Intelligent response hydrogel sustained-release microneedle and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses an intelligent response hydrogel sustained-release microneedle and a preparation method thereof. The intelligent response hydrogel sustained-release microneedle comprises a response microneedle and a sustained-release microneedle; the response microneedle is prepared by using a cross-linking agent, thrombin-sensitive peptide, a photoinitiator and methacrylic acid hyaluronic acid as raw materials through ultraviolet-induced cross-linking. PVP and PVA are used as matrix materials of the sustained-release microneedle, a hydrogel microneedle structure is formed under the crosslinking action of citric acid, and a blood coagulation factor compound is loaded. According to the intelligent response hydrogel sustained-release microneedle and the preparation method thereof, the hydrogel sustained-release microneedle is adopted for transdermal delivery, and FVIII is protected from being degraded, so that the bioavailability of the hydrogel sustained-release microneedle is improved, a basis is provided for microneedle-mediated efficient transdermal drug delivery, the problem of intravenous injection drug delivery is solved, and the application prospect is wide. The blood coagulation factor activity fluctuation in the body of the patient is large, the blood coagulation factor inhibitor is generated, the bleeding risk is high, and side effects exist.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an intelligent responsive hydrogel sustained-release microneedle and a preparation method thereof. Background Art

[0002] Hemophilia is one of the most common bleeding disorders, characterized by coagulation disorders caused by the deficiency of coagulation factor VIII (hemophilia A) or factor IX (hemophilia B). In my country, the incidence of hemophilia is about 1 / 10,000-1 / 20,000, and it is known as a common disease among rare diseases. The most typical symptom of hemophilia is repeated spontaneous bleeding in joints and muscles, which affects the ability to move and brings great pain to patients. Lifelong treatment also imposes a huge economic burden on families and society.

[0003] Preventive treatment is the standard treatment for managing hemophilia. Patients need to regularly supplement coagulation factors to maintain the activity of FⅧ in the blood to prevent bleeding. At present, intravenous infusion is used clinically for FⅧ administration. Although it can effectively supplement FⅧ, intravenous injection of coagulation factors can cause a large fluctuation in the activity of coagulation factors in the body, leading to a disorder of the balance between bleeding and coagulation in the body; and short-term exposure to high concentrations of coagulation factors can easily induce an immune response in hemophilia patients, produce antibodies against coagulation factors, namely coagulation factor inhibitors, and inactivate coagulation factors, seriously affecting the effect of preventive treatment. This results in repeated spontaneous bleeding in patients even after strict intravenous coagulation factor treatment, resulting in continuous damage to joints and muscles, and eventually paralysis. At the same time, intravenous injection also has problems such as high cost, high risk of bleeding, allergic reactions and side effects. Intravenous injection cannot replenish coagulation factors in time outside the hospital according to the needs of the body. Repeated intravenous injection increases the complexity and inconvenience of treatment, and also puts great psychological pressure on patients, which may lead to a decrease in treatment compliance and affect overall health status.

[0004] Therefore, there is an urgent need to explore new strategies for the administration of coagulation factors with sustained and controlled release capabilities to prevent complications and improve preventive and therapeutic effects. Microneedles are a type of safe, painless, and easy-to-use drug delivery carrier with the advantages of good biocompatibility, long-lasting and controllable drug release. This not only reduces the risk of infection, but also improves patient compliance, helps reduce biological waste, improves drug delivery efficiency, and has broad application prospects in the biomedical field. In addition, traditional microneedle materials and preparation methods will affect the stability of FⅧ. If the microneedle material is unstable or interacts with FⅧ, it may cause the degradation of FⅧ; when the drug is quickly released, the high local concentration may lead to accelerated degradation. At the same time, microneedles that do not have a sustained release function have a small drug loading capacity and may not be able to meet the needs of larger doses of FⅧ, especially in acute bleeding events; and it is difficult to achieve consistency in the dosage of each administration. Therefore, in order to solve the above technical problems, the present invention provides a smart responsive hydrogel sustained-release microneedle and a preparation method. Summary of the invention

[0005] The purpose of the present invention is to provide an intelligent responsive hydrogel sustained-release microneedle and a preparation method thereof. The hydrogel sustained-release microneedle performs transdermal delivery to protect FⅧ from degradation, thereby improving its bioavailability, providing a basis for microneedle-mediated efficient transdermal drug delivery, and solving the problems of intravenous administration, large fluctuations in the activity of coagulation factors in the patient's body, the production of coagulation factor inhibitors, high risk of bleeding, and side effects.

[0006] To achieve the above-mentioned purpose, the present invention provides an intelligent responsive hydrogel sustained-release microneedle, comprising a responsive microneedle and a sustained-release microneedle; the responsive microneedle is a responsive hydrogel microneedle, which is prepared by cross-linking with a cross-linker, a thrombin-sensitive peptide, a photoinitiator and methacrylated hyaluronic acid via ultraviolet light initiation; the sustained-release microneedle is a sustained-release hydrogel microneedle, which is prepared by cross-linking with PVP and PVA as matrix materials, forming a hydrogel microneedle structure under the cross-linking action of citric acid, and loading a coagulation factor complex.

[0007] Furthermore, the height of the smart responsive sustained-release hydrogel microneedles is 800 μm, the bottom diameter is 300 μm, the spacing is 600 μm, and the array is 10×10. The sustained-release hydrogel microneedles and responsive hydrogel microneedles are distributed in a double ring on the substrate.

[0008] The present invention also provides a method for preparing the above-mentioned smart responsive hydrogel sustained-release microneedle, comprising the following steps: Preparation of hydrogel microneedle tips: 1) Mix 8%~15% polyvinyl pyrrolidone PVP, 8%~15% polyvinyl alcohol PVA and 5% cross-linking agent evenly and pour into the needle tip area of ​​PDMS mold; 2) Dry at room temperature for 36-48 h, then physically crosslink at a high temperature of 90-130 °C, while the crosslinker chemically crosslinks the hydrogel; 3) Mix 1%~5% crosslinker, 0.5-1 mg / mL thrombin-sensitive peptide, 0.5%~1% photoinitiator, and 10%~15% methacrylated HA, then load the mixed solution into the remaining blank tip area of ​​the PDMS mold and centrifuge at 3600 r / min for 2 min; 4) Chemical cross-linking under ultraviolet light to spontaneously form a hydrogel; Preparation of microneedle substrate: 5) Add 8%~15% polyvinyl alcohol (PVA) base solution to the PDMS mold, and demold after drying and curing; Package FⅧ: 6) Soaking the needle tip in the FⅧ complex solution, and the microneedle absorbs and encapsulates the FⅧ complex solution; 7) Drying is performed to obtain smart responsive hydrogel sustained-release microneedles.

[0009] Furthermore, in step 1), polyvinyl pyrrolidone PVP, 8% to 15% polyvinyl alcohol PVA and 5% cross-linking agent are mixed in equal volumes.

[0010] Furthermore, in step 1), the cross-linking agent is citric acid CA.

[0011] Furthermore, in step 2), physical cross-linking is performed for 3-10 min.

[0012] Furthermore, in step 3), the cross-linking agent, thrombin-sensitive peptide, photoinitiator, and methacrylated HA are mixed in equal volumes, and the cross-linking agent is a chemical cross-linking agent.

[0013] Furthermore, in step 4), chemical cross-linking is performed for 10 to 20 min.

[0014] Further, in step 6), the needle tip is immersed in the FⅧ complex solution for 10-15 min.

[0015] The present invention also provides a smart response hydrogel sustained-release microneedle system, comprising the above-mentioned smart response hydrogel sustained-release microneedle.

[0016] The present invention also provides the use of the above-mentioned smart response hydrogel sustained-release microneedle system in constructing a drug delivery platform.

[0017] The advantages and positive effects of the smart responsive hydrogel sustained-release microneedle and preparation method described in the present invention are: 1. The present invention provides an intelligent responsive hydrogel sustained-release microneedle system, which delivers coagulation factors through intelligent responsive sustained-release microneedles for the prevention and treatment of hemophilia. The coagulation factor complex is constructed by combining von Willebrand Factor (VWF) and FVIII for delivery, thereby preventing FVIII from binding to the extravascular matrix and degrading, and improving the bioavailability of FVIII.

[0018] 2. The intelligent responsive sustained-release microneedles loaded with coagulation factor complexes consist of sustained-release microneedles and responsive microneedles. The sustained-release microneedles achieve long-term and stable release of coagulation factor complexes by optimizing the proportion of hydrogel components and cross-linking structure; the responsive microneedles can respond to the thrombin produced by microbleeding in the body and accelerate decomposition, thereby achieving controlled release of FⅧ during bleeding.

[0019] 3. Microneedle-mediated intelligent FⅧ delivery can provide the body with a stable and controllable level of coagulation factors, avoid the production of coagulation factor inhibitors, and reduce the risk of joint bleeding and other complications. Intelligent responsive sustained-release microneedles have the advantages of stable sustained-release and responsive controlled-release, which can effectively overcome the difficulties currently faced by intravenous administration, provide a new paradigm for the prevention and treatment of hemophilia, and have important clinical significance.

[0020] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a process flow chart of the controllable manufacturing of the smart responsive hydrogel sustained-release microneedles in an embodiment of the present invention; Figure 2 Schematic diagram of the transdermal delivery of coagulation factors by the smart responsive hydrogel sustained-release microneedle in an embodiment of the present invention; Figure 3 Schematic diagram of the release of coagulation factors from the smart responsive hydrogel sustained-release microneedles in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0023] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0024] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0025] The present invention compounds VWF with FⅧ, avoiding the binding of FⅧ with phospholipids, thereby improving the bioavailability of subcutaneous administration of coagulation factors. The compounding of VWF and FⅧ is studied by immunoprotein imprinting technology and immunoprecipitation technology. A 96-well plate is coated with an extracellular matrix complex, and FⅧ alone or a FⅧ complex compounded with VWF is added thereto for incubation, and then the FⅧ concentration in the supernatant is determined by ELISA. FⅧ or the FⅧ complex is hydrolyzed by human neutrophil elastase, and the degradation level of FⅧ is detected by immunoblotting experiment after treatment. Different proportions of coagulation factor complexes are subcutaneously injected into animals, blood is taken at different time points after injection, and the blood FⅧ level is measured to determine the optimal compounding conditions and effects.

[0026] A sustained-release hydrogel microneedle based on PVP and PVA, which achieves the sustained release of coagulation factor VIII (FVIII) by regulating the proportion of cross-linking components and the amount of cross-linking agent through the action of the cross-linking agent citric acid (CA), meets the demand for drug delivery in the treatment of coagulation disorders such as hemophilia.

[0027] The sustained-release hydrogel microneedles were prepared by the following method: using different proportions of PVP and PVA as matrix materials, forming a hydrogel microneedle structure under the cross-linking action of citric acid (CA), and loading the coagulation factor complex. The prepared sustained-release microneedles were placed in a solution containing 10% fetal bovine serum (FBS) to simulate the in vivo environment, and the FⅧ loading amount in the microneedles and the release level over time were determined by the FⅧ determination kit. By adjusting the ratio of PVP and PVA in the hydrogel matrix material and the amount of citric acid, the drug loading capacity of the hydrogel and the release rate of FⅧ can be adjusted, and the hydrogel microneedle structure that meets the FⅧ delivery requirements can be screened out.

[0028] A responsive hydrogel microneedle is prepared by mixing a crosslinker, a thrombin-sensitive peptide, a photoinitiator and a methacrylated hyaluronic acid (m-HA) solution, and then crosslinking by ultraviolet light. The hydrogel microneedle can respond to thrombin in a microbleeding environment, thereby achieving the directional release of coagulation factor FⅧ. In a 10% FBS solution containing or not containing thrombin, microbleeding in vivo or a normal environment is simulated, and the responsive hydrogel microneedle loaded with coagulation factor is incubated, and the release of FⅧ is measured at different time points. By using m-HA of different molecular weights and thrombin-sensitive peptides of different proportions, the responsive release ability of the responsive hydrogel microneedle can be further regulated.

[0029] In order to solve the problems of large fluctuations in the activity of coagulation factors in patients with intravenous administration, the production of coagulation factor inhibitors, high bleeding risk and side effects, the present invention prepares smart responsive hydrogel sustained-release microneedles, and the preparation method comprises the following steps: Preparation of hydrogel microneedle tips: (1) Mix 8%-15% polyvinyl pyrrolidone (PVP), 8%-15% polyvinyl alcohol (PVA), and 5% cross-linking agent citric acid (CA) in equal volumes and pour into the needle tip area of ​​the PDMS mold; (2) Drying at room temperature for 36-48 h, and then physically crosslinking the polymer at a high temperature of 90-130 °C for 3-10 min, while citric acid chemically crosslinks the hydrogel; (3) 0.5-1 mg / mL thrombin-sensitive peptide, 0.5%-1% photoinitiator, 1%-5% chemical crosslinker, and 10%-15% methacrylated HA (m-HA) were mixed in equal volumes and then loaded into the remaining blank tip area of ​​the PDMS mold, and then centrifuged at 3600 r / min for 2 min; (4) Chemical cross-linking under ultraviolet light for 5-10 minutes to spontaneously form a hydrogel; Preparation of microneedle substrate: (5) Add 8% to 15% polyvinyl alcohol (PVA) base solution to the mold, and demold after drying and curing; Package FⅧ: (6) Soak the needle tip in the FⅧ complex solution for 10-15 min, and the microneedle absorbs and encapsulates the FⅧ complex solution; (7) After drying, smart responsive sustained-release hydrogel microneedles are manufactured.

[0030] The height of the smart responsive sustained-release hydrogel microneedle is 800 μm, the bottom diameter is 300 μm, the spacing is 600 μm, and the array is 10×10. The sustained-release hydrogel microneedle and the responsive hydrogel microneedle are distributed in a double ring on the substrate.

[0031] Example 1 A method for preparing a smart responsive hydrogel sustained-release microneedle is as follows ( Figure 1 shown): Preparation of hydrogel microneedle tips: (1) Mix 12% polyvinyl pyrrolidone (PVP), 12% polyvinyl alcohol (PVA), and 5% cross-linking agent citric acid (CA) in equal volumes and pour into the needle tip area of ​​the PDMS mold; (2) Drying at room temperature for 48 h, and then physically crosslinking the polymer at 130 °C for 3-10 min, while citric acid chemically crosslinks the hydrogel; (3) 0.75 mg / mL thrombin-sensitive peptide, 0.75% photoinitiator, 3% cross-linker, and 12.5% ​​methacrylated HA (m-HA) were mixed in equal volumes and then loaded into the remaining blank tip area of ​​the PDMS mold and centrifuged at 3600 r / min for 2 min. (4) Chemical cross-linking under ultraviolet light for 5-10 minutes to spontaneously form a hydrogel; Preparation of microneedle substrate: (5) Add 12% polyvinyl alcohol (PVA) base solution to the mold, and demold after drying and curing; Package FⅧ: (6) Soak the needle tip in the FⅧ complex solution for 10 min, and the microneedle absorbs and encapsulates the FⅧ complex solution; (7) After drying, smart responsive sustained-release hydrogel microneedles are manufactured.

[0032] The prepared smart responsive hydrogel sustained-release microneedles are conical in shape with a height of 500-1000 μm.

[0033] In the present invention, the coagulation factor complex of VWF and FⅧ is used for transdermal delivery, which protects FⅧ from degradation, thereby improving its bioavailability, providing a basis for efficient transdermal drug delivery mediated by microneedles, and solving the problem that FⅧ will be degraded and inactivated by direct subcutaneous injection. Figure 2 shown.

[0034] The smart responsive sustained-release hydrogel microneedles in the present invention have the properties of sustained-release and controlled-release drug delivery. The sustained-release microneedles absorb interstitial fluid from the skin to enlarge the cross-linked grid, thereby continuously releasing the FVIII complex, which can maintain the FVIII activity at the desired level for a long time. The responsive microneedles are based on the specificity of the enzymatic hydrolysis reaction of thrombin, and by introducing thrombin-sensitive peptides into the cross-linked structure of the hydrogel to dynamically detect the coagulation state, the controlled release of the FVIII complex is achieved. This solves the problems of large fluctuations in factor activity, easy production of coagulation factor inhibitors, side effects, and complexity and inconvenience of treatment when using intravenous injection. The schematic diagram of the release of coagulation factors by smart microneedles is shown below. Figure 3 shown.

[0035] The present invention optimizes the geometric structure of the microneedle through modeling and finite element simulation, and realizes the controllable manufacturing of the smart microneedle with the help of a combination of photopolymerization technology, micromold casting process and high temperature / normal temperature preparation process, thereby solving the problems of inactivation or denaturation of FⅧ during the microneedle preparation process, and the influence of microneedle materials and preparation methods on the stability of FⅧ.

[0036] Therefore, the present invention adopts the above-mentioned smart response hydrogel sustained-release microneedle and preparation method, and the hydrogel sustained-release microneedle is used for transdermal delivery to protect FⅧ from degradation, thereby improving its bioavailability, providing a basis for microneedle-mediated efficient transdermal drug delivery, and solving the problems of intravenous administration, large fluctuations in the activity of coagulation factors in the patient's body, the production of coagulation factor inhibitors, high risk of bleeding and side effects.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A smart responsive hydrogel sustained-release microneedle, characterized in that: It includes responsive microneedles and sustained-release microneedles; the responsive microneedles are responsive hydrogel microneedles, which are prepared by cross-linking with a cross-linking agent, a thrombin-sensitive peptide, a photoinitiator and methacrylated hyaluronic acid through ultraviolet light initiation; the sustained-release microneedles are sustained-release ... PVP and PVA as matrix materials, forming a hydrogel microneedle structure under the cross-linking action of citric acid, and loading a coagulation factor complex.

2. The method for preparing a smart responsive hydrogel sustained-release microneedle according to claim 1, characterized in that: The following steps are involved: Preparation of hydrogel microneedle tips: 1) Mix 8%~15% polyvinyl pyrrolidone PVP, 8%~15% polyvinyl alcohol PVA and 5% cross-linking agent evenly and pour into the needle tip area of ​​PDMS mold; 2) Dry at room temperature for 36-48 h, then physically crosslink at a high temperature of 90-130°C, while the crosslinker chemically crosslinks the hydrogel; 3) Mix 1%~5% crosslinker, 0.5-1 mg / mL thrombin-sensitive peptide, 0.5%~1% photoinitiator, and 10%~15% methacrylated HA, then load the mixed solution into the remaining blank tip area of ​​the PDMS mold and centrifuge at 3600 r / min for 2 min; 4) Chemical cross-linking under ultraviolet light to spontaneously form a hydrogel; Preparation of microneedle substrate: 5) Add 8%~15% polyvinyl alcohol (PVA) base solution to the PDMS mold, and demold after drying and curing; Package FⅧ: 6) Soaking the needle tip in the FⅧ complex solution, and the microneedle absorbs and encapsulates the FⅧ complex solution; 7) Drying is performed to obtain smart responsive hydrogel sustained-release microneedles.

3. The preparation method according to claim 2, characterized in that: In step 1), polyvinyl pyrrolidone (PVP), 8% to 15% polyvinyl alcohol (PVA) and 5% cross-linking agent are mixed in equal volumes.

4. The preparation method according to claim 2, characterized in that: In step 1), the cross-linking agent is citric acid CA.

5. The preparation method according to claim 2, characterized in that: In step 2), physical crosslinking is performed for 3-10 min.

6. The preparation method according to claim 2, characterized in that: In step 3), the cross-linking agent, thrombin-sensitive peptide, photoinitiator, and methacrylated HA are mixed in equal volumes, and the cross-linking agent is a chemical cross-linking agent.

7. The preparation method according to claim 2, characterized in that: In step 4), chemical cross-linking is performed for 5 to 10 minutes.

8. The preparation method according to claim 2, characterized in that: Step 6) Soak the needle tip in the FⅧ complex solution for 10-15 min.

9. A smart responsive hydrogel sustained-release microneedle system, characterized in that: The invention comprises the smart responsive hydrogel sustained-release microneedle as described in claim 1.

10. Use of the smart responsive hydrogel sustained-release microneedle system as claimed in claim 9 in constructing a drug delivery platform.

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