Sorafenib-based carbon dot nanozyme with nucleolus-targeted oxidative stress amplification for tumor immune microenvironment remodeling and cancer theranostics

Sorafenib-based carbon dot nanozyme with nucleolus-targeted oxidative stress amplification for tumor immune microenvironment remodeling and cancer theranostics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sorafenib-based carbon dot nanozyme with nucleolus-targeted oxidative stress amplification for tumor immune microenvironment remodeling and cancer theranostics Huixi Guo, Chunmei Lai, Weiji Chen, Shaohua He This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9469655/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Carbon dots (CDs) are promising nanozymes with natural enzyme-like activities, yet their clinical translation for hepatocellular carcinoma (HCC) is limited by biosafety and efficacy concerns. Developing low-toxicity, FDA-precursor-derived CD nanozymes is thus an attractive strategy for advancing cancer therapy. Methods: Herein, we rationally designed a sorafenib-derived carbon dot ( SF-CDs ) nanozyme, leveraging the FDA-approved HCC drug sorafenib as the precursor. SF-CDs were fabricated and characterized to exhibit dynamic nucleolus-targeted activity, with the goal of amplifying oxidative stress, inducing ferroptosis, and remodeling the immunosuppressive tumor microenvironment. Results: SF-CDs exerted potent glutathione oxidase-like activity, disrupting the GPX4-mediated lipid peroxidation repair pathway to trigger robust cancer cell ferroptosis. In vivo , image-guided interventional injection of SF-CDs significantly suppressed tumor growth in both subcutaneous and orthotopic H22 mouse models, with no observed systemic toxicity. Moreover, SF-CDs markedly enhanced the infiltration of immune effector cells, converting “cold” HCC tumors into immunogenic “hot” tumors and eliciting systemic antitumor immunity. Conclusion: This study establishes SF-CDs as a promising nucleolus-targeted nanozyme platform for HCC theranostics, combining ferroptosis induction and tumor immune remodeling. The work provides a biosafe and effective strategy for advancing CD-based nanozyme therapy, addressing critical unmet needs in HCC treatment. Conclusions SF -CDs function as a drug-derived CDs nanozyme that induces ferroptosis through glutathione depletion and oxidative stress amplification while concurrently reshaping the tumor immune microenvironment. These findings support the potential of SF -CDs as a theranostic platform for HCC. Sorafenib Carbon dots Nanozyme Ferroptosis Immune activation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The remarkable catalytic activity and substrate specificity of natural enzymes position them as superior catalysts for a wide range of biomedical applications. One example is horseradish peroxidase, which is widely used in enzymatic sensing assays for the detection of biomarkers, viruses and bacterial entities [1]. Catalase, which plays a key role in breaking down hydrogen peroxide into water and oxygen, is used strategically to increase the effectiveness of radiotherapy [2], sonodynamic therapy [3], and photodynamic therapy in the fight against tumors [4, 5]. This enhanced anti-tumor activity is attributed to the enzyme’s ability to alleviate the hypoxic constraints characteristic of the tumor microenvironment (TME). Glutathione oxidase, an enzyme involved in the regulation of cellular redox balance, has been used to treat several conditions characterized by oxidative stress [6, 7]. GSH oxidase, which is critical for cellular redox homeostasis, is being investigated for its ability to improve chemotherapeutic and radiotherapeutic outcomes by increasing ROS in cancer cells and inducing cell death [8]. The investigation of GSH oxidase for therapeutic applications represents an emerging area of research aimed at optimizing its medical potential and understanding its role in a range of pathological scenarios. However, the majority of natural enzymes have intrinsic limitations, including susceptibility to denaturation, high production costs, complicated preparation processes and challenges associated with scalable manufacturing processes [9]. To overcome these obstacles, synthetic enzymes have emerged as substantial and cost-effective alternatives to naturally occurring enzymes [10]. Among these, nanomaterials with enzyme-like activities, known as nanozymes, have revolutionized our perspective on nanoscale enzyme mimics and have garnered widespread interest for their capacity to address the intrinsic shortcomings of natural enzymes [11]. Currently, a plethora of nanomaterials have been creatively engineered to exhibit robust enzymatic functions, either by replicating the active sites of natural enzymes or by incorporating various elements into their nanostructures. However, the heterogeneous compositions and structures of nanozymes have given rise to a myriad of complex catalytic reactions, making it difficult to pinpoint active sites and presenting hurdles to the rational engineering of nanozymes with tailored catalytic efficacy and selectivity [12]. Nanozymes with enzyme-like properties represent a promising avenue for the advancement of enzyme-mediated therapeutics [13]. Their nanoscale structure gives them not only enhanced catalytic potency, but also superior stability and easier up-scaling compared to natural enzymes, making them particularly advantageous for applications in diagnostic medicine, targeted therapeutics and biosensing [14, 15]. However, the practical application of existing nanozymes is hampered by the complexity of their synthesis, the exorbitant costs associated with their production and suboptimal biocompatibility. In particular, metal-based nanozymes, including those containing Au, Mn, Pd, Cd, Pt and Ag, which are frequently cited in the literature, have raised several health concerns regarding their use in translational medicine [16]. There is an urgent need to discover nanozymes made from innovative, biocompatible materials, especially those derived from abundant, edible resources, which hold great promise for future biomedical applications. Carbon nanomaterials (CNMs), endowed with unique electronic and morphological properties, have demonstrated the ability to mimic the catalytic activities of natural enzymes. CNMs-derived nanozymes are emerging as viable natural enzyme substitutes in biomedical fields due to their distinctive, unique physicochemical properties [17]. CDs, a class of luminescent nanomaterials, have attracted considerable attention in the last decade due to their unique properties [18, 19]. These CDs exhibit several advantages, including their nanoscale dimensions, facile synthesis, affordability, tunable photoluminescence, robust photostability and enhanced biocompatibility, which outperform luminescent entities such as quantum dots, metallic nanoclusters and rare earth nanoparticles [20, 21]. In addition, the variety of oxygen-rich functional groups present on the surface of CDs, including ketone, carboxylic acid and alcohol moieties, gives them exceptional quantum efficiency and enhanced biological capabilities, effectively mimicking various enzymatic sites of catalytic activity. Emerging evidence highlights the ability of CDs-based nanozymes to mimic the architecture and operating characteristics of native enzymes such as catalase and superoxide dismutase, which can induce oxidative stress and subsequent damage to biological systems. Ferroptosis, defined by its oxidative stress-induced cell death process, is gaining momentum in the scientific community. Mechanistically, ferroptosis is largely controlled by the GSH redox mechanism, which plays a key role in its regulation [22–24]. GSH depletion leads to GPX4 dysfunction, which ultimately causes ROS levels to rise through lipid peroxidation, triggering ferroptosis [25, 26]. In particular, cancer cells undergoing ferroptosis can trigger an immune response by releasing molecules that indicate cellular stress (DAMPs) and alarmins, which activate the immune system [27–29]. Therefore, ferroptosis could serve as a potent tactic in cancer therapeutics. Research to date has mainly focused on the catalytic functions of CDs, but there is still a lack of investigation into the engineering of CDs nanozymes to induce ferroptosis and enhance the anti-tumor immune response. Herein, inspired by ChA CQDs derived from the pyrolysis of roasted coffee beans, we prepared CDs using a straightforward hydrothermal synthesis with sorafenib, a breakthrough drug approved by the FDA in 2007 for the initial systemic treatment of HCC. Through such a facile assembly procedure, the pertinent product can be imbued with a multitude of exceptional attributes: 1) in contrast to sorafenib, whose clinical application is limited by its suboptimal water solubility and limited bioavailability, SF-CDs are nanoscale that can be easily functionalized for targeted delivery, thereby enhancing their bioavailability and tumor penetration; 2) Sorafenib, due to its broad kinase inhibition, can lead to dose-limiting toxicities. In contrast, SF-CDs , mimic GSH oxidase, specifically targeting cancer cells with high oxidative stress. This reduces off-target effects and systemic toxicity, while inducing ferroptosis through GSH oxidation and GPX4 inactivation; 3) SF-CDs exhibit dynamic nucleolus-to-mitochondria subcellular localization, selectively accumulating in cancer cell mitochondria to amplify oxidative stress and trigger ferroptosis, a cell death pathway far less resistant than the apoptosis primarily induced by sorafenib; 4) Compared to sorafenib, SF-CDs induce immunogenic cell death and release of damage-associated molecular patterns (DAMPs), which activate antitumor immunity and convert immunosuppressive “cold” tumors into immunoreactive “hot” ones (Scheme 1 ). Collectively, our groundbreaking preliminary research has revealed the potential of SF-CDs nanozymes with nucleolus targeting capacity to serve as potent instigators of tumor ferroptosis, thereby revitalizing the immune microenvironment within malignant tumors and highlighting the significant therapeutic potential of SF-CDs in the field of translational medicine. Materials and Methods Materials SF, Dimethylsulfoxide (DMSO), and DTNB (5,5’-dithiobis(2-nitrobenzoic acid)) were all obtained from Aladdin Chemical Co., Ltd. (Shanghai, China). GSH, oxidized glutathione (GSSG), FBS, DCFH-DA, 3,3’,5,5’-Tetramethylbenzidine (TMB), Dil (cell membrane probes), Mito-tracker, Calcein-AM/PI viability assay kit were all purchased from MeiLunBio. The H&E staining kit was supplied by Beyotime Biotech (Nantong, Jiangsu, China). Affinity purified antibodies recognizing NRF2, COX-2, GPX4, and GAPDH were procured from Abcam (Cambridge, UK). The MTT was acquired from Life Technologies GmbH. The carcinoma cell lines HepG2 (RRID:CVCL_0043), 4T1 (RRID:CVCL_0218), HeLa (RRID:CVCL_0030), and H22 (RRID:CVCL_0220), along with the normal liver cell line L02 (RRID:CVCL_0220), were all procured from the Cell Bank of the Chinese Academy of Medical Sciences in Beijing, China. All cell lines were confirmed to be free of contamination. All cell culture components were purchased exclusively from Life Technologies GmbH, with the exception of those specifically mentioned otherwise. Cell culture The HepG2 cell line was cultured in DMEM medium containing 10% fetal bovine serum, along with a 1% mixture of penicillin and streptomycin. Meanwhile, L02 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. All cell cultures were maintained under strict environmental parameters in an incubation system at 37℃ supplemented with a 5% CO 2 atmosphere to maintain a stable and regulated environment throughout the experimental period. In addition, female BALB/c rodents aged 5 to 7 weeks were obtained from Fuzhou Wushi Laboratory Animal Supply. The Institutional Animal Care and Use Committee of Fujian Agriculture and Forestry University (PZCASFAFU25100) thoroughly reviewed and approved all animal protocols, ensuring that the research was conducted in accordance with ethical and humane standards. Cell imaging To assess the uptake of SF-CDs , HepG2 cells were plated in 12-well plates at a density of 1.0×10⁵ cells per well. To determine cellular uptake, HepG2 cells were plated on glass slides in 12-well plates at 1.0×10 5 per well. The cells were subsequently treated with 1 mL of DMEM containing 10% FBS and supplemented with SF-CDs at a dose of 100 µg for intervals of 0, 5, 10, 15, and 30 min. Excess SF-CDs were removed by rinsing the cells twice with DMEM. Cell uptake was then assessed using a Leica Microsystems (Wetzlar, Germany) SP8 CLSM. To assess the efflux of SF-CDs , HepG2 cells were incubated with SF-CDs for 30 min. The supernatant was gently aspirated, and the cells were rinsed and then cultured in DMEM supplemented with 10% FBS for further incubation. Cell movements were then examined at 15, 30 and 60 min using a high-resolution SP8 CLSM (Leica Microsystems, Wetzlar, Germany) operating at an excitation wavelength of 365 nm. Transmission electron microscopy assessment For the TEM study, SF-CDs were suspended in a thin aqueous solution and applied to carbon-covered copper grids by the drop-coating technique. Their morphology and structure were carefully examined using a HT7800 manufactured by Hitachi at 200 kV. The images obtained were processed and evaluated for detailed and advanced analysis. Atomic force microscopy investigations AFM was used to determine the thickness of the SF-CDs , using a Dimension FastScan ATM atomic force microscope (Bruker, Karlsruhe, Germany) to meticulously measure the size characteristics of the samples. A precisely prepared 0.01 mg/mL aqueous solution of SF-CDs was sonicated for 3 min to achieve complete dispersion. This solution was delicately applied to a mica substrate. The resulting images were meticulously scrutinized. Fluorescence spectral analysis For the colorimetric assessment of highly luminescent SF-CDs , they were carefully prepared to a 10 mg/mL concentration within an optical quartz cell. Spectrofluorometric analysis was performed using a FluoroMax-4 instrument set to an excitation wavelength of 365 nm. Fourier transform infrared spectroscopy evaluation To perform the FT-IR spectroscopy, a precise mixture of 20 mg of KBr was prepared with a 0.5 mg portion of the sample material, thoroughly ground to ensure homogeneity, and then compressed at 10 MPa to form a disc for analysis. The spectral data were collected by scanning the wave numbers from 4000 to 400 cm − 1 . GSH oxidase-like activity assay The evaluation of GSH oxidase-like activity was conducted using DTNB as the substrate, spanning a spectrum of GSH concentrations [30]. Yellow complex formation from DTNB-GSH reactions was monitored spectrophotometrically at 422 nm with readings taken every 5 min using the spectrophotometer. For kinetic assays, experiments used 150 µL of PBS adjusted to contain 100 µg/mL SF-CDs , using a DTNB concentration of 100 µM and varying GSH concentrations between 0 and 4 mM. Reactive oxygen species assay HepG2 cell cultures were cultured in DMEM to which SF-CDs were added at concentrations of 25, 50 and 100 µg/mL for 4h. The cells were subsequently incubated with DCFH-DA at a concentration of 0.2 µM for 15 min at 37°C in darkness to promote the conversion of DCFH-DA into its fluorescent derivative, which allows for the measurement of ROS levels [31, 32]. After removal of unbound probe with PBS, cells were examined by CLSM with an emission wavelength set at 531 nm after excitation at 506 nm. Determination of GSH levels within cells using ellman’s method Intracellular GSH levels were determined by the Ellman test, which quantifies the light absorption of a yellow substance resulting from the interaction of DTNB with GSH. HepG2 cell cultures in plate wells were exposed to various concentrations of SF-CDs (25, 50 and 100 µg/mL) for 12h. Lysed cells were mixed with 50 µL of 10 mM DTNB solution to identify the yellow complex. The levels of GSH were determined at a wavelength of 422 nm using an F50 plate reader to measure optical density. Assessing cell membrane integrity The Dil staining protocol was used to assess the integrity of the plasma membrane. HepG2 cells were incubated with SF-CDs at doses of 25, 50 and 100 µg/mL for 24h. After a PBS wash to remove excess material, the cells were incubated with 5 µM Dil for 15 min at 37℃ in an opaque chamber to promote the intracellular conversion of Dil to its fluorescent derivative. Fluorescence microscopy was then performed using CLSM with an excitation wavelength of 549 nm and an emission of 565 nm. Analysis of mitochondrial structure In the Mito-tracker assay [6], HepG2 cells were exposed to SF-CDs at a concentration of 100 µg/mL for 24h. Cells were subsequently treated with 200 nM Mito-tracker for half an hour at 37°C in the dark. Fluorescence was examined using a CLSM with an excitation wavelength of 490 nm and an emission at 516 nm. For TEM analysis, HepG2 cells were treated with SF-CDs at a concentration of 100 µg/mL for 24h, followed by fixation in 2% glutaraldehyde at 4℃ for 8h. They were dehydrated through an acetone gradient, followed by infiltration, embedding and polymerization, and ultrathin 0.5 µm sections were prepared. After rinsing with PBS, the samples were examined at 200 kV. In vitro anti-proliferation In vitro cytotoxicity was assessed by culturing L02 cells, HepG2 cells, HeLa cells and 4T1 cells in 96-well plates to 80% confluence, followed by exposure to SF-CDs at doses ranging from 0 to 10 µg/mL for 24h. The viability of cells was evaluated with the CCK-8 assay kit, adhering to the method detailed in the referred [33]. Western blotting analysis After treatment of HepG2 cells with SF-CDs , protein lysates were isolated. From each sample, 50 µg of total protein was loaded onto a 10% SDS-PAGE gel, resolved, and subsequently transferred to a nitrocellulose membrane for blotting. The blot was probed with specific primary antibodies recognizing pro-caspase-3 (CST, diluted 1:1,500) and PARP-1 (CST, diluted 1:1,500) for 1h at 25℃, subsequently, the samples were incubated with a horseradish peroxidase-conjugated secondary antibody (zsBio, diluted at a ratio of 1:6000) for an additional 60 min. Assessment of lipid peroxidation in vitro The malondialdehyde (MDA) assay kit was utilized to quantify in vitro lipid peroxidation (LPO) levels [34]. HepG2 cells were initially plated in 6-well plates and incubated at 37°C overnight. Subsequently, the spent medium was replaced with fresh medium containing various treatments: medium only, erastin and SF-CDs with diverse concentrations (25, 50, and 100 µg/mL), respectively. These cells were then cultured for an additional 24h. Following this period, the lipid peroxidation levels were assessed using the TBA method provided by the MDA assay kit. Subcutaneous tumor model A total of 1×10 6 H22 cells in a physiological saline solution were injected beneath the skin on the right side of BALB/c mice. When the tumor volumes grew to around 80 mm³, the mice were randomly divided into three distinct groups. In the SF-CDs treatment groups, mice received intra-tumoral injections of SF-CDs and administered 10 mg/kg of body weight every 48h over a period of seven treatment cycles. Body weight and tumor volume measurements were recorded at two-day intervals. The calculation of tumor volume was based on the equation: V = 1/2×L×W 2 , where (V) represents volume, (L) is the length, and (W) is the width of the tumor. All animal-related procedures were carried out with the approval of the Institutional Animal Care and Use Committee at Fujian Agriculture and Forestry University (Approval Number: PZCASFAFU25100). In vivo anti-tumor activity of SF-CDs in orthotopic H22 tumor xenograft mouse model Female BALB/c mice, five weeks old were used to establish H22 orthotopic xenograft models. Briefly, 100 million H22 cells were suspended in 50 µL PBS and combined with an equal volume of Matrigel. The cell-Matrigel mixture was injected orthotopically into the left hepatic lobe of the mice under ultrasound guidance, with a total of five intra-tumoral injections. After disinfection with 75% ethanol, the mice were warmed, isolated and monitored until they regained full consciousness. After tumor inoculation, the mice were randomized into three groups of five animals each, following a three-day post-tumor implantation period: the saline control, SF and SF-CDs groups. They were treated every three days for a total of six doses over a period of 15d. On day 40, all mice were euthanized. Liver and tumor tissues were then harvested and measured. In the mouse survival study, the procedures outlined above were strictly followed, with a cohort of five mice per group, and any mortality were documented upon the experiment’s conclusion. Histological and biosafety analysis After the intervention, a thorough blood analysis was performed to carefully measure biochemical indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and MDA. Cardiopulmonary, liver, spleen and kidney tissues were then carefully collected, immersed in 4% formaldehyde solution for one day and incorporated into paraffin blocks for subsequent H&E staining and immunohistochemical (IHC) analysis. Major organs were also collected and specimens were preserved in 4% formaldehyde for an extended period of time and then mounted in paraffin for H&E staining and comprehensive IHC. In vivo immune response A suspension of H22 cells was administered intradermally to the left flank of mice to establish a unilateral H22 tumor model. Once the tumors had grown to a volume of about 80 mm 3 , the rodents were subjected to intravenous injections of PBS, SF and SF-CD (10 mg/kg of SF and an equivalent concentration of SF-CDs ). Statistical Analysis The results are clearly presented as averages with standard deviation. The statistical threshold for significance is P < 0.05. In vitro assays were performed three times, each time independently. Experimental procedures with animal models were initiated after randomization, with a quintet of mice per group for in vivo studies. Statistical evaluations were facilitated by using Student’s t-test for comparative analyses, with details provided in the figure captions. Box plots range from the 25th to the 75th percentile, including the interquartile range. Statistical Significance is indicated by one, two or three asterisks for P values less than 0.05, 0.01 and 0.001 respectively, while disparities in animal survival were determined by Kaplan-Meier estimation, with the log-rank test determining the P value. Result and discussion Preparation and characterization of SF-CDs nanozyme SF-CDs were meticulously synthesized using a well-established protocol as previously described [15]. 27 mg of SF was first combined with 27 mL of ddH 2 O and mixed for a duration of 20 min before being transferred to Teflon-lined autoclaves. These were then heated in an air oven at a constant temperature of 180°C for 6h (Fig. 1 A). When the temperature of the mixture reached ambient conditions, the thick solution was spun at 10,000 rpm for 5 min to carefully collect the supernatant. The product was then dialyzed to promote exchange with ultrapure water for 48h. Finally, the purified SF-CDs were lyophilized for convenient storage at 4°C and prepared for future applications. Inspired by our previous findings, we observed that the SF-derived dialyzed solution emitted a vivid blue fluorescence under UV radiation at 365 nm. TEM images confirmed that the SF-CDs were evenly distributed and had a mean size of 4.6 ± 0.4 nm, as shown in Fig. 1 B. The size distribution, shown in detail in Supplementary Fig. S1 , is indicative of synthesized particle genesis throughout the hydrothermal reaction. Their compact size allows for easy passage through the cell membrane via endocytosis and efficient penetration of the mitochondrial membrane’s double lipid bilayer, leading to accumulation within the mitochondrial matrix [35]. After a 6h hydrothermal process at 180°C, the products at concentrations of 25, 50, 100 and 200 µg/mL showed intense blue fluorescence under UV light at 365 nm ( Fig. S2 ). This result confirms the efficient production of SF-CDs from SF precursors. Given the recognized role of SF as a multi-kinase inhibitor, which has attracted considerable attention for its potential to inhibit tumourigenesis and progression and improve survival rates, our subsequent investigations focused on SF-CDs [36]. AFM imaging delineated the fine structure of SF-CDs , confirming their uniform distribution and consistent thickness between 5 and 10 nm (Fig. 1 C and D ). To thoroughly evaluate the photoluminescence properties of SF-CDs , we examined the 3D fluorescence emission spectrum under different excitation conditions (Fig. 1 E). The luminescence spectrum of SF-CDs has been found to have specific characteristics and shifted from 360 nm to 500 nm as the excitation wavelength increased from 280 to 430 nm, highlighting the characteristic excitation-dependent photoluminescence (Fig. 1 F). Given their remarkable photoluminescence, we have investigated the fluorescence properties of SF-CDs . Upon 365 nm UV light, the “FAFU” characters and the emblem of Fujian Agriculture and Forestry University etched with SF-CDs ink were visibly altered ( Fig. S3 ). A broad diffraction feature centered at approximately 30° was observed in the XRD pattern (Fig. 1 G), which is characteristic of the low-degree graphitization and predominantly amorphous nature of the as-prepared SF-CDs . This broad reflection arises from the disordered stacking of aromatic carbon domains and is consistent with the typical architecture of carbon dots, which commonly feature heterogeneous oligomeric fragments intertwined with weakly graphitized carbon cores. Structural characterization of SF-CDs To further elucidate the intricate structure of SF-CDs , mass spectrometry (MS) was employed for compositional analysis. A dominant signal centered at approximately 640 Da was observed in the mass spectrum, accompanied by a characteristic fragment at m/z 465. This observation supports the oligomeric nature of the molecular components within SF-CDs , consistent with the heterogeneous structural features of carbon-based nanodots (Fig. 1 H). Furthermore, FT-IR analysis identified absorption bands for -OH (3420 cm − 1 ), C-H (2950 cm − 1 ), CO = NH (1670 cm − 1 ), C-O-C (910 cm − 1 ) and C = N (1590 cm − 1 ) (Fig. 1 I), confirming the incorporation of hydroxyl, carboxyl and carbonyl functionalities within the SF-CDs framework. Zeta potential analysis of SF-CDs revealed a negatively charged surface with an average potential of -1.21 ± 0.25 mV (Fig. 1 J). XPS analysis revealed the presence of carbon and oxygen in the SF-CDs , with two distinct peaks observed in the spectrum, as shown in Fig. 1 K-N. Specifically, the fine resolution C 1s XPS spectrum was resolved into two distinct peaks: a pronounced peak for the C-O bond at 286 eV and a distinct peak for the C = O bond at 289 eV (Fig. 1 L). This finding further supports the presence of C = O or C-O-C moieties on the surface of the SF-CDs. The functional groups on SF-CDs , which facilitate electrostatic and hydrogen bonding with the positively charged biomolecules within the nucleus or the FG nucleoporins in the nuclear pore complex [37]. Consequently, this interaction enhances the internalization and accumulation of SF-CDs within the nucleus. GSH oxidase mimetic properties of SF-CDs A number of nanoparticles have recently been shown to have enzyme-like properties [36–40]. We therefore investigated whether SF-CDs possess the typical GSH oxidase-like activity using the Ellman assay (Fig. 2 A). The DTNB assay showed a remarkable ability of SF-CDs to scavenge GSH. When GSH at concentrations ranging from 0.5 to 4 mM was mixed with SF-CDs (100 µg/mL) and DTNB (100 µM), an absorbance maximum at 422 nm was detected that correlated with both reaction time and GSH concentration (Fig. 2 B). To further rule out the potential influence of reactive oxygen species, we established control groups containing only H 2 O 2 or ·OH. The results indicated that in the absence of SF-CDs , neither H 2 O 2 nor ·OH alone significantly oxidized GSH. This observation underscores the detoxifying capacity of SF-CDs towards GSH. Subsequently, quantitative analysis of the enzyme kinetics suggested that SF-CDs exhibited a typical Michaelis-Menten kinetics property in the DTNB colorimetric reaction (Fig. 2 C). Moreover, the K M and V max value of SF-CDs were determined to be 4.272 and 2.923 in a double reciprocal plot (Fig. 2 D). In addition, UV -vis spectroscopic analysis accurately verified the conversion of GSH to GSSG following interaction with SF-CDs , as shown in Fig. 2 E. To quantify oxygen depletion in the SF-CDs mediated reaction, ABTS was used as an indicator of oxidative stress. The results indicated a marked escalation in the conversion of ABTS to ox -ABTS in the presence of SF-CDs under atmospheric oxygen, as shown in Fig. 2 F. The catalysis reaction was further enhanced in an atmosphere with oxygen levels of approximately 100% and was inhibited in an oxygen free N 2 atmosphere, highlighting the central role of oxygen in SF-CDs -driven reactions. Besides, the generation of H 2 O 2 was determined by an Amplex red probe ( Fig. S11 ). As a result, the fluorescence intensity at 520 nm gradually increased with SF-CDs concentration, confirming that SF-CDs possessed oxidase-like activity. Adopting the well-recognized four-electron (4e − ) reduction mechanism, we postulated that SF-CDs might be able to reduce oxygen to H 2 O 2 and subsequently generated .OH radicals to accelerate the oxidative conversion of GSH to GSSG. Meanwhile, electron spin resonance (ESR) analysis also verified the formation of ·OH in the presence of H 2 O 2 and SF-CDs (Fig. 2 G). Taken together, these results indicate that SF-CDs exhibit oxidase-like behavior in the presence of GSH, as shown in Fig. 2 H. Capacity of SF-CDs for cellular uptake and release In our investigation, we examined the endocytic ability of SF-CDs in HepG2 cells, assessing uptake efficiency by measuring particle-emitted luminescence. Cellular uptake dynamics indicated rapid internalization of SF-CDs by HepG2 cells in less than 10 min. The fluorescence intensity persisted for at least 30 min, highlighting the enhanced biocompatibility of SF-CDs and their efficacy in cellular internalization (Fig. 3 A). To investigate the in vitro cytotoxic capabilities of SF-CDs , an analysis was conducted using the CCK8 assay method to assess the viability of both normal and carcinoma cells after incubation with SF-CDs . The cytotoxicity of SF-CDs over a range of concentrations on the normal hepatocyte line (L02) and the carcinoma cells such as HepG2, HeLa and 4T1 is shown in Fig. S4 . From which one can observe that the L02 cells exhibit a survival rate exceeding 80% at SF-CDs concentrations all the way up to 10 µg/mL. In comparison, HepG2 cell viability dropped to 26.6% at 10 µg/mL of SF-CDs , while 4T1 and HeLa cells showed 42% and 40.5% viability, respectively, at the same dose. This observation suggests a significant selective cytotoxicity of SF-CD s against malignant cells as opposed to their normal counterparts. Cellular distribution of SF-CDs The intracellular trafficking of nanodrugs is a vital factor in influencing their anticancer effects. Utilizing FITC-labeled SF-CDs (green fluorescence), HepG2 cell lines exposed to SF-CDs demonstrate colocalization (lime green and white) of nucleus (violet) and mitochondria (blue fluorescence), suggesting that a majority of the intake SF-CDs is situated in the nucleoli region (Fig. 3 B). Furthermore, the cyan colocalization fluorescence of SF-CDs demonstrated 88% overlap with nucleolus (pink) and only 3% with mitochondria (blue) ( Fig. S5 ). These distribution patterns indicated that SF-CDs were taken up by the cells and predominantly accumulated in the nucleolar of HepG2 cells. Additionally, Mito-tracker staining revealed that the mitochondria exhibited distorted cristae and a fragmented morphology in response to SF-CDs . SF-CDs trigger ferroptosis through GSH consumption dependent ROS generation We next investigated the GSH depleting effect of SF-CDs on HepG2 cells. As shown in Fig. S6 , exposure to SF-CDs caused a significant decrease in GSH levels, presumably through the oxidase-like conversion of GSH to GSSG. This depletion disrupts the redox balance and impairs the function of GPX4, which is a key enzyme in the mitochondria responsible for preventing lipid peroxidation. Consequently, the accumulation of SF-CDs within the mitochondria is further enhanced due to their catalytic activity, as they effectively target the nucleolus and disrupt the redox homeostasis. Given the relationship between GSH depletion and increased oxidative stress, we assessed the generation of ROS in cells using the DCFH-DA fluorescence assay. Accordingly, SF-CDs elicited a strong green fluorescence response in HepG2 cells (Fig. 4 A), confirming the generation of hydroxyl radicals (·OH). To further substantiate this finding, the formation of ·OH was investigated using a TMB + H₂O₂ system. As depicted in Fig. S7 , there is a significant color change from colorless to blue in the TMB solution upon the addition of both SF-CDs and H₂O₂. This phenomenon was not observed with TMB alone, nor with the mixtures of TMB + H₂O₂, TMB + SF-CDs , and H₂O₂+S F-CDs . This color change was quantitatively measured using UV-visible spectroscopy, showing an increase in absorbance at 652 nm, likely due to the formation of ·OH through a Fenton-like process. We also assessed the expression levels of key ferroptosis markers, namely GPX4, NRF2, SLC7A11 and COX-2, by Western blotting. The SF-CDs decreased the levels of GPX4, NRF2 and SLC7A11, while increasing the levels of COX-2, with these effects escalating with increasing concentrations of SF-CDs from 25 to 100 µg/mL (Fig. 4 B). This suggests that SF-CDs may specifically induce cell death through a ferroptosis-mediated pathway. LPO promotion is another key marker on ferroptosis pathway [38]. To further verify that SF-CDs could induce ferroptosis, we assessed intracellular LPO levels using an MDA assay kit, as MDA is a reliable indicator of LPO. Our results revealed that the MDA levels in cells treated with erastin and various concentrations of SF-CDs (25, 50, and 100 µg/mL) were significantly higher than those in the control group (Fig. 4 C). Additionally, we observed a marked increase in LPO levels as the concentration of SF-CDs was incrementally increased from 25 to 100 µg/mL, suggesting that SF-CDs can significantly promote the generation of LPO. Overall, these findings indicate that SF-CDs are capable of inducing ferroptosis. Given the importance of mitochondria in ferroptosis, we further investigated the process by examining mitochondrial morphology. As shown in Fig. 5 A-D, in contrast to cells treated with culture medium alone (Fig. 5 B), the ferroptosis-positive control cells exposed to SF (Fig. 5 C) and those treated with SF-CDs nanozymes (Fig. 5 D) exhibited varying degrees of mitochondrial damage, changing from an elongated to a punctate pattern, suggesting a compacted mitochondrial membrane and reduced size. In addition, Mito-tracker staining revealed mitochondrial cristae disintegration and mitochondrial fragmentation upon exposure to SF-CDs . Specifically, in the control group, HepG2 cells display mitochondria that are typically elongated and tubular, featuring well-defined and evenly spaced cristae. Conversely, cells treated with SF-CDs display a punctate pattern, indicative of mitochondrial membrane compaction and a reduction in size. Moreover, the cristae within these mitochondria appear disrupted, taking on a less organized and more diffuse appearance, suggesting disintegration. Additionally, the overall structure of the mitochondria in SF-CDs treated cells is fragmented, characterized by smaller, disconnected mitochondrial fragments rather than a continuous network (Fig. 5 E). Furthermore, depleted GSH levels were observed to enhance cellular injury by accelerating lipid peroxidation. We therefore used the lipid-soluble fluorescent agent Dil to assess the continuity of the cell membrane. As demonstrated in Fig. 4 D, treatment with SF-CDs caused a marked reduction in red fluorescence in HepG2 cells, indicating a disrupted cell membrane structure, which increased with increasing concentrations of SF-CDs . Accordingly, these findings are consistent with the initiation of ferroptosis in HepG2 cells. The LIVE/DEAD cell staining assay confirmed that SF-CDs induced the most pronounced red fluorescence, signifying the strongest cytotoxic effect on HepG2 cells (Fig. 4 E). Annexin V-FITC and PI staining assays were then performed to clarify the anti-proliferative mechanism of SF-CDs mediated by enzymatic-like activities and oxidative stress enhancement (Fig. 4 F). Flow cytometry analysis revealed that erastin-induced ferroptosis resulted in an apoptosis rate of 34.82%, whereas SF-CDs at concentrations of 25, 50 and 100 µg/mL induced apoptosis rates of 6.50%, 36.99% and 45.85%, respectively. Based on these observations, we propose that SF-CDs in the TME convert the overexpressed GSH to GSSG, and the resulting GSH depletion inactivates GPX4, leading to lipid peroxidation, ROS accumulation, and ultimately ferroptosis (Fig. 4 G). Taken together, these findings indicate that SF-CDs induce ferroptosis via a ROS generation pathway dependent on GSH depletion. Therapeutic tumor-suppressing capabilities of SF-CDs in vivo We then investigated the tumor inhibitory properties of SF-CDs in a subcutaneous xenograft model using BALB/c mice inoculated with H22 cells. SF, a multi-kinase inhibitor and the standard of care for HCC study, is widely used in cancer treatment and is known to induce ferroptosis, making it a positive control in our experiment [39]. In this study, SF-CDs were administered intratumorally administered at a dose of 10 mg/kg fortnightly for 14d (Fig. 6 A). Subsequently, a marked suppression of tumorigenesis was associated with SF-CDs therapy (Fig. 6 B). In addition, the absence of significant weight loss in the mice throughout the treatment period indicated the minimal toxicity of SF-CDs (Fig. 6 C). After 14d of treatment, the tumors were excised, their mass measured and photographed (Fig. 6 D). In contrast to the PBS-treated group, a decrease in tumor mass was observed in all other treatment groups. Biosafety estimation of SF-CDs Considering the potential biological hazards of nanomaterials as they move from research to clinical settings, we evaluated the treatments’ biocompatibility by determining serum AST, ALT, BUN, and MDA levels in the treated mice (Fig. 6 E). Serum markers remained stable in all treatment groups, comparable to healthy controls, indicating a lack of apparent acute toxicity. Additionally, we performed an in vivo analysis of H&E-stained tissue sections. The histological examination via H&E staining indicated subtle renal side effects from SF treatment (Fig. 6 F), with features of glomerulosclerosis and high serum creatinine, corroborating earlier research [31]. In contrast, SF-CDs and their comparators showed a similar safety profile in all major organs. Taken together, these results underscore the favorable biosafety profile of SF-CDs , which, combined with their significant anticancer efficacy, positions them as promising candidates for translational medical applications. SF-CDs boost immune cell infiltration Due to its robust immunogenicity, ferroptosis facilitates the presentation of antigens, we investigated the impact of SF-CDs on the modulation of anti-tumor immunity. For an in-depth analysis of the modulation of biological responses by SF-CDs within the tumor immune microenvironment (TIME), this investigation focused on the infiltration of immune cells, particularly T cells, in tumors that are typically considered immunologically “cold”. Figure 6 G and H showed that SF-CDs significantly increased the presence of CD4 + and CD8 + T cells within the tumor tissue, a phenomenon not reflected by SF alone. Furthermore, SF-CDs also increased the levels of CD56 and F4-80 in the TME, confirming previous findings [[40]]. Collectively, our results demonstrated that SF-CDs can transform immunologically “cold” tumors into “hot” ones by promoting the infiltration of T cells, NK cells and macrophages. Following our initial observations, we proceeded to conduct a detailed quantitative analysis of key tumor markers, specifically examining the expression levels of CD4, CD8, CD56, and F4/80 within mouse tumor tissues. For this analysis, we employed the Immunoway Quadruple-Fluorescence immunohistochemical mouse/rabbit kit (pH 9.0) to meticulously apply primary and secondary antibody staining to sections of mouse tumor tissues. The distinct colors assigned to each marker corresponded to specific cellular components: blue for DAPI, green for FITC, yellow for Cy3, red for Cy5, and magenta for SpRed. The CLSM results indicated that the control group exhibited standard baseline expression levels of the immune cell markers. The SF group, however, showed an increase in the intensity of green and magenta fluorescence, suggesting an upregulation of CD4 and F4/80 expression. This upregulation is likely a result of the treatment’s impact on the TME. Most notably, the SF-CDs treated group demonstrated a significant enhancement in fluorescence intensity across all channels (Fig. 7 A). This increase in fluorescence intensity suggests that SF-CDs have the potential to transform immunologically “cold” tumors, which are generally less responsive to immune-based therapies, into “hot” ones. This transformation is characterized by an increased infiltration of T cells, NK cells, and macrophages, thereby modifying the immune landscape of the tumor and potentially enhancing its responsiveness to immunotherapy. To evaluate the effect of SF-CDs -mediated ICD on immune cell responses, we utilized flow cytometry to analyze the expression profiles of CD4, CD8, CD3, and CD11b among various treatment groups, examining the distribution of these cell populations. The data demonstrated that groups receiving SF-CDs treatment exhibited markedly enhanced T cell activation rates when compared with other treatment groups. The percentages of CD4 + and CD8 + cells were recorded at 10.4% and 35.4%, respectively (Fig. 7 B and C ). Similarly, both the SF and SF-CDs treated groups displayed elevated CD11b expression levels, with percentages of 19.5% and 23.2%, respectively (Fig. 7 D). Taken together, these findings confirmed that SF-CDs effectively induce ICD, stimulate the activation of T cells and macrophages, and consequently bolster the efficacy of tumor immunotherapy. In vivo antitumor evaluation of orthotopic H22 xenograft mouse model Inspired by the remarkable success of a subcutaneous xenograft tumor model, we proceeded to investigate the therapeutic potential of SF-CDs within an orthotopic HCC tumor model to obtain a more authentic assessment of their antitumor capabilities, as shown in Fig. 8 A, which illustrates the structural design of SF-CDs and the hypothesized therapeutic mechanism. Capitalizing on the prevalent clinical practice of image-guided intra-tumoral injections for HCC treatment, we integrated this approach into our experimental protocol [41]. The mice were then implanted with orthotopic HCC tumors derived from H22 cells using ultrasound-guided injections (Fig. 8 B) and the corresponding video is displayed in Fig. S8 . After orthotopic tumor implantation, mice were divided into three cohorts to receive intravenous injections of saline, SF or SF-CD , as shown in Fig. 8 C, and after tumor establishment they underwent a sequence of six injections every three days. Notably, the SF-CDs treatment resulted in the most pronounced inhibition of tumor growth compared to the saline and SF treatments (Fig. 8 D), with mean tumor weights of 337.0 ± 29.07, 241.4 ± 8.1 and 156.4 ± 21.6 mg for the control, SF and SF-CDs groups, respectively (Fig. 8 E). In addition, the SF-CDs group had the lowest liver weight, highlighting the improved therapeutic efficacy of this intervention (Fig. 8 F). At the same time, the survival rate of SF-CDs -treated tumor-bearing mice increased significantly, reaching almost 80% at day 40 (Fig. 8 G). Throughout the biosafety evaluation, minimal variations in body weight were observed in all treatment groups, as shown in Fig. 8 H. Conclusion In conclusion, we have successfully fabricated a nucleolus-targeted oxidative stress-amplifying SF-CDs nanozyme derived from FDA-approved SF and processed via a facile hydrogel treatment. SF-CDs , which embody the attributes of a multifaceted CNMs, stand out as a paradigmatic anticancer nanozyme, characterized by their excellent ability to mimic GSH oxidase. Importantly, SF-CDs were shown to significantly suppress tumor growth in both subcutaneous and orthotopic HCC models in vivo , while maintaining a favorable safety profile with no adverse effects or toxicity. Significantly, our research emphasizes the significant therapeutic potential of nanozymes derived from frontline HCC treatments that effectively induce ferroptosis and stimulate the TIME pathway with minimal toxicity. Abbreviations ABTS: 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); AFM: atomic force microscopy; ALT: alanine aminotransferase; AST: aspartate aminotransferase; BUN: blood urea nitrogen; CCK-8: Cell Counting Kit-8; CDs: carbon dots; CLSM: confocal laser scanning microscopy; CNMs: carbon nanomaterials; DAMPs: damage-associated molecular patterns; DCFH-DA: 2′,7′-dichlorodihydrofluorescein diacetate; DMEM: Dulbecco’s modified Eagle medium; DMSO: dimethyl sulfoxide; DTNB: 5,5′-dithiobis(2-nitrobenzoic acid); ESR: electron spin resonance; FBS: fetal bovine serum; FDA: Food and Drug Administration; FITC: fluorescein isothiocyanate; FT-IR: Fourier transform infrared spectroscopy; GPX4: glutathione peroxidase 4; GSH: glutathione; GSH oxidase: glutathione oxidase; GSSG: oxidized glutathione; HCC: hepatocellular carcinoma; H&E: hematoxylin and eosin; IHC: immunohistochemistry; K M : Michaelis constant; LPO: lipid peroxidation; MDA: malondialdehyde; MS: mass spectrometry; NRF2: nuclear factor erythroid 2-related factor 2; PARP-1: poly(ADP-ribose) polymerase 1; PBS: phosphate-buffered saline; PI: propidium iodide; ROS: reactive oxygen species; RPMI-1640: Roswell Park Memorial Institute 1640 medium; RRID: Research Resource Identifier; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SF: sorafenib; SF-CDs: sorafenib-derived carbon dots; SLC7A11: solute carrier family 7 member 11; TEM: transmission electron microscopy; TMB: 3,3′,5,5′-tetramethylbenzidine; TME: tumor microenvironment; UV: ultraviolet; UV-vis: ultraviolet-visible; Vmax: maximum reaction velocity; XPS: X-ray photoelectron spectroscopy; XRD: X-ray diffraction. Declarations Ethics approval and consent to participate All care and handling of animals were performed with the approval of the Institutional Animal Care and Use Committee of Fujian Agriculture and Forestry University (PZCASFAFU26011). Consent for publication All authors have consented to submit this article for publication. Competing interests The authors declare no competing interests. Funding This work was supported by the Fujian Provincial Joint Fund for Science and Technology Innovation (2025Y9713), Major Science and Technology Project of Fujian Province (2024NZ029029) and Fujian Provincial Natural Science Foundation of China (2023J011173). Author Contribution CRediT authorship contribution statementHuixi Guo: Investigation, Data curation, Formal analysis, Visualization, Funding, Writing - original draft.Chunmei Lai: Conceptualization, Methodology, Formal analysis, Funding, Writing - review & editing.Weiji Chen: Investigation, Validation, Resources, Data curation.Shaohua He: Conceptualization, Supervision, Project administration, Funding acquisition, Resources, Funding and Writing - review & editing. Acknowledgement Not applicable. Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. References M. Naddaf, Science in 2025: the events to watch for in the coming year, Nature. 2025;637(8044):9-11. Z. Liu, L. Lei, Z. Zhang, M. Du, Z. Chen, Ultrasound-responsive engineered bacteria mediated specific controlled expression of catalase and efficient radiotherapy, Mater. Today Bio. 2025;31:101620. L. Gebicka, J. Krych-Madej, The role of catalases in the prevention/promotion of oxidative stress, J. Inorg. Biochem. 2019;197:110699. X. Li, J.F. Lovell, J. Yoon, X. Chen, Clinical development and potential of photothermal and photodynamic therapies for cancer, Nat. Rev. Clin. Oncol. 2020;17(11):657-674. J. Yu, J. Wu, J. Huang, C. Xu, M. Xu, C.Z.H. Koh, K. Pu, Y. Zhang, Hypoxia-tolerant polymeric photosensitizer prodrug for cancer photo-immunotherapy, Nat. Commun. 2025;16(1):153. N. Gong, X. Ma, X. Ye, Q. Zhou, X. Chen, X. Tan, S. Yao, S. Huo, T. Zhang, S. Chen, X. Teng, X. Hu, J. Yu, Y. Gan, H. Jiang, J. Li, X.J. Liang, Carbon-dot-supported atomically dispersed gold as a mitochondrial oxidative stress amplifier for cancer treatment, Nat. Nanotechnol. 2019;14(4):379-387. M. Wen, J. Ouyang, C. Wei, H. Li, W. Chen, Y.N. Liu, Artificial enzyme catalyzed cascade reactions: antitumor immunotherapy reinforced by NIR-II light, Angew. Chem., Int. Ed. Engl. 2019;58(48):17425-17432. H. Zhang, T. Yang, W. Mu, X. Peng, T. Liu, L. Weng, H. Wang, Y. Zhang, X. Chen, Effective amplification of oxidative stress and calcium manipulation mediated mitochondrial dysfunction based on engineered nanozyme for primary and metastatic breast cancer therapy, Small (Weinheim an der Bergstrasse, Germany). 2025;21(14):e2411299. B. Niu, K. Liao, Y. Zhou, T. Wen, G. Quan, X. Pan, C. Wu, Application of glutathione depletion in cancer therapy: Enhanced ROS-based therapy, ferroptosis, and chemotherapy, Biomaterials 2021;277:121110. Q. Xin, F. Tian, H. Qiao, G. Li, Y. Wang, L. Li, L. Zhang, N. Song, K. Chen, Y. Yan, S. Li, H. Ma, H. Liu, H. Wang, J. Yang, S. Sun, Y. Li, X.-D. Zhang, Machine learning assisted engineering of atomically precise cluster-derived artificial hydrolases for lipid metabolism diseases, Mater. Today. 2026;95:103294. Q. Huang, Q. Wu, L. Feng, F. Lei, Y. Zhang, F. Wang, Recent advances in crystalline porous framework-derived Nanozymes for emerging chemical contaminants remediation, Coord. Chem. Rev. 2026;559:217856. M.R. Chiang, C.W. Hsu, W.C. Pan, N.T. Tran, Y.S. Lee, W.H. Chiang, Y.C. Liu, Y.W. Chen, S.H. Chiou, S.H. Hu, Reprogramming dysfunctional dendritic cells by a versatile catalytic dual oxide antigen-captured nanosponge for remotely enhancing lung metastasis immunotherapy, ACS nano. 2025;19(2):2117-2135. J. Wu, X. Wang, Q. Wang, Z. Lou, S. Li, Y. Zhu, L. Qin, H. Wei, Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II), Chem. Soc. Rev. 2019;48(4):1004-1076. Y. Wang, M. Mu, R. Fan, H. Chen, B. Han, G. Guo, Single-atom enzymes: from nano-enzyme evolution to the biomedical frontier of precision catalysis, Coord. Chem. Rev. 2026;549:217317. C.-M. Lai, J. Xu, B.-C. Zhang, S.-H. He, J.-W. Shao, A natural product-derived nanozyme regulator induced chemo-ferroptosis dual therapy in remodeling of the tumor immune microenvironment of hepatocellular carcinoma, Chem. Eng. J. 2024;482:148976. Q. Liu, A. Zhang, R. Wang, Q. Zhang, D. Cui, A review on metal- and metal oxide-based nanozymes: properties, mechanisms, and applications, Nano-Micro Lett. 2021;13(1):154. W. Gao, J. He, L. Chen, X. Meng, Y. Ma, L. Cheng, K. Tu, X. Gao, C. Liu, M. Zhang, K. Fan, D.W. Pang, X. Yan, Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme, Nat. Commun. 2023;14(1):160. L. Yang, M. Li, Y. Peng, Y. Zhou, J. Zou, H. Liu, W. Zhang, J. Liu, P. He, F. Dai, J. Zheng, J. Lin, S. Qu, Z. Lu, G. Liu, Engineering NIR-II carbon dots through aniline extension with graphene and nitrogen enrichment for hepatobiliary theranostics, Nat. Commun. 2026. J. Wang, Y. Fu, Z. Gu, H. Pan, P. Zhou, Q. Gan, Y. Yuan, C. Liu, Multifunctional carbon dots for biomedical applications: diagnosis, therapy, and theranostic, Small. 2024;20(3):2303773. E.S. Grant, J.F. Olorunyomi, S.C. Scholten, I.O. Robertson, A.N. Abraham, N.H. Srikantamurthy, B.J. Murdoch, E.L.H. Maye, B. del Rosal, A.J. Healey, C.M. Doherty, P. Reineck, X. Mulet, J.-P. Tetienne, D.A. Broadway, Spin-dependent photoluminescence in carbon-based quantum dots, Adv. Mater. 2026;38(17):e18572. Y. Liu, Z. Cai, X. Sun, H. Zhang, Y. Hao, J. Wu, Z. Yan, X. Wu, Y. Liang, L. Wang, S. Qu, Establishing a model precursor system: over a decade of research on carbon dots from the citric acid-urea system, Adv. Funct. Mater. 2026;n/a(n/a):e32189. J. Wang, D. Guo, S. Jiang, W. Wu, X. Gao, Targeting ferroptosis in cancer: from mechanistic insights to therapeutic approaches, Mol. Biomed. 2026;7(1):20. X. Chen, Decoding GPX4 regulation in ferroptosis: mechanisms and therapeutic implications, Trends Mol. Med. 2026. Q. Chen, F. Liu, Y. Zhang, L. Yan, Y. Wu, D. Xu, P. Wu, H. Yuan, K. Jiang, Ferroptosis in cancer toward molecular insights and clinical translation in pancreatic cancer, Mol. Cancer. 2026;25(1):48. X. Fang, H. Ardehali, J. Min, F. Wang, The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease, Nat. Rev. Cardiol. 2023;20(1):7-23. Z. Wang, Z. Wang, D. Li, C. Yang, Q. Zhang, M. Chen, H. Gao, L. Wei, High-quality semiconductor fibres via mechanical design, Nature. 2024;626(7997):72-78. W. Wang, M. Green, J.E. Choi, M. Gijón, P.D. Kennedy, J.K. Johnson, P. Liao, X. Lang, I. Kryczek, A. Sell, H. Xia, J. Zhou, G. Li, J. Li, W. Li, S. Wei, L. Vatan, H. Zhang, W. Szeliga, W. Gu, R. Liu, T.S. Lawrence, C. Lamb, Y. Tanno, M. Cieslik, E. Stone, G. Georgiou, T.A. Chan, A. Chinnaiyan, W. Zou, CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy, Nature. 2019;569(7755):270-274. X. Chen, R. Kang, G. Kroemer, D. Tang, Broadening horizons: the role of ferroptosis in cancer, Nat. Rev. Clin. Oncol. 2021;18(5):280-296. C.M. Lai, W.J. Chen, Y. Qin, D. Xu, Y.K. Lai, S.H. He, Innovative hydrogel design: tailoring immunomodulation for optimal chronic wound recovery, Adv. Sci. (Weinh) 2025;12(2):e2412360. I. Rahman, A. Kode, S.K. Biswas, Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method, Nat. Protoc. 2006;1(6):3159-3165. M.P. Murphy, H. Bayir, V. Belousov, C.J. Chang, K.J.A. Davies, M.J. Davies, T.P. Dick, T. Finkel, H.J. Forman, Y. Janssen-Heininger, D. Gems, V.E. Kagan, B. Kalyanaraman, N.G. Larsson, G.L. Milne, T. Nyström, H.E. Poulsen, R. Radi, H. Van Remmen, P.T. Schumacker, P.J. Thornalley, S. Toyokuni, C.C. Winterbourn, H. Yin, B. Halliwell, Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo, Nat. Metab. 2022;4(6):651-662. X. Yao, W. Hu, Y. Li, J. Li, F. Kang, J. Zhang, S. Dong, Dual dynamic crosslinked hydrogel patch embodied with anti-bacterial and macrophage regulatory properties for synergistic prevention of peritendinous adhesion, Adv. Funct. Mater. 2024;34(34):2400660. L. Ouyang, J. Wang, B. Liu, M. Xie, L. Wang, C. Fan, J. Chao, Parameter pool-assisted centrifugation sorter for multiscale higher-order DNA nanomaterials, ACS nano. 2025;19(3):3830-3838. Y. Xiong, Z. Yong, S. Li, Q. Wang, X. Chen, Z. Zhang, Q. Zhao, Q. Deng, X. Yang, Z. Li, Self-Reliant nanomedicine with long-lasting glutathione depletion ability disrupts adaptive redox homeostasis and suppresses cancer stem cells, Adv. Funct. Mater. 2024;34(8):2310158. W. He, H. Gao, W. Wu, Nanomedicine biointeractions during body trafficking, Adv. Drug Delivery Rev. 2024;209:115324. J.M. Llovet, R. Pinyol, R.K. Kelley, A. El-Khoueiry, H.L. Reeves, X.W. Wang, G.J. Gores, A. Villanueva, Molecular pathogenesis and systemic therapies for hepatocellular carcinoma, Nat. Cancer 2022;3(4):386-401. N. Blal, G. Bardi, P.P. Pompa, D. Guarnieri, Nano-biointeractions of functional nanomaterials: the emerging role of inter-organelle contact sites, Targeting, and Signaling, Adv. Funct. Mater. 2024;34(52):2408436. Q. Zhou, Y. Meng, D. Li, L. Yao, J. Le, Y. Liu, Y. Sun, F. Zeng, X. Chen, G. Deng, Ferroptosis in cancer: From molecular mechanisms to therapeutic strategies, Signal Transduction Targeted Ther. 2024;9(1):55. X. Li, B. Zhang, C. Jiang, J. Zhang, X. Ning, Z. An, X. Chen, Y. Chen, P. Chen, Long cycle lifespan of flexible rechargeable zinc-air batteries based on porous sodium hyaluronate/polyacrylamide-based hydrogel electrolyte, J. Power Sources. 2025. X. Li, B. Liu, J. Wang, S. Li, X. Zhen, J. Zhi, J. Zou, B. Li, Z. Shen, X. Zhang, S. Zhang, C.-W. Nan, High-temperature capacitive energy storage in polymer nanocomposites through nanoconfinement, Nat. Commun. 2024;15(1):6655. A. Som, J.G. Rosenboom, A. Chandler, R.A. Sheth, E. Wehrenberg-Klee, Image-guided intratumoral immunotherapy: Developing a clinically practical technology, Adv. Drug Delivery Rev. 2022;189:114505. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supportinginformation1.docx floatimage1.png Scheme 1. A. Schematic representation of the SF-CDs synthesis procedure and B. The features for mitochondria targeting, enzyme-like activities induce ferroptosis and subsequent immunotherapy. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9469655","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629963956,"identity":"448adb9f-e176-4f75-800c-c2188c1d396c","order_by":0,"name":"Huixi Guo","email":"","orcid":"","institution":"Shengli Clinical Medical College of Fujian Medical University \u0026 Fuzhou University Affiliated Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huixi","middleName":"","lastName":"Guo","suffix":""},{"id":629963957,"identity":"d1786327-3282-4d71-87b4-8d9cc348c878","order_by":1,"name":"Chunmei Lai","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Chunmei","middleName":"","lastName":"Lai","suffix":""},{"id":629963958,"identity":"7872e890-b36e-4aa0-a813-b8b77389ac8e","order_by":2,"name":"Weiji Chen","email":"","orcid":"","institution":"Shengli Clinical Medical College of Fujian Medical University \u0026 Fuzhou University Affiliated Provincial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiji","middleName":"","lastName":"Chen","suffix":""},{"id":629963959,"identity":"13d595fc-1059-4120-bee2-601ba6fb44ee","order_by":3,"name":"Shaohua He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFACxgfGDAwH5NjY2w8Qq4XZAKTFmI/nTALxWpiBWhLnSTgYEKeBf3YzQ3HBnzvpbRIMCQw/KrYR1iJx5zCD8cy2Z7lt0o0HGHvO3CasxUAi/4Axb8Ph3DaZAwnMjG1EaUlmMOb5czidTSLBgBQtbIcTiNcicQOohbftsGEbMJAPEuUX/hnJbCCHycu3tx988KOCCC1AwAaPjwNEqQcC5gfEqhwFo2AUjIIRCgDhOjn97VS8OQAAAABJRU5ErkJggg==","orcid":"","institution":"Shengli Clinical Medical College of Fujian Medical University \u0026 Fuzhou University Affiliated Provincial Hospital","correspondingAuthor":true,"prefix":"","firstName":"Shaohua","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2026-04-20 09:09:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9469655/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9469655/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108406253,"identity":"9f95caef-f430-4950-bc1b-cca36f3736ca","added_by":"auto","created_at":"2026-05-04 09:41:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":736312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Illustration of \u003cem\u003eSF-CDs\u003c/em\u003e, which have a graphite-like core surrounded by chemically reactive groups; \u003cstrong\u003eB\u003c/strong\u003e. TEM image of \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eC \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e. AFM image showing the variation in thickness of \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eE\u003c/strong\u003e. 3D fluorescence intensity profiles of \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eF\u003c/strong\u003e. Photometric luminescence of \u003cem\u003eSF-CDs \u003c/em\u003edepends on excitation wavelength; \u003cstrong\u003eG\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eXRD analysis pattern of \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eH\u003c/strong\u003e. MALDI-TOF MS analysis of \u003cem\u003eSF-CDs\u003c/em\u003eacross an extensive m/z range with a consistent interval of 465 Da; \u003cstrong\u003eI\u003c/strong\u003e. FT-IR spectra of \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eJ\u003c/strong\u003e. The zeta potentials of \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eK-N\u003c/strong\u003e. High-resolution XPS spectrum of \u003cem\u003eSF-CDs\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/3a4a525a3c7e6b30a13ee94f.png"},{"id":108406263,"identity":"28485fba-dc64-4aac-b111-39874219e4aa","added_by":"auto","created_at":"2026-05-04 09:41:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":512947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Schematic depiction of GSH sensing via DTNB colorimetric method; \u003cstrong\u003eB\u003c/strong\u003e. Time-dependent absorbance with the introduction of \u003cem\u003eSF-CDs\u003c/em\u003e (100 μg/mL) and GSH concentrations ranging from 0.5 to 4 mM; \u003cstrong\u003eC\u003c/strong\u003e. Steady state kinetic assay; \u003cstrong\u003eD\u003c/strong\u003e. Lineweaver-burk plot for \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eE\u003c/strong\u003e. UV-visible absorption spectrum of GSH, GSSG and the resulting products of the \u003cem\u003eSF-CDs\u003c/em\u003ecatalyzed reaction with GSH; \u003cstrong\u003eF\u003c/strong\u003e. Nitrogen, ambient air and oxygen-rich environments were used to study the direct oxidation of ABTS by \u003cem\u003eSF-CDs\u003c/em\u003e; \u003cstrong\u003eG\u003c/strong\u003e. The ESR spectra captured the formation of ·OH when \u003cem\u003eSF-CDs \u003c/em\u003ewere treated with DMPO; \u003cstrong\u003eH\u003c/strong\u003e. Diagram of GSH oxidase-like activity of \u003cem\u003eSF-CDs\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/7792651f8a13244031c11cc1.png"},{"id":108406255,"identity":"5a97389e-bbea-4f73-89d7-6bcfa044fe08","added_by":"auto","created_at":"2026-05-04 09:41:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1024486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Time-based luminescence analysis of HepG2 cells exposed to\u003cem\u003e SF-CDs\u003c/em\u003e(100 μg/mL). The scale bar corresponds to 10 μm; \u003cstrong\u003eB\u003c/strong\u003e. CLSM images of HepG2 cells exposed to different concentrations of \u003cem\u003eSF-CDs\u003c/em\u003e (25, 50 and 100 μg/mL) for 4h. BF indicates bright field. Mitochondria stained with Mito-Tracker appear in blue, lysosomes stained with Lyso-Tracker appear in red, and the green “bars” or “lines” indicate FITC-labelled \u003cem\u003eSF-CDs \u003c/em\u003e(Note: All images were captured at the same magnification).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/b3d44ea9a32bdc516a0f918e.png"},{"id":108492562,"identity":"5314a8da-92d9-46ac-865b-c6a947f1f59f","added_by":"auto","created_at":"2026-05-05 09:58:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":832114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eFluorescence microscopy imaging of HepG2 cells labelled with DCFH-DA after exposure to \u003cem\u003eSF-CDs\u003c/em\u003e at concentrations of 25, 50 and 100 μg/mL for a period of 6h; \u003cstrong\u003eB\u003c/strong\u003e. \u003cem\u003eSF-CDs\u003c/em\u003esuppressed the expression of GPX4, NRF2, and SLC7A11 and stimulated COX-2 expression; \u003cstrong\u003eC\u003c/strong\u003e. The intracellular LPO levels (n=3); \u003cstrong\u003eD\u003c/strong\u003e. CLSM images showing Dil-labelled HepG2 cells after exposure to \u003cem\u003eSF-CDs\u003c/em\u003e at concentrations of 25, 50 and 100 μg/mL for 12h; \u003cstrong\u003eE\u003c/strong\u003e. LIVE/DEAD assay of HepG2 cells following 24h incubation with\u003cem\u003e SF-CDs\u003c/em\u003e. Scale bar, 100 μm; \u003cstrong\u003eF\u003c/strong\u003e. Flow cytometry assessment of HepG2 cell apoptosis following 48h treatment with various formulations; \u003cstrong\u003eG\u003c/strong\u003e. Diagrammatic representation of the tumor-inhibiting mechanism of \u003cem\u003eSF-CDs\u003c/em\u003e through the initiation of ferroptosis.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/57676a535555a806b123f93c.png"},{"id":108406257,"identity":"ed2ec285-dd7c-4c5b-8312-12da418c3177","added_by":"auto","created_at":"2026-05-04 09:41:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":610086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Schematic representation of mitochondrial damage; \u003cstrong\u003eB\u003c/strong\u003e. Bio-TEM image of untreated HepG2 cells; \u003cstrong\u003eC\u003c/strong\u003e. Bio-TEM image of HepG2 cells treated with SF and \u003cstrong\u003eD\u003c/strong\u003e. \u003cem\u003eSF-CDs\u003c/em\u003e, respectively； \u003cstrong\u003eE\u003c/strong\u003e. The effect of\u003cem\u003e SF-CDs\u003c/em\u003eon HepG2 cells, showing mitochondrial fragmentation via Mito-tracker staining.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/f4153a8423fac8d9af25f011.png"},{"id":108406259,"identity":"26d501a9-a55e-4315-ae99-e2a0ab8f3002","added_by":"auto","created_at":"2026-05-04 09:41:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":969048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eSchematic representation of \u003cem\u003eSF-CDs\u003c/em\u003e for suppression of tumor expansion; \u003cstrong\u003eB\u003c/strong\u003e. Tumor progression curve for tumors treated with saline, SF and\u003cem\u003e SF-CDs\u003c/em\u003e; \u003cstrong\u003eC\u003c/strong\u003e. The changes in body weight of mice after \u003cem\u003eSF-CDs\u003c/em\u003e treatment administered twice a day until day 14; \u003cstrong\u003eD\u003c/strong\u003e. Photographs of tumors harvested on day 21; \u003cstrong\u003eE\u003c/strong\u003e. Serum concentrations of AST, ALT, BUN and MDA for statistical analysis, data are presented as mean ± SEM. SF (10 mg/kg) was used as a positive control; \u003cstrong\u003eF\u003c/strong\u003e. H\u0026amp;E staining of tumors under different treatment regimens; \u003cstrong\u003eG\u003c/strong\u003e. Schematic representation of the hypothesized mechanism for transformation of immunologically cold tumors into hot ones via \u003cem\u003eSF-CDs\u003c/em\u003e. SF (10 mg/kg) served as a positive control; \u003cstrong\u003eH\u003c/strong\u003e. IHC staining for CD4, CD8, CD56 and F4-80 in H22 tumor tissue;\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/5fc7081602148c6363c12d54.png"},{"id":108492558,"identity":"5dff31a4-aefd-47e5-bd8a-6e3d3c969556","added_by":"auto","created_at":"2026-05-05 09:58:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1210687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Multicolor fluorescence assays demonstrated the expression levels of immune cells treated with PBS, SF, and \u003cem\u003eSF-CDs\u003c/em\u003e, respectively; \u003cstrong\u003eB-D\u003c/strong\u003e. Flow cytometry results showing the T cells (CD4\u003csup\u003e+\u003c/sup\u003e, CD8\u003csup\u003e+\u003c/sup\u003e) and macrophages (CD11b) after H22 cells were treated in different ways.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/bd7d7892c7ad3886bab753de.png"},{"id":108406261,"identity":"e3270cc0-308e-46d7-9271-713c5fcb7152","added_by":"auto","created_at":"2026-05-04 09:41:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":887461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Diagram illustrating the operating procedures and mechanism of\u003cem\u003e SF-CDs\u003c/em\u003e; \u003cstrong\u003eB\u003c/strong\u003e. Representative ultrasound-guided modeling photographs of the tumor and the tumors were outlined by red dashed lines; \u003cstrong\u003eC\u003c/strong\u003e. Outline of the dosing regimen. Three days after tumor implantation, subjects were randomly assigned to three groups for direct intra tumoral injections navigated by ultrasound imaging. A vehicle control comprising saline solution was utilized in the untreated condition, whereas SF was applied to serve as a positive control to elicit ferroptosis. The dose of \u003cem\u003eSF-CDs\u003c/em\u003e administered to each mouse was 10 mg/kg, with injections spaced 3 days apart; \u003cstrong\u003eD\u003c/strong\u003e. Representative images of livers after treatment, with tumor regions delineated by white dotted lines; \u003cstrong\u003eE\u003c/strong\u003e. Weight of tumors after different treatments (n=5); \u003cstrong\u003eF\u003c/strong\u003e. Liver weights after treatment (n=5);\u003cstrong\u003e G\u003c/strong\u003e. Disease-free survival rates in different treatment groups. Survival outcomes were assessed by Kaplan-Meier estimation, then analyzed for comparative statistical significance using the log-rank test (survival study was performed in a separate experiment alongside tumor inhibition assessment, n=5); \u003cstrong\u003eH\u003c/strong\u003e. Chronological variations in mouse body mass in different therapeutic cohorts (n=5). Data are presented as mean ± SEM;\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/748109b83b700b2155272884.png"},{"id":109069045,"identity":"695dd467-d035-42ab-b571-24d4dcd7dd14","added_by":"auto","created_at":"2026-05-12 10:19:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7217942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/48ccdba2-cd82-4cfd-add0-1a6cc8c4036e.pdf"},{"id":109067455,"identity":"4479b69e-3a9a-41d7-87d6-e4042b009fcc","added_by":"auto","created_at":"2026-05-12 09:52:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":61722750,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/a1c7e3c232106822133e68f9.docx"},{"id":108493412,"identity":"2aec53d6-73d2-43c1-9c8d-89368a17252b","added_by":"auto","created_at":"2026-05-05 10:00:19","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1006126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e. Schematic representation of the \u003cem\u003eSF-CDs\u003c/em\u003e synthesis procedure and \u003cstrong\u003eB\u003c/strong\u003e. The features for mitochondria targeting, enzyme-like activities induce ferroptosis and subsequent immunotherapy.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9469655/v1/c5d75cde9b1463ba3ee11a72.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sorafenib-based carbon dot nanozyme with nucleolus-targeted oxidative stress amplification for tumor immune microenvironment remodeling and cancer theranostics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe remarkable catalytic activity and substrate specificity of natural enzymes position them as superior catalysts for a wide range of biomedical applications. One example is horseradish peroxidase, which is widely used in enzymatic sensing assays for the detection of biomarkers, viruses and bacterial entities [1]. Catalase, which plays a key role in breaking down hydrogen peroxide into water and oxygen, is used strategically to increase the effectiveness of radiotherapy [2], sonodynamic therapy [3], and photodynamic therapy in the fight against tumors [4, 5]. This enhanced anti-tumor activity is attributed to the enzyme\u0026rsquo;s ability to alleviate the hypoxic constraints characteristic of the tumor microenvironment (TME). Glutathione oxidase, an enzyme involved in the regulation of cellular redox balance, has been used to treat several conditions characterized by oxidative stress [6, 7]. GSH oxidase, which is critical for cellular redox homeostasis, is being investigated for its ability to improve chemotherapeutic and radiotherapeutic outcomes by increasing ROS in cancer cells and inducing cell death [8]. The investigation of GSH oxidase for therapeutic applications represents an emerging area of research aimed at optimizing its medical potential and understanding its role in a range of pathological scenarios. However, the majority of natural enzymes have intrinsic limitations, including susceptibility to denaturation, high production costs, complicated preparation processes and challenges associated with scalable manufacturing processes [9]. To overcome these obstacles, synthetic enzymes have emerged as substantial and cost-effective alternatives to naturally occurring enzymes [10]. Among these, nanomaterials with enzyme-like activities, known as nanozymes, have revolutionized our perspective on nanoscale enzyme mimics and have garnered widespread interest for their capacity to address the intrinsic shortcomings of natural enzymes [11]. Currently, a plethora of nanomaterials have been creatively engineered to exhibit robust enzymatic functions, either by replicating the active sites of natural enzymes or by incorporating various elements into their nanostructures. However, the heterogeneous compositions and structures of nanozymes have given rise to a myriad of complex catalytic reactions, making it difficult to pinpoint active sites and presenting hurdles to the rational engineering of nanozymes with tailored catalytic efficacy and selectivity [12]. Nanozymes with enzyme-like properties represent a promising avenue for the advancement of enzyme-mediated therapeutics [13]. Their nanoscale structure gives them not only enhanced catalytic potency, but also superior stability and easier up-scaling compared to natural enzymes, making them particularly advantageous for applications in diagnostic medicine, targeted therapeutics and biosensing [14, 15]. However, the practical application of existing nanozymes is hampered by the complexity of their synthesis, the exorbitant costs associated with their production and suboptimal biocompatibility. In particular, metal-based nanozymes, including those containing Au, Mn, Pd, Cd, Pt and Ag, which are frequently cited in the literature, have raised several health concerns regarding their use in translational medicine [16]. There is an urgent need to discover nanozymes made from innovative, biocompatible materials, especially those derived from abundant, edible resources, which hold great promise for future biomedical applications.\u003c/p\u003e \u003cp\u003eCarbon nanomaterials (CNMs), endowed with unique electronic and morphological properties, have demonstrated the ability to mimic the catalytic activities of natural enzymes. CNMs-derived nanozymes are emerging as viable natural enzyme substitutes in biomedical fields due to their distinctive, unique physicochemical properties [17]. CDs, a class of luminescent nanomaterials, have attracted considerable attention in the last decade due to their unique properties [18, 19]. These CDs exhibit several advantages, including their nanoscale dimensions, facile synthesis, affordability, tunable photoluminescence, robust photostability and enhanced biocompatibility, which outperform luminescent entities such as quantum dots, metallic nanoclusters and rare earth nanoparticles [20, 21]. In addition, the variety of oxygen-rich functional groups present on the surface of CDs, including ketone, carboxylic acid and alcohol moieties, gives them exceptional quantum efficiency and enhanced biological capabilities, effectively mimicking various enzymatic sites of catalytic activity. Emerging evidence highlights the ability of CDs-based nanozymes to mimic the architecture and operating characteristics of native enzymes such as catalase and superoxide dismutase, which can induce oxidative stress and subsequent damage to biological systems.\u003c/p\u003e \u003cp\u003eFerroptosis, defined by its oxidative stress-induced cell death process, is gaining momentum in the scientific community. Mechanistically, ferroptosis is largely controlled by the GSH redox mechanism, which plays a key role in its regulation [22\u0026ndash;24]. GSH depletion leads to GPX4 dysfunction, which ultimately causes ROS levels to rise through lipid peroxidation, triggering ferroptosis [25, 26]. In particular, cancer cells undergoing ferroptosis can trigger an immune response by releasing molecules that indicate cellular stress (DAMPs) and alarmins, which activate the immune system [27\u0026ndash;29]. Therefore, ferroptosis could serve as a potent tactic in cancer therapeutics. Research to date has mainly focused on the catalytic functions of CDs, but there is still a lack of investigation into the engineering of CDs nanozymes to induce ferroptosis and enhance the anti-tumor immune response.\u003c/p\u003e \u003cp\u003eHerein, inspired by ChA CQDs derived from the pyrolysis of roasted coffee beans, we prepared CDs using a straightforward hydrothermal synthesis with sorafenib, a breakthrough drug approved by the FDA in 2007 for the initial systemic treatment of HCC. Through such a facile assembly procedure, the pertinent product can be imbued with a multitude of exceptional attributes: 1) in contrast to sorafenib, whose clinical application is limited by its suboptimal water solubility and limited bioavailability, \u003cem\u003eSF-CDs\u003c/em\u003e are nanoscale that can be easily functionalized for targeted delivery, thereby enhancing their bioavailability and tumor penetration; 2) Sorafenib, due to its broad kinase inhibition, can lead to dose-limiting toxicities. In contrast, \u003cem\u003eSF-CDs\u003c/em\u003e, mimic GSH oxidase, specifically targeting cancer cells with high oxidative stress. This reduces off-target effects and systemic toxicity, while inducing ferroptosis through GSH oxidation and GPX4 inactivation; 3) \u003cem\u003eSF-CDs\u003c/em\u003e exhibit dynamic nucleolus-to-mitochondria subcellular localization, selectively accumulating in cancer cell mitochondria to amplify oxidative stress and trigger ferroptosis, a cell death pathway far less resistant than the apoptosis primarily induced by sorafenib; 4) Compared to sorafenib, \u003cem\u003eSF-CDs\u003c/em\u003e induce immunogenic cell death and release of damage-associated molecular patterns (DAMPs), which activate antitumor immunity and convert immunosuppressive \u0026ldquo;cold\u0026rdquo; tumors into immunoreactive \u0026ldquo;hot\u0026rdquo; ones (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Collectively, our groundbreaking preliminary research has revealed the potential of \u003cem\u003eSF-CDs\u003c/em\u003e nanozymes with nucleolus targeting capacity to serve as potent instigators of tumor ferroptosis, thereby revitalizing the immune microenvironment within malignant tumors and highlighting the significant therapeutic potential of \u003cem\u003eSF-CDs\u003c/em\u003e in the field of translational medicine.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eSF, Dimethylsulfoxide (DMSO), and DTNB (5,5’-dithiobis(2-nitrobenzoic acid)) were all obtained from Aladdin Chemical Co., Ltd. (Shanghai, China). GSH, oxidized glutathione (GSSG), FBS, DCFH-DA, 3,3’,5,5’-Tetramethylbenzidine (TMB), Dil (cell membrane probes), Mito-tracker, Calcein-AM/PI viability assay kit were all purchased from MeiLunBio. The H\u0026amp;E staining kit was supplied by Beyotime Biotech (Nantong, Jiangsu, China). Affinity purified antibodies recognizing NRF2, COX-2, GPX4, and GAPDH were procured from Abcam (Cambridge, UK). The MTT was acquired from Life Technologies GmbH. The carcinoma cell lines HepG2 (RRID:CVCL_0043), 4T1 (RRID:CVCL_0218), HeLa (RRID:CVCL_0030), and H22 (RRID:CVCL_0220), along with the normal liver cell line L02 (RRID:CVCL_0220), were all procured from the Cell Bank of the Chinese Academy of Medical Sciences in Beijing, China. All cell lines were confirmed to be free of contamination. All cell culture components were purchased exclusively from Life Technologies GmbH, with the exception of those specifically mentioned otherwise.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eThe HepG2 cell line was cultured in DMEM medium containing 10% fetal bovine serum, along with a 1% mixture of penicillin and streptomycin. Meanwhile, L02 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. All cell cultures were maintained under strict environmental parameters in an incubation system at 37℃ supplemented with a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere to maintain a stable and regulated environment throughout the experimental period. In addition, female BALB/c rodents aged 5 to 7 weeks were obtained from Fuzhou Wushi Laboratory Animal Supply. The Institutional Animal Care and Use Committee of Fujian Agriculture and Forestry University (PZCASFAFU25100) thoroughly reviewed and approved all animal protocols, ensuring that the research was conducted in accordance with ethical and humane standards.\u003c/p\u003e\n\u003ch3\u003eCell imaging\u003c/h3\u003e\n\u003cp\u003eTo assess the uptake of \u003cem\u003eSF-CDs\u003c/em\u003e, HepG2 cells were plated in 12-well plates at a density of 1.0×10⁵ cells per well. To determine cellular uptake, HepG2 cells were plated on glass slides in 12-well plates at 1.0×10\u003csup\u003e5\u003c/sup\u003e per well. The cells were subsequently treated with 1 mL of DMEM containing 10% FBS and supplemented with \u003cem\u003eSF-CDs\u003c/em\u003e at a dose of 100 µg for intervals of 0, 5, 10, 15, and 30 min. Excess \u003cem\u003eSF-CDs\u003c/em\u003e were removed by rinsing the cells twice with DMEM. Cell uptake was then assessed using a Leica Microsystems (Wetzlar, Germany) SP8 CLSM.\u003c/p\u003e \u003cp\u003eTo assess the efflux of \u003cem\u003eSF-CDs\u003c/em\u003e, HepG2 cells were incubated with \u003cem\u003eSF-CDs\u003c/em\u003e for 30 min. The supernatant was gently aspirated, and the cells were rinsed and then cultured in DMEM supplemented with 10% FBS for further incubation. Cell movements were then examined at 15, 30 and 60 min using a high-resolution SP8 CLSM (Leica Microsystems, Wetzlar, Germany) operating at an excitation wavelength of 365 nm.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy assessment\u003c/h3\u003e\n\u003cp\u003eFor the TEM study, \u003cem\u003eSF-CDs\u003c/em\u003e were suspended in a thin aqueous solution and applied to carbon-covered copper grids by the drop-coating technique. Their morphology and structure were carefully examined using a HT7800 manufactured by Hitachi at 200 kV. The images obtained were processed and evaluated for detailed and advanced analysis.\u003c/p\u003e\n\u003ch3\u003eAtomic force microscopy investigations\u003c/h3\u003e\n\u003cp\u003eAFM was used to determine the thickness of the \u003cem\u003eSF-CDs\u003c/em\u003e, using a Dimension FastScan ATM atomic force microscope (Bruker, Karlsruhe, Germany) to meticulously measure the size characteristics of the samples. A precisely prepared 0.01 mg/mL aqueous solution of \u003cem\u003eSF-CDs\u003c/em\u003e was sonicated for 3 min to achieve complete dispersion. This solution was delicately applied to a mica substrate. The resulting images were meticulously scrutinized.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence spectral analysis\u003c/h2\u003e \u003cp\u003eFor the colorimetric assessment of highly luminescent \u003cem\u003eSF-CDs\u003c/em\u003e, they were carefully prepared to a 10 mg/mL concentration within an optical quartz cell. Spectrofluorometric analysis was performed using a FluoroMax-4 instrument set to an excitation wavelength of 365 nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFourier transform infrared spectroscopy evaluation\u003c/h3\u003e\n\u003cp\u003eTo perform the FT-IR spectroscopy, a precise mixture of 20 mg of KBr was prepared with a 0.5 mg portion of the sample material, thoroughly ground to ensure homogeneity, and then compressed at 10 MPa to form a disc for analysis. The spectral data were collected by scanning the wave numbers from 4000 to 400 cm\u003csup\u003e− 1\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eGSH oxidase-like activity assay\u003c/h3\u003e\n\u003cp\u003eThe evaluation of GSH oxidase-like activity was conducted using DTNB as the substrate, spanning a spectrum of GSH concentrations [30]. Yellow complex formation from DTNB-GSH reactions was monitored spectrophotometrically at 422 nm with readings taken every 5 min using the spectrophotometer. For kinetic assays, experiments used 150 µL of PBS adjusted to contain 100 µg/mL \u003cem\u003eSF-CDs\u003c/em\u003e, using a DTNB concentration of 100 µM and varying GSH concentrations between 0 and 4 mM.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eReactive oxygen species assay\u003c/h2\u003e \u003cp\u003eHepG2 cell cultures were cultured in DMEM to which \u003cem\u003eSF-CDs\u003c/em\u003e were added at concentrations of 25, 50 and 100 µg/mL for 4h. The cells were subsequently incubated with DCFH-DA at a concentration of 0.2 µM for 15 min at 37°C in darkness to promote the conversion of DCFH-DA into its fluorescent derivative, which allows for the measurement of ROS levels [31, 32]. After removal of unbound probe with PBS, cells were examined by CLSM with an emission wavelength set at 531 nm after excitation at 506 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of GSH levels within cells using ellman’s method\u003c/h2\u003e \u003cp\u003eIntracellular GSH levels were determined by the Ellman test, which quantifies the light absorption of a yellow substance resulting from the interaction of DTNB with GSH. HepG2 cell cultures in plate wells were exposed to various concentrations of \u003cem\u003eSF-CDs\u003c/em\u003e (25, 50 and 100 µg/mL) for 12h. Lysed cells were mixed with 50 µL of 10 mM DTNB solution to identify the yellow complex. The levels of GSH were determined at a wavelength of 422 nm using an F50 plate reader to measure optical density.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessing cell membrane integrity\u003c/h2\u003e \u003cp\u003eThe Dil staining protocol was used to assess the integrity of the plasma membrane. HepG2 cells were incubated with \u003cem\u003eSF-CDs\u003c/em\u003e at doses of 25, 50 and 100 µg/mL for 24h. After a PBS wash to remove excess material, the cells were incubated with 5 µM Dil for 15 min at 37℃ in an opaque chamber to promote the intracellular conversion of Dil to its fluorescent derivative. Fluorescence microscopy was then performed using CLSM with an excitation wavelength of 549 nm and an emission of 565 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of mitochondrial structure\u003c/h2\u003e \u003cp\u003eIn the Mito-tracker assay [6], HepG2 cells were exposed to \u003cem\u003eSF-CDs\u003c/em\u003e at a concentration of 100 µg/mL for 24h. Cells were subsequently treated with 200 nM Mito-tracker for half an hour at 37°C in the dark. Fluorescence was examined using a CLSM with an excitation wavelength of 490 nm and an emission at 516 nm.\u003c/p\u003e \u003cp\u003eFor TEM analysis, HepG2 cells were treated with \u003cem\u003eSF-CDs\u003c/em\u003e at a concentration of 100 µg/mL for 24h, followed by fixation in 2% glutaraldehyde at 4℃ for 8h. They were dehydrated through an acetone gradient, followed by infiltration, embedding and polymerization, and ultrathin 0.5 µm sections were prepared. After rinsing with PBS, the samples were examined at 200 kV.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanti-proliferation\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity was assessed by culturing L02 cells, HepG2 cells, HeLa cells and 4T1 cells in 96-well plates to 80% confluence, followed by exposure to \u003cem\u003eSF-CDs\u003c/em\u003e at doses ranging from 0 to 10 µg/mL for 24h. The viability of cells was evaluated with the CCK-8 assay kit, adhering to the method detailed in the referred [33].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eAfter treatment of HepG2 cells with \u003cem\u003eSF-CDs\u003c/em\u003e, protein lysates were isolated. From each sample, 50 µg of total protein was loaded onto a 10% SDS-PAGE gel, resolved, and subsequently transferred to a nitrocellulose membrane for blotting. The blot was probed with specific primary antibodies recognizing pro-caspase-3 (CST, diluted 1:1,500) and PARP-1 (CST, diluted 1:1,500) for 1h at 25℃, subsequently, the samples were incubated with a horseradish peroxidase-conjugated secondary antibody (zsBio, diluted at a ratio of 1:6000) for an additional 60 min.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of lipid peroxidation\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe malondialdehyde (MDA) assay kit was utilized to quantify \u003cem\u003ein vitro\u003c/em\u003e lipid peroxidation (LPO) levels [34]. HepG2 cells were initially plated in 6-well plates and incubated at 37°C overnight. Subsequently, the spent medium was replaced with fresh medium containing various treatments: medium only, erastin and \u003cem\u003eSF-CDs\u003c/em\u003e with diverse concentrations (25, 50, and 100 µg/mL), respectively. These cells were then cultured for an additional 24h. Following this period, the lipid peroxidation levels were assessed using the TBA method provided by the MDA assay kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSubcutaneous tumor model\u003c/h2\u003e \u003cp\u003eA total of 1×10\u003csup\u003e6\u003c/sup\u003e H22 cells in a physiological saline solution were injected beneath the skin on the right side of BALB/c mice. When the tumor volumes grew to around 80 mm³, the mice were randomly divided into three distinct groups.\u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eSF-CDs\u003c/em\u003e treatment groups, mice received intra-tumoral injections of \u003cem\u003eSF-CDs\u003c/em\u003e and administered 10 mg/kg of body weight every 48h over a period of seven treatment cycles. Body weight and tumor volume measurements were recorded at two-day intervals. The calculation of tumor volume was based on the equation: V = 1/2×L×W\u003csup\u003e2\u003c/sup\u003e, where (V) represents volume, (L) is the length, and (W) is the width of the tumor. All animal-related procedures were carried out with the approval of the Institutional Animal Care and Use Committee at Fujian Agriculture and Forestry University (Approval Number: PZCASFAFU25100).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanti-tumor activity of\u003c/b\u003e \u003cb\u003eSF-CDs\u003c/b\u003e \u003cb\u003ein orthotopic H22 tumor xenograft mouse model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFemale BALB/c mice, five weeks old were used to establish H22 orthotopic xenograft models. Briefly, 100\u0026nbsp;million H22 cells were suspended in 50 µL PBS and combined with an equal volume of Matrigel. The cell-Matrigel mixture was injected orthotopically into the left hepatic lobe of the mice under ultrasound guidance, with a total of five intra-tumoral injections. After disinfection with 75% ethanol, the mice were warmed, isolated and monitored until they regained full consciousness.\u003c/p\u003e \u003cp\u003eAfter tumor inoculation, the mice were randomized into three groups of five animals each, following a three-day post-tumor implantation period: the saline control, SF and \u003cem\u003eSF-CDs\u003c/em\u003e groups. They were treated every three days for a total of six doses over a period of 15d. On day 40, all mice were euthanized. Liver and tumor tissues were then harvested and measured. In the mouse survival study, the procedures outlined above were strictly followed, with a cohort of five mice per group, and any mortality were documented upon the experiment’s conclusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eHistological and biosafety analysis\u003c/h2\u003e \u003cp\u003eAfter the intervention, a thorough blood analysis was performed to carefully measure biochemical indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and MDA. Cardiopulmonary, liver, spleen and kidney tissues were then carefully collected, immersed in 4% formaldehyde solution for one day and incorporated into paraffin blocks for subsequent H\u0026amp;E staining and immunohistochemical (IHC) analysis. Major organs were also collected and specimens were preserved in 4% formaldehyde for an extended period of time and then mounted in paraffin for H\u0026amp;E staining and comprehensive IHC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eimmune response\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA suspension of H22 cells was administered intradermally to the left flank of mice to establish a unilateral H22 tumor model. Once the tumors had grown to a volume of about 80 mm\u003csup\u003e3\u003c/sup\u003e, the rodents were subjected to intravenous injections of PBS, SF and \u003cem\u003eSF-CD\u003c/em\u003e (10 mg/kg of SF and an equivalent concentration of \u003cem\u003eSF-CDs\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe results are clearly presented as averages with standard deviation. The statistical threshold for significance is P \u0026lt; 0.05. \u003cem\u003eIn vitro\u003c/em\u003e assays were performed three times, each time independently. Experimental procedures with animal models were initiated after randomization, with a quintet of mice per group for \u003cem\u003ein vivo\u003c/em\u003e studies. Statistical evaluations were facilitated by using Student’s t-test for comparative analyses, with details provided in the figure captions. Box plots range from the 25th to the 75th percentile, including the interquartile range. Statistical Significance is indicated by one, two or three asterisks for P values less than 0.05, 0.01 and 0.001 respectively, while disparities in animal survival were determined by Kaplan-Meier estimation, with the log-rank test determining the P value.\u003c/p\u003e \u003c/div\u003e "},{"header":"Result and discussion","content":"\u003ch2\u003ePreparation and characterization of SF-CDs nanozyme\u003c/h2\u003e\u003cp\u003e \u003cem\u003eSF-CDs\u003c/em\u003e were meticulously synthesized using a well-established protocol as previously described [15]. 27 mg of SF was first combined with 27 mL of ddH\u003csub\u003e2\u003c/sub\u003eO and mixed for a duration of 20 min before being transferred to Teflon-lined autoclaves. These were then heated in an air oven at a constant temperature of 180°C for 6h (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). When the temperature of the mixture reached ambient conditions, the thick solution was spun at 10,000 rpm for 5 min to carefully collect the supernatant. The product was then dialyzed to promote exchange with ultrapure water for 48h. Finally, the purified \u003cem\u003eSF-CDs\u003c/em\u003e were lyophilized for convenient storage at 4°C and prepared for future applications.\u003c/p\u003e\u003cp\u003eInspired by our previous findings, we observed that the SF-derived dialyzed solution emitted a vivid blue fluorescence under UV radiation at 365 nm. TEM images confirmed that the \u003cem\u003eSF-CDs\u003c/em\u003e were evenly distributed and had a mean size of 4.6 ± 0.4 nm, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB. The size distribution, shown in detail in Supplementary \u003cb\u003eFig. S1\u003c/b\u003e, is indicative of synthesized particle genesis throughout the hydrothermal reaction. Their compact size allows for easy passage through the cell membrane via endocytosis and efficient penetration of the mitochondrial membrane’s double lipid bilayer, leading to accumulation within the mitochondrial matrix [35]. After a 6h hydrothermal process at 180°C, the products at concentrations of 25, 50, 100 and 200 µg/mL showed intense blue fluorescence under UV light at 365 nm (\u003cb\u003eFig. S2\u003c/b\u003e). This result confirms the efficient production of \u003cem\u003eSF-CDs\u003c/em\u003e from SF precursors. Given the recognized role of SF as a multi-kinase inhibitor, which has attracted considerable attention for its potential to inhibit tumourigenesis and progression and improve survival rates, our subsequent investigations focused on \u003cem\u003eSF-CDs\u003c/em\u003e [36]. AFM imaging delineated the fine structure of \u003cem\u003eSF-CDs\u003c/em\u003e, confirming their uniform distribution and consistent thickness between 5 and 10 nm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cb\u003eand D\u003c/b\u003e). To thoroughly evaluate the photoluminescence properties of \u003cem\u003eSF-CDs\u003c/em\u003e, we examined the 3D fluorescence emission spectrum under different excitation conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). The luminescence spectrum of \u003cem\u003eSF-CDs\u003c/em\u003e has been found to have specific characteristics and shifted from 360 nm to 500 nm as the excitation wavelength increased from 280 to 430 nm, highlighting the characteristic excitation-dependent photoluminescence (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF). Given their remarkable photoluminescence, we have investigated the fluorescence properties of \u003cem\u003eSF-CDs\u003c/em\u003e. Upon 365 nm UV light, the “FAFU” characters and the emblem of Fujian Agriculture and Forestry University etched with \u003cem\u003eSF-CDs\u003c/em\u003e ink were visibly altered (\u003cb\u003eFig. S3\u003c/b\u003e). A broad diffraction feature centered at approximately 30° was observed in the XRD pattern (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG), which is characteristic of the low-degree graphitization and predominantly amorphous nature of the as-prepared \u003cem\u003eSF-CDs\u003c/em\u003e. This broad reflection arises from the disordered stacking of aromatic carbon domains and is consistent with the typical architecture of carbon dots, which commonly feature heterogeneous oligomeric fragments intertwined with weakly graphitized carbon cores.\u003c/p\u003e\u003cp\u003e \u003cb\u003eStructural characterization of\u003c/b\u003e \u003cb\u003eSF-CDs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further elucidate the intricate structure of \u003cem\u003eSF-CDs\u003c/em\u003e, mass spectrometry (MS) was employed for compositional analysis. A dominant signal centered at approximately 640 Da was observed in the mass spectrum, accompanied by a characteristic fragment at m/z 465. This observation supports the oligomeric nature of the molecular components within \u003cem\u003eSF-CDs\u003c/em\u003e, consistent with the heterogeneous structural features of carbon-based nanodots (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). Furthermore, FT-IR analysis identified absorption bands for -OH (3420 cm\u003csup\u003e− 1\u003c/sup\u003e), C-H (2950 cm\u003csup\u003e− 1\u003c/sup\u003e), CO = NH (1670 cm\u003csup\u003e− 1\u003c/sup\u003e), C-O-C (910 cm\u003csup\u003e− 1\u003c/sup\u003e) and C = N (1590 cm\u003csup\u003e− 1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI), confirming the incorporation of hydroxyl, carboxyl and carbonyl functionalities within the \u003cem\u003eSF-CDs\u003c/em\u003e framework. Zeta potential analysis of \u003cem\u003eSF-CDs\u003c/em\u003e revealed a negatively charged surface with an average potential of -1.21 ± 0.25 mV (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eJ). XPS analysis revealed the presence of carbon and oxygen in the \u003cem\u003eSF-CDs\u003c/em\u003e, with two distinct peaks observed in the spectrum, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eK-N. Specifically, the fine resolution C 1s XPS spectrum was resolved into two distinct peaks: a pronounced peak for the C-O bond at 286 eV and a distinct peak for the C = O bond at 289 eV (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eL). This finding further supports the presence of C = O or C-O-C moieties on the surface of the \u003cem\u003eSF-CDs.\u003c/em\u003e The functional groups on \u003cem\u003eSF-CDs\u003c/em\u003e, which facilitate electrostatic and hydrogen bonding with the positively charged biomolecules within the nucleus or the FG nucleoporins in the nuclear pore complex [37]. Consequently, this interaction enhances the internalization and accumulation of \u003cem\u003eSF-CDs\u003c/em\u003e within the nucleus.\u003c/p\u003e\u003cp\u003e \u003cb\u003eGSH oxidase mimetic properties of\u003c/b\u003e \u003cb\u003eSF-CDs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA number of nanoparticles have recently been shown to have enzyme-like properties [36–40]. We therefore investigated whether \u003cem\u003eSF-CDs\u003c/em\u003e possess the typical GSH oxidase-like activity using the Ellman assay (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). The DTNB assay showed a remarkable ability of \u003cem\u003eSF-CDs\u003c/em\u003e to scavenge GSH. When GSH at concentrations ranging from 0.5 to 4 mM was mixed with \u003cem\u003eSF-CDs\u003c/em\u003e (100 µg/mL) and DTNB (100 µM), an absorbance maximum at 422 nm was detected that correlated with both reaction time and GSH concentration (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). To further rule out the potential influence of reactive oxygen species, we established control groups containing only H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or ·OH. The results indicated that in the absence of \u003cem\u003eSF-CDs\u003c/em\u003e, neither H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e nor ·OH alone significantly oxidized GSH. This observation underscores the detoxifying capacity of \u003cem\u003eSF-CDs\u003c/em\u003e towards GSH. Subsequently, quantitative analysis of the enzyme kinetics suggested that \u003cem\u003eSF-CDs\u003c/em\u003e exhibited a typical Michaelis-Menten kinetics property in the DTNB colorimetric reaction (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Moreover, the K\u003csub\u003eM\u003c/sub\u003e and V\u003csub\u003emax\u003c/sub\u003e value of \u003cem\u003eSF-CDs\u003c/em\u003e were determined to be 4.272 and 2.923 in a double reciprocal plot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). In addition, UV\u003cem\u003e-vis\u003c/em\u003e spectroscopic analysis accurately verified the conversion of GSH to GSSG following interaction with \u003cem\u003eSF-CDs\u003c/em\u003e, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE.\u003c/p\u003e\u003cp\u003eTo quantify oxygen depletion in the \u003cem\u003eSF-CDs\u003c/em\u003e mediated reaction, ABTS was used as an indicator of oxidative stress. The results indicated a marked escalation in the conversion of ABTS to \u003cem\u003eox\u003c/em\u003e-ABTS in the presence of \u003cem\u003eSF-CDs\u003c/em\u003e under atmospheric oxygen, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF. The catalysis reaction was further enhanced in an atmosphere with oxygen levels of approximately 100% and was inhibited in an oxygen free N\u003csub\u003e2\u003c/sub\u003e atmosphere, highlighting the central role of oxygen in \u003cem\u003eSF-CDs\u003c/em\u003e-driven reactions. Besides, the generation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was determined by an Amplex red probe (\u003cb\u003eFig. S11\u003c/b\u003e). As a result, the fluorescence intensity at 520 nm gradually increased with \u003cem\u003eSF-CDs\u003c/em\u003e concentration, confirming that \u003cem\u003eSF-CDs\u003c/em\u003e possessed oxidase-like activity. Adopting the well-recognized four-electron (4e\u003csup\u003e−\u003c/sup\u003e) reduction mechanism, we postulated that \u003cem\u003eSF-CDs\u003c/em\u003e might be able to reduce oxygen to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and subsequently generated .OH radicals to accelerate the oxidative conversion of GSH to GSSG. Meanwhile, electron spin resonance (ESR) analysis also verified the formation of ·OH in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and \u003cem\u003eSF-CDs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). Taken together, these results indicate that \u003cem\u003eSF-CDs\u003c/em\u003e exhibit oxidase-like behavior in the presence of GSH, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH.\u003c/p\u003e\u003ch2\u003eCapacity of SF-CDs for cellular uptake and release\u003c/h2\u003e\u003cp\u003eIn our investigation, we examined the endocytic ability of \u003cem\u003eSF-CDs\u003c/em\u003e in HepG2 cells, assessing uptake efficiency by measuring particle-emitted luminescence. Cellular uptake dynamics indicated rapid internalization of \u003cem\u003eSF-CDs\u003c/em\u003e by HepG2 cells in less than 10 min. The fluorescence intensity persisted for at least 30 min, highlighting the enhanced biocompatibility of \u003cem\u003eSF-CDs\u003c/em\u003e and their efficacy in cellular internalization (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). To investigate the \u003cem\u003ein vitro\u003c/em\u003e cytotoxic capabilities of \u003cem\u003eSF-CDs\u003c/em\u003e, an analysis was conducted using the CCK8 assay method to assess the viability of both normal and carcinoma cells after incubation with \u003cem\u003eSF-CDs\u003c/em\u003e. The cytotoxicity of \u003cem\u003eSF-CDs\u003c/em\u003e over a range of concentrations on the normal hepatocyte line (L02) and the carcinoma cells such as HepG2, HeLa and 4T1 is shown in \u003cb\u003eFig. S4\u003c/b\u003e. From which one can observe that the L02 cells exhibit a survival rate exceeding 80% at \u003cem\u003eSF-CDs\u003c/em\u003e concentrations all the way up to 10 µg/mL. In comparison, HepG2 cell viability dropped to 26.6% at 10 µg/mL of \u003cem\u003eSF-CDs\u003c/em\u003e, while 4T1 and HeLa cells showed 42% and 40.5% viability, respectively, at the same dose. This observation suggests a significant selective cytotoxicity of \u003cem\u003eSF-CD\u003c/em\u003es against malignant cells as opposed to their normal counterparts.\u003c/p\u003e\u003cp\u003e \u003cb\u003eCellular distribution of\u003c/b\u003e \u003cb\u003eSF-CDs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe intracellular trafficking of nanodrugs is a vital factor in influencing their anticancer effects. Utilizing FITC-labeled \u003cem\u003eSF-CDs\u003c/em\u003e (green fluorescence), HepG2 cell lines exposed to \u003cem\u003eSF-CDs\u003c/em\u003e demonstrate colocalization (lime green and white) of nucleus (violet) and mitochondria (blue fluorescence), suggesting that a majority of the intake \u003cem\u003eSF-CDs\u003c/em\u003e is situated in the nucleoli region (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, the cyan colocalization fluorescence of \u003cem\u003eSF-CDs\u003c/em\u003e demonstrated 88% overlap with nucleolus (pink) and only 3% with mitochondria (blue) (\u003cb\u003eFig. S5\u003c/b\u003e). These distribution patterns indicated that \u003cem\u003eSF-CDs\u003c/em\u003e were taken up by the cells and predominantly accumulated in the nucleolar of HepG2 cells. Additionally, Mito-tracker staining revealed that the mitochondria exhibited distorted cristae and a fragmented morphology in response to \u003cem\u003eSF-CDs\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSF-CDs\u003c/b\u003e \u003cb\u003etrigger ferroptosis through GSH consumption dependent ROS generation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next investigated the GSH depleting effect of \u003cem\u003eSF-CDs\u003c/em\u003e on HepG2 cells. As shown in \u003cb\u003eFig. S6\u003c/b\u003e, exposure to \u003cem\u003eSF-CDs\u003c/em\u003e caused a significant decrease in GSH levels, presumably through the oxidase-like conversion of GSH to GSSG. This depletion disrupts the redox balance and impairs the function of GPX4, which is a key enzyme in the mitochondria responsible for preventing lipid peroxidation. Consequently, the accumulation of \u003cem\u003eSF-CDs\u003c/em\u003e within the mitochondria is further enhanced due to their catalytic activity, as they effectively target the nucleolus and disrupt the redox homeostasis. Given the relationship between GSH depletion and increased oxidative stress, we assessed the generation of ROS in cells using the DCFH-DA fluorescence assay. Accordingly, \u003cem\u003eSF-CDs\u003c/em\u003e elicited a strong green fluorescence response in HepG2 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), confirming the generation of hydroxyl radicals (·OH). To further substantiate this finding, the formation of ·OH was investigated using a TMB + H₂O₂ system. As depicted in \u003cb\u003eFig. S7\u003c/b\u003e, there is a significant color change from colorless to blue in the TMB solution upon the addition of both \u003cem\u003eSF-CDs\u003c/em\u003e and H₂O₂. This phenomenon was not observed with TMB alone, nor with the mixtures of TMB + H₂O₂, TMB + \u003cem\u003eSF-CDs\u003c/em\u003e, and H₂O₂+S\u003cem\u003eF-CDs\u003c/em\u003e. This color change was quantitatively measured using UV-visible spectroscopy, showing an increase in absorbance at 652 nm, likely due to the formation of ·OH through a Fenton-like process. We also assessed the expression levels of key ferroptosis markers, namely GPX4, NRF2, SLC7A11 and COX-2, by Western blotting. The \u003cem\u003eSF-CDs\u003c/em\u003e decreased the levels of GPX4, NRF2 and SLC7A11, while increasing the levels of COX-2, with these effects escalating with increasing concentrations of \u003cem\u003eSF-CDs\u003c/em\u003e from 25 to 100 µg/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). This suggests that \u003cem\u003eSF-CDs\u003c/em\u003e may specifically induce cell death through a ferroptosis-mediated pathway.\u003c/p\u003e\u003cp\u003eLPO promotion is another key marker on ferroptosis pathway [38]. To further verify that \u003cem\u003eSF-CDs\u003c/em\u003e could induce ferroptosis, we assessed intracellular LPO levels using an MDA assay kit, as MDA is a reliable indicator of LPO. Our results revealed that the MDA levels in cells treated with erastin and various concentrations of \u003cem\u003eSF-CDs\u003c/em\u003e (25, 50, and 100 µg/mL) were significantly higher than those in the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Additionally, we observed a marked increase in LPO levels as the concentration of \u003cem\u003eSF-CDs\u003c/em\u003e was incrementally increased from 25 to 100 µg/mL, suggesting that \u003cem\u003eSF-CDs\u003c/em\u003e can significantly promote the generation of LPO. Overall, these findings indicate that \u003cem\u003eSF-CDs\u003c/em\u003e are capable of inducing ferroptosis.\u003c/p\u003e\u003cp\u003eGiven the importance of mitochondria in ferroptosis, we further investigated the process by examining mitochondrial morphology. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-D, in contrast to cells treated with culture medium alone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), the ferroptosis-positive control cells exposed to SF (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC) and those treated with \u003cem\u003eSF-CDs\u003c/em\u003e nanozymes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) exhibited varying degrees of mitochondrial damage, changing from an elongated to a punctate pattern, suggesting a compacted mitochondrial membrane and reduced size. In addition, Mito-tracker staining revealed mitochondrial cristae disintegration and mitochondrial fragmentation upon exposure to \u003cem\u003eSF-CDs\u003c/em\u003e. Specifically, in the control group, HepG2 cells display mitochondria that are typically elongated and tubular, featuring well-defined and evenly spaced cristae. Conversely, cells treated with \u003cem\u003eSF-CDs\u003c/em\u003e display a punctate pattern, indicative of mitochondrial membrane compaction and a reduction in size. Moreover, the cristae within these mitochondria appear disrupted, taking on a less organized and more diffuse appearance, suggesting disintegration. Additionally, the overall structure of the mitochondria in \u003cem\u003eSF-CDs\u003c/em\u003e treated cells is fragmented, characterized by smaller, disconnected mitochondrial fragments rather than a continuous network (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, depleted GSH levels were observed to enhance cellular injury by accelerating lipid peroxidation. We therefore used the lipid-soluble fluorescent agent Dil to assess the continuity of the cell membrane. As demonstrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, treatment with \u003cem\u003eSF-CDs\u003c/em\u003e caused a marked reduction in red fluorescence in HepG2 cells, indicating a disrupted cell membrane structure, which increased with increasing concentrations of \u003cem\u003eSF-CDs\u003c/em\u003e. Accordingly, these findings are consistent with the initiation of ferroptosis in HepG2 cells. The LIVE/DEAD cell staining assay confirmed that \u003cem\u003eSF-CDs\u003c/em\u003e induced the most pronounced red fluorescence, signifying the strongest cytotoxic effect on HepG2 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE). Annexin V-FITC and PI staining assays were then performed to clarify the anti-proliferative mechanism of \u003cem\u003eSF-CDs\u003c/em\u003e mediated by enzymatic-like activities and oxidative stress enhancement (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF). Flow cytometry analysis revealed that erastin-induced ferroptosis resulted in an apoptosis rate of 34.82%, whereas \u003cem\u003eSF-CDs\u003c/em\u003e at concentrations of 25, 50 and 100 µg/mL induced apoptosis rates of 6.50%, 36.99% and 45.85%, respectively. Based on these observations, we propose that \u003cem\u003eSF-CDs\u003c/em\u003e in the TME convert the overexpressed GSH to GSSG, and the resulting GSH depletion inactivates GPX4, leading to lipid peroxidation, ROS accumulation, and ultimately ferroptosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG). Taken together, these findings indicate that \u003cem\u003eSF-CDs\u003c/em\u003e induce ferroptosis via a ROS generation pathway dependent on GSH depletion.\u003c/p\u003e\u003cp\u003e \u003cb\u003eTherapeutic tumor-suppressing capabilities of\u003c/b\u003e \u003cb\u003eSF-CDs in vivo\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe then investigated the tumor inhibitory properties of \u003cem\u003eSF-CDs\u003c/em\u003e in a subcutaneous xenograft model using BALB/c mice inoculated with H22 cells. SF, a multi-kinase inhibitor and the standard of care for HCC study, is widely used in cancer treatment and is known to induce ferroptosis, making it a positive control in our experiment [39]. In this study, \u003cem\u003eSF-CDs\u003c/em\u003e were administered intratumorally administered at a dose of 10 mg/kg fortnightly for 14d (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequently, a marked suppression of tumorigenesis was associated with \u003cem\u003eSF-CDs\u003c/em\u003e therapy (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). In addition, the absence of significant weight loss in the mice throughout the treatment period indicated the minimal toxicity of \u003cem\u003eSF-CDs\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). After 14d of treatment, the tumors were excised, their mass measured and photographed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). In contrast to the PBS-treated group, a decrease in tumor mass was observed in all other treatment groups.\u003c/p\u003e\u003cp\u003e \u003cb\u003eBiosafety estimation of\u003c/b\u003e \u003cb\u003eSF-CDs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsidering the potential biological hazards of nanomaterials as they move from research to clinical settings, we evaluated the treatments’ biocompatibility by determining serum AST, ALT, BUN, and MDA levels in the treated mice (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). Serum markers remained stable in all treatment groups, comparable to healthy controls, indicating a lack of apparent acute toxicity. Additionally, we performed an \u003cem\u003ein vivo\u003c/em\u003e analysis of H\u0026amp;E-stained tissue sections. The histological examination via H\u0026amp;E staining indicated subtle renal side effects from SF treatment (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF), with features of glomerulosclerosis and high serum creatinine, corroborating earlier research [31]. In contrast, \u003cem\u003eSF-CDs\u003c/em\u003e and their comparators showed a similar safety profile in all major organs. Taken together, these results underscore the favorable biosafety profile of \u003cem\u003eSF-CDs\u003c/em\u003e, which, combined with their significant anticancer efficacy, positions them as promising candidates for translational medical applications.\u003c/p\u003e\u003cp\u003e \u003cb\u003eSF-CDs\u003c/b\u003e \u003cb\u003eboost immune cell infiltration\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDue to its robust immunogenicity, ferroptosis facilitates the presentation of antigens, we investigated the impact of \u003cem\u003eSF-CDs\u003c/em\u003e on the modulation of anti-tumor immunity. For an in-depth analysis of the modulation of biological responses by \u003cem\u003eSF-CDs\u003c/em\u003e within the tumor immune microenvironment (TIME), this investigation focused on the infiltration of immune cells, particularly T cells, in tumors that are typically considered immunologically “cold”. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cb\u003eH\u003c/b\u003e showed that \u003cem\u003eSF-CDs\u003c/em\u003e significantly increased the presence of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells within the tumor tissue, a phenomenon not reflected by SF alone. Furthermore, \u003cem\u003eSF-CDs\u003c/em\u003e also increased the levels of CD56 and F4-80 in the TME, confirming previous findings [[40]]. Collectively, our results demonstrated that \u003cem\u003eSF-CDs\u003c/em\u003e can transform immunologically “cold” tumors into “hot” ones by promoting the infiltration of T cells, NK cells and macrophages.\u003c/p\u003e\u003cp\u003eFollowing our initial observations, we proceeded to conduct a detailed quantitative analysis of key tumor markers, specifically examining the expression levels of CD4, CD8, CD56, and F4/80 within mouse tumor tissues. For this analysis, we employed the Immunoway Quadruple-Fluorescence immunohistochemical mouse/rabbit kit (pH 9.0) to meticulously apply primary and secondary antibody staining to sections of mouse tumor tissues. The distinct colors assigned to each marker corresponded to specific cellular components: blue for DAPI, green for FITC, yellow for Cy3, red for Cy5, and magenta for SpRed. The CLSM results indicated that the control group exhibited standard baseline expression levels of the immune cell markers. The SF group, however, showed an increase in the intensity of green and magenta fluorescence, suggesting an upregulation of CD4 and F4/80 expression. This upregulation is likely a result of the treatment’s impact on the TME. Most notably, the \u003cem\u003eSF-CDs\u003c/em\u003e treated group demonstrated a significant enhancement in fluorescence intensity across all channels (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). This increase in fluorescence intensity suggests that \u003cem\u003eSF-CDs\u003c/em\u003e have the potential to transform immunologically “cold” tumors, which are generally less responsive to immune-based therapies, into “hot” ones. This transformation is characterized by an increased infiltration of T cells, NK cells, and macrophages, thereby modifying the immune landscape of the tumor and potentially enhancing its responsiveness to immunotherapy.\u003c/p\u003e\u003cp\u003eTo evaluate the effect of \u003cem\u003eSF-CDs\u003c/em\u003e-mediated ICD on immune cell responses, we utilized flow cytometry to analyze the expression profiles of CD4, CD8, CD3, and CD11b among various treatment groups, examining the distribution of these cell populations. The data demonstrated that groups receiving \u003cem\u003eSF-CDs\u003c/em\u003e treatment exhibited markedly enhanced T cell activation rates when compared with other treatment groups. The percentages of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e cells were recorded at 10.4% and 35.4%, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cb\u003eC\u003c/b\u003e). Similarly, both the SF and \u003cem\u003eSF-CDs\u003c/em\u003e treated groups displayed elevated CD11b expression levels, with percentages of 19.5% and 23.2%, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD). Taken together, these findings confirmed that \u003cem\u003eSF-CDs\u003c/em\u003e effectively induce ICD, stimulate the activation of T cells and macrophages, and consequently bolster the efficacy of tumor immunotherapy.\u003c/p\u003e\u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eantitumor evaluation of orthotopic H22 xenograft mouse model\u003c/b\u003e\u003c/p\u003e\u003cp\u003eInspired by the remarkable success of a subcutaneous xenograft tumor model, we proceeded to investigate the therapeutic potential of \u003cem\u003eSF-CDs\u003c/em\u003e within an orthotopic HCC tumor model to obtain a more authentic assessment of their antitumor capabilities, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA, which illustrates the structural design of \u003cem\u003eSF-CDs\u003c/em\u003e and the hypothesized therapeutic mechanism. Capitalizing on the prevalent clinical practice of image-guided intra-tumoral injections for HCC treatment, we integrated this approach into our experimental protocol [41]. The mice were then implanted with orthotopic HCC tumors derived from H22 cells using ultrasound-guided injections (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eB) and the corresponding video is displayed in \u003cb\u003eFig. S8\u003c/b\u003e. After orthotopic tumor implantation, mice were divided into three cohorts to receive intravenous injections of saline, SF or \u003cem\u003eSF-CD\u003c/em\u003e, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC, and after tumor establishment they underwent a sequence of six injections every three days. Notably, the \u003cem\u003eSF-CDs\u003c/em\u003e treatment resulted in the most pronounced inhibition of tumor growth compared to the saline and SF treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eD), with mean tumor weights of 337.0 ± 29.07, 241.4 ± 8.1 and 156.4 ± 21.6 mg for the control, SF and \u003cem\u003eSF-CDs\u003c/em\u003e groups, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE). In addition, the \u003cem\u003eSF-CDs\u003c/em\u003e group had the lowest liver weight, highlighting the improved therapeutic efficacy of this intervention (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eF). At the same time, the survival rate of \u003cem\u003eSF-CDs\u003c/em\u003e-treated tumor-bearing mice increased significantly, reaching almost 80% at day 40 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG). Throughout the biosafety evaluation, minimal variations in body weight were observed in all treatment groups, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eH.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we have successfully fabricated a nucleolus-targeted oxidative stress-amplifying \u003cem\u003eSF-CDs\u003c/em\u003e nanozyme derived from FDA-approved SF and processed via a facile hydrogel treatment. \u003cem\u003eSF-CDs\u003c/em\u003e, which embody the attributes of a multifaceted CNMs, stand out as a paradigmatic anticancer nanozyme, characterized by their excellent ability to mimic GSH oxidase. Importantly, \u003cem\u003eSF-CDs\u003c/em\u003e were shown to significantly suppress tumor growth in both subcutaneous and orthotopic HCC models \u003cem\u003ein vivo\u003c/em\u003e, while maintaining a favorable safety profile with no adverse effects or toxicity. Significantly, our research emphasizes the significant therapeutic potential of nanozymes derived from frontline HCC treatments that effectively induce ferroptosis and stimulate the TIME pathway with minimal toxicity.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eABTS: 2,2\u0026prime;-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); AFM: atomic force microscopy; ALT: alanine aminotransferase; AST: aspartate aminotransferase; BUN: blood urea nitrogen; CCK-8: Cell Counting Kit-8; CDs: carbon dots; CLSM: confocal laser scanning microscopy; CNMs: carbon nanomaterials; DAMPs: damage-associated molecular patterns; DCFH-DA: 2\u0026prime;,7\u0026prime;-dichlorodihydrofluorescein diacetate; DMEM: Dulbecco\u0026rsquo;s modified Eagle medium; DMSO: dimethyl sulfoxide; DTNB: 5,5\u0026prime;-dithiobis(2-nitrobenzoic acid); ESR: electron spin resonance; FBS: fetal bovine serum; FDA: Food and Drug Administration; FITC: fluorescein isothiocyanate; FT-IR: Fourier transform infrared spectroscopy; GPX4: glutathione peroxidase 4; GSH: glutathione; GSH oxidase: glutathione oxidase; GSSG: oxidized glutathione; HCC: hepatocellular carcinoma; H\u0026amp;E: hematoxylin and eosin; IHC: immunohistochemistry; K\u003csub\u003eM\u003c/sub\u003e: Michaelis constant; LPO: lipid peroxidation; MDA: malondialdehyde; MS: mass spectrometry; NRF2: nuclear factor erythroid 2-related factor 2; PARP-1: poly(ADP-ribose) polymerase 1; PBS: phosphate-buffered saline; PI: propidium iodide; ROS: reactive oxygen species; RPMI-1640: Roswell Park Memorial Institute 1640 medium; RRID: Research Resource Identifier; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SF: sorafenib; SF-CDs: sorafenib-derived carbon dots; SLC7A11: solute carrier family 7 member 11; TEM: transmission electron microscopy; TMB: 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine; TME: tumor microenvironment; UV: ultraviolet; UV-vis: ultraviolet-visible; Vmax: maximum reaction velocity; XPS: X-ray photoelectron spectroscopy; XRD: X-ray diffraction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003e All care and handling of animals were performed with the approval of the Institutional Animal Care and Use Committee of Fujian Agriculture and Forestry University (PZCASFAFU26011).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors have consented to submit this article for publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Fujian Provincial Joint Fund for Science and Technology Innovation (2025Y9713), Major Science and Technology Project of Fujian Province (2024NZ029029) and Fujian Provincial Natural Science Foundation of China (2023J011173).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCRediT authorship contribution statementHuixi Guo: Investigation, Data curation, Formal analysis, Visualization, Funding, Writing - original draft.Chunmei Lai: Conceptualization, Methodology, Formal analysis, Funding, Writing - review \u0026amp; editing.Weiji Chen: Investigation, Validation, Resources, Data curation.Shaohua He: Conceptualization, Supervision, Project administration, Funding acquisition, Resources, Funding and Writing - review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM. Naddaf, Science in 2025: the events to watch for in the coming year, Nature. 2025;637(8044):9-11.\u003c/li\u003e\n\u003cli\u003eZ. Liu, L. Lei, Z. Zhang, M. Du, Z. Chen, Ultrasound-responsive engineered bacteria mediated specific controlled expression of catalase and efficient radiotherapy, Mater. Today Bio. 2025;31:101620.\u003c/li\u003e\n\u003cli\u003eL. Gebicka, J. Krych-Madej, The role of catalases in the prevention/promotion of oxidative stress, J. Inorg. Biochem. 2019;197:110699.\u003c/li\u003e\n\u003cli\u003eX. Li, J.F. Lovell, J. Yoon, X. Chen, Clinical development and potential of photothermal and photodynamic therapies for cancer, Nat. Rev. Clin. Oncol. 2020;17(11):657-674.\u003c/li\u003e\n\u003cli\u003eJ. Yu, J. Wu, J. Huang, C. Xu, M. Xu, C.Z.H. Koh, K. Pu, Y. Zhang, Hypoxia-tolerant polymeric photosensitizer prodrug for cancer photo-immunotherapy, Nat. Commun. 2025;16(1):153.\u003c/li\u003e\n\u003cli\u003eN. Gong, X. Ma, X. Ye, Q. Zhou, X. Chen, X. Tan, S. Yao, S. Huo, T. Zhang, S. Chen, X. Teng, X. Hu, J. Yu, Y. Gan, H. Jiang, J. Li, X.J. Liang, Carbon-dot-supported atomically dispersed gold as a mitochondrial oxidative stress amplifier for cancer treatment, Nat. Nanotechnol. 2019;14(4):379-387.\u003c/li\u003e\n\u003cli\u003eM. Wen, J. Ouyang, C. Wei, H. Li, W. Chen, Y.N. Liu, Artificial enzyme catalyzed cascade reactions: antitumor immunotherapy reinforced by NIR-II light, Angew. Chem., Int. Ed. Engl. 2019;58(48):17425-17432.\u003c/li\u003e\n\u003cli\u003eH. Zhang, T. Yang, W. Mu, X. Peng, T. Liu, L. Weng, H. Wang, Y. Zhang, X. Chen, Effective amplification of oxidative stress and calcium manipulation mediated mitochondrial dysfunction based on engineered nanozyme for primary and metastatic breast cancer therapy, Small (Weinheim an der Bergstrasse, Germany). 2025;21(14):e2411299.\u003c/li\u003e\n\u003cli\u003eB. Niu, K. Liao, Y. Zhou, T. Wen, G. Quan, X. Pan, C. Wu, Application of glutathione depletion in cancer therapy: Enhanced ROS-based therapy, ferroptosis, and chemotherapy, Biomaterials 2021;277:121110.\u003c/li\u003e\n\u003cli\u003eQ. Xin, F. Tian, H. Qiao, G. Li, Y. Wang, L. Li, L. Zhang, N. Song, K. Chen, Y. Yan, S. Li, H. Ma, H. Liu, H. Wang, J. Yang, S. Sun, Y. Li, X.-D. Zhang, Machine learning assisted engineering of atomically precise cluster-derived artificial hydrolases for lipid metabolism diseases, Mater. Today. 2026;95:103294.\u003c/li\u003e\n\u003cli\u003eQ. Huang, Q. Wu, L. Feng, F. Lei, Y. Zhang, F. Wang, Recent advances in crystalline porous framework-derived Nanozymes for emerging chemical contaminants remediation, Coord. Chem. Rev. 2026;559:217856.\u003c/li\u003e\n\u003cli\u003eM.R. Chiang, C.W. Hsu, W.C. Pan, N.T. Tran, Y.S. Lee, W.H. Chiang, Y.C. Liu, Y.W. Chen, S.H. Chiou, S.H. Hu, Reprogramming dysfunctional dendritic cells by a versatile catalytic dual oxide antigen-captured nanosponge for remotely enhancing lung metastasis immunotherapy, ACS nano. 2025;19(2):2117-2135.\u003c/li\u003e\n\u003cli\u003eJ. Wu, X. Wang, Q. Wang, Z. Lou, S. Li, Y. Zhu, L. Qin, H. Wei, Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes (II), Chem. Soc. Rev. 2019;48(4):1004-1076.\u003c/li\u003e\n\u003cli\u003eY. Wang, M. Mu, R. Fan, H. Chen, B. Han, G. Guo, Single-atom enzymes: from nano-enzyme evolution to the biomedical frontier of precision catalysis, Coord. Chem. Rev. 2026;549:217317.\u003c/li\u003e\n\u003cli\u003eC.-M. Lai, J. Xu, B.-C. Zhang, S.-H. He, J.-W. Shao, A natural product-derived nanozyme regulator induced chemo-ferroptosis dual therapy in remodeling of the tumor immune microenvironment of hepatocellular carcinoma, Chem. Eng. J. 2024;482:148976.\u003c/li\u003e\n\u003cli\u003eQ. Liu, A. Zhang, R. Wang, Q. Zhang, D. Cui, A review on metal- and metal oxide-based nanozymes: properties, mechanisms, and applications, Nano-Micro Lett. 2021;13(1):154.\u003c/li\u003e\n\u003cli\u003eW. Gao, J. He, L. Chen, X. Meng, Y. Ma, L. Cheng, K. Tu, X. Gao, C. Liu, M. Zhang, K. Fan, D.W. Pang, X. Yan, Deciphering the catalytic mechanism of superoxide dismutase activity of carbon dot nanozyme, Nat. Commun. 2023;14(1):160.\u003c/li\u003e\n\u003cli\u003eL. Yang, M. Li, Y. Peng, Y. Zhou, J. Zou, H. Liu, W. Zhang, J. Liu, P. He, F. Dai, J. Zheng, J. Lin, S. Qu, Z. Lu, G. Liu, Engineering NIR-II carbon dots through aniline extension with graphene and nitrogen enrichment for hepatobiliary theranostics, Nat. Commun. 2026.\u003c/li\u003e\n\u003cli\u003eJ. Wang, Y. Fu, Z. Gu, H. Pan, P. Zhou, Q. Gan, Y. Yuan, C. Liu, Multifunctional carbon dots for biomedical applications: diagnosis, therapy, and theranostic, Small. 2024;20(3):2303773.\u003c/li\u003e\n\u003cli\u003eE.S. Grant, J.F. Olorunyomi, S.C. Scholten, I.O. Robertson, A.N. Abraham, N.H. Srikantamurthy, B.J. Murdoch, E.L.H. Maye, B. del Rosal, A.J. Healey, C.M. Doherty, P. Reineck, X. Mulet, J.-P. Tetienne, D.A. Broadway, Spin-dependent photoluminescence in carbon-based quantum dots, Adv. Mater. 2026;38(17):e18572.\u003c/li\u003e\n\u003cli\u003eY. Liu, Z. Cai, X. Sun, H. Zhang, Y. Hao, J. Wu, Z. Yan, X. Wu, Y. Liang, L. Wang, S. Qu, Establishing a model precursor system: over a decade of research on carbon dots from the citric acid-urea system, Adv. Funct. Mater. 2026;n/a(n/a):e32189.\u003c/li\u003e\n\u003cli\u003eJ. Wang, D. Guo, S. Jiang, W. Wu, X. Gao, Targeting ferroptosis in cancer: from mechanistic insights to therapeutic approaches, Mol. Biomed. 2026;7(1):20.\u003c/li\u003e\n\u003cli\u003eX. Chen, Decoding GPX4 regulation in ferroptosis: mechanisms and therapeutic implications, Trends Mol. Med. 2026.\u003c/li\u003e\n\u003cli\u003eQ. Chen, F. Liu, Y. Zhang, L. Yan, Y. Wu, D. Xu, P. Wu, H. Yuan, K. Jiang, Ferroptosis in cancer toward molecular insights and clinical translation in pancreatic cancer, Mol. Cancer. 2026;25(1):48.\u003c/li\u003e\n\u003cli\u003eX. Fang, H. Ardehali, J. Min, F. Wang, The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease, Nat. Rev. Cardiol. 2023;20(1):7-23.\u003c/li\u003e\n\u003cli\u003eZ. Wang, Z. Wang, D. Li, C. Yang, Q. Zhang, M. Chen, H. Gao, L. Wei, High-quality semiconductor fibres via mechanical design, Nature. 2024;626(7997):72-78.\u003c/li\u003e\n\u003cli\u003eW. Wang, M. Green, J.E. Choi, M. Gij\u0026oacute;n, P.D. Kennedy, J.K. Johnson, P. Liao, X. Lang, I. Kryczek, A. Sell, H. Xia, J. Zhou, G. Li, J. Li, W. Li, S. Wei, L. Vatan, H. Zhang, W. Szeliga, W. Gu, R. Liu, T.S. Lawrence, C. Lamb, Y. Tanno, M. Cieslik, E. Stone, G. Georgiou, T.A. Chan, A. Chinnaiyan, W. Zou, CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy, Nature. 2019;569(7755):270-274.\u003c/li\u003e\n\u003cli\u003eX. Chen, R. Kang, G. Kroemer, D. Tang, Broadening horizons: the role of ferroptosis in cancer, Nat. Rev. Clin. Oncol. 2021;18(5):280-296.\u003c/li\u003e\n\u003cli\u003eC.M. Lai, W.J. Chen, Y. Qin, D. Xu, Y.K. Lai, S.H. He, Innovative hydrogel design: tailoring immunomodulation for optimal chronic wound recovery, Adv. Sci. (Weinh) 2025;12(2):e2412360.\u003c/li\u003e\n\u003cli\u003eI. Rahman, A. Kode, S.K. Biswas, Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method, Nat. Protoc. 2006;1(6):3159-3165.\u003c/li\u003e\n\u003cli\u003eM.P. Murphy, H. Bayir, V. Belousov, C.J. Chang, K.J.A. Davies, M.J. Davies, T.P. Dick, T. Finkel, H.J. Forman, Y. Janssen-Heininger, D. Gems, V.E. Kagan, B. Kalyanaraman, N.G. Larsson, G.L. Milne, T. Nystr\u0026ouml;m, H.E. Poulsen, R. Radi, H. Van Remmen, P.T. Schumacker, P.J. Thornalley, S. Toyokuni, C.C. Winterbourn, H. Yin, B. Halliwell, Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo, Nat. Metab. 2022;4(6):651-662.\u003c/li\u003e\n\u003cli\u003eX. Yao, W. Hu, Y. Li, J. Li, F. Kang, J. Zhang, S. Dong, Dual dynamic crosslinked hydrogel patch embodied with anti-bacterial and macrophage regulatory properties for synergistic prevention of peritendinous adhesion, Adv. Funct. Mater. 2024;34(34):2400660.\u003c/li\u003e\n\u003cli\u003eL. Ouyang, J. Wang, B. Liu, M. Xie, L. Wang, C. Fan, J. Chao, Parameter pool-assisted centrifugation sorter for multiscale higher-order DNA nanomaterials, ACS nano. 2025;19(3):3830-3838.\u003c/li\u003e\n\u003cli\u003eY. Xiong, Z. Yong, S. Li, Q. Wang, X. Chen, Z. Zhang, Q. Zhao, Q. Deng, X. Yang, Z. Li, Self-Reliant nanomedicine with long-lasting glutathione depletion ability disrupts adaptive redox homeostasis and suppresses cancer stem cells, Adv. Funct. Mater. 2024;34(8):2310158.\u003c/li\u003e\n\u003cli\u003eW. He, H. Gao, W. Wu, Nanomedicine biointeractions during body trafficking, Adv. Drug Delivery Rev. 2024;209:115324.\u003c/li\u003e\n\u003cli\u003eJ.M. Llovet, R. Pinyol, R.K. Kelley, A. El-Khoueiry, H.L. Reeves, X.W. Wang, G.J. Gores, A. Villanueva, Molecular pathogenesis and systemic therapies for hepatocellular carcinoma, Nat. Cancer 2022;3(4):386-401.\u003c/li\u003e\n\u003cli\u003eN. Blal, G. Bardi, P.P. Pompa, D. Guarnieri, Nano-biointeractions of functional nanomaterials: the emerging role of inter-organelle contact sites, Targeting, and Signaling, Adv. Funct. Mater. 2024;34(52):2408436.\u003c/li\u003e\n\u003cli\u003eQ. Zhou, Y. Meng, D. Li, L. Yao, J. Le, Y. Liu, Y. Sun, F. Zeng, X. Chen, G. Deng, Ferroptosis in cancer: From molecular mechanisms to therapeutic strategies, Signal Transduction Targeted Ther. 2024;9(1):55.\u003c/li\u003e\n\u003cli\u003eX. Li, B. Zhang, C. Jiang, J. Zhang, X. Ning, Z. An, X. Chen, Y. Chen, P. Chen, Long cycle lifespan of flexible rechargeable zinc-air batteries based on porous sodium hyaluronate/polyacrylamide-based hydrogel electrolyte, J. Power Sources. 2025.\u003c/li\u003e\n\u003cli\u003eX. Li, B. Liu, J. Wang, S. Li, X. Zhen, J. Zhi, J. Zou, B. Li, Z. Shen, X. Zhang, S. Zhang, C.-W. Nan, High-temperature capacitive energy storage in polymer nanocomposites through nanoconfinement, Nat. Commun. 2024;15(1):6655.\u003c/li\u003e\n\u003cli\u003eA. Som, J.G. Rosenboom, A. Chandler, R.A. Sheth, E. Wehrenberg-Klee, Image-guided intratumoral immunotherapy: Developing a clinically practical technology, Adv. Drug Delivery Rev. 2022;189:114505.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sorafenib, Carbon dots, Nanozyme, Ferroptosis, Immune activation","lastPublishedDoi":"10.21203/rs.3.rs-9469655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9469655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eCarbon dots (CDs) are promising nanozymes with natural enzyme-like activities, yet their clinical translation for hepatocellular carcinoma (HCC) is limited by biosafety and efficacy concerns. Developing low-toxicity, FDA-precursor-derived CD nanozymes is thus an attractive strategy for advancing cancer therapy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Herein, we rationally designed a sorafenib-derived carbon dot (\u003cem\u003eSF-CDs\u003c/em\u003e) nanozyme, leveraging the FDA-approved HCC drug sorafenib as the precursor. \u003cem\u003eSF-CDs\u003c/em\u003e were fabricated and characterized to exhibit dynamic nucleolus-targeted activity, with the goal of amplifying oxidative stress, inducing ferroptosis, and remodeling the immunosuppressive tumor microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e \u003cem\u003eSF-CDs\u003c/em\u003e exerted potent glutathione oxidase-like activity, disrupting the GPX4-mediated lipid peroxidation repair pathway to trigger robust cancer cell ferroptosis. \u003cem\u003eIn vivo\u003c/em\u003e, image-guided interventional injection of \u003cem\u003eSF-CDs\u003c/em\u003e significantly suppressed tumor growth in both subcutaneous and orthotopic H22 mouse models, with no observed systemic toxicity. Moreover, \u003cem\u003eSF-CDs \u003c/em\u003emarkedly enhanced the infiltration of immune effector cells, converting “cold” HCC tumors into immunogenic “hot” tumors and eliciting systemic antitumor immunity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e This study establishes \u003cem\u003eSF-CDs \u003c/em\u003eas a promising nucleolus-targeted nanozyme platform for HCC theranostics, combining ferroptosis induction and tumor immune remodeling. The work provides a biosafe and effective strategy for advancing CD-based nanozyme therapy, addressing critical unmet needs in HCC treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSF\u003c/em\u003e-CDs function as a drug-derived CDs nanozyme that induces ferroptosis through glutathione depletion and oxidative stress amplification while concurrently reshaping the tumor immune microenvironment. These findings support the potential of \u003cem\u003eSF\u003c/em\u003e-CDs as a theranostic platform for HCC.\u003c/p\u003e","manuscriptTitle":"Sorafenib-based carbon dot nanozyme with nucleolus-targeted oxidative stress amplification for tumor immune microenvironment remodeling and cancer theranostics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 09:41:39","doi":"10.21203/rs.3.rs-9469655/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"adabd643-29bf-4f38-a39c-181d68d306fe","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-08T06:09:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T15:34:01+00:00","index":24,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T06:25:42+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 09:41:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9469655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9469655","identity":"rs-9469655","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}