Hydrogel microneedle patch as well as preparation method and application thereof

Abstract

The invention discloses a hydrogel microneedle patch and a preparation method and application thereof.The hydrogel microneedle patch is characterized in that a gel main body is obtained by mixing PEG600 and PEG1000 according to the mass ratio of 3: 4, erythromycin, vaccarin and 7-demethylated corticin are loaded, a temperature response type gel microneedle is prepared, DG is in a solid needle shape at the room temperature, and the temperature response type gel microneedle patch is a hydrogel microneedle patch. Under the action of external force, the medicine can penetrate through a biological membrane secreted by bacteria and then is quickly melted into liquid by virtue of body temperature, and loaded medicine is released; in the invention, the wound healing is promoted through the synergistic effect between the hydrogel and the medicine. The hydrogel microneedle patch prepared by the invention can remarkably promote rapid healing of a staphylococcus aureus infected wound surface and can remarkably promote rapid healing of a back wound surface of a diabetic rat.

Description

Technical Field

[0001] The invention relates to a hydrogel microneedle patch and a preparation method and application thereof, belonging to the field of pharmaceutical preparations. Background Art

[0002] Chronic wounds are difficult to heal due to persistent infection, persistent inflammation, and impaired angiogenesis. Many patients with chronic wounds also suffer from underlying diseases such as hypertension, diabetes, and wound infection. These patients often suffer from bacterial infections, mainly Gram-positive cocci and Staphylococcus aureus. Therefore, targeting bacterial infection, maintaining a moist wound environment, and regulating inflammation are essential for the treatment of chronic wounds. Although a large number of nanomaterials have been reported to have strong antibacterial effects, topical antibiotics are still the first choice for the treatment of local infections in chronic wounds. When microorganisms proliferate in the wound, their colonies are wrapped by autocrine matrices to form a protective biofilm, which hinders the penetration of antimicrobial agents, produces drug resistance, and delays wound healing. Biofilms are associated with approximately 60% of chronic wounds. Therefore, a delivery method that can penetrate the biofilm and release drugs directly to the site of infection without causing additional damage is an ideal method for the treatment of chronic wounds.

[0003] Microneedle patches are a transdermal drug delivery route that has developed rapidly in recent years. By adjusting the size and height of the microneedles, they can penetrate the stratum corneum of the skin without damaging the nerve endings and capillaries in the dermis, without causing tingling or bleeding. They are easy to use and have high safety. Among them, soluble microneedles are microneedles made of water-soluble or biodegradable polymer materials or sugars. They can completely dissolve or degrade in the skin, and the loaded drugs are released along with the dissolution of the microneedles. Compared with other microneedles, soluble microneedles can eliminate secondary contamination of needles. However, the existing microneedle patches cannot provide the necessary conditions for effective chronic wound healing in a more comprehensive manner when treating chronic wounds, such as solving the problems of wound infection, inflammatory phase blockade and angiogenesis at the same time. Summary of the invention

[0004] In order to solve the deficiencies of the prior art, the present invention prepares a multifunctional microneedle patch for chronic wound healing. The microneedle patch prepared by the present invention has a significant healing-promoting effect in the treatment of diabetic wounds and infectious wounds.

[0005] The present invention is achieved through the following technical solutions: The first object of the present invention is to provide a hydrogel microneedle patch, comprising a backing hydrogel and a hydrogel microneedle (DG) adhered to the backing hydrogel (BHG); the hydrogel microneedle is prepared by mixing PEG600 and PEG1000 in a mass ratio of 3:4 to obtain a gel body, and loading erythromycin, fenugreek flavonoid glycosides and 7-demethyl scutellariae.

[0006] In one embodiment of the present invention, in the hydrogel microneedles, 0.95%-1.05% erythromycin, 0.095%-0.105% vaccariae flavonoid glycosides and 0.095%-0.105% 7-demethylsuberin are loaded respectively by mass ratio.

[0007] The present invention prepares a temperature-responsive gel by using PEG600 and PEG1000 in a certain ratio. At room temperature, DG is in a solid needle-like form, and can penetrate the biofilm secreted by bacteria under the action of external force, and then rapidly melt into liquid by body temperature to release the loaded drug; in the present invention, wound healing is promoted by the synergistic effect between the hydrogel and the drug. First, the solid needle-like DG gel penetrates the biofilm secreted by bacteria under the action of external force, and rapidly melts into liquid by body temperature, releasing the loaded erythromycin for antibacterial, 7-demethyl cork pepper to regulate the inflammatory microenvironment of the wound surface to promote the secretion of anti-inflammatory factors, and then promotes angiogenesis through fenugreek flavonoid glycosides to transport oxygen, nutrients and growth factors to the wound site, and the DG of the present invention can regulate the inflammatory microenvironment of the wound surface by promoting the polarization of macrophages on the wound surface to M2 type; in addition, the PEG in DG can provide a moist environment for the wound surface, which is conducive to the regeneration of wound tissue and accelerates wound healing.

[0008] In one embodiment of the present invention, the hydrogel microneedles are also loaded with cyanidin.

[0009] In one embodiment of the present invention, the loading amount of cyanidin is 0.095%-0.105% by mass ratio.

[0010] In the present invention, cyanidin is loaded. On the one hand, cyanidin has the effects of anti-inflammatory and scavenging DPPH free radicals on the wound surface, which can further enhance the therapeutic effect of the gel microneedle; on the other hand, cyanidin can also be colored according to the pH of the wound tissue fluid, intuitively showing the progress of wound healing.

[0011] In one embodiment of the present invention, the backing hydrogel is prepared by cross-linking polyvinyl alcohol, sodium polyacrylate and glycerol.

[0012] In one embodiment of the present invention, the backing hydrogel is prepared by the following method: S01, dissolving polyvinyl alcohol in water to prepare a polyvinyl alcohol aqueous solution with a concentration of 0.8-0.9 g / mL; S02, dissolving sodium polyacrylate in glycerol to prepare a sodium polyacrylate glycerol solution with a mass concentration of 0.095-0.105 g / g; S03. Slowly pour the sodium polyacrylate glycerol solution into the polyvinyl alcohol aqueous solution at a mass ratio of 0.5-0.6, and stir the reaction to obtain the backing hydrogel.

[0013] Specifically, in the present invention, the preparation method of the backing hydrogel can be: 2.5 g is dissolved in 30 mL of water and soaked overnight, and then stirred at 95° C. for 1.5 h to completely dissolve the polyvinyl alcohol in the water in the form of a solution, which is solution A. Then 1.5 g of sodium polyacrylate is dissolved in 15 g of glycerol at 60° C. and stirred for 30 min as solution B. Finally, solution B is slowly poured into solution A as solution C. After stirring for 20 min, solution C is slowly poured into a flat plate and allowed to stand at room temperature for 24 h to obtain.

[0014] In the present invention, the backing hydrogel prepared according to the method of the present invention has, on the one hand, relatively good adhesion properties of the backing hydrogel, and can repeatedly bond the gel microneedles, and has a good adhesion effect on the gel microneedles; on the other hand, the backing hydrogel also has good water absorption, and can absorb the exudate of the wound surface, providing a better healing environment for the infected wound surface.

[0015] The second object of the present invention is to provide a method for preparing the hydrogel microneedle patch, comprising the following steps: S1. After melting PEG600 and PEG1000, mix them evenly in a mass ratio of 3:4 to obtain matrix glue; S2. Add 0.95%-1.05% erythromycin, 0.095%-0.105% vaccariae flavonoid glycosides and 0.095%-0.105% 7-demethyl cyperidin, or add 0.095%-0.105% cyanidin to the matrix gel according to the mass ratio, and mix well to prepare a microneedle gel system; S3, placing the liquid microneedle gel system in a needle-shaped mold, and attaching the backing gel to the back of the mold. After the microneedle gel system is cooled and solidified, the hydrogel microneedles are taken out by using the adhesive force of the backing gel to obtain the hydrogel microneedle patch.

[0016] In one embodiment of the present invention, the backing hydrogel is prepared by the following method: S01, dissolving polyvinyl alcohol in water to prepare a polyvinyl alcohol aqueous solution with a concentration of 0.8-0.9 g / mL; S02, dissolving sodium polyacrylate in glycerol to prepare a sodium polyacrylate glycerol solution with a mass concentration of 0.095-0.105 g / g; S03. Slowly pour the sodium polyacrylate glycerol solution into the polyvinyl alcohol aqueous solution at a mass ratio of 0.5-0.6, and stir the reaction to obtain the backing hydrogel.

[0017] In one embodiment of the present invention, the PEG600 and PEG1000 are melted at 55-65°C.

[0018] The third object of the present invention is to provide the use of the hydrogel microneedle patch in the preparation of a product for treating skin wounds infected by Staphylococcus aureus.

[0019] The fourth object of the present invention is to provide the use of the hydrogel microneedle patch in the preparation of a product for treating chronic diabetic wounds.

[0020] Beneficial effects of the present invention: The present invention prepares temperature-responsive gel microneedles by loading erythromycin, vaccariae flavonoid glycosides and 7-demethyl scutellariae ... First, the solid needle-shaped DG glue penetrates the biofilm secreted by bacteria under the action of external force, and quickly melts into liquid with body temperature, releasing the loaded erythromycin for antibacterial, 7-demethyl cork pepper to regulate the inflammatory microenvironment of the wound surface to promote the secretion of anti-inflammatory factors, and then promotes angiogenesis through flavonoid glycosides of Vaccaria segetalis to deliver oxygen, nutrients and growth factors to the wound site, and the DG of the present invention can regulate the inflammatory microenvironment of the wound surface by promoting the polarization of macrophages in the wound surface to M2 type; in addition, the PEG in DG can provide a moist environment for the wound surface, which is conducive to the regeneration of wound tissue and accelerates wound healing. The hydrogel microneedle patch prepared by the present invention can significantly promote the rapid healing of wounds infected with Staphylococcus aureus, and can significantly promote the rapid healing of back wounds in diabetic rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 . (A) Schematic diagram of the preparation process, temperature response and pH response of the DG@BHG microneedle patch; (B) Schematic diagram of the regulation of macrophage polarization; (C) Schematic diagram of promoting the healing of infected wounds and diabetic wounds; Figure 2 .Schematic diagram of pH response of DG, schematic diagram of temperature response and detection of water absorption and swelling rate of BHG; Figure 3 . DG in vitro DPPH scavenging ability test; Figure 4. DG@BHG microneedle patch promotes healing of infected wounds; Figure 5 . DG@BHG microneedle patch promotes wound healing on the back of diabetic rats; Figure 6 . DG@BHG microneedle patch promotes skin angiogenesis on the back wound surface of diabetic rats; Figure 7 . DG@BHG microneedle patch regulates the inflammatory microenvironment of the wound; Figure 8 . DG@BHG microneedle patch regulates wound surface to promote macrophage polarization toward M2 phenotype. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with specific examples. These implementation cases are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the claims attached to the application.

[0024] Source of raw materials: Polyethylene glycol 1000 (PEG1000) and polyethylene glycol 600 (PEG600) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0025] In the present application, a hydrogel microneedle patch is mainly provided, comprising a backing hydrogel and hydrogel microneedles adhered to the backing hydrogel; the hydrogel microneedles are prepared by mixing PEG600 and PEG1000 in a mass ratio of 3:4 to obtain a gel body, loaded with erythromycin, fenugreek flavonoid glycosides and 7-demethylcorticosterone.

[0026] Among them, the inventors conducted a lot of experimental explorations before preparing the hydrogel microneedles, and finally found that the melting point of the gel obtained by mixing PEG600 and PEG1000 in a mass ratio of 3:4 was 36°C, which is closer to the temperature of the human body. The melting points of the gels prepared with different PEG ratios are shown in Table 1. Further, the inventors found that when loading the required drugs, the appropriate drugs and concentration ranges were selected, preferably by mass ratio, to load 0.95%-1.05% erythromycin, 0.095%-0.105% flavonoid glycosides of Vaccaria segetalis, 0.095%-0.105% 7-demethylcork pepper and 0.095%-0.105% cyanidin. The melting point of the gel can be further reduced by 1~2°C, which is more suitable for the use of epidermal wounds.

[0027] Table 1 Melting points of PEG600 and PEG1000 in different ratios

[0028] In the present application, the backing hydrogel is prepared by the following method: S01, dissolving polyvinyl alcohol in water to prepare a polyvinyl alcohol aqueous solution with a concentration of 0.8-0.9 g / mL; S02, dissolving sodium polyacrylate in glycerol to prepare a sodium polyacrylate glycerol solution with a mass concentration of 0.095-0.105 g / g; S03. Slowly pour the sodium polyacrylate glycerol solution into the polyvinyl alcohol aqueous solution at a mass ratio of 0.5-0.6, and stir the reaction to obtain the backing hydrogel.

[0029] In the present invention, other gels with good bonding properties and good water absorption can also be used as a substitute, but the backing hydrogel prepared according to the method of the present application has a good bonding effect on the gel microneedles prepared in the present application, and can be well removed from the mold; the water absorption performance of the backing hydrogel prepared by the method of the present application also has a good synergistic effect on the therapeutic effect of the present invention, and will not destroy the moist environment provided by the gel microneedles, but can well absorb wound exudate.

[0030] The technical solution of the present invention is described in detail below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased from commercial sources, or prepared by conventional methods, or are commonly used in the industry.

[0031] Example 1: Preparation of DG@BHG microneedle patch First, DG was prepared: PEG600 and PEG1000 were melted at 60°C and mixed evenly in a mass ratio of 3:4 to obtain matrix gel (MG). Then, 1% erythromycin, 0.1% vaccariae flavonoid glycosides, 0.1% 7-demethyl suberin, and 0.1% cyanidin were added according to the mass ratio and mixed evenly to prepare DG. DG was prepared into a needle shape using a silicone microneedle mold. The melting point of DG is 34°C and it is solid at room temperature (25°C).

[0032] Then prepare BHG: BHG is prepared by cross-linking polyvinyl alcohol with sodium polyacrylate and glycerol. First, dissolve 2.5g of polyvinyl alcohol in 30mL of water and soak overnight, then stir at 95℃ for 1.5h to make the polyvinyl alcohol completely dissolved in water in the form of a solution, which is solution A. Then dissolve 1.5g of sodium polyacrylate in 15g of glycerol at 60℃ and stir for 30min as solution B. Finally, slowly pour solution B into solution A as solution C. After stirring for 20min, slowly pour solution C into a plate and let stand at room temperature for 24h to obtain BHG.

[0033] Finally, DG@BHG was prepared: first, the liquefied DG was placed in a needle-shaped silicone mold, and then BHG was attached to the back of the mold. After the DG was cooled to solid, the DG was taken out by the adhesive force of BHG to obtain a DG@BHG microneedle patch ( Figure 1 ).

[0034] Example 2: pH response and temperature response of DG and water absorption swelling rate of BHG Temperature response of DG: The melting point of DG was detected by differential scanning calorimeter (DSC Q200, Waters). 5 mg / MG of DG was added to the crucible, and the sample was heated from 0°C to 100°C (10°C / min) with nitrogen as the protective gas. The temperature-heat flux density curves of DG and MG were obtained as shown in Figure 2 As shown. Figure 2 It can be seen from Figure AD that DG is liquid above 34°C and solid at 25°C.

[0035] Swelling rate test: Take the freeze-dried BHG and measure the weight M1. After 5, 10, 20, 30, 40, 60, and 120 minutes, place it in a 37°C aqueous solution and weigh the weight Mt. The swelling rate of BHG is determined by the following formula: Swelling rate (%) = (Mt-M0) / M0*100%. Figure 2 As can be seen in Figure E, the swelling ratio of BHG is close to 600% after 5 minutes, and close to 1600% after 40 minutes.

[0036] pH response of DG: DG@BHG was first dissolved in solutions with pH values ​​of 5, 6, 7, 8, and 9. After DG was completely dissolved in the solution, the absorption spectrum of DG@BHG under visible light was detected using a full-function microplate reader (Synergy H4, Berten Instruments, USA). Figure 2 As can be seen in Figure F, as the pH value increases, the color of DG in the solution gradually changes from light red to dark brown.

[0037] Example 3: Detection of the scavenging ability of DG against DPPH Mix the DG solution with the DPPH working solution as solution A, and set up a control group. Let it stand at room temperature for 30 minutes, centrifuge it at 4000rpm for 5 minutes, and detect the absorbance of solution A and each control solution at a wavelength of 517nm. The DPPH free radical scavenging rate is calculated as follows: DPPH scavenging rate (%) = (1- (A test-A control) / A blank) * 100%. Figure 3 It can be seen that as the proportion (weight) of DG increases, its scavenging ability for DPPH▪ becomes stronger.

[0038] Example 4: DG@BHG microneedle patch promotes healing of infected wounds In vivo antibacterial activity of DG@BHG: First, mice were anesthetized with isoflurane, the hair on the back of the mice was shaved, and a full-thickness skin wound with a diameter of 10 mm was constructed on the back of the mice. Then, Staphylococcus aureus (1×10 5 CFU, 20μL), the mice were divided into 4 groups (6 in each group): normal control group, infection group was divided into saline group, MG@BHG group, DG@BHG group. The wound condition of ICR mice was observed in the next 8 days. On the 8th day after wound formation, the mice were euthanized with CO2 and the wound tissue was collected. The tissue was homogenized, cultured, and the number of CFU of Staphylococcus aureus was counted. Figure 4 It can be seen that DG@BHG can significantly promote the rapid healing of Staphylococcus aureus infected wounds.

[0039] Example 5: DG@BHG microneedle patch promotes wound healing on the back of diabetic rats Diabetic rat wound model: A wound model was established in 6-week-old male Sprague-Dawley rats by a single intraperitoneal injection of STZ solution 70 mg / kg. The model was considered successful when the blood glucose concentration exceeded 300 mg / dL for two consecutive times and the rats showed symptoms such as polydipsia, polyuria, and weight loss. Then, the hair on the back of the diabetic rats was removed, and a full-thickness skin excision wound model with a diameter of 1 cm was made on the back of the rats. The 36 rats with successful modeling were divided into 4 groups: model group, MG@BHG group, DG@BHG group, and positive drug Centella asiatica cream (AC) group. The model group was given 0.2 mL of normal saline, the MG@BHG and DG@BHG groups were given a patch once a day, and the AC group was given 0.2 mL once a day. On the 4th, 8th, and 13th days after the wound, the wound skin tissue was taken for pathological study. Centella asiatica cream (AC) is a commercially available drug that promotes wound healing and has a certain therapeutic effect on trauma, surgical wounds, and diabetic ulcers. By Figure 5 It can be seen that the DG@BHG microneedle patch can significantly promote the healing of back wounds in diabetic rats.

[0040] Example 6: DG@BHG microneedle patch promotes skin angiogenesis on the back wound surface of diabetic rats The skin slices of the back of the diabetic rats on the 13th day after the rats were injured in Example 5 were baked, debonded with xylene, hydrated with graded alcohol, repaired with citric acid for 10 minutes, inactivated with 3% hydrogen peroxide for 10 minutes, blocked with BSA for 15 minutes, incubated with CD31 primary antibody in a refrigerator at 4°C overnight, and incubated with secondary antibody at 37°C for 15 minutes. The skin was developed in time and the color development was terminated. Finally, the skin was stained with hematoxylin for 3 minutes, dehydrated, sealed with neutral glue, and observed and photographed under a microscope. Figure 6 It can be seen that the DG@BHG microneedle patch can significantly promote the regeneration of blood vessels in the skin of the back wound of diabetic rats.

[0041] Example 7: DG@BHG microneedle patch regulates wound surface to promote macrophage polarization to M2 phenotype and regulates wound surface inflammatory microenvironment The specific implementation method is the same as that in Example 6, except that the CD31 antibody is replaced with IL-10, TGF-β, CD86 and CD206 to stain the wound skin, and the wound skin is obtained in turn after taking pictures. Figure 7 and Figure 8 .Depend on Figure 7 It can be seen that as the wound heals, the expression of IL-10, TGF-β and CD206 in the wound skin of the DG@BHG microneedle patch group gradually increases and is significantly greater than that of the model group and the MG@BHG group. The expression of CD86 gradually decreases, indicating that the MG@BHG microneedle patch can regulate the polarization of wound macrophages to M2 type and regulate the inflammatory microenvironment of the wound site.

[0042] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A hydrogel microneedle patch, characterized in that: It comprises a backing hydrogel and a hydrogel microneedle adhered to the backing hydrogel; the hydrogel microneedle is prepared by mixing PEG600 and PEG1000 in a mass ratio of 3:4 to obtain a gel body, and loading erythromycin, vaccaria flavonoid glycosides and 7-demethyl scutellaria baicalensis.

2. The hydrogel microneedle patch according to claim 1, characterized in that: In the hydrogel microneedles, 0.95%-1.05% erythromycin, 0.095%-0.105% vaccariae flavonoid glycosides and 0.095%-0.105% 7-demethylcorticosterone are loaded respectively according to mass ratio.

3. The hydrogel microneedle patch according to claim 1, characterized in that: The hydrogel microneedles are also loaded with cyanidin.

4. The hydrogel microneedle patch according to claim 3, characterized in that: By mass ratio, the loading amount of cyanidin is 0.095%-0.105%.

5. The hydrogel microneedle patch according to claim 1, characterized in that: The backing hydrogel is prepared by cross-linking polyvinyl alcohol, sodium polyacrylate and glycerol.

6. The hydrogel microneedle patch according to claim 5, characterized in that: The backing hydrogel is prepared by the following method: S01, dissolving polyvinyl alcohol in water to prepare a polyvinyl alcohol aqueous solution with a concentration of 0.8-0.9 g / mL; S02, dissolving sodium polyacrylate in glycerol to prepare a sodium polyacrylate glycerol solution with a mass concentration of 0.095-0.105 g / g; S03. Slowly pour the sodium polyacrylate glycerol solution into the polyvinyl alcohol aqueous solution at a mass ratio of 0.5-0.6, and stir the reaction to obtain the backing hydrogel.

7. A method for preparing the hydrogel microneedle patch according to any one of claims 1 to 6, characterized in that: The steps include: S1. After melting PEG600 and PEG1000, mix them evenly in a mass ratio of 3:4 to obtain matrix glue; S2. Add 0.95%-1.05% erythromycin, 0.095%-0.105% vaccariae flavonoid glycosides and 0.095%-0.105% 7-demethyl cyperidin, or add 0.095%-0.105% cyanidin to the matrix gel according to the mass ratio, and mix well to prepare a microneedle gel system; S3, placing the liquid microneedle gel system in a needle-shaped mold, and attaching the backing gel to the back of the mold. After the microneedle gel system is cooled and solidified, the hydrogel microneedles are taken out by using the adhesive force of the backing gel to obtain the hydrogel microneedle patch.

8. The preparation method according to claim 7, characterized in that: The backing hydrogel is prepared by the following method: S01, dissolving polyvinyl alcohol in water to prepare a polyvinyl alcohol aqueous solution with a concentration of 0.8-0.9 g / mL; S02, dissolving sodium polyacrylate in glycerol to prepare a sodium polyacrylate glycerol solution with a mass concentration of 0.095-0.105 g / g; S03. Slowly pour the sodium polyacrylate glycerol solution into the polyvinyl alcohol aqueous solution at a mass ratio of 0.5-0.6, and stir the reaction to obtain the backing hydrogel.

9. Use of the hydrogel microneedle patch according to any one of claims 1 to 6 in preparing a product for treating skin wounds infected with Staphylococcus aureus.

10. Use of the hydrogel microneedle patch according to any one of claims 1 to 6 in preparing a product for treating chronic diabetic wounds.

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