Rational Design of Cyclometalated Iridium(III) Complexes for Three-Photon Phosphorescence Bioimaging

Compared to 2PE (two-photon excitation) microscopy, 3PE microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference. The design of suitable agents with a large Stokes shift, good three-photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is challenging. Now, two Ir^III complexes (3PAIr1 and 3PAIr2) were developed as efficient three-photon phosphorescence (3PP) agents. Calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three-photon transition probability. Confocal and lifetime imaging of mitochondria using Ir^III complexes as 3PP agents is shown. The complexes exhibit low working concentration (50 nm ), fast uptake (5 min), and low threshold for three-photon excitation power (0.5 mW at 980 nm). The impressive tissue penetration depth (ca. 450 μm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen.     

Theranostic Iridium(III) Complexes as One‐ and Two‐Photon Phosphorescent Trackers to Monitor Autophagic Lysosomes

During autophagy, the intracellular components are captured in autophagosomes and delivered to lysosomes for degradation and recycling. Changes in lysosomal trafficking and contents are key events in the regulation of autophagy, which has been implicated in many physiological and pathological processes. In this work, two iridium(III) complexes ( LysoIr1 and LysoIr2 ) are developed as theranostic agents to monitor autophagic lysosomes. These complexes display lysosome‐activated phosphorescence and can specifically label lysosomes with high photostability. Simultaneously, they can induce autophagy potently without initiating an apoptosis response. We demonstrate that LysoIr2 can effectively implement two functions, namely autophagy induction and lysosomal tracking, in the visualization of autophagosomal–lysosomal fusion. More importantly, they display strong two‐photon excited fluorescence (TPEF), which is favorable for live cell imaging and in vivo applications.     

Iridium(III) Anthraquinone Complexes as Two‐Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia

In the present study, four mitochondria‐specific and two‐photon phosphorescence iridium(III) complexes, Ir1 – Ir4 , were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia‐sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two‐electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant enhancement in phosphorescence intensity was observed under one‐ (λ=405 nm) and two‐photon (λ=720 nm) excitation, with a two‐photon absorption cross section of 76–153 GM at λ=720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two‐photon phosphorescence imaging.     

Cyclometalated Iridium(III) Complexes with Deoxyribose Substituents

Fundamental study of enzymatic nucleoside transport suffers for lack of optical probes that can be tracked noninvasively. Nucleoside transporters are integral membrane glycoproteins that mediate the salvage of nucleosides and their passage across cell membranes. The substrate recognition site is the deoxyribose sugar, often with little distinction among nucleobases. Reported here are nucleoside analogues in which emissive, cyclometalated iridium(III) complexes are “clicked” to C‐1 of deoxyribose in place of canonical nucleobases. The resulting complexes show visible luminescence at room temperature and 77 K with microsecond‐length triplet lifetimes. A representative complex is crystallographically characterized. Transport and luminescence are demonstrated in cultured human carcinoma (KB3‐1) cells.     

Cyclometalated Iridium(III) Complexes as Two‐Photon Phosphorescent Probes for Specific Mitochondrial Dynamics Tracking in Living Cells

Five cyclometalated iridium(III) complexes with 2‐phenylimidazo[4,5‐f][1,10]phenanthroline derivatives ( IrL1 – IrL5 ) were synthesized and developed to image and track mitochondria in living cells under two‐photon (750 nm) excitation, with two‐photon absorption cross‐sections of 48.8–65.5 GM at 750 nm. Confocal microscopy and inductive coupled plasma‐mass spectrometry (ICP‐MS) demonstrated that these complexes selectively accumulate in mitochondria within 5 min, without needing additional reagents for membrane permeabilization, or replacement of the culture medium. In addition, photobleaching experiments and luminescence measurements confirmed the photostability of these complexes under continuous laser irradiation and physiological pH resistance. Moreover, results using 3D multicellular spheroids demonstrate the proficiency of these two‐photon luminescent complexes in deep penetration imaging. Two‐photon excitation using such novel complexes of iridium(III) for exclusive visualization of mitochondria in living cells may substantially enhance practical applications of bioimaging and tracking.     

FerriIridium: A Lysosome‐Targeting Iron(III)‐Activated Iridium(III) Prodrug for Chemotherapy in Gastric Cancer Cells

Reported is the Fe^III‐activated lysosome‐targeting prodrug FerriIridium for gastric cancer theranostics. It contains a meta‐imino catechol group that can selectively bond to, and be oxidized by, free Fe^III inside the cell. Subsequent oxidative rearrangement releases Fe^II and hydrolyses the amine bond under acidic conditions, forming an aminobipyridyl Ir complex and 2‐hydroxybenzoquinone. Thus, Fe^II catalyzes the Fenton reaction, transforming hydrogen peroxide into hydroxyl radicals, the benzoquinone compounds interfere with the respiratory chain, and conversion of the prodrug into the Ir complex leads to an increase in phosphorescence and toxicity. These properties, combined with the high Fe^III content and acidity of cancer cells, make FerriIridium a selective and efficient theranostic agent (IC₅₀=9.22 μm for AGS cells vs. >200 μm for LO2 cells). FerriIridium is the first metal‐based compound that has been developed for chemotherapy using Fe^III to enhance both selectivity and potency.     

Carboranes Tuning the Phosphorescence of Iridium Tetrazolate Complexes

New iridium tetrazolate complexes containing o‐, m‐, or p‐carboranyl substitution in different positions of a phenylpyridine ligand have been prepared. The carborane isomers and the effect of their substitution position in the tuning of optical properties have been examined. The neutral complexes with the carboranyl substituent on the phenyl ring in meta position relative to the metal exhibit redshifted emission bands in contrast to blueshifts for those with carboranyl in para position. All cationic complexes display evidently blueshifted dual‐peak emission compared with the carborane‐free complex (c‐ TZ ) with a broad single‐peak emission. Introduction of carborane leads to a blueshift over 70 nm relative to c‐ TZ . Carboranes also significantly improve phosphorescence efficiency (Φ_P) and lifetime (τ), that is, Φ_P=0.64 versus 0.21 (c‐ TZ ) and τ=880 ns versus 241 ns (c‐ TZ ). The unique hydrophilic nido‐carborane‐based Ir^III complex nido‐o‐ 1 shows the largest phosphorescence efficiency (abs Φ_P=0.57) among known water‐soluble iridium complexes, long emission lifetime (τ=4.38 μs), as well as varying emission efficiency and lifetime with O₂ content in aqueous solution. Therefore, nido‐o‐ 1 has been used as an excellent oxygen‐sensitive phosphor for intracellular O₂ sensing and hypoxia imaging.     

Manipulation of Phosphorescence Efficiency of Cyclometalated Iridium Complexes by Substituted o‐Carboranes

A series of [(C^N)₂Ir(acac)] complexes [{5‐(2‐R‐CB)ppy}₂Ir(acac)] ( 3 a – 3 g ; acac=acetylacetonate, CB=o‐carboran‐1‐yl, ppy=2‐phenylpyridine; R=H ( 3 a ), Me ( 3 b ), iPr ( 3 c ), iBu ( 3 d ), Ph ( 3 e ), CF₃C₆H₄ ( 3 f ), C₆F₅ ( 3 g )) with various 2‐R‐substituted o‐carboranes at the 5‐position in the phenyl ring of the ppy ligand were prepared. X‐ray diffraction studies revealed that the carboranyl C?C bond length increases with increasing steric and electron‐withdrawing effects from the 2‐R substituents. Although the absorption and emission wavelengths of the complexes are almost invariant to the change of 2‐R group, the phosphorescence quantum efficiency varies from highly emissive (Φ_PL≈0.80 for R=H, alkyl) to poorly emissive (R=aryl) depending on the 2‐R group and the polarity of the medium. Theoretical studies suggest that 1) the almost nonemissive nature of the 2‐aryl‐substituted complexes is mainly attributable to the large contribution to the LUMO in the S₁ excited state from an o‐carborane unit and 2) the variation in the C?C bond length between the S₀ and T₁ state structures increases with increasing steric (2‐alkyl) and electronic effects (2‐aryl) of the 2‐R substituent and the polarity of the solvent. The solution‐processed electroluminescence (EL) devices that incorporated 3 b and 3 d as emitters displayed higher performance than the device based on the parent [(ppy)₂Ir(acac)] complex. Along with the high phosphorescence efficiency, the bulkiness of the 2‐R‐o‐carborane unit is shown to play an important role in improving device performance.     

Unveiling Photodeactivation Pathways for a New Iridium(III) Cyclometalated Complex

We report the synthesis and characterization of a neutral heteroleptic Ir^III complex bearing 6‐fluoro‐2‐phenylbenzo[d]thiazole as cyclometalating ligand and (Z)‐6‐(9H‐carbazol‐9‐yl)‐5‐hydroxy‐2,2‐dimethylhex‐4‐en‐3‐one as ancillary ligand. The photodeactivation mechanisms have been elucidated through extensive density functional theory (DFT) calculations. The active role of metal‐centered (³MC) triplet excited states in the nonradiative deactivation pathways is, for first time, confirmed in such complexes.     

Lysosome‐targeted Cyclometalated Iridium (III) Anticancer Complexes Bearing Phosphine‐Sulfonate Ligands

The synthesis, characterization and biological activity of four cyclometalated Ir (III) complexes ( Ir1 ‐ Ir4 ) containing different phosphine‐sulfonate ligands are reported. Most of these complexes showed good activity against A549 cancer cell lines and the human HeLa cervical cell lines. Spectroscopic properties study displays that all four complexes show rich fluorescence with emission maxima in the range of 474–510 nm. Fluorescence property of these complexes provides a tool to investigate the microscopic mechanism by confocal microscopy. Notably, the typical Ir (III) complex Ir4 can specially localize to lysosome, damage it and induce cell death via apoptosis. In addition, Ir4 enters into A549 cancer cells dominantly through energy‐dependent pathway.     

Cyclometalated Iridium (III) complexes: Recent advances in phosphorescence bioimaging and sensing applications

The phosphorescence bioimaging and sensing applications of Iridium (III) complexes, in particular to subcellular organelle staining as well as sensing of biologically important analytes, have been reviewed here. The bioimaging applications of the metal complexes provide an attractive alternative to fluorescent organic compounds in the construction of biosensors and biolabels because of having certain advantages.     

Cyanine-Like Boronic Acid-Derived Salicylidenehydrazone Complexes (Cy-BASHY) for Bioimaging Applications

Boronic acid-derived salicylidenehydrazone complex (BASHY) dyes with a polymethine backbone were designed to yield efficient red-emitting and two-photon absorbing fluorophores that can be used as markers for astrocytes. The dyes are chemically stable in aqueous solution and do not undergo photodecomposition. Their photophysical properties can be electronically fine-tuned and thereby adapted to potentially different imaging situations and requirements.     

Development of Strong Visible-Light-Absorbing Cyclometalated Iridium(III) Complexes for Robust and Efficient Light-Driven Hydrogen Production

Weak light absorption of common Ir(III) complexes (e. g., using phenylpyridine as the ligand) has hindered their applications in photocatalytic hydrogen generation from water as an efficient photosensitizer. To address this issue, a series of cyclometalated Ir(III) complexes (Ir1–Ir5), featuring different electron-donating substituents to enhance the absorptivity, have been synthesized and studied as photosensitizers (PSs) for light-driven hydrogen production from water. Ir6–Ir7 were prepared as fundamental systems for comparisons. Electron donors, including 9-phenylcarbazole, triphenylamine, 4,4′-dimethoxytriphenylamine, 4,4′-di(N-hexylcarbazole)triphenylamine moieties were introduced on 6-(thiophen-2-yl)phenanthridine-based cyclometalating (C^N) ligands to explore the donor effect on the hydrogen evolution performance of these cationic Ir(III) complexes. Remarkably, Ir4 with 4,4′-dimethoxytriphenylamine achieved the highest turn-over number (TON) of 12 300 and initial turnover frequency (TOF_i) of 394 h⁻¹, with initial activity (activity_i) of 547 000 μmol g⁻¹ h⁻¹ and initial apparent quantum yield (AQY_i) of 9.59 %, under the illumination of blue light-emitting diodes (LEDs) for 105 hours, which demonstrated a stable three-component photocatalytic system with high efficiency. The TON (based on n(H₂)/n(PSr)) in this study is the highest value reported to date among the similar photocatalytic systems using Ir(III) complexes with Pt nanoparticles as catalyst. The great potential of using triphenylamine-based Ir(III) PSs in boosting photocatalytic performance has also been shown.     

Emissive Iridium(III) Complexes with Phosphorous‐Containing Ancillary

Ir(III) metal complexes and related emitters bearing all kind of cyclometalated chromophoric chelates and non‐chromophoric ancillary are extensively studied during the past three decades. Many of them have been found to display bright room temperature phosphorescence from triplet excited states in both solution and solid states, offering a possible application in contemporary optoelectronic technologies, including organic light emitting diodes, electrochemiluminescence, biological imaging and chemical sensing. Among reported materials, there are Ir(III) complexes with at least one phosphorus (P)‐containing ligand and/or ancillary chelate, together with cyclometalates or equivalents that are in control of the actual emission energy. Particularly, possession of P‐based donor can lead to divergent structural and photophysical properties compared to the traditional designs. This review aims to provide a literature overview as well as the authors’ personal account to the development of relevant Ir(III) based phosphors bearing these P‐donors. To the readers’ convenience, the contents are subdivided into six sessions, according to whether or not they are charge natural, or with mono‐ or dianionic electronic character, and in accordance to their divergent bonding modes, i. e. monodentate, bidentate and tripodal coordination. In many cases, the P‐based ancillaries offer an easy accessible route to the formation of efficient sky‐blue and true‐blue emitters due to their π‐accepting property, together with enlarged emission energy gap and destabilized upper lying quenching state.     

Bimetallic Cyclometalated Iridium(III) Diastereomers with Non‐Innocent Bridging Ligands for High‐Efficiency Phosphorescent OLEDs

Two phosphorescent dinuclear iridium(III) diastereomers (ΛΔ/ΔΛ) and (ΛΛ/ΔΔ) are readily separated by making use of their different solubilities in hot hexane. The bridging diarylhydrazide ligand plays an important role in the electrochemistry and photophysics of the complexes. Organic light‐emitting devices (OLEDs) that use these complexes as the green‐emissive dopants in solution‐processable single‐active‐layer architectures feature electroluminescence efficiencies that are remarkably high for dinuclear metal complexes, achieving maximum values of 37 cd A^?1, 14 lm W^?1, and 11 % external quantum efficiency.     

Cyclometalated Iridium(III)–Polyamine Complexes with Intense and Long‐Lived Multicolor Phosphorescence: Synthesis,Crystal Structure,Photophysical Behavior,Cellular Uptake,and Transfection Properties

A new class of phosphorescent cyclometalated iridium(III)–polyamine complexes [{Ir(N^C)₂}_n(bPEI)](PF₆)_n (bPEI=branched poly(ethyleneimine), average M_w=25 kDa, n=15.6–27.4; HN^C=2‐phenylpyridine Hppy ( 1 a ), 2‐((1,1′‐biphenyl)‐4‐yl)pyridine Hpppy ( 2 a ), 2‐phenylquinoline Hpq ( 3 a ), 2‐phenylbenzothiazole Hbt ( 4 a ), 2‐(1‐naphthyl)benzothiazole Hbsn ( 5 a )) and [Ir(N^C)₂(en)](PF₆) (en=ethylenediamine; HN^C=Hppy ( 1 b ), Hpppy ( 2 b ), Hpq ( 3 b ), Hbt ( 4 b ), Hbsn ( 5 b )) have been synthesized and characterized. The X‐ray crystal structure of complex 5 b was also determined. All of these complexes showed a reversible iridium(IV/III) oxidation couple at +1.01 to +1.26 V and a quasi‐reversible ligand‐based reduction couple at ?1.54 to ?2.08 V (versus SCE). Upon photoexcitation, the complexes displayed intense and long‐lived green to orange–red emission in fluid solutions at room temperature and in low‐temperature glass. Lipophilicity measurements indicated that bPEI played a dominant role in the polar nature of complexes 1 a – 5 a , thus rendering them very soluble in aqueous solutions. Inductively coupled plasma–mass spectrometry (ICP‐MS) and confocal laser scanning microscopy (CLSM) data indicated that an energy‐requiring process, such as endocytosis, was involved in the cellular uptake of all of the complexes. In addition, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) and human embryonic kidney 293T (HEK293T) cell‐lines has been evaluated by the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The DNA‐binding properties of complex 5 a have been investigated by gel‐retardation assays and the polyplexes that were formed from this complex with plasmid DNA (pDNA) were studied by zeta‐potential measurements and particle‐size estimation. Furthermore, complex 5 a was grafted with poly(ethylene glycol) (PEG, average M_w=2 kDa) to different extents, thereby yielding the phosphorescent copolymers PEG_12.3‐g‐5 a , PEG_25.4‐g‐5 a , and PEG_62.1‐g‐5 a . Interestingly, these copolymers showed enhanced transfection activity, as revealed by in vitro transfection experiments with tissue‐culture‐based luciferase assays.     

Smart Poly(N‐isopropylacrylamide) Containing Iridium(III) Complexes as Water‐Soluble Phosphorescent Probe for Sensing and Bioimaging of Homocysteine and Cysteine

Homocysteine (Hcy) and cysteine (Cys) are two important kinds of amino acids in human bodies. Herein, we synthesized an iridium(III) complex‐functionalized poly(N‐isopropylacrylamide) and its hydrogel, which could be used as the excellent phosphorescent bioprobe for sensing Hcy and Cys. Their detection can be realized in aqueous system through the variations in absorption and photoluminescence spectra. Furthermore, living cell imaging experiments demonstrate that the phosphorescent bioprobe is membrane permeable and can monitor the changes of Hcy and Cys within living cells. In addition, the probe is also thermoresponsive, and its photoluminescence intensified with increasing temperature. These results suggests that this bioprobe has promising application in biomedical fields.