Dual Natural Enzyme-Tuned Biomineralized Nanoflowers for Boosting Cascade Catalytic Antibacterial Therapy and Relieving Inflammation

Dual Natural Enzyme-Tuned Biomineralized Nanoflowers for Boosting Cascade Catalytic Antibacterial Therapy and Relieving Inflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dual Natural Enzyme-Tuned Biomineralized Nanoflowers for Boosting Cascade Catalytic Antibacterial Therapy and Relieving Inflammation Hanyu Zhang, Meng Hao, Xiao Dong, Gemeng Liang, Jinshuo Zou, Yongxin Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5177757/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Dec, 2024 Read the published version in Advanced Composites and Hybrid Materials → Version 1 posted 9 You are reading this latest preprint version Abstract The development of the non-antibiotic agents that clinically safe remains a huge challenge in combating bacterial infections. Herein, we report the construction of dual natural enzymes bromelain (Bro) and glucose oxidase (Gox)-based peroxidase-like nanoflowers through copper phosphate biomineralization for synergistic antibacterial/anti-inflammatory therapy. The hybrid nanoflowers firstly exert the Gox activity for catalyzing the oxidation of glucose to produce H 2 O 2 , which is subsequently converted into highly reactive ·OH through their peroxidase-like activity. This cascade enzymatic activity endows nanoflowers with excellent antibacterial efficiencies, inhibiting the growth of Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) by 99% without the addition of exogenous H 2 O 2 , thus greatly reducing toxic side effects. Meanwhile, the nanoflowers downregulate the secretion of pro-inflammatory cytokines and inhibit the inflammatory response through the release of Bro, significantly accelerate the healing of bacteria-infected wounds. Besides, the nanoflowers utilize the biomolecules and endogenous metal species as building blocks, together with a green and simple synthesis method, guaranteeing their biosafety in practical applications. Overall, the unparalleled biocompatibility and robust antibacterial/anti-inflammatory ability make the nanoflowers a highly promising candidate for the treatment of bacterial infections in future clinical applications. natural enzyme biomineralization nanozyme antibacterial anti-inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Bacterial infections, as one of the leading causes of morbidity and mortality, still poses a serious threat to public health all over the world [ 1 , 2 ]. While the discovery and development of antibiotics offer effective approach to combat pathogens, the abuse of antibiotics has led to increasing bacterial resistance, making the treatment of bacterial infectious diseases more challenging [ 3 , 4 ]. Thus, it is of great significance to develop new bactericidal strategies for solving the problem of antibiotic resistance. Reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 ·− ), singlet oxygen ( 1 O 2 ), and hydroxyl radicals (·OH), have gained considerable attention for their little multi-drug resistance and optimal efficacy in eradicating bacteria [ 5 , 6 ]. Typically, the recently developed chemodynamic therapy (CDT) employs transition metal (e.g., Fe, Cu, Mn, Co, Ni)-based nanomaterials or carbon-based nanomaterials to catalyze the Fenton or Fenton-like reaction with H 2 O 2 to generate highly toxic ·OH, thereby achieving notable antibacterial and bactericidal effects [ 7 – 9 ]. These nanomaterials with enzyme-like activity, known as nanozyme, exhibits superior structural stability, adjustable activity levels and low cost compared to natural enzyme [ 10 , 11 ]. Although promising, the inherent characteristics of these reported nanozymes often limit their further biomedical application and clinical transformation. For example, most of metal oxides-, metal chalcogenides- and carbon-nanozyme often suffer from complex synthetic procedures, indistinct metabolism and difficult degradation in the body [ 12 – 14 ]. Additionally, major shortcomings of metal-organic frameworks (MOFs) nanozyme, like MIL-100 and ZIF-67, are their low drug loading capacity and possible toxicity caused by the extra carriers [ 15 , 16 ]. Furthermore, while the burgeoning supramolecular nanozymes based on the biomolecular self-assembly address the biosafety concerns, they exhibit the poor biostability and bioavailability [ 17 – 19 ]. More importantly, the low physiological H 2 O 2 concentration limits the sufficient ·OH generation by these nanozymes to effectively kill bacteria [ 20 ], while addition of extra H 2 O 2 inevitably leads to some toxic side effects on normal tissues. Therefore, it is urgent to construct biologically stable nanozymes with high ·OH generation efficiency and remarkably antibacterial activity by using bio-derived reagents and endogenous metal species as building blocks. Another challenge for antibacterial therapy is inflammation, which is triggered by the immune system in response to bacterial infections [ 21 , 22 ]. Prolonged inflammation has been reported to cause cell death and tissue damage, and eventually delaying wound healing and promoting excessive scarring [ 23 , 24 ]. Hence, inhibiting proinflammatory response in early stages of wounds could prevent the subsequent excessive and persistent inflammation pattern and foster supportive environment for wound healing [ 25 , 26 ]. However, most antibacterial agents often overlook considering the anti-inflammatory activity. As such, an ideal nanozyme for wound healing requires not only suppressing the growth of bacteria but also regulating abnormal inflammation. Bromelain (Bro), a bioactive proteolytic enzyme obtained from pineapple, has been widely applied for treating high-risk burn wounds due to its anti-inflammatory and immunomodulatory properties [ 27 , 28 ]. Bro has been used clinically as anti-inflammatory agents in rheumatoid arthritis, soft tissue injuries, colonic inflammation, chronic pain and asthma [ 29 , 30 ]. Further combined with the advantages of the safety and low cost, Bro could serve as anti-inflammatory role in the development of antibacterial nanozyme. Given all this, herein we developed the dual enzymes-based hybrid organic-inorganic nanoflowers with intrinsic peroxidase-like activity via biomimetic mineralization for synergistic antibacterial/anti-inflammatory therapy to promote wound healing (Scheme 1 ). The flower-like nanozymes (Cu/Bro/Gox) were constructed via a facile and mild aqueous synthesis using two natural enzymes glucose oxidase (Gox) and Bro as nucleation template and copper ions as the inorganic component, guaranteeing the high level of biocompatibility and biosafety. Gox can catalyze the glucose to produce gluconic acid and H 2 O 2 , which can activate and improve the Cu 2+ -mediated peroxidase activity in the cascade catalytic production of abundant ·OH, leading to significant antibacterial effects. Meanwhile, the Bro can downregulate the expression of inflammatory factors and induce macrophage polarization, which promotes wound healing. Thus, Cu/Bro/Gox can simultaneously achieve bactericidal and anti-inflammatory responses, facilitating the rapid recovery of the infected site while eliminating potential adverse side effects. In summary, our work demonstrated the development of multifunctional antimicrobial materials through utilizing biomineralization strategy and multiple natural biomacromolecules with different functionality as building blocks, presenting the enormous prospect in the clinical treatment of infectious diseases. 2. Experimental Section 2.1 Materials and instruments Glucose oxidase (Gox) and bromelain (Bro) were purchased from Macklin. Copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) was bought by Sinopharm Chemical Reagent Co., Ltd. UV-Vis spectra and X-ray diffraction (XRD) were taken on Hitachi UH5300 UV-Vis-NIR spectrophotometer and Ultima IV, respectively. Fourier transforms infrared (FT-IR) spectra were obtained using a Nicolai iS10. Inductively coupled plasma optical emission spectrometer (ICP-OES) was detected by PerkinElmer 8300. Zeta potentials of nanoflowers were detected by Zetasizer Nano (Malvern Instruments, Malvern, UK). The morphology information of nanostructures was characterized with transmission electron microscopy (TEM) (JSM-2100 Plus) and scanning electron microscope (SEM) (JSM-6390V). X-ray photoelectron spectroscopy (XPS, PHI5000 Versaprobe III) was used to analyze the composition. 2.2 Synthesis of Cu/Bro/Gox nanoflowers Typically, 100 µL of CuSO 4 ⋅5H 2 O aqueous solution (100 mM) was dropwise added into 10 mL PBS (1×, pH = 7.4) containing different concentrations of Bro and Gox, the mixture was incubated at 4 ℃ for 24 h. The blue precipitates were collected by centrifugation at 4 ℃ (8000 rpm, 5 min) and then washed with distilled water for three times to remove the excess compound. 2.3 Encapsulation efficiency of Bro and Gox The encapsulation efficiency (EE) of Bro and Gox were calculated according to the equation: EE (%) = (C 0 - C A )/C 0 × 100%. C 0 and C A are the initial concentration of enzyme and the residual concentration of enzyme in the supernatant, respectively. The enzyme in the supernatant was obtained by the centrifugation at a rotational speed of 8000 rpm for 5 min, and the concentrations of Bro and Gox in the supernatant were measured by high performance liquid chromatography (HPLC) (Agilent 1260 Infinity Ⅱ with GF-250/450 SEC column) and pre-established calibration curve. 2.4 Peroxidase-like activity and Gox activity of Cu/Bro/Gox nanoflowers The peroxidase-mimicking activity of Cu/Bro/Gox was investigated through the oxidation of TMB and degradation of MB. For the oxidation of TMB, 100 µg mL − 1 Cu/Bro/Gox was mixed with 5 mg mL − 1 glucose, then were added into sodium acetate buffer solution (pH 5.0) with 500 µg mL − 1 TMB. Then the absorbance of 3,3′,5,5′-tetramethylbenzidine (TMB) at 652 nm was measured by UV-Vis spectrophotometer. For the degradation of MB, 20 µg mL − 1 Cu/Bro/Gox was mixed with 5 mg mL − 1 glucose, then were added to 10 µg mL − 1 of methylene blue (MB) solution. The generation of ·OH was reflected by the decrease in absorbance of MB at 665 nm. The kinetic assays of Cu/Bro/Gox with H 2 O 2 or glucose as a substrate were carried out. The Cu/Bro/Gox (50 µg mL − 1 ) was mixed with H 2 O 2 with different concentrations (3.75, 7.5, 15, 30, 60, 120 mM) or glucose with different concentrations (4.625, 9.25, 13.875, 18.5, 27.75, 46.25 mM) and TMB (2 mM). The absorbance generated from oxidized TMB at 652 nm of different reactions was monitored at different time points. The Michaelis-Menten constant ( K m ) and maximal velocity ( V max ) were calculated through the fitting data to Michaelis-Menten saturation curve. 2.5 The detection of gluconic acid generation The Cu/Bro/Gox (500 µg mL − 1 ) was dispersed in 5 mL glucose solution (10 mg mL − 1 ), and the pH values of the solution at different times were measured with a pH meter. 2.6 Quantitative measurement of O 2 consumption The Cu/Bro/Gox (500 µg mL − 1 ) was dispersed in 5 mL glucose solution (10 mg mL − 1 ). The change of oxygen level with time was measured by dissolved oxygen meter. 2.7 Bacterial culture and in vitro bacterial experiment Escherichia coli ( E. coli ) and Staphylococcus aureus ( S. aureus ) single colonies were transferred to Luria-Bertani (LB) medium and shaken at 160 rpm for 16 h at 37 ℃. The bacteria were then diluted to 1 × 10 5 CFU mL − 1 with LB medium. Subsequently, the prepared bacterial suspensions were divided into seven groups and treated with (1) PBS, (2) Bro (10.1 µg mL − 1 ), (3) Gox (33.7 µg mL − 1 ), (4) Cu 2+ ([Cu 2+ ] = 6.2 µg mL − 1 ), (5) Cu/Bro ([Cu 2+ ] = 6.2 µg mL − 1 ), (6) Cu/Gox ([Cu 2+ ] = 6.2 µg mL − 1 ) and (7) Cu/Bro/Gox (50 µg mL − 1 ) in the presence of glucose (10 mg mL − 1 ), respectively. For colony count experiment, the bacterial suspensions after the different treatments were spread onto solid medium for subsequent incubation at 37 ℃ for 24 h. For SEM morphology analysis, the bacterial suspensions with different treatments were harvested by centrifugation (5000 rpm for 3 min) and fixed with glutaraldehyde solution (2.5%) at 4 ℃ for 4 h. Then bacteria were dehydrated using sequential ethanol concentrations of 30%, 50%,70%, 80%, 90% and 100%. Finally, the dried bacteria samples were observed using SEM. 2.8 Anti-inflammatory activity in vitro RAW264.7 macrophages (1 mL, approximately 10 5 per well) were seeded into 24-well plates, and then stimulated with lipopolysaccharide (LPS) (100 ng mL − 1 ) for 2 h to establish the cell inflammation model. Subsequently, the RAW264.7 macrophages were incubated with DMEM medium containing Bro (10.1 µg mL − 1 ), Gox (33.7 µg mL − l ), Cu/Bro ([Cu 2+ ] = 6.2 µg mL − 1 ), Cu/Gox ([Cu 2+ ] = 6.2 µg mL − 1 ) and Cu/Bro/Gox (50 µg mL − l ) for 24 h. The control group did not receive any treatment. Finally, the supernatant was collected, and the expression of inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected using an enzyme-linked immunosorbent assay kit. 2.9 Evaluation of wound healing in vivo All the experiments in vivo were ratified by the Committee of Medical College of Qingdao University. The infected wound model was created by slashing the backs of SD rats (6–8 weeks) and injecting them with 100 µL of 1 × 10 8 CFU mL − 1 S. aureus . The S. aureus -infected rats were divided into seven groups randomly and treated with 100 µL (1) PBS, (2) Bro (101 µg mL − 1 ), (3) Gox (337 µg mL − 1 ), (4) Cu 2+ ([Cu 2+ ] = 62 µg mL − 1 ), (5) Cu/Bro ([Cu 2+ ] = 62 µg mL − 1 ), (6) Cu/Gox ([Cu 2+ ] = 62 µg mL − 1 ) and (7) Cu/Bro/Gox (500 µg mL − 1 ) in the presence of glucose (10 mg mL − 1 ), respectively. Each group had at least three rats. The wound size of each rat was photographed and the body weight was recorded every day during the treatment process. The wound area of rats in all treatment groups was also obtained by ImageJ software. After 7 days of post-treatment, the wound tissues in each group were harvested and immersed in sterile PBS at 37 ℃ overnight. The residual live bacteria were extracted by the homogenate counting method and quantified by a plate counting assay. To further assess the anti-infective properties and inflammatory responses of various treatments, the hematoxylin and eosin (H&E) and immunohistochemical staining assays of wound tissue slices were performed. 2.10 Hemolysis test The freshly obtained blood cells from SD rats washed with PBS three times and then incubated with different formulations at room temperature. Negative control and positive control samples were treated with PBS and pure water, respectively. After 2 h, the supernatant was collected by centrifugation and the absorbance at 545 nm was measured to calculate the percentage of hemolysis. 3. Results and Discussion 3.1 Synthesis and characterization of Cu/Bro/Gox The Cu/Bro/Gox was simply prepared in one pot by dropwise adding 100 µL of CuSO 4 ⋅5H 2 O aqueous solution (100 mM) to 10 mL of PBS solution containing Bro and Gox. After the incubation at 4 ℃ for 24 h, the blue precipitates were collected for further characterizations. Figure 1 a- 1 i presented the morphologies of Cu/Bro/Gox synthesized under different concentrations of Bro and Gox. The well-defined flower-like structures with a uniform diameter of ~ 2.5 µm assembled from hundreds of nanoplates were observed at the Gox and Bro concentrations of 0.5 mg mL − 1 and 0.15 mg mL − 1 , respectively. When the concentration of Gox was fixed and the Bro concentration was gradually increased, the morphology of the nanoflowers became incomplete and disappeared completely at a concentration of 0.5 mg mL − 1 , indicating that excessive Bro hindered the formation of nanoflowers structures. Thus, the optimum Bro concentration for the synthesis of Cu/Bro/Gox nanoflowers was 0.15 mg mL − 1 , and the encapsulation efficiency of Bro in Cu/Bro/Gox was calculated to be over 70%. In contrast, when the concentration of Bro was fixed at 0.15 mg mL − 1 , nanoflowers structures of Cu/Bro/Gox exhibited the negligible change with the increasing the Gox concentration (0.5-1.0 mg mL − 1 ), but excessive addition of Gox led to the dramatic decrease of encapsulation efficiency (Table S1 ). Besides, Cu/Gox also exhibited the monodispersed nanoflowers with diameter of ~ 2.5 µm at Gox concentration ranging from 0.1 to 1.5 mg mL − 1 , while the nanoflowers were only obtained for Cu/Bro when Bro concentration was below 0.15 mg mL − 1 . It was probably attributed to the lower molecular weights of Bro, and the number of nucleation sites increased at higher concentration of Bro, resulting in the abundant formation of separate petals instead of nanoflowers. Considering the morphology and encapsulation yield of enzyme, the Cu/Bro/Gox nanoflowers prepared at Bro concentration of 0.15 mg mL − 1 and Gox concentration of 0.5 mg mL − 1 were selected further evaluation and applications. The energy dispersive spectroscopy (EDS) mapping images demonstrated the uniform distributions of C, O, P and Cu elements in Cu/Bro/Gox (Fig. 1 j). The transmission electron microscope (TEM) clearly reveals the uniform flower-like morphology of the Cu/Bro/Gox nanoflowers, as well as the dendritic microstructures at the edges and their relatively narrow size distribution (Fig. S1 ). The zeta potential of Cu/Bro/Gox was determined to be -24.5 mV through dynamic light scattering (DLS). Furthermore, the XRD patterns of Cu/Bro/Gox matched well with those of the crystal planes of Cu 3 (PO 4 ) 2 [ 31 ] (Fig. 1 k), indicating the high crystallinity. Additionally, FT-IR spectra revealed the presence of characteristics peak at 2937 cm − 1 in Cu/Bro/Gox and Cu/Gox (Fig. 1 l), which can be attributed to the absorption peak of methylene originating from Gox. The characteristics peaks belonging to the N-H bond groups of amine compound (3414 cm − 1 ) and sulfur aggregates (1384 cm − 1 ) of Bro were observed in the FT-IR spectra of Cu/Bro/Gox. The X-ray photoelectron spectroscopy (XPS) showed clear C, O, P, N and Cu signals in Cu/Bro/Gox (Fig. 1 m and Fig. S2 ). Specifically, the high resolution XPS of Cu 2p showed two main spin-orbit peaks for Cu 2p3/2 and Cu 2p1/2 at 962 eV and 971 eV, respectively. Thus, the above results confirmed the formation of Cu/Bro/Gox nanoflowers. Moreover, the storage stability of the Cu/Bro/Gox was evaluated through DLS measurement ( Fig. S3 ). No detectable alterations were observed even after 30 days of storage, with the aqueous dispersion remaining clear and stable, demonstrating its long-term stability. 3.2 Verification of cascade catalytic activity of Cu/Bro/Gox The cascade catalytic reaction of Cu/Bro/Gox was evaluated by 3,3′,5,5′-tetramethylbenzidine (TMB), which can be oxidized by ·OH to produce blue-colored oxTMB with a characteristic absorbance at 652 nm. As shown in Fig. 2 a, an obvious increase of the absorbance at 652 nm was observed over incubation time when TMB and Cu/Bro/Gox were incubated with glucose. In contrast, there was no discernible change in the absorbance at 652 nm of TMB in the absence of glucose in the Cu/Bro/Gox solution(Fig. 2 b). Moreover, methylene blue (MB) was also selected as indicator for identifying ·OH. Similarly, Cu/Bro/Gox can degrade MB in the presecnce of glucose, and the degradation process of MB over time is shown in Fig. 2 c. Mere Cu/Bro with glucose or Cu/Bro/Gox without glucose cannot degrade MB ( Fig. S4 ). The catalytic capabilities of Gox in Cu/Bro/Gox and glucose consumption were assessed based on gluconic acid generation and O 2 consumption. The pH of the Cu/Bro/Gox solution kept close to 7.4, and a significant pH decline from 7.4 to 4.4 occurred after treatment with glucose, suggesting the gluconic acid generation(Fig. 2 d). The O 2 content decreased from 7.4 to 0.3 mg L − 1 in 400 s with the existence of Cu/Bro/Gox and glucose, while no noticeable drop in O 2 content was observed in the solution without glucose(Fig. 2 e). The above results confirmed the cascade catalytic reaction of Cu/Bro/Gox, which first exhibited the Gox activity for oxidizing glucose to produce gluconic acid and H 2 O 2 , then efficiently catalyzed H 2 O 2 into abundent ·OH through peroxidase-like activity. The catalytic reaction kinetics of Cu/Bro/Gox were further studied. Typical Michaelis-Menten and Lineweaver-Burk plots were obtained using H 2 O 2 (Fig. 2 f and 2 g) and glucose (Fig. 2 h and 2 i) as substrates. The Michaelis-Menten constant ( K m ) value of Cu/Bro/Gox with H 2 O 2 as substrate was 1.95 mM, which was lower than that of HRP (3.70 mM). The maximum initial velocity ( V m ) of Cu/Bro/Gox (14.1 × 10 − 8 M s − 1 ) was obviously higher than that of HRP ( V m = 8.71 × 10 − 8 M s − 1 ). When glucose was used as the substrate, the K m and the V m value of Cu/Bro/Gox value were calculated to be 1.60 mM and 2.3 × 10 − 8 M s − 1 , respectively. 3.3 Antibacterial properties and anti-inflammatory activity of Cu/Bro/Gox in vitro Encouraged by the remarkable cascade catalytic ability of Cu/Bro/Gox, the antibacterial activities of Cu/Bro/Gox against Gram-negative E. coli and Gram-positive S. aureus were investigated, respectively. As depicted in Fig. 3 a, both of Bro and Cu 2+ demonstrated mild antibacterial effects. Gox can slightly kill bacteria due to the catalytic generation of H 2 O 2 . The bacterial colonies obviously reduced in the presence of Cu/Bro, which is likely attributed to the combined antibacterial activity of Bro and Cu 2+ . However, Cu/Gox exhibited more significant antibacterial effects compared to the Cu/Bro, indicating that Gox powered the cascade catalytic reaction to generate toxic ·OH. More satisfactorily, almost no colonies were observed in Cu/Bro/Gox-treated group. The results of the quantitative analysis on bacterial survival rate further confirmed the robust antibacterial activity of Cu/Bro/Gox (Fig. 3 b and 3 c). After treatment with Cu/Bro/Gox, the bacterial survival rates were only 0.82% for S. aureus and 2.57% for E. coli . To further visualize the effect of anti-bacterial effect of Cu/Bro/Gox, SEM analysis was employed to observe the morphological changes of S. aureus and E. coli after different treatments (Fig. 3 d). The E. coli and S. aureus in control group presented the regular rod and spherical shape with smooth surface and intact cell walls, respectively. The morphology of E. coli and S. aureus showed slight change after incubation with Bro, Gox or Cu 2+ individually, indicating the modest antibacterial effect of Bro, Gox and Cu 2+ . However, the surfaces of bacteria in Cu/Bro, Cu/Gox and Cu/Bro/Gox groups were rough and wrinkled, indicating the destroyed bacterial cellular integrity. Remarkably, most serious surface damage was observed when the bacteria were exposed to the Cu/Bro/Gox, further confirming the high catalytic efficiency of Cu/Bro/Gox in conversion of glucose to highly toxic ·OH, thereby enhancing antibacterial efficacy. In addition, the anti-inflammatory effect of Cu/Bro/Gox was investigated in RAW264.7 macrophages. LPS was utilized to stimulate macrophages to establish the inflammatory cell model. The inflammatory response of RAW264.7 was assessed through detecting the expression levels of TNF-α and IL-6. As shown in Fig. 3 e and 3 f, the secretion of TNF-α and IL-6 was significantly decreased after treatment with Cu/Bro or Cu/Bro/Gox compared to Cu/Gox, most likely due to the anti-inflammatory roles of Bro itself. 3.4 Wound healing-promoting effects of Cu/Bro/Gox in vivo Considering the favorable antibacterial properties and anti-inflammatory effects of Cu/Bro/Gox in vitro, we further evaluated their efficacy in promoting infected wound healing in vivo. S. aureus was injected into the wounds made on the back of rats to establish the bacteria-infected model. The infected rats were randomly divided into seven different groups. As shown in Fig. 4 a, the treatments with Cu/Bro, Cu/Gox and Cu/Bro/Gox groups exhibited a significantly accelerated wound healing rate compared to other groups. In addition, the bacteria around the wounds on day 7 were cultured and counted, the smallest number of colonies was found in the Cu/Bro/Gox group, verified their excellent antibacterial performances ((Fig. 4 b)). In particular, the wound area of rats treated with Cu/Bro/Gox reduced to 1.06 ± 0.5%, demonstrating almost complete healing (Fig. 4 c). Moreover, as shown in Fig. 4 d, during the treatment period, there was no significant decrease in body weight among the rats in all groups. Both qualitative and quantitative results indicate that the hemolysis percentage of Cu/Bro/Gox is below 1%, demonstrating that these nanoflowers do not even cause slight damage to red blood cells (Fig. 4 e). All these important findings validate the overall safety of Cu/Bro/Gox in vivo. Histological analyses using hematoxylin and eosin (H&E) staining were performed to evaluate the wound-healing efficacy. As shown in Fig. 5 a, In the control group, a large number of inflammatory cells and an incomplete skin epidermal layer were observed, with severe tissue edema. Although the Gox and Cu 2+ treatment groups showed some new skin tissue regeneration and a slight reduction in inflammatory cells, there was still capillary formation. In contrast, the Bro, Cu/Bro, and Cu/Gox exhibited gradual appearance of new hair follicles and epidermis with reduced inflammation, but inflammatory cell infiltration was still observable. In the Cu/Bro/Gox, however, only a few inflammatory cells were present, and new hair follicles were visible. Additionally, there was an almost complete epidermal structure with nearly normal thickness. Thus, the prepared Cu/Bro/Gox nanoflowers demonstrate excellent antibacterial activity and can promote the healing and regeneration of skin in wounds infected by bacteria. Subsequently, we further analyzed the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in wound tissue, which are key inflammatory markers, through immunohistochemistry (IHC) assessment. As shown in Fig. 5 b and 5 c, similar to the trends observed with H&E staining, the relevant inflammatory factors are stained brown. The control group, Gox, Cu 2+ , and Cu/Gox group all exhibited widespread TNF-α and IL-6 positive expression cells, indicating severe inflammatory responses. In contrast, the expression of these pro-inflammatory cytokines was somewhat suppressed in the Bro and Cu/Bro, likely due to the presence of Bro. Particularly, for the Cu/Bro/Gox, the expression of pro-inflammatory factors TNF-α and IL-6 was significantly reduced, showing a marked decrease in inflammation and contributing to enhanced wound healing. Using CD68 as a reliable marker for pro-inflammatory macrophages, consistent results were observed across the skin tissue slices of each group (Fig. 5 d). Similarly, the quantitative analysis of immunohistochemistry clearly shows that, compared to the other treatment groups, the Cu/Bro/Gox group exhibited the lowest expression levels of TNF-α and IL-6 or CD68 on day 7, indicating effective alleviation of inflammation (Fig. 5 e- 5 g). Based on these experimental results, we can conclude that treating rat wounds with Cu/Bro/Gox nanoflowers effectively eliminates local pathogens, modulates inflammatory responses, and promotes wound healing. 4. Conclusion In summary, two natural enzymes bromelain (Bro) and glucose oxidase (Gox)-based nanoflowers (Cu/Bro/Gox) with cascade catalytic antibacterial and anti-inflammatory activities were successfully constructed through a facile, economic and green biomimetic mineralization strategy. The presence of Gox catalyzed the conversion of glucose into gluconic acid and H 2 O 2 , which served as substrates for peroxidase-like Cu/Bro/Gox to generate highly toxic ·OH for killing bacteria. The Cu/Bro/Gox exhibited remarkable antibacterial activity both in vivo and in vitro in the presence of glucose, effectively countering the potential toxic effects linked to the direct introduction of high concentrations of H 2 O 2 . Simultaneously, the Cu/Bro/Gox significantly inhibited the expression of related inflammatory factors (TNF-𝛼 and IL-6) and pro-inflammatory activity of macrophage, thereby protecting normal cells from inflammatory reactions and facilitating wound healing. More importantly, the Cu/Bro/Gox exhibited high biocompatibility and low systemic toxicity. Thus, the nanoflowers with antibacterial/anti-inflammatory activity using biomolecules and endogenous metal species as building blocks hold great promise as antibiotic alternatives for further treatment of bacterial infections applications. Declarations Competing interests: The authors declare no competing interests. Funding The authors received financial support for this work from the National Natural Science Foundation of China (82203742). Author Contribution All authors have made contributions to this study work. Hanyu Zhang, Meng Hao, Xiao Dong and Yongxin Li wrote the manuscript. Hanyu Zhang, Meng Hao, Xiao Dong, Gemeng Liang, Jinshuo Zou, Yongxin Li and Peitao Xie all made contributions to experiment, data collection, and data analysis. Xiao Dong gave financial support for this work. All authors read and approved the final manuscript. Data availability statement Data can be made available on request to the corresponding authors. 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Adv Sci 11:2305183 Sun Y, Sun X, Li X, Li W, Li C, Zhou Y, Wang L, Dong B (2021) A versatile nanocomposite based on nanoceria for antibacterial enhancement and protection from aPDT-aggravated inflammation via modulation of macrophage polarization. Biomaterials 268:120614 Li R, Liu K, Huang X, Li D, Ding J, Liu B, Chen X (2022) Bioactive materials promote wound healing through modulation of cell behaviors. Adv Sci 9:2105152 Xu Z, Liang B, Tian J, Wu J (2021) Anti-inflammation biomaterial platforms for chronic wound healing. Biomater Sci 9:4388–4409 Cai Y, Chen K, Liu C, Qu X (2023) Harnessing strategies for enhancing diabetic wound healing from the perspective of spatial inflammation patterns. Bioact Mater 28:243–254 Secor E, Carson W, Cloutier M, Guernsey L, Schramm C, Wu C, Thrall R (2005) Bromelain exerts anti-inflammatory effects in an ovalbumin-induced murine model of allergic airway disease. Cell Immunol 237:68–75 Kwatra B (2019) A review on potential properties and therapeutic applications of bromelain. World J Pharm Pharm Sci 8:488–500 Hasannasab M, Nourmohammadi J, Dehghan M, Ghaee A (2021) Immobilization of bromelain and ZnO nanoparticles on silk fibroin nanofibers as an antibacterial and anti-inflammatory burn dressing. Int J Pharm 610:121227 Kumar V, Mangla B, Javed S, Ahsan W, Kumar P, Garga V, Dureja H (2023) Bromelain: a review of its mechanisms, pharmacological effects and potential applications. Food Funct 14:8101 Jiang W, Wang X, Yang J, Han H, Li Q, Tang J (2018) Lipase-inorganic hybrid nanoflower constructed through biomimetic mineralization: A new support for biodiesel synthesis. J Colloid Inter Sci 514:102–107 Xianga G, Wanga B, Zhang W et al (2024) A Zn-MOF-GOx-based cascade nanoreactor promotes diabetic infected wound healing by NO release and microenvironment regulation. Acta Biomater 182:245–259 Mittal R, Gupta S, Sharma V, Gupta N (2024) Synthesis and antibacterial activities of bromelain stabilized fluorescent copper nanoclusters against E. coli and S. aureus. J Photoch Photobiol A 450:115426 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupportingInformationZHY.docx TOCachm.docx scheme1.jpg Scheme 1Schematic diagram of synthesis of Cu/Bro/Gox and the combined antibacterial and anti-inflammatorytherapy of bacterial infections. Cite Share Download PDF Status: Published Journal Publication published 07 Dec, 2024 Read the published version in Advanced Composites and Hybrid Materials → Version 1 posted Editorial decision: Revision requested 23 Oct, 2024 Reviews received at journal 23 Oct, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviews received at journal 23 Oct, 2024 Reviewers agreed at journal 22 Oct, 2024 Reviewers invited by journal 22 Oct, 2024 Editor assigned by journal 22 Oct, 2024 Submission checks completed at journal 21 Oct, 2024 First submitted to journal 30 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5177757","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":369751416,"identity":"1e183b0f-5586-4e18-b33a-f994f10baef8","order_by":0,"name":"Hanyu Zhang","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Hanyu","middleName":"","lastName":"Zhang","suffix":""},{"id":369751417,"identity":"1779ba30-c283-47f4-9d55-5b9fd9c3c969","order_by":1,"name":"Meng Hao","email":"","orcid":"","institution":"Qingdao Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Meng","middleName":"","lastName":"Hao","suffix":""},{"id":369751418,"identity":"976232f8-86ad-4064-b518-782edc1eda5c","order_by":2,"name":"Xiao Dong","email":"","orcid":"","institution":"Shanghai First People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Dong","suffix":""},{"id":369751419,"identity":"320c780b-0ea1-4d58-b4d6-a1e1447b078d","order_by":3,"name":"Gemeng Liang","email":"","orcid":"","institution":"The University of Adelaide","correspondingAuthor":false,"prefix":"","firstName":"Gemeng","middleName":"","lastName":"Liang","suffix":""},{"id":369751420,"identity":"2ba920ce-c756-454c-90ba-2dee9ab053c4","order_by":4,"name":"Jinshuo Zou","email":"","orcid":"","institution":"The University of Adelaide","correspondingAuthor":false,"prefix":"","firstName":"Jinshuo","middleName":"","lastName":"Zou","suffix":""},{"id":369751421,"identity":"b5701441-2091-4931-badd-b015b9ee580c","order_by":5,"name":"Yongxin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACPmYg8YEBRDIYEKeFDaiYcQZpWoCYmYc0Lew8ZtI2ZdaJDezN2yQYau4Q4zAeY+Occ+mJDTzHyiQYjj0jSovh49y2w4kNEjlmEowNh4nSYnDYEqRF/g3xWgwfM4Jt4SFaC1uxYc+5dOM2nrRii4RjRGjh5z+8TeJHmbVsP/vhjTc+1BChhYGBwwAcO6AIYkggRgMDA/sDqPpRMApGwSgYBTgAAPD8LnWZrU6xAAAAAElFTkSuQmCC","orcid":"","institution":"Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Yongxin","middleName":"","lastName":"Li","suffix":""},{"id":369751422,"identity":"77077a87-1152-4b9c-ac41-0c61f25e18c0","order_by":6,"name":"Peitao Xie","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Peitao","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2024-09-30 04:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5177757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5177757/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s42114-024-01086-z","type":"published","date":"2024-12-07T15:56:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69793762,"identity":"e16aad53-c952-4826-90c7-fb847f590863","added_by":"auto","created_at":"2024-11-25 09:57:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":419385,"visible":true,"origin":"","legend":"\u003cp\u003eSEM images of Cu/Bro/Gox prepared at (a) 0.15 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro and 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e of Gox, (b) 0.3 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro and 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e Gox, (c) 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro and 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e Gox, (d) 0.15 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro and 0.75 mg mL\u003csup\u003e-1\u003c/sup\u003e of Gox, (e) 0.15 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro and 1.0 mg mL\u003csup\u003e-1\u003c/sup\u003e of Gox. SEM images of Cu/Gox prepared at (f) 0.1 mg mL\u003csup\u003e-1\u003c/sup\u003e of Gox, (g) 1.5 mg mL\u003csup\u003e-1\u003c/sup\u003e of Gox. SEM images of Cu/Bro prepared at (h) 0.15 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro, (i) 0.3 mg mL\u003csup\u003e-1\u003c/sup\u003e of Bro. (j) SEM image coupled with EDS elemental mapping of Cu/Bro/Gox. (k) XRD pattern of Cu/Bro/Gox, Cu/Bro and Cu/Gox. (l) FT-IR spectra of Cu/Bro/Gox, Cu/Bro and Cu/Gox. (m) High-resolution Cu 2p XPS of Cu/Bro/Gox.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/af27e398594c338e5b9eab7f.jpg"},{"id":69791607,"identity":"bb096d8f-0faf-48f5-ac49-cbe7c3c1ccca","added_by":"auto","created_at":"2024-11-25 09:49:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109290,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Time-dependent absorbance changes of TMB in the presence of Cu/Bro/Gox and glucose. (b) Time-dependent absorbance changes of TMB in the presence of only Cu/Bro/Gox. (c) The degradation of MB with time in the presence of Cu/Bro/Gox and glucose. (d) pH value changes of Cu/Bro/Gox solution with or without glucose. (e) O\u003csub\u003e2\u003c/sub\u003e concentration variations of Cu/Bro/Gox solution with or without glucose. (f) Michaelis-Menten kinetics and (g) Lineweaver-Burk plots of Cu/Bro/Gox with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e substrate (n = 3). (h) Michaelis-Menten kinetics and (i) Lineweaver-Burk plots of Cu/Bro/Gox with glucose substrate (n = 3).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/68d7b198e5bf7ac8a7411bd0.jpg"},{"id":69791339,"identity":"b9aff03a-c9da-49c8-a0ef-3f899381fdaa","added_by":"auto","created_at":"2024-11-25 09:41:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":250875,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photographs of the colonies formed by \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e after indicated treatments. Survival rate of (b) \u003cem\u003eS. aureus\u003c/em\u003e and (c)\u003cem\u003e E. coli \u003c/em\u003eafter different treatments. (d) SEM images of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e after exposure to indicated formulations. Expression of inflammatory cytokines (e) TNF-α and (f) IL-6 in LPS-pretreated macrophage RAW264.7 cells in different groups. Data were presented as mean ± SD and statistical difference was determined by Student’s t test. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/bf40415dcd07f89b8c2e815f.jpg"},{"id":69791332,"identity":"702df0ed-8f19-4077-9ec4-8596a7cb3dfe","added_by":"auto","created_at":"2024-11-25 09:41:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101247,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Photographs of \u003cem\u003eS. aureus\u003c/em\u003e-infected wounds of rats with different treatments on day 0, 1, 3, 5, and 7. (b) Photographs of bacterial colonies from tissues of different treatment groups. (c) Wound area of different treatment groups after 7 days. (d) Body weights of rats after different treatments. (e) Data were presented as mean ± SD and statistical difference was determined by Student’s t test. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/60885c0c5d23c9b3a35e1275.jpg"},{"id":69791335,"identity":"9278430f-3560-40e8-957a-a1e25293bd40","added_by":"auto","created_at":"2024-11-25 09:41:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":110990,"visible":true,"origin":"","legend":"\u003cp\u003eHistological and immunohistochemical evaluation of skin tissues after 7-day treatments. (a)H\u0026amp;E staining images of the infected skins harvested from different rats on seventh day post-treatment. Immunohistochemical staining for (b) TNF-α and (c) IL-6 in the skin tissues of the infected areas. (d)Immunostaining images of CD68 expressed in the phagocytic macrophages. The corresponding quantification of positive cells with (e) TNF-α, (f) IL-6, (g) CD68.Data were presented as mean ± SD and statistical difference was determined by Student’s t test. *\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05; **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/5e5a10476f10866eac4ecae4.jpg"},{"id":70964576,"identity":"b53b788b-243b-4dc7-83b0-ee2f7d5a43a0","added_by":"auto","created_at":"2024-12-09 16:08:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1617863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/c8c42f9a-94d5-491d-8c9e-e9a35d098f3e.pdf"},{"id":69791338,"identity":"de6c76f7-9ce9-48df-a305-301d86924bdd","added_by":"auto","created_at":"2024-11-25 09:41:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":588299,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformationZHY.docx","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/3ba1b9445d26e23344625f54.docx"},{"id":69791609,"identity":"49f394bd-10ce-481c-9d20-2e1ed7094a08","added_by":"auto","created_at":"2024-11-25 09:49:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":668486,"visible":true,"origin":"","legend":"","description":"","filename":"TOCachm.docx","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/ed6631a1a89e2221f0017846.docx"},{"id":69791606,"identity":"4d24a6fa-751c-4af5-9769-83d9d40cc9a4","added_by":"auto","created_at":"2024-11-25 09:49:24","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":72668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003eSchematic diagram of synthesis of Cu/Bro/Gox and the combined antibacterial and anti-inflammatorytherapy of bacterial infections.\u003c/p\u003e","description":"","filename":"scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5177757/v1/b885de93cc2ae598aa582a1d.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dual Natural Enzyme-Tuned Biomineralized Nanoflowers for Boosting Cascade Catalytic Antibacterial Therapy and Relieving Inflammation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBacterial infections, as one of the leading causes of morbidity and mortality, still poses a serious threat to public health all over the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While the discovery and development of antibiotics offer effective approach to combat pathogens, the abuse of antibiotics has led to increasing bacterial resistance, making the treatment of bacterial infectious diseases more challenging [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, it is of great significance to develop new bactericidal strategies for solving the problem of antibiotic resistance.\u003c/p\u003e \u003cp\u003eReactive oxygen species (ROS), including hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026middot;\u0026minus;\u003c/sup\u003e), singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e), and hydroxyl radicals (\u0026middot;OH), have gained considerable attention for their little multi-drug resistance and optimal efficacy in eradicating bacteria [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Typically, the recently developed chemodynamic therapy (CDT) employs transition metal (e.g., Fe, Cu, Mn, Co, Ni)-based nanomaterials or carbon-based nanomaterials to catalyze the Fenton or Fenton-like reaction with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate highly toxic \u0026middot;OH, thereby achieving notable antibacterial and bactericidal effects [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These nanomaterials with enzyme-like activity, known as nanozyme, exhibits superior structural stability, adjustable activity levels and low cost compared to natural enzyme [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although promising, the inherent characteristics of these reported nanozymes often limit their further biomedical application and clinical transformation. For example, most of metal oxides-, metal chalcogenides- and carbon-nanozyme often suffer from complex synthetic procedures, indistinct metabolism and difficult degradation in the body [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, major shortcomings of metal-organic frameworks (MOFs) nanozyme, like MIL-100 and ZIF-67, are their low drug loading capacity and possible toxicity caused by the extra carriers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, while the burgeoning supramolecular nanozymes based on the biomolecular self-assembly address the biosafety concerns, they exhibit the poor biostability and bioavailability [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. More importantly, the low physiological H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration limits the sufficient \u0026middot;OH generation by these nanozymes to effectively kill bacteria [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], while addition of extra H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e inevitably leads to some toxic side effects on normal tissues. Therefore, it is urgent to construct biologically stable nanozymes with high \u0026middot;OH generation efficiency and remarkably antibacterial activity by using bio-derived reagents and endogenous metal species as building blocks.\u003c/p\u003e \u003cp\u003eAnother challenge for antibacterial therapy is inflammation, which is triggered by the immune system in response to bacterial infections [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Prolonged inflammation has been reported to cause cell death and tissue damage, and eventually delaying wound healing and promoting excessive scarring [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Hence, inhibiting proinflammatory response in early stages of wounds could prevent the subsequent excessive and persistent inflammation pattern and foster supportive environment for wound healing [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, most antibacterial agents often overlook considering the anti-inflammatory activity. As such, an ideal nanozyme for wound healing requires not only suppressing the growth of bacteria but also regulating abnormal inflammation. Bromelain (Bro), a bioactive proteolytic enzyme obtained from pineapple, has been widely applied for treating high-risk burn wounds due to its anti-inflammatory and immunomodulatory properties [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Bro has been used clinically as anti-inflammatory agents in rheumatoid arthritis, soft tissue injuries, colonic inflammation, chronic pain and asthma [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Further combined with the advantages of the safety and low cost, Bro could serve as anti-inflammatory role in the development of antibacterial nanozyme.\u003c/p\u003e \u003cp\u003eGiven all this, herein we developed the dual enzymes-based hybrid organic-inorganic nanoflowers with intrinsic peroxidase-like activity via biomimetic mineralization for synergistic antibacterial/anti-inflammatory therapy to promote wound healing (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The flower-like nanozymes (Cu/Bro/Gox) were constructed via a facile and mild aqueous synthesis using two natural enzymes glucose oxidase (Gox) and Bro as nucleation template and copper ions as the inorganic component, guaranteeing the high level of biocompatibility and biosafety. Gox can catalyze the glucose to produce gluconic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which can activate and improve the Cu\u003csup\u003e2+\u003c/sup\u003e-mediated peroxidase activity in the cascade catalytic production of abundant \u0026middot;OH, leading to significant antibacterial effects. Meanwhile, the Bro can downregulate the expression of inflammatory factors and induce macrophage polarization, which promotes wound healing. Thus, Cu/Bro/Gox can simultaneously achieve bactericidal and anti-inflammatory responses, facilitating the rapid recovery of the infected site while eliminating potential adverse side effects. In summary, our work demonstrated the development of multifunctional antimicrobial materials through utilizing biomineralization strategy and multiple natural biomacromolecules with different functionality as building blocks, presenting the enormous prospect in the clinical treatment of infectious diseases.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental Section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and instruments\u003c/h2\u003e \u003cp\u003eGlucose oxidase (Gox) and bromelain (Bro) were purchased from Macklin. Copper (II) sulfate pentahydrate (CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO) was bought by Sinopharm Chemical Reagent Co., Ltd. UV-Vis spectra and X-ray diffraction (XRD) were taken on Hitachi UH5300 UV-Vis-NIR spectrophotometer and Ultima IV, respectively. Fourier transforms infrared (FT-IR) spectra were obtained using a Nicolai iS10. Inductively coupled plasma optical emission spectrometer (ICP-OES) was detected by PerkinElmer 8300. Zeta potentials of nanoflowers were detected by Zetasizer Nano (Malvern Instruments, Malvern, UK). The morphology information of nanostructures was characterized with transmission electron microscopy (TEM) (JSM-2100 Plus) and scanning electron microscope (SEM) (JSM-6390V). X-ray photoelectron spectroscopy (XPS, PHI5000 Versaprobe III) was used to analyze the composition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis of Cu/Bro/Gox nanoflowers\u003c/h2\u003e \u003cp\u003eTypically, 100 \u0026micro;L of CuSO\u003csub\u003e4\u003c/sub\u003e\u0026sdot;5H\u003csub\u003e2\u003c/sub\u003eO aqueous solution (100 mM) was dropwise added into 10 mL PBS (1\u0026times;, pH\u0026thinsp;=\u0026thinsp;7.4) containing different concentrations of Bro and Gox, the mixture was incubated at 4 ℃ for 24 h. The blue precipitates were collected by centrifugation at 4 ℃ (8000 rpm, 5 min) and then washed with distilled water for three times to remove the excess compound.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Encapsulation efficiency of Bro and Gox\u003c/h2\u003e \u003cp\u003eThe encapsulation efficiency (EE) of Bro and Gox were calculated according to the equation: EE (%) = (C\u003csub\u003e0\u003c/sub\u003e - C\u003csub\u003eA\u003c/sub\u003e)/C\u003csub\u003e0\u003c/sub\u003e \u0026times; 100%. C\u003csub\u003e0\u003c/sub\u003e and C\u003csub\u003eA\u003c/sub\u003e are the initial concentration of enzyme and the residual concentration of enzyme in the supernatant, respectively. The enzyme in the supernatant was obtained by the centrifugation at a rotational speed of 8000 rpm for 5 min, and the concentrations of Bro and Gox in the supernatant were measured by high performance liquid chromatography (HPLC) (Agilent 1260 Infinity Ⅱ with GF-250/450 SEC column) and pre-established calibration curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Peroxidase-like activity and Gox activity of Cu/Bro/Gox nanoflowers\u003c/h2\u003e \u003cp\u003eThe peroxidase-mimicking activity of Cu/Bro/Gox was investigated through the oxidation of TMB and degradation of MB. For the oxidation of TMB, 100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Cu/Bro/Gox was mixed with 5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose, then were added into sodium acetate buffer solution (pH 5.0) with 500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e TMB. Then the absorbance of 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB) at 652 nm was measured by UV-Vis spectrophotometer. For the degradation of MB, 20 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Cu/Bro/Gox was mixed with 5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose, then were added to 10 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of methylene blue (MB) solution. The generation of \u0026middot;OH was reflected by the decrease in absorbance of MB at 665 nm. The kinetic assays of Cu/Bro/Gox with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e or glucose as a substrate were carried out. The Cu/Bro/Gox (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was mixed with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e with different concentrations (3.75, 7.5, 15, 30, 60, 120 mM) or glucose with different concentrations (4.625, 9.25, 13.875, 18.5, 27.75, 46.25 mM) and TMB (2 mM). The absorbance generated from oxidized TMB at 652 nm of different reactions was monitored at different time points. The Michaelis-Menten constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) and maximal velocity (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003emax\u003c/em\u003e\u003c/sub\u003e) were calculated through the fitting data to Michaelis-Menten saturation curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 The detection of gluconic acid generation\u003c/h2\u003e \u003cp\u003eThe Cu/Bro/Gox (500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was dispersed in 5 mL glucose solution (10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and the pH values of the solution at different times were measured with a pH meter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Quantitative measurement of O\u003csub\u003e2\u003c/sub\u003e consumption\u003c/h2\u003e \u003cp\u003eThe Cu/Bro/Gox (500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was dispersed in 5 mL glucose solution (10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The change of oxygen level with time was measured by dissolved oxygen meter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Bacterial culture and in vitro bacterial experiment\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) single colonies were transferred to Luria-Bertani (LB) medium and shaken at 160 rpm for 16 h at 37 ℃. The bacteria were then diluted to 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with LB medium. Subsequently, the prepared bacterial suspensions were divided into seven groups and treated with (1) PBS, (2) Bro (10.1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (3) Gox (33.7 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (4) Cu\u003csup\u003e2+\u003c/sup\u003e ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;6.2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (5) Cu/Bro ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;6.2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (6) Cu/Gox ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;6.2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and (7) Cu/Bro/Gox (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the presence of glucose (10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), respectively. For colony count experiment, the bacterial suspensions after the different treatments were spread onto solid medium for subsequent incubation at 37 ℃ for 24 h. For SEM morphology analysis, the bacterial suspensions with different treatments were harvested by centrifugation (5000 rpm for 3 min) and fixed with glutaraldehyde solution (2.5%) at 4 ℃ for 4 h. Then bacteria were dehydrated using sequential ethanol concentrations of 30%, 50%,70%, 80%, 90% and 100%. Finally, the dried bacteria samples were observed using SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Anti-inflammatory activity in vitro\u003c/h2\u003e \u003cp\u003eRAW264.7 macrophages (1 mL, approximately 10\u003csup\u003e5\u003c/sup\u003e per well) were seeded into 24-well plates, and then stimulated with lipopolysaccharide (LPS) (100 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 2 h to establish the cell inflammation model. Subsequently, the RAW264.7 macrophages were incubated with DMEM medium containing Bro (10.1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Gox (33.7 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;l\u003c/sup\u003e), Cu/Bro ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;6.2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), Cu/Gox ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;6.2 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and Cu/Bro/Gox (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;l\u003c/sup\u003e) for 24 h. The control group did not receive any treatment. Finally, the supernatant was collected, and the expression of inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected using an enzyme-linked immunosorbent assay kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Evaluation of wound healing in vivo\u003c/h2\u003e \u003cp\u003e All the experiments in vivo were ratified by the Committee of Medical College of Qingdao University. The infected wound model was created by slashing the backs of SD rats (6\u0026ndash;8 weeks) and injecting them with 100 \u0026micro;L of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003cem\u003eS. aureus\u003c/em\u003e. The \u003cem\u003eS. aureus\u003c/em\u003e-infected rats were divided into seven groups randomly and treated with 100 \u0026micro;L (1) PBS, (2) Bro (101 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (3) Gox (337 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ), (4) Cu\u003csup\u003e2+\u003c/sup\u003e ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;62 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (5) Cu/Bro ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;62 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), (6) Cu/Gox ([Cu\u003csup\u003e2+\u003c/sup\u003e]\u0026thinsp;=\u0026thinsp;62 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and (7) Cu/Bro/Gox (500 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the presence of glucose (10 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), respectively. Each group had at least three rats. The wound size of each rat was photographed and the body weight was recorded every day during the treatment process. The wound area of rats in all treatment groups was also obtained by ImageJ software. After 7 days of post-treatment, the wound tissues in each group were harvested and immersed in sterile PBS at 37 ℃ overnight. The residual live bacteria were extracted by the homogenate counting method and quantified by a plate counting assay. To further assess the anti-infective properties and inflammatory responses of various treatments, the hematoxylin and eosin (H\u0026amp;E) and immunohistochemical staining assays of wound tissue slices were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Hemolysis test\u003c/h2\u003e \u003cp\u003eThe freshly obtained blood cells from SD rats washed with PBS three times and then incubated with different formulations at room temperature. Negative control and positive control samples were treated with PBS and pure water, respectively. After 2 h, the supernatant was collected by centrifugation and the absorbance at 545 nm was measured to calculate the percentage of hemolysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Synthesis and characterization of Cu/Bro/Gox\u003c/h2\u003e \u003cp\u003eThe Cu/Bro/Gox was simply prepared in one pot by dropwise adding 100 \u0026micro;L of CuSO\u003csub\u003e4\u003c/sub\u003e\u0026sdot;5H\u003csub\u003e2\u003c/sub\u003eO aqueous solution (100 mM) to 10 mL of PBS solution containing Bro and Gox. After the incubation at 4 ℃ for 24 h, the blue precipitates were collected for further characterizations. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei presented the morphologies of Cu/Bro/Gox synthesized under different concentrations of Bro and Gox. The well-defined flower-like structures with a uniform diameter of ~\u0026thinsp;2.5 \u0026micro;m assembled from hundreds of nanoplates were observed at the Gox and Bro concentrations of 0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.15 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. When the concentration of Gox was fixed and the Bro concentration was gradually increased, the morphology of the nanoflowers became incomplete and disappeared completely at a concentration of 0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, indicating that excessive Bro hindered the formation of nanoflowers structures. Thus, the optimum Bro concentration for the synthesis of Cu/Bro/Gox nanoflowers was 0.15 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the encapsulation efficiency of Bro in Cu/Bro/Gox was calculated to be over 70%. In contrast, when the concentration of Bro was fixed at 0.15 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, nanoflowers structures of Cu/Bro/Gox exhibited the negligible change with the increasing the Gox concentration (0.5-1.0 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), but excessive addition of Gox led to the dramatic decrease of encapsulation efficiency (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Besides, Cu/Gox also exhibited the monodispersed nanoflowers with diameter of ~\u0026thinsp;2.5 \u0026micro;m at Gox concentration ranging from 0.1 to 1.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while the nanoflowers were only obtained for Cu/Bro when Bro concentration was below 0.15 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. It was probably attributed to the lower molecular weights of Bro, and the number of nucleation sites increased at higher concentration of Bro, resulting in the abundant formation of separate petals instead of nanoflowers. Considering the morphology and encapsulation yield of enzyme, the Cu/Bro/Gox nanoflowers prepared at Bro concentration of 0.15 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and Gox concentration of 0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were selected further evaluation and applications. The energy dispersive spectroscopy (EDS) mapping images demonstrated the uniform distributions of C, O, P and Cu elements in Cu/Bro/Gox (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). The transmission electron microscope (TEM) clearly reveals the uniform flower-like morphology of the Cu/Bro/Gox nanoflowers, as well as the dendritic microstructures at the edges and their relatively narrow size distribution \u003cb\u003e(Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The zeta potential of Cu/Bro/Gox was determined to be -24.5 mV through dynamic light scattering (DLS). Furthermore, the XRD patterns of Cu/Bro/Gox matched well with those of the crystal planes of Cu\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek), indicating the high crystallinity. Additionally, FT-IR spectra revealed the presence of characteristics peak at 2937 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in Cu/Bro/Gox and Cu/Gox (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el), which can be attributed to the absorption peak of methylene originating from Gox. The characteristics peaks belonging to the N-H bond groups of amine compound (3414 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and sulfur aggregates (1384 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of Bro were observed in the FT-IR spectra of Cu/Bro/Gox. The X-ray photoelectron spectroscopy (XPS) showed clear C, O, P, N and Cu signals in Cu/Bro/Gox (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003em and \u003cb\u003eFig. S2\u003c/b\u003e). Specifically, the high resolution XPS of Cu 2p showed two main spin-orbit peaks for Cu 2p3/2 and Cu 2p1/2 at 962 eV and 971 eV, respectively. Thus, the above results confirmed the formation of Cu/Bro/Gox nanoflowers. Moreover, the storage stability of the Cu/Bro/Gox was evaluated through DLS measurement (\u003cb\u003eFig. S3\u003c/b\u003e). No detectable alterations were observed even after 30 days of storage, with the aqueous dispersion remaining clear and stable, demonstrating its long-term stability.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Verification of cascade catalytic activity of Cu/Bro/Gox\u003c/h2\u003e \u003cp\u003eThe cascade catalytic reaction of Cu/Bro/Gox was evaluated by 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB), which can be oxidized by \u0026middot;OH to produce blue-colored oxTMB with a characteristic absorbance at 652 nm. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, an obvious increase of the absorbance at 652 nm was observed over incubation time when TMB and Cu/Bro/Gox were incubated with glucose. In contrast, there was no discernible change in the absorbance at 652 nm of TMB in the absence of glucose in the Cu/Bro/Gox solution(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, methylene blue (MB) was also selected as indicator for identifying \u0026middot;OH. Similarly, Cu/Bro/Gox can degrade MB in the presecnce of glucose, and the degradation process of MB over time is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. Mere Cu/Bro with glucose or Cu/Bro/Gox without glucose cannot degrade MB (\u003cb\u003eFig. S4\u003c/b\u003e). The catalytic capabilities of Gox in Cu/Bro/Gox and glucose consumption were assessed based on gluconic acid generation and O\u003csub\u003e2\u003c/sub\u003e consumption. The pH of the Cu/Bro/Gox solution kept close to 7.4, and a significant pH decline from 7.4 to 4.4 occurred after treatment with glucose, suggesting the gluconic acid generation(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The O\u003csub\u003e2\u003c/sub\u003e content decreased from 7.4 to 0.3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in 400 s with the existence of Cu/Bro/Gox and glucose, while no noticeable drop in O\u003csub\u003e2\u003c/sub\u003e content was observed in the solution without glucose(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The above results confirmed the cascade catalytic reaction of Cu/Bro/Gox, which first exhibited the Gox activity for oxidizing glucose to produce gluconic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, then efficiently catalyzed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e into abundent \u0026middot;OH through peroxidase-like activity. The catalytic reaction kinetics of Cu/Bro/Gox were further studied. Typical Michaelis-Menten and Lineweaver-Burk plots were obtained using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg) and glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei) as substrates. The Michaelis-Menten constant (\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) value of Cu/Bro/Gox with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as substrate was 1.95 mM, which was lower than that of HRP (3.70 mM). The maximum initial velocity (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) of Cu/Bro/Gox (14.1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was obviously higher than that of HRP (\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e = 8.71 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). When glucose was used as the substrate, the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e and the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e value of Cu/Bro/Gox value were calculated to be 1.60 mM and 2.3 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e M s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Antibacterial properties and anti-inflammatory activity of Cu/Bro/Gox in vitro\u003c/h2\u003e \u003cp\u003eEncouraged by the remarkable cascade catalytic ability of Cu/Bro/Gox, the antibacterial activities of Cu/Bro/Gox against Gram-negative \u003cem\u003eE. coli\u003c/em\u003e and Gram-positive \u003cem\u003eS. aureus\u003c/em\u003e were investigated, respectively. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, both of Bro and Cu\u003csup\u003e2+\u003c/sup\u003e demonstrated mild antibacterial effects. Gox can slightly kill bacteria due to the catalytic generation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The bacterial colonies obviously reduced in the presence of Cu/Bro, which is likely attributed to the combined antibacterial activity of Bro and Cu\u003csup\u003e2+\u003c/sup\u003e. However, Cu/Gox exhibited more significant antibacterial effects compared to the Cu/Bro, indicating that Gox powered the cascade catalytic reaction to generate toxic \u0026middot;OH. More satisfactorily, almost no colonies were observed in Cu/Bro/Gox-treated group. The results of the quantitative analysis on bacterial survival rate further confirmed the robust antibacterial activity of Cu/Bro/Gox (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). After treatment with Cu/Bro/Gox, the bacterial survival rates were only 0.82% for \u003cem\u003eS. aureus\u003c/em\u003e and 2.57% for \u003cem\u003eE. coli\u003c/em\u003e. To further visualize the effect of anti-bacterial effect of Cu/Bro/Gox, SEM analysis was employed to observe the morphological changes of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e after different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e in control group presented the regular rod and spherical shape with smooth surface and intact cell walls, respectively. The morphology of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e showed slight change after incubation with Bro, Gox or Cu\u003csup\u003e2+\u003c/sup\u003e individually, indicating the modest antibacterial effect of Bro, Gox and Cu\u003csup\u003e2+\u003c/sup\u003e. However, the surfaces of bacteria in Cu/Bro, Cu/Gox and Cu/Bro/Gox groups were rough and wrinkled, indicating the destroyed bacterial cellular integrity. Remarkably, most serious surface damage was observed when the bacteria were exposed to the Cu/Bro/Gox, further confirming the high catalytic efficiency of Cu/Bro/Gox in conversion of glucose to highly toxic \u0026middot;OH, thereby enhancing antibacterial efficacy. In addition, the anti-inflammatory effect of Cu/Bro/Gox was investigated in RAW264.7 macrophages. LPS was utilized to stimulate macrophages to establish the inflammatory cell model. The inflammatory response of RAW264.7 was assessed through detecting the expression levels of TNF-α and IL-6. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, the secretion of TNF-α and IL-6 was significantly decreased after treatment with Cu/Bro or Cu/Bro/Gox compared to Cu/Gox, most likely due to the anti-inflammatory roles of Bro itself.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Wound healing-promoting effects of Cu/Bro/Gox in vivo\u003c/h2\u003e \u003cp\u003eConsidering the favorable antibacterial properties and anti-inflammatory effects of Cu/Bro/Gox in vitro, we further evaluated their efficacy in promoting infected wound healing in vivo. \u003cem\u003eS. aureus\u003c/em\u003e was injected into the wounds made on the back of rats to establish the bacteria-infected model. The infected rats were randomly divided into seven different groups. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the treatments with Cu/Bro, Cu/Gox and Cu/Bro/Gox groups exhibited a significantly accelerated wound healing rate compared to other groups. In addition, the bacteria around the wounds on day 7 were cultured and counted, the smallest number of colonies was found in the Cu/Bro/Gox group, verified their excellent antibacterial performances ((Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb)). In particular, the wound area of rats treated with Cu/Bro/Gox reduced to 1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%, demonstrating almost complete healing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eMoreover, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed, during the treatment period, there was no significant decrease in body weight among the rats in all groups. Both qualitative and quantitative results indicate that the hemolysis percentage of Cu/Bro/Gox is below 1%, demonstrating that these nanoflowers do not even cause slight damage to red blood cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). All these important findings validate the overall safety of Cu/Bro/Gox in vivo.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistological analyses using hematoxylin and eosin (H\u0026amp;E) staining were performed to evaluate the wound-healing efficacy. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, In the control group, a large number of inflammatory cells and an incomplete skin epidermal layer were observed, with severe tissue edema. Although the Gox and Cu\u003csup\u003e2+\u003c/sup\u003e treatment groups showed some new skin tissue regeneration and a slight reduction in inflammatory cells, there was still capillary formation. In contrast, the Bro, Cu/Bro, and Cu/Gox exhibited gradual appearance of new hair follicles and epidermis with reduced inflammation, but inflammatory cell infiltration was still observable. In the Cu/Bro/Gox, however, only a few inflammatory cells were present, and new hair follicles were visible. Additionally, there was an almost complete epidermal structure with nearly normal thickness. Thus, the prepared Cu/Bro/Gox nanoflowers demonstrate excellent antibacterial activity and can promote the healing and regeneration of skin in wounds infected by bacteria.\u003c/p\u003e \u003cp\u003eSubsequently, we further analyzed the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in wound tissue, which are key inflammatory markers, through immunohistochemistry (IHC) assessment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, similar to the trends observed with H\u0026amp;E staining, the relevant inflammatory factors are stained brown. The control group, Gox, Cu\u003csup\u003e2+\u003c/sup\u003e, and Cu/Gox group all exhibited widespread TNF-α and IL-6 positive expression cells, indicating severe inflammatory responses. In contrast, the expression of these pro-inflammatory cytokines was somewhat suppressed in the Bro and Cu/Bro, likely due to the presence of Bro. Particularly, for the Cu/Bro/Gox, the expression of pro-inflammatory factors TNF-α and IL-6 was significantly reduced, showing a marked decrease in inflammation and contributing to enhanced wound healing. Using CD68 as a reliable marker for pro-inflammatory macrophages, consistent results were observed across the skin tissue slices of each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Similarly, the quantitative analysis of immunohistochemistry clearly shows that, compared to the other treatment groups, the Cu/Bro/Gox group exhibited the lowest expression levels of TNF-α and IL-6 or CD68 on day 7, indicating effective alleviation of inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eBased on these experimental results, we can conclude that treating rat wounds with Cu/Bro/Gox nanoflowers effectively eliminates local pathogens, modulates inflammatory responses, and promotes wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn summary, two natural enzymes bromelain (Bro) and glucose oxidase (Gox)-based nanoflowers (Cu/Bro/Gox) with cascade catalytic antibacterial and anti-inflammatory activities were successfully constructed through a facile, economic and green biomimetic mineralization strategy. The presence of Gox catalyzed the conversion of glucose into gluconic acid and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which served as substrates for peroxidase-like Cu/Bro/Gox to generate highly toxic \u0026middot;OH for killing bacteria. The Cu/Bro/Gox exhibited remarkable antibacterial activity both in vivo and in vitro in the presence of glucose, effectively countering the potential toxic effects linked to the direct introduction of high concentrations of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Simultaneously, the Cu/Bro/Gox significantly inhibited the expression of related inflammatory factors (TNF-\u0026#120572; and IL-6) and pro-inflammatory activity of macrophage, thereby protecting normal cells from inflammatory reactions and facilitating wound healing. More importantly, the Cu/Bro/Gox exhibited high biocompatibility and low systemic toxicity. Thus, the nanoflowers with antibacterial/anti-inflammatory activity using biomolecules and endogenous metal species as building blocks hold great promise as antibiotic alternatives for further treatment of bacterial infections applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting interests:\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe authors received financial support for this work from the National Natural Science Foundation of China (82203742).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors have made contributions to this study work. Hanyu Zhang, Meng Hao, Xiao Dong and Yongxin Li wrote the manuscript. Hanyu Zhang, Meng Hao, Xiao Dong, Gemeng Liang, Jinshuo Zou, Yongxin Li and Peitao Xie all made contributions to experiment, data collection, and data analysis. Xiao Dong gave financial support for this work. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eData can be made available on request to the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhao F, Su Y, Wang J, Romanova S, DiMaio DJ, Xie J, Zhao S (2023) A highly efficacious electrical biofilm treatment system for combating chronic wound bacterial Infections. Adv Mater 35:2208069\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurray CJL, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G, Gray A, Han C, Bisignano C, Rao P, Wool E et al (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. 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J Photoch Photobiol A 450:115426\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"advanced-composites-and-hybrid-materials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"achm","sideBox":"Learn more about [Advanced Composites and Hybrid Materials](https://link.springer.com/journal/42114)","snPcode":"42114","submissionUrl":"https://submission.nature.com/new-submission/42114/3","title":"Advanced Composites and Hybrid Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"natural enzyme, biomineralization, nanozyme, antibacterial, anti-inflammation","lastPublishedDoi":"10.21203/rs.3.rs-5177757/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5177757/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of the non-antibiotic agents that clinically safe remains a huge challenge in combating bacterial infections. Herein, we report the construction of dual natural enzymes bromelain (Bro) and glucose oxidase (Gox)-based peroxidase-like nanoflowers through copper phosphate biomineralization for synergistic antibacterial/anti-inflammatory therapy. The hybrid nanoflowers firstly exert the Gox activity for catalyzing the oxidation of glucose to produce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, which is subsequently converted into highly reactive \u0026middot;OH through their peroxidase-like activity. This cascade enzymatic activity endows nanoflowers with excellent antibacterial efficiencies, inhibiting the growth of \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (\u003cem\u003eS. aureus\u003c/em\u003e) by 99% without the addition of exogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, thus greatly reducing toxic side effects. Meanwhile, the nanoflowers downregulate the secretion of pro-inflammatory cytokines and inhibit the inflammatory response through the release of Bro, significantly accelerate the healing of bacteria-infected wounds. Besides, the nanoflowers utilize the biomolecules and endogenous metal species as building blocks, together with a green and simple synthesis method, guaranteeing their biosafety in practical applications. Overall, the unparalleled biocompatibility and robust antibacterial/anti-inflammatory ability make the nanoflowers a highly promising candidate for the treatment of bacterial infections in future clinical applications.\u003c/p\u003e","manuscriptTitle":"Dual Natural Enzyme-Tuned Biomineralized Nanoflowers for Boosting Cascade Catalytic Antibacterial Therapy and Relieving Inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-25 09:41:19","doi":"10.21203/rs.3.rs-5177757/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-24T02:15:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-24T01:21:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158974164654437768981536179838747437204","date":"2024-10-24T01:19:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-24T01:06:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44790569194737383816389612862688047949","date":"2024-10-22T05:58:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-22T05:39:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-22T05:24:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-22T02:44:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Advanced Composites and Hybrid Materials","date":"2024-09-30T04:23:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"advanced-composites-and-hybrid-materials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"achm","sideBox":"Learn more about [Advanced Composites and Hybrid Materials](https://link.springer.com/journal/42114)","snPcode":"42114","submissionUrl":"https://submission.nature.com/new-submission/42114/3","title":"Advanced Composites and Hybrid Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ddba883-e3dd-42f1-8a52-af018395fce8","owner":[],"postedDate":"November 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T15:58:52+00:00","versionOfRecord":{"articleIdentity":"rs-5177757","link":"https://doi.org/10.1007/s42114-024-01086-z","journal":{"identity":"advanced-composites-and-hybrid-materials","isVorOnly":false,"title":"Advanced Composites and Hybrid Materials"},"publishedOn":"2024-12-07 15:56:56","publishedOnDateReadable":"December 7th, 2024"},"versionCreatedAt":"2024-11-25 09:41:19","video":"","vorDoi":"10.1007/s42114-024-01086-z","vorDoiUrl":"https://doi.org/10.1007/s42114-024-01086-z","workflowStages":[]},"version":"v1","identity":"rs-5177757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5177757","identity":"rs-5177757","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}