Complexes with a Modified Acetylacetonato Ligand - American

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Intracellular and in Vivo Oxygen Sensing Using Phosphorescent Ir(III) Complexes with a Modified Acetylacetonato Ligand Toshitada Yoshihara,† Masahiro Hosaka,‡ Motoki Terata,‡ Kazuki Ichikawa,† Saori Murayama,† Asami Tanaka,† Masanobu Mori,§ Hideyuki Itabashi,§ Toshiyuki Takeuchi,∥ and Seiji Tobita*,† †

Department of Chemistry and Chemical Biology, Gunma University, Kiryu, Gunma 376-8515, Japan Department of Biotechnology, Akita Prefectural University, Shimoshinjo, Akita 010-0195, Japan § Department of Environmental Engineering Science, Gunma University, Kiryu, Gunma 376-8515, Japan ∥ Gunma University, Maebashi, Gunma 371-8510, Japan ‡

S Supporting Information *

ABSTRACT: Small luminescent molecular probes based on the iridium(III) complex BTP, (btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone) have been developed for sensing intracellular and in vivo O2. These compounds are BTPSA (containing an anionic carboxyl group), BTPNH2 (containing a cationic amino group), and BTPDM1 (containing a cationic dimethylamino group); all substituents are incorporated into the ancillary acetylacetonato ligand of BTP. Introduction of the cationic dimethylamino group resulted in an almost 20-fold increase in cellular uptake efficiency of BTPDM1 by HeLa cells compared with BTP. The phosphorescence intensity of BTPDM1 internalized in living cells provided a visual representation of the O2 gradient produced by placing a coverslip over cultured monolayer cells. The intracellular O2 levels (pO2) inside and outside the edge of the coverslip could be evaluated by measuring the phosphorescence lifetime of BTPDM1. Furthermore, intravenous administration of 25 nmol BTPDM1 to tumor-bearing mice allowed the tumor region to be visualized by BTPDM1 phosphorescence. The lifetime of BTPDM1 phosphorescence from tumor regions was much longer than that from extratumor regions, thereby demonstrating tumor hypoxia (pO2 = 6.1 mmHg for tumor and 50 mmHg for extratumor epidermal tissue). Tissue distribution studies showed that 2 h after injection of BTPDM1 into a mouse, the highest distribution was in liver and kidney, while after 24 h, BTPDM1 was excreted in the feces. These results demonstrate that BTPDM1 can be used as a small molecular probe for measuring intracellular O2 levels in both cultured cells and specific tissues and organs.

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shell,20,21 or embedding it in a suitable polymer.22 Porphyrins have high absorption and emission efficiencies, and provide near-infrared absorption and emission that can penetrate tissue deeper than visible light. Pt(II)- and Pd(II)-porphyrins with a dendrimeric shell have been successfully used to assess intravascular O2 tension in animal models.23,24 To increase the efficiency of intracellular O2 measurements, metal porphyrins have been conjugated with cell-penetrating substituents to improve intracellular delivery,25,26 and O2 in multicellular spheroids has been imaged by phosphorescence lifetime measurements using metal porphyrin derivatives.27,28 Ru(II) complexes phosphoresce at room temperature, but their phosphorescence lifetimes are rather short (0.1−5 μs), so their O2 sensitivity is much lower than that of metal porphyrins. To improve O2 sensitivity and lipophilicity, Ru(II) complexes have been modified with a hydrophobic ligand such as pyrene.14

olecular oxygen (O2) plays a crucial role as a key molecule in cellular metabolism and bioenergetics. Consequently, the ability to sense O2 at the cellular and tissue level is essential in physiology, pathophysiology and cancer therapy, as well as in cell biology.1−3 Phosphorescence quenching methods, recently developed as intracellular and in vivo O2 sensing techniques,4−9 offer several advantages over other techniques, including high spatial resolution, good sensitivity, and the possibility of real-time O2 measurements in both cells and tissues. However, phosphorescence based O2 sensing generally requires exogenous probes that emit strong phosphorescence at room temperature. A limited number of compounds exhibit strong phosphorescence, of which Pt(II)and Pd(II)-porphyrins,10−12 Ru(II) complexes13−15 and Ir(III) complexes9,16−18 have been extensively studied as biological O2 probes. Pt(II)- and Pd(II)-porphyrins have relatively long phosphorescence lifetimes (10−1000 μs) that permit measurements of very low concentrations of O2. The sensitivity of the porphyrin complex to O2 can be tuned by conjugating it with a macromolecular carrier,19 coating it with a dendrimeric © XXXX American Chemical Society

Received: October 19, 2014 Accepted: January 29, 2015

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DOI: 10.1021/ac5040067 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. (A) Chemical structures of BTP, BTPSA, BTPNH2, BTPDM1 and BTPDM2. (B) Absorption and phosphorescence spectra of (a) BTP and (b) BTPDM1 in tetrahydrofuran at room temperature. The phosphorescence spectra were taken in both degassed and aerated solutions.

Ir(III) complexes with moderately long lifetimes (1−15 μs) have also been reported as luminescent dyes incorporated into nanoparticle oxygen sensors and the capability for sensing oxygen in extracellular fluids such as blood and interstitial fluids has been studied.16,17 On the other hand, we have recently reported that Ir(III) complexes serve as small luminescent molecular probes for both cellular and in vivo O2 sensing.29−33 Ir(III) complexes have excellent emission tunability,29,34 from blue to the near-infrared region, and a high phosphorescence quantum yield (>0.1) across this range. They are readily transported into living cells; furthermore, the ligand structures can be easily modified using typical synthetic techniques, thus allowing facile tailoring of their optical and cellular properties. We have shown that a red-emitting Ir complex, BTP (acetylacetonatobis[2-(2′-benzothienyl)pyridinato-κN, κC3′]iridium(III)), Figure 1A) has excellent potential for imaging hypoxic lesions such as tumor tissues.29 The spectral properties of BTP are determined almost exclusively by the benzothienylpyridinato groups; the acetylacetone moiety (ancillary ligand) has little influence on the spectral properties of the complex. The physicochemical properties of BTP can thus be

improved by introducing an appropriate substituent into the ancillary ligand while minimally perturbing the original photophysical properties. Various probes for intracellular or in vivo oxygen measurements have previously been developed, but few O2 probes can penetrate the capillary vessel wall and sense the intracellular O2 levels of living animals. Here we describe small molecular probes which can act as intracellular O2 sensor in various tissues. First, we designed and synthesized BTP derivatives bearing a carboxyl group (BTPSA), an amino group (BTPNH2), or a dimethylamino group (BTPDM1) in the acetylacetonato ligand (Figure 1A). Under physiological conditions, BTPSA is likely to be in the anionic form, generated by deprotonation of the carboxyl group, whereas BTPNH2 and BTPDM1 are likely in the cationic form, produced by protonation of the amino and dimethylamino group, respectively. Substituting the ancillary ligand in BTP allows the overall charge of the complex, and thus its hydrophobicity or lipophilicity, to be tuned. We also synthesized a fourth BTP derivative, BTPDM2 (Figure 1A), in which a dimethylamino group is substituted into the B


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Council of Gunma University (13-031) and Akita Prefectural University (12-01). Naive 6- to 7-week old female athymic BALB/c nude mice (nu/nu) from Charles River Laboratories Japan, Inc. were used as transplant recipients. Tumor transplants were established in nude mice by injection of 5 × 106 SCC-7 cells. Experiments with tumor-bearing mice were performed 2 weeks after injection of the tumor cells. In Vivo Imaging. Luminescence image measurements of the mice were performed as described previously.29 Luminescence images were obtained using the Maestro 2 in vivo imaging system (CRi, Inc.). Excitation was provided using a blue filter (435−480 nm) and emission using a green filter (>560 nm). The tunable filter was automatically stepped in 10 nm increments from 560 to 750 nm, and the camera captured images at each wavelength interval using a 100 ms exposure. Spectral fluorescence images consisting of autofluorescence spectra and Ir spectra were obtained and then unmixed using the Maestro software. The spectra of unmixed images are shown in Figure 5 (sky-blue is autofluorescence, red is the Ir complex spectra).

benzothienylpyridinato ligand through a methylene group. We next compared the cellular entry, subcellular localization and in vivo localizations of these Ir complexes in order to develop higher performance O2 probes. We propose an analytical method for evaluating intracellular O2 levels based on phosphorescence lifetime measurements with our probes using a time-correlated single-photon counting (TCSPC) system.

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EXPERIMENTAL SECTION Materials and Instruments. BTP, BTPSA, BTPNH2, BTPDM1 and BTPDM2 were synthesized and identified according to the methods described in the Supporting Information. Tetrahydrofuran (THF; Kanto, spectroscopic grade) was used as received. Absorption and emission spectra were recorded on a UV/vis spectrophotometer (Jasco, Ubest-V550) and a photonic multichannel analyzer PMA-12 (Hamamatsu, C10027-01) equipped with a monochromatized Xe arc lamp, respectively. The emission spectrum was corrected for spectral sensitivity. Phosphorescence lifetimes of the Ir complexes in solution were measured with a TCSPC system (Hamamatsu, Quantaurus-Tau C11367). Phosphorescence lifetimes of cultured cells were measured by using an inverted microscope (Olympus IX71) equipped with an O2 concentration-changeable multigas incubator (TOKAI HIT INUB-ONICS-F1-H2, GM-8000), a laser diode (TOPTICA PHOTONICS iBeam smart-S 488-S: 488 nm; pulse width, 25 or 50 ns; repetition rate, 40 kHz) as the excitation light source and a TCSPC system (Hamamatsu, Quantaurus-Tau C11367). In vivo lifetime measurements were made by using the same lifetime measurement system combined with a bifurcated fiber that was used to irradiate the skin surface (about 3 mm diameter) of a mouse and collect the emission from the irradiated area. Phosphorescence quantum yield was measured with an absolute photoluminescence quantum yield system (Hamamatsu, C992001)35 consisting of a Xe arc lamp, a monochromator, an integrating sphere, and a multichannel detector. Cell Culture and Imaging. Human uterine cancer-derived HeLa cells and human breast cancer-derived MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM). Mouse oral squamous carcinoma-derived SCC-7 cells and Chinese hamster ovary-derived CHO cells were cultured in RPMI-1640 and F-12 medium, respectively. These media were supplemented with 10% fetal bovine serum, penicillin (50 units/mL) and streptomycin (50 μg/mL). All cells were grown at 37 °C under a 5% CO2 atmosphere. For 2.5% O2 incubation of the cells, an O2 concentration-changeable multigas incubator (Wakenyaku Ex 9200) was used. To image living cells, the cells were seeded on glassbottomed dishes, allowed to adhere for 24 h and then incubated with Ir (III) complex at the indicated concentrations and for the indicated durations. An inverted microscope (Olympus IX71) equipped with a 100× oil-immersion objective lens and an electron multiplying CCD camera (Evolve 512, PHOTOMETRICS) driven by MetaMorph software were used to obtain luminescence microscope images. Samples were excited using a 100 W mercury lamp and imaged using custom filter settings (excitation: a bandpass filter (400−440 nm), emission: a long-pass filter (>590 nm)). Tumor Transplantation into Nude Mice. All studies were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals of the Medical Research

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RESULTS AND DISCUSSION Photophysical Properties of BTP and BTP Derivatives. We first elucidated the photophysical properties of BTP and modified BTPs in THF. The absorption and emission spectra of BTP and BTP derivatives in THF exhibited very similar spectral shapes and positions, as illustrated with BTP and BTPDM1 in Figure 1 (see also Figure S1, Supporting Information). The absorption band with a maximum at around 485 nm can be assigned to the electronic transition of the first singlet metal-to-ligand charge transfer (1MLCT) state, which has a moderately large absorption coefficient: 6700 M−1 cm−1 at 486 nm and 6800 M−1cm−1 at 485 nm for BTP and BTPDM1, respectively. Emission spectra with a maximum at around 615 nm are attributable to phosphorescence and are largely due to ligand (btp)-centered triplet ππ* transitions with significant contributions from triplet metal-to-ligand charge transfer (3MLCT) (dπ(Ir)→π*(btp)).36 The phosphorescence quantum yield (Φ0p) and lifetime (τ0p) of BTPDM1 in deaerated THF at room temperature were 0.29 and 5.6 μs, respectively, similar to those of BTP (Φ0p = 0.30 and τ0p = 5.7 μs). Because the quantum yield of the S1→T1 intersystem crossing of typical Ir complexes can be assumed to be unity,37 the phosphorescence radiative rate constants (kp) of BTP and BTPDM1 can be calculated to be 5.3 × 104 and 5.2 × 104 s−1, respectively, from the Φ0p and τ0p values by using the relation kp = Φ0p/τ0p. The extremely large kp values of BTP and BTPDM1 compared with those of typical aromatic molecules arise from the significantly larger spin−orbit coupling of BTP and BTPDM1 in the phosphorescent state. The large emission quantum yield arising from the large kp value makes these compounds advantageous for use as biological luminescent probes. The absorption and phosphorescence spectra, phosphorescence quantum yields, and lifetimes of the BTP derivatives shown in Figure 1A were very close to those of BTP (Table 1 and Figure S1 (Supporting Information)), indicating that the substitution in the acetylacetonato ligand has little influence on both the relaxation properties and spectral properties of BTP. Furthermore, as shown in Table 1, the introduction of a dimethylamino group into the benzothienylpyridinato ligand of BTP through a methylene bond to generate BTPDM2 has little influence on the photophysical properties. As can be seen from Table 1, BTP and BTP derivatives possess suitable spectral and C


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Analytical Chemistry Table 1. Maximum Absorption Wavelength (λmax abs ), Maximum Phosphorescence Wavelength (λmax phos), Phosphorescence Quantum Yield and Lifetime of BTP Derivatives in THF at Room Temperature compound

λmax abs (nm)

λmax phos (nm)

Φ0p

τ0p (μs)

τp (ns)

BTP BTPSA BTPNH2 BTPDM1 BTPDM2

486 485 485 485 488

615 615 616 616 619

0.30 0.28 0.28 0.29 0.30

5.72 5.67 5.53 5.62 5.01

112 114 117 108 124

Φ0p and τ0p denote the phosphorescence quantum yield and lifetime taken for degassed solutions, and τp stands for the phosphorescence lifetime taken for aerated solutions.

photophysical characteristics as cellular and in vivo luminescent probes, although the BTP derivatives are inferior in brightness and spectral characteristics compared to some known probes such as Pt and Pd porphyrins.10−12 The phosphorescence of BTP and BTPDM1 in THF was significantly quenched by dissolved O2. The quenching due to molecular O2 could be quantified by using the Stern−Volmer equation and the bimolecular quenching rate constants (kq) of BTP and BTPDM1 were obtained to be 6.3 × 104 and 5.6 × 104 mmHg−1 s−1, respectively. The O2 quenching of BTP and BTPDM1 phosphorescence was also examined in a lipid bilayer membrane, as these lipophilic probes are likely to accumulate in organelle membranes when they pass through the plasma membrane of living cells. The kq values in DMPC (dimyristoylL-α-phosphatidylcholine) model membranes in Tris−HCl buffer at 35 °C were determined to be 1.2 × 104 and 9.1 × 103 mmHg−1 s−1 for BTP29 and BTPDM1 (Figure S3, Supporting Information), respectively. We previously reported that the phosphorescence spectrum, quantum yield and lifetime of BTP incorporated into DMPC liposomes in buffered solutions are essentially unaffected by changes in the pH of the solution within the physiological pH range.32 Cellular Uptake and O2 Response in Living Cells. We examined the cellular uptake of BTP and the BTP derivatives into human uterine cancer-derived HeLa cells by phosphorescence microscopy (Figure 2A). The photographs taken with an exposure time of 500 ms show that BTPSA gives a much weaker image than BTP. In contrast, the phosphorescence images of BTPNH2, BTPDM1 and BTPDM2, taken with an exposure time of 500 ms were much brighter than that of BTP (Figure 2A shows the images taken with an exposure time of 200 ms). These findings suggest that cellular uptake efficiency for BTP is remarkably enhanced by introducing a cationic amino or dimethylamino group into the ligand. To confirm the higher cellular uptake efficiencies of BTP derivatives containing a cationic group, we performed ICP-MS analyses of the Ir complexes internalized into HeLa cells using the same probe concentration and incubation conditions (see the Supporting Information for experimental details). ICP-MS enables us to determine the content of individual heavy metals in biological cells and tissues. The average amount of BTPNH2 (5.9 fmol), BTPDM1 (18.5 fmol) and BTPDM2 (9.1 fmol) in a single HeLa cell was much higher than that of BTP (1.0 fmol). In particular, the cellular uptake efficiency for BTPDM1 was about 20 times higher than that for BTP. In contrast to the cationic BTP derivatives, the intracellular content of anionic BTPSA was only 0.65 fmol. These results from ICP-MS analyses are in good agreement with the relative intensities of

Figure 2. (A) Emission images of HeLa cells incubated with BTP or BTP derivatives (5 μM) for 2 h at 37 °C. Exposure times of the CCD camera were 500 or 200 ms. Scale bar, 20 μm. (B) Emission images of HeLa cells incubated with BTP or BTPSA (5 μM for 6 h), or BTPNH2, BTPDM1 or BTPDM2 (1 μM for 6 h) at 37 °C. The HeLa cells were placed in 21% and 2.5% O2.

the microscopic phosphorescence images of HeLa cells shown in Figure 2A. Because the cellular uptake efficiency of BTPDM1 and BTPDM2 seems to be facilitated by cooperation between the cationic charge and lipophilicity, we measured the octanol/ water partition coefficient (expressed in log Po/w) of BTP, BTPSA, BTPNH2, BTPDM1 and BTPDM2 and obtained log Po/w values of 1.38, 1.14, 0.94, 1.16 and 0.96, respectively (see the Supporting Information for experimental details). As expected, BTPDM1 gave the largest log Po/w, i.e., the highest lipophilicity, of the three cationic BTP derivatives. These results indicate that the cellular uptake of Ir complexes is significantly affected by the overall charge and lipophilicity of the complex.38,39 It should be noted that cellular uptake of cationic complexes could benefit from the potential gradient across the plasma membrane. BTP and its derivatives exhibited significant O2 responses in HeLa cells (Figure 2B): the brightness of the phosphorescence image was significantly enhanced when the O2 pressure in the incubator was reduced from normoxia (21%) to hypoxia (2.5%). We next investigated the cellular uptake and O2 response of BTP and BTPDM1 by using other cell lines: Chinese hamster ovary (CHO)-derived CHO, mouse oral squamous carcinomaderived SCC-7, and human mammary adenocarcinoma-derived MCF-7 (Figure S3, Supporting Information). The phosphorescence images of BTPDM1 were much brighter than those of BTP in these cell lines, suggesting significantly higher cellular uptake efficiency of BTPDM1 compared to BTP (Figure S3A, Supporting Information). Remarkable O2 responses were also seen for BTPDM1 and BTP phosphorescence in the CHO, SCC-7 and MCF-7 cells (Figure S3B,C, Supporting Information). At least for these cell lines, no significant cell-type dependency was observed for cellular uptake efficiency and O2 response of BTP and BTPDM1. We then examined the intracellular localization of the BTP derivatives in living cells by comparing the phosphorescence image of BTP derivatives with the fluorescence images of organelle-specific trackers (Figure S4, Supporting Information); BTPNH2, BTPDM1 and BTPDM2 were found to be mainly localized to the lysosome, in contrast with the specific endoplasmic reticulum (ER) D


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Analytical Chemistry localization of BTPSA and BTP in HeLa cells.29 These results indicate that the cellular uptake efficiency and the intracellular localization of Ir complexes can be modified by incorporating a suitable substituent into the ligand. Recently, mitochondriatargeted oxygen sensing has been reported by using phosphorescent oxygen nanosensors.40 We further compared the photostability and cellular uptake of BTPDM1 with those of a well-known Ru complex oxygen probe Ru(dpp)32+ (dpp = 4,7-diphenyl-1,10-phenanthroline)16,17 (Figure S5, Supporting Information). BTPDM1 was photochemically less stable than Ru(dpp)32+ in HeLa cells, whereas the cellular uptake efficiency of BTPDM1 was much higher than that of Ru(dpp)32+, demonstrating the excellent property of BTPDM1 as an intracellular oxygen sensor. Evaluation of O2 Levels in Monolayer Cultured Cells. The oxygen level of cells can be evaluated from the phosphorescence lifetime of probes delivered into cells if suitable calibration measurements are made. However, determining the intracellular O2 level of even cultured cells is not straightforward. The oxygen level of cultured cells depends on various factors, such as the pO2 in the incubator, the total oxygen consumption rate of cells (which is related to the respiratory status of each cell), the cell density, and the volume of medium.41 Furthermore, O2 molecules are not homogeneously distributed in a living cell: the O2 concentration of the hydrophilic cytosol is likely lower than that of hydrophobic organelle membranes. A suitable calibration method is therefore required to convert the phosphorescence lifetime of probe molecules internalized in living cells into the oxygen level inside the cells. Here we used the phosphorescence lifetime of BTPDM1 internalized into cultured SCC-7 cells as a standard to construct the calibration curves.8,42 The cells were cultured in an O2 concentration-changeable multigas incubator equipped with an inverted fluorescent microscope that was connected to the lifetime measurement system. The cell densities seeded in glassbottom dishes were 75−90% confluence, and the temperature was maintained at 37 °C. Under these conditions, the phosphorescence lifetime of BTPDM1 was only slightly affected by the addition of 10 μM antimycin A (an inhibitor of mitochondrial respiration) into the medium, suggesting that diffusional oxygen delivery from the atmosphere to the mitochondria in cells is sufficiently fast. Figure 3A shows the phosphorescence decay curves of BTPDM1 in SCC-7 cells measured under different pO2 in an incubator. The decay curves in Figure 3A could be fitted to biexponential decay functions with the lifetimes τ1 = 760 ns and τ2 = 1.69 μs and the preexponential factors A1 = 0.76 and A2 = 0.24 when the pO2 in the incubator is 160 mmHg. As described above, the hydrophobic BTPDM1 molecules in cells are expected to mainly localize in the lysosome membranes. The observed two-component phosphorescence lifetime suggests that some probe molecules are bound to proteins in the membrane, thereby providing longer lifetimes due to restricted access to oxygen molecules. The amplitudeaveraged lifetimes (⟨τp⟩), calculated using ⟨τp⟩ = A1τ1 + A2τ2 for different pO2, were used to construct the calibration curve based on Stern−Volmer analyses. 1 τp

=

1 τp0

Figure 3. (A) Phosphorescence decay curves of BTPDM1 in SCC-7 cells at 37 °C taken under different pO2 conditions using an O2 concentration-changeable multigas incubator. Lifetimes were measured 90 min after setting the pO2 to allow equilibration with the O2/N2 mixed gas. (B) Stern−Volmer plot of (⟨τp⟩)−1 of BTPDM1 in cells versus the pO2 of the incubator at 37 °C.

The Stern−Volmer plot of (⟨τp⟩)−1 versus pO2 (Figure 3B) could be fitted to a straight line, although the average lifetimes were used for the analysis. From the slope and intercept of the straight line, the apparent quenching rate constant (kq) and the average phosphorescence lifetime at pO2 = 0 (⟨τ0p⟩) were determined to be 5.42 × 103 mmHg−1 s−1 and 4.55 μs, respectively. It can be seen that the kq value in cells (5.42 × 103 mmHg−1 s−1) is much smaller than that in DMPC membrane (9.8 × 103 mmHg−1 s−1), probably due to the interaction of BTPDM1 with membrane proteins. Using these kq and ⟨τ0p⟩ values, the pO2 of cells can be evaluated by measuring the phosphorescence lifetime ⟨τp⟩. We therefore attempted to evaluate the pO2 level in monolayer cultured cells by applying eq 1. An O2 gradient was produced by gently placing a thin round coverslip (15 mm diameter, 0.17 mm thickness) onto a monolayer of SCC-7 cells (Figure 4A), thereby preventing O2 diffusion from the top surface of the cell layer.43 The bright-field image of the peripheral region of the round coverslip (Figure 4B) clearly shows the edge of the glass plate, and the phosphorescence microscopic image clearly shows much brighter emission from under the glass plate, i.e., there is an O2 concentration gradient from the edge of the coverslip toward the inner region due to restricted O2 diffusion from the edge to the inside of the covered area. Figure 4C displays the phosphorescence decay curves of cells located ∼2 mm inside (a) and ∼1 mm outside (b) the edge of the coverslip. These lifetime measurements were made 30 min after placing the coverslip on the cells. Both decays could be fitted to double-exponential decays; the

+ kqpO2 (1) E


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luminescence images were obtained 2 h after injection. The tumor region clearly provided a brighter luminescence image due to BTPDM1 compared with the extratumor regions (Figure 5A). The luminescence spectrum measured from the

Figure 5. (A) In vivo imaging with BTPDM1. A tumor-bearing nude mouse under anesthesia was injected via the tail vein with 25 nmol BTPDM1 in 250 μL 1% DMSO/saline mixed solvent. Phosphorescence was observed at 2 h after BTPDM1 injection using a Maestro 2 in vivo imaging system (excitation/emission at 435−480 nm/560− 750 nm). (B) Phosphorescence decay profiles of BTPDM1 in the center of a tumor, and in normal lower thigh tissue opposite the tumor. The decay curves could be fitted to double-exponential decay functions with lifetimes of 1.27 μs (A1 = 0.59) and 3.14 μs (A2 = 0.41) for normal tissue, and 1.71 μs (A1 = 0.28) and 4.82 μs (A2 = 0.72) for tumor. The average lifetimes (⟨τp⟩) were calculated to be 2.04 μs (normal tissue) and 3.95 μs (tumor) using the equation (⟨τp⟩ = A1τ1 + A2τ2).

Figure 4. (A) Normal and hypoxic monolayer cells in a glassbottomed dish. Hypoxic cells were produced by covering with a coverslip. (B) Left: bright-field image of SCC-7 cells in the vicinity of the coverslip edge. Scale bar, 200 μm. Right: phosphorescence microscopic image of the same field of view. (C) Phosphorescence decay curves of BTPDM1 in SCC-7 cells at 37 °C taken ∼2 mm inside (a) and ∼1 mm outside (b) the edge of the coverslip.

tumors was essentially identical with that of BTPDM1 taken in solution (Figure 1B). The tumor mass could be selectively imaged by administrating 25 nmol BTPDM1, a dosage about 1 order of magnitude lower than that required for BTP imaging.29 It should be noted here that the luminescence of the tumor region does not originate from accumulation of BTPDM1 in the tumor tissues, but rather is due to the hypoxic nature of tumor tissues. The phosphorescence decay profiles of tumor and extratumor regions (Figure 5B) support the low O2 status of tumor tissues: the average decay lifetime (3.95 μs) of a tumor tissue is remarkably increased as compared with that (2.04 μs) of extratumor tissue. These elongated lifetime values allowed verification of the lower O2 levels in the tumor tissues. Since cellular BTPDM1 uptake is extremely high, we believe that the decay rate of BTPDM1 phosphorescence indicates the

average lifetimes obtained were 3.89 μs and 893 ns, respectively. Using the values kq (= 5.42 × 103 mmHg−1 s−1) and ⟨τ0p⟩ (=4.55 μs) and the measured lifetimes, the pO2 of the cells inside and outside the covered area were determined to be 6.9 and 166 mmHg, respectively. The pO2 (6.9 mmHg) of cells inside the covered area clearly shows that the SCC-7 cells become hypoxic (low oxygen) within 30 min of being covered by the coverslip because of respiration in the mitochondria. Tumor Imaging Using BTPDM1. SCC-7 cells were transplanted into the lower thigh of tumor-bearing nude mice. BTPDM1 (25 nmol in 1.0% DMSO/saline/250 μL) was injected into the tail vein under pentobarbital anesthesia. Each mouse was placed in a darkbox (Maestro 2) for in vivo imaging; F



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CONCLUSIONS The aim of the present study was to develop small molecular probes for sensing both intracellular and in vivo oxygen, and to propose an analytical method to quantify the oxygen levels in living cells and tissues based on the lifetime measurements of these probes. We therefore designed and synthesized BTP derivatives that incorporate a cationic amino group (BTPNH2), a cationic dimethyl amino group (BTPDM1), or an anionic carboxyl group (BTPSA) into the acetylacetonato ligand, and a cationic dimethyl amino group into the benzothienylpyridinato ligand through a methylene group (BTPDM2). In this study, we evaluated the optical, cellular, and in vivo properties of these BTP derivatives. BTPDM1 was found to exhibit the best properties as a cellular and in vivo O2 probe. The introduction of the dimethylamino group significantly improved the properties of BTP as a biological O2 probe: cellular uptake efficiency was improved by almost a factor of 20 compared with that of BTP, and showed lysosomal distribution. Hypoxic tumor regions of tumor-bearing mice could be visualized by intravenous administration of 25 nmol BTPDM1. The wholebody distribution of BTP and BTPDM1 determined by ICPMS measurements showed that these complexes migrate from blood to muscles and organs within ∼10 min of injection, and are gradually excreted into the feces. The phosphorescence lifetimes of BTPDM1 internalized in SCC-7 cells were calibrated for the pO2 of an incubator according to Stern− Volmer analyses, providing the oxygen quenching rate constant (kq) and the average lifetime in the absence of oxygen (⟨τ0p⟩). Using these kq and ⟨τ0p⟩ values, the oxygen level of SCC-7 cells and tumor tissues of mice were evaluated by lifetime measurements. The results suggest that Ir complexes have vast potential as small molecular probes for determining intracellular O2 levels in both cultured cells and tissues.

average O2 level of the intracellular milieu in the tumor tissues. This decay measurement allows tumor-specific hypoxic luminescence to be distinguished from normal tissue-dependent background luminescence. Using eq 1 and the kq and ⟨τ0p⟩ values determined for cultured SCC-7 cells provides pO2 levels of 6.1 and 50 mmHg for tumor and extratumor tissues, respectively. The observed O2 level (6.1 mmHg) of tumor tissue is consistent with reported tumor hypoxia.2 Distribution of BTP and BTPDM1 in a Tumor-Bearing Mouse. To assess the in vivo distribution of BTP and BTPDM1, each probe (250 nmol in 10% DMSO/saline/250 μL) was injected into the tail vein of different tumor-bearing mice (Figure S6, Supporting Information). The Ir contents in muscle and blood were quantified at the indicated time after administration of BTP or BTPDM1 using ICP-MS (Figure S7, Supporting Information). In BTPDM1 administered mice, the Ir content in muscle became maximum within 10 min, then decreased gradually over a period of 12 h after probe injection. In BTP administered mice, the Ir content was much lower than that in BTPDM1 administered mice. The Ir content in blood after injection of BTPDM1 rapidly decreased, whereas that of BTP exhibited a maximum at 10 min after injection and then fell rapidly. These results suggest that the localization of BTP and BTPDM1 migrates from blood to tissues within a short period. The distribution of BTP and BTPDM1 in organs is illustrated in Figure 6. At 2 h after injection of the probes, BTP

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 6. Ir content of organs. The Ir content of organs from tumor− bearing mice was measured by ICP-MS. Each tumor-bearing mouse was injected with either 250 nmol BTPDM1 or BTP, and then the organs were resected at 2 h. After extracting Ir from an organ, the Ir content of that organ was measured by ICP-MS. Values are the mean ± SEM of at least five independent experiments.

AUTHOR INFORMATION

Corresponding Author

*S. Tobita. Tel.: +81 277 30 1210. Fax: +81 277 30 1213. Email: [email protected]. Notes

The authors declare no competing financial interest.

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and BTPDM1 give very different organ distributions: BTP shows the highest distribution in spleen and lung, whereas BTPDM1 shows the highest in liver and kidney. At 24 h after probe injection, the Ir content decreased remarkably and showed somewhat different distributions (Figure S8A, Supporting Information). The distribution of BTP and BTPDM1 at 2 h after injection (Figure 6) shows that muscle (normal tissue) and tumor have similar probe molecule distributions, although slightly higher accumulation is observed in tumors with BTPDM1 at 24 h after administration. Both Ir complexes were excreted into the feces (Figure S8B, Supporting Information). The specific organ accumulation of the probe soon after its administration suggests that these probes can be utilized to examine O2 levels of a specific organ such as liver and kidney. As far as we are aware, there are few oxygen probes that can directly sense intracellular oxygen levels of animal tissues.

ACKNOWLEDGMENTS The authors thank Kiryu Bureau of Waterworks for help with ICP-MS measurements. S.T. acknowledges support from a Grant-in-Aid for Industry-Academia Collaborative R&D Programs (In vivo Molecular Imaging: Towards Biophotonics Innovations in Medicine) from the Japan Science and Technology Agency, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 23310157). T.Y. was financially supported by a Grant-in-Aid from The Canon Foundation. M.H. thanks support from Akita Prefectural University President’s Research Project Fund.

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