Anti-scar frozen microneedle patch as well as preparation method and application thereof

Abstract

The invention relates to an anti-scar frozen microneedle patch as well as a preparation method and application thereof. The anti-scar freezing microneedle patch comprises a substrate layer and a microneedle array, the material of the microneedle comprises polyvinyl alcohol; the substrate layer and the microneedle array are obtained through liquid nitrogen freezing. The invention also provides a preparation method of the anti-scar frozen microneedle patch, which comprises the following steps: mixing the substrate layer material with the curing agent, pouring, heating in vacuum and standing to obtain the substrate layer; injecting a polyvinyl alcohol solution onto the substrate layer, freezing and forming to obtain the microneedle patch; and freezing the microneedle patch in liquid nitrogen to obtain the anti-scar frozen microneedle patch. The invention further provides application of the anti-scar frozen microneedle patch in preparation of drugs for treating hypertrophic scars. The invention solves the problems of unsatisfactory treatment effect, large side effect, high price and high technical requirement in the scar treatment method.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to an anti-scar freezing microneedle patch and a preparation method and application thereof. Background Art

[0002] After the skin is traumatized, scars are often left during the healing process. The formation of these scars is caused by excessive collagen proliferation during the skin repair process. How to effectively treat these scars and prevent excessive scar proliferation has become a common problem in clinical medicine. Despite this, there is currently no method that can completely cure scars. At present, the main treatment methods used in clinical practice include surgery, intra-scar drug injections, and laser therapy. However, these treatments have disadvantages such as high prices, large side effects, and the need for professional equipment. In addition, a single treatment method is often difficult to achieve satisfactory therapeutic effects. Therefore, there is an urgent need to develop new treatment strategies and combined treatment methods.

[0003] In recent years, physical therapy has gradually been applied to the treatment of hypertrophic scars. Among them, intra-scar cryotherapy, as a new type of physical therapy for scars, has achieved certain therapeutic effects. However, the disadvantage of this treatment method is that it is more invasive to deeper tissues and is only applicable to isolated lesions. In addition, cryotherapy requires professional equipment and operators, which to some extent limits its widespread application. In addition, the effect of single cryotherapy is still limited and difficult to meet clinical needs.

[0004] As an emerging scar treatment method, microneedle therapy has the advantages of minimally invasive, simple operation, and drug delivery. Microneedles create tiny channels on the skin surface to promote the absorption of drugs, thereby achieving the purpose of scar treatment. This method not only reduces the pain of patients, but also improves the convenience and safety of treatment. However, microneedle therapy also has some limitations, such as high technical requirements for operators, and in some cases it may need to be used in combination with other treatments to improve efficacy.

[0005] In summary, although the current treatment methods can alleviate scar problems to a certain extent, there are still many shortcomings. Therefore, developing new treatment strategies and combined treatment methods, as well as improving existing technologies, are important directions for future medical research. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an anti-scar cryo-microneedle patch and a preparation method and application thereof, so as to solve the problems of unsatisfactory treatment effect, large side effects, high price and high technical requirements in scar treatment methods.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: An anti-scar freezing microneedle patch, comprising a base layer and a microneedle array; The material of the microneedle includes polyvinyl alcohol (PVA); The base layer and the microneedle array are both obtained by freezing with liquid nitrogen.

[0008] Preferably, the material of the microneedle further includes triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu).

[0009] By adding triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) to the microneedles, the anti-scar cryo-microneedle patch TA+5-Fu@cryoMNs can achieve invasive, painless, low-cost, non-toxic and easy-to-operate treatment of hypertrophic scars; secondly, the cryo-microneedle integrates cryotherapy, microneedle mechanical microenvironment regulation therapy and drug therapy, effectively improving the treatment effect. In addition, the cryo-microneedle can ensure the physical and chemical properties of the drug, while effectively piercing the epidermis and dissolving it appropriately to release the drug, thereby improving the treatment efficacy.

[0010] Preferably, the content of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the microneedle is 2-4 μg / needle.

[0011] Preferably, the content of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the microneedle is 3 μg / needle.

[0012] Preferably, the material of the microneedle also includes human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos).

[0013] By adding human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) to the microneedles, the anti-scar cryo-microneedle patch HUMSC-exos@cryoMNs can achieve invasive, painless, low-cost, non-toxic and easy-to-operate treatment of hypertrophic scars; secondly, the cryo-microneedle integrates cryotherapy, microneedle mechanical microenvironment regulation and bioactive substance treatment, effectively improving the treatment effect. In addition, the cryo-microneedle can protect the activity of bioactive substances, while effectively piercing the epidermis and dissolving appropriately, releasing bioactive substances, thereby improving the treatment effect.

[0014] Preferably, the content of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the microneedle is (3-4) x10 6 Grain / root.

[0015] Preferably, the content of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the microneedle is 3.2x10 6 Grain / root.

[0016] Preferably, the material of the base layer is selected from polydimethylsiloxane (PDMS).

[0017] Preferably, in the microneedle array, the needle tip height is 1.5 mm, the needle tip spacing is 0.69 mm, the needle width is 0.3 mm, the array is 15 x 15, and the bottom diameter of a single needle is 300 μm.

[0018] Preferably, the components in the microneedles also include water.

[0019] Preferably, the maximum load rupture force of the microneedle is 0.5 N / needle.

[0020] Preferably, the water in the microneedles is selected from RO water.

[0021] Among them, RO water refers to water that has been treated by reverse osmosis technology. Reverse osmosis is a filtration process that can remove impurities, bacteria, viruses, minerals, and salts from water to obtain very pure water.

[0022] The present invention also provides a method for preparing the anti-scar cryo-microneedle patch according to the present invention, comprising the following steps: S1, mixing the base layer material and the curing agent, pouring, vacuum heating and standing to obtain the base layer; S2, injecting polyvinyl alcohol (PVA) solution onto the basal layer and freezing to form a microneedle patch; S3. Freeze the microneedle patch in liquid nitrogen to obtain an anti-scar frozen microneedle patch.

[0023] Preferably, the step S2 comprises: dissolving triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a dimethyl sulfoxide (DMSO) solution to obtain a first mixed solution; The first mixed solution and the polyvinyl alcohol (PVA) solution are mixed to obtain a second mixed solution, and the second mixed solution is injected onto the base layer and freeze-formed to obtain a microneedle patch.

[0024] Preferably, S2 comprises: dissolving human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in a polyvinyl alcohol (PVA) solution to obtain a third mixed solution, injecting the third mixed solution onto the basal layer, and freeze-molding to obtain a microneedle patch.

[0025] Preferably, the mass concentration of polyvinyl alcohol (PVA) in the polyvinyl alcohol (PVA) solution is 10%.

[0026] Preferably, the concentrations of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the first mixed solution are both 50 mg / mL.

[0027] Preferably, the mass concentration of dimethyl sulfoxide (DMSO) in the dimethyl sulfoxide (DMSO) solution is 5%.

[0028] Preferably, the concentration of human umbilical cord mesenchymal stem cell exosomes in the third mixed solution is 9 x 10 10 Particles / mL.

[0029] Preferably, S1 comprises: mixing polydimethylsiloxane (PDMS) and a curing agent in a mass ratio of 10:1, i.e. Sylgard 184, and then pouring it into a microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x15), slightly vibrating, standing in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placing it in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer, i.e., a PDMS mold.

[0030] Among them, Sylgard 184 is a two-component silicone rubber composed of polydimethylsiloxane (PDMS) and curing agent, and polydimethylsiloxane (PDMS) and curing agent are mixed in a weight ratio of 10:1.

[0031] Preferably, S2 includes: dissolving 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; injecting the polyvinyl alcohol (PVA) solution into a PDMS mold, centrifuging at a speed of 4000r / min for 3 min to completely fill the needle cavity with the polyvinyl alcohol (PVA) solution, and then filling the remaining space of the substrate with the polyvinyl alcohol (PVA) solution, freezing at a temperature of -20°C for 2 h, and after complete forming, separating the microneedle patch from the PDMS mold, and freezing at a temperature of -80°C for 2 h after demolding to obtain a microneedle patch.

[0032] Preferably, S2 comprises: dissolving 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts for multiple times, heating and stirring continuously until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolving triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a dimethyl sulfoxide (DMSO) solution with a mass percentage of 5% to obtain a first mixed solution in which the concentrations of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) are both 50 mg / mL; mixing the first mixed solution and the polyvinyl alcohol (PVA) solution in a volume ratio of 7:1 to obtain a second mixed solution, injecting the second mixed solution into a PDMS mold, centrifuging at a speed of 4000 r / min for 3 min to completely fill the needle cavity with the second mixed solution, removing the liquid outside the needle body, and then injecting the polyvinyl alcohol (PVA) solution into the mold to form a substrate, freezing at a temperature of -20 ° C for 2 h. After being fully formed, the microneedle patch was separated from the PDMS mold and, after demolding, was frozen at -80 °C for 2 h to obtain a microneedle patch.

[0033] Preferably, S2 comprises: dissolving 10 g of polyvinyl alcohol (PVA) (molecular weight of 195,000) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolving human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the polyvinyl alcohol (PVA) solution to obtain a third mixed solution, wherein the concentration of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the third mixed solution is 9 x 10 10 The third mixed solution was injected into the PDMS mold, and centrifuged at a speed of 4000r / min for 3min to completely fill the needle cavity with the third mixed liquid. The liquid outside the needle body was removed, and then the polyvinyl alcohol (PVA) solution was injected into the mold to form a substrate, which was frozen at -20°C for 2h. After complete forming, the microneedle patch was separated from the PDMS mold, and after demolding, it was frozen at -80°C for 2h to obtain a microneedle patch.

[0034] The present invention also provides an application of the anti-scar cryomicroneedle patch of the present invention in the preparation of a drug for treating hypertrophic scars, wherein the anti-scar cryomicroneedle patch has the functions of reducing inflammation, remodeling the mechanical microenvironment and regulating metabolism.

[0035] Beneficial effects of the present invention: The anti-scar cryo-microneedle patch of the present invention realizes the integration of cryotherapy, mechanical microenvironment regulation of microneedles and delivery of therapeutic factors by adopting microneedles in combination with freezing, thereby enhancing the therapeutic efficacy. Through the synergistic treatment of cryotherapy and mechanical microenvironment regulation of microneedles, it not only achieves the destruction of abnormal blood vessels in scar tissue and causes scar tissue necrosis, but also reduces scar inflammation, improves metabolism, and reduces mechanical stress in the scar area, which is beneficial to the treatment of hypertrophic scars. Cryo-microneedles affect hypertrophic scars in multiple dimensions such as inflammation, metabolism, and mechanical force. Through the combination of microneedles and freezing, the cryotherapy of scars within scars is improved, avoiding the limitations of deep tissue invasion, single lesion limitation, and the need for professional equipment, and has promotion and application value in the field of biomedical material technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a physical picture of the anti-scar cryomicroneedle patch cryoMN-10% prepared in Example 1; Figure 2 This is a physical picture of the anti-scar cryomicroneedle patch TA+5-Fu@cryoMNs prepared in Example 4; Figure 3 This is a physical picture of the anti-scar cryomicroneedle patch HUMSC-exos@cryoMNs prepared in Example 6; Figure 4 The bar graph of the melting time of cryo-microneedles prepared with different concentrations of PVA; Figure 5 Schematic diagram of the melting process of the anti-scar cryo-microneedle patch at room temperature and on the back of a rat; Figure 6 This is a HE staining picture of the anti-scar cryomicroneedle patch cryoMN-10% prepared in Example 1 piercing the mouse skin; Figure 7 This is a picture showing the morphological changes of rabbit ear scars after treatment with the anti-scar cryo-microneedle patch cryoMN-10% (HS is the hypertrophic scar group, and cryoMN is the cryo-microneedle treatment group); Figure 8 This is a picture showing the color, thickness and hardness of scar tissue after the rabbit ear scar was treated with the anti-scar cryomicroneedle patch cryoMN-10%; Fig. 9 This is the HE, Masson, Sirius Red, α-SMA and CD31 staining results of scar tissue after the rabbit ear scar was treated with the anti-scar cryomicroneedle patch cryoMN-10%; Fig.10 The results of immunofluorescence staining of pathological sections of inflammatory-related genes (IL-1β, IL-6, CCL2, TGFβ1, NFkB) in rabbit ear scars after treatment with the anti-scar cryomicroneedle patch cryoMN-10%; Fig.11 The results of immunofluorescence staining of pathological sections for mechanical response genes (YAP1, TAZ) and key genes for retinoic acid uptake (STRA6) in rabbit ear scars after treatment with the anti-scar cryomicroneedle patch cryoMN-10%; Fig.12 The morphological changes of rabbit ear scars before and after treatment with TA+5-Fu@cryoMNs (HS is the hypertrophic scar group, cryoMNs is the cryomicroneedle treatment, TA+5-Fu@cryoMNs is the cryomicroneedle treatment loaded with TA and 5-Fu); Fig.13 This is the color, thickness and hardness of scar tissue after the rabbit ear scar was treated with TA+5-Fu@cryoMNs; Fig.14 The results of HE, Masson, Sirius Red, α-SMA and CD31 staining of scar tissue after the rabbit ear scar was treated with TA+5-Fu@cryoMNs; Fig.15 The morphological changes of rabbit ear scars after treatment with HUMSC-exos@cryoMNs (HS is the hypertrophic scar group, cryoMNs is the cryomicroneedle treatment, and HUMSC-exos@cryoMN is the cryomicroneedle treatment with human umbilical cord mesenchymal stem cell exosomes); Fig.16 The color, thickness and hardness of scar tissue after the rabbit ear scar was treated with HUMSC-exos@cryoMNs; Fig.17 These are the results of HE, Masson, Sirius Red, α-SMA and CD31 staining of scar tissue after rabbit ear scar was treated with HUMSC-exos@cryoMNs. DETAILED DESCRIPTION

[0037] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0038] It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and thus the drawings only show components related to the present invention rather than being drawn according to the number, shape and size of components in actual implementation. In actual implementation, the type, quantity and proportion of each component may be changed arbitrarily, and the component layout may also be more complicated.

[0039] The present invention intends to disclose an anti-scar cryo-microneedle patch and a preparation method and application thereof, so as to solve the problems of unsatisfactory treatment effect, large side effects, high price and high technical requirements in scar treatment methods.

[0040] Among them, the anti-scar cryo-microneedle patch includes a base layer and a microneedle array; the material of the microneedle includes polyvinyl alcohol (PVA); the base layer and the microneedle array are both obtained by freezing with liquid nitrogen.

[0041] In some embodiments, the material of the microneedle further includes triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu).

[0042] By adding triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) to the microneedles, the anti-scar cryo-microneedle patch TA+5-Fu@cryoMNs can achieve invasive, painless, low-cost, non-toxic and easy-to-operate treatment of hypertrophic scars; secondly, the cryo-microneedle integrates cryotherapy, microneedle mechanical microenvironment regulation therapy and drug therapy, effectively improving the treatment effect. In addition, the cryo-microneedle can ensure the physical and chemical properties of the drug, while effectively piercing the epidermis and dissolving it appropriately to release the drug, thereby improving the treatment efficacy.

[0043] In some embodiments, the content of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the microneedle is 2-4 μg / microneedle.

[0044] Exemplarily, the content of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the microneedle is 3 μg / needle.

[0045] In some embodiments, the material of the microneedle also includes human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos).

[0046] By adding human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) to the microneedles, the anti-scar cryo-microneedle patch HUMSC-exos@cryoMNs can achieve invasive, painless, low-cost, non-toxic and easy-to-operate treatment of hypertrophic scars; secondly, the cryo-microneedle integrates cryotherapy, microneedle mechanical microenvironment regulation and bioactive substance treatment, effectively improving the treatment effect. In addition, the cryo-microneedle can protect the activity of bioactive substances, while effectively piercing the epidermis and dissolving appropriately, releasing bioactive substances, thereby improving the treatment effect.

[0047] In some embodiments, the content of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the microneedle is 3.2x10 6 Grain / root.

[0048] In some embodiments, the material of the base layer is selected from polydimethylsiloxane (PDMS).

[0049] In some embodiments, in the microneedle array, the needle tip height is 1.5 mm, the needle tip spacing is 0.69 mm, the needle width is 0.3 mm, the array is 15 x15, and the bottom diameter of a single needle is 300 μm.

[0050] Exemplarily, the components in the microneedles also include water.

[0051] Exemplarily, the maximum load rupture force of the microneedle is 0.5 N / needle.

[0052] Exemplarily, the water in the microneedles is selected from RO water.

[0053] Among them, RO water refers to water that has been treated by reverse osmosis technology. Reverse osmosis is a filtration process that can remove impurities, bacteria, viruses, minerals, and salts from water to obtain very pure water.

[0054] In some embodiments, a method for preparing the anti-scar cryo-microneedle patch according to the present invention is also provided, comprising the following steps: S1, mixing the base layer material with the curing agent, i.e. Sylgard 184, pouring, vacuum heating and standing to obtain the base layer; S2, injecting polyvinyl alcohol (PVA) solution onto the basal layer and freezing to form a microneedle patch; S3. Freeze the microneedle patch in liquid nitrogen to obtain an anti-scar frozen microneedle patch.

[0055] In some embodiments, S2 includes: dissolving triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a dimethyl sulfoxide (DMSO) solution to obtain a first mixed solution; The first mixed solution and the polyvinyl alcohol (PVA) solution are mixed to obtain a second mixed solution, and the second mixed solution is injected onto the base layer and freeze-formed to obtain a microneedle patch.

[0056] In some embodiments, S2 includes: dissolving human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in a polyvinyl alcohol (PVA) solution to obtain a third mixed solution, injecting the third mixed solution onto the basal layer, freeze-molding, and obtaining a microneedle patch.

[0057] Exemplarily, the mass concentration of polyvinyl alcohol (PVA) in the polyvinyl alcohol (PVA) solution is 10%.

[0058] Exemplarily, the concentrations of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the first mixed solution are both 50 mg / mL.

[0059] Exemplarily, the mass concentration of dimethyl sulfoxide (DMSO) in the dimethyl sulfoxide (DMSO) solution is 5%.

[0060] Exemplarily, the concentration of human umbilical cord mesenchymal stem cell exosomes in the third mixed solution is 9 x 10 10 Particles / mL.

[0061] The exemplary S1 includes: mixing polydimethylsiloxane (PDMS) and a curing agent in a mass ratio of 10:1, namely Sylgard 184, pouring it into a microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x15), slightly vibrating, standing in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placing it in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer, namely the PDMS mold.

[0062] Exemplarily, S2 includes: dissolving 10 g of polyvinyl alcohol (PVA) (molecular weight less than 195000) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; injecting the polyvinyl alcohol (PVA) solution into the PDMS mold, centrifuging at a speed of 4000r / min for 3 min to completely fill the needle cavity with the polyvinyl alcohol (PVA) solution, and then filling the remaining space of the substrate with the polyvinyl alcohol (PVA) solution, freezing at a temperature of -20°C for 2 h, and after complete forming, separating the microneedle patch from the PDMS mold, and freezing at a temperature of -80°C for 2 h after demolding to obtain a microneedle patch.

[0063] Exemplarily, S2 includes: dissolving 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolving triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a dimethyl sulfoxide (DMSO) solution with a mass percentage of 5% to obtain a first mixed solution in which the concentrations of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) are both 50 mg / mL; mixing the first mixed solution and the polyvinyl alcohol (PVA) solution in a volume ratio of 7:1 to obtain a second mixed solution, injecting the second mixed solution into a PDMS mold, centrifuging at a speed of 4000 r / min for 3 min to completely fill the needle cavity with the second mixed solution, removing the liquid outside the needle body, and then injecting the polyvinyl alcohol (PVA) solution into the mold to form a substrate, freezing at a temperature of -20 ° C for 2 h. After being fully formed, the microneedle patch was separated from the PDMS mold and, after demolding, was frozen at -80 °C for 2 h to obtain a microneedle patch.

[0064] Exemplarily, S2 includes: dissolving 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolving human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the polyvinyl alcohol (PVA) solution to obtain a third mixed solution, wherein the concentration of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the third mixed solution is 9 x 10 10 The third mixed solution was injected into the PDMS mold, and centrifuged at a speed of 4000r / min for 3min to completely fill the needle cavity with the third mixed liquid. The liquid outside the needle body was removed, and then the polyvinyl alcohol (PVA) solution was injected into the mold to form a substrate, which was frozen at -20°C for 2h. After complete forming, the microneedle patch was separated from the PDMS mold, and after demolding, it was frozen at -80°C for 2h to obtain a microneedle patch.

[0065] In order to make the technical problems, technical solutions and beneficial effects solved by the present application clearer, the preparation method of the anti-scar cryo-microneedle patch of the present invention will be further described in detail below in combination with specific embodiments and drawings. Obviously, the specific embodiments described are only part of the embodiments of the present application, not all of them. The following description of at least one exemplary embodiment is actually only illustrative and is by no means a limitation to the present application and its application. Based on the specific embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present application.

[0066] If no specific techniques or conditions are specified in the specific embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0067] Example 1 like Figure 1 As shown, a method for preparing an anti-scar cryo-microneedle patch (cryoMN) comprises the following steps: S1. According to the Sylgard 184 method, polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, and then poured into the microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x 15), slightly vibrated, and placed in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placed in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer, i.e., a PDMS mold; S2. Dissolve 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and several times, heat and stir continuously until the PVA powder is completely dissolved to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; inject the polyvinyl alcohol (PVA) solution into the PDMS mold, centrifuge at a speed of 4000r / min for 3 min to completely fill the needle cavity with the polyvinyl alcohol (PVA) solution, and then fill the remaining space of the substrate with the polyvinyl alcohol (PVA) solution, freeze at a temperature of -20 ℃ for 2 h, and after complete molding, separate the microneedle patch from the PDMS mold, and freeze at a temperature of -80 ℃ for 2 h after demolding to obtain a microneedle patch; S3. Freeze the microneedle patch obtained in S2 in liquid nitrogen to make the microneedle patch have higher mechanical strength, thereby obtaining an anti-scar frozen microneedle patch, which is recorded as cryoMN (10% PVA).

[0068] Example 2 A method for preparing anti-scar cryomicroneedle patches cryoMN-5% (5% PVA) and cryoMN-15% (15% PVA), comprising the following steps: S1. According to the Sylgard 184 method, polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, and then poured into the microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x 15), slightly vibrated, and placed in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placed in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer, i.e., a PDMS mold; S2. Dissolve 5 g and 15 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and several times, heat and stir continuously until the PVA powder is completely dissolved, and obtain polyvinyl alcohol (PVA) solutions with a mass percentage of 5% and 15%, respectively; inject polyvinyl alcohol (PVA) solutions with different mass percentages into the PDMS mold, centrifuge at a speed of 4000r / min for 3 min, so that the polyvinyl alcohol (PVA) solution completely fills the needle cavity, and then fill the remaining space of the substrate with polyvinyl alcohol (PVA) solution with the same mass percentage, freeze at -20 °C for 2 h, and after complete molding, separate the microneedle patch from the PDMS mold, and freeze at -80 °C for 2 h after demolding to obtain microneedle patches, which are recorded as cryoMN-5% (5% PVA) and cryoMN-15% (15% PVA); S3. The microneedle patches obtained in S2 were frozen in liquid nitrogen to make the microneedle patches have higher mechanical strength, thereby obtaining anti-scar frozen microneedle patches, which were respectively named cryoMN-5% (5% PVA) and cryoMN-15% (15% PVA).

[0069] Example 3 like Figure 2 As shown, a method for preparing an anti-scar cryo-microneedle patch (TA+5-Fu@cryoMNs) comprises the following steps: S1. According to the Sylgard 184 method, polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, and then poured into the microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x 15), slightly vibrated, and placed in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placed in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer, i.e., a PDMS mold; S2. Dissolve 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and several times, heat and stir continuously until the PVA powder is completely dissolved to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolve triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a 5% dimethyl sulfoxide (DMSO) solution to obtain a first mixed solution with a concentration of 50 mg / mL of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu); mix the first mixed solution and the polyvinyl alcohol (PVA) solution in a volume ratio of 7:1 to obtain a second mixed solution, inject the second mixed solution into the PDMS mold, centrifuge at a speed of 4000 r / min for 3 min to completely fill the needle cavity with the second mixed solution, remove the liquid outside the needle body, and then inject the polyvinyl alcohol (PVA) solution into the mold to form a substrate, and freeze it at -20 °C for 2 hours. h, after being fully formed, the microneedle patch was separated from the PDMS mold, and after demolding, it was frozen at -80 °C for 2 h to obtain a microneedle patch; S3. The microneedle patch obtained in S2 is frozen in liquid nitrogen to make the microneedle patch have higher mechanical strength, thereby obtaining an anti-scar frozen microneedle patch, which is recorded as TA+5-Fu@cryoMNs.

[0070] Example 4 like Figure 3 As shown, a method for preparing an anti-scar cryo-microneedle patch (HUMSC-exos@cryoMN) comprises the following steps: S1. According to the Sylgard 184 method, polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, and then poured into the microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x 15), slightly vibrated, and placed in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placed in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer, i.e., a PDMS mold; S2. Dissolve 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and several times, heat and stir continuously until the PVA powder is completely dissolved to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%. 11 The human umbilical cord mesenchymal stem cell exosome (HUMSC-exos) solution with a concentration of 9 x 10 10The third mixed solution was injected into the PDMS mold, and centrifuged at a speed of 4000r / min for 3 min to completely fill the needle cavity with the third mixed liquid. The liquid outside the needle body was removed, and then polyvinyl alcohol (PVA) solution was injected into the mold to form a substrate, and frozen at a temperature of -20 ℃ for 2 h. After complete molding, the microneedle patch was separated from the PDMS mold, and after demolding, it was frozen at a temperature of -80 ℃ for 2 h to obtain a microneedle patch; S3. The microneedle patch obtained in S2 is frozen in liquid nitrogen to make the microneedle patch have higher mechanical strength, thereby obtaining an anti-scar frozen microneedle patch, which is recorded as HUMSC-exos@cryoMNs.

[0071] Comparative Example 1 A method for preparing a cryo-microneedle patch comprises the following steps: S1. According to the Sylgard 184 method, polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, and then poured into a microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x 15), slightly vibrated, and placed in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placed in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer; S2. Freeze the basal layer obtained in S1 in liquid nitrogen to obtain a frozen microneedle patch, which is designated as cryoMN-0.

[0072] Comparative Example 2 A method for preparing a cryo-microneedle patch comprises the following steps: S1. According to the Sylgard 184 method, polydimethylsiloxane (PDMS) and curing agent were mixed at a mass ratio of 10:1, and then poured into a microneedle master mold (wherein, needle tip height: 1.5 mm, needle tip spacing: 0.69 mm, needle width: 0.3 mm, array: 15 x 15), slightly vibrated, and placed in a refrigerator at a temperature of 4 ° C for 30 min to remove bubbles, and then placed in a vacuum oven at a temperature of 80 ° C for 2 h to obtain a base layer; S2. Dissolve 10 g of polyvinyl alcohol (PVA) (molecular weight 195000) in 100 mL of deionized water in small amounts and several times, heat and stir continuously until the PVA powder is completely dissolved to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; inject the polyvinyl alcohol (PVA) solution into the PDMS mold, centrifuge at a speed of 4000r / min for 3 min to completely fill the needle cavity with the polyvinyl alcohol (PVA) solution, and then fill the remaining space of the substrate with the polyvinyl alcohol (PVA) solution, freeze at a temperature of -20 ℃ for 2 h, and after complete forming, separate the microneedle patch from the PDMS mold to obtain a microneedle patch.

[0073] Detection and analysis 1) Melting test The specific steps are: The anti-scar cryo-microneedle patches with different mass percentages of PVA prepared in Examples 1 and 2, and the cryo-microneedle patch without PVA added prepared in Control Example 1 were subjected to melting tests at room temperature and on the back of rats.

[0074] The results are as follows Figure 4 shown.

[0075] from Figure 4 The analysis shows that 10% PVA can not only significantly increase the melting time of the microneedle tip, but also save materials compared with 15% PVA. Therefore, 10% PVA is the most suitable.

[0076] The anti-scar cryo-microneedle patch prepared in Example 1 was subjected to melting tests at room temperature and on the needle tip of the rat back. The melting process was as follows: Figure 5 shown.

[0077] from Figure 5 The analysis showed that the microneedle tip melted completely within 360 s at room temperature and completely melted within 140 s on the rat's back.

[0078] HE staining picture of the anti-scar cryomicroneedle patch cryoMN-10% prepared in Example 1 piercing the mouse skin is as follows Figure 6 shown.

[0079] from Figure 6 The analysis showed that the cryo-microneedles could penetrate the epidermis of mice and reach the dermis.

[0080] 2) Treatment testing for hypertrophic scars The therapeutic effect of the anti-scar cryomicroneedle patch cryoMN-10% prepared in Example 1 on hypertrophic scars in rabbit ears The specific operation steps are as follows: After female New Zealand white rabbits weighing about 2.0-2.5 kg were raised for one week, four full-thickness skin wounds with a diameter of 1 cm were cut on each auricle, the perichondrium at the bottom of the wound was completely removed with a scalpel, and the scab was repeatedly removed to delay wound re-epithelialization and promote the proliferation of granulation tissue and fibrous tissue to form a raised scar. 30 days after the wound was created, the wound was completely healed, forming a rabbit ear hypertrophic scar model.

[0081] The anti-scar cryomicroneedle patch cryoMN-10% prepared in Example 1 was inserted into the scar area of ​​the rabbit ear 31 days after surgery and pressed for 2 minutes to completely melt the cryomicroneedle tip. The untreated group served as a blank control. The rabbit ear scar was photographed and recorded 7 days and 14 days after treatment to observe the treatment status. Experimental results: Figure 7 , Figure 8 and Fig. 9 shown.

[0082] from Figure 7 , Figure 8 and Fig. 9 The analysis showed that the anti-scar cryo-microneedle patch cryoMN-10% prepared in Example 1 could effectively treat hypertrophic scars in rabbit ears. On the 14th day of treatment, the scars in the cryo-microneedle treatment group were reduced compared with those in the untreated control group (HS) and the non-frozen microneedles (DryMNs).

[0083] At the same time, the genes related to inflammation, mechanical response pathway and retinoic acid metabolism pathway were immunofluorescently stained on pathological sections. Fig.10 and Fig.11 shown.

[0084] from Fig.10 and Fig.11 The analysis showed that the anti-scar cryomicroneedle patch cryoMN-10% prepared in Example 1 could significantly reduce the expression of inflammation-related genes (IL-1β, IL-6, CCL2, TGFβ1, NFkB) and mechanical response genes (YAP1, TAZ), and increase the expression of the key gene for retinoic acid uptake (STRA6).

[0085] The therapeutic effect of the anti-scar cryomicroneedle patch TA+5-Fu@cryoMNs prepared in Example 4 on hypertrophic scars in rabbit ears The specific operation steps are as follows: After female New Zealand white rabbits weighing about 2.0-2.5 kg were raised for one week, four full-thickness skin wounds with a diameter of 1 cm were cut on each auricle, the perichondrium at the bottom of the wound was completely removed with a scalpel, and the scab was repeatedly removed to delay wound re-epithelialization and promote the proliferation of granulation tissue and fibrous tissue to form a raised scar. 30 days after the wound was created, the wound was completely healed, forming a rabbit ear hypertrophic scar model.

[0086] The anti-scar cryomicroneedle patch TA+5-Fu@cryoMNs prepared in Example 4 was inserted into the scar area of ​​the rabbit ear 31 days after surgery and pressed for 2 minutes to completely melt the cryomicroneedle tip. The untreated group served as a blank control. The rabbit ear scar was photographed and recorded 7 days and 14 days after treatment to observe the treatment status. Experimental results: Fig.12 , Fig.13 and Fig.14 shown.

[0087] from Fig.12 , Fig.13 and Fig.14 The analysis showed that the anti-scar cryomicroneedle patch TA+5-Fu@cryoMNs prepared in Example 4 can effectively treat hypertrophic scars in rabbit ears. On the 14th day of treatment, compared with the HS control group, the scars in the TA+5-Fu@cryoMNs group were significantly alleviated, and the treatment effect was better than that of the cryoMNs treatment group in Example 1.

[0088] The therapeutic effect of the anti-scar cryomicroneedle patch HUMSC-exos@cryoMNs prepared in Example 6 on hypertrophic scars in rabbit ears The specific operation steps are as follows: After female New Zealand white rabbits weighing about 2.0-2.5 kg were raised for one week, four full-thickness skin wounds with a diameter of 1 cm were cut on each auricle, the perichondrium at the bottom of the wound was completely removed with a scalpel, and the scab was repeatedly removed to delay wound re-epithelialization and promote the proliferation of granulation tissue and fibrous tissue to form a raised scar. 30 days after the wound was created, the wound was completely healed, forming a rabbit ear hypertrophic scar model.

[0089] The anti-scar cryomicroneedle patch HUMSC-exos@cryoMNs prepared in Example 6 was inserted into the scar area of ​​the rabbit ear 31 days after surgery and pressed for 2 minutes to completely melt the cryomicroneedle tip. The untreated group served as a blank control. The rabbit ear scar was photographed and recorded 7 days and 14 days after treatment to observe the treatment status. Experimental results: Fig.15 , Fig.16 and Fig.17 as shown. from Fig.15 , Fig.16 and Fig.17 The analysis showed that the anti-scar cryomicroneedle patch HUMSC-exos@cryoMN prepared in Example 6 can effectively treat hypertrophic scars in rabbit ears. On the 14th day of treatment, compared with the HS control group, the scars in the HUMSC-exos@cryoMN group were significantly alleviated, and the treatment effect was better than that of the cryoMNs treatment group in Example 1.

[0090] In summary, the anti-scar cryo-microneedle patch of the present invention, by adopting microneedles combined with freezing, realizes the integration of cryotherapy, mechanical microenvironment regulation of microneedles and delivery of therapeutic factors, thereby enhancing the therapeutic efficacy. Through the synergistic treatment of cryotherapy and mechanical microenvironment regulation of microneedles, it not only achieves the destruction of abnormal blood vessels in scar tissue and causes scar tissue necrosis, but also reduces scar inflammation, improves metabolism, and reduces mechanical stress in the scar area, which is beneficial to the treatment of hypertrophic scars. Cryo-microneedles affect hypertrophic scars in multiple dimensions such as inflammation, metabolism, and mechanical force. Through the combination of microneedles and freezing, the cryotherapy of scars within scars is improved, avoiding the limitations of deep tissue invasion, single lesion limitation, and the need for professional equipment, and has promotion and application value in the field of biomedical materials technology.

[0091] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. An anti-scar freezing microneedle patch, characterized in that: including a basal layer and a microneedle array; The material of the microneedle includes polyvinyl alcohol (PVA); The base layer and the microneedle array are both obtained by freezing with liquid nitrogen.

2. The anti-scar freezing microneedle patch according to claim 1, characterized in that: The materials of the microneedle also include triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu).

3. The anti-scar freezing microneedle patch according to claim 2, characterized in that: The content of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the microneedle is 2-4 μg / needle.

4. The anti-scar freezing microneedle patch according to claim 1, characterized in that: The material of the microneedle also includes human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos).

5. The anti-scar freezing microneedle patch according to claim 4, characterized in that: The content of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the microneedle is (3~4)x10 6 Grain / root.

6. The anti-scar freezing microneedle patch according to claim 1, characterized in that: The material of the base layer is selected from polydimethylsiloxane (PDMS); And / or, in the microneedle array, the needle tip height is 1.5 mm, the needle tip spacing is 0.69 mm, the needle width is 0.3 mm, the array is 15 x 15, and the bottom diameter of a single needle is 300 μm; And / or, the components in the microneedles also include water; And / or, the maximum load rupture force of the microneedle is 0.5N / microneedle.

7. A method for preparing the anti-scar cryo-microneedle patch according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, mixing the base layer material and the curing agent, pouring, vacuum heating and standing to obtain the base layer; S2, injecting polyvinyl alcohol (PVA) solution onto the basal layer and freezing to form a microneedle patch; S3. Freeze the microneedle patch in liquid nitrogen to obtain an anti-scar frozen microneedle patch.

8. The preparation method according to claim 7, characterized in that: The step S2 includes: dissolving triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a dimethyl sulfoxide (DMSO) solution to obtain a first mixed solution; The first mixed solution and the polyvinyl alcohol (PVA) solution are mixed to obtain a second mixed solution, and the second mixed solution is injected onto the base layer and freeze-formed to obtain a microneedle patch; And / or, S2 includes: dissolving human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in a polyvinyl alcohol (PVA) solution to obtain a third mixed solution, injecting the third mixed solution onto the basal layer, and freeze-molding to obtain a microneedle patch.

9. The preparation method according to claim 8, characterized in that: The mass concentration of polyvinyl alcohol (PVA) in the polyvinyl alcohol (PVA) solution is 10%; and / or, the concentrations of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in the first mixed solution are both 50 mg / mL; And / or, the mass concentration of dimethyl sulfoxide (DMSO) in the dimethyl sulfoxide (DMSO) solution is 5%; And / or, the concentration of human umbilical cord mesenchymal stem cell exosomes in the third mixed solution is 9 x 10 10 Particles / mL; And / or, S1 includes: mixing polydimethylsiloxane (PDMS) and a curing agent in a mass ratio of 10:1, namely Sylgard 184, pouring into a microneedle master mold, slightly vibrating, standing in a refrigerator at a temperature of 4°C for 30 minutes to remove bubbles, and then placing in a vacuum oven at a temperature of 80°C for 2 hours to obtain a base layer, namely a PDMS mold; The S2 comprises: dissolving 10 g of polyvinyl alcohol (PVA) in 100 mL of water, heating and stirring until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; injecting the polyvinyl alcohol (PVA) solution into a PDMS mold, centrifuging at a speed of 4000 r / min for 3 min to completely fill the needle cavity with the polyvinyl alcohol (PVA) solution, and then filling the remaining space of the substrate with the polyvinyl alcohol (PVA) solution, freezing at a temperature of -20 ° C for 2 h, and after complete forming, separating the microneedle patch from the PDMS mold, and freezing at a temperature of -80 ° C for 2 h after demolding to obtain a microneedle patch; Alternatively, S2 comprises: dissolving 10 g of polyvinyl alcohol (PVA) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolving triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) in a dimethyl sulfoxide (DMSO) solution with a mass percentage of 5% to obtain a first mixed solution in which the concentrations of triamcinolone acetonide (TA) and 5-fluorouracil (5-Fu) are both 50 mg / mL; mixing the first mixed solution and the polyvinyl alcohol (PVA) solution in a volume ratio of 7:1 to obtain a second mixed solution, injecting the second mixed solution into a PDMS mold, centrifuging at a speed of 4000 r / min for 3 min to completely fill the needle cavity with the second mixed solution, removing the liquid outside the needle body, and then injecting the polyvinyl alcohol (PVA) solution into the mold to form a substrate, freezing at a temperature of -20 °C for 2 h, after being fully formed, the microneedle patch was separated from the PDMS mold, and after demolding, it was frozen at -80 °C for 2 h to obtain a microneedle patch; Alternatively, S2 comprises: dissolving 10 g of polyvinyl alcohol (PVA) in 100 mL of deionized water in small amounts and multiple times, heating and stirring continuously until the PVA powder is completely dissolved, to obtain a polyvinyl alcohol (PVA) solution with a mass percentage of 10%; dissolving human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the polyvinyl alcohol (PVA) solution to obtain a third mixed solution, wherein the concentration of human umbilical cord mesenchymal stem cell exosomes (HUMSC-exos) in the third mixed solution is 9 x 10 10 The third mixed solution was injected into the PDMS mold, and centrifuged at a speed of 4000r / min for 3 min to completely fill the needle cavity with the third mixed liquid. The liquid outside the needle body was removed, and then the polyvinyl alcohol (PVA) solution was injected into the mold to form a substrate, which was frozen at -20°C for 2 h. After complete forming, the microneedle patch was separated from the PDMS mold, and after demolding, it was frozen at -80°C for 2 h to obtain a microneedle patch.

10. An application of the anti-scar freezing microneedle patch according to any one of claims 1 to 6, characterized in that: The anti-scar cryomicroneedle patch is used in the preparation of a drug for treating hypertrophic scars. The anti-scar cryomicroneedle patch has the functions of reducing inflammation, remodeling the mechanical microenvironment and regulating metabolism.

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