Mol. Cells 2021; 44(10): 736-745
Published online October 15, 2021
https://doi.org/10.14348/molcells.2021.2167
© The Korean Society for Molecular and Cellular Biology
Correspondence to : OhES@ewha.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Although various marine ingredients have been exploited for the development of cosmetic products, no previous study has examined the potential of seaweed extracellular vesicles (EV) in such applications. Our results revealed that EV from Codium fragile and Sargassum fusiforme effectively decreased α-MSH-mediated melanin synthesis in MNT-1 human melanoma cells, associated with downregulation of MITF (microphthalmia-associated transcription factor), tyrosinase and TRP1 (tyrosinase-related proteins 1). The most effective inhibitory concentrations of EV were 250 μg/ml for S. fusiforme and 25 μg/ml for C. fragile, without affecting the viability of MNT-1 cells. Both EV reduced melanin synthesis in the epidermal basal layer of a three-dimensional model of human epidermis. Moreover, the application of the prototype cream containing C. fragile EV (final 5 μg/ml) yielded 1.31% improvement in skin brightness in a clinical trial. Together, these results suggest that EV from C. fragile and S. fusiforme reduce melanin synthesis and may be potential therapeutic and/or supplementary whitening agents.
Keywords extracellular vesicles, melanin synthesis, seaweed, skin epidermis
Melanin, which is produced in the melanosomes of melanocytes, is a pigment that determines the color of skin, eyes, and hair (Hearing, 2011). Epithelial melanin is transported to neighboring keratinocytes and protects the skin from UV damage (Archambault et al., 1995; Brenner and Hearing, 2008), and abnormal melanogenesis is closely associated with various disorders of hyperpigmentation (e.g., melasma, postinflammatory hyperpigmentation, freckles, and lentigines) and hypopigmentation (e.g., vitiligo and albinism) (Bastonini et al., 2016; Nicolaidou and Katsambas, 2014).
Various systemic agents, such as arbutin, kojic acid, hydroquinone, etc., are commonly used to cure these disorders (Parvez et al., 2006). Unfortunately, topical treatments show limited efficacy. The dermal epidermis has five major keratinocyte layers: the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum (Baroni et al., 2012). The thick structure of the skin epidermis limits the ability of topical agents to penetrate/absorb into the skin, accounting for their generally low efficacy. In addition, these drugs have been associated with negative side effects, such as dryness, redness, inflammation, and even a risk of cancer (Pillaiyar et al., 2017; Solano et al., 2006). Therefore, it would be beneficial to develop efficient and safe skin-permeating agents. In this context, many researchers have become interested in using extracellular vesicles (EVs) as an alternative agent (Azmi et al., 2013; van den Boorn et al., 2011).
EVs are nano-size membrane vesicles that carry various biomolecules, such as lipids, proteins, and RNAs (Naval and Chandra, 2019). Most forms of life, from microorganisms to higher eukaryotes, produce these vesicles and use them as a means of communicating between cells (Valadi et al., 2007) like intracellular-cellular vesicle in trafficking (Kwon et al., 2020). The physical properties of EVs make them useful carriers of biomolecules. In fact, recent studies have shown that EVs and/or EV-inspired vesicles can be therapeutically effective as natural drug delivery vehicles (Antimisiaris et al., 2018; Gomari et al., 2018). Therefore, it is very likely that EVs could improve the skin penetration of active ingredients.
Marine resources and compounds have been found to have various biological activities, including anti-cancer and anti-inflammatory activities. For example,
The polyclonal antibody against tyrosinase and TRP-1, and the monoclonal antibody against β-actin were purchased from Santa Cruz Biotechnology (USA). The polyclonal antibody against MITF was purchased from Proteintech (USA). The α-MSH, L-DOPA, kojic acid, and arbutin were purchased from Sigma (USA).
MNT-1 cells (a human melanoma cell line) were cultured in minimal essential medium (MEM; WelGene, Korea) supplemented with 20% fetal bovine serum (Gibco, USA), 10% Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, USA), 20 mM HEPES, and 50 μg/ml gentamicin (Sigma). The cells were maintained at 37°C in a humidified 5% CO2 atmosphere.
EVs were purified using ultracentrifugation following a standard protocol (Lane et al., 2015). Briefly, 1× phosphate-buffered saline (PBS) was added to semi-dried and sliced seaweeds (1:10, v:w) and hand-held food blenders were used to thoroughly disrupt tissue connections. The samples were centrifuged at 5,000 ×
Melanin contents were measured as described in a previous study (Jung et al., 2016). Cells were washed twice with PBS, detached by incubation with trypsin/EDTA, and collected by centrifugation at 1,000 x
The intracellular tyrosinase activity assay was performed as previously described. Briefly, cells were plated to coverslips in 12-well plates, fixed with 3.5% paraformaldehyde for 10 min, washed with PBS, and incubated in sodium phosphate buffer with 10 mM L-DOPA for 3 h at 37°C. The cells were then washed with PBS, the coverslips were mounted on glass slides, and the slides were observed by microscopy (Jung et al., 2014).
Cells were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, and 2 mM Na3VO4, pH 8.0) containing several protease inhibitors (1 μg/ml aprotinin, 1 μg/ml antipain, 1 mM dithiothreitol, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, and 20 μg/ml phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 13,000 rpm for 15 min at 4°C, denatured with sample buffer, boiled, and analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose blotting membranes (Amersham Biosciences, USA) and probed with the appropriate antibodies. Signals were detected with an Odyssey CLx imager (LI-COR Biosciences, USA) and analyzed using the Image Studio Lite software (LI-COR Biosciences).
Cell proliferation was measured using the MTT [3-(4,5-dimethythiazol-2-yl)2,5-diphenyltetrazolium bromide] assay. Briefly, MNT-1 cells were harvested with 0.05% trypsin/EDTA and seeded to 96-well plates at 5 × 103 cells/well. After incubation for 48 h, medium containing 0.5 mg/ml MTT (100 μl; Sigma) was added to each well, and the cells were incubated for 1 h. The medium was then removed and 100 μl of acidic isopropanol (90% isopropanol, 0.5% SDS, 25 mM NaCl) was added to each well. The mean concentration of absorbance at 570 nm in each sample set was measured using a 96-well microtiter plate reader (Dynatech, USA).
MelanoDerm (MEL-300-B) and the maintenance medium for EPI-100-NMM-113 were purchased from MatTek Corporation (USA). To measure melanin content, MelanoDerm tissue was incubated in 12-well plates containing the pre-warmed maintenance medium according to the manufacturer’s recommendations. The medium was changed every other day for 14 days (Park et al., 2018). For H&E staining, a fresh scalpel blade was used to separate the MelanoDerm tissues from the inserts at specified time points. The tissues were immediately fixed in 4% formalin and embedded in paraffin. Each paraffin block was sequentially sectioned at 4 μm, and the sections were mounted on slides and stained using H&E (Abcam, UK). The images were captured at a magnification of ×40, and a minimum of 20 fields per section was assessed using a color image analyzer (Leica DM1000 LED; Leica Microsystems, Germany) (Park et al., 2018).
Healthy Korean (n = 35) between the ages of 29 and 59 years (average 47) were enrolled in the study (Korea Institute of Dermatological Sciences, Korea). For the irritation test, a skin patch containing the test sample was applied to the test site for 24 h. The skin patch was then removed, and the test site was graded for irritation at 30 min, 24 h, and 48 h post-removal using the irritation grading scale of Evaluation followed the SOP of Korea Institute of Dermatological Sciences (Dores and Baron, 2011; Jin et al., 2018; Spínola et al., 2013). Healthy Korean women (n = 21) between the ages of 20 and 50 years (average 46) were enrolled in the study (Korea Institute of Dermatological Sciences). For the skin whitening efficacy test, the test products were each applied to half of the participant’s face (on the right for placebo cream, on the left for test cream) once a day for 4 weeks. The skin was imaged by VISIA-CA (VISIA Complexion Analysis; Canfield Scientific, Inc., USA) and the skin whitening was evaluated with a spectrophotometer (CM-2600D; Minolta, Japan).
Data are presented as the means from three independent experiments. Statistical analyses were performed using the unpaired Student’s
We first isolated EVs from five different Korean seaweeds:
To further characterize the isolated EVs from
Next, we investigated whether EVs from
L-3,4-dihydroxyphenylalanine (L-DOPA) staining showed that the level of intracellular tyrosinase activity was decreased in seaweed EV-treated MNT-1 cells (Fig. 3B). α-MSH treatment increased the presence of dark spots (pigment, induced by active tyrosinase) in MNT-1 cells, but EVs derived from
To confirm the anti-melanogenic activity of the EVs, we used an artificial skin model that closely mimics human skin (Fig. 4). MelanoDerm tissues models were treated with 50 μl of PBS (control), 50 µg of
To further confirm the anti-melanogenic activity of
To further investigate the contribution of the EV to the anti-melanogenic effect, a crude extract of
Many tyrosinase inhibitors have been developed as whitening agents, but some are limited in their usefulness due to low potency and the potential for side effects. For example,
We do not know yet how
In summary, we herein show for the first time that
This work was supported by the project titled ‘development of skin functional material using seaweed extracellular vesicle’, funded by the Ministry of Oceans and Fisheries, Korea (20190067). We thank the electron microscopy core facility at the ConveRgence mEDIcine research cenTer (CREDIT), Asan Medical Center, Seoul, Korea, for support and instrumentation. This electron microscopy was performed by the core facility at the ConveRgence mEDIcine research cenTer (CREDIT), Asan Medical Center, Seoul, Korea.
Written informed consent was obtained from the participants for publication of this article and accompanying images.
H.C. (Heesung Chung), B.J., S.Y., and E.S.O. wrote the manuscript. H.C. (Heesung Chung), B.J., H.J, H.K.S., E.P., and K.J. performed the experiments. H.C. (Han Choe) and H.S.C. helped analyzed EV. S.Y. provided reagents. H.C. (Heesung Chung), B.J., S.Y., and E.S.O. designed the experiments and expertise and feedback.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2021; 44(10): 736-745
Published online October 31, 2021 https://doi.org/10.14348/molcells.2021.2167
Copyright © The Korean Society for Molecular and Cellular Biology.
Bohee Jang1,5 , Heesung Chung1,5
, Hyejung Jung1
, Hyun-Kuk Song1
, Eunhye Park1
, Hack Sun Choi2
, Kyuhyun Jung3
, Han Choe4
, Sanghwa Yang3
, and Eok-Soo Oh1,*
1Department of Life Sciences, The Research Center for Cellular Homeostasis, Ewha Womans University, Seoul 03760, Korea, 2Subtropical/Tropical Organism Gene Bank, Jeju National University, Jeju 63243, Korea, 3ExoMed, Inc., Seoul 01795, Korea, 4Department of Physiology, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea, 5These authors contributed equally to this work.
Correspondence to:OhES@ewha.ac.kr
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Although various marine ingredients have been exploited for the development of cosmetic products, no previous study has examined the potential of seaweed extracellular vesicles (EV) in such applications. Our results revealed that EV from Codium fragile and Sargassum fusiforme effectively decreased α-MSH-mediated melanin synthesis in MNT-1 human melanoma cells, associated with downregulation of MITF (microphthalmia-associated transcription factor), tyrosinase and TRP1 (tyrosinase-related proteins 1). The most effective inhibitory concentrations of EV were 250 μg/ml for S. fusiforme and 25 μg/ml for C. fragile, without affecting the viability of MNT-1 cells. Both EV reduced melanin synthesis in the epidermal basal layer of a three-dimensional model of human epidermis. Moreover, the application of the prototype cream containing C. fragile EV (final 5 μg/ml) yielded 1.31% improvement in skin brightness in a clinical trial. Together, these results suggest that EV from C. fragile and S. fusiforme reduce melanin synthesis and may be potential therapeutic and/or supplementary whitening agents.
Keywords: extracellular vesicles, melanin synthesis, seaweed, skin epidermis
Melanin, which is produced in the melanosomes of melanocytes, is a pigment that determines the color of skin, eyes, and hair (Hearing, 2011). Epithelial melanin is transported to neighboring keratinocytes and protects the skin from UV damage (Archambault et al., 1995; Brenner and Hearing, 2008), and abnormal melanogenesis is closely associated with various disorders of hyperpigmentation (e.g., melasma, postinflammatory hyperpigmentation, freckles, and lentigines) and hypopigmentation (e.g., vitiligo and albinism) (Bastonini et al., 2016; Nicolaidou and Katsambas, 2014).
Various systemic agents, such as arbutin, kojic acid, hydroquinone, etc., are commonly used to cure these disorders (Parvez et al., 2006). Unfortunately, topical treatments show limited efficacy. The dermal epidermis has five major keratinocyte layers: the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum (Baroni et al., 2012). The thick structure of the skin epidermis limits the ability of topical agents to penetrate/absorb into the skin, accounting for their generally low efficacy. In addition, these drugs have been associated with negative side effects, such as dryness, redness, inflammation, and even a risk of cancer (Pillaiyar et al., 2017; Solano et al., 2006). Therefore, it would be beneficial to develop efficient and safe skin-permeating agents. In this context, many researchers have become interested in using extracellular vesicles (EVs) as an alternative agent (Azmi et al., 2013; van den Boorn et al., 2011).
EVs are nano-size membrane vesicles that carry various biomolecules, such as lipids, proteins, and RNAs (Naval and Chandra, 2019). Most forms of life, from microorganisms to higher eukaryotes, produce these vesicles and use them as a means of communicating between cells (Valadi et al., 2007) like intracellular-cellular vesicle in trafficking (Kwon et al., 2020). The physical properties of EVs make them useful carriers of biomolecules. In fact, recent studies have shown that EVs and/or EV-inspired vesicles can be therapeutically effective as natural drug delivery vehicles (Antimisiaris et al., 2018; Gomari et al., 2018). Therefore, it is very likely that EVs could improve the skin penetration of active ingredients.
Marine resources and compounds have been found to have various biological activities, including anti-cancer and anti-inflammatory activities. For example,
The polyclonal antibody against tyrosinase and TRP-1, and the monoclonal antibody against β-actin were purchased from Santa Cruz Biotechnology (USA). The polyclonal antibody against MITF was purchased from Proteintech (USA). The α-MSH, L-DOPA, kojic acid, and arbutin were purchased from Sigma (USA).
MNT-1 cells (a human melanoma cell line) were cultured in minimal essential medium (MEM; WelGene, Korea) supplemented with 20% fetal bovine serum (Gibco, USA), 10% Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, USA), 20 mM HEPES, and 50 μg/ml gentamicin (Sigma). The cells were maintained at 37°C in a humidified 5% CO2 atmosphere.
EVs were purified using ultracentrifugation following a standard protocol (Lane et al., 2015). Briefly, 1× phosphate-buffered saline (PBS) was added to semi-dried and sliced seaweeds (1:10, v:w) and hand-held food blenders were used to thoroughly disrupt tissue connections. The samples were centrifuged at 5,000 ×
Melanin contents were measured as described in a previous study (Jung et al., 2016). Cells were washed twice with PBS, detached by incubation with trypsin/EDTA, and collected by centrifugation at 1,000 x
The intracellular tyrosinase activity assay was performed as previously described. Briefly, cells were plated to coverslips in 12-well plates, fixed with 3.5% paraformaldehyde for 10 min, washed with PBS, and incubated in sodium phosphate buffer with 10 mM L-DOPA for 3 h at 37°C. The cells were then washed with PBS, the coverslips were mounted on glass slides, and the slides were observed by microscopy (Jung et al., 2014).
Cells were lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, and 2 mM Na3VO4, pH 8.0) containing several protease inhibitors (1 μg/ml aprotinin, 1 μg/ml antipain, 1 mM dithiothreitol, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, and 20 μg/ml phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 13,000 rpm for 15 min at 4°C, denatured with sample buffer, boiled, and analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose blotting membranes (Amersham Biosciences, USA) and probed with the appropriate antibodies. Signals were detected with an Odyssey CLx imager (LI-COR Biosciences, USA) and analyzed using the Image Studio Lite software (LI-COR Biosciences).
Cell proliferation was measured using the MTT [3-(4,5-dimethythiazol-2-yl)2,5-diphenyltetrazolium bromide] assay. Briefly, MNT-1 cells were harvested with 0.05% trypsin/EDTA and seeded to 96-well plates at 5 × 103 cells/well. After incubation for 48 h, medium containing 0.5 mg/ml MTT (100 μl; Sigma) was added to each well, and the cells were incubated for 1 h. The medium was then removed and 100 μl of acidic isopropanol (90% isopropanol, 0.5% SDS, 25 mM NaCl) was added to each well. The mean concentration of absorbance at 570 nm in each sample set was measured using a 96-well microtiter plate reader (Dynatech, USA).
MelanoDerm (MEL-300-B) and the maintenance medium for EPI-100-NMM-113 were purchased from MatTek Corporation (USA). To measure melanin content, MelanoDerm tissue was incubated in 12-well plates containing the pre-warmed maintenance medium according to the manufacturer’s recommendations. The medium was changed every other day for 14 days (Park et al., 2018). For H&E staining, a fresh scalpel blade was used to separate the MelanoDerm tissues from the inserts at specified time points. The tissues were immediately fixed in 4% formalin and embedded in paraffin. Each paraffin block was sequentially sectioned at 4 μm, and the sections were mounted on slides and stained using H&E (Abcam, UK). The images were captured at a magnification of ×40, and a minimum of 20 fields per section was assessed using a color image analyzer (Leica DM1000 LED; Leica Microsystems, Germany) (Park et al., 2018).
Healthy Korean (n = 35) between the ages of 29 and 59 years (average 47) were enrolled in the study (Korea Institute of Dermatological Sciences, Korea). For the irritation test, a skin patch containing the test sample was applied to the test site for 24 h. The skin patch was then removed, and the test site was graded for irritation at 30 min, 24 h, and 48 h post-removal using the irritation grading scale of Evaluation followed the SOP of Korea Institute of Dermatological Sciences (Dores and Baron, 2011; Jin et al., 2018; Spínola et al., 2013). Healthy Korean women (n = 21) between the ages of 20 and 50 years (average 46) were enrolled in the study (Korea Institute of Dermatological Sciences). For the skin whitening efficacy test, the test products were each applied to half of the participant’s face (on the right for placebo cream, on the left for test cream) once a day for 4 weeks. The skin was imaged by VISIA-CA (VISIA Complexion Analysis; Canfield Scientific, Inc., USA) and the skin whitening was evaluated with a spectrophotometer (CM-2600D; Minolta, Japan).
Data are presented as the means from three independent experiments. Statistical analyses were performed using the unpaired Student’s
We first isolated EVs from five different Korean seaweeds:
To further characterize the isolated EVs from
Next, we investigated whether EVs from
L-3,4-dihydroxyphenylalanine (L-DOPA) staining showed that the level of intracellular tyrosinase activity was decreased in seaweed EV-treated MNT-1 cells (Fig. 3B). α-MSH treatment increased the presence of dark spots (pigment, induced by active tyrosinase) in MNT-1 cells, but EVs derived from
To confirm the anti-melanogenic activity of the EVs, we used an artificial skin model that closely mimics human skin (Fig. 4). MelanoDerm tissues models were treated with 50 μl of PBS (control), 50 µg of
To further confirm the anti-melanogenic activity of
To further investigate the contribution of the EV to the anti-melanogenic effect, a crude extract of
Many tyrosinase inhibitors have been developed as whitening agents, but some are limited in their usefulness due to low potency and the potential for side effects. For example,
We do not know yet how
In summary, we herein show for the first time that
This work was supported by the project titled ‘development of skin functional material using seaweed extracellular vesicle’, funded by the Ministry of Oceans and Fisheries, Korea (20190067). We thank the electron microscopy core facility at the ConveRgence mEDIcine research cenTer (CREDIT), Asan Medical Center, Seoul, Korea, for support and instrumentation. This electron microscopy was performed by the core facility at the ConveRgence mEDIcine research cenTer (CREDIT), Asan Medical Center, Seoul, Korea.
Written informed consent was obtained from the participants for publication of this article and accompanying images.
H.C. (Heesung Chung), B.J., S.Y., and E.S.O. wrote the manuscript. H.C. (Heesung Chung), B.J., H.J, H.K.S., E.P., and K.J. performed the experiments. H.C. (Han Choe) and H.S.C. helped analyzed EV. S.Y. provided reagents. H.C. (Heesung Chung), B.J., S.Y., and E.S.O. designed the experiments and expertise and feedback.
The authors have no potential conflicts of interest to disclose.
Shunbun Kita and Iichiro Shimomura
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