Preparation method and application of cell-derived nano vesicles

Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of cell-derived nanovesicles. The preparation method comprises the following steps: physically crushing umbilical cord mesenchymal stem cells to obtain a cell crushing solution; mixing the cell disruption liquid and the extracting solution according to the volume ratio of 3: 1-6: 1, and standing for 10-30 hours; and centrifuging, and collecting the precipitate to obtain the cell-derived nano-vesicles. The extracting solution comprises PEG-6000, sodium chloride and a protective additive, and the solvent is a PBS (Phosphate Buffer Solution). According to the method, purification is carried out by combining physical crushing with the extracting solution containing the specific protective additive, the nano-vesicles with a complete membrane structure and high biological activity can be efficiently prepared under the mild condition, and the defects that a traditional ultracentrifugation method is small in scale and low in efficiency, and vesicles are prone to being damaged are effectively overcome. The invention also provides the nano-vesicles obtained by the method, and application of the nano-vesicles in preparation of pharmaceutical compositions, skin care products and freeze-drying preparations for promoting skin wound healing.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing cell-derived nanovesicles and their applications. Background Technology

[0002] Extracellular vesicles (EVs) are nanoscale vesicles with a lipid bilayer structure actively released by cells, ranging in diameter from tens of nanometers to several micrometers. As key carriers of intercellular communication, EVs can carry and deliver bioactive substances such as proteins, nucleic acids, and lipids, demonstrating enormous application potential in tissue repair, immune regulation, disease diagnosis, and treatment. In particular, EVs derived from mesenchymal stem cells, due to their similar biological functions to their parent cells and their low immunogenicity and high safety, have been widely studied and used in biomedical and cosmetic fields such as promoting skin wound healing and anti-aging.

[0003] Currently, isolating and purifying vesicles (EVs) from complex biological systems, especially obtaining specific subpopulations (such as 30-150 nm EVs), remains a technical challenge. Ultracentrifugation, the most commonly used method for EV purification, can yield relatively enriched vesicle populations, but it suffers from limitations such as small processing scale, lengthy processing time, and the risk of mechanical damage to the vesicle membrane structure from repeated centrifugation steps, leading to vesicle rupture, fusion, or leakage of contents, ultimately reducing product yield and functional integrity. Other methods, such as density gradient centrifugation, size exclusion chromatography, and ultrafiltration, also often struggle to achieve both high-purity separation and efficient preservation of vesicle structure / activity on a large scale.

[0004] Therefore, given the common problems in existing EV preparation technologies, such as low efficiency, limited scale, susceptibility to vesicle damage, and difficulty in balancing high yield and high stability, there is an urgent need to develop a novel preparation method that can be operated on a large scale, has a mild process, and can effectively protect the structural integrity and bioactivity of vesicles. The purpose of this invention is to provide such an improved method for preparing cell-derived nanovesicles (NVs) to overcome the aforementioned shortcomings of existing technologies and meet the growing application demands in fields such as medicine and cosmetics. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing cell-derived nanovesicles and their uses.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing cell-derived nanovesicles includes the following steps: (1) Physically disrupt umbilical cord mesenchymal stem cells to obtain cell disruption fluid; (2) Mix the cell lysate and the extract at a volume ratio of 3:1 to 6:1 and let stand for 10 to 30 hours; (3) Centrifuge at 4℃, collect the precipitate, and obtain cell-derived nanovesicles; The extract includes: PEG-6000: 8-12% (w / v); Sodium chloride: 0.4-0.8% (w / v); Protective additives: at least one of squalane, ceramide, and antioxidant peptides; The buffer solution is PBS with a pH of 6.8-7.4 and an osmotic pressure of 250-400 mOsm / kg.

[0007] Preferably, the ceramide includes any one of phytosphingosine, N-lauroyl-D-sphingosine, and N-hydroxyethylsphingosine derivatives.

[0008] The physical crushing process is selected from one of ultrasonic crushing, high-pressure homogenization crushing, and tissue grinding crushing.

[0009] Ultrasonic conditions include: power of 300-1000W, ultrasound time of 1-10min, and intermittent ultrasound mode.

[0010] A cell-derived nanovesicle was obtained by the preparation method described above.

[0011] A pharmaceutical composition or skin care product comprising the aforementioned cell-derived nanovesicles and a pharmaceutically or skin care-acceptable carrier.

[0012] The pharmaceutical composition is used to promote skin wound healing, and the skin care product is used for skin care, repair, or anti-aging.

[0013] A skincare product comprising the following components: cell-derived nanovesicles; a moisturizer, a thickener, and a skin-care active ingredient; wherein the cell-derived nanovesicles are present in a concentration of 5-100 μg / mL.

[0014] An essence containing stem cell nanovesicles is composed of the following raw materials in weight percentages: 0.1-0.5 wt% sodium hyaluronate, 0.05-0.2 wt% xanthan gum, 0.05-0.3 wt% polyglutamic acid, 1-4 wt% nicotinamide, 2-6 wt% 1,3-butanediol, 1-3 wt% 1,2-hexanediol, 5-100 μg / mL of the cell-derived nanovesicles of claim 4, and the balance being water.

[0015] A lyophilized formulation comprising the aforementioned cell-derived nanovesicles and a lyophilization protectant comprising trehalose, and optionally mannitol and / or glycine.

[0016] The freeze-drying protectant comprises trehalose, and optionally mannitol and / or glycine, and is reconstituted using a sodium hyaluronate solution with a molecular weight of 5-10 kDa.

[0017] The beneficial effects of this invention are: (1) The vesicles obtained by this invention have clear biological activity and can significantly promote the proliferation and migration of human skin fibroblasts (HSF), revealing their direct role in promoting tissue repair. Freeze-drying formulation tests have demonstrated that the protective agent system of this invention can improve the freeze-drying shape and clarity of vesicles after reconstitution, avoid protein aggregation, and maintain vesicle activity.

[0018] (2) This invention employs a mild medium-speed centrifugation (8,000-12,000 g) combined with a PEG-sodium chloride precipitation system for vesicle enrichment, successfully replacing the traditional method that relies on ultra-high-speed centrifugation (100,000 g). This process significantly reduces mechanical shear force and better protects the integrity of vesicles while greatly improving sedimentation efficiency and throughput, providing a reliable and economical solution for large-scale preparation.

[0019] (3) This invention innovatively introduces squalane and ceramide lipid protectants into the extraction system to construct a stable system that can simulate the natural biomembrane environment. This system can effectively inhibit membrane rupture, fusion and leakage of contents during the preparation process of vesicles, thereby significantly improving the membrane integrity, particle size uniformity and storage stability of vesicle products, laying the foundation for obtaining high-activity, low-aggregation high-quality vesicles.

[0020] (4) The cell-derived nanovesicles prepared in this invention have good skin biocompatibility and safety. Animal experiments showed that there was no obvious inflammatory response after local injection of nanovesicles, and the levels of inflammation-related factors remained basically stable; wound model experiments showed that nanovesicles can promote wound repair and show a trend of tissue regeneration, which has good application prospects.

[0021] (5) The nanovesicles prepared in this invention, based on their clear activity in promoting the proliferation and migration of human skin fibroblasts, their effective wound repair effects in animals, and their good biocompatibility and stability, are expected to be used in pharmaceutical compositions, skin repair preparations, and cosmetics. They can also be compounded with skin care ingredients such as sodium hyaluronate, polyglutamic acid, and niacinamide to form a stable serum system. The serum containing vesicles shows significant advantages in improving skin texture, reducing fine lines, and enhancing skin radiance, thus meeting the dual needs of skin barrier repair and anti-aging. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a comparison diagram of the particle size distribution of NVs and EVs prepared after ultrasonic cell disruption according to the present invention. Figure 2 Transmission electron micrographs of NVs and EVs prepared after ultrasonic cell disruption according to the present invention. Figure 3 Transmission electron microscopy images of NVs prepared after cell disruption under different homogeneous pressures according to the present invention. Figure 4 The figure shows the NTA analysis results of NVs prepared by different cell disruption methods according to the present invention. A represents NVs prepared after homogenization at 10 bar; B represents NVs prepared after homogenization at 20 bar; C represents NVs prepared after homogenization at 50 bar; D represents NVs prepared after ultrasonic disruption; and E represents EVs. Figure 5 This figure shows the effect of different concentrations of NVs on HSF cell proliferation. Cell proliferation rate is expressed as mean ± SD (n=3). Compared with the control group (CK), This means p < 0.001; Figure 6 The figures show representative results of the HSF cell scratch assay using NVs prepared by different cell disruption methods at a concentration of 5 μg / ml. A1 represents the control group (CK); A2 represents the NVs group prepared after ultrasonic cell disruption; A3 represents the NVs prepared after grinding and disruption (complete culture); and A4 represents the EVs group. Compared with the control group (CK), This means p < 0.05; Figure 7 The graph shows the quantitative analysis of the scratch healing rate of HSF cells by NVs prepared using different cell disruption methods at a concentration of 5 μg / ml. The healing rate is expressed as mean ± SD (n=3), compared with the control group (CK). This means p < 0.05; Figure 8 A representative image showing the effect of NVs and EVs prepared by the homogenized cell disruption method of this invention at 5 μg / ml on the Transwell migration of HSF cells; Figure 9A quantitative analysis of the 570 nm absorbance values ​​of NVs and EVs prepared by the homogenized cell disruption method of this invention at a concentration of 5 μg / ml on HSF cell Transwell migration. Figure 10 Representative photographs of wound healing at 0, 3, 7, and 14 days post-surgery in a mouse model of full-thickness skin defects prepared by ultrasonic cell disruption according to the present invention using NVs local drug delivery. Figure 11 The image shows the serum TNF-α levels at different time points after subcutaneous injection of NVs (non-toxic cells) prepared by ultrasonic disruption of the present invention into SD rats. TNF-α concentration is expressed as mean ± SD (n=3), compared with the control group (CK). This means p < 0.01; Figure 12 The image shows the serum IL-10 levels at different time points after subcutaneous injection of NVs (non-vitamin A cells) prepared by ultrasonic disruption of the present invention into SD rats. IL-10 concentration is expressed as mean ± SD (n=3). This means p < 0.05. This means p < 0.01; Figure 13 The figure shows the effect of NVs prepared by ultrasonic cell disruption of the present invention on the proliferation activity of HSF cells after being placed at 2-8℃ for different times. The cell proliferation rate is expressed as mean ± SD (n=3), compared with the control group (CK). This means p < 0.05. This means p < 0.01. This means p < 0.001; Figure 14 Comparative photos showing how stem cell-containing nanovesicles improve the appearance of photoaged nude mice skin. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this should not be construed as limiting the scope of protection of the present invention to the following embodiments. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention still fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, all raw materials used in the following examples are commercially available analytical grade reagents; unless otherwise specified, all experimental methods are conventional experimental methods.

[0026] 1. Experimental materials PBS (HyClone Laboratories, Inc.), DMEM (HyClone Laboratories, Inc.), Bovine Serum Albumin (Bovogen Biologicals Pty Ltd.), Human Umbilical Cord Mesenchymal Stem Cells (Jiangsu Sairuit Biotechnology Co., Ltd.), Recombinant Anti-TSG101 Antibody (Abcam), Anti-GAPDH Rabbit Polyclonal Antibody (Kangwei Century Biotechnology Co., Ltd.), Goat Anti-Rabbit IgG (H+L), HRP (Kangwei Century Biotechnology Co., Ltd.), Human Skin Fibroblasts (Nanjing Zhitai Biomedical Technology Co., Ltd.), 4% Paraformaldehyde Fixative (Shanghai Beyotimes Biotechnology Co., Ltd.), Crystal Violet Staining Solution (Shanghai Beyotimes Biotechnology Co., Ltd.), BCA Detection Kit (Enhanced) (Shanghai Beyotimes Biotechnology Co., Ltd.), RIPA Protein Lysis Buffer (Strong) (Shanghai Beyotimes Biotechnology Co., Ltd.), PMSF Protease Inhibitor (Shanghai Beyotimes Biotechnology Co., Ltd.), Western blot Kits (Shanghai Beyotime Biotechnology Co., Ltd.), Enhanced CCK-8 Kit (Shanghai Beyotime Biotechnology Co., Ltd.), SD rats and BALB / c mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.), Lipopolysaccharide (LPS) (Sigma-Aldrich LLC.), Rat tail collagen, Recombinant Mouse EGF (Sino Biological Inc.), Anti-SD rat TNF-α ELISA kit / Anti-SD rat IL10 ELISA kit (Hangzhou Lianke Biotechnology Co., Ltd.).

[0027] 2. Experimental Methods 2.1 Cell Culture 2.1.1 Culture and Harvesting of Human Umbilical Cord Mesenchymal Stem Cells P4 generation umbilical cord mesenchymal stem cells were cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin at a rate of 5000 cells / cm². 2 The cells were seeded at a density in culture dishes and incubated at 37°C with 5% CO2 saturated humidity for 3-4 days. When the cell confluence reached 90%-95%, the culture medium was discarded, and the cells were washed twice with sterile PBS. Then, a 0.25% trypsin-0.02% EDTA digestion solution was added, and the cells were digested at 37°C for about 2 minutes. After observing under a microscope that most of the cells had shrunk and become rounded, α-MEM culture medium containing 10% FBS was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in an appropriate amount of PBS solution.

[0028] 2.1.2 Starvation culture of human umbilical cord mesenchymal stem cells P4 generation umbilical cord mesenchymal stem cells were collected and cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin at a density of 5000 cells / cm². 2 The cells were seeded at a density in culture dishes and incubated at 37°C with 5% CO2 saturated humidity for about 3 days. When the cell confluence reached 80-85%, the medium was replaced with serum-free α-MEM and cultured at 37°C with 5% CO2 saturated humidity for another 48 hours. The supernatant was then collected for later use.

[0029] 2.1.3 Human skin fibroblast culture HSF cells were harvested and cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin at a density of 5000 cells / cm². 2 Density seeding was performed, and cells were incubated at 37°C with 5% CO2 saturated humidity for approximately 4 days. When cells reached 80%-90% confluence, the culture supernatant was discarded, and the cells were washed twice with PBS. Then, 0.25% trypsin and 0.02% EDTA were added, and the cells were digested at 37°C for approximately 2 minutes. After observing under a microscope that most cells had shrunk and become rounded, DMEM culture medium containing 10% FBS was added to terminate the digestion. The cells were centrifuged at 1200 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in an appropriate amount of PBS solution or fresh culture medium.

[0030] 2.2 Preparation of cell-derived nanovesicles by physical disruption method 2.2.1 Preparation of NVs by ultrasonic disruption method With a concentration of 1×10 7 Cells / mL of umbilical cord mesenchymal stem cells were transferred into 4mL centrifuge tubes and then subjected to ultrasonic disruption in an ice-water mixture. Ultrasonic conditions: Amplitude rod model The probe is placed 1cm below the liquid surface, with a power of 900W×40%. The working mode is ultrasonic for 2 seconds, with a 2-second interval, and a total processing time of 4 minutes. The temperature is controlled to not exceed 25℃ throughout the process.

[0031] Preparation of the extraction solution: First, weigh 8-12% (w / v) PEG-6000 and 0.4-0.8% (w / v) sodium chloride, dissolve them in PBS buffer at pH 6.8-7.4, and after complete dissolution, prepare the basic extraction solution; then add 0.01%-0.1% (v / v) squalane, 0.001%-0.05% (w / v) N-lauroyl-D-sphingosine or 0.001%-0.05% (w / v) glutathione to the basic solution, and vortex to fully disperse it; finally, filter the mixture through a 0.22 μm filter membrane for sterilization and store at 2-8℃.

[0032] The cell lysate obtained by sonication was mixed with the above extract at a volume ratio of 4:1 and placed in a refrigerator at 2-8℃ for 16 hours. Then, the mixture was centrifuged at 10,000g for 60 minutes at 4℃, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended with an appropriate amount of PBS to obtain the purified NVs (hereinafter referred to as sonicated NVs), which were aliquoted into 50μL tubes and stored at -80℃.

[0033] 2.2.2 Preparation of NVs by High-Pressure Homogenizer Crushing Method At 4-6℃, 50 mL of a 1×10⁻⁶ solution was prepared. 7 The suspension of umbilical cord mesenchymal stem cells was transferred into the material cup of a high-pressure homogenizer. The homogenization pressure was set, and the cells were homogenized three times at a frequency of 50 Hz. The homogenized liquid was collected as the cell lysate.

[0034] Mix the homogenized cell lysate with the extract at a volume ratio of 4:1 and let stand at 2-8℃ for 16 hours. Centrifuge, collect the precipitate, resuspend the precipitate with an appropriate amount of PBS to obtain the purified NVs (hereinafter referred to as homogenized NVs), aliquot into 50μL tubes and store at -80℃.

[0035] 2.2.3 Preparation of NVs by tissue grinding and disruption method 1 mL of a concentration of 1×10 7 Transfer the cell / mL suspension of umbilical cord mesenchymal stem cells to a 2mL centrifuge tube. Add sterile quartz sand at a ratio of 6:1 (v / w) of cell suspension to quartz sand. Place the centrifuge tube in a pre-cooled adapter at -20℃ and then place it in a tissue homogenizer. Set the homogenization parameters: frequency 55 Hz, homogenization time 60 seconds per cycle, repeat twice to complete cell disruption.

[0036] Mix the cell lysate obtained by grinding with the extract at a volume ratio of 4:1 and let stand at 2-8℃ for 16 hours. Centrifuge, collect the precipitate, resuspend the precipitate with an appropriate amount of PBS to obtain the purified NVs (hereinafter referred to as ground NVs), aliquot into 50μL tubes and store at -80℃.

[0037] 2.3 Preparation of EVs by Ultracentrifugation The cell supernatant after 48 hours of starvation culture was centrifuged at 300g for 10 minutes at 4°C and collected; then centrifuged at 16500g for 20 minutes at 4°C and collected again; after filtration through a 0.22μm filter membrane, the supernatant was transferred to an ultrafiltration tube and centrifuged at 100000g for 2 hours at 4°C and the supernatant was discarded; the precipitate was resuspended in PBS to obtain ultrapure EVs, which were aliquoted into 50μL tubes and stored at -80°C.

[0038] 2.4 Physicochemical Properties of Cell-Derived Nanovesicles 2.4.1 Determination of protein concentration by BCA method According to the BCA assay kit instructions, mix 50 volumes of BCA reagent A and 1 volume of BCA reagent B to prepare the working solution. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard solution to the standard wells of a 96-well plate, and bring the total volume to 20 μL with standard diluent. Add an appropriate volume of NVs / EVs sample to the sample wells, and bring the total volume to 20 μL with standard diluent. Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve.

[0039] 2.4.2 Particle size distribution analysis 2.4.2.1 Dynamic Light Scattering (DLS) Detection The NVs / EVs samples were prepared into 1 mL solutions of appropriate concentration, and the particle size distribution was detected using a nanoparticle size analyzer.

[0040] 2.4.2.2 Nanoparticle tracking analysis (NTA) Nanjing Fomax Biotechnology Co., Ltd. was commissioned to use NTA to detect the concentration and particle size distribution of the samples.

[0041] 2.4.3 Morphological observation using transmission electron microscopy (TEM) Take 20 μL of NVs / EVs sample and drop it onto the sealing film. Float a copper mesh on the droplet with the coated side facing the droplet. Let it stand at room temperature for 10 minutes, then blot away excess liquid with filter paper. Negatively stain with 2% phosphotungstic acid solution for 2 minutes, blot away excess liquid with filter paper, and dry under an infrared baking lamp. Observe the morphology of the vesicles using a transmission electron microscope and take electron microscope images.

[0042] 2.4.4 Western blot detection of characteristic proteins Mix NVs / EVs samples with 5× loading buffer at a ratio of 4:1, denature at 100℃ for 5 min, add to the sample wells of a 12% SDS-PAGE gel, with a protein content of 20 μg per well, stack the gel at a constant voltage of 90V, separate the gel at a constant voltage of 120V, and electrophoresis for 1 hour.

[0043] After electrophoresis, the gel was removed, and proteins were electrotransferred to a PVDF membrane under ice-water bath conditions. The PVDF membrane was blocked with blocking buffer at room temperature for 15 min, and washed three times with 1×TBST for 10 min each time. After cutting the membrane, it was placed in the corresponding primary antibody dilution buffer and incubated overnight at 2-8℃. It was then washed three times with 1×TBST for 10 min each time, and incubated for 2 h at room temperature with HRP-labeled secondary antibody dilution buffer. It was then washed three times with 1×TBST for 10 min each time. The membrane was developed with ultrasensitive ECL chemiluminescence reagent for 1 min, and images were acquired using a chemiluminescence imager.

[0044] 2.5 Effect of nanovesicles on HSF cell proliferation as determined by CCK-8 assay HSF cells in logarithmic growth phase were harvested and resuspended in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. They were seeded at a density of 1800 cells / well in 96-well plates (100 μL per well) and incubated at 37°C with 5% CO2 saturated humidity for 48 h. The medium was then replaced with low-serum medium (DMEM medium containing 1% FBS) containing different concentrations of nanovesicles (0, 25, 50, 100 μg / L), and cultured for another 48 h. 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 3 h. The absorbance at 450 nm was then measured using a microplate reader.

[0045] 2.6 Scratch assay to detect the effect of nanovesicles on HSF cell migration HSF cells in the logarithmic growth phase were harvested and resuspended in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, at a concentration of 1×10⁻⁶ cells / mL. 4 cells / cm 2 Cells were seeded at a density of 500 μL per well in 24-well plates and incubated at 37°C with 5% CO2 and saturated humidity until complete confluence. A 1 mL pipette tip was used to streak the cell layer horizontally, creating uniformly wide scratches. After washing twice with PBS, the medium was replaced with low-serum medium (DMEM medium containing 1% FBS) containing different concentrations of nanovesicles (5, 25, 50, 100 μg / L). Scratch images were captured using a high-content viability analyzer at 0, 24, 48, and 72 h. Scratch distance was measured using ImageJ software, with three measurement points for each scratch. The confluence rate was calculated using the formula: Confluence rate at T = (Edge distance at T0 - Edge distance at T) / Edge distance at T0 × 100%.

[0046] 2.7 Transwell migration assay to detect the effect of nanovesicles on HSF cell migration HSF cells in logarithmic growth phase were harvested, resuspended in serum-free DMEM medium, and the cell density was adjusted to 1×10⁶ cells / year. 5 Cells / mL, 200 μL was added to the upper chamber of the Transwell, and serum-free DMEM medium containing different concentrations of nanovesicles (0, 5, 20 μg / mL) was added to the lower chamber. The cells were incubated at 37°C and 5% CO2 saturated humidity for 2 days. The Transwell chamber was removed and the liquid inside was discarded. The cells were washed twice with PBS, and unmigrated cells in the upper chamber were wiped away with a cotton swab. Cells on the lower surface of the upper chamber filter membrane were fixed with 4% paraformaldehyde for 10 min, washed twice with PBS, stained with crystal violet for 10 min, washed with purified water, dried, and observed and photographed under an upright fluorescence microscope.

[0047] 3. Experimental study on the application of cell-derived nanovesicles in skin wound healing 3.1 Experiment on the effect of nanovesicles on wound healing after full-thickness skin resection in Balb / c mice Eight male Balb / c mice, 4-6 weeks old, were housed under constant temperature and standard conditions for one week. The day before the experiment, the mice were anesthetized and the surgical area on their backs was prepared. On the day of the experiment, the anesthetized mice were fixed to a sterile operating table, and a full-thickness skin defect with a diameter of 1 cm was created in the center of their backs. Eight mice were randomly divided into five groups: (1) PBS control group (n = 2), with 100 μL of PBS solution applied to the wound; (2) EVs group (n = 2), with 100 μL of EVs at a concentration of 100 μg / mL applied to the wound; (3) Ultrasound NVs group (n = 2), with 100 μL of ultrasound-prepared NVs at a concentration of 100 μg / mL applied to the wound; and (4) Ultrasound NVs + Collagen + EGF group (n = 2), with 100 μL of a mixed solution containing 100 μg / mL ultrasound-prepared NVs, 100 μg / mL mouse tail collagen, and 25 μg / mL Recombinant Mouse EGF applied to the wound. On postoperative day 0 and day 1, 100 μL of the corresponding treatment factor was applied to the wound, and the wound was bandaged with gauze, absorbent cotton gauze, and medical tape. The mice were housed separately according to their groups. On postoperative days 0, 3, 7, and 14, photographs were taken to observe and measure the degree of wound healing.

[0048] Postoperatively, medication was administered once on day 0 and once on day 1. After each administration, the wound was covered with petroleum jelly gauze and secured with absorbent cotton gauze and medical tape. Wound images were recorded and acquired on postoperative days 0, 3, 7, and 14. The wound area was measured using ImageJ software, and the wound healing rate on days 7 and 14 relative to day 3 was calculated using the following formula: Healing rate (%) = [(Wound area on day 3 - Unhealed area at a specific time point) / Wound area on day 3] × 100%.

[0049] 3.2 In vivo safety evaluation of nanovesicles Nine male SD rats, aged 4-6 weeks, were randomly divided into three groups (n=3 per group) after being kept under constant temperature and standard conditions for one week: (1) saline control group; (2) ultrasound NVs group (1 mg / mL); and (3) LPS positive control group (5 mg / mL). One day before the experiment, the SD rats in each group were anesthetized and the surgical area on their backs was prepared. On the day of the experiment, the anesthetized rats were fixed on a sterile operating table, and 200 μL of the corresponding reagent was injected subcutaneously at multiple points in the hairless area on their backs. Blood samples were collected from the tail vein at 0, 3, 6, 24, and 48 hours after injection for ELISA to detect the levels of TNF-α and IL-10 in serum. Simultaneously, local reactions such as redness and swelling at the injection site were observed, and the rats' behavior and physiological state were recorded.

[0050] 3.3 nanovesicle stability test at 2-8℃ Ultrasound NVs stored at -80℃ were divided into groups of three and stored at 2-8℃ for 0, 1, 3, 5, and 7 days, respectively. The proliferative activity of NVs on HSF cells at each time point was detected by the CCK-8 assay.

[0051] 4 Results and Analysis 4.1 Particle size distribution characteristics of nanovesicles prepared by fragmentation method Dynamic light scattering results show ( Figure 1 The NVs prepared by ultrasonic disruption exhibit a more uniform particle size distribution, concentrated in the range of 70-100 nm; while the EVs used as a control have a relatively wider particle size distribution, ranging from 50-150 nm. This data confirms that the process of this invention can effectively control the physical size of vesicles, obtaining nanovesicle products with a narrower particle size distribution and better uniformity.

[0052] 4.2 Transmission electron microscopy analysis of nanovesicles prepared by fragmentation method Transmission electron microscopy observation results ( Figure 2 The results showed that both the ultrasonically disrupted NVs and the control EVs exhibited typical vesicle structures, with no significant morphological differences observed. Specifically, both were round or elliptical membrane vesicle structures with distinct double membrane boundaries and a typical "cup-shaped" morphology. This result confirms that the ultrasonic disruption process effectively reduces the vesicle particle size distribution width while completely preserving its basic membrane structure and vesicle morphology.

[0053] 4.3 Particle size analysis and transmission electron microscopy analysis of nanovesicles prepared by different fragmentation methods Transmission electron microscopy observation results ( Figure 3 The results showed that classic cup-shaped spherical vesicles could be observed in NVs prepared by homogenization pressure at 10 bar and 50 bar, and that homogenization pressure of 10-50 bar could effectively produce structurally intact nanovesicles. NTA analysis results showed ( Figure 4 The nanovesicles, ultrasonic NVs, and EVs prepared by homogenization under different homogeneous pressures ranging from 5 to 50 bar have particle sizes mainly distributed in the range of 80-150 nm. These data confirm that the NVs prepared in this invention possess the key physical properties of EVs.

[0054] 4.4 Effects of Nanovesicles Prepared by Different Disruption Methods on HSF Cell Proliferation The results of the CCK-8 experiment showed that ( Figure 5 Within the concentration range of 5-100 μg / mL, both the ultrasonic NVs and milled NVs treatment groups showed statistically significant increases in cell proliferation efficiency compared to the blank control group (p<0.05). Notably, within this concentration range, no obvious dose-dependent effect was observed in the proliferation-promoting effect of the two NVs. This phenomenon suggests that the nanovesicles possess the potential advantage of low effective concentrations.

[0055] 4.5 Effects of Nanovesicles Prepared by Different Disruption Methods on HSF Cell Migration The scratch test results show that ( Figure 6-7 At a concentration of 5 μg / mL, all nanovesicle treatment groups showed a trend of promoting HSF cell migration and accelerating scratch closure within 24-72 hours. Transwell migration assay results showed ( Figure 8-9 At a concentration of 5 μg / mL, both homogenized NVs and EVs significantly promoted the chemotactic migration of HSF cells compared to the blank control group. These results, from different methodological perspectives, collectively confirm that the nanovesicles prepared in this invention can effectively enhance the migration ability of HSF cells.

[0056] 4.6 Effects of different nanovesicles on skin wound healing Preliminary evidence from in vivo animal experiments suggests that ( Figure 10 On day 14 post-surgery in mice, the wound closure morphology of the ultrasound NVs group, the ultrasound NVs combined with EGF group, and the EVs group all showed a trend of accelerated healing compared to the PBS control group. These results preliminarily confirm at the animal level that the nanovesicles prepared in this invention possess the potential function of promoting wound healing.

[0057] 4.7 Safety evaluation of nanovesicles No redness or swelling was observed at the injection site after subcutaneous injection of nanovesicles in rats, and no abnormal reaction symptoms were observed in the rats.

[0058] Analysis of inflammation-related factors showed that ( Figure 11-12Regarding pro-inflammatory factors, the serum TNF-α (a key factor mediating acute inflammatory responses) levels in the ultrasound NVs group were significantly lower than those in the saline control group at 0 and 3 hours after injection (P < 0.01), subsequently returning to baseline. This indicates that the material not only did not induce harmful inflammatory activation but may also have a slight inhibitory effect on the underlying inflammatory state. Regarding anti-inflammatory factors, the IL-10 level in the ultrasound NVs group (responsible for inhibiting excessive inflammation and maintaining immune homeostasis) showed no statistically significant difference compared to the control group throughout the observation period, remaining stable within the physiological range, suggesting that it did not disrupt the body's own immune balance. Compared to the LPS positive control (which exhibits a typical inflammatory response characterized by a sharp increase in TNF-α and compensatory upregulation of IL-10 after injection), the nanovesicles of this invention exhibit a distinctly different and more mild and safe response pattern, confirming its potential anti-inflammatory regulatory properties.

[0059] Stability of 4.8 nanometer vesicles stored at 2-8℃ Stability experiment analysis shows that ( Figure 13 The ultrasound NVs were stored at 2-8℃ for one week. Compared with the blank control group, the ultrasound NVs maintained significant proliferative activity against HSF cells throughout the storage period (P < 0.05). This result indicates that the nanovesicles prepared in this invention have excellent short-term storage stability under refrigeration conditions.

[0060] 5. Final Conclusion The nanovesicles prepared in this invention are highly similar to natural EVs in morphology and particle size, with a more concentrated particle size distribution and better batch-to-batch consistency. This material can effectively enhance the migration ability of HSF cells, which is a key link in promoting tissue repair. Animal experiments further show that this material tends to accelerate the healing of wounds in full-thickness skin defect models without inducing harmful inflammatory responses, and even shows potential anti-inflammatory regulatory properties, laying an important safety foundation for its biomedical applications. In addition, after being stored at 2-8°C for one week, the core biological activity of the nanovesicles is not significantly lost, proving that it has good short-term stability under recommended storage conditions and meets the basic requirements for practical storage and transportation.

[0061] In summary, this invention provides a nanovesicle with controllable process, stable structure, well-defined activity, and high safety, as well as a method for its preparation. These nanovesicles accelerate wound healing by promoting the proliferation and migration of repair-related cells, demonstrating significant application potential and industrialization prospects in tissue engineering and regenerative medicine, particularly in the development of products for acute and chronic wound repair.

[0062] 6. Application of stem cell-derived nanovesicles in skincare products Example 1 The serum, which does not contain stem cell nanovesicles, is composed of the following ingredients by weight percentage: 0.3wt% sodium hyaluronate, 0.1wt% xanthan gum, 0.1wt% polyglutamic acid, 2wt% niacinamide, 3wt% 1,3-butanediol, 2wt% 1,2-hexanediol, and the balance being water.

[0063] Example 2 The essence containing stem cell nanovesicles is composed of the following ingredients by weight percentage: 0.3wt% sodium hyaluronate, 0.1wt% xanthan gum, 0.1wt% polyglutamic acid, 2wt% nicotinamide, 3wt% 1,3-butanediol, 2wt% 1,2-hexanediol, 50μg / mL of the cell-derived nanovesicles, and the balance being water.

[0064] The method for preparing the cell-derived nanovesicles is as follows: Preparation of NVs by ultrasonic disruption: NVs with a concentration of 1×10⁻⁶ 7 Cells / mL of umbilical cord mesenchymal stem cells were transferred into 4mL centrifuge tubes and then subjected to ultrasonic disruption in an ice-water mixture. Ultrasonic conditions: Amplitude rod model The probe is placed 1cm below the liquid surface, with a power of 900W×40%. The working mode is ultrasonic for 2 seconds, with a 2-second interval, and a total processing time of 4 minutes. The temperature is controlled to not exceed 25℃ throughout the process.

[0065] Preparation of the extraction solution: First, weigh 10% (w / v) PEG-6000 and 0.6% (w / v) sodium chloride, dissolve them in PBS buffer at pH 7.2, and after complete dissolution, prepare the basic extraction solution; then add 0.06% (v / v) squalane and 0.02% (w / v) N-lauroyl-D-sphingosine to the basic solution, and vortex to disperse them thoroughly; finally, filter the mixture through a 0.22 μm filter membrane for sterilization and store at 2-8℃.

[0066] The cell lysate from ultrasonic disruption was mixed with the above extract at a volume ratio of 4:1 and placed in a 4°C refrigerator for 16 hours. Then, the mixture was centrifuged at 10,000g for 60 minutes at 4°C, the supernatant was discarded and the precipitate was collected. The precipitate was resuspended with an appropriate amount of PBS to obtain the ultrasonic NVs purified from the extract, which was stored at -80°C.

[0067] Anti-wrinkle experiment: Six healthy female nude mice (5-7 weeks old, weighing 18-22g) were selected and acclimatized for one week. A skin photoaging model was established by irradiating the backs of the mice with a UVA / UVB composite light source. Irradiation was performed three times a week for eight consecutive weeks. The irradiation dose was increased incrementally, with a cumulative dose of approximately 77 J / cm² for UVA. 2 UVB 19 J / cm 2After the modeling was completed, 6 nude mice were randomly divided into two groups (n=3): (1) Control group: The essence of Example 1 without stem cell nanovesicles was applied to the irradiated area daily; (2) Treatment group: The essence of Example 2 containing stem cell nanovesicles was applied to the irradiated area daily. Both groups were administered the medication once daily for 4 consecutive weeks.

[0068] The efficacy evaluation was conducted after the administration was completed. Two observers, unaware of the group assignments, directly observed and compared the skin condition of the irradiated areas on the backs of the two groups of nude mice.

[0069] After 8 weeks of UV irradiation, all nude mice showed varying degrees of increased wrinkles, roughness, and skin laxity on their backs. After 4 weeks of treatment, direct observation showed ( Figure 14 Compared with the control group, the treatment group treated with the stem cell nanovesicle essence of Example 2 showed a significant reduction in back wrinkles, smoother skin surface, and improved skin firmness. The results indicate that the stem cell nanovesicle essence provided in Example 2 of this invention can effectively improve the appearance of skin photoaging induced by UV irradiation.

[0070] Example 3 The process is basically the same as in Example 2, except for the following: Preparation of the extraction solution: First, weigh 10% (w / v) PEG-6000 and 0.6% (w / v) sodium chloride, dissolve them in PBS buffer at pH 7.2, and after complete dissolution, prepare the basic extraction solution; then add an equal mass of 0.06% (v / v) squalane to the basic solution and vortex it to disperse it fully; finally, filter the mixture through a 0.22 μm filter membrane for sterilization and store it at 2-8℃.

[0071] Example 4 The process is basically the same as in Example 2, except for the following: Preparation of the extraction solution: First, weigh 10% (w / v) PEG-6000 and 0.6% (w / v) sodium chloride, dissolve them in PBS buffer at pH 7.2, and after complete dissolution, prepare the basic extraction solution; then add an equal mass of 0.02% (w / v) N-lauroyl-D-sphingosine to the basic solution and vortex it to disperse it fully; finally, filter the mixture through a 0.22 μm filter membrane for sterilization and store it at 2-8℃.

[0072] Example 5 The process is basically the same as in Example 2, except that no protective agent was added. The preparation of the extract is as follows: First, weigh 10% (w / v) PEG-6000 and 0.6% (w / v) sodium chloride, dissolve them in PBS buffer at pH 7.2, and after complete dissolution, filter through a 0.22 μm filter membrane for sterilization and store at 2-8℃.

[0073] Anti-wrinkle test: Forty volunteers with wrinkles and dark circles around the eyes were randomly selected for testing. Volunteer criteria included: female, aged 30-50; no use of hormonal drugs or immunosuppressants in the past 30 days; no participation in other clinical trials on the test site in the past 30 days; and agreement not to use any cosmetics, drugs, or health products that could affect the results during the trial. Ten volunteers were divided into groups, and each group was tested with the stem cell nanovesicle-containing essences from Examples 1-4. The test results were the average of each group.

[0074] Test Method: Volunteers applied a pea-sized amount of the above-mentioned serum containing stem cell nanovesicles to their eye area every morning and evening after cleansing, for 90 consecutive days. Facial images of the subjects were captured using Canfield Visia-CR to analyze changes in wrinkles and melanin. The subjects were photographed without makeup. The test results are shown in Table 1.

[0075] Skin radiance was measured using SkinGlossMeter, SGM2008. The higher the value, the more radiant the skin. Values ​​were measured for each group of volunteers before using the skincare product and the average was taken. Values ​​were recorded after 3 months of product use.

[0076] Table 1 Results of wrinkle depth reduction test

[0077] As shown in Table 1, with the gradual addition of the protective agents squalane and N-lauroyl-D-sphingosine, the overall anti-wrinkle effect, skin radiance, and skin smoothness of the serum all showed an improving trend. The wrinkle area reduction rate in Examples 2-4 was significantly higher than that in Example 5 without the protective agent, indicating that the protective agent can enhance the stability and penetration efficiency of the nanovesicles in the formulation. Squalane can mimic the skin's lipid environment, improving the flexibility and skin affinity of the vesicle membrane, while N-lauroyl-D-sphingosine, as a ceramide, can promote barrier repair and increase the retention of vesicles in the stratum corneum, allowing the active substances carried by the vesicles to be released more efficiently to damaged tissues.

[0078] Therefore, the addition of the protectant improves the preservation rate of vesicle activity, strengthens the skin barrier structure, reduces epidermal moisture loss, and promotes fibroblast repair and collagen synthesis, thereby reducing wrinkle depth, decreasing skin roughness, and improving skin radiance. Ultimately, Example 2 achieves its optimal state, with wrinkles reduced by 32.8% and radiance increased to 65.8.

[0079] Example 6: Preparation and Reconstitution Performance Evaluation of Stem Cell-Derived Nanovesicle Lyophilized Powder Prepare the lyophilized powder according to the following steps: Take 200 μg of the nanovesicles prepared in this invention, add trehalose (final concentration of 0.5 wt% by total volume) as a lyophilization protectant, and adjust the volume to 2.00 mL with physiological saline. Mix well to make the final protein concentration of the nanovesicles 100 μg / mL. Filter the above solution through a 0.45 μm microporous membrane for sterilization, and pre-freeze at -80℃ for 2 hours to complete the sample solidification before freeze-drying.

[0080] The freeze-drying process was carried out under vacuum conditions, and the program was set as follows: first, maintain at -45℃ and 10-15 Pa for 4 hours; then maintain at -35℃ for 4 hours; then raise the temperature to -20℃ within 10 hours and maintain at that temperature for 2 hours; then raise the temperature to 5℃ within 5 hours; finally, maintain the vacuum degree below 10 Pa until the water content of the sample is less than 3%.

[0081] After freeze-drying, the freeze-dried nanovesicle powder is hollow and has a full and complete morphology. Using 0.15wt% sodium hyaluronate with a molecular weight of 7kDa as a resolvent, the freeze-dried powder can be quickly and completely redissolved. The resulting solution is clear and transparent, without any visible precipitates or suspended particles, indicating that the freeze-drying process effectively maintains the stability and redispersibility of the nanovesicles.

Claims

1. A method for preparing cell-derived nanovesicles, characterized in that, Includes the following steps: (1) Physically disrupt umbilical cord mesenchymal stem cells to obtain cell disruption fluid; (2) Mix the cell lysate and the extract at a volume ratio of 3:1 to 6:1 and let stand for 10 to 30 hours; (3) Centrifuge, collect the precipitate, and obtain cell-derived nanovesicles; The extract includes: PEG-6000: 8-12% (w / v); Sodium chloride: 0.4-0.8% (w / v); Protective additives: at least one of squalane, ceramide, and antioxidant peptides; The buffer solution is PBS with a pH of 6.8-7.4 and an osmotic pressure of 250-400 mOsm / kg; The ceramide includes any one of phytosphingosine, N-lauroyl-D-sphingosine, and N-hydroxyethylsphingosine derivatives.

2. The method for preparing cell-derived nanovesicles according to claim 1, characterized in that, The physical crushing process is selected from at least one of ultrasonic crushing, high-pressure homogenization crushing, and tissue grinding crushing.

3. The method for preparing cell-derived nanovesicles according to claim 1, characterized in that, Ultrasonic conditions include: power of 300-1000W, ultrasound time of 1-10min, and intermittent ultrasound mode.

4. A cell-derived nanovesicle, characterized in that, It is obtained by the preparation method described in any one of claims 1-3.

5. A pharmaceutical composition or skin care product, characterized in that, It includes the cell-derived nanovesicles as described in claim 4 and pharmaceutically or skincare-acceptable carriers.

6. The pharmaceutical composition or skin care product according to claim 5, characterized in that, The pharmaceutical composition is used to promote skin wound healing, and the skin care product is used for skin care, repair, or anti-aging.

7. A skincare product, characterized in that, It comprises the following components: the cell-derived nanovesicles as described in claim 4; a moisturizer, a thickener, and a skin care active ingredient; wherein the cell-derived nanovesicles contain 5-100 μg / mL.

8. An essence containing stem cell nanovesicles, characterized in that, It is composed of the following raw materials in weight percentages: 0.1-0.5 wt% sodium hyaluronate, 0.05-0.2 wt% xanthan gum, 0.05-0.3 wt% polyglutamic acid, 1-4 wt% nicotinamide, 2-6 wt% 1,3-butanediol, 1-3 wt% 1,2-hexanediol, 5-100 μg / mL of the cell-derived nanovesicles as described in claim 4, and the balance being water.

9. A lyophilized formulation, characterized in that, The product comprises the cell-derived nanovesicles of claim 4 and a lyophilization protectant, the lyophilization protectant comprising trehalose, and optionally mannitol and / or glycine.

10. The lyophilized formulation according to claim 9, characterized in that, The freeze-drying protectant comprises trehalose, and optionally mannitol and / or glycine, and is reconstituted using a sodium hyaluronate solution with a molecular weight of 5-10 kDa.

Images