A cobalt-doped iron oxide nanozyme as a highly active peroxidase for renal tumor catalytic therapy

The Fe3O4 nanozyme, the first reported nanozyme with intrinsic peroxidase-like activity, has been successfully employed for various diagnostic applications. However, only a few studies have been reported on the therapeutic applications of the Fe3O4 nanozyme partly due to its low affinity to the substrate H2O2. Herein, we report a new strategy for improving the peroxidase-like activity and affinity of the Fe3O4 nanozyme to H2O2 to generate reactive oxygen species (ROS) for kidney tumor catalytic therapy. We showed that cobalt-doped Fe3O4 (Co@Fe3O4) nanozymes possessed stronger peroxidase activity and a 100-fold higher affinity to H2O2 than the Fe3O4 nanozymes. The lysosome localization properties of Co@Fe3O4 enable Co@Fe3O4 to catalyze the decomposition of H2O2 at ultralow doses for the generation of ROS bursts to effectively kill human renal tumor cells both in vitro and in vivo. Moreover, our study provides the first evidence that the Co@Fe3O4 nanozyme is a powerful nanozyme for the generation of ROS bursts upon the addition of H2O2 at ultralow doses, presenting a potential novel avenue for tumor nanozyme catalytic therapy.


Introduction
Nanozymes are a class of nanomaterials with intrinsic enzymelike activities. [1][2][3] Over the last decade, a wide variety of nanomaterials have been reported to possess natural enzyme-like activities. [1][2][3][4][5] The biochemical reactions catalyzed by these types of nanozymes exhibit similar enzymatic kinetics as in the case of natural enzymes. Nanozymes exhibit comparable enzymatic activity but with much higher stability and lower cost as compared to natural enzymes. In addition, their activities are tunable, and they can be easily integrated with nanosystems to achieve multifunctionality; 6,7 therefore, nanozymes possess signicant potential for a wide range of applications in biomedicine such as in immunoassays, biosensors, and antibacterial and antibiolm agents. 4,8,9 As a classical magnetic nanomaterial, iron oxide (Fe 3 O 4 ) nanoparticles are the rst reported nanozyme with intrinsic peroxidase-like activity. 10,11 Fe 3 O 4 nanozymes with intrinsic magnetic properties have been extensively used for biological applications including magnetic resonance imaging, magnetic drug delivery, magnetic hyperthermia and magnetic separation. [12][13][14] Based on its newly discovered catalytic properties, the Fe 3 O 4 nanozyme can act as a multifunctional enzyme mimetic for versatile biomedical applications. 12 Recently, signicant efforts have been made to explore the feasibility of application of nanozymes in in vivo clinical diagnosis and therapy. 9,[15][16][17][18] As the rst well-studied nanozyme, Fe 3 O 4 nanozymes have already been evaluated in tumor catalytic therapy for catalyzing the decomposition of hydrogen peroxide to generate ROS. 16,19,20 However, because of the low affinity of the Fe 3 O 4 nanozymes to H 2 O 2 , Fe 3 O 4 nanozyme-based catalytic therapy typically requires an additional high dose of H 2 O 2 (approximately 10 À3 to 10 À4 M); 19,20 this makes this nanozyme-based catalytic tumor therapy strategy unviable for practical application.
Some heterogeneous oxide nanomaterials, such as ZnFeO 3 21 and NiFeO 4 22 , formed by iron and other metals have been reported to exhibit enhanced peroxidase-like behavior; this indicates that transition metal doping of Fe 3 O 4 nanozymes may be an effective way to improve the enzymatic activity of these nanoenzymes; 23 interestingly, Chen et al. have reported that Fe-Co bimetallic alloy nanoparticles also exhibit high peroxidase-like activity. 24

Materials
Chemicals and materials were supplied by Sigma-Aldrich (St. Louis, MO) unless otherwise specied.

Synthesis and characterization of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes
The Fe 3 O 4 nanozymes and Co-doped Fe 3 O 4 nanozymes were synthesized according to the solvothermal method reported in the literature 10,26 with some modications. Briey, for the Fe 3 O 4 nanozymes, FeCl 3 $6H 2 O (0.82 g) was dissolved in 40 mL ethylene glycol. When the solution became clear, NaAc (3.6 g) was added under continuous vigorous stirring for 30 min. The mixture was sonicated for 10 min, then transferred to a 50 mL Teon-lined stainless-steel autoclave and reacted at 200 C for 12 h. Aer the reaction was completed, the autoclave was cooled down to room temperature. Then, the products obtained were washed several times with ethanol and dried at 60 C.
The Co@Fe 3 O 4 nanozymes were also synthesized using the same procedure but extra Co(NO 3 ) 3 $6H 2 O (0.82 g) was added to the reaction system.
The morphology and structure of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes were characterized by transmission electron microscopy (TEM, JEOL JEM-1400 120 kV), scanning electron microscopy (SEM, Zeiss Supra55) and dynamic light scattering (DLS, DynaPro Titan). Energy dispersive X-ray spectroscopy (EDX) of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes was conducted using the Tecnai G2 F30 instrument. X-ray diffraction (XRD) measurements were performed using the X'Pert pro Philips X-ray powder diffractometer. X-ray photoelectron spectroscopy (XPS) was performed by the ESCALab220i-XL high-performance electron spectrometer with a monochromatic Al Ka source.

Kinetic analysis of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes
The kinetic parameters of the Fe 3  with H 2 O 2 as the substrate was performed by varying the concentrations of H 2 O 2 with 0.8 mM TMB and vice versa. The absorbance (652 nm) changes were calculated relative to the changes in the molar concentration of TMB using the molar absorption coefficient of 39 000 M À1 cm À1 for the TMBderived oxidation products according to the Beer-Lambert law. 27 All the measurements were performed at least in triplicate, and the values were then averaged. The results are provided as mean AE the standard deviation (SD). The Michaelis-Menten constant was calculated using the Lineweaver-Burk plots of the double reciprocal of the Michaelis-

ESR spectroscopy measurements
The ESR measurements were carried out using a Bruker electron spin resonance (ESR) spectrometer (A300-10/12, Germany) at ambient temperature. Herein, y microliter aliquots of the control or sample solutions were put in glass capillary tubes with the internal diameters of 1 mm and sealed. The capillary tubes were then inserted into the ESR cavity, and the spectra were obtained at selected times. The instrument settings are as follows: 1 G eld modulation, 100 G scan range, and a 20 mW microwave power for the detection of spin adducts using spin traps. The spin trap BMPO was employed to verify the formation of hydroxyl radicals (OHc) during the degradation of H 2 O 2 in the presence of the Fe 3 O 4 or Co@Fe 3 O 4 nanozymes under the same conditions. The amount of hydroxyl radicals was quantitatively estimated by the ESR signal intensity of the hydroxyl radical spin adduct (BMPO/OHc) using the peak-to-peak height of the second line of the ESR spectrum.

Cell viability assay
The cytotoxicity of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes with the addition of 10 nM H 2 O 2 was determined using the CCK-8 cell viability assay kit (Dojindo Molecular Technologies). Briey, A-498 cells (Human renal cancer cell, ATCC, HTB-44) were plated in 96-well plates (BD Biosciences) with the density of 5 Â 10 3 cells per well and cultured in 100 mL EMEM (Catalog No. 30-2003) for 1 day before the addition of Fe 3 O 4 ,Co@Fe 3 O 4 nanozymes, or only the buffer as a control. On each plate, blank wells (n ¼ 6) with media were dened as 0% viability. Moreover, the wells with only PBS-treated cells (n ¼ 6) were dened as 100% viability. The dilutions of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes were prepared using a buffer containing 10 nM H 2 O 2 . The cells were then exposed to the Fe 3 O 4 or Co@Fe 3 O 4 nanozymes at a series of concentrations (from 0 to 0.2 mg mL À1 ) for 24 hours. Aer stimulation, a 10 mL CCK-8 solution was added to each well. The plates were then incubated for 4 h at 37 C. Aer this, the absorbance was determined at 450 nm using the Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Inc.). The results presented herein are the average of those obtained via three independent experiments.

Localization of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes in cytoplasm
The cellular uptake and distribution of Fe 3 O 4 or Co@Fe 3 O 4 nanozymes in human renal tumor cells were investigated by a confocal laser scanning microscope. Briey, the A-498 cells were plated on poly-L-lysine-treated coverslips (BD Biosciences) and cultured in a six-well plate (Corning) for 12 h before use. Aer stimulation for 48 h with the Alexa-488labeled Fe 3 O 4 or Co@Fe 3 O 4 nanozymes (0.2 mg mL À1 ), the cells were washed with PBS, xed in 4% cold formaldehyde in PBS for 5 min, and then permeabilized with 0.1% Triton X-100. Aer being washed with PBS, the cells were blocked in a 5% normal goat serum for 30 min at room temperature. To visualize the lysosomes, the cells were incubated with anti-Lamp1 mAb (1 : 200, clone H4A3; Invitrogen) at 37 C for 1 h. The cells were then washed three times with PBS and incubated with goat anti-mouse IgG1 conjugated with Alexa-555 (1 : 500; Invitrogen) for 1 h at 37 C. Finally, the nuclei of the cells were stained with 4 0 ,6 0 -diamidino-2-phenylindole (DAPI, 1 mg mL À1 , Roche Applied Science) for 10 min at room temperature. The samples were examined using a confocal laser scanning microscope (Olympus FluoView FV-1000, Tokyo, Japan).

Intracellular ROS assay
The uorescent probe 2 0 ,7 0 -dichlorouorescin diacetate (H 2 DCFDA, Sigma-Aldrich, D6883) was used to measure the intracellular generation of ROS by the Fe 3 O 4 or Co@Fe 3 O 4 nanozymes. Briey, the conuent A-498 cells on the coverslips (BD Biosciences) were incubated with Fe 3 O 4 or Co@Fe 3 O 4 nanozymes (0.2 mg mL À1 ) for 4 hours. Aer being washed with PBS, the cells were incubated with 10 mM H 2 DCFDA in a serumfree DMEM for 20 min at 37 C in the dark. The uorescence intensities of H 2 DCFDA were measured by a confocal laser scanning microscope (Olympus FluoView FV-1000, Tokyo, Japan).

Apoptosis analysis
The apoptosis analysis of the treated tumor cells was conducted by PI and annexin V staining and ow cytometry (FACSCali-burTM, Becton Dickinson, Franklin Lakes, NJ, USA). Briey, the Fe 3 O 4 and Co@Fe 3 O 4 (0.2 mg mL À1 ) nanozymes were incubated with the A-498 tumor cell lines for 24 h. Aer trypsinization, the treated A-498 tumor cells were incubated with annexin V and PI for 15 min to achieve nuclear staining. Aer this, the cells were xed and incubated with streptavidin-uorescein (5 mg mL À1 ) (Sigma, USA) for 15 min. Cell death was evaluated by the quantication of annexin-stained apoptotic cells and PI-stained necrotic cells using ow cytometry.

Therapy studies
Herein, eighteen female BALB/c nude mice bearing A-498 tumors were randomly assigned to four groups (n ¼ 6 mice per group). All the mice were intratumorally treated with a single dose of Fe 3 O 4 and Co@Fe 3 O 4 nanozymes (3 mg mL À1 , 100 mL) with 10 nM H 2 O 2 when the diameter of the tumors was about 100 mm 3 . For the controls, PBS was administered. The tumor size was measured 3 times a week. The tumor size was calculated as volume [mm 3 ] ¼ length Â width 2 Â p/6. The measured values are presented as mean AE SD.

Characterization of the Co@Fe 3 O 4 nanozymes
The Fe 3 O 4 nanozymes and Co-doped Fe 3 O 4 nanozymes (Co@Fe 3 O 4 ) used in this study were synthesized by the solvothermal method. To study the composition of the asprepared nanozymes, the EDX analysis was performed. As shown in Fig. S1, † the EDX spectrum of the Co@Fe 3 O 4 nanozymes indicated that the Fe and Co elements were present in the nanoparticles. Based on the EDX mapping analysis, the content of Fe and Co in the Co@Fe 3 O 4 nanozymes were determined as 33.48% and 16.23%, respectively (Table S1 †). In conclusion, herein, the synthesized Co@Fe 3 O 4 nanozymes contained Fe and Co with the ratio of approximately 2 : 1; this conrmed that Co was successfully doped into the Fe 3 O 4 nanozymes by the simple solvothermal method.
To characterize the structure of the Co@Fe 3 O 4 nanozymes, TEM, SEM, DLS and X-ray diffraction (XRD) analysis were performed. The TEM images of the as-prepared Fe 3 O 4 and Co@Fe 3 O 4 nanozymes are shown in Fig. 1A and B, respectively. The SEM images of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes are presented in Fig. S2A S2C and D †), respectively. The XRD patterns of the asprepared nanozymes are shown in Fig. 1C  To characterize the oxidation state of cobalt in the Co@Fe 3 O 4 nanozyme, we further performed XPS analysis of the as-prepared Co@Fe 3 O 4 nanozyme. The high-resolution XPS spectrum of Co 2p is shown in Fig. 2A. The Co 2p XPS peak at 780.8 eV was assigned to Co (2p 3/2 ), with a shake-up satellite peak at 785.9 eV. In addition, the Co 2p XPS peak at 797.2 eV was attributed to Co (2p 1/2 ), with a satellite peak at 803.0 eV. 28 These characteristic and satellites peaks conrm that Co 2+ is present in the Co@Fe 3 O 4 nanozyme. Moreover, as shown in Fig. 2B, the Fe 2p XPS spectrum exhibited characteristic peaks with the binding energy values at 711.0 and 724.0 eV, assigned to the Fe (2p 3/2 ) and Fe (2p 1/2 ) peaks, 29 respectively. Since the atomic radius of iron (140 pm) is similar to that of the cobalt atom (135 pm), these results suggest that the cobalt atoms are probably located only at the lattice positions of the Fe 3 O 4 crystal structure.

Peroxidase-like activity and steady-state kinetic assay of the Co@Fe 3 O 4 nanozymes
To directly compare the peroxidase-like activity of the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes, we performed typical catalytic experiments using the peroxidase substrate 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) and H 2 O 2 as previously reported. 11 The results showed that both the Fe 3 O 4 and Co@Fe 3 O 4 nanozymes catalyzed the oxidation of TMB with H 2 O 2 to produce blue color products with absorption at 652 nm (Fig. 3A). Moreover, the results demonstrated that the Co@Fe 3 O 4 nanozymes exhibited a signicant improvement in the peroxidase-like activity as compared to the Fe 3 O 4 nanozymes; this indicated that a signicant improvement in the nanozyme activity was achieved by Co doping of the Fe 3 O 4 nanozymes.
The mechanism of action of the Co@Fe 3 O 4 nanozymes was investigated using the ESR method. As shown in Fig. 3B, similar to the previously reported Fe 3 O 4 nanozymes, the Co@Fe 3 O 4 nanozymes signicantly enhanced the generation  of hydroxyl radicals under acidic conditions. Importantly, the Co@Fe 3 O 4 nanozymes generated more hydroxyl radicals than the Fe 3 O 4 nanozymes under the same conditions; this further conrmed that Co doping signicantly improved the peroxidase-like activity of the Fe 3 O 4 nanozymes.
To obtain the apparent kinetic parameters of the Co@Fe 3 O 4 nanozymes, the Michaelis-Menten experiments were performed. Fig. 3C

Anti-tumor activities and mechanistic study of the Co@Fe 3 O 4 nanozymes
Tumor cells typically possess higher levels of endogenous H 2 O 2 and reactive oxygen species (ROS) than normal cells. 9,20 The balance of the ROS determines the fate of the tumor cells. It has been previously shown that stimulation of ROS is a common strategy for cancer chemotherapy. 30   As is well-known, the Fe 3 O 4 nanozymes exhibit peroxidase-like activity only under acidic conditions. 12 Since the Co@Fe 3 O 4 nanozymes exhibit signicant antitumor activity, we infer that the Co@Fe 3 O 4 nanozymes localize in the lysosome (pH 4-5) aer incubation with the tumor cells.
To verify this hypothesis, we labeled the nanozymes with Alexa Fluor 488 to track their intracellular localization. As shown in Fig. 4B, we found that aer incubation with tumor cells for 4 hours, most of the internalized Fe 3 O 4 nanozymes co-localized with lysosomes. Similar to the Fe 3 O 4 nanozymes, nearly all of the internalized Co@Fe 3 O 4 nanozymes localized in the lysosomes, the highly acidic microenvironment of which would favor the peroxidase-like activities. Thus, the colocalization analysis of the nanozymes and lysosomes demonstrated that the nanozyme-based tumor catalytic therapy strategy is feasible.
In our hypothesis, the antitumor activities of the Co@Fe 3 O 4 nanozymes are attributed to the catalytic generation of ROS by the decomposition of hydrogen peroxide, resulting in oxidative stress in the tumor cells. To verify this hypothesis, the intracellular ROS levels in the tumor cells were detected by employing 2 0 ,7 0 -dichlorouorescein diacetate (H 2 DCFDA), a typical ROS uorescent dye. As shown in Fig. 4C nanozymes only partially inhibited the renal tumor growth due to their relative low peroxidase activity and low binding affinity to H 2 O 2 ; 11 this was consistent with previous studies. 9 Overall, these results provide strong evidence that the Co@Fe 3 O 4 nanozymes possess the ability to regulate intracellular ROS upon the addition of H 2 O 2 at ultralow concentrations. Once located in the acidic microenvironment of lysosomes, these nanozymes induce cell death by boosting the level of ROS. The Co@Fe 3 O 4 nanozymes exhibited signicant antitumor activities against human renal tumor both in vitro and in vivo.

Discussion and conclusion
ROS-induced apoptosis is a popular strategy for cancer therapy. [32][33][34] The tumor therapy strategies utilizing nanozymes mainly act by stimulating the production of ROS. 9 The Fe 3 O 4 nanozymes can simulate peroxidase and thereby efficiently catalyze the decomposition of H 2 O 2 to generate ROS to inhibit tumors in vivo. However, the low binding affinity of the Fe 3 O 4 nanozyme to H 2 O 2 and its relatively low catalytic activity limit the development of the Fe 3 O 4 nanozyme-based tumor catalytic therapy.
Transition metal doping has been demonstrated to be an effective and easy way to improve the peroxidase-like activity of Fe 3 O 4 nanozymes. 23 Among the transition metals, cobalt, a nonnoble metal, has been proven to be a promising dopant to enhance the enzymatic activity of the Fe 3 O 4 nanozyme. 25 Importantly, Chen et al. have systematically studied the effects of doping Fe/Co at different ratios on the enzymatic activity of the Fe 3 O 4 nanozyme. They have demonstrated that when the ratio of Fe/Co is around 2 : 1, the peroxidase-like activity of the Co-doped Fe 3 O 4 nanozyme is the best enzymatic activity. 24 In this study, by employing a simple solvothermal method, we fabricated the Co@Fe 3 O 4 nanozyme with the ratio of Fe/Co around 2 : 1. Compared with the case of other strategies, including metal doping, biomimetic coating, and C-dot modi-cation methods, that signicantly improved the peroxidaselike activity of the Fe 3 O 4 nanozyme, our Co@Fe 3 O 4 nanozyme exhibited the best binding affinity to H 2 O 2 (Table S2 †).
The XPS and EDX analysis of the Co@Fe 3 O 4 nanozyme demonstrated that the cobalt atoms were probably located only at the lattice positions of the Fe 3 O 4 crystal structure. Although the Co atom possesses a similar size as the Fe atom, the Co atoms doped into the Fe 3 O 4 crystal may still slightly change the surface physical environment, 35 resulting in an improved binding affinity of the nanozyme to H 2 O 2 . In addition, the Co dopant may produce more catalytically active sites and substrate-binding sites on the surface of the Co@Fe 3 O 4 nanozyme when compared with the case of the Fe 3 O 4 nanozyme. 36 Moreover, the higher redox potential of Co 3+ /Co 2+ (1.30 V) as compared to that of Fe 3+ /Fe 2+ (0.771 V) in the Fe 3 O 4 nanozyme may be another reason for the improvement in the peroxidaselike activities of Co@Fe 3 O 4 . 37,38 In conclusion, using a simple solvothermal method, we successfully synthesized Co-doped Fe 3 O 4 (Co@Fe 3 O 4 ) nanozymes that contained Fe and Co at the ratio of approximately 2 : 1. The well-crystallized Co@Fe 3 O 4 nanozymes exhibited excellent peroxidase-like activity. More importantly, Co doping makes the Co@Fe 3 O 4 nanozymes exhibit a 50-fold and 100-fold higher affinity to H 2 O 2 than that of the HRP and  This journal is © The Royal Society of Chemistry 2019 generate an ROS burst. Importantly, the Co@Fe 3 O 4 nanozymes exhibited excellent antitumor activities both in vitro and in vivo for kidney tumor catalytic therapy.

Conflicts of interest
There are no conicts to declare.