Materials and methods

6. Cellular uptake assays

Internalization of EVs into cells was monitored by first staining LEVs and SEVs with lipophilic DiI (MOP-D -3911) (Invitrogen, Carlsbad, CA, USA). After treating cells with stained EVs for 1, 3, 6, 12, 24, and 48 h, the growth medium was removed and cells were fixed with 4% paraformaldehyde (Wako, Japan). Hoechst 33342 (Invitrogen) was added and the cells were incubated for 15 min at room temperature to stain the nuclei (blue). Finally, cells were washed with PBS containing 1% bovine serum albumin, and fluorescence (red and blue) was observed under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). At least three fields were selected and analyzed using ImageJ software.

7. Measurement of melanin content

To assess melanin content, B16BL6 melanoma cells were first inoculated in 24-well plates (5 × 104 cells/well) in a volume of 500 μL. After 24 h of incubation, cells were treated with 100 nM α-MSH (Sigma-Aldrich, St. Louis, MO) alone or 50 μL LEVs and SEVs at 1, 5, and 10 µg/mL for 48 hours. After treatment, the cells were washed with PBS and then incubated with 2.5% trypsin (Gibco, Thermo Fisher Scientific) for isolation. Cell microspheres were dissolved in 1n sodium hydroxide solution (Sigma-Aldrich) containing 1% DMSO (Sigma-Aldrich) for 1 hour at 80°C. The cell lysates were transferred to a 96-well plate and the melanin content was determined by measuring the absorbance at 405 nm using an enzyme marker (BioTek). Melanin content was determined using a melanin standard curve constructed from 0-100 µg/mL synthetic melanin solution (Sigma-Aldrich) (extracellular vesicles Figure as follows). Melanin content was calculated by comparison with controls.

8. Tyrosinase activity assay

TYR activity was measured using L-DOPA oxidase activity. B16BL6 cells were inoculated in α-MEM medium containing 100 nM Sigma-Aldrich α-MSH (500 μL) and cultured at a density of 1 × 105 cells/well in 24-well culture plates. After treating cells with 50 μL of LEVs and SEVs at concentrations of 10, 50, and 100 µg/mL for 24 h, cells were washed with PBS and lysed with 1% Triton X-100 (Sigma-Aldrich). The lysed cells were incubated at - 80°C for 30 min and then lyophilized and stored at RT for 10 min. The resulting samples were clarified by centrifugation at 12,000 × g for 15 min, after which 2 mg/mL L-DOPA (Sigma-Aldrich) was added and incubated at 37°C for 1 h. Absorbance was then measured at 490 nm using an enzyme marker (BioTek). The absorbance was then measured at 490 nm using BioTek.

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9. Western blot analysis

Protein levels associated with the anti-melanogenic pathway were determined intracellularly by Western blot analysis of whole cell extracts. A volume of 500 μL of B16BL6 mouse melanoma cells was inoculated in 24-well plates (1 × 105 cells/well). Cells were lysed by incubation in RIPA buffer (Thermo Fisher Scientific) for 20 min in the presence of protease inhibitor mixture (Roche, Germany) and treated with 50 μL of 10, 50, and 100 μ g/mL of EVs for 24 h. The cell lysates were centrifuged at 17 709 g for 15 min and the protein concentration in the resulting lysates was determined using a BCA assay kit (Thermo Fisher Scientific). Equal amounts of protein (10-20 μg/sample) were loaded into the wells of Bolt 4-12% Bis-Tris Plus gels (Invitrogen) and electrotransferred to PVDF (polyvinylidene fluoride) membranes (GE Healthcare, Chicago, IL, USA). The membranes were washed with Tris-buffered saline containing 0.2% (v/v) ten -20 (TBST), closed with TBST containing 5% (w/v) skim milk (Gibco, Thermo Fisher Scientific) for 1 h at RT, and then incubated at 4°C in a primary antibody solution diluted with 1% (w/v) skim milk. Incubation was carried out overnight. Primary antibodies used included anti-TRP-1 (1:1000), anti-TRP-2 (1:1000), and anti-MITF(1:1000) antibodies from Abcam, Cambridge, UK; anti-TYR (1:500) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin antibody (1:500; Santa Cruz) was used for standardization at the protein level. Primary antibodies were detected with horseradish peroxidase (HRP)-labeled secondary antibody from Genetex (Irvine, CA, USA). Membranes were washed with TBST and incubated for 1 h at RT with a 1:2000 dilution of the secondary antibody in TBST. The signal generated after washing the membrane with TBST and incubation with enhanced chemiluminescence (ECL) reagent (GE Healthcare) was detected using an ImageQuant 350 gel imaging system (GE Healthcare).

10. Electron microscopy detection of intracellular melanin production

Cells were processed with LEVs or SEVs and incubated for 48 h. Following treatment, cells were fixed by incubation with 200 mM cacodylate buffer containing 8% glutaraldehyde and 20% paraformaldehyde (Wako). Once dehydrated with ethanol, ultra-thin sections were prepared using a Leica EM UC7 microtome (Leica) and collected on a 200-mesh copper grid. Sections were stained with 1% uranyl acetate and lead citrate and images were acquired with a JEOL JEM 1010 transmission electron microscope (JEOL) at 80 kV.

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11. Measurement of whitening effect using a human skin model

The derm-me (Tego Science, Seoul, Korea) human skin model was used to test the whitening effect of LEVs. Human skin tissue was treated with 10 μ g/mL of lev for 7 d as a negative control. The concentration of arbutin was 70 µg/mL. Human skin tissue was dissolved in 1 N NaOH and the absorbance at 405 nm was measured using an enzyme marker (BioTek) to determine the melanin content. On day 7, skin pigmentation was compared by microscopic analysis of Fontana - mason stained samples. For Fontana-Masson staining, skin samples were fixed overnight at RT in 4% paraformaldehyde (Waco), sectioned, and embedded in paraffin wax. Paraffin-embedded sections were then heated in an oven at 60°C for 1 h to dry. The sections were then soaked three times in xylene, twice in 95% ethanol, and twice in 100% ethanol, and then washed with distilled water. Fontana-Masson staining was performed using the ammoniacal silver solution at 56°C and washed with distilled water. The slides were then fixed in 0.2% gold chloride solution and immersed in 5% sodium thiosulphate solution at room temperature. Finally, the slides were dehydrated in fresh alcohol.

12. Statistical analysis

The data obtained from the experiments are expressed as mean ± SEM. We performed experiments with four batches of activity, melanin content, and TYR activity (Supplementary Fig. S3). One-way analysis of variance (ANOVA) and Dunnett's test were performed using GraphPad Prism (GraphPad Prism Software Inc., San Diego, CA, USA). p < differences; 0.05, p < 0.01, p < 0.001 were considered statistically significant.

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Discussion

In contrast to most eukaryotic cells, plants have a complex cell wall, which poses an important barrier to the movement of exosomes. As a result, plant cells release exosomes through a series of multivesicular bodies fused to the plasma membrane. The secretory products released by plant secretions are deposited within the periplasmic gap adjacent to the plasma membrane; as they accumulate, they generate a pressure that allows the secretion to cross the cell wall barrier. As a result, secreted material, including exosomes, can be released without the need for energy. Plant-derived EVs are similar in size to or larger than naturally occurring animal cell exosomes and are similar to those observed in sunflower exosomal fluid (50-200 nm) and Arabidopsis EVs (50-300 nm). Cellular uptake efficiency is considered to be a key determinant of efficacy, as the targets of many therapeutic agents are located intracellularly. Therefore, we determined the optimal timing and concentration of uptake by melanoma cells and observed rapid transfer of LEVs and SEVs to melanoma cells within 12 hours.

TYR is a glycoprotein that catalyzes the conversion of l -tyrosinase to L-DOPA, the rate-limiting step in melanin synthesis. Both EVs exhibit an anti-melanogenic effect, but the anti-melanogenic effect of LEV is significantly greater than that of SEV.

Melanin production is regulated by α-MSH-MC1R, and inhibition of PKC is known to reduce skin and hair pigmentation. However, many genetic, biochemical, and pharmacological studies suggest that the α-MSH-MC1R signaling pathway is a major driver of melanogenesis. Although our experiments did not show a direct interaction between MITF and TYR, it was demonstrated that LEVs inhibit melanogenesis by reducing MITF expression through the UV-dependent α-MSHMC1R pathway, which in turn reduces the expression of TYR, TRP-1, and TRP-2. We hypothesize that the TRP family proteins are indirectly linked to each other, but further experiments may be required to demonstrate a direct interaction between them. Furthermore, reconstructed human skin models show that UV-induced melanin synthesis and melanin content are effectively inhibited by LEVs.

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Conclusion

Our findings suggest that LEVs are candidates for novel anti-melanogenic drugs that reduce melanogenesis by inhibiting the expression of melanin-related proteins. The results confirmed that LEVs reduced MITF and thus down-regulated TRPs in B16BL6 melanoma cells. levs and arbutin inhibited TRY by 66% and 67%, respectively. The melanin content of LEVs-treated reconstructed skin was reduced by 43% and 28% compared to the untreated negative control and arbutin as the positive control, respectively.

Based on the early findings, we believe that plant-derived EVs could be useful as an anti-melanogenic agent for the development of natural cosmetics. Further research is needed in the future to develop plant-derived EVs as an effective ingredient for skin improvement and it is necessary to demonstrate the safety of plant-derived EVs on human skin models. Our results suggest that EV can be used to reduce melanin and thus brighten the skin. However, EV may have toxic effects on the skin, and other experiments such as allergy tests or Ames tests should be performed.

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