Chitosan‐Stabilized PtAu Nanoparticles with Multienzyme‐Like Activity for Mixed Bacteria Infection Wound Healing and Insights into Its Antibacterial Mechanism

Traditional antibacterial agents are often observed to be ineffective because bacteria evolved to strains with greater antibiotic resistance. Here, vigorous chitosan‐stabilized PtAu nanoparticles (CSPA) with multienzyme‐like activity are successfully fabricated, which serve an effective artificial nanozyme to enhance antibacterial activity for mixed bacterial infection wound treatment. Ultrasmall size CSPA exhibits excellent hydrophilicity and biocompatibility, possesses strong oxidase‐ and peroxidase‐like activity generating a substantial amount of ROS ( O2−$$ {\mathrm{O}}_{2}^{-}$$ , 1O2, ·OH) to cause oxidative damage to bacteria, also demonstrates nicotinamide adenine dinucleotide dehydrogenase‐like activity disrupting the bacterial respiratory chains, and subsequently impedes adenosine triphosphate production. CSPA exhibits favorable broad‐spectrum antibacterial activity at very low concentrations, prevents bacterial resistance, and completely inhibits bacterial biofilm formation. Antibacterial Mechanism of CSPA by the transcriptomics is further revealed that CSPA can induce bacterial oxidative stress, hinder bacterial energy metabolism, and disrupt the synthesis and function of bacterial cell walls and cell membranes. In vivo, CSPA inhibits the mixed bacterial population at the wound site and promotes wound healing in rats. This study introduces a novel antibacterial approach, providing important insight into the antibacterial mechanism of CSPA nanozymes and promoting the advancement of nanocatalytic materials in biomedical applications.


Introduction
Bacterial infections have long posed a threat to human life and health by causing numerous grave ailments, including purulent wound infections, [1] sepsis, [2] pneumonia, [3] and infectious meningitis. [4]Wound infection results from the invasion of pathogenic bacteria into damaged tissue.If the number of bacteria in the tissue exceeds 10 5 CFU g À1 , [5] it can cause severe suppuration, which may lead to inflammatory oxidative stress reactions, and even result in systemic infection. [6]Over time, due to the overuse of antibiotics, the bacteria's sensitivity to existing antibiotics has reduced and developed resistance.Acquired resistance is deemed a significant factor in the development of bacterial drug resistance, [7] and it even results in the emergence of multiresistant "superbugs," [8] thereby worsening the issue of bacterial infections.Nowadays, bacterial resistance has emerged as a worldwide public health concern, [9] particularly with respect to pathogenic bacteria's resistance.Thus, scientists must explore novel antibacterial substances to counter the bacteria's continual mutations and evolutions toward resistance.
Organisms infected with bacteria can employ biocatalysts to restrict bacterial growth.For instance, the innate oxidase and peroxidase in the organism catalyzes a range of substrates, producing reactive oxygen species (ROS) to counter bacterial invasion. [10]However, the properties of proteins in most natural enzymes lead to low stability and potential immunogenicity, [11] which restricts their use in antibacterialfields.Nanozymes as a kind of nanomaterials with Traditional antibacterial agents are often observed to be ineffective because bacteria evolved to strains with greater antibiotic resistance.Here, vigorous chitosan-stabilized PtAu nanoparticles (CSPA) with multienzyme-like activity are successfully fabricated, which serve an effective artificial nanozyme to enhance antibacterial activity for mixed bacterial infection wound treatment.Ultrasmall size CSPA exhibits excellent hydrophilicity and biocompatibility, possesses strong oxidase-and peroxidase-like activity generating a substantial amount of ROS (O À 2 , 1 O 2 , •OH) to cause oxidative damage to bacteria, also demonstrates nicotinamide adenine dinucleotide dehydrogenase-like activity disrupting the bacterial respiratory chains, and subsequently impedes adenosine triphosphate production.CSPA exhibits favorable broad-spectrum antibacterial activity at very low concentrations, prevents bacterial resistance, and completely inhibits bacterial biofilm formation.Antibacterial Mechanism of CSPA by the transcriptomics is further revealed that CSPA can induce bacterial oxidative stress, hinder bacterial energy metabolism, and disrupt the synthesis and function of bacterial cell walls and cell membranes.In vivo, CSPA inhibits the mixed bacterial population at the wound site and promotes wound healing in rats.This study introduces a novel antibacterial approach, providing important insight into the antibacterial mechanism of CSPA nanozymes and promoting the advancement of nanocatalytic materials in biomedical applications.enzymatic activity possess benefits afforded by both natural enzymes and nanomaterials, resulting in characteristics such as easy storage, adjustable size, and high catalytic activity.The inception of nanozymes can be traced back to 2004 when Manea et al. reported the utilization of ligand functionalized gold nanoparticles (NPs) loaded with Zn 2þ for catalyzing the cleavage of phosphate diesters. [12]Later on, Gao et al. discovered that Fe 3 O 4 magnetic NPs displayed intrinsic enzyme mimicking activity akin to natural peroxidase, [13] marking a new epoch for nanozymes.Currently, the implementation of nanozymes in areas such as biosensing, identifying diseases, mitigating inflammation, impeding tumorous growth, and preventing bacterial infections [14] is progressively becoming more sophisticated.Particularly, nanozymes, as antibacterial agents, are less likely than traditional antibiotics to cause genetic mutations in bacteria at the genetic level, and possess broad-spectrum and long-lasting antibacterial properties. [15]Development of novel antibacterial agents based on nanozymes provides an effective means to tackle the issue of bacterial infections.However, their antibacterial concentrations are often high, necessitating increased drug dosages to effectively inhibit bacterial growth and reproduction. [16]dditionally, they may prove to be toxic to mammalian cells, posing a challenge to their clinical application.Thus, successful implementation of nanozyme antibacterial strategies demands heightened efforts.
Platinum (Pt)-based nanomaterials show outstanding antibacterial activities and catalytic capabilities. [17]The antibacterial rate of the prepared Pt-based nanozyme hydrogels increased significantly with the increase of platinum loading. [18]Platinum trimetallic nanoalloys demonstrated multiple enzymatic activities, making them effective in assessing the antioxidant capacity of natural substances. [19]In addition, bimetal catalytic technology has become a new catalyst for breaking the boundary of the linear scaling relations of individual metal centers.By exploiting the synergistic effect of the two metals, composite metal nanomaterials can improve antibacterial efficacy. [20]An alloy of platinum with other metals, such as silver, copper, and zinc, may be able to achieve these properties.However, it is important to note that these metals have certain safety issues.Silver was an early metallic material used for antibacterial purposes, but it has been found to be environmentally unfriendly, toxic, and unstable. [21]oreover, research has shown that the toxicity of silver NPs may be greater than that of the silver ions themselves. [22]In recent years, gold has been widely used in antimicrobial applications due to its high antipathogens efficacy. [23]Gold NPs are surface-inert and exhibit excellent stability in complex biological environments, making them superior to silver in terms of their good biosafety and stability. [24]Additionally, a recently reported gold nanocluster has high biosafety and excellent antimicrobial activity against a wide range of drug-resistant bacteria. [25]ompounds containing gold can replace Zn 2þ cofactors at active sites and irreversibly suppress the efficacy of drug-resistant enzymes, resulting in reduced bacterial resistance. [26]Gold NPs have a high surface charge density, thereby improving their interaction with bacterial cell membranes and promoting bacterial eradication. [27]Nevertheless, single metal particles have limited antibacterial performance.The collaboration of Pt and Au has a synergistic effect that can enhance nanozymes' catalytic activity. [28]Although nanozymes have made remarkable progress in antibacterial activity, their low specific binding capacity to bacteria and short lifetime severely limit the clinical application.Chitosan (CS), a natural positively charged biopolymer with good biocompatibility, excellent degradability, and nontoxicity, can be utilized for anti-infection, hemostasis, and wound healing promotion. [29]CS possesses the ability to bind with metal, and the amino groups present in its molecules provide high affinity binding sites that can coordinate with metals, thereby leading to metal adsorption on CS. [30] This further stabilizes the morphology of metal NPs and reduces the harmful side effects caused by metal ions. [31]Accordingly, CS can be used to stabilize artificial nanozymes for achieving high-efficiency, low-cost, eco-friendly antibacterial agents, ultimately for sterilization.
In this work, CS-stabilized PtAu (CSPA) nanoparticles (NPs) were designed and synthesized for antibacterial research.The stability of the NPs was improved while also enhancing the antibacterial properties of the CS ligands.CSPA exhibited robust activity of oxidase-like (OXD-like), peroxidase-like (POD-like), and nicotinamide adenine dinucleotide (NADH) dehydrogenaselike (NDH-like) enzymes.In its capacity as an OXD-and PODlike enzyme, CSPA was capable of catalyzing the production of substantial ROS from substrates including oxygen and hydrogen peroxide.As a NDH-like enzyme, it facilitated the degradation of NADH.
In vitro antibacterial experiments demonstrated that CSPA displayed a range of enzyme activities, generated ROS to destroy bacterial cell structures, consumed intracellular NADH leading respiratory chain disruption, reduced adenosine triphosphate (ATP) production, had a lower antibacterial concentration, exhibited superior antibacterial properties in comparison with CS, and was able to resist bacterial resistance.Preliminary biological findings suggested that CSPA prompts bacterial production of a significant amount of ROS, leading to a decrease in ATP content and consumption of glutathione (GSH).Additionally, the antibacterial molecular mechanism of CSPA had been uncovered through transcriptomics, analyzing differentially expressed genes (DEGs).Finally, an in vivo wound infection model had been developed to investigate CSPA's antibacterial effectiveness and its ability to promote wound healing (Scheme 1).CSPA demonstrated remarkable therapeutic effects, offering insights for the advancement of nanozyme antimicrobial agents.S1, Supporting Information), the addition of CS resulted in a homogeneous and well-dispersed product, whereas the product without CS almost settled at the bottom.The morphology and size were observed by the transmission electron microscopy (TEM).The TEM image (Figure 1A) depicted the shape of CSPA as the circular particles with distinct lattice showing in the high-resolution TEM (HRTEM).Figure 1B shows the size distribution of CSPA NPs, and the average diameter of these particles was 1.98 AE 0.41 nm.In addition, Figure S2, Supporting Information, shows that CSPA was positively charged at the surface with a potential of 31.18AE 1.60 mV.The X-ray diffraction (XRD) pattern in Figure 1C showed multiple peaks.A broad peak at 2θ = 24.4°wasobserved, which was attributed to the carbon phase resulting from the binding effect of metal and CS.Additionally, the diffraction peaks at 39.02°, 45.039°, 66.22°, and 79.205°correspond to the (200), (220), and (311) crystal planes of standard cards Pt (PDF 87-0636) and Au (PDF 04-0784), respectively.The four diffraction peaks were located between Pt and Au and appeared symmetrical.Reduction in the simultaneous presence of both metals could lead to the creation of alloying NPs. [20]The diffraction angle changed, with the diffraction peak of CSPA shifting left with respect to Pt and right with respect to Au.This resulted in the diffraction peaks being located between the two, suggesting the formation of an alloying structure of the two metals. [32]The element mapping pattern (Figure 1D) also showed that Pt and Au are evenly distributed throughout the NPs, providing further evidence of the alloy structure.And the presence of N, representing CS, was mainly concentrated on CSPA, suggesting that CS was distributed on the surfaces of the NPs to play the role of modification and stabilization.The elemental composition of CSPA was examined using X-ray photoelectron spectroscopy (XPS).Figure S3A, Supporting Information, presents elemental peaks visible at 285.3, 532.41, 400.76, 70.78, and 83.64 eV, which stand for C 1s, O 1s, N 1s, Pt 4f, and Au 4f, respectively, with atomic percentages of C, O, N, Pt, and Au amounting to 37.59%, 58.2%, 3.49%, 0.34%, and 0.39%.Further peak-fitting analyses were performed for each element.The XPS spectra with high-resolution of C 1s (Figure S3B, Supporting Information) displayed split peaks at 287.90, 286.16, and 284.69 eV, which, respectively, indicate the existence of C─N, C─O, and C─C bonds. [33]And the highresolution O 1s spectra (Figure S3C, Supporting Information) showed split peaks at 536.48, 533.00, and 532.03 eV, which corresponded to the signals of C─O─C, C=O, and C─O. [34]he high-resolution N 1s spectra (Figure S3D, Supporting Information) comprised split peaks at 401.65 and 399.62 eV, which associate with C=N─C and N─(C) n . [35]The highresolution XPS spectra of Pt 4f (Figure 1E) were obtained by fitting two pairs of characteristic double peaks, corresponding to Pt 4f 5/2 and Pt 4f 7/2 .One pair of peaks at 75.57 and 71.428 eV corresponds to Pt 2þ 4f 5/2 and Pt 2þ 4f 7/2 , and the other pair of peaks at 74.14 and 70.64 eV corresponds to Pt 0 4f 5/2 and Pt 0 4f 7/2 , indicating complete reduction of H 2 PtCl 6 and predominantly the presence of Pt 0 and Pt 2þ forms in the prepared NPs. [36]The high-resolution XPS spectra of Au 4f (Figure 1F) yielded a pairs of characteristic double peaks.The peaks loaded at 87.28 and 83.67 eV correspond to the singlet state of gold (Au 0 4f 5/2 and Au 0 4f 7/2 ). [37]It was evident that Au 0 is the primary form of the element present in the NPs.The surface functional groups of this NPs were characterized via Fourier-transform infrared (FTIR) spectroscopy.Figure 1C illustrates the FTIR spectra of CS and CSPA.Owing CS, the absorption peak of CS at 3435 cm À1 was attributed to υ O─H , the band at 2921 cm À1 was due to υ C─H , the characteristic peak at 1646 cm À1 was owing to υ C=O , and the absorption peak at 610 cm À1 corresponds to δ NH .Comparing with the raw material, the FTIR spectra of CSPA indicated that some absorption peaks remain, while the υ C─H peak disappeared.The spectra after interaction with CS were prone to changes in the amino band, [38] including a weakening of the intensity of the υ C=O peak and vanishing of the δ NH peak due to the high affinity of metal ions for amino groups.Above all, it was indicated that CS interacts with the surface of the metal NPs and played a role in the process of nanoparticle formation.

Multienzyme-Like Activity of CSPA
2.2.1.OXD-Like Activity of CSPA 3,3',5,5'-Tetramethylbenzidine (TMB) was selected as the reaction substrate to evaluate the OXD-like activity of CSPA.When oxidized, it generates a blue oxidized TMB (TMB ox ) with a typical absorption peak at 652 nm.This can be used to evaluate the catalytic performance of the nanozyme.As shown in Figure 2B, the TMB þ CSPA group showed a distinct absorption peak, while there is no absorption peak-only TMB.The absorption peak of the TMB þ CSPA group reduced after N 2 blowing.When the oxygen content was low, the quantity of TMB ox decreased considerably, suggesting that oxygen dissolved in the solution played a role in the oxidation process of TMB.So CSPA possessed OXD-like properties, which catalyzed oxygen to produce ROS, and hence oxidized TMB and performed color rendering.The UV absorption of CSP, CSA, and CSPA (Figure S4, Supporting Information) after interaction with TMB indicated that CSPA catalyzed the production of more ROS and exhibited better OXD-like activity.Electron spin resonance (ESR) was utilized to identify the types of ROS produced in the reaction system, in order to investigate the reaction mechanism of CSPA, which exhibited OXD activity.As depicted in Figure 2D,E, it was evident to see the 1:1:1:1 signal of •O À 2 and the 1:1:1 signal of 1 O 2 .Ordinarily, O 2 acted as an electron acceptor generally produced •O 2 À when acted upon by oxidase.
However, in acidic systems, the signal of [39] The signal of 1 O 2 increased significantly over time, whereas •O 2 À signal enhancement was not significant, likely due to the reaction producing 1 O 2 .The specific reaction was: This demonstrated that CSPA possessed strong OXD-like activity and could catalyze O 2 to yield abundant ROS.Furthermore, the steady-state kinetics were investigated by varying the concentration of TMB.As illustrated in Figure S6A,B, Supporting Information, the reaction rate was affected by alterations in TMB concentration.The Michaelis-Menten equation was utilized to calculate K m , which equaled 0.5391 mM, while V max was 0.6188 Â 10 À6 M s À1 .As shown in (Table S1, Supporting Information), the OXD-like catalytic kinetic constants of CSPA were compared with those of other reported OXD simulations.The lower K m value of CSPA indicates the stronger enzyme affinity toward the substrate.

POD-Like Activity of CSPA
The POD-like activity of CSPA also utilizes TMB as a substrate for the color reaction.The reaction buffer system was prepurged with N 2 to remove dissolved oxygen and reduce the impact of this reaction.As depicted in Figure 2C, the TMB group and TMB þ H 2 O 2 group showed no absorption peaks.The TMB þ CSPA group exhibited a low absorption peak, likely due to the presence of a small quantity of oxygen in the reaction system.Conversely, the TMB þ H 2 O 2 þ CSPA group demonstrated a robust absorption peak, suggesting that CSPA possesses POD-like activity that catalyzes the production of abundant ROS from H 2 O 2 to oxidize TMB.The absorption of CSP, CSA, and CSPA after interaction with TMB and H 2 O 2 was further examined and assessed for POD-like activity as shown in Figure S4, Supporting Information.The results indicated that CSPA exhibited the highest activity.Similarly, the ROS types produced in the reaction system were identified by using 5,5-Dimethyl-1pyrroline-N-oxide (DMPO) as the capture agent.The ESR spectrum (Figure 2F) indicated that the addition of H 2 O 2 to the reaction system produced a 1:2:2:1 signal, which increased over time, suggesting that CSPA catalyzes H 2 O 2 to generate •OH, thus verifying its POD-like activity.By altering the reaction substrate concentrations of TMB and H 2 O 2 , we analyzed the steady-state kinetics.The reaction rate was illustrated in Figure S6C-F, Supporting Information and varies depending on the concentrations of TMB and H 2 O 2 .The performance of CSPA in POD-like activity was also evaluated using Michaelis-Menten equation calculations.The K m was calculated to be 0.2763 mM for TMB substrate and 0.6984 mM for H 2 O 2 substrate.Meanwhile, the V max was calculated to be 0.3256 Â10 À6 M s À1 for TMB substrate and 0.3656 Â 10 À6 M s À1 for H 2 O 2 substrate.A lower K m value corresponded to stronger enzyme affinity toward the substrate, as depicted in (Table S2, Supporting Information).CSPA, as a nanozyme, had a lower K m than some reported nanozymes, revealing that it displays superior substrate TMB or H 2 O 2 affinity and higher enzyme activity.Furthermore, as a POD-like enzyme, CSPA exhibited a lower K m compared to natural horseradish peroxidase and a higher V max .This suggested that it can achieve superior catalytic performance compared to natural enzymes and has significant potential for application.

NADH Dehydrogenase-Like Activity of CSPA
NADH is an important cofactor in the regulation of cellular metabolism and energy production, participating in many biological redox reactions and maintaining intracellular redox balance. [40]Under the action of NADH dehydrogenase, NADH donates electrons and undergoes oxidative dehydrogenation to form to NAD þ .When there is insufficient NADH in the cell, ATP synthesis is hindered, leading to insufficient energy supply. [41]In order to investigate the NDH-like activity of CSPA, the degradation of NADH and the generation of NAD þ catalyzed by CSPA were measured in an acetic acid sodium acetate buffer system.NADH displays distinctive absorption at 340 and 260 nm wavelengths, whereas its oxidative dehydrogenation derivative NAD þ displays distinctive absorption at 260 nm.Compared to the group without CSPA addition, Figure 2G shows significant changes in the UV spectrum of NADH after the addition of CSPA.Specifically, the absorption peak at 340 nm decreases, while the absorption peak at 260 nm increases.Additionally, Figure S7A,B, Supporting Information, also reflects the changes in the absorption peaks of the two groups at different time points.The two typical absorption peaks of NADH alone remained constant for 30 min, indicating that NADH did not undergo selfoxidative dehydrogenation in the buffer system, and there was no alteration in the overall amount of NADH present.Upon adding CSPA, the absorption peak at 340 nm gradually decreased with time, while the absorption peak at 260 nm gradually increased.This indicated that CSPA can catalyze the conversion of NADH to NAD þ with NDH-like activity.The comparison of NADH degradation by CSP, CSA, and CSPA is shown in Figure S8A, Supporting Information.The UV absorption of the NADH treated by CSPA was the most severely reduced, indicating its superior NDH-like activity compared to CSP and CSA.The quantitative graph (Figure S8A, Supporting Information) further showed this finding.Moreover, the degradation of NADH increased with the concentration of CSPA and NADH in the reaction system, as illustrated in Figure 2H,I, indicating a concentration-dependent rate of oxidative dehydrogenation of NADH.
Namely, CSPA possessed OXD-and POD-like activity and NDH-like multienzyme activity (Figure 2A), which are essential for ROS-mediated oxidative damage to bacteria and obstruction of ATP synthesis to achieve antibacterial.

In Vitro Antibacterial Activities of CSPA
16b,42] The present study highlighted that CSPA displayed excellent OXDand POD-like activities, facilitating the production of hazardous ROS using O 2 and H 2 O 2 in the environment.Bacteria produce endogenous H 2 O 2 as a result of their metabolism. [43]This serves as a substrate for enzyme catalysis, which subsequently generates ROS and effectively enhances their antibacterial effects.Furthermore, it was anticipated that the NDH-like activity of CSPA would result in the depletion of NADH in bacteria, leading to disturbance of their redox equilibrium, influencing their respiratory chain and inhibiting metabolism, consequently producing antibacterial effects.Accordingly, the study utilized the dilution coating plate method to examine the impact of varying concentrations of CS and CSPA on the viability of Escherichia coli and Staphylococcus aureus.The antibacterial efficacy of these substances was determined by calculating colony counts across all groups after a 24 h incubation period.As illustrated in Figure 3A-D, with an increase in the concentration of CSPA, there was a gradual decrease in the number of colonies on the agar plate.When the treatment concentration is 2.0 μg mL À1 for E. coli, no bacterial growth was observed in the agar culture dish, whereas almost no colonies are found in the agar culture dish for S. aureus with a treatment concentration of 20.0 μg mL À1 .However, the number of colonies after treatment with CS at the same concentration was significantly more than that after treatment with CSPA, indicating that that CSPA has superior antibacterial activity to CS. Above all, this suggested that CSPA possessed greater bactericidal abilities and exhibited more potent antibacterial effects against E. coli at the same concentration.In addition, this study compared the impact of CSP, CSA, and CSPA on bacterial survival rates at the same concentration.As shown in Figure S9, Supporting Information, CSP and CSA had almost no effect on E. coli and could kill a small amount of S. aureus.In contrast, the survival rate of both E. coli and S. aureus decreased to less than 10% when exposed to CSPA, demonstrating its superior antibacterial activity compared to the other two.The time-dependent bacteriostasis was carried out at different concentrations of CSPA by measuring the OD 600 (Figure S10, Supporting Information).The OD 600 values of the E. coli and S. aureus were very small at the initial 6 and 8 h with or without the addition of CSPA, respectively, indicating slow bacterial growth before 6 and 8 h.After the corresponding time point, the OD 600 values were significantly lower with CSPA at different concentrations compared with the CS, indicating that CSPA has significant inhibitory effects on the two selected bacterial species.The OD 600 value decreased with the increase in the concentration of CSPA, indicating that CSPA was bacteriostatic in a concentration-dependent manner.Furthermore, CSPA possessed different inhibition capabilities toward E. coli and S. aureus with the complete inhibition concentration of 4 and 10 μg mL À1 , respectively.And the results of the microbroth method for different bacteria were consistent with the calculating colony counts.In addition, different treatments of E. coli and S. aureus were tested for live and dead bacteria using Syto9/ propidium iodide (PI) live/dead dyes, and observed under an inverted fluorescence microscope, where green fluorescence represented live bacteria and red fluorescence represented dead bacteria.As shown in Figure 3E,F, the control group and CS group exhibited predominantly green fluorescence, indicating bacterial survival.Contrastingly, the bacteria exhibited pronounced red fluorescence, indicating massive bacterial mortality.These findings corroborated previous antibacterial tests, underscoring the remarkable antibacterial efficacy of CSPA.
Antibiotics are highly susceptible to bacterial resistance with long-term use, so there is an urgent need to develop antibacterial agents with low or no resistance.The influence of CSPA on bacterial-induced resistance was assessed by comparing the resistance curve of CSPA with antibiotics regarding their ability to counteract resistance development in bacteria over extended exposure.As demonstrated in Figure 3G, using vancomycin hydrochloride (Van) as the control, there was no notable resistance of S. aureus to CSPA detected during 16 passages of treatment, and the fold change in minimum bactericidal concentration (MBC) remained constant.In contrast, S. aureus demonstrated an increase in resistance to Van during the third passage, and this resistance sharply escalated after the 11th passage.Furthermore, we conducted a study on the antibacterial activity of CSPA against induced vancomycin-resistant S. aureus.Figure 3H displays that CSPA preserved its original antibacterial activity against drug-resistant bacteria.The results indicated that it is not particularly easy for S. aureus to evolve resistance to the CSPA.It might be because the antimicrobial mechanism of CSPA was through electrostatic contact and then entered the bacteria to stimulate intracellular ROS production.The interacting behavior of CSPA through electrostatic binding rather than specifically on bacterial subunits might not induce the bacterial acquisition antimicrobial resistance evolution.
Biofilms shield bacteria from environmental factors, heightening their resilience and susceptibility to chronic infections and drug-resistant strains.Improving drug permeability and restraining biofilms formation were essential antibacterial approaches.In order to study the ability of CSPA to resist biofilms formation, the formation of bacterial biofilms was observed by crystal violet (CV) staining after 48 h following treatment with varying concentrations of CSPA and CS.As illustrated in Figure S11, Supporting Information, and Figure 3I, an increase in concentration of CSPA significantly inhibited S. aureus biofilms formation, resulting in a decrease in purple color.At a concentration of 150 μg mL À1 , the biofilms content in the CSPA group was merely 2.72%.Conversely, the corresponding biofilms content after CS treatment was as high as 80.61%.Furthermore, the 3D fluorescence images (Figure 3J) revealed that a thick layer of biofilm developed in the control group.In the CS group, the thickness of the biofilm reduced, and some dead bacteria were observed.After the CSPA treatment, the biofilm almost disappeared, with only a few dead bacteria remaining.This observation indicated that CSPA had a more substantial capability to completely inhibit biofilms formation, making it more effective in preventing bacterial resistance.

Antibacterial Mechanism of CSPA by the Cellular Levels and Transcriptome Sequence Analysis
For the further development and clinical application of CSPA, the research on its antibacterial mechanism is urgent.In this case, we have systematically studied its antibacterial mechanism at the cellular level and transcriptome sequence analysis.
Cellular levels of the bactericidal mechanism were investigated.To visually observe the bacterial damage by the synthesized nanozyme, the morphology and membrane integrity of the bacteria were observed via scanning electron microscopy (SEM) and TEM.As depicted in Figure 4A, the surface of both E. coli and S. aureus in the control group appeared flat, and their cell structures exhibited normal.After being treated with CS, the surface of E. coli appeared rough, but they still maintain their typical bacterial morphology.Meanwhile, S. aureus retained their regular morphology.After exposure to CSPA, E. coli experienced significant harm, including potential cell lysis and loss of membrane integrity.A similar phenomenon was observed by TEM (Figure 4B), where CSPA NPs adhered to the surface of E. coli, resulting in cell cavities, lysis, and the outflow of a significant amount of cell contents.A substantial quantity of NPs affixed to the surface of S. aureus, but no obvious bacterial damage was observed.S. aureus had numerous NPs attached to its surface, leading to slight cavitation and exudation of contents.Furthermore, certain S. aureus exhibited a midcell septum, impeding their mitotic process and preventing cell division.As a result, the proliferation process was abnormal, and CSPA detrimentally impacted cell division.
ROS possess potent oxidizing properties capable of inflicting irreversible damage to bacterial cell walls, membranes, DNA, proteins, and other components, ultimately leading to bacterial death.The synthesized CSPA in this study demonstrated competent OXD-and POD-like activity, facilitating the production of abundant ROS.The NPs attached to the surface of bacteria and had the ability to convert the endogenous H 2 O 2 of the bacteria into highly toxic •OH resulting in improved antibacterial effects.To verify this, 2',7'-dichlorofluorescein diacetate (DCFH-DA) was employed to detect the production of ROS in bacteria.As illustrated in Figure 5A, in comparison with the control group, the bacteria subjected to CSPA treatment exhibited a considerable emission of green fluorescence, suggesting an overproduction of ROS.Further investigation into the levels of GSH content in bacteria treated with CSPA was conducted.GSH played a crucial role in the biological system as an antioxidant, which can prevent the damage caused by ROS and was critical in maintaining bacterial redox homeostasis.Figure 5B,C illustrates that the increase in CSPA concentration corresponded with an increase in GSH loss, resulting in the loss of bacterial antioxidant protection and oxidative damage.ATP supplies energy for different biological activities in organisms.As illustrated in Figure 5D,E, following CSPA's impact on bacteria, their internal ATP level steadily declined as the dosage concentration increases.This suggested that CSPA exhibited NDH-like activity, transforming NADH within bacteria into NAD þ , which impacted the functioning of the bacterial respiratory chain and obstructed ATP synthesis.The aforementioned findings indicated that CSPA eradicated bacteria by creating ROS, consuming GSH to cause redox imbalance and disrupting their energy supply.
To further investigate the underlying mechanism of the excellent antibacterial activity by CSPA, we analyzed its impact on bacterial transcriptome through RNA sequencing technology.The expression levels of genes were compared between the control group and the CSPA group based on transcriptome sequencing results.Statistically significant DEGs were chosen for analysis with a statistical analysis criterion of p-value <0.05 and |log2 (fold change) |>0.As shown in Figure 6A, compared to the control group, the CSPA-treated E. coli samples revealed 718 DEGs, of which 361 were downregulated and 357 were upregulated.The DEGs between the control and CSPA groups of E. coli were further analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) databases.The adjusted p-values (p adj ) were used to screen the GO terms and KEGG pathways.The top 20 KEGG pathways for upregulation and downregulation were mapped in Figure 6B,C, correspondingly.GO analysis was categorized into biological process (BP), cellular component, and molecular function (MF).The top ten terms in each component of upregulation and downregulation were chosen for plotting, as shown in Figure 6D,E.Subsequently, we examined the particular pathways and corresponding DEGs to clarify the antibacterial mechanism of CSPA from a molecular standpoint.
ROS is the principal active chemical during cellular redox reactions.It acts as a signal molecule, facilitating intracellular signaling, gene transcription regulation, and cellular homeostasis. [44]xcessive ROS can disrupt the internal redox equilibrium of bacteria, leading to oxidative stress, destruction of the bacterial cell wall and membrane structure, and deterioration of intracellular biological macromolecules.This results in bacterial cell rupture and dysfunction.For downregulated DEGs, GO enrichment analysis showed that the most significant term in BP is oxidation-reduction process, with cell redox homeostasis ranking higher.Significant terms in MF include oxidoreductase activity and antioxidant activity, indicating an effect on the redox level of bacteria treated with CSPA.CnoX encodes the chaperedoxin of E. coli, which has a redox protection function.This protects bacterial cells from irreversible substrate oxidation and it also serves as a folding factor that protects the protein folding system. [45]uperoxide and hydrogen peroxide are natural byproducts of bacterial metabolism, [43a] which can result in the generation of extremely toxic •OH.Superoxide dismutase (SOD) can decrease the levels of superoxide.While superoxide does not actively react with biomolecules like proteins and DNA, it can cause the release of ferrous ions from a variety of cellular substances and catalyze the formation of extremely reactive •OH. [46]Thus, downregulation of SOD gene may lead to the toxicity of •OH on bacteria, resulting in oxidative stress.Catalase (KatG) has the capability of employing organic reducing agents, including GSH and ascorbic acid, to initiate the decomposition of hydrogen peroxide into water and oxygen.This process averts the negative effects of oxygen toxicity triggered by hydrogen peroxide.GSH reductase (gorA) is a crucial factor in preserving the balance between reducing and oxidizing GSH in bacteria, [47] and can resist oxidative stress suffered by bacteria.The cluster heat map (Figure 6F) analysis revealed that genes associated with antioxidant activity, particularly CnoX, sodA, KatG, and gorA, had experienced downregulation.This evidence suggested that CSPA triggered its antibacterial properties by producing ROS, which led to an internal redox mismatch in bacteria and ultimately attenuates antioxidant enzyme activity.
The consumption of energy is a prerequisite for any biological activity of bacteria, as it is essential for their physiological function, growth, and development.The core of energy metabolism is to convert external energy sources into ATP necessary for vital processes.Previous experiments observed a significant reduction of intracellular ATP levels in bacteria after treatment with CSPA, which implies an inhibition of their energy metabolism.KEGG enrichment analysis revealed significant impact on pathways including carbon metabolism, citrate cycle (TCA), glycolysis, pyruvate metabolism, and oxidative phosphorylation.The clustered heat map (Figure 6G) displayed the genes related to these pathways.Among them, genes related to oxidative phosphorylation (sdhD, nuoC, sdhA, cyoB, cyoA, cydB, cydA) and genes related to TCA (nifJ, acnA, sdhD, acnB, aceE, sdhA, fumC) were significantly downregulated.Bacteria metabolized substrates, like sugars, proteins, and lipids, to produce ATP for energy generation via these metabolic pathways.Nevertheless, subsequent to exposure to CSPA, bacterial metabolic activity reduced, halting energy supply and leading to bacterial demise.
The bacterial cell wall and membrane play crucial roles in maintaining the basic morphology of bacterial cells, safeguarding them from toxic substances and osmotic pressure.Moreover, they serve as essential structures for cell growth, division, and movement.The cell wall and membrane of Gram-negative bacteria are mainly composed of peptidoglycans, lipopolysaccharides, lipid bilayer, and proteins.Pathways involved in cell wall synthesis, including peptidoglycan biosynthesis, O-antigen nucleotide sugar biosynthesis, lipopolysaccharide biosynthesis, and biosynthesis of nucleotide sugar, exhibited significant upregulation as revealed by KEGG enrichment analysis (Figure 6B).Additionally, the glycerophospholipid metabolism and glycerolipid metabolism, which are associated with cell membranes, were significantly impacted.It was also found that phospholipid metabolism process in GO enrichment analysis was affected.The genes related were notably upregulated in comparison with the control group (Figure 6H), suggesting that the bacterial cell wall and membrane function was partially influenced by the CSPA treatment.Changes in the external environment can impact the growth and physiological functions of bacteria, resulting in environmental stress.Bacteria perceive these changes and respond quickly through corresponding physiological and genetic mechanisms to maintain their survival.Previous experiments had shown that CSPA caused significant damage to the cell wall and membrane of E. coli, resulting in cell lysis.In order to cope with this stress, bacteria must increase the synthesis of their cell wall and membrane and respond at the genetic level by activating genes related to their synthesis and enhancing their defense capabilities.A sizeable genetic cluster on the E. coli gene map incorporates seven genes (murC, murD, murE, murF, murG, mraY, ddlB) that encode proteins associated with peptidoglycan synthesis and cell division.The marked increase in the expression of these genes could function as a safeguarding approach for bacteria during exposure to CSPA.
These results by the cellular levels and transcriptomics provided very important insights into the antibacterial mechanism of CSPA and further provide a basis for the deep application of CSPA in biomedicine.

Biocompatibility Evaluation of CSPA
This study examined the cytotoxic effects of CSPA via MTT assays.Figure S12, Supporting Information, illustrates the treatment of HACAT and L929 cells with varying concentrations of CSPA for 24 h.Compared to the control group, both HACAT and L929 cells demonstrated 91% and 85% cell viability, respectively, when exposed to 500 μg mL À1 of CSPA, implying that cell activity remained strong.As the concentration increases, CSPA displayed a minor cytotoxic effect, revealing that it was mostly nontoxic to cells within a specific concentration range, rendering it safe for use.Furthermore, intracellular ROS were detected using DCFH-DA after the action of CSPA, as shown in Figure S13, Supporting Information.The positive group exhibited bright green fluorescence, while CSPA only produced a small amount of ROS which did not pose serious toxicity to the cells.CS, being a biocompatible natural biomolecule, could reduce the toxicity of the NPs and minimize damage to normal cells.In addition, due to the rich H 2 O 2 and weakly acidic environment of bacterial infection, CSPA exhibited exceptional enzyme activity under acidic conditions, resulting in the production of a plethora of ROS.Conversely, the neutral environment of normal cells limited CSPA's ability to produce ROS, allowing it to kill bacteria to treat infected wounds and avoid the toxicity to normal cells.The safety of CSPA underwent further investigation through in vitro hemolysis experiments to evaluate its impact on red blood cells.As shown in Figure S14, Supporting Information, pure water and saline were chosen as positive and negative controls, respectively.The erythrocyte solution in pure water exhibited a vivid red hue, and the cells were essentially ruptured, whereas the regular erythrocytes remained stable in saline.The hemolysis rate of red blood cells treated with saline solution containing various concentrations of CSPA remained less than 5% even at the highest concentration.This indicated that CSPA was not accountable for serious hemolysis within physiological conditions and exhibits good biological safety.The safety of CSPA was assessed in vivo using the rat epidermal wound administration method.Weight fluctuations, blood profiles, liver and kidney functions, and H&E staining sections of normal organ tissues were measured throughout the administration period to evaluate its in vivo safety.As depicted in Figure S15, Supporting Information, the weight of rats in both the control and experimental groups exhibited an increase compared to their weight before treatment, and the trend of weight change was consistent.Furthermore, Figure S16, Supporting Information, displays the results of the blood routine test, which indicated that all indicators in the control and experimental groups remained within the normal reference range.These findings suggest that CSPA was unlikely to result in any significant changes in blood components or had adverse effects on the blood system.Liver and renal function tests shown in Figure S17, S18, Supporting Information, demonstrated that CSPA did not result in any adverse reactions.In addition, the H&E staining of the major organs of the rat, including the heart, liver, spleen, lungs, and kidneys, is displayed in Figure S19, Supporting Information, and there was no evidence of pathological changes in the organs.These results suggested that CSPA possessed good biocompatibility and presented a favorable in vivo safety profile, and can be utilized for further in vivo treatments.

In Vivo Mixed Bacteria-Infected Wound Healing of CSPA
Based on the exceptional in vitro antibacterial and safety qualities of CSPA, we explored the effectiveness of CSPA in treating bacterial infection wounds in vivo.Bacterial infections, especially mixed bacterial infections, may hinder wound healing and even cause serious consequences.A Sprague Dawley (SD) rat model of skin excision wound infected by multiple bacteria (E. coli and S. aureus) was established to explore the anti-infection effect of CSPA.As illustrated in the schematic diagram (Figure 7A), first, an 18 mm diameter wound was created on the back of SD rats.A mixture of E. coli and S. aureus bacteria was introduced to the wound for 2 days, creating an in vivo bacterial infection model.As depicted in Figure 7F, following infection, the wound tissues in each group contained a significant quantity of bacterial colonies.H&E staining analysis (Figure S20A, Supporting Information) revealed that the infected tissue displayed a significant influx of inflammatory cells, losing the structure of normal tissue (as observed in the control tissue).Masson staining analysis (Figure S20B, Supporting Information) confirmed a substantial loss of collagen in the infected tissue relative to the control.The following treatment groups were established: control group, Van group, CS group, and CSPA group.As shown in Figure 7B,C, the representative photographs of the wound size during the treatment period of each group revealed that CS showed a slight wound accelerated healing effect within 4 days compared with the control group.However, the wound closure was greatly enhanced after the treatment of CSPA group, followed by the Van group.Figure 7D displays the changes in wound area over time.The wounds in each group exhibited a certain degree of shrinkage over time.In general, both the Van and CSPA treatment groups demonstrated more prominent wound healing effects.Additionally, the CSPA group displayed the smallest wound size and highest wound shrinkage rate after 8 days of treatment, suggesting a faster healing pace and superior treatment outcome.Mixed bacteria-infected wounds frequently lead to inflammation and impede the process of wound healing.As illustrated in Figure 7F, the growth of bacterial colonies in the wound tissue decreased in each group after treatment compared to the time of infection.The CSPA group and Van group exhibited significantly fewer bacterial colonies, indicating a strong antibacterial effect.This indicated that CSPA had the potential to generate antibacterial effects in vivo that were comparable to those of antibiotics.
To assess wound recovery after treatment, the skin of each group underwent H&E and Masson staining for pathological analysis.The H&E staining (Figure 8A) revealed impaired wound skin in the control group, showing a significant inflammatory response characterized by a large number of blue-purple neutrophils at the wound site.The CS group had an increased number of neutrophils.The group treated with Van and CSPA showed a more complete organizational structure, including new hair follicles and fewer neutrophils.The inflammation situation experienced a notable improvement.Masson staining (Figure 8B) revealed a larger blue area in the wound tissue after CSPA treatment when compared to both the control group and CS group.This indicated a higher amount of collagen deposition.Therefore, CSPA had potent in vivo antibacterial properties that promoted efficient healing of infected wounds, and provided an effective treatment option against bacterial infections.

Conclusion
In summary, this study synthesized an ultrasmall-sized CSPA nanozyme with better water dispersion, excellent biocompatibility nanozyme, and strong multienzyme-like activity which exhibited activity of OXD-, POD-, and NDH-like enzymes.It generated excessive ROS to induce oxidative stress, consume coenzyme NADH in the respiratory chain to interfere with ATP synthesis, and thus exerted antibacterial effects.The antibacterial property without acquired drug resistance implied that CSPA may be an effective and promising strategy for antibacterial capability.Transcriptomics further clarified that CSPA primarily targeted pathways related to oxidative stress, energy metabolism, and cell wall and membrane synthesis, revealing their mechanisms of action.The results of transcriptome sequence analysis provided important insights into the antibacterial mechanism of CSPA.In vivo CSPA could effectively kill mixed bacteria in wound infections and accelerate wound tissue healing, promote the generation of pores and collagen fibers, which played a vital role in wound infection treatment.Overall, CSPA exhibited favorable biocompatibility, demonstrating efficacy in both in vivo and in vitro antibacterial activities.This provided valuable insights for the potential application of CSPA nanozyme antibacterial agents.Oxidase-and Peroxidase-Like Activity Studies: The OXD-like catalytic activity of CSPA was studied based on the 3TMB whose oxidation product has an obvious absorption peak at 652 nm.Specifically, the experiment was divided into three groups: a) TMB; b) TMB þ CSPA (N 2 ); and c) TMB þ CSPA. 25 μL of CSPA solution was added into 200 μL of acetate buffer (10 mM, pH = 4) involving 25 μL of TMB solution (8 mM) and reacted for 5 min at 37 °C.The group c was pursed with nitrogen (N 2 ) in advance to remove most of dissolved oxygen.After the reaction, the absorbance of various group was performed on UV spectrophotometer (UV-vis, UV-2450).The POD-like catalytic activity of CSPA was also studied using TMB.In short, there were four groups: a) TMB

Experimental Section
Each group was pursed with nitrogen (N 2 ) in advance.25 μL CSPA solution was added into 175 μL acetate buffer (10 mM, pH = 4) involving 25 μL TMB (8 mM) and 25 μL H 2 O 2 kept for 5 min at 37 °C for absorbance collection.The CSP and CSA underwent UV absorbance measurements using the method described above.
The steady-state kinetic analyses of CSPA were evaluated by incubating with different concentrations of TMB and H 2 O 2 .The kinetic parameters of catalytic reaction were calculated by the Michaelis-Menten equation as follows: where V, V max , K m , and [S] represent the initial reaction velocity, maximal reaction velocity, Michaelis-Menten constant, and concentration of substrate, respectively.The ESR was used for detecting the types of ROS produced in the catalytic system.NADH Dehydrogenase-Like Activity Studies: The NADH dehydrogenaselike (NDH-like) activity of CSPA was investigated by utilizing NADH as the substrate.To achieve this, 25 μL of CSPA (75 μg mL À1 ) solution and 25 μL NADH (2 mM) solution were added to 200 μL of acetic acid sodium acetate buffer (10 mM) and uniformly mixed.Then, the reaction was carried out at 37 °C for varying periods (0, 5, 10, 15, 20, 25, and 30 min), and their distinctive absorption at 260 and 340 nm was measured using a UV spectrophotometer.The purified water was used instead of CSPA solution to set the control group and follow the instructions provided for measurement.Additionally, we investigated the consumption of NADH in catalytic reactions by altering the concentrations of both CSPA and NADH.The consumption of NADH was determined by calculating the absorbance difference at 340 nm prior to and postreaction, ΔA = A n min À A 0 min.The CSP and CSA underwent UV absorbance measurements using the method described above.
In Vitro Antibacterial Evaluation: E. coli and S. aureus were selected as experimental bacteria for evaluating the antibacterial activity.The bacteria (1 Â 10 6 CFU mL À1 ) were treated with varying concentrations of CS or CSPA at 37 °C for 2 h.Following this, the bacterial suspension was diluted 50 times, and 50 μL of the diluted bacterial solution was applied onto the agar medium and cultured at 37°Cfor 24 h.Then, the number of colonies in the ager medium was counted.Additionally, bacteria treated with different concentrations of CS or CSPA were cultured in Mueller-Hinton (MH) broth.Their absorbance at 600 nm was measured every 2 h using a microplate reader to observe the growth trend of the bacteria and plot corresponding growth curves.The survival of bacteria after incubation with the same concentration of CSP, CSA, and CSPA in MH broth was compared by measuring the absorbance at 600 nm.
Inhibiting Biofilms Formation: S. aureus (1 Â 10 6 CFU mL À1 ) was cultured in MH broth containing varying concentrations of CS or CSPA for 48 h.The formed biofilms were fixed with methanol, stained with 0.1% CV solution, and treated with 33.3% acetic acid solution.Thereafter, the final solution containing the stained biofilms was added to a 96-well plate to measure the amount of biofilms formation by determining its absorbance at 570 nm using a microplate reader.Moreover, the formed biofilms were dyed with Syto9 and PI in a dark for 30 min.The biofilm thickness and staining were observed using confocal laser scanning microscopy.
Drug Resistance Test: Using Van as a control, the MBC of CSPA or Van against bacteria of different generations was determined.First-generation S. aureus (1 Â 10 6 CFU mL À1 ) was treated with a range of concentrations of CSPA or Van and inoculated into MH broth at 37 °C for 24 h.The bacterial at the highest treatment concentration showing bacterial growth was selected for the next round of cultivation to observe bacterial resistance to CSPA or Van.
Morphological Observation of the Bacteria: E. coli and S. aureus were cultured in MH broth for 24 h.The culture was discarded via centrifugation (3000 rpm, 15 min).The bacterial precipitate obtained by centrifugation was resuspended in phosphate buffered saline (PBS) until the OD 600 was 0.5.After being treated with different groups (PBS, CS, CSPA) for 10 h, the bacteria were fixed with electron microscopy fixative overnight and then fixed with 1% osmic acid solution for 2 h.The bacteria were then dehydrated with a concentration gradient of ethanol or acetone.The structure and morphology of bacteria were observed by the use of SEM and TEM.
Live/Dead Staining Test: E. coli and S. aureus were cultured until the logarithmic growth phase.Following 4 h of treatment with CS or CSPA, the bacteria were washed 3 times.Subsequently, they were treated with Syto9 and PI dye in a dark environment for 30 min.The bacteria were rinsed thrice to eliminate any unbound dyes and then observed under a fluorescence microscope.
ROS Staining Test: The production of ROS in bacterial cells were detected using DCFH-DA.E. coli and S. aureus were treated with CS or CSPA for 4 h, then centrifuged, washed, and resuspended in physiological saline to create bacterial suspensions.Next, DCFH-DA was added to the bacterial suspensions and incubate in the dark at 37 °C for 30 min.The bacterial samples were washed with physiological saline to remove excess dye and the fluorescence signal at 488/525 nm (λ ex /λ eM ) was measured.Furthermore, the bacteria were observation under a fluorescence microscope.
Transcriptome Analysis: E. coli was chosen for transcriptomic sequencing analysis.The bacteria were cultured until the logarithmic growth phase, and subsequently inoculated into MH medium with CSPA (control group) and MH medium without CSPA (experimental group) for further 8 h.Three replicate experiments were carried out.The control group comprised C1, C2, and C3, while the experimental group comprised E1, E2, and E3.The processed bacteria underwent RNA extraction through the use of the RNAprep Pure culture cell/bacterial total RNA extraction kit.RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).Then cDNA libraries were constructed and sequenced on the Illumina platform by Novogene Co. Ltd.The DESeq2 R package (1.20.0) was employed to assess the differential gene expression.
Cytotoxicity Studies: The MTT assay was carried on to evaluate the cell cytotoxicity.HACAT and L929 cells with a density of 5 Â 10 4 mL À1 were incubated with 100 μL per well in a 96 well plate for 24 h.After adhesion, the cells were incubated with the culture medium containing different concentration of CSPA for 24 h.Then, the culture medium was discarded and the 96 well was added with 100 μL 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl tetrazolium bromide thiazole blue (MTT).After 4 h of treatment, the MTT supernatant was removed, and the dimethyl sulfoxide was added with 150 μL per well.The absorbance at 490 or 570 nm was measured to evaluate the cell survival rate.The production of ROS in cells after treatment of CSPA and positive control were detected using DCFH-DA.
The SD rat blood sample was used for hemolysis test.Fresh blood samples were centrifuged at 1500 rpm for 15 min to collect erythrocytes, and washed 3 times with physiological saline to prepare a 20% erythrocyte dispersion.The dispersion was treated with different concentration of CSPA for 3 h at 37 °C and then centrifuged (12 000 rmp, 15 min).The absorbance at 540 nm of supernatant was measured.Saline and purified water containing erythrocyte dispersion were used as negative and positive controls, respectively.The hemolysis rate was calculated as follows: In Vivo Antibacterial and Wound-Healing Evaluation: Animal experiments were guided of guideline and approved of the Animal Care and Use Committee of Fujian Medical University (No. FJMU IACUC 2019-0119).SD male rats (6 weeks, 180-200 g) were anesthetized with 7% chloral hydrate and the back of the rats was shaved.A circular wound with a diameter of approximately 18 mm was built by cutting off the entire layer of skin.Then 200 μL E. coli and S. aureus mixed bacterial suspension (1 Â 10 8 CFU mL À1 ) was added on the wound.After 48 h of infection, a wound model was established.All rats were randomly divided into four groups with different treatments: PBS, Van, CS, and CSPA at a dose of 1 mg kg À1 each.During the treatment period, medication was administered once a day and the wound size of each group was measured and recorded for healing.After treatment, all rats were euthanized and their wound tissues were collected.The wound tissue was homogenized and 50 μL of the homogenized tissue diluted 100 times was applied to an agar plate.After 24 h of cultivation at 37 °C, the bacterial content in each group of tissues was observed.In addition, the wound tissues of each group were fixed in 4% paraformaldehyde, followed by paraffin embedding for H&E staining and Masson staining for histological analysis.
Statistical Analysis: Statistical analysis of experimental data was performed using SPSS 27 software, and the quantitative data of each experimental group were presented as mean AE standard deviation (mean AE SD).Comparison between groups was conducted using one-way analysis of variance and least significant difference comparison test.p < 0.05 indicates statistical significance.Among them, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and the figure notes for each data plot provided the sample size (n).

Figure 1 .
Figure 1.Characterization of CSPA.A) TEM image (inset: HRTEM image).B) Size distribution.C) XRD spectra of CSPA.D) HAADF-STEM (high-angle annular dark-field imaging in scanning transmission electron microscopy) and elemental mapping images of CSPA.Scale bar = 2 nm.High-resolution XPS spectra of E) Pt 4f and F) Au 4f.G) FTIR spectra of CS and CSPA.

Figure 2 .
Figure 2. Multienzyme-like activity of CSPA.A) Schematic diagram illustrating the mechanism of enzyme-like catalytic reactions.B) OXD-like activity.C) POD-like activity.D) ESR spectra of •O À 2 captured by DMPO.E) 1 O 2 captured by TEMP.F) •OH captured by DMPO.G) NDH-like activity.H) Timedependent degradation of NADH in the presence of different concentrations of CSPA and I) NADH (n = 3).

Figure 3 .
Figure 3.In vitro antibacterial activity of CSPA.A) Colonies growth of E. coli after CS, CSPA treatment on agar plates and B) colonies statistics (n = 5).C) Colonies growth of S. aureus after CS, CSPA treatment on agar plates and D) colonies statistics (n = 5).Live/dead bacterial cells staining images of E) E. coli and F) S. aureus after different treatments.Scale bar = 50 μm.G) S. aureus resistance curves to Van and CSPA (n = 3).H) Survival rate of Van-resistant S. aureus treated with different concentration of CSPA (n = 3).I) Quantitative analysis of S. aureus biofilms treated with different concentration of CS and CSPA (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).J) Confocal laser scanning microscope photographs of S. aureus biofilm incubated with CSPA and CS, respectively.Green florescence: live bacteria stained by Syto9.Red fluorescence: dead bacteria stained by PI.Scale bar = 50 μm.

Figure 4 .
Figure 4. A) SEM and B) TEM images of E. coli and S. aureus after different treatments.Scale bar = 1 μm.

Figure 5 .
Figure 5. A) Fluorescence images of bacteria stained with DCFH-DA in control and CSPA group.Scale bar = 50 μm).Loss of GSH in B) E. coli and C) S. aureus after treatment with different concentrations of CSPA.The intracellular ATP content of D) E. coli and E) S. aureus treated with different concentrations of CSPA (n = 3).

Figure 6 .
Figure 6.RNA-Seq gene expression profiles of CSPA-treated E. coli compared with control group.A) Volcano plot of DEGs.KEGG enrichment analysis of B) upregulated DEGs and C) downregulated DEGs.GO enrichment analysis of D) upregulated DEGs and E) downregulated DEGs.Heat map of typical DEGs associated with F) oxidative stress, G) energy metabolism, and H) cell wall and membrane.Red represents upregulated genes while blue represents downregulated genes.

Figure 7 .
Figure 7.In vivo antibacterial activity of CSPA.A) Model construction and treatment diagram of infection wound.B) Photographs of E. coli and S. aureus infected wounds after various treatments.Scale bar = 10 mm.C) Traces of wound healing in different groups.D) Change in relative wound area of different groups on different days.E) Wounds contraction rate in different groups after completion of treatments.F) Colonies growth in tissues of different groups after infection and treatment.n = 6, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8 .
Figure 8. Histological analysis of wound tissues in different groups after completion of treatments.A) H&E and B) Masson staining of wound tissue.Scale bar of origin images (below) = 200 μm.Scale bar of magnified images (above) = 100 μm.