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Biomaterials 34 (2013) 1223e1234

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

The cellular uptake and localization of non-emissive iridium(III) complexes ascellular reaction-based luminescence probes

Chunyan Li 1, Yi Liu 1, Yongquan Wu1, Yun Sun 1, Fuyou Li*

Department of Chemistry & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, 220 Handan Road, Shanghai 200433, PR China

a r t i c l e i n f o

Article history:Received 25 August 2012Accepted 10 September 2012Available online 3 November 2012

Keywords:Iridium(III) complexNucleus stainFluorescent bioimagingStructureeactivity relationship

* Corresponding author. Fax: þ86 21 55664185.E-mail address: fyli@fudan.edu.cn (F. Li).

1 Fax: þ86 21 55664185.

0142-9612/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2012.09.014

a b s t r a c t

Improvement of cellular uptake and subcellular resolution remains a major obstacle in the successful andbroad application of cellular optical probes. In this context, we design and synthesize seven non-emissivecyclometalated iridium(III) solvent complexes [Ir(C

ˇ

N)2(solv)2]þL� (LIr2eLIr8, in which C

ˇ

N ¼ 2-phenylpyridine (ppy) or its derivative; solv ¼ DMSO, H2O or CH3CN; L� ¼ PF�6 or OTf�) applicable inlive cell imaging to facilitate selective visualization of cellular structures. Based on the above variations(including different counter ions, solvent ligands, and C

ˇ

N ligands), structureeactivity relationshipanalyses reveal a number of clear correlations: (1) variations in counter anions and solvent ligands ofiridium(III) complexes do not affect cellular imaging behavior, and (2) length of the side carbon chain inC

ˇ

N ligands has significant effects on cellular uptake and localization/accumulation of iridium complexesin living cells. Moreover, investigation of the uptake mechanism via low-temperature and metabolisminhibitor assays reveal that [Ir(4-Meppy)2(CH3CN)2]

þOTf� (LIr5) with 2-phenylpyridine derivative withside-chain of methyl group at the 4-position as C

ˇN ligand permeates the outer and nuclear membranes

of living cells through an energy-dependent, non-endocytic entry pathway, and translocation of thecomplex from the cell periphery towards the perinuclear region possibly occurs through a microtubule-dependent transport pathway. Nuclear pore complexes (NPCs) appear to selectively control the transportof iridium(III) complexes between the cytoplasm and nucleus. A generalization of trends in behavior andstructureeactivity relationships is presented, which should provide further insights into the design andoptimization of future probes.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Cells are the basic structural and functional units of all livingorganisms, and consist of subcellular organelles, including mito-chondria, Golgi apparatus, lysosomes, endoplasmic reticulum, andnucleus, which play important roles in numerous cellular events[1]. In particular, the nucleus houses chromosomes, and controlsmetabolism, heredity, and reproduction. Although the structure ofthe nucleus was discovered over two centuries ago [2], its biologicalroles remain to be fully elucidated. Studies of nuclear-relatedprocesses often require visualization of nuclei.

Fluorescent microscopy techniques using molecular probesprovide a unique approach for real-time and in situ observationof nuclear-related events owing to high temporal and spatial

All rights reserved.

resolution [3], and development of fluorescent nuclear stains hasattracted increasing attention in recent years. Currently, themost extensively used commercial nuclear imaging agents,Hoechst 33342 and 40,6-diamidino-2-phenylindole (DAPI),highlight the nuclear region via binding to or intercalatingbetween the stacked base pairs of nucleic acids [4]. However,these small-molecule organic dyes require ultraviolet (UV) lightas an excitation source, which causes photobleaching of thefluorescent stain and significant autofluorescence backgroundfrom biosamples [5].

To overcome the interference of autofluorescence backgroundfrom biological samples, time-gated luminescence bioimagingtechniques have been developed that promote the rapid develop-ment of long-lived luminescence probes [6e9]. Heavy-metalcomplexes are interesting candidates, possessing relatively longmicrosecond (ms) and millisecond (ms) lifetimes [6e9], in additionto other advantageous photophysical properties for bioimaging,such as enhanced photostability and large Stokes shifts for easydistinction between emission and excitation [10e26]. In recentyears, luminescent terbium, europium, ruthenium, iridium and

C. Li et al. / Biomaterials 34 (2013) 1223e12341224

platinum complexes have been applied in the cell nucleus imagingstudies. For example, Parker et al. [27] reported the effective use ofa Tb(III) complex with two trans-related azaxanthone chromo-phores to stain the nuclei of HeLa cells and monitor nuclear DNAchanges in dividing cells undergoing mitosis. The same groupdeveloped a europium complex that selectively stains the nucle-olus, both in live and fixed cells [28]. Lo et al. [29] reported thatIr(III) dipyridoquinoxaline complexes could stain the nucleoli of theMDCK cells after a long period of time. Barton and co-workersdemonstrated that a phosphorescent ruthenium complex conju-gated with a cell-penetrating peptide facilitates entry into thenucleus [30]. Furthermore, a dinuclear ruthenium(II) polypyridylcomplex was developed by Thomas et al. as a luminescent agentthat stained nuclear DNA of living cells [31]. These nuclear dyedesigns are focused on heavy-metal complexes that bond withnucleic acids and trigger an accompanying luminescent enhance-ment response.

In contrast to the above “light-switch” strategy of nucleicacid-bonding, we recently developed a non-emissive cyclo-metalated iridium(III) solvent complex [Ir(ppy)2(DMSO)2]þPF�6(LIr1) as a reaction-based luminescence turn-on agent specificfor nuclei of living cells [32]. LIr1, a non-nucleic acid-bondingprobe, is specifically concentrated in the nuclei of living cells andreacts with histidine/histidine-containing proteins to forma luminescent emissive product. Such a cyclometalated iridiu-m(III) complex was also developed as a highly selective lumi-nescent switch-on probe for protein staining [33]. To elucidatethe structural relationship with a view to optimizing efficiency ofcellular uptake and nuclear accumulation, we systematicallydesigned and synthesized a series of non-emissive cyclo-metalated iridium(III) solvent complex derivatives with modifi-cations in counter ions, solvent ligands, and C

ˇN ligands,

respectively (Chart 1). All seven iridium(III) solvent complexesshowed turn-on luminescence responses upon the addition ofhistidine and bovine serum albumin (BSA). The structureebehavioral activity relationship (with regard to counter ions,solvent ligands and C

ˇ

N ligands) for this class of metal complexesused as cellular reaction-based luminescence probes was inves-tigated in detail.

Chart 1. Chemical structures of the iridium(III

2. Materials and methods

2.1. Materials and general instruments

2-Phenylpyridine (ppy), 2-(4-methylphenyl)pyridine (4-Meppy), 2-(4-ethylphenyl)pyridine (4-Etppy), 2-(4-isopropylphenyl)pyridine (4-iPrppy), 2-(4-tert-butylphenyl)pyridine (4-Butppy) and 2-ethoxyethanol were obtained from FCHGroup (Ukraine). Phosphate-buffered saline (PBS), DMSO, AgOTf, L-alanine (Ala),L-arginine (Arg), L-asparagine (Asn), L-aspartic acid (Asp) L-glutamine (Gln), L-glycine(Gly), L-isoleucine (Ile), L-leucine (Leu), L-lysine (Lys), L-phenylalanine (Phe),L-proline (Pro), L-serine (Ser), L-threonine (Thr), L-tryptophan (Try), L-tyrosine (Tyr),L-valine (Val), L-glutamic acid (Glu), L-cysteine (Cys), L-methionine (Met), L-histidine(His), bovine serum albumin (BSA), deoxyribonucleotide triphosphate (dNTP) andCT DNA were obtained from Acros. IrCl3$3H2O was an industrial product and usedwithout further purification. Hoechst 33258, propidium iodide (PI), TubulineTrackerRed and cell culture reagents were purchased from Invitrogen.

1H NMR spectra were recorded with a Bruker spectrometer at 400 MHz. Elec-trospray ionization mass spectra (ESIeMS) were measured on a Micromass LCTTMsystem. UVevisible spectra were recorded on a Shimadzu UV-2550 spectrometer.Steady-state emission experiments at room temperature were measured on anEdinburgh instrument FL-900 spectrometer with Xe lamp as excitation source.Luminescence lifetime studies were performed with an Edinburgh FL-900 photo-counting systemwith a hydrogen-filled lamp as the excitation source. Luminescencequantum yields of LIrs-histidine in aerated solution were measured with referenceto quinine sulfate (0.55 in 0.05 M H2SO4).

2.2. Synthesis of iridium(III) complex LIrs

The complexes LIr2eLIr8 were synthesized according to previously reportedmethods [32,33]. Briefly, a mixture of 2-ethoxyethanol andwater (3:1, v/v) was addedto a flask containing IrCl3�3H2O (1.0 mmol) and the derivative ligands of2-phenylpyridine (2.5 mmol). The mixture was refluxed for 24 h. After cooling, theyellowsolid precipitatewasfiltered to give crude cyclometalated Ir(III) chloro-bridgeddimmer [34,35]. The chloro-bridged dimer (0.20 mmol) and AgOTf (0.44 mmol) wereplaced in the50mL roundbottomedflask.15mL solvent ligand (DMSO,H2OorCH3CN)was added, and the resulting slurry was stirred for 24 h. The solution was filteredthrough Celite and the precipitate was washed three times (2mL) with solvent ligand(DMSO, H2O or CH3CN). The filtrate and washings were combined and reduced byevaporation to a volumeof 1mL. This solutionwas cooled to 0 �C, and 25mL etherwasslowly addedwhile stirring. The bright yellowprecipitatewhich formedwas collectedby filtration and washed with ether and pentane, and then dried under vacuum for24 h to obtain the products (yield 80e85%) [34,35]. Then, the iridium complexeswerefurther characterized by 1H NMR and ESIeMS measurement.

LIr2: 1H NMR (400 MHz, d6-DMSO) d 9.50 (d, J ¼ 5.7 Hz, 2H), 8.40 (d, J ¼ 8.4 Hz,2H), 8.27 (t, J ¼ 7.7 Hz, 2H), 7.89 (d, J ¼ 7.8 Hz, 2H), 7.71 (t, J ¼ 6.6 Hz, 2H), 7.05(t, J ¼ 7.5 Hz, 2H), 6.92 (t, J ¼ 7.4 Hz, 2H), 5.91 (d, J ¼ 7.6 Hz, 2H), 2.54 (d, J ¼ 1.0 Hz,12H); (ESIeMS): Calcd. For C26H28IrN2O2S2 657.1 [M]þ, found 656.7 [M]þ.

) solvent complexes studied in this work.

C. Li et al. / Biomaterials 34 (2013) 1223e1234 1225

LIr3: 1H NMR (400 MHz, D2O) d 8.87 (d, J ¼ 5.0 Hz, 2H), 8.07 (d, J ¼ 8.1 Hz, 2H),7.98 (t, J ¼ 7.6 Hz, 2H), 7.67 (d, J ¼ 7.9 Hz, 2H), 7.46 (t, J ¼ 8.0 Hz, 2H), 6.88(t, J¼ 6.7 Hz, 2H), 6.71 (t, J¼ 7.5 Hz, 2H), 6.18 (d, J¼ 7.5 Hz, 2H); (ESIeMS): Calcd. ForC22H20IrN2O2 537.1 [M]þ, found 501.1 [Me2H2O]þ.

LIr4: 1H NMR (400 MHz, CDCl3) d 9.09 (d, J¼ 5.6 Hz, 2H), 7.91 (dd, J¼ 6.4, 1.4 Hz,4H), 7.54 (d, J¼ 6.9 Hz, 2H), 7.42 (td, J¼ 6.2, 2.5 Hz, 2H), 6.89 (dd, J¼ 10.8, 4.2 Hz, 2H),6.75 (dd, J¼ 7.4, 6.3 Hz, 2H), 6.08 (d, J¼ 7.6 Hz, 2H), 2.38 (s, 6H); (ESIeMS): Calcd. ForC26H22IrN4 583.2 [M]þ, found 583.2 [M]þ.

LIr5: 1H NMR (400 MHz, CDCl3) d 9.06 (d, J ¼ 5.6 Hz, 2H), 7.89 (dd, J ¼ 14.2,7.7 Hz, 4H), 7.46 (d, J ¼ 7.9 Hz, 2H), 7.40 (dd, J ¼ 9.4, 3.5 Hz, 2H), 6.74 (d, J ¼ 7.8 Hz,2H), 5.89 (s, 2H), 2.40 (s, 6H), 2.06 (s, 6H); (ESIeMS) Calcd. For C28H26IrN4 611.2 [M]þ,found 610.4 [M � H]þ.

LIr6: 1H NMR (400MHz, CDCl3) d 9.05 (d, J¼ 5.6 Hz, 2H), 7.94e7.79 (m, 4H), 7.45(d, J¼ 7.8 Hz, 2H), 7.41e7.35 (m, 2H), 6.75 (d, J¼ 7.9 Hz, 2H), 5.88 (s, 2H), 2.39 (s, 6H),2.36e2.30 (m, 4H), 0.98 (t, J ¼ 7.5 Hz, 6H); (ESIeMS): Calcd. For C30H30IrN4 639.2[M]þ, found 639.2 [M]þ.

LIr7: 1H NMR (400MHz, CDCl3) d 9.07 (d, J¼ 5.2 Hz, 2H), 7.91e7.83 (m, 4H), 7.44(d, J ¼ 7.8 Hz, 2H), 7.41e7.34 (m, 2H), 6.76 (d, J ¼ 7.7 Hz, 2H), 5.90 (s, 2H), 2.62e2.46(m, 2H), 2.39 (s, 6H), 1.11e0.78 (m, 12H).; (ESIeMS): Calcd. For C32H34IrN4 667.2[M]þ, found 667.2 [M]þ.

LIr8: 1H NMR (400 MHz, CDCl3) d 9.09 (d, J ¼ 5.6 Hz, 2H), 7.87 (dd, J ¼ 10.0,8.8 Hz, 4H), 7.40 (dd, J ¼ 10.9, 7.7 Hz, 4H), 6.91 (dd, J ¼ 8.2, 1.8 Hz, 2H), 6.05(d, J ¼ 1.7 Hz, 2H), 2.40 (s, 6H), 1.01 (s, 18H); (ESIeMS): Calcd. For C34H38IrN4 695.3[M]þ, found 695.3 [M]þ.

2.3. Interaction of LIrs with biomolecules

The interaction of LIrs with amino acids, bovine serum albumin (BSA), deoxy-ribonucleotide triphosphate (dNTP) and CT DNA have been investigated by lumi-nescent emission titration. Herein, L-alanine (Ala), L-arginine (Arg), L-asparagine(Asn), L-aspartic acid (Asp) L-glutamine (Gln), L-glycine (Gly), L-isoleucine (Ile),L-leucine (Leu), L-lysine (Lys), L-phenylalanine (Phe), L-proline (Pro), L-serine (Ser),L-threonine (Thr), L-tryptophan (Try), L-tyrosine (Tyr), L-valine (Val), L-glutamic acid(Glu), L-cysteine (Cys), L-methionine (Met) and L-histidine (His) were used asexamples of amino acids. In particular, the absorption and emission responses of LIrsto different amount of histidine were studied in detail.

2.4. Amphiphilicity measurement

The octanol/water partition coefficient, Po/w (or logPo/w), is a measure of theamphiphilicity of a material. It represents the relative solubilities of a given materialin oil and water. The octanol/water partition coefficient Po/w of LIrs was measured onan HY-4 oscillator according to a classical method. Equal amounts of n-octanol andphosphate-buffered saline (PBS) were thoroughly mixed in the oscillator for 24 h.The mixture was then left to separate for a further 24 h so as to yield water andoctanol phases, each saturated with the other. Complexes were carefully dissolved inPBS (concentration denoted as Co) and PBS saturated with octanol to form a 20 mmsolution, respectively. The latter was then mixed with an equal amount of octanol(saturated with water) and shaken again as described above. After separation, thefinal concentration of the complex in water was denoted as Cw. Both Co and Cwwere measured by an inductively coupled plasma atomic emission spectroscopy(ICPeAEC), and the partition coefficient (Po/w) for the complex was calculatedaccording to the equation: Po/w ¼ (Co�Cw)/Cw.

2.5. Cell culture

Human cervical carcinoma HeLa cells and human hepatocytes LO2 cells wereprovided by the Institute of Biochemistry and Cell Biology, Shanghai Institutes forBiological Sciences (SIBS), Chinese Academy of Sciences (CAS) (China). Primarymesenchymal stem cells (MSCs) was presented by the Jiangsu Stem Cell Bank. TheHeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supple-mented with 10% Fetal Bovine Serum (FBS). The LO2 cells were grown in RPMI 1640supplemented with 10% FBS. MSCs were grown in DMEM/F12 supplement with 10%FBS. All cells culture was at 37 �C under 5% CO2.

2.6. Luminescence imaging

2.6.1. Live cell imagingCells (5 � 108/L) were plated on 14 mm glass coverslips and allowed to adhere

for 24 h. The cells were washed with PBS and then incubated solely with 10 mmiridium(III) complexes in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 or 4 �C. Cellimaging was then carried out after washing the cells with PBS.

2.6.2. Co-staining of fixed cellsThe cells were detached from the culture and were fixed with 4% para-

formaldehyde at room temperature for 20 min. After washing with PBS, the fixedcells were incubated with 10 mm LIrs in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at37 �C, and then further stained with Hoechst 33258 for another 20 min. After

washing with PBS, the coverslips were separated from the chamber, and the cellswere mounted with 10% glycerol and sealed with nail varnish on a glass substrate.

Luminescence imaging, including xy-scan, lambda-scan, T-scan, and time-lapseimaging, was performed with an Olympus FluoView FV1000 confocal fluorescencemicroscope and a 60 � oil-immersion objective lens. Cells incubated with LIrs wereexcited at 488 nm with a semiconductor laser, and the emission was collected at520 � 20 nm. Quantization by line plots was accomplished using the softwarepackage provided by Olympus instruments. Hoechst 33258 was excited using a laserat 405 nm, and the emission was collected at 460 � 20 nm. PI was excited usinga laser at 488 nm, and the emission was collected at 620 � 20 nm. DiI dye wasexcited at 543 nm and emission was detected at 600 � 20 nm.

2.7. Protein isolation and electrophoresis

HeLa cells were maintained in DMEM supplemented with 10% fetal bovineserum (FBS) at 37 �C and 5% CO2. The cells were harvested and isolation of nuclearand cytoplasmic proteins was performed according to the Nuclear and CytoplasmicProtein Extraction Kit (Beyotime Biotech, China). The extraction proteins were dis-solved in SDS-sample buffer, heated according to the manufacturer’s instruction,and then separated using a 5% stacking gel and 12% resolving gel. After electro-phoresis, the gels were stained with complex LIrs (LIr5, LIr6 and LIr8, 50 mM) for20 min. The staining of the gels was measured on the BioeRad Gel Doc imagingsystem.

2.8. Cellular uptake and localization degree

HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) sup-plemented with 10% Fetal Bovine Serum (FBS) at 37 �C and 5% CO2. After beingdigested by TrypsineEDTA solution, the cells were counted and divided into fourparts. Each part (2 � 106 cells) were seeded in 75 cm2 culture flasks and allowed toadhere for 12 h before changing the culture medium to PBS solution with 10 mMiridium(III) complexes (LIr5, LIr6 and LIr8), respectively. The cells were incubatedwith the complex for 10 min at 37 �C under 5% CO2 followed by carefully washingcells with PBS solution. Then the nuclei and cytoplasms were extracted from HeLacells and were digested with a solution of HNO3 (0.25 mL, w15.7 M), H2O2 (0.05 mL,30%) and HF (0.02 mL, 5%) for 2 days, respectively. A solution of H3BO3 (0.1 mL, 0.4 M)was added and the mixture diluted with HNO3 (4.4 mL, 2%). The iridium concen-tration in the samples was determined using ICPeAEC analysis.

2.9. Temperature and inhibitor studies

2 � 106 HeLa cells seeded in a 75 cm2 culture flask were treated with 50 mM

2-deoxy-D-glucose and 5 mM oligomycin, 100 mM chloroquine, 50 mM NH4Cl, 1 mM

chlorpromazine, 20 mg/mL filipin, or 5 mg/mL cytochalasin D in serum-free DMEM for1 h before incubation of 10 mM iridium(III) complexes (LIr5, LIr6 and LIr8) withinhibitor in the fresh media for 15 min at 4 �C or 37 �C. After exposure to iridium(III)complexes and inhibitors for the desired time, the cells were washed with PBSsolution and then trypsinized and processed for ICPeAEC analysis as described inTheCellular Uptake and Localization Degree or carried out confocal luminescence imaging.

2.10. In vitro cytotoxicity

HeLa cells growing in log phase were seeded into 96-well cell culture plate at1 � 104/well and allowed to adhere for 24 h. The iridium complex (100 mL/well) atconcentration of 20 mM was added to the wells of the treatment group, and 100 mL/well DMSO diluted in DMEM at final concentration of 0.2% to the negative controlgroup, respectively. The cells were incubated for 24 h at 37 �C under 5% CO2. Thecombined MTT/PBS solution was added to each well of the 96-well assay plate, andincubated for an additional 4 h. An enzyme-linked immunosorbent assay (ELISA)reader (infinite M200, Tecan, Austria) was used to measure the OD570 (Absorbancevalue) of each well referenced at 690 nm. The following formula was used tocalculate the viability of cell growth:

Viabilityð%Þ ¼ ðmean of Absorbance value of treatment group=mean Absorbance

value of controlÞ$100

3. Results and discussion

3.1. Synthesis and photophysical properties of LIrs

The synthesis procedure for LIr2eLIr8 is outlined in Scheme S1.C

ˇ

N ligands were synthesized with good yield (w95%) via Suzukicoupling reaction of substituted phenylboronic acid with2-bromopyridine. The iridium(III) complex [Ir(C

ˇ

N)2(solv)2]þOTf�

(LIr2eLIr8) containing C

ˇ

N ligand and solvent ligand (DMSO, H2Oor CH3CN) was prepared with a two-step reaction using iridium(III)

Table 1Octanol/water partition coefficients of LIr2eLIr8 at room temperature.

Complex logPo/w

LIr2 �0.11LIr3 �0.09LIr4 �0.10LIr5 0.11LIr6 0.51LIr7 1.17LIr8 2.12

C. Li et al. / Biomaterials 34 (2013) 1223e12341226

m-chloro-bridged dimer complexes [Ir(C

ˇ

N)2Cl]2, according toa conventional procedure [35]. Firstly, [Ir(C

ˇ

N)2Cl]2 was synthesizedaccording to the method of Nonoyama [34]. Next, LIr2eLIr8 wasgenerated via a bridge-splitting reaction of crude [Ir(C

ˇ

N)2Cl]2 withAgOTf and subsequent complexation with the requisite solventligand (DMSO, H2O or CH3CN), with trifluoromethanesulfonic acidanion (OTf�) as the counterion. LIr2eLIr8 complexes were ob-tained with good yield (80%), and were further confirmed usingelectrospray ionization mass (ESIeMS) and nuclear magneticresonance (NMR) spectroscopy (Figs. S1eS14).

The photophysical properties of LIr2eLIr8 were investigatedusing spectroscopic techniques. The UV/vis absorption spectra ofLIr2eLIr8 in DMSO/PBS (pH 7.4, 1:99, v/v) buffer solution arepresented in Fig. S15. The complexes displayed intense high-energyabsorption bands in the region of 250e325 nm, and weak bands inthe 330e470 nm region, assigned to mixed singlet and tripletmetal-to-ligand charge-transfer (1MLCT and 3MLCT) and intra-ligand (pep*) transitions (C

ˇ

N). Similar to the solvent complex LIr1[32], the LIr2eLIr8 complexes exhibited negligible luminescence inboth solution and solid states at room temperature (Fig. 1), witha luminescence quantum yield of <0.1%.

3.2. Amphiphilicity test

To investigate the possible effectors of the cellular internaliza-tion process, lipophilicities of a range of iridium(III) complexeswere evaluated using the 1-octanol/water partition coefficient(logPo/w). As shown in Table 1, themeasured partition coefficients ofiridium(III) complexes ranged from hydrophilic (�0.11 for LIr2) tolipophilic (þ2.12 for LIr8). The lipid-water partition coefficients ofLIr3 and LIr4 are very close proximity to that of LIr2, while withintroducing side groups in the phenyl moiety, such as methyl, ethyl,isopropyl, and tertiary butyl groups at the 4-position, these iri-dium(III) complexes show more hydrophobic property.

3.3. Correlation of complex structures and cellular imagingbehavior

To design probes suitable for region-specific cellular imaging, itis important to understand the relationships between the chemical

Fig. 1. Images of the brightfield and room temperature luminescent emissions ofLIr2eLIr8 (100 mM) in DMSO/PBS (pH 7.4, 1:99, v/v) and their adducts with histidine,with [Ru(bpy)3]Cl2 as a reference (R). The excitation wavelength was set as 365 nmfrom a portable light source.

structures of iridium(III) complexes and their cellular uptakebehavior. Several structural aspects affect the intracellular distri-bution of these cyclometalated iridium(III) solvent complexes[Ir(C

ˇ

N)2(solv)2]þL�, such as the counterion, solvent ligand, and C

ˇ

Nligand components.

3.3.1. Effect of counter ionsIn consideration of the counter ions most commonly employed

in cation complexes, we initially modified the counterion of thereported complex [Ir(ppy)2(DMSO)2]þPF�6 (LIr1), and elucidatedthe potential effects on cell imaging. The anion component ofLIr1, PF�6 , was replaced with OTf� to form another complex,[Ir(ppy)2(DMSO)2]þOTf� (LIr2), and its interactions with livingHeLa cells were investigated using confocal luminescence micros-copy. As shown in Fig. 2, after incubationwith 10 mM LIr2 for 10min,intense luminescence was detected in the nuclear region underexcitation at 488 nm, while that in the cytoplasm was extremelyweak, similar to the imaging result obtained with LIr1. Comparablecell imaging phenomena were observed for other groups of[Ir(ppy)2(H2O)2]þPF�6 [32] and [Ir(ppy)2(H2O)2]þOTf� (LIr3). Thisfinding is not surprising, since these types of solvent complexes caneasily dissociate into cationic and anionic forms in aqueous solu-tion, whereby the cation portion of the complex is responsible forimaging behavior in living cells. Our observations clearly suggestthat the variations in counter anions do not affect the distributionof iridium complexes within cells, and consequently, cell imagingfindings.

3.3.2. Effect of solvent ligandsTo determine the effects of solvent ligands on cellular localiza-

tion and regional staining, solvent iridium(III) complexes contain-ing different auxiliary ligands, including DMSO, H2O, and CH3CN,were investigated in detail. Cell images of the three complexes,[Ir(ppy)2(DMSO)2]þOTf� (LIr2), [Ir(ppy)2(H2O)2]þOTf� (LIr3), and[Ir(ppy)2(CH3CN)2]þOTf� (LIr4), containing only one variable factorare presented in Fig. 2. Quantification of the luminescence intensityprofiles of HeLa cells treated with LIr2, LIr3 and LIr4 revealedextremely high luminescence signal-to-noise ratio between thenucleus and cytoplasm (Inucleus/Icytoplasm > 200), indicating exclu-sive nuclear staining (Fig. S16). This finding was further confirmedby complete intracellular colocalization with the nuclear counter-stain Hoechst 33258 (Fig. S17). Based on these observations, wepropose that solvent ligands play a negligible role in promoting theexclusive nuclear localization of these solvent iridium(III)complexes.

3.3.3. Effect of C

ˇ

N ligandsTo evaluate the effects of cyclometallated C

ˇ

N ligands in iridiumcomplexes on cell imaging behavior, 2-phenylpyridine (ppy) wasemployed as a starting matrix of the C

ˇ

N ligand, and subsequentlymodified by introducing side groups (changes in the number ofcarbon atoms) in the phenyl moiety. Consequently, four solventiridium (III) complexes [Ir(C

ˇ

N)2(CH3CN)2]þOTf� (LIr5eLIr8) with

Fig. 2. Confocal luminescence images, brightfield imagings and their overlay of living HeLa cells incubated with 10 mM LIr1eLIr8 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 37 �C,respectively. (lex ¼ 488 nm, lem ¼ 520 � 20 nm). Also, the amplification of their overlay was shown.

C. Li et al. / Biomaterials 34 (2013) 1223e1234 1227

side-chains of methyl, ethyl, isopropyl, and tertiary butyl groups atthe 4-position as C

ˇ

N ligands were synthesized, and cell imaginganalyses were performed to evaluate the correlation between C

ˇ

Nligand structures and cellular imaging behavior.

As shown in Fig. 2, efficient cellular uptake and nuclear stainingwere observed for [Ir(4-Meppy)2(CH3CN)2]þOTf� (LIr5), with ahigh signal ratio between the nucleus and cytoplasm (Inucleus/Icytoplasm > 200), as shown in Fig. S18. Upon incubation with[Ir(4-Etppy)2(CH3CN)2]þOTf� (LIr6), a generalized diffuse whole-cell staining pattern in both nuclei and cytoplasm with a lumines-cence turn-on signal was detected. With a further increase in theside carbon chain to three and four carbon atoms, i.e., [Ir(4-iPrppy)2(CH3CN)2]þOTf� (LIr7) and [Ir(4-Butppy)2(CH3CN)2]þOTf�

(LIr8), luminescence signals of the complexes were mainly local-ized in the cytoplasm and negligible in the nucleus (Icytoplasm/

Fig. 3. Confocal luminescence images and overlap rates of (a) living HeLa cells incubated withat 37 �C, followed by Hoechst 33258, and (b) fixed HeLa cells stained with LIr5 and Hoech

Inucleus > 100 for LIr7, Icytoplasm/Inucleus > 150 for LIr8) (Fig. S18), incontrast to the imaging behavior of the complex matrix[Ir(ppy)2(CH3CN)2]þOTf� (LIr4). Additionally, Z series imaging,performed to construct three-dimensional (3D) representations,indicated that LIr5 and LIr8 are localized in the nuclei and cyto-plasm of living cells, respectively (Fig. 4). Our results suggest thatcomplexes with increasing lengths of side carbon chain andbranched chains at the 4-position of the phenyl ring tend totranslocate from the nucleus and accumulate in the cytoplasm.

It should be noted that specific cellular region staining by LIrswith a luminescence enhancement effect is not restricted to cancercell lines, but cell-specific effects are operative. For example, theprimary cell lines, MSC and LO2, exhibit intense luminescence inspecific regions upon incubationwith LIrs (Fig. S19 and Fig. S20). Toensure that cells remained viable over the course of treatment at

10 mm [Ir(4-Meppy)2(CH3CN)2]þOTf� (LIr5) in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 minst 33258 under similar conditions.

Fig. 4. Three-dimensional (3D) luminescence images of live HeLa cells loaded with 10 mM LIr5 or LIr8 in DMSO/PBS (pH 7.4, 1:99, v/v) for 10 min at 25 �C. (a) The nucleuswas stained green color with [Ir(4-Meppy)2(CH3CN)2]þOTf� (LIr5) and the cell membrane stained red with DiI. (b) The cytoplasm was stained green color with[Ir(4-Butppy)2(CH3CN)2]þOTf� (LIr8) and the nucleus stained blue color with Hoechst 33258. (For interpretation of the references to color in this figure legend, the reader is referredto the web version of this article.)

C. Li et al. / Biomaterials 34 (2013) 1223e12341228

the concentrations of iridium complexes used, we quantified cellviability using trypan blue exclusion in separate experiments undersimilar conditions. Our results showed that about 96% of the cellsremained healthy. This observation was further confirmed by thelack of significant luminescence from propidium iodide (PI) signalupon co-staining of LIr-treated cells with PI.

3.4. Kinetic tracking of cellular uptake

To obtain further insight into the dynamic process ofcellular uptake of iridium(III) complexes, three compounds[Ir(4-Meppy)2(CH3CN)2]þOTf� (LIr5), [Ir(4-Etppy)2(CH3CN)2]þOTf�

(LIr6) and [Ir(4-Butppy)2(CH3CN)2]þOTf� (LIr8) were selected asexamples of real-time imaging of specific localization in thenucleus, cytoplasm and in both nuclei and cytoplasm in living cells.Initially, very weak luminescence was observed in the cells, prior toincubation with the iridium(III) complex. Subsequently, nuclear orcytoplasmic luminescence was detected within a very short periodof time (T0 < 30 s) for cells entering the focal plane of the objectivelens after incubation with LIr5 or LIr8, respectively (Fig. 5).Thereafter, a continuous increase in luminescence was observedwith increasing incubation times, and intensities saturated within6e8 min. Interestingly, luminescence enhancement was observedin both the cytoplasm and nucleus when living HeLa cells wereincubated with LIr6. Our results clearly indicate that iridium(III)complexes rapidly and selectively highlight specific regions ofliving cells, leading to significant luminescence enhancement.

3.5. Time-lapse imaging of LIrs in living cells

Since cells are living systems, continuous exchange of matterand energy occurs throughout the entire life process. To determinethe long-term kinetics of complex internalization, time-lapseimaging was carried out to monitor the progression of LIrs (LIr5,LIr6 and LIr8 as examples) in HeLa cells via a Live Cell Workstation.After incubation with specific LIrs complex for 10 min, the LIr-containing PBS solution was replaced with fresh DMEM for furtherculture. Confocal images were acquired after 15 min, 30 min, 6 h,12 h, and 24 h, respectively. In addition, commercially availablepropidium iodide (PI) was employed to evaluate the state of HeLacells. As shown in Fig. 6, we observed no significant changes in thelocalization of LIr5, LIr6 and LIr8 in HeLa cells, while luminescentemission became gradually weaker with time. Moreover, cellsstained negative for PI, indicated that LIr-treated cells remainedviable over the experimental time-course.

3.6. Interactions of LIrs with biomolecules

Since solvent iridium complexes are weakly emissive in bothsolution and solid states at room temperature, numerous substancesin the cell, including various amino acids, deoxyribonucleotide(dNTP) proteins and DNA, were examined to determine the keyfactors involved in the luminescence increase upon cellular stainingusing luminescent analysis technology. In the presence of histidine(His) or bovine serum albumin (BSA), all solvent complexesexhibited intense photoluminescence emission, with a maximalwavelength lmax range from 500 to 525 nm (Fig. S21eS27).The luminescence quantum yields of these adducts of solventiridiumcomplexeswere estimated as 3.43%e7.35% in theDMSO/PBSmixture (pH 7.4, 1:99, v/v) (Table S1). For example, interactionsof LIr6 with histidine resulted in emission enhancement of >200-fold (Fig. S28), corresponding to a quantum yield of 4.24% for theadduct. The luminescent lifetime of the LIr6 adduct withhistidinewasmeasured asw0.46 ms in the presence of oxygen in PBSsolution (Table S2), indicative of the phosphorescent nature of theemission. In contrast, weak luminescence changes were observedfor the other amino acids, deoxyribonucleotide triphosphate (dNTP)and CT DNA (Fig. 7). These results are consistent with previousfindings [32].

3.7. Protein electrophoresis

To investigate whether the differences in proteins within cellscontribute to cellular staining patterns, nuclear and cytoplasmicproteins of HeLa cellswere isolated and subjected to electrophoresis.Gelswere subsequently stainedwithcomplex LIrs (LIr5, LIr6, LIr7, orLIr8) (50 mM) for 20 min, using Coomassie Brilliant Blue as a refer-ence. As shown in Fig. 8, we observed luminescence enhancement ofboth nuclear and cytoplasmic proteinswith LIr5, whichmatched theCoomassie Brilliant Blue staining patterns, but displayed slightdifferences in terms of signal intensity. This result was in agreementwith imaging results showing that fixed cells incubated with LIr5display generalized diffuse whole-cell staining (Fig. 3). In addition,we investigated the interactions between proteins within cells andthe iridium complexes, LIr6, LIr7, and LIr8, using SDSePAGE gelanalysis (Fig. S29). Our findings were consistent with data obtainedfor LIr5. TEMeEDX analyses were performed, and the amounts ofiridium in the cytoplasm and nuclei of LIrs-stained cells werequantified via ICPeAEC measurement. These results discloseda correlation of complex distributionwith cellular imaging behavior(Fig. S30 and Table S2). Accordingly, we propose that intracellular

Fig. 5. Real-time monitoring of nuclear staining with LIr5, LIr6 and LIr8, respectively. (a) Luminescence images of living HeLa cells incubated with 10 mM complexes in DMSO/PBS(pH 7.4, 1:99, v/v) at 37 �C at selected time points (lex ¼ 488 nm, lem ¼ 520 � 20 nm). (b) Overlay of luminescence and bright-field images of living cells incubated with LIr6.(c) Time course of luminescence intensity in the nucleus (regions 1, 4 and 8), cytoplasm (regions 2, 5 and 7) and background (regions 3, 6 and 9).

C. Li et al. / Biomaterials 34 (2013) 1223e1234 1229

luminescence enhancement is mainly attributed to the distributionand accumulation of LIrs in cells, rather than differences in proteincomposition between the nucleus and cytoplasm.

3.8. Mechanisms of cellular internalization

To determine the key factors involved in iridium(III) complexinternalization, a series of uptake inhibition experiments werecarried out under conditions of different temperatures and meta-bolic and endocytic inhibitors. The results obtained are presentedin Fig. 9.

Firstly, to ascertain whether LIr5, LIr6 and LIr8 are taken up viaa passive or active transport mechanism, HeLa cells were incubatedwith the complexes at 4 �C. As shown in Fig. 9a, incubation of cellswith the complexes at this temperature led to inhibition of cellularuptake by 28%, 27% and 22%, respectively, indicating that thecomplexes enter cells through an energy-dependent pathway. Thisconclusion further supports earlier results obtained upon pre-treatment of cells with the metabolic inhibitors, 2-deoxy-D-glucose and oligomycin [36e38].

Endocytosis, a general entry mechanism for various extracel-lular materials, is an energy-dependent process [39]. To clearlydelineate the specific endocytotic pathways involved in cellular

internalization of iridium complexes, HeLa cells were pretreatedwith the inhibitors, chlorpromazine (clathrin-mediated) and filipin(caveolin-mediated). Co-incubation of cells with LIr5, LIr6 and LIr8and specific endocytosis inhibitors led to no effects on the inter-nalization ability of the complexes, as shown in Fig. 9a, suggestingthat these complexes are taken up by living cells via an energy-dependent, non-endocytotic pathway.

The nucleus of the cell is centrally important to an organism,serving to house chromosomes and control metabolism, heredity,and reproduction. Generally, small molecules taken up by livingcells need to cross two biologic barriers (cellular and nuclearmembranes) to eventually reach the nucleus. Limited informationis currently available about the interactions of iridium complexeswith intracellular organelles, proteins and transport behaviorwithin living cells. Here, we further examined the intracellularbehavior of LIr5 in the cytoplasm of living cells. Initially, chloro-quine and NH4Cl, two lysosomotropic agents that disrupt thelysosome acidification process, were employed to determine themechanism underlying nuclear accumulation of LIr5. After pre-incubation with chloroquine or NH4Cl at 37 �C for 1 h, LIr5 dis-played intense luminescence in the nuclei of living cells (Fig. 9b).No visible differences were detected, compared with the untreatedgroup, indicating that nuclear localization of LIr5 does not occur via

Fig. 6. Time-lapse imaging in situ of HeLa cells pre-incubated with 10 mM LIrs (LIr5, LIr6 and LIr8) for 10 min, and further cultured in fresh DMEM. Images were acquired after15 min, 30 min, 6 h, 12 h and 24 h, respectively. Inset: Propidium iodide (PI) staining is included to indicate cell viability.

Fig. 7. Luminescence intensity changes of 10 mM LIr5, LIr6 and LIr8 in the absence and presence of 50 mM various amino acids, BSA, 20-deoxyadenosine-50-triphosphate (dATP)20-deoxycytidine-50-triphosphate (dCTP), 20-deoxyguanosine-50-triphosphate (dGTP), 20-deoxythymidine-50-triphosphate (dTTP), and CT DNA in DMSO/PBS (pH 7.4, 1:99, v/v).Excitation and emission wavelengths were set at 365 and 520 nm, respectively.

C. Li et al. / Biomaterials 34 (2013) 1223e12341230

Fig. 8. SDSePAGE analysis of proteins from HeLa cells upon staining with Coomassiebrilliant blue complex G-250 and LIr5. (a) Coomassie brilliant blue G-250 staining.Lanes AeC present cytoplasm proteins, nuclear proteins, and BSA, respectively. (b) LIr5staining under ultraviolet transillumination (lex ¼ 365 nm). Lanes A1eC1 stand forcytoplasm proteins, nuclear proteins, and BSA, respectively. “M” denotes proteinmarkers.

C. Li et al. / Biomaterials 34 (2013) 1223e1234 1231

the lysosome pathway. Subsequently, cytochalasin D and colchicine(small molecules that disrupt actin filaments or microtubules) wereused to establish whether the microtubuleemicrofilament systemis involved in LIr5 delivery. Interestingly, treatment with microtu-bule and microfilament inhibitors caused significant inhibition ofspecific nuclear staining, in contrast to the untreated group

Fig. 9. Probing themechanism of cellular uptake of iridium(III) complexes (LIr5, LIr6 and LIr8)incubated with the indicated inhibitors at different temperatures, followed by 10 mM iridium(Iiridium(III) complexes by HeLa cells at 4 �C and 37 �C. Cells were pretreated with metabolic inchlorpromazine or 200 mg/mL filipin). (b) Confocal images of living HeLa cells pretreated withincubation with 10 mM iridium(III) complexes for 15 min. Percent internalization was normaliz

(Fig. 9b), suggesting that the translocation of LIr5 from the cellperiphery towards the perinuclear region is caused by themicrotubule-dependent transport pathway, an active processmediated by molecular motors, such as dyneins [40,41]. The ulti-mate localization of LIr5 in the nucleus may be attributed to theselectivity of nuclear pore complexes (NPCs), such as proteinimportin b, for nuclear transport [42].

To examine this theory, we performed cell cycle synchronizationexperiments where HeLa cells were delayed at the mitosis meta-phase (M phase) via treatment with colchicine. Generally, thestructure of the nuclear envelope is intact, mainly consisting ofnuclear lamina and nuclear pore complexes that mediate nucleo-cytoplasmic exchange [43]. The nuclear membrane temporarilydisintegrates (within a very short period of time in the cellcycle,w1 h) when the cell undergoes mitosis, and reappears in latemitosis. The LIr8 complex that specifically stains the cytoplasm ofliving cells was employed to investigate the selective permeableeffect of the nuclear envelope on nuclear entry for different struc-tural iridium complexes. After pre-incubation with 0.5 mg/mLcolchicine solution for 6 h, HeLa cells were blocked at the M phase.Colchicine-containing DMEM was replaced with fresh DMEM forfurther culture, and cells returned to the normal cell cycle.Following release from colchicine, HeLa cells were collected atdifferent time-points (M: 0.5 h, G1: 5.5 h, S: 16.5 h and G2: 21 h),and the efficiency of cell cycle synchronization confirmed via flowcytometry analysis (Fig. 10a and Fig. S31). Interestingly, duringmitosis of HeLa cells (whereby chromosomes congressed onto theequatorial plate), LIr8 displayed a generalized diffuse whole-cellstaining pattern, as shown in Fig. 10b. In contrast, cytoplasmicstaining was observed at the other stages (G1, S and G2) of the cellcycle, consistent with previous studies (Fig. 2). These observationssuggest that the nuclear envelope (in particular, NPC) plays animportant role in the process of penetration of solvent iridium(III)complexes into the nucleus, and may determine the ultimatelocalization of different structural complexes. Further research todetermine the precise mechanism of nuclear transport is ongoing.

On the basis of the results above, we propose a possiblegeneralization of trends in cellular staining behavior andstructureeactivity relationships of [Ir(C

ˇ

N)2(solv)2]þL� (LIrs) as

using inhibitors ofmetabolismand endocytosis at different temperatures. HeLa cellswereII) complexes for 15 min. (a) Temperature dependence and inhibitor studies of uptake ofhibitors (50 mM 2-deoxy-D-glucose and 5 mM oligomycin) or endocytosis inhibitors (1 mM100 mM chloroquine, 50 mM NH4Cl, 5 mg/mL cytochalasin D or 1 mM colchicine, followed byed to iridium(III) complex uptake in the absence of inhibitors at 37 �C.

Fig. 10. (a) Flow cytometric histogram profile of cell cycle synchronization. (b) Confocal luminescence images of living HeLa cells incubated with 10 mM LIr8 in DMSO/PBS (pH 7.4,1:99, v/v) for 10 min at 37 �C after release from colchicine at different time points (M: 0.5 h, G1: 5.5 h, S: 16.5 h and G2: 21 h) (LIr8: lex ¼ 488 nm, lem ¼ 520 � 20 nm, Hoechst33258: lexc ¼ 405 nm, lem ¼ 460 � 20 nm, TubulineTracker Red: lex ¼ 543 nm, lem ¼ 570 � 20 nm).

C. Li et al. / Biomaterials 34 (2013) 1223e1234 1233

follows: (1) variations in counter anions and solvent ligands ofiridium(III) complexes do not affect cellular imaging behavior, and(2) the number of carbon atoms in the side-chain of C

ˇ

N ligands hasa drastic effect on cellular uptake and localization of iridiumcomplexes in living cells. LIr5 translocation from the cell peripherytowards the perinuclear region is possibly stimulated by a micro-tubule-dependent transport pathway, and nuclear porecomplexes (NPCs) appear to selectively control the transport ofdifferent structural iridium(III) complexes between the cytoplasmand nucleus. However, the specific proteins that facilitate crossingof the nuclear membrane by LIrs are yet to be identified. (3)Replacement of leaving groups (solvent ligands) with histidine/histidine-containing proteins promotes luminescence turn-onprocesses in cells. (4) Hydrophobicity and molecular size of iri-dium(III) complexes are additional important factors in the cellularuptake and localization of LIrs.

3.9. Cytotoxicity of iridium(III) complexes

An ideal cellular probe for practical application should mini-mally perturb living systems at the concentrations employed.Accordingly, the cytotoxicities of solvent iridium(III) complexeswere determined using the MTT assay. In general, at a complexconcentration of 20 mM, cellular viability was estimated as greaterthan 90% after incubation for 24 h (Fig. S32). The results indicatethat iridium complexes generally present low toxicity for lumi-nescence cell imaging under the conditions applied (incubationtime of <10 min, iridium complex concentration of 10 mM).

4. Conclusion

We have demonstrated the design concept and practical appli-cation of cyclometalated iridium(III) solvent complexes LIrs ascellular reaction-based luminescence probes for regional stainingin living cells. LIrs are rapidly internalized and selectively accu-mulate within living cells, giving rise to significant luminescenceenhancement via binding to histidine/histidine-containingproteins. A major outcome of the present study is the revelationthat the number of carbon atoms in the side-chain of C

ˇ

N ligandsexert a significant effect on the cellular uptake and localization ofiridium complexes in living cells, while variations in counter anionsand solvent ligands of the complexes do not affect cellular imagingbehavior. There is evidence to indicate that the microtubule systemparticipates in the transportation of iridium complexes in thecytoplasm, and nuclear pore complexes (NPCs) selectively controlthe transport of iridium complexes between the cytoplasm andnucleus. Most importantly, through systematic investigation of thecorrelation between complex structures and cellular imagingbehaviors offers an insight into the possible inherent mechanismsunderlying the destiny of these complexes for effective use ascellular reaction-based luminescence probes, which should facili-tate the introduction of new strategies for the development ofluminescent agents in living cell-related studies.

Conflict of interest

No financial conflict of interest was reported by the authors ofthis paper.

Funding

This work was financially supported by NSFC (20825101,21231004 and 91027004), Shanghai Sci. Tech. Comm.(11XD1400200 and 10431903100), and SLADP (B108).

Appendix A. Supplementary data

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.biomaterials.2012.09.014.

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