Unraveling Ros Conversion Through Enhanced Enzyme‐Like Activity with Copper‐Doped Cerium Oxide for Tumor Nanocatalytic Therapy

Abstract Nanozyme catalytic therapy for cancer treatments has become one of the heated topics, and the therapeutic efficacy is highly correlated with their catalytic efficiency. In this work, three copper‐doped CeO2 supports with various structures as well as crystal facets are developed to realize dual enzyme‐mimic catalytic activities, that is superoxide dismutase (SOD) to reduce superoxide radicals to H2O2 and peroxidase (POD) to transform H2O2 to ∙OH. The wire‐shaped CeO2/Cu‐W has the richest surface oxygen vacancies, and a low level of oxygen vacancy (Vo) formation energy, which allows for the elimination of intracellular reactive oxygen spieces (ROS) and continuous transformation to ∙OH with cascade reaction. Moreover, the wire‐shaped CeO2/Cu‐W displays the highest toxic ∙OH production capacity in an acidic intracellular environment, inducing breast cancer cell death and pro‐apoptotic autophagy. Therefore, wire‐shaped CeO2/Cu nanoparticles as an artificial enzyme system can have great potential in the intervention of intracellular ROS in cancer cells, achieving efficacious nanocatalytic therapy.


Structure characterizations
All materials were of analytical grade and used without further purification.
A field emission scanning electron microscopy (FESEM; ZEISS-Merlin), a transmission electron microscopy (TEM, JEOL-2010) with energy dispersive X-ray spectroscopy (EDX), and a high-resolution TEM (HRTEM, JEOL-2010) were used to characterize the morphology and composition of samples.XRD curves of samples were recorded on Rigaku at 40 kV and 40 mA, and X-ray photoelectron spectroscopy (XPS) curves were obtained on a PHI Quantera SXM (ULVAC-PHI) instrument to determine the compositions and the valence states of the elements in the samples.All electrochemical measurements were performed on a CHI 760E electrochemical work station with a typical three-electrode setting at room temperature.A graphite rod and an Ag/AgCl electrode were selected as a counter and reference electrode, respectively.The self-supporting array grown on carbon cloth (1×1 cm, mass loading ~ 2.5 mg/cm -2 ) was directly used as a working electrode.The electrochemical data were collected in an electrolyte of 1.0 M KOH.

Synthesis of CeO2 wires (CeO2-W) and cubes (CeO2-C).
Generally, a solution composed of 3.472 g Ce(NO3)3• 6H2O (Aladdin) and 20 mL deionized (DI) water was mixed with the other solution composed of 38.4 g NaOH and 140 mL DI water.
After the obtained solution was continually stirred for 0.5 h, it was moved to a 250 mL stainless steel autoclave with a Teflon liner, followed by hydrothermal treatment at 100 °C for 24 h to obtain CeO2-W, and at 180 °C for 24 h to obtain CeO2-C.The precipitation was then purified by centrifugation.After it was rinsed using DI water (until pH = 7) and pure ethanol three times, the final ceria samples were obtained by drying at 80 °C for 8 h and calcination in air at 400 °C for 4 h.

Synthesis of CeO2 octahedrons (CeO2-O).
A solution composed of 1.716 g Ce(NO3)3• 6H2O and 20 mL DI water was mixed with the other solution composed of 0.015 g Na3PO4 and 140 mL DI water.After the obtained solution was continually stirred for half an hour, it was moved into a 250 mL autoclave and hydrothermally treated at 170 °C for 10 h.Finally, the formed solids were separated, dried and calcined by following the same procedure as CeO2-W.

Preparation of CeO2/Cu catalysts.
The CeO2-supported Cu catalysts were prepared by an incipient wetness impregnation method.The loadings of the Cu metal for these catalysts were kept to be 4.0 wt%.After impregnation, the samples were dried slowly in vacuum at 40 °C and then at 120 °C in an oven for 12 h.These dried samples were calcined in N2 at 300 °C for 5 h and finally reduced in H2 at 120 °C for 1 h and then at 250 °C for 0.5 h at a temperature ramping rate of 5 °C min -1 .The Cu contents in these sample was analyzed by ICP (TJA IRIS 1000), which were 4.03 wt% for CeO2/Cu-W, 4.08 wt% for CeO2/Cu-C, and 4.15 wt% for CeO2/Cu-O, respectively.

Calculation details
In this work, the DFT calculations were performed using the Vienna ab initio simulation package (VASP). [1,2] he projection augmented wave (PAW) method with a frozen-core approximation was used for the description of the interaction between electrons and ions, [3,4] and the electron exchange and correlation were treated within the generalized gradient approximation with Perdew-Burke-Ernzerhof (GGA-PBE). [5,6] he cut-off energy is set at 450 eV.The Brillouin zone integrations were approximated by k-points chosen using the Monkhorst-Pack grid.A self-consistent field energy tolerance was set at 10 -4 eV, and the maximum force tolerance of 0.02 eV/Å was used for geometries optimization.The DFT+U approach was used, and the value of U=5 eV was used for Ce 4f electrons. [7]e formation energy of an O vacancy can be calculated by

Acridine orange (AO) staining
MDA-MB-231 cells were seeded in 8-well chambered coverglass system and then incubated with CeO2/Cu nanoparticles (100 μg mL -1 ).As a positive control, rapamycin (Rapa) was added at a final concentration of 50 nM at the same time.After 24 h, the cells were stained with AO (1 μM) for 10 min at 37 ℃ and detected by a confocal laser scanning microscope (CLSM, N-SIM S, Nikon, Japan) with an excitation wavelength at 488 nm and emission wavelengths from 505 to 525 nm (green) and from 610 to 640 nm (red).To quantitatively determine the red/green ratio in AO staining cells, these MDA-MB-231 cells after treatment with CeO2/Cu nanoparticles were harvested for flow cytometry analysis.The mean red/green fluorescence ratio in the cells was calculated using FlowJo software.All the experiments were performed in triplicate.

Observation of fluorescent LC3 dot formation
MDA-MB-231 cells were seeded in confocal dishes at a density of 1 × 10 4 per well.After reaching 30% confluence, the cells were transfected with mCherry-GFP-LC3 adenovirus at a multiplicity of infection (MOI) value of 50.After infection for 12-16 h, the virus-containing media were removed and replaced with 400 μL of fresh media.
To analyze the autophagic flux, mCherry-GFP-LC3-expressing MDA-MB-231 cells were treated with different CeO2/Cu nanoparticles for 24 h and imaged with a CLSM.The fluorescent LC3 dot formation was quantified by counting 500 cells and expressed as the ratio of GFP positive cells and mCherry positive cells over the total number of cells.

Bio-transmission electron microscopy (Bio-TEM)
MDA-MB-231 cells were seeded in 6-well plates.After reaching 50% confluence, the cells were treated with CeO2/Cu nanoparticles for 24 h.Cells were collected by trypsinization, washed with PBS three times and then fixed in 0.01 M PBS (pH 7.4) containing 0.5% glutaraldehyde for 10 min at 4 ℃.After centrifugation at 12000 g for 15 min, the cells pellets were fixed in 3% glutaraldehyde solution at 4 ℃ and sent to Chengdu Lilai Biotechnology Co.

Cellular uptake
MDA-MB-231 cells (2 × 10 5 per well) were seeded in 6-well plates and cultured for 24 h.The cells were treated with CeO2/Cu nanoparticles (50 μg mL -1 ) for 6 h.After washing with PBS 3 times, the cells were collected, digested with 70% w/v nitric acid and heated to 120 ℃ to remove the vapors.After cooling to room temperature, the residues were dissolved in 5 mL of deionized water and the samples were filtered through a 0.45 μm hydrophilic membrane.
ICP-MS was performed to measure the content of cerium.

Cytotoxicity Testing
The cytotoxicity of CeO2/Cu nanoparticles was assessed by the CCK-8 assay.MDA-MB-231 cells were seeded in 96-well plates (5 × 10 3 cells per well) and cultured for 24 h.After incubation with CeO2/Cu nanoparticles for 24 or 48 h, the cells were washed with PBS and incubated with 100 μL of FBS-free medium containing 10% CCK-8 reagent (Dojindo Laboratories, Japan) for another 2 h.The absorbance at 450 nm was measured using a microplate reader.The relative cell viability was calculated according to the equation: cell viability = (ODsample-ODbackground)/(ODcontrol-ODbackground)×100%.

Reactive oxygen species (ROS) measurement
To probe the generation of ROS inside cells, MDA-MB-231 cells were seeded in 96-well plates and treated with PBS, CeO2/Cu-W, CeO2/Cu-C or CeO2/Cu-O nanoparticles for 3, 6, 12 and 24 h after they reached a confluence of about 70%.The cells were then exposed to FBSfree medium containing 10 μM DCFH-DA for 30 min and the absorbance at 510 nm was read via a microplate reader.The cells treated with PBS was used as a control.To directly monitor the intracellular ROS generation process, the cells were seeded on glass-bottomed dishes and treated with CeO2/Cu nanoparticles for 12 h.After staining with DCFH-DA for 30 min and Hoechst 33342 for 15 min, the cells were imaged with a CLSM at an excitation of 488 nm and an emission of 505-525 nm.

In vivo antitumor efficacy
A tumor model was established by subcutaneously injecting 1 × 10 6 MDA-MB-231 cells into the right flank of BALB/c nude mice (4 weeks, female).When the tumor volume reached about 50 mm 3 , the mice were randomly divided into 8 groups.DOX• HCl were intravenously administered at a dose of 5 mg DOX per kg body weight and CeO2/Cu nanoparticles were intratumorally injected at a dose of 10 mg per kg body weight on day 0, 4, 8 and 12.The tumor volume (V = lw 2 /2, w: width, l: length) and body weight were recorded every 2 days by a caliper.
The significant difference was evaluated by the student T test.At the end of the treatment course, the mice were sacrificed and the tumors were excised for immunohistochemistry analysis and hematoxylin and eosin (H&E) staining.All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Sichuan University and the experiments were approved by the ethics committee of Sichuan University.

Statistical analysis
Data are shown as mean ± standard deviation (SD).Statistical analysis was carried out using GraphPad Prism Software.For two groups, statistical significance was analyzed using twotailed Student's t-tests.In comparisons among multiple groups, a one-way ANOVA with Tukey's post-hoc test was used for between-group comparisons.In all cases, significant difference was defined as p ≤ 0.05.

Figure S2 .
Figure S2.SEM images of CeO2 with different morphology.

Figure S3 .
Figure S3.TEM images of CeO2 with different morphology.

Figure S6 .
Figure S6.(a−c) Cu K-edge FT-EXAFS spectra in the R-space of the CeO2/Cu samples.

Figure S9 .
Figure S9.(a, b) The feasibility of applying the above detection method at the conditions of pH 6.0 and pH 7.4.

Figure S10 .
Figure S10.Cell viabilities of 4T1 cells after treatment with CeO2/Cu nanoparticles for 24 h.Data are presented as mean ± SD (n = 5).

Figure S11 .
Figure S11.Cell viabilities of A549 cells after treatment with CeO2/Cu nanoparticles for 24 h.Data are presented as mean ± SD (n = 5).

Figure S12 .
Figure S12.Confocal images of intracellular ROS in cells after incubation for 12 h with CeO2/Cu nanoparticles in the presence N-acety-L-cysteine (NAC), a ROS scavenger, NAC was added two hours before the addition of Nano-C60.DCF fluorescence (green) for intracellular ROS and Hoechst 33342 (blue) for cell nuclei.

Table S2 .
Binding energies of deconvoluted plots of Ce 3d spectra for the catalysts along with the contents of Ce 3+ over the catalyst surfaces.