Soluble microneedle loaded with artificial platelet exosome and polyphenol as well as preparation method and application of soluble microneedle

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a soluble microneedle loaded with an artificial platelet exosome and polyphenol as well as a preparation method and application of the soluble microneedle. The microneedle comprises a back lining loaded with polyphenol and a needle body which is positioned on the back lining and is formed by compounding a soluble high-molecular polymer with an artificial platelet exosome and a protein protective agent. The soluble microneedle loaded with the artificial platelet exosome and the polyphenol can be used in the fields of alopecia, wound healing and the like, and growth factors rich in the platelet exosome can promote hair follicle regeneration, wound healing and the like. Artificial platelet exosome, polyphenol and a high-molecular polymer are compounded to prepare the soluble microneedle with enough hardness, the soluble microneedle can effectively pierce the cuticle of the skin surface layer, the needle body entering the skin can achieve rapid dissolution, active substances such as growth factors are guided into the dermis layer of the skin through the microneedle, and the microneedle is good in absorption effect, safe, reliable and free of side effects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a soluble microneedle loaded with artificial platelet exosomes and polyphenols, and a preparation method and application thereof. Background Art

[0002] Exosomes are nanoscale membranous vesicles actively secreted by cells to the extracellular space. They are a medium for cell-to-cell communication, transporting active substances between nearby cells and distant tissue cells through body fluids, and participating in processes such as regulating protein expression, tissue regeneration, and cell communication. Different molecules contained in them play different roles, including promoting cell proliferation and migration, reducing oxidative stress, etc., and play an important role in all stages of wound healing. Platelet exosomes are nanoscale vesicles with a diameter of about 30 - 150 nm, which are rich in various growth factors including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor (IGF), etc. In addition to growth factors, exosomes also contain substances such as proteins, lipids, and mRNA. Due to their procoagulant activity and effects such as hemostasis, angiogenesis, immune regulation, and promoting cell proliferation, they have great efficacy in tissue regeneration, but natural platelet exosomes are not easily obtained. Natural polyphenols have anti-inflammatory, antioxidant, antibacterial, angiogenic, and antifibrotic effects. They can reduce the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors in lipopolysaccharide-induced macrophages in vitro, and induce macrophages to polarize towards an anti-inflammatory phenotype. Therefore, polyphenols can be used to improve the local inflammatory response, angiogenesis defects, and up-regulation of pro-inflammatory cytokine expression caused by scald wounds.

[0003] Microneedles are needle-like protrusions with micron-scale dimensions (usually in the length range of 25 μm - 2000 μm), which are typically assembled onto a support substrate or patch in an array form. Microneedles can pierce the skin epidermis without causing significant bleeding or pain, and can be applied in the fields of transdermal drug delivery, cell delivery, or biosensors. The concept of microneedles was first proposed in the 1970s and was studied and demonstrated in detail in the 1990s. In the past three decades, according to literature reports, a large number of materials have been proven to be applicable to the preparation of microneedles or microneedle arrays, including silicon materials, metal materials, organic materials, polymer materials, ceramics, or glass, etc. Among them, polymer microneedles are widely used due to their advantages such as simple preparation, easy mass production, and good biocompatibility. Polymer microneedle materials can be classified into two categories according to whether they are water-soluble; the first is soluble microneedles, and the preparation materials are water-soluble polymer materials such as polyvinyl alcohol, chitosan, hyaluronic acid, etc. The other is polymer solid microneedles, which usually do not contain drugs and cannot be dissolved subcutaneously. Microneedle drug delivery is a new concept and method of transdermal drug delivery. Since microneedles deliver drugs between the epidermis and the dermis and do not touch the nervous system, patients will not feel pain or discomfort, which increases patient compliance. After the microneedle patch is applied to the skin surface, the drug will be slowly released into the body, without causing the phenomenon of excessive local drug concentration. Using microneedle patches for transdermal drug delivery can achieve environmental protection and low cost. Summary of the Invention

[0004] In view of the problems existing in the current prior art, the present invention provides a complex of artificial platelet-derived exosomes (aPexos) and polyphenols and its application, as well as a soluble microneedle loaded with artificial platelet-derived exosomes and polyphenols and its preparation method. The soluble microneedles loaded with artificial platelet-derived exosomes and polyphenols (i.e., soluble microneedles complexed with aPexos and polyphenols) prepared by this method have good mechanical strength, can pierce the skin barrier, and enter the dermis of the mouse skin. The growth factors rich in aPexos and the anti-inflammatory and antioxidant properties of polyphenols have a good effect on the wound healing of scalded rats.

[0005] In order to achieve the above invention purposes, the specific technical solutions of the present invention are as follows:

[0006] One object of the present invention is to provide an application of a complex of artificial platelet-derived exosomes and polyphenols in the preparation of microneedles for wound healing and / or hair loss treatment.

[0007] Furthermore, in the above application, the wound is various chronic or difficult-to-heal wounds, such as scald wounds, diabetic wounds, etc.

[0008] The second object of the present invention is to provide a soluble microneedle loaded with artificial platelet exosomes and polyphenols. The microneedle includes a backing (with an addition amount of 300 - 750 mg) loaded with polyphenols (with an addition amount of 1 - 5 mg), and a needle body located on the backing, which is formed by a soluble polymer (with an addition amount of 10 - 50 mg) compounded with aPexos (with an addition amount of 0.1 - 0.5 mL) and a protein protectant (with an addition amount of 1 - 5 mg).

[0009] Preferably, in the soluble microneedle, the backing (material) loaded with polyphenols is selected from any one or several of gelatin, carboxymethyl chitosan, polyvinyl alcohol, polyvinylpyrrolidone, sodium hyaluronate with a molecular weight of 10K - 1000KD, and carboxymethyl cellulose; the polyphenol is a natural polyphenol, such as tea polyphenols.

[0010] Preferably, in the soluble microneedle, the soluble polymer is selected from any one or several mixtures of sodium hyaluronate with a molecular weight of 10K - 1000KD, chitosan, polyvinylpyrrolidone, and polyvinyl alcohol.

[0011] Preferably, in the soluble microneedle, the protein protectant is selected from any one or several mixtures of sucrose, trehalose, mannitol, lactose, and glucose.

[0012] The third object of the present invention is to protect the preparation method of the above-mentioned soluble microneedle loaded with artificial platelet exosomes and polyphenols, which includes the following steps:

[0013] S1. Preparation of artificial platelet exosomes (aPexos):

[0014] Take concentrated platelets, ultrasonicate them with an ultrasonic cell disruptor and then perform gradient centrifugation to obtain artificial platelet exosomes;

[0015] S2. Preparation of the needle body: Dissolve the soluble polymer in normal saline or absolute ethanol, then add the protein protectant thereto, and after dissolution, add artificial platelet exosomes to obtain the needle body;

[0016] S3. Preparation of the backing: Add the material used for the backing to normal saline or absolute ethanol, heat it in a water bath to form a mixed solution; dissolve the polyphenol in deionized water, and after complete dissolution, obtain a polyphenol solution; mix the mixed solution and the polyphenol solution to obtain a backing solution;

[0017] S4. Pour the needles obtained in S2 into the microneedle mold, evacuate to vacuum and maintain for a period of time at room temperature. Take out the mold and scrape off the excess needles on the upper layer. Then place the mold in a dryer for drying. After drying, pour in the backing solution, and finally place the mold in the dryer to dry overnight until it naturally demolds to obtain soluble microneedles loaded with artificial platelet exosomes and polyphenols.

[0018] Preferably, in S1 of the preparation method of the soluble microneedles, the power of the ultrasonic cell disruptor is 30%, 10 s on, 10 s off, and the ultrasonic time is 1 min 20 s.

[0019] The specific operation of gradient centrifugation is as follows: Centrifuge at 3000 g for 20 minutes at 4 °C, take the supernatant; centrifuge at 20000 g for 20 min at 4 °C, discard the supernatant; resuspend the precipitate with 2 wt% trehalose PBS solution and store at -40 °C for later use.

[0020] Preferably, in S2 of the preparation method of the soluble microneedles, the final concentration of the soluble high molecular polymer dissolved in physiological saline or absolute ethanol is 10 - 50 mg / mL; the temperature of water bath heating is 37 °C; the addition amount of aPexos is 0.1 - 0.5 mL.

[0021] Preferably, in S3 of the preparation method of the soluble microneedles, add the material used for the backing into physiological saline or absolute ethanol, and the temperature of water bath heating is 55 °C; the added polyphenol solution is 2 - 10 mg of polyphenol dissolved in 0.1 mL of deionized water, and the addition amount of polyphenol is 2 - 10 mg.

[0022] More preferably, S3. The preparation steps of the backing material are as follows: Add 100 - 250 mg of gelatin and 200 - 500 mg of carboxymethyl chitosan powder in a mass ratio of 1:2 to 5 mL of physiological saline, and heat in a water bath to form a mixed solution as the backing solution for blank MN. Dissolve 2 - 10 mg of polyphenol in 0.1 mL of deionized water, and after complete dissolution, add it to 4.9 mL of the blank MN backing solution and mix evenly as the drug-loaded backing solution.

[0023] Preferably, in S4 of the preparation method of the soluble microneedles, evacuate the needles poured into the microneedle mold to -0.07 MPa and maintain at room temperature for 5 - 30 min; the drying time of the mold in the dryer is 2 - 4 h, the temperature in the dryer is maintained at 25 ± 2 °C, and the humidity is maintained at 20%.

[0024] The fourth object of the present invention is to protect the soluble microneedles prepared by any of the above - described methods. The aPexos composite microneedles prepared by the above method have good mechanical strength, can pierce the skin barrier and enter the dermis of the mouse skin. The growth factors and polyphenols rich in aPexos have good anti - inflammatory and antioxidant properties and have a good effect on the wound healing of scalded rats.

[0025] Compared with the existing technologies, the beneficial effects of the present invention are as follows:

[0026] (1). The artificial platelet - derived exosomes prepared by ultrasonic treatment combined with gradient centrifugation have a much higher number per unit volume and protein concentration than natural platelet - derived exosomes. They can be well combined with other raw materials of soluble microneedles to prepare microneedles with good mechanical strength and good wound - healing effect on scalded rats.

[0027] (2). In this application, artificial platelet - derived exosomes, polyphenols and polymer are combined to prepare microneedles. On the one hand, the microneedles have sufficient mechanical strength, and on the other hand, the microneedles are rich in various growth factors and proteins of platelets and have anti - inflammatory and antioxidant properties of polyphenols. Through the evaluation of the wound - healing effect of scalded rats, the platelet - derived exosome - polyphenol composite microneedle patch has a good therapeutic effect on the wounds of scalded rats.

[0028] (3). The soluble microneedles loaded with artificial platelet - derived exosomes and polyphenols of the present invention can be used in the fields of alopecia treatment, wound healing, etc. The growth factors rich in platelet - derived exosomes can promote hair follicle regeneration, wound healing, etc. The soluble microneedles prepared by combining artificial platelet - derived exosomes, polyphenols and polymer with sufficient hardness can effectively pierce the stratum corneum of the skin surface layer. The microneedles entering the skin can be rapidly dissolved, and active substances such as growth factors are introduced into the dermis of the skin through the microneedles, with good absorption effect, safety and reliability, and no side effects. Description of the Drawings

[0029] Figure 1 Transmission electron microscope pictures of aPexos and natural platelet - derived exosomes

[0030] Figure 2 Relative protein levels of natural platelet - derived exosomes and aPexos in the present invention (***P < 0.001)

[0031] Figure 3 Photographs of each group of microneedles under a high - definition microscope

[0032] Figure 4 Mechanical property curves of each group of microneedles

[0033] Figure 5Representative optical images of the wounds of SD rats at different time points;

[0034] Figure 6 For the wound healing of scalded rats, specifically the wound healing rate of SD rats (scale = 1 cm, x±SD, n = 10); *P < 0.05; **P < 0.01; ***P < 0.001;

[0035] Figure 7 For the histopathological results of scalded SD rats in different groups on the 17th day, where A is the HE staining of the wound tissue of SD rats; B is the Masson staining of the wound tissue of SD rats;

[0036] Figure 8 For the wound collagen deposition rate of scalded SD rats in different groups on the 17th day (x±SD, n = 3) *P < 0.05; **P < 0.01; ***P < 0.001. Detailed implementation manners

[0037] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0038] Any feature disclosed in this specification (including the claims and abstract), unless specifically stated, can be replaced by other equivalent or similar-purpose alternative features. That is, unless specifically stated, each feature is only an example of a series of equivalent or similar features.

[0039] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0040] The technologies and characterization means adopted in the embodiments of the present invention include the preparation of artificial platelet exosomes and the detection of their particle size, concentration, and growth factor content, the preparation of artificial platelet exosome and polyphenol composite microneedles, the characterization of the needle body height and morphology, the mechanical property detection, the effect of microneedles on the wound healing of scalded rats, etc.

[0041] The polyphenol used in the following embodiments is epigallocatechin gallate (EGCG).

[0042] Example 1:

[0043] S1. Weigh a certain amount of methacrylated chitosan (CSMA) and dissolve it in physiological saline; successively prepare clear solutions with mass concentrations of 10 mg / mL, 20 mg / mL, 30 mg / mL, and 40 mg / mL; the preparation of the drug-loaded microneedle matrix loaded with artificial platelet exosomes is similar to the above process, except that 100 μL of artificial platelet exosomes is added after the CSMA is dissolved.

[0044] S2. Add gelatin and carboxymethyl chitosan (CMCS) powders into physiological saline at a mass ratio of 1:1, and heat in a water bath at 55 °C to form a mixed solution as the backing solution for blank MNs. Dissolve 2 mg of polyphenols in 0.1 mL of deionized water, and after complete dissolution, add it into 4.9 mL of the blank MN backing solution and mix well as the drug-loaded backing solution.

[0045] S3. Pour 200 μL of the needle body solution obtained in S1 into the microneedle mold, evacuate to -0.07 MPa, maintain for 10 min at room temperature, take out the mold and scrape off the excess needle body material on the upper layer, then place the mold in a dryer and dry for 2 h, with the temperature in the dryer maintained at 25 ± 2 °C and the humidity maintained at 20%;

[0046] S4. After the needle body is dried, pour about 300 μL of the backing solution obtained in S2, and finally place the mold in the dryer and dry overnight until it naturally demolds.

[0047] Example 2 Preparation of Artificial Platelet Exosomes

[0048] Use the concentrated platelets stored for 5 days (prepared by the blood center), ultrasonicate with a 30% power of the ultrasonic cell disruptor (10 s on, 10 s off) for 1 min 20 s, then centrifuge at 3000 g for 20 min at 4 °C, take the supernatant, centrifuge at 20000 g for 20 min at 4 °C, discard the supernatant, resuspend the precipitate with 2 wt% trehalose PBS solution, and store at -40 °C for later use.

[0049] Comparative Example 1: Preparation of Natural Platelet Exosomes

[0050] Use the concentrated platelets stored for 5 days, without using ultrasonication, and the gradient centrifugation steps are the same as in Example 2.

[0051] Example 3 Characterization of Artificial Platelet Exosomes and Natural Platelet Exosomes

[0052] Take 10 μL of the reconstituted artificial platelet exosomes (prepared in Example 2) and natural platelet exosomes (prepared in Comparative Example 1), dilute 200000 times with PBS, and then measure their particle size and concentration using a nanoparticle tracking analyzer (NTA); detect the total protein content of artificial and natural platelet exosomes by the BCA method; detect the contents of human transforming growth factor β1 (TGF-β1), human vascular endothelial growth factor (VEGF), and human platelet-derived growth factor BB (PDGF-BB) in artificial and natural platelet exosomes using an ELISA kit; regard the total protein and growth factor contents of natural exosomes as 1 part, and the ratio of the content of artificial exosomes to that of natural exosomes is the relative protein level; observe the morphology of platelet exosomes using a transmission electron microscope. The morphology of platelet exosomes is as Figure 1As shown, both artificial exosomes and natural exosomes showed a cup-shaped structure under the electron microscope, with a diameter less than 150 nm. Figure 2 For the relative protein levels of natural platelet exosomes and aPexos (***P < 0.001). As shown by Figure 2 Table 1, the average particle size of artificial platelet exosomes was slightly higher than that of natural platelet exosomes, but the concentration of artificial platelet exosomes was much higher than that of natural platelet exosomes, and the total protein and growth factor contents of aPexos were also significantly higher than those of natural platelet exosomes.

[0053] Table 1 Particle size and concentration of artificial platelet exosomes and natural platelet exosomes

[0054] Average particle size (nm) Concentration (per mL) Artificial platelet exosomes 139.4±13.1 <![CDATA[(2.88±0.36)*10 13 > Natural platelet exosomes 114.0±3.7 <![CDATA[(12.48±0.03)*10 11 >

[0055] Example 4 Preparation of soluble microneedles loaded with artificial platelet exosomes (aPexos) combined with polyphenols

[0056] S1. Preparation of the needle body: Weigh 1 mg of the photoinitiator 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (I2959), and then dissolve it in physiological saline containing 0.01 mol / L HCl to obtain a 1% (w / v) I2959 solution, protected from light. Dissolve 35 mg of CSMA in 0.9 mL of the I2959 solution, place it in a water bath at 37 °C to obtain a 3.5% (w / v) CSMA solution, and then add 100 μL of the artificial platelet exosomes prepared in Example 2 to 0.9 mL of the CSMA solution containing I2959 to obtain the needle body solution.

[0057] S2. Preparation of the backing: Dissolve 250 mg of gelatin and 500 mg of CMCS in 5 mL of physiological saline, and heat it in a water bath at 55 °C to form a mixed solution as the backing solution for blank microneedles. Dissolve 2 mg of polyphenols in 0.1 mL of deionized water, and after complete dissolution, add it to 4.9 mL of the blank microneedle backing solution and mix well as the drug-loaded backing solution.

[0058] S3. Pour 50 μL of the needle body material obtained in S1 into the microneedle mold, evacuate to -0.07 MPa, maintain it at room temperature for 10 min, take out the mold and scrape off the excess needle body material on the upper layer, add another 50 μL of the needle body solution, and repeat the operation once. Subsequently, irradiate it with ultraviolet light at 365 nm for 5 min. Then add 250 μL of the backing solution prepared in S2, and finally place the mold in a dryer and dry it overnight until it naturally demolds to obtain the artificial platelet exosome and polyphenol composite microneedles.

[0059] The morphology of the microneedles was observed through a microscope, and the height and width of the needle body were measured. As Figure 3 shown, the artificial platelet exosome-polyphenol composite microneedle array was neat and the needle shape was intact. The height of the needle body was approximately 553.00 ± 5.96 μm.

[0060] Preparation of blank microneedles in Comparative Example 2

[0061] S1. Preparation of the needle body material: Weigh 1 mg of photoinitiator I2959 and dissolve it in physiological saline containing 0.01 mol / L HCl to obtain a 1% (w / v) I2959 solution, protected from light. Dissolve 35 mg of CSMA in 0.9 mL of the I2959 solution and place it in a water bath at 37°C to obtain a CSMA solution with a concentration of 3.5% (w / v), which is the needle body material for the blank microneedles.

[0062] S2. Preparation of the backing material: Dissolve 250 mg of gelatin and 500 mg of CMCS in 5 mL of physiological saline and heat it in a water bath at 55°C to form a mixed solution, which serves as the backing solution for the blank microneedles.

[0063] S3. Pour the needle body material obtained in S1 into the microneedle mold, evacuate to -0.07 MPa, maintain at room temperature for 10 min, take out the mold and scrape off the excess needle body material on the upper layer. Then pour in approximately 250 μL of the backing material obtained in S2 and place the mold in a desiccator to dry overnight.

[0064] Observe the needle body morphology and needle body height using a high-definition microscope, as Figure 3 shown.

[0065] Preparation of artificial platelet exosome microneedles in Comparative Example 3

[0066] S1. The preparation of the needle body material is the same as S1 in Example 4.

[0067] S2. The preparation of the backing material is the same as S2 in Comparative Example 2.

[0068] S3. Pour 50 μL of the needle body material obtained in S1 into the microneedle mold, evacuate to -0.07 MPa, maintain at room temperature for 10 min, take out the mold and scrape off the excess needle body material on the upper layer. Then add another 50 μL of the needle body solution and repeat the operation once. Subsequently, irradiate with ultraviolet light at 365 nm for 5 min. Then add 250 μL of the prepared backing solution in S2, and finally place the mold in a desiccator to dry overnight and wait for it to naturally demold to obtain the artificial platelet exosome microneedles.

[0069] Observe the needle body morphology and needle body height using a high-definition microscope and measure the mechanical properties of the microneedles. Specifically, as Figure 3 、 Figure 4 and Table 2 show. Figure 2 are the photos of each group of microneedles under the high-definition microscope;Figure 4 Mechanical property curves of each group of microneedles.

[0070] Table 2 Characterization results of different microneedles (x±s, n = 5)

[0071]

[0072] From Figure 3 and Figure 4 it can be seen that the three groups of microneedles are all neatly arrayed, with complete needle shapes and smooth surfaces. Compared with the blank microneedles, the tip height, tip bottom diameter and mechanical properties of the aPexos microneedles and aPexos-EGCG microneedles are slightly reduced. However, the mechanical property of each microneedle in the three groups is about 0.16 N on average, which is greater than the minimum pressure (0.045 N) required to penetrate the stratum corneum reported in the literature, indicating that the microneedles prepared from platelet exosomes have sufficient mechanical strength to penetrate the skin.

[0073] Example 6 Evaluation of wound healing effect in scalded rats

[0074] S1: Establishment of scalded rat model: Before scalding, rats were anesthetized with 2% sodium pentobarbital at a dose of 2 mL / kg. Scalding was induced using a pressing iron, with the probe temperature at 90 °C, a diameter of 1.5 cm, the pressure of contacting the skin being its own gravity, and the contact time between the probe and the skin being 20 s. Necrotic tissue was removed the next day.

[0075] S2: Evaluation of wound healing effect in scalded rats: ① Twenty rats were divided into four groups, namely the aPexos-EGCG microneedle treatment group, the aPexos microneedle treatment group, the blank microneedle treatment group, and the control group without treatment. After treatment, they were bandaged with a polyurethane fixed dressing and a bandage. The wound healing progress was monitored by photography at days 3, 7, 10, 14, and 17. ImageJ software was used to determine the wound size at each time point, and the wound healing area was calculated using the following formula. At = (1 - Ad / A0) * 100. Among them, Ad represents the wound area at days 3, 7, 10, 14, and 17, while A0 represents the wound area at 0 d, and At is the wound healing rate at time point t. ② After euthanizing the experimental animals on day 17, the skin was fixed with 4% paraformaldehyde. Then the tissue was embedded in paraffin, sectioned and stained with H&E and Masson's trichrome. The results of the wound healing rate are as Figure 5 and Figure 6 shown, Figure 5 Representative optical pictures of the scald wounds of SD rats healing at different time points; Figure 6 is the wound healing rate of SD rats (scale bar = 1 cm, x±SD, n = 10); *P < 0.05; **P < 0.01; ***P < 0.001.

[0076] From Figure 5 andFigure 6 As is known, the wound healing rate of the rats in the aPexos-EGCG microneedle group prepared in Example 4 on the 3rd day was higher than that of the blank microneedle group in Comparative Example 1 and the aPexos microneedle group in Comparative Example 2, which were (36.28±7.97)% vs (19.33±13.9)% vs (28.45±5.72)% respectively.

[0077] HE staining was performed on the wound tissues of the rats on the 17th day, and the results were as Figure 7 and Figure 8 shown. Figure 7 The following are the histopathological results of the scalded SD rats in different groups on the 17th day. Among them, A is the HE staining of the wound tissues of SD rats; B is the Masson staining of the wound tissues of SD rats. Figure 8 The collagen deposition rate of the wound of SD rats (x±SD, n = 3)*P < 0.05; **P < 0.01; ***P < 0.001.

[0078] Compared with the blank microneedle group, the skin structure and tissue of the aPexos-EGCG group were more complete and clear, with the least infiltration of inflammatory cells and the most newly formed capillaries. The Masson staining results of the rats' wounds were as Figure 7 shown. Compared with the blank microneedle group and the aPexos group, the aPexos-EGCG group had more collagen deposition, more regular arrangement of collagen, more uniform distribution, and was more mature. Figure 8 The results showed that there was a statistical difference in the proportion of collagen area deposition in the aPexos-EGCG group compared with the other three groups, which was significantly higher than that of the blank microneedle group and the aPexos group.

[0079] The above-described embodiments only represent the specific implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application.

[0080] This background technology section is provided to generally present the context of the present invention. The work of the currently named inventors, to the extent described in this background technology section, and aspects that are not prior art at the time of filing this application are neither expressly nor impliedly admitted to be prior art of the present invention.

Claims

1. Use of a complex of artificial platelet exosomes and polyphenols in the preparation of microneedles for wound healing and / or hair loss treatment.

2. The use according to claim 1, characterized in that: The wounds include chronic or difficult-to-heal wounds.

3. A soluble microneedle loaded with artificial platelet exosomes and polyphenols, characterized in that: The microneedle comprises a backing loaded with polyphenols, and a needle body formed by a soluble high molecular polymer composite artificial platelet exosomes and a protein protective agent on the backing; wherein, the backing loaded with polyphenols has a mass ratio of polyphenols to the backing of 1-5:300-750; the ratio of the mass mg of the soluble high molecular polymer to the volume mL of the artificial platelet exosomes is 10-50:0.1-0.5; and the mass ratio of the polyphenols to the protein protective agent is 1-5:1-5.

4. The soluble microneedle according to claim 3, characterized in that: The backing loaded with polyphenols is selected from any one of gelatin, carboxymethyl chitosan, polyvinyl alcohol, polyvinyl pyrrolidone, sodium hyaluronate with a molecular weight of 10K-1000KD, carboxymethyl cellulose, or a mixture of any one or more of natural polyphenols; the soluble high molecular polymer is selected from any one of sodium hyaluronate with a molecular weight of 10K-1000KD, chitosan, polyvinyl pyrrolidone, polyvinyl alcohol, or a mixture of any one of several; the protein protective agent is selected from any one of sucrose, trehalose, mannitol, lactose, and glucose, or a mixture of any one of several.

5. The method for preparing the soluble microneedle according to claim 3 or 4, characterized in that The following steps are involved: S1. Preparation of artificial platelet exosomes: Concentrated platelets were taken, sonicated with an ultrasonic cell crusher, and then gradient centrifuged to obtain artificial platelet exosomes; S2. Preparation of needle body: dissolving a soluble high molecular polymer in physiological saline or anhydrous ethanol, adding a protein protective agent thereto, and then adding artificial platelet exosomes after dissolution to obtain a needle body; S3. Preparation of backing: The material used for the backing is added to physiological saline or anhydrous ethanol and heated in a water bath to form a mixed solution; Dissolving polyphenol in deionized water to obtain a polyphenol solution after the polyphenol is completely dissolved; mixing the mixed solution with the polyphenol solution to obtain a backing solution; S4. Pour the needle body obtained in S2 into a microneedle mold, evacuate and maintain at room temperature for a period of time, take out the mold and scrape off the excess needle body on the upper layer, and then place the mold in a dryer to dry; after drying, pour the backing solution, and finally place the mold in a dryer to dry overnight, wait for it to be naturally demolded, and obtain soluble microneedles loaded with artificial platelet exosomes and polyphenols.

6. The method for preparing the soluble microneedle according to claim 5, characterized in that: In S1, the power of the ultrasonic cell disruptor was 30%, 10s on, 10s off, and the ultrasonic time was 1min20s; The specific operation of gradient centrifugation is: centrifuge at 4°C, 3000g for 20 minutes, remove the supernatant; centrifuge at 4°C, 20000g for 20 minutes, remove the supernatant; resuspend the precipitate with 2wt% trehalose PBS solution and store at -40°C for later use.

7. The method for preparing the soluble microneedle according to claim 5, characterized in that: In S2, the soluble high molecular weight polymer is dissolved in physiological saline or anhydrous ethanol at a final concentration of 10-50 mg / mL; the temperature of the water bath heating is 37°C.

8. The method for preparing soluble microneedles according to claim 5, characterized in that: In S3, the water bath heating temperature is 55°C; the ratio of the mass mg of the backing to the volume mL of physiological saline or anhydrous ethanol is 300-750:5; the ratio of the mass mg of the polyphenol to the volume mL of water is 2-10:0.

1.

9. The method for preparing soluble microneedles according to claim 5, characterized in that: In S4, the needle body is poured into the microneedle mold and evacuated to -0.07 MPa, and maintained at room temperature for 5-30 minutes; the mold is dried in a dryer for 2-4 hours, and the temperature and humidity in the dryer are maintained at 25±2°C and 20%.

Images