A double-crosslinked hydrogel microneedle patch, and a preparation method and application thereof

Abstract

The application discloses a double-crosslinked hydrogel microneedle patch, which comprises a substrate layer and a needle tip part, wherein the needle tip part is made of an active oxygen responsive polypeptide prodrug, an ester bond functionalized small molecule prodrug, a polypeptide diacetylene and a double bond functionalized macromolecule, and the active oxygen responsive polypeptide prodrug, the ester bond functionalized small molecule prodrug and the polypeptide diacetylene are loaded on the double bond functionalized macromolecule through coupling covalent action; the active oxygen responsive polypeptide prodrug and the ester bond functionalized small molecule prodrug release drug molecules slowly through responding to an active oxygen microenvironment and ester bond hydrolysis respectively. The needle tip of the microneedle patch obtained by the application has good mechanical properties and can break through the cuticle barrier of the skin; on the basis of the above-mentioned physical properties, the needle tip still retains the cell activity of the bioactive polypeptide; in addition, the selected polypeptide type of the application can also inhibit the expression of fibrosis related proteins of cells, and thus an unexpected technical effect is achieved.

Description

A double cross-linked hydrogel microneedle patch, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a double cross-linked hydrogel microneedle patch, its preparation method, and its application. Background Technology

[0002] Hypertrophic scars are the marks left by wound healing and the ultimate result of excessive tissue proliferation. They are caused by inflammation, activation of myofibroblasts, and excessive collagen fiber proliferation, and can persist in the body for months to years. Current traditional treatments are limited to surgical excision, laser therapy, and subcutaneous drug injections, which usually require postoperative care and are prone to pain and drug side effects, making them insufficient to meet current needs. Therefore, designing drug delivery platforms that target the pathological microenvironment of scars to achieve sustained drug release has become a research hotspot.

[0003] In the treatment of hypertrophic scars, painless drug delivery is a significant trend in recent years. Microneedling is a novel physical penetration enhancement technology, consisting of multiple micron-sized needles connected in an array on a base. The needles are typically 10–2000 μm high and 10–50 μm wide. The length, size, and shape of the microneedles can be individually designed according to treatment needs. Microneedles can penetrate the stratum corneum barrier, creating micron-sized mechanical channels that deliver medication directly to the epidermis or upper dermis, participating in subcutaneous treatment without passing through the stratum corneum to exert a pharmacological response and achieve painless drug delivery for hypertrophic scars.

[0004] Compared to metal microneedles, hydrogel microneedles have higher drug loading capacity, better biocompatibility, and leave no polymer residue after use, showing great promise for applications. However, current hydrogel microneedles generally struggle to overcome the mechanical strength required to overcome the skin barrier of scars, typically necessitating chemical or physical cross-linking to enhance their mechanical properties. Therefore, there is an urgent need to develop hydrogel microneedles with better mechanical strength, easier fabrication, and superior drug release performance for scar treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a double cross-linked hydrogel microneedle patch that solves the problems of poor mechanical strength and drug sustained-release performance, as well as the complexity of the preparation process.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: a double-crosslinked hydrogel microneedle patch, comprising a base layer and a needle tip, wherein the needle tip is made of reactive oxygen species (ROS) responsive peptide prodrug, ester bond-functionalized small molecule prodrug, peptide diacetylene, and double bond-functionalized macromolecule, and the ROS responsive peptide prodrug, ester bond-functionalized small molecule prodrug, and peptide diacetylene are loaded onto the double bond-functionalized macromolecule through coupling covalent interaction; the ROS responsive peptide prodrug and ester bond-functionalized small molecule prodrug slowly release drug molecules in response to the ROS microenvironment and ester bond hydrolysis, respectively.

[0007] Preferably, the reactive oxygen species responsive peptide prodrug and peptide diacetylene are modified with cysteine ​​and form a cross-linked network through double bond and thiol click chemistry; the peptide diacetylene further undergoes topological polymerization to form a second cross-linked network.

[0008] Preferably, the reactive oxygen species (ROS) responsive peptide prodrug has anti-fibrotic function; the ester bond-functionalized small molecule prodrug has anti-inflammatory function; the anti-fibrotic active ingredient in the ROS responsive peptide prodrug is selected from 5-fluorouracil, pirfenidone, nintedanib, pembrolizumab, ramitinol, stiotron, teriprost, deluximate, vemurafenib, selmetinib, imatinib, gefitinib, erlotinib, sorafenib, dasatinib, afatinib, budesonide, and colchicine. The active ingredient in the ester-functionalized small molecule prodrug is selected from one or more of the following: alkali, apatinib, sunitinib, and vesmogi; the active ingredient in the prodrug is selected from one or more of the following: triamcinolone acetonide, prednisone, methylprednisolone, hydrocortisone, dexamethasone, ibuprofen, naproxen, meloxicam, celecoxib, diclofenac, indomethacin, adalimumab, etanercept, infliximab, tocilizumab, sulfasalazine, azathioprine, cyclosporine, tacrolimus, methotrexate, and leflunomide.

[0009] Preferably, the anti-inflammatory active ingredient is modified with an anhydride to change the functional group from hydroxyl to carboxyl, and the ester bond functionalized small molecule prodrug is loaded onto the double bond functionalized macromolecule through an amidation reaction.

[0010] Preferably, the acid anhydride is one of succinic anhydride, glutaric anhydride, phthalic anhydride, maleic anhydride, succinic anhydride, pyridine-2,3-dicarboxylic anhydride, thiophene-2,3-dicarboxylic anhydride, and benzophenone tetracarboxylic dianhydride.

[0011] Preferably, the ester-functionalized small molecule prodrug is obtained by the following method:

[0012] The anti-inflammatory active ingredient, acid anhydride, and catalyst are dissolved together in an organic solvent and stirred at 10℃-50℃ for 6-24 h. After the reaction is complete, more than twice the volume of a highly polar reagent is added to precipitate the product, thus obtaining an ester-functionalized small molecule prodrug.

[0013] Preferably, the catalyst comprises at least one of 4-dimethylaminopyridine, triethylamine, dibutyltin dilaurate, stannous octoate, p-toluenesulfonic acid, and trifluoromethanesulfonic acid; the organic solvent comprises at least one of pyridine, acetone, tetrahydrofuran, dichloromethane, toluene, chloroform, ethyl acetate, ethers, or halogenated hydrocarbons; and the highly polar reagent comprises at least one of water, methanol, ethanol, acetonitrile, N,N-dimethylformamide, formic acid, acetic acid, and ethylene glycol.

[0014] Preferably, the polypeptide diacetylene comprises two bioactive peptides, which have anti-fibrotic and assembly functions, respectively. Preferably, the anti-fibrotic bioactive peptide is selected from one or more of the following: targeting the TGF-β / Smad pathway, regulating ECM degradation, mechanotransduction, and regulating metabolism. Preferably, it includes at least one of CXCL9(74-103), KP1, C53, M10, B7-33, E4, AF38Pep, ADP355, Thymosin β4 (Tβ4), SDKP, THR-184, and XFB-19. The assembly bioactive peptide is selected from at least one of AAVV, EAK16, RADA16, FF, LL, WG, and G4DD. The assembly morphology of the polypeptide diacetylene is one of nanoparticles, nanofibers, and nanoribbons.

[0015] Preferably, the diacetylene in the polypeptide diacetylene is derived from diacetic acid compounds, specifically selected from at least one of butynedioic acid, 2,4-hexadiyne-1,6-dicarboxylic acid, 3,5-heptadiyne-1,7-dicarboxylic acid, and 4,6-nonadiyne-1,9-dicarboxylic acid.

[0016] Preferably, the double-bond functionalized macromolecule is selected from at least one of the following: methacrylamide gelatin, methacrylamide collagen, allylated sodium alginate, methacrylamide fucoidan, acrylamide konjac glucomannan, methacrylamide xylo-glucan, allylamide inulin, methacrylamide konjac mannan, acrylamide elastin, methacrylamide fibrinogen, allylamide sericin, methacrylamide keratin, acrylamide lactoferrin, methacrylamide transferrin, methacrylamide phytosterol, allylamide ceramide, and methacrylamide resveratrol.

[0017] Another technical solution of the present invention is achieved as follows: the preparation method of the above-mentioned double cross-linked hydrogel microneedle patch specifically includes the following steps:

[0018] S1. After the polysiloxane compound and the curing agent are mixed evenly, the mixture is poured onto the microneedle template. After degassing, drying and curing, the mixture is demolded to obtain the microneedle array mold.

[0019] S2. Dissolve the raw materials, including reactive oxygen species responsive peptide prodrug, ester bond functionalized small molecule prodrug, peptide diacetylene and double bond functionalized macromolecule and initiator, in deionized water or buffer solution to prepare microneedle prepolymer solution. Pour the prepolymer solution into the microneedle array mold, remove air bubbles and then cure with ultraviolet light to obtain a microneedle array mold containing shaped needles.

[0020] S3. Dissolve the base material in deionized water or buffer solution to prepare a base solution. Pour the solution onto the microneedle array mold containing the shaped needles obtained in step (2). After removing air bubbles and drying and curing at room temperature in the dark, demold the microneedle patch to obtain the peptide drug-loaded microneedle patch.

[0021] Preferably, the double-bond functionalized macromolecule has a molecular weight of 10kDa-300kDa and a content of 5%-40%, the initiator content is 2%-10%, and the amounts of the reactive oxygen species responsive peptide prodrug, ester bond functionalized small molecule prodrug, and peptide diacetylene are 0.5%-10%. The initiator is preferably selected from at least one of 1-hydroxy-cyclohexylphenyl ketone, 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholino-1-propanone, and 2,4-diethylthioxanthrone.

[0022] The third technical solution of the present invention is achieved as follows: the application of the above-mentioned double cross-linked hydrogel microneedle patch or the double cross-linked hydrogel microneedle patch obtained by the above-mentioned preparation method in transdermal treatment of skin diseases.

[0023] Compared with existing technologies, this invention achieves a hydrogel microneedle patch with a double crosslinked network by click chemical crosslinking of a bioactive macromolecule and a polypeptide diacetylene (the polypeptide diacetylene contains cysteine ​​residues at both ends, and the bioactive macromolecule contains an unsaturated methacryloyl group), followed by topological polymerization of the polypeptide diacetylene itself. The tip of this microneedle patch has excellent mechanical properties and can penetrate the skin's stratum corneum barrier. In addition to the aforementioned physical properties, the tip retains the cellular activity of the bioactive polypeptide. By using the preferred polypeptide type of this invention, it can also inhibit the expression of fibrosis-related proteins in cells, achieving unexpected technical effects. Attached Figure Description

[0024] Figure 1 is a schematic diagram of the structure of the doubly cross-linked responsive acetylene-hydrogel microneedle patch obtained in an embodiment of the present invention;

[0025] Figure 2 is a schematic diagram of the chemical structure of the polypeptide diacetylene in an embodiment of the present invention;

[0026] Figure 3 is a schematic diagram of the chemical structure of the reactive oxygen species responsive peptide prodrug in an embodiment of the present invention;

[0027] Figure 4 is an optical image of the doubly cross-linked responsive acetylene-hydrogel microneedle patch obtained in an embodiment of the present invention;

[0028] Figure 5 shows the mechanical properties of the doubly cross-linked responsive acetylene-hydrogel microneedle patch obtained in the embodiment of the present invention.

[0029] Figure 6 shows the transdermal depth of the doubly cross-linked responsive acetylene-hydrogel microneedle patch obtained in the embodiment of the present invention;

[0030] Figure 7 shows the therapeutic effect of the doubly cross-linked responsive acetylene-hydrogel microneedle patch obtained in the embodiment of the present invention on a hypertrophic scar disease model. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0032] This invention provides a double-crosslinked hydrogel microneedle patch, comprising a base layer and a needle tip. The needle tip is made of reactive oxygen species (ROS) responsive peptide prodrugs, ester-functionalized small molecule prodrugs, peptide diacetylene, and double-bond functionalized macromolecules. The ROS responsive peptide prodrugs, ester-functionalized small molecule prodrugs, and peptide diacetylene are covalently loaded onto the double-bond functionalized macromolecules. The ROS responsive peptide prodrugs and ester-functionalized small molecule prodrugs slowly release drug molecules in response to the ROS microenvironment and ester bond hydrolysis, respectively.

[0033] In the specific implementation process, the reactive oxygen species responsive peptide prodrug and peptide diacetylene are modified with cysteine ​​and form a cross-linked network through double bond and thiol click chemical reaction; the peptide diacetylene further undergoes topological chemical polymerization to form a second cross-linked network.

[0034] In the specific implementation process, the reactive oxygen species responsive peptide prodrug has anti-fibrotic function; the ester bond-functionalized small molecule prodrug has anti-inflammatory function; the anti-fibrotic active ingredient in the reactive oxygen species responsive peptide prodrug is selected from 5-fluorouracil, pirfenidone, nintedanib, pembrolizumab, ramitinol, stiotiro, teriprost, deluximate, vemurafenib, selmetinib, imatinib, gefitinib, erlotinib, sorafenib, dasatinib, afatinib, budesonide, etc. Colchicine, apatinib, sunitinib, and vesmogi are selected from one or more of these drugs; the anti-inflammatory active ingredient in the ester-functionalized small molecule prodrug is selected from one or more of the following drugs: triamcinolone, prednisone, methylprednisolone, hydrocortisone, dexamethasone, ibuprofen, naproxen, meloxicam, celecoxib, diclofenac, indomethacin, adalimumab, etanercept, infliximab, tocilizumab, sulfasalazine, azathioprine, cyclosporine, tacrolimus, methotrexate, and leflunomide.

[0035] In the specific implementation process, the anti-inflammatory active drug is modified by acid anhydride to change the functional group from hydroxyl to carboxyl, and the ester bond functionalized small molecule prodrug is loaded onto the double bond functionalized macromolecule through an amidation reaction.

[0036] In the specific implementation process, the acid anhydride is one of succinic anhydride, glutaric anhydride, phthalic anhydride, maleic anhydride, succinic anhydride, pyridine-2,3-dicarboxylic anhydride, thiophene-2,3-dicarboxylic anhydride, and benzophenone tetracarboxylic dianhydride.

[0037] In the specific implementation process, the ester bond-functionalized small molecule prodrug is obtained through the following method:

[0038] The anti-inflammatory active ingredient, acid anhydride, and catalyst are dissolved together in an organic solvent and stirred at 10℃-50℃ for 6-24 h. After the reaction is complete, more than twice the volume of a highly polar reagent is added to precipitate the product, thus obtaining an ester-functionalized small molecule prodrug.

[0039] In the specific implementation process, the catalyst includes at least one of 4-dimethylaminopyridine, triethylamine, dibutyltin dilaurate, stannous octoate, p-toluenesulfonic acid, and trifluoromethanesulfonic acid; the organic solvent includes at least one of pyridine, acetone, tetrahydrofuran, dichloromethane, toluene, chloroform, ethyl acetate, ethers, or halogenated hydrocarbons; and the highly polar reagent includes at least one of water, methanol, ethanol, acetonitrile, N,N-dimethylformamide, formic acid, acetic acid, and ethylene glycol.

[0040] In the specific implementation process, the polypeptide diacetylene contains two bioactive peptides, which have anti-fibrotic and assembly functions, respectively. Preferably, the anti-fibrotic bioactive peptide is selected from one or more of the following: targeting the TGF-β / Smad pathway, regulating ECM degradation, mechanotransduction, and regulating metabolism. Preferably, it includes at least one of CXCL9(74-103), KP1, C53, M10, B7-33, E4, AF38Pep, ADP355, Thymosin β4 (Tβ4), SDKP, THR-184, and XFB-19. The assembly bioactive peptide is selected from at least one of AAVV, EAK16, RADA16, FF, LL, WG, and G4DD. The assembly form of the polypeptide diacetylene is one of nanoparticles, nanofibers, and nanoribbons.

[0041] In the specific implementation process, the diacetylene in the polypeptide diacetylene is derived from diacetic acid compounds, specifically selected from at least one of butynedioic acid, 2,4-hexadiyne-1,6-dicarboxylic acid, 3,5-heptadiyne-1,7-dicarboxylic acid, and 4,6-nonadiyne-1,9-dicarboxylic acid.

[0042] In the specific implementation process, the double-bond functionalized macromolecules are selected from at least one of the following: methacrylamide gelatin, methacrylamide collagen, allylated sodium alginate, methacrylamide fucoidan, acrylamide konjac glucomannan, methacrylamide xylo-glucan, allylamide inulin, methacrylamide konjac mannan, acrylamide elastin, methacrylamide fibrinogen, allylamide sericin, methacrylamide keratin, acrylamide lactoferrin, methacrylamide transferrin, methacrylamide phytosterol, allylamide ceramide, and methacrylamide resveratrol.

[0043] This invention also provides a method for preparing the above-mentioned double crosslinked hydrogel microneedle patch, which specifically includes the following steps:

[0044] S1. After the polysiloxane compound and the curing agent are mixed evenly, the mixture is poured onto the microneedle template. After degassing, drying and curing, the mixture is demolded to obtain the microneedle array mold.

[0045] S2. Dissolve the raw materials, including reactive oxygen species responsive peptide prodrug, ester bond functionalized small molecule prodrug, peptide diacetylene and double bond functionalized macromolecule and initiator, in deionized water or buffer solution to prepare microneedle prepolymer solution. Pour the prepolymer solution into the microneedle array mold, remove air bubbles and then cure with ultraviolet light to obtain a microneedle array mold containing shaped needles.

[0046] S3. Dissolve the base material in deionized water or buffer solution to prepare a base solution. Pour the solution onto the microneedle array mold containing the shaped needles obtained in step (2). After removing air bubbles and drying and curing at room temperature in the dark, demold the microneedle patch to obtain the peptide drug-loaded microneedle patch.

[0047] In the specific implementation process, the molecular weight of the double bond modified macromolecule is 10kDa-300kDa, and the content is 5%-40%. The content of the initiator is 2%-10%. The amount of the reactive oxygen species responsive peptide prodrug, ester bond functionalized small molecule prodrug, and peptide diacetylene is 0.5%-10%. The initiator is preferably selected from at least one of 1-hydroxy-cyclohexylphenyl ketone, 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2,4,6-trimethylbenzoyl-diphenylphosphine oxide, bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide, 2-methyl-1-[4-(methylthio)phenyl]-2-morpholino-1-propanone, and 2,4-diethylthioxanthrone.

[0048] In the specific implementation process, the polypeptide diacetylene is obtained through the following steps:

[0049] Step 1: Weigh 1.00 g of resin into the peptide synthesis apparatus, add analytical grade N,N-dimethylformamide to swell the resin for 2 h, then purge the solvent under argon pressure. Next, deprotect the resin with 10 ml of N,N-dimethylformamide solution containing 20% ​​piperidine, reacting twice, 20 min each time. After the reaction, wash the resin three times with N,N-dimethylformamide. Then, place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin color changes from white to purple. If so, the resin deprotection is successful and the amino acid coupling reaction can proceed. Otherwise, the deprotection reaction needs to be continued.

[0050] Step 2: Weigh 1.61 g Fmoc-Cys(Trt)-OH, 0.37 g 1-hydroxybenzotriazole (HOBT), and 1.04 g benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), dissolve them in 10 mL N,N-dimethylformamide, and then transfer them to the polypeptide synthesis apparatus containing the treated resin from step (1). Add 960 μL diisopropylethylamine (DIPEA) to the system and react at room temperature for 4 h. Then wash the resin three times with 10 mL N,N-dimethylformamide. Take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat it to boiling, and observe whether the resin changes color. If so, the first amino acid has been successfully coupled to the resin. Otherwise, the coupling reaction needs to be continued.

[0051] Step 3: Deprotect the first amino acid on the resin with 10 mL of N,N-dimethylformamide solution containing 20% ​​4-methylpiperidine. Repeat the reaction twice, each time for 20 minutes. After the reaction, wash the resin three times with N,N-dimethylformamide. Then, take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe the resin color change from white to purple. This indicates successful deprotection of the first amino acid, and subsequent amino acid coupling reactions can proceed. Otherwise, the deprotection reaction needs to be continued.

[0052] Step 4: Referring to Steps 2 and 3, sequentially condense Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, and DCDA.

[0053] Step 5: Wash the resin three times each with 10 mL of dichloromethane, methanol, and dichloromethane in sequence, and then dry the resin with a vacuum oil pump for 1 hour.

[0054] Step 6: Add 9.5 mL of trifluoroacetic acid, 0.25 mL of triisopropylsilane, and 0.25 mL of deionized water to the peptide reaction apparatus. React at room temperature for 3 h to separate the peptide from the resin. Slowly add the cutting solution dropwise to ice-cold isopropyl ether, and a white precipitate will appear. Then centrifuge at 3000 rpm for 10 min to separate the crude product, discard the supernatant, dissolve the precipitate in an appropriate amount of methanol, purify the product using a medium-pressure purification chromatography system, and finally freeze-dry to collect the white powder. The amino acid sequence of the obtained peptide diacetylene is Cys-Pro-Lys-Asp-Ser-Val-Val-Ala-Ala-DCDA-Ala-Ala-Val-Val-Ser-Asp-Lys-Pro-Cys, and its chemical structure is shown in Figure 2.

[0055] In the specific implementation process, the reactive oxygen species responsive peptide prodrug is obtained through the following steps:

[0056] Step 1: Following the synthesis steps of polypeptide diacetylene, sequentially condense Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Phe-OH, Fmoc-

[0057] Lys(Alloc)-OH, Fmoc-Phe-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Phe-OH, Fmoc-

[0058] Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Trt)-OH.

[0059] Step 2: Dissolve 130 μL of acetic anhydride in 10 mL of N,N-dimethylformamide, then transfer it to the peptide synthesis apparatus containing the resin. Add 960 μL of diisopropylethylamine (DIPEA) to the system and react at room temperature for 45 min. Wash the resin twice with 10 mL of DMF, and react again for 45 min using the same ratio to ensure complete end-capping. Finally, wash the resin three times with 10 mL of DMF. Place a small amount of resin in an ethanol solution of ninhydrin and phenol, heat to boiling, and observe if the resin does not change color; otherwise, the reaction needs to be repeated.

[0060] Step 3: Weigh 0.16 g of tetrakis(triphenylphosphine)palladium, dissolve it in 10 mL of dichloromethane (DCM), and then transfer the solution to the peptide synthesis apparatus containing the treated resin from Step 2. Add 4.06 mL of phenylsilane to the system to remove the protecting group of the lysine side chain. Repeat the reaction twice, 30 min each time. After the reaction, wash the resin three times with DMF. Then, take a small amount of resin and place it in an ethanol solution of ninhydrin and phenol, heat it to boiling, and observe whether the resin color changes from white to purple. This indicates that the lysine side chain has been successfully deprotected, and the subsequent amino acid coupling reaction can proceed. Otherwise, the deprotection reaction needs to be continued.

[0061] Step 4: Following the synthesis steps of polypeptide diacetylene, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH are sequentially condensed on the lysine side chain to functionalize 5-fluorouracil (5-FUA) with a carboxyl group.

[0062] Step 5, the cutting and purification steps are the same as those for the synthesis of polypeptide diacetylene. The amino acid sequence of the obtained reactive oxygen species responsive polypeptide prodrug is Ac-Cys-Lys-Lys-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys-Lys, and its chemical structure is shown in Figure 3.

[0063] In the specific implementation process, the ester bond-functionalized small molecule prodrug is obtained through the following steps:

[0064] 503.29 mg of triamcinolone acetonide, 342.43 mg of succinic anhydride, and 14.80 mg of dimethylaminopyridine were weighed and dissolved in 15 mL of pyridine. The mixture was stirred at room temperature for 24 h, and the reaction progress was monitored by thin-layer chromatography. After the reaction was complete, most of the solvent was removed by rotary evaporation. Deionized water was added to precipitate the product, which was stirred for 20 min, centrifuged at 3000 rpm for 10 min, filtered, and the filtered solid was dried at room temperature. The white powder obtained was ester-functionalized triamcinolone acetonide.

[0065] In the specific implementation process, the double-bond functionalized macromolecular gelatin is obtained through the following steps:

[0066] Weigh 5 g of pigskin gelatin and dissolve it in 50 mL of deionized water. Slowly add 1 mL of methacrylic anhydride to the above system and stir the reaction at 40 °C for 2 h. Transfer the mixture to a dialysis bag (8~14 kDa) and dialyze it with deionized water for two days, changing the water every 12 h to remove excess methacrylic anhydride. After dialysis, freeze dry the mixture to obtain a white, fluffy solid, i.e., double-bond functionalized gelatin.

[0067] In the specific implementation process, gelatin grafted with ester bonds to functionalize small molecule prodrugs is obtained through the following steps:

[0068] Weigh 76.0 mg of the ester-functionalized small molecule prodrug prepared above and dissolve it in 1 mL of dimethyl sulfoxide. Weigh 54.3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 61.7 mg of N-hydroxysuccinimide sodium sulfonate (Sulfo-NHS) and dissolve them in 1 mL of deionized water. Mix the two liquids and activate for 30 minutes. Weigh 1 g of the double-bond functionalized gelatin prepared in step 5 and dissolve it in 5 mL of deionized water. Mix the solution with the activated solution and add dimethyl sulfoxide to a final volume of V. DMSO V H2O = 3:1, stirred at 45℃ for 7 h, after which the reaction was transferred to a dialysis bag (8~14 kDa), and dialyzed with deionized water for one day, with the water changed every 12 h to remove residual ester bond functionalized small molecule prodrug. After dialysis, the gelatin was obtained by freeze drying in a freeze dryer as a white, fluffy solid, which is the gelatin grafted with ester bond functionalized small molecule prodrug.

[0069] The following is a specific embodiment 1

[0070] The doubly cross-linked responsive acetylene-hydrogel microneedle patch provided in Example 1 is obtained through the following steps:

[0071] S1, Polydimethylsiloxane (PDMS) and curing agent (Sylgard) 184Mix the prepolymer at a ratio of 10:1 until homogeneous, then pour the mixture onto the surface of the pyramid-shaped microneedle array positive mold. Degas the mold at room temperature under a vacuum of -0.08 MPa for 1 h using a vacuum degassing device, then heat and cure at 80℃ for 5 h. After cooling to room temperature, demold to obtain a polydimethylsiloxane microneedle array negative mold with a needle length of 650 μm, a needle spacing of 500 μm, and a needle quantity of 10×10 needles / patch.

[0072] S2. Weigh 5.0 mg of the obtained polypeptide diacetylene, 20.7 mg of the obtained reactive oxygen species-responsive polypeptide prodrug, 100 mg of the obtained grafted ester bond functionalized small molecule prodrug gelatin, and 2.5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (IRGACURE 2959) and dissolve them in 0.5 mL of PBS (pH=7.2 ~ 7.4) until fully dissolved to obtain a microneedle prepolymer solution. Pour the microneedle prepolymer solution into the polydimethylsiloxane microneedle array negative mold obtained in S1, and then degas it three times at 40℃ and -0.08 MPa vacuum using a vacuum degassing device for 15 min each time to help the prepolymer solution tightly fill the microneedle array negative mold. After removing excess prepolymer solution, the microneedle array negative mold tightly filled with prepolymer solution was irradiated with a UV light source with a wavelength of 365 nm and an optical power of 10 W for 1 min. Then, the microneedle array negative mold tightly filled with prepolymer solution was irradiated with a UV light source with a wavelength of 254 nm and an optical power of 8 W for 1 min to obtain a doubly cross-linked responsive acetylene-hydrogel microneedle array.

[0073] S3. Weigh 1 g of gelatin and dissolve it in 2.5 mL of deionized water to prepare a microneedle substrate solution with a solid content of 40%. Pour the microneedle substrate solution into the polydimethylsiloxane microneedle array negative mold containing a doubly crosslinked responsive diacetylene-hydrogel microneedle array obtained in S2. Then remove excess substrate solution and dry at room temperature in the dark for 24 min. Finally, peel off the polydimethylsiloxane microneedle array negative mold to obtain the doubly crosslinked responsive diacetylene-hydrogel microneedle array patch.

[0074] The obtained doubly cross-linked responsive acetylene-hydrogel microneedle array patch was observed under a dermoscope, as shown in Figure 4. The microneedles were arranged in a regular, uniform shape, and had sharp tips, forming a square pyramid shape. The results indicate that the microneedle patch was successfully prepared.

[0075] The following is a specific embodiment 2.

[0076] The difference between Example 2 and Example 1 lies in the concentration of the polypeptide diacetylene in the microneedle prepolymer solution. Specifically, 2.5 mg of the polypeptide diacetylene described in Example 1, 20.7 mg of the reactive oxygen species-responsive polypeptide prodrug, 100 mg of the gelatin of the grafted ester-functionalized small molecule prodrug, and 2.5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (IRGACURE 2959) were weighed and dissolved in 0.5 mL of PBS (pH = 7.2 ~ 7.4) to obtain the microneedle prepolymer solution.

[0077] The following is a specific embodiment 3

[0078] The difference between Example 3 and Example 1 lies in the concentration of the polypeptide diacetylene in the microneedle prepolymer solution. Specifically, 20.7 mg of the reactive oxygen species-responsive polypeptide prodrug described in Example 1, 100 mg of the gelatin containing the grafted ester bond functionalized small molecule prodrug, and 2.5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (IRGACURE2959) were weighed and dissolved in 0.5 mL of PBS (pH = 7.2 ~ 7.4) to obtain the microneedle prepolymer solution.

[0079] The following is a specific embodiment 4.

[0080] The difference between Example 4 and Example 1 lies in the prodrug component in the microneedle prepolymer solution. Specifically, 5.0 mg of the polypeptide diacetylene described in Example 1, 20.7 mg of the reactive oxygen species responsive polypeptide prodrug, 100 mg of the double-bond functionalized gelatin, and 2.5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (IRGACURE2959) were weighed and dissolved in 0.5 mL of PBS (pH = 7.2 ~ 7.4) to obtain the microneedle prepolymer solution.

[0081] The following is a specific embodiment 5.

[0082] The difference between Example 5 and Example 1 lies in the prodrug component in the microneedle prepolymer solution. Specifically, 5.0 mg of the polypeptide diacetylene described in Example 1, 100 mg of the gelatin containing the grafted ester functionalized small molecule prodrug, and 2.5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (IRGACURE 2959) were weighed and dissolved in 0.5 mL of PBS (pH = 7.2 ~ 7.4) to obtain the microneedle prepolymer solution.

[0083] The following are application test cases.

[0084] 1. Mechanical strength testing of microneedle patches used for hypertrophic scar treatment:

[0085] The mechanical properties of the microneedle patches in Examples 1-3 were tested using an in-situ tensile testing machine. In short, the microneedle patch was placed upwards on a stainless steel plate and compressed at a rate of 1 mm / min, with a maximum loading force of 250 N. The changes in force as displacement changed were recorded.

[0086] 2. Transdermal depth testing of microneedle patches for treating hypertrophic scars:

[0087] In Example 1, 1 mg / mL of Rhodamine B was added to the microneedle prepolymer solution to induce fluorescence. The subsequent microneedle patch preparation method was the same as in Example 1. Fresh pigskin was cleaned of surface oil, and the Rhodamine B-loaded microneedle patch was vertically pressed into the fresh pigskin for 5 minutes. The microneedle patch was carefully removed, and the pigskin was placed under a confocal laser scanning microscope. After determining the xy-plane with the highest and lowest fluorescence intensities using the confocal laser scanning microscope, fluorescence images were obtained from the xy-plane and continuously scanned along the z-axis at 20 μm intervals to determine the transdermal depth of the microneedle patch.

[0088] 3. Evaluation of the therapeutic effect of doubly cross-linked responsive acetylene-hydrogel microneedle patches on a hypertrophic scar disease model:

[0089] The microneedle patches obtained in Examples 1 and 4-5 were pressed onto the proliferative scar lesions on the ventral side of rabbit ears for 5 minutes. The base was then removed, leaving the needles embedded in the skin to slowly release the medication. Treatment was administered once a week for 4 consecutive weeks from the start of the experiment. The treatment effects were observed and recorded, and the differences between different microneedle patch treatment groups were compared.

[0090] Figure 5 shows the mechanical properties of the doubly cross-linked responsive diacetylene-hydrogel microneedle patch obtained in Examples 1-3 of the present invention. As can be seen from Figure 5, as the concentration of polypeptide diacetylene in the microneedle prepolymer solution increases, the breaking strength of the microneedle increases from 106 N to 127 N. At the same time, when the same compression ratio is reached, the microneedle containing a high concentration of polypeptide diacetylene can withstand greater pressure.

[0091] Figure 6 shows the transdermal depth of the doubly cross-linked responsive diacetylene-hydrogel microneedle patch obtained in Example 1 of the present invention. As can be seen from Figure 6, the fluorescence signal of Rhodamine B completely disappears at 300 μm below the skin, indicating that the microneedle patch can deliver the drug to the deep lesion and has strong penetration ability.

[0092] Figure 7 shows the therapeutic effects of the doubly cross-linked responsive acetylene-hydrogel microneedle patches obtained in Examples 1 and 4-5 of this invention on a hypertrophic scar disease model. As can be seen from Figure 7, the microneedle patch treatment group in Example 1 showed significantly better therapeutic effects than the microneedle patch treatment groups in Examples 4-5, and also better than the drug injection treatment group. Specifically, the microneedle patch treatment group in Example 1 exhibited a smoother scar tissue surface, reduced redness and swelling, and less distinct scar boundaries, indicating that the combined treatment of the two prodrugs was more effective. Simultaneously, the animals did not experience significant pain during drug administration compared to the injection group, demonstrating that this invention is a safe, effective, and humane drug delivery technology. This invention can responsively release drugs at the lesion site, reducing the frequency of drug administration and drug toxicity, improving drug bioavailability, and achieving long-term therapeutic effects, thus possessing broad prospects for clinical translation.

[0093] In summary, 1) This invention obtains a hydrogel microneedle patch with a double crosslinked network by click chemical crosslinking of a bioactive macromolecule and a polypeptide diacetylene (the polypeptide diacetylene contains cysteine ​​residues at both ends, and the bioactive macromolecule contains unsaturated methacryloyl groups), and then by topological polymerization of the polypeptide diacetylene itself. The needle tip of this microneedle patch has good mechanical properties and can penetrate the skin's keratin barrier. In addition to the above-mentioned physical properties, the needle tip still retains the cellular activity of the bioactive polypeptide. By using the preferred polypeptide type of this invention, it can also inhibit the expression of fibrosis-related cells. 1) The protein yielded unexpected technical results; 2) The reactive oxygen species-responsive peptide prodrug and ester bond-functionalized small molecule prodrug in this invention release drug molecules through response to the reactive oxygen species microenvironment and ester bond hydrolysis, respectively. This not only reduces the toxic side effects of the drug itself through sustained release but also simultaneously inhibits excessive fibroblast proliferation and has anti-inflammatory effects, accelerating the remodeling of the pathological microenvironment of proliferative scars; 3) The loaded drugs used in this invention can be widely selected to meet the needs of different users; 4) The bioactive peptides used in this invention are all synthesized from natural amino acids in a solid phase, which is simple to synthesize, low in cost, and easy to modify. Furthermore, the peptides have high bioactivity, few systemic side effects, and are easily degraded and metabolized in vivo.

[0094] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A double cross-linked hydrogel microneedle patch, characterized in that, The needle comprises a base layer and a tip, wherein the tip is made of a reactive oxygen species (ROS) responsive peptide prodrug, an ester bond-functionalized small molecule prodrug, a peptide diacetylene, and double bond-functionalized gelatin; the amino acid sequence of the ROS responsive peptide prodrug is Ac-Cys-Lys-Lys-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys(-Pro-Pro-Pro-5-FUA)-Phe-Lys(-Pro-Pro-Pro-5-FUA)-Phe -Lys-Lys; The ester-functionalized small molecule prodrug is obtained by the following method: 503.29 mg of triamcinolone acetonide, 342.43 mg of succinic anhydride, and 14.80 mg of dimethylaminopyridine are weighed and dissolved in 15 mL of pyridine. The mixture is stirred at room temperature for 24 h, and the reaction progress is monitored by thin-layer chromatography. After the reaction is complete, most of the solvent is removed by rotary evaporation, deionized water is added to precipitate the product, the mixture is stirred for 20 min, centrifuged at 3000 rpm for 10 min, filtered, and the filtered solid is dried at room temperature. The white powder obtained is the ester-functionalized triamcinolone acetonide. The amino acid sequence of the polypeptide diacetylene is Cys-Pro-Lys- Asp-Ser-Val-Val-Ala-Ala-DCDA-Ala-Ala-Val-Val-Ser-Asp-Lys-Pro-Cys; The double-bond functionalized gelatin is obtained by the following method: 5g of pigskin gelatin is dissolved in 50mL of deionized water, and 1mL of methacrylic anhydride is slowly added dropwise to the above system. The mixture is stirred and reacted at 40°C for 2 hours. The mixture is then transferred to a dialysis bag and dialyzed with deionized water for two days, changing the water every 12 hours to remove excess methacrylic anhydride. After dialysis, the mixture is freeze-dried to obtain a white, fluffy solid, which is the double-bond functionalized gelatin. The double-crosslinked hydrogel microneedle patch is obtained by the following steps: S1, Polydimethylsiloxane and curing agent are mixed evenly at a ratio of 10:1, and then the mixed prepolymer is poured into a pyramid-shaped microneedle array. The surface of the positive mold is degassed for 1 hour at -0.08 MPa vacuum using a vacuum degassing device at room temperature, then cured at 80°C for 5 hours. After cooling to room temperature, demolding yields a polydimethylsiloxane microneedle array negative mold with a needle length of 650 μm, a needle spacing of 500 μm, and a needle quantity of 10 × 10 needles / patch. S2: Weigh 2.5 mg or 5.0 mg of the aforementioned polypeptide diacetylene, 20.7 mg of the aforementioned reactive oxygen species responsive polypeptide prodrug, 100 mg of gelatin grafted with ester-functionalized small molecule prodrug, and 2.5 mg of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone and dissolve them in 0.5 mL of PBS to obtain a microneedle prepolymer solution. Pour the microneedle prepolymer solution onto the polydimethylsiloxane microneedle array negative mold obtained in S1, and then heat at 40°C and -0.08 MPa for 1 hour.The microneedle array was degassed three times under a vacuum of 0.8 MPa for 15 minutes each time, with the prepolymer solution tightly filling the microneedle array negative mold. After removing excess prepolymer solution, the microneedle array negative mold tightly filled with prepolymer solution was irradiated with a UV light source with a wavelength of 365 nm and a light power of 10 W for 1 minute. Then, the microneedle array negative mold tightly filled with prepolymer solution was irradiated with a UV light source with a wavelength of 254 nm and a light power of 8 W for 1 minute. This yielded polydimethylsiloxane microneedles containing a doubly cross-linked responsive diathyne-hydrogel microneedle array. An array of negative molds; wherein, the gelatin grafted with ester-functionalized small molecule prodrug is obtained through the following steps: 76.0 mg of the ester-functionalized small molecule prodrug is weighed and dissolved in 1 mL of dimethyl sulfoxide; 54.3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 61.7 mg of N-hydroxysuccinimide sodium sulfonate are weighed and dissolved in 1 mL of deionized water; the two liquids are mixed and activated for 30 minutes; 1 g of the double-bond functionalized gelatin is weighed and dissolved in 5 mL of deionized water, mixed with the activated solution, and dimethyl sulfoxide is added to a final volume of V. DMSO V H2O The mixture was stirred at 45°C for 7 hours with a ratio of 3:

1. After the reaction, it was transferred to a dialysis bag and dialyzed with deionized water for one day, changing the water every 12 hours to remove residual ester-functionalized small molecule prodrugs. After dialysis, it was freeze-dried to obtain a white, fluffy solid, namely gelatin grafted with ester-functionalized small molecule prodrugs. S3: 1 g of gelatin was weighed and dissolved in 2.5 mL of deionized water to prepare a microneedle base solution with a solid content of 40%. The microneedle base solution was poured into the polydimethylsiloxane microneedle array negative mold containing a doubly cross-linked responsive diacetylene-hydrogel microneedle array obtained in S2. Then, the excess base solution was removed and dried at room temperature in the dark for 24 min. Finally, the polydimethylsiloxane microneedle array negative mold was peeled off to obtain a doubly cross-linked hydrogel microneedle patch.

2. The double cross-linked hydrogel microneedle patch according to claim 1, characterized in that, The reactive oxygen species responsive peptide prodrug and peptide diacetylene are modified with cysteine ​​and form a cross-linked network through double bond and thiol click chemistry; the peptide diacetylene further undergoes topological polymerization to form a second cross-linked network.

3. The double cross-linked hydrogel microneedle patch according to claim 2, characterized in that, The triamcinolone acetonide in the ester-functionalized small molecule prodrug is modified with an anhydride to change the functional group from hydroxyl to carboxyl, and the ester-functionalized small molecule prodrug is loaded onto the double-bond functionalized gelatin through an amidation reaction.

4. The use of the double cross-linked hydrogel microneedle patch according to any one of claims 1-3 in the preparation of a transdermal drug for the treatment of hypertrophic scars.

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