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. 2025 Feb 25;31:101608. doi: 10.1016/j.mtbio.2025.101608

Nutrient transporter-oriented nanoinhibitor counteracts intracellular metabolic reprogramming for RT-resistant HCC treatment

Yuehua Wang a,b,1, Zhenjie Wang c,1, Mengnan Liu a, Chaojie Chen a, Qiye Xi d, Jingwen Tang a,b, Zhiqiang Yu e, Shengtao Wang f,g,⁎, Ling Yu h,⁎⁎, Meng Yu d,⁎⁎⁎
PMCID: PMC11914751  PMID: 40104639

Abstract

Radiotherapy (RT) is the primary treatment modality for hepatocellular carcinoma (HCC). Inevitably, the X-ray exposure also increases the metabolic stress and energy demands in surviving tumor cells, which leads to metabolic reprogramming that reduces the sensitivity of HCC to clinical treatments including RT. Nevertheless, the current research in tumor metabolic therapy predominantly focuses on inhibiting glycolytic pathways, and the consequent metabolic compensation behavior of tumor cells exacerbates the risks of drug resistance and recurrence. To address this challenge, we innovatively proposed a tumor-specific multi-metabolic pathway regulation strategy navigated by tumor cell surface nutrient transporter (2-DG/BP MRs), which can be triggered by X-ray radiation to achieve dual blockade of glycolysis and glutamine metabolism pathways. Thus, this nanosystem reconfigured metabolic pathways within tumor cells to counteract RT-induced metabolic reprogramming through dual metabolic inhibition (glycolysis and glutamine metabolism). This approach disrupted the essential energy supply required for cancer cell proliferation without causing metabolic disorders in normal cells, thereby sensitizing HCC to RT. This tumor cell-specific metabolic intervention strategy provides a safe and effective approach for combination therapy in clinically RT-resistant tumors.

Keywords: ROS, Tumor therapy, Responsive drug release, Nanomedicine, Tumor microenvironment

Graphical abstract

Scheme 1. Schematic illustration of 2-DG/BP MRs construction and the mechanism of radio-metabolism regulation for HCC treatment.In this research, for the first time, we have developed a metabolic regulator capable of simultaneously sensitizing RT and blocking the metabolic reprogramming of HCC (2-DG/BP MRs), based on Se-containing polymer materials, loaded with the glycolysis inhibitor 2-DG and the glutaminase inhibitor BP, to construct self-assembled nanomedicine and release drugs in response to RT. As illustrated in Schematic 1, the glycolysis inhibitor 2-DG on the surface of 2-DG/BP MRs specifically recognizes and competitively binds to the glucose transporter GLUT1 on HCC cells, significantly enhancing the efficiency of nanodrug delivery. Furthermore, upon exposure to X-ray irradiation, the Se-containing nanoparticles sensitize radiotherapy by amplifying the ROS killing effect. Subsequently, the breaking of Se-Se bonds in response to ROS leads to the disintegration of nanoparticles and drug release. The released 2-DG and BP synergistically intervened in glucose and glutamine metabolism pathways within tumor cells through a combination therapy strategy. This treatment strategy was able to sensitize RT meanwhile blocking the energy supply of tumor cells through multi-pathway metabolic regulation, offering a new perspective for improving the treatment of RT-resistant HCC.

Image 1

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Highlights

  • •

    2-DG/BP MRs is a targeted delivery of metabolic regulation against radiotherapy (RT)-induced HCC resistance.

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    X-ray exposure triggeted ROS-responsive disintegration, releasing metabolic inhibitors.

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    2-DG/BP MRs sensitized RT and starved tumor cells by blocking metabolic pathways.

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    Nanomicelles reconfigured tumor metabolic pathways, countering RT-induced reprogramming via dual inhibition.

1. Introduction

Hepatocellular carcinoma (HCC) is one of the most well-known malignancies and the leading cause of direct cancer-related death worldwide [1,2]. The close correlation between incidence and mortality (830,000 deaths per year) underscores the bleak prognosis associated with this disease [3]. Radiotherapy (RT) is currently one of the standard therapies for treating HCC patients, which kills cancer cells by directly causing DNA double-strand breaks (DSBs) or indirectly generating cytotoxic reactive oxygen species (ROS) [4,5]. In general, liver tumors and tumors arising from bone marrow and lymphatic tissue are extremely sensitive to radiation therapy [6]. Nevertheless, liver, as the body's largest metabolic organ, is involved in almost all central metabolic pathways, and thus liver tumorigenesis or progression absolutely leads to metabolic reprogramming process [7,8]. Many researchers suggested that drug-resistant liver cancer cells exhibit strong performance in many metabolic pathways while providing the necessary energy for cell activity and drug resistance, such as a marked increase in glycolysis, which is typically associated with reduced apoptosis [9,10], resulting in the failure of multiple traditional antitumor strategies, including RT, in the HCC treatment. Meanwhile, RT directly affects the metabolic pathways of tumor cells, increasing metabolic stress and energy requirement, while exacerbating metabolic imbalance through the increasing of oxidative stress [11]. In turn, the metabolic imbalance in tumor cells further diminishes their sensitivity to RT, contributing to RT resistance [12]. Therefore, fully considering the variations and roles of tumor metabolic behavior during the RT process is crucial for RT-resistant tumor treatment.

The characteristic metabolic features of tumor cells include vigorous aerobic glycolysis and inhibited oxidative phosphorylation, which rapidly supply the energy required for their rapid proliferation [13]. However, pre-clinical and clinical studies only showed limited effectiveness of monotherapy by blocking tumor glycolysis [14,15]. This is due to the activation of metabolic compensatory mechanisms within tumors further complicate the multiple energy supply pathways [16]. Consequently, regulating merely one metabolic pathway, such as glycolysis or glutamine metabolism, is insufficient to achieve significant tumor suppression effects [17]. For instance, inhibition of glucose utilization in cancer cells might lead to glutamine becoming an alternative energy and bio-supply [18]. Once the glycolytic pathway is blocked, cancer cells may switch to rely heavily on glutamine for survival or proliferation [19]. Thus, glutamine metabolism has also received considerable attention as a metabolic regulation target [20]. A potent and selective Glutaminase GLS1, BPTES (BP), has been employed to suppress tumor growth by inhibiting the uptake of glutamine by cancer cells [21]. Studies have shown that tumor cells exhibited significant metabolic flexibility and could adapt to nutrient deprivation by activating alternative pathways such as fatty acid oxidation, amino acid catabolism, mitochondrial respiration, and even utilizing alternative carbon sources present in the tumor microenvironment [22]. This metabolic flexibility was a hallmark of cancer and helped tumor cells resist therapeutic interventions that targeted specific metabolic processes. Therefore, considering that tumor cells can continuously obtain energy for proliferation through engaging metabolic compensatory mechanisms, development of combined metabolic therapy is crucial to combat RT-induced metabolic reprogramming in HCC.

As RT-induced metabolic reprogramming of tumor cells, which in turn significantly reduces the sensitivity of tumor cells to RT and leads to resistance, it is imperative that we continuously explore therapeutic strategies that can concurrently enhance RT sensitivity and regulate metabolism during the RT process. Selenium-based materials have been paid more attention because of their advantages in amplifying the ROS killing effect and constructing ROS-responsive disintegrable nanocarriers [23]. As an indispensable trace element, selenium plays an essential role in cellular antioxidant defense against ROS and amplifies the cytotoxic effects of RT by blocking DNA repair mechanisms [24]. It has been used to develop effective Se-engineered radiosensitizers for cancer RT and immunotherapy [25]. Further studies have demonstrated the applications of Se-containing nanoparticles in multimodal imaging, as well as in photothermal therapy [26,27]. Notably, due to the ROS-responsive cleavage advantage of Se-Se bonds, Se-based polymers have been utilized to develop nanocarriers that release drugs in response to ROS generated during RT, which are further used for the development of anti-tumor strategies combined with RT [28]. Therefore, using Se-containing nanocarriers to deliver metabolic regulatory drugs for combined treatment with RT, not only leverages the radio-sensitizing effect of Se-materials for tumor killing, but also facilitates controlled release of drugs under ROS to block the metabolic reprogramming of HCC. This approach has a unique advantage in combating drug-resistant tumors related to RT-induced metabolic abnormalities.

In this research, for the first time, we have developed a metabolic regulator capable of simultaneously sensitizing RT and blocking the metabolic reprogramming of HCC (2-DG/BP MRs), based on Se-containing polymer materials, loaded with the glycolysis inhibitor 2-DG and the glutaminase inhibitor BP, to construct self-assembled nanomedicine and release drugs in response to RT. As illustrated in Scheme 1), the glycolysis inhibitor 2-DG on the surface of 2-DG/BP MRs specifically recognizes and competitively binds to the glucose transporter GLUT1 on HCC cells, significantly enhancing the efficiency of nanodrug delivery. Furthermore, upon exposure to X-ray irradiation, the Se-containing nanoparticles sensitize radiotherapy by amplifying the ROS killing effect. Subsequently, the breaking of Se-Se bonds in response to ROS leads to the disintegration of nanoparticles and drug release. The released 2-DG and BP synergistically intervened in glucose and glutamine metabolism pathways within tumor cells through a combination therapy strategy. This treatment strategy was able to sensitize RT meanwhile blocking the energy supply of tumor cells through multi-pathway metabolic regulation, providing new suggestions for improving the treatment of RT-resistant HCC.

Scheme 1.

Scheme 1

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Schematic illustration of 2-DG/BP MRs construction and the mechanism of radio-metabolism regulation for HCC treatment.

2. Methods and materials

2.1. Materials

DSPE-PEG2000 and cholesterol were supplied by AVT (Shanghai) Pharmaceutical Tech Co., Ltd. The MTT and Annexin V-FITC/PI apoptosis detection kits were obtained from Solarbio Life Sciences. 2-Deoxy-D-glucose (2-DG) and BPTES inhibitors were supplied by MedChemExpress (MCE). Anti-GLS1 and Anti-mTOR antibodies obtained from Elabscience Biotechnology Co., Ltd. Anti-GAPDH antibody was supplied by Biosynthesis Biotechnology Inc. (Beijing, China). Meanwhile, TNF-α ELISA Kit was supplied by ELK Biotechnology. IFN-γ and IL-6R ELISA Kit (Mouse) were provided by Sino Biological Inc. Cyanine5.5 was supplied by Nanjing Goyoo Biotech Co., Ltd. Cell culture-related consumables were provided from NEST Biotechnology.

2.2. Cell lines and animals

Human HCC line cells HepG2 and mouse HCC cells line H22 were supplied by Chinese Academy of Medical Sciences. The female BALB/c mice aged 4−6 weeks were provided by Guangdong Medical Laboratory Animal Center, China. Animal specimens were obtained according to the “Research Protocol Approval” (LAEC-2020-093) approved by the “Guidelines for Clinical Research and Animal Testing” (ICE) of the Animal Experimental Center of Southern Medical University.

2.3. Characterization of DSPE-Se-Se-PEG2000-2-DG

At room temperature, succinic anhydride (1.8 g, 13.4 mmol) was dissolved in anhydrous THF (40 mL) and DSPE (10.0 g, 13.3 mmol) was added. Stir the mixture at room temperature for 2 h, reducing pressure to remove solvent. Then recrystallized and filtered in anhydrous ethanol (30 mL), the product 4-(2-(((2, 3-bis (stearacyloxy) propoxy) (hydroxyl) phosphoryl) oxy) ethyl amino) -4-oxybutyric acid 8.7 g (79 % yield) was obtained. Next, the above product (8.7 g, 10.3 mmol) was dissolved in anhydrous DMF (50 mL) and followed by HATU (5.8 g, 15.4 mmol) and DIEA (3.6 mL, 20.6 mmol). Selenocysteamine (5.0 g, 20.6 mmol) was added to the reaction solution when the reaction mixture was stirred for 10 min. It was then recrystallized and filtered in MTBE (30 mL) to obtain 3-((2-(4-((2-((2-aminoethyl) didecyl) ethyl) amino) -4-oxy-butanamide) ethoxy) (hydroxyl) phosphoryl) oxy) propane-1, 2-distearate diester 5.3 g (48 % yield). Then, the dicarboxylate polyethylene glycol (10 g, 5 mmol) was dissolved in anhydrous DMF (25 mL), followed by HATU (2.9 g, 7.6 mmol) and DIEA (1.8 mL, 10.3 mmol). The carboxylic acid derivative was then recrystallized in MTBE (30 mL) and filtered to 6.8 g (45 % yield). In the end, DCC (679 mg, 3.3 mmol) was successively added to dichloromethane (20 mL) of carboxylic acid derivatives (6.8 g, 2.2 mmol) and d-2-deoxyglucose (360 mg, 2.2 mmol). Subsequently, DMAP (536 mg, 4.4 mmol) was immediately added to the above reaction system. After the reaction was complete, the solid was filtered out and washed with ether to obtain a final product of 2.6g (38 % yield). The structure of DSPE-Se-Se-PEG2000-2-DG was verified by 1 H NMR and UV–Vis.

2.4. Preparation and characterization of 2-DG/BP MRs

1.0 mg DSPE-Se-Se-PEG2000-2-DG, 0.5 mg BPTES and 4.0 mg DSPE-PEG2000 were simultaneously dissolved in 200 μL dimethyl sulfoxide (DMSO), then slowly dropped into 2 mL water with rapid stirring. DMSO was volatilized by continuous stirring in 37 °C water baths. In this way, the nanoparticle 2-DG/BP MRs was obtained. Blank MRs and 2-DG MRs were prepared by the same method. The morphology of nanomedicines was investigated by transmission electron microscopy (TEM). The size and zeta potential of the nanomedicals were detected using dynamic light scattering (DLS) technique via the Zetasizer Nano ZS90.

2.5. In vitro release of BP and 2-DG from 2-DG/BP MRs

Load 1 mL 2-DG/BP MRs solution into a dialysis bags (MWCO = 3500), then soak dialysis bags in 30 mL PBS (normal physiological (pH 7.4) or under RT (100 μM H2O2) conditions) with continuously shaking at 37 °C for 80 rpm. HPLC and UV–vis were used to verify contents of BP and 2-DG released. Calculate the relative percentage of released BP and 2-DG as a mathematical function of time.

2.6. Cells viability assay

HepG2 cells (5 × 104/well) were divided into 96-well plates and cultured overnight. Furthermore, 100 μL of DMEM containing various concentrations of different drugs (Blank MRs + RT, 2-DG MRs + RT, Free BP + RT, 2-DG/BP MRs + RT) were added and cultured for 24 h or 48 h. After the time had passed, 20 μL MTT (5 mg/mL) was added to each well for 4 h. After the medium was discarded, 150 μL DMSO was added, and the absorbance was measured at 570 nm, then cells viability was calculated.

2.7. Cellular absorption

HepG2 cells were inoculated containing cell slides with 5 × 105 cells/well and cultured for 24 h. Then, the used DMEM medium was then replaced with fresh medium containing Non-sensitive-2-DG MRs/BP + RT (100 μg/mL, composed of DSPE-PEG2000 and DSPE-PEG-COOH) and 2-DG MRs/BP + RT (100 μg/mL, composed of DSPE-Se-Se-PEG-2-DG). Non-sensitive-2-DG MRs/BP and 2-DG MRs/BP with Cy5.5 (250 μg/mL) were prepared by precipitation method. After incubation for 6 h, it was washed 3 times with PBS and fixed with 4 % paraformaldehyde for 15 min. DAPI staining of nuclei for 10 min. Intracellular uptake images were visualized with confocal laser scanning microscopy (CLSM). Meanwhile, flow cytometry (BD) can also detect intracellular uptake of nanomedicines in HCC cells.

2.8. Intracellular ROS detection

The cells were inoculated in confocal dishes (5 × 105 cells/well) and incubated for 24 h. The cells were cultured with Non-sensitive-2-DG/BP MRs + RT and 2-DG/BP MRs + RT containing Cy5.5 (25 μg/mL) for 4 h. Untreated cells served as controls. After washing with PBS for 3 times, FITC-labeled DCFH-DA (10 μM) culture medium was added and cultured for 20 min. Further, cell uptake was examined with CLSM and flow cytometer.

2.9. Western blot method

The cells were inoculated at a density of 5 × 105 cells/well, and incubated with Blank MRs + RT, 2-DG MRs + RT, Free BP + RT, 2-DG/BP MRs + RT solution for 24 h. After incubation, the cells were digested with ice cold RIPA buffer for 20 min, collected and centrifuged (12,000 rpm, 4 °C, 20 min) for the subsequent protein extraction. Furthermore, the same amounts of protein samples and loading buffer were incubated at 95 °C for 10 min, added into electrophoresis and electro-transferred to PVDF membranes. The membranes were enclosed in TBST (TBS/0.1 % Tween 20) containing 5 % (w/v) nonfat milk at room temperature for 2 h. The PVDF membranes were then incubated at 4 °C overnight with anti-GLS1 (1:1000), anti-mTOR (1:1000) and anti-GAPDH antibodies (1:1000), followed by incubation at room temperature with secondary antibody for 1 h. The bands were detected using a chemiluminescence imaging system (Tanon 4200).

2.10. Anti-apoptosis activity of 2-DG/BP MRs

HepG2 cells were cultured (5 × 105 cells/well) for 24 h. Then, they were added with different formulations for another 24 h: (1) PBS, (2) 200 μg/mL Blank MRs + RT, (3) 200 μg/mL 2-DG MRs + RT, (4) 200 μg/mL Free BP + RT, (5) 200 μg/mL 2-DG/BP MRs + RT. Moreover, cells were collected, stained with Annexin V-FITC/PI apoptosis detection kit, and examined by flow cytometer.

2.11. Biodistribution and imaging of 2-DG/BP MRs in vivo

H22 cells (1 × 106/mL) from 100 μL PBS were injected into the right side of BALB/c female mice to establish a subcutaneous tumor model of H22. When tumor volume reached 100 mm3, Cy5.5-labeled 2-DG/BP MRs + RT and non-sensitive 2-DG/BP MRs + RT were administered intravenously to H22 tumor-bearing mice, followed by 4Gy irradiation 6 h later. Then mice were killed, major organs and tumors were taken, and the distributions of 2-DG/BP MRs + RT was imaged by the IVIS imaging system.

2.12. Antitumor study in vivo

H22 cells (1 × 106) in PBS were inoculated into the right side of each female BALB/c mice. When the tumor volume was 100∼150 mm3, the mice were randomly divided into 6 groups (n = 5). Then, mice were treated with 100 μL of PBS, RT, Blank MRs + RT, 2-DG MRs + RT, Free BP + RT, 2-DG/BP MRs + RT (at an equivalent BP dose of 5 mg/kg body weight) through intravenous injection once every two days. Weight and tumor size were measured every 2 days during treatment. After treatment, tumor tissue was photographed and weighed. H&E and TUNEL were used to evaluate the histological damage of tumors and major organs.

2.13. Biochemistry analysis of mouse serum

After treatment with different nanomedicine, eyeball blood was collected and stored in EDTA tube for hematological detection. Cytotoxicity in the blood was reflected by biomarker levels, including aspartate aminotransferase (AST), urea nitrogen (BUN), alkaline phosphatase (ALP), creatinine (CRE), alanine aminotransferase (ALT), and globulin (GLB), measured by a biochemical analyzer (Trilogy, France).

2.14. Metabolomics analysis of LC-MS

To perform RNA-seq analysis, mice tumors were collected from the 2-DG/BP MRs + RT and control group after 14 days of treatment and frozen with liquid nitrogen. RNA concentration was detected by Nanodrop 2000c (Thermo Scientific, USA). Online analysis of the data via BioDeep were carried out at PANOMIX Biomedical Technologies Co., LTD. (Suzhou, China).

2.15. Statistical analysis

All data were expressed as mean ± standard deviation (SD). The error bars represented the standard error of the mean analyzed in the experiments. The two-tailed Student's t-test was used to calculate statistical significance. According to p value, ∗ for p < 0.05, ∗∗ for p < 0.01, and ∗∗∗ for p < 0.001.

3. Results and discussion

3.1. Characterization of 2-DG/BP MRs nanosystems

As shown in Fig. 1A, for efficient loading and concurrent delivery of the metabolic modulating drugs 2-DG and BP, the 2-DG prodrug was initially synthesized by conjugating 2-DG molecule to a dicarboxylic polyethylene glycol (PEG) backbone, and then connected it to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) through RT-responsive cleaved Se-Se bonds. The structure of obtained DSPE-Se-Se-PEG-2-DG was confirmed by 1H NMR and MALDI-TOF-MS (Figs. S1–S3, Supporting Information). Subsequently, amphiphilic 2-DG prodrug polymer self-assembled in water to form 2-DG MRs, or with another metabolic drug BP being encapsulated into the hydrophobic core during this self-assembly process, resulting in the dual metabolic drug-loaded nanoparticles (2-DG/BP MRs). As shown in Fig. 1B, the transmission electron microscopy (TEM) image clearly revealed that 2-DG/BP MRs nanoparticles were roughly spherical. Briefly, elemental mapping in Fig. 1C indicated that characteristic elements including C, O, F, Se, and S were uniformly distributed within the nanoparticles present.

Fig. 1.

Fig. 1

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Characterization of self-assembled 2-DG/BP MRs. (A) Preparation and RT-responsive degradation of 2-DG/BP MRs. TEM (B) and elemental mapping (C) images of 2-DG/BP MRs. (D) The sizes of Blank MRs and 2-DG/BP MRs nanoparticles measured by DLS. (E) The ratio of PDI to size of Blank MRs and 2-DG/BP MRs in PBS (pH 7.4). (F) UV–vis absorption of Free BP and 2-DG/BP MRs. (G) Cumulative release curves of Free BP (a) and Free 2-DG (b) from 2-DG/BP MRs under RT (100 μM H2O2) conditions and normal physiological (pH 7.4), respectively. Data were expressed as mean ± SD (n = 3). ∗∗∗p < 0.001.

The hydrodynamic sizes of 2-DG/BP MRs and Blank MRs micelles were examined by DLS (Fig. 1D). After loading with 2-DG and BP, the particle size of 2-DG/BP MRs increased dramatically to about 160 nm from the 20–25 nm of blank MR micelles. The increase in size might be attributed to the conjugation of highly water-soluble molecule 2-DG to the DSPE-Se-Se-PEG polymer chain and the incorporation of BP into the internal hydrophobic region, which was consistent with the afore-mentioned TEM results. Accordingly, the zeta potential values of 2-DG/BP MRs and Blank MRs were approximately −9.43 mV and −6.03 mV, respectively, confirming that the surface charge of 2-DG/BP MRs contributed to their stability and biocompatibility (Fig. S4, Supporting Information). As shown in Fig. 1E, the PDI/Size ratio showed no significant changes during storage under physiological conditions, reflecting good uniformity and stability in the nanoparticle size distribution of 2-DG/BP MRs. Considering the smaller particle size and PDI of 2-DG/BP MRs, the combined dose of 2-DG/BP MRs (DSPE-Se-Se-PEG-2-DG: DSPE-PEG2000 = 1:2) was selected for follow-up experiments (Fig. S5, Supporting Information). As shown in the UV–vis–NIR spectra in Fig. 1, Fig. 2 and 2-DG/BP MRs exhibited typical characteristic absorption peaks at approximately 230 nm and 280 nm, corresponding to BP and 2-DG, respectively, further validating the success of preparation of nanoplatform. The susceptible Se-Se bond in 2-DG prodrug polymer facilitated the release of 2-DG upon exposure to a high quantity of ROS generated during RT, via ROS-responsive cleavage. In vitro RT and ROS-triggered release kinetics of the 2-DG/BP MRs showed that under simulated RT conditions (100 μM H2O2), more than 70 % of BP and 2-DG molecules was released within 24 h (Fig. 1G). In contrast, the release of BP and 2-DG molecules from the nanoparticles was limited under normal physiological conditions (pH 7.4), with less than 40 % released within 24 h. This H2O2-simulated RT treatment-triggered drug release behavior of 2-DG/BP MRs confirmed the feasibility of RT-responsive drug release mechanism.

Fig. 2.

Fig. 2

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Metabolic intervention of 2-DG/BP MRs by simultaneously inhibiting intracellular metabolism. (A) Relative cellular viability of HepG2 cells treated with different formulations (Blank MRs, Free BP, 2-DG MRs, and 2-DG/BP MRs) combined with RT (4Gy) for 24 h (left) and 48 h (right), respectively. (B) CLSM detection of cellular internalization and ROS generation in HepG2 cells incubated with Cy5.5-labeled nanoparticles during RT. ROS: DCFA-DA. Nuclei: DAPI. (C) Cell uptake and ROS production of HepG2 cells after incubation with different solutions for 6 h were detected by flow cytometry. (D) Western blot analysis of GLS1 and mTOR expression after different treatments in HepG2 cells. (E) The mechanism of dual metabolic inhibition by 2-DG/BP MRs in HepG2 cells. (F) Flow cytometry analysis for the HepG2 cells apoptosis for 24 h after different treatments by Annexin V-FITC/PI. Data were expressed as mean ± SD (n = 3). ∗∗∗p < 0.001.

3.2. Cellular localization and metabolic intervention of 2-DG/BP MRs

The Se-Se motifs in polymers could be sensitively cleaved during the RT process, leading to the disassembly of the 2-DG/BP MRs and the targeted release of 2-DG during RT, remodeling cellular metabolism through inhibiting the glucose and glutamine pathways, respectively. Therefore, the 2-DG/BP MRs could play a role in reshaping cellular metabolism accompanied with RT, synergistically enhancing the antitumor efficacy. Encouraged by these findings, we evaluated the cytotoxicity of 2-DG/BP MRs against HepG2 cells upon 4Gy X-ray radiation using MTT assay. As shown in Fig. 2A, 2-DG/BP MRs showed higher cytotoxicity against HepG2 cells than 2-DG MRs under RT, possibly due to the presence of BP inhibiting GLUT1 activity in cellular glucose metabolism pathway. Concurrently, 2-DG/BP MRs exhibited an enhanced anticancer activity compared to Free BP, with a 4-fold reduction in the half-maximal inhibitory concentration (IC50 = 96.3 μM). This improvement may result from the responsive disassembly and drug release capability of nanocarriers under RT, significantly increasing the tumor-targeted accumulation of metabolic regulators. This trend became more pronounced as the incubation period was extended from 24 h to 48 h.

To visualize and quantify the uptake of nanoparticles and the accumulation of active components in tumor cells, the fluorescent probe Cy5.5 was encapsulated into RT-sensitive 2-DG/BP MRs and Non-sensitive-2-DG/BP MRs to indicate the intracellular distribution of the drugs. After RT treatment (4Gy), the HepG2 cells were co-cultured with various drugs an additional for 6 h for confocal laser scanning microscope (CLSM) observation. We utilized the fluorescent probe (DCFH-DA) to detect the generation of intracellular ROS during RT and to examine its association with drug release behavior (Fig. 2B). CLSM images revealed that all cells exhibited green fluorescence represented for ROS following RT treatment, suggesting that RT could produce large amount of ROS for tumor cytotoxicity. Further, HepG2 cells displayed strong Cy5.5 fluorescence signals treated with 2-DG/BP MRs, whereas only negligible signals were observed in cells treated with non-sensitive 2-DG/BP MRs. Additionally, the quantitative fluorescence intensity of Cy5.5 in tumor cells also demonstrated the more significant targeting and uptake of 2-DG/BP MRs within the cells in Fig. S6 (Supporting Information). As shown in Fig. 2C and Fig. S7, the flow cytometry analysis further revealed a significant increase in nanoparticle uptake and glucose targeting in HepG2 cells affected by the 2-DG/BP MRs, compared to Non-sensitive 2-DG MRs/BP. This enhanced uptake was attributed to the glucose-targeting capability of 2-DG, which allows the 2-DG/BP MRs to be preferentially internalized by HepG2 cells through glucose transporters (GLUTs) that are overexpressed in cancer cells.

Tumor cells exhibit a propensity to consume large amounts of glucose, 2-DG could intervene TCA cycle by inhibiting oxidative phosphorylation. By impeding the conversion of glucose to glucose 6-phosphate, pyruvate, and lactate through the suppression of Hexokinase-2, 2-DG disrupts the glycolytic pathway and leads to tumor cell apoptosis. Additionally, tumor cells can escape from the inhibition of a single glycolysis pathway by converting glutamine to glutamate, thereby enabling a multi-pathway metabolic energy supply. This metabolic versatility allows tumor cells to sustain the energy supply required for cell proliferation even when challenged with traditional antitumor treatments, including chemotherapy and radiotherapy, leading to failure of treatments. Therefore, we examined the expression levels of the representative proteins GLS1 and mTOR involved in glutamine metabolism and tumor cell anabolism to better understand the molecular mechanisms by which 2-DG/BP MRs modulate multi-pathway metabolic regulation in combating resistant tumors. As depicted in Fig. 2D and Fig. S8 (Supporting Information), 2-DG/BP MRs treatment resulted in a remarkable decrease in the expression of GLS1 and mTOR, whereas RT alone and Blank MRs did not induce significant changes. The presence of BP (both Free BP and 2-DG/BP MRs) significantly inhibited glutaminase GLS1, thus blocking intracellular glutamine metabolism to counteract drug resistance induced by RT (Fig. 2E). Moreover, the co-delivery of BP and 2-DG by 2-DG/BP MRs allowed for a dual blockade of glutamine metabolism and glycolysis pathways, subjecting cancer cells to more severe nutritional deprivation.

To further explore the tumor cell injury induced by RT under dual metabolic suppression, the apoptosis rate of HepG2 cells was analyzed by flow cytometry (Fig. 2F). Specifically, apoptosis rates were determined based on the percentage of early apoptotic cells (Annexin V-FITC+/PI−), as well as late apoptotic cells (Annexin V-FITC+/PI+). The apoptosis rate for RT alone was merely 16.09 %, while when combined with 2-DG/BP MRs this data was sharply increased up to 44.76 %, which was significantly higher than that of single metabolic regulation with 2-DG MRs (25.5 %) and Free BP (30.41 %). Therefore, as expected, 2-DG/BP MRs could effectively target and enhance the damaging effect of RT on tumor cells, primarily due to its dual metabolic inhibition of glutamine and glycolysis metabolism.

3.3. Transcriptome analysis of metabolic pathways

Principal component analysis (PCA) showed significant differences in transcriptomic characteristics between the treatment group (2-DG/BP MRs + RT) and the normal group (PBS) (Fig. S9, Supporting Information). Then, we identified 1351 differentially expressed genes using a stringent threshold, which involved a fold change ≥2 and p < 0.05 between 2-DG/BP MRs + RT and PBS groups. These findings underscored the significant metabolic reprogramming occurring in the 2-DG/BP MRs + RT group, as evidenced by the distinctly altered gene expression profiles. As illustrated in Fig. 3A, the functional genomic analysis highlighted the substantial variation in the expression of genes associated with metabolic pathways in HepG2 cells, which can be attributed to the gene mutation of 2-DG/BP MRs. Simultaneously, the pivotal intermediates of glucose metabolism, including fructose 1,6-bisphosphate and succinic acid, were also decreased significantly during 2-DG/BP MRs + RT treatment. These results indicated that 2-DG/BP MRs + RT treatment could interfere with the glucose metabolic pathway in tumor cells, leading to starvation of tumors and subsequent induction of tumor cell apoptosis.

Fig. 3.

Fig. 3

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Metabolic transcriptomic analysis of HepG2 cells treated with 2-DG/BP MRs + RT. (A) Heat maps of metabolism-related differentially expressed genes in HepG2 cells before and after treatment. (B) KEGG enrichment analysis of cellular metabolic pathways after 2-DG/BP MRs + RT treatment. (C) The enrichment bubble patterns in the KEGG enrichment analysis treated with 2-DG/BP MRs + RT. (D) Concentrations of various metabolites including glutamine, glutamate, G6P, glucose, pyruvate, lactate, α-ketoglutaric acid, acetyl-coA, adenosine, and uracil in HepG2 cells. The red box showed metabolite changes in glutamine metabolism, the blue box showed metabolite changes in glucose metabolism and TCA cycle, and green box showed metabolite changes in amino acid synthesis. Data were expressed as mean ± SD (n = 3). ∗∗p < 0.01, and ∗∗∗p < 0.001.

As shown in Fig. 3B, the GO and KEGG analysis have emphasized significant alterations in the genes involved in amino acids biosynthesis, cancer central carbon metabolism and TCA cycle processes, indicating that 2-DG/BP MRs + RT caused tumors starvation by weakening metabolic pathways. The Venn diagram (Fig. S10, Supporting Information) indicated that a total of 1038 genes were commonly expressed between the two groups, while 732 genes were specifically expressed in the 2-DG/BP MRs-treated group. As expected, the enrichment bubble patterns from the KEGG enrichment analysis indicated that the upregulated proteins in the 2-DG/BP MRs-treated cohort were predominantly enriched in metabolic processes and biosynthesis pathways (Fig. 3C). These variations in RNA sequences strongly supported the simultaneous interference in glycolysis and amino acid metabolism by 2-DG/BP MRs.

We compared HepG2 metabolite concentrations (2-DG/BP MRs + RT) before and after therapy. Intracellular glutamine accumulation was significantly improved, whereas glutamate was significantly reduced after incubation for 48 h with 2-DG/BP MRs, compared with the control group (Fig. 3D). Concurrently, pyruvate levels related to glucose metabolism experienced a significant reduction, attributable to the impediment of glucose oxidase by 2-DG in glucose metabolic pathway. Furthermore, the protein concentrations of α-Ketoglutaric acid and lactate within TCA cycle were also reduced, suggesting 2-DG and BP incorporated in nanoparticles synergistically attenuated both glycolysis and glutamine metabolism. Taken together, these results evidenced that 2-DG/BP MRs could effectively induce a state of metabolic starvation in tumor cells by concurrently suppressing glycolysis and glutamine metabolism, which was postulated to arise from the inhibition of key enzymatic activities of glutaminase and glucose oxidase by BP and 2-DG, respectively.

3.4. In vivo targeting behavior and pharmacokinetics of 2-DG/BP MRs

Initial in vitro studies have indicated that 2-DG/BP MRs exhibited synergistic anticancer properties and notable tumor-targeting capability, suggesting their suitability for in vivo application. Therefore, we extended our study to an in vivo H22 xenograft tumor model to assess the potential of 2-DG/BP MRs to maximize therapeutic effects through efficient tumor targeting, aiming to address the deficiencies caused by deficient drug concentration inherent to conventional tumor therapies. To investigate the tumor targeting efficacy, Cy5.5-labeled 2-DG/BP MRs and Non-sensitive 2-DG/BP MRs were intravenously administered to H22 tumor-bearing mice, followed by 4Gy irradiation 6 h later. Sequential fluorescence imaging was performed at different time intervals post-injection to evaluate the tumor-targeting efficiency of the nano-formulations.

As illustrated in Fig. 4A, the Cy5.5 fluorescence from 2-DG/BP MRs accumulated rapidly in the tumor, attributed to the superficial 2-DG modification on the nanoparticles that actively target the glucose receptors of tumor cells, achieving efficient drug enrichment in the tumor. In addition, the fluorescence intensity of the tumor site increased significantly with the passing of time, and reached a peak at 12 h, indicating that 12 h after injection was the optimal time points for X-ray exposure. At the same time point, the fluorescence signals from the Non-sensitive group and free Cy5.5 were significantly lower, indicating the superior tumor-targeting efficiency of 2-DG/BP MRs. This enhanced accumulation can be attributed to the RT-induced changes in the tumor microenvironment, such as increased vascular permeability and enhanced permeability and retention (EPR) effects, which facilitated the preferential delivery of nanoparticles to the tumor site. Additionally, the nanoparticles not only help to efficiently deliver the drug to the tumor site but also benefit from the RT-responsive drug release mechanism of 2-DG/BP MRs, which facilitated the burst release of the drug at the lesion site, rapidly reaching therapeutic concentrations to kill tumor cells. Moreover, the residual fluorescence signal was still detectable even after 24 h, showing that the delivery of 2-DG/BP MRs helped the drug to have good tumor retention potential. This prolonged retention not only maximized the therapeutic impact but also underscored the ability of RT to create a favorable microenvironment for sustained drug activity.

Fig. 4.

Fig. 4

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Targeting behavior and pharmacokinetics of 2-DG/BP MRs in vivo. (A) In vivo imaging and biodistribution of Free Cy5.5 (5 mg/kg), Cy5.5-labeled Non-sensitive-2-DG/BP MRs (5 mg/kg) and 2-DG/BP MRs (5 mg/kg) after intravenous injection to H22 tumor-bearing mice. (B) Ex vivo fluorescence images of major organs and tumors at 24 h post-intravenous injection. The corresponding quantification of fluorescence intensity of tumor at different time points (C) and major organs at 24 h (D) after intravenous injection of various solutions. Fluorescence images (E) and corresponding quantitative analysis (F) of the blood collected at different time points post injection of various solutions. Error bars reflect standard deviation (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

We also conducted in vitro biodistribution studies, dissecting and imaging tumors and major organs 24 h after administration of Cy5.5-labeled non-sensitive or ROS-sensitive 2-DG/BP MRs. Fig. 4B revealed that both nanomedicines selectively accumulated in tumor tissues, efficiently clearing all major organs except the liver. Compared to mice treated with Free Cy5.5 and non-sensitive nanoparticles, the tumor in the 2-DG/BP MRs group showed stronger quantified Cy5.5 fluorescence (approximately a 2.1-fold higher increase in fluorescence intensity compared with Non-sensitive-2-DG/BP MRs group) (Fig. 4C and D). These results once again proved that 2-DG/BP MRs with RT-responsive drug release capability exhibited excellent tumor-targeting delivery behavior.

As illustrated in Fig. 4E, the blood fluorescence intensity of 2-DG/BP MRs was significantly higher than that of Non-sensitive-2-DG/BP MRs and Free Cy5.5 groups. The calculation results showed that the blood circulation time of 2-DG/BP MRs nanomaterials was 6.5 ± 0.54 h, 1.7 times that of free dyes (Fig. 4F). This suggested that nanomaterials can prolong the half-life in vivo, thus extending the treatment time, which was beneficial for tumor treatment. Apparently, 2-DG facilitated the ability of 2-DG/BP MRs nanoparticles to effectively active target glucose receptors on the cell surface and antagonize it. They were also capable of responding to specific release drug under RT conditions to rapidly increase the effective therapeutic drug concentration within tumor tissues. Moreover, the application of this nanocarrier also facilitated the comprehensive regulatory effect of drug through effectively prolonging the in vivo half-life.

3.5. In vivo antitumor performance of 2-DG/BP MRs combined with RT

Subsequently, to evaluate the anti-tumor performance of 2-DG/BP MRs combined with RT in vivo, a unilateral H22 cell subcutaneous xenograft model was constructed on BALB/c mouse (Fig. 5A). After 7 days of subcutaneous injection of H22 cells on the right side of BALB/c mice, solid tumors formed mass (100–150 mm3), and the mice were then randomly divided into 6 groups. Mice were given different nanomedicine intravenously at a dose of BP 5 mg/kg. RT therapy with 4Gy was administered on day 2, and the tumor volume and body weight of mice were also measured.

Fig. 5.

Fig. 5

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In vivo improved antitumor effect of 2-DG/BP MRs in RT treatment. (A) Schematic illustration of 2-DG/BP MRs-mediated synergistic medication process combined with RT (X-ray with 4Gy) the next day post the intravenous administration. (B) Tumor photographs collected from different groups at the end of antitumor therapy. (C) Tumor growth curves of individual mice in each treatment group (n = 5). (D) Tumor volume changes of the mice over 14 days of treatment (n = 5). (E) The average tumor weights of mice from different groups at the end of treatment (n = 5). (F) Body weight changes of the mice over 14 days of treatment (n = 5). (G) The histological and immunofluorescent analysis of tumor tissues 14 d after treatment. (H) The statistical analysis of IFN-γ, TNF-α, IL-6 expression in tumor tissues harvested from mice after different treatments. Data were shown as mean ± SD (n = 5). ∗∗p < 0.01, ∗∗∗p < 0.001.

Due to the unique metabolic pathways of tumor cells that allow it to evade damage from RT, monotherapy of RT only showed negligible tumor growth inhibition (Fig. 5B–D). Both individual and average tumor growth curves showed that additional treatment with single metabolic regulator (2-DG MRs or Free BP) by inhibiting glucose or glutamine metabolism, could enhance the tumor killing effect of RT to a certain extent. However, due to the presence of multiple metabolic compensation mechanisms in tumor cells, single metabolic pathway blockade therapies cannot completely cut off the energy supply of tumor cells, thus have limited anti-tumor effects. Therefore, a dual metabolic intervention strategy blocking both glucose and glutamate-based pathways (2-DG/BP MRs + RT) can effectively conquer compensatory energy metabolic pathways in tumors, showing the most significant enhancement of anti-tumor activity, and effectively inhibiting the development of RT-resistant HCC. Tumors were excised and weighed after treatment, as shown in Fig. 5E, the average tumor weight also confirmed that the dual metabolic intervention strategy of 2-DG/BP MRs could synergize with RT to exert the optimal anti-tumor effect.

Furthermore, the biosafety during the treatment process is also an important criterion for evaluating anti-tumor strategies. Mice did not demonstrate obvious body weight loss during the various treatments (Fig. 5F), and blood biochemical markers remained within the normal ranges after the medication process (Fig. S11, Supporting Information), indicating that the applied therapeutic methods did not cause systemic toxicity such as liver and kidney function damage. No significant pathological damage was found in the major organs of the group receiving 2-DG/BP MRs + RT treatment (Fig. S12, Supporting Information), confirming the good biocompatibility of 2-DG/BP MRs.

To verify the synergetic therapeutic effect of 2-DG/BP MRs combined with RT, H&E and TUNEL staining were performed on tumor tissue sections (Fig. 5G). H&E staining images indicated varying degrees of damage to tumor cells in different groups, with the 2-DG/BP MRs + RT exhibiting the greatest extent of necrosis and apoptosis. Moreover, the strongest TUNEL staining and nearly invisible Ki67 staining signals indicated that the combined therapy of 2-DG/BP MRs + RT could effectively promote apoptosis in tumor tissues while inhibiting tumor cell proliferation. To further verify the mechanisms of the synergistic effect of metabolic regulation by 2-DG/BP MRs and RT, we also evaluated the expression of energy metabolism-related markers at the tumor tissue level. HCC cells are known to rely heavily on glutamine metabolism, making GLS1 a key target for metabolic modulation. After BP (Free BP and 2-DG/BP MRs) regulation, GLS1 expression levels in tumor tissues were reduced compared with other groups, demonstrating that BP affected the blocking of glutamine metabolic pathways. In HCC, mTOR signaling is frequently dysregulated, driving metabolic reprogramming and resistance to therapy. Moreover, Fig. 5G showed that mTOR expression descended in the 2-DG MRs and 2-DG/BP MRs groups, indicating that 2-DG was involved in the inhibition of pyruvate dehydrogenase activity during glycolysis. As shown in Fig. 5H, the combined treatment was proven to be capable of promoting the secretion of specific effector cytokines within the tumor, including IFN-γ and TNF-α, further confirming that 2-DG/BP MRs not only directly regulated tumor cell energy metabolism but also indirectly promoted tumor cell death. These results validated the enhanced anti-tumor effects of 2-DG/BP MRs, making it a promising nanomaterial to inhibit tumor cells growth though reprogramming cell glutamine and glucose metabolism.

3.6. RNA sequencing analysis to understand the metabolic modulation by 2-DG/BP MRs in H22 tumor model

To further delve into the underlying biological mechanisms of 2-DG/BP MRs, Metabolome Sequencing (MS) was conducted that subjected to various treatments. Orthogonal partial least squares discriminant analysis (OPLS-DA) was utilized to differentiate samples, establishing a predictive model that associated metabolite expression with categories. The OPLS results depicted in Fig. 6A indicated minimal distinction between the sample categories of 2-DG/BP MRs + RT and PBS, despite significant differences were observed in the transcriptome analysis. As illustrated in Fig. 6B, the Venn diagram displayed the relationship between gene expression in the 2-DG/BP MRs + RT and PBS groups, revealing that there were 1761 genes were commonly expressed, while 823 genes exhibited differential expression under the influence of 2-DG/BP MRs + RT.

Fig. 6.

Fig. 6

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2-DG/BP MRs reshaped metabolic pathways in H22 tumor model. (A) OPLS based on differentially expressed genes of 2-DG/BP MRs + RT and PBS groups. (B) The differentially expressed genes of 2-DG/BP MRs + RT and PBS groups were shown by Venn diagram. (C)The up-regulation and down-regulation of metabolic pathways-related genes after 2-DG/BP MRs + RT treatment were analyzed by heat map. (D) Functional association networks of genes involved in metabolic pathways. (E) KEGG pathway analysis for the identified differentially expressed genes in vivo. (F) Differential metabolite association analysis between 2-DG/BP MRs + RT and PBS groups.

In addition, the levels of specific metabolite were measured by liquid chromatography-mass spectrometry (LC-MS) (Fig. 6C) and significant differences were found in various metabolites levels between 2-DG/BP MRs + RT and control groups. When treated with 2-DG/BP MRs + RT, the expressions of Glutathione, Beta-Leucine, 2-Amino-2-deoxy-D-glucose, and Oxoglutaric acid were remarkably reduced, suggesting the inhibition of critical metabolic pathways in tumors, particularly glycolysis pathways and TCA cycle, which were essential for energy production and biosynthetic processes in cancer cells. Furthermore, the concentrations of 2-Phenylacetamide and N-Acetylglutamic acid were notably decreased, indicating their close association with the glutamine cycle and tumor cell proliferation. This observation underscored the role of 2-DG/BP MRs + RT in disrupting glutamine metabolism, a pathway crucial for sustaining rapid tumor growth. Additionally, the levels of UDP, AMP and CMP were significantly reduced, indicating a substantial depletion of essential nucleotides required for DNA/RNA synthesis and other metabolic processes. Moreover, the expression levels of 2-Phenylacetamide and 2-Methylserine were decreased, potentially impacting various critical processes related to signaling functions and cellular transformation, which were closely associated with the tumorigenesis. These findings collectively highlighted the multifaceted impact of 2-DG/BP MRs + RT on tumor metabolism, targeting not only energy production but also nucleotide synthesis, amino acid metabolism, and cellular signaling pathways critical for cancer progression.

Notably, the difference in metabolites after 2-DG/BP MRs + RT treatment were significant compared to the control group (Fig. S13, Supporting Information). Additionally, functional association networks of metabolism-related genes were established as depicted in Fig. 6D. Within these co-expressed genes, Phosphoenolpyruvic acid exhibited a negative correlation with central carbon metabolism, Oxoadipic acid and Saccharopine displayed negative correlations with biosynthesis of amino acid in tumors, while ADP and FMN showed positive correlations with oxidative phosphorylation. These findings suggested that 2-DG/BP MRs possessed the capability to effectively regulate the metabolic microenvironment within tumors, influencing processes such as biosynthesis and metabolic modulation, ultimately enhancing its therapeutic efficacy against tumors.

KEGG analysis elucidated the metabolic pathway changes in the 2-DG/BP MRs Group, among which the most significant changes were mainly related to amino acid biosynthesis, citric acid cycle (TCA cycle), and glutamate metabolism (Fig. 6E). It was further observed that amino acids biosynthesis, metabolic pathways, protein digestion and absorption, and carbon metabolism were all significantly weakened, and were highly correlated with 2-DG/BP MRs treatment (Fig. S14, Supporting Information). As illustrated in Fig. 6F, the differential metabolite association analysis following 2-DG/BP MRs + RT treatment demonstrated a high level of consistency in the trends of changes among metabolites, which was achieved by calculating the Pearson correlation coefficient or Spearman grade correlation coefficient between all metabolites. Metabolite correlations frequently revealed the synchronized changes between metabolites: red indicated a value close to 1 (positive correlation) while blue indicated a value close to −1 (negative correlation). Ultimately, 2-DG/BP MRs significantly reshaped intra-tumoral metabolism by reducing the levels of multiple metabolites involved in glycolysis.

4. Conclusions

In summary, we have designed a Se-containing metabolic nanoregulator 2-DG/BP MRs that selectively sensitize HCC to RT through synergistically interfering with intracellular glucose and glutamine metabolism, leading to comprehensive tumor eradication. The RT-triggered 2-DG release from 2-DG/BP MRs played a crucial role by consuming the glucose transporter GLUT1 in tumor cells, thereby inhibiting glucose uptake and subsequently blocking the ATP production through glycolysis. The metabolic inhibitor BP simultaneously released inhibited intracellular glutamine metabolism by effectively inhibiting glutaminase GLS1 and preventing the conversion of glutamine to glutamate. The synergistic role on glucose and glutamine reshaped the metabolic pathways within tumor cells, thus sensitizing the sensitivity of HCC to RT, achieving significant anti-tumor performance and fewer side effects. Therefore, this study highlights the significant potential of rationally designed a metabolic nanoregulator as potent RT-adjuvant strategy for treatment of resistant HCC.

CRediT authorship contribution statement

Yuehua Wang: Writing – original draft, Funding acquisition, Formal analysis. Zhenjie Wang: Investigation, Data curation. Mengnan Liu: Methodology, Data curation. Chaojie Chen: Software, Project administration. Qiye Xi: Software, Methodology. Jingwen Tang: Validation, Methodology. Zhiqiang Yu: Supervision, Resources. Shengtao Wang: Visualization, Supervision, Software. Ling Yu: Validation, Supervision. Meng Yu: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by Shanghai Sailing Program (No. 23YF1441300), the Joint Project of Science and Technology Committee of Yangpu District and Health Commission of Shanghai Yangpu District (No. YPQ202304) and the hospital research grant awarded in 2023 (No. YJZD02) from Yangpu District Shidong hospital. The authors also appreciate the support by the Guangdong Basic and Applied Basic Research Foundation (2023A1515030291) and the Dongguan Science and Technology of Social Development Program (20211800905282). Animal specimens were obtained according to the guidelines approved by ICE for Clinical Research and Animal Trials of Southern Medical University Animal Laboratory Center Approval Letter for Research Protocol (LAEC-2020-093).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.101608.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1
mmc1.docx (3.8MB, docx)

Data availability

Data will be made available on request.

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