# In vitro study on anti-Helicobacter pylori effects of DL-3-n-butylphthalide-loaded silk fibroin nanoparticles

## TL;DR

This study shows that DL-3-n-butylphthalide (NBP) in a nanoparticle form effectively fights Helicobacter pylori, a bacteria linked to stomach cancer, by disrupting its structure and reducing its harmful effects.

## Contribution

The study introduces a novel silk fibroin nanoparticle delivery system for NBP, demonstrating enhanced antibacterial activity and multi-mechanistic effects against H. pylori.

## Key findings

- NBP-loaded silk fibroin nanoparticles showed improved antibacterial activity with lower MIC and MBC values compared to free NBP.
- NBP and its nanoparticles reduced H. pylori virulence by downregulating key toxin and motility-related genes.
- Metabolomics and network pharmacology identified five key metabolic pathways and seven core targets involved in NBP's anti-H. pylori action.

## Abstract

Helicobacter pylori (H. pylori) primarily colonizes the gastric mucosal epithelium in humans and is considered the strongest risk factor for gastric cancer. Current clinical eradication regimens rely on proton pump inhibitor (PPI)-based triple or quadruple antibiotic therapies. However, rising antibiotic resistance and reinfection rates greatly compromise their efficacy, underscoring the need for effective alternative treatments. This study identified potent in vitro anti-H. pylori activity of DL-3-n-Butylphthalide (NBP). Using silk fibroin as a carrier, a nanoparticle delivery system was constructed to further investigate the antibacterial potential and mechanism of NBP. Uniform and stable nanoparticles were successfully prepared, exhibiting an encapsulation efficiency of 45.80 ± 1.5% and drug loading of 15.27 ± 0.8%. Subsequently, Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) were performed, and the results collectively confirmed the successful encapsulation of NBP. The minimum inhibitory concentrations (MICs) of NBP against standard strains and clinical isolates of H. pylori ranged from 4 to 16 μg/mL, and the minimum bactericidal concentrations (MBCs) were between 8 and 32 μg/mL. The nanoparticles exhibited enhanced antibacterial and bactericidal activities, with MICs ranging from 2.5–10 μg/mL and MBCs from 5 to 20 μg/mL. When used in combination with four antibiotics, the interaction showed additive, irrelevant effects with no antagonistic phenomenon. Both NBP and its nanoparticles downregulated the expression of cytotoxin-related genes, vacuolating toxin (cagA, vacA), flagellum genes (flaA, flaB), and urease genes (ureA-B, ureE-H, nixA) mRNA, inhibited H. pylori motility and urease activity, destroyed the bacterial structure, and significantly reduced the expression of relevant virulence proteins. Integrated untargeted metabolomics and network pharmacology analysis further revealed five key metabolic pathways and seven core targets underlying the anti-H. pylori action of NBP. These findings highlight the promising role of NBP, particularly in a nanoformulation, as a potential multi-mechanistic therapeutic agent against H. pylori infection.

Diagram illustrating the preparation and effects of NBP nanoparticles on H. pylori. It shows a person with a magnified view of the stomach, nanoparticles preparation using Bombyx mori cocoons, and the self-assembly process into NBP-NPs. The process involves disassembling and destroying H. pylori structure with a microscopic image showing damaged bacteria. Below, it depicts metabolomic analysis, differential metabolite screening, and KEGG enrichment analysis, with charts showing data on metabolite sets and metabolic pathways.