Chlorine-Coordinated Iron Single-Atom Nanozymes for Amplified Ferroptosis in Triple-Negative Breast Cancer Therapy

Chlorine-Coordinated Iron Single-Atom Nanozymes for Amplified Ferroptosis in Triple-Negative Breast Cancer Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chlorine-Coordinated Iron Single-Atom Nanozymes for Amplified Ferroptosis in Triple-Negative Breast Cancer Therapy Mingming Yin, Bing-Hao Wang, Huijuan Wang, Jie Ouyang, Xingsheng Hu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7165005/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract Triple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype with limited therapeutic options and poor prognosis. Although single-atom nanozymes (SAzymes) show promise in cancer therapy, their ferroptosis-inducing capability remains limited. Herein, we present a rationally designed iron-based SAzyme with axial chlorine coordination (FeN₄Cl) that integrates catalytic and metabolic functions to enhance ferroptosis in TNBC. The engineered Fe-Cl coordination strategically modulates the d-band center relative to the Fermi level, resulting in significantly enhanced peroxidase-like activity (2.0-fold increase) and glutathione oxidase-like activity (3.2-fold increase) activities compared to conventional FeN₄ structures. Importantly, this electronic modulation triggers NCOA4-mediated ferritinophagy, establishing an autonomous iron supply mechanism that elevates intracellular labile Fe²⁺ levels. The synergistic disruption of redox homeostasis coupled with amplified Fenton reactions creates a potent feedback loop that induces cell death. By incorporating this SAzyme into a red blood cell membrane-based biomimetic delivery system (FeN₄Cl/RBC), we achieved enhanced biocompatibility and tumor targeting. In vivo studies demonstrated significant tumor suppression, presenting a promising approach for developing clinically relevant nanozyme-based therapeutics. Single-atom nanozyme Chlorine coordination Ferroptosis Ferritinophagy Triple-negative breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Triple-negative breast cancer (TNBC) is one of the most aggressive molecular subtypes of breast cancer, characterized by a high rate of metastasis, early recurrence, and poor prognosis. Due to the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2) amplification, TNBC does not respond to endocrine or targeted therapies, leaving cytotoxic chemotherapy as the primary—though often inadequate—treatment option.[ 1 – 3 ] According to the latest global cancer statistics, the global burden of breast cancer continues to rise. For patients with advanced or metastatic TNBC, the five-year survival rate drops below 30%, underscoring the urgent need for more effective therapeutic strategies.[ 4 ] This clinical challenge has sparked significant interest in targeting alternative cell death pathways. Among them, ferroptosis—an iron-dependent form of programmed cell death driven by lipid peroxidation—has emerged as a promising approach to overcoming TNBC's therapeutic resistance.[ 5 – 7 ] The canonical ferroptotic cascade involves glutathione (GSH) depletion, subsequent inactivation of glutathione peroxidase 4 (GPX4), and excessive accumulation of reactive oxygen species (ROS), culminating in fatal disruption of cellular redox homeostasis.[ 8 ] Intriguingly, the efficacy of ferroptosis is closely intertwined with that of autophagy, particularly ferritinophagy.[ 9 – 11 ] This process, which is mediated by nuclear receptor coactivator 4 (NCOA4), degrades the iron-storage protein ferritin, thereby releasing labile iron that can fuel the Fenton reaction, amplify ROS production, and potentiate ferroptosis.[ 12 – 14 ] Single-atom nanozymes (SAzymes), which have maximized atomic utilization, well-defined active sites, and potent catalytic activities, are exceptional candidates for inducing ferroptosis.[ 15 – 18 ] The catalytic performance of metal‒nitrogen‒carbon (M‒N‒C) materials is dictated by the electronic structure of the metal center. The local coordination environment directly modulates d-orbital occupancy, which in turn governs the adsorption energies of key reaction intermediates (e.g., ⋅OH, GSH) and thus the overall enzymatic activity.[ 19 , 20 ] Consequently, engineering the axial coordination of the central metal atom has been identified as a powerful strategy to tune the d-band center (E d ) relative to the Fermi level (E f ) and thereby optimize the catalytic function.[ 21 – 26 ] However, a significant hurdle persists: conventional synthesis methods, such as postmodification doping, often yield inhomogeneous coordination environments and irreproducible active sites, particularly struggling to achieve precise and uniform control over axial coordination, which limits rational design and consistent performance. Despite their promise, the application of SAzymes in ferroptosis therapy has been largely confined to a single dimension: mimicking peroxidase activity for ROS generation.[ 27 ] This singular focus overlooks the synergistic potential of a more holistic approach. An ideal ferroptosis-inducing agent must orchestrate a multipronged attack: not only generating ROS but also concurrently dismantling the cell's antioxidant defenses (e.g., via GSH depletion) and, most critically, modulating intracellular iron metabolism to self-amplify its therapeutic effect.[ 28 ] The activation of ferritinophagy to increase the labile iron pool is a key yet largely unaddressed component for maximizing ferroptotic efficacy, as most SAzyme designs lack the precise electronic and structural features—such as an optimally tuned d-band center and charge distribution—required to initiate this complex metabolic signaling.[ 29 ] In this study, we developed a multifunctional biomaterial platform designed to overcome current limitations in ferroptosis-based cancer therapy (Scheme 1 ). First, we synthesized an advanced single-atom nanozyme (FeN₄Cl) via a precoordination anchoring strategy, enabling atomic-level integration of an axial Cl ligand onto the FeN₄ active site. This unique axial Fe–Cl coordination significantly modulates the electronic structure, enhancing both peroxidase- and glutathione oxidase-like catalytic activities. Notably, for the first time, we demonstrated that this electronic modulation can activate NCOA4-mediated ferritinophagy, initiating a self-amplifying ferroptosis cycle. Finally, the FeN₄Cl nanozyme was encapsulated within biomimetic red blood cell membranes (FeN₄Cl/RBC), yielding excellent biocompatibility, enhanced tumor-targeting capability, and potent synergistic therapeutic effects against TNBC. 2. Materials and methods 2.1. Chemicals Zinc nitrate hexahydrate (Zn(NO₃)₂•6H₂O), iron(III) nitrate nonahydrate (Fe(NO₃)₃•9H₂O), and methanol (MeOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). 2-Methylimidazole (MeIM), hemin chloride, 3,3′,5,5′-tetramethylbenzidine (TMB), glutathione (GSH), and o-phenylenediamine (OPD) were obtained from Aladdin Industrial Corp. (Shanghai, China). The Cell Counting Kit-8 (CCK-8), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), Live/Dead Cell Staining Kit, malondialdehyde (MDA) assay kits, and mitochondrial membrane potential assay kits using JC-1 were acquired from Beyotime Biotechnology (Shanghai, China). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and Liperfluo probes were sourced from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). The anti-Glutathione Peroxidase 4 (GPX4) antibody was obtained from Abcam (Cambridge, UK), and primary antibodies against LC3, SQSTM1/p62, NCOA4, FTH1, and GAPDH were acquired from Cell Signaling Technology (Massachusetts, USA). 2.2. Synthesis of Hemin Cl/ZIF-8 Hemin Cl/ZIF-8 was synthesized following a procedure similar to that used for pristine ZIF-8, with one key modification: 90 mg of hemin Cl was added to Solution A prior to mixing with Solution B. The resulting composite was then dried in an oven at 70°C overnight. For comparison, Fe(NO₃)₃·9H₂O (55.8 mg, containing an equivalent amount of iron to 90 mg of hemin Cl) was used in place of hemin chloride to synthesize the FeN₄ SAC.[ 30 ] 2.3. Synthesis of N-C, FeN 4 and FeN 4 Cl Powdered samples of ZIF-8, Fe(NO₃)₃·9H₂O/ZIF-8, and hemin Cl/ZIF-8 were individually placed into ceramic boats and subjected to thermal activation in a tube furnace. The thermal treatment was carried out at 1000°C for 2 hours under a continuous nitrogen (N₂) flow, with a controlled heating rate of 5°C/min. The resulting carbon-based materials were designated as N–C, FeN₄, and FeN₄Cl, respectively. 2.4. Synthesis of FeN 4 Cl/RBC Mouse whole blood was centrifuged at 1,500 rpm for 5 minutes to isolate red blood cells (RBCs), which were washed three times with saline. RBCs were then lysed in 0.25× PBS under hypotonic conditions and centrifuged at 14,000 rpm for 10 minutes at 4°C to remove cytoplasmic contents. This process was repeated until the supernatant became colorless, indicating complete hemoglobin removal. For membrane coating, 10 mg of FeN₄Cl nanoparticles were co-sonicated with 10 mg of RBC membranes in PBS. The amphiphilic phospholipid bilayer facilitated hydrophobic interactions with FeN₄Cl surfaces. After three centrifugation–wash cycles to remove excess components, the mixture was extruded through a 200 nm polycarbonate membrane and stored at 4°C. 2.5. The peroxidase-like activities assay The peroxidase-like activity of FeN₄Cl nanozymes was assessed using a TMB–H₂O₂ chromogenic system, where FeN₄Cl catalyzed the oxidation of TMB to blue oxTMB, yielding an absorbance peak at 652 nm. For the assay, FeN₄Cl (5–30 µg/mL), 20 µL TMB (5 mg/mL), 20 µL 3% H₂O₂, and PBS were mixed to a final volume of 2 mL. After 5 minutes at 25°C, absorbance at 652 nm was measured by UV–vis spectroscopy. For kinetic analysis, mixtures contained 10 µL FeN₄Cl, 20 µL 3% H₂O₂, 150 µL HAc–NaAc buffer (pH 4.0), and 20 µL of TMB at varying concentrations (TMB kinetics), or varying H₂O₂ concentrations with fixed TMB (H₂O₂ kinetics). The peroxidase-catalyzed reaction was initiated by Kinetic parameters including the maximum reaction velocity ( V ₘₐₓ) and Michaelis-Menten constant ( K ₘ) were derived through linear regression analysis of Lineweaver-Burk plots. 2.6. The glutathione oxidase-like activities assay The glutathione oxidase (GSHOx)-like activity of FeN₄Cl was assessed using a coupled chromogenic system with reduced glutathione (GSH) as the substrate and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) as the thiol-detecting probe. Specifically, 0.1 mg of FeN₄Cl was added to 2 mL PBS buffer (pH 6.5) containing 0.25 mM DTNB and varying GSH concentrations (0.02–0.16 mM) to initiate the reaction. The formation of 5-thio-2-nitrobenzoic acid (TNB) was monitored at 412 nm via time-dependent UV–vis spectroscopy. Kinetic parameters were determined using Michaelis–Menten analysis based on initial reaction velocities measured within the first 3 minutes under pseudo-steady-state conditions. 2.7. Ti(SO 4 ) 2 terminational assay The Ti(SO₄)₂ method was used to assess H₂O₂ decomposition by NC, FeN₄, and FeN₄Cl under varying H₂O₂ concentrations.[ 31 ] Before testing, all catalysts were purged with N₂ for 30 minutes, a step repeated in all subsequent experiments. Residual H₂O₂ reacts with Ti(SO₄)₂ in H₂SO₄ to form a yellow complex with absorbance at 425 nm. In each assay, 25 µL of 10 mM H₂O₂ and 25 µL of 150 µg/mL catalyst were incubated in ultrapure water, then 50 µL of the mixture was added to 100 µL of Ti(SO₄)₂ solution (prepared from 1.33 mL of 24% Ti(SO₄)₂ and 8.33 mL of H₂SO₄ in 50 mL water). Absorbance at 425 nm was measured, and H₂O₂ decomposition efficiency was calculated using Eq. ( 1 ). $$\:{H}_{2}{O}_{2}\left(\text{%}\right)=\frac{{A}_{t}}{{A}_{0}}\times\:100\text{%}$$ 1 2.8. Quantified detection of hydroxyl radical by MB decay assays The Fenton catalytic activities of NC, FeN₄, and FeN₄Cl were assessed using methylene blue (MB) as a model substrate. MB (12.5 µg mL⁻¹), each catalyst (100 µg mL⁻¹), and H₂O₂ (0, 5, 10, or 20 mM) were mixed in a cuvette to initiate the reaction. Absorbance at 664 nm was recorded at 0, 5, 10, 20, and 30 minutes to monitor MB degradation. Degradation rates were calculated using Eq. ( 2 ).[ 32 ] $$\:MB\:degradation\:rate\left(\text{%}\right)=\left(1-\frac{{A}_{0}-{A}_{t}}{{A}_{0}}\right)\times\:100\text{%}$$ 2 2.9. Detection of •OH O-phenylenediamine (OPDA) was used to detect •OH radicals. FeN₄Cl (100 µg mL⁻¹) and 40 mM OPDA were incubated in pH 4.0 NaAc–HAc buffer (0.2 M) for 10 minutes. The luminescence spectrum was measured by fluorescence spectrophotometry, with intensity indicating •OH levels. 2.10. Cell culture MDA-MB-231 (human breast cancer), MCF-7 (human breast adenocarcinoma), 4T1 (mouse breast cancer), and HEK-293 (human embryonic kidney) cell lines used in this study were obtained from the Cancer Research Institute, Central South University (Changsha, Hunan, China). Cells were cultured in DMEM or RPMI (Gibco), supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Genview), and maintained at 37°C in a humidified incubator with 5% CO₂. 2.11. Hemolysis evaluation Fresh mouse blood (100 µL) was mixed with FeN₄Cl (1 mL) at concentrations of 12.5–200 µg mL⁻¹. Water and PBS were used as negative and positive controls, respectively. After 4 hours at 37°C, samples were centrifuged, and absorbance of the supernatants was measured at 570 nm. The hemolysis rate (%) was calculated as: Hemolysis rate (%) = (sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance) × 100%. 2.12. Cellular uptake MDA-MB-231 cells were incubated in confocal dishes for 24 hours, followed by a 4-hour incubation with Cy5.5-labeled FeN₄Cl. After three PBS washes and 10-minute Hoechst staining, samples were imaged using a confocal microscope (LSM980, Zeiss). 2.13. Cell viability assay Nanozyme cytotoxicity was assessed using a CCK-8 assay. MDA-MB-231 cells were cultured in RPMI-1640 with 10% FBS at 37°C and 5% CO₂, seeded into 96-well plates (1×10⁴ cells/well), and incubated for 24 hours. The medium was then replaced with fresh medium containing nanozymes (0–150 µg mL⁻¹) and incubated for another 24 hours. Cells were rinsed twice with cold PBS, followed by CCK-8 assay. Absorbance was measured at 450 nm. Live/dead staining was performed using calcein-AM and propidium iodide (PI). Cells were seeded in confocal dishes, treated with nanozymes, stained with calcein-AM and PI (300 µL each), and imaged using a confocal microscope (LSM980, Zeiss). 2.14. Intracellular ROS Detection Intracellular ROS generated by the nanozyme was detected using DCFH-DA. MDA-MB-231 cells were seeded in confocal dishes and incubated for 24 hours, then treated with 50 µg mL⁻¹ nanozyme for 6 hours. DCFH-DA was added and incubated for 30 minutes at 37°C in the dark. After washing with PBS, cells were imaged using a confocal microscope (LSM980, Zeiss). 2.15. Bio-transmission Electron Microscopy (bio-TEM) For bio-TEM analysis, MDA-MB-231 cells were seeded into culture plates at 2 × 10⁶ cells per dish and treated for 12 hours with (a) PBS, (b) FeN₄ (50 µg mL⁻¹), (c) FeN₄Cl (50 µg mL⁻¹), or (d) FeN₄Cl/RBC (50 µg mL⁻¹). After treatment, cells were washed with PBS, digested with trypsin, and collected by centrifugation (1000 rpm, 5 min). The cell pellets were fixed in 2.5% glutaraldehyde (3 mL) for 30 minutes at room temperature in the dark, then processed for bio-TEM imaging. 2.16. Lipid Peroxidation (LPO) Detection Remove adherent cells, discard the medium, and wash with PBS. Add Liperfluo premixed in serum-free medium, and incubate at 37°C in the dark for 30 minutes. Wash three times with PBS, then digest with trypsin and collect the cell suspension in a 1.5 mL tube. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend cells in 150 µL PBS. Keep on ice in the dark until analysis. Perform flow cytometry, collecting 10,000 cells per sample. 2.17. Animal model All animal procedures were approved by the HNU Animal Ethics Committee (Approval No.: HNU-IACUC-2024-110). Female BALB/c nude mice (4 weeks old) were obtained from Jiangsu Jiyuan Yaokang Biotechnology Co., Ltd. MDA-MB-231 cells (1×10⁶ in 50 µL PBS) were subcutaneously injected into the dorsal region. Treatment began when tumor volume reached ~ 100 mm³. Mice were randomly divided into four groups and treated at 10 mg/kg. Body weight and tumor volume were monitored every other day. At the study’s end, mice were euthanized, tumors were weighed, and major organs and tumor tissues were collected for H&E staining and immunohistochemistry. 2.18. Tumor study When tumor volume reached 100 mm³, nanozymes were intravenously administered at 10 mg kg⁻¹. Control mice received PBS (n = 5). Body weight and tumor volume—calculated as (length × width²) / 2—were monitored throughout. After 14 days, mice were euthanized via cervical dislocation. Blood was collected from the orbital sinus for hematological analysis. Tumors and major organs (heart, liver, spleen, lung, kidney) were fixed in 10% formalin or collected for pathological examination. 2.19. Statistical analysis Data are presented as mean ± standard deviation (SD). Statistical analysis was performed using Student’s t-test or one-way ANOVA with GraphPad Prism 9.0. Significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001. 3. Results and Discussion 3.1. Synthesis and characterization of FeN₄Cl SAzymes The FeN₄Cl SAzymes were fabricated via a precoordination anchoring strategy designed to achieve precise axial chlorine ligation (Fig. 1 a). Initially, hemin Cl was trapped in situ during the self-assembly of ZIF-8, yielding a hemin Cl/ZIF-8 precursor.[ 30 ] This precursor retained the characteristic rhombic dodecahedral morphology and crystal structure of the parent ZIF-8 (Figures S1 , S2). Following high-temperature pyrolysis, the resulting FeN₄Cl SAzyme preserved the polyhedral morphology of its precursor (Fig. 1 b). X-ray diffraction (XRD) patterns of FeN₄Cl revealed only two broad peaks at 25° and 44°, corresponding to the (002) and (101) planes of graphitic carbon, with a notable absence of any peaks corresponding to metallic iron or other iron-based crystalline phases (Figure S3). This confirms the successful conversion of the precursor into a carbonaceous material without the formation of iron nanoparticles. To verify the atomic-level structure, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was employed. The images of FeN₄Cl displayed a high density of isolated bright spots uniformly distributed across the carbon support, which are characteristic of individual heavy atoms, confirming the single-atom dispersion of iron (Figs. 1 d, 1 e). Similarly, energy-dispersive X-ray spectroscopy (EDS) mapping revealed a homogeneous distribution of C, N, and Fe and, critically, Cl throughout the FeN₄Cl structure (Fig. 1 c). In contrast, the control sample (FeN₄), prepared without hemin chloride, showed no Cl signal (Figure S4). The iron contents in FeN₄Cl and FeN₄ were quantified by inductively coupled plasma‒mass spectrometry (ICP-MS) to be 0.28 wt% and 0.32 wt%, respectively. X-ray photoelectron spectroscopy (XPS) was then conducted to investigate the surface chemical states and bonding configurations. The high-resolution N 1 s spectra for both FeN₄Cl and FeN₄ were deconvoluted into pyridinic N, pyrrolic N, graphitic N, oxidized N, and Fe–N peaks (Figure S6), confirming the successful formation of Fe–N coordination sites. Notably, the FeN₄Cl SAzyme had a higher content of pyridinic N (23.82%) than did FeN₄ (21.48%) (Table S1 ), which is considered beneficial for oxidase-like catalytic activities.[ 33 , 34 ] Direct evidence for the axial ligand came from the Cl 2p spectrum of FeN₄Cl, where a peak corresponding to Fe‒Cl bonds was observed at approximately 198 eV (Figure S7). Furthermore, the high-resolution Fe 2p spectrum of FeN₄Cl clearly shifted to higher binding energies than those of FeN₄ (Figure S8). This positive shift indicates a lower electron density on the Fe centers in FeN₄Cl, which is attributed to the strong electron-withdrawing effect of the highly electronegative axial Cl ligand. Finally, to elucidate the local coordination environment of the iron atoms, X-ray absorption fine structure (XAFS) measurements were performed. The Fe K-edge X-ray absorption near-edge structure (XANES) spectrum of FeN₄Cl exhibited an absorption edge at 7122.47 eV, which was positioned between those of the Fe foil and Fe₂O₃ (Fig. 1 f). This corresponds to an average Fe oxidation state of approximately + 2.23 (Fig. 1 g, Table S2), confirming the cationic nature of the iron centers. The k²-weighted Fourier transform extended X-ray absorption fine structure (EXAFS) spectrum provided more detailed structural information (Fig. 1 h). The spectrum is dominated by a primary peak at ~ 1.5 Å, which corresponds to the Fe–N scattering path. Critically, a second, smaller peak at ~ 1.7 Å is also present, which is assigned to the Fe‒Cl scattering path. The complete absence of any Fe‒Fe signal at ~ 2.2 Å, further corroborated by wavelet transform (WT) analysis (Fig. 1 j), provides unequivocal evidence for the atomic dispersion of iron. Quantitative fitting of the EXAFS spectrum yielded average coordination numbers of approximately 3.3 for Fe-N and 0.8 for Fe-Cl (Fig. 1 i). These results collectively confirm the successful synthesis of the target FeN₄Cl structure, where a single iron atom is coordinated by nitrogen atoms in the basal plane and one chlorine atom in the axial position. 3.2. Dual Enzyme-Mimicking Activities of FeN₄Cl SAzymes Having established the unique FeN₄Cl atomic structure, we next evaluated its capacity to function as a dual-action nanozyme capable of both generating ROS and depleting glutathione (GSH), two key events for initiating ferroptosis (Fig. 2 a). We first assessed peroxidase (POD)-like activity via a 3,3’,5,5'-tetramethylbenzidine (TMB) colorimetric assay. After optimizing the synthesis and reaction conditions (Figure S9), we found that the FeN₄Cl SAzyme exhibited robust catalytic activity that was highly dependent on pH, with maximal performance under acidic conditions (pH 4.0–5.0) characteristic of the tumor microenvironment (Figure S10). The activity of FeN₄Cl, which was dependent on both the catalyst concentration and time (Figs. 2 c, 2 d), was significantly greater than the negligible activity of the metal-free carbon support (NC), confirming that the atomically dispersed Fe sites are the catalytic centers (Fig. 2 b). A steady-state kinetic analysis revealed that the POD-like activity of FeN₄Cl followed classic Michaelis‒Menten kinetics for both the TMB and H₂O₂ substrates (Figs. 2 e, 2 f). Critically, axial Cl- coordination imparted a substantial catalytic advantage over the conventional FeN₄ analog. FeN₄Cl demonstrated a 2.0-fold higher catalytic constant (k cat ) and a 2.2-fold greater catalytic efficiency (k cat /K m ) for TMB oxidation. Furthermore, its Michaelis constant (K m ) for H₂O₂ was lower than that of FeN₄ (4.23 mM vs. 4.77 mM), indicating a stronger binding affinity for the H₂O₂ substrate. This superior catalytic performance, particularly the high catalytic efficiency (k cat /K m ), places FeN₄Cl among the top-performing iron-based peroxidase mimics reported to date (Table S3), especially under acidic conditions relevant to the tumor microenvironment, underscoring its therapeutic potential. To further confirm the reaction mechanism, poisoning experiments with thiocyanate (KSCN), a strong Fe-coordinating agent, led to significant suppression of POD-like activity, verifying the essential role of the single-atom Fe centers (Fig. 2 g). We also confirmed that the catalysis proceeded via the generation of hydroxyl radicals (•OH), as evidenced by the consumption of H₂O₂ (Fig. 2 h) and the positive detection of •OH via both the o-phenylenediamine (OPDA) probe and a methylene blue (MB) degradation assay (Figures S11, S12). In addition to generating ROS, an effective ferroptosis-inducing agent must dismantle the cell's primary antioxidant defense system, which is maintained by high levels of intracellular GSH. We therefore investigated the glutathione oxidase (GSHOx)-like activity of the nanozymes. Using a 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) assay, we found that FeN₄Cl was a remarkably potent catalyst for GSH depletion, far outperforming the FeN₄ control (Fig. 2 i). The GSH consumption rate was positively correlated with both the FeN₄Cl concentration and reaction time (Fig. 2 j, 2 k) and followed Michaelis–Menten kinetics (Fig. 2 l). Compared with FeN₄, FeN₄Cl exhibited a 2.79-fold greater maximum reaction velocity (V max ), demonstrating its vastly superior ability to rapidly deplete GSH. This potent GSHOx-like activity, in concert with its enhanced POD-like function, equips the FeN₄Cl SAzyme with a powerful two-pronged attack to disrupt cellular redox homeostasis and trigger robust ferroptosis. Axial Fe-Cl-Coordinated Single-Atom Nanozymes Amplify Ferritinophagy-Dependent Ferroptosis for TNBC Therapy. 3.3. Theoretical Insights into the Enhanced Catalytic Activity via DFT To elucidate the underlying mechanism for the enhanced peroxidase-like activity, we performed density functional theory (DFT) calculations. Our analysis revealed that the axial coordination of a single Cl atom profoundly modulates the electronic properties of the FeN₄ center. Charge density difference analysis revealed significant electronic rearrangement around the Fe active site upon Cl coordination (Figure S13). This resulted in the d-band center of FeN₄Cl shifting upward from − 1.09 eV to -0.93 eV relative to the Fermi level (Fig. 3 a). According to d-band theory, this upward shift closer to the Fermi level optimizes the adsorption energy of key reaction intermediates, which is a hallmark of enhanced catalytic activity. This electronic modulation was further quantified by Bader charge analysis, which revealed that the charge on the Fe atom increased from + 1.09 |e| in FeN₄ to + 1.11 |e| in FeN₄Cl, confirming the strong electron-withdrawing effect of the axial Cl ligand. We then calculated the Gibbs free energy profiles to map the reaction pathways for POD-like activity (Fig. 3 b, Figure S14). The calculations confirmed that the decomposition of H₂O₂ on the FeN₄Cl surface proceeded via an efficient Fenton-like mechanism. The process begins with the adsorption of H₂O₂, followed by homolytic cleavage into two adsorbed hydroxyl (*OH) groups. One *OH group desorbs as a free hydroxyl radical (•OH), whereas the other is protonated to form a water molecule, regenerating the catalytic site. Critically, the FeN₄Cl structure significantly lowers the activation energy barrier for the rate-determining step—the homolytic cleavage of H₂O₂ (H₂O₂ → 2OH). This energy barrier was calculated to be only 1.17 eV for FeN₄Cl, which is substantially lower than the 1.48 eV required for the FeN₄ system (Fig. 3 c). This theoretical result provides a direct explanation for the experimentally observed increase in POD-like activity. Finally, we analyzed the projected density of states (PDOS) to understand how orbital interactions contribute to this enhancement. The PDOS plots show that upon Cl coordination, the density of states for FeN₄Cl near the Fermi level increases significantly, and new d-p hybridized electronic states appear (Figs. 3 d, 3 e). This indicates that the axial Cl ligand not only induces charge redistribution but also promotes stronger orbital coupling between the Fe 3d orbitals and the substrate molecules. This enhanced orbital overlap facilitates more efficient interfacial charge transfer, thereby accelerating the overall catalytic kinetics. Collectively, these DFT results provide a robust theoretical foundation, demonstrating that axial Cl coordination is a highly effective strategy for rationally designing SAzymes with superior catalytic performance. 3.4. In vitro therapeutic efficacy and biocompatibility Inspired by the superior dual-enzyme activities of FeN₄Cl, we investigated its therapeutic potential against TNBC cells in vitro. To enhance their biocompatibility and physiological stability, the FeN₄Cl nanozymes were camouflaged with red blood cell membranes to form FeN₄Cl/RBCs. Successful coating was confirmed by SDS‒PAGE analysis, which revealed the characteristic protein bands of the RBC membranes, as well as a reversal of the surface zeta potential (Figures S15, S17). The resulting FeN₄Cl/RBC nanoparticles were well dispersed with a hydrodynamic diameter of approximately 150 nm (Figure S16) and exhibited excellent blood safety, with a hemolysis rate below the 5% international safety threshold even at high concentrations (Figure S18). Confocal microscopy tracking of Cy5.5-labeled FeN₄Cl/RBC revealed efficient internalization by MDA-MB-231 cells within 4 hours, with significant accumulation in lysosomes (Fig. 4 a). This lysosomal localization was further corroborated by biological TEM images (Fig. 4 b). Having confirmed cellular uptake, we assessed cytotoxicity via a CCK-8 assay. FeN₄Cl/RBC induced potent, dose-dependent cell death in MDA-MB-231 cells, reducing viability to below 10% at a concentration of 150 µg/mL (Fig. 4 c). This potent anticancer effect was also observed in other breast cancer cell lines, including 4T1 and MCF-7 cells (Fig. 4 d), and was visually confirmed by live/dead cell staining (Fig. 4 e). Crucially, FeN₄Cl/RBC exhibited high selectivity, showing negligible toxicity toward normal HEK293 cells (Figure S19), likely due to the reliance of its catalytic activity on the acidic and H₂O₂-rich microenvironment characteristic of tumor cells. 3.5. Elucidation of the Ferroptosis Induction Mechanism Having established the potent and selective cytotoxicity of FeN₄Cl/RBC, we next dissected its molecular mechanism of action. As hypothesized, treatment with FeN₄Cl/RBC induced a massive burst of intracellular ROS in MDA-MB-231 cells, as indicated by the strong fluorescence of the H₂DCFDA probe (Fig. 5 a, 5 b). This surge in oxidative stress led to extensive lipid peroxidation (LPO), a hallmark of ferroptosis. This was confirmed by multiple lines of evidence: a significant increase in the LPO end-product malondialdehyde (MDA) (Fig. 5 c) and intense green fluorescence from the LPO-sensitive probe BODIPY 581/591 C11 (Fig. 5 e) were detected. Concurrently, the expression of glutathione peroxidase 4 (GPX4), the key enzyme that repairs lipid peroxides, was dramatically downregulated in FeN₄Cl/RBC-treated cells (Fig. 5 d). This inactivation is a direct consequence of the potent GSHOx-like activity of the nanozyme, which depletes the GPX4 cofactor GSH. To definitively confirm that ferroptosis is a type of cell death, we cotreated cells with the specific ferroptosis inhibitor ferrostatin-1 (Fer-1). As expected, Fer-1 markedly attenuated the LPO accumulation induced by FeN₄Cl/RBC (Fig. 5 e, 5 g), confirming that the cell death cascade is indeed ferroptosis. In support of this conclusion, FeN₄Cl/RBC treatment caused severe mitochondrial dysfunction, another characteristic feature of ferroptosis,[ 16 ] as evidenced by a collapse in the mitochondrial membrane potential (JC-1 assay, Fig. 5 f) and a 44% reduction in cellular ATP production (Fig. 5 h). 3.6. Activation of Ferritinophagy Synergistically Amplifies Ferroptosis In addition to directly triggering ferroptosis, we investigated whether FeN₄Cl/RBCs could activate synergistic pathways. TEM analysis of treated cells revealed a marked accumulation of autophagosomes (Fig. 6 a), and Western blot assays confirmed an increase in autophagic flux, as shown by decreased p62 and increased lipidated LC3-II levels (Fig. 6 b, 6 c). We then specifically probed for ferritinophagy, the selective autophagy of the iron-storage protein ferritin. Strikingly, treatment with FeN₄Cl/RBC led to significant downregulation of ferritin heavy chain 1 (FTH1) and corresponding upregulation of its cargo receptor, NCOA4 (Fig. 6 d, 6 e). These findings provide clear evidence for the activation of NCOA4-mediated ferritinophagy. The functional consequence of this process—the degradation of ferritin—was a significant increase in the intracellular labile iron (Fe²⁺) pool (Fig. 6 f). Collectively, these results reveal a sophisticated and highly effective dual-pathway mechanism (Fig. 6 g). The FeN₄Cl/RBC nanozyme not only initiates ferroptosis by disrupting the GPX4-GSH axis but also synergistically amplifies it by activating ferritinophagy to self-supply the catalytic Fe²⁺ required to fuel the ROS-generating Fenton reaction, creating a deadly feedback loop within the cancer cell. 3.7. In vivo antitumor efficacy and biosafety Encouraged by the potent in vitro cytotoxicity, we proceeded to evaluate the therapeutic efficacy of FeN₄Cl/RBC in a murine xenograft model of human TNBC (MDA-MB-231). Following intravenous administration, real-time fluorescence imaging of Cy5.5-labeled FeN₄Cl/RBCs revealed significant and sustained accumulation at the tumor site for up to 48 hours. This biomimetic camouflage endows the nanoparticles with a 'stealth' capability, significantly prolonging their blood circulation time and thereby maximizing their opportunity for passive accumulation at the tumor site via the enhanced permeability and retention (EPR) effect (Figure S20). For the therapeutic study, tumor-bearing mice were randomized into four groups and received intravenous injections of PBS, FeN₄, FeN₄Cl, or FeN₄Cl/RBC (Fig. 7 a). The FeN₄Cl/RBC group exhibited profound tumor suppression, significantly outperforming all the other treatment groups and leading to nearly complete tumor regression by the end of the study (Fig. 7 c- 7 f). The potent in vivo efficacy of our FeN₄Cl/RBC nanoplatform compares favorably with that of other state-of-the-art ferroptosis-inducing nanobiomaterials recently reported for cancer therapy (Table S4), underscoring the superiority of our synergistic catalytic-metabolic design. Histological analysis of tumors from the FeN₄Cl/RBC group via H&E staining revealed extensive tissue damage, including vacuolar degeneration and nuclear pyknosis, which was corroborated by TUNEL staining, indicating widespread cell death (Fig. 7 g). To confirm that this therapeutic effect was driven by the intended mechanism in vivo, we performed immunohistochemical staining on the tumor tissues. As observed in vitro, tumors treated with FeN₄Cl/RBC presented markedly downregulated GPX4 expression and upregulated LC3 levels, confirming the induction of ferroptosis and autophagy within the tumor microenvironment (Fig. 7 h). Finally, a comprehensive systemic safety evaluation was conducted. Throughout the treatment period, no significant body weight fluctuations were observed in any group (Figure S21). Posttreatment H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) revealed no signs of pathological damage or inflammation (Figure S22). Furthermore, hematological and serum biochemical analyses confirmed that treatment with FeN₄Cl/RBC did not induce any adverse effects on liver or kidney function (Figure S23). Collectively, these results demonstrate that FeN₄Cl/RBC is a highly potent and safe nanotherapeutic agent for TNBC treatment in vivo. 4. Conclusions In conclusion, we constructed a novel single-atom nanozyme (FeN₄Cl) with axial Fe–Cl coordination that enables precise electronic modulation and substantially enhances dual enzyme-mimicking activities. The axial Cl ligand elevates the d-band center of the Fe site, thereby promoting both peroxidase- and glutathione oxidase-like catalytic performance. Mechanistically, the increased oxidative stress induced by FeN₄Cl activates NCOA4-mediated ferritinophagy, establishing a self-reinforcing ferroptotic loop through metabolic iron mobilization. Furthermore, red blood cell (RBC) membrane cloaking endows the nanozyme with excellent biocompatibility, prolonged circulation, and tumor-targeting capacity, resulting in potent ferroptosis-mediated antitumor efficacy in vivo without systemic toxicity. This study provides a paradigm for atomic-level nanozyme engineering coupled with metabolic reprogramming, offering promising translational potential for the treatment of TNBC. Declarations Acknowledgment s # Mingming Yin and Bing-Hao Wang contributed equally to this work. This work was financially supported by the National Natural Science Foundation of China (No. 22478103), the Key Project of the Hunan Provincial Natural Science Foundation of China (No. 2025JJ90010), the Key Project of the Hunan Provincial Department of Education (No. 23A0301), the Open Project of the State Key Laboratory of Chemical Biosensing and Chemometrics, Hunan University (No. 20240600). Author Contributions Mingming Yin : Investigation, Conceptualization, Formal analysis, Methodology, Investigation, Writing – original draft. Bing-Hao Wang: Formal analysis, Methodology, Writing – original draft. Huijuan Wang: Investigation, Methodology. Jie Ouyang: Formal analysis. Xingsheng Hu: Formal analysis. Xiong Wang: Formal analysis. Yongping Liu: Validation. Fenghua Xu: Conceptualization, Writing – review & editing. Yi Chen : Project administration, Supervision, Writing – review & editing, Funding acquisition. Shuang-Feng Yin: Project administration, Supervision, Writing – review & editing, Funding acquisition. Data Availability The datasets supporting the conclusions of this article are included within the article and its supplementary information files. Ethics approval and consent to participate All animal experiments were conducted in accordance with the guidelines of the Hunan University Animal Care and Use Committee (Approval No. HNU-IACUC-2024-110). Consent for publication All authors have approved the manuscript and agree for the submission. Competing interests The authors declare no competing interests. Supplementary Information The online version contains supplementary material available at DOI: to be assigned upon publication. (See Supplementary Information file) References Bianchini G, De Angelis C, Licata L, Gianni L. 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Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Revision requested 30 Oct, 2025 Reviews received at journal 30 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviewers agreed at journal 08 Oct, 2025 Reviewers agreed at journal 06 Oct, 2025 Reviewers invited by journal 23 Sep, 2025 Editor assigned by journal 22 Jul, 2025 Submission checks completed at journal 22 Jul, 2025 First submitted to journal 19 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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15:38:39","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166782,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/fbc5383ac945b0ae8e1ea362.png"},{"id":92880524,"identity":"bb988148-c777-4ace-b28c-8013576dd36b","added_by":"auto","created_at":"2025-10-06 15:30:39","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":192480,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/0aec7f1d233354d91a5c7565.png"},{"id":92881744,"identity":"bc03ccea-9936-4456-9a72-a19cd2a8249c","added_by":"auto","created_at":"2025-10-06 15:38:40","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130383,"visible":true,"origin":"","legend":"","description":"","filename":"0ac13794cb654f8db6fe51beb17a52f91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/ed10528cbd7c420842a55a8c.xml"},{"id":92880537,"identity":"19823a9a-bc2c-4162-877c-af2fbb64635a","added_by":"auto","created_at":"2025-10-06 15:30:40","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":137494,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/a4d4692595ef3814be5e9cec.html"},{"id":92881734,"identity":"8be8cfbe-6dde-401b-8a81-3c62ea3e81d7","added_by":"auto","created_at":"2025-10-06 15:38:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1175301,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis and Structural Characterization of FeN₄Cl. (a) Schematic illustration of the synthesis process for FeN₄Cl. (b) TEM image of FeN₄Cl. (c) Elemental mapping of C, N, Cl, and Fe in FeN₄Cl. (d, e) HAADF-STEM images of FeN₄Cl showing atomically dispersed Fe single-atom sites as bright dots (yellow circles indicate individual atoms). (f) Fe K-edge XANES spectra. (g) Comparison of the average oxidation state of Fe in FeN₄Cl. (h) EXAFS fitting curve of FeN₄Cl. (i) EXAFS fitting curve of FeN₄Cl in k space. (j) Wavelet transform (WT) contour plots of FeN₄Cl compared with those of Fe foil, Fe₂O₃, and Fe₃O₄.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/0e71dae68f74c20aab399b7a.png"},{"id":92880515,"identity":"b90e1e19-4cdf-441c-8a1b-450f9f5beca9","added_by":"auto","created_at":"2025-10-06 15:30:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":461866,"visible":true,"origin":"","legend":"\u003cp\u003eCatalytic and Enzymatic Activity Analysis of FeN₄Cl SAzyme. (a) Schematic representation of the peroxidase-like activity of FeN₄Cl SAzyme. (b) Comparison of peroxidase (POD) activity across different groups. (c) Concentration-dependent and (d) time-dependent oxidation of TMB by FeN₄Cl. (e, f) Steady-state kinetic analysis of FeN₄Cl using (e) TMB and (f) H₂O₂ as substrates. (g) Catalytic activity of NC, FeN₄, and FeN₄Cl with or without KSCN in the presence of H₂O₂ and a Ti(SO₄)₂ mixture. (h) Corresponding absorption spectra of H₂O₂–Ti(SO₄)₂ solutions with NC, FeN₄, and FeN₄Cl. (i) Comparison of glutathione oxidase (GSHOx) activity across different groups. (j) FeN₄Cl concentration-dependent and (k) reaction time-dependent GSH consumption. (l) GSHOx-like enzyme kinetics of FeN₄ and FeN₄Cl.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/68db950732f350f5b834e518.png"},{"id":92880517,"identity":"7c5ecaa5-198e-4206-b308-484d4093edbf","added_by":"auto","created_at":"2025-10-06 15:30:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":323855,"visible":true,"origin":"","legend":"\u003cp\u003eDFT Calculations of the Peroxidase-like Activity of FeN₄Cl. (a) Computed partial density of states (PDOS) of the Fe d orbitals for FeN₄ and FeN₄Cl. (b) Proposed catalytic mechanism of FeN₄Cl for peroxidase (POD)-like reactions. (c) Corresponding free energy diagram for POD-like reactions on FeN₄ and FeN₄Cl. (d, e) Projected density of states (PDOS) for (d) FeN₄ and (e) FeN₄Cl.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/959e5b5f3ce04cfcebd4f76e.png"},{"id":92881737,"identity":"64b31dd4-2476-4292-ac81-9b83c8003b50","added_by":"auto","created_at":"2025-10-06 15:38:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1024837,"visible":true,"origin":"","legend":"\u003cp\u003eIntracellular Localization and Cytotoxicity Assessment of FeN₄Cl/RBC SAzyme. (a) Intracellular localization of the FeN₄Cl/RBC SAzyme. (b) Bio-TEM images of MDA-MB-231 cells after 12 h of coincubation with FeN₄Cl/RBC. Red arrows indicate localization within endosomes/lysosomes; yellow arrows highlight nanoparticles within the cytoplasm. (c) Cell viability of MDA-MB-231 cells following treatment with FeN₄, FeN₄Cl, or FeN₄Cl/RBC. (d) Cell viability of various breast cancer cell lines after coculture with FeN₄Cl/RBC. (e) Fluorescence imaging and quantitative analysis of live/dead MDA-MB-231 cells. Data are presented as mean ± SD (n = 3). Statistical significance was determined by one-way ANOVA. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/0a69cfde399770aa1371fd9d.png"},{"id":92880520,"identity":"e8f40f57-4999-4163-bab1-21a6ce47d872","added_by":"auto","created_at":"2025-10-06 15:30:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":733637,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of Ferroptosis Induction by FeN₄Cl-Based SAzymes in MDA-MB-231 Cells. (a) CLSM images of DCFH-DA–stained MDA-MB-231 cells. (b) Quantitative analysis of intracellular ROS levels. (c) Measurement of MDA production. (d) Western blot analysis and quantitative evaluation of GPX4 protein expression in MDA-MB-231 cells after 24 h of treatment with various formulations. (e) Quantification of intracellular LPO levels. (f) CLSM images of MDA-MB-231 cells stained with JC-1 to assess mitochondrial membrane potential. (g) Quantitative analysis of intracellular LPO levels across different treatment groups: (i) Control, (ii) FeN₄, (iii) FeN₄Cl, (iv) FeN₄Cl/RBC, and (v) FeN₄Cl/RBC + Fer-1. (h) Measurement of intracellular ATP content in MDA-MB-231 cells following various treatments. (i) Schematic diagram illustrating the proposed ferroptosis pathway. Data are presented as mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA. ∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/a142f229341f99144742a7a1.png"},{"id":92881742,"identity":"89585612-ab5b-465a-85f9-6073e1d2b360","added_by":"auto","created_at":"2025-10-06 15:38:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":938429,"visible":true,"origin":"","legend":"\u003cp\u003eInvestigation of Autophagy-Mediated Ferroptosis in MDA-MB-231 Cells Treated with FeN₄Cl/RBC. (a) Representative TEM images of MDA-MB-231 cells treated with FeN₄Cl/RBC. Red arrows indicate mitochondria; white arrows indicate autophagosomes; yellow arrows indicate autophagolysosomes. (b) Western blot analysis and (c) quantitative evaluation of LC3-II and p62 protein expression in MDA-MB-231 cells after 24 h of treatment with different formulations. (d) Western blot analysis and (e) quantitative evaluation of FTH1 and NCOA4 protein expression in MDA-MB-231 cells after 24 h of treatment. (f) Detection of intracellular ferrous iron levels using the FerroOrange probe. (g) Schematic illustration of the proposed autophagy-mediated ferroptosis pathway. Statistical significance: ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ∗∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/62effee8ef19a3ac2b630cf3.png"},{"id":92882620,"identity":"af787c4c-98c2-4182-971c-24737eefc165","added_by":"auto","created_at":"2025-10-06 15:46:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1059056,"visible":true,"origin":"","legend":"\u003cp\u003eIn Vivo Antitumor Efficacy of FeN₄Cl/RBC in MDA-MB-231 Tumor-Bearing Mice. (a) Schematic illustration of the therapeutic strategy involving intravenous (i.v.) administration of FeN₄Cl/RBC in MDA-MB-231 tumor-bearing mice. (b) Individual tumor growth curves of MDA-MB-231 xenografts in mice treated with PBS, FeN₄, FeN₄Cl, or FeN₄Cl/RBC. (c) Tumor volume changes over time following i.v. injection in the control, FeN₄, FeN₄Cl, and FeN₄Cl/RBC groups. (d) Representative digital images of excised tumors from each group: (i) Control, (ii) FeN₄, (iii) FeN₄Cl, (iv) FeN₄Cl/RBC. (e) Tumor weight measurements across different treatment groups (mean ± SD, n = 5). (f) Quantitative evaluation of tumor inhibition in each group (n = 5). (g) H\u0026amp;E, TUNEL, and immunohistochemical staining (LC3, p62, and GPX4) of tumor tissue sections. Data are presented as mean ± SD (n = 5). Statistical significance was determined using one-way ANOVA. ∗∗∗\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/001150219878c9f702aeb7fc.png"},{"id":103765646,"identity":"83abe0b4-2bac-47a1-934f-2d65046f4f4e","added_by":"auto","created_at":"2026-03-02 16:06:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6402439,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/a85e3818-31a2-4243-9057-3b6dd3e420d2.pdf"},{"id":92880530,"identity":"74935eb5-82af-4d9e-86eb-53e83498b521","added_by":"auto","created_at":"2025-10-06 15:30:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4653316,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/7e3806fd76d838f1471a7c11.docx"},{"id":92881735,"identity":"a4a5314b-ccdb-4064-9487-27967e1c52a2","added_by":"auto","created_at":"2025-10-06 15:38:39","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1791131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Schematic illustration of the formation and d-band center upshift of FeN₄Cl, and the cascade catalytic mechanism of FeN₄Cl/RBC SAzyme inducing ferroptosis in TNBC.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7165005/v1/2e5a62ddf71c6cb860ced873.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chlorine-Coordinated Iron Single-Atom Nanozymes for Amplified Ferroptosis in Triple-Negative Breast Cancer Therapy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTriple-negative breast cancer (TNBC) is one of the most aggressive molecular subtypes of breast cancer, characterized by a high rate of metastasis, early recurrence, and poor prognosis. Due to the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2) amplification, TNBC does not respond to endocrine or targeted therapies, leaving cytotoxic chemotherapy as the primary\u0026mdash;though often inadequate\u0026mdash;treatment option.[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] According to the latest global cancer statistics, the global burden of breast cancer continues to rise. For patients with advanced or metastatic TNBC, the five-year survival rate drops below 30%, underscoring the urgent need for more effective therapeutic strategies.[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThis clinical challenge has sparked significant interest in targeting alternative cell death pathways. Among them, ferroptosis\u0026mdash;an iron-dependent form of programmed cell death driven by lipid peroxidation\u0026mdash;has emerged as a promising approach to overcoming TNBC's therapeutic resistance.[\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] The canonical ferroptotic cascade involves glutathione (GSH) depletion, subsequent inactivation of glutathione peroxidase 4 (GPX4), and excessive accumulation of reactive oxygen species (ROS), culminating in fatal disruption of cellular redox homeostasis.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] Intriguingly, the efficacy of ferroptosis is closely intertwined with that of autophagy, particularly ferritinophagy.[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] This process, which is mediated by nuclear receptor coactivator 4 (NCOA4), degrades the iron-storage protein ferritin, thereby releasing labile iron that can fuel the Fenton reaction, amplify ROS production, and potentiate ferroptosis.[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eSingle-atom nanozymes (SAzymes), which have maximized atomic utilization, well-defined active sites, and potent catalytic activities, are exceptional candidates for inducing ferroptosis.[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] The catalytic performance of metal‒nitrogen‒carbon (M‒N‒C) materials is dictated by the electronic structure of the metal center. The local coordination environment directly modulates d-orbital occupancy, which in turn governs the adsorption energies of key reaction intermediates (e.g., \u0026sdot;OH, GSH) and thus the overall enzymatic activity.[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Consequently, engineering the axial coordination of the central metal atom has been identified as a powerful strategy to tune the d-band center (E\u003csub\u003ed\u003c/sub\u003e) relative to the Fermi level (E\u003csub\u003ef\u003c/sub\u003e) and thereby optimize the catalytic function.[\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] However, a significant hurdle persists: conventional synthesis methods, such as postmodification doping, often yield inhomogeneous coordination environments and irreproducible active sites, particularly struggling to achieve precise and uniform control over axial coordination, which limits rational design and consistent performance.\u003c/p\u003e\u003cp\u003eDespite their promise, the application of SAzymes in ferroptosis therapy has been largely confined to a single dimension: mimicking peroxidase activity for ROS generation.[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] This singular focus overlooks the synergistic potential of a more holistic approach. An ideal ferroptosis-inducing agent must orchestrate a multipronged attack: not only generating ROS but also concurrently dismantling the cell's antioxidant defenses (e.g., via GSH depletion) and, most critically, modulating intracellular iron metabolism to self-amplify its therapeutic effect.[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] The activation of ferritinophagy to increase the labile iron pool is a key yet largely unaddressed component for maximizing ferroptotic efficacy, as most SAzyme designs lack the precise electronic and structural features\u0026mdash;such as an optimally tuned d-band center and charge distribution\u0026mdash;required to initiate this complex metabolic signaling.[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eIn this study, we developed a multifunctional biomaterial platform designed to overcome current limitations in ferroptosis-based cancer therapy (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). First, we synthesized an advanced single-atom nanozyme (FeN₄Cl) via a precoordination anchoring strategy, enabling atomic-level integration of an axial Cl ligand onto the FeN₄ active site. This unique axial Fe\u0026ndash;Cl coordination significantly modulates the electronic structure, enhancing both peroxidase- and glutathione oxidase-like catalytic activities. Notably, for the first time, we demonstrated that this electronic modulation can activate NCOA4-mediated ferritinophagy, initiating a self-amplifying ferroptosis cycle. Finally, the FeN₄Cl nanozyme was encapsulated within biomimetic red blood cell membranes (FeN₄Cl/RBC), yielding excellent biocompatibility, enhanced tumor-targeting capability, and potent synergistic therapeutic effects against TNBC.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Chemicals\u003c/h2\u003e\u003cp\u003eZinc nitrate hexahydrate (Zn(NO₃)₂\u0026bull;6H₂O), iron(III) nitrate nonahydrate (Fe(NO₃)₃\u0026bull;9H₂O), and methanol (MeOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). 2-Methylimidazole (MeIM), hemin chloride, 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB), glutathione (GSH), and o-phenylenediamine (OPD) were obtained from Aladdin Industrial Corp. (Shanghai, China). The Cell Counting Kit-8 (CCK-8), 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate (DCFH-DA), Live/Dead Cell Staining Kit, malondialdehyde (MDA) assay kits, and mitochondrial membrane potential assay kits using JC-1 were acquired from Beyotime Biotechnology (Shanghai, China). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and Liperfluo probes were sourced from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen, Carlsbad, CA, USA). The anti-Glutathione Peroxidase 4 (GPX4) antibody was obtained from Abcam (Cambridge, UK), and primary antibodies against LC3, SQSTM1/p62, NCOA4, FTH1, and GAPDH were acquired from Cell Signaling Technology (Massachusetts, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Synthesis of Hemin Cl/ZIF-8\u003c/h2\u003e\u003cp\u003eHemin Cl/ZIF-8 was synthesized following a procedure similar to that used for pristine ZIF-8, with one key modification: 90 mg of hemin Cl was added to Solution A prior to mixing with Solution B. The resulting composite was then dried in an oven at 70\u0026deg;C overnight. For comparison, Fe(NO₃)₃\u0026middot;9H₂O (55.8 mg, containing an equivalent amount of iron to 90 mg of hemin Cl) was used in place of hemin chloride to synthesize the FeN₄ SAC.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Synthesis of N-C, FeN\u003csub\u003e4\u003c/sub\u003e and FeN\u003csub\u003e4\u003c/sub\u003eCl\u003c/h2\u003e\u003cp\u003ePowdered samples of ZIF-8, Fe(NO₃)₃\u0026middot;9H₂O/ZIF-8, and hemin Cl/ZIF-8 were individually placed into ceramic boats and subjected to thermal activation in a tube furnace. The thermal treatment was carried out at 1000\u0026deg;C for 2 hours under a continuous nitrogen (N₂) flow, with a controlled heating rate of 5\u0026deg;C/min. The resulting carbon-based materials were designated as N\u0026ndash;C, FeN₄, and FeN₄Cl, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Synthesis of FeN\u003csub\u003e4\u003c/sub\u003eCl/RBC\u003c/h2\u003e\u003cp\u003eMouse whole blood was centrifuged at 1,500 rpm for 5 minutes to isolate red blood cells (RBCs), which were washed three times with saline. RBCs were then lysed in 0.25\u0026times; PBS under hypotonic conditions and centrifuged at 14,000 rpm for 10 minutes at 4\u0026deg;C to remove cytoplasmic contents. This process was repeated until the supernatant became colorless, indicating complete hemoglobin removal. For membrane coating, 10 mg of FeN₄Cl nanoparticles were co-sonicated with 10 mg of RBC membranes in PBS. The amphiphilic phospholipid bilayer facilitated hydrophobic interactions with FeN₄Cl surfaces. After three centrifugation\u0026ndash;wash cycles to remove excess components, the mixture was extruded through a 200 nm polycarbonate membrane and stored at 4\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. The peroxidase-like activities assay\u003c/h2\u003e\u003cp\u003eThe peroxidase-like activity of FeN₄Cl nanozymes was assessed using a TMB\u0026ndash;H₂O₂ chromogenic system, where FeN₄Cl catalyzed the oxidation of TMB to blue oxTMB, yielding an absorbance peak at 652 nm. For the assay, FeN₄Cl (5\u0026ndash;30 \u0026micro;g/mL), 20 \u0026micro;L TMB (5 mg/mL), 20 \u0026micro;L 3% H₂O₂, and PBS were mixed to a final volume of 2 mL. After 5 minutes at 25\u0026deg;C, absorbance at 652 nm was measured by UV\u0026ndash;vis spectroscopy. For kinetic analysis, mixtures contained 10 \u0026micro;L FeN₄Cl, 20 \u0026micro;L 3% H₂O₂, 150 \u0026micro;L HAc\u0026ndash;NaAc buffer (pH 4.0), and 20 \u0026micro;L of TMB at varying concentrations (TMB kinetics), or varying H₂O₂ concentrations with fixed TMB (H₂O₂ kinetics). The peroxidase-catalyzed reaction was initiated by Kinetic parameters including the maximum reaction velocity (\u003cem\u003eV\u003c/em\u003eₘₐₓ) and Michaelis-Menten constant (\u003cem\u003eK\u003c/em\u003eₘ) were derived through linear regression analysis of Lineweaver-Burk plots.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. The glutathione oxidase-like activities assay\u003c/h2\u003e\u003cp\u003eThe glutathione oxidase (GSHOx)-like activity of FeN₄Cl was assessed using a coupled chromogenic system with reduced glutathione (GSH) as the substrate and 5,5\u0026prime;-dithiobis(2-nitrobenzoic acid) (DTNB) as the thiol-detecting probe. Specifically, 0.1 mg of FeN₄Cl was added to 2 mL PBS buffer (pH 6.5) containing 0.25 mM DTNB and varying GSH concentrations (0.02\u0026ndash;0.16 mM) to initiate the reaction. The formation of 5-thio-2-nitrobenzoic acid (TNB) was monitored at 412 nm via time-dependent UV\u0026ndash;vis spectroscopy. Kinetic parameters were determined using Michaelis\u0026ndash;Menten analysis based on initial reaction velocities measured within the first 3 minutes under pseudo-steady-state conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Ti(SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e terminational assay\u003c/h2\u003e\u003cp\u003eThe Ti(SO₄)₂ method was used to assess H₂O₂ decomposition by NC, FeN₄, and FeN₄Cl under varying H₂O₂ concentrations.[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] Before testing, all catalysts were purged with N₂ for 30 minutes, a step repeated in all subsequent experiments. Residual H₂O₂ reacts with Ti(SO₄)₂ in H₂SO₄ to form a yellow complex with absorbance at 425 nm. In each assay, 25 \u0026micro;L of 10 mM H₂O₂ and 25 \u0026micro;L of 150 \u0026micro;g/mL catalyst were incubated in ultrapure water, then 50 \u0026micro;L of the mixture was added to 100 \u0026micro;L of Ti(SO₄)₂ solution (prepared from 1.33 mL of 24% Ti(SO₄)₂ and 8.33 mL of H₂SO₄ in 50 mL water). Absorbance at 425 nm was measured, and H₂O₂ decomposition efficiency was calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{H}_{2}{O}_{2}\\left(\\text{%}\\right)=\\frac{{A}_{t}}{{A}_{0}}\\times\\:100\\text{%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Quantified detection of hydroxyl radical by MB decay assays\u003c/h2\u003e\u003cp\u003eThe Fenton catalytic activities of NC, FeN₄, and FeN₄Cl were assessed using methylene blue (MB) as a model substrate. MB (12.5 \u0026micro;g mL⁻\u0026sup1;), each catalyst (100 \u0026micro;g mL⁻\u0026sup1;), and H₂O₂ (0, 5, 10, or 20 mM) were mixed in a cuvette to initiate the reaction. Absorbance at 664 nm was recorded at 0, 5, 10, 20, and 30 minutes to monitor MB degradation. Degradation rates were calculated using Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:MB\\:degradation\\:rate\\left(\\text{%}\\right)=\\left(1-\\frac{{A}_{0}-{A}_{t}}{{A}_{0}}\\right)\\times\\:100\\text{%}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Detection of \u0026bull;OH\u003c/h2\u003e\u003cp\u003eO-phenylenediamine (OPDA) was used to detect \u0026bull;OH radicals. FeN₄Cl (100 \u0026micro;g mL⁻\u0026sup1;) and 40 mM OPDA were incubated in pH 4.0 NaAc\u0026ndash;HAc buffer (0.2 M) for 10 minutes. The luminescence spectrum was measured by fluorescence spectrophotometry, with intensity indicating \u0026bull;OH levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Cell culture\u003c/h2\u003e\u003cp\u003eMDA-MB-231 (human breast cancer), MCF-7 (human breast adenocarcinoma), 4T1 (mouse breast cancer), and HEK-293 (human embryonic kidney) cell lines used in this study were obtained from the Cancer Research Institute, Central South University (Changsha, Hunan, China). Cells were cultured in DMEM or RPMI (Gibco), supplemented with 10% fetal bovine serum and 1% penicillin\u0026ndash;streptomycin (Genview), and maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Hemolysis evaluation\u003c/h2\u003e\u003cp\u003eFresh mouse blood (100 \u0026micro;L) was mixed with FeN₄Cl (1 mL) at concentrations of 12.5\u0026ndash;200 \u0026micro;g mL⁻\u0026sup1;. Water and PBS were used as negative and positive controls, respectively. After 4 hours at 37\u0026deg;C, samples were centrifuged, and absorbance of the supernatants was measured at 570 nm. The hemolysis rate (%) was calculated as: Hemolysis rate (%) = (sample absorbance\u0026thinsp;\u0026minus;\u0026thinsp;negative control absorbance)/(positive control absorbance\u0026thinsp;\u0026minus;\u0026thinsp;negative control absorbance) \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Cellular uptake\u003c/h2\u003e\u003cp\u003eMDA-MB-231 cells were incubated in confocal dishes for 24 hours, followed by a 4-hour incubation with Cy5.5-labeled FeN₄Cl. After three PBS washes and 10-minute Hoechst staining, samples were imaged using a confocal microscope (LSM980, Zeiss).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13. Cell viability assay\u003c/h2\u003e\u003cp\u003eNanozyme cytotoxicity was assessed using a CCK-8 assay. MDA-MB-231 cells were cultured in RPMI-1640 with 10% FBS at 37\u0026deg;C and 5% CO₂, seeded into 96-well plates (1\u0026times;10⁴ cells/well), and incubated for 24 hours. The medium was then replaced with fresh medium containing nanozymes (0\u0026ndash;150 \u0026micro;g mL⁻\u0026sup1;) and incubated for another 24 hours. Cells were rinsed twice with cold PBS, followed by CCK-8 assay. Absorbance was measured at 450 nm.\u003c/p\u003e\u003cp\u003eLive/dead staining was performed using calcein-AM and propidium iodide (PI). Cells were seeded in confocal dishes, treated with nanozymes, stained with calcein-AM and PI (300 \u0026micro;L each), and imaged using a confocal microscope (LSM980, Zeiss).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14. Intracellular ROS Detection\u003c/h2\u003e\u003cp\u003eIntracellular ROS generated by the nanozyme was detected using DCFH-DA. MDA-MB-231 cells were seeded in confocal dishes and incubated for 24 hours, then treated with 50 \u0026micro;g mL⁻\u0026sup1; nanozyme for 6 hours. DCFH-DA was added and incubated for 30 minutes at 37\u0026deg;C in the dark. After washing with PBS, cells were imaged using a confocal microscope (LSM980, Zeiss).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15. Bio-transmission Electron Microscopy (bio-TEM)\u003c/h2\u003e\u003cp\u003eFor bio-TEM analysis, MDA-MB-231 cells were seeded into culture plates at 2 \u0026times; 10⁶ cells per dish and treated for 12 hours with (a) PBS, (b) FeN₄ (50 \u0026micro;g mL⁻\u0026sup1;), (c) FeN₄Cl (50 \u0026micro;g mL⁻\u0026sup1;), or (d) FeN₄Cl/RBC (50 \u0026micro;g mL⁻\u0026sup1;). After treatment, cells were washed with PBS, digested with trypsin, and collected by centrifugation (1000 rpm, 5 min). The cell pellets were fixed in 2.5% glutaraldehyde (3 mL) for 30 minutes at room temperature in the dark, then processed for bio-TEM imaging.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e2.16. Lipid Peroxidation (LPO) Detection\u003c/h2\u003e\u003cp\u003eRemove adherent cells, discard the medium, and wash with PBS. Add Liperfluo premixed in serum-free medium, and incubate at 37\u0026deg;C in the dark for 30 minutes. Wash three times with PBS, then digest with trypsin and collect the cell suspension in a 1.5 mL tube. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend cells in 150 \u0026micro;L PBS. Keep on ice in the dark until analysis. Perform flow cytometry, collecting 10,000 cells per sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e2.17. Animal model\u003c/h2\u003e\u003cp\u003e All animal procedures were approved by the HNU Animal Ethics Committee (Approval No.: HNU-IACUC-2024-110). Female BALB/c nude mice (4 weeks old) were obtained from Jiangsu Jiyuan Yaokang Biotechnology Co., Ltd. MDA-MB-231 cells (1\u0026times;10⁶ in 50 \u0026micro;L PBS) were subcutaneously injected into the dorsal region. Treatment began when tumor volume reached\u0026thinsp;~\u0026thinsp;100 mm\u0026sup3;. Mice were randomly divided into four groups and treated at 10 mg/kg. Body weight and tumor volume were monitored every other day. At the study\u0026rsquo;s end, mice were euthanized, tumors were weighed, and major organs and tumor tissues were collected for H\u0026amp;E staining and immunohistochemistry.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.18. Tumor study\u003c/h2\u003e\u003cp\u003eWhen tumor volume reached 100 mm\u0026sup3;, nanozymes were intravenously administered at 10 mg kg⁻\u0026sup1;. Control mice received PBS (n\u0026thinsp;=\u0026thinsp;5). Body weight and tumor volume\u0026mdash;calculated as (length \u0026times; width\u0026sup2;) / 2\u0026mdash;were monitored throughout. After 14 days, mice were euthanized via cervical dislocation. Blood was collected from the orbital sinus for hematological analysis. Tumors and major organs (heart, liver, spleen, lung, kidney) were fixed in 10% formalin or collected for pathological examination.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e2.19. Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using Student\u0026rsquo;s t-test or one-way ANOVA with GraphPad Prism 9.0. Significance was defined as \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Synthesis and characterization of FeN₄Cl SAzymes\u003c/h2\u003e\u003cp\u003eThe FeN₄Cl SAzymes were fabricated via a precoordination anchoring strategy designed to achieve precise axial chlorine ligation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Initially, hemin Cl was trapped in situ during the self-assembly of ZIF-8, yielding a hemin Cl/ZIF-8 precursor.[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] This precursor retained the characteristic rhombic dodecahedral morphology and crystal structure of the parent ZIF-8 (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2). Following high-temperature pyrolysis, the resulting FeN₄Cl SAzyme preserved the polyhedral morphology of its precursor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). X-ray diffraction (XRD) patterns of FeN₄Cl revealed only two broad peaks at 25\u0026deg; and 44\u0026deg;, corresponding to the (002) and (101) planes of graphitic carbon, with a notable absence of any peaks corresponding to metallic iron or other iron-based crystalline phases (Figure S3). This confirms the successful conversion of the precursor into a carbonaceous material without the formation of iron nanoparticles.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo verify the atomic-level structure, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) was employed. The images of FeN₄Cl displayed a high density of isolated bright spots uniformly distributed across the carbon support, which are characteristic of individual heavy atoms, confirming the single-atom dispersion of iron (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). Similarly, energy-dispersive X-ray spectroscopy (EDS) mapping revealed a homogeneous distribution of C, N, and Fe and, critically, Cl throughout the FeN₄Cl structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In contrast, the control sample (FeN₄), prepared without hemin chloride, showed no Cl signal (Figure S4). The iron contents in FeN₄Cl and FeN₄ were quantified by inductively coupled plasma‒mass spectrometry (ICP-MS) to be 0.28 wt% and 0.32 wt%, respectively.\u003c/p\u003e\u003cp\u003eX-ray photoelectron spectroscopy (XPS) was then conducted to investigate the surface chemical states and bonding configurations. The high-resolution N 1 s spectra for both FeN₄Cl and FeN₄ were deconvoluted into pyridinic N, pyrrolic N, graphitic N, oxidized N, and Fe\u0026ndash;N peaks (Figure S6), confirming the successful formation of Fe\u0026ndash;N coordination sites. Notably, the FeN₄Cl SAzyme had a higher content of pyridinic N (23.82%) than did FeN₄ (21.48%) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which is considered beneficial for oxidase-like catalytic activities.[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] Direct evidence for the axial ligand came from the Cl 2p spectrum of FeN₄Cl, where a peak corresponding to Fe‒Cl bonds was observed at approximately 198 eV (Figure S7). Furthermore, the high-resolution Fe 2p spectrum of FeN₄Cl clearly shifted to higher binding energies than those of FeN₄ (Figure S8). This positive shift indicates a lower electron density on the Fe centers in FeN₄Cl, which is attributed to the strong electron-withdrawing effect of the highly electronegative axial Cl ligand.\u003c/p\u003e\u003cp\u003eFinally, to elucidate the local coordination environment of the iron atoms, X-ray absorption fine structure (XAFS) measurements were performed. The Fe K-edge X-ray absorption near-edge structure (XANES) spectrum of FeN₄Cl exhibited an absorption edge at 7122.47 eV, which was positioned between those of the Fe foil and Fe₂O₃ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). This corresponds to an average Fe oxidation state of approximately\u0026thinsp;+\u0026thinsp;2.23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, Table S2), confirming the cationic nature of the iron centers. The k\u0026sup2;-weighted Fourier transform extended X-ray absorption fine structure (EXAFS) spectrum provided more detailed structural information (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). The spectrum is dominated by a primary peak at ~\u0026thinsp;1.5 \u0026Aring;, which corresponds to the Fe\u0026ndash;N scattering path. Critically, a second, smaller peak at ~\u0026thinsp;1.7 \u0026Aring; is also present, which is assigned to the Fe‒Cl scattering path. The complete absence of any Fe‒Fe signal at ~\u0026thinsp;2.2 \u0026Aring;, further corroborated by wavelet transform (WT) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej), provides unequivocal evidence for the atomic dispersion of iron. Quantitative fitting of the EXAFS spectrum yielded average coordination numbers of approximately 3.3 for Fe-N and 0.8 for Fe-Cl (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei). These results collectively confirm the successful synthesis of the target FeN₄Cl structure, where a single iron atom is coordinated by nitrogen atoms in the basal plane and one chlorine atom in the axial position.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Dual Enzyme-Mimicking Activities of FeN₄Cl SAzymes\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHaving established the unique FeN₄Cl atomic structure, we next evaluated its capacity to function as a dual-action nanozyme capable of both generating ROS and depleting glutathione (GSH), two key events for initiating ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We first assessed peroxidase (POD)-like activity via a 3,3\u0026rsquo;,5,5'-tetramethylbenzidine (TMB) colorimetric assay. After optimizing the synthesis and reaction conditions (Figure S9), we found that the FeN₄Cl SAzyme exhibited robust catalytic activity that was highly dependent on pH, with maximal performance under acidic conditions (pH 4.0\u0026ndash;5.0) characteristic of the tumor microenvironment (Figure S10). The activity of FeN₄Cl, which was dependent on both the catalyst concentration and time (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), was significantly greater than the negligible activity of the metal-free carbon support (NC), confirming that the atomically dispersed Fe sites are the catalytic centers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eA steady-state kinetic analysis revealed that the POD-like activity of FeN₄Cl followed classic Michaelis‒Menten kinetics for both the TMB and H₂O₂ substrates (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Critically, axial Cl- coordination imparted a substantial catalytic advantage over the conventional FeN₄ analog. FeN₄Cl demonstrated a 2.0-fold higher catalytic constant (k\u003csub\u003ecat\u003c/sub\u003e) and a 2.2-fold greater catalytic efficiency (k\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e) for TMB oxidation. Furthermore, its Michaelis constant (K\u003csub\u003em\u003c/sub\u003e) for H₂O₂ was lower than that of FeN₄ (4.23 mM vs. 4.77 mM), indicating a stronger binding affinity for the H₂O₂ substrate. This superior catalytic performance, particularly the high catalytic efficiency (k\u003csub\u003ecat\u003c/sub\u003e/K\u003csub\u003em\u003c/sub\u003e), places FeN₄Cl among the top-performing iron-based peroxidase mimics reported to date (Table S3), especially under acidic conditions relevant to the tumor microenvironment, underscoring its therapeutic potential. To further confirm the reaction mechanism, poisoning experiments with thiocyanate (KSCN), a strong Fe-coordinating agent, led to significant suppression of POD-like activity, verifying the essential role of the single-atom Fe centers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). We also confirmed that the catalysis proceeded via the generation of hydroxyl radicals (\u0026bull;OH), as evidenced by the consumption of H₂O₂ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh) and the positive detection of \u0026bull;OH via both the o-phenylenediamine (OPDA) probe and a methylene blue (MB) degradation assay (Figures S11, S12).\u003c/p\u003e\u003cp\u003eIn addition to generating ROS, an effective ferroptosis-inducing agent must dismantle the cell's primary antioxidant defense system, which is maintained by high levels of intracellular GSH. We therefore investigated the glutathione oxidase (GSHOx)-like activity of the nanozymes. Using a 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) assay, we found that FeN₄Cl was a remarkably potent catalyst for GSH depletion, far outperforming the FeN₄ control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). The GSH consumption rate was positively correlated with both the FeN₄Cl concentration and reaction time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek) and followed Michaelis\u0026ndash;Menten kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). Compared with FeN₄, FeN₄Cl exhibited a 2.79-fold greater maximum reaction velocity (V\u003csub\u003emax\u003c/sub\u003e), demonstrating its vastly superior ability to rapidly deplete GSH. This potent GSHOx-like activity, in concert with its enhanced POD-like function, equips the FeN₄Cl SAzyme with a powerful two-pronged attack to disrupt cellular redox homeostasis and trigger robust ferroptosis. Axial Fe-Cl-Coordinated Single-Atom Nanozymes Amplify Ferritinophagy-Dependent Ferroptosis for TNBC Therapy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Theoretical Insights into the Enhanced Catalytic Activity via DFT\u003c/h2\u003e\u003cp\u003eTo elucidate the underlying mechanism for the enhanced peroxidase-like activity, we performed density functional theory (DFT) calculations. Our analysis revealed that the axial coordination of a single Cl atom profoundly modulates the electronic properties of the FeN₄ center. Charge density difference analysis revealed significant electronic rearrangement around the Fe active site upon Cl coordination (Figure S13). This resulted in the d-band center of FeN₄Cl shifting upward from \u0026minus;\u0026thinsp;1.09 eV to -0.93 eV relative to the Fermi level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). According to d-band theory, this upward shift closer to the Fermi level optimizes the adsorption energy of key reaction intermediates, which is a hallmark of enhanced catalytic activity. This electronic modulation was further quantified by Bader charge analysis, which revealed that the charge on the Fe atom increased from +\u0026thinsp;1.09 |e| in FeN₄ to +\u0026thinsp;1.11 |e| in FeN₄Cl, confirming the strong electron-withdrawing effect of the axial Cl ligand.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then calculated the Gibbs free energy profiles to map the reaction pathways for POD-like activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Figure S14). The calculations confirmed that the decomposition of H₂O₂ on the FeN₄Cl surface proceeded via an efficient Fenton-like mechanism. The process begins with the adsorption of H₂O₂, followed by homolytic cleavage into two adsorbed hydroxyl (*OH) groups. One *OH group desorbs as a free hydroxyl radical (\u0026bull;OH), whereas the other is protonated to form a water molecule, regenerating the catalytic site. Critically, the FeN₄Cl structure significantly lowers the activation energy barrier for the rate-determining step\u0026mdash;the homolytic cleavage of H₂O₂ (H₂O₂ \u0026rarr; 2OH). This energy barrier was calculated to be only 1.17 eV for FeN₄Cl, which is substantially lower than the 1.48 eV required for the FeN₄ system (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This theoretical result provides a direct explanation for the experimentally observed increase in POD-like activity.\u003c/p\u003e\u003cp\u003eFinally, we analyzed the projected density of states (PDOS) to understand how orbital interactions contribute to this enhancement. The PDOS plots show that upon Cl coordination, the density of states for FeN₄Cl near the Fermi level increases significantly, and new d-p hybridized electronic states appear (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). This indicates that the axial Cl ligand not only induces charge redistribution but also promotes stronger orbital coupling between the Fe 3d orbitals and the substrate molecules. This enhanced orbital overlap facilitates more efficient interfacial charge transfer, thereby accelerating the overall catalytic kinetics. Collectively, these DFT results provide a robust theoretical foundation, demonstrating that axial Cl coordination is a highly effective strategy for rationally designing SAzymes with superior catalytic performance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.4. In vitro therapeutic efficacy and biocompatibility\u003c/h2\u003e\u003cp\u003eInspired by the superior dual-enzyme activities of FeN₄Cl, we investigated its therapeutic potential against TNBC cells in vitro. To enhance their biocompatibility and physiological stability, the FeN₄Cl nanozymes were camouflaged with red blood cell membranes to form FeN₄Cl/RBCs. Successful coating was confirmed by SDS‒PAGE analysis, which revealed the characteristic protein bands of the RBC membranes, as well as a reversal of the surface zeta potential (Figures S15, S17). The resulting FeN₄Cl/RBC nanoparticles were well dispersed with a hydrodynamic diameter of approximately 150 nm (Figure S16) and exhibited excellent blood safety, with a hemolysis rate below the 5% international safety threshold even at high concentrations (Figure S18).\u003c/p\u003e\u003cp\u003eConfocal microscopy tracking of Cy5.5-labeled FeN₄Cl/RBC revealed efficient internalization by MDA-MB-231 cells within 4 hours, with significant accumulation in lysosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This lysosomal localization was further corroborated by biological TEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Having confirmed cellular uptake, we assessed cytotoxicity via a CCK-8 assay. FeN₄Cl/RBC induced potent, dose-dependent cell death in MDA-MB-231 cells, reducing viability to below 10% at a concentration of 150 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). This potent anticancer effect was also observed in other breast cancer cell lines, including 4T1 and MCF-7 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), and was visually confirmed by live/dead cell staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Crucially, FeN₄Cl/RBC exhibited high selectivity, showing negligible toxicity toward normal HEK293 cells (Figure S19), likely due to the reliance of its catalytic activity on the acidic and H₂O₂-rich microenvironment characteristic of tumor cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Elucidation of the Ferroptosis Induction Mechanism\u003c/h2\u003e\u003cp\u003eHaving established the potent and selective cytotoxicity of FeN₄Cl/RBC, we next dissected its molecular mechanism of action. As hypothesized, treatment with FeN₄Cl/RBC induced a massive burst of intracellular ROS in MDA-MB-231 cells, as indicated by the strong fluorescence of the H₂DCFDA probe (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This surge in oxidative stress led to extensive lipid peroxidation (LPO), a hallmark of ferroptosis. This was confirmed by multiple lines of evidence: a significant increase in the LPO end-product malondialdehyde (MDA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) and intense green fluorescence from the LPO-sensitive probe BODIPY 581/591 C11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) were detected. Concurrently, the expression of glutathione peroxidase 4 (GPX4), the key enzyme that repairs lipid peroxides, was dramatically downregulated in FeN₄Cl/RBC-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). This inactivation is a direct consequence of the potent GSHOx-like activity of the nanozyme, which depletes the GPX4 cofactor GSH.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo definitively confirm that ferroptosis is a type of cell death, we cotreated cells with the specific ferroptosis inhibitor ferrostatin-1 (Fer-1). As expected, Fer-1 markedly attenuated the LPO accumulation induced by FeN₄Cl/RBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg), confirming that the cell death cascade is indeed ferroptosis. In support of this conclusion, FeN₄Cl/RBC treatment caused severe mitochondrial dysfunction, another characteristic feature of ferroptosis,[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] as evidenced by a collapse in the mitochondrial membrane potential (JC-1 assay, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) and a 44% reduction in cellular ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Activation of Ferritinophagy Synergistically Amplifies Ferroptosis\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition to directly triggering ferroptosis, we investigated whether FeN₄Cl/RBCs could activate synergistic pathways. TEM analysis of treated cells revealed a marked accumulation of autophagosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), and Western blot assays confirmed an increase in autophagic flux, as shown by decreased p62 and increased lipidated LC3-II levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). We then specifically probed for ferritinophagy, the selective autophagy of the iron-storage protein ferritin. Strikingly, treatment with FeN₄Cl/RBC led to significant downregulation of ferritin heavy chain 1 (FTH1) and corresponding upregulation of its cargo receptor, NCOA4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). These findings provide clear evidence for the activation of NCOA4-mediated ferritinophagy. The functional consequence of this process\u0026mdash;the degradation of ferritin\u0026mdash;was a significant increase in the intracellular labile iron (Fe\u0026sup2;⁺) pool (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003eCollectively, these results reveal a sophisticated and highly effective dual-pathway mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). The FeN₄Cl/RBC nanozyme not only initiates ferroptosis by disrupting the GPX4-GSH axis but also synergistically amplifies it by activating ferritinophagy to self-supply the catalytic Fe\u0026sup2;⁺ required to fuel the ROS-generating Fenton reaction, creating a deadly feedback loop within the cancer cell.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.7. In vivo antitumor efficacy and biosafety\u003c/h2\u003e\u003cp\u003eEncouraged by the potent in vitro cytotoxicity, we proceeded to evaluate the therapeutic efficacy of FeN₄Cl/RBC in a murine xenograft model of human TNBC (MDA-MB-231). Following intravenous administration, real-time fluorescence imaging of Cy5.5-labeled FeN₄Cl/RBCs revealed significant and sustained accumulation at the tumor site for up to 48 hours. This biomimetic camouflage endows the nanoparticles with a 'stealth' capability, significantly prolonging their blood circulation time and thereby maximizing their opportunity for passive accumulation at the tumor site via the enhanced permeability and retention (EPR) effect (Figure S20).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor the therapeutic study, tumor-bearing mice were randomized into four groups and received intravenous injections of PBS, FeN₄, FeN₄Cl, or FeN₄Cl/RBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The FeN₄Cl/RBC group exhibited profound tumor suppression, significantly outperforming all the other treatment groups and leading to nearly complete tumor regression by the end of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef). The potent in vivo efficacy of our FeN₄Cl/RBC nanoplatform compares favorably with that of other state-of-the-art ferroptosis-inducing nanobiomaterials recently reported for cancer therapy (Table S4), underscoring the superiority of our synergistic catalytic-metabolic design. Histological analysis of tumors from the FeN₄Cl/RBC group via H\u0026amp;E staining revealed extensive tissue damage, including vacuolar degeneration and nuclear pyknosis, which was corroborated by TUNEL staining, indicating widespread cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). To confirm that this therapeutic effect was driven by the intended mechanism in vivo, we performed immunohistochemical staining on the tumor tissues. As observed in vitro, tumors treated with FeN₄Cl/RBC presented markedly downregulated GPX4 expression and upregulated LC3 levels, confirming the induction of ferroptosis and autophagy within the tumor microenvironment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eh).\u003c/p\u003e\u003cp\u003eFinally, a comprehensive systemic safety evaluation was conducted. Throughout the treatment period, no significant body weight fluctuations were observed in any group (Figure S21). Posttreatment H\u0026amp;E staining of major organs (heart, liver, spleen, lungs, and kidneys) revealed no signs of pathological damage or inflammation (Figure S22). Furthermore, hematological and serum biochemical analyses confirmed that treatment with FeN₄Cl/RBC did not induce any adverse effects on liver or kidney function (Figure S23). Collectively, these results demonstrate that FeN₄Cl/RBC is a highly potent and safe nanotherapeutic agent for TNBC treatment in vivo.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn conclusion, we constructed a novel single-atom nanozyme (FeN₄Cl) with axial Fe\u0026ndash;Cl coordination that enables precise electronic modulation and substantially enhances dual enzyme-mimicking activities. The axial Cl ligand elevates the d-band center of the Fe site, thereby promoting both peroxidase- and glutathione oxidase-like catalytic performance. Mechanistically, the increased oxidative stress induced by FeN₄Cl activates NCOA4-mediated ferritinophagy, establishing a self-reinforcing ferroptotic loop through metabolic iron mobilization. Furthermore, red blood cell (RBC) membrane cloaking endows the nanozyme with excellent biocompatibility, prolonged circulation, and tumor-targeting capacity, resulting in potent ferroptosis-mediated antitumor efficacy in vivo without systemic toxicity. This study provides a paradigm for atomic-level nanozyme engineering coupled with metabolic reprogramming, offering promising translational potential for the treatment of TNBC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e# Mingming Yin and Bing-Hao Wang contributed equally to this work. This work was financially supported by the National Natural Science Foundation of China (No. 22478103), the Key Project of the Hunan Provincial Natural Science Foundation of China (No. 2025JJ90010), the Key Project of the Hunan Provincial Department of Education (No. 23A0301), the Open Project of the State Key Laboratory of Chemical Biosensing and Chemometrics, Hunan University (No. 20240600).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMingming Yin\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003eInvestigation, Conceptualization, Formal analysis, Methodology, Investigation, Writing \u0026ndash; original draft. Bing-Hao Wang: Formal analysis, Methodology, Writing \u0026ndash; original draft.\u0026nbsp;Huijuan Wang: Investigation, Methodology.\u0026nbsp;Jie Ouyang: Formal analysis.\u0026nbsp;Xingsheng Hu: Formal analysis.\u0026nbsp;Xiong Wang: Formal analysis.\u0026nbsp;Yongping Liu: Validation.\u0026nbsp;Fenghua Xu: Conceptualization, Writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;Yi Chen\u003cstrong\u003e:\u003c/strong\u003e Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition. Shuang-Feng Yin: Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the guidelines of the Hunan University Animal Care and Use Committee (Approval No. HNU-IACUC-2024-110).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have approved the manuscript and agree for the submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at DOI: to be assigned upon publication. (See Supplementary Information file)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBianchini G, De Angelis C, Licata L, Gianni L. Treatment landscape of triple-negative breast cancer - expanded options, evolving needs. Nat Rev Clin Oncol. 2022;19(2):91\u0026ndash;113.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWellmann RM, Ghafouri SN, McAndrew NP, Hurvitz SA. Chemotherapy-related outcomes in triple-negative breast cancer. J Clin Oncol. 2019;37(15):e12049.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarrido-Castro AC, Lin NU, Polyak K. Insights into Molecular Classifications of Triple-Negative Breast Cancer: Improving Patient Selection for Treatment. Cancer Discov. 2019;9(2):176\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKim J, Harper A, Mccormack V, Sung HYA, Houssami N, Morgan E, Mutebi M, Garvey G, Soerjomataram I. 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Nat Commun. 2023;14(1):7127.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Single-atom nanozyme, Chlorine coordination, Ferroptosis, Ferritinophagy, Triple-negative breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-7165005/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7165005/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTriple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype with limited therapeutic options and poor prognosis. Although single-atom nanozymes (SAzymes) show promise in cancer therapy, their ferroptosis-inducing capability remains limited. Herein, we present a rationally designed iron-based SAzyme with axial chlorine coordination (FeN₄Cl) that integrates catalytic and metabolic functions to enhance ferroptosis in TNBC. The engineered Fe-Cl coordination strategically modulates the d-band center relative to the Fermi level, resulting in significantly enhanced peroxidase-like activity (2.0-fold increase) and glutathione oxidase-like activity (3.2-fold increase) activities compared to conventional FeN₄ structures. Importantly, this electronic modulation triggers NCOA4-mediated ferritinophagy, establishing an autonomous iron supply mechanism that elevates intracellular labile Fe\u0026sup2;⁺ levels. The synergistic disruption of redox homeostasis coupled with amplified Fenton reactions creates a potent feedback loop that induces cell death. By incorporating this SAzyme into a red blood cell membrane-based biomimetic delivery system (FeN₄Cl/RBC), we achieved enhanced biocompatibility and tumor targeting. In vivo studies demonstrated significant tumor suppression, presenting a promising approach for developing clinically relevant nanozyme-based therapeutics.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Chlorine-Coordinated Iron Single-Atom Nanozymes for Amplified Ferroptosis in Triple-Negative Breast Cancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 15:30:35","doi":"10.21203/rs.3.rs-7165005/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-30T09:59:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T07:11:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T11:32:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T08:32:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311995680072329232006466765415126158556","date":"2025-10-10T02:11:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89147148465150957251435728618543005571","date":"2025-10-09T00:55:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169448298775791383871430855373651092664","date":"2025-10-06T15:11:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-23T15:31:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-22T08:13:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-22T08:10:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-07-19T14:17:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00d5d1d5-c1bb-4ccb-b9c5-51b3746a31eb","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:03:10+00:00","versionOfRecord":{"articleIdentity":"rs-7165005","link":"https://doi.org/10.1186/s12951-026-04096-9","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2026-02-24 15:57:46","publishedOnDateReadable":"February 24th, 2026"},"versionCreatedAt":"2025-10-06 15:30:35","video":"","vorDoi":"10.1186/s12951-026-04096-9","vorDoiUrl":"https://doi.org/10.1186/s12951-026-04096-9","workflowStages":[]},"version":"v1","identity":"rs-7165005","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7165005","identity":"rs-7165005","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}