Polypeptide microneedle dressing and preparation method thereof

Abstract

The invention provides a polypeptide microneedle dressing and a preparation method, and belongs to the technical field of polypeptide excipients, the dressing comprises a microneedle substrate and a needle body array arranged on the microneedle substrate, the needle body is composed of polypeptide microgel and carboxymethyl chitosan hydrogel, and the microneedle substrate is carboxymethyl chitosan hydrogel; and collagen active ingredients are delivered to subcutaneous tissues, so that the purpose of promoting skin cell regeneration or proliferation is achieved. Active molecules with a positive effect on the skin are loaded on the polypeptide microgel through heparin molecules, so that the growth and proliferation of skin bottom cells are promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide auxiliary materials, and specifically relates to a polypeptide microneedle dressing and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body, including the epidermis, dermis and subcutaneous tissue. The structure of the dermis is composed of collagen, elastic fibers and matrix. During the growth of a person, ultraviolet rays can penetrate the skin to reach the dermis, activating matrix metalloproteinases in the body that induce excessive degradation of collagen. At the same time, the sugar ingested by the human body will also react with the collagen in the skin, causing the collagen to decompose. When the synthesis and loss rate of collagen in the skin no longer maintain a balanced state, the skin will lack elasticity and lose support, resulting in wrinkles and other skin aging problems.

[0003] Collagen is an important component in the dermis of human skin, especially type I and type III collagen, which are the main structural proteins in human skin and play a role in maintaining the normal physiological functions of the skin. Therefore, non-toxic and non-irritating collagen is usually injected into the dermis (as a dermal filler) to offset skin wrinkles and restore skin fullness. However, collagen cannot be directly absorbed through the skin barrier, so new drug delivery methods have become a difficult problem to solve.

[0004] Microneedle patches are composed of tiny microneedles and a substrate, can carry active ingredients, act on the skin in a minimally invasive manner, and are an invasive treatment tool for introducing drugs into the skin. Compared with traditional skin dressings, microneedle patches have many advantages: ① The microneedle administration method is simple and easy to operate; ② The microneedle delivers the active ingredients to specific locations under the skin through puncture, thereby improving the transdermal efficiency of the active ingredients. Therefore, it is particularly important to obtain a collagen microneedle to deliver the collagen active ingredient to the subcutaneous tissue to achieve the purpose of promoting skin cell regeneration or proliferation. Summary of the invention

[0005] In order to solve the above defects and shortcomings in the prior art, the present invention provides a polypeptide microneedle dressing and a preparation method thereof.

[0006] To solve the above technical problems: The first object of the present invention is to provide a polypeptide microneedle dressing, which comprises a microneedle substrate and a needle array arranged on the microneedle substrate, wherein the needle array is composed of polypeptide microgel and carboxymethyl chitosan hydrogel, and the microneedle substrate is carboxymethyl chitosan hydrogel; preferably, the polypeptide microgel is loaded with active molecules having a positive effect on the skin through heparin molecules; Preferably, the polypeptide is one of animal collagen, recombinant collagen, recombinant human collagen, gelatin or recombinant peptide; more preferably, the polypeptide is recombinant human collagen with a molecular weight of 100-300Kda; most preferably, the recombinant human collagen is recombinant human type I collagen or recombinant human type III collagen.

[0007] The above-mentioned animal-derived collagen is derived from cattle, pigs or fish, the recombinant collagen and recombinant human-derived collagen are derived from mammalian expression systems, plant expression systems, insect expression systems or microbial expression systems; the gelatin is extracted from animal bones, skins, and connective tissues; the recombinant peptides are derived from bacterial expression systems, yeast expression systems, and mammalian cell expression systems; the molecular weights of the animal-derived collagen and recombinant collagen are 50-300 KDa, and the molecular weight of the gelatin is 1-30 Kda.

[0008] Furthermore, the active ingredient is basic fibroblast growth factor, antimicrobial peptide with repair and regeneration function, nicotinamide for treating skin inflammation or lidocaine for local anesthesia.

[0009] The second object of the present invention is to provide a method for preparing a polypeptide microneedle dressing, comprising the following steps: Step 1, preparation of peptide microgel: Adding methacrylic acid to the first polypeptide solution to prepare a methacrylic anhydride first polypeptide solution, collecting the supernatant by centrifugation, diluting, filtering, and dialyzing for 3-4 days, and freeze-drying to obtain a solid sample, and dissolving the solid sample in a photoinitiator to obtain a solution A; As a preferred solution, the first polypeptide is recombinant human type I collagen, methacrylic acid is added to the recombinant human type I collagen solution, and the mixture is reacted for 3-5 hours under magnetic stirring to prepare a methacrylic anhydride recombinant human type I collagen solution (RHCⅠ-MA solution), and then the supernatant is 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, and then the freeze-dried RHCⅠ-MA is dissolved in a certain amount of photoinitiator to obtain "solution A"; The second polypeptide solution is treated in the same manner to obtain solution B; wherein the second polypeptide is recombinant human type III collagen; Preferably, the volume ratio of methacrylic acid added to the recombinant human type I / III collagen solution is 1%-15%; more preferably, 3%-10%.

[0010] Solution A and solution B are fully mixed to obtain solution C, and solution C is slowly added dropwise to a mixed solution of paraffin and surfactant span 80 at a rotation speed of 1000-5000 rpm, stirred, irradiated with 405 nm ultraviolet light for 3-10 minutes, centrifuged to remove the supernatant, added PBS buffer and mixed evenly, and the oil phase is removed, and the process is repeated 3-5 times until the oil phase is completely removed, and the precipitate is collected by filtration to obtain 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, and the particle size of the microgel is inversely proportional to the rotation speed, and the faster the rotation speed, the smaller the particle size of the obtained microgel.

[0011] Step 2, coupling heparin molecules: dissolve heparin sodium salt powder in EDC solution, add the polypeptide microgel prepared in step 1, shake and react for 4-6 hours at room temperature, rinse with PBS buffer solution after the reaction, and obtain polypeptide microgel-heparin molecule complex; preferably, the EDC concentration is 0.1 M-0.3 M. The concentration of EDC affects the cross-linking between polypeptide microgel and heparin molecules. When the concentration of EDC is low, the heparin molecules cannot be fully coupled with the polypeptide microgel, and when the concentration is too high, it will affect the biological activity of the heparin molecules. Therefore, the applicant found that when the concentration of EDC is 0.1M-0.3M, it does not affect the biological activity of the heparin molecules while ensuring that the heparin molecules and polypeptide microgels are fully coupled.

[0012] The active molecule is dissolved in PBS buffer, and the above-mentioned polypeptide microgel-heparin molecule complex is added, and incubated at 4°C for 10-12 h to obtain a polypeptide microgel-heparin molecule-active molecule complex as a needle body; Preferably, 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. The polypeptide microgel is loaded with active molecules through heparin. On the one hand, the polypeptide as an effective ingredient can supplement the deficiency in the skin.

[0013] Step 3: Preparation of microneedle substrate: Carboxymethyl chitosan is dissolved in PBS buffer solution to obtain carboxymethyl chitosan hydrogel, and the polypeptide microgel-heparin molecule-active molecule complex of step three is added and mixed evenly, and bubbles are removed by negative pressure to obtain a microneedle preparation; carboxymethyl chitosan is used to prepare the microneedle substrate, firstly, the introduction of organic solvents is avoided; secondly, the carboxymethyl chitosan hydrogel has good biodegradability and biocompatibility, can be naturally degraded in the body, and its degradation products are harmless to the organism, and it itself will not cause immune response or inflammation.

[0014] The microneedle preparation is injected into the PDMS microneedle negative 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 polypeptide microneedle dressing.

[0015] Furthermore, the volume ratio of the carboxymethyl chitosan hydrogel to the polypeptide microgel-heparin molecule-active molecule complex is 1:5-5:1.

[0016] 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%.

[0017] Further, the volume ratio of solution A to solution B is 1:10-10:1 Beneficial technical effects achieved by the present invention: The present invention provides a polypeptide microneedle dressing and a preparation method thereof, the dressing comprising a microneedle substrate and a needle array arranged on the microneedle substrate, the needle body consisting of polypeptide microgel and carboxymethyl chitosan hydrogel, the microneedle substrate being carboxymethyl chitosan hydrogel; delivering collagen active ingredients to the subcutaneous tissue to achieve the purpose of promoting skin cell regeneration or proliferation. Active molecules having a positive effect on the skin are loaded on the polypeptide microgel through heparin molecules to promote the growth and proliferation of skin basal cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow chart of the preparation method of the polypeptide microneedle dressing of the present invention; Figure 2 Electron microscopic images of microgels prepared at different rotation speeds in the embodiments of the present invention; Figure 3 This is a panoramic photo of the polypeptide composite microneedle loaded with basic fibroblast growth factor in Example 15 of the present invention; Figure 4 This is a SEM image of the polypeptide composite microneedle loaded with basic fibroblast growth factor in Example 16 of the present invention; Figure 5 This is a panoramic photo of the polypeptide composite microneedle loaded with basic fibroblast growth factor in Example 17 of the present invention; Figure 6 This is a SEM image of the polypeptide composite microneedle loaded with antimicrobial peptide LL-37 in Example 18 of the present invention; Figure 7 These are cell live / dead fluorescence staining micrographs of human skin fibroblasts in the test examples of the present invention cultured in the mixed system environment of the polypeptide microgel loaded with basic fibroblast growth factor and carboxymethyl chitosan hydrogel in the present invention and in the complete medium environment for 24 h and 48 h. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0020] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0021] Recombinant human type I collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.; Recombinant human type III collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.; Carboxymethyl chitosan was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0022] Heparin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Fibroblast growth factor b-FGF was purchased from Yacoin Biotechnology Co., Ltd. Others were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The present invention provides a polypeptide microneedle dressing, which comprises a microneedle substrate and a needle body array arranged on the microneedle substrate, wherein the needle body is composed of polypeptide microgel and carboxymethyl chitosan hydrogel, and the microneedle substrate is carboxymethyl chitosan hydrogel; specifically, the polypeptide microgel is loaded with active molecules having a positive effect on the skin through heparin molecules; Example 1

[0023] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, the human collagen is derived from a Pichia pastoris expression system, has a molecular weight of 100Kda, and the active molecule is basic fibroblast growth factor. Example 2

[0024] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, the human collagen is derived from a mammalian expression system, has a molecular weight of 200Kda, and the active molecule is an antimicrobial peptide. Example 3

[0025] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, the human collagen is derived from a plant expression system, has a molecular weight of 300Kda, and the active molecule is basic fibroblast growth factor. Example 4

[0026] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, the human collagen is derived from a mammalian expression system, has a molecular weight of 300Kda, and the active molecule is an antimicrobial peptide. Example 5

[0027] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, the human collagen is derived from an insect expression system, has a molecular weight of 200Kda, and the active molecule is lidocaine. Example 6

[0028] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, the human collagen is derived from a microbial expression system, has a molecular weight of 200Kda, and the active molecule is basic fibroblast growth factor. Example 7

[0029] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pig skin or fish skin, with a molecular weight of 50Kda, and the active molecule is basic fibroblast growth factor. Example 8

[0030] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pig skin or fish skin, with a molecular weight of 200Kda, and the active molecule is basic fibroblast growth factor. Example 9

[0031] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pig skin or fish skin, with a molecular weight of 300Kda, and the active molecule is basic fibroblast growth factor. Example 10

[0032] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pig skin or fish skin, with a molecular weight of 200Kda, and the active molecule is an antimicrobial peptide. Embodiment 11

[0033] A polypeptide microneedle dressing, wherein the polypeptide microgel is animal-derived collagen extracted from cowhide, pig skin or fish skin, with a molecular weight of 200Kda, and the active molecule is lidocaine. Example 12

[0034] A polypeptide microneedle dressing, wherein the polypeptide microgel is gelatin extracted from animal bones, skin or connective tissue, with a molecular weight of 1Kda, and the active molecule is lidocaine. Embodiment 13

[0035] A polypeptide microneedle dressing, wherein the polypeptide microgel is gelatin extracted from animal bones, skin or connective tissue, with a molecular weight of 20Kda, and the active molecule is basic fibroblast growth factor. Embodiment 14

[0036] A polypeptide microneedle dressing, wherein the polypeptide microgel is recombinant human collagen, and the preparation method thereof is as follows: First, prepare a microneedle PDMS mold as follows: (1) Prepare a 5×5 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 1.5 mm, and a spacing of 1 mm between microneedles.

[0037] (2) The metal positive mold prepared above is placed in a container, and then polydimethylsiloxane (PDMS) prepolymer is poured into the container. The container is then dried in an oven at 70 °C for 3 h. Finally, the metal positive mold is separated from the PDMS to obtain the PDMS microneedle negative mold.

[0038] Next, prepare the microneedle preparation filled in the microneedle PDMS mold: (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 600ul of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 3-5 hours to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute, filter through a 0.22 um disposable syringe filter, dialyze through a dialysis bag for 3-4 days, and freeze-dry to obtain a 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". Use recombinant human type III collagen to prepare "Solution B" in the same way as above. (4) "Solution A" and "Solution B" were mixed evenly at a volume ratio of 4:1 to obtain "Solution C", which was then irradiated with 405 nm ultraviolet light for 5 min to prepare a polypeptide hydrogel; (5) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL PBS buffer under magnetic stirring at room temperature to prepare a carboxymethyl chitosan hydrogel with a concentration of 10% (w / v). Then, mix the carboxymethyl chitosan hydrogel with the polypeptide hydrogel prepared in the above step (4) at a volume ratio of 1:5, and remove bubbles under negative pressure to obtain a microneedle preparation. (6) Pour the microneedle preparation prepared in step (5) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube, and then centrifuge at 3500 rpm for 15 min to inject the microneedle preparation into the PDMS microneedle negative mold. Finally, pour carboxymethyl chitosan hydrogel into it to form a base with a thickness of 0.5 mm-1 mm. Then dry it in a constant temperature drying oven at a temperature of 25 ° C and a humidity of 10% for 3 h. Remove the PDMS mold to obtain a polypeptide microneedle dressing. Embodiment 15

[0039] like Figure 1 As shown, a preparation method of a polypeptide microneedle dressing is different from Example 13 in that: the microneedle preparation is loaded with an active molecule, and the active molecule is basic fibroblast growth factor; the specific process of the microneedle preparation is as follows: (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 600ul of methacrylic acid to the recombinant human type I collagen solution and react under magnetic stirring for 3-5 hours to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute, filter through a 0.22 um disposable syringe filter, dialyze through a dialysis bag for 3-4 days, and freeze-dry to obtain a 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". Use recombinant human type III collagen to prepare "Solution B" in the same way as above. (4) Solution A and solution B were mixed evenly at a volume ratio of 4:1 to obtain solution C. Solution C was then slowly added dropwise to a mixed solution 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, and stirred for 3 min. After irradiation with 405 nm ultraviolet light for 5 min, the supernatant was removed by centrifugation, PBS was added and shaken to remove the remaining oil phase, and PBS buffer was added three times to remove the oil phase. Finally, PBS buffer was added to rinse the mixture, and the polypeptide microgel was collected by filtration. Figure 2 shown; from Figure 2 It can be seen that as the rotation speed increases, the particle size of the obtained polypeptide microgel becomes smaller and smaller. The smaller the particle size of the polypeptide microgel, the more conducive it is to absorption.

[0040] (5) Weigh heparin sodium salt powder and dissolve it in 0.1 M EDC solution to prepare a heparin solution with a dissolution amount of 1 mg / ml. Add the polypeptide microgel prepared in step (4) to 100 mL of the heparin solution and continue shaking the reaction at room temperature for 4 h. After the reaction is completed, add PBS buffer to rinse the microgel three times to obtain polypeptide microgels coupled with heparin molecules. (6) Weigh a certain amount of basic fibroblast growth factor freeze-dried 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 the growth factor solution, and incubate at 4°C for 10 h to obtain polypeptide microgel loaded with basic fibroblast growth factor; (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL PBS buffer under magnetic stirring at room temperature to prepare a carboxymethyl chitosan hydrogel with a concentration of 10% (w / v). Then, mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic dermal fibroblast growth factor-loaded polypeptide microgel prepared in step (6) at a volume ratio of 1:5, and remove bubbles under negative pressure to obtain a microneedle preparation. (8) Pour the microneedle preparation prepared in step (7) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube, and then centrifuge at 3500 rpm for 15 min to inject the microneedle preparation into the PDMS microneedle negative mold. Finally, pour carboxymethyl chitosan hydrogel into the tube to form a base with a thickness of 0.5 mm to 1 mm. Then, dry the tube in a constant temperature drying oven at 25 °C and 10% humidity for 3 h. Remove the PDMS mold to obtain a polypeptide microneedle dressing. Example 16

[0041] A method for preparing a polypeptide microneedle dressing, the specific process is as follows: First, prepare a microneedle PDMS mold as follows: (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 spacing of 2 mm between microneedles.

[0042] (2) The metal positive mold prepared above is placed in a container, and then polydimethylsiloxane (PDMS) prepolymer is poured into the container. The container is then dried in an oven at 70 °C for 4 h. Finally, the metal positive mold is separated from the PDMS to obtain the PDMS microneedle negative mold.

[0043] Next, prepare the microneedle preparation filled in the microneedle PDMS mold: (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 ul 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, filter through a 0.22 um disposable syringe filter, dialyze through a dialysis bag for 3-4 days, and freeze-dry to obtain a 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". Use recombinant human type III collagen to prepare "Solution B" in the same way as above. (4) Solution A and solution B were mixed evenly in a volume ratio of 1:1 to obtain solution C. Solution C was then slowly added dropwise into a mixed solution of liquid paraffin (oil phase) and surfactant span 80 in a volume ratio of 10:2 at 2000 rpm through a syringe and stirred for 3 min. After irradiation with 405 nm ultraviolet light for 6 min, the supernatant was removed by centrifugation, PBS was added and shaken to remove the remaining oil phase, PBS buffer was added three times to remove the oil phase, and PBS buffer was added to rinse the mixture clean. The polypeptide microgel was collected by filtration.

[0044] (5) Weigh heparin sodium salt powder at 2 mg / ml and dissolve it in 0.2 M EDC solution to prepare a heparin solution. Add the polypeptide microgel prepared in step (4) to 100 mL of the heparin solution and shake continuously at room temperature for 4 h. After the reaction, add PBS buffer to rinse the microgel three times to obtain polypeptide microgel coupled with heparin molecules. (6) Weigh a certain amount of basic fibroblast growth factor freeze-dried 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 the growth factor solution and incubate at 4°C for 10 h to obtain polypeptide microgel loaded with basic fibroblast growth factor; (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL PBS buffer under magnetic stirring at room temperature to prepare a carboxymethyl chitosan hydrogel with a concentration of 20% (w / v). Then, mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic dermal fibroblast growth factor-loaded polypeptide microgel prepared in step (6) in a volume ratio of 2:1, and remove bubbles under negative pressure to obtain a microneedle preparation. (8) Pour the microneedle preparation prepared in step (7) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube, and then centrifuge at 3500 rpm for 15 min to inject the microneedle preparation into the PDMS microneedle negative mold. Finally, pour carboxymethyl chitosan hydrogel into the base to form a base with a thickness of 0.5 mm to 1 mm. Then, dry it in a constant temperature drying oven at a temperature of 30 ° C and a humidity of 10% for 4 h. Remove the PDMS mold to obtain a polypeptide microneedle dressing, such as Figure 3 shown. Embodiment 17

[0045] A method for preparing a polypeptide microneedle dressing, the specific process is as follows: First, prepare a microneedle PDMS mold as follows: (1) Prepare a metal positive mold of an 8×8 microneedle array 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.

[0046] (2) The metal positive mold prepared above is placed in a container, and then polydimethylsiloxane (PDMS) prepolymer is poured into the container. The container is then dried in an oven at 70 °C for 4 h. Finally, the metal positive mold is separated from the PDMS to obtain the PDMS microneedle negative mold.

[0047] Next, prepare the microneedle preparation filled in the microneedle PDMS mold: (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 ul 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, filter through a 0.22 um disposable syringe filter, dialyze through a dialysis bag for 3-4 days, and freeze-dry to obtain a 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". Use recombinant human type III collagen to prepare "solution B" in the same way as above. (4) The polypeptide microgel was prepared according to the method of step (4) of Example 15, wherein the volume ratio of "solution A" to "solution B" was 1:10.

[0048] (5) Weigh heparin sodium salt powder and dissolve it in 0.2 M EDC solution at 3 mg / ml to prepare a heparin solution. Add the polypeptide microgel prepared in step (4) to 100 mL of the heparin solution and shake continuously at room temperature for 5 h. After the reaction, add PBS buffer to rinse the microgel three times to obtain polypeptide microgel coupled with heparin molecules. (6) Weigh a certain amount of basic fibroblast growth factor freeze-dried 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 the growth factor solution and incubate at 4°C for 11 h to obtain polypeptide microgel loaded with basic fibroblast growth factor. (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL PBS buffer under magnetic stirring at room temperature to prepare a carboxymethyl chitosan hydrogel with a concentration of 20% (w / v). Then, mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic dermal fibroblast growth factor-loaded polypeptide microgel prepared in step (6) in a volume ratio of 3:2, and remove bubbles under negative pressure to obtain a microneedle preparation. (8) Pour the microneedle preparation prepared in step (7) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube, and then centrifuge at 3500 rpm for 15 min to inject the microneedle preparation into the PDMS microneedle negative mold. Finally, pour carboxymethyl chitosan hydrogel into the tube to form a base with a thickness of 0.5 mm to 1 mm. Then, place the entire mold in a constant temperature drying oven at a temperature of 40°C and a humidity of 15% and dry it for 5 h. After removing the PDMS mold, a polypeptide microneedle dressing can be obtained. Figure 4 shown. Embodiment 18

[0049] A method for preparing a polypeptide microneedle dressing, the specific process is as follows: First, prepare a microneedle PDMS mold as follows: (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.

[0050] (2) The metal positive mold prepared above is placed in a container, and then polydimethylsiloxane (PDMS) prepolymer is poured into the container. The container is then dried in an oven at 70 °C for 5 h. Finally, the metal positive mold is separated from the PDMS to obtain the PDMS microneedle negative mold.

[0051] Next, prepare the microneedle preparation filled in the microneedle PDMS mold: (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 ul of methacrylic acid to the recombinant human type I collagen solution and react it under magnetic stirring for 5 h to prepare RHCⅠ-MA solution. Then collect the supernatant by centrifugation, dilute, filter through a 0.22 um disposable syringe filter, dialyze through a dialysis bag for 3-4 days, and freeze-dry to obtain a RHCⅠ-MA sponge-like solid sample. Then, the freeze-dried RHCⅠ-MA is dissolved in a 1% (w / v) photoinitiator irgacure 2959 solution to obtain "solution A". Use recombinant human type III collagen to prepare "solution B" in the same way as above. (4) Prepare polypeptide microgels according to the method of step (4) of Example 15, wherein the volume ratio of "solution A" to "solution B" is 10:1.

[0052] (5) Weigh heparin sodium salt powder at 3 mg / ml and dissolve it in 0.3 M EDC solution to prepare a heparin solution. Add the polypeptide microgel prepared in step (4) to 100 mL of the heparin solution and continue shaking the reaction at room temperature for 6 h. After the reaction, add PBS buffer to rinse the microgel three times to obtain polypeptide microgels coupled with heparin molecules. (6) Weigh a certain amount of basic fibroblast growth factor freeze-dried 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 the growth factor solution and incubate at 4°C for 12 h to obtain polypeptide microgel loaded with basic fibroblast growth factor; (7) Weigh a certain amount of carboxymethyl chitosan powder and dissolve it in 10 mL PBS buffer under magnetic stirring at room temperature to prepare a carboxymethyl chitosan hydrogel with a concentration of 20% (w / v). Then, mix the carboxymethyl chitosan hydrogel with the heparin-coupled and basic dermal fibroblast growth factor-loaded polypeptide microgel prepared in step (6) at a volume ratio of 5:1, and remove bubbles under negative pressure to obtain a microneedle preparation. (8) Pour the microneedle preparation prepared in step (7) evenly onto the PDMS microneedle negative mold, place the negative mold in a centrifuge tube, and then centrifuge at 3500 rpm for 15 min to inject the microneedle preparation into the PDMS microneedle negative mold. Finally, pour carboxymethyl chitosan hydrogel into the tube to form a base with a thickness of 0.5 mm to 1 mm. Then, place the entire mold in a constant temperature drying oven at a temperature of 40°C and a humidity of 20% and dry it for 6 h. After removing the PDMS mold, a polypeptide microneedle dressing can be obtained. Figure 5 shown. Embodiment 19

[0053] A method for preparing a polypeptide microneedle dressing, the specific process is as follows: First, prepare a microneedle PDMS mold: Prepare a microneedle mold according to the method steps of Example 17; Next, prepare the microneedle preparation: Prepare "Solution A" and "Solution B" according to the method steps of step (3) of Example 17; According to step (4) of Example 17, a polypeptide microgel having a volume ratio of type I to type III collagen of 10:1 was prepared; The polypeptide microgel coupled with heparin molecules was prepared according to step (5) of Example 17; A certain amount of lyophilized powder of the antimicrobial peptide LL-37 was weighed and dissolved in a PBS solution to prepare an antimicrobial peptide LL-37 solution with a concentration of 100 ng / ml. Then, the heparin-coupled polypeptide microgel prepared in step (4) was added to 100 mL of the antimicrobial peptide LL-37 solution and incubated at 4 °C for 12 h to obtain polypeptide microgel loaded with the antimicrobial peptide LL-37. A certain amount of carboxymethyl chitosan powder was weighed and dissolved in 10 mL PBS buffer under magnetic stirring at room temperature to prepare a carboxymethyl chitosan hydrogel with a concentration of 20% (w / v). The carboxymethyl chitosan hydrogel was then mixed with the heparin-coupled and antimicrobial peptide LL-37-loaded polypeptide microgel prepared in step (5) at a volume ratio of 5:1, and the bubbles were removed by negative pressure to obtain a microneedle preparation. The microneedle preparation prepared in step (6) is evenly poured onto the PDMS microneedle negative mold, and the negative mold is placed in a centrifuge tube and then centrifuged at 3500 rpm for 15 min to allow the microneedle preparation to be injected into the PDMS microneedle negative mold. Finally, carboxymethyl chitosan hydrogel is poured into the tube to form a base with a thickness of 0.5 mm to 1 mm. The mold is then placed in a constant temperature drying oven at a temperature of 40 ° C and a humidity of 20% for 6 h and the PDMS mold is removed to obtain a polypeptide microneedle dressing, such as Figure 6 shown.

[0054] Test Example 1 Human dermal fibroblasts (HFFs) were cultured in a basic fibroblast growth factor-loaded peptide microgel-carboxymethyl chitosan hybrid system.

[0055] HFFs cells were cultured in endothelial cell culture medium "ScienCell 1001", which contained 500 mL basal culture medium, 25 mL fetal bovine serum, 5 mL endothelial cell growth factor and 5 mL penicillin / streptomycin solution. HFFs cells were resuspended in this culture medium and the resuspended cells were incubated at 10 5 The cells were inoculated at a density of 10 cells / medium in a cell culture dish. The experimental group added 5 mL of the above medium and the basic fibroblast growth factor-loaded polypeptide microgel and carboxymethyl chitosan hydrogel mixed system prepared in Example 15 and mixed evenly. The control group was filled with complete medium. Then, the cells were placed in a 37°C, 5% CO 2 After 24 h of culture, the cells were taken out, washed with PBS, stained with acridine orange / ethidium bromide, and observed under a fluorescent inverted microscope.

[0056] The results are as follows Figure 7 As shown, HFFs cells were regular green pebble-shaped with good morphology and normal condition. Compared with the control group, the cells in the experimental group had normal morphology and significantly increased number after 48 hours of culture, indicating that the microneedle preparation had excellent biocompatibility and was beneficial to cell proliferation.

[0057] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A polypeptide microneedle dressing, characterized in that: The dressing comprises a microneedle substrate and a needle array arranged on the microneedle substrate, wherein the needles are composed of polypeptide microgel and carboxymethyl chitosan hydrogel, and the microneedle substrate is carboxymethyl chitosan hydrogel; the polypeptide microgel is loaded with active molecules having a positive effect on the skin through heparin molecules; The polypeptide is one of animal collagen, recombinant collagen, recombinant human collagen, gelatin or recombinant peptide; the animal collagen is derived from cattle, pigs or fish, the recombinant collagen and recombinant human collagen are derived from mammalian expression system, plant expression system, insect expression system or microbial expression system; the gelatin is extracted from animal bones, skin and connective tissue; the recombinant peptide is derived from bacterial expression system, yeast expression system and mammalian cell expression system.

2. The polypeptide microneedle dressing according to claim 1, characterized in that: The molecular weights of the animal-derived collagen and the recombinant collagen are 50-300 KDa, and the molecular weight of the gelatin is 1-30 KDa.

3. The polypeptide microneedle dressing according to claim 1, characterized in that: The polypeptide is recombinant human collagen with a molecular weight of 100-300Kda.

4. The polypeptide microneedle dressing according to claim 1, characterized in that: The active ingredients are basic fibroblast growth factor, antimicrobial peptide with repair and regeneration functions, nicotinamide for treating skin inflammation or lidocaine for local anesthesia.

5. A method for preparing a polypeptide microneedle dressing, characterized in that: The following steps are involved: Step 1, preparation of peptide microgel: Adding methacrylic acid to the first polypeptide solution to prepare a methacrylic anhydride first polypeptide solution, collecting the supernatant by centrifugation, diluting, filtering, and dialyzing for 3-4 days, and freeze-drying to obtain a solid sample, and dissolving the solid sample in a photoinitiator to obtain a solution A; The second polypeptide solution was treated in the same manner to obtain solution B; Solution A and solution B are fully mixed to obtain solution C, and solution C is slowly added dropwise to the mixed solution of paraffin and surfactant at a speed of 1000-5000 rpm, stirred, irradiated with ultraviolet light, centrifuged to remove the supernatant, PBS buffer is added and mixed evenly, and the oil phase is removed, and the process is repeated 3-5 times until the oil phase is completely removed, and the precipitate is collected by filtration to obtain the polypeptide microgel; Step 2, coupling heparin molecules: dissolving heparin sodium salt powder in EDC solution, adding the polypeptide microgel prepared in step 1, shaking and reacting at room temperature for 4-6 hours, and washing with PBS buffer solution after the reaction to obtain polypeptide microgel-heparin molecule complex; The active molecule is dissolved in PBS buffer, and the above-mentioned polypeptide microgel-heparin molecule complex is added, and incubated at 4°C for 10-12 h to obtain a polypeptide microgel-heparin molecule-active molecule complex as a needle body; Step 3: Preparation of microneedle substrate: Carboxymethyl chitosan is dissolved in PBS buffer solution to obtain carboxymethyl chitosan hydrogel, and the polypeptide microgel-heparin molecule-active molecule complex prepared in step 3 is added and mixed evenly, and bubbles are removed by negative pressure to obtain a microneedle preparation; The microneedle preparation is injected into the PDMS microneedle negative 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 polypeptide microneedle dressing.

6. The method for preparing the polypeptide microneedle dressing according to claim 5, characterized in that: The first polypeptide is recombinant human type I collagen; the second polypeptide is recombinant human type III collagen.

7. The method for preparing the polypeptide microneedle dressing according to claim 5, characterized in that: The active ingredients are basic fibroblast growth factor, antimicrobial peptide with repair and regeneration functions, nicotinamide for treating skin inflammation or lidocaine for local anesthesia.

8. The method for preparing the polypeptide microneedle dressing according to claim 5, characterized in that: The volume ratio of the carboxymethyl chitosan hydrogel to the polypeptide microgel-heparin molecule-active molecule complex is 1:5-5:

1.

9. The method for preparing the polypeptide microneedle dressing according to claim 5, 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%.

10. The method for preparing the polypeptide microneedle dressing according to claim 5, characterized in that: The volume ratio of solution A to solution B is 1:10-10:1.

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