A polypeptide microneedle dressing and a preparation method thereof

Abstract

The application provides a polypeptide microneedle dressing and a preparation method, and belongs to the technical field of polypeptide accessories, and the dressing comprises a microneedle base and a needle body array arranged on the microneedle base, the needle body is composed of polypeptide microgel and carboxymethyl chitosan hydrogel, and the microneedle base is carboxymethyl chitosan hydrogel; collagen effective components are delivered to the subcutaneous layer, so that the purpose of promoting skin cell regeneration or proliferation is achieved. Active molecules having a positive effect on the skin are loaded on the polypeptide microgel through heparin molecules, so that the growth and proliferation of skin dermal cells are promoted.

Description

Technical Field

[0001] This invention belongs to the technical field of polypeptide excipients, specifically relating to a polypeptide microneedle dressing and its preparation method. Background Technology

[0002] The skin is the largest organ in the human body, comprising the epidermis, dermis, and subcutaneous tissue. The dermis is composed of collagen, elastin fibers, and matrix. During human growth, ultraviolet radiation can penetrate the skin to reach the dermis, activating matrix metalloproteinases that induce excessive collagen degradation. Simultaneously, ingested sugars react with collagen in the skin, causing it to break down. When the rate of collagen synthesis and loss in the skin is no longer balanced, the skin loses elasticity and support, leading to wrinkles and other signs of aging.

[0003] Collagen is an important component of the dermis in human skin, especially type I and type III collagen, which are the main structural proteins in human skin and play a role in maintaining normal skin physiological functions. Therefore, non-toxic and non-irritating collagen is often used for dermal injection (as a dermal filler) to reduce wrinkles and restore skin plumpness. However, collagen cannot be directly absorbed transdermally through the skin barrier, making novel delivery methods a key challenge.

[0004] Microneedle patches are invasive therapeutic tools composed of tiny microneedles and a base, capable of carrying active ingredients and acting on the skin in a minimally invasive manner to deliver medication. Compared to traditional skin dressings, microneedle patches have many advantages: ① microneedles offer a simple and convenient drug delivery method; ② microneedles deliver active ingredients to specific subcutaneous locations through puncture, improving the transdermal efficiency of active ingredients. Therefore, developing collagen microneedles to deliver active collagen ingredients subcutaneously and promote skin cell regeneration or proliferation is particularly important. Summary of the Invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a polypeptide microneedle dressing and its preparation method.

[0006] To solve the above technical problems:

[0007] The first objective of this invention is to provide a polypeptide microneedle dressing, comprising a microneedle base and an array of needles arranged on the microneedle base, wherein the needles are composed of polypeptide microgels and carboxymethyl chitosan hydrogels, and the microneedle base is a carboxymethyl chitosan hydrogel; preferably, the polypeptide microgels are loaded with active molecules that have a positive effect on the skin via heparin molecules.

[0008] Preferably, the polypeptide is one of animal-derived collagen, recombinant collagen, recombinant human collagen, gelatin, or recombinant peptide; more preferably, the polypeptide is recombinant human collagen with a molecular weight of 100-300 kDa; most preferably, the recombinant human collagen is recombinant human type I collagen or recombinant human type III collagen.

[0009] The aforementioned animal-derived collagen is derived from cattle, pigs, or fish; the recombinant collagen and recombinant human collagen are derived from mammalian expression systems, plant expression systems, insect expression systems, or microbial expression systems; the gelatin is extracted from animal bones, skin, and connective tissue; the recombinant peptides are derived from bacterial expression systems, yeast expression systems, and mammalian cell expression systems; the molecular weight of the animal-derived collagen and recombinant collagen is 50-300 kDa, and the molecular weight of the gelatin is 1-30 kDa.

[0010] Furthermore, the active ingredient is basic fibroblast growth factor, antimicrobial peptides with repair and regeneration functions, nicotinamide for treating skin inflammation, or lidocaine for local anesthesia.

[0011] A second objective of this invention is to provide a method for preparing a polypeptide microneedle dressing, comprising the following steps:

[0012] Step 1, Preparation of peptide microgels:

[0013] Methacrylic acid was added to the first polypeptide solution to prepare a methacrylic anhydride-modified first polypeptide solution. The supernatant was collected by centrifugation, diluted, filtered, dialyzed for 3-4 days, and freeze-dried to obtain a solid sample. The solid sample was then dissolved in a photoinitiator to obtain solution A.

[0014] As a preferred embodiment, the first polypeptide is recombinant human type I collagen. Methacrylic acid is added to the recombinant human type I collagen solution, and the reaction is carried out under magnetic stirring for 3-5 hours to prepare a methacrylic anhydride-modified recombinant human type I collagen solution (RHCⅠ-MA solution). The supernatant is then collected by centrifugation, diluted, filtered, dialyzed through a dialysis bag for 3-4 days, and freeze-dried to obtain a sponge-like solid sample of RHCⅠ-MA. Subsequently, the freeze-dried RHCⅠ-MA is dissolved in a certain amount of photoinitiator to obtain "solution A".

[0015] The second polypeptide solution was treated in the same way to obtain solution B; wherein the second polypeptide was recombinant human type III collagen.

[0016] Preferably, the volume ratio of added methacrylic acid to the recombinant human type I / III collagen solution is 1%-15%; more preferably, it is 3%-10%.

[0017] Solution A and solution B are thoroughly mixed to obtain solution C. Solution C is slowly added dropwise to a mixture of paraffin and surfactant Span 80 at a speed of 1000-5000 rpm. After stirring, the mixture is irradiated with 405 nm ultraviolet light for 3-10 min, centrifuged to remove the supernatant, PBS buffer is added and mixed evenly to remove the oil phase. This process is repeated 3-5 times until the oil phase is completely removed. The precipitate is then collected by filtration, which yields the polypeptide microgel. Preferably, the volume ratio of liquid paraffin (oil phase) to surfactant Span 80 is 10:2. The rotation speed is 2000 rpm. The particle size of the microgel is inversely proportional to the rotation speed; the faster the rotation speed, the smaller the particle size of the microgel.

[0018] Step 2: Heparin Molecular Coupling: Dissolve heparin sodium salt powder in EDC solution, add the peptide microgel prepared in Step 1, and react with shaking at room temperature for 4-6 hours. After the reaction, wash with PBS buffer solution to obtain the peptide microgel-heparin molecule complex. Preferably, the EDC concentration is 0.1 M-0.3 M. The concentration of EDC affects the cross-linking between the peptide microgel and heparin molecules. When the EDC concentration is too low, the heparin molecules cannot be fully coupled with the peptide microgel, while when the concentration is too high, it will affect the biological activity of the heparin molecules. Therefore, the applicant's research found that when the EDC concentration is 0.1 M-0.3 M, it ensures sufficient coupling between the heparin molecules and the peptide microgel without affecting the biological activity of the heparin molecules.

[0019] The active molecule was dissolved in PBS buffer, and the above peptide microgel-heparin molecule complex was added. The mixture was incubated at 4°C for 10-12 h to obtain the peptide microgel-heparin molecule-active molecule complex, which was used as the needle body.

[0020] Preferably, the active ingredient is basic fibroblast growth factor, an antimicrobial peptide with repair and regeneration functions, nicotinamide for treating skin inflammation, or lidocaine for local anesthesia. The polypeptide microgel uses heparin to load the active molecules, allowing the polypeptide, as an effective ingredient, to replenish deficiencies in the skin.

[0021] Step 3: Microneedle substrate preparation:

[0022] Carboxymethyl chitosan was dissolved in PBS buffer solution to obtain carboxymethyl chitosan hydrogel. The polypeptide microgel-heparin molecule-active molecule complex from step three was added and mixed evenly. The mixture was then degassed under negative pressure to obtain a microneedle formulation. Carboxymethyl chitosan was used to prepare the microneedle base. First, this avoids the introduction of organic solvents. Second, carboxymethyl chitosan hydrogel has good biodegradability and biocompatibility. It can be naturally degraded in vivo, and its degradation products are harmless to organisms and will not cause immune responses or inflammation.

[0023] The microneedle formulation is injected into the PDMS microneedle analgesic membrane, and then carboxymethyl chitosan hydrogel is added to form the microneedle base. The mold is dried and the PDMS mold is removed to obtain the peptide microneedle dressing.

[0024] Furthermore, the volume ratio of carboxymethyl chitosan hydrogel to polypeptide microgel-heparin molecule-active molecule complex is 1:5-5:1.

[0025] Furthermore, the photoinitiator is LAP or irgacure 2959; wherein the mass concentration of LAP is 0.1%-1% and the mass concentration of irgacure 2959 is 0.1%-5%.

[0026] Furthermore, the volume ratio of solution A to solution B is 1:10-10:1.

[0027] The beneficial technical effects achieved by this invention are as follows: This invention provides a polypeptide microneedle dressing and its preparation method. The dressing includes a microneedle base and an array of needles arranged on the microneedle base. The needles are composed of polypeptide microgels and carboxymethyl chitosan hydrogels, and the microneedle base is a carboxymethyl chitosan hydrogel. It delivers effective collagen components subcutaneously, achieving the purpose of promoting skin cell regeneration or proliferation. Heparin molecules are loaded onto the polypeptide microgel to carry active molecules with positive effects on the skin, promoting the growth and proliferation of basal skin cells. Attached Figure Description

[0028] Figure 1 This is a flowchart of the preparation method of the polypeptide microneedle dressing in this invention;

[0029] Figure 2 These are electron micrographs of microgels prepared at different rotation speeds in the embodiments of the present invention;

[0030] Figure 3 This is a panoramic image of the polypeptide composite microneedles loaded with basic fibroblast growth factor in Example 15 of the present invention.

[0031] Figure 4 This is a SEM image of the polypeptide composite microneedles loaded with basic fibroblast growth factor in Example 16 of the present invention.

[0032] Figure 5 This is a panoramic image of the polypeptide composite microneedles loaded with basic fibroblast growth factor in Example 17 of the present invention.

[0033] Figure 6 This is a SEM image of the polypeptide composite microneedles loaded with antimicrobial peptide LL-37 in Example 18 of the present invention.

[0034] Figure 7These are fluorescence staining micrographs of human skin fibroblasts in the experimental examples of this invention, cultured for 24 h and 48 h in a mixed system of polypeptide microgel loaded with basic fibroblast growth factor and carboxymethyl chitosan hydrogel, and in a complete culture medium environment. Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Recombinant human type I collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0038] Recombinant human type III collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0039] Carboxymethyl chitosan was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0040] Heparin, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0041] Fibroblast growth factor b-FGF was purchased from Asiacoine Biotechnology Co., Ltd.

[0042] All other items were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0043] This invention provides a polypeptide microneedle dressing, which includes a microneedle base and an array of needles arranged on the microneedle base. The needles are composed of polypeptide microgels and carboxymethyl chitosan hydrogels, and the microneedle base is a carboxymethyl chitosan hydrogel. Specifically, the polypeptide microgels are loaded with active molecules that have a positive effect on the skin via heparin molecules. Example 1

[0044] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, which is derived from the Pichia pastoris expression system and has a molecular weight of 100 kDa, and the active molecule is basic fibroblast growth factor. Example 2

[0045] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, which is derived from a mammalian expression system and has a molecular weight of 200 kDa, and the active molecule is an antimicrobial peptide. Example 3

[0046] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, which is derived from a plant expression system and has a molecular weight of 300 kDa, and the active molecule is basic fibroblast growth factor. Example 4

[0047] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, which is derived from a mammalian expression system and has a molecular weight of 300 kDa, and the active molecule is an antimicrobial peptide. Example 5

[0048] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen derived from an insect expression system, has a molecular weight of 200 kDa, and the active molecule is lidocaine. Example 6

[0049] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, which is derived from a microbial expression system and has a molecular weight of 200 kDa, and the active molecule is basic fibroblast growth factor. Example 7

[0050] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pigskin or fish skin, with a molecular weight of 50 kDa, and the active molecule is basic fibroblast growth factor. Example 8

[0051] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pigskin or fish skin, with a molecular weight of 200 kDa, and the active molecule is basic fibroblast growth factor. Example 9

[0052] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pigskin or fish skin, with a molecular weight of 300 kDa, and the active molecule is basic fibroblast growth factor. Example 10

[0053] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pigskin or fish skin, with a molecular weight of 200 kDa, and the active molecule is an antimicrobial peptide. Example 11

[0054] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pigskin or fish skin, with a molecular weight of 200 kDa, and the active molecule is lidocaine. Example 12

[0055] A polypeptide microneedle dressing, wherein the polypeptide microgel is gelatin, which is extracted from animal bones, skin or connective tissue, has a molecular weight of 1 kDa, and the active molecule is lidocaine. Example 13

[0056] A polypeptide microneedle dressing, wherein the polypeptide microgel is gelatin, which is extracted from animal bones, skin or connective tissue, and has a molecular weight of 20 kDa, and the active molecule is basic fibroblast growth factor. Example 14

[0057] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, is prepared by the following method:

[0058] First, prepare a microneedle PDMS mold using the following method:

[0059] (1) Prepare a 5×5 microneedle array metal positive mold with a microneedle cone bottom length of 1 mm, a height from the needle tip to the microneedle cone bottom of 1.5 mm, and a spacing of 1 mm between microneedles.

[0060] (2) Place the metal positive mold prepared above in a container, then pour polydimethylsiloxane (PDMS) prepolymer into the container, and then dry it in an oven at 70 °C for 3 h. Finally, separate the metal positive mold from PDMS to obtain the PDMS microneedle negative mold.

[0061] Next, the microneedle formulation was prepared and filled into the microneedle PDMS mold:

[0062] (3) Weigh a certain amount of recombinant human type I collagen (RHCⅠ) powder and dissolve it in PBS buffer to prepare a 10% (w / v) recombinant human type I collagen solution. Add 600 μL of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 3-5 h to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute it, filter it through a 0.22 μm disposable syringe filter, dialyze it through a dialysis bag for 3-4 days, and freeze-dry it to obtain a sponge-like solid sample of RHCⅠ-MA. Then dissolve the freeze-dried RHCⅠ-MA in a 0.1% (w / v) photoinitiator LAP solution to obtain "solution A". "solution B" is prepared by recombinant human type III collagen using the same method as above.

[0063] (4) Mix "solution A" and "solution B" evenly at a volume ratio of 4:1 to obtain "solution C", and then irradiate with 405 nm ultraviolet light for 5 min to prepare a polypeptide hydrogel;

[0064] (5) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL of PBS buffer under magnetic stirring at room temperature to prepare a 10% (w / v) carboxymethyl chitosan hydrogel. Then mix the carboxymethyl chitosan hydrogel with the polypeptide hydrogel prepared in step (4) at a volume ratio of 1:5. Remove the air bubbles under negative pressure to obtain the microneedle preparation.

[0065] (6) Pour the microneedle formulation prepared in step (5) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube and centrifuge at 3500 rpm for 15 min to inject the microneedle formulation into the PDMS microneedle negative mold, and finally pour in carboxymethyl chitosan hydrogel to form a base with a thickness of 0.5 mm-1 mm. Then dry it in a constant temperature drying oven at 25 ℃ and 10% humidity for 3 h. Remove the PDMS mold to obtain a polypeptide microneedle dressing. Example 15

[0066] like Figure 1 As shown, a method for preparing a polypeptide microneedle dressing differs from Example 13 in that the microneedle formulation is loaded with an active molecule, which is basic fibroblast growth factor. The specific process for preparing the microneedle formulation is as follows:

[0067] (3) Weigh a certain amount of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 10% (w / v) recombinant human type I collagen solution. Add 600 μL of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 3-5 h to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute it, filter it through a 0.22 μm disposable syringe filter, dialyze it through a dialysis bag for 3-4 days, and freeze-dry it to obtain RHCⅠ-MA sponge-like solid sample. Then dissolve the freeze-dried RHCⅠ-MA in a 0.1% (w / v) photoinitiator LAP solution to obtain "solution A". "solution B" is prepared by recombinant human type III collagen using the same method as above.

[0068] (4) Mix "Solution A" and "Solution B" at a volume ratio of 4:1 to obtain "Solution C". Then, slowly add "Solution C" dropwise through a syringe to a mixture of liquid paraffin (oil phase) and surfactant Span 80 at a volume ratio of 10:2 at 600 rpm, 1600 rpm, and 2000 rpm, respectively. Stir for 3 min, irradiate with 405 nm ultraviolet light for 5 min, centrifuge to remove the supernatant, add PBS and shake well to remove the remaining oil phase. Repeat the addition of PBS buffer 3 times to remove the oil phase. Finally, rinse with PBS buffer and collect by filtration to obtain the polypeptide microgel. Figure 2 As shown; from Figure 2It can be seen that as the rotation speed increases, the particle size of the obtained polypeptide microgels becomes smaller and smaller. The smaller the particle size of the polypeptide microgels, the more conducive they are to absorption.

[0069] (5) Weigh heparin sodium salt powder and dissolve it in 0.1 M EDC solution to prepare heparin solution with a dissolution amount of 1 mg / ml. Add the polypeptide microgel prepared in step (4) to 100 mL of heparin solution and shake continuously at room temperature for 4 h. After the reaction is completed, add PBS buffer to wash the microgel 3 times to obtain polypeptide microgel coupled with heparin molecules.

[0070] (6) Weigh a certain amount of basic fibroblast growth factor lyophilized powder and dissolve it in PBS solution to prepare a 100 ng / ml basic fibroblast growth factor solution. Then add the heparin-coupled polypeptide microgel prepared in step (5) to 100 mL of growth factor solution and incubate at 4 °C for 10 h to obtain a polypeptide microgel loaded with basic fibroblast growth factor.

[0071] (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL of PBS buffer under magnetic stirring at room temperature to prepare a 10% (w / v) carboxymethyl chitosan hydrogel. Then mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic skin fibroblast growth factor-loaded polypeptide microgel prepared in step (6) at a volume ratio of 1:5. Remove the air bubbles under negative pressure to obtain the microneedle preparation.

[0072] (8) Pour the microneedle formulation prepared in step (7) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube and centrifuge at 3500 rpm for 15 min to inject the microneedle formulation into the PDMS microneedle negative mold, and finally pour in carboxymethyl chitosan hydrogel to form a base with a thickness of 0.5 mm-1 mm. Then dry it in a constant temperature drying oven at 25 ℃ and 10% humidity for 3 h. Remove the PDMS mold to obtain a polypeptide microneedle dressing. Example 16

[0073] A method for preparing a polypeptide microneedle dressing, the specific process of which is as follows:

[0074] First, prepare a microneedle PDMS mold using the following method:

[0075] (1) Prepare a 10×10 microneedle array metal positive mold with a microneedle conical bottom length of 1 mm, a height from the needle tip to the microneedle conical bottom of 2 mm, and a microneedle spacing of 2 mm.

[0076] (2) Place the metal positive mold prepared above in a container, then pour polydimethylsiloxane (PDMS) prepolymer into the container, and then dry it in an oven at 70 °C for 4 h. Finally, separate the metal positive mold from PDMS to obtain the PDMS microneedle negative mold.

[0077] Next, the microneedle formulation was prepared and filled into the microneedle PDMS mold:

[0078] (3) Weigh a certain amount of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type I collagen solution. Add 300 μL of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 3 h to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute it, filter it through a 0.22 μm disposable syringe filter, dialyze it through a dialysis bag for 3-4 days, and freeze-dry it to obtain RHCⅠ-MA sponge-like solid sample. Then dissolve the freeze-dried RHCⅠ-MA in a 0.5% (w / v) photoinitiator LAP solution to obtain "solution A". "solution B" is prepared by recombinant human type III collagen using the same method as above.

[0079] (4) Mix "Solution A" and "Solution B" evenly at a volume ratio of 1:1 to obtain "Solution C". Then, at a speed of 2000 rpm, slowly add "Solution C" dropwise through a syringe to a mixed solution of liquid paraffin (oil phase) and surfactant Span 80 at a volume ratio of 10:2 and stir for 3 min. After irradiation with 405 nm ultraviolet light for 6 min, centrifuge to remove the supernatant, add PBS and shake evenly to remove the remaining oil phase. Repeat the addition of PBS buffer 3 times to remove the oil phase. Finally, add PBS buffer to rinse clean and collect the peptide microgel by filtration.

[0080] (5) Weigh heparin sodium salt powder and dissolve it in 0.2 M EDC solution at 2 mg / ml to prepare heparin solution. Add the polypeptide microgel prepared in step (4) to 100 mL of heparin solution and shake continuously at room temperature for 4 h. After the reaction is completed, add PBS buffer to wash the microgel 3 times to obtain polypeptide microgel coupled with heparin molecules.

[0081] (6) Weigh a certain amount of basic fibroblast growth factor lyophilized powder and dissolve it in PBS solution to prepare a basic fibroblast growth factor solution with a concentration of 200 ng / ml. Then add the heparin-coupled polypeptide microgel prepared in step (5) to 100 mL of growth factor solution and incubate at 4 ℃ for 10 h to obtain a polypeptide microgel loaded with basic fibroblast growth factor.

[0082] (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL of PBS buffer under magnetic stirring at room temperature to prepare a 20% (w / v) carboxymethyl chitosan hydrogel. Then mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic skin fibroblast growth factor-loaded polypeptide microgel prepared in step (6) at a volume ratio of 2:1. Remove the air bubbles under negative pressure to obtain the microneedle preparation.

[0083] (8) The microneedle formulation prepared in step (7) is evenly poured onto the PDMS microneedle negative mold. The negative mold is placed in a centrifuge tube and centrifuged at 3500 rpm for 15 min to inject the microneedle formulation into the PDMS microneedle negative mold. Finally, carboxymethyl chitosan hydrogel is poured in to form a base with a thickness of 0.5 mm-1 mm. Then, it is dried in a constant temperature drying oven at 30 ℃ and 10% humidity for 4 h. After removing the PDMS mold, a polypeptide microneedle dressing is obtained, such as... Figure 3 As shown. Example 17

[0084] A method for preparing a polypeptide microneedle dressing, the specific process of which is as follows:

[0085] First, prepare a microneedle PDMS mold using the following method:

[0086] (1) Prepare an 8×8 microneedle array metal positive mold with a microneedle conical bottom length of 0.5 mm, a height from the needle tip to the microneedle conical bottom of 1 mm, and a spacing of 1 mm between microneedles.

[0087] (2) Place the metal positive mold prepared above in a container, then pour polydimethylsiloxane (PDMS) prepolymer into the container, and then dry it in an oven at 70 °C for 4 h. Finally, separate the metal positive mold from PDMS to obtain the PDMS microneedle negative mold.

[0088] Next, the microneedle formulation was prepared and filled into the microneedle PDMS mold:

[0089] (3) Weigh a certain amount of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type I collagen solution. Add 300 μL of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 4 h to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute it, filter it through a 0.22 μm disposable syringe filter, dialyze it through a dialysis bag for 3-4 days, and freeze-dry it to obtain RHCⅠ-MA sponge-like solid sample. Then dissolve the freeze-dried RHCⅠ-MA in a 0.5% (w / v) photoinitiator irgacure 2959 solution to obtain "solution A". "solution B" is prepared by recombinant human type III collagen using the same method.

[0090] (4) Prepare polypeptide microgels according to the method of step (4) in Example 15, wherein the volume ratio of "solution A" to "solution B" is 1:10.

[0091] (5) Weigh heparin sodium salt powder and dissolve it in 0.2 M EDC solution at 3 mg / ml to prepare heparin solution. Add the polypeptide microgel prepared in step (4) to 100 mL of heparin solution and shake continuously at room temperature for 5 h. After the reaction, add PBS buffer to wash the microgel 3 times to obtain polypeptide microgel coupled with heparin molecules.

[0092] (6) Weigh a certain amount of basic fibroblast growth factor lyophilized powder and dissolve it in PBS solution to prepare a basic fibroblast growth factor solution with a concentration of 300 ng / ml. Then add the heparin-coupled polypeptide microgel prepared in step (5) to 100 mL of growth factor solution and incubate at 4 ℃ for 11 h to obtain a polypeptide microgel loaded with basic fibroblast growth factor.

[0093] (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL of PBS buffer under magnetic stirring at room temperature to prepare a 20% (w / v) carboxymethyl chitosan hydrogel. Then mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic skin fibroblast growth factor-loaded polypeptide microgel prepared in step (6) at a volume ratio of 3:2. Remove the air bubbles under negative pressure to obtain the microneedle preparation.

[0094] (8) The microneedle formulation prepared in step (7) is evenly poured onto the PDMS microneedle negative mold. The negative mold is placed in a centrifuge tube and centrifuged at 3500 rpm for 15 min to inject the microneedle formulation into the PDMS microneedle negative mold. Finally, carboxymethyl chitosan hydrogel is poured in to form a base with a thickness of 0.5 mm-1 mm. The mold is then placed in a constant temperature drying oven at 40℃ and 15% humidity for 5 h to dry. After removing the PDMS mold, a polypeptide microneedle dressing is obtained, such as... Figure 4 As shown. Example 18

[0095] A method for preparing a polypeptide microneedle dressing, the specific process of which is as follows:

[0096] First, prepare a microneedle PDMS mold using the following method:

[0097] (1) Prepare a 10×10 microneedle array metal positive mold with a microneedle conical bottom length of 0.5 mm, a height from the needle tip to the microneedle conical bottom of 1 mm, and a spacing of 1 mm between microneedles.

[0098] (2) Place the metal positive mold prepared above in a container, then pour polydimethylsiloxane (PDMS) prepolymer into the container, and then dry it in an oven at 70 °C for 5 h. Finally, separate the metal positive mold from PDMS to obtain the PDMS microneedle negative mold.

[0099] Next, the microneedle formulation was prepared and filled into the microneedle PDMS mold:

[0100] (3) Weigh a certain amount of recombinant human type I collagen powder and dissolve it in PBS buffer to prepare a 20% (w / v) recombinant human type I collagen solution. Add 300 μL of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 5 h to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute it, filter it through a 0.22 μm disposable syringe filter, dialyze it through a dialysis bag for 3-4 days, and freeze-dry it to obtain RHCⅠ-MA sponge-like solid sample. Then dissolve the freeze-dried RHCⅠ-MA in a 1% (w / v) photoinitiator irgacure 2959 solution to obtain "solution A". "Solution B" is prepared by recombinant human type III collagen using the same method as above.

[0101] (4) Prepare polypeptide microgels according to the method of step (4) in Example 15, wherein the volume ratio of "solution A" to "solution B" is 10:1.

[0102] (5) Weigh heparin sodium salt powder and dissolve it in 0.3 M EDC solution at 3 mg / ml to prepare heparin solution. Add the polypeptide microgel prepared in step (4) to 100 mL of heparin solution and shake continuously at room temperature for 6 h. After the reaction, add PBS buffer to wash the microgel 3 times to obtain polypeptide microgel coupled with heparin molecules.

[0103] (6) Weigh a certain amount of basic fibroblast growth factor lyophilized powder and dissolve it in PBS solution to prepare a basic fibroblast growth factor solution with a concentration of 300 ng / ml. Then add the heparin-coupled polypeptide microgel prepared in step (5) to 100 mL of growth factor solution and incubate at 4 ℃ for 12 h to obtain a polypeptide microgel loaded with basic fibroblast growth factor.

[0104] (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL of PBS buffer under magnetic stirring at room temperature to prepare a 20% (w / v) carboxymethyl chitosan hydrogel. Then mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic skin fibroblast growth factor-loaded polypeptide microgel prepared in step (6) at a volume ratio of 5:1. Remove the air bubbles under negative pressure to obtain the microneedle preparation.

[0105] (8) The microneedle formulation prepared in step (7) is evenly poured onto the PDMS microneedle negative mold. The negative mold is placed in a centrifuge tube and centrifuged at 3500 rpm for 15 min to inject the microneedle formulation into the PDMS microneedle negative mold. Finally, carboxymethyl chitosan hydrogel is poured in to form a base with a thickness of 0.5 mm-1 mm. The mold is then placed in a constant temperature drying oven at 40℃ and 20% humidity for 6 h to dry. After removing the PDMS mold, a polypeptide microneedle dressing is obtained, such as... Figure 5 As shown. Example 19

[0106] A method for preparing a polypeptide microneedle dressing, the specific process of which is as follows:

[0107] First, a microneedle PDMS mold is prepared:

[0108] The microneedle mold was prepared according to the method steps of Example 17;

[0109] Next, prepare the microneedle formulation:

[0110] "Solution A" and "Solution B" are prepared according to the method steps of step (3) in Example 17;

[0111] Following step (4) of Example 17, a polypeptide microgel with a volume ratio of type I to type III collagen of 10:1 was prepared.

[0112] According to step (5) of Example 17, a polypeptide microgel with conjugated heparin molecules was prepared;

[0113] Weigh a certain amount of lyophilized antimicrobial peptide LL-37 powder and dissolve it in PBS solution to prepare an antimicrobial peptide LL-37 solution with a concentration of 100 ng / ml. Then, add the heparin-coupled polypeptide microgel prepared in step (4) to 100 mL of the antimicrobial peptide LL-37 solution and incubate at 4 °C for 12 h to obtain a polypeptide microgel loaded with antimicrobial peptide LL-37.

[0114] Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL of PBS buffer under magnetic stirring at room temperature to prepare a 20% (w / v) carboxymethyl chitosan hydrogel. Then, mix the carboxymethyl chitosan hydrogel with the heparin-coupled and antimicrobial peptide LL-37-loaded peptide microgel prepared in step (5) at a volume ratio of 5:1. Remove the air bubbles under negative pressure to obtain the microneedle formulation.

[0115] The microneedle formulation prepared in step (6) was evenly poured onto a PDMS microneedle negative mold. The negative mold was placed in a centrifuge tube and centrifuged at 3500 rpm for 15 min to inject the microneedle formulation into the PDMS microneedle negative mold. Finally, carboxymethyl chitosan hydrogel was poured in to form a base with a thickness of 0.5 mm-1 mm. The mold was then placed in a constant temperature drying oven at 40 ℃ and 20% humidity for 6 h to dry. After removing the PDMS mold, a polypeptide microneedle dressing was obtained. Figure 6 As shown.

[0116] Experimental Example 1

[0117] Human skin fibroblasts (HFFs) were cultured in a polypeptide microgel-carboxymethyl chitosan mixture loaded with basic fibroblast growth factor.

[0118] HFFs cells were cultured using ScienCell 1001 endothelial cell culture medium, which consisted of 500 mL basal medium, 25 mL fetal bovine serum, 5 mL endothelial growth factor, and 5 mL penicillin / streptomycin solution. The HFFs cells were resuspended in this medium and then incubated at 10 °C. 5 Cells were seeded at a density of 100 cells / medium in culture dishes containing cells. For the experimental group, 5 mL of the above-mentioned culture medium was added and mixed thoroughly with the polypeptide microgel loaded with basic fibroblast growth factor and carboxymethyl chitosan hydrogel prepared in Example 15. The control group received complete culture medium. The mixtures were then incubated at 37°C in a 5% CO2 incubator. After 24 h of incubation, the cells were removed, washed with PBS, stained with acridine orange / ethidium bromide, and observed under a fluorescence inverted microscope.

[0119] The results are as follows Figure 7 As shown, HFFs cells are regular green pebble-shaped, with good morphology and normal condition. Compared with the control group, the cells in the experimental group have normal morphology and significantly increased number after 48 hours of culture, indicating that the microneedle preparation has excellent biocompatibility and is conducive to cell proliferation.

[0120] The present invention has been disclosed above with reference to preferred embodiments, but it is not intended to limit the present invention. All technical solutions obtained by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide microneedle dressing, characterized in that, Includes the following steps: Step 1, Preparation of peptide microgels: Methacrylic acid was added to the first polypeptide solution to prepare a methacrylic anhydride-modified first polypeptide solution. The supernatant was collected by centrifugation, diluted, filtered, dialyzed for 3-4 days, and freeze-dried to obtain a solid sample. The solid sample was then dissolved in a photoinitiator to obtain solution A. The second polypeptide solution was treated using the same method to obtain solution B; Solution A and solution B are thoroughly mixed to obtain solution C. Solution C is slowly added dropwise to a mixture of paraffin and surfactant at 1000-5000 rpm. After stirring and irradiation with ultraviolet light, the supernatant is removed by centrifugation. PBS buffer is added and mixed evenly to remove the oil phase. This process is repeated 3-5 times until the oil phase is completely removed. The precipitate is then filtered and collected to obtain the polypeptide microgel. Step 2, Heparin molecule coupling: Dissolve heparin sodium salt powder in EDC solution, add the polypeptide microgel prepared in Step 1, shake and react at room temperature for 4-6 hours, wash with PBS buffer solution after the reaction to obtain polypeptide microgel-heparin molecule complex. The active molecule was dissolved in PBS buffer, and the above peptide microgel-heparin molecule complex was added. The mixture was incubated at 4°C for 10-12 h to obtain the peptide microgel-heparin molecule-active molecule complex, which was used as the needle body. Step 3: Microneedle substrate preparation: Carboxymethyl chitosan was dissolved in PBS buffer solution to obtain carboxymethyl chitosan hydrogel. The polypeptide microgel-heparin molecule-active molecule complex from step three was added and mixed evenly. The air bubbles were removed under negative pressure to obtain the microneedle formulation. The microneedle formulation is injected into the PDMS microneedle analgesic membrane, and then carboxymethyl chitosan hydrogel is added to form a microneedle base. The mold is dried and the PDMS mold is removed to obtain the peptide microneedle dressing. The first polypeptide is recombinant human type I collagen; the second polypeptide is recombinant human type III collagen; the active molecule is basic fibroblast growth factor, an antimicrobial peptide with repair and regeneration functions, nicotinamide for treating skin inflammation, or lidocaine for local anesthesia; the volume ratio of carboxymethyl chitosan hydrogel to polypeptide microgel-heparin molecule-active molecule complex is 1:5-5:

1.

2. The method for preparing the polypeptide microneedle dressing according to claim 1, characterized in that: The photoinitiator is LAP or irgacure 2959; the mass concentration of LAP is 0.1%-1%, and the mass concentration of irgacure 2959 is 0.1%-5%.

3. The method for preparing the polypeptide microneedle dressing according to claim 1, characterized in that: The volume ratio of solution A to solution B is 1:10-10:

1.

4. A polypeptide microneedle dressing, wherein the polypeptide microneedle dressing is obtained by the preparation method according to any one of claims 1-3.

5. The polypeptide microneedle dressing according to claim 4, characterized in that: The recombinant human collagen has a molecular weight of 100-300 kDa.

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