Over the past two decades, luminescent cyclometalated iridium(III) complexes have attracted great attention as bioimaging agents and oxygen sensors in biological objects [1–5]. Currently, a wide number of iridium-containing luminophores of both neutral and ionic structures are known [6–13]. The photophysical properties of neutral complexes are determined mainly by the nature of cyclometalating ligands bound with iridium. The variation of such ligands makes it possible to regulate the color of luminescence and luminescence efficiency of iridium luminophores [6–9]. The nature of diimine ligands, incorporated into cationic complexes, also has a noticeable effect on the photophysical characteristics of iridium emitters [10–13].

Iridium complexes emitting in the red and near-infrared spectral regions are of special interest, since such radiation penetrates most deeply into biological tissues. Red light-emitting iridium luminophores have been successfully used as luminescent markers and oxygen sensors in various biological objects [5, 14, 15]. One of the main requirements for iridium biomarkers and sensors is a sufficiently high solubility in water. Two approaches were used to increase the solubility of luminescent iridium complexes. The first is the functionalization of cyclometalating and ancillary ligands in iridium complexes by hydrophilic groups [16–20]. According to the second approach, luminescent iridium complexes are embedded in a water-soluble polymer matrix [21–24]. Water-soluble polymeric iridium luminophores synthesized using the second approach are the most attractive, since such polymeric biomarkers, in addition to high solubility in water and efficient luminescent characteristics, reveal the ability to accumulate in tumors due to the so-called enhanced permeability and retention (EPR) effect [25–27]. In the course of recent studies, we synthesized functionalized luminescent iridium-containing polynorbornenes and demonstrated the possibility of their use as phosphorescent sensors of molecular oxygen in living tumor cells and tissues [28, 29].

This paper reports the synthesis of a new norbornene-substituted iridium(III) cationic complex and the preparation of water-soluble polymers based on it by the ROMP method. The resulting polymers exhibit intense oxygen-dependent phosphorescence in the orange region of the spectrum and show a high cytotoxicity with respect to human colorectal cancer cells.

To obtain the target iridium-containing polymers, oxanorbornene monomers 1 and 2 with oligoether and amino acid groups, and also norbornene monomer 3 containing the iridium(III) cationic complex were used (Scheme 1).

Scheme
scheme 1

1.

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Well-known organic monomers 1 and 2 were obtained by published methods [30, 31]. New iridium-containing monomer 3 was synthesized in accordance with Scheme 2.

Scheme
scheme 2

2.

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Monomer 3 was isolated as an air-stable solid substance of red color, soluble in THF, CH2Cl2, and CHCl3 and insoluble in hexane. According to 1H NMR data, the compound is a mixture of endo- and exo-isomers in a ratio of 85:15.

In further studies, it was found that iridium-containing monomer 3 enters into a copolymerization metathesis reaction with organic monomers 1 and 2 to form luminescent water-soluble polymers P1 and P2 (Scheme 3).

Scheme
scheme 3

3.

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The ROMP reactions involving monomers 1-3 occur in the presence of a third-generation Grubbs catalyst (monomers : catalyst = 100 : 1). Polymer products have been isolated with high yields in the form of brown gummy substances stable in air, soluble in THF, CH2Cl2, CHCl3, and H2O and insoluble in hexane. Compounds P1 and P2 were identified by elemental analysis, IR and NMR spectroscopy, and also GPC. Polymers P1 and P2 have medium molecular weights (Mw 26000-28100 Da) and a narrow molecular weight distribution (Mw/Mn = 1.21‒1.36, Fig. S1, see Supplementary materials).

The dynamic light scattering method revealed that in aqueous solutions at a concentration of 0.1– 0.2 g/L, polymer compounds form nanoparticles with average sizes of 21 (P1) and 15 nm (P2). The particle size distribution is shown in Fig. S2 (see Supplementary materials). Polymer particles, apparently, are micelles with the shell consisting of oligoether and amino acid groups, and with the core including side chains containing iridium complexes.

The absorption spectrum of iridium-containing monomer 3 (Fig. S3a, see Supplementary materials) contains intense bands in the region of 250–360 nm related to intra-ligand 1(π→π*) transitions in 1-phenylisoquinoline and phenylimidazophenanthroline ligands (Table 1) [32, 33]. Bands of lower intensity in the region of 400‒500 nm refer to metal-to-ligand charge transfer transitions (MLCT) mixed with ligand-ligand charge transfer (LLCT) [32, 33]. The absorption spectra of polymers P1 and P2 (Fig. 1 and Table 1) contain bands characteristic of iridium complexes associated with a polymer chain.

Fig. 1.
figure 1

Absorption spectra of polymers P1 and P2 in (a) methylene chloride and (b) water.

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Table 1. Photophysical characteristics of monomer 3 and polymers P1, P2
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The photoluminescence spectra of monomer 3 (Fig. S3b, see Additional Materials) and polymers P1 and P2 (Fig. 2) show wide bands with maxima at 587 and 626 nm (shoulder), related to 3MLCT/3LLCT transitions (Table 1) [32, 33]. The photoluminescence chromaticity coordinates of monomer 3 and polymers P1 and P2 in the CIE diagram (Commision Internationalede l’Eclairage) (Table 1) match the orange color.

Fig. 2.
figure 2

Photoluminescence spectra of polymers P1 and P2 in (a, b) methylene chloride solution and (c, d) aqueous solution at room temperature, λex 360 nm. (1) Degassed solution, (2) aerated solution.

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The photoluminescence intensity and quantum yields of monomer 3 and polymers P1 and P2 in aerated solutions are significantly lower compared to degassed solutions (Table 1, Fig. 2, S3b, see Supplementary materials). The reason for this is that oxygen is an active quencher of phosphorescence of cyclometallic iridium(III) complexes [34], and, as a result, the photoluminescence intensity and quantum yields of iridium-containing polymers in aerated solutions are significantly reduced.

The phosphorescence lifetimes of iridium-containing polymers P1 and P2, measured by phosphorescence lifetime imaging method (PLIM) in aerated aqueous solutions (2.2 µs) are also significantly shorter compared to degassed solutions (3.2 µs). Phosphorescent images of solutions of polymers P1 and P2 and phosphorescence decay curves are presented in Fig. 3.

Fig. 3.
figure 3

Phosphorescence of polymers P1 and P2 in aerated and degassed aqueous solutions: (a) macro-PLIM images of polymer solutions in tubes under excitation at a wavelength of 375 nm; (b) Phosphorescence decay curves in degassed (green curve) and aerated (red curve) solutions. Monoexponential decay. Image size 4.5×4.5 mm.

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It is known that a number of luminescent cyclometalated iridium(III) complexes are toxic to tumor cells, which makes it possible to consider them not only as bioimaging agents, but also as potential antitumor agents [35]. In this regard, it seemed reasonable to determine the degree of cytotoxicity of polymers P1 and P2 on the example of human colorectal cancer cells HCT116. Using the MTT test, it was found that the synthesized polymers exhibit high cytotoxicity. The values of semi-inhibitory IC50 concentrations were 10 (P1) and 0.5 µmol (P2) (Fig. 4). It should be noted that the cytotoxicity of polymers P1 and P2 is comparable to the cytotoxicity of widely used drugs for cancer chemotherapy, such as cisplatin/oxaliplatin and 5-fluorouracil [36, 37].

Fig. 4.
figure 4

Analysis of the effect of P1 and P2 polymers on the viability of cancer cells CT26 by the MTT test. Mean ± standard deviation: (*, #) statistically significant difference from a control, p ≤ 0.05, n = 3.

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Thus, a new norbornene-substituted cyclometalated iridium(III) cationic complex was synthesized and water-soluble polymers P1 and P2 exhibiting oxygen-dependent orange phosphorescence were obtained using the ROMP method. By the MTT test method, it was found that polymer products have high cytotoxicity with respect to HCT116 cancer cells. The physicochemical and biological properties of the obtained polymers allow us to consider them as potential oxygen sensors and antitumor agents.

EXPERIMENTAL

All operations with easily oxidized and hydrolyzed substances were carried out in vacuum or in argon using the standard Schlenk technique. The dimeric iridium chloride [Ir2(piq)4Cl2] [38], norbornene substituted phenylimidazophenanthroline NBE(CH2)5phphen [39], 7-oxanorbornene monomers with oligoether groups (1) [30] amino acid fragments (2) [31], and (H2IMes)(3-Br-py)2(Cl)2Ru=CHPh (Grubbs catalyst of the III generation) [40, 41] were synthesized as described in the literature.

Elemental C,H,N analysis was performed on an Elementar Vario EL cube automatic elemental analyzer. 1H and 13C NMR spectra were recorded on a Bruker Avance Neo 300 spectrometer. Chemical shifts are indicated relative to the signal of the residual protons of the deuterated solvent. IR spectra were recorded on an FSM 1201 IR Fourier spectrometer. Samples of compounds were prepared in the form of thin films between KBr plates.

The molecular weight distribution of polymers was determined by gel permeation chromatography (GPC) on a Knauer chromatograph with a Smartline RID 2300 differential refractometer as a detector, and a set of two Phenomenex columns with Phenogel sorbent with a pore size of 104 and 105 Å (eluent—THF, 2 mL/min, 40°C, sample volume 20 µL, sample concentration 10 mg/mL). The columns were calibrated using 13 polystyrene standards. The sizes of polymer particles in aqueous solutions were determined by dynamic light scattering on a Brookhaven NanoBrook Omni device.

The electronic absorption spectra of polymers in CH2Cl2 and H2O solutions were taken on a Perkin Elmer Lambda 25 spectrometer. Photoluminescence spectra were recorded on a Perkin Elmer LS55 fluorescence spectrometer. The photoluminescence quantum yields of iridium-containing monomer 3 in a CH2Cl2 solution and of polymeric products P1 and P2 in CH2Cl2 and H2O solutions were determined at room temperature, the excitation wavelength 360 nm. The values of quantum yields were calculated relative to Rhodamine B in ethanol (Φf 0.70) [42] according to the method described in [43].

The sensitivity of phosphorescent polymers P1 and P2 to oxygen in solutions was estimated using a two-channel FLIM/PLIM confocal macroscanner (Becker&Hickl, Germany) [44]. Sealed ampoules with aerated (21% O2) and degassed (0% O2) aqueous solutions of the studied phosphorescent polymers P1 and P2 were placed on a macro scanner slide table. The complexes were excited at a wavelength of 375 nm in a single-photon mode using a BDL-375-SMC picosecond laser (Becker&Hickl, Germany) with a power of 8 mW. The signal accumulation time was 60 s. The resulting images were processed in the SPCImage 8.5 program (Becker&Hickl, Germany). The phosphorescence decay curves were approximated by a one-exponential function with an acceptable χ2 (from 0.8 to 1.2). The lifetime of phosphorescence was calculated using the maximum likelihood (MLE) method.

The cytotoxicity of the studied polymers P1 and P2 was estimated on the library cell culture HCT116 (human colorectal cancer) by the MTT test. The cells were cultured in a full growth medium DMEM (Dulbecco’s Modified Eagle’s Medium) with the addition of L-glutamine and 10% fetal bovine serum at 37°C, 5%CO2. To estimate cytotoxicity, HCT116 cells were seeded on a 96-well plate in the amount of 1×104 cells per well. After 24 h, after cell adhesion, the DMEM nutrient medium was replaced with solutions of the studied polymers P1 and P2 in concentrations: 0.5, 1.0, 2.5, 5.0, 10.0, 20.0, and 50.0 mmol in 3 repetitions of 10 wells for each concentration, and incubated for 24 h. The MTT reagent (0.5 mg/mL) (Alfa Aesar, USA) was added to the culture medium and incubated for 4 h. Optical density was measured spectrophotometrically on a multimodal tablet reader (SynergyTM Mx, BioTek® Instruments, Inc.) with absorption at wavelengths of 570 and 630 nm, then the amount of living cells in the presence of the complex was calculated as a percentage of the control. Cells without the addition of polymers were used as a control.

Iridium-containing monomer 3. To a suspension of [Ir2CI2(piq)4] (0.0962 g, 0.076 mmol) in a mixture of methanol (4 mL) and dichloromethane (8 mL), NBE(CH2)5phph (0.0684 g, 0.149 mmol) was added, and the reaction mixture were boiled for 8 h. The reaction solution was cooled to room temperature, NH4PF6 (0.12 g, 0.74 mmol) was added, the solution was stirred for 2 h at room temperature, then separated from the precipitate by centrifugation. The solvent was removed in vacuum, the solid residue was purified by chromatography on silica gel (CH2Cl2–MeOH, 10 : 1 vol). After removing the solvent, the residue was dried for 1 h in vacuum at 50°C. Monomer 3 (0.0885 g, 49%) was obtained in the form of an air-stable solid of red color. IR spectrum, ν, cm–1: 3041w, 2957 v.s, 2934 v.s, 2852 v.s, 1739 w, 1598 md, 1461 s, 1380 s, 1260 md, 1156 md, 1040 md, 843 s, 731 md, and 559 md. 1Н NMR spectrum (CDCl3), δ, ppm (J, Hz): 0.30–0.42 m (1Н, Alk), 0.58–1.00 m (5Н, Alk), 1.08–1.16 g (5Н, Alk), 1.86–1.96 m (2Н, Alk), 2.58–2.70 m (2Н, Alk), 4.64–4.90 m (2Н, Alk), 5.76–5.81 m (0.85Н, =CH endo), 5.86–5.97 m (0.30Н, =CH exo), 6.01–6.06 m (0.85Н, =CH endo), 6.41 d (2Н, Ar, J 7.6), 6.93–7.01 m (2Н, Ar), 7.14–7.18 m (3Н, Ar), 7.29 s (2Н, Ar), 7.57–7.63 m (3Н, Ar), 7.68–7.80 m (8Н, Ar), 7.81–7.88 m (2Н, Ar), 7.96–8.03 m (1Н, Ar), 8.07 t (2Н, Ar, J 5.2), 8.32 d (2Н, Ar, J 8.2), 8.97 t (2Н, Ar, J 9.5), 9.06 d (1Н, Ar, J 8.4), and 9.25 d (1Н, Ar, J 8.2). 13C NMR spectrum (CDCl3), δC, ppm: 26.3, 27.8, 29.7, 30.1, 30.9, 32.3, 34.4, 38.5, 42.5, 45.3, 46.8, 49.5, 121.6, 122.6, 126.4, 126.8, 127.3, 127.4, 127.7, 128.6, 128.8, 129.1, 129.4, 129.9, 131.2, 132.3, 132.5, 132.6, 136.9, 137.1, 139.9, 140.6, 141.4, 145.4, 145.8, 148.8, and 153.3. Found, %: C 60.68; Н 4.35; N 6.57. C61H50F6IrN6P. Calculated, %: C 60.84; Н 4.18; N 6.98.

Polymer P1. To the solution of monomer 1 (0.1023 g, 0.2146 mmol) and monomer 3 (0.0065 g, 0.0054 mmol) in 1 mL of THF, a solution of the Grubbs catalyst of the III generation (0.0019 g, 0.0022 mmol) in 1 mL of THF was added. The mixture was stirred at 40°C. The course of polymerization was controlled by thin-layer chromatography. The polymerization time was 10 h. After the reaction was completed, several drops of ethylvinyl ether were added to the resulting mixture to decompose the catalyst, and the mixture was additionally stirred for 20 min at room temperature. The resulting polymer was precipitated with hexane (30 mL) and dried in vacuum at 40°C to a constant weight. The yield is 0.1034 g (95%), a brown gummy substance. IR spectrum, ν, cm–1: 2920 v.s, 2882 v.s, 2825 s, 1741 v.s, 1453 md, 1384 md, 1355 md, 1287 md, 1200 s, 1109 v.s, 1032 md, 847 md, and 762 v.s. 1H NMR spectrum (CDCl3), δ, ppm: 1.22–1.28 m (1H, Alk), 1.82–1.88 m (7H, Alk), 2.20–2.40 m (3H, Alk), 3.00–3.20 m (82H, Alk), 3.48–3.56 m (160H, Alk), 4.10–4.30 m (160H, Alk), 3.30–3.40 m (242H, Alk), 3.58–3.70 m (640H, Alk), 4.55–4.75 m (40H, Alk), 4.90–5.20 m (42H, Alk), 5.35–5.65 m (42H, =CH), 5.75– 5.95 m (40H, =CH), 6.30–6.45 (2H, Ar), 6.80–7.00 m (4H, Ar), 7.10–7.20 m (8H, Ar), 7.55–7.70 (3H, Ar), 7.70–7.80 m (6H, Ar), 7.90–8.15 m (2H, Ar), 8.20– 8.40 m (2H, Ar), and 8.90–9.10 m (2H, Ar). Found, %: C 55.45; H 7.40; N 0.43. C941H1490F6IrN6O440P. Calculated, %: C 55.76; H 7.43; N 0.41. Mw 28100, Mn 20600, Mw/Mn = 1.36.

Polymer P2 was obtained similarly from monomer 1 (0.1121 g, 0.2352 mmol), monomer 2 (0.0343 g, 0.0782 mmol), monomer 3 (0.0093 g, 0.0077 mmol) in 2 mL of THF, and a solution of the Grubbs catalyst of the III generation (0.0027 g, 0.0031mmol) in 1 mL of THF. The yield is 0.1448 g (93%), a brown gummy substance. IR spectrum, ν, cm–1: 3324 m, 2952 v.s, 2877 v.s, 2821 s, 1744 v.s, 1683 md, 1538 md, 1451 md, 1352 md, 1285 s, 1250 s, 1202 v.s, 1109 v.s, 1032 s, 980 md, 845 md, and 746 v.w. 1Н NMR spectrum (CDCl3), δ, ppm: 0.80–0.98 (120H, Alk), 1.18–1.28 (6H, Alk), 1.52–1.80 (67H, Alk), 3.00–3.20 m (84H, Alk), 3.30–3.40 m (200H, Alk), 3.48–3.56 m (120H, Alk), 3.58–3.75 m (540H, Alk), 4.10– 4.30 m (140H, Alk), 4.55–4.80 m (40H, Alk), 4.90– 5.20 m (42H, Alk), 5.40–5.70 m (42H, =CH), 5.75– 5.95 m (40H, =CH), 6.30–6.45 (2H, Ar), 6.80–7.00 m (4H, Ar), 7.10–7.20 m (8H, Ar), 7.55–7.65 (3H, Ar), 7.70–7.85 m (6H, Ar), 7.95–8.15 m (2H, Ar), 8.20– 8.40 m (2H, Ar), and 8.90–9.10 m (2H, Ar). Found, %: C 56.50; Н 7.36; N 1.78. C941H1470F6IrN26O400P. Calculated, %: C 56.83; Н 7.47; N 1.83. Mw 26000, Mn 21400, Mw/Mn = 1.21.