Aggregation-Induced Emission-Active Ruthenium(II) Complex of 4,7

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Aggregation-Induced Emission-Active Ruthenium(II) Complex of 4,7Dichloro Phenanthroline for Selective Luminescent Detection and Ribosomal RNA Imaging Sanjoy Kumar Sheet,†,§ Bhaskar Sen,†,§ Sumit Kumar Patra,† Monosh Rabha,† Kripamoy Aguan,‡ and Snehadrinarayan Khatua*,† †

Centre for Advanced Studies, Department of Chemistry, and ‡Department of Biotechnology and Bioinformatics, North Eastern Hill University, Shillong, Meghalaya 793022, India S Supporting Information *

ABSTRACT: The development of red emissive aggregation-induced emission (AIE) active probes for organelle-specific imaging is of great importance. Construction of metal complex-based AIE-active materials with metal-to-ligand charge transfer (MLCT), ligand-to-meal charge transfer (LMCT) emission together with the ligand-centered and intraligand (LC/ILCT) emission is a challenging task. We developed a red emissive ruthenium(II) complex, 1[PF6]2, and its perchlorate analogues of the 4,7-dichloro phenanthroline ligand. 1[PF6]2 has been characterized by spectroscopic and single-crystal Xray diffraction. Complex 1 showed AIE enhancement in water, highly dense polyethylene glycol media, and also in the solid state. The possible reason behind the AIE property may be the weak supramolecular π···π, C−H···π, and C−Cl···H interactions between neighboring phen ligands as well as C−Cl···O halogen bonding (XB). The crystal structures of the two perchlorate analogues revealed C−Cl···O distances shorter than the sum of the van der Waals radii, which confirmed the XB interaction. The AIE property was supported by scanning electron microscopy, transmission electron microscopy, dynamic light scattering, and atomic force microscopy studies. Most importantly, the probe was found to be low cytotoxicity and to efficiently permeate the cell membrane. The cell-imaging experiments revealed rapid staining of the nucleolus in HeLa cells via the interaction with nucleolar ribosomal ribonucleic acid (rRNA). It is expected that the supramolecular interactions as well as C−Cl···O XB interaction with rRNA is the origin of aggregation and possible photoluminescence enhancement. To the best of our knowledge, this is the first report of red emissive ruthenium(II) complex-based probes with AIE characteristics for selective rRNA detection and nucleolar imaging. KEYWORDS: ruthenium(II) complex, aggregation-induced emission, halogen bonding, bioimaging, ribosomal ribonucleic acid

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INTRODUCTION Construction of light−emitting metal complexes is the recent hot scientific research theme because of its potential practical applications in various research areas such as molecular sensors, bioimaging probes, organic light-emitting diodes, optoelectronic devices, and in photocatalysis.1−3 In general, organic and metal complex-based luminophores exhibit bright luminescence in dilute nonaqueous solutions but emit very weakly in the aggregated state or in the solid state because of aggregationcaused quenching (ACQ).4,5 It is highly desirable to develop non-ACQ metal complexes which can resolve this problem. In 2001, Tang et al. reported a new phenomenon which is absolutely opposite to ACQ and termed as the “aggregationinduced emission (AIE)” effect.6 AIE-active materials are weakly emissive or nonemissive when dissolved in good solvents, but these materials can emit strongly in the nanoaggregated state or in the solid state.7−16 The key reasons behind the AIE effect are restricted intramolecular rotation and intermolecular π−π stacking interactions.17,18 Besides these, a © XXXX American Chemical Society

number of other mechanistic pathways are involved such as planarization, internal charge transfer, twisted intramolecular charge transfer and excited-state intramolecular proton transfer, J-aggregate formation, and E−Z isomerization.19−22 AIEgens have a wide range of applications in sensing, cell organellespecific imaging, chemo and photodynamic therapy, and so forth. Although scientists are debating on elucidating the mechanism of the AIE effect, there is an immense scope to develop AIE-active materials through a smart strategic molecular design. Halogen bonding (XB) is a directional, noncovalently attractive interaction between the positive site of the halogen atom and the negative sites of a Lewis base-like O/N/S/P, chalcogens, and π-electron donors. The halogen atom connected to a carbon (C−X bond) leads to the development Received: December 19, 2017 Accepted: April 10, 2018

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DOI: 10.1021/acsami.7b19290 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Synthesis of 1[PF6]2 and 1[ClO4]2

Figure 1. ORTEP plot of (a) 1[PF6]2, (b) 1[ClO4]2, and (c) 1[ClO4]2(CH3CN)(H2O) (thermal ellipsoids were drawn at the 30% probability level). Only non-carbon and non-hydrogen atoms are labeled here.

spatial-temporal transportation and processing of RNA in live cells. Although there are three major types of RNA, ribosomal RNA (rRNA) is extremely important for synthesis of protein and comprises ∼70% of all RNAs in the cell.50 rRNA is mainly accumulated in the nucleolar region of nucleus and evidently visible by fluorescence microscopy as dense and dark region of the nucleus.51,52 Although there are many reported literature studies on the selective imaging of cellular organelles, for instance, nucleus,53 mitochondria,54 lysozome,55 endoplasmic reticulum,56 selective RNA detection and nucleolus staining probes are still limited.57 Recently, we published Ag+-assisted rRNA detection and imaging by a bisheteroleptic Ru(II) complex.58 In continuation of our research on Ru(II)-based probes for organelle-specific imaging, herein, we report red emissive bisheteroleptic ruthenium(II) compounds (1[PF6]2 and 1[ClO4]2) of 4,7dichloro phenanthroline ligand and investigate the AIE property, X-ray crystal structure, density functional theory (DFT) calculations, and efficient light-up detection of rRNA and the specific nucleolus imaging in live cells. The effect of weak supramolecular and C−Cl···O XB interactions in crystal packing and aggregate formation in solution as well as rRNA imaging and detection are also discussed herein. Although there is a recent report on organic AIEgen for RNA imaging, the probe is not highly specific to RNA as it also stains the mitochondria.59 To the best of our knowledge, this is the first successful report of the AIE-active ruthenium(II) complex, which is utilized as a selective rRNA imaging probe through the in vitro photoluminescence (PL) spectroscopy and in vivo investigation.

of a positive electrostatic potential because of the dislocation of electron density.23−25 While in the anion recognition field the charge-assisted XB interactions for selective anion recognition is successfully employed, the XB interaction-assisted aggregation and development of smart luminescent bioimaging probe is still uncommon.26−28 In contrast to fluorescent organic dye, development of deep red emissive probes is favored in bioimaging and in vivo bioassay because of reduced light scattering, background autofluorescence, photodamage to biosamples, and better tissue penetration.29−31 Ruthenium(II) polypyridyl complexes of substituted 2,2′-bipyridine (bpy) or 1,10-phenanthroline (phen) have extensive application in chemistry and biology such as sensors, photodynamic therapy, cellular imaging, and deoxyribonucleic acid (DNA)/ribonucleic acid (RNA)-responsive photoswitchable probes because of its superior photophysical/chemical properties such as visible-light absorption and deep red or near-infrared emission, significant stokes shifts, high stability in aqueous media compared with pure organic luminophores, low cytotoxicity, and good cell permeability.32−35 Luminescence of ruthenium(II) complexes primarily originated from the metal-to-ligand charge transfer (1MLCT/3MLCT) states. As the charge transfer emission is afforded by the involvement of diimine ligands, the exclusive photophysical properties can be readily maneuvered by proper modification of the diimine ligand framework. Albeit there are several reports on organic compound-based AIEgens36−39 and metal complexes,40,41 utilization of ruthenium(II) complexes as AIE-active materials is extremely rare.8,42 Fluorescent probes for organelle-specific live cell imaging have become an imperative part of biomedical research in the last few years, as they allow to monitor biological functions of a specific molecule in a cellular system.43−45 RNA molecules in living cells are primarily responsible for every vital biological function, counting physical transportation, regulation of the gene expression, protein production, and some crucial biocatalytic activities.46−49 Therefore, it is highly desirable to develop RNA-selective probes to gain the information about

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RESULTS AND DISCUSSION Synthesis and Characterization. The ligand, 4,7-dichloro phenanthroline (L), was synthesized according to the reported literature procedure and characterized by 1H NMR spectroscopy and electrospray ionization-mass spectrometry (ESI-MS) (Figures S1 and S2).60 The synthetic routes of two B


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Figure 2. Weak supramolecular interactions (C−H···π and π−π stacking), C−Cl···H interaction, and C−Cl···O XB interaction in (a) 1[PF6]2, (b) 1[ClO4]2, and (c) 1[ClO4]2(CH3CN)(H2O), respectively. View of the two-dimensional (2D) crystal packing in (d) compounds 1[PF6]2, (e) 1[ClO4]2, and (f) 1[ClO4]2(CH3CN)(H2O). Solvent molecules and counter ions are omitted for clarity.

which suggests the better π-acceptor ability of the 4,7-dichloro phenanthroline ligand than phen ligand. Crystal Structure. Compounds 1[PF6]2 and 1[ClO4]2 were found to crystallize in the triclinic and monoclinic space groups P1̅ and P21/n, respectively. Crystal structures reveal that the ruthenium centers adopt a similar octahedral geometry through the coordination of two ancillary phenanthrolines and a 4,7-dichloro phenanthroline (L) ligand (Figure 1a,b). The unit cell of 1[PF6]2 contains two PF6 anions and chloroform and acetone as the solvent of crystallization, whereas 1[ClO4]2 consists two ClO4 anions and three disordered water molecules which were further removed by PLATON SQUEEZE program during refinement. The perchlorate analogue was further crystallized (space group P1̅) from the CH3CN solvent. The asymmetric unit of 1[ClO4]2(CH3CN)(H2O) contains complex 1, two perchlorate ions, water, and acetonitrile as the solvent of crystallization (Figure 1c). The Ru−N bond distances in 1[PF6]2, 1[ClO4]2 and 1[ClO4]2(CH3CN)(H2O) are in the range of 2.058(4)−2.078(6), with trans N−Ru−N angles in the range of 172.20(2)−174.19(15)°. Analysis of the crystal packing in three compounds reveals that the complexes form noncovalent 2D networks (Figure 2) which are held together by weak C−H···π and C−Cl···H interactions as well as by π−π stacking interactions of two phenanthrolines of neighboring complexes. In addition, the occurrence of C− H···F and C−H···O hydrogen bonds indicates that the counter anions PF6− and ClO4− play a crucial role in formation of the 2D packing. Most interestingly, the chlorine atom of 4,7dichloro phenanthroline ligand in 1[ClO4]2 forms an XB interaction with oxygen atoms of ClO4 ions [C3−Cl1···O3 (3.018 Å, 158°) and C8−Cl2···O4 (3.246 Å, 156°)] (Figure 2c). The C−Cl···O halogen bond also exists in the compound, 1[ClO4]2(CH3CN)(H2O), and the XB interaction is much more directional than in the compound 1[ClO4]2 [C8−Cl2··· O1(3.015 Å; 174°)] (Figure 2c). The electron deficiency on

bisheteroleptic ruthenium(II) compounds 1[PF6]2 and 1[ClO4]2 are outlined in Scheme 1. Compound 1[PF6]2 was prepared by refluxing cis-[Ru(phen)2Cl2] and L in EtOH/H2O (3:1) solution for 24 h under N2 atmosphere followed by PF6 anion exchange and isolated in good yield (59%) (Scheme 1). Dark red crystals of 1[PF6]2 were obtained from CH3COCH3/ CHCl3 (6:1). Compound 1[ClO4]2 was prepared from 1[PF6]2 by stirring in dry acetone with excess tetra-n-butyl ammonium perchlorate. Shiny red crystals were obtained from the CH3COCH3/H2O (3:2) mixture. 1[PF6]2 was fully characterized by various spectroscopies, namely, 1D NMR (1H, 13C) and 2D NMR (1H−1H COSY, 1H−13C HSQC, and 1H−13C HMBC) spectroscopy (Figures S3−S7 in the Supporting Information), ESI-MS (Figure S8 in the Supporting Information), and elemental analysis. The solid-state structure of 1[PF6]2 was determined by single-crystal X-ray diffraction (Figure 1). The 1D and 2D NMR spectra of 1[PF6]2 were recorded in acetone-d6 at room temperature, which clearly show all expected peaks of phenanthroline and 4,7-dichloro phenanthroline ligand. All carbon peaks are fully assigned by 13 C NMR with the help of HSQC and HMBC. The ESI-MS spectra of 1[PF6]2 clearly show a peak at m/z = 854.99 (calcd 854.99), which corresponds to singly charged 1·(PF6)+. 1[ClO4]2 was characterized by 1H NMR (Figure S9), ESIMS (Figure S10), elemental analysis, and single-crystal X-ray diffraction (Figure 1).The electrochemical properties of 1[PF6]2 were examined by cyclic voltammetry (CV) using a Pt working electrode in dry and degassed CH3CN under N2 atmosphere with ferrocene as the internal standard (Eox 1/2 = +0.400 V vs SCE). Compound 1[PF6]2 shows a single quasireversible (ΔEp = 80 mV) Ru2+/Ru3+ redox couple at Eox 1/2 = +0.950 V (Epa = 0.990 V and Epc = 0.910 V) versus Fc+/Fc (Figure S11). The redox wave is anodically shifted by 39 mV in + comparison to [Ru(phen)3]2+ (Eox 1/2 = +0.911 V vs Fc /Fc), C


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demonstrates a sharp band at λ = 263 nm (ε = 8.9 × 104), which is assigned to intraligand (IL) π−π* transitions within phen and 4,7-dichloro phenanthroline ligands. In addition, a broad MLCT absorption is observed in the 340−500 nm range. For 1[PF6]2, a band at λmax = 435 nm (ε = 1.4 × 104) and a shoulder at λ = 470 nm (ε = 1.0 × 104) are observed. A timedependent DFT (TD-DFT) calculation was performed to assign those bands (vide infra). 1[PF6]2 (10 μM) in CH3CN furnishes a broad emission band with a maxima at 615 nm (Φ = 0.011) under the excitation of 435 nm (Figure 3a) with a mega stokes shift of 180 nm. For the perchlorate analogue, 1[ClO4]2, a similar spectral feature is observed but with a slightly higher quantum yield (Φ = 0.012). The outline of the PL spectra and the emission maxima (λem) is independent on the excitation wavelength (435−470 nm), which suggests that the emission arises from the same MLCT excited state. Both 1[PF6]2 and 1[ClO4]2 in the solid and thin film (for 1[PF6]2) show a similar PL band and deep red luminescence under a laboratory UV lamp, though the intensity is brighter in the case of 1[ClO4]2 (Figure 3b). It is well-established that the solid-state luminescence depends on the crystal packing and intermolecular interactions.61 Therefore, herein, we presume that the more intense luminescence of 1[ClO4]2 may be due to the better crystal packing through the additional C−Cl····O XB interaction. DFT calculation was performed to obtain the energyminimized structure of complex 1[PF6]2. The optimized structure, selected bond lengths and angles, and Cartesian coordinates are given in Figure S12 and Tables S1 and S2 in the Supporting Information. Theoretically generated molecular orbitals for the ground-state geometry of 1[PF6]2 are given in Figure S13. The highest occupied molecular orbital (HOMO) is associated to the ruthenium(II) dz orbital, while HOMO − 2 and HOMO − 3 are allied to the ruthenium(II) t2g set. The lowest unoccupied molecular orbitals (LUMOs) of 1[PF6]2 mainly focused on the ancillary phen ligand, except the LUMO and LUMO + 1 orbital, which are based on the chloro phenanthroline ligand. The TD-DFT calculation on optimized

the chlorine center of the 4,7-dichloro phenanthroline ligand may be significantly increased as it is coordinated to a positive metal center such as Ru2+, and the development of the δ+ charge facilitates the halogen bond formation. Photophysical Properties and DFT Calculations. The absorption and PL spectra of 1[PF6]2 in CH3CN (10 μM) are provided in Figure 3a. The UV−vis spectrum of 1[PF6]2

Figure 3. (a) UV−vis and PL spectra of 1[PF6]2 in CH3CN (10 μM) solid and thin films at room temperature (λex = 435 nm) and (b) image of 1[PF6]2 and 1[ClO4]2 in CH3CN (10 μM) and solid thinfilm emission under UV irradiation.

Figure 4. PL spectra of (a) 1[PF6]2 (10 μM) and (b) 1[ClO4]2 in the CH3CN−water mixture (0−90%) (λex = 435 nm). (inset) Plot of emission intensity vs water fraction. (c) Photographs of the luminescence of 1[PF6]2 in different water fractions (0−90%) under 365 nm UV illumination. D


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ACS Applied Materials & Interfaces geometry of 1[PF6]2 in CH3CN was performed to explicate the electronic transitions responsible for the absorption spectrum (Figure S14). The transitions ranging from 340 to 500 nm are assigned to various MLCT bands in the singlet state (1MLCT). TD-DFT calculations indicate that the experimental MLCT band at λmax = 435 nm (2.85 eV) is due to the transitions from HOMO → LUMO + 5 (f = 0.057) (383 nm, 3.93 eV) and HOMO − 3 → LUMO + 4 ( f = 0.050) (372 nm, 3.99 eV). Further, a broad band at λmax = 470 nm (2.64 eV) arises from transitions characterized as HOMO → LUMO + 1 (f = 0.127) (419 nm, 3.61 eV), HOMO − 2 → LUMO + 1 (f = 0.093) (404 nm, 3.71 eV), HOMO − 3 → LUMO + 1 (f = 0.060) (401 nm, 3.71 eV), and HOMO − 2 → LUMO + 2 (f = 0.102) (401 nm, 3.83 eV) (Table S3). AIE Properties. Solid crystalline 1[PF6]2 reveals bright red luminescence under illumination of a 365 nm UV lamp, whereas the emission intensity of diluted 1[PF6]2 (10 μM) in CH3CN is relatively weak. This outcome is similar to the previously reported materials which possess the AIE behavior.7−16 To corroborate the AIE of 1[PF6]2 and also 1[ClO4]2, the PL spectra were recorded in CH3CN with increasing water fractions ( f w) (0−90% of CH3CN−water). The gradual increasing fraction of water in CH3CN solution of 1[PF6]2 (10 μM) displays the regular increment of PL intensity. The PL intensity of 1[PF6]2 increases ∼2.5 times in 90% water fraction (Φ = 0.034) (Figure 4a,c), but for 1[ClO4]2, the PL intensity (Φ = 0.047) is enhanced ∼2.8 times (Figure 4b). Further, the luminescence of 1[PF6]2 in diluted mixtures of CH3CN with different polyethylene glycol (PEG) ( f PEG) fractions (0−90% CH3CN−PEG) was studied. For 1[PF6]2, the PL intensity increases ∼3.2 times in the presence of 90% of PEG in CH3CN which again indicates that 1[PF6]2 holds AIE activity and forms nanoaggregates at higher f PEG (Figure S15). Additionally, the overall shift of the UV−vis spectral baseline of 1[PF6]2 (10 and 20 μM, CH3CN) with the increasing amount of water and PEG (0−90%) is ascribed to the scattering of light by the aggregated structures in solution (Figures S16−S18). In the dilute solution, molecules exist in a nonaggregated state and upon excitation release energy via a nonradiative relaxation pathway through interacting solvents and consequently quench the luminescence. Conversely, in the aggregated state, the weak intermolecular interactions such as C−H···π and π−π stacking, C−Cl····H hydrogen bonding between phenanthroline groups, and C−Cl····O XB (for 1[ClO4]2) interactions between neighboring phen units restrict the intramolecular motions, which inhibit the nonradiative decay from the MLCT excited state and intensify the photoluminescence of 1[PF6]2 and 1[ClO4]2. Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Atomic Force Microscopy (AFM) Studies. The aggregation in the CH3CN−water mixture is supported by the DLS measurements study. The hydrodynamic diameters of the particles at f w = 90% range from 220 to 615 nm with a Z average of 372.4 nm (Figure 5). Further, to explore the morphological property of aggregated 1[PF6]2, SEM, TEM, and AFM measurements were done in 90% of water and 10% of the CH3CN mixture at 10 μM. SEM and TEM images clearly validate the nanoaggregate formation (Figure 6a,b). In SEM images, the diameter of spherical nanoaggregates range from ∼250 to ∼550 nm, while the TEM images demonstrated that nanorange particles are in the ∼150−∼500 nm range (Figure 6a,b). To show the three-dimensional surface of nano-

Figure 5. Particle size distribution of 1[PF6]2 (10 μM) in the CH3CN−water (1:9) mixture.

aggregates, AFM images of 1[PF6]2 (10 μM) in 90% of water (10% CH3CN) were captured which show a root mean square roughness of 103.8 nm, and the result validates the aggregation formation (Figure 6c,d). Cytotoxicity, RNA Imaging, and RNA Binding Studies. We evaluated the cytotoxicity of 1[PF6]2 in HeLa cells for 24 h. The cell viability remains high at probe concentrations as high as 10 μM and decreased slightly from the concentration range 25−100 μM. More than 84% of cells survived at ≤100 μM of 1[PF6]2 after 24 h of treatment, suggesting low cytotoxicity and good biocompatibility of 1[PF 6 ] 2 at the 5−100 μM concentration range (Figure 7). For the initial cell imaging experiment, the 1.0−10 μM concentration of 1[PF6]2 was used as the cell viability was >96% at the 10 μM concentration. Further, to assess the potential biological applications of the probe 1[PF6]2 in living samples, cell imaging experiments were carried out in HeLa cells. At first, HeLa cells were treated with different concentrations of 1[PF6]2 and incubated for 20 min. The probe was found to be cell-permeable, and it stained the cell nucleoli as well as the cytoplasm (Figure 8a) at an optimal concentration at 10 μM. We performed imaging experiments in HeLa cells at different time points, and as shown in Figure 8b,c for 1[PF6]2, to obtain clear red luminescence in the nucleoli and cytoplasm, ∼20 min incubation is essential. Although a long incubation time is reported for selective nucleoli imaging in live cells,62 for 1[PF6]2, we observed the bright red luminescence from all over the cytoplasm after ∼1 h incubation. To establish whether the probe 1[PF6]2 binds to cellular DNA or RNA, the DNase and RNase digestion tests were performed (Figure 9). The DNase hydrolyzes only DNA without affecting RNA in the cells, whereas only RNA substrates are hydrolyzed in the RNase digest test. As expected, the cell nucleoli stained with probe 1[PF6]2 shows significant red luminescence after the DNase treatment, though the luminescence intensity in the nucleoli is slightly decreased in some cells. By contrast, in the RNase-treated cells, the bright red luminescence of the nucleoli is diminished significantly (Figure 9), and also the luminescence intensity in the cytoplasm is decreased to some extent. The cells treated with both the DNase and RNase show less intense luminescence from the cytoplasm, and the luminescence in the nucleolus is vanished completely. For the nuclear staining dye DAPI, the fluorescence in the nucleolus almost vanished in the presence of DNase, whereas the blue fluorescence was mostly unaffected upon treatment with RNase (Figure 9). The DNase RNase digest experiment results demonstrated that 1[PF6]2 stains nucleolus via the interaction with nucleolar RNA. It is amply E


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Figure 6. (a) SEM and (b) TEM images of 1[PF6]2 (10 μM) in CH3CN−H2O mixtures (1:9). AFM images of 1[PF6]2 formed in CH3CN−H2O mixtures with 90% water fraction. (c) 2D image and (d) 3D image.

Figure 9. Fluorescence images of fixed HeLa cells during DNase and RNase digest experiments with 1[PF6]2 and nucleus staining dye DAPI.

Figure 7. Cell viability of HeLa cells in the presence of different concentrations of 1[PF6]2 for 24 h. Data are expressed as the mean value for four separate trials. Asterisks denote cell viability significantly different from the control (0 μM of 1[PF6]2) at p ≤ 0.05.

Figure 8. Fluorescence microscopy images of HeLa cells (a) stained with different concentrations of 1[PF6]2 (1.0−10 μM) and (b) at different time points (1[PF6]2 at 10 μM) in aqueous phosphate buffered saline (PBS) buffer. (c) Intensity profile within the regions of interest [white line in (A− D)] of 1[PF6]2 across HeLa cells. F


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Figure 10. (a) PL spectra of 1[PF6]2 (10 μM) upon the addition of RNA, ctDNA, ssDNA, BSA, and G-quadruplex DNA (4 fold) in CH3CN/PBS buffer (v/v, 1:1; pH 7.4). (b) PL titration of 1[PF6]2 with rRNA (0−4 fold) in CH3CN/PBS buffer (v/v, 1:1; pH 7.4). (Inset) Photograph showing the luminescence enhancement of 1[PF6]2 (10 μM) upon the addition of rRNA (40 μM). (c) DLS plot of 1[PF6]2 (10 μM) in 50% mixed aqueous buffer and in the presence of rRNA (40 μM).

1[PF6]2 the C−H−π, π−π stacking interaction and XB interaction (in 1[ClO4]2) are the key reasons for aggregation and PL enhancement. In addition, the extended π clouds in three phen ligands may facilitate π−π stacking with purine and pyrimidine bases of RNA. Ho et al. reported several halogen (Br) containing DNA crystal structures with XB interactions in the four-stranded intermediate DNA Holliday junctions.28,65−67 As numerous experimental evidences are reported regarding the XB interaction between chlorinated drugs and nucleotides,68−70 here, we speculated that in addition to π−π stacking with purine and pyrimidine bases, the XB interaction between chlorine atoms of the 4,7-dichloro phenanthroline ligand of 1[PF6]2 and oxygen of the cytosine (C), guanine (G), and uracil (U) of single-stranded RNA may also be responsible for RNA binding, aggregation, and PL enhancement (Schemes S1 and S2). To check AIE property, we did the DLS experiment to measure the particle size of blank in 50% aqueous buffer and with rRNA. The average particle diameter is increased from 211.9 to 355.7 nm in the presence of rRNA (Figure 10c), which implies possible aggregation in the presence of rRNA and PL enhancement.

known that compounds with positive charge have the potential to target the negatively charged RNA because of its phosphodiester backbone.63 Thomas et al. studied the effect of charge on the metal complexes for the nuclear uptake capability.45,64 In 1[PF6]2, the ruthenium(II) center carries two positive charges and has a tendency to interact with singlestrand RNA. In the living system, DNA exists as a doublestranded helix, and single-stranded DNAs (ssDNA) are very rarely formed and found. Because the rRNA is synthesized and assembled in the nucleolar region of the nucleus and ∼70% total cellular RNA is rRNA, we therefore assume that probe 1[PF6]2 mostly binds rRNA during imaging. We further investigated the interactions of 1[PF6]2 with calfthymus DNA (ctDNA), ssDNA1, G-quadruplex DNA, bovine serum albumin (BSA), and RNA (from Saccharomyces cerevisiae; contains >70% of rRNA) in the PL spectroscopy in mixed buffer media. As expected, the PL intensities of 1[PF6]2 enhanced (λem = 619 nm; λex = 435 nm) significantly only in the presence of rRNA, implying that 1[PF6]2 selectively interacts with rRNA (Figure 10a). Although both ssDNA1 and rRNA are single-stranded oligonucleotides, enhancement of PL with rRNA but not with ssDNA1 (primers of 21 base pair) may indicate that there might be some restriction of the secondary structure formation and interactions with complex and probable aggregation in the case of ssDNA1. The PL titration of 1[PF6]2 with 4-fold excess RNA shows a gradual PL enhancement at 619 nm (Figure 10b). As the probe form aggregates in the presence of water, a PL titration of 1[PF6]2 with gradual addition of 4-fold excess aqueous buffer was performed (Figure S19). The PL is unaffected upon the aqueous buffer addition, which implies that the PL enhancement with rRNA purely due to the rRNA probe interaction. In

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CONCLUSIONS In summary, we successfully synthesized deep red emissive bisheteroleptic ruthenium(II) compounds (1[PF6]2 and 1[ClO4]2) and characterized by spectroscopy as well as singlecrystal X-ray diffraction. Compounds show the AIE property in the polar solvent (water) and high density PEG media. The Xray structure demonstrated that the C−H····π, π−π stacking C−Cl····H hydrogen bonding interactions between neighbouring phen ligands and C−Cl····O XB interaction (for 1[ClO4]2) G


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Synthesis of 1[ClO4]2. 1[PF6]2 (0.112 g, 0.100 mmol) and tetra-nbutyl ammonium perchlorate (0.205 g, 6.0 mmol) were stirred for 30 min in 6 mL of distilled acetone. The bright red precipitate was filtered and washed by distilled acetone. Subsequent crystallization from the CH3COCH3/H2O (3:2) mixture provided 1[ClO4]2 as shiny bright red crystals in 99% (0.096 g) yield. Anal. calcd for C36H22Cl4N6O8Ru· 3H2O (Mw = 963.52) C, 44.88; H, 2.93; N, 8.72; C, 44.96; H, 2.84; N, 8.71. FT-IR in KBr disk (ν/cm−1): 3449, 1718, 1632, 1427, 1410, 1178, 1120, 928. ESI-MS [C36H22Cl3O4N6Ru]+: calcd: m/z, 810.03; found: m/z, 810.07; C36H22Cl2N6Ru]2+: calcd: m/z, 355.02; found: m/ z, 355.01; 1H NMR (400 MHz, D2O): δ (ppm) 8.66−8.58 (m, 3H, H4,7,d), 8.25 (s, 2H, H5,6), 8.11 (d, J = 5.3 Hz, 1H, H2), 8.04 (d, J = 5.7 Hz, 2H, Ha,9), 7.79 (d, J = 5.7 Hz, 1H, Hb), 7.64 (m, 2H, H3,8). Electrochemistry. Electrochemical experiments were performed at 25 °C using a CHI 600C electrochemical workstation (CH Instruments) instrument. The cell contained a Pt working electrode, a Pt wire auxiliary electrode, and an Ag wire as a pseudo-reference electrode. Experiments were carried out on a 1.0 mM solution of 1[PF6]2 in a dry and degassed acetonitrile with 0.1 M tetra-nbutylammonium perchlorate (Bu4NClO4) as the supporting electrolyte. For comparison, the electrochemical data of [Ru(phen)3]2+ were also collected under the same experimental conditions. After each experiment, the electrochemical potential window was calibrated using ferrocene as the internal standard. The redox potential of the ferrocene/ferrocenium (Fc/Fc+) couple was taken as 0.0 V versus Ag wire electrode. All the reported potentials were measured at a scan rate of 100 mV s−1. Cytotoxicity Study. The cytotoxicity of the compound 1[PF6]2 against HeLa cells (human cervical adenocarcinoma epithelial cell line) was determined by the colorimetric cell cytotoxicity assay kit ab112118 (abcam) in a 96-well cell culture plate. HeLa cells were seeded in a 96-well plate at a density of 5 × 103 cells/well and incubated at 37 °C in a 5% CO2 incubator. At 70% confluency, cells were treated with different concentrations of 1[PF6]2 (5, 10, 25, 50, and 100 μM) and incubated for 24 h. Assay solution was thawed and warmed at 37 °C, and 20 μL (1/5 volume) was added into each well. The reagents were mixed by shaking the plate gently for 40 s and incubated at 37 °C in a 5% CO2 incubator. The absorbance change was monitored at 570 and 605 nm using a microplate reader. The ratio of OD570 to OD605 was calculated to determine the cell viability in each well. The cell viability is proportional to the increase in OD570 and decrease in OD605. Readings were taken in quadruplet. The percentage of cell viability was calculated for samples and controls based on the following formula

in the solid can efficiently improve the QYs in polar and high dense solution. DLS, SEM TEM, and AFM studies successfully supported the aggregation property. The compound shows a large Stokes shift, very low cytotoxicity, and good cell permeability. The cell-imaging experiments reveal that the compound at low concentrations rapidly stains the nucleolus in HeLa cells through the binding with RNA. The weak supramolecular interaction together with C−Cl····O XB interaction in the presence of RNA forms aggregate and enhances the PL. This work may provide an opportunity for the construction of halogen containing red emissive AIE-based metal complexes for RNA specific nucleolar imaging.

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EXPERIMENTAL SECTION

Materials and Physical Measurements. All chemicals were used as received from commercial suppliers (Aldrich, Alfa Aesar and spectrochem India). The 1H and 13C NMR spectra were measured on a Bruker AVANCE II (400 MHz) spectrometer, and chemical shifts were expressed in ppm using solvent residual as the internal standard. ESI-MS was performed with a Waters ZQ-4000 mass spectrometer. Infrared spectra were recorded using a PerkinElmer FT-IR spectrometer with KBr pellets in the range of 4000−400 cm−1. Elemental analysis measurements were done using the PerkinElmer 2500 series II elemental analyzer. UV−visible and PL spectra were recorded on a PerkinElmer Lambda 25 UV−vis scanning spectrophotometer and Hitachi F-4500 and PerkinElmer LS55 fluorescence spectrophotometers with a quartz cuvette (path length = 1 cm). All spectroscopic measurements of 1[PF6]2 and 1[ClO4]2 were performed in a distilled organic solvent, Millipore water, and mixed aqueous buffer solution (CH3CN/0.1 M PBS buffer solution; pH 7.4). Excitation and the emission slit were set to 10 mm and the PMT volt at 700. DLS measurements were performed using Malvern Zen 3690. The SEM and TEM images were captured using Carl Zeiss SUPRA 55VP FESEM and JEOL JSM 100CX, respectively. The AFM images were taken in Nano surf Easyscan 2 in the dynamic mode. Synthesis of 4,7-Dichloro Phenanthroline (L). Ligand L was synthesized according to the reported literature procedure60 and was used for the synthesis of ruthenium complex, 1[PF6]2. Melting point (mp) 246−249 °C (lit. 249−250 °C); ESI-MS [C12H6Cl2N2+H]+: calcd, m/z = 249.00; found, m/z = 249.09. 1H NMR (400 MHz, CDCl3): δ (ppm) 9.07 (d, J = 4.8 Hz, 1H), 8.33 (s, 1H), 7.75 (d, J = 4.8 Hz, 1H). Synthesis of 1[PF6]2. A mixture of cis-[Ru(phen)2Cl2] (0.270 g, 0.500 mmol) and 4,7-dichloro phenanthroline (0.126 g, 0.500 mmol) was refluxed in 20 mL of EtOH/H2O (3:1) solution for 24 h under N2 atmosphere at 90 °C. After that, the reaction mixture was cooled down to room temperature, and the solution was concentrated under reduced pressure. An excess of NH4PF6 was added to the mixture. After the workup crude product was purified by silica gel column chromatography (CH3CN/H2O/saturated KNO3; 8.5:1:0.5), and the first dark red band was collected as a nitrate salt of 1[PF6]2. The excess of NH4PF6 was added to the collected portion and volume was reduced in a rotary evaporator. The dark red precipitate was filtered and washed by distilled water. Subsequent crystallization from acetone and CHCl3 (6:1) mixture provided 1[PF6]2 as shiny dark red crystals in 59% (0.360 g) yield. Melting point (mp) >300 °C. Anal. calcd for C36H22Cl2F12N6P2Ru·CHCl3 (Mw = 1119.88): C, 39.68; H, 2.07; N, 7.50. Found: C, 39.71; H, 2.02; N, 7.48. FT-IR in KBr disk (ν/cm−1): 3453, 1718, 1632, 1429, 1412, 1186, 1121, 836, 557. ESI-MS [C36H22Cl2F6N6PRu]+: calcd: m/z, 854.99; found: m/z, 854.99; 1H NMR (400 MHz, acetone-d6): δ (ppm) 8.80 (t, J = 7.6 Hz, 2H, H4,7), 8.70 (s, 1H, Hd), 8.53 (d, J = 5.0 Hz, 1H, H2), 8.46 (d, J = 5.7 Hz, 1H, Ha), 8.42 (s, 2H, H5,6), 8.37 (d, J = 5.0 Hz, 1H, H9), 7.96 (d, J = 5.7 Hz, 1H, Hb), 7.82−7.78 (m, 2H, H3,8).13C NMR (100 MHz, acetoned6): δ (ppm) 154.8 (1C, C2), 154.4 (1C, Ca), 154.0 (1C, C9), 149.9 (1C, Ce), 148.8 (2C, C11,12), 143.9 (1C, Cc), 138.0 (2C, C4,7), 132.0 (2C, C13,14), 130.4 (1C, Cf), 129.1 (1C, C5), 129.0 (1C, C6), 127.7 (1C, Cb), 127.1 (2C, C3,8), 126.6 (1C, Cd).

%cell viability = 100 × (R sample − R 0)/(R ctrl − R 0) where Rsample is the absorbance ratio of OD570/OD605 in the presence of the sample. Rctrl is the absorbance ratio of OD570/OD605 in the absence of the sample (vehicle control). R0 is the averaged background (noncell control) absorbance ratio of OD570/OD605. Cell Culture and Imaging. A fluorescence inverted microscope (Leica DMI4000B) was used to visualize the fluorescence of the cells following the addition of the respective compound with 20× objective lens. Fluorescence detection was carried out using an excitation filter BP 515-560 for 1[PF6]2. HeLa cells were cultured in Dulbecco’s modified Eagle medium containing low glucose (Invitrogen) with 10% fetal bovine serum (Invitrogen) at 37 °C in a 5% CO2 incubator chamber. One day before imaging, cells were seeded in 24-well flatbottomed plates. After 24 h cell growth, cells washed with PBS and fresh PBS (500 μL) were added in two successive wells. The wells were treated with 0−10 μM of 1[PF6]2 and incubated for 5−25 min. DNase and RNase Digest Experiments. For DNase and RNase digest experiments, cultured cells grown on a special confocal microscope dish were fixed by precooled methanol (−20 °C) for 10 min and washed with PBS for 5 min twice. For DNase and RNase digest tests, four sets of pretreated HeLa cells were stained with 1[PF6]2 (10 μM) and DAPI (1 μg/mL) and incubated for 20 min. After that, cells washed with PBS and fresh PBS (500 μL) were added in four successive wells. A total of 100 μL clean PBS (as control H


Research Article

ACS Applied Materials & Interfaces Table 1. Sequences of the Oligonucleotides Used in This Work name

sequence

structure

ssDNA1 ctDNA HUM24 22AG1 22AG2 22AG3 GQ-7090 GQ-268 RNA

5′-CCAGTTCGTAGTAAC3GACC-3′ from calf thymus 5′-GTTAG3TTAG3TTAG3TTAGG-3′ 5′-AG3TTAG3TTAG3TTAG3-3′ 5′-AG3TTAG3TTAG3TTAG3TTAG3-3′ 5′-AG3TTAG3TTAG3TTAG3TTAG3TTAG3-3′ 5′-AACTG5ATG4TGTGTG3TTAG3TGGCG4CAGG-3′ 5′-CTCCG3AGCG4CG4CGAA-3′ ∼70% of 16S- and 23S-ribosomal from backer yeast

single-strand double-strand hybrid-type G4 hybrid-type G4 hybrid-type G4 hybrid-type G4 Unknown Unknown single-strand

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experiment), 30 μg/mL DNase (Thermo Fisher Scientific, USA), 30 μg/mL DNase-Free RNase (Thermo Fisher Scientific, USA) and both DNase and RNase (30 μg/mL each) were added into the three adjacent wells and incubated at 37 °C in 5% CO2 for 2 h. Cells were rinsed by clean PBS twice before imaging. For each test, the fluorescent imaging pictures were obtained with an equal parameter for control. For the digest test using DAPI, the fluorescence detection was carried out using excitation filter BP 340-380. Images were processed by ImageJ software version 1.51J8. General Method for in Vitro RNA Binding Study. All oligonucleotides, ctDNA, ssDNA1, and RNA (from Baker’s yeast) used in this study were purchased from Integrated DNA Technologies, Merck, and Alfa Aesar, respectively (Table 1). Stock solutions of ctDNA and RNA were prepared by dissolving them in DNase- and RNase-free Millipore water. The concentrations of ctDNA and RNA were determined spectrophotometrically using the molar absorption coefficients of ε260nm = 6600 and 7800 M−1 cm−1, respectively. To obtain G-quadruplex formation, oligonucleotides were annealed in relevant buffer containing KCl (100 mM) by heating to 95 °C for 5 min, followed by gradual cooling to room temperature. The 1.0 mM stock solutions of 1[PF6]2 were prepared in distilled CH3CN for the PL measurements. In the emission titration study, the solution of 1[PF6]2 (10 μM) is titrated with aliquots of RNA solution using a PerkinElmer LS55 fluorescence spectrophotometer with a 1 mL quartz cuvette (path length = 1 cm). The concentration of RNA in working solutions was 1.0 mM. The PBS (pH = 7.4, 20 mM) was prepared using the DNase- and RNase-free Millipore water.

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ACKNOWLEDGMENTS This work was financially supported by DST, India (no. SB/ FT/CS/115/2012). We thank the DST Purse program for single-crystal X-ray diffraction facility at NEHU and Sophisticated Analytical and Instrumentation Facility (SAIF), North Eastern Hill University for NMR, MS, and TEM data. J. M. Baruah and Dr. Jyoti Narayan, School of Basic Science, NEHU, India, are gratefully acknowledged for the AFM study. We thank Dr. L. R. Singh of School of Nanotechnology, NEHU, India, for the solid state fluorescence data. S.K.S. and B.S. thank RGNF and North Eastern Hill University for their research fellowship. Also, we would like to thank the reviewers for their comments and suggestions.

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DEDICATION The present work is dedicated to Prof. Mihir Kanti Choudhuri on the occasion of his 70th birthday.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b19290.

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REFERENCES

1D and 2D NMR, ESI-MS, CV, UV−vis and PL data, DFT calculations for 1[PF6]2, associated figures, and crystallographic tables for three compounds (PDF) Crystallographic information for 1[PF6]2 (CIF) Crystallographic information for 1[ClO4]2 (CIF) Crystallographic information for 1[ClO4]2 (CH3CN)(H2O) (CIF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ORCID

Snehadrinarayan Khatua: 0000-0003-0992-4800 Author Contributions §

S.K.S. and B.S. contributed equally.

Notes

The authors declare no competing financial interest. I


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