CN112336749A

CN112336749A - Stem cell exosome microneedle patch for removing freckles and wrinkles and preparation method thereof

Info

Publication number: CN112336749A
Application number: CN202011148113.8A
Authority: CN
Inventors: 唐忆琳; 华佩君; 陈巧林
Original assignee: Zhongke Cell Technology Guangzhou Co ltd
Current assignee: Zhongke Cell Technology Guangzhou Co ltd
Priority date: 2020-10-23
Filing date: 2020-10-23
Publication date: 2021-02-09
Anticipated expiration: 2040-10-23
Also published as: CN112336749B

Abstract

The invention discloses a stem cell exosome microneedle patch for removing freckles and wrinkles, which comprises microneedles and a waterproof backing layer for fixing the microneedles; the microneedle is obtained by solidifying a needle tip solution and a needle body solution; the needle point solution comprises stem cell exosome, astaxanthin, trehalose, chitosan, bovine serum albumin and phosphate buffer solution; the needle solution comprises PVP K30, PEG400, squalane and sterile water. The microneedle patch disclosed by the invention can penetrate the stratum corneum barrier of the skin, provides a novel administration way for macromolecular drugs, and solves the problem of transdermal absorption of exosome repair liquid; the micro-needle patch can slowly release the exosome repairing liquid, so that the absorption rate of an organism is improved; the microneedle patch has good puncture property, solubility and biological activity, is simple to operate, is easy to store and can be produced in a large scale.

Description

Stem cell exosome microneedle patch for removing freckles and wrinkles and preparation method thereof

Technical Field

The invention relates to the field of stem cell microneedle cosmetology, in particular to a stem cell exosome microneedle patch for removing freckles and wrinkles and a preparation method thereof.

Background

Facial anti-aging is an increasingly hot subject of cosmetic surgery at present. The appearance of wrinkles and color spots is the most direct embodiment of facial aging, and therefore, wrinkle removal and freckle removal are also topics which are more and more concerned. At present, medicinal cosmetology and surgical cosmetology are temporary measures for treating symptoms but not root causes, and have the problems of high risk, complications, side effects, pain, bruise and the like. The microneedle injection technology is favored by the mass beauty lovers because of the advantages of minimal invasion, no pain, quick recovery and good effect.

The skin, particularly the stratum corneum, has a barrier effect on the transdermal absorption of the drugs and can block the absorption of the drugs, the micro-needles can pierce the stratum corneum barrier of the skin to generate micro-channels to increase the permeation of the drugs, and the drugs permeate into a capillary vascular network of the dermis layer of the skin through the pores of the micro-needles and directly reach a treatment target position along with blood circulation, so that the purpose of sufficient and rapid drug delivery can be achieved, the dosage of the drugs is greatly reduced, and the curative effect of the drugs is improved. Because the micro-needle is thin and sharp, the general puncture depth is only in the stratum corneum and the upper layer of the dermis, and the micro-needle does not contact nerve endings, thereby reducing the damage degree to the organism tissue, simultaneously stimulating the dermis and promoting the collagen hyperplasia through the self-healing capability of the skin.

Mesenchymal Stem Cells (MSCs) are important members of the stem cell family, are derived from the mesoderm, and belong to pluripotent stem cells. MSCs secrete a variety of growth factors (e.g., epidermal growth factor, vascular endothelial growth factor, collagen, fibronectin, etc.) and inflammatory factors (e.g., interferon gamma, interleukin 9, tumor necrosis factor alpha, etc.), and have significant anti-aging effects in inhibiting skin aging, whitening skin, assisting fat transplantation, promoting hair regeneration, etc. In recent years, with the research and application of MSCs becoming more and more intensive, the function of the exosomes of mesenchymal stem cells is gradually becoming a new research hotspot. The exosome is a nano-scale lipid bilayer membrane vesicle released into an extracellular environment after fusion of a multivesicular endosome of a eukaryotic cell and a cell membrane, can be secreted by various cells, contains different types of substances with biological activity, such as protein, lipid, mRNA, microRNA, micromolecule and the like, and delivers the active substances to a receptor cell after fusion with the cell membrane of an adjacent cell, thereby realizing intercellular information transfer. Clinically, the stem cell exosomes are mainly used for treating burns, scalds and skin ulcers and regenerating healthy skin; the skin care product is mainly used for repairing the skin which is damaged by trauma and is aged and poisoned, comprehensively conditioning the skin, improving the skin quality and restoring the skin to a young and healthy state. However, when the stem cell exosomes are directly smeared on the surface of the skin, most exosomes cannot enter target cells in the inner layer of the skin due to the blocking effect of the horny layer of the surface skin, but once the skin is broken by a micro-needle mediated method, the exosomes can directly reach the muscle bottom cells along a cell channel through micro-needle pores, and the skin-care and skin-nourishing effects of the exosomes are achieved. Therefore, the micro-needle cosmetic technology combined with the stem cell exosome can effectively prevent and resist aging.

Microneedle cosmetics are popular due to their advantages of high efficiency, safety, easy operation, and quick recovery, and are highly popular with beauty-seeking people. Most microneedle patches in the market at present directly adopt stem cells or growth factors as material liquid, have single active ingredients, low individual pertinence, poor product effect and serious product homogenization. The stem cell exosome can provide substances required by skin growth, improvement and repair, and activate a repair system of a human body. Therefore, the development of the stem cell exosome microneedle patch which can efficiently remove freckles and wrinkles and has the effects of preventing and resisting aging has important market application value in the field of beauty.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, comprehensively utilizes the stem cell exosome transfer and microneedle transdermal patch administration technology and provides a stem cell exosome microneedle patch for resisting wrinkles, removing freckles and beautifying. The invention can increase the conveying efficiency of the exosome by utilizing the micro-needle transdermal patch technology, directly reaches the part of the skin needing beauty, and can directly convey the effective components to the required skin tissue in a fixed-point, fixed-layer and quantitative manner.

The first purpose of the invention is to provide a stem cell exosome microneedle patch for removing freckles and wrinkles.

The second purpose of the invention is to provide a preparation method of the stem cell exosome microneedle patch for removing freckles and wrinkles.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention claims a stem cell exosome microneedle patch for removing freckles and wrinkles, which comprises microneedles and a waterproof backing layer for fixing the microneedles; the micro-needle is obtained by solidifying a needle point solution and a needle body solution, the needle point solution is solidified to obtain a needle tip part of the micro-needle, and the needle body solution is solidified to obtain a needle body part of the micro-needle;

the needle tip solution comprises stem cell exosomes, astaxanthin, trehalose, chitosan, bovine serum albumin and a phosphate buffer solution;

the needle solution comprises PVP K30, PEG400, squalane and sterile water.

Preferably, the volume ratio of the needle tip solution to the needle body solution is 1-3: 1 to 5.

Preferably, the volume ratio of the dosage of the needle tip solution to the dosage of the needle body solution is 1: 4.

preferably, the concentration of each component of the needle tip solution is: stem cell exosomes 1 x 10¹⁴～5×10¹⁴L, astaxanthin 10-20 g/L, trehalose 20-60 g/L, chitosan 5-10g/L, bovine serum albumin 2-3 g/L, and phosphate buffer 0.01-0.02 mol/L.

Preferably, the stem cell exosomes are umbilical cord mesenchymal stem cell exosomes.

More preferably, the mesenchymal stem cell exosome is obtained by culturing mesenchymal stem cells of P5-P7 generation tissues.

Preferably, the Ph of the phosphate buffer is 7.2.

Preferably, the concentration of each component of the needle body solution is as follows: PVP K301000-3000 g/L, PEG 40010-30 g/L, squalane 15-25 g/L and sterile water. .

More preferably, the needle solution is formulated from 1500g/L PVP K30, 30g/LPEG400, 20g/L squalane and 500 μ L sterile water.

Preferably, the waterproof backing layer comprises a waterproof layer and a backing layer.

Preferably, the backing layer is prepared from a backing layer composition comprising polyvinyl alcohol, sodium carboxymethyl cellulose, glycerol and water.

More preferably, the formulation of the backing layer composition is: 4-8 parts of film material polyvinyl alcohol, 2-4 parts of thickener sodium carboxymethyl cellulose, 2-8 parts of plasticizer glycerol and 100 parts of water.

More preferably, the backing layer is prepared by the following method: adding the polyvinyl alcohol, the glycerol and the purified water into a beaker, stirring at a constant temperature of 80 ℃, keeping the temperature for 5min to remove partial bubbles after completely dissolving and uniformly mixing, centrifuging at 3000r/min for 4-10 min, pouring the mixture on a 75mm glass plate after completely removing bubbles, quickly leveling the mixture by using a 2 mm-specification film coater, and horizontally placing the glass plate in a 40 ℃ oven for drying for 0.5 h.

Preferably, the waterproof layer is prepared from an ethanol solution of ethyl cellulose.

More preferably, the preparation method of the waterproof layer specifically comprises the following steps: an ethyl cellulose solution (10mL of absolute ethanol, 1g of ethyl cellulose, 10mL in total) was prepared at a concentration of 10% (w/v) using ethanol as a solvent. The glass was poured onto the glass of the backing layer, dried in an oven at 40 ℃ for 1h horizontally, and after the film had been shaped, peeled off the glass plate.

The invention also claims a preparation method of the stem cell exosome microneedle patch, which comprises the following steps:

s1, adding the needle point solution into a groove of a mold by adopting a vacuum filling method, drying, filling the needle point solution into the needle-shaped groove of the mold, and pressurizing;

s2, sticking a waterproof backing layer on the surface of the microneedle mould with the waterproof layer facing upwards to enable the backing layer to be tightly attached to the tip part of the microneedle;

and S3, drying and demolding to obtain the product.

Preferably, the vacuum filling method in step S1 is to continuously evacuate for 1h by using a vacuum pump.

Preferably, the drying in step S1 is horizontal drying for 10 min.

Preferably, the pressurizing in step S1 is performed by a simple pressurizing operation using a syringe.

Preferably, the drying and demolding method of the step S3 is to demold the whole system after drying for 24h at 20 ℃ to constant weight.

Preferably, the side length of the microneedle patch is 2-50 mm square, 1-1000 microneedles are used in total, and the distance between every two adjacent microneedles is 0.5-4 mm.

Most preferably, the patch comprises a microneedle and a waterproof backing layer for fixing the microneedle; the micro-needle is obtained by solidifying a needle point solution and a needle body solution, the needle point solution is solidified to obtain a needle tip part of the micro-needle, and the needle body solution is solidified to obtain a needle body part of the micro-needle; the side length is 2-50 mm, 1-1000 microneedles are totally arranged, and the distance between every two adjacent microneedles is 0.5-4 mm.

Therefore, the invention claims any one of the stem cell exosome microneedle patches and a preparation method thereof.

Compared with the prior art, the invention has the following beneficial effects:

1. the stem cell exosome contained in the stem cell exosome microneedle patch has the functions of removing color spots and wrinkles, can promote fibroblast metabolism and collagen formation, induces and activates immune cells through inflammatory factors, reduces the residues of pigments and dead cells and the like, and accumulates and secretes synthetic collagen fibers, so that the wrinkles are reduced and eliminated, the color spots and the pigmentation are eliminated, and the skin is tender, white and pure.

2. The astaxanthin contained in the needle point solution of the stem cell exosome microneedle patch has an antioxidation effect, has fat solubility and water solubility, is easy to be absorbed by a human body, has the antioxidation capability 500-1000 times that of vitamin E, can delay skin aging, and has the main action mechanism that: (1) can directly remove peroxide and free radicals, and has antioxidant effect; (2) inhibiting lipid peroxide generation, removing chloasma, inhibiting tyrosinase activity, and reducing melanin; (3) protect the skin from ultraviolet rays and pollution, and reduce the deposition of scars and pigments.

3. The tip solution of the stem cell exosome microneedle patch also contains trehalose, chitosan, bovine serum albumin and a phosphate buffer solution, wherein the trehalose can form a protective film on the surface of exosome to prevent the inactivation of protein on the surface of exosome, and the chitosan has an antiseptic property; bovine serum albumin can provide a proper protein concentration environment for exosome, so that the exosome can keep biological activity for a long time; the phosphate buffer solution can adjust the osmotic pressure and the ionic strength of the exosome preservation solution.

4. The microneedle patch can penetrate the stratum corneum barrier of the skin, provides a novel administration way for macromolecular drugs, and solves the problem of transdermal absorption of exosome repair liquid; the micro-needle patch can slowly release the exosome repairing liquid, so that the absorption rate of an organism is improved; the microneedle patch has good puncture property, solubility and biological activity, is simple to operate, is easy to store and can be produced in a large scale.

Drawings

Fig. 1 is a structural diagram of a stem cell exosome microneedle patch.

Figure 2 is umbilical cord mesenchymal stem cell morphology (40 ×).

FIG. 3 is the surface marker of the mesenchymal stem cell expressed by the separated umbilical cord mesenchymal stem cell, and the pictures A-D are respectively the control group CD73⁺、CD90⁺、CD105⁺Expression of surface markers and CD45⁺、CD34⁺、CD11b⁺、CD19⁺And HLA-DR⁺Negative expression of (a); expression of surface markers and CD45⁺、CD34⁺、CD11b⁺、CD19⁺And HLA-DR⁺Negative expression of (a); FIGS. E-H are the experimental group CD73, respectively⁺、CD90⁺、CD105⁺Expression of surface markers and CD45⁺、CD34⁺、CD11b⁺、CD19⁺And HLA-DR⁺Negative expression of (4).

FIG. 4 shows the expression of exosome CD63 and TSG101 signature proteins.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 preparation of Stem cell exosomes

1. Umbilical cord mesenchymal stem cell isolated culture

Taking a newborn umbilical cord with the length of 15-20cm, and requiring a healthy full-term cesarean delivery fetus in obstetrics and gynecology department, wherein the pregnant woman has negative detection of HBV antigen, anti-HCV antibody, anti-HIV antibody, anti-treponema pallidum antibody, mycoplasma, anti-cytomegalovirus antibody and the like. Treating for 6h, washing with PBS under aseptic condition until no blood stain exists, longitudinally cutting, removing blood vessel, cutting into tissue blocks with size of 1mm × 1mm × 1mm, adding 0.5ml umbilical cord tissue suspension into 75cm2 culture flask, adding 10ml serum-free complete culture medium, and culturing at 37 deg.C in 5% CO2 culture box. Half the liquid change on day 3 and full liquid change on day 7, observed under an inverted microscope, when the cells grow to 80% confluence, digested with 0.05% trypsin, and mixed according to a ratio of 1: and 5 passages, and carrying out exosome culture on P5-P7 generation cells with good growth state.

2. Exosome culture

Mesenchymal stem cells of generation P5-P7Inoculating after trypsinization, wherein the culture medium is DMEM culture medium containing 10% (v/v) fetal bovine serum, and placing at 37 ℃ and CO after inoculation₂Culturing in an incubator with the volume concentration ratio of 5%, when the fusion degree reaches 80-90%, washing by using PBS, continuously culturing for 72 hours by using a DMEM (DMEM) culture medium without phenol red and serum, and collecting the culture medium supernatant for later use;

3. exosome collection

Centrifuging the culture medium supernatant at 300 × g for 10min and 2000 × g for 20min, filtering with 0.45 μm filter to remove cell debris, collecting supernatant, and centrifuging at 100000 × g for 90min at ultra high speed at 4 deg.C; discarding the supernatant, taking the precipitate, resuspending with PBS to remove macromolecular protein complex, repeating the centrifugation at ultra high speed for 1 time, discarding the supernatant, collecting the exosome, and storing in a refrigerator at-80 ℃ for later use.

Example 2 identification of Stem cells and detection of exosomes

1. Identification of umbilical cord mesenchymal stem cells

(1) Taking 1 bottle of the umbilical cord mesenchymal stem cells (the number of the cells is 1 multiplied by 10) with the confluence degree of 80 to 90 percent of the T175 bottle⁷Respectively), digesting with 5ml of 0.05% pancreatin for 3-5 min; when most of cells are shrunk and rounded under a microscope, adding 2mL of serum-containing culture medium to terminate digestion, blowing and beating the cells, transferring the cells into a 15mL centrifuge tube, and centrifuging at 800rpm for 5 min;

(2) cell preparation: discarding the supernatant, adding 2mL of PBS to suspend the precipitate, and counting the cells; according to 1X 10 of each tube⁵Evenly distributing the cell suspension into 1.5mL Ep tubes, and centrifuging at 800rpm for 5 min; discarding the supernatant, adding 100 μ L PBS into the precipitate, and suspending;

(3) antibody incubation: adding 1 mu L of corresponding flow type antibody into each tube, incubating for 40min in the dark, washing by PBS after the incubation is finished, centrifuging, finally adding 300 mu LPBS suspension cells, taking the human umbilical cord mesenchymal stem cells without any antibody as negative control, and carrying out cell flow type identification on the sample by using a flow cytometer.

(4) Results are shown in FIGS. 2 and 3: the stem cells isolated from umbilical cord are polygonal or fusiform, and the cells have strong refractivity (figure 1); through flow identification, the separated cells strongly express the surface markers of CD29+, CD73+, CD90+ and negatively express CD45R + and HLA-DR + (figure 2), have the characteristics of composite mesenchymal stem cells, and confirm that the cells separated from the umbilical cord are umbilical cord mesenchymal stem cells.

2. Exosome detection

(1) Detecting the expression of the exosome characteristic protein by using an exosome CD63& TSG101 protein detection kit (comprising exosome protein special lysis solution, a CD63 antibody, a Tsg101 antibody and Goat anti-Mo mu se IgG antibody (HRP)), collecting cell supernatants and exosome samples, lysing the samples by using the exosome protein special lysis solution, centrifuging for 5min at 12000g at 4 ℃, and taking the supernatants. SDS-PAGE gels (10% separation gels) were prepared and the protein concentration of each sample was determined. After electrohydraulic and conjunctival occlusion, the cells were incubated with CD63 and TSG101 primary antibody, respectively, overnight at 4 ℃. The next day, the membrane was reacted with HRP-labeled secondary antibody at room temperature for 50min, and then a chemiluminescent substrate was added for color development and observation.

(2) Results as shown in fig. 4, the sample was able to express exosome CD63 and TSG101 characteristic proteins, with exosome characteristics, confirming that the collected sample was mesenchymal stem cell exosomes.

Example 3 preparation of Stem cell exosome microneedle Patches

1. Preparation of needle tip solution (exosome repairing liquid)

(1) Consists of the following components:

exosome (2.5X 10)¹⁴/L)；

Astaxanthin (15 g/L);

trehalose (40 g/L);

chitosan (7.5 g/L);

bovine serum albumin (2 g/L);

the pH was adjusted to 7.2 with phosphate buffer (0.015 mol/L).

(2) And uniformly mixing the solution prepared in the step according to a formula to prepare an exosome repairing solution, and filtering by using a 500-mesh cell screen to obtain a microneedle tip solution of the microneedle.

2. Preparation of needle body solution

The needle body solution is prepared from PVP K30(2000g/L), PEG400(20g/L), squalane (20g/L) and 500 mu L of sterile water.

3. Preparation of a waterproof backing layer

(1) The backing layer comprises the following components in percentage by weight: 4-8 parts of film material polyvinyl alcohol, 2-4 parts of thickener sodium carboxymethyl cellulose, 2-8 parts of plasticizer glycerol and 100 parts of water.

(2) Preparation of the backing layer: adding PVA, glycerol and purified water into a beaker, stirring at a constant temperature of 80 ℃, preserving heat and standing for 5min to remove partial bubbles after completely dissolving and uniformly mixing, centrifuging for 4-10 min at 3000r/min, pouring onto a 75mm glass plate after completely removing bubbles, and quickly scraping by using a 2 mm-sized film coating device; the glass plate was horizontally placed in an oven at 40 ℃ for drying for 0.5 h.

(3) Preparing a waterproof layer: a10% (w/v) ethyl cellulose solution (10mL of absolute ethanol, 1g of ethyl cellulose, 10mL in total) was prepared using ethanol as a solvent, poured onto glass of the backing layer, dried in an oven horizontally at 40 ℃ for 1 hour, and peeled from the glass plate after film formation.

4. Preparation of anti-wrinkle freckle-removing stem cell microneedle patch

(1) The solution is fully filled into a microneedle mould made of PTFE material by adopting a vacuum filling method, wherein the microneedle mould is a square with the side length of 2-50 mm and the inclusion density of 15 multiplied by 15/cm²Needle-shaped groove with depth of 1mm and bottom diameter of 300 μm), adding 30 μ L of needle tip solution into the groove of the mold, standing and drying for 10 min;

(2) adding 120 mu L of needle body solution, and continuously vacuumizing for 1h by using a vacuum pump to enable the needle body solution to enter and fill the needle-shaped grooves of the mold;

(3) cutting a proper waterproof backing layer (the waterproof layer is upward) on the surface of the mould to enable the backing layer to be tightly attached to the needle tip part; one side of the obtained waterproof layer can be waterproof.

(4) And drying the whole system at 20 ℃ for 24h to constant weight, and then demoulding to obtain the complete microneedle patch.

As shown in figure 1, the prepared microneedle patch is square with the side length of 2-50 mm, 1-1000 microneedles are arranged in total, and the distance between every two adjacent microneedles is 0.5-4 mm.

Example 4 test effect of microneedle patch facial skin treatment

Firstly, testing steps

1. 60 volunteers were recruited to test for wrinkles and spots on the facial skin due to skin inflammation, aging or scarring. The volunteers are prohibited from eating spicy and irritating foods, quitting smoking and abstaining from alcohol in the clinical observation period. The volunteers did not use hormone drugs and antiallergic drugs and auxiliary cosmetics from 2 weeks before clinic. Volunteers volunteer to participate in the experiment and sign informed consent.

The 60 volunteers were divided on average into three groups: the group A is a group which uses the stem cell exosome alone, the group B is a group which uses the stem cell exosome repairing liquid, and the group C is a group which uses the stem cell exosome microneedle patch. Group a, male 3, female 7, mean age (37.87 ± 7.21); group B males 4, females 6, mean age (38.13 ± 8.34); group C men 3, women 7, mean age (39.25 ± 8.09).

2. Application method

(1) Methods of using the stem cell exosomes alone: the prepared stem cell exosome is smeared on the positions with sunken wrinkles at nasolabial folds, raised lines, glabellar lines and crow's feet and (or) the positions with spots at the cheek and forehead, and one time per day is taken, and 30 times are continuously taken as a treatment course.

(2) The application method of the stem cell exosome repairing liquid comprises the following steps: the prepared stem cell exosome restoration solution (needle tip solution) is applied to the positions with sunken wrinkles at nasolabial folds, raised lines, glabellar lines and fishtail lines and/or the positions with spots at the cheek parts and the forehead parts, once a day, and 30 times are continuously taken as a treatment course.

(3) The use method of the stem cell exosome microneedle patch comprises the following steps: the prepared stem cell exosome micro-needle patch is attached to a part with sunken wrinkles at the positions of nasolabial folds, raised lines, glabellar lines and fishtail lines and/or a part with spots at the cheek part and the forehead part. The patch is applied once a day for 30 times as a treatment course.

3. The test method comprises the following steps: the VISIA skin tester consists of 3 light sources of natural light, polarized light and ultraviolet light, quantitatively analyzes skin aging indexes such as textures, pores, spots and wrinkles by using a Mirror medical imaging system, a circulating facial photography technology and a small magnifier tool, and accurately displays the improvement progress by using numerical values. VISIA skin tests were performed on the patient's face before and after treatment, and the patient's skin was examined for pores, spots and wrinkles and accurately recorded for characteristic counts.

4. The satisfaction degrees of three groups of doctors and doctors are evaluated by adopting self-made questionnaires respectively, the doctors answer questions according to self conditions, the higher the score is, the higher the satisfaction degree is, and the total score is 10;

second, test results

1. The facial pore profile of the patient's skin, the results are shown in table 1:

TABLE 1 comparison of three groups of pore facial VISIA skin test results before and after treatment

The result shows that the pores of the microneedle patch group for the stem cell exosomes are obviously improved after treatment, and the curative effect is superior to that of the group A using the stem cell exosomes alone and that of the group B using the stem cell exosome repairing liquid.

2. Facial speckle condition of the patient's skin, results are shown in table 2:

TABLE 2 comparison of three groups of Spot facial VISIA skin test results before and after treatment

The result shows that spots are obviously improved after the stem cell exosome micro-needle patch group is treated, and the curative effect is superior to that of the group A using the stem cell exosome alone and that of the group B using the stem cell exosome repairing liquid.

3. The facial wrinkle condition of the patient's skin, the results are shown in table 3:

TABLE 3 comparison of three groups of facial VISIA skin test results for treatment of wrinkles before and after treatment

The results show that the wrinkles are obviously improved after the stem cell exosome microneedle patch group is used for treating, and the curative effect is superior to that of the group A using the stem cell exosome alone and that of the group B using the stem cell exosome repairing liquid.

4. The results of sensory evaluation by the patients and the physicians are shown in Table 4:

TABLE 4 sensory evaluation score comparison of three groups of patients and physicians

The results show that the satisfaction of the stem cell exosome microneedle patch group for the doctor and the doctor is higher than that of the group A using the stem cell exosome alone and that of the group B using the stem cell exosome repairing liquid.

In conclusion, the stem cell exosome microneedle patch has an obvious effect in facial rejuvenation treatment, can obviously improve the skin aging symptoms of the patients, improves the postoperative satisfaction degree, and is worthy of wide popularization and application.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A stem cell exosome microneedle patch for removing freckles and wrinkles is characterized by comprising microneedles and a waterproof backing layer for fixing the microneedles; the micro-needle is obtained by solidifying a needle point solution and a needle body solution, the needle point solution is solidified to obtain a needle tip part of the micro-needle, and the needle body solution is solidified to obtain a needle body part of the micro-needle;

the needle solution comprises PVP K30, PEG400, squalane and sterile water.

2. The stem cell exosome microneedle patch according to claim 1, wherein the volume ratio of the needle tip solution to the needle body solution is 1-3: 1 to 5.

3. The stem cell exosome microneedle patch according to claim 2, wherein the volume ratio of the needle tip solution to the needle body solution is 1: 4.

4. the stem cell exosome microneedle patch according to claim 1, wherein the concentration of each component of the needle tip solution is: stem cell exosomes 1 x 10¹⁴～5×10¹⁴The anti-aging coating comprises the following components, by mass, 10-20 g/L of astaxanthin, 20-60 g/L of trehalose, 5-10 g/L of chitosan, 2-3 g/L of bovine serum albumin and 0.01-0.02 mol/L of phosphate buffer solution.

5. A stem cell exosome microneedle patch according to any one of claim 1 or claim 2, wherein the stem cell exosomes are umbilical cord mesenchymal stem cell exosomes.

6. The stem cell exosome microneedle patch according to claim 1, wherein the concentration of each component of the needle body solution is: PVP K301000-3000 g/L, PEG 40010-30 g/L, squalane 15-25 g/L and sterile water.

7. The stem cell exosome microneedle patch according to claim 1, wherein said water-proof backing layer comprises a backing layer and a water-proof layer.

8. A stem cell exosome microneedle patch according to claim 7, wherein the backing layer is prepared from a backing layer composition comprising polyvinyl alcohol, sodium carboxymethyl cellulose, glycerol and water.

9. The stem cell exosome microneedle patch according to claim 7, wherein the water-repellent layer is prepared from an ethanol solution of ethyl cellulose.

10. A method for preparing a stem cell exosome microneedle patch according to any one of claims 1 to 9, characterized by comprising the steps of:

and S3, drying and demolding to obtain the product.