Mitochondria-targeted iridium(III) complexes encapsulated in liposome induce cell death through ferroptosis and gasdermin-mediated pyroptosis.

{"full_text": " European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nMitochondria-targeted iridium(III) complexes encapsulated in liposome\ninduce cell death through ferroptosis and gasdermin-mediated pyroptosis\nChunxia Huang a, 1, Yuhan Yuan a, b, 1, Gechang Li a, Shuang Tian a, Huiyan Hu a, Jing Chen a,\nLijuan Liang a, Yi Wang a, Yunjun Liu a, *\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, China\nb\n Foshan women and children hospital, Foshan, 528000, China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: This paper unveils a novel perspective on synthesis and characterization of the ligand 5-bromo-2-amino-2\u2019-\nIridium(III) complexes (phenyl-1H-imidazo[4,5-f][1,10]phenanthroline) (BAPIP), and its iridium(III) complexes [Ir(PPY\u2212 )2(BAPIP)]\nApoptosis (PF6) (1a, with PPY\u2212 as deprotonated 2-phenylpyridine), [Ir(PIQ\u2212 )2(BAPIP)](PF6) (1b, piq\u2212 denoting deproto\u00ad\nFerroptosis\n nated 1-phenylisoquinoline), and [Ir(BZQ\u2212 )2(BAPIP)](PF6) (1c, bzq\u2212 signifying deprotonated benzo[h]quino\u00ad\nPyroptosis\nIn vivo antitumor\n line). Systematic evaluation of the cytotoxicity of 1a, 1b, and 1c across diverse cell lines encompassing B16,\nRNA-Sequence HCT116, HepG2, A549, HeLa, and LO2 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\n (MTT) method. Unexpectedly, compounds 1b and 1c demonstrated no cytotoxicity against the above cell lines.\n Motivated by the pursuit of heightened anti-proliferative potential, a strategic encapsulation approach yielded\n liposomes 1alip, 1blip, and 1clip. As expectation, 1alip, 1blip, and 1clip displayed remarkable anti-proliferative\n efficacy, particularly noteworthy in A549 cells, exhibiting IC50 values of 4.9 \u00b1 1.0, 5.9 \u00b1 0.1, and 7.6 \u00b1 0.2 \u03bcM,\n respectively. Moreover, our investigation illuminated the mitochondrial accumulation of these liposomal en\u00ad\n tities, 1alip, 1blip, and 1clip, evoking apoptosis through the mitochondrial dysfunction mediated by reactive\n oxygen species (ROS). The ferroptosis was confirmed by decrease in glutathione (GSH) concentrations, the\n downregulation of glutathione peroxidase 4 (GPX4), increase of high mobility group protein 1 (HMGB1), and\n lipid peroxidation. Simultaneously, pyroptosis as another mode of cell death was undertaken. RNA-sequencing\n was employed to investigate intricate signalling pathways. In vivo examination provided tangible evidence of\n 1alip in effectively curbing tumor growth. Collectively, this study provides a multifaceted mode of cellular\n demise orchestrated by 1a, 1alip, 1blip, and 1clip, involving pathways encompassing apoptosis, ferroptosis, and\n pyroptosis.\n\n\n\n\n1. Introduction toxicities persist as an imposing impediment to effective chemotherapy.\n Thus, the scientific community\u2019s focus pivots towards conceiving novel\n Malignant neoplasms pose a formidable threat to human well-being, antineoplastic chemotherapies and negligible side effects [5]. Cisplatin\u2019s\nengendering a reduction in the quality of life and, at times, culminating clinical deployment have been limited by its systemic toxicity, precipi\u00ad\nin mortality. The challenge of combating cancer is monumental, given tating deleterious adversities such as profound emesis, auditory debili\u00ad\nits proclivity for recurrence, metastasis, dearth of safe and efficacious tation, and renal compromise. Consequently, the scientific vanguard\ntherapies, and the annual escalation in morbidity and mortality rates embarks on a quest for alternative metal complexes as surrogates for\n[1]. In the wake of swift advancements in detection methodologies and cisplatin [6,7]. Cyclometalated iridium(III) complexes, an intriguing\ntreatment modalities, immunotherapy, gene therapy, and chemotherapy subset of compounds, have attracted considerable attention [8,9]. The\nemerge as valid clinical strategies for a treatment of malignancies [2\u20134]. cyclometalated complexes constituted by iridium(III) as the nucleus,\nAlthough myriad antineoplastic agents have been used to address their ensconced between two cyclic ligands and a bidentate ligand, the\nrespective malignancies, the multifaceted resistance and systemic chemical properties of which can be adjusted by changing the chemical\n\n\n * Corresponding author.\n E-mail address: lyjche@gdpu.edu.cn (Y. Liu).\n 1\n These authors contribute equally.\n\nhttps://doi.org/10.1016/j.ejmech.2023.116112\nReceived 5 November 2023; Received in revised form 25 December 2023; Accepted 29 December 2023\nAvailable online 1 January 2024\n0223-5234/\u00a9 2024 Elsevier Masson SAS. All rights reserved.\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\nstructure, which is more diverse than the typical planar structure of quinoline) were synthesized and characterized. The ligand BAPIP was\nplatinum complexes [10]. The distinctive attributes encompass an formed by the replacement of a hydrogen atom at 4-position of apip with\nextended phosphorescence lifespan, pronounced photostability, and a bromide atom, poor solubility makes it tough for 1a, 1b, 1c to enter the\ncellular ingress capability, endowing it with potential not only as an cell. Therefore, 1a, 1b and 1c were wrapped into liposomes, which\nanticancer agent but also as an innovative biological probe [11\u201318]. facilitated the passage of the drug through the cell membrane and thus\nAlthough iridium(III) complexes have strong anticancer effects, many exhibited excellent anticancer efficacy.\nproblems remain to be solved, including incomplete metabolism and\npoor water solubility. Nanosystems are capable of passive targeting the 2. Results and discussion\ntumor tissue, resulting in the accumulation of nanoparticles near tumor\ncells and the release of drugs [19\u201321]. Liposomes, one of the most 2.1. Chemistry\nfascinating organic nanostructures, are synthetic lipid vesicles with a\nhydrophilic cavity surrounded by an amphiphilic phospholipid bilayer The ligand BAPIP was synthesized through a reaction involving\n[22]. Hence, liposomes have been intensively investigated as a lipid phenanthroline-5,6-dione, 2-amino-4-bromobenzaldehydedione and\nbilayer structure for their diagnostic and drug-delivery functions [23, NH4Ac in glacial acetic acid (Scheme 1). Subsequently, compounds 1a,\n24]. Several studies in recent years have described liposome-loaded 1b, and 1c were prepared by refluxing [Ir(PPY\u2212 )2Cl]2, [Ir(PIQ\u2212 )2Cl]2\niridium complex-based drug delivery systems [25\u201327]. The iridium and [Ir(BZQ\u2212 )2Cl]2 with BAPIP using methanol and dichloromethane as\n(III) complexes encapsulated in polyethylene-glycollated liposomes can a mixed solvent. The characterization of 1a, 1b, and 1c involved High-\nbe used as cancer therapeutics. Resolution Mass Spectroscopy (HRMS) and nuclear magnetic reso\u00ad\n In our previous work, we found that iridium(III) complexes with nance (NMR) spectroscopy. In HRMS, the determined molecular weights\npolypyridyl ligands apip (2-aminophenylimidazo[4,5-f][1,10]phenan\u00ad were consistent with the expected values. Notably, the 1H NMR spectra\nthroline), maip (3-aminophenylimidazo[4,5-f][1,10]phenanthroline) revealed signals corresponding to \u2013NH2 resonances at 4.49 ppm for 1a\nand paip (4-aminophenylimidazo[4,5-f][1,10]phenanthroline) con\u00ad and 4.48 ppm for 1c. Additionally, owing to probably rapid exchange,\ntaining \u2013NH2 as substitute group show high anticancer effect on A549 the peak of the proton bonded to nitrogen in the imidazole ring was not\nand B16 cells [28]. Moreover, we also found that the polypyridyl iridium discovered in the 1H NMR spectra.\n(III) complex [Ir(ppy)2(BBIP)](PF6) (BBIP = 2-(7-bromo-2H-benzo[d] The purities of 1a, 1b, and 1c were assessed via high performance\nimidazole-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) with Br atom liquid chromatography (HPLC) using a mobile phase consisting of\nas substitute group also displays strong ability to inhibit A549 (IC50 = methanol and water in ratios of 72:28, 72:28, and 70:30 for 1a, 1b, and\n4.9 \u00b1 0.5 \u03bcM) and HeLa (IC50 = 9.4 \u00b1 0.5 \u03bcM) cell proliferation [29]. 1c, respectively. The purities of 1a, 1b and 1c were determined as 96.56\nAdditionally, halogen atoms are among the most popular substituents %, 98.11 %, and 96.85 %, respectively (Fig. S1 and SI).\nutilized for the optimization of the properties of biologically active Furthermore, the encapsulation of 1a, 1b, and 1c within liposomes,\ncompounds, moreover, the halogen atoms can form both hydrophobic denoted as 1alip, 1blip, and 1clip, was achieved through a film disper\u00ad\nand electrostatic interactions with biological targets [30]. The litera\u00ad sion method, resulting in encapsulation efficiency (EE) values of 87.6 %,\ntures reported that the compounds with Br atom as substitute group 88.3 %, and 85.4 %, respectively. These outcomes indicate successful\nreveal high anticancer activity [31\u201333]. Therefore, based on the above encapsulation of the complexes within the liposomal structures. The\nfindings and reports, in this article, a new ligand 5-bromo-2-ami\u00ad prepared 1alip, 1blip, and 1clip exhibited a yellow hue (Fig. S2a and SI).\nno-2\u2019-(phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (BAPIP) with Their particle sizes were determined as 109.1 nm (\u00b12.4), 95.5 nm\n\u2013NH2 and Br atom as substitute groups and iridium(III) complexes [Ir (\u00b11.9), and 90.7 nm (\u00b11.3), respectively (Fig. S2b and SI). The \u03b6-po\u00ad\n(PPY\u2212 )2(BAPIP)](PF6) (1a, PPY\u2212 = deprotonated 2-phenylpyridine), [Ir tential measurements unveiled surface mean charges of \u2212 21.80 mV,\n(PIQ\u2212 )2(BAPIP)](PF6) (1b, PIQ\u2212 = deprotonated 1-phenylisoquinoline) \u2212 23.68 mV, and \u2212 23.93 mV for 1alip, 1blip, and 1clip, respectively\nand [Ir(BZQ\u2212 )2(BAPIP)](PF6) (1c, BZQ\u2212 = deprotonated benzo[h] (Fig. S2c and SI), confirming the stability of these complexes-loaded\n\n\n\n\n Scheme 1. Synthetic route for ligand BAPIP and complexes 1a, 1b, 1c.\n\n 2\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\nliposomes. Additionally, the stability of 1alip, 1blip and 1clip was 2.3. Cellular uptake, co-location, ROS, mitochondrial membrane\nexamined by UV\u2013Vis spectra, as shown in Fig. S2d (SI), we observed no potential, Ca2+ concentration\nchange in the peak shape at 0 h and 48 h, indicating that the liposome-\nloaded complexes are stability. The morphology of 1clip was examined The requisite for the manifestation of the anticancer activity of\nby transmission electron microscope (TEM), as seen from Fig. S2e (SI), complexes resides in their cellular uptake. In this context, an uptaken\n1clip displays a spherical particle size. amount of 1a, 1alip, 1blip, and 1clip by A549 cells was undertaken\n Subsequently, the release kinetics of 1a, 1b, and 1c from 1alip, 1blip, through laser scanning confocal microscopy. As discerned from Fig. S3a\nand 1clip were monitored over 72 h. As illustrated in Fig. S2f (SI), the (SI), the treated groups exhibited green fluorescence emanating from the\ninitial release percentages were observed to be 34.74 % for 1a, 19.94 % complexes, while the cell nuclei were distinctly stained blue. Upon\nfor 1b, and 43.87 % for 1c. At 72 h, the release rates increased to 70.14 scrutinizing the merged images, it becomes apparent that 1a, 1alip,\n% for 1a, 41.32 % for 1b, and 88.93 % for 1c. Therefore, the release of 1blip, and 1clip enter the cells and focus on the cytoplasmic locale, while\n1a, 1b and 1c from 1alip, 1blip and 1clip was carried out slowly. in the 1b, 1c-treated groups, the green fluorescence is the same as that in\n the control, indicating almost no complexes entering the cells. The re\u00ad\n sults obtained from that cellular uptake are agreement with those of\n2.2. Evaluation of cytotoxic activity\n cytotoxic activity.\n Given that mitochondria are pivotal in apoptosis, their involvement\n The assessment of cytotoxicity for compounds 1a, 1b, 1c, 1alip, 1blip\n in the cellular context was explored. Notably, the co-localization of red\nand 1clip was conducted using the 3-(4,5-dimethylthiazol-2-yl)-2,5-\n fluorescence (indicative of mitochondria) and green fluorescence (cor\u00ad\ndiphenyltetrazolium bromide (MTT) method [34]. The cytotoxic effi\u00ad\n responding to the complex or complex-loaded liposomes) became\ncacy of these compounds on A549, HTC-116, HepG2, B16, HeLa, and\n evident after the entry of 1a, 1alip, 1blip, and 1clip into the cells for 4 h,\nnon-cancer LO2 cells was quantified in terms of half maximum inhibi\u00ad\n as depicted in Fig. S3b (SI). This concurrence of signals underscores the\ntory concentration (IC50) values. See from Tables 1 and 1a exhibited a\n convergence of these complexes at the mitochondria.\nmoderate level of cytotoxicity. Intriguingly, compounds 1b and 1c dis\u00ad\n Mitochondrial dysfunction, a salient hallmark of apoptosis, materi\u00ad\nplayed no discernible cytotoxicity. This phenomenon could likely be\n alizes through the diminution of mitochondrial membrane potential\nattributed to the limited solubility of 1b and 1c, impeding their effective\n (MMP) and the simultaneous opening of the mitochondrial permeability\ncellular internalisation and consequently resulting in the absence of\n transition pore (MPTP) [36,37]. To gauge alterations in MMP, the flu\u00ad\ncytotoxic activity for these two compounds. To amplify the intracellular\n orophore JC-1 (5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetrethylbenzimida\u00ad\nuptaken amounts of 1a, 1b, and 1c, these compounds were encapsulated\n lylcarbocyanine iodide) was harnessed. The transition of JC-1 from a\nwithin liposomal carriers, yielding 1alip, 1blip, and 1clip. Notably, the\n red-fluorescent aggregate to a green-fluorescent monomer within the\ncytotoxicity of 1alip towards A549 cells was amplified by a factor of 2.94\n mitochondria corroborates changes in MMP [38]. Exposure of A549\ncompared to that of 1a. The IC50 values for 1alip, 1blip, and 1clip against\n cells to 1a, 1alip, 1blip, and 1clip for 24 h occasioned a diminution in the\nA549 cells were determined as 4.9 \u00b1 1.0, 5.9 \u00b1 0.1, and 7.6 \u00b1 0.2 \u03bcM,\n red fluorescence (indicative of high MMP) alongside a concomitant\nrespectively. The anti-proliferative efficacy of 1alip, 1blip, and 1clip\n elevation in green fluorescence (indicative of low MMP), reflective of a\nsurpassed that of cisplatin against B16, HCT116, and HepG2 cells, but\n decline in the mitochondrial membrane potential, as presented in\nlower than liposome entrapped cisplatin (CDDPlip) toward B16 and\n Fig. S3c (SI). The quantification of red and green fluorescence is\nA549 cells. Their cytotoxicity is comparable with that of\n graphically elucidated in Fig. S3d (SI), 0.5 \u00d7 IC50 concentration of 1a,\nliposome-encapsulated [Ir(bzq\u2212 )2(DIPH)](PF6) against HeLa cells (IC50\n 1alip, 1blip and 1clip show low effect on the change of mitochondrial\n= 6.3 \u00b1 0.2 \u03bcM) [27]. Additionally, we also examined the cytotoxic\n membrane potential, while A549 cells were treated with 2 \u00d7 IC50 con\u00ad\nactivity of blank liposome against A549 cells, we found that the blank\n centration of 1a, 1alip, 1blip and 1clip for 24 h, an obvious change in the\nliposomes have no cytotoxic activity toward A549 cells, this is agree\u00ad\n ratio of red/green fluorescence was uncovered. These observations\nment with the results reported in the literature [35]. According to the\n further underscore the propensity of 1a, 1alip, 1blip and 1clip to induce\nIC50 values, we discovered that 1alip, 1blip and 1clip show the highest\n a decline in MMP with a concentration-dependent manner.\nanticancer activity towards A549, HeLa and HepG2 cells, respectively,\n Concurrent with changes in MMP, the sustained opening of mito\u00ad\nwhich demonstrates that the liposome-loaded different compounds\n chondrial permeability transition pore (MPTP) transpires. This phe\u00ad\nexhibit different anticancer efficacy toward different cancer cells. This\n nomenon is orchestrated by the protein tyrosine phosphatase (PTP),\ncollective evidence substantiates the notion that liposome-encapsulated\n which ensures continuous pore opening when the mitochondrial matrix\ncomplexes engender a substantial augmentation in their anticancer\n undergoes elevated osmotic pressure. The outcome encompasses mito\u00ad\npotential.\n chondrial membrane depolarisation and a surge in reactive oxygen\n species (ROS), ultimately culminating in apoptosis [39,40]. Apparent\nTable 1 from Fig. S3e (SI) is the robust green fluorescence in the control and\nIC50 values (\u03bcM) of 1a, 1b, 1c, 1alip, 1blip, 1clip and liposome-loaded cisplatin\n negative groups, indicative of normal MPTP function. Conversely,\n(CDDPlip) towards the selected cancer cells for 48 h.\n treatment of A549 cells with 1a, 1alip, 1blip, and 1clip ushered in a\n Complex B16 HCT116 HepG2 A549 HeLa LO2 substantial reduction in green fluorescence, substantiating their poten\u00ad\n 1a 16.6 \u00b1 14.3 \u00b1 33.8 \u00b1 14.4 \u00b1 15.3 \u00b1 43.8 \u00b1 tial to induce continuous MPTP opening.\n 0.2 0.2 2.2 0.5 3.5 1.3 Given the intertwined relationship between ROS and mitochondrial\n 1b >200 >200 >200 >200 >200 >200 dysfunction, intracellular ROS content was evaluated using 2,7\u2032-\n 1c >200 >200 >200 >200 >200 >200\n 1alip 5.7 \u00b1 6.3 \u00b1 0.1 5.0 \u00b1 4.9 \u00b1 6.9 \u00b1 8.7 \u00b1\n dichlorodihydrofluorescein diacetate (DCFH-DA) as a fluorescence\n 0.3 0.4 1.0 0.2 1.0 probe. The treatment of A549 cells with 1a, 1alip, 1blip, and 1clip\n 1blip 5.1 \u00b1 6.2 \u00b1 0.3 9.9 \u00b1 5.9 \u00b1 4.4 \u00b1 7.8 \u00b1 yielded heightened green fluorescence, an effect quantitatively corrob\u00ad\n 0.1 0.2 0.1 0.1 0.2 orated and presented in Fig. S3f (SI). Notably, the fluorescence intensity\n 1clip 8.8 \u00b1 7.5 \u00b1 0.2 5.9 \u00b1 7.6 \u00b1 6.8 \u00b1 10.3 \u00b1\n in cells treated with 1a exhibited a moderate increment, while 1alip,\n 0.4 0.2 0.2 0.1 0.2\n cisplatin 18.8 \u00b1 14.5 \u00b1 9.1 \u00b1 6.6 \u00b1 5.7 \u00b1 18.3 \u00b1 1blip, and 1clip induced increments of 2.81, 2.21, and 2.98 times\n 1.2 2.2 1.4 0.7 1.1 2.4 compared to the control, respectively (Fig. 3g and SI). These results\n CDDPlip 3.6 \u00b1 nd nd 1.5 \u00b1 nd 3.7 \u00b1 collectively underscore the propensity of 1a, 1alip, 1blip, and 1clip to\n 0.1 0.1 1.5 elicit an elevated level of ROS production.\nnd: not determination. Calcium ions (Ca2+) function as pivotal second messengers\n\n 3\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\ngoverning diverse cellular processes encompassing gene transcription, various cancer-associated signalling pathways as evidenced by the\ncell proliferation, migration, and apoptosis [41]. The elevation in Ca2+ KEGG pathway analysis in Fig. 1c. These pathways encompass TNF,\nlevel imparts impetus to ROS production, diminishes mitochondrial PI3K/AKT, and calcium ion signalling pathways, collectively orches\u00ad\nmembrane potential, and triggers sustained MPTP opening, thereby trating cell apoptosis and demise. The GO enrichment analysis, as\nengendering apoptosis [42]. As illustrated in Fig. S3h (SI), the control illustrated in Fig. 1d, further corroborates the enrichment of biological\nand 1a-treated groups exhibited modest green fluorescence, whereas in processes, molecular functions, and cellular components underpinning\nthe groups treated with 1alip, 1blip, and 1clip, a marked increase in the observed effects.\nfluorescence intensity was discovered. Notably, the enhancement was A deeper exploration of the apoptotic mechanisms was pursued to\nquantified as 2.95, 1.85, and 2.33 times than that of the initial intensity, corroborate the findings derived from RNA sequencing. The apoptotic\nas illustrated in Fig. S3i (SI). This discernible increase underscores the cascade is fundamentally directed through two primary pathways: the\nrole of 1a, 1alip, 1blip, and 1clip in eliciting a release of Ca2+, thereby mitochondrial and death receptor pathways [45]. These pathways\nfurther exacerbating mitochondrial dysfunction and hastening the depend on the activation of caspases (cysteinyl aspartate-specific pro\u00ad\napoptotic cascade. Based on the above results, we consider that the teases), which subsequently cleave numerous proteins, thereby\ncomplexes-loaded liposomes induce apoptosis in A549 cells via a executing apoptosis and culminating in cellular demise, accompanied by\nROS-mediated mitochondrial dysfunction pathway. distinctive biochemical hallmarks [46]. Caspase 3, operating as an\n effector caspase, plays a central role in this process, ultimately precipi\u00ad\n2.4. Cell cycle distribution and apoptosis tating cellular death by cleaving death substrates such as poly-ADP\n ribose polymerase (PARP) [45], and cleaved PARP serves as a func\u00ad\n In eukaryotic organisms, cellular proliferation constitutes a pivotal tional indicator of apoptosis [47]. In the presented study, the modula\u00ad\nprocess underpinning cell growth, development, and tissue regeneration tion of apoptotic markers including caspases and Bcl-2 family proteins is\n[43]. Therefore, impeding the progression of the cell cycle emerges as a evident, as depicted in Fig. 1e and f. The upregulation of Bax, concurrent\nfundamental strategy within the anticancer paradigm, whereby the re\u00ad with the downregulation of Bcl-2, PI3K, AKT, and caspase 3 expression,\nstraint of cellular proliferation thwarts the advancement of malignancy. is apparent in response to 1a, 1alip, 1blip, and 1clip treatments. The\nAs portrayed in Fig. S4a (SI), examining A549 cell populations subjected cumulative evidence substantiates that 1a, 1alip, 1blip, and 1clip\nto 2 \u00d7 IC50 concentration of 1a (IV), 1alip (VII), 1blip (X), and 1clip orchestrate apoptosis by modulating Bcl-2 family protein expression and\n(XIII) treatments reveals a decline in cell numbers within the G0/G1 and hindering the PI3K/AKT pathway, a conclusion harmonious with the\nG2/M phases. Correspondingly, a significant increase is observed in the RNA sequencing analysis.\nS-phase population, manifesting as a 19.73 % increase for 1a, 26.82 %\nfor 1alip, 23.30 % for 1blip and 22.02 % for 1clip (Table 2). These results 2.6. Cell ferroptosis studies\nsubstantiate that 1a, 1alip, 1blip, and 1clip effectively suppress cell\nproliferation during the S-phase with a concentration-dependent Lipid peroxidation is an intricate autoxidation process instigated by\nmanner. Furthermore, it is pertinent to note that 1alip displays a supe\u00ad the assault of free radicals, contributing significantly to the orchestra\u00ad\nrior capacity in inhibiting cell proliferation compared to its corre\u00ad tion of cell demise [48]. Notably, glutathione (GSH) holds a pivotal\nsponding complex 1a. regulatory role in the facets of cell differentiation, proliferation, and\n The influence of the complexes and their encapsulated liposomes on apoptosis [49]. To Illustrating this, Fig. 2a portrays the discernible\ncellular proliferation inevitably entails an induction of cell death. reduction in GSH levels after a 24-h exposure of A549 cells to 1a, 1alip,\nWithin this context, apoptosis emerges as a pivotal mechanism for 1blip, and 1clip. This observation lucidly underscores the efficacious\neliminating senescent cells [44]. As depicted in Fig. S4b (SI), after A549 capability of 1a, 1alip, 1blip, and 1clip to reduce GSH amounts, ulti\u00ad\ncells (I) were incubated with 2 \u00d7 IC50 concentration of 1a (IV), 1alip mately culminating in cellular demise. Conversely, in the context of\n(VII), 1blip (X) and 1clip (XIII) for 24 h, the proportion of cells in early elevated ROS levels, GSH interacts with ROS, resulting in the formation\napoptosis (Q3) rises from 1.32 % in the control group to 11.5 % for 1a, of glutathione disulfide (GSSG). As depicted in Fig. 2b, the diminishment\n16.8 % for 1alip, 18.3 % for 1blip, and 20.1 % for 1clip-treated groups, in the GSH/GSSG ratio affirms the reduction in GSH content.\nrespectively (Table 2). These outcomes collectively affirm that 1a, 1alip, On a parallel note, malondialdehyde (MDA), a product of lipid per\u00ad\n1blip, and 1clip can induce apoptosis with a concentration-dependent oxidation, is serves as an apt gauge of oxidative stress-mediated cellular\nmanner. Notably, the liposome-encapsulated complex 1alip exhibits an injury [50]. The depiction in Fig. 2c notably showcases the conspicuous\nenhanced apoptotic efficacy compared with 1a. elevation in intracellular MDA content upon treating A549 cells with 1a,\n 1alip, 1blip, and 1clip. This discernible elevation in MDA content cor\u00ad\n2.5. RNA-sequence, apoptosis-related protein expression roborates that 1a, 1alip, 1blip, and 1clip can induce lipid peroxidation.\n As a programmed cell death, ferroptosis assumes a significant role\n Moving forward, the exploration of RNA sequencing and an assess\u00ad characterized by its intricate interplay with lipid peroxidation, facili\u00ad\nment of apoptosis-related protein expression were carried out. RNA tated through glutathione peroxidase 4 (GPX4) as the principal pathway\nsequencing stands as an insightful technique capable of unveiling the [51]. Inactivation of GPX4 results in the accumulation of lipid peroxides,\npathways through which compounds orchestrate cell death. In this re\u00ad thereby instigating heightened ROS levels [52]. Notably, ferroptosis can\ngard, an RNA sequencing experiment unveiled the biological processes be induced by activating high mobility group protein 1 (HMGB1), which\nunderpinning the antitumor activity of interest. The heatmap and gene functions as both a damage-associated molecule and a nexus between\ndistribution profiles depicted in Fig. 1a and b elucidate that 1alip elicits ferroptosis and pyroptosis [53,54]. Fig. 2d shows that 1a, 1alip, 1blip,\nthe downregulation of 367 genes and the upregulation of 534 genes. and 1clip downregulated GPX4 expression in comparison to the control,\nThis cascade of gene expression modulation leads to the enrichment of while concurrently, an augmented expression of HMGB1 is evident\n\nTable 2\nThe percentage of cell at the S phase and early apoptosis after A549 cells exposed to different concentration (\u03bcM) of 1a, 1alip, 1blip and 1clip for 24 h.\n Control 1a 1alip 1blip 1clip\n\n Concentration 8.3 16.6 33.2 2.85 5.7 11.4 2.55 5.1 10.2 4.4 8.8 17.5\n Percentage at S phase (%) 23.29 41.58 42.90 43.12 47.30 48.46 50.21 42.58 44.71 46.69 43.13 44.36 45.41\n apoptosis (%) 1.32 1.53 3.44 11.5 6.35 7.47 16.8 6.76 7.94 18.3 7.14 10.40 20.1\n\n\n 4\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\n\n\nFig. 1. RNA-sequencing assay of 1alip, (a) heatmap, (b) number of up or down-regulated genes, (c) KEGG Enrichment, (d) GO Enrichment, (e) expression of\napoptosis-related proteins after a treatment of 24 h of A549 cells with 2 \u00d7 IC50 concentration of 1alip, (f) gray values.\n\n\nacross the treatment groups encompassing 1a, 1alip, 1blip, and 1clip. treatment groups encompassing 1a, 1alip, 1blip, and 1clip. Moreover,\nThe interplay of these molecular expressions substantiates the inference the reduction in the red/green fluorescence ratio, as depicted in Fig. 2g,\nthat 1a, 1alip, 1blip, and 1clip stimulate ferroptosis via GPX4 down\u00ad further substantiates the proclivity of 1a, 1alip, 1blip, and 1clip to cause\nregulation and HMGB1 upregulation. lipid peroxidation.\n To further elucidate the influence of 1a, 1alip, 1blip, and 1clip on\nferroptosis, we examined the cell viability in the presence of ferrostatin- 2.7. Pyroptosis studies\n1 (Fer-1), a recognized antioxidant capable of inhibiting ferroptosis.\nFig. 2e underscores that the presence of Fer-1 correlates with enhanced Pyroptosis, recognized as a distinct form of programmed cell death\ncell viability, conclusively demonstrating the potential of 1a, 1alip, characterized by its inflammatory nature, manifests its execution\n1blip, and 1clip to induce ferroptosis. In addition, we also examined the contingent upon pro-inflammatory caspase 1, accompanied by the\ncytotoxic activity of Fer-1 (6.25 \u2192 400 nM) towards A549 cells during participation of other cysteine aspartame caspases, most notably caspase\n48 h and found that Fer-1 has no cytotoxicity against A549 cells. 3, engendering a conspicuous release of inflammatory mediators [56]. A\n In the realm of assessing lipid peroxidation, 4,4-difluoro-5-(4- hallmark of pyroptotic cells lies in their distinctive morphological at\u00ad\nphenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic tributes, marked by cellular enlargement and discernible balloon-like\nacid (C11-BODIPY581/591) emerges as a probe to determine the lipid protrusions along the cell membrane [57,58]. The observations depic\u00ad\nperoxidation [55]. Fig. 2f reveals a marked decrease in red fluorescence ted in Fig. 3a illuminate these features following a 24-h exposure of\n(indicative of non-oxidized species) accompanied by a concurrent in\u00ad A549 cells to 1a, 1alip, 1blip, 1clip. Notably, a meticulous examination\ncrease in green fluorescence (indicative of oxidized species) across the reveals that 1a leads to fewer cells adopting the pyroptotic phenotype\n\n 5\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\n\n\nFig. 2. (a) Intracellular GSH content, (b) ratio of GSH/GSSG, (c) MDA content, (d) expression of HMGB1 and GPX4, (e) cell viability assay in the presence Fer-1 (100\nnM), (f) lipid peroxidation detection using C11-BODIPY581/591 as a fluorescence dye, (g) ratio of red/green fluorescence, in the above assays, A549 cells were treated\nwith IC50 concentration of 1a, 1alip, 1blip and 1clip for 24 h.\n\n\n\n\nFig. 3. Pyroptosis assay: (a) cell morphology, (b) LDH release (positive: LDH release reagent), (c) expression of NF-\u03baB, caspase 1 and GSDME while a 24 h-treatment\nof A549 cells with IC50 concentration of 1a, 1alip, 1blip and 1clip.\n\n\nwhen contrasted with 1alip, 1blip, and 1clip. cascading to catalyze caspase 1 activation and GSDMD cleavage, en\u00ad\n The lactate dehydrogenase (LDH) release assay, esteemed as a direct genders cell membrane perforation, membrane swelling, and thereby\ngauge of pyroptosis, operates on the premise of quantifying cell mem\u00ad underpins the pyroptosis.\nbrane integrity. In the assay of pyroptosis, the release of LDH from cells\nis emblematic of compromised cell membrane integrity, thereby 2.8. Antitumor in vivo studies\nresulting in an increase in LDH content [56]. The findings in Fig. 3b\nunmistakably illustrate an increase in LDH content, augmenting by Subcutaneous injection of vigorously proliferative A549 cells into\nfactors of 2.23, 4.15, 4.06, and 4.86 in the groups treated with 1a, 1alip, BALB/c experimental mice facilitated the development of spherical tu\u00ad\n1blip, and 1clip, respectively. This empirical evidence underscores the mors. During the drug administration phase, mice were subjected to\npivotal role of 1a, 1alip, 1blip, and 1clip in eliciting pyroptosis. intravenous injections of 1alip at doses of 1.4 mg/kg and 2.1 mg/kg. In\n Pyroptosis, by nature, constitutes an inflammatory cell death pro\u00ad tandem, daily monitoring was conducted to assess body weight fluctu\u00ad\ngram that enlists the participation of NF-\u03baB protein, wielding substantial ations and tumor dimensions. After a duration of 9 days of drug\ninfluence over the inflammatory cascade [59]. A pivotal function of administration, the application of 1alip was terminated in both treat\u00ad\nNF-\u03baB protein resides in its orchestration of downstream protein ment cohorts. The characteristic morphology and dimensions of the\nexpression, culminating in the assembly of inflammatory vesicles. This, tumors are expounded in Fig. 4a, revealing a substantial reduction in\nin turn, sets in motion the activation of caspase 1, a pro-inflammatory tumor size within the groups treated with 1.4 mg/kg and 2.1 mg/kg of\nentity, ultimately culminating in the cleavage of gasdermin D 1alip compared with that in control (Fig. 4b). Notably, the tumor-\n(GSDMD) proteins. The resultant cleaved N-terminal of GSDMD serves suppressive efficacy of 1.4 mg/kg and 2.1 mg/kg of 1alip was quanti\u00ad\nas a fulcrum, initiating perforations within the cellular membrane. The fied at 37.4 % and 70.4 %, respectively (Fig. 4c). On the 15th day, a\nconsequences encompass compromised substance exchange regulation, comprehensive depiction of tumor dimensions is illustrated in Fig. 4d.\ncoupled with the emergence of vacuoles that decisively usher in the This analysis evinced no significant deviations in the body weight of\ninstantiation of pyroptosis [56,57,60]. Concurrently, GSDMD can also mice throughout the 9-day treatment period. However, a modest change\nbe subject to cleavage by the activated caspase 3 [25]. Fig. 3c meticu\u00ad in the body weight of mice within the 2.1 mg/kg 1alip group was dis\u00ad\nlously outlines these intricate interactions, wherein the expression of cerned. Further insight was garnered from the assessment of relative\nNF-\u03baB is enhanced in the 1a, 1alip, 1blip, and 1clip treatment. Converse tumor volumes, which exhibited a decline within the 1alip-treated\nto this, caspase 1 is demonstrably downregulated. The culminating ef\u00ad cohort (Fig. 4e). Moreover, upon discontinuation of treatment (days\nfect materializes in the cleavage of GSDMD under the catalytic influence 10\u201315), the body weights of mice exhibited no conspicuous alterations\nof activated caspase 1, engendering the generation of active N-terminal (Fig. 4f). In contrast, the relative tumor volume in the 1.4 mg/kg 1alip\nGSDMD-F. In summation, the orchestration of NF-\u03baB activation, group exhibited a swift resurgence, approximating values observed in\n\n 6\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\n\n\nFig. 4. (a) Tumor obtained on 9th day, (b) mean tumor weight, (c) inhibitory rate, (d) tumor obtained on 15th day, (e) mean weight of mice, (f) relative tumor\nvolume, (h) H&E staining tissues obtained on the 9th day, (i) H&E staining tissues obtained on the 15th day.\n\n\nthe control group. In contrast, the relative tumor volume in the 2.1 mg/ juxtaposed with comparatively minor and subdued instances of hae\u00ad\nkg 1alip group exhibited marginal variation post-cessation of adminis\u00ad morrhage within the 1alip-treated groups. Tumors within the control\ntration. Significantly, no instances of mortality were documented within group showed substantial mass expansion with pronounced cellular\nthe experimental cohort. proliferation. In contrast, within the 1.4 mg/kg and 2.1 mg/kg 1alip\n Detailed analysis through H&E staining (Fig. 4h) of murine tissues groups, distinctive features of lamellar necrosis and a diminished tumor\nover the 9-day treatment interval underscored that the control group cell population interspersed with expansive necrotic regions were\nexhibited pronounced haemorrhage within the pulmonary regions, evident. Notably, no discernible deviations in the structural architecture\n\n\n 7\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\nof the liver, brain, spleen, heart, and kidney between the control and at a frequency of 500 MHz. Dimethyl sulfoxide (DMSO\u2011d6) was\n1alip-treated groups were observed, reaffirming no side effects associ\u00ad employed as the solvent, while tetramethylsilane (TMS) served as an\nated with 1alip administration. internal standard. High-resolution mass spectra (HRMS) were obtained\n Upon cessation of drug administration, an analysis of H&E-stained through direct injection using a Waters Xevo G2-XS QTof mass analyzer.\ntissues is presented in Fig. 4i. Both the control group and the two 1alip-\ntreated cohorts exhibited pulmonary damage after drug withdrawal. 4.2. Synthesis of ligand and complexes\nMorphological scrutiny of tumor tissues in the control group exposed\nvigorous growth patterns, contrasted by sparse malignant cell pop\u00ad 4.2.1. Preparation of ligand BAPIP\nulations within the low-concentration 1alip group, and a scarcity of 2-amino-4-bromobenzaldehyde (0.198 g, 1.0 mmol), 1,10-phenan\u00ad\nmalignant cells within the high-concentration 1alip group. Notably, the throline-5,6-dione (0.21 g, 1.0 mmol) [61], and NH4Ac (1.54 g, 20\nstructural and functional integrity of the experimental groups displayed mmol) were meticulously placed into the flask. The mixture was dis\u00ad\nminimal variance both prior to and following the discontinuation of solved in 25 mL of glacial acetic acid and refluxed for 2 h. Upon\ndrug administration. completion of the reflux, the reaction mixture was allowed to cool down.\n The solution was neutralized with concentrated NH3\u22c5H2O and the\n3. Conclusions gained precipitate was thoroughly washed using ice H2O, yielding a\n distinctive yellow-hued powder. Yield: 82 %. Analytical calculations for\n The cellular uptake reveals that 1a, 1alip, 1blip and 1clip can enter the compound C19H12BrN5: C, 58.48 %; H, 3.10 %; N, 17.95 %. Exper\u00ad\nthe cells, while it is difficult for 1b and 1c to enter the cells. 1a, 1alip, imental measurement values: C, 58.65 %; H, 3.23 %; N, 17.78 %. HRMS\n1blip and 1clip locate at the mitochondria, increase the content of (CH3CN): m/z = 387.4650.\nintracellular ROS and Ca2+, reduce the mitochondrial membrane po\u00ad\ntential. The apoptosis and cell cycle distribution show that 1a, 1alip, 4.2.2. Synthesis of [Ir(PPY\u2212 )2(BAPIP)](PF6) (1a)\n1blip and 1clip can cause apoptosis and inhibit A549 cell growth at the S Cis-[Ir(PPY\u2212 )2Cl]2 (0.175 g, 0.15 mmol) [62] and BAPIP (0.117 g,\nphase. RNA-sequence studies show that 1alip downregulates the 0.3 mmol) were dissolved in 15 mL of methanol and 30 mL of\nexpression of 367 genes and the upregulates the expression of 534 genes dichloromethane. Then the above solution was refluxed under an argon\nand induce cell death through TNF, PI3K/AKT, and calcium ion sig\u00ad atmosphere for 6 h. After the designated reaction time, an excess of\nnalling pathways. Additionally, a decrease of GSH, an increase of MDA, NH4PF6 (1 g) was introduced into the mixture and stirred for 1 h. Sub\u00ad\nupregulation of HMGB1 and downregulation of GPX4, the increase of sequently, the solution underwent filtration and concentration, leading\ncell viability in the presence of Fer-1 and C11-BODIPY581/591 dyed ex\u00ad to the formation of an orange-hued solid. To purify the product, crude\nperiments suggest that 1a, 1alip, 1blip and 1clip can cause ferroptosis. material was subjected to neutral alumina column chromatography. The\nThe pyroptotic characteristic in the cell morphology, LDH release, the elution process employed a dichloromethane-acetone solvent mixture in\nupregulation of NF-\u03baB, downregulation of caspase 1 and the trans\u00ad a 5:1 (v/v) ratio. The orange band was collected and concentrated to\nformation from GSDMD-F to GSDMD-N indicate that 1a, 1alip, 1blip and yield a yellow powder, yield: 84 %. The molecular formula of the\n1clip can induce pyroptosis. Antitumor in vivo shows that 1alip can compound was identified as C41H28BrF6PN7Ir. 1H NMR (DMSO\u2011d6, 500\nvalidly inhibit the tumor growth with a high inhibitory rate of 70.4 %, MHz, Fig. S5a and SI): \u03b4 8.26 (d, J = 8.5 Hz, 2H), 8.15 (dd, J = 1.5, J =\nmoreover, 1alip has no side effect on the lung, liver, kidney, heart, brain 5.0 Hz, 2H), 8.10\u20138.07 (m, 2H), 7.96 (d, J = 8.0 Hz, 4H), 7.88 (t, J = 7.5\nand spleen. Taken together, 1a, 1alip, 1blip and 1clip induce cell death Hz, 2H), 7.51 (d, J = 5.5 Hz, 2H), 7.16 (d, J = 7.0 Hz, 1H), 7.07 (t, J =\nvia a ROS-mediated mitochondrial dysfunction pathway, ferroptosis and 7.0, 2H), 7.00\u20136.93 (m, 6H), 6.30 (d, J = 7.5 Hz, 2H), 4.49 (s, 2H). 13C\npyroptosis (Fig. 5). NMR (DMSO\u2011d6, 125 MHz, Fig. 5b and SI): 167.38, 153.72, 153.37,\n 150.85, 149.87, 149.63, 148.83, 144.51, 139.17, 132.93, 131.70,\n4. Experimental 130.74, 129.63, 127.46, 126.03, 125.55, 124.69, 124.31, 122.85,\n 120.45, 118.71, 118.19. HRMS (CH3CN): m/z = 890.1215 ([M \u2212 PF6]+).\n4.1. Materials and methods\n 4.2.3. Synthesis of [Ir(PIQ\u2212 )2(BAPIP)] (PF6) (1b)\n A selection of cell lines including B16 mouse melanoma cells, A549 Synthesis of [Ir(PIQ\u2212 )2(BAPIP)](PF6) (1b) is closely similar to 1a. In\nhuman lung carcinoma cells, HeLa human cervical cancer cells, HepG2 this case, the starting material cis-[Ir(PIQ\u2212 )2Cl]2 [62] replaced cis-[Ir\nhuman hepatocellular carcinoma cells, HCT116 human colon cancer (PPY\u2212 )2Cl]2, yield: 80 %. Molecular formula: C49H32BrF6PN7Ir. 1H NMR\ncells, and normal LO2 human liver cells was procured from Sun Yat-Sen (DMSO\u2011d6, 500 MHz, Fig. S6a and SI): \u03b4 9.09 (d, J = 9.0 Hz, 2H), 9.02 (d,\nUniversity (Guangzhou). Nuclear Magnetic Resonance (NMR) spectra J = 8.0, 2H), 8.40 (d, J = 8.0 Hz, 2H), 8.01\u20137.99 (m, 2H), 7.90\u20137.84 (m,\nwere meticulously recorded using a Varian-500 spectrometer operating 9H), 7.44 (d, J = 6.5 Hz, 2H), 7.37 (d, J = 6.5 Hz, 2H), 7.17 (t, J = 7.0\n Hz, 2H), 7.01\u20136.96 (m, 4H), 6.31 (d, J = 7.5 Hz, 2H). 13C NMR\n (DMSO\u2011d6, 125 MHz, Fig. S6b and SI): 172.52, 168.31, 155.21, 154.39,\n 153.74, 146.81, 145.82, 144.16, 141.24, 136.96, 132.92, 132.50,\n 132.16, 131.10, 131.02, 129.85, 129.70, 128.16, 127.32, 126.91,\n 126.01, 122.80, 122.66, 121.57, 118.57, 118.10. HRMS (CH3CN): m/z\n = 990.1518 ([M \u2212 PF6]+).\n\n 4.2.4. Synthesis of [Ir(BZQ\u2212 )2(BAPIP)] (PF6) (1c)\n The synthesis procedure for [Ir(BZQ\u2212 )2(BAPIP)] (PF6) (1c) paral\u00ad\n leled that of 1a, with the substitution of cis-[Ir(PPY\u2212 )2Cl]2 with cis-[Ir\n (BZQ\u2212 )2Cl]2 [62], yield: 78 %. Molecular formula: C45H28BrF6PN7Ir. 1H\n NMR (DMSO\u2011d6, 500 MHz, Fig. S7a and SI): \u03b4 9.29 (d, J = 8.0 Hz, 2H),\n 8.53 (dd, J = 1.0, J = 5.0 Hz, 2H), 8.13 (d, J = 4.5 Hz, 2H), 8.04\u20137.94\n (m, 7H), 7.92\u20137.88 (m, 2H), 7.60 (d, J = 7.0 Hz, 2H), 7.45 (q, 2H), 7.24\n (t, J = 7.0 Hz, 2H), 7.15 (d, J = 2.0 Hz, 1H), 6.93 (dd, J = 2.0, J = 8.5 Hz,\n 1H), 6.33 (d, J = 7.0 Hz, 2H), 4.48 (s, 2H). 13C NMR (DMSO\u2011d6, 125\n Fig. 5. Mechanism of 1alip inducing cell death in A549. MHz, Fig. 7b and SI): 158.44, 154.89, 151.41, 150.89, 149.19, 146.48,\n\n 8\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\n142.38, 139.57, 135.78, 134.57, 131.76, 131.52, 131.12, 130.57, buffer. The resulting lysate was then centrifuged at 12,000 g for a\n129.05, 128.73, 126.24, 124.77, 122.38, 120.23, 119.73. HRMS duration of 20 min. Determination of protein concentration within the\n(CH3CN): m/z = 938.1209 ([M \u2212 PF6]+). supernatant was accomplished using the BCA kit (Beyotime Biotech,\n Shanghai, China). Equal quantities of proteins were subjected to frac\u00ad\n4.3. Preparation of 1alip, 1blip and 1clip tionation through SDS-polyacrylamide gel electrophoresis (SDS-PAGE).\n Subsequently, the proteins were transferred to polyvinylidene difluoride\n Liposomes were prepared based on the thin film hydration technique (PVDF) membranes (Millipore, MA, USA), which were subsequently\n[63]. A mixture of PC-98T 30 mg, CHO-HP 6 mg, DSPE-mPEG2000 5 mg blocked using a 5 % non-fat milk solution in TBST (20 mM Tris-HCl, 150\nand iridium(III) complex 1 mg was dissolved in 6 mL ethanol/chloro\u00ad mM NaCl, 0.05 % Tween 20, pH 8.0) buffer for a duration of 1 h. The\nform (1:2, v/v). The mixture was then transferred to a pear-shaped flask. membranes were then subjected to an overnight hybridization with\nThe organic reagent was dried in a 328 K water bath using a rotary primary antibodies. This was followed by an additional incubation with\nvacuum evaporator and wholly removed under vacuum for 3 h. In 328 K an appropriate secondary antibody conjugated with horseradish\nwater bath, 5 mL phosphate-buffered saline (PBS) was used to hydrate peroxidase. Finally, the protein expression levels were visualized uti\u00ad\nthe dried lipid membrane. In addition, the liposome suspension was lizing a chemiluminescence (ECL) detection kit.\nultrasonically treated with a 150W ultrasonic cell grinder for 6 min.\nThen the liposome was centrifuged at 11000 rpm for 10 min. Finally, the 4.8. Intracellular lipid peroxidation\nsupernatant was collected as liposomes and stored in 277 K\nrefrigerators. A549 cells were seeded in a 6-well plate overnight. Then the cells\n Note: The experimental procedures for determination of encapsula\u00ad were incubated with 1a, 1alip, 1blip and 1clip for a period of 24 h. Post-\ntion efficiency, liposome size and zeta potential, drug release, cell cul\u00ad treatment, the cells underwent triple washing with PBS and were then\nture, cellular uptake, mitochondrial localization, mitochondrial stained with BODIPY-C11 581/591 (2.5 \u03bcM) for 30 min at 37 \u25e6 C. The\nmembrane potential, mitochondrial permeability transition pore, subsequent imaging of cells was gained under a fluorescence micro\u00ad\nintracellular ROS, Ca2+ levels, RNA sequencing analysis, glutathione scope, while the determination of fluorescence intensity was conducted\n(GSH) and malondialdehyde (MDA) measurement can be found in the using flow cytometry.\nsupplementary materials.\n 4.9. Ferrostatin-1 (Fer-1) assay\n4.4. In vitro cytotoxicity assessment\n In the Fer-1 assay, A549 cells were initially cultured in a 96-well\n To evaluate the cytotoxic potential of 1a, 1b, 1c, 1alip, 1blip, and plate for a duration of 24 h. The cells within the 96-well microarray\n1clip, a widely employed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte\u00ad plate were subsequently divided into distinct groups, including control,\ntrazolium bromide (MTT) assay [34] was conducted. Cancer cell lines control + Fer-1, 1a, 1a + Fer-1, 1alip, 1alip + Fer-1, 1b, 1blip + Fer-1,\nwere seeded into 96-well microarray plates at a density of 1 \u00d7 104 cells 1cip, and 1clip + Fer-1. Following a 24-h interval, the cells were treated\nper well. A range of distinct concentrations of the complexes were added with MTT for 4 h. Eventually, 100 \u03bcL dimethyl sulfoxide was introduced\nto the plates. Following 48 h of incubation, a fresh serum-free medi\u00ad to dissolve the residual MTT, determining the absorbance at 490 nm.\num-MTT (9:1, v/v) solution was added to each well. The cells were\nsubsequently incubated at 37 \u25e6 C for an additional 4 h. Finally, Dimethyl 4.10. Cell morphology and lactate dehydrogenase release\nsulfoxide was applied to dissolve MTT followed by measurement of the\nabsorbance at 490 nm. In the pursuit of cell morphology and lactate dehydrogenase (LDH)\n release investigations, A549 cells were initially seeded in a 6-well plate.\n4.5. Cell cycle arrest studies This was followed by an overnight incubation at 37 \u25e6 C. The cells were\n then treated with 1a, 1alip, 1blip, and 1clip for 24 h. Afterwards, the cell\n A549 cells were seeded in a 6-well plate at a density of 5 \u00d7 105 cells morphology was meticulously observed under an inverted light\nper well and allowed to adhere overnight. Subsequently, the cells were microscope.\ntreated with 1a, 1alip, 1blip, and 1clip for a duration of 24 h. Following For LDH release studies, cells cultured in a 96-well plate were co-\ntreatment, the cells were collected through trypsinization, then rinsed incubated with 1a, 1alip, 1blip, and 1clip for a period of 24 h.\nwith cold PBS, and finally fixed with 70 % ethanol overnight. The next Following this treatment, the control groups were exposed to an LDH-\nsteps involved the addition of 0.1 % Triton X-100, 20 \u03bcL of RNAse (0.2 release solution for an hour. Thereafter, the supernatant from all wells\nmg/mL), and 20 \u03bcL of PI (propidium iodide) solution (0.02 mg/mL) to was transferred to new wells and mixed with LDH detection working\nthe cell suspensions. This mixture was incubated at 37 \u25e6 C for 30 min, solution at 25 \u25e6 C for 30 min, with precautions taken to avoid light. The\nafter which the cells were analyzed utilizing FACSCalibur flow absorbance was measured at 490 nm.\ncytometry.\n 4.11. In vivo anti-tumor experiment\n4.6. Apoptosis assay using Annexin V-FITC/PI staining\n Given the evident toxicity of 1alip to A549 cells in vitro, the in vivo\n A549 cells were cultured into a 6-well plate overnight, the cells were antitumor efficiency of 1alip was investigated using an A549 xenografts\nexposed to 1a, 1alip, 1blip, and 1clip for a duration of 24 h. Post- model. Experimental mice were provided by Guangdong Medical Lab\u00ad\ntreatment, the cells were detached through trypsinization, followed by oratory Animal Center (Guangzhou, China). All experimental proced\u00ad\nstaining using the Annexin V-FITC/PI Assay Kit. Flow cytometry analysis ures abided by the rules and regulations drawn up by the Animal Ethics\n(Beckman Instruments, NJ) was then employed to evaluate the stained Committee of Guangdong Pharmaceutical University. The cells were\ncells. cultivated in a thermostatic incubator at 37 \u25e6 C under 5 % CO2 and 100 %\n humidity. The mice were randomized into three groups (n = 6). To\n4.7. Western blotting analysis establish the tumor model, A549 cells were subcutaneously injected into\n the flank of each mouse. Post 7 days, intraperitoneal (i.p.) administra\u00ad\n Following treatment with 1a, 1alip, 1blip, and 1clip, exponentially tion of 1alip (2.1 mg/kg and 1.4 mg/kg) was initiated, spanning from\ngrowing A549 cells underwent a series of methodical steps. The cells days 1\u20139. The control group received injections of normal saline\nwere initially washed with cold PBS and subsequently lysed using lysis exclusively. The tumor size and the mice\u2019s weights were measured and\n\n 9\n\fC. Huang et al. European Journal of Medicinal Chemistry 265 (2024) 116112\n\n\nrecorded daily. Upon completion of the administration period, half of [13] X.C. Liu, Z. Wang, X.R. Zhang, X.C. Lv, Y. Song, R.X. Dong, G.X. Li, X.Y. Ren, Z.\n Y. Ji, X.A. Yuan, Z. Liu, Configurationally regulated half-sandwich iridium(III)-\nthe mice from each group were euthanized, while the remaining mice\n ferrocene heteronuclear metal complexes: potential anticancer agents, J. Inorg.\nwere observed daily, and the tumor size and body weight of mice were Biochem. 249 (2023) 112393.\nrecorded. The lung, heart, liver, brain, and spleen were collected. [14] S.Y. Guo, F.M. Wei, J. Karges, Y.K. Zhao, L.N. Ji, H. Chao, Cancer cell membrane-\n camouflaged and h2o2-activatable nanocomposites for synergistic chemotherapy\n and two-photon photodynamic therapy against melanoma, Inorg. Chem. Front. 10\n4.12. Data analysis (2023) 2716\u20132730.\n [15] J. Chen, H.M. Liu, Y.C. Chen, H.Y. Hu, C.X. Huang, Y. Wang, L.J. Liang, Y.J. Liu,\n Iridium(III) complexes inhibit the proliferation and migration of bel-7402 cells\n Data were presented as mean \u00b1 SD. Statistical treatment was per\u00ad through the pi3k/akt/mtor signaling pathway, J. Inorg. Biochem. 241 (2023)\nformed using a t-test. Differences were considered significant at *p < 112145.\n0.05. [16] J. Bonelli, E. Ortega-Forte, G. Vigueras, M. Bosch, N. Cutillas, J. Rocas, J. Ruiz,\n V. Marcha\u0301n, Polyurethane-polyurea hybrid nanocapsules as efficient delivery\n systems of anticancer Ir(III) metallodrugs, Inorg. Chem. Front. 9 (2022)\nCRediT authorship contribution statement 2123\u20132138.\n [17] Y.H. Yuan, Y.Y. Zhang, J. Chen, C.X. Huang, H.M. Liu, W.L. Li, L.J. Liang, Y. Wang,\n Y.J. Liu, Synthesis, biological evaluation of novel iridium(III) complexes targeting\n Chunxia Huang: Writing \u2013 original draft, Methodology, Investiga\u00ad mitochondria toward melanoma b16 cells, Eur. J. Med. Chem. 247 (2023) 115046.\ntion. Yuhan Yuan: Methodology, Investigation. Gechang Li: Formal [18] A.R. Rubio, J. Fidalgo, J. Martin-Vargas, C. Pe\u0301rez-Arnaiz, S.R. 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