Ir(III) Complexes with AIE Characteristics for Biological Applications

Both biological process detection and disease diagnosis on the basis of luminescence technology can provide comprehensive insights into the mechanisms of life and disease pathogenesis and also accurately guide therapeutics. As a family of prominent luminescent materials, Ir(III) complexes with aggregation-induced emission (AIE) tendency have been recently explored at a tremendous pace for biological applications, by virtue of their various distinct advantages, such as great stability in biological media, excellent fluorescence properties and distinctive photosensitizing features. Significant breakthroughs of AIE-active Ir(III) complexes have been achieved in the past few years and great progress has been witnessed in the construction of novel AIE-active Ir(III) complexes and their applications in organelle-specific targeting imaging, multiphoton imaging, biomarker-responsive bioimaging, as well as theranostics. This review systematically summarizes the basic concepts, seminal studies, recent trends and perspectives in this area.


Introduction
Iridium(III) complexes have attracted significant scientific interest in various fields, ranging from photoelectric devices, catalysis and chemical sensing to biological applications, benefitting from their prominent photophysical properties. Ir(III) has a higher spinorbit coupling (SOC) constant (4430 cm −1 ) than other transition metals (Ru(II): 990 cm −1 , Rh(III): 1425 cm −1 , Os(II): 3531 cm −1 , Pt(II): 4000 cm −1 ) and the strong SOC effect can accelerate the intersystem crossing (ISC) process [1,2]. Moreover, Ir(III) can easily form multiple coordination compounds. The structures of auxiliary ligands that coordinate with the metal center are rich and diverse, resulting in efficient metal to ligand charge transfer (MLCT), ligand-centered (LC) and ligand to ligand charge transfer (LLCT) [3][4][5], which causes strong phosphorescent emission and long excited-state lifetimes [6]. Meanwhile, Ir(III) complexes possess tunable photophysical properties, satisfactory photostability and large Stokes shifts. On the basis of those features, in particular, Ir(III) complexes are receiving great attention as promising candidates for optical bioimaging and phototherapy [7][8][9].
Photodynamic therapy (PDT) has accurately eradicated tumors and reduced side effects in healthy tissues by controlling the area of light irradiation. In general, the tumor apoptosis caused by PDT goes through photochemical reactions between excited triplet states of photosensitizers (PSs) and cellular substrates or oxygen to generate cytotoxic reactive oxygen species (ROS). Typically, the activated PSs may migrate to their triplet excited state via ISC. After that, the triple-activated PSs further react with oxygen to afford ROS via electron transfer (type I) or energy transfer (type II) processes [10]. Of particular interest is the heavy atom effect of Ir atoms, which plays an important role in efficiently enhancing the ability of ISC and effectively improving the generation efficiency of ROS [11][12][13]. However, the aggregation-caused quenching (ACQ) effect caused by the aggregation of Ir(III) complexes in an aqueous physiological environment leads to a reduction in luminescence emission and the generation capacity of ROS, which further limits their practical applications in the biomedical field [14,15].
Fortunately, aggregation-induced emission (AIE) was proposed in 2001 to solve the problem of unsatisfactory emission of luminescent materials in the aggregated state [16]. The main mechanism of the AIE phenomenon is the restricted intramolecular motion (RIM), thereby blocking the nonradiative energy decay pathway and boosting the radiative channel [17]. In recent years, AIE luminogens (AIEgens) have attracted extensive attention in the field of bioimaging, sensing and therapy applications, thanks to their distinct properties, including excellent photobleaching resistance and reliable output signal in an aggregated state [18][19][20]. In particular, AIEgens have unique aggregation-enhanced theranostics (AET) properties, referring to the advantages of enhancements in ROS generation, nonlinear optical effects under aggregation-induced effects [21,22]. On the other hand, many cases of Ir(III) complexes with AIE properties have been reported to date [23][24][25][26] and the following design strategies are generally recommended: (i) introducing active units with AIE properties into Ir(III) complexes and (ii) restricting intramolecular motion of flexible rotating units (such as imine bonds of Schiff bases) through coordination with the central metal iridium. Ir(III) complexes are potential phosphorescent molecules for the construction of AIE materials via strategic modification of ligands [27]. Therefore, constructing AIE phosphorescent materials based on Ir(III) complexes is one of the most promising candidates in the field of photo-triggered theranostics.
For bioimaging applications, Ir(III) complexes with the AIE property encounter aggregation spontaneously in a physiological environment, providing good signal-to-noise ratio and high sensitivity in specific responses to analytes. In addition, general Ir(III) complex PSs mainly exhibit two challenges in the field of PDT: (i) the original short excitation wavelength (300-450 nm) causes poor tissue penetration and adverse to deep tumor treatment [28,29]; (ii) ROS have a limited area of action region (20~40 nm) and short lifetime (<40 ns), so organelle-targeting is a desirable strategy to further enhance the efficacy of PDT [30][31][32]. At present, AIE-active Ir(III) complexes are still under development in the field of biological applications. Nonetheless, considerable progress has been made on AIE materials based on Ir(III) complexes to address the above two issues. For example, the excellent optical properties of Ir(III) complexes can overcome the problem of penetration depth of PSs through two-photon excitation [33][34][35][36]. Meanwhile, the positive charge and lipophilicity of the cationic Ir(III) complexes exhibit potential affinity to mitochondria [37][38][39][40]. This mini-review summarizes and presents the development of AIE-active Ir(III) complexes (Scheme 1) for bioimaging, sensing and therapy applications, in order to stimulate more researchers to enter this field. Biosensors 2022, 12, x FOR PEER REVIEW 3 of 21 Scheme 1. Chemical structures of Ir(III) complexes with AIE properties in biological field.

Improving Photophysical Property of Ir(III) Complexes with AIE Activity
The main drawback of Ir-based PSs is that they are usually excited by UV/Vis light, which causes shallower penetration and limits the treatment of thicker and larger tumors [2,41]. Therefore, many concerns have focused on improving excitation and emission spectra of Ir(III) complexes [42].

Improving Photophysical Property of Ir(III) Complexes with AIE Activity
The main drawback of Ir-based PSs is that they are usually excited by UV/Vis light, which causes shallower penetration and limits the treatment of thicker and larger tumors [2,41]. Therefore, many concerns have focused on improving excitation and emission spectra of Ir(III) complexes [42].

Red-Shift Emission
Making the emission red shift is a good way to reduce the interference of background antofluorescence and increase the depth of penetration [43]. Yang et al. [44] presented complex 1, featuring AIE for image-guided PDT for cancer ( Figure 1A). Complex 1 exhibited an absorption peak at~384 nm ( Figure 1B), with phosphorescence peaking at~630 nm ( Figure 1B). When the EtOH fraction was more than 20%, 1 exhibited weak phosphorescence intensity. Furthermore, the solid of 1 also demonstrated bright-red phosphorescence (in the inset of Figure 1D) and the solid-state phosphorescence quantum yield was 5.5%. Hence, 1 showed typical AIE features. It was observed that the ROS genergation efficiency of 1 was much better than that of Rose Bengal (RB), which is a commercially available and popular PS ( Figure 1E). MTT assessment revealed that 1 displayed both high phototoxicity and low dark cytotoxicity ( Figure 1F). Moreover, a cell-imaging experiment was conducted via the incubation of HeLa cells with 1, as illustrated in Figure 1G. Cells can be clearly visualized with excellent image contrast to the cell background.

Red-Shift Emission
Making the emission red shift is a good way to reduce the interference of background antofluorescence and increase the depth of penetration [43]. Yang et al. [44] presented complex 1, featuring AIE for image-guided PDT for cancer ( Figure 1A). Complex 1 exhibited an absorption peak at ~384 nm ( Figure 1B), with phosphorescence peaking at ~630 nm ( Figure 1B). When the EtOH fraction was more than 20%, 1 exhibited weak phosphorescence intensity. Furthermore, the solid of 1 also demonstrated bright-red phosphorescence (in the inset of Figure 1D) and the solid-state phosphorescence quantum yield was 5.5%. Hence, 1 showed typical AIE features. It was observed that the ROS genergation efficiency of 1 was much better than that of Rose Bengal (RB), which is a commercially available and popular PS ( Figure 1E). MTT assessment revealed that 1 displayed both high phototoxicity and low dark cytotoxicity ( Figure 1F). Moreover, a cell-imaging experiment was conducted via the incubation of HeLa cells with 1, as illustrated in Figure 1G. Cells can be clearly visualized with excellent image contrast to the cell background.  Compared with red emission, near-infrared (NIR) emission has better performance in overcoming autofluorescence interference, minimizing photodamage to cells and improving the signal-to-noise ratio (SNR) of imaging [45,46]. Li et al. designed and synthesized mono-and tri-nuclear NIR AIE cationic Ir(III) complexes and corresponding nanoparticles (NPs) 2 and 3 by self-assembling ( Figure 2A) [47]. Further, 2 and 3 showed bright NIR emission at 710 nm and 730 nm when the water fraction reached 90%, showing typical AIE character ( Figure 2B,C). The photoluminescence quantum yields (PLQYs) of 2 NPs and 3 NPs were 25% and 22% in water, which were higher than those of 2 and 3 (6% and 4%, respectively). Remarkably, the NPs possessed similar emission peaks while PL intensities were obviously enhanced. The greater biological NIR emission was valuable for efficient biological applications. Intracellular NIR emission was monitored, showing that 3 NPs exhibited excellent imaging performance ( Figure 2D). The 1 O 2 production was evaluated by using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator and brightgreen fluorescence was observed with 3 and 3 NPs upon light irradiation ( Figure 2E). These results proved that 3 NPs could be applied as an efficient PS for imaging-guided PDT. Compared with red emission, near-infrared (NIR) emission has better performance in overcoming autofluorescence interference, minimizing photodamage to cells and improving the signal-to-noise ratio (SNR) of imaging [45,46]. Li et al. designed and synthesized mono-and tri-nuclear NIR AIE cationic Ir(III) complexes and corresponding nanoparticles (NPs) 2 and 3 by self-assembling ( Figure 2A) [47]. Further, 2 and 3 showed bright NIR emission at 710 nm and 730 nm when the water fraction reached 90%, showing typical AIE character ( Figure 2B,C). The photoluminescence quantum yields (PLQYs) of 2 NPs and 3 NPs were 25% and 22% in water, which were higher than those of 2 and 3 (6% and 4%, respectively). Remarkably, the NPs possessed similar emission peaks while PL intensities were obviously enhanced. The greater biological NIR emission was valuable for efficient biological applications. Intracellular NIR emission was monitored, showing that 3 NPs exhibited excellent imaging performance ( Figure 2D). The 1 O2 production was evaluated by using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as an indicator and bright-green fluorescence was observed with 3 and 3 NPs upon light irradiation ( Figure 2E). These results proved that 3 NPs could be applied as an efficient PS for imaging-guided PDT.   [47], copyright 2020, Royal Society of Chemistry.

Enhancing Molar Absorption Coefficient
In general, the upper limit of the output signal depends on the absorption capacity of the PSs. Actually, high brightness can be achieved by increasing the molar absorption coef- ficients. Therefore, improving absorptive capacity has been the focus of many efforts [48]. Zhang et al. proposed, for the first time, that additional metal centers further resulted in an increase in the molar absorption coefficient by increasing 3 MLCT [49]. They designed the first example of an AIE-active multinuclear Ir(III) complex. Adding Ir(III) metal centers, the molar absorption coefficient increased ( Figure 3A,B). Thus, 4 NPs, 5 NPs and 6 NPs showed higher PLQYs (33%, 15% and 35%, respectively) in water than those of 4, 5 and 6 (11%, 5%, and 8%, respectively) in THF-water mixtures. Confocal laser scanning microscopy (CLSM) demonstrated the cellular uptake of PS and 6 NPs showed red fluorescence in cells ( Figure 3C). The enhanced cellular uptake was highly advantageous to improve PDT. With light irradiation, green emission was observed from cells, indicating the presence of 1 O 2 generation ( Figure 3D). This study offered a new method to improve the efficiency of PSs for clinical cancer treatments. cells treated with 3 and 3 NPs under light. Reprinted with permission from [47], copyright 2020, Royal Society of Chemistry.

Enhancing Molar Absorption Coefficient
In general, the upper limit of the output signal depends on the absorption capacity of the PSs. Actually, high brightness can be achieved by increasing the molar absorption coefficients. Therefore, improving absorptive capacity has been the focus of many efforts [48]. Zhang et al. proposed, for the first time, that additional metal centers further resulted in an increase in the molar absorption coefficient by increasing 3 MLCT [49]. They designed the first example of an AIE-active multinuclear Ir(III) complex. Adding Ir(III) metal centers, the molar absorption coefficient increased ( Figure 3A,B). Thus, 4 NPs, 5 NPs and 6 NPs showed higher PLQYs (33%, 15% and 35%, respectively) in water than those of 4 ,5 and 6 (11%, 5%, and 8%, respectively) in THF-water mixtures. Confocal laser scanning microscopy (CLSM) demonstrated the cellular uptake of PS and 6 NPs showed red fluorescence in cells ( Figure 3C). The enhanced cellular uptake was highly advantageous to improve PDT. With light irradiation, green emission was observed from cells, indicating the presence of 1 O2 generation ( Figure 3D). This study offered a new method to improve the efficiency of PSs for clinical cancer treatments. NPs. Reprinted with permission from [49], copyright 2019, Wiley-VCH.

With the UCNPs
Upconversion nanoparticles (UCNPs) are desirable photoconversion materials for biosensing and biomedicine on account of their capability to transform the NIR photons to UV/visible photons [50,51]. Therefore, the conjugation of PSs to UCNPs presents an alternative to achieve NIR light-triggered generation of ROS. With this idea in mind, Liu et al.

With the UCNPs
Upconversion nanoparticles (UCNPs) are desirable photoconversion materials for biosensing and biomedicine on account of their capability to transform the NIR photons to UV/visible photons [50,51]. Therefore, the conjugation of PSs to UCNPs presents an alternative to achieve NIR light-triggered generation of ROS. With this idea in mind, Liu et al. [52] employed AIE-active non-charged Ir(III) complexes integrated with UCNPs for the first time ( Figure 4A). In UV-vis absorption spectra, 7 and 8 exhibited absorption peaks at~425 nm. The PL spectra showed that 7 emitted at 640 nm and 8 emitted at 660 nm ( Figure 4B), while the PLQYs were 16.6% and 18.9%, respectively. When the water fraction reached 50%, the emission intensity of 8 was greatly enhanced, which demonstrated that 8 had typical AIE features ( Figure 4C). Further, 8 was then encapsulated with UCNPs within d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) to construct UCNPs@8 as PSs for the ensuing in vitro experiments. The UV-vis absorption spectra of UCNPs@8 showed a significant absorption at about 425 nm, corresponding to that of 8 ( Figure 4D). To ensure that the energy transferred between 8 and UCNPs can be realized and UCNPs@8 could be irradiated by NIR light, the PL spectrum of UCNPs was measured under laser irradiation (980 nm). The absorption band of 8 was in the UV and blue regions, overlapping with the emission bands of UCNPs ( Figure 4E). Encouraged by the 1 O 2 generation ability of NIR-irradiated UCNPs@8, their ability to kill cancer cells through PDT was investigated. MTT assays indicated that UCNPs@8 had considerable cytotoxicity ( Figure 4F). To further evaluate the therapeutic effect of UCNPs@8, Calcein AM and propidium iodide (PI) costaining assay showed that a number of cells died under 980 nm irradiation ( Figure 4G). The cells treated by UCNPs@8 exhibited strong green fluorescence ( Figure 4I). All the results illustrated the feasible strategy that UCNPs and AIE-active 8 were encapsulated together to achieve effective energy transfer and exerted their respective advantages to construct NIR-irradiated PSs, UCNPs@8, which generated toxic ROS and thereby killed the cancer cells. This approach effectively overcame the problems of Ir(III) complexes, which own short-wavelength absorption [52].
[52] employed AIE-active non-charged Ir(III) complexes integrated with UCNPs for the first time ( Figure 4A). In UV-vis absorption spectra, 7 and 8 exhibited absorption peaks at ~425 nm. The PL spectra showed that 7 emitted at 640 nm and 8 emitted at 660 nm ( Figure  4B), while the PLQYs were 16.6% and 18.9%, respectively. When the water fraction reached 50%, the emission intensity of 8 was greatly enhanced, which demonstrated that 8 had typical AIE features ( Figure 4C). Further, 8 was then encapsulated with UCNPs within d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) to construct UCNPs@8 as PSs for the ensuing in vitro experiments. The UV-vis absorption spectra of UCNPs@8 showed a significant absorption at about 425 nm, corresponding to that of 8 ( Figure 4D). To ensure that the energy transferred between 8 and UCNPs can be realized and UCNPs@8 could be irradiated by NIR light, the PL spectrum of UCNPs was measured under laser irradiation (980 nm). The absorption band of 8 was in the UV and blue regions, overlapping with the emission bands of UCNPs ( Figure 4E). Encouraged by the 1 O2 generation ability of NIR-irradiated UCNPs@8, their ability to kill cancer cells through PDT was investigated. MTT assays indicated that UCNPs@8 had considerable cytotoxicity ( Figure 4F). To further evaluate the therapeutic effect of UCNPs@8, Calcein AM and propidium iodide (PI) co-staining assay showed that a number of cells died under 980 nm irradiation ( Figure 4G). The cells treated by UCNPs@8 exhibited strong green fluorescence ( Figure 4I). All the results illustrated the feasible strategy that UCNPs and AIE-active 8 were encapsulated together to achieve effective energy transfer and exerted their respective advantages to construct NIR-irradiated PSs, UCNPs@8, which generated toxic ROS and thereby killed the cancer cells. This approach effectively overcame the problems of Ir(III) complexes, which own short-wavelength absorption [52].

Two-Photon Absorption (TPA)
In recent years, two-photon absorption (TPA) has emerged as an attractive protocol in bioimaging and PDT. Two-photon photodynamic therapy (TP-PDT) activates PSs by contemporaneously absorbing two photons [53][54][55][56]. It provides photodamage and phototoxicity as well as deeper penetration, weaker autofluorescence and lower photobleaching [57,58]. Developing Ir(III) complexes with TPA is a new strategy to enhance the efficiency of Ir-PSs. However, conventional PSs suffer from ACQ and reduced photocytotoxicity, resulting in inferior imaging and PDT efficacy. In summary, it is very prospective to construct Ir(III) complexes as AIE-active TPA PSs [59]. Liu et al. [60] conjugated triphenylamine with Ir(III) complexes to achieve the goals of AIE and TPA ( Figure 5A). The PLQYs of 9 were 0.1% in DMSO. Further, 9 performed obvious AIE behavior and the TPA cross-sections (ð 2 ) were better than the previous reported TPA organometallic molecular structure ( Figure 5B,C). As anticipated, the 1 O 2 emission intensity enhanced as the water ratio of the mixture gradually increased from 0 to 90%, i.e., the strongest AIE effect ( Figure 5D). Under two-photon laser irradiation, for the majority of cells treated with 9 ( Figure 5E,F), the fluorescence intensity enhanced and significant annexin V-FITC (AV) and PI signals were detected, revealing that 9 possessed the highest potency of being an efficient TPA-PDT agent.

Two-Photon Absorption (TPA)
In recent years, two-photon absorption (TPA) has emerged as an attractive protocol in bioimaging and PDT. Two-photon photodynamic therapy (TP-PDT) activates PSs by contemporaneously absorbing two photons [53][54][55][56]. It provides photodamage and phototoxicity as well as deeper penetration, weaker autofluorescence and lower photobleaching [57,58]. Developing Ir(III) complexes with TPA is a new strategy to enhance the efficiency of Ir-PSs. However, conventional PSs suffer from ACQ and reduced photocytotoxicity, resulting in inferior imaging and PDT efficacy. In summary, it is very prospective to construct Ir(III) complexes as AIE-active TPA PSs [59]. Liu et al. [60] conjugated triphenylamine with Ir(III) complexes to achieve the goals of AIE and TPA ( Figure 5A). The PLQYs of 9 were 0.1% in DMSO. Further, 9 performed obvious AIE behavior and the TPA crosssections (ð2) were better than the previous reported TPA organometallic molecular structure ( Figure 5B,C). As anticipated, the 1 O2 emission intensity enhanced as the water ratio of the mixture gradually increased from 0 to 90%, i.e., the strongest AIE effect ( Figure 5D). Under two-photon laser irradiation, for the majority of cells treated with 9 ( Figure 5E,F), the fluorescence intensity enhanced and significant annexin V-FITC (AV) and PI signals were detected, revealing that 9 possessed the highest potency of being an efficient TPA-PDT agent. However, TP-AIE-active Ir(III) complexes still have two main disadvantages: (i) Most activated TP-PSs undergo type II photochemical reactions to generate ROS. Their therapeutic effect is still limited by hypoxia. (ii) Poor water solubility makes it difficult to use in the clinic [61]. To address these issues, Jiang et al. designed the first type-I AIE PSs with the TP-PDT strategy, named TPABP and 12 ( Figure 6A). To enhance biocompatibility and structural stability of 12, bovine serum albumin (BSA) was added to formulate 12@BSA However, TP-AIE-active Ir(III) complexes still have two main disadvantages: (i) Most activated TP-PSs undergo type II photochemical reactions to generate ROS. Their therapeutic effect is still limited by hypoxia. (ii) Poor water solubility makes it difficult to use in the clinic [61]. To address these issues, Jiang et al. designed the first type-I AIE PSs with the TP-PDT strategy, named TPABP and 12 ( Figure 6A). To enhance biocompatibility and structural stability of 12, bovine serum albumin (BSA) was added to formulate 12@BSA NPs. TPABP and 12 exhibited the balance between the AIE feature and the typical TICT effect. When the water fraction was 0%, the PLQYs of 12 were about 9%. σ 2 of 12@BSA NPs was 340 GM at 880 nm ( Figure 6B,C). The ROS generation of 12 was better than RB and Ce6 ( Figure 6D). Under two-photon irradiation, a 3D tumor model was constructed to evaluate the penetrativity of 12@BSA NPs. A penetration depth of 270 µm was measured by z-scanning ( Figure 6E,F), which showed 12@BSA NPs performed well [62]. Similarly, Cai et al. [63] synthesized two Ir(III) complexes (13 and 14) ( Figure 6G) and 13 possessed the AIE feature ( Figure 6H). Encapsulating 13 to DSPE-mPEG 2000 to form nanoparticles (13 NPs) is a strategy to improve biocompatibility and the NP carrier did not influence the photophysical properties of 13 ( Figure 6I). The cellular imaging capability of 13 NPs was investigated using CLSM. Upon two-photon excitation, the red phosphorescent signal was stonger than that upon one-photon excitation ( Figure 6J).
NPs. TPABP and 12 exhibited the balance between the AIE feature and the typical TICT effect. When the water fraction was 0%, the PLQYs of 12 were about 9%. σ2 of 12@BSA NPs was 340 GM at 880 nm ( Figure 6B,C). The ROS generation of 12 was better than RB and Ce6 ( Figure 6D). Under two-photon irradiation, a 3D tumor model was constructed to evaluate the penetrativity of 12@BSA NPs. A penetration depth of 270 μm was measured by z-scanning ( Figure 6E,F), which showed 12@BSA NPs performed well [62]. Similarly, Cai et al. [63] synthesized two Ir(III) complexes (13 and 14) ( Figure 6G) and 13 possessed the AIE feature ( Figure 6H). Encapsulating 13 to DSPE-mPEG2000 to form nanoparticles (13 NPs) is a strategy to improve biocompatibility and the NP carrier did not influence the photophysical properties of 13 ( Figure 6I). The cellular imaging capability of 13 NPs was investigated using CLSM. Upon two-photon excitation, the red phosphorescent signal was stonger than that upon one-photon excitation ( Figure 6J).

Multiphoton Absorption (MPA)
Compared with TPA, multiphoton absorption (MPA) improves the conversion efficiency through simultaneously absorbing several infrared photons. The two primary benefits of MPA have attracted attention: excitation wavelengths are in the near infrared (NIR) (700-900 nm) and the relationship between multiphoton absorption activity and excitation intensity is nonlinear [55]. Nevertheless, Ir(III) complexes activated by MPA are still very rare. Zhang et al. [64] combined the distinct merits of excellent photostability of infrared Ir(III) complexes, religious antibacterial ability of organotin(IV) compounds and MPA materials to design 15 and it showed obvious AIE behavior ( Figure 7A,B). Using [Ru(byp)3]Cl2 as the standard, the PLQYs of 15 were 0.2 in water. The 2PA and 3PA crosssections of 15 were 6.25 GM and 1.5 × 10 −81 cm 6 s 2 per photon 2 ( Figure 7C-F). Under multiphoton confocal and STED microscopies, 15 showed superstability in nucleolus, which

Multiphoton Absorption (MPA)
Compared with TPA, multiphoton absorption (MPA) improves the conversion efficiency through simultaneously absorbing several infrared photons. The two primary benefits of MPA have attracted attention: excitation wavelengths are in the near infrared (NIR) (700-900 nm) and the relationship between multiphoton absorption activity and excitation intensity is nonlinear [55]. Nevertheless, Ir(III) complexes activated by MPA are still very rare. Zhang et al. [64] combined the distinct merits of excellent photostability of infrared Ir(III) complexes, religious antibacterial ability of organotin(IV) compounds and MPA materials to design 15 and it showed obvious AIE behavior ( Figure 7A,B). Using [Ru(byp) 3 ]Cl 2 as the standard, the PLQYs of 15 were 0.2 in water. The 2PA and 3PA cross-sections of 15 were 6.25 GM and 1.5 × 10 −81 cm 6 s 2 per photon 2 ( Figure 7C-F). Under multiphoton confocal and STED microscopies, 15 showed superstability in nucleolus, which displayed that 15 had high signal-to-noise ratio ( Figure 7G). Recently, Lu et al. [65] designed and synthesized a series of 3PA-Ir(III) complexes, named 16, 17 and 18 ( Figure 7H). Only 16, which did not have a long alkoxy chain, showed AIE activity and excellent three-photon absorption properties ( Figure 7I,J). Further, 16 also revealed the PLQYs, which were 1.4%. They displayed strong red emission under two-photon laser irradiation (820 nm), indicating the effectiveness of 16 in inducing cell death ( Figure 7K). displayed that 15 had high signal-to-noise ratio ( Figure 7G). Recently, Lu et al. [65] designed and synthesized a series of 3PA-Ir(III) complexes, named 16, 17 and 18 ( Figure 7H). Only 16, which did not have a long alkoxy chain, showed AIE activity and excellent threephoton absorption properties ( Figure 7I,J). Further, 16 also revealed the PLQYs, which were 1.4%. They displayed strong red emission under two-photon laser irradiation (820 nm), indicating the effectiveness of 16 in inducing cell death ( Figure 7K).

Targeting Mitochondria
It is well known that mitochondria are one of the most essential organelles. As the power workshop of cells, mitochondria provide most of the cellular ATP through pathways, such as pyruvate oxidation, fatty acid oxidation, tricarboxylic acid cycle (TCA cycle), and so on. On the other hand, mitochondria are the key to the apoptotic pathway. For instance, in extrinsic apoptosis, the mitochondrial outer membrane opens after signals, including intracellular Ca 2+ overload and oxidation, which leads to the release of proapoptotic factors [66]. PSs act on mitochondria and make mitochondrial ROS burst, so mitochondria will be destroyed and then Cytochrome C, kinds of pro-apoptotic factors, will release, resulting in cancer cell death [67]. As a result, mitochondria-targeted PDT becomes an effective method to improve the treatment of cancer. The agents with delocalized positive charge are preferable to go through the mitochondrial lipid bilayer membrane due to the highly negative inner-membrane potential of mitochondria. Ir(III) complexes are demonstrated as mitochondrial targeting molecules applied in PDT, due to their lipophilic cationic properties.
Jiang et al. [62] proposed an Ir(III) complex with AIE characteristics, which was called 12. Due to the feature that 12 was a lipophilic cation, there was an electrostatic interaction

Targeting Mitochondria
It is well known that mitochondria are one of the most essential organelles. As the power workshop of cells, mitochondria provide most of the cellular ATP through pathways, such as pyruvate oxidation, fatty acid oxidation, tricarboxylic acid cycle (TCA cycle), and so on. On the other hand, mitochondria are the key to the apoptotic pathway. For instance, in extrinsic apoptosis, the mitochondrial outer membrane opens after signals, including intracellular Ca 2+ overload and oxidation, which leads to the release of pro-apoptotic factors [66]. PSs act on mitochondria and make mitochondrial ROS burst, so mitochondria will be destroyed and then Cytochrome C, kinds of pro-apoptotic factors, will release, resulting in cancer cell death [67]. As a result, mitochondria-targeted PDT becomes an effective method to improve the treatment of cancer. The agents with delocalized positive charge are preferable to go through the mitochondrial lipid bilayer membrane due to the highly negative inner-membrane potential of mitochondria. Ir(III) complexes are demonstrated as mitochondrial targeting molecules applied in PDT, due to their lipophilic cationic properties.
Jiang et al. [62] proposed an Ir(III) complex with AIE characteristics, which was called 12. Due to the feature that 12 was a lipophilic cation, there was an electrostatic interaction between 12 NPs and mitochondria, holding a highly negative inner-membrane potential, which caused 12 NPs to penetrate lipid bilayers and accumulate inside mitochondria ( Figure 6I). Cai et al. [63] reported two Ir(III) complexes and both of them possessed AIE properties, good photosensitivity and mitochondria-targeting ability. Further, 13 was encapsulated with DSPE-mPEG 2000 so that nanoparticles called 13 NPs were formed. Taken up via caveolae-mediated endocytosis, 13 NPs targeted mitochondria, which benefited from damaging mitochondria and enhanced antitumor effect ( Figure 6M). Liu et al. [60] reported three different structural Ir(III) complexes 9-11, which were PSs with AIE property and mitochondria-targeting capability, achieved though the endocytic pathway ( Figure 5G). Interestingly, 9-11 exhibited more preferential photocytotoxicity towards HeLa cells than L02 cells.
In addition, the damaged mitochondria can be removed via mitophagy, a form of autophagy, which provides healthy surroundings in cells. During the autophagy process, a membrane structure forms the phagophore, which gradually expands and engulfs a portion of the cytosol or specific cargos and delivers them to the vacuole/lysosome for degradation [68]. The physiological changes are related to several diseases, so it is indispensable to monitor the mitophagy process. Jin et al. [69] synthesized five Ir(III) complexes, which possessed the AIE property ( Figure 8A,B). These Ir(III) complexes, which were named 19-23, were certified as specifically accumulated in mitochondria through mitochondrial colocalization imaging experiments with MitoTracker Green (MTG) and ICP-MS experiments ( Figure 8C,D). The mechanisms of cellular uptake also proved that 19 used a non-endocytic energy-dependent active transport to cross the plasma membrane ( Figure 8G). Thus, 19 successfully fulfilled the real-time monitoring of the mitophagy process, which was caused by CCCP ( Figure 8H). In addition, 19-23 owned excellent stability during 10 scans, with a total irradiation time of~150 s, as well as in a pH range from 4.0 to 8.0 ( Figure 8E,F). Further, 19-23 were expected to be used as mitochondrion-specific probes in biological imaging and dynamic monitoring studies. sulated with DSPE-mPEG2000 so that nanoparticles called 13 NPs were formed. Taken up via caveolae-mediated endocytosis, 13 NPs targeted mitochondria, which benefited from damaging mitochondria and enhanced antitumor effect ( Figure 6M). Liu et al. [60] reported three different structural Ir(III) complexes 9-11, which were PSs with AIE property and mitochondria-targeting capability, achieved though the endocytic pathway ( Figure  5G). Interestingly, 9-11 exhibited more preferential photocytotoxicity towards HeLa cells than L02 cells.
In addition, the damaged mitochondria can be removed via mitophagy, a form of autophagy, which provides healthy surroundings in cells. During the autophagy process, a membrane structure forms the phagophore, which gradually expands and engulfs a portion of the cytosol or specific cargos and delivers them to the vacuole/lysosome for degradation [68]. The physiological changes are related to several diseases, so it is indispensable to monitor the mitophagy process. Jin et al. [69] synthesized five Ir(III) complexes, which possessed the AIE property ( Figure 8A,B). These Ir(III) complexes, which were named 19-23, were certified as specifically accumulated in mitochondria through mitochondrial colocalization imaging experiments with MitoTracker Green (MTG) and ICP-MS experiments ( Figure 8C,D). The mechanisms of cellular uptake also proved that 19 used a nonendocytic energy-dependent active transport to cross the plasma membrane ( Figure 8G). Thus, 19 successfully fulfilled the real-time monitoring of the mitophagy process, which was caused by CCCP ( Figure 8H). In addition, 19-23 owned excellent stability during 10 scans, with a total irradiation time of ~150 s, as well as in a pH range from 4.0 to 8.0 ( Figure  8E,F). Further, 19-23 were expected to be used as mitochondrion-specific probes in biological imaging and dynamic monitoring studies.  Ir complexes can be applied in mitochondrial staining simply. Alam et al. [70] proposed two AIE-active Ir(III) complexes ( Figure 9A-C,E). The PLQYs for 25 were tested and found to be 6.03%. With poor ROS production capacity, 25 had lower cytotoxicity ( Figure 9F) and 25 was useful for bioimaging. When 25 was incubated with HOS cells, the good fluorescence of 25 was only observed in mitochondria ( Figure 9D). With AIE characteristics, low cytotoxicity and mitochondrion-targeted capability, 25 owned potential applications in bioimaging, particularly in mitochondrial staining. stained with 19 (500 nM, orange) and LTG (100 nM, green) in the presence of CCCP (10 μM). Th regions (right) indicated in white boxes are enlarged from the shown area of this cell (left). Th location that the white arrow pointed out indicated the occurrence of mitophagy. Reprinted wit permission from [69], copyright 2016, Springer Nature.
Ir complexes can be applied in mitochondrial staining simply. Alam et al. [70] pro posed two AIE-active Ir(III) complexes ( Figure 9A-C,E). The PLQYs for 25 were tested and found to be 6.03%. With poor ROS production capacity, 25 had lower cytotoxicity ( Figure 9F) and 25 was useful for bioimaging. When 25 was incubated with HOS cells, th good fluorescence of 25 was only observed in mitochondria ( Figure 9D). With AIE char acteristics, low cytotoxicity and mitochondrion-targeted capability, 25 owned potentia applications in bioimaging, particularly in mitochondrial staining.

Targeting Nucleolus
Ribonucleic acid (RNA) exists in biological cells and some viruses, as a viroid geneti information carrier. As the basis of life, RNA takes part in biological processes, such a gene transcription, post-transcriptional regulation of gene expression, protein synthesi based on gene information, etc. Thereinto, rRNA is responsible for protein synthesizing rRNA gathers in the nucleolar region, which is a dark region under fluorescence micros copy. In recent years, the exploration of rRNA-selective probes for the nucleolar region becomes a meaningful topic. Holding more intense interaction with DNA than rRNA small molecules are difficult to make use of as rRNA-selective probes. Ir(III) complexe have potential to construct a kind of rRNA-selective probe due to their greater interaction with intra-nuclear proteins [9].

Targeting Nucleolus
Ribonucleic acid (RNA) exists in biological cells and some viruses, as a viroid genetic information carrier. As the basis of life, RNA takes part in biological processes, such as gene transcription, post-transcriptional regulation of gene expression, protein synthesis based on gene information, etc. Thereinto, rRNA is responsible for protein synthesizing. rRNA gathers in the nucleolar region, which is a dark region under fluorescence microscopy. In recent years, the exploration of rRNA-selective probes for the nucleolar region becomes a meaningful topic. Holding more intense interaction with DNA than rRNA, small molecules are difficult to make use of as rRNA-selective probes. Ir(III) complexes have potential to construct a kind of rRNA-selective probe due to their greater interaction with intra-nuclear proteins [9].
Based on the previous work, Sheet et al. [71] synthesized the 26 Ir(III) complex with benzimidazole-substituted 1,2,3-triazole-pyridine ligand (BiPT), which showed green luminescence ( Figure 10A). The mechanisms of the AIE property of 26 are that 26 formed into aggregated particles in a CH 3 CN-water mixture and were verified by fluorescence, DLS and SEM images (Figure 10B,E-G). The PLQYs of 26 were found to be 0.016 in CH 3 CN at the same time. Noteworthily, a large portion of 26 was found to enter the cell nucleoli region after being incubated with Hela cells in co-staining experiments while faint green luminescence was still observed in the cytoplasm, which meant that 26 selectively targeted the nucleoli where most RNA was assembled ( Figure 10I). Compared with ctDNA, ssDNA, G-quadruplex DNA and BSA, there was stronger interaction with 26 and RNA ( Figure 10C). The results of Autodock Tools suggested that 26 combined with the purine and pyrimidine bases of RNA via supramolecular N-H···π and π-π interactions ( Figure 10H). Exhilaratingly, PL intensity of 26 elevated gradually with an enhancement in rRNA concentration ( Figure 10D). Thereout, 26 was the first Ir(III) complex reported as an rRNA-selective probe.
iosensors 2022, 12, x FOR PEER REVIEW 13 of 21 luminescence ( Figure 10A). The mechanisms of the AIE property of 26 are that 26 formed into aggregated particles in a CH3CN-water mixture and were verified by fluorescence, DLS and SEM images (Figure 10B,E-G). The PLQYs of 26 were found to be 0.016 in CH3CN at the same time. Noteworthily, a large portion of 26 was found to enter the cell nucleoli region after being incubated with Hela cells in co-staining experiments while faint green luminescence was still observed in the cytoplasm, which meant that 26 selectively targeted the nucleoli where most RNA was assembled ( Figure 10I). Compared with ctDNA, ssDNA, G-quadruplex DNA and BSA, there was stronger interaction with 26 and RNA ( Figure 10C). The results of Autodock Tools suggested that 26 combined with the purine and pyrimidine bases of RNA via supramolecular N-H•••π and π-π interactions ( Figure  10H). Exhilaratingly, PL intensity of 26 elevated gradually with an enhancement in rRNA concentration ( Figure 10D). Thereout, 26 was the first Ir(Ⅲ) complex reported as an rRNAselective probe.

Selective Toxicity of Cells
Selective anticancer activity is one of the standards to estimate anticancer drugs. Damaging cancer cells and keeping normal cells well simultaneously are what researchers are trying to achieve. Ir(III) metalla-rectangles 27 and 28, which consist of a BODIPY-based linker, were reported by Gupta et al. (Figure 11A). The white arrows showed 28 aggregated in the form of tiny granules, namely the AIE phenomenon ( Figure 11B). Through initial biological screening, the selective toxicities of 27 against MCF-7 and 28 against U87 cells were proved ( Figure 11B,C). The toxicity against cancer cell lines of 27 and 28 was stronger than that against normal cells. The complexes had different activities to different cell lines, attributed to various intracellular molecular targets, which demands further study [72].

Selective Toxicity of Cells
Selective anticancer activity is one of the standards to estimate anticancer drugs. Damaging cancer cells and keeping normal cells well simultaneously are what researchers are trying to achieve. Ir(III) metalla-rectangles 27 and 28, which consist of a BODIPY-based linker, were reported by Gupta et al. (Figure 11A). The white arrows showed 28 aggregated in the form of tiny granules, namely the AIE phenomenon ( Figure 11B). Through initial biological screening, the selective toxicities of 27 against MCF-7 and 28 against U87 cells were proved ( Figure 11B,C). The toxicity against cancer cell lines of 27 and 28 was stronger than that against normal cells. The complexes had different activities to different cell lines, attributed to various intracellular molecular targets, which demands further study [72].

Specific Response of Ir(III) Complexes with AIE Activity
In recent years, Ir(III) complexes with AIE properties have attracted more attention as photoluminescent probes in bioimaging, owing to their high photophysical performance (such as large Stokes shifts, tunable photophysical properties, long excited-states lifetime, satisfactory photostability, etc.). So far, several specific responsive probes based on Ir(III) complexes with AIE activity have been reported for bioimaging applications. The detailed structures and photophysical properties of these probes are summarized and discussed in the following sections.

Perchlorate (ClO4 − )
Perchlorate (ClO4 − ) salts are widespread and relatively stable in foods and water samples [73]. ClO4 − competitively disrupts thyroid uptake of iodine and accumulates in the human body due to its similar ionic radius with iodide, resulting in adverse consequences, such as iodide deficiency and goiter, even brain impairment [74]. Hence, it is necessary to detect ClO4 − in water and monitor it in cells. An AIE-active complex 29 based on the Ir(III) complex for specific sensing of ClO4 − in cells reported by Chao et al. in 2016 was rationally designed by introducing the thioacetal group [75]. The phosphorescence quantum yield of 29 in solution was 1%, which increased to 2% in the solid state due to the aggregation of nanoparticles ( Figure 12B). In particular, 29 exhibited a specific response to ClO4 − in HEPES buffer ( Figure 12C,D) and the luminescence turn-on response was not interfered with by Hg 2+ , although the thioacetal group can undergo a thioacetal deprotection reaction [76]. Further, 29 and ClO4 − lead to enhanced-emissive nanoparticles in water, while the reaction of 29 and Hg 2+ generated weak emission ( Figure 12E). Moreover, the binding rate of 29 and ClO4 − was faster than the thioacetal deprotection reaction of Hg 2+ . The application of 29 in imaging ClO4 − in living cells was further investigated, where a strong red phosphorescent emission was observed in the cytoplasm.

Specific Response of Ir(III) Complexes with AIE Activity
In recent years, Ir(III) complexes with AIE properties have attracted more attention as photoluminescent probes in bioimaging, owing to their high photophysical performance (such as large Stokes shifts, tunable photophysical properties, long excited-states lifetime, satisfactory photostability, etc.). So far, several specific responsive probes based on Ir(III) complexes with AIE activity have been reported for bioimaging applications. The detailed structures and photophysical properties of these probes are summarized and discussed in the following sections.

Perchlorate (ClO 4 − )
Perchlorate (ClO 4 − ) salts are widespread and relatively stable in foods and water samples [73]. ClO 4 − competitively disrupts thyroid uptake of iodine and accumulates in the human body due to its similar ionic radius with iodide, resulting in adverse consequences, such as iodide deficiency and goiter, even brain impairment [74]. Hence, it is necessary to detect ClO 4 − in water and monitor it in cells. An AIE-active complex 29 based on the Ir(III) complex for specific sensing of ClO 4 − in cells reported by Chao et al. in 2016 was rationally designed by introducing the thioacetal group [75]. The phosphorescence quantum yield of 29 in solution was 1%, which increased to 2% in the solid state due to the aggregation of nanoparticles ( Figure 12B). In particular, 29 exhibited a specific response to ClO 4 − in HEPES buffer ( Figure 12C,D) and the luminescence turn-on response was not interfered with by Hg 2+ , although the thioacetal group can undergo a thioacetal deprotection reaction [76]. Further, 29 and ClO 4 − lead to enhanced-emissive nanoparticles in water, while the reaction of 29 and Hg 2+ generated weak emission ( Figure 12E). Moreover, the binding rate of 29 and ClO 4 − was faster than the thioacetal deprotection reaction of Hg 2+ . The application of 29 in imaging ClO 4 − in living cells was further investigated, where a strong red phosphorescent emission was observed in the cytoplasm.
In 2017, Chao et al. [77] first reported a water-soluble Ir(III) complex as a probe of ClO 4 − based on the AIE phenomenon. Complex 30 had two imidazolium-like groups to improve positive charges and solubility ( Figure 13A). A possible mechanism was presented that ClO 4 − induced 30 and aggregated in water due to the exchange between Cl − and ClO 4 − , resulting in an enhanced emission ( Figure 13B). Furthermore, 30 was used for cell imaging and showed strong red emission in the presence of ClO 4 − in HeLa cells ( Figure 13D). In 2017, Chao et al. [77] first reported a water-soluble Ir(III) complex as a probe of ClO4 − based on the AIE phenomenon. Complex 30 had two imidazolium-like groups to improve positive charges and solubility ( Figure 13A). A possible mechanism was presented that ClO4 − induced 30 and aggregated in water due to the exchange between Cl − and ClO4 − , resulting in an enhanced emission ( Figure 13B). Furthermore, 30 was used for cell imaging and showed strong red emission in the presence of ClO4 − in HeLa cells ( Figure  13D).  In 2017, Chao et al. [77] first reported a water-soluble Ir(III) complex as a probe of ClO4 − based on the AIE phenomenon. Complex 30 had two imidazolium-like groups to improve positive charges and solubility ( Figure 13A). A possible mechanism was presented that ClO4 − induced 30 and aggregated in water due to the exchange between Cl − and ClO4 − , resulting in an enhanced emission ( Figure 13B). Furthermore, 30 was used for cell imaging and showed strong red emission in the presence of ClO4 − in HeLa cells ( Figure  13D).

BSA
BSA is a globular protein and has an important effect on biophysical functions, such as blood circulation and drug binding [78,79]. Two Ir(III) complexes, namely 31 and 32 ( Figure 14A), were reported by Laskar et al. [80]. The emission intensity of 31 raised with the increasing concentration of water ( Figure 14B). Compared with 32, only 31 had AIE properties due to the restriction of intramolecular motion of the picolinate group ( Figure 14C). Moreover, the interaction of 31 with BSA resulted in green emission ( Figure 14D). The mechanism of 31 as a specific probe for sensing BSA proved that the auxiliary ligand may bind to the protein molecule through molecular docking studies ( Figure 14E).

BSA
BSA is a globular protein and has an important effect on biophysical functions, such as blood circulation and drug binding [78,79]. Two Ir(III) complexes, namely 31 and 32 ( Figure 14A), were reported by Laskar et al. [80]. The emission intensity of 31 raised with the increasing concentration of water ( Figure 14B). Compared with 32, only 31 had AIE properties due to the restriction of intramolecular motion of the picolinate group ( Figure  14C). Moreover, the interaction of 31 with BSA resulted in green emission ( Figure 14D). The mechanism of 31 as a specific probe for sensing BSA proved that the auxiliary ligand may bind to the protein molecule through molecular docking studies ( Figure 14E).  [80], copyright 2020, Royal Society of Chemistry.

Drug-Resistant Bacteria
Antimicrobial resistance (AMR) has developed rapidly due to the overuse of medicines, leading to contaminating water bodies and posing a serious threat to public health [81,82]. Therefore, there is an urgent need for monitoring and eliminating drug-resistant bacteria to control waterborne diseases [83]. The work of Sasmal et al. is a good example of developing Ir(III) complexes with AIE properties for detecting and inhibiting drug-resistant bacteria [84]. Three cationic Ir(III) complexes (33)(34)(35) were synthesized as bacterial theranostic agents ( Figure 15A). These complexes contained the same cyclometalated ligands and different auxiliary ligands. Further, 33 had two ethyl ester moieties in the auxiliary ligand, whereas 34 and 35 had bis-polyethylene glycol (PEG) to improve the biocompatibility and aqueous solubility. Both 33 and 34 were found to possess AIE active and highly selective for detecting LPS/LTA ( Figure 15D). The PLQYs for 33, 34 and 35 were determined to be 0.0072, 0.0091 and 0.0084, respectively. Moreover, the mechanisms of bacterial cells damaged by 33 and 34 suggest the disintegration of membrane integrity Reprinted with permission from [80], copyright 2020, Royal Society of Chemistry.

Drug-Resistant Bacteria
Antimicrobial resistance (AMR) has developed rapidly due to the overuse of medicines, leading to contaminating water bodies and posing a serious threat to public health [81,82]. Therefore, there is an urgent need for monitoring and eliminating drug-resistant bacteria to control waterborne diseases [83]. The work of Sasmal et al. is a good example of developing Ir(III) complexes with AIE properties for detecting and inhibiting drug-resistant bacteria [84]. Three cationic Ir(III) complexes (33)(34)(35) were synthesized as bacterial theranostic agents ( Figure 15A). These complexes contained the same cyclometalated ligands and different auxiliary ligands. Further, 33 had two ethyl ester moieties in the auxiliary ligand, whereas 34 and 35 had bis-polyethylene glycol (PEG) to improve the biocompatibility and aqueous solubility. Both 33 and 34 were found to possess AIE active and highly selective for detecting LPS/LTA ( Figure 15D). The PLQYs for 33, 34 and 35 were determined to be 0.0072, 0.0091 and 0.0084, respectively. Moreover, the mechanisms of bacterial cells damaged by 33 and 34 suggest the disintegration of membrane integrity and generation of ROS ( Figure 15E). This work is a platform that integrates the detection and elimination of bacteria into AIE-active Ir(III) complexes. and generation of ROS ( Figure 15E). This work is a platform that integrates the detection and elimination of bacteria into AIE-active Ir(III) complexes.

Conclusions
This review provides an overview of Ir(III) complexes with AIE properties for bioimaging, sensing and therapy applications in terms of extended absorption wavelength, precise targeting and specific response, respectively. By introducing various ligands into Ir(III) complexes, the photophysical properties could be improved to enhance the effect of bioimaging and therapeutics. These results are of great importance and promote the development of AIE materials based on Ir(III) complexes for biomedical applications.
However, there are still many limitations in the design strategy of efficient AIE-active Ir(III) complexes. Herein, we propose research directions where concerted efforts are required for AIEgens based on Ir(III) complexes in bioimaging and therapeutics. First, extending the absorption of Ir(III) complexes to NIR II will further improve the tissue-penetration depth and reduce side effects. Second, more attention should be placed on enhancing the targeting ability of AIE-active Ir(III) complexes and selectively releasing in the tumor microenvironment (TME) to improve the efficacy of tumor treatment. Third, the synergistic therapy of AIEgens could be further increased via exploiting the rational design of the Ir(III) complexes. We believe that AIE materials based on Ir(III) complexes would become a powerful tool for the biomedical field in the future.

Conclusions
This review provides an overview of Ir(III) complexes with AIE properties for bioimaging, sensing and therapy applications in terms of extended absorption wavelength, precise targeting and specific response, respectively. By introducing various ligands into Ir(III) complexes, the photophysical properties could be improved to enhance the effect of bioimaging and therapeutics. These results are of great importance and promote the development of AIE materials based on Ir(III) complexes for biomedical applications.
However, there are still many limitations in the design strategy of efficient AIE-active Ir(III) complexes. Herein, we propose research directions where concerted efforts are required for AIEgens based on Ir(III) complexes in bioimaging and therapeutics. First, extending the absorption of Ir(III) complexes to NIR II will further improve the tissuepenetration depth and reduce side effects. Second, more attention should be placed on enhancing the targeting ability of AIE-active Ir(III) complexes and selectively releasing in the tumor microenvironment (TME) to improve the efficacy of tumor treatment. Third, the synergistic therapy of AIEgens could be further increased via exploiting the rational design of the Ir(III) complexes. We believe that AIE materials based on Ir(III) complexes would become a powerful tool for the biomedical field in the future.