Luminescent ruthenium(II) complex bearing bipyridine and N-heterocyclic carbene-based C∧N∧C pincer ligand for live-cell imaging of endocytosis.

Luminescent ruthenium(II)-cyanide complex with N-heterocyclic carbene pincer ligand C(∧)N(∧)C = 2,6-bis(1-butylimidazol-2-ylidene)pyridine and 2,2'-bipyridine (bpy) shows minimal cytotoxicity to both human breast carcinoma cell (MCF-7) and human retinal pigmented epithelium cell (RPE) in a wide range of concentration (0.1-500 μM), and can be used for the luminescent imaging of endocytosis of the complex in these cells.

Endocytosis, a process that materials are internalized into cells without passing through the cell membranes, represents one of the most common mechanisms for cells to interact with their environments1. This mechanism is known in many vital cellular processes including nutrient acquisition, receptor desensitization, and antigen presentation2. Visualization of endocytosis would not only allow investigation of many physiological processes, but may also provide insight on the rational design of new therapeutic agents34. In this regard, designing luminescent molecules and visualizing how they are trafficked in cells upon they have been endocytosed using microscopic techniques would be a powerful way to understand endocytosis.

In these two decades, a new class of structurally robust and easily accessible complexes has been intensively studied on their potentials in biomedical applications. A reservoir of successful cases has been documented in dozens of reviews567891011. Yet, only a few examples have been reported with n class="Chemical">Ru(II)-NHC complexes and these studies are mainly focused on cytotoxicity evaluation12131415161718192021. In this regard, development of luminescent Ru(II)-NHC compounds for probing cellular processes may provide some insights for biomedical practice of coordination compounds.

In these years, our group have initiated a program to develop new luminescent organometallic n class="Chemical">Ru(II)- and Os(II)-bipyridine complexes2223242526272829303132. Recently, we have developed luminescent Ru(II)-bipyridine complexes bearing neutral tridentate NHC-based pincer ligands 2,6-bis(1-butylimidazol-2-ylidene)pyridine (C∧N∧C)30. We envision that Ru(II) complexes in the form of [RuII(C∧N∧C)(bpy)L]n+ would be a valuable system for the design of luminescent molecular probes for live-cell imaging, as the C∧N∧C and bpy ligands on the [RuII(C∧N∧C)(bpy)L]n+ are inert towards ligand substitution, therefore would in principle minimize the chance of complicating any biological pathways via ligand dissociation. However, the [RuII(C∧N∧C)(bpy)L]n+ complexes we reported previously (L = Cl−, n = 1; L = CH3CN, t-BuNC, n = 2) suffer from low luminescent quantum yields (10−5–10−3), and the ligand L is substitutionally labile in the case of L = CH3CN, making them imperfect molecular systems for live-cell imaging probes. We herein report the synthesis of [RuII(C∧N∧C)(bpy)(C≡N)]+ (1), a complex which has improved quantum yield and non-labile auxiliary ligand (L = −CN). It is not only non-toxic for both human breast carcinoma cell (MCF-7) and immortalized noncancerous human retinal pigmented epithelium cell (RPE), but can also be used for the luminescent imaging of endocytosis of the complex in these cells.

The design of Ru(II) complex [RuII(C∧N∧C)(bpy)(C≡N)]+ (1) is a rational modification of our previously reported [RuII(C∧N∧C)(bpy)L]n+ system (L = Cl−, n = 1; L = CH3CN, t-BuNC, n = 2). For complexes bearing [RuII(bpy)] moiety, it is well-known that the presence of thermally-populated metal-centered 3dd excited state serve as an efficient deactivation pathway for the emissive triplet Ru-to-bpy metal-to-ligand charge transfer excited state (3MLCT)3334353637383940. One promising approach to improve the population of the 3MLCT state is the incorporation of a stronger donor to elevate the 3dd state, and we therefore employ cyanide as L in this work.

Results

Synthesis

Complex 1 was synthesized by reacting [RuII(C∧n class="Chemical">N∧C)(bpy)(OH2)]2+ with KCN in refluxing H2O. Its 1H and 13C signals signify the presence of a plane of symmetry on NMR time scale at room temperature (e.g. 16 sets of aromatic signals in 13C spectrum). The 13C NMR signal at 194.7 ppm is typical for metalated N-heterocyclic carbene on Ru(II) complexes. Molecular structure of 1 was determined by X-ray crystallography and the perspective view is depicted in Figure 1. The Ru-center adopts a distorted octahedral geometry with C∧N∧C-pincer chelating meridionally in an almost planar configuration. The bond distances of Ru–CNHC and Ru–CCN are 2.054(3)–2.063(3) and 2.002(3) Å respectively. These Ru–CNHC distances are similar to those in chloride-ligated complexes [RuII(C∧N∧C)(N∧N)Cl]+ (N∧N = 2,2′-bipyridine-like diimine ligands, Ru–CNHC = 2.048(2)–2.062(4) Å)30.

Figure 1

(a) Chemical structure and labeling scheme for the H and C atoms in complex 1. (b) Perspective view of complex 1 at 30% probability ellipsoids; hydrogen atoms are omitted for clarity. The butyl chains on the Ĉ[Ncirc]C ligand are disordered over two positions (labeled as A and B, occupancy ratio ~ 0.7:0.3). Selected bond lengths (Å) and angles (deg): Ru(1)–C(1) 2.002(3), C(1)–N(1) 1.160(3), Ru(1)–C(2) 2.054(3), Ru(1)–C(10) 2.063(3), Ru(1)–N(4) 2.014(2), C(2)–Ru(1)–N(4) 77.22(10), C(10)–Ru(1)–N(4) 77.35(10).

Photophysical properties

The absorption and emission spectra for 1 are depicted in Figure 2a. With reference to our previous spectroscopic studies on the [RuII(C∧n class="Chemical">N∧C)(bpy)L]n+ system, the lowest-energy absorption band (λmax = 464 nm, εmax = 7.1 × 103 dm3 mol−1 cm−1) is assigned as dπ(RuII) → π*(bpy) MLCT transition30. The assignment is further supported by time-dependent density functional theory (TD-DFT) calculation on model complex 1′, where its metal core is the same as that in 1 except the butyl chains on the C∧N∧C are replaced by methyl groups to reduce the computational cost. The simulated spectrum (Figure 2b) is in qualitative agreement with its experimental spectrum. The lowest energy transition for 1′ is a net dπ(RuII) → π*(bpy) charge transfer, which is clearly shown by an electronic difference density plot for 1′ (generated by taking the difference in the excited-state electron density and ground-state electron density) in its lowest energy excited state (marked with * in Figure 2). Complex 1 is emissive with λem at 659 nm in CH3CN, and 648 nm in CH3CN-H2O 1:1 v/v mixture, upon photoexcitation at its lowest-energy absorption band. Importantly, its emission quantum yield (8.23 × 10−3 in degassed CH3CN, 2.14 × 10−3 in non-degassed CH3CN-H2O mixture) and lifetime (1.71 μs in degassed CH3CN, 0.34 μs in non-degassed CH3CN-H2O mixture) are more superior to its Cl−, CH3CN−, and t-BuNC-ligated analogues (≤2.98 × 10−3 and ≤0.32 μs respectively in degassed CH3CN)31.

Figure 2

(a) Absorption (solid line) and emission (dash line, λex = 450 nm) spectra of 1 in degassed CH3CN at 298 K. (b) TD-DFT calculated absorption spectrum for model complex 1′ in CH3CN. Excitation energies and oscillator strengths are shown by the blue vertical lines; spectrum (in black) is convoluted with a Gaussian function having a full width at half-maximum of 3000 cm−1. Insert shows the electronic difference density plot for 1′ at the vertical transition marked with * (isodensity value = 0.004 au).

Bio-imaging study

The interactions of complex 1 with mammalian cells were investigated by using the n class="Species">human mammary carcinoma cell line, MCF-7, and immortalized noncancerous human retinal pigmented epithelium (RPE) cell line. The toxicity of the complex to these cell lines was examined by employing the Prestoblue reagent, and the results showed that the complex was not cytotoxic to both cell lines in the concentration range of 0.1–500 μM (Figures S1 and S2). The luminescent bioimaging potential of 1 was evaluated using MCF-7 cells and RPE cells, the latter of which have a much flatter morphology than MCF-7 cells and thus allow easier observation of the transport of the complex in the cytoplasm of the cells. The localization of the luminescent signals of the complex in RPE cells was studied at complex concentration of 50, 100, 250, and 500 μM for 4 h, and surprisingly, the intensities of the luminescent signals presented in the cytoplasm of the cells were quite similar. Most of the signal appeared as spots under the laser confocal microscope and they could be mapped within the vesicles presented in the cytoplasm of the treated cells (Figure 3, lower row). The same observation was also obtained when the complex was incubated with MCF-7 cells (Figure 3, upper row). We also observed that complex 1 in the form of vesicles seemed to be more concentrated closer to the Golgi apparatus and nuclei with time of incubation. To confirm this observation, time-lapse luminescent micrographs were taken over an hour on the complex-treated RPE cells. The luminescent signals in the form of vesicles were observed to translocate from the distal end of the cell towards the nucleus (Figure S3), suggesting that the complex was engulfed from the medium (too weak to be recorded under confocal microscope), entered the cells via endocytosis in the form of endocytic vesicles, and transported along the microtubules towards the Golgi area, where the complex may then be processed or broken down in the lysosomes.

Figure 3

Luminescent images, differential interference contrast (DIC) images, and the overlay of these images (merged) of the live MCF-7 cells (A) and RPE cells (B) cells incubated with 1.

Two examples of the mapping of luminescent signals to vesicles present in the cytoplasm are indicated by arrows. Scale bars represent 25 μm.

Discussion

The results of this study show that the luminescent complex [RuII(C∧n class="Chemical">N∧C)(bpy)(C≡N)]+ can be used as a bioimaging tool to illuminate the pathway that how molecules present in the surrounding enter the cells via the formation of endocytic vesicles and be processed along the endomembrane systems in the cells. Progress will be made to search for molecules that can change their emission wavelength by a change in pH, which would be very useful in showing when the vesicles merge with the acidic content of the lysosomes during the later stage of the endocytic pathway.

Methods

General Procedure

All reactions were performed under an argon atmosphere using standard Schlenk techniques unless otherwise stated. All reagents and solvents were used as received. Complex [n class="Chemical">Ru(C∧N∧C)(bpy)(OH2)](ClO4)2 was prepared according to literature method30. 1H, 13C{1H}, DEPT-135, 1H–1H COSY, and 1H–13C HSQC NMR spectra were recorded on Bruker 400 DRX FT-NMR spectrometer. Peak positions were calibrated with solvent residue peaks as internal standard. Electrospray mass spectrometry was performed on a PE-SCIEX API 3000 triple quadrupole mass spectrometer. Infrared spectra were recorded as KBr plates on an Avatar 360 FTIR spectrometer. UV–visible spectra were recorded on a Shimadzu UV-1700 spectrophotometer. Elemental analyses were done on an Elementar Vario Micro Cube carbon–hydrogen–nitrogen elemental micro-analyzer. Steady-state emission spectra were obtained on a Jobin Yvon Fluorolog-3-TCSPC spectrophotometer. Sample and standard solutions were degassed with at least three freeze-pump-thaw cycles. The emission quantum yields were measured by the method of Demas and Crosby41 with [Ru(bpy)3](PF6)2 in degassed CH3CN as standard (Φr = 0.062) and calculated by Φs = Φr(Br/Bs)(ns/nr)2(Ds/Dr), where the subscripts s and r refer to sample and reference standard solution, respectively, n is the refractive index of the solvents, D is the integrated intensity, and Φ is the luminescence quantum yield. The quantity B is calculated by B = 1 − 10−AL, where A is the absorbance at the excitation wavelength and L is the optical path length.

[Ru(C A mixture of [n class="Chemical">Ru(C∧N∧C)(bpy)(OH2)](ClO4)2 (0.125 mmol) and KCN (0.25 mmol) was refluxed in water (30 mL) under argon for 2 h. Upon cooling to room temperature, a saturated NaClO4 solution (5 mL) was added into the reaction mixture to give bright orange solids. The solid was filtered and washed with EtOH/Et2O mixture (1:10, v/v) (2 × 5 mL) and Et2O (2 × 5 mL), and was recrystallized by slow diffusion of Et2O into CH3CN solution to give bright orange crystal. Yield 81 mg, 92%. Anal. Calcd for C30H33N8RuClO4: C, 51.03; H, 4.71; N, 15.87. Found: C, 50.97; H, 5.00; N, 15.92. 1H NMR (400 MHz, CD3CN): δ 0.57–0.86 (m, 10H, Bu), 0.88–1.04 (m, 2H, Bu), 1.25–1.43 (m, 2H, Bu), 3.20–3.37 (m, 4H, Bu), 7.04–7.09 (m, 1H, Hg), 7.13 (d, J = 2.4 Hz, 2H, Hk/Hl), 7.23–7.28 (m, 1H, Hh), 7.62–7.70 (m, 3H, Hb + Hj), 7.77–7.84 (m, 1H, Hf), 7.94 (d, J = 2.4 Hz, 2H, Hk/Hl), 7.99–8.10 (m, 2H, Hc + Hi), 8.29–8.35 (m, 1H, He), 8.41–8.47 (m, 1H, Hd), 10.10–10.17 (m, 1H, Ha). 13C NMR (100.6 MHz, CD3CN): δ 13.75, 20.23, 34.31, 50.78 (Bu), 106.71 (Cj), 118.09 (Ck/Cl), 123.59 (Ce), 123.90 (Ck/Cl), 124.44 (Cd), 126.51 (Cg), 127.12 (Cb), 135.65 (Cc), 136.89 (Cf), 139.51 (Ci), 143.66 (C≡N), 150.07 (Ch), 154.47 (quaternary carbon), 155.05 (Ca), 155.84, 156.33 (quaternary carbons), 194.73 (Ru–CNHC). IR (KBr, cm−1): νCN = 2063. ESI-MS: m/z 607.5 [M+].

X-ray Crystallography

X-ray diffraction data for 1(ClO4) was collected on an Oxford Diffraction Gemini S Ultra X-ray single crystal diffractometer with Mo Kα radiation (λ = 0.71073 Å) at 173 K. The data was processed using CrysAlis. The stn class="Chemical">ructure was solved by Patterson method, and refined by full-matrix least-squares based on F2 with program SHELXS-97 and SHELXL-97 within WinGX. All non-hydrogen atoms were refined anisotropically in the final stage of least-squares refinement. The positions of H atoms were calculated based on riding mode with thermal parameters equal to 1.2 times that of the associated C atoms. The butyl chains on the C∧N∧C ligand are disordered over two positions, and split model was applied. CCDC 1043253 contains the supplementary crystallographic data for this paper, which can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Computational Methodology

DFT and TD-DFT calculations were performed on model complex 1′ using the ORCA software package (version 3.0.0). Its electronic ground state was optimized using the PBE0 functional4243 accompanied with (i) the zero-order regular approximation (ZORA)444546 to account for relativistic effects, (ii) the conductor-like screening model (COSMO)47 to model solvation in CH3CN, and (iii) atom-pairwise dispersion correction with n class="Disease">Becke-Johnson damping4849. The def2-SVP basis sets were used for the H, C, and N atoms, while the def2-TZVP(-f) basis set was used for the Ru atom50. Auxiliary basis sets, used to expand the electron density in the calculations, were chosen to match the orbital basis sets5152. The combination of the resolution of the identity and the “chain of spheres exchange” algorithms (RIJCOSX)535455 was used to accelerate all DFT and TD-DFT calculations. Tight SCF convergence criteria (1 × 10−8 Eh in energy, 1 × 10−7 Eh in the density charge, and 1 × 10−7 in the maximum element of the DIIS error vector) were used throughout. The vertical transition energies for 1′ in CH3CN was computed using TD-DFT method with the density functional and basis sets aforementioned, and with the Tamm-Dancoff approximation.

Cell culture and cytotoxicity assay

Human n class="Disease">breast cancer cell, MCF-7 and the human retinal pigmented epithelium cell line, RPE, were cultured at 37°C in 5% CO2 with Dulbecco's modified Eagle medium (DMEM) and in the presence of 10% fetal bovine serum (FBS) until the confluency reached 50% to 60%. Cell viability was measured by using the PrestoBlue reagent according to the manufacturer's instruction (Invitrogen, USA).

Author Contributions

W.K.T., L.H.C. and M.M.K.W. carried out all the experiments, W.H.T., H.S.L. and Y.L. performed the data analysis; C.H.L., D.L.M., S.K.C. and C.Y.W. designed the experiments, analyzed the results and wrote the manuscript. * W.K.T. and L.H.C. contributed equally to this work.