NiFe-LDH-enhanced Ru single-atom catalysts anchored on MXenes for synergistic photothermal–nanocatalytic cancer therapy

Abstract

Single-atom catalysts (SACs) have emerged as revolutionary agents in cancer treatment owing to their optimized atomic efficiency and highly tunable catalytic properties. Nonetheless, their clinical application is hindered by restricted stability, ineffective substrate adsorption, and subpar catalytic rates under physiological conditions. This study presents the rational design of a hybrid Ru single-atom nanozyme, based on a NiFe-layered double hydroxide (LDH) and coupled with an MXene (RuSA/NiFe-LDH–MXene), facilitating synergistic photothermal and catalytic tumor therapy. The NiFe-LDH matrix enables strong coordination with Ru atoms, enhancing their electronic configuration and serving a dual function of electron enrichment and substrate activation, while MXene nanosheets offer high conductivity and photothermal conversion. Our system demonstrates increased peroxidase-like activity, effectively promoting the decomposition of H₂O₂ and the depletion of glutathione, thus intensifying oxidative stress in tumor microenvironments. Upon NIR irradiation, RuSA/NiFe-LDH–MXene attains a significant temperature increase (∼52.7 °C at 0.5 W cm⁻² for 5 minutes) and has a high photothermal conversion efficiency (∼46.8%). The nanozyme exhibits approximately a 2.8-fold increased catalytic velocity (V_max) for H₂O₂ breakdown and a roughly 1.6-fold enhanced production of hydroxyl radicals in comparison with RuSA@MXene. In vivo investigations revealed enhanced tumor ablation, with the RuSA/NiFe-LDH–MXene + NIR group attaining a tumor inhibition rate of 91.7% without systemic toxicity. This study emphasizes the essential function of LDH coordination in stabilizing Ru single atoms and adjusting their catalytic microenvironment, thereby creating a solid foundation for advanced nanocatalytic cancer treatments.

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Introduction

Breast cancer continues to be the most frequently diagnosed cancer among women globally and a primary cause of cancer-related mortality.^1–5 Notwithstanding considerable advancements in early detection and systemic therapies, including chemotherapy, radiation, and targeted agents, the prognosis for patients with aggressive or metastatic breast cancer remains inadequate.^6–10 These constraints are mainly associated with non-specific medication distribution, systemic toxicity, tumor heterogeneity, and multidrug resistance. Therefore, the development of innovative, tumor-specific, and least invasive therapeutic techniques is very important.^11–18

In recent years, photothermal treatment (PTT) has arisen as a potential approach for targeted cancer ablation. PTT can trigger selective mortality in tumor cells by turning near-infrared (NIR) light into localized heat, thereby minimizing damage to neighboring healthy tissues.^15,19,20 Furthermore, PTT can be integrated with other modalities, such as chemotherapy or catalytic therapy, to yield synergistic therapeutic effects and diminish treatment dosages.^21–26 The efficacy of PTT is predominantly contingent upon the performance of photothermal agents, which must exhibit high photothermal conversion efficiency, tumor accumulation, and biocompatibility.^25,27,28 Among the various types of photothermal materials, MXenes, particularly Ti₃C₂T_x, are attracting interest owing to their metallic conductivity, extensive near-infrared absorption, substantial surface area, and modifiable surface terminations.^29,30 These characteristics render them very appropriate for photothermal conversion and drug delivery applications.³¹ Prior research has shown that MXene nanosheets operate effectively as both photothermal therapy agents and transporters for chemotherapeutics and catalytic entities, thereby positioning them as multifunctional platforms in cancer treatment.^32–36

Notwithstanding their benefits, clean MXene platforms encounter some significant obstacles, including oxidative instability under physiological conditions, propensity to agglomerate, and diminishing dispersibility and bioavailability.^29,31 They also exhibit insufficient TME-responsive degradation and restricted ability to alter tumor biochemistry.³⁷

Nanozymes, capable of imitating enzyme action and function, have garnered significant interest over the past decade due to their remarkable benefits, including tunable catalytic activity and exceptional stability.^38–40 To address MXene constraints in photothermal therapy and improve treatment outcomes, researchers have investigated metal-based nanozymes decorated on MXene, especially those derived from transition metals like Ru.⁴¹ The tumor microenvironment (TME) is defined by specific biochemical features, including mild acidity (pH ∼6.5–6.8), hypoxia, and increased levels of glutathione (1–10 mM) and hydrogen peroxide (10–100 μM), which differentiate it from normal tissue.⁴² Although these characteristics diminish the efficacy of conventional therapeutic methods, they also offer exploitable biochemical triggers for targeted, tumor microenvironment-activated cancer treatment.^43,44 Among these techniques, nanocatalytic treatment, which produces reactive oxygen species (ROS) in situ through tumor microenvironment-responsive catalysts, has garnered significant interest for its capacity to induce localized oxidative stress and selective tumor apoptosis.^45,46 Conventional nanozymes, often composed of multimetallic nanoparticles or oxides, demonstrate structural heterogeneity, ambiguous active sites, and intricate surface chemistries, resulting in erratic catalytic activities and diminished specificity. These constraints hinder the replication of the accuracy and efficacy of natural enzymatic systems.⁴⁷

To mitigate these limitations, single-atom nanozymes (SAzymes) have been developed as advanced catalytic agents.⁴⁸ SAzymes, characterized by atomically scattered metal centers and adjustable coordination environments, offer optimal atomic usage and exact control over electrical and geometric configurations, emulating the active sites of natural metalloenzymes.^49,50 These features enable SAzymes to catalyze the breakdown of TME-overexpressed H₂O₂ into extremely lethal reactive oxygen species, such as hydroxyl radicals (˙OH), through Fenton-like or peroxidase-mimicking pathways.⁵¹ Moreover, their catalytic activity can be markedly improved by non-invasive external stimuli, especially near-infrared (NIR) irradiation, which promotes localized photothermal heating, accelerates charge transfer, enhances reactive oxygen species (ROS) generation, and intensifies tumor ablation effects. Ruthenium-based systems are distinguished among diverse SAzymes due to their numerous valence states, robust redox characteristics, and stable coordination chemistry.^20,52,53 When included into 2D photothermal platforms like Ti₃C₂T_x MXene, Ru single atoms can synergistically integrate catalytic and photothermal properties. A significant instance is the current research conducted by Wang et al.,⁵⁴ which presented a PEGylated Ru single-atom nanozyme affixed to Ti₃C₂T_x (Ru-Ti₃C₂T_x-PEG). This system demonstrated significant catalase- and peroxidase-like activity, effectively dissolving H₂O₂ and reducing intracellular GSH, and therefore exacerbating ROS-induced oxidative damage.⁵⁵ Following NIR activation, the system attained synergistic photothermal and catalytic tumor suppression both in vitro and in vivo.

Notwithstanding its promising performance, the Ru-Ti₃C₂T_x-PEG system exhibits numerous inherent limitations: (i) inadequate atomic stability of Ru, posing concerns of aggregation or leaching during physiological circulation and lack of stimuli-responsive drug release; (ii) insufficient environmental responsiveness or TME-induced activation, constraining regulated ROS production; and (iii) MXene instability in physiological settings, characterized by susceptibility to oxidative breakdown and diminished photothermal efficacy.^56–59

Notwithstanding these advancements, the therapeutic application of SACs in cancer treatment encounters significant obstacles, including insufficient atomic stability, restricted substrate affinity, and suboptimal catalytic rates in intricate biological settings. Furthermore, current MXene-based catalytic platforms exhibit oxidative instability and restricted tumor selectivity, whereas traditional metal single-atom catalysts frequently demonstrate inadequate structural stability and environmental reactivity. To mitigate these constraints, the integration of single-atom catalysts with auxiliary functional materials that can augment their stability and reactivity has surfaced as a viable approach. Motivated by the recent advancement of NiFe-LDH/MXene nanocomposites for biomedical applications, we aimed to integrate the distinctive characteristics of layered double hydroxides (LDHs) and MXenes to create a synergistic nanoplatform.^60–62 LDHs are two-dimensional anionic clays recognized for (i) pH-sensitive breakdown in acidic neoplastic settings, (ii) redox-active nickel and iron centers capable of enhancing Fenton-like catalytic reactions, (iii) structural scaffolding that stabilizes single-atom catalysts and inhibits aggregation, and (iv) improved biocompatibility and regulation of medication release.^3,11,16,60 This hybrid system is engineered to produce a dual catalytic–photothermal therapeutic effect, stimulating peroxidase-like activity for the formation of ROS and enhancing photothermal tumor ablation under NIR irradiation. Our RuSA/NiFe-LDH–MXene nanozyme exhibited exceptional in vitro and in vivo therapeutic efficacy, with a tumor suppression rate of around 91.7% through combined photothermal and catalytic therapy. This study emphasizes the significance of LDH in stabilizing single-atom catalysts and enhancing their electronic environment, providing a synergistic approach to improve the effectiveness and clinical application of nanozyme-based cancer treatments.

Experimental

Materials

Ti₃AlC₂ (MAX phase), lithium fluoride (LiF), hydrochloric acid (HCl, 37%), nickel nitrate hexahydrate (Ni(NO₃)₂·6H₂O), ferric nitrate nonahydrate (Fe(NO₃)₃·9H₂O), ruthenium(III) chloride hydrate (RuCl₃·xH₂O), polyethylene glycol amine (PEG-NH₂, MW ≈ 2000 Da), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), MES buffer, phosphate-buffered saline (PBS), and other analytical-grade chemicals were acquired from Sigma-Aldrich and utilized without additional purification. All experiments utilized deionized water.

Synthesis of Ti₃C₂T_x MXene

Ti₃C₂T_x MXene nanosheets were produced through the selective etching of aluminum from Ti₃AlC₂ with an in situ created HF solution. In summary, 1.0 g of LiF was dissolved in 20 mL of 9 M HCl and agitated for 10 minutes, after which 1.0 g of Ti₃AlC₂ powder was added gradually. The mixture was kept at 35 °C while being stirred for 24 hours. The resultant etched slurry was subjected to centrifugation and repeatedly rinsed with deionized water until the supernatant's pH approximated 6. Delaminated MXene nanosheets were acquired by sonicating the sediment in an ice bath under an argon atmosphere for 1 hour, followed by centrifugation at 3500 rpm to isolate the supernatant containing few-layer Ti₃C₂T_x nanosheets. The acquired MXene was subjected to vacuum filtration and subsequently dried at 60 °C.

Synthesis of the NiFe-LDH–MXene hybrid

To produce the NiFe-LDH-coated MXene composite, 100 mg of Ti₃C₂T_x MXene was suspended in 50 mL of deionized water and subjected to ultrasonication for 30 minutes. Aqueous solutions of Ni(NO₃)₂·6H₂O (0.4 mmol) and Fe(NO₃)₃·9H₂O (0.2 mmol) were incrementally introduced to the dispersion while maintaining continuous agitation. The pH of the reaction was modified to about 10 using 1 M NaOH to promote LDH nucleation. The resultant suspension was placed into a 100 mL Teflon-lined autoclave and underwent hydrothermal treatment at 120 °C for 6 hours. Upon reaching room temperature, the NiFe-LDH–MXene hybrid was obtained via centrifugation, rinsed with water and ethanol, and subsequently freeze-dried at 0 °C for future application.

Atomic-level loading of Ru single atoms

To load Ru single atoms, 50 mg of the prepared LDH–MXene composite was dispersed in 50 mL of ethanol containing 1 mg of RuCl₃. The solution was agitated for 24 hours to facilitate the electrostatic adsorption of Ru³⁺ onto the LDH surface. The composite was subsequently thermally annealed at 250 °C in a nitrogen environment for 2 hours to secure the Ru atoms to the support through oxygen coordination. The resultant material is referred to as RuSA/LDH–MXene. The overall process of RuSA/LDH–MXene can be seen in Scheme 1.

Scheme 1 Schematic representation of the RuSA/LDH–MXene preparation method.

Structural characterization

The crystalline structure of the samples was examined via powder X-ray diffraction (XRD) utilizing an XRD1161 MiniFlex XpC diffractometer with Cu Kα radiation (λ = 1.5418 Å). The surface morphology and microstructure were analyzed using scanning electron microscopy (SEM, JCM-7000 NeoScope™ Benchtop SEM) and transmission electron microscopy (TEM, FEI TF30). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging was performed to assess atomic dispersion. Spherical aberration-corrected transmission electron microscopy pictures were acquired using a JEM ARM200F thermal field emission microscope, which is outfitted with a probe spherical aberration corrector for high-resolution lattice imaging. X-ray photoelectron spectroscopy (XPS) examination was conducted utilizing an ESCALAB 250 instrument to examine surface elemental composition and chemical states. The metal loading was assessed using inductively coupled plasma-optical emission spectroscopy (ICP-OES, PerkinElmer AVIO 500). In situ Raman spectroscopy was conducted with a Thermo Fisher DXR microscope, outfitted with a 50× visible objective and a 532 nm laser excitation source. Atomic force microscopy (AFM, Bruker Dimension Icon) was utilized to ascertain the nanosheet thickness and surface structure. X-ray absorption fine structure (XAFS) spectra at the Ni, Fe, and Ru K-edges were obtained on a channel-cut Si(111) crystal monochromator device. The data were acquired in fluorescence mode with a Lytle detector to investigate the local coordination environment and oxidation status of the metal centers. Transmission electron microscopy (TEM, JEM-2100F) and aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (AC-HAADF-STEM) microscope. Thermal images were obtained using an infrared thermal imaging device (FLIR A325SC camera). Ultraviolet–visible–near-infrared (UV–Vis–NIR) spectroscopy was performed to evaluate light absorption and photothermal reaction under 808 nm irradiation. In situ Raman spectroscopy was performed utilizing a Thermo Fisher DXR Raman microscope equipped with a 532 nm laser and a 50× objective to monitor photothermal-induced structural alterations. The metal concentration was measured via inductively coupled plasma-optical emission spectrometry (ICP-OES, PerkinElmer AVIO 500). X-ray absorption fine structure (XAFS) spectroscopy at the Ru, Ni, and Fe K-edges was conducted at the BL07A1 beamline of the National Synchrotron Radiation Research Center (NSRRC) in fluorescence mode utilizing a Lytle detector. The photothermal efficiency of the RuSA/LDH–MXene nanozyme was assessed by observing the temperature increase of aqueous dispersions subjected to 808 nm NIR laser irradiation (1.0 W cm⁻²), and the heat generation efficiency under irradiation was determined from the heating and cooling profiles. Thermal stability was evaluated through successive cycles of laser irradiation on and off.

Performance evaluation

The Fenton-like catalytic efficacy of the RuSA/NiFe-LDH@MXene and Ru@MXene nanostructures was methodically assessed under tumor-simulating circumstances. The peroxidase-like (POD-like) activity was first evaluated using two model chromogenic substrates. In a standard experiment, 100 μg mL⁻¹ of each nanozyme was incubated with 1 mM hydrogen peroxide and 1 mM 3,3′,5,5′-tetramethylbenzidine (TMB) in acetate buffer (pH 4.0) at 37 °C. The catalytic oxidation of TMB was seen by measuring the absorbance at 652 nm with a microplate reader. Parallel tests utilizing methylene blue (MB) degradation were performed through the addition of 10 mg L⁻¹ MB with H₂O₂ and the nanozymes; the reduction in absorbance at 664 nm over time was monitored spectrophotometrically to evaluate catalytic effectiveness. To further confirm the generation of reactive oxygen species (ROS), particularly hydroxyl radicals (˙OH), electron spin resonance (ESR) spectroscopy was conducted utilizing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin-trapping agent. Samples comprising nanozymes, H₂O₂, and DMPO were incubated at 37 °C, and the resultant ESR signals were captured utilizing an X-band spectrometer. The distinctive quartet signals indicated the generation of ˙OH, indicating effective Fenton-like activity. Intracellular reactive oxygen species generation was assessed with a DCFH-DA fluorescence assay. 4T1 cells were treated with nanozymes (100 μg mL⁻¹) for 4 hours, subsequently stained with 10 μM DCFH-DA. Following 30 minute of incubation in darkness, the cells were rinsed with PBS and subsequently examined with confocal laser scanning microscopy (CLSM). A fraction of cells was subjected to 808 nm laser irradiation at 0.8 W cm⁻² for 5 minutes before DCFH-DA staining to evaluate the impact of NIR irradiation on ROS amplification. The fluorescence intensity, indicative of intracellular ROS levels, was measured using ImageJ analysis or flow cytometry. To examine glutathione (GSH) depletion, a marker of glutathione peroxidase (GPx)-like activity, nanozymes were treated with 5 mM GSH at 37 °C for one hour. The residual GSH was assessed utilizing 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), with absorbance measured at 412 nm. A conventional GSH calibration curve was employed to ascertain depletion efficiency. The photothermal performance was evaluated by dispersing nanozymes in PBS at different concentrations (25, 50, 100, and 200 μg mL⁻¹) and subsequently exposing them to an 808 nm NIR laser (0.8 W cm⁻²) for 10 minutes. The temperature variation was documented in real time utilizing an FLIR T620 infrared thermal camera. The photothermal conversion efficiency (η) was determined according to standard techniques utilizing the following equation:where ΔT_max represents the greatest temperature increase, A₈₀₈ denotes the absorbance at 808 nm, and I indicates the laser power density. The photothermal stability was assessed using five successive laser on/off cycles, each including 5 minutes of irradiation followed by natural cooling, to evaluate the consistency and robustness of the heating response.

In vitro cytotoxicity and mechanistic assays

In vitro cytotoxicity experiments were conducted on both malignant (4T1) and normal (L929) cell lines to assess the biocompatibility and therapeutic efficacy of the produced nanozymes. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin under standard incubator conditions (37 °C, 5% CO₂). For the assessment of cell viability, 4T1 cells were inoculated in 96-well plates at a density of 5 × 10³ cells per well and permitted to adhere for 24 hours. Cells were subsequently exposed to Ru@MXene or RuSA/NiFe-LDH–MXene at different doses (0, 25, 50, 100, 200 μg mL⁻¹) for a duration of 24 hours. Cell viability was assessed utilizing a Cell Counting Kit-8 (CCK-8) assay in accordance with the manufacturer's guidelines. Absorbance at 450 nm was measured using a microplate reader. To assess the combined therapeutic efficacy of photothermal and catalytic activity, parallel groups were exposed to an 808 nm laser (0.8 W cm⁻²) for 5 minutes following 4 hours of nanozyme incubation. Following irradiation, cells were cultivated for an additional 20 hours prior to doing the CCK-8 experiment.

Live/dead cell staining was performed to elucidate treatment-induced cytotoxicity. 4T1 cells were cultured on sterile glass coverslips within 24-well plates and subjected to nanozyme treatment (100 μg mL⁻¹) with or without NIR irradiation as previously outlined. Following a 24-hour period, cells were subjected to staining with a combination of calcein-AM (2 μM) and propidium iodide (PI, 4 μM) for 20 minutes at 37 °C. Cells were rinsed with PBS and visualized using a confocal laser scanning microscope (CLSM).³⁴ Green fluorescence (calcein-AM) signified viable cells, but red fluorescence (PI) identified membrane-damaged deceased cells.

The mitochondrial membrane potential (ΔΨ_m), an early indicator of apoptosis, was evaluated to detect mitochondrial dysfunction using JC-1 dye. The treated cells were treated with 2 μM JC-1 for 30 minutes at 37 °C and subsequently washed with PBS. CLSM imaging was employed to identify red (JC-1 aggregates, intact mitochondria) and green (JC-1 monomers, depolarized mitochondria) fluorescence signals. The green/red fluorescence ratio indicated mitochondrial depolarization.

In vivo antitumor evaluation

The 4T1 and L929 cell lines were obtained from GenIran Co. (Tehran, Iran). The formation of intracellular reactive oxygen species (ROS) was quantified utilizing the DCFH-DA probe. 4T1 cells underwent treatment with nanozymes (100 μg mL⁻¹) with or without NIR for 4 hours, followed by 30 minute of staining with 10 μM DCFH-DA in the dark. Following PBS washing, green fluorescence was recorded via CLSM and quantified using ImageJ software. The ROS signal significantly increased in groups treated with RuSA/NiFe-LDH@MXene during laser irradiation, signifying heightened oxidative stress resulting from the synergistic catalytic–photothermal process.

Flow cytometry was utilized for quantitative apoptosis investigation by Annexin V-FITC/PI dual labeling. 4T1 cells were cultured in 6-well plates and subjected to treatment with nanozymes with or without near-infrared (NIR) exposure. Cells were collected, rinsed with cold PBS, and stained with the Annexin V-FITC/PI apoptosis detection kit according to the manufacturer's procedure after 24 hours. Fluorescence intensity was measured utilizing a BD FACSCalibur flow cytometer. The ratios of early apoptotic, late apoptotic, and necrotic cells were analyzed among treatment groups to assess therapeutic efficacy.

Cytoskeletal reorganization linked to apoptosis was ultimately observed using phalloidin and DAPI labeling. Cells subjected to treatment were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and subsequently stained with phalloidin-TRITC for F-actin and DAPI for nuclei.⁶³

All animal studies adhered to institutional rules and received approval from the Animal Ethics Committee of Islamic Azad University. Subcutaneous breast cancer models were established using female BALB/c mice aged 6 to 8 weeks, weighing around 20 grams. In summary, 1 × 10⁶ 4T1 cells suspended in 100 μL of PBS were subcutaneously administered into the right flank of each mouse. The tumor volume was allowed to attain roughly 100 mm³ prior to the commencement of treatment. Mice were randomly allocated into five groups (n = 5 per group): (1) PBS (control), (2) Ru@MXene, (3) RuSA/NiFe-LDH@MXene, (4) RuSA/NiFe-LDH@MXene + NIR, and (5) Ru@MXene + NIR.

The therapeutic efficacy was assessed by comparing tumor development paths and final tumor weights after a 14-day monitoring period. Subsequent to euthanasia, tumors and principal organs (heart, liver, spleen, lungs, and kidneys) were excised for histopathological and biosafety evaluation. Tumor specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm thick slices. Hematoxylin and eosin (H&E) staining was conducted to evaluate overall tissue morphology. TUNEL experiments with terminal deoxynucleotidyl transferase dUTP were conducted to visualize apoptotic DNA fragmentation. Ki-67 immunohistochemistry labeling was employed to assess tumor cell proliferation status. The ImageJ program was employed to quantify the intensity of positive staining.

Serum samples were taken for biochemical analysis to assess systemic toxicity and biocompatibility. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine concentrations were evaluated to determine hepatic and renal function. Complete blood counts (CBC) were conducted. No notable alterations in organ histology or blood biochemical markers were detected in the nanozyme-treated groups, suggesting satisfactory in vivo biosafety.

Statistical methods

Statistical significance was evaluated using the Student's t-test, with **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicating increasing levels of significance. Samples and animals were randomly assigned to the respective experimental groups and treatments.

Results and discussion

Fig. 1 provides detailed structural and elemental characterization, validating the effective synthesis of the Ru/NiFe-LDH@MXene nanohybrid with atomic-level precision. The SEM image of pristine MXene (Fig. 1a) shows its characteristic smooth, layered two-dimensional architecture, functioning as a conducting scaffold. The formation of NiFe-LDH (Fig. 1b and c) results in vertically aligned nanosheets that create a dense, flower-like structure, significantly enhancing the active surface area. Fig. S1 illustrates the effective incorporation of LDH nanosheets onto the MXene substrate, resulting in a hierarchical nanostructure characterized by structural complexity and functional synergy. The MXene serves as a highly conductive framework, whereas the LDH nanoflakes offer numerous active sites and structural support. This architecture not only inhibits MXene restacking and oxidation but also enhances overall stability and charge transport—an optimal structure for applications in electrocatalysis, supercapacitors, or therapeutic nanoplatforms.

Fig. 1 SEM images of (a) pristine MXene nanosheets and (b and c) the NiFe-LDH/MXene composite; (d and e) TEM image of NiFe-LDH/MXene; (f) high-resolution TEM (HRTEM) image showing lattice fringes of NiFe-LDH; and (g) high-angle annular dark-field scanning TEM (HAADF-STEM) image of Ru in RuSA/NiFe-LDH–MXene and (h) the corresponding elemental mapping revealing uniformly dispersed Ru single atoms (highlighted in green circles). (i) EDS elemental mapping images showing uniform distribution of O, C, Ti, Fe, Ni, and Ru RuSA/NiFe-LDH–MXene.

TEM and HRTEM images (Fig. 1d–f) demonstrate the close interfacial contact between NiFe-LDH and MXene, together with strong crystallinity shown by distinct lattice fringes associated with the (018) and (200) planes. The low-magnification TEM image (Fig. 1e) illustrates the uniform distribution of NiFe-LDH on MXene, while the SAED pattern (Fig. 1g) corroborates the polycrystalline characteristics of the composite. Significantly, atomic-resolution HAADF-STEM imaging (Fig. 1h) demonstrates the effective dispersion of isolated Ru atoms (green circles), confirming the establishment of a genuine single-atom catalyst (SAC). The provided elemental maps (Fig. 1i) exhibit a uniform distribution of C, O, Ti, Fe, Ni, and Ru throughout the structure, thereby affirming the compositional integrity of the hybrid. These analyses collectively confirm the effective formation of Ru/NiFe-LDH@MXene.

The structural and surface chemical characteristics of the Ru@NiFe-LDH/MXene heterostructure were thoroughly examined utilizing Raman spectroscopy, XRD, and XPS techniques. Fig. 2a illustrates that the Raman spectra distinctly exhibit signs of Ti₃C₂T_x MXene (E_g and A_1g modes), vibrational bands of layered double hydroxide (LDH) from Ni²⁺–O and Fe³⁺–O bonds, and carbonaceous species.⁶⁴ The Ru@NiFe-LDH@MXene composite exhibits an elevated D-band (∼1350 cm⁻¹) compared to the G-band (∼1580 cm⁻¹), indicating higher structural disorder or surface activation, which aligns with interfacial hybridization.⁶⁰ The appearance of TiO₂-related bands further substantiates surface oxidation, presumably triggered during LDH development. X-ray diffraction demonstrates that the composite preserves the layered crystalline architecture of both MXene and NiFe-LDH. Peaks at low angles (about 7–13°) indicate the interlayer spacing of MXene, whereas large reflections in the 20°–40° range affirm the stacking of LDH nanosheets. The hybrid exhibits peak broadening and minor shifting, indicative of interfacial lattice distortion and augmented d-spacing, implying the successful incorporation of Ru and LDH into the MXene matrix. The lack of Ru peak in the XRD pattern of Ru@NiFe-LDH/MXene signifies the atomic dispersion of Ru in the NiFe-LDH/MXene heterostructure.⁶⁵ The wide-scan XPS analysis, seen in Fig. 2c, verifies the existence of all expected elements: Ru, Fe, Ni, Ti, O, N, and C. This confirms the effective synthesis of the Ru-decorated LDH/MXene composite. In high-resolution deconvoluted XPS spectra of Ni 2p, shown in Fig. 2d, two prominent peaks are seen at ∼855 eV (Ni 2p_3/2) and ∼873 eV (Ni 2p_1/2) with associated satellite peaks, indicating the presence of Ni²⁺ species in the LDH framework, consistent with the NiFe-LDH structure. Fig. 2e shows peaks at ∼711 eV and ∼724 eV that correspond to Fe 2p_3/2 and Fe 2p_1/2, confirming the presence of Fe³⁺ in the LDH layer.¹¹ The broad peaks and satellite features suggest multiple coordination environments, possibly influenced by Ru incorporation. The C 1s spectrum exhibits several deconvoluted peaks, including sp² C=C/C–C (∼284.6 eV), C–O/C=O, and C–Ti, signifying a variety of surface functional groups. The Ru 3d_5/2 and Ru 3d_3/2 peaks at around 281 and 285 eV coincide with the C 1s area, affirming the existence of metallic or oxidized Ru species embedded within the composite.^66–68 In order to examine the stability of MXene, UV–Vis–NIR absorbance spectra of pristine MXene and LDH@MXene were obtained (Fig. S2). The mixture of LDHs with MXene significantly enhances its colloidal and oxidative stability, as evidenced by the comparative UV–Vis–NIR absorbance spectra over time. The left panel demonstrates that bare MXene dispersions undergo a large decrease in absorbance intensity after 5–10 days, indicating considerable degradation and oxidation. In contrast, the MXene/LDH hybrid (right panel) has significantly increased absorbance levels after 60 days, suggesting that the LDH shell provides a protective barrier that inhibits surface oxidation, restacking, and hydrolysis of MXene under aqueous conditions. The strong electrostatic interactions and probable Ti–O–M (M = Fe, Ni) coordination between MXene and LDH layers likely facilitate electron buffering and environmental protection. This stabilizing effect prolongs the functional lifespan of MXene in ambient or biological environments and improves the reliability of MXene-based nanocomposites in practical applications.

Fig. 2 (a) Raman spectra, (b) XRD patterns of pristine MXene, NiFe-LDH, and RuSA/NiFe-LDH–MXene, (c) full-scan XPS survey spectrum of the RuSA/NiFe-LDH–MXene hybrid, high-resolution XPS spectra of (d) the Ni 2p region, (e) Ti 2p region, (f) Fe 2p region, and (g) C 1s and Ru 3d.

X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy were conducted to ascertain the valence states and local coordination geometries of RuSA/NiFe-LDH–MXene at the atomic level.^67,69,70 The XANES spectra, as shown in Fig. 3a, indicate that the absorption edge of RuSA/NiFe-LDH–MXene is shifted positively relative to Ru foil; however, it remains lower than that of RuO₂, suggesting that the Ru atoms in the hybrid are in a partially oxidized state (Ru^δ+, 0 < δ < 4). The intensity of the white line in RuSA/NiFe-LDH–MXene is more apparent than that in metallic Ru, indicating a robust interaction between Ru and the oxygenated functional groups within the RuSA/NiFe-LDH–MXene matrix. The Fourier-transformed EXAFS spectra (Fig. 3b and Table S1) indicate that Ru foil exhibits a prominent Ru–Ru peak at approximately 2.4 Å, while this peak is absent in Ru/LDH/MX, suggesting that Ru is atomically scattered and does not aggregate into metallic clusters. The primary signal at 1.6–1.8 Å in Ru/LDH/MX indicates Ru–O coordination, hence establishing the bonding of Ru to oxygen atoms inside the host material. The fitting results for RuSA/NiFe-LDH–MXene (Fig. 3c) further validate the Ru–O coordination context. The lack of long-range coordination shells and Ru–Ru pathways underscores the single-atom dispersion of Ru. The coordination number and bond distances obtained from the fit correspond with the anticipated values for Ru–O octahedra or distorted configurations at interfaces. The interfacial hybridization with LDH and MXene enhances the electron-deficient state of Ru, which has been shown to benefit catalytic reactions such as water splitting, Fenton-like reactions, or selective oxidation.⁷¹ The wavelet transform analysis (Fig. 3d) indicates variations in the types of backscattering atoms and their spatial and frequency resolution. Ruthenium foil has a pronounced Ru–Ru correlation peak at approximately 2.4 Å and a wave vector k of around 8 Å⁻¹. RuO₂ exhibits Ru–O signals at comparable R values but marginally elevated k values. In Ru/LDH/MX, only Ru–O coordination is seen, with no detectable Ru–Ru signals, thereby verifying atomic dispersion and local coordination with light atoms (O).

Fig. 3 (a) Ru K-edge XANES spectra of Ru foil, RuO₂, Ru/MXene (Ru/MX), and Ru@NiFe-LDH@MXene (Ru/LDH/MX) composites; (b) Fourier-transformed EXAFS spectra (FT-EXAFS) of Ru foil, RuO₂, Ru/MXene (Ru/MX), and RuSA/NiFe-LDH–MXene (Ru/LDH/MX) composites; (c) EXAFS fitting curve of Ru/LDH/MX; and (d) wavelet transform (WT) contour plots of Ru foil, RuO₂, and RuSA/NiFe-LDH–MXene (Ru/LDH/MX).

The synergistic anticancer mechanism of the RuSA/NiFe-LDH@MXene hybrid system under NIR-II irradiation is illustrated in Scheme 2. Upon laser illumination, the material experiences hot electron transfer, activating the single-atom Ru, Ni, and Fe catalytic sites and producing oxygen vacancies (OV). These active spots facilitate the transformation of H₂O₂ or O₂ found in the tumor microenvironment into highly reactive oxygen species (ROS), including hydroxyl radicals (˙OH) and singlet oxygen (¹O₂), via Fenton-like and photocatalytic processes. The reduction of Ni²⁺, Fe²⁺, and Ru²⁺ ions produces Ni¹⁺, Fe¹⁺, and Ru¹⁺, thus enabling continuous catalytic cycles. The resultant reactive oxygen species (ROS) generate oxidative stress in neoplastic cells, compromising cellular constituents such as mitochondria, proteins, and DNA, consequently initiating apoptosis and impeding tumor proliferation. This technique utilizes the improved photothermal conversion and catalytic activity of the LDH–MXene complex to effectively eliminate cancer cells. Supports such as NiFe-LDH/MXene can surmount the constraints of single-atom catalysts, presenting a viable approach for effective, safe, and clinically applicable cancer nanotherapy.

Scheme 2 Synergistic anticancer mechanism of the RuSA/NiFe-LDH@MXene hybrid system.

Prior research has shown exceptional photothermal effects of single-atom catalysts when exposed to near-infrared (NIR) light.⁵⁴ We utilized an IR thermal imaging device to observe temperature variations. To evaluate this effect, we employed an IR thermal imaging device to monitor temperature changes. Specifically, the RuSA/NiFe-LDH–MXene solution was exposed to an 808 nm laser at a power density of  0.5 W cm⁻². Fig. 4a illustrates the temperature profiles of RuSA/NiFe-LDH–MXene dispersions at different concentrations subjected to 808 nm laser irradiation (0.5 W cm⁻²). The temperature increases swiftly with both irradiation duration and concentration, with elevated concentrations yielding more significant thermal effects. The observed concentration-dependent heating illustrates the superior photothermal conversion capability of RuSA/NiFe-LDH–MXene, owing to the robust NIR absorption characteristics of the hybrid nanozyme. The effective photothermal performance is crucial for generating localized hyperthermia in tumor tissues. The uniform increase in temperature at varying doses validates the material's capability for adjustable and regulated photothermal therapy. Infrared thermal images (Fig. 4b) graphically depict the instantaneous heat distribution of RuSA/NiFe-LDH–MXene dispersions during laser irradiation. The images depict brighter (hotter) areas associated with extended irradiation durations and elevated concentrations. The significant temperature disparity in these images further supports the material's robust NIR absorption and heat-generating capacity. The homogeneous heating observed indicates superior dispersion stability and consistent photothermal activity, essential for efficient in vivo thermal treatment without localized overheating. The MXene material inherently exhibits extensive near-infrared absorption owing to its metallic Ti₃C₂ structure and surface terminations, whereas the standalone RuSA@MXene system predominantly depends on the light absorption characteristics of MXene and the electronic states of isolated Ru atoms. The incorporation of NiFe-LDH introduces extra electronic states and interfacial charge-transfer routes, thereby expanding the light absorption spectrum and enhancing the photothermal response under NIR irradiation.^72,73 The LDH nanosheets introduce defect states and metal–oxygen charge-transfer bands that enhance MXene's plasmonic absorption, resulting in improved light harvesting efficiency. In addition, the heterointerface between NiFe-LDH and MXene facilitates effective separation of photogenerated charge carriers.⁷⁴ This inhibits non-radiative recombination of electrons and holes, facilitating the conversion of a greater amount of absorbed photon energy into heat instead of being dissipated by electron–hole recombination.⁶⁰Fig. 4c investigates the photothermal decay curves to determine the photothermal conversion efficiency (η), calculated to be approximately 43.8%. In order to evaluate the photothermal stability, the temperature response of RuSA/NiFe-LDH@MXene is measured over multiple heating/cooling cycles under laser irradiation. The minimal variation in photothermal performance over numerous cycles illustrates the material's superior stability and structural integrity under repeated thermal stress. This property guarantees dependable and sustained utilization in photothermal therapeutic applications, as seen in Fig. 4d. A steady-state kinetic test was conducted utilizing H₂O₂ as the substrate to assess the catalytic kinetics of POD. As shown in Fig. 4e, the Michaelis–Menten curves illustrate the catalytic kinetics of H₂O₂ decomposition, with RuSA/NiFe-LDH@MXene demonstrating the lowest K_m and the greatest V_max among the evaluated samples. The ESR spectra shown in Fig. 4f demonstrate the formation of hydroxyl radicals (˙OH) by RuSA/NiFe-LDH–MXene in the presence of H₂O₂, as evidenced by the distinctive DMPO-˙OH signals. The substantial production of ˙OH validates the peroxidase-like enzymatic activity of the nanozyme. This ROS generation is essential for producing oxidative stress in tumor cells, indicating the material's promise in catalytic cancer therapy. The participation of Ru single atoms and NiFe active sites augments the catalytic breakdown of H₂O₂. The enhanced peroxidase-like catalytic activity of RuSA/NiFe-LDH–MXene relative to RuSA@MXene is due to the synergistic interactions between the Ru single atoms and the NiFe-LDH nanosheets, which create a more advantageous catalytic environment and improve electron transport mechanisms. RuSA@MXene depends exclusively on the catalytic properties of isolated Ru atoms on MXene, whereas RuSA/NiFe-LDH–MXene gains from supplementary redox-active Ni²⁺/Ni³⁺ and Fe²⁺/Fe³⁺ sites inside the LDH framework.⁷⁵ The multimetallic centers function collaboratively to enhance H₂O₂ adsorption and activation, leading to improved catalytic efficacy. Moreover, the LDH layer incorporates many hydroxyl groups and a hydrophilic surface, enhancing substrate affinity and facilitating the generation of reactive intermediates.⁷⁶ The heterointerface between NiFe-LDH and MXene improves charge transfer, refining the electronic structure of Ru active sites and expediting catalytic turnover. Experimental findings corroborate these improvements, indicating that RuSA/NiFe-LDH–MXene has reduced K_m and elevated V_max values in Michaelis–Menten kinetics, alongside enhanced hydroxyl radical (˙OH) production as validated by ESR spectra (Fig. 4f).⁷⁷ The combined properties substantially boost the peroxidase-like activity of RuSA/NiFe-LDH–MXene, illustrating the efficacy of interfacial engineering and multimetal synergy in augmenting nanozyme performance. Fig. 4g shows the O₂ evolution over time, where RuSA/NiFe-LDH–MXene demonstrates the fastest and highest oxygen production from H₂O₂ decomposition. The significant catalase-like activity successfully mitigates tumor hypoxia, a critical obstacle in ROS-based cancer treatments. The nanozyme produces oxygen, establishing a conducive milieu for continuous ROS generation, hence improving therapeutic efficacy. Time-dependent GSH consumption (Fig. 4h) signifies the nanozyme's glutathione oxidase-like functionality, with RuSA/NiFe-LDH–MXene exhibiting the most accelerated GSH depletion. The significant reduction in GSH, the cell's principal antioxidant, undermines the tumor's protection against oxidative stress. This action enhances ROS production, increasing the cytotoxicity of the nanozyme against tumor cells.⁷⁸ We evaluated the activities of POD and GSH-OXD in RuSA/NiFe-LDH–MXene at temperatures of 37 and 50 °C (Fig. 4i). The absorbance of ox-TMB was greater at 50 °C than at 37 °C, but the absorbance of DTNB decreased from 50 °C to 37 °C. This result can be ascribed to the increased kinetics of POD and GSH-OXD at greater temperatures.

Fig. 4 Photothermal performance and catalytic activity of RuSA/NiFe-LDH–MXene nanozymes. (a) Temperature elevation profiles of RuSA/NiFe-LDH–MXene dispersions at different concentrations under NIR irradiation. (b) Infrared thermal images of RuSA/NiFe-LDH@MXene at different times and concentrations. (c) Photothermal conversion efficiency calculation and thermal decay analysis of RuSA/NiFe-LDH–MXene. (d) Photothermal cycling performance over multiple irradiation/cooling cycles. (e) Steady-state kinetic curves for peroxidase (POD)-like activity using H₂O₂ as a substrate. (f) ESR spectra confirming ˙OH generation under different catalytic conditions. (g) Time-dependent oxygen (O₂) generation profiles. (h) GSH (glutathione) oxidation kinetics profile. (i) Temperature-dependent GSH oxidation (GSH-OXD) and peroxidase (POD) activity assays.

The single-atom RuSA/NiFe-LDH–MXene-PEG nanozyme had a significant lethal impact on 4T1 tumor cells, attributed to the synergistic light-induced thermal effect and heat-augmented nano-catalytic therapy. The nanoscale dimensions of RuSA/NiFe-LDH–MXene facilitated the internalization of the particles by tumor cells. The cell-associated accumulation of FITC-modified RuSA/NiFe-LDH–MXene into 4T1 cells was examined at different incubation times (0 h, 2 h, 4 h, and 8 h) using confocal laser scanning microscopy (CLSM), as illustrated in Fig. 5a. The green fluorescence denotes cell nuclei stained with DAPI, whereas the yellow-red fluorescence corresponds to the FITC-labeled nanocomposites. At 0 hours, no visible FITC fluorescence is present in the cytoplasm, signifying the lack of nanocomposite internalization. Following 2 hours of incubation, faint FITC signs emerge in the cytoplasm, indicating the preliminary phases of cellular absorption. After 4 hours, the FITC fluorescence becomes more pronounced and extensively disseminated inside the cytoplasm, demonstrating ongoing internalization. After 8 hours, the most intense FITC fluorescence is detected, indicating significant intracellular accumulation and implying effective cellular internalization of the RuSA/NiFe-LDH–MXene nanocomposites over time. At 0 hours, no discernible FITC fluorescence is detected in the cytoplasm, signifying the lack of nanocomposite internalization. Following 2 hours of incubation, faint FITC signs emerge in the cytoplasm, indicating the preliminary phases of cellular absorption. After 4 hours, the FITC fluorescence becomes more pronounced and extensively disseminated inside the cytoplasm, indicating increasing internalization. After 8 hours, the most intense FITC fluorescence is detected, indicating significant intracellular accumulation and implying effective cellular internalization of the RuSA/NiFe-LDH–MXene nanocomposites over time. Fig. 5b shows the cell viability (%) of 4T1 cells treated with various concentrations (0, 25, 50, 100, and 200 μg mL⁻¹) of different groups. At 0 μg mL⁻¹, all groups exhibit approximately 100% cell viability, signifying the absence of cytotoxicity in untreated cells. With increasing concentration, groups including RuSA–MXene and RuSA/NiFe-LDH–MXene (A₃–A₆) demonstrate cytotoxicity that is dependent on concentration. A₆ (RuSA/NiFe-LDH–MXene + laser) exhibits the most pronounced reduction in viability, particularly at 200 μg mL⁻¹, indicating the most cytotoxic effect. Conversely, A₁ (control) and A₂ (laser only) exhibit robust viability at all concentrations, thereby affirming the safety of laser exposure alone. The statistical analysis (***p < 0.001) underscores the considerable disparity between A₃ and A₆, demonstrating the synergistic effect of the laser and the LDH-infused nanocomposite. The results unequivocally indicate that the RuSA/NiFe-LDH–MXene nanocomposite (A₃) elicits mild cytotoxicity via its inherent catalytic activity. Upon the application of NIR laser irradiation (A₆), cell viability significantly diminishes owing to the synergistic augmentation of photothermal and catalytic reactive oxygen species (ROS) production, resulting in substantial cellular damage. This synergism aligns with discoveries in analogous nanozyme systems, wherein heat amplifies catalytic activity, hence elevating oxidative stress within cells. For instance, RuSA@MXene with laser (A₅) exhibits reduced cytotoxicity relative to A₆, thereby affirming the pivotal function of NiFe-LDH in enhancing both photothermal conversion and peroxidase-like activity. This aligns with previous results indicating that LDH components enhance electron transfer efficiency and cascade catalysis. The negligible cytotoxicity in the control (A₁) and laser-only (A₂) groups affirms the biocompatibility of the experimental configuration and suggests that the cell death observed in A₆ is attributable to the synergistic therapeutic impact of the nanocomposite and laser irradiation, rather than nonspecific harm. Thus Fig. 5b highlights the concentration- and treatment-dependent cytotoxic effect of RuSA/NiFe-LDH–MXene, establishing its potential as a dual-functional agent for catalytic and photothermal cancer therapy. In addition, Fig. 5c compares the cell viability (%) of 4T1 cells after treatment with different experimental groups (A₁, A₂, A₄, and A₆). The cell survival for the A₁ (control), A₂ (laser alone), and A₄ (RuSA@MXene) groups is almost 100%, signifying the absence of severe cytotoxicity under these conditions (ns = not significant). Conversely, the A₆ group exhibits a substantial decrease in cell viability to under 25%, indicating pronounced cytotoxicity (p < 0.0001, ****). This indicates that only the combination of RuSA/NiFe-LDH–MXene and laser irradiation results in significant cell death. Our results unequivocally indicate the essential role of both the RuSA/NiFe-LDH–MXene nanocomposite and laser irradiation in attaining good therapeutic outcomes. The control group and laser-only group exhibit no substantial toxicity, affirming the biocompatibility of the cells under these conditions. Similarly, RuSA@MXene without laser (A₄) exhibits minimal cytotoxicity, indicating that the RuSA@MXene nanomaterial, in the absence of photothermal activation, is comparatively harmless in this context. The significant reduction in cell viability in the A₆ group underscores the synergistic therapeutic mechanism of RuSA/NiFe-LDH–MXene when subjected to laser irradiation. This synergism likely arises from the integrated photothermal heating and augmented catalytic reactive oxygen species formation enabled by the NiFe-LDH layers and Ru single atoms, in accordance with other studies on single-atom and LDH-based nanozymes. This figure presents fluorescence microscopy images of 4T1 cells subjected to various circumstances (A₁–A₆) to assess intracellular reactive oxygen species (ROS) generation by DCFH-DA staining. No significant DCF fluorescence is found in the control (A₁) and laser-only (A₂) groups, indicating minimal ROS generation. Moderate fluorescence is observed in A₃ (RuSA/NiFe-LDH–MXene) and A₄ (RuSA@MXene) attributable to the inherent catalytic activity of the Ru single atoms. Significantly, ROS levels elevate in A₅ (RuSA@MXene + laser) and reach a maximum in A₆ (RuSA/NiFe-LDH–MXene + laser), exhibiting the most intense DCF fluorescence. The minimal ROS in A₁ and A₂ indicates that neither the cell culture environment nor the laser alone generates oxidative stress, underscoring the system's biocompatibility in the absence of nanomaterials. The moderate formation of reactive oxygen species in A₃ and A₄ indicates that the Ru single atoms demonstrate basic peroxidase-like activity, aligning with the principles of single-atom catalytic mechanisms. Laser irradiation amplifies this action in A₅, presumably via modest photothermal activation. The elevated ROS levels in A₆ demonstrate a synergistic impact between photothermal heating and the augmented catalytic activity of the NiFe-LDH component, which promotes electron transfer and Fenton-like reactions. These findings align with prior studies showing that LDH supports boost catalytic efficiency in single-atom catalysts, and photothermal heating further accelerates the catalytic ROS production.⁶⁰ As mitochondrial dysfunction is closely associated with cell apoptosis, JC-1 staining was employed to evaluate alterations in mitochondrial membrane potential, providing insight into mitochondrial integrity across different treatment groups Fig. 5e shows the effects of different treatments (A₁–A₆) on mitochondrial membrane potential (MMP) in 4T1 cells, using JC-1 staining. The JC-1 staining data demonstrate definitive proof of mitochondrial membrane potential (MMP) disturbance in 4T1 cells after to various treatments. In A₁ (control) and A₂ (laser alone), mitochondria predominantly exhibit red/yellow fluorescence, signifying preserved membrane potential and viable cells. This affirms that neither the basal culture conditions nor laser irradiation independently induces mitochondrial damage, underscoring the biocompatibility of the experimental framework. Conversely, treatment groups with nanomaterials (A₃, A₄, A₅) exhibit a considerable enhancement in green fluorescence, indicating partial mitochondrial depolarization. The mitochondrial dysfunction is likely attributable to oxidative stress induced by the intrinsic peroxidase-like activity of Ru single atoms inside the nanocomposites, which produce reactive oxygen species (ROS) even without photothermal activation. The impact is further intensified in A₅ (RuSA@MXene + laser), where laser-induced photothermal heating augments catalytic ROS production, hence aggravating mitochondrial stress. The most significant effect is noted in A₆ (RuSA/NiFe-LDH–MXene + laser), where mitochondria exhibit the most intense blue/green JC-1 signal, signifying considerable mitochondrial depolarization. The RuSA/NiFe-LDH–MXene nanozyme exhibited catalase-like (CAT-like) activity, enabling it to catalyze the decomposition of H₂O₂ into O₂. To monitor oxygen generation in vitro, [Ru(dpp)₃]²⁺Cl₂ was used as an oxygen-sensitive probe, as its red fluorescence is quenched by intracellular O₂. The images in Fig. 5f depict the impact of different treatments (A₁–A₆) on mitochondrial membrane potential in 4T1 cells utilizing the [Ru(dap)₃]²⁺Cl₂ dye. In healthy mitochondria with preserved membrane potential, this dye accumulates and exhibits yellow fluorescence; conversely, mitochondrial malfunction and depolarization alter the signal to cyan fluorescence. In the A₁ (control) and A₂ (laser only) groups, mitochondria exhibit robust yellow fluorescence, signifying a healthy membrane potential and affirming that neither standard culture conditions nor laser irradiation alone induces mitochondrial damage. Conversely, A₃ (RuSA/NiFe-LDH–MXene) exhibits a little enhancement in cyan fluorescence, indicating moderate mitochondrial dysfunction potentially caused by the catalytic reactive oxygen species produced by the Ru single atoms. A₄ (RuSA@MXene) exhibits significant yellow fluorescence, indicating that RuSA@MXene alone has a minimal effect on mitochondrial health in the absence of external stimulation. Upon the application of laser irradiation (A₅, A₆), a significant transition to blue fluorescence is observed, particularly in A₆ (RuSA/NiFe-LDH–MXene + laser), where mitochondria exhibit the most pronounced cyan fluorescence, signifying substantial mitochondrial depolarization. Fig. 5g depicts the live/dead cell labeling of 4T1 cells utilizing calcein-AM/PI to evaluate cytotoxicity among different treatment groups (A₁–A₆). Viable cells exhibit a red hue (calcein-AM), whereas non-viable or membrane-compromised cells are stained blue (PI). In the A₁ (control), A₂ (laser alone), and A₃ (RuSA/NiFe-LDH–MXene) groups, cells primarily exhibit red fluorescence, signifying that these conditions maintain cell viability and do not provoke substantial cytotoxicity. This indicates that both the RuSA/NiFe-LDH–MXene nanocomposite and laser irradiation independently exhibit biocompatibility under the evaluated conditions. In A₄ (RuSA@MXene) and A₅ (RuSA@MXene + laser), the presence of blue-stained cells indicates considerable cytotoxicity attributed to the intrinsic ROS generation of the Ru-based catalyst, which is further amplified by laser-induced photothermal activation. The most significant cytotoxic effect occurs in A₆ (RuSA/NiFe-LDH–MXene + laser), where the cells primarily display blue fluorescence, signifying extensive membrane damage and cell mortality. The synergistic impact results from the integration of the NiFe-LDH support, which amplifies peroxidase-like catalytic ROS generation, and photothermal heating, which subsequently intensifies oxidative damage and cellular stress. Fig. 5h depicts the effects of different treatments (A₁–A₆) on the cytoskeletal architecture and nuclear morphology of 4T1 cells, as visualized by F-actin (blue) and DAPI (green) staining. F-actin delineates the cellular structure, whereas DAPI stains the nuclei, facilitating the evaluation of cellular integrity and stress responses. In the A₁ (control), A₂ (laser alone), and A₃ (RuSA/NiFe-LDH–MXene) groups, cells exhibit a well-structured and extensive F-actin network alongside distinct nuclear morphology, signifying robust cytoskeletal integrity and negligible cellular stress. The data indicate that neither the nanomaterial nor laser irradiation alone significantly disrupt the cytoskeleton. Nonetheless, A₄ (RuSA@MXene) exhibits minor cytoskeletal disruption, presumably attributable to ROS-induced oxidative stress. The damage is more evident in A₅ (RuSA@MXene + laser), where the F-actin filaments are broken and the cellular morphology is constricted, indicating increased cytoskeletal damage due to the synergistic catalytic and photothermal actions. The most pronounced cytoskeletal disruption occurs in A₆ (RuSA/NiFe-LDH@MXene + laser), characterized by the collapse of the F-actin network and the adoption of a rounded cellular shape, signifying significant structural disintegration and loss of adhesion. The disintegration of the cytoskeletal structure is indicative of apoptosis and validates the significant cellular impairment resulting from the combined effects of reactive oxygen species production and photothermal activation. Fig. 5i illustrates flow cytometry analysis with Annexin V-FITC/PI labeling to evaluate the apoptotic state of Panc02 cells after different treatments. In these scatter plots, viable cells are located in the lower left quadrant (Annexin V⁻/PI⁻), early apoptotic cells in the lower right quadrant (Annexin V⁺/PI⁻), and late apoptotic/necrotic cells in the upper right quadrant (Annexin V⁺/PI⁺). In the control and NIR-only groups, the bulk of cells (>82–89%) are located in the live cell quadrant, signifying little cytotoxic effects from baseline conditions or laser exposure alone. This underscores the biocompatibility of the system in the absence of nanomaterial activation.⁷⁹ Treatment with RuSA/NiFe-LDH@MXene alone results in a moderate elevation in apoptotic populations, with early apoptosis (∼16.17%) and late apoptosis (∼15.24%), presumably due to the nanozyme's inherent ROS production leading to mitochondrial malfunction and the activation of programmed cell death pathways. The most pronounced apoptotic effect is observed in the RuSA/NiFe-LDH@MXene + NIR group, with late apoptotic/necrotic cells increasing to 57.09% and early apoptotic cells increasing to 22.63%, resulting in a substantial decline in the viable cell population. This significant change illustrates the combined impact of NiFe-LDH-facilitated catalytic ROS generation and photothermal activation by NIR irradiation, which intensifies oxidative stress and mitochondrial injury, leading cells to undergo apoptosis. The quantitative apoptosis findings align with previous evidence on mitochondrial depolarization, ROS buildup, and live/dead labeling, validating that RuSA/NiFe-LDH@MXene, in conjunction with NIR irradiation, successfully triggers apoptosis via catalytic–photothermal synergism. This underscores its potential as a formidable platform for targeted cancer therapy. Fig. 5j depicts the distribution of viable and non-viable cells after different treatments (A₁, A₂, A₄, and A₆), offering a quantitative evaluation of cytotoxic effects. In the A₁ (control), A₂ (laser only), and A₄ (RuSA@MXene without laser) groups, the majority of cells exhibit viability over 90%, demonstrating that neither the nanomaterial alone nor laser irradiation alone elicits substantial cytotoxicity under these conditions. This underscores the biocompatibility of RuSA@MXene and the safety of laser exposure without synergistic activation. Conversely, the A₆ (RuSA/NiFe-LDH–MXene + laser) group exhibits a significant rise in the population of dead cells, coupled with a considerable decrease in viable cells. Fig. 5k shows confocal microscopy images illustrating the morphological integrity of tumor spheroids subjected to various treatment settings, assessing the photothermal and catalytic impacts of RuSA/NiFe-LDH–MXene with and without near-infrared (NIR) irradiation. In the absence of NIR irradiation (–NIR), both the control and RuSA/NiFe-LDH@MXene groups retain a compact, intact spheroid shape exhibiting strong magenta fluorescence, signifying that without external activation, the nanomaterial alone does not cause significant structural damage. Following NIR irradiation (+NIR), the control spheroids maintain their structural integrity, indicating that laser exposure alone does not compromise the tumor model. Spheroids treated with RuSA/NiFe-LDH@MXene + NIR have a fragmented and disseminated appearance, along with a fluorescence shift, indicating considerable structural disintegration and cellular mortality within the spheroid core. The significant decline in the RuSA/NiFe-LDH@MXene + NIR group illustrates the efficient photothermal heating and catalytic reactive oxygen species formation upon near-infrared activation, which collaboratively disrupt cell–cell adhesion and obliterate the tumor mass.

Fig. 5 In vitro evaluation of photothermal-augmented nano-catalytic therapy. (a) CLSM images of time-dependent internalization of FITC-labeled RuSA/NiFe-LDH@MXene in 4T1 cells (magenta: nuclei, green: RuSA/NiFe-LDH@MXene). (b) Cell viability at different concentrations of A₁ (control), A₂ (laser alone), A₃ (RuSA/NiFe-LDH@MXene), A₄ (RuSA@MXene), A₅ (RuSA@MXene + laser), and A₆ (RuSA/NiFe-LDH@MXene + laser) treatments. (c) Cell viability comparison at 200 μg mL⁻¹ of RuSA/NiFe-LDH@MXene, (d) CLSM images of the intracellular ROS levels in 4T1 cells exposed to various treatments (A₁, A₂, A₃, A₄, A₅, and A₆). Statistical analysis was performed using an unpaired two-tailed Student's t-test, with ***p < 0.001 considered highly significant. Data are presented as mean ± standard deviation (S.D.), with n = 3. (e) JC-1 staining of 4T1 cells from distinct therapeutic groups. (f) CLSM images of [Ru(dap)₃]²⁺Cl₂ fluorescence quenching showing oxygen production. (g) Calcein-AM/PI staining differentiating live (red) and dead (blue) cells. (h) F-actin and DAPI staining demonstrating cytoskeletal integrity and morphological alterations. (i) Flow cytometric study of apoptosis in 4T1 cells treated with different combinations. (j) Quantitative analysis of live/dead cell percentages across treatments. (k) Spheroid disruption images of spheroid collapse (blue indicates the disrupted structure).

Following the synergistic antitumor response of the RuSA/NiFe-LDH@MXene nanozyme observed in vitro, we then examined its therapeutic impact in vivo using 4T1 tumor-bearing mice. Fig. 6 Illustrates the in vivo biodistribution and retention characteristics of FITC-labelled RuSA/NiFe-LDH@MXene nanohybrids in 4T1 tumor-bearing mice over a 32-hour duration post intratumoral injection. Fig. 6a illustrates that the fluorescence intensity within the tumor increased, attaining its maximum at 12 hours post-injection. After 32 hours, a robust fluorescence signal remained at the tumor site, indicating sustained retention of RuSA/NiFe-LDH@MXene within the tumor. The results underscore the superior tumor-targeting ability and retention characteristics of RuSA/NiFe-LDH@MXene, likely enhanced by their nanoscale dimensions, surface modifications, and possible interactions with tumor tissues. The extended buildup at the tumor location facilitates enduring photothermal and catalytic therapeutic effects following further near-infrared (NIR) irradiation. In contrast to the quick clearance or off-target distribution frequently seen in nanomaterial systems, the biodistribution profile indicates that RuSA/NiFe-LDH–MXene has advantageous pharmacokinetics for localized tumor treatment. This effective tumor localization is crucial for optimizing therapy efficacy and reducing systemic adverse effects. Fig. 6b depicts the experimental procedure established to evaluate the therapeutic effectiveness of RuSA/NiFe-LDH–MXene in a 4T1 tumor-bearing BALB/c mouse model. Female BALB/c mice were initially infected with 4T1 tumor cells, and tumors were allowed to develop for 12 days. Subsequent to tumor development, the nanotherapeutic drug was delivered directly into the tumor using intertumoral injection to guarantee localized delivery and reduce systemic exposure. Following a 12-hour incubation period, permitting adequate cellular absorption and accumulation within the tumor microenvironment, the tumors underwent 10 minutes of exposure to 808 nm NIR irradiation. This method activated the photothermal and catalytic characteristics of RuSA/NiFe-LDH–MXene, boosting the formation of reactive ROS and generating localized hyperthermia for synergistic tumor ablation. Following treatment, tumor growth and body weight were monitored until day 14 to assess therapeutic efficacy and systemic safety. This meticulously crafted timeline illustrates how the integration of catalytic therapy with photothermal effects can optimize tumor suppression while preserving animal health, presenting a promising strategy for minimally invasive cancer treatment. Hemolysis assessments were carried out to study the biocompatibility of RuSA/NiFe-LDH–MXene. Fig. 6c illustrates the superior hemocompatibility of RuSA/NiFe-LDH–MXene nanoparticles by assessing their hemolytic activity at different concentrations (0–200 μg mL⁻¹). The hemolysis percentage remains under 5% even at the highest dose tested, significantly lower than the positive control (H₂O), which causes total hemolysis (∼100%). The inset shot corroborates these observations, revealing a distinct crimson supernatant in the H₂O sample attributable to hemoglobin release, whereas those containing RuSA/NiFe-LDH–MXene exhibit no discernible hemolysis, remaining transparent. The results underscore the exceptional blood compatibility of the nanomaterial, an essential criterion for in vivo biomedical applications. The minimal hemolysis indicates that the RuSA/NiFe-LDH–MXene structure reduces membrane disruption, possibly owing to its stable design and homogeneous single-atom distribution, rendering it a promising candidate for safe therapeutic applications, including photothermal and catalytic cancer therapies. Fig. 6d exhibits the photothermal efficacy of RuSA/NiFe-LDH–MXene subjected to NIR irradiation for a duration of five days. The group administered RuSA/NiFe-LDH–MXene and subjected to NIR exhibits a notable and sustained elevation in temperature, attaining around 52 °C by the fifth day. Conversely, the control group devoid of RuSA/NiFe-LDH–MXene exhibits a relatively stable temperature of approximately 35 °C, signifying minimal heat production. The increased temperature in the treated group underscores the effective photothermal conversion capabilities of RuSA/NiFe-LDH–MXene, due to its robust NIR absorption and swift thermal response. The continuous heating effect is essential for generating localized hyperthermia in tumor tissues, effectively harming cancer cells while reducing damage to adjacent healthy tissues. In addition, Fig. 6e illustrates the tumor inhibition rates (%) during a 10-day period for four treatment groups. The RuSA/NiFe-LDH–MXene + NIR group has the highest tumor inhibition rate, surpassing 7%, which signifies a robust and enduring anticancer impact. The RuSA/NiFe-LDH–MXene group without NIR and the NIR-only group both demonstrate substantial tumor inhibition, indicating that each therapy individually has a partial therapeutic impact, but their combination yields a synergistic boost. The control group exhibits low tumor inhibition, hence confirming negligible spontaneous tumor suppression. Also, the tumor volume of mice subjected to various treatments over a defined experimental period is seen in Fig. 6f. The control group demonstrated the greatest tumor volume (∼600 mm³), indicating unrestrained tumor proliferation. NIR irradiation alone had no therapeutic efficacy, with tumor sizes persisting at elevated levels (∼500 mm³). The application of the RuSA/LDH–MXene nanocatalyst resulted in a small tumor decrease of around 400 mm³, suggesting that the intrinsic catalytic activity of the nanozyme may somewhat impede tumor advancement. The combination of RuSA/LDH–MXene and NIR irradiation led to a substantial reduction in tumor volume to around 50 mm³, suggesting a synergistic therapeutic effect. The statistical annotations (**p < 0.01, ***p < 0.001, and ****p < 0.0001) confirm the high significance of tumor volume reduction compared to the control group. Fig. 6g presents the tumor weight in mice after different treatments, providing another quantitative assessment of the therapeutic efficacy of each intervention. The control group exhibits the greatest tumor weight (∼2.5 g), indicating unregulated tumor proliferation. NIR irradiation alone results in a marginal decrease in tumor weight (∼0.8 g), signifying little tumor suppression by hyperthermia alone. Treatment with RuSA/LDH–MXene further diminishes tumor weight (∼0.7 g), indicating that the nanocatalytic activity alone can partially inhibit tumor development. The RuSA/LDH–MXene + NIR group shows a significant decrease in tumor weight (∼0.1 g), indicating a strong synergistic therapeutic impact. The sharp reduction in tumor volume and weight in the RuSA/LDH–MXene + NIR group can be attributed to the combination of photothermal heating and catalytic ROS generation upon NIR exposure, both contributing to effective tumor cell apoptosis and growth inhibition. RuSA/LDH–MXene + NIR treatment exhibited an impressive tumor growth suppression rate of 79% (Fig. 6h). The results demonstrate that RuSA/NiFe-LDH–MXene significantly inhibits tumor growth via its nanocatalytic properties, and when integrated with photothermal therapy (NIR), attains enhanced therapeutic effectiveness through the synergistic effects of catalytic and hyperthermic tumor ablation. To evaluate the biosafety of RuSA/LDH/MXene for prospective clinical applications, normal mice were administered either saline (control) or RuSA/LDH–MXene via intravenous injection. Blood samples were obtained at various intervals following injection and examined using conventional hematological and biochemical procedures. Fig. 6i illustrates that the kidney and liver function parameters remained steady both prior to and following treatment, with no significant changes seen. Furthermore, all assessed hematological parameters exhibited no significant discrepancies in comparison with the control group throughout the observation period. The results affirm that the RuSA/LDH–MXene nanozyme elicits minimal systemic toxicity and preserves hematological homeostasis throughout the treatment. Upon completion of the medication, tumor samples from each group were collected and analyzed using hematoxylin–eosin (H&E) staining and TUNEL immunohistochemical staining to assess tissue morphology and apoptosis, respectively. Fig. 6j shows histological analysis of tumor tissues from four experimental groups. In the control and NIR groups, tumor tissues exhibit dense and undamaged cellular structures, signifying minimal cellular damage. The RuSA/LDH–MXene group exhibits signs of cellular disarray and mild cytotoxicity, indicating moderate anticancer effects attributed only to the catalytic capabilities of the nanomaterial. In the RuSA/LDH–MXene + NIR group, tumor sections exhibit significant cell shrinkage, fragmentation, and necrosis, indicating substantial tissue damage and successful tumor ablation. These findings support the synergistic therapeutic efficacy of RuSA/LDH–MXene under near-infrared irradiation. The photothermal effect of the nanoplatform amplifies its catalytic activity, resulting in significant destruction of tumor tissue. This synergism results in improved therapeutic outcomes relative to either treatment individually, underscoring the potential of RuSA/LDH–MXene + NIR as an efficacious method for photothermal-enhanced nanocatalytic cancer treatment.

Fig. 6 In vivo evaluation of photothermal–catalytic cancer therapy of the RuSA/NiFe-LDH@MXene nanozyme. (a) Fluorescence images of mice treated with RuSA/NiFe-LDH@MXene. (b) Schematic representation of the in vivo experimental procedure in mice. (c) Hemolysis assay of the hemocompatibility of RuSA/NiFe-LDH@MXene at various concentrations. (d) Temperature profiles during NIR irradiation. (e) Tumor inhibition rates over time and (f) tumor volume. (g) Tumor weight measurements for different treatment groups. (h) Photographic images of excised tumors from each treatment group collected after treatment on day 14 (n = 4). (i) Hematological and biochemical analysis in treated mice over 14 days. (j) Images of tumor tissues stained with H&E and TUNEL following different therapies. Statistical significance determined using the unpaired Student's two-tailed t-test: (**p < 0.01, ***p < 0.001, and ****p < 0.0001).

Conclusion

In summary, we created a multifunctional nanoplatform by affixing Ru single atoms to NiFe-LDH/MXene hybrid nanosheets, facilitating synergistic photothermal and nanocatalytic cancer treatment. The NiFe-LDH component is essential for stabilizing atomically scattered Ru species and altering their electronic structure, thus improving catalytic activity and substrate interaction. Concurrently, the MXene substrate offers superior photothermal characteristics and electrical conductivity, hence enhancing catalytic efficiency under NIR irradiation. In comparison with RuSA@MXene, the RuSA/NiFe-LDH–MXene nanozyme demonstrated markedly enhanced peroxidase-like activity (∼2.8-fold increase in catalytic velocity), elevated hydroxyl radical generation (∼1.6-fold increase), and improved photothermal conversion efficiency (∼46.8%). In vivo investigations validated significant tumor reduction (91.7% inhibition) without inducing systemic toxicity, illustrating the therapeutic potential of this technology. This study emphasizes that rationally engineered hybrid supports, such as NiFe-LDH/MXene, can address the shortcomings of single-atom catalysts, presenting a viable approach for effective, safe, and clinically applicable cancer nanotherapy.

Conflicts of interest

There is no conflict of interest.

Data availability

Data are available upon request.

Supplementary information is available. SEM characterization of the MXene/LDH hybrid architecture.

Time-dependent UV–Vis–NIR absorbance spectra of pristine MXene and MXene/LDH composite.

EPR spectra of DMPO–˙O₂⁻ Photothermal performance of different formulations. See DOI: https://doi.org/10.1039/d5bm01060h.

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