Abstract

   Overcoming apoptosis resistance is necessary to ensure an effective
   cancer treatment; however, it is currently very difficult to achieve. A
   desirable alternative for cancer treatment is the targeted activation
   of pyroptosis, a unique type of programmed cell death. However, the
   pyroptosis inducers that are efficient for cancer therapy are limited.
   This work reports the engineering of 2D NiCoO [x] nanosheets as
   inducers of the production of harmful reactive oxygen species (ROS),
   which promote intense cell pyroptosis, and that can be applied to
   ultrasound (US)‐augmented catalytic tumor nanotherapy. The main
   therapeutic task is carried out by the 2D NiCoO [x] nanosheets, which
   have four multienzyme‐mimicking activities: peroxidase‐ (POD), oxidase‐
   (OXD), glutathione peroxidase‐ (GPx), and catalase‐ (CAT) mimicking
   activities. These activities induce the reversal of the hypoxic
   microenvironment, endogenous glutathione depletion, and a continuous
   ROS output. The ROS‐induced pyroptosis process is carried out via the
   ROS‐NLRP3‐GSDMD pathway, and the exogenous US activation boosts the
   multienzyme‐mimicking activities and favors the incremental ROS
   generation, thus inducing mitochondrial dysfunction. The anti‐cancer
   experimental results support the dominance of NiCoO [x]
   nanosheet‐induced pyroptosis. This work expands on the biomedical
   applications of engineering 2D materials for US‐augmented catalytic
   breast cancer nanotherapy and deepens the understanding of the
   multienzyme activities of nanomaterials.

   Keywords: 2D nanosheets, catalytic therapy, multienzyme‐mimicking,
   pyroptosis, ultrasound therapy
     __________________________________________________________________

   The 2D multienzyme‐mimicking NiCoO [x] nanosheets as proptosis inducers
   have been developed to increase the rate of deleterious reactive oxygen
   species generation for the ultrasound‐augmented catalytic nanotherapy
   of breast cancer.

   graphic file with name ADVS-10-2301279-g001.jpg

1. Introduction

   According to the laws of enzyme kinetics under physiological
   conditions, nanoenzymes are nanomaterials that can catalyze specific
   chemical reactions and that have enzyme‐mimicking properties.^[ [36]^1
   ^] Nanoenzymes are similar to natural enzymes in that they share some
   nanomaterial properties, such as high stability, low preparation costs,
   straightforward production and purification processes, high catalytic
   activity, and the capacity to speed up biochemical reactions.^[ [37]^2
   ^] These nanoenzymes also offer exceptional advantages for medicinal
   applications, particularly for cancer treatment.^[ [38]^3 ^]
   Representatively, nanozymes with peroxidase (POD)‐mimicking and oxidase
   (OXD)‐like catalytic capabilities can catalyze the disintegration of
   hydrogen peroxide (H[2]O[2]) or oxygen (O[2]) and generate highly toxic
   ROS that can induce the apoptosis or necrosis of cancer cells.^[ [39]^4
   ^] Thus, the application potential of nanoenzymes for ROS‐mediated
   tumor‐nanocatalytic treatment is promising.^[ [40]^5 ^] Unfortunately,
   the highly complex tumor microenvironment (TME) influences the effect
   of the nanoenzyme‐enabled catalytic procedures, such as hypoxia, mildly
   acidic environment, and overexpression of H[2]O[2] and glutathione
   (GSH). To overcome these limitations, H[2]O[2]‐responsive nanoenzymes
   with dual‐ or multienzyme‐mimicking activities have been explored with
   broad interest. Besides the above two types of enzyme activities, the
   glutathione peroxidase (GPx)‐ and catalase (CAT)‐like activities also
   play important roles. In recent years, nanoenzymes that catalyze
   reactions of multivalent metal ions (such as Fe^2+/3+,^[ [41]^6 ^]
   Co^2+/3+,^[ [42]^7 ^] Sn^2+/Sn^4+,^[ [43]^8 ^] Ni^2+/3+,^[ [44]^9 ^]
   among others) mimic the function of natural enzymes and play a
   significant role in regulating the TME, thus enhancing their
   therapeutic effect. CoFe[2]O[4] nanoflowers can act as POD‐ and
   CAT‐mimicking nanoenzymes to catalyze the decomposition of H[2]O[2]
   into hydroxyl radicals (•OH) and O[2] for sonodynamic therapy (SDT) and
   chemodynamic therapy (CDT).^[ [45]^10 ^] Zhang and co‐workers prepared
   MnFe[2]O[4] nanoenzymes with GPx‐ and CAT‐like activities that can
   overcome tumor hypoxia and consume GSH by continuously catalyzing
   H[2]O[2] to generate O[2], to achieve a better therapeutic effect.^[
   [46]^11 ^] However, nanozymes that concurrently have POD‐, OXD‐, CAT‐,
   and GPx‐mimicking activities under weak acidic conditions have rarely
   been explored.

   At present, nanoenzyme‐induced cell death has been applied to multiple
   processes, including apoptosis,^[ [47]^12 ^] ferroptosis,^[ [48]^13 ^]
   pyroptosis,^[ [49]^14 ^] and so on. Pyroptosis is a form of necrosis
   that regulates cell death and is performed by the gasdermin (GSDM)
   family.^[ [50]^15 ^] Although GSDMs are expressed in an inactive form,
   some proteinases activate them via proteolysis. Cleaved gasdermin D
   (GSDMD) releases the N‐terminal fragment (N‐GSDMD), which forms
   membrane pores, and then releases pro‐inflammatory molecules into the
   extracellular environment, thus triggering inflammation and an
   immunological reaction.^[ [51]^16 ^] Ultrasound (US), which is a
   non‐invasive sound wave that can penetrate deep tissues, possesses
   acoustic cavitation effects.^[ [52]^17 ^] Therefore, the mass diffusion
   of the nanoenzyme can be improved through US, which significantly
   improves the enzyme‐like activity and corresponding reaction rates.^[
   [53]^18 ^] Therefore, appropriately integrating the US‐enhanced
   enzyme‐like activities into one nanoenzyme can effectively inhibit
   tumor growth and recurrence.^[ [54]^19 ^] Overall, pyroptotic cell
   death is caused by the activation of multiple cellular protein
   complexes called inflammasomes; for example, the nucleotide binding
   oligomerization domain‐like receptor protein 3 (NLRP3) inflammasome
   acts as a regulatory factor of pyroptosis.^[ [55]^20 ^] The activated
   NLRP3 inflammasomes then transform protoproteinase‐1 into caspase‐1. In
   contrast with apoptosis, pyroptosis and ferroptosis are immunogenic
   programmed cell death (PCD) processes, constituting powerful
   anti‐cancer strategies because they can initiate antitumor immune
   responses by delivering adequate cytokines and damage‐associated
   molecular patterns (DAMPs).^[ [56]^21 ^] US‐induced pyroptosis can
   maximize the significant control of tumor growth, thus generating a
   strong and lasting antitumor response.

   2D nanomaterials possess exceptional intrinsic characteristics, such as
   an ultrathin layer, a unique nanosheet structure and surface chemistry,
   a high surface area and quantum size effect.^[ [57]^22 ^] In
   particular, 2D nanomaterials have become a promising nanoplatform for
   various biomedical applications due to their specific physical and
   chemical properties and high drug loading capacity.^[ [58]^23 ^] By
   taking advantage of the interactions between the TME and 2D
   nanomaterials, an efficient TME‐targeted tumor treatment can be
   actualized by developing TME‐responsive 2D nanomaterials, which would
   enable the application of 2D nanomaterials to catalytic medicine and to
   efficient tumor treatments. To demonstrate our points, herein,
   morphology‐controlled 2D NiCoO [x] nanozymes were successfully
   constructed using the hard template method.^[ [59]^24 ^] The 2D NiCoO
   [x] nanosheets act as multienzyme‐mimicking pyroptosis inducers for
   US‐augmented catalytic breast cancer nanotherapy without complicated
   side effects (Scheme [60]1 ). The strong catalytic activity of 2D NiCoO
   [x] nanozymes in acidic conditions and upon US simulation was further
   enhanced to achieve a desirable US‐enhanced effect and
   pyrolysis‐inducing effects for promoting cancer treatment. Furthermore,
   the intrinsic Ni^2+/Ni^3+ and Co^2+/Co^3+ redox couple can produce
   highly toxic ROS via enzyme‐like activity reactions, thus enhancing its
   antitumor efficiency. The ROS‐NLRP3‐GSDMD pathway was progressively
   demonstrated to induce pyroptosis based on the results of transcriptome
   sequencing, western blot analysis, and enzyme‐linked immunosorbent
   assays. More importantly, this research provides a new perspective on
   the design of biocompatible and efficient nanoenzymes for catalyzing
   cascade cancer therapy and pyroptosis with low side effects. Therefore,
   this study is highly expected to open a door for tumor treatment and
   ignite the further exploration and application of nanoenzymes to
   diverse tumor nanotherapies.

Scheme 1.

   Scheme 1
   [61]Open in a new tab

   a) The scheme of the synthetic production of the 2D NiCoO [x]
   nanosheets. b) Schematic illustration of the underlying mechanism of
   how the 2D NiCoO [x] nanosheets lead to US‐augmented cell pyroptosis
   and catalytic tumor nanotherapy.

2. Results and Discussion

2.1. Synthesis and Characterization of the 2D NiCoO [x] Nanosheets

   The detailed process of the synthetic production of the 2D NiCoO [x]
   nanosheets is displayed in Figure [62]1a. First, the 2D NiCoO[x]
   nanosheets were fabricated using a three‐step synthesis process,
   including the reaction of Co(NO[3])[2]·6H[2]O with Ni(NO[3])[2]·6H[2]O,
   the addition of hydrophilic polyvinylpyrrolidone (PVP) and urea, and
   the lyophilization and calcination of the Ni‐Co precursor.^[ [63]^24 ^]
   The morphology of the 2D NiCoO [x] nanosheets according to the
   transmission electron microscopy (TEM) observations is presented in
   Figure [64]1b. The 2D NiCoO [x] nanosheets have an ultrathin
   sheet‐shaped structure.^[ [65]^24 ^] The TEM image of the 2D NiCoO [x]
   nanosheets shows that they exhibit high dispersibility, with an average
   particle size of 178 nm, according to the statistical analysis (Figure
   [66]S1, Supporting Information). The lattice distance of 0.202 nm
   corresponds to the (400) diffraction plane of the 2D NiCoO [x] sheets,
   as observed from the high‐resolution TEM (HRTEM) images
   (Figure [67]1c,d). As shown in Figure [68]1e,f, X‐ray energy‐dispersive
   spectrometer (EDS) analysis and the corresponding elemental mapping of
   the 2D NiCoO [x] nanosheets show the coexistence of the Ni, Co, and O
   elements, demonstrating the successful fabrication of the 2D NiCoO [x]
   nanosheets. Moreover, X‐ray diffraction (XRD) analysis was performed to
   determine the components, purity, and crystal structure of the
   fabricated 2D NiCoO [x] nanosheets (Figure [69]1g), in which the
   characteristic diffraction peaks were found to be well indexed to the
   standard structure. The pure 2D NiCoO [x] nanosheets featured three
   peaks at 2θ values of 38.4°, 44.62°, and 64.98°, which were indexed to
   the (311), (400), and (440) planes, respectively, and were consistent
   with the standard values corresponding to NiCo[2]O[4] (JCPDS 20–0781).
   The band gap value of the 2D NiCoO [x] sheets recorded by the
   derivative Tauc's plot was around 1.43 eV (Figure [70]1h). Furthermore,
   the X‐ray photoelectron spectroscopy (XPS) valence‐band spectrum was
   utilized to demonstrate the location of the valence band, which was
   located at 0.5 eV (Figure [71]1i). Therefore, the ROS generation
   capability is likely to be induced by US radiation.

Figure 1.

   Figure 1
   [72]Open in a new tab

   Synthesis and characterization of the 2D NiCoO [x] nanosheets. a) The
   scheme of the synthetic production of the 2D NiCoO [x] nanosheets. b)
   TEM image of the 2D NiCoO [x] nanosheets. c) HRTEM image and d) the
   corresponding selected area electron diffraction pattern of the 2D
   NiCoO [x] nanosheets. e) Corresponding area‐elemental mapping of the 2D
   NiCoO [x] nanosheets. f) Energy‐dispersive spectrometer (EDS) spectrum.
   g) XRD pattern of the 2D NiCoO [x] nanosheets. h) Energy bandgap and i)
   valence XPS spectra of the 2D NiCoO [x] nanosheets. j) XPS spectrum of
   the 2D NiCoO [x] nanosheets. High‐resolution k) Ni 2p, l) Co 2p, and m)
   O 1s XPS data obtained from the 2D NiCoO [x] nanosheets.

   Furthermore, to investigate the mixed valence of the components in the
   NiCoO [x] nanosheets, the XPS spectroscopy spectrum of the 2D NiCoO [x]
   nanosheets was recorded. The XPS survey spectrum shows that the
   nanosheets are composed of the Ni, Co, and O elements (Figure [73]1j).
   The analysis of Ni 2p and Co 2p was conducted using high‐resolution XPS
   (Figures [74]1k,l). The Ni 2p spectrum (Figure [75]1k) can be fitted to
   two shakeup satellites and two prominent 2p[3/2] and 2p[1/2] spin‐orbit
   peaks. In detail, the Ni 2p[3/2] peak is composed of Ni^2+
   (853.6 eV)/Ni^3+ (855.5 eV).^[ [76]^24 ^] The high‐resolution Co 2p
   spectrum can also be fitted to two satellites and two spin‐orbit peaks
   with binding energies, presented in the valence state of Co^2+
   (779.3 eV)/Co^3+ (781.2 eV), respectively (Figure [77]1l). The
   existence of the Ni^2+/Ni^3+ and Co^2+/Co^3+ redox couple provides
   tremendous potential for the POD‐, CAT‐, OXD‐, and GPx‐like
   multienzyme‐mimicking activities. Moreover, the spectrum in
   Figure [78]1m shows three main peaks at 529.5, 531.1, and 532 eV, which
   are derived from O 1s and are assigned to metal‐oxygen bonds, surface
   hydroxyl groups, or defects with low oxygen coordination states, and
   adsorbed oxygen, respectively.^[ [79]^24 ^]

2.2. Enzymatic Activities of the 2D NiCoO [x] Nanosheets

   Taking into consideration the intimate relationship between the enzyme
   activities and anti‐cancer properties, we explored whether the 2D NiCoO
   [x] nanosheets possess multienzyme‐mimicking activities. The POD‐like
   activity of NiCoO [x] was revealed by the oxidation of
   tetramethylbenzidine (TMB) upon adding H[2]O[2] (Figure [80]2b).^[
   [81]^25 ^] This oxidant can react with the TMB substrate to generate a
   blue‐colored product (oxTMB) with a maximum absorption peak at 652 nm.
   The absorbance at 652 nm in the NiCoO [x] + H[2]O[2] group was
   significantly higher than in the NiCoO [x] or H[2]O[2] alone groups,
   validating the dramatic oxidation of TMB (Figure [82]2c; Figure [83]S2,
   Supporting Information). Additionally, the POD‐like activity was
   enhanced by US irradiation (Figure [84]S3, Supporting Information). US
   can enhance the production of •OH on application of the 2D NiCoO [x]
   nanozymes. Considering the characteristics of the acidic TME, the
   POD‐mimicking activity of NiCoO[x] in phosphate buffered saline with
   different pH values was studied to evaluate the TME adaptability of the
   engineered nanoenzyme systems. The 2D NiCoO [x] nanosheets exhibited
   only marginal POD‐like activity at neutral pH, while their enzymatic
   activity was significantly enhanced in weak acidic conditions
   (Figure [85]2d). To further investigate the quantity of ROS production
   under US stimulation and in the presence of NiCoO [x] ,
   1,3‐diphenylisobenzofuran (DPBF) was used to detect the superoxide
   anion radical (·O[2] ^−) and singlet oxygen (^1O[2]) after US
   irradiation (1.2 W cm^−2, 50% duty cycle, 1 min). The characteristic
   peak of DPBF at 416 nm at different NiCoO [x] concentrations decreased
   gradually (Figure [86]S4, Supporting Information). Similarly, the
   absorption of DPBF did not remarkably decline without NiCoO [x] (Figure
   [87]S5, Supporting Information). Subsequently, the US‐augmented
   OXD‐mimicking activity was also detected for a prolonged time
   (Figure [88]2e). 2D NiCoO [x] nanosheets possess pH‐responsive
   catalytic activity, showing a more obvious OXD‐mimicking activity in
   weak acidic environments (Figures [89]2b; Figure [90]S6, Supporting
   Information).

Figure 2.

   Figure 2
   [91]Open in a new tab

   Multienzyme activities of the 2D NiCoO [x] nanosheets. a) Catalytic
   mechanism of the 2D NiCoO [x] nanosheets. b) Schematic diagram of the
   POD‐mimicking catalytic process of the 2D NiCoO [x] nanosheets. c)
   POD‐mimicking activity of the 2D NiCoO [x] nanosheets, as tested using
   a TMB probe with or without NiCoO [x] , H[2]O[2], and US. d) The UV–vis
   absorbance alterations at 652 nm at different pH values (4.4, 5.4, 6.0,
   6.5, and 7.4) originating from TMB oxidation. e) The time‐dependent
   oxidation of the 2D NiCoO [x] nanosheets using DPBF as the trapping
   agent for demonstrating the production of ^1O[2], which is enhanced
   under US irradiation. f) The CAT‐mimicking activity of 2D NiCoO [x] ,
   as determined by measuring the dissolved O[2] in the presence of
   different concentrations of H[2]O[2] (0, 25, 50, and 100 µm). g)
   GPx‐mimicking activity of the 2D NiCoO [x] nanosheets as measured based
   on the UV–vis absorbance alterations at 340 nm using a GPx assay kit.
   h) The ESR spectra of the detection of •OH, which was trapped using
   DMPO, for the different groups. i) The ESR spectra of the detection of
   ^1O[2] in different groups using TEMP (US‐involved group: 1 MHz; 1.2 W
   cm^−2; 50% duty cycle; and 1 min).

   Conversely, CAT is an enzyme that catalyzes the decomposition of
   H[2]O[2] to water and O[2].^[ [92]^26 ^] The CAT‐mimicking activity of
   the 2D NiCoO [x] nanosheets was further evaluated by measuring the
   dissolved oxygen level under the following conditions: 100 µg mL^−1 of
   the 2D NiCoO [x] nanosheets and 0–100 µm of H[2]O[2]. Similar to the
   natural catalase, the 2D NiCoO [x] nanosheets can efficiently catalyze
   the conversion of H[2]O[2] to O[2], resulting in an increased O[2]
   concentration in solution (Figure [93]2f), confirming the CAT activity
   of the 2D NiCoO [x] nanosheets. Moreover, the generation of O[2] can
   relieve tumor hypoxia. GSH, an important antioxidant that is highly
   expressed in tumor cells, decreased the efficacy of the ROS‐based
   treatments. We confirmed whether the synthesized 2D NiCoO [x]
   nanosheets have a GSH depletion capability, which could overcome the
   tumor resistance to oxidation stress and improve the treatment
   efficacy. Therefore, the GSH depletion capability was quantitatively
   analyzed using a reduced GSH kit, and the decrease in the NADPH level
   in the presence of NiCoO [x] at 340 nm was measured
   spectrophotometrically in real time (Figure [94]2g).^[ [95]^27 ^] With
   an extended time, the characteristic absorbance at 340 nm decreased,
   indicating that the 2D NiCoO [x] nanosheets can undergo a redox
   reaction associated with GSH depletion (Figure [96]2g). Moreover, the
   consumption of GSH increases as the H[2]O[2] concentration rises, which
   is reflected in a decreased absorption value (Figure [97]S7, Supporting
   Information).

   Furthermore, the generation of •OH and ^1O[2] was quantitatively
   evaluated based on the electron spin resonance (ESR) measurement
   (Figure [98]2h,i), in which •OH was trapped by 5,5‐dimethyl‐1‐pyrroline
   N‐oxide (DMPO), and 2,2,6,6‐tetra‐methylpiperidine (TEMP) was utilized
   to capture ^1O[2].^[ [99]^25 , [100]^28 ^] As shown in
   Figure [101]2h,i, the 2D NiCoO [x] nanosheets cannot produce •OH and
   ^1O[2] solely. Unlike other groups, the NiCoO[x] + H[2]O[2] or US
   groups showed strong characteristic signals, indicating the presence of
   •OH and ^1O[2], with the strongest signals being emitted by the NiCoO
   [x] + H[2]O[2] + US group. Notably, the introduction of US increases
   the production rate of •OH, as revealed by the enhanced ESR signal
   strength, which further demonstrates that the external US stimulus
   (Figures [102]S8 and S[103]9, Supporting Information) combined with the
   internal cavitation effect can improve the reaction degree and release
   energy to accelerate the reaction rate, promoting the generation of
   more ROS and ultimately amplifying the therapeutic efficacy.

   These in vitro assays solidly demonstrated the POD‐, OXD‐, CAT‐, and
   GPx‐like catalytic activities of the 2D NiCoO [x] nanosheets
   (Figure [104]2a). These activities are further enhanced under weak
   acidic conditions (Figures [105]S6 and [106]S7, Supporting
   Information). The ROS family typically contains •OH, ·O[2] ^−, and
   ^1O[2], among which •OH is typically assumed to be the strongest one
   that can be generated by a Fenton reaction in the presence of H[2]O[2]
   and Fenton reagent for US‐mediated cascade catalysis.^[ [107]^29 ^] The
   conversion efficiency of Ni^3+ to Ni^2+ and of Co^3+ to Co^2+ could be
   expedited by means of US irradiation, and the produced Ni^2+ and Co^2+
   further interacts with H[2]O[2] to make more •OH free radicals
   available. The use of US in conjunction with powerful shock waves
   produced from the cavitation bubbles generated by US could trigger
   partial violent turbulence and increase the mass transfer rate of the
   nanosystems. Therefore, the engineered 2D NiCoO [x] nanosheets with
   special multienzyme‐mimicking performance could be employed for
   generating ROS and killing tumor cells, further improving the
   therapeutic effect induced by US irradiation.

2.3. In Vitro Therapeutic Assessment at the Cellular Level

   Based on its intrinsic non‐toxicity, desirable biodegradability, and
   water‐solubility, bovine serum albumin (BSA) is the main available
   albumin extensively used in nanobiomedicine.^[ [108]^30 ^] The Fourier
   transform infrared (FTIR) spectra showed that BSA was successfully
   grafted onto the surface of the 2D NiCoO [x] nanosheets (abbreviated as
   NiCoO [x] @BSA, Figure [109]S10, Supporting Information). The measured
   zeta potential of NiCoO [x] @BSA was around −17.4 mV (Figure [110]S11,
   Supporting Information). In the cell and animal experiments performed,
   we used NiCoO [x] @BSA because of its improved stability under
   physiological conditions. The results of the multienzyme‐mimicking
   activities of the 2D NiCoO [x] nanosheets and the synergistic effect
   enhanced by US were initially evaluated at the cellular level. The
   cellular uptake of the 2D NiCoO [x] nanosheets was first measured. The
   cellular phagocytosis of this 2D NiCoO [x] nanozyme was determined in a
   breast cancer cell line (mouse 4T1 cells) using confocal laser scanning
   microscopy (CLSM). As shown in Figure [111]3a, the Rhodamine B
   (RB)‐labeled NiCoO [x] nanosheets entered the 4T1 cells for each of the
   different culture durations (1, 2, 4, and 8 h), which can be
   demonstrated by the red fluorescence emitted by RB and the
   corresponding linear‐scan spectrogram. The intensity of the red
   fluorescence gradually increased as the incubation duration increased.

Figure 3.

   Figure 3
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   In vitro therapeutic assessment results. a) Representative CLSM images
   and corresponding linear‐scan profiles of the 4T1 cells after
   co‐incubation with Rhodamine‐B‐labeled NiCoO [x] nanozymes (100 µg
   mL^−1) for 1, 2, 4, and 8 h. b) Schematic diagram illustrating the ROS
   release from the 4T1 cells that causes apoptosis, which is triggered by
   the US‐enhanced multienzyme activities of the 2D NiCoO [x] nanosheets.
   c) CLSM images of the intracellular ROS level of the 4T1 cells after
   the different treatments at pH = 6.5 (for the groups involving US:
   1 MHz; 1.2 W cm^−2; 50% duty cycle; 1 min). d) Quantitative ROS
   production in 4T1 cells treated with the various treatments at pH 6.5
   and pH 7.4, respectively. e) Cytotoxicity of the 2D NiCoO [x]
   nanosheets toward 4T1 cells at different concentrations and pHs (6.5
   and 7.4) (n = 3, mean ± SD). f) Cell viability of the 4T1 cells
   incubated with different concentrations of 2D NiCoO [x] nanosheets at
   various pHs (6.5 and 7.4) and H[2]O[2] concentrations (50 and 100 µm)
   (n = 3, mean ± SD). g) Relative cell viabilities of the 4T1 cancer
   cells after incubation with the 2D NiCoO [x] nanosheets at various
   concentrations and H[2]O[2] (100 µm) at pH 6.5 using different US
   irradiation settings (1 MHz; 1.2 W cm^−2; 50% duty cycle; and 1, 2, and
   3 min) (n = 4, mean ± SD). h) CLSM images of the 4T1 cancer cells
   stained with calcein AM/PI at pH 6.5 after the different treatments
   were performed (for the groups involving US: 1 MHz; 1.2 W cm^−2; 50%
   duty cycle; and 1 min) and flow cytometric analysis results of the 4T1
   tumor cells after the different treatments at pH 6.5 (for the groups
   involving US: 1 MHz; 1.2 W cm^−2; 50% duty cycle; and 1 min). i) The
   relative quantitative analysis of the 4T1 cancer cells after the
   various treatments by staining cells with Annexin‐V‐FTIC/PI at pH 6.5.
   j) Confocal images of the 4T1 tumor cells after co‐incubation with
   Thiol Tracker violet (green, GSH detection kit) and after the various
   treatments at pH 6.5 (for the groups involving US: 1 MHz; 1.2 W cm^−2;
   50% duty cycle; and 1 min). The data are shown as the mean ± standard
   deviation (SD) as calculated using a Student's t‐test with *p < 0.05,
   **p < 0.01, and ***p < 0.001.

   Based on the excellent aforementioned properties of the
   multienzyme‐activated cytotoxic ROS production, to further investigate
   the intracellular •OH generation at the cellular level, the ROS levels
   in cells under acidic (pH 6.5) (Figure [113]3c) and neutral conditions
   (pH 7.4) (Figure [114]S12, Supporting Information) were determined
   based on the fluorescence of a chemical detector,
   2′,7′‐dichlorofluorescin diacetate (DCFH‐DA). The single treatments,
   such as using US, H[2]O[2], and NiCoO [x] , only produce weak
   fluorescence intensities. Notably, the obvious green fluorescence
   observed in 4T1 cells indicated that the 2D NiCoO [x] nanosheets induce
   the production of small amounts of ROS under US irradiation, while the
   further combination with H[2]O[2] under acidic conditions significantly
   increases the intracellular ROS levels. Therefore, the chemodynamic
   effect generated by the 2D NiCoO [x] nanosheets and US under acidic
   conditions (pH 6.5) can markedly improve the generation efficiency of
   •OH (Figure [115]3c,d).

   The cytocompatibility of 4T1 cells was investigated using a typical
   cell counting kit‐8 (CCK‐8). As shown in Figure [116]3e, with the
   increase in the concentration of the NiCoO [x] nanosheets, the
   viability of 4T1 cells decreased. Next, we investigated the effect of
   different concentrations of H[2]O[2] and of the US time on the
   cytotoxicity. Tumor cells featured higher endogenous H[2]O[2] levels
   than normal cells. To further model the TME and more comprehensively
   understand the cytotoxic properties of the 2D NiCoO [x] nanosheets, 4T1
   cancer cells were treated using different NiCoO [x] concentrations (0,
   12.5, 25, 50, 100, and 200 µg mL^−1) under neutral (pH 7.4) and acidic
   (pH 6.5) environmental conditions in the presence of H[2]O[2] (50 or
   100 µm) for 24 h (Figure [117]3f). In an acidic medium, the
   cytotoxicity assays showed that the cell viability decreased
   significantly with an increasing sample concentration; however, under
   neutral conditions, the cell viability did not prominently decline for
   the same concentration values. By further adding H[2]O[2], the
   viability of the 4T1 cancer cells subjected to the 100 µg mL^−1 NiCoO
   [x] treatment decreased to 44.1% (pH = 6.5, H[2]O[2] = 50 µm) and 31.9%
   (pH = 6.5, H[2]O[2] = 100 µm), respectively (Figure [118]3f). For the
   US‐amplified assessment, we further studied the effect of the US time
   on cell viability (Figure [119]3g). As the US irradiation time
   increased, the activity of 4T1 cells decreased significantly,
   indicating that US could significantly improve the nanoenzyme‐initiated
   therapeutic efficiency (Figure [120]3g).

   Subsequently, the US‐amplified effects of the 2D NiCoO [x] nanosheets
   on tumor cells were further confirmed using calcein‐AM and propidium
   iodide (PI) staining experiments (Figure [121]3h; Figure [122]S13,
   Supporting Information). Notably, almost all cells of the NiCoO [x] +
   H[2]O[2] + US group died under acidic conditions due to the increased
   •OH and ^1O[2] production during the US‐amplified catalytic procedure
   (Figure [123]3b). The fluorescence results highlight the remarkable US
   coupling system results. Meanwhile, the apoptosis of 4T1 cancer cells
   stained with annexin V‐FITC and PI after treatment was measured using
   flow cytometry (Figure [124]3i; Figure [125]S14, Supporting
   Information). A small amount of 4T1 cancer cells died in the 2D NiCoO
   [x] group (Q2 + Q3 quadrant, 26.69%), and the combination of NiCoO [x]
   + H[2]O[2] and US stimulation effectively amplified the
   multienzyme‐mimicking activities of the 2D NiCoO [x] nanozymes, leading
   to apoptosis progress revulsion (Q2 + Q3 quadrant, 72.7% for pH = 6.5)
   (Figure [126]3j; Figure [127]S15, Supporting Information). Obviously,
   the US‐induced 2D NiCoO [x] treatment regimen had the most prominent
   tumor cell‐killing capability, killing almost all 4T1 cancer cells,
   demonstrating that the 2D NiCoO [x] nanosheets induced high
   chemokinetic toxicity and that they could be used as an effective
   therapeutic agent for the US‐enhanced multienzyme‐catalyzed therapy of
   malignant tumors.

   GSH, which is an important cellular antioxidant, plays an irreplaceable
   role in protecting cells from intracellular oxidative stress. A
   superior US‐augmented enzymatic activity was achieved using the 2D
   NiCoO [x] nanosheets. The aforementioned catalysis of the 2D NiCoO [x]
   nanosheets during GSH consumption prompted us to investigate the
   cellular GSH levels after performing different treatments. The
   commercial Thiol Tracker Violet intracellular GSH probe was used to
   visualize the GSH in the 4T1 cells. The control group cells emitted
   distinct green fluorescence (pH 6.5), whereas the fluorescence
   intensity observed in the NiCoO[x] + H[2]O[2] and NiCoO [x] + H[2]O[2]
   groups was lower than that found in the control group, and that
   observed in the NiCoO[x] + US + H[2]O[2] group was almost non‐existent,
   indicating that the 2D NiCoO [x] nanosheets have an excellent
   capability of scavenging the intracellular GSH through their
   US‐augmented enzymatic activity (Figure [128]3k; Figure [129]S16,
   Supporting Information). Therefore, the 2D NiCoO [x] nanosheets with
   multienzyme properties deplete cells of GSH through their US‐augmented
   enzymatic activity.

2.4. Analysis of the In Vitro Pyroptosis‐Like Cancer Cell Death

   Cell apoptosis is closely related to mitochondrial dysfunction.^[
   [130]^31 ^] Next, we used JC‐1 staining to detect the mitochondrial
   membrane potential in the different treatment groups to illustrate the
   relationship between apoptosis and mitochondrial dysfunction (Figure
   [131]4a; Figure [132]S17, Supporting Information). Red JC‐1 aggregates
   represent healthy high‐potential polarized mitochondria, whereas green
   JC‐1 monomers represent unhealthy low‐potential depolarized
   mitochondria (Figure [133]4b). As shown in Figure [134]4a, compared
   with that of the control group, the JC‐1 dye in the NiCoO [x] +
   H[2]O[2]+ US group was completely transformed from red aggregates to
   green monomers under acidic conditions (pH 6.5), which is
   representative of mitochondrial membrane disruption. Consequently, the
   2D NiCoO [x] nanosheets were significantly cytotoxic in acidic medium,
   whereas they did not lead to significant cell death in neutral medium
   (Figure [135]S17, Supporting Information). Quantitative fluorescence
   analysis further confirmed this (Figure [136]S18, Supporting
   Information). These in vitro results suggest that the US‐induced 2D
   NiCoO [x] nanosystems can achieve GSH depletion and amplify the
   oxidative stress, thereby promoting US‐augmented cancer cell killing.
   The 2D NiCoO [x] nanosheets cause obvious mitochondrial damage, which
   could be attributed to ROS production. Additional significant upstream
   processes connected to NLRP3 activation include mitochondrial
   malfunction and the release of ROS into the cytosol.^[ [137]^32 ^]
   Considering the desirable US‐amplified performance of the 2D NiCoO [x]
   nanosheets, these are speculated to be able to induce pyroptosis. The
   formation of transmembrane pores is the main feature of pyrolysis,
   which leads to the swelling and expansion of cells and to bubble‐like
   protrusions.^[ [138]^33 ^] To verify our hypothesis, we used an
   inverted microscope to examine the morphology of the 4T1 cells of the
   different treatment groups. Compared with the results of the control
   group, a larger number of vesicles and distinct balloon‐like cells were
   observed in the NiCoO [x] + US group, which demonstrates that the NiCoO
   [x] + US treatment could efficiently induce pyroptosis
   (Figure [139]4d).

Figure 4.

   Figure 4
   [140]Open in a new tab

   Cellular interaction and the pyroptosis‐inducing performance of the
   multienzyme‐mimicking 2D NiCoO [x] nanosheets. a) JC‐1 assay of the 4T1
   tumor cells after coincubation with NiCoO [x] (100 µg mL^−1) via
   various treatments at pH 6.5 (for the groups involving US: 1 MHz; 1.2 W
   cm^−2; 50% duty cycle; and 1 min). b) Diagram of the mitochondrial
   membrane potential variation. c) Schematic diagram of the molecular
   mechanism of cancer cell pyroptosis induced by multienzyme‐mimicking
   NiCoO [x] under US irradiation. d) Representative microscopy images
   (bright‐field) of 4T1 cancer cells after the various treatments. Each
   arrow tip indicates pyroptotic cells (scale bar: 200 µm). e) The NLRP3,
   cleaved caspase‐1, and N‐GSDMD protein expression in the various groups
   of 4T1 tumor cells, as observed in the western blot analysis (for the
   groups involving US: 1 MHz; 1.2 W cm^−2; 50% duty cycle; and 1 min). f)
   Relative contents of NLRP3, cleaved caspase‐1, and N‐GSDMD protein
   expression. g) Results of the assay analyzing the amount of IL‐1β
   released by the 4T1 tumor cells after the different treatments. h)
   Results of the assay analyzing the amount of LDH released by the 4T1
   tumor cells after the different treatments (for the groups involving
   US: 1 MHz; 1.2 W cm^−2; 50% duty cycle; and 1 min). The data are shown
   as the mean ± standard deviation (SD), as calculated using a Student's
   t‐test with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.001.

   GSDMs are a family of intracellular proteins with a pore‐forming effect
   that can induce cell membrane permeability and pyroptosis. GSDMs have a
   poisonousness N‐terminal structure (N‐GSDMD) and a C‐terminal repressor
   structure (C‐GSDMD), which can be proteolytically activated by certain
   proteases.^[ [141]^34 ^] The released N‐GSDMD forms large oligomeric
   pores in the plasma membrane, rupturing the cell membrane. The cells
   undergoing pyrolysis release various pro‐inflammatory cytokines into
   the extracellular environment, thus triggering inflammation and an
   immune response (Figure [142]4c). To further understand the 2D NiCoO
   [x]‐mediated pyroptosis mechanism, western blot analysis of the related
   pyrolytic core proteins was performed (Figure [143]4e). The expression
   of pyrolysis biomarkers, including NLRP3 and N‐GSDMD, was significantly
   increased in the 4T1 cells of the NiCoO [x] group. Under US irradiation
   (NiCoO [x] + US), their pyroptosis capability could be further improved
   (Figure [144]4f; Figure [145]S19, Supporting Information). Furthermore,
   we also observed that the expression of cleaved caspase‐1 in the NiCoO
   [x] + US group was enhanced. Caspase‐1 could cleave GSDMD, and the
   formed pores led to cell pyroptosis and released DAMPs.^[ [146]^35 ^]
   Another characteristic of GSDMD‐dependent pyroptosis is that it
   promotes the non‐conventional secretion of inflammatory molecules and
   cell contents, thus causing intense inflammation.^[ [147]^36 ^] After
   the NiCoO [x] + US treatment, the concentrations of lactic
   dehydrogenase (LDH) and interleukin‐1β (IL‐1β) in the 4T1 cells
   apparently increased; however, no significant release of LDH or IL‐1β
   was observed in the control group, indicating that the NiCoO [x] + US
   treatment highly induces pyroptosis (Figure [148]4g,h). All of these
   results confirm that the NiCoO [x] + US treatment can lead to
   pyroptosis.

2.5. RNA Sequencing and Analysis of the Therapeutic Mechanism

   To further clarify the underlying cellular therapeutic mechanism of
   US‐induced NiCoO[x] multienzyme‐mimicking nanosystems, transcriptome
   analysis was performed to compare the changes in the messenger RNA
   (mRNA) expression in 4T1 cancer cells treated with 2D NiCoO [x] in the
   presence of H[2]O[2]. In total, 1786 genes were differentially
   expressed from those expressed in the US‐treated NiCoO [x] group,
   including 1104 up‐regulated mRNAs and 682 down‐regulated mRNAs (Figure
   [149]5a; Table [150]S1, Supporting Information). Based on the diversity
   of the RNA sequencing results, we performed Gene Ontology (GO) and
   Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to explain the
   changes in the mRNA biological function and the relevant pathways of
   influence (Figure [151]5b; Table [152]S2, Supporting Information). The
   information acquired from the aforementioned analysis results
   (Figure [153]5c) suggests that the differentially expressed genes
   observed after combining the NiCoO[x] with the US treatment were
   enriched in inflammatory signaling pathways, such as cytokine‐cytokine
   receptor interaction, MAPK signaling pathway, NOD‐like receptor
   signaling pathway, TNF signaling pathway, IL‐17 signaling pathway, and
   NF‐kappa B signaling pathway, which verifies that the NiCoO [x] + US
   treatment efficiently kills cancer cells and induces inflammation
   through 2D NiCoO [x] ‐induced pyroptosis. Interestingly, the HIF‐1
   signaling pathway was also enriched for those genes, indicating that
   the oxygen level of the tumor tissues could change after the treatment
   (Figure [154]5c). The up‐ and down‐regulated pathway‐related genes are
   more simply reported in the loop diagram (Figure [155]5d). Notably, the
   results affirm that the association of NiCoO [x] with US aggravates
   inflammation, which is reflected in the upregulation of the NOD‐like
   receptor signaling pathway, confirming the superimposed effect of the
   combined application of NiCoO [x] and US.

Figure 5.

   Figure 5
   [156]Open in a new tab

   Exploration of the therapeutic mechanism of the US‐induced 2D NiCoO [x]
   nanosheets on the 4T1 cells via transcriptome high‐throughput
   sequencing. a) The volcano plot of the up‐ and down‐regulated genes in
   the US‐induced NiCoO [x] group (1 MHz, 1.2 W cm^−2, 50% duty cycle, and
   1 min) and comparison with that of the control group. b) Gene ontology
   (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the
   differentially expressed genes in the US‐induced NiCoO [x] group and
   control group (BP, biological process; CC, cellular components; MF,
   molecular function). c) KEGG pathway enrichment analysis of the
   identified differentially expressed genes in the US‐induced NiCoO [x]
   group and control group. d) The circle plot of GO and KEGG analysis. e)
   Enriched chord plot of the KEGG pathways. f) The heat map of the
   differentially expressed genes. g) Protein–protein interaction network
   of the 40 functional intersecting genes.

   After determining the gene expression differences, the specific
   regulatory genes corresponding to the main influencing pathways were
   presented in a chord diagram after the KEGG pathway analysis. Notably,
   the most significantly regulated genes belonged to the NOD‐like
   receptor signaling pathway (Figure [157]5e; Table [158]S3, Supporting
   Information). The gene set enrichment analysis results also confirmed
   that the significantly differentially expressed genes were involved in
   the NOD‐like signaling pathway (Figure [159]S20, Supporting
   Information). To further explore the underlying mechanism, a heatmap
   and protein–protein interaction system of the differentially expressed
   genes related to PCD (including inflammatory response and pyroptosis)
   were generated (Figure [160]5f,g). Notably, NLRP3 was remarkably
   up‐regulated, which is consistent with the western blot results
   (Figure [161]4e,f). For the nuclear factor‐k‐gene binding
   (NF‐kB)‐related genes (Nfkbib, Nfkb1, Ripk2, and Nfkbia), tumor
   necrosis factor (TNF)‐related genes (Tnf, Traf6, and Tnfaip3), the
   antiviral protein genes (Oas1a, Oas3, and Oas2), interferon‐related
   genes (Tyk2, Gbp2b, Stat1, Gbp5, Irf9, Stat2, Gbp7, Irf7, and Gbp2),
   and chemokine genes (Cxcl3, Cxcl1, Cxcl2, Ccl2, and Ccl5), the obvious
   appearance of these up‐regulated gene changes confirmed that the 2D
   NiCoO [x] nanosheets can activate a pyroptosis‐binding inflammatory
   reaction. These findings demonstrate that the US‐induced 2D NiCoO [x]
   nanosheets can alleviate tumor hypoxia and further induce pyroptosis.

2.6. In Vivo Antitumor Effect

   Considering the favorable catalytic and cellular therapeutic results
   obtained using the 2D NiCoO [x] nanosheets, we further evaluated their
   antitumor therapeutic effect in vivo. Female BALB/c mice aged 3–4 weeks
   were subcutaneously injected with 4T1 cells on the right side to
   establish a breast cancer model, and the tumor‐bearing mice were
   irregularly divided into four groups (n = 5 per group): 1) control
   group; 2) US group; 3) NiCoO [x] group; 4) NiCoO [x] + US group
   (intravenous injection (i.v.)). The detailed treatment protocol of the
   tumor‐bearing mice is shown in Figure [162]6a. After intravenous
   injection of NiCoO [x] nanosheets, the majority of Ni was distributed
   in the spleen and liver tissues of the 4T1 tumor‐bearing mice, in
   addition to their efficient accumulation at tumor sites (Figure
   [163]S21, Supporting Information). The tumor was irradiated with
   assisted US irradiation (1 MHz, 1.2 W cm^−2, 50% duty cycle, and
   10 min) 1, 3, 7, and 11 days after tail vein injection of a NiCoO [x]
   aqueous solution for 15 min and 4 h. The body weight and tumor volume
   of the mice were monitored every 2 days during the whole treatment
   period. As expected, no apparent body weight changes were observed for
   any of the groups, which show the high therapeutic biosafety of the
   treatment (Figure [164]6d). Concurrently, compared with the control
   group results, the results of the hematoxylin and eosin (H&E) staining
   of the main organs (heart, liver, lungs, spleen, and kidneys) of the
   mice further showed no obvious pathological abnormalities or
   inflammatory lesions in the treatment group, confirming the desirable
   histocompatibility of the 2D NiCoO[x] nanosheets (Figure [165]S22,
   Supporting Information).

Figure 6.

   Figure 6
   [166]Open in a new tab

   Evaluation of the in vivo antitumor efficacy. a) Schematic diagram of
   the timeline of the assay performed to evaluate the effectiveness of 2D
   NiCoO [x] nanoenzymes for antitumor therapy. b) Individual tumor growth
   curve of the tumor‐bearing mice in the various treatment groups after
   being exposed to the US‐amplified multienzyme effect for tumor
   treatment (n = 5). c) Tumor volume and d) relative tumor volume of the
   mice after undergoing diverse treatments (n = 5). e) Body weight of
   mice during the 14 days of treatment. f) Relative 4T1‐tumor inhibition
   rate of the treatment group and comparison with that of the control
   group. g) H&E, h) TUNEL, i) Ki‐67, and j) cleaved caspase‐1 staining
   images of the tumor slices obtained from the various groups (control,
   US, NiCoO [x] , and NiCoO [x] + US) of mice.

   Regarding tumor inhibition evaluation, the tumor growth of each group
   during the 14 days treatment period was recorded in detail
   (Figure [167]6b–e). Compared with the control group mice, for which a
   continuous growth of the tumor volume was observed, the mice
   administered NiCoO [x] showed slight tumor inhibition (average volume
   of 288 mm^3), which is attributed to the catalytic therapeutic effect
   of the 2D NiCoO [x] nanozymes (Figure [168]6b). A more effective 4T1
   breast tumor inhibition was observed in the group treated with NiCoO
   [x]+ US, especially when NiCoO [x] was administered intravenously.
   After 14 days of treatment, the average tumor volume of mice decreased
   to 107 mm^3 (Figure [169]6c), highlighting how US magnifies the
   simulated activity of the 2D NiCoO[x] nanozymes with POD‐, OXD‐, GPx‐,
   and CAT‐mimicking activities. This satisfactory tumor inhibition effect
   is attributed to the synergistic effect of US‐enhanced nanocatalytic
   therapy, which is enabled by the 2D NiCoO [x] nanozymes. Furthermore,
   we calculated the tumor growth inhibition (TGI) rate to quantitatively
   evaluate the desirable antitumor therapeutic efficiency of the
   nanoenzymes enhanced by US. Compared with that of the NiCoO [x] group
   (the relative TGI rate was 0.14%) at the end of day 14, the TGI rate of
   the US‐induced NiCoO [x] group was 71.8%, surpassing that of the US
   group (−0.04%) (Figure [170]6f). Moreover, the average weight of the
   tumors collected after treatment based on relevant visualized tumor
   photographs further confirmed the high antitumor efficacy (Figure
   [171]S23, Supporting Information).

   Additionally, the results of H&E and TdT‐mediated dUTP nick‐end
   labeling (TUNEL) staining of the tumor slices after treatment further
   suggest that the tumors in the NiCoO [x] + US group show serious
   apoptosis (Figure [172]6g,h; Figure [173]S24, Supporting Information).
   Furthermore, the ROS level in NiCoO [x] + US‐treated tumors is
   considerably higher than that in NiCoO [x] and US‐treated tumors,
   deriving from the in vivo O[2] ^− overproduction triggered by NiCoO [x]
   + US (Figure [174]S25 Supporting Information). And the tumor cell
   proliferation inhibition effect was confirmed by the Ki‐67
   immunohistochemistry staining assay results. As expected, compared with
   those of the control and US groups, an effective inhibitory effect on
   tumor proliferation, characterized by a strong blue signal, was
   observed in the mice injected with the 2D NiCoO [x] nanozymes under US
   stimulation (Figure [175]6i; Figure [176]S26, Supporting Information).
   To further confirm the pyroptosis effect, we also carried out
   immunohistochemical examination of cleaved caspase‐1 expression in the
   tumor tissue (Figure [177]6j; Figure [178]S27, Supporting Information).
   The expression of this protein was increased in the NiCoO[x] group, and
   its effect could be further drastically enhanced under US irradiation,
   demonstrating that the NiCoO [x] + US treatment can effectively induce
   cancer cell pyroptosis. Taken together, we can conclude that the
   NiCoO[x] + US‐mediated pyroptosis could effectively induce a lasting
   antitumor effect.

3. Conclusions

   In summary, we developed specific US‐augmented multienzyme‐mimicking
   strategies for an efficient tumor nanotherapy by engineering distinct
   2D NiCoO nanosheets/nanoenzymes to improve the rate of harmful ROS
   generation for catalytic tumor nanotherapy and cell pyroptosis. A
   variety of multienzyme‐mimicking activities were observed in the
   designed/fabricated 2D NiCoO [x] nanosheets, including ROS‐associated
   POD‐ and OXD‐mimicking activities, hypoxia reversal involving a
   CAT‐like activity, and a GSH‐consumption capability involving GPx‐like
   activity. US stimulation improved the multienzyme‐like activities and
   enhanced ROS production, which caused mitochondrial dysfunction. The
   pyroptosis process can be specifically induced by the ROS generated by
   the multienzyme‐mimicking with US activation, which was demonstrated by
   caspase‐1 activation and the release of N‐GSDMD and LDH/IL‐1β. The
   subsequent transcriptome sequencing analysis revealed that pyroptosis
   was caused by the ROS‐NLRP3‐GSDMD pathway. Importantly, the
   US‐augmented catalytic tumor nanotherapy produced remarkable
   therapeutic results, as evidenced by the systematic in vitro and in
   vivo evaluations (4T1 breast tumor model). Collectively, our results
   show that the engineered 2D nanosheets/nanoenzymes with US‐augmented
   multienzyme‐mimicking activities constitute a novel strategy to induce
   pyroptosis, which enables a highly efficient catalytic tumor
   nanotherapy.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [179]Click here for additional data file.^ (1.9MB, pdf)

Acknowledgements