Confinement of Singlet Oxygen Generated from Ruthenium Complex

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Confinement of Singlet Oxygen Generated from Ruthenium Complex-Based Oxygen Sensor in the Pores of Mesoporous Silica Nanoparticles Natsuko Kitajima,† Yui Umehara,† Aoi Son,† Teruyuki Kondo,*,† and Kazuhito Tanabe*,‡ †

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Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto, 615-8510, Japan ‡ Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara, 252-5258, Japan S Supporting Information *

ABSTRACT: We synthesized mesoporous silica nanoparticles bearing ruthenium complexes in their pores (MSN-Ru) and characterized their photochemical properties. The ruthenium complexes that were immobilized in the pores showed oxygen-dependent phosphorescence, similar to the complexes that were not tethered to nanoparticles. Cellular imaging and in vivo experiments revealed that hypoxic cells and tissues could be visualized by monitoring the phosphorescence of MSN-Ru. Our most important finding was that the toxic effect of singlet oxygen (1O2), which was generated by excitation of the complexes, was effectively suppressed by the deactivation before leaking out from the pores. In addition, we observed a negligible toxic effect of the ruthenium complexes themselves due to the blockage of their direct interaction with intracellular biomolecules. Thus, MSN-Ru is a promising molecular probe of oxygen levels in living cells and tissues.

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cells, and the second is the generation of singlet oxygen (1O2) from excited state of the metal complexes via an energy transfer process. The 1O2 is a typical reactive oxygen species that causes the destruction of cells and tissues, leading to serious cytotoxic effects.31 Therefore, it is essential to avoid direct contact of the metal complexes and 1O2 with biomolecules in living systems, while maintaining the function of the metal complexes to exhibit oxygen-dependent phosphorescence. These research contexts urged us to construct an oxygen probe consisting of mesoporous silica nanoparticles (MSN), that were functionalized by metal complexes. We employed the ruthenium complexes with phenanthroline ligand,23−26 which showed robust phosphorescence and superior properties for chemical modification in our previous studies, and introduced them into the pores of MSN.32,33 Recently, we demonstrated that MSN modified with photosensitizers within their pores produced 1O2, which was confined within the pore.34 In the system, the 1O2 produced was deactivated before leaking out from the pores, and thus there is negligible damage in living cells. Therefore, we expected that the modification of the pores by ruthenium complexes led to 1O2, which was generated by the energy transfer from the excited state of ruthenium complexes, would be quenched in the pores and does not come out as 1O2. Thus, the damage of the cells by 1O2 would be diminished. In addition, we predicted that modification of the

INTRODUCTION Oxygen is an essential molecule for living organisms, as it maintains their vital activity.1 Oxygen is key for generating energy, and its distribution and supply in the whole body are strictly controlled.2,3 On the other hand, oxygen deficiency in cells and tissues is related to pathological conditions such as cardiac ischemia,4,5 inflammatory diseases,6 and solid tumors.7,8 As the early detection of these disease states is important for providing appropriate medical treatment, various techniques for visualizing oxygen status in living organisms have been designed and developed using functional fluorophores,9−18 radioactive species,19 oxygen-responsive electrodes,20 and magnetic resonance imaging using molecular probes.21,22 One of the most useful methods for detecting oxygen levels is the use of phosphorescence emission from metal complexes. We and others have reported several metal complexes, such as ruthenium, 23−26 iridium, 27 and porphyrin-metal complexes,28−30 that showed robust phosphorescence. In general, the phosphorescence from these complexes was emitted under hypoxic conditions, while it was quenched by molecular oxygen via the energy transfer between the triplet excited state of the metal complexes and the triplet ground state of oxygen. Energy transfer occurred instantaneously; therefore, the oxygen status could be monitored in real time by phosphorescence emission. However, for the development of versatile systems, two problems require an immediate solution. One is the toxicity of the complexes themselves in the living © XXXX American Chemical Society

Received: November 10, 2018

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DOI: 10.1021/acs.bioconjchem.8b00811 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

487 ± 54 nm (Figure S3). In MSN-Ru, the ruthenium complexes were located mainly in the inner pores. The ratio of the inner surface area to the total surface area of the nanoparticles was calculated to be 99% (eqs 1 and 2; see Experimental Section). Because the large majority of the thiol groups were located in the inner pores, the modification by ruthenium complexes occurred predominantly in the inner pores. Using absorption spectra, we estimated that the number of ruthenium complexes incorporated onto the surface of MSN was 59.8 nmol/mg. We measured the phosphorescence emission of MSN-Ru using excitation at 452 nm. As shown in Figure 2A, a robust phosphorescence emission around 600 nm was observed under hypoxic conditions, while an increase in oxygen concentration resulted in a decrease in emission because of the quenching effect of molecular oxygen. We also found that this oxygendependent emission was reversible (Figure 2B). The emission wavelength of MSN-Ru was indistinguishable from that of the naked ruthenium complexes,23−25 indicating that incorporation of the complexes into nanoparticles did not affect the triplet energy levels of the complexes. The responses of phosphorescence intensity to oxygen concentration obeyed Stern−Volmer relationships. As shown in Figure 2C, the oxygen concentration was proportional to the ratio of the phosphorescence intensities of MSN-Ru (I0/I, where I0 is the intensity under anoxic conditions and I is the intensity under the indicated oxygen concentration). The Stern−Volmer constant (Ksv), which is an index of the responsiveness to the oxygen concentration, was estimated to be 2649 M −1. Given that the Ksv values of a series of ruthenium complexes that we developed previously, such as Ru-Me2 (Figure S1),23 were 2000−3000 M−1, the ruthenium complex synthesized in this study maintained a high oxygendependent response even if it was immobilized in the pores of MSN. We next investigated the applicability of MSN-Ru to the imaging of oxygen status in human cervix epithelial adenocarcinoma HeLa cells. MSN-Ru was incubated with HeLa cells and its emission was monitored. The cells were incubated with MSN-Ru for 24 h at 37 °C, followed by replacement of the medium with fresh medium, which was maintained under either hypoxic (0.3% O2) or aerobic (20% O2) conditions, to prepare hypoxic or aerobic cells. After replacement of the medium, the cells were subjected to confocal microscopy. As shown in Figure 3, we observed bright emission from the ruthenium complex in the hypoxic cells, while the addition of oxygen promptly decreased the emission intensity. The measurement of the atomic absorption of cell lysates revealed that the amount of ruthenium complexes on MSN-Ru in cells was 0.22 or 1.52 fmol/cell, when the cells were incubated with 0.067 or 0.67 mg/mL of MSN-Ru, respectively. Thus, MSN-Ru was able to penetrate the cell membrane and allowed real-time visualization of the fluctuation of oxygen levels in living cells. The intracellular localization of MSN-Ru was evaluated by using several tracking reagents to perform color imaging. We examined the merged images of the phosphorescence of MSNRu and cytoplasmic organelle-specific fluorescent molecules, which targeted mitochondria, lysosome, endoplasmic reticulum (ER), and Golgi body. We observed a merged yellow color of MSN-Ru and fluorescent agents in lysosomes (Figure 4), whereas the other fluorescent molecules did not give any merged images (Figure S4), indicating that MSN-Ru had

pores would suppress the contact between the ruthenium complexes and biomolecules, leading to the suppression of the cytotoxic effects of the ruthenium complexes themselves. Previously, ruthenium complexes have been introduced into nanoparticles including MSN, and utilized as photoactivated drugs35 or oxygen sensors.36,37 However, to the best of our knowledge, there are no reports for the detoxification of ruthenium complex by means of nanoparticles. In this study, we prepared ruthenium complexes-modified MSN (MSN-Ru) and characterized their properties in living cells and tissues. Eventually, MSN-Ru functioned as biological oxygen probes without cytotoxic effects, even under photoirradiation conditions.

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RESULTS AND DISCUSSION Scheme 1 outlines the synthesis of MSN-Ru by coupling of ruthenium complex bearing maleimide unit (Ru-M, see Figure Scheme 1a

a

Reagents and Conditions: (a) Ru-M, DMF, sodium phosphate (pH 8.4), 7%.

S1)26,38 with thiol-modified MSN with a pore size of 4 nm (MSN-SH). To confirm the incorporation of ruthenium complexes into MSN, we measured the absorption spectra. Monodisperse MSN-Ru exhibited an absorption typical of ruthenium complexes between 400 and 500 nm, which was also observed for Ru-M (Figure S2), while MSN-SH showed no absorption at these wavelengths (Figure 1A). We also

Figure 1. (A) Absorption spectra of MSN-Ru (0.5 mg/mL, solid line) and MSN-SH (0.5 mg/mL, dashed line) in DMSO. (B) IR spectra of MSN-Ru and MSN-SH.

measured the IR spectra of MSN-Ru (Figure 1B). MSN-Ru showed a strong band around 1100 cm−1, which seemed to be a signal from the structure of the silica nanoparticles.39−41 It is striking that the bands detected around 1700 cm−1 observed for MSN-Ru were attributed to the CO stretching vibration of the maleimide unit,42 indicating that the nanoparticles incorporated the ruthenium complexes. Transmission electron microscopy images revealed that the size of the MSN-Ru was B


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Figure 2. (A) Emission spectra of MSN-Ru (10 μM: concentration of ruthenium complexes) in PBS containging 1% DMSO under different oxygen concentrations (0% O2, red line; 2% O2, orange line; 20% O2, green line; 50% O2, blue line; 100% O2, purple line). The spectra were measured with excitation at 452 nm. (B) Stern−Volmer plot of relative phosphorescence intensity (I0/I) in the presence of oxygen as a quencher. The relative emission intensity of MSN-Ru (10 μM: concentration of ruthenium complexes) was plotted against oxygen concentration [O2]. (C) Reversible responses of phosphorescence (610 nm) of MSN-Ru (Concentration of ruthenium complex: 10 μM) to alternating changes of oxygen concentration (0% and 20%).

Figure 3. (A,B) Emission images of HeLa cells as incubated with MSN-Ru. The cells were incubated with MSN-Ru (0.02 mg/mL) for 24 h at 37 °C and then the medium was replaced by fresh medium which were kept under the different oxygen concentrations (A: 0% O2, B: 20% O2). After the replacement, the images were immediately taken by means of microscopy (excitation at 450 nm and emission at 600−700 nm). (C) Cellular uptake of MSN-Ru. After incubation of HeLa cells with MSN-Ru (0.02 mg/mL) for 24 h at 37 °C, the cell lysate was harvested. The amount of ruthenium complexes in the cell lysate was measured by atomic absorption photometer.

Figure 4. (A−D) Localization of MSN-Ru in HeLa cells. The cells were incubated with MSN-Ru (0.05 mg/mL) and then lysosomes were stained by organelle markers, Lyso tracker. (A) Bright field. (B) Emission of MSN-Ru (excitation at 450 nm, emission at 600−700 nm). (C) Emission of Lyso tracker (excitation at 488 nm, emission at 498−520 nm). (D) Merged pictures. (E,F) Trafficking of MSN-Ru into HeLa cells. The cells were incubated with MSN-Ru at 37 °C (E) or 4 °C (F).

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Figure 5. (A) Photoinduced cytotoxic effect of MSN-Ru (gray) or Ru-Py (white). The HeLa cells were photoirradiated (450 nm) for 0, 10, or 20 min at 37 °C in the presence of MSN-Ru (0.1 mg/mL) or Ru-Py (6 μM). The cell viability was estimated by WST assay. (B) Time dependence of phosphorescence emission (1270 nm) of 1O2 generated from MSN-Ru (red) or Ru-Me2 (black). (C) Cytotoxicity of MSN-Ru and Ru-Py toward HeLa cells without photoirradiation. The cells were incubated with MSN-Ru (gray) or Ru-Py (white) for 24 h and the cell viability was estimated by WST assay. The concentration of ruthenium complexes (6 or 60 μM) immobilized on MSN-Ru (0.1 or 1.0 mg/mL) were identical to that of Ru-Py (6 or 60 μM).

D). SOSG was so small that it could enter the pores of MSN, and therefore, SOSG reacted with 1O2 generated even in the pores of MSN-Ru. Thus, 1O2 was produced in cells upon photoirradiation in the presence of these oxygen probes, despite the fact that photoinduced cytotoxicity was undetectable for MSN-Ru. Second, we conducted the reaction of 1O2 in the presence of anthracene derivatives, which were immobilized in the pores of MSN (MSN-Anth). Active 1O2 was detected by means of anthracene molecules in MSN-Anth, the absorption of which was reduced by the [4 + 2] cycloaddition of 1O2.34 Because anthracene units tethered in the pores of MSN-Anth could not enter the pores of MSN-Ru, the 1O2 species generated inside the pores of MSN-Ru could not react with the anthracene units. Thus, we expected that the presence of 1O2 outside the pores of MSN-Ru could be tracked by monitoring the absorption of MSN-Anth. We photoirradiated a mixture of MSN-Anth and MSN-Ru and confirmed that the change in absorption of the anthracene units at 356 nm was limited. In a control reaction, we compared the photoreaction of ruthenium complex (Ru-Me2, Figure S1), which were not tethered to MSN, in the presence of MSN-Anth and confirmed that photoirradiation of Ru-Me2 efficiently reduced absorption of anthracene units in MSN-Anth (Figure S7). These results support that 1O2 species generated in the MSN-Ru were mainly confined to the pores. Third, we compared the lifetime of the 1O2 generated in the pores of MSN-Ru with that of the 1 O2 generated from the ruthenium complex (Ru-Me2) in DMSO. As shown in Figure 5B, the 1O2 generated from MSNRu showed shorter lifetime (4.03 ± 0.01 μs) compared with Ru-Me2 (4.96 ± 0.01 μs). It is well-known that the lifetime of 1 O2 is affected by its surrounding environment.34 Thus, these experimental results strongly indicate that 1O2 was generated mainly in the pores in MSN-Ru but was inactivated before leaking out because of its shortened lifetime, leading to the reduced phototoxicity of MSN-Ru. In addition, the introduction of ruthenium complexes into the pores of MSN reduced the cytotoxicity of the ruthenium complexes themselves. The administration of a large amount of Ru-Py in cells resulted in high cytotoxicity; however, the administration of MSN-Ru, which had the same amount of ruthenium complexes, showed decreased cytotoxicity (Figure 5C). Thus, immobilization of ruthenium complexes in the pores of MSN resulted in the great advantage of decreased cytotoxicity.

efficient access to the lysosome in the cytoplasm. To verify MSN-Ru trafficking, HeLa cells were incubated at 4 °C, after the administration of MSN-Ru, and the uptake of nanoparticles was monitored. As seen in Figure 4E and F, the incubation of cells at 4 °C led to a decrease in nanoparticle internalization, suggesting the inhibition of endocytosis.43 In addition, colocalization experiments were conducted using MSN-Ru and fluorescein dextran (F-Dex), which was used as a bulkphase endocytic marker.44,45 We confirmed that these molecules showed a concordant distribution in cells (Figure S5). These results strongly indicate that MSN-Ru is likely to enter cells through endocytosis. An attempt was made to demonstrate the photochemical function of this system using HeLa cells. Initially, we compared the cytotoxic properties of MSN-Ru under photoirradiation conditions with those of our conventional oxygen probe, RuPy.23 Ru-Py showed the highest performance as an oxygen probe among our previous studies. After the administration of Ru-Py to HeLa cells and their incubation to allow the penetration of the probe into cells, we exposed the cells to photoirradiation. As shown in Figure 5, a negligible cytotoxic effect was observed in the presence of Ru-Py under conditions without photoirradiation. On the other hand, photoirradiation of the cells that had been incubated with Ru-Py resulted in a significant decrease in cell viability, indicating the generation of 1 O2 by photoirradiation of Ru-Py. We next conducted similar cellular experiments using MSN-Ru. In the experiments, the number of ruthenium complexes in MSN-Ru that were administered to cells was equalized to that of Ru-Py, which were administered to cells. In contrast to the significant cytotoxic effects of Ru-Py, MSN-Ru showed almost no cytotoxic effects in cells, even upon photoirradiation. We also conducted similar experiments using an iridium complexbased oxygen sensor, LOX-1, which is a commercially available phosphorescence probe, and confirmed its photoinduced cytotoxic effect, which was caused by the generation of 1O2 (Figure S6). To elucidate the behavior of MSN-Ru, we next assessed the generation and reaction of 1O2 and its lifetime. First, we verified the generation of 1O2 in the cells using Singlet Oxygen Sensor Green (SOSG), which is a fluorescent indicator of 1O2. The cells to which MSN-Ru, Ru-Py, or LOX-1 were administered, incubated with SOSG, and photoirradiated for 5 min. We observed bright emission of SOSG in all cells to which the oxygen probes had been administered (Figure S6BD



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EXPERIMENTAL SECTION General Procedures. 1,10-Phenanthroline maleimide, ethanol, disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, Dulbecco’s modified minimum essential medium (DMEM), and Dulbecco’s phosphate buffer saline (PBS) were purchased from Nacalai Tesque Inc. (Kyoto, Japan). cis-Dichlorobis(2,2′-bipyridine)ruthenium(II) dehydrate and N,N-dimethylformamide were purchased from Wako pure chemical Industries (Osaka, Japan). Propylthiolfunctionalized mesoporous silica nanoparticles (MSN-SH), fetal bovine serium (FBS), trypsin-EDTA, penicillin, and streptomycin were purchased from Sigma-Aldrich Japan K.K (Tokyo, Japan). Cell counting kit-8 was purchased from Dojindo laboratories (Kumamoto, Japan). Bis(2,2′bipyridine)(5-maleimide-1,10-phenanthroline)ruthenium(II) was prepared as described previously.26,35 All reagents were used without further purification unless otherwise stated. UV− vis absorption spectra were recorded on a HITACHI U-3010 spectrophotometer with a 1 cm quartz cell. All phosphorescence spectra were recorded on a JASCO EF-6300 spectrofluorophotometer with a 1 cm quartz cell. ESI-TOF mass spectra were recorded on a Bruker Daltonics microTOF focus-KE mass spectrometer. The concentration of ruthenium complexes in HeLa cells was determined with atomic absorption spectrometry (AAS; Z-2710, Hitachi Ltd., Tokyo, Japan). The spectrophotometer (iMark Microplate reader, Bio Rad laboratories Inc., California, USA) was used to evaluate a cytotoxic effect of the compounds. A confocal microscopy (Zeiss LSM710) and Xenogen in vivo Imaging System (IVIS; Xenogen) were used for in vitro and in vivo phosphorescence imaging studies. BALB/c nu/nu mice were purchased from SLC (Shizuoka, Japan). The mice were housed at the Institute of Laboratory Animals at Kyoto University Graduate School of Medicine. All studies and procedures were approved by Animal Research Committee of Kyoto University Graduate School of Medicine. All animal experiments were performed according to the Institutional Guidance of Kyoto University on Animal Experimentation and under permission by the animal experiment committee of Kyoto University. Preparation of Mesoporous Silica Nanoparticles Bearing Bis(2,2′-bipyridine) (5-maleimide-1,10-phenanthroline) Ruthenium(II) (MSN-Ru). Propylthiol-functionalized silica (MSN, 27 mg) and bis(2,2′-bipyridine) (5maleimide-1,10-phenanthroline) ruthenium(II) (27.4 mg, 39.8 μmol) were dissolved in DMF (1.5 mL) containing phosphate buffer (pH 8.4), and the resulting mixture was stirred for 12 h at room temperature in the dark. After the reaction, the mixture was washed by water and centrifuged (5000 rcf) for 5 min. Wash of the precipitate with water tentimes gave MSN-Ru as a brown solid (48.7 mg, 90%). The formation of MSN-Ru was identified by measurement of UV and IR spectra (see Figure 1). Amount of ruthenium complex introduced in MSN was measured by absorption spectra of MSN-Ru. Using Beer− Lambert Law, we calculated the absorption coefficient of ruthenium complexes (ε = 7.92 × 103 L/mol.cm) and estimated the number of ruthenium complexes incorporated onto the surface of MSN (59.8 μmol/mg). Measurement of Phosphorescence Spectra of MSNRu. Phosphorescence spectra of MSN-Ru in PBS (including DMSO in a ratio of 1%) were measured after 5 min bubbling

Based on the emission and cytotoxic properties of MSN-Ru described above, further attempts were made to monitor the fluctuation of the oxygen concentration in vivo. After the injection of MSN-Ru intramuscularly into the left leg of mice under pentobarbital anesthesia, optical imaging was performed. As shown in Figure 6, phosphorescent emission was observed

Figure 6. Optical imaging of ischemia-based hypoxia in vivo. After MSN-Ru were injected intramuscularly into left leg, the emission were monitored by in vivo imaging system (excitation at 445−490 nm, emission at 575−600 nm) (A). Then, hypoxic status at left leg was constructed by ligature, and the emissions were monitored (B).

in the left leg. Reducing the blood flow by ligaturing the left leg to establish hypoxic status resulted in an enhancement of emission intensity in the left leg. In a separate experiment, rhodamine 6G, the fluorescence emission of which is not affected by molecular oxygen, was administered intramuscularly and the emission intensity was monitored in a similar manner, to examine whether the emission of MSN-Ru was dependent on oxygen concentration in the legs of mice. As expected, the emission intensity of rhodamine 6G from the leg without ligature was similar to that from the leg with a ligature (Figure S8). These results indicate that the phosphorescence of MSN-Ru was effectively quenched by sufficient oxygen, while MSN-Ru showed bright phosphorescence in the hypoxic leg, which was established by ligation. Thus, it is reasonable to conclude that MSN-Ru is an oxygen status indicator that acts effectively in vivo.

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CONCLUSION In this study, we synthesized a nanoparticle-type sensor to visualize oxygen status in living cells and tissues. We prepared mesoporous silica nanoparticles in which phosphorescent ruthenium complexes were immobilized in the pores (MSNRu) to reduce the cytotoxicity of the metal complexes. Ruthenium complexes showed their original phosphorescence even in the pores of MSN, while the singlet oxygen generated by their photoexcitation was confined to the nanometer-sized pore. Singlet oxygen was deactivated to form ground state triplet oxygen before leaking from the pore, and thus the damage by the singlet oxygen was diminished dramatically. In addition, the suppression of direct contact with biological molecules in cells led to a decrease in the cytotoxicity of ruthenium complexes themselves when immobilized on MSN. Thus, the cytotoxicity was successfully reduced, whereas the oxygen-dependent emission of ruthenium complexes in cells and tissues was maintained. We thus showed that MSN-Ru is a promising phosphorescent oxygen probe for bioimaging. E


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laser-scanning microscope at high magnification using a 63× oil objective. Cellular Uptake of MSN-Ru to HeLa Cells (Quantification of MSN-Ru in the Cells). HeLa cells (5.0 × 105 cells/ cm2) were seeded on 6-multiwell cell culture plate and incubated for 24 h at 37 °C. After addition of MSN-Ru, the cells were further incubated for 24 h at 37 °C. Then, the cells were washed by PBS and were peeled off by trypsin EDTA. After the cell membranes were destroyed by addition of nitric acid (1 M, including triton in a ratio of 1%), the concentration of ruthenium complexes was determined by measurement of atomic absorption spectrometry (Z-2710, Hitachi Ltd., Tokyo, Japan). Cellular Localization of MSN-Ru. HeLa cells (5.0 × 104 cells) were plated into 35 mm glass bottom dish and cultured for 24 h at 37 °C. After addition of MSN-Ru, the cells were further incubated for 4 h at 37 °C. To monitor the intracellular localization of MSN-Ru, lysosome was stained by LysoTracker (80 nM), after the incubation in the presence of MSN-Ru. Then, the cells were subjected to confocal microscopy. Phosphorescent images of MSN-Ru in HeLa cells were acquired using an argon laser (458 nm) for excitation and a 600−700 nm band-pass filter for emission. Fluorescent images of lysosome were acquired using an argon laser (488 nm) for excitation and a 498−520 nm band-pass filter for emission. Endocytosis Inhibition. To HeLa cells (5.0 × 104 cells) cultured in 35 mm glass bottom dish for 24 h, was added MSN-Ru and then the cells were incubated for 4 h at 4 °C. After the incubation and wash of the cells, the medium was replaced by the new medium, and immediately the cells were subjected to confocal microscopy. Cytotoxic Effect of MSN-Ru and Previous Phosphorescent Probes (Ru-Py or LOX-1). After HeLa cells were seeded on 96-multiwell cell culture plates (2.7 × 103 cells/ cm2) and incubated for 24 h at 37 °C, MSN-Ru or previous phosphorescent probes were added to the medium and incubated for 24 h at 37 °C. After wash, living cells were stained by WST-8 and the absorbance at 450 nm was measured by the spectrophotometer. To evaluate the cytotoxic effect under photoirradiation conditions, 450 nm light was irradiated to the cells for 10 or 20 min after addition of the reagents (MSN-Ru or phosphorescent probes) and wash of the cells. Then, living cells were stained by WST-8. Detection of Singlet Oxygen Generation in the Cells. HeLa cells (3.0 × 104 cells) were plated into 35 mm glass bottom dish and cultured for 24 h at 37 °C. MSN-Ru or Ru-Py were added to the medium and incubated for 4 h at 37 °C. To stain singlet oxygen, Singlet Oxygen Sensor Green (SOSG, 2.5 μM) was added and the cells were further incubated for 3 h. After wash of the cells, 450 nm light was irradiated for 10 min and fluorescent images of SOSG were acquired using 488 nm excitation. In Vivo Imaging of Nude Mice. Before scanning, female BALB/cSlc-nu/nu mice were anesthetized with pentobarbital through i.p. injection. MSN-Ru (0.7125 mg/mL) in PBS (including DMSO in a ratio of 1%, 100 μL) were intramuscularly injected at the left leg and whole-body images were acquired. Then, the left leg was tied by thread and wholebody images were acquired again.

of gases consisted of 0% O2, 2% O2, 20% O2, 50% O2, and 100% O2. Determination of Ksv Values. The Stern−Volmer coefficient (Ksv) of MSN-Ru was estimated by using the following equations: I0/I = 1 + K sv[O2 ]

where I0 stands for relative emission intensity at 0% O2, I for emission intensity at arbitrary O2 concentrations, and Ksv for Stern−Volmer coefficient. Stern−Volmer plot ([O2] versus [I0/I]) was used to determine Ksv value of a MSN-Ru. Calculation of Inner and Outer Area of MSN Surface. Inner and outer MSN surface area were calculated by eq 1 and eq 2, respectively.34 Assuming that MSN (radius: 244 nm) composes of tubes (radius: 2.0 nm) hexagonally arranged in 61 layers (≈244/4) from the center, the inner and outer area were expressed in the following equations. ii 1 yy Inner area = 1 × 2πr × 2 R2 − jjjr jjj1 − zzzzzz 2 {{ kk

2

61

+

∑ (6(n − 1)) × 2πr

ii 1 yy × 2 R2 − jjjr jjjn − zzzzzz 2 {{ kk n=2

Outer area = 4πR2

2

(1) (2)

where R = MSN radius (= 244 nm) and r = the pore radius (= 2.0 nm). Consequently, the inner area and outer area are 6.3 × 107 nm2 and 7.5 × 106 nm2 per particle, respectively. Therefore, the ratio of the inner area to the total area is approximately 99%. Measurement of Emission of Singlet Oxygen. Phosphorescence emission of singlet oxygen at 1270 nm, which was obtained from photoirradiation of MSN-Ru and RuMe2, were measured by means of spectrometer consisted of XeCl excimer laser (Lambda Physik Complex102, 308 nm, Pulse width 200 ns) equipped with photomultiplier tube (Hamamatsu Photonics H10330B-45). The samples in DMSO were prepared and subjected to spectrometer. HeLa Cells Culture. The HeLa cells were cultured at 37 °C in 5% CO2 in 75 cm2 flasks containing Dulbecco’s modified minimum essential medium (DMEM) supplemented with 10% fetal bovine serium (FBS), 1% 10000 U/mL Penicillin and 10 mg/mL Streptomycin. Cellular Imaging of MSN-Ru. To HeLa cells (3.0 × 104 cells) cultured in 35 mm glass bottom dish for 24 h, was added MSN-Ru (0.02 mg/mL) and then the cells were incubated for 24 h at 37 °C under normoxic conditions. After the incubation, the medium was replaced by new medium under aerobic conditions (20% O2), and immediately, phosphorescent images were taken by confocal microscopy (images under aerobic conditions). On the other hand, for the phosphorescent images of hypoxic cells, the medium was replaced by new medium under hypoxic conditions (0% O2) and images were taken (images under hypoxic conditions) immediately. The medium for imaging of hypoxic cells was prepared by standing under hypoxic conditions for 24 h. Phosphorescent images of MSN-Ru in HeLa cells were acquired using an argon laser (458 nm) for excitation and a 600−700 nm band-pass filter for emission. The specimens were viewed with a confocal F


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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/acs.bioconjchem.8b00811. Chemical structures, absorption spectra, TEM images, localization and trafficking of MSN-Ru in HeLa cells, photo-induced cytotoxic effects, optical imaging of mice (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +81-75-383-7055. FAX: +81-75-383-2504. *E-mail: [email protected]. Phone: +81-42759-6229. FAX: +81-42-759-6493. ORCID

Teruyuki Kondo: 0000-0003-1213-2337 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We sincerely thank Professor Tadashi Suzuki (Aoyama Gakuin University) for the measurement of emission and lifetime of singlet oxygen. This work was supported in part by Grant-inAid for Scientific Research (for K.T. Grant number 23113508, and for T.K. Grant numbers 18K05353 and 17H05528), Grant-in-Aid for Challenging Exploratory Research (for A.S. Grant number 16K12876), Grant-in-Aid for JSPS Fellows (for Y.U. Grant number 17J03866) and the Innovative TechnoHub for Integrated Medical Bio-Imaging of the Project for Developing Innovation Systems from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. T.K. and A.S. acknowledge financial support from the Acceleration Transformative Research for Medical Innovation (ACT-M) from the Japan Agency for Medical Research and Development (AMED).

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Confinement of Singlet Oxygen Generated from Ruthenium Complex

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