Gallium‐Doped MXene Nanozymes Protect Liver Through Multi‐Death Pathway Blockade and Hepatocyte Regeneration

ABSTRACT

Acetaminophen‐induced liver injury (AILI) is a major cause of acute liver failure, yet single‐target therapies like N‐acetylcysteine remain inadequate due to its complex pathogenesis. To address this challenge, we propose a dual‐action defense‐regeneration strategy that concurrently blocks multi‐death pathways and promotes hepatocyte regeneration. Specifically, the therapeutic metal gallium is doped into V₂C MXene nanozymes (Ga‐V₂C) to surpass conventional nanozymes by integrating sustained antioxidant activity for death signaling blockade, multi‐pathway regulation of cell death networks, and activation of pro‐regenerative molecules. In vivo, Ga‐V₂C nanozymes exhibited superior protective efficacy over N‐acetylcysteine against AILI. Mechanistic investigations revealed that the Ga‐V₂C nanozymes disrupt the synergistic amplification of liver injury by simultaneously inhibiting three key death pathways: oxidative stress (via ROS scavenging, reduce JNK phosphorylation, and activated Nrf2/HO‐1), apoptosis (via restored Bcl‐2/Bax balance), and ferroptosis (by suppressed iron‐dependent lipid peroxidation and upregulated SLC7A11/FTH1/FTL1). Notably, Ga‐V₂C nanozymes fostered a pro‐regenerative microenvironment by activating Wnt/βCAT pathways signaling and key cell cycle drivers (CCND1, MYC, PCNA), thereby enhancing hepatocyte regeneration. This work not only offers a promising therapeutic approach for AILI but also significantly expands the scope of nanozyme‐based therapeutics for complex diseases requiring multi‐target intervention.

This study develops gallium‐doped V₂C MXene nanozymes (Ga‐V₂C) to treat acetaminophen‐induced liver injury through multi‐death pathway blockade and hepatocyte regeneration. Unlike conventional single‐target therapies like N‐acetylcysteine, Ga‐V₂C nanozymes enable oxidative stress suppression, apoptosis, and ferroptosis inhibition, and enhanced hepatocyte regeneration. In vivo studies demonstrate that Ga‐V₂C nanozymes surpass conventional therapies, offering a promising strategy for complex diseases treatment.

1. Introduction

Acetaminophen (APAP)‐induced liver injury (AILI) is a leading cause of acute liver failure [1], accounting for approximately 50% of acute liver failure cases globally, particularly in the USA and parts of Europe [2, 3]. AILI pathogenesis involves multidimensional and crosstalking pathological mechanisms [4]. APAP metabolites, notably N‐acetyl‐p‐benzoquinone imine (NAPQI), deplete glutathione (GSH), triggering oxidative stress and subsequent c‐Jun N‐terminal kinase (JNK) phosphorylation, which exacerbates mitochondrial dysfunction and necroptosis. Recent studies reveal that oxidative stress dynamically interacts with programmed cell death networks during AILI progression [5]: sustained JNK activation not only amplifies necrotic cell death but also induces apoptosis via Bcl‐2/Bax imbalance, while iron‐driven lipid peroxidation and GPX4 inactivation synergistically drive ferroptosis. Furthermore, impaired hepatocyte regeneration exacerbates injury. Although the clinical first‐line antidote, N‐acetylcysteine (NAC), alleviates oxidative stress by replenishing GSH and protects hepatocytes from necrosis and apoptosis, it fails to address crosstalk among multiple cell death pathways or promote tissue regeneration [6, 7]. This significant therapeutic gap underscores the urgent need for novel multi‐target therapeutic strategies.

To address the complex pathology of AILI, this study proposes a dual‐action defense‐regeneration therapeutic strategy. The defense component requires systemic blockade of the interconnected programmed cell death networks, necessitating an agent capable of simultaneously intervening in crosstalk pathways, including necrosis, apoptosis, and ferroptosis. Conventional antioxidants, which merely neutralize reactive oxygen species (ROS), fail to suppress downstream JNK‐Bax‐mediated mitochondrial apoptosis or ferroptosis execution. Similarly, specific apoptosis inhibitors cannot mitigate iron‐driven lipid peroxidation. Concurrently, the regeneration component aims to activate intrinsic hepatocyte proliferation by modulating cell cycle drivers (e.g., CCND1, MYC) and proliferation markers (e.g., PCNA) to achieve structural repair. Thus, realizing this dual‐action defense‐regeneration therapeutic strategy requires a novel therapeutic agent integrating three key functions: sustained antioxidant activity to block death signaling, multi‐target regulation of cell death networks, and activation of pro‐regenerative molecules.

Nanozymes surpass natural enzymes in terms of superior stability, cost‐effectiveness, reusability, and durability under extreme conditions, leading to widespread use in treating diseases, including cancer, bacterial infections, neurodegenerative disorders, and cardiovascular diseases [8, 9, 10, 11, 12, 13, 14]. As a result, they hold great promise for addressing oxidative stress and ROS‐related diseases [15, 16, 17, 18], serving as effective alternatives to NAC for AILI treatment. However, conventional nanozyme systems are limited by single‐pathway mechanisms insufficient to modulate complex inter‐pathway crosstalk and lack pro‐regenerative functions beyond basic ROS scavenging. Recent advances highlight MXene‐based nanozymes, particularly V₂C MXene nanozymes, as promising candidates due to their high ROS scavenging efficiency, facile functionalization, multifunctional nature, and 2D structure‐enhanced membrane permeability [19, 20, 21, 22]. Furthermore, metal doping technology shows potential to enhance these MXene systems by overcoming restrictions from single‐metal composition, enabling broader applications in complex pathological environments [23].

Based on the above considerations, this study chose to dope the therapeutic metal gallium into the structure of V₂C, thereby facilitating the synchronized action and distribution of the two active components in vivo. The developed gallium‐doped V₂C MXene nanozymes (Ga‐V₂C) were expected to protect the liver through a dual‐action defense‐regeneration therapeutic strategy of multi‐death pathway blockade and hepatic regeneration (Scheme 1): (1) Ga‐V₂C MXene nanozymes were expected to exhibit sustained antioxidant activity by scavenging excessive ROS, thereby disrupting downstream death signaling pathways (e.g., JNK phosphorylation, Bcl‐2/Bax imbalance, and ferroptosis‐related lipid peroxidation) and enhancing endogenous antioxidant defenses (e.g., Nrf2/HO‐1 activation). (2) Due to analogous ionic radii and coordination chemistry, Ga³⁺ competes with Fe³⁺ in biological systems, displacing it on transferrin to reduce the availability of free Fe²⁺ for Fenton reaction‐driven •OH generation, thereby modulating oxidative processes and exhibiting anti‐inflammatory effects [24]. (3) Ga‐V₂C MXene nanozymes inhibit crosstalk pathways by simultaneously suppressing necrosis, apoptosis, and ferroptosis, thereby interrupting their synergistic exacerbation of liver injury. (4) Ga‐V₂C nanozymes may enhance hepatocyte proliferation by activating Wnt/β‐CAT and cell cycle drivers (CCND1, MYC, PCNA) and proliferation‐related signaling pathways (e.g., Nrf2), thereby creating a pro‐regenerative microenvironment.

SCHEME 1.

Ga‐V₂C MXene nanozymes alleviate APAP‐induced liver injury through multi‐death pathway blockade and hepatocyte regeneration. Ga‐V₂C demonstrates: (A) multienzyme‐mimetic activities: SOD, POD, GPx, and CAT‐like activities reduce oxidative stress through ROS scavenging, JNK phosphorylation reduction, and Nrf2/HO‐1 activation; (B) synergistic regulation of cell death: Restoring the Bcl‐2/Bax balance (anti‐apoptosis) and inhibiting iron‐dependent lipid peroxidation (anti‐ferroptosis) effectively block cell death; (C) enhanced hepatocyte regeneration: Activation of cell‐cycle drivers (CCND1, MYC, PCNA) promotes hepatocyte restoration and repair.

2. Results and Discussion

2.1. Preparation and Characterization of Ga‐V₂C MXene Nanozymes

The synthesis of Ga‐V₂C MXene nanozymes involved a modified multistep procedure (Figure 1A) [25]. Briefly, V₂C nanosheets were fabricated through selective etching of the aluminum (Al) layers from the V₂AlC MAX phase using a mixing solution of lithium fluoride (LiF) and hydrochloric acid (HCl). The single‐ or few‐layered MXene nanosheets were then obtained after ultrasonication‐assisted exfoliation. Subsequently, a thermal sintering process was employed to incorporate gallium (Ga) onto the V₂C MXene nanosheets. To characterize the basic structural features of the samples, dynamic light scattering (DLS) measurements were carried out to determine their zeta potential and size distribution. As depicted in Figure 1B,C, both V₂C and Ga‐V₂C nanozymes exhibit a similar negative surface charge with an average hydrodynamic diameter of approximately 400 nm. These findings suggest that the doping of Ga has minimal impact on the morphology of the nanozymes.

FIGURE 1.

Preparation and characterization of Ga‐V₂C MXene nanozymes. (A) Schematic illustration of the synthetic process for Ga‐V₂C nanozymes. Zeta potentials (B) and hydrodynamic diameters (C) of V₂C and Ga‐V₂C nanozymes. AFM (D), TEM (E), and HRTEM (F) images of Ga‐V₂C nanozymes. (G) TEM‐EDS mapping images of Ga‐V₂C nanozymes. XPS spectra of Ga‐V₂C (H), and corresponding high‐resolution XPS spectra of V 2p (I), C 1s (J), Ga 2p (K), Ga 3d (L), and O 1s region (M).

The morphology of Ga‐V₂C nanozymes was further investigated through the utilization of atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and high‐resolution transmission electron microscopy (HRTEM) measurements. The images revealed that the Ga‐V₂C nanozymes were composed of single‐layer or few‐layer structures with a uniform thickness around 2 nm (Figure 1D,E; Figure S1). According to the HRTEM image, the as‐prepared Ga‐V₂C nanozymes have high‐degree crystallinity asrevealed by crystalline lattice, indicating that the Ga doping would not significantly influence the crystallized structure of V₂C MXene. In addition, energy dispersive spectroscopy (EDS) demonstrated that elements of V, C, Ga, and O were uniformly dispersed throughout the Ga‐V₂C nanozymes (Figure 1G; Figure S2).

Furthermore, the chemical nature of Ga‐V₂C MXene was characterized by the typical X‐ray photoelectron spectroscopy (XPS). The survey XPS spectra of Ga‐V₂C showed distinct peaks for the V, C, Ga, and O elements present in the nanozymes, while the disappearance of Al‐indexed peaks in the spectrum indicated the complete removal of the Al component from the MAX‐phase ceramic after the etching process (Figure 1H). Additionally, the high‐resolution spectrum of the V 2p region in XPS was fitted by components corresponding to a group of peaks of V²⁺ (513.2 and 520.2 eV), V³⁺ (514.5 and 522.2 eV), and V⁴⁺ (516.5 and 523.9 eV) (Figure 1I). The C 1 s spectrum was deconvoluted into four peaks of C─V, C─C, C─O, and C═O, located at 282.0, 284.4, 285.2, and 288.2 eV, respectively (Figure 1J). In the case of Ga, the main peaks of Ga are located at 1117.7 eV for Ga 2p1/2, 1144.5 eV for Ga 2p3/2, 20.4 eV for Ga 3d3/2, and 18.9 eV for Ga 3d5/2, respectively (Figure 1K,L). The XPS analysis indicated that Ga predominantly existed in the form of Ga³⁺, which is the most stable form of oxidized Ga [26, 27]. The O 1s spectra could be fitted with four peaks at 529.6, 530.8, 531.9, and 533.0 eV corresponding to V─O_x, V─C─O, C─V─(OH)_x, and H₂O (Figure 1M), indicating a slight oxidation of the Ga‐V₂C nanosheet surface as well as the presence of oxygen and hydroxyl functional groups along with interlayer water. Moreover, we utilized inductively coupled plasma optical emission spectroscopy (ICP‐OES) to analyze the Ga content within the Ga‐V₂C nanozymes, which showed that the Ga loading capacity of the Ga‐V₂C nanozymes reached 26.1%.

2.2. Multienzyme Mimetic Activities of the Ga‐V₂C Nanozymes

In AILI, APAP overdose can lead to the production of large amounts of the toxic metabolite NAPQI in hepatocytes, causing oxidative stress and mitochondrial dysfunction, which in turn leads to hepatocyte death [28, 29]. Therefore, the capacity and efficiency of Ga‐V₂C MXene nanozymes in scavenging ROS reflect their potential for use in attenuating oxidative damage and promoting hepatocyte recovery. Subsequently, we evaluated the multienzyme mimetic activities of Ga‐V₂C nanozymes under physiological conditions by selecting substrates typically utilized by natural enzymes (Figure 2A).

FIGURE 2.

Multienzyme mimetic activities of the Ga‐V₂C MXene nanozymes. (A) Schematic illustration of enzyme‐mimetic activities of Ga‐V₂C nanozymes. (B) SOD‐like activity of Ga‐V₂C nanozymes. (C) POD‐like activity of Ga‐V₂C nanozymes at various concentrations of H₂O₂ (left) and corresponding Michaelis‐Menten curve (right). (D) Schematic diagram depicting the GPx‐like activity of Ga‐V₂C nanozymes in the presence of GSH, and recycling by GR in a coupled reaction (left), and GPx‐mimetic activity of Ga‐V₂C nanozymes (right) (Control: 300 µM NADPH). (E) CAT‐mimetic activity of Ga‐V₂C nanozymes at various concentrations of H₂O₂ (left) and corresponding Michaelis‐Menten curve (right).

The superoxide dismutase (SOD)‐like activity of Ga‐V₂C nanozymes was initially investigated, as SOD serves as a crucial intracellular enzyme for cellular antioxidant defense. This enzyme mitigates ROS by catalyzing the dismutation of the superoxide anion (O₂ ^•−) into hydrogen peroxide (H₂O₂) and molecular oxygen (O₂). The SOD‐like activity of Ga‐V₂C nanozymes is considered very important in AILI treatment, because O₂ ^•− is a highly reactive oxygen radical capable of damaging cellular components. To quantitatively assess the SOD‐like activity of Ga‐V₂C nanozymes, a commercial SOD assay kit was employed. The assay involved the use of a water‐soluble tetrazolium salt (WST‐1) to react with O₂ ^•− produced by the reaction of xanthine oxidase with xanthine to form spectrophotometrically identifiable formazan [30]. The reduction of this reaction in the presence of Ga‐V₂C nanozymes confirmed their SOD‐like activity. Furthermore, increasing the concentration of Ga‐V₂C nanozymes resulted in a higher inhibition ratio of formazan formation (Figure 2B), demonstrating a dose‐dependent SOD‐like activity. The nanozymes exhibit high SOD‐like activity, and the half‐maximal inhibitory concentration (IC₅₀) value for the inhibition of formazan formation was determined to be 73.6 ± 0.1 µg/µL.

Peroxidase (POD) is primarily used to detoxify H₂O₂ by converting it into harmless H₂O [31]. To evaluate the POD‐like activity of Ga‐V₂C nanozymes, we employed 3,3′,5,5′‐tetramethyl‐benzidine (TMB) and H₂O₂ as substrates [32]. As H₂O₂ was added, Ga‐V₂C nanozymes facilitated the oxidation of TMB into blue oxTMB, and the extent of conversion notably depended on the reaction duration and H₂O₂ concentration (Figure 2C). Subsequently, we investigated the kinetic parameters of Ga‐V₂C nanozymes, which coincided with the Michaelis–Menten model [33]. The K _M value, which indicates the affinity of the nanozyme for the substrate, was found to be 0.80 mM. Concurrently, the V _max, which represents the catalytic activity of the nanozyme, was determined to be 2.49 × 10⁻⁷ M/s for the Ga‐V₂C nanozymes.

GPx is essential for maintaining intracellular redox balance [34]. GPx catalyzes the conversion of H₂O₂ into H₂O and O₂ by oxidizing reduced glutathione (GSH) to glutathione disulfide (GSSG), and this process is balanced by the reduction of GSSG back to GSH in the presence of glutathione reductase (GR) and nicotinamide adenine dinucleotide phosphate (NADPH) (Figure 2D) [35]. The GPx‐like activity of Ga‐V₂C nanozymes was determined by monitoring the absorbance changes of NADPH. As shown in Figure 2D, the rapid decrease in NADPH absorbance over time confirms the efficient GPx‐like activity of Ga‐V₂C nanozymes.

Catalase (CAT) is recruited by cells to catalyze the decomposition of H₂O₂ into H₂O and O₂, thereby averting excessive accumulation of H₂O₂, which may contribute to pathological conditions and inflammatory processes [36]. To determine the CAT‐like activity of Ga‐V₂C nanozymes upon H₂O₂ decomposition, we monitored O₂ production using a dissolved oxygen meter. The addition of Ga‐V₂C nanozymes resulted in the observation of O₂ bubbles, and it was found that the production of O₂ increased as the concentration of H₂O₂ increased. This provides direct evidence that Ga‐V₂C nanozymes can act as an efficient CAT mimic to scavenge H₂O₂ (Figure 2E). Moreover, The K _M value of Ga‐V₂C nanozymes with H₂O₂ as the substrate was 19.67 mM, and the V _max value was 0.07 M/s  [37].

In the context of treating AILI, the dominant role of these activities likely depends on the pathological ROS profile. SOD‐like activity may be particularly critical due to the central role of •O₂ ⁻ in initiating oxidative cascades during APAP hepatotoxicity, as evidenced by studies showing mitochondrial superoxide overproduction as a key driver of APAP‐induced damage [5, 38, 39]. However, the synergistic combination of these enzymatic activities may collectively contribute to comprehensive ROS scavenging.

Considering the susceptibility of V₂C MXene to oxidation [19, 20, 21, 22], it is essential to store V₂C‐based Ga‐V₂C nanozymes in an inert atmosphere (e.g., nitrogen or argon) to maintain their enzyme‐mimetic activity and prevent oxidative degradation. To further enhance the stability of these nanozymes, optimal storage conditions should include low temperatures and protection from light. Stability studies indicate that Ga‐V₂C nanozymes maintain their enzyme‐mimetic activities and size for a minimum of one month when stored at 4°C under nitrogen. Moreover, storage at −20°C is anticipated to extend the shelf life to over 1 year while preserving efficacy.

2.3. Protective Efficacy of Ga‐V₂C Nanozymes Against AILI In Vitro and In Vivo

Since Ga‐V₂C MXene nanozymes have been demonstrated as outstanding multi‐enzyme analogues, we further explored their potential to safeguard the liver from AILI. First, we evaluated the uptake ability of Hep G2 cells (mimicking hepatocytes) and human umbilical vein endothelial cells (HUVECs, mimicking vascular endothelial cells) for Ga‐V₂C nanozymes. Hep G2 and HUVEC cells were observed to effectively take up Ga‐V₂C nanozymes, thereby confirming that the nanomedicine with a two‐dimensional structure can penetrate the cell membrane (Figure S3A). To evaluate the in vivo biodistribution profile, Ga‐V₂C nanozymes were administered at a dosage of 5 mg/kg body weight via caudal vein injection, and their tissue concentrations over 28 days were subsequently measured using inductively coupled plasma mass spectrometry (ICP‐MS) (Figure 3A,B). Remarkably, Ga‐V₂C demonstrated rapid accumulation in the liver and lungs within 4 h postinjection. Although Ga‐V₂C was predominantly cleared from the lungs within 7 days, it showed prolonged retention in the liver for up to 28 days. This significant and sustained hepatic accumulation, in conjunction with the known susceptibility of V₂C‐based MXenes to oxidative degradation and the established renal and fecal clearance pathways for other MXene systems [40, 41], highlights its potential as a long‐acting therapeutic agent for liver diseases. Therefore, these remarkable characteristics of Ga‐V₂C nanozymes motivated us to evaluate its protective effect against AILI.

FIGURE 3.

Protective efficacy of Ga‐V₂C nanozymes against AILI. (A) Schematic illustration of the distribution and clearance of Ga‐V₂C nanozymes in mice (Created with BioRender.com). (B) The content of Ga‐V₂C nanozymes in the heart, liver, spleen, lung, and kidney at different time points (n = 3). (C) Live/dead cell evaluation in APAP‐induced Hep G2 cell injury after various treatments (Created with BioRender.com). (D and E) The proportion of dead cells was evaluated by flow cytometry (n = 3). (F) Experimental protocol for V₂C or Ga‐V₂C nanozyme treatment studies in AILI mice (Created with BioRender.com). (G) Serum ALT and AST levels were measured in all groups of mice at 8 h after APAP administration (n = 5). (H) The morphological differences and histological changes in the liver of mice at 8 h after APAP administration (n =  5). (I) The liver damage area in all groups of mice at 8 h after APAP administration (n = 5). All data are expressed as mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

The Hep G2 cell line, derived from human hepatoblastoma, is the standard hepatocyte model for studies of liver metabolism, drug biotransformation, and toxicology [42, 43, 44]. In vitro, we observed that both V₂C and Ga‐V₂C nanozymes attenuated APAP‐induced Hep G2 death (p < 0.001), with the effect of Ga‐V₂C nanozymes being more significant than that of V₂C (p < 0.001) (Figure 3C–E). This finding inspired us to explore the protective effects of these two nanomedicines in vivo.

As shown in Figure 3F, healthy mice were fasted for 12 h prior to APAP administration. Subsequently, APAP was injected intraperitoneally into the mice to induce acute liver injury. At 3 h postinjection, the mice were intravenously injected with V₂C, Ga‐V₂C, or NAC, respectively. NAC is the standard treatment for APAP overdose, and the 3 h timepoint falls outside its optimal therapeutic window. Blood and liver samples from various treatment groups were collected at 6, 8, 12, and 24 h to assess the extent of injury.

Compared to AILI mice at different times points, those treated with V₂C, Ga‐V₂C, or NAC consistently showed lower levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), suggesting that these compounds conferred protection against AILI (Figure 3G; Figure S3B). Our previous research found that AILI mice experienced the most severe liver damage between 8 to 10 h [45], so we specifically isolated livers at 8 h for morphological assessment. It was observed that there was dark red or reddish‐brown discoloration on the liver surface in the control group, which was markedly attenuated following treatment (Figure 3H). Notably, V₂C and Ga‐V₂C appeared to offer a superior preventive effect compared to the APAP + NAC group, potentially due to reduced hepatocyte lesions. Microscopic examination of the sample collected at 6, 8, 12, and 24 h further revealed that AILI mice showed hepatocyte vacuolar degeneration or death near the hepatic lobules, whereas treatment with V₂C and Ga‐V₂C nanozymes significantly mitigated these pathological alterations via their intrinsic properties (Figure 3H,I; Figure S3C,D). Moreover, we conducted a survival study in which mice were challenged with a lethal dose of APAP (800 mg/kg). The results showed that Ga‐V₂C treatment remarkably enhanced survival rates compared to the NAC treatment groups (Figure S3E).

The results collectively suggest that both V₂C and Ga‐V₂C nanozymes confer protection against AILI. Notably, Ga‐V₂C exhibits superior protective effects, as evidenced by the most significant reduction in ALT and AST levels (Figure 3G; Figure S3B) and the smallest area of tissue damage at each time point after APAP injection (Figure 3H,I; Figure S3C,D).

2.4. Mechanisms of Ga‐V₂C Nanozymes Alleviating Oxidative Stress in AILI Mice

In AILI mice, oxidative stress induces excessive ROS accumulation and mitochondrial dysfunction, which leads to hepatocyte necrosis. To further demonstrate the therapeutic advantages of Ga‐V₂C nanozymes, we conducted an in‐depth comparison of the mechanisms by which V₂C or Ga‐V₂C nanozymes alleviate oxidative stress. Using DCFH‐DA dye to evaluate ROS levels, we found that upon administering V₂C and Ga‐V₂C nanozymes, ROS levels decreased significantly, similar to NAC (Figure 4A,B). Notably, Ga‐V₂C nanozymes were more effective than V₂C in reducing intracellular ROS levels (p < 0.01), indicating the potential of Ga‐V₂C nanozymes to counteract the oxidative stress caused by APAP.

FIGURE 4.

Mechanisms of Ga‐V₂C nanozymes alleviating oxidative stress in AILI mice. (A‐B) Detection of ROS in APAP‐induced Hep G2 cell injury after various intervention (n = 3). (C) The contents of GSH, GPx, SOD, and MDA in the liver sample were examined at 8 h after APAP administration (n = 5). (D and E) Protein expressions of p‐JNK, JNK, Nrf2, HO‐1, SOD1, and GPX4 in the liver were evaluated by Western blot at 8 h after APAP administration (n = 3). (F) The contents of GPX4 in the liver were examined at 8 h after APAP administration (n = 5). (G) The contents of IL‐1β and TNF‐α in the liver were examined by ELISA (n = 5). (H) Schematic illustration of the mechanisms of Ga‐V₂C nanozymes alleviating oxidative stress and inflammation. The values are expressed as mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

To further validate these findings, we quantified oxidative stress markers in mice. As shown in Figure 4C, AILI mice exhibited typical necrotic features induced by oxidative stress, as evidenced by the highest malondialdehyde (MDA) levels, a lipid peroxidation end‐product reflecting oxidative damage. In contrast, the levels of antioxidant‐related enzymes (GSH, GPx, and SOD) in mice treated with V₂C or Ga‐V₂C nanozymes were higher than those in the APAP groups (p < 0.05). These results support that V₂C or Ga‐V₂C nanozymes improved the antioxidant capacity and could reduce oxidative stress‐mediated hepatocyte necrosis.

As is well‐known, APAP obstructs the mitochondrial oxidative respiration process, resulting in the production of ROS. Subsequently, ROS that leaks into the cytoplasm activates c‐Jun N‐terminal kinase (JNK), which further stimulates ROS production [2]. The nuclear factor E2‐related factor (Nrf2) /heme oxygenase‐1 (HO‐1) pathway, being the most common endogenous antioxidant pathway, has been found to play a role in protecting against AILI [46]. Therefore, we further investigated whether V₂C or Ga‐V₂C nanozymes alleviated oxidative stress in AILI mice by affecting the JNK and Nrf2/HO‐1 pathway by Western blot. We found that JNK phosphorylation decreased and Nrf2/HO‐1 pathway activation increased after V₂C or Ga‐V₂C nanozymes application (p < 0.05, Figure 4D, E). Particularly, a significant increase in SOD and GPX4 levels was observed in the livers of mice treated with Ga‐V₂C nanozymes. GPX4 activity also showed the same change (Figure 4F). Moreover, we used immunohistochemical (IHC) to evaluate the expressions of Nrf2, HO‐1, SOD, and GPX4 (Figure S4A), and the results were mostly consistent with those of Western blot. Finally, we found that both V₂C and Ga‐V₂C nanozymes could alleviate the inflammatory response, including the pro‐inflammatory cytokines TNF‐α and IL‐1β (Figure 4G). Therefore, we concluded that both V₂C and Ga‐V₂C nanozymes reduced oxidative stress and inflammation in AILI mice by inhibiting the JNK phosphorylation and enhancing the antioxidant capacity of mice through the Nrf2/HO‐1 pathway (Figure 4H).

Studies indicate that certain nanozymes demonstrate antioxidant and anti‐inflammatory effects in hepatic and colonic inflammation through Nrf2 pathway activation [39, 47]. While iron plays crucial roles in cellular processes, its redox‐active excess amplifies ROS via Fenton reactions. Notably, gallium has been validated as an oxidative process modulator and anti‐inflammatory agent [5, 24]. This unique characteristic likely potentiates the antioxidant and anti‐inflammatory capacity of Ga‐V₂C nanozymes compared to V₂C alone (p < 0.05, Figure 4D–G). The enhanced biochemical markers correlate with the superior potential Ga‐V₂C nanozymes in mitigating drug‐induced liver injury relative to unmodified V₂C nanozymes.

2.5. Mechanisms of Ga‐V₂C Nanozymes Inhibiting Apoptosis and Ferroptosis in AILI Mice

ROS plays a crucial role in cellular signaling and stress response. It acts as a regulator of crosstalk between different types of cell death, such as necroptosis, apoptosis, and ferroptosis [48]. This suggests that Ga‐V₂C nanozymes may influence multiple hepatocyte death pathways in AILI mice, beyond the previously identified modulation of oxidative stress‐mediated necroptosis.

Apoptosis has been verified as a key component of AILI pathology [45]. We found that APAP exposure induced apoptosis in HepG2 cells, and the level of apoptosis decreased following intervention with various agents, with Ga‐V₂C and NAC showing the most significant inhibitory effects on apoptosis (p < 0.001) (Figure 5A). Subsequently, TUNEL staining was used to evaluate hepatocyte apoptosis in mice livers. As shown in Figure 5B, TUNEL staining of liver tissues revealed intense apoptotic signals in APAP‐treated mice, while the V₂C and Ga‐V₂C groups showed diminished staining intensity. Besides, we tested the anti‐apoptosis Bcl‐2 and the apoptosis‐promoter Bax/C‐caspase‐3 via Western blot and IHC (Figure 5C and D; Figure S4B). We observed that the expression of Bcl‐2 was significantly elevated in the Ga‐V₂C group (p < 0.001), while the levels of Bax and C‐caspase‐3 were notably reduced in the same group (p < 0.001). These findings confirm the superior antiapoptotic efficacy of Ga‐V₂C nanozymes compared to V₂C alone. The data suggest that Ga‐V₂C can inhibit apoptosis by modulating Bcl‐2/Bax expression to restore their balance, thereby alleviating APAP‐induced hepatocyte apoptosis.

FIGURE 5.

Mechanisms of Ga‐V₂C nanozymes inhibiting apoptosis and ferroptosis in AILI mice. (A) Apoptotic cells were detected and analyzed by flow cytometry in Hep G2. (B) TUNEL staining was evaluated using immunohistochemical staining (n = 5). (C) Representative western blotting analysis of Bcl‐2, Bax, C‐caspase‐3, 4‐HNE, SLC7A11, FTH1, and FTL1 for every group, while GAPDH was used as the loading control. (D) Protein expressions of Bcl‐2, Bax, and C‐caspase‐3 in the liver were evaluated by Western blot (n = 3). (E) The levels of LPO in the liver were measured (n = 5). (F) Mitochondria in the liver were observed using TEM (n = 3). (G) Protein expressions of 4‐HNE, SLC7A11, FTH1, and FTL1 in the liver were evaluated by Western blot (n = 3). (H) Tissue iron content evaluation at 8 h after APAP administration (n =  5). (I) Schematic illustration of the mechanisms of Ga‐V₂C nanozymes inhibiting ferroptosis and apoptosis. The values are expressed as mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Ferroptosis, characterized by iron homeostasis imbalance, represents another critical mechanism in AILI progression [49]. Studies have shown that ferroptosis is closely related to lipid peroxidation. We found that V₂C or Ga‐V₂C both decreased the LPO and 4‐HNE level in mouse liver, and the introduction of gallium made Ga‐V₂C most significantly decrease the lipid peroxidation markers (p < 0.001 Figure 5C,E,G). Because the features of ferroptosis include morphological changes such as a decrease in the mitochondrial volume, mitochondrial rupture, and the disappearance of mitochondrial cristae, we observed the liver of mice via TEM. We observed that the APAP group showed that mitochondria were significantly reduced and mitochondrial cristae disappeared, and V₂C or Ga‐V₂C reversed these changes (Figure 5F). Furthermore, IHC and Western blot were used to evaluate the expression of anti‐ferroptosis proteins, including SLC7A11, FTH1, and FTL1. We found that the mice had the lowest expression in the AILI group, but the mice had the highest expression in the Ga‐V₂C group (p < 0.05) (Figure 5C, G; Figure S4C). As we described above, Ga is similar to iron and can interfere with iron metabolism [24]. We further studied that the liver iron contents of mice treated with Ga‐V₂C were significantly lower than those of the APAP group, suggesting effective blockade of iron uptake (Figure 5H). Further temporal analysis revealed a progressive decline in hepatic iron content at 8, 12, and 24 h (Figure S4D). This decrease inversely correlated with the concurrent accumulation of Ga‐V₂C in the liver over the same period (Figure 3B). Thus, it is reasonable that Ga‐V₂C nanozymes with multienzyme mimetic activities may be involved in ferroptosis in AILI, and gallium has an inhibitory effect on ferroptosis. Collectively, these findings collectively suggest that Ga‐V₂C nanozymes can suppress ferroptosis by reducing lipid peroxidation, reversing mitochondrial damage, upregulating anti‐ferroptosis proteins, and decreasing hepatic iron content via gallium‐mediated iron metabolism interference (Figure 5I).

In summary, these studies demonstrate the capacity of Ga‐V₂C nanozymes to concurrently inhibit apoptosis and ferroptosis in AILI mice, with gallium's unique properties enhancing therapeutic outcomes compared to V₂C alone.

2.6. Mechanisms of Ga‐V₂C Nanozymes Promoting Hepatocyte Regeneration

Hepatocytes renewal and regeneration take place in non‐severe liver injuries with the assistance of signal mediators and nonparenchymal cells. Cytokines secreted by immune cells, hepatic stellate cells, and endothelial cells facilitate the regeneration of hepatocytes in AILI [2]. Previous studies have revealed that the gallium complex can not only regulate the production of cytokines in macrophages but also induce the osteogenic differentiation of stem cells [50, 51]. Meanwhile, Thompson et al. found that gallium citrate led to degrees of reepithelization along with granulation tissue formation in skin wounds infected with K. pneumoniae [52]. Recent studies indicate that certain MXene materials may contribute indirectly to tissue regeneration by exhibiting antioxidant properties [53]. Therefore, Ga‐V₂C nanozyme is likely responsible for promoting hepatocyte regeneration primarily through the intrinsic pro‐regenerative activity of gallium, with the structural characteristics (e.g., ROS‐scavenging capability) of the MXene component playing a supportive role. These findings motivated us to further evaluate the effect of V₂C or Ga‐V₂C nanozymes on hepatocyte regeneration.

Firstly, we observed that V₂C and Ga‐V₂C nanozymes increased the relative cell viability in APAP‐induced Hep G2 cell injury (Figure 6A), and the single cell cloning experiment verified this finding (Figure 6B,C). Ga‐V₂C nanozymes exhibited stronger therapeutic effects than V₂C in both experiments (p < 0.001). Subsequently, we measured the expression of Wnt/βCAT pathway, CCND1, MYC, and PCNA, which are drivers of cell growth and proliferation, in all groups of mice using Western blot (Figure 6D). Mice treated with Ga‐V₂C nanozymes exhibited a significant increase in p‐βCAT, CCND1, MYC, and PCNA (p < 0.05), while there was only a slight increase in p‐βCAT, MYC, and PCNA in mice treated with V₂C nanozymes. This demonstrates the advantage of Ga‐V₂C nanozymes in promoting hepatocyte regeneration due to the presence of gallium. To further validate these findings, we performed IHC analysis. Ki‐67 immunostaining, a specific marker for cell proliferation, showed an increase in positively stained cells within the Ga‐V₂C treated groups. Additionally, the elevated expression levels of CCND1, MYC, and PCNA observed following Ga‐V₂C treatment were consistent with the Western blot results (Figure S5).

FIGURE 6.

Mechanisms of Ga‐V₂C nanozymes in promoting hepatocyte regeneration. (A) V₂C and Ga‐V₂C nanozymes increased the relative cell viability in APAP‐induced Hep G2 cell injury (n = 6). Images (B) and quantitative analysis (C) show that V₂C and Ga‐V₂C nanozymes promoted the formation of single cell cloning (n = 3). (D and E) Protein expressions of p‐βCAT, CCND1, MYC and PCNA were evaluated by Western blot (n = 3). (F) Schematic illustration of the mechanisms of Ga‐V₂C nanozymes promoting hepatocyte regeneration. The values are expressed as mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Overall, both V₂C and Ga‐V₂C nanozymes promoted hepatocyte regeneration, with Ga‐V₂C nanozymes showing a more pronounced effect. Ga‐V₂C nanozymes had a better performance in promoting hepatocyte regeneration compared to V₂C nanozymes by activating Wnt/βCAT pathway and proliferation drivers CCND1, MYC, and PCNA (Figure 6E). This may be the result of the collective activation of antioxidant, anti‐apoptosis, anti‐ferroptosis pathways, and proliferation‐related signaling pathways like Nrf2, thereby creating a pro‐regenerative microenvironment [54, 55, 56].

2.7. Biosafety of Ga‐V₂C Nanozymes In Vitro and In Vivo

To assess the biosafety of Ga‐V₂C nanozymes, we conducted comprehensive cytocompatibility and biocompatibility assessments. Figure 7A demonstrated that V₂C exhibited good biocompatibility, with over 90% of cells remaining alive after treatments at different concentrations, and gallium doping did not significantly reduce the cytocompatibility of Ga‐V₂C nanozymes in Hep G2 and HUVEC cells.

FIGURE 7.

Biosafety evaluation of Ga‐V₂C nanozymes in vitro and in vivo. (A) Viability of Hep G2 and HUVEC cells was recorded after 24 h exposure to an increasing concentration of V₂C or Ga‐V₂C nanozymes (n = 5). (B) Schematic diagram illustrating the biosafety examinations conducted on mice (Created with BioRender.com). (C) H&E staining of major organs was evaluated after 8 h, 7, and 28 days of indicated treatments (n = 4). (D) Blood biochemistry (ALT, AST, BUN, Cr, CK, and LDH) of mice was assessed following intravenous administration of V₂C or Ga‐V₂C nanozymes (n = 4). The values are expressed as mean ± SEM. ns, not significant with p > 0.05.

Subsequently, the short‐ and long‐term potential side effects of various samples were evaluated in mice (Figure 7B). The major organs, including the liver, heart, spleen, lung, and kidney, exhibited normal histological morphology without any signs of inflammation or histopathological abnormalities (Figure 7C). Similarly, blood analysis revealed that the levels of ALT, AST, creatinine (Cr), urea nitrogen (BUN), and lactate dehydrogenase (LDH) were within the normal range after 8 h, 7 and 28 days of treatment (Figure 7D), indicating that V₂C or Ga‐V₂C nanozymes had no adverse effects on liver, kidney, and heart function in mice. Overall, these results supported the high level of biosafety exhibited by Ga‐V₂C nanozymes and its potential for clinical translation.

3. Conclusions

In summary, we designed and successfully developed a multifunctional Ga‐V₂C MXene Nanozymes for effectively prevention of AILI. Ga‐V₂C nanozymes possess multienzyme mimetic activities, including SOD, POD, GPx, and CAT. Both in vivo and in vitro studies demonstrated that Ga‐V₂C nanozymes outperform NAC in mitigating AILI due to their multifaceted mechanisms: (1) scavenging reactive oxygen species (ROS) to suppress JNK phosphorylation and enhance Nrf2/HO‐1‐mediated antioxidant defenses; (2) competing with Fe³⁺ via Ga³⁺ to inhibit iron‐driven lipid peroxidation and ferroptosis; (3) restoring the balance between Bcl‐2 and Bax to block apoptosis; (4) activating Wnt/βCAT pathway and proliferation markers such as CCND1, MYC, and PCNA to promote hepatocyte regeneration. The Ga‐based MXene nanozymes strategy exhibits excellent therapeutic efficacy against AILI with negligible toxicity.

Our work may provide a promising approach for AILI treatment and broaden the applicability of nanozyme‐based therapeutics to complex diseases. The significance of this study lies in its contribution to advancing nanozyme‐based therapeutics for complex diseases, offering a foundation for developing multifunctional, metal‐based MXene nanozymes with tailored mechanisms. By addressing these challenges, Ga‐V₂C MXene nanozymes may unlock new opportunities in precision medicine and inspire innovative solutions for oxidative stress‐related pathologies.

Given the promising outcomes of this study, future research must address the potential challenges in the clinical translation of MXene nanozymes, such as Ga‐V₂C. Key obstacles include addressing toxicological concerns and ensuring long‐term biosafety through comprehensive preclinical evaluations. Optimizing surface engineering strategies to enhance catalytic activity and selectivity while minimizing off‐target effects remains critical. Furthermore, standardized protocols for large‐scale production and rigorous clinical validation will be essential for successful therapeutic implementation [57, 58]. Future research should focus on bridging the gap between laboratory‐scale achievements and large‐scale biomedical applications, particularly in therapeutic, diagnostic, and theranostic contexts.

4. Experimental Section

4.1. Materials

V₂AlC powder was purchased from Forsman Scientific Co., Ltd. (Beijing, China). LiF, TPAOH (40%), and gallium were both purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Calcein‐AM/PI double staining kit and FITC/PI double stain kit were obtained from YEASEN company (Shanghai, China). ROS assay kits and RIPA lysis buffer and was purchased from Beyotime company (Shanghai, China). Primary antibodies against FTL1, SLC7A11, Bcl‐2, Bax, CCND1, MYC, JNK, and PCNA were purchased from ProteinTech Group (Chicago, IL, USA). Primary antibodies against p‐JNK, FTH1 were purchased from Cell Signaling Technology (Boston, USA). The other primary antibodies against Nrf2, HO‐1, GPX4, SOD, GAPDH, and secondary antibodies were purchased from Abcam (Cambridge, UK). The iron assay kit was obtained from Nanjing Jiancheng Bioengineering Institute Co., Ltd. (Nanjing, China). All other chemicals were purchased as analytical grade and used without additional purification.

4.2. Cell Lines and Animals

Hep G2 and HUVEC cell lines were from our laboratory preservation and were grown in a DMEM culture medium with 10% fetal bovine serum in a humidified 5% CO₂ atmosphere at 37°C. Male C57BL/6 mice (18–22 g) were purchased from Guangdong Medical Laboratory Animal Center. The mice were maintained in suitable living conditions with a 12 h light/dark cycle and had ad libitum access to food and water. The mice were adapted for 1 week before the experiment. All experimental protocols met the requirements of the National Institutes of Health guidelines and were approved by the Animal Care and Use Committee of Jinan University (ID: 20231215‐0003).

4.3. Fabrication of Ga‐V₂C MXene Nanozymes

The fabrication of Ga‐V₂C MXene nanozymes was achieved through a multi‐step process. Initially, 1.0 g of bulk MAX‐phase V₂AlC ceramics was carefully added to a 10 mL aqueous solution of 6 M HCl containing 6.0 g of LiF, under a nitrogen atmosphere. This mixture was stirred for 72 h at 50°C to facilitate the removal of the intermediate Al layer. The resulting acidic suspension underwent repeated washing with nitrogen‐deaerated water and was subsequently exfoliated through sonication. The sample was then subjected to centrifugation and vacuum drying to yield single‐ or few‐layered V₂C MXene nanosheets.

To achieve gallium doping, a mixture comprising V₂C powder and liquid gallium was prepared at a mass ratio of 1:2 at 45°C, and then calcined in a muffle furnace at 400°C for 3 h. Excess gallium was removed by washing the sample with 6 M HCl. The resultant product was then immersed in nitrogen‐deaerated water, placed into a Teflon‐lined stainless steel autoclave, and subjected to heating at 120°C for 6 h.

4.4. Characterization of Ga‐V₂C MXene Nanozymes

Zeta potential and hydrodynamic diameter of nanozymes were measured by DLS on a Zetasizer Nano ZS90 (Malvern Instruments, UK). The thickness and surface morphology of nanozymes were examined by an AFM (NTEGRA Solaris, NT‐MDT, Russia) at ambient conditions. The morphology and microstructure of the samples were investigated by TEM and HRTEM images and elemental mapping images using a JEOL‐2100F microscope (JEOL, Tokyo, Japan). XPS was conducted using an ESCAlab250 electron spectrometer (Thermo VG, Waltham, MA, USA) for the qualitative analysis of the surface element and valence states of the samples. The gallium loading capacity was determined by measuring the gallium content in Ga‐V₂C nanozymes using an Agilent 5110 ICP‐OES (Agilent, Santa Clara, CA, USA) following acid digestion with a 1:1 HNO₃ to HCl mixture and dilution with ultrapure water.

4.5. Multienzyme Mimetic Activity Assay of Ga‐V₂C MXene Nanozymes

The SOD activity of Ga‐V₂C nanozymes was assessed by measuring their ability to quench O₂ ^•−, as indicated by formazan formation using a WST‐1 assay kit (S311‐10, Dojindo, Japan). Briefly, xanthine, xanthine oxidase, and WST‐1 were mixed with suspensions of Ga‐V₂C at varying concentrations (0, 12.5, 25, 50, 100, 200, and 400 µg/mL). The absorption of formazan was subsequently measured at 450 nm using a SpectraMax M2 microplate reader. The IC₅₀ value, which denotes the concentration of Ga‐V₂C nanozymes required to inhibit 50% of formazan formation, was determined through regression curve fitting analysis using GraphPad Prism software.

The POD‐like activity assay of the prepared Ga‐V₂C nanozymes was assessed utilizing TMB (Solaibao, Beijing, China) as a substrate with the assistance of H₂O₂. A suspension of Ga‐V₂C nanozymes at a concentration of 100 µg/mL was combined with TMB at 0.8 mM and varying concentrations of H₂O₂ (0, 1, 5, 10, and 15 mM) under ambient conditions in an acetate buffer solution. The progression of the reaction was monitored by measuring the absorbance change at 652 nm in real‐time using a time‐scan mode. The steady‐state kinetic analyses were conducted following a previously established method [19, 59]. The K _M and V _max values were calculated utilizing the Michaelis–Menten equation through nonlinear regression analysis performed with GraphPad Prism software.

Standard GR‐coupled assay was employed to evaluate the GPx‐like activity of Ga‐V₂C nanozymes, employing the GPx assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. Briefly, GSH was oxidized to GSSG with the addition of H₂O₂ (5 mM) and Ga‐V₂C nanozymes (200 µg/mL), and then GSSG was reduced to GSH by GR, accompanied with NADPH (300 µM). The decrease in the absorbance of NADPH at 340 nm was monitored and quantified spectrophotometrically using a time‐driven mode to determine the catalytic reaction rate of Ga‐V₂C nanozymes. All assays were conducted at a temperature of 25°C.

To study the CAT‐like activity of Ga‐V₂C nanozymes, a suspension of Ga‐V₂C nanozymes at a final concentration of 100 µg/mL was introduced into PBS at pH 7.4 with H₂O₂ serving as the substrate at concentrations of 12.5, 25, 50, 100, and 200 mM. The dissolved oxygen levels were measured with a portable dissolved oxygen meter (JPSJ‐606L, Leici, China) at various time intervals during the reaction. The data were fit to the Michaelis–Menten equation using non‐linear regression to determine the apparent K _M and V _max parameters using GraphPad Prism.

4.6. Phagocytosis of Ga‐V₂C Nanozymes In Vitro

Hep G2 and HUVEC cells were seeded in immunofluorescence dishes at a concentration of 1.5 × 10⁵ cells per dish and cultured overnight. Then, Ga‐V₂C nanozymes at concentrations of 0, 50, and 100 µg mL⁻¹ were added to the culture for 12 h at 37°C. One milliliter Live Cell Nucleus Staining Solution Hoechst (Beyotime, Shanghai, China, 33342) was added to each dish, incubated for 30 min in the dark, and observed under a confocal microscope after washing with PBS.

4.7. Estimation of Live/Dead Cells

Hep G2 cells were seeded in 12‐well plates at 5 × 10⁴ cells/well. After 24 h of attachment, the cells were subjected to oxidative stress by treatment with 10 mmol L⁻¹ APAP (#HY‐66005, MCE, USA) for 24 h. Then, the cells were treated with V₂C, Ga‐V₂C, or NAC (25 µg mL⁻¹) for addtional 24 h. Cell viability was assessed using a Calcein‐AM/PI double staining kit and observed via flow cytometry.

4.8. Estimation of Intracellular ROS

Hep G2 cells were seeded into 12‐well plates at a density of 5 × 10⁴ cells/well. According to the above method, APAP (10 mmol L⁻¹) and V₂C/Ga‐V₂C/NAC (25 µg mL⁻¹) were co‐incubated sequentially, then the intracellular ROS levels in Hep G2 cells were evaluated using ROS assay kits. Finally, fluorescence was detected using a fluorescence microscope.

4.9. Estimation of Apoptotic Cells

Hep G2 cells were seeded into 12‐well culture plates at an initial density of 5 × 10⁴ cells per well. According to the above method, APAP and V₂C/Ga‐V₂C/NAC were co‐incubated sequentially, then cellular apoptosis was assessed using a FITC/PI double stain kit. Flow cytometry was subsequently performed.

4.10. Estimation of Cell Proliferation

Cell proliferation experiments were performed using a cell viability assay and a single‐cell cloning assay. Firstly, Hep G2 cells were incubated with APAP (10 mmol L⁻¹) and V₂C/Ga‐V₂C nanozymes (25 µg mL⁻¹) for 24 h in turn. Then, the cells were seeded into 96‐well and 12‐well plates at a density of 1 × 10³ cells/well. CCK‐8 Kits were used for cell viability at different time points (0, 24, 48, and 72 h). Hep G2 cells in the 12‐well plates were stained with crystal violet on the 7th day.

4.11. Measurement of Ga‐V₂C Nanozymes Biodistribution

Mice were administered a dose of Ga‐V₂C nanozymes at 5 mg/kg·BW via caudal vein injection. At 4 h, 1, 3, 7, 14, and 28 days after administration, liver, heart, spleen, lung, and kidney were collected from mice (n = 3). Tissue homogenates were then analyzed by ICP‐MS to determine vanadium and Ga‐V₂C nanozymes content.

4.12. Measurement of Liver Injury In Vivo

Mice were randomly divided into 5 groups as follows (n = 5): (1) Control, (2) APAP (MCE, Monmouth Junction, NJ, USA), (3) APAP+V₂C, (4) APAP+Ga‐V₂C, (5) APAP+NAC (MCE, Monmouth Junction, NJ, USA). Mice in groups 2, 3, 4, and 5 were given a peritoneal injection of 500 mg/kg APAP to induce liver injury. The mice were fasted for 12 h before APAP administration. Three hours after APAP administration, mice in groups 3, 4, and 5 were administered V₂C (5 mg/kg), Ga‐V₂C (5 mg/kg), and NAC (150 mg/kg) via caudal vein injection, respectively. At 6, 8, 12, and 24 h after APAP administration, blood and liver tissue were collected for further examination and analysis. Serum ALT and AST levels, as well as H&E staining of liver tissue, were performed to evaluate the protective efficacy of V₂C and Ga‐V₂C nanozymes.

4.13. Survival Rate Assessment

Mice were randomly divided into 5 groups as follows (n = 10): (1) Control, (2) APAP, (3) APAP+V₂C, (4) APAP+Ga‐V₂C, (5) APAP+NAC. Mice in groups 2, 3, 4, and 5 received an intraperitoneal injection of 800 mg/kg APAP. Three hours after APAP administration, mice in groups 3, 4, and 5 were administered V₂C (5 mg/kg), Ga‐V₂C (5 mg/kg), and NAC (150 mg/kg), respectively, via tail vein injection. The survival status of mice was monitored at 0, 4, 8, 12, 24, 36, 48, and 54 h.

4.14. Oxidase and Inflammatory Factors Measurement in Liver Tissue

Mouse liver tissue at 8 h after APAP administration was homogenized and centrifuged at 3000 × g for 20 min. Supernatants were assayed for GSH, GPx, SOD, and MDA levels using kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, China). Inflammatory markers (TNF‐α and IL‐1β) were quantified according to manufacturer instructions using kits (Solarbio Life Sciences Co., Ltd., Beijing, China).

4.15. Protein Expression Analysis by Western Blot

Proteins of the liver were lysed using RIPA lysis buffer (containing 1% PMSF) to extract the total proteins. Next, the proteins were fractionated and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The separated proteins were subsequently probed with primary antibodies, followed by incubation with their corresponding secondary antibodies. For the expression of different proteins in the same blots, partly blotted membranes were incubated with Western blot fast stripping buffer (EpiZyme) followed by several washes, and treated as mentioned above. Protein bands were visualized and quantified using NIH ImageJ software. For quantitative analysis, the levels of specific proteins were normalized to the levels of GAPDH detected on the same membrane [60]. The detailed information of antibodies is shown in Table S1.

4.16. Protein Distribution and Expression Analysis by IHC

To further clarify the changes in the position and total amount of proteins in the liver tissues, we performed immunohistochemical staining. Using the liver samples embedded in paraffin, we evaluated these proteins as follows: Nrf2, HO‐1, GPX4, SOD, SLC7A11, FTH1, FTL1, Bcl‐2, Bax, Ki‐67, CCND1, MYC, and PCNA. The distribution and expression of these proteins were detected under a light microscope.

4.17. TUNEL Assay

TUNEL analysis was performed using commercial kits according to the manufacturer's instructions. In brief, 5 µm thick sections were deparaffinized, dehydrated, and permeabilized for 15 min at room temperature. Samples were then incubated with the TUNEL reaction mixture in a humidified atmosphere for 1 h at 37°C. Sections were mounted with DAB chromogenic reagent and visualized using a microscopy. The positive apoptosis cells developed by DAB reagent have brown–yellow nucleus.

4.18. Ultrastructure Evaluation by TEM

Fresh liver tissues were perfused with 2.5% glutaraldehyde and 1% osmium tetroxide. Then, the tissues were sectioned (70–90 nm) and stained with 2% uranyl acetate. Ultrastructural images of the liver were observed and imaged with TEM (Hitachi H‐7650, Tokyo, Japan).

4.19. Detection of Iron Contents of Liver Tissue

Liver tissue was homogenized and centrifuged at 30 mg for 10 min. The supernatants were measured using the tissue iron assay kit.

4.20. In Vivo Short‐ and Long‐Term Safety Evaluation of V₂C and Ga‐V₂C Nanozymes

Mice were given a dose of V₂C and Ga‐V₂C nanozymes at 5 mg/kg·BW by caudal injection for 8 h, 7, and 28 days (n = 4). Then, serous ALT, AST, Cr, BUN, and LDH testing kits were used to evaluate the toxicity of V₂C and Ga‐V₂C nanozymes. Furthermore, pathological evaluations of different organs were made by H&E.

4.21. Statistical Analysis

The data were expressed as mean ± SEM. Statistical analyses between the two groups were examined using a two‐tailed Student's t‐test. Statistical analyses were conducted using GraphPad Prism. Statistical significance of p‐values was indicated as: ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Author Contributions

The study was conceptualized by Guilan Quan, Weilin Wang, Min Zhou, and Chao Lu. Xiaopeng Cai, Jingwen Deng, and Liqing Wang contributed to methodology, data curation, and co‐writing the original draft. Junjie Su contributed to data curation and investigation. Dongyi Xian, Yilang Yan, and Xinyu Yang conducted formal analysis and visualization. Chuanbin Wu, Tingting Peng, and Yuan Ding provided methodological guidance and supervision throughout the research. Guilan Quan, Weilin Wang, Min Zhou, and Chao Lu supervised the project, reviewed and edited the manuscript. Funding acquisition was secured by Xiaopeng Cai, Jingwen Deng and Chao Lu. All authors reviewed the final manuscript and approved its submission.

Ethics Statement

All experimental protocols met the requirements of the National Institutes of Health guidelines and were approved by the Animal Care and Use Committee of Jinan University (ID: 20231215‐0003).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: advs73637‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.