also observed for 4, but the effect was weaker because of the fact that there is only one disulfide-linked ferrocenyl moiety in 4 and the noncleaved hydrazone-linked ferrocenyl unit still partially quenches the fluorescence. Nevertheless, the cleavage was confirmed and the rate was monitored by HPLC. In the presence of 40 mM DTT,

Figure 2. (a) Changes in fluorescence intensity of 4 (4 μM) with time at pH 6.0, 6.8, or 7.4 and in the presence of 2 μM or 10 mM DTT in PBS with 0.5% Cremophor EL. (b) Comparison of the rate of photodegradation of DPBF sensitized by 4 under these conditions.

attained at higher DTT concentration (10 mM > 2 μM) and lower pH (6.0 > 6.8 > 7.4). At 10 mM DTT and pH 6.0, the extent of cleavage reached 28% after 6 h, 51% after 10 h, and 76% after 24 h as determined by HPLC analysis. It is clear that the compound is responsive toward both stimuli. For 5 and 6, their fluorescence intensity only responded to a single stimulus 4090

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Figure 3. (a) Fluorescence images of MCF-7 cells after sequential incubation with DTT (0, 2 μM, or 4 mM) for 1 h, nigericin (25 μM) at pH 5.0 or 7.4 for 30 min, and 4 (1 μM) for 1 h. (b) Fluorescence images of MCF-7 cells after incubation with nigericin (25 μM) at pH 5.0, 6.0, or 7.4 for 30 min, followed by incubation with 5 (1 μM) for 1 h. The corresponding bright field images are shown in the upper row. (c) Comparison of the relative intracellular fluorescence intensity of 4 at different DTT concentrations and pH values. Significant difference, compared with the intensity at 0 or 2 μM DTT at the same pH, is denoted by ∗, P < 0.001. (d) Comparison of the relative intracellular fluorescence intensity of 5 at different pH values. Significant differences are denoted by ∗, P < 0.001, and #, P < 0.001, when compared with the intensity at pH 6.0 or 7.4 and with that at pH 5.0 or 7.4, respectively. Data are expressed as the mean ± standard deviation (number of cells, 30).

In Vitro Studies. The DTT- and pH-dependent fluorescence emission of 4 was also examined at the cellular level. In this study, MCF-7 human breast cancer cells were sequentially incubated with DTT (0, 2 μM, or 4 mM), the ionophore nigericin which can equilibrate the intracellular and extracellular pH (at pH 5.0 or 7.4),43 and 4, and then their fluorescence images were captured and the intracellular fluorescence intensities were determined (Figure 3a and Figure 3c). To better illustrate the extent of pH effect, the extremes of a wider range (at pH 5.0 and 7.4) were used. At pH 7.4, very weak fluorescence was observed in the absence or presence of 2 μM DTT. However, the fluorescence intensity increased by about 3-fold when the DTT concentration was increased to 4 mM. This can be attributed to the cleavage of the disulfide linker. At pH 5.0, the cells showed moderately strong intracellular fluorescence in the absence or presence of 2 μM DTT due to the cleavage of the acid-labile hydrazone linker. Under these conditions, the intracellular fluorescence was stronger than that at pH 7.4 and 4 mM DTT. This also supported that the hydrazone-linked ferrocenyl moiety is a stronger quencher than the disulfide-linked analogue as inferred by comparing the fluorescence quantum yields of 5 and 6 (Table 1). The fluorescence intensity was particularly strong when the cells were exposed to 4 mM DTT at pH 5.0, under which both the disulfide and hydrazone linkages are cleaved, rendering the compound to be fully “turned on”. For the bis(hydrazone-linked) derivative 5, a similar increase in intracellular fluorescence was also observed at lower pH. The intensity increased by about 10-fold when the pH decreased from 7.4 to 5.0 (Figure 3b and Figure 3d). The effect was

(either pH or DTT) as shown in Figure S7 (Supporting Information). Apart from the fluorescence intensity, the effects of DTT and pH on the singlet oxygen generation efficiency of these compounds were also examined. Figure S8 (Supporting Information) compares the rate of photodegradation of DPBF sensitized by 4−6 upon exposure to 2 μM or 10 mM DTT in PBS. All these compounds could not trigger the formation of singlet oxygen in the dark (data not shown). Upon illumination and at 2 μM DTT, all these compounds could produce a small amount of singlet oxygen. In the presence of 10 mM DTT, the singlet oxygen generation efficiency increased significantly and followed the order 5 < 4 < 6, which again could be attributed to the different extent of disulfide cleavage. Related studies were performed at different pH (4.5, 6.0, and 7.4). Similarly, under all these conditions, singlet oxygen could not be generated in the dark (data not shown). Upon illumination, the singlet oxygen generation efficiency followed the trend 6 < 4 < 5, and for 4 and 5, the efficiency increased at lower pH as shown in Figure S9 (Supporting Information) reflecting the different extent of hydrazone cleavage. Figure 2b shows the combined DTT and pH effect on the singlet oxygen generation efficiency of 4. The results also showed that 4 can be dual-activated. The highest efficiency was attained at pH 6.0 and 10 mM DTT. When there was only one favorable condition (i.e., either pH 6.0 or 10 mM DTT), the efficiency was relatively lower but still higher than that under the nonactivating condition (i.e., pH 7.4 and 2 μM DTT). As shown in Figure S10 (Supporting Information), 5 and 6 were only responsive to either pH (for 5) or DTT (for 6). 4091

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significantly when the DTT concentration is increased to 4 mM. The extent of decrease in IC50 value is slightly smaller than that for 6, which can be attributed to the presence of one additional disulfide-linked ferrocenyl moiety in 6. Compound 5 is also highly potent with IC50 values in the range of 73−76 nM, which are not affected by the DTT concentration because the compound does not contain DTT-cleavable disulfide linker. Attempts to study the pH-dependent photocytotoxicity of these compounds were not successful because most of the cells were killed after being incubated with nigericin at pH 6.0 for 30 min and left overnight for cell viability measurements. The subcellular localization of 4 in MCF-7 cells, after the treatment with DTT (4 mM), was further investigated by confocal microscopy. As shown in Figure 5, the compound can target the endoplasmic reticulum but not the mitochondria and lysosomes of the cells. Similar results were observed for 6,38 but they were contrary to those for our previously reported silicon(IV) phthalocyanines which were localized in mitochondria44−46 or lysosomes.35,47−50 Thus, the axial ligands play a crucial role in controlling the subcellular localization of these photosensitizers. In Vivo Studies. Finally, the activation of 4 was demonstrated in vivo. Nude mice bearing a HT29 human colorectal carcinoma were treated with an intratumoral dose (1 μmol per kg body weight) of 4 or the noncleavable analogue of 6, in which the disulfide linker is replaced with an ethylene group.38 Their whole-body fluorescence images were then monitored and quantified continuously for 24 h (Figure 6). The results clearly showed that the fluorescence of 4 was greatly increased within the first 10 h, while that of the control was negligible, showing that the fluorescence of 4 was restored inside the tumor likely due to the cleavage of the linkers. In addition, this compound could also effectively inhibit the growth of tumor upon illumination as shown in Figure 7.

stronger than that of 4, which only exhibited a 4-fold enhancement. As the extracellular pH of tumors is slightly acidic (6.4−6.8) while the normal tissues are generally neutral,39 compound 5 is a potential fluorescent probe for tumor. The photodynamic activities of 4 and 5 were also evaluated against MCF-7 cells. To reveal the effect of DTT on their cytotoxicity, the cells were pretreated with different concentrations of DTT (0, 2 μM, or 4 mM) prior to incubation with the phthalocyanines. Figure 4 shows the corresponding dose-



CONCLUSIONS In summary, we have prepared and characterized two novel silicon(IV) phthalocyanines. Compound 4 functions as a dual pH- and redox-responsive photosensitizer. The fluorescence intensity, singlet oxygen generation efficiency, intracellular fluorescence, and in vitro photodynamic activity of this compound are enhanced in a slightly acidic environment (pH = 4.5−6.8) or in the presence of DTT (in millimolar range). The greatest enhancement can be attained when both of these two conditions occur, which can roughly mimic the acidic and reducing environment of tumor tissues. The fluorescence and photodynamic activity of this compound can also be activated in vivo. Compound 5 shows an almost 10-fold increase in intracellular fluorescence intensity when the pH drops from 7.4 to 5.0. Therefore, it also serves as a promising fluorescent probe for tumor.

Figure 4. Cytotoxic effects of (a) 4 and (b) 5 on MCF-7 cells without pretreatment with DTT (squares) or pretreated with 2 μM (circles) or 4 mM (triangles) DTT for 1 h prior to drug incubation for 6 h in the absence (closed symbols) and presence (open symbols) of light (λ > 610 nm, 40 mW cm−2, 48 J cm−2). Data are expressed as the mean ± standard error of the mean of three independent experiments, each performed in quadruplicate.

dependent survival curves in the absence and presence of light. Both compounds were noncytotoxic in the dark regardless of the concentration of DTT but exhibited a high potency upon illumination. The IC50 values are summarized in Table 2, which also includes the data for 6 for comparison.38 It can be seen that the photocytotoxicity of 4 remains nearly unchanged in the absence and presence of 2 μM DTT but is enhanced



Table 2. Half Maximal Inhibitory Concentration (IC50) Values of 4−6 against MCF-7 Cellsa IC50 (nM) compd

no DTT

2 μM DTT

4 mM DTT

4 5 638

100 ± 2 75 ± 4 160 ± 9

105 ± 6 76 ± 3 150 ± 8

64 ± 1* 73 ± 5 81 ± 9*

EXPERIMENTAL SECTION

General. All the reactions were performed under an atmosphere of nitrogen. DMF was dried over barium oxide and distilled under reduced pressure. Tetrahydrofuran (THF), toluene, and pyridine were distilled from sodium benzophenone ketyl, sodium, and calcium hydride, respectively. Chromatographic purifications were performed on silica gel (Macherey-Nagel, 230−400 mesh) columns with the indicated eluents. All other solvents and reagents were of reagent grade and used as received. Compounds 2,38 7,51 10,52 14,38 and the noncleavable analogue of 638 were prepared as described. 1 H and 13C{1H} NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer (1H, 400 MHz; 13C, 100.6 MHz) in

Significant difference, compared with the data for no DTT or 2 μM DTT, is denoted by ∗, P < 0.001. a

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Figure 5. Visualization of intracellular fluorescence for MCF-7 cells by using filter sets specific for 4 (in red, column 2) and ER-Tracker, MitoTracker, or LysoTracker (in green, column 3). The corresponding bright field and merged images are shown in column 1 and column 4, respectively. CDCl3. Spectra were referenced internally by using the residual solvent (1H, δ = 7.26) or solvent (13C, δ = 77.0) resonances relative to SiMe4. Electrospray ionization (ESI) mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer. UV−vis and steady-state fluorescence spectra were taken on a Cary 5G UV−vis− NIR spectrophotometer and a Hitachi F-7000 spectrofluorometer, respectively. The fluorescence quantum yields (ΦF) of the samples (in DMF) were determined by the equation: ΦF(sample) = (Fsample/ Fref)(Aref/Asample)(n2sample/n2ref)ΦF(ref),53 where F, A, and n are the measured fluorescence (area under the emission peak), the absorbance at the excitation position (610 nm), and the refractive index of the solvent, respectively. ZnPc in DMF was used as the reference [ΦF(ref) = 0.28].54 The singlet oxygen quantum yields (ΦΔ) were measured in DMF by the method of chemical quenching of DPBF by using ZnPc as the reference (ΦΔ = 0.56).55 The purity of compounds 4 and 5 was determined by HPLC and 1 H NMR spectroscopy, respectively, and was found to be ≥95%. Reversed-phase analytical HPLC experiments were performed by using an Apollo-C18 column (5 μm, 4.6 mm × 250 mm) and a Waters Breeze 2 HPLC system with a 2998 photodiode array detector. The conditions were set as follows: solvent A = 5% DMSO in acetonitrile; solvent B = methanol. The gradient was 40% A + 60% B in the first 5 min, then changed to 100% A + 0% B in 25 min, kept under these conditions for 10 min, and finally changed to 40% A + 60% B and maintained under these conditions for a further 5 min. The flow rate was fixed at 1.5 mL min−1. Preparation of 9. A mixture of 3,5-dihydroxybenzyl alcohol (7) (2.50 g, 17.84 mmol), ethyl bromoacetate (8) (2.98 g, 17.84 mmol), and anhydrous K2CO3 (2.47 g, 17.87 mmol) in acetone (10 mL) was heated under reflux for 6 h. The volatiles were evaporated under reduced pressure. The residue was mixed with water, and then the pH of the mixture was adjusted to ∼5 with 3 M HCl. The mixture was then evaporated under reduced pressure at 80 °C. The residue was redissolved in THF, and the resulting mixture was filtered to remove

the inorganic salt. The crude product was then subject to column chromatography using CHCl3 and then CHCl3/EtOH (50:1 v/v) as the eluents. The product was isolated as a pale yellow oily liquid (1.21 g, 30%). Rf [CHCl3/EtOH (10:1 v/v)] = 0.32. 1H NMR (CDCl3): δ = 6.39 (s, 1 H, ArH), 6.33 (s, 1 H, ArH), 6.25 (s, 1 H, ArH), 4.48 (s, 2 H, CH2), 4.43 (s, 2 H, CH2), 4.21 (q, J = 7.2 Hz, 2 H, CO2CH2), 3.74 (s, 1 H, OH), 1.26 (t, J = 7.2 Hz, 3 H, CH3). 13C{1H} NMR (CDCl3): δ = 169.6, 158.9, 157.4, 143.2, 107.6, 105.0, 101.5, 65.1, 64.6, 61.7, 14.0. MS (ESI): m/z 249 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C11H14NaO5 [M + Na]+, 249.0733; found, 249.0741. Preparation of 11. A mixture of 9 (0.80 g, 3.54 mmol), tosylate 10 (1.24 g, 3.89 mmol), and anhydrous K2CO3 (1.47 g, 10.64 mmol) in acetone (20 mL) was heated under reflux overnight. The mixture was then cooled to room temperature and filtered. The crude product was subject to column chromatography using CHCl3 and then CHCl3/EtOH (50:1 v/v) as the eluents. The product was isolated as a colorless oily liquid (1.03 g, 78%). Rf [CHCl3/EtOH (50:1 v/v)] = 0.29. 1H NMR (CDCl3): δ = 6.58 (s, 1 H, ArH), 6.51 (s, 1 H, ArH), 6.42 (s, 1 H, ArH), 4.61 (d, J = 6.0 Hz, 2 H, CH2), 4.59 (s, 2 H, CH2), 4.26 (q, J = 7.2 Hz, CO2CH2), 4.11 (t, J = 4.8 Hz, 2 H, CH2), 3.83 (t, J = 4.8 Hz, 2 H, CH2), 3.71−3.73 (m, 2 H, CH2), 3.63−3.68 (m, 4 H, CH2), 3.53−3.55 (m, 2 H, CH2), 3.37 (s, 3 H, OCH3), 1.30 (t, J = 7.2 Hz, 3 H, CH3). 13C{1H} NMR (CDCl3): δ = 168.8, 160.2, 159.1, 143.6, 106.3, 105.2, 101.1, 71.9, 70.8, 70.7, 70.5, 69.7, 67.6, 65.4, 65.1, 61.4, 59.0, 14.1. MS (ESI): m/z 395 [M + Na]+ (100%). HRMS (ESI): m/z calcd for C18H28NaO8 [M + Na]+, 395.1676; found, 395.1676. Preparation of 12. A mixture of hydrazine hydrate (80%, 2 mL) and 11 (0.41 g, 1.10 mmol) in ethanol (2 mL) was stirred at room temperature for 30 min. The mixture was then evaporated under reduced pressure at 60 °C. The product was obtained as a colorless oily liquid (0.34 g, 86%). 1H NMR (CDCl3): δ = 7.90 (br s, 1 H, NH), 6.57 (s, 1 H, ArH), 6.49 (s, 1 H, ArH), 6.35 (s, 1 H, ArH), 4.59 (s, 2 H, CH2), 4.48 (s, 2 H, CH2), 4.08 (t, J = 4.4 Hz, 2 H, CH2), 3.82 (t, J 4093

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chromatography using CHCl3 and then CHCl3/MeOH (50:1 v/v) as the eluents. The product was isolated as an orange oily liquid (0.45 g, 64%). Rf [CHCl3/MeOH (10:1 v/v)] = 0.29. 1H NMR (CDCl3): δ = 9.27 (s, 1 H, CONH), 8.12 (s, 1 H, NCH), 6.63 (s, 1 H, ArH), 6.59 (s, 1 H, ArH), 6.46 (s, 1 H, ArH), 4.70 (s, 2 H, FcH), 4.65 (d, J = 5.6 Hz, 2 H, CH2), 4.59 (s, 2 H, CH2), 4.42 (s, 2 H, FcH), 4.21 (s, 5 H, FcH), 4.12−4.14 (m, 2 H, CH2), 3.84−3.86 (m, 2 H, CH2), 3.72− 3.74 (m, 2 H, CH2), 3.64−3.69 (m, 4 H, CH2), 3.54−3.56 (m, 2 H, CH2), 3.38 (s, 3 H, CH3). 13C{1H} NMR (CDCl3): δ = 163.8, 160.2, 158.2, 151.4, 144.3, 106.5, 105.3, 101.0, 77.9, 71.9, 70.7, 70.6, 70.4, 69.6, 69.3, 69.2, 68.3, 67.7, 67.6, 64.7, 58.9. MS (ESI): m/z 555 [M + H]+ (100%). HRMS (ESI): m/z calcd for C27H35FeN2O7 [M + H]+, 555.1788; found, 555.1784. Preparation of 4. A mixture of silicon(IV) phthalocyanine dichloride (1) (0.12 g, 0.20 mmol), alcohol 2 (0.13 g, 0.25 mmol), and pyridine (1 mL, 12.36 mmol) in toluene (10 mL) was heated under reflux for 2 h. To this reaction mixture, a solution of alcohol 3 (0.13 g, 0.23 mmol) and pyridine (0.5 mL, 6.18 mmol) in toluene (10 mL) was added, and the mixture was kept stirring under reflux for a further 4 h. After evaporation of the volatiles in vacuo, the residue was redissolved in CHCl3 to give a blackish green mixture, which was then filtered through a bed of Celite to remove the insoluble black solid. The greenish blue filtrate was evaporated, and the residue was chromatographed using CHCl3 and then CHCl3/MeOH (50:1 v/v) as the eluents. The two greenish blue bands were collected separately and evaporated. The crude products were recrystallized from CHCl3/ hexane to afford 4 (48 mg, 15%) and 5 (0.14 g, 43%) both as a blue solid. Rf [CHCl3/MeOH (10:1 v/v)] = 0.52. 1H NMR (CDCl3): δ = 9.61−9.63 (m, 8 H, Pc-Hα), 8.74 (s, 1 H, CONH), 8.33−8.35 (m, 8 H, Pc-Hβ), 8.00 (s, 1 H, NCH), 7.91 (d, J = 8.8 Hz, 2 H, ArH), 7.73 (d, J = 15.6 Hz, 1 H, CCH), 7.08 (d, J = 15.6 Hz, 1 H, CCH), 6.81 (d, J = 8.8 Hz, 2 H, ArH), 5.64 (s, 1 H, ArH), 4.73 (br s, 2 H, FcH), 4.58 (br s, 2 H, FcH), 4.47 (br s, 2 H, FcH), 4.45 (br s, 2 H, FcH), 4.39 (s, 2 H, CH2), 4.22 (s, 5 H, FcH), 4.16 (s, 5 H, FcH), 3.83 (s, 2 H, CH2), 3.56−3.63 (m, 8 H, CH2), 3.51−3.54 (m, 2 H, CH2), 3.41− 3.46 (m, 4 H, CH2), 3.36 (s, 3 H, CH3), 3.09 (virtual t, J = 4.8 Hz, 2 H, ArH), −0.38 (t, J = 6.0 Hz, 2 H, CH2), −0.70 (s, 2 H, CH2), −1.77 (t, J = 6.0 Hz, 2 H, CH2). The signal of one set of SCH2 protons was overlapped with the water signal at δ 1.56. 13C{1H} NMR (CDCl3): δ = 187.3, 167.3, 163.5, 159.7, 157.8, 156.1, 150.4, 149.4, 146.1, 141.7, 135.9, 133.5, 131.1, 130.5, 123.7, 118.8, 114.3, 102.2, 100.2, 79.3, 71.9, 71.3, 70.8, 70.7, 70.6, 70.5, 69.8, 69.3, 69.0, 68.4, 66.6, 66.5, 64.7, 62.5, 59.1, 53.8, 38.7, 35.8 (some of the signals were overlapped). MS (ESI): m/z 1620 [M + H]+ (21%). HRMS (ESI): m/z calcd for C84H75Fe2N10O12S2Si [M + H]+, 1620.3504; found, 1620.3507. Preparation of 5. A mixture of silicon(IV) phthalocyanine dichloride (1) (63 mg, 0.10 mmol), alcohol 3 (0.12 g, 0.22 mmol), and pyridine (1 mL, 12.36 mmol) in toluene (15 mL) was heated under reflux for 6 h. The volatiles were evaporated in vacuo. The residue was redissolved in CHCl3 to give a blackish green mixture, which was then filtered through a bed of Celite to remove the insoluble black solid. The greenish blue filtrate was evaporated, and the residue was chromatographed using CHCl3 and then CHCl3/MeOH (10:1 v/v) as the eluents. The crude product was further purified by size exclusion chromatography on Bio-Beads S-X1 beads (200−400 mesh) using THF as the eluent. It was then chromatographed again on a silica gel column using CHCl3 and then CHCl3/MeOH (10:1 v/v) as the eluents. The crude product was finally recrystallized from CHCl3/hexane to afford a blue solid (0.13 g, 79%). Rf [CHCl3/MeOH (10:1 v/v)] = 0.44. 1H NMR (CDCl3): δ = 9.59−9.61 (m, 8 H, PcHα), 8.75 (s, 2 H, CONH), 8.33−8.35 (m, 8 H, Pc-Hβ), 7.97 (s, 2 H, NCH), 5.64 (s, 2 H, ArH), 4.72 (br s, 4 H, FcH), 4.44 (br s, 4 H, FcH), 4.21 (s, 10 H, FcH), 3.81 (s, 4 H, CH2), 3.58−3.62 (m, 12 H, CH2), 3.52−3.54 (m, 4 H, CH2), 3.42−3.45 (m, 8 H, CH2), 3.36 (s, 6 H, CH3), 3.06 (virtual t, J = 3.6 Hz, 4 H, ArH), −0.70 (s, 4 H, CH2). 13 C{1H} NMR (CDCl3): δ = 163.2, 158.4, 156.2, 150.8, 149.4, 142.1, 135.9, 131.1, 130.9, 123.6, 102.2, 99.5, 71.9, 70.7, 70.6, 70.5, 69.3, 68.3, 66.5, 66.2, 59.0, 58.1 (some of the signals were overlapped). MS (ESI): m/z 1648 [M + H] + (5%). HRMS (ESI): m/z calcd for C86H83Fe2N12O14Si [M + H]+, 1648.4643; found, 1648.4640.

Figure 6. (a) Fluorescence images of tumor-bearing nude mice before and after intratumoral injection of 4 or the noncleavable control (1 μmol per kg body weight) over 24 h. (b) Corresponding changes in fluorescence intensity per unit area of the tumor. Five mice were used for each compound.

Figure 7. Tumor growth delay after photodynamic treatment with 4. Each of the five mice was treated with an intratumoral dose of 4 (1 μmol per kg body weight) followed by illumination with laser light at 7 and 48 h after injection (675 nm, 30 J cm−2). The mice for the control (n = 4) were not treated with 4 and light. Data are expressed as the mean ± standard derivation. = 4.4 Hz, 2 H, CH2), 3.70−3.72 (m, 2 H, CH2), 3.62−3.67 (m, 4 H, CH2), 3.52−3.55 (m, 2 H, CH2), 3.36 (s, 3 H, CH3). 13C{1H} NMR (CDCl3): δ = 168.4, 160.2, 158.2, 144.1, 106.3, 105.1, 100.8, 71.8, 70.7, 70.6, 70.5, 69.6, 67.5, 66.8, 64.7, 59.0. MS (ESI): m/z 359 [M + H]+ (100%). HRMS (ESI): m/z calcd for C16H27N2O7 [M + H]+, 359.1813; found, 359.1813. Preparation of 3. A mixture of ferrocenecarboxyaldehyde (13) (0.32 g, 1.50 mmol) and 12 (0.45 g, 1.26 mmol) in ethanol (10 mL) was heated under reflux for 2 h. The volatiles were evaporated under reduced pressure. The crude product was subject to column 4094

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Study of DTT- and pH-Responsive Fluorescence Emission. Phthalocyanines 4−6 were dissolved in DMF to give 1 mM solutions, which were diluted to 4 μM with PBS in the presence of 0.5% Cremophor EL (v/v). DTT was dissolved in deionized water to give a 1 M solution. To study the effect of DTT on the fluorescence intensity, a mixture of the phthalocyanine (4 μM) with DTT (2 μM, 10 mM, or 40 mM) or without DTT in PBS at pH 7.4 was prepared. To examine the effect of pH on the fluorescence intensity, the study was started readily after the phthalocyanine solution (1 mM in DMF) was diluted to 4 μM with PBS at different pH (4.5, 6.0, or 7.4). To study the combined effect of DTT and pH on the fluorescence intensity, a mixture of the phthalocyanine (4 μM) with DTT (2 μM or 10 mM) in PBS at pH 6.0 or 7.4 was prepared. All these solutions were stirred continuously at room temperature during the kinetic study, and the fluorescence spectra (λex = 610 nm, λem = 630−800 nm) were recorded at different time intervals. Study of DTT- and pH-Responsive Singlet Oxygen Generation. DPBF was dissolved in DMF to give a 10 mM solution. To study the effect of DTT on the singlet oxygen generation efficiency, a mixture of phthalocyanine (4 μM) with DTT (2 μM or 10 mM) in PBS at pH 7.4 was prepared. To examine the effect of pH, the study was carried out after the phthalocyanine solution (1 mM in DMF) was diluted to 4 μM with PBS at different pH (4.5, 6.0, or 7.4). To study the combined effect of pH and DTT, a mixture of the phthalocyanine (4 μM) with DTT (2 μM or 10 mM) in PBS at pH 6.0 or 7.4 was prepared. The above phthalocyanine solutions were stirred continuously at room temperature for 8 h. These solutions (3 mL) were then mixed with the DPBF solution (10 mM, 21 μL), followed by illumination with red light coming from a 100 W halogen lamp after passing through a water tank for cooling and a color glass filter (Newport, cut-on at 610 nm). The decay of DPBF at 411 nm was monitored with time. Cell Lines and Culture Conditions. The MCF-7 human breast cancer cells (ATCC, no. HTB-22) were maintained in RPMI 1640 medium (Invitrogen, no. 223400-21) supplemented with fetal calf serum (10%), sodium pyruvate (1 mM), and penicillin−streptomycin (100 units mL−1 and 100 μg mL−1, respectively). The HT29 human colorectal carcinoma cells (ATCC, no. HTB-38) were maintained in Dulbecco’s modified Eagle medium (Invitrogen, no.10313-021) supplemented with fetal calf serum (10%), penicillin−streptomycin (100 units mL−1 and 100 μg mL−1, respectively), L-glutamine (2 mM), and transferrin (10 μg mL−1). Approximately 3 × 104 cells per well in the media were inoculated in 96-multiwell plates and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. Study of DTT- and pH-Responsive Intracellular Fluorescence. Phthalocyanines 4 and 5 were first dissolved in DMF to give 1.6 mM solutions, which were diluted to 80 μM with the culture medium in the presence of 0.5% Cremophor EL (v/v). These solutions were then further diluted with the culture medium for the following studies. About 6 × 104 MCF-7 cells were seeded on a coverslip and incubated overnight at 37 °C under 5% CO2. The medium was removed and rinsed with PBS. For the study of 4, the cells were first incubated with DTT (2 μM or 4 mM) or without DTT in PBS (2 mL) for 1 h under the same conditions. The cells were rinsed with PBS and then incubated with nigericin (Sigma) in PBS (25 μM, 2 mL) at different pH (5.0 or 7.4) for 30 min. The cells were then rinsed with PBS and incubated with a solution of 4 in the medium (1 μM, 2 mL) for 1 h. For the study of 5, the cells were incubated with nigericin in PBS (25 μM, 2 mL) at different pH (5.0, 6.0, or 7.4) for 30 min. They were then rinsed with PBS, followed by incubation with a solution of 5 in the medium (1 μM, 2 mL) for 1 h. The cells were then rinsed with PBS twice and then viewed with a Leica SP5 confocal laser scanning microscope equipped with a 633 nm helium−neon laser. The emission signals from 650 to 760 nm were collected, and the images were digitized and analyzed by Leica Application Suite Advanced Fluorescence software. The average intracellular fluorescence intensities (for a total of 30 cells in each sample) were also determined.

Study of Photocytotoxicity. DTT was dissolved in deionized water to give a 1 M stock solution, which was diluted with the culture medium to give 2 μM and 4 mM DTT solutions. Approximately 3 × 104 cells per well in the culture medium were inoculated in 96multiwell plates and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. The cells were rinsed with PBS and incubated with DTT solution (2 μM or 4 mM, 100 μL) or the culture medium (without DTT) (100 μL) for 1 h under the same conditions. The cells, after being rinsed with PBS twice, were incubated with 100 μL of the above drug solutions for 6 h at 37 °C under 5% CO2. The cells were then rinsed again with PBS and refilled with 100 μL of the culture medium before being illuminated at ambient temperature. The light source consisted of a 300 W halogen lamp, a water tank for cooling, and a color glass filter (Newport) cut-on 610 nm. The fluence rate (λ > 610 nm) was 40 mW cm−2. Illumination of 20 min led to a total fluence of 48 J cm−2. Cell viability was determined by means of the colorimetric MTT assay.56 After illumination, the cells were incubated at 37 °C under 5% CO2 overnight. An MTT (Sigma) solution in PBS (3 mg mL−1, 50 μL) was added to each well followed by incubation for 2 h under the same environment. A solution of sodium dodecyl sulfate (Sigma, 10% by weight, 50 μL) was then added to each well. The plate was incubated in an oven at 60 °C for 30 min, and then 80 μL of isopropanol was added to each well. The plate was agitated on a BioRad microplate reader at ambient temperature for 10 s before the absorbance at 540 nm for each well was taken. The average absorbance of the blank wells, which did not contain the cells, was subtracted from the readings of the other wells. The cell viability was then determined by the following equation: % viability = [(Ai/Acontrol) × 100]/n, where Ai is the absorbance of the ith data (i = 1, 2, ..., n), Acontrol is the average absorbance of the control wells in which the phthalocyanine was absent, and n (= 4) is the number of the data points. Subcellular Localization Studies. Approximately 6 × 104 MCF-7 cells in RPMI 1640 medium (2 mL) were seeded on a coverslip and incubated overnight at 37 °C with 5% CO2. After removal of the medium, the cells were first incubated with DTT in PBS (4 mM, 2 mL) for 1 h. They were then rinsed with PBS and incubated with a solution of 4 in the medium (2 μM, 2 mL) for a further 2 h under the same conditions. The medium was removed, and the cells were incubated with ER-Tracker Green (Molecular Probes, 0.2 μM in PBS), MitoTracker Green FM (Molecular Probes, 0.25 μM in the medium), or LysoTracker Green DND 26 (Molecular Probes, 2.0 μM in the medium) for a further 20 min. For all the cases, the cells were then rinsed with PBS and viewed with an Olympus FV1000 IX81-SIM confocal microscope equipped with a 488 nm multi-argon laser and a 635 nm diode laser. All the trackers were excited at 488 nm and monitored at 510−560 nm, while 4 was excited at 635 nm and monitored at 650−750 nm. The images were digitized and analyzed using the FV10-ASW software. The subcellular localization of 4 was revealed by comparing the intracellular fluorescence images caused by the ER-Tracker, MitoTracker, or LysoTracker and 4. In Vivo Imaging. Female Balb/c nude mice (20−25 g) were obtained from the Laboratory Animal Services Centre at The Chinese University of Hong Kong. All animal experiments had been approved by the Animal Experimentation Ethics Committee of the University. The mice were kept under pathogen-free conditions with free access to food and water. HT29 cells (1 × 107 cells in 200 μL) were inoculated subcutaneously at the back of the mice. Once the tumors had grown to a size of 60−100 mm3, the mice were fed with low fluorescence diet (TestDiet, no. AIN-93M) for 4 days. Phthalocyanine 4 or the noncleavable analogue of 6, in which the disulfide linker is replaced with an ethylene group (200 nmol), was first dissolved in dimethylsulfoxide (15 μL) and Tween 80 (1 μL). The solutions were then diluted to 1 nmol μL−1 with distilled water (184 μL). These phthalocyanine solutions (25 μL) were injected intratumorally to the tumor-bearing mice. In vivo fluorescence imaging was performed before and after the injection (at different time points) with an Odyssey infrared imaging system (excitation wavelength of 680 nm, emission wavelength of ≥700 nm). The images were digitized and 4095

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analyzed by the Odyssey imaging system software (no. 9201-500). Five mice were used for each compound. In Vivo Photodynamic Treatment. The length, width, and thickness of the tumors were measured by a micrometer digital caliper (SCITOP Systems). The tumor volume (mm3) was calculated using the following formula: tumor volume = π(length × width × thickness)/6. Once the tumors had grown to a size of 80−100 mm3, the mice were injected intratumorally with a solution of 4 prepared as described above (1 nmol μL−1, 25 μL). At 7 h after injection, the tumor was illuminated with a diode laser (Biolitec Ceralas) at 675 nm operating at 0.1 W. Illumination on a spot size of 1.0 cm2 for 5 min led to a total fluence of 30 J cm−2. The illumination was repeated at 48 h after injection. The tumor sizes of the nude mice were monitored periodically for a duration of 10 d. The tumor volumes were compared with a control group of mice without the drug and light treatment. Five mice were used for the photodynamic treatment with 4, while four mice were used for the control. Statistical Analyses. Data were analyzed with unpaired t test using GraphPad QuickCalcs Online. Differences were considered statistically significant at P < 0.01.



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ASSOCIATED CONTENT

S Supporting Information *

Synthetic scheme for 3, 1H NMR and 1H−1H COSY spectra of 4 in CDCl3, UV−vis spectra of 4 in DMF or RPMI 1640 culture medium, fluorescence spectra of 4−6 and 14 in the culture medium, time-dependent changes in fluorescecne intensity of 4−6 in the presence of different concentrations of DTT and/or at different pH in PBS, and comparison of the rate of photodegradation of DPBF sensitized by 4−6 upon exposure to different concentrations of DTT and/or at different pH in PBS. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*P.-C.L.: phone, +852-3943-1326; fax, +852-2603-5057; e-mail, [email protected]. *D.K.P.N.: phone, +852-3943-6375; fax, +852-2603-5057; email, [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Prof. W.-P. Fong and P.-W. Choi for help in the confocal microscopic study. This work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (Project No. 402211).



ABBREVIATIONS USED DMF, N,N-dimethylformamide; DPBF, 1,3-diphenylisobenzofuran; DTT, dithiothreitol; ESI, electrospray ionization; PBS, phosphate buffered saline; PDT, photodynamic therapy; THF, tetrahydrofuran; ZnPc, zinc(II) phthalocyanine; ΦF, fluorescence quantum yield; ΦΔ, singlet oxygen quantum yield



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dx.doi.org/10.1021/jm500456e | J. Med. Chem. 2014, 57, 4088−4097

A dual activatable photosensitizer toward targeted photodynamic therapy.

An unsymmetrical bisferrocenyl silicon(IV) phthalocyanine has been prepared in which the disulfide and hydrazone linkers can be cleaved by dithiothrei...
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