Oxygen imaging of living cells and tissues using luminescent molecular probes

Accepted Manuscript Title: Oxygen imaging of living cells and tissues using luminescent molecular probes Authors: Toshitada Yoshihara, Yosuke Hirakawa, Masahiro Hosaka, Masaomi Nangaku, Seiji Tobita PII: DOI: Reference:

S1389-5567(16)30083-1 http://dx.doi.org/doi:10.1016/j.jphotochemrev.2017.01.001 JPR 255

To appear in: Reviews

Journal of Photochemistry and Photobiology C: Photochemistry

Received date: Accepted date:

14-11-2016 16-1-2017

Please cite this article as: Toshitada Yoshihara, Yosuke Hirakawa, Masahiro Hosaka, Masaomi Nangaku, Seiji Tobita, Oxygen imaging of living cells and tissues using luminescent molecular probes, Journal of Photochemistry and Photobiology C:Photochemistry Reviews http://dx.doi.org/10.1016/j.jphotochemrev.2017.01.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Oxygen imaging of living cells and tissues using luminescent molecular probes

Toshitada Yoshihara, a,* Yosuke Hirakawa,b Masahiro Hosaka,c Masaomi Nangaku,b Seiji Tobitaa,* a

Department of Chemistry and Chemical Biology, Gunma University, Kiryu, Gunma 3768515, Japan b Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan c Department of Biotechnology, Akita Prefectural University, Shimoshinjo, Akita 0100195, Japan

Highlights

Biological oxygen imaging using luminescent molecular probes is reviewed. Metal complexes serve as an oxygen sensing probe for living cells and tissues. Phosphorescence quenching mechanism of metal complexes by oxygen is discussed. Lifetime-based measurements allow tissue oxygen imaging and hypoxic tumor imaging.

Abstract Oxygen imaging of biological cells and tissues is becoming increasingly important in cell biology and in the pathophysiology of various hypoxia-related diseases. The optical oxygen-sensing method using luminescent probes provides very useful, high spatial resolution information regarding oxygen distribution in living cells and tissues. This 1

review focuses on recent advances in biological oxygen measurements based on the phosphorescence quenching of probe molecules by oxygen, and on hypoxia-sensitive fluorescent probes. Special attention is devoted to metal complex probes, Pt(II)- and Pd(II)-porphyrins, Ru(II) complexes, and Ir(III) complexes. Current knowledge regarding the mechanism of phosphorescence quenching of metal complexes by oxygen is described in relation to the oxygen sensitivity of the probes, and recent advances in optical oxygen probes and detection techniques for intracellular and tissue oxygen measurements are reviewed, emphasizing the usefulness of chemical modifications for improving probe properties. Tissue oxygen imaging and hypoxic tumor imaging using these metal complex probes demonstrate the vast potential of optical oxygen-sensing methods using luminescent probes.

Contents 1. Introduction 2. Importance of oxygen for living organisms 3. Principle of oxygen sensing using luminescent molecules 3.1. Oxygen sensing based on phosphorescence quenching 3.1.1. Quenching of excited metal complexes by oxygen 3.1.2. Intensity- and lifetime-based methods 3.1.3. Ratiometric methods 3.2. Hypoxia sensitive fluorescent probes 4. Basic metal complexes for biological oxygen sensing 5. Chemical modification of metal complexes 5.1. Small molecule probes 5.1.1. Metalloporphyrins 5.1.2. Ru(II) complexes 5.1.3. Ir(III) complexes 5.1.4. Protoporphyrin IX 5.2. Dendron-coated porphyrins 2

6. Intracellular oxygen sensing 6.1 Monolayer cultured cells 6.2 Multicellular spheroids 7. Tissue oxygen imaging 7.1. General importance and difficulty in tissue oxygen imaging 7.2. Kidney and liver 7.3. Brain 7.4. Bone marrow 7.5. Retina 8. Hypoxic tumor imaging 8.1. Oxygen needle electrodes 8.2. Immunohistochemical staining 8.3. Positron emission tomography (PET) 8.4. Optical imaging 9. Summary and future outlook Acknowledgements References

1. Introduction Oxygen plays essential roles in aerobic organisms, especially as a terminal electron acceptor in the electron transport chain of mitochondria in cells [1,2]. The intracellular oxygen concentration of the entire body is strictly regulated by the microcirculation system. The disruption of oxygen homeostasis in the body often results in serious diseases such as cardiovascular diseases and stroke, and thus considerable effort has been devoted in recent years to the development of suitable techniques allowing the detection of intracellular and tissue oxygen levels. A variety of modalities have been developed to date for this purpose, and include positron emission tomography (PET) imaging [3,4], magnetic resonance imaging (MRI) [3,5], electron paramagnetic resonance (EPR) oximetry [6,7], optical methods [8,9], and conventional electrochemical methods using 3

various electrodes [10,11]. Over the last few years the optical imaging method has become an integral part of intracellular and in vivo oxygen measurements [12-18] because optical oxygen-sensing using luminescent probes has various advantages, including (1) high O2 sensitivity, (2) the feasibility of quantitative oxygen measurements in real time based on emission lifetime or by using ratiometric probes, (3) high spatial resolution at the single-cell level, and (4) the possibility of multicolor imaging which enables, for example, the simultaneous detection of more than one species. Optical probes and detection techniques constitute two pillars of optical oxygen measurement techniques. Recent progress in fluorescence microscopy involving confocal microscopy, two-photon laser microscopy, and fluorescence lifetime imaging microscopy, as well as in vivo imaging techniques, has accelerated the development of biological oxygen measurement techniques based on the optical method [19,20]. It has, therefore, become increasingly important to develop excellent probes for use with these sophisticated detection techniques. Optical oxygen probes can be classified as exogenous and endogenous probes. Exogenous probes include oxygen-sensitive molecular probes and nanoparticle-type probes containing an oxygen-sensitive unit, and endogenous oxygen probes (or indicators) include biomolecules such as hemoglobin, nicotinamide adenine dinucleotide (NADH), and protoporphyrin IX. Exogenous probes have the significant advantage of being amenable to chemical modification based on rational design to provide probes suited for specific purposes. This review focusses on exogenous molecular oxygen probes that can be directly delivered into cells or tissues. In the next section, the importance of oxygen for living organisms is briefly described, together with the cellular response to oxygen deprivation (hypoxia). Section 3 deals with the fundamental principles of optical oxygen sensing, 4

with particular attention paid to the phosphorescence quenching of metal complexes by molecular oxygen. Sections 4 and 5 highlight representative oxygen probe molecules for biological use and their chemical modification for various purposes. Section 6 summarizes recent studies on the intracellular oxygen sensing of cultured cells, spheroids, and tissue models. Sections 7 and 8 provide overviews of recent applications of optical oxygen probes in tissue oxygen measurements and for hypoxic tumor. Finally, a summary and future outlooks is provided in Section 9.

2. Importance of oxygen for living organisms Oxygen is vital for aerobic respiration, and thus maintaining oxygen homeostasis is a critical issue for organisms. Mammals have a complex system for oxygen delivery, and once a hypoxic condition (insufficient oxygenation of arterial blood) occurs, cellular responses to address this decrease in oxygen are activated. Such cellular responses include, for example, angiogenesis, increasing the hemoglobin level in blood, promoting glycolysis, and producing anti-oxidant factors [21]. These cellular reactions are regulated by hypoxia inducible factors (HIFs) (Fig. 1). HIFs are considered the “master regulator” of cellular and tissue reactions against hypoxia [21]. HIFs were originally discovered as enhancers of the erythropoietin gene which promotes erythropoiesis [22], and thereafter came to be known as transcription factors binding to the promoter regions of many genes that are upregulated in hypoxia [23,24]. HIFs consist of two subunits, HIF-α and HIF-β. There are three subtypes of HIF-α (HIF1α, HIF-2α, HIF-3α) while there is only one subtype of HIF-β. The concentration of HIFα is regulated by oxygen-dependent hydroxylation followed by ubiquitination at the 5

hydroxylated site. In the hypoxic state, HIF-α is not hydroxylated and HIF-α accumulates in the cytosol. Then, the increased levels of HIF-α and HIF-β form a dimer and move into the nucleus and function as a transcription factor. This is why HIFs are called the “master regulator” against hypoxia. The rate limiting reaction of this HIF degradation process is hydroxylation by proline hydroxylase domains (PHDs). Since PHDs require molecular oxygen for their enzymatic activity, they work as an oxygen sensor in metazoans. HIF-α is hydroxylated in the cytosol and thus intracellular oxygen tension is related to activation of the HIF pathway. Therapeutic approaches focusing on these enzymes are being developed, and PHD inhibitors with the potential to activate HIF are currently in clinical trial against renal anemia [25]. There is also research indicating that PHD inhibitors ameliorate pathologic processes [26-28]. Progress on these fronts has drawn considerable attention to the detection of hypoxia and the targeting of hypoxia to provide clues regarding effective medical treatments.

3. Principles of oxygen sensing using luminescent molecules As described above, intracellular and tissue oxygen status are crucial for maintaining normal cellular functions and life. In this section, the theoretical basis of the oxygensensing method based on optical probes is reviewed.

3.1. Oxygen sensing based on phosphorescence quenching 3.1.1. Quenching of excited metal complexes by oxygen Oxygen sensing using luminescent molecules (usually phosphorescent molecules) is generally based on the bimolecular quenching of excited molecules by molecular oxygen

6

[29]. An energy state diagram that includes oxygen quenching of the excited triplet state for a molecule M is shown in Fig. 2, together with the rate constants of the dominant relaxation processes. As described below, oxygen quenching is related to two competing pathways from the lowest excited triplet state: non-charge transfer (n-CT) channels, and CT channels. The kinetics of oxygen-induced quenching in solution follows the SternVolmer equation (Eq. (1)) that gives the relation between lifetime (or quantum yield) and oxygen partial pressure (or concentration):

 0p p



τ p0 τp

 1  k q τ p0 p O 2  1  K SV p O 2

(1)

 1  k q ' τ p0 [O 2 ]  1  K SV '[O 2 ]

0 0 where p and τ p , respectively, are the phosphorescence quantum yield and lifetime in

the absence of oxygen, p and τ p are these variables at an oxygen partial pressure of pO2 or at an oxygen concentration [O2], and kq (or kq’) and Ksv (or Ksv’) are the bimolecular quenching rate constant and Stern-Volmer constant, respectively. If phosphorescence quenching due to oxygen dissolved in water proceeds with the diffusion-controlled rate constant kd, the kq’ value is estimated to be 7.4 × 109 M-1s-1 at 298 K from the viscosity of the solvent [30]. Since the dissolved oxygen concentration in aerated water under 1 atm at 298 K is estimated to be ~0.27 mM [30], it can be expected that phosphorescence with a lifetime longer than approximately 1 s is significantly quenched by the dissolved oxygen. In usual organic solvents, or in lipid bilayer membranes such as those comprising phosphatidylcholine, oxygen quenching can occur more efficiently than in water because of the higher solubility of oxygen in these media 7

and/or a larger diffusion rate. A small number of metal complexes are known to exhibit distinct phosphorescence in solution at room temperature [31-34] due to the heavy atom effect of the central metal ion. These include Ru(II), Ir(III), Pt(II), Re(II), and Os(II) complexes, and metalloporphyrins with Pt(II) or Pd(II) as the central metal ion. Of these complexes, Pt(II) and Pd(II) porphyrins and Ru(II) and Ir(III) complexes are most typically used as biological oxygen probes (see Sec. 4). The rational design and development of useful biological oxygen probes using phosphorescent molecules would benefit from understanding the phosphorescence quenching mechanism of O2, as this would reveal the physical factors governing quenching efficiency. Numerous studies have been performed over the past several decades to clarify the mechanism by which oxygen quenches the excited triplet state of metal complexes [35-40] and aromatic organic molecules [41-46]. Bimolecular collisions between excited triplet molecules (M(T1)) and ground-state oxygen (O2(3g-)) competitively produce singlet, triplet and quintet encounter pairs according to the spin-conservation rule (Fig. 3). These pairs can be denoted by 1(T1 3), 3

(T1 3), and 5(T1 3), and their formation rate constants are given by (1/9)kd, (3/9)kd, and

(5/9)kd, respectively. Here, the diffusion-controlled quenching rate constant (kd) of aromatic molecules or metal complexes by O2 in acetonitrile is estimated to be (3.0-5.0) × 1010 M-1s-1 [30] according to the Smoluchowski theory [47] of diffusion-controlled reactions expressed by

kd = 4NA (DM + DO2)(rM+ rO2)

(2)

where DM and rM are the diffusion coefficient and molecular radius of molecule M, 8

respectively, DO2 and rO2 are these variables for molecular oxygen, and NA is Avogadro’s constant. Figure 3 schematically shows the quenching processes of M(T1) by O2(3g-) for systems in which the triplet energy is 1.78 eV and CT quenching is not involved, i.e., for systems in which the contribution of the CT channel in Fig. 2 can be neglected. Since the quintet encounter pair 5(T1 3) cannot lead to the ground-state molecule (M(S0)), and the internal conversion rate from the triplet encounter pair 3(T1 3) to 3(S0 3) is relatively slow owing to the large energy gap, the observed oxygen quenching rate constant is generally less than (1/9)kd. In the non-CT mechanism, phosphorescence quenching is caused by energy transfer from M(T1) to O2(3g-) to produce singlet oxygen O2(1g). The decay rate of the resultant O2(1g) is usually dominated by nonradiative transitions (rate 1

 constant knr ) because of the strictly forbidden nature of the electric dipole (a1g → X3g- )

transition [45,48-50]. In solvents with C–H bonds, singlet oxygen (a1g) deactivates rapidly owing to electronic-to-vibrational energy transfer [45], thereby regenerating ground-state oxygen (X3g-). In living cells, reactions of singlet oxygen with biological substances cannot be neglected; moreover, intense and continuous light irradiation to probes may have toxic effects on them. However, under low or moderate irradiation, almost complete recovery of ground-state oxygen enables reversible and real-time oxygen measurements with minimum perturbation to cellular functions. This is in contrast with conventional oxygen measurements based on a capillary electrode, in which oxygen is consumed by electrochemical reactions [11]. In compounds with a relatively low oxidation potential and high triplet energy (ET), CT (or electron transfer) from M(T1) to O2 becomes possible [35,51]. The contribution of the CT channel can be evaluated from the Gibbs energy change (Gel) for the electrontransfer reaction that is usually estimated from the standard Rehm–Weller treatment [52] 9

as

Gel = E ox (M) − E red (O2) − ET + C

(3)

where Eox (M), Ered (O2), and C are the oxidation potential of donor molecule M, the reduction potential of O2, and the electrostatic interaction energy (−0.06 and −0.12 eV for neutral and monocationic complexes, respectively, in CH3CN). Oxygen quenching rate constants reported for several Ir(III) complexes [35], Ru(II) complexes [53-57] and metalloporphyrins [58] are plotted as a function of Gel in Fig. 4: Ir(III) complexes give a much wider range of Gel values compared with Ru(II) complexes and metalloporphyrins because the triplet energy and oxidation potential of Ir(III) complexes can be tuned over a wide range by controlling the ligand type and substituents. For complexes with Gel ≲ -0.2 eV, the kq value increases with the exothermicity of the electron transfer, e.g., (ppy)2Ir(acac) (ppy = phenyl pyridine, acac = acetylacetone) with Gel = -0.81 eV gives a very large kq value (2.0 × 1010 M-1s-1[35]). The Gel dependence of the logkq values indicates that the CT channel is involved in the quenching mechanism of Ir(III) complexes as shown in Fig. 2, in addition to the energy transfer to O2(3g-) to produce singlet oxygen [35]. In contrast, the phosphorescence quenching of Ru(II) complexes and metalloporphyrins shown in Fig. 4 proceeds primarily through a non-CT channel, which results in smaller kq values as compared with Ir(III) complexes. The oxygen sensitivity of these complexes can be evaluated by the Stern-Volmer 0 0 constant Ksv, i.e., the product of kq and τ p . Typical lifetimes ( τ p ) of Pd(II) and Pt(II)

porphyrins range from 10-1000 s, which are much longer than those of Ru(II) complexes 10

(0.5-5 s) and Ir (III) complexes (1-20 s). Metalloporphyrins are, therefore, capable of measuring very low O2 concentrations, although their extremely long lifetimes are not favorable for emission decay measurements with a high repetition rate. The large oxygen quenching rate constants and moderately long phosphorescence lifetimes of Ir(III) complexes are suitable for oxygen measurements using a high-repetition-rate laser. It has also been reported that the quenching efficiency is affected by ligand structure [59], as shielding of the lowest unoccupied molecular orbital of the ligand reduces the kq value. Dendritic protection of metalloporphyrins has also been shown to control quenching efficiency [60].

3.1.2 Intensity- and lifetime-based methods Equation (1) predicts that compounds with a relatively large Ksv will exhibit a decreased phosphorescence quantum yield (or phosphorescence intensity) under constant excitation light intensity as the oxygen partial pressure (pO2) or concentration ([O2]) in 0 solution increases. The phosphorescence quantum yield is reduced to half the p value

when the pO2 is equal to 1/Ksv, for instance, for compounds with a Ksv value of 0.025 0 mmHg-1, p is reduced to half the p value at pO2 = 40 mmHg. In a similar manner,

the increase in the phosphorescence intensity of probes introduced into cells or tissues demonstrates the reduction in the oxygen concentration, as long as the excitation light intensity and probe distribution are kept constant. Observation of the emission intensity change enables us to examine variation in the oxygen concentration in real-time both in vitro and in vivo, although the absolute oxygen concentration cannot be evaluated from the intensity-based measurements. If the probe molecules distribute uniformly in the

11

target tissues, high resolution oxygen measurements become possible by combining a suitable oxygen probe and a luminescence microscope. For example, Paxian et al. [61] have reported the high-resolution visualization of oxygen distribution in rat liver using a Ru(II) complex as a luminescent oxygen probe. As described above, phosphorescence quenching of metal complexes by O2 in water or organic solvents follows the Stern-Volmer equation (1), and thus the oxygen level expressed by the pO2 or [O2] of the solution can be accurately determined by 0 phosphorescence lifetime measurements using the kq (kq’) and τ p values measured in

advance in the same solvent and at the same temperature. In contrast, in cells or tissues, oxygen quenching is not necessarily in accordance with Eq. (1) and the apparent kq value is often lower as compared with that in solution because the probe molecules exist in complex heterogeneous environments and can interact with endogenous biological components such as proteins. The interaction of oxygen probes with biological substances is particularly important in the case of small molecule probes that are not protected from the external environment. In biological environments, it is often observed that the emission decays of small molecule probes do not follow a single exponential decay, suggesting that some of the probe molecules interact with proteins and/or that the probes are distributed non-homogeneously in cells or tissues. To resolve this complexity, the emission decay is often approximately fitted to a bi-exponential decay function with lifetimes of 1 and 2 and relative amplitudes A1 and A2. The intensity-averaged lifetime ( τpint ) or the amplitude-averaged lifetime ( τp amp ), derived as τpint = (A112 + A222)/( A11 + A22) and τp amp = (A11 + A22)/( A1 + A2) [62], can be used to quantify the biological oxygen level. In cellular oxygen measurements, the phosphorescence 12

intensity at two delay times after pulsed excitation is often used to evaluate the lifetime as= (t2-t1)/(ln(F1/F2)), where F1 and F2 are the phosphorescence intensities at delay times t1 and t2 [63,64]. This method is particularly useful for real-time oxygen measurements using a microplate reader, although the accuracy of the lifetime measurements is inferior to the time-correlated single photon counting (TCSPC) method, which uses hundreds of channels. The phosphorescence lifetimes of probe molecules taken up into cells can be calibrated against the oxygen partial pressure pO2 by measuring the lifetime of probes loaded into cultured cells under different pO2 conditions in an incubator [65,66]. In this case, it is preferable to add an inhibitor of respiration, such as Antimycin A, into the medium to block oxygen consumption by cellular respiration. On the other hand, in the case of nanoparticle oxygen probes which contain phosphorescent molecules, the phosphorescent molecules are prevented from interacting with biological substances by the host particle, allowing the construction of more reliable calibration curves for the lifetime. In the same way, in dendron-coated porphyrins, the oxygen-sensitive metalloporphyrin is protected from proteins, and these probes are very useful for accurate measurements of the intravascular oxygen levels of small animals (see Sec. 7). Recent advances in phosphorescence lifetime imaging microscopy (PLIM) using a confocal laser scanning microscope enabled intracellular and tissue oxygen imaging with high spatial resolution [67-69].

3.1.3 Ratiometric methods Lifetime-based measurements provide quantitative information about the oxygen concentration or partial pressure of cells and tissues but require a special instrument, 13

generally constructed from a pulsed excitation source and a lifetime measurement system. The need for complex instrumentation can be circumvented by using ratiometric oxygen probes [70,71] that do not require lifetime measurements. There are two types of ratiometric oxygen probes: small molecule-based ratiometric probes, and nanoparticlebased ratiometric probes. Small molecule-based ratiometric probes consist of an oxygeninsensitive fluorophore and an oxygen-sensitive phosphor. These luminophors are connected by a rigid linker, such as a peptide with an optimum length, because part of the excitation energy in the fluorophore is transferred to the phosphor by singlet-singlet energy transfer. The ratio between the fluorescence and phosphorescence intensities correlates with the oxygen partial pressure as

RI0  1  K SV p O 2 RI

(4)

0

0

where RI0 is the intensity ratio between the fluorescence and phosphorescence ( IP /If ) under deaerated conditions and RI is the ratio ( Ip / If ) under the oxygen partial pressure pO2. It is assumed here that fluorescence quenching due to oxygen is negligibly small. If the plot of RI0 / RI vs. pO2 follows the linear relation in Eq. (4), then Ksv is determined from the slope of the straight line and the oxygen partial pressure in solution can be estimated from the Ksv value.

3.2. Hypoxia sensitive fluorescent probes Bioreductive hypoxia markers comprising nitroimidazole derivatives have been widely utilized as a conventional method to detect hypoxic tissues [72-75]. Nitroimidazole 14

derivatives, as exemplified by pimonidazole (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2propanolol), form covalent bonds with cellular macromolecules at oxygen levels below 10 mmHg. The probe molecules fixed in poorly oxygenated regions in histological sections can be visualized by immunohistochemical staining. Immunostaining of the reduction product enables visualization of hypoxic areas at a specific time, but this approach is not applicable to real-time oxygen measurements of living cells or tissues. This drawback has been addressed by developing a similar type of hypoxia marker that does not require immunostaining: bioreductive nitroimidazoles conjugated with a fluorophore, i.e., hypoxia-sensitive fluorescent probes (Fig. 5A). Nagasawa and coworkers [76] have designed and synthesized a near-infrared (NIR) fluorescent probe, GPU-167, comprising 2-nitroimidazole and tricarbocyanine. They demonstrated that GPU-167 administered into a tumor-bearing mouse can specifically visualize tumor hypoxia in vivo. Smith, Zhu and coworkers [77] reported that a similar NIR fluorescent probe consisting of 4-nitroimidazole and indocyanine is retained in hypoxic tumors, allowing imaging of tumor hypoxia. Jayagopal and coworkers [78] have synthesized fluorescein-based conjugates of 2-nitroimidazole (HYPOX-1) and pimonidazole (HYPOX-2). These imaging probes are capable of detecting hypoxic retinal cells and tissues with high specificity and sensitivity. Recently, they developed a new nitroimidazole-based fluorescent probe, HYPOX-4, which allows the detection of retinalhypoxia in living animals [79]. The bioreductive properties of hypoxic cells and tissues have also been exploited to develop activatable hypoxia probes in which a fluorophore and a quenching unit are connected with a bond that undergoes reductive cleavage under hypoxia conditions (Fig. 5B). Under normoxia conditions, the emission from the fluorescing moiety is quenched 15

by the quenching unit by different mechanisms, including intramolecular electron transfer [80, 81] (or dark-state quenching [82]), Förster resonance energy transfer (FRET) [83, 84], and structural change around an azo bond [85]. Reduction of the quenching unit in hypoxic environments results in isolation of the fluorophore and thus remarkable enhancement of fluorescence intensity. One drawback of the above-mentioned hypoxia-sensing fluorescent probes is the lack of reversibility in oxygen-level measurements. To address this, Hanaoka and coworkers [86] have developed a reversible hypoxia-sensing fluorescent probe, RHyCy5, whose fluorescence is turned on and off depending on redox reactions under hypoxia and normoxia (Fig. 5C). RHyCy5 consists of a cyanine dye, Cy5, as a fluorophore and a quenching unit, QSY-21, which acts as an energy acceptor in FRET. Weakly-fluorescent QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, thus becoming strongly fluorescent, and the radical is oxidized to QSY-21 upon recovery of normoxia. This probe can be used to detect repeated cycles of hypoxia-normoxia in live cells.

4. Basic metal complexes for biological oxygen sensing The phosphorescence quenching method is extremely useful for quantitative oxygen measurements, as described in Section 3.1. Strongly phosphorescent compounds, including Pt(II)- and Pd(II)-porphyrins, and several d6 transition metal complexes as exemplified by Ru(II)- and Ir(III) complexes, exhibit favorable characteristics for intracellular and in vivo oxygen measurements based on phosphorescence quenching. Depending on the target (for example, intracellular oxygen of cultured cells, or intracellular or intravascular oxygen in vivo), probe molecules should be optimized for various properties, including (1) photophysical properties such as absorption and 16

emission wavelengths, absorption efficiency, emission quantum yield, and lifetime, (2) intracellular properties such as cellular uptake efficiency, subcellular localization, and cytotoxicity, and (3) physicochemical properties such as hydrophilicity, chemical stability and interactions with endogenous species such as proteins. Figure 6 shows the basic structures of representative metal complexes previously used as phosphorescent probes for intracellular and in vivo oxygen measurements. The photophysical characteristics of these complexes are summarized in Table 1, along with relevant references [87-96]. Pt(II)- and Pd(II)-porphyrins have been widely used as biological oxygen probes since the pioneering research of Wilson and coworkers [87,97,98] on tissue oxygen measurements using palladium(II) coproporphyrin (PdCP) and palladium meso-tetra-(4-carboxyphenyl)porphine (PdTCPP). Papkovsky and coworkers [99] used a PtCP-bovine serum albumin (BSA) conjugate for cell viability screening. Pt(II)- and Pd(II)-porphyrins exhibit significantly longer phosphorescence lifetimes compared with Ru(II) and Ir(III) complexes: 30-100 s for Pt(II)-porphyrins and 100-1000 s for Pd(II)-porphyrins. These long lifetimes are advantageous for measuring very low oxygen concentrations. As seen from Table 1, metalloporphyrins have two characteristic absorption bands: one in the visible region (500-550 nm), called the Q band, and the other in the near ultraviolet region (380-420 nm), called the B or Soret band. The molar absorption coefficient () of the B band is extremely large and its absorption peak is sharp and narrow. Because of their large  and moderately large p, metalloporphyrins give strong phosphorescence in the red to near infrared regions (630750 nm) under deoxygenated conditions. Perfluorophenyl-substituted porphyrin (PtTFPP) has been used as a highly photostable oxygen-sensing dye in different types of matrices [100,101]. This dye exhibits a moderately long lifetime (60 s), strong 17

absorption bands in the visible to near ultraviolet regions, and intense phosphoresce in deaerated organic solvents. In addition to Pt(II)- and Pd(II)-porphyrins, luminescent Ru(II) polypyridyl complexes have also been used for intracellular oxygen measurements. Ruthenium tris(2,2’-dipyridyl) ion [Ru(bpy)3]2+ produced from ruthenium tris(2,2’-dipyridyl) dichloride hydrate (RTDP) is the prototype polypyridine metal complex [102]. [Ru(bpy)3]2+ gives a broad absorption band with the maximum around 460 nm. This band has a relatively large absorption coefficient, and is assigned to the singlet metal-to-ligand charge transfer (1MLCT) transition which involves the promotion of an electron from the 5d orbital of the Ru2+ ion to the * orbital of the dipyridyl ligand. [Ru(bpy)3]2+ exhibits a broad phosphorescence band with the maximum at 630 nm which originates from the triplet MLCT state (3MLCT). Gerritsen et al. [103] imaged the oxygen distribution in J774 macrophages from the phosphorescence lifetime distribution of RTDP delivered into the cells. Mycek and coworkers [104-106] have performed fluorescence lifetime imaging microscopy (FLIM) studies of live cells by using RTDP as an oxygen probe and a gated CCD camera to record lifetime images. As seen from Table 1, the phosphorescence lifetime (ca. 1.0 s) of [Ru(bpy)3]2+ is much shorter than that of Pt(II)- or Pd(II)-porphyrins, indicating that [Ru(bpy)3]2+ has a much lower oxygen sensitivity. The oxygen sensitivity and phosphorescence intensity are improved in [Ru(dpp)3]2+, which has a relatively long phosphorescence lifetime (6.4 s) and high quantum yield (0.37) under deoxygenated conditions. Because of its high lipophilicity, [Ru(dpp)3]2+ has mostly been used as an oxygen-sensing unit embedded into a nanoparticle probe [70]. A less lipophilic [Ru(dpp)3]2+ analogue, [Ru(phen)3]2+, has been used as a luminescent probe for high-resolution visualization of oxygen distribution 18

in the liver [61]. It was recently reported that a highly phosphorescent Ir(III) complex, BTP, [(btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone)] serves as an oxygensensing molecular probe for living cells and tissues [107]. BTP has a relatively long lifetime (5.7 s) and high quantum yield (p = 0.30) in degassed THF, and a large oxygenquenching rate constant (kq = 6.2×109 M-1s-1 in acetonitrile). BTP emits red phosphorescence with the maximum at 615 nm. Since the original work on biological oxygen sensing using BTP [107], various chemical modifications have been made to BTP and other cyclometalated Ir(III) complexes, as described in Section 5.1 [108]. The chemically stable and highly phosphorescent natures of Ir(III) complexes originally received extensive attention in the field of organic light emitting diodes [109-111]. These properties, together with the moderately long phosphorescence lifetimes (1-20 s) and tunabilities of the emission wavelengths of these compounds are very useful for their use as biological oxygen probes. The moderately long lifetimes of Ir(III) complexes afford sufficiently high oxygen sensitivity, and are advantageous for phosphorescence decay measurements with a high repetition rate such as the TCSPC method because high signal acquisition rates can be attained. The first absorption band of most Ir(III) complexes is in the visible region. The absorption maximum and molar absorption coefficient () depend significantly on the structures of the aromatic ligands [112]. The absorption coefficient (,700 dm3 mol-1cm-1 at 486 nm) of BTP is somewhat smaller than that of Ru(II) complexes. The introduction of ligands with high absorptivity can improve the absorption efficiency of complexes; for example, an iridium complex with coumarin ligands, Ir(Cs)2(acac) (Fig. 6), has a large  value (92,800 dm3 mol-1 cm-1 at 472 nm) although its absorption and emission wavelengths 19

are rather short. The phosphorescence wavelengths of Ir(III) complexes also depend remarkably on the structures of the ligands, indicating that emission color can be tuned by designing the ligand structure [113]. This property is useful for multicolor cellular imaging using a color CCD camera. Moreover, the high phosphorescence quantum yields and large kq values of Ir(III) complexes can compensate for their relatively small absorption efficiencies.

5. Chemical modification of metal complexes An important advantage of metal complexes is the ease of chemical modification to optimize the probe for various oxygen measurements. In the following subsections, we divide metal complex probes into small molecule probes and dendritic probes, depending on the size of the probe. Recent advances in probe development and the applications of probes to biological oxygen measurements are briefly described in the following sections.

5.1 Small molecule probes Small molecule probes have several attributes, including synthetic ease, high cellular uptake efficiency, and clear subcellular localization. They are, therefore, particularly suitable for intracellular oxygen measurements both in cultured cells and in tissues. To date, a variety of small molecule oxygen probes based on phosphorescent metal complexes have been designed and synthesized that exhibit improved photophysical, intracellular, and physicochemical properties. Here we review the oxygen probes utilizing the most common metal complexes, metalloporphyrins, ruthenium(II) complexes, iridium(III) complexes, and protoporphyrin IX.

20

5.1.1 Metalloporphyrins Papkovsky and coworkers have developed diverse metalloporphyrin derivatives that are especially useful for intracellular oxygen measurements. In their early studies on intracellular oxygen sensing using PtCP and Pt(II)- and Pd(II)-coproporphyrin ketones [99,114,115], probe molecules were conjugated with BSA to increase their solubility in aqueous medium. These probes required transfection reagents such as Endo-porter and Escort III for loading into mammalian cells [65]. To improve the cellular uptake efficiency of PtCP, the peripheral carboxy groups were modified with small substituents containing primary amino, aliphatic hydroxy, and hydrophobic ethyl ester group(s) [116]. Although the phosphorescence intensity signal of PtCP-NH2, substituted with an amino group (NHCH2CH2NH2), was higher than that of PtCP conjugated with BSA, a transfection reagent was still needed to evaluate changes in the intracellular oxygen level. The cell-penetration ability of PtCP has been further improved by linking cellpenetrating peptides, such as the trans-activator of transcription (TAT) sequence or oligoarginine derivatives known to facilitate cellular uptake [117,118]. Papkovsky and coworkers [119] have synthesized various intracellular oxygen sensing probes based on mono- or tetrasubstituted PtCP derivatives conjugated with a TAT peptide, a bactenecin 7 peptide fragment [120], or arginine-rich peptides [121] (Fig. 7A). Recently, several new cell-penetrating phosphorescent probes consisting of PtTFPP dye and monosaccharide substituents such as glucose and galactose, or short arginine peptides, were developed by the same group. Glucose and galactose conjugates of PtPFPP demonstrated good potential for high-resolution oxygen imaging of 3D tissue models and related biomaterials [122] (Fig. 7A). These probes were synthesized by click-modification with thiols. Koren et al. [123,124] synthesized a new group of metalloporphyrins, Ir(III) 21

octaethylporphyrin (Ir-OEP), which can bear two axial ligands such as histidinetetraarginine peptide (Ir1), a truncated fragment of bactenecin 7 peptide (Ir2), or a peptide with the RGD sequence (Ir3) [124]. The Ir1 and Ir2 ligands were advantageous for detecting change in oxygen levels in living cells. Metal chelate coupling of Pt(II) porphyrins to peptides, proteins, and protein nanoparticles was investigated by using PtCP and PtTFPP derivatives bearing the nitrilotriacetate (NTA) moiety [125]. It was shown that a PtTFPP-NTA conjugate with a histidine-containing peptide can facilitate intracellular loading. PdCP and PdTCPP were also utilized in early studies on tissue oxygen sensing [97,98,126-128]. However, oxygen measurements using PdTCPP phosphorescence were limited to the surface regions of animal tissues because the main absorption band of PdTCPP is at relatively short wavelengths in the visible region (see Table 1). To overcome this limitation, Vinogrdov et al. [129,130] focused on the Pd(II)-meso-tetraphenyltetrabenzoporphyrin structure (Fig. 7B), which has an extended -electronic structure compared with PdTCPP. A water soluble derivative of Pd(II)-tetrabenzoporphyrin, Pd(II) meso-tetra(sulphophenyl)tetrabenzoporphyrin (Green 2W), exhibits the Q band at 636 nm ( = 5.1×104 dm3 mol-1cm-1) and its phosphorescence maximum at 790 nm. The use of Green 2w allowed minimally invasive in vivo imaging of oxygen distribution in the vascular systems of mice. Vinogradov and coworkers have further improved the probe and succeeded in developing a dendron-coated Pd(II)-tetrabenzoporphyrin (see Sec. 5.2).

5.1.2 Ru(II) complexes Two typical Ru(II) polypyridyl complexes, [Ru(bpy)3]2+ and [Ru(phen)3]2+, have been used for the oxygen sensing of living cells [103,131] and tissues [61,132]. However, these 22

probes are inferior to metalloporphyrins in cellular uptake efficiency and oxygen sensitivity because of their high hydrophilicity and short emission lifetime (< 1s). These disadvantages were addressed by synthesizing [Ru(bpy-pyr)(bpy)2]2+ [133] (Fig. 8A) and Ru-Py [134] (Fig. 8B), which have a lipophilic pyrenyl group in the bipyridyl and Nalkylaminophenanthroline ligand, respectively. These compounds exhibited enhanced cell membrane permeability and oxygen response. Ru-Py has a very long phosphorescence lifetime (17.5 s) in phosphate buffer (pH 7.4) solution. This prolonged lifetime is attributed to reversible triplet-triplet energy transfer between the Ru(II) polypyridyl and pyrene moieties, since pyrene and Ru(II) polypyridyl moieties have the lowest triplet states with * and MLCT character, respectively, in close proximity [135]. A water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit, Ru-Cou 1 [136], was synthesized for the ratiometric sensing of oxygen levels in living cells. Ru-Cou 1 showed dual emission both in solution and in living cells, and allowed monitoring of changes in oxygenation resulting from stimulation by valinomycin or antimycin A. Cell-penetrating peptides are useful for increasing cellular uptake efficiency and controlling subcellular localization. Keyes and coworkers synthesized [Ru(bpy)2(pic)]2+ (pic=2-(4-carboxyphenyl)imidazo[4,5-f][1,10]phenanthroline) with penta (Ru-Ahx-R5) or octa (Ru-Ahx-R8) arginine peptide substituents [137]. Ru-Ahx-R8 was transported passively through the cell membrane and accumulated in myeloma cells. They also developed a peptide-bridged dinuclear Ru(II) complex that localized in mitochondria [138]. Recently, Ru(II) polypyridyl complexes were modified for hypoxia imaging in vivo. Zhang et al. [139] designed and synthesized Ru(II) anthraquinone complexes as 23

reversible two-photon luminescent probes for cycling hypoxia images of cells, tumor spheroids, and zebrafish (Figure 9). These probes consisted of a luminescent Ru (II) complex, and a redox-active anthraquinone moiety which acts as a quencher. Under hypoxic conditions, the quinone moiety is reduced to hydroquinone, thus interrupting electron transfer quenching of the Ru(II) complex and significantly enhancing emission intensity. When the oxygen levels in cells or tissues recover, hydroquinone is reoxidized to quinone and emission of the Ru(II) complex is quenched again. Son et al. [140] developed Ru(II) complexes with a 2-nitroimidazole (NI) unit, Ru-NI 1, which can be used as an exogenous marker for hypoxic tissues (Fig. 8B).

5.1.3 Ir(III) complexes BTP (Fig. 6) was the first Ir(III) complex to be delivered directly into living cells and tissues as a small O2 probe, allowing detection of the intracellular and in vivo oxygen status [107]. Synthetic modification of Ir(III) complexes can improve their probe characteristics, including cellular uptake, intracellular localization, and absorption and emission properties. Figure 10 shows typical chemical modifications of BTP that provide improved intracellular and tissue oxygen measurements. For example, the cellular uptake efficiency of BTPDM1 was significantly improved over that of BTP due to introduction of a cationic dimethylamino group into the acac ligand of BTP [66]. BTPDM1 taken up into cells is mainly localized to lysosomes, in contrast with the specific endoplasmic reticulum localization of BTP in Hela cells. The mitochondria-specific oxygen sensing probe BTP-Mito was developed by introducing a triphenylphosphonium cation group, which is known to facilitate mitochondrial accumulation, into the acac ligand of BTP [95]. This probe indeed specifically localized in the mitochondria. BTP-Mito is the first 24

mitochondria-specific oxygen probe based on an Ir(III) complex. Similar mitochondriatargeting oxygen probes emitting in the red and near infrared region were synthesized using 5-amino-1,10-phenanthroline derivatives as ligands [141]. Recently, Liu et al. [142] synthesized the nucleus-targeting Ir(III) complex (ppy)2Ir(tpphz) and used it to monitor intranuclear oxygen levels. BTP and its acac-substituted derivatives have their absorption and emission bands in the visible region, thus limiting in vivo oxygen measurements to the surface layer of the tissue. In order to improve tissue penetrance, an Ir(III) complex with longer absorption and emission wavelengths, BTPHSA, was designed and synthesized by extending the electronic system of the ligand [107,143]. The first absorption maximum of BTPHSA appears at 550 nm and the phosphorescence maximum is observed at 720 nm. BTPHSA has a large emission quantum yield (0.32) and a relatively long lifetime (2.1 s) in deaerated aqueous solution, and allowed imaging of a tumor transplanted 6 to 7 mm below the abdominal skin surface [107]. Furthermore, BTPHSA is useful as a two-photon absorbing phosphorescent probe at the 1030 nm wavelength of a femtosecond fiber laser [144]. Ratiometric oxygen probes connecting an oxygen-sensitive Ir(III) complex and an oxygen-insensitive fluorophore have been designed and synthesized for the quantitative oxygen sensing of living cells. For example, C343-Pro4-BTP [71] consists of a blue fluorescent coumarin (C343) and a red phosphorescent BTP connected with a tetraproline linker (Fig. 10). This probe exhibits oxygen-dependent dual emission in aqueous solution, lipid membrane, and also in living HeLa cells. However, the oxygen concentration gradient in cultured cells could not be obtained due to its low cellular uptake efficiency. The cell membrane permeability of C343-Pro4-BTP was improved by developing similar 25

ratiometric oxygen probes using a cationic Ir(III) complex [145].

5.1.4 Protoporphyrin IX The use of naked phosphor as an oxygen probe has been hampered by its low cell membrane permeability, poor solubility, and non-specific interactions with biomolecules. Mik and co-workers developed a unique oxygen monitoring method using protoporphyrin IX (PpIX), an endogenous porphyrin that is biosynthesized from 5-aminolevulinic acid inside the mitochondria [146]. PpIX shows delayed fluorescence in the red wavelength region. Since this delayed fluorescence has an extremely long lifetime (1.2 ms in 2% BSA solution at pH 7.4), its emission is easily quenched by molecular oxygen. The oxygen partial pressures of rat liver [147,148], rat heart [149] and rat skin [150,151], as well as of mitochondria in cultured cells [146], were estimated based on lifetime measurements of the delayed fluorescence of PpIX.

5.2 Dendron-coated porphyrins Although small molecule probes have various advantages, unexpected interactions with biological substances such as proteins, as well as self-aggregation, might be unavoidable in living organisms. Moreover, the emission properties of the probes can also be affected by microenvironmental variables such as polarity, pH, and ionic strength. To address these problems, Vinogradov and co-workers have designed and synthesized dendritic oxygen probes (Fig. 11). The probes were developed to specifically quantify oxygen distribution in the vasculature or interstitial space of tissues. Oxyphors R2 and G2 consist of a meso-substituted Pd(II)-porphyrin and -benzoporphyrin core, respectively, and second-generation polyglutamic dendrons [152]. The dendritic polyglutamic chains 26

protect the phosphor from interference by biological components, pH, and ionic strength, and also reduce quenching by molecular oxygen. Oxyphors R2 and G2 were used to measure oxygen partial pressures in animal tissues and subcutaneous solid tumors (see Chapter 8). Moreover, Vinogradov and coworkers developed three intravascular oxygen sensing probes, Oxyphors G3 [153], R4, and G4 [154] (Fig. 11) in which Pd(II) porphyrin was

encapsulated

into

Gen

2

poly(arylglycine)

dendrons

coated

with

a

poly(ethyleneglycol) (PEG) layer. Since these probes have a hydrophilic PEG layer on the surface, their solubility in water is significantly enhanced, thereby preventing aggregation and interactions with biological macromolecules, including albumin. Similar dendritic phosphorescent probes with water-soluble poly(arylglycine) dendrons were reported by the same group [155]. Recently, Evans and coworkers designed and synthesized a click-assembled oxygensensing (CAOS) nanoconjugate called G3 CAOS, which can penetrate cells and be used for oxygen sensing inside 3D spheroids [156]. G3 CAOS comprises a Pd(II)benzoporphyrin core surrounded by poly(amidoamine) dendrons, providing it with a polycationic character under physiological pH conditions. The oxygen sensitivity of G3 CAOS in aqueous solution in the presence of 2% BSA was of the same order of magnitude as that of Oxyphor G2. G3 CAOS could be incorporated into multicellular spheroids and showed enhanced emission under hypoxic conditions. They also developed a Pd(II)porphyrin-based dendritic oxygen sensor called Clickaphor Red G2 using a fast and highly efficient azide–alkyne click reaction [157]. Clickaphor Red G2 was embedded with Coumarin 500 in a liquid bandage matrix formulation and was successfully applied to biomedical imaging of tissue oxygenation. One-photon-absorbing oxygen probes have an inherent depth limitation even if red 27

emission light is used. To achieve oxygen images of deeper tissues, Vinogradov and coworkers [158-161] developed novel two-photon excitable probes based on the dendritic molecular structure, as illustrated in Fig. 12. The two-photon dendritic probe PtP-C343 comprised a Pt(II)-porphyrin core decorated with G3 poly(arylglycine) chains terminated with a poly(ethyleneglycol) layer and several coumarin 343 moieties which act as twophoton absorbers [162]. The excitation energy harvested by the coumarin 343 antenna was transferred to the central Pt(II)-porphyrin core by FRET. It was recently reported that a new two-photon antenna-core oxygen probe, PtTCHP-C307, shows high FRET efficiency and a significant increase in phosphorescence quantum yield due to reduced phosphorescence quenching by electron transfer [163]. The combination of these probes and two-photon phosphorescence lifetime microscopy enabled non-invasive 3D measurements of the oxygen distribution in cerebral vessels [164-168] and bone marrow [163,169] of living animals (see Sec. 7).

6. Intracellular oxygen sensing 6.1. Monolayer cultured cells The elucidation of cellular oxygen levels is associated with various important functions of living cells such as mitochondrial function and dysfunction, and cell metabolism, including biological responses to hypoxic conditions. Recent progress in optical oxygen probes and time-resolved fluorescence microscopy has made possible minimally invasive techniques for measuring cellular oxygen levels. Papkovsky and coworkers have measured the oxygen levels of cultured cells with a real-time oxygen sensing method using a commercially available microplate reader equipped with a time-resolved emission measurement module, together with various 28

Pt(II)-porphyrin-based probes with cell-penetrating abilities [170]. Lifetime was determined from the phosphorescence intensities at two distinct delay times after flash lamp excitation. Each lifetime was converted to oxygen concentration based on a calibration curve constructed in advance by using cultured cells loaded with the same probe and the respiratory inhibitor antimycin A. The plate reader was placed in a hypoxia chamber pre-set at different oxygen levels. The oxygen levels of different types of living cells were measured following treatment with metabolic effectors such as a respiratory inhibitor and an uncoupler, and a saccharide [13,171,172]. For example, addition of the mitochondrial

uncoupler

FCCP

(carbonyl

cyanide-4-

(trifluoromethoxy)phenylhydrazone) to monolayer cultured cells significantly decreased the intracellular oxygen concentration, indicating an increase in cellular respiration. Hypoxia is known to influence many key biological functions. Prior et al. detected intracellular hypoxia using an Ir(III) complex BTP as an oxygen probe [173]. The welldifferentiated cancer cell lines LNCaP and MCF-7 showed intracellular hypoxia without exogenous hypoxia, whereas the poorly-differentiated cancer cell lines PC-3 and MDAMB231 exhibited intracellular normoxia because of low oxygen consumption. Since most oxygen transported into cells is consumed in the mitochondria, it is generally accepted that mitochondria act as an oxygen sink by generating a flow of oxygen from the cell periphery toward the mitochondria. The oxygen distribution in a single cell is, therefore, expected to be determined by two main quantities: the oxygen consumption rate in mitochondria, and the diffusion rate of molecular oxygen in cells. Mycek and coworkers [104-106] performed time-resolved optical imaging of living cells using the Ru(II)-based oxygen probe RTDP. A gated CCD camera was used to obtain lifetime images of the cells. Intracellular oxygen concentrations evaluated from the 29

lifetime measurements were in good agreement with those determined by ERP oximetry [104]. Similar oxygen imaging experiments in single living cells were performed by Okura and coworkers [174] using MH134 cells incubated with PtTCPP and a gated CCD camera as a detector. They reported that the phosphorescence lifetime of PtTCPP in the cytoplasm was slightly shorter than that in the plasma membrane, although the spatial resolutions of the lifetime images were insufficient to resolve cellular compartments. Recently, laser scanning confocal PLIM has been developed to visualize oxygen distribution in a single cell with higher spatial resolution [175]. Kamachi and coworkers [176] obtained phosphorescence lifetime images of Pt(II)-porphyrin PtTCPP internalized into MKN45 cells using a PLIM system. The phosphorescence lifetimes of cell interiors were longer than that of the plasma membrane, suggesting that the oxygen concentration inside cells is lower than that of the periphery region. Recently, Papkovsky and coworkers [177] demonstrated the presence of oxygen gradients in giant umbrella cells (diameter > 100 m) using the cell-penetrating phosphorescent probe Pt-Glc. A confocal PLIM system was used to obtain lifetime images. Distinct regions with very low oxygen concentrations were found in a single cell, corresponding to the areas enriched with mitochondria. Lifetime-based oxygen measurements in living cells based on the TCSPC method were also performed using the Ir(III) complex BTPDM1 [66]. An oxygen concentration gradient in cultured cells was produced by placing a coverslip on a monolayer of cells, thereby preventing oxygen diffusion from the top surface of the cell layer. The oxygen tension of cells inside the covered area was 6.9 mmHg, indicating that the environment inside this area was hypoxic due to respiration by the cells. As described in Sec. 3.1.2., the phosphorescence decay profile of metal complexes 30

incorporated into cells frequently deviates from single exponential decay because of the possibility of the occurrence of several binding environments and configurations. Compared

with

intravascular

oxygen

measurements,

accurate

concentration

measurement are more difficult to perform because of the complexity of calibrating oxygen sensors within the cellular environment. In this respect, calibration of the measured lifetime in living cells remains challenging.

6.2. Multicellular spheroids Two-dimensional (2D) monolayer cultured cells are commonly used as a model cell system in cell biological and biomedical studies and are useful for studying the metabolic functions of individual cells. However, in living tissues, cells exist in 3D microenvironments

and

experience

intercellular

and

cell-matrix

interactions.

Multicellular 3D spheroids are therefore regarded as more accurate models of living tissues, although not of microvascular systems. The oxygen concentration of cells in the core of a spheroid may be lower than that of cells in the peripheral region because the oxygen supply from the environment by diffusional motion may be insufficient to compensate for oxygen consumed by the cells. Dmitriev et al. [178] have reported the 2D mapping of oxygen concentrations in neurospheres and tumor spheroids using NanO2 [101], a cell-penetrating nanoparticle oxygen probe containing PtTFPP dye. The 2D lifetime images obtained using a gated CCD camera demonstrated remarkable deoxygenation of the core region. Dmitriev et al. acquired high-resolution 2D and 3D distribution profiles of oxygen in spheroids by performing confocal PLIM experiments using NanO2 as an oxygen probe, and twophoton ratiometric intensity measurements using MM2, a ratiometric nanoparticle probe 31

[179]. The 3D oxygen images also showed a hypoxic core in the spheroids, but the distribution of these probes in neurospheres and spheroids was not uniform. To improve the uptake efficiency of the probe into spheroids, they developed the small molecule oxygen probes Pt-Glc and Pt-Gal containing saccharide vectors [122] (Fig. 7). The PtGlc probe accumulated more rapidly in neurospheres, showed uniform distribution, and provided deeper staining compared with the nanoparticle oxygen probe NanO2. Using PtGlc and a confocal PLIM system, they clarified the dependence of oxygen distribution on neurosphere size by 2D lifetime measurements (Fig. 13). Evans and coworkers demonstrated phosphorescence intensity images of 3D ovarian cancer spheroids using a cell-permeable polycationic dendrimer (G3 CAOS) and confocal microscopy [156]. The spheroids were treated with 2 M G3 CAOS for 4 h. The phosphorescence signal of G3 CAOS could be observed throughout each spheroid at depths exceeding 100 m; this indicates that G3 CAOS penetrated hundreds of micrometers into the spheroids. They also confirmed the oxygen response of the phosphorescence

intensity

of

G3

CAOS

incorporated

into

spheroids;

the

phosphorescence intensity increased when the culture dish was purged with nitrogen gas.

7. Tissue oxygen imaging 7.1. General importance of and difficulty in imaging tissue oxygen Inflammation and fibrosis are major hallmarks of many human diseases, such as chronic kidney disease and liver fibrosis [180,181]. Hypoxia is closely related to inflammation, which can serve to trigger fibrosis [182-184]. Many inflammatory responses in mammals start with the transmigration of neutrophils, which consume the local O2. In addition, interleukin 1β, a well-known inflammatory cytokine, induces 32

CCAAT/enhancer-binding protein δ, resulting in an increase in HIF-1α transcription [185]. These findings indicate that inflammation is accompanied by hypoxia, and cellular responses to hypoxia are augmented. In turn, HIFs are required for inflammation [186], and thus hypoxia and inflammation are mutually facilitated. Both inflammation and hypoxia are augmenting factors of fibrosis [187-189]; consequently, assessing hypoxia is imperative when researching inflammation and fibrosis. There are also many diseases related to angiogenesis, such as diabetic retinopathy, age-related macular degeneration, and chronic kidney disease [190,191]. VEGF (vascular endothelial growth factor), a wellknown angiogenic factor, is strongly upregulated by the activation of HIFs [192], reinforcing the need to detect hypoxia in living organs. A key consideration for tissue oxygen imaging other than tumor imaging is that the extent of “hypoxia” differs between hypoxic tumors and non-tumorous living organs. The oxygen tension in hypoxic tumor is far lower than that of non-tumorous organs, even in pathological conditions. Thus, hypoxia-detecting methods used with non-tumor tissues must be more sensitive and therefore quantitative methods are preferable [193]. Representative quantitative oxygen-sensing techniques and their characteristics, such as measurement site, advantages, and limitations, are listed in Table 2.

7.2. Kidney and Liver Chronic kidney disease (CKD) is a major health problem worldwide. Renal hypoxia occurs with the progression of CKD, and in turn accelerates CKD progression irrespective of the cause of CKD [189]. Drug therapies against renal hypoxia show promise [194], thus increasing the importance of assessing renal hypoxia in vivo. Although the kidneys receive up to 20% of the cardiac output, renal oxygen tension is comparatively low 33

[191,195,196], in part due to the unique vascular system of the kidneys: the arterial and venous blood vessels run close together and parallel, facilitating oxygen transfer. This phenomenon is called the “arterial-to-venous oxygen shunt”. In the absence of disease, this low oxygen tension is “physioxia” (partial oxygen pressure under normal physiological conditions) in kidney [197], but oxygen levels are decreased further in CKD due to several mechanisms, including low hemoglobin level, loss of capillaries, and increased metabolic demand [191]. Techniques often used to assess renal oxygenation include pimonidazole protein adduct immunohistochemistry, needle electrode measurements, and blood oxygen level dependent

MRI

(BOLD-MRI)

[198].

Pimonidazole

protein

adduct

immunohistochemistry studies require a paraffin section and thus this technique cannot be used with living organs. Needle electrode measurements are considered the “gold standard” for quantitating oxygen partial pressures and are often used to assess oxygenation in both healthy and diseased kidney [199,200]. However, in addition to its high invasiveness, this technique does not allow accurate identification of the oxygen tension measurement site. This is particularly problematic if we want to distinguish intracellular oxygen tension from intravascular oxygen tension. BOLD-MRI is a promising technique for use in clinical patients since it is minimally invasive [201-203], but it only measures the concentration of deoxyhemoglobin in veins. We therefore cannot determine the oxygen concentration or oxygen saturation of hemoglobin because the measurement results are highly affected by the hemoglobin concentration in the vein [204,205]. In an attempt to overcome this disadvantage, the relaxation time T1 in kidney was assessed in rats as this parameter is directly affected by intracellular oxygen tension [206-208]. 34

Another approach for assessing renal oxygenation is the phosphorescence-based determination of oxygen tension. Mik et al. have been developing approaches for determining renal oxygenation using Oxyphor G2, a phosphorescent probe that remains in blood [152]. Using this dye, they determined the oxygen tension in renal veins and showed alterations in oxygen tension in response to acute hemodynamic changes [209]. They also simultaneously measured oxygen tension on the renal surface and deep inside the kidney [210]. This is a very important advance because there is an oxygen gradient in kidney [196]. The biggest drawback of these techniques is that they assess only oxygen tension in blood, or extracellular oxygen tension, and cannot assess intracellular oxygen tension directly. Our group recently reported a cationic lipophilic phosphorescent probe, BTPDM1, which can be used as an intracellular oxygen tension indicator in healthy and diseased kidney [211]. Interestingly, this probe distributes only inside tubules, allowing the specific assessment of oxygen tension inside tubules. By using a lifetime measurement system connected with a bifurcated fiber, we could detect intracellular hypoxia in mice of chronic kidney disease model (Fig. 14). Hypoxia in tubular cells is closely related to renal fibrosis [191] and thus this technique should be useful for investigating renal hypoxia. Liver is believed to be sensitive to hypoxia due to its unique vascularization: hepatocellular cells receive blood both from the hepatic artery and the portal vein [212]. A liver unit, the hepatic lobule, is surrounded by the terminal liver arteriole and the terminal portal vein, with the central vein in the center of the hepatic lobule. This vasculature has two hallmarks with regards to oxygen tension. First, the oxygen tension of the terminal portal vein, one of the vessels feeding the hepatic lobule, is lower than the 35

arterial oxygen tension. Second, the intracellular oxygen tension of hepatocytes is heterogeneous: the oxygen tension of hepatocytes in the periportal area is higher than that in the perivenous area. Indeed, Paxian et al. showed this intracellular oxygen gradient with high resolution using the ruthenium-based phosphorescent probe [Ru(phen)3]2+ [62]. These two characteristics contribute to the relatively low oxygen tension and oxygen gradient in liver tissue, which results in high sensitivity to hypoxia [213]. This low oxygen tension and oxygen gradient is similar to kidney, and HIFs play an important role in pathophysiology of liver. Indeed, HIF activation in liver occurs at a higher hemoglobin level compared with brain and kidney [214]. Furthermore, HIFs affect fibrogenesis, angiogenesis, and lipid metabolism in liver diseases such as non-alcoholic fatty liver diseases and hepatitis-B virus infection, and subsequent liver fibrosis [215-217]. MRI-based studies are used to detect and assess both liver and kidney hypoxia, such as BOLD-MRI, a T2*-based technique that can be used to quantitate the deoxyhemoglobin concentration in veins, which in turn reflects oxygen tension in the vein [218]. Another approach is to assess the relaxation rate R1, a T1-based technique that directly measures tissue oxygen tension [206-208]. Indeed, the latter technique succeeded in detecting changes in liver tissue oxygen tension during hyperoxic breathing [207]. This MR-based technique can be used with patients. Bodmer et al. reported a technique to determine microvascular and mitochondrial oxygen tension in liver using Oxyphor G2 and protoporphyrin IX [148]. This technique can detect changes in oxygen tension when the fraction of inspired oxygen is changed; furthermore, because of their high sensitivity, phosphorescence or fluorescence lifetime measurements are useful in researching liver hypoxia.

36

7.3. Brain Hypoxia in brain is critically important in the context of brain tumor and stroke, and it was recently reported to also be related to various other diseases such as migraine and Alzheimer disease [219,220]. Thus, research on hypoxia in brain is becoming more intriguing. BOLD-MRI is most frequently used to detect brain hypoxia [221-223]. Measuring the concentration of oxyhemoglobin and deoxyhemoglobin by near-infrared spectroscopy (NIRS) is another technique for detecting hypoxia in brain [224-226]. The advantage of these two techniques is that a chemical probe does not need to be administered because they use hemoglobin as an endogenous oxygen-sensitive probe. Importantly, recent phosphorescence lifetime measurements have been conducted to accurately determine the intraarterial oxygen tension gradient in brain using PtP-C343 [165,227]. Other studies report measurement of the oxygen concentration using Oxyphor G4 in the microvasculature of epileptic mice or pigs under resuscitation [228,229]. However, intravascular distributing phosphorescent probes measure the oxygen tension in vessels and not the intracellular oxygen tension. Recently, Muir et al. reported measurement of the intracellular oxygen tension in brain using a T1-based MRI technique typically used in kidney and liver [230]. This is the only report assessing intracellular oxygen tension in brain, and thus an oxygen indicator which achieves intracellular distribution is required to establish other intracellular oxygen tension measurement techniques in brain. Dmitriev et al. reported anionic cell-permeable phosphorescent nanoparticles which distribute inside cells; in combination with phosphorescence lifetime imaging microscopy, they could detect oxygen concentration heterogeneity in neural spheroid cells [231]. These probes hold promise for use in living animals. 37

7.4. Bone marrow Hematopoietic stem cells reside in bone marrow, a hypoxic tissue due to its unique vasculature system [232]. Nutrient arteries of bone marrow enter the medullary canal penetrating the cortical bone and are subdivided into arterial capillaries. These capillaries are connected to sinusoids, which are fenestrated and loosely organized. Hematopoietic stem cells are located in endosteal zones, which closely interact with sinusoids and nonsinusoidal microvessels [233]. This complex system limits the perfusion of endosteal zones and thus hematopoietic stem cells live in a hypoxic microenvironment. Recently, Spencer et al. directly measured the oxygen concentration in arteries penetrating cortical bones and proved that the intraarterial oxygen concentration decreases from 31.8 mmHg to 22.2 mmHg after entering the medullary canal [169]. These measurements employed PtP-C343 to conduct phosphorescence lifetime measurements with a two-photon microscope. Prior to this research, hypoxia in bone marrow had been confirmed only by pimonidazole staining and HIF-α activation [233], neither of which provide direct measurements of oxygen, nor are they indicative of oxygen concentration.

7.5. Retina Hypoxia in retina is believed to be related to retinal diseases such as age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy, in part because these diseases are related with disturbed retinal blood flow. Age-related macular degeneration is closely related to abnormality in autophagy, and retinopathy of prematurity and diabetic retinopathy result from undesirable neovasculization. Autophagy and neovasculization are downstream of HIF, further supporting the 38

importance of hypoxia in these diseases [234-236]. Two techniques are frequently used to detect retinal hypoxia: oximetry, and phosphorescence lifetime measurements using metalloporphyrins. Oximetry is a technique to determine oxyhemoglobin and deoxyhemoglobin levels by different absorption spectra [237]. This technique is unique in that endogenous substances are used as the oxygen sensor and thus it is easily applied to research in human since the retina can be easily observed noninvasively. Oximetry differs from NIRS in brain research in that it utilizes optical wavelengths and thus the target organ must be visible on the surface of the body. There are multiple reports of the use of oximetry in healthy volunteers [238-240]. This technique allows the oxygen saturation of hemoglobin (SatO2) to be determined in arteries and veins. SatO2 is strongly correlated to the partial pressure of oxygen, but can be influenced by other microenvironmental factors such as pH or the partial pressure of carbon dioxide (Bohr effect). Phosphorescence lifetime measurement using metalloporphyrins is another major technique for detecting retinal hypoxia [241] but requires the administration of phosphorescent

metalloporphyrins.

Phosphorescence

lifetime

measurements

of

metalloporphyrins allowed the 3D mapping of oxygen tension in rats [242] and of VEGF changes in retinal oxygen delivery [243], demonstrating that these methods are applicable for detecting intravascular hypoxia. An investigation of intracellular hypoxia in rat retina by Piao et al. succeeded in detecting retinal intracellular hypoxia using a green fluorophore containing a hypoxiasensitive azo-bond [85]. Recently, Uddin et al. also reported the detection of retinal hypoxia using a near-infrared imaging agent coupled to a dark quencher containing a hypoxia-sensitive azo-bond [244]. These azo-bonds are cleaved by azoreductase inside 39

the cell, allowing these probes to provide a high signal-to-noise ratio and intracellular distribution even in living animal. Intracellular hypoxia is clearly important in the retina and thus further research is expected in the future.

8. Hypoxic tumor imaging Hypoxia is involved in many human pathologies, including stroke, ischemic heart disease, and cancer. Tumor hypoxia is a condition in which the tumor region of a tissue is deprived of sufficient oxygen due to explosive growth of the tumor beyond the means supportable by the oxygen supply delivered by blood flow. The oxygen levels in normoxic tissues are in the range 5-10% O2, whereas the oxygen levels in hypoxic tumor are less than 1.3% O2 [245,246]. A study of the difference in radio-sensitivity of tumors and normal/healthy tissues published in 1953 first suggested hypoxia in tumor [247]. The discovery of HIFs led to hypoxia being considered a key regulatory factor in tumor growth, invasion, angiogenesis, apoptosis, metastasis [248], and radiation resistance [249]. Accordingly, understanding the degree and area of hypoxic tissue in tumor is becoming invaluable for both basic oncology and clinical treatment. A number of chemical compounds and methods have been developed to measure the degree and area of hypoxia, including oxygen needle electrodes, immunohistochemical staining, PET, single photon emission computed tomography (SPECT), MRI, and optical imaging. In this section, we provide an overview of practical research conducted using oxygen needle electrodes, immunohistochemical staining of hypoxic tumor, PET, and optical imaging. In particular, this section focuses on recent progress in phosphorescent probes as emerging agents for noninvasive optical-imaging of tumor hypoxia.

40

8.1. Oxygen needle electrodes Oxygen needle electrodes are commercially available and have been extensively used to directly determine pO2 in both human and animal tumor studies [250,251]. These oxygen electrodes are generally based on the classical Clark electrode [252] which involves a noble metal (e.g., silver, gold, or platinum) for reducing oxygen to provide the negative polarizing voltage. The voltage between the reference electrode (anode) and the measuring electrode (cathode) is in proportion to the amount of oxygen reduced on the cathode. The oxygen needle electrode is the only technique that directly measures the oxygen concentration of tissues and has thus long been considered the gold standard [253256] for such measurements. However, there are several disadvantages to using oxygen needle electrodes for measuring pO2 in tumors: (1) the needle itself is slightly invasive and potentially damages the tissue [257], (2) movement of the electrode through tissues of different densities (e.g., between tumors and normal/healthy tissues) can induce high pressure at the tip of the needle, resulting in inaccurate pO2 values [256], (3) a single point measurement by an oxygen needle electrode cannot adequately describe intra-tumoral heterogeneity in pO2 values [257,258], and (4) the signal-to-noise ratio (a measure of signal strength relative to background noise) is relatively low and can result in unreliable pO2 values [256]. Thus, the oxygen needle electrode is increasingly being used to complement recently developed noninvasive methods.

8.2. Immunohistochemical staining Immunohistochemical methods are based on the antibody detection of exogenous or endogenous hypoxia markers. Nitroimidazole derivatives, such as misonidazole (1(alpha-methoxymethylethanol)-2-nitroimidazole) [259], pimonidazole [260,261], and 41

EF5 (2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide) [262264], are well-known exogenous markers for hypoxic tumors. In most animal studies, a nitroimidazole

derivative

is

intravenously

administered,

followed

by

immunohistochemical detection. Nitroimidazole derivatives are reduced and bind to thiol groups of proteins in the hypoxic areas. Immunohistochemical staining with nitroimidazole derivatives is also useful for human biopsy, and radiolabeled nitroimidazole derivatives have been used for noninvasive PET imaging of hypoxia in vivo [265]. Hypoxia changes the expression of genes and proteins as an adaptation response to reduced O2 availability [266]. This cellular response to hypoxia is mainly controlled by the HIF family, the most intensively studied hypoxia-signaling pathways in molecular biology (see Sec. 2). HIF-1 regulates the expression of genes such as VEGF, MMP2 (matrix metallopoteinase 2), cytochrome c, and the glucose transporters (GLUTs) involved in angiogenesis, invasion, metastasis, and apoptosis cell metabolism. Further, HIF-1α is highly expressed in a wide variety of tumors [267-269]. Due to the widespread expression of HIF-1α, and the influence of HIFs on multiple hypoxia functions, HIF-1α is used as an endogenous hypoxia-related marker. Moreover, HIF-1α is also believed to be useful for detecting hypoxic tumor tissues.

8.3. Positron emission tomography (PET) PET is a nuclear medicine imaging technique that uses small amounts of radioactive tracers. When applied to imaging tumors, the tracers are designed to be integrated into the tumor. 18F-FDG PET utilizes the increased glucose uptake and glycolysis of cancer cells to visualize tumor tissues using a PET scanner [270]. PET imaging is extensively 42

used because it enables three-dimensional assessment of tumors in the whole body [271,272]. Many PET tracers have been developed and tested to target hypoxic tumors and microenvironments in preclinical and clinical trials; these tracers include nitroimidazole derivatives and Cu-labelled diacetyl bis(N4-methylthiosemicarbazone) (ATSM) [272-274]. 14

C-Misonidazole is a member of the nitroimidazole family and was the first tracer

used to detect hypoxic tumors [259]. Later, two important types of radioactive probes, 18

F-labelled nitroimidazole derivatives and 60,61,62,64Cu-ATSM, were developed to detect

hypoxic tumors using PET. PET imaging requires that the hypoxia tracers nonspecifically enter cells and leave cells in the presence of proper oxygen concentrations (more than 5% O2). Nitroimidazole derivatives diffuse into biological cells. In normoxic conditions, the anion of the nitroimidazole derivative is rapidly reoxidized by reductases that are ubiquitously expressed, and the anion can diffuse out of the cell. In hypoxic conditions, the nitroimidazole derivative is further reduced, resulting in binding to thiols in macromolecules and subsequent accumulation in the cells. A new complex of Cu with ATSM has been developed as a PET tracer for hypoxia that exhibits enhanced membrane permeability [275,276]. Many PET tracers have been developed for the detection of hypoxic tissues and tumors in live animals (for review see refs. 271 and 272).

8.4. Optical imaging Optical tumor imaging using hypoxia-sensitive luminescent probes requires intravenous injection of probes into small animals under anesthesia. Phosphorescent probes distribute throughout the body, usually without specific accumulation in tumor tissue. If the probes distribute throughout normal and tumor tissues essentially uniformly, 43

the tumor tissue should provide a brighter emission image compared with normal tissue because of the lower oxygen level of tumor tissues. Even if the probe distribution is not uniform, the lifetime-based measurements described in Sec. 3.1.2 can image the tumor region specifically by difference in the lifetimes: i.e., longer lifetime in the tumor relative to that in the peripheral region. Lifetime can also be used to assess the oxygen concentration or partial pressure of the tumor. Pt(II)- and Pd(II)-porphyrins have been developed to image the vascular (blood plasma) and interstitial space of tissues. Initially, metalloporphyrin probes were chemically synthesized as water-soluble porphyrin derivatives with a Pd(II) ion (PdCP and PdTCPP in Fig. 6) to measure oxygen distribution in perfused tissue [87,98]. These probes were bound to BSA before injection into animals in order to enhance their hydrophilicity and prevent their interaction with biological molecules. However, two disadvantages

of early

metalloporphyrins

had to

be

addressed:

(1) these

metalloporphyrins were too sensitive to oxygen quenching when used as oxygen sensors, due to their extremely long phosphorescence lifetime, and (2) the additional albumin may have caused toxicity and immunogenic responses in live animals. Therefore, metalloporphyrins have subsequently been chemically modified with polyglutamic acid and poly(arylglycine) dendrons to adjust the access of oxygen to the porphyrin [60,152,154,158,277]. Esipova et al. reported a new series of dendron-coated metalloporphyrins (Oxyphors R4 and G4) which are highly soluble in aqueous environments and do not permeate biological membranes [154]. Using these sophisticated oxygen probes and a lifetime imaging system, they could clearly visualize intravascular oxygenation in a mouse, including tumor hypoxia (Fig. 15). A phosphorescent Ru(II) complex with a 2-nitroimidazole unit, Ru-NI 1 (Fig. 8B), 44

was developed to sense oxygen fluctuation in cancerous tumors [140]. The nitroimidazole unit was introduced to promote specific accumulation of the probe in hypoxic tumor tissues, and the phosphorescent Ru(II) complex worked as a reporter of the oxygen status of tumor tissues. Ru-NI 1 (500 nmol) was intraperitoneally injected into tumor-bearing nude mice, accumulated at the hypoxic tumor tissue, and its phosphorescence intensity in tumor varied, reflecting tissue oxygen levels. The red-phosphorescence-emitting iridium complex BTP and its derivatives were applied to in vivo oxygen imaging [66,107]. These probes exhibited oxygen-dependent red emission in a tumor-bearing mouse, indicating that Ir(III) complexes have vast potential for imaging hypoxic lesions such as tumor tissues. In contrast to the vascular localization of metalloporphyrins, iridium complexes localize in the cell [66,71,107,108]; furthermore, iridium complexes are relatively small (Mw: 600 – 800 Da) and lipophilic, and thus they permeate the cell membrane and diffuse into the cell. It is important to quantify the intracellular oxygen level in tumors because hypoxia is believed to promote the expression of regulatory factors for growth, invasion, angiogenesis, apoptosis, and metastasis [248]. One of the drawbacks of in vivo optical imaging is the penetration limit of luminescence in tissue due to light absorption by hemoglobin and water, and to light scattering. BTPH was therefore synthesized and has an extended -electronic system compared with the benzothienyl-pyridinato group of BTP. The aqueous solubility of BTPH was extremely low and thus a carboxy derivative of BTPH, BTPHSA, was synthesized that exhibits improved solubility (Fig. 10). The absorption maximum of BTPHSA is located at around 550 nm and the phosphorescence-emission maximum is at 720 nm, i.e., in the NIR region [107]. BTPHSA was used to visualize tumors transplanted 45

6 to 7 mm below the skin surface. The cellular uptake efficiency of BTP was increased by designing and synthesizing BTP derivatives incorporating a cationic amino group (BTPNH2), a cationic dimethyl amino group (BTPDM1), or an anionic carboxyl group (BTPSA) into the acetylacetonato ligand [66] (Fig. 10). Introduction of a dimethylamino group increased cellular uptake efficiency by a factor of 20 compared with that of BTP. BTPDM1 allowed imaging of the tumor region in a mouse following administration of a much smaller amount of probe (25 nmol) compared with BTP (250 nmol) [66]. The lifetimes of BTPDM1 phosphorescence from tumor and extratumor regions demonstrated tumor hypoxia (pO2 = 6.1 mmHg in tumor vs. 50 mmHg in healthy epidermal tissue). Systemic administration of BTP and its derivatives to a mouse showed that these complexes are distributed in different organs and exhibit phosphorescence lifetime change depending on the oxygen level. These results show the potential of iridium complexes as cellular or in vivo oxygen probes. In contrast to the oxygen-sensitive property of phosphorescence, fluorescence is generally insensitive to oxygen under physiological conditions, as expected from the short fluorescence lifetimes (typically 1-10 ns) (see discussion in Sec. 3.1). Fluorescent chromophores have been used to image tumor or tissue hypoxia by conjugating them with molecules that express their activity in hypoxic conditions. As mentioned in Sec. 2, HIF1α has been a key target for measuring hypoxic activity in tumor or tissue cells. HIF-1α comprises 826 amino acids and contains an oxygen-dependent degradation (ODD) domain (residues 401-603) which is hydroxylated by proline-hydroxylase-2 (PHD-2) under normoxia [278]. Prolyl hydroxylation leads to the von Hippel Lindau protein (pVHL)-dependent ubiquitination and rapid proteasomal degradation of HIF-1α [279]. An imaging probe for HIF-1-active cells has been developed comprising a protein 46

transduction domain (PTD) to provide membrane penetrating activity and an ODD domain. This probe, PTD-ODD fusion protein, is expected to co-localize with endogenous HIF-1α [280-284]. The utility of this fusion protein for imaging and therapy was examined by either attaching a fluorescent chromophore (e.g., GFP or Cy5.5) or a functional protein (e.g., procaspase 3 or β-galactosidase). As expected, the PTD-ODD fusion probe permeated through the cell membrane and escaped from intracellular degradation in hypoxic environments. The probes survived in hypoxic tumor cells and could delineate the tumor area following conjugation with fluorescent chromophores. Moreover, conjugation of the PTD-ODD probes with procaspase 3 resulted in tumor cell apoptosis. The number of protein-based probes incorporating domains of HIF-1α or other proteins (e.g., antibody against CA9 or VEGF [285]) is increasing and they show promising results in preclinical studies, although their toxicities and pharmacokinetics remain unknown.

9. Summary and outlook Phosphorescent transition metal complexes have attracted increasing interest as biological oxygen probes owing to their advantageous photophysical properties. Since long-lifetime phosphorescence is significantly quenched by ambient oxygen, metal complexes have been successfully used in biological oxygen sensing. In this review, we focused on Pt(II)- and Pd(II)-porphyrins, Ru(II) complexes, and Ir(III) complexes as exogenous molecular probes for intracellular and in vivo oxygen imaging. Phosphorescence quenching of these metal complexes by molecular oxygen proceeds through two competing channels: non-CT channels to produce singlet oxygen via energy transfer and/or CT channels involving charge transfer (or electron transfer) reactions. The 47

magnitude of the quenching rate constant is correlated with the Gibbs free energy change Gel for the electron-transfer reaction. In Pt(II)- and Pd(II)-porphyrins and Ru(II) complexes, oxygen quenching mainly proceeds via the non-CT mechanism, whereas in Ir(III) complexes, the CT mechanism involving charge transfer is often involved in oxygen quenching because the Gel value for Ir(III) complex/O2 systems can be negative. As a result, Ir(III) complexes generally exhibit larger oxygen quenching rate constants compared with metalloporphyrins and Ru(II) complexes. In order to refine the characteristics of these complexes for biological oxygen sensing, various chemical modifications have been made in the ligands. These complexes exhibit diverse photophysical properties. Pd(II)- and Pt(II)-porphyrins have very long phosphorescence lifetimes (10–1000 s) and thereby enable the detection of low levels of oxygen. This feature is also favorable for lifetime measurements using a conventional flash lamp with relatively long pulse widths (~s) and low repetition frequencies as the excitation source. On the other hand, Ir(III) complexes that have moderately long lifetimes (1–20 s) and large oxygen quenching rate constants are advantageous for lifetime imaging that uses high-frequency pulsed laser. The brightness of a probe is usually evaluated by multiplying the molar absorption coefficient with emission quantum yield  0p . The excitation of Pt(II)- or Pd(II)-porphyrins in the Soret band can produce very bright phosphorescence; however, the Soret band is sharp and appears at relatively short wavelengths (~450 nm). In contrast, Pd(II) meso-tetraphenyl-tetrabenzoporphyrin derivatives exhibit strong absorption, which originates from the Q band at much longer wavelengths (~630 nm); thus, these compounds can be ideal probes for in vivo oxygen measurements. For Pd(II)- and Pt(II)-porphyrins and Ir(III) complexes, NIR emission with moderately large p values can be realized by extending the-electronic systems, 48

whereas in Ru(II) complexes, extension of the emission wavelength to the NIR region seems to significantly decrease the emission quantum yield and lifetime. As for intracellular oxygen measurements, Ir(III) complexes generally exhibit excellent characteristics such as a high cellular-uptake efficiency and specific subcellular localization. In the case of metalloporphyrins and Ru(II) complexes, chemical modifications of ligands are needed to increase cell-membrane permeability. These complexes have provided excellent performance with respect to different analytical tasks. For intracellular oxygen sensing, various Pt(II)-porphyrin-based probes with the cell-penetrating ability have been successfully developed to monitor the oxygen status of living cells. A small molecule oxygen probe called Pt-Glc, which contains glucose units in the PtTFPP derivatives, is taken up rapidly into neurospheres; this enables the visualization of oxygen distribution in the cell spheroid. Early Ru(II) complex-based oxygen probes had relatively short lifetimes and low cell permeability. These drawbacks are being overcome by chemically modifying the ligand; introduction of a lipophilic pyrenyl group into the N-alkylaminophenanthroline ligand in Ru–Py has increased cell membrane permeability and oxygen response. Chemical modification works very effectively for Ir(III) complexes. A cationic BTP derivative, BTPDM1, is easily taken up into living cells; this enables the visualization of oxygen distribution in monolayer cultured cells through phosphorescence lifetime imaging. In tissue oxygen measurements, especially in intravascular oxygen measurements, dendron-coated porphyrins are becoming the standard probe because of their excellent analytical characteristics such as high oxygen sensitivity, NIR emission, and two-photon excitation. One of the great advantages of optical oxygen-sensing methods is that they allow oxygen imaging at the cellular level. The combination of suitable probes with 49

phosphorescence lifetime imaging microscopy will facilitate further studies on the oxygen dependence of cellular metabolism. Although the use of phosphorescent metal complex probes and time-resolved luminescence microscope have opened up a doorway to oxygen imaging at the cellular level, many challenges still need to be overcome. One particular challenge is the development of in vivo oxygen probes that rapidly pass through blood-vessel walls and reflect intracellular oxygen levels, thereby allowing the measurement of tissue oxygen tension on a spatial scale similar to the O2-diffusion distance (70–100 m). Furthermore, detailed analysis of the phosphorescence decay profile into various cells is required for the design and synthesis of suitable oxygen probes to improve the accuracy and sensitivity of intracellular oxygen-concentration measurements.

Acknowledgements This study was supported by Grants-in-Aid for Scientific Research on Innovative Areas (No. 26111003 to M.N., No. 26111012 to S.T.) and for Young Scientists (A) (No. 26702011 to T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a Grant-in-Aid for Industry-Academia Collaborative R&D Programs (In vivo Molecular Imaging: Towards Biophotonics Innovations in Medicine) from

the

Japan

Agency

for

Medical

Research

and

Development

(No.

15im0402008h0005).

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Toshitada Yoshihara received his PhD degree from Gunma University in 2001. He was a postdoctoral researcher at Max-Planck-Institut für Biophysikalische Chemie (2001-2002) and National Institute of Advanced Industrial Science and Technology (AIST) (2002-2004). Since 2004, he worked at Department of Chemistry of Gunma University and became an associate professor in 2016. His current research interests focus on photophysics of bioimaging probes and measurement of intracellular and in vivo oxygen by using optical method.

Yosuke Hirakawa is a medical doctor and received his PhD degree in the University of Tokyo Graduate School of Medicine in 2016. In his PhD thesis he established a new method to quantify intracellular oxygen tension in renal tubular cells in vivo in the working group of Prof. Nangaku in collaboration with Prof. Tobita’s group. He is working as an assistant professor in the Division of Nephrology and Endocrinology, the University of Tokyo Hospital. His research interest includes in vivo oxygen imaging and relation to chronic kidney disease.

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Dr. Masahiro Hosaka received his PhD degree in 1995 at University of Tsukuba under the supervision of Kazuhisa Nakayama. He moved to the United States in 1995, where he began postdoctoral training at University of Texas Southwestern Medical Center at Dallas, Texas, under the supervision of Thomas C. Südhof. Hosaka finished his postdoctoral training in 1999 and moved to Gunma University in Japan. In 2011, he moved to Akita Prefectural University as a professor. During his research, he focused on the molecular and cellular mechanism about protein transport in nerve and endocrine cells for over 25 years. Currently, his research interests are; metabolisms induced by physiological oxygen environments, development of hypoxia-specific luminescent probe for identifying cancers, and molecular imaging in living animals.

Masaomi Nangaku received his PhD degree in the University of Tokyo Graduate School of Medicine in 1996. Now he is the head and the professor of Division of Nephrology and Endocrinology in the University of Tokyo Graduate School of Medicine from 2012. He works as a vice president of the University of Tokyo Hospital. He is an associate editor of Kidney International. His research interest includes pathophysiology of chronic kidney disease, renal hypoxia, and complement dysregulation. He is also currently working as a lead researcher in OXYGEN BIOLOGY, which is supported by Japan Society for the Promotion of Science.

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Prof. Seiji Tobita received his Doctoral degree in physical chemistry from Tokyo Institute of Technology in 1983. In 1990-1991, he worked in Université de Paris-Sud in France and Freie Universität Berlin as invited scientist to study the formation and fragmentation of monocations and dications of polycyclic aromatic hydrocarbons. He joined the Faculty of Engineering, Gunma University, as an associate professor in 1992. Currently he is a professor of chemistry and chemical biology at Gunma University. His main research interests are photophysics and photochemistry of aromatic molecules and metal complexes, and development of luminescent probes and techniques for molecular imaging.

Figure captions Fig. 1 The mechanism of HIF regulation by oxygen. When a cell is in the normoxic condition, HIF- is hydroxylated by PHD using an oxygen molecule. The hydroxylated HIF is degraded after ubiquitination. In contrast, when a cell is in the hypoxic condition, the hydroxylation of HIF- occurs less frequently, resulting in more HIF- in the cytosol. HIF- then forms a heterodimer with HIF-, an oxygen-independent subunit, and functions as a transcription factor. Abbreviations: HIF, hypoxia-inducible factor; PHD, prolyl hydroxylase domain Fig. 2 Energy state diagram, including oxygen quenching of the excited triplet state for a molecule M. Fig. 3 Phosphorescence quenching pathways of Ir(III) complex/O2 systems with Gel ≳ −0.2 eV. Fig. 4 Rehm-Weller plots of logkq vs. Gel for Ir(III) complexes/O2, Ru(II) complexes/O2, and Pt(II) and Pd(II) porphyrins/O2 systems (green, blue and red circles, respectively)

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[35,53-58]. The dotted lines show (a) logkd, (b) log(4/9kd), and (c) log(1/9kd). Fig. 5 Design concept behind (A) a hypoxia-sensitive fluorescent probe with a bioreductive nitroimidazole unit, (B) an activatable fluorescent hypoxia probe in which a fluorophore and a quencher unit are connected by a bond which undergoes reductive cleavage under hypoxia, and (C) a reversible off/on hypoxia-sensing fluorescent probe. Fig. 6 Chemical structures of typical phosphorescent metal complexes used for oxygen sensing. Fig. 7 (A) Pt(II)-porphyrin derivatives synthesized to improve cellular uptake efficiency. (B) Chemical structure of Pd(II)-meso-tetraphenyl-tetrabenzoporphyrin. Fig. 8 (A) Chemical structure of [Ru(bpy-pyr)(bpy)2]2+. (B) Chemically modified Ru(II) complexes for biological oxygen sensing. Fig. 9 Two-photon excitable off/on luminescent oxygen probe based on a Ru(II) complex. Fig. 10 Chemical modification of BTP for intracellular and in vivo oxygen sensing. Fig. 11 Chemical structure of Oxyphor G4 and schematic representation of dendrimercoated probes. Fig. 12 Schematic representation of dendrimer-coated two-photon probes. Fig. 13 (A) PLIM images of the ~60 and 250 mm large neurospheres from cortices of embryonic (E18) rat brain staining with Pt-Glc. (B) Phosphorescence lifetime (, blue lines) of Pt-Glc and oxygen concentration profiles (red lines) for the cross-section of the ~60 and 250 mm large neurospheres. Scale bar is in m. Reprinted with permission from Ref. [122]. Copyright 2014 Royal Society of Chemistry.

Fig. 14 (A) Schematic diagram of the phosphorescence lifetime measurement system for

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living mouse. (B) Picture of phosphorescence lifetime measurements of kidneys in a living mouse without black-out curtain. Liver (L), gut (G) and kidney (K) were seen. B is a tip of bifurcated fiber. The excitation laser was spotted on the kidney and the diameter of the spotted area was approximately 3 mm. (C) Experimental scheme of unilateral ischemia-reperfusion (I/R) model. Average of phosphorescence of I/R injured and contralateral kidneys in five mice. Values under the graph show oxygen partial pressure (pO2). #P < 0.05 by two-tailed paired t-test.

Fig. 15 Imaging of intravascular oxygenation in a mouse using Oxyphor G4. (A) Photograph of an anesthetized mouse on a heating pad, and the zoomed-in region, as imaged by an ICCD camera. (B) Phosphorescence lifetime (left panel) and oxygen partial pressure (right panel) images of the mouse using Oxyphor G4. Reprinted with permission from Ref. [154]. Copyright 2016 American Chemical Society.

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Table 1 Spectral and photophysical properties of representative luminophores used in biological oxygen sensing compound PdCP

solvent

PtCP

2% albumin aqueous solution phosphate buffer

PdTCPP

H2O

PdTCPP

2% albumin aqueous solution CH2Cl2

PtTFPP

[Ru(bpy)3]2+ (RTDP) Ru[(phen)3] 2+ [Ru(dpp)3]2+ BTP Ir(Cs)2(acac)

EtOH:MeOH (4:1 v/v) EtOH:MeOH (4:1 v/v) EtOH:MeOH (4:1 v/v) THF CHCl3

max abs

(nm),  (dm3mol-1cm-1) 380

max phos

(nm)

0 p

(s)

ref

p a

667

1200

0.17

87,88

650

52

0.23

89

701

300

0.01

90

687

637 b

0.06

91

535, 503, 380, 523, 413, 523 416 538, 504, 390, 450,

18,000 15,000 120,000 21,000 270,000

29,400 23,200 323,000 14,300

647

60

0.088

92

630

1.15

0.089

93

445, 463, 486, 472, 444,

20,000 28,600 6,700 92,800 86,800

595 618 615 563

0.45 6.40 5.7 11.3

0.019 0.366 0.30 0.54

94 94 95 96

a) glycerol:phosphate buffer (9:1 v/v), pH 8.0; b) 2% albumin aqueous solution at pH 7.4, 38 °C

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Table 2 Characteristics of representative intravital and quantitative oxygen measurement techniques Technique

Oxygen sensor

Measured substance Oxygen molecule

Measurement site Microcirculation

Invasiveness

Advantage

Limitation

High invasiveness

Highly indicative of oxygen tension

High invasiveness Need proficiency

Needle electrode

Polarographic electrode

BOLD-MRI

Hemoglobin

Deoxyhemoglobin Intravenous

None

Applicable to clinical research Deep tissues are observable

Affected by hemoglobin concentration

NIRS/oximetry

Hemoglobin

Oxyhemoglobin/ Intravascular Deoxyhemoglobin

None

Applicable to clinical research

Only sites near the body surface are observable

Fluorescence measurement using PpIX

Protoporphyrin IX Oxygen molecule (PpIX)

Intracellular

Probe toxicity

Highly indicative of intracellular oxygen tension Applicable to clinical research of skin

Need several hours to accumulate PpIX Phototoxicity

Phosphorescence measurement using small molecule

Phosphorescence probe

Oxygen molecule

Intracellular

Probe toxicity

Highly indicative of intracellular oxygen tension

Probe toxicity

Phosphorescence measurement using dendrimer

Phosphorescence probe

Oxygen molecule

Intravascular Interstitial

Probe toxicity

Highly indicative of oxygen tension

Probe toxicity

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81