Materials and reagents

Ciprofloxacin (Cip, 97%) was purchased from Bidepharm (Shanghai, China). Ellagic acid (EA) was purchased from J&K Scientific (Beijing, China). H₂O₂, •OH, •O₂^– kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Nile red (95%), ammonium persulfate (97%), 2,2-Diphenyl-1-picrylhydrazyl (DPPH, 95%), N, N, N′, N′-Tetramethylethylenediamine (TEMED, 95%) were purchased from Macklin (Shanghai, China). SDS-PAGE, Hoechst 33,342, and 4′,6-diamidino-2-phenylindole (DAPI, 95%) were purchased from Thermo Fisher (Shanghai, China). Dil (95%), bicinchoninic acid (BCA) kits, 30% Acr-Bis (29:1), sodium 1-dodecanesulfonate (SDS), RIPA lysate, coomassie blue, and cell counting kit-8 (CCK-8) were purchased from Beyotime (Shanghai, China). Cy5 (Sulfo-Cyanine5, 95%) was purchased from MedChemExpress (New Jersey, USA). Anti-mouse CD16/32 antibody (Clone: 93), anti-mouse CD3-FITC (Clone: 17A2), anti-mouse CD4-BV421 (Clone: RM4-4), anti-mouse CD8a-APC (Clone: 53-6.7), anti-mouse Foxp3-PE (Clone: MF-14), anti-mouse CD19-APC (Clone: 6D5), anti-mouse CD138-APC (Clone: 281-2), anti-mouse CD86-BV421 (Clone: GL-1), anti-mouse CD80-APC (Clone: 16-10A1), anti-mouse CD11c-FITC (Clone: N418), anti-mouse CD206-FITC (Clone: C068C2), anti-mouse CD86-BV421 (Clone: GL-1) and anti-mouse F4/80-APC (Clone: BM8) were purchased from BioLegend (California, USA). Dulbecco’s Modified Eagle Medium (DMEM), penicillin-streptomycin, phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco Life Technologies, Inc. (Grand Island, USA). Mouse TNF-α, IL-1β, IL-6, IL-10 ELISA kits, and macrophage colony-stimulating factor 1 (M-CSF) were purchased from iCell Bioscience Inc. (Shanghai, China). Bovine serum albumin (BSA) and Triton-X-100 were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).

Bacterial strains

S. aureus WH^GFP, E. coli Xen14 (PerkinElmer Inc., Waltham, MA, USA), E. coli DH5α, and S. typhimurium 15,649 were employed in this study. S. typhimurium 15,649 was kindly provided by Zhou Lab at the First Affiliated Hospital of Wenzhou Medical University. For experiments, one colony of E. coli DH5α on lysogeny broth (LB, OXOID, Basingstoke, UK) plates was inoculated into 10 mL of LB at 37 °C for 24 h in ambient air. Preculture was diluted 1:20 in 200 mL LB and grown statically (37 °C, 16 h) for the main culture to harvest bacteria. S. aureus WH^GFP with green fluorescence and E. coli Xen14 with bioluminescence are multidrug-resistant strains cultured at 37 °C under ambient atmosphere in tryptone soy broth (TSB, OXOID, Basingstoke, UK) or LB medium, respectively. Bacteria were cultured using the same protocol described above, with plate growth and preculture performed in medium containing strain-specific antibiotics: 10 μg/mL tetracycline for S. aureus WH^GFP or 30 μg/mL kanamycin for E. coli Xen14. S. typhimurium 15,649 was incubated for 24 h at 37 °C, followed by main culture using the same protocol described above. Finally, bacteria were harvested by centrifugation at 5000 g for 5 min. The collected bacteria were washed twice in PBS (10 mL, 5 mM K₂HPO₄, 5 mM KH₂PO₄, and 150 mM NaCl, pH 7.4), and bacterial concentration was determined in a Bürker-Türk counting chamber.

Cell lines

Murine macrophage cell line (RAW264.7) and mouse fibroblast (L929) cells were involved in this study and were purchased from the American Type Culture Collection (ATCC). RAW264.7 and L929 cells were cultured in DMEM containing 10% FBS and 1% penicillin−streptomycin solution.

Animals

Female BALB/c mice (6–8 weeks) were provided by Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The mice were housed under controlled conditions (20 ± 2 °C, 50% humidity, and 12/12 light cycle). Experiments followed the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. All animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at Wenzhou Institute, University of Chinese Academy of Sciences (No. WIUCAS22081605, WIUCAS25010207).

Preparation of bacterial membrane

The collected and lyophilized E. coli DH5α (2 g) was dispersed in Tris-HCl buffer (40 mL, 20 mM, pH 8.0) containing lysozyme (15 mg/mL). The samples were incubated at 37 °C under constant shaking for 3 h. Subsequently, the bacterial solution was incubated with 400 mg SDS and disrupted using a probe sonicator while cooling in an ice/water bath before being lyophilized. The resulting dried bacteria lysate was suspended in a mixture of chloroform-methanol-water (5 mL, 30:15:1, v/v/v), placed on a shaker at 37 °C for 1 h, and then filtered by Millipore HVLP Durapore® membrane (0.45 μm). The residue was extracted by chloroform-methanol-water three more times, and then the filtrate was combined and dried under vacuum to yield BMV (0.58 g, yield: 29%). The yield (%) was determined by dividing the weight of BMV by the total bacterial weight. Finally, the protein concentration of the bacterial membrane was determined using the BCA assay.

Synthesis of Cip-pba-EA prodrug

The phenylboronic acid-functionalized ciprofloxacin (Cip-pba) was synthesized according to our previous report²⁴. Briefly, ciprofloxacin (Cip, 252 mg, 0.73 mmol) and K₂CO₃ (300 mg, 2.17 mmol) were dissolved in DMF/H₂O (4.0 mL, v/v = 2/1), followed by the addition of 4-(bromomethyl)-phenylboronic acid (163 mg, 1.35 mmol). The reaction mixture was allowed to react overnight. The pH of the solution was adjusted to 7.4 using an HCl solution. Subsequently, the precipitation was filtered and dried under a vacuum to obtain the white powder. ¹H NMR (400 MHz, DMSO-d₆) of Cip-pba: 1H NMR (400 MHz, DMSO) δ 15.18 (s, 1H), 8.67 (s, 1H), 8.01 (s, 2H), 7.91 (d, J = 13.3 Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.58 (d, J = 7.4 Hz, 1H), 7.33 (d, J = 7.7 Hz, 2H), 3.83 (s, 1H), 3.59 (s, 2H), 2.62 (s, 4H), 1.33 (d, J = 6.4 Hz, 2H), 1.19 (s, 2H).

The conjugated prodrug, Cip-pba-Ea was synthesized by a dynamic covalent bond. Briefly, Cip-pba (5.0 mg, 10.0 μmol) and Ea (1.5 mg, 5.0 μmol) were reacted in DMSO (500 μL) under stirring at 25 °C for 12 h. The resulting product is denoted as CpE. The chemical structure of CpE was verified by ¹H–¹H COSY and ¹H–¹H NOSY spectra using NMR spectroscopy (AVANCE III 400 MHz, Bruker, Germany) in methanol-d₄, mass spectrum (Agilent 6520Q, California, USA), Fourier infrared spectrometer (Bruker Tensor II, Karlsruhe, Germany) and UV-vis spectrum (UV-1900i, Shimadzu, Japan).

Preparation of CpE@BMV

Cip-pba (5.0 mg, 10.0 μmol) and Ea (1.5 mg, 5.0 μmol) were reacted in DMSO (500 μL) under stirring at 25 °C for 12 h. The resulting product CpE was coated with BMV through extrusion. In brief, CpE (100 μL, 13 mg/mL) and BMV (900 μL, 500 μg/mL in ultrapure water) were mixed, and a noticeable Tyndall effect was observed immediately. Then, the mixture of BMV and CpE was extruded 20 times successively through a 200 nm polycarbonate porous membrane with an Avanti mini extruder to obtain the CpE@BMV, and the CpE@BMV was kept at 4 °C for further use.

In addition, the Dil-labeled CpE@BMV (CpE@BMV^Dil) was prepared by adding Dil (3%, w/w of BMV) to the BMV, followed by mixing with CpE according to a similar protocol as described above. The free Dil was removed by centrifugation. For the preparation of Cy5- or Nile Red-labeled CpE@BMV (CpE_Cy5@BMV or ^{Nile red}CpE@BMV), Cy5 (3%, w/w of Cip-pba) or Nile Red (3%, w/w of Cip-pba) was added during CpE formation, and a similar protocol was used to prepare CpE_Cy5@BMV or ^{Nile Red}CpE@BMV. Free Cy5 or Nile Red was removed by centrifugation. The CpE_Cy5@BMV^Dil was prepared by adding Dil (3%, w/w of BMV) to the BMV, followed by mixing with Cy5-labeled CpE (3%, w/w) using a similar protocol. Free Cy5 and Dil were removed by centrifugation.

The loading content and encapsulation efficiency of Cip and Ea were determined by HPLC.

Characterization of CpE@BMV

The size distribution, colloidal stability, and zeta potential were measured using Malvern Nano ZS Zen3600 (Malvern, UK). For size and Zeta potential measurement, CpE and CpE@BMV were diluted with phosphate buffer (PB, pH 7.4, 10 mM) to a final concentration of approximately 0.1 mg/mL. The sample morphology was observed by transmission electron microscope (TEM, FEI Talos-F200S, Hillsboro, USA) with an accelerating voltage of 120 kV. EDS mapping was used to analyze the spatial distribution of various elements.

To determine the BMV coating, the membrane proteins of CpE@BMV were identified through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In brief, the obtained CpE@BMV was incubated with RIPA lysate (1:100, v/v) for 30 min on ice and subsequently centrifuged (15,000 g, 4 °C) for 20 min to isolate the membrane protein. Total protein concentration was determined using the BCA protein assay kit. Equivalent amounts of total proteins from BMV and CpE@BMV were denatured by boiling in an SDS loading buffer at 99 °C for 15 min, and CpE was treated similarly as a control. All samples were separated by electrophoresis on a 10% SDS-PAGE gel, further stained with Coomassie blue for 2 h, and decolorized overnight before being photographed using a mobile phone camera. In addition, CpE_Cy5@BMV, CpE@BMV^Dil, or CpE_Cy5@BMV^Dil were analyzed by flow cytometry (Beckman CytoFlex, California, USA) to prove the BMV coating.

Molecular dynamics simulation

In order to understand the experimental results from a microscopic perspective, all-atom simulations were carried out to investigate the self-assembly of CpE molecules and their interactions with the outer membrane of Gram-negative bacteria. Similar to our previous work⁶⁴, we used the ATB tool⁶⁵ to build an all-atom model of the CpE molecule, employed the CHARMM-GUI tool⁶⁶ to build a model of the outer membrane of Gram-negative bacteria⁶⁷, and then adopted the combination rule to construct the hybrid system of these two parts.

To prepare the initial configurations for self-assembly simulations, the Gromacs insert-molecules tool was employed. Fifty CpE molecules were randomly positioned within a periodic cubic box with a side length of 15 nm. The system was then solvated with TIP3P water molecules to mimic a realistic biological environment. To maintain the electrical neutrality of the system, calcium and chloride ions were strategically added by replacing water molecules at random until a concentration of 0.1 M was achieved. Finally, to promote the formation of the most stable self-assembled structure, an annealing simulation was conducted for 100 ns. During this process, the temperature of the system was gradually decreased from 450 K to 298.15 K. Nose-Hoover thermostat and isotropic Parrinello–Rahman barostat were utilized throughout the simulation to maintain the desired temperature and pressure conditions.

To study how CpE molecules interact with the outer membrane of Gram-negative bacteria, they were initially positioned under the outer membrane. Then the system evolves freely for 100 ns under constant temperature and pressure conditions. The conformational changes were examined and analyzed after the simulation. The simulations were run with Gromacs version 2021.4 and analyzed with VMD software^68,69.

Evaluation of ROS scavenging

Hydrogen peroxide (H₂O₂) scavenging assay: the H₂O₂ scavenging capacity of CpE@BMV was evaluated using a hydrogen peroxide assay kit. Briefly, Amplite IR peroxidase substrate and peroxidase were mixed to prepare the working solution according to the protocol. Then, CpE@BMV (50 μL, at different concentrations of 16, 31, 62, 125, and 250 μg/mL) was added to the working solution (50 μL) at room temperature. After 30 min, the solution was measured using a microplate reader with the fluorescence mode (Ex/Em: 640 nm/680 nm). The inhibition rate was calculated based on an H₂O₂ standard curve.

Hydroxyl radical (•OH) scavenging test: a hydroxyl free radical assay kit was used to study the •OH scavenging capacity of CpE@BMV. Briefly, CpE@BMV (200 μL, at different concentrations of 16, 31, 62, 125, and 250 μg/mL) was added into a fluorescence solution (200 μL) and incubated for 1 min at 37 °C. Then, hydroxyl radical initiator and Fenton reagent were added and incubated for 20 min at 25 °C. The absorbance of samples at 550 nm was recorded with a microplate reader. The inhibition rate was calculated based on a standard curve.

Superoxide anion (•O₂^–) scavenging assay: the •O₂^– scavenging capacity of CpE@BMV was evaluated using a superoxide anion assay kit. Briefly, CpE@BMV (20 μL, at different concentrations of 16, 31, 62, 125, and 250 μg/mL) was mixed with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) working solution (200 μL). Subsequently, xanthine oxidase solution (20 μL) was added to each tube and incubated for 10 min at 37 °C. The absorbance of samples at 550 nm was recorded with a microplate reader. The inhibition rate was calculated based on a standard curve.

ABTS•·radical scavenging activity: ABTS• was added to potassium persulfate to produce an ABTS radical cation (ABTS + •). Then, CpE@BMV nanoparticles (100 μL, at different concentrations of 16, 31, 62, 125, and 250 μg/mL) were added to the ABTS + • solution. The absorbance of both ABTS + • solution (A_B) and the mixture solution containing ABTS + • and CpE@BMV nanoparticles (A_E) was measured at 734 nm. The percentage scavenging of ABTS + • was calculated as follows:

DPPH• scavenging assay: CpE@BMV nanoparticles (100 μL, at different concentrations of 16, 31, 62, 125, and 250 μg/mL) were incubated with DPPH ethanol solution (0.1 mM, 100 μL) for 30 min. The absorbance of DPPH• solution (A_c), CpE@BMV nanoparticles (A_d), and the mixture solution containing DPPH• and CpE@BMV nanoparticles (A_s) was measured at 517 nm. The percentage scavenging of DPPH• was calculated as follows:

ROS scavenging capability of CpE@BMV in vitro

RAW264.7 cells (1 × 10⁵ cells/well) were plated in 24-well plates and cultured for 24 h (37 °C, 5% CO₂). After PBS washing, the cells were cultured with LPS (200 ng/mL, 24 h). The cells were treated with Cip (4.4 μg/mL), CpE (8.2 μg/mL), BMV (2.8 μg/mL), and CpE@BMV (with Cip concentration of 4.4 μg/mL) for 24 h, and further incubated with DCFH-DA (10 μM) for 30 min. Meanwhile, the cell nuclei were stained with Hoechest33324 and imaged on a confocal laser scanning microscope (CLSM, Nikon A1, Japan), with the excitation of 405/488 nm, collecting fluorescence at 420–460 nm (DAPI) and 500–535 nm (DCFH-DA). In parallel experiments, culture supernatants were analyzed for inflammatory cytokines of IL-6, IL-1β, TNF-α, and IL-10 using ELISA kits according to the protocols.

Cell-protective ability of CpE@BMV against LPS and H₂O₂ damage

RAW264.7 cells were plated in 96-well plates (1 × 10⁴ cells/well) and cultured overnight. The LPS (2 μg/mL) or H₂O₂ (1 mM) was added to cells for 2 h. The cells were then treated with Cip (4.4 μg/mL), Ea (1.9 μg/mL), BMV (2.8 μg/mL), and CpE@BMV (with Cip concentration of 4.4 μg/mL) for 24 h. The treated RAW264.7 cells of all groups were harvested, and the cell viability was assessed using CCK-8 kits according to the protocols.

Red blood cell hemolysis analysis

Mouse whole blood (1 mL) was diluted in PBS (1:10), centrifuged (4000 × g, 15 min), and washed with PBS (10 mL × 5) to collect red blood cells (RBCs). The RBCs were resuspended to 4% (v/v) in PBS. Serial dilutions of CpE@BMV (4–400 μg/mL) were incubated with 4% RBC suspensions (1:1 v/v) for 3 h at 37 °C. After centrifugation (3000 × g, 15 min), supernatant absorbance (OD_treated, 545 nm) was measured to calculate hemolysis. The PBS was used as a negative control (OD _{negative control}) and 0.1% Triton-X as a positive control (OD _{positive control}). The percentage of hemolysis was determined using the formula:

Cip and Ea release from CpE@BMV

The release profile of Cip and Ea from CpE@BMV was evaluated by dialysis and measured by a High-performance liquid chromatography system (HPLC, Agilent, US). A solution of CpE@BMV (1 mL, [Cip] = 350 μg/mL, [Ea] = 150 μg/mL) was transferred into a dialysis bag (MWCO 3000 Da), and suspended in different buffers at 37 °C in an incubator: PBS (20 mL, 10 mM) at pH 7.4, PBS (20 mL, 10 mM) at pH 6.5, PBS (20 mL, 10 mM) at pH 7.4 containing H₂O₂ (100 μM), PBS (20 mL, 10 mM) at pH 6.5 containing H₂O₂ (100 μM). The elevated concentration of H₂O₂ at the infection site, which can reach up to 1 mM, is a result of the oxidative burst generated by immune cells to combat invading pathogens. This serves as a critical component of the host’s defense mechanism^64,70. At predetermined intervals, samples (3 mL) were withdrawn from the buffer solution and replaced with fresh isopycnic phosphate buffer. All collected solutions were lyophilized and resolved in DMSO to measure the content of Cip and Ea using the HPLC. HPLC analysis was conducted using an Agilent 1260 Infinity II HPLC system (Agilent, US) with an Alltima C18 column (4.6 × 100 mm, 2.7 μm). The UV detection was set at 280 nm, with a mobile phase of 2% acetic acid in water/ACN (84:16, v/v). The flow rate was 0.7 mL/min, and the injection volume was 5 μL.

MIC and MBC determination

We tested minimum inhibitory concentration (MIC) as well as bactericidal concentration (MBC) values of the tested formulations according to our published protocal^71,72. Briefly, Cip, Ea, CpE, and CpE@BMV (100 μL) with an equivalent amount of Cip ranging from 0 to 7.0 μg/mL and an equivalent amount of Ea ranging from 0 to 3.0 μg/mL were applied to suspensions of S. aureus WH^GFP (in TSB), E. coli Xen14, and S. typhimurium 15,649 (in LB) (100 μL, 2 × 10⁵ bacteria/mL). The MIC values represented the lowest antibiotic concentrations preventing visible growth. The MBC values were tested by plating aliquots (10 μL) from wells showing no visible growth on agar plates after incubation for 24 h at 37 °C, and the lowest concentration at which colony formation remained absent was considered as the MBC value.

Macrophage phenotypic regulation of CpE@BMV in vitro

RAW264.7 cells were plated in 24-well plates (5 × 10⁴ cells/well) and cultured overnight. Treatments included PBS, Cip (4.4 μg/mL), Ea (1.9 μg/mL), and CpE@BMV (with the concentration of Cip 4.4 μg/mL and Ea 1.9 μg/mL) for 12 h. The treated RAW264.7 cells of all groups were harvested and blocked with anti-mouse CD16/32 antibody (10 μL, 0.5 mg/mL) for Fc receptors. Subsequently, the cell surface was stained with anti-mouse F4/80-APC (10 μL, 0.5 mg/mL) and anti-mouse CD86-BV421 (10 μL, 0.5 mg/mL), while the intracellular staining was performed using anti-mouse CD206-FITC (10 μL, 0.5 mg/mL) for flow cytometry (Beckman CytoFlex, California, USA). In addition, cytokine levels of IL-6, IL-1β, TNF-α, and IL-10 in the cell supernatant were measured using ELISA kits according to the protocols.

Cellular uptake of CpE@BMV by S. aureus WH^GFP infected RAW264.7 cells in vitro

The cell uptake of CpE, CpE@BMV by S. aureus WH^GFP infected RAW264.7 cells was studied. Briefly, RAW264.7 cells were plated in six-well plates and grown for 12 h at 37 °C (4 × 10⁵ cells/mL). The cell was mixed with S. aureus WH^GFP (8 × 10⁶ bacteria/well) for 4 h. Gentamicin (100 μg/mL) was then added to inhibit extracellular growth during 24 h of coculture. Following coculture, wells were gently washed once with PBS to remove extracellular S. aureus WH^GFP. The freshly prepared Nile red-loaded CpE or CpE@BMV (with the concentration of Cip 0.44 μg/mL) were cocultured with cells for another 2 h. Cells were washed with PBS (1 mL/well), fixed with 4% paraformaldehyde (15 min, room temperature), and stained with 4′,6-diamidino-2-phenylindole (10 μM, 30 min, in the dark). RAW264.7 cells containing internalized red-fluorescent CpE or CpE@BMV were visualized using confocal laser scanning microscopy (A1, Nikon, Japan), with the excitation of 405/525 nm, collecting fluorescence at 420–460 nm (DAPI) and 560–625 nm (Nile red). Furthermore, the red fluorescence intensity in infected macrophages was detected using flow cytometry (Beckman CytoFlex, California, USA).

Live/dead staining of bacteria in vitro

E. coli Xen14 was seeded onto a confocal dish and incubated with Cip, CpE, and CpE@BMV (with the concentration of Cip 0.44 μg/mL) for 6 h. Subsequently, the bacteria were stained with SYTO 9 (green, for live bacteria) and PI (red, for dead bacteria) for 30 min before being observed using confocal laser scanning microscopy (A1, Nikon, Japan), with the excitation of 488/525 nm, collecting fluorescence at 420–460 nm (SYTO 9) and 560–625 nm (PI). Meanwhile, the bacteria were treated with the treatment of PBS as the control group.

Antibacterial activity against extracellular and intracellular bacteria in RAW264.7 cells in vitro

For extracellular bacteria eradication in vitro: RAW264.7 cells were seeded at a 1.5 × 10⁵ cells/well density in 24-well plates and grown for 24 h at 37 °C. The cells were infected by S. aureus WH^GFP or E. coli Xen14 (3 × 10⁶ bacteria/well) for 2 h. After washing the cells with PBS (1 mL × 3), the infected macrophage cells were treated with Cip, Cip-pba, CpE, and CpE@BMV (with the concentration of Cip 0.44 μg/mL) for 12 h, 24 h, and 48 h, respectively. The supernatant was collected, and the extracellular S. aureus WH^GFP or E. coli was measured by serial dilution and plated on TSB or LB agar for c.f.u. enumeration.

For intracellular bacteria eradication in vitro: RAW264.7 cells were seeded at a 1.5 × 10⁵ cells/well density in 24-well plates and grown for 24 h at 37 °C. RAW264.7 cells were infected with S. aureus WH^GFP or E. coli Xen14 (3 × 10⁶ bacteria/well) for 2 h. The cells were washed with PBS (1 mL × 3) to remove the extracellular bacteria. The infected macrophage cells were treated with Cip, Cip-pba, CpE, and CpE@BMV (with the concentration of Cip 0.44 μg/mL) for 12 h, 24 h, and 48 h, respectively. Cells were collected through centrifugation (1000 × g, 3 min) and lysed in PBS supplemented with 0.1% bovine serum albumin and 0.1% Triton-X. The harvested lysate was diluted serially and plated on TSB or LB agar for c.f.u. enumeration. The detection limit for c.f.u. was 200, using 100 µL of the undiluted suspension.

Phagocytosis assay induced by CpE@BMV in vitro

RAW264.7 cells were seeded in 24-well plates (1.5 × 10⁵ cells/well) and cultured for 24 h. The cells were pretreated with Cip, Ea, and CpE@BMV ([Cip] = 7.0 μg/mL, [Ea] = 3.0 μg/mL, [CpE@BMV] = 17.5 μg/mL in which the concentration of Cip is 7.0 μg/mL) for 24 h. Subsequently, the cells were cocultured with SYTO 9-labeled S. aureus WH^GFP (1.5 × 10⁶ bacteria/well) for 60 min, followed by washing with PBS and staining with Dil (10 μM). Red fluorescence of RAW264.7 and green fluorescence of S. aureus WH^GFP were detected using flow cytometry (Beckman CytoFlex, California, USA).

Cytotoxicity of CpE@BMV in vitro

L929 cells and RAW264.7 cells were cultured in a 96-well plate (1 × 10⁴ cells/well) overnight. The cells were incubated with CpE@BMV at different concentrations (0.1–400 μg/mL) for 24 h. The cell cytotoxicity was assessed using a CCK-8 kit according to the protocol.

Antibacterial efficacy of CpE@BMV in a pneumonia model

BALB/c mice were intratracheally administered with E. coli Xen14 (1 × 10⁹ bacteria/mL, 50 μL per mouse) to establish a pneumonia model. The mice were randomly allocated into six groups (n = 7): Control group (healthy mice), the E. coli Xen14 infected mice with different formulations using equivalent doses of Cip, Ea, and BMV were 1.76 mg/kg, 0.76 mg/kg, and 1.12 mg/kg, respectively. Different formulations were treated via intratracheal instillation. Body weights were monitored every 48 h during treatment. At day 3 and day 7 post-treatment, the blood was collected from the eyes (n = 5) to measure the major blood parameters using Mindray, Shenzhen, China, and the serum was further collected and applied for cytokine quantification (TNF-α, IL-6, IL-β, IL-10) with ELISA Kits. The mice were sacrificed on day 7. The heart, liver, spleen, lung, and kidney were harvested (n = 3) for H&E staining according to the protocol. The frozen slices of lung tissue were prepared, and the immunofluorescence staining was performed to analyze macrophage phenotypes labeled with CD86 and CD206. The lung tissue with PBS-treated and CpE@BMV-treated groups was also collected for RNA sequencing (n = 3) and 2bRAD sequencing for microbiome (n = 3). Additionally, the lung tissue (n = 4) was homogenized in PBS and centrifuged to collect supernatants, 10 μL supernatants were diluted serially and plated on LB agar for c.f.u. enumeration, and the other 10 μL supernatants were applied for cytokine quantification (TNF-α, IL-6, IL-β, IL-10) with ELISA Kits.

Antibacterial efficacy of CpE@BMV in a peritonitis model

BALB/c mice were randomly allocated into six groups (n = 5) and intraperitoneally injected with E. coli Xen14 solution (100 μL, 1 × 10⁸ bacteria/mL) on day 0, 7, and 14. After 4 h of infection, the mice were intraperitoneally administered with different formulations using equivalent doses of Cip, Ea, and BMV were 1.76 mg/kg, 0.76 mg/kg, and 1.12 mg/kg, respectively, on days 0, 7, and 14. Bioluminescence of bacteria in the mouse peritoneal cavity was recorded at 0, 4, 8, and 24 h after the first treatment, using a bio-optical imaging system (IVIS®, 45 s exposure time, medium binning, 1F/Stop, Open Emission Filter).

On day 15, the blood of mice was collected from eyes (n = 5) to measure the major blood parameters using (Mindray, Shenzhen, China), and the serum was further collected and applied for cytokine quantification (TNF-α, IL-6, IL-β, IL-10) with ELISA Kits. The mice were sacrificed on day 15. The peritoneal fluid of mice was collected and split for c.f.u. counting (n = 4), and cytokine quantification (TNF-α, IL-6, IL-β, IL-10) with ELISA Kits, the spleen tissues were harvested and homogenized in PBS to obtain the supernatant for cytokine quantification with ELISA Kits. The major organs of mice (n = 4) were homogenized in PBS and centrifuged to collect supernatants, 10 μL supernatants were diluted serially and plated on LB agar for c.f.u. enumeration.

Infiltrating immune cells analysis in vivo

Immune cells in lung and spleen tissues at different time points (pneumonia model: days 3 and 7; peritonitis model: day 15) were analyzed by multiparameter flow cytometry following various treatments. The excised lung/spleen tissue was mechanically disrupted using the tip of a syringe, then 4 mL of digestion solution (Sigma, Germany) was added, and the mixture was put on a shaker (150 rpm) for 1 h to ensure complete digestion. Finally, the suspension was filtered through a 200-mesh sieve and centrifuged at 450 × g for 5 min to obtain a single-cell suspension. The single-cell suspension was subsequently stained with antibodies and analyzed using flow cytometry. Firstly, a single-cell suspension was incubated with anti-mouse CD16/32 (10 μL, 0.5 mg/mL) at 4 °C for 15 min to block Fc receptors. For macrophages, cells were stained with anti-mouse CD45-BV605 (10 μL, 0.2 mg/mL), anti-mouse CD11b-FITC (10 μL, 0.2 mg/mL), anti-mouse F4/80-BV421 (10 μL, 0.2 mg/mL), anti-mouse CD86-APC (10 μL, 0.2 mg/mL), anti-mouse CD206-PE (10 μL, 0.2 mg/mL) for 30 min; For T cells, cells were stained with anti-mouse CD45-BV605 (10 μL, 0.2 mg/mL), anti-mouse CD3-FITC (10 μL, 0.5 mg/mL), anti-mouse CD4-BV421 (10 μL, 0.2 mg/mL), anti-mouse CD8-APC (10 μL, 0.2 mg/mL), anti-mouse Foxp3-PE (10 μL, 0.2 mg/mL) for 30 min; For B cells, cells were stained with anti-mouse CD45-BV605 (10 μL, 0.2 mg/mL), anti-mouse CD19-BV421 (10 μL, 0.2 mg/mL), anti-mouse CD138-APC (10 μL, 0.2 mg/mL) for 30 min. Flow cytometry was performed by Beckman Coulter (CytoFlex, California, USA), and the result was analyzed using FlowJo software. Besides, the infection-draining lymph nodes (IDLN) were separated to collect immune cells, and the cells were stained with anti-mouse CD16/32 antibody (10 μL, 0.5 mg/mL), anti-mouse CD45-BV605 (10 μL, 0.2 mg/mL), anti-mouse CD11c-BV421 (10 μL, 0.2 mg/mL), anti-mouse CD86-APC (10 μL, 0.2 mg/mL), and anti-mouse CD80-FITC (10 μL, 0.2 mg/mL) for 30 min, and analyzed by Beckman Coulter.

RNA sequencing

Total RNA in lung tissue was extracted using TRIzol according to the user guide. Next, the purity and quantification of the collected RNA were measured by a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was checked by an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The transcriptome library was built using a VAHTS Universal V5 RNA-seq Library Prep Kit. The sequencing was conducted using an Illumina Novaseq 6000 sequencing platform. DESeq2 software was employed to analyze differentially expressed genes (DEG). The DEG was further investigated by GO and KEGG Pathway enrichment based on the hypergeometric distribution algorithm to identify the enriched GO and KEGG terms. RNA sequencing was conducted at the OE Biotech Co., Ltd. (Qingdao, China).

2bRAD-M sequencing for microbiome

The 2bRAD-M library construction was based on the method reported by Mikhail V Matz et al.⁷³. Genomic DNA of lung tissue was extracted using a MagPure Soil DNA KF Kit (MGBio, China), and the collected DNA (1 pg–200 ng) was further treated with the enzyme BcgI (4 U, NEB) for three hours. After ligating adapters to the DNA fragments by mixing 10 μL digested DNA with a master mix (10 μL, with 0.2 μL adapters, and 800 U T4 DNA ligase) at 4 °C overnight, the mixture was amplified using an 8% polyacrylamide gel, in which the bands around 100 bp were excised and DNA was collected. Sample-specific barcodes were incorporated via PCR using platform-compatible indexing primers, which were further purified by a QIAquick PCR purification kit. The 2BRAD-M sequencing was performed by the Illumina Nova PE150 platform.

The databases of GTDB and Ensembl were applied to identify the species-level of 2bRAD-M markers. The set of 2bRAD tags derived from each genome was assigned under the corresponding GCF number, along with the taxonomic information associated with the whole genome. At the end, all single-copy 2bRAD tags (occurring once per genome) from each GCF were compared across all genomes to develop species-specific 2bRAD markers. The sequenced 2bRAD tags were mapped against the database to calculate relative abundance. The relative abundance of a specific species was determined by the proportion of microbial individuals in the species relative to the total number of detectable species in a sample.

Statistical analysis

All result data were presented as mean ± standard deviation (s.d.). Unless otherwise stated, all experiments used biological replication. Statistical analyses were analyzed using an unpaired Student’s t-test (two-tailed) for two groups, and one-way ANOVA with Tukey’s multiple comparisons was used for three or more groups. Significance levels were defined as follows: n.s., no significant difference, p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; p-values were calculated by GraphPad Prism v8.3.0 (GraphPad Software) and marked on the figures.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.