Drug-loaded multi-stimulation response type hydrogel microneedle and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a drug-loaded multi-stimulation response type hydrogel microneedle and a preparation method thereof. The drug-loaded multi-stimulus responsive hydrogel microneedle is prepared from phenylboronic acid grafted methacrylated hyaluronic acid, methacrylated gelatin, drug molecules and strontium-doped hydroxyapatite modified by polyaspartic acid. The drug-loaded multi-stimulus-responsive hydrogel microneedle has pH and ROS dual responses at the same time, can efficiently release drug molecules in a specific acid environment or ROS environment, and has good biodegradability and biocompatibility, so that the drug-loaded multi-stimulus-responsive hydrogel microneedle can be used for preparing the drug-loaded multi-stimulus-responsive hydrogel microneedle, and the drug-loaded multi-stimulus-responsive hydrogel microneedle can be used for preparing the drug-loaded multi-stimulus-responsive hydrogel microneedle. The application of the hydrogel microneedle in the field of drug delivery is expanded, and the hydrogel microneedle has important medical significance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a drug-loaded multi-stimulus responsive hydrogel microneedle and a preparation method thereof. Background Art

[0002] As an emerging drug delivery method, microneedles can not only avoid the pain and discomfort caused by traditional injections, but also achieve efficient and accurate drug delivery through micron-sized needle tips penetrating the surface of the skin. Therefore, they show broad application prospects in the fields of local drug delivery, vaccination, skin treatment, etc. At present, the research on microneedles mainly focuses on solid microneedles, hollow microneedles, and hydrogel microneedles developed in recent years. Among them, solid microneedles are complicated to operate, hollow microneedles are easy to break, and these two types of microneedles are mostly made of materials such as metals and silicon, and have poor biocompatibility.

[0003] In contrast, hydrogel microneedles have become a major research hotspot in the field of microneedles due to their good biodegradability.

[0004] The Chinese patent technology with publication number CN 117860657 A discloses a responsive high-strength hydrogel microneedle, preparation method and application. The responsive high-strength hydrogel microneedle is prepared from raw materials including acrylonitrile, acrylic acid and polyethylene glycol diacrylate, has pH responsiveness and can release drugs under specific pH conditions. However, the responsive high-strength hydrogel microneedle in the patent only has a single pH responsiveness and lacks multiple stimulus responsiveness (for example, it has more than two responses such as pH and reactive oxygen species at the same time), which greatly limits the application of hydrogel microneedles in the field of drug delivery.

[0005] Therefore, developing a responsive hydrogel microneedle that can simultaneously respond to multiple stimuli (such as pH, reactive oxygen species, etc.) is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0006] The present invention provides a drug-loaded multi-stimulus-responsive hydrogel microneedle. The drug-loaded multi-stimulus-responsive hydrogel microneedle has dual responses to pH and reactive oxygen species (ROS), can efficiently release drug molecules in a specific acidic environment or a ROS environment, and has good biodegradability and biocompatibility, which is conducive to expanding the application of hydrogel microneedles in the field of drug delivery and has important medical significance.

[0007] The present invention also provides a method for preparing a drug-loaded multi-stimulus responsive hydrogel microneedle, by which the drug-loaded multi-stimulus responsive hydrogel microneedle having dual responses to pH and reactive oxygen species (ROS) and having good biodegradability and biocompatibility can be prepared.

[0008] The first aspect of the present invention provides a drug-loaded multi-stimuli responsive hydrogel microneedle, and its preparation raw materials include phenylboronic acid grafted methacrylated hyaluronic acid, methacrylated gelatin, drug molecules, and strontium-doped hydroxyapatite modified with polyaspartic acid.

[0009] For the drug-loaded multi-stimuli responsive hydrogel microneedle as described above, in the drug-loaded multi-stimuli responsive hydrogel microneedle, the mass ratio of the phenylboronic acid grafted methacrylated hyaluronic acid, the methacrylated gelatin, the drug molecules, and the strontium-doped hydroxyapatite modified with polyaspartic acid is (10 - 20):(10 - 20):1:(0.2 - 0.4).

[0010] For the drug-loaded multi-stimuli responsive hydrogel microneedle as described above, the preparation process of the strontium-doped hydroxyapatite modified with polyaspartic acid is as follows:

[0011] Dissolve polyaspartic acid in the first deionized water to obtain an aqueous polyaspartic acid solution; use the aqueous polyaspartic acid solution to impregnate the strontium-doped hydroxyapatite, and the precipitate obtained after the first centrifugation treatment is the strontium-doped hydroxyapatite modified with polyaspartic acid.

[0012] For the drug-loaded multi-stimuli responsive hydrogel microneedle as described above, in the aqueous polyaspartic acid solution, the mass concentration of polyaspartic acid is 0.1 - 0.4 g / mL, and the mass ratio of polyaspartic acid to the strontium-doped hydroxyapatite is (10 - 40):1.

[0013] For the drug-loaded multi-stimuli responsive hydrogel microneedle as described above, the drug molecules include dihydromyricetin.

[0014] The second aspect of the present invention provides a preparation method of the above-mentioned drug-loaded multi-stimuli responsive hydrogel microneedle, including the following steps:

[0015] Dissolve the phenylboronic acid grafted methacrylated hyaluronic acid and the methacrylated gelatin in the second deionized water, and add the strontium-doped hydroxyapatite modified with polyaspartic acid after the first stirring treatment to obtain a gel solution for the microneedle body;

[0016] Dissolve polyvinylpyrrolidone and polyvinyl alcohol in the third deionized water, and obtain a solution for the microneedle substrate after the second stirring treatment;

[0017] Dissolve the drug molecules in ethanol to obtain a drug solution, and add the drug solution to the gel solution of the microneedle body to obtain a gel solution containing the drug;

[0018] Mix the drug-containing gel solution with the photoinitiator LAP, pour the mixture into a microneedle mold, add the solution of the microneedle substrate after the second centrifugation treatment, irradiate it under ultraviolet light, and obtain the drug-loaded multi-stimuli responsive hydrogel microneedles after drying treatment.

[0019] For the preparation method of the drug-loaded multi-stimuli responsive hydrogel microneedles as described above, in the solution of the microneedle substrate, the dosage ratio of the polyvinylpyrrolidone, the polyvinyl alcohol and the third deionized water is (1 - 2 g):(0.5 - 1 g):(5 - 10 mL);

[0020] And / or, in the drug solution, the mass concentration of the drug molecules is 40 - 50 mg / mL.

[0021] For the preparation method of the drug-loaded multi-stimuli responsive hydrogel microneedles as described above, the irradiation under ultraviolet light includes: irradiating under ultraviolet light with a wavelength of 365 - 405 nm for 30 s.

[0022] For the preparation method of the drug-loaded multi-stimuli responsive hydrogel microneedles as described above, the preparation process of the phenylboronic acid grafted methacrylated hyaluronic acid is as follows:

[0023] Dissolve hyaluronic acid in the fourth deionized water, then add methacrylic anhydride to obtain a first raw material system, adjust the pH value of the first raw material system and then carry out a stirring reaction to obtain a first product; place the first product in a first dialysis bag and carry out a first dialysis treatment with the fifth deionized water, and obtain methacrylated hyaluronic acid after the first freeze-drying;

[0024] Dissolve the methacrylated hyaluronic acid in the sixth deionized water and add DMTMM, then add 3-aminophenylboronic acid to obtain a second raw material system, carry out a light-shielding reaction on the second raw material system to obtain a second product; place the second product in a second dialysis bag and carry out a second dialysis treatment with the seventh deionized water, and obtain the phenylboronic acid grafted methacrylated hyaluronic acid after the second freeze-drying.

[0025] For the preparation method of the drug-loaded multi-stimuli responsive hydrogel microneedles as described above, in the first raw material system, the dosage ratio of the hyaluronic acid, the fourth deionized water and the methacrylic anhydride is 1 g:100 mL:1 mL;

[0026] And / or, in the second raw material system, the dosage ratio of the methacrylated hyaluronic acid, the sixth deionized water, the DMTMM and the 3-aminophenylboronic acid is 1 g:100 mL:1.4 g:0.11 g;

[0027] And / or, adjusting the pH value of the first raw material system includes: adjusting the pH value of the first raw material system to 8.0 - 8.5;

[0028] And / or, the first dialysis bag is a dialysis bag with a molecular weight cut-off of 10,000 Da - 14,000 Da;

[0029] And / or, the second dialysis bag is a dialysis bag with a molecular weight cut-off of 10,000 Da - 14,000 Da.

[0030] Compared with the prior art, the solution of the present invention has at least the following effects:

[0031] The multi-stimuli responsive hydrogel microneedles loaded with drugs provided by the present invention are prepared from raw materials including phenylboronic acid grafted methacrylated hyaluronic acid, methacrylated gelatin, drug molecules, and strontium-doped hydroxyapatite modified with polyaspartic acid. The multi-stimuli responsive hydrogel microneedles loaded with drugs have both pH and reactive oxygen species (ROS) dual responses, can efficiently release drug molecules in a specific acidic environment or ROS environment, and also have good biodegradability and biocompatibility, which is beneficial to expanding the application of hydrogel microneedles in the field of drug delivery and has important medical significance. Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0033] Figure 1 1H NMR spectra of hyaluronic acid (HA), methacrylated hyaluronic acid (HAMA), and phenylboronic acid grafted methacrylated hyaluronic acid (HAMA-PBA) in Example 1 of the present invention;

[0034] Figure 2 Scanning electron microscope image of strontium-doped hydroxyapatite in Example 1 of the present invention;

[0035] Figure 3 Scanning electron microscope image of strontium-doped hydroxyapatite modified with polyaspartic acid in Example 1 of the present invention;

[0036] Figure 4 Scanning electron microscope image of multiple needle tips in the multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention;

[0037] Figure 5 Cell viability test result graph of the present invention;

[0038] Figure 6 This is the degradation result diagram of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Example 3 in a solution with pH = 5.6;

[0039] Figure 7 This is the degradation result diagram of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Example 3 in a solution with a concentration of 1 mmol / L H2O2;

[0040] Figure 8 This is the in vitro drug release curve of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Examples 1-2 in a solution with pH = 5.6;

[0041] Figure 9 This is the in vitro drug release curve of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Examples 1-2 in a solution with a concentration of 1 mmol / L H2O2. Detailed implementation manners

[0042] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0043] It should be noted that the descriptions involving "first", "second", "third", "fourth", "fifth", "sixth", "seventh", etc. in the present invention are used to distinguish similar objects, rather than to describe a specific order or sequence, and thus should not be construed as a limitation to the present invention.

[0044] The first aspect of the present invention provides a drug-loaded multi-stimuli responsive hydrogel microneedle, and its preparation raw materials include phenylboronic acid grafted methacrylated hyaluronic acid, methacrylated gelatin, drug molecules, and strontium-doped hydroxyapatite modified with polyaspartic acid.

[0045] Specifically, the object of the present invention for preparation is a multi-stimuli responsive hydrogel microneedle loaded with drugs. The present invention prepares a multi-stimuli responsive hydrogel microneedle loaded with drugs from raw materials including phenylboronic acid grafted methacrylated hyaluronic acid, methacrylated gelatin, drug molecules, and strontium-doped hydroxyapatite modified with polyaspartic acid. The multi-stimuli responsive hydrogel microneedle loaded with drugs has dual responses to pH and reactive oxygen species (ROS), can efficiently release drug molecules in a specific acidic environment or ROS environment, and also has good biodegradability and biocompatibility.

[0046] The principle of the present invention is explained as follows: The construction of this dual response system is based on the synergistic effect of two functionalized biomaterials. First, the phenylboronic acid ester bond introduced in phenylboronic acid grafted methacrylated hyaluronic acid has sensitivity to reactive oxygen species (ROS). When the concentration of ROS such as hydrogen peroxide (H2O2) or hydroxyl radical (·OH) in the environment increases, the ester bond will undergo oxidative cleavage, triggering the specific dissociation of the carrier. Second, methacrylated gelatin (GelMA) has pH response characteristics through the protonation effect of amino groups. Under weakly acidic conditions, the charge state and swelling degree of its three-dimensional network will change significantly, realizing drug release triggered by the acidic environment touch. Therefore, the multi-stimuli responsive hydrogel microneedle loaded with drugs provided by the present invention has a ROS / pH dual response mechanism, which can accurately match the dynamic microenvironment characteristics of pathological tissues and provide a spatiotemporally controllable release strategy for the intelligent drug delivery system.

[0047] The present invention does not particularly limit the specific method for preparing methacrylated gelatin, and it can be prepared according to the methods known in the art. In some embodiments, the preparation process of methacrylated gelatin includes:

[0048] Dissolve gelatin in phosphate buffer solution, then add methacrylic anhydride for reaction, place the reaction product in a dialysis bag and perform dialysis treatment with deionized water, and obtain methacrylated gelatin after freeze-drying.

[0049] In a specific embodiment, in the multi-stimuli responsive hydrogel microneedle loaded with drugs, the mass ratio of the phenylboronic acid grafted methacrylated hyaluronic acid, the methacrylated gelatin, the drug molecules, and the strontium-doped hydroxyapatite modified with polyaspartic acid is (10 - 20):(10 - 20):1:(0.2 - 0.4).

[0050] When the parameters of the mass ratio of phenylboronic acid grafted methacrylated hyaluronic acid, methacrylated gelatin, drug molecules, and strontium-doped hydroxyapatite modified with polyaspartic acid in the above-mentioned drug-loaded multi-stimuli responsive hydrogel microneedles are within the above ranges, the composite matrix of phenylboronic acid grafted hyaluronic acid and methacrylated gelatin can precisely release drugs in a high reactive oxygen / acidic microenvironment. At the same time, strontium-doped hydroxyapatite modified with polyaspartic acid has the effect of promoting alveolar bone regeneration, which is beneficial to preparing a drug-loaded multi-stimuli responsive hydrogel microneedle with dual responses to pH and reactive oxygen species (ROS), capable of efficiently releasing drug molecules in a specific acidic environment or ROS environment, and having good biodegradability and biocompatibility.

[0051] In a specific embodiment, the preparation process of the strontium-doped hydroxyapatite modified with polyaspartic acid is as follows:

[0052] Dissolve polyaspartic acid in deionized water for the first time to obtain an aqueous polyaspartic acid solution; use the aqueous polyaspartic acid solution to impregnate strontium-doped hydroxyapatite, and the precipitate obtained after the first centrifugation treatment is the strontium-doped hydroxyapatite modified with polyaspartic acid.

[0053] Specifically, in the present invention, polyaspartic acid is dissolved in deionized water for the first time to obtain an aqueous polyaspartic acid solution; then the strontium-doped hydroxyapatite is impregnated with the aqueous polyaspartic acid solution, so that the polyaspartic acid in the aqueous polyaspartic acid solution modifies the strontium-doped hydroxyapatite and is loaded on at least part of the pores and / or at least part of the surface of the strontium-doped hydroxyapatite to obtain the solution after the impregnation treatment; finally, the solution after the impregnation treatment is subjected to the first centrifugation treatment, and the precipitate obtained after the first centrifugation treatment is the strontium-doped hydroxyapatite modified with polyaspartic acid, which can be used to prepare a drug-loaded multi-stimuli responsive hydrogel microneedle with dual responses to pH and reactive oxygen species (ROS), capable of efficiently releasing drug molecules in a specific acidic environment or ROS environment, and having good biodegradability and biocompatibility.

[0054] The present invention does not particularly limit the specific method for preparing strontium-doped hydroxyapatite, and it can be prepared according to the methods well-known to those skilled in the art. In some embodiments, the preparation method of the strontium-doped hydroxyapatite of the present invention includes:

[0055] Dissolve diammonium hydrogen phosphate in the eighth deionized water, adjust the pH to 10 - 11 to obtain an aqueous solution of diammonium hydrogen phosphate; dissolve calcium nitrate tetrahydrate and strontium nitrate in the ninth deionized water to form a mixed solution, heat the mixed solution, add the aqueous solution of diammonium hydrogen phosphate to the heated mixed solution under stirring, and then add polyethylene glycol - 400 for reaction to obtain a reacted solution; successively carry out precipitation aging, centrifugation, washing, drying and grinding on the reacted solution to obtain strontium - doped hydroxyapatite.

[0056] In a specific embodiment, in the aqueous solution of polyaspartic acid, the mass concentration of polyaspartic acid is 0.1 - 0.4 g / mL, and the mass ratio of polyaspartic acid to the strontium - doped hydroxyapatite is (10 - 40):1.

[0057] In a specific embodiment, the drug molecule includes dihydromyricetin.

[0058] The drug - loaded multi - stimulus - responsive hydrogel microneedles of the present invention can efficiently release drug molecules in a specific acidic environment or ROS environment. When the drug molecule in the drug - loaded multi - stimulus - responsive hydrogel microneedles is dihydromyricetin, the release process of dihydromyricetin can be regulated to play a sustained - release role.

[0059] The second aspect of the present invention provides a preparation method of the above - mentioned drug - loaded multi - stimulus - responsive hydrogel microneedles, including the following steps:

[0060] Dissolve phenylboronic acid - grafted methacrylated hyaluronic acid and methacrylated gelatin in the second deionized water, add strontium - doped hydroxyapatite modified with polyaspartic acid after the first stirring treatment to obtain a gel solution for the microneedle body;

[0061] Dissolve polyvinylpyrrolidone and polyvinyl alcohol in the third deionized water to obtain a solution for the microneedle substrate after the second stirring treatment;

[0062] Dissolve the drug molecule in ethanol to obtain a drug solution, and add the drug solution to the gel solution of the microneedle body to obtain a gel solution containing the drug;

[0063] Mix the gel solution containing the drug with the photoinitiator LAP, pour it into a microneedle mold, add the solution of the microneedle substrate after the second centrifugation treatment, irradiate it under ultraviolet light, and obtain the drug - loaded multi - stimulus - responsive hydrogel microneedles after drying treatment.

[0064] The present invention does not make special limitations on the specific dosage and specific source of the used photoinitiator LAP, and it can be selected according to specific needs.

[0065] Through the above preparation method, the present invention can prepare a drug-loaded multi-stimuli responsive hydrogel microneedle with dual responses to pH and reactive oxygen species (ROS), which can efficiently release drug molecules in a specific acidic environment or ROS environment and has good biodegradability and biocompatibility.

[0066] In a specific embodiment, in the solution of the microneedle substrate, the dosage ratio of polyvinylpyrrolidone, polyvinyl alcohol and the third deionized water is (1 - 2 g):(0.5 - 1 g):(5 - 10 mL).

[0067] When the parameters of the dosage ratio of polyvinylpyrrolidone, polyvinyl alcohol and the third deionized water in the solution of the microneedle substrate are within the above ranges, it can enable better matching and interaction among polyvinylpyrrolidone, polyvinyl alcohol and the third deionized water, thereby obtaining the solution of the microneedle substrate.

[0068] In a specific embodiment, in the drug solution, the mass concentration of the drug molecule is 40 - 50 mg / mL.

[0069] When the mass concentration of the drug molecule in the above drug solution is within the above range, it can enable the drug molecule to be better loaded into the hydrogel microneedle, obtaining a drug-loaded multi-stimuli responsive hydrogel microneedle that can efficiently release drug molecules in a specific acidic environment or ROS environment.

[0070] In a specific embodiment, the irradiation under ultraviolet light includes: irradiating for 30 s under ultraviolet light with a wavelength of 365 - 405 nm.

[0071] When irradiating for 30 s under ultraviolet light with a wavelength of 365 nm - 405 nm, it can effectively excite the photoinitiator LAP, thereby initiating the cross-linking reaction between the gel solution containing the drug and the solution of the microneedle substrate.

[0072] In a specific embodiment, the preparation process of phenylboronic acid grafted methacrylated hyaluronic acid is as follows:

[0073] Dissolve hyaluronic acid in the fourth deionized water, then add methacrylic anhydride to obtain a first raw material system, adjust the pH value of the first raw material system and then carry out a stirring reaction to obtain a first product; place the first product in a first dialysis bag and carry out a first dialysis treatment with the fifth deionized water, and obtain methacrylated hyaluronic acid after the first freeze-drying;

[0074] Dissolve the methacrylated hyaluronic acid in deionized water for the sixth time and add DMTMM, then add 3-aminophenylboronic acid to obtain a second raw material system, and carry out a light-shielded reaction on the second raw material system to obtain a second product; place the second product in a second dialysis bag and perform a second dialysis treatment with deionized water for the seventh time, and obtain the phenylboronic acid-grafted methacrylated hyaluronic acid after the second freeze-drying.

[0075] The present invention does not particularly limit the specific parameters of the temperature and time of the above stirring reaction, and can be selected according to specific needs. In a specific embodiment, the temperature of the stirring reaction can be 4 °C and the time can be 24 h.

[0076] The present invention does not particularly limit the specific parameters of the temperature and time of the above light-shielded reaction, and can be selected according to specific needs. In a specific embodiment, the temperature of the light-shielded reaction can be 25 °C and the time can be 24 h.

[0077] The present invention does not particularly limit the amounts of deionized water for the fifth time and deionized water for the seventh time, and can be selected according to actual needs.

[0078] The present invention can prepare phenylboronic acid-grafted methacrylated hyaluronic acid through the above preparation process. This phenylboronic acid-grafted methacrylated hyaluronic acid can be used to prepare drug-loaded multi-stimuli responsive hydrogel microneedles with dual responses to pH and reactive oxygen species (ROS), which can efficiently release drug molecules in a specific acidic environment or ROS environment, and have good biodegradability and biocompatibility.

[0079] In a specific embodiment, in the first raw material system, the dosage ratio of the hyaluronic acid, the deionized water for the fourth time, and the methacrylic anhydride is 1 g: 100 mL: 1 mL.

[0080] When the parameters of the dosage ratio of the hyaluronic acid, the deionized water for the fourth time, and the methacrylic anhydride in the above first raw material system are within the above range, the grafting rate and photo-crosslinking efficiency of the methacrylated hyaluronic acid can be optimized, while maintaining the flexibility and biological activity of the polymer chain, and balancing the mechanical properties and biological functions.

[0081] In a specific embodiment, in the second raw material system, the dosage ratio of the methacrylated hyaluronic acid, the deionized water for the sixth time, the DMTMM, and the 3-aminophenylboronic acid is 1 g: 100 mL: 1.4 g: 0.11 g.

[0082] When the parameters of the dosage ratios of the methacryloylated hyaluronic acid, the sixth deionized water, the DMTMM, and the 3-aminophenylboronic acid in the above second raw material system are within the above ranges, a high grafting rate can be achieved while maintaining the backbone stability of HAMA; the reaction efficiency is improved under the catalysis of DMTMM, and the residue of by-products is avoided. The density of the phenylborate ester bond is precisely regulated to balance the ROS response rate and the photocrosslinking performance.

[0083] In a specific embodiment, adjusting the pH value of the first raw material system includes: adjusting the pH value of the first raw material system to 8.0 - 8.5.

[0084] When the pH value of the first raw material system is within the above range, maintaining the pH value at 8.0 - 8.5 can preferentially activate the primary amino group of hyaluronic acid, promote the directional grafting of methacrylic anhydride, and at the same time inhibit the side reaction of ester bond hydrolysis, ensure the integrity of the polymer chain and the photocrosslinking stability, and retain the natural receptor-binding activity of hyaluronic acid.

[0085] In a specific embodiment, the first dialysis bag is a dialysis bag with a molecular weight cut-off of 10000Da - 14000Da.

[0086] When the molecular weight cut-off of the first dialysis bag is within the above range, unreacted monomers (hyaluronic acid, methacrylic anhydride) and small molecule by-products can be efficiently removed, while the backbone structure with a molecular weight greater than 14kDa is retained, avoiding the loss of high molecular weight active ingredients, and maintaining the stability of the grafting group density.

[0087] In a specific embodiment, the second dialysis bag is a dialysis bag with a molecular weight cut-off of 10000Da - 14000Da.

[0088] When the molecular weight cut-off of the second dialysis bag is within the above range, unreacted monomers (methacryloylated hyaluronic acid, 3-aminophenylboronic acid) and small molecule by-products can be efficiently removed, while the backbone structure with a molecular weight greater than 14kDa is retained, avoiding the loss of high molecular weight active ingredients, and maintaining the stability of the grafting group density.

[0089] The following is a detailed description of a drug-loaded multi-stimuli responsive hydrogel microneedle and its preparation method provided by the present invention in combination with examples, but they should not be construed as limiting the protection scope of the present invention.

[0090] Example 1

[0091] The drug-loaded multi-stimuli responsive hydrogel microneedle of this example is prepared by a method including the following steps:

[0092] (1) Preparation of phenylboronic acid-grafted methacryloylated hyaluronic acid

[0093] Dissolve 1 g of hyaluronic acid in 100 mL of deionized water, then add 1 mL of methacrylic anhydride to obtain the first raw material system. After adjusting the pH value of the first raw material system to 8.5 with 1 mol / L sodium hydroxide solution, carry out a stirring reaction at 4 °C for 24 h to obtain the first product; place the first product in a dialysis bag with a molecular weight cut-off of 10,000 Da - 14,000 Da and perform dialysis treatment with deionized water for 96 hours (h), and obtain methacryloylated hyaluronic acid after freeze-drying.

[0094] Dissolve 1 g of methacryloylated hyaluronic acid in 100 mL of deionized water and add 1.4 g of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM) to activate the carboxyl groups on hyaluronic acid, then add 0.11 g of 3-aminophenylboronic acid to obtain the second raw material system, and carry out a light-shielding reaction on the second raw material system at 25 °C for 24 h to obtain the second product; place the second product in a dialysis bag with a molecular weight cut-off of 10,000 Da - 14,000 Da and perform dialysis treatment with deionized water for 72 hours, and obtain phenylboronic acid-grafted methacryloylated hyaluronic acid after freeze-drying.

[0095] (2) Preparation of methacryloylated gelatin

[0096] Dissolve 2 g of gelatin in phosphate buffer solution (pH = 7.5) at 50 °C to obtain a gelatin solution; then add 4 mL of methacrylic anhydride to the gelatin solution under stirring of a magnetic stirrer and react for 3 hours. Place the reacted product in a dialysis bag and perform dialysis treatment with deionized water at 40 °C for 72 hours, and obtain methacryloylated gelatin after freeze-drying.

[0097] (3) Preparation of strontium-doped hydroxyapatite modified with polyaspartic acid

[0098] Preparation of strontium-doped hydroxyapatite:

[0099] Dissolve 3.96 g of diammonium hydrogen phosphate in 50 mL of deionized water and adjust the pH to 10.5 with ammonia water to obtain an aqueous solution of diammonium hydrogen phosphate; dissolve 10.62 g of calcium nitrate tetrahydrate and 1.06 g of strontium nitrate in 50 mL of deionized water to form a mixed solution, heat the mixed solution to 45 °C using a water bath, add the aqueous solution of diammonium hydrogen phosphate to the heated mixed solution under stirring, then add 3.47 g of polyethylene glycol-400 and react at 45 °C for 1 hour to obtain the reacted solution; carry out precipitation aging on the reacted solution at room temperature for 24 hours, then centrifuge, wash with absolute ethanol, dry in a vacuum oven at 80 °C for 24 hours and grind to obtain strontium-doped hydroxyapatite.

[0100] Preparation of polyaspartic acid-modified strontium-doped hydroxyapatite:

[0101] Dissolve 2 g of polyaspartic acid in 10 mL of deionized water to obtain an aqueous polyaspartic acid solution; use the aqueous polyaspartic acid solution to impregnate 50 mg of strontium-doped hydroxyapatite, and the precipitate obtained after centrifugation is polyaspartic acid-modified strontium-doped hydroxyapatite.

[0102] (4) Preparation of drug-loaded multi-responsive hydrogel microneedles

[0103] Dissolve 0.5 g of phenylboronic acid-grafted methacrylated hyaluronic acid and 0.5 g of methacrylated gelatin in 10 mL of deionized water, add 10 mg of polyaspartic acid-modified strontium-doped hydroxyapatite after stirring to obtain a gel solution for the microneedle body;

[0104] Dissolve 2 g of polyvinylpyrrolidone and 0.5 g of polyvinyl alcohol in 7.5 mL of deionized water, and obtain a solution for the microneedle substrate after stirring;

[0105] Dissolve 50 mg of dihydromyricetin in 1 mL of ethanol to obtain a dihydromyricetin solution, and add the dihydromyricetin solution to the gel solution of the microneedle body to obtain a gel solution containing dihydromyricetin;

[0106] Mix the gel solution containing dihydromyricetin with 5 μL of photoinitiator LAP (CAS No.: 85073-19-4), pour it into a microneedle mold, centrifuge it at a speed of 3500 rpm for 5 min using a horizontal rotor centrifuge, scrape off the excess solution after centrifugation with a spatula, and then irradiate it with ultraviolet light at a wavelength of 405 nm for 30 s. After drying, drug-loaded multi-responsive hydrogel microneedles are obtained.

[0107] Example 2

[0108] The preparation method of the drug-loaded multi-responsive hydrogel microneedles provided in this example is basically the same as that in Example 1, except that:

[0109] (4) Preparation of drug-loaded multi-responsive hydrogel microneedles

[0110] Dissolve 1 g of phenylboronic acid-grafted methacrylated hyaluronic acid and 1 g of methacrylated gelatin in 10 mL of deionized water, add 10 mg of polyaspartic acid-modified strontium-doped hydroxyapatite after stirring to obtain a gel solution for the microneedle body;

[0111] Dissolve 2 g of polyvinylpyrrolidone and 0.5 g of polyvinyl alcohol in 7.5 mL of deionized water, and obtain a solution for the microneedle substrate after stirring;

[0112] Dissolve 50 mg of dihydromyricetin in 1 mL of ethanol to obtain a dihydromyricetin solution, and add the dihydromyricetin solution to the gel solution of the microneedle body to obtain a gel solution containing dihydromyricetin;

[0113] Mix the gel solution containing dihydromyricetin with 5 μL of photoinitiator LAP, pour the mixture into a microneedle mold, centrifuge it at a speed of 3500 rpm for 5 min using a horizontal rotor centrifuge, scrape off the excess solution after centrifugation with a spatula, and then irradiate it with ultraviolet light at a wavelength of 405 nm for 30 s. After drying, a multi-stimuli responsive hydrogel microneedle loaded with the drug is obtained.

[0114] Comparative Example 1 (without adding phenylboronic acid-grafted methacrylated hyaluronic acid)

[0115] The preparation method of the hydrogel microneedle loaded with the drug provided in this comparative example is basically the same as that of Example 1, except that:

[0116] (4) Preparation of multi-stimuli responsive hydrogel microneedles loaded with drugs

[0117] Dissolve 0.5 g of methacrylated gelatin in 10 mL of deionized water, add 1 mg of strontium-doped hydroxyapatite modified with polyaspartic acid after stirring to obtain a gel solution of the microneedle body;

[0118] Dissolve 2 g of polyvinylpyrrolidone and 0.5 g of polyvinyl alcohol in 7.5 mL of deionized water, and obtain a solution of the microneedle substrate after stirring;

[0119] Dissolve 50 mg of dihydromyricetin in 1 mL of ethanol to obtain a dihydromyricetin solution, and add the dihydromyricetin solution to the gel solution of the microneedle body to obtain a gel solution containing dihydromyricetin;

[0120] Mix the gel solution containing dihydromyricetin with 5 μL of photoinitiator LAP, pour the mixture into a microneedle mold, centrifuge it at a speed of 3500 rpm for 5 min using a horizontal rotor centrifuge, scrape off the excess solution after centrifugation with a spatula, and then irradiate it with ultraviolet light at a wavelength of 405 nm for 30 s. After drying, a multi-stimuli responsive hydrogel microneedle loaded with the drug is obtained.

[0121] Comparative Example 2 (without adding methacrylated gelatin)

[0122] The preparation method of the hydrogel microneedle loaded with the drug provided in this comparative example is basically the same as that of Example 1, except that:

[0123] (4) Preparation of multi-stimuli responsive hydrogel microneedles loaded with drugs

[0124] Dissolve 0.5 g of phenylboronic acid grafted methacrylated hyaluronic acid in 10 mL of deionized water. After stirring, add 10 mg of strontium-doped hydroxyapatite modified with polyaspartic acid to obtain the gel solution for the microneedle body.

[0125] Dissolve 2 g of polyvinylpyrrolidone and 0.5 g of polyvinyl alcohol in 7.5 mL of deionized water. After stirring, obtain the solution for the microneedle substrate.

[0126] Dissolve 50 mg of dihydromyricetin in 1 mL of ethanol to obtain a dihydromyricetin solution. Add the dihydromyricetin solution to the gel solution of the microneedle body to obtain a gel solution containing dihydromyricetin.

[0127] Mix the gel solution containing dihydromyricetin with 5 μL of photoinitiator LAP and pour it into a microneedle mold. After centrifuging at a speed of 3500 rpm for 5 min using a horizontal rotor centrifuge, scrape off the excess solution after centrifugation. Then, irradiate it with ultraviolet light at a wavelength of 405 nm for 30 s. After drying, obtain the multi-stimuli responsive hydrogel microneedles loaded with drugs.

[0128] Comparative Example 3 (without adding strontium-doped hydroxyapatite modified with polyaspartic acid)

[0129] The preparation method of the hydrogel microneedles loaded with drugs provided in this comparative example is basically the same as that of Example 1, except that:

[0130] (4) Preparation of multi-stimuli responsive hydrogel microneedles loaded with drugs

[0131] Dissolve 0.5 g of phenylboronic acid grafted methacrylated hyaluronic acid and 0.5 g of methacrylated gelatin in 10 mL of deionized water. After stirring, obtain the gel solution for the microneedle body.

[0132] Dissolve 2 g of polyvinylpyrrolidone and 0.5 g of polyvinyl alcohol in 7.5 mL of deionized water. After stirring, obtain the solution for the microneedle substrate.

[0133] Dissolve 50 mg of dihydromyricetin in 1 mL of ethanol to obtain a dihydromyricetin solution. Add the dihydromyricetin solution to the gel solution of the microneedle body to obtain a gel solution containing dihydromyricetin.

[0134] Mix the gel solution containing dihydromyricetin with 5 μL of photoinitiator LAP and pour it into a microneedle mold. After centrifuging at a speed of 3500 rpm for 5 min using a horizontal rotor centrifuge, scrape off the excess solution after centrifugation. Then, irradiate it with ultraviolet light at a wavelength of 405 nm for 30 s. After drying, obtain the multi-stimuli responsive hydrogel microneedles loaded with drugs.

[0135] Result description

[0136] 1. The proton nuclear magnetic resonance spectra of hyaluronic acid (HA), methacrylated hyaluronic acid (HAMA), and phenylboronic acid grafted methacrylated hyaluronic acid (HAMA-PBA) in Example 1 of the present invention were tested respectively, and the test results are as Figure 1 shown.

[0137] As Figure 1 can be seen, the chemical shift at 7.5 - 8.0 ppm corresponds to the hydrogen atoms on the benzene ring in the phenylboronic acid group, and the characteristic peaks of the hydrogen atoms on the double bond are at 5.59 ppm and 6.22 ppm, which proves the successful synthesis of phenylboronic acid grafted methacrylated hyaluronic acid (HAMA-PBA).

[0138] 2. The strontium-doped hydroxyapatite and strontium-doped hydroxyapatite modified with polyaspartic acid in Example 1 of the present invention were tested by scanning electron microscopy respectively; Figure 2 This is the scanning electron micrograph of the strontium-doped hydroxyapatite in Example 1 of the present invention, Figure 3 This is the scanning electron micrograph of the strontium-doped hydroxyapatite modified with polyaspartic acid in Example 1 of the present invention.

[0139] As Figure 2 and Figure 3 can be seen, in the strontium-doped hydroxyapatite modified with polyaspartic acid in Example 1 of the present invention, polyaspartic acid is adsorbed on the surface of the strontium-doped hydroxyapatite.

[0140] 3. The multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention were tested by scanning electron microscopy, Figure 4 This is the scanning electron micrograph of multiple needle tips in the multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention.

[0141] As Figure 4 can be seen, the multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention have regular morphology, uniform needle tip morphology, and the needle tip is a conical structure.

[0142] 4. Cytotoxicity evaluation

[0143] The cell viability test was carried out on the multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention.

[0144] Experimental method: The effect of drug-loaded multi-stimuli responsive hydrogel microneedles on the cytotoxicity of mouse fibroblasts (L929) was determined by the CCK-8 method. Before the cytotoxicity test, L929 cells in the logarithmic growth phase were collected in advance. The L929 cells were seeded in a 24-well plate at an inoculation density of 20,000 cells per well, and supplemented with complete medium (by mass fraction, the complete medium consists of 89% high-glucose DMEM medium, 10% fetal bovine serum (FBS), and 1% triple antibody (penicillin-streptomycin-amphotericin B solution)) at 1 mL per well. After culturing for 24 h, the cells were allowed to adhere and grow completely. Then, the pre-sterilized by ultraviolet drug-loaded multi-stimuli responsive hydrogel microneedles were added to the 24-well plate. The group without adding the drug-loaded multi-stimuli responsive hydrogel microneedles was used as the blank control group. After co-incubating for 24 h, the medium was removed, washed with PBS solution, 100 μL of complete medium containing 10 μL of CCK-8 was added, and after co-incubating for 2 h, 100 μL of the medium was aspirated from each well to a new 96-well plate, and the absorbance was measured using a microplate reader at a wavelength of 450 nm, and the cell viability was calculated.

[0145] According to the above experimental method, the drug-loaded multi-stimuli responsive hydrogel microneedles with 10, 20, and 30 needle tips in Example 1 were respectively taken for cell viability tests, and the results are as Figure 5 shown;

[0146] It can be Figure 5 seen that after treating L929 cells with the drug-loaded multi-stimuli responsive hydrogel microneedles provided in the examples of the present invention, the L929 cells still have high cell viability, indicating that the drug-loaded multi-stimuli responsive hydrogel microneedles provided in the examples of the present invention have good biocompatibility and no cytotoxicity.

[0147] 5. Biodegradability test

[0148] The drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Example 3 were respectively immersed in a solution with pH = 5.6 and a solution with a concentration of 1 mmol / L H2O2 for biodegradability tests; Figure 6 is the degradation result diagram of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Example 3 in a solution with pH = 5.6, Figure 7 is the degradation result diagram of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention and the drug-loaded hydrogel microneedles in Comparative Example 3 in a solution with a concentration of 1 mmol / L H2O2.

[0149] It can be Figure 6 and Figure 7It can be seen that in the PBS buffer solution with pH = 5.6 and in the PBS buffer solution with a concentration of 1 mmol / L H2O2, the multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention will gradually degrade and release strontium-doped hydroxyapatite within 60 minutes, while the hydrogel microneedles loaded with drugs in Comparative Example 3 do not release strontium-doped hydroxyapatite. The above results illustrate that the multi-stimuli responsive hydrogel microneedles loaded with drugs provided in the examples of the present invention have good biodegradability.

[0150] 6. In vitro release experiment

[0151] (1) pH responsiveness

[0152] The multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention and the hydrogel microneedles loaded with drugs in Comparative Examples 1-2 were respectively immersed in 10 mL of a solution with pH = 5.6, and then placed in a shaking incubator at 37 °C and 100 rpm for shaking. At regular time intervals, 1 mL of the release solution was taken, and 1 mL of fresh solution (solution with pH = 5.6) was added. The absorbance of the released drug (dihydromyricetin) was measured using a microplate reader, and the cumulative release amount of the drug (cumulative release) was calculated according to the release standard curve. The results are as Figure 8 shown.

[0153] As Figure 8 can be seen, compared with Comparative Examples 1-2, the multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention have the largest cumulative drug release amount within 72 hours in the solution with pH = 5.6, indicating that the multi-stimuli responsive hydrogel microneedles loaded with drugs provided by the present invention have excellent pH responsiveness and can efficiently release drugs in a specific acidic environment.

[0154] (2) ROS responsiveness

[0155] The multi-stimuli responsive hydrogel microneedles loaded with drugs in Example 1 of the present invention and the hydrogel microneedles loaded with drugs in Comparative Examples 1-2 were respectively immersed in 10 mL of a solution with a concentration of 1 mmol / L H2O2, and then placed in a shaking incubator at 37 °C and 100 rpm for shaking. At regular time intervals, 1 mL of the release solution was taken, and 1 mL of fresh solution (solution with a concentration of 1 mmol / L H2O2) was added. The absorbance of the released drug (dihydromyricetin) was measured using a microplate reader, and the cumulative release amount of the drug (cumulative release) was calculated according to the release standard curve. The results are as Figure 9 shown.

[0156] As Figure 9It can be seen that, compared with Comparative Examples 1-2, in the solution with a concentration of 1 mmol / L H2O2, the drug cumulative release amount of the drug-loaded multi-stimuli responsive hydrogel microneedles in Example 1 of the present invention is the largest, indicating that the drug-loaded multi-stimuli responsive hydrogel microneedles provided by the present invention have excellent ROS responsiveness and can efficiently release drugs in a specific ROS environment.

[0157] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A drug-loaded multi-stimulus responsive hydrogel microneedle, characterized in that: The preparation raw materials include phenylboronic acid grafted methacryloyl hyaluronic acid, methacryloyl gelatin, drug molecules and polyaspartic acid modified strontium-doped hydroxyapatite.

2. The drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 1, characterized in that: In the drug-loaded multi-stimulus responsive hydrogel microneedle, the mass ratio of the phenylboronic acid-grafted methacryloyl hyaluronic acid, the methacryloyl gelatin, the drug molecule and the polyaspartic acid-modified strontium-doped hydroxyapatite is (10-20): (10-20): 1: (0.2-0.4).

3. The drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 1, characterized in that: The preparation process of the polyaspartic acid-modified strontium-doped hydroxyapatite is as follows: Dissolving polyaspartic acid in first deionized water to obtain a polyaspartic acid aqueous solution; using the polyaspartic acid aqueous solution to perform an impregnation treatment on strontium-doped hydroxyapatite, and obtaining a precipitate after a first centrifugal treatment, namely the polyaspartic acid-modified strontium-doped hydroxyapatite.

4. The drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 3, characterized in that: In the polyaspartic acid aqueous solution, the mass concentration of polyaspartic acid is 0.1-0.4 g / mL, and the mass ratio of the polyaspartic acid to the strontium-doped hydroxyapatite is (10-40):

1.

5. The drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 1, characterized in that: The drug molecules include dihydromyricetin.

6. A method for preparing the drug-loaded multi-stimulus responsive hydrogel microneedle according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dissolving phenylboronic acid-grafted methacryloyl hyaluronic acid and methacryloyl gelatin in a second deionized water, adding polyaspartic acid-modified strontium-doped hydroxyapatite after a first stirring treatment to obtain a gel solution of a microneedle body; Dissolving polyvinyl pyrrolidone and polyvinyl alcohol in a third deionized water, and obtaining a solution of a microneedle substrate after a second stirring treatment; Dissolving drug molecules in ethanol to obtain a drug solution, and adding the drug solution to the gel solution of the microneedle body to obtain a gel solution containing the drug; The drug-containing gel solution is mixed with the photoinitiator LAP and poured into a microneedle mold. After a second centrifugation treatment, the solution of the microneedle substrate is added, irradiated under ultraviolet light, and dried to obtain the drug-loaded multi-stimulus responsive hydrogel microneedles.

7. The method for preparing the drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 6, characterized in that: In the solution of the microneedle substrate, the amount ratio of the polyvinyl pyrrolidone, the polyvinyl alcohol and the third deionized water is (1-2 g): (0.5-1 g): (5-10 mL); And / or, in the drug solution, the mass concentration of the drug molecules is 40-50 mg / mL.

8. The method for preparing the drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 6, characterized in that: The irradiating under ultraviolet light includes: irradiating under ultraviolet light with a wavelength of 365-405nm for 30s.

9. The method for preparing the drug-loaded multi-stimulus responsive hydrogel microneedle according to claim 6, characterized in that: The preparation process of the phenylboronic acid grafted methacryloyl hyaluronic acid is as follows: Dissolving hyaluronic acid in fourth deionized water, then adding methacrylic anhydride to obtain a first raw material system, adjusting the pH value of the first raw material system and stirring the reaction to obtain a first product; placing the first product in a first dialysis bag and performing a first dialysis treatment with fifth deionized water, and performing a first freeze-drying to obtain methacryloyl hyaluronic acid; The methacryloyl hyaluronic acid is dissolved in the sixth deionized water and DMTMM is added, and then 3-aminophenylboronic acid is added to obtain a second raw material system, and the second raw material system is subjected to a light-proof reaction to obtain a second product; the second product is placed in a second dialysis bag and subjected to a second dialysis treatment with the seventh deionized water, and the phenylboronic acid-grafted methacryloyl hyaluronic acid is obtained after a second freeze-drying.

10. The method for preparing drug-loaded multi-stimulus responsive hydrogel microneedles according to claim 9, characterized in that: In the first raw material system, the usage ratio of the hyaluronic acid, the fourth deionized water and the methacrylic anhydride is 1 g: 100 mL: 1 mL; and / or, in the second raw material system, the usage ratio of the methacryloyl hyaluronic acid, the sixth deionized water, the DMTMM and the 3-aminophenylboronic acid is 1 g: 100 mL: 1.4 g: 0.11 g; and / or, adjusting the pH value of the first raw material system, including: adjusting the pH value of the first raw material system to 8.0-8.5; And / or, the first dialysis bag is a dialysis bag with a molecular weight cut-off of 10000Da-14000Da; And / or, the second dialysis bag is a dialysis bag with a molecular weight cutoff of 10000Da-14000Da.

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