Stability of Plant Leaf-Derived Extracellular Vesicles According to Preservative and Storage Temperature.

Plant-derived extracellular vesicles (EVs) are capable of efficiency delivering mRNAs, miRNAs, bioactive lipids, and proteins to mammalian cells. Plant-derived EVs critically contribute to the ability of plants to defend against pathogen attacks at the plant cell surface. They also represent a novel candidate natural substance that shows potential to be developed for food, cosmetic, and pharmaceutical products. However, although plant-derived EVs are acknowledged as having potential for various industrial applications, little is known about how their stability is affected by storage conditions. In this study, we evaluated the stability of Dendropanax morbifera leaf-derived extracellular vesicles (LEVs) alone or combined with the preservatives, 1,3-butylene glycol (to yield LEVs-1,3-BG) or TMO (LEVs-TMO). We stored these formulations at -20, 4, 25, and 45 °C for up to 4 weeks, and compared the stability of fresh and stored LEVs. We also assessed the effect of freeze-thawing cycles on the quantity and morphology of the LEVs. We found that different storage temperatures and number of freeze-thawing cycles altered the stability, size distribution, protein content, surface charge, and cellular uptake of LEVs compared to those of freshly isolated LEVs. LEVs-TMO showed higher stability when stored at 4 °C, compared to LEVs and LEVs-1,3-BG. Our study provides comprehensive information on how storage conditions affect LEVs and suggests that the potential industrial applications of plant-derived EVs may be broadened by the use of preservatives.

Anti-melanogenic effects of extracellular vesicles derived from plant leaves and stems in mouse melanoma cells and human healthy skin.

Consumer interest in cosmetic industry products that produce whitening effects has increased demand for agents that decrease melanin production. Many such anti-melanogenic agents are associated with side effects, such as contact dermatitis and high toxicity, and also exhibit poor skin penetration. Considerable recent research has focused on plant-derived products as alternatives to chemotherapeutic agents that possess fewer side effects. In the current study, we investigated the anti-melanogenic effects of extracellular vesicles (EVs) extracted from leaves and stems of Dendropanax morbifera. Using spectrophotometric and biochemical approaches, we found that leaf-derived extracellular vesicles (LEVs) and stem-derived extracellular vesicles (SEVs) reduced melanin content and tyrosinase (TYR) activity in the B16BL6 mouse melanoma cell line in a concentration-dependent manner. An electron microscopy analysis further confirmed that LEVs and SEVs induce a concentration-dependent decrease in melanin content in melanoma cells. Both LEVs and SEVs exerted a greater whitening effect on melanoma cells than arbutin, used as a positive control, with LEVs producing the greater effect. Notably, neither LEVs nor SEVs induced significant cytotoxicity. We also examined the effects of plant-derived EVs on the expression of tyrosinase-related proteins (TRPs) in melanoma cells. LEVs inhibited expression of melanogenesis-related genes and proteins, including microphthalmia-associated transcription factor (MITF), TYR, TRP-1 and TRP-2. In a human epidermis model, LEVs exerted a stronger inhibitory effect on melanin production than arbutin. Collectively, our data suggest that LEVs from D. morbifera may be a novel candidate natural substance for use as an anti-melanogenic agent in cosmeceutical formulations.