Abstract Modulating mitochondrial activity to regulate cancer cell homeostatic recycling presents a promising approach to overcome tumor resistance. Consequently, there is an urgent need for novel mitochondria-targeting agents and innovative strategies. We have developed [((η^5-Cp∗)Ir(rhod)]^2+2PF[6]^− (Ir-rhod), a new mitochondria-targeted iridium complex that exhibits greater cytotoxicity towards A549R (cisplatin-resistant human lung cancer) cells compared to the ligand rhod. Ir-rhod's mitochondrial targeting ability stems from both rhodamine's inherent mitochondrial affinity and the complex's positive bivalent nature. The positively charged Ir-rhod enters cells and is drawn to mitochondria due to the high transmembrane potential in tumor cells. Notably, rhodamine enables real-time observation of Ir-rhod's dynamic distribution in vivo. Ir-rhod influences mitochondrial function, triggering tumor cell ferroptosis and apoptosis by modulating ACSL4 and GPX4. The targeting effect of Ir-rhod reduces its systemic toxicity in vivo, enhancing its biosafety profile. To our knowledge, Ir-rhod is an effective mitochondria-targeted Ir complex capable of inducing tumor cell death by disrupting mitochondrial function, offering a potent strategy to suppress cisplatin resistance in non-small cell lung cancer. Keywords: Mitochondria-targeted, Iridium complex, Ferroptosis, Mitochondrial function, Cisplatin-resistant non-small cell lung cancer Highlights * • Iridium complex Ir-rhod with rhodamine as the ligand, which possesses imaging and mitochondrial targeting capabilities. * • The iridium complex Ir-rhod regulating mitochondrial function by upregulating the expression and activity of ACSL4. * • The Ir-rhod suppresses cisplatin-resistant non-small-cell lung cancer by triggering ferroptosis and apoptosis. 1. Introduction Non-small cell lung cancer (NSCLC) is the dominant type of lung cancer as well as the leading cause of cancer-related fatalities worldwide [[33]1]. Despite significant advancements in NSCLC treatment over the past decades, including improved quality of life and symptom alleviation through chemotherapy (particularly with cisplatin or platinum drugs), antitumor drug resistance persists as a major challenge [[34]2]. The resistance is responsible for approximately 90 % of cancer patient deaths, indicating that despite initial remission following intensive chemotherapy, drug-resistant tumors often lead to NSCLC relapse [[35]3]. The development of new therapeutic agents to against drug resistance and the mechanisms are thus critical areas of research [[36][4], [37][5], [38][6]]. Targeting mitochondria has emerged as a promising therapeutic strategy against cancer, particularly in overcoming drug resistance [[39]7,[40]8]. Some mitochondria-targeted molecules have entered clinical trials, exerting anticancer effects through mitochondrial-dependent cell death pathways and potentially overcoming cancer resistance [[41][9], [42][10], [43][11]]. Additionally, researchers have synthesized fluorescent mitochondria-targeted molecules with novel anticancer mechanisms to investigate and monitor the behavior of specific compounds within mitochondria over time, such as assessing the suppression of cancer cell metabolism through the use of rhenium (I) complexes that are anchored to the mitochondria [[44]12,[45]13]. These investigations have inspired the strategic design of emissive mitochondria-targeted multifunctional diagnostic and therapeutic agents to overcome cancer resistance, which can monitor mitochondrial changes during treatment and provide insights into mechanisms for overcoming chemotherapy resistance [[46][14], [47][15], [48][16], [49][17]]. Rhodamine compounds have garnered sustained interest for their selective uptake by cancer mitochondria and their ability to disrupt cancer cell metabolism or activate apoptotic cell death processes [[50]18,[51]19]. Rhodamine-123, a fluorescent molecule targeting mitochondria, has been commercialized for its ability to specifically identify mitochondrial membrane potential. Additionally, mitochondrial damage in cancer cells may increase their susceptibility to certain drugs, leading to cell death signaling. Tang et al. developed a mitochondria-targeted nano radiosensitizer that enhanced radiation therapy by activating reactive oxygen species (ROS) [[52]20]. However, recent rhodamine-based drug designs primarily focus on mitochondrial detection rather than utilizing their mitochondrial targeting function to overcome chemotherapy resistance. The efficacy of platinum-based cancer therapeutics has led to the development of non-platinum metallodrugs [[53][21], [54][22], [55][23], [56][24], [57][25]]. Organometallic iridium (Ir) compounds have emerged as promising cancer therapeutic candidates, demonstrating comparable or superior anticancer activity to cisplatin [[58]26,[59]27]. Motivated by these discoveries, we have resolved to conduct a comprehensive in vitro examination of Ir^III complex bearing a derivative of the rhodamine ligand, namely, [((η^5-Cp∗)Ir(rhod)]^2+2PF[6]^− (Ir-rhod). Cp∗ = pentamethylcyclopentadienyl; rhod = rhodamine B-based fluorescence probe ([60]Scheme 1); Complex Ir-rhod not only take advantage of optical probes to mitochondria, but also the tridentate N^N^O ligand tunes the compound as lipophilic cations that contribute to the mitochondria affinity due to proton gradient considerations. In this study, the iridium complex Ir-rhod was found to be effectively absorbed by A549R cells, selectively localizing in mitochondria. Ir-rhod not only elevated ROS levels but also disrupted mitochondrial structure and function, as evidenced by mitochondrial fragmentation, altered mitochondrial translation, and dysregulation of mitochondrial respiratory electron transport. Furthermore, Ir-rhod activated long-chain acyl-CoA synthetase 4 (ACSL4) and inactivated glutathione peroxidase 4 (GPX4), resulting in lethal lipid peroxidation (LPO) accumulation and inducing ferroptosis. The mitochondria-targeted Ir-rhod caused mitochondrial fragmentation and induced cell death through combined apoptosis and ferroptosis pathways, effectively overcoming tumor resistance ([61]Scheme 1). Biodistribution and general tissue toxicity studies suggest that Ir-rhod shows promise for treating cisplatin-resistant lung cancer. Scheme 1. [62]Scheme 1 [63]Open in a new tab Proposed anticancer mechanism of iridium (Ir) complex Ir-rhod that induces mitochondrial metabolic dysregulation by specifically targeting mitochondria, resulting in a decrease in the expression of GPX4 and an increase in the expression of ACSL4 within cells. This cascade of events culminates in the accumulation of lipid hydroperoxides, triggering dual-regulation of ferroptosis and apoptosis, and thereby mitigating drug resistance in NSCLC. 2. Results 2.1. Synthesis and characterization of Ir-rhod complex In the present work, we synthesized rhodamine-based hydrazide as tridentate N^N^O ligand to form at metal-complex, [((η^5-Cp∗)Ir(rhod)]^2+2PF[6]^− (Ir-rhod), in which the tridentate N^N^O ligand was rhod = rhodamine B-based fluorescence probe, and the Cp∗ was pentamethylcyclopentadienyl. The complex Ir-rhod was synthesized by treating the dinuclear dichloro-bridged complex [(η^5-Cp∗)IrCl[2]][2] (dimer 1) with 2.1 equivalents of rhod in methanol (MeOH), followed by the substitution of the anionic component with NH[4]PF[6]. Rhod was prepared utilizing literature procedures [[64]28]. The complex Ir-rhod was obtained as show in [65]Fig. 1A. The complex was purified through recrystallization and identified by ^1H NMR spectroscopy ([66]Fig. S1−S4). Complex Ir-rhod was identified using X-ray crystallography that were formed by gradually introducing hexane into a concentrated dichloromethane solution containing the complex. The configurations and corresponding atomic labeling are depicted in [67]Fig. 1B. The asymmetric unit of Ir-rhod consists of a [((η^5-Cp∗)Ir(rhod)]^2+cation, hexafluorophosphate anions and the complex exhibits the anticipated pseudo-octahedral geometry, reminiscent of a half-sandwich piano stool. The crystallographic data and specific bond angles and lengths are detailed in [68]Table S1−S2, respectively. As shown in [69]Table S1, Complex Ir-rhod was organized within the triclinic crystal system, belonging to the P-1 space group. The separation between the iridium core and the center of the η^5-cyclopentadienyl ring is 1.8229 Å. The N1– Ir –N2 angles, N1– Ir –O and N2– Ir –O angles, formed between the nitrogen atoms of the ligand of pyridine rings or the oxygen atoms of rhodamine b to the Ir (III) center, are in the typical range (76.8(11), 88.9(8) and 75.9(9) respectively). Ir-rhod shows intense absorption in the range of 250–350 nm attributed to spin-allowed ^1π−π∗ ^1LC transitions, and exhibit weak absorption between 350 and 450 nm, which can be ascribed to a combination of ^1MLCT, ^1LLCT, ^3MLCT, ^3LLCT, and ^3π−π∗ LC transitions and the strong absorption at 500–600 nm assigned to the rhodamine B ([70]Fig. 1C). Upon excitation at 594 nm, Ir-rhod emits red (500−700 nm) light. ([71]Fig. 1D). The Ir-rhod complex remained stable for a minimum of 12 h in PBS (containing 1 % DMSO) at ambient temperature, as observed through UV–visible spectroscopic analysis. Fig. 1. [72]Fig. 1 [73]Open in a new tab Synthesis and Characterization of Ir-rhod complex. (A) Synthesis of ligands and half-sandwich Ir-rhod complex. (B) The X-ray crystal structure of complex Ir-rhod. The PF[6]^− counterion and the H atoms have been omitted for clarity. (C) UV/vis absorption spectra of complex Ir-rhod (5 × 10^−5 M) in aqueous solution. (D) Fluorescence response of Ir-rhod (2 × 10^−5 M) are measured in MDSO. The excitation wavelength is 594 nm. (E)UV–vis traces of complex Ir-rhod in PBS (1 % DMSO) from 0 to 12 h indicating its prolonged stability. These experiments of UV–vis and Fluorescence spectra were repeated three times independently with similar results. 2.2. In vitro biological evaluation The anticancer efficacy of Ir-rhod complex, rhod ligand, and CDDP (Cisplatin) against A549R cells (a cisplatin-resistant line maintained at 1 μg/mL cisplatin) was evaluated utilizing the CCK-8 assay. As depicted in [74]Fig. 2A, Ir-rhod demonstrated superior anticancer effects on A549R cells compared to rhod and CDDP, exhibiting significantly stronger antiproliferative activity than CDDP. Ir-rhod's inhibitory potency was enhanced in A549R (IC[50] = 2.34 μM) relative to the rhod ligand (IC[50] = 23.7 μM). These findings indicate that the positive electric effect substantially improves Ir-rhod's antiproliferative activity, particularly in cisplatin-resistant tumor cells, validating the complex's effectiveness in promoting anticancer activity and reversing cisplatin resistance. The A549 cell line is recognized as a dependable model for the study of NSCLC. Conversely, the cisplatin-resistant NCI–H460 cell line, which is frequently employed in lung cancer research, serves as an additional model. The IC[50] of Ir-rhod was tested against A549, NCI–H460, and NCI–H460R cells (a cisplatin-resistant line maintained at 1 μg/mL cisplatin). The IC[50] of Ir-rhod for A549 cells was determined to be 15.56 μM, whereas for the A549R cells, it was 2.34 μM, indicating a 6.65-fold increase in toxicity compared to the non-resistant A549 cells. The IC[50] for NCI–H460 cells was 5.03 μM, and for NCI–H460R cells, it was 1.72, exhibiting toxicity 2.92 times higher than the non-resistant NCI–H460 cells ([75]Fig. S5). It is important to note that the Ir-rhod exhibits low toxicity to normal lung cells (MRC-5, human embryo lung cells, [76]Fig. S5 E). Flow cytometry analysis revealed that the Ir-rhod's uptake in MRC-5 cells is lower compared to A549R cells and A549 cells. This may be attributed to the higher mitochondrial membrane potential in A549R and A549 tumor cells, which facilitates the Ir-rhod's absorption ([77]Fig. S5 D, F). To examine Ir-rhod's impact on tumor cell proliferation, A549R cells underwent a cell clone formation trial. After treating cells with five different concentrations for 10 days, cell colonies were counted ([78]Fig. 2B and C). The CDDP and rhod ligand groups showed the highest colony numbers, suggesting that the rhod ligand does not impede cell proliferation. Conversely, the Ir-rhod complex group exhibited a significant, concentration-dependent decrease in colony numbers. Notably, the Ir-rhod complex group (5 μM) produced the fewest colonies, indicating substantial inhibition of tumor cell proliferation. Cell destruction caused by Ir-rhod was further assessed using Calcein-AM/propidium iodide (PI) fluorescence imaging ([79]Fig. 2D and E). The strong red fluorescence from PI entering dead cell nuclei demonstrated Ir-rhod's ability to effectively kill cells in a manner that increases with the dose administered. Annexin V-FITC/PI co-staining assay ([80]Fig. 2F and G) revealed dose-dependent apoptosis in A549R cells: 11.6 % at 1.25 μM Ir-rhod, 34.6 % at 2.5 μM, and 51.2 % at 5 μM. Ir-rhod's effect on cell invasion suppression was also investigated. In a Matrigel-coated transwell invasion assay, Ir-rhod treatments significantly reduced A549R cells' invasive ability in a manner that increased with the dose when compared to the control group ([81]Fig. 2H and [82]Fig. S6A). The HUVECs tubular structures assay showed a notable, dose-dependent inhibition by Ir-rhod compared to the control group ([83]Fig. 2I). A549R cell migration towards an extracellular matrix was examined under various conditions ([84]Fig. S6B and C). After 24 h, the control group displayed the highest wound healing ratio, while the Ir-rhod groups at 1.25, 2.5, and 5 μM showed ratios of 63.6 %, 40.9 %, and 18.2 %, respectively. It should be noted that the cell marks in the complex Ir-rhod treatment group were wider than other groups, indicating that the complex Ir-rhod could weaken the ability of tumor cells to migrate to their greatest extent with a dose-dependent manner. To ascertain whether the anti-migration and invasion effects of Ir-rhod are due to cell death induction or the inhibition of invasive properties of A549R cells, we examined the expression of migration and invasion-related proteins E-Cadherin and N-Cadherin. We observed that the drug upregulated E-Cadherin and downregulated N-Cadherin ([85]Fig. 2J and K). Additionally, the inclusion of ferroptosis and apoptosis inhibitors revealed that the drug still significantly inhibited cell migration and invasion ([86]Fig. S7). This suggests that the Ir-rhod inhibits the invasive characteristics of A549R cells. Collectively, these in vitro experiments indicated that the complex Ir-rhod has promising antitumor efficacy on A549R cells ([87]Fig. 2 L). Fig. 2. [88]Fig. 2 [89]Open in a new tab The cytotoxicity analysis for the complex Ir-rhod in vitro for A549R cells. (A)Effects of CDDP, rhod and complex Ir-rhod at varied concentrations on the A549R cells. (B–C) Photograph and numbers of the surviving fractions of A549R cells after the different treatments (repeated measurement two-way ANOVA, n = 3 each group). (D) Cell viability detection images measured by Calcein-AM/PI (Calcein-AM: λ[ex] = 488 nm, λ[em] = 500−550 nm; PI: λ[ex] = 561 nm, λ[em] = 580−630 nm; scale bars: 120 μm). (E) Statistical analysis of PI fluorescence intensity of D (repeated measurement two-way ANOVA, n = 3 each group). n.s., not significant, ∗P < 0.05, ∗ ∗P < 0.01, ∗ ∗ ∗P < 0.001. (F) Flow cytometry analysis results (Annexin V-FITC and PI staining) of A549R cells after treatment with different formulations for 72 h. (G) The apoptotic rates of the groups were calculated by Flow cytometry (repeated measurement two-way ANOVA, n = 3 each group). n.s., not significant, ∗P < 0.05, ∗ ∗P < 0.01, ∗ ∗ ∗P < 0.001. (H) Representative images of migrated A549R cells. Scale bars:100 μm. (I) Images of HUVEC tube formation on Matrigel in each group. Scale bar: 100 μm. (J) Western blot analysis of expression level of E-Cadherin and N-Cadherin in A549R cells after 72 h of Ir-rhod treatment at indicated concentrations. (K) Statistics showing the expression level of E-Cadherin after Ir-rhod treatment at indicated concentrations. (L) Schematic diagram of Ir-rhod has promising antitumor efficacy on A549R cells. 2.3. Cellular uptake and localization Drug absorption in cells is a complex, dynamic process encompassing dissolution, diffusion, transport mechanisms, intracellular distribution, and the balance between absorption and elimination ([90]Fig. 3 A) [[91]29]. The speed and degree of drug absorption can fluctuate over time and are affected by the drug's characteristics. Grasping these elements is essential for enhancing drug therapy and achieving effective treatment results [[92]30]. Confocal microscopy and flow cytometry were employed for quantitative analysis of the uptake levels of the Ir-rhod complex in A549R cells. The findings show that after 0.5 h of incubation, the Ir-rhod complex efficiently enters A549R cells and remains there for 12 h ([93]Fig. 3 B). Furthermore, to investigate how concentration and time influence the cellular uptake levels of complex Ir-rhod, flow cytometry was first employed to examine changes in drug distribution over time, revealing minimal temporal effects ([94]Fig. S8). Subsequently, fluorescence changes were observed after 24 h exposure to 1.25 μM, 2.5 μM, and 5 μM concentrations, indicating that cellular uptake levels increased with drug concentration ([95]Fig. 3C). To further validate the distribution of the Ir-rhod within the mitochondria, we incubated A549R cells with Ir-rhod for a duration of 2 h, followed by mitochondrial extraction and subsequent ICP-MS quantification of the Ir-rhod's mitochondrial concentration. The study found that the concentration of iridium in the mitochondria increases with the elevation of drug concentration ([96]Fig. 3 D and [97]Fig. S9). The inherent fluorescence of the Ir-rhod complex allows for easy analysis of its subcellular localization using confocal microscopy ([98]Fig. 3 E). These collective results demonstrate that Ir-rhod specifically localizes to mitochondria in A549R cells. Following approximately 16 h of incubation, small red dots of the Ir-rhod complex gradually appeared, indicating the process of complex Ir-rhod being removed from the mitochondria and transferred into the nucleus, which became more pronounced after 24 h of incubation. Fig. 3. [99]Fig. 3 [100]Open in a new tab Cellular uptake and Colocalization with mitochondria of the complex Ir-rhod. (A) Schematic diagram of Ir-rhod targeting mitochondria through its unique positive bivalent nature and rhodamine's inherent mitochondrial affinity. (B) Fluorescence confocal microscopy images of A549R cells incubated with complex Ir-rhod (6 μM; 0.5 h, 6 h, 12 h) at 37 °C. Scale bar:100 μm. (C) The cellular uptake of the complex Ir-rhod (24h; 1.25 μM, 2.5 μM, 5 μM) incubated with A549R cells by flow cytometer. (D) The mitochondrial iridium content in A549R cells was determined following incubation with different concentrations of the Ir-rhod, as assessed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). (E) Long-term tracking of mitochondrial morphology by Ir-rhod. A549R cells were colabeled with Ir-rhod (6 μM) and MTDR (mitochondrial tracker) (100 nM), and then the cells were imaged by confocal microscopy at the indicated time intervals. Ir-rhod: λ[ex] = 594 nm; λ[em] = 610 ± 20 nm. MTDR: λ[ex] = 490 nm; λ[em] = 520 ± 30 nm; Scale bar:100 μm. 2.4. Mitochondrial targeting and stress induction of Ir-rhod The ability of complex Ir-rhod to induce mitochondrial dysfunction was examined due to its selective localization in mitochondria ([101]Fig. 4 A). Mitochondrial dysfunction typically involves a loss of mitochondrial membrane potential (MMP), which was assessed using flow cytometry with JC-1 staining [[102]31]. [103]Fig. 4 B shows that control cells primarily exhibited red fluorescence, indicating high membrane potentials. However, cells treated with Ir-rhod for 72 h displayed a notable dose-responsive shift from red to green, signifying a substantial decrease in MMP. The proportion of cells exhibiting depolarized mitochondrial membranes climbed from 0.86 % in the control group to 64.92 % at a concentration of 1.25 μM, 70.73 % at 2.5 μM, and 93.78 % at 5 μM. [104]Fig. S10 illustrates the JC-1 red/green fluorescence intensity ratios in cells treated with Ir-rhod, demonstrating time-dependent MMP loss. Organometallic complexes have been shown to function as potent oxidants for cancer treatment, such as the process of converting NADH to NAD ^+ under neutral to basic conditions affects cellular redox states and signaling pathways. The catalytic performance of Ir-rhod (1 μM) with NADH (150 μM) in a MeOH/H[2]O (1:1) mixture was tracked by UV–Vis spectroscopy at ambient temperature, as depicted in [105]Fig. 4C. The turnover number (TON), determined from the UV absorption change at 339 nm, achieved a value of 41 after 8-h period for 150 μM NADH. This surpasses the performance of the previously reported Ir(iii) half-sandwich catalyst [(η^5-Cp^xbiph)Ir(phpy)py]PF[6], which had a TON of 7.6 after 20 h [[106]32]. UV/Vis spectroscopy data indicated that Ir-rhod can catalyze hydride transfer from NADH. As anticipated, the NADH/NAD ^+ ratio in A549R cells decreased significantly with increasing Ir-rhod concentration ([107]Fig. 4 D). Mitochondria are crucial for cell power generation, ROS production, and apoptosis regulation through the mitochondrial permeation transition pore (mtPTP). [108]Fig. 4 E-F shows that cells treated with Ir-rhod for 72 h exhibited increased red fluorescence in a manner that is directly proportional to the dose administered, indicating elevated ROS generation. Similar results were observed using confocal immunofluorescence imaging ([109]Fig. S11). As depicted in [110]Fig. 4 G, upon incubation of the Ir-rhod with A549R cells for 10 min, the mitochondria exhibit a well-organized and structured appearance within the cellular mitochondrial network. However, when the Ir-rhod is co-incubated with A549R cells for 24 h, confocal microscopy reveals that the normally tubular or filamentous mitochondrial structures appear fragmented into smaller, punctate structures, indicating damage to the mitochondria ([111]Fig. 4H). To more clearly characterize the structural changes in mitochondria, transmission electron microscopy (TEM) was employed to analyze the mitochondrial ultrastructure of A549R cells. [112]Fig. 4 I reveals that untreated cells have predominantly round mitochondria. In contrast, Ir-rhod-treated cells showed significant changes in mitochondrial morphology, with numerous vacuoles appearing, some mitochondrial membranes disappearing, and some cristae damaged or distorted. Additionally, TMT-based proteomics was performed on Ir-rhod-treated and untreated A549R cells. As shown in [113]Fig. 4 J, Ir-rhod treatment mainly influenced mitochondrial translation and dysregulated mitochondrial respiratory electron transport, as revealed by proteomics reactome pathway enrichment analysis of differentially expressed proteins induced by Ir-rhod, suggesting mitochondrial dysfunction after Ir-rhod treatment. These findings suggest that alterations in mitochondrial structure could impact the MMP and cellular energy metabolism. Fig. 4. [114]Fig. 4 [115]Open in a new tab The complex Ir-rhod induces mitochondrial damage in A549R cell. (A) Schematic diagram of Ir-rhod targeting mitochondria to induce mitochondrial dysfunction. (B) Effects of Ir-rhod on MMP measured by JC-1 staining (λ[ex] = 488 nm, λ[em] = 525 ± 30 nm (JC-1 monomers) and 585 ± 30 nm (JC-1aggregates)) and flow cytometry. A549R cells were treated with Ir-rhod at the indicated concentrations for 72 h. (C) Catalytic oxidation of NADH (150 μM) by complex Ir-rhod (1 μM) in PBS, as monitored by ultraviolet–visible spectroscopy. The colored lines represent spectra recorded every 5 min for 8 h. The direction of change in absorbance with time is indicated by red arrows. (D) Relative NADH/NAD ^+ level in A549R cells after exposed to Ir-rhod with specified concentration for 72 h (repeated measurement two-way ANOVA, n = 3 each group). (E) Flow cytometric analysis of ROS levels. A549R cells were exposed to Ir-rhod for 72 h before staining DHE. Fluorescence values of DHE were collected at 610 nm upon excited at 535 nm. (F) Quantification of ROS levels in E (repeated measurement two-way ANOVA, n = 3 each group). (G) Ir-rhod (6 μM) was incubated with A549R cells for 10 min to observe mitochondrial morphology. (H) Ir-rhod (6 μM) was incubated with A549R cells for 24 h to observe mitochondrial morphology. MTDR (mitochondrial tracker) (100 nM) was incubated with A549R cells for 30 min, Ir-rhod: λ[ex] = 594 nm; λ[em] = 610 ± 20 nm. MTDR: λ[ex] = 490 nm; λ[em] = 520 ± 30 nm; Scale bar:100 μm. (I) TEM images of A549R cells treated with vehicle (1 % DMSO) or Ir-rhod (6 μM, 72 h). white rectangles represent the region enlarged. (J) Proteomics reactome pathway enrichment of differentially expressed proteins induced by Ir-rhod (5 μM, 72 h). 2.5. The mechanisms for the complex Ir-rhod regulation effects of ACSL4 Additionally, TMT-based proteomics analysis was performed on A549R cells that were treated with Ir-rhod and those that were not. Using rigorous selection criteria, including the presence of at least two unique peptides per protein, 1512 proteins with altered expression in the treated cells were identified. The expression values of these proteins were logarithmically transformed and depicted as volcano plots and heatmaps, revealing 724 proteins with increased expression and 788 with decreased expression ([116]Fig. 5A, [117]Fig. S12). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was then exercised on these differentially expressed proteins to provide significant insights into their functional roles. As depicted in [118]Fig. 5B, C and [119]Figs. S13–14, Ir-rhod treatment mainly influenced the Fatty acid biosynthesis and chemical carcinogenesis-racctive oxygen species. Analysis of proteins in the fatty acid biosynthesis pathway has revealed that the complex Ir-rhod primarily affects ACSL4, DHB8, ACSL3, and ACSL1 proteins, all of which are implicated in ferroptosis, with ACSL4 being particularly significant ([120]Fig. 5D and E) [[121]33,[122]34]. The upregulation of the expression level of ACSL4 after treatment with Ir-rhod was confirmed using Western blot analysis ([123]Fig. 5F and G). Concurrently, the enzymatic activity of the protein ACSL4, as illustrated in [124]Fig. 5H and [125]Fig. S15, exhibited a similar upward trend. This observation indicates that the activity of ACSL4, which is involved in the synthesis of long-chain fatty acids, is increasing over the course of the experiment. The upward trend could be attributed to the metal complex Ir-rhod that enhances the activity of ACSL4. An increase in ACSL4 enzymatic activity can significantly influence lipid metabolism by enhancing de novo lipogenesis, activating other lipogenic enzymes, supporting metastasis, regulating ferroptosis, and impacting metabolic reprogramming and antitumor immunity. These functions highlight the importance of ACSL4 in the lipid metabolism of cancer cells and its potential as a therapeutic target. The docking score energy of the metal complex Ir-rhod with ACSL4 is −9.20 kcal/mol. Subsequently, the binding details were analyzed, and the interaction between ACSL4 and the metal complex Ir-rhod was depicted as shown in [126]Fig. 5I and J. It can be observed that a hydrogen bond exists between the -O- of the metal complex Ir-rhod and the OH of SER353, with a distance of 3.2 Å, and a hydrogen bond formed between the main chain NH of K116 and N, with a distance of 3.40 Å. Additionally, a possible coordination interaction between the COO of E681 and the Ir is also observed; F115, T183, P345, L346, K354, R380, T679, P680, and F689 form van der Waals interactions with the heterocycles of the metal complex Ir-rhod. In summary, the metal complex Ir-rhod exhibits a strong binding affinity with the protein ACSL4, which leads to an increase in the expression and enzymatic activity of ACSL4. Fig. 5. [127]Fig. 5 [128]Open in a new tab Proteomics analysis reveals the mechanisms for the complex Ir-rhod regulation effects of ACSL4. (A) Schematic of the TMT-based proteomics of the complex Ir-rhod in A549R cells. (B) Heatmap for the differentially expressed proteins between the Ir-rhod and control. (C) Chord diagram for the differential expressed proteins between the Ir-rhod and control. (D) The top enriched KEGG pathways of differentially expressed proteins between the Ir-rhod and control. A KEGG enrichment analysis was performed using a two-tailed Fisher's exact test to identify significantly enriched pathways. (E) Heatmap for the proteins of fatty acid biosynthesis. (F) Western blot analysis of expression level of ACSL4 in A549R cells after 72 h of Ir-rhod treatment at indicated concentrations. (G) Statistics showing the expression level of ACSL4 after Ir-rhod treatment at indicated concentrations (n = 3, repeated measurement two-way ANOVA). (H) A549R cells was incubated with different concentrations of Ir-rhod for 72 h at 37 °C. The enzymatic activity of ACSL4 was analyzed by the formation of the protocol described in Material and Methods. (I) Global binding figure: The overall interaction between the protein and the metal complex, with the protein represented in a cartoon model and the key amino acids involved in the interaction displayed in either stick or wireframe models. (J) Local binding detail in 3D: The binding details of the optimal structure, with the metal complex depicted in a stick model. Hydrogen bonds are indicated by red dashed lines, van der Waals forces/hydrophobic interactions by yellow dashed lines, and coordination bonds by black dashed lines. 2.6. The complex Ir-rhod regulates ferroptosis in A549R cells through ACSL4 Our investigation extended to examining whether Ir-rhod induces ferroptosis. The results revealed that the Ir-rhod-treated group exhibited reduced GPX4 protein expression compared to the control group, with this effect being dose-dependent ([129]Fig. 6A and B) [[130]35]. We hypothesize that upregulation of ACSL4 enhances lipid peroxidation, requiring higher Gpx4 levels, and that Gpx4 downregulation is a cellular response to the stress of increased lipid peroxidation. Studies suggest that ACSL4 is part of a positive feedback loop involving PKCβII that promotes ferroptosis by increasing PUFA incorporation into PLs, potentially exacerbating lipid peroxidation and leading to Gpx4 downregulation. To ascertain whether the cell death elicited by Ir-rhod in chemo-resistant cells proceeds via both apoptotic and ferroptotic pathways, the effects of ferroptosis and apoptosis inhibitors on the proliferation of A549R cells were evaluated using a clonogenic assay. As depicted in [131]Fig. 6C, it was observed that the proliferation-inhibiting effect of Ir-rhod on A549R cells was attenuated by the addition of the apoptosis inhibitor Z-VAD-FMK. Similarly, the ferroptosis inhibitor Pioglitazone yielded comparable results. When both ferroptosis and apoptosis inhibitors were co-administered, the inhibitory effect of Ir-rhod on A549R cell proliferation was markedly diminished, suggesting that the drug induces cell death via both apoptotic and ferroptotic pathways. The flow cytometry results further confirmed this conclusion ([132]Fig. 6 D). LPOs accumulation, a key ferroptosis indicator, can trigger this process. We employed C11-BODIPY as an LPOs probe to assess their levels in cancer cells. This probe accumulates on cell membranes and shifts from red to green fluorescence upon oxidation. [133]Fig. 6 E-F shows prominent green fluorescence post co-treatment with 2.5 μM Ir-rhod, while 5 μM Ir-rhod resulted in the weakest red and strongest green fluorescence, indicating substantial LPOs formation with concentration variability. Quantitative analysis in [134]Fig. 6G and H corroborated these findings. We also evaluated intracellular GSH levels following treatment with various formulations. [135]Fig. 6I shows a notable decrease in the GSH/GSSG ratio after Ir-rhod treatment, suggesting the complex's ability to modulate ferroptosis. Intracellular malondialdehyde (MDA), another crucial ferroptosis marker, was also measured. As shown in [136]Fig. 6J, the Ir-rhod group exhibited elevated MDA levels compared to control cells. To substantiate the induction of ferroptosis by Ir-rhod, additional evaluations of biomarkers associated with ferroptosis were conducted subsequent to the administration of both ferroptosis inhibitor pioglitazone and apoptosis inhibitors Z-VAD-FMK. The results indicated that Ir-rhod triggered an elevation in MDA and this increase was mitigated by the presence of the ferroptosis inhibitor ([137]Fig. 6K). Due to iron serving as a catalyst in the Fenton reaction, which generates hydroxyl radicals that can initiate lipid peroxidation, a key event in ferroptosis, we observed that iron levels followed a similar trend to MDA ([138]Fig. 6L). As shown in [139]Fig. 6M, the addition of the ferroptosis inhibitor pioglitazone reversed the drug-induced elevation of intracellular iron levels, indicating that the inhibitor mitigated the iron overload caused by the Ir-rhod treatment. This additional evidence supports the notion that the drug induces cell death via both apoptotic and ferroptotic pathways. Lipid reactive oxygen species levels, as anticipated, increased following Ir-rhod treatment ([140]Fig. 6N-O). In the execution of ferroptosis, phospholipids containing polyunsaturated fatty acid chains (PUFA-PLs) serve as the primary substrates for lipid peroxidation, a process that is contingent upon ROS, phospholipids with polyunsaturated fatty acid chains, and transition metal iron. Under the catalysis of iron, a substantial amount of ROS is generated through Fenton reactions or similar processes, triggering lipid peroxidation and inducing ferroptosis [[141]36,[142]37]. As depicted in [143]Fig. 6P, the drug-induced increase in intracellular phospholipids was significantly reduced upon the addition of ferroptosis inhibitors, suggesting that the drug-induced augmentation of phospholipids affects the activity of key enzymes ACSL4, alters the content of PUFA-phospholipids in the cell membrane, and thereby influences the extent of lipid peroxidation, ultimately leading to the onset of ferroptosis. Additionally, the inclusion of ferroptosis and apoptosis inhibitors revealed that the drug still significantly inhibited cell invasion ([144]Fig. 6Q) that further suggests the Ir-rhod inhibits the invasive characteristics of A549R cells. Collectively, this evidence indicates that the Ir-rhod complex induces ferroptosis by regulating ACSL4 ([145]Fig. 6R). Fig. 6. [146]Fig. 6 [147]Open in a new tab The complex Ir-rhod regulates ferroptosis in A549R cells through ACSL4. (A) Western blot analysis of expression level of GPX4 in A549R cells after 72 h of Ir-rhod treatment at indicated concentrations. (B) Statistics showing the expression level of GPX4 after Ir-rhod treatment at indicated concentrations. ∗ ∗P < 0.01 (mean ± SEM, repeated measurement two-way ANOVA, n = 3 each group). (C) Photograph and numbers of the surviving fractions of A549Rcells after the different treatments. (D) Flow cytometry analysis results (Annexin V-FITC and PI staining) of A549R cells after treatment with different formulations for 72 h. (E) Confocal immunofluorescence image of C11-BODIPY-stained A549R cells after treatment with different formulations, Scale bar: 100 μm. (F) Quantitative analysis of green fluorescence/red fluorescence intensity of C11-BODIPY in C (repeated measurement two-way ANOVA, n = 3 each group). (G) Flow cytometry analysis results (C11-BODIPY staining) of A549R cells after treatment with different formulations for 72 h. (H) Quantitative analysis of green fluorescence intensity of oxidized C11-BODIPY in G (repeated measurement two-way ANOVA, n = 3 each group). (I) Relatively GSH/GSSG ratio of A549R cells after treatment with different formulations for 72 h (repeated measurement two-way ANOVA, n = 3 each group). (J) Quantitative analysis of MDA level of A549R cells after treatment with different formulations for 72 h (repeated measurement two-way ANOVA, n = 3 each group). (K) Quantitative analysis of MDA level of A549R cells after treatment with different formulations for 4 days. (L) Quantitative analysis of Fe level of A549R cells after treatment with different formulations for 72 h (repeated measurement two-way ANOVA, n = 3 each group). (M) Quantitative analysis of Fe level of A549R cells after treatment with different formulations for 4 days (repeated measurement two-way ANOVA, n = 3 each group). (N–O) Flow cytometric analysis and quantitative analysis of ROS levels. A549R cells were exposed to Ir-rhod for 72 h before staining H2DCF-DA. Fluorescence values of DCF were collected at 530 nm upon excited at 488 nm (repeated measurement two-way ANOVA, n = 3 each group). n.s., not significant, ∗P < 0.05, ∗ ∗P < 0.01, ∗ ∗ ∗P < 0.001. (P) Quantitative analysis of PLIP level of A549 cells after treatment with different formulations for 72 h (repeated measurement two-way ANOVA, n = 3 each group). n.s., not significant, ∗P < 0.05, ∗ ∗P < 0.01, ∗ ∗ ∗P < 0.001. (Q) Representative images of migrated A549R cells after treatment with the complex Ir-rhod at indicated concentrations. Scale bars:100 μm. (R) Schematic illustration of the complex Ir-rhod regulates ferroptosis in A549R cells through ACSL4. To establish that Ir-rhod treatment induces ferroptosis in A549R cells, we conducted validation using A549 cells, which are known to be strongly resistant to ferroptosis, as a control. We have conducted cell viability on A549 cells, with IC[50] = 15.56 μM, and on A549R, with IC[50] = 2.34 μM. The inhibitory effect of proliferation on A549R is 6.65 times higher than the non-resistant A549 cells. The results of the flow cytometry demonstrate that Ir-rhod induces apoptosis in A549 cells, with an apoptotic rate of 96.3 %. After the addition of the apoptosis inhibitor Z-VAD-FMK, the apoptosis rate is reduced to 48.3 %. The addition of the ferroptosis inhibitor Pioglitazone has no effect on apoptosis, proving that Ir-rhod induces apoptosis in A549 cells ([148]Fig. S16 A). Meanwhile, MMP was evaluated via flow cytometry employing JC-1 staining. The proportion of cells exhibiting depolarized mitochondrial membranes climbed from 13.5 ± 1.5 % in the control group to 17.3 ± 2.1 % at a concentration of 15.6 μM, 93.6 ± 0.5 % at 30.2 μM, and 97.7 ± 1.5 % at 60.4 μM ([149]Fig. S16 B). To further prove whether Ir-rhod induces ferroptosis in A549 cells, we detected the relevant indicators of ferroptosis, and the results showed that the drug could not change the levels of MDA and iron levels within the cells. Moreover, the treatment of Ir-rhod increased the level of PLIP that is critical for ferroptosis susceptibility ([150]Figure S17 A-C). Furthermore, our experiments revealed that the Ir-rhod induces cell death in A549 cells through apoptosis. 2.7. The therapeutic effect of the complex Ir-rhod in mice We examined the in vivo antitumor effectiveness of Ir-rhod through intravenous administration. Once tumor volumes reached 80–100 mm^3, A549R tumor-bearing mice were sorted into four groups (n = 6): PBS, Ir-rhod (100 μL, 4 mg/kg), Ir-rhod (100 μL, 8 mg/kg), and cisplatin (100 μL, 5 mg/kg) as a positive control, all administered via tail vein injection. After 18 days, the mice were euthanized for tumor and major organ collection ([151]Fig. 7A). [152]Fig. 7B and C demonstrate that Ir-rhod (100 μL, 4 mg/kg) effectively suppressed or eliminated A549R tumor volume following intravenous administration. The PBS group's tumor weight was approximately 6.5 times that of the Ir-rhod (100 μL, 4 mg/kg) group ([153]Fig. 7D). The tumor inhibition rate for Ir-rhod (100 μL, 4 mg/kg) was about 87.6 ± 2.31 %, notably higher than the Ir-rhod (100 μL, 2 mg/kg) group at 72.1 ± 8.10 % ([154]Fig. 7E). Notably, the cisplatin group showed no significant antitumor effect, as evidenced by minimal impact on tumor growth post-injection. No substantial weight changes were observed in Ir-rhod-treated groups ([155]Fig. S18), suggesting good in vivo biocompatibility. Ki67 and TUNEL staining further investigated Ir-rhod's antitumor proliferation effects ([156]Fig. 7F). The Ir-rhod (100 μL, 4 mg/kg) group markedly reduced Ki67 expression and induced more tumor cell death. These findings indicate Ir-rhod's considerable in vivo antitumor efficacy. We also assessed tumor ACSL4 and GPX4 expression to verify in vivo ferroptosis. [157]Fig. 7G shows that both Ir-rhod (100 μL, 2 mg/kg) and Ir-rhod (100 μL, 4 mg/kg) groups increased ACSL4 and decreased GPX4 expression levels. The cisplatin-treated group exhibited minor changes in ACSL4 and GPX4 expression. These results further confirm that Ir-rhod induces ferroptosis by modulating mitochondrial function, effectively overcoming tumor resistance in vivo. Similar outcomes were observed in serum analysis ([158]Fig. 7H–J). The Ir-rhod (100 μL, 4 mg/kg) group significantly increased MDA generation while decreasing GSH-PX and T-GSH expression, promoting tumor cell ferroptosis. To evaluate Ir-rhod's safety, H&E staining of major organs and serum biochemical markers were analyzed across treatment groups. H&E staining revealed no significant changes in major organs of Ir-rhod-treated groups ([159]Fig. S19), preliminarily indicating lower toxicity compared to cisplatin. Biochemical markers γ-GT, T-CHO, CR, and uric acid content in Ir-rhod treatment groups showed no significant differences compared to control group mice ([160]Fig. 7K-N). Hence, Ir-rhod did not cause any hepatic or renal damage following seven consecutive treatments. Fig. 7. [161]Fig. 7 [162]Open in a new tab The in vivo therapeutic efficacy of the complex Ir-rhod in A549R -bearing mice. (A) Schematic illustration of the experiment design in mice models to assess the antitumor efficacy mediated by Ir-rhod. (B) The photographs of the tumor were collected at the end of the therapeutic period. (C) The volume changes of tumors. (D) The weight of the tumors after dissection (n = 6, repeated measurement two-way ANOVA). (E) The tumor inhibition rate in mice (n = 5, repeated measurement two-way ANOVA). (F) Tunel, Ki67, ACSL4 and GPX4 immunofluorescence staining of tumor tissue. Scale Bar: 100 μm. (G) Statistical analysis of Tunel, Ki67, ACSL4 and GPX4 immunofluorescence staining of tumor tissue (n = 3, repeated measurement two-way ANOVA). (H) MDA analysis in serum (n = 3, repeated measurement two-way ANOVA). (I) GSH-PX analysis in serum (n = 3, repeated measurement two-way ANOVA). (J) T-GSH analysis in serum (n = 3, repeated measurement two-way ANOVA) (K–N) Analysis of blood biochemistry indicators after different treatment (n = 3. γ-GT: γ--glutamyltranspeptidase, T-CHO: cholesterol, CR: creatinine, UA: Uric Acid). 3. Discussion Targeting mitochondria is deemed a promising strategy for cancer treatment, potentially effective even in patients who are unresponsive to chemoradiotherapy [[163]38]. Some compounds targeting mitochondria have already attended clinical trials. Drugs that target mitochondria can fight cancer by disrupting the systems that produce energy within the mitochondria and by disturbing the balance of redox reactions. This leads to the triggering of cell death mechanisms that are depend on mitochondria, potentially bypassing resistance to cancer treatment [[164]39,[165]40]. In the present study, we have developed a novel mitochondria-targeted iridium complex Ir-rhod, which is more cytotoxic to A549R (cisplatin-resistant human lung cancer) than the ligand of rhod. The capacity to target mitochondria of iridium complex Ir-rhod not only take advantage of rhodamine's ability to target mitochondria, but also come from the positive bivalent of the novel iridium complex [((η^5-Cp∗)Ir(rhod)]^2+cation. The positively charged agent of iridium complex Ir-rhod enters the cell and, driven by the high mitochondrial transmembrane potential of the tumor cell, is targeted to the mitochondria. More interestingly, rhodamine assisted the real-time observation of dynamic distribution of complex Ir-rhod in vivo, and iridium complex Ir-rhod regulates mitochondrial function to induce tumor cell ferroptosis and apoptosis by regulating ACSL4. Concurrently, the systemic toxicity of the iridium complex Ir-rhod was mitigated in vivo due to its targeted action, enhancing its biosafety profile. As far as we know, the iridium complex Ir-rhod is an effective mitochondria-targeted Ir complex that can trigger mitochondrial metabolism-imbalanced to induce tumor cell death for efficiently overcome tumor resistance. It's important to highlight that half-sandwich iridium (III) complexes with potent anticancer properties have been identified, and their mechanisms of action are distinct from those of platinum-based drugs such as cisplatin. This difference in mechanism is significant as it may provide alternative treatment options for cancers that have developed resistance to traditional chemotherapy. Additionally, the anticancer efficacy of these complexes is influenced by their structural features, allowing for the fine-tuning of their activity against cancer cells. Complexes featuring a structure where all three legs of the piano-stool are occupied by a single tridentate ligand have been less extensively investigated compared to those with three individual monodentate ligands or a mix of bidentate and monodentate ligands. Conversely, a variety of fluorescent molecules that target mitochondria have been created to detect specific chemicals within these organelles and monitor their changes over time. Building on these studies, there is a strong interest in the strategic development of luminescent mitochondria-targeted multifunctional theranostic agents for cancer therapy. Thus, mitochondrial-targeting rhodamine B-based fluorescence probe moiety that links aldehyde pyridine forms a tridentate ligand. Iridium has been recognized as a metal suitable for the development of novel half-sandwich metallodrugs with a valence of +2, which exhibit significant anticancer activity. These iridium (III) complexes are designed to target mitochondria, a strategy that has shown promise in cancer treatment and may be effective in patients who are resistant to traditional chemoradiotherapy approaches. As expected, rhodamine and the charge of plus two of [((η^5-Cp∗)Ir(rhod)]^2+ played a synergistic role, further improving the mitochondrial targeting ability of the iridium complex Ir-rhod and contributing to overcoming chemotherapy resistance. The compound Ir-rhod has been shown to disrupt the mitochondrial energy production system and the cellular redox balance, which in turn can enhance the killing of tumor cells. This effect may be associated with the complex's ability to trigger cell death pathways that are dependent on mitochondria. Mitochondria-targeted Ir-rhod primarily triggers a cascade of events that are reliant on mitochondria, including mitochondrial injury, ROS level elevation, and inducing tumor cell ferroptosis and apoptosis. Rhodamine assisted the real-time observation of dynamic distribution of complex Ir-rhod in vivo and which could completely eradicate solid tumors in immunodeficient mice models, suggesting that Ir-rhod has excellent anti-tumor effects. Moreover, the iridium complex Ir-rhod demonstrated a reduced systemic toxicity in vivo, a consequence of its targeted action, which in turn enhanced its biosafety profile. This suggests that targeting mitochondria and mitochondrial metabolism for chemotherapy resistance cancer is extremely promising and that our novel mitochondria-targeted iridium complex Ir-rhod holds great promise for clinical application. 4. Conclusion In conclusion, we have created a novel mitochondria-targeted iridium complex, Ir-rhod, that demonstrates highly efficient anti-tumor effects against cisplatin-resistant human lung cancer by triggering mitochondrial dysfunction and inducing tumor cell ferroptosis and apoptosis through ACSL4 and GPX4 regulation. Ir-rhod's mitochondrial targeting ability stems from both rhodamine's inherent targeting capacity and the complex's positive bivalent nature. Mechanistic studies reveal that Ir-rhod primarily induces a range of events that depend on mitochondria, including mitochondrial damage, ROS elevation, and redox imbalance. The superior fluorescence of Ir-rhod enables real-time monitoring of the morphological changes in mitochondria induced by Ir (III). The complex serves as an improved theranostic agent for tracking therapeutic effects in situ, remaining well-fixed within mitochondria even after damage occurs. Notably, Ir-rhod completely eradicated solid tumors in immunodeficient mice models, indicating excellent anti-tumor effects and low toxicity to vital organs. This research shows that focusing on mitochondria and their metabolic processes is a potent strategy for cancer treatment and provides a unique design perspective for developing anticancer agents with multifunctional theranostic capabilities that target mitochondria. 5. Materials and methods Experimental reagents and animals: The brands, item numbers, or origins of the major experimental reagents and instruments used in this experiment are detailed in the Supplementary Material. Animal studies were sanctioned by the university's animal ethics committee at Guide Medical Technology Co., Ltd. Nude mice (4–5 weeks old, ∼20 g) were obtained from Guangdong Yaokang Biotechnology Co., Ltd. and kept in SPF conditions. The Animal Ethics Approval number is IACUC-DK-2024-02-26. At the end of the experiment, the remaining mice were executed by cervical dislocation. Synthesis of rhodamine B hydrazid (L1): L1 was produced through a single-step reaction between rhodamine B and hydrazine hydrate in ethanol. The literature provides a detailed report on the synthesis of the ligand L1 [[166]41]. Rhodamine B hydrazid: Yield: 2.82 g (58 %). ^1H NMR (500 MHz, DMSO) δ 7.77–7.74 (m, 1H), 7.50–7.45 (m, 2H), 6.98 (dd, J = 5.9, 2.2 Hz, 1H), 6.34 (dt, J = 11.5, 5.3 Hz, 6H), 4.26 (s, 2H), 3.34 (s, 2H), 3.30 (d, J = 6.9 Hz, 6H), 1.08 (t, J = 7.0 Hz, 12H). Synthesis of the ligands (rhod): Rhodamine B hydrazide (0.856 g, 2 mmol) was dissolved in 30 mL dichloromethane, and pyridine-2-aldoxime (0.396 g, 2 mmol) was added. Formic acid was added dropwise, and the mixture was refluxed for 24 h. After cooling, the solvent was evaporated to obtain a purple solid, which was filtered and purified by methanol washing [[167]42]. Rhod:^1H NMR (500 MHz, DMSO) δ 8.93 (s, 1H), 8.54 (d, J = 6.0 Hz, 2H), 7.93 (d, J = 7.5 Hz, 1H), 7.66 (t, J = 7.4 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.35 (d, J = 6.0 Hz, 2H), 7.15 (d, J = 7.6 Hz, 1H), 6.45 (d, J = 2.5 Hz, 2H), 6.41 (d, J = 8.9 Hz, 2H), 6.35–6.32 (m, 2H), 3.30 (s, 8H), 1.07 (s, 12H). Synthesis of complex Ir-rhod: The Ir-rhod complex was made by reacting Rhod (0.10 mmol) with [(η^5-Cp∗)IrCl[2]][2] (0.05 mmol) in MeOH (30 mL) under reflux for 12 h until a red residue formed. The solution was centrifuged, the residue was dissolved in hot methanol, and a saturated NH[4]PF[6] methanolic solution was added to precipitate the red solid. The precipitate was dissolved in methylene chloride, filtered through a kieselguhr column, and recrystallized from ether to yield red crystals. Ir-rhod: ^1H NMR (500 MHz, DMSO) δ 10.23 (d, 1H), 8.05 (d, 1H), 7.98 (m, 1H), 7.79 (m, 1H), 7.62 (m, 2H), 7.52 (m, 1H), 7.25 (d, 2H), 7.14 (m, 1H), 6.98 (d, 1H), 6.94 (m, 1H), 6.89 (d, 1H), 6.73 (d, 1H), 6.45 (d, 1H), 4.46 (d, 2H), 4.37 (d, 2H), 4.09 (dd, 2H), 3.68 (d, 7H), 3.17 (d, 2H), 1.91 (s, 1H), 1.63 (s, 1H), 1.47 (s, 10H), 1.25 (d, 8H). ^13C NMR (126 MHz, CDCl[3]) δ 157.93 (s), 155.58 (s), 132.07 (s), 130.29 (s), 129.09 (s), 114.38 (s), 113.11 (s), 96.51 (s), 46.23 (s), 29.70 (s), 12.71 (s). Crystal Structure Analysis: X-ray diffraction data were acquired utilizing a Bruker Smart Apex CCD detector with Mo Kα radiation, which was passed through a graphite monochromator. The SADABS program was employed for absorption corrections. Unlocalized electron density was eliminated during final refinement using the SQUEEZE option. Structures were determined via direct methods with SHELXS (TREF), and additional atoms were identified through Fourier methods. SHELXL was used for refinement against F2, with hydrogen atoms positioned at theoretically calculated locations and refined such that they were attached to their parent atoms. Crystallographic data for Ir-rhod complex are presented in [168]Fig. 1, [169]Tables S1 and S2, and have been submitted to the Cambridge Crystallographic Data Centre (CCDC 2386979). The rhod complex was crystallized through slow hexane diffusion into a concentrated dichloromethane solution. CIF format crystallographic data are accessible from the Cambridge Crystallographic Data Centre. Nuclear Magnetic Resonance Analysis:^1H NMR spectra were obtained at 298 K on a Bruker DPX 500 (500.13 MHz) using 5 mm tubes. Chemical shifts for DMSO-d^6 and chloroform-d1 were internally referenced to (CHD[2])(CD[3])SO (2.50 ppm) and CHCl[3] (7.26 ppm). Data was processed with XWIN-NMR 3.6 (Bruker UK Ltd.). Ultraviolet–Visible Spectroscopy: A TU-1901 UV spectrophotometer with 1 cm path-length quartz cuvettes (3 mL) was used to obtain UV–Vis spectra of the Ir-rhod complex. UV Winlab software was utilized for spectral processing. Unless stated otherwise, experiments were conducted at 298 K. NADH Reaction Study: The reaction between the complex (approximately 1 μM) and NADH (approximately 150 μM) in a solvent mixture of 20 % methanol and 80 % water by volume was investigated by UV–Vis spectroscopy at a temperature of 298 K at different time points. The TON was evaluated by dividing the decrease in NADH level past a period of 8 h by the level of iridium catalyst used. The consumption of NADH was determined using its molar absorptivity value (ε[339] = 6220 M^^−1cm^^−1) at 339 nm. Cultivation of cells: A549R cells were purchased from IMMOCELL (Xiamen, Fujian, China). The culture conditions were 90 % DMEM supplemented with 10 % FBS and PS containing 1 μg/mL cisplatin. NCI–H460R (Cat No. FH1207) was provided by Shanghai Fuheng Biotechnology Co., LTD. The culture conditions were RPMI1640 supplemented with 10 % FBS and 1 % P/S containing 1 μg/mL cisplatin. A549 cells were cultured in Dulbecco's minimum essential medium (DMEM) and NCI–H460 cells were cultured in RPMI1640 supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S) at 37 °C in an incubator with a humidified atmosphere of 20 % O[2]and 5 % CO[2]. Cell toxicity assay: We used the CCK-8 to test the toxicity of the drugs to the cells in order to determine the concentration to be used. Firstly, after the cells were fully appended to the wall in the 96-well plate species, they were incubated with cell culture medium containing different concentrations of drugs for as long as required for the experiment. At the end of incubation, the original medium was discarded, the pre-prepared CCK-8 reagent was added, and the 96-well plate was placed in a cell incubator. Absorbance was measured after 40 min and cell viability was calculated from it. Cellular Uptake Analysis: A549R cells were exposed to Ir-rhod complex (6 μM) for 0.5 h, 6 h, and 12 h at 37 °C. After washing thrice with cold PBS, cells were visualized using a confocal microscope (LSM 710, Carl Zeiss, Göttingen, Germany). Emission was detected at 610 ± 20 nm following excitation at 594 nm. Colocalization Experiment: A549R cells were treated with 6 μM Ir-rhod complex and 200 nM MTDR at 37 °C for 30 min. After washing with PBS, cells were examined under a confocal microscope. Excitation wavelengths were 594 nm for Ir-rhod and 490 nm for MTDR. Emission was collected at 610 ± 20 nm (Ir-rhod) and 520 ± 30 nm (MTDR). Annexin V/PI Assay: Following supplier guidelines, A549R cells were grown in 6-well plates and exposed to varying concentrations of Ir-rhod complex for 72 h. Post-treatment, cells were collected and stained with Annexin V and PI for 15 min in the dark. Flow cytometry was performed at 488 nm, excluding the Ir-rhod complex absorbance. Data was analyzed with FlowJo software. Mitochondrial Dysfunction: A549R cells exposed to the Ir-rhod complex for a period of 72 h. Subsequently, the cells were collected, re-suspended at a density of 1 × 10^^6 cells/mL in tempered PBS that contained 5 μg/mL of JC-1, and then incubated for 30 min at a temperature of 37 °C and washed with PBS, and analyzed using flow cytometry. The fluorescence was assessed by measuring the emission of JC-1's monomer (at 527 nm; green color) and aggregate (at 590 nm; red color) forms after excitation at 488 nm. Transmission Electron Microscopy: A549R cells exposed to different concentrations of the Ir-rhod complex for a period of 72 h. After treatment, cells were fixed in a 2.5 % glutaraldehyde solution at 4 °C overnight. After osmium tetroxide post-fixation and staining with uranyl acetate and lead citrate, samples were observed under a transmission electron microscope (JEM 100 CX, JEOL, Tokyo, Japan). Wound Healing Assay: The procedure was conducted as previously outlined. A549R cells were placed in a 6-well plate. At 90 % confluence, cross lines were drawn with a pipette tip. Cells were incubated with Ir-rhod complex in serum-free medium at 37 °C for 24 h. Cross line images were taken at the beginning and end using an inverted microscope. Transwell Invasion Assay: Cell invasion was evaluated using an 8 μm pore Transwell chamber from Millipore, pre-coated with Matrigel for over 12 h. A549R cells were fixed with 4 % PFA and stained with crystal violet. Non-invading cells were removed, and invaded cells were counted under a microscope using ImageJ software from five random fields per sample. Tube Formation Assay: HUVECs were grown on Matrigel-coated 24-well plates at 8 × 10^^4 cells/well and treated with the complex. Tubular structure formation was assessed under a microscope. Tube lengths were measured using AngioSys 2.0 software, as per established protocols. In vivo Anti-tumor Evaluation of Ir-rhod: A549R tumor models were created through injecting beneath the skin with 5 × 10^6 A549R cells. When tumors reached 80–100 mm^3, mice were randomly divided into four groups (n = 5): PBS, Ir-rhod (100 L, 2 mg/kg), Ir-rhod (100 L, 4 mg/kg) administered via tail vein injection (i.v.), and cisplatin as a positive control (i.v.). Treatments were given every 3 days, totaling seven administrations. Body weight was recorded every two days. ACSL4 activity assay: This ACSL4 ELISA kit is intended Laboratory for Research use only and is not for use in diagnostic or therapeutic procedures. The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of ACSL4 in the sample, this ACSL4 ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus ACSL4 concentration. The concentration of ACSL4 in the samples is then determined by comparing the O.D. of the samples to the standard curve. Follow the instructions of the Human ACSL4 ELISA Kit for operation (JM-4973H2). Phospholipids: Phospholipids are hydrolyzed by phospholipase D to generate choline and phosphatidic acid. Choline is subsequently oxidized by choline oxidase to produce hydrogen peroxide. This hydrogen peroxide reacts with 4-aminoantipyrine and DAOS to form a blue-colored dye. The dye exhibits a maximum absorption peak at 600 nm, and the absorbance intensity is directly proportional to the phospholipid content in the sample. The procedure is carried out according to the instructions provided in the Phospholipid (PLIP) (Oxidase Method) Content Test Kit manual (ADS-W-D034). Detection of intracellular Fe: Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Take 5 million bacteria or cells and add to 1 mL of extraction solution. Sonicate the bacteria or cells (on ice, power at 20 % or 200W, sonicate for 3 s, interval for 10 s, repeat for 30 cycles). Centrifuge at 4 °C, 12000 g for 10 min, and collect the supernatant for analysis. In an acidic medium, iron is released from complexes, reduced to ferrous iron by a reducing agent, and forms a purple-red complex with ferrozine. This colored complex has a characteristic absorption peak at 562 nm, from which the iron content is calculated. Follow the instructions of the ferrozine-based iron assay kit for operation (ADS-W-D007). TMT-based proteomics analysis method: Sample Pretreatment, Protein Extraction, and Quality Control. Enzymatic Digestion and Desalting: Take an appropriate amount of protein and add a final concentration of 5 mM DTT, incubate at 37 °C for 1 h, then allow the sample to return to room temperature. Add a final concentration of 10 mM iodoacetamide and incubate in the dark at room temperature for 45 min. Dilute the sample with 25 mM ammonium bicarbonate to a 4-fold concentration and add trypsin at a protein-to-trypsin ratio of 50:1, then incubate overnight at 37 °C. On the second day, adjust the pH to less than 3 with formic acid to terminate the enzymatic digestion. Desalting with a C18 Column: Activate the C18 desalting column with 100 % acetonitrile and equilibrate the column with 0.1 % formic acid. Load the sample onto the column, then wash the column with 0.1 % formic acid to remove impurities. Elute with 70 % acetonitrile, collect the flow-through, and lyophilize. Sample Labeling: Take the TMT reagent out of the freezer and let it thaw at room temperature, then open the cap and add 41 μl of acetonitrile. Vortex for 5 min and centrifuge. Add the TMT reagent to 100 μg of the enzymatically digested sample and react at room temperature for 1 h. Terminate the reaction with ammonia water. Mix the labeled samples, vortex, and centrifuge to the bottom of the tube. Vacuum freeze-dry the samples. Fractionation: Dissolve the mixed labeled samples with 100 μl of mobile phase A. Centrifuge at 14,000g for 20 min, take the supernatant, and use high-performance liquid chromatography (HPLC) for fractionation. The flow rate is 0.7 ml/min. Mass Spectrometry Analysis: Prepare mobile phase A (100 % water, 0.1 % formic acid) and phase B (80 % acetonitrile, 0.1 % formic acid). Dissolve the freeze-dried powder with 10 μL of phase A, centrifuge at 4 °C at 14,000g for 20 min, and take the supernatant. Inject 1 μg of the sample into the liquid chromatography-mass spectrometry (LC-MS) system for analysis. Use a Q Exactive HF-X mass spectrometer with a Nanospray Flex™ (NSI) ion source. Set the ion spray voltage to 2.2 kV, the ion transfer tube temperature to 320 °C, and use a data-dependent acquisition mode. The full scan range of the mass spectrometer is set to m/z 350–1800, with a resolution of 60,000 (at 200 m/z), AGC set to 3 × 10^^6, and the maximum C-trap injection time to 50 ms. Select the top 15 parent ions with the highest intensity in the full scan for fragmentation using high-energy collisional dissociation (HCD) for MS/MS analysis. Set the MS/MS resolution to 45,000 (at 200 m/z), AGC to 2 × 10^^5, and the maximum injection time to 120 ms. Set the peptide fragmentation collision energy to 34 % to generate raw mass spectrometry data (.raw). Database Selection: The selection of the database is based on the species of interest, the completeness of database annotation, and the reliability of the sequence. When selecting a database, follow these principles: if the organism has been sequenced, use the species-specific database directly; if the organism has not been sequenced, choose the most relevant broad protein database that is closest to the species being analyzed. Histological and Immunofluorescence Analysis: By day 18, animals were euthanized, and tumors and organs were collected. Sections were stained with H&E. Tumor sections (6 μm) were acetone-fixed, permeabilized with Triton X-100, blocked with BSA, and incubated with primary and secondary antibodies. Nuclei were stained with Hoechst 33342, and images were captured by CLSM. TUNEL assay was used to assess apoptosis, and slides were examined using Nikon CLSM. Statistical Analysis: Statistical analysis was performed using SPSS 22.0 software. The experiments were performed with 3 or more replicates, and the results are presented as the mean standard deviation (mean ± SD). ANOVA were used to determine significant differences between treatment groups, with P values of <0.05 (∗), <0.01 (∗∗), <0.001 (∗∗∗), and <0.0001 (∗∗∗∗). CRediT authorship contribution statement Juanjuan Li: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. Guibin Gao: Conceptualization, Data curation, Formal analysis, Project administration, Resources, Software, Validation. Wenrui Ouyang: Data curation, Formal analysis, Software, Visualization. Jinkun Huang: Project administration, Supervision. Hongxing Liu: Conceptualization, Funding acquisition, Project administration. Jin Li: Supervision. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Declaration of competing interest The authors declare no conflict of interest. Acknowledgements