Drug-loaded hydrogel microneedle as well as preparation method and application thereof

Abstract

The invention discloses a drug-loaded hydrogel microneedle and a preparation method and application thereof, and the drug-loaded hydrogel microneedle comprises drug-loaded nanoparticles comprising a protein hypoglycemic drug loaded on a phenylboronic acid modified cationic polymer; the hydrogel microneedle is prepared from methacrylic acid chitosan modified by natural polyphenol and a diol diacrylic acid polymer, the drug-loaded nanoparticles are loaded on the hydrogel microneedle. The drug-loaded hydrogel microneedle has excellent biocompatibility, can effectively reduce inflammatory response through a triple therapy strategy of breaking a bacterial biofilm and improving oxidative stress response and hyperglycemia level of a wound surface, can be used for delivering different types of protein hypoglycemic drugs for promoting wound healing, and can be applied to the field of medical treatment through intelligent drug delivery. The repairing of blood vessels and tissues of wound surfaces is promoted, and a series of problems of poor bioavailability, poor treatment effect and the like of the existing hydrogel dressing are effectively solved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a drug-loaded hydrogel microneedle and its preparation method and application. Background Art

[0002] As one of the most serious complications of diabetes, diabetic foot ulcer (DFU) affects over 30 million patients globally, imposing an annual medical burden of approximately $13 billion and a high amputation rate of 20 - 30%, posing a major challenge in the field of global public health. Research shows that persistent hyperglycemia and highly expressed reactive oxygen species (ROS) induce severe inflammatory responses, along with external bacterial infections, which are the key factors causing chronic wounds such as diabetic foot ulcer (DFU) to be difficult to heal. However, existing drug delivery systems (such as traditional hydrogels) have many defects, such as poor drug permeability, poor biofilm disruption performance, uncontrollable drug release, and poor comprehensive microenvironment regulation performance, making it difficult to fundamentally improve clinical efficacy. Therefore, it is urgent to develop a new drug delivery system that can integrally solve the above dilemmas.

[0003] In recent years, hydrogel-formed microneedles (HMN) have shown great potential in the treatment of DFU due to their advantages such as minimally invasive penetration of the stratum corneum (needle length 100 - 1000 μm), targeted drug delivery to the dermis layer (diffusion rate increased by 4 orders of magnitude), and micropore-assisted biofilm clearance (destruction rate > 90%). However, on the one hand, the mechanical properties of existing HMN systems are limited, unable to penetrate the bacterial biofilm and deeply regulate the wound microenvironment inside the tissue. On the other hand, the drug delivery function is single, making it difficult to achieve multi-factor synergistic regulation. Summary of the Invention

[0004] The present invention aims to at least solve one of the above technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a drug-loaded hydrogel microneedle and its preparation method and application.

[0005] To achieve the above purpose, the technical solutions adopted by the present invention are as follows:

[0006] In the first aspect of the present invention, a drug-loaded hydrogel microneedle is provided, comprising:

[0007] Drug-loaded nanoparticles, comprising a protein-based hypoglycemic drug loaded on a phenylboronic acid-modified cationic polymer;

[0008] Hydrogel microneedles, comprising methylacrylated chitosan modified with natural polyphenols and a diol diacrylate polymer;

[0009] The drug-loaded nanoparticles are loaded on the hydrogel microneedles.

[0010] In the present invention, loading protein-based hypoglycemic drugs onto phenylboronic acid-modified cationic polymers can protect the activity of drug molecules and avoid the toxic and side effects caused by the initial burst release of the drug; the mechanical properties of diol dimethacrylate polymers can be precisely regulated, for example, the mechanical properties can be precisely regulated by the types and ratios of monomers and reaction conditions; methylacrylated chitosan modified with natural polyphenols has antibacterial and antioxidant activities. Hybridizing the two to form hydrogel microneedles endows the tip structure of the microneedle array with mechanical properties, thereby breaking through the bacterial biofilm, which can not only improve bacterial infection and oxidative stress injury, but also release drugs deep into the tissue to achieve precise regulation of high blood sugar levels and reshape the immune microenvironment of DFU.

[0011] In some embodiments of the present invention, the drug-loaded nanoparticles are loaded on the tips of the hydrogel microneedles.

[0012] In some embodiments of the present invention, the microneedle body is a three-dimensional needle-like structure, such as a cone, a quadrangular pyramid or a cuboid. The height of the microneedle body is 500-2000 μm, such as 500 μm, 1200 μm or 1500 μm; when the microneedle body is conical, the bottom diameter of the microneedle body can be 200-800 μm, such as 500 μm. When the microneedle body is a quadrangular pyramid, the side length of the bottom surface of the microneedle body is 200-800 μm, such as 500 μm; the base is a cylinder, the height of the base is 0.01-2 mm, and the diameter of the base is 0.5-5 cm; when there are multiple microneedle bodies, the tip spacing of the microneedle bodies can be the conventional tip spacing in the art, for example, 100-1000 μm, and for another example, 200 μm, 700 μm or 800 μm.

[0013] In some embodiments of the present invention, the mass ratio of the drug-loaded nanoparticles to the hydrogel microneedles is 1:1-2000, such as 1:1-1700, 1:10-1500, 1:50-1000, 1:80-800, 1:100-500, 1:100-300, 1:100-200, etc.

[0014] In some embodiments of the present invention, in the drug-loaded nanoparticles, the mass ratio of the phenylboronic acid-modified cationic polymer to the protein-based hypoglycemic drug is 1-100:1, such as 20-80:1, 30-70:1, 40-60:1. If the dosage of the phenylboronic acid-modified cationic polymer is too high, it is easy to cause cytotoxicity. If the dosage is too low, the drug loading amount is low or the drug loading effect is poor, and the therapeutic effect is poor.

[0015] In some embodiments of the present invention, the phenylboronic acid-modified cationic polymer is obtained by grafting carboxyphenylboronic acid onto a cationic polymer through a grafting reaction. By mass, the mass ratio of the carboxyphenylboronic acid to the cationic polymer is 1:(0.1 - 20), such as 1:1 - 20, 1:1 - 10, 5:6, etc.

[0016] In some embodiments of the present invention, in the hydrogel microneedles, the mass ratio of the natural polyphenol-modified methacrylated chitosan to the diol diacrylate polymer is 1:3 - 10, such as 1:4 - 9, 1:5 - 9, 1:6 - 8.

[0017] In some embodiments of the present invention, in the natural polyphenol-modified methacrylated chitosan, the mass ratio of the natural polyphenol to the methacrylated chitosan is 1 - 10:10 - 1, such as 1 - 5:5 - 1, 3:2.

[0018] In some embodiments of the present invention, the cationic polymer in the phenylboronic acid-modified cationic polymer includes at least one of polylysine (such as ε-polylysine), polyglutamic acid, polyethyleneimine, polypropyleneimine, polyamidoamine, polyester amide, and poly(ethyl methacrylate 2-(dimethylamine)).

[0019] In some embodiments of the present invention, the protein-based hypoglycemic drugs include insulin and / or GLP-1 receptor agonists. Among them, the GLP-1 receptor agonists are selected from at least one of exenatide, lysine exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, taspoglutide, benaglutide, dulaglutide, loxisenatide, and semaglutide, such as exenatide, exenatide microspheres, lysine exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, taspoglutide, benaglutide, or dulaglutide, and also such as liraglutide or dulaglutide; as long as the hypoglycemic effect can be achieved.

[0020] In some embodiments of the present invention, the natural polyphenols include at least one of gallic acid, epigallocatechin gallate, epicatechin, catechin, tea polyphenols, protocatechuic acid, chlorogenic acid, caffeic acid, rutin, and tannic acid.

[0021] In some embodiments of the present invention, the diol diacrylate polymers include at least one of polyethylene glycol acrylate, polyethylene glycol methacrylate, polyethylene glycol dimethacrylate, polyethylene glycol diacrylate, poly(dipropylene glycol) diacrylate, and poly(diethylene glycol) dimethacrylate.

[0022] The second aspect of the present invention provides a method for preparing the above-mentioned drug-loaded hydrogel microneedles, which includes the following steps:

[0023] Mix the methacrylated chitosan modified with natural polyphenols and the diol diacrylate polymer, add the drug-loaded nanoparticles and a photoinitiator, and prepare the drug-loaded hydrogel microneedles after a photocrosslinking reaction.

[0024] In some embodiments of the present invention, the method for preparing the drug-loaded hydrogel microneedles includes the following steps: Mix the methacrylated chitosan modified with natural polyphenols and the diol diacrylate polymer to prepare a hydrogel microneedle matrix; add the drug-loaded nanoparticles and a photoinitiator to obtain a mixed solution, transfer the mixed solution to a microneedle mold, and centrifuge to enrich the drug-loaded nanoparticles at the tips of the microneedles, and prepare the drug-loaded hydrogel microneedles after a photocrosslinking reaction.

[0025] In some embodiments of the present invention, the centrifugation rate is 1000 rpm to 10000 rpm; such as 2000 to 7000 rpm, 3000 to 6000 rpm, 4000 rpm.

[0026] In some embodiments of the present invention, the concentration of the methacrylated chitosan modified with natural polyphenols is 0.01 to 1000 mg / mL, such as 0.1 to 100 mg / mL, 1 to 50 mg / mL, 10 to 30 mg / mL, 20 mg / mL.

[0027] In some embodiments of the present invention, the concentration of the diol diacrylate polymer is 0.01 to 1000 mg / mL, such as 0.1 to 500 mg / mL, 10 to 300 mg / mL, 50 to 200 mg / mL, 150 mg / mL.

[0028] In some embodiments of the present invention, the concentration of the hydrogel microneedle matrix is 0.1 to 1000 mg / mL, such as 0.1 to 500 mg / mL, 10 to 300 mg / mL, 1 to 200 mg / mL.

[0029] In some embodiments of the present invention, the mass ratio of the hydrogel microneedle matrix to the drug-loaded nanoparticles is 1 to 500:1, such as 10 to 400:1, 50 to 300:1, 100 to 200:1, 170:1.

[0030] In some embodiments of the present invention, the mass-volume concentration of the photoinitiator is 0.1% to 10%, such as 0.5% to 1.5%, 1%; the photoinitiator includes photoinitiator 2959.

[0031] In some embodiments of the present invention, the photocrosslinking is in-situ photocrosslinking; in the present invention, the drug-loaded hydrogel microneedles are prepared by an in-situ photocrosslinking reaction, which can avoid cumbersome preparation processes.

[0032] In some embodiments of the present invention, the photocrosslinking is carried out under ultraviolet light irradiation; the intensity of the ultraviolet light irradiation is 10 - 1000 W, such as 100 - 800 W, 200 - 700 W, 300 - 600 W; the reaction time of the photocrosslinking reaction is 10 - 600 s; such as 10 - 90 s, 20 - 80 s, 30 - 70 s, etc. If the ultraviolet light irradiation time is too long (>600 s), the activity of the loaded drug is easily damaged; if the ultraviolet light irradiation time is too short (<10 s), it is difficult to form a hydrogel or the mechanical strength is poor.

[0033] In some embodiments of the present invention, the method for preparing the drug-loaded nanoparticles includes mixing a cationic polymer modified with phenylboronic acid and a protein hypoglycemic drug to obtain the drug-loaded nanoparticles; the concentration of the protein hypoglycemic drug is 0.001 - 10 mg / mL, such as 0.01 - 10 mg / mL, 0.05 - 1 mg / mL, 0.1 mg / mL.

[0034] In some embodiments of the present invention, the method for preparing the phenylboronic acid-modified cationic polymer includes the following steps: activating carboxylphenylboronic acid and then adding a cationic polymer for reaction to obtain the phenylboronic acid-modified cationic polymer; the temperature of the reaction is 20 - 35 °C; the reaction time is 12 - 48 h; the activating agent used for the activation includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); the method for preparing the phenylboronic acid-modified cationic polymer further includes purifying the reaction product; the specific operation of the purification treatment includes dialysis and freeze-drying.

[0035] In some embodiments of the present invention, the method for preparing the natural polyphenol-modified methacrylated chitosan includes the following steps: activating the natural polyphenol and then adding it to react with methacrylated chitosan to obtain the natural polyphenol-modified methacrylated chitosan; the temperature of the reaction is 20 - 35 °C; the reaction time is 12 - 48 h; the activating agent used for the activation includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); the method for preparing the natural polyphenol-modified methacrylated chitosan further includes purifying the reaction product; the specific operation of the purification treatment includes dialysis and freeze-drying.

[0036] In the third aspect of the present invention, a microneedle patch is proposed, which includes a plurality of the drug-loaded hydrogel microneedles and a plate on which the drug-loaded hydrogel microneedles stand and are arranged.

[0037] Among them, the materials of the hydrogel microneedles and the plate in the drug-loaded hydrogel microneedles can be the same or different.

[0038] The polymeric material forming the plate can be hydrophilic or water-soluble under certain conditions, but forms a water-insoluble cross-linked structure after physical or chemical cross-linking treatment.

[0039] Among them, in the microneedle sheet, the distance between the tips of the microneedles can be the conventional distance between the tips of microneedles in the art, such as 100-1000 μm, and for example, 200 μm, 700 μm or 800 μm.

[0040] In some embodiments of the present invention, the microneedle shafts are arranged in an array on the plate.

[0041] In some embodiments of the present invention, the array density of the microneedle shafts on the plate is 10-800 needles / (cm 2 plate).

[0042] In a fourth aspect of the present invention, there is provided an application of the medicated hydrogel microneedles or the microneedle sheet described above in the preparation of diabetes drugs.

[0043] In some embodiments of the present invention, the diabetes drugs include diabetes wound repair materials.

[0044] In some embodiments of the present invention, the diabetes wounds include diabetes skin ulcer wounds, such as diabetes chronic skin ulcer wounds.

[0045] The beneficial effects of the present invention are as follows:

[0046] The medicated hydrogel microneedles of the present invention have excellent biocompatibility. Through the triple therapy strategy of breaking bacterial biofilms, improving the oxidative stress response and high blood sugar levels in the wound, the inflammatory response can be effectively reduced. It can be used to deliver different types of protein-based hypoglycemic drugs that promote wound healing. Through intelligent drug delivery, it can promote the repair of blood vessels and tissues in the wound, and effectively solve a series of problems such as poor bioavailability and poor treatment effect of existing hydrogel dressings.

[0047] The present invention is an extension and expansion of the in-depth research on hydrogel dressings in the field of DFU treatment. HMN can effectively remove deep bacterial biofilms and play an antioxidant role. It has excellent DPPH free radical scavenging ability, can significantly improve the intracellular ROS level, provides a good microenvironment, and the microchannels formed by the microneedles greatly improve the problem of poor permeability of traditional dressings, significantly improving the bioavailability of drugs, greatly enhancing the treatment effect of DFU, and is expected to provide new ideas and new preparations for the efficient repair of DFU, with important scientific research and clinical significance.

[0048] The novel hydrogel microneedle preparation obtained by the present invention can also effectively break bacterial biofilms, significantly inhibit the growth and proliferation of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and is of great significance for the healing of severely infected skin ulcer wounds. Compared with conventional antibacterial drug microneedles, the microneedle matrix polymer molecules themselves have antibacterial activity, enabling a longer-lasting antibacterial effect. In addition, through the cross-linking of dual polymer molecules, the mechanical strength of its microneedles can be controlled by the polymer ratio rather than by adjusting the drying time (which can reduce the risk of drug molecule inactivation caused by too long drying process), making it more operable.

[0049] The preparation method of the drug-loaded hydrogel microneedles of the present invention has the advantages of good gel-forming performance in the reaction, simple preparation process, easy implementation, green and safe, etc., can be widely promoted and applied on a large scale, and has excellent application prospects in the preparation of repair materials for diabetic skin ulcer wounds. Description of the Drawings

[0050] Figure 1 is the microscopic morphology diagram of the hydrogel microneedles of the present invention (scale bar is 1000 μm).

[0051] Figure 2 is the microscopic morphology diagram of the hydrogel microneedles of the present invention (scale bar is 250 μm).

[0052] Figure 3 is the hemolysis experiment of the hydrogel microneedles of Example 1 and Comparative Examples 2 and 3 of the present invention.

[0053] Figure 4 is the transdermal performance of the hydrogel microneedles of Example 1 of the present invention.

[0054] Figure 5 is the drug-loading performance of the hydrogel microneedles of Comparative Example 2 of the present invention.

[0055] Figure 6 is the drug release performance of the hydrogel microneedles of Example 1 of the present invention.

[0056] Figure 7 is the antioxidant performance of the components of the hydrogel microneedles of the present invention.

[0057] Figure 8 is the antibacterial performance of the hydrogel microneedles of Example 1 and Comparative Examples 2 and 3 of the present invention.

[0058] Figure 9 is the anti-biofilm performance of the hydrogel microneedles of Example 1 and the hydrogel dressing of Comparative Example 4 of the present invention.

[0059] Figure 3 and Figure 9 In, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001. Detailed implementation manners

[0060] The content of the present invention will be further described in detail through specific embodiments below. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0061] Example 1

[0062] In this example, a drug-loaded hydrogel microneedle was prepared. The specific process was as follows:

[0063] S1: Insulin was dissolved in 0.1 M dilute hydrochloric acid and mixed with phenylboronic acid-modified polylysine molecules (PLPBA) according to a mass ratio of 1:50 to obtain drug-loaded nanoparticles (NPs).

[0064] S2: Gallic acid-modified methacrylated chitosan (CMG) and polyethylene glycol diacrylate (PEGDA) were dissolved in purified water and mixed according to a mass ratio of 20:150, and the concentration of PEGDA was controlled to be 150 mg / mL to obtain a hydrogel microneedle matrix mixture solution.

[0065] S3: The NPs obtained in step S1 were mixed with the mixture solution obtained in S2 according to a mass ratio of 1:170 to obtain a hydrogel microneedle precursor solution. A photoinitiator 2959 (dissolved in absolute ethanol) with a mass-volume concentration of 0.1% was added so that the final concentration of insulin was 0.1 mg / mL. The mixture solution was transferred to a microneedle plastic mold, centrifuged at 4000 rpm to enrich the NPs at the tip of the microneedles, the air bubbles were removed by vacuum, and irradiated with 400 W ultraviolet light for 60 s to obtain a drug-loaded hydrogel microneedle preparation (CSMAGA / PEGDA@PLPBA@insulin, abbreviated as CMGPN).

[0066] Among them, the specific preparation method of the phenylboronic acid-modified polylysine molecule (PLPBA) was as follows: 5 g of 4-carboxyphenylboronic acid was dissolved in 60 mL of dimethyl sulfoxide. Then, 6.5 g of EDC and 3.45 g of NHS were respectively dissolved in 20 ml of dimethyl sulfoxide and added to the former solution for amino group activation for 1 h. The activated solution was slowly added dropwise to the ε-polylysine solution under ice bath conditions and stirred at room temperature (20 °C) for 24 h. Finally, it was dialyzed in deionized water for 3 d (500 Da) and freeze-dried to obtain the product.

[0067] The specific preparation method of gallic acid-modified methacrylated chitosan (CMG) is as follows: Dissolve 3 g of gallic acid in 120 mL of 2-morpholinoethanesulfonic acid buffer solution (0.1 M, pH = 5.5), and dissolve 3.3 g of EDC and 2.1 g of NHS in the former solution for 2 h of activation of ester bond groups. Slowly add the activated solution dropwise to the methacrylated chitosan solution, and stir and react at room temperature (20 °C) for 24 h. Finally, dialyze in deionized water for 5 d (14 kDa), and freeze-dry to obtain the product.

[0068] Example 2

[0069] In this example, a drug-loaded hydrogel microneedle was prepared, and the specific process was as follows:

[0070] S1: Dissolve insulin with 0.1 M dilute hydrochloric acid, and mix it with phenylboronic acid-modified polylysine molecules (PLPBA) according to a mass ratio of 1:50 to obtain drug-loaded nanoparticles (NPs);

[0071] S2: Dissolve gallic acid-modified methacrylated chitosan (CMG) and polyethylene glycol diacrylate (PEGDA) with purified water, and mix them according to a mass ratio of 20:100 to obtain a hydrogel microneedle matrix mixture solution;

[0072] S3: Mix the NPs obtained in step S1 with the mixture solution obtained in S2 according to a mass ratio of 1:120 to obtain a hydrogel microneedle precursor solution. Add a photoinitiator 2959 with a mass-volume concentration of 0.1% (dissolved in absolute ethanol) to make the final concentration of insulin 0.1 mg / mL. Transfer the mixture solution to a plastic mold, centrifuge at 4000 rpm to enrich the NPs at the tip of the microneedles, evacuate to remove air bubbles, and irradiate with ultraviolet light (400 W) for 60 s to obtain the drug-loaded hydrogel microneedle preparation (CSMAGA / PEGDA@PLPBA@insulin, abbreviated as CMGPN-1).

[0073] Example 3

[0074] In this example, a drug-loaded hydrogel microneedle was prepared, and the specific process was as follows:

[0075] S1: Dissolve insulin with 0.1 M dilute hydrochloric acid, and mix it with phenylboronic acid-modified polylysine molecules (PLPBA) according to a mass ratio of 1:50 to obtain drug-loaded nanoparticles (NPs);

[0076] S2: Dissolve gallic acid-modified methacrylated chitosan (CMG) and polyethylene glycol diacrylate (PEGDA) with purified water, and mix them according to a mass ratio of 10:150 to obtain a hydrogel microneedle matrix mixture solution;

[0077] S3: Mix the NPs obtained in step S1 and the mixture solution obtained in S2 at a mass ratio of 1:160 to obtain a hydrogel microneedle precursor solution. Add a photoinitiator 2959 with a mass-volume concentration of 0.1% (dissolved in absolute ethanol) to make the final concentration of insulin 0.1 mg / mL. Transfer the mixture solution to a plastic mold, centrifuge at 4000 rpm to enrich the NPs at the tip of the microneedles, evacuate to remove air bubbles, and irradiate with ultraviolet light (400 W) for 60 s to obtain a drug-loaded hydrogel microneedle preparation (CSMAGA / PEGDA@PLPBA@insulin, abbreviated as CMGPN-2).

[0078] Comparative Example 1

[0079] A hydrogel microneedle (HMN) was prepared in this comparative example. The difference from Example 1 was that insulin was not added, and the rest of the preparation method was the same as that of Example 1. The specific process was as follows:

[0080] S1: Dissolve the polylysine molecule modified with phenylboronic acid (PLPBA) in 0.1 M dilute hydrochloric acid and mix;

[0081] S2: Dissolve chitosan methacrylate modified with gallic acid (CMG) and polyethylene glycol diacrylate (PEGDA) in purified water, and mix at a mass ratio of 20:150 to obtain a hydrogel microneedle matrix mixture solution;

[0082] S3: Mix the mixture solutions obtained in steps S1 and S2 at a mass ratio of 1:170 to obtain a hydrogel microneedle precursor solution. Add a photoinitiator 2959 with a mass-volume concentration of 0.1% (dissolved in absolute ethanol) to make the final concentration of PLPBA 5 mg / mL. Transfer the mixture solution to a microneedle plastic mold, centrifuge at 4000 rpm to enrich the PLPBA at the tip of the microneedles, evacuate to remove air bubbles, and irradiate with ultraviolet light for 60 s to obtain a hydrogel microneedle preparation (CSMAGA / PEGDA@PLPBA, abbreviated as CMGPP).

[0083] Comparative Example 2

[0084] A hydrogel microneedle was prepared in this comparative example. The difference from Example 1 was that insulin and polylysine modified with phenylboronic acid were not added, and the rest of the preparation method was the same as that of Example 1. The specific process was as follows:

[0085] S1: Dissolve chitosan methacrylate modified with gallic acid (CMG) and polyethylene glycol diacrylate (PEGDA), and mix at a mass ratio of 150:20 to obtain a hydrogel microneedle matrix mixture solution;

[0086] S2: Add photoinitiator 2959 with a mass - volume concentration of 0.1% (dissolved in absolute ethanol) to the hydrogel microneedle matrix mixture solution. Transfer the mixture solution to a microneedle plastic mold, evacuate to remove air bubbles, and irradiate with ultraviolet light for 60 s to obtain a hydrogel microneedle preparation (CSMAGA / PEGDA, abbreviated as CMGP).

[0087] Comparative Example 3

[0088] A hydrogel microneedle was prepared in this comparative example. The difference from Example 1 is that insulin, polylysine modified with phenylboronic acid, and polyethylene glycol diacrylate are not added, and the rest of the preparation method is the same as that of Example 1. The specific process is as follows:

[0089] S1: Dissolve methylacrylated chitosan modified with gallic acid (CMG) to obtain a hydrogel microneedle matrix mixture solution;

[0090] S2: Add photoinitiator 2959 with a mass - volume concentration of 0.1% (dissolved in absolute ethanol) to the hydrogel microneedle matrix mixture solution so that the final concentration of CSMAGA is 100 mg / mL. Transfer the mixture solution to a microneedle plastic mold, evacuate to remove air bubbles, and irradiate with ultraviolet light for 60 s to obtain a hydrogel microneedle preparation (CSMAGA, abbreviated as CMG).

[0091] Comparative Example 4

[0092] A hydrogel dressing was prepared in this comparative example. The difference from Example 1 is that a microneedle mold is not used, and the rest of the preparation method is the same as that of Example 1. The specific process is as follows:

[0093] S1: Dissolve insulin with 0.1 M dilute hydrochloric acid, and mix it with polylysine molecules modified with phenylboronic acid (PLPBA) according to a mass ratio of 1:50 to obtain drug - loaded nanoparticles (NPs);

[0094] S2: Dissolve methylacrylated chitosan modified with gallic acid (CMG) and polyethylene glycol diacrylate (PEGDA) with purified water, mix them according to a mass ratio of 20:150, and control the concentration of PEGDA to be 150 mg / mL to obtain a hydrogel microneedle matrix mixture solution;

[0095] S3: Mix the NPs obtained in step S1 with the mixture solution obtained in S2 according to a mass ratio of 1:170 to obtain a hydrogel microneedle precursor solution. Add photoinitiator 2959 with a mass - volume concentration of 0.1% (dissolved in absolute ethanol) so that the final concentration of insulin is 0.1 mg / mL. Transfer the mixture solution to a flat plastic mold, evacuate to remove air bubbles, and irradiate with ultraviolet light for 60 s to obtain a hydrogel preparation (CSMAGA / PEGDA@PLPBA, abbreviated as CMGPN gel).

[0096] Test Example 1

[0097] In this test example, the microscopic morphology of the hydrogel microneedles was characterized. The specific process was as follows:

[0098] Observation and research were carried out using a scanning electron microscope (SEM).

[0099] The microscopic array structure of the hydrogel microneedles is one of the key factors to ensure their efficient penetration of the barrier, disruption of the bacterial biofilm, and improvement of bioavailability. The SEM images of the drug-loaded hydrogel microneedles prepared in Example 1 are as shown in Figure 1 and Figure 2 It can be seen that the tip structure of the hydrogel microneedle preparation is complete and uniform, and there is no collapse or fracture phenomenon. This may be attributed to the high-strength cross-linking of gallic acid-modified methacrylated chitosan (CMG) and polyethylene glycol diacrylate (PEGDA), which endows the hydrogel microneedles with better mechanical strength. For this hydrogel microneedle system, on the one hand, drug loading does not affect the microscopic structure and gel-forming properties of the hydrogel microneedles; on the other hand, it also shows great promise in disrupting the biofilm and improving bioavailability by the deep penetration of the tip part into the deep tissue and the synergistic antibacterial groups.

[0100] Test Example 2

[0101] In this test example, the biocompatibility of the hydrogel microneedles was evaluated. The specific process was as follows:

[0102] The hydrogel microneedles prepared in Example 1 and Comparative Example 3, and methacrylated chitosan (CM) were respectively mixed with 20 μL of blood cells. Incubated at 37 °C for 12 h, and then centrifuged at 3000 rpm for 15 min to evaluate the effect of the hydrogel microneedles on red blood cell hemolysis. PBS and water were used as negative and positive control groups respectively.

[0103] The red blood cell hemolysis test is one of the important means to evaluate the blood compatibility of drug delivery systems. In this test example, the hemolysis of red blood cells by the hydrogel microneedle system within 12 h at room temperature was evaluated. The results are as shown in Figure 3 As shown, like the negative control, the hemolysis rate of each group of hydrogel microneedles was within 5%, indicating that it did not cause red blood cell hemolysis. This may be because the HMN system was constructed using polymer molecules with excellent biocompatibility and the usage concentration was within the safe range. It can be seen that the HMN system of the present invention has excellent blood compatibility and has significant advantages in deep tissue administration as a drug delivery system.

[0104] Test Example 3

[0105] In this test example, the transdermal performance of the hydrogel microneedles was evaluated. The specific process was as follows:

[0106] Take the rat epithelium, use forceps to pick up the hydrogel microneedles prepared in Example 1 and lay them flat on the skin surface with the needle tips facing the skin. Press the microneedles loaded with methylene blue against the skin with a force of about 10 N for 10 min, and observe through a stereomicroscope whether the rat skin is successfully punctured by the microneedles.

[0107] The transdermal performance of the hydrogel microneedles is a key performance for breaking through the bacterial biofilm and improving bioavailability. In this test example, rat skin was used as a model to preliminarily evaluate the transdermal performance of the hydrogel microneedles. The results are as Figure 4 shown. The hydrogel microneedles can effectively break through the blocking barrier of the skin stratum corneum and deliver the dye loaded at the needle tips to the deep tissue. This may be due to the excellent mechanical properties of the hydrogel microneedles and the combined effect of a suitable needle structure. It can be speculated that the hydrogel microneedles of the present invention have great prospects in loading drug-loaded nanoparticles to break through the bacterial biofilm and forming a drug release channel to release drugs deep into the wound internal environment.

[0108] Test Example 4

[0109] In this test example, the drug loading performance of the hydrogel microneedles was evaluated. The specific process was as follows:

[0110] To verify that PLPBA@insulin NPs are effectively loaded into the tips of HMNs, in this test example, FITC-labeled insulin was used as a model drug, and the drug loading performance of HMNs was evaluated by fluorescence visualization of NPs. HMNs were prepared according to Example 1. Subsequently, a stereomicroscope was used to observe the fluorescence loading.

[0111] The results are as Figure 5 shown. Obvious green can be seen inside the hydrogel microneedles, indicating that PLPBA@insulin was effectively loaded inside the hydrogel microneedles and located at the needle tip rather than the base.

[0112] Application Example 1

[0113] In this application example, the drug release performance of the hydrogel microneedles was evaluated. The specific process was as follows:

[0114] Covalently bind FITC fluorescent small molecules to insulin in Example 1, and then perform subsequent operations to prepare the corresponding hydrogel microneedles, which were respectively placed in the corresponding solutions (pH = 5.5, 10 mM H2O2, 5 mg / mL Glu), and the fluorescence intensity changes were observed after 24 h.

[0115] The results are as Figure 6As shown, it can be seen that after being placed in the solution (pH = 5.5, 10 mM H2O2, 5 mg / mL Glu) for 24 h, a significant decrease in fluorescence intensity was observed for HMN, indicating that the hydrogel microneedles can trigger drug release in an environment of weak acid, high ROS, and high sugar. Thus, it can be known that the hydrogel microneedle preparation has great potential in the treatment of diseases such as chronic diabetic skin wounds.

[0116] Application Example 2

[0117] In this application example, the antioxidant performance of the hydrogel microneedles was evaluated. The specific process was as follows:

[0118] The antioxidant ability of the hydrogel was evaluated by the DPPH scavenging experiment. The hydrogel microneedle sample of Example 1, methacrylated chitosan (CM), and gallic acid (GA) were placed in a centrifuge tube containing 5 mL of DPPH solution and reacted for 30 min under dark conditions at room temperature. Using absolute ethanol as a blank control, the absorbance of the solution after the reaction was measured at a wavelength of 517 nm using a UV-visible spectrophotometer.

[0119] The results are as Figure 7 shown. It can be seen that methacrylated chitosan (CM) without gallic acid modification hardly has antioxidant activity; both gallic acid monomer (GA) and the hydrogel microneedles of Example 1 (CMGPN) exhibit excellent antioxidant activity, significantly reducing the color of DPPH radicals. Thus, it can be known that the modification of gallic acid further endows the hydrogel microneedle matrix with antioxidant activity.

[0120] Application Example 3

[0121] In this application example, the antibacterial performance of the hydrogel microneedles was evaluated. The specific process was as follows:

[0122] The colonies on the agar culture dishes treated with the hydrogel microneedles prepared in Example 1 and Comparative Example 3 and methacrylated chitosan (CM) were gently washed with PBS solution. (Remove HMN and take the treated bacteria for staining).

[0123] SYTO 9 (1.5 μL) and PI (1.5 μL) were diluted in PBS solution (1 mL), and the colonies were stained with the mixed dye in the dark for 30 min.

[0124] Then, after washing three times with PBS solution, the fluorescence images of the colonies were recorded using a fluorescence microscope, and the obtained fluorescence images were analyzed using Leica software.

[0125] SYTO 9 can label all bacteria in a population, including bacteria with intact membranes and damaged membranes; conversely, when two dyes are added to the system, PI can only penetrate into damaged membranes, and the insertion of PI will cause a decrease in the fluorescence of SYTO 9 staining. The results are as Figure 8 shown. The total number of bacteria in the hydrogel microneedles was effectively inhibited (the inhibition rate exceeded 99%), and the proportion of dead bacteria also increased successively with the increase of the components, further indicating that the hydrogel microneedles have excellent antibacterial properties.

[0126] Application Example 4

[0127] This application example evaluates the anti-biofilm performance of the hydrogel microneedles. The specific process is as follows:

[0128] The coverslips with biofilms grown on them were taken out from the 24-well culture plates, washed twice with PBS solution to remove unattached bacteria, and then treated with the hydrogel microneedles of Example 1 (CMGPN HMN) and the hydrogel dressing of Comparative Example 4 (CMGPN gel). Then, the coverslips were placed into new sterile 24-well culture plates, and the biofilms were washed twice with PBS solution to remove floating bacteria and substances. 500 μL of anhydrous methanol was added to each well of the biofilms for fixation for 30 min. Then, 0.1% CV dye was added to each well for staining for 15 min. After staining was completed, the biofilms in each well were gently washed twice with PBS solution and dried at room temperature. Finally, the CV bound to the biofilms was dissolved in anhydrous ethanol and incubated for 30 min. The extract was transferred to a 96-well plate and the ultraviolet-visible absorbance of all samples at 550 nm was measured to quantify the biomass of the biofilms. A camera was used to record the color of the CV bound to the biofilms in each treatment group.

[0129] The results are as Figure 9 shown. The biofilm clearance rates of the hydrogel microneedles and the hydrogel dressing were 71.27 ± 6.021% and 37.10 ± 9.615% respectively, indicating that the hydrogel microneedles have better biofilm clearance ability compared to the gel.

[0130] The above results show that: the hydrogel microneedle preparation of the present invention has excellent biocompatibility, combines multiple functions such as breaking bacterial biofilms, antioxidant and anti-infection, maximizes the therapeutic effect, and can encapsulate different types of protein hypoglycemic drugs that promote wound healing, thereby significantly improving the bioavailability of the drugs, reducing adverse reactions and toxic side effects, and having an excellent effect of promoting the repair of chronic wounds in vivo, which also confirms the effectiveness of the design of the multifunctional hydrogel microneedle preparation of the present invention.

[0131] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A drug-loaded hydrogel microneedle, characterized in that: Comprising: Drug-loaded nanoparticles, including protein hypoglycemic drugs loaded on cationic polymers modified with phenylboronic acid; Hydrogel microneedles, including methylacrylated chitosan modified with natural polyphenols and diol diacrylate polymers; The drug-loaded nanoparticles are loaded on the hydrogel microneedles.

2. The medicated hydrogel microneedle according to claim 1, wherein: The drug-loaded nanoparticles are loaded on the tips of the hydrogel microneedles.

3. The medicated hydrogel microneedle according to claim 1, characterized in that: The mass ratio of the drug-loaded nanoparticles to the hydrogel microneedles is 1:1 to 2000.

4. The medicated hydrogel microneedle according to claim 1, wherein: In the drug-loaded nanoparticles, the mass ratio of the cationic polymer modified with phenylboronic acid to the protein hypoglycemic drug is 1 to 100:1; and / or, in the cationic polymer modified with phenylboronic acid, the cationic polymer includes at least one of polylysine, polyglutamic acid, polyethyleneimine, polypropyleneimine, polyamidoamine, polyester amide, and polyethyl methacrylate 2-(dimethylamine); and / or, the protein hypoglycemic drug includes insulin and / or a GLP-1 receptor agonist, wherein the GLP-1 receptor agonist is selected from at least one of exenatide, lysine, lixisenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, taspoglutide, benaglutide, dulaglutide, loxisenatide, and semaglutide.

5. The medicated hydrogel microneedle according to claim 1, wherein: In the hydrogel microneedles, the mass ratio of the methylacrylated chitosan modified with natural polyphenols to the diol diacrylate polymer is 1:3 to 10; and / or, in the methylacrylated chitosan modified with natural polyphenols, the mass ratio of the natural polyphenols to the methylacrylated chitosan is 1 to 10:10 to 1; and / or, the natural polyphenols include at least one of gallic acid, epigallocatechin gallate, epicatechin, catechin, tea polyphenols, protocatechuic acid, chlorogenic acid, caffeic acid, rutin, and tannic acid; and / or, the diol diacrylate polymer includes at least one of polyethylene glycol acrylate, polyethylene glycol methacrylate, polyethylene glycol dimethacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, and polyethylene glycol dimethacrylate.

6. A preparation method of the medicated hydrogel microneedles according to any one of claims 1 to 5, characterized in that: Including the following steps: Mix the methylacrylated chitosan modified with natural polyphenols and the diol diacrylate polymer, add the drug-loaded nanoparticles and a photoinitiator, and prepare the drug-loaded hydrogel microneedles after a photocrosslinking reaction.

7. The preparation method of the drug-loaded hydrogel microneedles according to claim 6, characterized in that: The preparation method of the drug-loaded hydrogel microneedles includes the following steps: Mix the methylacrylated chitosan modified with natural polyphenols and the diol diacrylate polymer to prepare a hydrogel microneedle matrix; add the drug-loaded nanoparticles and a photoinitiator to obtain a mixed solution, transfer the mixed solution to a microneedle mold, centrifuge to enrich the drug-loaded nanoparticles at the tips of the microneedles, and prepare the drug-loaded hydrogel microneedles after a photocrosslinking reaction.

8. A microneedle patch, characterized in that: Including several drug-loaded hydrogel microneedles according to any one of claims 1 to 5 and a plate on which the drug-loaded hydrogel microneedles stand and are arranged.

9. Use of a drug-loaded hydrogel microneedle according to any one of claims 1 to 5 or a microneedle sheet according to claim 8 in the preparation of a diabetes drug.

10. The application according to claim 9, wherein: The diabetes drug includes a diabetes wound repair material.

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