Helicobacter pylori (Hp) infection causes several gastric disorders and accounts for 90% of all diagnosed gastric cancers, the 5th most common and 4th deadliest cancer worldwide1. Hp is a source of concern for the World Health Organization, as the available antibiotherapy presents high failure rate (10-40%), mainly due to mounting antibiotic resistance2. The use of antimicrobial peptides (AMPs) is gaining relevance as an alternative to antibiotics. PexigananA, MSI-78A, is an analog of Pexiganan with reported bactericidal activity against Hp3. AMPs immobilization onto biomaterials allow to protect AMPs of proteolytic degradation and aggregation with proteins, while maintaining the bactericidal effect4.

Here, a versatile, cost-effective, and environmentally friendly “one-pot” microfluidics system suitable for nanoparticles (NPs) production and bioconjugation of any ligand containing a thiol group (e.g., cysteine amino acid) is proposed. MSI-78A was directly grafted onto chitosan nanoparticles (AMP-NP, 113 ± 2 nm) surface using an innovative microfluidic system that allows peptide bioconjugation in situ using the Thiol–Norbornene “Photoclick” Chemistry. The reaction yield was ~85% (UV/vis spectroscopy – indirect method), and grafting was confirmed by Fourier-transform infrared spectroscopy, where the characteristic absorption bands of the AMP appeared at 1660 cm-1 (amide I) and 1530 cm-1 (amide II).

AMP-NP in a concentration of 1011 NP/mL had a fast-bactericidal effect against H. pylori 26695 strain, reaching full eradication in 30min, while for the H. pylori J99 strain (human highly pathogenic strain), the same bactericidal effect was achieved after 24h. These results demonstrated that MSI-78A maintained its activity after surface grafted onto NP, in which the amount of grafted peptide (210 µg/mL) was lower than the minimal inhibitory concentration (MIC) & minimal bactericidal concentration (MBC) of the free peptide (256 µg/mL). The high Hp–chitosan affinity4 could further improve the killing effect. Bacteria morphology was also evaluated by Transmission Electron Microscopy, demonstrating that after exposure to AMP (AMP-NP), Hp membrane had irregularities, such as the formation of vesicles and changes/release in the cytoplasm.

Furthermore, AMP-NP at bactericidal concentration were cytocompatible against human gastric adenocarcinoma cell lines (AGS & MKN74. ATCC®), in accordance with ISO 10,993–5;12.

Overall, the designed AMP-NPs boosted the activity of MSI-78A and are promising for Hp eradication.

Also, a straightforward system to obtain AMP-conjugated chitosan nanoparticles stable in gastric conditions was developed and its main advantage is the possibility to simultaneously produce, crosslink and perform the immobilization of different thiolated-compounds (due to a thiol-ene chemistry) in the same device. This system is also versatile and can be explored with other biomaterials.

References

[1] Rawla et al. Prz Gastroenterol., 2019, 14(1):26-38

[2] Sung et al. CA Cancer J Clin., 2020, 71:209–249

[3] Neshani et al. Helicobacter, 2019, p. 24

[4] Fonseca et. al. Acta Biomaterialia, 2022, 137:186-198