Mitochondria-targeted cyclometalated iridium-β-carboline complexes as potent non-small cell lung cancer therapeutic agents.

{"full_text": " Metallomics, 15, 2023, mfad035\n https://doi.org/10.1093/mtomcs/mfad035\n Advance access publication date: 18 May 2023\n Paper\n\n\n\nMitochondria-targeted cyclometalated iridium-\u03b2-\ncarboline complexes as potent non-small cell lung\ncancer therapeutic agents\nJincan Chen1 ,2 ,3 ,\u2021 , Xinhua Guo1 ,2 ,\u2021 , Dunhui Li4 ,5 , Hong Tang1 ,2 ,3 , Jie Gao1 ,2 , Wenzhu Yu1 ,2 ,3 , Xufeng Zhu2 ,3 , Zirong Sun1 ,\nZunnan Huang1 ,\u2217 and Lanmei Chen 1 ,2 ,\u2217\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\n1\n Key Laboratory of Computer-Aided Drug Design of Dongguan City, Key Laboratory for Research and Development of Natural Drugs of Guangdong Province,\nSchool of Pharmacy, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China, 2 The Marine Biomedical Research Institute, Guangdong Medical\nUniversity, Zhanjiang, Guangdong 524023, P.R. China, 3 The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang, Guangdong 524023, P.R.\nChina, 4 Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Perth, Western Australia 6150, Australia and 5 College of Nursing and\nHealth, Zhengzhou University, Zhengzhou 450001, P.R. China\n\u2217\n Correspondence: Key Laboratory of Computer-Aided Drug Design of Dongguan City, Key Laboratory for Research and Development of Natural Drugs of\nGuangdong Province, School of Pharmacy, Guangdong Medical University, Dongguan, Guangdong 523808, P.R. China. (+86)0759-2388568; E-mail:\nzn_huang@gdmu.edu.cn (Z. Huang), lanmeichen@126.com (L. Chen).\n\u2021\n These authors contributed equally to this work.\n\n\n\nAbstract\nNatural products and metals play a crucial role in cancer research and the development of antitumor drugs. We designed and syn-\nthesized three new carboline-based cyclometalated iridium complexes [Ir(C-N)2 (PP\u03b2C)](PF6 ), where PP\u03b2C = N-(1,10-phenanthrolin-\n5-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-carboxamide, C-N = 2-phenylpyridine (ppy, Ir1), 2-(2,4-difluorophenyl) pyridine (dfppy, Ir2),\n7,8-benzoquinoline (bzq, Ir3), by combining iridium with \u03b2-carboline derivative. These iridium complexes exhibited high potential\nantitumor effects after being promptly taken up by A549 cells. Accumulating in mitochondria rapidly and preferentially, Ir1-3 caused\na series of changes in mitochondrial events, including the loss of mitochondrial membrane potential, the depletion of cellular ATP,\nand the elevation of reactive oxygen species, leading to significant death of A549 cells. Moreover, the activation of intracellular caspase\npathway and apoptosis was further validated to contribute to iridium complexes-induced cytotoxicity. These novel iridium complexes\nexerted a prominent inhibitory effect on tumor growth in a three-dimensional multicellular tumor spheroid model.\n\nKeywords: Iridium-\u03b2-carboline complexes, Mitochondria-targeted, Mitochondrial dysfunction; Apoptosis\n\n\nGraphical abstract\n NAC,GSH\n Ir complexes MTR Merge\n\n\n Ir1 H\n L N\n N\n R=0.87 H ROS\n Ir\n N N\n Mitochondria-targeted\n O\n Ir2 N\n R=0.89\n L\n Iridium-\u03b2-carboline complexes MMP\n Ir3\n R=0.92\n\n\n\n\n Ir1-3 localized in mitochondria ATP\n\n Control Ir1 Ir2 Ir3\n\n\n PI\n\n\n\n Calcein AM\n\n\n\n Overlay\n 3D MCTSs\n Antitumor effect of Ir1-3 on 3D MCTSs Apoptosis\n\n\n\n\nThree novel cyclometalated iridium complexes Ir1-3 by combining iridium with \u03b2-carboline derivative were designed and synthe-\nsized, which preferentially accumulated in mitochondria, caused a series of changes in mitochondrial events, and induced A549 cell\napoptosis. Meanwhile, Ir1-3 also exerted a prominent inhibitory effect on 3D A549 multicellular tumor spheroids (MCTSs).\n\n\n\nIntroduction after chemotherapies.2 For example, platinum-based anticancer\n drugs have serious drawbacks, such as renal toxicity, gastroin-\nCancer is a major cause of death worldwide and is a significant\n testinal reaction, and drug resistance. Therefore, there is an ur-\nbarrier to patients\u2019 quality of life.1 Under carcinogenic condi-\n gent and unmet need to develop novel and safer metal-based\ntions, proto-oncogenes in local tissues are activated, and tumor\n anticancer drugs with high activity. Some of the non-platinum\nsuppressor genes are inactivated, thereby losing the regulation\n metal complexes containing ruthenium (Ru), iridium (Ir), copper\nof normal cell growth and apoptosis and leading to primary tu-\n (Cu), and rhenium (Re) possess high antitumor activities, which\nmors. Surgery, chemotherapy, and radiation therapy are the main-\n are of great potential to be developed as substitutes for platinum\nstream treatment options for malignant tumors, but there is\n drugs.3\u20137 In recent years, iridium complexes have attracted ex-\nstill room for improvement especially in reducing side effects\n tensive attention. Many studies showed that iridium complexes\n\nReceived: March 29, 2023. Accepted: May 17, 2023\n\u00a9 The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com\n\f2 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\nFig. 1 Chemical structures of Ir1-4, P\u03b2CA, and PP\u03b2C.\n\n\n\nexert anticancer activities through various mechanisms, includ- by conjugating \u03b2-carboline derivative ligands and investigating\ning DNA binding,8\u201311 protein\u2013protein interactions,12 , 13 photody- their biological functions. It is widely accepted that slight\nnamic effects,14\u201316 and targeting subcellular organelles.3 , 7 , 17 , 18 modifications in molecular geometry can significantly im-\n Natural products are an important source of novel drugs, drug pact the biological activities of compounds. Following this\nleads, and chemical entities.19 , 20 It is reported that more than logic, in this work, with N-(1,10-phenanthrolin-5-yl)-1-phenyl-9H-\n60 natural marine compounds, including leukomycin bipyridyl pyrido[3,4-b]indole-3-carboxamide (PP\u03b2C) as a main ligand, three\nalkaloids, cyanobacterial bipyridyl glycosides, and polycyclic new cyclometalated Ir(III) complexes [Ir(ppy)2 (PP\u03b2C)](PF6 ) (Ir1),\ntetrameric acid macrochlorolactam have been isolated and iden- [Ir(dfppy)2 (PP\u03b2C)](PF6 ) (Ir2), and [Ir(bzq)2 (PP\u03b2C)](PF6 ) (Ir3) (Fig. 1)\ntified from marine-derived cyanobacteria WH1-2216-6.4 Some of were designed and synthesized. Ir1-3 that combine iridium with\nthese compounds exhibit cytotoxic, antibacterial, or immunosup- \u03b2-carboline derivative are more effective than the other iridium\npressive activity; however, only a few marine antitumor drugs complexes reported in the literature.35 To elucidate the anticancer\nhave entered the clinic.21 , 22 Alkaloids are a class of important functions and molecular mechanisms, the cellular uptake, the ac-\nnatural compounds, with complex structures and superior bio- cumulation site, and the antitumor effects of these three iridium\nlogical activities, widely present in plants, animals, and marine complexes were investigated. Ir1-3 induced severe mitochondria\norganisms. Natural \u03b2-carboline alkaloids are mainly distributed damage in A549 cells, such as MMP reduction, ATP inhibition, reac-\nin plants and marine organisms (sponges and soft corals). They tive oxygen species (ROS) production, and mitochondria morphol-\nhave good biochemical activities, such as antithrombotic, antivi- ogy alteration, resulting in typical apoptosis for a large portion of\nral, bactericidal, and anti-inflammatory effects,23\u201325 making them tumor cells. Moreover, these novel iridium complexes exerted a\nbroad application prospects in antitumor research. However, the prominent inhibitory effect on tumor growth in 3D multicellular\namount of \u03b2-carboline alkaloids obtained directly from marine or- tumor spheroids (MCTSs) of A549 cells.\nganisms is very small and the extraction process is complicated,\nwhich brings difficulties to their further research. Modern synthe-\nsis technology can solve this problem.\n Mitochondria, commonly known as the cellular \u2018powerhouses\u2019, Results and discussions\nare responsible for various crucial functions in cellular processes Synthesis, characterization, and stability\nand signaling pathways, such as energy production, generation The synthetic route is shown in Scheme S1 in supporting informa-\nof reactive oxygen species (ROS), biosynthetic metabolism, reg- tion. L-tryptophan methyl ester was obtained from the amino acid\nulation of cell death, etc.26\u201328 Tumor cells depend on glycolysis L-tryptophan by methyl esterification.36 After Pictect\u2013Spengler cy-\nand mitochondrial metabolism to generate macromolecules, in- clization, the obtained product can be carried out to the next step\ncluding nucleotides, lipids, amino acids, and ATP, which are es- without purification and acylated with toluenesulfonyl chloride to\nsential for their survival and proliferation.29 Due to the altered obtain P-N-Ts-4H-\u03b2C, which is eliminated and aromatized under\nmitochondrial function in cancer cells, including mitochondrial alkaline conditions to obtain compound 5 (P\u03b2C). This step is a se-\nmembrane potential (MMP) and oxidative stress, mitochondria ries of elimination and aromatization reactions with mild reaction\nhave emerged as an attractive pharmacological target for selec- conditions and high yield. After hydrolysis, the natural product\ntively killing cancer cells. In the last few years, mitochondria- 1-phenyl-9H-pyrido[3,4-b]indole-3-carboxylic acid (P\u03b2CA) is ob-\ntargeted iridium complexes have received increasing attention as tained. The main ligand PP\u03b2C is obtained by condensing P\u03b2C with\nanticancer or photodynamic therapy.24 , 30\u201332 1,10-phenanthroline-5-amino.20 , 37 Finally, the iridium precursor\n Our previous studies found that Ru-\u03b2-carboline derivatives [Ir(C-N)2 Cl2 ]\u22121 (C-N = ppy, dfppy, bzq) and the main ligand PP\u03b2C\ncomplexes show antitumor effects.33 , 34 To enhance the an- are reacted to get the target cyclometalated iridium-\u03b2-carboline\nticancer effects, we aimed to develop new Ir(III) complexes complexes Ir1-3 (Fig. 1).\n\f Paper | 3\n\n\nTable 1. Cytotoxicity assay in vitro (IC50 a ) and LogPo/w values\n\n IC50 (\u00b5M)\n\nCompounds A549 Hela HepG-2 MCF-7 BEAS-2B SIb LogPO/W\n\nIr1 1.0 \u00b1 0.01 2.2 \u00b1 0.04 3.4 \u00b1 0. 1 5.2 \u00b1 0.3 10.4 \u00b1 1.4 10.4 1.9 \u00b1 0.2\nIr2 0.7 \u00b1 0.02 1.9 \u00b1 0.01 2.1 \u00b1 0.6 4.7 \u00b1 0.9 8.8 \u00b1 0.7 12.6 2.3 \u00b1 0.6\nIr3 0.5 \u00b1 0.01 1.1 \u00b1 0.02 1.6 \u00b1 0.4 3.7 \u00b1 0.3 7.5 \u00b1 0.7 15.0 3.2 \u00b1 0.4\nIr4c 15.6 \u00b1 0.6 21.5 \u00b1 1.3 \u2013 17.4 \u00b1 0.3 \u2013 \u2013 \u2013\nPP\u03b2C 5.1 \u00b1 0.1 6.3 \u00b1 0.21 5.7 \u00b1 0.09 5.4 \u00b1 0.1 8.1 \u00b1 1.3 1.6 \u2013\nP\u03b2CA 54.8 \u00b1 1.7 56.9 \u00b1 1.5 65.4 \u00b1 2.4 79.3 \u00b1 1.6 83.4 \u00b1 3.1 1.5 \u2013\nCisplatin 25.3 \u00b1 2.4 27.4 \u00b1 2.1 32.1 \u00b1 2.5 21.4 \u00b1 2.1 25.5 \u00b1 2.5 1.0 \u22124.2 \u00b1 0.9\n\na\n Cell viability was determined by MTT assay after treating the illustrated cells for 48 h. The data are expressed as mean \u00b1 standard deviation (Mean \u00b1 SD).\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\nb\n SI (selectivity index) = IC50 (BEAS-2B)/IC50 (A549).\nc\n The IC50 value of Ir4 are from ref.34\n\n\n\n The UV absorption and fluorescence spectra of Ir1-3 are shown the natural product P\u03b2CA and the main ligand PP\u03b2C, all iridium\nin Figures S1\u2013S3. In the UV absorption spectra, the peak intensity complexes showed a significantly improved broad-spectrum\nbefore 300 nm is caused by the intra-ligand charge transfer, and antitumor activity against most cancer cell lines. Among iridium\nthe broad peak around 350 nm is caused by the charge trans- complexes, Ir3 was determined to be the most effective as demon-\nfer transition from the metal center to the ligand. Under the ex- strated by nearly 110 times higher than that of the natural product\ncitation of 405 nm in the fluorescence spectra, the maximum P\u03b2CA and 10 times higher than that of the main ligand PP\u03b2C\nemission wavelengths of Ir1-3 are 586, 525, and 587 nm. At last, against A549 cells. Moreover, Ir1-3 are more effective than clinical\nthe complexes Ir1-3 and the main ligand PP\u03b2C were character- cisplatin and the iridium complex [Ir(ppy)2 (phen)](PF6 ) (Ir4) with-\nized by elemental analysis, ESI-MS (Figs. S4\u20137), 1 H NMR (Figs. out \u03b2-carboline derivative as the main ligand against all the test\nS8\u2013S11), 13 C NMR (Figs. S12\u201314), 1 H-1 H COSY (Figs. S15\u201317). The cancer cell lines, suggesting the main ligand PP\u03b2C plays a vital role\nESI-MS spectrum of PP\u03b2C showed a characteristic peak at m/z in vitro activity of iridium complexes. Compared with cisplatin,\n466.1787([M + H]+ ) (Fig. S4). For Ir1-3, 966.2849, 1038.2535, and PP\u03b2C, and P\u03b2CA, complexes Ir1-3 exhibited much lower toxicity\n1014.2789 [M-PF6 ]+ cationic peaks were detected, respectively towards normal BEAS-2B cells. These findings suggest that Ir1-3\n(Figs. S5\u20137). These experimental values were consistent with the possess a preferable therapeutic profile against cancer, especially\ntheoretical values, indicating the successful synthesis of above lung cancer cells. The selectivity index (SI) assay, as shown in\ncomplexes. In the 1 H NMR spectrum of PP\u03b2C, proton of the amide Table 1, further supported this observation, revealing a clear\nbond at 11.16 ppm is clearly observable, indicating the successful trend in which Ir3 (15.0) displayed the highest selectivity in-\ncondensation of the amide bond (Fig. S8). The purity of Ir1-3 was dex, followed by Ir2 (12.6), Ir1 (10.4), PP\u03b2C (1.6), P\u03b2CA (1.5), and\nhigher than 97% calculated based on the chromatographic peak Cisplatin (1.0). Since complexes Ir1-3 showed better antitumor\narea ratio by reversed-phase high performance liquid chromatog- activity in lung cancer cell line A549, this cell line was chosen as\nraphy (RP-HPLC) (Fig. S18). the follow-up research object.\n Drug stability plays a crucial role in cellular internalization It is well known that the lipophilicity of an anticancer drug\nand target binding. Thus, the time-dependent absorption spectra plays a crucial role in its cytotoxicity, where increased lipophilic-\nof iridium complexes in PBS were analysed. UV-Vis absorption ity leads to enhanced cellular uptake and, thus, more profound\nspectra of Ir1-3 did not change significantly within 48 h (Fig. cytotoxic effects.38\u201340 The oil\u2013water partition coefficients of the\nS19). It is important to note that upon systemic administration, three iridium complexes are 1.9, 2.3, and 3.2, respectively, as listed\niridium complexes have the ability to either precipitate or form in Table 1. The highest lipophilicity of Ir3 may explain its promi-\nbonds with specific biological molecules, such as various plasma nent antitumor effects compared to the other two iridium com-\nproteins. This can hinder their ability to reach high-concentration plexes. To further evaluate the antitumor effect of Ir3, EdU as-\nbiological targets. To investigate the potential binding of iridium says were performed to assess the cell proliferation rate after the\ncomplexes to such internal molecules, the absorption spectra treatment of Ir3. As shown in Fig. S22, the intensity of red fluores-\nof Ir1-3 were monitored using UV-Vis spectra at 298 K, while cence of Ir3-treated A549 cells was reduced in a concentration-\nbeing incubated in aqueous solutions containing bovine serum dependent manner, indicating a reduced DNA replication rate and\nalbumin (BSA). The results, as shown in Fig. S20, indicate that a decreased proliferation of A549 cells, showing that Ir3 can effec-\nthere was no significant change in absorbance in the UV-Vis tively inhibit the proliferation of A549 cells, which is consistent\nspectra, suggesting no prominent binding of iridium complexes with the cytotoxicity results.\nwith plasma proteins. The inductively coupled plasma mass\nspectrometry (ICP-MS) results also confirmed that the Ir1-3 did\nnot bind to the BSA (Fig. S21). Intracellular uptake and localization of iridium\n complexes\n Earlier research has demonstrated that the cellular localization\nCytotoxicity of iridium complexes in vitro of metal complexes and their mechanisms of action can be\nThe cytotoxicity of three iridium complexes against four cancer influenced by their lipophilicity.41\u201346 Metal complexes that are\ncell lines (A549, HeLa, HepG2, and MCF-7) and a normal human more lipophilic tend to accumulate and localize in the mito-\ncell line (BEAS-2B) were determined by 3-(4,5-dimethylthiazol- chondria, while those that are more hydrophilic tend to prefer\n2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with clinical the nucleus.44\u201346 The cellular uptake characteristics of transi-\ncisplatin as a positive control. As seen in Table 1, compared to tion metal-based drugs are also a crucial factor influencing their\n\f4 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\nFig. 2 (A) Flow cytometry analysis of different concentrations of Ir1-3 uptake levels in A549 cells. (B) Subcellular distribution of Ir content in A549 cells\nafter incubation with 0.5 \u03bcM Ir1-3 for 24 h. (C) Cellular Ir contents were determined in A549 cells incubated with 0.5 \u03bcM Ir3 for 3 h. (D) Confocal\nmicroscopy excited phosphorescent images of A549 cells co-labeled with Ir1-3 (1 \u03bcM, 1.5 h) and MTR (20 nM, 20 min). (MTR) \u03bbex = 561 nm,\n\u03bbem = 617 \u00b1 36 nm, (Ir1 and Ir3) \u03bbex = 405 nm, \u03bbem = 595 \u00b1 20 nm, (Ir2) \u03bbex = 405 nm, \u03bbem = 525 \u00b1 25 nm. (E) Confocal microscopy excited\nphosphorescent images of A549 cells co-labeled with Ir1-3 (1 \u03bcM, 1.5 h) and LTR (50 nM, 0.5 h). (LTR) \u03bbex = 561 nm, \u03bbem = 617 \u00b1 36 nm, (Ir1 and Ir3) \u03bbex\n= 405 nm, \u03bbem = 595 \u00b1 20 nm, (Ir2) \u03bbex = 405 nm, \u03bbem = 525 \u00b1 25 nm. (F) Cellular uptake mechanisms of Ir1-3. A549 cells were incubated with Ir1, Ir2,\nand Ir3 (1 \u03bcM, 30 min), respectively, under different temperatures, pretreated with CCCP (20 \u03bcM, 1 h) or CQ (50 \u03bcM, 1 h). (Ir1 and Ir3) \u03bbex = 405 nm,\n\u03bbem = 595 \u00b1 20 nm, (Ir2) \u03bbex = 405 nm, \u03bbem = 525 \u00b1 25 nm.\n\n\ncytotoxicity.35 , 45 , 47 As shown in Fig. 2A, Ir1-3 entered cells in a occurred between the Ir3 and MTR channels, with a Pearson\u2019s\nconcentration-dependent manner as measured by flow cytome- coefficient (R) of 0.92. Despite this, it was observed that the ac-\ntry. ICP-MS was further used to study the cellular uptake of irid- cumulation of Ir1-3 complexes in LTR channels was poor, in-\nium complexes and their intracellular distribution. We detected dicating that a significant portion of Ir1-3 was localized within\nthe distribution of Ir1-3 in cells using a mitochondrial extrac- the mitochondria after being internalized by the cells (Fig. 2E).\ntion kit. As shown in Fig. 2B, the content of Ir1-3 in mitochon- To explore the mechanisms underlying iridium complexes enter-\ndria was significantly higher than in the nucleus and cytoplasm. ing cells, A549 cells were treated with an energy inhibitor car-\nFigure 2C showed that the intracellular Ir3 content reached the bonyl cyanide 3-chlorophenyl hydrazone (CCCP), and an endocy-\nhighest value at about 2 h. Compared to other reported metal tosis inhibitor chloroquine (CQ), respectively. As shown in Fig. 2F,\ncomplexes, the rate of iridium complexes entering the cells is the fluorescence intensity was significantly reduced in A549 cells\nsignificantly fast.48 , 49 The colocalization was further analysed after being treated with CCCP compared to the control group\nby confocal laser scanning microscopy (CLSM). All three iridium (37\u00b0C), indicating the existence of an energy-dependent mecha-\ncomplexes were co-stained with Mito-Tracker Red (MTR) or Lyso- nism. However, no significant changes in cellular uptake in cells\nTracker Red (LTR) in A549 cells, respectively. Figure 2D and E pretreated with CQ. These results suggest that iridium complexes\ndemonstrate that the highest degree of superposition patterns enter cancer cells through an energy-dependent non-endocytosis\n\f Paper | 5\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\nFig. 3 (A) Generation of mitochondrial ROS caused by Ir1-3 treatment. A549 cells were treated with Ir1-3 at 0.5 \u03bcM for 3 h, respectively. Then, the cells\nwere co-stained with H2 DCFDA and MTR for CLSM observation. (DCF: \u03bbex = 488 nm; \u03bbem = 525 \u00b1 25 nm; MTR: \u03bbex = 561 nm; \u03bbem = 617 \u00b1 36 nm).\n(B) Cell viability assay after A549 cells treatment with 1 \u03bcM Ir3 for 24 h in the absence or presence of GSH or NAC. GSH: 5 mM; NAC: 10 mM. (C) CLSM\nanalysis of cellular MMP level by JC-1 staining after treatment with Ir3 at 0.5, 1 \u03bcM for 6 or 12 h, respectively. (JC-1 aggregate, \u03bbex = 585 nm,\n\u03bbem = 595 \u00b1 20 nm; JC-1 monomer, \u03bbex = 488 nm, \u03bbem = 525 \u00b1 25 nm). (D) Intracellular ATP levels after treatment with Ir3 at the indicated\nconcentrations. (E) Representative TEM images showing the morphological features in A549 cells after treatment with 0.5 \u03bcM Ir3 for 24 h. Red arrows:\nimpaired mitochondria.\n\n\npathway, consistent with previously reported other iridium ROS in mitochondria was detected by CLSM. As demonstrated\ncomplexes.7 , 50 , 51 in Fig. 3A, the fluorescence of DCF overlapped well with that of\n MTR, indicating that mitochondria are the major ROS-generating\nProduction of reactive oxygen species and sites. Figure S23 showed the changes of ROS measured by the mi-\nmitochondrial dysfunction croplate reader, indicating that the intracellular ROS after irid-\n ium complexes treatment increases in a time- and concentration-\nMitochondria, which are the powerhouse of cells, participate\n dependent manner. Three iridium complexes Ir1-3 caused an ele-\nin several crucial cellular processes, including cell differentia-\n vated production of ROS, and it was further found that this ROS is\ntion, information transmission, and apoptosis, and are the pri-\n mainly produced from mitochondria (Fig. 3A). To demonstrate the\nmary source of ROS, which is closely related to mitochondrial\n effect of ROS on cell viability, two antioxidants N-acetyl-L-cysteine\ndamage.52\u201356 Using the ROS probe H2 DCF-DA, the production of\n\f6 | Metallomics\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\nFig. 4 (A) Representative confocal images of annexin V labeled A549 cells after treatment with 0.5 or 1 \u03bcM complex Ir3 or 50 \u03bcM cisplatin for 24 h.\n(Annexin V: \u03bbex = 488 nm; \u03bbem = 525 \u00b1 25 nm). (B) Caspase activities were measured by using specific fluorescent substrates for caspase-3/7. (C) The\nexpression levels of PARP and cleaved caspase-3/9 and cleaved PARP were evaluated with 0.5 \u03bcM Ir1-3 treatment for 24 h, and GAPDH was used as an\ninternal control. (D) Cell viability detection after treatment with 0.5 \u03bcM Ir3 for 24 h in the absence or presence of various inhibitors. 3-MA: 1 mM;\nZ-VAD-FMK: 25 \u03bcM; Necrostatin-1: 60 \u03bcm.\n\n\n\n\n(NAC) and glutathione (GSH) were used. As evidenced in Fig. 3B, Ir3 Underlying mechanisms of Ir3-induced cell death\ninduced significant cell death in a concentration-dependent man- The potent anticancer activity of iridium complexes has\nner and the cell death was rescued by antioxidants. prompted us to explore the underlying mechanisms. To in-\n We have previously found that iridium complexes Ir1-3 can ac- vestigate whether Ir3 induces apoptosis of A549 cells, AO/EB\ncumulate in mitochondria, and the changes in MMP can be de- double staining was applied (Fig. S24). AO is a critical dye that ex-\ntected after staining live cells with 5,5\u0003 ,6,6\u0003 -tetrachloro-1,1\u0003 ,3,3- hibits green fluorescence and has the ability to stain both live and\ntetraethylbenzimidalylcarbo cyanine iodide (JC-1) as a fluorescent dead cells; EB, as a nuclear dye, cannot penetrate normal cells.\nprobe.57 , 58 Compared to the control, the Ir3 treatment caused a Only when the permeability of the cell membrane is changed\nred-to-green color shift in most of the treated cells, indicating the can EB enter the cells and emit red fluorescence.59 Control cells\nloss of MMP (Fig. 3C). exhibited normal morphology with a uniform green fluorescence,\n Considering the role of mitochondria in cellular energy pro- while preincubation of cells with Ir3 complex resulted in the\nduction and the effects of loss of membrane potential on mi- observation of orange or red fluorescence, which were distinct\ntochondrial ATP production, ATP assays were performed. We morphological features of apoptosis. The AO/EB double staining\nfound that intracellular ATP levels were significantly decreased results suggested that the Ir3 complex could induce apoptosis of\nconcentration-dependent (Fig. 3D). The ultrastructural changes of A549 cells.\nIr3-treated A549 cells were further analyzed using transmission In addition, to further study if there are Ir3-induced early apop-\nelectron microscopy (TEM). Figure 3E illustrated that the chro- tosis events, Annexin V staining was conducted, which can bind to\nmatin of A549 cells treated with Ir3 did not change significantly; phosphatidylserine (PS) exposed to extracellular space. As shown\nhowever, the mitochondrial structure was swollen, the cristae in Fig. 4A, PS of both Ir3 and cisplatin-treated cells were stained,\nfolds weakened, and the overall rounded and shortened, indicat- indicating that both complex Ir3 and cisplatin induced A549 cells\ning that mitochondrial damage caused structural and morpho- apoptosis. Furthermore, since caspase plays a crucial role in the\nlogical changes. These findings suggested that Ir3 preferentially initiation and performance of apoptosis, and caspase activation is\naccumulates in the mitochondria and can rapidly and effectively considered one of the essential features of apoptosis,60 , 61 to fur-\naffect the mitochondria\u2019s functions in ROS and cellular ATP pro- ther confirm cell apoptosis induced by Ir3, the activity of caspase-\nduction and alter the mitochondrial morphology. 3/7 was examined using specific fluorescent substrates. Figure 4B\n\f Paper | 7\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\nFig. 5 (A) Formation of A549 tumor spheroids within 3 days. (B) MCTSs were treated with 0.5 \u03bcM Ir1-3 for 10 days, respectively. (C) After treatment with\n0.5 \u03bcM Ir1-3, A549 MCTSs were routinely monitored for regrowth inhibition. (D) Calcein AM and PI dual-staining of MCTSs after treated with 0.5 \u03bc\u039c\nIr1-3 for 48 h. (E) Z-stack images (top) and 3D z-stacks (bottom) of Calcein-AM (\u03bbex = 488 nm, \u03bbem = 525 \u00b1 25 nm)/PI (\u03bbex = 535 nm, \u03bbem = 617 \u00b1 36 nm)\ndouble staining observed by CLSM after incubation of A549 MCTSs with 0.25 \u03bcM Ir1-3 over 48 h, respectively. Z-axis image scan from the top to the\nbottom of an intact spheroid every 30 mm.\n\n\n\nindicates that the pre-incubation of A549 cells with Ir3 complex could prevent Ir3-induced apoptosis; however, adding Z-VAD-FMK\nresulted in a significant and concentration-dependent increase in increased the cell survival rate. Thus, the above results confirmed\ncaspase-3/7 activities. Notably, 1 \u03bcM Ir3 activated caspase-3/7 lev- that iridium complexes induce cell death through the apoptosis\nels comparable to 100 \u03bcM cisplatin. pathway.\n To further study the signaling pathway of apoptosis induced\nby the synthesized iridium complexes, the effects of the three\n Iridium complexes-induced antitumor effects on\niridium complexes on the protein expression levels of PARP, and\n 3D multicellular tumor spheroids (MCTSs)\ncleaved PARP, cleaved caspase-3/9, were studied by western blot-\n Multicellular tumor spheroids (MCTSs) possess the ability to de-\nting assays. As shown in Fig. 4C, compared to the control group,\n velop a necrotic core, a quiescent intermediate region, and a pro-\n0.5 \u03bcM Ir1-3 complexes increased the protein expression levels\n liferating periphery region, which allows them to closely resemble\nof cleaved-PARP and cleaved caspase-3/9 in A549 cells after 24 h\n the microenvironment of a tumor when compared to a monolayer\ntreatment. The results further verified that the three iridium com-\n cell system62 , 63 . As a result, the use of three-dimensional MCTS\nplexes could induce the apoptosis pathway in A549 cells.\n models for drug screening has gained significant attention.64 To\n To exclude that non-programmed cell death is involved in Ir3-\n further investigate the biological effect of Ir1-3 on 3D MCTSs,\ninduced cell death, a necrosis inhibitor (Necrostatin-1), an au-\n A549 MCTSs with a diameter of approximately 200 \u03bcm were suc-\ntophagy inhibitor (3-MA), and an apoptosis inhibitor (Z-VAD-FMK)\n cessfully constructed (Fig. 5A). Then, the growth rate of MCTSs\nwere used. As shown in Fig. 4D, neither Necrostatin-1 nor 3-MA\n after the treatment of Ir1-3 at 0.5 \u03bc\u039c was monitored. The cell\n\f8 | Metallomics\n\n\nspheroid treated with Ir1-3 were significantly smaller than the washed with petroleum ether, and filtered with suction to obtain\ncontrol group within 10 days (Fig. 5B). Unlike other groups, obvi- compound 4, a yellow solid yielding 86.8%.\nous cell debris appeared around the spheroids after Ir3 treatment.\n (4) Synthesis of compound 5 (P\u03b2C)\nBesides, as shown in Fig. 5C, the diameter of the cell spheroid\nchanged slowly, displaying that the growth of the cell spheroid Substance 4 (1.84 g, 4 mmol) was dissolved in dimethyl sulfox-\nwas significantly inhibited. To further assess the cytotoxicity of ide, potassium carbonate (0.69 g, 5 mmol) was added, and the mix-\ncomplexes Ir1-3\u2013A549 MCTSs, A549 MCTSs were stained with ture was heated to reflux at 100\u00b0C for 5 h. After the reaction was\nCalcein-AM/propidium iodide (PI) after treatment with 0.5 \u03bc\u039c completed, the liquid was cooled to room temperature, 100 mL\nIr1-3, respectively. As shown in Fig. 5D, untreated MCTSs, com- of water was added, stirred and suction filtered, washed with wa-\nposed mainly of viable cells, were observed to emit extensive ter, and dried to obtain the product, compound 5, with a yield of\ngreen fluorescence in A549 cells. Conversely, incubation with 89.1%.\n0.5 \u03bcM Ir1-3 resulted in significant enhancement of PI red and\n (5) Synthesis of compound 6 (P\u03b2CA)\nweak green fluorescence in MCTSs, indicating severe cellular\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\ndamage in a majority of cancer cells. When the concentration of Compound 5 (1.21 g, 4 mmol) was dissolved in a mixed solution\ncomplexes was reduced to 0.25 \u03bcM, the red fluorescence of Ir3- of ethanol and water (30 mL, 2:1, v/v), sodium hydroxide (0.40 g,\ntreated A549 MCTSs was still strong (Fig. 5E), suggesting that com- 12 mmol) was added, and refluxed at 100\u00b0C. The pH of the reacted\nplex Ir3 exhibited good antitumor activity even at low concentra- solution was adjusted to 5 with 5 M HCl, suction filtered, and dried\ntion. to obtain a yellow solid with a yield of 85.5%.\n\n (6) Synthesis of compound 7 (PP\u03b2C)\nConclusions\nIn summary, we designed and synthesized three novel Ir(III) com- Compound 7 was prepared following a published\nplexes Ir1-3 with marine-derived \u03b2-carboline as the parent af- procedure.67 Compound 6 (1.15 g, 4 mmol) was added\nter structural modification and optimization. Complexes Ir1-3 to 1-Hydroxybenzotriazole (0.81 g, 6 mmol), N-Ethyl-N-(1-\nshowed significant anticancer activities, among which Ir3 was methylethyl)-2-propanamine (400 uL), and 1,10-phenanthroline-\nthe most prominent. Synthesized Ir(III) complexes Ir1-3 can enter 5-amino (0.78 g, 4 mmol). After 2 h of reaction at room tempera-\nA549 cells through an energy-dependent non-endocytosis path- ture, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlo-\nway, and preferentially accumulate in the mitochondria and im- ride (1.15 g, 6 mmol) was added, and the reaction was continued\npair mitochondrial functions as evidenced by the loss of MMP, the at room temperature for 21 h. The solvent was removed, washed\nreduction of cellular ATP production, and the increase of ROS in with water, and the resulting yellow precipitate was filtered. The\nmitochondria. These iridium complexes can induce remarkable crude product was applied to a silica gel column for a white solid.\ncell death through the caspase-mediated apoptosis pathway in PP\u03b2C: 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 12.04 (s, 1H), 11.16 (s, 1H),\nvitro. Using the 3D MCTSs model to simulate the conditions of 9.17 (dd, J = 4.3, 1.6 Hz, 1H), 9.08 (dd, J = 4.3, 1.7 Hz, 1H), 9.04 (s,\nsolid tumor proliferation in vivo, the results showed that Ir1-3 had 1H), 8.55 (ddd, J = 10.3, 8.2, 1.7 Hz, 2H), 8.50 (d, J = 7.9 Hz, 1H), 8.46\na notable effect against the growth tumor spheres. Our findings (s, 1H), 8.39\u20148.31 (m, 2H), 7.87 (dd, J = 8.4, 4.2 Hz, 1H), 7.79 (dd,\nindicated that the synthesized novel three iridium complexes tar- J = 8.1, 4.3 Hz, 1H), 7.73 (q, J = 8.1, 7.7 Hz, 3H), 7.64 (q, J = 7.5 Hz,\ngeted mitochondria, significantly inhibited tumor growth, and had 2H), 7.37 (t, J = 7.5 Hz, 1H).\nsubstantial application prospects in targeted therapy of tumors. (7) Synthesis of compound Ir2 (L)4 Cl2\n\n We followed the procedure of an established synthesis68 , 69 and\nExperimental section obtained a pure yellow crystal of the dichloro-bridged Ir2 (L)4 Cl2\nSynthesis and characterization dimer (L = 2-phenylpyridine, 2-(2,4-difluorophenyl) pyridine, 7,8-\nSynthesis of complexes benzoquinoline).\n (1) Synthesis of compound 2 (Trp-OCH3 ) (8) Synthesis of compound Ir1-3\n 65\n Compound 2 was prepared following a published protocol. Ir2 (L)4 Cl2 (2 mmol) and PP\u03b2C (1.86 g, 4 mmol) were dissolved\n (2) Synthesis of compound 3 (P-4H-\u03b2C) in a mixed solution of dichloromethane: methanol (2:1, v/v) and\n refluxed for 4 h under argon protection. After the reaction, the\n Compound 3 was synthesized according to references65 , 66 ; i.e. solution was cooled to room temperature, CH2 Cl2 was removed,\ncompound 2 (1.019 g, 4 mmol) was added to the reaction tube, fol- and 20 mL of KPF6-saturated aqueous solution was added. After\nlowed by benzaldehyde (408 \u03bcL, 4 mmol) and dissolved in 15 mL standing, filter, vacuum dry, and purify to obtain Ir1-3. The yellow\nof isopropanol. Under argon protection, the mixture was heated precipitate was filtered, washed with water and dried in a vacuum.\nto reflux at 90\u00b0C for 10 h, the obtained liquid was distilled under Ir1: 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 12.11 (s, 1H), 11.36 (s, 1H),\nreduced pressure to remove the solvent, stirred with benzene, suc- 9.08 (s, 1H), 8.94 (dd, J = 8.4, 1.4 Hz, 1H), 8.92\u20148.88 (m, 1H), 8.87 (s,\ntion filtered, and dried to obtain a pale-yellow solid with a yield of 1H), 8.52 (d, J = 7.9 Hz, 1H), 8.34\u20148.25 (m, 5H), 8.17\u20148.15 (m, 1H),\n92.3%. 8.15\u20148.11 (m, 1H), 8.04 (dd, J = 8.3, 5.0 Hz, 1H), 7.98 (d, J = 7.8 Hz,\n 2H), 7.93\u20147.85 (m, 4H), 7.75 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 7.3 Hz,\n (3) Synthesis of compound 4 (P -N-Ts-4H-\u03b2C)\n 1H), 7.52 (t, J = 4.6 Hz, 2H), 7.37 (s, 2H), 7.11\u20147.06 (m, 2H), 7.03\n Compound 3 (1.23 g, 4 mmol) was dissolved in (t, J = 6.7 Hz, 2H), 7.00\u20146.94 (m, 2H), 6.33 (d, J = 7.4 Hz, 2H).\ndichloromethane, 350 \u03bcL of pyridine and p-toluenesulfonyl ESI-MS(CH3 CN): m/z calc. for ([M-PF6 ]+ ) 966.25; found: 966.28. 13 C\nchloride (0.76 g, 4 mmol) was added at \u22128\u00b0C, then stirred at NMR (101 MHz, DMSO-d6 ) \u03b4 167.37, 164.84, 151.31, 150.54, 150.27,\nroom temperature for 4 h, and the solvent was distilled off under 150.19, 149.57, 149.53, 147.04, 144.53, 144.49, 142.24, 141.49, 139.23,\nreduced pressure. It was washed with 10 mL of 10% potassium 138.95, 138.82, 137.75, 135.26, 134.94, 134.58, 131.79, 131.33, 130.74,\ncarbonate solution, dried over anhydrous magnesium sulphate, 130.68, 129.75, 129.39, 128.14, 127.79, 127.21, 125.58, 124.47, 124.40,\n\f Paper | 9\n\n\n124.32, 122.90, 122.68, 121.66, 121.01, 120.52, 120.47, 114.54, 113.36. well. After a 4 h incubation period, DMSO (150 \u03bcL/well) was used\nUV-Vis (\u03bb/nm, \u03b5/M\u22121 \u2022cm\u22121 ) (PBS): 280 (69 000), 348 (32 150). Ele- to solubilize the fresh formazan. The optical density of each well\nmental analysis calcd (%) for C52 H35 F6 IrN7 OP: C, 56.15; H, 3.15; N, was then measured at a wavelength of 570 nm using a microplate\n8.82; found: C, 54.18; H, 3.16; N, 8.84. spectrophotometer. The analysis of absorbance data provided the\n Ir2: 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 12.09 (s, 1H), 11.40 (s, 1H), IC50 values.\n9.08 (s, 1H), 8.97 (dd, J = 13.1, 8.3 Hz, 2H), 8.90 (s, 1H), 8.51 (d,\nJ = 7.9 Hz, 1H), 8.38 (s, 1H), 8.34 (d, J = 7.4 Hz, 4H), 8.29\u20148.24 (m, EdU staining to detect cell proliferation\n1H), 8.14 (dd, J = 8.5, 5.1 Hz, 1H), 8.00 (d, J = 7.1 Hz, 3H), 7.75 (s, EdU is a thymidine analog, a new nucleoside marker that can re-\n1H), 7.73\u20147.62 (m, 4H), 7.59 (d, J = 6.1 Hz, 2H), 7.38 (t, J = 7.4 Hz, place thymidine (T) during DNA replication. After a cell cycle of\n1H), 7.16\u20147.08 (m, 2H), 7.04 (s, 2H), 5.75 (dd, J = 8.3, 2.4 Hz, 2H). EdU culture, EdU will enter all cells and act as proliferating cells-\nESI-MS (CH3 CN): m/z calc. for ([M-PF6 ]+ ) 1038.22; found: 1038.25. sensitive fluorescent markers. A549 cells were evenly seeded in a\n13\n C NMR (101 MHz, DMSO-d6 ) \u03b4 164.84, 164.65, 164.54, 164.52, confocal dish, and the complex was added after the cells were at-\n163.38, 163.31, 162.60, 162.47, 162.13, 162.11, 162.00, 160.02, 159.89, tached to the dish for 12 h. These cells were stained with EDU,\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\n154.65, 154.59, 154.37, 154.31, 151.97, 150.77, 150.28, 146.72, 144.16, followed by infiltration with 0.5% Triton X-100 for 20 min and\n142.25, 141.52, 140.45, 139.35, 138.92, 137.75, 135.50, 135.28, 134.67, washing with 3% BSA. Hoechst 33 342 was stained in the dark for\n131.47, 130.68, 129.75, 129.41, 128.33, 128.23, 128.08, 127.52, 124.99, 10 min, washed with PBS, and observed and photographed under\n124.90, 123.94, 123.92, 123.77, 123.74, 122.65, 121.66, 121.00, 120.53, an inverted fluorescence microscope.\n114.55, 114.09, 113.93, 113.37, 99.92, 99.64, 99.38. UV-Vis (\u03bb/nm,\n\u03b5/M\u22121 \u2022cm\u22121 ) (PBS): 281 (57 950), 348(27 950). Elemental analysis Lipophilicity measurements\ncalcd (%) for C52 H31 F10 IrN7 OP: C, 52.74; H, 2.62; N, 8.28; found: C, LogPo/w values were determined using a protocol that was pre-\n52.71; H, 2.61; N, 8.30. viously described.70 Equal amounts of H2 O and octanol were\n Ir3: 1 H NMR (400 MHz, DMSO-d6 ) \u03b4 12.08 (s, 1H), 11.39 (s, 1H), mutually saturated for 12 h. Ir1-3 were added to a mixture of\n9.07 (s, 1H), 8.90 (s, 3H), 8.57\u20148.50 (m, 3H), 8.34\u20148.31 (m, 2H), octanol/H2 O aqueous solution (1:1, v/v), respectively, and then\n8.26 (dd, J = 5.0, 1.2 Hz, 1H), 8.13 (dd, J = 5.1, 1.3 Hz, 1H), 8.04\u2014 shaken at 37 \u00b0C, 200 rpm for 24 h. The oil and water phases were\n7.99 (m, 5H), 7.96\u20147.93 (m, 1H), 7.91 (d, J = 3.2 Hz, 1H), 7.89 (d, separated by centrifugation (3000 rpm, 10 min) and collected. To\nJ = 3.3 Hz, 1H), 7.75 (s, 1H), 7.68 (d, J = 7.6 Hz, 3H), 7.60 (d, J = 7.7 Hz, prepare the samples, 100 \u03bcL of the aqueous phase and 100 \u03bcL of\n3H), 7.48 (ddd, J = 8.1, 5.4, 1.3 Hz, 2H), 7.40\u20147.36 (m, 1H), 7.25 (s, the oil phase were taken and treated with 10% HNO3 and 20%\n2H), 6.35 (dd, J = 7.2, 3.6 Hz, 2H). ESI-MS(CH3 CN): m/z calc. for H2 O2 , respectively, overnight. The resulting solutions were then\n([M-PF6 ]+ ) 1014.25; found: 1014.28. 13 C NMR (101 MHz, DMSO) \u03b4 brought up to a final volume of 5 mL each. The ICP-MS (NEXION-\n164.79, 156.89, 151.82, 150.66, 149.31, 147.47, 147.34, 147.05, 144.92, 300X, PerkinElmer, USA) was utilized to measure the Ir(III) content\n142.24, 141.48, 140.89, 140.84, 138.93, 138.80, 138.09, 137.74, 135.27, in the aqueous and oil phases. LogPo/w values were calculated us-\n134.83, 134.55, 134.25, 131.34, 130.67, 130.21, 130.00, 129.74, 129.40, ing the equation LogPo/w = Log ([Ir]o /[Ir]w ).\n129.36, 129.13, 128.06, 127.75, 127.23, 127.20, 124.71, 123.29, 123.22,\n122.65, 121.65, 120.99, 120.91, 120.28, 114.53, 113.35. UV-Vis (\u03bb/nm, Cellular uptake\n\u03b5/M\u22121 \u2022cm\u22121 ) (PBS): 260 (76 000), 340(34 050). Elemental analysis A549 cells were seeded in 60 mm tissue culture dishes and incu-\ncalcd (%) for C56 H35 F6 IrN7 OP: C, 57.97; H, 3.02; N, 8.45; found: C, bated for 24 h before treatment with Ir3. Following treatment, the\n57.99; H, 3.01; N, 8.43. cells were trypsinized, collected in centrifugal tubes, and washed\n thrice with cold PBS. The cell pellets were digested in a mixture of\nUV-vis and fluorescence spectroscopy analysis HNO3 and H2 O2 for 24 h and diluted to 4 mL with ultrapure water.\nThe UV-Vis absorption spectra of Ir1-3 at 293 K were obtained us- The amount of iridium taken up by the A549 cells was determined\ning PBS as the solvent, and recorded on a Perkin Elmer Lambda by ICP-MS using a 100 ng/mL iridium standard solution.\n850 spectrophotometer. The samples were excited at 405 nm, and\nthe emission wavelengths were recorded on a Perkin Elmer LS-55 Subcellular distribution of ir(III) complexes\nspectrofluorometer, ranging from 450 to 750 nm. A549 cells were seeded in 60 mm tissue culture dishes and incu-\n bated for 12 h before treatment with Ir1-3 for 24 h, respectively.\nStability assay Cellular fractions, including nuclear, mitochondrial, and cytoplas-\nThe stability of the Ir1-3 complexes was measured by UV-Vis spec- mic fractions, were extracted using the Cell Mitochondria Isola-\ntroscopy and ICP-MS according to our previous reports.48 , 70 tion Kit (Beyotime).71 , 72 The harvested cells were resuspended in\n mitochondrial isolation buffer, homogenized, and centrifuged at\nCell lines and culture conditions 600 g for 10 min to obtain the pellet (nucleus fraction). The re-\nThe Experimental Animal Center of Sun Yat-Sen University maining supernatant was centrifuged at 11 000 g for 10 min at\n(Guangzhou, China) provided four cancer cell lines, A549, HepG2, 4 \u00b0C to obtain the supernatant (cytoplasmic fraction) and pellet\nMCF-7, and HeLa, as well as a normal cell line, BEAS-2B. All the (mitochondrial fraction). The fractions were then digested using\ncell lines were cultured in DMEM media supplemented with 10% HNO3 and H2 O2 for 24 h, diluted to 4 mL with ultrapure water.\nFBS and incubated in a 5% CO2 incubator at 37 \u00baC. The iridium content in each fraction was determined by ICP-MS,\n using a 100 ng/mL iridium standard solution.\nIn vitro cytotoxicity assay\nTo assess the cytotoxicity of Ir1-3 and other control compounds, Colocalization assay\nthe MTT assay was utilized. The cells were seeded overnight in 96- A549 cells were initially seeded into 35 mm confocal dishes and\nwell plates at a density of 4 \u00d7 103 cells per well. Next, the tested incubated at 37 \u00b0C for 12 h. Afterward, Ir1-3 (1 \u03bcM) was added,\ncompounds, Ir1-3, and the same amount of DMSO as the negative respectively, and the cells were incubated for an additional 3 h.\ncontrol were added to the wells. The plates were incubated for 48 h Subsequently, the cells were washed with PBS twice and in-\nbefore the addition of MTT dye stock solution (10 \u03bcL, 1 mM) to each cubated with fluorescent probes, Mito-Tracker Red (MTR), and\n\f10 | Metallomics\n\n\nLyso-Tracker Red (LTR), at 37 \u00b0C for 30 min each. The cells were Caspase-3/7 activity assay\nthen rewashed with PBS and observed using CLSM with a 60\u00d7 oil- A549 cells were seeded in white-walled nontransparent bottomed\nimmersion objective lens. The excitation/emission wavelengths 96-well plates and treated with Ir3 at different concentrations\nfor Ir1 and Ir3 were set at 405 nm/595 \u00b1 20 nm, while for Ir2, (0.5, 1.0, 2.0 \u03bcM). Following 24 h of incubation, the activity of\nthey were set at 405 nm/525 \u00b1 25 nm. The excitation/emission caspase-3/7 was measured using the Caspase Glo\u00ae 3/7 Assay kit\nwavelengths for MTR and LTR were set at 561 nm/617 \u00b1 36 nm. protocol, and luminescence was measured using a microplate\n reader (infinite M200 PRO, TECAN, Switzerland).\nCellular uptake mechanism study\nTo investigate the cellular uptake mechanism of Ir1-3, CLSM\n Western blotting analysis\nimaging was used. After being pretreated with various inhibitors\n The expression levels of A549 cells protein (cleaved caspase 3/9,\nunder different conditions, A549 cells were incubated with Ir1-3.\n PARP, cleaved-PARP) after the treatment with iridium complexes\nThen, the cells were washed with PBS three times and observed\n were detected by western blot following published methods.70 , 74\nusing CLSM. For the temperature influence study, HeLa cells were\n The protein bands were visualized using ChemiDocTM XRS \u00fe Imag-\n\n\n\n\n Downloaded from https://academic.oup.com/metallomics/article/15/6/mfad035/7172870 by guest on 12 May 2026\ntreated with Ir1-3 (0.5 \u03bcM) in the dark for 2 h at 37 \u00b0C and 4 \u00b0C,\n ing System (Bio-Rad, USA).\nrespectively. In the metabolic inhibition study, A549 cells were pre-\ntreated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP,\n20 mM) or chloroquine (CQ, 50 \u03bcM) for 1 h and then exposed to 3D multicellular tumor spheroids (MCTSs) formation\nIr1-3 (0.5 \u03bcM) in the dark for 2 h at 37 \u00b0C. A sterilized PBS solution containing 0.75% agarose was added to\n each well of a 96-well plate at a volume of 50 mL per well. The\nCellular ROS detection plate was then cooled for 4 h under UV light irradiation. A cell\nA549 cells were treated with the iridium complexes at the indi- suspension of A549 (3 \u00d7 103 cells per well) was seeded on top of\ncated concentrations for 3 h. The cells were harvested and incu- the agarose and incubated for 1\u20132 days until MCTSs were formed.\nbated with 10 \u03bcM H2 DCFDA and MTR for 15 min at 37\u00b0C in the The generated MCTSs were cultured and stored in a cell incubator\ndark and then photographed using an inverted fluorescence mi- at 37 \u00b0C and 5% CO2, with the medium replaced every 2 days. Im-\ncroscope. age J software was used to calculate the diameter of each tumor\n spheroid.\nMMP measurement\nMMP was analyzed using CLSM. A549 cells were incubated with Calcein AM/PI staining assay\ndifferent concentrations (0.5, 1.0 \u03bcM) of Ir3 at different time points Ir1-3 to MCTSs was evaluated using Calcein AM/PI double stain-\n(6, 12 h). Cells were incubated in a complete medium containing ing. MCTSs were prepared as described previously and treated\n10 mg/mL JC-1 for 30 min and washed twice with PBS; images of with iridium complexes for 48 h. The treated MCTSs were then\nthe cells were photographed immediately by CLSM. stained with Calcein AM (\u03bbex = 488 nm, \u03bbem = 525 \u00b1 25 nm), and\n PI (\u03bbex = 535 nm, \u03bbem = 617 \u00b1 36 nm). Imaging was performed\nIntracellular ATP detection using CLSM.\nThe experimental procedure was conducted by a previously pub-\nlished method.73 The Cell Titer-Glo\u00ae Luminescent Cell Viability\nAssay kit (G7570, Promega, USA) was used to measure the cellular Supplementary material\nATP level according to the manufacturer\u2019s instructions.\n Supplementary data are available at Metallomics online.\nTransmission electron microscopy (TEM) detection\nA549 cells were grown in 60 mm tissue culture dishes for 12 h,\n Acknowledgments\nfollowed by treatment with Ir3 (0.5 \u03bcM). After 24 h of incubation,\nthe cells were collected and fixed overnight in PBS containing 2.5% This research was funded by the National Natural Science Foun-\nglutaraldehyde (pH = 7.4) at 4\u00b0C. The cells were then treated with dation of China (No. 21701034, 82101505), the Discipline Construc-\nosmium tetroxide, stained with uranyl acetate and lead citrate so- tion Project of Guangdong Medical University (No. 4SG23004G),\nlution, and finally observed under a transmission electron micro- the Science and Technology Program of Guangdong Province\nscope (JEM-1400, JEOL, Japan). (No. 2019B090905011), the Science and Technology Program of\n Zhanjiang (No. 2021A05044, 2021A05242), the Medical Scien-\nAO/EB staining assay tific Research Foundation of Guangdong Province of China (No.\nA549 cells were cultured in 35 mm confocal dishes and treated A2022026), the Zhanjiang Marine Young Talent Innovation Project\nwith or without iridium complexes for 24 h. After incubation, the (No. 2022E05013) and the University Student Innovation Experi-\ncells were washed twice with cold PBS, stained with AO/EB solu- ment Program (No. S202210571061, ZZZF006). We thank the Public\ntions at a concentration of 100 \u03bcg/mL, and observed using an in- Service Platform of South China Sea for R&D Marine Biomedicine\nverted fluorescence microscope. Resources for support.\n\nAnnexin V staining assay\nThe experiment was carried out following the manufacturer\u2019s in- Data availability\nstructions. A549 cells were seeded in 35 mm confocal dishes and All data are incorporated into the article and its online supple-\ntreated with various concentrations of Ir3 for 24 h. The cells were mentary material.\nthen collected and stained with Annexin V and PI at room tem-\nperature in the dark for 15 min before being imaged by CLSM us-\ning a 100 \u00d7 oil-immersion objective lens. 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Cao, M. F. Zhang, C. P. Tan, L. N.\n Ji and Z. W. Mao, Cyclometalated iridium(iii) complexes induce\n\n\n\n\nReceived: March 29, 2023. Accepted: May 17, 2023\n\u00a9 The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com\n\f", "pages_extracted": 13, "text_length": 92069}