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. 2023 Feb 9:14:1083330.
doi: 10.3389/fmicb.2023.1083330. eCollection 2023.

Amoxicillin-docosahexaenoic acid encapsulated chitosan-alginate nanoparticles as a delivery system with enhanced biocidal activities against Helicobacter pylori and improved ulcer healing

Saeed Khoshnood  1 , Babak Negahdari  2 , Vahab Hassan Kaviar  1 , Nourkhoda Sadeghifard  1 , Mohd Azmuddin Abdullah  3 , Mohamed El-Shazly  4 , Mohammad Hossein Haddadi  1
Affiliations

Amoxicillin-docosahexaenoic acid encapsulated chitosan-alginate nanoparticles as a delivery system with enhanced biocidal activities against Helicobacter pylori and improved ulcer healing

Saeed Khoshnood et al. Front Microbiol. .

Abstract

Encapsulation of amoxicillin (AMX) for drug delivery against Helicobacter pylori infection and aspirin-induced ulcers in rat's stomachs was performed using docosahexaenoic acid (DHA)-loaded chitosan/alginate (CA) nanoparticles (NPs) developed by ionotropic gelation method. The physicochemical analyses of the composite NPs were performed by scanning electron microscopy, Fourier transform infrared spectroscopy, zeta potential, X-ray diffraction, and atomic force microscopy. The encapsulation efficiency of AMX was increased to 76% by incorporating DHA, which resulted in a reduction in the particle size. The formed CA-DHA-AMX NPs effectively adhered to the bacteria and rat gastric mucosa. Their antibacterial properties were more potent than those of the single AMX and CA-DHA NPs as demonstrated by the in vivo assay. The composite NPs attained higher mucoadhesive potential during food intake than during fasting (p = 0.029). At 10 and 20 mg/kg AMX, the CA-AMX-DHA showed more potent activities against H. pylori than the CA-AMX, CA-DHA, and single AMX. The in vivo study showed that the effective dose of AMX was lower when DHA was included, indicating better drug delivery and stability of the encapsulated AMX. Both mucosal thickening and ulcer index were significantly higher in the groups receiving CA-DHA-AMX than in the groups receiving CA-AMX and single AMX. The presence of DHA declines the pro-inflammatory cytokines including IL-1β, IL-6, and IL-17A. The synergistic effects of AMX and the CA-DHA formulation increased the biocidal activities against H. pylori infection and improved ulcer healing properties.

Keywords: Helicobacter pylori; amoxicillin; biocidal activities; chitosan; chitosan-DHA nanoparticles; docosahexaenoic; encapsulation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Physicochemical properties of CA-based NPs. (A) SEM micrograph of CA NPs and CA-DHA-AMX NPs (CA = 1%, DHA = 100 μM, 2% v/v, and AMX = 60 mg/ml). (B) FTIR spectra of chitosan, alginate, DHA (100 μM), AMX, and CA-DHA-AMX NP. (C) XRD patterns of chitosan (a), alginate (b), DHA (c), and CA-DHA (d). (D,E) AFM analysis of the surface topography of the unloaded and DHA-loaded NPs. (F,G) Zeta potentials of unloaded and DHA-loaded NPs (2% v/v) at various pH levels.
Figure 2
Figure 2
The antibacterial activity of DHA and chitosan alone and in combination. (A) The growth inhibition rate of CA NPs with different concentrations of chitosan (0.1, 0.5, and 1.0%). (B) The antibacterial activity of DHA with different concentrations (25, 50 and 100 μM). (C) The antibacterial activity of DHA -loaded NPs at different loaded concentrations of DHA (CA 0.1%, DHA 1, 1.5 and 2%).
Figure 3
Figure 3
The growth inhibition activity of composite NPs. (A) The antibacterial activity of CA-NPs containing different concentrations of DHA and AMX. (B) The growth inhibition rate of CA-DHA (100 μM)-AMX (60 mg/ml) composite NPs at different time intervals 6, 12, and 24 h. (C,D) CA-DHA (100 μM)-AMX (60 mg/ml) at different pH values after 6 h incubation was investigated for their growth inhibition activity and release of drugs. (E) The in vivo antibacterial activity of chitosan, DHA and CA-DHA. (F) In vivo antibacterial activity of various formulations of composite NPs, including CA-DHA, CA-AMX, and CA-DHA-AMX compared with the untreated group. (G) Antibacterial activity was tested at a dose of 10 and 20 mg/kg AMX in different formulations. Data are presented as mean ± SEM.
Figure 4
Figure 4
The bacterial binding capacity of composite NPs. (A) Bacterial adhesion of CA-DHA NPs and unloaded NPs at different concentrations of DHA. (B) The effect of different formulations of NPs on bacterial adhesion at two different time intervals; 2 h and 4 h.
Figure 5
Figure 5
The mucoadhesion potential of composite NPs. (A) The in vitro assessment of mucoadhesive potential. Results of the unloaded NPs were similar to CA-AMX (data not shown). (B) In vivo mucoadhesive activity was evaluated by fluorescence microscopy under two distinct diet conditions; fasted (2 h before feeding) and fed (2 h after feeding, scale bar 500 μM). (C) The in vivo mucoadhesive activity was performed under two feeding conditions, fasting and feeding. Data are presented as mean ± SEM (n = 3).
Figure 6
Figure 6
A histopathological evaluation of ASA-induced ulcers in rats infected with H. pylori. (A) The bacterial count of biopsies in treatment groups at day 14. (B) The phenotypic assessment of the rat stomach in different examined groups at day 14. (C–F) The analysis of ulcer index, histopathological features, ulcer area, and mucosa thickness of treated groups compared with control (untreated group). Data are presented as mean ± SEM (n = 3).
Figure 7
Figure 7
The distribution of macrophage (MQ), neutrophil and fibroblast cells. (A) The microscopic distribution of macrophage, neutrophil and fibroblast cells in four different group untreated group, CA-AMX (20 mg/kg), CA-DHA-AMX (10 mg/kg), and CA-DHA, AMX (20 mg/kg) at 14. Hematoxylin and Eosin staining. (B) The comparison of macrophage, neutrophil and fibroblast cells at day 14 among all treated groups. Data are presented as mean ± SEM (n = 3).
Figure 8
Figure 8
The histopathological evaluation and the pro-inflammatory cytokine production of gastric biopsies. (A) The levels of pro-inflammatory cytokines including IL-1β, IL-6, and IL-17A was conducted using ELISA method. (B) The collagen accumulation among different treated groups. (C) Masson’s trichrome staining was used to evaluate the accumulation of collagen (yellow arrow), the blue and red are responsible for collagen and muscle fibers. Immunohistochemical staining for Collagen I (yellow arrow), high accumulation of collagen I in glands and endothelia observed in untreated group, while the accumulation was reduced in treated groups, the lowest accumulation was observed in CA-DHA-AMX (20 mg/kg). Data are presented as mean ± SEM (n = 3).
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