Super-loaded dissoluble anti-decline microneedle patch as well as preparation method and application of super-loaded dissoluble anti-decline microneedle patch

Abstract

The invention provides a super-loaded dissoluble anti-decline microneedle patch as well as a preparation method and application thereof, and belongs to the technical field of medical materials. According to the application, the application of the extracellular particle super in resisting skin light aging or aging is firstly provided, the extracellular particle super is used as a non-vesicular extracellular particle with the particle size smaller than 50 nm, aging of DF cells can be effectively slowed down, and proliferation of the DF cells and collagen synthesis are promoted; meanwhile, the extracellular particle super-polymer is further loaded in the microneedle patch, the administration mode of the extracellular particle super-polymer is optimized, the extracellular particle super-polymer can directly and uniformly act on the corium layer, the skin light aging resistance or aging resistance effect of the extracellular particle super-polymer is improved, and the microneedle patch has important clinical transformation significance.

Description

Technical Field

[0001] The present invention relates to a dissolvable anti-aging microneedle patch loaded with supermere and its preparation method and application, belonging to the technical field of medical materials. Background Art

[0002] Skin aging is a complex evolutionary process, affected by both internal factors (such as hormones, age, etc.) and external factors (such as polluted air, ultraviolet rays, etc.). Among them, skin aging caused by ultraviolet (UV) irradiation is called photoaging, accounting for 80% of external factors and being the most concerned phenomenon in skin health problems. Clinical manifestations of skin photoaging include epidermal thickening, deep and thick wrinkles, telangiectasia, etc. This not only leads to a decline in skin aesthetics but also is accompanied by the destruction of skin barrier integrity, significantly increasing the risk of suffering from various skin inflammations and skin malignancies. Therefore, preventing skin aging is not only limited to anti-aging and beauty but also helps prevent various malignant skin diseases, achieving the combination of medical treatment and prevention. At present, although medical aesthetic means such as botulinum toxin injection and skin filling surgery claim to have the effect of improving damaged aging skin, their action time is short, and the relatively serious side effects are not acceptable to the public. Therefore, finding new methods for preventing and delaying skin photoaging has important clinical transformation significance.

[0003] In recent years, stem cell therapies represented by mesenchymal stem cells (MSCs) have attracted much attention in tissue regeneration medicine research. Currently, a large number of studies have shown that human umbilical cord mesenchymal stem cells (hucMSCs) can promote tissue injury repair through extracellular particles (EPs) in the paracrine group; and EPs include non-vesicular extracellular particles (NVEP) and extracellular vesicles (EV), and small extracellular vesicles (sEV) in EV are vesicular bodies with a bilayer membrane structure and a particle size of about 150 nm, which have been proven to effectively defend against skin photoaging; while there is less research on NVEP at present. Among them, extracellular particles supermere (supergranule) with a particle size less than 50 nm has only been reported in terms of biomarkers, and whether it can play a role similar to that of sEV in tissue injury repair remains to be further explored.

[0004] Dissolvable microneedle patches are more capable of achieving self - administration, miniaturization, painless drug delivery, being gentle and safe, and avoiding medical waste pollution. Compared with transdermal enhancers, soluble microneedles can help functional particle molecules penetrate through the epidermis to reach the dermis, achieving precise drug delivery of functional particles. Some studies have shown that soluble microneedles can improve the tissue distribution of drugs and prolong the retention time of drugs in the skin. Currently, there is no report on the application of soluble microneedles loaded with supermere in skin aging. If supermere is precisely delivered through microneedles, it can achieve the prevention or treatment of skin photoaging, which is of great significance for the clinical intervention of skin photoaging. Summary of the Invention

[0005] Aiming at some deficiencies in the existing technology, the present invention provides a dissolvable anti - aging microneedle patch loaded with supermere, its preparation method and application.

[0006] To achieve the above - mentioned technical objectives, the present invention provides the following technical solutions:

[0007] The present invention first provides the application of extracellular particle supermere in anti - skin aging, anti - skin photoaging, photoaged skin repair or promoting collagen synthesis.

[0008] Furthermore, the source of the extracellular particle supermere includes hucMSC cells.

[0009] Furthermore, the extracellular particle supermere is a non - vesicular extracellular particle with a particle size less than 50 nm.

[0010] Furthermore, the application includes the application in the preparation of products for anti - skin aging, anti - skin photoaging, photoaged skin repair or promoting collagen synthesis.

[0011] Furthermore, the product is a cosmetic or medical aesthetic product, which is used for the skin or skin appendages.

[0012] Furthermore, the product includes a dissolvable anti - aging microneedle patch.

[0013] The present invention also provides a dissolvable anti - aging microneedle patch, and the microneedle patch is loaded with extracellular particle supermere.

[0014] Furthermore, the microneedle patch includes a needle body and a backing; the needle body material includes hyaluronic acid and extracellular particle supermere, and the backing material includes polyvinyl alcohol (PVA). Preferably, the concentration of hyaluronic acid in the needle body is 20 wt%; the concentration of PVA in the backing is 20 wt%, and the solvent is water.

[0015] The present invention also provides a preparation method of the above - mentioned dissolvable anti - aging microneedle patch, including:

[0016] (1) Mix hyaluronic acid with extracellular particle supermere, stir, and allow it to swell naturally until completely dissolved without bubbles to obtain the needle body material;

[0017] (2) Mix polyvinyl alcohol (PVA) powder with water, stir, and allow it to swell in a water bath until there are no bubbles to obtain the backing material;

[0018] (3) Add the needle body material obtained in step (1) to fill the PDMS microneedle mold, evacuate, centrifuge, remove the excess needle body material, then add the backing material obtained in step (2) to fill the mold, dry, and demold to obtain the dissolvable anti-aging microneedle patch.

[0019] Further, in step (1), the final concentration of hyaluronic acid in the needle body material is 20 wt%, with a molecular weight of 30 - 45 kDa.

[0020] Further, in step (2), the final concentration of PVA in the backing material is 20 wt%, and the solvent is water.

[0021] Further, in step (3), the evacuation step is at room temperature - 0.07 Mpa, hold for 3 min and then restore atmospheric pressure, repeat 3 times; the centrifugation is at 4°C, 2000 g for 5 min; the drying is at room temperature for 18 h.

[0022] The present invention also provides the application of extracellular particle supermere in the preparation of drugs for preventing and / or treating diseases caused by photoaging.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The present invention first proposes that supermere has the effect of anti-skin photoaging. Through experimental verification, supermere can delay the senescence of DF cells, promote the proliferation of DF cells and the expression of COL1A1, thereby further promoting the synthesis of collagen, and the effect produced by supermere is significantly better than that produced by sEV; at the same time, supermere can better inhibit the reduction of collagen and the generation of erythema in the skin of mice caused by UV irradiation than sEV; in addition, through CCK8 cytotoxicity detection, supermere does not have cytotoxicity. Therefore, the anti-skin photoaging effect of supermere provides a safer and more effective new strategy for anti-skin photoaging.

[0025] (2) The present invention further provides a microneedle patch loaded with supermere, which optimizes the administration method of supermere; by loading supermere into the microneedles (needle bodies) of the microneedle patch, it promotes supermere to directly act on the dermis layer. While ensuring a high transdermal rate of supermere, supermere is evenly distributed subcutaneously and does not cause too much pain. Description of the Drawings

[0026] Figure 1 Morphological diagrams of P0 and P3 generation hucMSC cells in Example 1. The left figure is P0 generation, and the right figure is P3 generation.

[0027] Figure 2 Transmission electron microscopy results of hucMSC-sEV and hucMSC-supermere in Example 1. The left figure is hucMSC-sEV, and the right figure is hucMSC-supermere.

[0028] Figure 3 Western Blot test result diagrams of positive markers Calnexin of hucMSC cells, CD9 of hucMSC-sEV, and ACE2 and AGO2 of hucMSC-supermere in Example 1.

[0029] Figure 4 Fluorescence microscopy identification result diagrams of positive markers α-SMA and Vimentin and negative marker CK10 of DF cells in Example 2. From left to right in the figure, they are the identification results of α-SMA, Vimentin, and CK10 in sequence. In the small figure on the left of each figure, from top to bottom, they are the microscope individual identification diagrams of the marker, cytoskeleton (Phalloidin staining), and cell nucleus (Hoechst staining), and the colors are the same as the text colors on the upper side of each figure; on the right side of each figure is the microscope comprehensive identification diagram of the marker, cytoskeleton (Phalloidin staining), and cell nucleus (Hoechst staining).

[0030] Figure 5 Fluorescence microscopy result diagrams of DF cells taking up hucMSC-sEV and hucMSC-supermere at 12, 24, and 48 h of incubation in Example 2; among them, green is the cytoskeleton (Phalloidin staining), blue is the cell nucleus (Hochest staining), and red is sEV (CM-DIL staining) and supermere (Alexa Fluor TM 647 staining).

[0031] Figure 6Graph showing the cytotoxicity detection results of hucMSC-sEV and hucMSC-supermere on DF cells in Example 2.

[0032] Figure 7 Graph showing the detection results of the number of senescent cells in the control group (Sham), UVA-treated group, hucMSC-sEV intervention group, and hucMSC-supermere intervention group in Example 2. The following figure is a partial enlarged view of the above figure.

[0033] Figure 8 Graph showing the cell proliferation detection results of the control group (Sham), UVA-treated group, hucMSC-sEV intervention group, and hucMSC-supermere intervention group in Example 2; in the figure, green represents newly synthesized DNA, which represents proliferating cells (EdU staining), and blue represents the cell nuclei of all cells (Hoechst staining).

[0034] Figure 9 Graph showing the detection results of the cell COL1A1 level in the control group (Sham), UVA-treated group, hucMSC-sEV intervention group, and hucMSC-supermere intervention group in Example 2. The upper figure is the single staining result of COL1A1, and the lower figure is the staining result of COL1A1, cytoskeleton (Phalloidin staining), and cell nuclei (Hoechst staining).

[0035] Figure 10 Flowchart showing the steps for preparing a dissolvable anti-aging microneedle patch using a PDMS mold in Example 3.

[0036] Figure 11 Structure diagram of the needle body of the dissolvable anti-aging microneedle patch after drying in Example 3. The left figure is a stereomicroscope image, and the right figure is a scanning electron microscope image.

[0037] Figure 12 Schematic diagram showing the usage method of a microneedle syringe in Example 3.

[0038] Figure 13 Graph showing the state of the needle body part of the microneedle patch at different dissolution times in Example 3; the upper figure is the dissolution state diagram of the needle body part, and the lower figure is the skin state diagram of the mouse at the corresponding time.

[0039] Figure 14 Graph showing the subcutaneous distribution of hucMSC-sEV and hucMSC-supermere under different administration methods in Example 3. The left figure is a fluorescence microscope image of the distribution, and the right figure is a three-dimensional heat map of the distribution.

[0040] Figure 15 Schematic diagram showing the construction process of a mouse skin photoaging intervention model in Example 4.

[0041] Figure 16 The figure showing the detection results of the mRNA levels of CXCL2, CXCL5, and CXCL10 in the CTR group, UV group, MN group, sEV group, and supermere group in Example 4.

[0042] Figure 17 The figure showing the dermoscopy results of the CTR group, UV group, MN group, sEV group, and supermere group in Example 4.

[0043] Figure 18 The figure showing the Masson staining results of the CTR group, UV group, MN group, sEV group, and supermere group in Example 4. Detailed implementation manners

[0044] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the protection scope of the present invention.

[0045] In the embodiments of the present invention, those not described in detail are all completed by conventional experimental methods. Those processes not described in detail in the embodiments are understandable and easy to implement by those skilled in the art according to the product specifications or basic knowledge in the art, and thus will not be described in detail.

[0046] In the embodiments of the present invention, α-MEM medium (purchased from Invitrogen), serum (purchased from Vazyme), CK10 antibody (purchased from SCBT), Vimentin antibody (purchased from SCBT), α-SMA antibody (purchased from SCBT), Calnexin antibody (purchased from CST), CD9 antibody (purchased from CST), ACE2 antibody (purchased from Proteintech), AGO2 antibody (purchased from Proteintech), β-galactosidase kit (purchased from CST), EdU-488 cell proliferation detection kit (purchased from Beyotime), Alexa Fluor TM 647 protein labeling kit (purchased from Invitrogen), hyaluronic acid (97%, 30 - 45 kDa: H924870, purchased from MACKLIN), PVA ( PVA - 203 polyvinyl alcohol: P119359, purchased from aladdin).

[0047] Example 1: Acquisition and identification of HucMSC-sEV and hucMSC-supermere

[0048] (1) Isolation and culture of hucMSC: According to the isolation method in Qiao Chun et al. Human mesenchymal stem cells isolated from the umbilical cord. Cell Biol Int. 2008 Jan; 32(1): 8-15, hucMSC was isolated from fresh human umbilical cord tissue (from the Fourth Affiliated Hospital of Jiangsu University), and the isolated hucMSC (denoted as primary umbilical cord mesenchymal stem cells) was cultured in an incubator at 37°C and 5% CO2 saturated humidity.

[0049] The cultured cells were observed under a microscope, and the results were as Figure 1 shown. The cells migrating out of the tissue block in the figure were denoted as P0 generation. The P0 generation cells were in a fish school shape around the tissue block. When further cultured and passaged to P3 generation, the hucMSC cells were spindle-shaped.

[0050] (2) Isolation of hucMSC-sEV and hucMSC-supermere: Using α-MEM complete medium containing 10% fetal bovine serum, the primary umbilical cord mesenchymal stem cells were cultured to P3 generation, digested with 0.25% trypsin for passage to obtain P4 generation hucMSC. When the cell density reached about 60%, the complete medium was removed, and after washing three times with PBS, the serum-free α-MEM medium was changed. When the cell density reached 100%, the cells were passaged and cultured, and the supernatants of P4 - P6 generations were collected; the collected supernatants were combined, centrifuged at 300g for 20 min at 4°C to remove dead cells, the supernatant was taken and centrifuged at 2000g for 20 min to remove cell debris, and then centrifuged at 10000g for 30 min to remove organelles and large extracellular vesicles. The supernatant was transferred to a 100-kDa MWCO ultrafiltration centrifugal tube and centrifuged at 2000g for 30 min for concentration. The concentrated solution at the bottom of the filter was retained, and the concentrated solution was centrifuged at 100000g for 120 min;

[0051] A. After centrifugation, the precipitate was taken, resuspended in PBS, and centrifuged again at 100000g for 120 min for washing. Finally, the precipitate was collected and denoted as hucMSC-sEV; the hucMSC-sEV precipitate was dissolved overnight with PBS and sterilized through a 0.22-μm filter. The obtained hucMSC-sEV solution was aliquoted and stored at -80°C after measuring the particle concentration using a nanoparticle tracking analysis instrument.

[0052] B. Take the supernatant and centrifuge it at 160,000 g for 16 h at 4°C. After centrifugation, centrifuge the supernatant again at 360,000 g for 16 h to obtain a precipitate, denoted as hucMSC - supermere. Dissolve the hucMSC - supermere precipitate overnight with PBS and then sterilize it through a 0.22 - μm filter. After measuring the particle concentration of the obtained hucMSC - supermere solution using a nanoparticle tracking analysis instrument, aliquot and store it at - 80°C.

[0053] (3) Identification of hucMSC - sEV and hucMSC - supermere: Drop the hucMSC - sEV or hucMSC - supermere solution obtained in step (2) onto a 400 - mesh carbon - coated copper grid, and let it stand at room temperature for 5 min. Use filter paper to absorb the residual liquid at the edge of the carbon - coated copper grid. Then invert the carbon - coated copper grid onto phosphotungstic acid (30 g / L) with a pH value of 6.8 for negative staining for 5 min. Then dry the carbon - coated copper grid under an incandescent lamp and observe and take pictures under a transmission electron microscope. Take an appropriate amount of hucMSC - sEV or hucMSC - supermere solution, dilute it with pure water, and use nanoparticle tracking analysis to analyze the particle size and concentration of sEV or supermere. Mix the hucMSC - sEV solution and hucMSC - supermere solution with RIPA protein lysate at a volume ratio of 1:1, then shake repeatedly, let it stand on ice, centrifuge at 12,000 g for 15 min at 4°C, retain the supernatant, measure the protein concentration of the supernatant by the BCA method, and then add protein buffer at a volume ratio of supernatant to buffer of 3:1, boil at 100°C for 10 min to obtain protein samples, and then perform SDS - PAGE electrophoresis to detect the markers ACE2, AGO2, CD9, and Calnexin of the protein samples.

[0054] The identification results are as follows: From Figure 2 the transmission electron microscope results, it can be seen that sEV shows a typical cup - shaped structure with a particle size of about 150 - 200 nm, and supermere shows a translucent granular shape with a particle size of about 25 - 50 nm; From Figure 3 the Western Blot detection results shown, it can be found that the hucMSC positive marker Calnexin is normally expressed, the sEV positive marker CD9 is normally expressed, and the supermere positive markers ACE2 and AGO2 are normally expressed, indicating the successful isolation of hucMSC - sEV and hucMSC - supermere.

[0055] Example 2: Evaluation of the anti - photoaging effect of HucMSC - supermere on DF

[0056] (1) Acquisition and identification of SD rat dermal fibroblasts (DF)

[0057] The primary dermal fibroblasts of rats (purchased from the Experimental Animal Center of Jiangsu University) were isolated and obtained according to the method in the existing patent (Application No.: 201410085659.1). 2×10 4 cells were seeded in a 12-well plate containing cell slides. After the cells adhered, 1 mL of 4% paraformaldehyde was added to each well and fixed at room temperature for 10 min. Then, 500 μL of 0.1% Triton X-100 in volume concentration was added to each well to permeabilize the membrane at room temperature for 10 min. After that, it was washed with PBS, and 5% BSA was added to block at room temperature for 2 h. Then, CK10 antibody, Vimentin antibody, and α-SMA antibody were added dropwise and incubated at 4°C for 14 - 18 h. The next day, the antibody was discarded, and after washing with PBS, the fluorescent secondary antibody was added dropwise and incubated at room temperature for 1 h. After washing with PBS, it was continuously incubated with phalloidin dye at room temperature for 30 min, washed with PBS, and incubated with Hoechst33342 dye at room temperature for 10 min. After sealing with an anti-fluorescence quencher, it was observed under a fluorescence microscope. The DF positive markers α-SMA and Vimentin and the negative marker CK10 were identified. The results were as Figure 4 shown. It can be seen from the figure that both α-SMA and Vimentin were expressed, and no CK10 expression was seen, indicating that the obtained cells were DF cells.

[0058] (2) Uptake of hucMSC-sEV and hucMSC-supermere by DF cells:

[0059] A. hucMSC-sEV was incubated with CM-Dil dye in the dark on a shaker at room temperature for 1 h; the dye-labeled sEV was transferred to a 100 kDa MWCO ultrafiltration centrifugal tube, and PBS was added and centrifuged at 2000 g at 4°C for 30 min to remove the unbound dye. The washed sEV collected was the dye-labeled hucMSC-sEV.

[0060] B. 0.5 mL of hucMSC-supermere with a protein concentration of 2 mg / mL was added to 50 μL of 1 M sodium bicarbonate. After mixing, it was added to the active dye bottle restored to room temperature in the Alexa Fluor TM 647 protein labeling kit. After inverting and mixing several times, it was magnetically stirred at room temperature for 1 h, and then transferred to the purification spin column in the kit. The column was placed in the collection tube and centrifuged at 1000 g for 2 min to remove the buffer. The column was transferred to a new collection tube, the lid was removed, and the reaction mixture (0.5 mL) was slowly applied to the center of the precipitated resin. The column tube assembly was centrifuged at 1000 g for 2 min to collect the sample, and the sample was in the collection tube, which was the dye-labeled hucMSC-supermere.

[0061] The dye-labeled hucMSC-supermere was dissolved in complete α-MEM medium after filtration and stored for later use, with the final concentration of the dye-labeled hucMSC-sEV or hucMSC-supermere being 1×10 10 cells / mL. The cell slides were pre-placed in 12-well plates. According to a density of 2×10 4 cells / well, DF cells were seeded into the 12-well plates. After the cells adhered, the supernatant was aspirated. Then, 1 mL of complete α-MEM medium containing the dye-labeled hucMSC-sEV or hucMSC-supermere was taken and co-incubated with DF cells at 37 °C and 5% CO2 in an incubator for 12, 24, and 48 h. After the incubation ended, the nutrient solution was removed, and the cells were washed with PBS. 1 mL of paraformaldehyde was added to each well and fixed at room temperature for 10 min. Then, 500 μL of 0.1% Triton X-100 was added to each well to permeabilize the membrane at room temperature for 10 min, followed by washing with PBS. 500 μL of 5% BSA was added to each well and blocked at room temperature for 30 min. Then, incubation was carried out with phalloidin dye in the dark for 30 min. Finally, Hoechst33342 dye was added and incubated in the dark at room temperature for 10 min. After washing 3 times with PBS in the dark, the cell slides in the wells were taken out and mounted with an anti-fluorescence quenching agent, and observed under a fluorescence microscope.

[0062] The results were as Figure 5 shown. It could be seen from the figure that both hucMSC-sEV and hucMSC-supermere extracellular particles could be taken up by DF cells, and a large amount of hucMSC-sEV and hucMSC-supermere had been taken up by DF cells at 24 h.

[0063] (3) Safety evaluation of hucMSC-sEV and hucMSC-supermere: 1×10 5 DF cells were seeded in each well of a six-well plate. After the cells adhered, hucMSC-sEV or hucMSC-supermere with a final concentration of 1×10 10 cells / mL were co-incubated with DF cells for 24 h respectively as the experimental groups (sEV group, supermere group), and then the cells were collected; DF cells without adding hucMSC-sEV and hucMSC-supermere were used as the control group (CTR group), and then the cells were collected; The cells of the control group and the experimental groups were seeded in 96-well plates at a density of 2×10 3 cells per well. After 24 h, 48 h, 72 h, and 96 h of adhesion, the culture medium was removed, and CCK8 detection reagent was added. After incubation in a 37 °C, 5% CO2 incubator in the dark for 2 h, the absorbance was measured at a wavelength of 450 nm.

[0064] The results were as Figure 6As shown, it can be found from the figure that the cell viability of the DF cells in the experimental groups supplemented with hucMSC-sEV and hucMSC-supermere did not show significant differences from that of the CTR group within 24, 48, 72, and 96 hours, indicating that hucMSC-sEV and hucMSC-supermere are non-toxic to DF cells.

[0065] (4) Establishment of a photoaging intervention model for DF cells

[0066] The DF cells obtained in step (1) were subcultured, and the P4-generation DF cells with strong proliferation ability and good growth state were selected. The experiment was divided into a normal control group, a UVA treatment group, a hucMSC-sEV intervention group, and a hucMSC-supermere intervention group. In the hucMSC-sEV intervention group and the hucMSC-supermere intervention group, 1×10 10 particle numbers of sEV and supermere were used to treat for 24 hours, and then a DF cell photoaging model was established using the method in the patent with the application number 202211004331.3. The specific operations are as follows:

[0067] After the treatment of the intervention groups, the cell culture medium of each group was removed, a thin layer of PBS was spread, and fresh cell culture medium was replaced. Each time during ultraviolet irradiation, it was placed under the ultraviolet lamp.

[0068] The cells in the UVA treatment group (UVA group), the hucMSC-sEV intervention group (sEV group), and the hucMSC-supermere intervention group (supermere group) were respectively irradiated with ultraviolet light. The cells in the control group (Sham group) were not irradiated with ultraviolet light, but the placement time at room temperature was the same as that of other groups.

[0069] The cells in the UVA group, the sEV group, and the supermere group were respectively placed 5 cm below the preheated ultraviolet lamp. The ultraviolet lamp was turned on for the first irradiation, and at the same time, the probe of the ultraviolet irradiance meter was placed to observe the irradiation dose in real time. The ultraviolet lamp was turned on for the first irradiation. When the irradiation dose reached 5 J / cm 2 ², the ultraviolet lamp was turned off, the PBS was removed, and fresh α-MEM complete culture medium was replaced. 24 hours after the first irradiation, the ultraviolet lamp was turned on for the second irradiation. When the irradiation dose reached 5 J / cm 2 ², the ultraviolet lamp was turned off, the PBS was removed, and fresh α-MEM complete culture medium was replaced. 48 hours after the first irradiation (i.e., 24 hours after the second irradiation), the ultraviolet lamp was turned on for the third irradiation. When the irradiation dose reached 5 J / cm 2When it was time, turn off the ultraviolet lamp, remove the PBS, replace it with fresh complete α-MEM culture medium, and continue culturing for 6 h. Then, detect the number of senescent cells, proliferating cells, and the collagen level of cells in each group.

[0070] Detection of senescent cells: After the establishment of the DF photoaging intervention model, that is, 6 h after the last ultraviolet lamp irradiation treatment in the UVA group, sEV group, and supermere group, remove the upper-layer culture medium of the cells in each group and wash them with PBS. Then, stain them with a β-galactosidase kit (senescent cells will be stained blue), incubate at 37 °C for 12 h, and observe under an inverted optical microscope.

[0071] The results are as Figure 7 shown. It can be found from the figure that the number of senescent cells in the supermere group is significantly lower than that in the UVA group and the sEV group, indicating that supermere can better reduce the number of senescent cells.

[0072] Detection of proliferating cells: After the establishment of the DF photoaging intervention model, that is, 6 h after the last ultraviolet lamp irradiation treatment in the UVA group, sEV group, and supermere group, remove the upper-layer culture medium of the DF cells in each group, wash them with PBS, then stain them with an EdU-488 cell proliferation detection kit, and observe under an inverted fluorescence microscope.

[0073] The results are as Figure 8 shown. It can be found from the figure that the cell proliferation level in the supermere group is only lower than that in the Sham group, but significantly higher than that in the UVA group and the sEV group, indicating that supermere can effectively promote the proliferation of DF cells.

[0074] Detection of cell collagen level: After the establishment of the DF photoaging intervention model, that is, 6 h after the last ultraviolet lamp irradiation treatment in the UVA group, sEV group, and supermere group, remove the upper-layer culture medium of the cells in each group and wash them with PBS. Add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 10 min. Then, add 500 μL of 0.1% Triton X-100 to each well and permeabilize at room temperature for 10 min. After that, wash with PBS, add 5% BSA and block at room temperature for 2 h. Then, add COL1A1 antibody dropwise and incubate at 4 °C for 14 - 18 h. The next day, discard the antibody, wash with PBS, add fluorescent secondary antibody dropwise and incubate at room temperature for 1 h. Wash with PBS, continue to incubate with phalloidin dye at room temperature for 30 min, wash with PBS, incubate with Hoechst33342 dye at room temperature for 10 min, and observe under a fluorescence microscope after sealing with an anti-fluorescence quencher.

[0075] The results are as Figure 9As shown, it can be found from the figure that the expression level of COL1A1 in the supermere group is the highest, indicating that supermere can increase the expression of COL1A1 and promote the synthesis of collagen.

[0076] Example 3: Preparation of dissolvable anti-aging microneedle patches loaded with hucMSC-sEV or hucMSC-supermere

[0077] Preparation of the needle body material: Hyaluronic acid (HA) powder was mixed with the hucMSC-sEV, hucMSC-supermere solution, and PBS solution obtained in Example 1 respectively, so that the final concentration of HA was 20 wt%, and after stirring, it was naturally swollen at 4 °C until completely dissolved without bubbles.

[0078] Preparation of the backing material: Polyvinyl alcohol (PVA) powder was mixed with pure water at a final concentration of 20 wt%, heated from room temperature to 90 °C in a water bath and continuously stirred until completely dissolved, and then cooled to room temperature.

[0079] Preparation of the microneedle patch using a PDMS microneedle mold (denoted as the PDMS mold in the figure): As Figure 10 shown, first, the needle body material was added and filled the mold. After maintaining at room temperature -0.07 Mpa for 3 min, the atmospheric pressure was restored, and this was repeated 3 times. Then, it was centrifuged at 4 °C and 2000 g for 5 min to remove the excess needle body material. The backing material was added to the mold until the entire mold was filled. Finally, it was placed in a dryer and dried at room temperature for 18 h. After demolding, a dissolvable anti-aging microneedle patch loaded with hucMSC-sEV or hucMSC-supermere was obtained. If the needle body material only included hyaluronic acid and the PBS solution, it was a microneedle patch without the loaded intervention drug.

[0080] The needle body structure of the dried dissolvable anti-aging microneedle patch loaded with hucMSC-supermere in this example was intact (as Figure 11 ). Using Figure 12 the microneedle syringe shown (purchased from Henan Micro-Nano Pentium Biotechnology Co., Ltd.), the dissolvable anti-aging microneedle patch loaded with hucMSC-supermere was ejected into the skin of mice. The needle body part of the dissolvable anti-aging microneedle patch loaded with hucMSC-supermere could dissolve 15 min after being pressed into the skin (as Figure 13 ). Prepare CM-DIL dye-labeled hucMSC-sEV and Alexa Fluor according to the method in Example 2 TM647 dye-labeled hucMSC-supermere, mixing the dye-labeled hucMSC-sEV or hucMSC-supermere solution with hyaluronic acid powder according to the method described in this example to prepare a dissolvable anti-aging microneedle patch, and comparing the subcutaneous distribution of hucMSC-sEV and hucMSC-supermere under three administration methods: application (Duab), ordinary subcutaneous injection (Needle injection), and dissolvable hyaluronic acid microneedle patch (HA MN). That is, in the application group, 1×10 10 particle numbers of dye-labeled hucMSC-sEV or hucMSC-supermere were evenly applied to the back skin of 6-8-week-old ICR female mice (purchased from the Experimental Animal Center of Jiangsu University) with a pipette tip. In the ordinary subcutaneous injection group, 1×10 10 particle numbers of dye-labeled hucMSC-sEV or hucMSC-supermere were injected subcutaneously at a single point on the back of the mice. In the dissolvable anti-aging microneedle patch group, the dissolvable anti-aging microneedle patch was pressed into the skin of the mice using a microneedle syringe. After the needle body dissolved in 20 min, the dissolvable anti-aging microneedle patch was removed. All mice were housed in a dark environment for 24 h. After euthanasia, the back skin of the mice was fixed with 4% paraformaldehyde, and then frozen sections were obtained. The sections were incubated with Hoechst33342 dye at room temperature for 10 min, washed 3 times with histochemical PBS, and sealed with an anti-fluorescence quencher and then observed under a fluorescence microscope.

[0081] The results are as Figure 14 shown. Compared with application and ordinary subcutaneous injection, the dissolvable anti-aging microneedle patch has a higher transdermal rate of hucMSC-sEV and hucMSC-supermere and is more evenly distributed subcutaneously.

[0082] Example 4: Evaluation of the effect of dissolvable anti-aging microneedle patch loaded with hucMSC-supermere on alleviating skin aging

[0083] (1) Establishment of a mouse skin photoaging intervention model: ICR female mice aged 6 - 8 weeks (purchased from the Experimental Animal Center of Jiangsu University) were randomly divided into a CTR group, a UV group, an MN group, an sEV group, and a supermere group. After one week of adaptive feeding, the hair on the back skin of all mice was removed using a razor and depilatory cream, and then washed clean with normal saline. Taking 7 days as a cycle: On the first day, all mice were anesthetized. In the MN group, sEV group, and supermere group, the microneedle patches without loaded intervention drugs in Example 3, the dissolvable anti-aging microneedle patches loaded with hucMSC-sEV, and the dissolvable anti-aging microneedle patches loaded with hucMSC-supermere were respectively pressed into the back skin. After 20 minutes of pressing the skin until the needles of the microneedle patches were completely dissolved, the microneedle patches were removed. The CTR group was not treated. From the second day to the sixth day, the mice in the UV group, MN group, sEV group, and supermere group were subjected to UV irradiation (UVA combined with UVB irradiation), that is, after the mice were anesthetized, the head and tail of the mice were covered with a light-shielding plate, and the depilated area on the back was exposed to UV irradiation. The mice in the CTR group were only anesthetized and not treated otherwise. On the seventh day, no treatment was given to each group.

[0084] The model had a total of 4 cycles, as shown specifically in Figure 15 : Cover the head and tail of the mice, and irradiate them with UVA and UVB simultaneously at a distance of 25 cm from the ultraviolet lamp tube. The UV irradiation dose was gradually increased according to the minimum erythema dose (MED), that is, the first cycle was 1 MED, the second cycle was 2 MED, and the third and fourth cycles were 3 MED. After the model establishment was completed, the cumulative doses of UVA and UVB were 24.165 J / cm 2 and 1.845 J / cm 2 .

[0085] (2) Detection of senescence-related molecules: After the establishment of the mouse skin photoaging intervention model in step (1), the mice were euthanized, and the back skin of the mice was immediately cut, ground in liquid nitrogen to obtain tissue powder, and the RNA of each group was extracted using an automatic nucleic acid extractor. The mRNA levels of CXCL2, CXCL5, and CXCL10 in each group were analyzed by qRT-PCT.

[0086] The results are as shown in Figure 16 : It can be found from the figure that hucMSC-supermere can better alleviate the increase in the level of skin inflammatory factors caused by UV irradiation than hucMSC-sEV, indicating that hucMSC-supermere has the effect of resisting or treating skin photoaging, and is superior to hucMSC-sEV.

[0087] (3) Skin collagen detection: After the construction of the mouse skin photoaging model was completed, the gross appearance of the skin of each group of mice was observed by dermoscopy. Then, the mice were euthanized, and the skin on the back of the mice was immediately excised and fixed in 4% paraformaldehyde. After paraffin embedding, sections were made and stained with Masson to detect the skin collagen level of each group of mice.

[0088] The dermoscopy results showed that hucMSC-supermere could better reduce erythema and dandruff caused by UV irradiation (such as Figure 17 ); Masson staining showed that hucMSC-supermere could better inhibit the reduction of skin collagen caused by UV irradiation than hucMSC-sEV (such as Figure 18 ).

[0089] The present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Application of supermere extracellular particles in anti-skin aging, anti-skin photoaging, photoaging skin repair or promoting collagen synthesis.

2. The use according to claim 1, characterized in that: The sources of the extracellular supermere particles include hucMSC cells.

3. The use according to claim 1, characterized in that: The supermere extracellular particles are non-vesicular extracellular particles with a particle size of less than 50 nm.

4. The use according to claim 1, characterized in that: The application includes application in preparing products for resisting skin aging, resisting skin photoaging, repairing photoaging skin or promoting collagen synthesis.

5. The use according to claim 4, characterized in that: The products include cosmetics or medical beauty products.

6. The use according to claim 5, characterized in that: The products include dissolvable anti-aging microneedle patches.

7. A dissolvable anti-aging microneedle patch, characterized in that: The microneedle patch is loaded with extracellular particles supermere.

8. The dissolvable anti-aging microneedle patch according to claim 7, characterized in that: The microneedle patch comprises a needle body and a backing; the needle body material comprises hyaluronic acid and extracellular particles supermere, and the backing material comprises polyvinyl alcohol.

9. The dissolvable anti-aging microneedle patch according to claim 7 or 8, characterized in that: The preparation method of the dissolvable anti-aging microneedle patch comprises: (1) mixing hyaluronic acid with extracellular supermere particles, stirring and allowing them to swell naturally until they are completely dissolved without bubbles, thereby obtaining a needle material; (2) mixing polyvinyl alcohol powder with water, stirring and swelling in a water bath until there are no bubbles, to obtain a backing material; (3) Add the needle body material obtained in step (1) to fill the PDMS microneedle mold, evacuate, centrifuge, remove excess needle body material, add the backing material obtained in step (2) to the PDMS microneedle mold and fill the mold, dry, and demold to obtain a soluble anti-aging microneedle patch.

10. Use of extracellular particles supermere in the preparation of drugs for preventing and / or treating diseases caused by photoaging.

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