Fernanda Araujo Fernandes^1,2, Julia Eduarda Charleaux ², Sarah Beatriz de Fucio Barros^1,2, Abel Sana^1,2, Marcel Ivan Ramirez^1,2

Introduction

Extracellular vesicles (EVs) are lipid-based nanoparticles secreted by various cell lines, playing a crucial role in intercellular communication and the modulation of physiological and pathological processes. Recently, there has been growing interest in plant-derived EVs due to their therapeutic potential. They are secreted by different parts of plants, including fruits, roots, and leaves, and contain lipids, proteins, RNAs, and other metabolites. Plant-derived extracellular vesicles (EVs) have emerged as promising natural nanocarriers due to their biocompatibility and potential anti-inflammatory and immunomodulatory properties. Ginger (Zingiber officinale) has shown therapeutic potential in models of intestinal inflammation, but other medicinal plants such as turmeric (Curcuma longa) and boldo (Peumus boldus) may also produce EVs with biological activity. We aimed to establish a reproducible method for isolating and characterising EVs from ginger that can be adapted for other plant sources. Additionally, we intend to test the functional effects of these vesicles in a 3D Caco-2 cell culture model established in our laboratory, focusing on their capacity to maintain or disrupt epithelial integrity during interactions between intestinal cells and pathogens.

Materials and Methods

Fresh ginger rhizomes (25 g) were peeled, washed, and macerated in 10 mL PBS, then hydrated for 30 min. The extract was filtered and subjected to sequential centrifugation (3,000 × g for 5 and 10 min at 10 °C; 4,000 × g for 30 min at 4 °C; 20,000 × g for 2 h at 4 °C), followed by 0.22 µm filtration. Final pellets were resuspended in 150 µL PBS. Vesicle size and concentration were analysed by NTA, and protein content was measured using the Qubit™ Protein Assay Kit. This workflow is being extended to turmeric and boldo, with vesicles applied to 3D Caco-2 cultures to assess effects on epithelial integrity (TEER, confocal microscopy) during pathogen interaction.

Results          

Ginger-derived EVs showed a concentration of 1.4 × 10⁸ particles per gram of fresh tissue. The average size was within the expected range for small EVs (exosome-like), and the protein content was 2.28 mg per gram of ginger, resulting in a calculated protein-to-vesicle ratio of ~1.6 × 10⁻⁵ µg per vesicle. The vesicles were stable in PBS and suitable for application in cell culture models. Initial microscopy data from Caco-2 3D cultures indicate good cellular uptake of the ginger EVs.

Conclusion

We successfully established a simple and effective method for isolating extracellular vesicles from Zingiber officinale. The approach yields stable, protein-rich vesicles, and is now being extended to other medicinal plants such as turmeric and boldo. Preliminary results suggest these vesicles are efficiently internalised by epithelial cells in 3D culture. Ongoing studies will determine whether these plant-derived EVs contribute to epithelial protection or disruption during host–pathogen interactions. This platform may support the development of novel plant-based therapeutic strategies for chronic intestinal inflammation and infection.

Agradecimentos: Universidade Federal do Paraná / Instituto Carlos Chagas - Fiocruz Paraná