Substrate-free anti-inflammatory hydrogel microneedle particle and preparation method thereof

Abstract

The invention provides a substrate-free anti-inflammatory hydrogel microneedle particle and a preparation method thereof. The microneedle particle comprises an anti-inflammatory active component and a microneedle substrate used for wrapping the anti-inflammatory active component, the anti-inflammatory active component is selected from any one of curcumin, celecoxib and aesculin; the hydrogel is prepared from hyaluronic acid. A substrate of a traditional microneedle is abandoned, and the problems of displacement and falling caused by sweat or skin deformation are avoided. The raw materials of the microneedle particles are safe, no chemical cross-linking agent is used during preparation, and the risks of inducing local allergy and aggravating oxidative stress are avoided. The substrate-free microneedle particles have higher flexibility and contact area, and can be better attached to the skin, so that the delivery efficiency of drugs can be improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and relates to microneedles for transdermal drug delivery methods, in particular to an inflammation-resistant hydrogel microneedle particle without a substrate and a preparation method thereof. BACKGROUND

[0002] Skin inflammatory diseases (such as atopic dermatitis, psoriasis) are caused by active oxygen imbalance and immune disorders, and the balance of oxidative and inflammatory regulation mechanisms is destroyed, leading to excessive release of pro-inflammatory factors and damage to the skin barrier. Existing therapies include topical external drugs (hormone cream, etc.), oral immunomodulators and subcutaneous injection of biological agents, but all have significant limitations: topical drugs are limited by the stratum corneum barrier, have low transdermal efficiency, and are difficult to reach an effective concentration in the deep layer; oral or systemic administration can easily cause liver toxicity, immunosuppression and other systemic side effects; subcutaneous injection causes pain and mechanical damage, and the patient's compliance is poor. Transdermal patches also have difficulty in delivering macromolecular drugs (such as proteins, nucleic acids). Therefore, there is an urgent need for a safer and more efficient strategy to treat skin inflammation.

[0003] Among different transdermal drug delivery methods, microneedles (MN) can successfully penetrate the stratum corneum with micrometer-sized needles (length 50-900 μm) in the form of a microneedle array, delivering drugs below the stratum corneum in a minimally invasive manner without damaging blood vessels and nerves in the dermis, improving patient compliance, and allowing drugs exposed in the epidermis or dermis to be rapidly absorbed by surrounding capillaries and lymph nodes. Because hydrogel microneedles have good biocompatibility and a three-dimensional cross-linked network that allows high drug loading, they are particularly suitable for the delivery of macromolecules (such as antibodies, nucleic acids) and hydrophilic drugs, and have become a multifunctional platform for the treatment of various skin inflammations.

[0004] In the application of skin inflammation treatment, the reported microneedles are mainly in the form of needle tip substrate integration for treatment. Due to the limitations of patch size and substrate flexibility, the drug delivery effect of microneedles in different affected areas is affected, and the drugs in the substrate cannot be fully utilized. In addition, the application of microneedles often requires long-term wear, and problems such as limb movement and sweat production can cause the microneedle patch to rebound, displace, and even fall off, and the low air permeability of the substrate material and the residual chemical cross-linking agent can induce local allergic reactions or infections, exacerbating the oxidative stress level in the inflammatory microenvironment. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a substrate-free anti-inflammatory hydrogel microneedle particle and a preparation method thereof, to solve the technical problems in the prior art that the microneedle adopts an integrated design of a needle tip and a substrate, but the size and flexibility of the substrate limit the drug delivery effect and utilization; long-term wearing causes the patch to rebound, displace or fall off due to movement and sweat; and the low air permeability of the material and residual crosslinking agent can induce allergy, infection and aggravate oxidative stress.

[0006] To solve the above technical problems, the present application adopts the following technical solutions: A substrate-free anti-inflammatory hydrogel microneedle particle comprises an anti-inflammatory active component and a hydrogel for wrapping the anti-inflammatory active component, and does not comprise a microneedle substrate; the anti-inflammatory active component is selected from any one of curcumin, celecoxib and aesculin; and the hydrogel is prepared from hyaluronic acid.

[0007] The present application also has the following technical features: Specifically, the mass ratio of curcumin to hyaluronic acid is 1:2630-5550; the mass ratio of celecoxib to hyaluronic acid is 1:2630-5550; and the mass ratio of aesculin to hyaluronic acid is 1:26.3-55.5.

[0008] Specifically, the molecular weight of hyaluronic acid is 200-400 kDa.

[0009] The present application also protects a preparation method of the substrate-free anti-inflammatory hydrogel microneedle particle as described above, characterized in that the method comprises the following steps: Step one, preparation of an anti-inflammatory active component solution: If the anti-inflammatory active component is curcumin or celecoxib, step one comprises: dissolving curcumin or celecoxib in anhydrous ethanol to obtain an anti-inflammatory active component mother liquor; then, adding the anti-inflammatory active component to water, and adding anhydrous ethanol thereto, and ultrasonically dissolving to prepare an anti-inflammatory active component solution, wherein the volume ratio of ethanol to water is 3:7; the concentration of curcumin in the anti-inflammatory active component solution is 20 μg / mL, and the concentration of celecoxib is 20 μg / mL.

[0010] If the anti-inflammatory active component is aesculin, step one comprises: dissolving aesculin in water, and stirring in a water bath at 37 ℃ until the aesculin is completely dissolved, to obtain an anti-inflammatory active component mother liquor; the concentration of aesculin is 2000 μg / mL.

[0011] Step two, preparation of an anti-inflammatory pre-gel solution: add hyaluronic acid powder to the anti-inflammatory active component solution prepared in step one, stir until the powder is fully dissolved, and stand until the bubbles disappear, to obtain an anti-inflammatory pre-gel solution; the mass fraction of hyaluronic acid in the anti-inflammatory pre-gel solution is 5 wt%-10 wt%.

[0012] Step three, preparation of the substrate-free anti-inflammatory hydrogel microneedle particles: inject the anti-inflammatory pre-gel solution prepared in step two into the mold, remove the excess solution on the surface, then perform vacuum suction to fill the solution, and then stand until the small bubbles disappear, and then dry and solidify; repeat the above processes of vacuum suction, standing, and dry solidification multiple times until the microneedle mold is completely filled under dry conditions; the standing temperature is 4 ℃-10 ℃, and the standing time is 2 h-3 h; the dry solidification temperature is 20 ℃-35 ℃, and the dry solidification time is 1 h-2 h.

[0013] Compared with the prior art, the present application has the following technical effects: (I) The substrate-free microneedle particles of the present application are in the shape of a quadrangular pyramid, are composed of hyaluronic acid and load anti-inflammatory active components. This design discards the substrate of traditional microneedles, avoiding displacement and shedding caused by sweat or skin deformation. The raw materials of the microneedle particles are safe, and no chemical crosslinking agent is used during preparation, eliminating the risk of inducing local allergy and aggravating oxidative stress. The substrate-free microneedle particles have higher flexibility and contact area, can better fit the skin, and thus can improve the delivery efficiency of drugs.

[0014] (II) In the preparation of the substrate-free microneedle particles, the present application uses a water-ethanol cosolvent system and heating method to increase the solubility of the drugs, thereby significantly improving the drug loading capacity of the microneedles; the obtained hydrogel microneedle particles not only have high drug loading capacity and good transdermal efficiency, but also have excellent biological safety and anti-inflammatory activity and other biological properties.

[0015] (III) The anti-inflammatory active components (curcumin, celecoxib, and aesculin) loaded in the microneedle particles of the present application can effectively inhibit the inflammatory signaling pathways of NF-κB and MAPK, reduce the release of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β, and down-regulate the expression of COX-2 and iNOS, and have excellent antioxidant activity. Therefore, the microneedle particles have wide application prospects in the biomedical field of skin inflammation treatment.

[0016] (IV) The preparation method of the present application is simple and easy to operate, and the reaction conditions are mild, without the need for complex instruments or additional crosslinking agents. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The preparation process flow chart of the substrate-free anti-inflammatory hydrogel microneedle particles of the present application.

[0018] Figure 2 The parameter optimization results of the preparation of the substrate-free anti-inflammatory hydrogel microneedle particles. Figure 2Fig. 1: (a) Pictures of microneedles prepared from different pre-gel solutions with different hyaluronic acid content, (b) Photos of hyaluronic acid solutions with different ethanol content, (c) Photos of 40% ethanol solution before and after drug precipitation during the preparation of microneedles, (d) UV-vis absorption spectra of curcumin in 30% ethanol solution, the inset is the dissolution state photos of 10, 20, 30 μg / mL curcumin solution.

[0019] Figure 3 Fig. 2: Optical micrographs of the substrate-free anti-inflammatory hydrogel microneedle particles, Figure 3 Fig. 3: (a) and (b) are optical micrographs of hyaluronic acid microneedles with a needle length of 650 μm and a base of 280 μm x 280 μm, (c) and (d) are optical micrographs of hyaluronic acid microneedles with a needle length of 200 μm and a base of 200 μm x 200 μm.

[0020] Figure 4 Fig. 4: Mechanical penetration force test curves of different substrate-free concentration of hyaluronic acid hydrogel microneedles; Figure 4 Fig. 5: (a) is a mechanical penetration force test curve of hyaluronic acid microneedles with a needle length of 650 μm, (b) is a mechanical penetration force test curve of hyaluronic acid microneedles with a needle length of 200 μm.

[0021] Figure 5 Fig. 6: In vitro drug release curve of substrate-free anti-inflammatory hydrogel microneedles; Figure 5 Fig. 7: (a) is the in vitro drug release curve of 5% mass fraction of hyaluronic acid curcumin-loaded microneedles, (b) is the in vitro drug release curve of 10% mass fraction of hyaluronic acid curcumin-loaded microneedles.

[0022] Figure 6 Fig. 8: In vitro transdermal simulation experiment of substrate-free anti-inflammatory hydrogel microneedle particles. Figure 6 Fig. 9: (a) is a 5% mass fraction of hyaluronic acid microneedle sealant penetration simulation test, (b) is a 10% mass fraction of hyaluronic acid microneedle sealant penetration simulation test, (c) is a pig skin transdermal confocal image of 5% mass fraction of hyaluronic acid microneedles, (d) is a pig skin transdermal confocal image of 10% mass fraction of hyaluronic acid microneedles.

[0023] Figure 7 Fig. 10: In vitro antioxidant activity characterization of the substrate-free anti-inflammatory hydrogel microneedle particles of the present application. Figure 7 Fig. 11: (a) is the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid cation radical (ABTS +• UV-vis absorption spectra of ABTS solution after treatment with different materials, inset is the photo of ABTS solution after treatment with different materials + • Photos of the solution after treatment with different materials; (b) is the scavenging efficiency of different materials on ABTS + • UV-vis absorption spectra of DPPH solution after treatment with different materials, inset is the photo of DPPH solution after treatment with different materials; (d) is the scavenging efficiency of different materials on DPPH·.

[0024] The specific content of the present application is further explained in detail in the following combined with examples. DETAILED DESCRIPTION

[0025] It should be noted that all the raw materials used in the present application are known in the art, and no special explanation is needed.

[0026] In the present application, the microneedle substrate refers to an adhesive flexible base (such as a medical tape).

[0027] The following gives specific examples of the present application, it should be noted that the present application is not limited to the following specific examples, any equivalent transformation based on the technical solutions of the present application falls within the protection scope of the present application.

[0028] Example 1: This example gives a kind of substrate-free anti-inflammatory hydrogel microneedle particles, which is composed of anti-inflammatory active components and hydrogel for wrapping anti-inflammatory active components. The hydrogel is prepared by using hyaluronic acid. The anti-inflammatory active component is selected from curcumin (as shown in formula I below), celecoxib (as shown in formula II below) or aesculin (as shown in formula III below): Formula I; Formula II; Formula III.

[0029] Example 2: This example gives a preparation method of the substrate-free anti-inflammatory hydrogel microneedle particles of example 1, as shown in the formula, the key of the method is to remove the microneedle substrate and keep the needle tip to form a microneedle particle with five sharp vertices. The method specifically includes the following steps: Figure 1 ​Step one, preparation of anti-inflammatory active component solution: 20 mg of curcumin was dissolved in 20 mL of absolute ethanol to obtain a curcumin ethanol stock solution of 1 mg / mL; then, 200 μL of the curcumin ethanol solution was added to 7 mL of ultrapure water, and 2.80 mL of absolute ethanol was added to the solution, which was ultrasonically dissolved to prepare a solution containing 20 μg / mL of curcumin (light yellow liquid; the solvent is a mixed system of water and ethanol, and the volume fraction of ethanol is 30%), and then placed at 4 ℃ for standby.

[0030] Step two, preparation of anti-inflammatory pre-gel solution: 526 mg of hyaluronic acid powder was added to 10 mL of the solution containing 20 μg / mL of curcumin prepared in step one, and stirred overnight to fully dissolve the powder. The pre-gel solution was left to stand at room temperature for 2 hours to remove bubbles, obtaining 10 mL of a curcumin pre-gel solution with a 5% mass fraction of hyaluronic acid.

[0031] Step three, preparation of anti-inflammatory hydrogel microneedle particles without substrate: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, and after removing the excess solution on the surface, vacuum suction was performed for 5 minutes to fill the solution. After 2 hours of cold storage in a 4 ℃ refrigerator to remove small bubbles, the hydrogel microneedle particles were demolded from the mold using adhesive tape, obtaining curcumin hyaluronic acid microneedle particles (Cur-5HA MN).

[0032] Example 3: This example gives a method for preparing the anti-inflammatory hydrogel microneedle particles without substrate of Example 1, which specifically comprises the following steps: Step one, preparation of anti-inflammatory active component solution: in this example, step one is exactly the same as step one of Example 2.

[0033] Step two, preparation of anti-inflammatory pre-gel solution: 1.11 g of hyaluronic acid was dissolved in 10 mL of a solution containing 20 μg / mL of curcumin (30% volume fraction of ethanol solution), and stirred overnight to fully dissolve the powder. The pre-gel solution was left to stand at room temperature for 2 hours to remove bubbles, obtaining 10 mL of a curcumin pre-gel solution with a 10% mass fraction of hyaluronic acid.

[0034] Step three, preparation of the substrate-free anti-inflammatory hydrogel microneedle particles: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, after removing the excess solution on the surface, vacuum suction was performed for 5 minutes to fill the solution, and after 2 hours of refrigeration in a 4 ℃ refrigerator to remove small bubbles, natural drying was performed at room temperature, the solidification time was 1 hour, the anti-inflammatory pre-gel solution was replenished, the excess pre-gel solution on the surface was removed, and vacuum suction and drying were performed again, and the process was repeated multiple times until the microneedle mold was completely filled under dry conditions. The hydrogel microneedle particles in the mold were demolded using adhesive tape to obtain curcumin hyaluronic acid microneedle particles (Cur-10HA MN).

[0035] In this embodiment, when the mass fraction of hyaluronic acid is increased to 10%, the viscosity of the hyaluronic acid solution becomes high, and it takes a longer time to fully dissolve and stand to obtain a stable pre-gel solution, and further obtain curcumin hyaluronic acid microneedle particles (Cur-10HA MN) with stronger mechanical properties.

[0036] Example 4: This embodiment gives a method for preparing the substrate-free anti-inflammatory hydrogel microneedle particles of Example 1, which specifically comprises the following steps: Step one, preparation of an anti-inflammatory active ingredient solution: 20 mg of celecoxib was dissolved in 20 mL of anhydrous ethanol to obtain a 1 mg / mL celecoxib stock solution; then, 200 μL of the celecoxib stock solution was added to 7 mL of ultrapure water, and 2.80 mL of anhydrous ethanol was added to the solution, which was ultrasonically dissolved to prepare a solution containing 20 μg / mL celecoxib (colorless transparent liquid; the solvent is a mixed system of water and ethanol, and the volume fraction of ethanol is 30%), which was then placed at 4 ℃ for standby.

[0037] Step two, preparation of an anti-inflammatory pre-gel solution: 526 mg of hyaluronic acid powder was added to 10 mL of the solution containing 20 μg / mL celecoxib prepared in step one, and the powder was fully dissolved after stirring overnight, and the pre-gel solution was placed at room temperature for 2 hours to remove bubbles, to obtain 10 mL of a celecoxib pre-gel solution with a 5% mass fraction of hyaluronic acid.

[0038] Step three, preparation of the substrate-free anti-inflammatory hydrogel microneedle particles: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, after removing the excess solution on the surface, vacuum suction was performed for 5 minutes to fill the solution, and after 2 hours of refrigeration in a 4 °C refrigerator to remove small bubbles, natural drying was performed at room temperature, the curing time was 1 hour, the anti-inflammatory pre-gel solution was replenished, the excess pre-gel solution on the surface was removed, and vacuum suction and drying were performed again, and the process was repeated multiple times until the microneedle mold was completely filled in a dry state. The hydrogel microneedle particles in the mold were demolded using adhesive tape to obtain the celcoxib hyaluronic acid microneedle particles (CXB-5HA MN).

[0039] Example 5: This example gives a method for preparing the substrate-free anti-inflammatory hydrogel microneedle particles of Example 1, which specifically comprises the following steps: Step one, preparation of the anti-inflammatory active component solution: in this example, step one is exactly the same as step one of Example 4.

[0040] Step two, preparation of the anti-inflammatory pre-gel solution: 1.11 g of hyaluronic acid was dissolved in 10 mL of a solution containing 20 μg / mL of celcoxib (30% volume fraction of ethanol solution), the powder was fully dissolved after stirring overnight, and the pre-gel solution was left to stand at room temperature for 2 hours to remove bubbles, obtaining 10 mL of a 10% hyaluronic acid mass fraction celcoxib pre-gel solution.

[0041] Step three, preparation of the substrate-free anti-inflammatory hydrogel microneedle particles: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, after removing the excess solution on the surface, vacuum suction was performed for 5 minutes to fill the solution, and after 2 hours of refrigeration in a 4 °C refrigerator to remove small bubbles, natural drying was performed at room temperature, the curing time was 1 hour, the anti-inflammatory pre-gel solution was replenished, the excess pre-gel solution on the surface was removed, and vacuum suction and drying were performed again, and the process was repeated multiple times until the microneedle mold was completely filled in a dry state. The hydrogel microneedle particles in the mold were demolded using adhesive tape to obtain the celcoxib hyaluronic acid microneedle particles (CXB-5HA MN).

[0042] Example 6: This example gives a method for preparing the substrate-free anti-inflammatory hydrogel microneedle particles of Example 1, which specifically comprises the following steps: Step one, preparation of the anti-inflammatory active component solution: 20 mg of aesculin was dissolved in 10 mL of ultrapure water, and the water bath was stirred at 37 °C until the aesculin was completely dissolved, obtaining a 2000 μg / mL aesculin stock solution.

[0043] Step two, preparation of anti-inflammatory pre-gel solution: 526 mg of hyaluronic acid powder was added to 10 mL of the 2000 μg / mL aescine mother liquor prepared in step one, and stirred overnight to fully dissolve the powder. The pre-gel solution was left to stand at room temperature for 2 hours to remove bubbles, obtaining 10 mL of aescine pre-gel solution with 5% hyaluronic acid (colorless and transparent liquid).

[0044] Step three, preparation of anti-inflammatory hydrogel microneedle particles without substrate: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, and after removing the excess solution on the surface, vacuum suction was performed for 5 minutes to fill the solution. After being left to stand in a 4 ℃ refrigerator for 2 hours to remove small bubbles, the hydrogel microneedle particles in the mold were demolded using adhesive tape, obtaining aescine hyaluronic acid microneedle particles (Esc-5HA MN).

[0045] Example 7: This example gives a method for preparing the anti-inflammatory hydrogel microneedle particles without substrate of Example 1, which specifically comprises the following steps: Step one, preparation of anti-inflammatory active component solution: in this example, step one is exactly the same as step one of Example 6.

[0046] Step two, preparation of anti-inflammatory pre-gel solution: 1.11 mg of hyaluronic acid powder was added to 10 mL of the 2000 μg / mL aescine solution prepared in step one, and stirred overnight to fully dissolve the powder. The pre-gel solution was left to stand at room temperature for 2 hours to remove bubbles, obtaining 10 mL of aescine pre-gel solution with 5% hyaluronic acid.

[0047] Step three, preparation of anti-inflammatory hydrogel microneedle particles without substrate: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, and after removing the excess solution on the surface, vacuum suction was performed for 5 minutes to fill the solution. After being left to stand in a 4 ℃ refrigerator for 2 hours to remove small bubbles, the hydrogel microneedle particles in the mold were demolded using adhesive tape, obtaining aescine hyaluronic acid microneedle particles (Esc-10HA MN).

[0048] Comparative Example 1: This comparative example gives a method for preparing hydrogel microneedle particles without substrate without active components, which specifically comprises the following steps: Step 1: Preparation of the pre-gel solution without substrate: 526 mg of hyaluronic acid powder was added to 10 mL of deionized water, stirred overnight to fully dissolve the powder, and left to stand at room temperature for 2 hours to remove bubbles in the pre-gel solution, obtaining a 10 mL pre-gel solution of 5% hyaluronic acid (without active ingredients).

[0049] Step 2: Preparation of the hydrogel microneedle particles without substrate: 300 μL of the pre-gel solution was injected into the PDMS mold, the excess solution on the surface was removed, vacuum suction was performed for 5 minutes to fill the solution, and after 2 hours of cold storage in the 4 ℃ refrigerator to remove small bubbles, natural drying was performed at room temperature, the solidification time was 1 hour, the anti-inflammatory pre-gel solution was replenished, the excess pre-gel solution on the surface was removed, and vacuum suction and drying were performed again, and repeated multiple times until the microneedle mold was completely filled under dry conditions. The hydrogel microneedle particles in the mold were demolded using adhesive tape, obtaining 5% hyaluronic acid microneedle particles (5HA MN).

[0050] Comparative Example 2: This comparative example gives a preparation method of hydrogel microneedle particles without substrate without active components, which specifically includes the following steps: Step 1: Preparation of the pre-gel solution without substrate: 1.11 g of hyaluronic acid powder was dissolved in 10 mL of deionized water, stirred overnight to fully dissolve the powder, and left to stand at room temperature for 2 hours to remove bubbles in the pre-gel solution, obtaining a 10 mL pre-gel solution of 10% hyaluronic acid.

[0051] Step 2: Preparation of the hydrogel microneedle particles without substrate: In this comparative example, the specific process of step 2 is basically the same as that of Comparative Example 1. Finally, 10% hyaluronic acid microneedle particles (10HA MN) were obtained.

[0052] Comparative Example 3: This comparative example gives a preparation method of hydrogel microneedle particles without substrate without active components, which specifically includes the following steps: Step 1: Preparation of the pre-gel solution without substrate: 1.76 g of hyaluronic acid powder was added to 10 mL of deionized water, stirred overnight to fully dissolve the powder, and left to stand at room temperature for 2 hours to remove bubbles in the pre-gel solution, obtaining a 10 mL pre-gel solution of 15% hyaluronic acid (without anti-inflammatory active ingredients).

[0053] Step 2: Preparation of the hydrogel microneedle particles without substrate: In this comparative example, the specific process of step 2 is basically the same as that of Comparative Example 1. Finally, 15% hyaluronic acid microneedle particles (15HA MN) were obtained.

[0054] Comparative Example 4: The present comparative example gives a preparation method of a substrate-free anti-inflammatory hydrogel microneedle particle, which is basically the same as that of Example 2, except that the content of ethanol in the solvent system is different in step one. The method specifically comprises the following steps: Step one, preparation of anti-inflammatory active component solution: 20 mg of curcumin is dissolved in 20 mL of anhydrous ethanol to obtain a curcumin ethanol stock solution of 1 mg / mL; then, 200 μL of the curcumin ethanol solution is added to 6 mL of ultrapure water, and 3.80 mL of anhydrous ethanol is further added to the solution, which is ultrasonically dissolved to prepare a solution containing 20 μg / mL of curcumin (light yellow liquid; the solvent is a mixed system of water and ethanol, and the volume fraction of ethanol is 40%), which is then placed at 4 ℃ for standby.

[0055] Step two, preparation of anti-inflammatory pre-gel solution: 526 mg of hyaluronic acid powder is added to 10 mL of the solution containing 20 μg / mL of curcumin prepared in step one, and the powder is fully dissolved by stirring overnight. The pre-gel solution is removed after standing at room temperature for 2 hours to remove bubbles, and 10 mL of a curcumin pre-gel solution containing 5% of hyaluronic acid is obtained.

[0056] Step three, preparation of substrate-free anti-inflammatory hydrogel microneedle particles: the anti-inflammatory pre-gel solution prepared in step two is injected into the PDMS mold, and the excess solution on the surface is removed. Vacuum suction is performed for 5 minutes to fill the solution, and the microneedle mold is filled completely after multiple repetitions of supplementing the anti-inflammatory pre-gel solution, removing the excess pre-gel solution on the surface, and vacuum suctioning and drying again. The hydrogel microneedle particles in the mold are demolded using adhesive tape with adhesion, and curcumin hyaluronic acid microneedle particles are obtained.

[0057] Comparative Example 5: The present comparative example gives a preparation method of a substrate-free anti-inflammatory hydrogel microneedle particle, which is basically the same as that of Example 2, except that the content of ethanol in the solvent system is different in step one. The method specifically comprises the following steps: Step one, preparation of anti-inflammatory active component solution: 20 mg of curcumin is dissolved in 20 mL of anhydrous ethanol to obtain a curcumin ethanol stock solution of 1 mg / mL; then, 200 μL of the curcumin ethanol solution is added to 6 mL of ultrapure water, and 3.80 mL of anhydrous ethanol is further added to the solution, which is ultrasonically dissolved to prepare a solution containing 20 μg / mL of curcumin (light yellow liquid; the solvent is a mixed system of water and ethanol, and the volume fraction of ethanol is 40%), which is then placed at 4 ℃ for standby.

[0058] Step two, preparation of anti-inflammatory pre-gel solution: 526 mg of hyaluronic acid powder was added to 10 mL of the solution containing 30 μg / mL curcumin prepared in step one, stirred overnight to fully dissolve the powder, and room temperature for 1 hour to remove the pre-gel solution gas bubble, to obtain 10 mL of curcumin pre-gel solution with a mass fraction of 5% of hyaluronic acid.

[0059] Step three, preparation of anti-inflammatory hydrogel microneedle particles without substrate: the anti-inflammatory pre-gel solution prepared in step two was injected into the PDMS mold, the excess solution on the surface was removed, vacuum suction was performed for 5 minutes to fill the solution, and after 2 hours of cold storage in the refrigerator at 4 ℃ to remove small bubbles, natural drying was performed at room temperature, the solidification time was 1 hour, the anti-inflammatory pre-gel solution was replenished, the excess pre-gel solution on the surface was removed, and vacuum suction and drying were performed again, and repeated multiple times until the microneedle mold was completely filled under dry conditions. The hydrogel microneedle particles in the mold were demolded using adhesive tape, and curcumin hyaluronic acid microneedle particles were obtained.

[0060] Effect verification: Figure 2 The photographs and graphs of the parameter optimization in the preparation process of the anti-inflammatory hydrogel microneedle particles without substrate in Comparative Examples 1, 2, 3, 4, and 5 are shown. Figure (a) shows that different pre-gel solution contents of hyaluronic acid have an effect on the morphology of the microneedles. Since the 15% hyaluronic acid solution is relatively viscous, it is difficult to remove bubbles during the drying process at room temperature, resulting in shrinkage and deformation of the substrate and the tip, affecting the morphology and mechanical properties of the microneedles. Figure (b) shows that different ethanol contents have an effect on hyaluronic acid. When the ethanol content in the water-ethanol cosolvent solution reaches 50%, the hyaluronic acid will deteriorate due to the presence of ethanol, and will present a milky white gel state. When the ethanol content is 40%, the curcumin hyaluronic acid pre-gel solution prepared will become turbid during the drying process of the microneedle preparation, and the curcumin will precipitate as the ethanol evaporates, resulting in uneven microneedles

【 Figure 2 Figure 2 (d) shows that the maximum solubility of curcumin with 30% ethanol is about 20 μg / mL, and the solution is clear and transparent. When the concentration reaches 30 μg / mL, the solution is turbid and a large number of drug particles are not dissolved.

[0061] Figure 3 The optical microscope images of the two kinds of microneedle particles with different lengths prepared in Comparative Example 1 are shown. The needle body is in the shape of a four-sided pyramid. Figure 3 (a) and (b) show that the microneedle particles do not rely on substrate support, have sharp tips, and have good dispersibility. The average longitudinal needle length of the microneedle particles is 646.3 μm, and the average lateral needle length at the bottom is 262.8 μm. Figure 3 ​(c), (d) are optical microscope images of short needle long microneedle particles, the average needle length of each angle is 190.9 μm, the microneedle particles shrink during the drying process, the size is slightly small, and it can be observed that the microneedle particles all have five sharp vertices.

[0062] Figure 4 is a mechanical puncture force test curve of the hyaluronic acid microneedles prepared in Comparative Examples 1 and 2 and Example 1. The breaking strength of the microneedles is detected by using a mechanical testing machine to ensure that they will not break during the penetration of the skin. Figure 4 It can be seen that the microneedles are not prone to brittle fracture to cause the instantaneous drop of the force-displacement curve and failure, the mechanical properties of the 5% hyaluronic acid microneedles are better in general, the microneedle tips of the 10% hyaluronic acid microneedles are more prone to bending under the action of force, the loaded drug has no obvious effect on the mechanical properties of the microneedles, and each group of microneedles can withstand the minimum force (0.0058 N) for penetrating the skin.

[0063] Figure 5 is an in vitro drug release curve of Cur-5HA MN and Cur-10HA MN prepared in Example 1 and Example 2. Figure 5 (a) is the UV-Vis absorption spectrum of Cur-5HA- MN, it can be seen that the microneedles are completely dissolved at about 50 min, at which time the microneedles release all the loaded drugs. Similarly, from Figure 5 (b) it can be seen that the microneedles completely release the loaded drugs at about 90 min. The increase of the content of hyaluronic acid slightly slows down the release of the drugs, since the cross-linking degree of the hydrogel formed by the self-crosslinking of hyaluronic acid is low, the microneedles quickly release the drugs after absorbing water.

[0064] Figure 6 is an in vitro transdermal simulation characterization of Cur-5HA MN and Cur-10HA MN prepared in Example 1, Example 2, Comparative Example 1 and Comparative Example 2. The microneedles leave clear holes on the front and back of the Parafilm® M film after penetrating the film, by counting the number of holes left by the microneedles after penetrating the Parafilm® M film with different layers, the depth and penetration rate of the microneedles can be obtained Figure 6(a). The microneedles easily penetrated the first two layers of Parafilm® M film, with a penetration rate of 95%; when the film thickness was 375 μm, the penetration rate of the microneedles decreased to about 20%, because the length of the microneedles was 600 μm and the Parafilm® M film had a certain elasticity, so the microneedles were difficult to penetrate; when the film thickness was 500 μm, the microneedles could hardly penetrate the Parafilm® M film. According to the penetration of the sealing film by the microneedles, it was found that the penetration depth of about 5% and 10% of the microneedles was about 300 μm, and the loaded drug had no obvious effect on the penetration depth. In the pig skin transdermal simulation, the microneedles dissolved rapidly within 5 min, and the fluorescence intensity at different depths of the pig skin was observed under a confocal laser microscope. The 5% hyaluronic acid microneedles could reach a depth of about 180 μm, and the 10% microneedles could reach a depth of about 330 μm.

[0065] Figure 7 is the in vitro antioxidant activity characterization of the substrate-free anti-inflammatory hydrogel microneedle particles prepared in Examples 1, 2, 3, and 4, as shown in Figure 7 The in vitro hydrogel microneedle antioxidant experiment showed that the material with a final concentration of 10 mg / mL had a certain ability to scavenge active nitrogen. The performance of the material in scavenging active nitrogen was analyzed by 1,1-diphenyl-2-trinitrobenzene hydrazine free radical (DPPH·) and 2,2-azobis-(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) methods. The ABTS method is another common method for evaluating the performance of materials in scavenging active nitrogen. ABTS + · free radical has a characteristic absorption peak at 734 nm. When ABTS + · reacts with substances with antioxidant activity, ABTS + · is reduced and the absorbance at 734 nm decreases, and the change in absorbance at 734 nm can be used to analyze the activity of the material in scavenging ABTS + ·. Cur-5HA MN can completely fade ABTS + · and the scavenging efficiency reaches 60.47%, while the color of the control solution does not change significantly, and the color of the CXB-HA MN solution changes from blue-green to light blue, with a weaker ability to scavenge active nitrogen, about 20%. DPPH· is a stable nitrogen-containing free radical that appears purple in an ethanol solution and has a distinct characteristic absorption peak at 520 nm. Because DPPH· contains a single electron, it can accept an electron to stabilize it. When DPPH· combines with a free radical scavenger, the single electron is captured and scavenged, causing the absorbance at 520 nm to decrease, and the degree of decrease is positively correlated with the ability to scavenge free radicals, so the change in absorbance at 520 nm can be used to quantitatively evaluate the activity of the material in scavenging DPPH·. Like the ABTS +· Results, DPPH· also showed similar trends, by UV-vis absorption spectrum analysis found that Figure 7 (c) The addition of Cur-HA MN reduced the UV absorption value of DPPH· free radicals at 517 nm from 0.59 to 0.21, and the quantitative analysis results showed that the clearance rate of DPPH· reached 49.85%

Figure 7(b)

Claims

1. A substrate-free anti-inflammatory hydrogel microneedle particle, characterized in that: The invention comprises an anti-inflammatory active component and a hydrogel for encapsulating the anti-inflammatory active component, but does not include a microneedle substrate; The anti-inflammatory active component is selected from any one of curcumin, celecoxib and aesculin; The hydrogel is prepared from hyaluronic acid.

2. The substrate-free anti-inflammatory hydrogel microneedle particle according to claim 1, wherein The mass ratio of curcumin to hyaluronic acid is 1:2630-5550; the mass ratio of celecoxib to hyaluronic acid is 1:2630-5550; and the mass ratio of aesculin to hyaluronic acid is 1:26.3-55.

5.

3. A method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 1 or 2, characterized in that: The method comprises the following steps: Step 1, preparation of anti-inflammatory active component solution; Step 2: Preparation of anti-inflammatory pre-gel solution: adding hyaluronic acid powder to the anti-inflammatory active component solution prepared in step 1, stirring until the powder is fully dissolved, and standing until bubbles disappear to obtain an anti-inflammatory pre-gel solution; Step 3. Preparation of substrate-free anti-inflammatory hydrogel microneedle particles: inject the anti-inflammatory pre-gel solution prepared in step 2 into the mold and fill it completely, and obtain substrate-free anti-inflammatory hydrogel microneedle particles after demoulding.

4. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 3, wherein: In step 1, the concentration of curcumin in the anti-inflammatory active component solution is 20 μg / mL, the concentration of celecoxib is 20 μg / mL, and the concentration of aesculin is 2000 μg / mL.

5. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 3, wherein: Step 1 comprises: dissolving curcumin or celecoxib in anhydrous ethanol to obtain an anti-inflammatory active component mother liquor; then, adding the anti-inflammatory active component to water, adding anhydrous ethanol thereto, and ultrasonically dissolving to prepare an anti-inflammatory active component solution, wherein the volume ratio of ethanol to water is 3:

7.

6. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 3, wherein: Step 1 comprises: dissolving aesculin in water, stirring in a water bath at 37° C. until the aesculin is completely dissolved, to obtain a mother solution of anti-inflammatory active components.

7. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 3, wherein: In step 2, the mass fraction of hyaluronic acid in the anti-inflammatory pre-gel solution is 5 wt% to 10 wt%.

8. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 3, wherein: In step three, the process of completely filling the anti-inflammatory pre-gel solution includes: removing excess solution on the surface and performing vacuum suction to fill the solution, then standing until small bubbles disappear, and then drying and curing; repeating the above vacuum suction, standing, drying and curing processes many times until the microneedle mold is completely filled in a dry state.

9. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 8, wherein: In step 3, the standing temperature is 4°C to 10°C, and the standing time is 2 h to 3 h.

10. The method for preparing substrate-free anti-inflammatory hydrogel microneedle particles according to claim 8, wherein: In step 3, the drying and curing temperature is 20°C to 35°C, and the drying and curing time is 1 h to 2 h.

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