Targeted delivery of a short antimicrobial peptide (CM11) against Helicobacter pylori gastric infection using concanavalin A-coated chitosan nanoparticles

Abstract

Helicobacter pylori is the cause of most cases of stomach ulcers and also causes some digestive cancers. The emergence and spread of antibiotic-resistant strains of H. pylori is one of the most important challenges in the treatment of its infections. The present study aims to develop a concanavalin A (ConA) coated chitosan (CS) nanocarrier-based drug delivery for the targeted release of peptides to the site of H. pylori infection. Accordingly, chitosan was used as an encapsulating agent for CM11 peptide delivery by applying ionotropic gelation method. Con-A was used for coating CS nanoparticles to target H. pylori. The CS NPs and ConA-CS NPs were characterized by FTIR, dynamic light scattering (DLS), and scanning electron microscopy (SEM). The MIC of CM11-loaded ConA-CS NPs against H. pylori SS1 strain was analyzed in vitro. In order to evaluate the treatment efficiency in vivo, a gastric infection model of H. pylori SS1 strain was established in mice and histopathological studies and IL-1β cytokine assay were performed. Based on the results, the size frequency for CS NPs and ConA-CS NPs was about 200 and 350 nm, respectively. The prepared CM11-loaded ConA-CS NPs exhibited antibacterial activity against H. pylori SS1 strain with a concentration of 32 µg/ml. The highest healing process was observed in synthesized CM11-loaded ConA-CS NPs treatments and a significant decrease in IL-1β was observed. Our findings highlight the potential of chitosan nanoparticles as a drug delivery vehicle in the treatment of gastric infection model of H. pylori SS1 strain.

Fig. 1

FTIR analysis of (A) ConA-CS NPs, (B) cancanavalin (A), and (C) chitosan samples. The spectrum was taken in the range of 400–4000 cm⁻¹

Fig. 2

Scanning electron microscope photography of ConA-CS NPs

Fig. 3

Diameter size (A) and zeta potential (B) of CS NPs (part I) and ConA-CS NPs (part II). Size frequency for CS NPs and ConA-CS NPs is about 200 nm and 350 nm, respectively. Zeta potential for CS NPs and ConA-CS NPs is 25.4 mV and 22.3 mV, respectively

Fig. 4

In vitro peptide release from CM11-loaded CS NPs and CM11-loaded ConA-CS NPs at two different pHs (5 and 7.4)

Fig. 5

The Time-kill assay of free CM11, CM11-loaded CS NPs, CM11-loaded ConA-CS NPs, clarithromycin and amoxicillin, and triple antibiotics mixture (omeprazole, metronidazole, and tetracycline) against amoxicillin and clarithromycin-resistant H. pylori SS1 strain

Fig. 6

Cell viability assay of human gastric adenocarcinoma cell-line (AGS cells) treated with free peptide, CM11-loaded CS NPs, and CM11-loaded ConA-CS NPs. A) Effects of 32 μg/ml concentration of the CM11 antimicrobial peptide on AGS cells viability (free peptide); B) Effects of 32 μg/ml concentration of the CM11 peptide encapsulated in chitosan nanoparticles on AGS cells viability; C) Effects of 32 μg/ml concentration of the CM11 peptide encapsulated in ConA-coated chitosan nanoparticles on AGS cells viability. Data are shown as the mean ± SEM

Fig. 7

Histopathology graph comparing different treated groups. A Infection without treatment showing high neutrophil infiltration as a marker for inflammation and disrupted epithelium cells; (B) Infection with triple antibiotic mixture treatment revealed lower inflammation with normal epithelium appearance; (C) Infection treated with CM11 antimicrobial peptide (free peptide) indicates neutrophil infiltration and epithelia disruption; (D) Infection treated with CM11-loaded chitosan nanoparticles revealed the inflammation with neutrophile accumulation. E Infection treated with CM11-loaded ConA-chitosan nanoparticles revealed less inflammation with normal epithelium appearance compared to other groups; (F) Number of neutrophils in each microscope field (10X). Data are shown as the mean ± SEM. **P < 0.01, ***P < 0.001 by post hoc one-way ANOVA statistical analysis

Fig. 8

IL-1β assay in gastric biopsies from different groups reveals inflammation. IL-1β level significantly decreased in the triple antibiotic mixture treatment group and CM11-loaded ConA-CS NPs treatment. Data are shown as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by post hoc one-way ANOVA statistical analysis