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

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-1ß 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.

Characterizing Kaempferia parviflora extracellular vesicles, a nanomedicine candidate.

Plant-derived extracellular vesicles (EVs) are a promising candidate for nanomedicine delivery due to their bioactive cargos, high biocompatibility to human cells, biodegradability, low cytotoxicity, and potential for large-scale production. However, the research on EVs derived from medicinal plants is very limited. In this study, Kaempferia parviflora extracellular vesicles (KPEVs) were isolated by differential and sucrose density gradient centrifugation, and their size, morphology, and surface charge were characterized using transmission electron microscopy and dynamic light scattering. The biological properties of KPEVs, including their bioactive compound composition, gastric uptake, cytotoxicity, acid tolerance, and storage stability, were also examined. In addition, KPEVs had an average and uniform size of 200-300 nm and a negative surface charge of 14.7 ± 3.61 mV. Moreover, 5,7-dimethoxyflavone, the major bioactive compound of KP, was packaged into KPEVs. Meanwhile, KPEVs were resistant to gastric digestion and stably maintained at -20°C and -80°C for 8 weeks with no freeze-thaw cycle. The lipid hydrolysis during EVs storage at room temperature and 4°C were also demonstrated for the first time. Furthermore, the labeled KPEVs were internalized into adenocarcinoma gastric cells, and the cell viability was reduced in a dose-dependent manner, according to the results of the thiazolyl blue tetrazolium assay. Our study supports the potential application of KPEVs as a vehicle for anticancer or oral drugs.

Ethyl acetate extract of Kaempferia parviflora inhibits Helicobacter pylori-associated mammalian cell inflammation by regulating proinflammatory cytokine expression and leukocyte chemotaxis.

BACKGROUND: Kaempferia parviflora (KP) has been used in traditional Thai medicine to cure gastrointestinal disorders since ancient times. Helicobacter pylori is an initiating factor in gastric pathogenesis via activation of massive inflammation, the cumulative effect of which leads to gastric disease progression, including gastric carcinogenesis. Accordingly, the effect of a crude ethyl acetate extract of KP (CEAE-KP) on proinflammatory cytokine production and cell chemotaxis was the focus of this study. METHODS: The cytotoxicity of CEAE-KP (8-128 µg/ml) on AGS (gastric adenocarcinoma) cells was determined at 6, 12 and 24 h using an MTT assay. The effect of CEAE-KP on H. pylori-induced interleukin (IL)-8 production by AGS cells was evaluated by ELISA and RT-PCR. The effect of CEAE-KP on monocyte and neutrophil chemotaxis to H. pylori soluble protein (sHP) and IL-8, respectively, was determined using a Boyden chamber assay with THP-1 or HL-60 cells. RESULTS: CEAE-KP reduced AGS cell viability in a concentration- and time-dependent manner, but at 8-16 µg/ml, it was not cytotoxic after 6-24 h of exposure. Coculture of AGS cells with CEAE-KP at a noncytotoxic concentration of 16 µg/ml and H. pylori reduced IL-8 secretion by ~ 60% at 12 h, which was consistent with the decreased level of mRNA expression, and inhibited neutrophil chemotaxis to IL-8. sHP (100 ng/ml) induced marked monocyte chemoattraction, and this was decreased by ~ 60% by CEAE-KP. CONCLUSION: CEAE-KP might serve as a potent alternative medicine to ameliorate the inflammation mediated by H. pylori infection.