Multifunctional Antibacterial Nanonets Attenuate Inflammatory Responses through Selective Trapping of Endotoxins and Pro-Inflammatory Cytokines

Abstract

Extracellular lipopolysaccharide (LPS) released from bacteria cells can enter the bloodstream and cause septic complications with excessive host inflammatory responses. Target-specific strategies to inactivate inflammation mediators have largely failed to improve the prognosis of septic patients in clinical trials. By utilizing their high density of positive charges, de novo designed peptide nanonets are shown to selectively entrap the negatively charged LPS and pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). This in turn enables the nanonets to suppress LPS-induced cytokine production by murine macrophage cell line and rescue the antimicrobial activity of the last-resort antibiotic, colistin, from LPS binding. Using an acute lung injury model in mice, it is demonstrated that intratracheal administration of the fibrillating peptides is effective at lowering local release of TNF-α and IL-6. Together with previously shown ability to simultaneously trap and kill pathogenic bacteria, the peptide nanonets display remarkable potential as a holistic, multifunctional anti-infective, and anti-septic biomaterial.

Keywords: anti-infective; anti-septics; inflammation; nanostructures; peptides.

Figure 1

Peptide nanonets entrap LPS via two different mechanisms. a) FITC‐LPS trapping index of peptide was computed by measuring fluorescence emission of FITC‐LPS in the supernatant (λ _EX = 488 nm and λ _EM = 520 nm) after 1 h of incubating LPS (2 µg mL⁻¹) with peptide at different concentrations. b) Confocal microscopy images of 64 µm peptide incubated with 32 µg mL⁻¹ FITC‐LPS for 1 h. Magnification = 200×. Scale bar = 20 µm. c) FITC‐LPS trapping index of pre‐formed nanonets was determined after 1 h pre‐incubation with LPS or LTA (2 µg mL⁻¹) followed by 1 h incubation with FITC‐LPS (2 µg mL⁻¹). For (a) and (c), three repeats were conducted in duplicates and data were expressed as mean ± SE, and data points were fitted to a non‐linear regression inhibitor concentration versus response model for computation of EC₅₀ values. d) Proposed dual mechanism of LPS trapping by the peptide nanonets via scaffold integration and post‐binding.

Figure 2

Peptide nanonets nullify the colistin‐inactivating effect of extracellular LPS. a) Proposed mechanism of fibrillating peptide rescuing the activity of colistin. b) MICs of BTT2‐4A and colistin against different bacteria strains were determined either in the absence or presence of 100 µg mL⁻¹ cell‐free LPS. c) MICs of colistin were determined in the presence of free LPS (100 µg mL⁻¹) and varying sub‐MIC concentrations of BTT2‐4A. MIC assays were conducted in duplicates in three independent experiments.

Figure 3

Peptide nanonets display selective binding toward pro‐inflammatory cytokines. a) Isoelectric point (PI) and net charge of the cytokines tested in this work. b) Cytokine trapping by LPS‐induced nanonets. After 1 h of incubating LPS (5 µg mL⁻¹) and cytokine (2 ng mL⁻¹) with peptide at different concentrations, sample was centrifuged and the cytokine remaining in the supernatant was quantified using ELISA. Results were normalized against peptide‐free controls subjected to identical processing steps. Three independent experiments were performed in duplicates and data are presented as mean ± SE. Statistical significance between samples were evaluated using one‐way analysis of variance (ANOVA) followed by Tukey post‐hoc test (*: p ≤ 0.05, **: p ≤ 0.01).

Figure 4

Peptide nanonets suppress LPS‐induced release of pro‐inflammatory cytokines by RAW cells. a) Effect of peptide post‐treatment on LPS‐induced TNF‐α and IL‐6 production by RAW 264.7 cells at different LPS concentrations. After 1 h of incubating the cells with LPS, peptide was added followed by 23 h incubation before cytokine in the culture media was quantified. Results were normalized against the peptide‐free negative control. Three independent experiments were conducted, and data are presented as mean ± SE. Statistical significance between samples were evaluated using one‐way analysis of variance (ANOVA) followed by Tukey post‐hoc test (*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001). b) Proposed anti‐inflammatory mechanisms of the fibrillating peptides: (1) LPS trapping by the nanonets, (2) cytokine trapping by the nanonets, and (3) intracellular effect by soluble peptide molecules. c) Quantification of immune cells and cytokine production in bronchoalveolar lavage fluids (BALF) collected from mice after 4 h treatment (n = 5–6 per group). Mice were given LPS only or saline as controls. Data are presented as mean ± SD. Statistical significance between samples were evaluated using one‐way ANOVA followed by Tukey post‐hoc test, and p values are shown in figure.