Construct an “immunogenic cell death” amplifier based on Fe-MOFs by accelerating Fe(III) reduction strategies for integration of tumor diagnosis, treatment, and prevention

Abstract

Traditional tumor treatments focus on treating the location of the lesion, while immunogenic cell death (ICD) triggers systemic anti-tumor immunity and inhibits tumor metastasis. Therefore, there is a need to develop an inducer that amplifies ICD. Here, methotrexate (MTX) and MoO₂ were loaded into a Cu²⁺-doped iron-based targeted metal–organic framework Fe–NH₂-MIL-101 with nano-enzymatic activity to establish a novel ICD amplifier. The photothermal agent MoO₂ generates heat under near-infrared (NIR) light excitation, inducing tumor ablation. Simultaneously, the released Mo⁺ combines with Fe²⁺ and Cu⁺ in the system, synergistically enhancing electron transfer efficiency based on the bimetallic system. Combined with thermal effects, this approach cooperatively elevates glutathione peroxidase (GPx)-like and peroxidase (POD)-like activities. This catalytic cascade depletes glutathione through Fenton-like reactions while amplifying hydroxyl radical (˙OH) generation, thereby remodeling the tumor microenvironment (TME), potentiating chemodynamic therapy (CDT), and triggering ICD. The chemotherapeutic agent MTX not only exerts direct cytotoxic effects but also serves as an inducer of ICD. In vitro and in vivo experiments have shown that the resulting synergistic treatment model based on the combination of CDT, photothermal therapy (PTT), and chemotherapy guided by T₂-MRI imaging will amplify the ICD effect, enhance tumor treatment, and is expected to achieve the prevention of metastasis and recurrence of tumors and to realize the integration of tumor diagnosis, treatment, and prevention.

---

1. Introduction

Metastasis and recurrence of tumors are the main causes of death in patients with advanced tumors.¹ Surgery, chemotherapy, and radiotherapy are commonly used treatments. However, these methods tend to focus on treating the location of the lesion and have the drawbacks of poor healing, high recurrence rate, etc. Compared with traditional treatment modalities, immunotherapy has a significant therapeutic effect on preventing tumor recurrence and metastasis by activating the body's immune system,² which has attracted more and more attention. Immunogenic cell death (ICD) is an important immune stimulus initiator that triggers systemic antitumor immunity,³ and inducing ICD can mediate the body's ability to generate an antitumor immune response and maintain long-term immunity to suppress tumor metastasis.⁴

Some studies have shown that some tumor treatment methods such as chemotherapy and radiotherapy can induce ICD,⁵ but serious side effects often occur in healthy tissues. Therefore, it is necessary to develop an ICD inducer that is not harmful to normal cells. PTT converts light energy into heat energy, which can cause cancer cell apoptosis, thus regulating anti-tumor immune response, but the PTT process is limited by the depth of tumor penetration,⁶ which produces a weak ICD effect. CDT converts H₂O₂ in tumor cells into ˙OH, which can stimulate the death of tumor cells and transform from non-immunogenicity to immunogenicity, inducing the ICD effect.⁷ The design of CDT-based ICD inducers and the anti-tumor applications have attracted the interest of scientists.

However, due to the immunosuppressive tumor microenvironment (TME), including slightly acidic environment (pH 5.5–6.8), high concentration of GSH (10–20 μM), insufficient H₂O₂ (0.5 nmol/10⁴ cell per h), and microenvironment hypoxia (Hypoxia),⁸ the proliferation and metastasis of tumors are accelerated, resulting in insufficient ICD effect of a single treatment mode and difficulty in stimulating effective systemic immunity.⁹ Therefore, there is an urgent need to develop synergistic strategies for different induced ICD generation to overcome the limitations of single strategies, amplify the ICD effect, and enhance immunotherapy.

Researchers have found that the design of nanoenzymes to mimic the catalytic activity of natural enzymes can effectively regulate TME to reverse immunosuppression, and at the same time convert H₂O₂ into hydroxyl radicals (˙OH) to achieve self-enhanced CDT, which is expected to improve the therapeutic effect and amplify the ICD effect.^10–12 Among kinds of artificial nanozymes, metal–organic frameworks (MOFs) have shown obvious advantages in the study of nanozymes due to their adjustable topological structure and metal sites, controllable surface functionalization, and pore structure:^13–15 high oxidation state of variable valence metal ions can simulate glutathione oxidase (GPx) activity and reduce GSH concentration; POD activity and catalase (CAT) activity are common in variable-valent metal ions. The former can convert H₂O₂ into ˙OH to achieve chemodynamic therapy and induce ICD. The latter decomposes H₂O₂ into O₂ to regulate TME hypoxia.

Since iron is one of the essential trace elements in the human body, and iron-based nanoparticles can be used as T₂-magnetic resonance imaging (MRI) contrast agents to provide diagnostic information on tumor tissue,¹⁶ the synthesis of bio-friendly iron-centered MOFs (Fe-MOFs) as multifunctional nanozymes for tumor diagnosis and therapy has become a hot spot. Fe-MOFs have abundant iron active sites that can catalyze H₂O₂ in tumor cells to produce ˙OH. The formation rate of ˙OH is determined by the cycle efficiency between Fe(III)/Fe(II):

Fe²⁺ + H₂O₂ → Fe³⁺ + ˙OH + OH⁻ (1)

Fe³⁺ + GSH → Fe²⁺ + GSSG (2)

However, in the Fe(III)/Fe(II) cycle, the oxidation rate of Fe(II) (reaction (1)) is 600 times higher than the reduction rate of Fe(III) (reaction (2)), which makes it difficult to achieve an efficient cycle of Fe(III)/Fe(II) and weakens the catalytic activity. As a result, the ICD effect is weakened because of insufficient production of ˙OH.

It has been found that bimetallic catalysts can achieve rapid reduction of Fe(III) through the synergistic effect between metal centers,¹⁷ and the reduction rate of Fe(III) can be accelerated through the doping of other transition metal ions in Fe-MOFs.¹⁸ The synergistic effect of bimetals can improve the catalytic performance. It has also been found that the combination of light, ultrasound, heat, and other energy-assisted methods can accelerate the reduction of Fe(III) to Fe(II) and enhance the catalytic efficiency.^19,20 Based on the synergistic strategy of “1 + 1 > 2”, the construction of iron-based bimetallic MOFs combined with energy-assisted methods can synergistically accelerate the reduction of Fe(III), realize the efficient cycle of Fe(III)/Fe(II), which improved the catalytic activity and triggered the ICD effect. Therefore, the introduction of photothermal agents into bimetallic MOFs can combine the energy effect with the bimetallic effect to synergistically increase the rate of Fe(III) reduction and greatly improve the catalytic performance, resulting in enough hydroxyl radicals. Besides, based on the previous research on drug loading and release of MOFs in our group,²¹ targeted MOFs can be designed and loaded with anticancer drugs. Combined with the target release of an anti-cancer drug to tumor cells, the ICD effect can be further amplified. Then, the composites of photothermal agents and anti-cancer loaded bimetal MOFs can be used as multifunctional nanozymes, which can not only regulate the TME to reverse the immunosuppression microenvironment but also combine the ICD effect of chemotherapy, PTT, and CDT to greatly stimulate the body's immunity to prevent tumor metastasis.

Inspired by this, a novel ICD amplifier NH₂-MIL-101(Fe/Cu)@MoO₂/MTX was designed, as shown in Scheme 1. Cu²⁺ was doped with targeted metal–organic framework Fe–NH₂-MIL-101, and the rapid reduction of Fe(III) was ensured by the synergistic effect of bimetallic centers. In addition, Cu²⁺ transforms into Cu⁺ with better catalytic activity, by consuming GSH in the tumor, thus possessing multi-enzyme activities such as POD-like and GPx-like to regulate the TME, further improving the catalytic efficiency and inducing ICD. Subsequently, the stabilized photothermal agent MoO₂²² was wrapped around the outer layer to form NH₂-MIL-101(Fe/Cu)@MoO₂. With near-infrared light irradiation, the reduction of Fe(III) will be further accelerated and other enzymatic activities will be enhanced. Besides, due to the reported ICD induction effect²³ of methotrexate (MTX), MTX was loaded to obtain NH₂-MIL-101(Fe/Cu)@MoO₂/MTX, which has a tumor targeting and combining with chemotherapy in a synergistic way to treat the tumor. The resulting T₂-MRI imaging-guided synergistic treatment modality based on the integration of chemotherapy, PTT, and CDT will amplify the ICD-induced effect and enhance the effectiveness of tumor treatment, which is expected to achieve the prevention of tumor metastasis and recurrence.

Scheme 1 Schematic illustrating the preparation and therapeutic mechanism of Cu/Fe–NH₂-MIL-101@MoO₂/MTX nanozymes.

2. Experimental section

2.1. Synthesis of Cu/Fe–NH₂-MIL-101

Cu/Fe–NH₂-MIL-101 was synthesized by hydrothermal method, and 0.3595 g Cu/Fe–NH₂-MIL-101 was obtained. FeCl₃·6H₂O and 0.1619 g Cu(NO₃)₂·3H₂O were dissolved in 15 mL DMF to form solution A, followed by 0.2175 g NH₂-BDC was dissolved in 15 mL DMF to form solution B. After mixing solution A and solution B, 0.3 g PVP was added to make the solution uniform. After stirring for 1 h until the solution is clarified, the solution is transferred to a Teflon-lined autoclave and reacted at 110 °C for 24 h, and Cu/Fe–NH₂-MIL-101 was collected by centrifugation and purified with DMF, water, and ethanol to remove excess reactants.

2.2. Synthesis of Cu/Fe–NH₂-MIL-101@MoO₂

Take 100 mg of the Cu/Fe–NH₂-MIL-101 dissolved in 15 mL of water, until it is completely dissolved to form solution A, weighing 0.0817 g of molybdenyl acetylacetonate dissolved in 15 mL of ethanol to form solution B, and then mix the two, stirring at room temperature for 1 h, and until transferred to Teflon-lined autoclave and reaction at 180 °C for 24 h, followed by washing and drying to obtain the final product.

2.3. Synthesis of Cu/Fe–NH₂-MIL-101@MoO₂/MTX

10 mg MTX was dissolved in 0.5% of 100 mL Na₂CO₃ solution. Then 10 mg of Cu/Fe–NH₂-MIL-101@MoO₂ was dissolved in 10 mL of MTX.

2.4. Catalytic properties

The detailed calculation is listed in the ESI.†

2.5. Photothermal properties

The detailed calculation is listed in the ESI.†

2.6. Enzymatic activity experiment

The detailed calculation is listed in the ESI.†

2.7. Drug loading and release

The detailed calculation is listed in the ESI.†

2.8. EPR experiment

All EPR measurements are carried out on Bruker EMX plus, and 1 mg mL⁻¹ is used for catalytic testing. Cu/Fe–NH₂-MIL-101@MoO₂/MTX reacted with 10 mM H₂O₂ to reach equilibrium, and 3 μL DMPO capture agent was added to it.

2.9. Cytotoxicity assay

The CCK-8 colorimetric method was used to examine the cytotoxicity of different materials with 4T1 and 293T cells. 4T1 or 293T cells were seeded in 96-well plates and incubated with different material groups for 24 h, then the medium was replaced with serum-free medium containing 10% CCK-8. After further incubation for 0.5 to 3 h, the absorbance at the wavelength of 450 nm was measured using a microplate reader. The percentage of cell viability was calculated according to the correlation formula.

2.10. DCFH-DA probe experiment

ROS intracellular assay was performed for the production of ˙OH in different materials in cells using a DCFH-DA probe. Cu/Fe–NH₂-MIL-101@MoO₂/MTX and Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR with 4T1 cells were incubated for 12 h, and the cells were stained with 10 μM DCFH-DA for 30 min. Subsequently, the cells were imaged by fluorescence confocal microscopy.

2.11. Flow cytometry

The apoptosis of cancer cells induced by Cu/Fe–NH₂-MIL-101@MoO₂/MTX was detected by flow cytometry. The 4T1 cells were seeded in 6-well plates and incubated for 24 h, then the culture medium was changed to incubate with Cu/Fe–NH₂-MIL-101@MoO₂/MTX and Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR for 12 h, and the cells were digested and collected. Then, the cells were analyzed by flow cytometry, and the apoptosis under NIR conditions was the same as the above experimental operation. The cells were irradiated with NIR (808 nm, 1 W cm⁻²) for 10 minutes during cell culture.

2.12. Live–dead dyeing experiment

AnnexinV-AbFlour™ 488/PI staining directly observed cancer cell death induced by Cu/Fe–NH₂-MIL-101@MoO₂/MTX. The 4T1 cells were seeded in a confocal culture dish for 24 h, and then treated with Cu/Fe–NH₂-MIL-101@MoO₂/MTX experimental group and Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR group for 24 h. The cells were stained with AnnexinV-AbFlour™ 488 (2 μg mL⁻¹) and PI (4.5 μg mL⁻¹) for 30 min, respectively. Excitation at wavelengths of 491 nm and 535 nm, and finally the cell morphology was observed under a confocal microscope.

2.13. MRI imaging experiment

100 μL of aqueous solution containing different concentrations (Fe: 0, 0.1, 0.2, 0.3, 0.4, 0.5 mM) was placed in a magnetic resonance magnet (Siemens Magnetometer Trio 3.0 T), which was used to detect the corresponding relaxation time. Ultimately, r₂ values were obtained by fitting a curve of 1/T₂ relaxation time (s⁻¹) versus Fe concentration. For in vivo magnetic resonance imaging, tumor-bearing mice anesthetized with the material (100 μL, 0.02 mg mL⁻¹) intravenously injected at different time intervals were placed on a magnetic resonance imager for imaging, with an additional unmedicated group as a control group.

2.14. In vivo therapy

Female mice were xenografted subcutaneously with 4T1 cells (10⁶ cells per mouse). When the tumor volume was ≈100 mm³, the mice were randomly divided into 2 groups of 6 mice each. The mice were then injected intravenously with 100 μL of different materials.

3. Results and discussion

3.1. Characterization of Cu/Fe–NH₂-MIL-101

The XRD diffraction pattern is shown in Fig. S1(a) (ESI†). The characteristic peaks of Cu–NH₂-MIL-101 are at 2θ = 10.3°, 11.8°, 16.8°, 24.7° and 43.3° and are consistent with those reported in the literature.²⁴ The successful synthesis of Cu–NH₂-MIL-101 was proved. The characteristic peaks of Fe–NH₂-MIL-101 are 2θ = 3.2°, 5.1°, 5.7°, 8.2°, 9.0°, 10.2° and 16.2°,²⁵ which proved the successful synthesis of Fe–NH₂-MIL-101. As shown in Fig. S1(b) (ESI†), with the increase of Cu²⁺ content, the two peaks at about 2θ = 10° gradually evolved into a single peak, which was close to Cu–NH₂-MIL-101. Successful doping of Cu²⁺ is demonstrated.

The FT-IR spectra of Cu–NH₂-MIL-101, Fe–NH₂-MIL-101 and Cu/Fe–NH₂-MIL-101 are shown in Fig. S1(c and d) (ESI†). It can be seen from the figure that the absorption peaks near 3454 cm⁻¹ and 3324 cm⁻¹ in Fe–NH₂-MIL-101 belong to the symmetric and asymmetric vibrations of NH₂.²⁶ The absorption peaks at 1578 cm⁻¹ and 1382 cm⁻¹ are attributed to the classical carboxyl vibration. The absorption peak at 1255 cm⁻¹ is caused by the stretching vibration of the benzene ring. The absorption peak at 769 cm⁻¹ is caused by the C–H bending vibration in the organic complex, and obvious Fe–O peaks are observed at 578 cm⁻¹ and 519 cm⁻¹, which are proved by the above. The successful synthesis of Fe–NH₂-MIL-101.

The morphology of different materials was observed by FESEM. As shown in Fig. S2(a) (ESI†), the Fe–NH₂-MIL-101 is an uniform octahedral shape and the particle size is about 1 μm. Cu–NH₂-MIL-101 has a sheet-like morphology and a particle size of about 1 μm (Fig. S2(f), ESI†). Fig. S2(b–e) (ESI†) shows that the doping morphology of Cu²⁺ has changed to a certain extent. When Fe : Cu = 3 : 1, the material is more aggregated, but with the increase of Cu content, the material is more and more dispersed. When Fe : Cu = 2 : 1, the particle size of the material is about 300 nm, and the morphology is uniform. When Fe : Cu = 1 : 1, the particle size of the material begins to increase, which is about 400 nm.

3.2. Catalytic performance and characterization of Cu/Fe–NH₂-MIL-101

The catalytic properties of different Cu/Fe–NH₂-MIL-101 were explored, with 3,3′,5,5′-tetramethylbenzidine (TMB) as an indicator for ˙OH detection, and the experimental results are shown in Fig. S3(a) (ESI†). Through recording the absorbance value of TMB at 650 nm, the quantitative analysis of ˙OH generation was conducted. In comparison with Fe–NH₂-MIL-101 or Fe–NH₂-MIL-101, the TMB with Cu/Fe–NH₂-MIL-101 exhibited larger absorbance, indicating the highest level of ˙OH being produced, which indicates that the best catalytic performance was obtained when Fe : Cu = 2 : 1, probably due to the doping of metal ions, which accelerated the electron transfer rate. Subsequently, the ratio of Fe : Cu = 2 : 1 was chosen to explore the effect of different pH, and the results are shown in Fig. S3(b) (ESI†), the blue color is the darkest at pH = 5.8, which proves that it produces the most ˙OH. In summary, the Cu/Fe–NH₂-MIL-101 can undergo the Fenton reaction under the weakly acidic condition and catalyze the production of more ˙OH from H₂O₂.

3.3. Characterization and performance of Cu/Fe–NH₂-MIL-101@MoO₂

The XRD patterns of different materials are presented in Fig. 1(a), and the characteristic diffraction peaks of MoO₂ are consistent with those of standard cards, particularly exhibiting distinct reflections at 2θ = 36.5° and 53.5°.²⁷ Notably, Cu/Fe–NH_2-MIL-101 demonstrates emerging diffraction signatures within the 2θ = 20–30° range after MoO₂ encapsulation, confirming the successful incorporation of MoO₂.²⁸ After loading MTX, some of the peaks of Cu/Fe–NH₂-MIL-101/MoO₂ were retained and new characteristic diffraction peaks appeared at 57.0°, which proved the successful synthesis of Cu/Fe–NH₂-MIL-101/MoO₂/MTX. FT-IR spectra of the synthesized materials are presented in Fig. 1(b). The characteristic peak Cu/Fe–NH₂-MIL-101/MoO₂ is red-shifted at wavelength 3400 cm⁻¹ compared to that of pure MoO₂. The absorption peaks of Cu/Fe–NH₂-MIL-101/MoO₂/MTX at 1655 cm⁻¹ and 3448 cm⁻¹, 1459 cm⁻¹ are attributed to C=O and N–H bonds. The characteristic peaks at 831 cm⁻¹ and 930 cm⁻¹ are attributed to Mo=O bonds. All the above proved the successful synthesis of Cu/Fe–NH₂-MIL-101/MoO₂/MTX. Besides, the N₂ adsorption–desorption isotherm curves and pore size distribution plots reveal that the specific surface area of Cu/Fe–NH₂-MIL-101/MoO₂ decreased from 334.497 m² g⁻¹ to 15.899 m² g⁻¹ after MTX loading (Fig. S4(a), ESI†). As shown in Fig. S4(b) (ESI†), the pore size decreased from 26.973 nm to 19.569 nm, while the pore volume reduced from 0.528 cm³ g⁻¹ to 0.0946 cm³ g⁻¹. The significant reductions in both specific surface area and pore volume following MTX loading corroborate the physical adsorption mechanism.

Fig. 1 (a) XRD of materials. (b) FT-IR of materials. (c) DLS of Cu/Fe–NH₂-MIL-101, Cu/Fe–NH₂-MIL-101/MoO₂ and Cu/Fe–NH₂-MIL-101/MoO₂/MTX. (d) Zeta potential of Cu/Fe–NH₂-MIL-101, Cu/Fe–NH₂-MIL-101/MoO₂ and Cu/Fe–NH₂-MIL-101/MoO₂/MTX. (e) Photothermal properties of different materials. (f) TMB color development experiment for different. FESEM image of materials (g) MoO₂, (h) Cu/Fe–NH₂-MIL-101/MoO₂ and (i) Cu/Fe–NH₂-MIL-101/MoO₂/MTX. TEM image of (j)–(l) Cu/Fe–NH₂-MIL-101/MoO₂/MTX.

Fig. 1(c and d) shows that the particle size and Zeta potential of Cu/Fe–NH₂-MIL-101 are 255 nm and 10.10 mV, respectively. Upon MoO₂ encapsulation, the composite Cu/Fe–NH₂-MIL-101/MoO₂ exhibited a significant size increase to 295 nm accompanied by a surface charge reversal (−8.48 mV). Subsequent MTX loading further enlarged the particle size to 342 nm while modulating the zeta potential to −5.79 mV. With the increase of particle size and the change of potential after layer-by-layer coating, it was proved that the composite material was successfully synthesized. The photothermal conversion efficiency of Cu/Fe–NH₂-MIL-101/MoO₂ was quantitatively evaluated under varying MoO₂ loadings (Fig. 1(e)). It can be seen that the photothermal performance is getting better and better with the increase of the MoO₂ ratio. Combined with the ability to produce ˙OH, as shown in Fig. 1(f), Cu/Fe–NH₂-MIL-101/MoO₂ has the best Fenton reaction performance and is better than pure MoO₂.

In addition, the morphological evolution during composite fabrication was systematically investigated. As shown in Fig. 1(g), the morphology of MoO₂ is a uniform sphere with an average diameter of ∼180 nm. In Fig. 1(h), Cu/Fe–NH₂-MIL-101/MoO₂ can be seen that the sphere is wrapped on Cu/Fe–NH₂-MIL-101, and the particle size is about 200 nm. As shown in Fig. 1(i), after MTX was loaded on Cu/Fe–NH₂-MIL-101/MoO₂, the morphology was uniform and the particle size was about 100 nm. This may be because the solvent added during the drug loading process affects the nucleation process of the material. The TEM characterization of Cu/Fe–NH₂-MIL-101/MoO₂/MTX is shown in Fig. 1(j–l). The results are consistent with FESEM, which proves the successful synthesis of the material.

Elemental spatial distribution analysis was performed to validate the composite architecture. As shown in Fig. S5(a) (ESI†), which further confirms the successful synthesis of the material. Mo, Fe, C and O are uniformly distributed on Cu/Fe–NH₂-MIL-101/MoO₂, and the Cu element is not obvious due to its low content. Further EDX characterization is shown in Fig. S5(b and c) (ESI†). The atomic ratios of C, O, Fe, Cu and Mo are 55.15%, 33.05%, 10.59%, 0.77% and 0.45%, respectively. The above research shows that the composite successfully combines Cu/Fe–NH₂-MIL-101 with MoO₂.

3.4. Photothermal performance of the Cu/Fe–NH₂-MIL-101@MoO₂

The photoresponsive heating behavior of Cu/Fe–NH₂-MIL-101@MoO₂ was systematically evaluated under 808 nm laser irradiation. Fig. 2(a) first explores the heating capacity of the material at different powers. According to the different final increase temperatures of the material at the same time, the power of 1 W cm⁻² was selected for subsequent photothermal experiments. Fig. 2(b) shows that the Cu/Fe–NH₂-MIL-101@MoO₂ aqueous solution temperature reached about 53 °C at 10 minutes after irradiation. However, the temperature of the same volume of water was essentially unchanged after irradiation. Compared to water baseline confirms efficient near-infrared-to-thermal energy transduction. The good photothermal capability of Cu/Fe–NH₂-MIL-101@MoO₂ is also verified by the corresponding thermal imaging maps of different concentrations in Fig. 2(c). Cyclic irradiation tests (Fig. 2(d)) revealed good photostability after 5 on/off cycles (808 nm, 1 W cm⁻², 10 min). Cu/Fe–NH₂-MIL-101@MoO₂ and H₂O were irradiated by a laser, respectively. The results are shown in Fig. 2(e and f). The photothermal conversion ability of the material was evaluated, and Cu/Fe–NH₂-MIL-101@MoO₂ was calculated according to the photothermal formula. The photothermal conversion efficiency is 39.4%, which is significantly better than that of other nanomaterials PEG@S-MoO_x (36.72%)²⁹ and Cu_2−xS@Au (32.03%).³⁰ In summary, the synthesized Cu/Fe–NH₂-MIL-101@MoO₂ has good photostability and photothermal conversion ability and has great application potential in cancer treatment.

Fig. 2 (a) Temperature changes of Cu/Fe–NH₂-MIL-101@MoO₂ (0.2 mg mL⁻¹) under 808 nm laser irradiation with different power densities for 10 min. (b) Temperature changes of Cu/Fe–NH₂-MIL-101@MoO₂ with different concentrations under 808 nm laser irradiation (1 W cm⁻²). (c) The infrared thermal images of Cu/Fe–NH₂-MIL-101@MoO₂ irradiated by 808 nm laser (1 W cm⁻²) at different aqueous concentrations. (d) Temperature variation of Cu/Fe–NH₂-MIL-101@MoO₂ with repeated 808 nm irradiation (1 W cm⁻²) on/off for five cycles. (e) and (f) Heating and cooling curves of Cu/Fe–NH₂-MIL-101@MoO₂ under the laser and corresponding cooling time constant (τ_s) calculation and photothermal conversion efficiency (η).

3.5. Analysis of the enzymatic activity of the Cu/Fe–NH₂-MIL-101@MoO₂

The synergistic effect of different metal ions can be proved by the impedance diagrams, as shown in Fig. S6 (ESI†). In the Nyquist plot, a smaller semicircle radius indicates faster electron transfer kinetics and higher catalytic degradation efficiency. As demonstrated by the experimental results, Cu/Fe–NH₂-MIL-101@MoO₂/MTX exhibits the smallest semicircle radius, confirming that the bimetallic MOF structure facilitates enhanced electron transfer.

X-ray photoelectron spectroscopy (XPS) analysis was performed to probe the valence state evolution of Cu/Fe–NH₂-MIL-101@MoO₂ during H₂O₂ and GSH treatments (Fig. 3). The survey spectrum (Fig. 3(a)) confirms the coexistence of Cu 2p, Fe 2p, O 1s, C 1s, N 1s, and Mo 3d core-level signals, validating the chemical composition of the synthesized composite. As shown in Fig. 3(b), both Mo 3d_5/2 (231.9 eV) and Mo 3d_3/2 (235.15 eV) are the peaks of Mo⁴⁺ detected in the XPS spectrum. After H₂O₂ treatment, these peaks shift to 232.9 eV and 236.1 eV, respectively, confirming oxidation to Mo⁶⁺. Post-GSH exposure (Fig. 3(c)) reveals mixed Mo⁴⁺/Mo⁶⁺ states, suggesting partial reduction of Mo⁶⁺ by thiol groups, which may be due to the high valence state of Mo, which is easily reduced. Fig. 3(d) shows that the peak of Fe element Fe 2p_3/2 in the XPS spectrum is 710.1 eV and 723.8 eV, and the peaks of Fe 2p_1/2 are 711.7 eV and 727.5 eV. Also, 718 eV and 733.2 eV are the satellite peaks of the Fe element.³¹ After the reaction with H₂O₂, the peak intensity of Fe²⁺ decreased and the position shifted, indicating that Fe²⁺ was oxidized to Fe³⁺ by H₂O₂. Conversely, GSH treatment (Fig. 3(e)) restores Fe²⁺ content, a part of Fe³⁺ peak intensity is weakened, indicating that Fe³⁺ is reduced to Fe²⁺ by GSH. As shown in Fig. 3(f), the peaks at 932.4 eV, 934.7 eV, and 942.4 eV in the Cu 2p_3/2 region correspond to Cu⁺, Cu²⁺, and satellite peaks, respectively. The peaks at 952.3 eV, 954.7 eV, and 962.7 eV belong to Cu⁺, Cu²⁺, and satellite peaks in the Cu 2p_1/2 region. By analyzing the changes in peak area and shifts in binding energy before and after the reaction, it can be observed that Cu²⁺ is reduced to Cu⁺ under the action of GSH.

Fig. 3 (a) XPS full spectrum of the material. (b) Valence change of Mo before and after reaction with H₂O₂. (c) Valence change of Mo before and after reaction with GSH. (d) Valence change of Fe before and after reaction with H₂O₂. (e) Valence change of Fe before and after reaction with GSH. (f) Valence change of Cu before and after reaction with GSH. (g) Color experiments of TMB under different concentrations of H₂O₂. (h) Michaelis–Menten kinetic analysis and (i) Lineweaver–Burk plots of H₂O₂ concentrations catalyzed by Fe–NH₂-MIL-101 or Fe–NH₂-MIL-101 + NIR. (j) Color experiments of TMB with Cu/Fe–NH₂-MIL-101@MoO₂ or Cu/Fe–NH₂-MIL-101@MoO₂ + NIR (k) Michaelis–Menten kinetic analysis and (l) Lineweaver–Burk plots of H₂O₂ concentrations catalyzed by Cu/Fe–NH₂-MIL-101@MoO₂ or Cu/Fe–NH₂-MIL-101@MoO₂ + NIR.

The POD-like activity of Fe–NH₂-MIL-101 and Cu/Fe–NH₂-MIL-101@MoO₂ nanozymes was systematically investigated via TMB oxidation assays. As shown in Fig. 3(g), the characteristic absorption peaks of oxidized TMB at 370 nm and 652 nm exhibited progressive intensification with increasing H₂O₂ concentration (0–10 mM), indicating H₂O₂-dependent ˙OH generation through nanozyme-mediated Fenton-like reactions. Steady-state kinetic analysis was performed by fixing the concentrations of Fe–NH₂-MIL-101 and Cu/Fe–NH₂-MIL-101@MoO₂ while varying H₂O₂ concentrations, as shown in Fig. 3(h and k). The enzymatic kinetic parameters, including maximum reaction velocity (V_m) and Michaelis–Menten constant (K_m), were calculated from the corresponding Lineweaver–Burk plots (Fig. 3(i and l)). The results are summarized in the Table S1 (ESI†).

For Fe–NH₂-MIL-101, both V_m and K_m remained nearly unchanged under both NIR(on) and NIR(off) conditions, indicating that NIR light alone cannot accelerate the reaction rate in the absence of the photothermal agent MoO₂. In contrast, Cu/Fe–NH₂-MIL-101@MoO₂ exhibited a decreased K_m and an increased V_m compared to Fe–NH₂-MIL-101 under NIR(off) conditions, demonstrating that the bimetallic system enhances charge transfer and catalytic efficiency. Only when both the photothermal agent and the Fe/Cu bimetallic system are present, under NIR irradiation (Fig. 3(j)), the K_m and V_max values of Cu/Fe–NH₂-MIL-101@MoO₂ were determined to be 0.55 mM and 6.00 × 10⁻⁹ M s⁻¹, respectively. The lower K_m reflects higher substrate affinity, while the higher V_m signifies enhanced enzymatic activity. These results clearly demonstrate that near-infrared (NIR) irradiation accelerates electron transfer between Cu/Fe/Mo centers, leading to a significant improvement in enzymatic performance. This conclusion was confirmed by XPS valence state analysis and is consistent with the EIS results.

The Cu/Fe–NH₂-MIL-101@MoO₂ nanozyme was studied by GSH and DTNB probe experiments. As demonstrated in Fig. S7(a and b) (ESI†), the nanozyme exhibits GSH concentration-dependent catalytic behavior, which significantly improved the GPx-like enzymatic activity under NIR irradiation. Kinetic analysis (Fig. S7(c), ESI†) reveals classical Michaelis–Menten saturation kinetics toward the GSH substrate. The Cu/Fe–NH₂-MIL-101@MoO₂ nanozyme conforms to the Michaelis–Menten model. K_m and V_max are calculated from the Lineweaver–Burk plot as shown in Fig. S7(d) (ESI†).

The EPR test was further performed on the material, as shown in Fig. S8 (ESI†). The EPR spectrum proves that the ability to produce ˙OH can be enhanced under the action of NIR (808 nm, 1 W cm⁻²), which lays a foundation for subsequent multimodal tumor therapy.

3.6. Drug loading and release of materials

The drug loading capacity of Cu/Fe–NH₂-MIL-101 and its MoO₂ composite was quantified via UV-vis spectroscopy based on the MTX standard curve (Fig. 4(a)). As shown in Fig. 4(b), the MTX loading efficiencies reached 9.3 mg g⁻¹ and 18.1 mg g⁻¹, respectively – values superior to most reported nanocarriers. pH/GSH dual-responsive release profiles were systematically investigated under simulated physiological (pH 7.4, 2 mM GSH) and tumor microenvironmental (pH 5.8, 10 mM GSH) conditions (Fig. 4(c)). The composite exhibited minimal MTX release under physiological conditions after 72 h, whereas tumor-mimicking conditions triggered accelerated release kinetics with cumulative release reaching 32.6% – attributed to pH-sensitive coordination bond cleavage and GSH-mediated redox dissolution.

Fig. 4 (a) The standard working curve of MTX. (b) The drug loading capacity of different materials to MTX. (c) Drug release rate at different pH and GSH concentrations. (d) The content of Fe³⁺ and Mo²⁺ ions in the supernatant under different conditions was tested by ICP-OES.

ICP-OES analysis (Fig. 4(d)) confirmed concomitant Mo ion release under acidic/high-GSH conditions, verifying the self-accelerating degradation mechanism. These results conclusively demonstrate the material's pH/GSH dual-responsive drug release behavior, aligning with tumor microenvironment characteristics for precision therapy applications.

3.7. Cytotoxicity and intracellular ROS detection

The in vitro biocompatibility and therapeutic efficacy were evaluated using CCK-8 assays (Fig. 5). As shown in Fig. 5(a), Cu/Fe–NH₂-MIL-101@MoO₂/MTX exhibited minimal cytotoxicity toward normal mouse fibroblast 293T cells, confirming its biosafety profile. For 4T1 murine breast cancer cells (Fig. 5(b)), the composite demonstrated concentration-dependent cytotoxicity, with cell viability decreasing progressively from 100% to 50% as material concentration increased from 25 to 200 μg L⁻¹.

Fig. 5 (a) Viability of L929 cell after 24 h of incubation with Cu/Fe–NH₂-MIL-101@MoO₂/MTX. (b) Cell viability of 4T1 cells after 24 h of incubation of Cu/Fe–NH₂-MIL-101, Cu/Fe–NH₂-MIL-101@MoO₂, Cu/Fe–NH₂-MIL-101@MoO₂/MTX and Cu/Fe–NH₂-MIL-101@MoO₂/MTX + laser. The error bar is the standard deviation of experimental data for three times. (c) CLSM images of DCFH-DA probe against 4T1 cells after treatment with different concentrations Cu/Fe–NH₂-MIL-101@MoO₂/MTX.

Remarkably, NIR irradiation (808 nm, 1 W cm⁻²) synergistically enhanced the therapeutic effect, reducing 4T1 cell viability to 35% under optimal conditions. This photothermal-chemotherapy combination outperformed individual treatment modalities, consistent with the material's previously demonstrated photothermal conversion efficiency and redox-responsive drug release behavior.

4T1 cells were incubated with fluorescent probe DCFH-DA to study the ability of different materials to produce –OH in 4T1 cells, and the results are shown in Fig. 5(c). DCFH-DA did not show any obvious green fluorescence in 4T1 cells of the control group, and the addition of stimulus in the control group only stimulated the weak green fluorescence of 4T1 cells. As the concentration of Cu/Fe–NH₂-MIL-101@MoO₂/MTX increased, the green fluorescence became brighter and brighter, and the brightest green fluorescence was produced after NIR irradiation.

The brightest green fluorescence was produced after NIR irradiation, indicating that Cu/Fe–NH₂-MIL-101@MoO₂/MTX can produce ˙OH in cancer cells, and under the condition of light and heat, it can produce more ˙OH, which is conducive to the realization of photothermal enhancement of catalytic reaction for the use of tumor therapy.

3.8. Intracellular CRT detection and flow cytometry analysis

The high expression of CRT was used as a marker of ROS-induced ICD to detect the ICD effect^32–34 of Cu/Fe–NH₂-MIL-101@MoO₂/MTX, as shown in Fig. 6(a). As depicted in the figure, after treatment with Cu/Fe–NH₂-MIL-101@MoO₂/MTX and NIR irradiation, CRT has a significant shift to the cell membrane, indicating the induction of ICD. Fig. 6(b) demonstrates that the apoptosis rate of Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR reached 33.23%, highlighting its potent ability to induce apoptosis of 4T1 cells compared with the control group + NIR. Fig. 6(c) presents live and dead cell staining fluorescence microscopy, which corroborated the apoptosis and cytotoxicity experimental results. Microscopic examination revealed extensive red staining, confirming its capacity to induce significant apoptosis.

Fig. 6 (a) CLSM images of cellular CRT against 4T1 cells after treatment with different samples. (b) Flow cytometry analysis of 4T1 cells after treatment with different samples. (c) Live dead cell staining analysis of 4T1 cells after treatment with different samples.

3.9. MRI magnetic resonance imaging

The pH-responsive decomposition of Cu/Fe–NH₂-MIL-101@MoO₂/MTX nanozymes was explored in the drug release properties, which would facilitate the release of Fe³⁺, a known T₂ imaging contrast agent. T₂-MRI images of Cu/Fe–NH₂-MIL-101@MoO₂/MTX solution were recorded with a 3.0 T MRI scanner as shown in Fig. 7(a). The longitudinal relaxation r₂ was measured to be 272.8 mM⁻¹ s⁻¹ for different Fe concentrations. The in vivo metabolism of Cu/Fe–NH₂-MIL-101@MoO₂/MTX nano-enzymes was then investigated at different times, Fig. 7(c) illustrates that the imaging region progressively darkened with increasing time, further confirming the superior MRI imaging capability of Cu/Fe–NH₂-MIL-101@MoO₂/MTX nanozymes. These findings suggest that Cu/Fe–NH₂-MIL-101@MoO₂/MTX holds significant potential for applications in tumor therapy and diagnosis.

Fig. 7 (a) The relaxation rate as a function of Fe concentrations and corresponding MRI images of purified Cu/Fe–NH₂-MIL-101@MoO₂/MTX. (b) Histogram of time and tumor MRI imaging intensity. (c) In vivo MRI images of tumors in the treatment groups.

3.10. Antitumous effect

The therapeutic effect on proximal and distal tumors was verified by mouse experiments, and the ability to prevent tumor recurrence can be speculated. The 4T1 mouse mammary cancer model was established by inoculating 4T1 cells subcutaneously in the thighs of Balb/c nude mice. When the tumor size reached approximately 100 mm³, the mice were treated by intravenous injection of different materials according to the groupings to evaluate their antitumor effects. Mice with double tumors were used to verify whether different treatment groups produced ICD effects triggering anti-tumor immune responses during the treatment of primary tumors, and the process is shown in Fig. S9 (ESI†).

In this experiment, the right tumor was designed as the original tumor to prove the synergistic therapeutic effect based on catalysis and chemotherapy, and the left tumor was designed as the distal tumor to test the ICD effect. Only the right-side tumor was subjected to NIR irradiation during the treatment. Fig. 8(a) shows that there was no significant change in the body weight of the mice during the treatment, indicating that different treatment groups did not affect the body weight of the mice.

Fig. 8 (a) Change curve of mouse body weight under PBS, Cu/Fe–NH₂-MIL-101, Cu/Fe–NH₂-MIL-101@MoO₂, Cu/Fe–NH₂-MIL-101@MoO₂/MTX and Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR respectively for 14 days. (b) The growth of 4T1 tumors in different groups of mice after various treatments in the primary tumor indicated. The relative tumor volumes were normalized to their initial sizes. (c) The growth of 4T1 tumors in different groups of mice after various treatments in the distant tumor indicated. (d) The size of the image of the primary tumor after 21 days. (e) The size of the image of the distant tumor after 21 days. (f) IR thermal images of 4T1 tumor-bear mice under the laser irradiation (808 nm, 1.0 W cm⁻²) taken at different time intervals. (g) Representative photos of mice after various different treatments. Data were presented as mean ± SD (n = 3).

Fig. 8(b and c), the relative volume of the primary and distal tumors was calculated, respectively. In the Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR group, 77% of the primary tumors were inhibited and 33.1% of the distant tumors were inhibited. It is proved that the ICD effect of tumors is efficiently activated under the synergistic treatment mode of chemotherapy, photothermal therapy and catalytic therapy.

Fig. 8(g) is the life diagram of mice, showing the tumor effect of different treatment groups in different time periods. The Cu/Fe–NH₂-MIL-101@MoO₂/MTX+ NIR group showed obvious double ablation of the original tumor and the distal tumor after 21 days of treatment, which confirmed that the material had a strong therapeutic effect and ability to activate immunity. When the mice were treated for 21 days, the results are shown in Fig. 8(d and e), the mice were executed with anesthetics, the tumor site was removed, and the weighing showed that the Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR group had the best therapeutic effect. Fig. 8(f) shows the photothermal imaging of the mice, the local warming of Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR compared with the control PBS + NIR group reached 51.2 °C, which also confirmed the good photothermal responsiveness of the material and certain tumor targeting effect.

After 21 days of treatment, the mice were sacrificed, and the tumor tissues were taken for H&E staining analysis. As shown in Fig. S10 (ESI†), the H&E staining experiments of the primary and distant tumors further confirmed that the Cu/Fe–NH₂-MIL-101@MoO₂/MTX + NIR group caused a large number of apoptosis of tumor cells. These conclusions have confirmed that Cu/Fe–NH₂-MIL-101@MoO₂/MTX can effectively activate ICD to induce an anti-tumor response. In addition to the treatment of primary tumors, it can also inhibit the growth of distant and metastatic tumors, which is expected to prevent tumor recurrence and metastasis.

4. Conclusion

In summary, we synthesized multifunctional nanozymes based on Fe–NH₂-MIL-101 for integrated diagnosis and prevention, which can both specifically accumulate in tumor cells and achieve synergistic treatment of tumors through chemotherapy, catalytic therapy, and photothermal therapy under the guidance of MRI. With the assistance of MTX, Cu/Fe–NH₂-MIL-101@MoO₂ can effectively accumulate in tumor cells. In addition, the photothermal properties of Cu/Fe–NH₂-MIL-101@MoO₂ can enhance the activity of nanozymes, which in turn leads to oxidative stress and apoptosis in the tumor microenvironment. Meanwhile, the combination of drug loading and photothermal can induce the ICD effect, which triggers the distal anti-tumor effect, thus producing effective inhibition of tumor metastasis.

Ethical statement

The authors declare that all experiments were performed in compliance with relevant laws and all experiments followed Hubei University guidelines. The authors declare that the experiments were designed in vivo, followed ethical and legal guidelines, and were approved by Tongji Hospital of Huazhong University of Science and Technology.

Data availability

All data that support the findings of this study are included within the article (and any ESI†)

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Natural Science Foundation of Hubei Province (Grant No. 2025AFD349), and the Hubei Key Laboratory for Precision Synthesis of Small Molecule Pharmaceuticals, also supported by the Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Function Molecules, Hubei University. The animal experiments were performed in accordance with the guidelines as stated by the National Institutes of Health and approved by the Institution Animal Care and Use Committee at Tongji Medical College, Huazhong University of Science and Technology.

References

1. L. L. Zhang, Q. B. Zhang and D. T. Hinojosa, et al., Multifunctional Magnetic Nanoclusters Can Induce Immunogenic Cell Death and Suppress Tumor Recurrence and Metastasis, ACS Nano, 2022, 16(11), 18538–18554.
2. H. Zhang, Y. Liu, Y. Cao, P. Song, W. Li, Z. Lv, S. Song, Y. Wang, H. Zhang and A. Porous, Organic Framework-Based Nanoplatform for Directional Destruction on Endoplasmic Reticulum to Enhance Tumor Immunotherapy, Adv. Funct. Mater., 2023, 33, 2307175.
3. B. Li, G. Hao, B. Sun, Z. Gu and Z. P. Xu, Engineering a Therapy-Induced “Immunogenic Cancer Cell Death” Amplifier to Boost Systemic Tumor Elimination, Adv. Funct. Mater., 2020, 30, 1909745.
4. Y. P. Wang, J. M. Qian and W. J. Xu, et al., Polysaccharide nanodonuts for photochemotherapy amplified immunogenic cell death to potentiate systemic antitumor immunity against hepatocellular carcinoma, Adv. Funct. Mater., 2022, 33, 2208486.
5. H. Barker, J. Paget and A. Khan, et al., The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence, Nat. Rev. Cancer, 2015, 15, 409–425.
6. S. S. You, S. Lu and H. Q. Gao, et al., Photothermal-enhanced multifunctional nanozyme for regulate GSH and H₂O₂ in tumor for improving catalytic therapy, Phys. Scr., 2024, 99(2), 025014.
7. X. L. Feng, T. Lin and D. Chen, et al., Mitochondria-associated ER stress evokes immunogenic cell death through the ROS-PERK-eIF2alpha pathway under PTT/CDT combined therapy, Acta Biomater., 2023, 160, 211–224.
8. S. Peng, F. Xiao, M. Chen and H. Gao, Tumor-Microenvironment-Responsive Nanomedicine for Enhanced Cancer Immunotherapy, Adv. Sci., 2022, 9, 2103836.
9. X. Shi, X. Wang and W. Yao, et al., Mechanism insights and therapeutic intervention of tumor metastasis: latest developments and perspectives, Signal Transduction Targeted Ther., 2024, 9, 192.
10. X. Niu, X. Li, Z. Lyu, J. Pan, S. Ding, X. Ruan, W. Zhu, D. Du and Y. Lin, Chem. Commun., 2020, 56, 11338,  10.1039/D0CC04890A.
11. Y. Huang, J. Ren and X. Qu, Chem. Rev., 2019, 119(6), 4357–4412,  DOI:10.1021/acs.chemrev.8b00672.
12. A. Li, Y. Jia, Y. He, M. Yuan, Z. Hao, Y. He, Y. Fu, J. Zhang, D. Gao, X. Zhang, X. Jiang and W. Tu, Natural MOF-Like Photocatalytic Nanozymes Alleviate Tumor Pressure for Enhanced Nanodrug Penetration, Adv. Healthcare Mater., 2024, 2400596.
13. X. Hu, R. Li, J. Liu, K. Fang, C. Dong and S. Shi, Engineering Dual-Responsive Prodrug-MOFs as Immunogenic Cell Death Initiator for Enhancing Cancer Immunotherapy, Adv. Healthcare Mater., 2024, 13, 2302333.
14. J. Wang, Y. Wang and X. Xiaohalati, et al., A Bioinspired Manganese-Organic Framework Ameliorates Ischemic Stroke through its Intrinsic Nanozyme Activity and Upregulating Endogenous Antioxidant Enzymes, Adv. Sci., 2023, 10(20), 2206854.
15. H. Pham, K. Ramos and A. Sua, et al., Tuning crystal structures of iron-based metal-organic frameworks for drug delivery applications, ACS Omega, 2020, 5, 3418–3427.
16. Z. Zhao, Z. Zhou and J. Bao, et al., Octapod iron oxide nanoparticles as high-performance T₂ contrast agents for magnetic resonance imaging, Nat. Commun., 2013, 4, 2266.
17. L. Liu and A. Corma, Bimetallic Sites for Catalysis: From Binuclear Metal Sites to Bimetallic Nanoclusters and Nanoparticles, Chem. Rev., 2023, 123(8), 4855–4933.
18. X. Li, J. Chen and Z. Liu, et al., Recognizing the relevance of non-radical peroxymonosulfate activation by co-doped Fe metal-organic framework for the high-efficient degradation of acetaminophen: Role of singlet oxygen and the enhancement of redox cycle, Chem. Eng. J., 2024, 156081.
19. Y. Zhu, R. Zhu, Y. Xi, J. Zhu, G. Zhu and H. He, Strategies for enhancing the heterogeneous Fenton catalytic reactivity: A review, Appl. Catal., B, 2019, 255, 117739.
20. N. Wang, T. Zheng, G. Zhang and P. Wang, A review on Fenton-like processes for organic wastewater treatment, J. Environ. Chem. Eng., 2016, 4(1), 762–787.
21. Q. Song, B. Chi and H. Gao, et al., Functionalized nanozyme with drug loading for enhanced tumour combination treatment of catalytic therapy and chemotherapy, J. Mater. Chem. B, 2023, 11, 6889–6895.
22. Y. X. Guo, Y. Li and W. L. Zhang, et al., Insights into the deep-tissue photothermal therapy in near-infrared II region based on tumor-targeted MoO2 nanoaggregates, Sci. China Mater., 2020, 63(6), 1085–1098.
23. E. Y. Xing, Y. Y. Du and J. J. Yin, et al., Multi-functional Nanodrug Based on a Three-dimensional Framework for Targeted Photo-chemo Synergetic Cancer Therapy, Adv. Healthcare Mater., 2021, 10(8), 2001874.
24. M. Rezki, N. L. W. Septiani, M. Iqbal, S. Harimurti, P. Sambegoro, D. R. Adhika and B. Yuliarto, Amine-functionalized Cu-MOF nanospheres towards label-free hepatitis B surface antigen electrochemical immunosensors, J. Mater. Chem. B, 2021, 9(28), 5711–5721.
25. J. Liu, J. Yang, S. An, M. Wen, X. Wang, R. Gong and Y. Nie, Synthesis and electromagnetic properties of NH2-MIL-88B(Fe) crystals with morphology and size controllable through synergistic effects of surfactant and water, J. Mater. Sci.: Mater. Electron., 2022, 33(17), 14228–14239.
26. L. Shao, Z. Yu, X. Li, X. Li, H. Zeng and X. Feng, Carbon nanodots anchored onto the metal-organic framework NH2-MIL-88B(Fe) as a novel visible light-driven photocatalyst: Photocatalytic performance and mechanism investigation, Appl. Surf. Sci., 2020, 505, 144616.
27. J. Zhou, N. S. Xu, S. Z. Deng, J. Chen and J. C. She, Synthesis of large-scaled MoO2 nanowire arrays, Chem. Phys. Lett., 2003, 382(3–4), 443–446.
28. W. Liu, X. Li, W. Li, Q. Zhang, H. Bai, J. Li and G. Xi, Highly stable molybdenum dioxide nanoparticles with strong plasmon resonance are promising in photothermal cancer therapy, Biomaterials, 2018, 163, 43–54.
29. Y. Wang, N. Gong, Y. Li, Q. Lu, X. Wang and J. Li, Atomic-Level Nanorings (A-NRs) Therapeutic Agent for Photoacoustic Imaging and Photothermal/Photodynamic Therapy of Cancer, J. Am. Chem. Soc., 2020, 142(4), 1735–1739.
30. L. Yang, B. Hu, A. H. Liu and Y. Zhang, A hollow-structured nanohybrid: Intelligent and visible drug delivery and photothermal therapy for cancer, Talanta, 2020, 215, 120893.
31. S. Koo, O. K. Park, J. Kim, S. I. Han, T. Y. Yoo, N. Lee, Y. G. Kim, H. Kim, C. Lim, J. S. Bae, J. Yoo, D. Kim, S. H. Choi and T. Hyeon, Enhanced Chemodynamic Therapy by Cu-Fe Peroxide Nanoparticles: Tumor Microenvironment-Mediated Synergistic Fenton Reaction, ACS Nano, 2022, 16(2), 2535–2545.
32. M. Obeid, A. Tesniere and F. Ghiringhelli, et al., Calreticulin exposure dictates the immunogenicity of cancer cell death, Nat. Med., 2007, 13, 54–61,  DOI:10.1038/nm1523.
33. J. Fucikova, R. Spisek and G. Kroemer, et al., Calreticulin and cancer, Cell Res., 2021, 31, 5–16,  DOI:10.1038/s41422-020-0383-9.
34. T. Panaretakis, et al., Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death, EMBO J., 2009, 28(5), 578,  DOI:10.1038/emboj.2009.1.

---

Footnotes

† Electronic supplementary information (ESI) available: Experimental procedures, characterization data, and related details. See DOI: https://doi.org/10.1039/d5tb00686d

‡ These authors contributed equally to this work.

---