A method for preparing extracellular vesicles derived from Tremella fuciformis fermentation broth and its application

Abstract

This invention discloses a method for preparing extracellular vesicles derived from Tremella fuciformis fermentation broth and its applications. The prepared extracellular vesicles promote cell proliferation, cell migration, and angiogenesis, and significantly promote wound healing. This indicates that extracellular vesicles derived from Tremella fuciformis fermentation broth have a wound-healing effect and can be applied in the fields of skin damage repair, medical aesthetics, and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for preparing extracellular vesicles derived from Tremella fuciformis fermentation broth and its application. Background Technology

[0002] A skin wound is a mechanical injury to the skin and its internal tissues and organs caused by external forces; it is an open wound. Skin wounds mainly result from external forces, burns, surgery, and complications from various diseases. After skin trauma, treatment should be initiated as soon as possible to promote wound healing. Without intervention, adverse reactions such as inflammation, ischemia or necrosis, scarring, pigmentation, and ulceration are highly likely. With rapid economic and technological development, the level of skin wound treatment has gradually improved. However, traditional skin healing therapies such as lasers, therapeutic dressings, negative pressure therapy, and skin grafts have significant limitations in clinical application. Rapid and scarless skin wound repair remains one of the challenges in clinical medicine.

[0003] Extracellular vesicles (EVs) are generally defined as cytoplasmic vesicles surrounded by a lipid bilayer, released from the cell into the extracellular space. EVs carry characteristic biological information molecules of the parent cell, such as proteins, lipids, DNA, and non-coding RNA, possessing natural molecular transport properties and good biocompatibility. They can mediate intercellular communication and serve as natural nanocarriers for endogenous cells, exhibiting very low cytotoxicity and immunogenicity. In recent years, increasing reports have revealed that plants and fungi can also secrete extracellular vesicles with significant activity, demonstrating great potential in disease treatment. Conventional methods of preparing extracellular vesicles through plant or animal cell extraction suffer from problems such as unstable raw material quality and limited material sources, resulting in high manufacturing costs and hindering industrialization.

[0004] Tremella fuciformis (TF) is the fruiting body of a fungus in the genus Tremella, also known as white fungus. As a traditional medicinal and edible fungus, Tremella fuciformis has high nutritional value and can be used in both medicine and food, earning it the title of "King of Fungi." Studies have shown that Tremella fuciformis possesses unique biological activities, exhibiting significant effects in anti-aging, anti-tumor, anti-inflammatory, antioxidant, and skin protection. Research has found that Tremella fuciformis fermentation broth obtained through liquid culture not only ensures the quality of the active ingredients in Tremella fuciformis but also enables industrial-scale production with a shortened growth cycle, offering advantages such as cost savings, small footprint, and ease of control.

[0005] Current research on Tremella fuciformis fermentation broth mainly focuses on active ingredients such as polysaccharides, phenolic acids, catechins, and flavonoids. There are no reports on whether Tremella fuciformis fermentation broth contains extracellular vesicles or whether it promotes wound healing. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an extracellular vesicle derived from Tremella fuciformis fermentation broth and its application.

[0007] This invention prepares extracellular vesicles (TFB-EVs) from the fermentation broth of Tremella fuciformis, which can promote cell proliferation and migration, and promote angiogenesis. This invention provides a new option for drugs or formulations for wound cell repair or special medical purposes.

[0008] The first objective of this invention is to provide the use of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote cell proliferation and / or migration.

[0009] A second objective of this invention is to provide the application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote angiogenesis.

[0010] A third objective of this invention is to provide the application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote skin wound healing.

[0011] The fourth objective of this invention is to provide a product that promotes the healing of skin wounds.

[0012] This invention claims protection for the following applications:

[0013] Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote cell proliferation and / or migration.

[0014] Preferably, the cells are skin tissue cells.

[0015] Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote angiogenesis.

[0016] Application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote skin wound healing.

[0017] Preferably, the strain number of the Tremella fuciformis is CGMCC 5.466 (China General Microbiological Culture Collection Center).

[0018] Preferably, the method for preparing extracellular vesicles from the Tremella fuciformis fermentation broth is as follows:

[0019] Remove the mycelium from the fermentation broth of Tremella fuciformis, centrifuge at 8000–15000×g for 25–35 min at 3–5℃, discard the precipitate and collect the supernatant, repeat the high-speed centrifugation and collect the supernatant; then, at 3–5℃, ultracentrifuge the supernatant at 90000–110000×g for 65–75 min, discard the supernatant, resuspend the precipitate in pre-cooled PBS, repeat the ultracentrifugation and collect the supernatant; the purified product is obtained.

[0020] More preferably, the method for preparing the extracellular vesicles derived from the Tremella fuciformis fermentation broth is as follows:

[0021] Remove the mycelium from the fermentation broth of Tremella fuciformis, centrifuge at 12000×g for 30 min at 4℃, discard the precipitate and collect the supernatant, repeat high-speed centrifugation to collect the supernatant; at 4℃, ultracentrifuge the supernatant at 110000×g for 70 min, discard the supernatant, resuspend the precipitate in pre-cooled PBS, repeat ultracentrifugation; after resuspending the precipitate in pre-cooled PBS, repeat ultracentrifugation to collect the supernatant; purification yields the final product.

[0022] Preferably, the purification process involves filtration sterilization at 0.22 μm.

[0023] More preferably, the method for removing mycelium is to filter with eight layers of gauze.

[0024] This invention also claims protection for a product that promotes the healing of skin wounds, containing extracellular vesicles derived from tremella fermentation liquid.

[0025] Preferably, the method for preparing the extracellular vesicles derived from the Tremella fuciformis fermentation broth is as follows:

[0026] Remove the mycelium from the fermentation broth of Tremella fuciformis, centrifuge at 8000–15000×g for 25–35 min at 3–5℃, discard the precipitate and collect the supernatant, repeat the high-speed centrifugation and collect the supernatant; then, at 3–5℃, ultracentrifuge the supernatant at 90000–110000×g for 65–75 min, discard the supernatant, resuspend the precipitate in pre-cooled PBS, repeat the ultracentrifugation and collect the supernatant; the purified product is obtained.

[0027] More preferably, the method for preparing the extracellular vesicles derived from the Tremella fuciformis fermentation broth is as follows:

[0028] Remove the mycelium from the fermentation broth of Tremella fuciformis, centrifuge at 12000×g for 30 min at 4℃, discard the precipitate and collect the supernatant, repeat high-speed centrifugation to collect the supernatant; at 4℃, ultracentrifuge the supernatant at 110000×g for 70 min, discard the supernatant, resuspend the precipitate in pre-cooled PBS, repeat ultracentrifugation; after resuspending the precipitate in pre-cooled PBS, repeat ultracentrifugation to collect the supernatant; purification yields the final product.

[0029] Preferably, the strain number of the tremella is CGMCC 5.466 (China General Microbiological Culture Collection Center).

[0030] Preferably, the method for removing mycelium is to filter with eight layers of gauze.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention provides extracellular vesicles derived from Tremella fuciformis fermentation broth and their applications. The prepared extracellular vesicles promote cell proliferation, cell migration, and angiogenesis, and significantly promote wound healing. This indicates that extracellular vesicles derived from Tremella fuciformis fermentation broth have a wound-healing effect and can be applied in the fields of skin damage repair, medical aesthetics, and cosmetics. Attached Figure Description

[0033] Figure 1 Identification of extracellular vesicles from the fermentation broth of Tremella fuciformis.

[0034] Figure 2 Effects of extracellular vesicles derived from the fermentation broth of Tremella fuciformis on the proliferation (a) and migration rate (b) of HaCaT cells (40×).

[0035] Figure 3 Effects of extracellular vesicles derived from Tremella fuciformis fermentation broth on the proliferation (a) and migration rate (b) of HUVEC cells (40×).

[0036] Figure 4 Results of HUVEC cell angiogenesis (40×).

[0037] Figure 5 The results show the wound healing of mice from 0 to 14 days. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0039] Preparation of culture medium:

[0040] PDA medium: 2g glucose, 0.3g KH2PO4, 0.15g MgSO4·7H2O, 1.5g agar, 100mL potato extract. Sterilize at 115℃ for 30min.

[0041] Liquid culture medium: 2g glucose, 0.5g yeast extract, 0.05g KH2PO4, 0.1g MgSO4·7H2O, 100mL purified water. After removing larger particles by filtration through a 0.22μm aqueous membrane, the culture medium is dispensed into 250mL Erlenmeyer flasks, sealed, and sterilized at 115℃ for 30min.

[0042] Human immortalized keratinocytes (HaCaT cells) are from Wuhan Shangen Biotechnology, and human umbilical vein endothelial cells (HUVEC cells) HaCaT cells are from Wuhan Shangen Biotechnology.

[0043] Example 1: Extraction of extracellular vesicles (TFB-EVs) from Tremella fuciformis fermentation broth

[0044] 1. Activation of microbial strains

[0045] In a clean bench, a strain of Tremella fuciformis (purchased from the China General Microbiological Culture Collection Center, strain number CGMCC 5.466) was inoculated into three petri dishes containing PDA medium using a sterile inoculation loop. The dishes were then placed in a 28°C incubator and incubated for 3 days to serve as the strain for subsequent experiments.

[0046] 2. Preparation of seed solution

[0047] In a clean bench, a piece of activated mycelium was picked up with a sterile inoculation loop and inoculated into an Erlenmeyer flask containing 100 mL of liquid for fermentation culture. The flask was cultured at 27 °C and 160 r / min for 3 days to prepare as a seed culture.

[0048] 3. Liquid fermentation of microbial cells

[0049] The seed culture was added to Erlenmeyer flasks containing liquid fermentation medium at a volume ratio of 2%, and cultured with shaking at 27°C and 160 r / min for 3 days to obtain the fermentation broth.

[0050] 4. The fermentation broth of Tremella fuciformis is derived from extracellular vesicles.

[0051] After 3 days of fermentation, the fermentation broth was collected. The mycelium was removed using 8 layers of gauze and the broth was placed into a 50 mL centrifuge tube. The tube was balanced and centrifuged at 4°C and 12000×g for 30 min. The supernatant was collected, and the centrifugation was repeated once. The supernatant was then placed in an ultracentrifuge bottle, balanced, and centrifuged at 4°C and 110000×g for 70 min. The supernatant was discarded, and the precipitate was resuspended in PBS. The ultracentrifugation was repeated once, and the precipitate was resuspended in 1 mL of PBS. The mixture was filtered through a 0.22 μm aqueous filter membrane, aliquoted, and stored at -80°C.

[0052] Example 2: Characterization of extracellular vesicles derived from Tremella fuciformis fermentation broth

[0053] I. Experimental Methods

[0054] 1. Transmission electron microscopy (TEM) detection

[0055] Using a pipette, 20 μL of the TFB-EVs sample obtained in Example 1 was dropped onto a carbon-supported copper mesh and left to stand for 3–5 min. Excess liquid was then blotted away with filter paper. Next, 2% phosphotungstic acid was dropped onto the carbon-supported copper mesh and left to stand for 1–2 min. Excess liquid was then blotted away with filter paper, and the sample was allowed to dry at room temperature. Finally, the sample was observed under a transmission electron microscope, and images were acquired and analyzed.

[0056] 2. NTA particle size analysis

[0057] The TFB-EVs vesicle sample obtained in Example 1 was appropriately diluted with 1×PBS buffer. The particle size and concentration of the outer vesicles were measured using nanoparticle tracking analysis (NTA) on a Zeta View PMX110 instrument and the corresponding software Zeta View 8.04.02 (the Zeta View system was calibrated with 110 nm polystyrene particles, and the temperature was maintained at approximately 23°C and 37°C).

[0058] II. Experimental Results

[0059] like Figure 1 The results of NTA analysis of TFB-EVs show that the average particle size of LEVs is 85.3 nm and the concentration is 2.35 × 10⁻⁶. 9 Particles / mL ( Figure 1 A) Morphological observation of LEVs using transmission electron microscopy, such as... Figure 1 As shown in B, the results indicate that TFB-EVs are spherical, bilayer-coated nanoparticles, consistent with the electron microscopy morphology of extracellular vesicles.

[0060] Example 3: Tremella fuciformis fermentation liquid promotes skin cell proliferation.

[0061] I. Experimental Methods

[0062] The TFB-EVs (0.2–0.3 mg / mL) obtained in Example 1 were administered according to the protein content after the protein concentration was determined using a BCA kit.

[0063] HaCaT cells and HUVEC cells in logarithmic growth phase were collected, and the cell density was adjusted to 5 × 10⁻⁶. 4Cells / mL, take a 96-well cell culture plate, add cell suspension with adjusted density to the 96-well cell culture plate, add 100 μL of cell suspension to each well, place the 96-well cell culture plate in a constant temperature incubator and culture for 24 h; set up experimental groups: blank well (culture medium), blank control group (cells + culture medium), vesicle drug administration group (cells + culture medium containing TFB-EVs, the concentration of TFB-EVs calculated by the amount of protein is: 5 μg / mL, 10 μg / mL, 20 μg / mL, respectively), and set up three replicates for each group.

[0064] The specific procedure is as follows: After discarding the original culture medium in the wells, add different concentrations of TFB-EVs culture medium to each well, and then place the 96-well cell culture plate back into the constant temperature incubator and incubate for 24 hours.

[0065] The number of viable cells was then detected using a CCK-8 assay kit, following the instructions. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Results analysis: Cell viability (%) = (OD value of cells in the drug-treated group - OD value of blank wells) / (OD value of blank control group - OD value of blank wells) × 100%.

[0066] II. Experimental Results

[0067] like Figure 2 a and Figure 3 As shown in a, compared with normal culture (blank control group), extracellular vesicles derived from Tremella fuciformis fermentation broth showed significant proliferative activity against HaCaT cells and HUVEC cells (p<0.05).

[0068] Example 4: Tremella fuciformis fermentation liquid promotes skin cell migration.

[0069] I. Experimental Methods

[0070] Prepare a 24-well plate. Using a marker, draw three horizontal lines across the bottom of each well, approximately every 0.5–1 cm. Collect cells in the logarithmic growth phase, such as HaCaT cells, at a rate of 5 × 10⁻⁶ cells / well. 5 HUVEC cells were seeded at a density of 3.5 × 10⁶ cells / mL into 24-well culture plates at a density of 500 μL per well; 5At a cell density of 500 μL / mL, cells were seeded into each well of a 24-well plate and cultured for one day until confluence. Subsequent experiments were then performed. Using a 200 μL pipette tip, the bottom of the confluent 24-well plate was scratched perpendicularly to the cell surface, with the scratches perpendicular to the horizontal lines on the back of the plate. After scratching, the old culture medium was discarded, and the cells were washed twice with PBS to remove cells in the center of the scratches. The intersections of the upper and lower edges of each line on the bottom of the 24-well plate with the scratches were used as observation points. Serum-free culture medium (normal group) and serum-free culture medium containing different concentrations of TFB-EVs obtained in Example 1 (drug-treated groups, with final TFB-EV concentrations of 5 μg / mL and 20 μg / mL) were added to the corresponding wells. Photos were taken after scratching, at time point 0h. The location of each observation point was recorded, and photos were taken at 0h, 24h, and 48h.

[0071] II. Experimental Results

[0072] Compared to the normal group, extracellular vesicles derived from the fermentation broth of *Tremella fuciformis* showed significant migration-promoting activity against HaCaT cells and HUVEC cells. Figure 2 b and Figure 3 b) Effect (p<0.05).

[0073] Example 5: Effect of extracellular vesicles derived from Tremella fuciformis fermentation broth on angiogenesis in HUVEC cells.

[0074] I. Experimental Methods

[0075] The melted matrix gel was diluted with pre-chilled serum-free culture medium at a ratio of 1:2, and 50 μL / well was added to pre-chilled 96-well cell culture plates using a pipette. The plates were then incubated for 40 minutes until the matrix gel solidified. While waiting for solidification, HUVEC cells were digested, and the cell density was adjusted to 2 × 10⁶ cells / well. 4 Cells were seeded per well in a 96-well cell culture plate containing matrix gel. The drug-treated group received TFB-EVs obtained in Example 1 at a final concentration of 20 μg / mL (calculated as protein concentration). The control group received no vesicles. Each group had three replicates. Cells were incubated for 4 hours, and cell tube formation was observed under a microscope. The results were analyzed using Image-J software.

[0076] II. Experimental Results

[0077] The results are as follows Figure 4 As shown, angiogenesis experiments demonstrated that extracellular vesicles derived from the fermentation broth of Tremella fuciformis can promote tube formation in HUVEC cells and significantly increase the vessel length of HUVEC cells (p<0.05).

[0078] Example 6: Tremella fuciformis fermentation broth-derived vesicles promote wound healing in mice.

[0079] I. Experimental Methods

[0080] Twelve 4-week-old KM mice were randomly selected, ear-tagged, and fed normally for one week. Then, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital at a rate of 1 mL / kg. After the mice were in a coma, the hair on their backs was removed. The mice were then separated into individual cages and wound modeling was performed the next day.

[0081] Modeling: Mice were randomly divided into three groups: control group (NC), positive control group (PC), and EVs group, with 4 mice in each group.

[0082] After grouping, the mice were anesthetized one by one. After the mice were unconscious, their backs were wiped with alcohol swabs and then a full-thickness skin excision was performed in the center of the mouse's back using a sterile circular punch to form a circular or nearly circular full-thickness skin wound. The size of the wound was recorded, and the wound at this time was recorded as Day 0.

[0083] Administration: The NC group was injected with 50 μL of PBS at the wound site, the PC group was injected with 50 μL of commercially available recombinant human epidermal growth factor topical solution (2000 IU / mL) at the wound site, and the EVs group was injected with 50 μL of TFB-EVs obtained in Example 1 at a concentration of 0.2 mg / mL.

[0084] After administration, the mouse wounds were bandaged with elastic bandages, and the wound size of each mouse was recorded on day 7 and day 14.

[0085] II. Experimental Results

[0086] like Figure 5 As shown, the wound area on day 7 (compared to a wound healing rate of 29% on Day 0) and on day 14 (compared to a wound healing rate of 3.8% on Day 0) in the EVs group were significantly smaller than those in the NC group at the same time points, but there was no significant difference compared to the PC group. The healing rate was calculated as the wound area on day 7 divided by the wound area on day 0. These results indicate that extracellular vesicles derived from *Tremella fuciformis* fermentation broth promoted wound healing in mice.

Claims

1. The application of extracellular vesicles derived from Tremella fuciformis fermentation broth in the preparation of products that promote skin wound healing, characterized in that, The method for preparing the extracellular vesicles derived from the Tremella fuciformis fermentation broth is as follows: The Tremella strain was inoculated into PDA medium and cultured at a constant temperature to obtain the activated strain. The mycelia of the activated strain were inoculated into liquid, fermented, and the cultured liquid was used as seed culture. The seed liquid was added to a liquid fermentation medium and cultured to obtain the tremella fermentation liquid; Remove the mycelium from the fermentation broth of Tremella fuciformis, centrifuge at 8000–15000×g for 25–35 min at 3–5℃, discard the precipitate and collect the supernatant, repeat the high-speed centrifugation and collect the supernatant; then, at 3–5℃, ultracentrifuge the supernatant at 90000–110000×g for 65–75 min, discard the supernatant, resuspend the precipitate in pre-cooled PBS, repeat the ultracentrifugation and collect the supernatant; the purified product is obtained.

2. The application according to claim 1, characterized in that, The method for preparing the extracellular vesicles derived from the Tremella fuciformis fermentation broth is as follows: Remove the mycelium from the fermentation broth of Tremella fuciformis, centrifuge at 12000×g for 30 min at 4℃, discard the precipitate and collect the supernatant, repeat high-speed centrifugation to collect the supernatant; at 4℃, ultracentrifuge the supernatant at 110000×g for 70 min, discard the supernatant, resuspend the precipitate in pre-cooled PBS, repeat ultracentrifugation; after resuspending the precipitate in pre-cooled PBS, repeat ultracentrifugation to collect the supernatant; purification yields the final product.

3. The application according to claim 1, characterized in that, The method for removing mycelium is to filter it using eight layers of gauze.

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