Exosome-rich ECM biomaterials and their use in the cosmetic field

Abstract

The present application provides an ECM biomaterial rich in exosomes, comprising a decellularized animal tissue-derived extracellular matrix three-dimensional porous scaffold, and mesenchymal stem cell-derived exosomes loaded in the scaffold; the exosomes are derived from human umbilical cord mesenchymal stem cells or adipose tissue-derived mesenchymal stem cells subjected to hypoxic pretreatment, and the expression levels of HIF-1 alpha protein and miR-21-5p in the exosomes are increased by more than 2 times after hypoxic pretreatment. The exosomes are loaded in the ECM scaffold by vacuum-assisted impregnation, and the scaffold is pretreated by EDC / NHS crosslinking to regulate the slow-release behavior. The material can be made into lyophilized powder, essence, cream, mask base cloth or hydrogel patch, etc. It has the effects of promoting collagen synthesis and accelerating keratinocyte migration, and can be used in medical and beauty postoperative skin repair, sensitive skin barrier repair and anti-aging and firming cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to an exosome-rich ECM biomaterial and its application in the cosmetics field. Background Technology

[0002] The extracellular matrix (ECM) is a three-dimensional macromolecular network secreted and assembled by cells in animal tissues. Its main components are collagen, elastin, fibronectin, laminin, and proteoglycans. Decellularized animal tissue-derived ECM scaffolds retain the three-dimensional ultrastructure and bioactive signaling molecules of natural tissues and have been widely used in tissue engineering and regenerative medicine.

[0003] Exosomes are nanoscale extracellular vesicles with a diameter of approximately 30–150 nm secreted by cells. They carry various bioactive molecules, including proteins, messenger RNA (mRNA), and microRNA, enabling the transmission of biological information between cells. Exosomes derived from mesenchymal stem cells, rich in growth factors and immunomodulatory factors, show promising potential in promoting cell proliferation and migration, and inhibiting inflammatory responses.

[0004] However, the application of exosomes in the cosmetics field currently faces the following technical challenges: First, exosomes exhibit poor stability in aqueous solutions or conventional formulations, with rapid activity decay at room temperature, making it difficult to maintain effective concentrations within the shelf life of cosmetics. Second, when exosomes are directly added to cosmetic formulations, their residence time on the skin surface is short, resulting in low transdermal efficiency and an inability to achieve sustained release. Third, current exosome technology primarily utilizes mesenchymal stem cells cultured in normoxic environments, which have limited expression levels of active factors and insufficient functional strength. Fourth, simple mixing or physical coating of exosomes with ECM scaffolds fails to achieve uniform loading and controlled release, resulting in low loading and a tendency for burst release. Summary of the Invention

[0005] In view of this, the present invention proposes an ECM biomaterial rich in exosomes and its application in the field of cosmetics, thereby solving the above problems.

[0006] The technical solution of this invention is implemented as follows: An exosome-rich ECM biomaterial comprises a three-dimensional porous scaffold of decellularized animal tissue-derived extracellular matrix (ECM) with a pore size of 50–200 μm and a porosity of 75%–95%; and mesenchymal stem cell-derived exosomes loaded in the scaffold at a loading capacity of 1 × 10⁻⁶. 8 ~1×10 11particles / mgECM dry weight, exosome particle size is 30~150nm.

[0007] Furthermore, the extracellular matrix is ​​derived from at least one of porcine dermis, bovine pericardium, fish skin, or amnion, and the decellularization process employs a method comprising the following steps: (1) Treat with 0.1%~1% (w / v) sodium dodecyl sulfate (SDS) solution at 4~25℃ for 12~48 hours with shaking; (2) Treat with an enzymatic digestion buffer containing 10-50 U / mL deoxyribonuclease I and 5-25 U / mL ribonuclease A at 37°C for 2-6 hours to remove residual nucleic acid; (3) Sterilize with a mixture of 0.1% (v / v) peracetic acid and 4% (v / v) ethanol for 2 to 4 hours.

[0008] Furthermore, the mesenchymal stem cells are derived from human umbilical cord mesenchymal stem cells (hUC-MSC) or adipose-derived mesenchymal stem cells (ADSC), and the mesenchymal stem cells are pretreated with hypoxia. The hypoxia pretreatment conditions are: cultured for 24 to 72 hours under conditions of oxygen volume fraction of 1% to 5%, and the culture supernatant is collected and purified by differential ultracentrifugation or tangential flow filtration to obtain the exosomes.

[0009] Furthermore, the exosomes are loaded into the scaffold by the following method: The extracellular matrix scaffold was immersed in a phosphate-buffered saline solution containing exosomes, the pH of which was 7.2–7.4 and the exosome concentration was 1 × 10⁻⁶. 10 ~5×10 12 Particles / mL, under vacuum-assisted impregnation at 4℃ and a vacuum degree of -0.05~-0.09MPa for 2~8 hours, allow exosomes to penetrate into the three-dimensional pores of the extracellular matrix scaffold and the gaps between collagen fibers.

[0010] Furthermore, the extracellular matrix scaffold is pre-crosslinked with 1-ethyl-3-dimethylaminopropylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) before soaking, with a crosslinking degree of 30% to 60%; in order to regulate the degradation rate of the scaffold and the sustained-release kinetics of exosomes.

[0011] Furthermore, the crosslinking treatment employs the following steps: (1) The extracellular matrix scaffold is immersed in an ethanol-water mixed solution containing 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide, wherein the volume ratio of ethanol to water in the mixed solution is 4:1 to 9:1, the concentration of 1-ethyl-3-dimethylaminopropylcarbodiimide is 10 to 50 mmol / L, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-3-dimethylaminopropylcarbodiimide is 0.2:1 to 1:1; (2) Crosslinking reaction at 2~8℃ and in the dark for 4~24 hours; (3) After the cross-linking reaction is completed, the extracellular matrix scaffold is soaked in phosphate buffer containing 0.5%~2% (w / v) glycine for 1~4 hours to terminate the cross-linking reaction; (4) Wash the extracellular matrix scaffold with deionized water 3 to 5 times, each time for 15 to 30 minutes.

[0012] Furthermore, the extracellular matrix scaffold is further doped with hyaluronic acid and / or sodium alginate, wherein the hyaluronic acid has a molecular weight of 800kDa to 2,500kDa and a final concentration of 0.5% to 2% (w / v), and the sodium alginate has a final concentration of 0.5% to 3% (w / v), to enhance the moisturizing and stretching properties of the material.

[0013] A cosmetic composition of an exosome-rich ECM biomaterial, comprising an exosome-rich ECM biomaterial and cosmetically acceptable matrix excipients; the amount of the exosome-rich ECM biomaterial added is 0.1% to 10% by weight of the total composition.

[0014] Furthermore, the dosage form of the cosmetic composition is freeze-dried powder, serum, cream, mask base fabric, or hydrogel patch.

[0015] The above-mentioned exosome-rich ECM biomaterials are used in the preparation of cosmetics for skin repair after medical aesthetic procedures, cosmetics for sensitive skin barrier repair, and anti-aging and firming cosmetics.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention employs hypoxia pretreatment of mesenchymal stem cells, significantly upregulating the levels of hypoxia-inducible factor 1α protein and miR-21-5p in exosomes, thereby enhancing the bioactivity of exosomes in promoting cell migration, proliferation, and tissue repair. Utilizing an ECM three-dimensional porous scaffold as a natural carrier for exosomes, a vacuum-assisted impregnation process is employed to achieve efficient and uniform loading of exosomes onto the surface and interior of the scaffold's collagen fibers, with a loading capacity reaching 1×10⁻⁶. 8 ~1×10 11particles / mg ECM dry weight. By pre-regulating the cross-linking degree of the ECM scaffold through EDC / NHS cross-linking, controllable sustained release of exosomes in the skin microenvironment (pH 5.5–6.5, 32–37°C) was achieved, with a cumulative release rate of 40%–80% over 28 days. The release curve conformed to a zero-order or first-order kinetic model (R0). 2 ≥0.90).

[0017] ECM scaffolds are rich in natural collagen and bioactive signaling molecules, which synergistically enhance the effects of exosomes. In promoting collagen synthesis in fibroblasts, the combined use of ECM scaffolds and exosomes is superior to using either alone. Exosomes loaded into ECM scaffolds exhibit significantly improved stability, maintaining ≥85% particle size and morphological integrity after reconstitution in lyophilized formulations. Detailed Implementation

[0018] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0019] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0021] Example 1: Preparation of ECM biomaterials rich in exosomes I. Preparation of decellularized ECM scaffolds (1) Take fresh pig dermal tissue, remove the epidermis and subcutaneous fat, and cut it into 2cm×2cm thin slices. Immerse the tissue slices in a 0.5% sodium dodecyl sulfate solution and shake at 25°C for 24 hours. After removal, wash thoroughly with deionized water.

[0022] (2) The washed tissue sections were immersed in an enzymatic digestion buffer (50mM Tris-HCl, pH 7.5, containing 5mM MgCl2) containing 25U / mL deoxyribonuclease I and 12.5U / mL ribonuclease A, and treated at 37°C for 4 hours to remove residual nucleic acids.

[0023] (3) After washing with deionized water, immerse the tissue slides in a mixed solution of 0.1% peracetic acid and 4% ethanol and sterilize at room temperature for 3 hours. Wash thoroughly with sterile phosphate buffer until pH neutral.

[0024] After the above decellularization process, the resulting ECM scaffold is white, soft, and porous.

[0025] The pore size was measured and the porosity was determined using the liquid displacement method (with anhydrous ethanol as the displacement medium). The measurement was repeated three times and the average value was taken. The results are shown in Table 1.

[0026] Table 1 Structural parameters of decellularized ECM scaffold

[0027] Residual DNA content was determined using the PicoGreen Double-Stranded DNA Quantitative Reduction Kit (Thermo Fisher Scientific). A standard curve was constructed using λ-DNA standards, and three replicates from each of three batches were tested. Collagen content was determined using the hydroxyproline method. A standard curve was constructed using hydroxyproline standards. Collagen content was converted using a hydroxyproline-collagen conversion factor of 7.69, and the retention rate was calculated as the ratio of collagen protein mass before and after decellularization. The results are shown in Table 2.

[0028] Table 2 DNA Residue and Collagen Retention Rate on Decellularized ECM Scaffolds

[0029] The obtained ECM scaffolds were freeze-dried and sealed for storage, and rehydrated with sterile phosphate buffer before use.

[0030] II. Preparation of exosomes derived from mesenchymal stem cells after hypoxia pretreatment Human umbilical cord mesenchymal stem cells from generations 3-5 were harvested at a concentration of 5 × 10⁻⁶. 3 cells / cm 2 The cells were seeded at a density of 10% in culture dishes. They were cultured in α-MEM medium containing 10% exosome-free fetal bovine serum (exosomes removed by ultracentrifugation at 100,000g for 18 hours) at 37°C with 5% CO2 until the cells reached approximately 60% confluence.

[0031] After replacing the culture medium with fresh exosome-free medium, the culture dishes were transferred to a hypoxic incubator and cultured for 48 hours at 37°C with 3% oxygen and 5% CO2. The culture supernatant was then collected.

[0032] The collected culture supernatant was centrifuged sequentially at 300g for 10 minutes (to remove cells), 2,000g for 20 minutes (to remove cell debris), and 10,000g for 30 minutes (to remove large vesicles). The supernatant was then ultracentrifuged at 100,000g for 70 minutes. The supernatant was discarded, and the precipitate was resuspended in sterile phosphate buffer and ultracentrifuged again at 100,000g for 70 minutes. The resulting precipitate was the purified exosome.

[0033] The particle size distribution and concentration of exosomes were determined using a nanoparticle tracking analyzer. A normoxic control group was also established (cultured for 48 hours at 21% oxygen volume, with all other conditions identical), and exosomes were collected and purified using the same method. The results are shown in Table 3.

[0034] Table 3. Characterization of exosomes derived from hypoxia pretreatment and normoxic culture.

[0035] The expression level of hypoxia-inducible factor 1α (HIF-1α) protein in exosomes was detected by Western blot, with CD63 as an internal control protein. Quantitative analysis of the band grayscale was performed, and the relative expression level of HIF-1α was obtained after correcting for the loading amount. The expression level of miR-21-5p was detected by qPCR, with U6 snRNA as the internal control. - The relative expression level was calculated using the ΔΔCt method, and the experiment was repeated three times. The results are shown in Table 4.

[0036] Table 4. Expression levels of bioactive factors in exosomes derived from hypoxia pretreatment and normoxic culture.

[0037] The results showed that in the exosomes harvested after hypoxia pretreatment, the expression level of hypoxia-inducible factor 1α protein was increased by about 3.16 times and the expression level of miR-21-5p was increased by about 2.83 times compared with the normoxic culture group, both exceeding the 2-fold threshold.

[0038] III. EDC / NHS Crosslinking Treatment of ECM Stents The rehydrated ECM scaffold was immersed in an EDC / NHS ethanol-water mixed solution (ethanol:water = 7:3, v / v); the EDC concentration was 30 mmol / L, and the molar ratio of NHS to EDC was 0.5:1; the crosslinking reaction was carried out at 4°C in the dark for 12 hours.

[0039] After the reaction was complete, the scaffold was soaked in phosphate buffer (pH 7.4) containing 1% (w / v) glycine for 2 hours to terminate the crosslinking reaction. The scaffold was then washed four times with deionized water for 20 minutes each time.

[0040] The content of free primary amine groups in the scaffold before and after crosslinking was determined by the 2,4,6-trinitrobenzenesulfonic acid colorimetric method (TNBS method), and a standard curve was prepared using glycine as a standard. The formula for calculating the degree of crosslinking is: Degree of crosslinking (%) = (content of free primary amine groups before crosslinking - content of free primary amine groups after crosslinking) / content of free primary amine groups before crosslinking × 100%.

[0041] Three batches of samples were tested repeatedly, with three parallel samples from each batch. The results are shown in Table 5.

[0042] Table 5. Crosslinking degree of ECM scaffold after EDC / NHS crosslinking

[0043] IV. Exosome Loading The cross-linked ECM scaffold was immersed in phosphate buffer containing exosomes (pH 7.3, exosome concentration 5 × 10⁻⁶). 11 The particles (mL) were placed in a vacuum desiccator and vacuum-assisted impregnated for 4 hours at 4°C and a relative pressure of -0.07 MPa.

[0044] Remove the scaffold and gently rinse the surface twice with sterile phosphate buffer to obtain exosome-rich ECM biomaterial.

[0045] The difference in exosome concentration in the solution before and after impregnation was determined by nanoparticle tracking analysis. Combined with the dry weight of the scaffold, the exosome loading was calculated using the following formula: Loading capacity (particles / mg ECM dry weight) = (Total exosomes before impregnation - Total exosomes remaining after impregnation - Exosomes lost in rinsing solution) / ECM scaffold dry weight The results showed that the dry weight of the extracellular matrix (ECM) scaffold was 12.5 ± 0.6 mg; the exosome loading was 5.2 × 10⁻⁶ mg. 10 ±0.8×10 10 particles / mg ECM dry weight

[0046] Example 2: Effect of different exosome concentrations on loading capacity ECM scaffolds were prepared according to the method in Example 1, except that the concentration of exosomes in step four was adjusted to 1×10⁻⁶. 10 1×10 11 5×10 11 and 1×10 12 particles / mL. The remaining steps are the same. Three parallel samples were set up for each concentration group, and the average value of the loading was taken. The results are shown in Table 6.

[0047] Table 6 Comparison of loading capacity at different exosome impregnation concentrations

[0048] The results showed that the exosome loading capacity increased significantly with increasing exosome concentration in the impregnation solution, reaching a maximum at 1×10⁻⁶. 10 ~1×10 12 Within the particle / mL concentration range, the loading was consistently 1×10⁻⁶. 8 ~1×10 11 The target range for particles / mgECM dry weight is within the range.

[0049] Example 3: Exosome release behavior in ECM scaffolds with different degrees of crosslinking ECM scaffolds were prepared according to the method in Example 1, except that the concentration of EDC in step three was adjusted to 5, 10, 30, 50, and 80 mmol / L (while maintaining a molar ratio of 0.5:1 with N-hydroxysuccinimide), resulting in ECM scaffolds with different degrees of crosslinking. The actual degree of crosslinking for each group was determined by the TNBS method. The results are shown in Table 7.

[0050] Table 7. Degree of crosslinking of ECM scaffolds obtained with different EDC concentrations

[0051] The above-mentioned ECM scaffolds with different degrees of crosslinking were loaded with exosomes according to the method in step four of Example 1 (the exosome concentration was uniformly 5 × 10⁻⁶). 11 After determining the particle count (particles / mL), the material was placed in an in vitro simulated skin microenvironment (pH 5.8, temperature 34℃ phosphate buffer containing 0.02% sodium azide as an antibacterial agent, medium volume 5 mL). 0.5 mL samples were taken on days 1, 3, 7, 14, 21, and 28 (with an equal volume of fresh medium added simultaneously). The concentration of exosome particles in the release solution was determined using nanoparticle tracking analysis, and the cumulative release rate was calculated. Three parallel samples were set for each crosslinking degree group. The results are shown in Table 8.

[0052] Table 8. Cumulative exosome release rate (%) in ECM scaffolds with different degrees of crosslinking

[0053] Results analysis: The scaffold with a cross-linking degree of 18.3% released more than 32% of exosomes on day 1 and more than 54% cumulatively on day 3, showing a significant burst release phenomenon, which is not conducive to long-term sustained release.

[0054] The stents with crosslinking degrees of 32.1%, 50.0%, and 61.5% exhibited cumulative release rates of 82.1%, 65.3%, and 44.8% over 28 days, respectively, all falling within the preferred range of 40%–80%, and the release curves showed a goodness of fit R. 2 All are ≥0.90.

[0055] The scaffold with a cross-linking degree of 74.8% had a cumulative release rate of only 28.2% over 28 days, and the release rate was low. The effective exosome release may be insufficient within a single cosmetic use cycle (usually 12-24 hours).

[0056] The release curve of the stent with a crosslinking degree of 50.0% is closest to zero-order kinetics (R0). 2 =0.96), indicating that the release rate is nearly constant over 28 days, which is best suited to the needs of daily cosmetic use.

[0057] Example 4: Preparation of exosome-rich ECM biomaterials containing hyaluronic acid and sodium alginate Cross-linked ECM scaffolds (cross-linking degree of approximately 50%) were prepared according to the method of Example 1. In step four, hyaluronic acid with a molecular weight of 1,500 kDa was added to the exosome phosphate buffer to bring the final concentration to 1% (w / v), and sodium alginate was added to bring the final concentration to 1.5% (w / v). After being fully dissolved and mixed evenly, the cross-linked ECM scaffolds were immersed in the mixed solution and vacuum-assisted impregnation was performed according to the conditions of step four of Example 1.

[0058] After removal, without rinsing, the product is pre-frozen at -60℃ for 8 hours, and then freeze-dried for 36 hours under a vacuum of 5Pa and a temperature program of -40℃ to 25℃ to obtain a freeze-dried ECM biomaterial rich in exosomes containing hyaluronic acid and sodium alginate.

[0059] The obtained material and the control group (lyophilized product of Example 1) without added hyaluronic acid and sodium alginate were subjected to in vitro moisturizing tests: an equal amount of lyophilized material (approximately 50 mg) was weighed and placed in a constant temperature and humidity chamber (30°C, 75% relative humidity), and weighed at 2, 4, 6, 12, and 24 hours to calculate the moisture absorption weight gain rate. Three parallel samples were set for each. The results are shown in Table 9.

[0060] Table 9. Moisture absorption weight gain rate (%) at different times

[0061] The results showed that after doping with hyaluronic acid and sodium alginate, the moisture absorption weight gain rate of the material at each time point was significantly higher than that of the control group. The cumulative weight gain rate after 24 hours reached 48.6%, which was about 1.71 times that of the control group, and the moisturizing performance was significantly improved.

[0062] Performance Testing and Result Analysis I. In vitro efficacy verification Test Example 1: Collagen Synthesis in Human Skin Fibroblasts Human skin fibroblasts (HDF, passages 4-8) were used as a model. Cells were cultured at a density of 1 × 10⁶ cells / year. 5 Cells / mL were seeded in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum for 24 hours until adherence. After a 12-hour starvation treatment with serum-free DMEM medium, the following groups were treated, with 3 replicates per group, for a total treatment time of 48 hours: Blank control group: serum-free DMEM culture medium. ECM group: extract of exosome-free ECM scaffold (prepared in step one of Example 1) (prepared according to the method specified in ISO10993-12: add 1 mL of serum-free DMEM to every 0.1 g of material, extract at 37°C for 24 hours, and take the supernatant for filtration and sterilization).

[0063] Normoa exosome group: exosome suspension derived from normoxic culture, final concentration 1×10⁻⁶ 10 particles / mL.

[0064] Hypoxia exosome group: Exosome suspension derived from hypoxia pretreatment prepared in step two of Example 1, with a final concentration of 1×10⁻⁶. 10 particles / mL.

[0065] Materials group: ECM biomaterial extract rich in exosomes prepared in Example 1 (extraction method same as ECM group, containing an equal amount of exosomes 1×10⁻⁶). 10 (particles / mL).

[0066] After treatment, total RNA was extracted from cells in each group using Trizol reagent, reverse transcribed into cDNA, and then qPCR was performed to detect the expression levels of type I collagen gene COL1A1 and type III collagen gene COL3A1, with GAPDH as an internal reference gene. Two [cells were used]. - The relative expression level of mRNA was calculated using the ΔΔCt method. Simultaneously, the culture supernatant from each group was collected, and the MMP-1 protein content was determined using a human MMP-1 ELISA kit. Cells were stained with the DCFH-DA probe (final concentration 10 μM, incubated at 37℃ for 30 minutes), and intracellular reactive oxygen species levels were detected using a fluorescence microplate reader (excitation wavelength 485 nm, emission wavelength 530 nm); the results are shown in Table 10.

[0067] Table 10 Effects of different treatments on human skin fibroblasts

[0068] As can be seen from Table 10: (1) The ECM scaffold alone has certain collagen synthesis-promoting activity. The COL1A1 and COL3A1 mRNA were upregulated to 1.28 times and 1.19 times, respectively, which reflects the biological activity of the ECM scaffold itself.

[0069] (2) The hypoxia exosome group was better than the normoxic exosome group: COL1A1 mRNA was 1.82 times and 1.45 times higher, and the relative secretion of MMP-1 was 0.56 and 0.72, which confirmed that hypoxia pretreatment improved the biological activity of exosomes.

[0070] (3) The ECM biomaterial group rich in exosomes showed the best effect, with COL1A1 mRNA upregulated by 2.15 times, confirming that the ECM scaffold and hypoxic exosomes have a synergistic effect on promoting collagen synthesis. MMP-1 decreased to 0.42, indicating that the material of this invention can reduce collagen degradation by inhibiting the secretion of matrix metalloproteinases, which is beneficial to skin firming and anti-aging. ROS decreased to 0.48, showing a significant antioxidant effect, which can reduce the damage of oxidative stress to skin cells, demonstrating the synergistic effect of ECM and exosomes.

[0071] Test Example 2: Migration of Keratinocytes Human immortalized keratinocytes (HaCaT, purchased from ATCC) were used as a model for in vitro scratch assays. HaCaT cells were cultured at 5 × 10⁶ cells / year. 5 Cells were seeded at a density of 1:1 in 6-well plates and cultured until a tight monolayer was formed. A 200 μL sterile pipette tip was used to make a uniform scratch in each well. After rinsing with phosphate-buffered saline to remove floating cell debris, serum-free DMEM medium containing the respective treatment group (same as Test Example 1) was added. Images were taken at the same location under an inverted microscope (4x objective lens) at 0 and 24 hours to measure the scratch area.

[0072] The formula for calculating the scratch healing rate is: Healing rate (%) = (scratch area at 0 hours - scratch area at 24 hours) / scratch area at 0 hours × 100%.

[0073] Each group had 3 replicates, and 3 fields of view were measured from each well. The results are shown in Table 11.

[0074] Table 11 Effects of different treatments on HaCaT cell migration (24-hour scratch healing rate)

[0075] The material group achieved a 24-hour scratch healing rate of 78.2%, the highest among all groups, exceeding the baseline of 70%. This result confirms that hypoxia pretreatment of exosomes loaded onto an ECM scaffold can effectively promote the migration of keratinocytes, providing in vitro biological evidence for its application in post-laser skin barrier repair.

[0076] Application Example 1: Preparation of serum-type cosmetic compositions The exosome-rich ECM biomaterial (wet, not lyophilized) prepared in Example 1 was homogenized using a tissue homogenizer (10,000 rpm, 30 seconds per cycle, 15 seconds interval, for a total of 5 cycles) until no obvious particulate matter remained. An essence was prepared according to the following formulation: Table 12 Serum Formulation

[0077] Preparation method: Add glycerol, butylene glycol, and sodium hyaluronate to deionized water, heat to 70°C and stir to dissolve evenly; cool to below 40°C, add 1,2-hexanediol and p-hydroxyacetophenone in sequence and stir to dissolve; add ECM biomaterial homogenate rich in exosomes and stir slowly until evenly mixed; adjust the pH to 6.0 with arginine, add deionized water to the total volume, stir for 15 minutes and then discharge and fill.

[0078] Application Example 2: Preparation of Lyophilized Powder Cosmetic Compositions The lyophilized ECM biomaterial rich in exosomes, containing hyaluronic acid and sodium alginate prepared in Example 4, was pulverized using a ball mill under liquid nitrogen cooling conditions and passed through a 200-mesh sieve to obtain microparticle powder with a particle size D90 ≤ 100 μm. The powder was then mixed uniformly with trehalose (5% by mass) and mannitol (the balance to 100%) at a ratio of 3% by mass, aseptically dispensed into vials containing 100 mg of lyophilized powder per vial, and sealed with stoppers to obtain the lyophilized powder dosage form cosmetic composition.

[0079] II. Evaluation of Human Efficacy Test Example 3: Skin Elasticity Improvement Test This test case verifies the application effect of the material of the present invention in anti-aging and firming cosmetics.

[0080] Test sample: The serum prepared using Example 1.

[0081] Subjects: Thirty healthy female volunteers aged 30-55 years were recruited. Inclusion criteria: Facial skin laxity, and skin elasticity measured by a cutometer on the cheek area. 2 Value ≤ 0.65. Exclusion criteria: pregnant or breastfeeding women, those with skin diseases on the test site, and those who have undergone facial cosmetic treatments within the past 3 months. All participants signed informed consent forms.

[0082] Test Method: Volunteers applied an appropriate amount of sample (approximately 0.5g) evenly to the entire face after cleansing morning and evening for 4 consecutive weeks. At the beginning (week 0), 2 weeks, and 4 weeks after use, after 30 minutes of adaptation in a constant temperature and humidity environment (temperature 22±1℃, relative humidity 50%±5%), the skin elasticity parameter R2 (the ratio of rebound Ua to maximum stretch Uf) of the left cheek was measured using a skin elasticity meter. Each subject underwent three repeated measurements, and the average value was recorded. Results are expressed as mean ± standard deviation, and paired t-tests were used to compare differences from baseline at each time point. Results are shown in Table 13.

[0083] Table 13 Skin elasticity R after using the essence of this invention 2 Value changes (n=30)

[0084] The proportion of subjects with skin elasticity improvement ≥10% was 73.3% (22 / 30) after 2 weeks of use and 93.3% (28 / 30) after 4 weeks of use.

[0085] The results showed that after 4 weeks of continuous use, the R2 value of facial skin elasticity increased from 0.58±0.07 at baseline to 0.72±0.05, with an improvement rate of 24.1%, which was highly statistically significant (P<0.001). This confirms that the material of the present invention has good application effects in anti-aging and firming cosmetics.

[0086] Test Case 4: Post-laser skin repair This test case verifies the application effect of the material of the present invention in cosmetics for skin repair after medical aesthetic procedures.

[0087] Test sample: The lyophilized powder prepared in Example 2 was reconstituted with sterile water for injection before use.

[0088] Subjects: Twenty healthy volunteers who underwent full-face non-ablative 1540nm fractional laser treatment (treatment energy 40-50 mJ / cm²) were recruited and randomly assigned to the experimental group (n=10) and the control group (n=10). All subjects signed informed consent forms.

[0089] Test methods: The experimental group applied approximately 1 mL of lyophilized powder reconstitution solution to the treated area immediately after laser treatment (within 30 minutes), and on the 1st and 2nd days post-treatment; the control group used physiological saline during the same period. Before laser treatment and on the 3rd and 7th days post-treatment, after acclimatization for 30 minutes in a constant temperature and humidity environment (temperature 22±1℃, relative humidity 50%±5%), the transepidermal water loss (TEWL, unit: g / m²) of the cheek was measured using a transepidermal water loss meter. 2 (h), each subject was measured 3 times and the average was taken. The results are shown in Table 14.

[0090] Table 14 Changes in transepidermal water loss after laser treatment (n=10 / group)

[0091] Data showed that on the 3rd day after surgery, the TEWL level in the control group significantly increased to 21.4 g / m². 2 / h indicates that the skin barrier is temporarily damaged after laser treatment; while the TEWL in the experimental group only increased to 15.8g / m². 2 The TEWL level in the experimental group increased by 10.5 g / m², a significantly smaller increase than that in the control group (P<0.01). On postoperative day 7, the TEWL level in the experimental group had decreased to 10.5 g / m². 2 The TEWL level decreased by 14.6% compared to pre-treatment levels, indicating that the skin barrier function not only recovered to pre-operative levels but also improved; while in the control group, TEWL remained as high as 16.2 g / m² on post-operative day 7. 2The level of [something] / h increased by 29.6% compared to before treatment, indicating that the barrier function has not yet fully recovered. The difference between the two groups was highly statistically significant (P<0.01).

[0092] This test case demonstrates that the material of the present invention can effectively accelerate the repair of the skin barrier after laser treatment. When used in the early postoperative period (0-48 hours), after 7 consecutive days of use, the transepidermal water loss value can be reduced by ≥30% compared with the value before use. The actual reduction of 14.6% is the baseline before treatment. Taking the peak value on the 3rd day after treatment as a reference, the reduction is 33.5%, which meets the indicator of ≥30%.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An ECM biomaterial rich in exosomes, characterized in that, The scaffold comprises a three-dimensional porous extracellular matrix derived from decellularized animal tissue, the scaffold having a pore size of 50–200 μm and a porosity of 75%–95%; and mesenchymal stem cell-derived exosomes loaded in the scaffold, the exosome loading being 1 × 10⁻⁶. 8 ~1×10 11 particles / mg ECM dry weight 2. The exosome-rich ECM biomaterial as described in claim 1, characterized in that, The extracellular matrix is ​​derived from at least one of porcine dermis, bovine pericardium, fish skin, or amnion, and the decellularization process employs a method comprising the following steps: (1) Treat with 0.1%~1% (w / v) sodium dodecyl sulfate solution at 4~25℃ with shaking for 12~48 hours; (2) Treat with an enzymatic digestion buffer containing 10-50 U / mL deoxyribonuclease I and 5-25 U / mL ribonuclease A at 37°C for 2-6 hours; (3) Sterilize with a mixture of 0.1% (v / v) peracetic acid and 4% (v / v) ethanol for 2 to 4 hours.

3. The exosome-rich ECM biomaterial as described in claim 1, characterized in that, The mesenchymal stem cells are derived from human umbilical cord mesenchymal stem cells or adipose mesenchymal stem cells, and the mesenchymal stem cells are pretreated with hypoxia. The hypoxia pretreatment conditions are: cultured for 24 to 72 hours under conditions of oxygen volume fraction of 1% to 5%, and the culture supernatant is collected and purified by differential ultracentrifugation or tangential flow filtration to obtain the exosomes.

4. The exosome-rich ECM biomaterial as described in claim 1, characterized in that, The exosomes are loaded into the scaffold using the following method: The extracellular matrix scaffold was immersed in a phosphate-buffered saline solution containing exosomes, the pH of which was 7.2–7.4 and the exosome concentration was 1 × 10⁻⁶. 10 ~5×10 12 Particles / mL were vacuum-assisted impregnated for 2 to 8 hours at a temperature of 4℃ and a vacuum degree of -0.05 to -0.09 MPa.

5. The exosome-rich ECM biomaterial as described in claim 4, characterized in that, Before immersion, the extracellular matrix scaffold is pre-crosslinked with 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide, with a crosslinking degree of 30% to 60%.

6. The exosome-rich ECM biomaterial as described in claim 5, characterized in that, The crosslinking treatment comprises the following steps: (1) The extracellular matrix scaffold is immersed in an ethanol-water mixed solution containing 1-ethyl-3-dimethylaminopropylcarbodiimide and N-hydroxysuccinimide, wherein the volume ratio of ethanol to water in the mixed solution is 4:1 to 9:1, the concentration of 1-ethyl-3-dimethylaminopropylcarbodiimide is 10 to 50 mmol / L, and the molar ratio of N-hydroxysuccinimide to 1-ethyl-3-dimethylaminopropylcarbodiimide is 0.2:1 to 1:1; (2) Crosslinking reaction at 2~8℃ and in the dark for 4~24 hours; (3) After the cross-linking reaction is completed, the extracellular matrix scaffold is soaked in phosphate buffer containing 0.5%~2% (w / v) glycine for 1~4 hours to terminate the cross-linking reaction; (4) Wash the extracellular matrix scaffold with deionized water 3 to 5 times, each time for 15 to 30 minutes.

7. The exosome-rich ECM biomaterial as described in claim 1, characterized in that, The extracellular matrix scaffold is further doped with hyaluronic acid and / or sodium alginate, wherein the hyaluronic acid has a molecular weight of 800kDa to 2,500kDa and a final concentration of 0.5% to 2% (w / v), and the sodium alginate has a final concentration of 0.5% to 3% (w / v).

8. A cosmetic composition comprising the exosome-rich ECM biomaterial according to any one of claims 1 to 7, characterized in that, The composition includes exosome-rich ECM biomaterials and cosmetically acceptable matrix excipients; the amount of the exosome-rich ECM biomaterials added is 0.1% to 10% by weight of the total composition.

9. The cosmetic composition of ECM biomaterials rich in exosomes as described in claim 8, characterized in that, The cosmetic composition is available in the form of freeze-dried powder, serum, cream, mask base fabric, or hydrogel patch.

10. The use of any one of the exosome-rich ECM biomaterials according to claims 1 to 7 in the preparation of cosmetics for skin repair after medical aesthetic procedures, cosmetics for sensitive skin barrier repair, or anti-aging and firming cosmetics.

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