Biotin-modified cyclometalated iridium-based photosensitizers as mitochondria-targeted theranostic agents for tumor photodynamic therapy in vitro and in vivo

{"full_text": " Dyes and Pigments 219 (2023) 111641\n\n\n Contents lists available at ScienceDirect\n\n\n Dyes and Pigments\n journal homepage: www.elsevier.com/locate/dyepig\n\n\n\n\nBiotin-modified cyclometalated iridium-based photosensitizers as\nmitochondria-targeted theranostic agents for tumor photodynamic therapy\nin vitro and in vivo\nLai Wei a, Xiangdong He a, Xinyue Shang a, Martha Kandawa-Shultz c, Guoqiang Shao b, **,\nYihong Wang a, *\na\n School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China\nb\n Department of Nuclear Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing, 211166, China\nc\n Department of Chemistry and Biochemistry, University of Namibia, Windhoek, 13301, Namibia\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: Photodynamic therapy (PDT) is a promising non-invasive treatment technique for various types of cancer.\nBiotin Photoluminescent cyclometalated Ir(III) complexes, a distinctive genre of photosensitizers (PSS), have garnered\nIr(III) complex significant interest due to their exceptional selective toxicity towards cancerous cells and minimal cytotoxicity\nMitochondria targeting\n towards normal cells. This study aimed to investigate the application of three newly designed biotin-modified\nPhotodynamic therapy\n cyclometalated Ir(III) complexes (Ir1-Ir3) for the treatment of cancer. The three complexes were found to\nApoptosis\n have good photocatalytic activity, with singlet oxygen (1O2) yields ranging from 0.24 to 0.65 in aqueous solu\u00ad\n tions. The results suggest that these complexes have the potential as phototherapeutic and photodiagnostic\n agents for cancer treatment. Additionally, the biotin fragments in the Ir1-Ir3 structures enable their selective\n targeting of tumor cells. Notably, Ir3 displayed negligible dark toxicity towards A549 human lung cancer cells\n (IC50 > 100 \u03bcM), but its value decreased to 0.25 \u03bcM after 15 min of 425 nm (40 mW/cm2) light exposure.\n Furthermore, these complexes exhibited low phototoxicity towards non-cancerous BHK mouse kidney cells.\n Further investigation demonstrated that Ir3 had potent targeting abilities for mitochondria, and could trigger cell\n apoptosis through the activation of ROS-mediated mitochondrial signaling pathways under 425 nm light irra\u00ad\n diation. In vivo studies revealed that Ir3 effectively suppressed tumor growth in A549 xenograft-bearing mice\n when exposed to 425 nm light. These results indicate that Ir3 has the potential to be a valuable photosensitizer\n drug for the photodynamic treatment of tumors.\n\n\n\n\n1. Introduction wavelengths and molecular oxygen (3O2). The synergistic action of these\n three harmless components can produce reactive oxygen species (ROS),\n In recent years, research on cancer treatment has been one of the which can disrupt the regular function of cancer cells and lead to\nhotspots in the field of life sciences [1,2]. Safer and more effective apoptosis [10,11]. In the process of PDT treatment for tumors, the least\ncancer treatment methods are needed due to the various side effects and dose of toxic photosensitizer is first administered to the patient\u2019s body.\nlimitations of traditional treatments such as surgery, chemotherapy, and Then, the tumor site is irradiated with light, causing the photosensitizer\nradiation therapy [3]. Photodynamic therapy (PDT) is a non-invasive and light to combine and produce reactive oxygen, which damages\ntreatment method that shows promise in treating a range of diseases, cellular functions and ultimately leads to cell death. It\u2019s important to\nsuch as cancer, skin diseases, ophthalmic diseases, and oral diseases. Its note that the photodynamic effect is limited to the irradiated area due to\ntherapeutic effects have been increasingly recognized, making it a the low diffusion rate and short lifespan of reactive oxygen species\nvaluable treatment option [4\u20139]. This therapy mainly relies on three [12\u201314]. Compared to traditional cancer treatment methods such as\nnon-toxic components: photosensitizers, light of appropriate chemotherapy, radiation therapy, and surgery, PDT has several\n\n\n\n * Corresponding author.\n ** Corresponding author.\n E-mail addresses: guoqiangshao@163.com (G. Shao), yihongwang@seu.edu.cn (Y. Wang).\n\nhttps://doi.org/10.1016/j.dyepig.2023.111641\nReceived 6 May 2023; Received in revised form 24 July 2023; Accepted 21 August 2023\nAvailable online 22 August 2023\n0143-7208/\u00a9 2023 Elsevier Ltd. All rights reserved.\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\nsignificant advantages, including non-invasiveness, local treatment, certain types of cancer cells overexpress SMVT [33]. Recently, it has\nrepeatability, tissue selectivity, no toxic side effects and no adverse ef\u00ad been reported that strategies utilizing the overexpression of biotin re\u00ad\nfects or discomfort to patients [15]. Currently, PDT has become a mature ceptors in cancer cells and the modification of drugs with biotin frag\u00ad\ntreatment method and has been widely applied in clinical medicine, ments have been applied in various fields, including bioimaging, cancer\nbiomedical research and the field of life sciences [16]. therapy, and biosensing [16,33\u201336]. Biotin-conjugated drugs utilize the\n Platinum-based chemotherapy drugs, such as cisplatin, carboplatin, highly specific binding between biotin and biotin receptors to accurately\noxaliplatin, nedaplatin and lobaplatin, have been extensively utilized in deliver drugs to tumor cells, achieving selective killing of tumor cells.\ncancer treatment. Nevertheless, these drugs have vast drawbacks, Biotin-conjugated drugs have many advantages in tumor treatment.\nincluding toxic side effects, drug resistance, high cost, and negative Firstly, they can achieve targeted delivery of drugs, reduce toxicity to\nimpact on patients\u2019 quality of life. For these reasons, the scientific normal cells, and minimize adverse reactions. Secondly, by designing\ncommunity turned its attention to explore other metal-based medicines and synthesizing different drug molecules containing biotin structures,\n[17]. Over the past few years, iridium-based complexes have emerged as diversification and personalization can be achieved, thereby improving\nattractive alternatives to platinum-based drugs due to their better the therapeutic effect [33,34,37].\nproperties [18\u201321]. Recent research has shown that cyclometalated Ir Mitochondria are responsible for producing ATP in most cells and\n(III) complexes have excellent photo-physical properties and remarkable play a significant role in tumor development and progression. When\nanti-cancer activity. Compared to platinum complexes, cyclometalated tumor cells are exposed to hypoxic conditions, it affects their mito\u00ad\nIr(III) complexes have better ligand substitution kinetics and more chondrial metabolism, making them more sensitive to oxidative stress or\ndiverse molecular structures, which results in better treatment outcomes reactive species [38]. Therefore, mitochondria represent an appropriate\nfor tumors [10,22,23]. Additionally, these complexes also exhibit subcellular target for PDT [39].\nseveral advantages, such as long fluorescence lifetimes (~ms), good In this study, we designed and prepared three biotinylated Ir(III)\nchemical stability, high quantum yield, efficient charge transfer ability, complexes (Ir1-Ir3) for tumor-targeted imaging and therapy (Scheme\ntunable optical and chemical properties, strong photobleaching resis\u00ad 1). Under 425 nm (40 mW/cm2) light irradiation, Ir1-Ir3 exhibited\ntance, excellent cell permeability, and the ability to generate ROS remarkable anti-tumor effects against various tumor cells in vitro, with\nthrough their interaction with molecules inside the cells in the tumor relatively low toxicity toward normal cells. The antitumor mechanism of\n[10,24,25]. Given the remarkable photophysical and photochemical Ir3 was elucidated, including subcellular localization, mitochondrial\nproperties of Ir(III) complexes, they can be utilized to design diverse, dysfunction, ROS production and cell apoptosis. Moreover, in vivo bio\u00ad\nhighly sensitive fluorescent probes for tumor imaging and diagnosis [26, logical evaluation of Ir3 was conducted in A549 xenograft-bearing mice,\n27]. In recent years, there has been a growing interest among re\u00ad demonstrating significant antitumor effects in the Ir3-treated group of\nsearchers in modifying the properties of cyclometalated Ir(III) com\u00ad mice exposed to 425 nm (40 mW/cm2) light irradiation (Scheme 1).\nplexes through the design of suitable ligands and structural\nmodifications. These efforts aim to improve the performance of these 2. Results and discussion\ncomplexes and broaden their potential applications [28\u201330].\n The majority of drugs used for tumor treatment cannot specifically 2.1. Ir(III) complexes synthesis and photophysical characterization\ntarget tumors, and typically only a small portion of these drugs can\neffectively act on the affected area after entering the body. This is the The synthetic route of the ligands and Ir(III) complexes Ir1-Ir3 and\nfundamental reason for the low efficacy of these drugs and the occur\u00ad Ir3-NB was displayed in Scheme 1 and Scheme S1. Their structures were\nrence of adverse drug reactions [31,32]. Biotin, also known as vitamin H confirmed by elemental analysis, ESI-MS, and 1H NMR (Figs. S1\u2013S14).\nor coenzyme R, is a water-soluble vitamin that is essential for the syn\u00ad The normalized UV\u2013Vis absorption spectra of Ir1-Ir3 in CH2Cl2, CH3OH\nthesis of vitamin C and for the normal metabolism of fats and proteins. and H2O were shown in Fig. S15. The relatively intense absorption\nBiotin is usually transported in mammalian cells via a Na+-dependent bands below 350 nm were ascribed to the \u03c0\u2192\u03c0* transitions, while the\nmultivitamin transporter (SMVT). To meet the demands of rapid growth, relatively intense absorption bands to 500 nm could be assigned to the\n\n\n\n\n Scheme 1. Schematic of the preparation process of Ir1-Ir3 and photodynamic antitumor mechanism of Ir3.\n\n 2\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\nmetal-to-ligand charge transfer (MLCT) and ligand-to-ligand charge molecular orbital (LUMO) part of Ir1-Ir3 was primarily concentrated on\ntransfer (LLCT) characters. Upon excitation at 425 nm, the fluorescence the ligand containing biotin fragment, while the highest occupied mo\u00ad\nemission spectra from 450 to 750 nm and the normalized fluorescence lecular orbital (HOMO) predominantly resided on the cyclometalated\nemission spectra from 500 to 750 nm of Ir1-Ir3 in CH2Cl2, CH3OH and ligands for Ir1 and Ir3 and the ligand containing biotin fragment for Ir2.\nH2O were presented in Fig. S16. Upon excitation at 425 nm, the Ir(III) In contrast to Ir1 (\u0394Eg = 3.33 eV) and Ir3 (\u0394Eg = 3.14 eV), Ir2 (\u0394Eg =\ncomplexes (Ir1-Ir3) exhibited a color change from green to red in these 3.46 eV) exhibited a distinct separation in the HOMO and LUMO dis\u00ad\nsolvents. Additionally, all complexes had large Stokes shifts, with Ir3 tributions. Additionally, we conducted further calculations to determine\nhaving the largest shift, followed by Ir1 and Ir2. The normalized fluo\u00ad the singlet and triplet energy levels of Ir1-Ir3 (Fig. 2B and Table S1). The\nrescence spectra revealed that Ir1-Ir3 exhibited two fluorescence emis\u00ad energy gap \u0394E(S1-T1) between the S1 and T1 states was measured to be\nsion peaks. As the solvent polarity increased, the fluorescence intensity 0.11 eV for Ir1, 0.76 eV for Ir2 and 0.44 eV for Ir3. Crossing from the\nof the three iridium complexes decreased, and the peak shifted towards lowest singlet excited state (S1) to the nearest triplet excited state (T1)\nlonger wavelengths (redshifted). This phenomenon can be attributed to within PSs after being stimulated by light, the excited electrons combine\nthe alteration in the local environment of the molecules caused by the with triplet oxygen (3O2) to generate singlet oxygen (1O2), which is\nincrease in solvent polarity, leading to changes in fluorescence intensity highly toxic to tumor cells. So the singlet oxygen (1O2) generation ca\u00ad\nand wavelength displacement. pacity in the whole process mainly depends on the ability of photosen\u00ad\n sitizer to intersystem crossing (ISC). The lower energy gap in Ir1-Ir3\n2.2. Singlet oxygen (1O2) generation rate facilitated the singlet-triplet intersystem crossing (ISC) process. Appar\u00ad\n ently, Ir1-Ir3 all show smaller \u0394E(S1-T1) values, leading to an effective\n 1\n To assess the potential of Ir1-Ir3 as photosensitizers against cancer O2 generation. Furthermore, the calculated results revealed a signifi\u00ad\ncells via photodynamic therapy (PDT), it is crucial to evaluate their cant difference in the excited state energy between Ir2 and Ir1 as well as\ncapacity to generate reactive oxygen species (ROS), with a particular Ir3. Notably, the fluorescence signal of Ir2 was barely detectable beyond\nfocus on singlet oxygen (1O2) generation [40]. The quantum yields (\u03a6\u0394) 450 nm, which aligns with the experimental findings indicating that Ir2\nof 1O2 produced from Ir1-Ir3 were determined in H2O, utilizing [Ru exhibited the weakest fluorescence (Table S1).\n(bpy)3]Cl2 as a standard, by monitoring the changes in the UV\u2013vis\nspectra of 9,10-Anthracenediyl-bis(methylene)dimalonic Acid (ABDA) 2.4. Lipophilicity\nat 380 nm. While the mixture solution of Ir1-Ir3 and ABDA was irra\u00ad\ndiated (425 nm, 40 mW/cm2), a remarkably decreased absorbance To determine the hydrophilicity and lipophilicity of Ir1-Ir3, the n-\noccurred. The \u03a6\u0394 values of Ir1-Ir3 and standard were determined as the octanol/water partition coefficient of Ir1-Ir3 were determined using the\nfollowing order: Ir2 (0.65) > Ir1 (0.37) > Ir3 (0.24) > [Ru(bpy)3]Cl2 shake-flask method. As shown in Fig. S18, the lipophilicity indices of the\n(0.18) (Fig. 1 and Fig. S17). These results indicate that Ir1-Ir3 can three complexes followed the order of Ir3 > Ir2 > Ir1. It is important to\nproduce 1O2 when exposed to light irradiation, making them a prom\u00ad note that the lipophilicity of a drug can significantly influence the rate\nising option for PDT applications. and mechanism of drug molecules traversing the cell membrane.\n Generally, drugs with higher lipophilicity have an enhanced ability to\n penetrate the lipid bilayer cell membrane and gain access to the interior\n2.3. Time-dependent density functional theory (TD-DFT) calculations\n of the cell. Ir3 has higher lipophilicity and therefore may be better taken\n up by cells.\n Time-dependent density functional theory (TD-DFT) calculations\nwere employed to further explain the generation mechanism of ROS of\nIr1-Ir3. As shown in Fig. 2A and Table S1, the lowest unoccupied\n\n\n\n\nFig. 1. The absorbance of ABDA (100 \u03bcM) after photodecomposition under visible light irradiation (425 nm, 40 mW/cm2) over different times in the case of (A) Ir1,\n(B) Ir2 and (C) Ir3 in H2O solution (20 \u03bcM). (D) The absorbance changes of ABDA at 380 nm with Ir1, Ir2, Ir3 and [Ru(bpy)3]Cl2 in H2O solution (20 \u03bcM) under\nvisible light irradiation (425 nm, 40 mW/cm2) over different times.\n\n 3\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\n\n\n Fig. 2. (A) HOMO\u2013LUMO distribution at S1 for Ir1-Ir3. (B) The energy gap between S1 and T1 for Ir1-Ir3 from TD-DFT (Gaussian/B3LYP/6-311G(d)).\n\n\n2.5. Cellular uptake 2.6. In vitro cytotoxicity and photocytotoxicity\n\n To establish the functional significance of the biotin moiety in To evaluate the impact of illumination time on the sensitivity of Ir3-\nfacilitating the localization of the Ir1-Ir3 to biotin receptor-enriched treated A549 cells, the viability of these cells was assessed via the MTT\ntumor cells, we conducted an incubation experiment in which the Ir1- assay across a range of illumination times (0 min, 5 min, 10 min,15 min\nIr3 were exposed to human lung cancer cells (A549) and mouse kidney and 20 min). As shown in Fig. S19, when the concentration of Ir3 was\ncells (BHK) for 3 h. A549 and BHK cells, as biotin-positive and negative constant, the viability of A549 cells decreased with increasing illumi\u00ad\ncell systems, respectively [41]. Flow cytometry was utilized to analyze nation time (0 min, 5 min, 10 min, and 15 min, 425 nm, 40 mW/cm2).\nthe internalization of cells and quantify the uptake of Ir1-Ir3. As shown However, a negligible decline in cell viability was observed for illumi\u00ad\nin Fig. 3A\u2013F, A549 cells treated with Ir1-Ir3 exhibited strong fluores\u00ad nation times exceeding 15 min (15 min, 20 min). Hence, 15 min was\ncence signals compared to control cells, approximately 13.2-fold for Ir1, chosen as the illumination time of PDT in this study.\n11.6-fold for Ir2, and 49.8-fold for Ir3. In contrast, negligible fluores\u00ad The cytotoxic effect and PDT activities of Ir1-Ir3 and cisplatin in\ncence was observed in BHK cells. These findings suggest that the A549, MDA-MB-231, MCF-7 and BHK cells were determined by MTT\namplified uptake of Ir1-Ir3 in A549 cells is primarily attributed to assay (Table 1). Under 425 nm, 40 mW/cm2 light irradiation for\nreceptor-mediated endocytosis through the biotin receptors, which is 15min, all three Ir(III) complexes-treated cancer cells exhibited high\nfacilitated by the interaction between the biotin moiety and the biotin phototoxicity. The cytotoxic effect of Ir1-Ir3 under dark conditions\nreceptor on the surface of tumor cells. was negligible with IC50 > 100 \u03bcM. Notably, Ir3 showed the highest\n Considering the favorable performance of Ir3, further experiments phototoxicity index (PI) > 416 in A549 cells. In addition, Ir1-Ir3\nwere conducted to determine the cellular uptake of this complex. The exhibited relatively low phototoxic effects in non-cancerous cells\nconfocal laser scanning microscope (CLSM) images showed no signifi\u00ad (BHK cells), which may be attributed to the low expression of biotin\ncant fluorescence signal in the normal cell group (BHK) after 3 h of Ir3 receptors that mediate cellular uptake of Ir1-Ir3 in non-cancerous\ntreatment (Fig. 3G). In contrast, the fluorescence intensity of Ir3 in cells (BHK cells). However, it should be noted that the phototox\u00ad\nA549 cells increased significantly with the incubation time (Fig. 3H). icity index (PI) of Ir1-Ir3 in non-cancerous cells (BHK cells),\nThe results of the flow cytometry showed that compared with the control although much smaller compared to cancer cells, reached values of\ngroup, the fluorescence signal of A549 cells increased significantly after Ir1 > 13.3, Ir2 > 16.4, and Ir3 > 17.9, indicating that the over\u00ad\nincubation with Ir3 for different times (15.1-fold for 1h, 30.4-fold for 2h expression of biotin receptors in cancer cells is a primary pathway\nand 49.2-fold for 3h, Fig. 3I and K). On the contrary, only negligible for Ir1-Ir3 uptake, a passive uptake mechanism cannot be\nfluorescence was observed in BHK cells even after 3 h of incubation with completely ruled out.\nIr3 (6-fold, Fig. 3J and K). These results further demonstrate that the Inspired by the above results, Ir(III) complex without biotin group\nbiotin-modified complex Ir3 promotes the uptake and selective accu\u00ad modification was synthesized (Ir3-NB) and further investigated for its\nmulation of Ir3 by overexpressing the biotin receptor in cancer cells anticancer activity by MTT assay. As shown in Tables 1 and Ir3-NB was\n(A549) through receptor-mediated endocytosis. At the same time, the more phototoxic to the selected cell lines than Ir1-Ir3, but Ir3-NB also\nhigher lipophilicity of Ir3 also promotes its uptake by cells. The effective showed high toxicity to the selected cancer cell lines and non-cancer cell\nuptake of the photosensitizer by cells may potentially improve PDT. lines under dark conditions. These results indicated that the selective\n phototoxicity of Ir1-Ir3 to tumor cells was enhanced by the modification\n of biotin groups.\n Notably, the photocytotoxicity of Ir1-Ir3 was significantly\n\n 4\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\n\n\nFig. 3. Flow cytometry analysis of BHK and A549 cells treated with (A, D) Ir1 (10 \u03bcM), (B, E) Ir2 (10 \u03bcM), and (C, F) Ir3 (10 \u03bcM) for 3 h. BHK cells treated with the\ncomplexes: green lines, A549 cells treated with the complexes: blue lines, untreated BHK cells: black lines, and untreated A549 cells: red lines. B means BHK cells, and\nA means A549 cells. Confocal microscopy images of BHK (G) and A549 (H) cells treated with Ir3 (10 \u03bcM) for 1, 2, and 3 h. The samples were excited at 400 nm and\ndetected at 590 \u00b1 20 nm. Scale bar = 20 \u03bcm. Flow cytometry analysis of BHK (I, K) and A549 (J, K) cells treated with Ir3 (10 \u03bcM) for 0.25, 0.5, 1, 2, and 3h. Data are\nrepresented as mean \u00b1 standard deviation. The single asterisk symbol (*) represents p < 0.05, and the double asterisk symbol (**) represents p < 0.01.\n\n\nirrelevant to the quantum yield of 1O 2 formation, implying that the under 425 nm (40 mW/cm2 ) light irradiation. Therefore, Ir3 was\nphotokinetic activity of these complexes may be influenced by the selected as a targeting compound to elucidate the mechanism of its\nlevel of cellular uptake, 1 O 2 production capacity and target binding anticancer effect.\naffinity. In addition, Ir3 was more effective against tumor cells\n\n\n 5\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\nTable 1\nIC50 Values (\u03bcM) and Photocytotoxicity Indices (PI) for Ir1-Ir3 and Ir3-NB from an MTT assay.\n IC50 (\u03bcM)\n\n A549 MDA-MB-231 MCF-7 BHK\n\n Complex Darka (Lightb) PIc Darka (Lightb) PIc Darka (Lightb) PIc Darka (Lightb) PIc\n\n Ir1 \uff1e100 (1.21 \u00b1 0.08) \uff1e82.6 \uff1e100 (1.09 \u00b1 0.12) \uff1e91.7 \uff1e100 (1.58 \u00b1 0.11) \uff1e63.3 \uff1e100 (7.50 \u00b1 0.21) \uff1e\n 13.3\n Ir2 \uff1e100 (1.35 \u00b1 0.11) \uff1e74.1 \uff1e100 (1.07 \u00b1 0.15) \uff1e93.5 \uff1e100 (1.15 \u00b1 0.14) \uff1e87.0 \uff1e100 (6.11 \u00b1 0.06) \uff1e\n 16.4\n Ir3 \uff1e100 (0.24 \u00b1 0.02) \uff1e \uff1e100 (0.38 \u00b1 0.06) \uff1e \uff1e100 (0.51 \u00b1 0.03) \uff1e \uff1e100 (5.60 \u00b1 0.09) \uff1e\n 416.7 263.2 196.1 17.9\n Ir3-NB 7.84 \u00b1 0.12 (0.13 \u00b1 0.01) 60.3 8.18 \u00b1 0.23 (0.16 \u00b1 0.03) 51.1 9.29 \u00b1 0.14 (0.18 \u00b1 0.02) 51.6 6.07 \u00b1 0.11 (0.14 \u00b1 0.05) 43.4\n Cisplatin 25.30 \u00b1 0.83 (24.70 \u00b1 1.0 13.43 \u00b1 0.09 (13.02 \u00b1 1.0 18.73 \u00b1 0.15 (17.96 \u00b1 1.0 36.20 \u00b1 2.10 (34.10 \u00b1 1.0\n 0.52) 0.32) 0.27) 1.10)\n a\n Cell lines were treated with Ir1-Ir3 and Ir3-NB for 24 h in the dark.\n b\n Cell lines were treated with Ir1-Ir3 and Ir3-NB for 3 h in the dark, and then exposed to light irradiation at 425 nm (40 mW/cm2) for 15 min.\n c\n PI = Phototoxicity index, which represents the ratio of IC50 values in the absence and presence of light irradiation.\n\n\n2.7. Cell cloning test 1) staining by flow cytometry. Mitochondrial depolarization can be\n revealed by a decrease in the ratio of red (JC-1 aggregates) to green (JC-\n The growth inhibition of A549 cells by various concentrations of Ir3 1 monomers) fluorescence intensity [43]. As shown in Fig. 6 and\ntreatment was assessed using the colony formation assay. As shown in Fig. S20, under 40 mW/cm2, 425 nm light irradiation for 15min, the\nFig. 4, under 425 nm (40 mW/cm2, 15min) light conditions, colony intensity ratio of red/green fluorescence in Ir3-treated A549 cells was\nformation of Ir3-treated A549 cells was dramatically reduced by reduced in a concentration-dependent manner. In contrast, the shift\nincreasing Ir3 concentration. The colony formation rates were 69% at from red to green fluorescence was barely observed in Ir3-treated A549\n0.125 \u03bcM, 24% at 0.25 \u03bcM and 2% at 0.5 \u03bcM, respectively. Conversely, cells under dark conditions. These results suggest that Ir3 tends to\nthe inhibitory effect of Ir3 treatment on colony formation could be impair the integrity of mitochondria in the light, but not in the dark.\nignored in the dark, with a colony formation rate of 95% at 0.5 \u03bcM. This\ntrend observed is consistent with that obtained from the MTT assay.\n 2.10. ROS detection\n\n2.8. Subcellular location experiments Photodynamic therapy (PDT) relies on photosensitizers to generate\n cytotoxic reactive oxygen species (ROS), leading to tumor cell apoptosis\n The intracellular localization of photosensitizers has a significant [11]. Numerous studies have suggested that mitochondrial dysfunction is\nimpact on the effectiveness of PDT [42]. Given that Ir3 exhibits strong associated with excessive accumulation of ROS, which plays a decisive role\nPDT activity in cancer cells, further investigations of its distribution in cell apoptosis [44]. 2\u2032,7\u2032-dichlorofluorescein diacetate (DCFH-DA) is a\nwithin cells can be conducted through the confocal laser scanning mi\u00ad widely used ROS indicator. In the presence of ROS, DCFH reacts with ROS\ncroscope (CLSM). The images (Fig. 5) obtained from the confocal mi\u00ad to form dichlorofluorescein (DCF), and the resulting fluorescence is pro\u00ad\ncroscopy revealed that after 3 h of incubation with A549 in the dark, the portional to ROS levels [45]. In this study, DCFH-DA was used as a fluo\u00ad\nPearson co-localization coefficient between Ir3 and the mitochondrial rescent probe to detect the level of ROS in Ir3-treated A549 cells using\nprobe Mito-Tracker Green was 0.96, while under the same conditions, confocal microscopy and flow cytometry. As shown in Fig. 7A and B, the\nthe Pearson co-localization coefficient between Ir3 and the lysosomal fluorescence intensity of Ir3-treated A549 cells increased in a\nprobe Lyso-Tracker Green was 0.41. These results revealed that Ir3 concentration-dependent manner with increasing Ir3 concentration when\npredominantly localized to the mitochondria, indicating that Ir3 may irradiated with 425 nm (40 mW/cm2, 15 min) light, indicating effective\ninduce cell death via the mitochondrial apoptosis pathway. ROS production. In contrast, under dark conditions, negligible green\n fluorescence was observed in Ir3-treated A549 cells. Flow cytometry re\u00ad\n2.9. Mitochondrial damage sults (Fig. 7C and D) showed that under 425 nm light irradiation, the\n average DCF fluorescence intensity in Ir3-treated A549 cells increased\n As presented above, Ir3 is mainly localized in mitochondria, its effect 6.1-fold, 7.7-fold, and 10.2-fold for 0.125 \u03bcM, 0.25 \u03bcM, and 0.5 \u03bcM of Ir3,\non the membrane integrity of mitochondrial was assessed via 5,5\u2032,6,6\u2032- respectively. In contrast, there was almost no increase in the DCF average\ntetrachloro-1,1\u2032,3,3\u2032- tetraethyl benzimidazolylcarbocyanine iodide (JC- fluorescence intensity in Ir3-treated A549 cells under dark conditions.\n\n\n\n\nFig. 4. (A) Colony formation of A549 cells at 10 days after treatment with Ir3 (0.125 \u03bcM, 0.25 \u03bcM and 0.5 \u03bcM) in the dark and with light irradiation. (B) The\nquantification analysis of (A). Data are represented as mean \u00b1 standard deviation. The single asterisk symbol (*) represents p < 0.05, the double asterisk symbol (**)\nrepresents p < 0.01 and the triple asterisk symbol (***) represents p < 0.001.\n\n 6\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\n\n\nFig. 5. CLSM co-localization images of Ir3 (10 \u03bcM, 3 h) with Mito-Tracker Green (Mito-Tracker; 100 nM, 30 min) and Lyso-Tracker Green (Lyso-Tracker; 50 nM, 30\nmin) in A549 cells. \u03bbex = 500 nm (Mito-Tracker and Lyso-Tracker) and 400 nm (Ir3); \u03bbem = 510 \u00b1 10 nm (Mito-Tracker and Lyso-Tracker) and 590 \u00b1 20 nm (Ir3). All\nimages share the same scale bar, 20 \u03bcm.\n\n\n\n\n Fig. 6. Flow cytometry analysis of the effect of Ir3 on the mitochondrial membrane potential of A549 cells (\u03bbex = 490 nm and \u03bbem = 530 nm).\n\n\n2.11. Apoptosis induction evaluated by wound healing assay. As shown in Fig. 9, cells were treated\n with different concentrations of Ir3 for 12 and 24 h in the dark or under\n To further validate the apoptotic pathway triggered by Ir3, we the indicated light conditions. Notably, cells treated with 0.125, 0.25,\nconducted experiments on A549 cells with different concentrations of and 0.5 \u03bcM of Ir3 under 425 nm (40 mW/cm2, 15 min) light exhibited\nIr3 treatments under both light and dark conditions. A double-staining significant time-dependent inhibition of wound healing integrity\ntechnique using FITC-Annexin V/PI was utilized to label the A549 compared to cells in the dark. The results indicate that Ir3 may exert an\ncells and the apoptotic rate was analyzed via flow cytometry. As shown important role in inhibiting the wound-healing process under light\nin Fig. 8 and Fig. S21, the percentages of early and late apoptotic cells in irradiation conditions.\nA549 cells treated with Ir3 increased in a concentration-dependent\nmanner under 425 nm light conditions, indicating that apoptosis 2.13. In vivo antitumor evaluation\noccurred in A549 cells. In contrast, under dark conditions, the per\u00ad\ncentages of early and late apoptotic cells in A549 cells treated with Ir3 The in vitro experimental findings demonstrate that Ir3 specifically\nremained almost unchanged. targets biotin receptors that are overexpressed in tumor cells and lo\u00ad\n calizes in mitochondria, exhibiting effective efficacy in photodynamic\n2.12. Inhibition of cell migration therapy (PDT). To further investigate the therapeutic effects of Ir3, an in\n vivo experiment was conducted using A549 nude mouse xenograft\n The effect of Ir3 on the horizontal migration of A549 cells was models (Fig. 10A). Tumor-bearing mice were randomly divided into four\n\n 7\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\n\n\nFig. 7. (A) Confocal microscopy images of ROS generation in different concentrations of Ir3 (3 h)-treated A549 cells and DCF (10 \u03bcM, 0.5 h, \u03bbex = 488 nm, \u03bbem = 530\nnm) before and after light irradiation. Scale bar = 20 \u03bcm. (B) The 3D image of (A). (C) Flow cytometry of ROS generation in different concentrations of Ir3 (3 h)-\ntreated A549 cells. (D) The quantification analysis of (C). Data are represented as mean \u00b1 standard deviation. The double asterisk symbol (**) represents p < 0.01\nand the triple asterisk symbol (***) represents p < 0.001.\n\n\ngroups, each containing four mice: the saline dark group, saline light lesions, cell death, or apoptosis were observed in the normal organs\ngroup, Ir3 dark group, and Ir3 light group. Mice in the Ir3 dark and Ir3 (Fig. 10F). In summary, these results demonstrate that Ir3 induces tumor\nlight groups were intratumorally injected with Ir3 at a dose of 10 mg/kg. cell death through PDT in vivo, and at the tested dose, it has no severe\nSubsequently, the tumor site of mice in the Ir3 light group was irradiated adverse effects on normal organs, making it an effective photosensitizer\nwith 425 nm at 40 mW/cm2 for 15 min, 3 h after injection. The saline for tumor photodynamic therapy.\ndark group and the saline light group were treated with an equivalent\ndose of saline and light intensity. The body weight and tumor volume 3. Conclusions\nvariation were recorded every three days to evaluate the therapeutic\neffect. The growth of tumors in the Ir3 light group was remarkably In summary, three novel Ir(III) complexes (Ir1-Ir3) incorporating\ninhibited, and the tumor volume significantly decreased over time. In biotin fragments were designed, synthesized, characterized, and evalu\u00ad\nstark contrast, the tumor volume in the other three groups increased ates their potential as photocatalysts for cancer therapy. These com\u00ad\nsharply during the same period (Fig. 10B). After 21 days, there was no plexes showed strong phototoxicity against the tested human cancer cell\nsignificant change in the weight of mice in all groups (Fig. 10C), lines with low phototoxicity toward normal mouse cells. Cell confocal\ndemonstrating the biological safety of Ir3. After sacrificing the mice on imaging revealed that Ir3 was preferentially taken up by A549 cancer\nday 21, tumor tissues were collected, photographed, and weighed. The cells through highly expressed biotin receptors and selectively targeted\ntumor volume reduction was visually observed in the Ir3 light group mitochondria. Ir3 severely disrupted the physiological function of\n(Fig. 10E), and the tumor weight in the Ir3 light group was significantly mitochondria, as evidenced by a decrease in mitochondrial membrane\nsmaller than that in the other three groups (Fig. 10D). The tumor and potential and an increase in ROS production and further led to apoptosis\nnormal organs (heart, liver, spleen, lung, and kidney) were subjected to under 425 nm (40 mW/cm2) light irradiation. The in vivo therapy results\nhistopathological analysis. Hematoxylin and eosin (H&E) staining of the indicated that Ir3 significantly inhibited the growth of A549 tumors in a\ntumor and normal organs showed that the structure of the tumor in the mouse xenograft model under the light. From this viewpoint, the biotin-\nIr3 light group was severely damaged, with deformed nuclei, intercel\u00ad modified Ir(III) complex, Ir3, could be a promising candidate for mo\u00ad\nlular edema, and overall tumor tissue atrophy. The structure of the lecular targeted photodynamic therapy (PDT). This study also highlights\ntumor in the Ir3 dark group was slightly damaged, with a slight decrease the potential of using this design strategy to develop multifunctional\nin cell density. In the other control groups, the tumor cells were densely anticancer drugs with reduced side effects.\narranged, and no significant pathological abnormalities or inflammatory\n\n\n 8\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\n\n\nFig. 8. Flow-cytometric quantification of double Annexin V/PI-labeled A549 cells treated with different concentrations of Ir3 in the dark and under light (425 nm,\n40 mW/cm2).\n\n\n Fig. 9. The wound healing assay was carried out on\n Ir3-treated A549 cells. (A) The cells were treated with\n 0.125, 0.25, and 0.5 \u03bcM of Ir3 for 0, 12 and 24 h in\n the dark. (B) The cells were treated with 0.125, 0.25,\n and 0.5 \u03bcM of Ir3 for 0, 12 and 24 h under 15 min of\n light irradiation at 425 nm. Scale bars, 200 \u03bcm. The\n results of wound healing assay for (C) dark and (D)\n light irradiation conditions. Wound closure (%) = [1 -\n (distance at indicated time)/(distance at 0 h)] \u00d7\n 100%. Data are represented as mean \u00b1 standard de\u00ad\n viation. The single asterisk symbol (*) represents p <\n 0.05, the double asterisk symbol (**) represents p <\n 0.01 and the triple asterisk symbol (***) represents p\n < 0.001.\n\n\n\n\n4. Experimental section acetic acid (80 mL). The resulting suspension was heated to reflux for 6\n h. After completion of the reaction, the reaction mixture was adjusted to\n4.1. Synthesis of 2-(4-nitrophenyl)-1H-imidazo[4,5-f][1,10]- pH = 7 with concentrated aqueous ammonia. The resulting precipitate\nphenanthroline (PIP-NO2) was collected by filtration, washed with water, and collected as a solid,\n which was then dried under the vacuum to get a bright yellow solid\n Ligand PIP-NO2 was synthesized with modifications according to the PIP-NO2. Yield: 86% (2.93 g, 8.59 mmol). Anal. Calcd(%) for\npreviously reported method [46]. 4-nitrobenzaldehyde (1.57 g, 10.39 C19H11N5O2: C, 66.86; H, 3.25; N, 20.52. Found: C, 66.80; H, 3.28; N,\nmmol), 1,10-phenanthroline-5,6-dione (2.12 g, 10.08 mmol), and 20.54. ESI-MS: m/z = 342.09964 ([M+H]+). 1H NMR (600 MHz, DMSO-\nammonium acetate (15.44 g, 200 mmol) were suspended in glacial d6): \u03b4 = 9.05 (dd, J = 4.3, 1.7 Hz, 2H), 8.93 (dd, J = 8.1, 1.8 Hz, 2H),\n\n 9\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\n\n\nFig. 10. Anti-proliferative activity of Ir3 in A549 xenograft-bearing mice. (A) Scheme of anti-tumor experimentation. (B) The average tumor volume, (C) body\nweight and (D) the average tumor weight of nude mice in saline control (in the dark and under the light), Ir3 (20 mg/kg, in the dark) and Ir3 (20 mg/kg, under the\nlight) groups. Each group consisted of five mice, and the data are presented as mean \u00b1 SD. The double asterisk symbol (**) represents p < 0.01 and the triple asterisk\nsymbol (***) represents p < 0.001. (E) Photographs of tumors removed from A549 xenograft-bearing mice. (F) Histological hematoxylin and eosin (H&E) analysis of\nthe tumor tissues and major organ tissues (heart, liver, spleen, lung, and kidney) collected from mice in the different groups at the end of treatment. Scale bars,\n100 \u03bcm.\n\n\n8.54 (d, J = 8.9 Hz, 2H), 8.48 (d, J = 8.8 Hz, 2H), 7.85 (dd, J = 8.1, 4.3 [1,10]-phenanthroline (PIP-NO2) (2.00 g, 5.86 mmol) was suspended in\nHz, 2H). 1,4-dioxane (60 mL) and heated to 80 \u25e6 C. Na2S\u22c59H2O (4.22 g, 17.57\n mmol) was dissolved in water (20 mL) and heated to 80 \u25e6 C. The warm\n4.2. Synthesis of 4-(1H-imidazo[4,5- f][1,10]phenanthroline-2-yl) Na2S solution was added to the yellow suspension, and the mixture was\naniline (PIP-NH2) stirred at 80 \u25e6 C for 4 h until the solid completely dissolved and the color\n of the solution changed from orange to red. The dioxane was evapo\u00ad\n Ligand PIP-NH2 was synthesized with modifications according to the rated, and the precipitate was collected and washed with water and\npreviously reported method [46]. 2-(4-nitrophenyl)-1H-imidazo[4,5-f] ether respectively. The product PIP-NH2 was dried under vacuum and\n\n 10\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\nobtained as an ochre solid in a yield of 80% (1.44 g, 4.63 mmol). Anal. [Ir(dfppy)2(PIP-Biotin)]PF6 (Ir2) Yield\uff1a61% (0.229g, 0.183 mmol).\nCalcd(%) for C19H13N5: C, 73.30; H, 4.21; N, 22.49. Found: C, 73.28; H, Anal. Calcd(%) for C51H39F10IrN9O2PS: C, 48.80; H, 3.13; N, 10.04.\n4.22; N, 22.51. ESI-MS: m/z = 312.12549 ([M+H]+). 1H NMR (600 Found: C, 48.82; H, 3.10; N, 10.02. ESI-MS: m/z = 1110.24815 ([M \u2212\nMHz, DMSO- d6): \u03b4 = 13.32 (s, 1H), 9.00 (dd, J = 4.3, 1.8 Hz, 2H), 8.89 PF6]+). 1H NMR (600 MHz, DMSO- d6): \u03b4 = 14.28 (s, 1H), 10.20 (s, 1H),\n(dd, J = 8.1, 1.8 Hz, 2H), 7.97 (d, J = 8.6 Hz, 2H), 7.81 (dd, J = 8.1, 4.3 9.21 (d, J = 8.3 Hz, 2H), 8.37\u20138.17 (m, 6H), 8.09 (s, 2H), 7.98 (t, J = 8.2\nHz, 2H), 6.74 (d, J = 8.6 Hz, 2H), 5.63 (s, 2H). Hz, 2H), 7.86 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 5.9 Hz, 2H), 7.13\u20136.98 (m,\n 4.3. Synthesis of N-(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl) 4H), 6.42 (d, J = 45.2 Hz, 2H), 5.72 (dd, J = 8.3, 2.4 Hz, 2H), 4.32 (dd, J\nphenyl)-5-(2-oxohexahydro-1H-thieno [3,4-d]imidazole-4-yl)pentanam = 7.7, 5.2 Hz, 1H), 4.19\u20134.12 (m, 1H), 3.17\u20133.12 (m, 1H), 2.84 (dd, J =\nide (PIP-Biotin) 12.4, 5.1 Hz, 1H), 2.59 (d, J = 12.4 Hz, 1H), 2.38 (t, J = 7.4 Hz, 2H),\n 4-(1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)aniline (PIP-NH2) 1.66 (dd, J = 14.6, 7.1 Hz, 3H), 1.54 (dd, J = 14.8, 6.0 Hz, 1H),\n(0.80 g, 2.56 mmol) and biotin (0.62 g, 2.56 mmol) were dissolved in 1.44\u20131.37 (m, 2H). Elemental Analysis: C, 55.18; H, 3.54; N, 11.35; O,\nanhydrous dichloromethane and cooled to 0 \u25e6 C. Then, 1-Ethyl-3-(3\u2032- 2.88.\ndimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.23 g, 6.40 [Ir(1-pq)2(PIP-Biotin)]PF6 (Ir3) Yield\uff1a64% (0.246g, 0.192 mmol).\nmmol) and 4-Dimethylaminopyridine (DMAP) (0.31 g, 2.56 mmol) were Anal. Calcd(%) for C59H47F6IrN9O2PS: C, 55.22; H, 3.69; N, 9.82.\nadded to the mixture. The reaction mixture was stirred at 0 \u25e6 C for 1 h and Found: C, 55.24; H, 3.67; N, 9.83. ESI-MS: m/z = 1138.32283 ([M \u2212\nthen slowly warmed to room temperature, and the reaction was allowed PF6]+). 1H NMR (600 MHz, DMSO- d6): \u03b4 = 14.26 (s, 1H), 10.23 (d, J =\nto continue for 12 h at room temperature. After the reaction was 8.7 Hz, 1H), 9.19 (d, J = 8.3 Hz, 2H), 9.03 (d, J = 8.2 Hz, 2H), 8.42 (d, J\ncompleted, the dichloromethane was removed and the residue was = 8.1 Hz, 2H), 8.26 (d, J = 8.3 Hz, 2H), 8.13\u20137.99 (m, 6H), 7.88 (dd, J =\ndissolved in water (50 mL), filtered and the precipitate collected. The 9.0, 4.3 Hz, 6H), 7.47\u20137.36 (m, 4H), 7.18 (q, J = 6.8, 6.0 Hz, 2H), 6.98\nprecipitate was then dispersed in acetonitrile, filtered and the precipi\u00ad (t, J = 7.7 Hz, 2H), 6.46 (s, 1H), 6.39 (s, 1H), 6.30 (d, J = 7.6 Hz, 2H),\ntate collected. After vacuum drying, a dark red PIP-Biotin product was 4.34\u20134.30 (m, 1H), 4.16 (dd, J = 7.5, 4.5 Hz, 1H), 3.14 (dt, J = 10.4, 5.0\nobtained with a yield of 83% (1.14 g, 2.12 mmol). Anal. Calcd(%) for Hz, 1H), 2.84 (dd, J = 12.4, 5.2 Hz, 1H), 2.58 (d, J = 12.4 Hz, 1H), 2.39\nC29H27N7O2S: C, 64.79; H, 5.06; N, 18.24. Found: C, 64.75; H, 5.10; N, (t, J = 7.4 Hz, 2H), 1.66 (dd, J = 14.6, 7.1 Hz, 3H), 1.53 (dd, J = 14.8,\n18.25. ESI-MS: m/z = 538.20522 ([M+H]+). 1H NMR (600 MHz, DMSO- 6.0 Hz, 1H), 1.46\u20131.37 (m, 2H).\nd6): \u03b4 = 13.67 (s, 1H), 10.17 (s, 1H), 9.04 (dd, J = 4.2, 1.8 Hz, 2H), 8.93 [Ir(1-pq)2(PIP)]PF6 (Ir3-NB) Yield\uff1a56% (0.212g, 0.203 mmol).\n(ddd, J = 11.3, 8.1, 1.8 Hz, 2H), 8.23 (d, J = 8.7 Hz, 2H), 7.91\u20137.77 (m, Anal. Calcd(%) for C49H32F6IrN6P: C, 56.48; H, 3.10; N, 8.07. Found: C,\n4H), 6.44 (d, J = 47.3 Hz, 2H), 4.33 (dd, J = 7.7, 5.0 Hz, 1H), 4.16 (ddd, 56.24; H, 3.07; N, 8.11. ESI-MS: m/z = 897.22456 ([M \u2212 PF6]+). 1H\nJ = 7.5, 4.5, 1.8 Hz, 1H), 3.15 (ddd, J = 8.6, 6.1, 4.4 Hz, 1H), 2.84 (dd, J NMR (600 MHz, DMSO- d6): \u03b4 = 9.15 (d, J = 8.2 Hz, 2H), 9.03 (d, J =\n= 12.4, 5.1 Hz, 1H), 2.60 (d, J = 12.4 Hz, 1H), 2.38 (td, J = 7.2, 2.1 Hz, 8.2 Hz, 2H), 8.38 (dd, J = 39.8, 7.9 Hz, 4H), 8.07\u20137.83 (m, 10H), 7.59 (t,\n2H), 1.71\u20131.36 (m, 6H). J = 7.6 Hz, 2H), 7.53\u20137.38 (m, 5H), 7.18 (t, J = 7.7 Hz, 2H), 6.97 (t, J =\n 7.4 Hz, 2H), 6.32 (d, J = 7.6 Hz, 2H).\n4.3. Synthesis of 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (PIP)\n CRediT authorship contribution statement\n The ligand PIP was synthesized according to the methods described\npreviously [47]. Lai Wei: Conceptualization, Data curation, Investigation, Method\u00ad\n ology, Writing \u2013 original draft. Xiangdong He: Methodology, Investi\u00ad\n4.4. Synthesis of Ir(III) dimer gation, Validation. Xinyue Shang: Conceptualization, Methodology.\n Martha Kandawa-Shultz: Conceptualization, Methodology. Guoqiang\n The cyclometalated Ir(III) chloro-bridged dimers [Ir(ppy)2Cl]2 (Ir Shao: Supervision, Resources. Yihong Wang: Resources, Supervision,\n(III) dimer-1), [Ir(dfppy)2Cl]2 (Ir(III) dimer-2), and [Ir(1-pq)2Cl]2 (Ir(III) Project administration, Writing \u2013 review & editing, All authors have\ndimer-3) were synthesized according to the methods described previ\u00ad approved the manuscript.\nously [48\u201350].\n Declaration of competing interest\n4.5. Synthesis of Ir complexes\n The authors declare that they have no known competing financial\n 0.15 mmol of Ir-dimer (1 eq.) and 0.30 mmol of PIP-Biotin or PIP (2 interests or personal relationships that could have appeared to influence\neq.) were dissolved in a mixture of 21 mL 2:1 CH2Cl2/CH3OH (v/v). The the work reported in this paper.\nmixture was refluxed under an argon atmosphere for 24 h in the dark.\nAfter completion of the reaction, the mixture was cooled to room tem\u00ad Data availability\nperature and 6-fold excess NH4PF6 was added under vigorous stirring for 2\nh to yield a precipitate. The precipitate was collected by filtration, washed Data will be made available on request.\nwith a small amount of ether, and dried under vacuum. The desired\nproduct was obtained by purifying the crude product through neutral Acknowledgements\nAl2O3 column chromatography using a flow of CH2Cl2/CH3OH (10:1, v/v).\n [Ir(ppy)2(PIP-Biotin)]PF6 (Ir1) Yield\uff1a58% (0.205g, 0.173 mmol). This work was supported by the National Natural Science Foundation\nAnal. Calcd(%) for C51H43F6IrN9O2PS: C, 51.77; H, 3.66; N, 10.65. of China (No. 81571812), A Project Funded by the Priority Academic\nFound: C, 51.75; H, 3.63; N, 10.64. ESI-MS: m/z = 1038.28712 ([M \u2212 Program Development of Jiangsu Higher Education Institutions\nPF6]+). 1H NMR (600 MHz, DMSO\u2011d6): \u03b4 = 14.30 (s, 1H), 10.18 (s, 1H), (1107047002), Jiangsu Provincial Medical Youth Talent\n9.18 (d, J = 8.3 Hz, 2H), 8.27 (d, J = 8.8 Hz, 4H), 8.13 (d, J = 4.9 Hz, (QNRC2016075), The Nanjing Medical Science and Technique Devel\u00ad\n2H), 8.06 (dd, J = 8.3, 5.1 Hz, 2H), 7.96 (dd, J = 8.0, 1.4 Hz, 2H), opment Foundation (ZKX19022) and Jiangsu Provincial High-level\n7.93\u20137.77 (m, 4H), 7.50 (dd, J = 5.9, 1.6 Hz, 2H), 7.07 (td, J = 7.6, 1.3 health talent \u201csix one project\u201d (LGY2019005).\nHz, 2H), 7.04\u20136.87 (m, 4H), 6.45 (s, 1H), 6.38 (s, 1H), 6.30 (dd, J = 7.6,\n1.2 Hz, 2H), 4.32 (dd, J = 7.8, 5.1 Hz, 1H), 4.16 (ddd, J = 7.4, 4.6, 1.9 Appendix A. Supplementary data\nHz, 1H), 3.14 (ddd, J = 8.6, 6.1, 4.5 Hz, 1H), 2.84 (dd, J = 12.4, 5.1 Hz,\n1H), 2.59 (d, J = 12.4 Hz, 1H), 2.38 (t, J = 7.1 Hz, 2H), 1.67 (dd, J = Supplementary data to this article can be found online at https://doi.\n14.6, 7.1 Hz, 3H), 1.55 (dd, J = 14.8, 6.0 Hz, 1H), 1.45\u20131.38 (m, 2H) org/10.1016/j.dyepig.2023.111641.\n\n 11\n\fL. Wei et al. Dyes and Pigments 219 (2023) 111641\n\n\nReferences [26] Yang X, Zhou X, Zhang YX, Li D, Li C, You C, et al. Blue phosphorescence and\n hyperluminescence generated from imidazo[4,5-b]pyridin-2-ylidene-based iridium\n (III) phosphors. Adv Sci 2022;9(25):2201150.\n [1] Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA A Cancer J\n [27] Wang L, Monro S, Cui P, Yin H, Liu B, Cameron CG, et al. Heteroleptic Ir(III)N(6)\n Clin 2023;73(1):17\u201348.\n complexes with long-lived triplet excited states and in vitro photobiological\n [2] Guo X, Yang N, Ji W, Zhang H, Dong X, Zhou Z, et al. 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Dalton Trans\n 2013;42(13):4479\u201386.\n\n\n\n\n 12\n\f", "pages_extracted": 12, "text_length": 75376}