A self-assembled and H2O2-activatable hybrid nanoprodrug for lung infection and wound healing therapy

Abstract

Background: The pursuit of effective antibacterial strategies aimed at mitigating pathogenic bacterial infections while minimising drug resistance remains of paramount importance. A combinational therapeutic strategy that integrates distinct treatment components can enhance overall efficacy and mitigate undesired effects, thereby exhibiting considerable promise in combating bacterial infections. Methods: In this study, a meticulously engineered self-assembling hybrid nanoprodrug (CPBP NPs) has been devised, functioning as a hybrid prodrug of Ciprofloxacin (Cip) and hydroxybenzyl alcohol (HBA). Results: CPBP molecules can generate nanoassemblies via self-assembly and subsequently undergo decomposition to synchronously release Cip and HBA upon hydrogen peroxide (H₂O₂) exposure. The CPBP NPs exert antibacterial and anti-inflammatory properties through the controlled release of Cip and HBA, while also facilitating the scavenging of reactive oxygen species. These CPBP NPs exhibit broad-spectrum antibacterial activity against both Gram-negative bacteria (E. coli, 98.4%) and Gram-positive bacteria (S. aureus, 98.5%). Notably, CPBP NPs not only accumulate in the lungs to facilitate organ-specific infection treatment but also expedite the healing process of infected wounds. Conclusions: Consequently, this H₂O₂-activatable hybrid nanoprodrug, possessing excellent biocompatibility, holds substantial promise for advancing clinical applications in managing bacterial infections.

Keywords: Lung infection; Nanoprodrug; Self-assemblies; Synergetic therapy; Wound healing.

Scheme 1

Schematic diagram of the construction and application of CPBP NPs. (A) The preparation process of CBPB NPs and schematic diagram of their antibacterial and anti-inflammatory mechanism. CPBP NPs had an effective therapeutic effect in (B) the lung infection model and (C) the infected skin wound healing model.

Figure 1

Characterization of CPBP NPs. (A) Size distribution histogram and Tindall effect of CPBP NPs solution. (B) SEM image of CPBP NPs. (C) DLS results regarding the size of CPBP NPs for 48 h in PBS and 10% FBS-F12 medium (n = 3). (D) Structural changes of initial CPBP molecules in the system during simulation and intermolecular interaction patterns in the CPBP system. (E) The RMSD changes of CPBP molecules in the system during simulation. (F) The total number of formed hydrogen bonds between CPBP molecules in the system during the self-assembly process. (G) Cumulative release of Cip from CPBP NPs (n = 3).

Figure 2

Antioxidant and anti-inflammatory activity of CPBP NPs. (A) Protective effects CPBP NPs on H₂O₂-stimulated RAW 264.7, MLE-12, and L-929 cells (n = 3), **P < 0.01. After 3 h of treatment with H₂O₂ (200 μM), cells were incubated with Cip+HBA or CPBP NPs. (B) OxiVision Green assay for determining the amount of H₂O₂ eliminated by adding Cip+HBA or CPBP NPs (n = 3), **P < 0.01. (C) Suppressive effects of Cip+HBA or CPBP NPs on the generation of intracellular ROS in H₂O₂-stimulated cells, DAPI: excitation wavelengths is 405 nm, DCFH-DA: excitation wavelengths is 488 nm. Suppressive effects on Cip+HBA or CPBP NPs on the expression of TNF-α (D), IL-1β (E), and IL-6 (F) in H₂O₂-stimulated cells (n = 3), **P < 0.01 and *P < 0.05.

Figure 3

Antibacterial activity of CPBP NPs. (A) Representative photographs and (B) their quantitative analysis of bacterial colonies formed on agar plates in different treatment groups (n = 3), P < 0.01. ^#P > 0.05. (C) Fluorescence images for S. aureus after various treatments, green and red represent live and dead bacteria respectively. (D) SEM images of S. aureus after various treatments. E) Biofilm biomass of S. aureus identified by crystal violet staining after various treatments and (F) the quantitative evaluation of biofilm clearance ratio (n = 3), **P < 0.01. ^#P > 0.05.

Figure 4

Therapeutic effect on lung infection with S. aureus. (A) Schematic diagram of lung infection treatment schedule in PBS, Cip+HBA, and CPBP NPs. (B) Representative images of lung sections H&E with various treatments. (C) Photographs of bacterial colonies obtained from infected lung tissues. (D) Wet/dry weight ratio of lungs (n= 5), **P < 0.01. (E) Survival curves of mice with bacterial pneumonia in various treatment groups (n = 8). (F) Immunofluorescence staining for ROS by DCFH-DA fluorescence probe (red is a false color) and (G) their relative intensity, **P < 0.01. (H) Immunofluorescence staining images of CD86 and CD206 in lung infection of mice after various treatments and their relative intensity of CD86 (I) and CD206 (J), **P < 0.01.

Figure 5

Transcriptomic analysis illustrating the therapeutic mechanism of CPBP NPs in bacterial pneumonia model infected by S. aureus. (A) Volcano plot showing DEGs in PBS and CPBP NPs groups. (B) Venn diagrams illustrating the number of DEGs in Normal versus PBS, Normal versus CPBP NPs, and PBS versus CPBP NPs. (C) Heatmap of hierarchical clustering of DEGs. GO enrichment analysis of the gene functions of (D) downregulated and (E) upregulated DEGs. (F) Downregulated and G) upregulated DEGs enriched in the KEGG pathway.

Figure 6

Therapeutic effect on wound healing with S. aureus. (A) Schematic diagram of wound healing model treatment schedule, the female BALB/c mice were created 5 mm wounds on the back, after 24 h, subcutaneously injected S. aureus suspension (50 μL, 3 × 10⁷ CFU/mL), and infected mice were treated with PBS, Cip+HBA, CPBP NPs, and CPBP NPs/G. (B) Representative photographs of infected wounds on days 0, 6, and 12. (C) Monitoring of wound closure during treatments in each group. (D) The relative wound area in different groups (n = 5), **P < 0.01. (E) Representative photographs of bacterial colonies in S. aureus-infected wounds. (F) Corresponding changes in body weight of the mice. (G) H&E and Masson staining images of the infected wound tissues of mice after 12 days of different treatments. Quantitative analysis of stained images acquired from different groups: (H) scar width and (I) college deposition. (n = 5), **P < 0.01.

Figure 7

Representative immunofluorescence images (A) and quantitative analysis (B) of ROS expression in infected wounds (n = 5), red is a false color. Representative immunofluorescence images of (C) CD206, CD86, and (D-E) their quantifications of vessel density (n = 5), **P < 0.01. Representative immunofluorescence images of (F) angiogenesis (CD31), and (G) their quantifications of vessel density (n = 5). Quantitative analysis of inflammatory cytokines at the site of infection including TNF-α (G) and IL-6 (H) after 6-days treatment in infected wounds of mice after different treatment groups (n = 5).