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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2026 Jul 15;152(9):184. doi: 10.1007/s00432-026-06538-1

Timosaponin AIII-based liposomes loaded with Auranofin for the induction of ferroptosis in anaplastic thyroid carcinoma

Xinyi Deng 1, Lei Yang 2, Dihua Li 2, Yan Wang 3, Qiang Jia 1, Ning Li 1, Zhaowei Meng 1,4,✉
PMCID: PMC13601440  PMID: 42455333

Abstract

Purpose:

Anaplastic thyroid carcinoma (ATC) is an exceptionally aggressive malignancy that, despite accounting for only 1%-1.5% of all thyroid cancers, is responsible for over half of thyroid cancer-related deaths. Characterized by limited treatment options and a poor prognosis, ATC presents a critical clinical challenge. In this study, we developed and evaluated an innovative liposomal system (T-AUF-LPs) co-loading Auranofin (AUF) and Timosaponin AIII (TAIII) aimed at inducing ferroptosis in ATC cells.

Methods:

T-AUF-LPs were prepared via the film-hydration method and their physicochemical properties were characterized. Cellular uptake, anti-proliferative effects, and ferroptosis-related mechanisms—including reactive oxygen species (ROS), iron levels, glutathione (GSH), GPX4, ACSL4, and NCOA4—were assessed in CAL-62 and 8505C ATC cell lines. In vivo antitumor efficacy and biosafety were evaluated using a BALB/c nude mouse xenograft model.

Results:

T-AUF-LPs showed superior physicochemical properties compared to conventional liposomes, including uniform spherical morphology, reduced particle size, and enhanced encapsulation efficiency. Cellular assays revealed significantly improved uptake of T-AUF-LPs in both ATC cell lines. The formulation inhibited cell proliferation in a dose-dependent manner, an effect effectively reversed by ferrostatin-1, confirming ferroptosis as the primary cell death mechanism. Mechanistic investigations indicated that T-AUF-LPs treatment increased intracellular ROS and iron levels, depleted GSH, and induced upregulation of ACSL4 and NCOA4 along with downregulation of GPX4. In vivo evaluation demonstrated effective tumor growth suppression with an excellent safety profile and no significant organ toxicity.

Conclusion:

T-AUF-LPs exert potent anti-tumor effects by inducing ferroptosis without causing systemic organ toxicity. They represent a promising therapeutic strategy for ATC, thereby addressing a critical unmet need in the management of this disease.

Keywords: Timosaponin AIII, Auranofin, Liposomes, Anaplastic thyroid carcinoma, Ferroptosis

Introduction

Anaplastic thyroid carcinoma (ATC) is the most aggressive and poorest-prognosis subtype of thyroid cancer, accounting for only 1–1.5% of all thyroid malignancies, yet responsible for over half of thyroid cancer-related mortality. Currently, there is a lack of effective treatment options for ATC (Maniakas et al. 2022). Available therapeutic strategies primarily include surgical resection, radiotherapy, chemotherapy, and multi-kinase inhibitors (Hamidi et al. 2025). However, ATC is largely insensitive to conventional radiotherapy and chemotherapy, leading to a very short median survival and poor overall survival rates, posing a significant clinical challenge (Abe and Lam 2021). Therefore, exploring novel therapeutic strategies for ATC, particularly those capable of overcoming traditional drug resistance mechanisms, is of great clinical importance.

In recent years, ferroptosis, a novel form of iron-dependent regulated cell death, has provided a new perspective for overcoming tumor therapy resistance. Its core hallmark is the excessive accumulation of lipid peroxidation products (LPO) (Zhou et al. 2024). This process involves three key aspects: firstly, the inactivation of the core antioxidant enzyme Glutathione Peroxidase 4 (GPX4) compromises the cellular antioxidant defense system (Zhang et al. 2024); Secondly, Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) provides easily oxidizable phospholipid substrates for LPO generation (Liang et al. 2022). Finally, Nuclear Receptor Coactivator 4 (NCOA4) increases labile iron pools via ferritinophagy, catalyzing LPO production (Tang et al. 2021; Huang et al. 2025). The upregulation of both ACSL4 and NCOA4 promotes LPO accumulation. The interplay among the above three aspects collectively drives the execution of ferroptosis. Studies indicate that various refractory tumor cells, including ATC, exhibit high susceptibility to ferroptosis, making it a promising new therapeutic target (Dos Santos et al. 2023). However, achieving tumor-specific and highly efficient induction of ferroptosis remains a significant challenge in current research.

AUF, a traditional anti-rheumatic drug, has recently been identified as a potent inhibitor of thioredoxin reductase 1 (TXNRD1). It can indirectly promote lipid peroxidation by inducing oxidative stress, thereby demonstrating potential for inducing ferroptosis (Yang et al. 2024a, b; Johnson et al. 2024). However, its efficacy as a monotherapy is limited and it carries certain toxicity concerns. On the other hand, TAIII is extracted from Anemarrhena asphodeloides, a traditional Chinese medicine with a history of use spanning over two thousand years. It has shown therapeutic effects in various diseases (Lin et al. 2020). Furthermore, research reports indicate that TAIII can promote ferroptosis through multiple molecular pathways. Its mechanisms involve downregulating GPX4 expression to weaken the tumor cell’s defense capacity and inducing the generation of reactive oxygen species (ROS), collectively leading to LPO accumulation and effectively triggering ferroptosis (Zhou et al. 2023; Shen et al. 2024a, b).

However, both drugs face challenges such as poor aqueous solubility and rapid metabolism when used as single agents. The simultaneous delivery of TAIII and AUF to tumor sites in vivo to achieve enhanced anti-tumor efficacy presents a significant hurdle (Wang et al. 2021). To overcome this challenge, liposomal drug delivery systems are considered ideal carriers due to their ability to improve drug solubility, good biocompatibility, and sustained release properties (Guimarães et al. 2021). However, conventional liposomes use cholesterol as a membrane stabilizer, whose oxidation products pose potential risks to human health (Wang et al. 2019). Existing reports demonstrate that saponin compounds can replace cholesterol to construct liposomes for drug delivery, further exerting potential anti-tumor effects (Hong et al. 2024, 2019; Zhu et al. 2023).

Therefore, we utilized TAIII to replace cholesterol in conventional liposomes. As an amphiphilic steroidal saponin, similar to the saponins mentioned above, TAIII can embed into the lipid bilayer, serving to stabilize the membrane structure (Zhang et al. 2021a, b). This approach avoids the potential risks associated with cholesterol oxidation products, significantly enhancing the safety profile of the formulation. Most importantly, this strategy transforms the carrier itself from merely a delivery vehicle into a pharmacologically active component, achieving functional integration of the carrier and the drug, providing a new strategy for enhancing anti-tumor efficacy.

Compared to previously reported nanocarriers for ferroptosis induction or AUF-based delivery systems, our TAIII-functionalized liposome system (T-AUF-LPs) presents several distinct features. First, while conventional cholesterol-based liposomes or other nanocarriers have been used to deliver ferroptosis inducers, most rely on a single agent and a single pathway (Yang et al. 2021; Jiang et al. 2024). In contrast, our system co-delivers TAIII and AUF to achieve multi-node regulation of ferroptosis (GPX4 downregulation, ACSL4 upregulation, NCOA4 upregulation). Second, unlike previously reported AUF-based systems that deliver AUF as a free drug or in simple carriers, our approach incorporates TAIII not merely as a co-loaded therapeutic but as a membrane-stabilizing component replacing cholesterol, thereby converting the carrier into an active participant in ferroptosis induction. Third, distinct from other saponin-based liposomes that primarily induce apoptosis, our system specifically triggers non-apoptotic ferroptosis, offering a potential strategy to overcome apoptosis resistance. These distinctions are further elaborated in the Discussion section.

In conclusion, this study aims to construct a TAIII-based functionalized liposome for the co-delivery of AUF and TAIII. Through systematic in vitro and in vivo experiments, we will thoroughly investigate the mechanism by which this delivery system induces ferroptosis in ATC cells, aiming to provide a new therapeutic strategy for ATC treatment.

Materials and methods

Materials

The human ATC cell lines CAL-62 and 8505C were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Female BALB/c nude mice (4–6 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Soybean lecithin and cholesterol are from Shanghai Aivito Medical Technology Co., Ltd. and Beijing Solarbio Science & Technology Co., Ltd., respectively. TAIII and AUF were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Coumarin 6 (Cou6) was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. The Annexin V-FITC/PI apoptosis detection kit was supplied by Shanghai Yisheng Biotechnology Co., Ltd. The DCFH-DA fluorescent probe for detecting ROS was purchased from Suzhou Ueland Biological Technology Co., Ltd. (Suzhou, China). C11-BODIPY 581/591 Lipid Peroxidation Sensor was purchased from Thermo Fisher Scientific Inc. Assay kits for glutathione (GSH) and iron (Fe) were procured from Beijing Solarbio Science & Technology Co., Ltd. Other key reagents included: Dulbecco's Modified Eagle Medium (DMEM) and trypsin were obtained from Gibco Laboratories, Thermo Fisher Scientific; fetal bovine serum (FBS) from Suzhou Shuangru Bio-technology Co., Ltd.; dimethyl sulfoxide (DMSO) from Beijing Solarbio Science & Technology Co.; cDNA synthesis kit and SYBR qPCR SuperMix Plus from Suzhou Genecreate Biotechnology Co., Ltd.

Methods

Preparation of liposomes

Liposomes were prepared using the thin-film hydration method. For the preparation of cholesterol-containing liposomes (C-AUF-LPs), soybean lecithin, cholesterol, and AUF were dissolved in a 1:1 (v/v) mixture of methanol and chloroform at a weight ratio of 10:3:1. Similarly, T-AUF-LPs were formulated by dissolving soybean lecithin, TAIII, and AUF in the same organic solvent mixture (methanol: chloroform, 1:1, v/v) at the same weight ratio (10:3:1). Briefly, 50 mg of soybean lecithin, 15 mg of cholesterol (for C-AUF-LPs) or TAIII (for T-AUF-LPs), and 5 mg of AUF were dissolved in 5 mL of the methanol: chloroform mixture (1:1, v/v). The resulting mixture was rotary-evaporated at 50 °C using a rotary evaporator (LOOYE, China) to form a lipid film. The lipid film was hydrated with 5 mL phosphate-buffered saline (PBS) at 50 °C for 30 min, followed by probe sonication (180 W, 20 kHz; Sonics & Materials, Inc.) in an ice bath for 30 min using a cycle of 3 s sonication and 2 s rest.

Characterization of liposomes

The prepared C-AUF-LPs and T-AUF-LPs were diluted to the appropriate concentration with PBS, and was then placed onto a specialized copper grid for transmission electron microscopy (TEM) (JEM-1200EX, Japan). Following this, 3% uranyl acetate was added to the grid, and negative staining was performed for approximately 2 min. After removing excess stain with filter paper and air-drying at room temperature, the grids were imaged by TEM.

The particle size, polydispersity index (PDI), and zeta potential were determined by dynamic light scattering (DLS) detector (Zetasizer Nano ZS90, UK).

High-Performance Liquid Chromatography (HPLC) was employed to determine the encapsulation efficiency (EE%) and loading efficiency (LE%) of liposomes (Hong et al. 2016). In summary, the amount of AUF retained in the liposomes was analyzed after removal of precipitated AUF through 0.8 µm membrane filtration. The chromatographic conditions were as follows: a YWG-C18 column (250 × 4.6 mm, 5 μm) was used, with a mobile phase consisting of methanol and water (70:30, v/v) containing 0.1% (v/v) acetic acid. The flow rate was set at 1 mL/min, and the injection volume was 10μL. Detection was performed at a wavelength of 210 nm.

Cell culture

CAL-62 and 8505C human ATC cells were cultured in DMEM and RPMI-1640 medium, respectively, each containing 10% FBS, at 37℃ and 5%CO2 in an incubator. When cells reach 70–80% confluence, the cells were passaged using 0.25% trypsin/EDTA or treated the cells with TAIII, AUF, AUF + TAIII (AT), C-AUF-LPs and T-AUF-LPs for 24 h before proceeding with subsequent experiments.

In vitro cellular uptake

To further investigate the cellular uptake of T-AUF-LPs, we conducted cellular uptake experiments in vitro. CAL-62 and 8505C cells were seeded in 6-well plates at densities of 2 × 105 and 2 × 10⁶ cells per well, respectively. After 24 h of incubation, CAL-62 and 8505C cells were treated with Coumarin 6-loaded C-LPs and T-LPs at a concentration of 500 ng/mL. The cells were then incubated at 37 °C for 4 h. Prior to the end of incubation, Hoechst 33,342 (1:1000) was added and incubated for 30 min protected from light. The medium was subsequently removed, and the cells were washed three times with PBS. Qualitative assessment and image acquisition were performed using an inverted fluorescence microscope. For quantitative analysis of the cellular uptake of C-LPs and T-LPs by CAL-62 and 8505C cells, the intracellular fluorescence intensity of Coumarin 6 was measured by flow cytometry. Three independent samples were used per group (n = 3), and data were presented as mean ± standard deviation (SD).

Cytotoxicity assay

We selectively determined the cytotoxicity of T-AUF-LPs against ATC cells in vitro using the CCK-8 assay. CAL-62 and 8505C cells were seeded in 96-well plates at densities of 5 × 104 and 1 × 105 cells per well, respectively. After 24 h of incubation, the cells were treated with various formulations including free AUF, AUF + TAIII, C-AUF-LPs, and T-AUF-LPs. AUF was administered at concentrations ranging from 0 to 10 μmol/mL, while TAIII was applied at concentrations ranging from 0 to 30 μmol/mL. After 24 h of incubation, the CCK-8 assay was performed according to the manufacturer's protocol. Briefly, 10 μL of CCK-8 solution was added to each well, and the optical density (OD) at 450 nm was measured using a NANOMEDICINE 1981 microplate reader (Tecan Trading AG, Switzerland). Cell viability was then calculated.

Apoptosis assay

Cell apoptosis was detected using an Annexin V-FITC/PI staining kit. CAL-62 and 8505C cells were seeded in 12-well plates (2 × 105 and 3 × 105 cells per well), respectively. Upon reaching 80% confluence, the cells were treated for 24 h with free TAIII, AUF, AT, C-AUF-LPs, or T-AUF-LPs. After treatment, the cells were harvested, washed with PBS, and resuspended in 200µL of 1 × Binding Buffer. Subsequently, 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) were added to the cell suspension. Following incubation at room temperature for 15 min in the dark, the samples were analyzed by flow cytometry (ACEA, USA).

Detection of intracellular ROS lipid peroxidation levels

The production of excessive reactive oxygen species (ROS) was detected using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (US Everbright, Suzhou, China). The brief procedure was as follows: 8505C cells were seeded in 24-well cell culture plates (5 × 104 cells per well) and incubated overnight at 37 °C. After cell attachment, the original medium was replaced with RPMI 1640 medium containing different formulations (AUF, TAIII, AT, C-AUF-LPs, and T-AUF-LPs) and cultured for an additional 7 h before discarding the cell supernatant. The cells were washed twice with PBS, followed by the addition of DCFH-DA to each well for 1 h of incubation. Finally, after washing the cells twice with PBS again, observations were carried out under a fluorescence microscope (Leica, Germany).

Detection of intracellular lipid peroxidation levels

To further investigate the underlying mechanism of reactive oxygen species (ROS) elevation, CAL-62 cells were seeded in 12-well plates at a density of 2 × 105 cells per well and treated with different drug formulations for 24 h. Subsequently, a lipid peroxidation fluorescent probe C11-BODIPY 581/591 was added to the cells and incubated at 37 °C in the dark for 30 min. Finally, cell images were captured under different excitation wavelengths using a Leica inverted fluorescence microscope (Leica, Germany), with fluorescence signals recorded through red and green channels for subsequent semi-quantitative analysis.

Cellular total iron detection experiment

CAL-62 and 8505C cells were inoculated into 12-well plates (5 × 105 cells per well). When they reached 70–80% confluence, various drug treatments were administered for 24 h. A total of 0.5 ml of lysis buffer was added, and the cells were disrupted using ultrasonic waves in an ice bath. The cell suspension was then centrifuged at 4 ℃ and 8000 g for 10 min. The supernatant was collected for measurement according to the manufacturer's protocol. Reagents were added and mixed, and the mixture was incubated at 25 ℃ for 10 min. The absorbance measured at a 510 nm using a microplate reader.

GSH assay experiment

CAL-62 and 8505C cells were seeded in 12-well plates (4 × 105 cells per well) and incubated overnight. After 24 h of drug treatment, the cells were harvested and centrifuged at low speed. The cell pellet was washed with PBS and then lysed with the proprietary lysis buffer provided in the GSH kit by ultrasonication on ice. The lysate was centrifuged at 12,000 g for 10 min at 4 °C to remove cellular debris. A predetermined volume of the resulting supernatant was then transferred to a 96-well plate and mixed with the assay reagents. The reaction mixture was incubated at room temperature for exactly 2 min, and the absorbance was immediately measured at 405 nm using a microplate reader The GSH concentration in each sample was calculated based on a standard curve generated with known concentrations of GSH.

Western blotting (WB)

Proteins were extracted from tissues using RIPA lysis buffer supplemented with protease inhibitors. The samples were disrupted by ultrasonication on ice and centrifuged at 12,000 g for 15 min at 4 °C to collect the supernatant. Protein concentration was determined using a BCA Protein Assay Kit (Solarbio, China). Equal amounts of protein (40 μg) were separated by SDS-PAGE and electrophoretically transferred to a PVDF membrane. The membrane was blocked with 5% milk in TBST for 2 h at room temperature and then incubated overnight at 4 °C with the indicated primary antibodies (all from HUABIO, China): anti-ACSL4 (1:1000, #ET7111-43), anti-NCOA4 (1:1000, #ET7111-43), anti-GPX4 (1:10,000, #ET1706-45), and anti-GAPDH (1:10,000, #ET1601-4). After three washes with TBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:5000, #AN19618, ANOLA) for 2 h at room temperature. After extensive washing with TBST, the bands were visualized with an Enhanced Chemiluminescence (ECL) kit and captured using a Tanon imaging system.

qPCR

Total RNA was extracted from cells or tissues using Trizol reagent, and first-strand cDNA was synthesized using a reverse transcription kit. mRNA levels of ACSL4, NCOA4, and GPX4 were assessed using the SYBR qPCR SuperMix Plus kit. The primer sequences were as follows: ACSL4 (5′-AGGTCTGTTTGCTGTTGCTGA-3′, 5′-GCAAGATATATTGGGGGAAGTCATT-3′), NCOA4 (5′-CTTGTGCCTTGGGGGTGTTC-3′, 5′-GTATTTCCGGGTAAGAGGCAGTT-3′), GPX4 (5′-AAGAACGGCTGCGTGGTG-3′, 5′-GCTGGGATTTTCGGGGTCTGC-3′), and GAPDH (5′-TCGGAGTCAACGGATTTGGT-3′, 5′-TTCCCGTTCTCAGCCTTGAC-3′). The amplification reaction included pre-denaturation: 95 °C for 3 min; PCR reaction: 95 °C for 15 s, 60 °C for 1 min, 40 cycles.

Animal assay

4–6 weeks old female BALB/c nude mice were acclimatized for 2 weeks prior to the experiment and maintained in an environment with a temperature of 16–25 °C. A total of 5 × 106 CAL-62 cells were inoculated subcutaneously into the dorsal area of the mice. The mice were randomly divided into 6 groups, with 3 mice in each group: control group (equal volume of PBS), TAIII group (10 mg/kg), AUF group (3 mg/kg), AT combination group (AUF 3 mg/kg + TAIII 10 mg/kg), C-AUF-LPs group (containing AUF 3 mg/kg), and T-AUF-LPs group (containing AUF 3 mg/kg and TAIII 10 mg/kg). Both TAIII and AUF were dissolved in PBS containing 2% DMSO, and the liposomal preparations were resuspended in PBS with 2% DMSO. When the tumor volume reached 90-100mm3, intraperitoneal injections were administered every three days. Body weight and tumor volume were recorded every three days over a 15-day period. After the experiment, tumor tissues were collected and stored in liquid nitrogen for subsequent studies. Tumor volume (V) was calculated using the formula: V = (length × width2) / 2. The experimental protocol strictly adhered to the ethical guidelines of the Animal Care and Use Committee of Tianjin Medical University General Hospital (Approval No.: IRB2024-DW-67) and followed the Guide for the Care and Use of Laboratory Animals.

H&E staining

To maintain the integrity of the tissue structure, the excised organs were fixed in 4% paraformaldehyde for 24 h. After fixation, the tissues were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. The embedded tissues were then sectioned into 3 μm-thick slices. The sections were stained with haematoxylin and eosin (H&E) and then examined under a light microscope (Leica, Germany).

Statistics

Statistical analyses were performed using GraphPad Prism software (v10.0, USA). Data obtained from three independent experiments are presented as the mean ± standard error of the mean (SEM). Differences between multiple groups were analyzed by one-way ANOVA or two-way ANOVA, as appropriate, followed by a suitable post-hoc test. The quantification of protein band intensity from Western blots and the fluorescence intensity of ROS was carried out using ImageJ software (v1.52a, USA). P < 0.05 was considered statistically significant.

Results

Characterization of T-AUF-LPs liposomes

Transmission electron microscopy (TEM) revealed that the prepared liposomes exhibited a uniform spherical morphology (Fig. 1A).The particle size of C-AUF-LPs was 167.00 ± 2.08 nm, while T-AUF-LPs exhibited a smaller size of 119.40 ± 3.11 nm. The polydispersity index (PDI) values for C-AUF-LPs and T-AUF-LPs were 0.11 ± 0.08 and 0.14 ± 0.08, respectively, indicating no significant difference between the two formulations (Table 1). The absolute zeta potential value of T-AUF-LPs (− 12.06 ± 0.38 mV) was higher than that of C-AUF-LPs (− 8.48 ± 0.54 mV). Throughout the 7-day storage stability study, both the particle size and PDI of C-AUF-LPs and T-AUF-LPs remained stable without notable changes, demonstrating their good stability (Fig. 1C). Furthermore, T-AUF-LPs showed slightly higher encapsulation efficiency (EE%) and loading efficiency (LE%) than C-AUF-LPs. Specifically, the EE% of T-AUF-LPs was 85.96 ± 0.89%, compared to 83.67 ± 2.02% for C-AUF-LPs, while the LE% was for 7.51 ± 4.51% for T-AUF-LPs and 3.62 ± 2.12% for C-AUF-LPs (Table 1).

Fig. 1.

Fig. 1

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Characterization of TAIII-loaded liposomes. A TEM images of T-AUF-LPs and C-AUF-LPs. B Hydrodynamic size distribution of T-AUF-LPs and C-AUF-LPs measured by dynamic light scattering (DLS). C Stability evaluation of C-AUF-LPs and T-AUF-LPs. The hydrodynamic size and polydispersity index (PDI) were monitored

Table 1.

Characteristics of different liposomes (n = 3; mean ± SD)

Size (nm) ± SD PDI ± SD Zeta(mV) ± SD EE% LE%
C-AUF-LPs 167.00 ± 2.08 0.11 ± 0.08 − 8.48 ± 0.54 83.67 ± 2.02 3.62 ± 2.12
T-AUF-LPs 119.40 ± 3.11 0.14 ± 0.08 − 12.06 ± 0.38 85.96 ± 0.89 7.51 ± 4.51
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Uptake of T-AUF-LPs liposomes by ATC cells

Confocal laser scanning microscopy (CLSM) images revealed a significantly more intense green fluorescence signal from Cou6-labeled T-Cou6-LPs in CAL-62 and 8505C cells compared to the C-Cou6-LPs group (Fig. 2A–B), indicating enhanced cellular uptake of the TAIII-modified liposomes. Flow cytometric quantitative analysis further confirmed these findings, demonstrating that the mean fluorescence intensity of T-Cou6-LPs was 1.4-fold and 1.6-fold higher than that of C-Cou6-LPs in CAL-62 and 8505C cells, respectively (P < 0.001), consistent with the observations from fluorescence microscopy (Fig. 2C–D).

Fig. 2.

Fig. 2

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Uptake of C-Cou6-LPs and T-Cou6-LPs in ATC Cells. A Confocal laser scanning microscopy (CLSM) images of CAL62 cells after incubation with C-Cou6-LPs or T-Cou6-LPs.Scale bar: 200 μm. B Confocal laser scanning microscopy (CLSM) images of 8505C cells after incubation with C-Cou6-LPs or T-Cou6-LPs. Scale bar: 200 μm. C Quantitative analysis of liposome uptake in CAL-62 cells by flow cytometry. D Quantitative analysis of liposome uptake in 8505C cells by flow cytometry

Effect of T-AUF-LPs liposomes on cell viability

To evaluate the antitumor efficacy of T-AUF-LPs, we conducted CCK-8 assays on CAL-62 and 8505C cells in vitro. Free AUF demonstrated a concentration-dependent inhibitory effect on tumor cell growth, which was further enhanced when loaded into C-LPs. Notably, T-AUF-LPs exhibited the strongest inhibitory activity, and in CAL-62 and 8505C cells, the half-maximal inhibitory concentration (IC₅₀) differed from that of C-AUF-LP by 3.6-fold and 3.4-fold, respectively (Fig. 3A–B). These results suggest that the incorporation of TAIII significantly enhances the antitumor efficacy of conventional AUF liposomes.

Fig. 3.

Fig. 3

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T-AUF-LPs suppress cell viability and induce cell death in ATC cells. A–B In vitro cytotoxicity of various formulations (free AUF, AT, C-AUF-LPs, T-AUF-LPs) in CAL-62 and 8505C cells. C–F Analysis of cell death by flow cytometry

Effect of T-AUF-LPs liposomes on ferroptosis

To investigate the mechanism by which T-AUF-LPs suppress cell viability, cell death was first examined using flow cytometry. Compared to the control group (CON, 3.4%), all drug-treated groups exhibited increased cell death: TAIII (5.5%), AUF (8.1%), AT combination (9.1%), C-AUF-LPs (11.0%), and T-AUF-LPs (14.7%) (P < 0.001), with T-AUF-LPs showing a particularly pronounced effect. Pretreatment with the ferroptosis inhibitor Ferrostatin-1 (Fer-1) for 2 h significantly attenuated cell death induced by both C-AUF-LPs and T-AUF-LPs, reducing the rates to 7.7% and 8.8%, respectively (P < 0.001) (Fig. 3C–D). A similar trend was observed in the 8505C cell line, where cell death rates were as follows: CON (7.9%), TAIII (8.3%), AUF (8.9%), AT combination (18.6%), C-AUF-LPs (19.8%), and T-AUF-LPs (25.6%) (P < 0.001). Again, Fer-1 pretreatment markedly reversed cell death induced by C-AUF-LPs and T-AUF-LPs, lowering the rates to 12.2% and 13.5%, respectively (P < 0.001) (Fig. 3E–F). These results suggest that ferroptosis serves as a key cell death mechanism.

To detect core indicators of ferroptosis, 8505C cells treated with various drug groups for 24 h were incubated with ROS probes at 37 °C for 30 min. Confocal microscopy results showed enhanced green fluorescence in the T-AUF-LPs treatment group (Fig. 4A). Semi-quantitative analysis confirmed a significant increase in mean fluorescence intensity in the T-AUF-LPs treatment group (Fig. 4B) (P < 0.05), indicating elevated intracellular ROS levels in 8505C cells.

Fig. 4.

Fig. 4

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Intracellular ROS and lipid peroxide levels in 8505C cells. A CLSM image of intracellular ROS of 8505C cells after incubation with TAIII, AUF, AT, C-AUF-LPs and T-AUF-LPs. Scale bar represents 100 μm. B Analysis of mean fluorescence intensity for intracellular ROS in 8505C cells (CLSM). C Representative confocal microscopy images of cells stained with C11-BODIPY 581/591 probe after treatment with various formulations for 24 h. Red fluorescence indicates reduced probe (non-oxidized), while green fluorescence indicates oxidized probe, reflecting LPO levels. Scale bar: 100 μm. D Lipid peroxidation analysis (fluorescence ratio, green/red) normalized to control *P < 0.05, **P < 0.01, ***P < 0.001

To further investigate the mechanism underlying ROS elevation, CAL-62 cells were similarly treated with different drug groups for 24 h and stained with C11-BODIPY 581/591 fluorescent probe, followed by 30-min incubation at 37 °C in darkness. Confocal microscopy observations (Fig. 4C) demonstrated reduced red fluorescence and enhanced green fluorescence in the T-AUF-LPs treatment group. Semi-quantitative analysis confirmed a significant increase in relative fluorescence intensity (Fig. 4D) (P < 0.05), indicating elevated levels of intracellular lipid peroxidation in CAL-62 cells.

Simultaneously, molecular level analysis demonstrated that T-AUF-LPs treatment significantly modulated the expression of key ferroptosis-related molecules in ATC cells. PCR results revealed that compared with the control group, T-AUF-LPs markedly upregulated mRNA levels of ACSL4 and NCOA4 (P < 0.001) while downregulating GPX4 mRNA expression (P < 0.05) (Fig. 5A–C). Furthermore, the T-AUF-LPs group exhibited significantly elevated intracellular iron levels (P < 0.001) (Fig. 5D) and substantially reduced GSH levels (P < 0.001) (Fig. 5E), collectively establishing the essential biochemical environment for ferroptosis induction. Among all experimental groups, T-AUF-LPs demonstrated the most pronounced regulatory effects.

Fig. 5.

Fig. 5

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T-AUF-LPs trigger ferroptosis in ATC cells. A–C The mRNA expression levels of ferroptosis-related genes (ACSL4, NCOA4, GPX4) were analyzed by RT-qPCR after T-AUF-LPs treatment for 24 h. D Intracellular iron content in ATC cells. E Intracellular GSH levels in ATC cells

Anti-tumor activity of T-AUF-LPs liposomes in vivo

T-AUF-LPs demonstrated significant antitumor efficacy in the xenograft model, with its mechanism closely associated with the activation of the ferroptosis pathway. Specifically, compared with the control group, T-AUF-LPs treatment markedly suppressed tumor growth (Fig. 6A–B) (P < 0.05) without causing significant reduction in mouse body weight (Fig. 6C), indicating both therapeutic effectiveness and preliminary safety. Further molecular investigations revealed that T-AUF-LPs effectively modulated key ferroptosis markers in tumor tissues: on one hand, they significantly upregulated the expression of ACSL4 and NCOA4 at both mRNA (P < 0.01, P < 0.001) and protein (P < 0.05) levels; on the other hand, they substantially suppressed GPX4 expression at both mRNA (P < 0.05) and protein (P < 0.05) levels (Fig. 6D–H). This concerted modulation of pivotal molecules creates a favorable microenvironment for ferroptosis induction, consistent with the in vitro findings. Moreover, histopathological analysis of major organs revealed no significant toxic damage (Fig. 7), ruling out the possibility of treatment-related tissue injury. Thus, this study confirms at the in vivo level that T-AUF-LPs can safely and effectively inhibit ATC growth by remodeling the regulatory network of tumor ferroptosis.

Fig. 6.

Fig. 6

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Inhibition of ATC tumor growth by T-AUF-LPs in vivo. A Images of CAL-62 tumors after receiving various treatments. B Relative tumor volume curves. C Body weight changes of mice. D–F mRNA expression levels of NCOA4, ACSL4, and GPX4 in tumor tissues. G–H Protein expression levels of NCOA4, ACSL4, and GPX4 in tumor tissues

Fig. 7.

Fig. 7

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Biosafety evaluation of T-AUF-LPs. Representative H&E-stained images of major organs from treated mice. Scale bar: 100 μm

Discussion

Anaplastic Thyroid Cancer (ATC) is a rare yet highly aggressive malignant tumor. Due to its rapid progression, strong invasiveness, and early metastasis, patients are often diagnosed at an advanced stage of the disease, missing the optimal window for surgical intervention, which poses significant challenges for clinical management (Maniakas et al. 2022). AUF, primarily known as a TXNRD1 inhibitor for treating rheumatoid arthritis, has also demonstrated promising efficacy against various other cancer types (Deben et al. 2024). Furthermore, a 2024 study by An et al. preliminarily suggested that AUF acts in ATC via the apoptosis pathway, although its deeper mechanisms remain unclear (An et al. 2024). To further explore its mechanism of action, we subsequently investigated whether AUF exerts its inhibitory effects on ATC cells through the ferroptosis pathway. However, the widespread application of high-dose AUF is limited by its severe systemic toxicity, which compromises its effectiveness as a chemotherapeutic agent. TAIII, a natural triterpenoid saponin, exhibits lower toxicity compared to traditional chemotherapeutic drugs. Substantial in vitro and in vivo data indicate its promising potential in cancer therapy (Tu et al. 2024). For instance, Hyun Min Ko et al. found that Timosaponin A3 (TA3) induces apoptosis in HCT116 colorectal cancer cells by suppressing c-Myc expression mediated by CNOT2 or MID1IP1(Ko et al. 2022). Zhang et al. reported that TAIII induces G2/M phase arrest and apoptosis in breast cancer cells by activating the ATM/Chk2 and p38 MAPK signaling pathways (Zhang et al. 2021a, b). Additionally, Zhou et al. demonstrated that TAIII promotes ferroptosis in non-small cell lung cancer by targeting and facilitating HSP90-mediated ubiquitination and degradation of GPX4 (Zhou et al. 2023). Shen et al. discovered that TAIII induces lipid peroxidation and ferroptosis in colorectal cancer cells by enhancing Rab7-mediated lipophagy (Shen et al. 2024a, b). Despite this promise, TAIII also faces the challenge of low bioavailability (Wang et al. 2021). To overcome the delivery limitations of single agents and enhance their efficacy, nanodelivery systems have emerged as a promising strategy. Recent studies show that liposomes, commonly used nanocarriers composed of lipid bilayers forming enclosed vesicles, can preferentially accumulate in tumor tissue through the Enhanced Permeability and Retention (EPR) effect and are effective for co-delivering various anticancer drugs (Guimarães et al. 2021). However, they also have limitations; traditional liposomes rely on cholesterol as a membrane stabilizer, which can oxidize producing toxic oxidative products. This not only compromises the integrity and stability of the liposomal membrane but can also cause damage to normal human tissues and organs (Wang et al. 2019). Notably, the hydrophobic polycyclic structure of TAIII allows it to effectively insert into the lipid bilayer. By enhancing membrane stability and reducing membrane fluidity, it provides advantageous conditions for drug delivery systems, thereby improving delivery efficiency (Zhang et al. 2021a, b).

To more clearly elucidate the innovativeness of this study, we compared the TAIII liposome system with previously reported saponin-based liposomes in the following aspects: First, differences in drug combinations. Previous saponin-based liposomes predominantly loaded conventional chemotherapeutic agents (e.g., paclitaxel) (Zhu et al. 2023) or other natural products (e.g., curcumin) (Liu et al. 2026). In this study, AUF (a thioredoxin reductase inhibitor) was co-loaded with TAIII into the saponin-based liposome system. Second, TAIII exerts antitumor effects via ferroptosis pathways. Previously reported saponin-based liposomes primarily rely on the action mechanisms of their loaded chemotherapeutic agents (e.g., inducing apoptosis) (Yu et al. 2025). In contrast, TAIII exerts antitumor effects through ferroptosis—a non-apoptotic cell death mechanism—manifested by downregulation of GPX4 expression, accumulation of lipid reactive oxygen species, and elevated levels of related metabolites (Zhou et al. 2023). In contrast to previously reported saponin-based liposomes (which exert anti-tumor effects primarily through apoptosis), and distinct from conventional cholesterol-based liposomes loaded with a single agent (which induce either apoptosis or ferroptosis via a single pathway), our T-AUF-LPs operate through a fundamentally different mechanism. Specifically, this system triggers non-apoptotic ferroptosis via a dual-axis synergistic action: downregulation of GPX4 (antioxidant defense), upregulation of ACSL4 (lipid peroxidation), and upregulation of NCOA4 (iron availability). This multi-node regulatory pattern—simultaneously targeting iron metabolism, antioxidant capacity, and lipid peroxidation—has not been achieved in previous liposomal ferroptosis therapies. This unique death mechanism not only distinguishes itself from conventional saponin-based liposome systems but also provides a novel strategy for overcoming tumor cell apoptosis resistance.

Building on this, our research utilized TAIII to replace cholesterol in traditional liposomes, constructing a bilayer phospholipid system using soybean lecithin. Physicochemical characterization results showed that T-AUF-LPs exhibited superior encapsulation efficiency and drug loading capacity compared to C-LPs. When TAIII is integrated into the liposomal bilayer replacing cholesterol, it enhances the intermolecular interactions between phospholipids, increasing the molecular packing order and density, thereby reducing membrane gaps. This ultimately leads to structural compression of the T-AUF-LPs, resulting in a significantly smaller particle size. Furthermore, over a 7-day storage period, T-AUF-LPs showed smaller fluctuations in particle size and Polydispersity Index (PDI) compared to conventional liposomes (C-GEM-LPs). Using the fluorescent marker coumarin 6 incorporated into the liposome phase, we observed that T-AUF-LPs exhibited stronger green fluorescence intensity in both CAL-62 and 8505C cells, indicating enhanced cellular uptake ability. Regarding the in vitro release behavior of T-AUF-LPs, we acknowledge that these data were not included in the current study. Notably, the release profile of TAIII-loaded liposomes has been characterized by Lu et al. (2018) under pH 7.4 conditions, and that study confirmed their sustained-release characteristics(Lu et al. 2018). The present study primarily focused on the in vivo antitumor efficacy and mechanistic validation of T-AUF-LPs; therefore, the in vitro release data were not incorporated into the main text. We recognize this as a limitation of our study and will conduct a systematic evaluation of the in vitro release profile of T-AUF-LPs under relevant conditions in our future work.

Cell-based experiments showed that after 24 hours of drug treatment, T-AUF-LPs exhibited the strongest antiproliferative effect on ATC cells, with the lowest half-maximal inhibitory concentration (IC₅₀). To elucidate the underlying mechanism of this potent effect, we first assessed the mode of cell death using flow cytometry. The results showed that T-AUF-LPs treatment significantly increased cell death, an effect that was markedly reversed by the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1), preliminarily suggesting a key role for ferroptosis. Ferroptosis is an iron-dependent form of regulated cell death characterized by the massive accumulation of LPO (Chen et al. 2021). To systematically validate its involvement in T-AUF-LPs-induced cell death, we conducted an in-depth investigation across three key aspects: iron metabolism, antioxidant defense, and lipid peroxidation. Regarding iron metabolism, the T-AUF-LPs treatment group showed a significant increase in intracellular total iron content (P=0.019) and significant upregulation of the key iron metabolism regulator NCOA4 at both the transcriptional and protein levels (P<0.001), indicating intracellular free iron accumulation, which provides the necessary ionic foundation for ferroptosis (Ohshima et al. 2022; Santana-Codina et al. 2022).In terms of the antioxidant system, T-AUF-LPs treatment led to a significant decrease in the mRNA and protein expression of the core antioxidant enzyme GPX4 (P<0.001), accompanied by depletion of intracellular reduced glutathione (GSH) (P<0.001), reflecting a severe weakening of the cell's intrinsic antioxidant defense system(Qiao et al. 2024; Liu et al. 2023).Furthermore, we focused on the core execution event of ferroptosis – the accumulation of LPO. Acyl-CoA Synthetase Long-Chain Family Member 4 (ACSL4) is a crucial promoter of lipid peroxidation. It catalyzes the activation of polyunsaturated fatty acids (PUFAs) like arachidonic acid (AA) and adrenic acid (AdA) into acyl-CoA esters (PUFA-CoAs). These are then incorporated into lysophosphatidylethanolamine (Lyso-PE) by lysophospholipid acyltransferases, generating lipid substrates prone to peroxidation (Chen et al. 2023; Cui et al. 2024). In this study, T-AUF-LPs treatment significantly upregulated ACSL4 mRNA and protein expression levels (P<0.001), indicating effective promotion of the biosynthesis of peroxidation-susceptible lipid substrates.

To directly observe changes in lipid peroxidation levels, we first detected an overall increase in reactive oxygen species (ROS) levels in 8505C cells using the DCFH-DA probe. Based on the premise that ROS is a key driver of lipid peroxidation, we further validated this in CAL-62 cells using the highly lipid peroxidation-sensitive C11-BODIPY 581/591 probe. Both confocal microscopy observation and semi-quantitative analysis consistently showed that in CAL-62 cells treated with T-AUF-LPs, the red fluorescence (non-oxidized state) of the probe was significantly weakened, while the green fluorescence (oxidized state) correspondingly increased, confirming a significant elevation in lipid peroxidation levels. However, the biological link from "ROS elevation" to "occurrence of lipid peroxidation" involves multi-layered complexity. First, although the DCFH-DA probe used in this study is widely employed, it responds to the total intracellular oxidative state, including various ROS such as hydrogen peroxide and peroxynitrite. The chemical activity of different ROS species and their efficiency in initiating lipid peroxidation vary greatly. Therefore, the fluorescence enhancement we observed more accurately reflects an overall change of the cellular oxidative stress state, which creates the necessary oxidative environment for downstream lipid peroxidation (Yang et al 2021, 2024a). Second, the C11-BODIPY probe detects the endpoint of lipid peroxidation, which is a chain amplification process itself. The initial ROS attack on polyunsaturated fatty acids generates lipid radicals, which can then trigger self-sustaining chain reactions. This means that the lipid peroxidation signal induced by T-AUF-LPs may stem not only from the direct attack of initial ROS but also includes the secondary oxidative damage triggered thereby (Shen et al 2024a, b). Furthermore, different cell lines (such as 8505C and CAL-62) may exhibit different ROS accumulation kinetics and lipid peroxidation thresholds in response to the same oxidative stimulus due to variations in their antioxidant defense systems (e.g., GSH levels, antioxidant enzyme activity). This is one of the reasons we conducted stepwise validation in different cells. In summary, our data clearly indicate that T-AUF-LPs can effectively trigger intense oxidative stress within tumor cells, ultimately leading to irreversible peroxidation damage to the critical biomacromolecules—lipids. This process may involve a complex dynamic balance between the specificity of ROS species, the chain propagation of oxidative damage, and the cell's own antioxidant compensatory mechanisms. By co-delivering AUF and TAIII, T-AUF-LPs likely disrupts this balance, effectively pushing tumor cells toward an oxidative damage-driven death pathway, which provides an important mechanistic explanation for its significant anti-tumor efficacy.

In animal model studies, we further validated the significant anti-tumor effect and excellent biosafety profile of T-AUF-LPs mediated through the induction of ferroptosis. Phenotypically, in vivo tumor inhibition results showed that compared to the control group, T-AUF-LPs treatment significantly suppressed tumor growth in nude mice, resulting in markedly reduced tumor volume, demonstrating the potent anti-tumor activity of this formulation in vivo. Subsequently, to elucidate the molecular pathway, we conducted systematic molecular biological detection on tumor tissues. The results showed that in the T-AUF-LPs treatment group, the mRNA and protein expression of the key ferroptosis regulator GPX4 were significantly downregulated, while the mRNA and protein expression of ACSL4 and NCOA4 were significantly upregulated. This expression profile change was highly consistent with our earlier cell-based findings, collectively revealing the core mechanism by which T-AUF-LPs induces ferroptosis: on one hand, by weakening the intracellular antioxidant defense system represented by GPX4, and on the other hand, by enhancing ACSL4-mediated lipid metabolic reprogramming and the NCOA4-regulated ferritinophagy process, thereby synergistically promoting lipid peroxide accumulation and increasing intracellular free iron levels, ultimately triggering iron-dependent cell death. Furthermore, this study systematically evaluated its biosafety. Throughout the experimental observation period, the body weight of the nude mice in all groups showed no significant decrease, suggesting the treatment regimen did not induce systemic toxic reactions. Further histopathological analysis of vital organs such as the heart, liver, lungs, and kidneys revealed no obvious structural damage or pathological alterations, fully demonstrating that the T-AUF-LPs formulation possesses good biocompatibility and safety alongside its efficient anti-tumor action.

Limitations of the present study

We acknowledge several limitations in the current study that should be addressed in future work. First, the present study did not directly compare the pharmacokinetic profile of the T-AUF-LPs formulation with that of free drugs, thus precluding direct evidence of improved in vivo delivery efficiency. This decision was primarily due to our initial focus on formulation development, physicochemical characterization, and validation of functional advantages in vitro and in vivo. Nevertheless, indirect evidence supports improved delivery: enhanced cellular uptake (using coumarin 6 as a fluorescent marker), lower IC₅₀ values, and more potent in vivo tumor suppression with concomitant alterations in ferroptosis-related markers (GPX4 downregulation, ACSL4 and NCOA4 upregulation). Systematic pharmacokinetic studies remain a critical future direction. Second, the in vitro release profile of T-AUF-LPs was not characterized in this study. While Lu et al. (2018) have demonstrated sustained-release characteristics of TAIII-loaded liposomes under pH 7.4 conditions, we recognize that systematic evaluation of the release behavior of our dual-agent formulation under relevant physiological conditions is warranted. Third, all in vivo experiments were conducted using immunodeficient nude mice, which do not fully recapitulate the intact immune microenvironment of human tumors. Given the emerging crosstalk between ferroptosis and anti-tumor immunity, further validation in immunocompetent models is necessary.

Clinical translation potential

Despite the above limitations, the T-AUF-LPs system demonstrates promising potential for clinical translation in the treatment of anaplastic thyroid cancer (ATC) and potentially other ferroptosis-sensitive malignancies.

Favorable safety profile. Compared to conventional chemotherapeutic agents and high-dose AUF, our formulation exhibits significantly reduced systemic toxicity. No significant body weight loss or histopathological abnormalities in major organs (heart, liver, lungs, kidneys) were observed in treated nude mice, supporting its favorable biocompatibility.

Carrier-as-therapy design. The replacement of cholesterol with TAIII—a natural product with over two thousand years of clinical use history in traditional Chinese medicine—not only avoids cholesterol oxidation-related toxicity but also transforms the carrier into an active therapeutic component. This dual-function strategy enhances efficacy while simplifying formulation composition.

Multi-node ferroptosis induction. By simultaneously targeting GPX4 (downregulation), ACSL4 (upregulation), and NCOA4 (upregulation), our system induces non-apoptotic ferroptosis, offering a potential strategy to overcome apoptosis resistance—a major obstacle in ATC and many refractory tumors.

Leveraging EPR effect. As a liposomal formulation, T-AUF-LPs can passively accumulate in tumor tissues via the enhanced permeability and retention (EPR) effect, potentially reducing off-target toxicity compared to free drugs.

However, several challenges must be addressed before clinical application. Scalable manufacturing: As a complex nano-formulation co-loading two components, robust and reproducible production processes must be established with rigorous quality control over particle size, encapsulation efficiency, drug ratio, and sterility (Inguva et al 2025).Patient stratification: Given that ferroptosis sensitivity varies among tumors, identifying predictive biomarkers (e.g., GPX4, ACSL4, or NCOA4 expression levels) will be crucial for selecting patients most likely to benefit from T-AUF-LPs therapy. Advanced preclinical models, such as patient-derived xenografts (PDXs) or organoids, as well as immunocompetent and humanized mouse models, are needed to further validate efficacy and guide biomarker development (Alawi et al 2025). Regulatory pathway: The use of a natural saponin (TAIII) as both a membrane component and a therapeutic agent represents a novel regulatory paradigm, requiring careful documentation of its pharmacokinetics, biodistribution, and long-term safety (Lambrechts et al 2018).

Future directions

To advance T-AUF-LPs toward clinical translation, future work should focus on: (1) conducting systematic pharmacokinetic and biodistribution studies; (2) evaluating in vitro release profiles under physiologically relevant conditions; (3) validating efficacy and mechanism in immunocompetent and patient-derived models; (4) developing a scalable, GMP-compliant manufacturing process; (5) performing comprehensive long-term toxicological and immunotoxicity assessments; and (6) exploring combination strategies with existing therapies (e.g., immune checkpoint inhibitors or radiotherapy) to further enhance anti-tumor efficacy.

In conclusion, this study successfully constructed a multifunctional liposomal system, T-AUF-LPs. This system not only serves as an efficient carrier enhancing drug accumulation at the tumor site, but its intrinsic TAIII component also exerts synergistic therapeutic effects. More importantly, from the perspective of inducing ferroptosis, we systematically elucidated the mechanism through which this system disrupts intracellular redox homeostasis, thereby significantly enhancing anti-tumor activity and overcoming drug resistance. This provides a new strategic direction for the clinical treatment of ATC.

Author contribution

Xinyi Deng wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.

Funding

National Natural Science Foundation of China (81971650); Tianjin Medical Key Discipline (Specialty) Construction Project Funding (TJYXZDXK-001A); Tianjin Municipal Science and Technology Commission Fund (21JCYBJC01820); Funded by Tianjin Key Medical Discipline Construction Project (Grant No.TJYXZDXK-3-003B); Tianjin Health Research Project (No. TJWJ2025RC011).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

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