A purslane-derived extracellular vesicle microneedle with anti-inflammatory soothing effect and a preparation method and application thereof

Abstract

The present application relates to a purslane-derived extracellular vesicle microneedle with anti-inflammatory soothing effect and a preparation method and application thereof, the preparation raw material of the microneedle includes matrix material and purslane-derived extracellular vesicle loaded in the matrix material; the matrix material includes purslane polysaccharide and hyaluronic acid or salt thereof. The present application extracts and purifies purslane-derived extracellular vesicles creatively, and studies the physiological effect thereof, finds that it has excellent anti-inflammatory effect in vivo and in vitro, and combines the purslane extracellular vesicles with the microneedle dosage form, providing a new idea for preparing anti-inflammatory soothing cosmetics or drugs. In addition, purslane polysaccharide and hyaluronic acid or salt thereof are used as the matrix of the microneedle, the purslane polysaccharide and the purslane extracellular vesicles can play the anti-inflammatory effect together, and the mechanical strength of the microneedle prepared by mixing hyaluronic acid or salt thereof and purslane polysaccharide is higher, and the physical stability is better.

Description

Technical Field

[0001] This invention belongs to the field of plant extracellular vesicle technology, and relates to a purslane-derived extracellular vesicle microneedle with anti-inflammatory and soothing effects, its preparation method and application. Background Technology

[0002] Extracellular vesicles (EVs) are 40-150 nm bio-nanoparticles produced by cells. They have a phospholipid bilayer membrane structure and contain active substances such as mRNA, proteins, and nucleic acids. EVs can transmit information between plants, animals, and microorganisms. Initially, EVs were considered cell debris, thus their value was overlooked. Today, however, EVs are recognized as key carriers for intercellular information transmission and regulation of cellular physiological functions. Plant EVs can serve as effective carriers for communication between plant and pathogen cells, participate in plant defense responses against pathogens, participate in the formation of plant cell wall components, and play an important role in regulating plant immunity.

[0003] Purslane (Portulaca oleracea L.) is an ancient Chinese plant used for both medicinal and edible purposes, possessing various biological activities such as antibacterial, anti-inflammatory, anti-diabetic, antioxidant, and anti-tumor effects. However, extracellular vesicles (EVs) derived from purslane have received little attention. Whether it is feasible to extract extracellular vesicles from purslane, and whether the extracted purslane-derived EVs possess biological activity, remains unknown. Therefore, it is significant to develop a method for extracting high-purity, high-quality extracellular vesicles from purslane and to conduct in-depth research on the functions of the extracted EVs to explore their potential applications in cosmetics or pharmaceuticals.

[0004] With the increasing number of people with sensitive skin, consumers have a growing demand for skin-soothing products (such as cosmetics or pharmaceuticals). Factors influencing skin irritation include internal factors such as race, age, skin health, and genetics, as well as external factors such as climate and inferior cosmetics. Skin irritation can cause dryness, tightness, redness, itching, peeling, burning and stinging sensations, or damage to the skin barrier, abnormal nerve function, and in severe cases, inflammatory reactions.

[0005] Atopic dermatitis (AD) is a chronic, recurrent inflammatory skin disease that can affect people of any age and has a genetic predisposition. It is characterized by intense itching and eczematous lesions. Most patients experience their first onset in infancy, and due to its chronic and recurrent nature, some patients may continue to have the disease into adulthood. Severe cases can significantly impact daily life and even lead to sleep disturbances. The main pathogenesis of AD is currently unclear, but it may involve immune system signaling imbalances, genetics, skin dysfunction, and psychological factors. Current treatments for atopic dermatitis primarily include topical corticosteroids, glucocorticoids, oral antihistamines, biologics, and ultraviolet therapy. However, these treatments all have drawbacks to varying degrees; for example, long-term use of corticosteroids can cause serious adverse reactions, and biologics are expensive. Therefore, the search for new drugs and treatments is particularly necessary.

[0006] Microneedling is a dosage form in which bioactive molecules are absorbed into the human circulation through the skin and capillaries at a certain rate. Compared with oral or subcutaneous injection methods, microneedling has advantages such as better patient compliance, avoidance of the first-pass effect, stable and controllable blood concentration, and high safety. It can penetrate the stratum corneum of the skin to form a duct, through which bioactive molecules diffuse directly into the dermal microcirculation, promoting the delivery of macromolecules (molecular weight > 500 Da), proteins, and nanoparticles in the body. It also has the advantages of being minimally invasive, painless, safe, and convenient to use. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a purslane-derived extracellular vesicle microneedle with anti-inflammatory and soothing effects, its preparation method, and its application. The present invention loads purslane-derived extracellular vesicles into soluble microneedles using purslane polysaccharides and hyaluronic acid or their salts as a matrix, thus preparing purslane-derived extracellular vesicle microneedles (POP-MNs-PELNs). The aim is to safely and effectively deliver purslane-derived extracellular vesicles into the skin in a painless manner using the microneedle as a minimally invasive delivery platform, achieving anti-inflammatory and soothing effects.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a purslane-derived extracellular vesicle microneedle with anti-inflammatory and soothing effects. The raw materials for preparing the purslane-derived extracellular vesicle microneedle include a matrix material and purslane-derived extracellular vesicles loaded in the matrix material. The matrix material includes purslane polysaccharide and hyaluronic acid or its salt.

[0010] The aforementioned hyaluronic acid or its salts refer to hyaluronic acid or sodium hyaluronate.

[0011] This invention creatively extracts and purifies extracellular vesicles derived from purslane and studies their physiological effects, discovering excellent in vivo and in vitro anti-inflammatory effects. Furthermore, combining purslane extracellular vesicles with microneedles provides a new approach for preparing cosmetics or pharmaceuticals with anti-inflammatory and soothing effects. In addition, using purslane polysaccharides and hyaluronic acid or their salts as a microneedle matrix has potential synergistic effects; purslane polysaccharides and purslane extracellular vesicles can jointly exert anti-inflammatory effects. Simultaneously, microneedles prepared from the mixture of hyaluronic acid or its salts and purslane polysaccharides have higher mechanical strength and better physical stability. Experimental studies show that this microneedle product has excellent effects in relieving skin sensitivity and irritation, as well as anti-inflammatory and anti-allergic effects. It can alleviate and treat skin lesions in a mouse model of atopic dermatitis, reduce the spleen index in a mouse model of atopic dermatitis, and reduce the expression levels of TNF-α and IL-4 in skin tissue.

[0012] Preferably, the loading of the purslane-derived extracellular vesicles is 0.1-3% based on protein concentration, such as 0.1%, 0.3%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 2%, 2.5%, 3%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0013] Preferably, the mass ratio of purslane polysaccharide to hyaluronic acid or its salt is 1:1 to 1:7, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, etc. Other specific values ​​within this range can be selected, and will not be elaborated here. More preferably, the ratio is 1:2 to 1:7.

[0014] When the mass ratio of purslane polysaccharide to hyaluronic acid or its salt is used in the specific mass ratio described above, the matrix solution is in the best state, exhibiting rheological properties. After preparation into microneedles, the microneedle backing is suitable, and the prepared microneedle tips have more complete morphology with no collapse around the edges. If the relative amount of purslane polysaccharide used is further increased, cavitation and breakage may occur in the dried microneedle tips, or the matrix solution may be too viscous and lack rheological properties, resulting in a harder microneedle backing and incomplete tip morphology. If the relative amount of purslane polysaccharide used is further reduced, the anti-inflammatory and soothing effects of the product will be weakened.

[0015] The purslane-derived extracellular vesicles involved in this invention can be prepared by any method known to those skilled in the art, but the following extraction method is preferred. The purslane-derived extracellular vesicles obtained by this method have higher purity and better anti-inflammatory and soothing effects.

[0016] Preferably, the method for preparing the purslane-derived extracellular vesicles includes ultracentrifugation or polyethylene glycol precipitation.

[0017] Preferably, the ultracentrifugation method specifically includes the following steps:

[0018] (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant;

[0019] (2) After filtering the supernatant, centrifuge it at ultraspeed, collect the precipitate and resuspend it to obtain the product;

[0020] Preferably, step (1) involves centrifugation and collection of the supernatant at least three times, with centrifugation speeds successively of 800-1800g (e.g., 800g, 900g, 1000g, 1200g, 1300g, 1400g, 1500g, 1600g, 1800g, etc.), 2000-5000g (e.g., 2000g, 2500g, 3000g, 3500g, 4000g, 4500g, 5000g, etc.), and 8000-15000g (e.g., 8000g, 9000g, 10000g, 11000g, 1200g, 1200g, etc.). Centrifugation times are 5-35 min (e.g., 5 min, 10 min, 15 min, 20 min, 25 min, 35 min, etc.), 40-90 min (e.g., 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, etc.) for 00g, 13000g, 15000g, etc., respectively; other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0021] Preferably, the ultracentrifugation speed in step (2) is 100,000-200,000g (e.g., 100,000g, 120,000g, 140,000g, 150,000g, 160,000g, 170,000g, 180,000g, 200,000g, etc.), and the time is 60-120min (e.g., 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc.); other specific point values ​​within the above ranges can be selected, and will not be elaborated here.

[0022] The centrifugation and ultracentrifugation processes described above will affect the purity and particle size of the purslane-derived extracellular vesicles to some extent. When extraction is performed under the specific preparation conditions described above, the purslane-derived extracellular vesicles obtained will have higher purity and smaller particle size.

[0023] Preferably, the polyethylene glycol precipitation method specifically includes the following steps:

[0024] (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant;

[0025] (2) After filtering the supernatant, mix it with polyethylene glycol, let it stand, centrifuge to collect the precipitate and resuspend it;

[0026] (3) The resuspension is purified to obtain the final product;

[0027] Preferably, step (1) involves centrifugation and collection of the supernatant at least three times, with centrifugation speeds successively of 3000-7000g (e.g., 3000g, 4000g, 4500g, 5000g, 5500g, 6000g, 7000g, etc.), 7000-10000g (e.g., 7000g, 7500g, 8000g, 8500g, 9000g, 9500g, 10000g, etc.), and 10000-15000g (e.g., 10000g, 11000g, 11500g, 12000g, 12500g, 13...). Centrifugation times are 5-20 min (e.g., 5 min, 10 min, 15 min, 20 min), 15-45 min (e.g., 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min), and 40-90 min (e.g., 40 min, 45 min, 50 min, 55 min, 60 min, 70 min, 80 min, 90 min), depending on the weight range of the above values. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0028] Preferably, the number average molecular weight of the polyethylene glycol is 4000-8000, such as 4000, 5000, 6000, 7000, 8000, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0029] Preferably, the final concentration of polyethylene glycol after mixing in step (2) is 5-15%, such as 5%, 6%, 7%, 8%, 10%, 12%, 13%, 15%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0030] The final concentration of polyethylene glycol and the centrifugation process settings mentioned above will affect the purity and particle size of the purslane-derived extracellular vesicles to a certain extent. When the final concentration is 6-9%, the purity of the purslane-derived extracellular vesicles is significantly improved and the particle size is significantly reduced when the specific centrifugation process is used.

[0031] Preferably, the settling time is 8-16 hours, such as 8 hours, 9 hours, 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0032] Preferably, the centrifugation speed in step (2) is 6000-10000g (e.g., 6000g, 7000g, 7500g, 8000g, 9000g, 10000g, etc.), and the time is 10-50min (e.g., 10min, 15min, 20min, 25min, 30min, 40min, 50min); other specific point values ​​within the above ranges can be selected, and will not be elaborated here.

[0033] Preferably, the purification method includes size exclusion chromatography.

[0034] Preferably, the purslane polysaccharide is prepared by a method comprising the following steps:

[0035] Purslane raw material is mixed with water and extracted by heating at 90-100℃ (e.g., 92℃, 93℃, 94℃, 95℃, 96℃, 98℃, 100℃, etc.) for 30-120 min (e.g., 30 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 120 min, etc.). The extract is then subjected to protein removal treatment, followed by alcohol extraction. After standing, centrifugation, precipitation, and freeze-drying, the purslane polysaccharide is obtained. Other specific values ​​within the above ranges can be selected and will not be elaborated further here.

[0036] Preferably, the protein removal is performed using Sevag reagent.

[0037] Preferably, the alcohol extraction process uses 75-95% ethanol.

[0038] Preferably, the standing is carried out at 2-8℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc.) for 12-48h (e.g., 12h, 14h, 18h, 24h, 30h, 36h, 48h, etc.).

[0039] Preferably, the centrifugation is performed at 2000-8000 rpm (e.g., 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, etc.) for 5-20 minutes (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, etc.).

[0040] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0041] In a second aspect, the present invention provides a method for preparing purslane-derived extracellular vesicle microneedles with anti-inflammatory and soothing effects according to the first aspect, the preparation method comprising the following steps:

[0042] Purslane polysaccharide, hyaluronic acid or its salts are mixed with purslane-derived extracellular vesicle suspension, the mixture is cast into a microneedle mold, left to stand under vacuum, dried, and demolded to obtain the purslane-derived extracellular vesicle microneedles.

[0043] The preparation method of the microneedle product involved in this invention is simple and easy to operate, and can be easily industrialized.

[0044] Preferably, the vacuum degree of the vacuum environment is -0.05 to -0.1 MPa, such as -0.05 MPa, -0.06 MPa, -0.07 MPa, -0.08 MPa, -0.09 MPa, -0.1 MPa, etc.

[0045] Preferably, the settling time is 5-30 minutes, such as 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, etc.

[0046] Preferably, the drying temperature is 25-45℃, such as 25℃, 30℃, 33℃, 35℃, 37℃, 40℃, 45℃, etc.

[0047] Other specific point values ​​within the above ranges can be selected, and will not be elaborated on here.

[0048] Thirdly, the present invention provides the application of purslane-derived extracellular vesicle microneedles with anti-inflammatory and soothing effects as described in the first aspect in the preparation of cosmetics or pharmaceuticals.

[0049] Preferably, the cosmetic is used to soothe sensitive skin, that is, the cosmetic has the effect of soothing sensitive skin.

[0050] Preferably, the medicine is used to prevent or treat inflammatory skin diseases, that is, the medicine is a medicine for preventing or treating inflammatory skin diseases; preferably, the inflammatory skin diseases include atopic dermatitis.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] This invention creatively extracts and purifies extracellular vesicles derived from purslane and studies their physiological effects, discovering excellent in vivo and in vitro anti-inflammatory effects. Furthermore, combining purslane extracellular vesicles with microneedles provides a new approach for preparing cosmetics or pharmaceuticals with anti-inflammatory and soothing effects. In addition, using purslane polysaccharides and hyaluronic acid or their salts as a microneedle matrix has potential synergistic effects; purslane polysaccharides and purslane extracellular vesicles can jointly exert anti-inflammatory effects. Simultaneously, microneedles prepared from the mixture of hyaluronic acid or its salts and purslane polysaccharides have higher mechanical strength and better physical stability. Experimental studies show that this microneedle product has excellent effects in relieving skin sensitivity and irritation, as well as anti-inflammatory and anti-allergic effects. It can alleviate and treat skin lesions in a mouse model of atopic dermatitis, reduce the spleen index in a mouse model of atopic dermatitis, and reduce the expression levels of TNF-α and IL-4 in skin tissue. Attached Figure Description

[0053] Figure 1 This is a transmission electron microscope image of the purslane-derived extracellular vesicles prepared in Example 1;

[0054] Figure 2 This is a particle size distribution diagram of the purslane-derived extracellular vesicles prepared in Example 1;

[0055] Figure 3 This is a potential characterization diagram of the purslane-derived extracellular vesicles prepared in Example 1;

[0056] Figure 4 This is a standard curve for BCA protein assay;

[0057] Figure 5 This is an SDS-PAGE image of the purslane-derived extracellular vesicles prepared in Example 1;

[0058] Figure 6 This is a transmission electron microscope image of the purslane-derived extracellular vesicles prepared in Example 2;

[0059] Figure 7 This is a particle size distribution diagram of the purslane-derived extracellular vesicles prepared in Example 2;

[0060] Figure 8 This is a potential characterization diagram of the purslane-derived extracellular vesicles prepared in Example 2;

[0061] Figure 9 This is an SDS-PAGE image of the purslane-derived extracellular vesicles prepared in Example 2;

[0062] Figure 10 This is a statistical graph showing the results of CCK8 assay for detecting the toxic effects of purslane-derived extracellular vesicles on macrophages;

[0063] Figure 11This is the infrared spectrum of purslane polysaccharide;

[0064] Figure 12 This is a standard curve for calculating the polysaccharide content of purslane;

[0065] Figure 13 This is a full-view image of the microneedles obtained in Example 1;

[0066] Figure 14 This is a microneedle array pattern obtained in Example 1 (scale bar = 1 mm);

[0067] Figure 15 This is a microneedle tip diagram obtained in Example 1 (scale bar = 500 μm);

[0068] Figure 16 This is a diagram of a single microneedle tip obtained in Example 1 (scale bar = 400 μm);

[0069] Figure 17 It is a flowchart of animal experiments;

[0070] Figure 18 These are images of skin lesions on the backs of mice in each group;

[0071] Figure 19 This is a graph showing the statistical results of the skin damage scores on the backs of mice in each group;

[0072] Figure 20 These are anatomical diagrams of the spleens of mice in each group;

[0073] Figure 21 This is a graph showing the statistical results of spleen indices in each group of mice;

[0074] Figure 22 These are HE sections of skin lesions on the backs of mice in each group (scale bar = 100 μm);

[0075] Figure 23 This is a graph showing the statistical results of the effects of each drug administration group on TNF-α in the skin tissue of mice with atopic dermatitis;

[0076] Figure 24 This is a graph showing the statistical results of the effects of each drug administration group on IL-4 in the skin tissue of mice with atopic dermatitis;

[0077] Figure 25 This is a graph showing the statistical results of the expression levels of TBIL, TP, ALT, AST, CR, and UR in the serum of mice in each group. Detailed Implementation

[0078] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the described embodiments are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0079] The purslane raw materials involved in the following preparation examples and embodiments have been identified as the dried aerial parts of the Portulacaceae plant Portulaca oleracea.

[0080] Preparation Example 1

[0081] Purslane-derived extracellular vesicles (PELNs) were extracted using ultracentrifugation.

[0082] (1) Take purslane raw material, wash it, cut it into sections, weigh it, soak it for 1 hour, and juice it according to the ratio of 150mL of pure water per 100g.

[0083] (2) Filter the juice through double-layer nylon gauze, take 1200g of juice, centrifuge at 20min and 4℃, then take the supernatant and centrifuge at 3000g, 20min and 4℃, and continue to take the supernatant and centrifuge at 10000g, 60min and 4℃.

[0084] (3) Filter the supernatant through a 0.8 μm filter membrane and centrifuge at 150,000 g for 90 min at 4 °C. Remove the supernatant and take the precipitate. Resuspend the precipitate with a small amount of supernatant to obtain purslane-derived extracellular vesicles (abbreviated as PELNs).

[0085] Test Example 1

[0086] Characterization of PELNs prepared in Example 1:

[0087] The morphology, particle size, potential, protein concentration, and protein molecular weight distribution of the PELNs extracted in Preparation Example 1 were characterized.

[0088] (1) Take PELNs solution, dilute it with an appropriate amount of pure water, add it dropwise onto a copper grid, let it dry naturally, stain with phosphotungstic acid, and observe the morphology under a transmission electron microscope. For example... Figure 1 As shown, the extracellular vesicles derived from purslane exhibit a typical teacup shape, with a distinct phospholipid bilayer. They are complete and plump, consistent with the morphology of general plant extracellular vesicles, and vary in size. Their particle size also falls within the range measured by the Malvern particle size analyzer.

[0089] (2) The particle size and potential distribution of PELNs were analyzed using a Malvern particle size analyzer, such as... Figure 2 and Figure 3 As shown, the average particle size of PELNs is 112.4±1.1 nm, the PDI is 0.345±0.037, and the average potential is -1.6±0.4 mV. Their particle size falls within the typical range of 30–150 nm for plant extracellular vesicles, and the negative charge of the particles is consistent with the charged properties of plant extracellular vesicles.

[0090] (3) Prepare samples according to the kit instructions and measure their absorbance. Establish a BCA standard curve and calculate the protein content by substituting the values ​​into the BCA standard curve. Plot a linear regression between absorbance value y and mass concentration x, as shown below. Figure 4 As shown, the protein concentration in the range of 0–2.0 μg / μL showed a good linear relationship with the absorbance value. Then, the protein content in PELNs was determined by the BCA kit method to characterize the amount of PELNs used. The protein concentration of PELNs was 1.54 ± 0.18 μg / μL.

[0091] (4) Protein expression levels in PELNs were analyzed by SDS-PAGE electrophoresis, such as... Figure 5 As shown in the figure, the molecular weight of its protein is mainly distributed below 75kDa.

[0092] Preparation Example 2

[0093] Extracellular vesicles (PELNs) derived from purslane were extracted using the polyethylene glycol precipitation method.

[0094] (1) Take purslane raw material, wash it, cut it into sections, weigh it, soak it for 1 hour, and juice it according to the ratio of 150mL of pure water per 100g.

[0095] (2) Let the juice stand for 2 hours. Filter the upper layer solution with a double layer of 400-mesh nylon sieve, and filter the lower layer solution with double layers of 100, 200 and 400-mesh nylon sieves in sequence. Mix the filtered upper and lower layers of solution, and centrifuge the filtrate in three steps: 5000g, 4℃, 10min; 8000g, 4℃, 30min; 10000g, 4℃, 1h. Collect the supernatant.

[0096] (3) Filter through a 0.8 μm filter membrane, transfer the supernatant to a new centrifuge tube, and add 16%, 20%, 24%, and 30% polyethylene glycol 6000 solution to the supernatant in equal proportions to make final concentrations of 8%, 10%, 12%, and 15%, respectively. Mix thoroughly and incubate vertically at 4°C for 12 hours. Then centrifuge at 8000g, 4°C, for 30 minutes, remove the supernatant, and dissolve the precipitate thoroughly with an appropriate amount of PBS buffer to form a suspension of purslane extracellular vesicles.

[0097] The yield, purity, and particle size of four purslane extracellular vesicle samples were evaluated (yield = mass of lyophilized purslane extracellular vesicles / mass of fresh product; purity = protein concentration × reconstitution volume / mass of lyophilized purslane extracellular vesicles). The results are shown in the table below:

[0098]

[0099]

[0100] Therefore, it can be concluded that the product has the best overall properties when the final concentration of polyethylene glycol is 8%.

[0101] (4) The extracellular vesicle suspension of purslane obtained with 8% PEG concentration was purified by agarose gel CL-2B filtration chromatography, specifically as follows:

[0102] Take 10 mL of gel and wash with an appropriate amount of ultrapure water to remove the 20% ethanol solvent used to preserve the gel. Add 3 times the volume of PBS buffer (pre-filtered through a 0.22 μm membrane) to homogenize the gel. Take the chromatography column, fix it on the iron stand, and wet the inside and bottom of the column with buffer, maintaining a small liquid level, ensuring there are no air bubbles at the bottom. Use a glass rod to guide the homogenate slowly and continuously pour it into the column along the inner wall, being careful not to generate air bubbles, allowing the gel to settle naturally under gravity. After complete sedimentation, slowly add PBS buffer from the top, open the column outlet, and equilibrate the two column volumes. Close the bottom outlet, slowly add the sample in a ring from the top, open the column outlet, and when the sample settles to the level of the liquid surface, slowly add about 10 mL of PBS elution buffer for separation and purification (gel column length 97 mm, column inner diameter 15 mm).

[0103] Test Example 2

[0104] Characterization of PELNs prepared in Example 2:

[0105] The morphology, particle size, potential, protein concentration, and protein molecular weight distribution of the PELNs extracted in Preparation Example 2 were characterized.

[0106] (1) Take PELNs solution, dilute it with an appropriate amount of pure water, add it dropwise onto a copper grid, let it dry naturally, stain with phosphotungstic acid, and observe the morphology under a transmission electron microscope. For example... Figure 6 As shown, the extracellular vesicles derived from purslane exhibit a typical teacup shape, with a distinct phospholipid bilayer. They are complete and plump, consistent with the morphology of general plant extracellular vesicles, and vary in size. Their particle size also falls within the range measured by the Malvern particle size analyzer.

[0107] (2) The particle size and potential distribution of PELNs were analyzed using a Malvern particle size analyzer, such as... Figure 7 and Figure 8 As shown, the average particle size of PELNs is 115.6±7.0 nm, the PDI is 0.436±0.036, and the average potential is -1.46±0.11 mV. Their particle size falls within the typical range of 30–150 nm for plant extracellular vesicles, and the negative charge of the particles is consistent with the charged properties of plant extracellular vesicles.

[0108] (3) Samples were prepared according to the kit instructions and their absorbance was measured. A BCA standard curve was established and the protein content was calculated by substituting the samples into the BCA standard curve. The results showed that the protein concentration of PELNs was 2.066±0.024μg / μL.

[0109] (4) Protein expression levels in PELNs were analyzed by SDS-PAGE electrophoresis, such as... Figure 9 As shown in the figure, the molecular weight of its protein is mainly distributed below 75kDa.

[0110] Test Example 3

[0111] Cytotoxic and anti-inflammatory effects of PELNs on RAW264.7 cells:

[0112] (1) Effect of PELNs on the proliferation of RAW264.7 cells was detected by CCK-8 assay: RAW264.7 cells were grown at a cell density of 1×10⁻⁶. 4 pcs / cm 2 Cells were seeded in 96-well plates. Different concentrations of PELNs (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL) were co-cultured with RAW264.7 cells. The absorbance of each group of cells was measured at 450 nm after 72 hours of culture. Figure 10 As shown, the results indicate that the selected concentrations of PELNs did not inhibit RAW264.7 cells, and the survival rate of each group was greater than 100%, indicating that PELNs did not produce cytotoxicity to RAW264.7 cells and had good biosafety.

[0113] (2) Evaluation of in vitro anti-inflammatory effect: RAW264.7 cells in logarithmic growth phase with good morphology were selected and administered at a concentration of 2×10⁻⁶ cells / cells. 5 pcs / cm 2 Cells were seeded at high density in 24-well plates and incubated for 24 h. A blank control group, an LPS-induced stimulation group, a positive control group (dexamethasone, dextromethorphan, 500 nM), and sample groups (50 μg / mL and 200 μg / mL protein concentrations) were set up and incubated at 37°C in a 5% CO2 incubator for 24 h. Except for the blank control group, LPS was added to each well and incubated for 24 h. Cell supernatants were analyzed according to the instructions of the ELISA kit (Thermo Fisher Scientific (China) Co., Ltd.) and the nitric oxide assay kit (Thermo Fisher Scientific (China) Co., Ltd.). The levels of TNF-α, IL-6, and NO in the collected cell supernatants were measured, and the inhibition rate of inflammatory factors in each group relative to the LPS-induced stimulation group was calculated. The results are shown in the table below.

[0114]

[0115] The data in the table above show that PELNs have good anti-inflammatory effects at the cellular level.

[0116] Preparation Example 3

[0117] Extraction of purslane polysaccharides:

[0118] Purslane raw material was mixed with water at a ratio of 1:10 g / mL and extracted at 95℃ for 90 min. The residue was then mixed with water at a ratio of 1:10 g / mL and extracted at 95℃ for 60 min. The residue was then mixed with water at a ratio of 1:10 g / mL and extracted at 95℃ for 30 min. The three extracts were combined and treated with Sevag reagent to remove proteins. The treated solution was then mixed with 95% ethanol to a final alcohol concentration of 75% and subjected to alcohol extraction. The mixture was placed in a 4℃ refrigerator for 24 h, centrifuged at 4000 rpm for 10 min, and the precipitate was collected and freeze-dried to obtain the purslane polysaccharide.

[0119] Test Example 4

[0120] (1) Infrared spectral analysis of purslane polysaccharides:

[0121] An appropriate amount of purslane polysaccharide powder prepared in Example 3 was compressed into tablets with potassium bromide and the infrared absorption spectrum was measured by scanning in the range of 4000 to 300 cm⁻¹ using a Fourier transform infrared spectrometer.

[0122] like Figure 11 As shown, at 3404cm -1 There is a strong -OH stretching vibration peak nearby, at 2938 cm⁻¹. -1 The value shown is the CH stretching vibration peak, at 1744 cm⁻¹. -1 The peak at 1618 cm⁻¹ is the symmetrical stretching vibration peak of C=O. -1 The left and right sides are the asymmetric stretching vibration peaks of C=O, 1411 cm⁻¹ -1 The peak at 1411 cm⁻¹ is the deformed absorption peak of =CH₂. -1 The nearby peak is the characteristic peak of the protonated carboxyl group in uronic acid, at 1244 cm⁻¹. -1 This is the vibrational peak of the unprotonated carboxyl group in uronic acid, 1111 cm⁻¹. -1 The absorption peak is the carbon-oxygen (C=O) on the ring, indicating that the substance has the characteristic structure of a polysaccharide.

[0123] (2) Determination of the content of purslane polysaccharides:

[0124] The polysaccharide content in the purslane powder extracted in Preparation Example 3 was determined using the phenol-sulfuric acid method. D-glucose standard solutions of 10, 20, 30, 40, 50, and 60 μg / mL were prepared by adding 2 mL of deionized water, with deionized water serving as a blank. 1 mL of 5% phenol solution was added sequentially, and after vortexing, 5 mL of concentrated sulfuric acid was added. The mixture was heated to boiling for 15 min, then removed and cooled to room temperature. The absorbance was measured at a wavelength of 490 nm. A standard curve was plotted by piecing absorbance against concentration. Figure 12 As shown.

[0125] Purslane polysaccharide powder was prepared into a 50 μg / mL solution with deionized water. The absorbance of the sample solution was measured using the same method, and the polysaccharide content was calculated by substituting the absorbance into the standard curve. The measured purslane polysaccharide content was 32.77 ± 0.22%.

[0126] Example 1

[0127] Preparation of Portulaca oleracea-derived extracellular vesicle microneedles (abbreviated as POP-MNs-PELNs):

[0128] Weigh 0.1 g of purslane polysaccharide powder and 0.2 g of sodium hyaluronate obtained in Preparation Example 3, add 1.5 mL of purslane-derived extracellular vesicle solution obtained in Preparation Example 1, and stir until completely dissolved. Pour the mixture into a clean microneedle mold, place it under a vacuum of -0.08 mPa for 15 min, scrape off the upper layer of unfilled matrix, and add new matrix, repeating the above steps 3 times. Dry completely in a 37℃ drying oven, and demold to obtain purslane extracellular vesicle microneedles. The overall microneedle image and transmission electron microscopy image of the microneedles are shown below. Figure 13 , Figure 14 , Figure 15 , Figure 16 As shown.

[0129] Test Example 5

[0130] Therapeutic effects of POP-MNs-PELNs on mice with atopic dermatitis:

[0131] (1) Modeling method: 2,4-Dinitrochlorobenzene (DNCB) was dissolved in a mixture of acetone and olive oil (4:1 volume ratio) to prepare 0.5% and 1% DNCB solutions. One day before model establishment, a 2cm x 2cm area of ​​hair was removed from the back of mice. On days 1, 2, 3, and 4, 200μL of 0.5% DNCB solution was evenly applied to the back to sensitize the mice. On days 5 and 7, 100μL of 1% DNCB solution was applied to the back to stimulate sensitization. This process was repeated every 2 days for a total of 4 times to establish a mouse atopic dermatitis model. 48 hours after the 4th stimulation, the back skin of the mice was harvested. (See schematic diagram below.) Figure 17 As shown.

[0132] (2) Grouping and Administration: Thirty-six healthy six-week-old BALB / c mice were used, divided into six groups of six each: a control group, a model group, a positive control group (Dex), a drug-loaded microneedle group (POP-MNs-PELNs prepared in Example 1), a blank microneedle group (POP-MNs, differing from Example 1 only in that they were not loaded with PELNs), and a free drug group (PELNs prepared in Example 1). Administration began on day 8. Mice in the control and model groups were treated with 200 μL of distilled water on their backs. Mice in the positive control group were treated with a pea-sized amount of dexamethasone acetate ointment on their backs. Mice in the drug-loaded microneedle group were injected with POP-MNs-PELNs on their backs. Mice in the control microneedle group were injected with POP-MNs on their backs. Mice in the free drug group were treated with 200 μL of PELNs (the same amount as the drug-loaded microneedle group based on protein concentration). Administration was once daily for 14 consecutive days. (See schematic diagram below.) Figure 17 As shown.

[0133] (3) Score of skin lesions on the back: Starting from day 7 of the experiment, the severity of skin lesions in each mouse was assessed every 3 days. Based on the Area and Severity Index of Dermatitis (EASI), a score standard for the severity of mouse skin lesions was established, and a total of 5 assessments were conducted. Finally, the cumulative total score changes of each group over 5 days were plotted, and the condition of the skin lesions on the back of the mice was photographed and recorded on days 7, 9, 11, 13, 15, 17, 19, and 21 of the experiment.

[0134] The scoring criteria for the severity of skin lesions in mice are as follows:

[0135] (3.1) Erythema / darkening of color

[0136] 0 points: No erythema observed; 1 point: A few erythema spots, light in color; 2 points: Patchy erythema, bright red in color; 3 points: Widespread erythema, bright red or purplish-red in color, with elevated skin temperature.

[0137] (3.2) Exudation / Scabbing

[0138] 0 points: No exudation; 1 point: Less exudation, accompanied by punctate crusts; 2 points: Exudation accompanied by patchy crusts; 3 points: More exudation, with obvious serous fluid and crusts.

[0139] (3.3) Drying / Desquamation

[0140] 0 points: No dryness / scaling observed; 1 point: Mild dryness / scaling of the epidermis; 2 points: Moderate dryness / scaling of the epidermis; 3 points: Severe dryness / scaling of the epidermis.

[0141] (3.4) Exfoliation / Erosion

[0142] 0 points: No skin peeling / erosion observed; 1 point: Spotted peeling / erosion visible on the skin lesion; 2 points: Skin peeling / erosion is relatively obvious, appearing as small scattered patches; 3 points: Skin peeling / erosion is obvious, distributed in large areas.

[0143] (3.5) Skin thickening

[0144] 0 points: No obvious thickening of the skin; 1 point: Partial thickening of the skin; 2 points: Significant thickening of the skin; 3 points: Large-area thickening of the skin.

[0145] The effects of each drug administration group on the dorsal skin lesions and dorsal dermatitis scores in mice with DNCB-induced atopic dermatitis were observed. Images of the dorsal skin lesions in each group are shown below. Figure 18 As shown in the figure, the skin lesion scoring charts on the backs of mice in each group are as follows. Figure 19 As shown, the results revealed that in the first week of the experiment, DNCB-induced atopic dermatitis in mice caused dry skin, erythema, epidermal shedding, and skin thickening, indicating that DNCB successfully induced atopic dermatitis in mice. Two weeks after administration, the skin of mice in the positive drug group was significantly thinner, and no erythema or epidermal shedding was observed. The POP-MNs-PELNs group, the POP-MNs group, and the PELNs group all showed varying degrees of improvement in the skin lesions of mice, with the POP-MNs-PELNs group showing the most significant improvement.

[0146] (4) Spleen index:

[0147] Weigh the spleen and body weight of each group of mice. Calculate the spleen index for each group using the formula: Spleen Index = Spleen weight (g) / Mouse body weight (g) × 100.

[0148] Anatomical diagrams of the spleen and statistical results of spleen index in each group of mice are shown below. Figure 20 and Figure 21 As shown in the figure (compare with the Control group, ###p<0.001, ####p<0.0001; compare with the Model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=6), after continuous DNCB stimulation, the spleen index of mice in the model group significantly increased, showing a significant difference compared with the normal group (p<0.0001). After treatment with the positive control drug dexamethasone cream, the spleen index of mice in the model group was significantly decreased compared with the model group (p<0.001). The spleen index of mice in the POP-MNs-PELNs group, POP-MNs group, and PELNs group were all decreased, showing significant differences compared with the model group (p<0.0001).

[0149] (5) Observation of HE staining of back skin pathological sections:

[0150] After drug administration, skin samples were cut from the lesions, fixed with perforated aluminum foil, and then immersed in formalin for fixation. After dehydration, the samples were embedded in paraffin to prepare 3 μm thick paraffin sections, stained with hematoxylin and eosin (HE), and mounted. The histopathological changes of the HE-stained sections in each group were observed under a microscope.

[0151] The results are as follows Figure 22 As shown in the HE results, the skin tissue of mice in the Control group was smooth with no inflammatory infiltration. Compared with the Control group, the AD model group showed significant thickening of the epidermis and dermis, with significant inflammatory cell infiltration in the dermis. Compared with the model group, the epidermis and dermis of mice in the positive control group, POP-MNs-PELNs group, POP-MNs group, and PELNs group were significantly thinner, accompanied by mild inflammatory cell infiltration. Applying microneedles loaded with purslane extracellular vesicles and applying purslane extracellular vesicles can alleviate skin inflammation in mice to some extent, with the drug-loaded microneedle group showing better efficacy.

[0152] (6) ELISA detection of TNF-α and IL-4 expression levels in skin tissue:

[0153] On day 21 of the experiment, the levels of IgE in the serum and TNF-α and IL-4 in the skin tissue of mice in each group were detected by ELISA kit.

[0154] The results are as follows Figure 23 , Figure 24 As shown in the figure (compared with the Control group, ###p<0.001, ####p<0.0001; compared with the Model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=6), it can be seen from the figure that compared with the normal group, the levels of TNF-α and IL-4 in the skin tissue of mice in the model group were significantly increased (p<0.0001); while compared with the model group, all drug-treated groups showed a decrease. Among them, except for the positive drug group, the application of drug-loaded microneedles and the application of PELNs could significantly reduce the content of TNF-α in the skin tissue (p<0.01). For IL-4, the application of drug-loaded microneedles had the best effect (p<0.0001).

[0155] (7) Serum biochemical markers determination:

[0156] 100 μL of serum was collected from each group of mice, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), creatinine (CR), urea (UR), and total protein concentration (TP) in the serum were detected using a fully automated biochemical analyzer. These serum biochemical indicators were used to evaluate the liver and kidney function of the mice after drug administration.

[0157] The results are as follows Figure 25 As shown in the figure (compared to the Control group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), among the serum biochemical indicators, TBIL and TP levels reflect bilirubin metabolism and liver synthesis function. Elevated TBIL and TP levels suggest potential liver damage. ALT and AST, mainly found in hepatocytes, are sensitive indicators for evaluating liver function; elevated levels of both indicate hepatotoxicity. CR and UR are important indicators for assessing kidney function. Except for the positive control group, no significant pathological changes were observed in any tissue of the mice in the other groups compared to the normal group. This suggests that attention should be paid to the hepatotoxicity and nephrotoxicity of dexamethasone cream when using it. Furthermore, this demonstrates the good biosafety of this novel drug delivery system.

[0158] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0159] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0160] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. The application of purslane-derived extracellular vesicle microneedles in the preparation of drugs for the prevention, relief, or treatment of atopic dermatitis, characterized in that... The raw materials for preparing the purslane-derived extracellular vesicle microneedles include a matrix material and purslane-derived extracellular vesicles loaded in the matrix material; the matrix material includes purslane polysaccharide and hyaluronic acid or its salts; The loading of the purslane-derived extracellular vesicles is 0.1-3% based on protein concentration; the mass ratio of the purslane polysaccharide to hyaluronic acid or its salt is 1:1-1:

7. The method for preparing the purslane-derived extracellular vesicles includes ultracentrifugation or polyethylene glycol precipitation, wherein the ultracentrifugation method specifically includes the following steps: (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant; (2) After filtering the supernatant, centrifuge it at ultraspeed, collect the precipitate and resuspend it to obtain the product; The polyethylene glycol precipitation method specifically includes the following steps: (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant at least three times. The centrifugation speeds are 3000-7000 g, 7000-10000 g, and 10000-15000 g respectively, and the centrifugation times are 5-20 min, 15-45 min, and 40-90 min respectively. (2) After filtering the supernatant, mix it with polyethylene glycol with a number average molecular weight of 4000-8000. The final concentration of polyethylene glycol after mixing is 6-9%. Let it stand, centrifuge to collect the precipitate and resuspend it. (3) The resuspension is purified to obtain the final product.

2. The application according to claim 1, characterized in that, In the ultracentrifugation method, step (1) of centrifuging and collecting the supernatant includes at least three centrifugations, with centrifugation speeds of 800-1800 g, 2000-5000 g, and 8000-15000 g in sequence, and centrifugation times of 5-35 min, 5-35 min, and 40-90 min in sequence.

3. The application according to claim 1, characterized in that, In the ultracentrifugation method, the ultracentrifugation speed in step (2) is 100,000-200,000 g, and the time is 60-120 min.

4. The application according to claim 1, characterized in that, In the polyethylene glycol precipitation method, the settling time is 8-16 h.

5. The application according to claim 1, characterized in that, In the polyethylene glycol precipitation method, the centrifugation speed in step (2) is 6000-10000 g and the time is 10-50 min.

6. The application according to claim 1, characterized in that, In the polyethylene glycol precipitation method, the purification method includes size exclusion chromatography.

7. The application according to claim 1, characterized in that, The purslane polysaccharide is prepared by the following method: The purslane raw material was mixed with water and heated at 90-100℃ for 30-120 min. After filtration, the extract was subjected to protein removal treatment, and the treated liquid was then subjected to alcohol extraction. After standing, centrifugation, precipitation, and freeze-drying, the purslane polysaccharide was obtained.

8. The application according to claim 7, characterized in that, The protein removal process uses Sevag reagent.

9. The application according to claim 7, characterized in that, The alcohol extraction process uses 75-95% ethanol.

10. The application according to claim 7, characterized in that, In the preparation method of the purslane polysaccharide, the standing time is 12-48 h at 2-8℃.

11. The application according to claim 7, characterized in that, In the method for preparing purslane polysaccharide, the centrifugation is performed at 2000-8000 rpm for 5-20 min.

12. The application according to claim 1, characterized in that, The purslane-derived extracellular vesicle microneedles are prepared by a method including the following steps: Purslane polysaccharide, hyaluronic acid or its salts are mixed with purslane-derived extracellular vesicle suspension, the mixture is cast into a microneedle mold, left to stand under vacuum, dried, and demolded to obtain the purslane-derived extracellular vesicle microneedles.

13. The application according to claim 12, characterized in that, The vacuum level of the vacuum environment is -0.05 to -0.1 MPa.

14. The application according to claim 12, characterized in that, In the preparation method of the purslane-derived extracellular vesicle microneedles, the standing time is 5-30 min.

15. The application according to claim 12, characterized in that, The drying temperature is 25-45℃.

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