Crossfire for Two-Photon Photodynamic Therapy ... - ACS Publications

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Crossfire for Two-Photon Photodynamic Therapy with Fluorinated Ruthenium (II) Photosensitizers Kangqiang Qiu,† Jinquan Wang,†,§ Cuilan Song,† Lili Wang,‡ Hongyi Zhu,† Huaiyi Huang,† Juanjuan Huang,† Hui Wang,‡ Liangnian Ji,† and Hui Chao*,† MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and ‡School of Physics, Sun Yat-Sen University, Guangzhou 510275, P. R. China § Guangdong Provincial Key Laboratory of Biotechnology Candidate Drug Research, Guangdong Pharmaceutical University, Guangzhou 510006, P. R. China Downloaded via UNIV OF SUSSEX on August 17, 2018 at 09:17:12 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: Synergistic photodynamic therapy (PDT) that combines photosensitizers (PSs) to attack different key sites in cancer cells is very attractive. However, the use of multiple PSs may increase dark cytotoxicity. Additionally, realizing the multiple vein passage of several PSs through dosing could be a challenge in clinical treatment. To address these issues, a novel strategy that enables a single PS to ablate two key sites (i.e., cytomembranes on the outside and mitochondria on the inside) of cancer cells synergistically was proposed. Five new fluorinated ruthenium (II) complexes (Ru1−Ru5), which possessed excellent two-photon properties and good singlet oxygen quantum yields, were designed and synthesized. When incubated with HeLa cells, the complexes were observed on the cytomembranes at first. With an extension of the treatment time, both the cytomembranes and mitochondria were lit up by the complexes. Under two-photon laser irradiation, the mitochondria and cytomembranes were ablated simultaneously, and the HeLa cells were destroyed effectively by the complexes, whether the cells were in a monolayer or in multicellular spheroids. With the largest phototoxicity index under the two-photon laser, Ru4 was used for two-photon PDT of in vivo xenograft tumors and successfully inhibited the growth of the tumors. Our results emphasized that the strategy of attacking two key sites with a single PS is an efficient method for PDT. KEYWORDS: Two-photon photodynamic therapy, ruthenium (II) complex, cytomembrane, mitochondria, fluorination

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cellular events, including apoptosis.10 Both of these contain O2. Therefore, combining PSs that act on the cytomembrane and the mitochondrion could potentially enhance the antitumor effect. However, compared with the single PS, synergistic PDT could increase the dark cytotoxicity caused by the multiple PSs. Additionally, realizing the multiple vein passage of several PSs through dosing could be a challenge in clinical treatment.11 To address these issues, a single PS that ablates both the cytomembranes (outside) and mitochondria (inside) cancer cells synergistically was developed in this work (Scheme 1a). Possessing rich visible light absorption and a reasonably longlived triplet excited state that allows the efficient generation of 1 O2, the Ru(II) polypyridyl complex has been recognized as an excellent PS candidate.12−24 These Ru(II) polypyridyl complexes could overcome some defects of traditional porphyrinbased PSs, such as low water solubility and slow clearance from the body.25,26 The preassociation of the complexes with the cationic nature of Ru(II) polypyridyl complexes,26 which aid

INTRODUCTION Cancer, the second leading cause of death, has been responsible for 15% of all deaths, and it is predicted that the number of new cancer patients will increase by ∼27 million by 2030.1,2 Among the various therapeutic technologies to combat cancer under study, photodynamic therapy (PDT) has attracted tremendous attention because it is a noninvasive medical technique that destroys cancer cells without systemic toxicity3 because the nontoxic photosensitizer (PS) becomes toxic only in response to light.4,5 Upon light irradiation, 1O2 and other cytotoxic reactive oxygen species (ROS) generated by the excited PS, ultimately, cause cancer cell damage.6 Besides, 1O2 is thought to be the primary cytotoxic ROS in PDT because 1O2 is a very reactive and toxic form of oxygen.7 Because of its high reactivity, the lifetime and diffusion of 1O2 is quite limited, and PDT, therefore, makes the induction of cell death with spatial and temporal control possible.8 The cytomembrane is the first protective barrier of cancer cells and the only channel for exchanging substances (i.e., O2) and information between the cancer cell and its environment.9 Furthermore, mitochondria play a critical role in the cellular energy production of cancer cells and regulate many other © 2017 American Chemical Society

Received: March 1, 2017 Accepted: May 5, 2017 Published: May 5, 2017 18482

DOI: 10.1021/acsami.7b02977 ACS Appl. Mater. Interfaces 2017, 9, 18482−18492

Research Article

ACS Applied Materials & Interfaces

Scheme 1. (a) Schematic Illustration of the Ru(II) Complexes Ablating the Cytomembrane (Outside) and the Mitochondria (Inside) Synergistically under Two-Photon Laser Irradiation and (b) Chemical Structures of Ru1−Ru5

electrospray ionization mass spectrometry (ESI-MS) (Figures S1−S10). Ru1−Ru5 (10 μM) showed good solubility, and their emission spectra and electronic absorption spectra were studied at 298 K. The photophysical properties are summarized in Table S1. In the absorption spectra, the bands at 350−550 nm in Figure S11 consisted of overlapping Ru(dπ) → dmp(π*) and Ru(dπ) → ligand(π*) transitions and were assigned to metal−ligand charge transfer (3MLCT).38 In the emission spectra, the emission peaks between 550 and 800 nm were obtained from the excitation of Ru1−Ru5 (Figure S12). Referenced to Ru(bpy)3Cl2 (6.2%),39 the relative quantum yields of the complexes were 5.2−6.5%. Furthermore, the luminescent lifetimes of the complexes were determined in both air-equilibrated and degassed acetonitrile. The lifetime values for the complexes were 120−150 ns in air-equilibrated acetonitrile and 480−520 ns in degassed acetonitrile. All of the lifetimes of the excited states of the complexes were strongly influenced by the presence of oxygen, indicating that the ground state of molecular oxygen could interact with the triplet excited states of the complexes.7 For studying the two-photon properties of Ru1−Ru5, their two-photon excited luminescence was investigated and rhodamine B was used as the reference.40 At 825 nm, the maximum two-photon action cross section of Ru1−Ru5 was estimated to be 163−191 Göppert−Mayer (GM) units (Figure 1, Table S1), which was larger than that for other well-known PSs (2.2 GM for H2TPP and 66 GM for [Ru(bpy)3]2+).31,41 The log−log linear relationships of the complexes between the incident power and the emission intensity were observed, with slopes of 2.10 for Ru1, 2.06 for Ru2, 2.09 for Ru3, 2.18 for Ru4, and 2.04 for Ru5 (Figure S13). Singlet Oxygen Production. The production of the singlet oxygen of Ru1−Ru5 under irradiation was confirmed by the electron spin resonance (ESR) spin trapping technique.42 Without light irradiation, no signals could be detected in the air-saturated CH3OH solutions of the complexes (20 μM) and 2,2′,6,6′-tetramethylpiperidine (TEMP, 1O2 trapper, 50 mM). After radiation at 450 nm for 5 min, three-line signals with 1:1:1 intensities were found

the cytomembrane through electrostatic attraction, is also advantageous to the complexes into the cell. Besides, Ru(II) polypyridyl complexes have large Stokes shifts, impressive photostability, and bright luminescence, which allows for PDT with these complexes to act as imaging guided therapy.27−29 Moreover, because of their excellent two-photon properties, Ru(II) polypyridyl complexes could be used for two-photon PDT under low-energy near-infrared or longer wavelength laser irradiation, which allows for deeper therapy and less damage to normal cells.30−33 No matter whether in nonpolar or polar environments, fluorinated compounds have a high phase-separation tendency. Therefore, the affinity of compounds for the cell membrane can be improved by fluorination.34−37 Herein, we designed and synthesized five new fluorinated Ru(II) complexes (Ru1−Ru5) (Scheme 1b) for two-photon PDT. The two-photon properties and the singlet oxygen quantum yields of these complexes were measured. The distribution and cellular uptake in the HeLa cells of the complexes were investigated. The complexes were found on the cytomembranes within 30 min of treatment of HeLa cells and in both the mitochondria and cytomembranes after 4 h. Besides, the abilities of the complexes to generate 1O2 in HeLa cells were confirmed and the changes in morphology of HeLa cells after two-photon PDT were investigated. The dark cytotoxicity and photocytotoxicity of the complexes were evaluated in monolayer cells and multicellular spheroids under one- and two-photon irradiation. With the largest phototoxicity index, Ru4 was used for in vivo studies of xenograft tumors.

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RESULTS AND DISCUSSION Synthesis and Photophysical Properties. Ru1−Ru5 were synthesized as in Scheme S1. On condensation with 4-tertbutylaniline, 1,10-phenanthroline-5,6-dione, and fluorinated benzaldehyde, the ligands (L) were obtained with good yield. The Ru(II) complexes were obtained by the reactions between Ru(dmp)2Cl2 (dmp = 4,7-dimethyl-1,10-phenanthroline) and L in a mixed solvent system of alcohol and water in yields ranging from 80 to 90%. The complexes were characterized by elemental analysis, 1H and 19F NMR spectroscopy and 18483


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Cellular Uptake and Localization. After confirming that Ru1−Ru5 could efficiently produce 1O2 upon light irradiation, we investigated their cellular localization and cellular uptake efficiency. The cellular uptake of PSs is important for PDT treatment efficacy and cellular imaging. It is known that the cellular uptake levels depend on the incubation time. Because of their excellent two-photon properties, cellular distributions of the complexes were investigated by the two-photon imaging technology of confocal laser scanning microscopy (CLSM). As shown in Figures 2a and S16, within 0.5 h treatment, more complexes were located on the cytomembranes. When the incubation time was extended, the complexes entered the cells, although some complexes were still found on the cytomembranes after 4 h incubation. Two-photon Z-stack imaging was used to confirm the presence of complexes on the cytomembranes and in the cells (Figures S17−S21). Furthermore, colocalization was determined by staining the organelles with DiO (a commercial cell membrane imaging dye) and MitoTracker Deep Red FM (MTDR). As shown in Figures 2b and S22, after 0.5 h incubation, by overlapping with the imaging region, the complexes were colocalized with DiO. After 4 h incubation, the red stains of the complexes were colocalized with DiO and MTDR (Figures 2c and S23). These results showed that Ru1−Ru5 were localized outside (on the cytomembranes) of HeLa cells after a short incubation time (0.5 h) and were localized both outside (on the cytomembranes) and inside (on the mitochondria) after 4 h incubation. The Ru contents were determined to be 11−14 ng per million cells by inductively coupled plasma mass spectrometry (ICPMS) after 4 h incubation (Figure S24). Because of the cationic nature of Ru1−Ru5, the complexes attach to the anionic cytomembrane through electrostatic

Figure 1. Two-photon action absorption cross sections of Ru1−Ru5 at different excitation wavelengths from 725 to 1050 nm.

(Figure S14) between 3480 and 3530 G, a characteristic of the adduct for TEMP and 1O2.43 To determine the efficiency of singlet oxygen generation, the 1 O2 quantum yields of the complexes were quantified by a direct method (Figure S15).44 On consideration of the shortlived singlet oxygen in aqueous solution (∼3.5 μs), the assay was performed in CD3CN (70−80 μs).30 Upon irradiation at 458 nm, and by direct comparison with the 1O2 emission areas of the well-known PS [Ru(bpy)3]2+ (0.56), the 1O2 generation quantum yields of Ru1−Ru5 were determined to be 0.53−0.59 in CD3CN (Table S1). The results indicated that Ru1−Ru5 were efficient PSs.

Figure 2. (a) Images of HeLa cells incubated with Ru1 at different times (10 μM, λex = 825 nm, λem = 630 ± 20 nm). (b) Images of HeLa cells colabeled with Ru1 (0.5 h) and DiO (5 μM, 20 min, λex = 488 nm, λem = 510 ± 10 nm). (c) Images of HeLa cells colabeled with Ru1 (4 h), DiO, and MTDR (50 nM, 0.5 h, λex = 633 nm, λem = 660 ± 10 nm). BF represents bright field. Overlay1 and Overlay2: Overlay of Ru1 and DiO. Overlay3: Overlay of Overlay2 and MTDR. Scale bar: 20 μm. 18484


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Figure 3. (a) Images of 1O2 generation in HeLa cells incubated with Ru1−Ru5 (10 μM, 4 h, λex = 458 nm, λem = 630 ± 20 nm) after one-photon light irradiation for 2 min. Overlay of complexes and DCF. Scale bar: 20 μm. (b−d) Images of 1O2 generation in three-dimensional (3D) MCTS incubated with Ru1 (10 μM, 6 h, λex = 825 nm, λem = 630 ± 20 nm) before and after two-photon laser irradiation, BI stands for before irradiation and AI stands for after irradiation. Scale bar: 200 μm. (c) The two-photon Z-stack images of Ru1 were taken of every 5 μm section from the top to bottom. (d) The Z-stack images of DCF were taken of every 5 μm section from the top to bottom. The images were taken under a 10× objective. DCF (10 μM, 0.5 h, λex = 488 nm, λem = 510 ± 10 nm). BF stands for bright field.

Table 1. (Photo)cytotoxicity (IC50, μM) of Tested Complexes toward HeLa Cells 0.5 h treatment with complexes complexes Ru1 Ru2 Ru3 Ru4 Ru5 cisplatin

a

dark 54.72 62.59 48.51 64.13 59.15 ndd

± ± ± ± ±

4.30 5.07 4.33 4.68 3.29

b

light 0.84 0.81 0.62 0.75 0.88 ndd

± ± ± ± ±

0.07 0.15 0.06 0.11 0.08

4 h treatment with complexes PI

c

dark

65.14 77.27 78.24 85.5 67.22 nae

28.88 35.10 24.58 35.85 32.33 26.48

± ± ± ± ± ±

a

1.91 3.42 2.33 1.68 2.95 4.29

lightb

PIc

0.63 ± 0.09 0.55 ± 0.08 0.46 ± 0.08 0.49 ± 0.13 0.64 ± 0.12 25.68 ± 3.12

45.84 63.82 53.43 73.16 50.52 1.03

a The IC50 values in the dark. bThe IC50 values upon light irradiation. cPI is the phototoxicity index, which is the ratio between the IC50 values in the dark and upon light irradiation. dNot determined. eNot applicable.

Treated with Ru1−Ru5 at 4 °C or pretreated with the metabolic inhibitors oligomycin and 2-deoxy-D-glucose, the energy-dependent (active transport, endocytosis) or the energy-independent (passive diffusion, facilitated diffusion) transport pathway for complexes into cells was determined.48 The suppressed cellular luminescence indicated Ru1−Ru5 uptake followed an energy-dependent pathway (Figure S26). Adenosine triphosphate (ATP) or temperature could affect endocytosis.49 Endocytic inhibitors NH4Cl and chloroquine were used to restrain endocytosis. The luminescence was not suppressed and the complexes were not taken up by endocytosis. This result means that the complexes were taken up via the active transport pathway. 1 O2 Generation and Phototoxicity in HeLa Cells under One-Photon Irradiation. The abilities of complexes to

attraction.13 No matter whether in nonpolar or polar environments, fluorinated compounds have a high phaseseparation tendency. The affinity of compounds for the cell membrane can be improved by fluorination.34−37 Therefore, it is reasonable that the complexes were found on the cytomembranes even after 4 h incubation. According to a theory of Horobin et al.,45−47 cations with log P values between 0 and 5 show a high probability of localization in the mitochondria; the targets of the lipophilic Ru(II) complexes (Figure S25) in cells were the mitochondria. Ru1−Ru5 were found on the cytomembranes after 0.5 h incubation, not in the cell. This phenomenon means the complexes cannot go freely across the cytomembranes. To determine how the complexes enter cells, the cellular uptake mechanisms of Ru1−Ru5 were investigated by CLSM. 18485


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Figure 4. Images of HeLa cells incubated with Ru1 (10 μM, λex = 825 nm, λem = 630 ± 20 nm) after two-photon light irradiation for different periods of time. (a) HeLa cells incubated with Ru1 30 min. (b) HeLa cells incubated with Ru1 4 h. BF represents bright field. Scale bar: 20 μm.

generate 1O2 within HeLa cells were investigated using the 2,7dichlorodi-hydrofluorescein diacetate (DCFH-DA) fluorescence assay. In the living cell, DCFH-DA is hydrolyzed and oxidized to 2,7-dichlorofluorescein (DCF) by 1O2, DCF is a fluorescent compound.31 HeLa cells were pretreated with Ru1−Ru5 and incubated with DCFH-DA. Before imaging, the cells were washed with phosphate-buffered saline (PBS) six times, after which the complexes were still found on the cytomembranes. After one-photon light irradiation, strong green intracellular signals were observed (Figure 3a), indicating the generation of 1O2 in cells. Under one-photon light irradiation, either on the outside or inside of the cells, the complexes could generate 1O2. The darkand photo-cytotoxicity of Ru1−Ru5 toward HeLa cells were investigated (Table 1). After treatment with the complexes in 0.5 or 4 h, the media were removed and replaced with fresh media without any washing. The complexes were slightly toxic in the dark, and the cytotoxicity was enhanced with the increased treatment time. Upon irradiation with 450 nm surface light for 10 min (12 J cm−2), the cytotoxicity of the complexes increased significantly, with nanomolar IC50 values ranging from 620 to 880 nM after 0.5 h treatment and from 460 to 630 nM after 4 h treatment. The low IC50 values in the 0.5 h treatment indicated that the complexes could effectively kill HeLa cells despite the short time of treatment with complexes. Because of the low dark cytotoxicity on the cytomembranes, the phototoxicity index (PI = IC50 in the dark/IC50 upon irradiation) after 0.5 h treatment was higher than the PI of 4 h. However, after 4 h treatment, the complexes could kill HeLa cells at lower concentrations due to the presence of the complexes on the cytomembrane and in the mitochondria, which facilitated damage to the cytomembranes and the mitochondria simultaneously upon irradiation. 1 O2 Generation and the Morphology Changes of HeLa Cells under Two-Photon Irradiation. Compared to that of the one-photon surface light source, the volume of the two-

photon laser light source is small because of the quadratic intensity dependence of two-photon excitation.50 As a result, it is difficult to obtain the IC50 values of two-photon cytotoxicity in a 96-well plate with monolayer cells. To observe the cytotoxic effect induced by two-photon light irradiation, the morphology changes of HeLa cells were tracked by CLSM. Before tracking, the complexes generated 1O2 in cells by twophoton light irradiation, which was confirmed by the green signals of DCF (Figure S27). After two-photon laser irradiation, as shown in Figures 4a and S28−S31, the morphology of HeLa cells pretreated with complexes for 30 min changed significantly and blebs were found. As the irradiation time was extended, cell shrinkage and more blebs were observed, and the complexes on the cytomembranes entered into the cytoplasm through the damaged cytomembranes. Because of the complexes damaging the cytomembranes and the mitochondria simultaneously upon two-photon light irradiation, the morphology of the HeLa cells pretreated with complexes for 4 h changed dramatically (Figures 4b and S32−S35). Severe cell shrinkage and blebs were observed after 2 min two-photon light irradiation. The morphology of the mitochondria in the cytoplasm changed from solid granules to hollow spheres, when the irradiation time was extended, which represent extremely swollen mitochondria.51 For comparison, the cells without complexes showed no significant damage under irradiation (Figure S36). Phototoxicity in HeLa 3D Multicellular Tumor Spheroids (MCTSs). To accurately quantify two-photon PDT, a model that could be irradiated completely by a two-photon laser light source was needed. Additionally, conventional twodimensional (2D) culture models present significant limitations in term of reproducing many of the important characteristics of in vivo solid tumors, and they often fail to predict the drug response in a tumor.52,53 Three-dimensional MCTSs, a validated 3D cancer model between cell monolayers and solid tumors, have been widely used for the assessment of anticancer drugs and have been exploited to evaluate two-photon PDT 18486


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ACS Applied Materials & Interfaces Table 2. (Photo)cytotoxicity (IC50, μM) of Complexes toward HeLa MCTSs with 6 h Treatment complexes Ru1 Ru2 Ru3 Ru4 Ru5 cisplatin

darka 41.3 59.4 34.9 62.0 50.4 72.6

± ± ± ± ± ±

3.1 4.2 4.7 5.7 4.0 6.2

OPb

PIc

± ± ± ± ± ±

21.0 31.8 24.2 37.1 26.4 1.05

1.97 1.87 1.44 1.67 1.91 69.4

0.31 0.30 0.28 0.21 0.25 7.9

TPd

PIe

± ± ± ± ± ±

39.7 83.7 67.1 101.6 53.1 1.03

1.04 0.71 0.52 0.61 0.95 70.8

0.12 0.17 0.10 0.11 0.14 5.4

a

The IC50 values in the dark. bThe IC50 values under one-photon light irradiation. cPI is the phototoxicity index, which is the ratio between the IC50 values in the dark and upon one-photon light irradiation. dThe IC50 values under two-photon light irradiation. eThe ratio between the IC50 values in the dark and upon two-photon light irradiation.

Figure 5. (a) Representative photographs of HeLa tumors in mice with four different treatments (physiological saline; physiological saline + laser; Ru4 only; Ru4 + laser). (b) Histological examination of tumors with four treatments after a postirradiation period of 12 h. (c) Tumor volumes with time of the mice under different treatments. (d) Body weight changes with time of the mice under different treatments.

effectively.31 Therefore, 3D MCTSs were used for assessing the two-photon PDT effect of Ru(II) complexes. The HeLa MCTS uptake of the Ru(II) complexes depending on the treatment time was investigated. Under two-photon light irradiation, the complexes gradually lit up the HeLa MCTSs with red luminescence. At 6 h, the red luminescence reached a maximum intensity (Figure S37). These results indicated that the complexes could overcome the extracellular matrix barrier, which may hinder drug delivery.54 For the long excitation wavelength at 700−900 nm, two-photon excitation

exhibited deeper tissue penetration depth than one-photon excitation. The penetration depth of two-photon excited luminescence for the Ru(II) complexes in HeLa MCTSs was ∼200 μm, compared to the ∼70 μm depth for one-photon excitation (Figures S38−S42). Before two-photon PDT, the abilities of the complexes to generate 1O2 in HeLa MCTSs in response to a two-photon laser were confirmed by the green signals of DCF (Figures 3b− d and S43−S46). After being incubated with the complexes for 6 h, the HeLa MCTSs were exposed to two-photon laser light 18487


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ACS Applied Materials & Interfaces (360 J cm−2) or one-photon surface light (12 J cm−2). After another 48 h incubation time, the IC50 values of the complexes were measured by the CellTiter-Glo 3D Cell Viability kit (Promega). The dark cytotoxicity of the complexes (Table 2) was slightly toxic. For deeper penetration depth, the IC50 values of two-photon excitation were lower than the values of onephoton excitation, and the PI of two-photon excitation was larger than that of one-photon excitation. With the largest PI (101.6 for Ru4) under two-photon laser light, Ru4 was chosen for the study of its two-photon PDT efficacy in in vivo xenograft tumors. Two-Photon PDT in Vivo. To evaluate the in vivo twophoton PDT efficacy of Ru4, the HeLa tumor-bearing mice with a xenograft tumor volume of 75 mm3 were randomized into four groups (eight mice per group): physiological saline as control (group 1), physiological saline and two-photon laser irradiation (group 2), Ru4-injected only (group 3), and Ru4injected and two-photon laser irradiation (group 4). The mice in group 4 were intratumorally injected with 65.8 μg kg−1 (25 μL, 40 μM) Ru4 and irradiated by an 800 nm femtosecond laser (1.18 W cm−2, 25 min) 6 h postinjection. The same laser dose and the same Ru4 dose were utilized with groups 2 and 3. After a postirradiation period of 12 h, the tumors were removed from one mouse in each group for histological examination by hematoxylin and eosin stain (H&E stain). No significant damage was found in groups 1−3. Besides, the tumor tissue of group 4 was noted to display irreversible pathological alterations. Pyknosis, blue cell nuclei, and pink proteins were observed in the tumor tissue of group 4, reflecting necrosis or apoptosis of the tumor cells (Figure 5a,b). The tumor growth rates were recorded every 3 days for 15 days. As shown in Figure 5c, compared to their original size, the tumors grew 12.7-fold for group 1, 12.1-fold for group 2, 11.6-fold for group 3, and 1.2-fold for group 4. Group 4 exhibited an apparent inhibition of tumor growth, and tumor volume inhibition efficiency was found to be ∼90.5%, whereas no significant difference was found in groups 3, 2, and 1. This result indicated that Ru4 had excellent therapeutic effects on in vivo xenograft tumors. Potential systemic toxicities of the treatments were also investigated. In addition to recording the body weight, the histology of the organs (heart, intestine, spleen, lung, ovary, brain, liver, and kidney) was also observed. Neither noticeable body weight loss (Figure 5d) nor significant organ damage (Figure S47) could be found in the four groups, which suggested that Ru4 possessed relatively low systemic toxicity. All data indicated that Ru4 was a promising PDT agent in vivo.

HeLa tumors, which indicated that Ru4 was a promising PDT agent in vivo. Overall, our results demonstrated a new strategy for PDT, that is, ablating two key sites in cancer cells (outside and inside) synergistically using a single PS to be an efficient method for anticancer therapy.

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MATERIALS AND METHODS

Materials. Without purification unless otherwise specified, the purchased reagents were used directly. Ruthenium chloride hydrate, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), cisplatin, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 4,7-dimethyl-1,10-phenanthroline (dmp) were obtained from Sigma-Aldrich. Mitotracker Deep Red FM (MTDR) and DiO were purchased from Life Technology. Prior to the experiments, Ru1−Ru5 were dissolved in dimethyl sulfoxide (DMSO), the final DMSO concentration was less than 1% (v/v). Physical Measurement. A Perkin-Elmer 240Q elemental analyzer was used for measuring microanalysis (C, H, and N). An LCQ system (Finnigan MAT) was used for recording the electrospray ionization mass spectrum (ESI-MS). A nuclear magnetic resonance spectrometer (Mercury-Plus 300, Varian) was used for recording the NMR spectrum and tetramethylsilane was used as the standard. A PerkinElmer Lambda 850 spectrophotometer was used for recording the UV−vis spectra and the n-octanol/water partition coefficients. A Perkin-Elmer LS 55 spectrofluorophotometer was used for recording the emission spectra and [Ru(bpy)3]2+ (φ = 0.062 in deaerated CH3CN)39 was used as the reference for calculating the quantum yield of phosphorescence. An FLS 920 combined fluorescence-lifetime and steady-state spectrometer (Edinburgh Instruments Itd) was used for time-resolved emission measurement and 1O2 production measurement, [Ru(bpy)3]2+ was used as the reference (0.56 in air-saturated CD3CN).55 The two-photon laser microscopy system consisted of a confocal microscope (Zeiss LSM 710 NLO) and a two-photon laser (COHERENT MRU X1). The system was manipulated by ZEN 2011 software (blue edition). The resolution of the image was 1024 × 1024. The images were processed by ZEN 2011 software (blue edition). A Bruker model A300 spectrometer was used for recording the ESR spectra and 2,2′,6,6′-tetramethylpiperidine (TEMP) was used as the spin trap for 1O2. All measurements were carried out at 25 °C. Synthesis. According to literature methods, 1,10-phenanthroline5,6-dione56 and cis-Ru(dmp)2Cl2·2H2O57 were prepared. 1-(4-(tert-Buty)phenyl)-2-(4-fluorophenyl)-1H-imidazo[4,5-f ][1,10]phenanthroline (L1). A mixture of glacial acetic acid (10 mL), 4(tert-butyl)aniline (0.149 g, 1 mmol), 4-fluorobenzaldehyde (0.124 g, 1 mmol), ammonium acetate (1.542 g, 20 mmol), and 1,10-phenanthroline-5,6-dione (0.21 g, 1 mmol) was refluxed under argon for 24 h. Neutralized with 25% NH3 solution, a yellow precipitate was collected and water was used to wash the precipitate. The product was recrystallized from CH2Cl2 and produced a green powder. (Yield: 74%). 1H NMR (300 MHz, CDCl3) δ 9.25−9.00 (m, 3H), 7.76 (dd, J = 8.1, 4.4 Hz, 1H), 7.67−7.52 (m, 4H), 7.49−7.38 (m, 3H), 7.30 (dd, J = 8.4, 4.2 Hz, 1H), 6.99 (t, J = 8.6 Hz, 2H), 1.46 (s, 9H). ESI-MS: m/z = 447.1 [M + H]+, 915.3 [2M + Na]+. 1-(4-(tert-Buty)phenyl)-2-(3,4-difluorophenyl)-1H-imidazo[4,5-f ][1,10]phenanthroline (L2). L2 was synthesized in a manner identical to that described for L1, except with 3,4-difluorobenzaldehyde (0.142 g, 1 mmol) in place of 4-fluorobenzaldehyde. (Yield: 76%). 1H NMR (300 MHz, CDCl3) δ 9.36−9.02 (m, 3H), 7.83 (dd, J = 8.1, 4.6 Hz, 1H), 7.68 (d, J = 8.3 Hz, 2H), 7.59−7.29 (m, 6H), 7.09 (dd, J = 17.6, 9.0 Hz, 1H), 1.47 (s, 9H). ESI-MS: m/z = 465.1 [M + H]+, 951.4 [2M + Na]+. 1-(4-(tert-Buty)phenyl)-2-(3,4,5-trifluorophenyl)-1H-imidazo[4,5f ][1,10]phenanthroline (L3). L3 was synthesized in a manner identical to that described for L1, except with 3,4,5-trifluorobenzaldehyde (0.160 g, 1 mmol) in place of 4-fluorobenzaldehyde. (Yield: 72%). 1H NMR (300 MHz, CDCl3) δ 9.39−9.14 (m, 3H), 7.91 (dd, J = 8.1, 4.9 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.53−7.39 (m, 4H), 7.25−7.14 (m, 2H), 1.49 (s, 9H). ESI-MS: m/z = 483.1 [M + H]+, 987.4 [2M + Na]+.

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CONCLUSIONS In summary, we have designed and synthesized five new fluorinated Ru(II) complexes, which possessed excellent twophoton properties and good singlet oxygen quantum yields, for the two-photon photodynamic and synergistic ablation of cytomembranes (outside) and mitochondria (inside) cancer cells. The complexes were found on the cytomembranes of HeLa cells after 30 min treatment and in both the mitochondria and cytomembranes after 4 h. Under two-photon laser irradiation, the mitochondria and cytomembranes were ablated simultaneously, and the HeLa cells were destroyed by the complexes effectively, both in monolayer cells and in multicellular spheroids. Because it had the largest PI under two-photon laser irradiation, Ru4 was used for a two-photon PDT in vivo study and successfully inhibited the growth of 18488


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Opolette 355II (pulse width ≤100 fs, 80 MHz repetition rate, tuning range 725−1050 nm, Spectra Physics Inc.) was used to acquire the two-photon luminescence data of the complexes in the fluorometric quartz cuvettes. The reabsorption can affect two-photon absorption measurements and was used to conduct the excitation and detection for the experimental luminescence. At an excitation wavelength of 825 nm, the quadratic relationships between the excitation power and the two-photon-induced luminescence intensity for the complexes were verified. The two-photon absorption cross sections of each wavelength for Ru1−Ru5 were calculated according to eq 1

1-(4-(tert-Buty)phenyl)-2-(2,3,5,6-tetrafluorophenyl)-1H-imidazo[4,5-f ][1,10]phenanthroline (L4). L4 was synthesized in a manner identical to that described for L1, except with 2,3,5,6-tetrafluorobenzaldehyde (0.178 g, 1 mmol) in place of 4-fluorobenzaldehyde. (Yield: 77%). 1H NMR (300 MHz, CDCl3) δ 9.41 (s, 1H), 9.22 (dd, J = 12.5, 6.2 Hz, 2H), 7.92 (dd, J = 7.9, 4.9 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.52 (dd, J = 8.4, 1.4 Hz, 1H), 7.46−7.38 (m, 3H), 7.18 (t, J = 8.4 Hz, 1H), 1.42 (s, 9H). ESI-MS: m/z = 501.1 [M + H]+, 1023.3 [2M + Na]+. 1-(4-(tert-Buty)phenyl)-2-(perfluorophenyl)-1H-imidazo[4,5-f ][1,10]phenanthroline (L5). L5 was synthesized in a manner identical to that described for L1, except with 2,3,4,5,6-pentafluorobenzaldehyde (0.196 g, 1 mmol) in place of 4-fluorobenzaldehyde. (Yield: 74%). 1H NMR (300 MHz, CDCl3) δ 9.32 (ddd, J = 27.7, 6.0, 3.3 Hz, 3H), 8.02 (dd, J = 8.0, 4.5 Hz, 1H), 7.62 (d, J = 8.6 Hz, 2H), 7.54− 7.46 (m, 2H), 7.39 (d, J = 8.2 Hz, 2H), 1.44 (s, 9H). ESI-MS: m/z = 519.1 [M + H]+, 1059.4 [2M + Na]+. Ru(dmp)2(L1)Cl2 (Ru1). A mixture of cis-Ru(dmp)2Cl2·2H2O (0.12 g, 0.2 mmol) and L1 (0.134 g, 0.3 mmol) in the mixture solvent of ethanol and water (10 mL, 9:1, v/v) was refluxed under argon for 8 h to give a clear red solution. The solvent was removed by rotary evaporation. Column chromatography was used to purify the crude product. Acetonitrile and ethanol were used as the eluents. (Yield: 85%). Anal. Calcd for C57H47Cl2FN8Ru: C, 66.15; H, 4.58; N, 10.83%. Found: C, 65.93; H, 4.90; N, 10.66%. 1H NMR (300 MHz, DMSO-d6) δ 9.16 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 9.6 Hz, 4H), 8.09 (d, J = 5.2 Hz, 1H), 7.92 (ddd, J = 21.7, 13.6, 6.4 Hz, 6H), 7.67 (ddd, J = 26.3, 21.8, 11.2 Hz, 10H), 7.52−7.47 (m, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.30 (t, J = 8.7 Hz, 2H), 2.91 (s, 12H), 1.39 (s, 9H). 19F NMR (300 MHz, DMSO-d6) δ −110.7 (1F). ESI-MS: m/z = 481.7 [M − 2Cl−]2+. Ru(dmp)2(L2)Cl2 (Ru2). Ru2 was synthesized in a manner identical to that described for Ru1, except with L2 (0.139 g, 0.3 mmol) in place of L1. (Yield: 83%). Anal. Calcd for C57H46Cl2F2N8Ru: C, 65.02; H, 4.40; N, 10.64%. Found: C, 64.87; H, 4.63; N, 10.53%. 1H NMR (300 MHz, DMSO-d6) δ 9.12 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 7.1 Hz, 4H), 8.06 (d, J = 5.3 Hz, 1H), 7.95−7.80 (m, 6H), 7.76−7.64 (m, 4H), 7.60−7.38 (m, 9H), 2.90 (s, 12H), 1.38 (s, 9H). 19F NMR (300 MHz, DMSO-d6) δ −135.8 (1F), −137.7 (1F). ESI-MS: m/z = 491.1 [M − 2Cl−]2+. Ru(dmp)2(L3)Cl2 (Ru3). Ru3 was synthesized in a manner identical to that described for Ru1, except with L3 (0.145 g, 0.3 mmol) in place of L1. (Yield: 87%). Anal. Calcd for C57H45Cl2F3N8Ru: C, 63.92; H, 4.24; N, 10.46%. Found: C, 63.64; H, 4.55; N, 10.39%. 1H NMR (300 MHz, DMSO-d6) δ 9.12 (dd, J = 8.2, 1.2 Hz, 1H), 8.44 (d, J = 6.9 Hz, 4H), 8.07 (dd, J = 5.3, 1.2 Hz, 1H), 7.96−7.82 (m, 6H), 7.79−7.66 (m, 4H), 7.58 (dt, J = 9.4, 5.5 Hz, 4H), 7.52−7.36 (m, 4H), 2.89 (s, 12H), 1.38 (s, 9H). 19F NMR (300 MHz, DMSO-d6) δ −134.1 (2F), −157.9 (1F). ESI-MS: m/z = 500.1 [M − 2Cl−]2+. Ru(dmp)2(L4)Cl2 (Ru4). Ru4 was synthesized in a manner identical to that described for Ru1, except with L4 (0.150 g, 0.3 mmol) in place of L1. (Yield: 84%). Anal. Calcd for C57H44Cl2F4N8Ru: C, 62.87; H, 4.07; N, 10.29%. Found: C, 62.63; H, 4.36; N, 10.12%. 1H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 6.6 Hz, 4H), 8.28−8.11 (m, 1H), 8.07 (d, J = 6.4 Hz, 1H), 7.99 (t, J = 3.3 Hz, 1H), 7.95−7.90 (m, 2H), 7.87−7.80 (m, 3H), 7.70−7.50 (m, 10H), 2.89 (s, 12H), 1.32 (s, 9H). 19F NMR (300 MHz, DMSO-d6) δ −137.8 (2F), −138.2 (2F). ESI-MS: m/z = 508.6 [M − 2Cl−]2+. Ru(dmp)2(L5)Cl2 (Ru5). Ru5 was synthesized in a manner identical to that described for Ru1, except with L5 (0.155 g, 0.3 mmol) in place of L1. (Yield: 85%). Anal. Calcd for C57H43Cl2F5N8Ru: C, 61.85; H, 3.92; N, 10.12%. Found: C, 61.55; H, 4.13; N, 10.04%. 1H NMR (300 MHz, DMSO-d6) δ 9.08 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 6.4 Hz, 4H), 8.08 (d, J = 5.3 Hz, 1H), 7.99 (d, J = 5.0 Hz, 1H), 7.93 (t, J = 4.9 Hz, 2H), 7.88−7.80 (m, 3H), 7.70−7.51 (m, 10H), 2.89 (s, 12H), 1.33 (s, 9H). 19F NMR (300 MHz, DMSO-d6) δ −137.9 (2F), −148.1 (1F), −160.4 (2F). ESI-MS: m/z = 517.0 [M − 2Cl−]2+. Determination of Two-Photon Absorption Cross Sections.58,59 To determine the two-photon absorption spectra of the ruthenium (II) complexes, rhodamine B in methanol as the standard and the typical two-photon induced luminscence method was used.

δ2 = δ1

ϕ1C1I2n2 ϕ2C 2I1n1

(1)

where ϕ is the quantum yield, n is the refractive index, C is the concentration, and I is the integrated luminescence intensity. Subscript “1” represents the reference, while “2” stands for the sample. Cell Culture Conditions. In a humidified incubator with an atmosphere of 95% air and 5% CO2 at a constant temperature of 37 °C, fetal bovine serum (10%, v/v) and Dulbecco’s modified Eagle medium (DMEM) were used to culture the HeLa cells, which were obtained from the Experimental Animal Center, Sun Yat-sen University (Guangzhou, China). Confocal Imaging of 2D HeLa Cells. Seeded in 35 mm glass bottom dishes (Corning) for 24 h, the HeLa cells were incubated with 10 μM complexes in the dark for 4 h. After the media was replaced, cell imaging was performed. For cytomembrane colocalization, after treatment with Ru(II) complexes for 10 min, DiO was added and further incubated for 20 min. For cytomembrane and mitochondrial colocalization, after incubation with Ru(II) complexes for 3.5 h, MTDR and DiO were added and further incubated for 0.5 h. For singlet oxygen detection, after incubation with Ru(II) complexes for 4 h, the cells were washed with PBS three times and incubated with DCFH-DA for 30 min. For morphology change tracking, the cells were washed with PBS, incubated with 10 μM of the Ru(II) complexes for 30 min or 4 h in the dark. To image the cells, a Zeiss LSM 710 NLO confocal microscope (63×/NA 1.4 oil immersion objective) was used. At 458 nm (for Ru1−Ru5), 488 nm (for DiO and DCF), 633 nm (for MTDR), or 825 nm (for two-photon imaging), the luminescence (fluorescence) was excited. At 630 ± 20 nm (for Ru1−Ru5), 510 ± 10 nm (for DiO and DCF) or 660 ± 10 nm (for MTDR), the emission signal was collected. Cellular Uptake (ICP-MS). Plated onto 10 cm tissue culture dishes for 48 h, the HeLa cells were treated with the complex (10 μM) for 4 h. After being washed with PBS, the cells were trypsinized, collected, counted, and digested with HNO3 (60%) for 1 day. An Agilent inductively coupled plasma mass spectrometer was used for measuring the concentration of ruthenium. Cell Uptake Mechanism. For normalized incubation and low temperature incubation, HeLa cells were treated with 10 μM complexes at 37 and 4 °C for 4 h, respectively. Pretreated with 5 μM oligomycin and 50 mM 2-deoxy-D-glucose for 1 h at 37 °C for metabolic inhibition, the cells were then incubated with 10 μM complexes at 37 °C for 4 h. Pretreated with chloroquine (50 μM), or endocytic inhibitors NH4Cl (50 mM) for 30 min, the cells were then incubated with 10 μM complexes at 37 °C for 4 h. After incubation with inhibitors and complexes, the cells were excited with 825 nm luminescence, and the emission signal was collected at 610−650 nm. Photocytotoxicity Test on 2D HeLa Cells. Plated onto 96-well plates for 24 h, the HeLa cells were incubated with increasing concentrations of the compounds in the dark for 0.5 or 4 h. Then, the media was removed and replaced with fresh media. For phototoxicity studies, the cells were irradiated for 10 min (12 J cm−2). Both the dark and light groups were incubated for an additional 48 h. MTT (5 mg mL−1) was used to stain the viable cells for 4 h. DMSO (200 μL) was added to each well after the media was aspirated. The optical density was measured at 595 nm by using a Tecan Infinite M200 monochromator-based multifunction microplate reader and the cell survival rate was 100% for the wells without complexes. 18489


Research Article

ACS Applied Materials & Interfaces Generation of 3D MCTSs. 3D MCTSs were obtained by a liquid overlay method. A 150 μL suspension with 6000 cells was added to the 96-well plates coated with 0.5 mg agarose (in 50 μL DMEM). Cultured in incubator, the MCTSs were 450−550 and 750−850 μm in diameter on day 3 and day 6, respectively. Confocal Imaging of 3D MCTSs. For investigating the uptake of Ru(II) complexes, 3D MCTSs of 750−850 μm diameter were treated with Ru(II) complexes (10 μM) for different times (1, 2, 3, 4, 5, and 6 h). For singlet oxygen detection, after treatment with Ru(II) complexes (10 μM) for 6 h, the 3D MCTSs were washed with PBS three times and incubated with DCFH-DA (10 μM) for 30 min. The images of spheroids were collected using a Zeiss LSM 710 NLO confocal microscope (10× objective). At 458 nm (for Ru1−Ru5), 488 nm (for DCF), or 825 nm (for two-photon imaging), the complexes became luminescent (fluorescent). The emission signal was collected at 630 ± 20 nm (for Ru1−Ru5) or 510 ± 10 nm (for DCF). Photocytotoxicity Test on 3D MTCSs. On incubation with increasing concentrations of the compounds for 6 h, the 3D MTCSs with 450−550 mm diameter were divided into three groups: the first group remained unchanged, the second group was subjected to onephoton irradiation (450 nm, 12 J cm−2), the third group was subjected to two-photon irradiation (825 nm, 360 J cm−2). The cytotoxicity of the complexes toward 3D MTCSs was measured by ATP concentration with the CellTiterGlo kit (Promega) on infinite M200 PRO equipment (TECAN). Two-Photon PDT in Vivo.60,61 BALB/c-(nu/nu) female nude mice aged 4−5 weeks were purchased and bred in the Traditional Chinese Medicine and Marine Medicine Laboratory, School of Life Sciences, Sun Yat-sen University. All experimental protocols were approved by the Sun Yat-sun University Animal Care and Use Committee. We then subcutaneously (s.c.) injected 2 × 106 cells into BALB/c-(nu/nu) female nude mice, and HeLa xenograft tumors were established. When the tumor volumes reached approximately 150 mm3, the well-grown tumors were cut into 1 mm3 fragments, and the fragments were transplanted (s.c.) into the other nude mice. The nude mice were allocated into four groups (eight mice per group) randomly when the tumor volumes of the mice reached approximately 75 mm3. The PDT process was as follows: Group 1 (physiological saline, control): physiological saline solution (25 μL) was intratumorally injected into mice; Group 2 (physiological saline + laser): physiological saline solution (25 μL) was intratumorally injected into mice, and the mice were irradiated by 800 nm femtosecond laser (1.18 W cm−2, 25 min); Group 3 (Ru4-injected only): mice were intratumorally injected with Ru4 (65.8 μg kg−1, 25 μL, 40 μM); Group 4 (Ru4-injected + laser): mice were intratumorally injected with Ru4 (65.8 μg kg−1, 25 μL, 40 μM) and irradiated by 800 nm femtosecond laser (1.18 W cm−2, 25 min) after 6 h postinjection. The mice were anesthetized by using Gas Anesthesia Systems (XGI8, XENOGEN). A caliper was used to measure the tumor sizes every 3 days. At day 0, day 5, and day 15, a digital color camera was used to photograph the mice. The following formula 2 was used for calculate the tumor volumes

analysis of variance and with the t-test for grouped data. Differences were considered significant at P less than 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b02977. NMR spectra, ESI-MS spectra, absorption spectra, emission spectra, logarithmic plots, ESR signals, 1O2 phosphorescence spectra, images at different times, Zstack images of cells, colocalization images with DiO, colocalization images with DiO and MTDR, cellular uptake efficiency, the n-octanol/water partition coefficients, cellular uptake mechanism, images of DCF in HeLa cells, images of HeLa cells after irradiation, images of spheroids with time increased, one- and two-photon images of spheroids, images of 1O2 generation in spheroids, histological examination, and photophysical data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-20-86110613. Fax: +86-20-84112245. ORCID

Lili Wang: 0000-0002-9273-0021 Hui Chao: 0000-0003-4153-5303 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 program (No. 2015CB856301), and the National Science Foundation of China (Nos. 21471164 and 21525105).

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REFERENCES

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