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Mar Drugs
2021 Oct 21;1911:. doi: 10.3390/md19110592.
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Characterization of Marine Organism Extracellular Matrix-Anchored Extracellular Vesicles and Their Biological Effect on the Alleviation of Pro-Inflammatory Cytokines.
Jo SH
,
Kim SH
,
Kim C
,
Park SH
.
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Representative marine materials such as biopolymers and bioceramics contain bioactive properties and are applied in regenerative medicine and tissue engineering. The marine organism-derived extracellular matrix (ECM), which consists of structural and functional molecules, has been studied as a biomaterial. It has been used to reconstruct tissues and improve biological functions. However, research on marine-derived extracellular vesicles (EVs) among marine functional materials is limited. Recent studies on marine-derived EVs were limited to eco-system studies using bacteria-released EVs. We aimed to expand the range of representative marine organisms such as fish, crustaceans, and echinoderms; establish the extraction process; and study the bioactivity capability of marine EVs. Results confirmed that marine organism ECM-anchored EVs (mEVs) have a similar morphology and cargos to those of EVs in land animals. To investigate physiological effects, lipopolysaccharide (LPS)-infected macrophages were treated with EVs derived from sea cucumber, fish, and shrimp. A comparison of the expression levels of inflammatory cytokine genes revealed that all types of mEVs alleviated pro-inflammatory cytokines, although to different degrees. Among them, the sea cucumber-derived EVs showed the strongest suppression ability. This study showed that research on EVs derived from various types of marine animals can lead to the development of high value-added therapeutics from discarded marine wastes.
Figure 1. The biologically active molecular components of marine organism extracellular matrix (ECM)-anchored extracellular vesicles (EVs).
Figure 2. Physical characterization of marine organism ECM-anchored EVs. (A) Morphology of mEVs identified using transmission electron microscope (TEM). Scale bar, 0.5 μm. (B) Morphology of mEVs identified using scanning electron microscope (SEM). Scale bar, 1 μm. (C) The size distribution of each mEV was measured using dynamic light scattering (DLS). Data are presented for the average size of three independent experiments.
Figure 3. Marine organism ECM-anchored EVs internal cargo. (A) The amount of genetic material per mEVs protein weight was calculated based on the absorbance values measured at 260 nm. (B) The amount of total protein was measured using Bradford assay. (C) The mEVs protein cargos were compared with sea cucumber-, fish-, and shrimp-derived EVs using SDS-PAGE. Data are presented as the mean ± SD from four independent experiments. (*** p < 0.001 as calculated using Student’s t-test).
Figure 4. Endocytosis and cytotoxicity assay using marine organism ECM-anchored EVs. (A) RAW-264.7 cells were treated with PKH-26-labeled mEVs (red) for 24 h and counterstained with DAPI (blue). Upper right panel represents the cell membrane-labeled group (control); upper left panel represents the labeled SEV uptake group; left lower panel represents the labeled FEV uptake group; and right lower panel represents the labeled ShEV uptake group. Yellow arrows point out representative PKH-26-labeled mEVs. Scale bar, 20 μm. (B) Live/dead cell viability assay of RAW-264.7 cells cultured mEVs (20 μg/mL) suspension medium for 24 h. The live and dead cells exhibited green and red fluorescence, respectively. Scale bar, 100 μm. (C) The RAW-264.7 cells were treated with several concentration of mEVs (0, 5, 10, 15, 20 μg/mL) for 24 h. Cell proliferation analysis using water-soluble tetrazolium salt. Data are presented as the mean ± SD from independent experiments (n = 4). (*** p < 0.001 as calculated using one-way ANOVA).
Figure 5. Marine organism ECM-anchored EVs have biological activity. (A) The effect of cell proliferation in inflammation environment was measured using water-soluble tetrazolium salt. The mEVs of several concentrations (0, 5, 10, 15, 20 μg/mL) were used to treat LPS (50 ng/mL)-induced inflammatory model for 24 h. Data are presented as the mean ± SD from independent experiments (n = 4). (* p < 0.001, ** p < 0.01, and *** p < 0.001, versus inflammation (*) calculated using one-way ANOVA) (B) Anti-inflammation activity of mEVs in LPS-induced model, pro-inflammatory cytokine mRNA (TNF-α, IL-1β, IL-6, MCP-1, and iNOS) expression was analyzed using RT-qPCR and normalized by using the GAPDH endogenous expression. Data are presented as the mean ± SD from independent experiments (n = 4). (*, # p < 0.001, **, ## p < 0.01 and ***, ### p < 0.001, versus inflammation (*) or normal groups (#) calculated using one-way ANOVA).
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