Preparation method and application of purslane extracellular vesicles

Abstract

The invention relates to application of a plant-derived extracellular vesicle and a compound thereof in preparation of a pharmaceutical composition for treating or preventing diseases caused by high colonization degree of gram-positive bacteria in skin, in particular to application of a purslane extracellular vesicle and a compound of the purslane extracellular vesicle and hydrogel.

Description

Technical Field

[0001] This invention relates to the field of natural product antibacterial agents, specifically to a method for preparing and identifying extracellular vesicles derived from fresh purslane and their application in inhibiting Staphylococcus aureus. Background Technology

[0002] Staphylococcus aureus (S. aureus) is a common clinical pathogen that can cause purulent skin infections, pneumonia, and other diseases, and its drug resistance is becoming an increasingly serious problem. The traditional Chinese medicine purslane (Portulaca oleracea L.) is recorded in ancient books such as *Xian Nian Ji* as a "treatment for sores and ulcers" when pounded and applied (Figure 1). Modern research has confirmed that its fresh product is significantly effective against skin diseases such as atopic dermatitis (AD) and also possesses antibacterial activity.

[0003] Extracellular vesicles (EVs) are nanoscale membrane vesicles rich in bioactive components, possessing advantages such as low immunogenicity and high biocompatibility. The inventors previously extracted extracellular vesicles (P-EVs) from fresh purslane, which showed excellent anti-Staphylococcus aureus activity; however, their standardized preparation and mechanism of action remain unclear. Summary of the Invention

[0004] This invention aims to provide an efficient and stable method for preparing extracellular vesicles from fresh purslane, establish systematic identification standards, and elucidate its application value in inhibiting Staphylococcus aureus.

[0005] This invention provides a method for preparing plant-derived extracellular vesicles, comprising the following steps:

[0006] (1) Clean the plants;

[0007] (2) Crush the plant and filter to obtain plant juice;

[0008] (3) Centrifuge at differential speed and collect the supernatant;

[0009] (4) The supernatant collected in step (3) is filtered;

[0010] (5) Centrifuge at high speed and collect the precipitate;

[0011] (6) Resuspension step (5) The final collected precipitate is used to obtain extracellular vesicles.

[0012] In one or more embodiments, the plant is a Portulaca family plant.

[0013] In one or more embodiments, the plant is a plant of the genus *Portulaca*.

[0014] In one or more embodiments, the plant is purslane.

[0015] In one or more embodiments, the plant-derived extracellular vesicles are purslane extracellular vesicles (P-EVs).

[0016] In one or more embodiments, the crushing in step (1) is carried out under the conditions of 2000W power and 20,000 rpm rotation speed.

[0017] In one or more embodiments, the cleaning in step (1) includes a preliminary cleaning with purified water followed by a rinse with PBS buffer. In one or more embodiments, the plant in step (1) is a fresh whole purslane plant.

[0018] In one or more embodiments, the crushing in step (2) includes: (i) cutting the plant into pieces, preferably into small pieces with a length of 10 to 20 mm, and (ii) adding sterile PBS buffer at a weight-to-volume ratio of 1:1 to 1:5 (g / mL) and crushing for 5 to 10 minutes.

[0019] In one or more embodiments, the disruption in step (2) includes: (i) cutting the plant into pieces of 10 to 20 mm in length, and (ii) adding sterile PBS buffer at a weight-to-volume ratio of 1:3 (g / mL) and disrupting for 5 to 10 minutes, preferably 7 minutes.

[0020] In one or more embodiments, the filtration in step (2) is: after crushing, filtering is performed using a 200-mesh screen.

[0021] In one or more embodiments, the differential centrifugation in step (3) is performed as follows: (i) centrifuging at 100-300×g (preferably 200×g) for 5-15 minutes and collecting the supernatant; (ii) centrifuging at 1000-3000×g (preferably 2000×g) for 10-30 minutes and collecting the supernatant; and (iii) centrifuging at 7000-15000×g for 20-40 minutes and collecting the supernatant.

[0022] In one or more embodiments, the differential centrifugation in step (3) is performed sequentially at 4±0.5°C as follows: (i) centrifugation at 200×g for 10 minutes, collecting the supernatant; (ii) centrifugation at 2000×g for 20 minutes, collecting the supernatant; and (iii) centrifugation at 10000×g for 30 minutes, collecting the supernatant.

[0023] In one or more embodiments, the processing filtration in step (4) is filtration using a 0.22-0.45 μm filter membrane.

[0024] In one or more embodiments, the treatment in step (4) is to filter the material sequentially using 0.45 μm and 0.22 μm filter membranes.

[0025] In one or more embodiments, the high-speed centrifugation in step (5) is 80,000-120,000 × g for 1 to 2 hours.

[0026] In one or more embodiments, the high-speed centrifugation in step (5) is: centrifuging at 100,000 × g for 1.05 hours at 4 ± 0.5 °C, and collecting the precipitate.

[0027] In one or more embodiments, the resuspension in step (6) is resuspension using PBS solution.

[0028] In one or more embodiments, the P-Evs particle size is 30-150 nm, preferably 50-100 nm, and more preferably 60-90 nm.

[0029] This invention provides a plant-derived extracellular vesicle prepared by the method described in any embodiment of this invention.

[0030] In one or more embodiments, the plant is a Portulaca family plant.

[0031] In one or more embodiments, the plant is a plant of the genus *Portulaca*.

[0032] In one or more embodiments, the plant is purslane.

[0033] In one or more embodiments, the plant-derived extracellular vesicles are purslane extracellular vesicles (P-EVs).

[0034] In one or more embodiments, the P-EVs have a particle size of 30-150 nm, preferably 50-100 nm, and more preferably 60-90 nm.

[0035] In one or more embodiments, the P-EVs have antibacterial activity against Gram-positive bacteria, preferably, the P-EVs have antibacterial activity against Staphylococcus aureus.

[0036] In one or more embodiments, the P-EVs achieve antibacterial effects by disrupting bacterial cell wall integrity and inhibiting respiratory chain complex I activity.

[0037] The present invention also provides a plant-derived extracellular vesicle complex comprising the plant-derived extracellular vesicles and carrier described in any embodiment herein.

[0038] In one or more embodiments, the plant is a Portulaca family plant.

[0039] In one or more embodiments, the plant is a plant of the genus *Portulaca*.

[0040] In one or more embodiments, the plant is purslane.

[0041] In one or more embodiments, the plant-derived extracellular vesicles are purslane extracellular vesicles (P-EVs).

[0042] In one or more embodiments, the carrier is a hydrogel.

[0043] In one or more embodiments, the hydrogel is selected from at least one of alginate, gelatin, matrix gel, collagen, chitosan, fibrin, polyethylene glycol, and methacrylic anhydride gelatin (GelMA) hydrogel.

[0044] In one or more embodiments, the hydrogel is a GelMA hydrogel.

[0045] In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel.

[0046] In one or more embodiments, the concentration of the P-EVs-GelMA hydrogel is 1 ug / ul.

[0047] This invention also provides a method for preparing a plant-derived extracellular vesicle complex as described in any embodiment of this invention, comprising:

[0048] (1) Prepare the initiator standard solution.

[0049] (2) Add the initiator described in step (1) to the solid GelMA to prepare a GelMA hydrogel solution.

[0050] (3) Extracellular vesicles were obtained using the plant-derived extracellular vesicle preparation method described in this article.

[0051] (4) Mix extracellular vesicles and hydrogel in a certain proportion to prepare a complex.

[0052] In one or more embodiments, the initiator concentration in step (1) is 0.25%.

[0053] In one or more embodiments, the concentration of the GelMA hydrogel solution in step (2) is 10%.

[0054] In one or more embodiments, the ratio of step (4) is 1 mg of vesicles to 1 mL of 10% GelMA hydrogel, and the preparation conditions are mixing at 37°C.

[0055] In one or more embodiments, the plant-derived extracellular vesicle complex is a P-EVs-GelMA hydrogel.

[0056] In one or more embodiments, the concentration of the P-EVs-GelMA hydrogel is 1 ug / ul.

[0057] The present invention also provides the use of plant-derived extracellular vesicles or complexes in the preparation of pharmaceutical compositions for treating or preventing diseases of skin barrier dysfunction, abnormal immune system response, skin microbiome imbalance, skin flora imbalance and / or skin inflammation, said pharmaceutical composition further comprising pharmaceutically acceptable excipients.

[0058] In one or more embodiments, the pharmaceutically acceptable excipients include carriers, diluents, and / or excipients.

[0059] In one or more embodiments, the pharmaceutical composition may be in the form of, but is not limited to, topical ointments, gels, and skin sprays.

[0060] In one or more embodiments, the plant is a Portulaca family plant.

[0061] In one or more embodiments, the plant is a plant of the genus *Portulaca*.

[0062] In one or more embodiments, the plant is purslane.

[0063] In one or more embodiments, the plant-derived extracellular vesicles are purslane extracellular vesicles (P-EVs).

[0064] In one or more embodiments, the plant-derived extracellular vesicle complex comprises plant-derived extracellular vesicles and a carrier.

[0065] In one or more embodiments, the carrier is a hydrogel, preferably a GelMA hydrogel.

[0066] In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel.

[0067] In one or more embodiments, the disease is a chronic skin condition, such as atopic dermatitis (AD), eczema, psoriasis, or pityriasis rosea.

[0068] In one or more embodiments, the plant-derived extracellular vesicles are prepared by the method described in any of the embodiments herein.

[0069] The present invention also provides the use of plant-derived extracellular vesicles or complexes in the preparation of pharmaceutical compositions for the treatment or prevention of diseases with high colonization of Gram-positive bacteria in the skin, said pharmaceutical compositions further comprising pharmaceutically acceptable excipients.

[0070] In one or more embodiments, the pharmaceutically acceptable excipients include carriers, diluents, and / or excipients.

[0071] In one or more embodiments, the pharmaceutical composition may be in the form of, but is not limited to, topical ointments, gels, and skin sprays.

[0072] In one or more embodiments, the plant is a Portulaca family plant.

[0073] In one or more embodiments, the plant is a plant of the genus *Portulaca*.

[0074] In one or more embodiments, the plant is purslane.

[0075] In one or more embodiments, the plant-derived extracellular vesicles are purslane extracellular vesicles (P-EVs).

[0076] In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel.

[0077] In one or more embodiments, in the disease, Gram-positive bacteria colonize the skin at a rate greater than 70%, preferably greater than 80% or 90%.

[0078] In one or more embodiments, the disease is a skin disease. In one or more embodiments, the Gram-positive antigen load in the skin reaches 10. 5 CFU / cm² or above, 10 6 CFU / cm² or above or 10 7 CFU / cm² or higher.

[0079] In one or more embodiments, the Gram-positive bacterium is Staphylococcus aureus.

[0080] In one or more embodiments, the disease is a disease in which Staphylococcus aureus colonizes the skin to a degree of 70%, preferably, the degree of colonization is greater than 80% or 90%.

[0081] The present invention also provides a method for inhibiting Gram-positive bacteria in vitro, comprising the step of contacting plant-derived extracellular vesicles or complexes thereof with a sample for which Gram-positive bacteria need to be inhibited.

[0082] In one or more embodiments, the extracellular vesicles are purslane extracellular vesicles.

[0083] In one or more embodiments, the extracellular vesicles are obtained by a preparation method as described in any embodiment herein.

[0084] In one or more embodiments, the Gram-positive bacterium is Staphylococcus aureus.

[0085] In one or more embodiments, the ratio of Gram-positive bacteria to extracellular vesicles is from 1*10E5 cfu / ml: 1-1.5 μg / μl to 9*10E5 cfu / ml: 1-1.5 μg / μl.

[0086] In one or more embodiments, the ratio of Gram-positive bacteria to extracellular vesicles is from 1*10E5 cfu / ml: 1.242 μg / μl to 9*10E5 cfu / ml: 1.242 μg / μl.

[0087] In one or more embodiments, the contact lasts for at least 2 hours.

[0088] The present invention also provides a Gram-positive bacteria inhibitor comprising plant-derived extracellular vesicles or complexes thereof.

[0089] In one or more embodiments, the extracellular vesicles are purslane extracellular vesicles.

[0090] In one or more embodiments, the extracellular vesicles are obtained by the preparation method described in any of the embodiments herein.

[0091] In one or more embodiments, the concentration of extracellular vesicles in the inhibitor is 1-1.5 μg / μl.

[0092] In one or more embodiments, the concentration of extracellular vesicles in the inhibitor is 1.242 μg / μl.

[0093] In one or more embodiments, the Gram-positive bacterium is Staphylococcus aureus.

[0094] The beneficial effects of this invention are:

[0095] This invention proposes for the first time a standardized preparation process for fresh purslane extracellular vesicles (P-EVs), clarifying the mechanism of action of P-EVs against Staphylococcus aureus. Compared with traditional extracts, P-EVs have advantages such as strong targeting and stable active ingredients, achieving an antibacterial rate of 70%. P-EVs have a wide range of raw material sources and a simple preparation process, and can be extended to the treatment of various Gram-positive bacterial infections. Attached Figure Description

[0096] Figure 1 This is a record of purslane treating sores and tinea in Volume 4, "Surgery," of the book "Xian Nian Ji" (a Tongdetang edition published in the 19th year of the Qianlong reign of the Qing Dynasty).

[0097] Figure 2 This is a schematic diagram of the P-EVs extraction process.

[0098] Figure 3 In the image, A is an electron microscope image of P-EVs, and B is the particle size distribution of P-EVs.

[0099] Figure 4 In the table, A shows the immunofluorescence of DiI-stained P-EVs on the back skin of AD mice treated with PBS; B shows DiI-stained Portulaca oleracea vesicle P-EVs applied externally to the skin of AD mice. Note: BF is bright field, without fluorescence; DAPI is blue, used to stain cell nuclei; DiI is red, used to stain Portulaca oleracea vesicle P-EVs; MERGE was used to co-localize DAPI and DiI.

[0100] Figure 5 The therapeutic effect of P-EVs on AD mice.

[0101] Figure 6 To observe the skin pathology of AD mice by P-EVs.

[0102] Figure 7 The graph shows the antibacterial effect of P-EVs on Staphylococcus aureus. In Figure A, the control group is in the first row, and the P-EVs treatment group is in the second row.

[0103] Figure 8 To observe the effects of P-EVs on the morphology and structure of Staphylococcus aureus under electron microscopy (×15000), Figure A shows the control group; Figure B shows the P-EVs treatment group. Detailed Implementation

[0104] This invention aims to provide an efficient and stable method for preparing extracellular vesicles from fresh purslane, establish systematic identification standards, and elucidate its application value in inhibiting Staphylococcus aureus.

[0105] As used herein, the term "extracellular vesicles (EVs)" refers to nanoscale vesicles with a lipid bilayer structure released by cells. These vesicles can carry bioactive molecules such as proteins, RNA, and DNA, and target specific cells through their membrane surface properties to regulate cellular function. The term "purslane extracellular vesicles (P-EVs)" refers to nanoscale lipid vesicles isolated and extracted from the plant purslane, which contain a variety of bioactive components of the plant (purslane).

[0106] This invention provides a method for producing plant-derived extracellular vesicles, comprising the following steps: (1) washing the plant with purified water and PBS; (2) crushing the plant at a power of 2000W and a rotation speed of 20,000 rpm, and filtering to obtain plant juice; (3) centrifuging at a differential speed and collecting the supernatant; (4) processing the supernatant collected in step (3); (5) centrifuging at a high speed and collecting the precipitate; (6) processing the precipitate collected in step (5) to obtain extracellular vesicles. In some embodiments, the plant is a Portulacaceae family and a Portulaca genus, and the plant-derived extracellular vesicles are Portulaca extracellular vesicles (P-EVs). Specifically, the cleaning in step (1) includes initial cleaning with purified water and rinsing with PBS buffer, wherein the plant is a fresh whole purslane plant; the crushing in step (2) includes first cutting the plant into small pieces with a length of 10-20 mm, then adding sterile PBS buffer at a weight-to-volume ratio of 1:3 (g / mL), and crushing in a juicer for 5-10 minutes, preferably 7 minutes; the filtration in step (2) is to filter using a 200-mesh screen; the differential centrifugation in step (3) is to centrifuge at 4±0.5℃ for 10 minutes at 200×g, 2000×g for 20 minutes, and 10000×g for 30 minutes, and collect the supernatant; the treatment in step (4) is to use 0.45μm and 0.22μm filters sequentially. The process involved membrane filtration; high-speed centrifugation in step (5) was performed at 100,000 × g for 1.05 hours at 4 ± 0.5 °C, and the precipitate was collected; the treatment in step (6) involved resuspending the precipitate in sterile PBS solution and bringing the volume to 50 mL. The extracellular vesicles collected by the above method were analyzed for concentration using the BCA method (the sample to be tested was exosome lysis buffer), and the protein concentration in the solution was found to be 1.24 μg / μl. In this invention, the BCA assay can obtain a reliable and quantifiable indicator (total protein concentration) for precise standardization of all subsequent experiments.

[0107] Furthermore, the present invention also identifies the P-EVs collected by the method, mainly including morphological observation, particle size analysis, and functional verification. In one or more embodiments, the extracellular vesicles are morphologically observed using a transmission electron microscope (Hitachi HT7800), including an electron microscope sample preparation step: 10 μL of vesicle suspension is added to a copper mesh, fixed with 2.5% glutaraldehyde, washed with double-distilled water, stained with 2% uranium acetate, and then air-dried. In some embodiments, the P-EVs appear as typical saucer-shaped or concave hemispherical shapes under a transmission electron microscope. In one or more embodiments, the particle size of the P-EVs is detected using nanoflow cytometry, with a particle size of 30-150 nm, preferably 50-100 nm, more preferably 60-90 nm, an average particle size of 86.13 nm, and a concentration of 2.6 × 10⁻⁶.11 Particles / mL.

[0108] As used in this invention, Staphylococcus aureus possesses an electron transfer chain (ETC), also commonly referred to as the respiratory chain. This chain is a series of protein complexes and electron carriers located on the inner mitochondrial membrane (or prokaryotic membrane). Its main function is to transfer high-energy electrons from one carrier to another through a series of redox reactions, and to use the released energy to pump protons out of the membrane, establishing an electrochemical gradient and ultimately driving ATP synthesis. Staphylococcus aureus is a prokaryote, and its ETC is located on its membrane. In addition, Staphylococcus aureus contains four transmembrane protein complexes: (1) Complex I: also known as NADH dehydrogenase, its function is to receive high-energy electrons from NADH and transfer them to ubiquinone. In this process, Complex I pumps 4 protons from the mitochondrial matrix to the intermembrane space; (2) Complex II: also known as succinate dehydrogenase, its function is to receive electrons from succinate (from the citric acid cycle) and transfer them to ubiquinone; (3) Complex III: also known as cytochrome c reductase, its function is to receive electrons from reduced ubiquinone and transfer them to cytochrome c. In this process, Complex III pumps 4 protons to the intermembrane space; (4) Complex IV: also known as cytochrome c oxidase, its function is to receive electrons from cytochrome c and ultimately transfer these electrons to oxygen molecules, reducing them to water (this process is the process by which oxygen is consumed in respiration). In this process, Complex IV pumps 2 protons to the intermembrane space.

[0109] In one or more embodiments, the P-EVs also have an inhibitory effect on Staphylococcus aureus, which achieves the antibacterial effect by disrupting the integrity of the bacterial cell wall and inhibiting the activity of respiratory chain complex I, with an antibacterial rate of ≥70% against Staphylococcus aureus.

[0110] Accordingly, the present invention also provides plant-derived extracellular vesicles produced by the method described above. Specifically, the plant is a plant of the Portulacaceae family and the Portulaca genus, and the plant-derived extracellular vesicles are Portulaca extracellular vesicles (P-EVs). In one or more embodiments, the P-EVs have antibacterial activity against Gram-positive bacteria, preferably, the P-EVs have antibacterial activity against Staphylococcus aureus. In one or more embodiments, the P-EVs achieve their antibacterial effect by disrupting the integrity of bacterial cell walls and inhibiting the activity of respiratory chain complex I.

[0111] In this article, the term "initiator" refers to a substance capable of initiating a specific chemical reaction. In a narrow sense, an initiator is a substance that can initiate the polymerization reaction of monomers, including free radicals, anionic compounds, cationic compounds, and coordination compounds. Initiators initiate polymerization reactions by homolytically cleaving covalent bonds under environmental changes (such as the effects of heat or light).

[0112] The term "hydrogel" refers to a class of soft materials with a three-dimensional (3D) network structure, characterized by a high degree of tunability in their physical and chemical properties. The starting materials for preparing polymeric hydrogel materials can be monomers (water-soluble or oil-soluble monomers), polymers (natural or synthetic polymers), or mixtures of monomers and polymers.

[0113] To deliver the extracellular vesicles described in this invention, a functionalized hydrogel prepared by chemical cross-linking is provided. This material exhibits excellent water absorption and retention capacity, stability of the three-dimensional network structure, and highly tunable physicochemical properties, as well as good biocompatibility and biodegradability. In some embodiments, the hydrogel components are selected from one or more of alginate, gelatin, matrix gum, collagen, chitosan, fibrin, polyethylene glycol, methacrylamide, and methacrylic acid hydrogels. As an example of delivering extracellular vesicles, the hydrogel is prepared from methacrylic acid gelatin (GelMA).

[0114] Therefore, the present invention also provides a plant-derived extracellular vesicle complex comprising the extracellular vesicles and carrier described in any embodiment herein. In one or more embodiments, the carrier is a hydrogel, preferably a zwitterionic hydrogel, more preferably a GelMA hydrogel. In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel with a concentration of 1 ug / ul.

[0115] Accordingly, a method for preparing the extracellular vesicle complex is also provided, comprising: (1) preparing an initiator standard solution; (2) adding the initiator from step (1) to GelMA solid to prepare a GelMA hydrogel solution; (3) obtaining extracellular vesicles by the method described herein; and (4) mixing the extracellular vesicles and hydrogel in a certain proportion to prepare the complex. Wherein, the concentration of the initiator in step (1) is 0.25%; the concentration of the GelMA hydrogel solution in step (2) is 10%; and the proportion in step (4) is 1 mg of vesicle mixed with 1 mL of 10% GelMA hydrogel, and the preparation conditions are mixing at 37°C. In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel, and the concentration of the P-EVs-GelMA hydrogel is 1 ug / ul.

[0116] This invention also provides a method for in vitro inhibition of Gram-positive bacteria, comprising the step of contacting a plant-derived extracellular vesicle or a complex thereof with a sample for which Gram-positive bacteria need to be inhibited. In one or more embodiments, the extracellular vesicle is a Portulaca oleracea extracellular vesicle. In one or more embodiments, the extracellular vesicle is obtained by the preparation method described in any embodiment herein. In one or more embodiments, the Gram-positive bacteria is Staphylococcus aureus. In one or more embodiments, the ratio of Gram-positive bacteria to extracellular vesicles is from 1*10E5 cfu / ml: 1-1.5 μg / μl to 9*10E5 cfu / ml: 1-1.5 μg / μl, preferably from 1*10E5 cfu / ml: 1.242 μg / μl to 9*10E5 cfu / ml: 1.242 μg / μl. In one or more embodiments, the contact lasts for at least 2 hours.

[0117] This invention also provides a Gram-positive bacteria inhibitor comprising plant-derived extracellular vesicles or complexes thereof. In one or more embodiments, the extracellular vesicles are purslane extracellular vesicles; the extracellular vesicles are obtained by the preparation method described in any embodiment herein. In one or more embodiments, the concentration of extracellular vesicles in the inhibitor is 1-1.5 μg / μl, preferably, the concentration of extracellular vesicles in the inhibitor is 1.242 μg / μl. In one or more embodiments, the Gram-positive bacterium is Staphylococcus aureus.

[0118] This invention provides the use of plant-derived extracellular vesicles or complexes in the preparation of pharmaceutical compositions for treating or preventing skin barrier dysfunction, abnormal immune system responses, skin microbiome imbalance, skin flora imbalance, and / or skin inflammation, said pharmaceutical compositions further comprising pharmaceutically acceptable excipients. The forms of said pharmaceutical compositions include, but are not limited to, topical ointments, gels, and skin sprays. In one or more embodiments, the plant is a plant of the genus *Portulaca* in the family Portulacaceae. In one or more embodiments, the plant-derived extracellular vesicles are *Portulaca oleracea* extracellular vesicles (P-EVs). In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel. In one or more embodiments, the pharmaceutical composition is used for the prevention or treatment of diseases of skin barrier dysfunction and / or abnormal immune system responses, preferably, the diseases are chronic skin diseases such as atopic dermatitis (AD), eczema, psoriasis, and pityriasis rosea.

[0119] This invention further provides the use of plant-derived extracellular vesicles or complexes in the preparation of pharmaceutical compositions for treating or preventing diseases with high colonization of Gram-positive bacteria in the skin, said pharmaceutical compositions further comprising pharmaceutically acceptable excipients. The forms of said pharmaceutical compositions include, but are not limited to, topical ointments, gels, and skin sprays. In one or more embodiments, the plant is a plant of the genus *Portulaca* in the family Portulacaceae. In one or more embodiments, the extracellular vesicles are *Portulaca oleracea* extracellular vesicles (P-EVs). In one or more embodiments, the extracellular vesicle complex is a P-EVs-GelMA hydrogel. In one or more embodiments, the Gram-positive bacteria are *Staphylococcus aureus*. In one or more embodiments, in said disease, the colonization of Gram-positive bacteria in the skin is greater than 70%, preferably greater than 80% or 90%. In one or more embodiments, said disease is a skin disease, and the Gram-positive bacteria load in the skin reaches 10... 5 CFU / cm². In one or more embodiments, the Gram-positive bacterium is Staphylococcus aureus, and the disease is a disease in which Staphylococcus aureus colonizes the skin to a degree of 70% (see: Global first microbiome-friendly standard for eczema-prone skin, published in 2025).

[0120] The pharmaceutical compositions described herein contain microvesicles obtained by the methods described herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic to the recipient at the doses and concentrations used. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining, or retaining, for example, the composition's pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeation. These substances are known in the art. The optimal pharmaceutical composition can be determined based on the intended route of administration, delivery method, and required dosage.

[0121] This invention also provides a method for treating patients (especially those with atopic dermatitis or skin infections caused by Staphylococcus aureus) by applying plant-derived extracellular vesicles, their complexes, or pharmaceutical compositions thereof as described in any embodiment of the invention. In this document, the terms “patient,” “subject,” “individual,” and “object” are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. “Treatment” refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., restoration of skin barrier dysfunction and / or reduction or prevention of abnormal immune system responses). Effective treatment regimens for patients can vary depending on various factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit a disease-fighting response in the subject.

[0122] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. Although the numerical ranges and parameter approximations shown in the broad scope of the invention are given, the values ​​shown in the specific embodiments are described as accurately as possible. However, any value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained herein.

[0123] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0124] Example

[0125] Example 1, Preparation of P-EVs

[0126] 1. Material selection: Select 1 kg of fresh purslane whole plant free from diseases and pests, rinse with purified water and then rinse 3 times with PBS.

[0127] 2. Pulping: Cut 15 mm segments, add PBS buffer at a ratio of 1:3, and crush in a juicer for 7 minutes (power 2000W, speed 20,000 rpm). Filter through a 200-mesh screen to obtain 500 mL of juice.

[0128] 3. Centrifugal purification:

[0129] Instrument Name Manufacturers model ultracentrifuge hitachi CP100MX Refrigerated centrifuge beckman mircofuge 20R

[0130] Differential centrifugation: At 4℃, centrifuge at 200×g for 10 minutes, 2000×g for 20 minutes, and 10000×g for 30 minutes in sequence, and take the supernatant after each centrifugation.

[0131] Ultracentrifugation: The supernatant obtained in the previous step was filtered through a 0.45 μm filter membrane, then filtered through a 0.22 μm filter membrane, and then centrifuged at 100000×g and 4℃ for 1.05 hours; the precipitate was collected and resuspended in 5 mL PBS.

[0132] 4. Storage: The protein concentration in the solution determined by the BCA method was 1.24 μg / μl (Table 1). The resuspended solution was aliquoted and stored at -80℃.

[0133] The specific steps for determining the protein concentration in a solution are as follows:

[0134] (1) Preparation of exosome lysis buffer: The extracted exosomes were first treated with a total protein extraction kit (KGB5303-100, Jiangsu Kaiji Biotechnology Co., Ltd., China) to obtain exosome lysis buffer. The sample to be tested is the exosome lysis buffer. Then, BCA was used for quantification;

[0135] (2) Protein quantification using the BCA method: The BCA protein content detection kit (KGB2101-250, Jiangsu Kaiji Biotechnology Co., Ltd., China) was used for detection;

[0136] 1) Plotting the standard curve: Take an ELISA plate and add samples according to the instructions.

[0137] 2) Based on the number of samples, prepare an appropriate amount of BCA working solution with the corresponding protein content (μg) by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mix thoroughly.

[0138] 3) Dilute the sample to a suitable concentration so that the total volume of the sample dilution solution is 20uL, and add 200uL of BCA working solution;

[0139] 4) Place the ELISA plate on a shaker and shake for 30 seconds, incubate at 37°C for 30 minutes, and then measure the absorbance at 562 nm. Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis.

[0140] 5) Calculate the corresponding protein content (μg) on ​​the standard curve based on the absorbance value of the measured sample, and divide it by the sample volume before dilution (uL).

[0141] Table 1: Determination of P-EVs protein concentration by BCA method

[0142] Sample number OD value Calibrate OD Concentration (μg / μl) purslane 0.2056 0.031 1.242

[0143] Example 2, Identification of P-EVs

[0144] 1. Morphological identification:

[0145] Sample preparation: 10 μL of vesicle suspension was dropped onto a copper mesh, fixed with 2.5% glutaraldehyde, washed with double-distilled water, stained with 2% uranium acetate, and air-dried. The sample was then observed using a Hitachi HT7800 electron microscope.

[0146] Under electron microscopy, a typical saucer-like structure is visible, with a clean background. Figure 3 A).

[0147] 2. Particle size analysis: The particle size of P-EVs was determined using a nanoflow cytometer (NanoFCM; N30E). The results showed that the particles exhibited a unimodal distribution, with an average particle size of 79.4 nm and a concentration of 1.85 × 10⁻⁶. 10 Particles / mL ( Figure 3 B).

[0148] Example 3: Application of P-EVs in treating skin diseases in AD mice

[0149] P-EVs were used to treat AD mice, with topical PBS serving as a control group. In addition to visual observation, in-depth detection was performed using DiI staining combined with immunofluorescence technology.

[0150] 1. Alzheimer's disease (AD) mouse model:

[0151] Animals: BALB / c female mice, 6-8 weeks old;

[0152] Modeling method: Before modeling, the body weight of each mouse was measured, and a 2 cm × 3 cm hair removal area was prepared on the back of the mouse. From day 1 to day 7 of the experiment, 100 μL of 1% dinitrochlorobenzene (DNCB) solution was evenly applied to the hair removal area on the back of the mouse daily to induce an AD-like response. Starting from week 2, 100 μL of 0.2% DNCB solution was applied twice a week to maintain the AD-like response for 3 weeks. After hair removal, an equal volume of solvent was applied to the control group mice. Note: During the modeling process, after administration, the lesion site was covered with gauze to avoid mechanical irritation caused by scratching by the mice.

[0153] 2. DiI staining of P-EVs: DiI (a red fluorescent probe for cell membranes, Beyotime C1036) was used.

[0154] The test revealed that purslane vesicles (P-EVs) crossed the skin barrier of AD mice and accumulated around hair follicles. Figure 4 P-EVs were used to treat AD mice, with topical PBS used as a control. Figure 5 ).

[0155] 3. Pathological staining of AD mouse skin: Normal group: Epidermis, dermis, subcutaneous tissue, hair follicles, and sebaceous glands were visible in the skin. The collagen fibers in the dermis were relatively well-arranged, and no other obvious pathological changes were observed; AD group: Epidermis, dermis, and hair follicles were visible in the skin. Epidermal hyperplasia and parakeratosis of the stratum corneum were observed. The collagen fibers in the dermis were disordered, and infiltrating inflammatory cells were visible; P-EVs group: Epidermis, dermis, subcutaneous tissue, hair follicles, and sebaceous glands were visible in the skin. The cell layers in the epidermis were well-arranged, the collagen fibers in the dermis were relatively well-arranged, and a small number of inflammatory cells were visible. Figure 6 ).

[0156] Example 4: Inhibitory effect of P-EVs on Staphylococcus aureus

[0157] 1. Antibacterial rate determination:

[0158] 1) Preparation of bacterial suspension: Take 0.5 ml of Staphylococcus aureus culture and 4.5 ml of 0.9% sterile sodium chloride solution, and serially dilute 10 times to a bacterial concentration of 50~150 cfu / ml.

[0159] 2) Preparation of experimental bacterial suspension groups: Each 1 ml of bacterial suspension (bacterial suspension concentration 1~9*10) 5 Add 20 μg (1.242 μg / μl) of exosomes to (cfu / ml) and shake on a shaker for 2 hours at room temperature to obtain the product.

[0160] 3) Antibacterial rate test: Take 1 ml of bacterial suspension and dilute it 10 times to a bacterial concentration of 50~150 cfu / ml as the control group. Take 1 ml of the control group and 1 ml of the experimental bacterial suspension and inject them into a Petri dish. Immediately pour in 15~20 ml of tryptic soy agar medium, mix well, and allow to solidify. Prepare three parallel Petri dishes and incubate at 30~35 ℃ for no more than 3 days. Figure 7 ).

[0161] 2. Electron microscopy observation and respiratory chain complex detection:

[0162] 1) Electron microscopy observation: The bacterial cell walls in the treatment group ruptured, and the cell contents leaked out. Figure 8 B), control group cells remained intact ( Figure 8 A).

[0163] 2) Respiratory chain detection: 20 μg / mL P-EVs can inhibit the activity of Staphylococcus aureus ETC (electron transfer chain) complex I to 48.54% (Table 2).

[0164] Table 2: Effects of P-EVs on the activity of Staphylococcus aureus ETC complex (x̄±s, n=3)

[0165] Group dose Complex I (%) Complex III (%) Complex IV (%) control group - 100.00±15.21 100.00±17.58 100.00±11.66 P-EVs processing group 20μg / ml 48.54±6.82*** 53.67±10.47** 45.96±12.74***

[0166] Note: *P<0.001, P<0.01 compared with the control group

[0167] Example 5: Preparation of P-EVs combined with GelMA hydrogel

[0168] 1. Prepare a 0.25% initiator standard solution.

[0169] 1) Take 20 mL of PBS and add it to a brown bottle containing the initiator LAP (containing 0.05 g LAP).

[0170] 2) Dissolve in a water bath at 40-50℃ for 15 minutes, shaking several times during the process. Store at 4℃ away from light.

[0171] 2. Prepare a 10% GelMA solution (GelMA; Gelatin Methacryloyl; Supplier: Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; Model: EFL-GM-30 / 60 / 90)

[0172] 1) Add 10 mL of the initiator standard solution to 1 g of GelMA;

[0173] 2) Dissolve in a water bath at 60-70℃ in the dark for 20-30 minutes, shaking several times during the process; if there is a large amount of solid GelMA, the dissolving time needs to be extended accordingly.

[0174] 3) After dissolving, centrifuge at 3000 rpm for 2 minutes to remove air bubbles;

[0175] 4) Immediately sterilize the GelMA solution using a 0.22 μm sterile syringe filter (to prevent low-temperature gelation). Store at -20°C for later use.

[0176] 3. Purslane vesicle P-EVs-GelMA hydrogel

[0177] 1) Mix 1 mg of vesicles with 1 mL of 10% GelMA hydrogel at 37°C to prepare 1 ug / ul purslane vesicle-hydrogel. Prepare as needed and store at -20°C for later use.

[0178] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this invention, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of plant-derived extracellular vesicles or a complex thereof in the preparation of a medicament for treating or preventing a disease in which the degree of colonization of the skin by Gram-positive bacteria is high, Preferably, the plant is a Portulacaceae plant, Preferably, the complex comprises plant-derived extracellular vesicles and a carrier, more preferably, the carrier is a hydrogel, Preferably, the disease is a disease in which the degree of colonization of the skin by Gram-positive bacteria is greater than 70%.

2. Use according to claim 1, characterized in that, The Gram-positive bacteria are Staphylococcus aureus.

3. Use of plant-derived extracellular vesicles or a complex thereof in the preparation of a medicament for treating or preventing a disease of the skin barrier dysfunction, abnormal reaction of the immune system, skin microbial imbalance, skin flora imbalance and / or skin inflammation, Preferably, the plant is a Portulacaceae plant, Preferably, the complex comprises plant-derived extracellular vesicles and a carrier, more preferably, the carrier is a hydrogel.

4. Use according to claim 3, wherein the disease is a chronic skin disease, such as atopic dermatitis, eczema, psoriasis, pityriasis rosea.

5. Use according to any one of claims 1-4, wherein, the medicament further comprises pharmaceutically acceptable adjuvants, including carriers, diluents and / or excipients, and / or the form of the pharmaceutical composition includes, but is not limited to, external ointments, gels and skin sprays.

6. A method for producing plant-derived extracellular vesicles, comprising the steps of: (1) washing the plant; (2) crushing the plant to obtain plant juice by filtration; (3) collecting the supernatant by differential centrifugation; (4) filtering the supernatant collected in step (3); (5) collecting the precipitate by high-speed centrifugation; (6) resuspending the precipitate collected in step (5) to obtain extracellular vesicles, Preferably, the washing in step (1) comprises preliminary washing with purified water and rinsing with PBS buffer, The breaking in step (2) includes: (i) cutting the plant into small pieces, preferably 10-20 mm in length, and (ii) adding sterile PBS buffer at a weight-to-volume ratio of 1:1-1:5 (g / mL), crushing for 5-10 minutes, preferably for 7 minutes, the filtration in step (2) is performed after crushing is completed using a 200-mesh screen, the differential centrifugation in step (3) comprises the following steps in sequence: (i) 100-300 x g centrifugation for 5-15 minutes; (ii) 1000-3000 x g centrifugation for 10-30 minutes; and (iii) 7000-15000 x g centrifugation for 20-40 minutes, the filtration in step (4) is performed using a 0.2-0.5 μm filter membrane, the high-speed centrifugation in step (5) is 80000-120000 x g centrifugation for 1-2 hours, the resuspension in step (6) is performed using PBS solution, Preferably, the plant is a Portulacaceae plant, Preferably, the extracellular vesicles have a particle size of 30-150 nm, more preferably 50-100 nm.

7. Extracellular vesicles produced according to the method of claim 6, Preferably, the extracellular vesicles are Portulacaceae extracellular vesicles, Preferably, the extracellular vesicle has a size of 30-150 nm, more preferably 50-100 nm.

8. A plant-derived extracellular vesicle complex comprising extracellular vesicles of a plant of the family Portulacaceae and a carrier, Preferably, the carrier is a hydrogel, more preferably the carrier is a GelMA hydrogel, Preferably, the extracellular vesicles are obtained by the method of claim 6.

9. A method for inhibiting gram-positive bacteria in vitro, comprising the step of contacting plant-derived extracellular vesicles or a complex thereof with a sample in which inhibition of gram-positive bacteria is desired, Preferably, the extracellular vesicles are Portulaca extracellular vesicles, Preferably, the extracellular vesicles are obtained by the method of claim 6, Preferably, the gram-positive bacteria are Staphylococcus aureus, Preferably, the ratio of gram-positive bacteria to extracellular vesicles is 1*10E5 cfu / ml : 1-1.5 pg / pL to 9*10E5 cfu / ml : 1-1.5 pg / pL, Preferably, the contacting is for at least 2 hours.

10. An inhibitor of gram-positive bacteria comprising plant-derived extracellular vesicles or a complex thereof, Preferably, the extracellular vesicles are Portulaca extracellular vesicles, Preferably, the extracellular vesicles are obtained by the method of claim 6, Preferably, the concentration of extracellular vesicles in the inhibitor is 1-1.5 pg / pL, Preferably, the gram-positive bacteria are Staphylococcus aureus.

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