A double-layer nested structure microneedle and application thereof

Abstract

The application provides a double-layer nested structure microneedle and application thereof, and belongs to the technical field of biomedical materials. The double-layer nested structure microneedle comprises a template microneedle and a nested layer. The nested layer covers the tip of the template microneedle. The nested layer comprises tea polysaccharide-manganese dioxide@polydopamine nanoparticles and a nested layer matrix.

Description

TECHNICAL FIELD

[0001] The application relates to a double-layer nested structure microneedle and application thereof, and belongs to the technical field of biomedical materials. BACKGROUND

[0002] Microneedle (MN) technology is an innovative drug delivery method that uses micron-sized fine needles to penetrate the stratum corneum in a nearly painless manner, delivering drugs directly to the dermis. The height of these microneedles is carefully controlled between 100-1000 mu m to ensure that they can effectively penetrate the stratum corneum without touching the pain nerves, thus achieving painless drug delivery. Compared with traditional injection methods, microneedle technology is difficult to be detected by the naked eye due to its small size. Moreover, the microneedle drug delivery system is easy to operate and can be performed by non-professional medical personnel, which is suitable for self-administration at home or community medical services. In addition, the design of the microneedle array enables drugs to penetrate the skin barrier more efficiently and directly act on the target area, thereby improving the therapeutic effect.

[0003] The advantages of painless, minimally invasive, high efficiency and patient friendliness of the microneedle drug delivery system have made it a research hotspot in the field of transdermal drug delivery, and the microneedle drug delivery system has shown great potential and broad application prospects in the field of transdermal drug delivery systems.

[0004] Hair loss is defined as partial hair loss on the head due to various causes such as drugs, stress and autoimmune diseases. SUMMARY

[0005] The application provides a double-layer nested structure microneedle and application thereof, which can effectively solve the above problems.

[0006] The application is implemented as follows:

[0007] A preparation method of tea polysaccharide-manganese dioxide@polydopamine nanoparticles, comprising:

[0008] Potassium permanganate solution and tea polysaccharide solution are mixed uniformly at room temperature, and reacted under stirring to obtain a tea polysaccharide-manganese dioxide nanoparticle solution;

[0009] An alkaline aqueous solution with a pH value of 8-9.5 is added to the tea polysaccharide-manganese dioxide nanoparticle solution, and after being uniformly mixed, a dopamine hydrochloride solution is added, and reacted under stirring. After the reaction is completed, the precipitate is centrifuged, washed and freeze-dried to obtain tea polysaccharide-manganese dioxide@polydopamine nanoparticles.

[0010] Tea polysaccharide-manganese dioxide@polydopamine nanoparticles prepared by the above method.

[0011] The tea polysaccharide-manganese dioxide@polydopamine nanoparticle is used for preparing a medicament for promoting hair growth.

[0012] A double-layer nested structure microneedle comprises a template microneedle and a nested layer covering the tip of the template microneedle, and the nested layer comprises the tea polysaccharide-manganese dioxide@polydopamine nanoparticle prepared by the method and a nested layer matrix.

[0013] A preparation method of the double-layer nested structure microneedle comprises the following steps: mixing a polysaccharide derivative, a solvent, a photoinitiator and the tea polysaccharide-manganese dioxide@polydopamine nanoparticle to obtain a mixed hydrogel.

[0014] The mixed hydrogel is placed in a microneedle concave mold, centrifugation is performed, the mixed hydrogel fills the needle cavity in the microneedle concave mold, and the excess mixed hydrogel is removed.

[0015] After the tip of the template microneedle is aligned with the needle cavity, the template microneedle is inserted, and then ultraviolet light curing is performed to obtain the double-layer nested structure microneedle.

[0016] The double-layer nested structure microneedle or the double-layer nested structure microneedle prepared by the method is used for preparing a medicament for promoting hair growth.

[0017] The tea polysaccharide-manganese dioxide@polydopamine nanoparticle prepared by the method has the following beneficial effects:

[0018] The preparation method of the tea polysaccharide-manganese dioxide@polydopamine nanoparticle provided by the application can slowly release the nanoparticles in the dermis, so that the purpose of step-by-step administration is achieved, and the released tea polysaccharide-manganese dioxide nanoparticles can remove excessive active oxygen around hair follicles.

[0019] The double-layer nested structure microneedle provided by the application can penetrate the dermis and insert and remain in the dermis, the nested layer comprises the tea polysaccharide-manganese dioxide@polydopamine nanoparticle, can remove excessive active oxygen around hair follicles, and the mechanical stress of the microneedle can stimulate hair follicles, so that the regeneration of hair is promoted.

[0020] The nested layer matrix provided by the application is a cross-linked hydrogel of polysaccharide derivatives, has good biocompatibility, and can become an ideal carrier for loading and releasing drugs, the nested layer matrix has a cross-linked network structure, can achieve slow release of drugs when applied to microneedles, and the reuse of various polysaccharide derivatives can improve the mechanical strength of the nested layer and the effect of the double-layer nested structure microneedle on penetrating the skin.

[0021] The micro-needle concave mold and the template micro-needle are prepared from acrylic resin by 3D printing technology, the geometric parameters of the template micro-needle can be designed according to different hair loss areas, appearances and degrees, so that the customization of the micro-needle is realized, and the manufacturing flexibility and efficient material utilization rate are achieved.

[0022] Meanwhile, the acrylic resin used in the micro-needle matrix is a biocompatible resin, which can cause appropriate reactions when implanted in vivo or in contact with biological tissues, and cannot cause excessive inflammatory reactions, rejection reactions or allergic reactions and other adverse phenomena. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0024] Figure 1 The preparation process schematic diagram of the double-layer nested structure micro-needle provided by the embodiments of the present application.

[0025] Figure 2 The particle size distribution diagram of the TPs-MnO2@PDA NPs provided by the embodiments of the present application.

[0026] Figure 3 In the figure, (a) is the SEM image of the template micro-needle provided by the embodiments of the present application; (b) is the SEM image of the double-layer nested structure micro-needle.

[0027] Figure 4 The comparison diagram of the hair growth effect of different schemes on mice for 14 days provided by the embodiments of the present application. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely in the following by combining the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0029] The embodiment of the present application provides a preparation method of tea polysaccharide-manganese dioxide@polydopamine nanoparticles, which comprises the following steps:

[0030] Potassium permanganate solution and tea polysaccharide solution are mixed uniformly at room temperature, and then the mixture is reacted under stirring to obtain a tea polysaccharide-manganese dioxide nanoparticle solution;

[0031] An alkaline aqueous solution with a pH value of 8-9.5 is added into the tea polysaccharide-manganese dioxide nanoparticle solution, and then a dopamine hydrochloride solution is added after uniform mixing, and the mixture is reacted under stirring; after the reaction is completed, the mixture is centrifuged to obtain a precipitate; the precipitate is washed and then freeze-dried to obtain tea polysaccharide-manganese dioxide@polydopamine nanoparticles.

[0032] A sufficient amount of alkaline aqueous solution is added to provide an alkaline environment and promote the reaction.

[0033] The dopamine hydrochloride can be self-polymerized to form PDA under alkaline conditions, and the PDA is wrapped on the surface of the tea polysaccharide-manganese dioxide.

[0034] In some embodiments, the alkaline aqueous solution is ammonia water or a sodium hydroxide aqueous solution.

[0035] In some embodiments, the alkaline aqueous solution is an alkaline buffer solution.

[0036] In some embodiments, the alkaline aqueous solution is a Tris buffer solution or a bicarbonate buffer solution.

[0037] In some embodiments, the mass ratio of the amount of potassium permanganate to the amount of tea polysaccharide is 0.09-0.2:1.

[0038] If the amount of potassium permanganate is too much, the tea polysaccharide-manganese dioxide nanoparticles cannot be obtained through the reaction; and if the amount of potassium permanganate is too little, the yield is affected.

[0039] In some embodiments, the mass ratio of the amount of dopamine hydrochloride to the amount of tea polysaccharide is 0.09-0.2:1.

[0040] Using the mass ratio, the nanoparticles prepared have good stability and uniform particle diameter.

[0041] If the amount of dopamine hydrochloride is too much, the nanoparticles will be aggregated together; and if the amount of dopamine hydrochloride is too little, the polydopamine cannot completely wrap the tea polysaccharide-manganese dioxide nanoparticles.

[0042] The embodiment of the present application provides tea polysaccharide-manganese dioxide@polydopamine nanoparticles prepared by the method described in any one of the above embodiments.

[0043] The structure of the nanoparticles is that of a polydopamine-coated surface of a theaflavonoid-manganese dioxide nanoparticle, and the polydopamine is slowly dissolved after absorbing interstitial fluid, and the theaflavonoid-manganese dioxide nanoparticle wrapped by the polydopamine is also released, achieving the effect of gradual release of drugs.

[0044] The theaflavonoid-modified manganese dioxide nanoparticle has good biocompatibility and hydrophilicity, and is uniformly and stably present in a solution.

[0045] The released theaflavonoid-manganese dioxide nanoparticle can scavenge excessive reactive oxygen species (ROS) around hair follicles.

[0046] The application of the theaflavonoid-manganese dioxide@polydopamine nanoparticle in the preparation of a drug for promoting hair growth is provided.

[0047] In some embodiments, the application is that of the theaflavonoid-manganese dioxide@polydopamine nanoparticle in the preparation of a drug for treating alopecia.

[0048] In some embodiments, the application is that of the theaflavonoid-manganese dioxide@polydopamine nanoparticle in the preparation of a drug for treating androgenetic alopecia.

[0049] The application provides a double-layer nested structure microneedle, which comprises a template microneedle and a nested layer, the nested layer covers the tip of the template microneedle, and the nested layer comprises theaflavonoid-manganese dioxide@polydopamine nanoparticles and a nested layer matrix.

[0050] The nested layer covers the tip of the template microneedle, and the two have an overlapping part, forming a double-layer nested structure.

[0051] In some embodiments, the double-layer nested structure microneedle has a conical or quadrangular conical shape, that is, the tip of the template microneedle and the nested layer both have a conical shape or both have a quadrangular conical shape. Both shapes can effectively penetrate the skin.

[0052] The use method of the double-layer nested structure microneedle is as follows: the double-layer nested structure microneedle is directly attached to the alopecia area, the back plate of the double-layer nested structure microneedle is pressed to make the tip of the double-layer nested structure microneedle penetrate the skin, the pressing is maintained for 1 min, and then the double-layer nested structure microneedle is fixed and retained for 4-5 min, so that the nested layer absorbs interstitial fluid and falls off, is separated from the template microneedle, and then the template microneedle is removed.

[0053] In use, the double-layer nested structure microneedle can effectively penetrate the skin to reach the dermis, after a period of action, the template microneedle is automatically separated from the nested layer, and the nested layer can remain in the dermis when the template microneedle is removed from the skin.

[0054] The nested layer comprises tea polysaccharide-manganese dioxide@polydopamine nanoparticles, and as described above, the polydopamine is slowly dissolved after absorbing interstitial fluid, and releases the tea polysaccharide-manganese dioxide nanoparticles wrapped therein, so that the drug is released in stages.

[0055] The released tea polysaccharide-manganese dioxide nanoparticles can remove excessive reactive oxygen species (ROS) around the hair follicle, and at the same time, the mechanical stress of the microneedle can stimulate the hair follicle, and the two work together to promote the regeneration of hair.

[0056] Compared with the traditional drug delivery method, the use of the double-layer nested structure microneedle reduces the drug delivery frequency, realizes the directional delivery of the drug, improves the therapeutic effect of the drug, and has great application prospect.

[0057] In some embodiments, the nested layer comprises a cross-linked hydrogel of polysaccharide derivatives.

[0058] The nested layer is quickly prepared by mixing the hydrogel of polysaccharide derivatives and nanoparticles and then being cured by ultraviolet light.

[0059] The application of the hydrogel of polysaccharide derivatives in the microneedle can achieve the slow release effect of the drug or nanoparticles.

[0060] After the nested layer reaches the dermis layer, the cross-linked hydrogel of polysaccharide derivatives is gradually dissolved, and then the tea polysaccharide-manganese dioxide@polydopamine nanoparticles are released in stages, so as to further achieve the effect of controlling the drug release time.

[0061] In some embodiments, the polysaccharide-based derivative is one or more of methacrylated gelatin, methacrylated hyaluronic acid, methacrylated dextran, methacrylated silk fibroin, and methacrylated chondroitin sulfate.

[0062] These materials not only have good biocompatibility, but also can be an ideal carrier for loading and releasing drugs or nanoparticles.

[0063] The hydrogel material after being cured by a single polysaccharide-based derivative has a fast dissolution rate, and the reuse of multiple polysaccharide-based derivatives forms a cross-linked network structure, and at the same time, the mechanical strength of the nested layer is improved, so that the effect of the double-layer nested structure microneedle penetrating the skin is improved.

[0064] The embodiment of the present application provides a preparation method of the double-layer nested structure microneedle described in any one of the above embodiments, comprising:

[0065] Mixing the polysaccharide-based derivative, the solvent, the photoinitiator, and the tea polysaccharide-manganese dioxide@polydopamine nanoparticles to obtain a mixed hydrogel;

[0066] placing the mixed hydrogel into a microneedle concave mold, centrifuging to fill the needle cavities in the microneedle concave mold with the mixed hydrogel, and removing excess mixed hydrogel;

[0067] aligning the tips of the template microneedles with the needle cavities, inserting the template microneedles, and then performing ultraviolet curing to obtain the double-layer nested structure microneedles.

[0068] The mixed hydrogel forms the nested layer after ultraviolet curing.

[0069] In some embodiments, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), the polysaccharide-based derivative is one or more of methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA), methacrylated dextran (DexMA), methacrylated silk fibroin (SFMA), and methacrylated chondroitin sulfate (CHSMA), and the solvent is a phosphate (PBS) buffer solution.

[0070] The photoinitiator LAP has good biocompatibility and can rapidly initiate the curing of photosensitive hydrogel materials under blue light (wavelength 405 nm), and is suitable for use in the field of biocompatible materials.

[0071] The use of a PBS buffer solution can maintain the pH value of the mixed hydrogel within its buffer range (usually 7.2-7.4), and can maintain the stability of the nanoparticles, so that they are uniformly distributed in the solution, thereby improving the reaction efficiency.

[0072] In some embodiments, a polysaccharide-based derivative with a molecular weight of 200-400 kDa is selected.

[0073] Polysaccharide-based derivatives with a small molecular weight are not easily cured under blue light, and those with a large molecular weight have poor solubility, so a medium molecular weight range is generally selected.

[0074] In some embodiments, the mass ratio of the polysaccharide-based derivative, the PBS buffer solution, and the photoinitiator LAP is 10-20:90-100:0.3-0.7.

[0075] In some embodiments, the mass ratio of the polysaccharide-based derivative, the PBS buffer solution, the photoinitiator LAP, and the TPs-MnO2@PDANPs is 10-20:90-100:0.3-0.7:0.01-1.

[0076] The double-layer nested structure microneedles obtained by the above preparation method have good formability, high mechanical strength, stable drug loading, and a simple process, and can be customized for individual hair loss conditions.

[0077] By adjusting the proportion of the polysaccharide derivative components in the nested layer and the specific composition of the polysaccharide derivative components, the swelling and degradation performance of the nested layer matrix in the dermis layer can be controlled, so as to control the release speed of the drug or the nanoparticles.

[0078] In some embodiments, the polysaccharide-based derivative is GelMA and HAMA.

[0079] In some embodiments, the mass ratio of GelMA to HAMA is 10:1.

[0080] By adjusting the proportion between the plurality of polysaccharide-based derivatives, the degradation time of the nested layer matrix can be controlled.

[0081] In some embodiments, the grafting rate of GelMA is 60%-80%, and the grafting rate of HAMA is 15%-20%.

[0082] Using polysaccharide derivatives with a higher grafting rate can improve the speed of cross-linking and curing under blue light, and obtain a cross-linked hydrogel of the polysaccharide derivative with better molding effect.

[0083] In some embodiments, the mass fraction of TPs-MnO2@PDA NPs in the mixed hydrogel is 0.01%-1%.

[0084] The drug loading capacity of the nested layer is controllable, and accordingly, the relative proportion of the nested layer matrix is also controllable, so as to realize the control of the release speed of the drug or the nanoparticles and the regional drug action amount of each tip.

[0085] In some embodiments, the mass fraction of TPs-MnO2@PDA NPs in the mixed hydrogel is 0.5%.

[0086] In some embodiments, the mass ratio of GelMA, HAMA, a photoinitiator LAP, and TPs-MnO2@PDA NPs is 200:20:5:0.5.

[0087] In some embodiments, the ultraviolet light curing time is 10-20 s.

[0088] In some embodiments, the height of the template microneedle is 600-605 μm, the height of the nested layer is 595-600 μm, and the height of the microneedle is 895-905 μm.

[0089] The nested layer covers the tip of the template microneedle to form a nested structure with an overlapping part.

[0090] In some embodiments, the preparation method of the template microneedle comprises:

[0091] The microneedle concave mold and the template microneedle are obtained by light-cured 3D printing, wherein the microneedle concave mold and the template microneedle respectively comprise a microneedle matrix, and the microneedle matrix comprises an acrylic resin.

[0092] The light-cured 3D printing specifically comprises the following steps:

[0093] (1) By analyzing the hair loss area and morphology, the geometric parameters of the template microneedle are designed by using 3D modeling software, and a microneedle concave mold with corresponding size is designed.

[0094] (2) The slicing software matched with the 3D printer is used for slicing, and the parameters and procedures of 3D printing are set.

[0095] (3) The acrylic resin is used as the material for 3D printing.

[0096] (4) Start light-cured 3D printing, and then obtain a microneedle array and a corresponding microneedle concave mold.

[0097] (5) The microneedle array obtained in step (4) is washed and placed under ultraviolet light for continuous irradiation for 2-3 min to enhance its mechanical properties, and the template microneedle is obtained.

[0098] The template microneedle comprises a back plate and a tip forming an array.

[0099] The template microneedle prepared by combining 3D printing technology and biocompatible resin has the advantages of manufacturing flexibility, high material utilization rate, significantly shortened research and development period, and good biocompatibility.

[0100] The preparation process by 3D printing is simple, and the size and shape can be finely customized, which is suitable for various regular and irregular skin surfaces and different degrees of hair loss, and can give a customized microneedle design scheme.

[0101] The microneedle matrix and the nested layer matrix both use biocompatible resin, which can cause appropriate reactions when implanted in the body or in contact with biological tissues, and will not cause adverse phenomena such as excessive inflammatory response, rejection reaction or allergic reaction.

[0102] Further, the acrylic resin used in the microneedle matrix has good biocompatibility, excellent antibacterial property, corrosion resistance, adjustable physical and chemical properties, and superior processing performance, which has significant advantages and wide application prospects in the medical and health fields compared with other biocompatible resins.

[0103] An acrylic resin suitable for 3D light-cured printing is selected.

[0104] In some embodiments, the acrylic resin is one or more of ethylene glycol diacrylate, poly(alkylene) glycol dimethacrylate, methyl methacrylate.

[0105] The application provides application of the double-layer nested structure microneedle in any of the above embodiments in preparation of a drug for promoting hair growth.

[0106] The application provides application of the double-layer nested structure microneedle prepared by the method in any of the above embodiments in preparation of a drug for promoting hair growth.

[0107] In some embodiments, the application is application of the double-layer nested structure microneedle in preparation of a drug for treating alopecia.

[0108] In some embodiments, the application is application of the double-layer nested structure microneedle in preparation of a drug for treating androgenetic alopecia.

[0109] Embodiment 1

[0110] Preparation of the double-layer nested structure microneedle:

[0111] 1. Preparation of the template microneedle and the microneedle concave mold:

[0112] As shown in the specific steps include: Figure 1

[0113] (1) By analyzing the area and morphology of alopecia, the geometric parameters of the template microneedle are designed by using 3D modeling software, and the microneedle concave mold with corresponding size is designed.

[0114] The geometric parameters of the template microneedle are specifically: height 600 μm, bottom diameter 200 μm, needle spacing 500 μm, array 15x15, 225 sharp tips, and the shape of the sharp tip is a quadrangular pyramid.

[0115] (2) The slicing software matched with the 3D printer is used for slicing, and the parameters and procedures of 3D printing are set.

[0116] (3) The biocompatible resin purchased from Shenzhen Zong Weicun Technology Co., Ltd. is used as the material for 3D printing. The main components of the resin include caprolactone acrylate, poly(alkylene) glycol dimethacrylate and polyethylene glycol diacrylate.

[0117] (4) Start light curing 3D printing, and then obtain the microneedle array and the corresponding microneedle concave mold.

[0118] (5) The microneedle array obtained in step (4) is washed and placed under ultraviolet light for continuous irradiation for 3 min, so as to enhance the mechanical properties, and the template microneedle is obtained.​

[0119] 2. Preparation of tea polysaccharide-manganese dioxide@polydopamine nanoparticles (TPs-MnO2@PDA NPs):

[0120] Specifically comprising the following steps:

[0121] (1) Using tea polysaccharide (TPs) extracted from green tea leaves, the TPs were dissolved in deionized water to prepare a 20 mg / mL solution for standby.

[0122] In addition, 10 mM potassium permanganate (KMnO4) solution, 20 mM Tris buffer solution with pH=8.8 and 1 mg / mL dopamine hydrochloride (DA) solution were prepared for standby.

[0123] (2) 10 mL of 10 mM KMnO4 solution and 5 mL of 20 mg / mL TPs solution were stirred at room temperature for 1 h to obtain TPs-MnO2 NPs.

[0124] KMnO4 will be reduced to MnO2 nanoparticles (NPs) by TPs, suspended in the solution, showing a colloidal state, and the solution will change from original purple red to yellow brown, proving that TPs-MnO2 NPs have been synthesized.

[0125] (3) 10 mL of 20 mM Tris buffer solution with pH=8.8 was added to the solution obtained in step (2), and finally 10 mL of 1 mg / mL DA solution was added, and the reaction was stirred for 24 h. After the reaction was completed, the solution was centrifuged at 8000 rpm for 10 min using a high-speed centrifuge to obtain TPs-MnO2@PDA NPs. The solution was washed with deionized water for 3 times until the supernatant was colorless, and the lower precipitate part was freeze-dried for standby.

[0126] As shown in Figure 2 , it can be seen that the average particle diameter of the prepared TPs-MnO2@PDA NPs is about 156.7±2.9 nm.

[0127] 3. Preparation of double-layer nested structure microneedle:

[0128] As shown in Figure 1 , specifically comprising the following steps:

[0129] (1) The TPs-MnO2@PDA NPs, phosphate buffer solution (PBS), methacrylated gelatin (GelMA), methacrylated hyaluronic acid (HAMA) and photoinitiator LAP prepared above are configured into a mixed hydrogel in a mass ratio of 0.5:774.5:200:20:5, wherein the concentration of TPs-MnO2@PDA NPs is 500 μg / mL.

[0130] The grafting rate of GelMA is 65%, and the grafting rate of HAMA is 18%.

[0131] (2) The mixed hydrogel is injected into the microneedle concave mold prepared above, and after centrifugation at 4000 rpm for 5 min, the mold needle cavity is completely filled, and the excess liquid in the mold is scraped off.

[0132] (3) The mold template microneedle prepared above is aligned and pressed into the drug-loaded mold needle cavity, and after adjusting the height of the mold template microneedle, it is placed under a UV lamp for 20 s for curing to form a double-layer nested structure microneedle. The double-layer nested structure microneedle is demolded and taken out.

[0133] 4. Microneedle morphology characterization:

[0134] The mold template microneedle and the double-layer nested structure microneedle prepared above are respectively placed on the sample table with conductive glue, gold spraying treatment is performed, and then the sample is placed in an electron scanning microscope. The machine is vacuumized, and after the machine is stable, the surface morphology of the sample is observed. The experimental results are shown in Figure 3 .

[0135] Figure 3 (a) is the SEM image of the mold template microneedle, the tip is 600 μm high, the bottom diameter is 200 μm, and the needle spacing is 500 μm. Figure 3 (b) is the SEM image of the double-layer nested structure microneedle, the total height is 900 μm, the bottom diameter is 200 μm, and the overlapping part of the nested layer and the mold template microneedle is 300 μm high.

[0136] Compared with the mold template microneedle, the double-layer nested structure microneedle is higher by a section, and has a obvious overlapping structure, indicating that the double-layer nested structure microneedle is successfully prepared.

[0137] Example 2

[0138] Evaluation of the efficacy of the double-layer nested structure microneedle for preparing drugs for promoting hair growth

[0139] The mice used in the examples of the present application are C57BL / 6 (male, 6 weeks old).

[0140] The mice were anesthetized with chloral hydrate (10%) and a 1 cm x 1 cm area was shaved on the back of the mice along the dorsal midline using a pet clipper. The depilatory cream was evenly applied to the area and washed off after 5 min with warm water.

[0141] After depilation, the mice were randomly divided into a model group, a blank microneedle group, and a double-layer nested structure microneedle group.

[0142] Then, 100 μL of a 0.5% testosterone solution (50% ethanol as a solvent, w / v) was applied daily to the depilated area for 28 days (one hair cycle) to inhibit the transition of hair follicles from the resting phase to the growth phase.

[0143] Model group: In addition to the daily application of the testosterone solution described above, no additional treatment was performed.

[0144] External nano-drug group: In addition to the daily application of the testosterone solution described above, 100 μL of a TPs-MnO2@PDA (100 μg / mL) solution was applied to the depilated area on days 1, 4, 7, 10, and 13 after depilation.

[0145] Blank microneedle group:

[0146] The difference between the blank microneedle and the double-layer nested structure microneedle in Example 1 is that:

[0147] The nested layer only includes the nested layer matrix, and no nanoparticles are loaded in the nested layer matrix.

[0148] Preparation of the blank microneedle:

[0149] The difference from Example 1 is that:

[0150] The PBS buffer solution, GelMA, HAMA, and photoinitiator LAP were configured into a mixed hydrogel at a mass ratio of 774.5:200:20:5, wherein the concentration of GelMA was 200 mg / mL.

[0151] The remaining steps are the same as in Example 1.

[0152] In addition to the daily application of the testosterone solution described above, one blank microneedle was administered to the depilated area on days 1, 4, 7, 10, and 13 after depilation. The tip of the microneedle was pressed into the skin with the thumb for 1 min, and then remained for 4 min before being removed.

[0153] Double-layer nested structure microneedle group: In addition to the daily application of the testosterone solution described above, one double-layer nested structure microneedle prepared in Example 1 was administered to the depilated area on days 1, 4, 7, 10, and 13 after depilation. The tip of the microneedle was pressed into the skin with the thumb for 1 min, and then remained for 4 min before being removed.

[0154] As shown in Figure 4 The skin of each group of mice was pink on the first day after depilation, indicating that the hair follicles in the depilation area of each group of mice were in the resting phase at this time. After different treatment regimens, on the 14th day after depilation, the model group of mice did not have hair growth, and the skin remained pink, indicating that the testosterone-induced androgenetic alopecia mouse model was basically established. The nano drug external use group, the blank microneedle group and the double-layer nested structure microneedle group all grew hair on the 14th day.

[0155] Compared with the model group, the nano drug external use group has a certain hair growth effect, which shows that the external use of TPs-MnO2@PDA NPs can release TPs-MnO2NPs, and TPs-MnO2NPs can remove excessive reactive oxygen species (ROS) around the hair follicle, and has a certain hair growth effect.

[0156] Compared with the model group, the blank microneedle group also has a certain hair growth effect, which is because the mechanical stress generated by the blank microneedle when it is pierced promotes the regeneration of the hair follicle.

[0157] And the hair density of the double-layer nested structure microneedle group on the 14th day is much higher than that of other groups, and the treatment effect is very significant, which shows that the double-layer nested structure microneedle has a good effect on treating androgenetic alopecia.

[0158] At the same time, the nano drug in the double-layer nested structure microneedle and the microneedle administration method have a synergistic effect.

[0159] On the 14th day, the new hair coverage rate of each group was counted by ImageJ, and it was found that the new hair coverage rate of the double-layer nested structure microneedle group (57.57%) was significantly higher than that of the model group (2.2%), the nano drug external use group (24.5%) and the blank microneedle group (33.2%).

[0160] The embodiment of the application provides a separable double-layer nested structure microneedle, compared with a traditional treatment scheme, the microneedle administration method has the advantages of minimally invasive, painless, low drug administration frequency, weak drug dependence, good treatment effect and the like. At the same time, due to the addition of the 3D printing technology, the flexibility of the microneedle design is greatly enhanced, the material utilization rate is improved, and the preparation period is shortened.

[0161] The double-layer nested structure microneedle provided by the embodiment of the application greatly enhances the flexibility of the microneedle design, improves the material utilization rate, and shortens the preparation period by using the 3D printing technology.

[0162] The double-layer nested structure microneedle improves the acceptance and compliance of patients by the painless and minimally invasive characteristics of the microneedle administration, and avoids the first-pass effect and gastrointestinal degradation of oral drugs, and ensures the bioavailability of the drugs. The double-layer nested structure microneedle also realizes the precise control and sustained release of the drugs, prolongs the drug action time, reduces the drug administration frequency, has the advantages of weak drug dependence and good treatment effect, and provides a more convenient, safe and effective drug administration method for the medical field.

[0163] The double-layer nested structure microneedle has good biocompatibility of the material, reduces the risk of infection, and improves the safety of treatment.

[0164] The nested layer of the double-layer nested structure microneedle can be completely separated after penetrating into the dermis, and only the nested layer remains in the dermis, so that the drug administration process is almost traceless.

[0165] Further, the treatment scheme of the double-layer nested structure microneedle can also be applied to other drugs.

[0166] The above only describes the preferred embodiments of the present application and is not used to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A preparation method of tea polysaccharide-manganese dioxide@polydopamine nanoparticles, characterized in that, Comprising: Mixing potassium permanganate solution and tea polysaccharide solution uniformly at room temperature, and reacting under stirring to obtain tea polysaccharide-manganese dioxide nanoparticle solution, the mass ratio of the amount of potassium permanganate to the amount of tea polysaccharide being 0.09-0.2:1; Adding an alkaline aqueous solution with pH=8-9.5 to the tea polysaccharide-manganese dioxide nanoparticle solution, mixing uniformly, then adding dopamine hydrochloride solution, and reacting under stirring, and obtaining a precipitate by centrifugation after the reaction is completed, and obtaining tea polysaccharide-manganese dioxide@polydopamine nanoparticles by freeze-drying after washing the precipitate, the mass ratio of the amount of dopamine hydrochloride to the amount of tea polysaccharide being 0.09-0.2:

1. 2.Tea polysaccharide-manganese dioxide@polydopamine nanoparticles prepared by the method of claim 1. 3.The use of the tea polysaccharide-manganese dioxide@polydopamine nanoparticles of claim 2 in the preparation of a drug for promoting hair growth.

4. A double-layer nested structure microneedle, characterized in that, The double-layer nested structure microneedle comprises a template microneedle and a nested layer, the nested layer covers the tip of the template microneedle, the nested layer comprises the tea polysaccharide-manganese dioxide@polydopamine nanoparticles prepared by the method of claim 1 and a nested layer matrix, and the nested layer matrix is one or more of methacrylated gelatin, methacrylated hyaluronic acid, methacrylated dextran, methacrylated silk fibroin and methacrylated chondroitin sulfate.

5. A method of making the double-layer nested structure microneedle of claim 4, wherein, Comprising: Mixing the nested layer matrix, a solvent, a photoinitiator and the tea polysaccharide-manganese dioxide@polydopamine nanoparticles to obtain a mixed hydrogel; Placing the mixed hydrogel in a microneedle concave mold, centrifuging to make the mixed hydrogel fill the needle cavities in the microneedle concave mold, and removing the excess mixed hydrogel; After aligning the tip of the template microneedle with the needle cavities, inserting the template microneedle, and then performing ultraviolet light curing, the double-layer nested structure microneedle is demolded.

6. The method of claim 5, wherein, The mass fraction of the tea polysaccharide-manganese dioxide@polydopamine nanoparticles in the mixed hydrogel is 0.01%-1%.

7. The method of claim 5, wherein, The photoinitiator is lithium phenyl(2, 4, 6-trimethylbenzoyl) phosphate, and the solvent is a phosphate buffer solution.

8. The method of claim 5, wherein, Comprising: The preparation method of the template microneedle comprises: The microneedle concave mold and the template microneedle are obtained by photocuring 3D printing, wherein the microneedle concave mold and the template microneedle respectively comprise a microneedle matrix, and the microneedle matrix comprises an acrylic resin. 9.The use of the double-layer nested structure microneedle of claim 4, or the double-layer nested structure microneedle prepared by the method of any one of claims 5-8 in the preparation of a drug for promoting hair growth.

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