Mild‐Photothermal Effect Induced High Efficiency Ferroptosis‐Boosted‐Cuproptosis Based on Cu2O@Mn3Cu3O8 Nanozyme

Abstract A core‐shell‐structured Cu2O@Mn3Cu3O8 (CMCO) nanozyme is constructed to serve as a tumor microenvironment (TME)‐activated copper ionophore to achieve safe and efficient cuproptosis. The Mn3Cu3O8 shell not only prevents exposure of normal tissues to the Cu2O core to reduce systemic toxicity but also exhibits enhanced enzyme‐mimicking activity owing to the better band continuity near the Fermi surface. The glutathione oxidase (GSHOx)‐like activity of CMCO depletes glutathione (GSH), which diminishes the ability to chelate Cu ions, thereby exerting Cu toxicity and inducing cuproptosis in cancer cells. The catalase (CAT)‐like activity catalyzes the overexpressed H2O2 in the TME, thereby generating O2 in the tricarboxylic acid (TCA) cycle to enhance cuproptosis. More importantly, the Fenton‐like reaction based on the release of Mn ions and the inactivation of glutathione peroxidase 4 induced by the elimination of GSH results in ferroptosis, accompanied by the accumulation of lipid peroxidation and reactive oxygen species that can cleave stress‐induced heat shock proteins to compromise their protective capacity of cancer cells and further sensitize cuproptosis. CMCO nanozymes are partially sulfurized by hydrogen sulfide in the colorectal TME, exhibiting excellent photothermal properties and enzyme‐mimicking activity. The mild photothermal effect enhances the enzyme‐mimicking activity of the CMCO nanozymes, thus inducing high‐efficiency ferroptosis‐boosted‐cuproptosis.

The CAT-like activity kinetic assays of Cu2O, CMO and CMCO with H2O2 as the substrate were performed.Cu2O/CMO/CMCO (50 μg•mL -1 ) and different concentrations (2,4,8,12,16, 20 mM) of H2O2 were added into PBS (pH = 6.5) for reacting 30 s.The real-time oxygen concentration was recorded using portable dissolved oxygen meter.For each H2O2 concentration, the initial reaction rates (V0) were decided from the real-time oxygen concentration changes.
The values of Km and Vmax can be calculated according to the initial reaction rates (V0) with different initial substrate ([S]) by Lineweaver-Bruke plot (equation 2).

Theoretical calculation
The calculations were based on density functional theory (DFT) using projector augmented wave (PAW) methods, as implemented in the Vienna ab initial simulation package (VASP).A plane-wave basis set with a kinetic-energy cut-off of 400 eV was used to expand the wave function of valence electrons.The generalized gradient approximation (GGA) with the Perdew-Burke-Ernzerhof (PBE) functional was used for describing the exchangecorrelation interactions.The structural relaxations were performed by computing the Hellmann-Feynman forces within the total energy and force convergences of 10 -5 eV and 10 -4 eV/Å, respectively.

Photothermal conversion efficiency of CMCO and Cu2O in the presence of NaHS
CMCO (100 μg•mL -1 ) reacted with NaHS•xH2O (200 μg•mL -1 ) for 5, 10, 20, 60 min, respectively, and the products were dispersed in the same volume of deionized water after centrifugation.The aqueous solution (1 mL) was irradiated upon 1064 nm laser (1 W•cm -2 ) for 780 s, and then turned off.In the meantime, the temperature was detected by infrared camera every 30 s.
The photothermal conversion efficiency (η) can be calculated according to the equation 3: Where h is the heat transfer coefficient, S is the surface area of the container.Here, hS can be obtained by the equation 4. Tmax is the maximum temperature of the solution, and Tsurr is the temperature of the surrounding.Qdis is the heat generated after water and container absorbs light, which is calculated by the equation 5.I is the laser power density, and A1064 is the absorption value of the material at 1064 nm.
To test the photothermal stability of CMCO in the presence of NaHS, the 1064 nm laser (1 W•cm -2 ) was turned on for 600 s and turned off for 600s to the products, which were reacted by CMCO (100 μg•mL -1 ) and NaHS•xH2O (200 μg•mL -1 ) for 10 min.Three cycles were repeated.
mD is the mass of water, and CD is the heat capacity of water (4.2 J•g -1 •°C -1 ).τs is the sample system time constant, which was calculated using the equation 6 and equation 7.
t is the time of the cooling process after irradiation, and T is the temperature of the solution at different time point during this process.

Enzymatic activities capacity of CMCO nanozymes partially sulfurized
The OPD as probe was used to assess the generation of •OH in the presence of H2O2.
After centrifugation, the supernatant were mixed with DTNB in PBS (pH = 6.5) for 10 min (complete reaction).The absorbance of the supernatant solution was recorded using UV-vis spectrophotometer.

Intracellular ROS detection
The intracellular ROS genaration was detected using ROS assay kit. 1 × 10 5 CT26 cells were seeded on coverslips in 24 well plates and grown for 24 h.And then, the cells were treated with PBS, Cu2O (20 μg•mL -1 ) and CMCO (20 μg•mL -1 ) for 4 h.After removing the culture medium, the cells were incubated with DCFH-DA and Hoechest for 20 min at 37 ℃.Washed using serum-free medium, the cells were further irradiated with or without 1064 nm laser (1 W•cm -2 , 5 min) and imaged by confocal microscopy.

Detection of intracellular GSH
The CT26 cells were seeded into culture dishs (D = 9 mm) and treated with PBS, NIR, CMCO and CMCO + NIR for 4 h.After incubation, the cells were counted and collected for 5 × 10 6 .The relative GSH contents of different groups were detected using the reduced GSH assay kit.

Detection of intracellular protein expression
The CT26 cells were seeded into 6 well plates for 24 h.To analyse GPX-4 expression, four experiment groups were designed as PBS, NIR, CMCO and CMCO + NIR.To analyse HSP70 expression, nine experiment groups were designed as PBS, NIR, 42℃, CMCO, CMCO measured every two days.The tumor volume (V) was calculated using the formula V = L×W 2 ×0.52.On day 15, the main organs (heart, liver, spleen, lung and kidney) and tumor tissues were removed after mice sacrifice under anesthesia.The organs were harvested and dissected to make paraffin section for further hematoxylin and eosin (H&E) staining.The excised tumors were harvested and dissected to make paraffin section for further H&E staining and terminal deoxynucleotidyl transferase dUTP nick labeling (TUNEL).In addition, the blood was collected from the eyeball for biochemistry assay.

Distribution and metabolism of CMCO in vivo
The healthy BALB/c mice were intravenously injected with CMCO (200 μg, 100 μL) and the products (200 μg, 100 μL) of CMCO sulfurized by NaHS for 1 h, respectively.Besides, the CT26 tumor-bearing mice were intravenously injected with CMCO (200 μg, 100 μL).The main organs were collected, weighed and dissolved in mixed solution of concentrated nitric acid and hydrogen peroxide (5:1).Mn and Cu elements of various samples were detected by ICP-MS.As shown in Table S1, the ratio of Cu and Mn was close to 1:1.However, due to the nickel oxide supporting film as the carrier stage, the O element in the blank could increase the proportion of O in the shell, so the true radio of O element can't be determined.
Furthermore, the corresponding chemical formula and crystal form was analyzed based on the XRD pattern of the nanozyme (Figure 1D), assuming a Cu-Mn ratio of 1:1, and the shell layer was finally determined to be Mn3Cu3O8.The structure of CMO was similar to CMCO, which both were core-shell (Figure S15A).
Differently, the shell of CMO was composed of monoclinic Mn5O8 (space group: C2/m) (Figure S15B).The elements of Cu, Mn and O were similarly distributed in different parts over the whole core-shell structure of CMO.O element homogeneously distributed on the core and shell, while Mn mainly distributed on the shell and Cu element mostly distributed on the core, respectively (Figure S15C).

Figure S4 .
Figure S4.TME images of nanoparticles generated by the reaction of Cu2O with KMnO4 for (A) 0.5 h, (B) 1 h, (C) 2 h, (D) 3 h and (E) 4 h and (F-J) the corresponding products after being sulfurated by NaHS solution for 10 min.

Figure S8 .
Figure S8.Elemental mapping of Cu, Mn and O of CMCO.

Figure S10 .
Figure S10.TME images of three batches of CMCO nanozymes for measuring the thickness of Mn3Cu3O8 shell layer.It was calculated to be about 25.7 ± 0.7 nm.

Figure S18 .
Figure S18.Michaelis-Menten kinetic analysis of CAT-like activities for (A) Cu2O, (C) CMO and (E) CMCO with H2O2 as a substrate.Lineweaver-Burk plot of CAT-like activities for (B) Cu2O, (D) CMO and (F) CMCO with H2O2 as a substrate.

Figure S20 .
Figure S20.(A) Baseband calculation results and (B) densities of states of Mn3Cu3O8 shell of nanozymes.

Figure S29 .
Figure S29.The size distribution of CMCO and the product of CMCO reacting with NaHS in PBS (pH = 6.5) for 10 min.

Figure S37 .
Figure S37.Cytotoxicity assessment on CSMC treated with different concentration of Cu2O and CMCO nanozymes.Dates are presented as mean ± SD (n = 6).

Figure S42 .
Figure S42.Cytotoxicity assessment on CT26 with different treatment.Dates are presented as mean ± SD (n = 6).

Figure S48 .
Figure S48.The tumor growth curves of CT26 tumor-bearing mice after different treatments with intratumor injection.

Figure S49 .
Figure S49.The body weights of CT26 tumor-bearing mice over time after different treatments with intratumor injection.Dates are presented as mean ± SD (n = 6).

Figure S50 .
Figure S50.Fluorescence images of CMCO and CMCO-FITC accumulated in the tumor site of CT26-bearing mice injected via the tail vein.

Figure S51 .
Figure S51.(A), (B)and (C) Tumor growth curves and (D) body weights of CT26 tumorbearing mice after different treatments with intravenous injection.Dates in (D) are presented as mean ± SD (n = 6).

Figure S55 .
Figure S55.(A) TEM and (B) XRD of the products of Cu2O reacting with NaHS for 1 min.

Figure S57 .
Figure S57.The metabolism of Mn and Cu from CT26 tumor-bearing mice through (A) urine and (B) feces after treated with CMCO + NIR.Dates are presented as mean ± SD (n = 3).

Table S1 .
The ratio of elements in the Mn3Cu3O8 shell from the areas of FigureS6C.