Kaempferia parviflora Extracellular Vesicle Loaded with Clarithromycin for the Treatment of Helicobacter pylori Infection

Abstract

Purpose

Kaempferia parviflora extracellular vesicles (KPEVs) have been reported as promising nanovesicles for drug delivery. This study aimed to load clarithromycin (CLA) into KPEVs (KPEVS-CLA) and determine the physical properties, drug-releasing efficiency, gastric cell uptake, anti-H. pylori activities, and anti-inflammatory responses in comparison with free CLA and KPEVs.

Methods

The size and surface charge of KPEVs-CLA were evaluated using dynamic light scattering and visualized using a transmission electron microscope. The encapsulation efficiency (EE%), loading capacity (LC%), and drug release of KPEVs-CLA were examined using HPLC. Anti-H. pylori growth and anti-adhesion were evaluated. IL-8 gene expression, NF-κB signaling proteins, and anti-inflammatory profiles were examined using qRT-PCR, Western blotting, and Bio-Plex immunoassay, respectively. Anti-chemotaxis was then examined using a Transwell assay.

Results

KPEVs-CLA were intact and showed a negative surface charge similar to that of KPEVs. However, slightly enlarged KPEVs were observed. CLA was successfully loaded into KPEVs with EE of 93.45% ± 2.43%, LC of 9.3% ± 3.02%. CLA release in the PBS and gastric mimic buffer with Fickian diffusion (n ≤ 0.43) according to Korsmeyer-Peppas kinetic model (R²=0.98). KPEVs-CLA was localized in the gastric cells’ cytoplasm and perinuclear region. Anti-H. pylori growth and anti-H. pylori adhesion of KPEVs-CLA were compared with those of free CLA with no cytotoxicity to adenocarcinoma gastric cells. KPEVs-CLA significantly reduced IL-8, G-CSF, MIP-1α, and MIP-1β levels. Moreover, KPEVs-CLA showed a superior effect over CLA in reducing G-CSF, MIP-1α, and NF-κB phosphorylation and monocyte chemotactic activities.

Conclusion

KPEVs serve as potential carriers of CLA. They exhibited a higher efficiency in inhibiting gastric cell inflammation mediated by H. pylori infection than free CLA. The establishment of KPEVs-CLA as a nanodrug delivery model for H. pylori treatment could be applied to other plant extracellular vesicles or loaded with other cancer drugs for gastric cancer treatment.

Keywords:

• extracellular vesicle
• clarithromycin
• Kaempferia parviflora
• Helicobacter pylori
• inflammation

Abbreviations

CFU, colony-forming unit; CLA, clarithromycin; CLSI, Clinical and Laboratory Standards Institute; EE, encapsulation efficiency; EVs, extracellular vesicles; FBS, fetal bovine serum; HP, Helicobacter pylori; HPLC, high-performance liquid chromatography; kDa, kilodalton; KP, Kaempferia parviflora; KPEVs, Kaempferia parviflora extracellular vesicles; KPEVs-CLA, Kaempferia parviflora extracellular vesicles loaded with CLA; LC, loading capacity; MBC, minimum bactericidal concentration; MOI, multiplicity of infection; MWCO, molecular weight cut off; PBS, phosphate-buffered saline; PDEVs, plant-derived extracellular vesicles; PDI, polydispersity index; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Acknowledgments

We would like to thank Professor Richard Ferrero for giving us the opportunity to participate in the research exchange program.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This work was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. program (Grant No. PHD/0217/2559), 90th Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund, GCUGR1125633043D) and Thailand Science Research and Innovation (TSRI) (CU_FRB640001_01_37_1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.