Multifunctional beauty microneedle for improving skin photoaging and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a multifunctional beauty microneedle for improving skin photoaging and a preparation method and application thereof. According to the invention, water-soluble hyaluronic acid and chitosan / beta-sodium glycerophosphate temperature-sensitive hydrogel are used as a polymer of a microneedle substrate layer, and the polymer comprises exosome of human adipose-derived mesenchymal stem cells and epigallocatechin gallate. When the microneedle is inserted into the skin, hyaluronic acid is quickly dissolved after contacting with tissue fluid, the needle tip is implanted into the skin, and the CS / beta-GP temperature-sensitive hydrogel is in a solid-gel-like state and can slowly release two cosmetic active ingredients in a long-acting manner under the influence of environment temperature and humidity; the facial mask has the effects of promoting tissue regeneration, reducing hyperpigmentation, replenishing water, preserving moisture, resisting oxidation, delaying skin aging and the like, so as to achieve the medical beauty effects of reducing wrinkles, whitening, preserving moisture, rejuvenating the face and the like. The microneedle provided by the invention can be further prepared into a patch, and is beneficial to treatment of wrinkles and stains at specific parts.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a multifunctional cosmetic microneedle for improving skin photoaging, and a preparation method and application thereof. Background Art

[0002] Aging is an inevitable process for everyone. The most obvious symptoms of human aging are first manifested as changes in skin appearance, such as skin sagging, loss of elasticity, and wrinkle formation. In addition to age, ultraviolet rays are an important factor causing exogenous skin aging, also known as photoaging. Ultraviolet rays upregulate the expression of matrix metalloproteinases and the secretion of proinflammatory cytokines by producing reactive oxygen species, induce fibroblast damage in the dermis, and then reduce the production of extracellular matrix. Not only that, long-term exposure to ultraviolet rays will cause excessive deposition of melanin in the skin, resulting in uneven pigmentation and freckles. Moreover, for women who pay special attention to their personal appearance, beauty and skin care have become their daily topics of discussion and concerns. Due to the growth of this demand, the corresponding beauty market has also been developed. Therefore, a variety of beauty products and beauty methods have appeared in people's sight, including oral beauty, smear beauty, laser beauty, etc. In recent years, microneedles have also appeared in the public eye as a new technology.

[0003] In order to make the active ingredients work on the skin, external preparations containing active ingredients are usually used, such as ointments, cream preparations, tape preparations, and patches. Through local application or attachment, the active ingredients are absorbed through the skin and work under the skin. The above preparations have the following disadvantages: during use, the preparations fall off or disappear due to sweating and washing. In particular, due to the blocking effect of the stratum corneum, it is difficult for the active ingredients to penetrate the stratum corneum and enter the deep part of the skin tissue; especially when the active ingredients are macromolecules or nanoparticles such as exosomes, it is more difficult to absorb and cannot play the desired role.

[0004] Microneedles (MNs) are a new type of non-surgical treatment method. They use microneedles to stimulate the skin, forming a large number of reversible microchannels, allowing the active cosmetic ingredients to reach the deep layer of the epidermis along the microchannels, diffuse into the dermis, and stimulate the proliferation of collagen and fibroblasts in the dermis. In addition, microneedles allow patients to administer medication by themselves, which brings great convenience. However, microneedles are a medical technology that uses fine needle-like instruments to implement mechanical or physical, minimally invasive damage stimulation to the skin soft tissue in order to achieve therapeutic or cosmetic effects. On the basis of improving skin problems, it is easy to cause skin damage and erythema. Moreover, the existing microneedle technology generally has a short efficacy time and needs to be repaired regularly, which also greatly increases the cost.

[0005] Therefore, the existing technology generally has problems such as short microneedle treatment effect and easy occurrence of erythema, and it is necessary to further optimize the microneedle technology. Summary of the invention

[0006] In response to the common problems in the prior art, the present invention provides a multifunctional cosmetic microneedle for improving skin photoaging, which simultaneously has multiple functions such as promoting tissue regeneration, reducing pigmentation, moisturizing, anti-oxidation and delaying skin aging. The improvement effect is mild, the efficacy is long, and the cost of skin care is effectively reduced.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A multifunctional cosmetic microneedle for improving skin photoaging comprises a patch base layer and a microneedle array. The patch base layer comprises a chitosan / sodium β-glycerophosphate complex and hyaluronic acid, and the microneedle array comprises exosomes of human adipose-derived mesenchymal stem cells and epigallocatechin gallate.

[0009] Hyaluronic acid is a substance found in the human body. It has good biocompatibility and no need to worry about rejection. It has strong viscoelasticity and hygroscopicity, and can play a unique role in protecting the skin. It can keep the skin moisturized, smooth, delicate, tender, and elastic. It has the functions of anti-wrinkle, anti-aging, beauty care, and restoring the physiological functions of the skin. Hyaluronic acid is used in the preparation process of soluble microneedles, which has excellent mechanical strength and can be used for multiple injections.

[0010] Mesenchymal stem cells (MSCs) are defined as multipotent stem cells with stronger self-renewal and differentiation abilities, which can be derived from a variety of tissue types, such as placenta, fat, bone, and umbilical cord. More and more studies have shown that mesenchymal stem cells have significant therapeutic potential in promoting wound healing, improving myocardial infarction, and repairing ischemic diseases. In addition, mesenchymal stem cells have also attracted much attention due to their great therapeutic potential in improving skin aging. Studies have shown that in a D-galactose-induced mouse skin aging model, human adipose-derived mesenchymal stem cells (ADSCs) can promote skin regeneration through glycosylation inhibition and anti-oxidation. Most importantly, the anti-aging application of MSCs may be due to the exosomes secreted by stem cells through paracrine effects. Epigallocatechin gallate (EGCG) contains multiple reducing phenolic hydroxyl groups and has a strong hydrogen supply capacity. It can scavenge harmful free radicals in the body and prevent damage to biological membranes, thereby delaying skin aging. It can also inhibit the activity of tyrosine and peroxidase in the human body, thereby inhibiting the synthesis of melanin and achieving a whitening effect.

[0011] The chitosan / β-sodium glycerophosphate complex (CS / β-GP) in the present invention is a hydrogel, containing a loose porous structure and having temperature sensitivity. During use, the chitosan / β-sodium glycerophosphate complex hydrogel will gradually cross-link with the change of temperature, thereby achieving a continuous and slow release of the drug. Therefore, it can be used as a drug sustained-release carrier. During use, the drug can be administered in a minimally invasive manner, and the cosmetic active ingredient can be implanted into the target tissue to obtain better results. Compared with traditional hydrogels, the thermosensitive property allows the drug to be evenly dispersed in the gel matrix and enables it to be delivered in any shape or by injection.

[0012] The present invention uses CS (chitosan) / β-GP (sodium β-glycerophosphate) thermosensitive hydrogel as the polymer of the microneedle needle tip, and encapsulates two cosmetic active ingredients, EGCG (epigallocatechin gallate) and ADSCs-Exo (exosomes of human adipose-derived mesenchymal stem cells, Ex), and uses water-soluble HA (hyaluronic acid) as the polymer of the microneedle base layer. When the microneedle is inserted into the skin, HA quickly dissolves after contacting the tissue fluid and implants the needle tip into the skin. Affected by the ambient temperature and humidity, the CS / β-GP thermosensitive hydrogel (CS / β-GP is a polymer designed for drug encapsulation in the needle tip layer and has a sustained-release effect) is in a solid gel-like state at this time and can release the two cosmetic active ingredients in a long-term sustained-release manner, and has the effects of promoting tissue regeneration, reducing pigmentation, replenishing water and moisturizing, anti-oxidation and delaying skin aging, so as to achieve medical and cosmetic effects such as reducing wrinkles, whitening and moisturizing, and facial rejuvenation. In addition, the E / Ex@CS-HA MNs patch (MNs is the English abbreviation of microneedles) has a local therapeutic effect, which is beneficial for the treatment of wrinkles and pigmentation in specific areas.

[0013] As can be seen from the above, in the present invention, microneedles are used to stimulate the skin to form a large number of reversible microchannels, so that the beauty active ingredients EGCG and ADSCs-Exo reach the deep layer of the epidermis along the microchannels, diffuse into the dermis, and stimulate the proliferation of collagen and fibroblasts in the dermis, thereby constructing a beauty microneedle with multiple functions including promoting tissue regeneration, reducing pigmentation, moisturizing, anti-oxidation and delaying skin aging. In addition, in terms of material design, a long-term transdermal drug delivery system is constructed with a thermosensitive chitosan hydrogel as a carrier, which is used for more effective and longer-lasting local drug delivery and functional factor delivery, which is beneficial to long-term skin care and reduces skin care ingredients.

[0014] Preferably, the hyaluronic acid has a molecular weight of 10 kDa and a concentration of 20-25%.

[0015] Preferably, the chitosan has a deacetylation degree of ≥95%, a viscosity of 100-200 mPa·s, a concentration of 1-3%, and is solubilized with acetic acid.

[0016] Preferably, the concentration of epigallocatechin gallate is ≥75 μg / mL.

[0017] The present invention also provides a method for preparing the multifunctional cosmetic microneedle, comprising the following steps:

[0018] S1. Extract and purify exosomes from human adipose-derived mesenchymal stem cells;

[0019] S2, dissolving hyaluronic acid powder and epigallocatechin gallate in water to prepare hyaluronic acid stock solution and epigallocatechin gallate solution respectively;

[0020] S3, dissolving chitosan with acetic acid in water to prepare a chitosan stock solution;

[0021] S4, accurately weighing β-sodium glycerophosphate powder and dissolving it in water to prepare a β-GP solution, and then slowly adding the solution to the chitosan stock solution prepared in step S3, continuously stirring and cross-linking to obtain a CS / β-GP thermosensitive hydrogel;

[0022] S5, placing the CS / β-GP thermosensitive hydrogel prepared in step S4 in an ice water bath, and then dropping the exosomes prepared in step S1 and the epigallocatechin gallate solution prepared in step S2 thereinto, mixing well, to obtain a mixture I;

[0023] S6. Inject the mixture I described in S5 into the microneedle mold, vacuum treat it, and then incubate it in an oven for cross-linking. On this basis, add the hyaluronic acid stock solution prepared in step S2, dry and demould to obtain.

[0024] The invention uses beta-glycerophosphate as a crosslinking agent for the chitosan hydrogel. After crosslinking, the chitosan hydrogel presents a sol state at low temperatures and can be quickly converted into a gel state at human body temperature.

[0025] Preferably, the concentration of acetic acid in step S3 is 0.05-0.15M.

[0026] Preferably, the incubation condition in the oven in step S6 is incubating at 35-37° C. for 2-3 hours; the microneedle mold is a PDMS microneedle mold, the needle body is cylindrical, arranged in a 10×10 array, and has a height of 600 μm.

[0027] The present invention also provides an application of the multifunctional cosmetic microneedle in preparing a skin care product for improving skin photoaging.

[0028] The CS / β-GP hydrogel prepared by the present invention has thermosensitivity and a loose porous structure. As a drug sustained-release carrier, it can effectively enhance drug targeting, control local drug concentration, improve drug utilization, and prolong drug action time. CS hydrogel can maintain the stability of proteins and miRNA under physiological conditions, greatly promoting the therapeutic effect of bioactive macromolecules.

[0029] The present invention successfully constructs a multifunctional soluble long-lasting transdermal beauty microneedle E / Ex@CS-HA MNs, which simultaneously has multiple functions such as promoting tissue regeneration, reducing pigmentation, replenishing water and moisturizing, anti-oxidation and delaying skin aging. The microneedle is divided into a dissolving layer and a sustained-release layer. The dissolving layer is also the needle tip layer, which can penetrate the skin to form a large number of reversible microchannels. The two cosmetic active ingredients loaded in the sustained-release layer, namely, exosomes that promote tissue regeneration and delay skin aging and epigallocatechin gallate that resists oxidation and reduces pigmentation, reach the deep layer of the epidermis along the microchannels and diffuse into the dermis; the chitosan / β-sodium glycerophosphate (CS / β-GP) complex hydrogel has a thermosensitive and loose porous structure, so that the two cosmetic active ingredients wrapped therein are efficiently, continuously and slowly released, which is beneficial to long-term skin care and reduces the cost of skin care.

[0030] Compared with the prior art, the present invention has the following advantages: the multifunctional cosmetic microneedle E / Ex@CS-HA MNs prepared by the present invention has a simple preparation method and has multiple functions such as promoting tissue regeneration, reducing pigmentation, moisturizing, anti-oxidation and delaying skin aging. The effect lasts for a long time, effectively reducing the cost of skin care. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 1 is a diagram of the preparation process of CS / β-GP thermosensitive hydrogel;

[0032] Figure 2 Figure 2 is the preparation process diagram of E / Ex@CS-HA MNs;

[0033] Figure 3 The structure diagram of the multifunctional cosmetic microneedle E / Ex@CS-HA MNs finally prepared;

[0034] Figure 4 , 5 This is the result diagram of the in vitro antioxidant damage effect of E / Ex@CS-HA MNs;

[0035] Figure 6 , 7 This is the result diagram of E / Ex@CS-HA MNs promoting the proliferation and migration of skin fibroblasts;

[0036] Figure 8This is the result diagram of the in vitro anti-ultraviolet damage effect of E / Ex@CS-HA MNs;

[0037] Fig. 9 This is the result diagram of the effect of E / Ex@CS-HA MNs on ROS generation in photoaging damaged cells;

[0038] Fig.10 , 11 This is the result diagram of the in vitro anti-melanin production effect of E / Ex@CS-HA MNs;

[0039] Fig.12 This is the result diagram of the therapeutic effect of E / Ex@CS-HA MNs on the guinea pig skin photoaging model;

[0040] Fig.13 This is the in vitro sustained release performance result diagram of E / Ex@CS-HA MNs;

[0041] Fig.14 This is the result diagram of the in vivo retention effect of E / Ex@CS-HA MNs. DETAILED DESCRIPTION

[0042] The technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. CS-HA, EGCG@CS-HA, Exo@CS-HA, and E / Ex@CS-HA involved in the following experiments are all prepared by methods similar to the present invention.

[0043] Example 1 Preparation of exosomes

[0044] The preparation process of the exosomes is as follows:

[0045] (1) Cell culture: Human adipose-derived mesenchymal stem cells (available from Huatuo Biotechnology) were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% double-antibody solution (anti-penicillin and anti-streptomycin mixed in a volume ratio of 1:1) at 37°C in a constant temperature incubator with 5% CO2;

[0046] (2) Cell passaging: When the cell growth density reaches about 80%, wash the cells three times with PBS, add trypsin EDTA solution (0.25%, purchased from Shanghai Aiyan Biotechnology Co., Ltd., catalog number S310) to digest the cells from the culture medium. After observing that the cells are detached from the culture dish, remove the trypsin EDTA solution, add 1 mL of culture medium to terminate the digestion process, wash the cells by blowing the culture medium, distribute them into a new cell culture dish, add 4 mL of culture medium and continue to culture for later experiments;

[0047] (3) Cell cryopreservation: After culturing until the cell density reaches 80%, wash 3 times with PBS, add trypsin EDTA solution for digestion, centrifuge the cell suspension at 1000 rpm for 5 min, remove the supernatant, add 1 mL of freezing solution, mix well, transfer to a cell cryopreservation tube and store at -80°C, or store in liquid nitrogen for a long time;

[0048] (4) Collection of exosomes: Thaw the stored human adipose-derived mesenchymal stem cells hADSCs and culture them in DMEM / F12 complete medium. When the cell density grows to about 70%, discard the original medium, rinse with PBS buffer 3 times, and continue to culture with serum-free DMEM / F12 medium for 48 hours. Collect the cell supernatant and store it in a -80°C refrigerator for subsequent exosome extraction;

[0049] (5) Extraction of exosomes: The supernatant of the collected cells was extracted by ultracentrifugation. The specific steps are as follows: centrifuge at 300×g for 10 min to remove cell debris and take the supernatant; then centrifuge at 2000×g for 10 min to remove macromolecules and take the supernatant; centrifuge at 10000×g for 30 min, take the supernatant, and use a 0.22μm filter to remove large particles; centrifuge at 100000×g for 90 min at 4°C, discard the supernatant, resuspend the precipitate with PBS, and centrifuge at 100000×g for 90 min again to collect and store in a -80°C refrigerator. The above steps were all centrifuged at 4°C. The exosomes obtained by high-speed centrifugation have higher purity, safety and effectiveness are guaranteed, and at the same time, issues related to stem cell ethics are avoided.

[0050] Example 2 Preparation of CS / β-GP Thermosensitive Hydrogel

[0051] The preparation process of the CS / β-GP thermosensitive hydrogel is as follows;

[0052] (1) Accurately weigh 2.0 g of chitosan powder and dissolve it in 100 mL of 0.1% acetic acid solution. Continue stirring until it is completely dissolved to prepare a 2% (w / v) chitosan (CS) solution, which is stored at 4°C for later use.

[0053] (2) Accurately weigh 5.6 g of sodium β-glycerophosphate powder and dissolve it in 10 mL of distilled water to prepare a 56% (w / v) β-GP solution;

[0054] (3) Take 7 mL of chitosan solution prepared in step (1) into a 50 mL beaker and place it in an ice water bath. Then slowly drop 3 mL of β-GP solution prepared in step (2) into the CS solution and continue stirring until the mixture becomes clear after the addition. If flocculent precipitation occurs, the test is considered to have failed and stored at 4°C. CS / β-GP can be cross-linked into a gel at 37°C. The finished product is shown in the figure below. Figure 1 As shown, Figure 1 In the figure, the left picture is the newly prepared CS / β-GP composite solution, which is cross-linked into a solid state after incubation at 37°C for 30 minutes, becoming the right picture. In the actual experimental process, the hydrogel needs to be prepared and used immediately.

[0055] Example 3 Preparation of multifunctional cosmetic microneedle E / Ex@CS-HA MNs

[0056] The preparation process of the multifunctional cosmetic microneedle E / Ex@CS-HA MNs is as follows (steps as Figure 2 ):

[0057] (1) 5 mL of CS / β-GP hydrogel (i.e., the CS / β-GP composite solution prepared in Example 1, without cross-linking) was continuously stirred in an ice water bath, and exosomes (Exo, Ex) of human adipose-derived mesenchymal stem cells and epigallocatechin gallate (EGCG) were added dropwise, respectively, so that the final drug concentration of the composite hydrogel was 75 μg / mL Exo and 100 μg / mL EGCG, and stored at 4°C, labeled A;

[0058] (2) 2.5 g of hyaluronic acid (HA) powder was accurately weighed and dissolved in 10 mL of distilled water. The mixture was stirred for 2 h to completely dissolve the HA. A 25% (w / v) HA hydrogel was prepared and stored at 4°C. The hydrogel was labeled B.

[0059] (3) taking 50 μL of the mixed solution A prepared in step (1) and adding it to the PDMS microneedle mold, vacuum treating it for 5 min to remove bubbles and excess liquid outside the needle tip, and incubating it at 37° C. for 30 min for cross-linking;

[0060] (4) Based on (3), 200 μL of solution B prepared in step (2) was added to the PDMS microneedle mold, vacuum treated for 5 min, bubbles and excess liquid outside the needle tip were removed, refrigerated and dried, and demolded for the next experiment. The final microneedle structure is shown in the figure below. Figure 3 shown.

[0061] Example 4 Antioxidant effect of E / Ex@CS-HA MNs in vitro

[0062] 1. Experimental samples: Blank group: The same cell concentration as each group, 100 μL of DMEM medium without any treatment; Model group: The same cell concentration as the blank group, 100 μL of DMEM medium (300 mmol / L H2O2) was added, incubated for 3 hours, and the H2O2 oxidative damage model was established; Experimental group: First, 50 μL of each component of E / Ex@CS-HA MNs (CS-HA, EGCG@CS-HA, Exo@CS-HA, E / Ex@CS-HA) was added and incubated with NIH3T3 for 24 hours, and then the model was established under the same conditions as the model group; Control group: First, 2% vitamin C was added and incubated with NIH3T3 for 24 hours, and then the model was established under the same conditions as the model group;

[0063] 2. Experimental process: A H2O2 oxidative damage model was established under in vitro conditions. Mouse embryonic fibroblasts (NIH-3T3) induced by hydrogen peroxide in vitro is a classic in vitro oxidative stress damage model. The specific construction process is as follows: NIH-3T3 cells were treated with 300 μM H2O2 for 3 h. When the cell survival rate was around 50%, it was confirmed that the oxidative damage model was successfully constructed.

[0066] NIH3T3 cells in the logarithmic growth phase (cell density in each well was 5×10 4 ) were inoculated in a 96-well plate, and when the cells were fused to 80%, 100 μL DMEM medium containing different groups was added and incubated for 24 hours. Except for the blank group, 300 mM H2O2 was added to each well. After incubation for 3 hours, the old medium was discarded and replaced with 100 μL DMEM medium. Under light-proof conditions, 10 μL CCK-8 was added to the cells in each well and incubated in an incubator for 60 minutes. The absorbance value of each well was measured at a wavelength of 450 nm using an enzyme reader.

[0067] NIH3T3 cells in the logarithmic growth phase (cell density per well was 2×10 5 ) were inoculated in 6-well plates, and when the cells were fused to 80%, 1 mL of DMEM medium containing different groups was added for 24 hours, and then 300 mM H2O2 was added to each well except the control group. After incubation for 3 hours, the old medium was discarded, and RIPA lysis buffer was added to collect cells after lysis, and the supernatant was centrifuged and taken as samples for testing. After the sample preparation was completed, the MDA content in the cells was determined according to the MDA detection kit (Nanjing Jiancheng Bioengineering Institute, Cat. No.: A003-1).

[0068] 3. Test results: Absorbance value results are as follows Figure 4 As shown, the MDA results are as follows Figure 5 As shown, it can be seen that Figure 4In the experiment, after 3h of H2O2 treatment, the cell survival rate of NIH3T3 cells dropped to about 50%. The addition of EGCG and positive control Vc could slightly increase the cell survival rate, but there was no statistical difference compared with the Model. In contrast, the cell survival rates of Exo@CS-HA and E / Exo@CS-HA groups were significantly improved, which may be related to the effect of Exo in promoting fibroblast proliferation. Among them, E / Exo@CS-HA had the best effect on improving NIH3T3 cells with oxidative damage. During the oxidative stress of cells, lipid peroxidation will occur. Further detection of intracellular MDA content, such as Figure 5 As shown, when cells were damaged by oxidation, the MDA content in the Model group increased significantly, and compared with the Model group, the MDA content in the treatment group decreased.

[0069] Example 5 Effect of E / Ex@CS-HA MNs on Proliferation and Migration of Skin Fibroblasts

[0070] 1. Test samples: EGCG@CS-HA, Exo@CS-HA, E / Ex@CS-HA;

[0071] 2. Experimental method: Cell proliferation: HDFs cells in the logarithmic growth phase (cell density in each well is 1×10 4 ) were inoculated in a 24-well plate (4 groups, 3 replicates per group), and a blank well was taken and 1 mL of fresh DMEM complete medium was added. It was placed in an incubator and incubated for 4 hours. After the cells adhered to the wall, 0.1 mL of CCK-8 reagent was added to each well and placed in an incubator for 2 hours. The absorbance of each well was measured at a wavelength of 450 nm using an enzyme marker, which was recorded as D0. The cells were then replaced with 1 mL of DMEM medium containing each component of E / Ex@CS-HA MNs (50 μL each of EGCG@CS-HA, Exo@CS-HA, and E / Ex@CS-HA) to continue culturing. On the 1st, 2nd, 3rd, 4th, and 5th day after the first measurement, CCK-8 reagent was added again and the OD value was measured. After each measurement, 1 mL of DMEM medium containing each component was replaced and continued to be cultured.

[0072] Cell migration: Adjust the cell concentration to 1×10 5The cells were inoculated at a density of 100 μL / well in a 6-well culture plate and placed in an incubator at 37°C and 5% CO2. After the cells were fused into a single layer, they were scratched with a 200 μL sterile pipette tip and carefully washed with sterile PBS buffer 3 times, 1 min each time. 2 mL of DMEM medium containing different groups (50 μL for each group of EGCG@CS-HA, Exo@CS-HA, and E / Ex@CS-HA) was added to each well for continued cultivation, and the scratched area was photographed at 0h, 24h, and 48h.

[0073] ImageJ software was used for statistical analysis, and the cell migration rate was calculated according to the following formula.

[0074]

[0075] Among them, S0, S t Refers to the area of ​​cell migration at 0h and th, respectively.

[0076] 3. Test results: Cell proliferation test results are as follows Figure 6 The cell migration results are shown in Figure 7 As shown. Figure 6 As shown, the CCK-8 results showed that compared with the untreated control group, the cell viability and proliferation rate of the Exo@CS-HA group and the E / Ex@CS-HA group were higher; Figure 7 As shown in the figure, compared with the untreated control group, the cells in the Exo@CS-HA group and the E / Ex@CS-HA group migrated faster, significantly improving the motility of skin fibroblasts; in summary, E / Ex@CS-HA MNs have good biocompatibility for skin fibroblasts, and can promote their proliferation and migration, have a certain repair effect on the skin, and maintain the normal physiological state of the skin. Example 6 Anti-ultraviolet damage effect of E / Ex@CS-HA MNs in vitro

[0077] 1. Experimental samples: Blank group: The same cell concentration as each group, 100 μL of DMEM medium without any treatment; Model group: The same cell concentration as the blank group, 100 μL of DMEM medium (300 mmol / L H2O2) was added, incubated for 3 hours, and the H2O2 oxidative damage model was established; Experimental group: 50 μL of each component of E / Ex@CS-HA MNs (CS-HA, EGCG@CS-HA, Exo@CS-HA, E / Ex@CS-HA) was first added and incubated with NIH3T3 for 24 hours, and then the model was established under the same conditions as the model group; Control group: 2% vitamin C was first added and incubated with HDFs cells for 24 hours, and then the model was established under the same conditions as the model group;

[0078] 2. Experimental process: A simulated UV damage model was established in vitro. The specific construction process was as follows: HDFs cells were cultured in DMEM complete medium. When the cells proliferated to 80%, each well except the control group was placed 10 cm below the UVB light source (311 nm) to irradiate with a cumulative radiation dose of 40 mJ / cm 2 , the original culture medium was replaced with an equal amount of PBS before UVB irradiation.

[0079] HDFs cells in logarithmic growth phase (cell density per well was 1.5×10 4 ) were inoculated into a 96-well culture plate, and when they proliferated to 80%, 100 μL of DMEM medium containing different groups was added for 24 h. Then, except for the control group, each well was placed 10 cm below a UVB light source (311 nm) to irradiate, so that the cumulative radiation dose was 40 mJ / cm 2 Before UVB irradiation, the original culture medium was replaced with an equal amount of PBS. When the cell survival rate was no higher than 50%, it proved that the UV damage model was successfully established.

[0080] Then, PBS was replaced with DMEM medium and cultured for 24 hours. The old medium was discarded and 100 μL DMEM medium was added again. Under light-proof conditions, 10 μL CCK-8 was added to each well of cells and incubated in an incubator for 60 minutes. The absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader, and the cell survival rate was calculated according to the following formula.

[0081]

[0082] 3. Test results: Specific test results are as follows: Figure 8 As shown in Figure 2, UVB has a significant inhibitory effect on HDFs cell proliferation. Through the exploration of the previous experimental conditions, we selected the irradiation dose for modeling at 40mJ / cm 2 At this time, the cell survival rate was 46.43%, close to the half lethality. After adding E / Exo@CS-HA, the cell survival rate was significantly improved, indicating that EGCG and Exo have a certain photoprotective effect.

[0083] Example 7 Effect of E / Ex@CS-HA MNs on ROS production in photoaging damaged cells

[0084] The more ROS are produced, the higher the degree of cell senescence. The anti-aging effect of each experimental group of materials can be reflected by detecting the ROS positive rate of each group of cells.

[0085] 1. Experimental samples: Blank group: The same cell concentration as each group, 100 μL of DMEM medium without any treatment; Model group: The same cell concentration as the blank group, 100 μL of DMEM medium (300 mmol / L H2O2) was added, incubated for 3 hours, and the H2O2 oxidative damage model was established; Experimental group: First, 50 μL of each component of E / Ex@CS-HA MNs (CS-HA, EGCG@CS-HA, Exo@CS-HA, E / Ex@CS-HA) was added and incubated with NIH3T3 for 24 hours, and then the model was established under the same conditions as the model group; Control group: First, 2% vitamin C was added and incubated with HDFs cells for 24 hours, and then the model was established under the same conditions as the model group;

[0086] 2. Experimental process: Adjust the HDFs cell concentration to 5×10 4 The cells were plated at a density of 10 μM / well and then inoculated into a 6-well plate. When the cells were fused to 80%, the experimental group cells were added to 1 mL of DMEM medium containing different groups 24 h before UVB irradiation. The rest of the treatments remained the same. On the day of UVB irradiation, the H2DCFDA probe was diluted with DMEM to a concentration of 10 μM and added to each well (added after irradiation, irradiation conditions were the same as in Example 6).

[0087] 1mL H2DCFDA probe was placed in a cell culture incubator and incubated in the dark for 30 minutes. Washed 3 times with DMEM to fully remove the H2DCFDA probe that was not loaded into the cells. Then, the experimental group cells were irradiated with UVB according to the method and conditions described in Example 5. After irradiation, trypsin-0.25% EDTA was used for digestion, and the cells were gently and repeatedly blown with a pipette to fully digest the cells. After fully blowing, the single cell suspension was collected in a flow tube, and the production content of ROS was detected using a flow cytometer.

[0088] 3. Test results: The test results of ROS are as follows Fig. 9 As shown in the figure, compared with the Control group, the intracellular ROS level increased significantly after UVB irradiation (64.7% vs 54.8%); compared with the Model group, the intracellular ROS level in the E / Exo@CS-HA group was significantly decreased (15.0% vs 64.7%). The above results indicate that the addition of E / Exo@CS-HA prevents the excessive production of ROS in HDFs cells induced by UVB.

[0089] Example 8 E / Ex@CS-HA MNs in vitro anti-melanin production effect

[0090] 1. Experimental samples: Blank group: The same cell concentration as each group, 100 μL of DMEM medium without any treatment; Model group: The same cell concentration as the blank group, 100 μL of DMEM medium (300 mmol / L H2O2) was added, incubated for 3 hours, and the H2O2 oxidative damage model was established; Experimental group: 50 μL of each component of E / Ex@CS-HA MNs (CS-HA, EGCG@CS-HA, Exo@CS-HA, E / Ex@CS-HA) was first added and incubated with NIH3T3 for 24 hours, and then the model was established under the same conditions as the model group; Control group: 2% vitamin C was first added and incubated with B16F10 cells for 24 hours, and then the model was established under the same conditions as the model group;

[0091] 2. Experimental process: Tyrosinase activity determination: A mouse melanoma cell model was established in vitro. The mouse melanoma cell model is a classic cell model for the efficacy of whitening active ingredients. Mouse melanoma cells (B16F10) have the characteristics of being able to be passaged multiple times, growing fast, and having relatively low requirements for culture medium conditions. Their melanin synthesis function is basically the same as that of normal human skin melanocytes, making them the preferred cells for screening whitening active ingredients. In the case of difficulty in culturing primary human skin melanocytes, the internationally used B16F10 cell line is usually used as the test cell in the experiment. On this basis, we established a melanoma cell model (the construction method can be referred to the webpage https: / / pro.biomart.cn / lab-web / method / 34bsbaqgo2elv.html).

[0092] B16F10 cells in the logarithmic growth phase (cell density in each well was 5×10 4 ) were inoculated in a 96-well plate, and when the cells were fused to 80%, 100 μL of DMEM medium containing different groups was added respectively, and the cells were incubated for 48 hours, then washed with PBS for 3 times, 100 μL of 1% TritonX-100 solution was added to each well, and then quickly placed in a -80°C refrigerator for 1 hour. After the cells were lysed at room temperature, 100 μL of 0.1% L-DOPA solution was added to each well, and the plates were incubated at 37°C for 2 hours. The absorbance was measured at a wavelength of 490 nm using an enzyme reader, and the tyrosinase activity of the cells was calculated by the following formula.

[0093]

[0094] Melanin content determination: adjust the cell concentration to 2×10 5The cells were inoculated at a density of 100 cells / well in a 24-well plate. When the cells were fused to 80%, 1 mL of DMEM culture medium containing different groups was added respectively. After incubation for 48 h, the cells were washed 3 times with PBS, digested and collected, and 500 μL of 1 mol / L NaOH solution containing 10% DMSO was added respectively. The cells were heated in an 80°C water bath for 60 min, centrifuged at 1500 rpm for 5 min, the supernatant was taken, and the absorbance was measured at a wavelength of 405 nm using an enzyme reader. The relative melanin content of the cells was calculated by the following formula.

[0095]

[0096] 3. Test results: Tyrosinase activity test results are as follows Fig.10 The relative content of melanin is shown in Fig.11 As shown by Fig.10 It can be seen that the tyrosinase activity of the EGCG@CS-HA group and the E / Exo@CS-HA group was 59.27% ​​and 58.87%, respectively, which significantly inhibited the tyrosinase activity compared with the control group, among which EGCG had the best effect. Fig.11 It can be seen that the EGCG@CS-HA group and the E / Exo@CS-HA group significantly reduced the melanin content, which is consistent with the changes in the above-mentioned tyrosinase activity.

[0097] Example 9 Therapeutic effect of E / Ex@CS-HA MNs on guinea pig skin photoaging model

[0098] 1. Test samples: CS-HA MNs, E / Ex@CS-HAMNs, vitamin C cream (Shanghai Kanggaoxiu Cosmetics Co., Ltd., vitamin C emulsion);

[0099] 2. Experimental animals: 15 healthy female guinea pigs, 3 in each group, were purchased from Guangdong Medical Experimental Animal Center (Experimental Animal Production License SCXK (Guangdong) 2019-0035) and kept in Guangdong Pharmaceutical University Medical Experimental Animal Center (Experimental Unit Use License SYXK (Guangdong) 2022-0125). The animals were adapted to laboratory conditions for more than 7 days, including the quarantine period, and were used when they were 7-9 weeks old. The relevant animal experiments were carried out in strict accordance with the Regulations on the Management and Use of Laboratory Animals of Guangdong Pharmaceutical University.

[0100] In this experiment, the experimental group of guinea pigs was subjected to ultraviolet irradiation for a total of 4 weeks. After 7 days of irradiation, the irradiation continued and the experimental group began a 21-day drug treatment. The specific experimental operations of each group are as follows:

[0101] (A) Blank control group: no intervention;

[0102] (B) Model group: modeling, no treatment;

[0103] (C) No-load CS-HAMNs group: 30 min before UV irradiation, the CS-HAMNs patch was inserted and fixed on the exposed skin on the back of the guinea pig using breathable tape for 10 min;

[0104] (D) E / Ex@CS-HAMNs group: 30 min before UV irradiation, the E / Ex@CS-HAMNs patch was inserted and fixed on the exposed skin on the back of the guinea pig using breathable tape for 10 min;

[0105] (E) Positive control Vc group: 30 minutes before UV irradiation, Vc emulsion was applied to keep the exposed skin of guinea pigs moist for 10 minutes.

[0106] 3. Experimental process: The normal control group (Control group) did not receive any treatment. The guinea pigs in the experimental group were placed in a homemade guinea pig holder to cover the important parts (head). One UVA (TL-K 40W / 10R, Philips) and two UVB (TL 20W / 01RS, Philips) were used to simulate sunlight to irradiate the back skin of the guinea pigs (the irradiation time was determined according to the minimum erythema dose). The irradiation distance was 20 cm. The lamp was preheated for 15 minutes before irradiation. According to the minimum erythema dose (MED), the radiation dose for each time in the first week was 70 J / cm 2 In the second week, the radiation dose was 2 times the minimum erythema dose, in the third week, the radiation dose was 3 times the minimum erythema dose, and in the fourth week, the radiation dose was 4 times the minimum erythema dose. Irradiate every day and pay close attention to the changes in the guinea pig's back skin. For 4 consecutive weeks, the total dose was 700 J / cm 2 The photoaging model was established. The photoaging model was successfully established by observing the appearance and state of guinea pig skin and subsequent related experiments.

[0107] After 7 days of irradiation, the model group continued irradiation, and the experimental group began a 21-day drug treatment, which included CS-HAMNs, E / Ex@CS-HAMNs, and Vc cream, 30 minutes before UV irradiation every day, and the drug treatment lasted for 10 minutes. The aging characteristics of the guinea pig's back skin were observed every day, including: pigmentation, wrinkles, erythema, etc., and photographed and recorded. The single-blind method was used to perform macroscopic scoring of the experimental guinea pig's back skin.

[0108] 3. Test results: The changes in the appearance of the back skin of guinea pigs in each group at different stages are as follows Fig.12As shown, on day 0, the skin texture of guinea pigs in each group was smooth, clear, and white. The Control group maintained its initial smooth and rosy state for 28 days, with occasional erythema and tiny wrinkles; on day 7, except for the Control group, the skin texture of each group showed roughly the same degree of disorder, swelling, and redness; from day 8 to day 28, the skin aging degree of the Model group gradually worsened, elasticity decreased, wrinkles became coarser and deeper, and a leather-like appearance appeared, accompanied by changes such as dandruff and pigmentation; the texture of the E / Ex@CS-HA MNs group gradually became clear, and skin elasticity was restored. Compared with the Model group, symptoms such as wrinkles, dandruff, and pigmentation were significantly alleviated, and the effect was better than that of the Positve group. The results of the analysis of skin macroscopic characteristics are shown in the figure. Fig.12 As shown in the data, the skin macroscopic scores of the Control group tended to be stable within 28 days, and the skin macroscopic scores of the Model group were 0.8825±0.0625, 4.9475±0.2755, 7.072±0.348, 7.7485±0.3145, and 8.686±0.117 on days 0, 7, 14, 21, and 28, respectively, with significant differences (P<0.05). During the establishment of the photoaging model, obvious erythema and wrinkles appeared on the back skin of the guinea pigs, and the skin color gradually deepened. The skin macroscopic scores of the E / Ex@CS-HAMNs group were 0.994±0.058, 5.1625±0.2725, 5.7805±0.2945, 3.346±0.278, and 1.9915±0.1385 on days 0, 7, 14, 21, and 28, respectively, with significant differences (P<0.001). The appearance characteristics of the guinea pig back skin, such as erythema, wrinkles, and pigmentation, gradually improved, and the effect was better than that of the Positive group. There was no significant improvement in the appearance characteristics of the guinea pig back skin, such as erythema, wrinkles, and pigmentation in the CS-HAMNs group.

[0109] Example 10 In vitro sustained release performance of E / Ex@CS-HA MNs

[0110] 1. Test samples: E / BSA@CS / β-GP MNs, E / Ex@CS-HA MNs;

[0111] 2. Experimental process: BSA was used as a model to simulate hADSCs-Exo macromolecular drugs for measurement. The cross-linked E / BSA@CS / β-GP (i.e., replacing Ex with BSA) gel was placed in the upper chamber of the Transwell of a 24-well plate. EGCG and BSA could freely pass through the semipermeable membrane of the Transwell. 1.2 mL of release medium (PBS solution at 37°C) was added to the lower chamber. It was placed in a 37°C 180rpm constant temperature shaker for continuous shaking. 400 μL of the medium solution (i.e., PBS solution at 37°C) was aspirated at 0.5, 1, 2, 4, 8, 12h and 1, 2, 3, 4, 5, 6, and 7d. At the same time, an equal amount of release medium was added, and free EGCG and BSA were used as controls. The standard curves and absorbances of EGCG and BSA were measured at wavelengths of 276nm and 562nm, respectively. The cumulative release rate of the drug at each time point was then calculated, and the drug release curve was drawn. The cumulative drug release curve was expressed as the percentage of total drug release.

[0112]

[0113] Where W0 is the weight of the microneedle, Cn and Cn-1 are the concentrations of extracted n and n-1 in the buffer solution, respectively.

[0114] 3. Test results: Specific test results are as follows: Fig.13 As shown in the figure, the free EGCG group showed a rapid release characteristic, with a cumulative release rate of 82.36±0.90% in 2 days, while the EGCG in the E / Ex@CS / β-GP gel showed a slow release characteristic, with a cumulative release rate of 78.78±1.99% in 7 days. The results showed that the CS / β-GP thermosensitive hydrogel had a good local retention capacity for Exo and EGCG, and could release Exo and EGCG to the outside world for a long time in the physiological environment of 37°C in vivo to exert its therapeutic effect.

[0115] Example 10 In vivo retention effect of E / Ex@CS-HA MNs

[0116] 1. Test samples: E / Ex@CS-HA MNs, E / Ex@HA MNs;

[0117] 2. Experimental animals: 9 guinea pigs, 3 guinea pigs in each group, divided into 3 groups.

[0118] 2. Experimental process: Exo was resuspended in sterile PBS and incubated with DiR working solution at 37°C for 30 minutes. Unbound dye was removed using a 300kDa ultrafiltration tube, and ultrafiltration was centrifuged and washed at 12000rpm for 5 minutes. The hair on the upper back of the guinea pig was shaved, and E / Ex@CS-HA MNs containing 100μg DiR-labeled Exo were inserted into the back skin of the guinea pig. At the same time, the same amount of E / Ex@HA MNs (as a control) was inserted and PBS was applied to the back skin as a control. The changes in the fluorescence of the back at different time points were detected (the guinea pigs were anesthetized by inhalation of ether 15 minutes before the detection time point), and the detection was performed on DAY1, DAY3, DAY5, DAY7, and DAY14 respectively.

[0119] 3. Test results: Specific test results are as follows: Fig.14 As shown, the retention of E / Ex@CS-HA MNs in the guinea pig skin was observed using in vivo imaging technology for visual analysis. Fig.14 As shown. DiR-labeled Exo E / Ex@HAMNs and E / Ex@CS-HA MNs were inserted into the back skin of guinea pigs. After 1 day, the fluorescence intensity of the E / Ex@HA MNs group was greatly weakened, and only weak fluorescence was observed after 3 days, indicating that Exo began to be released in large quantities after the HA MNs were dissolved after being inserted into the skin. In contrast, high fluorescence expression sites were visible in the E / Ex@CS-HAMNs group within 5 days, and partial fluorescence was still observed on the 7th day, indicating that under the application of CS / β-GP thermosensitive hydrogel, E / Ex@CS-HA MNs increased the retention time of the cosmetic active ingredients EGCG and Exo in the skin, improved the low retention rate and instability of stem cell-derived exosomes in clinical applications, and at the same time, the long-term sustained release of the two cosmetic active ingredients effectively reduced the cost of skin care.

[0120] Finally, it should be noted that the above embodiments are only illustrative of the principles, performance and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A multifunctional cosmetic microneedle for improving skin photoaging, comprising a patch base layer and a microneedle array, characterized in that: The patch base layer includes chitosan / sodium beta-glycerophosphate complex and hyaluronic acid, and the microneedle array includes exosomes of human adipose-derived mesenchymal stem cells and epigallocatechin gallate.

2. The multifunctional cosmetic microneedle according to claim 1, characterized in that: The molecular weight of the hyaluronic acid is 10 kDa, and the concentration is 20-25%.

3. The multifunctional cosmetic needle according to claim 1, characterized in that: The chitosan has a deacetylation degree of ≥95%, a viscosity of 100-200 mPa·s, a concentration of 1-3%, and is solubilized with acetic acid.

4. The multifunctional cosmetic needle according to claim 1, characterized in that: The concentration of epigallocatechin gallate is ≥75 μg / mL.

5. A method for preparing the multifunctional cosmetic microneedle according to any one of claims 1 to 4, characterized in that: The process includes: S1. Extract and purify exosomes from human adipose-derived mesenchymal stem cells; S2, dissolving hyaluronic acid powder and epigallocatechin gallate in water to prepare hyaluronic acid stock solution and epigallocatechin gallate solution respectively; S3, dissolving chitosan with acetic acid in water to prepare a chitosan stock solution; S4, accurately weighing β-sodium glycerophosphate powder and dissolving it in water to prepare a β-GP solution, and then slowly adding the solution to the chitosan stock solution prepared in step S3, continuously stirring and cross-linking to obtain a CS / β-GP thermosensitive hydrogel; S5, placing the CS / β-GP thermosensitive hydrogel prepared in step S4 in an ice water bath, and then dropping the exosomes prepared in step S1 and the epigallocatechin gallate solution prepared in step S2 thereinto, mixing well, to obtain a mixture I; S6. Inject the mixture I described in S5 into the microneedle mold, vacuum treat it, and then incubate it in an oven for cross-linking. On this basis, add the hyaluronic acid stock solution prepared in step S2, dry and demould to obtain.

6. The preparation method according to claim 5, characterized in that: The acetic acid concentration in step S3 is 0.05-0.15M.

7. The preparation method according to claim 5, characterized in that: The incubation condition in the oven described in step S6 is incubating at 35-37° C. for 2-3 hours; the microneedle mold is a PDMS microneedle mold, and the needle body is cylindrical.

8. Use of the multifunctional cosmetic microneedle as claimed in claim 1 in preparing skin care products for improving skin photoaging.

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