A holothurian body wall extracellular vesicle, and a preparation method and application thereof

Abstract

This invention discloses an extracellular vesicle of the sea cucumber body wall, its preparation method, and its applications, belonging to the field of marine biotechnology. Targeting the dense and highly collagenized structure of the sea cucumber body wall, this invention first uses acidic pepsin for low-temperature pretreatment to break the collagen cross-linking network. Then, switching to a neutral system, collagenase and neutral protease are used for synergistic enzymatic hydrolysis, with EDTA chelation termination technology to inhibit enzyme interference. Finally, high-purity vesicles are obtained through fractional centrifugation. The obtained vesicles have a main peak particle size of 100–180 nm, are uniformly distributed, and have intact morphology. CCK-8 experiments show that the vesicles prepared by this invention are non-toxic to RAW264.7 cells at concentrations of 5–500 μg / mL and exhibit significant proliferative activity, indicating broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of marine biotechnology, specifically relating to an extracellular vesicle of the sea cucumber body wall, its preparation method, and its application. Background Technology

[0002] Extracellular vesicles, as key nanocarriers mediating intercellular signal transduction and substance transport, possess a unique lipid bilayer structure that effectively protects endogenous bioactive substances from degradation. The sea cucumber body wall, as its core physiological defense barrier and main medicinal and edible active component, contains abundant biomolecules. Extracting extracellular vesicles from its body wall is of significant value for developing novel bioactive substances. However, the sea cucumber body wall is a highly collagenized, dense connective tissue containing a large amount of acidic mucopolysaccharides, making it difficult to directly apply current mainstream methods for extracting extracellular vesicles from mammalian body fluids or plant sap. Existing technologies for processing solid matrices like the sea cucumber body wall generally face technical bottlenecks such as incomplete tissue dissociation, severe interference from extracellular matrix impurities, osmotic pressure imbalance leading to vesicle rupture, low extract purity, and loss of bioactivity. Therefore, considering the unique histological characteristics of the sea cucumber body wall, there is an urgent need to establish an extraction method that can efficiently degrade tissue, maintain the integrity of vesicle structure, and accurately remove complex matrix background interference. Summary of the Invention

[0003] The purpose of this invention is to provide an extracellular vesicle of the sea cucumber body wall, a method for preparing the extracellular vesicle of the sea cucumber body wall, and further, the application of the extracellular vesicle of the sea cucumber body wall.

[0004] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows: An extracellular vesicle derived from the body wall of a sea cucumber, with a particle size ranging from 100 to 180 nm, exhibits a typical saucer-like morphology.

[0005] The method for preparing the extracellular vesicles of the sea cucumber body wall includes the following steps: (1) Pretreatment and preliminary degradation: The sea cucumber body wall tissue was mixed with an acidic protease system and subjected to low-temperature stirring treatment; (2) Neutralization and system conversion: Adjust the pH of the system obtained in step (1) to neutral using alkaline solution, and add a buffer solution containing calcium ions; (3) Compound enzymatic hydrolysis promotes the release of extracellular vesicles: Add compound enzyme solution to the system and perform isothermal shaking enzymatic hydrolysis; (4) Enzymatic termination and separation: Add a stop solution to terminate the enzymatic hydrolysis reaction and perform fractionation and centrifugation extraction.

[0006] Further, in step (1), the mass-to-volume ratio of the sea cucumber body wall tissue to the acidic protease system is 1:5 to 1:10 (g:mL), preferably 1:8; the acidic protease system consists of 0.4 to 0.6 M acetic acid solution and 2% to 8% ( w / w The composition of the pepsin solution is based on the wet weight of the original sea cucumber tissue; preferably, the sodium concentration of the acetic acid solution is 0.5 M, and the mass fraction of the pepsin solution is 5%. w / w (Based on the wet weight of the original sea cucumber tissue).

[0007] Furthermore, in step (1), the low-temperature stirring treatment is a continuous stirring treatment at 2 to 6°C for 12 to 16 hours, so that the dense collagen fiber framework of the body wall undergoes initial cross-linking and breakage.

[0008] Further, in step (2), the alkaline solution is 10 M NaOH, and the pH is adjusted to 7.4 ~ 7.6, preferably 7.5; the buffer solution is Hanks balanced salt solution (HBSS buffer), and the final concentration of CaCl2 in the system is 1 ~ 4 mM, preferably 2 mM.

[0009] Further, in step (3), the composite enzyme solution includes collagenase and neutral protease; wherein the amount of collagenase added is 0.5% to 3.0% ( w / w (Based on the wet weight of the original sea cucumber tissue), the amount of neutral protease added is 0.5% ~ 2.0% ( w / w (Based on the wet weight of the original sea cucumber tissue). Preferably, the amounts of collagenase and neutral protease added are 2 mg / g and 1 mg / g, respectively (based on the wet weight of the original sea cucumber tissue).

[0010] Furthermore, in step (3), the enzymatic hydrolysis temperature is 35 ~ 38℃ and the time is 1.5 ~ 2.5 h; preferably, the enzymatic hydrolysis is carried out at 37℃ for 2 h.

[0011] Further, in step (4), the termination solution is HBSS buffer containing EDTA, preferably, the concentration of EDTA is 5 ~ 10 mM.

[0012] Further, in step (4), the graded centrifugation is carried out at 2~8℃, preferably 4℃; including low-speed centrifugation of 500~2000 g for 15 min, high-speed centrifugation of 10,000~15,000 g for 30 min and ultra-high-speed centrifugation of 80,000~120,000 g or more for 30 min.

[0013] The application of the extracellular vesicles of the sea cucumber body wall in promoting cell proliferation.

[0014] The application of the extracellular vesicles of the sea cucumber body wall in the preparation of immunomodulatory products.

[0015] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention provides a method for extracting extracellular vesicles from the body wall of sea cucumbers. Low-temperature acidic pepsin pretreatment breaks down the unique collagen cross-linking network of the sea cucumber body wall, solving the problem of conventional methods' difficulty in penetrating deep tissue layers and significantly improving the mechanical release rate of the vesicles. The combination of low-temperature pretreatment and key EDTA chelation termination technology effectively inhibits the damage of endogenous hydrolases to vesicle membrane proteins, ensuring the bioactivity and structural integrity of the extracted product. The precise stepwise combination of pepsin, collagenase, and neutral protease can specifically degrade mucopolysaccharides and fibrous components that interfere with separation, significantly reducing sample viscosity and improving sedimentation purity and particle size distribution uniformity during ultracentrifugation.

[0016] The proposed method for isolating extracellular vesicles from the sea cucumber body wall is stable, effective, and simple to operate. The resulting product is rich in content, has low background noise, and exhibits excellent biocompatibility. It provides high-quality biological samples for the subsequent functional development of extracellular vesicles from the sea cucumber body wall and has promising application prospects. Attached Figure Description

[0017] Figure 1 Figure showing the particle size analysis results of extracellular vesicles in the body wall of sea cucumber in Example 1.

[0018] Figure 2 Figure showing the particle size analysis results of extracellular vesicles in the body wall of sea cucumber in Example 2.

[0019] Figure 3 Figure showing the particle size analysis results of extracellular vesicles in the body wall of sea cucumber in Example 3.

[0020] Figure 4 Transmission electron micrograph of extracellular vesicles in the body wall of sea cucumber in Example 1.

[0021] Figure 5 Transmission electron microscopy image of extracellular vesicles in the body wall of sea cucumber in Example 2.

[0022] Figure 6 Transmission electron micrograph of extracellular vesicles in the body wall of sea cucumber in Example 3.

[0023] Figure 7 The effect of different concentrations of extracellular vesicles from the body wall of sea cucumber on cell survival rate in Example 3. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention will be described in detail below with reference to specific embodiments. The following embodiments are only for explaining the present invention and do not constitute a limitation on the scope of protection of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1: This embodiment provides a method for preparing extracellular vesicles of sea cucumber body wall using a conventional enzymatic hydrolysis process. The specific steps are as follows: (1) Fresh sea cucumber body walls were selected as raw materials, and after being embrittled by liquid nitrogen at low temperature, they were ground into powder; according to a ratio of 1:8 ( w / v Add pre-cooled HBSS buffer (containing 2 mM CaCl2) to the solid-liquid ratio of the sea cucumber, mix thoroughly, and obtain the original extract of the sea cucumber body wall.

[0027] (2) Add 50 mL of compound enzyme solution (the solute components are 2% collagenase and 1% neutral protease) to the above extract. w / w (Based on the wet weight of the original sea cucumber tissue); the mixture was incubated in a constant temperature environment of 37°C for 2 h.

[0028] (3) Fractional purification: The enzymatic hydrolysis product was transferred to centrifuge tubes and purified by differential centrifugation. Specifically, the supernatant was collected by centrifugation at 4℃ and 2000×g for 15 min; then further centrifuged at 4℃ and 10000×g for 30 min to remove cell debris and large particulate impurities. The supernatant was initially filtered through a 0.22 μm filter membrane, and then ultracentrifuged at 4℃ and 100000×g for 30 min. The supernatant was discarded, and the bottom precipitate was collected and resuspended in PBS buffer. Finally, it was filtered through a 0.45 μm filter membrane to obtain the extracellular vesicles of the sea cucumber body wall.

[0029] Example 2: This embodiment introduces a reaction termination step based on Embodiment 1, as follows: (1) Take the fresh sea cucumber body wall after it has been pulverized with liquid nitrogen, and mix it at a ratio of 1:8 ( w / v Add HBSS buffer containing 2 mM CaCl2; add 50 mL of complex enzyme solution (2% collagenase + 1% neutral protease). w / w (Based on the wet weight of the original sea cucumber tissue), incubated at 37°C for 2 h.

[0030] (2) After the enzymatic hydrolysis reaction is completed, an equal volume of pre-cooled EDTA-HBSS buffer is immediately added to the system. The chelation of metal ions by EDTA rapidly inactivates the protease, preventing its excessive degradation of vesicle membrane proteins, and yields the terminated extract.

[0031] (3) The subsequent centrifugation purification steps are the same as in Example 1, namely, after preliminary impurity removal at 2000×g and 10000×g, filtration through a 0.22 μm filter membrane, and ultracentrifugation at 100000×g, the target extracellular vesicles are obtained by resuspending with PBS and filtration through a 0.45 μm filter membrane.

[0032] Example 3: This embodiment targets the dense collagen fiber structure of the sea cucumber body wall and employs a stepwise acid and enzyme degradation process. The specific steps are as follows: (1) Take fresh sea cucumber body wall, pulverize it with liquid nitrogen, and mix it at a ratio of 1:5 ( w / v The following ratio was added to the acidic pepsin system (composed of 0.5 M acetic acid solution and 5% ( w / w The collagen fibers were digested (based on the wet weight of the original sea cucumber tissue) for 16 hours at 4°C. This step aims to induce the initial dissociation of the collagen fiber skeleton through an acidic environment.

[0033] (2) Slowly adjust the pH of the system to 7.5 using 10 M NaOH solution; then add 50 mL of compound enzyme solution (2% collagenase + 1% neutral protease) in proportion. w / w The original sea cucumber tissue was incubated at 37°C for 2 h using HBSS buffer containing 2 mM CaCl2 as the solvent (based on wet weight of the original sea cucumber tissue). The tissue was then incubated for further degradation of the extracellular matrix by the synergistic effect of collagenase and neutral protease.

[0034] (3) After the reaction is complete, add an equal volume of pre-cooled EDTA-HBSS buffer to terminate the enzymatic digestion.

[0035] (4) The subsequent centrifugation purification steps are the same as in Example 1, namely, after preliminary impurity removal at 2000×g and 10000×g, filtration through a 0.22 μm filter membrane, and ultracentrifugation at 100000×g, the target extracellular vesicles are obtained by resuspending with PBS and filtration through a 0.45 μm filter membrane.

[0036] Example 4: This embodiment provides a comprehensive quality evaluation of the extracellular vesicles derived from the body wall of sea cucumbers prepared in the above-described manner.

[0037] (1) Size distribution of extracellular vesicles in the body wall of sea cucumber The particle size of the extracellular vesicles of the sea cucumber body wall prepared in Examples 1-3 was detected using a nano particle size analyzer (Nano ZS90). Specifically, the particle concentration of the extracellular vesicles of the sea cucumber body wall was diluted to an appropriate concentration, filtered through a 0.22 μm filter membrane, and then injected into the nano particle size analyzer for detection.

[0038] Depend on Figures 1-3 The nanoparticle size analysis results show that the particle size distribution curves of Examples 1 and 2 are relatively flat with obvious tailing, and multiple impurity peaks exist in the 10-100 nm range. This indicates that without acid pretreatment, due to incomplete dissociation of the collagen matrix of the sea cucumber body wall, the extract contains a large amount of tissue debris or protein aggregates, and due to the interference of impurities, the characteristic peak intensity of the target vesicles is at a low level.

[0039] In Example 3, the particle size distribution curve exhibits a highly significant single-peak distribution, with the main peak positioned between 100 and 180 nm, consistent with the typical particle size range of extracellular vesicles. Compared to Examples 1 and 2, the curve in Example 3 shows a more stable baseline, symmetrical peak shape, and a several-fold increase in peak intensity.

[0040] (2) Microscopic morphological observation of sea cucumber body wall The morphology of extracellular vesicles in the body wall of sea cucumbers prepared in Examples 1-3 was observed using transmission electron microscopy. The specific steps were as follows: 10 μL of vesicle sample was taken, fixed in situ with glutaraldehyde, dropped onto a copper grid and allowed to stand for 5 min for adsorption, then stained with saturated uranium acetate-acetic acid solution for 1 min. After the sample was dried at room temperature, the micromorphology of the vesicles was observed using transmission electron microscopy under an accelerating voltage of 80 kV.

[0041] The results are as follows Figures 4-6 As shown, Figure 4 and Figure 5 The background of the vesicles was cluttered, with many protein aggregates or cell debris present. Figure 6 The number of vesicles increased significantly, and their morphology was typical of a saucer-shaped teacup. The structure was intact, the background was clean, and the distribution was uniform. Figure 6 The method involves first using pepsin to dissociate collagen fibers, followed by simultaneous incubation with two enzymes. The two enzymes exhibit a significant synergistic effect when acting on the substrate, which can more thoroughly degrade impurity proteins and mucopolysaccharides, thereby releasing more high-purity vesicles.

[0042] Taking into account the particle size and morphological characteristics of the vesicles, Example 3 is the optimal implementation scheme of the present invention.

[0043] Example 5: This embodiment evaluates the cell activity of extracellular vesicles in the body wall of sea cucumbers obtained in Example 3.

[0044] (1) Cell culture Mouse macrophages RAW264.7 were cultured in DMEM medium containing 10% fetal bovine serum and passaged in a 37°C, 5% CO2 incubator.

[0045] (2) Measurement of cell viability The effect of extracellular vesicles in the body wall of sea cucumbers on cell viability was investigated using the CCK-8 assay. RAW264.7 cells were cultured at 1×10⁻⁶ cells / cells. 5 Sea cucumbers were seeded at a density of 100 μL / mL in 96-well plates. After pre-culturing at 37℃ and 5% CO2 for 24 h, the original culture medium was discarded. Culture medium containing different concentrations (5, 10, 50, 100, 200, 500 μg / mL) of sea cucumber body wall extracellular vesicles was added, and incubation was continued for 24 h. 10 μL of CCK-8 solution was added to each well, and the plates were incubated in the dark for 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader, and cell viability was calculated according to formula (1): Cell viability = [OD(experimental) - OD(blank)] / [OD(normal) - OD(blank)] × 100% (1) In the formula: OD(experimental) is the absorbance of cell groups with different concentrations of vesicles added; OD(blank) is the absorbance of cell-free culture medium wells; OD(normal) is the absorbance of normal cell groups without vesicle treatment.

[0046] The results are as follows Figure 7 As shown in the figure. Within a concentration range of 5–500 μg / mL, the extracellular vesicles extracted from the sea cucumber body wall in this invention showed no toxicity to RAW264.7 cells. The cell viability in the experimental groups was significantly higher than 100%, indicating that the vesicles not only have good biosafety within the experimental concentration range but also significantly promote the proliferation of mouse macrophages. Cell viability reached its peak at a concentration of approximately 50 μg / mL. This further verifies that the extraction method of this invention can effectively preserve the bioactivity of the extracellular vesicles from the sea cucumber body wall.

[0047] This invention addresses the structural characteristics of sea cucumber body wall tissue, which is dense, high in collagen, and contains a large amount of mucopolysaccharides. It utilizes a multi-enzyme combined stepwise degradation process to solve the technical problems of efficient release of extracellular vesicles from solid matrices and difficulty in impurity removal using traditional methods. This results in sea cucumber body wall extracellular vesicles with high integrity and high yield. Furthermore, this invention provides sea cucumber body wall extracellular vesicles with high biocompatibility and cell proliferation-promoting activity, supporting their application in the fields of biomedicine and functional foods.

[0048] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

[0049] Finally, although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A holothurian epithelial extracellular vesicle, characterized in that, These extracellular vesicles originate from the sea cucumber body wall, with a particle size ranging from 100 to 180 nm, and exhibit a typical saucer-like morphology.

2. The method for preparing extracellular vesicles of the sea cucumber body wall according to claim 1, characterized in that, Includes the following steps: (1) Pretreatment and preliminary degradation: The sea cucumber body wall tissue was mixed with an acidic protease system and subjected to low-temperature stirring treatment; (2) Neutralization and system conversion: Adjust the pH of the system obtained in step (1) to neutral using alkaline solution, and add a buffer solution containing calcium ions; (3) Compound enzymatic hydrolysis promotes the release of extracellular vesicles: Add compound enzyme solution to the system and perform isothermal shaking enzymatic hydrolysis; (4) Enzymatic termination and separation: Add a stop solution to terminate the enzymatic hydrolysis reaction and perform fractionation and centrifugation extraction.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass-to-volume ratio of the sea cucumber body wall tissue to the acidic protease system is 1:5 to 1:10; the acidic protease system is composed of 0.4 to 0.6 M acetic acid solution and 2 wt% to 8 wt% pepsin.

4. The preparation method according to claim 2, characterized in that, In step (1), the low-temperature stirring treatment is a continuous stirring treatment at 2 ~ 6℃ for 12 ~ 16 h.

5. The preparation method according to claim 2, characterized in that, In step (2), the alkaline solution is 10 M NaOH, and the pH is adjusted to 7.4 ~ 7.6; the buffer solution is Hanks' equilibrium salt solution, and the final concentration of CaCl2 in the system is 1 ~ 4 mM.

6. The preparation method according to claim 2, characterized in that, In step (3), the compound enzyme solution includes collagenase and neutral protease; wherein the amount of collagenase added is 0.5wt% to 3.0wt% based on the wet weight of the original sea cucumber tissue, and the amount of neutral protease added is 0.5wt% to 2.0wt% based on the wet weight of the original sea cucumber tissue; the enzymatic hydrolysis temperature is 35 to 38℃, and the time is 1.5 to 2.5 h.

7. The preparation method according to claim 2, characterized in that, In step (4), the termination solution is HBSS buffer containing EDTA, and the concentration of EDTA is 5 ~ 10 mM.

8. The preparation method according to claim 2, characterized in that, In step (4), the graded centrifugation is carried out at 2~8℃; including low-speed centrifugation of 500~2000 g, high-speed centrifugation of 10,000~15,000 g and ultra-speed centrifugation of 80,000~120,000 g and above.

9. The application of the extracellular vesicles of the sea cucumber body wall as described in claim 1 in promoting cell proliferation.

10. The use of the extracellular vesicles of the sea cucumber body wall as described in claim 1 in the preparation of immunomodulatory products.

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