A bottle brush-like polypeptide-modified hyaluronic acid hydrogel, preparation and application thereof

Abstract

The application discloses a bottlebrush-shaped polypeptide modified hyaluronic acid hydrogel as well as a preparation method and application thereof, and belongs to the field of biomedical materials. The polypeptide modified hyaluronic acid hydrogel disclosed by the application not only has antibacterial effect on various bacteria causing acne, but also can relieve inflammation and repair skin. The soluble microneedle based on the bottlebrush-shaped polypeptide modified hyaluronic acid hydrogel can pierce the cuticle of the skin to form a microchannel and has excellent transdermal absorption effect, meanwhile, the hyaluronic acid can absorb tissue fluid of the skin to be dissolved, and then deliver active components to the subcutaneous tissue, so that the active components remaining in the subcutaneous tissue have good acne-removing and skin-protection effect on the skin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and more particularly relates to a hyaluronic acid-based hydrogel modified by bottlebrush-like polypeptide as well as a preparation method and application thereof. BACKGROUND

[0002] Acne vulgaris is a common inflammatory skin disease. Up to 80% of adolescents (even adults) are affected by acne, which can leave permanent scars and even affect the psychological development of adolescents. According to the latest classification, anaerobic bacteria Propionibacterium acnes (P. acnes) is now called Cutibacterium acnes (C. acnes) and is considered to play a major role in the pathogenesis of acne. Due to excessive secretion of skin lipids, the pores are blocked, the skin microenvironment is hypoxic, and thus the C. acnes proliferates in the skin microbiota, damaging normal tissues. C. acnes is also believed to promote the development of acne symptoms by inducing some inflammatory responses. Therefore, finding suitable drugs to inhibit acne or reduce inflammation caused by acne may be a direction for treating acne vulgaris.

[0003] In addition, although some drugs with good acne inhibition effect have appeared on the market, the preparation process of the above drugs is complex, and most of the use methods are through the use of traditional smearing methods, which have poor transdermal penetration, and long-term use by patients can cause irritation, redness and scarring or melanin deposition, and even cause skin allergies. As a new type of transdermal drug delivery means, microneedles can pierce the skin and form microchannels in the stratum corneum, allowing drugs and cosmetic active ingredients to reach the deep layer of the skin along the microchannels and diffuse into the dermis, combining the dual advantages of injection and traditional transdermal drug delivery, and can achieve rapid effect, painless and no trace.

[0004] Compared with metal microneedles, hydrogel microneedles have higher drug loading capacity, stronger biological properties, and no residual polymer after use, which have great application prospects. However, the general hydrogel molecules currently used in preparation almost use crosslinking agents, which to some extent aggravate environmental pollution, and the current hydrogel microneedles also have problems such as uncontrollable release of drugs in skin cells, poor targeting for different skin conditions, etc. Therefore, it is urgent to develop a hydrogel acne-removing microneedle with better biocompatibility, simple preparation and better drug carrying performance. SUMMARY

[0005] This invention firstly utilizes biomimetic design of hydrogels, specifically by synthesizing hydrogels with excellent biocompatibility using hyaluronic acid and antibacterial peptides, which can effectively inhibit acne. Then, it employs a microneedle template to fabricate the hydrogel into soluble solid microneedles, integrating microneedles and drugs into one, thus effectively solving the aforementioned technical problems.

[0006] The technical solution of the present invention is as follows:

[0007] This invention first provides a bottle-brush-shaped hyaluronic acid hydrogel based on peptide modification, as shown in formula (1):

[0008]

[0009] In the formula (1), R represents a functional polypeptide, which is at least one of a self-complementary polypeptide with a single thiol group, an antimicrobial peptide with two or more thiol groups, a targeting peptide, or a cell adhesion peptide.

[0010] x represents the grafting rate of the functional polypeptide, x = 0.1-0.4, and n represents the degree of polymerization of the hydrogel, n is greater than or equal to 30.

[0011] According to one embodiment of the present invention, the self-complementary polypeptide is a polypeptide with a cysteine ​​residue at the N-terminus and a polypeptide sequence with alternating positive and negative charges at the C-terminus, wherein the polypeptide sequence is one of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, and SEQ ID No:5.

[0012] The present invention also provides a method for preparing the above-mentioned hydrogel, the steps of which are as follows:

[0013] (1) Dissolve hyaluronic acid in water to obtain an aqueous solution of hyaluronic acid;

[0014] (2) Dissolve the activator and condensing agent in a polar aprotic organic solvent to prepare a solution of the activating condensing agent;

[0015] (3) Mix and stir the solutions from steps (1) and (2) to activate them;

[0016] (4) Add the functionalized small molecules to the mixed solution of step (3), stir and react overnight at room temperature. After the reaction is complete, dialyze the mixed solution in water and freeze dry to obtain functionalized hyaluronic acid.

[0017] (5) Take the lyophilized functionalized hyaluronic acid and functional peptides and dissolve them in buffer solution, adjust the pH to 7-7.5, mix and stir to obtain sol;

[0018] (6) Add antimicrobial peptides, anti-inflammatory peptides and cosmetic peptides to the sol in step (5), continue stirring, and obtain a bottle-brush-shaped peptide-modified hyaluronic acid hydrogel through electrostatic adsorption self-assembly.

[0019] According to one embodiment of the present invention, the concentration of hyaluronic acid in step (1) is 2wt%-3wt%, and the molecular weight is 10kDa-30kDa.

[0020] According to one embodiment of the present invention, in step (2), the activator and the condensing agent are sodium N-hydroxysuccinimide sulfonate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, respectively.

[0021] According to one embodiment of the present invention, the functionalized small molecule in step (4) is one or more of maleimide and its derivatives, methacrylic anhydride, glycidyl methacrylate, glycidyl methacrylate ether, 2-aminoethyl methacrylate, vinyl polyethylene glycol and its derivatives.

[0022] According to one embodiment of the present invention, the antimicrobial peptide is one of the hydrophilic-hydrophobic alternating antimicrobial peptides (KX)n, wherein X = Ile, Leu, or Phe, n = 3, 4, 5, or 6, and the concentration of the hydrophilic-hydrophobic alternating antimicrobial peptide is 30-100 μg / mL; the anti-inflammatory polypeptide is one or more of palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, and anti-inflammatory agent-1; the cosmetic polypeptide is one or more of palmitoyl tripeptide-1, palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, carnosine, palmitoyl tripeptide-5, palmitoyl oligopeptide, hexapeptide-9, hexapeptide-11, and palmitoyl hexapeptide-6, and the concentration of the cosmetic polypeptide is 50-100 μg / mL.

[0023] According to another aspect of the present invention, the present invention also provides the application of the above-described hydrogel in the preparation of acne-inhibiting microneedles or acne-repairing microneedles, comprising the following steps:

[0024] The hydrogel is injected into the microneedle mold, and vacuum degassing is performed to ensure that the needle tip and base are completely filled with hydrogel. After the microneedles dry and are demolded, the soluble acne-repairing microneedles are obtained.

[0025] According to one embodiment of the present invention, the above-mentioned injection degassing operation is to inject the hydrogel, degas it for 15 minutes under vacuum at -0.08 kPa and 25°C, remove the substrate bubbles, and then repeatedly re-inject and degas until the bubbles no longer appear.

[0026] According to one embodiment of the present invention, the hydrogel microneedles are in a 10×10 array, the height of the microneedle tip is 650±15μm, the base width is 220±8μm, and the distance between adjacent tips is 500±14μm.

[0027] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0028] (1) The bottle-brush-shaped peptide-modified hyaluronic acid hydrogel prepared in this invention uses hyaluronic acid and peptides with excellent biocompatibility. Hyaluronic acid can be dissolved under the action of body fluids, will not cause inflammatory reactions, is not harmful to the human body, and has a moisturizing effect in the skin, which can enhance the beauty effect. Meanwhile, self-complementary peptides have diverse functions, high responsiveness in the body, and simple and easy synthesis and modification methods.

[0029] (2) The bottle-brush peptide-modified hyaluronic acid hydrogel prepared in this invention is obtained by a Michael addition reaction between the double bonds on the double-bond functionalized hyaluronic acid and an amphoteric self-complementary peptide containing thiol functional groups. The reaction conditions are simple and mild. The positive and negative charges at the C-terminus of the self-complementary peptide are attracted by electrostatic attraction, thereby physically crosslinking the bottle-brush polymer to obtain the hydrogel. This hydrogel avoids the use of chemical crosslinking agents. The bottle-brush peptide-modified hyaluronic acid, with its extracellular matrix proteoglycan-like structure, endows it with good biocompatibility and the function of promoting collagen production.

[0030] (3) The bottle-brush-like peptide-modified hyaluronic acid prepared in this invention can form microchannels in the stratum corneum of the skin, allowing the drug and its active ingredients to reach the deep layers of the skin along the microchannels. When it is in a strongly acidic skin pathological environment, the charge of the amino acids on the peptide can change, thereby destroying the cross-linked structure of the hydrogel and allowing the drug to be released rapidly. When it is in a weakly acidic or near-neutral skin pathological environment, the hydrogel will rapidly absorb body fluids and swell, producing continuous and unobstructed hydrogel conduits, allowing the drug to penetrate into the skin and thus achieve sustained release. Therefore, it can achieve a rapid onset of action, painless and scarless controllable drug release effect, providing a better treatment method for inhibiting acne. Attached Figure Description

[0031] Figure 1 The image shows the gelation process of the hydrogel prepared in Example 1.

[0032] Figure 2 The NMR characterization image of the hydrogel prepared in Example 1.

[0033] Figure 3 The image shows a scanning electron microscope (SEM) image of the hydrogel prepared in Example 1.

[0034] Figure 4 The image shows the macroscopic morphology of the hydrogel microneedles prepared in Example 1.

[0035] Figure 5 Scanning electron microscopy image of the antibacterial active ingredient in the hydrogel microneedles prepared in Example 1 against Propionibacterium acnes.

[0036] Figure 6 The minimum inhibitory concentration of the antibacterial active ingredient in the hydrogel microneedles prepared in Example 1 against Propionibacterium acnes.

[0037] Figure 7 The diagram shows the bactericidal effect of the antimicrobial peptides in the hydrogel microneedles prepared in Examples 1-5 against Propionibacterium acnes and Staphylococcus aureus.

[0038] Figure 8 Biocompatibility test diagrams of hydrogel microneedles prepared in Examples 1-5.

[0039] Figure 9 The images show the effect of scratch experiments on promoting wound healing in the preparation of hydrogel microneedles in Examples 1-5. Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, those skilled in the art can easily understand that the following description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0041] This invention first provides a bottle-brush-shaped hyaluronic acid hydrogel based on peptide modification, as shown in formula (1):

[0042]

[0043] In some embodiments, x = 0.1-0.4, n represents the degree of polymerization of the hydrogel, and n is greater than or equal to 25.

[0044] In some embodiments, R represents a functional polypeptide, which is at least one of a self-complementary polypeptide having a single thiol group, an antimicrobial peptide having two or more thiol groups, a targeting peptide, or a cell adhesion peptide.

[0045] In some embodiments, the self-complementary polypeptide in step (5) is a polypeptide sequence containing a cysteine ​​residue at the N-terminus and a continuous alternation of positive and negative charges at the C-terminus. The sequence of the self-complementary polypeptide is one of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, and SEQ ID No:5, and its sequence and structural formula are shown in the table below:

[0046]

[0047] The present invention also provides a method for preparing the above-mentioned hydrogel, the steps of which are as follows:

[0048] (1) Dissolve hyaluronic acid in water to obtain an aqueous solution of hyaluronic acid;

[0049] (2) Dissolve the activator and condensing agent in a polar aprotic organic solvent to prepare a solution of the activating condensing agent;

[0050] (3) Mix and stir the solutions from steps (1) and (2) to activate them;

[0051] (4) Add the functionalized small molecules to the mixed solution of step (3), stir and react overnight at room temperature. After the reaction is complete, dialyze the mixed solution in water and freeze dry to obtain functionalized hyaluronic acid.

[0052] (5) Take the lyophilized functionalized hyaluronic acid and functional peptides and dissolve them in buffer solution, adjust the pH, mix and stir to obtain sol;

[0053] (6) Add antimicrobial peptides, anti-inflammatory peptides and cosmetic peptides to the sol in step (5), continue stirring, and obtain a bottle-brush-shaped peptide-modified hyaluronic acid hydrogel through electrostatic adsorption self-assembly.

[0054] In some embodiments, the concentration of hyaluronic acid in step (1) is 2wt%-3wt%, and the molecular weight is 10kDa-30kDa.

[0055] In some embodiments, in step (2), the activator and condensing agent are preferably sodium N-hydroxysuccinimide sulfonate to neutralize 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0056] In some embodiments, the functionalized small molecule in step (4) is one or more of maleimide and its derivatives, methacrylic anhydride, glycidyl methacrylate, glycidyl methacrylate ether, 2-aminoethyl methacrylate, vinyl polyethylene glycol and its derivatives.

[0057] In some embodiments, in step (5), the buffer solution may be a PBS buffer solution, and the pH range of the buffer solution is 7-7.5.

[0058] In some embodiments, in step (6), the stirring time is 8-12 hours.

[0059] In some embodiments, the antimicrobial peptide is one of the alternating hydrophilic and hydrophobic antimicrobial peptides (KX)n, wherein X = Ile, Leu, or Phe, n = 3, 4, 5, or 6, and the concentration of the alternating hydrophilic and hydrophobic antimicrobial peptide is 30-100 μg / mL; the anti-inflammatory polypeptide is one or more of palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, and anti-inflammatory agent-1; the cosmetic polypeptide is one or more of palmitoyl tripeptide-1, palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, carnosine, palmitoyl tripeptide-5, palmitoyl oligopeptide, hexapeptide-9, hexapeptide-11, and palmitoyl hexapeptide-6, and the concentration of the cosmetic polypeptide is 50-100 μg / mL.

[0060] The present invention also provides the application of the above-mentioned hydrogel in inhibiting or treating acne: (1) the above-mentioned hydrogel is made into hydrogel microneedles; (2) the hydrogel microneedles are attached to the wound, wherein the preparation method of the hydrogel microneedles in step 1 is as follows: the above-mentioned hydrogel is injected into the microneedle mold, vacuum degassing is performed so that the needle tip and the base are completely filled with hydrogel, and the hydrogel microneedles are obtained after the microneedles are dried and demolded.

[0061] In some embodiments, the above-mentioned injection degassing operation involves injecting the hydrogel, degassing it for 15 minutes under a vacuum of -0.08 kPa and 25°C, removing the substrate bubbles, and then repeatedly re-injecting and degassing until the bubbles no longer appear.

[0062] In some embodiments, the hydrogel microneedles comprise a 10×10 array, with a microneedle tip height of 650±15μm, a base width of 220±8μm, and a distance of 500±14μm between adjacent tips.

[0063] The following are specific examples:

[0064] Example 1

[0065] The preparation method of the acne-removing microneedles based on bottle-brush-shaped peptide-modified hyaluronic acid hydrogel in this embodiment includes the following steps:

[0066] (1) Weigh 200 mg of 10 kDa sodium hyaluronate, stir and dissolve it in 10 mL of deionized water to obtain an aqueous solution of hyaluronic acid; weigh 110 mg of activator N-hydroxysuccinimide sodium salt (Sulfo-NHS) and 96 mg of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dissolve them in 10 mL of DMSO to obtain a solution of activator and condensing agent;

[0067] (2) Mix the aqueous solution of hyaluronic acid and the solution of activator condensing agent from step (1), and stir to activate for 30 min;

[0068] (3) Weigh 90 mg of N-(2-aminoethyl)maleimide hydrochloride and add it to the solution in step (2). Stir the reaction at room temperature overnight. After the reaction is complete, dialyze the solution in deionized water for 3-5 days using a dialysis bag with a molecular weight cutoff of 3500. After dialysis, freeze dry the solution to obtain maleimide-modified hyaluronic acid (HAMAL).

[0069] (4) Weigh 40 mg of the self-complementary polypeptide with sequence SEQ ID No:1 and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5; weigh 40 mg of HAMAL from step (3) and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5. Mix the two solutions and stir at 120 rpm for 6 h at room temperature to obtain a sol of polypeptide-modified hyaluronic acid.

[0070] (5) Add 30 μg / mL of hydrophilic-hydrophobic alternating antimicrobial peptide (amino acid sequence KIKIKIKI-NH2), 50 μg / mL of anti-inflammatory peptide anti-inflammatory-1 (amino acid sequence MQMKKVLDS-OH), and 50 μg / mL of cosmetic peptide palmitoyl tripeptide-1 (amino acid sequence Palmitoyl-GHK-OH) to the sol obtained in step (4), and continue stirring overnight to obtain a peptide-modified hyaluronic acid hydrogel;

[0071] (6) Weigh 200 mg of 10 kDa hyaluronic acid and stir to dissolve it in 10 mL of deionized water to obtain a 2 wt% hyaluronic acid solution as the base part of the microneedles.

[0072] (7) Take 100 μL of hydrogel loaded with peptides and inject it into a tetrahedral microneedle mold of polydimethylsiloxane. Defoam in vacuum at -0.08 kPa and 25°C for 15 min. After scraping off the surface bubbles, repeatedly inject the hydrogel and defoam in vacuum. Finally, take 100 μL of 2 wt% hyaluronic acid solution from step (6) to fill the microneedle base. After drying for 48 h, demold to obtain hydrogel acne-removing microneedles.

[0073] Results and Discussion:

[0074] 1. From Figure 1 As can be seen from the macroscopic diagram of the gelation of the peptide-modified hyaluronic acid hydrogel, when the concentration of the brush polymer grafted with CVAEK peptide and hyaluronic acid is 1 wt%, the brush polymer is still in solution; when the concentration increases to 2 wt%, the brush polymer begins to assemble into a gel, indicating the successful synthesis of the hydrogel in Example 1.

[0075] 2. From Figure 2The NMR spectrum of the peptide-modified hyaluronic acid hydrogel shows a characteristic absorption peak of hydrogen on the double bond of maleimide at a chemical shift of 6.84 ppm, indicating the successful synthesis of maleimide-modified hyaluronic acid. The grafting rate of maleimide was calculated to be 33%. After peptide grafting, the characteristic absorption peak of hydrogen on the double bond of maleimide at a chemical shift of 6.84 ppm disappeared, and a characteristic absorption peak of hydrogen in the peptide appeared in the range of 0.6-1.5 ppm, indicating that the peptide in this embodiment was successfully grafted with hyaluronic acid.

[0076] 3. Scanning electron micrograph of peptide-modified hyaluronic acid hydrogel.

[0077] In this embodiment, scanning electron microscope (SEM) images of lyophilized peptide-grafted hyaluronic acid at different concentrations are shown below. Figure 3 As shown, at a concentration of 1 wt%, a network structure is formed because no hydrogel is formed, while at 2 wt% and 3 wt%, a porous structure is formed. Although the surface of the 3 wt% structure still has a porous structure, the pores on the surface are relatively rough.

[0078] 4. Macroscopic morphological observation of microneedles used for acne treatment and repair.

[0079] The microneedles prepared in Example 1 for acne treatment and repair were placed on a white background, and macroscopic images of the microneedle patch were taken using a dermoscope, as shown below. Figure 4 As shown, the prepared microneedles are square, with pyramidal microneedles distributed in the middle, indicating the successful preparation of the microneedles.

[0080] 5. Scanning electron micrographs of the antibacterial active ingredients in microneedles used for acne treatment and repair against Propionibacterium acnes, such as... Figure 5 As shown.

[0081] 10 8 CFU / mL bacterial culture was added to 24-well plates coated with cell spreaders and incubated for 48 hours. Then, antimicrobial peptide (KI) 4 or an equal volume of PBS (Control group) was added to a final concentration of 30 μg / mL, and the plates were incubated for 4-6 hours. After fixation with glutaraldehyde, graded dehydration with alcohol, and gold sputtering, the morphology of *Propionibacterium acnes* in different samples was observed using SEM. The bacteria in the Control group exhibited intact cell morphology and a regular rod-shaped form. However, treatment with the antimicrobial peptide disrupted the normal bacterial structure, leading to bacterial cell membrane contraction and even rupture.

[0082] Example 2

[0083] The preparation method of the acne-removing microneedles based on bottle-brush-shaped peptide-modified hyaluronic acid hydrogel in this embodiment includes the following steps:

[0084] (1) Weigh 200 mg of 10 kDa sodium hyaluronate, stir and dissolve it in 10 mL of deionized water to obtain an aqueous solution of hyaluronic acid; weigh 110 mg of activator N-hydroxysuccinimide sodium salt (Sulfo-NHS) and 96 mg of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dissolve them in 10 mL of DMSO to obtain a solution of activator and condensing agent;

[0085] (2) Mix the aqueous solution of hyaluronic acid and the solution of activator condensing agent from step (1), and stir to activate for 30 min;

[0086] (3) Weigh 90 mg of N-(2-aminoethyl)maleimide hydrochloride and add it to the solution in step (2). Stir the reaction at room temperature overnight. After the reaction is complete, dialyze the solution in deionized water for 3-5 days using a dialysis bag with a molecular weight cutoff of 3500. After dialysis, freeze dry the solution to obtain maleimide-modified hyaluronic acid (HAMAL).

[0087] (4) Weigh 40 mg of the self-complementary polypeptide with sequence SEQ ID No:2 and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5; weigh 40 mg of HAMAL from step (3) and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5. Mix the two solutions and stir at 120 rpm for 6 h at room temperature to obtain a bottle-brush-like polypeptide-modified hyaluronic acid sol.

[0088] (5) Add 50 μg / mL of hydrophilic-hydrophobic alternating antimicrobial peptide (amino acid sequence KFKFKFKFKF-NH2), 50 μg / mL of anti-inflammatory peptide anti-inflammatory-1 (amino acid sequence MQMKKVLDS-OH), and 50 μg / mL of cosmetic peptide palmitoyl tripeptide-1 (amino acid sequence Palmitoyl-GHK-OH) to the sol obtained in step (4), and continue stirring overnight to obtain bottle-brush-like peptide-modified hyaluronic acid hydrogel;

[0089] (6) Weigh 200 mg of 10 kDa hyaluronic acid and stir to dissolve it in 10 mL of deionized water to obtain a 2 wt% hyaluronic acid solution as the base part of the microneedles.

[0090] (7) Take 100 μL of hydrogel loaded with peptides and inject it into the PDMS pyramidal microneedle mold. Defoam in vacuum at -0.08 kPa and 25°C for 15 min. After scraping off the surface bubbles, repeatedly inject the hydrogel and defoam in vacuum. Finally, take 100 μL of the 2 wt% hyaluronic acid solution from step (6) to fill the microneedle base. After drying for 48 h, demold to obtain hydrogel acne-removing microneedles.

[0091] Example 3

[0092] The preparation method of the acne-treating microneedles made from bottle-brush-shaped peptide-modified hyaluronic acid hydrogel in this embodiment includes the following steps:

[0093] (1) Weigh 200 mg of 10 kDa sodium hyaluronate, stir and dissolve it in 10 mL of deionized water to obtain an aqueous solution of hyaluronic acid; weigh 110 mg of activator N-hydroxysuccinimide sodium salt (Sulfo-NHS) and 96 mg of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dissolve them in 10 mL of DMSO to obtain a solution of activator and condensing agent;

[0094] (2) Mix the aqueous solution of hyaluronic acid and the solution of activator condensing agent from step (1), and stir to activate for 30 min;

[0095] (3) Weigh 90 mg of N-(2-aminoethyl)maleimide hydrochloride and add it to the solution in step (2). Stir the reaction at room temperature overnight. After the reaction is complete, dialyze the solution in deionized water for 3-5 days using a dialysis bag with a molecular weight cutoff of 3500. After dialysis, freeze dry the solution to obtain maleimide-modified hyaluronic acid (HAMAL).

[0096] (4) Weigh 40 mg of the self-complementary polypeptide with sequence SEQ ID No:3 and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5; weigh 40 mg of HAMAL from step (3) and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5. Mix the two solutions and stir at 120 rpm for 6 h at room temperature to obtain a sol of polypeptide-modified hyaluronic acid.

[0097] (5) Add 50 μg / mL of hydrophilic-hydrophobic alternating antimicrobial peptide (amino acid sequence: HFHFHFHFHF-NH2), 50 μg / mL of anti-inflammatory peptide palmitoyl tetrapeptide-7 (amino acid sequence: Palmitoyl-GQPR-OH), and 50 μg / mL of cosmetic peptide palmitoyl tripeptide-1 (amino acid sequence: Palmitoyl-GHK-OH) to the sol obtained in step (4), and continue stirring overnight to obtain peptide-modified hyaluronic acid hydrogel;

[0098] (6) Weigh 300 mg of 10 kDa hyaluronic acid and stir to dissolve it in 10 mL of deionized water to obtain a 3 wt% hyaluronic acid solution as the base part of the microneedles.

[0099] (7) Take 100 μL of hydrogel loaded with peptides and inject it into the PDMS pyramidal microneedle mold. Defoam in vacuum at -0.08 kPa and 25°C for 15 min. After scraping off the surface bubbles, repeatedly inject the hydrogel and defoam in vacuum. Finally, take 100 μL of the 3 wt% hyaluronic acid solution from step (6) to fill the microneedle base. After drying for 48 h, demold to obtain hydrogel acne-removing microneedles.

[0100] Example 4

[0101] The preparation method of the acne-removing microneedles based on bottle-brush-shaped peptide-modified hyaluronic acid hydrogel in this embodiment includes the following steps:

[0102] (1) Weigh 100 mg of 10 kDa sodium hyaluronate, stir and dissolve it in 5 mL of deionized water to obtain an aqueous solution of hyaluronic acid; weigh 55 mg of activator N-hydroxysuccinimide sodium salt (Sulfo-NHS) and 48 mg of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dissolve them in 5 mL of DMSO to obtain a solution of activator and condensing agent;

[0103] (2) Mix the aqueous solution of hyaluronic acid and the solution of activator condensing agent from step (1), and stir to activate for 30 min;

[0104] (3) Weigh 90 mg of N-(2-aminoethyl)maleimide hydrochloride and add it to the solution in step (2). Stir the reaction at room temperature overnight. After the reaction is complete, dialyze the solution in deionized water for 3-5 days using a dialysis bag with a molecular weight cutoff of 3500. After dialysis, freeze dry the solution to obtain maleimide-modified hyaluronic acid (HAMAL).

[0105] (4) Weigh 60 mg of the self-complementary polypeptide with sequence SEQ ID No:4 and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5; weigh 60 mg of HAMAL from step (3) and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5. Mix the two solutions and stir at 120 rpm for 6 h at room temperature to obtain a sol of polypeptide-modified hyaluronic acid.

[0106] (5) Add 100 μg / mL of hydrophilic-hydrophobic alternating antimicrobial peptide (amino acid sequence KLKLKLKLKL-NH2), 50 μg / mL of anti-inflammatory peptide anti-inflammatory-1 (amino acid sequence MQMKKVLDS-OH), and 50 μg / mL of cosmetic peptide palmitoyl tripeptide-1 (amino acid sequence Palmitoyl-GHK-OH) to the sol obtained in step (4), and continue stirring overnight to obtain peptide-modified hyaluronic acid hydrogel;

[0107] (6) Weigh 300 mg of 10 kDa hyaluronic acid and stir to dissolve it in 10 mL of deionized water to obtain a 3 wt% hyaluronic acid solution as the base part of the microneedles.

[0108] (7) Take 100 μL of hydrogel loaded with peptides and inject it into the PDMS pyramidal microneedle mold. Defoam in vacuum at -0.08 kPa and 25°C for 15 min. After scraping off the surface bubbles, repeatedly inject the hydrogel and defoam in vacuum. Finally, take 100 μL of the 5 wt% hyaluronic acid solution from step (6) to fill the microneedle base. After drying for 48 h, demold to obtain hydrogel acne-removing microneedles.

[0109] Example 5

[0110] The preparation method of the acne-removing microneedles based on bottle-brush-shaped peptide-modified hyaluronic acid hydrogel in this embodiment includes the following steps:

[0111] (1) Weigh 200 mg of 10 kDa sodium hyaluronate, stir and dissolve it in 10 mL of deionized water to obtain an aqueous solution of hyaluronic acid; weigh 110 mg of activator N-hydroxysuccinimide sodium salt (Sulfo-NHS) and 96 mg of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and dissolve them in 10 mL of DMSO to obtain a solution of activator and condensing agent;

[0112] (2) Mix the aqueous solution of hyaluronic acid and the solution of activator condensing agent from step (1), and stir to activate for 30 min;

[0113] (3) Weigh 90 mg of N-(2-aminoethyl)maleimide hydrochloride and add it to the solution in step (2). Stir the reaction at room temperature overnight. After the reaction is complete, dialyze the solution in deionized water for 3-5 days using a dialysis bag with a molecular weight cutoff of 3500. After dialysis, freeze dry the solution to obtain maleimide-modified hyaluronic acid (HAMAL).

[0114] (4) Weigh 660 mg of the self-complementary polypeptide with sequence SEQ ID No:3 and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5; weigh 60 mg of HAMAL from step (3) and dissolve it in 1 mL of deionized water, and adjust the pH to 7.0-7.5. Mix the two solutions and stir at 120 rpm for 6 h at room temperature to obtain a sol of polypeptide-modified hyaluronic acid.

[0115] (5) Add 100 μg / mL of hydrophilic-hydrophobic alternating antimicrobial peptide (amino acid sequence RLRLRLRLRL-NH2), 50 μg / mL of anti-inflammatory peptide palmitoyl tetrapeptide-7 (amino acid sequence Palmitoyl-GQPR-OH), and 50 μg / mL of cosmetic peptide palmitoyl tripeptide-5 (amino acid sequence Palmitoyl-KVK-OH) to the sol obtained in step (4), and continue stirring overnight to obtain peptide-modified hyaluronic acid hydrogel;

[0116] (6) Weigh 200 mg of 10 kDa hyaluronic acid and stir to dissolve it in 10 mL of deionized water to obtain a 2 wt% hyaluronic acid solution as the base part of the microneedles.

[0117] (7) Take 100 μL of hydrogel loaded with peptides and inject it into the PDMS pyramidal microneedle mold. Defoam in vacuum at -0.08 kPa and 25°C for 15 min. After scraping off the surface bubbles, repeatedly inject the hydrogel and defoam in vacuum. Finally, take 100 μL of the 2 wt% hyaluronic acid solution from step (6) to fill the microneedle base. After drying for 48 h, demold to obtain hydrogel acne-removing microneedles.

[0118] Application test cases

[0119] 1. The minimum inhibitory concentration of antimicrobial peptides in microneedles used for acne treatment and repair against Propionibacterium acnes and Staphylococcus aureus.

[0120] The minimum inhibitory concentration (MIC) of the antimicrobial peptides in the microneedles of Examples 1-5 against Propionibacterium acnes and Staphylococcus aureus was tested using a two-fold dilution method. The test steps are as follows:

[0121] 1) Revive and activate the bacteria, and prepare a solution with a concentration of 2×10⁻⁶. 6 Prepare a bacterial suspension of CFU / mL for later use;

[0122] 2) Take a sterile 96-well plate and add 100 μL of bacterial basal culture medium to each well;

[0123] 3) Add 100 μL of an antimicrobial peptide at a concentration of 4096 μg / mL to the first well and mix well. Add 100 μL from the first well to the second well and mix well. Add 100 μL from the second well to the third well, and so on, discarding 100 μL from the last well. At this point, the concentrations of the antimicrobial peptide in each well are 2048, 1024, 512, 256, 128, 64, 32, 16, 8, and 4 μg / mL, respectively.

[0124] 4) Add 100 μL of the prepared 2×10⁻⁶ solution to each well. 6 A bacterial suspension with CFU / mL, at this point the bacterial suspension concentration is 1×10⁻⁶. 6CFU / mL, the concentrations of antimicrobial peptides in each well were 1024, 512, 256, 128, 64, 32, 16, 8, 4, and 2 μg / mL;

[0125] 5) The negative control consisted of 200 μL of LB medium, the positive control consisted of 100 μL of LB medium and 100 μL of bacterial suspension;

[0126] 6) Place the inoculated 96-well plate in a 37°C incubator for 24 hours. After 24 hours, measure the absorbance at OD600nm using an ELISA reader. Calculate the inhibition rate of each well based on the absorbance. The minimum concentration of the antimicrobial peptide that achieves an inhibition rate of 90% is defined as the minimum concentration of the antimicrobial peptide that inhibits the growth of the bacterial strain.

[0127] By measuring the absorbance at OD600nm, it can be seen that an antimicrobial peptide concentration of less than 100μg / mL is sufficient to inhibit bacterial growth, indicating that the hydrophilic-hydrophobic alternating antimicrobial peptides in Examples 1-5 all have good antimicrobial effects.

[0128] Results and Discussion:

[0129] 1. Figure 6 By testing the minimum inhibitory concentration of the antimicrobial peptide in the acne-removing microneedles of Examples 1-5, it can be seen that the hydrophilic-hydrophobic alternating type antimicrobial peptide can achieve the effect of inhibiting 90% of bacterial growth at a low concentration.

[0130] 2. By Figure 7 As shown in the bactericidal effect diagrams of Examples 1-5 against Propionibacterium acnes and Staphylococcus aureus, the bottle-brush-shaped peptide-modified hyaluronic acid hydrogel can effectively release antimicrobial peptides, achieving a good antimicrobial effect.

[0131] 3. By Figure 8 As can be seen from the biocompatibility test diagrams of Examples 1-5, the bottle-brush-like peptide-modified hyaluronic acid hydrogel has good biocompatibility.

[0132] 4. By Figure 9 As can be seen from the scratch test results of Examples 1-5 on promoting wound healing, Examples 1-5 can all promote the migration of fibroblasts in mice, thus having a certain effect on promoting wound healing.

[0133] 5. The mechanical strength of the microneedles in Examples 1-5 was tested. As shown in Table 1, the insertion force of Examples 1-5 was greater than the required insertion force (0.098 N / needle) for microneedle insertion into the skin.

[0134] Table 1. Strength of hydrogels prepared in Examples 1-5

[0135]

[0136]

[0137] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0138]

[0139]

Claims

1. A method for preparing a hydrogel, characterized in that, Includes the following steps: (1) Dissolve hyaluronic acid in water to obtain an aqueous solution of hyaluronic acid; (2) Dissolve the activator and condensing agent in a polar aprotic organic solvent to prepare a solution of the activating condensing agent; (3) Mix and stir the solutions from steps (1) and (2) to activate them; (4) Add the functionalized small molecules to the mixed solution of step (3), stir and react overnight at room temperature. After the reaction is complete, dialyze the mixed solution in water and freeze dry to obtain functionalized hyaluronic acid. (5) Take the lyophilized functionalized hyaluronic acid and functional peptides and dissolve them in buffer solution, adjust the pH to 7-7.5, mix and stir to obtain sol; (6) Add antimicrobial peptides, anti-inflammatory peptides and cosmetic peptides to the sol in step (5), continue stirring, and obtain a bottle-brush-shaped peptide-modified hyaluronic acid hydrogel through electrostatic adsorption self-assembly. The grafting rate of the functional polypeptide is 0.1-0.4, and the degree of polymerization of the hydrogel is greater than or equal to 30; the sequence of the functional polypeptide is one of SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, and SEQ ID No:

5. The antimicrobial peptide is a hydrophilic-hydrophobic alternating type antimicrobial peptide (KX). n One of the following, wherein X = Ile, Leu or Phe, n = 3, 4, 5 or 6, and the anti-inflammatory polypeptide is one or more of palmitoyl tripeptide-8, palmitoyl tetrapeptide-7, and anti-inflammatory agent-1.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of hyaluronic acid is 2wt%-3wt%, and the molecular weight is 10kDa-30kDa.

3. The preparation method according to claim 1, characterized in that, In step (2), the activator and condensing agent are sodium N-hydroxysuccinimide sulfonate and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, respectively.

4. The preparation method according to claim 1, characterized in that, In step (4), the functionalized small molecule is one or more of maleimide, methacrylic anhydride, glycidyl methacrylate, glycidyl methacrylate ether, 2-aminoethyl methacrylate, and vinyl polyethylene glycol.

5. The preparation method according to any one of claims 1-4, characterized in that, The concentration of the hydrophilic-hydrophobic alternating antimicrobial peptide is 30-100 μg / mL; the cosmetic polypeptide is one or more of palmitoyl tripeptide-1, palmitoyl pentapeptide-4, palmitoyl tetrapeptide-7, carnosine, palmitoyl tripeptide-5, palmitoyl oligopeptide, hexapeptide-9, hexapeptide-11, and palmitoyl hexapeptide-6, and the concentration of the cosmetic polypeptide is 50-100 μg / mL.

6. The application of a hydrogel obtained by the preparation method according to any one of claims 1-5 in the preparation of acne-inhibiting microneedles or acne-repairing microneedles, characterized in that, Includes the following steps: The hydrogel is injected into the microneedle mold, and vacuum degassing is performed so that the needle tip and the base are completely filled with hydrogel. After the microneedles dry and are demolded, the acne-inhibiting microneedles or acne-repairing microneedles can be obtained.

7. The application according to claim 6, characterized in that, The degassing procedure involves injecting the hydrogel, followed by degassing under a vacuum of -0.08 kPa and 25°C for 15 minutes to remove the substrate bubbles. This process is repeated until no more bubbles appear.

8. The application according to claim 6 or 7, characterized in that, The hydrogel microneedles are arranged in a 10×10 array, with a needle tip height of 650±15μm, a base width of 220±8μm, and a distance of 500±14μm between adjacent needle tips.

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