Rapid bubble separation type responsive hydrogel microneedle and preparation method thereof

Abstract

The invention discloses a quick bubble separation type responsive hydrogel microneedle and a preparation method thereof. The microneedle structurally comprises a needle tip layer, a separation layer and a substrate layer, the needle tip layer is prepared from ROS (reactive oxygen species) responsive hydrogel formed by spontaneous crosslinking of modified beta-cyclodextrin, a high-molecular polymer with polyhydroxy and a synthesized micromolecular crosslinking agent; the separation layer is prepared from a high-molecular polymer with a polar group and small-molecular weak acid; the substrate layer is made of a high-molecular polymer material; bubbles exist between the needle point layer and the substrate layer. According to the design of the bubble microneedle, bubbles appear between the needle tip layer and the substrate layer to realize rapid separation, stimulation and anaphylaxis caused by long-time adhesion of a traditional microneedle to the skin are avoided, and the needle tip is effectively retained in the skin to become a responsive drug release reservoir. The bubble separation type responsive hydrogel microneedle disclosed by the invention can improve the solubility and the stability of drugs, and is beneficial to delivery of insoluble drugs and unstable protein drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of new biological medicine preparations, and in particular to a rapid bubble separation type responsive hydrogel microneedle and a preparation method thereof. BACKGROUND

[0002] Proteins and nucleic acid biological macromolecule drugs are increasingly widely used in the medical field, however, their clinical application is limited by various factors. First, such drugs are susceptible to first-pass effect, endogenous enzyme degradation and physiological pH environment, resulting in reduced biological activity. Second, exogenous macromolecules have certain immunogenicity, and direct blood entry can cause acute allergic reactions. In addition, some small molecule drugs have poor solubility, narrow therapeutic window and significant adverse reactions when administered systemically.

[0003] As the most promising new drug delivery technology, microneedle transdermal drug delivery system has the advantages of high bioavailability of injection drug delivery and safety, convenience and patient compliance of traditional transdermal drug delivery. This technology can significantly improve the efficiency of transdermal drug delivery by penetrating the stratum corneum barrier. The advantages of the microneedle system mainly include the following aspects: (1) its length can be precisely controlled, forming a micron-scale channel that only acts on the epidermis, avoiding stimulation of dermal nerve endings, so the pain is significantly reduced compared with traditional injection; (2) easy to operate, no need for professional medical personnel assistance; (3) prepared from biocompatible polymer materials, with mild process conditions, and drug loaded in solid form, which is conducive to maintaining the stability of biological macromolecules; (4) by adjusting the composition and ratio of responsive cross-linked polymer materials, loading pathological related proteins or enzymes (such as uricase and glucose oxidase, etc.), enzyme decomposing physiological small molecules in blood, such as uric acid and glucose, releasing H2O2 to promote the degradation of responsive cross-linked polymers, achieving pathological response release of drugs, and achieving real-time control of diseases.

[0004] The invention patent with application number 201610080970.6 discloses an active separation type soluble microneedle, which is provided with an intermediate layer between the tip of the traditional microneedle and the base layer. When the microneedle is used, the intermediate layer contacts the moisture on the surface of the skin and a small amount of tissue fluid exuded from the skin, the intermediate layer dissolves, the tip can actively separate from the base, and remains in the skin, reaches the dermis and releases the drug into the skin below the stratum corneum.

[0005] However, the scheme still has the problem of low efficiency of traditional soluble microneedle delivery of drugs. Because the skin has elasticity, the needle body is difficult to fully penetrate the skin, the pores caused by the microneedle are closed due to the self-healing of the skin in a short time, the drugs remaining on the surface of the skin are lost or cannot penetrate into the body due to external contact, resulting in waste of drugs, reduced efficiency of transdermal drug delivery, and inaccurate drug dosage. And when used on a relatively dry skin surface, the amount of interstitial fluid exuded from the skin is limited, which may prolong the dissolution rate of the intermediate layer. Moreover, the scheme does not design the needle body remaining in the skin to control the slow release of drugs.

[0006] The invention patent with the application number 200980139874.2 discloses a phase inversion polymer microneedle, proteins, polypeptide genes or other water-soluble drug substances are dispersed in a hydrophilic polymer material, which is in a glass state when dry, and the microneedle absorbs body fluid to change into a hydrogel state after piercing the epidermis, achieving the purpose of sustained and controlled release of drugs.

[0007] However, the microneedle of the scheme only uses a hydrophilic polymer, and still has the problem of poor stability of poorly soluble drugs in the hydrophilic polymer. It is also difficult to co-load two drugs with different physicochemical properties for collaborative treatment of diseases. SUMMARY

[0008] In order to overcome the shortcomings of the prior art, the present application provides a rapid bubble separation type responsive hydrogel microneedle which is beneficial to improve the solubility of drugs, stabilize drugs and release drugs in response.

[0009] The first aspect of the present application provides a bubble separation type responsive hydrogel microneedle, the structure of the microneedle includes a needle tip layer, a separation layer and a base layer, the needle tip layer is dispersed with a drug-loaded inclusion compound, the drug-loaded inclusion compound is formed by inclusion of drugs after modification of β-cyclodextrin;

[0010] The separation layer is prepared from a high molecular polymer with a polar group and a small molecule weak acid;

[0011] The base layer is a high molecular polymer material;

[0012] There is a bubble between the needle tip layer and the base layer.

[0013] Preferably, the structure of the microneedle from the needle tip layer to the separation layer is a cone, a cylinder in turn, and the base layer covers the other bottom surface of the cylinder; further preferably, the height of the cylinder is 300-600 μm, the height of the cone is 300-800 μm, and the total height of the cone and the cylinder is not more than 1200 μm; in some specific embodiments of the present application, the diameter of the cone and the cylinder is 300 μm, and the height of the cone and the cylinder is 600 μm.

[0014] Preferably, the needle tip layer is formed by a drug-loaded inclusion complex and a polymer material crosslinked by a crosslinking agent; the drug-loaded inclusion complex is formed by a β-cyclodextrin modified by maleic anhydride and dopamine and then loaded with a small molecule drug.

[0015] The needle tip layer can also be formed by a drug-loaded inclusion complex and a polymer material crosslinked by a crosslinking agent and then loaded with a ROS-responsive drug.

[0016] The needle tip layer is formed by a polymer material to form a crosslinked network structure, and the inclusion complex can together achieve the responsive release of the drug. The small molecule drug is dispersed in the needle tip layer, and the needle tip layer can also be loaded with a responsive drug. The inclusion complex can improve the solubility of the drug and keep it stable, which is beneficial to the delivery of poorly soluble drugs and unstable protein drugs, and at the same time, the responsive release is achieved.

[0017] Further preferably, the needle tip layer is formed by a drug-loaded inclusion complex and polyvinyl alcohol (PVA) crosslinked by a crosslinking agent.

[0018] In some specific embodiments of the present application, the small molecule drug is colchicine (Col), and the ROS-responsive drug includes at least one of uricase and glucose oxidase.

[0019] Further preferably, the molar ratio of the β-cyclodextrin to the dopamine is 1:(1.5-2.5); and the molar ratio of the small molecule drug to the β-cyclodextrin modified by maleic anhydride and dopamine is (0.8-1.2):1.

[0020] Preferably, the separation layer is prepared from polyvinylpyrrolidone and anhydrous citric acid.

[0021] Further preferably, the mass ratio of the polyvinylpyrrolidone to the anhydrous citric acid is (3-5):1.

[0022] Preferably, the base layer material includes a monomer polymer or a copolymer of polyvinylpyrrolidone and its derivatives; in some preferred embodiments of the present application, the base layer material is PVP K90.

[0023] The second aspect of the present application provides a preparation method of the bubble separation type responsive hydrogel microneedle, including the following steps:

[0024] (1) Preparation of a drug-loaded inclusion complex: after β-cyclodextrin is modified by maleic anhydride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dopamine and N-hydroxysuccinimide are added, and the reaction is carried out under light shielding and stirring to obtain β-CD-DA, and then a small molecule drug is added and stirred to obtain a drug-loaded inclusion complex;

[0025] Preparation of the tip layer solution: the drug-loaded inclusion compound is prepared into a solution, a crosslinking agent and a polymer material are added and stirred to dissolve, and the tip layer solution is obtained;

[0026] Preparation of the separation layer solution: a polymer material with a polar group, a small molecule weak acid and anhydrous ethanol are mixed and stirred to obtain the separation layer solution;

[0027] Preparation of the substrate layer solution: a polymer material is mixed with anhydrous ethanol or water and stirred uniformly to obtain the substrate layer solution;

[0028] (2) the tip layer solution is added to the microneedle negative mold, and the first centrifugation is performed at room temperature, the excess tip layer solution is scraped off, and the second centrifugation is performed at room temperature, and then drying is performed;

[0029] (3) step (2) is repeated once, and then drying is performed at room temperature after the end;

[0030] (4) the separation layer solution is added, the first centrifugation is performed at room temperature, the excess separation layer solution is scraped off, the second centrifugation is performed at room temperature, and then drying is performed;

[0031] (5) the substrate layer solution is added, and centrifugation is performed at room temperature;

[0032] (6) the entire microneedle negative mold is dried at room temperature, and then the bubble separation type responsive hydrogel microneedle is obtained.

[0033] In some specific embodiments of the present application, the microneedle negative mold has a shape of a combination of a cone and a cylinder, and contains microneedle concave structures arranged in an array in the groove, and the microneedle concave structures are matched with the shape of the microneedle body. The single-piece microneedle contains 144 microneedles, and the array is arranged in 12x12, the tip spacing is 0.80 mm, and the microneedle body is arranged in order.

[0034] In the present application, after the microneedle is prepared, the bubble structure appears between the tip layer and the substrate layer, the microneedle has sufficient hardness to penetrate the skin in a dry state, the bubble structure can break the tip when the microneedle is applied to the skin and the substrate layer is peeled off, and the drug-containing tip is separated from the substrate layer quickly and retained in the skin.

[0035] Preferably, in step (1), in the preparation of the tip layer solution, the mass ratio of the drug-loaded inclusion compound, the crosslinking agent and the polymer material is (30-80):1:1; further preferably, the mass ratio of the drug-loaded inclusion compound, the crosslinking agent and the polymer material is (30-60):1:1.

[0036] Preferably, in step (2), the first centrifugation and the second centrifugation have the same speed, and the centrifugal speed is 4000-5000 rpm; the time of the first centrifugation is 5-15 min, and the time of the second centrifugation is 30-90 min.

[0037] Preferably, in step (4), the first centrifugation and the second centrifugation have the same speed, and the centrifugal speed is 1000-4000 rpm; the time of the first centrifugation is 5-15 min, and the time of the second centrifugation is 20-40 min.

[0038] Preferably, in step (5), the centrifugal speed is the same as that of the first centrifugation in step (4).

[0039] Compared with the prior art, the application has the following beneficial effects:

[0040] The design of the separated microneedle of the application causes bubbles to appear between the tip layer and the base layer, so that rapid separation can be achieved, and irritation and allergic reactions caused by the long-term adhesion of traditional microneedles to the skin are avoided, and the tip effectively remains in the skin as a drug-responsive release reservoir. The bubble-separated responsive hydrogel microneedle can improve the solubility of drugs and keep them stable, which is beneficial to the delivery of poorly soluble drugs and unstable protein drugs. By adjusting the types and proportions of the polymer materials of the tip, different drugs such as enzymes can be loaded to achieve different drug-responsive release effects. The inclusion compound of the tip layer and the tip layer can jointly carry different drugs, thereby improving the drug loading capacity and achieving the purpose of synergistically delivering drugs with different properties. The rapid bubble-separated responsive hydrogel microneedle of the application has the advantages of biocompatibility, controllable release, and local targeting, compared with traditional separable dissolvable microneedles, the difficulty of using microneedles for local sustained-release delivery of unstable drugs is solved, and the drug loading capacity is effectively improved.

[0041] The rapid bubble-separated responsive hydrogel microneedle of the application, by designing a microneedle structure including a separation layer and a tip drug-loaded layer, and selecting a hydrophilic polymer composite material for the separation layer, using a composite of a high molecular polymer with a polar group and a small molecule acid, a bubble structure can be formed between the tip layer and the base layer during the preparation of the microneedle, which is beneficial to the rapid breaking of the tip and the separation from the base layer during application, and the high molecular polymer has a large affinity for water and is easily wetted by water, which can accelerate the dissolution speed of the support separation layer, so that the tip drug-loaded layer can be quickly separated from the base. Through the above scheme, the problems of low drug delivery efficiency and skin irritation caused by the long-term wearing of traditional dissolvable microneedles are solved, and the safety of the microneedle drug delivery system is improved.

[0042] The quick separable responsive hydrogel composite microneedle of the application solves the problem of pathological responsive release of local drug delivery, realizes on-demand drug release, and improves drug safety. After the polymer material of the needle tip layer is crosslinked by the crosslinking agent, the needle tip layer forms a biocompatible hydrogel network structure. After the base layer is peeled off, the needle tip retained in the skin does not dissolve quickly, but slowly swells after absorbing the skin moisture and interstitial fluid. Then, the pathological related enzymes or proteins in the needle tip layer decompose pathological small molecules (such as uric acid or glucose), promote the degradation of the hydrogel network structure, and realize the responsive release of the drug-loaded inclusion compound. In addition, the design of the inclusion compound can further slow down the drug release rate.

[0043] The quick separable responsive hydrogel microneedle of the application adds pathological related enzyme active proteins to form a composite microneedle. The hydrophobic drug is loaded in the cavity by using the double parent property of the cavity of β-cyclodextrin, which can be used as a carrier for biological macromolecular drugs, unstable drugs and poorly soluble drugs, thereby increasing the solubility of the drug and improving the drug loading capacity. The microneedle avoids the decrease in activity of the drug caused by endogenous enzyme cutting in the body, loss or rapid clearance by the immune system of the body, and prevents the decrease in activity of the drug in the unstable state in the body environment. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The mold design drawing and physical drawing of the example;

[0045] Figure 2 The overall morphology of the blank bubble separable microneedle under SFM and the microneedle base drawing after the needle body and the base are directly peeled off;

[0046] Figure 3 The preparation method schematic diagram of the drug-loaded bubble separable hydrogel sustained-release microneedle;

[0047] Figure 4 The preparation and characterization evaluation of the responsive hydrogel PVA-TPA-β-CD-DA in the bubble separable hydrogel sustained-release microneedle in Example 2;

[0048] Figure 5 The bright field and fluorescence microscopic images of the bubble separable microneedle prepared in Example 2 under SFM and FM observation (black arrows indicate the separation layer bubbles);

[0049] Figure 6 The microneedle base drawing observed by SFM after the needle body of the whole bubble separable microneedle is peeled off after penetrating into the skin;

[0050] Figure 7 The image of the bubble separable microneedle separated in water under FM;

[0051] Figure 8 Mechanical strength curve of the bubble-separated microneedle prepared for three pieces of Example 2;

[0052] Figure 9 Picture of skin puncture test for the bubble-separated microneedle;

[0053] Figure 10 Image of the frozen section of the skin of a lactating pig implanted with the drug-loaded bubble-separated hydrogel sustained-release microneedle under a fluorescence microscope;

[0054] Figure 11 Picture of the uric acid-responsive release evaluation of the drug-loaded bubble-separated responsive hydrogel microneedle body;

[0055] Figure 12 Image of the drug-loaded bubble-separated responsive hydrogel microneedle prepared by different preparation methods under SFM and the corresponding mechanical strength of Comparative Example 1;

[0056] Figure 13 Mechanical strength of the bubble-separated microneedle prepared by different preparation methods of Comparative Example 1;

[0057] Figure 14 Schematic diagram of the structural design of the bubble-separated microneedle with different structures of Comparative Example 2;

[0058] Figure 15 Image of the bubble-separated microneedle prepared by different microneedle structure designs and different preparation methods under SFM of Comparative Example 2. DETAILED DESCRIPTION

[0059] The specific embodiments of the present application will be further described below. It should be noted that the description of these embodiments is used to help understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0060] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0061] Example 1

[0062] The embodiment provides a blank gas-bubble separable hydrogel sustained-release microneedle, which comprises a needle tip layer, a separation layer and a substrate layer, the separation layer is located between the needle tip layer and the substrate layer, the needle tip layer contains a blank inclusion compound modified by dopamine; the separation layer of the microneedle is composed of polyvinylpyrrolidone and anhydrous citric acid, and the ratio (w:w) of polyvinylpyrrolidone to anhydrous citric acid is 4:1; the substrate layer of the microneedle is composed of PVP K90; and the needle tip layer of the microneedle is formed by cross-linking polyvinyl alcohol with a cross-linking agent N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diaminiμm (TPA).

[0063] The preparation method comprises the following steps:

[0064] 1) Preparation of a blank inclusion compound

[0065] A certain amount of β-CD and MAH (maleic anhydride) is weighed and dissolved in DMF at a mass ratio of 2.3:1, and after stirring and reaction, it is cooled to room temperature, chloroform is added to precipitate the precipitate, and after washing with acetone, β-CD-MAH powder is obtained after drying. β-CD-MAH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), DA (dopamine) and N-hydroxysuccinimide (NHS) are dispersed in a MES buffer solution at a mass ratio of 2:1:0.7:0.6, stirred, protected from light and argon, and after the reaction is completed, acetone is added to precipitate the product and washed with acetone, and β-CD-DA powder is obtained after drying.

[0066] 2) Synthesis of TPA

[0067] About 0.20g of N,N,N',N'-tetramethyl-1,3-propanediamine and 1.0g of 4-(bromoethyl)phenylboronic acid are precisely weighed and dissolved in 10mL of DMF solution, and stirred under an oil bath at 60°C for 16h. After the reaction is completed, 80mL of ice THF is added to the generated mixture, and placed in a-20°C refrigerator overnight to precipitate white solids. Centrifugation is performed at a speed of 3000rpm for 5min at 4°C, the supernatant is discarded, centrifugation is repeated twice, and then the collected white solids are placed in a vacuum drying box and dried for 24h to obtain white solid powder.

[0068] 3) Preparation of a needle tip layer solution

[0069] A certain amount of β-CD-DA powder was precisely weighed, dissolved in a certain amount of borate buffer to prepare a 1.0 g / mL blank inclusion complex solution, 100 mg / mL TPA solution and 100 mg / mL PVA solution were added in turn, mixed according to the volume ratio TPA:PVA:blank inclusion complex = 1:1:6, diluted 6 times (volume ratio) with water after mixing to obtain a hydrogel precursor solution, and rhodamine B was added for dyeing (mother liquor concentration 1%), so that the final concentration was 0.4 ‰.

[0070] 4) Preparation of separation layer solution

[0071] A certain amount of polyvinylpyrrolidone and anhydrous citric acid were weighed, added with anhydrous ethanol and stirred to dissolve to obtain a separation layer solution, wherein PVP: citric acid: ethanol = 4:1:24 (mass ratio).

[0072] 5) Preparation of base layer solution

[0073] A certain amount of PVP K90 was weighed, added with anhydrous ethanol and stirred to dissolve to obtain a base layer solution, PVP K90: ethanol = 1:6 (mass ratio).

[0074] 6) Preparation of blank gas-bubble separable sustained-release microneedle

[0075] Microneedle mold: The microneedle mold in the embodiment is as shown in Figure 1 a is a microneedle mold design drawing, b is a physical drawing of a male mold, and c is a physical drawing of a female mold.

[0076] The microneedle female mold has a combination of a cone and a cylinder, and contains microneedle concave structures arranged in an array in the groove, which are matched with the shape of the microneedle body. A single microneedle sheet contains 144 microneedles, arranged in an array of 12x12, with a needle tip spacing of 0.80 mm, and the needle bodies are arranged in order;

[0077] The needle tip layer solution is added to the microneedle negative mold which can be matched with the shape of the microneedle, and centrifuged at a speed of 4500 rpm for 10 min at a temperature of 25℃, the excess needle tip solution is scraped off, the solution is just filled in the conical cavity of the concave structure of the microneedle negative mold, and the centrifugation is continued at a speed of 4500 rpm for 60 min at a temperature of 25℃; the above steps are repeated once after centrifugation; the needle tip layer is dried at room temperature overnight; the separation layer solution is added, and centrifuged at a speed of 3000 rpm for 10 min at a temperature of 25℃, the excess separation layer solution is scraped off, the solution is just filled in the cylindrical cavity of the concave structure of the microneedle negative mold, and the centrifugation is continued at a speed of 3000 rpm for 30 min at a temperature of 25℃; the base layer solution is added, and centrifuged at a speed of 3000 rpm for 10 min at a temperature of 25℃; the whole microneedle negative mold is placed in a normal temperature dryer for drying for 24 h, and the microneedle is taken out from the microneedle negative mold after drying, to obtain the blank bubble separation type sustained-release microneedle.

[0078] The morphology of the microneedle and the base after separation are observed under a stereofluorescence microscope and a fluorescence microscope, as shown in Figure 2 , a is the morphology of the blank bubble separation type sustained-release microneedle under the FM; b is the base after the needle body and the base are directly peeled off under the SFM. It can be seen that the blank bubble separation type hydrogel sustained-release microneedle prepared in Example 1 has a full and regular needle body shape, and there is no defect and fracture, the rhodamine B is mainly distributed in the needle tip layer, the structure of each layer is clear, and the bubble structure of the separation layer is obvious; and after the needle body and the base are directly peeled off, the base hole is complete, which indicates that the needle body can be easily and quickly peeled off from the base, and the bubble structure plays a significant role.

[0079] Example 2

[0080] The present embodiment provides a drug-loaded bubble separation type composite hydrogel sustained-release microneedle, the microneedle comprises a needle tip layer, a separation layer and a base layer, the separation layer is located between the needle tip layer and the base layer; the separation layer of the microneedle is composed of polyvinylpyrrolidone and anhydrous citric acid, and the ratio (w:w) of polyvinylpyrrolidone to anhydrous citric acid is 4:1; the base layer of the microneedle is composed of PVPK90; the needle tip layer of the microneedle is crosslinked by PVA, and the hydrogel solution obtained by mixing a drug-loaded inclusion compound, TPA and PVA at a ratio of 1:1:6 and diluting with an appropriate amount of water by 6 times is used as the needle tip layer material; the drug-loaded inclusion compound is obtained by inclusion of a drug after modification of β-cyclodextrin by DA, and the molar ratio of β-CD to DA is 1:2, and the molar ratio of the drug to β-CD-DA is 1:1; the drug loaded in the inclusion compound of the microneedle is colchicine, and the drug loaded in the needle tip layer is uricase.

[0081] The preparation method comprises the following steps:

[0082] 1) Preparation of drug-loaded inclusion compound

[0083] The same procedure as in Example 1 was used to prepare a blank inclusion complex β-CD-DA powder. β-CD-DA and colchicine were added to a phosphate buffer (PBS) at a molar ratio of 1:1, stirred at room temperature, and then centrifuged to obtain a supernatant, which was dried under vacuum to obtain a drug-loaded inclusion complex Col / β-CD-DA.

[0084] 2) Preparation of the tip layer solution

[0085] A certain amount of drug-loaded inclusion complex was precisely weighed and dissolved in a certain amount of borate buffer to prepare a drug-loaded inclusion complex solution of 1.0 g / mL. Then, 100 mg / mL of TPA solution and 100 mg / mL of PVA solution were added in sequence to obtain a hydrogel precursor solution. Uricase (UAO) was added for dissolution to obtain a tip layer solution. After adding the gel precursor solution, the final concentration of uricase was 4 mg / mL. A responsive hydrogel PVA-TPA-β-CD-DA in the tip layer solution was prepared. Finally, a rhodamine B solution was added for staining, and the final concentration was 0.4 ‰. The amounts of TPA, PVA, Col / β-CD-DA, and water were as shown in Table 1 below.

[0086] Table 1

[0087]

[0088] 3) Preparation of the separation layer solution

[0089] The same procedure as in Example 1 was used to prepare the separation layer solution.

[0090] 4) Preparation of the base layer solution

[0091] The same procedure as in Example 1 was used to prepare the base layer solution.

[0092] 5) Preparation of the gas bubble separable hydrogel composite sustained-release microneedle

[0093] The preparation method of Example 2 was the same as that of Example 1, and the structure of the microneedle negative mold was also basically the same as that of Example 1. According to the results in Table 1, the tip layer prepared according to the scheme of sample No. 3 was selected, and the details are as follows:

[0094] As Figure 3As shown, the tip solution was added to the microneedle negative mold described in Example 1, and low-speed centrifugation was performed at a temperature of 25°C and a speed of 4500 rpm for 10 min, the excess tip drug-loaded solution was scraped off, the solution was just filled into the conical cavity in the concave structure of the microneedle negative mold, and the solution was dried by continuing centrifugation at a temperature of 25°C and a speed of 4500 rpm for 60 min; the above steps were repeated once after centrifugation; the tip layer was dried at room temperature overnight; the separation layer solution was added, and low-speed centrifugation was performed at a temperature of 25°C and a speed of 3000 rpm for 10 min, the excess separation layer solution was scraped off, the solution was just filled into the cylindrical cavity of the concave structure of the microneedle negative mold, and the solution was centrifuged at a temperature of 25°C and a speed of 3000 rpm for 30 min; the base layer solution was added, and 3000 rpm centrifugation was performed at 25°C for 10 min again; the entire microneedle negative mold was placed in a normal temperature dryer and dried for 24 h, and the microneedle was taken out from the microneedle negative mold after drying, thereby obtaining the bubble-separable composite hydrogel sustained-release microneedle (named Col / β-CD-DA@UAO-rMNs).

[0095] Referring to the preparation process of Col / β-CD-DA@UAO-rMNs, Col and β-CD-DA were directly added to the preparation of the tip layer solution in step 2) instead of preparing the drug-loaded inclusion compound Col / β-CD-DA, and other experimental conditions were the same as those in the preparation of Col / β-CD-DA@UAO-rMNs, thereby obtaining the bubble-separable composite hydrogel sustained-release microneedle Col+β-CD-DA@UAO-rMNs.

[0096] Referring to the preparation process of Col / β-CD-DA@UAO-rMNs, UAO was replaced by IgG, thereby obtaining the bubble-separable composite hydrogel sustained-release microneedle Col / β-CD-DA@IgG-rMNs.

[0097] Effect verification

[0098] 1. Preparation and characterization of responsive hydrogel PVA-TPA-β-CD-DA in the tip layer solution

[0099] The preparation and characterization of the bubble-separable responsive hydrogel PVA-TPA-β-CD-DA of Example 2 were as shown in Figure 4 , wherein, Figure 4 (a) is a schematic diagram of the preparation of PVA-TPA-β-CD-DA; Figure 4 (b) is the ultraviolet spectrum of β-CD, β-CD-DA, Col and Col / β-CD-DA, whether the prepared inclusion compound is formed can be determined by ultraviolet-visible absorption spectrometry in the range of 320-400 nm; Figure 4(c) The molar ratio of β-CD-DA to Col is linearly related to the inclusion ability of the inclusion complex, and the inclusion effect of the inclusion complex is positively correlated with the molar ratio of β-CD-DA and Col; Figure 4 (d) The FTIR results of Col, β-CD, β-CD-DA, physical mixture (Col and β-CD-DA mixed at a molar ratio of 1:1) and Col / β-CD-DA show that DA successfully modified β-CD; Figure 4 (e) The XRD results of Col, β-CD-DA, physical mixture and Col / β-CD-DA show that the drug-loaded inclusion complex is formed; Figure 4 (f) The graph of the change of the elastic modulus (G') and the viscosity (G') of the hydrogel with time, Figure 4 (g) The frequency spectrum graph of the g' and g' modulus of the hydrogel, Figure 4 (f) and (g) show the formation of a solid-like gel; Figure 5 (h) The pictures of the hydrogel PVA-TPA-β-CD-DA prepared by different schemes, including the results of 0h (top view), 0h (EP tube inverted), 24h (EP tube inverted), and the final selection of No. 3, i.e. the ratio of 1:1:6, as the best hydrogel prescription.

[0100] 2. Morphological observation of microneedles

[0101] The drug-loaded bubble-separable hydrogel sustained-release microneedles prepared using rhodamine B dyeing needle tip layer solution were observed by SFM, and the results are shown in Figure 6 It can be seen that the drug-loaded bubble-separable hydrogel sustained-release microneedles prepared in Example 2 have basically consistent needle tip length, full and regular shape, and no defects and fractures. Rhodamine B is mainly distributed in the needle tip layer, and the structure is clear, and the bubble structure of the separation layer is obvious.

[0102] 3. Base layer peeling of microneedles

[0103] The bubble-separable hydrogel sustained-release microneedles were prepared according to the steps described in Example 2, and the microneedle tip was directly and quickly peeled off from the base. The base after peeling was observed by SFM, and the results are shown in Figure 7 It can be seen that after the needle tip of the drug-loaded bubble-separable hydrogel sustained-release microneedles prepared in Example 2 is peeled off from the base, the hole of the base is complete, clear and obvious.

[0104] 4. Water separation test of microneedles

[0105] The drug-loaded bubble-separable hydrogel sustained-release microneedles prepared using rhodamine B dyeing needle tip layer solution were cut into 1x12 shape, adhered to a glass slide, and placed in a culture dish. Ultra-pure water was added to the culture dish, and FM was used for observation. Pictures were taken every 3s, and the results are shown in Figure 8The separation layer was separated from the base layer at the bubble after about 9 s of the microneedle being put into water, and the needle tip drug-containing layer was completely separated from the base layer.

[0106] 5. Mechanical strength determination of microneedles

[0107] The mechanical strength of the bubble-separable hydrogel sustained-release microneedles prepared according to the steps described in Example 2 was detected by a texture analyzer, and the mechanical strength curve results of three pieces of sample No. 3 bubble-separable microneedles prepared by the process are shown in Figure 9 It can be seen that the mechanical strength of the drug-loaded bubble-separable hydrogel sustained-release microneedles prepared in Example 2 meets the requirements of insertion into the skin.

[0108] 6. Skin penetration test of microneedles

[0109] The rat abdominal skin was taken, and drug-loaded bubble-separable hydrogel sustained-release microneedles were prepared using a rhodamine B dyeing needle tip layer solution. After pressing for 3 min, the base layer was torn off, and SFM was used for observation. The results are shown in Figure 10 It can be seen that after the separation layer is quickly broken and dissolved, most of the microneedle tips are retained in the skin. Further, the microneedles were inserted into the piglet skin, and the piglet skin with the retained needle tips was frozen sectioned. The results are shown in NO. Under the fluorescence microscope, it can be seen that the microneedles successfully penetrated the piglet skin and retained the needle tips in the skin.

[0110] 7. Uric acid-responsive release evaluation

[0111] Drug loading determination: Two different microneedles, Col / β-CD-DA@UAO-rMNs and Col+β-CD-DA@UAO-rMNs, were prepared, and 3 pieces of MNs were taken from each group and dissolved in 6 mL of PBS containing 1% H2O2. After centrifugation of the solution, 10.0 μL of the supernatant was taken for HPLC quantitative analysis.

[0112] In vitro transdermal penetration test: In order to evaluate the uric acid response performance of Col / β-CD-DA@UAO-rMNs, the in vitro transdermal penetration behavior of different formula microneedles in different transdermal diffusion buffer was studied. Drug transdermal diffusion instrument was used for determination, and two kinds of transdermal diffusion medium were configured: a) PBS (pH 7.4); b) PBS containing uric acid (uric acid concentration is 7 mg / dL). The experiment was divided into three groups, as shown in Table 2 below. Specific steps: a) implant MNs conforming to the shape and size of Franz diffusion cell instrument into the back skin of pre-treated rats; b) after pressing for 30 s, place the MN-containing skin on the Franz diffusion cell instrument for fixation, with the dermis facing the bottom of the diffusion cell; c) inject PBS or PBS containing uric acid into the diffusion cell as the diffusion medium, and place the diffusion cell in a 32°C water bath for incubation with stirring at 250 rpm; d) at different time points (2, 6, 12, 24, 48, 72, 120, 168 and 240 h), take 1 mL of the diffusion medium for determination of Col concentration by HPLC method, and add 1 mL of fresh diffusion medium to the diffusion cell.

[0113] Table 2 Grouping of in vitro transdermal diffusion

[0114] MNs Transdermal diffusion medium Col / β-CD-DA@UAO-rMNs 1 PBS, pH 7.4 Col / β-CD-DA@UAO-rMNs 2 PBS containing 7 mg / dL uric acid Col / β-CD-DA@IgG-MNs 3 PBS containing 7 mg / dL uric acid Figure 11

[0115] In vivo drug treatment test: In order to better evaluate the uric acid response ability of Col / β-CD-DA@UAO-rMNs, a hyperuricemia rat model was established to study their drug retention characteristics in the skin. Rhodamine 6G (R6G) was used as a fluorescent dye to replace Col to prepare inclusion compounds with β-CD-DA, and then microneedles R6G / β-CD-DA@IgG-rMNs and R6G / β-CD-DA@UAO-MNs were prepared. Specific steps: a) 9 male healthy adult SD rats, weighing 200±40 g, were randomly divided into 3 groups. Among them, the 2nd and 3rd groups of mice were given Potassium oxonate (PO, 750 mg / kg / d, ig) 3 weeks in advance to establish a hyperuricemia model; b) when the drug was given, press the microneedles at the knee joint of the rat for 30 s, and remove the basal layer after 3 min; among them, the 2nd group was given R6G / β-CD-DA@IgG-rMNs, and the 1st and 3rd groups were given R6G / β-CD-DA@UAO-MNs; c) use The spectral in vivo imaging system recorded the fluorescence images at specific time points (1, 3, 5 and 7 d), and calculated the fluorescence intensity.

[0116] Uric acid-responsive release evaluation of drug-loaded bubble-separable responsive hydrogel microneedle bodies as shown in Figure 11 , wherein, Figure 11 (a) is a responsive hydrogel responsive release mechanism diagram,Figure 11 (b) Drug loading of microneedles prepared for Col inclusion complex (Col / β-CD-DA@UAO-rMNs) or physical mixture (Col + β-CD-DA@UAO-rMNs) showed that β-CD-DA inclusion of Col could increase the drug loading of Col by 3 times; Figure 11 (c) In vitro drug permeation experiment results of Col / β-CD-DA@IgG-MNs in uric acid (MA) solution, Col / β-CD-DA@UAO-rMNs in PBS and Col / β-CD-DA@UAO-rMNs in MA solution, in vitro transdermal diffusion experiment results showed that the microneedles could achieve uric acid-responsive release in vitro; Figure 11 (d) is a schematic diagram of in vivo retention experiment scheme; Figure 11 (e) is the meaning of G1-G3; Figure 11 (f) is the fluorescence image of rhodamine 6G-containing MNs acting on the knee skin of rats at different time points, Figure 11 (g) is the in vivo drug retention of R6G / β-CD-DA@UAO-rMNs without PO / ig, R6G / β-CD-DA@IgG-MNs with PO / ig and R6G / β-CD-DA@UAO-rMNs with PO / ig; Figure 12 (d)-(f) proved that the microneedles could achieve uric acid-responsive release in vivo by using a mouse live imaging system in a high uric acid model.

[0117] Comparative Example 1

[0118] The present embodiment provides a separable microneedle and a preparation method thereof with the same prescription, the same microneedle structure design and different preparation methods.

[0119] The microneedle comprises a base layer, a separation layer and a tip layer, wherein the microneedle separation layer is composed of polyvinylpyrrolidone and anhydrous citric acid, and the ratio (w:w) of polyvinylpyrrolidone to anhydrous citric acid is 4:1; the microneedle base layer is composed of PVPK90; and the microneedle tip layer is crosslinked from a high molecular material PVA. The preparation method comprises the following steps:

[0120] 1) Preparation of tip layer solution, separation layer solution and base layer solution

[0121] The preparation method of the tip layer solution, the separation layer solution and the base layer solution of Comparative Example 1 is the same as that of Embodiment 2.

[0122] 2) Preparation of separable microneedle with the same prescription, the same microneedle structure design and different preparation methods

[0123] The microneedle negative mold structure of Comparative Example 1 is the same as that of Embodiment 1, and the specific preparation method is as follows:

[0124] The needle tip solution was added to the microneedle negative mold described in Example 1, centrifuged at a speed of 4500 rpm for 10 min at a temperature of 25℃, the excess needle tip drug-loaded solution was scraped off, the solution was just filled into the conical cavity in the concave structure of the microneedle negative mold, and the centrifugation was continued at a speed of 4500 rpm for 60 min at a temperature of 25℃; the above steps were repeated once after centrifugation; the needle tip layer was dried at room temperature overnight; the separation layer solution was added, centrifuged at a speed of 1000 rpm, 2000 rpm, 3000 rpm and 4000 rpm for 10 min at a temperature of 25℃, respectively, the excess separation layer solution was scraped off, the solution was just filled into the cylindrical cavity of the concave structure of the microneedle negative mold, and the centrifugation was continued for 30 min at the same speed as the filling of the separation layer solution at a temperature of 25℃; the base layer solution was added, centrifuged at a speed corresponding to the same speed as the preparation of the separation layer at a temperature of 25℃ for 10 min; the entire microneedle negative mold was placed in a normal temperature dryer for drying for 24 h, and the microneedles were taken out from the microneedle negative mold after drying, thereby obtaining the microneedles with the same prescription, the same microneedle structure design and different preparation methods.

[0125] The results of the prepared microneedles of different shapes were observed by SFM, and the mechanical strength of the microneedles was detected by a texture analyzer. Figure 13 The mechanical strength of the microneedles prepared by different methods was compared. Figure 14 It can be seen that the lower the centrifugal speed during the preparation of the separation layer and the base layer, the more obvious the bubble structure of the separation layer, but the more significant the broken needle condition of the microneedle patch after demolding, and the microneedle array is not complete; and the more obvious the bubble structure of the separation layer, the more prone the needle tip layer to break and separate from the base, and the lower the mechanical strength, which cannot meet the requirement of insertion into the skin. When the centrifugal speed is 3000 rpm, the bubble structure of the separation layer is relatively obvious, and the microneedle array is relatively complete, and the mechanical strength can meet the requirement of insertion into the skin.

[0126] Comparative Example 2

[0127] The present embodiment provides a microneedle with the same prescription, different microneedle structure design and different preparation method, and a preparation method thereof.

[0128] The microneedle comprises a base layer, a separation layer and a needle tip layer, wherein the microneedle separation layer is composed of polyvinylpyrrolidone and anhydrous citric acid, and the ratio (w:w) of polyvinylpyrrolidone to anhydrous citric acid is 4:1; the microneedle base layer is composed of PVPK90; and the microneedle needle tip layer is crosslinked from a high molecular material PVA. The preparation method comprises the following steps:

[0129] 1) Preparation of solutions of the needle tip layer, the separation layer and the base layer

[0130] The preparation method of the solutions of the needle tip layer, the separation layer and the base layer of Comparative Example 2 is the same as that of Example 2.

[0131] 2) The preparation of detachable microneedles with the same formulation, different microneedle structure design and different preparation method

[0132] The microneedle negative mold structure of Comparative Example 2 is two different conical shapes, and the preparation method and results are shown in Figure 15 , and the specific preparation method is as follows:

[0133] The tip solution was added to the microneedle negative mold with different conical shapes, and centrifuged at a speed of 4500 rpm for 10 min at a temperature of 25°C. The excess tip drug-loaded solution was scraped off, and the solution was filled into the bottom cavity of the microneedle negative mold concave structure. The solution was further dried by centrifugation at a speed of 4500 rpm for 60 min at a temperature of 25°C. After centrifugation, the above steps were repeated once. The tip layer was dried overnight at room temperature. The separation layer solution was added, and centrifuged at a speed of 2000 rpm and 3000 rpm for 10 min at a temperature of 25°C. The excess separation layer solution was scraped off, and the solution was further filled into the cavity of the microneedle negative mold concave structure. The solution was dried by centrifugation at the same speed as the separation layer solution for 30 min at a temperature of 25°C. The base layer solution was added, and centrifuged at a speed corresponding to the same speed as the separation layer for 10 min at a temperature of 25°C. The entire microneedle negative mold was placed in a normal temperature dryer and dried for 24 h. After drying, the microneedles were removed from the microneedle negative mold, and the detachable microneedles with the same formulation, different microneedle structure design and different preparation method were obtained.

[0134] The results of the different microneedle morphologies prepared by Comparative Example 2 were observed by SFM, as shown in ​ . It can be seen that in the microneedles prepared by conical bodies, the increase in centrifugal speed increases the integrity of the microneedle array demolding, but the generation of bubbles is reduced. That is, in the microneedles with conical shape, this preparation method can generate bubbles under suitable conditions, but it is difficult to successfully demold the tip, resulting in incomplete microneedle array. Therefore, it can be concluded that different microneedle structure designs have a significant impact on the formation and maintenance of bubble structures in the separation layer. The remaining needle body of the microneedles prepared in Comparative Example 2 has no bubble structure and the needle breaking is obvious, and the microneedle array is incomplete, while the microneedle structure design of Example 2 is obviously beneficial to the appearance and maintenance of bubble structure, and is beneficial to the rapid separation of needle body and base during microneedle application.

[0135] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of these embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A bubble separation type responsive hydrogel microneedle, characterized by, The microneedle structure comprises a needle tip layer, a separation layer and a substrate layer, the needle tip layer is dispersed with drug-loaded inclusion compound, the drug-loaded inclusion compound is formed by β-cyclodextrin modified and then inclusion drug; The separation layer is prepared by high molecular polymer with polar groups and small molecule weak acid; The substrate layer is high molecular polymer material; There is bubble between the needle tip layer and the substrate layer.

2. The bubble-separated responsive hydrogel microneedle according to claim 1, wherein, The structure of the microneedle from the needle tip layer to the separation layer is conical body, cylinder body in turn, and the substrate layer covers the other bottom surface of the cylinder body.

3. The bubble-separated responsive hydrogel microneedle according to claim 1, wherein, The needle tip layer is crosslinked by drug-loaded inclusion compound and high molecular material through crosslinking agent; the drug-loaded inclusion compound is formed by β-cyclodextrin modified by maleic anhydride and dopamine and then inclusion small molecule drug; The needle tip layer can also be formed by drug-loaded inclusion compound, high molecular material and crosslinking agent, and then loaded with ROS responsive drug.

4. The bubble-separated responsive hydrogel microneedle according to claim 3, wherein, The molar ratio of β-cyclodextrin to dopamine is 1:(1.5-2.5); the molar ratio of small molecule drug to β-cyclodextrin modified by maleic anhydride and dopamine is (0.8-1.2):

1.

5. The bubble-separated responsive hydrogel microneedle according to claim 1, wherein, The separation layer is prepared by polyvinylpyrrolidone and anhydrous citric acid.

6. The bubble-separated responsive hydrogel microneedle according to claim 5, wherein, The mass ratio of polyvinylpyrrolidone to anhydrous citric acid is (3-5):

1.

7. The bubble-separated responsive hydrogel microneedle according to claim 1, wherein The substrate layer material includes monomer polymer or copolymer of polyvinylpyrrolidone and its derivatives.

8. The method of claim 1-7, wherein the preparation method of the bubble separation type responsive hydrogel microneedle is characterized by, It comprises the following steps: (1) Preparation of drug-loaded inclusion compound: β-cyclodextrin is modified by maleic anhydride, then 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, dopamine and N-hydroxysuccinimide are added, and the reaction is carried out under light shielding and stirring to obtain β-CD-DA, then small molecule drug is added and stirred to obtain drug-loaded inclusion compound; Preparation of needle tip layer solution: the drug-loaded inclusion compound is prepared into solution, crosslinking agent and high molecular material are added and stirred to dissolve, and needle tip layer solution is obtained; Preparation of separation layer solution: high molecular polymer with polar groups, small molecule weak acid and anhydrous ethanol are mixed and stirred to obtain separation layer solution; Preparation of substrate layer solution: high molecular polymer material is mixed with anhydrous ethanol or water and stirred uniformly to obtain substrate layer solution; (2) The needle tip layer solution is added to the microneedle negative mold, and the first centrifugation is carried out at room temperature, the excess needle tip drug solution is scraped off, and the second centrifugation is carried out at room temperature, and then dried; (3) Repeat step (2) once, and dry at room temperature after finishing; (4) Add the separation layer solution, carry out the first centrifugation at room temperature, scrape off the excess separation layer solution, carry out the second centrifugation at room temperature, and dry; (5) Add the substrate layer solution and centrifuge at room temperature; (6) After drying the whole microneedle negative mold at room temperature, it is taken out, and the bubble separation type responsive hydrogel microneedle is obtained.

9. The production method according to claim 8, characterized by, In step (2), the rotation speed of the first centrifugation is the same as that of the second centrifugation, the rotation speed is 4000-5000 rpm; the time of the first centrifugation is 5-15 min, and the time of the second centrifugation is 30-90 min.

10. The preparation method according to claim 8, characterized in that In step (4), the first centrifugation and the second centrifugation have the same rotation speed, and the rotation speed is 1000-4000 rpm; the time of the first centrifugation is 5-15 min, and the time of the second centrifugation is 20-40 min. In step (4), the first centrifugation and the second centrifugation have the same rotation speed, and the rotation speed is 1000-4000 rpm; the time of the first centrifugation is 5-15 min, and the time of the second centrifugation is 20-40

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