CN118831037B - A multifunctional microneedle loaded with microspheres and nanozymes and its preparation method and application - Google Patents

Abstract

The invention relates to the technical field of biological material preparation, in particular to a multifunctional microneedle loaded with microspheres and nano enzymes, and a preparation method and application thereof. The multifunctional microneedle comprises a substrate and a needle point, wherein the substrate and the needle point substrate are both hyaluronic acid, the needle point is loaded with microspheres and cerium dioxide nano-enzyme, and the microspheres are methacryloyl sulfonated chitosan microspheres containing VEGF. The multifunctional microneedle patch has good biocompatibility, angiogenesis promotion and anti-inflammatory properties, and provides a new treatment strategy for diabetic wounds.

Description

Multifunctional microneedle loaded with microsphere and nano enzyme and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological material preparation, in particular to a multifunctional microneedle loaded with microspheres and nano enzymes, and a preparation method and application thereof.

Background

Diabetic Chronic Wound (DCWs) is a serious complication of diabetes. Generally, the hyperglycemic environment of diabetes can lead to various local lesions of the wound microenvironment, such as impaired angiogenesis, overexpression of Reactive Oxygen Species (ROS), proliferation of pro-inflammatory M1 macrophages, and the like, so as to delay wound healing. In newly formed wounds, the nutrients and oxygen required for wound healing depend on the transport of new and normal blood vessels. Impaired angiogenesis limits nutrient and oxygen transport to the wound site and induces persistent inflammation. Overexpression of ROS and massive differentiation of pro-inflammatory M1 macrophages lead to elevated levels of oxidative stress in the wound environment. This can impair normal cellular function and trigger deleterious processes such as necrosis, inflammation and fibrosis. Existing methods of treating diabetic wounds include drugs, hyperbaric oxygen therapy, and stem cell therapy.

However, existing therapies are monotherapy, which is expensive and has side effects. Whereas diabetic wound microenvironments are interrelated, the single effect of current treatment methods will limit the effectiveness of the treatment. Thus, a strategy to address the above-mentioned key points by combining the functions of multiple therapies together is a promising direction for DCWs treatments. Thus, new therapeutic approaches are urgently needed to promote angiogenesis and reduce ROS production in hyperglycemic microenvironments, thereby accelerating diabetic wound healing.

Disclosure of Invention

In order to solve the problems, the invention provides a multifunctional microneedle loaded with microspheres and nano enzymes, wherein the base material of the microneedle is Hyaluronic Acid (HA), and the methacrylic acylated Sulfonated Chitosan (SCSMA) microspheres containing Vascular Endothelial Growth Factor (VEGF) and the ceria nano enzymes (CONPs) are loaded in the tip of the microneedle for wound healing, so that a novel treatment strategy is provided for diabetic wounds.

The invention provides a multifunctional microneedle for loading microspheres and nano enzymes, which comprises a substrate and a needle tip, wherein the substrate and the needle tip substrate are both hyaluronic acid, the needle tip is loaded with the microspheres and the cerium dioxide nano enzymes, and the microspheres are methacryloylated sulfonated chitosan microspheres containing VEGF.

Chitosan Sulfate (SCS) is a heparan biomaterial, and electrostatic interaction exists between sulfonic acid groups and basic amino acids in the growth factor VEGF, so that the activity of the growth factor can be protected. Thereby achieving the effect of promoting angiogenesis. The methacryloylated Sulfonated Chitosan (SCSMA) is obtained by methacryloylating the microsphere and can be prepared into microspheres with slow release effect.

Ceria has a wide range of enzymatic activities including Catalase (CAT) and superoxide dismutase (SOD) mimetic activities, making ceria have antioxidant, anti-inflammatory and upgraded protective effects.

In the scheme of the invention, the methacryloyl sulfonated chitosan microsphere containing VEGF and the cerium dioxide nano enzyme are combined, CONPs is quickly released in a pre-existing period to reduce ROS, thereby eliminating tissue injury and inflammatory reaction in a diabetes wound, and in a later period, the SCSMA microsphere is slowly degraded, the loaded VEGF is gradually released, the angiogenesis is stimulated, and the nutrition and oxygen supply of the wound environment are restored. And the combined use can enhance the phenotypic polarization of M2 macrophages.

Preferably, the mass ratio of Ce ³⁺ to Ce ⁴⁺ on the surface of the cerium oxide nano-enzyme is Ce ³⁺/Ce⁴⁺ =60-70/30-40, such as 60:40,65:35,68:32,70:30, etc.

Preferably, the particle size of the cerium oxide nano enzyme is 100-400nm, the PDI is 0.01-0.05, and further the particle size is 100-200nm, and the PDI is 0.01-0.03.

Preferably, the synthesis of the VEGF-containing methacryloyl sulfonated chitosan microsphere comprises the steps of adopting a capillary microfluidic device, taking a mixed solution of methacryloyl sulfonated chitosan, a photoinitiator and VEGF as an internal phase liquid, taking microdroplet generated oil as an external phase liquid, forming monodisperse liquid drops, and then exposing and polymerizing the monodisperse liquid drops into the microsphere in UV light.

Capillary microfluidic devices are typically assembled from two coaxially arranged circular capillaries in a square capillary, with the junction sealed, such as with an injection needle and transparent epoxy. The inner phase liquid and the outer phase liquid are respectively injected into the micro-channel, the inner phase liquid is to be dispersed liquid, the outer phase liquid is usually oil phase as water phase, conventional microdroplet generating oil is selected, and the dispersed phase forms monodisperse liquid drops of water-in-oil W/O under the pushing of two injection pumps due to the shearing force. By adjusting the flow rate of the two phases, droplets of different size distributions can be obtained. Because the liquid drops contain the photoinitiator, the collected W/O emulsion can be exposed and polymerized into microspheres in UV light to generate monodisperse liquid drops, and after the liquid drops are collected, a micro-liquid drop demulsifier is added for careful flushing to remove covering oil, so that the microspheres can be obtained.

The droplet-forming oil is of a conventional commercially available type, such as fluorinated oil.

In one example, the internal phase fluid contained 5wt% SCSM, 0.25% LAP, and 20 μg/ml VEGF, and the external phase fluid was 2% fluorinated oil.

Preferably, the methacryloyl sulfonated chitosan is prepared by the following steps:

S1, preparing a chitosan solution, namely adding formamide and methanol into chitosan, stirring to fully dissolve the chitosan to obtain the chitosan solution, wherein preferably, in the chitosan solution, the chitosan comprises formamide and methanol=2.5 g and 50mL and 2mL;

S2, sulfonation, namely slowly dripping a sulfonation reagent into the chitosan solution under the conditions of stirring and nitrogen protection, and performing post-treatment on the product after the reaction to obtain sulfonated chitosan, wherein the sulfonation reagent is prepared by dripping chlorosulfonic acid into DMF (N, N-dimethylformamide) solution under the ice bath condition of 0-4 ℃;

preferably, in the sulfonation reagent, the volume ratio of chlorosulfonic acid to DMF is 1:10, and the sulfonation reaction condition is 60 ℃ for 2 hours;

Preferably, the post-treatment comprises removing unreacted chitosan by suction filtration after sulfonation reaction, adding absolute ethyl alcohol into the residual supernatant, precipitating to obtain a solid product, washing the solid product with absolute ethyl alcohol, dissolving the washed solid in deionized water, adjusting pH to 7-8 (1M hydrochloric acid or 1M sodium hydroxide can be used for the like), centrifuging, and retaining the supernatant;

S3, methacryloylation, namely, methacrylic Anhydride (MA) is dripped into the Sulfonated Chitosan (SCS) solution prepared in the step S2, the mixture is stirred at room temperature overnight, and then the solution is filtered and freeze-dried to obtain the methacryloylated Sulfonated Chitosan (SCSMA).

Preferably, the sulfonated chitosan solution concentration is 1.5% (w/v), methacrylic anhydride: sulfonated chitosan solution = 900 μl:25mL.

In another aspect of the present invention, there is also provided a method for preparing the above-mentioned multifunctional microneedle, comprising the steps of:

(1) Preparing a microneedle tip, namely dispersing the VEGF-containing methacrylic acylated sulfonated chitosan microspheres and cerium oxide nano enzyme in a hyaluronic acid solution, adding the solution into a mould, vacuumizing, concentrating, drying and forming to obtain the microneedle tip;

(2) And (3) preparing the microneedle substrate, namely adding the hyaluronic acid solution into a mould, vacuumizing, concentrating, drying and forming to obtain the microneedle substrate, and separating the mould to obtain the complete multifunctional microneedle.

Preferably, in the step (1), the microsphere/ceria nano enzyme/hyaluronic acid solution=20mg:20μg/1 mL, and the concentration of the hyaluronic acid solution is 10% w/v.

In still another aspect, the invention further provides application of the multifunctional microneedle in repairing diabetic wounds.

Compared with the prior art, the invention has the following beneficial effects:

In contrast to conventional hypodermic needles, the microneedles of the present invention bypass the skin barrier, effectively delivering macromolecules into the array of microscale needles with limited pain. When the microneedle tips are inserted into the wound site of the skin, the microspheres and the ceria nanoenzyme are released because the hyaluronic acid is rapidly dissolved deep into the dermis layer. The rapid release of ceria nanoenzymes during the pre-existing period results in a reduction of ROS, thereby eliminating tissue damage and inflammatory reactions within the diabetic wound. In the latter stage, SCSMA microspheres slowly degrade, and the loaded VEGF is gradually released, stimulating angiogenesis and restoring nutrition and oxygen supply to the wound environment. The multifunctional microneedle patch has good biocompatibility, angiogenesis promotion and anti-inflammatory properties. Therefore, the multifunctional microneedle provides a new treatment strategy for the diabetic wound surface.

Drawings

FIG. 1 is a TEM image of CeO ₂, B) the size distribution of CeO ₂, C) elemental energy spectrum analysis of CeO ₂, D) XPS image of CeO ₂, E) FT-IR of SCSMA, F) 1H NMR spectrum of SCSMA, G) the size distribution of microspheres, H) the optical microscope image of microspheres, and I) SEM image of microspheres;

FIG. 2 shows the results of microneedle characterization and antioxidant performance measurements, A) SEM image of MN, B) pressure applied to the needle tip when the sensor is close to the needle tip (n=3), C) MN image loaded with DOX, D) MN image punctured by pig skin, E) subcutaneous drug release results of MN loaded with DOX, F) scavenging capacity of different component micro-towards H2O2 (n=3), G) scavenging capacity of different component micro-towards active oxygen (n=3), H) activity of different component micro-towards SOD enzyme (n=3), I) different component micro-towards hydrogenoxidase (CAT) like activity (n=3);

FIG. 3 shows the results of microneedle-induced ROS reduction and in vitro M2 polarization assays, A) determination of ROS after 24h treatment of HUVECs cells with different concentrations of microneedles and normal cell culture medium (control group), DCFH-DA staining (green), B) flow cytometry analysis of CD86 and CD206 expression (n=3), C) quantitative analysis of CD206 positive cells based on flow cytometry results, D) M1/M2 values (n=3), and E) quantitative statistics of ROS fluorescence intensity (n=3);

FIG. 4 shows the results of in vitro cell migration and angiogenesis test by microneedles, A) HUVECs optical images for cell scoring experiments, B) in vitro tube formation of HUVECs cultured by different microneedles, C) quantitative assessment of total tube length, D) number of nodes, E) number of connections in vascularized network structure (n=3), F) quantitative cell mobility (n=3);

fig. 5 shows the observation result of the therapeutic effect of the microneedle-targeted treatment on diabetic wounds, wherein the images and schematic diagrams of the change of the wound areas of the rats in the 5 th group on days 0, 3,7, 10 and 14, the quantitative analysis of the wound closure rate (n=3), the H & E staining of the wound on day 14, and the quantitative analysis of the width of granulation tissue (n=3) are shown in the following formula.

Detailed Description

The following examples are intended to further illustrate the application but are not meant to limit it. It is important to note that the examples are presented for the purpose of further illustration only and are not to be construed as limiting the scope of the present application. Furthermore, it is to be understood that after reading the teachings of the present application, those skilled in the art may make various insubstantial modifications and adaptations in light of the above teachings, which are intended to be within the scope of the application as defined in the claims appended hereto.

The reagents, materials and equipment used in the embodiments of the invention are all commercially available sources unless otherwise specified, and can be carried out by referring to conventional techniques for process parameters which are not specifically noted.

The cerium oxide nanoenzyme (CONP _S) used in the examples was obtained from Sigma-Aldrich.

EXAMPLE 1 Synthesis of VEGF-loaded Methacryloylated sulfonated Chitosan microspheres

1.1 Synthesis of methacryloylated Sulfonated Chitosan (SCSMA)

In the first step, a sulfonating reagent is prepared.

50ML of N, N-Dimethylformamide (DMF) solution was taken into a 500mL three-necked flask, placed in an ice bath cooled to 0-4℃and then 5mL of chlorosulfonic acid was added dropwise to the solution. The reaction mixture (dmf.so3) was cooled to room temperature with stirring. The reaction process is protected by inert gas.

And secondly, preparing Sulfonated Chitosan (SCS).

2.5G of chitosan was added to a 500mL three-necked flask, followed by 50mL of formamide and 2mL of methanol, and stirring was performed to dissolve the chitosan sufficiently. The sulfonation reagent prepared in the first step is slowly dripped into a three-necked flask under the conditions of magnetic stirring and nitrogen protection, and the reaction is carried out for 2 hours at 60 ℃.

After the reaction is finished, unreacted chitosan is removed by suction filtration, 500-1000 mL of absolute ethyl alcohol is added into the supernatant, and the product is obtained by precipitation. The solid product was washed 3 times with absolute ethanol. Dissolving the washed solid in deionized water, adjusting pH to 7-8 with 1M hydrochloric acid or 1M sodium hydroxide, centrifuging for 15min, and retaining supernatant. The supernatant was then subjected to water dialysis for 3 days with a dialysis membrane having a molecular weight of 14000 Da. After lyophilization, SCS was obtained.

The third step is methacryloylation.

The SCS synthesized in the second step is prepared into 1.5% (W/V) SCS solution, 900 mu L of MA (methacrylic anhydride) is measured and added into 25mL of SCS solution dropwise, the solution is stirred at room temperature overnight, and then the solution is filtered and freeze-dried to obtain the methacryloyl sulfonated chitosan.

1.2 Synthesis of SCSMA microspheres

The capillary microfluidic device is formed by assembling two coaxially arranged circular capillaries in a square capillary, and all joints are sealed by an injection needle and transparent epoxy resin.

SCSMA microsphere

5Wt% SCSM A was prepared and 0.25% LAP was added as the inner phase liquid and 2% fluorinated oil as the outer phase liquid. Two phases are respectively injected into a micro channel by using a micro fluidic device, and the dispersed phase forms monodisperse liquid drops under the action of shearing force under the pushing of two injection pumps. By adjusting the flow rate of the two phases, droplets of different size distributions can be obtained. The collected W/O emulsion was polymerized into microspheres after 60s exposure to UV light at 405nm,25mw/cm ², resulting in monodisperse droplets, which were collected and carefully rinsed with a micro-droplet demulsifier to remove the coating oil to give SCSMA Microspheres (MP).

SCSMA microsphere-V@MP loaded with VEGF

Referring to the above procedure, microspheres loaded with VEGF SCSMA were obtained with 5wt% SCSM A, 0.25% LAP and 20. Mu.g/ml VEGF as the inner phase fluid and 2% fluorinated oil as the outer phase fluid, designated V@MP.

Example 2 preparation of microneedles

2.1 Hyaluronic acid microneedles-MN microneedles

First, 100. Mu.L 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 vacuum was applied at 25℃until the air bubbles were completely removed, and the solution was concentrated and dried at room temperature, and during the concentration, 300. Mu.L of HA solution was continuously added dropwise until the microneedle patch was completely dried and molded, to obtain a microneedle formed entirely of hyaluronic acid, which was designated as MN microneedle.

2.2 Microneedle Supported by cerium oxide or microsphere-C@MN and V@MP@MN microneedle

20Mg of microspheres or 20 mug of ceria are dispersed in 1mL of HA solution, the HA solution containing the microspheres or ceria is added to a PDMS MN mold, then the mold is placed in a vacuum chamber, vacuum is applied at 25 ℃ until bubbles are completely removed, the mixture is concentrated and dried at room temperature to form a needle tip, then the HA solution is added to form a substrate, and finally the microspheres loaded with ceria (designated as c@mn) and the microspheres loaded with microspheres (designated as v@mp@mn) are obtained.

2.3 Micropins loaded with microspheres and cerium oxide-V@MP/C@MN

Referring to 2.2, 20mg of microspheres and 20 μg of ceria were dispersed in 1mL of HA solution, the HA solution containing the microspheres and ceria was added to a PDMS MN mold, and then the mold was placed in a vacuum chamber, and vacuum was applied at 25 ℃ until the bubbles were completely removed, forming a needle tip first, and then forming a substrate, to obtain a microsphere-and ceria-loaded microneedle, designated v@mp/c@mn.

3. Experimental example

3.1 Raw materials testing and characterization

Morphology, particle size and XPS analysis were performed on CeO ₂ nano-enzyme (CONPs) loaded in the microneedles, and infrared, nuclear magnetic and size morphology analysis was performed on the SCSMA microspheres synthesized in example 1. The results are shown in FIG. 1.

Morphology CONPs (fig. 1A) was observed by Transmission Electron Microscopy (TEM), and CONPs was found to be lamellar in structure. The DLS results of CONPs (FIG. 1B) showed that CONPs had a hydrated particle size of 125.77.+ -. 1.05nm, a PDI of 0.03, and a normal distribution of particle sizes of CONPs. The smaller the particle size, the more favorable CONPs is the loading at the tip. By energy spectrum analysis (EDS), we determined that Ce and O were uniformly distributed on the CONPs surface (fig. 1C). The valence state of CONPs surface was then analyzed by X-ray photoelectron spectroscopy (XPS) (fig. 1D), which shows that the existence of Ce ³⁺ and Ce ⁴⁺ on CONPs surface coexists with a Ce ³⁺ content of 63.2% and a Ce ⁴⁺ content of 36.8%. It is well known that the Ce ³⁺/Ce⁴⁺ surface ratio is a main determinant of SOD mimic activity, and that the high Ce ³⁺/Ce⁴⁺ surface ratio has very good scavenging effect on ROS.

Since Sulfonated Chitosan (SCS) has heparin-like structure and can effectively maintain VEGF activity, SCSMA is used as a base material for preparing microspheres. The structure of SCSMA prepared was analyzed using fourier transform near infrared (FT-IR) spectroscopy (fig. 1E). In the FT-IR spectra of SCS and SCSMA, the characteristic absorption peak at the 830cm ^-1 bond had a distinct absorption peak due to C-O-S stretching vibration. The successful introduction of C-O-S groups into chitosan shows that SCS synthesis is successful. The peak at SCSMA cm ^-1 is due to the displacement of the amide bond III. In the 1H NMR spectrum of SCSMA, two new chemical shift peaks can be observed at the positions 5.35 and 5.58ppm (FIG. 1F), due to the methacryloylmethylene in SCSMA. This indicates that the methacryloyl groups have been grafted efficiently to the SCS, indicating that methacrylic anhydride has successfully modified the SCS and synthesized SCSMA.

The particle size distribution of VEGF-loaded microspheres V@MP was 35.34.+ -. 1.66 μm (FIG. 1G). The morphology is shown in FIG. 1H. Scanning Electron Microscope (SEM) images showed many holes on the surface of microsphere MPs (fig. 1I), indicating that drug can be released through these holes. The results show that these MPs have good sphericity, uniformity and high monochromaticity.

3.2 Microneedle sample characterization and antioxidant Property determination

For the four different microneedle samples prepared in example 2, namely, HA MN alone (labeled MN), HA MN containing 20 μ g CONPs (labeled C@MN), HA MN containing 20mg of VEGF-loaded microspheres (labeled V@MP@MN), and HA MN containing 20 μ g CONPs and 20mg of VEGF-loaded microspheres (labeled V@MP/C@MN), a topography test, a penetration test, and an oxidation resistance test were performed.

These microneedles are each composed of a 10 x 10 array of tips, and SEM images (fig. 2A and 2B) show that the tips of the microneedles are pyramid shaped and aligned on the substrate layer. This sharp taper ensures that the MN can be inserted into the deep layer of the skin quickly, noninvasively and accurately.

The application of a displacement pressure resistance of 0.4mm to v@mp@mn, c@mn and v@mp/c@mn (fig. 2B) all experienced forces lower than MN, which may be related to drug disintegration, resulting in voids inside the hydrogel network. But a tolerance of greater than 0.3N/pin (minimum force to puncture the skin, indicating that v@mp/c@mn can penetrate the skin without breaking, indicating its potential ability to penetrate the skin.

The structure of the pigskin is similar to human skin. We implanted the dox-containing MN patch into porcine skin (FIGS. 2C, D) to evaluate its transdermal delivery capacity. To visualize the distribution and penetration of the drug in the local skin, fluorescence microscopy images were taken perpendicular to the skin surface at different depths using Confocal Laser Scanning Microscopy (CLSM) (fig. 4E). Red fluorescence was visible below 390 μm, indicating that MN was administered at a depth of at least 390 μm. The depth of administration of soluble MN is typically between 150-400 μm, and similar depths of administration were achieved for MN prepared in this study.

SOD and CAT enzymes are antioxidant enzymes, CONPs having the activity of both enzymes. H ₂O₂ detection kit (FIG. 2F) showed that the clearance of H ₂O₂ after incorporation was better for C@MN and V@MP/C@MN, unlike the control group (P < 0.05). The DPPH method measures the free radical scavenging capacity (figure 2G), and the results show that the C@MN and the V@MP/C@MN have higher free radical scavenging capacities which respectively reach 45.02% and 60.84%. WST-8 experiments showed (FIG. 2H) that C@MN and V@MP/C@MN had better SOD mimic activity than the control. The dissolved oxygen experiment shows (FIG. 2I) that the microneedle catalyzes H ₂O₂ to generate oxygen (FIG. 2B) with good CAT enzyme activity.

These results demonstrate that the V@MP/C@MN microneedle of the example of the present invention has the ability to scavenge active oxygen.

3.3 Microneedle-induced ROS reduction and in vitro M2 polarization capability test.

In general, the inflammatory phase is the most deregulated stage of wound healing in diabetics, and excessive ROS can hinder critical healing processes such as neovascular, epithelial regeneration and ECM remodeling. To study the reduction of ROS by V@MP/C@MN, we selected pma-stimulated HUVEC as the oxidative stress injury model and measured intracellular ROS levels using DCFH-DA probes (FIG. 3A). The level of ROS in the PMa-treated HUVECs cells was significantly increased, and the addition of the V@MP/C@MN and C@MN extracts to the cells significantly inhibited the level of PMa-induced oxidative stress, and the fluorescence intensity (FIG. 3C) was significantly reduced.

After confirming the advantage of v@mp/c@mn in reducing ROS, we further studied its function of inducing macrophage phenotype changes. Wherein, M1 macrophage (CD 86 positive) can secrete pro-inflammatory cytokine to induce inflammation, and M2 macrophage (CD 206 positive) can generate anti-inflammatory medium and therapeutic factor, thereby reducing inflammation and promoting repair. After LPS stimulation, RAW246.7 cells were transformed to M1 phenotype with a 39.4% proportion of CD86 positive cells. In V@MP/C@MN treated cells, the M2 phenotype was as high as 16.4% (FIG. B, D, E). This is due to CONPs's ability to modulate inflammatory responses, and SCSMA to reduce IL-6 secretion, increase IL-4 and TGF- β1 secretion in chronic diabetic wounds, thereby reducing macrophage polarization to M1, reducing inflammatory levels. In addition, there was no significant difference between the v@mp@mn group M2 phenotype and the c@mn group M2 phenotype, indicating that SCSMA also promoted M2 polarization. These results all demonstrate that V@MP/C@MN microneedles have anti-inflammatory capabilities.

3.4 Microneedles were tested for cell migration and angiogenic capacity in vitro.

The new blood vessel plays a key role in the wound repair process, and continuously transmits oxygen and nutrient substances to the wound to promote the wound healing. Therefore, migration of HUVEC and tube formation are important steps in determining the effectiveness of diabetic wound healing during angiogenesis. Cell proliferation was lower within 24h when serum concentration in the medium was maintained at a lower level. As incubation time was extended, microneedle array cells gradually migrated to the scratch and the blank area gradually decreased (fig. 4a, d). After 24h, the mobility of V@MP/C@MN (89.96.+ -. 1.73%) was higher than that of the control group (60.8.+ -. 1.81%). The differences were statistically significant compared to the control group. To assess angiogenesis, HUVECs were incubated with normal cell culture medium (control), MN solution, c@mn, v@mp@mn and v@mp/c@mn extracts, respectively, for 4 hours. The total number of lymph nodes, tube lengths and vascular connections were quantified for the different groups (fig. 3C, D, E). After 4 hours, no angiogenesis was seen in the control group, and there was a tendency to form a network structure (fig. 4B). After V@MP/C@MN and V@MP@MN were treated, the tube lengths increased by 3.74 times and 2.94 times, respectively. Experiments prove that SCSMA microspheres can maintain VEGF activity, promote VEGF to combine with endothelial cells and induce angiogenesis. In conclusion, the advantage of V@MP/C@MN in promoting cell migration in vitro is verified, and the V@MP/C@MN is expected to promote angiogenesis and tissue regeneration in vivo.

3.5 Observing the curative effect of the microneedle targeted treatment on the diabetic wound surface.

To evaluate the therapeutic effect of V@MP/C@MN microneedles, a model of full-thickness skin lesions of diabetic rats was established and subjected to different treatments. The wound healing effect is evaluated by adopting PBS, MN, C@MN, V@MP@MN and V@MP/C@MN groups. To vividly demonstrate the healing process, we photographed the wound once on days 0, 3, 7, 10, 14, respectively, and recorded the change in wound area.

The test results are shown in fig. 5. We found that the wound defect area of the rats in the V@MP/C@MN group decreased most rapidly and significantly in all groups. There was also a difference in wound healing rate between the control and MN groups during the first 7 days, probably because MN can promote the generation of tissue repair by disrupting collagen chains of the dermis and inducing subcutaneous collagen synthesis. The repair of microneedles in vivo was observed by a series of histopathological analyses. Granulation tissue formation is a critical stage in the healing of skin wounds. As collagen fibers in the granulation tissue increase, the skin wound gradually shrinks, and the width of the granulation tissue decreases by 45. Thus, after HE staining, the width of granulation tissue (healing band width) was measured and wound healing was assessed from a histological level. The epithelialization process was studied by HE staining (fig. 5C), with v@mp/c@mn microneedle treated for 14 days with minimal rat granulation tissue, consistent with the results of previous wound healing rates.

3.6 Experimental examples description of the specific methods of each experiment

3.6.1 Methods for testing reactive oxygen species scavenging

HUVEC cells were seeded into 24-well cell culture plates, after 24 hours the medium was removed and fresh medium containing PMA (1. Mu.g/ml) was added. Cells were incubated for 30min and washed 2 times with PBS. Finally, incubating the whole culture solution containing PBS, MN, C@MN, V@MP@MN and V@MP/C@MN with the cells for 24 hours respectively, and taking out the culture medium after incubation is completed. The DCFH-DA probe was melted with the staining solution of Hoechst 33342, the DCFH-DA was diluted with serum-free medium 1:1000 under the dark condition, and the Hoechst 33342 was diluted with serum-free medium 1:1000 and mixed 1:1. The cells were covered with a suitable amount of staining solution added to each well, incubated for 20min, the staining solution was removed and discarded, the cells were rinsed 1 time with PBS, and photographed under an inverted fluorescence microscope (Leica DMi 8C).

3.6.2 Cell scratch experiments

HUVEC cells in logarithmic growth phase were digested and inoculated into 6-well plates, cultured at 37℃under 5% CO ₂, and grown to about 90% of the whole plates. After scraping at the bottom of the well, the old medium was removed with a 1ml tip, and washed three times with PBS to clear the scraped cells. Finally, the whole culture solution containing PBS, MN, C@MN, V@MP@MN and V@MP/C@MN is respectively incubated with the cells, a photo is taken by a Leika DMi8C microscope, and migration conditions of the cells at time points of 0h, 12h and 24h are recorded. Scratch areas were calculated using Image J software and scratch healing rates were calculated as in equation (2).

Cell mobility (%) = (a ₁-A₂)/A₁ x 100% (2)

Where a ₁ is a scratch area at t=0h, and a ₂ is a scratch area at a predetermined time.

3.6.3 Test tube formation test

Notably, HUVECs were used in vitro tube formation assays. Matrigel (reduced growth factor) was thawed on ice at 4 ℃ overnight and cured in 48-well plates at 37 ℃. Subsequently, HUVECs with a density of 1X 105 cells were uniformly seeded onto Matrigel and incubated with microneedle extracts of different composition for 4 hours. The tubular formation of each well was observed under bright field using an inverted fluorescence microscope (Leika DMi 8C) and photographed. The angiogenic activity was analyzed by calculating the knot number and the total tube length using ImageJ software.

Polarization of 3.6.4 macrophages

RAW264.7 cells were seeded in 24-well plates, each group of cells was pretreated with 1000ng mL-1LPS for 6h, LPS group was cultured with DMEM whole medium, and the other group was cultured with microneedle extract. After 24h incubation of each group, cells were collected and stained for anti-cd 86 antibodies and anti-cd 206 antibodies, and analyzed by flow cytometry.

3.6.5 Diabetes wound model and wound healing effect experiment

A 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 7d and rats with blood glucose above 300mg dL-1 were anesthetized with sodium pentobarbital. A circular full-thickness wound (diameter: 1 cm) was then made on the scraped rat back skin. Rats were then randomly divided into 5 groups of 5 rats each. The control group was not treated at all. For the MN group, rats received HA microneedles only. For the c@mn group, ceria loaded microneedles were added directly onto the wound. Rats in the V@MP@MN group were treated with microsphere-loaded microneedles. The V@MP/C@MN group was treated with a microneedle loaded with ceria and microspheres. The wound areas were recorded with a digital camera at days 0, 3, 7, and 14 of treatment.

In conclusion, the multifunctional microneedle patch provided by the embodiment of the invention has good biocompatibility, angiogenesis promotion and anti-inflammatory properties, and provides a new treatment strategy for diabetic wounds.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (8)

1. The multifunctional microneedle comprises a substrate and a needle tip, and is characterized in that the substrate and the needle tip substrate are both hyaluronic acid, and the needle tip is loaded with microspheres and cerium dioxide nano-enzyme, and the microspheres are methacryloylated sulfonated chitosan microspheres containing VEGF;

the synthesis of the VEGF-containing methacryloyl sulfonated chitosan microsphere comprises the following steps:

adopting a capillary microfluidic device, taking a mixed solution of a methacryloyl sulfonated chitosan solution, a photoinitiator and VEGF as an internal phase liquid, taking microdroplet generated oil as an external phase liquid, forming monodisperse liquid drops, and then exposing and polymerizing the monodisperse liquid drops into microspheres in UV light;

the methacryloyl sulfonated chitosan is prepared by the following steps:

S1, preparing a chitosan solution, namely adding formamide and methanol into chitosan, and stirring to fully dissolve the chitosan to obtain the chitosan solution;

s2, sulfonation, namely slowly dripping a sulfonation reagent into the chitosan solution under the conditions of stirring and nitrogen protection, and performing post-treatment on the product after the reaction to obtain sulfonated chitosan, wherein the sulfonation reagent is prepared by dripping chlorosulfonic acid into a DMF solution under the ice bath condition of 0-4 ℃;

S3, methylacryloylating, namely dropwise adding methacrylic anhydride into the sulfonated chitosan solution prepared in the step S2, stirring overnight at room temperature, filtering and freeze-drying the solution to obtain the methylacryloylated sulfonated chitosan.

2. The multifunctional microneedle according to claim 1, wherein the mass ratio of Ce ³⁺ to Ce ⁴⁺ on the surface of the ceria nanoenzyme is Ce ³⁺/Ce⁴⁺ =60-70/30-40.

3. The multifunctional microneedle of claim 1, wherein the internal phase solution comprises 5 wt% methacryloylated sulfonated chitosan, 0.25% LAP, and 20 μg/ml VEGF, and the external phase solution is 2% fluorinated oil.

4. The multifunctional microneedle according to claim 1, wherein in the step S1, chitosan in a solution of formamide, methanol=2.5 g, 50 mL, 2mL, in the step S2, chlorosulfonic acid in DMF in a volume ratio of 1:10, in a sulfonation reaction condition of 60 ℃ for 2 hours, in the step S3, the concentration of the sulfonated chitosan solution is 1.5% w/V, and methacrylic anhydride in a sulfonated chitosan solution=900 μl, 25mL.

5. The multifunctional microneedle according to claim 1, wherein in the step S2, the post-treatment comprises removing unreacted chitosan by suction filtration after the sulfonation reaction is completed, adding absolute ethyl alcohol into the remaining supernatant, precipitating to obtain a solid product, washing the solid product with absolute ethyl alcohol, dissolving the washed solid in deionized water, adjusting the pH to 7-8, centrifuging, retaining the supernatant, dialyzing the supernatant with water by using a dialysis membrane with a molecular weight of 14000 Da, and finally lyophilizing to obtain the sulfonated chitosan.

6. The method for preparing the multifunctional microneedle according to claim 1, comprising the steps of:

(1) Preparing a microneedle tip, namely dispersing the VEGF-containing methacrylic acylated sulfonated chitosan microspheres and cerium dioxide nano enzyme in a hyaluronic acid solution, adding the solution into a mould, vacuumizing, concentrating, drying and forming to obtain the microneedle tip;

(2) And (3) preparing the microneedle substrate, namely adding the hyaluronic acid solution into a mould, vacuumizing, concentrating, drying and forming to obtain the microneedle substrate, and separating the mould to obtain the complete multifunctional microneedle.

7. The method of claim 6, wherein in the step (1), the microsphere/ceria nanoenzyme/hyaluronic acid solution=20mg:20μg/1 mL, and the concentration of 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 the preparation of a medicament for repairing a diabetic wound.