Recombinant human thymosin beta4 microneedle patch as well as preparation method and application thereof in wound healing

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a recombinant human thymosin beta4 microneedle patch as well as a preparation method and application thereof. The method comprises the following steps: obtaining a microneedle solution and a supporting layer solution; adding the microneedle solution into a microneedle mold, and then performing exhaust treatment; after exhaust treatment, adding the supporting layer solution into a microneedle mold to obtain a molded body; and drying the molded body, and demolding to obtain the recombinant human thymosin beta4 microneedle patch. The drying temperature is lower than 40 DEG C. The recombinant human thymosin beta4 microneedle patch prepared by the invention is high in drug loading capacity, good in forming and high in mechanical strength, can quickly and continuously release most of drugs in a short time, and is beneficial to percutaneous penetration of Tbeta4 drugs; the recombinant human thymosin beta4 microneedle patch prepared by the invention has a good promotion effect on wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant human thymosin β4 microneedle patch, a preparation method thereof, and an application thereof in wound healing. Background Art

[0002] Recombinant human thymosin β4 (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 4960.5Da 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. Its anti-inflammatory ability also helps promote wound healing after skin and eye injuries.

[0003] As a non-invasive route of administration, transdermal administration is an attractive non-intestinal administration method because it can avoid enzymatic degradation in the gastrointestinal tract and liver caused by oral administration, thereby obtaining higher bioavailability, and can also avoid the large fluctuations in blood drug concentration caused by food during oral administration. However, hydrophobic drugs with a molecular weight greater than 500 Da and biomacromolecule drugs (including protein drugs) are difficult to penetrate the skin, which greatly limits the application of transdermal delivery of protein drugs.

[0004] Microneedles are an emerging and popular drug delivery method that is attracting more and more attention from researchers. The needle body length is between 10 and 2000 μm, and the tip diameter is less than tens of microns. It can effectively pierce the stratum corneum of the skin, forming a drug delivery channel on the skin surface, allowing the drug to reach a specified depth in the skin and enter the subcutaneous capillary network to be absorbed. It can better solve the problem that large molecule drugs cannot penetrate the skin, and it has the advantages of being painless, minimally invasive, and improving bioavailability.

[0005] However, the existing conventional microneedles are prepared at high temperatures, and the preparation process will destroy the activity of the protein drug (Tβ4 molecule), so the resulting protein drug microneedles have the disadvantages of poor drug stability and low drug loading. Summary of the invention

[0006] The purpose of the present invention is to provide a recombinant human thymosin β4 (Tβ4) microneedle patch and a preparation method thereof and application in wound healing. The recombinant human thymosin β4 microneedle patch prepared by the present invention has a high drug loading capacity, good molding, high mechanical strength, can quickly and continuously release most of the drugs in a short time, and is beneficial to the transdermal penetration of the Tβ4 drug; the recombinant human thymosin β4 microneedle patch prepared by the present invention has a good promoting effect on wound healing.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a recombinant human thymosin β4 microneedle patch, comprising the following steps:

[0009] Mixing recombinant human thymosin β4, a first auxiliary material and water to obtain a microneedle solution; the first auxiliary material includes at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose;

[0010] Mixing a second auxiliary material and water 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;

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

[0012] The molded body is dried and demoulded to obtain the recombinant human thymosin β4 microneedle patch; the drying temperature is less than 40°C.

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

[0014] Preferably, the mass concentration of recombinant human thymosin β4 in the microneedle solution is 10 to 400 mg / mL.

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

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

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

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

[0019] Preferably, 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 microneedle structure is in the shape of a hollow cone, the bottom diameter of the microneedle structure is 100 to 500 μm, and the height is 300 to 1000 μm; the distance between the tops of any two adjacent microneedle structures is 500 to 1200 μm.

[0020] The present invention provides a recombinant human thymosin β4 microneedle patch prepared by the preparation method described in the above technical scheme, comprising a support layer and microneedles arrayed on the surface of the support layer, the microneedles comprising recombinant human thymosin β4 and a first auxiliary material, the first auxiliary material comprising at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; the support layer comprises a second auxiliary material, the second auxiliary material comprising at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose.

[0021] The present invention provides the use of the recombinant human thymosin β4 microneedle patch described in the above technical solution in the manufacture of medical devices for wound healing.

[0022] The invention provides a method for preparing a recombinant human thymosin β4 microneedle patch, comprising the following steps: mixing recombinant human thymosin β4, a first auxiliary material and water 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; mixing a second auxiliary material and water to obtain a supporting layer solution; the second auxiliary material comprises at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; adding the microneedle solution to a microneedle mold, and then performing an exhaust treatment; after the exhaust treatment, adding the supporting layer solution to the microneedle mold to obtain a molded body; drying the molded body, and demolding to obtain the recombinant human thymosin β4 microneedle patch; the drying temperature is less than 40°C. The present invention reasonably arranges the types of auxiliary materials in the microneedle solution and the supporting layer solution, and adopts low temperature conditions of temperature less than 40°C for drying, thereby effectively avoiding the problem that recombinant human thymosin β4 may lose activity or undergo structural changes under high temperature conditions, resulting in loss of efficacy. The prepared recombinant human thymosin β4 microneedle patch has a high drug loading capacity; at the same time, the obtained recombinant human thymosin β4 microneedle patch has good molding and high mechanical strength, can quickly and continuously release most of the drugs in a short time, and is beneficial to the transdermal penetration of the Tβ4 drug. It can be seen from the results of the embodiments that the recombinant human thymosin β4 microneedle patch prepared by the present invention is well-formed, has a good needle body morphology and appearance, has no empty needle phenomenon, and has no defect or breakage at the tip, and has a sharp needle tip; the recombinant human thymosin β4 microneedle patch prepared by the present invention can easily penetrate the aluminum foil and has sufficient mechanical strength. Each single needle can withstand a force of 0.47N, and the force required to penetrate the skin is usually less than 0.1N / needle, so the microneedle can easily puncture the skin and has good mechanical properties; the drug loading of the recombinant human thymosin β4 microneedle patch prepared by the present invention is 200 to 1000 μg per microneedle; the recombinant human thymosin β4 microneedle patch prepared by the present invention can quickly and continuously release most of the drugs within 30 minutes, and the rapid and continuous release is conducive to the transdermal penetration of the drugs. The recombinant human thymosin β4 microneedle patch prepared by the present invention has good safety; the recombinant human thymosin β4 microneedle patch prepared by the present invention has a good promoting effect on wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a physical picture of the overall structure of the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0024] Figure 2 This is a microneedle morphology diagram of the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0025] Figure 3 This is a graph showing the aluminum foil puncture test results of the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0026] Figure 4 This is a graph showing the results of a mouse skin puncture experiment using a recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0027] Figure 5 This is a force-displacement curve of the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0028] Figure 6 This is a drug release diagram of the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0029] Figure 7 The skin recovery results of the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0030] Figure 8 This is a graph showing the results of the mouse full-thickness cortical injury model using the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0031] Fig. 9 This is a picture of the wound healing of mice with the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0032] Fig.10 This is a graph showing the recovery rate of mouse wounds using the recombinant human thymosin β4 microneedle patch prepared in Example 1 of the present invention;

[0033] Fig.11 This is a force-displacement curve of the recombinant human thymosin β4 microneedle patch prepared in Example 2 of the present invention;

[0034] Fig.12 This is a drug release diagram of the recombinant human thymosin β4 microneedle patch prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing a recombinant human thymosin β4 microneedle patch, comprising the following steps:

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

[0037] Mixing a second auxiliary material and water 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 an exhaust treatment is performed; after the exhaust treatment, the support layer solution is added to the microneedle mold to obtain a molded body;

[0039] The molded body is dried and demoulded to obtain the recombinant human thymosin β4 microneedle patch; the drying temperature is less than 40°C.

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

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

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

[0043] The present invention mixes the second auxiliary material and water to obtain a supporting layer solution; the second auxiliary material includes at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose. In the present invention, the second auxiliary material is preferably chondroitin sulfate and sucrose. Or the second auxiliary material is preferably hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol. The water is specifically preferably ultrapure water. When the second auxiliary material is preferably chondroitin sulfate and sucrose, the content of chondroitin sulfate in the supporting layer solution is preferably 10-100w / v%, more preferably 20-80w / v%; the content of sucrose is preferably 0.1-10w / v%, more preferably 1-8w / v%. When the second auxiliary material is preferably hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol, the content of hydroxypropyl-β-cyclodextrin in the supporting layer solution is preferably 10-100w / v%, more preferably 20-80w / v%; the content of Tween 80 is preferably 0.1-10w / v%, more preferably 1-8w / v%; the content of trehalose is preferably 0.1-30w / v%, more preferably 1-25w / v%; the content of polyvinyl alcohol is preferably 10-100w / v%, more preferably 20-80w / v%.

[0044] After obtaining the microneedle solution, the present invention adds the microneedle solution to the microneedle mold and then performs exhaust treatment. In the present invention, the material of the microneedle mold is preferably polydimethylsiloxane. The microneedle mold includes a supporting layer structure and a microneedle structure arranged on the surface of the supporting layer structure, and the microneedle structure is arranged in an array on the surface of the supporting layer structure; the shape of the microneedle structure is a hollow cone, the bottom diameter of the microneedle structure is preferably 100 to 500 μm, more preferably 200 to 400 μm, and the height is preferably 300 to 1000 μm, more preferably 300 to 1000 μm; the top spacing between any two adjacent microneedle structures is preferably 500 to 1200 μm, more preferably 600 to 1000 μm. In the present invention, the supporting layer structure of the microneedle mold is a square, and the long side of the square supporting layer structure is 10 to 50 mm. In a specific embodiment of the present invention, the size of the support layer structure of the microneedle abrasive tool may be 10.5 mm×10.5 mm or 50 mm×50 mm, and the microneedle structures are preferably arranged in a 10×10 square array on the surface of the support layer structure.

[0045] In the present invention, the exhaust treatment is preferably carried out under vacuum conditions, and the vacuum degree is preferably -0.09 to -0.1 MPa. In a specific embodiment of the present invention, the exhaust treatment is preferably carried out in a vacuum drying oven. The exhaust treatment is preferably carried out in a cycle, and the steps of a single exhaust treatment include: evacuating the exhaust treatment environment to -0.09 to -0.1 MPa, maintaining for 1 to 5 minutes, and then degassing to normal pressure. The exhaust treatment is preferably cycled 1 to 5 times.

[0046] After the exhaust treatment and after obtaining the support layer solution, the present invention adds the support layer solution to the microneedle mold to obtain a molded body. Before adding the support layer solution to the microneedle mold, the present invention preferably scrapes off the excess microneedle solution located in the support layer structure.

[0047] After obtaining the molded body, the present invention dries the molded body, and after demolding, the recombinant human thymosin β4 microneedle patch is obtained; the drying temperature is less than 40°C, more preferably 2-30°C, and further preferably 2-15°C. In the present invention, the drying time is preferably ≥36h, more preferably 36-48h. The drying is preferably performed in a dryer.

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

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

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

[0051] In the present invention, 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 invention, the shape of the microneedles of the recombinant human thymosin β4 microneedle patch is preferably a cone, the bottom diameter of the microneedles is preferably 100-500 μm, more preferably 200-400 μm, the height is preferably 300-1000 μm, more preferably 800-1000 μm; the distance between the tops of any two adjacent microneedles is preferably 500-1200 μm, more preferably 600-1000 μm.

[0053] In an embodiment of the present invention, the support layer of the recombinant human thymosin β4 microneedle patch is in a square shape with a side length of 1 to 15 cm, preferably 1 to 5 cm. The size of the support layer of the recombinant human thymosin β4 microneedle patch is specifically 1 cm×1 cm or 5 cm×5 cm.

[0054] In an embodiment of the present invention, the microneedles of the recombinant human thymosin β4 microneedle patch are arranged in a 10×10 to 20×20 square array on the surface of the support layer.

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

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

[0057] Example 1

[0058] 1. Dissolve Tβ4 in ultrapure water to prepare a Tβ4 solution, add chondroitin sulfate and sucrose to prepare a microneedle solution, in which: 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, wherein the content of chondroitin sulfate in the support layer solution is 50% (w / v) and the content of sucrose is 1% (w / v).

[0060] 3. Add the microneedle solution to the polydimethylsiloxane microneedle mold. The microneedle mold 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 1cm×1cm or 5cm×5cm. The microneedle structure is arranged in a 10×10 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 350μm and the height is 1000μm. The top spacing of any two adjacent microneedle structures is 800μm. Then put it into a vacuum drying oven and evacuate it to -0.1MPa, keep it for 3min and then release it to remove bubbles. Vacuum it to -0.1MPa again and keep it for 2min and then release it.

[0061] 4. Scrape off the excess microneedle solution and add the support layer solution.

[0062] 5. Place the microneedle mold containing the microneedle solution and the support layer solution in step 4 in a dryer and dry it at 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 Patch

[0064] The morphology of the Tβ4 drug-loaded microneedle patch was observed using a microscope. Figure 1 and Figure 2 As shown. 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 breaks, and has a sharp needle tip.

[0065] Test Example 2: Hardness test of Tβ4 drug-loaded microneedle patch

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

[0067] (1) Aluminum foil puncture test

[0068] When piercing the aluminum foil, use a certain force to vertically pierce the microneedle into the aluminum foil and maintain it for 2 minutes. Observe whether the microneedle can pierce the aluminum foil and evaluate the hardness of the microneedle. Figure 3 ,Depend on Figure 3 The results show that the microneedles of the Tβ4 drug-loaded microneedle patch prepared in Example 1 can easily penetrate the aluminum foil, indicating that the Tβ4 drug-loaded microneedle patch prepared in Example 1 has sufficient mechanical strength.

[0069] (2) In vitro rat skin puncture test

[0070] Treatment of ex vivo skin

[0071] Healthy SD rats were taken and killed by dislocating the cervical vertebrae. The hair on the abdomen was carefully shaved, the skin was removed, the fat tissue and fascia were peeled off, and the rats were repeatedly washed with saline, dried with filter paper, wrapped with tin foil, and stored in a -80℃ refrigerator. When used, the skin was first soaked in 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 healthy, undamaged rat skin. The microneedles were removed after 10 minutes, and the skin was immediately stained with a 0.4% trypan blue solution. After 15 minutes, the excess dye on the skin surface was removed with isopropanol, and the skin was cleaned with saline to observe the stained holes on the skin surface. After trypan blue staining, clearly visible holes can be seen on the rat skin. Figure 4 . Figure 4 The results show that the Tβ4 drug-loaded microneedle patch prepared in Example 1 has sufficient mechanical strength and can penetrate the ex vivo skin, meeting the test requirements.

[0074] Test Example 3: Mechanical properties test of Tβ4 drug-loaded microneedles

[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, the mechanical strength of the Tβ4 drug-loaded microneedle patch prepared in Example 1 was evaluated using a texture analyzer. The test speed was set to 0.05 mm / s, and the mechanical properties of the microneedle were evaluated by comparing the relationship between the force exerted on the microneedle tip during the probe pressing process and the probe displacement.

[0076] The results are as follows Figure 5 As shown, when the force reaches 47N, the force-displacement curve suddenly drops, indicating that the needle tip is broken. It can be concluded that each single needle can withstand a force of 0.47N, and the force required to penetrate the skin is usually less than 0.1N / needle. Therefore, the microneedle can easily puncture the skin and has good mechanical properties.

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

[0078] Microneedle drug loading determination

[0079] pH 7.4 PBS was used as the release medium, and the Tβ4 drug-loaded microneedle patch prepared in Example 1 was placed in the release medium, and the sample was taken by ultrasonication for 3 minutes. The above operation was performed in parallel for 3 groups. Liquid chromatography was used for detection.

[0080] The average value of the test results showed that the drug loading per microneedle was 248.15 μg.

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

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

[0083]

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

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

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

[0087] Time / min Release rate (%) 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 rate of the Tβ4 drug-loaded microneedle patch prepared in Example 1 is shown in FIG. Figure 6 As shown. Figure 6 The results showed that the cumulative release of drug-loaded microneedles was 62.10% at 1 minute, 86.67% at 30 minutes, and 99.70% at 45 minutes. The results showed that Tβ4 drug-loaded microneedles could release most of the drugs rapidly and continuously within 45 minutes, and the rapid release of Tβ4 microneedles was beneficial to the transdermal penetration of drugs.

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

[0090] Microneedles may break during use and remain in the skin for a long time, so the safety of the material is of vital importance. The irritation test is to observe whether the blood vessels, muscles, skin, mucous membranes and other parts of the animal cause local reactions such as redness, swelling, congestion, exudation, degeneration or necrosis after contact with the test substance.

[0091] SD rats were anesthetized by intraperitoneal injection of 150 μL 4% chloral hydrate, and the abdominal hair was removed. The microneedles were attached to the abdominal skin of the rats for 10 minutes and then the skin status was recorded. The time was recorded as 0 minutes. The skin recovery status was recorded at 2 minutes, 4 minutes, 6 minutes, 8 minutes, 10 minutes, 30 minutes, 1 hour, and 2 hours. The skin recovery results are shown in the figure. Figure 7 .

[0092] like Figure 7 As shown in the figure, at 0 minutes, the holes left by the microneedles were clear, with no bleeding or redness. Within 2 minutes, the holes on the skin gradually blurred, and after 10 minutes, the marks on the skin almost disappeared. After 1 hour, the skin returned to normal, and the marks completely disappeared. The safety of microneedles was found to be good through the degree of erythema and edema of the skin after microneedling.

[0093] Test Example 7: Study on the application of Tβ4 drug-loaded microneedles in wound healing

[0094] (1) Establishment of the mouse full-thickness cortical injury model

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

[0096] (2) Experimental animal grouping and treatment

[0097] The trauma-modeling mice were randomly divided into two groups: the experimental group had the back wounds covered with Tβ4-loaded microneedles and fixed with pressure-sensitive tape; the blank control group had no microneedle coverage. There were 10 mice in each group, which were housed in separate cages and the microneedles were replaced every day.

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

[0099] During the experiment, the wounds were photographed and sampled every day (with a ruler for comparison), the wound healing conditions were compared and recorded, and the wound area of ​​the mice in a specific period was calculated using Image J software to calculate the wound healing rate. The calculation formula for the wound healing rate is shown in Formula 3:

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

[0101] Fig. 9 This is a diagram of wound healing. Fig.10 Wound recovery rate chart

[0102] According to the results of animal experiments ( Fig. 9 and Fig.10 ) It can be seen that the wound healing rate of the Tβ4 microneedle experimental group was significantly different from that of the blank group from the 4th day. On the 12th day of the experiment, the wound healing rate of the microneedle group reached 95.65%, which was almost healed, while the recovery rate of the control group was only 86.44%. On the 14th day, the microneedle group had basically returned to normal, while the control group was still in the healing process. This shows that Tβ4 microneedles have a promoting effect on wound healing.

[0103] Example 2

[0104] 1. Dissolve Tβ4 in ultrapure water to prepare a Tβ4 solution, add hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol to prepare a microneedle solution, wherein 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 to ultrapure water to prepare a supporting layer solution, in which the content of hydroxypropyl-β-cyclodextrin, the content of Tween 80, the content of trehalose and the content of polyvinyl alcohol are 10% (w / v), 10% (w / v), and 10% (w / v) respectively.

[0106] 3. Add the microneedle solution to the polydimethylsiloxane microneedle mold. The microneedle mold 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 10.5mm×10.5mm. The microneedle structure is arranged in an 8×8 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 350μm and the height is 1000μm. The top spacing of any two adjacent microneedle structures is 800μm. Then put it into a vacuum drying oven and evacuate it to -0.1MPa, keep it for 3min and then release it to remove bubbles. Vacuum it to -0.1MPa again and keep it for 2min and then release it.

[0107] 4. Scrape off the excess microneedle solution and add the support layer solution.

[0108] 5. Place the microneedle mold containing the microneedle solution and the support layer solution in step 4 in a dryer and dry it at 2-4° C. for 48 hours to obtain a Tβ4 drug-loaded microneedle patch.

[0109] The Tβ4 drug-loaded microneedle patch prepared in Example 2 was subjected to a mechanical strength performance test according to the method described in the above-mentioned "Test Example 3: Mechanical Performance Test of Tβ4 Drug-Loaded Microneedle". The test results are as follows: Fig.11 As shown, Figure 1 The horizontal coordinate X is the displacement, unit is mm, and the vertical coordinate Y is the force, unit is N, which is given by Fig.11 It can be seen that the average mechanical strength of the four needles of the Tβ4 drug-loaded microneedle patch prepared in Example 2 is: 1.05±0.03N, which is greater than 0.2N and meets the requirements.

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

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

[0112]

[0113]

[0114] The drug release rate of the Tβ4 drug-loaded microneedle patch prepared in Example 2 is shown in FIG. Fig.12 As shown. Fig.12 It was shown that the wound healing performance of the Tβ4 drug-loaded microneedle patch prepared in Example 2 was similar to that of Example 1.

[0115] Comparative Example 1

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

[0117] Determination of drug loading of Tβ4 drug-loaded microneedle patch prepared in Comparative Example 1

[0118] 5 mL of PBS with pH = 7.4 was used as the release medium, and the mold with the needle tip part filled with the needle tip solution was placed in the release medium, and the sample was taken by ultrasonication for 3 minutes. The above operation was performed in 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 parallel chromatograms were 9.252, 11.877, and 10.876, respectively. Substituting them into the linear standard curve equation 1, the drug loading of the microneedles prepared in Comparative Example 1 was obtained, and the average drug loading of each microneedle was 95.126 μg. Compared with the drug loading of the Tβ4 drug-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 is much lower than that of the microneedle patch prepared by the low-temperature drying method in Example 1.

[0120] From the above examples, it can be seen that the present invention uses chondroitin sulfate and sucrose as auxiliary materials of recombinant human thymosin β4, and uses low temperature conditions of temperature ≤4°C for drying, thereby effectively avoiding the problem that recombinant human thymosin β4 may lose activity or undergo structural changes under high temperature conditions, resulting in loss of efficacy. From the results of the examples, it can be seen that the recombinant human thymosin β4 microneedle patch prepared by the present invention is well-formed, the needle body has a good appearance, there is no empty needle phenomenon, and the tip has no defect or breakage phenomenon, and has a sharp needle tip; the recombinant human thymosin β4 microneedle patch prepared by the present invention can easily penetrate the aluminum foil and has sufficient mechanical strength. Each single needle can withstand a force of 0.47N, and the force required to penetrate the skin is usually less than 0.1N / needle, so the microneedle can easily puncture the skin and has good mechanical properties; the drug loading of the recombinant human thymosin β4 microneedle patch prepared by the present invention is 248.15μg per microneedle; the recombinant human thymosin β4 microneedle patch prepared by the present invention can quickly and continuously release most of the drugs within 20min, and the rapid release is conducive to the transdermal penetration of the drug. The recombinant human thymosin β4 microneedle patch prepared by the present invention has good safety; the recombinant human thymosin β4 microneedle patch prepared by the present invention has a good promoting effect on wound healing.

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a recombinant human thymosin β4 microneedle patch, characterized in that: The following steps are involved: Mixing recombinant human thymosin β4, a first auxiliary material and water to obtain a microneedle solution; the first auxiliary material includes at least two of hydroxypropyl-β-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-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; adding the microneedle solution into a microneedle mold, and then performing an exhaust treatment; After the exhaust treatment, the support layer solution is added to the microneedle mold to obtain a molded body; The molded body is dried and demoulded to obtain the recombinant human thymosin β4 microneedle patch; the drying temperature is less than 40°C.

2. The preparation method according to claim 1, characterized in that: The drying temperature is ≤35°C and the drying time is ≥36h.

3. The preparation method according to claim 1, characterized in that: The mass concentration of recombinant human thymosin β4 in the microneedle solution is 10-400 mg / mL.

4. The preparation method according to claim 1 or 3, characterized in that: The first auxiliary material is chondroitin sulfate and sucrose. In the microneedle solution, the content of chondroitin sulfate is 10-100 w / v%, and the content of sucrose is 0.1-10 w / v%.

5. The preparation method according to claim 1 or 3, characterized in that: The first auxiliary material is hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol. In the microneedle solution, the content of hydroxypropyl-β-cyclodextrin is 10-100w / v%, the content of Tween 80 is 0.1-10w / v%, the content of trehalose is 0.1-30w / v%, and the content of polyvinyl alcohol is 10-100w / v%.

6. The preparation method according to claim 1, characterized in that: The second auxiliary material is chondroitin sulfate and sucrose. In the support layer solution, the content of chondroitin sulfate is 10-100w / v%, and the content of sucrose is 0.1-10w / v%.

7. The preparation method according to claim 1, characterized in that: The second auxiliary material is hydroxypropyl-β-cyclodextrin, Tween 80, trehalose and polyvinyl alcohol. In the supporting layer solution, the content of hydroxypropyl-β-cyclodextrin is 10-100w / v%, the content of Tween 80 is 0.1-10w / v%, the content of trehalose is 0.1-30w / v%, and the content of polyvinyl alcohol is 10-100w / v%.

8. The preparation method according to any one of claims 1 to 3, characterized in that: 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 microneedle structure is in the shape of a hollow cone, the bottom diameter of the microneedle structure is 100 to 500 μm, and the height is 300 to 1000 μm; the top spacing between any two adjacent microneedle structures is 500 to 1200 μm.

9. The recombinant human thymosin β4 microneedle patch prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The invention comprises a supporting layer and microneedles arrayed on the surface of the supporting layer, wherein the microneedles comprise recombinant human thymosin β4 and a first auxiliary material, wherein the first auxiliary material comprises at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose; and the supporting layer comprises a second auxiliary material, wherein the second auxiliary material comprises at least two of hydroxypropyl-β-cyclodextrin, Tween 80, trehalose, polyvinyl alcohol, chondroitin sulfate and sucrose.

10. Use of the recombinant human thymosin β4 microneedle patch according to claim 9 in the manufacture of medical devices for wound healing.

Images