A Co-catalytic nanosystem based on molybdenum
disulfide and Prussian blue for synergistic
chemodynamic and photothermal therapy through
mitochondrial damage and ferroptosis

Yilin He, Chunxu Lv, Nolwenn Le Breton, Haijun Peng, Tengfei Wang, Paolo Samorì, Sylvie Choua, Alberto Bianco, Baojin Ma, Cécilia Ménard-Moyon

► To cite this version:

Yilin He, Chunxu Lv, Nolwenn Le Breton, Haijun Peng, Tengfei Wang, et al.. A Co-catalytic nanosys-
tem based on molybdenum disulfide and Prussian blue for synergistic chemodynamic and photothermal
therapy through mitochondrial damage and ferroptosis. Acta Biomaterialia, 2025, 204, pp.518-533.
10.1016/j.actbio.2025.07.037 . hal-05247212

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# A Co-catalytic nanosystem based on molybdenum disulfide and Prussian blue for synergistic chemodynamic and photothermal therapy through mitochondrial damage and ferroptosis

<figure><img src="image_3.png" /></figure>

Yilin He<sup>a,1</sup>, Chunxu Lv<sup>b,1</sup>, Nolwenn Le Breton<sup>c</sup>, Haijun Peng<sup>d</sup>, Tengfei Wang<sup>a</sup>, Paolo Samorì<sup>d</sup>, Sylvie Choua<sup>c</sup>, Alberto Bianco<sup>a,* ID</sup>, Baojin Ma<sup>b,* ID</sup>, Cécilia Ménard-Moyon<sup>a,e,* ID</sup>

## ARTICLE INFO

**Keywords:**

Biomedical

Cancer therapy

Fenton reaction

Inorganic nanoparticles

Molybdenum disulfide

Reactive oxygen species

## ABSTRACT

Chemodynamic therapy (CDT) is regarded as an emerging strategy with high specificity for tumor therapy by producing highly toxic reactive oxygen species (ROS) in tumor cells by a Fenton or Fenton-like reaction. Excessive ROS can cause mitochondrial damage and induce ferroptosis in cells, leading to the death of cancer cells. However, the generally low efficiency of the Fenton reaction has limited the effectiveness of CDT. Herein, two-dimensional MoS₂ decorated with PB NPs is used as a co-catalyst to promote Fe³⁺/Fe<fnref n="1" />⁺ conversion and thus enhance the efficiency of the Fenton reaction. The photothermal properties of both MoS₂ and PB NPs further enhance the Fenton reaction, eventually producing a large amount of ROS. Mitochondrial damage and ferroptosis caused by ROS are evidenced *in vitro* and in a tumor-bearing mouse model and jointly lead to a decrease in heat shock protein content, further enhancing the photothermal effect of PB NP/MoS₂ nanosystem. This chemo-dynamic/photothermal synergistic therapy allows achieving good anticancer therapeutic effect.

**Statement of significance:** CDT is a promising cancer treatment that selectively generates toxic ROS to eliminate tumor cells. Nevertheless, its efficacy is often limited by the low efficiency of the Fenton reaction. This study presents a nanocomposite composed of MoS₂ nanosheets decorated with PB NPs, which enhances CDT by improving Fe³⁺/Fe<fnref n="1" />⁺ conversion and increasing ROS production. In addition, the photothermal properties of the material further amplify its therapeutic effects. In cell and animal models, this synergistic approach effectively induces mitochondrial damage and ferroptosis, thereby weakening the defenses of the cancer cells. This work provides a significant advancement in CDT, offering a more potent strategy for cancer therapy.

## 1. Introduction

Cancer is an ever increasing health threat that continuously demands new and innovative approaches to curb its spread. To overcome the shortcomings of traditional cancer treatment, such as low selectivity, long-term side effects, drug resistance, and high cost, advanced therapeutic strategies have been explored [1,2]. Among them, chemodynamic therapy (CDT) relies on the generation of highly toxic *OH by the Fenton or the Fenton-like reactions under high-level of endogenous H₂O₂ in

cancer cells, resulting in their selective elimination [1,3]. Interestingly, ROS generation through the Fenton reaction can trigger lipid peroxidation accumulation and induce irreversible damage to mitochondria, which can cause ferroptosis, thereby enhancing the lethality to cancer cells [4–7]. Although significant progress has been made in improving current CDT strategies, further development is needed before they can progress to clinical trials [8]. Indeed, some limitations of CDT weaken the therapeutic efficiency. For example, the rate of Fe³⁺/Fe<fnref n="1" />⁺ conversion is very slow, which has been recognized as the rate-limiting step in the

<footnote><sup>a</sup> CNRS, Immunology, Immunopathology and Therapeutic Chemistry, UPR 3572, University of Strasbourg, ISIS, 67000 Strasbourg, France</footnote>

<footnote><sup>b</sup> Department of Periodontology & Tissue Engineering and Regeneration, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, 250012, China</footnote>

<footnote><sup>c</sup> Institute of Chemistry, UMR 7177, Université de Strasbourg, 67000 Strasbourg, France</footnote>

<footnote><sup>d</sup> Université de Strasbourg, CNRS, ISIS UMR 7006, 8 allée Gaspard Monge, 67000 Strasbourg, France</footnote>

<footnote><sup>e</sup> University of Strasbourg Institute of Advanced Study (USIAS), 67000 Strasbourg, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: a.bianco@ibmc-cnrs.unistra.fr (A. Bianco), baojinma@sdu.edu.cn (B. Ma), c.menard@ibmc-cnrs.unistra.fr (C. Ménard-Moyon).</footnote>

<footnote n="1">¹ These authors contributed to this work equally.</footnote>
Fenton reaction [9]. Therefore, there is an urgent need to design a strategy that can generate a large amount of ROS through a high Fenton reaction rate, achieving effective CDT and ferroptosis. Furthermore, a single therapy is generally not efficient enough to completely eradicate tumors and metastases [2]. Whereupon, synergistic therapies are thriving currently, which could integrate the advantages and minimize the disadvantages of single therapies [10,11].

Molybdenum disulfide (MoS₂), a two-dimensional material known for its rapid charge recombination and relatively low quantum efficiency, holds promise as a catalyst and carrier [12,13]. Recently, MoS₂ has been identified as an effective co-catalyst, significantly enhancing the Fenton efficiency by promoting Fe³⁺/Fe²⁺ conversion with the assistance of Mo⁴⁺/Mo⁵⁺ couple [9,14]. The photocatalytic Fenton process involving MoS₂/Fe has shown success in degrading organic pollutants [15]. However, how to prepare in situ-grown Fe/MoS₂ nanosystem with good dispersibility and targeting capability for cancer therapy is still a challenge and limits biological applications. Hence, it is essential to explore smart and effective MoS₂/Fe composites to enhance CDT.

PB NPs, with the ideal formula of Fe<sup>II</sup><sub>4</sub>[Fe<sup>II</sup>(CN)<sub>6</sub>]₃·nH<sub>2</sub>O, have shown promising potential for biomedical applications [16]. Although PB NPs

can be converted to Prussian green (PG) in the presence of H₂O₂ through the Fenton reaction, their application in CDT remains largely unexplored, primarily due to the limited efficiency of the Fenton reaction rate [17–19]. Therefore, if MoS₂ can be employed as a co-catalyst to accelerate the Fe³⁺/Fe²⁺ conversion of PB in the Fenton reaction, it may potentially enhance the efficiency of the reaction and facilitate the generation of ROS. Besides, it is worth noting that PB NPs show good photothermal conversion efficiency stemming from the charge transfer in the Fe³⁺-CN-Fe²⁺ bond [20]. The good photothermal effect can enhance the Fenton efficiency, as demonstrated by previous studies by our team and other researchers [21–23]. Therefore, endowing a CDT nanoplatform with photothermal properties could achieve a synergistic effect. However, photothermal therapy (PTT) is limited by the self-protective nature of cells, which upregulate the production of heat shock proteins (HSPs) upon hyperthermia [24–26]. This self-protective mechanism makes cancer cells thermotolerant, thereby reducing the efficacy of PTT. Interestingly, recent studies have found that mitochondrial damage and ferroptosis can inhibit HSP production [27–30]. Indeed, intracellular ROS and lipid peroxidation can disrupt the structure of HSPs, thus reducing the stress protective capacity of cancer cells,

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** (a) Preparation route of MoS₂-PB-TEG-FA (MPF) NSs. (b) Schematic illustration of the co-catalytic mechanism between MoS₂ NSs and PB NPs. (c) Schematic illustration of the mechanism of mutually boosted combined CDT and PTT.</figcaption></figure>
thereby enhancing the photothermal effect [31].

In this study, we propose a strategy to synthesize a MoS₂-PB (named MP) nanohybrid with MoS₂ nanosheets (NSs) decorated with PB NPs (Fig. 1a). MoS₂ NSs were produced by liquid-phase exfoliation and then PB NPs were grown on the surface. To impart targeting capability, folic acid (FA) was conjugated onto PB NPs via a triethylene glycol (TEG) linker to obtain MoS₂-PB-TEG-FA (named MPF). Under acidic conditions and in the presence of endogenous H₂O₂, MPF NSs generated a substantial amount of ROS through a co-catalytic Fenton reaction between MoS₂ NSs and PB NPs (Fig. 1b). As expected, the photothermal effect of MPF NSs can synergistically promote the Fenton reaction. The generated ROS can induce ferroptosis to amplify cancer cell death. Moreover, we expect ROS to also cause mitochondrial damage and decrease the expression of HSPs, thereby reducing tumor heat resistance and resulting in a CDT-enhanced PTT (Fig. 1c).

## 2. Experimental section

### 2.1. Materials

Triethylene glycol diamine (NH₂-TEG-NH₂), N-hydroxysuccinimide, K₃[Fe(CN)₆]·3H₂O, FeCl₃, citric acid, vitamin E, 5-dimethyl-1-pyrroline N-oxide (DMPO), 1-ethyl-3-diaminopropyl carbodiimide, ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1) and intracellular GPX test kit were ordered from Sigma-Aldrich. FA and rhodamine B (RhB) were ordered from Alfa Aesar. MoS₂ was purchased from Acros. JC-1 (5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethyl benzimidazolylcarbocyanine iodide) was ordered from Solarbio. Methylene blue (MB) was ordered from Fluka. RPMI 1640 medium, Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Corning Incorporated. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and intracellular glutathione (GSH) test kit were bought from Promega Corporation. The 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA), C11-BODIPY and Live/Dead™ cytotoxicity kit were ordered from Thermo Fisher Scientific. Filtration membranes (0.1 µm pore size) were purchased from Merck Millipore. Bicinchoninic acid (BCA) protein test kit was bought from Solarbio Life Sciences, China. BBoxiProbe®O13 probe, a cell-permeable oxidative-sensitive fluorescent dye, was purchased from Bestbio, China. Elma model Elmasonic P was used to sonicate and disperse the materials. Centrifugation was performed using Eppendorf centrifuge 5418 R.

### 2.2. Preparation of MPF NSs

MoS₂ NSs were firstly prepared through liquid-phase exfoliation in an ethanol/water (45 v%) mixture [32]. Briefly, 500 mg MoS₂ was dispersed in 91.6 mL of H₂O and 75 mL of ethanol, followed by bath sonication at 37 MHz for 5 h at a temperature below 30 °C. Then, the dispersion was centrifuged at 1610 g for 30 min. The supernatant was collected and filtered using a 0.1 µm membrane. The solid was dispersed in water just before use. The thickness of the MoS₂ NSs is expected to be around 2–4 nm, corresponding to 3–4 monolayers, as previously reported, [32] since the thickness of an MoS₂ monolayer is about 0.9–1.2 nm [33].

PB NPs were grown on MoS₂ NS by adding FeCl₃ (9.73 mg), K₃[Fe(CN)₆]·3H₂O (11.83 mg), citric acid (200 mg) and MoS₂ NSs (10 mg) in 100 mL of water at pH 2 and 60 °C for 3 h. The mass ratio of FeCl₃/K₃[Fe(CN)₆]·3H₂O/citric acid/MoS₂ NSs was 9.7:11.8:200:10. The mixture was filtered using a 0.1 µm membrane, the solid was washed using 300 mL of water for three times and the solid was dispersed in 10 mL of water. Different conditions were studied to optimize the growth of PB on MoS₂. The experiments are detailed in the Supplementary materials.

PB NPs alone as the control group were synthesized with slight modifications following a protocol reported in the literature [34]. Briefly, a 20 mL 1.0 mM FeCl₃ solution containing 0.5 mmol of citric

acid was prepared and slowly added into a 20 mL 1.0 mM K₄[Fe(CN)₆]·3H₂O solution containing 0.5 mmol of citric acid. The mixture was stirred vigorously at 60 °C for 10 min, then stirred at room temperature for 30 min. Then, the NPs were collected by centrifugation and washed with water three times (14,500 g).

NH₂-TEG-NH₂ was derivatized with FA by amidation following our previous protocol [35]. Then, NH₂-TEG-FA was conjugated onto the carboxyl groups of citric acid on the surface of MP NSs through amidation. Briefly, MP NSs (9.6 mg) were dispersed in 10 mL of water, followed by the addition of 1-ethyl-3-diaminopropyl carbodiimide (4 mg), N-hydroxysuccinimide (2.5 mg) and NH₂-TEG-FA (10 mg). After magnetic stirring for 24 h, the suspension was filtered and washed several times.

### 2.3. Characterization

UV–Vis–NIR absorption spectra were recorded on UV–Vis–NIR spectrophotometer (Cary 5000). Transmission electron microscopy (TEM) images were collected on a Hitachi 7500. Dynamic light scattering (DLS) and zeta potential measurements were done on a Zetasizer Lab (Malvern Panalytical, Worcestershire, UK) supplied with ZS Xplorer v2.01 software at 25 °C. A high-resolution TEM (2100F, JEOL) under 200 kV coupled to an energy-dispersive X-ray spectroscopy (EDX) spectrometer was employed to assess the chemical composition of the materials. Their crystal structure was studied using an X-ray diffractometer (Bruker D8 X-ray diffractometer). Scanning electron microscopy (SEM) images were recorded with a FEI Quanta FEG 250 instrument S3 (FEI corporate, Hillsboro, Oregon, USA). Fourier-transform infrared spectroscopy (FTIR) spectra were collected on a FT-IR 4700 Fourier Transform Infrared Spectrometer (JASCO) equipped with ATR Diamond. Thermogravimetric analyses (TGA) were performed by a TGA1 Mettler Toledo equipment using platinum pans under nitrogen atmosphere (50 mL/min) and a temperature ramp of 10 °C/min. X-ray photoelectron spectroscopy (XPS) analyses were performed on a Thermo Scientific KAlpha X-ray photoelectron spectrometer. The XPS experiments are detailed in the Supplementary materials. Electron paramagnetic resonance (EPR) spectroscopy was performed on an EMX plus spectrometer (Bruker Biospin GmbH) equipped with a high sensitivity resonator (4119-HSW, Bruker Biospin GmbH) and a variable temperature unit (ER4131VT, Bruker Biospin GmbH). The following parameters were used: microwave power of 5 mW, modulation amplitude of 0.2 mT at a frequency of 100 kHz. The field was swept over 120 G with 1001 points, a conversion time of 60 ms and a time constant of 10.24 ms. In the biological experiments, the quantitative analysis of fluorescent cell images was performed using ImageJ software. Nuclei were quantified by manually counting the stained cells, while cytoplasmic staining intensity was measured by analyzing the grayscale values of the fluorescence signal. The quantitative analysis was carried out based on more than three sets of fluorescent images.

### 2.4. Stability in cell culture medium

The stability of MPF NSs was assessed in the RPMI 1640 medium with 10 % FBS at 37 °C. Digital images, UV–Vis–NIR spectra, zeta potential and DLS data were collected before and after incubation.

### 2.5. Photothermal performance and generation of •OH by the Fenton reaction

The photothermal performance of MPF NSs was evaluated under irradiation at 808 nm laser (1.0 W/cm²). MoS₂ NSs and PB NPs were also studied as control groups. The photothermal conversion efficiency was calculated as reported in our previous work (calculations are shown in the Supplementary materials) [36].

To evaluate the •OH generation by the Fenton reaction of MPF NSs, colorimetric assays were performed. Firstly, the decolorization of MB
was measured. Briefly, 100 µg of MPF NSs were dispersed in 980 µL of PBS (pH 7.4 and 5.5). Then, 10 µL of 100 mM H₂O₂ and 10 µL of 10 mg/mL MB aqueous solution were added. The suspensions were analyzed by UV–Vis spectroscopy at different time points after centrifugation. The absorbance at 665 nm (corresponding to the maximum absorbance of MB) was measured. The effect of the temperature on the Fenton reaction was studied at 45 °C using a water bath. MoS₂ NSs and PB NPs were also studied as control groups under the same conditions. Using an 808-nm laser to perform this experiment for reaching 45 °C would have been challenging because of laser power limitations and durability of the laser system. Indeed, continuous use of an 808-nm laser for 1.5 h requires stable power output over extended periods. But, many laser systems are designed for short operational times. Therefore, a continuous irradiation for an extended period (like 1.5 h) could lead to laser power fluctuation or overheating of the laser device, reducing its effectiveness and potentially damaging the equipment. Additionally, the MB degradation was also conducted under different conditions: with/without H₂O₂ and under neutral/acidic conditions. Besides, the decoloration of RhB was also measured. For this purpose, 100 µg of MPF NSs were dispersed in 980 µL of PBS (pH 7.4 and 5.5). Then, 10 µL of 100 mM H₂O₂ and 10 µL of 10 mg/mL RhB were added. The absorbance of the suspensions was measured by UV–Vis spectroscopy at different time points after centrifugation.

To further investigate the Fenton reaction, the specific spin-trapping agent DMPO was used. The DMPO·*OH adduct was characterized by EPR spectroscopy. In detail, stock solutions were mixed to obtain a final solution of 1 mL at a concentration of 100 µg/mL of MPF NSs, 50 mM of DMPO and 2 mM H₂O₂. Then, the solution was introduced in a one-end sealed capillary (50 µL, Hirschmann) for EPR measurements.

## 2.6. Cell culture and cell viability

MCF-7 cells and HEK 293 cells were cultured in RPMI 1640 cell culture medium and DMEM and 10 % FBS. Briefly, MCF-7 cells and HEK 293 cells were seeded in 96-well plates at a density of ~8000 cells per well and ~2 × 10⁴ cells per well, respectively. After incubation with the materials, the cells were washed with PBS twice. The MTS assay was performed following the manufacturer's description.

## 2.7. Cellular uptake

MCF-7 cells were cultured in 12-well plates at a density of ~1 × 10⁴ cells per well. Then, 100 µg/mL of MPF NSs were incubated with cells for 4 h. Competition experiments were performed following our previous literature to confirm the receptor-mediated uptake [36,37]. Firstly, 10 mg of FA was added to 1 mL of PBS. After sonication for 10 min, the solution was centrifuged at 13,000 g for 20 min. Then, the cells were incubated with 50 µL of the supernatant for 2 h before adding MPF NSs. After incubation, the cells were washed with PBS twice and collected for ICP-MS analysis.

TEM observation was performed to assess the cellular uptake of MPF NSs. The TEM grids were prepared following the same protocol from our previous work [38]. The experiment is detailed in the Supplementary materials.

## 2.8. In vitro combined CDT/PTT

MCF-7 cells were seeded in 96-well plates at a density of 8000 cells per well, and cultured for 24 h. Then, four groups were set to study the combined CDT/PTT: (1) MPF, (2) MPF + Vitamin E, (3) MPF (NIR), and (4) MPF + Vitamin E (NIR). After incubation for 8 h, all the groups were washed with PBS. Groups 3 and 4 were irradiated by an 808-nm laser at a power density of 1.0 W/cm² for 10 min. Then all the groups were kept culturing to reach 24 h. After incubation with the materials, the cells were washed with PBS twice. Cells with and without irradiation were set as control groups. The cell viability was measured by the MTS assay kit,

and live/dead cell staining was performed using the live/dead staining kit.

## 2.9. Intracellular ROS generation

The intracellular ROS generation was evaluated by DCFH-DA assay. MCF-7 cells were treated with MPF NSs for 8 h. Then, irradiation by an 808-nm laser was performed at a power density of 1.0 W/cm² for 10 min. The cells incubated with MPF NSs without irradiation were set as the control group. The cells were stained with 10 µM DCFH-DA solution for 20 min. Following two additional washing with PBS, the plates were observed under a confocal scanning microscope with a Microscope Zeiss Axio Observer Z1 Confocal Spinning disk using a 488-nm laser and recording in the green channel (505–555 nm).

## 2.10. Mitochondrial membrane potential analysis and TEM of mitochondrial damage

Mitochondrial membrane potential (ΔΨm) analysis was performed to assess the impact of the treatments on mitochondria. MCF-7 cells were treated with 100 µg/mL of MPF NSs for 6 h and then irradiated with an 808-nm laser with a power density of 1.0 W/cm² for 10 min. The cells without treatment were set as control group. The cells were washed with PBS, followed by incubation with JC-1 in the dark at 37 °C for 30 min. Then, the cells were washed with PBS twice and observed by fluorescence microscopy.

Besides, the mitochondrial damage was observed by TEM. MCF-7 cells were cultured in 12-well plates at a density of ~10 × 10⁴ cells per well. Then, 100 µg/mL of MPF NSs were incubated with the cells for 24 h. The cells without MPF NSs served as control. The TEM grids were prepared as previously described.

## 2.11. Lipid peroxidation assay

Lipid peroxidation was evaluated using the C11-BODIPY fluorescence probe. MCF-7 cells were seeded in 96-well plates at a density of 8000 cells per well and cultured for 24 h. Then, MPF NSs were added to the MCF-7 cells and the incubation was performed for 8 h at 37 °C. The cells were washed by PBS and irradiated by an 808-nm laser at a power density of 1.0 W/cm² for 10 min. Afterwards, the cells were washed with PBS twice and incubated with C11-BODIPY (5.0 µM) for 20 min. The cells treated with MPF NSs without irradiation were set as control group.

## 2.12. Glutathione peroxidase 4 activity assay

The glutathione peroxidase 4 (GPX4) activity was assessed by a cellular GPX test kit. Briefly, MCF-7 cells were seeded in 96-well plates with 8000 cells per well. After 24 h, MPF NSs were added to the MCF-7 cells and the incubation was performed for 8 h at 37 °C. Then, the cells were washed with PBS and irradiated by an 808-nm laser at a power density of 1.0 W/cm² for 10 min. The cells treated with MPF without irradiation were set as control group. Then, the GPX4 activity was measured according to the kit instructions.

## 2.13. GSH level measurement

The GSH level was determined using the cellular GSH test kit. Briefly, MCF-7 cells were seeded in 96-well plates at a density of 8000 cells per well, and cultured for 24 h. MPF NSs were added to the MCF-7 cells and the incubation was performed for 8 h at 37 °C. Then, the cells were washed with PBS and irradiated by an 808-nm laser at a power density of 1.0 W/cm² for 10 min. The cells treated with MPF NSs without irradiation were set as control group. The GSH level was measured according to the kit instructions.
## 2.14. Cell viability under ferroptosis inhibition

MCF-7 cells were seeded in 96-well plates at a density of 8000 cells per well, and cultured for 24 h. Then, ferroptosis inhibitors, Fer-1 and liproxstatin-1, were added at different concentrations together with 100 µg/mL of MPF NSs. All groups were kept culturing for 24 h. The cell viability was measured using the MTS assay kit.

## 2.15. Western blot

To assess the intracellular expression of HSP70, the MCF-7 cells were seeded in 6-well plates at a density of $2 \times 10^4$ cells per well. After overnight incubation, the cells were treated as follows: no treatment (blank), incubation at 45 °C, MPF NSs (100 µg/mL), and MPF NSs (100 µg/mL) combined with 808 nm laser irradiation (1.0 W/cm², 10 min). Then, the cells were collected by trypsin and lysed in a lysis buffer. Subsequently, the proteins were extracted and concentrated by a BCA protein analysis according to the manufacturer's description. The Western blotting was carried out to detect the expression of HSP70 (Abcam) following the standard protocols.

## 2.16. Hematoxylin and eosin staining assay

Tissue sections were prepared by cutting and pasting the glass slide, and deparaffinized by immersing the slides in xylene for hematoxylin and eosin (H&E) staining. The tissues were then rehydrated by immersing the slides in descending concentrations of alcohol. The rehydrated tissues were stained with hematoxylin for 5 min and rinsed with water to remove excess hematoxylin. Subsequently, the tissues were stained with eosin for 1 min. The tissue slides were immersed in alcohol with increasing concentration to dehydrate the tissue, and then immersed in xylene to clean the tissue. The cover glasses were stuck on the glass slide with neutral glue for the next observation.

## 2.17. Immunohistochemical staining assay

Fresh tissue samples were fixed in a formalin solution, and then the tissue was embedded in paraffin. The section of the tissue was cut into 4 µm sections and placed on a slide. Deparaffinization of the slide was performed by soaking it in xylene, then with a graded series of ethanol solutions. Antigen repair was carried out by heating the slide in a citrate buffer. To minimize non-specific binding, the same kind of serum-blocking agent as the second antibody was used to block the slide. Primary antibodies (Ki67, CD31 and GPX4) were applied and the slides were cultured in a damp chamber for about 1 h. The glass slide was incubated with the second antibody bound with a reporter enzyme. 3,3'-diaminobenzidine tetrahydrochloride was added and the slide was incubated for 5 min. Then, the slide was soaked in hematoxylin for 2 min. The slide was washed in distilled water and dehydrated with gradient ethanol solution (50–100 %). Finally, the slide was mounted using a neutral gum before covering it with a glass cover slip.

## 2.18. TUNEL staining assay

The tissue sections were collected to be stained and fixed in 4 % paraformaldehyde for 30 min. Then, the fixed samples were washed with PBS three times, 5 min each. Following this, the samples were treated with an 0.1 % Triton X-100 solution in PBS at room temperature. Then, the samples were washed with PBS for 5 min, to remove any residual solution. The TdT enzyme, buffer and dUTP label in a ratio of 1:5:50 were mixed to prepare the TUNEL reaction solution, which was used to incubate the sections for 1 h at 37 °C in the dark. After washing, a counterstain was performed with 4',6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Finally, the samples were washed with PBS and mounted on slides using an anti-quenching mounting medium for observation by a fluorescent microscope.

## 2.19. Tissue ROS staining assay

Tumor tissues were harvested and cut into 10 µm sections after being frozen at the optimal cutting temperature. Tissue ROS levels were detected by using an O13 probe. After incubation in the dark for 30 min at 37 °C, the samples were washed again with PBS. A counterstain with DAPI was performed at room temperature for 5 min. The fluorescence intensity was detected at an excitation/emission of 510/610 nm.

## 2.20. Statistical analysis

All experiments were conducted at least three times and expressed as mean ± standard deviation. The statistical differences among samples versus control cells were calculated by one-way ANOVA (Bonferroni's test).

# 3. Results and discussion

## 3.1. Preparation and characterization

MoS₂ NSs were prepared via surfactant-free liquid-phase exfoliation. The average lateral size was assessed by TEM using Image J software, showing a value around 300 nm (Fig. S1a and S1c). Then, PB NPs were grown onto MoS₂ NSs in the presence of citric acid, which acted as a mild reducing agent to facilitate $Fe^{3+}$ reduction, [39] controlled PB size through iron chelation, [40] and stabilized the PB NPs by surface capping enhancing colloidal stability in aqueous solutions. During the growth of PB NPs on the MoS₂ NSs, $K_3[Fe(CN)_6]$ is likely reduced by MoS₂ to $K_4[Fe(CN)_6]$, which can adsorb on the surface of MoS₂, followed by the fast reaction between $[Fe(CN)_6]^{4-}$ and $FeCl_3$ to form PB NPs [41]. We optimized the conditions by controlling the amount of $FeCl_3$ and $K_3[Fe(CN)_6] \cdot 3H_2O$ versus MoS₂ (see Supplementary materials and Fig. S2). PB NPs in the absence of MoS₂ NSs were synthesized as control (Fig. S1b). TEM and SEM studies revealed that when MoS₂ NSs were used as seed or scaffold, cubic PB NPs were formed (Fig. 2a and 2b). High-resolution TEM showed that the size of PB NPs ranged from 50 to 80 nm (Fig. S3a). An analysis by EDX coupled to SEM and an elemental mapping confirmed the existence of Mo and S contributed by MoS₂ NSs and Fe, C and N from PB NPs (Fig. S3b and 2c). The UV–Vis–NIR spectra of as-prepared MoS₂ NSs and MP NSs showed the two characteristic excitonic absorption bands of MoS₂ NSs (Fig. 2d), consistent with previous reports [42]. After being modified with PB NPs, the wide absorption band contributed by the charge transfer between $Fe^{II}-CN-Fe^{III}$ at 700–800 nm appeared. The XRD patterns of MP NSs displayed new peaks (200), (220), (400), (420) corresponding to the characteristic crystal diffraction peaks of standard PB NPs (JCPDS card No 73–0687) (Fig. 2e) [43]. MP NSs were subsequently functionalized with FA to target the cancer cells. For this purpose, FA was derivatized with TEG diamine and the $NH_2-TEG-FA$ derivative was conjugated to PB NPs through amidation between the COOH groups of citric acid and $NH_2$ to obtain MPF NSs. The peak at $2060 \text{ cm}^{-1}$ assigned to the stretching of the $-C=O$ N-bond in the FTIR spectrum of PB NPs was also observed in the spectrum of MP and MPF NSs (Fig. 2f and S4). Furthermore, the peaks at $1480 \text{ cm}^{-1}$ and $1080 \text{ cm}^{-1}$ attributed to the C–H bending and C–O stretching in the TEG chain, were also present in the spectrum of MPF NSs, proving the conjugation of $NH_2-TEG-FA$ on MP NSs. In addition, TGA provided strong evidences for the functionalization of MP NSs with $NH_2-TEG-FA$. The weight loss of MoS₂ NSs under nitrogen atmosphere was negligible (Fig. 2g). The weight loss of MP NSs between 40 °C and 200 °C and between 280 °C and 400 °C was attributed to the loss of PB crystal water and decomposition of the metal-organic framework, respectively [44]. We also observed that the weight loss of MPF NSs was increased after 400 °C in comparison to MP NSs, indicating the pyrolysis of $NH_2-TEG-FA$. Besides, the size distribution after functionalization was measured by DLS. Although DLS is not appropriate for non-spherical materials, it allows monitoring the change in the size distribution
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Characterization of MPF NSs. (a) TEM images of MoS₂ (left) and MP NSs (right). (b) SEM image of MP NSs. (c) EDX elemental mapping of MP NSs. (d) UV–Vis–NIR spectra and (e) XRD patterns of MoS₂ and MP NSs. (f) FTIR spectra of MP NSs, MPF NSs and NH₂-TEG-FA (panel 2f is shown with annotation in the Supplementary materials as Fig. S4). (g) TGA of MoS₂, MP and MPF NSs, and NH₂-TEG-FA under nitrogen atmosphere. (h) DLS and (i) zeta potential of MoS₂, MP and MPF NSs.</figcaption></figure>

before and after the different treatments to assess whether the treatments could lead to aggregation. The final material, MPF NSs, showed a size around 270 nm with good dispersibility (PDI < 0.3) (Fig. 2h). The zeta potential of MP NSs was higher than MoS₂ NSs, which could be attributed to the growth of PB on MoS₂ surface. With the functionalization of the COOH groups with folic acid in MPF NSs, the zeta potential became less negative, increasing from –27.9 eV to –16.0 eV (Fig. 2i). This increase is due to the conversion of the carboxylic acids into amides during the reaction with the targeting agent containing an amine

function. Taken together, all the above results confirmed the successful preparation of MPF NSs.

## 3.2. *Photothermal property and Fenton activity*

As PB NPs and MoS₂ have photothermal properties, we investigated the photothermal performance of MPF NSs [16,45]. Fig. 3a shows the temperature increase of an aqueous suspension of MPF NSs with different concentrations upon irradiation with an 808-nm laser at a
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** (a) Temperature increase of an aqueous suspension of MPF NSs at different concentrations over irradiation with an 808-nm laser (1.0 W/cm²). (b) Temperature change of an aqueous suspension of MPF NSs (50 µg/mL) over three ON/OFF cycles under 808-nm laser irradiation (1.0 W/cm²). (c) Temperature increase of water and an aqueous suspension of MPF NSs, PB NPs and MoS₂ NSs (100 µg/mL) over irradiation with 808-nm laser (1.0 W/cm²). (d) UV–Vis–NIR absorption spectra and corresponding digital photos of a solution of RhB in water after treatment with MPF NSs (100 µg/mL) in the presence of 1 mM H₂O₂ and PBS (pH 5.5) at room temperature. (e) UV–Vis absorption spectra and corresponding digital photos of a solution of MB in water after treatment with MPF NSs (100 µg/mL) in the presence of 1 mM H₂O₂ and PBS (pH 5.5) at room temperature. (f) UV–Vis absorption spectra and corresponding digital photos of a solution of MB in water after treatment with MPF NSs (100 µg/mL) in the presence of 1 mM H₂O₂ and PBS (pH 5.5) at 45 °C. (g) MB degradation rate by MPF NSs (100 µg/mL) under different treatments at room temperature. (h) MB degradation rate by MoS₂ NSs, PB NPs and MPF NSs (concentration of 100 µg/mL) after 30 min in the presence of 1 mM H₂O₂ and PBS (pH 5.5). The A/A₀ represents the ratio of the absorbance at 665 nm to the initial absorbance at 665 nm. (i) EPR spectra of 100 µg/mL MPF NSs in the presence of 50 mM DMPO and 2 mM H₂O₂ after 3 min and at different pH and temperatures.</figcaption></figure>

power density of 1.0 W/cm² for 10 min. A concentration-dependent temperature increase was observed, with the temperature difference reaching 39 °C at the highest concentration of 100 µg/mL. After irradiation for 3 cycles, the photothermal performance of MPF NSs was maintained, showing good photostability (Fig. 3b). Moreover, MPF NSs exhibited a better photothermal effect than MoS₂ NSs and PB NPs at the same concentration (100 µg/mL) (Fig. 3c). The photothermal conversion efficiency of MPF NSs was determined to be 47.5 %, higher than MoS₂ NSs and PB NPs (14.5 % and 24.1 %, respectively) (Fig. S5). MPF NSs displayed a stronger photothermal performance because they showed an enhanced NIR absorbance compared to MoS₂ NSs (Fig. 2d) allowing more efficient light energy harvesting leading to greater total photon-to-heat conversion. The higher NIR absorbance could be due to intimate interactions between MoS₂ and PB NPs, which could improve charge carrier separation and transfer, and promote efficient heat generation through rapid electron-phonon interactions [46,47].

Next, the Fenton reaction performance of MPF NSs was assessed

using the color change of MB and RhB, dyes that have been extensively used for evaluating the efficacy of the Fenton reaction through the degradation by •OH. The acidic condition (pH = 5.5) was used to mimic the tumor environment. As shown in Fig. 3d and 3e, the absorbance of RhB and MB decreased significantly over time in the presence of MPF NSs and H₂O₂ under acidic conditions at room temperature. In the MB degradation experiments, after 90 min, MB almost completely degraded into a colorless solution. It has been reported that the efficiency of the Fenton reaction can be enhanced by increasing the temperature [21–23]. Therefore, MB degradation experiments were conducted at a temperature of 45 °C (Fig. 3f), which was chosen to mimic the photothermal effect in mild PTT [48,49]. In this case, the absorbance of MB decreased more rapidly than at room temperature. Indeed, within 30 min, the MB nearly completely degraded into a colorless solution, demonstrating that the photothermal effect significantly promoted the rate of the Fenton reaction. In contrast, almost no Fenton efficiency was observed under neutral conditions or in the absence of H₂O₂ (Fig. 3g).
This property imparts selectivity to MPF NSs for generating hydroxyl radicals specifically in the tumor. In addition, we also investigated the Fenton capacity of the control MoS₂ NSs and PB NPs (Fig. 3h). After 30 min, only a slight degradation of MB was observed in the two control groups, whereas a significant degradation of MB was evident for MPF NSs. This result proved that a co-catalytic process likely occurred between MoS₂ NSs and PB NPs, thereby achieving a higher Fenton efficiency, which was also confirmed by EPR spectroscopy using DMPO as a probe. As shown in Fig. 3i, EPR signals specific to DMPO that reacted with hydroxyl radicals were detected at pH 5.5, [50] whereas a less

intense signal was obtained at pH 7.4, confirming that a large amount of *OH was generated by MPF NSs only in acidic conditions. Furthermore, a notably higher EPR signal was observed at 45 °C, consistent with the results of the MB colorimetric assay, evidencing that increasing the temperature improved the efficiency of the Fenton reaction. Taken together, these results provide evidence that the association of PB NPs with MoS₂ NSs leads to an increase in the Fenton reaction rate, thus overcoming the low efficiency of PB-mediated Fenton reaction [17–19].

In the next step, we investigated further the co-catalytic process between MoS₂ NSs and PB NPs, as MoS₂ NSs were reported to act as a co-

<figure><img src="image_7.png" /><figcaption>Fig. 4. (a) XPS survey, (b) Mo 3d and (c) Fe 2p spectra of MPF NSs. (d) XPS survey, (e) Mo 3d and (f) Fe 2p spectra of MPF NSs after the Fenton reaction. (g) Proposed mechanism of the Fenton reaction induced by MoS₂ catalyzed-PB.</figcaption></figure>
catalyst to promote iron-based Fenton reaction [9,14]. XPS measurements were conducted on MPF NSs before and after the Fenton reaction. Despite the *in situ* growth of PB NPs on MoS₂ NSs, the oxidation state of Fe, Mo and S remained the same compared to MoS₂ NSs before the decoration or to the control PB NPs alone (Fig. S6). Hence, the *in situ* growth strategy had no impact on the oxidation state of the different elements. After the reaction of MPF NSs with H₂O₂ under acidic condition, the survey spectra revealed an increase in the oxygen content, accompanied by the appearance of a new Mo VI peak at 236 eV in the Mo 3d spectrum (Fig. 4a, 4b, 4d and 4e). The percentages of Fe and Mo in each valence state before and after the Fenton reaction were quantified,

showing an increased amount of Mo VI (Table S1), indicating a partial oxidation of Mo during the reaction. Additionally, there were no obvious changes in the Fe 2p spectra after the reaction (Fig. 4c and 4f).

To verify the electron transfer from MoS₂ NSs to PG and the reduction of iron, we synthesized PG and simply mixed PG with MoS₂ NSs without H₂O₂. We observed a new peak at 236.5 eV corresponding to Mo VI in the Mo 3d spectrum after mixing, indicating the oxidation of Mo (Fig. S7). In the Fe 2p spectra, part of Fe III after mixing was reduced to Fe II, leading to the conversion from PG to PB. Besides, the characterization by UV-Vis-NIR spectroscopy confirmed the reduction of PG to PB by MoS₂ NSs (Fig. S8). All the above results demonstrated that MoS₂

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** TEM images of MCF-7 cells (a) without MPF NSs and (b) after incubation with MPF NSs at a concentration of 50 µg/mL for 4 h. (c) Fe and Mo content in the MCF-7 cells after incubation with MPF NSs (100 µg/mL, 4 h) assessed by ICP-MS. “Control” corresponds to the untreated cells. A saturated FA solution was used to treat the cells before incubation and set as a control group (red bars). Cell viability of the MCF-7 cells after incubation with MPF NSs at different concentrations for 8 h (d) without and (e) with irradiation using an 808-nm laser (1.0 W/cm²). (f) Fluorescence microscopy images of the MCF-7 cells incubated with MPF NSs (Live/Dead assay). (*, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001).</figcaption></figure>
NSs can accelerate the PG/PB conversion through electron transfer.

Based on these results, we propose a mechanism based on the oxidation of PB to PG during the Fenton reaction (Eq. (1)), followed by the reduction to PB induced by MoS₂ NSs (oxidation of MoIV to MoVI), thereby significantly enhancing the rate of the Fenton reaction at the limiting step (Fig. 4g): [9,13,17,51].

$$KFe^{III}Fe^{II}(CN)_{6} (PB) + H_{2}O_{2} \rightarrow KFe^{III}Fe^{III}(CN)_{6} (PG) + \cdot OH + OH^{-} \quad (1)$$

$$KFe^{III}Fe^{III}(CN)_{6} (PG) + \frac{1}{2} Mo^{IV} \rightarrow KFe^{III}Fe^{II}(CN)_{6} (PB) + \frac{1}{2} Mo^{VI} \quad (2)$$

$$Mo^{VI} + H_{2}O_{2} \rightarrow Mo^{IV} + H_{2}O + \frac{1}{2} O_{2} \quad (3)$$

Since the standard redox potentials of PG/PB and MoVI/MoIV are 1.4 V and 0.65 V (versus the standard hydrogen electrode), respectively, the reduction of iron can be boosted by MoS₂ NSs (Eq. (2)) [14,52]. Subsequently, MoVI can be reduced back to MoIV in the presence of H₂O₂, ensuring the catalytic cycle (Eq. (3)).

Therefore, XPS, MB assay and EPR analysis provided direct catalytic experimental evidence supporting each step of the proposed mechanism depicted in Fig. 4g, highlighting the role of MoS₂ NSs in efficiently accelerating the rate-limiting step of the Fenton reaction by the reduction of PG back to PB.

### 3.3. In vitro cytotoxicity and combined PTT/CDT

We evaluated the colloidal stability of MPF NSs. For this purpose, MPF NSs were dispersed in a cell culture medium for 24 h, followed by the characterization by DLS, zeta potential and UV–Vis–NIR spectroscopy (Fig. S9). No significant change in size, zeta potential and absorbance was observed after 24 h, indicating a good stability of MPF NS suspension. Then, we evaluated the cellular uptake of MPF NSs by TEM after incubation with the human breast cancer cells MCF-7 at a concentration of 50 µg/mL for 4 h. TEM images of the treated MCF-7 cells showed that MPF NSs were internalized and some were localized in intracellular vesicles (Fig. 5a and 5b). Besides, ICP-MS analysis was conducted to quantify the intracellular concentrations of Mo and Fe. In this assay, MCF-7 cells were pre-incubated with free folic acid to saturate the folate receptors and competitively inhibit receptor-mediated uptake. This competition-based strategy is widely recognized for validating folate receptor-specific internalization, as previously reported [53]. Instead of employing additional cell lines with low folate receptor expression, this approach was adopted to directly assess the targeting specificity of MPF NSs. The ICP-MS analysis of the cells treated with MPF NSs indicated that the content of Mo and Fe was significantly higher compared to the control groups (i.e., untreated cells and cells treated with free FA 2 h before incubation with MPF NSs in order to saturate the folate receptor and inhibit internalization), which confirmed that the internalization predominantly occurred via folate receptor-mediated endocytosis (Fig. 5c). The cytotoxicity of MPF NSs, MoS₂ NSs and PB NPs was evaluated on normal cells (human embryonic kidney HEK 293 cells) (containing a low level of endogenous H₂O₂) using the MTS assay. The nanohybrid, as well as the control MoS₂ NSs and PB NPs, showed satisfactory cell viability even at the highest concentration of 100 µg/mL (Fig. S10a and S10b). In contrast, the cell viability of the MCF-7 cells decreased after incubation with MPF NSs, with 52 % survival at 100 µg/mL (Fig. 5d). It is noteworthy that, in contrast to MPF NSs, both MoS₂ NSs and PB NPs exhibited minimal cytotoxicity towards the MCF-7 cells (Fig. S10c), proving that the chemodynamic effect cannot be achieved by either MoS₂ NSs or PB NPs alone. In the MPF group with the NIR laser treatment, a high decrease in the cell viability was observed, particularly at concentrations above 50 µg/mL (Fig. 5e). At a concentration of 25 µg/mL the cytotoxicity was moderate, whereas the temperature increase observed at this concentration was nearly 30 °C in solution (Fig. 3a). The temperature increase in cells is expected to be lower than that observed in solution, even under the same irradiation conditions and concentration. This difference is due to two main factors. First, the cells scatter and

absorb light, thus reducing the effective light intensity that can be absorbed by the MPF NSs. Secondly, while the MPF NSs are evenly dispersed in water and freely diffuse, they are confined in cellular compartments (vide supra). Therefore, in solution there is no barrier to heat propagation leading to efficient and widespread heat transfer, whereas in cells, the local heat diffusion is limited resulting in more localized heating, which does not significantly increase the bulk temperature of the sample. Additionally, NIR light irradiation alone did not affect cell viability (Fig. 5e,f). Indeed, cells exposed to NIR light in the absence of MPF NSs showed similar viability levels to the untreated control (i.e., cells without treatment with MPF NSs or light) (Fig. 5d), confirming that the irradiation parameters used are non-toxic. These results validate that the therapeutic effects observed in our study can be attributed specifically to the photothermal effect induced by MPF NSs, rather than any cytotoxicity from the NIR light itself.

Furthermore, consistent results were obtained from the fluorescence images using the Live/Dead assay (live cells show a bright green fluorescence, while dead cells display a red fluorescence). As shown in Fig. 5f, with increasing concentration, the number of live cells gradually decreased. The number of live cells in the NIR group was lower than in the dark group. At the highest concentration in the NIR group, there were almost no live cells. The related cell viability, calculated by measuring the fluorescence intensity of the live cells by microplate reader, showed similar results to the MTS assay (Fig. S11). However, the red fluorescence arising from dead cells at high concentration appeared negligible. This is due to the fact that the cells were not fixed; thereby, most of the dead cells were washed away during the washing step with PBS. Based on the above results, our therapeutic strategy relying on synergistic CDT/PTT mediated by MPF NSs led to an optimal lethality to cancer cells.

### 3.4. In vitro therapeutic mechanism

MPF NSs achieved an efficient elimination of cancer cells through a CDT/PTT combined treatment. This outcome is likely not just due to the additive effects of PTT and CDT, but may also be due to a potential mutual synergistic enhancement. To explore the synergy between CDT and PTT, we investigated the underlying mechanism. Considering the Fenton activity and high CDT effect on cancer cells, intracellular ROS generation was first evaluated using DCFH-DA. After incubating the MCF-7 cells with MPF NSs, a noticeable bright green fluorescence was observed, particularly with increased intensity after NIR light irradiation (Fig. 6a and S12a). The related fluorescence intensity was also evaluated by flow cytometry, showing a similar trend, confirming the amplified ROS generation in the cells due to the photothermal effect (Fig. 6b). This suggests that although intracellular ROS might be reduced by the consumption of H₂O₂ by MoVI species, this does not appear to significantly reduce the intratumoral H₂O₂ level. Notably, the intracellular ROS level remain sufficient to support the MPF-mediated Fenton reaction. An excessive intracellular ROS amount can trigger oxidative stress, leading to mitochondria-mediated damage with the decrease of ΔΨm [54,55]. For this purpose, JC-1 was employed to evaluate ΔΨm, as this dye loses its ability to aggregate in mitochondria when ΔΨm decreases and thus is in the form of monomers. Fig. 6c shows that MPF NSs could significantly reduce the amount of JC-1 aggregates in the mitochondria and increase the formation of monomers. In the irradiation group, the amounts of monomers increased even more. The mitochondrial damage induced by ROS was confirmed by TEM. Indeed, destroyed mitochondrial cristae were observed in the treated cells (Fig. 6d). As is widely recognized, mitochondria play a critical role in regulating cellular energy. Therefore, we examined the ATP content and found that the ATP generation was decreased as expected, especially in the irradiation group (Fig. 6e). All these pieces of evidence indicate that the combined CDT/PTT can induce the mitochondrial damage, thereby leading to cell death. This is a promising result, especially given that most studies on photothermal enhancement of CDT do not provide
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** (a) Confocal microscopy images and (b) flow cytometry analysis of DCFH-DA stained MCF-7 cells treated with MPF NSs with or without NIR light irradiation. (c) Effect of MPF NSs on ΔΨm changes detected by JC-1 and the related quantitative analysis of the red fluorescence, green fluorescence and the red/green florescence ratio. (d) TEM images of the MCF-7 cells before (left) and after treatment with MPF NSs showing destroyed mitochondria (middle and right). (e) ATP content of the MCF-7 cells treated with MPF NSs with or without NIR light irradiation. MCF-7 cells without treatment were set as control group. The MPF+NIR group was irradiated with an 808-nm laser for 10 min (1.0 W/cm²).</figcaption></figure>
mechanistic details about the biological pathways involved in the combined treatment, and only few studies have explored the induction of ferroptosis and mitochondrial damage by ROS for tumor treatment so far [27–30,56,57].

The accumulation of ROS, the mitochondrial morphological changes and the decrease of ATP are the main markers of ferroptosis [58,59]. An excessive ROS amount can also induce lipid peroxidation [60]. Thereby, lipid peroxidation was assessed using the C11-BODIPY 581/591 dye, for which the fluorescence shifts to green upon oxidation. After treatment with MPF NSs, the cell membrane exhibited a green fluorescence in comparison with the untreated cells, suggesting ferroptosis (Fig. 7a, 7b and S12b). Importantly, the cells treated with MPF NSs under irradiation displayed a stronger green fluorescence intensity, indicating a photothermal-enhanced ferroptosis. GPX4 is a key regulator of ferroptosis, which can be inactivated by reduced intracellular GSH [61]. As shown in Fig. 7c and 7d, the GPX4 level and GSH content were substantially reduced in the cells after the treatment with MPF NSs and even

more when combined with NIR light irradiation. GSH reacts with ROS to protect cellular components from oxidative damage. During the Fenton reaction, the intracellular GSH level in the cancer cells decreased, as GSH acts as an antioxidant that neutralizes ROS, including hydroxyl radicals. As a result, the intracellular GSH level diminished [62]. Additionally, ferroptosis inhibitors Fer-1 and Lip-1 were incubated with the cells at different concentrations together with MPF NSs (100 µg/mL) (Fig. 7e). We observed an increase in cell viability with increasing concentrations of the ferroptosis inhibitors, indicating that the ferroptosis was induced during the CDT treatment. Besides, vitamin E, a ROS scavenger, was alternatively used to inhibit ferroptosis, allowing to evaluate the efficiency of PTT alone for the cells treated with MPF NSs and vitamin E and irradiated with an 808-nm laser (Fig. S13). We observed that in the irradiation group treated with MPF NSs and viamine E, the predominant effect observed was the photothermal effect. Taken together, these results confirmed that ferroptosis is induced by the CDT treatment and the therapeutic effect is enhanced under NIR light

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** (a) Confocal microscopy images and (b) flow cytometry analysis of C11-BODIPY stained MCF-7 cells treated with MPF NSs (100 µg/mL for 8 h) with and without NIR light irradiation. (c) GPX4 activity and (d) GSH content of the MCF-7 cells treated with MPF NSs with and without NIR light irradiation. (e) Cell viability of the MCF-7 cells treated with different concentrations of Lip-1 or Fer-1 in the presence of MPF NSs (100 µg/mL, incubation for 8 h). (f) Western blot to assess the intracellular expression of HSP70. As control, the cells were heated at 45 °C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control antibody for Western blot to normalize the levels of protein. (g) Quantitative analysis of the Western blot for HSP70 expression. In the above experiments, the cells without treatment were used as control group. The MPF + NIR group was irradiated with an 808-nm laser for 10 min (1.0 W/cm²). (*, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001).</figcaption></figure>
exposure.

Noticeably, the overexpression of HSP in cancer cells can induce heat resistance, thus reducing the PTT effect on the cell viability. Interestingly, mitochondrial damage and ferroptosis can inhibit the expression of HSP, which is considered an effective approach to enhance the PTT efficacy [24,25,29,63]. Hence, we evaluated the content of HSP70

(Fig. 7f, 7g and Fig. S14). Compared with the control group, the expression level of HSP70 in the cells treated with MPF NSs with and without irradiation decreased to 45.5 % and 64.9 %, respectively. The higher inhibition of HSP70 expression by MPF NSs under light irradiation could be due to the induction of more mitochondrial damage and ferroptosis, and production of a significant amount of ROS, leading to

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** (a) Digital photos of tumors after 10 days of treatment. (b) Tumor size changes during the therapy. (c) Average weight of the tumors after the treatment. (d) H&E staining of tumor sections after the treatment. (e) Ki67 and CD31 immunohistochemistry of tumor tissue after treatment. (f) ROS staining, (g) GPX4 staining and (h) TUNEL staining of tumor tissues, with related quantitative analysis (right panels). The tumor tissues were excised from the CAL-27 tumor-bearing mice. (*, p ≤ 0.05; **, p ≤ 0.01, ***, p ≤ 0.001).</figcaption></figure>
the inhibition of HSP70. These results suggested that the PTT efficiency was boosted by the inhibition of the expression of HSP70. This mutual promotion between CDT and PTT led to favourable *in vitro* therapeutic efficacy.

### 3.5. *In vivo* anticancer efficiency

The antitumor effect of MPF NSs *in vivo* was studied in the tumor-bearing BALB/c nude mice (tongue squamous carcinoma cell line) xenograft. When the tumor reached 200 mm³, the mice were injected with saline (control group) or MPF NSs in saline (4 mg/mL, 50 µL) intratumorally and the tumor was irradiated (or not for the control groups) 24 h after, followed by sequences of injection and irradiation every 48 h (Fig. S15). MoS₂ NSs could not be used as a control group, because of their low colloidal stability in aqueous solutions, hampering their administration *in vivo*. In contrast, the growth of PB NPs allowed increasing the colloidal stability of MoS₂ NSs, as demonstrated by the analysis of MPF NSs dispersed in a cell culture medium for 24 h by DLS, zeta potential and UV–Vis–NIR spectroscopy (Fig. S9). In this study, MPF NSs were administered via intratumoral injection in CAL-27 (a human tumor cell line derived from squamous cell carcinomas of the tongue) tumor-bearing mice. The intratumoral administration was reported in our previous study as an effective strategy for solid tumor treatment [64]. This local delivery route holds clinical relevance for oral cancers, including tongue squamous cell carcinoma, as it allows direct delivery of therapeutic agents to the tumor site. Compared to systemic administration, intratumoral injection facilitates higher local accumulation of nanoparticles while minimizing systemic exposure, thereby enhancing therapeutic efficacy and reducing potential side effects. Although the current study focuses on local administration, future investigations using animal tumor models that require intravenous injection will be essential to further evaluate the systemic behavior, biodistribution and therapeutic performance of MPF NSs. The timeline of the treatment is shown in Fig. S15. In detail, the tumor was irradiated (or not for the control groups) 24 h after the administration of MPF with NIR light (808 nm, 1.0 W/cm², 10 min). Intratumoral injection followed by NIR light irradiation (or not for the control groups) 1 h after was then performed every 48 h (day 3, day 5, day 7 and day 9). The 24-h interval between the injection and the first NIR light irradiation was chosen to allow sufficient nanoparticle distribution in the tumor and uptake in the cancer cells, ensuring an optimal and a uniform photothermal efficacy upon laser treatment. After 10 days, the tumor size decreased significantly in the MPF-treated group, while the irradiation further reduced the size compared to the saline group (Fig. 8a, 8b and S16). Meanwhile, the MPF treatment reduced the tumor weight by ~50%, with the weight loss reaching around 80% after irradiation (Fig. 8c). The above results demonstrated that MPF NSs showed efficient tumor inhibition effect by CDT alone, and when combined with PTT, they led to a pronounced tumor therapeutic effect. H&E staining of the tumor tissues revealed that the mice treated with MPF NSs under irradiation exhibited numerous apoptotic cells, while the control group contained many live cells (Fig. 8d). In the immunohistochemical staining images, we observed that MPF NSs reduced noticeably the number of Ki67+ cells (Ki67 is a protein present in the cell nucleus, which is a crucial indicator for assessing the activity level of tumor cells) and CD31+ cells (CD31 is a platelet endothelial cell adhesion molecule, which is an important marker for endothelial differentiation) in the tumor tissues. These results indicate that the proliferation of cancer cells and tumor angiogenesis were effectively inhibited (Fig. 8e), which further inhibited the tumor growth. To prove the ROS generation in the tumor, a staining of the tumors with the O13 probe was performed [65]. Compared with the control group, the level of ROS in the tumor treated by MPF NSs was significantly increased, and it was further greatly enhanced under NIR laser irradiation (Fig. 8f), which indicated that the PTT treatment could

significantly enhance the CDT efficacy. Furthermore, an immunostaining was done to assess the expression level of GPX4 (Fig. 8g). The MPF with NIR-treated group exhibited the lowest GPX4 expression, suggesting the occurrence of ferroptosis in the tumor site. In addition, a TUNEL staining analysis confirmed that the treatment with MPF NSs effectively induced apoptosis, and there were more apoptotic cells after irradiation (Fig. 8h). Finally, the biosafety of MPF NSs was evaluated in healthy mice. A haematological analysis revealed that MPF NSs did not impact erythrocytes or platelet markers (Fig. S17). Similarly, H&E staining of the major organs (e.g., heart, liver, spleen, lung and kidney) showed no apparent pathological abnormalities following the injection of MPF NSs, demonstrating a good biocompatibility of MPF NSs (Fig. S18). To further explore the therapeutic potential of MPF in cancer treatment, it would be essential to investigate the biodegradability of the MPF NSs. For MoS₂, our previous study on the degradation of MoS₂ nanosheets elucidated the mechanisms involving the intracellular transformation and extracellular vesicle-mediated clearance [66]. In the case of PB NPs, a comprehensive study evaluated the risks and biological fate of citric acid-modified PB NPs after intravenous injection in mice [67]. This study showed that PB NPs caused no obvious toxicity in mice (at a dose of 5 or 10 mg/kg), while a higher dose of 20 mg/kg led to a temporary loss of appetite and weight without acute or subacute toxicity. PB NPs were rapidly cleared from the blood, primarily accumulated in the liver and lungs, and were gradually eliminated from the liver and lung tissues over time. Therefore, a dedicated and systematic investigation into the degradation characteristics and clearance pathways of MPF NSs will be necessary in future research.

### 4. Conclusion

We developed a co-catalytic PB/MoS₂ hybrid nanosystem by *in situ* growing PB NPs on MoS₂ nanosheets for mutual-promoted chemo-dynamic/photothermal synergistic therapy. MoS₂ NSs acted as a co-catalyst to promote the conversion of Prussian green to PB (Fe³⁺/Fe²⁺), thereby leading to an efficient Fenton reaction. The high generation of ROS in the tumor environment resulted in mitochondrial damage and ferroptosis, which inhibited the expression of HSP and thus enhanced the PTT. Meanwhile, the photothermal effect simultaneously improved the efficiency of the Fenton reaction. This combined therapeutic strategy allowed achieving an efficient elimination of the cancer cells both *in vitro* and *in vivo*, providing meaningful insight for potential future anticancer therapy, in particular to assess the long-term effect of the treatment in a larger cohort of animals.

### Data availability

Data will be made available on request.

### CRediT authorship contribution statement

**Yilin He:** Writing – original draft, Methodology, Investigation, Data curation. **Chunxu Lv:** Writing – original draft, Investigation, Data curation. **Nolwenn Le Breton:** Investigation, Data curation. **Haijun Peng:** Investigation, Data curation. **Tengfei Wang:** Investigation, Data curation. **Paolo Samori:** Supervision. **Sylvie Choua:** Supervision. **Alberto Bianco:** Writing – review & editing, Supervision. **Baojin Ma:** Writing – review & editing, Supervision. **Cécilia Ménard-Moyon:** Writing – review & editing, Supervision.

### 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 the Centre National de la Recherche Scientifique (CNRS), by the Agence Nationale de la Recherche (ANR) through the Interdisciplinary Thematic Institute SysChem via IdEx Unistra (ANR-10-INDEX-0002) within the program Investissement d'Avenir and by Jean-Marie Lehn Foundation. This work was also supported by the National Natural Science Foundation of China (No. 82100974). The French research infrastructure INFRANALYTICS FR2054 is acknowledged for its financial support for EPR spectroscopy experiments. The authors also wish to thank Cathy Royer from the "Plateforme Imagerie *In Vitro* de l'ITI Neurostra" CNRS UAR 3156, University of Strasbourg (Strasbourg, France) for TEM analysis, Dr. Dris Ihiawakrim from the Transmission Electron Microscopy Platform at IPCMS (Strasbourg, France) for the high-resolution TEM analyses. Yilin He is indebted to the Chinese Scholarship Council for supporting her PhD.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2025.07.037.

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