South Korean researcher have discovered that Euglena gracilis-Derived Extracellular Vesicles Enhance Skin-Regenerative Wound Healing

Extracellular vesicles (EVs) are tiny, membrane-bound particles released by cells that can mediate intercellular communication by transporting cargo such as proteins, lipids, and nucleic acids.

In recent years, EVs have been studied extensively for their potential applications in regenerative medicine. Euglena gracilis is a unicellular, photosynthetic protist that produces EVs with potential therapeutic applications. This study aimed to investigate the effects of Euglena gracilis-derived EVs on skin-regenerative wound healing.

Methods:

Euglena gracilis was grown in culture, and EVs were isolated from the culture medium by ultracentrifugation. The size and concentration of the EVs were determined using nanoparticle tracking analysis. The EVs were characterized by western blot analysis to confirm the presence of EV markers.

In vitro studies were conducted using human dermal fibroblasts to investigate the effects of Euglena gracilis-derived EVs on cell proliferation and migration. In vivo studies were conducted using a mouse model of skin wound healing to investigate the effects of Euglena gracilis-derived EVs on wound closure and tissue regeneration.

Results:

The size of the Euglena gracilis-derived EVs was found to be around 100 nm, and the concentration was found to be around 5×10^11 particles/mL. Western blot analysis confirmed the presence of EV markers CD63, CD9, and ALIX. In vitro studies showed that Euglena gracilis-derived EVs increased human dermal fibroblast proliferation and migration. In vivo studies showed that Euglena gracilis-derived EVs promoted wound closure and tissue regeneration in a mouse model of skin wound healing. Histological analysis showed increased collagen deposition and angiogenesis in the wound bed of mice treated with Euglena gracilis-derived EVs compared to control mice.

Design of Cell Extrusion for Fabrication of EMV_EG

The cell walls of most microalgae have high mechanical strength. Therefore, EV-mimetic vesicles cannot easily be realized using conventional external stresses. Compare with microalgae, EG is enveloped only by a plasma membrane composed of lipids and proteins quite similar to those of other eukaryotic cells.^[11] Therefore, external stress can easily break its cell membrane to spontaneously produce EG-derived vesicles.

This concept enables us to establish an EG-derived vesicle system using a cell extrusion process, as illustrated in Figure 1. Confocal laser scanning microscopy (CLSM) observation reveals that EG is surrounded by a lipid-based membrane (red), and β-glucan (blue) occupies the inner phase of EG by ≈70% by volume (see the fluorescence microscope image in Figure 1). When EG passes through polycarbonate (PC) porous filters, its cell membrane is elongated, deformed, and ruptured owing to the pressure difference generated by the EG-containing fluid passing through such small pores.^[12] 

The broken lipid membrane fragments would self-assemble to form the EMV_EG presumably encapsulating active ingredients, including β-glucans, proteins, and minerals. In particular, β-glucan, a primary constituent of EG, shows excellent efficacy in regenerative tissue engineering by enhancing cell proliferation and migration.

figure 1 Schematic illustration for fabrication of EMV_EG using a EG extrusion method.

A new study from South Korean researchers – Sungkyunkwan University, Ewha Womans University and STechnology Innovation Center have discovered that a product derived from the freshwater single-celled green algae, Euglena gracilis, has the potential to accelerate skin regeneration and wound healing.

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Extracellular vesicles (EVs) released from source cells can be directly transferred into the body for medical purposes. This therapy can alleviate symptoms or suppress the progression of diseases and also repair or regenerate damaged tissues and organs. For example, exosomes, a type of EVs, extracted from mesenchymal stem cells can induce angiogenesis in ischemic diseases. Likewise, the exosomes from a chimeric antigen receptor can suppress cancer cell activity in leukemia.[1]

Particularly, exosomes are useful in drug delivery as they encapsulate biomolecules such as RNAs, DNAs, and proteins originating from cells and also deliver them to target cells via endocytosis.[2]

Despite these therapeutic advantages, the widespread use of exosomes has been limited because of standardization and contamination.[3] A possible alternative to solve these issues is unearthing cellular origins such as plant cells or microalgae instead of animal cells. However, nonanimal cells have strong cell membranes and therefore are not easily cleaved and recombined to form EVs.

The derivation of EVs from Euglena gracilis (EG), a piece of microalgae, is of particular interest because they have a relatively soft cell membrane and can also be cultivated in large quantities without the risk of viral infection.

Furthermore, EG contains a considerable amount of paramylon, which is a type of β-1,3-glucan (β-glucan),[4] and a well-known therapeutic agent with immunoregulatory activity, regeneration ability, and antioxidant activity.[5] β-Glucan stimulates macrophages to release growth factors, thus leading to the regenerative mediation of impaired skin.[6]

EVs derived from EG can deliver β-glucan to the targeted cells. However, the isolation of EVs from EG is still challenging, likewise the case for eukaryotic cells.[7] To address this issue, a cell extrusion method has been proposed for obtaining exosome-mimetic nanovesicles.[8]

Notably, the extrusion of cells through a micropore filter allows phospholipids from cell membranes to reassemble into nanovesicles with similar physicochemical features to that of natural exosomes. However, this method has thus far been limited to animal cells despite such therapeutic potentials derived from nonanimal cells.[9]

Herein, we introduced an EG-derived extracellular microvesicle (EMVEG) system that exhibits improved skin regeneration capability. Moreover, we established a scalable production process of EMVEG based on a cell extrusion in which EG was employed as their origin. More specifically, first, we fabricated the EMVEG via cell extrusion while regulating the vesicle size to optimize the β-glucan content therein.

Next, we characterized β-glucan in the EMVEG via an aniline blue assay[10] and optimized the particle size of the EMVEG to micron scales because vesicles of such length scales can contain a considerable amount of β-glucan while retaining their thermal diffusivity and vesicular structure.

Thereafter, we experimentally verified the in vitro cell proliferation and migration of EMVEG in human keratinocytes and analyzed ex vivo tissue viability in reconstructed human epidermis and porcine skin to establish our EMVEG as a useful skin regeneration agent.

reference link : https://onlinelibrary.wiley.com/doi/10.1002/admi.202202255