Double-layer microneedle loaded with gingerol and shogaol as well as preparation method and application of double-layer microneedle

Abstract

The invention relates to a gingerol and shogaol loaded double-layer microneedle and a preparation method and application thereof.The double-layer microneedle comprises a microneedle base and a microneedle body, and the microneedle body is prepared from gingerol, shogaol, a base material and a medicinal solubilizer. The double-layer microneedle provided by the invention realizes simultaneous loading of gingerol and shogaol, solves the problems of poor stability and low water solubility of gingerol and shogaol, and realizes transdermal transmission of drugs. The gingerol and the enol are jointly loaded in the microneedle body of the double-layer microneedle, and the gingerol and the enol have a synergistic effect, so that the treatment effect of the microneedle on the diabetic wound surface can be further improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a double-layer microneedle loaded with gingerol and shogaol, and a preparation method and application thereof. Background Art

[0002] Refractory diabetic wounds are one of the common complications of diabetes, with the characteristics of long-term non-healing and recurrent attacks. When the skin of diabetic patients is damaged, the immune disorder caused by diabetes causes the wound to be in a high-level inflammatory environment for a long time, gradually turning into a chronic ulcer that is refractory. Improper or untimely treatment of chronic diabetic wounds may lead to amputation or even life-threatening. At present, wound dressings and hydrogels have made great progress in the treatment of diabetic wounds, but they still have the disadvantages of single function, difficulty in drug penetration into deep parts, limited repair effect, and secondary trauma caused by peeling off sticky dressings from the wound. Therefore, there is an urgent need to develop an effective treatment for diabetic wounds.

[0003] CN118415960A discloses a microneedle patch and a preparation method for promoting the healing of diabetic wounds, the microneedle patch includes a microneedle array and a base, both of which are made of a water-soluble matrix material and a recombinant adipose stem cell exosome liposome suspension with a mass volume ratio of 5 to 10 g: 100 mL. The microneedle patch can release recombinant adipose stem cell exosome liposomes directly into the diabetic ulcer wound, and the stem cell exosomes can penetrate into the wound tissue through osmotic absorption, improve the wound microenvironment, promote wound tissue growth and angiogenesis, and accelerate the healing of ulcerated wounds.

[0004] CN115708817A discloses a microneedle patch for promoting healing of diabetic wounds, comprising a backing, graphene oxide adsorbing cobalt ions dispersed inside the backing, microneedles arranged on the backing, and hydrogen-releasing nanoparticles dispersed inside the microneedles, wherein the nano hydrogen-releasing particles are mesoporous silica nanoparticles loaded with hydrogen prodrug ammonia borane. By combining microneedle transdermal drug administration and nanomaterials, hydrogen is continuously released locally on the wound surface to relieve inflammation, while hydrogen reduces graphene oxide to release cobalt ions to promote angiogenesis, which has excellent photothermal antibacterial effect.

[0005] Ginger is the dried rhizome of ginger, a plant of the Zingiberaceae family. It is one of the important natural medicinal plants. The rhizome of ginger and its extracts are also widely used as medicines. The oleoresin extracted from the rhizome of ginger contains a variety of irritating components such as gingerol and shogaol, and has been shown to have a variety of pharmacological activities. However, due to its poor transdermal absorption and poor stability, its further clinical application is limited.

[0006] Therefore, how to improve the transdermal absorption effect and stability of gingerol and shogaol so that they can better exert their efficacy and promote the healing effect of diabetic wounds has become a technical problem that needs to be solved urgently. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention aims to provide a double-layer microneedle loaded with gingerol and shogaol, and a preparation method and application thereof. The double-layer microneedle loaded with gingerol and shogaol can realize the simultaneous loading of gingerol and shogaol, solve the problems of poor stability and low water solubility of gingerol and shogaol, realize transdermal transmission of drugs, and can improve the therapeutic effect on diabetic wounds.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a double-layer microneedle loaded with gingerol and shogaol, wherein the double-layer microneedle comprises a microneedle base and a microneedle body, and the raw materials for preparing the microneedle body comprise gingerol, shogaol, a matrix material and a pharmaceutical solubilizer.

[0010] Gingerol is a class of active substances extracted from ginger rhizomes, and its structure contains gingerol structural units; shogaol is an aromatic compound composed of a benzene ring and a cyclohexene ring, and its structure contains a carbon-carbon double bond and a phenolic hydroxyl group. Gingerol and shogaol are chemically unstable and have poor transdermal absorption. The double-layer microneedles provided by the present invention achieve simultaneous loading of gingerol and shogaol, solve the problems of poor stability and low water solubility of gingerol and shogaol, and achieve transdermal transmission of drugs.

[0011] At the same time, gingerol and enol, as two different types of substances, are co-loaded in the microneedle body of the double-layer microneedle. The synergistic effect of the two can further improve the therapeutic effect of microneedles on diabetic wounds.

[0012] Preferably, the mass ratio of gingerol to shogaol is 1:(0.1-10).

[0013] Among them, the specific point values ​​between 0.1 and 10 can be selected from 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0014] Preferably, the gingerol includes any one of 6-gingerol, 8-gingerol or 10-gingerol, or a combination of at least two of them.

[0015] Preferably, the shogaol includes any one of 6-shogaol, 8-shogaol or 10-shogaol, or a combination of at least two thereof.

[0016] Preferably, the matrix material includes a first polymer material and a second polymer material, the first polymer material includes any one of gelatin, peach gum or locust bean gum or a combination of at least two thereof, and the second polymer material includes hyaluronic acid.

[0017] Preferably, the first polymer material includes gelatin, peach gum and locust bean gum.

[0018] Gelatin is a high molecular weight polypeptide polymer obtained by partial degradation of collagen in connective tissues such as animal skin, bone, and fascia. Its main component is protein and it contains a variety of amino acids. It has good biocompatibility and degradability, and its degradation products can be absorbed and metabolized by the human body. Peach gum is a colloid, translucent polysaccharide natural gum secreted from the bark of peach or mountain peach in the Rosaceae family. Its main component is polysaccharide and it also contains a small amount of protein, minerals, etc. Locust bean gum comes from the endosperm part of the bean kernel in the pods of the Robinia tree in the Leguminosae family, and its main component is galactomannan.

[0019] The present invention creatively discovers that gelatin, peach gum and locust bean gum together constitute the main components of the microneedle needle body matrix material, and the formed microneedles have a better pore structure, which can improve the loading efficiency of gingerol and shogaol. At the same time, the prepared microneedle needle body can slowly dissolve, control the drug to release more persistently on the wound surface, and increase the residence time of the drug at the site of action.

[0020] Hyaluronic acid has extremely strong water absorption. Adding hyaluronic acid to the base material of the microneedle body can increase the transdermal absorption function of the microneedle and improve the transdermal absorption efficiency of gingerol and shogaol.

[0021] Preferably, the mass ratio of the gelatin, peach gum and locust bean gum is 1:(0.1-0.3):(0.2-0.6).

[0022] Among them, the specific point values ​​in 0.1-0.3 can be selected from 0.1, 0.15, 0.2, 0.25, 0.3, etc., and the specific point values ​​in 0.2-0.6 can be selected from 0.2, 0.3, 0.4, 0.5, 0.6, etc.

[0023] Based on the potential interaction among gelatin, peach gum and locust bean gum in improving drug loading and release effect, when the three meet the above-mentioned specific ratio relationship, the drug release effect can be better improved.

[0024] Preferably, the mass ratio of the first polymer material to the second polymer material is 1:(0.01-0.5).

[0025] Among them, the specific point values ​​between 0.01 and 0.5 can be selected as 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, etc.

[0026] Preferably, the pharmaceutical solubilizer includes any one of sodium carboxymethylcellulose, sodium carboxymethyl starch, hydroxypropyl cellulose or cross-linked polyvinylpyrrolidone, or a combination of at least two thereof.

[0027] Preferably, the pharmaceutically acceptable solubilizer comprises sodium carboxymethylcellulose, sodium carboxymethyl starch and hydroxypropyl cellulose.

[0028] The present invention creatively finds that sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose work synergistically to further improve the solubility of gingerol and shogaol, thereby increasing the drug loading capacity of the microneedle body.

[0029] Preferably, the mass ratio of sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose is 1:(0.1-0.5):(0.5-1.5).

[0030] Among them, the specific point values ​​in 0.1-0.5 can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, etc., and the specific point values ​​in 0.5-1.5 can be selected from 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, etc.

[0031] Preferably, the raw material for preparing the microneedle base includes any one of polyvinyl alcohol, polyhydroxyethyl methacrylate or polyvinyl pyrrolidone, or a combination of at least two thereof.

[0032] Preferably, the microneedle body is conical, with a height of 500-700 μm and a bottom diameter of 300-500 μm.

[0033] Preferably, the center distance between the microneedle bodies is 800-1200 μm.

[0034] Among them, the specific point values ​​in 500-700μm can be selected from 500μm, 550μm, 600μm, 650μm, 700μm, etc., the specific point values ​​in 300-500μm can be selected from 300μm, 350μm, 400μm, 450μm, 500μm, etc., and the specific point values ​​in 800-1200μm can be selected from 800μm, 900μm, 1000μm, 1100μm, 1200μm, etc.

[0035] In a second aspect, the present invention provides a method for preparing a double-layer microneedle loaded with gingerol and shogaol as described in the first aspect, the preparation method comprising the following steps:

[0036] The mixed liquid of raw materials for preparing the microneedle body is added into the microneedle mold and dried to obtain the microneedle body; the mixed liquid of raw materials for preparing the microneedle base is added into the mold with the microneedle body, dried, and demolded to obtain the double-layer microneedles loaded with gingerol and shogaol.

[0037] Preferably, the method for preparing the raw material mixture for preparing the microneedle body comprises the following steps:

[0038] (1) mixing a first polymer material and a second polymer material in a solvent to obtain a needle base material mixed solution; mixing gingerol, shogaol and an aqueous solution of a pharmaceutical solubilizer to obtain a drug mixed solution;

[0039] (2) Mixing the needle body base material mixture liquid and the drug mixture liquid to obtain a raw material mixture liquid for preparing the microneedle body.

[0040] Preferably, in step (1), the temperature for mixing the first polymer material and the second polymer material in the solvent is 20 to 30° C., and the mixing time is 5 to 20 min.

[0041] Preferably, the temperature of mixing the aqueous solution of gingerol, shogaol and the pharmaceutical solubilizer in step (1) is 20 to 30° C., and the mixing time is 5 to 20 minutes.

[0042] Preferably, the mixing temperature in step (2) is 20 to 30° C., and the mixing time is 5 to 20 min.

[0043] Among them, the specific point values ​​in 20-30℃ can be selected from 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc., and the specific point values ​​in 5-20min can be selected from 5min, 10min, 15min, 20min, etc.

[0044] Preferably, the mixed liquid of raw materials for preparing the microneedle base is an aqueous solution of raw materials for preparing the microneedle base.

[0045] Preferably, in the mixed solution of raw materials for preparing the microneedle base, the mass-volume percentage of the raw materials for preparing the microneedle base is 2-10%.

[0046] Among them, the specific point values ​​between 2% and 10% can be selected from 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.

[0047] In a third aspect, the present invention provides a use of the double-layer microneedles loaded with gingerol and shogaol as described in the first aspect in preparing a healing drug for diabetic wounds.

[0048] The present application creatively discovered that gingerol and shogaol can promote the transformation of macrophages from M1 type to M2 type, thereby improving the healing effect of diabetic wounds. The double-layer microneedles loaded with gingerol and shogaol prepared by the present invention can maintain the stability of gingerol and shogaol, thereby achieving the effect of promoting the healing of diabetic wounds.

[0049] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The double-layer microneedles loaded with gingerol and shogaol developed by the present invention realize the simultaneous loading of gingerol and shogaol, solve the problems of poor stability and low water solubility of gingerol and shogaol, realize the transdermal transmission of drugs, and improve the therapeutic effect of microneedles on diabetic wounds. Gingerol and shogaol are co-loaded in the microneedle body of the double-layer microneedle, and the two synergize to further improve the therapeutic effect of microneedles on diabetic wounds.

[0052] Furthermore, gelatin, peach gum and locust bean gum are combined to form the main components of the microneedle body matrix material. The formed microneedles have a better pore structure, which can improve the loading efficiency of gingerol and shogaol. At the same time, the prepared microneedle body can slowly dissolve, control the drug release on the wound surface for a longer time, and increase the residence time of the drug at the site of action.

[0053] Furthermore, sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose are combined as pharmaceutical solubilizers. The three synergistically enhance the solubility of gingerol and shogaol, thereby increasing the drug loading capacity of the microneedle body. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is a schematic diagram of the preparation process of 6-G+6-S@MN prepared in Example 1;

[0055] Figure 2 This is a physical picture of 6-G+6-S@MN prepared in Example 1;

[0056] Figure 3 This is the immunofluorescence image of 6-G+6-S@MN prepared in Example 1;

[0057] Figure 4 This is a comparison of infrared spectra of different microneedles in Verification Example 1;

[0058] Figure 5 is a graph showing the results of Western blot analysis of different experimental groups in Verification Example 2;

[0059] Figure 6 This is the HE staining result of different test groups in Test Example 2;

[0060] Figure 7 is the immunofluorescence staining result of CD31 expression in different test groups of Test Example 2;

[0061] Figure 8 This is a diagram of microneedle degradation and skin surface changes in test example 3;

[0062] Fig. 9 This is a comparison chart of cell survival rates in different test groups in Test Example 3. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] The sources of the components / raw materials in the following embodiments are shown in the following table:

[0065]

[0066]

[0067] Example 1

[0068] This embodiment provides a double-layer microneedle loaded with 6-gingerol and 6-shogaol, which is prepared by the following method:

[0069] (1) Preparation of microneedle body:

[0070] The mass-volume percentage of each raw material in the microneedle body is as follows:

[0071] Calculated by mass-volume percentage, the raw materials for preparing the microneedle body include: 5% of the first polymer material, 0.5% of the second polymer material, 0.5% of the medicinal solubilizer, 1% of 6-gingerol, 1% of 6-shogaol, and the balance is water.

[0072] Preparation method:

[0073] ① Mix the first polymer material (gelatin powder, peach gum powder and locust bean gum powder in a mass ratio of 1:0.2:0.4) and the second polymer material (hyaluronic acid), add double distilled water, and dissolve in a 37°C water bath until completely dissolved to obtain a needle base material mixed solution.

[0074] ② A pharmaceutical solubilizer (sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 1:0.3:1) is mixed and dissolved in water, and 6-gingerol (6-G) powder and 6-shogaol (6-S) powder are dissolved in the aqueous solution of the pharmaceutical solubilizer to obtain a drug mixture.

[0075] ③ Mix the needle body substrate mixture with the drug mixture, take 1 ml and pour it into a polydimethylsiloxane (PDMS) mold, centrifuge at 4000 rpm for 15 minutes, and dry in a fume hood at 25°C for 1 day to form a microneedle body.

[0076] (2) Preparation of microneedle base:

[0077] The polyvinyl alcohol (PVA) powder was dissolved in double distilled water, stirred evenly, and placed in a 37° C. water bath until completely dissolved to obtain a PVA solution with a mass-volume percentage of 6%.

[0078] (3) Preparation of double-layer microneedles:

[0079] Pour 1 ml of 6% PVA solution into the PDMS mold with the microneedle body, and continue to dry in a fume hood at 25°C for 1 day. After drying, peel off the PDMS mold to obtain a double-layer microneedle loaded with 6-G and 6-S (hereinafter abbreviated as 6-G+6-S@MN).

[0080] The schematic diagram of the preparation process of double-layer microneedles loaded with 6-gingerol and 6-shogaol is shown in Figure 1 shown.

[0081] Example 2

[0082] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is prepared by the following method:

[0083] (1) Preparation of microneedle body:

[0084] The mass-volume percentage of each raw material in the microneedle body is as follows:

[0085] Calculated by mass-volume percentage, the raw materials for preparing the microneedle body include: 7% of the first polymer material, 3% of the second polymer material, 0.8% of the medicinal solubilizer, 1.2% of 8-gingerol, 0.8% of 8-shogaol, and the balance is water.

[0086] Preparation method:

[0087] ① Mix the first polymer material (gelatin powder, peach gum powder and locust bean gum powder in a mass ratio of 1:0.1:0.5) and the second polymer material (hyaluronic acid), add double distilled water, and dissolve in a 30°C water bath until completely dissolved to obtain a needle base material mixed liquid.

[0088] ② A pharmaceutical solubilizer (sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 1:0.1:0.5) is mixed and dissolved in water, and 8-gingerol (8-G) powder and 8-shogaol (8-S) powder are dissolved in the aqueous solution of the pharmaceutical solubilizer to obtain a drug mixture.

[0089] ③ Mix the needle base material mixture with the drug mixture, take 1 ml and pour it into a polydimethylsiloxane (PDMS) mold, centrifuge at 7000 rpm for 10 minutes, and dry in a fume hood at 25°C for 1 day to form a microneedle body.

[0090] (2) Preparation of microneedle base:

[0091] The polyvinyl alcohol (PVA) powder was dissolved in double distilled water, stirred evenly, and placed in a 30° C. water bath until completely dissolved to obtain a PVA solution with a mass-volume percentage of 4%.

[0092] (3) Preparation of double-layer microneedles:

[0093] 1 ml of 4% PVA solution was poured into the PDMS mold with the microneedle body, and continued to dry in a fume hood at 25°C for 1 day. After drying, the PDMS mold was peeled off to obtain a double-layer microneedle loaded with 8-G and 8-S.

[0094] Example 3

[0095] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is prepared by the following method:

[0096] (1) Preparation of microneedle body:

[0097] The mass-volume percentage of each raw material in the microneedle body is as follows:

[0098] Calculated by mass-volume percentage, the raw materials for preparing the microneedle body include: 10% of the first polymer material, 1% of the second polymer material, 1.5% of the medicinal solubilizer, 0.8% of 10-gingerol, 1.2% of 10-shogaol, and the balance is water.

[0099] Preparation method:

[0100] ① Mix the first polymer material (gelatin powder, peach gum powder and locust bean gum powder in a mass ratio of 1:0.3:0.6) and the second polymer material (hyaluronic acid), add double distilled water, and dissolve in a 45°C water bath until completely dissolved to obtain a needle base material mixed liquid.

[0101] ② A pharmaceutical solubilizer (sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 1:0.5:1.2) was mixed and dissolved in water, and 10-gingerol (10-G) powder and 10-shogaol (10-S) powder were dissolved in the aqueous solution of the pharmaceutical solubilizer to obtain a drug mixture.

[0102] ③ Mix the needle base material mixture with the drug mixture, take 1 ml and pour it into a polydimethylsiloxane (PDMS) mold, centrifuge at 5000 rpm for 13 minutes, and dry in a fume hood at 25°C for 1 day to form a microneedle body.

[0103] (2) Preparation of microneedle base:

[0104] The polyvinyl alcohol (PVA) powder was dissolved in double distilled water, stirred evenly, and placed in a 45° C. water bath until completely dissolved to obtain a PVA solution with a mass-volume percentage of 8%.

[0105] (3) Preparation of double-layer microneedles:

[0106] 1 ml of 8% PVA solution was poured into the PDMS mold with the microneedle body, and continued to dry in a fume hood at 25°C for 1 day. After drying, the PDMS mold was peeled off to obtain a double-layer microneedle loaded with 10-G and 10-S.

[0107] Example 4

[0108] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is different from Example 1 only in that the gelatin powder, peach gum powder and locust bean gum powder in a mass ratio of 1:0.2:0.4 are replaced with gelatin powder and peach gum powder in a mass ratio of 1:0.2, the total mass of the first polymer material remains unchanged, and the remaining steps are consistent with Example 1.

[0109] Example 5

[0110] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is different from Example 1 only in that the gelatin powder, peach gum powder and locust bean gum powder in a mass ratio of 1:0.2:0.4 are replaced with gelatin powder and locust bean gum powder in a mass ratio of 1:0.4, the total mass of the first polymer material remains unchanged, and the remaining steps are consistent with Example 1.

[0111] Example 6

[0112] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is different from Example 1 only in that the gelatin powder, peach gum powder and locust bean gum powder in a mass ratio of 1:0.2:0.4 are replaced with peach gum powder and locust bean gum powder in a mass ratio of 0.2:0.4, the total mass of the first polymer material remains unchanged, and the remaining steps are consistent with Example 1.

[0113] Example 7

[0114] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is different from Example 1 only in that sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 1:0.3:1 are replaced with sodium carboxymethyl cellulose and sodium carboxymethyl starch in a mass ratio of 1:0.3, the total mass of the pharmaceutical solubilizer remains unchanged, and the remaining steps are consistent with Example 1.

[0115] Example 8

[0116] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is different from Example 1 only in that sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 1:0.3:1 are replaced with sodium carboxymethyl cellulose and hydroxypropyl cellulose in a mass ratio of 1:1, the total mass of the pharmaceutical solubilizer remains unchanged, and the remaining steps are consistent with Example 1.

[0117] Example 9

[0118] This embodiment provides a double-layer microneedle loaded with gingerol and shogaol, which is different from Example 1 only in that sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 1:0.3:1 are replaced with sodium carboxymethyl starch and hydroxypropyl cellulose in a mass ratio of 0.3:1, the total mass of the pharmaceutical solubilizer remains unchanged, and the remaining steps are consistent with Example 1.

[0119] Example 10

[0120] This embodiment provides an integrated microneedle loaded with gingerol and shogaol, which is prepared by the following method:

[0121] (1) The mass-volume percentage of each raw material in the microneedle body is consistent with that in Example 1. The needle body substrate mixture and the drug mixture are prepared by the same steps as steps (1) ① to ② in Example 1. The PVA solution with a mass-volume percentage of 6% is prepared by the same steps as step (2) in Example 1.

[0122] (2) The needle substrate mixed solution, drug mixed solution, and PVA solution were mixed evenly, poured into a PDMS mold, and dried in a fume hood at 25°C for 1 day. After drying, the PDMS mold was peeled off to obtain an integrated microneedle loaded with 6-G and 6-S.

[0123] Comparative Example 1

[0124] This comparative example provides a double-layer microneedle loaded with gingerol, which differs from Example 1 only in that 6-shogaol is not added, and the mass-volume percentage of 6-gingerol is adjusted to 2%. Accordingly, in the preparation process of the microneedle body, only 6-gingerol powder is dissolved in an aqueous solution of a pharmaceutical solubilizer to obtain a drug mixture, and the remaining steps are consistent with Example 1.

[0125] Comparative Example 2

[0126] This comparative example provides a double-layer microneedle loaded with gingerol, which differs from Example 1 only in that 6-gingerol is not added, and the mass-volume percentage content of 6-shogaol is adjusted to 2%. Accordingly, in the preparation process of the microneedle body, only 6-shogaol powder is dissolved in an aqueous solution of a pharmaceutical solubilizer to obtain a drug mixture, and the remaining steps are consistent with Example 1.

[0127] Comparative Example 3

[0128] This comparative example provides a non-drug-loaded double-layer microneedle, which differs from Example 1 only in that 6-gingerol and 6-shogaol are not added. Accordingly, in the preparation process of the microneedle body, steps (1)-② are adjusted to dissolve the medicinal solubilizer in water to obtain a mixed solution without drugs; steps (1)-③ are adjusted to mix the needle body base material mixed solution with the mixed solution without drugs. The remaining steps are consistent with Example 1, and a non-drug-loaded double-layer microneedle (hereinafter referred to as MN) is prepared.

[0129] Verification Example 1

[0130] This verification example characterizes the structure of the double-layer microneedles loaded with 6-G and 6-S prepared in Example 1.

[0131] (1) The physical picture of 6-G+6-S@MN prepared in Example 1 is as follows Figure 2 As shown, the microneedle body is conical, with a height of about 600 μm, a bottom diameter of about 400 μm, and a center distance of about 1000 μm. The microneedle body has a certain hardness, is neatly arranged, and has an upright needle tip, and can be used to pierce the skin. The immunofluorescence image of the 6-G+6-S@MN prepared in Example 1 is shown in FIG. Figure 3 As shown, it is shown that the microneedles are loaded with drugs 6-G and 6-S and are evenly distributed, which is conducive to the stable release of the drugs.

[0132] (2) Infrared spectroscopy was performed on the 6-G+6-S@MN prepared in Example 1, the non-drug-loaded double-layer microneedle (MN) prepared in Comparative Example 3, and 6-gingerol and 6-shogaol. The results are as follows: Figure 4 As shown in the figure, compared with the infrared spectrum of blank MN, the 6-G+6-S@MN is located at 3445cm -2 The characteristic peak of -OH of 6-G appears at 1000cm -2 The characteristic peak of CH of 6-S appeared. These evidences proved that 6-gingerol and 6-shogaol were successfully loaded on the microneedles.

[0133] Verification Example 2

[0134] This example verifies the effects of 6-G and 6-S on macrophage polarization.

[0135] (1) Trial grouping and intervention method:

[0136] RAW264.7 cells were seeded in 6-well plates and divided into control group, model group, 6-G group, 6-S group and 6-G+6-S group. Lipopolysaccharide LPS (10 ng / ml) was used to induce modeling in the model group, 6-G group, 6-S group and 6-G+6-S group.

[0137] 40 μM 6-G, 40 μM 6-S and 20 μM 6-G + 20 μM 6-S were added to the 6-G group, 6-S group and 6-G + 6-S group, respectively, and equal volumes of PBS buffer were added to the model group and the control group.

[0138] After 24 hours, RAW264.7 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis buffer, and the total protein concentration was determined using a biuret acid (BCA) kit. After separation by gel electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk powder for 1 hour, followed by the addition of primary antibodies against tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) and CD11b, and incubated overnight at 4°C. Afterwards, the corresponding species secondary antibody was added and incubated at room temperature for 1 hour, and the expression of the target protein was detected by ECL luminescence. The results of Western blot analysis of TNF-α, iNOS and CD11b protein expression are shown in Figure 2. Figure 5 As shown, it can be seen that in the 6-G group, 6-S group and 6-G+6-S group, the expression levels of TNF-α, iNOS and CD11b proteins were reduced to varying degrees, indicating that 6-G and 6-S have good anti-inflammatory effects and can affect the transformation of macrophages from M1 to M2, that is, from a pro-inflammatory state to an anti-inflammatory and tissue repair state, which is beneficial to the disappearance of inflammation and the recovery of diabetic wound tissue.

[0139] Test Example 1

[0140] This test example tests the wound healing effect of microneedles.

[0141] (1) Construction of diabetic mouse model: 8-week-old male C57BL / 6 mice (purchased from the Experimental Animal Center of Chongqing Medical University) were selected and first fed a high-fat diet for 4 weeks, then fasted overnight and intraperitoneally injected with streptozotocin (STZ) for 3 consecutive days. The injection dose of STZ was 120 mg / kg, and blood glucose levels were monitored within 1 to 2 days after injection. One week later, plasma glucose levels were randomly measured in three blood glucose tests, and mice with plasma glucose ≥16.7 mM were identified as diabetic mice. 42 mice with successfully constructed diabetic models were selected and divided into 14 groups, with 3 mice in each group, including 13 experimental groups and 1 model group. The mice were anesthetized with isoflurane, the hair on the back was shaved, and a full-thickness skin defect with a diameter of 0.8 cm was prepared.

[0142] (2) Trial grouping and intervention method:

[0143] The microneedles prepared in the examples and comparative examples were cut into circular patches with a diameter of 0.8 cm, sterilized with ultraviolet rays and then used for later use.

[0144] Experimental group: The microneedles prepared in the example and comparative example were sterilized and sequentially attached to the skin defect surfaces of diabetic mice in each experimental group, and fixed around the wound with Tegaderm film to ensure that the patch was stable during the experiment;

[0145] Model group: No microneedle patch was used on the skin defect surface of diabetic mice in the model group.

[0146] The average skin wound area of ​​3 mice in each test group was counted on the 0th day (before patching), the 7th day and the 14th day. The results are shown in the following table:

[0147]

[0148]

[0149] From the data comparison of Example 1 and Comparative Examples 1 to 3, it can be seen that the double-layer microneedles loaded with gingerol and shogaol of the present invention can promote wound healing. At the same time, gingerol and shogaol have significant synergistic effect in promoting wound healing.

[0150] From the data of Examples 1 and 4 to 6, it can be seen that gelatin, peach gum and locust bean gum together constitute the main components of the microneedle needle body matrix material. The three synergistically enhance the effect, and the prepared microneedles can better promote the drug release of gingerol and shogaol and improve the wound healing effect.

[0151] It can be seen from the data of Examples 1 and 7 to 9 that the synergistic effect of sodium carboxymethylcellulose, sodium carboxymethyl starch and hydroxypropyl cellulose can further improve the solubility of gingerol and shogaol, and better promote the healing of skin wounds.

[0152] It can be seen from the data of Example 1 and Example 10 that, compared with the integrated microneedle, the double-layer microneedle involved in the present invention can better promote the release of gingerol and shogaol in the skin wound surface and promote faster wound healing.

[0153] Test Example 2

[0154] This test example tests the repair effect of microneedles on wound tissue.

[0155] The same batch of mice as in Test Example 1 were selected. On the 14th day after the microneedle patch was applied, the mice in the group with Example 1, Comparative Example 3 (blank microneedle group) and the model group without microneedle patch (diabetes model group) were anesthetized by inhalation of isoflurane and killed. Skin samples (1×1 cm 2 ), fixed with 4% paraformaldehyde (PFA), and stained for observation.

[0156] HE staining results Figure 6 As shown, it can be observed that the skin tissue of mice affixed with the microneedle patch of Example 1 (6-G+6-S@MN) undergoes obvious healing.

[0157] The immunofluorescence staining results of CD31 expression and the statistical results of the relative fluorescence intensity of immunofluorescence staining are shown in Figure 7 As shown, it shows that 6-G+6-S@MN microneedle patch can effectively promote angiogenesis in diabetic wounds.

[0158] Test Example 3

[0159] This test example tests the safety of the double-layer microneedles loaded with 6-G and 6-S prepared in Example 1.

[0160] (1) Double-layer microneedle safety test

[0161] The double-layer microneedles loaded with 6-G and 6-S prepared in Example 1 were sterilized and attached to the skin defect surface of diabetic mice to observe the degradation of the microneedles and the changes in the skin surface. Figure 8 As shown, it can be observed that the 6-G+6-S@MN microneedle patch can be effectively degraded within three days, and no redness, swelling or inflammation occurs around the tissue, indicating the safety of the material itself.

[0162] (2) In vitro biocompatibility and cytotoxicity tests

[0163] Trial group and intervention method:

[0164] 6-G+6-S@MN group: The 6-G+6-S@MN prepared in Example 1 was extracted with complete culture medium (α-MEM) to obtain a 6-G+6-S@MN sample solution; RAW264.7 and NIH / 3T3 cells were inoculated in the sample solution, respectively. When the cell density reached 80%, the cells were digested with trypsin containing 0.25% ethylenediaminetetraacetic acid (EDTA), and the cells were inoculated in a 24-well plate (1×105 cells were added to each well) and cultured for 24 h;

[0165] Blank microneedle group: The MN prepared in Comparative Example 3 was extracted with complete culture medium (α-MEM) to obtain a MN sample solution; RAW264.7 and NIH / 3T3 cells were inoculated in the sample solution, respectively. When the cell density reached 80%, the cells were digested with trypsin containing 0.25% ethylenediaminetetraacetic acid (EDTA), and the cells were inoculated in a 24-well plate (1×105 cells were added to each well) and cultured for 24 hours;

[0166] Control group: RAW264.7 and NIH / 3T3 cells were inoculated in the same amount of culture medium as that of the 6-G+6-S@MN group and the MN group, respectively. When the cell density reached 80%, the cells were digested with trypsin containing 0.25% ethylenediaminetetraacetic acid (EDTA), and the cells were inoculated in a 24-well plate (1×105 cells were added to each well) and cultured for 24 h.

[0167] The cell counting kit-8 (CCK-8, Bio-Time, Shanghai, China) was used to analyze the cell viability of the cells in each experimental group within 4, 12, and 24 hours. The results are shown in Fig. 9 As shown, it can be seen that at different culture periods, the cell activities of the 6-G+6-S@MN group, the MN group and the control group are at the same level, indicating that the 6-G+6-S@MN prepared in Example 1 will not affect the activity of the cells and has good biocompatibility.

[0168] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0169] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0170] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A double-layer microneedle loaded with gingerol and shogaol, characterized in that: The double-layer microneedle comprises a microneedle base and a microneedle body. The raw materials for preparing the microneedle body comprise gingerol, shogaol, a matrix material and a pharmaceutical solubilizer.

2. The double-layer microneedle loaded with gingerol and shogaol according to claim 1, characterized in that: The mass ratio of gingerol to shogaol is 1:(0.1-10); Preferably, the gingerol includes any one of 6-gingerol, 8-gingerol or 10-gingerol, or a combination of at least two thereof; Preferably, the shogaol includes any one of 6-shogaol, 8-shogaol or 10-shogaol or a combination of at least two thereof; Preferably, the matrix material comprises a first polymer material and a second polymer material, the first polymer material comprises any one or a combination of at least two of gelatin, peach gum or locust bean gum, and the second polymer material comprises hyaluronic acid; Preferably, the first polymer material includes gelatin, peach gum and locust bean gum; Preferably, the mass ratio of the gelatin, peach gum and locust bean gum is 1:(0.1-0.3):(0.2-0.6); Preferably, the mass ratio of the first polymer material to the second polymer material is 1:(0.01-0.5).

3. The double-layer microneedle loaded with gingerol and shogaol according to claim 1 or 2, characterized in that: The pharmaceutical solubilizer includes any one of sodium carboxymethylcellulose, sodium carboxymethyl starch, hydroxypropyl cellulose or cross-linked polyvinylpyrrolidone, or a combination of at least two thereof; Preferably, the pharmaceutical solubilizer comprises sodium carboxymethylcellulose, sodium carboxymethyl starch and hydroxypropyl cellulose; Preferably, the mass ratio of sodium carboxymethyl cellulose, sodium carboxymethyl starch and hydroxypropyl cellulose is 1:(0.1-0.5):(0.5-1.5).

4. The double-layer microneedle loaded with gingerol and shogaol according to any one of claims 1 to 3, characterized in that: The raw material for preparing the microneedle base includes any one of polyvinyl alcohol, polyhydroxyethyl methacrylate or polyvinyl pyrrolidone or a combination of at least two thereof.

5. The double-layer microneedle loaded with gingerol and shogaol according to any one of claims 1 to 4, characterized in that: The microneedle body is conical, with a height of 500-700 μm and a bottom diameter of 300-500 μm; Preferably, the center distance between the microneedle bodies is 800-1200 μm.

6. A method for preparing a double-layer microneedle loaded with gingerol and shogaol as claimed in any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: The mixed liquid of raw materials for preparing the microneedle body is added into the microneedle mold and dried to obtain the microneedle body; the mixed liquid of raw materials for preparing the microneedle base is added into the mold with the microneedle body, dried, and demolded to obtain the double-layer microneedles loaded with gingerol and shogaol.

7. The preparation method according to claim 6, characterized in that: The method for preparing the raw material mixture for preparing the microneedle body comprises the following steps: (1) mixing a first polymer material and a second polymer material in a solvent to obtain a needle base material mixed solution; mixing gingerol, shogaol and an aqueous solution of a pharmaceutical solubilizer to obtain a drug mixed solution; (2) Mixing the needle body base material mixture liquid and the drug mixture liquid to obtain a raw material mixture liquid for preparing the microneedle body.

8. The preparation method according to claim 7, characterized in that: Step (1) The temperature of mixing the first polymer material and the second polymer material in a solvent is 20 to 30° C. and the mixing time is 5 to 20 minutes; Preferably, in step (1), the temperature of mixing the aqueous solution of gingerol, shogaol and the pharmaceutical solubilizer is 20 to 30° C., and the mixing time is 5 to 20 min; Preferably, the mixing temperature in step (2) is 20 to 30° C., and the mixing time is 5 to 20 min.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The mixed solution of raw materials for preparing the microneedle base is an aqueous solution of raw materials for preparing the microneedle base; Preferably, in the mixed solution of raw materials for preparing the microneedle base, the mass-volume percentage of the raw materials for preparing the microneedle base is 2-10%.

10. Use of the double-layer microneedle loaded with gingerol and shogaol as claimed in any one of claims 1 to 5 in preparing a healing drug for diabetic wounds.

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