Abstract

   Hematoma, a risk factor of implant‐associated infections (IAIs),
   creates a Fe‐rich environment following implantation, which
   proliferates the growth of pathogenic bacteria. Fe metabolism is a
   major vulnerability for pathogens and is crucial for several
   fundamental physiological processes. Herein, a deferiprone (DFP)‐loaded
   layered double hydroxide (LDH)‐based nanomedicine (DFP@Ga‐LDH) that
   targets the Fe‐rich environments of IAIs is reported. In response to
   acidic changes at the infection site, DFP@Ga‐LDH systematically
   interferes with bacterial Fe metabolism via the substitution of Ga^3+
   and Fe scavenging by DFP. DFP@Ga‐LDH effectively reverses the Fe/Ga
   ratio in Pseudomonas aeruginosa, causing comprehensive interference in
   various Fe‐associated targets, including transcription and substance
   metabolism. In addition to its favorable antibacterial properties,
   DFP@Ga‐LDH functions as a nano‐adjuvant capable of delaying the
   emergence of antibiotic resistance. Accordingly, DFP@Ga‐LDH is loaded
   with a siderophore antibiotic (cefiderocol, Cefi) to achieve the
   antibacterial nanodrug DFP@Ga‐LDH‐Cefi. Antimicrobial and biosafety
   efficacies of DFP@Ga‐LDH‐Cefi are validated using ex vivo human skin
   and mouse IAI models. The pivotal role of the hematoma‐created Fe‐rich
   environment of IAIs is highlighted, and a nanoplatform that efficiently
   interferes with bacterial Fe metabolism is developed. The findings of
   the study provide promising guidance for future research on the
   exploration of nano‐adjuvants as antibacterial agents.

   Keywords: antibiotic adjuvant, biofilm, implant‐associated infection,
   iron metabolism interference
     __________________________________________________________________

   A DFP@Ga‐LDH‐Cefi nanomedicine is designed to target the
   hematoma‐created Fe‐rich environments of IAIs by bacterial Fe
   metabolism intervention. Functioning as a nano‐adjuvant,
   DFP@Ga‐LDH‐Cefi realizes favorable antibacterial properties while
   delaying the emergence of drug‐resistant bacteria. These findings
   provide promising guidance for future research on the exploration of
   antibiotic nanoadjuvants and the complete treatment of IAIs.

   graphic file with name ADVS-11-2400862-g005.jpg

1. Introduction

   Medical implants, particularly joint prostheses, have significantly
   transformed medical practice. Notably, infections originating from
   implanted medical devices are increasing.^[ [36]^1 ^] Pathogenic
   bacteria in IAIs tend to colonize implants as tough biofilms.^[ [37]^2
   ^] These biofilms consist of extracellular polymeric substances,
   rendering them highly resistant to antibiotics.^[ [38]^3 ^]
   Accordingly, treatment of IAIs necessitates extended durations and high
   doses of antibiotics, thus leading to the emergence of
   antibiotic‐resistant bacteria.^[ [39]^4 ^] Presently, devising
   antimicrobial strategies for the comprehensive treatment of IAIs is
   necessary.

   The intervention of the bacterial metabolism holds notable potential
   for achieving effective sterilization while inhibiting the emergence of
   drug‐resistant strains.^[ [40]^5 ^] Among various bacterial metabolic
   processes, Fe metabolism plays a pivotal role in the growth and
   proliferation of bacteria,^[ [41]^6 ^] where Fe serves as a vital
   cofactor for several enzymes.^[ [42]^7 ^] Reduction of Fe concentration
   by the immune system is crucial in the host‐pathogen battle.^[ [43]^8
   ^] Moreover, inhibition of bacterial Fe acquisition could mitigate the
   emergence of antibiotic resistance.^[ [44]^9 ^]

   Surgical implantation invariably causes bleeding, leading to the
   development of a hematoma. Closed suction drainage is essential in
   preventing hematoma formation.^[ [45]^10 ^] However, after the
   abandonment of closed suction drainage and the widespread adoption of
   anticoagulants, the prevalence and severity of hematoma are on the
   rise.^[ [46]^11 ^] When the accumulation of Fe from hematoma surpasses
   the macrophages’ ability to process it, it significantly increases the
   occurrence and severity of IAIs.^[ [47]^12 ^] Additionally, a Fe‐rich
   environment plays a role in the development of antibiotic‐resistant
   bacteria.^[ [48]^13 ^] Consequently, antimicrobial strategies targeting
   bacterial Fe metabolism in hematoma‐created Fe‐rich environments are
   highly promising approaches for tackling IAIs.

   Element substitution has been demonstrated to efficiently disrupt
   bacterial Fe metabolism. Our previous studies have revealed that Se can
   substitute for S in bacterial hydrogen sulfide metabolism, thereby
   perturbing bacterial metabolic processes.^[ [49]^14 ^] Ga^3+, as a Fe
   contender, competes with Fe^3+ to bind to the vital enzymes that
   normally bind to Fe^3+.^[ [50]^15 ^] Moreover, Ga‐containing
   pharmaceuticals are relatively safe as gallium nitrate has been
   approved by the Food and Drug Administration (FDA) for malignant
   hypercalcemia.^[ [51]^16 ^] Nevertheless, the efficacy of substituting
   bacterial Fe with Ga can be attenuated by excessively high Fe
   concentrations and limited distribution of gallium into the infected
   site. Fe chelators could augment the effectiveness of Ga substitution
   in hematoma‐created Fe‐rich environments.^[ [52]^17 ^] Deferiprone
   (DFP), an oral Fe chelator, has been approved by the FDA for
   Fe‐overloaded diseases such as hemochromatosis and thalassemia. By
   scavenging bacterial Fe, DFP could treat Gram‐negative bacterial
   infections.^[ [53]^18 ^] However, it needs an effective delivery
   strategy for the targeted enrichment of DFP and Ga^3+ at the site of
   infection. Among nanomaterials for drug delivery, layered double
   hydroxides (LDHs) are ideal due to their chemical stability,
   biocompatibility, and pH‐dependent solubility. The lamellar structure
   and high surface‐to‐volume ratio of LDHs allow therapeutic agents to be
   intercalated.^[ [54]^19 ^] We hypothesized that doping LDH with Ga^3+
   is a promising DFP delivery system for therapeutic agents and gallium,
   thus achieving accumulation of DFP and Ga^3+ in the infected area. This
   design can reduce systemic side effects of ga‐based antibacterial
   agents and increase the accumulation of gallium at the infection site.

   Herein, we report a systematic Fe metabolism interference strategy
   based on a DFP‐loaded Ga‐doped LDH (Ga‐LDH) nanomedicine (DFP@Ga‐LDH)
   for the treatment of IAIs (Scheme [55]1 ). In response to the acidic
   microenvironment at the infection site, DFP@Ga‐LDH is dissociated to
   release Ga^3+ and DFP, thereby chelating Fe and reversing the
   intracellular Fe and Ga levels. Our experiments indicated that
   200 µg/mL of DFP@Ga‐LDH could significantly reduce Pseudomonas (P.)
   aeruginosa’s Fe content to 19.34%. Multiple Fe‐related targets in P.
   aeruginosa, such as siderophore synthesis and transcription, were
   intervened. Additionally, DFP@Ga‐LDH, as a powerful antibiotic
   adjuvant, demonstrated considerable synergistic antibacterial activity
   with the siderophore antibiotic Cefi for preventing resistant bacteria
   from evolving. Thus, we subsequently introduced Cefi into DFP@Ga‐LDH to
   obtain DFP@Ga‐LDH‐Cefi. Through in vitro, in vivo, and ex vivo
   evaluations, it has been demonstrated that DFP@Ga‐LDH‐Cefi possesses
   superior biocompatibility and antimicrobial efficacy. This work
   highlighted the pivotal role of the hematoma‐created Fe‐rich
   environment of IAIs and developed a nanoplatform that efficiently
   interfered with bacterial Fe metabolism. In addition to its favorable
   antibacterial properties, DFP@Ga‐LDH functions as a nano‐adjuvant
   capable of combating antibiotic resistance, which presents significant
   therapeutic potential in IAIs treatments.

Scheme 1.

   Scheme 1
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   A) The synthesis process of DFP@Ga‐LDH‐Cefi. B) The hematoma formation
   after implantation creating a Fe‐rich environment. Macrophages
   phagocytose RBCs from hematomas, which results in the release of Fe and
   heme from hemoglobin. Heme and Fe could be absorbed by bacteria, thus
   facilitating bacterial proliferation and contributing to the
   development and severity of IAIs. C) The therapeutic mechanism of
   DFP@Ga‐LDH‐Cefi for eradicating IAIs by bacterial Fe metabolism
   intervention. DFP@Ga‐LDH‐Cefi exhibited controlled releases of Ga^3+,
   DFP, and Cefi in acidic IAI environments. The synergistic action of
   Ga^3+ and DFP led to effective element substitution and Fe scavenging,
   causing intervention in bacterial Fe‐related targets such as
   transcription, substance metabolism, and ROS defense.

2. Results and Discussion

2.1. Synthesis and Characterization of DFP@Ga‐LDH‐Cefi

   Schematics of the preparation and antibacterial therapeutic performance
   of DFP@Ga‐LDH‐Cefi are shown in Scheme [57]1. Ga‐doped LDH nanosheets
   were fabricated via a “bottom‐up” approach, in which Ga^3+ was doped in
   LDH host layers (mass content: 11.2%) to realize the relative
   replacement of Fe^3+ by Ga^3+ in the biofilm microenvironment. Specific
   surface areas of Ga‐LDH nanosheets were calculated to be 267.4 m/g
   (Table [58]S1, Supporting Information), offering the possibility of
   efficient loading of small molecules into these nanosheets. In this
   study, Ga‐LDH nanosheets were loaded with DFP to acquire DFP@Ga‐LDH.
   Transmission electron microscopy (TEM) image revealed well‐dispersed
   sheet structures of Ga‐LDH and DFP@Ga‐LDH nanosheets (Figure [59]1A;
   Figure [60]S1, Supporting Information). Elemental mapping demonstrated
   uniform distributions of Mg, Al, Ga, and O in these nanosheets,
   confirming the successful formation of Ga‐LDH nanosheets
   (Figure [61]1B). Moreover, X‐ray photoelectron spectroscopy (XPS)
   profiles indicated two Al^3+ satellite peaks at 74.5 eV and a Mg^2+
   satellite peak at 1303.6 eV, indexed to Al^3+ and Mg^2+ in LDH,
   respectively (Figure [62]S2, Supporting Information). Furthermore, the
   characteristic peaks of Ga 2p at 1117.8 and 1144.6 eV were assigned to
   2p3 and 2p1 of Ga^3+ (Figure [63]1C,D), respectively, suggesting
   successful doping of Ga^3+ into the LDH laminate. Hydrodynamic
   diameters of Ga‐LDH nanosheets were 113.3 ± 3.5 nm, and they increased
   to 119.6 ± 2.8 nm after DFP loading. Zeta potentials of the Ga‐LDH
   nanosheets decreased from 39.7 to 18.7 mV after DFP loading
   (DFP@Ga‐LDH), which was ascribed to the loading of negatively charged
   DFP into these nanosheets (Table [64]S1, Supporting Information).

Figure 1.

   Figure 1
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   Characterization and antibacterial efficacy of Ga‐LDH nanosheets and
   DFP@Ga‐LDH‐Cefi. A) TEM image of Ga‐LDH nanosheets. B) HAADF‐STEM and
   elemental mapping images of Mg, Al, Ga, and O in Ga‐LDH nanosheets. XPS
   data showing the C) survey and D) Ga 2p spectra. E) In vitro cumulative
   release of DFP from DFP@Ga‐LDH‐Cefi at different pH values. F) The
   growth‐inhibitory effects of Ga‐LDH after being supplemented with Fe
   (FeCl[3]). G) Checkerboard microdilution assay for the combination
   between DFP and Ga‐LDH against P. aeruginosa. H) Isobologram of the
   combination between Ga‐LDH and DFP/DFO/DFX. I) The growth inhibition of
   P. aeruginosa after treatment with Ga‐LDH and DFP@Ga‐LDH. J) Division
   times of the attached bacteria with incubation of Ga‐LDH and
   DFP@Ga‐LDH. K) Fe uptake of P. aeruginosa after treatment with Ga‐LDH
   and DFP@Ga‐LDH. L) The Fe/Ga ratio of P. aeruginosa after incubation
   with DFP@Ga‐LDH. M) 3D reconstructions of fluorescence‐labeled P.
   aeruginosa and S. aureus biofilms (Scale bars: 50 µm). N)
   High‐resolution SEM images of P. aeruginosa and S. aureus biofilms
   (Scale bars: 1 µm.) (^** p < 0.01 and ^*** p < 0.001; Data are
   expressed in mean ± SD).

   Moreover, Cefi was conjugated to DFP@Ga‐LDH via a pH‐sensitive imine
   covalent bond, thus yielding the representative antibacterial nanodrug
   DFP@Ga‐LDH‐Cefi. The conjugation of Cefi to DFP@Ga‐LDH exhibited no
   substantial influence on the hydrodynamic diameter of DFP@Ga‐LDH (Table
   [66]S1, Supporting Information). Brunauer–Emmett–Teller (BET) surface
   areas of the Ga‐LDH nanosheets considerably decreased after DFP loading
   and subsequent Cefi conjugation. Changes in the zeta potentials of the
   Ga‐LDH nanosheets after DFP loading and subsequent Cefi conjugation
   further verified the effective preparation of DFP@Ga‐LDH‐Cefi.
   Furthermore, the rate of DFP and Cefi was evaluated by
   thermogravimetric (TG) analysis. The results indicate that the weight
   losses of Ga‐LDH, DFP@Ga‐LDH, DFP@Ga‐LDH‐CHO, and DFP@Ga‐LDH‐Cefi were
   37.33%, 52.45%, 60.82%, and 66.12%, respectively (Figure [67]S3,
   Supporting Information). Thus, the rates of DFP and Cefi in the
   DFP@Ga‐LDH‐Cefi were calculated to be 15.12% and 5.3%, respectively.

   LDH‐based nanomaterials with pH‐responsive biodegradabilities have been
   extensively investigated as pH‐responsive nanocarriers for drug
   delivery. Therefore, we examined the DFP release behavior of
   DFP@Ga‐LDH‐Cefi at different pH values. At pH 7.4, the amount of DFP
   released from DFP@Ga‐LDH‐Cefi was less than 10% after 24 h, and almost
   no Ga^3+ was released from DFP@Ga‐LDH‐Cefi (Figure [68]1E; Figure
   [69]S4, Supporting Information), suggesting high stability of
   DFP@Ga‐LDH‐Cefi in a normal physiological environment. In contrast, at
   pH 6.5 and 5.0, the amounts of released DFP increased to 44.5% and
   68.9% within 8 h. Additionally, the release pattern of Ga^3+ was highly
   pH‐dependent, which was attributed to the dissolution of the LDH
   carrier under acidic conditions. These results further confirmed the
   spatial specificity of DFP@Ga‐LDH‐Cefi for DFP and Ga^3+ released into
   the bacterial microenvironment.

2.2. Significant Antibiofilm Efficacy of DFP@Ga‐LDH Due to Fe Chelation and
Element Substitution

   Ga‐LDH nanosheets exhibit considerable antibacterial effects, as
   evidenced by the MIC against P. aeruginosa is 96 µg mL^−1.
   Bacteriostatic assay further demonstrated that the Ga‐LDH nanosheets
   effectively inhibited the growth of PAO1 in a dose‐dependent manner
   (Figure [70]S5A, Supporting Information). The 24‐h bactericidal assay
   indicated that 250 µg mL^−1 Ga‐LDH nanosheets eradicated 99% of
   stationary‐phase bacteria (Figure [71]S5B, Supporting Information). As
   the antibacterial properties of Ga^3+ relied on its ability to
   substitute bacterial Fe^3+, our study aimed to investigate the impact
   of Fe concentration on the antibacterial efficacies of Ga‐LDH
   nanosheets. FeCl[3] and lactoferrin, a natural chelating agent, were
   separately introduced into Ga‐LDH nanosheets to modify their Fe
   concentrations. Results revealed a notable decline in the antibacterial
   properties of the Ga‐LDH nanosheets upon the addition of FeCl[3]
   (Figure [72]S5C, Supporting Information). Contrarily, lactoferrin
   significantly enhanced the antibacterial properties of the Ga‐LDH
   nanosheets (Figure [73]1F). These findings demonstrate that the
   antibacterial efficiencies of Ga‐LDH nanosheets are influenced by Fe
   concentration, suggesting the potential of utilizing Fe chelators to
   enhance the antibacterial efficacies of Ga‐LDH nanosheets. Defersirox
   (DFX), deferoxamine (DFO), and DFP, which are FDA‐approved Fe
   chelators, are extensively used to treat Fe overload diseases,^[
   [74]^20 ^] and possess inherent antimicrobial activities.^[ [75]^21 ^]
   Checkerboard microdilution assays were performed to analyze the
   synergistic effects of Ga‐LDH nanosheets and Fe chelators, which
   revealed that the combination of Ga‐LDH nanosheets and DFP exhibited
   the highest level of antibacterial activity (Figure [76]1G,H; Figure
   [77]S6, Supporting Information). Therefore, herein, a nanomaterial
   platform, DFP@Ga‐LDH, was constructed by loading Ga‐LDH nanosheets with
   DFP. DFP@Ga‐LDH demonstrated a MIC of 48 µg/mL against P. aeruginosa.
   Moreover, the bactericidal assay verified the significant antibacterial
   activity of DFP@Ga‐LDH as compared to those of Ga‐LDH nanosheets
   (Figure [78]1I; Figure [79]S5D, Supporting Information). These results
   imply superior antibacterial efficacy of DFP@Ga‐LDH to those of Ga‐LDH
   nanosheets.

   Interventions in bacterial Fe metabolism can impede bacterial
   proliferation.^[ [80]^22 ^] Notably, both DFP@Ga‐LDH and Ga‐LDH
   nanosheets demonstrated inhibitory effects on the growth of P.
   aeruginosa, and DFP@Ga‐LDH exhibited more considerable growth
   inhibition (Figure [81]1J). Speculating that DFP@Ga‐LDH intervened in
   Fe metabolism via Fe scavenging and element substitution, we conducted
   the following experiments. ICP‐MS data demonstrated
   concentration‐dependent inhibitory effects of both Ga‐LDH nanosheets
   and DFP@Ga‐LDH on bacterial Fe content. Remarkably, DFP@Ga‐LDH
   exhibited more prominent inhibitory effects on bacterial Fe content
   than those of Ga‐LDH nanosheets (Figure [82]1K). Further investigations
   were performed to assess Fe substitution by DFP@Ga‐LDH. After
   co‐cultivation with DFP@GaLDH and P.aeruginosa, DFP@Ga‐LDH did not
   adhere to bacterial cells(Figure [83]S7, Supporting Information).With a
   gradual increase in the concentration of DFP@Ga‐LDH, the bacterial Fe
   load progressively reduced, whereas the Ga load concomitantly increased
   (Figure [84]1L). These findings validate the effectiveness of chelating
   Fe^3+ and substituting Fe^3+ with Ga^3+ using DFP@Ga‐LDH.

   Inhibiting the adhesion of bacteria to biomaterials can effectively
   suppress biofilm formation, which is a critical aspect of IAIs
   treatment. Effective interventions in bacterial Fe metabolism can
   impede bacterial adhesion.^[ [85]^23 ^] Our results revealed that
   DFP@Ga‐LDH effectively inhibited bacterial adhesion and biofilm
   formation(Figures [86]S8–S10, Supporting Information). Subsequently,
   scanning electron microscopy (SEM) and confocal laser scanning
   microscopy (CLSM) were performed to analyze the effects of DFP@Ga‐LDH
   on biofilm formation. After treatment with DFP@Ga‐LDH, the biofilm was
   significantly disrupted with thin and dispersed features
   (Figure [87]1M). In contrast, in the cases of the control and LDH
   groups, the 3D structures of biofilms remained intact and the bacteria
   were highly active. SEM revealed substantial bacterial densities and
   well‐preserved bacterial morphologies for both the control and LDH
   groups. Nevertheless, the majority of P. aeruginosa and Staphylococcus
   (S.) aureus within the biofilm demonstrated shriveling, distortion, or
   complete lysis after incubation with DFP@Ga‐LDH (Figure [88]1N).
   Antibiofilm efficacy of DFP@Ga‐LDH was evaluated against both P.
   aeruginosa and S. aureus, and a standard crystal violet assay of the
   biofilm biomass indicated that DFP@Ga‐LDH efficiently inhibited biofilm
   formation and eradicated the preformed biofilm (Figures [89]S11, and
   [90]S12, Supporting Information).

2.3. Simultaneous Intervention of DFP@Ga‐LDH in Multiple Fe‐Related Targets
Within P. aeruginosa

   The abovementioned findings confirm the effectiveness of DFP@Ga‐LDH in
   efficiently chelating Fe^3+ and substituting Fe^3+ with Ga^3+. In the
   following investigations, we aimed to elucidate the intervention of
   DFP@Ga‐LDH in precise Fe‐related targets. At first, to investigate the
   global nature of P. aeruginosa following treatment with DFP@Ga‐LDH,
   transcriptomic analysis was conducted(Figure [91]S13A‐C, Supporting
   Information). Compared to the control group, the DFP@Ga‐LDH group
   demonstrated significant differential expression, with 458 upregulated
   and 523 downregulated genes (Figure [92]2A). Gene Ontology (GO) and
   Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, which are
   widely utilized functional enrichment analyses in transcriptomics, were
   employed to predict the potential targets intervened by DFP@Ga‐LDH.
   Notably, GO analysis revealed the substantial effects of DFP@Ga‐LDH on
   crucial biological processes, for example, siderophore biosynthesis,
   ribosomes, metabolism, and electron transport chain, in bacteria
   (Figure [93]2B). Furthermore, KEGG analysis indicated the significant
   impacts of DFP@Ga‐LDH on important pathways, including ribosomes,
   citrate cycle, siderophore biosynthesis, and oxidative phosphorylation,
   in bacteria (Figure [94]2C). These results implied that siderophore
   synthesis, transcription, substance metabolism, and oxidative
   phosphorylation were potential targets for DFP@Ga‐LDH.

Figure 2.

   Figure 2
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   DFP@Ga‐LDH alters multiple Fe‐dependent targets. A) Volcano plots of
   differentially expressed genes (gray: genes that are not significantly
   different; red: upregulated genes; and blue: downregulated genes). B)
   GO enrichment analysis of transcriptomics. C) KEGG enrichment analysis
   of transcriptomics. D) Heat map for the relative abundance of
   metabolites in P. aeruginosa upon DFP@Ga‐LDH treatment. E) KEGG
   enrichment analysis of metabolomics. F) Analysis of mRNA levels of P.
   aeruginosa treated with or without DFP@Ga‐LDH. G) Real‐time changes in
   the oxygen consumption rate of P. aeruginosa after treatment with
   DFP@Ga‐LDH. H) ATP content of P. aeruginosa under DFP@Ga‐LDH treatment.
   I) The activity of Catalase and SOD after treatment with DFP@Ga‐LDH. J)
   Schematic of the Fe‐related targets of DFP@Ga‐LDH. (^* p < 0.05, ^**
   p < 0.01, and ^*** p < 0.001; ns means no significance. Data are
   expressed in mean ± SD).

   Alterations in substance metabolism can influence the efficiencies of
   antimicrobial agents.^[ [96]^24 ^] Metabolomics was employed to examine
   alterations in the metabolite abundance of P. aeruginosa induced by
   DFP@Ga‐LDH(Figure 13D‐E, Supporting Information). After a 4‐h
   intervention, the DFP@Ga‐LDH‐treated group exhibited a significant
   increase in the abundance of diverse metabolites (Figure [97]2D),
   accompanied by a concomitant elevation in intracellular adenosine
   triphosphate (ATP) levels (Figure [98]2H). The observed accumulation of
   multiple metabolites signified that metabolic rate and energy
   utilization decreased after DFP@Ga‐LDH treatment. Pathway enrichment
   analysis confirmed the substantial suppression of substance metabolism
   by DFP@Ga‐LDH (Figure [99]2E).

   Ribonucleic acid (RNA) polymerase, a Fe‐containing enzyme, is possibly
   responsible for the observed transcriptional repression.^[ [100]^25 ^]
   The reduced expression of housekeeping genes in P. aeruginosa confirmed
   the transcriptional inhibition caused by DFP@Ga‐LDH, as shown in
   Figure [101]2F. This target is the same as that of the canonical
   antibiotic rifampicin. Oxygen consumption rate (OCR) was used as an
   indicator of electron transport chain activity because oxygen is the
   terminal electron acceptor. DFP@Ga‐LDH supplementation progressively
   decreased the OCR of P. aeruginosa (Figure [102]2G), indicating
   inhibition of bacterial respiratory activity. Bacterial reactive oxygen
   species (ROS)‐scavenging enzymes, such as Fe‐superoxide dismutase and
   catalases, play critical roles as antioxidant enzymes. The addition of
   DFP@Ga‐LDH resulted in a 70% decrease in bacterial catalase activity,
   however, no significant alteration was noticed in bacterial superoxide
   dismutase activity (Figure [103]2I). These findings suggest that
   DFP@Ga‐LDH can weaken the bacterial ROS defense mechanism, rendering
   the bacterium more susceptible to oxidative stress, and various
   antibiotics, clinical oxidants, and photodynamic therapy demonstrate
   the same mechanism.^[ [104]^26 ^]

   According to the preceding discussion, a schematic depicts the observed
   outcomes, which reveals that DFP@Ga‐LDH exerts a discernible impact on
   multiple Fe‐related processes, including transcription, substance
   metabolism, oxidative phosphorylation, siderophore synthesis, and
   defense against ROS, in P. aeruginosa (Figure [105]2J).

2.4. DFP@Ga‐LDH: A Nano‐Adjuvant with Considerable Antiresistant Activity

   Prolonged treatment of IAIs facilitates the emergence of drug‐resistant
   bacteria, which commonly lead to treatment failure.^[ [106]^27 ^] The
   aforementioned findings provide compelling evidence that DFP@Ga‐LDH
   simultaneously targets multiple Fe‐related processes. This mode of
   intervention targeting bacterial Fe metabolism holds the potential in
   delaying the progression of antibiotic resistance.^[ [107]^28 ^]
   Subsequent experiments demonstrated the ability of DFP@Ga‐LDH to
   effectively suppress the emergence of drug‐resistant bacteria (Figure
   [108]3A). Spontaneous resistance refers to the ability of bacteria to
   naturally develop drug resistance. Our findings revealed that ≈1 in 80
   million P. aeruginosa spontaneously developed resistance to DFP@Ga‐LDH,
   which was lower than the resistance frequencies observed for several
   tested classical antibiotics (Figure [109]3B). Moreover, serial passage
   offers valuable insights into the dynamics of drug resistance
   development.^[ [110]^29 ^] Notably, after serial passage, the
   development of resistance to DFP@Ga‐LDH was substantially slower than
   those in the cases of conventional antibiotics (Figure [111]3C). The
   combination of DFP@Ga‐LDH with Cefi, tazobactam, and tobramycin
   significantly reduced the prevalence of resistant bacteria
   (Figure [112]3D; Figure [113]S15, Supporting Information). Next, a
   checkerboard microdilution assay was used to investigate the combined
   antibacterial effects of DFP@Ga‐LDH and the tested antibiotics. The
   combination of DFP@Ga‐LDH with Cefi and tazobactam exhibited
   synergistic effects, and this observation was supported by the
   time‐kill curves. However, DFP@Ga‐LDH demonstrated antagonistic effects
   against tobramycin (Figure [114]3E–G; Figure [115]S16, Supporting
   Information). Oxidizing agents are extensively employed in skin
   disinfection and debridement surgeries.^[ [116]^30 ^] In accordance
   with the previous findings substantiating the inhibitory effect of
   DFP@Ga‐LDH on catalase activity, hydrogen peroxide (H[2]O[2]) exhibited
   high sensitivity against P. aeruginosa in the presence of DFP@Ga‐LDH.
   Contrarily, DFP@Ga‐LDH did not demonstrate high sensitivity to
   tert‐butyl (Figure [117]3H).

Figure 3.

   Figure 3
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   DFP@Ga‐LDH functions as a nano adjuvant capable of delaying the
   emergence of drug resistance and sensitizing antibacterial antibiotics.
   A) Schematic illustrating the mechanism of DFP@Ga‐LDH as a
   resistance‐modifying nano‐adjuvant. B) Spontaneous resistance of
   DFP@Ga‐LDH and classic antibiotics. C,D) Resistance acquisition curves
   with the subinhibitory concentration of representative antibiotics (C)
   or combination of representative antibiotics and DFP@Ga‐LDH (D) against
   P. aeruginosa. (MIC test was performed every two passages.) E)
   Isobolograms of the combinations between antibiotics and DFP@Ga‐LDH. F)
   Checkerboard microdilution assay for the combinations between
   DFP@Ga‐LDH and representative antibiotics. G) The synergistic effects
   of DFP@Ga‐LDH in combination with cefiderocol and tobramycin. H) Effect
   of DFP@Ga‐LDH on the sensitivity of P. aeruginosa to H[2]O[2] and
   tert‐butyl. (^*** p < 0.001; ns means no significance).

   In conclusion, DFP@Ga‐LDH as an antibiotic adjuvant suppressed the
   emergence of drug‐resistant bacteria and enhanced the bactericidal
   efficiencies of conventional antibiotics and clinical oxidants.

2.5. Validation of Antimicrobial Efficacy and Biosafety of DFP@Ga‐LDH‐Cefi

   Among the antibiotics tested herein, DFP@Ga‐LDH exhibited a prominent
   synergistic effect with the novel siderophore cephalosporin Cefi. This
   synergistic effect can be attributed to the intervention in bacterial
   Fe metabolism by DFP@Ga‐LDH, which enhanced siderophore synthesis
   (Figure [119]S14, Supporting Information). Utilizing the considerable
   drug‐loading capacities of LDH materials, we successfully conjugated
   Cefi to DFP@Ga‐LDH, resulting in DFP@Ga‐LDH‐Cefi.

   In vitro experiments demonstrated that DFP@Ga‐LDH‐Cefi outperformed
   DFP@Ga‐LDH in combating both planktonic bacteria and biofilms (Figure
   [120]4A–C,E; Figures [121]S17–S19, Supporting Information). Bacterial
   adhesion is an initial and crucial step in biofilm formation, and the
   effective inhibition of bacterial adhesion is paramount for preventing
   IAIs.^[ [122]^31 ^] DFP@Ga‐LDH‐Cefi substantially decreased the
   adhesion of both P. aeruginosa and S. aureus to the biomaterials
   (Figure [123]4D). An ex vivo human skin model was established to
   evaluate the antimicrobial efficacy and biocompatibility of
   DFP@Ga‐LDH‐Cefi.^[ [124]^32 ^] The experimental procedure is outlined
   in Figure [125]4F. The DFP@Ga‐LDH‐Cefi group demonstrated a comparable
   percentage of viable cells to the control group, indicating the
   biosafety of DFP@Ga‐LDH‐Cefi (Figure [126]4J). SEM images clearly
   indicated that treatment with DFP@Ga‐LDH‐Cefi effectively suppressed
   biofilm formation, as evidenced by the reduced presence of S. aureus
   and diminished extracellular matrix as compared to the case of the
   control group (Figure [127]4G). After culturing the excised skin wounds
   under aseptic conditions, hematoxylin and eosin (H&E) and Masson
   stainings were conducted, and results revealed that the control group
   exhibited no signs of wound re‐epithelialization due to severe S.
   aureus infection. In contrast, the DFP@Ga‐LDH‐Cefi group, in which S.
   aureus infection was effectively suppressed, demonstrated noticeable
   wound re‐epithelialization (Figure [128]4H,I). The antibacterial
   efficacy of DFP@Ga‐LDH‐Cefi was confirmed using the plate counting
   method, which involved the detection of viable bacteria in wound tissue
   homogenates. Results indicated that the bacterial load in the
   DFP@Ga‐LDH‐Cefi group was significantly low as compared to that in the
   control group (Figure [129]4K,L). Expression levels of the inflammatory
   factors in skin tissue serve as indicators of the severity of bacterial
   infection. Following treatment with DFP@Ga‐LDH‐Cefi, the skin tissue
   exhibited substantially reduced expression levels of tumor necrosis
   factor‐α (TNF‐α), interleukin (IL)−6, and IL‐8 as compared to those in
   the case of the control group. This suggests that DFP@Ga‐LDH‐Cefi
   effectively inhibits bacterial infection in the skin tissue. Healing of
   infected wounds reflects the efficacy of the drug in controlling skin
   infections. Notably, the expression levels of transforming growth
   factor‐beta (TGF‐β), keratin (Krt)17, and Krt16 in the DFP@Ga‐LDH‐Cefi
   group were significantly high, further highlighting the positive impact
   of DFP@Ga‐LDH‐Cefi on wound healing and infection control
   (Figure [130]4M–O). The preceding discussion indicates outstanding
   biocompatibility and antibacterial properties of DFP@Ga‐LDH‐Cefi in the
   ex vivo human skin model. To ensure the biocompatibility of
   DFP@Ga‐LDH‐Cefi for further applications, the toxicities of DFP@Ga‐LDH
   and DFP@Ga‐LDH‐Cefi were systematically assessed.

Figure 4.

   Figure 4
   [131]Open in a new tab

   Validation of the antimicrobial efficacy and biosafety of
   DFP@Ga‐LDH‐Cefi. A) Representative images of 3D reconstructions of P.
   aeruginosa biofilm stained with SYTO 9/PI (Scale bar: 50 µm). B)
   Representative SEM images of P. aeruginosa and S. aureus biofilms. C)
   24‐h killing assays of P. aeruginosa and S. aureus conducted using
   DFP@Ga‐LDH‐Cefi. D) Number of adherent viable bacteria after incubation
   with DFP@Ga‐LDH‐Cefi. E) DFP@Ga‐LDH‐Cefi inhibits P. aeruginosa growth
   in a concentration‐dependent manner. F) Schematics for the preparation
   of an ex vivo human skin model and experimental process. G) SEM images
   of wound bed after various treatments (Scale bar: 5 µm). H,I) H&E
   staining (H) and Masson staining (I) of the wound bed after several
   treatments (Scale bar: 250 µm). J) Cell viability of skin tissue after
   treatment with DFP@Ga‐LDH‐Cefi. K) Quantification of viable bacteria.
   L) Representative images of SPM from S. aureus after DFP@Ga‐LDH‐Cefi
   treatment. M) Gene expression of pro‐inflammatory and pro‐reparative
   cytokines. N) Quantification of TNF‐α and O) TGF‐β proteins in culture
   media. (^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^****
   p < 0.0001; ns means no significance. Data are expressed in mean ± SD).

   Immune cells, especially macrophages, could take up gallium. These
   facts raise concern that DFP@Ga‐LDH could negatively affect the
   efficiency of antimicrobial immunity. Our findings indicate that the
   antimicrobial immunity was not adversely impacted by DFP@Ga‐LDH. The
   viability of human monocyte‐derived macrophages (HMDM) was not reduced
   by DFP@Ga‐LDH treatment (Figure [132]S20A, Supporting Information). We
   also investigated the impact of continuous exposure to DFP@Ga‐LDH
   (1 mg mL^−1) for 24 h on macrophage antimicrobial activity. Our
   findings indicate that this treatment sightly enhances the ability of
   macrophage in P. aeruginosa killing (Figure [133]S20B, Supporting
   Information). In addition, DFP@Ga‐LDH had a modest impact on M1
   polarization (Figure [134]S20C, Supporting Information). In vitro
   cytotoxicity tests and in vivo toxicity evaluations revealed no
   substantial toxicities of DFP@Ga‐LDH and DFP@Ga‐LDH‐Cefi (Figures
   [135]S21–S27, Supporting Information).

2.6. In Vivo Anti‐Infection Evaluations of DFP@Ga‐LDH‐Cefi Using a Mouse IAI
Model

   Following implantation, the Fe concentration around the implant
   elevates to 141.8 µg g^−1 tissue, while in normal tissues it is
   48.3 µg/g tissue. This indicates the existence of a hematoma‐created
   Fe‐rich environment (Figure [136]S28, Supporting Information). The
   abovementioned experiments demonstrated the excellent antibacterial
   ability of DFP@Ga‐LDH‐Cefi, which prompted us to further investigate
   the antibiofilm efficacy of DFP@Ga‐LDH‐Cefi in a mouse model of IAIs. A
   schematic illustrating the development of the mouse IAIs model and
   corresponding treatment is depicted in Figure [137]5A. On day −2,
   sterile polyetheretherketone (PEEK) sheets were immersed in a
   suspension of P. aeruginosa to facilitate biofilm synthesis. On day 0,
   the PEEK sheets coated with biofilms were implanted into all mice and
   then randomly divided into four experimental groups: control, Ga‐LDH,
   DFP@Ga‐LDH, and DFP@Ga‐LDH‐Cefi groups. Different types of
   nanomedicines were administered on days 0, 3, and 5 for treatment.
   After sacrificing the mice, tissue samples were acquired from the
   infection site for histological and quantitative analyses. On 7 and 14
   days after PEEK sheet implantation, macroscopic images of the skin
   tissue with subcutaneous implants were obtained (Figure [138]5B). In
   the control and Ga‐LDH groups, considerable swelling and suppuration
   were observed, indicating inflammatory responses at the abscess sites.
   In contrast, the DFP@Ga‐LDH group exhibited minimal swelling of the
   scabby skin tissue. Notably, the DFP@Ga‐LDH‐cefi group demonstrated no
   swelling, suppuration, or wound festering, implying its superior
   antibiofilm activity. Blood tests were performed to analyze the
   inflammatory markers in the mouse blood (Figure [139]5C). Compared with
   the normal group, the control group exhibited elevated levels of white
   blood cells, lymphocytes, monocytes, and neutrophil granulocytes.
   Nevertheless, the DFP@Ga‐LDH‐Cefi group demonstrated slightly elevated
   levels of these cells, indicating alleviation of the systemic
   inflammatory response. Bacterial loads of PEEK plates and peripheral
   tissues after the sacrifice of mice were evaluated by estimating the
   number of colonies of viable bacteria. As expected, the numbers of
   bacterial colonies in the DFP@Ga‐LDH and DFP@Ga‐LDH‐Cefi groups were
   substantially lower than those in the other two groups
   (Figure [140]5D). To assess the involvement of neutrophils in
   inflammation development tissue repair and inflammatory recovery, the
   two inflammatory biomarkers myeloperoxidase (MPO) and
   n‐acetylglucosaminidase (NAG) were quantified in the tissue,
   respectively. Results revealed that the DFP@Ga‐LDH‐Cefi group exhibited
   the highest activities of macrophages, indicating the presence of
   tissue repair processes, and minimal inflammatory reactions were
   observed in this group (Figure [141]5E). The H&E staining revealed
   significant neutrophil infiltration in the tissue surrounding the
   implant in the control group, indicating a pronounced inflammatory
   response induced by bacterial infection. However, this inflammatory
   response was effectively inhibited by the treatment of DFP@Ga‐LDH‐Cefi
   (Figure [142]5F). Giemsa staining revealed the presence of bacteria in
   tissues. In the DFP@Ga‐LDH‐Cefi group, the stained bacteria were
   significantly fewer than those in the other groups (Figure [143]5G). In
   the DFP@Ga‐LDH‐Cefi group, the peri‐implant soft tissue demonstrated
   complete healing without infiltration of macrophages and neutrophils,
   as evidenced by tissue immunofluorescence analysis (Figure [144]5H).
   Overall, our findings confirmed that DFP@Ga‐LDH and DFP@Ga‐LDH‐Cefi
   effectively alleviated IAIs.

Figure 5.

   Figure 5
   [145]Open in a new tab

   In vivo anti‐infection evaluations of nanodrugs using the mouse IAI
   model. A) Timeline illustration of the mouse IAI model. B)
   Representative images of the local infection site at different time
   points (Scale bar: 25 mm). C) Routine blood test at 7 and 14 days. D)
   Quantification of viable bacteria in peri‐implant soft tissues and
   implants. E) Quantification of the inflammatory cell biomarkers in
   peri‐implant soft tissues. F) Representative H&E staining images of the
   histologic sections of wounds in various groups at 14 days (Scale bar:
   200 µm). G) Giemsa staining of peri‐implant soft tissues visually
   indicated the matched bacterial load (Scale bar: 200 µm). H)
   Representative images of the immunofluorescence‐stained sections of
   peri‐implant soft tissues presenting different functional cells (Scale
   bar: 100 µm, Green: MPO for neutrophils, Red: F480 for macrophages, and
   Blue: DAPI for nuclei) (^** p < 0.01 and ^*** p < 0.001. Data are
   expressed in mean ± SD).

3. Conclusion

   In summary, herein, we successfully prepared DFP@Ga‐LDH and its
   Cefi‐loaded counterpart, DFP@Ga‐LDH‐Cefi. DFP@Ga‐LDH‐Cefi exhibited
   controlled releases of Ga^3+, DFP, and Cefi in acidic IAI environments.
   The synergistic action of Ga^3+ and DFP led to effective Fe
   substitution and Fe scavenging, causing intervention in bacterial
   Fe‐related targets such as transcription and substance metabolism.
   Moreover, the considerable antimicrobial activity of DFP@Ga‐LDH‐Cefi
   along with its ability to counteract drug resistance renders it highly
   promising for the treatment of IAIs. We anticipate that the
   investigation of bacterial Fe metabolism intervention in the Fe‐rich
   environments around implants along with the conceptualization of
   pharmaceutical design involving element chelation and substitution will
   serve as a source of inspiration in the fields of pharmaceutical
   sciences, materials engineering, and nanomedicine.

4. Experimental Section

Clinical Samples

   Skin samples were acquired from patients undergoing surgery at Shanghai
   Sixth People's Hospital. Only remnant clinically healthy skin that was
   not needed for diagnostic purposes was analyzed after obtaining written
   informed consent from the patients and approval from the Ethics
   Committee of Shanghai Sixth People's Hospital in accordance with
   current regulations and institutional guidelines. The approval number
   is 2022‐KY‐139(K).

Synthesis of Ga‐LDH Nanosheets

   Ga‐LDH nanosheets were synthesized by a bottom‐up method. Briefly,
   Al(NO[3])[3]·9H[2]O (3 mmol), Mg(NO[3])[2]·6H[2]O (6 mmol), and
   Gd(NO[3])[3]·6H[2]O (0.1 mmol) were dissolved in deionized water
   (10 mL). Then, NaNO[3] solution (1.5 mmol, 25% formamide) and 30 mL
   NaOH solution (1.5 mm) were slowly added to the resulting solution
   followed by stirring for 30 min. The resulting mixture was centrifuged
   (13 000 rpm) and washed twice with deionized water to obtain the Ga‐LDH
   nanosheets.

Preparation of DFP@Ga‐LDH and DFP@Ga‐LDH‐Cefi

   For surface modification, the as‐synthesized Ga‐LDH nanosheets (20 mg)
   were mixed with polyethylene glycol‐aldehyde (PEG‐CHO) aqueous solution
   (10 mL, molecular weight (MW): 3000) under vigorous stirring for 2 h.
   To remove excess and unbound PEG‐CHO, the resulting product Ga‐LDH‐CHO
   was acquired by centrifugation and washed several times with water and
   ethanol. For DFP loading, Ga‐LDH‐CHO dispersion was mixed with DFP
   solution at various DFP/Ga‐LDH‐CHO molar ratios (from 1:2 to 2:1)
   followed by stirring at room temperature for 12 h. The resulting
   product DFP@Ga‐LDH was obtained by centrifugation and then washed three
   times with deionized water, followed by redispersion in water for
   further use. Thereafter, DFP@Ga‐LDH was conjugated with Cefi via
   pH‐sensitive imine covalent bonds according to the previous study.^[
   [146]^33 ^] Initially, Cefi (5 mg) was pre‐dissolved in water (10 mL)
   followed by the addition of
   1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (20 mg) and
   N‐hydroxysuccinimide (45 mg) under ultrasonication. After being stirred
   for 24 h, the mixture was dialyzed against water (cutoff MW: 3500 Da)
   for 3 days and redispersed in 5 mL ultrapure water. Subsequently, the
   as‐prepared DFP@Ga‐LDH dispersion and activated Cefi solution were
   mixed followed by the addition of NaOH (1 m) to adjust the pH value to
   8.5 and stirring for 24 h. After centrifugation at 13 000 rpm three
   times, DFP@Ga‐LDH‐Cefi was achieved and redispersed for further use.
   Moreover, in vitro DFP release from DFP@Ga‐LDH was analyzed at varying
   pH values (7.4, 6.5, and 5.0) by a dialysis method. The absorbance of
   the released solution at 278 nm was measured. Additionally, 1 mL of
   another sample was acquired and examined using ICP‐OES to quantify the
   released Ga^3+.

Resistance Development by Sequential Passaging

   Continuous passaging was performed in 96‐well plates, with one
   replicate per plate, to avoid cross‐contamination. BM2 medium was
   prepared using each agent at eight concentrations in twofold dilutions,
   and 5 µL PAO1 was introduced into each well. After overnight
   incubation, the highest concentration of the agent that allowed
   bacterial growth was recorded. A 5 µL aliquot of the culture from this
   well was transferred to a fresh medium containing the agent, and this
   process was repeated for 12 days. Control replicates were passed
   without this agent. A total of 12 replicates were passaged for each
   agent for 12 days.

Mutation Rate Assays

   In this experiment, PAO1 was grown to the mid‐log phase and then
   resuspended in a fresh medium. Aliquots of successive dilutions of the
   overnight cultures grown in LB media were placed on BM2 plates without
   and with different antibacterial agents at concentrations four times
   MIC for each agent. After 24 h of incubation, the number of colonies
   growing on the plates was counted. Thereafter, mutation frequency was
   calculated as the fraction of resistant mutant cells relative to the
   total number of cells.

Ex Vivo Human Skin Model of Wound Infection

   Skin samples were washed with 3xABAM and phosphate‐buffered saline
   (PBS) after the removal of adipose tissue. Subsequently, circular
   wounds were created using 3 mm diameter biopsy punch tools, and the
   wound and rim of the tissue were excised using 8 mm punch tools. After
   being washed twice with Dulbecco's modified Eagle medium (DMEM)
   supplemented with 10% fetal bovine serum (FBS), the skin explants were
   individually cultured in 1 mL culture medium, and the epithelium was
   maintained at the air/liquid interface. DMEM supplemented with 10% FBS
   was used as the culture medium. S. aureus was incubated at 37 °C in
   tryptic soy broth normalized to a concentration of 10^6 colony‐forming
   units (CFUs) mL^−1. Thereafter, the skin wounds were inoculated with
   10 µL bacterial inoculum (10 000 CFUs) and various nanodrugs. The skin
   explants were cultured for 48 h, and the medium was changed every 12 h
   to facilitate biofilm formation and select adherent bacteria. After
   48 h, the skin wounds were obtained for analysis.

Bacterial Enumeration in Skin

   At first, a quarter of each wound was placed in sterile PBS and
   homogenized on ice using a Tissue‐Tearor at maximum speed for 25 s.
   Then, the resulting homogenate was serially diluted in triplicate from
   10^−1 to 10^−5. The diluted samples were placed on tryptic soy agar
   plates followed by incubation at 37 °C for 24 h. Colonies that formed
   on the plates were counted, and CFUs were quantified by
   back‐calculation based on the dilution factor. CFU represents the
   estimated number of viable bacteria in the original wound sample.

Assessment of Tissue Viability

   In this experiment, wound tissues were incubated ex vivo for 48 h and
   then minced using surgical scissors. The resulting tissue fragments
   were treated with dispase II solution at 37 °C for 30 min, followed by
   incubation with 2 mg mL^−1 collagenase D under constant agitation for
   3 h at 37 °C to obtain single‐cell suspensions. The resulting cell
   suspensions were washed with DMEM supplemented with 10% FBS,
   L‐glutamine (2 mm) 0.15% hydrogen carbonate, sodium pyruvate (1 mm),
   nonessential amino acids, and 100 µg/mL gentamycin. The cells were
   labeled using the LIVE/DEAD Fixable Yellow Dead Cell Stain Kit to
   distinguish between live and dead cells. The percentage of live cells
   was determined by acquiring ≈2 00 000 events from each sample using a
   BD Fortessa flow cytometer equipped with lasers emitting at 405, 488,
   642, and 785 nm. These data provide valuable information on the
   viability of cells in wound tissue samples.

Relative Messenger RNA (mRNA) Expression

   The sample was crushed, and RNA was extracted according to the protocol
   of the Qiagen RNeasy Kit. After extraction and purification,
   complementary deoxyribonucleic acid (cDNA) was generated using the
   High‐Capacity cDNA Reverse Transcription Kit (Applied Biosystems,
   Foster City, CA) and SuperScript III Reverse Transcriptase Kit
   (Invitrogen, Carlsbad, CA). Changes in gene expression were determined
   using the CFZX384 Touch Real‐Time Fluorescence Quantitative PCR
   Detection System.

Preparation of PEEK Sheets

   Briefly, at first, PEEK was cut into circular sheets with diameters of
   10 mm and thicknesses of 0.5 mm. These circular sheets were sterilized
   and transferred to a six‐well plate. Next, a 2 mL suspension of P.
   aeruginosa (10^4 CFU/mL) was introduced onto the aforementioned plate.
   Subsequently, the plate was incubated under conditions of 5% CO[2] and
   a temperature of 37 °C for a duration of 2 days in order to facilitate
   the growth of bacterial biofilm. The medium was refreshed daily. After
   2 days, the PEEK sheets were acquired and used for the construction of
   a mouse IAI model.

Mouse IAI Model

   Animal experiments for this study were approved by the Animal Care and
   Experiment Committee of Shanghai Jiao Tong University Affiliated Sixth
   People's Hospital. The assigned approval/accreditation number of the
   laboratory/investigator is DWLL2021‐0674. Herein, 32 BALB/c mice were
   equally and randomly divided into the following four groups: control,
   Ga‐LDH, DFP@Ga‐LDH, and DFP@Ga‐LDH‐Cefi groups. A mouse IAI model was
   used to evaluate the in vivo antibiofilm ability of DFP@Ga‐LDH‐Cefi.
   Briefly, the mice were anesthetized with 1% pentobarbital sodium, and
   the skin on their backs was shaved, disinfected, and incised.
   Subsequently, the prepared PEEK sheets with biofilms were
   subcutaneously implanted, and the skin wounds were immediately sutured.
   After surgical implantation, the mice in different groups were injected
   with 1 mL of the corresponding solution via the tail vein (1 mL saline
   for the control group and 1 mL corresponding nanodrugs for the other
   three groups) at a dose of 2 mg/100 g body weight. The injection was
   repeated on days 3 and 5 after the first injection. Changes at the
   infection site were daily observed until the mice were sacrificed.
   Images of the infected sites were acquired on days 7 and 14. All mice
   were sacrificed on day 14.

Statistical Analysis

   In all the experiments, at least three replicates were included, and
   data were expressed as mean ± standard error of the mean (SEM). The
   differences between the two groups were analyzed by independent sample
   t‐test. Differences among multiple groups were examined using a one‐way
   analysis of variance followed by a Tukey post‐test. Statistical
   significance was indicated as p < 0.05 and expressed as ^* p < 0.05,
   ^** p < 0.01, and ^*** p < 0.001.

Conflict of Interest

   The authors declare no conflict of interest.

Supporting information

   Supporting Information
   [147]ADVS-11-2400862-s001.pdf^ (2.4MB, pdf)

Acknowledgements