Staphylococcus epidermidis outer vesicle, and preparation method and application thereof

Abstract

The present application relates to a kind of staphylococcus epidermidis outer vesicle and its preparation method and application, the preparation method of the staphylococcus epidermidis outer vesicle includes the following steps: S1, staphylococcus epidermidis CCSMS0776 is inoculated in TSB culture medium and is cultivated, obtains staphylococcus epidermidis CCSMS0776 culture;S2, staphylococcus epidermidis CCSMS0776 culture obtained in S1 step is centrifuged, and supernatant is collected to ultrafiltration, obtains concentrated solution;S3, the concentrated solution obtained in S2 step is centrifuged, and supernatant is filtered, and then the filtrate is carried out ultra-high speed centrifugation, and the collection precipitate is resuspended in PBS, obtains staphylococcus epidermidis outer vesicle.Compared with prior art, the bacterial outer vesicle prepared in the present application shows good anti-inflammatory and skin barrier repair effect, and has wide application prospect in the protection of light damage and the treatment of inflammatory skin diseases and soothing skin care product.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to an epidermal Staphylococcus aureus exovesicle, its preparation method and application. Background Technology

[0002] The skin, the largest organ in the human body, is also a complex ecosystem, home to a vast number of microorganisms, including bacteria, fungi, and viruses, on its surface and in its hair follicles. The skin microbiota plays a vital role in maintaining skin homeostasis, regulating immunity, resisting pathogens, and promoting the skin barrier. Numerous scientific studies have confirmed that the skin microbiota not only acts as a biological barrier but also participates in physical, chemical, and immune barriers, forming a comprehensive and multi-layered barrier protection function. Staphylococcus epidermidis is the most abundant bacterium on the skin, also known as a sentinel bacterium, and plays a crucial role in maintaining skin health. Staphylococcus epidermidis secretes sphingomyelinase, which hydrolyzes sphingomyelin into ceramides, enhancing the integrity of the skin barrier. Staphylococcus epidermidis produces short-chain fatty acids (such as butyrate), downregulating UVB-induced IL-6 and modulating inflammatory responses. Staphylococcus epidermidis produces phenol-soluble protein γ / δ (PSMγ / δ), which combats pathogens and synergistically enhances immune defense with antimicrobial peptides. Staphylococcus epidermidis can also inhibit the activation of the aryl hydrocarbon receptor (AHR) pathway, reduce reactive oxygen species (ROS), and inhibit the secretion of inflammatory cytokines. Although there is no clear definition of skin probiotics, Staphylococcus epidermidis has been recognized as a "guardian" of skin health and is expected to be a highly promising candidate strain of skin probiotics.

[0003] Extracellular vesicles (EVs) are a class of cell-secreted, lipid bilayer-structured nanoscale vesicles widely found in eukaryotic and prokaryotic cells. EVs contain various proteins, phospholipids, and nucleic acids (DNA and RNA), and possess multiple functions including transmembrane transport, biological signal transduction, and immune regulation. They play a crucial role in various bacterial-host biological processes, including virulence, horizontal gene transfer, cellular metabolite export, phage infection, and intercellular communication. Research has found that EVs, with their nanoscale size, non-replicability, and enhanced safety, exhibit unique advantages in immune regulation, antioxidant activity, and tissue repair, providing safe, efficient, and engineerable new strategies for the treatment of skin diseases.

[0004] Skin exposed to the elements is susceptible to external stimuli, including physical damage, microbial invasion, inflammation, and ultraviolet (UV) radiation, leading to skin damage accompanied by inflammatory responses and disruption of the skin barrier. Inflammatory skin diseases such as atopic dermatitis, acne, seborrheic dermatitis, sensitive skin, and dandruff all involve inflammation and impaired skin barrier function, exacerbating clinical symptoms. While Staphylococcus epidermidis plays a crucial role in maintaining the skin barrier and managing inflammation, whether its outer vesicles possess anti-inflammatory and barrier-protective functions remains to be thoroughly investigated. Summary of the Invention

[0005] The purpose of this invention is to provide Staphylococcus epidermidis exovesicles, their preparation method, and their applications.

[0006] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a method for preparing Staphylococcus epidermidis exovesicles, comprising the following steps: S1. Staphylococcus epidermidis CCSMS0776 was inoculated into TSB medium and cultured to obtain Staphylococcus epidermidis CCSMS0776 culture. S2. Centrifuge the Staphylococcus epidermidis CCSMS0776 culture obtained in step S1, collect the supernatant and ultrafilter it to obtain a concentrated solution. S3. Centrifuge the ultrafiltration concentrate obtained in step S2, collect the supernatant and filter it, then centrifuge the filtrate at ultra-high speed, collect the precipitate and resuspend it in PBS to obtain Staphylococcus epidermidis exovesicles.

[0007] In some specific implementations, step S1 involves the following culture conditions: 37°C, 160 r / min shaking culture for 16-24 h.

[0008] In some specific implementations, the centrifugation parameters for step S2 are: (9000 g~10000 g), centrifugation at 4℃ (15 min~25 min).

[0009] In some specific embodiments, in step S2, the ultrafiltration parameters are as follows: the supernatant is first filtered sequentially through vacuum filters with pore sizes of 0.45 µm and 0.22 µm, and then the filtrate is concentrated to 1 / 50 of the initial volume using a Vivaflow 200 laboratory crossflow chamber and a 100 kDa hydrogel membrane.

[0010] In some specific implementations, the centrifugation parameters for step S3 are: (12000 g~17000 g), centrifugation at 4℃ (20 min~40 min).

[0011] In some specific embodiments, step S3 involves filtering the supernatant using a vacuum filter with a pore size of 0.22 µm.

[0012] In some specific implementations, the parameters for ultra-high speed centrifugation in step S3 are: (120,000 g~170,000 g), centrifugation at 4°C (2.5 h~3.5 h).

[0013] The second technical solution of the present invention is to provide a Staphylococcus epidermidis exovesicle, which is obtained by the preparation method described in one of the above technical solutions. The Staphylococcus epidermidis exovesicle is a spherical structure wrapped by a lipid membrane, with a particle size of 180-190 nm and a zeta potential of -25.00 to -28.00 mV.

[0014] The third technical solution of the present invention is to provide the application of Staphylococcus epidermidis external vesicles as described in the second technical solution above in the preparation of drugs for treating inflammatory skin diseases and / or skin care products.

[0015] In some specific embodiments, the inflammatory skin disease is an inflammatory skin disease caused by UVB.

[0016] The fourth technical solution of the present invention is to provide the application of Staphylococcus epidermidis exovesicles as described in the second technical solution above in the preparation of skin anti-photodamage drugs and / or skin care products.

[0017] In some specific embodiments, the drug and / or skin care product is a drug and / or skin care product for protecting the skin from UVB photodamage.

[0018] In some specific embodiments, the drug and / or skin care product are drugs and / or skin care products used for skin repair and strengthening the skin barrier.

[0019] Compared with the prior art, the present invention has the following advantages: (1) The present invention prepares Staphylococcus epidermidis CCSMS0776 exovesicles by ultrafiltration combined with ultra-high speed centrifugation, which has good anti-inflammatory and barrier protection effects and can be used as a metabiotic for preventing photodamage and treating inflammatory skin diseases as well as for soothing skin care products.

[0020] (2) The present invention has been proven by experiments that the extracellular vesicles of Staphylococcus epidermidis CCSMS0776 present a complete and regular spherical structure with a lipid membrane, and the particle size and zeta potential are 180-190 nm and -25.00 ~ -28.00 mV, respectively, and have good stability.

[0021] (3) The extracellular vesicles of Staphylococcus epidermidis CCSMS0776 of the present invention can reduce the expression of inflammatory factors in HaCaT cells induced by UVB. Specifically, the extracellular vesicles of Staphylococcus epidermidis CCSMS0776 at a concentration of 25 μg / mL can significantly reduce the expression of inflammatory factors TNF-α, IL-6 and IL-1β in HaCaT cells induced by UVB, showing a good anti-inflammatory effect.

[0022] (4) The extracellular vesicles of Staphylococcus epidermidis CCSMS0776 of the present invention can significantly enhance UVB-induced HaCaT cell migration and TEER value, enhance skin barrier function, and have a good barrier protection effect.

[0023] (5) The extracellular vesicles of Staphylococcus epidermidis CCSMS0776 of the present invention show good anti-inflammatory and skin barrier repair effects, filling the gap in the field of symbiotic skin microorganisms in the field of photodamage prevention and anti-inflammation, and have broad application prospects in photodamage prevention, treatment of inflammatory skin diseases and soothing skin care products. Attached Figure Description

[0024] Figure 1 The particle size distribution of extracellular vesicles in Staphylococcus epidermidis CCSMS0776.

[0025] Figure 2 The ZETA potential is that of extracellular vesicles of Staphylococcus epidermidis CCSMS0776.

[0026] Figure 3 Transmission electron micrograph of extracellular vesicles of Staphylococcus epidermidis CCSMS0776.

[0027] Figure 4 This is a CLSM image of extracellular vesicles of Staphylococcus epidermidis CCSMS0776.

[0028] Figure 5 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on cell viability.

[0029] Figure 6 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on UVB-induced TNF-α expression levels in HaCaT cells.

[0030] Figure 7 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on UVB-induced IL-6 expression levels in HaCaT cells.

[0031] Figure 8 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on UVB-induced IL-1β expression levels in HaCaT cells.

[0032] Figure 9 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on UVB-induced HaCaT cell migration (representative image).

[0033] Figure 10 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on UVB-induced HaCaT cell migration.

[0034] Figure 11 The effect of extracellular vesicles of Staphylococcus epidermidis CCSMS0776 on UVB-induced TEER in HaCaT cells. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0037] The materials used in the following embodiments include: (1) Staphylococcus epidermidis CCSMS0776, which was deposited at the China General Microbiological Culture Collection Center on June 5, 2024, with the accession number CGMCC 30873.

[0038] (2) Tryptic soybean liquid culture medium (TSB): 15 g / L tryptic, 5 g / L soybean papain hydrolysate, sodium chloride, autoclaved at 121℃ for 15 min, ready for use.

[0039] In the following embodiments, unless otherwise specified, the materials and processes used are conventional materials and processes employed in the art to achieve the corresponding functions.

[0040] Example 1 This embodiment provides an extracellular vesicle from Staphylococcus epidermidis CCSMS0776, the preparation method of which includes the following steps: Staphylococcus epidermidis CCSMS0776 was inoculated into TSB medium and cultured at 37°C with shaking at 160 r / min for approximately 20 h to obtain a culture of Staphylococcus epidermidis CCSMS0776. First, the culture was centrifuged for 20 min (4°C, 10000 g). Then, the culture supernatant was filtered sequentially through bacterial filters with pore sizes of 0.45 µm and 0.22 µm to obtain the filtrate. Next, the filtrate was concentrated to 1 / 50 of its initial volume using ultrafiltration (Vivaflow 200 tangential flow filtration coupled with a 100 kDa hydrogel membrane) to obtain the concentrate. The concentrate was centrifuged for 30 min (4°C, 15000 g) and then filtered through a vacuum filter with a pore size of 0.22 μm. Finally, the concentrate was centrifuged for 3 h (4°C, 150,000 g), and the precipitate was collected to obtain extracellular vesicles of Staphylococcus epidermidis CCSMS0776 (named SE 776-EVs). After washing in PBS (4°C, 150,000 g centrifuged for 3 h), the precipitate was resuspended in PBS and stored at -80°C.

[0041] The prepared Staphylococcus epidermidis CCSMS0776 extracellular vesicles SE 776-EVs were subjected to the following tests: (1) Characterization Ten mL of the SE 776-EVs prepared in Example 1 was deposited onto a carbon-coated copper grid. After 30 min, the grid was dried with filter paper and then stained with 10 mL of 1% phosphotungstic acid for 5 min. The shape of the SE 776-EVs prepared in Example 1 was observed by transmission electron microscopy (TEM). The SE 776-EVs prepared in Example 1 were characterized by nanoparticle tracking analysis (NTA) to obtain the particle size, particle concentration, and surface charge (zeta potential, in mV) of the SE 776-EVs prepared in Example 1. Subsequently, the grid was air-dried and observed by transmission electron microscopy at 100 kV. SE 776-EVs were labeled with 0.1% (w / w) (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) DiI for 30 min, followed by washing three times with PBS through a 100 kDa ultrafiltration tube to remove unbound dye. DiI-stained SE 776-EVs were observed using confocal laser scanning microscopy (CLSM).

[0042] from Figure 1 , Figure 2 , Figure 3 and Figure 4 It can be seen that the extracellular vesicles SE 776-EVs of Staphylococcus epidermidis CCSM0776 prepared in Example 1 exhibit a complete and regular spherical structure with a lipid membrane. The particle size and zeta potential of SE 776-EVs are 186.5 ± 3.62 nm and -28.00 ± 0.63 mV, respectively.

[0043] (2) Protein content The protein concentration of SE776-EVs prepared in Example 1 was determined using the ASPEN BCA Protein Concentration Assay Kit (catalog number: AS1086).

[0044] That is, the SE 776-EVs prepared in Example 1 were diluted 10 times, and the OD... 562 The value of 0.5228 is substituted into the standard equation Y = -0.21394222 + 0.99152752X, where Y represents the protein concentration of the standard curve and X represents OD. 562 After conversion, the concentration of SE 776-EVs prepared in Example 1 was found to be 3.044 mg / mL.

[0045] (3) Cell viability and anti-inflammatory effects (3-1) Culture of HaCat cells (human immortalized epidermal cells) HaCaT cells were purchased from iCell Bioscience (catalog number iCell-h066). Cells were cultured at 37°C in DMEM medium containing 4.5 g / L of D-glucose with 10% fetal bovine serum at 5% CO2. Cell confluence in plates reached 80-90%, and the cells were used for further experiments.

[0046] (3-2) Cell viability experiment Cell viability was assessed using a CCK-8 assay kit (Biosharp, catalog number BS350b) according to the manufacturer's instructions. Specifically, HaCaT cells were cultured at a concentration of 2 × 10⁻⁶ cells / mL. 4 Cells were seeded in 96-well plates and treated with different concentrations of SE 776-EVs prepared in Example 1 for 24 h. CCK-8 solution was then added to all wells, and the plates were incubated for another 2 h. Absorbance at 450 nm was measured using a microplate reader. Untreated cells served as the control group, and cell-free culture medium served as the blank group.

[0047] Depend on Figure 5It can be seen that, within the concentration range of 0–50 μg / mL, the cell viability of the SE 776-EVs prepared in Example 1 showed a trend of first increasing and then decreasing with increasing concentration, without exhibiting cytotoxicity. The highest cell viability (141.75%) was observed when the concentration of SE 776-EVs prepared in Example 1 reached 25 μg / mL. Although the cell viability of the SE 776-EVs prepared in Example 1 showed a decreasing trend within the concentration range of 25–50 μg / mL, the cell viability of the SE 776-EVs prepared in Example 1 at a concentration of 50 μg / mL was 113.65%, still higher than that of the blank control group (100%).

[0048] (3-3) Detection of inflammatory factors The effects of SE 776-EVs prepared in Example 1 on the expression levels of inflammatory cytokines in HaCaT cells exposed to UVB irradiation were measured using an ELISA kit containing TNF-α (ELK Biotechnology, catalog number: ELK1190), IL-6 (ELK Biotechnology, catalog number: ELK1156), and IL-1β (ELK Biotechnology, catalog number: ELK1270). The UVB irradiation protocol was as follows: a 311 nm UVB lamp (PHILIPS, PL-S 9W / 01, Poland) was used at 100 μw / cm². 2 At the given irradiation dose, the exposure time is 300 s, and the irradiation dose is calculated using the formula: 100 μw / cm². 2 × 300 s = 30mJ / cm 2 .

[0049] HaCaT cells exposed to UVB irradiation were treated with different concentrations of SE 776-EVS for 24 h, and the supernatant was collected to determine the levels of TNF-α, IL-1β and IL-6.

[0050] Figure 6 , Figure 7 and Figure 8 The figures represent the effects of SE 776-EVs prepared in Example 1 on the expression levels of TNF-α, IL-6, and IL-1β in HaCaT cells exposed to UVB. Compared with the control group, UVB irradiation caused a significant increase in TNF-α, IL-6, and IL-1β, reaching 103.74, 136.02, and 60.46 pg / mL, respectively. P< 0.0001), and after treatment with SE 776-EVs prepared in Example 1, the expression levels of the above three inflammatory cytokines showed a gradual decreasing trend with increasing concentration. Compared with the UVB treatment group, when HaCaT cells irradiated by UVB were treated with SE 776-EVs prepared in Example 1 at a concentration of 25 μg / mL, the expression levels of TNF-α, IL-6, and IL-1β decreased to 67.72 pg / mL ( P < 0.0001), 76.59 pg / mL ( P < 0.0001) and 28.24 pg / mL ( P < 0.0001). When the concentration of SE 776-EVs prepared in Example 1 reached 50 μg / mL, the expression levels of inflammatory factors in HaCaT cells decreased to the lowest level, with TNF-α, IL-6, and IL-1β decreasing to 56.17 pg / mL, 54.11 pg / mL, and 24.01 pg / mL, respectively, all showing significant differences. P < 0.0001.

[0051] (4) Cell migration Cell migration was assessed using a cell scratch assay. HaCaT cells were seeded at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and incubated until a confluent monolayer formed. A line was then drawn at the bottom of the culture dish using the tip of a sterile 200 μL pipette. Then, [the solution was applied] with 30 mJ / cm [fluid]. 2 UVB or 30 mJ / cm 2 Scratch cells were treated with UVB and 25 μg / mL SE 776-EVs. The scratch cells were gently rinsed with PBS, and images of the scratch were taken under a microscope at the 0-hour time point. Incubation continued, and the wound was imaged at 12-h and 24-h time points using a microscope (OLYMPUS, IX51, Japan). The migration area was then quantified using an imaging system (Q-IMAGING, MicroPublisher, CAN) to assess wound healing rate. The experiment included three independent biological replicates. Cell migration rate was calculated as follows: Cell migration rate (%) = [(Initial scratch width - Final scratch width) / Initial scratch width] × 100.

[0052] Figure 9 and Figure 10 These represent the effects of SE 776-EVs prepared in Example 1 on UVB-induced HaCaT cell migration.

[0053] Depend on Figure 9It can be seen that, compared with HaCaT cells that are only exposed to UVB, SE 776-EVs significantly promoted the migration of UVB-induced HaCaT cells.

[0054] Depend on Figure 10 It was found that at 12 h, the migration rate of HaCaT cells induced by UVB alone was 21.99%, while the migration rate of HaCaT cells induced by UVB treated with SE 776-EVs was 31.26%. P < 0.0001). At 24 h, the difference in cell migration rate was more pronounced; the migration rate of HaCaT cells treated with UVB alone was 36.16%, while the migration rate of HaCaT cells treated with SE 776-EVs and treated with UVB was 55.32%. P < 0.0001). The results indicate that SE 776-EVs have a skin-repairing effect.

[0055] (5) Transepithelial electrical resistance (TEER) effect HaCaT cells were used at a rate of 1 × 10⁻⁶ per well. 5 Cells were seeded at a density of 1,000 cells per 1,000 cm² on 24-well polycarbonate transporous membranes (Corning, 3422, USA), with an average pore size of 8.0 μm and a surface area of ​​0.33 cm². 2 Using 30 mJ / cm 2 UVB or 30 mJ / cm 2 Cells were treated with UVB and 25 μg / mL SE 776-EVs. After 24 h, resistance values ​​were recorded using a resistance system (Millicell, ERS2, USA). Resistance values ​​of culture plates without cultured cells were also recorded as a control group. The actual TEER of HaCaT cells in each group was calculated using the formula: TEER (Ω·cm) 2 = (Measured resistance - Blank resistance) × Effective film area (0.33 cm²) 2 ).

[0056] Figure 11 This demonstrates the effect of SE 776-EVs prepared in Example 1 on UVB-induced TEER in HaCaT cells. Figure 11 It can be seen that when HaCaT cells were exposed to UVB irradiation, the TEER value at 24 h was 119.03 Ω·cm. 2 After treatment with SE776-EVs, the TEER value significantly increased to 181.7 Ω·cm. 2 ( P< 0.0001). The results indicate that SE 776-EVs have the effect of enhancing the skin barrier.

[0057] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing Staphylococcus epidermidis exovesicles, characterized in that, Includes the following steps: S1. Staphylococcus epidermidis CCSMS0776 was inoculated into TSB medium and cultured to obtain Staphylococcus epidermidis CCSMS0776 culture. S2. Centrifuge the Staphylococcus epidermidis CCSMS0776 culture obtained in step S1, collect the supernatant and ultrafilter it to obtain a concentrated solution. S3. Centrifuge the ultrafiltration concentrate obtained in step S2, collect the supernatant and filter it, then centrifuge the filtrate at ultra-high speed, collect the precipitate and resuspend it in PBS to obtain Staphylococcus epidermidis exovesicles.

2. The method for preparing Staphylococcus epidermidis exovesicles according to claim 1, characterized in that, Step S1, culture conditions are: 37℃, 160 r / min shaking culture for 16-24 h.

3. The method for preparing Staphylococcus epidermidis exovesicles according to claim 1, characterized in that, In step S2, the centrifugation parameters are: (9000 g~11000 g), centrifugation at 4℃ (15 min~25 min).

4. The method for preparing Staphylococcus epidermidis exovesicles according to claim 1, characterized in that, In step S2, the ultrafiltration parameters are as follows: the supernatant is first filtered sequentially through vacuum filters with pore sizes of 0.45 µm and 0.22 µm, and then the filtrate is concentrated to 1 / 50 of the initial volume using a Vivaflow 200 laboratory crossflow chamber and a 100 kDa hydrogel membrane.

5. The method for preparing Staphylococcus epidermidis exovesicles according to claim 1, characterized in that, Step S3, centrifugation parameters: (12000 g ~ 17000 g), centrifugation at 4℃ (20 min ~ 40 min).

6. The method for preparing Staphylococcus epidermidis exovesicles according to claim 1, characterized in that, In step S3, the filtration parameters are as follows: the supernatant is filtered using a vacuum filter with a pore size of 0.22 µm.

7. The method for preparing Staphylococcus epidermidis exovesicles according to claim 1, characterized in that, Step S3, ultra-high speed centrifugation parameters: (120000 g~170000 g), centrifugation at 4℃ (2.5 h~3.5 h).

8. A Staphylococcus epidermidis exovesicle, characterized in that, The preparation method described in any one of claims 1 to 6 yields Staphylococcus epidermidis extravesicles, which are spherical structures enclosed by a lipid membrane, with a particle size of 180-190 nm and a zeta potential of -25.00 to -28.00 mV.

9. The use of Staphylococcus epidermidis vesicles as described in claim 7 in the preparation of medicaments for treating inflammatory skin diseases and / or skin care products.

10. The use of Staphylococcus epidermidis exovesicles as described in claim 7 in the preparation of skin protection drugs against photodamage and / or skin care products.

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