Biomimetic metal-drug coordination nanoplatform to counteract drug resistance in Pseudomonas aeruginosa via energy disruption

Domain: neuroscience
Published: 2026-02-15
Editorial outcome: minor_revision

Abstract
The emergence of antimicrobial resistance is a critical global health challenge that renders conventional antibiotics ineffective. Developing innovative strategies to resensitize drug-resistant pathogens to existing antibiotics represents a promising therapeutic approach. Here, we present a biomimetic nanoplatform (Ce-Car@EVNPs) through the rational integration of ginger-derived extracellular vesicles (EVs) and a pH-responsive cerium-carbenicillin coordination nanoparticles (Ce-CarNCPs). This design enables prolonged circulation and targeted degradation in acidic infection sites, releasing Ce4+ ions and carbenicillin. The released Ce4+ ions penetrate the bacterial cells, where they disrupt ATP synthesis, impede oxidative phosphorylation, and inhibit the activity of efflux pump. By depleting ATP, blocking efflux pumps, and thereby reversing bacterial resistance, Ce4+ ions act as a potent adjuvant to carbenicillin. Here we show that this strategy effectively restores carbenicillin efficacy against drug-resistant Pseudomonas aeruginosa both in vitro and in vivo, establishing a therapeutic strategy leveraging metallic adjuvants to counteract antimicrobial resistance.

Introduction
Infections caused by pathogenic bacteria can result in various diseases including pulmonary infections, septic wounds, sepsis, and bacterial meningitis, posing a severe threat to global health1–4. Although antibiotics-mediated therapeutic strategies are regarded as the standard treatment for bacterial infections, their overuse and misuse have accelerated the development of antimicrobial resistant (AMR) bacterial strains, particularly in Gram-negative bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), a high-priority pathogen known for its intrinsic resistance mechanisms5. Typically, P. aeruginosa is the leading pathogenic bacteria of respiratory infections, especially in immunocompromised individuals, such as cystic fibrosis patients, which ranks among the top respiratory pathogens in clinical settings according to the 2022 CHINET report6,7. Current treatments for P. aeruginosa infection rely on β-lactams, aminoglycosides, and fluoroquinolones8. However, the low outer membrane permeability and high efflux pump expression of pathogens contribute to both intrinsic and acquired resistance, resulting in the impairment of their therapeutic efficacy9. Notably, little progress in developing new antibiotics targeting Gram-negative bacteria has been made since the 1980s10, thus emphasizing the urgent need for optimized strategies to improve the clinical efficacy of existing antibiotics.

As the universal energy currency of bacteria, adenosine triphosphate (ATP) serves as the most immediate energy source for essential cellular functions, including DNA replication, protein synthesis, and active transport11. Importantly, ATP plays a dual pivotal role in bacterial antibiotic resistance, acting as both the core energy currency driving drug efflux via efflux pumps and a key signaling molecule regulating the expression of resistance genes12,13. This mechanistic insight reveals a promising therapeutic opportunity by achieving targeted depletion of intracellular ATP through an energy deprivation strategy, which can simultaneously inhibit efflux pump function and disrupt the resistance gene network, thereby resensitizing drug-resistant pathogens to conventional antibiotics and reversing bacterial resistance. Recent research has verified that cerium, a redox-active lanthanide metal, exhibits phosphatase-mimicking activity due to its multivalent states, demonstrating the capacity to selectively hydrolyze ATP and ADP14,15. This unique property enables the design of cerium-based nanoplatforms to effectively deplete intracellular ATP, providing potent energy-deprivation antibacterial strategies.

Although metal-based antimicrobial agents have broad-spectrum targeting capacity, their potential cytotoxicity has limited their development and clinical applications16. On the other hand, combining metal-based agents with conventional antibiotics offers a promising strategy to overcome the potential toxicity of metal compounds when treating drug-resistant bacterial infections17,18. For example, an orally administered bismuth compound in combination with N-acetylcysteine is currently undergoing phase II/III clinical trials as part of an anti-coronavirus cocktail therapy19. However, there are still many challenges in achieving optimal drug loading efficiency and precisely controlled release for targeted delivery. In this context, nanotechnology-based drug delivery strategies emerge as a promising solution to address these limitations. In particular, nanoscale coordination polymers (NCPs), consisting of metal ions and organic linkers, have gained attention as versatile delivery carriers due to their high porosity, tunable architecture, and structural flexibility20–22. Metal-drug coordination nanoplatform using drug as the organic linkers with the capacity to respond to weak acidic microenvironment provides a valuable opportunity for antibacterial applications, showing the efficient drug loading and controlled release23–25. Generally, nanoparticle clearance by the mononuclear phagocytic system limits drug delivery efficiency to infection sites26. Recently, biomimetic extracellular vesicle (EV)-coated nanoplatforms have received significant attention. Based on our previous finding that ginger-derived EVs enhance nanoparticle circulation and bacterial uptake27, we developed a pH-responsive Ce-Car@EV nanoplatform that combines high drug-loading metal-organic coordination polymers (NCPs) with EV-based immune evasion.

Results
Preparation and characterization of Ce-Car@EV nanoplatforms
Car is a typical β-lactam antibiotic, whose two carboxyl groups can coordinate with various metal ions28. Hence, Car was utilized as organic bridge to connect with Ce(IV) ions for the construction of Ce-Car coordination polymers (Ce-Car NCPs) via a one-pot self-assembly of Ce(IV) and Car through coordination interactions (Fig. 2a). Ginger-derived EVs (G-EVs) were extracted via differential and density gradient centrifugation, subsequently encapsulating Ce-Car NCPs under the treatment of ultrasonication to form Ce-Car@EV nanoparticles (Ce-Car@EV NPs) (Fig. 2a). As shown in Fig. 2b, transmission electron microscopy (TEM) images reveal that Ce-Car NCPs exhibited a homogeneous spherical structure with an average diameter of about 10 nm, while EVs displayed a typical saucer-like shape. Notably, TEM images of Ce-Car@EV NPs show that Ce-Car NCPs were primarily encapsulated within G-EVs, confirming the successful preparation of the Ce-Car@EV hybrid nanoplatform. X-ray photoelectron spectroscopy (XPS) analysis verified the presence of distinct Ce 3d, O 1s, and N 1s peaks in the structure of Ce-Car@EV NPs (Supplementary Fig. 1), while the high-resolution XPS spectra revealed the presence of both Ce(IV) and Ce(III) species at a ratio of 71.5:28.5 (Supplementary Fig. 2). Various studies have shown that the ligand-to-metal or metal-to-ligand charge transfer processes may occur in metal complexes, and smaller energy gaps usually indicate that charge transfer is more likely to occur within the complex molecule29. According to the simulation results, the energy gap between HOMO (the highest occupied molecular orbital) and LUMO (the lowest unoccupied molecular orbital) of Ce-Car NCPs is 1.393 eV, indicating that Ce(IV) is successfully coordinated to Car, and the charge transfer can occur between the metal node and organic ligand (Fig. 2c). In addition, Fourier-transform infrared spectroscopy provides additional evidence for the successful formation of Ce–O bonds between Ce(IV) and Car in the coordination structure of Ce-Car NCPs (Fig. 2d). The hydrodynamic size distribution assessed by dynamic light scattering (DLS) analysis showed a profile consistent with the TEM findings (Supplementary Fig. 3). The zeta potential of Ce-Car NCPs decreased from approximately −2.6 to −16.2 mV upon the encapsulation of G-EVs, further confirming the successful construction of Ce-Car@EV NPs (Fig. 2e). Additionally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis reveals that the resultant Ce-Car@EV NPs retained the surface proteins of biologically active G-EVs (Fig. 2g). Furthermore, as demonstrated in Supplementary Fig. 4, Western blot image indicates that the exosomal marker protein CD9 is present on Ce-Car@EVs NPs, which are broadly consistent with SDS-PAGE findings. Therefore, these results demonstrate the successful synthesis of Ce-Car NCPs and G-EVs coating of Ce-Car@EV NPs.

As the important component of Ce-Car@EV NPs with antibacterial activity, the contents of Ce(IV) and Car in the structure were calculated to be 27.2 and 58.4% via inductively coupled plasma mass spectrometry analysis. Structurally, protonation of these carboxyl groups under acidic conditions disrupts the metal coordination, triggering structural dissociation and subsequent Car release30. To evaluate the pH-responsive antibiotic release behavior of Ce-Car@EV NPs, we performed in vitro release studies under physiological (pH 7.4) and acidic (pH 5.5) conditions. As shown in Fig. 2f, at pH 5.5, the release efficiency of Car exceeded 70% within 12 h, whereas less than 15% of the antibiotic was released under physiological conditions, demonstrating that the drug release was in a pH-controlled manner. Furthermore, TEM images in Supplementary Fig. 5 reveal the structural disruption of Ce-Car@EV NPs at pH 5.5 over 24 h, along with complete disintegration and disappearance of Ce-Car NCPs within ginger-derived EVs. These results indicate that Ce-Car@EV NPs have the potential for a rapid response to the acidic microenvironment of infection and induce the efficient release of Car, thereby significantly avoiding damage to normal tissues.

Penicillin-binding protein 3 (PBP3) serves as a primary target for various β-lactam antibiotics. By binding to PBP3, Car can effectively inhibit the synthesis of the bacterial cell wall, thereby exerting its antibacterial activity. Notably, the antibacterial efficacy of metal-coordination nanomaterials involves both intact nanoparticles and their degradation products31,32. As Ce-Car@EV NPs degrade in acidic infectious microenvironments, they specifically and targeted release bioactive Ce(IV) and carbenicillin. Therefore, we evaluated the effect of Ce(IV) on the binding mode of Car to PBP3 via molecular docking. As shown in Fig. 2h, in the absence of Ce(IV), Car forms hydrogen bonds with residues Arg483, Asn345, and π–π interactions with Phe401 to produce a binding energy of −6.93 kcal/mol. In the presence of Ce(IV), the binding mode has significantly changed. Ce(IV) forms coordination bonds with the carboxylate group of Glu286 and the backbone oxygen of Arg483, bringing the Car molecule closer to the binding site of Ce(IV). This shift facilitates the formation of additional hydrogen bonds between Car and Tyr524, enhancing the overall binding affinity (Fig. 2i). Meanwhile, the introduction of Ce(IV) reduces the binding energy to −7.84 kcal/mol, suggesting that the electrostatic attraction of Ce(IV) strengthens the interaction between Car and PBP3. These results indicate that Ce(IV) could not only enhance the binding affinity of Car to its target protein but also potentially stabilize the drug-protein complex, thereby augmenting its antibacterial efficacy.

In vitro antibacterial activity and drug resistance studies of Ce-Car@EV NPs
In vitro antibacterial activity of Ce-Car@EV NPs was evaluated using P. aeruginosa as a model bacterium. Considering the distinct microenvironments of normal physiological conditions and infected tissues, the bacterial viability of P. aeruginosa treated by Ce-Car@EV NPs was assessed in vitro under different pH conditions, and the cell counting kit-8 (CCK-8) assay was employed to quantitatively measure the bacterial growth inhibition. At a concentration of 32 μg/mL, the survival rate of bacteria at pH 5.5 was reduced to 3.19%, while the survival rate remained above 80% at pH 7.4 (Fig. 3a). These results clearly demonstrated the significantly enhanced antibacterial activity of Ce-Car@EV NPs in acidic environments, which was attributed to the pH-triggered degradation of Ce-Car@EV NPs and subsequent release of Car. The pH-responsive antibacterial activity of Ce-Car@EV NPs was further verified through a live/dead double staining assay. Under physiological conditions (pH 7.4), P. aeruginosa showed predominantly green fluorescence stained by SYTO-9, associated with living bacteria. In contrast, obvious red fluorescence-associated with dead bacteria was observed under acidic conditions (pH 5.5) in the presence of Ce-Car@EV NPs (Fig. 3b). These results were further supported by scanning electron microscopy (SEM) imaging, which revealed severe structural deformation and membrane disruption of P. aeruginosa treated by Ce-Car@EV NPs at pH 5.5, in contrast to the intact morphology of untreated bacteria (Fig. 3c). To further investigate the interaction between Ce(IV) and Car, the fractional inhibitory concentration index (FICI) was determined using a checkerboard dilution assay. According to the standard classification criteria (FICI ≤ 0.5: synergistic effect; 0.5 < FICI ≤ 1: additive effect; FICI > 1: antagonistic effect), the combination of Ce(IV) and Car exhibited a synergistic effect with FICI of 0.5 (Fig. 3d). These results demonstrate that the two key components of Ce(IV) and Car acted synergistically to enhance antibacterial efficacy.

Continued use of antibiotics can drive the emergence of resistant pathogens. To determine whether the treatment of Ce-Car@EV NPs could induce bacterial resistance comparable to that of free Car, P. aeruginosa was cultured with free Car or Ce-Car@EV NPs at sub-inhibitory concentrations (1/2 MIC) for 30 generations, respectively (Fig. 3e). Under free Car incubation, the 30th-generation MIC increased 256-fold, indicating the rapid development of resistance (designated PA-30R). In contrast, the Ce-Car@EV NPs-treated P. aeruginosa (designated PA-30M) exhibited significantly delayed and a lower degree of resistance. Furthermore, the antibacterial efficacy of Ce-Car@EV NPs against Car-resistant P. aeruginosa (PA-30R) was subsequently assessed, and Fig. 3f revealed that the combination of Ce(IV) and Car maintained a synergistic effect on combating resistant bacteria with a FICI of 0.5. The results of the colony formation assay in Fig. 3g revealed substantial bacterial growth even at free Car up to 2048 μg/mL. In contrast, Ce-Car@EV NPs at 16 μg/mL significantly reduced colony size. Meanwhile, quantitative colony counting demonstrated that the bacterial survival was more than 70% at a Car concentration of 16 μg/mL, while the survival rate dropped to only 0.19% with the treatment of Ce-Car@EV NPs at the equivalent concentration (Fig. 3h, i). These results validate the pH-responsive properties and potent bactericidal capability of Ce-Car@EV NPs due to the synergistic effect between Ce(IV) and Car. Meanwhile, the wide-spectrum antibacterial performance of Ce-Car@EV NPs was also investigated using Klebsiella pneumoniae (K. pneumoniae), another formidable Gram-negative bacterium frequently implicated in pneumonia, as a model. Ce-Car@EV NPs displayed complete bactericidal activity against drug-resistant K. pneumoniae at a concentration of 128 μg/mL following an 8-h co-incubation (Supplementary Fig. 6), which might be attributed to the core antibacterial mechanism, operating through pH-responsive payload release and bacterial energy metabolism disruption. These results indicate the effective elimination of Ce-Car@EV NPs to diverse Gram-negative pathogens, suggesting their considerable potential for applying a wide range of clinically relevant bacterial infections.

Mechanism study of Ce-Car@EV NPs in overcoming drug resistance
To further investigate the underlying mechanisms of Ce-Car@EV NPs to reverse the bacterial resistance, RNA sequencing (RNA-seq) transcriptomics was performed on P. aeruginosa, the corresponding PA-30R, and PA-30M. The degree of correlation between gene expression levels in the three experimental groups was reflected by the Pearson correlation coefficient. Significant changes in gene expression levels were observed in PA-30R cells, which may be related to their high antibiotic resistance (Supplementary Fig. 7). The Venn diagram illustrating differences in differentially expressed genes (DEGs) between the three groups showed that PA-30R exhibited more DEGs than PA-30M in comparisons with PA (Fig. 4a). Meanwhile, volcano plot analysis revealed that 599 DEGs were upregulated and 126 were downregulated in the group of PA-30M compared to those of PA-30R (Fig. 4b). Compared to the pure P. aeruginosa, 360 DEGs in PA-30R were identified, with 112 upregulated and 248 downregulated genes, while PA-30M showed only 182 DEGs, with 163 upregulated and 19 downregulated types (Fig. 4b). The magnitude of transcriptional alterations in PA-30M relative to pure P. aeruginosa was substantially attenuated compared to that in PA-30R.

Furthermore, gene ontology (GO) analysis in Fig. 4c indicates that DEGs between PA-30M and PA-30R were involved in biological processes (BP), cellular components (CC), and molecular functions (MF). The BP related to the gene expression changes were obviously regulated, including biological regulation, cellular processes, and metabolic processes, et al., while several important CC, such as cellular anatomical entities and protein-containing complexes, were also significantly altered. Additionally, various MF of proteins, such as binding/catalytic activity and transporter activity, exhibited significant changes. The results of GO enrichment analysis reveal that Ce-Car@EV NPs could also affect the bacterial respiratory chain activity and interfere with the bacterial energy metabolism (Supplementary Fig. 8). As shown in Fig. 4d, kyoto encyclopedia of genes and genomes (KEGG) pathway analysis further identifies key enriched pathways between PA-30M and PA-30R, including carbon metabolism, oxidative phosphorylation, glycolysis/gluconeogenesis, pyruvate metabolism, and the citric acid cycle (TCA cycle). Moreover, the comparison of PA-30R and PA-30M with PA revealed that these DEGs were also associated with the metabolic processes, including amino acid metabolism (e.g., the degradation of valine, leucine, and isoleucine, Supplementary Fig. 9). These results indicate that Ce-Car@EV NPs possess the potential to target the regulation of bacterial energy metabolism, including the TCA cycle and oxidative phosphorylation.

To reveal the mechanism of Ce-Car@EV NPs participating in bacterial resistance reversal, genes associated with multidrug efflux were analyzed. The heatmap in Fig. 4e shows significant upregulation of major efflux pump genes, including mexA, mexB, oprM, and PA3228 in PA-30R compared with PA. These genes encoded the MexAB-OprM system, a member of the resistance-nodulation-cell-division (RND) superfamily and the ATP-binding cassette (ABC) superfamily, both of which could promote the rapid efflux of Car33,34. In contrast, the expression of these upregulated genes in PA-30M were restored to normal levels or even downregulated, suggesting that Ce-Car@EV NPs could inhibit antibiotic efflux, thereby contributing to the reversal of antibiotic resistance. Beyond inhibiting efflux pump activity, the transcriptomic analysis in Supplementary Fig. 11 also revealed that Ce-Car@EV NPs significantly modulate the expression behaviors of genes associated with biofilm formation, verifying their capacity to disrupt this critical adaptive resistance mechanism, which is frequently responsible for therapeutic tolerance in persistent infections35. Further investigation into bacterial energy metabolism revealed that Ce-Car@EV NPs dramatically reduced the expression level of intracellular ATP, a vital energy source for cells, which was closely linked to oxidative phosphorylation and the electron transport chain. As shown in Fig. 4f, genes encoding complex I (nuoE, nuoG, nuoI, nuoN) and IV (ccoN2, coxB, cyoD) were significantly downregulated in the PA-30M group. This downregulation directly leads to proton motive force (PMF) dissipation36,37, indicating that Ce(IV) has the capacity to interfere with the bacterial respiration. Additionally, genes involving in the glycolysis and TCA cycle, such as pyruvate kinase (pykA), α-ketoglutarate dehydrogenase (sucB), and aconitase (acnA), were downregulated, suggesting the suppression of bacterial energy metabolism induced by treatment of Ce-Car@EV NPs (Fig. 4g). Meanwhile, the expression levels of genes closely related to P. aeruginosa virulence are also shown in the heatmap (Supplementary Fig. 10), indicating that most of the genes encoding pathogenesis-associated toxins were down-regulated. Collectively, these multi-faceted transcriptional alterations- spanning virulence attenuation, energy metabolism disruption, efflux pump suppression, and biofilm formation inhibition- converge into a unified resistance-reversal mechanism, thereby significantly enhancing the potential to combat complex resistance profiles in P. aeruginosa. Figure 4h provides a detailed summary of the mechanism by which Ce-Car@EV NPs overcome P. aeruginosa resistance. After the treatment of Ce-Car@EV NPs, the bacterial glycolysis was severely inhibited, as evidenced by the significant down-regulated expression of genes related to the conversion between glyceraldehyde-3-phosphate and glycerate-3-phosphate in this pathway. In addition, genes associated with aconitase, isocitrate dehydrogenase and succinyl-CoA synthetase were also significantly down-regulated in the TCA cycle. Critically, this metabolic perturbation extends to compensatory pathways, evidenced by genes associated with the synthesis and metabolism of glyoxylate were also down-regulated, a key anaplerotic circuit that bypasses decarboxylation steps in the TCA cycle38,39. The resultant energy crisis is further amplified by the Ce(IV)-mediated down-regulation of respiratory complex I and complex IV genes, which directly dissipates PMF. Furthermore, the respiratory chain activities of complex I and complex IV were also significantly reduced in Car-resistant P. aeruginosa after Ce-Car@EV NPs treatment, confirming that Ce(IV) could disrupt bacterial energy metabolism and prevent the operation of energy-dependent efflux pumps (Fig. 4k). ATP hydrolysis assay in Fig. 4i demonstrates that Ce-Car@EV NPs could greatly decrease the content of intracellular ATP in a concentration-dependent manner (Fig. 4j), achieving a 60% reduction at 100 μg/mL and revealing their strong potential to effectively deplete bacterial ATP. To functionally assess inhibition capacity to the efflux pump, ethidium bromide (EtBr), a well-characterized fluorescent substrate for RND-type efflux pumps in P. aeruginosa, was utilized to verify the impaired pump activity via direct monitoring of intracellular fluorescence accumulation40,41. Fig. 4l demonstrates that Ce-Car@EV NPs significantly inhibited the efflux pump activity in a concentration-dependent manner, further reinforcing their role in reversing bacterial resistance by blocking the energy supply for efflux pumps. By coordinately normalizing efflux pump transcription, disrupting energy metabolism, and functionally compromising efflux pumps, Ce-Car@EV NPs establishes a powerful multi-mode mechanism that reverses carbenicillin resistance of drug-resistant bacteria at both transcriptional and functional levels.

Biocompatibility of Ce-Car@EV NPs in vitro and in vivo
The potential toxicity and biocompatibility assessment of biomaterials is of paramount importance to ensure their feasibility for clinical and biomedical applications. Therefore, the cytotoxicity and hemolytic activity of Ce-Car@EV NPs were systematically evaluated. The cytotoxicity of Ce-Car@EV NPs was systematically evaluated using three representative cell types, including mouse fibroblast L929, murine macrophage RAW264.7, and human umbilical vein endothelial cells (HUVECs). Cell viability in all three lineages remained above 80% even after 24 h of co-culture with Ce-Car@EV NPs at a concentration of 512 μg/mL, indicating negligible cytotoxicity (Supplementary Fig. 12a–c). Additionally, the hemolytic activity of Ce-Car@EV NPs was measured using mouse red blood cells (RBCs). When the extracted RBCs were incubated with 512 μg/mL of Ce-Car@EV NPs for 3 h, the hemolysis ratio was less than 5%, demonstrating the favorable hemocompatibility of Ce-Car@EV NPs (Supplementary Fig. 12d). Furthermore, the in vivo biocompatibility of Ce-Car@EV NPs was examined. Mice injected intravenously with Ce-Car@EV NPs displayed a weight gain trajectory similar to that of untreated control mice over 7 days, along with stable body temperatures (Supplementary Fig. 13), indicating negligible adverse effects of Ce-Car@EV NPs on mouse growth. Meanwhile, routine blood tests and serum biochemical analyses showed no significant differences between Ce-Car@EV NPs-treated and healthy mice (Supplementary Fig. 14). Histopathological examination of major organs, including the heart, liver, spleen, lungs, kidneys, and brain, revealed negligible pathological abnormalities after the injection of Ce-Car@EV NPs compared to controls after 7 days (Supplementary Fig. 15). Oxidative stress is recognized as a primary concern contributing to nanoparticle-induced neurotoxicity42,43. To assess the potential neurotoxic effects of Ce-Car@EV NPs, several key oxidative stress markers in the mouse central nervous system, including superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA), were examined. After 7 days of treatment with Ce-Car@EV NPs, the cerebral cortex showed insignificant alterations in the levels of SOD, GSH, or MDA relative to the control group (Supplementary Fig. 16). These results suggest that Ce-Car@EV NPs administration provoke negligible substantial oxidative injury within neural tissues. These results demonstrate that Ce-Car@EV NPs displayed favorable biocompatibility and minimal biotoxicity both in vitro and in vivo, underscoring their potential as a safe and effective therapeutic platform for bacterial infections.

In vivo pharmacokinetics, biodistribution and metabolism of Ce-Car@EV NPs
The robust in vitro antimicrobial capacity and good biocompatibility of Ce-Car@EV NPs suggest their potential for combating bacterial infections in vivo. After intravenous injection, numerous studies demonstrate that natural EV camouflage can extend the blood circulation time and enhance preferential accumulation of nanomaterials at the infection sites44. To validate the extended blood circulation capability conferred by G-EVs coating, we compared the cellular uptake of Ce-Car NCPs and Ce-Car@EV NPs by RAW264.7 macrophages in vitro. Confocal laser scanning microscopy (CLSM) images confirmed that the rational encapsulation of G-EVs significantly reduced the phagocytosis of Ce-Car@EV NPs compared to the bare Ce-Car NCPs (Supplementary Fig. 17). This diminished immunocellular clearance capacity is crucial for prolonging the systemic circulation of the nanoplatform. Upon reaching the pulmonary vasculature, most of the traditional nanoparticles initially accumulate through the EPR effect resulting from infection-induced vascular hyperpermeability45,46. Furthermore, this targeted enhanced accumulation is augmented by the complementary biological mechanisms. The infected pulmonary microenvironment typically exhibits substantial infiltration of M1-polarized macrophages, demonstrating heightened phagocytic activity toward nano-sized structures. Additionally, tremendous studies on plant-derived EVs document their inherent targeted capability to the inflammatory site, including the potential affinity toward M1 macrophages47,48. These coordinated mechanisms, including prolonged circulation, passive EPR-mediated accumulation, and potential active targeted processes, may collectively contribute to the enhanced targeting efficacy of Ce-Car@EV NPs. To validate these properties, Cy7-labeled Ce-Car@EV NPs were intravenously administered to mice with drug-resistant P. aeruginosa-infected lungs, and their biodistribution was tracked at various time points using fluorescence imaging (Fig. 5a). As shown in Fig. 5b, Cy7-related fluorescence signals in mice injected with bare Ce-Car NCPs were predominantly localized in the liver, with minimal accumulation in the infected lung. These fluorescence signals obviously diminished after 8 h, probably due to the rapid immune clearance. In contrast, mice with lung infections treated with Ce-Car@EV NPs exhibited significantly stronger and more sustained fluorescence signals in the infected lung, indicating enhanced and prolonged retention. Quantitative fluorescence analysis demonstrates that Ce-Car@EV NPs began accumulating in the infected lungs within 2 h post-injection, and peaked at 6 h, with significantly higher accumulation than bare Ce-Car NCPs (Fig. 5c). These findings were further confirmed by fluorescence imaging and quantitative analysis of isolated organs at 6 h post-injection, demonstrating the efficient and selective accumulation of Ce-Car@EV NPs in infected lungs, along with the prolonged retention (Fig. 5d, e). Interestingly, the targeted accumulation of Ce-Car@EV NPs depending on the specific pathological microenvironment of bacterial infection was unequivocally verified through control experiments in healthy mice. The intravenous administration of Ce-Car@EV NPs in healthy mice resulted in negligible pulmonary fluorescence signals, exhibiting a stark contrast with the substantial accumulation in the lungs of pneumonic mice (Supplementary Fig. 18). These results demonstrate that the targeted efficiency of Ce-Car@EV NPs is intrinsically associated with the inflammatory microenvironment, rather than the nonspecific distribution. Taken together, these results suggest that the biomimetic camouflage with ginger-derived EVs effectively promotes the targeted delivery of Ce-Car@EV NPs to bacterial infection sites.

To quantitatively track the in vivo trajectory of Ce-Car@EV NPs, we systematically investigated its pharmacokinetic profile and metabolic fate. Following the intravenous administration of Ce-Car@EV NPs in healthy mice, blood samples were collected at predetermined time points and subjected to quantitative analysis of cerium ions. Pharmacokinetic data via a non-compartmental model determined the circulating half-life of Ce-Car@EV NPs to be 6.68 h (Fig. 5f), providing a critical kinetic foundation for the enhanced accumulation at the infection site. Meanwhile, the excretory pathways of the nanoplatform were traced by monitoring cerium levels in urine and feces. As summarized in Fig. 5g, h, Ce-Car@EV NPs underwent dual clearance routes involving hepatic metabolism and renal excretion, showing a near-complete elimination within 7 days, which was consistent to the quantification of residual cerium in major organs with only minimal retention (Supplementary Fig. 19). These results indicate a good pharmacokinetic profile characterized by extended circulation, defined clearance mechanisms, and negligible long-term organ accumulation. This profile affirms the robust biodegradability and biosafety of Ce-Car@EV NPs, underscoring their potential as a translatable antimicrobial agent.

In vivo therapeutic performance of Ce-Car@EV NPs for P. aeruginosa-induced lung infection
Encouraged by the potent antibacterial properties, good biosafety, and efficient targeted accumulation at the infection sites, the in vivo antimicrobial efficacy of Ce-Car@EV nanoplatforms was assessed using drug-resistant P. aeruginosa-infected lung model in mice. Commercial mice were intratracheally inoculated with 50 μL of drug-resistant P. aeruginosa, and neutrophil reduction was induced by cyclophosphamide. Two hours post-infection, the infected mice were randomly divided into four groups and respectively treated with the intravenous injections of saline (control), free Car, Ce-Car NCPs, and Ce-Car@EV NPs (Fig. 6a), while untreated mice served as the healthy group. Survival rates were monitored over 36-h, alongside physiological measurements of body temperatures and body weight. As shown in Fig. 6b, all infected mice treated with saline, Car, or Ce-Car NCPs died within 36 h, whereas those injected with Ce-Car@EV NPs exhibited a satisfactory survival rate, indicating their therapeutic potential against P. aeruginosa lung infection. Additionally, the body temperatures and weight of Ce-Car@EV NPs-treated mice remained stable, comparable to healthy controls, suggesting negligible side effects (Fig. 6c, d). Clinical scores on six typical parameters, including activity, coat condition, eyes, breathing, posture, and isolation, were also significantly improved in the Ce-Car@EV NPs group (Fig. 6f). These results indicate that Ce-Car@EV NPs are highly effective against acute lung infections when administered intravenously, without adverse effects on mouse health.

Furthermore, lungs from mice receiving different treatments were collected and analyzed at 12 and 60 h post-treatment. As shown in Fig. 6e, visual inspection of the lung tissues revealed clear differences in treatment efficacy between the Ce-Car@EV NPs group and other groups. After the treatment of saline, Car, or Ce-Car NCPs, the lung tissues were red and mottled, exhibiting congestion and focal infections. In contrast, lungs from Ce-Car@EV NPs-treated mice appeared pink with fewer focal infections, demonstrating effective treatment of P. aeruginosa infection. Quantification of residual bacteria in lung homogenates and bronchoalveolar lavage fluid (BALF) further confirmed the antibacterial efficacy of Ce-Car@EV NPs. As shown in Fig. 6g and Supplementary Fig. 20a, bacterial counts in Ce-Car@EV NPs-treated lungs decreased by over two orders of magnitude versus the other groups, with the lowest bacterial counts observed in BALF (Fig. 6h and Supplementary Fig. 20b). At 60 h post-treatment, bacterial load in both lung homogenates and BALF of the Ce-Car@EV NPs-treated mice decreased by one order of magnitude compared to those at 12 h post-treatment (Supplementary Fig. 21). In contrast, saline, Car, or bare Ce-Car NCPs-treated mice showed bacterial spread to heart, liver, spleen, and kidneys at 12 h post-treatment, indicating systemic infection. Throughout treatment, Ce-Car@EV NPs-treated mice maintained minimal bacterial loads in all major organs (Supplementary Fig. 22). Notably, by the 60 h post-treatment time point, Ce-Car@EV NPs-treated mice showed further reductions or undetectable bacterial loads in all major organs compared to the 12 h post-treatment time point (Supplementary Fig. 23), demonstrating the great potential for targeted antibacterial treatment of the infected lung. Fluorescence images of P. aeruginosa-stained lung sections confirmed obviously lower bacterial loads in Ce-Car@EV NPs-treated lungs compared with other groups (Fig. 6i). These results demonstrate that Ce-Car@EV NPs effectively inactivated drug-resistant P. aeruginosa in both infected lungs and distant organs, establishing their therapeutic potential for the treatment of multidrug-resistant bacterial infections.

The lung wet-to-dry (W/D) ratio, a well-established indicator of pulmonary edema, was measured to evaluate the extent of lung tissue damage, specifically assessing the influx of protein-rich edema fluid into alveolar spaces caused by increased capillary-endothelial-alveolar epithelial barrier permeability49. As shown in Fig. 7a, the control group exhibited the highest lung W/D ratio, indicating significant pulmonary edema. In contrast, the W/D ratio of the lung treated by Ce-Car@EV NPs was significantly lower than those in the Car and Ce-Car NCPs groups, suggesting that Ce-Car@EV NPs treatment effectively alleviated lung edema. Further analysis of the total protein concentration and lactate dehydrogenase (LDH) activity in BALF revealed that Ce-Car@EV NPs-treated lungs displayed low levels of edema fluid and minimal tissue damage (Fig. 7b, c). Additionally, the assessment of lung lesion severity using the lung index was consistent with these findings, showing an obvious reduction in lesion extents in Ce-Car@EV NPs-treated mice (Fig. 7d). Taken together, these results demonstrate that Ce-Car@EV NPs could effectively inhibit the influx of protein-rich edema fluid into the alveoli, mitigate lung injury, and reduce mortality in bacterially infected mice.

Moreover, hematoxylin and eosin (H&E) staining and Masson staining were employed to evaluate the pathological changes in lung sections following different treatments. As shown in Fig. 7e, H&E staining images revealed noticeable inflammatory cell infiltration in the control group, along alveolar septum thickening, alveolar wall changes, and partial alveolar shrinkage, as well as extensive regional consolidation. In contrast, histological lesions in Car- and Ce-Car NCPs-treated mice were alleviated to some extent, though significant abnormalities persisted. Notably, lung tissues from Ce-Car@EV NPs-treated mice exhibited nearly normal structures. Masson staining in Fig. 7f further verified these observations, showing the relatively intact alveolar structures and minimal fibrosis in Ce-Car@EV NPs-treated mice, whereas other treatment groups showed pronounced lung fibrosis changes. Collectively, these results demonstrate that Ce-Car@EV NPs significantly enhanced the in vivo antibacterial efficacy of Car and provided significantly therapeutic benefits for acute lung infections, facilitating the recovery of lung structure and function.

Methods
Materials and reagents
Ceric ammonium nitrate, sucrose, and cyclophosphamide monohydrate were purchased from Energy Chemical (Shanghai, China). Polyvinylpyrrolidone (PVP, MW=55000), ethidium bromide and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Dimethylsulfoxide (DMSO, >90%) and ethanol (>99.7%) were provided by Beijing Chemical Works (China). Cyanine7 amine (Cy7-NH2) and carbenicillin disodium were purchased from Shanghai Aladdin Biochemical Technology Co. (China). Glutaraldehyde (GA) was obtained from Sinopharm Group Co., Ltd (China). The LIVE/DEAD BacLight Bacterial Viability Kit (L7012) was purchased from Invitrogen (Life-Technologies, USA). Anti-Pseudomonas outer membrane porin F antibody (Abcam, catalog no. ab316973, clone: EPR28629-91) and CD9 rabbit pAb (Bioss, catalog no. bs-2489R) were obtained from the respective manufacturers and used at dilutions of 1:100 and 1:500, respectively. The lysogeny broth (LB) solution was autoclaved for 20 min at 120 °C before use. The strains of P. aeruginosa (ATCC 27853) was provided by Promega (Madison, USA). The drug-resistant K. pneumoniae strain was a clinical isolate obtained from the Chinese PLA General Hospital. Milli-Q Ultrapure water (18.2 MΩ) was used in all experiments unless otherwise stated.

Isolation and purification of ginger-derived extracellular vesicles (G-EVs)
G-EVs were extracted from fresh ginger according to the previous literatures52,53. Fresh ginger was purchased from a local shop and washed repeatedly with deionized water and saline. Ginger was cut into small pieces, ground in a juicer, and immersed in saline to collect the juice. Ginger outer membrane vesicles were first coarsely extracted by differential centrifugation at 200 × g for 10 min, 2000 × g for 20 min, and 10,000 × g for 30 min to remove large particles as well as precipitated cells and debris. The supernatant was collected and centrifuged at 150,000 × g in an ultracentrifuge for 2 h. The precipitate was collected and resuspended in saline. Ginger outer membrane vesicles were further purified by sucrose gradient centrifugation, and the precipitate resuspended in saline was transferred to a sucrose gradient (8%, 20%, 30%, 45%) and centrifuged at 150,000 × g for 2 h. Ginger outer membrane vesicles were finally floated in different sucrose bands. All the processes were carried out at 4 °C. The bands between 8/30 and 30/45% were collected separately and washed with saline. Finally, the precipitates were taken by centrifugation at 150,000 × g for 1 h and resuspended in saline, quantified by the BCA protein quantification kit, and stored at −80 °C for backup.

Synthesis of Ce-Car@EV NPs
Briefly, Ce(NH4)2(NO3)6 (16.272 mg) and PVP (48.816 mg) were mixed in a glass vial containing 3 mL of deionized water. The mixture was stirred at 700 rpm for 2 h at room temperature to obtain a homogeneous light-yellow solution. Subsequently, carbenicillin sodium (15.204 mg) was added dropwise to the light-yellow solution, and the mixed solution was stirred for another 10 min to obtain a final light-yellow suspension. The light-yellow suspension was centrifuged at 20,000 rpm for 30 min, washed three times and resuspended in deionized water to obtain Ce-Car NCPs. Subsequently, the prepared Ce-Car NCPs were transferred to a centrifuge tube, and 6 mg of purified ginger-derived EVs were added and sonicated in an ice bath. Finally, the product was incubated at 37 °C for 1 h and then centrifuged at 10,000 rpm for 30 min, collected and resuspended in saline to obtain Ce-Car@EV NPs.

Checkerboard analysis and FIC index determination
Synergistic interaction between Ce(IV) and Car was assessed by checkerboard analysis. Briefly, the solution containing Ce(IV) and Car was placed in 96-well plates at 2-fold serial dilution to form an 8 × 8 matrix. The final volume of each well was 150 μL containing 1 × 105 CFU/mL bacteria. After 24 h of incubation at 37 °C on a shaker, the absorbance value at 600 nm of each well was determined. The synergistic effect of Ce(IV) and Car was evaluated by calculating the FIC index. The calculation formula was presented as follows.

FIC index = FICA + FICB (1)

where FICA was the MIC of Ce(IV) in combination/MIC of Ce(IV) alone, and FICB was the MIC of Car in combination/MIC of Car alone.

Resistance studies
Changes in resistance of P. aeruginosa ATCC 27853 were investigated by sequential passaging assay under sub-MIC conditions of sequential exposure to Car and Ce-Car@EV NPs according to the previous literature54. Firstly, MIC of Car and Ce-Car@EV NPs were determined via a microbroth dilution assay. For subsequent passages, 1 × 105 CFU/mL of bacteria were inoculated into medium containing serial dilutions of Car or Ce-Car@EV NPs, and passages were performed at 24-h intervals. The absorbance values at 600 nm were measured by a microplate detector to determine the MIC of the bacteria after different treatments. Bacteria co-cultured with 1/2 MIC value of each group was used for passaging, and the above steps were repeated for 30 days. The MIC of each passage was recorded and compared with that of the first passage. Under the same culture protocol, a distinct carbenicillin-resistant strain (PA-15R) was generated through 15 sequential passages of the parental ATCC 27853 strain in the standard and undiluted LB medium containing carbenicillin, which was utilized for subsequent antibacterial efficacy evaluation.

Bactericidal properties of Ce-Car@EV NPs
In vitro antimicrobial activity of Ce-Car@EV NPs was systematically assessed by CCK-8 assay, CLSM, SEM and standard plate counting method. Firstly, the bacteria were inoculated into LB culture medium on a shaker at 37 °C overnight and cultured to logarithmic phase. The antibacterial assays were performed in 10 mM tris-HCl buffer at two distinct pH values (5.5 and 7.4), with the pH carefully adjusted using hydrochloric acid. Diluted log-phase bacteria (8 × 106 CFU/mL) were treated with Ce-Car@EV NPs (0, 1, 2, 4, 8, 16, and 32 μg/mL) dispersed in the respective buffers. After 2 h of co-culture, bacterial viability of each group was calculated using the CCK-8 method.

Furthermore, CLSM images were employed to visually observe the antibacterial effect of Ce-Car@EV NPs under different pH conditions using the live/dead double staining method. Bacterial suspensions (108 CFU/mL) were dispersed in the buffers containing Ce-Car@EV NPs (512 μg/mL) under different pH conditions (5.5 or 7.4) and co-cultivated for 6 h. Bacterial suspensions receiving different treatments were centrifuged, washed, and collected. The bacterial suspension (109 CFU/mL) was then double-stained with a mixture of SYTO-9/PI and maintained in the dark for 15 min. Then, the stained bacteria were observed by CLSM, and the bacteria treated by PBS were regarded as controls. Finally, the morphology of bacteria after different treatments were also characterized by SEM imaging.

Antimicrobial effect of Ce-Car@EV NPs under different experimental conditions were further quantified by the standard plate counting method. Bacterial suspensions (105 CFU/mL) were respectively treated by Car or Ce-Car@EV NPs at different concentrations (0, 2, 4, 8, and 16 μg/mL) for 4 h, and aliquots were taken out of the suspensions and serially diluted. Then, 50 μL of diluted bacterial suspensions from each group were inoculated onto LB agar plates, and the bacterial survival rates were counted and calculated after 18 h of incubation.

In vivo biodistribution of Ce-Car@EV NPs
Female Balb/c mice (8 weeks old), an inbred strain, were acquired from Beijing Vital River Laboratory Animal Technology Co. (China) and housed with a 12 h light/dark cycle under controlled ambient temperature (22 °C) and humidity (30–70%). Given the anticipated applicability of findings to both sexes, and that gender was not a study variable, female mice were selected to align with standard models. In vivo biodistribution of Ce-Car@EV NPs was evaluated using in vivo fluorescent imaging. The mice were fasted overnight prior to the experiment, and the chests and abdomens of the mice were exposed. After the mice were anesthetized with isoflurane, 50 μL of drug-resistant P. aeruginosa (7 × 107 CFU/mL) was injected into the lungs of healthy mice via the trachea to establish a mouse model of acute pneumonia induced by drug-resistant P. aeruginosa. After 2 h, drug-resistant P. aeruginosa-infected mice were randomly divided into two groups (n = 3) and intravenously injected with 100 μL of Cy7-labeled Ce-Car NCPs and Cy7-labeled Ce-Car@EV NPs (the concentration of Ce-Car NCPs was 4 mg/mL), respectively. Fluorescence images of mouse lungs were acquired at predetermined time points (0, 2, 4, 6, 8, 12, and 24 h) post-injection by the Xenogen IVIS spectrum imaging system (Caliper Life Sciences, America). To further assess the distribution of nanoparticles in isolated organs, mice were sacrificed 6 h post-administration and major organs (heart, liver, spleen, lungs, and kidneys) were collected for fluorescence imaging. The fluorescence signals from these major organs were qualitatively observed and quantitatively analyzed.

Establishment of mouse lung infection model
Neutropenia and granulocytopenia were induced in mice by administering 150 and 100 mg/kg of cyclophosphamide 4 days prior to infection and 1 day prior to infection, respectively55. Bacterial lung infections were induced by anesthetizing mice with isoflurane, removing hair from the backs of the mice, and intratracheal nebulized inoculation of mice with drug-resistant P. aeruginosa (50 μL, 5 × 107 CFU/mL). Two hours post-infection, the infected mice were randomly divided into four experimental groups (n = 5), with healthy mice serving as the negative control. The mice received intravenous injection with 100 μL of saline, Car (carbenicillin concentration = 1.168 mg/mL), Ce-Car NCPs (containing Car = 1.168 mg/mL), and Ce-Car@EV NPs (containing Car = 1.168 mg/mL), respectively. Treatments were administered every 12 h for a total of three times. Animal temperature and body weight were monitored every 2 h for survival studies. A portion of mice was sacrificed 12 and 60 h after different treatments, and pre-cooled sterile saline (1 mL) was slowly injected from the trachea. BALF was collected by repeatedly aspirating the lung solution three times to ensure that the recovery of BALF was greater than 80%. The BALF was further tested for bacterial enumeration using the standard plate counting method. The remaining BALF was then centrifuged, and the total protein concentration in BALF was determined using a BCA protein assay kit, and the LDH concentration in the lung lavage fluid was determined using an LDH assay kit (Nanjing Jiancheng Bioengineering Institute). Lung tissues were photographed and accurately weighed on an electronic balance, which was defined as the wet weight, and then dried in an oven at 65 °C for 72 h until the weight did not change, which was defined as the dry weight, to calculate the wet/dry (W/D) ratio of the lungs. Mouse lung tissues and major organ samples (heart, liver, spleen, and kidney) after different treatments were collected, homogenized and diluted in plates to determine the colony counts. Then, the bacterial load in the lungs of infected mice was quantified as the total number of bacteria in the lung homogenates and BALF.

Lung histology and immunofluorescence staining
The collected lungs were fixed in 4% tissue fixative for over 24 h. Then, the lungs were embedded in paraffin, cut into thin slices, stained with H&E and Masson, and visualized by microscopy. For immunofluorescence detection of P. aeruginosa, lung sections were stained with primary antibody (anti-P. aeruginosa antibody, Abcam, act. no. ab316973) and fluorescently labeled secondary antibody according to the manufacturer’s instructions. All sections were finally stained with DAPI and imaged using a CLSM.

Clinical scores of mice
Mice were scored in six categories, including fur, activity, eyes, respiration, posture, and isolation, with scores of 0, 1, and 2 for each category, containing (A) coat (0, no fur erection; 1, slight fur erection; 2, significant fur erection), (B) activity (0, mice displaying normal levels of activity including exploratory behaviors and locomotion; 1, slightly reduced activity, but still responding to stimuli; 2, markedly reduced activity and reduced response to stimuli), (C) eyes (0, clear, bright eyes with no discharge; 1, slight periocular discharge or slight eyelid swelling; 2, marked periocular discharge, eyes half-closed), (D) breathing (0, normal, even rhythmic respiration; 1, slight increase in respiratory rate or slight abnormal respiration; 2, marked respiratory distress, such as nasal flaring, shoulder movement), (E) posture (0, normal, with limbs supporting the body and no abnormal postures; 1, obvious postural irregularities, such as hunching, or squatting most of the time; 2, abnormal postures, such as reclining, inability to stand properly), and (F) isolation (0, normal social interactions, including interaction and playfulness with peers; 1, slight decrease in socialization, and will still engage in some peer interaction; 2, obvious isolating behavior, often avoiding peer interaction).

Biosafety of Ce-Car@EV NPs
Briefly, healthy mice were randomly divided into two groups (n = 3), and the hair on the back of the mice was shaved. Ce-Car@EV NPs (100 μL, 4 mg/mL) were injected intravenously, and the untreated healthy mice were employed as controls. The body temperature and body weight of the animals were monitored once a day. After 7 days, the major organs (heart, liver, spleen, lung, kidney, and brain) of mice were collected for H&E staining. For blood biochemical analysis, expression levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were determined. In the complete blood count test, contents of white blood cells (WBC), RBC, neutrophil (Neut), lymphocyte (Lymph), mean corpuscular volume (MCV), and erythrocyte corpuscle (HCT) were determined.

Evaluation of cytotoxicity in vitro
L929 cells were inoculated into 96-well plates at a cell density of 1 × 104/well and cultured in an incubator containing 5% CO2 at 37 °C for 24 h. Subsequently, Ce-Car@EV NPs at different concentrations (8, 16, 32, 64, 128, 256, and 512 μg/mL) were co-cultured with L929 cells. After 24 h the medium was removed, and the prepared MTT solution (100 μL, 0.5 mg/mL) was added, and further incubated at 37 °C for 4 h. Finally, the medium was removed, and 100 μL of DMSO was added to each well and shaken at low speed for 10 min. The absorbance value of each well at 490 nm was measured and recorded by a microplate detector.

Blood was collected from Balb/c mice receiving different treatments, and then centrifuged at 4 °C for 15 min to obtain precipitated RBCs. The erythrocytes were rinsed with saline to make the supernatant colorless, and the erythrocyte suspension was diluted to 4% v/v with saline. The erythrocyte suspension was co-incubated with Ce-Car@EV NPs at different concentrations (2, 4, 8, 16, 32, 64, 128, 256, and 512 μg/mL) for 3 h at 37 °C. Saline and Triton-X were used as a negative and positive control, respectively. Then, the mixture of erythrocytes and Ce-Car@EV NPs was centrifuged at 2000 rpm for 15 min. Finally, the absorbance value of the supernatant at 540 nm were recorded using a microplate detector and the percentage of hemolysis was calculated. The formula for calculating the percentage of hemolysis is presented as follows.

Hemolysis (%) = (ODM − ODN) / (ODP − ODN) × 100% (2)

Where ODM, ODP, and ODN present the absorbance values of Ce-Car@EV NPs-treated groups, positive control group and negative control group, respectively.

Statistical analysis
Data were presented as means ± standard deviation and are from at least three independent experiments. The differences were calculated by using two-tailed Student’s t test for two groups or one-way ANOVA for three or more groups. The statistical analysis were carried out with the software of Excel 2019 or GraphPad Prism. The level of significance in all statistical analyses was set at P < 0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Ye and colleagues introduce Ce-Car@EV, a nanoparticle made from a cerium-carbenicillin complex and ginger-derived\nvesicles, emphasizing its potential for treating bacterial pneumonia. This nanoparticle demonstrates effective bactericidal\nproperties and in vitro/in vivo biocompatibility, targeting the lungs via the EPR effect to combat Pseudomonas aeruginosa.\nThe study is well-structured, including antibacterial experiments against drug-resistant Pseudomonas aeruginosa,\nconstruction and sequencing of drug-resistant strains, and an exploration of bacterial resistance mechanisms. However, the\nstudy focuses solely on Pseudomonas aeruginosa, raising concerns about its broad-spectrum antibacterial efficacy. The\nantibacterial effectiveness against other key pathogens causing bacterial pneumonia, such as Streptococcus pneumoniae\nand Klebsiella pneumoniae, remains unexplored, limiting its clinical applicability. Additionally, the mechanism by which\nginger vesicles, as an external substance, extend their time in the bloodstream is not fully addressed. While the combination\nof cerium-based nanoparticles and ginger vesicles is innovative, the research lacks significant innovation in material\nsynthesis and mechanism exploration. The study does not provide groundbreaking insights into material preparation or\nbiomedical issues. Given the innovative nature of the topic and the experimental data, it is challenging to meet the standards\nof Nature Communications. It is recommended to either reject the paper or submit it to Communications Materials.\n1. Given that this paper builds on your lab's prior research, the choice of ginger-derived vesicles is understandable.\nHowever, it seems counterintuitive to claim that these vesicles, as exogenous substances, can evade the immune system.\nCompared to mammalian cells, ginger vesicles and other plant vesicles, as foreign entities, are more likely to be recognized\nand engulfed by immune cells, potentially affecting their effectiveness. The authors should conduct relevant experiments\nand provide reliable data to support this claim, such as immune cell phagocytosis assays or bacterial phagocytosis assays.\nAdditionally, considering the recent extensive studies on natural product-derived vesicles, most of which are believed to\nhave immunomodulatory properties, it would be valuable to investigate whether the ginger-derived vesicles used in this\npaper have other effects during the treatment of bacterial infections. Additionally, offer reasonable justifications for selecting\nginger vesicles over vesicles from other plants.\n2. The Pseudomonas aeruginosa strains used in both in vitro and in vivo experiments should be clearly specified. According\nto Figure 3e and 3f, the initial MIC of ATCC27853 for Cars is 8 μg/mL. However, the MIC for Car in Figure 3b is 64 μg/mL,\nindicating these are not the same strain. Please detail the specific strains and their antimicrobial resistance profiles.\nAdditionally, is the Ce-Car@EV, designed and synthesized in this study, effective against various mechanisms of drug-\nresistant Pseudomonas aeruginosa or other bacterial species like Streptococcus pneumoniae and Klebsiella pneumoniae,\ncommonly found in pneumonia cases?\n3. Could a clearer and more explicit TEM image be provided in Figure 1b? Additionally, in line 129, you mentioned that\n'TEM images of Ce-Car@EV NPs show that Ce-Car NPs were uniformly distributed on the surface of G-EVs,' which\ncontradicts the schematic diagram in Figure 1a, where Ce-Car NPs are depicted as encapsulated inside G-EVs, and the\nstatement 'Ce-Car NPs within ginger-derived EVs' in line 163. Could you clarify the positional relationship between G-EVs\nand Ce-Car NPs in Ce-Car@EV NPs? Given the description of nanoparticle sizes, it is recommended to include dynamic\nlight scattering (DLS) size data to support the TEM findings.\n4. In addition to its two carboxyl groups that facilitate binding to cerium ions, are there other reasons the authors chose\ncarbenicillin? Given that drug-resistant bacteria often exhibit high resistance to penicillin antibiotics, could carboxypenicillin\nbe replaced by other antibiotics with stronger antibacterial effects?\n5. In line 153, the ICP-MS experimental results are cited to demonstrate the ratio of Ce and Car in Ce-Car@EV. The\ncorresponding raw data should be provided for clarity.\n6. In line 267, you concluded that 'These results demonstrate that the treatment of Ce-Car@EV could attenuate\ntranscriptional alterations in Car-induced genes, suggesting reversal of Car-induced bacterial resistance at the\ntranscriptional level.' However, considering the larger number of differentially expressed genes (DEGs) between PA-30R\nand PA30M, the conclusion based solely on the volcano plot analysis seems insufficient to support this result.\n7. Can you clarify the difference between Figure 4i and Figure 4j? This information is not fully provided in the article.\n8. Since this study employs Ce-Car@EV for treating bacterial pneumonia in subsequent animal experiments, it is\ninappropriate to use only the mouse fibroblast cell line L929 in the in vitro biocompatibility tests. Regardless of the suitability\nof the chosen cell line, at least two cell lines should be used to confirm in vitro biocompatibility.\n9. In the fluorescence targeting experiments depicted in Figure 5, the authors noted that the EPR effect caused material\naggregation in the inflamed lung. However, they did not include an intravenous injection of Ce-Car@EV in healthy mice as a\ncontrol to demonstrate the absence of significant Ce-Car@EV aggregation in non-inflamed lung tissue. Additionally, Ce-\nCar@EV lacks specific targeting modifications for bacterial infection sites, and the EPR effect alone cannot account for the\ntargeting of infected lungs as shown in the experimental results in Figure 5. A more robust explanation is needed for this\nobservation.\n10. There is a citation error in the reference at line 639. The original text of the cited part is: 'Mice were made neutropenic\nwith two subcutaneous injections of cyclophosphamide: 150 mg/kg at 4 days before PAO1 infection and 100 mg/kg at 1 day\nbefore PAO1 infection.'\n11. In line 427, there is a missing space in 'withCe-Car@EV' (it should be 'with Ce-Car@EV')..\n12. The abbreviations in the manuscript should be standardized. Ce-Car@EV is referred to both as 'nanoscale coordination\npolymers (NCPs)' and 'nanoparticles (NPs)', and this inconsistency should be resolved by using a single term.\n13. The Discussion section is underdeveloped, both in length and content. It only summarizes the experimental findings\nwithout placing them in the context of previous ginger vesicle studies, including those using the same nanoparticle\ncomponents. Additionally, the section lacks any discussion of the study's limitations.\n14. It is advisable to review and correct formatting issues throughout the manuscript, including inconsistent line spacing\nwithin paragraphs and variations in font size..\n15. The Ce-Car obtained is a light yellow substance. However, in the CCK8 experiment, the formazan produced by active\nbacteria is also yellow. It is advisable to include an image of the 32μg/mL material to illustrate whether it interferes with the\nformazan detection.\n16. In the in vitro antibacterial experiments, the authors utilized a buffer with a pH of 5.5. Please indicate the type of buffer\nand the method to adjust the pH.\n17. In Figures 3b-d, the sequence and description of the images are unclear. It is suggested to reorder the images and\nprovide a clear narrative.\n18. The authors should depict the metabolic pathways and half-lives of cerium metal in living organisms. The synthetic Ce-\nCar@EV is released in a slightly acidic environment. Under these conditions, will the released cerium enter nearby immune\ncells at the infection site and inhibit the energy metabolism of human cells?", "reviewer_2": "This manuscript presents a highly innovative and well-executed study on the development and evaluation of a biomimetic\nnanoplatform (Ce-Car@EV) for combating drug-resistant P. aeruginosa. The work addresses the critical global challenge of\nantimicrobial resistance (AMR) through a sophisticated strategy combining pH-responsive metal-drug coordination\nnanoparticles (i.e. Ce-Car NPs) with ginger-derived extracellular vesicles (EVs). Extensive physicochemical\ncharacterization (TEM, XPS, FTIR, zeta potential, SDS-PAGE) confirmed successful synthesis, EV coating, and pH-\nresponsive degradation. Transcriptomics (RNA-seq) and pathway analysis (KEGG/GO) convincingly demonstrated\ndownregulation of efflux pumps (mexAB-oprM), oxidative phosphorylation, and TCA cycle genes, linking Ce(IV) to energy\ndisruption. The nanoplatform significantly improves survival, reduces bacterial loads in lungs/organs, alleviates lung injury,\nand demonstrates targeted accumulation via EV-mediated evasion of immune clearance. However, these authors need to\naddress the following issues:\n1. Safety of Ce(IV): While biocompatibility is shown, long-term biodistribution/accumulation of cerium in organs should be\naddressed (e.g., via ICP-MS in tissues beyond 7 days, selectivity between bacterial cells and mammalian cells).\n2. Direct evidence such as efflux assay with fluorescent substrates like Hoechst 33342 to complement transcriptomic data,\nshall be added.\n3. Figure 2C: It should be “LUMO” instead of “HUMO”\n4. Line 215-219: “According to the standard classification criteria (FICI ≤0.5: synergistic effect; 0.5 < FICI ≤ 1: additive effect;\nFICI > 1: no interaction), the combination of Ce(IV) and Car exhibited a strong synergistic effect with FICI of 0.5.” According\nto your description, FICI 0.5 can not be defined as “Strong synergistic effect”, and it should be additive effect between your\ntwo drugs. This exaggeration occurs many times and describes the mechanism reluctantly. Also, FICI>1 should be\nantagonistic effect instead of no interaction.\n5. Figure 3a and 3f, the bacterial viability of Ce-Car@EV seems not constant. Why resistant strains even demonstrated lower\nbacterial viability (0%) under the treatment of 16 μg/ml CE-Car@EV compared with sensitive strains (20%). Please clarify\nthe bacterial strains used in each figure in figure captions.\n6. The Ce-Car@EV platform combines innovative design, mechanistic rigor, and compelling in vivo validation, however, the\nsynergistic effect seems reluctant and overall the mechanism is confusing. These authors shall clarify it.\n7. These authors presented the use of Ce-Car@EV nanoplatform for AMR. However, in the docking simulation, they use Car\nand Ce(IV) ions. They shall address the gap between nano materials and small molecules and ions (metabolism and/or\ndecomposition). Key references shall be cited: Nat Rev Chem. 2023,7, 202-224; J. Am Chem Soc. 2025, 147, 12361-12380.", "reviewer_3": "In this work, Ye et al. developed ginger-derived extracellular vesicle (EV)-encapsulated cerium-carbenicillin nanoplatforms\n(Ce-Car@EV) with the antibacterial capacity by the disruption of bacterial energy metabolism. The nanoplatform utilizes the\nCe⁴⁺ ions to induce ATP depletion and proton motive force dissipation to reverse efflux pump-driven resistance, which can\nsignificantly enhance carbenicillin efficacy against drug-resistant P. aeruginosa. Experimental results in vivo demonstrate\nthe reduced bacterial burden and improved survival in a murine pneumonia model. However, there are still some issues in\nthe manuscript that need to be addressed. Therefore, I think this article is worthy of being published in Nature\nCommunications after minor revisions.\n1. The biological role of G-EVs as drug carriers requires more detailed biochemical validation. Current characterizations via\nTEM and SDS-PAGE cannot fully confirm their exosomal properties. Western blotting analysis should be performed to\nassess conserved exosome markers and verify vesicle integrity.\n2. Compared with mammalian cell-derived EVs, plant-derived edible EVs exhibit lower immunogenicity, along with\nadvantages in sustainability and renewability. The yield of ginger-derived exosomes requires quantification.\n3. The cytotoxicity assessment in murine fibroblasts provides preliminary evidence of biocompatibility; however, evaluation\nacross additional cell lines is needed to obtain a comprehensive safety profile.\n4. Although Supplementary Figures 9-11 confirm systemic biocompatibility at therapeutic concentrations in vivo, the\npotential neurotoxicity of cerium ions requires specific evaluation in neural tissues.\n5. Several graphical elements require clarification. Specifically, in Fig. 2c, the label 'HUMO' should be corrected to 'LUMO'\n(lowest unoccupied molecular orbital) to ensure accurate representation of molecular orbitals.\n6. Reference formatting requires consistent verification. For example, the volume and page numbers for Reference No. 5\nshould be provided.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Dear Reviewers,\nThank you very much for the comments on the present work. Those comments are\nvaluable and very helpful for revising and improving our paper, as well as the important\nguiding significance to our research. In the following, we make a point-to-point\nresponse to all the comments.\nReviewer 1:\nYe and colleagues introduce Ce-Car@EV, a nanoparticle made from a cerium-\ncarbenicillin complex and ginger-derived vesicles, emphasizing its potential for\ntreating bacterial pneumonia. This nanoparticle demonstrates effective bactericidal\nproperties and in vitro/in vivo biocompatibility, targeting the lungs via the EPR effect\nto combat Pseudomonas aeruginosa. The study is well-structured, including\nantibacterial experiments against drug-resistant Pseudomonas aeruginosa,\nconstruction and sequencing of drug-resistant strains, and an exploration of bacterial\nresistance mechanisms. However, the study focuses solely on Pseudomonas aeruginosa,\nraising concerns about its broad-spectrum antibacterial efficacy. The antibacterial\neffectiveness against other key pathogens causing bacterial pneumonia, such as\nStreptococcus pneumoniae and Klebsiella pneumoniae, remains unexplored, limiting\nits clinical applicability. Additionally, the mechanism by which ginger vesicles, as an\nexternal substance, extend their time in the bloodstream is not fully addressed. While\nthe combination of cerium-based nanoparticles and ginger vesicles is innovative, the\nresearch lacks significant innovation in material synthesis and mechanism exploration.\nThe study does not provide groundbreaking insights into material preparation or\nbiomedical issues. Given the innovative nature of the topic and the experimental data,\nit is challenging to meet the standards of Nature Communications. It is recommended\nto either reject the paper or submit it to Communications Materials.\nResponse: We sincerely appreciate the reviewer's valuable comments. The manuscript\nhas been carefully revised according to each comment from the reviewer, and its overall\nquality has been significantly enhanced to meet the stringent requirements of Nature\nCommunications.\nIn response to the comment on the broad-spectrum antimicrobial efficacy, in revision,\nwe have supplemented the assessment of Ce-Car@EV NPs against Klebsiella\npneumoniae, a primary pathogen causing pneumonia, as presented in Supplementary\nFig. 6. The results indicate that after 8 h of co-culture, Ce-Car@EV NPs exhibits\ncomplete bactericidal activity at a concentration of 128 μg/mL. These results confirm\nthat Ce-Car@EV NPs can effectively eliminate multiple Gram-negative pathogens,\ndemonstrating significant potential for addressing a broader range of clinically relevant\nbacterial infections.\nWe also thank the reviewer for this insightful comment regarding the mechanism of\nimmune evasion. The prolonged circulation of ginger-derived extracellular vesicles (G-\nEVs) is attributed to their inherent biogenic properties, which are significantly distinct\nfrom synthetic nanocarriers. This is directly supported by our newly added in vitro\nmacrophage uptake assay, which shows a substantial reduction in the internalization of\nCe-Car@EV NPs compared to Ce-Car NCPs (Supplementary Fig. 17). Furthermore,\nupon entering the bloodstream, natural vesicles are known to form a specific protein\ncorona, which further minimizes opsonization and subsequent clearance in vivo (Nat.\nCell. Biol. 2022, 24, 1322-1325; Cell. Commun. Signal. 2023, 21, 64; J. Extracell.\nVesicles 2025, 14, e70124.). In vivo biodistribution data also demonstrates markedly\nextended blood circulation and enhanced lung accumulation of Ce-Car@EV NPs (Fig.\n5). In conclusion, the long-circulating property is a well-substantiated functional\ncharacteristic of Ce-Car@EV NPs platform, underpinned by both established literature\nand our experimental validation.\nFrom the perspective of material design, cerium-carbenicillin coordination polymer\nrepresents a departure from the conventional nanocarrier systems. Rather than simply\nloading drugs into pre-formed particles, we constructed an integrated metal-antibiotic\nnetwork, in which the therapeutic agent forms an inherent part of the nanostructure,\nenabling simultaneous effective delivery of the antibiotic and components with\ncatalytic function. Mechanistically, we have identified a comprehensive “energy\ndisruption” strategy by utilizing Ce-Car@EV NPs. Transcriptomic and functional\nanalyses demonstrate that our platform concurrently disrupts ATP production,\ndissipates proton motive force, and inhibits efflux pump activity. These effects,\ncombined with the capacity to suppress biofilm formation and attenuate virulence,\ncollectively establish a unified resistance-reversal mechanism effective against the\ncomplex resistance profiles of drug-resistant P. aeruginosa. Furthermore, we validated\nthe versatility of Ce-Car@EV NPs against other clinically relevant pulmonary\npathogens (e.g., K. pneumoniae), confirming the broad applicability and translational\npotential of our coordination-based strategy beyond initial model organisms.\nWe hope that the provided clarifications and additional data adequately highlight the\nnovelty and mechanistic insights of our study. The changes and detailed descriptions in\nthe revised manuscript were highlighted by red font.\nSupplementary Fig. 6. a Corresponding plate-counting data of drug-resistant K.\npneumoniae after treatment with Ce-Car@EV NPs at different concentrations. b\nViability of drug-resistant K. pneumoniae after 8 h of treatments with Ce-Car@EV NPs\nat different concentrations. Data are presented as mean values ± SD (n = 5 independent\nsamples).\nFig. 5. In vivo pharmacokinetics, biodistribution and metabolism of Ce-Car@EV NPs.\na Schematic illustration of in vivo imaging procedure of Ce-Car@EV NPs. b\nRepresentative in vivo fluorescent images of drug-resistant P. aeruginosa pneumonia\nmice at various time points (0, 2, 4, 6, 8, 12, and 24 h) after tail vein injection of either\nCe-Car NCPs or Ce-Car@EV NPs. c Quantitative analysis of fluorescence signal\nintensities in infected lung regions at different time points after post-injection (n = 3\nindependent samples). Fluorescence images (d) and corresponding quantitative\nanalysis (e) of fluorescence intensities in major organs and infected lung tissues at 6 h\npost-injection (n = 3 independent samples). f Cerium concentrations in blood were\nquantified at designated time points (0.167, 1, 2, 4, 6, 8, 12, 24, and 48 h) after\nintravenous administration to healthy mice, with the circulation half-life determined via\nnon-compartmental modeling (n = 3 independent samples). Mass of cerium in feces (g)\nand urine (h) at different intervals within 7 days post-injection (n = 3 independent\nsamples). Data are presented as mean values ± SD. One-way analysis of variance\n(ANOVA) was used to calculate statistical significance.\nSupplementary Fig. 17. CLSM images of RAW 264.7 cells respectively co-cultured\nby Ce-Car NCPs and Ce-Car@EV NPs.\n1. Given that this paper builds on your lab's prior research, the choice of ginger-derived\nvesicles is understandable. However, it seems counterintuitive to claim that these\nvesicles, as exogenous substances, can evade the immune system. Compared to\nmammalian cells, ginger vesicles and other plant vesicles, as foreign entities, are more\nlikely to be recognized and engulfed by immune cells, potentially affecting their\neffectiveness. The authors should conduct relevant experiments and provide reliable\ndata to support this claim, such as immune cell phagocytosis assays or bacterial\nphagocytosis assays.\nResponse: We thank the reviewer for this insightful comment regarding the immune\nrecognition and response of plant-derived vesicles. Tremendous studies document that\nmammalian extracellular vesicles still pose inherent immunogenicity risks, even in\nallogeneic applications. Of particular concern is the accelerated blood clearance\nobserved upon repeated administration of small extracellular vesicles derived from\nallogeneic sources. This phenomenon is attributed to immune recognition, thus leading\nto potential adverse effects. Generally, mammalian vesicles carry various immunogenic\ncomponents such as MHC class I/II proteins that can trigger specific immune responses\nand further exacerbate this clearance phenomenon (Adv. Mater. 2024, 36, 2403199;\nExpert Opin. Drug Del. 2025, 22, 787–803.) In contrast, plant-derived extracellular\nvesicles lack these animal-specific immunogenic markers, substantially lowering their\nrisk of immunogenicity and further avoiding complications such as anti-drug antibody\nresponses (Adv. Mater. 2023, 35, 2207826; Asian J. Pharm. Sci. 2025, 20, 101052.). In\nour work, this theoretical advantage is corroborated by pharmacokinetic studies, which\ndemonstrate that plant-derived vesicles can achieve circulation half-lives extending to\nseveral hours, far exceeding the minute-scale half-lives of mammalian counterparts\n(Nat. Rev. Mater. 2021, 6, 103–106; J. Extracell. Vesicles 2015, 4, 26238; ACS Appl.\nMater. Interfaces 2021, 13, 25575–25588; Eur. J. Pharm. Sci. 2017, 96, 316–322; Nat.\nCommun. 2013, 4, 1867.). Additionally, systematic macrophage phagocytosis assays\nhave been conducted, which clearly show that the as-prepared Ce-Car@EV NPs exhibit\nlower cellular uptake efficiency compared to that of the bare Ce-Car NCPs without\nginger-derived extracellular vesicles (G-EVs) coating (Supplementary Fig. 17).\nCombined with the proven stability, scalable production potential, and absence of\nzoonotic risks, these characteristics collectively provide grounds for the selection of\nplant-derived vesicles (Adv. Drug Deliver. Rev. 2022, 182, 114108; Mol. Ther. 2021, 29,\n13–31.). Relevant changes and detailed descriptions in the revised manuscript and\nSupporting Materials were presented as follows.\n“After intravenous injection……To validate the extended blood circulation capability\nconferred by G-EVs coating, we compared the cellular uptake of Ce-Car NCPs and Ce-\nCar@EV NPs by RAW 264.7 macrophages in vitro. CLSM images confirmed that the\nrational encapsulation of G-EVs significantly reduced the phagocytosis of Ce-Car@EV\nNPs compared to the bare Ce-Car NCPs (Supplementary Fig. 17). This diminished\nimmunocellular clearance capacity is crucial for prolonging the systemic circulation of\nthe nanoplatform.” (Manuscript Page 17, Lines 429-435)\nSupplementary Fig. 17. CLSM images of RAW 264.7 cells respectively co-cultured\nby Ce-Car NCPs and Ce-Car@EV NPs.\nAdditionally, considering the recent extensive studies on natural product-derived\nvesicles, most of which are believed to have immunomodulatory properties, it would be\nvaluable to investigate whether the ginger-derived vesicles used in this paper have\nother effects during the treatment of bacterial infections.\nResponse: We thank the reviewer for this valuable suggestion regarding the\nimmunomodulatory potential of natural product-derived vesicles. We agree that this\nrepresents an interesting dimension of their biological activity. In response, we\nspecifically addressed this issue in the “Discussion” section of the revised Manuscript,\nwhere we acknowledge the immunomodulatory properties of ginger-derived vesicles\nand their potential relevance in the context of infection treatment. While the primary\nfocus of this study remains on establishing the fundamental antibacterial efficacy and\ndrug delivery capability of our engineered system, we fully recognize the importance\nof immune modulation as a complementary therapeutic mechanism. Therefore, the\npotential immunomodulatory effects of ginger-derived vesicles represent a compelling\navenue for future investigation, which we are committed to exploring further in our\nsubsequent research.\nAdditionally, offer reasonable justifications for selecting ginger vesicles over vesicles\nfrom other plants.\nResponse: We appreciate the reviewer for this insightful question regarding the\nselection of ginger-derived vesicles (G-EVs). Our previously published work\nestablished the efficacy of G-EVs in the efficient delivery of therapeutic cargo,\nproviding a strong rationale for their use in the current platform (Nat. Commun. 2022,\n13, 7164; Adv. Sci., 2024, 11, 2408442.).Various studies have consistently demonstrated\nthat G-EVs exhibit superior stability and a natural tropism for inflammatory sites,\nmaking them particularly suitable for targeted drug delivery to the specific infection\nmicroenvironments (J. Adv. Res. 2025, 69, 1-15; Adv. Sci. 2025, 12, e05208). Moreover,\nginger is widely documented as a scientifically established safe edible plant (Pharmacol.\nRes. 2023, 187, 106610; Semin. Cancer Biol. 2021, 69, 140-149). While extracellular\nvesicles derived from other plant sources are also under investigation, our selection of\nG-EVs as the ideal vector was guided by our team’s research experience, their\ndemonstrated inflammatory targeting capability, and established safety profile.\n2. The Pseudomonas aeruginosa strains used in both in vitro and in vivo experiments\nshould be clearly specified. According to Figure 3e and 3f, the initial MIC of\nATCC27853 for Cars is 8 μg/mL. However, the MIC for Car in Figure 3b is 64 μg/mL,\nindicating these are not the same strain. Please detail the specific strains and their\nantimicrobial resistance profiles.\nResponse: We thank the reviewer for these insightful questions regarding the bacterial\nstrains and the broader applicability of our platform. In Fig. 3d, the experiments are\ncarried on the standard strain P. aeruginosa (ATCC 27853), which exhibits intrinsic\nresistance to carbenicillin as reflected by the MIC value of 64 μg/mL. In Fig. 3f and all\nsubsequent in vivo antibacterial experiments, we employed a laboratory-generated and\ncarbenicillin-resistant strain, designated PA-15R. This drug-resistant strain was derived\nfrom parental ATCC 27853 through 15 sequential passages in standard, undiluted LB\nmedium containing carbenicillin, resulting in a high-level resistant strain (MIC = 2048\nμg/mL). The separate 30-generation resistance study in Fig. 3e and the corresponding\ntranscriptomic analysis utilized a distinct strain lineage (PA-30R) that was passaged in\n5-fold diluted LB medium throughout the extended experimental protocol. Relevant\nchanges and detailed descriptions in the revised manuscript and Supporting Materials\nwere presented as follows.\nFig. 3. In vitro antibacterial performance of Ce-Car@EV NPs. a Viability of standard\nP. aeruginosa (ATCC 27853) following 2 h of treatment with Ce-Car@EV NPs at\nvarious concentrations at pH of 5.5 and 7.4 (n = 3 independent samples). b Live/dead\nstaining of P. aeruginosa treated with Ce-Car@EV NPs under different pH conditions\n(pH 5.5 and pH 7.4). Scale bar = 25 µm. c SEM images of P. aeruginosa after treatment\nwith Ce-Car@EV NPs under different pH conditions. Scale bar = 500 nm. d\nCheckerboard dilution assay of the synergistic effects between Car and Ce(IV) against\nP. aeruginosa (n = 3 independent samples). e Development of resistance to Car in P.\naeruginosa over 30 generations induced by Ce-Car@EV NPs (n = 3 independent\nsamples). f Checkerboard dilution assay of synergistic effects between Car and Ce(IV)\nagainst drug-resistant P. aeruginosa (PA-15R) (n = 3 independent samples). g\nAntibacterial effects of Car and Ce-Car@EV NPs on drug-resistant P. aeruginosa (PA-\n15R) (n = 3 independent samples). h Viability of drug-resistant P. aeruginosa (PA-15R)\nafter 4 h of co-incubation with Car and Ce-Car@EV NPs at different concentrations (n\n= 3 independent samples). i Corresponding plate-counting data of drug-resistant P.\naeruginosa (PA-15R) after treatment with Car and Ce-Car@EV NPs at different\nconcentrations (n = 3 independent samples). Data are presented as mean values ± SD.\nTwo-tailed Student’s t-test was used to calculate statistical significance.\n“Bacteria co-cultured with 1/2 MIC value of each group was used for passaging, and\nthe above steps were repeated for 30 days. The MIC of each passage was recorded and\ncompared with that of the first passage. Under the same culture protocol, a distinct\ncarbenicillin-resistant strain (PA-15R) was generated through 15 sequential passages\nof the parental ATCC 27853 strain in the standard and undiluted LB medium containing\ncarbenicillin, which was utilized for subsequent antibacterial efficacy evaluation.”\n(Manuscript Page 29, Lines 732-735)\nAdditionally, is the Ce-Car@EV, designed and synthesized in this study, effective\nagainst various mechanisms of drug-resistant Pseudomonas aeruginosa or other\nbacterial species like Streptococcus pneumoniae and Klebsiella pneumoniae,\ncommonly found in pneumonia cases?\nResponse: Transcriptomic analysis provides compelling evidence that the antibacterial\neffect of Ce-Car@EV NPs is achieved through the disruption of multiple resistance\npathways. Beyond the initially demonstrated efflux pump inhibition, as shown in the\nrevised Supplementary Fig. 11, antibacterial treatment of Ce-Car@EV significantly\naffects genes associated with biofilm formation, demonstrating its ability to counter this\nkey adaptive resistance mechanism. This multi-target action against both intrinsic and\nadaptive resistance mechanisms strengthen the potential of our nanoplatform for\ncombating complex resistance profiles in P. aeruginosa. Concerning activity against\nother pneumonia-relevant pathogens, we have evaluated Ce-Car@EV NPs against\nKlebsiella pneumoniae as suggested. Results demonstrate excellent bactericidal activity\nat a concentration of 128 μg/mL following an 8-hour co-culture (Supplementary Fig.\n6), confirming their efficacy against this typical Gram-negative pathogen and multiple\npneumonia-associated pathogens. Relevant changes and detailed descriptions in the\nrevised manuscript and Supporting Materials were presented as follows.\nSupplementary Fig. 11. Heatmap of bacterial biofilm formation-related genes in PA,\nPA-30R and PA-30M cells.\n“…….suggesting that Ce-Car@EV NPs could inhibit antibiotic efflux, thereby\ncontributing to the reversal of antibiotic resistance. Beyond inhibiting efflux pump\nactivity, the transcriptomic analysis in Supplementary Fig. 11 also revealed that Ce-\nCar@EV NPs significantly modulate the expression behaviors of genes associated with\nbiofilm formation, verifying their capacity to disrupt this critical adaptive resistance\nmechanism, which is frequently responsible for therapeutic tolerance in persistent\ninfections35…Collectively, these multi-faceted transcriptional alterations - spanning\nvirulence attenuation, energy metabolism disruption, efflux pump suppression, and\nbiofilm formation inhibition - converge into a unified resistance-reversal mechanism,\nthereby significantly enhancing the potential to combat complex resistance profiles in\nP. aeruginosa.” (Manuscript Page 13-14, Lines 319-341)\nSupplementary Fig. 6. a Corresponding plate-counting data of drug-resistant K.\npneumoniae after treatment with Ce-Car@EV NPs at different concentrations. b\nViability of drug-resistant K. pneumoniae after 8 h of treatments with Ce-Car@EV NPs\nat different concentrations. Data are presented as mean values ± SD (n = 5 independent\nsamples).\n“Meanwhile, the wide-spectrum antibacterial performance of Ce-Car@EV NPs was\nalso investigated using Klebsiella pneumoniae (K. pneumoniae), another formidable\nGram-negative bacterium frequently implicated in pneumonia, as a model. Ce-\nCar@EV NPs displayed complete bactericidal activity against drug-resistant K.\npneumoniae at a concentration of 128 μg/mL following an 8-hour co-incubation\n(Supplementary Fig. 6), which might be attributed to the core antibacterial mechanism,\noperating through pH-responsive payload release and bacterial energy metabolism\ndisruption. These results indicate the effective elimination of Ce-Car@EV NPs to\ndiverse Gram-negative pathogens, suggesting their considerable potential for applying\na wide range of clinically relevant bacterial infections.” (Manuscript Page 10, Lines\n246-256)\n“The antibacterial effectiveness of Ce-Car@EV NPs against diverse Gram-negative\npathogens, including both P. aeruginosa and K. pneumoniae, stems from its unique\ndual-mechanism approach. Both pathogens possess penicillin-binding proteins, which\nserve as targets for carbenicillin’s inhibition of cell wall biosynthesis. Consequently,\nCe(IV) ions concurrently induce energy deprivation through ATP hydrolysis and\ndissipation of the proton motive force (Fig. 4h-l). Overall, this coordinated attack on\nstructural integrity and energy metabolism enables effective suppression of diverse\nresistant pathogens.” (Manuscript Page 27, Lines 646-653)\n3. Could a clearer and more explicit TEM image be provided in Figure 1b?\nResponse: We appreciate the reviewer's insightful comments regarding the structural\ncharacterization of Ce-Car@EV NPs. In revision, we have replaced the TEM image in\nFig. 1b with a high-resolution photograph, which more clearly reveals the structural\ndetails of Ce-Car NCPs, G-EVs, and Ce-Car@EV NPs. Relevant changes and detailed\ndescriptions in the revised manuscript were presented as follows.\nFig. 2. Preparation and characterization of Ce-Car@EV NPs. a Schematic diagram of\nthe preparation process of Ce-Car@EV NPs. b TEM images of Ce-Car NCPs, G-EVs\nand Ce-Car@EV NPs. c Partial charge density plot of the HOMO and LUMO levels of\nCe-Car NCPs. d FTIR spectra of Car and Ce-Car NCPs. e Zeta potential of Ce-Car\nNCPs, G-EVs and Ce-Car@EV NPs (n = 3 independent samples). f Release profiles of\nCar from Ce-Car@EV NPs in Tris-HCl at pH of 5.5 and 7.4 (n = 3 independent samples).\ng Protein analysis of G-EVs and Ce-Car@EV NPs using SDS-PAGE. Molecular\ndocking simulations of Car binding to Penicillin-Binding Protein 3 (PBP3) in the\nabsence (h) or presence (i) of Ce(IV). The protein is shown as a cartoon model, while\nCar is depicted as a stick model. Hydrogen bonds, π-π interactions, van der\nWaals/hydrophobic interactions, and electrostatic interactions are represented by red,\nblue, yellow, and purple dashed lines, respectively. Data are presented as mean values\n± SD.\nAdditionally, in line 129, you mentioned that 'TEM images of Ce-Car@EV NPs show\nthat Ce-Car NPs were uniformly distributed on the surface of G-EVs,' which\ncontradicts the schematic diagram in Figure 1a, where Ce-Car NPs are depicted as\nencapsulated inside G-EVs, and the statement 'Ce-Car NPs within ginger-derived EVs'\nin line 163. Could you clarify the positional relationship between G-EVs and Ce-Car\nNPs in Ce-Car@EV NPs?\nResponse: We sincerely appreciate the reviewer for identifying the inconsistency in\ndescribing the spatial relationship between Ce-Car NCPs and G-EVs. The Ce-Car@EV\nNPs involve the encapsulation of Ce-Car NCPs within the lumen of G-EVs. We have\nchanged the related description, and the relevant changes in the revised manuscript were\npresented as follows.\n“Notably, TEM images of Ce-Car@EV NPs show that Ce-Car NCPs were primarily\nencapsulated within G-EVs, confirming the successful preparation of the Ce-Car@EV\nhybrid nanoplatform.” (Manuscript Page 6, Lines 129-131)\nGiven the description of nanoparticle sizes, it is recommended to include dynamic light\nscattering (DLS) size data to support the TEM findings.\nResponse: We sincerely appreciate the reviewer's valuable comments. In revision, we\nhave performed dynamic light scattering (DLS) measurements to complement the size\ncharacterization of the TEM image. Relevant changes and detailed descriptions in the\nrevised manuscript and Supporting Materials were presented as follows.\n“…between Ce(IV) and Car in the coordination structure of Ce-Car NCPs (Fig. 2d).\nThe hydrodynamic size distribution assessed by dynamic light scattering (DLS) analysis\nshowed a profile consistent with the TEM fundings (Supplementary Fig. 3).”\n(Manuscript Page 6, Lines 144-146)\nSupplementary Fig. 3. Size distribution of Ce-Car NCPs, G-EVs, and Ce-Car@EV\nNPs.\n4. In addition to its two carboxyl groups that facilitate binding to cerium ions, are there\nother reasons the authors chose carbenicillin?\nResponse: We thank the reviewer for this insightful question regarding our antibiotic\nselection. The choice of carbenicillin is primarily based on its molecular structure,\nwhich contains two carboxyl groups that provide effective coordination sites for cerium\nions. These enable the formation of a metal-drug coordination framework, serving as\nthe core of our platform design.\nGiven that drug-resistant bacteria often exhibit high resistance to penicillin antibiotics,\ncould carboxypenicillin be replaced by other antibiotics with stronger antibacterial\neffects?\nResponse: The design principle of our platform indeed demonstrates the good\nversatility beyond the specific carbenicillin-cerium system presented in this work. In\nrevision, we have conducted additional experiments, demonstrating that this fabrication\nstrategy of metal-drug coordination can be successfully extended to other antibiotic\nclasses. Specifically, we synthesized a vancomycin-cerium coordination polymer (Fig.\nR1) and confirmed its potent antibacterial efficacy against K. pneumoniae (Fig. R2).\nThese results indicate that this strategy for preparing the metal-antibiotic coordination\nprovides a generalizable platform adaptable to various antibiotic classes, significantly\nbroadening its potential application range.\nFig. R1. TEM image of vancomycin-cerium coordination polymer.\nFig. R2. Antibacterial effect of pure vancomycin and vancomycin-cerium coordination\npolymer on K. pneumoniae.\n5. In line 153, the ICP-MS experimental results are cited to demonstrate the ratio of Ce\nand Car in Ce-Car@EV. The corresponding raw data should be provided for clarity.\nResponse: We thank the reviewer for requesting the raw data to support ICP-MS results.\nThe original ICP-MS data files quantifying Ce and Car content in Ce-Car@EV NPs are\nprovided in the Source Data file accompanying this manuscript.\n6. In line 267, you concluded that 'These results demonstrate that the treatment of Ce-\nCar@EV could attenuate transcriptional alterations in Car-induced genes, suggesting\nreversal of Car-induced bacterial resistance at the transcriptional level.' However,\nconsidering the larger number of differentially expressed genes (DEGs) between PA-\n30R and PA30M, the conclusion based solely on the volcano plot analysis seems\ninsufficient to support this result.\nResponse: We thank the reviewer for this thoughtful comment regarding the evidence\nsupporting our conclusion on transcriptional reversal of resistance. We agree that the\noriginal presentation gave the impression that the conclusion was primarily based on\nthe volcano plot analysis. In fact, this interpretation was supported by transcriptomic\ndata presented in Fig. 4, which extends beyond the initial DEG identification. The key\nevidence resides in the subsequent functional analysis of specific gene categories. As\nmore clearly articulated statements in the revised manuscript, these conclusions are\nindeed confirmed by the downregulation of key resistance gene transcription levels.\nThe downregulation of efflux pump genes (such as mexA, mexB, and oprM) and the\ncoordinated downregulation of central energy metabolism pathways (TCA cycle,\noxidative phosphorylation) are included, as well as the attenuation of virulence factors.\nRelevant changes and detailed descriptions in the revised manuscript were presented as\nfollows.\n“The magnitude of transcriptional alterations in PA-30M relative to pure P. aeruginosa\nwas substantially attenuated compared to that in PA-30R.” (Manuscript Page 12,\nLines 288-290)\n“By coordinately normalizing efflux pump transcription, disrupting energy metabolism,\nand functionally compromising efflux pumps, Ce-Car@EV NPs establishes a powerful\nmulti-mode mechanism that reverses carbenicillin resistance of drug-resistant bacteria\nat both transcriptional and functional levels.” (Manuscript Page 14, Lines 367-371)\n7. Can you clarify the difference between Figure 4i and Figure 4j? This information is\nnot fully provided in the article.\nResponse: Thank you for your useful comment. In revision, the figure legends and\ncorresponding labels in Figs. 4i and 4j have been changed to more clearly distinguish\nbetween the ATP hydrolysis assay and the measurement of intracellular ATP levels in\nbacterial cells. Relevant changes and detailed descriptions in the revised manuscript\nwere presented as follows.\nFig. 4. Mechanism of Ce-Car@EV NPs to overcome drug resistance of P. aeruginosa.\na Venn diagram of DEGs in the three groups. b Volcano plot of DEGs in bacteria after\ndifferent treatments. c Gene ontology (GO) annotation analysis of DEGs between PA-\n30M and PA-30R cells. d KEGG enrichment analysis of DEGs between PA-30M and\nPA-30R cells. The size of the bubbles represents the number of genes and the color of\nthe bubbles corresponds to different Q-value ranges. e Cluster analysis of bacterial\nmultidrug efflux-related genes based on expression patterns. f Heatmap of oxidative\nphosphorylation-related genes in PA, PA-30R, and PA-30M cells. g Heatmap of genes\nrelated to TCA cycle and glycolysis in PA, PA-30R, and PA-30M cells. h Schematic\nrepresentation of the mechanism of Ce-Car@EV NPs on the oxidative phosphorylation\nprocess. i ATP content after direct incubation with Ce-Car@EV NPs at different\nconcentrations (n = 4 independent samples). j Intracellular ATP content in P. aeruginosa\nafter the treatment of Ce-Car@EV NPs at different concentrations after 24 h (n = 4\nindependent samples). k Effect of Ce-Car NCPs on the activity of bacterial respiratory\nchain complexes I and IV (n = 4 independent samples). l Effect of Ce-Car@EV NPs at\ndifferent concentrations on activity of drug efflux pumps in drug-resistant P. aeruginosa\n(n = 3 independent samples). Data are presented as mean values ± SD. Statistical\nsignificance testing in i, j and l was calculated by One-way analysis of variance\n(ANOVA), whereas that of k were calculated by two-tailed Student’s t-test.\n8. Since this study employs Ce-Car@EV for treating bacterial pneumonia in subsequent\nanimal experiments, it is inappropriate to use only the mouse fibroblast cell line L929\nin the in vitro biocompatibility tests. Regardless of the suitability of the chosen cell line,\nat least two cell lines should be used to confirm in vitro biocompatibility.\nResponse: We appreciate the reviewer's valuable suggestion. In revision, cytotoxicity\nassays have been evaluated using murine macrophage cells (RAW 264.7) and human\numbilical vein endothelial cells (HUVECs), alongside the original L929 fibroblast.\nThe changes and detailed descriptions in the revised manuscript and Supporting Materials\nwere presented as follows.\n“……The cytotoxicity of Ce-Car@EV NPs was systematically evaluated using three\nrepresentative cell types, including mouse fibroblast L929, murine macrophage RAW\n264.7, and human umbilical vein endothelial cells (HUVECs). Cell viability in all three\nlineages remained above 80% even after 24 h of co-culture with Ce-Car@EV NPs at a\nconcentration of 512 μg/mL, indicating negligible cytotoxicity (Supplementary Fig.\n12a-c).” (Manuscript Page 16, Lines 394-399)\nSupplementary Fig. 12. a Relative cell viability of L929 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n3 independent samples). b Relative cell viability of RAW 264.7 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n5 independent samples). c Relative cell viability of human umbilical vein endothelial\ncells (HUVECs) treated with Ce-Car@EV NPs at different concentrations. Data are\npresented as mean values ± SD (n = 5 independent samples). d Relative hemolysis ratio\nfor Ce-Car@EV NPs at different concentrations. The red blood cells treated with saline\nand Triton-X presented as negative and positive controls. The insets are the\ncorresponding photographs. Data are presented as mean values ± SD (n = 3 independent\nsamples).\n9. In the fluorescence targeting experiments depicted in Figure 5, the authors noted that\nthe EPR effect caused material aggregation in the inflamed lung. However, they did not\ninclude an intravenous injection of Ce-Car@EV in healthy mice as a control to\ndemonstrate the absence of significant Ce-Car@EV aggregation in non-inflamed lung\ntissue.\nResponse: We thank the reviewer for this insightful comment regarding the targeting\nmechanism of Ce-Car@EV NPs in vivo. In revision, we have performed the control\nexperiment involving intravenous injection of Ce-Car@EV NPs in healthy mice as\nrecommended by the reviewer, and corresponding fluorescence images are presented in\nSupplementary Fig. 18. These results clearly demonstrate the absence of a significant\nfluorescence signal in the lungs of healthy animals, which contrasts obviously with the\npronounced accumulation in the lungs of mice with bacterial pneumonia. This critical\nexperiment confirms that the accumulation of Ce-Car@EV NPs is indeed specific to\nthe inflamed pulmonary tissue. Relevant changes and detailed descriptions in the\nrevised manuscript and Supporting Materials were presented as follows.\n“Interestingly, the targeted accumulation of Ce-Car@EV NPs depending on specific\npathological microenvironment of bacterial infection was unequivocally verified\nthrough control experiments in healthy mice. The intravenous administration of Ce-\nCar@EV NPs in healthy mice resulted in negligible pulmonary fluorescence signals,\nexhibiting a stark contrast with the substantial accumulation in the lungs of pneumonic\nmice (Supplementary Fig. 18). These results demonstrate that the targeted efficiency\nof Ce-Car@EV NPs is intrinsically associated with the inflammatory microenvironment,\nrather than the nonspecific distribution.” (Manuscript Page 18, Lines 461-468)\nSupplementary Fig. 18. a Fluorescent images in vivo of healthy mice at various time\npoints (0, 2, 4, 6, 8, 12, and 24 h) after tail vein injection of Ce-Car@EV NPs. b\nQuantitative analysis of fluorescence signal intensities in lung regions at different time\npoints of post-injection. Data are presented as mean values ± SD (n = 4 independent\nsamples). One-way analysis of variance (ANOVA) was used to calculate statistical\nsignificance.\nAdditionally, Ce-Car@EV lacks specific targeting modifications for bacterial infection\nsites, and the EPR effect alone cannot account for the targeting of infected lungs as\nshown in the experimental results in Figure 5. A more robust explanation is needed for\nthis observation.\nResponse: We thank the reviewer for this insightful comment regarding the targeting\nmechanism of Ce-Car@EV NPs in vivo. Tremendous observations support the intrinsic\ninflammatory tendency of plant-derived extracellular vesicles, as exemplified by\nhoneysuckle-derived nanovesicles that accumulate preferentially in the inflamed colon\nof inflammatory bowel disease mice compared to healthy tissues (Adv. Sci. 2025, 12,\ne05208.). Notably, nanoparticles similar to ginger-derived exosomes have also been\nreported to exhibit targeting potential toward M1-type macrophages (J. Adv. Res. 2025,\n69, 1-15). In the infected pulmonary microenvironment, substantial infiltration and\npolarization of macrophages toward the M1 phenotype occur. We propose that the\nginger-derived vesicles in Ce-Car@EV NPs are susceptible to this enhanced\nphagocytosis, thereby promoting their sequestration and retention within the inflamed\nlung tissue. Relevant changes and detailed descriptions in the revised manuscript and\nSupporting Materials were presented as follows.\n“Upon reaching the pulmonary vasculature, most of the traditional nanoparticles\ninitially accumulate through the enhanced permeability and retention (EPR) effect\nresulting from infection-induced vascular hyperpermeability.45, 46 Furthermore, this\ntargeted enhanced accumulation is augmented by the complementary biological\nmechanisms. The infected pulmonary microenvironment typically exhibits substantial\ninfiltration of M1-polarized macrophages, demonstrating heightened phagocytic\nactivity toward nano-sized structures. Additionally, tremendous studies on plant-\nderived EVs documents their inherent targeted capability to the inflammatory site,\nincluding the potential affinity toward M1 macrophages.47, 48 These coordinated\nmechanisms, including prolonged circulation, passive EPR-mediated accumulation,\nand potential active targeted processes, may collectively contribute to the enhanced\ntargeting efficacy of Ce-Car@EV NPs.” (Manuscript Page 17, Lines 435-446)\n10. There is a citation error in the reference at line 639. The original text of the cited\npart is: ‘Mice were made neutropenic with two subcutaneous injections of\ncyclophosphamide: 150 mg/kg at 4 days before PAO1 infection and 100 mg/kg at 1 day\nbefore PAO1 infection.’\nResponse: We sincerely thank the reviewer for identifying this error in our\nmethodology description. In revision, the error has been corrected, ensuring to precisely\nreflect the established method. Relevant changes and detailed descriptions in the\nrevised manuscript were presented as follows.\n“Neutropenia and granulocytopenia were induced in mice by administering 150 mg/kg\nand 100 mg/kg of cyclophosphamide four days prior to infection and one day prior to\ninfection, respectively.” (Manuscript Page 31, Lines 784-786)\nFig. 6. In vivo therapeutic performance of Ce-Car@EV NPs against drug-resistant P.\naeruginosa-induced lung infection. a Experimental design for establishing a drug-\nresistant P. aeruginosa-induced acute lung infection model and treatment schedule. b\nSurvival rates of infected mice treated with Car, Ce-Car NCPs, or Ce-Car@EV NPs\nover 36 h (n = 5 independent samples). Changes in body temperature (c) and body\nweight (d) of infected mice receiving different treatment over a 12 h period (n = 5\nindependent samples). e Digital images of lung tissues from mice after different\ntreatments. f Clinical scores assessing six parameters: activity, coat condition, eye\nappearance, breathing, posture, and isolation (n = 5 independent samples).\nQuantification of surviving bacteria in lung homogenates (g) and bronchoalveolar\nlavage fluid (BALF) (h) at 12 h post-treatment (n = 5 independent samples). i\nImmunofluorescence images of lung sections stained with antibodies against drug-\nresistant P. aeruginosa. Data are presented as mean values ± SD. One-way analysis of\nvariance (ANOVA) was used to calculate statistical significance.\n11. In line 427, there is a missing space in 'withCe-Car@EV' (it should be 'with Ce-\nCar@EV').\nResponse: We sincerely thank the reviewer for their meticulous reading and for\nidentifying this typographical error. The missing space in “withCe-Car@EV” has been\ncorrected to “with Ce-Car@EV” in the revised manuscript. Furthermore, we have\nperformed a comprehensive proofreading of the whole manuscript to ensure proper\nformatting. Relevant changes and detailed descriptions in the revised manuscript were\npresented as follows.\n“……whereas those injected with Ce-Car@EV NPs exhibited a satisfactory survival\nrate, indicating their therapeutic potential against P. aeruginosa lung infection.”\n(Manuscript Page 21, Lines 513-515)\n12. The abbreviations in the manuscript should be standardized. Ce-Car@EV is\nreferred to both as 'nanoscale coordination polymers (NCPs)' and 'nanoparticles (NPs)',\nand this inconsistency should be resolved by using a single term.\nResponse: We thank the reviewer for this insightful comment regarding terminology\nstandardization. In response, we have thoroughly revised the manuscript to ensure\nstandardized and uniform terminology applied in the whole text.\n13. The Discussion section is underdeveloped, both in length and content. It only\nsummarizes the experimental findings without placing them in the context of previous\nginger vesicle studies, including those using the same nanoparticle components.\nAdditionally, the section lacks any discussion of the study's limitations.\nResponse: We sincerely thank the reviewer for this insightful comment regarding the\ndevelopment of the Discussion section. In revision, we have substantially expanded and\nrefined the Discussion section to provide a more comprehensive and contextualized\ninterpretation of our findings. Relevant changes and detailed descriptions in the revised\nmanuscript were presented as follows.\n“The rapid emergence and global spread of antimicrobial resistance (AMR) have\nescalated into one of the most pressing public health crises of our time. This alarming\ntrend is progressively rendering conventional bactericidal agents ineffective against\nvarious pathogenic bacteria, particularly Gram-negative types such as Pseudomonas\naeruginosa (P. aeruginosa). Notably, little progress has been made in developing novel\nantibiotics targeting Gram-negative bacteria since the 1980s. Therefore, developing\ninnovative strategies to resensitize drug-resistant pathogens to the existing antibiotics\nhas emerged as an urgent therapeutic imperative. In this work, a novel biomimetic pH-\nresponsive Ce-Car@EV nanoplatform is proposed by synergistically integrating Ce-\nCar NCPs with G-EVs to combat and reverse AMR in Gram-negative pathogens. Our\ncomprehensive experimental results demonstrate that this innovative system achieves\nremarkable antibacterial efficacy through collaborative enhancement mechanisms via\nthe depletion of intracellular ATP and disruption of efflux pump activity. Meanwhile,\ntargeted delivery approach of G-EVs with the combination of high drug loading\ncapacity and pH-triggered release behavior of Ce-Car NCPs, can significantly enhance\nintracellular antibiotic accumulation and antibacterial efficacy (Fig. 3). Encouraged\nby these in vitro favorable properties, the therapeutic efficacy of our platform is further\nunequivocally demonstrated in a mouse model of drug-resistant P. aeruginosa\npneumonia. The treatment of Ce-Car@EV NPs significantly improves survival rates\nand reduces bacterial loads not only in the lungs but also in other major organs (Fig.\n6), including the heart, liver, spleen, and kidney (Supplementary Fig. 22-23). These\ncompelling in vivo antibacterial results, combined with the excellent biocompatibility\nprofile in safety assessments, strongly support the clinical potential of this\nnanoplatform.\nThe strategic design of our platform builds upon yet substantially extends previous work\non both plant-derived vesicles and metal-drug coordination systems. While previous\nstudies have established the fundamental drug delivery capabilities of G-EVs,27, 44, 50\nour research demonstrates their capacity to effectively and target-deliver sophisticated\nmetal-antibiotic coordination polymers while maintaining their innate biological\nfunctions. This design represents a distinct advance beyond systems relying on external\nenergy, which directly targets the core physiological basis of resistance through energy\ndisruption and pathway interdiction. Crucially, the biomimetic properties of G-EVs\ncoating have been found to confer superior targeting capability intrinsically depending\non the pathological inflammatory microenvironment, as confirmed by our in vivo\nbiodistribution studies (Fig. 5). Furthermore, this rational integration predicts a\nmeaningful advancement over the conventional nanocarrier systems, as it not only\npreserves the structural integrity of both components but also ensures a high local\nconcentration of therapeutic agents precisely at the desired site, thereby enhancing\nantibacterial efficacy. Beyond their established role as delivery vehicles, the\nimmunomodulatory potential of G-EVs adds another dimension to our platform’s\ntherapeutic profile. Although a detailed investigation of immune modulation is beyond\nthe scope of this study, the emerging observations suggest that plant-derived vesicles\ncan interact with immune cells and modulate inflammatory responses,48, 51 thereby\nextending their potential in clinical practices. This characteristic, combined with the\nexcellent antibacterial activity of our platform, provides a comprehensive approach to\ninfection management, which warrants further exploration in future studies.\nThe antibacterial effectiveness of Ce-Car@EV NPs against diverse Gram-negative\npathogens, including both P. aeruginosa and K. pneumoniae, stems from its unique\ndual-mechanism approach. Both pathogens possess penicillin-binding proteins, which\nserve as targets for carbenicillin’s inhibition of cell wall biosynthesis. Consequently,\nCe(IV) ions concurrently induce energy deprivation through ATP hydrolysis and\ndissipation of the proton motive force (Fig. 4h-l). Overall, this coordinated attack on\nstructural integrity and energy metabolism enables effective suppression of diverse\nresistant pathogens. Although our results are compelling, several frontiers remain to be\nexplored. The relative contribution of each targeting mechanism (e.g., passive EPR\neffect vs. active biomimetic homing) warrants further deconvolution through more\nsophisticated disease models. Additionally, the immunomodulatory potential of G-EVs\nand a comprehensive investigation into their interaction with host immune cells may\nreveal their powerful adjuvant effects, which can transform our nanoplatform from a\nmere bactericidal agent into an immunotherapeutic one. In conclusion, the Ce-\nCar@EV platform-mediated AMR antibacterial therapy presents a promising strategy\nthrough the intelligent and rational integration of natural biostructures and synthetic\nmaterials. This capability to simultaneously disrupt energy metabolism, inhibit efflux\npumps, and enable targeted delivery of agents allows it to effectively reverse bacterial\nresistance and kill the AMR pathogenic bacteria, offering a promising avenue to combat\nmultidrug-resistant bacterial infections in clinics.” (Manuscript Page 25-27, Lines\n601-665)\n14. It is advisable to review and correct formatting issues throughout the manuscript,\nincluding inconsistent line spacing within paragraphs and variations in font size.\nResponse: We appreciate the reviewer's attention to these formatting inconsistencies.\nWe have carefully reviewed the entire manuscript and conducted a thorough revision to\nensure uniform formatting throughout the document. We believe that the current type\nof this manuscript fully adheres to the high formatting standards for the publication,\nenhancing its overall readability and clarity.\n15. The Ce-Car obtained is a light yellow substance. However, in the CCK8 experiment,\nthe formazan produced by active bacteria is also yellow. It is advisable to include an\nimage of the 32μg/mL material to illustrate whether it interferes with the formazan\ndetection.\nResponse: We thank the reviewer for this valid methodological concern regarding the\npotential interference of the light yellow Ce-Car NCPs with the yellow formazan\nproduct in the CCK-8 assay. We fully acknowledge that color overlap could, in principle,\naffect the accuracy of bacterial viability readings. The Ce-Car@EV NPs solution at a\nconcentration of 32 μg/mL is visually clear (Fig. R3), demonstrating that the intrinsic\ncolor of the Ce-Car@EV NPs does not interfere with formazan detection.\nFig. R3. Photograph of aqueous solution containing Ce-Car@EV at a concentration of\n32 μg/mL.\n16. In the in vitro antibacterial experiments, the authors utilized a buffer with a pH of\n5.5. Please indicate the type of buffer and the method to adjust the pH.\nResponse: We thank the reviewer for requesting clarification regarding the buffer\nsystem. In antibacterial assays in vitro at pH 5.5, the acidic microenvironment was\nestablished using a 10 mM Tris buffer, which was precisely adjusted to the target pH of\n5.5 by titration with hydrochloric acid, thereby obtaining the resultant Tris-HCl buffer\nused in our study. Relevant changes and detailed descriptions in the revised manuscript\nwere presented as follows.\n“The antibacterial assays were performed in 10 mM of Tris-HCl buffer at two distinct\npH values (5.5 and 7.4), with the pH carefully adjusted using hydrochloric acid. Diluted\nlog phase bacteria (8 × 106 CFU/mL) were treated with Ce-Car@EV NPs (0, 1, 2, 4, 8,\n16, and 32 µg/mL) dispersed in the respective buffers.” (Manuscript Page 30, Lines\n741-744)\n17. In Figures 3b-d, the sequence and description of the images are unclear. It is\nsuggested to reorder the images and provide a clear narrative.\nResponse: Thank you for this valuable suggestion regarding the presentation of data in\nFigs. 3b-d. In the revised manuscript, we have reorganized the panel sequence in\nFigures 3b-d to establish a clearer logical progression and enhance the narrative\ncoherence. Relevant changes and detailed descriptions in the revised manuscript were\npresented as follows.\nFig. 3. In vitro antibacterial performance of Ce-Car@EV NPs. a Viability of standard\nP. aeruginosa (ATCC 27853) following 2 h of treatment with Ce-Car@EV NPs at\nvarious concentrations at pH of 5.5 and 7.4 (n = 3 independent samples). b Live/dead\nstaining of P. aeruginosa treated with Ce-Car@EV NPs under different pH conditions\n(pH 5.5 and pH 7.4). Scale bar = 25 µm. c SEM images of P. aeruginosa after treatment\nwith Ce-Car@EV NPs under different pH conditions. Scale bar = 500 nm. d\nCheckerboard dilution assay of the synergistic effects between Car and Ce(IV) against\nP. aeruginosa (n = 3 independent samples). e Development of resistance to Car in P.\naeruginosa over 30 generations induced by Ce-Car@EV NPs (n = 3 independent\nsamples). f Checkerboard dilution assay of synergistic effects between Car and Ce(IV)\nagainst drug-resistant P. aeruginosa (PA-15R) (n = 3 independent samples). g\nAntibacterial effects of Car and Ce-Car@EV NPs on drug-resistant P. aeruginosa (PA-\n15R) (n = 3 independent samples). h Viability of drug-resistant P. aeruginosa (PA-15R)\nafter 4 h of co-incubation with Car and Ce-Car@EV NPs at different concentrations (n\n= 3 independent samples). i Corresponding plate-counting data of drug-resistant P.\naeruginosa (PA-15R) after treatment with Car and Ce-Car@EV NPs at different\nconcentrations (n = 3 independent samples). Data are presented as mean values ± SD.\nTwo-tailed Student’s t-test was used to calculate statistical significance.\n18. The authors should depict the metabolic pathways and half-lives of cerium metal in\nliving organisms.\nResponse: We appreciate the reviewer for raising these important questions regarding\nthe metabolic fate of cerium. In revision, we have performed a comprehensive set of\nexperiments to elucidate the pharmacokinetics, biodistribution, and metabolic behavior\nof the nanoplatform.\nOur results demonstrate that the cerium released from Ce-Car@EV NPs exhibits a\nwell-defined pharmacokinetic profile, with a circulating half-life of 6.68 hours (Fig. 5f)\nand dual clearance via hepatic and renal pathways, leading to near-complete elimination\nfrom the body within 7 days (Fig. 5g-h). Importantly, quantification of residual cerium\nin major organs after 7 days revealed only minimal retention, including in organs rich\nin immune cells (Supplementary Fig. 19). These findings indicate that the released\ncerium is efficiently cleared from the body with negligible long-term accumulation.\nRelevant changes and detailed descriptions in the revised manuscript and Supporting\nMaterials were presented as follows.\n“To quantitatively track the in vivo trajectory of Ce-Car@EV NPs, we systematically\ninvestigated its pharmacokinetic profile and metabolic fate. Following the intravenous\nadministration of Ce-Car@EV NPs in healthy mice, blood samples were collected at\npredetermined time points and subjected to quantitative analysis of cerium ions.\nPharmacokinetic data via a non-compartmental model determined the circulating half-\nlife of Ce-Car@EV NPs to be 6.68 h (Fig. 5f), providing a critical kinetic foundation\nfor the enhanced accumulation at the infection site. Meanwhile, the excretory pathways\nof the nanoplatform were traced by monitoring cerium levels in urine and feces. As\nsummarized in Fig. 5g-h, Ce-Car@EV NPs underwent dual clearance routes involving\nhepatic metabolism and renal excretion, showing a near-complete elimination within 7\ndays, which was consistent to the quantification of residual cerium in major organs\nwith only minimal retention (Supplementary Fig. 19). These results indicate a good\npharmacokinetic profile characterized by extended circulation, defined clearance\nmechanisms, and negligible long-term organ accumulation. This profile affirms the\nrobust biodegradability and biosafety of Ce-Car@EV NPs, underscoring their potential\nas a translatable antimicrobial agent.” (Manuscript Page 18-19, Lines 471-486)\nFig. 5. In vivo pharmacokinetics, biodistribution and metabolism of Ce-Car@EV NPs.\na Schematic illustration of in vivo imaging procedure of Ce-Car@EV NPs. b\nRepresentative in vivo fluorescent images of drug-resistant P. aeruginosa pneumonia\nmice at various time points (0, 2, 4, 6, 8, 12, and 24 h) after tail vein injection of either\nCe-Car NCPs or Ce-Car@EV NPs. c Quantitative analysis of fluorescence signal\nintensities in infected lung regions at different time points after post-injection (n = 3\nindependent samples). Fluorescence images (d) and corresponding quantitative\nanalysis (e) of fluorescence intensities in major organs and infected lung tissues at 6 h\npost-injection (n = 3 independent samples). f Cerium concentrations in blood were\nquantified at designated time points (0.167, 1, 2, 4, 6, 8, 12, 24, and 48 h) after\nintravenous administration to healthy mice, with the circulation half-life determined via\nnon-compartmental modeling (n = 3 independent samples). Mass of cerium in feces (g)\nand urine (h) at different intervals within 7 days post-injection (n = 3 independent\nsamples). Data are presented as mean values ± SD. One-way analysis of variance\n(ANOVA) was used to calculate statistical significance.\nSupplementary Fig. 19. The mass of cerium in major organs measured via ICP-MS at\n7 days following a single intravenous injection of Ce-Car@EV NPs in mice. Data are\npresented as mean values ± SD (n = 3 independent samples).\nThe synthetic Ce-Car@EV is released in a slightly acidic environment. Under these\nconditions, will the released cerium enter nearby immune cells at the infection site and\ninhibit the energy metabolism of human cells?\nResponse: We thank the reviewer for this insightful question regarding the potential\noff-target effects of released cerium on host immune cells. Some previous studies and\nour experimental results indicate that the cerium ions do not significantly impair the\nenergy metabolism of mammalian cells, even in the acidic infectious microenvironment.\nFirst, the structure and regulation of ATP synthesis differ fundamentally between\nbacterial and mammalian systems. Bacterial oxidative phosphorylation occurs at the\nplasma membrane and is more accessible to external perturbations (Nat. Rev. Microbiol.\n2021, 19, 319-330.). In contrast, mammalian ATP production is compartmentalized\nwithin mitochondria, which are protected by a double membrane and a more complex\nset of enzymatic regulatory mechanisms (Science 2011, 33, 1109-1112; Sig. Transduct.\nTarget. Ther. 2024, 9, 268.), likely conferring a lower susceptibility to energy disruption.\nFurthermore, the cytotoxicity assessment on the three typical cell lines directly\naddressed this concern. Even at high concentrations, Ce-Car@EV NPs exhibited\nlimited toxicity toward RAW 264.7 macrophages, a representative immune cell type,\nwith cell viability above 80% (Supplementary Fig. 12b). These results strongly\nsuggests that the metabolic integrity and ATP-generating capacity of immune cells are\nnot compromised. Coupled with the pharmacokinetic data showing the rapid systemic\nclearance of cerium, the release of cerium ions in an acidic infectious microenvironment\nhas little effect on the energy metabolism of mammalian cells. Relevant changes and\ndetailed descriptions in the revised manuscript and Supporting Materials were\npresented as follows.\nSupplementary Fig. 12. a Relative cell viability of L929 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n3 independent samples). b Relative cell viability of RAW 264.7 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n5 independent samples). c Relative cell viability of human umbilical vein endothelial\ncells (HUVECs) treated with Ce-Car@EV NPs at different concentrations. Data are\npresented as mean values ± SD (n = 5 independent samples). d Relative hemolysis ratio\nfor Ce-Car@EV NPs at different concentrations. The red blood cells treated with saline\nand Triton-X presented as negative and positive controls. The insets are the\ncorresponding photographs. Data are presented as mean values ± SD (n = 3 independent\nsamples).\nReviewer 2:\nThis manuscript presents a highly innovative and well-executed study on the\ndevelopment and evaluation of a biomimetic nanoplatform (Ce-Car@EV) for\ncombating drug-resistant P. aeruginosa. The work addresses the critical global\nchallenge of antimicrobial resistance (AMR) through a sophisticated strategy\ncombining pH-responsive metal-drug coordination nanoparticles (i.e. Ce-Car NPs)\nwith ginger-derived extracellular vesicles (EVs). Extensive physicochemical\ncharacterization (TEM, XPS, FTIR, zeta potential, SDS-PAGE) confirmed successful\nsynthesis, EV coating, and pH-responsive degradation. Transcriptomics (RNA-seq) and\npathway analysis (KEGG/GO) convincingly demonstrated downregulation of efflux\npumps (mexAB-oprM), oxidative phosphorylation, and TCA cycle genes, linking Ce(IV)\nto energy disruption. The nanoplatform significantly improves survival, reduces\nbacterial loads in lungs/organs, alleviates lung injury, and demonstrates targeted\naccumulation via EV-mediated evasion of immune clearance. However, authors need\nto address the following issues.\nResponse: We sincerely appreciate the reviewer’s valuable comments. The manuscript\nhas been carefully revised based on each comment from the reviewer, and the overall\nquality has been significantly enhanced to meet the stringent requirements of Nature\nCommunications. The changes and detailed descriptions in the revised manuscript and\nSupporting Materials were highlighted by red font.\n1. Safety of Ce(IV): While biocompatibility is shown, long-term\nbiodistribution/accumulation of cerium in organs should be addressed (e.g., via ICP-\nMS in tissues beyond 7 days, selectivity between bacterial cells and mammalian cells).\nResponse: We thank the reviewer for their insightful comments regarding the long-\nterm biodistribution and cellular selectivity of cerium. In revision, we have conducted\nthe systematic investigations to thoroughly address these important aspects of our\nnanoplatform’s biosafety profile.\nOur pharmacokinetic evaluation reveals that cerium released from Ce-Car@EV\nNPs displays a well-defined in vivo profile, and circulating half-life of 6.68 hours (Fig.\n5f). The assessment of elimination pathways demonstrated the dual clearance of Ce-\nCar@EV NPs through hepatic metabolism and renal excretion, resulting in near-\ncomplete systemic elimination within 7 days (Fig. 5g-h). Meanwhile, the quantification\nof residual cerium in major organs after 7 days detected only minimal retention\n(Supplementary Fig. 19), supporting negligible long-term tissue accumulation.\nConcerning cellular selectivity, multiple independent lines of evidence indicate the\npreferential antibacterial activity of Ce-Car@EV NPs while preserving mammalian cell\nfunction. The fundamental structures and regulatory distinctions in energy metabolism\nbetween bacterial and mammalian systems provide a mechanistic foundation for this\nselectivity. While bacterial oxidative phosphorylation occurs at the plasma membrane,\nrendering it potentially more susceptible to external challenges (Nat. Rev. Microbiol.\n2021, 19, 319-330.), mammalian ATP production is compartmentalized within\nmitochondria protected by both physical barriers and sophisticated regulatory networks\n(Science 2011, 33, 1109-1112; Sig. Transduct. Target. Ther. 2024, 9, 268.).\nCe-Car@EV NPs exhibited no significant cytotoxicity across multiple mammalian\ncell types, including RAW 264.7 macrophages, L929 fibroblasts, and HUVECs, with\ncell viability above 80% even at high concentrations (Supplementary Figure 12a-c).\nThe high cell viability strongly suggests that the metabolic integrity of mammalian cells\nremains substantially intact under treatment of Ce-Car@EV NPs. In summary, the\ncombination of efficient systemic clearance, minimal long-term organ retention, and\nhigh mammalian cell viability provides compelling evidence for the favorable biosafety\ncharacteristics and selective antibacterial action of our nanoplatform. Relevant changes\nand detailed descriptions in the revised manuscript and Supporting Materials were\npresented as follows.\n“To quantitatively track the in vivo trajectory of Ce-Car@EV NPs, we systematically\ninvestigated its pharmacokinetic profile and metabolic fate. Following the intravenous\nadministration of Ce-Car@EV NPs in healthy mice, blood samples were collected at\npredetermined time points and subjected to quantitative analysis of cerium ions.\nPharmacokinetic data via a non-compartmental model determined the circulating half-\nlife of Ce-Car@EV NPs to be 6.68 h (Fig. 5f), providing a critical kinetic foundation\nfor the enhanced accumulation at the infection site. Meanwhile, the excretory pathways\nof the nanoplatform were traced by monitoring cerium levels in urine and feces. As\nsummarized in Fig. 5g-h, Ce-Car@EV NPs underwent dual clearance routes involving\nhepatic metabolism and renal excretion, showing a near-complete elimination within 7\ndays, which was consistent to the quantification of residual cerium in major organs\nwith only minimal retention (Supplementary Fig. 19). These results indicate a good\npharmacokinetic profile characterized by extended circulation, defined clearance\nmechanisms, and negligible long-term organ accumulation. This profile affirms the\nrobust biodegradability and biosafety of Ce-Car@EV NPs, underscoring their potential\nas a translatable antimicrobial agent.” (Manuscript Page 18-19, Lines 471-486)\nFig. 5. In vivo pharmacokinetics, biodistribution and metabolism of Ce-Car@EV NPs.\na Schematic illustration of in vivo imaging procedure of Ce-Car@EV NPs. b\nRepresentative in vivo fluorescent images of drug-resistant P. aeruginosa pneumonia\nmice at various time points (0, 2, 4, 6, 8, 12, and 24 h) after tail vein injection of either\nCe-Car NCPs or Ce-Car@EV NPs. c Quantitative analysis of fluorescence signal\nintensities in infected lung regions at different time points after post-injection (n = 3\nindependent samples). Fluorescence images (d) and corresponding quantitative\nanalysis (e) of fluorescence intensities in major organs and infected lung tissues at 6 h\npost-injection (n = 3 independent samples). f Cerium concentrations in blood were\nquantified at designated time points (0.167, 1, 2, 4, 6, 8, 12, 24, and 48 h) after\nintravenous administration to healthy mice, with the circulation half-life determined via\nnon-compartmental modeling (n = 3 independent samples). Mass of cerium in feces (g)\nand urine (h) at different intervals within 7 days post-injection (n = 3 independent\nsamples). Data are presented as mean values ± SD. One-way analysis of variance\n(ANOVA) was used to calculate statistical significance.\nSupplementary Fig. 12. a Relative cell viability of L929 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n3 independent samples). b Relative cell viability of RAW 264.7 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n5 independent samples). c Relative cell viability of human umbilical vein endothelial\ncells (HUVECs) treated with Ce-Car@EV NPs at different concentrations. Data are\npresented as mean values ± SD (n = 5 independent samples). d Relative hemolysis ratio\nfor Ce-Car@EV NPs at different concentrations. The red blood cells treated with saline\nand Triton-X presented as negative and positive controls. The insets are the\ncorresponding photographs. Data are presented as mean values ± SD (n = 3 independent\nsamples).\nSupplementary Fig. 19. The mass of cerium in major organs measured via ICP-MS at\n7 days following a single intravenous injection of Ce-Car@EV NPs in mice. Data are\npresented as mean values ± SD (n = 3 independent samples).\n2. Direct evidence such as efflux assay with fluorescent substrates like Hoechst 33342\nto complement transcriptomic data, shall be added.\nResponse: We sincerely appreciate the reviewer's valuable comments. In our original\nsubmission, Fig. 4l presents the results of an efflux assay via ethidium bromide (EtBr),\nwhich serves as an established fluorescent substrate for assessing efflux function in P.\naeruginosa (Nat Commun. 2022, 13, 3731; Microb. Pathog. 2023, 185, 106397.). These\nresults provided the data support for transcriptomic findings, showing downregulation\nof efflux pump-associated genes. While we recognize that Hoechst 33342 represents\nanother valid substrate, the EtBr assay already provides direct evidence of\ncompromised efflux capability. The changes and detailed descriptions in the revised\nmanuscript and Supporting Materials were presented as follows.\n“To functionally assess inhibition capacity to efflux pump, ethidium bromide (EtBr),\na well-characterized fluorescent substrate for RND-type efflux pumps in P. aeruginosa,\nwas utilized to verify the impaired pump activity via direct monitoring of intracellular\nfluorescence accumulation.40, 41” (Manuscript Page 14, Lines 361-364)\nFig. 4. Mechanism of Ce-Car@EV NPs to overcome drug resistance of P. aeruginosa.\na Venn diagram of DEGs in the three groups. b Volcano plot of DEGs in bacteria after\ndifferent treatments. c Gene ontology (GO) annotation analysis of DEGs between PA-\n30M and PA-30R cells. d KEGG enrichment analysis of DEGs between PA-30M and\nPA-30R cells. The size of the bubbles represents the number of genes and the color of\nthe bubbles corresponds to different Q-value ranges. e Cluster analysis of bacterial\nmultidrug efflux-related genes based on expression patterns. f Heatmap of oxidative\nphosphorylation-related genes in PA, PA-30R, and PA-30M cells. g Heatmap of genes\nrelated to TCA cycle and glycolysis in PA, PA-30R, and PA-30M cells. h Schematic\nrepresentation of the mechanism of Ce-Car@EV NPs on the oxidative phosphorylation\nprocess. i ATP content after direct incubation with Ce-Car@EV NPs at different\nconcentrations (n = 4 independent samples). j Intracellular ATP content in P. aeruginosa\nafter the treatment of Ce-Car@EV NPs at different concentrations after 24 h (n = 4\nindependent samples). k Effect of Ce-Car NCPs on the activity of bacterial respiratory\nchain complexes I and IV (n = 4 independent samples). l Effect of Ce-Car@EV NPs at\ndifferent concentrations on activity of drug efflux pumps in drug-resistant P. aeruginosa\n(n = 3 independent samples). Data are presented as mean values ± SD. Statistical\nsignificance testing in i, j and l was calculated by One-way analysis of variance\n(ANOVA), whereas that of k were calculated by two-tailed Student’s t-test.\n3. Figure 2C: It should be “LUMO” instead of “HUMO”\nResponse: Thank you for your kind comment. In revision, the label of “HUMO” in Fig.\n2c has been corrected to “LUMO”. We sincerely apologize for this oversight and have\nconducted a comprehensive check of our manuscript and Supporting Materials to\nensure the accuracy of all technical terminology throughout the text.\nFig. 2. Preparation and characterization of Ce-Car@EV NPs. a Schematic diagram of\nthe preparation process of Ce-Car@EV NPs. b TEM images of Ce-Car NCPs, G-EVs\nand Ce-Car@EV NPs. c Partial charge density plot of the HOMO and LUMO levels of\nCe-Car NCPs. d FTIR spectra of Car and Ce-Car NCPs. e Zeta potential of Ce-Car\nNCPs, G-EVs and Ce-Car@EV NPs (n = 3 independent samples). f Release profiles of\nCar from Ce-Car@EV NPs in Tris-HCl at pH of 5.5 and 7.4 (n = 3 independent samples).\ng Protein analysis of G-EVs and Ce-Car@EV NPs using SDS-PAGE. Molecular\ndocking simulations of Car binding to Penicillin-Binding Protein 3 (PBP3) in the\nabsence (h) or presence (i) of Ce(IV). The protein is shown as a cartoon model, while\nCar is depicted as a stick model. Hydrogen bonds, π-π interactions, van der\nWaals/hydrophobic interactions, and electrostatic interactions are represented by red,\nblue, yellow, and purple dashed lines, respectively. Data are presented as mean values\n± SD.\n4. Line 215-219: “According to the standard classification criteria (FICI ≤0.5:\nsynergistic effect; 0.5 < FICI ≤ 1: additive effect; FICI > 1: no interaction), the\ncombination of Ce(IV) and Car exhibited a strong synergistic effect with FICI of 0.5.”\nAccording to your description, FICI 0.5 can not be defined as “Strong synergistic\neffect”, and it should be additive effect between your two drugs. This exaggeration\noccurs many times and describes the mechanism reluctantly. Also, FICI>1 should be\nantagonistic effect instead of no interaction.\nResponse: We appreciate the reviewer's insightful comment regarding the\ninterpretation of the FICI value. In revision, the description has been corrected\nthroughout the whole manuscript to ensure precise and accurate terminology. The\nphrase “strong synergistic effect” has been replaced by the standard classification\n“synergistic effect” for FICI of 0.5. Furthermore, the criterion “FICI > 1” has been\ncorrectly updated to “antagonistic effect” in the relevant statements. These corrections\nensure that the pharmacological interactions between Ce(IV) and carbenicillin are\ndescribed in strict accordance with the established scientific conventions. The changes\nand detailed descriptions in the revised manuscript and Supporting Materials were\npresented as follows.\n“……To further investigate the interaction between Ce(IV) and Car, the fractional\ninhibitory concentration index (FICI) was determined using a checkerboard dilution\nassay. According to the standard classification criteria (FICI ≤ 0.5: synergistic effect;\n0.5 < FICI ≤ 1: additive effect; FICI > 1: antagonistic effect), the combination of Ce(IV)\nand Car exhibited a synergistic effect with FICI of 0.5 (Fig. 3d).” (Manuscript Page\n9, Lines 222-226)\n5. Figure 3a and 3f, the bacterial viability of Ce-Car@EV seems not constant. Why\nresistant strains even demonstrated lower bacterial viability (0%) under the treatment\nof 16 μg/ml Ce-Car@EV compared with sensitive strains (20%). Please clarify the\nbacterial strains used in each figure in figure captions.\nResponse: We thank the reviewer for the careful examination of the antibacterial data\nin Fig. 3a and 3h. The observed difference in bacterial viability between these two\nexperimental groups directly stems from distinct experimental parameters (bacterial\nstrain type and exposure duration). In Fig. 3a, antibacterial activity was assessed\nfollowing the co-incubation with the standard Pseudomonas aeruginosa strain (ATCC\n27853) for 2 h. In contrast, the experiment depicted in Fig. 3h employed a laboratory-\nconstructed Carbenicillin-resistant strain (PA-15R). Exposure of this resistant strain to\nCe-Car@EV NPs was extended to 4 h to fully evaluate the extent of resistance reversal.\nThe prolonged exposure time enabled Ce-Car@EV NPs to more thoroughly disrupt the\nmetabolic system of the drug-resistant bacteria, thereby accounting for the enhanced\nbactericidal efficacy observed in the resistant group at 16 μg/mL. We have\nsupplemented the figure legends with details of the bacterial strains. The changes and\ndetailed descriptions in the revised manuscript were presented as follows.\n“……a Viability of standard P. aeruginosa (ATCC 27853) following 2 h of treatment\nwith Ce-Car@EV NPs at various concentrations at pH of 5.5 and 7.4 (n = 3\nindependent samples)……. h Viability of drug-resistant P. aeruginosa (PA-15R) after 4\nh of co-incubation with Car and Ce-Car@EV NPs at different concentrations (n = 3\nindependent samples).” (Manuscript Page 11, Lines 258-270)\nFig. 3. In vitro antibacterial performance of Ce-Car@EV NPs. a Viability of standard\nP. aeruginosa (ATCC 27853) following 2 h of treatment with Ce-Car@EV NPs at\nvarious concentrations at pH of 5.5 and 7.4 (n = 3 independent samples). b Live/dead\nstaining of P. aeruginosa treated with Ce-Car@EV NPs under different pH conditions\n(pH 5.5 and pH 7.4). Scale bar = 25 µm. c SEM images of P. aeruginosa after treatment\nwith Ce-Car@EV NPs under different pH conditions. Scale bar = 500 nm. d\nCheckerboard dilution assay of the synergistic effects between Car and Ce(IV) against\nP. aeruginosa (n = 3 independent samples). e Development of resistance to Car in P.\naeruginosa over 30 generations induced by Ce-Car@EV NPs (n = 3 independent\nsamples). f Checkerboard dilution assay of synergistic effects between Car and Ce(IV)\nagainst drug-resistant P. aeruginosa (PA-15R) (n = 3 independent samples). g\nAntibacterial effects of Car and Ce-Car@EV NPs on drug-resistant P. aeruginosa (PA-\n15R) (n = 3 independent samples). h Viability of drug-resistant P. aeruginosa (PA-15R)\nafter 4 h of co-incubation with Car and Ce-Car@EV NPs at different concentrations (n\n= 3 independent samples). i Corresponding plate-counting data of drug-resistant P.\naeruginosa (PA-15R) after treatment with Car and Ce-Car@EV NPs at different\nconcentrations (n = 3 independent samples). Data are presented as mean values ± SD.\nTwo-tailed Student’s t-test was used to calculate statistical significance.\n6. The Ce-Car@EV platform combines innovative design, mechanistic rigor, and\ncompelling in vivo validation, however, the synergistic effect seems reluctant and\noverall the mechanism is confusing. These authors shall clarify it.\nResponse: We appreciate the reviewer’s valuable comment. The process is initiated by\npH-responsive degradation of Ce-Car@EV NPs in the acidic infectious\nmicroenvironment. The pivotal role of Ce(IV) ions in antibacterial performance is then\nmanifested through their direct disruption of bacterial energy homeostasis, which\ninvolves depleting intracellular ATP pools and impairing oxidative phosphorylation.\nThis energy deprivation directly compromises the function of energy-dependent efflux\npumps, which are critical for antibiotic resistance. Consequently, the intracellular\nconcentration and retention of carbenicillin are significantly enhanced. The synergistic\neffect between Ce(IV) and carbenicillin is further unequivocally supported by\ntranscriptomic data, which demonstrate a concerted downregulation of efflux pump\ngenes and key metabolic pathways, effectively reversing the established resistance\nphenotype. This coordinated mechanism, progressing from targeted activation to\nenergy disruption and culminating in efflux pump inhibition, provides a compelling\napproach for restoring antibiotic sensitivity in resistant bacterial strains.\n7. These authors presented the use of Ce-Car@EV nanoplatform for AMR. However, in\nthe docking simulation, they use Car and Ce(IV) ions. They shall address the gap\nbetween nano materials and small molecules and ions (metabolism and/or\ndecomposition). Key references shall be cited: Nat Rev Chem. 2023,7, 202-224; J. Am\nChem Soc. 2025, 147, 12361-12380.\nResponse: We thank the reviewer for this valuable advice. The molecular docking study\nwith carbenicillin and Ce(IV) ions is based on the pH-responsive degradation of Ce-\nCar@EV NPs. This platform is stable in circulation but breaks down in acidic infection\nsites. This releases the active components, allowing them to interact synergistically with\nthe bacterial target PBP3. Our docking models this specific scenario. We have revised\nthe text to clarify that the simulations reflect the activated state of Ce-Car@EV NPs\nafter its dissociation, not the original nanoparticle. The changes and detailed\ndescriptions in the revised manuscript were presented as follows.\n“……Notably, the antibacterial efficacy of metal-coordination nanomaterials involves\nboth intact nanoparticles and their degradation products.31, 32 As Ce-Car@EV NPs\ndegrade in acidic infectious microenvironments, they specifically and targeted release\nbioactive Ce(IV) and carbenicillin. Therefore, we evaluated the effect of Ce(IV) on the\nbinding mode of Car to PBP3 via molecular docking. As shown in Fig.\n2h……”(Manuscript Page 7, Lines 174-178)\nReviewer 3:\nIn this work, Ye et al. developed ginger-derived extracellular vesicle (EV)-encapsulated\ncerium-carbenicillin nanoplatforms (Ce-Car@EV) with the antibacterial capacity by\nthe disruption of bacterial energy metabolism. The nanoplatform utilizes the Ce⁴⁺ ions\nto induce ATP depletion and proton motive force dissipation to reverse efflux pump-\ndriven resistance, which can significantly enhance carbenicillin efficacy against drug-\nresistant P. aeruginosa. Experimental results in vivo demonstrate the reduced bacterial\nburden and improved survival in a murine pneumonia model. However, there are still\nsome issues in the manuscript that need to be addressed. Therefore, I think this article\nis worthy of being published in Nature Communications after minor revisions.\nResponse: We sincerely appreciate the reviewer's valuable comments. The manuscript\nhas been carefully revised based on each comment from the reviewer, and the overall\nquality has been significantly enhanced to meet the stringent requirements of Nature\nCommunications. The changes and detailed descriptions in the revised manuscript\nwere highlighted by red font.\n1. The biological role of G-EVs as drug carriers requires more detailed biochemical\nvalidation. Current characterizations via TEM and SDS-PAGE cannot fully confirm\ntheir exosomal properties. Western blotting analysis should be performed to assess\nconserved exosome markers and verify vesicle integrity.\nResponse: We thank the reviewer for suggesting further biochemical validation of G-\nEVs. In revision, the Western blot assay has been employed to assess the exosomal\ncharacteristics of G-EVs. The changes and detailed descriptions in the revised\nmanuscript and Supporting Materials were presented as follows.\n“Additionally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)\nanalysis reveals that the resultant Ce-Car@EV NPs retained the surface proteins of\nbiologically active G-EVs (Fig. 2g). Furthermore, as demonstrated in Supplementary\nFig. 4, Western blot image indicates that the exosomal marker protein CD9 is present\non Ce-Car@EVs NPs, which are broadly consistent with SDS-PAGE findings. Therefore,\nthese results demonstrate the successful synthesis of Ce-Car NCPs and G-EVs coating\nof Ce-Car@EV NPs.” (Manuscript Page 6, Lines 151-155)\nSupplementary Fig. 4. Characterization of the surface marker protein CD9 on G-EVs\nand Ce-Car@EV NPs via Western blot analysis.\n2. Compared with mammalian cell-derived EVs, plant-derived edible EVs exhibit lower\nimmunogenicity, along with advantages in sustainability and renewability. The yield of\nginger-derived exosomes requires quantification.\nResponse: We thank the reviewer for this constructive comment regarding the\nquantification of ginger-derived exosome yield. In this work, the production yield of\nginger-derived extracellular vesicles has been quantified. Based on the established\npurification protocol, we consistently obtained approximately 70-80 mg of EVs from\none kilogram of fresh ginger rhizome.\n3. The cytotoxicity assessment in murine fibroblasts provides preliminary evidence of\nbiocompatibility; however, evaluation across additional cell lines is needed to obtain a\ncomprehensive safety profile.\nResponse: Thank you for your valuable suggestion. In revision, cytotoxicity assays\nhave been evaluated using murine macrophage cells (RAW 264.7) and human umbilical\nvein endothelial cells (HUVECs), alongside the L929 fibroblast. The changes and\ndetailed descriptions in the revised manuscript and Supporting Materials were\npresented as follows.\n“……The cytotoxicity of Ce-Car@EV NPs was systematically evaluated using three\nrepresentative cell types, including mouse fibroblast L929, murine macrophage RAW\n264.7, and human umbilical vein endothelial cells (HUVECs). Cell viability in all three\nlineages remained above 80% even after 24 h of co-culture with Ce-Car@EV NPs at a\nconcentration of 512 μg/mL, indicating negligible cytotoxicity (Supplementary Fig.\n12a-c).” (Manuscript Page 16, Lines 394-399)\nSupplementary Fig. 12. a Relative cell viability of L929 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n3 independent samples). b Relative cell viability of RAW 264.7 cells treated with Ce-\nCar@EV NPs at different concentrations. Data are presented as mean values ± SD (n =\n5 independent samples). c Relative cell viability of human umbilical vein endothelial\ncells (HUVECs) treated with Ce-Car@EV NPs at different concentrations. Data are\npresented as mean values ± SD (n = 5 independent samples). d Relative hemolysis ratio\nfor Ce-Car@EV NPs at different concentrations. The red blood cells treated with saline\nand Triton-X presented as negative and positive controls. The insets are the\ncorresponding photographs. Data are presented as mean values ± SD (n = 3 independent\nsamples).\n4. Although Supplementary Figures 9-11 confirm systemic biocompatibility at\ntherapeutic concentrations in vivo, the potential neurotoxicity of cerium ions requires\nspecific evaluation in neural tissues.\nResponse: We appreciate the reviewer’s pertinent concern regarding the neurotoxic\npotential of cerium ions. In revision, we specifically assessed oxidative stress markers\nin brain tissues, given the established role of oxidative damage as a primary concern in\nnanoparticle-related neurotoxicity. Several key oxidative stress markers in the mouse\ncentral nervous system, including superoxide dismutase (SOD), glutathione (GSH), and\nmalondialdehyde (MDA) have been examined. The changes and detailed descriptions\nin the revised manuscript and Supporting Materials were presented as follows.\n“Oxidative stress is recognized as a primary concern contributing to nanoparticle-\ninduced neurotoxicity42, 43. To assess the potential neurotoxic effects of Ce-Car@EV\nNPs, several key oxidative stress markers in the mouse central nervous system,\nincluding superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde\n(MDA) were examined. After 7 days of treatment with Ce-Car@EV NPs, the cerebral\ncortex showed insignificant alterations in the levels of SOD, GSH, or MDA relative to\ncontrol group (Supplementary Fig. 16). These results suggest that Ce-Car@EV NPs\nadministration provoke negligible substantial oxidative injury within neural tissues.”\n(Manuscript Page 16-17, Lines 412-420)\nSupplementary Fig. 16. Activity of GSH (a), SOD (b), and MDA (c) level in cerebral\ncortex of mouse treated with Ce-Car@EV NPs after 7 days. Data are presented as mean\nvalues ± SD (n = 5 independent samples). Two-tailed Student’s t-test is used to calculate\nstatistical significance.\n5. Several graphical elements require clarification. Specifically, in Fig. 2c, the label\n'HUMO' should be corrected to 'LUMO' (lowest unoccupied molecular orbital) to\nensure accurate representation of molecular orbitals.\nResponse: Thank you for your valuable advice. In revision, the label of “HUMO” in\nFig. 2c has been corrected to “LUMO”. The changes and detailed descriptions in the\nrevised manuscript were presented as follows.\nFig. 2. Preparation and characterization of Ce-Car@EV NPs. a Schematic diagram of\nthe preparation process of Ce-Car@EV NPs. b TEM images of Ce-Car NCPs, G-EVs\nand Ce-Car@EV NPs. c Partial charge density plot of the HOMO and LUMO levels of\nCe-Car NCPs. d FTIR spectra of Car and Ce-Car NCPs. e Zeta potential of Ce-Car\nNCPs, G-EVs and Ce-Car@EV NPs (n = 3 independent samples). f Release profiles of\nCar from Ce-Car@EV NPs in Tris-HCl at pH of 5.5 and 7.4 (n = 3 independent samples).\ng Protein analysis of G-EVs and Ce-Car@EV NPs using SDS-PAGE. Molecular\ndocking simulations of Car binding to Penicillin-Binding Protein 3 (PBP3) in the\nabsence (h) or presence (i) of Ce(IV). The protein is shown as a cartoon model, while\nCar is depicted as a stick model. Hydrogen bonds, π-π interactions, van der\nWaals/hydrophobic interactions, and electrostatic interactions are represented by red,\nblue, yellow, and purple dashed lines, respectively. Data are presented as mean values\n± SD.\n6. Reference formatting requires consistent verification. For example, the volume and\npage numbers for Reference No. 5 should be provided.\nResponse: We thank the reviewer valuable advice. In revision, the volume and page\nnumbers for Ref. 5 were corrected. Additionally, a comprehensive verification of all\nreference entries in the manuscript has been performed to ensure consistent formatting\nthroughout the reference list.\nReviewer 4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We sincerely appreciate the reviewer’s valuable comments. The manuscript\nhas been carefully revised based on each comment from the reviewer, and the overall\nquality has been significantly enhanced to meet the stringent requirements of Nature\nCommunications. The changes and detailed descriptions in the revised manuscript\nwere highlighted by red font.\nSincerely,\nFu-Jian Xu\nProfessor, Beijing University of Chemical Technology\nDear Reviewers,\nThank you very much for the comments on the present work. Those comments are\nvaluable and very helpful for revising and improving our paper, as well as providing\nimportant guidance for our research. In the following, we make a point-to-point\nresponse to all the comments.\nReviewer 1:\nThe authors have systematically revised the manuscript in response to the previous\nreviewer comments. The overall quality of the paper has been significantly improved,\nmeeting the standards of Nature Communications. Following substantial revisions, the\nauthors ’ research has been constructively refined, presenting an excellent\nantibacterial material derived from ginger-derived vesicles with considerable\ntranslational potential. After careful consideration, we recommend accepting this\nmanuscript and look forward to the subsequent outstanding research outputs from Prof.\nXu’s lab.\nResponse: We appreciate the positive comment from the reviewer.\nReviewer 2:\nThese authors have made major revision as requested. They have addressed almost all\nof my concerns. This reviewer has minor comments for the authors to consider.\nResponse: We sincerely thank you for your constructive feedback and for\nacknowledging the revisions made to our manuscript. We are pleased that the key\nconcerns have been addressed to your satisfaction. Below, we have carefully considered\nyour remaining minor comments and have incorporated corresponding clarifications\nand amendments into the manuscript.\n1. Molecular docking was used to demonstrate the binding of Ce(IV) and Car to PBP3.\nThey showed the \"Ce(IV) forms coordination bonds with the carboxylate of Glu286 and\nthe backbone oxygen of Arg483, bringing the Car molecule closer to the binding site of\nCe(IV).\" Since Ce(IV) normally coordinates at least 6 donor atoms, do these authors\nhave any comments on coordination of Ce(IV) to any other residues and/or water in\naddition to Glu286 and Arg483.\nResponse: We thank the reviewer for raising this insightful point. We agree that, in\nsolution or under actual protein environments, Ce(IV) typically exhibits a high\ncoordination number, which may involve water molecules and oxygen atoms from other\nresidues serving as ligands. The molecular docking results presented in this study\nprimarily focus on elucidating the most direct and functionally significant coordination\ninteractions between Ce(IV) and the PBP3 binding pocket, the coordination bonds\nformed with the carboxylate group of Glu286 and the backbone oxygen of Arg483.\nThese two types of interactions are the most prominent both energetically and spatially\nand represent the key mechanism driving the directional positioning of the Car.\nCertainly, the complete physiological coordination environment may indeed involve\nadditional coordination contributions from surrounding residues or water molecules.\nThe current simulation presents the most stable coordination mode within the docked\nconformation, which serves as a critical entry point for understanding the functional\nmechanism. In follow-up studies, we will further investigate the complete coordination\nnetwork that Ce(IV) may form at the binding site through more refined theoretical\ncalculations, such as molecular dynamics simulations. We appreciate the reviewer for\nhighlighting this direction worthy of in-depth exploration.\n2. Fig. 6g and 6h: Log10 is normally used for CFU in the y-axis. Or they may use\n\"...(x107 CFU) so that the y-axis can simply change to 0, 5, 10, 15.\nResponse: We thank the reviewer for the constructive suggestion regarding the\npresentation of the colony‑forming unit (CFU) data. As recommended, we have revised\nFig. 6g-h by adopting the scaling “……(×10⁷ CFU)” and adjusting the y‑axis\naccordingly.\nFig. 6. In vivo therapeutic performance of Ce-Car@EV NPs against drug-resistant\nP. aeruginosa-induced lung infection. a Experimental design for establishing a drug-\nresistant P. aeruginosa-induced acute lung infection model and treatment schedule. b\nSurvival rates of infected mice treated with Car, Ce-Car NCPs, or Ce-Car@EV NPs\nover 36 h (n = 5 independent samples). Changes in body temperature (c) and body\nweight (d) of infected mice receiving different treatments over a 12 h period (n = 5\nindependent samples). e Digital images of lung tissues from mice after different\ntreatments. f Clinical scores assessing six parameters: activity, coat condition, eye\nappearance, breathing, posture, and isolation (n = 5 independent samples).\nQuantification of surviving bacteria in lung homogenates (g) and bronchoalveolar\nlavage fluid (BALF) (h) at 12 h post-treatment (n = 5 independent samples). i\nImmunofluorescence images of lung sections stained with antibodies against drug-\nresistant P. aeruginosa. Data are presented as mean values ± SD. Statistical significance\nwas calculated by one-way ANOVA using the Tukey post-test. Source data are provided\nas a Source Data file.\nReviewer 3:\nThe authors have adequately responded to my concerns.\nResponse: We appreciate the reviewer's positive comment.\nReviewer 4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts..\nResponse: We sincerely appreciate the reviewer’s valuable comments.\nWe greatly thank all the reviewers’ valuable comments, which help us substantially\nimproved our manuscript.\nSincerely,\nFu-Jian Xu\nProfessor, Beijing University of Chemical Technology"}]

Decision letter