De novo design of a pH-triggered self-assembled β-hairpin nanopeptide with the dual biological functions for antibacterial and entrapment

Abstract

Background: Acid-tolerant enteric pathogens can evade small intestinal acid barriers, colonize and infect the intestinal tract. However, broad-spectrum antibiotics are not the best therapeutic strategy because of the disruption of intestinal flora caused by its indiscriminate antimicrobial activity against beneficial and harmful bacteria. So that is what inspired us to combine pH regulation with nanotechnology to develop a pH-triggered site-targeted antimicrobial peptide with entrapping function.

Results: A pH-triggered dual biological functional self-assembled peptide (SAP) was designed according to the features of amino-acid building blocks and the diagonal cation-π interaction principle. The results of characterization experiments showed that changes in pH conditions could trigger microstructural transformation of the nanopeptide from nanospheres to nanofibers. The subsequent antibacterial and toxicity experiments determined that SAP had great antimicrobial activity against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, and Bacillus cereus above 15.6 μg/mL under acidic conditions by disrupting bacterial membrane integrity, excellent biocompatibility in vitro even at 250 μg/mL and high tolerance in physical environment. Moreover, at peptide concentrations greater than 62.5 μg/mL, SAP showed the entrapment property, which played an important role in phagocytic clearance in infection forces. Meanwhile, the in vivo results revealed that SAP possessed excellent therapeutic effect and good biosafety.

Conclusions: Our study revealed the antibacterial activity of a short β-hairpin forming self-assembled peptide, and established an innovative design strategy for peptide-based nanomaterials and a new treatment strategy for gastrointestinal bacterial infections.

Keywords: Antimicrobial activity; Entrapment property; Histidine functioned; pH-triggered self-assembled peptide; β-hairpin structure.

Scheme 1

Schematic illustration of the pH-triggered microstructural transformation of the β-sheet self-assembled peptide and its biological functions

Fig. 1

a Schematic of the SAP. b The three-dimensional structure of SAP modeling by http://zhanglab.ccmb.med.umich.edu/I-TASSER. The hydrophobicity spectrum: red-white-blue (− 4.5 ~  + 4.5). c Concentration-dependent self-assembly of the SAP. ANS fluorescence binding assay for the SAP at different concentrations at pH 5.0, 6.0 and 7.0. d The critical aggregation concentrations of the SAP at pH 5.0, 6.0 and 7.0. e TEM images and f CD spectra of SAP nanoparticles under three pH conditions

Fig. 2

a Antibacterial activity of the SAP against E. coli ATCC 25922, S. typhimurium ATCC 14028, Listeria monocytogenes CGMCC 1.10753 and Bacillus cereus CGMCC 1.932 in three pH environments (mean ± SD, n = 3). Compared to control: * p < 0.05, ** p < 0.01, *** p < 0.001. b Antibacterial activity of the SAP and melittin at pH 6.0 against E. coli ATCC 25922 after incubation with trypsin and chymotrypsin at mass ratios of 2:1, 1:1, 0.5:1 and 0.25:1 (enzyme: peptide), respectively for 1 h at 37 °C (mean ± SD, n = 3). # indicated no colony formation. c Antibacterial activity of the SAP against E. coli ATCC 25922 in the presence of 100 mM and 150 mM NaCl at pH 6.0 (mean ± SD, n = 3) Compared to the control: ** p < 0.01. d Cytoplasmic membrane potential variations of E. coli ATCC 25922 treated by SAP and melittin at different concentrations. e SEM micrographs of E. coli ATCC 25922 treated with 250 μg/mL SAP for 30 min at pH 6.0 or not. f TEM micrographs of E. coli ATCC 25922 treated with 250 μg/mL SAP for 30 min at pH 6.0 or not

Fig. 3

Cytotoxicity of various concentrations of the SAP and melittin against RAW264.7 cells, HEK293T cells and RBCs under three pH conditions (mean ± SD, n = 3)

Fig. 4

a The entrapment phenomenon caused by SAP. b Plots of colony forming units (CFU/mL) of the supernatant of E. coli ATCC 25,922 treated with 250 μg/mL SAP for 8 h in HEPES buffer at pH = 5.0, 6.0 and 7.0. c Plots of colony forming units (CFU/mL) of bacterial supernatant treated with different concentrations of SAP for 8 h in HEPES buffer at pH = 6.0. d Deltavision OMX SR fluorescence microscopic images analysis of E. coli strain BL21 consisting of PET-28a-EGFP plasmid treated with 250 μg/mL SAP or not. e Fluorescence microscopic images analysis of phagocytosis of E. coli (green) particles pretreated with 250 μg/mL SAP for 1 h by RAW 264.7 cells (DAPI, blue) or not

Fig. 5

a Schematic illustration of the experimental protocol for efficacy measurement. b Bacterial burdens in liver, spleen, kidney and peritioneal cavity after infection of E. coli and treatment with saline, SAP and colistin (mean ± SD, n = 6, p < 0.05). c The levels of TNF-α, IL-6, IL-1β, IL-4 and IL-10 after injection of saline and infection of E. coli and treatment with saline, SAP and colistin (mean ± SD, n = 6, p < 0.05). d Histopathological H&E staining of liver, spleen and kidney tissue after injection of saline and infection of E. coli and treatment with saline, SAP and colistin

Fig. 6

a Schematic illustration of the experimental protocol for biocompatibility measurement. b Curve of weight change, c relative organ index, d renal function parameters and e hepatic function parameters of mouse injected with 10 mg/kg, 20 mg/kg, 40 mg/kg and saline in four days (mean ± SD, n = 6, p > 0.05). f Histopathological H&E staining of liver and kidney tissue after injection of SAP and saline