A recombinant human thymosin beta 4 microneedle patch, a preparation method thereof and application in wound healing

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a recombinant human thymosin beta 4 microneedle patch, a preparation method thereof and application. After a microneedle solution and a support layer solution are obtained, the microneedle solution is added into a microneedle mold, and then exhaust treatment is performed; after the exhaust treatment, the support layer solution is added into the microneedle mold to obtain a formed body; the formed body is dried, and after demolding, the recombinant human thymosin beta 4 microneedle patch is obtained; the temperature of the drying is < 40 DEG C. The recombinant human thymosin beta 4 microneedle patch prepared by the application has high drug loading, good forming, and high mechanical strength, can rapidly and continuously release most of the drugs in a short time, is beneficial to the percutaneous penetration of the Tbeta4 drug, and has a good promoting effect on wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a recombinant human thymosin beta 4 microneedle patch, a preparation method thereof and application in wound healing. BACKGROUND

[0002] Recombinant human thymosin beta 4 (rhTβ4, Tβ4) is an oligopeptide composed of 43 amino acids, widely distributed in various tissues and cells of the human body, with a molecular weight of 4960.5 Da and an isoelectric point of 4.6. It is a water-soluble peptide. As an angiogenic factor, Tβ4 promotes wound healing by stimulating endothelial cell migration, angiogenesis, collagen deposition, and increasing protease activity, and has anti-inflammatory ability, which also helps to promote wound healing after skin and eye injury.

[0003] Transdermal administration as a non-invasive administration route is an attractive parenteral administration method, because transdermal administration can not only avoid the degradation of the gastrointestinal tract and liver enzymes caused by oral administration, thereby obtaining higher bioavailability, but also avoid the large fluctuation of blood drug concentration caused by food when orally administered. However, hydrophobic drugs with a molecular weight greater than 500 Da and biological macromolecular drugs (including protein drugs) are difficult to penetrate the skin, thereby greatly limiting the application of protein drug transdermal delivery.

[0004] Micro-needles, as a new and popular drug delivery route, are increasingly attracting the attention of researchers. The needle body length is 10-2000 μm, and the tip diameter is less than several tens of microns, which can effectively penetrate the stratum corneum of the skin, form a drug delivery channel on the surface of the skin, and make the drug reach the designated depth of the skin and enter the subcutaneous capillary network for absorption, which can better solve the problem of macromolecular drugs that cannot penetrate the skin, and has the advantages of painless, minimally invasive, and improved bioavailability.

[0005] However, the existing conventional micro-needles are prepared by high temperature, and the preparation process destroys the activity of the protein drug (Tβ4 molecule), so that the protein drug micro-needles obtained have the disadvantages of poor drug stability and low drug loading. SUMMARY

[0006] The present application provides a recombinant human thymosin beta 4 (Tβ4) microneedle patch and a preparation method thereof and application in wound healing. The recombinant human thymosin beta 4 microneedle patch prepared by the present application has high drug loading, good forming, and high mechanical strength, and can rapidly and continuously release most of the drug in a short time, which is conducive to the transdermal penetration of Tβ4 drug. The recombinant human thymosin beta 4 microneedle patch prepared by the present application has a good promoting effect on wound healing.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0008] The application provides a preparation method of a recombinant human thymosin beta 4 microneedle patch, comprising the following steps:

[0009] The recombinant human thymosin beta 4, the first auxiliary material and water are mixed to obtain a microneedle solution; the first auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;

[0010] The second auxiliary material and water are mixed to obtain a support layer solution; the second auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;

[0011] The microneedle solution is added into a microneedle mold, and then exhaust treatment is performed; after the exhaust treatment, the support layer solution is added into the microneedle mold to obtain a formed body;

[0012] The formed body is dried, and the recombinant human thymosin beta 4 microneedle patch is obtained after demolding; the temperature of the drying is < 40 DEG C.

[0013] Preferably, the temperature of the drying is ≤ 35 DEG C, and the time is ≥ 36 h.

[0014] Preferably, the mass concentration of the recombinant human thymosin beta 4 in the microneedle solution is 10-400 mg / mL.

[0015] Preferably, the first auxiliary material is chondroitin sulfate and sucrose, and the content of chondroitin sulfate in the microneedle solution is 10-100 w / v%, and the content of sucrose is 0.1-10 w / v%.

[0016] Preferably, the first auxiliary material is hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol, and the content of hydroxypropyl-beta-cyclodextrin in the microneedle solution is 10-100 w / v%, the content of Tween 80 is 0.1-10 w / v%, the content of trehalose is 0.1-30 w / v%, and the content of polyvinyl alcohol is 10-100 w / v%.

[0017] Preferably, the second auxiliary material is chondroitin sulfate and sucrose, and the content of chondroitin sulfate in the support layer solution is 10-100 w / v%, and the content of sucrose is 0.1-10 w / v%.

[0018] Preferably, the second auxiliary material is hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol, and the content of hydroxypropyl-beta-cyclodextrin in the support layer solution is 10-100 w / v%, the content of Tween 80 is 0.1-10 w / v%, the content of trehalose is 0.1-30 w / v%, and the content of polyvinyl alcohol is 10-100 w / v%.

[0019] Preferably, the microneedle mold comprises a support layer structure and a microneedle structure arranged on the surface of the support layer structure, the microneedle structure is arranged in an array on the surface of the support layer structure; the microneedle structure is in the shape of a hollow cone, the bottom diameter of the microneedle structure is 100-500 μm, and the height is 300-1000 μm; the distance between the top ends of any two adjacent microneedle structures is 500-1200 μm.

[0020] The application provides a recombinant human thymosin beta 4 microneedle patch prepared by the preparation method, comprising a support layer and microneedles arranged in an array on the surface of the support layer, wherein the microneedles comprise recombinant human thymosin beta 4 and a first excipient, the first excipient comprises at least two of hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; and the support layer comprises a second excipient, the second excipient comprises at least two of hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose.

[0021] The application provides an application of the recombinant human thymosin beta 4 microneedle patch in the manufacture of a medical device for wound healing.

[0022] The application provides a preparation method of a recombinant human thymosin beta 4 microneedle patch, comprising the following steps: mixing a recombinant human thymosin beta 4, a first auxiliary material and water to obtain a microneedle solution; the first auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; mixing a second auxiliary material and water to obtain a support layer solution; the second auxiliary material comprises at least two of hydroxypropyl-beta-cyclodextrin, tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; adding the microneedle solution into a microneedle mold, and then performing exhaust treatment; after the exhaust treatment, the support layer solution is added into the microneedle mold to obtain a formed body; the formed body is dried, and after demolding, the recombinant human thymosin beta 4 microneedle patch is obtained; and the temperature of the drying is < 40 DEG C. According to the application, the types of the auxiliary materials in the microneedle solution and the support layer solution are reasonably set, and the low-temperature drying condition with a temperature < 40 DEG C is adopted, so that the problem that the recombinant human thymosin beta 4 may lose activity or structural change under high-temperature conditions to cause the loss of drug efficacy can be effectively avoided, the prepared recombinant human thymosin beta 4 microneedle patch has high drug loading capacity; the obtained recombinant human thymosin beta 4 microneedle patch is well formed, has high mechanical strength, can rapidly and continuously release most of the drugs in a short time, and is beneficial to the percutaneous penetration of the Tβ4 drug. According to the results of the examples, the recombinant human thymosin beta 4 microneedle patch prepared by the application is well formed, has a good needle body appearance, has no empty needle phenomenon, and has a sharp needle tip without damage or fracture phenomenon; the recombinant human thymosin beta 4 microneedle patch prepared by the application can easily penetrate the aluminum foil, has sufficient mechanical strength, and each single needle can withstand a force of 0.47 N, so the force required for penetrating the skin is usually less than 0.1 N / needle, so the microneedle can easily penetrate the skin and has good mechanical properties; the drug loading capacity of the recombinant human thymosin beta 4 microneedle patch prepared by the application is 200-1000 μg per microneedle patch; the recombinant human thymosin beta 4 microneedle patch prepared by the application can rapidly and continuously release most of the drugs in 30 min, and the rapid and continuous release is beneficial to the percutaneous penetration of the drug; the recombinant human thymosin beta 4 microneedle patch prepared by the application has good safety; and the recombinant human thymosin beta 4 microneedle patch prepared by the application has a good promoting effect on wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a whole structure of the recombinant human thymosin beta 4 microneedle patch prepared by the example 1 of the application;

[0024] Figure 2 It is a microneedle morphology of the recombinant human thymosin beta 4 microneedle patch prepared by the example 1 of the application;

[0025] Figure 3 It is an aluminum foil puncture experiment result of the recombinant human thymosin beta 4 microneedle patch prepared by the example 1 of the application;

[0026] Figure 4 The mouse skin puncture experiment result graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0027] Figure 5 The force-displacement curve graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0028] Figure 6 The drug release degree graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0029] Figure 7 The skin recovery result of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0030] Figure 8 The mouse full skin layer injury model result graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0031] Figure 9 The mouse wound healing graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0032] Figure 10 The mouse wound recovery rate graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 1 of the present application;

[0033] Figure 11 The force-displacement curve graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 2 of the present application;

[0034] Figure 12 The drug release degree graph of the recombinant human thymosin β4 microneedle patch prepared for the embodiment 2 of the present application. DETAILED DESCRIPTION

[0035] The present application provides a preparation method of a recombinant human thymosin β4 microneedle patch, comprising the following steps:

[0036] The recombinant human thymosin β4, the first auxiliary material and water are mixed to obtain a microneedle solution; the first auxiliary material comprises at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;

[0037] The second auxiliary material and water are mixed to obtain a support layer solution; the second auxiliary material comprises at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;

[0038] The microneedle solution is added to a microneedle mold, and then exhaust treatment is performed; after the exhaust treatment, the support layer solution is added to the microneedle mold to obtain a shaped body;

[0039] The formed body is dried, and after demolding, the recombinant human thymosin β4 microneedle patch is obtained; the drying temperature is < 40℃.

[0040] In the present application, all the preparation raw materials / components are commercially available products well known to those skilled in the art, unless otherwise specified.

[0041] The present application mixes recombinant human thymosin β4, a first excipient and water to obtain a microneedle solution; the first excipient includes at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose. In the present application, the first excipient is preferably chondroitin sulfate and sucrose. Or the first excipient is preferably hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol. The water is particularly preferably ultrapure water. The mass concentration of recombinant human thymosin β4 in the microneedle solution is preferably 10-400 mg / mL, more preferably 10-150 mg / mL, and more preferably 50-100 mg / mL. When the first excipient is chondroitin sulfate and sucrose, the content of chondroitin sulfate in the microneedle solution is preferably 10-100 w / v%, more preferably 20-80 w / v%; the content of sucrose is preferably 0.1-10 w / v%, more preferably 1-8 w / v%. When the first excipient is hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol, the content of hydroxypropyl-β-cyclodextrin in the microneedle solution is preferably 10-100 w / v%, more preferably 20-80 w / v%; the content of Tween 80 is preferably 0.1-10 w / v%, more preferably 1-8 w / v%; the content of trehalose is preferably 0.1-30 w / v%, more preferably 1-25 w / v%; the content of polyvinyl alcohol is preferably 10-100 w / v%, more preferably 20-80 w / v%.

[0042] In the present application, the mixing of the recombinant human thymosin β4, the first excipient and water preferably includes: dissolving the recombinant human thymosin β4 in water to obtain a recombinant human thymosin β4 aqueous solution; adding the first excipient to the recombinant human thymosin β4 aqueous solution.

[0043] The second auxiliary material includes at least two of hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose. In the present application, the second auxiliary material is preferably chondroitin sulfate and sucrose. Alternatively, the second auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol. The water is particularly preferably ultrapure water. When the second auxiliary material is preferably chondroitin sulfate and sucrose, the content of chondroitin sulfate in the support layer solution is preferably 10-100 w / v%, more preferably 20-80 w / v%; the content of sucrose is preferably 0.1-10 w / v%, more preferably 1-8 w / v%. When the second auxiliary material is preferably hydroxypropyl-beta-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol, the content of hydroxypropyl-beta-cyclodextrin in the support layer solution is preferably 10-100 w / v%, more preferably 20-80 w / v%; the content of Tween 80 is preferably 0.1-10 w / v%, more preferably 1-8 w / v%; the content of trehalose is preferably 0.1-30 w / v%, more preferably 1-25 w / v%; the content of polyvinyl alcohol is preferably 10-100 w / v%, more preferably 20-80 w / v%.

[0044] After obtaining the microneedle solution, the microneedle solution is added to the microneedle mold, and then the exhaust treatment is performed. In the present application, the material of the microneedle mold is preferably polydimethylsiloxane. The microneedle mold includes a support layer structure and a microneedle structure arranged on the surface of the support layer structure, and the microneedle structure is arranged in an array on the surface of the support layer structure; the shape of the microneedle structure is a hollow cone, the bottom diameter of the microneedle structure is preferably 100-500 μm, more preferably 200-400 μm, and the height is preferably 300-1000 μm, more preferably 300-1000 μm; the distance between the top ends of any two adjacent microneedle structures is preferably 500-1200 μm, more preferably 600-1000 μm. In the present application, the support layer structure of the microneedle mold is a square, and the long side of the square support layer structure is 10-50 mm. In a specific embodiment of the present application, the size of the support layer structure of the microneedle mold can be 10.5 mm x 10.5 mm or 50 mm x 50 mm, and the microneedle structure is preferably arranged in a 10 x 10 square array on the surface of the support layer structure.

[0045] In the present application, the exhaust treatment is preferably performed under vacuum conditions, and the vacuum degree is preferably -0.09 to -0.1 MPa. In a specific embodiment of the present application, the exhaust treatment is preferably performed in a vacuum drying box. The exhaust treatment is preferably performed cyclically, and a single exhaust treatment step includes: vacuumizing the environment for exhaust treatment to -0.09 to -0.1 MPa, maintaining for 1-5 min, and then releasing the air to normal pressure. The exhaust treatment is preferably cycled 1-5 times.

[0046] After exhaust treatment, and after obtaining the support layer solution, the present application adds the support layer solution into the microneedle mold to obtain a shaped body. Before adding the support layer solution into the microneedle mold, the present application preferably scrapes off the excess microneedle solution located at the support layer structure part.

[0047] After obtaining the shaped body, the present application dries the shaped body to obtain the recombinant human thymosin β4 microneedle patch after demolding; the drying temperature is < 40℃, more preferably 2-30℃, and further preferably 2-15℃. In the present application, the drying time is preferably ≥ 36h, and more preferably 36-48h. The drying is preferably performed in a dryer.

[0048] The present application provides a recombinant human thymosin β4 microneedle patch prepared by the preparation method described in the above technical solution, comprising a support layer and microneedles arranged in an array on the surface of the support layer, the microneedles comprising recombinant human thymosin β4 and a first excipient, the first excipient comprising at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate, and sucrose; and the support layer comprising a second excipient, the second excipient comprising at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate, and sucrose.

[0049] In the present application, the first excipient is preferably chondroitin sulfate and sucrose. Alternatively, the first excipient is preferably hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, and polyvinyl alcohol.

[0050] In the present application, the second excipient is preferably chondroitin sulfate and sucrose. Alternatively, the second excipient is preferably hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, and polyvinyl alcohol.

[0051] In the present application, the active pharmaceutical ingredient of the recombinant human thymosin β4 microneedle patch is recombinant human thymosin β4. The recombinant human thymosin β4 microneedle patch is a soluble drug-loaded microneedle patch.

[0052] In the present application, the shape of the microneedle of the recombinant human thymosin β4 microneedle patch is preferably a cone, the bottom diameter of the microneedle is preferably 100-500μm, more preferably 200-400μm, and the height is preferably 300-1000μm, more preferably 800-1000μm; and the distance between the top ends of any two adjacent microneedles is preferably 500-1200μm, more preferably 600-1000μm.

[0053] In the embodiments of the present application, the support layer of the recombinant human thymosin β4 microneedle patch has a square shape with a side length of 1-15 cm, preferably 1-5 cm. The size of the support layer of the recombinant human thymosin β4 microneedle patch is specifically 1 cm x 1 cm or 5 cm x 5 cm.

[0054] In the embodiments of the present application, the microneedles of the recombinant human thymosin β4 microneedle patch are arranged in a 10 x 10-20 x 20 square array on the surface of the support layer.

[0055] The present application provides the use of the recombinant human thymosin β4 microneedle patch described in the above technical solution in the manufacture of a medical device for wound healing. In the present application, the medical device for wound healing is specifically preferably a microneedle patch.

[0056] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0057] Example 1

[0058] 1. Dissolve Tβ4 in ultrapure water to prepare a Tβ4 solution, and add chondroitin sulfate and sucrose to prepare a microneedle solution. In the microneedle solution, the mass concentration of Tβ4 is 80 mg / mL, the content of chondroitin sulfate is 50% (w / v), and the content of sucrose is 1% (w / v).

[0059] 2. Add chondroitin sulfate and sucrose to ultrapure water to prepare a support layer solution. In the support layer solution, the content of chondroitin sulfate is 50% (w / v), and the content of sucrose is 1% (w / v).

[0060] 3. Add the microneedle solution to a polydimethylsiloxane microneedle mold, which includes a support layer structure and a microneedle structure arranged on the surface of the support layer structure. The size of the support layer structure is 1 cm x 1 cm or 5 cm x 5 cm. The microneedle structure is arranged in a 10 x 10 square array on the surface of the support layer structure. The microneedle structure has a hollow conical shape with a bottom diameter of 350 μm and a height of 1000 μm. The distance between the top ends of any two adjacent microneedle structures is 800 μm. Then, place it in a vacuum drying oven and vacuumize to -0.1 MPa. Keep it for 3 min, release the vacuum, remove the bubbles, vacuumize again to -0.1 MPa, and keep it for 2 min, and then release the vacuum.

[0061] 4. After scraping off the excess microneedle solution, add the support layer solution.

[0062] 5. Place the microneedle mold containing the microneedle solution and the support layer solution in a desiccator and dry it in an environment of 2-4°C for 48 hours to obtain a Tβ4 drug-loaded microneedle patch.

[0063] Test Example 1: Morphological Characterization of Tβ4 Drug-Loaded Microneedle Patches

[0064] The morphology of the Tβ4 drug-loaded microneedle patch was observed under a microscope, and the results are as follows: Figure 1 and Figure 2 As shown. By Figure 1 and Figure 2 It can be seen that the Tβ4 drug-loaded microneedle patch prepared in Example 1 is well formed, the needle body has a good appearance, there is no empty needle phenomenon, and the tip has no defects or breakage, and has a sharp needle tip.

[0065] Test Example 2: Hardness Assessment of Tβ4 Drug-Loaded Microneedle Patches

[0066] The mechanical properties of the microneedles in the Tβ4 drug-loaded microneedle patch were investigated using aluminum foil puncture test and isolated rat skin puncture test, respectively.

[0067] (1) Aluminum foil puncture test

[0068] During aluminum foil puncture, a certain force was applied to vertically insert the microneedle into the aluminum foil and maintained for 2 minutes. The ability of the microneedle to pierce the aluminum foil was observed, and the hardness of the microneedle was evaluated. Results are shown below. Figure 3 ,Depend on Figure 3 The results showed that the microneedles of the Tβ4 drug-loaded microneedle patch prepared in Example 1 could easily penetrate the aluminum foil, indicating that the Tβ4 drug-loaded microneedle patch prepared in Example 1 had sufficient mechanical strength.

[0069] (2) Ex vivo rat skin puncture test

[0070] Treatment of ex vivo skin

[0071] Healthy SD rats were euthanized by cervical dislocation. The abdominal hair was carefully removed, the skin was taken, and the adipose tissue and fascia were separated. The rats were repeatedly washed with physiological saline, blotted dry with filter paper, wrapped in aluminum foil, and stored at -80°C. Before use, the skin was first soaked in physiological saline for 30 minutes.

[0072] Ex vivo rat skin puncture

[0073] The microneedles of the Tβ4 drug-loaded microneedle patch prepared in Example 1 were vertically inserted into the excised skin of healthy, undamaged rats using a certain force. After 10 minutes, the microneedles were removed, and the skin was immediately stained with 0.4% trypan blue solution. After 15 minutes, excess staining agent on the skin surface was removed with isopropanol, and the skin was rinsed with physiological saline. The condition of the stained pores on the skin surface was observed. Clearly visible pores could be seen on the rat skin after trypan blue staining. Figure 4 . Figure 4 The results show that the Tβ4 drug-loaded microneedle patch prepared in Example 1 has sufficient mechanical strength to penetrate detached skin and meets the experimental requirements.

[0074] Test Example 3: Mechanical property test of Tβ4-loaded microneedle

[0075] The microneedle needs to have a certain mechanical strength to ensure that it can pierce the skin barrier without breaking or bending. In order to detect the mechanical strength of the microneedle, a texture analyzer was used to evaluate the mechanical strength of the Tβ4-loaded microneedle patch prepared in Example 1. The test speed was set to 0.05 mm / s, and the mechanical properties of the microneedle were evaluated by comparing the force and displacement of the microneedle tip during the compression process.

[0076] The results are shown in Figure 5 When the force reached 47 N, the force-displacement curve showed a sudden drop, indicating that the tip broke. It was concluded that each single needle could withstand a force of 0.47 N, and the force required to penetrate the skin was usually less than 0.1 N / needle. Therefore, the microneedle could easily penetrate the skin and had good mechanical properties.

[0077] Test Example 4: Drug content test of Tβ4-loaded microneedle patch

[0078] Drug content determination of microneedle

[0079] pH 7.4 PBS was used as the release medium, and the Tβ4-loaded microneedle patch prepared in Example 1 was placed in the release medium. The sample was taken after 3 min of ultrasonic treatment, and the above operation was repeated for 3 groups in parallel. Liquid chromatography was used for detection.

[0080] The average drug content of each microneedle was 248.15 μg.

[0081] Test Example 5: In vitro release experiment of Tβ4-loaded microneedle

[0082] pH 7.4 PBS was used as the release medium and added to a small beaker. The Tβ4-loaded microneedle patch prepared in Example 1 was placed in the release medium, and the small beaker was placed in a constant temperature shaker. The temperature was set to 37°C, and the rotation speed was 100 rpm. Samples of 400 μL were taken at 15 s, 30 s, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 45 min, 60 min, 120 min, 180 min, 240 min, 300 min, 360 min, and 420 min, and the same volume of release medium was added quickly. The above experiment was repeated 3 times (n = 3), and the drug content in each time period was determined by high performance liquid chromatography. The chromatographic detection conditions were the same as those in Test Example 3, and the cumulative release amount (CRA) and cumulative release rate (CRR) were calculated. The formula for calculating CRA is shown in Formula 2:

[0083]

[0084] In formula 2: V is the volume of release medium, Vi is the volume of each sampling, Ci and Ci-1 are the drug concentration in the release medium at the i-th and i-1-th sampling, respectively.

[0085] Cumulative release = cumulative release amount / total drug content x 100%.

[0086] Table 1 Drug release results of Tβ4-loaded microneedle patches prepared in Example 1

[0087] Time / min Release (%) 0.25 36.54±8.91 0.5 56.62±1.36 1 62.10±6.36 2 69.07±13.74 5 70.45±20.03 10 70.70±17.04 20 77.83±10.86 30 86.67±4.42 45 99.70±0.23 60 93.92±0.87 120 91.71±3.38 180 89.69±0.46 240 86.02±4.45 300 84.12±2.70 360 85.75±3.45 420 86.09±1.61

[0088] The drug release profile of Tβ4-loaded microneedle patches prepared in Example 1 is shown in Figure 6 As shown in Figure 6 , the cumulative release of the drug-loaded microneedles was 62.10% at 1 min, 86.67% at 30 min, and 99.70% at 45 min. The results demonstrate that the Tβ4-loaded microneedles can rapidly and continuously release most of the drug within 45 min, and the rapid release of Tβ4 microneedles is conducive to the transdermal penetration of the drug.

[0089] Test Example 6: Safety evaluation of Tβ4-loaded microneedles

[0090] The microneedles may break during use and remain in the skin for a long time, so the safety of the material is crucial. The irritation test is to observe whether the local reactions such as redness, hyperemia, exudation, degeneration or necrosis are caused in the blood vessels, muscles, skin, mucous membranes and other parts of the animals after contacting the test substance.

[0091] SD rats were anesthetized by intraperitoneal injection of 150 μL of 4% chloral hydrate and the abdominal hair was removed. The microneedle patch was placed on the rat abdominal skin for 10 min and then removed to take a photo to record the skin condition. At this time, 0 min was recorded, and the skin recovery state was recorded at 2 min, 4 min, 6 min, 8 min, 10 min, 30 min, 1 h, and 2 h, respectively. The skin recovery results are shown in Figure 7 .

[0092] As shown in Figure 7 , at 0 min, the hole marks caused by the microneedle penetration were clear, and there was no bleeding or redness. Within 2 min, the holes on the skin gradually blurred, and after 10 min, the marks on the skin almost disappeared. After 1 h, the skin returned to normal and the marks completely disappeared. Through the degree of skin erythema and edema after the application of the microneedle, it was found that the microneedle had good safety.

[0093] Test Example 7: Study on Tβ4-loaded microneedles for wound healing

[0094] (1) The mouse full-thickness wound model is established

[0095] Twenty C57 mice are selected, anesthetized with 4% chloral hydrate (100 μL / 10 g), fixed in a prone position, the hair on the back is shaved, and the hair is removed with depilatory cream. After the surgical site is disinfected, the full-thickness skin is cut with tissue scissors to prepare a circular wound (about 8 mm in diameter) and sutured with a healing pad. The mouse full-thickness wound model is successfully established. Figure 8 The mouse full-thickness wound model is established.

[0096] (2) Experimental animal grouping and treatment

[0097] The wound modeling mice are randomly divided into two groups: the experimental group mice are covered with Tβ4-loaded microneedles on the back wound, and are fixed with pressure-sensitive tape; the blank control group is not covered with microneedles. Each group has 10 mice, which are raised in separate cages, and the microneedles are replaced every day.

[0098] (3) Wound observation and wound healing rate

[0099] During the experiment, the wound is photographed and sampled every day (using a ruler for comparison), the healing of the wound is compared and recorded, and the Image J software is used to calculate the wound area of the mouse at a specific period, and the healing rate of the wound is calculated. The calculation formula of the healing rate of the wound is shown in formula 3:

[0100] Healing rate = (wound area on day 0 - wound area on the measurement day) / wound area on day 0 x 100% formula 3

[0101] Figure 9 The wound healing graph is shown. Figure 10 The wound recovery rate graph is shown

[0102] From the animal experiment results ( Figure 9 and Figure 10 ), it can be seen that the wound healing rate of the Tβ4 microneedle experimental group has a significant difference compared with the blank group from the 4th day, on the 12th day of the experiment, the wound healing rate of the microneedle group reaches 95.65%, almost healed, and the recovery rate of the control group is only 86.44%, on the 14th day, the microneedle group has basically recovered to normal, and the control group is still in the healing process. It shows that Tβ4 microneedles have a promoting effect on wound healing.

[0103] Example 2

[0104] 1. Tβ4 is dissolved in ultrapure water to prepare a Tβ4 solution, and hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol are added to prepare a microneedle solution. In the microneedle solution, the mass concentration of Tβ4 is 80 mg / mL, the content of hydroxypropyl-β-cyclodextrin is 10% (w / v), the content of Tween 80 is 0.1% (w / v), the content of trehalose is 10% (w / v), and the content of polyvinyl alcohol is 10% (w / v).

[0105] 2. Add hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol into ultrapure water to prepare a support layer solution, the content of hydroxypropyl-β-cyclodextrin in the support layer solution is 10% (w / v), the content of Tween 80 is 0.1% (w / v), the content of trehalose is 10% (w / v), and the content of polyvinyl alcohol is 10% (w / v).

[0106] 3. Add the microneedle solution into the polydimethylsiloxane microneedle mold, the microneedle mold comprises a support layer structure and a microneedle structure arranged on the surface of the support layer structure, the size of the support layer structure is 10.5 mm x 10.5 mm, the microneedle structure is arranged in an 8 x 8 square array on the surface of the support layer structure, the shape of the microneedle structure is a hollow cone, the bottom diameter of the microneedle structure is 350 μm, and the height is 1000 μm; the distance between the top ends of any two adjacent microneedle structures is 800 μm. Then, vacuumize to -0.1 MPa, keep for 3 min, release the vacuum, remove the bubbles, vacuumize again to -0.1 MPa, keep for 2 min, and release the vacuum.

[0107] 4. After scraping off the excess microneedle solution, add the support layer solution.

[0108] 5. Place the microneedle mold containing the microneedle solution and the support layer solution in a desiccator, dry in an environment of 2-4°C for 48 hours, and obtain the Tβ4 drug-loaded microneedle patch.

[0109] The Tβ4 drug-loaded microneedle patch prepared in Example 2 is subjected to mechanical strength performance test according to the method described in the above “Test Example 3: Mechanical property test of Tβ4 drug-loaded microneedle”, and the test results are shown in Figure 11 Figure 1 The horizontal coordinate X in Figure 11 is displacement, unit: mm, and the vertical coordinate Y is force, unit: N, and it can be known from

[0110] The Tβ4 drug-loaded microneedle patch prepared in Example 2 is subjected to in vitro release experiment according to the method described in the above “Test Example 5: In vitro release experiment of Tβ4 drug-loaded microneedle”, and the release degree of the Tβ4 drug-loaded microneedle patch prepared in Example 2 is shown in Table 2.

[0111] Table 2 Release degree results of the Tβ4 drug-loaded microneedle patch prepared in Example 2

[0112]

[0113]

[0114] ​The drug release profile of the Tβ4-loaded microneedle patch prepared in Example 2 is shown in FIG. 2. As shown in FIG. 2, the performance of the Tβ4-loaded microneedle patch prepared in Example 2 in wound healing is similar to the performance of Example 1 in wound healing. Figure 12 Figure 12

[0115] Comparative Example 1

[0116] The preparation method of Example 1 was basically the same, except that the drying method in step 5 was as follows: drying in a 40°C air-drying oven for 24 hours.

[0117] Drug loading amount determination of the Tβ4-loaded microneedle patch prepared in Comparative Example 1

[0118] 5 mL of PBS with pH = 7.4 was used as the release medium, the mold with the needle tip part filled with the needle tip solution was placed in the release medium, and sampling was performed by ultrasonic treatment for 3 min. The above operation was repeated for 3 groups in parallel (n = 3), and the chromatographic detection conditions were the same as those in Test Example 4.

[0119] The peak areas of the three groups in parallel were 9.252, 11.877, and 10.876, respectively. Substituting these values into the linear standard curve equation 1, the drug loading amount of the microneedle prepared in Comparative Example 1 was obtained, and the average value was 95.126 μg per microneedle. Compared with the drug loading amount of the Tβ4-loaded microneedle patch prepared in Example 1 obtained in Test Example 4, it can be seen that the drug content of the microneedle patch prepared in Comparative Example 1 was much lower than that of the microneedle patch prepared in Example 1 by the low-temperature drying method.

[0120] ​​From the above examples, it can be seen that the present application uses chondroitin sulfate and sucrose as the excipient of recombinant human thymosin beta 4, and at the same time, low-temperature conditions of temperature ≤ 4℃ are used for drying, so as to effectively avoid the problem that the recombinant human thymosin beta 4 may lose activity or structural changes under high-temperature conditions, resulting in loss of drug efficacy. From the results of the examples, it can be seen that the recombinant human thymosin beta 4 microneedle patch prepared by the present application has good forming, the needle body appearance is good, there is no empty needle phenomenon, and the sharp end has no defect or fracture phenomenon, and has a sharp needle tip; the recombinant human thymosin beta 4 microneedle patch prepared by the present application can easily penetrate the aluminum foil, has sufficient mechanical strength, each single needle can withstand 0.47N force, and the force required to penetrate the skin is usually less than 0.1N / needle, so the microneedle can easily penetrate the skin, and has good mechanical properties; the drug loading of the recombinant human thymosin beta 4 microneedle patch prepared by the present application is 248.15μg per microneedle; the recombinant human thymosin beta 4 microneedle patch prepared by the present application can rapidly and continuously release most of the drug within 20min, and the rapid release is conducive to the transdermal penetration of the drug; the recombinant human thymosin beta 4 microneedle patch prepared by the present application has good safety; the recombinant human thymosin beta 4 microneedle patch prepared by the present application has good promoting effect on wound healing.

[0121] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. A method for preparing a recombinant human thymosin β4 microneedle patch, characterized in that, Includes the following steps: Recombinant human thymosin β4, the first excipient, and water were mixed to obtain a microneedle solution; the microneedle solution contained recombinant human thymosin β4 at a mass concentration of 80 mg / mL, hydroxypropyl-β-cyclodextrin at a content of 10% (w / v), Tween 80 at a content of 0.1% (w / v), trehalose at a content of 10% (w / v), and polyvinyl alcohol at a content of 10% (w / v). The second excipient is mixed with water to obtain a support layer solution; the support layer solution contains 10% (w / v) hydroxypropyl-β-cyclodextrin, 0.1% (w / v) Tween 80, 10% (w / v) trehalose, and 10% (w / v) polyvinyl alcohol. The microneedle solution is added to the microneedle mold, and then the air is removed. After the venting process, the support layer solution is added to the microneedle mold to obtain the molded body; The molded body is dried and demolded to obtain the recombinant human thymosin β4 microneedle patch; the drying temperature is 2~4℃. The drying time is ≥36 hours; The microneedle mold includes a support layer structure and microneedle structures disposed on the surface of the support layer structure. The microneedle structures are arranged in an array on the surface of the support layer structure. The shape of the microneedle structure is a hollow cone. The bottom diameter of the microneedle structure is 100~500μm and the height is 300~1000μm. The distance between the top ends of any two adjacent microneedle structures is 500~1200μm.

2. The recombinant human thymosin β4 microneedle patch prepared by the preparation method according to claim 1.

3. The use of the recombinant human thymosin β4 microneedle patch according to claim 2 in the manufacture of medical devices for wound healing.

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