A multifunctional microneedle loaded with microspheres and nanozymes and its preparation method and application

Abstract

The present invention relates to the technical field of biomaterial preparation, and in particular to a multifunctional microneedle loaded with microspheres and nanoenzymes, and a preparation method and application thereof. The multifunctional microneedle comprises a substrate and a needle tip, wherein both the substrate and the needle tip substrate are hyaluronic acid, and the needle tip is loaded with microspheres and cerium dioxide nanoenzymes, wherein the microspheres are methacryl sulfonated chitosan microspheres containing VEGF. The multifunctional microneedle patch has good biocompatibility, angiogenesis-promoting and anti-inflammatory properties, and provides a new treatment strategy for diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial preparation, and in particular to a multifunctional microneedle loaded with microspheres and nanoenzymes, and a preparation method and application thereof. Background Art

[0002] Diabetic chronic wounds (DCWs) are a serious complication of diabetes. In general, the hyperglycemic environment of diabetes can lead to multiple local lesions in the wound microenvironment, such as impaired angiogenesis, overexpression of reactive oxygen species (ROS), and proliferation of proinflammatory M1 macrophages, thereby delaying wound healing. In newly formed wounds, the nutrients and oxygen required for wound healing depend on the delivery of new and normal blood vessels. Impaired angiogenesis limits the transport of nutrients and oxygen to the wound site and induces persistent inflammation. Overexpression of ROS and massive differentiation of proinflammatory M1 macrophages lead to elevated levels of oxidative stress in the wound environment. This impairs normal cellular function and triggers harmful processes such as necrosis, inflammation, and fibrosis. Existing treatments for diabetic wounds include drugs, hyperbaric oxygen therapy, and stem cell therapy.

[0003] However, existing therapies are all single therapies, which are expensive and have side effects. The diabetic wound microenvironment is interconnected, and the single effect of current treatments will limit the effectiveness of the treatment. Therefore, a strategy to address the above key points by combining the functions of multiple therapies is a promising direction for DCWs treatment. Therefore, new treatments are urgently needed to promote angiogenesis and reduce ROS production in the hyperglycemic microenvironment, thereby accelerating diabetic wound healing. Summary of the invention

[0004] In order to solve the above problems, the present invention proposes a multifunctional microneedle loaded with microspheres and nanozymes. The basic material of the microneedle is hyaluronic acid (HA), and methacryloyl sulfonated chitosan (SCSMA) microspheres containing vascular endothelial growth factor (VEGF) and ceria nanozymes (CONPs) are loaded in the microneedle tip for wound healing, providing a new treatment strategy for diabetic wounds.

[0005] The first aspect of the present invention provides a multifunctional microneedle loaded with microspheres and nanozymes, comprising a substrate and a needle tip, wherein the substrates of the substrate and the needle tip are both hyaluronic acid, and the needle tip is loaded with microspheres and cerium dioxide nanozymes, wherein the microspheres are methacryloyl sulfonated chitosan microspheres containing VEGF.

[0006] Chitosan sulfate (SCS) is a heparin-like biomaterial. The electrostatic interaction between the sulfonic acid group in the growth factor VEGF and the basic amino acid can protect the activity of the growth factor, thereby promoting angiogenesis. Methacrylating it to obtain methacrylylated sulfonated chitosan (SCSMA) and preparing it into microspheres can have a sustained release effect.

[0007] Ceria has a wide range of enzyme activities, including catalase (CAT) and superoxidase (SOD) mimetic activities, which endow Ceria with antioxidant, anti-inflammatory and upregulated protective effects.

[0008] In the scheme of the present invention, methacrylylated sulfonated chitosan microspheres containing VEGF and ceria nanozymes are used together. The rapid release of CONPs in the pre-existing period leads to the reduction of ROS, thereby eliminating tissue damage and inflammatory response in diabetic wounds. In the later stage, SCSMA microspheres slowly degrade, and the loaded VEGF is gradually released, stimulating angiogenesis and restoring the nutrition and oxygen supply of the wound environment. Moreover, the combined use can enhance the phenotypic polarization of M2 macrophages.

[0009] Preferably, the Ce on the surface of the ceria nanozyme 3+ and Ce 4+ Mass ratio is Ce 3+ / Ce 4+ =60-70 / 30-40, such as 60:40, 65:35, 68:32, 70:30, etc.

[0010] Preferably, the particle size of the ceria nanozyme is 100-400 nm, and the PDI is 0.01-0.05; further, the particle size is 100-200 nm, and the PDI is 0.01-0.03.

[0011] Preferably, the synthesis of the methacryloyl sulfonated chitosan microspheres containing VEGF comprises the following steps: using a capillary microfluidic device, using a mixture of methacryloyl sulfonated chitosan, a photoinitiator and VEGF as the inner phase liquid, and using droplet-generating oil as the outer phase liquid to form monodisperse droplets, which are then exposed to UV light to polymerize into microspheres.

[0012] Capillary microfluidic devices are usually assembled by two coaxially arranged circular capillaries in a square capillary, and the joints are sealed, such as by using a syringe needle and a transparent epoxy resin. The inner phase liquid and the outer phase liquid are injected into the microchannels respectively. The inner phase liquid is the liquid to be dispersed, which serves as the aqueous phase. The outer phase liquid is usually the oil phase. A conventional droplet generation oil is selected. Under the push of two injection pumps, the dispersed phase forms monodisperse droplets of water-in-oil W / O due to shear force. By adjusting the flow rate of the two phases, droplets of different size distributions can be obtained. Since the droplets contain photoinitiators, the collected W / O emulsion can be exposed to UV light to polymerize into microspheres, producing monodisperse droplets. After collection, a microdroplet demulsifier is added and carefully rinsed to remove the covering oil to obtain microspheres.

[0013] The droplet-forming oil may be any conventional commercially available oil, such as fluorinated oil.

[0014] In one embodiment, the inner phase liquid contains 5 wt % SCSMA, 0.25 % LAP and 20 μg / ml VEGF; the outer phase liquid is 2 % fluorinated oil.

[0015] Preferably, the methacrylylated sulfonated chitosan is prepared by the following steps:

[0016] S1: preparing a chitosan solution: adding formamide and methanol to chitosan, stirring to fully dissolve the chitosan, and obtaining the chitosan solution; preferably, in the chitosan solution, chitosan: formamide: methanol = 2.5 g: 50 mL: 2 mL;

[0017] S2 sulfonation: under stirring and nitrogen protection, slowly dripping a sulfonation reagent into the chitosan solution, and after the reaction, post-treating the product to obtain sulfonated chitosan; the sulfonation reagent is prepared by dripping chlorosulfonic acid into a DMF (N,N-dimethylformamide) solution under an ice bath at 0-4°C;

[0018] Preferably, in the sulfonation reagent, the volume ratio of chlorosulfonic acid to DMF is 1:10; the sulfonation reaction conditions are: 60° C., 2 h;

[0019] Preferably, the post-treatment includes: after the sulfonation reaction is completed, unreacted chitosan is removed by suction filtration, anhydrous ethanol is added to the remaining supernatant, and a solid product is precipitated, the solid product is washed with anhydrous ethanol, and then the washed solid is dissolved in deionized water, the pH is adjusted to 7-8 (1M hydrochloric acid or 1M sodium hydroxide can be used), centrifuged, and the supernatant is retained; then the supernatant is dialyzed with water using a dialysis membrane with a molecular weight of 14000Da, and finally lyophilized to obtain sulfonated chitosan;

[0020] S3 Methacrylation: Methacrylic anhydride (MA) is added dropwise to the sulfonated chitosan (SCS) solution prepared in step S2, and stirred at room temperature overnight. The solution is then filtered and freeze-dried to obtain methacrylated sulfonated chitosan (SCSMA).

[0021] Preferably, the concentration of the sulfonated chitosan solution is 1.5% (w / v), methacrylic anhydride: sulfonated chitosan solution = 900 μL: 25 mL.

[0022] Another aspect of the present invention further provides a method for preparing the multifunctional microneedle, comprising the steps of:

[0023] (1) Preparation of microneedle tips: dispersing the methacrylylated sulfonated chitosan microspheres containing VEGF and the cerium oxide nanozyme in a hyaluronic acid solution, then adding the solution into a mold, evacuating, concentrating and drying to obtain the microneedle tips;

[0024] (2) Preparation of microneedle substrate: Add hyaluronic acid solution into a mold, vacuum concentrate and dry to obtain the microneedle substrate, and separate the mold to obtain the complete multifunctional microneedle.

[0025] Preferably, in step (1), microspheres: ceria nanozyme: hyaluronic acid solution = 20 mg: 20 μg: 1 mL, and the concentration of the hyaluronic acid solution is 10% w / v.

[0026] Another aspect of the present invention provides the use of the multifunctional microneedle in repairing diabetic wounds.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Compared with traditional hypodermic needles, the microneedles of the present invention can bypass the skin barrier and effectively deliver macromolecules to the microscale needle array with limited pain. When the microneedle tip is inserted into the wound site of the skin, the microspheres and ceria nanozymes are released because the hyaluronic acid quickly dissolves deep into the dermis. The rapid release of ceria nanozymes in the pre-existing period leads to a reduction in ROS, thereby eliminating tissue damage and inflammatory responses within diabetic wounds. In the later stage, the SCSMA microspheres slowly degrade, and the loaded VEGF is gradually released, stimulating angiogenesis and restoring the nutrition and oxygen supply of the wound environment. The multifunctional microneedle patch has good biocompatibility, pro-angiogenesis and anti-inflammatory properties. Therefore, the multifunctional microneedle provides a new treatment strategy for diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Characterization and test results of microneedle raw materials; A) TEM image of CeO2, B) size distribution of CeO2, C) elemental energy spectrum analysis of CeO2, D) XPS image of CeO2, E) FT-IR of SCSMA, F) 1H NMR spectrum of SCSMA, G) size distribution of microspheres, H) optical microscope image of microspheres, I) SEM image of microspheres;

[0030] Figure 2 Characterization of microneedles and determination of antioxidant properties; A) SEM image of MN, B) Pressure applied to the needle tip when the sensor is close to the needle tip (n=3), C) Image of MN loaded with DOX, D) Image of MN after pig skin puncture, E) Subcutaneous drug release results of MN loaded with DOX, F) Scavenging ability of microneedles with different components on H2O2 (n=3), G) Scavenging ability of microneedles with different components on reactive oxygen species (n=3), H) SOD enzyme activity of microneedles with different components (n=3), I) Hydrogen oxidase (CAT)-like activity of microneedles with different components (n=3);

[0031] Figure 3 Results of microneedle-induced ROS reduction and in vitro M2 polarization test, A) Determination of ROS after HUVECs cells were treated with different concentrations of microneedles and normal cell culture medium (control group) for 24 hours, DCFH-DA staining (green), B) Flow cytometric analysis of macrophage CD86 and CD206 expression (n=3), C) Quantitative analysis of CD206-positive cells based on flow cytometric results, D) M1 / M2 value (n=3), E) Quantitative statistics of ROS fluorescence intensity (n=3);

[0032] Figure 4 Results of the in vitro detection of cell migration and angiogenesis ability of microneedles; A) Optical image of HUVECs used in cell scratch assay, B) In vitro tube formation of HUVECs cultured with different microneedles, C) Quantitative evaluation of total tube length, D) Number of nodes, E) Number of connections in the vascularized network structure (n=3), F) Quantification of cell migration rate (n=3);

[0033] Figure 5 Results of the efficacy observation of microneedle targeted therapy for diabetic wounds; A) images and schematic diagram of wound area changes in 5 groups of rats on days 0, 3, 7, 10, and 14, B) quantitative analysis of wound closure rate (n=3), C) H&E staining of wounds on day 14, D) quantitative analysis of granulation tissue width (n=3). DETAILED DESCRIPTION

[0034] The following examples are used to further illustrate the present invention, but are not intended to limit the present invention. It is necessary to point out that the examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. In addition, it should be understood that after reading the content of the present invention, a person skilled in the art in the field can make some non-essential improvements and adjustments based on the content of the present invention, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

[0035] Unless otherwise specified, the reagents, materials, and equipment used in the examples of the present invention are all commercially available; for process parameters not specifically specified, conventional techniques can be used.

[0036] The ceria nanozyme (CONP S )Source: Purchased from Sigma-Aldrich.

[0037] Example 1 Synthesis of VEGF-loaded methacrylylated sulfonated chitosan microspheres

[0038] 1.1 Synthesis of methacrylylated sulfonated chitosan (SCSMA)

[0039] Step 1: Preparation of sulfonation reagent.

[0040] Take 50mL of N,N-dimethylformamide (DMF) solution and add it to a 500mL three-necked flask, put it in an ice bath cooled to 0-4℃, and then drop 5mL of chlorosulfonic acid into the solution. Stir the reaction mixture (DMF·SO3) and cool it to room temperature. The reaction process is protected by inert gas.

[0041] Step 2: Preparation of sulfonated chitosan (SCS).

[0042] 2.5 g of chitosan was added to a 500 mL three-necked flask, followed by 50 mL of formamide and 2 mL of methanol, and stirred to fully dissolve the chitosan. Under magnetic stirring and nitrogen protection, the sulfonation reagent prepared in the first step was slowly dripped into the three-necked flask and reacted at 60°C for 2 h.

[0043] After the reaction is completed, the unreacted chitosan is removed by filtration, and 500-1000 mL of anhydrous ethanol is added to the supernatant to precipitate the product. The solid product is washed 3 times with anhydrous ethanol. The washed solid is dissolved in deionized water, the pH is adjusted to 7-8 with 1M hydrochloric acid or 1M sodium hydroxide, centrifuged for 15 minutes, and the supernatant is retained. The supernatant is then dialyzed with water for 3 days using a dialysis membrane with a molecular weight of 14000Da. SCS is obtained after freeze-drying.

[0044] The third step is methacrylation.

[0045] The SCS synthesized in the second step was prepared into a 1.5% (W / V) SCS solution, 900 μL MA (methacrylic anhydride) was added dropwise to 25 mL SCS solution, and stirred at room temperature overnight. The solution was then filtered and freeze-dried to obtain methacrylylated sulfonated chitosan.

[0046] 1.2 Synthesis of SCSMA microspheres

[0047] A capillary microfluidic device composed of two coaxially arranged circular capillaries assembled inside a square capillary was used, and all junctions were sealed with injection needles and clear epoxy resin.

[0048] SCSMA Microspheres

[0049] 5wt% SCSMA was prepared and 0.25% LAP was added as the inner phase liquid; 2% fluorinated oil was used as the outer phase liquid. The two phases were injected into the microchannel separately using a microfluidic device. Under the push of two injection pumps, the dispersed phase formed monodisperse droplets due to shear force. By adjusting the flow rate of the two phases, droplets of different size distributions can be obtained. The collected W / O emulsion was at 405nm, 25mW / cm 2 After exposure to UV light for 60 s, the samples were polymerized into microspheres to produce monodisperse droplets, which were collected and carefully rinsed with a microdroplet demulsifier to remove the covering oil, thereby obtaining SCSMA microspheres (MP).

[0050] VEGF-loaded SCSMA microspheres - V@MP

[0051] Referring to the above method, 5wt% SCSMA, 0.25% LAP and 20μg / ml VEGF were used as the inner phase liquid; 2% fluorinated oil was used as the outer phase liquid to obtain SCSMA microspheres loaded with VEGF, which were recorded as V@MP.

[0052] Example 2 Preparation of microneedles

[0053] 2.1 Hyaluronic acid microneedle - MN microneedle

[0054] First, 100 μL of 10% (w / v) HA solution was added to a polydimethylsiloxane (PDMS) MN mold, and then the mold was placed in a vacuum box and evacuated at 25°C until the bubbles were completely removed, and concentrated and dried at room temperature. During the concentration process, 300 μL of HA solution was continuously added until the microneedle patch was completely dried and formed, thereby obtaining microneedles formed entirely of hyaluronic acid, which were recorded as MN microneedles.

[0055] 2.2 Microneedles loaded with ceria or microspheres—C@MN and V@MP@MN microneedles

[0056] 20 mg of microspheres or 20 μg of ceria were dispersed in 1 mL of HA solution. The HA solution containing microspheres or ceria was added to the PDMS MN mold. The mold was then placed in a vacuum chamber and evacuated at 25°C until the bubbles were completely removed. The mold was first concentrated and dried at room temperature to form a needle tip. Then HA solution was added to form a substrate. Finally, microneedles loaded with ceria (denoted as C@MN) and microspheres (denoted as V@MP@MN) were obtained.

[0057] 2.3 Microneedles loaded with microspheres and ceria—V@MP / C@MN

[0058] Referring to 2.2, 20 mg of microspheres and 20 μg of ceria were dispersed in 1 mL of HA solution, the HA solution containing microspheres and ceria was added to the PDMS MN mold, and then the mold was placed in a vacuum chamber and evacuated at 25 °C until the bubbles were completely removed, forming the needle tip first and then the base, to obtain microneedles loaded with microspheres and ceria, denoted as V@MP / C@MN.

[0059] 3. Experimental examples

[0060] 3.1 Raw material testing and characterization

[0061] The morphology, particle size, and XPS analysis of CeO2 nanozymes (CONPs) loaded in the microneedles were performed, and the IR, NMR, and size morphology analysis of the SCSMA microspheres synthesized in Example 1 were performed. Figure 1 shown.

[0062] The morphology of CONPs was observed by transmission electron microscopy (TEM). Figure 1 A), CONPs were found to have a lamellar structure. DLS results of CONPs ( Figure 1 B) shows that the hydrated particle size of CONPs is 125.77±1.05nm, the PDI is 0.03, and the particle size of CONPs shows a normal distribution trend. The smaller the particle size, the more conducive it is to the loading of CONPs on the tip. Through energy spectrum analysis (EDS), we determined that Ce and O are evenly distributed on the surface of CONPs ( Figure 1 C). X-ray photoelectron spectroscopy (XPS) was then used to analyze the valence states of the CONPs surface ( Figure 1 D), the results show that there is Ce on the surface of CONPs 3+ and Ce 4+ Coexistence, Ce 3+ The content is 63.2%, Ce 4+ The content is 36.8%. As we all know, Ce 3+ / Ce 4+ The surface area ratio is the main determinant of SOD mimetic activity. 3+ / Ce4+ The surface ratio has a very good scavenging effect on ROS.

[0063] Since sulfonated chitosan (SCS) has a heparin-like structure and can effectively maintain VEGF activity, SCSMA is used as the basic material for preparing microspheres. The structure of the prepared SCSMA was analyzed by Fourier transform near infrared (FT-IR) spectroscopy ( Figure 1 E). In the FT-IR spectra of SCS and SCSMA, 830 cm-1 is due to COS stretching vibration. -1 The characteristic absorption peak at the bond has an obvious absorption peak. The COS group was successfully introduced into chitosan, indicating that the synthesis of SCS was successful. SCSMA is at 1245cm -1 The peak at is attributed to the shift of amide bond III. In the 1H NMR spectrum of SCSMA, two new chemical shift peaks can be observed at 5.35 and 5.58 ppm ( Figure 1 F), which is attributed to the methacryloyl methylene in SCSMA. This indicates that the methacryloyl group has been effectively grafted onto SCS, indicating that methacrylic anhydride has successfully modified SCS and synthesized SCSMA.

[0064] The particle size distribution of VEGF-loaded microspheres V@MP is 35.34±1.66μm ( Figure 1 G). Its shape is as follows Figure 1 H. Scanning electron microscope (SEM) images show that there are many holes on the surface of microsphere MPs ( Figure 1 I), indicating that drugs can be released through these pores. The results show that these MPs have good sphericity, uniformity and high monodispersity.

[0065] 3.2 Microneedle sample characterization and antioxidant performance determination

[0066] For the four different microneedle samples prepared in Example 2, namely: HA MN alone (labeled as MN), HA MN containing 20 μg CONPs (labeled as C@MN), HA MN containing 20 mg VEGF-loaded microspheres (labeled as V@MP@MN) and HA MN containing 20 μg CONPs and 20 mg VEGF-loaded microspheres (labeled as V@MP / C@MN), morphology tests, penetration ability tests and antioxidant tests were performed.

[0067] These microneedles are composed of 10×10 needle tip arrays, SEM images ( Figure 2 A and Figure 2 B) shows that the tip of the microneedle is pyramid-shaped and aligned on the substrate layer. This sharp cone structure ensures that the MN can be inserted into the deep layer of the skin quickly, non-invasively, and accurately.

[0068] Apply 0.4mm displacement pressure resistance to V@MP@MN, C@MN and V@MP / C@MN ( Figure 2 B), the forces they withstand are all lower than those of MN, which may be related to drug disintegration, resulting in voids inside the hydrogel network. However, the tolerable force is greater than 0.3N / pin (the minimum force to pierce the skin), indicating that V@MP / C@MN can penetrate the skin without rupture, indicating its potential ability to penetrate the skin.

[0069] The structure of pig skin is similar to that of human skin. We implanted MN patches containing dox into pig skin ( Figure 2 C, D) to evaluate its transdermal drug delivery ability. In order to visualize the distribution and penetration of the drug in the local skin, confocal laser scanning microscopy (CLSM) was used to take fluorescence microscopy images perpendicular to the skin surface at different depths ( Figure 4 E). Red fluorescence was visible below 390 μm, indicating that the administration depth of MN was at least above 390 μm. The administration depth of soluble MN is usually between 150-400 μm, and the MN prepared in this study also achieved a similar administration depth.

[0070] SOD and CAT are antioxidant enzymes, and CONPs have the activity of these two enzymes. Figure 2 F) showed that C@MN and V@MP / C@MN had better scavenging ability for H2O2 after incorporation, which was different from the control group (P<0.05). The DPPH method was used to determine their free radical scavenging ability ( Figure 2 G), the results showed that C@MN and V@MP / C@MN had high free radical scavenging capacity, reaching 45.02% and 60.84% ​​respectively. WST-8 experiment showed ( Figure 2 H) C@MN and V@MP / C@MN have better SOD mimicking activity than the control. Dissolved oxygen experiments showed that ( Figure 2 I), microneedles catalyze H2O2 to generate oxygen ( Figure 2 B), has good CAT enzyme activity.

[0071] These results show that the V@MP / C@MN microneedles of the present invention have the ability to scavenge active oxygen.

[0072] 3.3 Microneedle-induced ROS reduction and in vitro M2 polarization ability test.

[0073] Typically, the inflammatory phase is the most dysregulated stage of wound healing in diabetic patients, and excessive ROS can hinder key healing processes such as neovascularization, epithelial regeneration, and ECM remodeling. To investigate the reduction of ROS by V@MP / C@MN, we selected PMA-stimulated HUVEC as an oxidative stress injury model and used the DCFH-DA probe to measure intracellular ROS levels ( Figure 3 A). The ROS level of HUVECs cells treated with pma was significantly increased. The addition of V@MP / C@MN and C@MN extracts to the cells significantly inhibited the oxidative stress level induced by pma. The fluorescence intensity ( Figure 3 C) significantly decreased.

[0074] After confirming the advantage of V@MP / C@MN in reducing ROS, we further studied its function of inducing changes in macrophage phenotypes. Among them, M1 macrophages (CD86 positive) can secrete proinflammatory cytokines to induce inflammation, and M2 macrophages (CD206 positive) can produce anti-inflammatory mediators and therapeutic factors to reduce inflammation and promote repair. After LPS stimulation, RAW246.7 cells were transformed into M1 phenotype, and the proportion of CD86 positive cells was 39.4%. In V@MP / C@MN treated cells, the M2 phenotype was as high as 16.4% (Figure B, D, E). This is attributed to CONPs regulating inflammatory responses and the ability of SCSMA to reduce IL-6 secretion and increase IL-4 and TGF-β1 secretion in chronic diabetic wounds, thereby reducing macrophage polarization to M1 and reducing the level of inflammation. In addition, there was no significant difference between the M2 phenotype of the V@MP@MN group and the M2 phenotype of the C@MN group, indicating that SCSMA also promoted M2 polarization. These results all prove that V@MP / C@MN microneedles have anti-inflammatory ability.

[0075] 3.4 Microneedle detection of cell migration and angiogenesis ability in vitro.

[0076] New blood vessels play a key role in the wound repair process. They continuously deliver oxygen and nutrients to the wound and promote wound healing. Therefore, the migration of HUVEC and the formation of tubes are important steps in the angiogenesis process that determine the healing effect of diabetic wounds. When the serum concentration in the culture medium is maintained at a low level, the cell proliferation is low within 24 hours. As the incubation time increases, the cells in the microneedle group gradually migrate to the scratch, and the blank area gradually decreases ( Figure 4 A, D). After 24 h, the migration rate of V@MP / C@MN (89.96±1.73%) was higher than that of the control group (60.8±1.81%). The difference was statistically significant compared with the control group. To evaluate angiogenesis, HUVECs were incubated with normal cell culture medium (control), MN solution, C@MN, V@MP@MN, and V@MP / C@MN extract for 4 h. The total number of lymph nodes, tube length, and vascular connections in different groups were quantified ( Figure 3 C, D, E). After 4 hours, no angiogenesis was observed in the control group, but there was a tendency to form a reticular structure ( Figure 4B). After treatment with V@MP / C@MN and V@MP@MN, the length of the test tube increased by 3.74 times and 2.94 times, respectively. The experiment proved that SCSMA microspheres can maintain VEGF activity, promote VEGF binding to endothelial cells, and induce angiogenesis. In summary, the advantage of V@MP / C@MN in promoting cell migration in vitro has been verified, and it is expected to promote angiogenesis and tissue regeneration in vivo.

[0077] 3.5 Observation on the efficacy of microneedle targeted treatment of diabetic wounds.

[0078] To evaluate the therapeutic effect of V@MP / C@MN microneedles, a full-thickness skin injury model of diabetic rats was established and different treatments were performed. The wound healing effect was evaluated using PBS, MN, C@MN, V@MP@MN, and V@MP / C@MN groups. In order to vividly demonstrate the healing process, we took photos of the wounds once on days 0, 3, 7, 10, and 14 to record the changes in wound area.

[0079] Test results such as Figure 5 As shown. We found that among all the groups, the wound defect area of ​​rats in the V@MP / C@MN group decreased the fastest and most significantly. In the first 7 days, there was also a difference in the wound healing rate between the control group and the MN group, which may be because MN can promote tissue repair by destroying the collagen chains in the surface layer of the dermis and inducing subcutaneous collagen synthesis. The repair effect of microneedles in vivo was observed through a series of histopathological analyses. The formation of granulation tissue is a key stage in the healing of skin wounds. With the increase of collagen fibers in the granulation tissue, the skin wound gradually shrinks and the width of the granulation tissue decreases by 45. Therefore, after HE staining, the width of the granulation tissue (healing zone width) was measured to evaluate the wound healing at the histological level. The epithelial formation process was studied by HE staining ( Figure 5 C), the granulation tissue of rats treated with V@MP / C@MN microneedles for 14 days was the shortest, which was consistent with the previous results of wound healing rate.

[0080] 3.6 Specific methods of each experiment in the experimental example

[0081] 3.6.1 Cellular reactive oxygen species scavenging test method

[0082] HUVEC cells were inoculated in a 24-well cell culture plate. After 24 hours, the culture medium was removed and fresh culture medium containing PMA (1 μg / ml) was added. The cells were incubated for 30 minutes and washed twice with PBS. Finally, the cells were incubated with the full culture medium containing PBS, MN, C@MN, V@MP@MN and V@MP / C@MN for 24 hours, and the culture medium was removed after the incubation. The DCFH-DA probe and the Hoechst 33342 staining solution were melted, and DCFH-DA was diluted 1:1000 with serum-free culture medium under light-proof conditions, and Hoechst 33342 was diluted 1:1000 with serum-free culture medium, and mixed 1:1. An appropriate amount of staining solution was added to each well to cover the cells. After incubation for 20 minutes, the staining solution was removed and discarded, and the cells were rinsed with PBS once, and placed under an inverted fluorescence microscope (Leica DMi8C) for observation.

[0083] 3.6.2 Cell scratch assay

[0084] HUVEC cells in the logarithmic growth phase were digested and inoculated into 6-well plates and cultured at 37°C and 5% CO2. The cells grew to about 90% of the entire plate. The old culture medium was removed after scraping the bottom of the well with a 1ml needle tip and washed three times with PBS to remove the scraped cells. Finally, the whole culture medium containing PBS, MN, C@MN, V@MP@MN and V@MP / C@MN was incubated with the cells respectively, and photos were taken with a Leica DMi8C microscope to record the migration of cells at 0h, 12h and 24h. The scratch area was calculated using Image J software, and the scratch healing rate was calculated according to formula (2).

[0085] Cell migration rate (%) = (A1-A2) / A1*100% (2)

[0086] Where A1 is the scratch area at t = 0h, and A2 is the scratch area at a specified time.

[0087] 3.6.3 Test tube formation test

[0088] It is worth noting that HUVECs were used for in vitro tube formation assays. Matrigel (reduced growth factors) was thawed on ice at 4°C overnight and solidified in a 48-well plate at 37°C. Subsequently, HUVECs at a density of 1×105 cells were evenly seeded on Matrigel and incubated with microneedle extracts of different compositions for 4 h. Tubular formations in each well were observed and photographed under bright field using an inverted fluorescence microscope (Leica DMi8C). The number of junctions and total tube length were calculated using ImageJ software to analyze angiogenic activity.

[0089] 3.6.4 Polarization of macrophages

[0090] RAW264.7 cells were seeded in 24-well plates, and each group of cells was pretreated with 1000 ng mL-1 LPS for 6 h. The LPS group was cultured with DMEM full medium, and the other groups were cultured with microneedle extracts. After 24 h of culture in each group, the cells were collected, stained with anti-CD86 antibodies and anti-CD206 antibodies, and analyzed by flow cytometry.

[0091] 3.6.5 Diabetic wound model and wound healing effect experiment

[0092] The diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ) buffer (60 mg kg-1 rat body weight). Blood glucose levels were monitored after 7 days, and rats with blood glucose higher than 300 mg dL-1 were anesthetized with sodium pentobarbital. Circular full-thickness wounds (diameter: 1 cm) were then made on the scraped dorsal skin of the rats. The rats were then randomly divided into 5 groups, with 5 rats in each group. The control group did not receive any treatment. For the MN group, rats only received HA microneedles. For the C@MN group, microneedles loaded with cerium dioxide were directly added to the wound. Rats in the V@MP@MN group were treated with microneedles loaded with microspheres. The V@MP / C@MN group was treated with microneedles loaded with cerium dioxide and microspheres. The wound area on days 0, 3, 7, and 14 of treatment was recorded with a digital camera.

[0093] In summary, it can be seen that the multifunctional microneedle patch of the embodiment of the present invention has good biocompatibility, angiogenesis-promoting and anti-inflammatory properties, and provides a new treatment strategy for diabetic wounds.

[0094] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0095] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A multifunctional microneedle loaded with microspheres and nanozymes, comprising a substrate and a needle tip, characterized in that: The substrate of the base and the needle tip are both hyaluronic acid, and the needle tip is loaded with microspheres and ceria nanozymes, wherein the microspheres are methacryl-sulfonated chitosan microspheres containing VEGF; The synthesis of the methacrylylated sulfonated chitosan microspheres containing VEGF comprises the following steps: A capillary microfluidic device was used to form monodisperse droplets with a mixture of methacrylylated sulfonated chitosan solution, photoinitiator and VEGF as the inner phase and droplet-generating oil as the outer phase. The droplets were then polymerized into microspheres under UV light. The methacrylylated sulfonated chitosan is prepared by the following steps: S1. preparing a chitosan solution: adding formamide and methanol to chitosan, stirring to fully dissolve the chitosan, and obtaining the chitosan solution; S2 Sulfonation: under stirring and nitrogen protection, slowly dripping a sulfonation reagent into the chitosan solution, and after the reaction, post-treating the product to obtain sulfonated chitosan; the sulfonation reagent is prepared by dripping chlorosulfonic acid into a DMF solution under an ice bath at 0-4°C; S3 Methacrylation: Methacrylic anhydride is added dropwise to the sulfonated chitosan solution prepared in step S2, and the mixture is stirred at room temperature overnight. The solution is then filtered and freeze-dried to obtain methacrylated sulfonated chitosan.

2. The multifunctional microneedle according to claim 1, characterized in that: The Ce on the surface of the ceria nanozyme 3+ and Ce 4+ Mass ratio is Ce 3+ / Ce 4+ =60-70 / 30-40.

3. The multifunctional microneedle according to claim 1, characterized in that: The inner phase liquid contains 5 wt% methacrylylated sulfonated chitosan, 0.25% LAP and 20 μg / ml VEGF; the outer phase liquid is 2% fluorinated oil.

4. The multifunctional microneedle according to claim 1, characterized in that: In the step S1, in the chitosan solution, chitosan: formamide: methanol = 2.5 g: 50 mL: 2 mL; in the step S2, in the sulfonation reagent, the volume ratio of chlorosulfonic acid: DMF is 1:10; the sulfonation reaction conditions are: 60° C., 2 h; in the step S3, the concentration of the sulfonated chitosan solution is 1.5% W / V, methacrylic anhydride: sulfonated chitosan solution = 900 μL: 25 mL.

5. The multifunctional microneedle according to claim 1, characterized in that: In the step S2, the post-treatment includes: after the sulfonation reaction is completed, the unreacted chitosan is removed by suction filtration, anhydrous ethanol is added to the remaining supernatant, and a solid product is precipitated, the solid product is washed with anhydrous ethanol, and then the washed solid is dissolved in deionized water, the pH is adjusted to 7-8, centrifuged, and the supernatant is retained; then the supernatant is dialyzed with water using a dialysis membrane with a molecular weight of 14000 Da, and finally lyophilized to obtain sulfonated chitosan.

6. The method for preparing the multifunctional microneedle according to claim 1, characterized in that: Includes steps: (1) Preparation of microneedle tips: dispersing the methacrylylated sulfonated chitosan microspheres containing VEGF and the ceria nanozyme in a hyaluronic acid solution, then adding the solution into a mold, evacuating, concentrating and drying to obtain the microneedle tips; (2) Preparation of microneedle substrate: Add hyaluronic acid solution into the mold, vacuum concentrate and dry to form the microneedle substrate, and separate the mold to obtain the complete multifunctional microneedle.

7. The method for preparing the multifunctional microneedle according to claim 6, characterized in that: In the step (1), microspheres: ceria nanozyme: hyaluronic acid solution = 20 mg: 20 μg: 1 mL, and the concentration of the hyaluronic acid solution is 10% w / v.

8. Use of the multifunctional microneedle according to any one of claims 1 to 5 or the multifunctional microneedle prepared by the preparation method according to any one of claims 6 to 7 in preparing a diabetic wound repair drug.

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