English

• Cutaneous melanoma (CM) is highly malignant and characterized by easy metastasis, poor prognosis and low long-term survival [1]. While surgery can remove primary tumors or those with local metastasis, adjuvant therapy is necessary to prevent recurrence. In cases of advanced CM, surgery and conventional chemotherapy are often ineffective. Although targeted therapies and checkpoint immune therapies have demonstrated promise, they are not universally effective [2]. Hence, there is an urgent need to develop novel therapeutic approaches that can effectively suppress both primary tumors and inhibit distant metastasis.

  Evidence suggests that increased cellular reactive oxygen species (ROS) is closely linked to cancer development [3]. High levels of ROS can both promote tumor progression and induce resistance to chemotherapy while also simultaneously causing damage to cellular components, which can trigger apoptosis. Obviously, regulating cellular ROS can be a potential strategy for cancer therapy and inflammatory diseases [4,5]. However, antioxidants have failed to suppress melanoma and even facilitated migration and metastasis [6]. Instead, increasing ROS in melanoma cells appears more promising, such as through a therapeutic platform that inhibits antioxidant enzyme [7]. Nanozymes are multifunctional, stable, and can withstand harsh conditions [8,9]. They can mimic oxidative enzymes to generate ROS for killing tumor cells or bacteria, thriving a research field termed as nanocatalytic medicine [10]. Recently, single-atom nanozymes (SAzymes) have gained attention for their high catalytic activities [11–13], but their performance is still inferior to natural enzymes, motivating scientists to design novel nanozymes to achieve higher catalytic performance. Single-atom alloy nanozymes, a kind of SAzyme with metal nanoparticles (NPs) as the support of the active centers, offer improved catalytic selectivity and efficiency [14]. Moreover, the metal support of single atom alloy nanozyme is more conducive to the electron transfer during the catalytic process to achieve stronger catalytic activities [15,16].

  Cu is a crucial trace element that serves as the active center of many enzymes of human beings. Cu NPs have shown potential in nanocatalytic medicine, but their performance is limited [17,18]. Cooperating Cu NPs with other metals, such as Pt which is known for catalyzing oxygen reduction reactions [19], holds great potential to enhance their catalytic activities [20,21].

  The present work reported such a PtCu alloy nanozyme (PtCu-zyme) that integrates single atom Pt and Pt clusters on the surface of Cu NPs, exhibiting remarkable catalase (CAT)-, peroxidase (POD)-, and oxidase (OXD)-like activities essential for potent CM therapy (Scheme 1). To enhance the mitochondrial targeting ability, triphenylphosphine (TPP) was functionalized on the surface of PtCu-zyme to obtain PtCu-TPP. Further, PtCu-TPP was mixed with hyaluronic acid (HA) analog, isoliquiritigenin grafted HA (HA-ISL), to prepare nanozyme loaded microneedles (PtCu-TPP@MNs) to further enhance the anti-CM efficacy. Upon application to the skin, PtCu-TPP@MNs dissolved, releasing the nanozymes. The residual HA-ISL on the surface of PtCu-TPP guided the nanozymes to enter into the tumor cells via HA-ISL-CD44-mediated endocytosis and TPP later harnessed the nanozymes to localize at mitochondria. PtCu-TPP@MNs effectively restrained tumor growth and metastasis not only through PtCu-TPP-mediated nanocatalytic therapy, but also via HA-ISL-mediated signaling regulation. Importantly, PtCu-TPP@MNs prolonged the lifespan of CM bearing mice and the retained PtCu-zyme in the tumor continually catalyzed ROS generation, thereby achieving a sustained nanocatalytic therapy.

  Scheme 1

  

  Scheme 1.  The preparation of PtCu-TPP@MNs and their possible anti-CM mechanisms. PtCu-zyme was first prepared and DSPE-PEG-TPP was later functionalized on the surface of the nanozyme to obtain PtCu-TPP. Subsequently, PtCu-TPP was mixed with HA-ISL to fabricate PtCu-TPP@MNs. Once being applicated on the skin, PtCu-TPP@MNs dissolved, releasing the nanozymes. The residual matrix HA-ISL on the surface of PtCu-TPP facilitated the internalization of nanozymes into the tumor cells via HA-ISL-CD44-mediated endocytosis. Following, the functionalized DSPE-PEG-TPP harnessed the nanozymes to localize at mitochondria to catalyze the generation of ROS. The high level of ROS caused damage to mitochondria, induced the release of cytochrome C (Cyt-c) and the phosphorylation of p38, resulting in the tumor cell apoptosis and necrosis.

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  The workflow for the synthesis of PtCu-zyme is shown in Fig. 1A. Firstly, nitrogen-doped carbon (NC), a support with an average size of ~140 nm, was prepared according to the previous publication (Fig. S1 in Supporting information) [11]. X-ray diffraction (XRD) analysis verified the graphitization of NC, showing clear peaks at 26° (002 plane) and 43° (101 plane) (Fig. S2 in Supporting information) [22]. Subsequently, Cu NPs with a diameter of ~15 nm were fabricated by a modified reduction method [23,24], which were later uniformly dispersed onto NC to yield Cu/NC. In comparison with Cu NPs (Fig. S3A in Supporting information), embedding Cu NPs onto NC significantly improved their dispersity (Fig. S3B in Supporting information). Ultimately, single atom Pt and Pt subnanoclusters were doped onto the Cu NPs within Cu/NC through the galvanic replacement (GR) reaction to obtain PtCu-zyme [25]. As expected, the loading of single atom Pt and Pt clusters did not significantly alter the morphology of Cu/NC (Fig. 1B) and Pt was uniformly distributed on Cu NPs embedded within the NC support (Fig. 1C). Subsequently, aberration-corrected high-angle-annular-dark-field scanning transmission electron microscopy (ac-HAADF-STEM) identified that Pt dispersed atomically or formed subnanoclusters (highlighted by red-square) in-between the lattice fringes of Cu NPs (marked by blue-circles) (Fig. 1D) [26].

  Figure 1

  

  Figure 1.  Preparation and characterization of PtCu-zyme. (A) Schematic illustration of the synthesis process. (B) TEM image. (C) Energy dispersive spectroscopy (EDS) mapping. (D) ac-HAADF-STEM image (the red boxes refer to single atom Pt or Pt subnanoculaters). (E) Pt L3-edge EXAFS Fourier-transform spectra of Pt foil, and PtCu-zyme. EXAFS wavelet transforms spectra of (F) Pt foil, (G) PtO₂ and (H) PtCu-zyme. (I) Time dependent O₂ release in the presence of H₂O₂ and various nanozymes (n = 3). (J) Absorbance of ox-TMB in the presence of H₂O₂ and various nanozymes (n = 3). (K) Ultraviolet–visible (UV–vis) spectra of OPD-ox oxidated by ^•OH with the presence of various nanozymes.

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  To verify the co-existence of single atom Pt or Pt subnanoclusters, we examined extended X-ray absorption fine structure (EXAFS) of Pt species in PtCu-zyme (Fig. 1E). A broad peak was observed at ~2.4 Å, suggesting that Pt species in PtCu-zyme occupied different coordination environments. In comparison to the Pt species in Pt foil (~2.5 Å), this peak shifted towards a shorter bond length, indicating the coordination of Pt with smaller Cu atoms. These results were further corroborated by wavelet transform analysis. The shift in the maximum intensity of PtCu-zyme plot revealed the presence of Pt-Pt and Pt-Cu coordinations (Figs. 1F–H). Two distinct scattering centers, positioned at ~7 Å⁻¹ and ~9 Å⁻¹, were attributed to Pt-Cu and Pt-Pt scattering, respectively. The fitting results further validated the coordination of Pt in PtCu-zyme with both Pt and Cu (Fig. S4 and Table S1 in Supporting information). Specifically, the coordination numbers of Pt-Cu and Pt-Pt were calculated to be 4.8 ± 1.1 and 7.1 ± 0.8, respectively (Table S1), demonstrating the occurrence of single atom Pt and Pt subnanoclusters [16,27]. The content of Pt and Cu in PtCu-zyme was determined to be 1.95 wt% and 35.02 wt% by inductively coupled plasma optical emission spectrometer (ICP-OES), respectively. These results were consistent with the previous studies that increasing the amount of Pt to ~2 wt% led to a mixture of single atom Pt and Pt subnanoclusters [15].

  XRD analysis confirmed the crystallographic properties of Cu NPs within PtCu-zyme, with peaks corresponding to Cu (PDF #85–1326, International Centre for Diffraction Data (ICDD)), Cu₂O (PDF #99–0041, ICDD), and CuO (PDF #45–0937, ICDD), but not CuPt, implying the highly dispersed Pt on the support (Fig. S5 in Supporting information). High resolution transmission electron microscope (HR-TEM) further identified the Cu (111) facet (Fig. S6 in Supporting information) [28]. The missing crystal of Pt (PDF #01–1194) can be explained by the low content of Pt subnanoclusters, which were hardly detected by XRD. X-ray photoelectron spectroscopy (XPS) revealed the elemental composition of the nanozymes (Figs. S7A and D in Supporting information) and demonstrated the presence of Cu/Cu⁺/Cu²⁺ and Pt in both +2 and +4 oxidation states (Figs. S7B and C in Supporting information) [29]. In comparison to Cu 2p spectra of Cu/NC, Cu/Cu⁺ 2p_1/2 and 2p_3/2 peaks of PtCu-zyme slightly shifted to higher binding energies, implying the electronic interaction between Pt and Cu (Figs. S7B and E in Supporting information) [30,31]. The ratios of Cu²⁺ to Cu/Cu⁺ and Pt²⁺ to Pt⁴⁺ were calculated to be 1.33 and 2.47, respectively. As XPS captured certain area of the sample surface, Pt(0) was not shown in the determined region.

  With the systematic investigations, we confirmed the successful fabrication of PtCu-zyme, where the integration of single atom Pt and Pt subnanoclusters enhanced electron transfer, potentially enhancing redox reactions. Cu NPs remained polycrystalline upon loading onto the NC support, with improved dispersity and increased air interaction, leading to the oxidation of Cu [32]. The co-existence of Cu/Cu⁺/Cu²⁺ and Pt²⁺/Pt⁴⁺ is the fundamental to the nanozymes that catalyzing redox reactions [33]. Due to the low content of Pt subnanoclusters or the limit detection capability of XRD, the Pt clusters were only seen from the EXAFS of PtCu-zyme.

  Similar to the other Cu- and Pt-based nanozymes, PtCu-zyme was supposed to mimic POD, CAT, and OXD (Fig. S8A in Supporting information) to promote ROS generation [34–36]. We first examined the CAT-like activity of PtCu-zyme. As seen from Fig. 1I, Cu NPs alone had minimal impact on O₂ generation, but they significantly accelerated O₂ release when dispersed on NC (Cu/NC), likely due to the improved dispersity and accessibility of catalytic sites (Fig. S3). The catalytic efficacy of Cu/NC was comparable to the commercial catalyst Pt/C (Fig. 1I). In agreement with the previous study, NC promoted O₂ release as well [11] but it was less effective than Cu/NC. After doping with Pt atoms, the CAT-like performance of the nanozyme PtCu-zyme was boosted and the maximum O₂ release was achieved within ~6 min, highlighting the superiority of the single atom Pt and Pt subnanoclusters in obtaining highly effective heterogeneous nanozymes. The kinetic study revealed that PtCu-zyme showed high affinity to H₂O₂ (Fig. S9 in Supporting information).

  OXD catalyzes O₂ to produce O₂^•− which oxidizes the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to generate blue ox-TMB [37]. We then incubated PtCu-zyme with TMB, the lack of significant absorbance at 652 nm suggested low OXD-like activity or insufficient O₂ (Fig. S8B in Supporting information). In the presence of H₂O₂, PtCu-zyme mimicked CAT and catalyzed the generation of O₂, amplifying the OXD-like performance of PtCu-zyme to catalyze the generation of, as evidenced by a pronounced absorbance at 652 nm that increased with concentration (Fig. 1J). The sextet signal in the electron paramagnetic resonance (EPR) spectra in Fig. S8C (Supporting information) specifically affirmed the generation of O₂^•−. Differently, the OXD-like activity of NC was as low as Cu NPs. The dispersion of Cu NPs onto NC (Cu/NC) significantly enhanced the OXD-like activity, which was further boosted after anchoring single atom Pt and Pt clusters onto Cu/NC to form PtCu-zyme.

  POD decomposes H₂O₂ into ^•OH, which oxidizes the colorless O-phenylenediamine (OPD) to yellow phenazine-2,3-diamine (OPD-ox) with the maximum absorbance between 420 nm and 450 nm [38]. After short co-incubation of nanozymes (Cu/NC, PtCu-zyme or Pt/C) with H₂O₂ and OPD, a noticeable absorbance of the supernatant in the 420–450 nm range confirmed their POD-like activity. More importantly, the POD-like activity of PtCu-zyme was superior to that of Cu NPs or NC or the commercial catalyst Pt/C (Fig. 1K). The generation of ^•OH by PtCu-zyme and Cu/NC was identified with EPR (Fig. S8D in Supporting information) as well [39]. The specific activity of POD-like activity of PtCu-zyme was determined to be 6.83 U/mg, which was lower than that of horseradish peroxidase (HRP, Fig. S10 in Supporting information).

  Our comprehensive research affirmed that PtCu-zyme exhibited moderate OXD-like activity, while showing potent CAT- and POD-like activities. The incorporation of single atom Pt and Pt subnanoclusters into PtCu-zyme significantly enhanced its enzyme-like activities. The content of Pt was a key determinant of PtCu-zyme's enzymatic activities (Fig. S11A and Table S2 in Supporting information). Furthermore, PtCu-zyme performed strong enzyme-like activities over a wide range of pH values and temperatures (Fig. S12 in Supporting information). Its enzyme-like activities and morphology were still maintained after multiple catalytic cycles, indicating excellent stability (Figs. S13 and S14 in Supporting information). These characteristics positions PtCu-zyme as a promising candidate for various catalytic applications.

  Encouraged by the impressive POD- and CAT-like activities of PtCu-zyme, we proposed that our nanozymes would exhibit remarkable anti-tumor activity by catalyzing the generation of more toxic ROS. Given that higher loading of Pt elevated the toxicity of nanozyme to normal cells (Fig. S11B in Supporting information), we selected PtCu-zyme with a moderate Pt loading (PtCu-zyme, 1.95%, Table S2) for further studies. To improve the biocompatibility and mitochondria-targeting ability of PtCu-zyme, a mitochondria-targeting ligand (TPP)-modified phospholipid polymer (DSPE-PEG, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine) was anchored onto the surface (Fig. S15A in Supporting information). The resulting PtCu-TPP still retained the morphology of PtCu-zyme but showed more obvious edges due to the attachment of DSPE-PEG-TPP (Fig. S15B in Supporting information), and the particle size increased to ~170 nm (Fig. S15C in Supporting information). The less negative zeta potential further verified the successful preparation of PtCu-TPP (Fig. S16 in Supporting information). As expected, the coating of DSPE-PEG-TPP shielded the toxicity of PtCu-zyme by reducing hemolysis (Fig. S17 in Supporting information) and exerted no significant influences on the proliferation of L929 cells at 20 µg/mL (Fig. S18 in Supporting information), demonstrating that PtCu-TPP was safe enough for in vivo applications.

  As an alternative to tail vein injection, we aimed to deliver the nanozymes by transdermal administration through MNs, which was quite suitable for the treatment of skin cancers and improved patient compliance. Toward this end, HA-ISL was first synthesized, characterized (Fig. S19 in Supporting information) and proven to suppress B16 cells proliferation and migration (Fig. S20 in Supporting information), which were probably achieved by interfering with HA-CD44 signaling [40,41]. HA-ISL was later mixed with PtCu-TPP to prepare PtCu-TPP@MNs via a micro-molding method (Fig. S21A in Supporting information, 10 × 10 array, 2.25 ± 0.096 µg/patch) [42], which were uniform and capable of penetrating mouse skin (Figs. S21B–E in Supporting information). A commercial anti-tumor drug DOX, which has proven to be effective for CM therapy [43,44], and unmodified HA were used to prepare DOX@HA-MNs as a positive control. Moreover, the rapid release of PtCu-TPP from MNs ensured swift access to tumor tissues (Fig. S21F in Supporting information).

  Next, we established B16 tumor bearing mice models to systematically evaluate the anti-tumor efficacy of PtCu-TPP@MNs with the approval of the Ethics Committee of School of Pharmaceutical Sciences, Zhengzhou University (approved No. yxyllsc20220066). The mice were randomly divided and administrated with MNs, PtCu-TPP@HA-MNs, PtCu-TPP@MNs, DOX@HA-MNs, or a mixture of PtCu-TPP and HA-ISL (HA-ISL+PtCu-TPP, intravenous (i.v.)) every two days, respectively (Fig. 2A). The continued increasing body weight of the mice during the treatment implied the biosafety of the administrations (Fig. 2B). MNs alone modestly reduced tumor volume, whereas the rest treatments all demonstrated a capacity to restrain tumor growth (Figs. 2C and F). Notably, PtCu-TPP@MNs exhibited a remarkable reduction in tumor growth, clearly underscoring the outstanding anti-tumor efficacy. Compared with the tail vein injection, the transdermal delivery of PtCu-TPP offered greater advantages in suppressing tumor volume. Additionally, the anti-tumor effects of PtCu-TPP were comparable to those of the chemotherapeutic drug DOX (Fig. 2C).

  Figure 2

  

  Figure 2.  Anti-tumor efficacy, metabolism and survival. (A) Overview of the animal experiments. Fluctuations of (B) body weight and (C) tumor volume during the treatment (n = 5). The insert figures in (B) and (C) present the body weight and tumor volume on day 15. (D) Tumor nodules on the surface of lungs at the end of the therapy (n = 4 or 5). (E) Quantification of ROS levels of tumor by Image J. (F) Photo of tumors. (G) Fluorescent images of major organs ex vivo (n = 3). (H) Survival of B16 tumor bearing mice after the listed treatments (n = 6). (I) Content of Pt in liver, kidney and tumor at the end of therapy (n = 2). Data are presented as mean ± standard deviation (SD). ^**P < 0.01, ^****P < 0.0001. ns, non-significant.

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  Moreover, the application of HA-ISL and the developed nanozymes demonstrated prominent effects on the inhibition of tumor migration, with a significantly reduced number of black dots on the lung surface (Fig. 2D and Fig. S22 in Supporting information). These results were further corroborated by the rare nodules in the lung sections of the treated mice (Fig. S23 in Supporting information). Importantly, MNs alone successfully restrained the tumor migration, regardless of its modest impact on tumor growth (Figs. 2C and D). The superior anti-metastatic effects of DOX@HA-MNs were attributed to a synergistic effect of DOX-mediated chemotherapy and low molecular weight HA induced cell apoptosis [41,45].

  Further studies revealed that transdermal administration of PtCu-zyme (PtCu-TPP@MNs and PtCu-TPP@HA-MNs) significantly elevated tumor ROS levels, which was more effective than tail vein injection of PtCu-TPP (Fig. 2E and Fig. S24 in Supporting information). Curiously, PtCu-TPP@MNs induced a greater ROS generation than PtCu-TPP@HA-MNs (Fig. 2E), possibly due to the ability of ISL to enhance cellular ROS [46]. Nevertheless, none of the treatments caused significant influences on the blood and histological morphology of organs including heart, liver, spleen, and kidney, underscoring the biosafety of these nanozymes (Figs. S25 and S26 in Supporting information).

  Biodistribution study confirmed that transdermal administration led to greater tumor accumulation of PtCu-TPP-fluorescein isothiocyanate (PtCu-TPP-FITC) until 24 h (Fig. 2G, Figs. S27A and B in Supporting information). PtCu-TPP-FITC was also observed in the lungs, which could aid in targeting metastatic tumor cells (Fig. 2G and Fig. S27A). This preferential tumor accumulation likely underlay the enhanced efficacy of PtCu-TPP@MNs. Survival analysis further confirmed the therapeutic potency of PtCu-TPP@MNs. After seven treatments with PtCu-TPP@MNs, the median survival time was significantly extended to 24 days, which was much longer than that of the Model group (15 days) (Fig. 2H). Although Pt was detectable in liver and kidney, much higher concentrations of Pt (~250 ng/g) were determined in tumors at the end of the therapy (Fig. 2I). The persisting nanozymes within the tumors continued to catalyze ROS generation (Fig. S27C in Supporting information), conferring the nanozymes with a sustainable anti-tumor effect.

  We then systematically explored the possible anti-tumor mechanisms of PtCu-TPP in vitro against B16 cells. Under neutral conditions, nanozymes such as NC, Cu NPs, and Cu/NC showed minimal influences on cell viability (Fig. S28A in Supporting information). However, PtCu-TPP and PtCu-zyme exhibited a significant inhibition on B16 cell proliferation at the same conditions, highlighting their potent anti-tumor activity (Fig. S28B in Supporting information). In acidic conditions simulating the tumor microenvironment, the anti-tumor efficacy of all nanozymes was enhanced, resulting in a remarkable reduction in cell viability (Fig. 3A and Fig. S28C in Supporting information). PtCu-TPP was especially effective, with B16 cell viability falling below 20% at a concentration of 20 µg/mL. The enhanced activity was likely due to the improved catalytic performance of PtCu-TPP in acidic conditions (Fig. S12B). Moreover, PtCu-zyme caused more cell death than Cu/NC but was less cytotoxic than PtCu-TPP (Fig. S29 in Supporting information), highlighting the pivotal role of Pt atoms and TPP in enhancing the anti-tumor activity of the nanozyme.

  Figure 3

  

  Figure 3.  Anti-tumor efficacy against B16 cells and possible mechanisms. (A) Effects of nanozymes on the viability of B16 cells under simulated tumor microenvironment (acidic conditions, n = 3). (B) Fluctuations of mitochondrial membrane potential after the treatment with various nanozymes (n = 3). (C) Western blot analysis of the cellular expression of Cyt-c and phosphorylated p38 MAPK (p-p38). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Flow cytometric analysis of the cellular ROS. Bio-TEM images of B16 cells with the treatment of PtCu-TPP: (E) Endocytosis; (F) mitochondrial damage.

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  Indeed, PtCu-TPP induced much higher ROS levels in B16 cells than other nanozymes (Fig. 3D and Fig. S30 in Supporting information), which was likely due to its enhanced mitochondria targeting ability (Fig. S31 in Supporting information). The elevated ROS led to a significant decrease in mitochondrial membrane potential in B16 cells (Fig. 3B and Fig. S32 in Supporting information). With the assistance of biological transmission electron microscopy (Bio-TEM), we clearly observed the endocytosis of PtCu-TPP (Fig. 3E) and the disordered mitochondrial matrix (Fig. 3F). These mitochondrial dysfunctions, in turn, triggered cell apoptosis with PtCu-TPP inducing the highest level of apoptosis/necrosis (~60%, Fig. S33 in Supporting information). Western blot analysis revealed that PtCu-zyme induced cell apoptosis through the upregulation of Cyt-c expression and the activation of the p38 signaling pathway by facilitating the phosphorylation of p38 (Fig. 3C and Fig. S34 in Supporting information).

  Moreover, PtCu-zyme and PtCu-TPP demonstrated great anti-tumor efficacy against a range of other tumor cells, including 4T1 (mouse breast cancer cells), Hepa 1–6 (mouse hepatocellular carcinoma cells), and Hep G2 (human hepatocellular carcinomas) (Fig. S35 and Table S3 in Supporting information). This broad-spectrum anti-tumor efficacy underscored the potential of the developed nanozymes as versatile therapeutic agents against various cancer types.

  Density functional theory (DFT) calculations were performed to theoretically understand the mechanisms of the enhanced POD-like activity of PtCu-zyme, which contributed most to their anti-tumor activity. According to ac-HAADF-STEM image (Fig. 1D) and EXAFS wavelet transforms (Fig. 1H), a model with a single atom Pt and Pt cluster sitting on Cu (111) was established, and the adsorption energy of H₂O₂ at different sites (Cu (111), single-atom Pt-doped Cu (111), and Cu (111) of PtCu-zyme) was calculated. The results confirmed that H₂O₂ preferred to adsorb at Cu atop of Cu (111) of PtCu-zyme with the lowest energy (Fig. 4A), demonstrating that the co-appearance of single atom Pt and Pt subnanoclusters was profitable for H₂O₂ binding. The possible reaction process under acidic conditions was speculated as follows: (i) Adsorption and activation of H₂O₂ on Cu atop sites; (ii) decomposition of H₂O₂ to generate ^•OH and OH* with OH* adsorbing on Pt clusters and ^•OH being released; (iii) approach of surrounding H⁺ to OH*; and (iv) desorption of H₂O to regenerate the catalyst (Fig. 4B). During this process, the active energy barrier for the degradation of H₂O₂ was as low as 0.27 eV, revealing that ^•OH was generated atomically at room temperature (Fig. 4C).

  Figure 4

  

  Figure 4.  Catalytic mechanisms. (A) Adsorption energy of H₂O₂ at Cu (111) of different models. (B) Reaction processes with Cu atop as the active center. (C) Variations of the free energy at each step.

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  Up-regulating cellular ROS has been proven to be a potent approach to treat cancer. Although vitamin C loaded PtCu nanoframes achieved anti-tumor therapy via ROS generation as well [47], the mechanism of ROS production was fundamentally different from the PtCu-zyme designed in the presented study. In the case of PtCu nanoframes, vitamin C must be firstly released from PtCu nanoframes under NIR irradiation, catalyzing the generation of H₂O₂ in the tumor microenvironment. Subsequently, the released Cu ions catalyzed the conversion of the newly generated H₂O₂ into ROS. In contrast, the PtCu-zyme designed in the presented study directly catalyzed H₂O₂ in the tumor microenvironment to generate ROS by mimicking multiple oxidant enzyme-like activities, which was independent of any additional inputs, minimizing possible harm to healthy cells.

  Single atom Pt and Pt subnanoclusters were dispersed on the surface of Cu NPs within Cu/NC as designed (Fig. 1), and PtCu-zyme effectively catalyzed H₂O₂ and/or O₂ to generate ROS by mimicking CAT, POD and OXD (Fig. 1). The higher enzyme-like activities of PtCu-zyme than that of Cu/NC highlighted the importance of the co-appearance of single atom Pt and Pt subnanoclusters. Moreover, the catalytic activities of PtCu-zyme were enhanced along with the increase of Pt content (Fig. S11A). Although SAzyme with bimetallic active centers enhanced the catalytic performance of nanozymes as well, the alloy nanozymes offered better catalytic selectivity and efficiency [14]. Furthermore, the functions of the two kinds of metals in PtCu-zyme were more specific, which were different from the previous designed SAzyme with Fe and Co as bi-active centers (FeCo SAzyme) [11]. For PtCu-zyme, substrates first were adsorbed onto Cu atop and ROS was generated on the site of Pt atoms (Fig. 4). Whereas both Fe and Co of FeCo/DIA were active centers for catalyzing ROS generation [11].

  To fully utilize the mitochondrial H₂O₂, the mitochondria-targeting ligand TPP was anchored on PtCu-zyme to obtain mitochondria targeting nanozyme PtCu-TPP (Fig. S15A). The resulting PtCu-TPP was mixed with HA-ISL to prepare PtCu-TPP@MNs for transdermal administration. The grafting ratio of ISL onto HA was too low (~8.0%, Fig. S19C) to influence the binding of the backbone HA to its receptor CD44 [48]. Therefore, we deduced that the residual HA-ISL on the surface of PtCu-TPP drove the nanozymes to get access to the cells through HA-ISL-CD44-mediated endocytosis after application of PtCu-TPP@MNs on the skin, and that TPP subsequently guided PtCu-zyme to specifically localize to mitochondria to boost the generation of ROS. Although microneedles have been proven to be very beneficial in the therapy of skin cancer[49], a combination of alloyed nanozyme and microneedles as therapeutics of CM has not been fully explored. Due to the outstanding catalytic activities of PtCu-zyme, PtCu-TPP@MNs exhibited excellent anti-tumor efficacy in vivo and successfully restrained the tumor growth and metastasis (Fig. 2). Moreover, the matrix HA-ISL suppressed tumor metastasis as well.

  Despite strong FITC signal in the kidney and liver observed in the first 24 h with single administration (Figs. S27A and B in Supporting information), 20-fold higher Pt concentration was detected in the tumor than in the liver and kidney after 7 administrations (Fig. 2I), implying that multiple administrations of PtCu-TPP reorganized the nanozyme distribution in vivo. More excitingly, the retained nanozymes in the tumor continued to catalyze ROS generation, highlighting the sustained anti-tumor efficacy of nanozymes. Cell studies revealed that the excellent anti-tumor efficacy of PtCu-TPP was closely related to an increase in cellular ROS, damage to mitochondria, up-regulated expression of Cyt-c, and phosphorylation of p38 [50].

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Yueru Wei: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Qi Miao: Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Miaomiao Zhang: Visualization, Methodology, Investigation, Formal analysis, Data curation. Wenying Zhang: Methodology, Investigation, Formal analysis, Data curation. Mengxiao Shi: Methodology, Investigation, Data curation. Rui Liu: Methodology, Investigation, Data curation. Jingjing Su: Visualization, Methodology. Pengchao Sun: Writing – review & editing, Writing – original draft, Supervision, Project administration, Conceptualization. Yongxing Zhao: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.

  Acknowledgment

  The present work was support by the National Natural Science Foundation of China (Nos. 82172591 and 81573011).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2025.111164.

  
  
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  Scheme 1  The preparation of PtCu-TPP@MNs and their possible anti-CM mechanisms. PtCu-zyme was first prepared and DSPE-PEG-TPP was later functionalized on the surface of the nanozyme to obtain PtCu-TPP. Subsequently, PtCu-TPP was mixed with HA-ISL to fabricate PtCu-TPP@MNs. Once being applicated on the skin, PtCu-TPP@MNs dissolved, releasing the nanozymes. The residual matrix HA-ISL on the surface of PtCu-TPP facilitated the internalization of nanozymes into the tumor cells via HA-ISL-CD44-mediated endocytosis. Following, the functionalized DSPE-PEG-TPP harnessed the nanozymes to localize at mitochondria to catalyze the generation of ROS. The high level of ROS caused damage to mitochondria, induced the release of cytochrome C (Cyt-c) and the phosphorylation of p38, resulting in the tumor cell apoptosis and necrosis.

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  Figure 1  Preparation and characterization of PtCu-zyme. (A) Schematic illustration of the synthesis process. (B) TEM image. (C) Energy dispersive spectroscopy (EDS) mapping. (D) ac-HAADF-STEM image (the red boxes refer to single atom Pt or Pt subnanoculaters). (E) Pt L3-edge EXAFS Fourier-transform spectra of Pt foil, and PtCu-zyme. EXAFS wavelet transforms spectra of (F) Pt foil, (G) PtO₂ and (H) PtCu-zyme. (I) Time dependent O₂ release in the presence of H₂O₂ and various nanozymes (n = 3). (J) Absorbance of ox-TMB in the presence of H₂O₂ and various nanozymes (n = 3). (K) Ultraviolet–visible (UV–vis) spectra of OPD-ox oxidated by ^•OH with the presence of various nanozymes.

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  Figure 2  Anti-tumor efficacy, metabolism and survival. (A) Overview of the animal experiments. Fluctuations of (B) body weight and (C) tumor volume during the treatment (n = 5). The insert figures in (B) and (C) present the body weight and tumor volume on day 15. (D) Tumor nodules on the surface of lungs at the end of the therapy (n = 4 or 5). (E) Quantification of ROS levels of tumor by Image J. (F) Photo of tumors. (G) Fluorescent images of major organs ex vivo (n = 3). (H) Survival of B16 tumor bearing mice after the listed treatments (n = 6). (I) Content of Pt in liver, kidney and tumor at the end of therapy (n = 2). Data are presented as mean ± standard deviation (SD). ^**P < 0.01, ^****P < 0.0001. ns, non-significant.

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  Figure 3  Anti-tumor efficacy against B16 cells and possible mechanisms. (A) Effects of nanozymes on the viability of B16 cells under simulated tumor microenvironment (acidic conditions, n = 3). (B) Fluctuations of mitochondrial membrane potential after the treatment with various nanozymes (n = 3). (C) Western blot analysis of the cellular expression of Cyt-c and phosphorylated p38 MAPK (p-p38). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Flow cytometric analysis of the cellular ROS. Bio-TEM images of B16 cells with the treatment of PtCu-TPP: (E) Endocytosis; (F) mitochondrial damage.

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  Figure 4  Catalytic mechanisms. (A) Adsorption energy of H₂O₂ at Cu (111) of different models. (B) Reaction processes with Cu atop as the active center. (C) Variations of the free energy at each step.

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