Endocytic Uptake of Self-Assembled Iridium(III) Nanoaggregates for Holistic Treatment of Metastatic 3D Triple-Negative Breast Tumor Spheroids.

{"full_text": "RESEARCH ARTICLE\n www.small-journal.com\n\n\nEndocytic Uptake of Self-Assembled Iridium(III)\nNanoaggregates for Holistic Treatment of Metastatic 3D\nTriple-Negative Breast Tumor Spheroids\nAyushi Chaudhary, Ashwini Kumar, Nikhil Swain, Kajal Chaudhary, Himanshu Sonker,\nSayari Dewan, Rutwik Anil Patil, and Ritika Gautam Singh*\n\n progesterone receptor, ER: estrogen recep-\nTriple-negative breast cancer (TNBC) presents a formidable challenge due to tor, or HER-2: human epidermal growth\nits aggressive behavior and limited array of treatment options available. This factor receptor-2 status is indispensable\nstudy focuses on employing nanoaggregate material of organometallic Ir(III) for the evaluation and management of\ncomplexes for treating TNBC cell line MDA-MB-231. In this approach, Ir(III) breast cancer treatment.[2] Triple-negative\n breast cancer (TNBC) represents a sub-\ncomplexes with enhanced cellular permeability are strategically designed and\n type characterized by its aggressive clin-\nachieved through the incorporation of COOMe groups into their structure. ical behavior compared to other breast\nThe lead compound, IrL1 , exhibits promiscuous nanoscale aggregation in cancer subtypes.[3] TNBC comprises tu-\nRPMI cell culture media, characterized by a stable hydrodynamic e\ufb00ective mors with negative expression status for\ndiameter ranging from 190 to 202 nm over 48 h. With excellent ER, PR, and HER-2 necessitating spe-\n cialized therapeutic inventions.[4,5] TNBC\nphoto-responsive contrast-enhanced cell imaging properties IrL1 exhibits an\n constitutes \u224810\u201320% of all breast cancer\noutstanding IC50, 48h value of 36.05\u00b1 0.03 nm when irradiated with 390 nm cases and is frequently detected in pa-\nlight in MDA-MB-231 (IC50, 48 h of Cisplatin is 5.29 \u03bc\u03bc). In cell, investigation tients aged 35 years or younger.[6] It rep-\ncon\ufb01rms that IrL1 nanoaggregates internalization via energy-dependent resents a unique form of breast cancer\nendocytosis undergo ferroptosis and ROS mediated cell death in with the poorest prognosis, contributing to\nMDA-MB-231 cells. Further, these in vivo studies using NOD-SCID mice 15% of all breast cancer diagnoses and ac-\n counting for 25% of related mortalities.[7]\ncon\ufb01rmed that IrL1 exhibits a tendency to ablate tumors inoculated in mice\n Owing to its notable propensity for\nmodels at therapeutically relevant doses. Thus, this comprehensive approach metastasis, resistance to traditional\nholds promise for expanding the repertoire of organometallic Ir(III) chemotherapy and endocrine treatments\nnanoaggregates with adaptable characteristics, thereby advancing their poses signi\ufb01cant clinical challenges. Small\nclinical utility of nanomedicine in the holistic treatment of metastatic molecule targeted therapy involves the\n development of therapeutic drugs tai-\n3D triple-negative breast tumor spheroids.\n lored at the molecular level to speci\ufb01cally\n target de\ufb01ned carcinogenic markers, fa-\n cilitating tumor cell death upon binding\n to these targets. Targeted treatment has\n1. Introduction emerged as a key focus in the treatment of TNBC, leading to\n the initiation of numerous targeted drug trials. Despite the rapid\nBreast cancer is one of the most widespread malignancies world-\n progress in these developments, the inherent drawback of single-\nwide, explicitly a\ufb00ecting women. Employing gene expression\n target drug discovery makes TNBC vulnerable to drug resis-\npro\ufb01ling and molecular biology techniques breast cancer can be\n tance. TNBC\u2019s tendency for recurrence and distant metastasis\ndivided into \ufb01ve distinct clinical subtypes based on histopatho-\n contributes to an unfavorable prognosis for patients.[8]\nlogical characteristics.[1] Tumor classi\ufb01cation based upon PR:\n Hence, a rising avenue to tackle this issue includes the de-\n velopment of combination drug strategies, including dual target\nA. Chaudhary, A. Kumar, N. Swain, K. Chaudhary, H. Sonker, S. Dewan, drugs (designed to address two speci\ufb01c targets simultaneously)\nR. A. Patil, R. G. Singh and combinations of di\ufb00erent drugs. Despite notable progress in\nDepartment of Chemistry apoptosis-based chemotherapy in recent decades,[9,10] the overall\nIIT Kanpur\n e\ufb03cacy of the current antitumor medications and survival rates\nKanpur, UP 208016, India\nE-mail: rgautam@iitk.ac.in of patients with malignant tumors still fall below expectations\n due to inherent apoptosis resistance observed in various tumor\n The ORCID identi\ufb01cation number(s) for the author(s) of this article types, notably TNBC. This underscores the urgent need for devel-\n can be found under https://doi.org/10.1002/smll.202406809 oping non-apoptotic treatment strategies to e\ufb00ectively eradicate\nDOI: 10.1002/smll.202406809 tumor cells.\n\n\nSmall 2025, 21, 2406809 2406809 (1 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 1. Previously reported Ir(III)-based medications with a potential to treat Triple-Negative Breast Cancer (TNBC).\n\n\n Ferroptosis, a recently recognized mode of regulated cell systemic toxicity. A variety of prodrug systems have been de-\ndeath (RCD) is characterized by the abnormal accumulation of veloped to address this challenge, including nanoparticles, lipo-\ncytotoxic-iron mediated lipid peroxides. Unlike extensively stud- somes, and polymers. Among these, polymeric systems are often\nied RCD forms like necrosis and apoptosis, ferroptosis has gar- favored due to their ability to incorporate numerous functional\nnered signi\ufb01cant attention for its potential in treating apoptosis- groups. This versatility enables them to mitigate issues related to\nresistant tumors.[11] poor pharmacokinetics, inappropriate distribution, and low solu-\n Recently, metal-based medications have demonstrated poten- bility, thereby enhancing drug clearance rates. To overcome these\ntial in treating TNBC, including several organometallic com- challenges, we designed, synthesized, and characterized a se-\npounds. Clinical trials predominantly explore the combinations ries of novel organometallic Ir(III) complexes-based nanoaggre-\nof conventional chemotherapy agents like cisplatin, carboplatin, gates with the inclusion of methyl ester (-COOMe) groups aims\noxaliplatin, gemcitabine, paclitaxel, or lxabepilone along with in- to enhance the lipophilicity and cell permeability of the com-\nhibitors targeting growth factor receptors such as Erlotinib, ki- plexes. Many FDA-approved drugs, such as oseltamivir, aspirin,\nnase inhibitors (like Everolimus), check-point inhibitors (such as and enalapril, utilize esteri\ufb01cation to improve bioavailability and\nImprime), or antibodies (such as Bevacizumab, Cetuximab).[12] absorption. Moreover, \ufb02uorescent dyes such as calcein-AM, \ufb02uo-\n Organometallic Ir(III) complexes have been extensively stud- rescein diacetate, and Fluo-3 AM are commonly used in esterase-\nied, for their potential as anticancer agents, utility in contrast- mediated delivery inside cells. Based on these well-established\nenhanced cell imaging, and labeling of peptides.[13\u201317] How- precedents, we designed our Iridium(III) complexes incorporat-\never, the e\ufb00ectiveness and mechanisms of iridium compounds ing methyl ester groups, with the expectation that they would\nin treating TNBC remain unclear. A preliminary study utiliz- contribute to enhanced cellular uptake and lipophilicity.[21b\u2013d] No-\ning organo-iridium (III) complexes synthesized by Sadler et. tably, these Ir(III) complexes exist as in situ generated nanoscale\nal exhibited enhanced anti-proliferative properties in TNBC aggregates in biological cell culture media. These nanoaggre-\ncell lines MDA-MB-468 and OCUB-M during screening with gates tend to co-localize in the endoplasmic reticulum and mito-\nthe National Cancer Institute (NCI) 60 cell line, particu- chondria and exhibit unique photophysical, biological, and phar-\nlarly the iridium-phenylazopyidine complex.[18] In 2021 He macological behavior that can be utilized for therapeutic advan-\net al. illustrated the combined impact of ferroptosis and apop- tage. Our study indicates that the core of Ir(III) nanoaggregates\ntosis in hindering the growth of triple-negative MDA-MB- is a highly conjugated system that absorbs light, enabling e\ufb03-\n231 cells by utilizing mitochondria-targeting Ir(III) cyclometa- cient energy transfer to generate ROS. These ROS with tumor or\nlated compounds.[19] In 2022, Chanda and coworkers synthe- subcellular organelle targeting properties can alter cellular up-\nsized half-sandwich cyclometalated Ir(III) complexes, inducing take, organ accumulation, and many aspects of pharmacokinet-\nmitochondrion-mediated apoptosis in the TNBC cell line MDA- ics and pharmacodynamics (PK/PD). The prodrug Ir(III) nanoag-\nMB-468.[20] A recent study by Brabec et al. in 2023 underscored gregates generated ROS depletes intracellular GSH and inhibits\nthe e\ufb00ectiveness of octahedral Ir(III) complexes in e\ufb00ectively in- glutathione peroxidase 4, a GSH-dependent hydroperoxidase, to\nhibiting metastatic processes in the triple-negative breast cancer cause lipid peroxidation and eventually act as a ferroptosis-based\ncell line MDA-MB-231 (Figure 1).[21a] cell death inducer in TNBC cell line MDA-MB-231. Addition-\n However, despite its several bene\ufb01ts, Iridium(III) based com- ally, the remarkable imaging potential of these Ir(III) nanoag-\nplexes face signi\ufb01cant challenges such as poor uptake, lack of gregates assists in monitoring the dynamic behavior of subcel-\nspeci\ufb01city, extensive biological distribution, brief half-life, and lular organelles and the changes in the microenvironment. The\n\n\nSmall 2025, 21, 2406809 2406809 (2 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 2. Schematic illustration of IrL1 nanoaggregates unveiling ferroptosis mediated cell death in 4T1 tumor-bearing mice.\n\n\nanticancer response of IrL1 has been well studied including the lipophilicity and cell permeability of the complexes.[24] Addi-\nmechanism of cellular uptake, its e\ufb00ect on other cellular or- tionally, these COOMe groups serve as functionalization han-\nganelles, and in vivo investigations (Figure 2) circumventing the dles, facilitating the attachment of targeting ligands that can\nlimitations associated with using doxorubicin in conventional speci\ufb01cally bind to receptors overexpressed on cancer cell sur-\nTNBC chemotherapy. Finally, our comprehensive research initia- faces, thereby minimizing o\ufb00-target e\ufb00ects, and enhancing ther-\ntive has capabilities to transcend the con\ufb01nes of our laboratory, apeutic e\ufb03cacy. MDA-MB-231 cells lack major receptors that\nheralding a pivotal step toward the ability to combine both ther- would directly interact with ester groups. However, these cells\napeutic and diagnostic capabilities into a single nanoaggregate- do express carboxylesterase enzymes capable of hydrolyzing es-\nbased complex and advancing the frontiers of nanomedicine with ter bonds, which could convert prodrugs into their active or inac-\na profound clinical implication. tive forms. Moreover, the integration of the phenanthroline moi-\n ety provides a versatile platform, endowing the complexes with\n2. Result and Discussion a high a\ufb03nity for DNA binding through intercalation or groove\n binding.[25] Detailed synthesis procedures and characterization\n2.1. Design and Synthesis of Ir(III) Complexes data for both ligands and complexes are provided in the sup-\n porting Information (Figures S1\u2013S24, Supporting Information).\nOur approach focused on the meticulous design and synthesis of All complexes (Figure 3) were thoroughly characterized by vari-\nIridium(III) cyclometalated complexes, motivated by their out- ous spectroscopic techniques, 1 H and 13 C{1 H} NMR, FT-IR, UV\u2013\nstanding anticancer activity. Additionally, we aimed to leverage vis, and ESI-MS, and their purity was assessed by RP-HPLC and\ntheir potential for precise organelle targeting and advanced in- determined to be > 95%. The FT-IR spectra of the metal com-\ntracellular imaging capabilities.[22,23] To accomplish this objec- plexes are shown in Figure S25, Supporting Information. The\ntive, we systematically synthesized a series of novel Ir(III) com- FT-IR spectra of Ir(III) complexes displayed bands at 550\u2013560\nplexes using L1 \u2013L8 ligands as shown in Figure 3. The signi\ufb01- cm\u22121 v(P\u2500F) 1400\u20131590 cm\u22121 \ud835\udf08(C\u2550Caromatic ), 2905\u20132925 cm\u22121\ncance of these synthesized complexes lies in their meticulously \ud835\udf08(C\u2550Npy ), 1715\u20131735 cm\u22121 \ud835\udf08(C\u2550O), 3380\u20133395 cm\u22121 \ud835\udf08(N\u2500H).\nchosen molecular moieties, as illustrated in Figure 3. The lig- Notably, the complexes exhibited solubility in common organic\nands were synthesized from Suzuki coupling using 2-bromo- solvents including DMSO, DMF, MeOH, CH3 CN, CH2 Cl2 , and\nisonicotinic acid and boronic acids with substituted groups at THF. Crystallographic data for ligand L3 and intermediate dimer\nX and Y. Within the ligand sca\ufb00old, various biologically rele- complex Ir2 Cl2 (L3 )4 are summarized in Tables S3 and S5 (Sup-\nvant groups were strategically incorporated. Notably, the inclu- porting Information) the selected bond angles and bond lengths\nsion of methyl ester (-COOMe) groups aims to enhance the are provided in Tables S4 and S6 (Supporting Information). The\n\n\nSmall 2025, 21, 2406809 2406809 (3 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 3. Design and synthesis scheme of Ir(III) complexes.\n\n\nDFT calculations have also been performed on all the complexes 550\u2013590 nm. The emission property indicates that Ir(III) com-\nand the molecular orbital (MO) distribution is represented in plexes can be utilized for intracellular imaging and as a diagnostic\nFigure 4. sensor.\n\n\n2.2. Photophysical Properties in Solution 2.3. Stability and Lipophilicity Pro\ufb01le\n\nThe absorption spectra of ligands and their corresponding com- The stability of Ir(III) complexes was assessed using UV\u2013vis ab-\nplexes in DMSO at 298 K are reported in Figures S26 and S27 sorption spectroscopy in a tris-bu\ufb00er solution at pH 7.4. As de-\n(Supporting Information). The 290\u2013350 nm spectral window dis- picted in Figure S29 (Supporting Information), the UV\u2013vis spec-\nplays the intense absorption bands (\ud835\udf16 \u2248 (0.5\u20132) \u00d7 105 M\u22121 cm\u22121 ) tra of IrL1 \u2013IrL8 showed a consistent pattern without any dis-\nthat represent spin-allowed ligand-centered (LC) \ud835\udf0b\u2013\ud835\udf0b* transi- cernible hypsochromic or bathochromic shifts observed at 0, 24,\ntions which involve both the (N\u02c4N) type ancillary ligands and and 48 h, suggesting that IrL1 \u2013IrL8 maintained stability in so-\nthe (C\u02c4N) cyclometalated ligands.[26,27] On the other hand, the lution at room temperature over 48 h. Given that along with\nbands at longer wavelengths (450\u2013480 nm; \ud835\udf16 \u2248 (1\u20135) \u00d7 104 M\u22121 stability, the cellular uptake of the drugs is signi\ufb01cantly in\ufb02u-\ncm\u22121 ) correspond to the metal-to-ligand charge transfer transi- enced by factors such as water solubility, alongside mechanisms\ntions (MLCT).[28] The emission spectra of the Ir(III) complexes of cellular uptake and molecular size,[29] we initially examined\n298 K in DMSO are shown in Figure S28 (Supporting Informa- the lipophilicity index of Ir(III) complexes before embarking on\ntion), and the emission spectra of ligands in DMSO and 1X PBS anticancer studies. The lipophilicity of these compounds was\nare depicted in Figure S26 in Supporting Information. The lu- quanti\ufb01ed by their log Po/w values (partition coe\ufb03cient in n-\nminescence properties and photophysical parameters of Ir(III) octanol/water system), as detailed in Table S2 (Supporting Infor-\ncomplexes and ligands are summarized in Table 1. All the Ir(III) mation). The log Po/w values suggest that the Ir(III) complexes\ncomplexes displayed green emission with maxima between are moderately lipophilic in nature.\n\n\nSmall 2025, 21, 2406809 2406809 (4 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 4. Illustrations of Frontier molecular orbitals, HOMOs, and LUMOs of Ir(III) complexes determined by DFT at B3LYP functional along with\n6\u201331G** and LANL2DZ for Ir(III).\n\n\n2.4. IrL1 Nanoaggregates Formation and Characterization sembles the nanodots.[30] The selected particles have a spacing\n of 0.28 nm (Figure 5c.) Energy-dispersive X-ray (EDX)-TEM and\nAfter the characterization of the monomeric IrL1 complexes, the EDX-SEM analysis further con\ufb01rmed the presence of IrL1 within\ncorresponding nanoaggregates condition and formation were ex- these particles (Figures S39 and S40, Supporting Information).\nplored. To comprehend the behavior of the nanoaggregate forma-\ntion in the cell culture media, we delved into the phenomenon\nof self-assembly and conducted Dynamic Light Scattering (DLS) 2.5. Anticancer Pro\ufb01le of Ir(III) Complexes\nmeasurements on a solution of IrL1 in RPMI media at a \ufb01-\nnal concentration of 3 \u03bc\u03bc. Remarkably, our \ufb01ndings unveiled Following detailed solid state and solution characterization of\nthe immediate formation of self-assembly of IrL1 , characterized Ir(III)-nanoaggregates, the complexes IrL1 \u2013IrL8 were screened\nby a hydrodynamic e\ufb00ective diameter ranging from 190 to 202 against a series of cancer cell lines namely, breast adenocarci-\nnm. Importantly, this self-assembled state remained unchanged noma cell lines MDA-MB-231 and MCF-7, Human lung adeno-\nover a period of 48 h (Figure 5). For a comprehensive under- carcinoma cell line A549, human prostate cancer cell line PC3,\nstanding of the aggregation, the solution of IrL1 in RPMI me- Human colon cancer cell line HCT-116, and normal human em-\ndia was examined using scanning electron microscopy (SEM) bryonic kidney cell line HEK-293, using FDA approved cisplatin\nand high-resolution transmission electron microscopy (HRTEM) as the positive control. Table 2 demonstrates the IC50,48h value of\ntechniques. In solution, IrL1 exhibited aggregated particles with the Ir(III) complexes determined by the MTT assay. In the an-\nan average size of \u224895\u2013140 nm (Figure 5b). The observed mor- tiproliferative assay IrL1 displayed highest cytotoxicity with IC50\nphology of IrL1 nanoaggregates that appeared in the HRTEM re- value on MDA-MB-231 (0.33 \u03bc\u03bc), A549 (0.82 \u03bc\u03bc), MCF-7 (0.64\n\nTable 1. Absorption and emission spectra of ligands and their corresponding Ir(III) complexes were recorded in DMSO at room temperature. The\nemission spectra of Ir(III) complexes were recorded upon excitation (400\u2013460 nm) in the MLCT region. Quantum yields (\u03a6) were measured in DMSO\nat room temperature using \ufb02uorescein (\u03a6 = 0.79, 0.1 m NaOH) as the reference.\n\nCompound \ud835\udf06abs, nm [\ud835\udf16/ 103 M\u22121 cm\u22121 ] \ud835\udf06em, nm Compound \ud835\udf06abs, nm [\ud835\udf16/ M\u22121 cm\u22121 ] \ud835\udf06em, nm \u03a6\n\nL1 302 (5.50) 356 IrL1 327 (1.70 \u00d7 105 ), 476 (5.0 \u00d7 104 ) 586 0.01\nL2 303 (5.01) 387 IrL2 331 (1.45 \u00d7 105 ), 475 (3.19 \u00d7 104 ) 581 0.01\nL3 296 (7.32) 362 IrL3 295 (1.53 \u00d7 105 ), 469 (2.64 \u00d7 104 ) 561 0.04\nL4 237 (6.42) 418 IrL4 325 (1.91 \u00d7 105 ), 477 (4.11 \u00d7 104 ) 589 0.02\nL5 303 (6.61) 392 IrL5 328 (5.84 \u00d7 104 ), 473 (1.32 \u00d7 104 ) 584 0.01\nL6 291 (7.78) 435 IrL6 329 (1.25 \u00d7 105 ), 475 (3.23 \u00d7 104 ) 581 0.09\nL7 291 (7.73) 393 IrL7 327 (1.33 \u00d7 105 ), 473 (3.45 \u00d7 104 ) 587 0.01\nL8 320 (5.31) 479 IrL8 327 (1.11 \u00d7 105 ), 477 (3.51 \u00d7 104 ) 558 0.0007\n\n\n\n\nSmall 2025, 21, 2406809 2406809 (5 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 5. DLS data at di\ufb00erent time intervals of a solution of IrL1 in RPMI cell culture media. (SEM images of IrL1 nanoaggregates and HRTEM images\nof IrL1 nanoaggregates are shown in Supporting Information).\n\nTable 2. Half-maximum (50%) Inhibitory concentrations (IC50 , \u03bc\u03bc) of IrL1 \u2013IrL8 determined by the MTT assay after exposure of Ir (III) complexes for\n48 h.\n\nCell growth inhibition, IC50 (\u03bc\u03bc), 48 h\n\nCompounds MDA-MB-231 MCF-7 A549 PC3 HCT-116 HEK-293\n\nIrL1 0.33 \u00b1 0.05 0.64 \u00b1 0.04 0.82 \u00b1 0.19 0.51 \u00b1 0.30 1.27 \u00b1 0.15 2.45 \u00b1 0.87\nIrL2 2.50 \u00b1 1.70 1.13 \u00b1 0.33 4.81 \u00b1 1.66 4.85 \u00b1 1.42 1.60 \u00b1 0.07 0.96 \u00b1 0.42\nIrL3 >25 >25 >25 >25 >25 >25\nIrL4 1.48 \u00b1 1.14 1.78 \u00b1 0.16 0.96 \u00b1 1.48 6.02 \u00b1 3.84 6.19 \u00b1 1.98 1.83 \u00b1 1.46\nIrL5 >25 >25 >25 >25 >25 >25\nIrL6 1.0 \u00b1 0.47 0.47 \u00b1 0.07 1.74 \u00b1 0.45 0.97 \u00b1 5.08 2.00 \u00b1 0.43 2.06 \u00b1 2.18\nIrL7 >25 >25 >25 >25 >25 >25\nIrL8 >25 >25 >25 >25 4.09 \u00b1 2.17 >25\nCisplatin 5.29 \u00b1 0.25 7.0 \u00b1 4.51 11.12 \u00b1 2.55 3.13 \u00b1 0.22 5.38 \u00b1 2.57 2.66 \u00b1 0.71\n\n\n\n\n\u03bc\u03bc), HCT-116 (1.27 \u03bc\u03bc) and PC3 (0.51 \u03bc\u03bc) compared to rest of same conditions of light irradiation on healthy kidney cell line\nthe Ir(III) complexes, indicating that IrL1 is more cytotoxic than HEK-293. The results obtained indicated no pronounced e\ufb00ect of\ncisplatin. IrL1 displayed signi\ufb01cant cytotoxicity on the MDA-MB- light irradiation on the HEK-293 cell line. However, the cytotoxic-\n231 cell line, compared to other screened cancer cells, indicating ity of the compound increased \u224810 times on MDA-MB-231 cells.\nits speci\ufb01city in cytotoxicity on the triple-negative breast cancer These results display the selectivity of the complex toward cancer\ncell line. The photocytotoxicity of IrL1 was also investigated on cells upon light irradiation. (Figure S52 and Table S8, Supporting\nMDA-MB-231 cells. Upon exposure to 390 nm light (0.363 mW Information). Conversely IrL1 showed comparable antiprolifera-\ncm\u22122 25 min), IrL1 demonstrated exceptional photocytotoxicity tive activity with cisplatin on HEK-293 (IrL1 , IC50, 48 h = 2.45 \u03bc\u03bc;\nwith an IC50,48 h value of 36.05 \u00b1 0.03 n\u03bc on MDA-MB-231 cells. Cisplatin, IC50, 48 h = 2.66 \u03bc\u03bc). Hence, IrL1 was solicited for fur-\n(Figure 6). We conducted the cytotoxicity experiment under the ther mechanistic studies (Figure S41, Supporting Information).\n The e\ufb03cacy of the drugs is a\ufb00ected by their ability to be\n taken up by cellular structures. We discovered that IrL1 had a\n more pronounced cytotoxic e\ufb00ect on MDA-MB-231 cells com-\n pared to other cell types. Consequently, for our further in-\n vestigations, we focused on the intracellular uptake of IrL1\n and IrCl3 .xH2 O in MDA-MB-231 cells using inductively cou-\n pled plasma mass spectrometry (ICP-MS). After an 8 h treat-\n ment with IrL1 , we observed a signi\ufb01cant increase in intracel-\n lular Iridium content in MDA-MB-231 cells compared to the\n control group and cells treated with IrCl3 .xH2 O (Figure 7b).\n The order of cellular uptake was IrL1 > IrCl3 .xH2 O > con-\n trol. These results clearly indicate that the cellular uptake of\n IrL1 is notably higher than that of the corresponding metal salt\n IrCl3 .xH2 O. Furthermore, our \ufb01ndings suggest a correlation be-\n tween the antiproliferative activity of IrL1 and its cellular uptake\nFigure 6. Percentage cell viability of MDA-MB-231 cells treated with IrL1 e\ufb03ciency, as seen in both cytotoxicity assays and cellular uptake\nunder light exposure (390 nm, 0.363 mW cm\u22122 , 25 min). measurements.\n\n\n\nSmall 2025, 21, 2406809 2406809 (6 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 7. The Cellular uptake mechanism of IrL1 . a) MDA-MB-231 cells were stained with Ir(III) complex (1.65 \u03bcm, 2 h) under di\ufb00erent conditions by\nvarying the temperature (4 \u00b0C and 37 \u00b0C) as well as the addition of metabolic inhibitor (CCCP) and endocytic inhibitors (chloroquine and NH4 Cl). Scale\nbar: 25 \u03bcm. b) ICP-MS assay of total cell uptake after treatment with 6.6 \u03bcm IrL1 and IrCl3 .xH2 O in MDA-MB-231 cell line for 8 h at 37 \u00b0C. The untreated\ncells were taken as the control. Each data point represents mean \u00b1 SD, n = 3. **p < 0.01.\n\n\n2.6. Mechanism of Cellular Uptake IrL6 were found to possess aggregation in the range 200\u2013400\n nm, whereas the remaining metal complexes possessed hydro-\nTo identify the speci\ufb01c uptake mechanism and to explore the dynamic diameter >1000 nm in the culture media. Also, in the\npathway by which the drug reaches its intended target within the previous cytotoxicity experiment conducted, we found that these\ncell we extended our study to investigate the mechanism of cel- complexes which are found to form nanoaggregates displayed\nlular uptake. Drugs can be taken up by di\ufb00erent pathways like signi\ufb01cant cytotoxicity compared to the remaining ones. Thus,\npassive di\ufb00usion, active transport, endocytosis, facilitated di\ufb00u- to investigate this reason so as to why these complexes displayed\nsion, and macropinocytosis but for nanoaggregates, they gener- such behavior, the DLS investigation of the active compounds in\nally enter cells by endocytosis via energy-dependent or energy- the culture medium in the absence of FBS was performed (Figure\nindependent pathways.[31a] Although we have incorporated dif- S49, Supporting Information), as it is documented in the litera-\nferent functional groups, the lipophilicity results indicate mod- ture that the serum proteins in the culture medium can stabilize\nerate lipophilicity of all the complexes. Thus, we ruled out their the nanoaggregates formation.[31b] The results obtained indicated\npossibility to enter the cells by passive di\ufb00usion. Therefore, we that in the absence of FBS, particles with hydrodynamic diame-\nmoved on to investigate the cellular uptake mechanism via active ter >1000 nm were formed, resulting in the precipitation of these\ntransport; energy-dependent uptake, and endocytosis. For this complexes, indicating the role of serum proteins in the formation\npurpose, we incubated MDA-MB-231 cells with IrL1 at 4 \u00b0C and and stabilization of the nanoaggregates. Thus, these nanoaggre-\ntreated them with the metabolic inhibitor (CCCP). It results in gates were formed through \ud835\udf0b\u2013\ud835\udf0b stacking and were stabilized in a\na decreased \ufb02uorescence of IrL1 compared with the IrL1 treated culture medium with the help of serum proteins, in the absence\ncells at 37 \u00b0C, this indicates the energy-dependent mechanism. of which these complexes precipitated.\nThe decreased \ufb02uorescence was also observed when cells were\npretreated with the endocytosis inhibitor NH4 Cl and chloroquine\n(Figure 7a). NH4 Cl and chloroquine are weak bases that can ac- 2.8. Rationale for Nanoaggregate Formation\ncumulate within the acidic vesicles and result in raising the pH\nof these vesicles. By increasing the pH both substances can inter- The formation of nanoaggregates is a complex process gov-\nfere with the normal acid-dependent processes that are crucial for erned by a range of non-covalent interactions, including hydro-\nendocytosis. As a result, the endocytosis was inhibited, and the gen bonding, hydrophobic forces, electrostatic interactions, \ud835\udf0b\u2013\ud835\udf0b\nresults obtained indicated that IrL1 enters MDA-MB-231 cells by stacking, and van der Waals forces.[31c,d] These forces collectively\nthe energy-dependent endocytosis pathway. drive the self-assembly of small molecules, with each type of in-\n teraction playing a dominant role depending on the molecular\n system under study. In the context of our research, the observed\n2.7. Self-Assembly Behavior di\ufb00erences in nanoaggregation behavior among the complexes\n IrL1 , IrL2 , IrL4 , IrL6 , and others can be primarily attributed to vari-\nTo better understand the self-assembly, nanoaggregates forma- ations in their hydrophobicity and \ud835\udf0b\u2013\ud835\udf0b interactions. Lipophilicity\ntion, and the role of media proteins in the stabilization of these data (Table S2, Supporting Information) indicate that IrL1 , IrL2 ,\nnanoaggregates, we performed DLS, HR-TEM, and DLS of all and IrL6 , containing unsubstituted, tert-butyl, and ethoxy func-\nthe IrL1 \u2013IrL8 complexes in RPMI media without FBS protein. As tional groups respectively, exhibit greater hydrophobicity com-\nshown by the DLS and TEM data provided in Figures S46\u2013S48 pared to the other complexes. In aqueous environments such\n(Supporting Information), the complexes IrL1 , IrL2 , IrL4 , and as cell culture media, these hydrophobic interactions drive the\n\n\nSmall 2025, 21, 2406809 2406809 (7 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\naggregation process, as the molecules tend to cluster to minimize of glutathione-dependent antioxidant enzyme glutathione perox-\ntheir exposure to the surrounding aqueous phase.[31e,f] On the idase 4 (GPX4).[32,33] We initiated our study by investigating the\nother hand, complexes such as IrL5 , IrL7 , and IrL8 are relatively electrochemical redox pro\ufb01le of IrL1 by cyclic voltammetry (CV)\nmore hydrophilic, likely due to the presence of polar functional in anhydrous acetonitrile. The compound IrL1 exhibited an irre-\ngroups, including formyl, hydroxyl, and methoxy moieties. Com- versible behavior during the anodic scans whereas it exhibited\nplexes IrL3 , IrL5 , IrL7 , and IrL8 displayed particle sizes exceeding reversible behavior during the cathodic scans. The irreversible\n1000 nm in the culture medium, possibly due to stronger inter- oxidation peak at +1.28 V was attributed to IrIV / IrIII as shown in\nmolecular hydrogen bonding, which resulted in poor solvation Figure S42 (Supporting Information).[34] The redox nature of IrL1\nand, consequently, larger aggregate formation. Hydrogen bond- was further con\ufb01rmed by ROS assay. As shown in the Figure 8c.\ning is known to have a dual role, potentially reducing or enhanc- IrL1 causes extensive ROS generation as observed by cell per-\ning drug dissolution rates depending on the system.[31g] The ten- meating dihydrodichloro\ufb02uorescein diacetate (DCFH-DA) that\ndency of IrL1 , IrL2 , IrL4 , and IrL6 to form nanoaggregates can be gets hydrolyzed intracellularly to produce a dihydrodichloro\ufb02uo-\nexplained by their higher lipophilicity and stronger \ud835\udf0b\u2013\ud835\udf0b stacking rescein (DCFH) probe, which is oxidized to produce the bright\ninteractions compared to the other complexes, thus rationalizing green, \ufb02uorescent product dichloro\ufb02uorescein (DCF). The in-\ntheir self-assembly behavior in our experimental conditions. crease in the DCF emission intensity was detected by the \ufb02uo-\n rescent cell imager after 3 h of IrL1 -treatment, suggesting ROS\n elevation with the increase in the concentration of IrL1 in the\n2.9. Intracellular Localization of IrL1 cells. However, no distinct increase in \ufb02uorescence intensity of\n DCF was detected by adding the ROS scavenger N-acetyl cysteine\nPhosphorescent iridium(III) complexes possess unique excited- (NAC) in advance, suggesting that IrL1 was indeed capable of\nstate properties including high luminescence quantum yields, generating ROS in the cells (Figure 8c; Figure S43, Supporting\ntunable phosphorescence wavelengths, large Stokes shifts, long Information). The BODIPY 581/591 C11 probe was used to de-\nphosphorescence lifetimes, and high chemical and photochemi- tect and quantify lipid peroxidation (LPO) using \ufb02uorescent mi-\ncal stability. This renders them suitable for a broad spectrum of croscopy and \ufb02ow cytometry. Oxidation of the polyunsaturated\napplications, encompassing cellular imaging reagents, labeling butadienyl portion of the dye results in a shift of the \ufb02uores-\nreagents, biosensors, cytotoxic drugs, and photosensitizers. To vi- cence emission peak from \u2248590 nm (red) to \u2248510 nm (green).[35]\nsualize the intracellular localization of IrL1 we utilize its emissive Notably, IrL1 caused increased lipid peroxidation as shown in\nproperty. Here, MDA-MB-231 cells were incubated with IrL1 , and Figure 8d,e. The antioxidant glutathione (GSH) is used as a de-\norganelle-speci\ufb01c staining dyes were utilized to examine intracel- fense tool by cancer cells as it can consume ROS to hinder cell\nlular localization (MitoTracker Deep Red, LysoTracker Deep Red, death.\nand ER-Tracker Red). The co-stain pattern of IrL1 matched with To measure the change in the GSH level in MDA-MB-231 af-\nthose of MitoTracker Deep Red, and ER-Tracker Red with the con- ter treatment with IrL1 , a GSH-Glo assay was performed. This\nsiderable value of Pearson correlation coe\ufb03cient (\u224890%) in the is the luminescence-based assay which is utilized for quanti\ufb01ca-\nscatter plot, which suggests preferential accumulation of IrL1 in tion and detection of GSH levels in cells. This assay relies on the\nthe mitochondria and endoplasmic reticulum (Figure 8a). conversion of luciferin derivative into luciferin in the presence\n of glutathione and this reaction is catalyzed by the glutathione\n S-transferase enzyme provided in the kit. The luciferin formed\n2.10. Investigation of Ferroptosis Mediated Cell Death Pathway is detected by its reaction with Ultra-Glo Recombinant luciferase\n (provided in the GSH-Glo Glutathione assay kit, Promega) that\nTo investigate the mode of cell death pathway, the cytotoxicity generates luminescence which is proportional to the amount of\nof IrL1 was determined in presence of various inhibitors mainly GSH present in the cell. As shown in Figure 8f, the GSH con-\nZ-VAD\u2013fmk (apoptosis inhibitor), cycloheximide (paraptosis in- centration was decreased signi\ufb01cantly in treated cells when com-\nhibitor), Necrostatin-1 (necroptosis inhibitor), NAC (ROS scav- pared to the control. Further to analyze the cause of ROS gener-\nenger), 3-methyl adenine (autophagy inhibitor), and ferrostatin-1 ation we detected the change in the expression of GPX4 by west-\n(ferroptosis inhibitor). The IC50, 24 h value of IrL1 increased from ern blot. GPX4, the glutathione-dependent enzyme, reduces lipid\n2.33 to 11.08 \u03bc\u03bc and 14.33 \u03bc\u03bc in the presence of ferrostatin-1 and hydroperoxides to lipid alcohol and limits the iron-dependent for-\nN-acetyl cysteine (NAC) respectively (Figure 8b), suggesting the mation of lipid alkoxy radicals from lipid hydroperoxides.[36] The\nferroptosis-mediated cell death mechanism. western blot result clearly shows the inhibition of GPX4 with the\n increasing concentration of IrL1, at the highest drug dose the\n GPX4 intensity was \u224890% less than that of the control, which\n2.11. Ferroptosis Triggered by IrL1 led to the accumulation of lipid ROS, and eventually cell death\n (Figure 6g).\nInvestigation of the mode of cell death by incubating IrL1 with\ndi\ufb00erent inhibitors suggests that none of the inhibitors dis-\nplayed a clear in\ufb02uence on reduction in cell viability except for 2.12. IrL1 Interplay With Cellular Organelles (Endoplasmic\nferrostatin-1 treated cells, indicating that IrL1 causes ferroptosis. Reticulum Stress and Mitochondrial Dysfunction)\nBased on these results, we thoroughly veri\ufb01ed the common fer-\nroptosis markers, namely reactive oxygen species (ROS) gener- The primary interaction between therapeutic agents and cells\nation, Glutathione depletion, lipid peroxidation, and inhibition is dependent on the subcellular target. The endoplasmic\n\n\nSmall 2025, 21, 2406809 2406809 (8 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 8. a) Co-localization of IrL1 by BIO-RAD ZOE \ufb02uorescent cell imager. MDA-MB-231 cells were incubated with IrL1 (3.3 \u03bcm, 3 h) and then re-\nspectively incubated with ER-Tracker Red, MitoTracker Deep Red FM, or LysoTracker Deep Red DND-26 for 0.5 h after being washed with PBS. Scale bar\n25 \u03bcm. Ferroptosis triggered by IrL1 . b) Cell viabilities of MDA-MB-231 with the coincubation of various inhibitors and IrL1 complex. Each data point\nrepresents mean \u00b1 SD, n =3. Signi\ufb01cantly compared to IrL1 : **p < 0.01, *p < 0.1 ns= non-signi\ufb01cant. c) Measurement of intracellular ROS levels by\nDCFH-DA assay. H2 O2 and untreated cells were taken as positive and negative controls respectively. Scale bar: 100 \u03bcm. d) Fluorescent images of C11-\nBODIPY dye\u2013stained MDA-MB-231 cells after being incubated with IrL1 IC50 for 0.5 h. Untreated cells were taken as the negative control. Scale bar: 25\n\u03bcm. The \ufb02uorescence was monitored by a BIO-RAD ZOE \ufb02uorescent cell imager. The mean \ufb02uorescence intensity was calculated using Image J software.\ne) Lipid peroxidation in MDA-MB-231 cells treated with 3 and 6 \u03bcm IrL1 with untreated cells as the control, determined by BODIPY-C11 staining via \ufb02ow\ncytometry after 3 h incubation () Decrease of cellular GSH levels in MDA-MB-231 cells incubated with IrL1 . The luminescence intensity was measured\nusing a microplate reader. Each data point represents mean \u00b1 SD, n =3. Signi\ufb01cantly compared to control: ****p < 0.0001, *p < 0.1. g) Analysis of\nexpression of GPX4 by western blot. The fold change in expression of GPX4 in MDA-MB-231 cells treated with IrL1 (0.11\u20139 \u03bcM) was calculated vs. the\nintensity of untreated cells and normalized based on the intensity of respective \ud835\udefd\u2013Actin bands.\n\n\n\nreticulum (ER) and mitochondria are closely linked within a very stress. The Endoplasmic reticulum (ER) is the largest cell or-\nshort distance, only a few nanometers (10\u201330 nm) apart, forming ganelle which is mainly responsible for protein regulation, intra-\njunctions where they are tethered together.[37,38] First, we delved cellular Ca2+ storage, and lipid biosynthesis.[39] Cell growth re-\ninto examining the potential impact of IrL1 on the endoplasmic quires proper ER function, however, in cancer cells, the ER func-\nreticulum (ER), speci\ufb01cally focusing on its role in inducing ER tion becomes dysregulated resulting in increased ER stress in\n\n\nSmall 2025, 21, 2406809 2406809 (9 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\ncancer cells. To cope with the ER stress, the unfolded protein re- (tetramethylrohdamine methyl ester) dye. The collapse in mito-\nsponse (UPR) pathway is upregulated by cancer cells, the mecha- chondrial membrane potential was characterized by the reduc-\nnism by which cells control endoplasmic reticulum (ER) protein tion in red \ufb02uorescence of the dye in the positive control H2 O2\nhomeostasis. The UPR remains inactivated under normal condi- and IrL1 treated cells in comparison to the control. Figure 9c rep-\ntions, however under stress conditions such as altered glycosyla- resents the concentration-dependent decrease in the red \ufb02uores-\ntion or hypoxia, UPR gets activated because of the accumulation cence. These results indicate that IrL1 can cause the loss of MMP.\nof unfolded proteins. UPR generally makes cancer cells resistant The mitochondrial damage may result in the alteration of energy\nto chemotherapy.[40] Regardless of the UPR\u2019s role in developing production pathways; therefore, we further explored the e\ufb00ect of\ncancer progression and resistance to chemotherapy, the chemi- IrL1 on mitochondrial energy metabolism status, mainly the in-\ncal induction of ER stress has gained attention as an anticancer tracellular ATP level. As shown in Figure 9d IrL1 treated cells rep-\nstrategy. Such examples include the FDA-approved drugs car\ufb01l- resent the dose-dependent reduction in the intracellular ATP lev-\nzomib and bortezomib, which induce ER stress by inhibiting the els as compared to the control. Notably, \u224855% decrease in ATP\ndegradation of unfolded proteins. The UPR consists of three ma- production was found at the highest concentration of IrL1 (200\njor pathways: PERK, IRE1, and ATF6.[41] IRE1, PERK, and ATF6 \u03bc\u03bc) when treated for 12 h. The combined results of MMP and ATP\nare the three ER transmembrane proteins that sense ER stress in assay suggest that IrL1 has the tendency to cause mitochondrial\nthe ER and become activated resulting in the regulation of a cas- dysfunction and metabolic inhibition. Damage or impairment of\ncade of signaling pathways collectively termed as the Unfolded mitochondria results in increased oxidative stress due to the gen-\nProtein Response (UPR). The main functions of UPR include the eration of reactive oxygen species (ROS).[46a] Subsequently, we\nreduction of protein translation and activation of degradation of employed Mitosox, a probe speci\ufb01cally targeting mitochondrial\nmisfolded and unfolded proteins.[42] The cancer cell will survive superoxide (O2 \u25cf- ), to assess this phenomenon. Figure 9e displays\nif the UPR can mitigate the ER stress encountered by it. When the signi\ufb01cant increase in O2 \u25cf- level treated with IrL1 .\nthe ER stress conditions are unresolvable then UPR fails to lower\nthe ER stress and restore ER homeostasis and thus promote cell\ndeath.[43] In this study, we explored the impact of varying con- 2.13. Propensity Between Ferroptosis Induction and Organelle\ncentrations of IrL1 on the activation of endoplasmic reticulum Targeting\n(ER) stress, speci\ufb01cally examining markers such as PERK and\nBiP through Western blot analysis. MDA-MB-231 cells were ex- To understand the precise mode of action between ferroptosis\nposed to \ufb01ve doses of IrL1 , determined relative to the IC50 values and organelle targeting further investigation was performed. Our\nobtained from the 48 h MTT experiment. The expression levels previous experiments mentioned in the manuscript displayed\nof ER markers were then compared to those of untreated cells, as that in the presence of Ferrostatin-1 (Fer-1), a known ferroptosis\nillustrated in Figure 9a,b. Under non-stressful conditions, the en- inhibitor, the cell viability of IrL1 was enhanced, suggesting that\ndoplasmic reticulum (ER) chaperone, immunoglobulin binding ferroptosis was the cell death mechanism induced by IrL1 . To fur-\nprotein (BiP), aids in protein folding by binding to protein kinase ther investigate the propensity between ferroptosis induction and\nR-like ER kinase (PERK), preventing its activity. During the ER organelle targeting, the impact of co-incubation of IrL1 with Fer-1\nstress, the BiP protein dissociates from the PERK protein and the on mitochondrial function was evaluated. Results indicated that\nPERK protein undergoes autophosphorylation to form phospho- changes in mitochondrial membrane potential, mitosox stain-\nPERK. This is known as the PERK activation.[43a] The phospho- ing, and ATP depletion still persisted as shown in the following\nrylation levels of PERK can be detected by the phospho-speci\ufb01c Figures (Figures S54\u2013S57, Supporting Information). The Fer-1 is\nPERK antibody.[43b] Thus, during ER stress, the downregulation unable to inhibit mitochondrial superoxide generation. 46b These\nof PERK protein expression and upregulation of phospho-PERK observations imply that while mitochondrial damage occurs, it is\nprotein expressions indicates PERK activation.[43c] In this west- not the primary cause of cell death when ferroptosis is inhibited.\nern blot experiment we determined the expression of PERK pro- This leads to the conclusion that IrL1 \u2019s targeting of mitochondria\ntein and not the phospho-PERK protein. Therefore, we observed is insu\ufb03cient to inhibit ferroptosis. Furthermore, previously, it\nthe PERK downregulation. The Western blot \ufb01ndings for PERK was observed that increased cell viability of IrL1 in the presence of\nand BiP substantiated PERK activation, as the protein expression NAC (an antioxidant), and enhanced lipid peroxidation observed\nmarkedly decreased with escalating drug concentrations. For in- by the C11-BODIPY probe, suggesting oxidative lipid damage as\nstance, at the highest drug dosage of 9 \u03bc\u03bc, PERK intensity was the mode of action of IrL1 . The inhibition of lipid peroxidation\napproximately >90% lower than the control. Conversely, BiP in- by Ferrostatin-1 is shown in Figure S56 (Supporting Informa-\ntensity increased many folds compared to the control, indicating tion). It is reported that while lipid peroxidation in lysosomal or\nits dissociation from PERK and the initiation of the unfolded pro- mitochondrial membranes can induce ferroptosis; the oxidative\ntein response (UPR).[44] Intense ER stress triggers the release of destruction of speci\ufb01c membrane lipids, particularly within the\ncalcium ions (Ca2+ ) from the endoplasmic reticulum (ER), stimu- endoplasmic reticulum dominantly triggers the ferroptosis.[46c,d]\nlating the \ufb02ux of Ca2+ between the ER and mitochondria, thereby\nleading to mitochondrial dysfunction.[45]\n Consequently, we conducted a mitochondrial membrane po- 2.14. In-vitro and In-silico Intercalative Mode of Binding of IrL1\ntential assay. The impact of IrL1 on the integrity of mitochon- with DNA\ndrial membrane potential was monitored by TMRM assay. The\nchanges in the mitochondrial membrane potential (MMP) were Considering the propensity of positively charged metal com-\ndetected by the \ufb02uorescence of the cells stained with TMRM plexes possessing an extended aromatic surface to engage in\n\n\nSmall 2025, 21, 2406809 2406809 (10 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 9. a,b) Analysis of various endoplasmic reticulum (ER) stress biomarkers by western blot. MDA-MB-231 cells were treated for 24 h with increasing\nconcentrations of IrL1 (0.11\u20139 \u03bcM). \ud835\udefd-Actin was used as a loading control. The fold change in protein expression was calculated vs. the intensity of\nuntreated cells and normalized based on the intensity of respective \ud835\udefd\u2013Actin bands. c) Change in Mitochondrial membrane potential (\u0394\u03a8m ) observed\nby \ufb02uorescent images of TMRM dye-stained MDA-MB-231 cells after being incubated with IrL1 for 0.5 h. The \ufb02uorescence was monitored by a BIO-RAD\nZOE \ufb02uorescent cell imager. Untreated cells were taken as the negative control, and H2 O2 was taken as the positive control. Scale bar: 25 \u03bcm. d) Mitosox\nassay for detection and quanti\ufb01cation of superoxide in IrL1 -treated MDA-MB-231. Cells. Untreated cells were taken as the negative control. Scale bar: 25\n\u03bcm. The \ufb02uorescence was monitored by a BIO-RAD ZOE \ufb02uorescent cell imager. The mean \ufb02uorescence intensity was calculated using Image J software.\n****p < 0.0001, **p < 0.01, ns= non-signi\ufb01cant. e) Decrease of cellular ATP levels in MDA-MB-231 cells incubated with IrL1 . Cells were treated with\nthe indicated concentrations of IrL1 for 12 h. The luminescence intensity was measured in a microplate reader. Untreated cells were taken as control.\nEach data point represents mean\u00b1SD, n =3. Signi\ufb01cantly compared to control: ****p < 0.0001, **p < 0.01, ns= non-signi\ufb01cant. Binding interaction\nof IrL1 with CT-DNA in Tris-HCl bu\ufb00er (pH=7.4) resulted in f) hyperchromism and isosbestic point in UV\u2013vis titration studies of IrL1 with CT-DNA.\ng) Circular dichroism (CD) spectra of CT-DNA with a \ufb01xed concentration of CT-DNA and increased concentrations of IrL1 (2 \u03bcM- 10 \u03bcm). Inset f) Plot\nof [DNA]/(\ud835\udf16 a \u2212\ud835\udf16 f ) vs. [DNA] for IrL1 . Molecular docking of IrL1 with DNA (PDB: 1BNA) h) ribbon view i) space \ufb01ll view.\n\n\n\nnon-covalent interactions with DNA,[47a] we speculated that nu- ported to target mitochondrial DNA.[47b] Taking this into account\nclear/mitochondrial DNA could be among the potential targets we explored the interaction of IrL1 with CT-DNA using UV\u2013vis\nof IrL1 . Owing to the compound\u2019s ability to intercalate with titrations study and Circular dichroism (CD) spectra. The UV\u2013\nDNA and mitochondrial localization, both the nuclear and mi- vis spectra shown in Figure 9f display a hyperchromic shift in\ntochondrial DNA can be suspected as the potential target. In- \ud835\udf0b\u2013\ud835\udf0b* region and a hypochromic shift at the MLCT region that\ndeed recently Ir(III) cyclometalated complexes have been re- resulted in the appearance of an isosbestic point. These spectral\n\n\nSmall 2025, 21, 2406809 2406809 (11 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n disruption in DNA replication that leads to S phase cell cycle\n arrest.\n\n\n 2.16. Synergistic Antitumor E\ufb00ect of Cisplatin and by\n Combination Index (CI) and Isobologram Analysis\n\n Combination therapy is being used for the management of many\n cancers as it o\ufb00ers the possibility of synergism between two\n drugs. Having the knowledge of a distinct mechanism of action\n of IrL1 on cancer cells as compared to cisplatin, we explored the\n e\ufb00ect of both drugs on cancer cells when used in combination\n with each other. Following the previously reported protocol49,\n MDA-MB-231 cells were treated with IrL1 and cisplatin in dif-\n ferent ratios for 48 h. The cell viability was determined by MTT\n assay. As shown in Figure 11a, the IC50 values obtained for dif-\n ferent combinations of IrL1 and cisplatin are considerably lower\n as compared to their IC50 values alone. We utilized the combina-\n tion index (CI) and isobologram methods to analyze whether the\n combination of these drugs resulted in synergistic, antagonistic,\n or additive e\ufb00ects. The following formula was used for calculat-\n ing combination index (CI): CI = CA /CXA + CB /CXB , where CA and\nFigure 10. Cell cycle distribution of MDA-MB-231 cells treated with IrL1 CB are the concentrations of drugs A and B required to achieve\nat indicated concentrations for 24 h. Untreated cells were taken as the a certain e\ufb00ect using a combination of A and B, and CXA and\ncontrol. CXB are the concentrations of drugs A and B alone. CI >1 im-\n plies antagonistic; CI = 1 implies additive; and CI < 1 implies\ncharacteristics indicate the interaction of IrL1 with DNA together synergistic e\ufb00ects.[50] As shown in Figure 11a, a strong synergis-\nthrough an intercalative mode of action.[20] The magnitude of the tic e\ufb00ect was observed when IrL1 and cisplatin were used in a 2:1\nbinding strength of IrL1 with CT-DNA was calculated via the in- ratio whereas the antagonistic e\ufb00ect was observed at other ratios.\ntrinsic binding constant Kb, where Kb = 3.51 \u00d7 105 M\u22121 . To To verify these results the experiment was repeated in triplicates\ncon\ufb01rm the results obtained by UV\u2013vis, circular dichroism (CD) in a set of three independent experiments. Next, we generated\nspectra were recorded. The positive band at 280 nm is due to left- the isolobogram by plotting the IC50 /48 h values obtained for in-\nhanded helicity (due to the common arrangement of base stack- dividual agents and for combinations. As shown in Figure 11b,\ning or nucleobases), while the negative band at 248 nm is due the isolobogram also showed the synergistic antitumor e\ufb00ect of\nto the right-handed helicity of DNA. The changes in the CD sig- cisplatin and IrL1 , which is consistent with our analysis using CI\nnals of DNA upon interaction with the drug can be assigned to values.\nchanges in the structure of DNA. The CD spectra obtained for\nCT-DNA and IrL1 in Figure 9g resulted in substantial changes\ndue to alterations in base stacking and helicity of DNA, indicat- 2.17. Intracellular Distribution of IrL1 within 3D-Multicellular\ning the intercalative mode of binding of IrL1 with DNA.[48] To Spheroids (MCTSs)\nfurther evaluate the interaction of IrL1 with DNA, computational\nmodeling was performed using the optimized structure of IrL1 . In drug delivery, it is essential to ensure prolonged circulation\nThe results obtained through docking also indicated the prefer- in the bloodstream and e\ufb03cient penetration into deep tumor re-\nential intercalation of IrL1 to B-DNA (Figure 9h,i). gions, aspects directly impacted by the size and structure of nano-\n materials. Considering the necessity for deep tumor penetration,\n multicellular spheroids (MCTSs) of MDA-MB-231 cells with a\n2.15. Cell Cycle Disruption by IrL1 diameter of 500\u2013600 \u03bcm were developed. MCTSs are character-\n ized by multilayered cell structures that show di\ufb00usional limits to\nTo gain an insight into the e\ufb00ect of IrL1 on cell cycle progres- drug penetration and MCTSs also develop the hypoxic core which\nsion, the cell cycle arrest experiment on MDA-MB-231 cells was can make the tumor cells resistant to cancer therapeutics.[51,52a]\nperformed by \ufb02ow cytometry. The cell cycle includes G0/G1, S, The 3D MDA-MB-231 spheroids were treated with varying con-\nand G2/M phases, halting cell division at any of these phases centrations of IrL1 . Figure 11c shows the di\ufb00usion e\ufb03cacy of\nleads to cell cycle arrest. As illustrated in Figure 10 the per- IrL1 nanoaggregates into the tumor spheroids. The IrL1 treated\ncentage of cells at the S phase after treatment with IrL1 at spheroids collapsed remarkably upon photoirradiation (390 nm,\nvarying concentrations for 24 h, increased with concentration 0.363 mW cm\u22122 , 25 min) showing a higher photodynamic e\ufb00ect\nof IrL1 from 14.9% (1X IC50 ) to 34.5% (4X IC50 ) compared compared to the dark conditions. The loss of integrity in treated\nto control (8.7%). These results suggest that IrL1 induces cell spheroids in the dark and via photoirradiation compared to con-\ncycle arrest during the S phase, a critical DNA synthesis pe- trol indicated the potential of IrL1 nanoaggregates to suppress\nriod. In conclusion, IrL1 \u2019s ability to intercalate with DNA causes hypoxic 3D solid tumors.\n\n\nSmall 2025, 21, 2406809 2406809 (12 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 11. a) IC50/48h values (\u03bcm) and CI values in MDA-MB-231 cells for cisplatin and IrL1 when used as a single agent or in combinations (48 h\nincubation MTT assay). b) Isobologram of cisplatin and IrL1 in MDA-MB-231 cells. c) Microscopic images of MDA-MB-231 Multicellular tumor spheroids\n(MCTSs). MCTSs were treated with di\ufb00erent concentrations of IrL1 with respect to IC50 obtained in 2D culture for 48 h. Photoirradiation was conducted\nby an LED light of 390 nm (0.363 mW/cm2 , 25 min). The images for 0 h were recorded before treatment. Scale bar: 100 \u03bcm. d) Hemolytic activity of IrL1\non human RBCs. 0.1% Triton X-100 was used as the positive control. Each data point represents mean \u00b1 SD, n =3. Signi\ufb01cantly compared to positive\ncontrol: ****p < 0.0001.\n\n\n2.18. Hemolysis Evaluation of IrL1 spleen, kidney, intestine, and lung) and tumor were harvested,\n and the tissues were examined by hematoxylin-eosin (H & E)\nPrior to exploring the translational potential of our compound staining. The photographs of the harvested tumor are repre-\nthrough in-vivo experiments, it is imperative to ensure the safety sented in Figure 12e. The inductively coupled mass spectrom-\nand e\ufb03cacy of the drug candidates. We focused on understand- etry (ICP-MS) data (Figure 12f) illustrates that IrL1 was accumu-\ning the impact of IrL1 , a potential therapeutic agent, on mam- lated majorly in the spleen of mice whereas the H & E staining\nmalian cells, particularly red blood cells (RBCs). Through metic- indicates no injuries or abnormalities in any organs including\nulous examination across a spectrum of concentrations, ranging the spleen (Figure 12m). On the other hand, signi\ufb01cant damage\nfrom 12.5 to 0.39 \u03bc\u03bc, we meticulously evaluated IrL1 \u2019s hemolytic was observed in IrL1 -treated tumor tissue compared to the con-\npotential. Our \ufb01ndings reveal that IrL1 demonstrates remark- trol. The morphological analysis of erythrocytes indicates no tox-\nable biocompatibility, with minimal hemolysis observed, even at icity of IrL1 on the erythrocytes (Figure 12g). The complete blood\nhigher concentrations, measuring \u22641%. Intriguingly, within its count is shown in Figure 12h\u2013l. Figure 12c,d illustrates the reduc-\nIC50 range, IrL1 showcases non-hemolytic behavior, with hemol- tion in tumor volume in IrL1 -treated mice compared to control.\nysis rates below 0.5% (Figure 11d). These results underscore the The above indices indicate the safety and e\ufb03cacy of IrL1 in mice\npromising safety pro\ufb01le of IrL1 , positioning it as a potentially models.\nviable therapeutic option. 0.1% Triton X-100, a positive control,\nelicited complete hemolysis. 3. Conclusion\n In summary, this research outlines the synthesis of a novel set of\n2.19. IrL1 Therapeutic E\ufb03cacy In vivo organometallic Ir(III) complexes, incorporating COOMe groups\n to augment cellular permeability. Notably, the lead compound,\nThe safety and e\ufb03cacy of IrL1 in blood further prompted us to IrL1 , exhibits the formation of nanoaggregates within cell culture\nexplore the therapeutic e\ufb03cacy of IrL1 4T1 xenograft NOD.CB17- media, thereby facilitating enhanced di\ufb00usion in deep-seated tis-\nPrkdcscid/NCrCrl mice. After 4T1 tumor establishment, the sue within 3D MDA-MB-231 tumor spheroids. Under 390 nm\nmice were randomly divided into two groups of four mice each, light exposure IrL1 exhibited remarkable photocytotoxicity with\ni.e., control, and drug-treated. The schematic in Figure 12a out- an IC50 value of 36.05 \u00b1 0.03 nm and exhibited a more pro-\nlines the experimental details. nounced e\ufb00ect on 3D MDA-MB-231 spheroids compared to dark\n The body weight and tumor volume of each mice were mon- conditions. Upon internalization via energy-dependent endocy-\nitored every two days a week revealing high biocompatibility of tosis, IrL1 selectively targets the endoplasmic reticulum and mi-\nIrL1 as mice did not lose any weight compared to control dur- tochondria, showcasing potent anti-tumor properties through\ning the treatment (Figure 12b). After 16 days of treatment, all ferroptosis-mediated cell death, ER stress induction, mitochon-\nmice were euthanized, blood and all major organs (heart, liver, drial dysfunction, and DNA intercalation. Importantly, IrL1\n\n\nSmall 2025, 21, 2406809 2406809 (13 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\n\n\n\nFigure 12. In vivo antitumor e\ufb03cacy of IrL1 in 4T1 tumor-bearing NOD.CB17-Prkdcscid/NCrCrl mice. a) Schematic detail of the treatment. b) Body\nweight of the animals during treatment. c) Relative tumor volume during the treatment period. d) Tumor volume post-treatment e) Photographs of the\ntumor harvested after 16 days of treatment. f) Biodistribution of IrL1 after 16 days of treatment. g) Erythrocyte images h) RBC count i) WBC count j)\nHemoglobin k) Platelets count from the blood collected after euthanization. l) Percentage of lymphocytes, monocytes, neutrophils, and eosinophils in\nthe blood collected from control and IrL1 -treated mice. m) Histological examination of di\ufb00erent organs with hematoxylin and eosin (H & E) stain from\nthe tissues harvested after euthanization.\n\n\n\n\nSmall 2025, 21, 2406809 2406809 (14 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\ndemonstrates minimal hemolytic activity, indicating a favorable or re\ufb02uxed at 120 \u00b0C for 24 h. The red precipitates obtained were then \ufb01l-\nsafety pro\ufb01le for therapeutic utilization. The Ir(III) complexes tered, washed with water and diethyl ether, and dried. Yield: 75\u201380%. The\nexisting in the literature for the treatment of TNBC displayed formation of the intermediates was characterized by ESI-MS and the next\n step proceeded without any further puri\ufb01cation.\nlimited cytotoxicity compared to IrL1 both in dark and light\n Synthesis of IrL1\u22128 : A mixture of [(C\u02c4 N) 4 Ir2 Cl2 ] (1 eq.) and (N\u02c4 N) lig-\nconditions. Uniquely, IrL1 nanoaggregates induce ferroptosis- and (2.2 eq.) in DCM: MeOH (1:1, v/v %) was re\ufb02uxed under for 12 h. The\nmediated cell death and cause mitochondrial dysfunction and ER solution was cooled down to room temperature and then NH4 PF6 (10 eq.)\nstress generation. The similar existing Ir(III) complexes in the lit- was added to it. The reaction mixture was stirred for the next 3 h at room\nerature induce ferroptosis-mediated cell death by multiple path- temperature and then \ufb01ltered. The \ufb01ltrate was concentrated to dryness.\nways such as ferroptosis-apoptosis, ferroptosis-autophagy, and The product obtained was puri\ufb01ed by silica gel column chromatography\n in acetone/DCM.\nferroptosis-pyroptosis pathways.[52b\u2013e] The reported molecules\n Purity Analysis of Ir(III) Complexes: The RP-HPLC analysis was utilized\nthat speci\ufb01cally induce ferroptosis were incorporated with the to determine the purity of IrL1 -IrL1 complexes. For this, the solution of\nferroptosis-causing agents in their such RSL3 and ferrocene,[52f,g] the complexes in 20/80 (v/v) acetonitrile/water was injected into the RP-\nwhereas IrL1 speci\ufb01cally induces ferroptosis only and without HPLC column. The detection wavelength was set as 254 nm. HPLC-grade\nthe incorporation of ferroptosis-inducing agents in its struc- acetonitrile and MQ water containing 0.1% TFA v/v were used as the mo-\nture. Furthermore, IrL1 exhibits low hemolysis in blood, indicat- bile phase in the experiment.\ning a high level of safety. Notably, no other ferroptosis-inducing Photophysical Properties in Solution: UV\u2013visible absorption spectra\n were obtained on a JASCO V-770 UV\u2013visible\u2013NIR spectrophotometer\nmolecules reported to date possess the distinctive capability to equipped at room temperature. All the photoluminescence spectra were\nundergo nanoaggregation, a novel feature that enhances ther- recorded on HORIBA Fluromax-4 at room temperature. Stock solutions of\napeutic speci\ufb01city and e\ufb00ectiveness, particularly against MDA- the compounds were prepared in DMSO. Photoluminescence quantum\nMB-231 cells. This combination of selective ferroptosis induction yields (\u03a6em or PLQYs) in solution were obtained from corrected spectra\namong other cell death pathways along with its safety and unique and measured according using \ufb02uorescein (\u03a6 = 0.79, in 0.1 m NaOH) as\nnanoaggregation property, underscores the novelty and signi\ufb01- standard. The quantum yield calculations were done using the equation:\n \u03a6 = \u03a6ref \u00d7 (Aref /A) \u00d7 (I/Iref ).\ncance of IrL1 compared to the existing molecules.\n Stability of Ir(III) complexes by UV\u2013vis Absorption Spectroscopy: The sta-\n Collectively, the utilization of nanoaggregates comprising bility of Ir(III) complexes was investigated by UV\u2013vis absorption spec-\norganometallic Ir(III) complexes, as corroborated by comprehen- troscopy in tris bu\ufb00er (pH = 7.4) as solvent. Brie\ufb02y, the Ir(III) complexes\nsive in vitro, in silico, and in vivo analyses, presents a promising were dissolved in DMSO and the absorption spectra were recorded in tris\nstrategy for enhancing delivery e\ufb03ciency and achieving profound bu\ufb00er for 0, 24, and 48 h at room temperature.\ntumor penetration, particularly in the context of challenging Lipophilicity by ICP-MS: The \ufb02ask-shaking method and ICP-MS anal-\n ysis were utilized to determine the lipophilicity (log Po/w ) of Ir(III) com-\ntriple-negative breast cancer. Consequently, this work unveils a\n plexes. An equivalent amount of 0.9% (w/v) NaCl solution, saturated with\nnovel Ir(III) complex with exceptional properties to form nanoag- octanol, was introduced into a 1 mL stock solution of the Ir(III) complexes\ngregates that can act as photodynamic therapy (PDT) agents, sug- in n-octanol. The resulting mixture was agitated for 24 h in an incubator set\ngesting a new promising strategy for its future study to eluci- at 37 \u00b0C. Following this, the mixtures underwent centrifugation at 4000 \u00d7\ndate its mode of action under light exposure. Future structural rpm for 10 min. Subsequently, the distinct oil and water phases were care-\nre\ufb01nements hold the potential for diversifying the repertoire of fully isolated. A total of 100 \u03bcL from each of the oil and water phases were\norganometallic Ir(III) nanoaggregates with adaptable character- subjected to dissolution using 65% HNO3 (300 \u03bcL) and subsequently di-\n luted to a \ufb01nal volume of 10 mL using distilled water. The concentration of\nistics, thereby advancing their clinical applications within the Ir(III) (Co or Cw ) was determined via the ICP-MS technique utilizing an in-\nrealm of oncology and the \ufb01eld of nanomedicine. ternal reference. The Po /w value is directly proportional to the ratio of the\n concentration of the organic phase (Co ) to the attention of the aqueous\n phase (Cw ).\n Cell Lines and Cell Culture Conditions: Breast adenocarcinoma cell lines\n4. Experimental Section MDA-MB-231 and MCF-7, Human lung adenocarcinoma cell line A549,\n Synthesis of C\u02c4N Ligands: To a solution of 2-Bromo-isonicotinic acid Human colon cancer cell line HCT-116, Human embryonic kidney cell\n(1eq.) and boronic acid (1.2 eq.) in THF: Water (3:5) was added K2 CO3 line HEK-293, and Human prostate cancer cell line PC3 was obtained\n(3.0 eq.) and the reaction mixture was stirred until both the reactants had from National Centre for Cell Science (NCCS Pune, India). The cell lines\ndissolved completely. Then Pd(PPh3 )2 Cl2 (0.05 eq.) was added, and the re- were maintained in RPMI 1640 (MDA-MB-231, MCF-7, A549, and PC3)\naction mixture was heated to 70 \u00b0C for 24 h. After the reaction completion, or DMEM medium (HCT-116, HEK-293), supplemented with 10% fetal\nthe reaction mixture was \ufb01ltered, THF was evaporated, and the remaining bovine serum, 100 Units mL\u22121 penicillin and 100 \u03bcg mL\u22121 streptomycin,\nsolution was extracted with DCM. The aqueous layer was acidi\ufb01ed with 1 at 37 \u00b0C humidi\ufb01ed atmosphere with 5% CO2 . All stock solutions of the\nm citric acid to pH 3 to get the precipitates. The precipitates were dried compound were prepared in DMSO.\nto get the product. To the round bottom \ufb02ask equipped with a magnetic MTT Assay: The cytotoxicity of the compounds was determined by the\nstirrer was added the product obtained (1 eq.) and dry MeOH (10 eq.). MTT assay. For each cell type, 0.35 \u00d7 106 cells were plated in a culture \ufb02ask.\nTo this was then added conc. H2 SO4 (0.2 eq.) and the reaction mixture The cells were harvested from culture \ufb02asks by trypsinization and seeded\nwas re\ufb02uxed for 8 h. Reaction completion was monitored by TLC. After re- into 96-well microculture plates at the seeding density of 2500 cells per\naction completion, the solvent was evaporated, and the reaction mixture well. After the cells were allowed to resume exponential growth for 24 h,\nwas extracted with DCM and saturated sodium bicarbonate solution and they were exposed to drugs at di\ufb00erent concentrations in media for 48 h.\nconcentrated to dryness to get the \ufb01nal product. Cisplatin (10 \u03bc\u03bc) and untreated cells were used as positive and negative\n Synthesis of N\u02c4N Ligand: The ligand dipyrido[3,2-a:2\u2032,3\u2032-c]phenazin- controls, respectively. After the exposure of 44 h, the cells were treated\n11-amine (adppz) was prepared according to the reported method.[53] with MTT solution (100 \u03bcLeach well, 5 mg mL\u22121 ) for 4 h in the dark, and\n Synthesis of [(C\u02c4 N) 4 Ir2 Cl2 ]: To the microwave vial equipped with a then absorbance was measured at 570 nm by SpectraMax M5e microplate\nmagnetic stirrer was added the (C\u02c4 N) ligand (2 eq.) and IrCl3 .xH2 O (1 reader. The photocytotoxicity was carried out by using Kessil PR160 LED\neq.). The solvent ethylene glycol: water (4:1) 5 mL was added, and the re- 390 nm, 0.363 mW cm\u22122 at a height of 60 cm. All procedures were car-\naction mixture was subject to microwave radiation for 60 min at 120 \u00b0C ried out in a triplicate of three independent experiments. IC50 values were\n\n\n\nSmall 2025, 21, 2406809 2406809 (15 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\ncalculated using GraphPad Prism software and the results were presented After 3 h of incubation, the media was removed, and the cells were washed\nas a mean \u00b1 SD. with 1X PBS. Then, a solution of 2\u2032,7\u2032-dichlorodihydro\ufb02uorescein diacetate\n Cellular Uptake by ICP-MS: MDA-MB-231 cells were plated in a 6-well (H2 DCF-DA; 10 \u03bc\u03bc, 100 \u03bcL) in 1X PBS was added to the cells and incubated\nplate (1 \u00d7 106 cells each well). After 24 h incubation, the medium was re- in the dark at 37 \u00b0C for 30 min. The cells were then washed thrice with\nplaced by a medium containing IrL1 and IrCl3 .xH2 O. After 8 h incubation, 1X PBS to remove excess H2 DCF-DA. Fluorescent images were captured\nthe culture medium was aspirated. Cells were trypsinized, counted, and immediately using a BIO-RAD ZOE \ufb02uorescent cell imager.\nwashed three times with PBS. Then, cells were centrifuged at 3000 rpm Lipid Peroxidation Assay: MCF-7 cells (2.5 \u00d7 104 cells each well) were\nat 4 \u00b0C for 10 min, and digested in HNO3 for two days. Each sample was plated in 96 well plates and allowed to adhere overnight. Afterward, the\ndiluted to 10 mL before the test. The amount of Ir in cells was determined cells were treated with Ir(III) complex IrL1 with its two-fold dilutions. Un-\nby ICP-MS. The experiment was performed in triplicates. treated cells were used as a negative control in this experiment. After 3\n Physiochemical Characterizations of Nanoaggregates: Dynamic light h of incubation, the media containing compounds was removed and the\nscattering (DLS) was conducted at room temperature on a Zetasizer Nano cells were washed with PBS. Then, a solution of C11-BODIPY (2.5 \u03bcm) in\nZS90 by Malvern. Scanning electron microscopy (SEM) and energy disper- media was added to the cells and incubated in the dark at 37 \u00b0C for 30 min.\nsive X-ray spectroscopy (EDS) elemental mapping images were taken on a The cells were then washed thrice with PBS to remove excess C11-BODIPY\nZeiss scanning electron microscope. High-resolution TEM (HRTEM) was and \ufb02uorescence microscopy images were captured immediately using a\nconducted on FEI-Titan G2 60\u2013300 KV TEM. BIO-RAD ZOE \ufb02uorescent cell imager. Following the same protocol, \ufb02u-\n Multicellular Tumor Spheroids (MCTSs) Formation: The spheroids were orescence detection was also performed by the FL-1H channel on a \ufb02ow\ncultured in 96-well plates coated with 1.5% agarose. 2500 cells each well cytometer (BD FACSCalibur).\nwere seeded per well and the spheroids were maintained in a humidi\ufb01ed Intracellular Glutathione Depletion Assay: The GSH-Glo Glutathione\ncell culture incubator at 37 \u00b0C with a 5% CO2 atmosphere. The integrity Assay is a luminescence-based assay for detecting and quantifying glu-\nof spheroids after treatment with IrL1 was monitored by a BIO-RAD ZOE tathione (GSH). The experiment was performed according to the assay\nmicroscope. The spheroids were irradiated by using Kessil PR160 LED 390 protocol. In the 96-well plate, 2500 MDA-MB-231 cells were seeded and\nnm, 0.363 mW cm\u22122 at a height of 60 cm. allowed to adhere overnight. The cells were treated with di\ufb00erent concen-\n Cellular Uptake Mechanism: MDA-MB-231 cells were stained with IrL1 trations of IrL1 and incubated for 24 h. Afterward, the media was aspirated,\ncomplex under di\ufb00erent conditions by varying the temperature (4 and 37 and cells of each well were treated with 100 \u03bcL of prepared 1X GSH-Glo\n\u00b0C) as well as pretreated with metabolic inhibitor (CCCP) and endocytic Reagent and incubated for 30 min at room temperature. Furthermore, 100\ninhibitors (chloroquine and NH4 Cl). The cells were observed under a BIO- \u03bcL of reconstituted Luciferin Detection Reagent was added to each well\nRAD ZOE \ufb02uorescent cell imager to determine the cellular uptake by their and incubated for 15 min. The luminescence was then recorded using a\n\ufb02uorescence. plate reader.\n Intracellular Localization: The MDA-MB-231 cells were incubated with Western Blot: MDA-MB-231 cells (1 \u00d7 106 ) were seeded in a 6-well\nIrL1 (3.3 \u03bc\u03bc) at 37 \u00b0C for 3 h and then co-incubated with ER-Tracker Red plate. After overnight incubation, the cells were treated with increasing\n(1 \u03bc\u03bc), MitoTracker Deep Red FM (500 nm), or LysoTracker Deep Red concentrations of IrL1 (0.11\u20139 \u03bc\u03bc) for 24 h. After 24 h treatment, the cells\nDND-26 (50 nm) at 37 \u00b0C for 0.5 h, then washed by PBS three times. were washed with 1X PBS and lysed using the lamellae bu\ufb00er. The cell\nCells were then immediately visualized by \ufb02uorescent microscope. The ex- lysates were scraped, collected, and stored at 4 \u00b0C. The protein concen-\ncitation wavelength for IrL1 was 460 nm, while the excitation wavelengths tration for each sample was measured by Bradford assay. Samples with\nof ER-Tracker Red, LysoTracker Deep Red DND-26, and MitoTracker Deep the same protein concentration (20 \u03bcg) were heated at 100 \u00b0C for 5 min.\nRed FM are 587, 647, and 644 nm. Emission \ufb01lter:(peak wavelength of IrL1 ) The protein mixtures were separated on SDS-PAGE gel (12%) and then\n\u00b1 20 nm respectively, and 615 \u00b1 20 nm for ER-Tracker Red, 668 \u00b1 20 nm transferred to the PVDF membrane. The membrane was blocked with 5%\nLysoTracker Deep Red DND-26, and 665 \u00b1 20 nm for MitoTracker Deep skimmed milk for 2 h and then incubated with the primary antibodies\nRed FM. overnight at 4 \u00b0C. The samples were also incubated with \ud835\udefd-actin antibody\n Determination of Mechanism of Cell Death in Presence of Inhibitors: The as a loading control. The membranes were washed with 1X TBST thrice\ncell viability of MDA-MB-231 cells was measured by MTT assay after 24 (5 min each) and incubated for 2 h with the secondary antibody. After in-\nh co-culture with a variety of inhibitors and IrL1 complex. MDA-MB-231 cubation, the membranes were again washed thrice with 1X TBST (5 min\ncells were seeded in a 96-well plate, with 2500 cells each well. Cells were each) and visualized using SuperSignal West Pico Plus Chemiluminescent\nallowed to adhere overnight. The cells were then pre-incubated for 30 min substrate and a chemiluminescence imaging machine (ChemiDoc Touch\nwith the inhibitors Z-VADfmk (15 \u03bc\u03bc), 3-methyladenine (100 \u03bc\u03bc), cyclohex- imaging system, BioRad). The fold change in each protein expression was\nimide (0.1 \u03bc\u03bc), Necrostatin-1 (50 \u03bc\u03bc), CCCP (40 \u03bc\u03bc), Ferrostatin-1 (50 \u03bc\u03bc) analyzed using Image J software.\nand NAC (1 mm) which. IrL1 was then incubated in the concentration of TMRM assay: MDA-MB-231 cells (1.5 \u00d7 104 cells each well) were\n0.8, 1.6, 3.12, and 6.25 \u03bc\u03bc for 24 h. Cisplatin was incubated at the concen- plated in 96 well plates and allowed to adhere overnight. Afterward, the\ntration of 5.3, 10.6, and 21.2 \u03bc\u03bc for 24 h. MTT assay was then performed cells were treated with Ir(III) complexes. Hydrogen peroxide (H2 O2 150\nto determine the % cell viability in the presence of these inhibitors. \u03bcm) was used as a positive control whereas untreated cells were used\n Electrochemical Pro\ufb01le: The electrochemical experiment was carried as a negative control in this experiment. After 3 h of incubation, the me-\nout with the CHI610E electrochemical analyzer. Cyclic voltammetry was dia containing compounds was removed and the cells were washed with\nperformed under an N2 atmosphere at room temperature using a cell PBS. Then, a solution of tetramethyl rhodamine methyl ester perchlorate\nequipped with three electrodes: glassy carbon as the working electrode, (TMRM; 50 nm, 100 \u03bcL) in RPMI-1640 media was added to the cells and\nplatinum wire as the counter electrode, and the Ag/AgNO3 (0.01 m in incubated in the dark at 37 \u00b0C for 30 min. The cells were then washed\nCH3 CN) as the reference electrode. The measurement was performed thrice to remove excess TMRM and \ufb02uorescence microscopy images were\nin dry CH3 CN solutions with 0.1 m tetrabutylammonium perchlorate captured immediately using a BIO-RAD ZOE \ufb02uorescent cell imager.\n[nBu4 N] [ClO4 ] as the supporting electrolyte, with 1 mm of the compound ATP Assay: ATP production was measured using the Cell Titer-Glo lu-\nto be investigated in it at a scan rate of 100 mV S\u22121 . The potential values minescence cell viability assay kit (Promega) according to the manufac-\nwere reported versus the potential of the Ag/Ag+ couple used as a refer- turer\u2019s instructions. MDA-MB-231 cells were cultured in an opaque-walled\nence. 96-well plate and treated with IrL1 at the indicated concentrations for 12\n ROS Generation in Cells: MDA-MB-231 cells (2.5 \u00d7 104 cells each well) h. 100 \u03bcL Cell Titer-Glo reagent was added to each well. The plate was\nwere seeded in 96 well plates and allowed to adhere overnight. Afterward, incubated at room temperature for 30 min. Luminescence intensity was\none set of the cells was pretreated with N-acetyl cysteine (1 mm) for 1 h. measured in a microplate reader.\nAfter 1 h both sets, with and without N-acetyl cysteine were treated with Mitosox Staining: To detect superoxide radicals, 2.5 \u00d7 104 MDA-MB-\ndi\ufb00erent concentrations of IrL1 . Hydrogen peroxide (H2 O2 ) was used as a 231 cells were seeded on the 96-well plate for 24 h. After 24 h the cells were\npositive control whereas untreated cells were used as a negative control. then treated with di\ufb00erent concentrations of IrL1 for 3 h. The cells were\n\n\n\nSmall 2025, 21, 2406809 2406809 (16 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\nwww.advancedsciencenews.com www.small-journal.com\n\nthen washed with 1X PBS and incubated with 1 \u03bcm mitosox (M36007) for In vivo Therapeutic Evaluation: The animal experimental protocols\n30 min at 37 \u00b0C. The cells were then washed thrice with to remove excess were approved by the Institutional Animal Ethics Committee (IAEC) of the\nMitosox and \ufb02uorescence microscopy images were captured immediately Indian Institute of Technology Kanpur with an approval number IEC Com-\nusing a BIO-RAD ZOE \ufb02uorescent cell imager. The mean \ufb02uorescence in- munication Number: IITK/IEC/2023-24/I/10 and: llTK/IAEC/2023/1186.\ntensity was calculated by Image J software. Eight-week-old female NOD/SCID mice were purchased from Hylasco\n DNA Binding Studies: The binding of IrL1 with CT-DNA was examined Biotechnology (India) Pvt. Ltd. Hyderabad. After \u224810 days of inoculation,\nvia UV\u2013vis absorption titrations and CD spectroscopy. The stock solution the 4T1 cells were injected subcutaneously into the right \ufb02anks of mice.\nof CT-DNA was prepared in tris bu\ufb00er (pH = 7.4). The DNA concentration After tumor establishment (day 0), mice were randomly divided into two\nwas monitored by UV absorption measurements using \ud835\udf16 260 nm = 6600 M\u22121 groups of four mice each. The control group (saline-treated) and the IrL1 -\ncm\u22121 . The titration study was performed by maintaining the concentra- treated group were intravenously injected with saline (100 \u03bcL) and IrL1 (5\ntion of IrL1 (8.6 \u03bc\u03bc) constant and increasing the concentration of CT-DNA mg Kg\u22121 dose, 100 \u03bcL). The \ufb01rst dose was recorded as day 1. The body\nfrom 0 to 6 \u03bc\u03bc. The absorbance was measured 10 min after the addition weight, length, and width of tumors were recorded every second day. The\nof CT-DNA. The intrinsic binding constant (Kb ) of IrL1 with CT-DNA was tumor volume was calculated by the formula 0.5 \u00d7 length \u00d7 (width)2. On\ndetermined by using the equation: [DNA]/(\ud835\udf16 a \u2212\ud835\udf16 f ) = [DNA]/ (\ud835\udf16 b \u2212\ud835\udf16 f ) + 1/ the 17th day, the mice were sacri\ufb01ced, the blood was collected, and the\nKb (\ud835\udf16 b \u2212\ud835\udf16 f ), where \ud835\udf16 a is the extinction coe\ufb03cient of the complex at a given morphology of erythrocytes were captured. The tumors were stripped out\nDNA concentration, \ud835\udf16 b is the extinction coe\ufb03cient of complex when fully for photograph and weight. Other organs (heart, liver, kidney, lungs, intes-\nbound to DNA, \ud835\udf16 f is the extinction coe\ufb03cient of the complex in free solu- tine, and spleen) were also harvested and \ufb01xed in 4% paraformaldehyde\ntion. The plot [DNA]/(\ud835\udf16 a \u2212\ud835\udf16 f ) versus [DNA] gave the slope 1/ (\ud835\udf16 b \u2212\ud835\udf16 f ) and for H & E staining. The organs were digested in aqua regia to determine\nintercept 1/ Kb (\ud835\udf16 b \u2212\ud835\udf16 f ), Kb was calculated as the ratio of slope to inter- the Ir content by ICP-MS.\ncept. The CD spectra of solutions were measured after 2 h incubation of\ncompounds at room temperature in the range from 220 to 320 nm on a\nJASCOJ-1500 spectrometer. The concentration of CT-DNA was \ufb01xed and Supporting Information\nthat of IrL1 was varied from 2 to 10 \u03bc\u03bc.\n Cell-Cycle Disruption: MDA-MB-231 cells were seeded at a density of Supporting Information is available from the Wiley Online Library or from\n106 cells each well in a 6-well plate. After 24 h incubation, the cells were the author.\ntreated with di\ufb00erent concentrations of IrL1 for 24 h. After 24 h the samples\nwere harvested, washed with 1X PBS, \ufb01xed with 70% ethanol, and kept\nat 4 \u00b0C overnight. Fixed cells were washed with 1X PBS and stained with\n Acknowledgements\npropidium iodide (20 \u03bcg mL\u22121 ) and 100 \u03bcg mL\u22121 RNase A for 30 min at 37 The authors express their gratitude to Madhu Verma (Prof. Sri Sivakumar\u2019s\n\u00b0C. The cell cycle pro\ufb01le was measured by a \ufb02ow cytometer. The analysis laboratory), for granting access to the \ufb02ow cytometer instrument. The au-\nwas performed by Flow Jo software using the Dean\u2013Jett\u2013fox method. thors are thankful to Prof. S. Ganesh\u2019s laboratory (BSBE department, IITK)\n Structure Optimization and Molecular Docking: The complexes were for granting access to the plate reader (SpectraMax M5) and ChemiDoc.\noptimized in the gas phase by DFT (Density Functional Theory)[54\u201356] us- The authors are also thankful to Prof. Sandeep Verma\u2019s Lab (Chemistry\ning Gaussian 16.[57] Calculations were performed using B3LYP functional Department, IITK) for granting access to the HPLC instrument. This work\nalong with 6\u201331G** basis set for C, H, N, O while LANL2DZ was used was supported by the DST SERB Start-up Research Grant (Grant 2021435),\nfor Ir with e\ufb00ective core pseudopotential of the metal.[58\u201360] The opti- INSPIRE Faculty Research Grant (Grant 2019295), and Prime Minister Re-\nmized structure of IrL1 was docked to DNA dodecamer structure (PDB: search Grant (PMRF-ID: 2300572).\n1BNA)[61] using Autodock, version 1.5.7. Binding poses were viewed by\nDiscovery Studio software.\n Cisplatin and IrL1 Combination Study: In a 96-well plate, MDA-MB-231 Con\ufb02ict of Interest\ncells were seeded at a density of 2500 cells per well and incubated for 24\nh. Stock solutions of IrL1 and cisplatin were serially diluted in a culture The authors declare no con\ufb02ict of interest.\nmedium. The cells were then treated with di\ufb00erent concentrations of IrL1\nand cisplatin in the following ratio (IrL1 : Cisplatin): 1:1, 1:2, 2:1, 1:4, 4:1,\n1:8, and 8:1. The plate was then incubated for 48 h. The % cell viability was Author Contributions\ndetermined by performing an MTT assay. The experiment was performed\nin triplicates in a set of three independent experiments. The manuscript was written through the contributions of all authors. All\n Hemolytic Assay: To investigate the induction of hemolysis by IrL1 , authors have given approval to the \ufb01nal version of the manuscript. A.C.,\nfresh human blood was used in this experiment. Brie\ufb02y, the blood was col- N.S., R.P., and S.D. executed the synthesis. A.C. executed all characteri-\nlected and centrifuged at 1500 rpm for 10 min to remove blood plasma. zations. N.S. performed and analyzed the CV experiment. A.C. and A.K.\nThe pellet of red blood cells (RBCs) were then washed thrice in PBS. Af- worked on all mammalian cell-based experiments and molecular docking\nterward, RBCs were resuspended in 4% v/v in the PBS bu\ufb00er. Compound studies. A.C., A.K., and K.C. worked on in-vivo experiments. A.C. executed\nIrL1 was dissolved in DMSO and serially diluted. In the 96-well plate, 20 \u03bcL DFT studies. H.S. analyzed the X-ray structures. The authors declare no\nof each concentration was added to the 100 \u03bcL of RBC suspension in each competing \ufb01nancial interest.\nwell, and a volume of 200 \u03bcL was achieved by adding PBS. The plate was\nimmediately kept for the incubation of 1 h. After incubation, the plates\nwere centrifuged again at 1500 rpm for 10 min, the supernatant (20 \u03bcL)\n Data Availability Statement\nwas added to 80 \u03bcL of PBS in a fresh 96-well plate and hemoglobin re- The data that support the \ufb01ndings of this study are available from the\nlease was measured. The optical density (OD) at 414 nm was measured corresponding author upon reasonable request.\nfor the hemoglobin release by using a SpectraMax M5e microplate reader.\nFor positive control, 0.1% Triton X-100 was used whereas untreated RBC\nsuspension was used as negative control. Percentage hemolysis was cal- Keywords\nculated using the formula:\n ferroptosis, iridium, photoresponsive, triple negative breast cancer, tumor\n\n% Hemolysis = [(OD of Sample \u2212 OD of untreated cells)\u2215 Received: August 7, 2024\n Revised: October 23, 2024\n (OD of Triton X \u2212 OD of untreated cells)] \u00d7 100 (1) Published online: November 28, 2024\n\n\nSmall 2025, 21, 2406809 2406809 (17 of 19) \u00a9 2024 Wiley-VCH GmbH\n\f 16136829, 2025, 13, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smll.202406809 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. 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