A core–shell‐structured zeolitic imidazolate framework@cationic antimicrobial agent templated silica nanocomposite for tackling antibiotic resistant bacteria infection

Bacterial infection is a major threat to public health. Nanotechnology offers a solution by combining nanomaterials with antibacterial agents. The development of an effective nanocomposite against drug‐resistant bacteria such as methicillin‐resistant Staphylococcus aureus (MRSA) is highly important yet challenging. Here, an anti‐MRSA core–shell structure is designed, containing antibacterial zeolitic imidazolate framework‐8 (ZIF‐8) as the core and bactericidal benzalkonium chloride (BAC) templated rough‐surface mesostructured silica nanocomposite (RMSN) as the shell. The resultant ZIF‐8@RMSN nanocomposite exhibits sustained release of BAC and zinc ions, effective disruption of the bacterial membrane, generation of oxidative damage of bacterial DNA, leakage of intracellular components, and finally bacterial death. Furthermore, the synergistic antibacterial mechanisms lead to enhanced biofilm elimination performance. In addition, the ZIF‐8@RMSN‐modified band‐aid effectively combats MRSA infection in vivo. This work has provided a promising nanocomposite against MRSA‐related infections.

the failure of antibacterial treatments. [7,10]Hence, there is an urgent need to develop effective strategies for tackling MRSA-related bacterial infections.
Recently, the combination of functional nanomaterials and antibiotics provides a feasible solution to combating multidrug-resistant bacteria and biofilms.[13][14] Various nanoparticles, including cationic polymers, [15,16] metal nanoparticles [17] and metal-organic frameworks (MOFs) [18,19] have been successfully employed in combination with antibiotics to boost antibacterial properties.As a subclass of MOFs, zeolitic imidazolate framework-8 (ZIF-8) has widespread biomedical applications, primarily due to its intrinsic porous structure, controllable morphology, and unique pH-responsive dissolution behavior. [20,21]In acidic microenvironments such as biofilms, ZIF-8 nanoparticles release zinc ions and exhibit antibacterial properties by generating reactive oxygen species (ROS); moreover, the content of secreted EPS is reduced. [22,23]In addition, the acid-responsive decomposition properties of ZIF-8 make it an excellent carrier for antibacterial drugs, giving drugs the function of targeting biofilms. [24,25]Nevertheless, the rapid release of zinc ions (Zn 2+ ) can induce potential cytotoxicity and the loss of active components. [26]his drawback can be addressed by strategies to enhance the stability of ZIF-8, e.g.[32] However, the introduced composition in composite materials is generally inert in antibacterial property, limiting the antibacterial effectiveness of the composites.
Benzalkonium chloride (BAC) is a quaternary ammonium compound (QAC) and one of the most common active ingredients in disinfectants used in residential, industrial, agricultural, and clinical settings due to its broad-spectrum bactericidal activity. [33,34]The anti-biofilm mechanism of QAC is that the positively charged groups can bind to the negatively charged biofilm and enhance the hydrophilicity of EPS, making the biofilm easy to remove. [35]However, with the evolution and development of drug-resistant bacteria, the sensitivity of bacteria to BAC has decreased, making it difficult for traditional BAC formulations to exert effective antibacterial activity and biofilm removal performance. [33]ecently, BAC has been used as a cationic surfactant template to synthesize mesostructured silica nanoparticles as bactericidal agents. [36,37]Nevertheless, due to the inert antibacterial nature of silica, the efficacy of BAC-silica composites towards drug-resistant bacteria such as MRSA is rarely reported.
Herein, we report the synthesis of a core-shell structure as a multifunctional antibacterial platform (Figure 1A), using ZIF-8 as the core and BAC-containing rough-surface mesostructured silica nanocomposite as the shell (named as ZIF-8@RMSN).Even though the core or the shell has been reported individually, the synergy of the core and the shell contributes to enhanced antibacterial and biofilm elimination properties in a drug-resistant bacterial model (MRSA).It is demonstrated that the core-shell structure exhibits sustained release of BAC and zinc ions, effective disruption of the bacterial membrane, generation of intracellular ROS for oxidative damage of bacterial DNA, all contributing to bacteria killing (Figure 1B).Moreover, ZIF-8@RMSN shows EPS reduction and MRSA biofilm elimination properties (Figure 1C).In addition, ZIF-8@RMSN modified antibacterial band-aid possesses excellent anti-MRSA performance both in vitro and in vivo (Figure 1D).Our work has revealed the potential application of ZIF-8@RMSN nanocomposites and provided a nanomaterial-based solution for tackling MRSA-related infections.

Synthesis of ZIF-8 nanoparticles
In a typical synthesis, ZIF-8 nanoparticles were synthesized in an aqueous solution at room temperature.Typically, 0.25 wt% of CTAB and 2-MIM were added to 70 mL of water and stirred until complete dissolution, followed by the addition of 10 mL of Zn(NO 3 )

Synthesis of ZIF-8@RMSN
ZIF-8@RMSN with a core-shell structure was synthesized using ZIF-8 as the core, BAC and NaSal as co-templates, and TEOS as the silica source for creating the silica shell. [36]In brief, 13.6 mg of TEA was added to a 5 mL solution containing uniformly dispersed ZIF-8 solution (10 g L −1 ) and stirred at 65 • C for 30 min.Then, 20 mg of NaSal was added and stirred at 65 • C for 1 h.Following this, 0.14 mL of 50% aqueous BAC was added to the above solution, and reaction continued for 1 h.Then 0.6 mL of TEOS was added to the solution for further stirring at 65 • C for 2 h.The precipitate was collected by centrifugation at 15,000 rpm for 5 min, washing with ethanol three times, and vacuum drying at 50

Characterization
Transmission electron microscopy (TEM) image was taken using J HT7700-EXALENS with an accelerated voltage of 80-100 kV.Samples were dispersed in ethanol and then dried on a copper grid.Scanning electron microscope (SEM) images were collected using a JEOL JSM 7800 field-emission scanning electron microscope (FE-SEM).Samples were dispersed in ethanol and then dried on the silicon wafer.Energy-dispersive X-ray (EDX) mapping was performed using a Hitachi HF5000 Cs-STEM/TEM.The chemical functional groups of the nanoparticles were characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectrometer equipped with Diamond ATR Crystal (Thermo Nicolet Nexus 6700).Dynamic light scattering (DLS) analysis and zeta potential were conducted using the Zetasizer Nano-ZS (Malvern Instruments).X-ray diffraction (XRD) analysis was performed on a Bruker D8 Advance with Cu Kα (λ = 0.154 nm, 40 kV, and 40 mA) as the X-ray source in the 2θ range of 5 • -60 • .X-ray photoelectron spectroscopy (XPS) measurements were performed on a PHI 5000 Versa Probe equipped with monochromatic Al-Kα radiation.A thermo scientific iCAP 6500 inductively coupled plasma atomic emission spectroscopy (ICP-OES) instrument was used to detect the zinc content.Quantitative determination of BAC and 2-MIM was achieved using a triple quadrupole mass spectrometer (Thermo Scientific TSQ Quantum Ultra) coupled to a Dionex high-performance liquid chromatography (HPLC) with an ultraviolet detector and fluorescence detectors.

Release study
To investigate the stability of ZIF-8 and ZIF-8@RMSN nanoparticles in different pH environments, the samples were dispersed in PBS (pH = 7.0 or 5.5) via sonication.The PBS with a pH = 5.5 was adjusted using an HCl solution. [40]hen the samples were incubated in a constantly shanking incubator at 25 • C at 100 rpm.At specified time intervals, 0.5 mL of the sample was taken out for centrifugation at 13,000 rpm for 5 min and then the supernatant was analyzed by LC-MS to determine the concentrations of BAC and 2-MIM in the solution.Due to the possible zinc phosphate precipitates formed by zinc ions in PBS buffer solution that might affect the experimental results, [21] the assessment of 2-MIM was used to gauge the decomposition rate of ZIF-8 in solution.

Antibacterial experiments
The antibacterial activity of ZIF-8@RMSN against MRSA was assessed using the Luria-Bertani (LB) agar plate assay.BAC, ZIF-8, a mixture of BAC and ZIF-8 (denoted as BAC/ZIF-8), and a mixture of BAC and zinc ions (denoted as BAC/Zn 2+ ) were selected as controls.All samples underwent sterilization and were dissolved in sterilized PBS, and each group maintained the same BAC concentrations of 2, 5, and 7 μg mL −1 .Meanwhile, the ZIF-8 and BAC/Zn 2+ groups maintained the same zinc ion content as the ZIF-8@RMSN group.The mixture of bacterial suspension (1.0 × 10 7 CFU mL −1 ), LB medium, and different samples were incubated in a 37 • C shaker at 220 rpm for 24 h, and the bacterial viability was examined using a minimum inhibitory assay by determining the optical density at 600 nm via a plate reader and LB-agar plate assay.Specifically, 200 μL of different sample-treated bacterial suspensions were spread on sterilized LB-agar plates.After overnight incubation at 37 • C, photographs were taken, and the bacterial colony growth in each plate was quantified to determine the bacterial viability.
The DNA damage of the MRSA incubated with ZIF-8@RMSN was investigated by measuring the concentration of DNA.MRSA was treated with different groups for 4 h, DNA was extracted from all the bacteria by using a Bacterial Genomic DNA Extraction Kit (ThermoFisher), and then the concentration of DNA was determined by using NanoDrop. [41]To further study the DNA damage, agarose gel electrophoresis was applied.Specifically, the solidified agarose gel was transferred into the gel tank and fully covered with TAE buffer. 2 μL of nucleic acid sample buffer (5×, Bio-Rad) was added into the extracted DNA solutions at a total volume of 10 μL loaded in each well.Electrophoresis was carried out at 80 V for 50 min, and the bands were visualized on a UV tans-illuminator (Bio-Rad).The leakage of protein within the bacteria was measured using the BCA kit (ThermoFisher) to reflect the damage to the bacterial cell membrane.
For the intracellular ROS quantification, treated MRSA was collected by centrifugation at 8000 rpm for 10 min.Then, nitro blue tetrazolium (NBT) solution was added to the bacteria pellet, and the mixture was incubated in the dark at room temperature for 1 h.The pellet was collected via centrifugation at 8000 rpm for 10 min and washed with PBS to remove the residual NBT solution.Subsequently, the pellet was resuspended in a 2 M KOH solution, and 50% DMSO was added for incubation at room temperature for 10 min to dissolve the formazan crystals.Then the ROS level was quantified based on the absorbance of the supernatant at 620 nm. [42]

SEM observation
After treatment with different groups, bacteria were fixed with 2.5% glutaraldehyde at 4 • C for 24 h, and then the bacterial solution was dropped on a round glass coverslip coated with poly-L-lysine.Different gradients (30%, 50%, 70%, 90%, and 100%) of ethanol were used to dehydrate the samples.Afterward, the samples were treated with 50% hexamethyldisilane (HDMS-ethanol) and 100% HMDS, dried and platinum sputter coated, then the SEM system is used to observe the samples.

Bacterial cell membrane experiment
SYTO9 and PI dyes were used to access bacterial cell membrane integrity.Briefly, bacterial suspensions (10 7 CFU mL −1 ) were treated with ZIF-8@RMSN, BAC/ZIF-8, BAC/Zn 2+ or an equal volume of PBS.After incubation for 24 h, the bacterial suspension was collected, centrifuged to remove the supernatant, and resuspended in 100 μL PBS.Afterward, co-incubated with PI and SYTO9 dye for 30 min at 37 • C in the dark condition.Finally, confocal laser scanning microscopy was used to observe live and dead bacteria. [43]

Anti-biofilm experiments
To develop mature biofilm, the prepared MRSA suspension (1.0 × 10 8 CFU mL −1 ) was mixed with 1 mL LB medium and added to a 24-well plate, then statically incubated at 37 • C for 48 h. [43]For the biofilm disruption experiment, mature biofilms were treated with blank LB medium, BAC/ZIF-8, BAC/Zn 2+ , and ZIF-8@RMSN (quantified based on 7 μg BAC mL −1 ), respectively.Following static co-incubation for 48 h, the solution was discarded and the residual biofilm was gently washed with PBS.Then the biofilms were stained with SYTO9 for 30 min, and characterized by confocal microscopy to visualize the 3D structure of the treated biofilm.The residual biofilm was also stained with crystal violet, photographed after drying, and dissolved with acetic acid, then the optical density at 590 nm (OD590) was measured to determine the residual biofilm.In addition, the residual biofilms were removed and spread on LB agar plates to determine the viable bacteria in the biofilms.
For the biofilm inhibition study, 200 μL of MRSA bacterial suspension (1.0 × 10 8 CFU mL −1 ) was mixed with different formulations for 24 h after static incubation.The biofilm inhibitory effect was evaluated by confocal microscopy, crystal violet staining, and agar plate counting as described above.
For EPS characterization, mature MRSA biofilms were treated with different formulations for 48 h, then the biofilms were washed with PBS and stained with 200 μL of 5 μg mL −1 WGA-AF488 for 2 h at 4 • C in the darkness.The unbound dye was removed by washing twice with PBS and the EPS was visualized by confocal microscopy. [44]For eDNA quantification, mature MRSA biofilms were treated with different groups, and the remaining biofilms were collected for centrifugation at 5000 g for 10 min.Then the supernatant was filtered using a 0.22 μm filter to quantify the eDNA content by NanoDrop. [7]

Preparation of ZIF-8@RMSN band-aid
The ZIF-8@RMSN-based band-aid was prepared based on a reported method. [45]One square gauze (1 cm × 1 cm) was mounted on a negative pressure suction filter.ZIF-8@RMSN, BAC/ZIF-8, and BAC/Zn 2+ solutions (all groups kept at the same BAC concentrations of 7 μg mL −1 , meanwhile, BAC/ZIF-8 and BAC/Zn 2+ groups maintained the same zinc ions content as the ZIF-8@RMSN group, and the solution volume of all groups was 100 μL) were added onto the gauze until all the solutions passed through the gauze, and ensured that the solutions were completely absorbed by the gauze.After vacuum drying at 50 • C, the ZIF-8@RMSN coated gauze was fixed to the sticker as the ZIF-8@RMSN bandaid, and making BAC/ZIF-8 and BAC/Zn 2+ band-aids in a similar way.Band-aid containing PBS was utilized as control, named as blank band-aid.To prevent excessive wound exudate produced on the first day to affect the antibacterial effect of the band-aid, the band-aid was replaced on the first day and then kept for another 72 h.Then the mice were sacrificed and the number of bacteria on the wound was quantified by the standard plate count method.For the long-term antibacterial experiment of ZIF-8@RMSN band-aid in vitro, the ZIF-8@RMSN coated band-aid patch was vacuum dried and sterilized by the ultraviolet-light radiation method, then placed in a mixture of bacterial suspension (1.0 × 10 7 CFU mL −1 ) and LB medium.After incubating for 3 days in a shaker at 37 • C at 150 rpm, the bacterial viability was determined by OD600.The BAC/ZIF-8, BAC/Zn 2+ , and blank band-aids were used as control groups.

Statistical analysis
Experimental data were presented as means ± standard deviation, and differences between two groups were calculated by using a two-tailed t-test.In all tests, P values less than 0.05 were considered statistically significant (*p<0.05,**p<0.01,***p<0.001,****p<0.0001).Statistical analysis was performed using the GraphPad Prism software.

In vivo antibacterial application of ZIF-8@RMSN band-aid
A mouse excisional wound model was built using female mice (BALB/c, 6 weeks old) created with an 8 mm diameter circular excisional wound on the dorsal area.100 μL of MRSA suspension in PBS (10 8 CFU/mL) was placed on the wound site.After infection for 24 h, the wound site was covered with ZIF-8@RMSN band-aid with blank Band Aid, BAC/ZIF-8, BAC/Zn 2+ as control groups.On day 1, the band-aid was replaced with a new ZIF-8@RMSN band-aid, and mice were then divided into two batches.Batch 1 mice were sacrificed on day 4, while other mice were left for the observation of wound healing.Tissue samples were harvested, and homogenized into 1 mL of PBS solution, and plated on an agar plate to determine bacterial counts.BAC/ZIF-8 band-aid, BAC/Zn 2+ band-aid, and blank band-aid were served as control groups.Photographs were taken within a set period to record the closure of the mouse wounds, and the body weight changes were detected.
All experiments involving animals were performed according to the protocol approved by the East China Normal University (ECNU) Animal Care and Use Committee (protocol ID: m20240103) and in direct accordance with the Ministry of Science and Technology of the People's Republic of China on Animal Care Guidelines.All mice were euthanized after the completion of the experiments.

Characterization of ZIF-8@RMSN nanocomposites
ZIF-8 nanoparticles were synthesized following a reported protocol. [46]As shown in the SEM image (Figure 2A), the synthesized ZIF-8 nanoparticles had a uniform cubic morphology and a smooth surface.The cubic morphology of ZIF-8 was further visualized in the TEM image (Figure S1), with an average particle size of 104.5 ± 10.9 nm by measuring 100 particles (Figure S2 and Table S1).Subsequently, a mesostructured silica-surfactant shell was coated on the ZIF-8 core via electrostatic interaction using a sol-gel method, [36,47] leading to the ZIF-8@RMSN nanocomposite as the final product.The cubic morphology originated from ZIF-8 core particles was well-maintained for ZIF-8@RMSN, as evidenced by TEM and SEM images (Figure 2B,C).Nevertheless, a porous shell with a rough surface feature and a thickness of ∼35 nm was observed (Figure S2).The average particle size increased to 174.1 ± 17.3 nm.The core-shell structure of ZIF-8@RMSN was evidenced by the line-scan EDX mapping (Figure 2D), showing a Zn-rich core and silicon-rich shell.
The crystalline structures of ZIF-8 and ZIF-8@RMSN were studied by XRD analysis (Figure 2E).The typical characteristic peaks of ZIF-8 were well remained for ZIF-8@RMSN, indicating the crystalline structure of the ZIF-8 core particle was preserved.In the FTIR spectra of ZIF-8 and ZIF-8@RMSN (Figure 2F), the bands at 2926, 1600, 1145, and 995 cm −1 can be attributed to the C─H, C═N, and C−N bonds in the imidazole ring, respectively.The peak at ∼1044 cm −1 observed only in the spectrum of ZIF-8@RMSN can be assigned to the asymmetric band of Si−O−Si. [48]BAC and ZIF-8@RMSN both exhibited a C−H stretching band at 2854 cm −1 (alkyl groups). [37]XPS was further applied to characterize the composition.The presence of characteristic peaks of Zn, O, N, C, and Si elements is in accordance with the composition of ZIF-8@RMSN (Figure 2G).The zeta potential was measured to study the surface charge change (Table S1).Compared to the positive surface charge of ZIF-8 (+22.6 mV), the zeta potential of ZIF-8@RMSN decreased to −21.3 mV due to the presence of Si−OH.It is suggested that the electrostatic attraction between positively charged ZIF-8 and negatively charged silica oligomers is the driving force for the formation of ZIF-8@RMSN with a core-shell structure. [47]Taken together, the above results demonstrate the successful synthesis of ZIF-8@RMSN nanocomposite.

Acid-responsive release performance of ZIF-8@RMSN
To study the release behavior of ZIF-8@RMSN under neutral and acidic conditions, ICP-OES was conducted for zinc content measurement in the composite and LC-MS for BAC/2-MIM content and release detection.As shown in Table S1, the zinc, BAC, and 2-MIM weight percentages were calculated to be 14.0%, 14.5% (Figure S4A) and 31.4% (Figure S4B), respectively.Further, the release test at pH 7.0 and 5.5 within 48 h was performed considering the acidic environment upon bacterial infection. [5,7]At pH value of 7.0, the decomposition percentages of ZIF-8 and ZIF-8@RMSN were 69.1% and 32.7%, respectively (Figure 2H).At pH 5.5, the decomposition percentages of ZIF-8 and ZIF-8@RMSN increased to 88.0% and 58.5%, respectively.In addition, a burst release within 2 h was observed, thus the samples after 2 h of incubation were collected for TEM characterization.As shown in Figure S5A, almost all ZIF-8 nanoparticles were decomposed with the initial regular morphology lost, while a yolk-shell morphology was observed for ZIF-8@RMSN (Figure S5B).The above results indicate that ZIF-8 and ZIF-8@RMSN exhibit acid-responsive release, and the core-shell structure of ZIF-8@RMSN slows down the fast degradation of ZIF-8, thus the stability of ZIF-8 core is significantly enhanced.
Furthermore, the BAC release under different pH values was investigated (Figure 2I).The acidic responsive release behavior was also observed, similar to the observation in Figure 2H.Although there is a burst release within 2 h, sustained release also exists until 48 h.The sustained release could be attributed to the protection of silica shell, while the higher release percentage of BAC at acidic pH could be explained by the accelerated degradation of silica shell and the release of BAC under acidic conditions rather than neutral. [36]The acid-responsive release characteristics of ZIF-8@RMSN are beneficial in an acidic microenvironment upon bacterial and biofilm infection.
The bactericidal capability was evaluated using the plate counting method (Figure 3B and Figure S7).The least colony was observed for ZIF-8@RMSN among all groups under test (Figure 3B), indicating excellent antibacterial activity of ZIF-8@RMSN, indicating a synergistic antibacterial effect of the BAC and Zn 2+ that are slowly released from the core-shell structure.In addition, cell viability was assessed in Human Embryonic Kidney (HEK239T) cells (Figure S8).Compared with other BAC-treated group (BAC/ZIF-8, BAC/Zn 2+ , and BAC), ZIF-8@RMSN nanocomposites exhibited higher cell viability at all dosages, indicating improved cytocompatiability of core-shell-structured ZIF-8@RMSN.
Subsequently, live/dead bacteria were characterized using SYTO9/PI fluorescence staining. [22]SYTO9 labels all bacteria, while PI can only label dead bacteria and exhibit red fluorescence.As shown in Figures 3C,E,F and S9, negligible or weak red signals were observed from untreated and free BAC-treated groups (BAC/ZIF-8 and BAC/Zn 2+ ), indicative of more live bacteria.In contrast, the ZIF-8@RMSN group possessed the strongest red fluorescence (Figure 3D), show-ing excellent bactericidal properties.In addition, PI can be used as an indicator of bacterial cell membrane integrity, [22] the highest red fluorescence intensity (Figure S10) in the case of ZIF-8@RMSN treatment group indicates that ZIF-8@RMSN significantly enhanced the permeability of the cell membrane.This may be due to the fact that both components, BAC and zinc ions, can be adsorbed on the bacterial surface through electrostatic interactions, causing cell membrane damage.

Antibacterial mechanism of ZIF-8@RMSN
To further study the cell membrane damage induced by ZIF-8@RMSN, SEM and TEM were performed to observe the morphological changes of bacteria with or without treatment.Compared with the intact spherical morphology in the untreated group (Figure 4A), severe bacterial cell membrane damage and cytoplasmic content leakage (indicated by a white arrow) were observed in the ZIF-8@RMSN group (Figure 4B).The morphology change and cell membrane damage in ZIF-8@RMSN group are more obvious than that in BAC/ZIF-8 (Figure 4C) and BAC/Zn 2+ groups (Figure 4D), indicative of more effective bacterial membrane rupture performance.The bacterial membrane damage was also evidenced from TEM image after ZIF-8@RMSN treatment (Figure S11).The BAC and zinc ions can bind to bacterial cell membranes through electronic interactions, causing cell membrane damage. [22,33]Moreover, bacterial membrane damage can prompt intracellular components such as proteins to leak into the surrounding environment from the cytoplasm, causing bacterial dysfunction and ultimately inducing bacterial death.To provide further evidence, BCA kit was used to evaluate the leaked protein amount. [43]The suspension of bacteria in the ZIF-8@RMSN treatment group had the highest protein content (Figure 4E), suggesting its strongest destruction of the bacteria membrane.
The zinc content per bacteria after treatment was also quantified by ICP-OES (Figure S12).The zinc content per bacteria in ZIF-8@RMSN group was 0.85 pg, almost four times that of BAC/ZIF-8 treated bacteria (0.24 pg) and ten times of the BAC/Zn 2+ group (0.083 pg).The above results indicate that the ZIF-8@RMSN group with a rough surface either has enhanced adhesion onto bacteria membrane than ZIF-8 nanoparticles with a smooth surface, or delivers more zinc ions into bacteria.The antibacterial mechanism of zinc ions has been reported to be related with intracellular ROS generation and subsequently damage to biomolecules. [21,22]In order to examine the oxidative stress effect of ZIF-8@RMSN on bacteria, nitro blue tetrazolium (NBT) solution was used to measure intracellular ROS generation. [42,49,50]As shown in Figure 4F, the ROS level was significantly increased in MRSA treated with the ZIF-8@RMSN group compared to other treatment groups and the control group.It is also reported that intracellular ROS generation can cause DNA damage, ultimately leading to cell death. [51]After treatment, DNA was extracted from bacteria and quantified using Nanodrop.As depicted in Figure 4G, the bacterial DNA content was significantly reduced after ZIF-8@RMSN treatment.The DNA damaging effect of ZIF-8@RMSN was further investigated through gel electrophoresis analysis. [52,53]The results are shown in Figure S13A.Compared with the clearly visible DNA bands of the untreated group and the BAC/ZIF-8 treated group, after ZIF-8@RMSN treatment, almost no band was observed.The residue DNA percentage after ZIF-8@RMSN treatment was calculated by Image J, estimated to be 10.2% in comparison with the PBS group (Figure S13B), [54] indicating severe DNA damage and fragmentation.Taken together, ZIF-8@RMSN realized the co-delivery of dual antibacterial components (BAC and zinc ions), which is conducive to cell membrane damage, leakage of cytoplasmic contents, the generation of intracellular ROS and DNA damage, ultimately inducing cell death (Figure 4H).The multifunctional antibacterial mechanisms at different levels of ZIF-8@RMSN may provide valuable insights for the development of advanced antibacterial strategies.

Biofilm inhibition and eradication performance of ZIF-8@RMSN
It has been reported that up to 80% of bacterial infections are caused by bacterial biofilms.During biofilm formation, bacterial cells transform from a planktonic form into bacterial aggregates surrounded by EPS, which poses a great challenge to the treatment of bacterial infections. [10,44]o investigate the potency of ZIF-8@RMSN on biofilm destruction, mature MRSA biofilm was constructed and incubated with different formulations, and then their biofilm eradication performance was monitored.For the in vivo wound healing experiment, a concentration of 7 μg BAC mL −1 was selected based on a literature report. [55]As shown in Figures 5A,B and S14, the colony counting method was utilized to quantitatively examine the number of colonies remaining in the biofilm after nanoparticle treatment.Fewer MRSA colonies were observed in the ZIF-8@RMSN group compared with the BAC/ZIF-8 and BAC/Zn 2+ groups, indicating improved performance of ZIF-8@RMSN in dispersing mature biofilms.In addition, crystal violet staining showed that ZIF-8@RMSN removed 76% of the mature biofilm (Figure 5D), higher than the BAC/ZIF-8 (31%) and BAC/Zn 2+ (26%) groups (Figure S14).To provide direct visualization, three-dimensional reconstructions of residual MRSA biofilm stained with SYTO9 (green, live bacteria) and PI (red, dead bacteria) were performed to characterize biofilm upon various treatments.For the control (Figure 5E), BAC/Zn 2+ (Figure 5F), and BAC/ZIF-8 (Figure 5G) groups, bright green fluorescence and negligible red fluorescence were observed, indicating intact bacterial biofilms.In contrast, the ZIF-8@RMSN group displayed apparent red fluorescence with little green fluorescence throughout the whole biofilm (Figure 5H), indicating excellent bacterial killing and effective biofilm eradication.The biofilm thickness was also measured (Figure 5C).The thickness of untreated biofilm was 31 μm, while a thinner biofilm was observed for BAC/ZIF-8 (18 μm), BAC/Zn 2+ (19 μm) and ZIF-8@RMSN (about 10 μm).The above results demonstrated the excellent eradication performance of ZIF-8@RMSN towards mature biofilm.
In addition, the performance of ZIF-8@RMSN in MRSA biofilm inhibition was also studied (Figure S15).In the ZIF-8@RMSN treatment group, the number of MRSA colonies on the agar plate was reduced by more than 99.9% (Figure S15A,C).Crystal violet staining evaluation results showed that ZIF-8@RMSN inhibited 90% of biofilm formation (Figure S15B,D).3D confocal microscopy also showed more MRSA inhibition and biofilm destruction in ZIF-8@RMSN-treated group (Figure S15E,J).
EPS acts as a physical/chemical barrier in biofilms, hindering antibacterial agents to achieve the antibacterial performance. [8,10]QACs have been reported to bind to the EPS through electrostatic interaction which, disrupt the EPS for easy removal of biofilms. [35]The destructive properties of ZIF-8@RMSN towards EPS were investigated by using wheat germ agglutinin-Alexa Fluor 488 (WGA-AF488) fluorescent conjugate, which specifically binds to poly-Nacetylglucosamine residues in the EPS of MRSA biofilms. [44] visualized via confocal microscopy (Figure 5I-L), following ZIF-8@RMSN treatment, EPS was destroyed into smaller fragments, demonstrating a substantially higher EPS matrix removal effect.EPS treated with BAC/ZIF-8 and BAC/Zn 2+ showed weaker damaging effects, in accordance with the observed biofilm removal performance difference.In addition, eDNA as the main component of EPS to maintain the structural stability of biofilm, [7,56] was also characterized.As depicted in Figure S16, ZIF-8@RMSN demonstrated a superior reduction in the content of eDNA compared with other treatment groups.Collectively, the ZIF-8@RMSN exhibited enhanced eDNA and EPS damage, antibacterial performance and excellent anti-biofilm capacities, and the elimination of biofilms.
Collectively, the above results underscore the excellent biofilm eradication performance of ZIF-8@RMSN.The dual antibacterial components (BAC and zinc ions), co-delivered by ZIF-8@RMSN, synergistically promote the destructive performance of eDNA and EPS, leading to the inhibition of biofilm formation and the elimination of mature biofilm.These findings highlight the potential of ZIF-8@RMSN as a promising strategy against biofilm-related bacterial infections.

Potential application of ZIF-8@RMSN
Due to its unique structure, sustained release profiles, and excellent antibacterial and anti-biofilm properties, ZIF-8@RMSN was loaded on blank sterile gauze through negative pressure filtration to form the ZIF-8@RMSN bandaid. [45]The ZIF-8@RMSN band-aid characterized by SEM and EDS.From the SEM image of the blank gauze (Figure S17A), a clean surface of fiber can be observed, there are almost no signals of silicon (Figure S17B) and zinc elements (Figure S17C) in the EDX mapping.The SEM image of ZIF-8@RMSN band-aid showed nanoparticles adhered to the fibers of the gauze (Figure S17D) with well distributed silicon (Figure S17E) and zinc signals (Figure S17F), indicating the successful preparation of ZIF-8@RMSN antibacterial band-aid.The content of BAC loaded on the band-aid was measured by LC-MS, showing BAC loading efficacy of >90% in all groups (Figure S17I).Meanwhile, the BAC release curve of the band-aid was tested (Figure S17G).The ZIF-8@RMSN band-aid group exhibited a lower BAC release percentage of 75.5% at 12 h than the other groups.The antibacterial performance of the ZIF-8@RMSN band-aid was also evaluated.As shown in Figure S17H, after treating bacteria with different groups for 3 days, the ZIF-8@RMSN band-aid exhibited the highest antibacterial effect at a concentration of 7 μg mL −1 among all groups, suggesting its potential as a long-term antibacterial band-aid.Then, the antibacterial effect of ZIF-8@RMSN band-aid was evaluated in vivo by constructing a MRSA-infected mouse excisional wound model. [44]To establish the infection model, 100 μL of 10 8 CFU mL −1 bacteria were inoculated at the 8 mm wound site and incubated for 24 h to induce infected wounds with MRSA biofilm (Figure 6A).For the in vivo wound healing experiment, a concentration of 7 μg BAC mL −1 was selected based on a literature report. [55]Band-aid with a diameter of ∼1 cm × 1 cm were prepared containing ZIF-8@RMSN, BAC/ZIF-8, and BAC/Zn 2+ at a concentration of 7 μg BAC mL −1 , respectively.Band-aid containing PBS was utilized as control.After the band-aid was replaced on the first day and kept for another 72 h, the mice were sacrificed, and the number of bacteria on the wound wasquantified by the standard plate count method.As shown in Figure 6B, the ZIF-8@RMSN band-aid can effectively reduce bacteria growth at the wound site.From the bacterial counting results (Figures 6C and S18), the number of bacterial colonies observed in the ZIF-8@RMSN group was significantly lower than that in other treatment groups.In order to evaluate the biosafety for ZIF-8@RMSN, the major organs were collected and analyzed by H&E staining after treatment for 4 days.For the ZIF-8@RMSN group, no remarkable pathological abnormalities were observed in H&E staining images of major organs (Figure 6D), suggesting good biocompatibility of ZIF-8@RMSN.The body weight of mice was monitored and recorded daily, with no obvious change in all groups (Figure S19).Meanwhile, the wound appearance in all mice was photographed in a time-dependent manner, showing a smaller wound area in the ZIF-8@RMSN treatment group compared to other treatment groups (Figure S20).

CONCLUSIONS
In summary, the core-shell structured ZIF-8@RMSN nanoparticles for the co-delivery of BAC and zinc ions components to achieve synergistic antibacterial applications were synthesized and demonstrated.The ZIF-8@RMSN exhibited the sustained release of zinc ions and BAC to kill bacteria, increased membrane adhesion and damage, enhanced oxidative stress and damage to DNA, which synergistically contributed to efficient bacterial killing and elimination of mature MRSA biofilms.Moreover, the ZIF-8@RMSN coated band-aid exhibited excellent antibacterial performance in vitro and in vivo against MRSA.However, there are several limitations to the current work.Firstly, the initial burst release of Zn 2+ and BAC may lead to local toxicity or reduced efficacy over time.The in vivo release of antibacterial agents and their long-term biosafety evaluation remain to be fully assessed.Furthermore, the detailed nano-bio interactions should be investigated in other cell lines, such as epithelial cells and immune cells, to better understand the mechanism of wound healing.By providing answers to the remaining questions, our strategy may provide a promising solution for killing drug-resistant bacteria, eliminating biofilm formation, and facilitating wound healing.

A C K N O W L E D G E M E N T S
This work was supported by the Australian Research Council, the National Natural Science Foundation of China (no.32171414), the Natural Science Foundation of Shanghai (no.23ZR1419500), and the Nature Science Foundation of Chongqing, China (no.CSTB2022NSCQ-MSX0461).This work used the Queensland node of the NCRIS-enabled Aus-tralian National Fabrication Facility (ANFF).The authors acknowledge the support from the Centre for Microscopy and Microanalysis, the University of Queensland, and the China Scholarship Council.The authors also thank the ECNU Multifunctional Platform for Innovation (011) for supporting the mouse experiments.

C O N F L I C T O F I N T E R E S T S S TAT E M E N T
The authors declare no conflict of interests.

F I G U R E 4
Antibacterial mechanism of ZIF-8@RMSN.SEM images of MRSA isolates after treatment with (A) PBS, (B) ZIF-8@RMSN, (C) BAC/ZIF-8, (D) BAC/Zn 2+ .(E) The protein leakage from MRSA after different treatments.(F) The ROS level in MRSA isolates after treatment with different groups.(G) The concentration of genomic DNA in MRSA isolates after treatment with different groups.(H) Schematic diagram of the antibacterial mechanism of ZIF-8@RMSN.The concentration of different treatment groups is 5 μg BAC mL −1 , and the same zinc ion concentration is maintained.

F I G U R E 6
In vivo assessments of the antibacterial performance of ZIF-8@RMSN band-aid.(A) Scheme of in vivo study of formulation band-aid in an established MRSA mouse excisional wound model.(B) Digital photos and (C) quantitative analysis of bacterial colonies from wound tissues of different groups on day 4. (D) H&E staining of the major organ sections from mice at day 4 under control and treatment with different groups, scale bar: 100 μm. 2 2