Biocompatible Phosphorescent O2 Sensors Based on Ir(III) Complexes for In Vivo Hypoxia Imaging

In this work, we obtained three new phosphorescent iridium complexes (Ir1–Ir3) of general stoichiometry [Ir(N^C)2(N^N)]Cl decorated with oligo(ethylene glycol) fragments to make them water-soluble and biocompatible, as well as to protect them from aggregation with biomolecules such as albumin. The major photophysical characteristics of these phosphorescent complexes are determined by the nature of two cyclometallating ligands (N^C) based on 2-pyridine-benzothiophene, since quantum chemical calculations revealed that the electronic transitions responsible for the excitation and emission are localized mainly at these fragments. However, the use of various diimine ligands (N^N) proved to affect the quantum yield of phosphorescence and allowed for changing the complexes’ sensitivity to oxygen, due to the variations in the steric accessibility of the chromophore center for O2 molecules. It was also found that the N^N ligands made it possible to tune the biocompatibility of the resulting compounds. The wavelengths of the Ir1–Ir3 emission maxima fell in the range of 630–650 nm, the quantum yields reached 17% (Ir1) in a deaerated solution, and sensitivity to molecular oxygen, estimated as the ratio of emission lifetime in deaerated and aerated water solutions, displayed the highest value, 8.2, for Ir1. The obtained complexes featured low toxicity, good water solubility and the absence of a significant effect of biological environment components on the parameters of their emission. Of the studied compounds, Ir1 and Ir2 were chosen for in vitro and in vivo biological experiments to estimate oxygen concentration in cell lines and tumors. These sensors have demonstrated their effectiveness for mapping the distribution of oxygen and for monitoring hypoxia in the biological objects studied.


Introduction
The development and application of transition metal phosphorescent complexes, such as non-invasive molecular oxygen sensors, is a topical area of research. The possibility of using such complexes in biomedical experiments is particularly important, since tracking changes in O 2 concentration is a fundamental issue in the studies of metabolic processes, as well as for diagnosing various pathologies and evaluating the efficiency of the therapy used [1][2][3][4]. The sensory response of phosphorescent complexes to the presence of molecular oxygen is based on the effective energy transfer from the excited triplet state of phosphors to the ground triplet state of O 2 molecules to give phosphorescence quenching, accompanied by a decrease in emission intensity and in the lifetime of the excited state [5][6][7].
In early studies, phosphorescence intensity was actively used as an analytical signal to determine oxygen concentration. In the ratiometric approach, the sensor response was quantified by comparison of the phosphorescence intensity with that of a certain oxygenindependent external or internal standard; fluorescence emission was commonly used as the latter. However, this approach makes analytical systems more complicated (at least two emitters have to be used in the measurements) and suffers from the dependence of the results on the optical properties of the samples under study, as well as the possible influence of different factors, other than oxygen concentration, on the luminescence of the reference emitter (pH, temperature, viscosity, etc.). The lifetime response of oxygen concentration variations is free from the above drawbacks, does not depend on the sensor concentration that makes phosphorescence lifetime measurements more reliable and results in a wide use of phosphoresce lifetime imaging (PLIM) in different analytical and biomedical applications, including oxygen sensing [7][8][9][10].
For the successful application of phosphorescent oxygen sensors in biology, they should be soluble in physiological (aqueous) media, demonstrate low toxicity and high stability and exhibit good photophysical characteristics (high quantum yield, sensitivity to the presence of molecular oxygen, emission and excitation in a required wavelength range). It is also worth noting that, in order to verify the practicable applicability of oxygen sensors in biomedical experiments, it is necessary to test them in various model biological media, since different factors, such as variations in pH values, temperature, salinity, viscosity, ions and the presence of biomacromolecules (primarily albumin, which has in its structure so-called "hydrophobic pockets"), can significantly affect the photophysical characteristics of the sensor.
Exploration of Ir(III) orthometallated complexes as oxygen sensors has a rather long and rich history, beginning with a key publication [39] demonstrating the applicability of this type of emitter for the detection of hypoxia in biomedical research. Later (see a mini-review [40] and recent review [41] for a complete list of references), in the works of Dr. S. Tobita et al. and several other research teams [36,[42][43][44][45][46][47], it has been demonstrated that Ir(III) molecular emitters can be successfully applied in in vitro and in vivo biomedical studies to qualitatively detect hypoxia in the tumor microenvironment [45,48] and to measure oxygen concentration in cell cultures by using a ratiometric approach [49] or timeresolved techniques, such as PLIM [50,51]. Moreover, in some cases, directed subcellular localization is possible; for example, in mitochondria [44,52], which makes it possible to track (especially with simultaneous FLIM imaging of the NADH and other coenzymes) metabolic processes and their variations with changes in nutritional conditions, oxygenation, etc. Significantly shorter values of the lifetimes of iridium complexes compared with the platinum and palladium porphyrins (by one or two orders of magnitude) allow for much faster signal accumulation in PLIM experiments, making it possible to increase the acquisition rate and/or real-time image resolution. Nevertheless, it must be mentioned that the iridium chromophores display lower brightness compared with the above-mentioned Pt and Pd porphyrins, which is determined not by their quantum yields (which are quite comparable), but by the lower extinction coefficient of these samples. Nevertheless, the versatility of the photophysical characteristics of iridium complexes achieved by variations in the donor properties of the ligands and simpler synthetic methods open the way for their further modification and vectorization, making Ir(III) complexes the compounds of choice for the preparation of oxygen sensors for particular biological studies.
As for many other phosphorescent transition metal complexes, their hydrophobicity and insolubility in aqueous media remain issues to be solved. One of the useful approaches consists of chromophore conjugation with synthetic [45] or natural [53,54] water-soluble polymers. Recently, we have designed and synthesized a number of oxygen sensors based on iridium complexes [52,[55][56][57][58][59][60][61][62][63][64][65], which exhibit good sensitivity to molecular oxygen. The chromophores in these compounds are shielded from interaction with the bio-environment by relatively short oligo(ethylene glycol) tails, which makes intracellular internalization possible and to simultaneously impart water solubility and increase the biocompatibility of these molecules.
In this article, we present the synthesis, characterization and photophysical study of three novel phosphorescent [Ir(NˆC) 2 (NˆN)]Cl complexes (Ir1-Ir3) containing the same NˆC metallated and different NˆN diimine ligands. The oxygen-sensing properties of the most effective emitters (Ir1 and Ir2) were investigated in model physiological media and in living cells, as well as through in vivo experiments in a mice tumor model by using time-resolved phosphorescent lifetime imaging.
Details of synthetic and separation procedures, photophysical experiments and computational studies, as well as data concerning the description of installations and methods for conducting biological experiments, are given in the Supplementary Materials.

Synthesis and Characterization
A derivative of pyridine-benzothiophene was chosen as a cyclometallating ligand, since it is known that iridium complexes based on it exhibit emissions in the red region of the spectrum [68][69][70], which can be useful for in vivo studies, since such luminescence will be within the transparency window of biological tissues (≥600 nm).
To impart water solubility, biocompatibility and low toxicity to the target compounds, as well as to protect them from nonspecific interactions with biomolecules, we introduced short-branched oligo(ethylene glycol) substituents into the structure of the cyclometallating and diimine ligands. The scheme of the corresponding modification and obtaining a new NˆC ligand is given below (Scheme 1), while the modification reactions of the corresponding NˆN ligands are described in the previous literature [52,60,62]. To impart water solubility, biocompatibility and low toxicity to the target compounds, as well as to protect them from nonspecific interactions with biomolecules, we introduced short-branched oligo(ethylene glycol) substituents into the structure of the cyclometallating and diimine ligands. The scheme of the corresponding modification and obtaining a new N^C ligand is given below (Scheme 1), while the modification reactions of the corresponding N^N ligands are described in the previous literature [52,60,62]. In the next stage of the synthesis (Scheme 2, upper part), upon the reaction of this cyclometallating ligand N^C and iridium(III) chloride, we isolated a new dimeric complex [Ir2(N^C)4Cl2], which we further used as a starting material in obtaining the target phosphorescent compounds, Ir1-Ir3. The synthesis of the target complexes (Scheme 2, bottom part) was carried out by replacing the labile chloride ligands in the [Ir2(N^C)4Cl2] dimer with diimine N^N chelates, with simultaneous dissociation of the dimer, to obtain the [Ir(N^C)2(N^N#)]Cl complexes in good preparative yields (59-95%), where the N^N# diimine ligands were also modified with oligo(ethylene glycol) residues. In the next stage of the synthesis (Scheme 2, upper part), upon the reaction of this cyclometallating ligand NˆC and iridium(III) chloride, we isolated a new dimeric complex [Ir 2 (NˆC) 4 Cl 2 ], which we further used as a starting material in obtaining the target phosphorescent compounds, Ir1-Ir3. The synthesis of the target complexes (Scheme 2, bottom part) was carried out by replacing the labile chloride ligands in the [Ir 2 (NˆC) 4 Cl 2 ] dimer with diimine NˆN chelates, with simultaneous dissociation of the dimer, to obtain the [Ir(NˆC) 2 (NˆN#)]Cl complexes in good preparative yields (59-95%), where the NˆN# diimine ligands were also modified with oligo(ethylene glycol) residues.
The compounds obtained (ligands, iridium(III) dimer and the target iridium complexes) were comprehensively characterized by NMR spectroscopy and mass spectrometry. It should be noted that, due to the presence of a large number of oligo(ethylene glycol) substituents in the structure of the target complexes, we failed to obtain these compounds either in the form of high-quality single crystals, suitable for X-ray diffraction analysis, or as a polycrystalline material, suitable for elemental analysis of the CHNS content. Nevertheless, the number, multiplicity, location and integral intensities of 1 H signals in 1D NMR spectra, as well as their cross-correlations in 2D 1 H-1 H COSY and NOESY NMR spectra, made it possible to reliably establish the structure and composition of these compounds (Figures S1-S17 in the Supplementary Materials). Additionally, these conclusions were confirmed by high-resolution ESI + mass spectrometry; see Figures S3-S18. In the obtained mass spectra, the main signals corresponded to the molecular ions of these complexes, both in pure form and with the addition of H + or Na + cations. The isotopic distribution patterns were also in excellent agreement with those calculated for these particles.
We also carried out quantum chemical calculations, which included optimization of the ground state structure (as an example, the optimized structure of the Ir1-0 complex is shown in Figure 1). Note that for the sake of simplicity in the optimization procedure, we used a model structural pattern, which differs from the structures of the Ir1-Ir3 compounds in that oligo(ethylene glycol) groups were replaced by methyl substituents to reduce the calculation time. It should be noted that such substitution is reasonable, since neither oligo(ethylene glycol) nor methyl substituents noticeably affect the central core structure and photophysical characteristics, being remote from the chromophoric center and having a similar nature from the viewpoint of donor-acceptor properties. The optimized structures for the Ir2 and Ir3 complexes are shown in Figures S22 and S23 in the Supplementary Materials. The obtained characteristics of the optimized structures (ligand disposition in the coordination octahedron, bond lengths and angles) are not exceptional and fit the structures of closely analogous complexes well, synthesized and characterized previously [68][69][70]. The obtained structural parameters of the optimized patterns are summarized in Tables S9-S11; see Supplementary Materials. It is also important to note that the optimized structural motifs of Ir1-Ir3 are in complete agreement with the data of proton NMR spectroscopy in terms of the ligand environment symmetry and intramolecular non-bonding proton contacts observed in the NOESY spectra, which additionally confirm the correctness of the complexes' composition and structure determination by this method. The compounds obtained (ligands, iridium(III) dimer and the target iridium complexes) were comprehensively characterized by NMR spectroscopy and mass spectrometry. It should be noted that, due to the presence of a large number of oligo(ethylene glycol) substituents in the structure of the target complexes, we failed to obtain these compounds either in the form of high-quality single crystals, suitable for X-ray diffraction analysis, or as a polycrystalline material, suitable for elemental analysis of the CHNS content. Nevertheless, the number, multiplicity, location and integral intensities of 1 H signals in 1D NMR spectra, as well as their cross-correlations in 2D 1 H-1 H COSY and NOESY NMR spectra, made it possible to reliably establish the structure and composition of these compounds (Figures S1-S17 in the Supplementary Materials). Additionally, these conclusions were confirmed by high-resolution ESI + mass spectrometry; see Figures S3-S18. In the obtained mass spectra, the main signals corresponded to the molecular ions of these complexes, both in pure form and with the addition of H + or Na + cations. The isotopic distribution patterns were also in excellent agreement with those calculated for these particles.

Photophysical Study
All compounds obtained exhibited luminescence in the red region of the visible spectrum. The absorption and emission spectra of Ir1-Ir3 are shown in Figure 2, and numerical spectroscopic data, together with emission quantum yields and lifetimes in aqueous aerated and deaerated solutions, are summarized in Table 1 and Table S1 ( previously [68][69][70]. The obtained structural parameters of the optimized patterns are sum-marized in Tables S9-S11; see Supplementary Materials. It is also important to note that the optimized structural motifs of Ir1-Ir3 are in complete agreement with the data of proton NMR spectroscopy in terms of the ligand environment symmetry and intramolecular non-bonding proton contacts observed in the NOESY spectra, which additionally confirm the correctness of the complexes' composition and structure determination by this method.

Photophysical Study
All compounds obtained exhibited luminescence in the red region of the visible spectrum. The absorption and emission spectra of Ir1-Ir3 are shown in Figure 2, and numerical spectroscopic data, together with emission quantum yields and lifetimes in aqueous aerated and deaerated solutions, are summarized in Tables 1 and S1 (see   The absorption spectra of the studied iridium complexes exhibit strong high-energy bands in the range 250-350 nm and low energy absorption at ca. 450 nm with the tail extending below 550 nm. DFT analysis of the absorption spectra (see Tables S3-S8) showed that the observed high-energy bands may be assigned to a combination of the transitions between the aromatic systems of the N^C and N^N ligands with some admixture of the metal to ligand charge transfers ( 1 MLCT). The low-energy bands at ca 450 nm may be associated with intraligand and ligand-to-ligand charge transfer ( 1 LLCT and 1 ILCT) localized at two N^C ligands with a minor admixture of 1 MLCT transitions to N^C to the N^C ligands. The lowest calculated S0 ⟶ S1 transition, which has a very low oscillator strength, is located well below 500 nm and is associated with electron density transfer to the N^N ligand from N^C ligands and iridium ion.
Ir1-Ir3 demonstrated luminesce in an aqueous solution in the red region of the visible spectrum, showing slightly structured emission bands with the maxima at 632, 638 and 655 nm, respectively; see Figure 2 and Table 1. These complexes displayed large Stokes shifts of ca. 17-200 nm, lifetimes in the microsecond domain and strong sensitivity to the presence of molecular oxygen, is indicative of the triplet nature of the emissive excited state, i.e., phosphorescence. Ir1 and Ir2 exhibit rather intense luminescence with the The absorption spectra of the studied iridium complexes exhibit strong high-energy bands in the range 250-350 nm and low energy absorption at ca. 450 nm with the tail extending below 550 nm. DFT analysis of the absorption spectra (see Tables S3-S8) showed that the observed high-energy bands may be assigned to a combination of the transitions between the aromatic systems of the NˆC and NˆN ligands with some admixture of the metal to ligand charge transfers ( 1 MLCT). The low-energy bands at ca 450 nm may be associated with intraligand and ligand-to-ligand charge transfer ( 1 LLCT and 1 ILCT) localized at two NˆC ligands with a minor admixture of 1 MLCT transitions to NˆC to the NˆC ligands. The lowest calculated S 0 → S 1 transition, which has a very low oscillator strength, is located well below 500 nm and is associated with electron density transfer to the NˆN ligand from NˆC ligands and iridium ion. Ir1-Ir3 demonstrated luminesce in an aqueous solution in the red region of the visible spectrum, showing slightly structured emission bands with the maxima at 632, 638 and 655 nm, respectively; see Figure 2 and Table 1. These complexes displayed large Stokes shifts of ca. 17-200 nm, lifetimes in the microsecond domain and strong sensitivity to the presence of molecular oxygen, is indicative of the triplet nature of the emissive excited state, i.e., phosphorescence. Ir1 and Ir2 exhibit rather intense luminescence with the quantum yields 17.3% and 8.5%, respectively, in a deaerated aqueous solution. On the contrary, Ir3 was a very weak emitter with an emission intensity two orders of magnitude lower compared to its congeners, which makes its application as an oxygen sensor in biological experiments impossible.
The DFT and TD-DFT calculations gave the emission wavelengths, which were in very good agreement with the experimental data (Table S2). Analysis of the nature of emissive transitions (T 1 → S 0 ) for the studied complexes showed that the character of Ir3 emission is essentially different from those revealed for Ir1 and Ir2; see Figure 3 and interfragment charge transfer Tables S4, S6 and S8. In the complexes Ir1 and Ir2, the relaxation processes occur through the 3 ILCT and 3 MLCT transitions associated with the NˆC cyclometallating ligand. For Ir3, the excited-state relaxation is mainly associated with the diimine NˆN ligand, and, as a consequence, 3 MLCT (NˆN → Ir) and 3 LLCT (NˆN → NˆC) transitions are observed. Such a significant difference in the nature of the phosphorescence processes is most probably responsible for the strong difference in emission quantum yield (of the order of 0.1% in deaerated water) of Ir3 as compared to Ir1 and Ir2. One of the possible explanations of this observation may be the higher contribution of rotational non-radiative channels to excited-state relaxation for Ir3 due to the presence of two {-C(O)NHR} substituents at the diimine ligand compared to only one substituent at the NˆC ligands in Ir1 and Ir2.
transitions are observed. Such a significant difference in the nature of the phosphorescence processes is most probably responsible for the strong difference in emission quantum yield (of the order of 0.1% in deaerated water) of Ir3 as compared to Ir1 and Ir2. One of the possible explanations of this observation may be the higher contribution of rotational non-radiative channels to excited-state relaxation for Ir3 due to the presence of two {-C(O)NHR} substituents at the diimine ligand compared to only one substituent at the N^C ligands in Ir1 and Ir2.

Ir1-0
Ir2-0 Ir3-0  The noticeably higher phosphorescence quantum yields of Ir1 and Ir2, as well as their high sensitivity to the variations in molecular oxygen concentration in solution, led us to choose these compounds for the further studies of their sensor properties and their applicability as luminescent oxygen probes in biosystems. To calibrate the dependence of Ir1 and Ir2 excited state lifetimes on oxygen concentration, we carried out the measurements in water and in model biological solutions containing typical components of intracellular media: PBS buffer with the addition of bovine serum albumin-BSA and DMEM with the addition of 10% fetal bovine serum-FBS; see Table 1 and Figure 4. The experiments in the model solutions make it possible to reveal the effect of salinity, pH and the presence of the main protein component of plasma (albumin) on the sensors' characteristics. The obtained data (Table S1) indicate lifetime independence on pH and media salinity. Aqueous solutions were studied for comparison. The noticeably higher phosphorescence quantum yields of Ir1 and Ir2, as well as their high sensitivity to the variations in molecular oxygen concentration in solution, led us to choose these compounds for the further studies of their sensor properties and their applicability as luminescent oxygen probes in biosystems. To calibrate the dependence of Ir1 and Ir2 excited state lifetimes on oxygen concentration, we carried out the measurements in water and in model biological solutions containing typical components of intracellular media: PBS buffer with the addition of bovine serum albumin-BSA and DMEM with the addition of 10% fetal bovine serum-FBS; see Table 1 and Figure 4. The experiments in the model solutions make it possible to reveal the effect of salinity, pH and the presence of the main protein component of plasma (albumin) on the sensors' characteristics. The obtained data (Table S1) indicate lifetime independence on pH and media salinity. Aqueous solutions were studied for comparison.  The obtained calibration curves ( Figure 4) indicated that the growth (DMEM + 10 % FBS) and model biological media (PBS with 70 mM BSA) had an almost identical effect on the behavior of these sensors. The slope of the Stern-Volmer curves in these media for both complexes coincided under the limits of experimental uncertainty and obviously differed from that in pure water. This observation can be explained by an increase in solution viscosity in the presence of biological macromolecules that reduces the rate of oxygen diffusion in the sample under study and causes a drop in the Stern-Volmer quenching constant. It is also impossible to exclude a reversible non-covalent interaction of the chromophores with hydrophobic pockets of albumin, which may shield the complexes from collisions with oxygen molecules that increase the observed excited state lifetime. The obtained data indicate that the oligo(ethylene glycol) moieties are unable to completely shield the chromophore from the effects of the bio-environment, which in turn implies the diffusion in the sample under study and causes a drop in the Stern-Volmer quenching constant. It is also impossible to exclude a reversible non-covalent interaction of the chromophores with hydrophobic pockets of albumin, which may shield the complexes from collisions with oxygen molecules that increase the observed excited state lifetime. The obtained data indicate that the oligo(ethylene glycol) moieties are unable to completely shield the chromophore from the effects of the bio-environment, which in turn implies the use of calibration curves obtained in model physiological media for the estimation of oxygen concentration in biological samples. It is worth noting that Ir1 and Ir2 demonstrated high dark stability and low photobleaching rate under irradiation with 355 and 365 nm.
Thus, the obtained data clearly indicate that the phosphorescent complexes Ir1 and Ir2 are suitable for application in functional bioimaging as promising oxygen sensors, since they exhibit appreciable quantum yields, high sensitivity to the presence of molecular oxygen and good solubility and stability in aqueous solutions, including model biological media.

Biological Experiments
Using the MTT assay, it was found that all the compounds tested were non-toxic for cultured cancer cells CT26 (murine colorectal carcinoma cell line) and HCT116 (human colorectal carcinoma cell line) in the concentration range studied. Upon incubation with the complexes for 24 h, more than 90% of tumor cells remained viable at concentrations of 150 µM and below ( Figure 5 and Figure S24 in Supplementary Materials).

Biological Experiments
Using the MTT assay, it was found that all the compounds tested were non-toxic for cultured cancer cells CT26 (murine colorectal carcinoma cell line) and HCT116 (human colorectal carcinoma cell line) in the concentration range studied. Upon incubation with the complexes for 24 h, more than 90% of tumor cells remained viable at concentrations of 150 μM and below ( Figures 5 and S24 in Supplementary Materials). Next, the ability of Ir1, Ir2 and Ir3 to accumulate in living cancer cells was investigated. As mentioned above, Ir3 featured a very low-emission quantum yield that gave an extremely weak luminescence signal inside cells, thus making this complex unsuitable for further biological testing. Using laser scanning microscopy, it was shown that Ir1 and Ir2 successfully penetrated into cultured cancer cells ( Figure 6); the phosphorescence intensity of both complexes increased stepwise in the time period from 1 to 24 h of incubation. Complex Ir2 displayed more intense luminescence compared to Ir1 under normoxic conditions, due to a higher extinction coefficient at 405 nm (excitation wavelength in cell internalization experiments). Inside the cells, the complexes were distributed heterogeneously as granules in the cytoplasm and also as a homogenous fraction in the cytosol. Next, the ability of Ir1, Ir2 and Ir3 to accumulate in living cancer cells was investigated. As mentioned above, Ir3 featured a very low-emission quantum yield that gave an extremely weak luminescence signal inside cells, thus making this complex unsuitable for further biological testing. Using laser scanning microscopy, it was shown that Ir1 and Ir2 successfully penetrated into cultured cancer cells ( Figure 6); the phosphorescence intensity of both complexes increased stepwise in the time period from 1 to 24 h of incubation. Complex Ir2 displayed more intense luminescence compared to Ir1 under normoxic conditions, due to a higher extinction coefficient at 405 nm (excitation wavelength in cell internalization experiments). Inside the cells, the complexes were distributed heterogeneously as granules in the cytoplasm and also as a homogenous fraction in the cytosol. After 3 h of incubation, the subcellular localization of Ir1 and Ir2 was analyzed using co-staining with organelle-specific dyes (Figure 7). It was found that both complexes colocalized moderately with lysosomes (M1 = 0.584 for Ir1 and M1 = 0.769 for Ir2) and almost did not colocalize with mitochondria (M1 = 0.236 for Ir1, M1 = 0.316 for Ir2).  After 3 h of incubation, the subcellular localization of Ir1 and Ir2 was analyzed using co-staining with organelle-specific dyes (Figure 7). It was found that both complexes colocalized moderately with lysosomes (M1 = 0.584 for Ir1 and M1 = 0.769 for Ir2) and almost did not colocalize with mitochondria (M1 = 0.236 for Ir1, M1 = 0.316 for Ir2). After 3 h of incubation, the subcellular localization of Ir1 and Ir2 was analyzed using co-staining with organelle-specific dyes (Figure 7). It was found that both complexes colocalized moderately with lysosomes (M1 = 0.584 for Ir1 and M1 = 0.769 for Ir2) and almost did not colocalize with mitochondria (M1 = 0.236 for Ir1, M1 = 0.316 for Ir2).  In order to assess the applicability of the Ir1 and Ir2 sensors for determination of the oxygen concentration inside cells, we conducted PLIM experiments, both under conditions of normal oxygenation and hypoxia simulation. In the course of the imaging experiments, we did not observe emission intensity degradation that is indicative of the sensors' stability in physiological media. These experiments demonstrated that upon modeling hypoxia, the phosphorescence lifetime of the Ir1 and Ir2 complexes increased by approximately two times-from 3.8 µs and 3.5 µs to 8.1 µs and 6.3 µs, respectively, indicating the high sensitivity of both complexes localized in cell cytoplasm to variations of oxygen content (Figure 8). It also should be noted that the difference between normoxic and hypoxic phosphorescence lifetimes were slightly more pronounced for Ir1 (4.3 µs) than for Ir2 (2.8 µs), and therefore the Ir1 complex was used for further in vivo testing on mouse tumor models.
Biosensors 2023, 13, x FOR PEER REVIEW 12 of 18 In order to assess the applicability of the Ir1 and Ir2 sensors for determination of the oxygen concentration inside cells, we conducted PLIM experiments, both under conditions of normal oxygenation and hypoxia simulation. In the course of the imaging experiments, we did not observe emission intensity degradation that is indicative of the sensors' stability in physiological media. These experiments demonstrated that upon modeling hypoxia, the phosphorescence lifetime of the Ir1 and Ir2 complexes increased by approximately two times-from 3.8 μs and 3.5 μs to 8.1 μs and 6.3 μs, respectively, indicating the high sensitivity of both complexes localized in cell cytoplasm to variations of oxygen content (Figure 8). It also should be noted that the difference between normoxic and hypoxic phosphorescence lifetimes were slightly more pronounced for Ir1 (4.3 μs) than for Ir2 (2.8 μs), and therefore the Ir1 complex was used for further in vivo testing on mouse tumor models. After 30 min of local administration of Ir1 into tumors in vivo at the concentration of 250 μM, the phosphorescence signal was detected primarily in the cytoplasm of tumor cells. Inside the cells, the complex was distributed more homogeneously in comparison with cultured cells in vitro. We also checked the intensity of phosphorescence after 3 h and 6h of local sensor administration ( Figure S26 in Supplementary Materials) and did not observe significant changes. Therefore, for greater convenience and reliability of the experiment, we used the smallest incubation interval-30 min.
In CT26 tumor cells in vivo, the phosphorescence lifetime of Ir1 was 7.4 ± 0.8 μs on average (Figure 9). In HCT116 tumor xenografts, the phosphorescence lifetime of Ir1 was 8.6 ± 0.5 μs, indicating their more hypoxic status ( Figure S25 in Supplementary Materials). After 30 min of local administration of Ir1 into tumors in vivo at the concentration of 250 µM, the phosphorescence signal was detected primarily in the cytoplasm of tumor cells. Inside the cells, the complex was distributed more homogeneously in comparison with cultured cells in vitro. We also checked the intensity of phosphorescence after 3 h and 6 h of local sensor administration ( Figure S26 in Supplementary Materials) and did not observe significant changes. Therefore, for greater convenience and reliability of the experiment, we used the smallest incubation interval-30 min.
In CT26 tumor cells in vivo, the phosphorescence lifetime of Ir1 was 7.4 ± 0.8 µs on average (Figure 9). In HCT116 tumor xenografts, the phosphorescence lifetime of Ir1 was 8.6 ± 0.5 µs, indicating their more hypoxic status ( Figure S25 in Supplementary Materials). However, within each CT26 tumor, a high heterogeneity of phosphorescence lifetime, and consequently oxygen distribution, was observed at the cellular level. In the same tumors, the phosphorescence lifetimes varied from ~6.8 μs to ~9.7 μs, which, in general, corresponded with the values of the phosphorescence lifetimes typical of different degrees of hypoxia and the lifetime data measured for this complex in cuvette experiments (Table 1).
It is important to note that the dose of the complex used did not induce any acute toxic effect on mice and did not change the typical histological structure of the tumor tissue, thus proving the potential biocompatibility of these phosphorescent O2 probes. Therefore, the conducted in vitro and in vivo experiments showed the high potential of the new Ir1 complex for assessing tissue oxygenation using PLIM.

Conclusions
We have obtained and comprehensively characterized three new target iridium complexes, [Ir(N^C)2(N^N#)]Cl, with various diimine N^N ligands in their composition. These complexes were decorated with short-branched oligo(ethylene glycol) groups to give them water solubility, biocompatibility and low toxicity.
These compounds exhibited oxygen-dependent phosphorescence. The study of their photophysical properties made it possible to determine the two most promising sensors for further biological testing. The quantum yields of these complexes were moderate and reached 17% in deaerated water. The emission wavelengths were in the transparency window of biological tissues.
Biological studies showed that these compounds have low toxicity. Cellular in vitro experiments proved that these sensors exhibit a significant lifetime response to changes in the oxygen concentration in the sample. Simulation of hypoxia in cells led to a two-and threefold increase of these values. The effectiveness of these sensors allowed their application in in vivo experiments on living mice tumor models. In these experiments, the sensors also made it possible to record the presence of significant hypoxia in tumors, as well as their heterogeneity. However, within each CT26 tumor, a high heterogeneity of phosphorescence lifetime, and consequently oxygen distribution, was observed at the cellular level. In the same tumors, the phosphorescence lifetimes varied from~6.8 µs to~9.7 µs, which, in general, corresponded with the values of the phosphorescence lifetimes typical of different degrees of hypoxia and the lifetime data measured for this complex in cuvette experiments (Table 1).
It is important to note that the dose of the complex used did not induce any acute toxic effect on mice and did not change the typical histological structure of the tumor tissue, thus proving the potential biocompatibility of these phosphorescent O 2 probes. Therefore, the conducted in vitro and in vivo experiments showed the high potential of the new Ir1 complex for assessing tissue oxygenation using PLIM.

Conclusions
We have obtained and comprehensively characterized three new target iridium complexes, [Ir(NˆC) 2 (NˆN#)]Cl, with various diimine NˆN ligands in their composition. These complexes were decorated with short-branched oligo(ethylene glycol) groups to give them water solubility, biocompatibility and low toxicity.
These compounds exhibited oxygen-dependent phosphorescence. The study of their photophysical properties made it possible to determine the two most promising sensors for further biological testing. The quantum yields of these complexes were moderate and reached 17% in deaerated water. The emission wavelengths were in the transparency window of biological tissues.
Biological studies showed that these compounds have low toxicity. Cellular in vitro experiments proved that these sensors exhibit a significant lifetime response to changes in the oxygen concentration in the sample. Simulation of hypoxia in cells led to a twoand threefold increase of these values. The effectiveness of these sensors allowed their application in in vivo experiments on living mice tumor models. In these experiments, the sensors also made it possible to record the presence of significant hypoxia in tumors, as well as their heterogeneity.
Thus, we have obtained and studied very promising new molecular oxygen sensors based on low-toxicity biocompatible phosphorescent iridium complexes.