Magnesium Hydride-Loaded Microneedles and Their Application in Wound Healing

Abstract

A microneedle patch, comprising a support layer and microneedles, one end of the microneedles is connected to the support layer, the needle body of the microneedles is made of a biodegradable material, and the tip contains MgH2. The microneedle patch of the present invention is used for transdermal delivery and sustained release of H2 and Mg ions (Mg 2+ ), and treats diabetic wounds in a minimally invasive manner. At the same time, the microneedles based on biodegradable materials maintain the percutaneous delivery of MgH2 with minimal invasiveness, protect MgH2 from contact with water, extend its service life, and allow MgH2 to be continuously released into the physiological microenvironment. Therefore, verified in vitro and in vivo, the microneedle patch loaded with MgH2 can promote the wound healing process, promote M2 polarization, enhance cell proliferation and migration, improve angiogenesis and reduce ROS production.

Description

Technical Field

[0001] The present invention relates to a medical device made of biological materials, and particularly to a patch with microneedles loaded with magnesium hydride for promoting wound healing, especially for diabetic patients. Background Art

[0002] In the past few decades, the incidence of people diagnosed with diabetes has been increasing, and the mortality and disability rates related to diabetes are experiencing an alarming increase. Diabetic foot ulcer (DFU) is one of the most common complications of diabetes, accounting for about 15 - 25% of diabetic patients. The hyperglycemic microenvironment caused by diabetes, such as the persistent polarization of M1 macrophages (pro-inflammatory), vascular deterioration, and ROS accumulation, will delay wound healing and even lead to gangrenous amputation. Despite the development of hypoglycemic drugs, blood glucose monitoring technologies, and insulin pumps in recent years, there are currently only limited methods to treat diabetic wounds and regulate their hyperglycemic microenvironment. Therefore, new treatment methods that can enhance the polarization of pro-healing M2 macrophages and angiogenesis and reduce ROS production in the hyperglycemic microenvironment are urgently needed to accelerate the healing of diabetic wounds.

[0003] Hydrogen (H 2 ) has been proven to have therapeutic effects and no cytotoxicity. H 2 , as an antioxidant, can resist oxidative stress in tissues and cells by neutralizing the strongest oxidant (·OH) without interfering with normal metabolic oxidation or interrupting other ROS functions in the cell signaling system. H 2 has the effect of reducing ROS production and is expected to become a therapeutic drug for changing the diabetic inflammatory microenvironment. Previously, inhaled H 2 has been used for antioxidant treatments such as treating brain injuries, vascular diseases, and tumors. However, H 2 has the characteristics of high diffusivity, low water solubility, and dose dependence, resulting in limited efficacy of inhalation therapy. To overcome these difficulties, stimulus-responsive nanomaterials such as photoactivated nanocatalysts and acid-responsive H 2 drug precursors have been developed, which mainly achieve site-specific release and release of H 2 through subcutaneous or intravenous injection to improve the therapeutic effect. However, it is still difficult to achieve long-term release of H 2 to obtain the best therapeutic effect.

[0004] Mg and its alloys, as biodegradable implants, have been widely used in clinical tissue repair and regenerative therapy. For example, poly(lactic-co-glycolic acid) (PLGA) microparticles containing Mg powder are placed in the muscle near the knee joint with osteoarthritis, but Mg powder itself is flammable and there are safety hazards during the sample preparation process. Summary of the Invention

[0005] One object of the present invention is to provide a microneedle patch, which uses a biodegradable material as a substrate, loads magnesium hydride, and is used for wounds to promote healing.

[0006] Another object of the present invention is to provide a microneedle patch, which uses PLGA as a substrate to load magnesium hydride, is used for wounds to promote healing.

[0007] Another object of the present invention is to provide a microneedle patch, which uses PLGA as a substrate to load magnesium hydride, delays the release of magnesium ions and hydrogen, reduces the generation of reactive oxygen species, promotes cell proliferation and migration, and enhances angiogenesis.

[0008] Another object of the present invention is to provide a microneedle patch, which is used as a medical device for diabetic foot ulcers to promote healing.

[0009] The present invention provides a microneedle patch, which contains a biodegradable material and loads a compound, has the effects of promoting cell proliferation and migration, promoting M2 polarization, enhancing angiogenesis, and promoting the healing of deep tissue wounds, and is used as a medical device for diabetic foot ulcers to promote healing.

[0010] A microneedle patch includes a support layer and microneedles, and one end of the microneedles is connected to the support layer. The needle body of the microneedles is made of a biodegradable material, such as PLGA. At the tip of the microneedles, there is MgH 2 . The material of the support layer is the same as or different from the material of the whole microneedles, or a non-biodegradable material is used.

[0011] The MgH 2 distributed in the biodegradable material is a particle with an average diameter of 8.1 μm and a purity of 98% - 99.9%.

[0012] A specific implementation of the microneedle patch is a 10×10 microneedle array, and the specification of each microneedle is a rectangular pyramid with dimensions of 200 μm×200 μm×500 μm (width×length×height).

[0013] Another specific implementation of the microneedle patch includes several microneedle patch units. Each unit is a 10×10 microneedle array, and the specification of each needle is a rectangular pyramid with dimensions of 200 μm×200 μm×500 μm (width×length×height).

[0014] Compared with Mg powder, MgH 2 can store and generate more H 2 , and is also more stable at room temperature. However, the application of MgH 2 in H 2 therapy is still very rare. In addition, Mg 2+It can promote the phenotypic change of macrophages into pro-healing M2 macrophages, enhance angiogenesis, and alleviate microvascular lesions caused by the hyperglycemic microenvironment. Therefore, MgH 2 is a promising compound for the treatment of diabetic wounds.

[0015] The microneedle patch of the present invention is used for percutaneous delivery and sustained release of H 2 and Mg ions (Mg 2+ ), and treats diabetic wounds in a minimally invasive manner. At the same time, the PLGA-based microneedles maintain the percutaneous delivery of MgH 2 with minimal invasiveness, protect MgH 2 from contact with water, extend its service life, and allow MgH 2 to be continuously released into the physiological microenvironment. Therefore, verified in vitro and in vivo, the microneedle patch loaded with MgH 2 (MN-MgH 2 ) can promote the wound healing process, promote M2 polarization, enhance cell proliferation and migration, improve angiogenesis and reduce ROS production.

[0016] MN-MgH 2 combines the therapeutic effects of Mg 2+ and H 2 , and has the effect of promoting the healing of deep tissue wounds, specifically manifested as: a) the MN-MgH 2 patch optimizes the therapeutic effect by continuously and efficiently releasing Mg 2+ and H 2 ; b) the H 2 treatment can reduce ROS; c) Mg 2+ promotes cell proliferation and migration and enhances angiogenesis; d) Mg 2+ enhances the polarization of the M2 macrophage phenotype. Verified, MN-MgH 2 has multiple functions, proving its effectiveness in vitro and in vivo. This MN-MgH 2 that integrates multiple therapeutic functions provides a new approach for improving diabetic wound healing. Brief Description of the Drawings

[0017] Figure 1 For the synthesis and characterization diagrams of MN-PLGA, MN-MgH 2 and MgH 2 ; among them, a is the schematic diagram of the preparation method of MN-MgH 2 , b is the SEM image of MgH 2 and its particle size analysis, c is the XRD result of MgH 2 , d is the TG and DSC of MgH 2 , e is the photo of MgH 2 , f is MN-MgH2 SEM images at different angles and magnification ratios and SEM size measurement results. g is MgH 2 and MN-MgH 2 In-situ and in-vivo release of Mg 2+ and H 2 Schematic diagram of the test method for 2 and MN-MgH 2 Release curve of Mg 2+ in simulated body fluid. i is MgH 2 and MN-MgH 2 Release curve of H 2 when immersed in simulated body fluid;

[0018] Figure 2 is the in-vitro immunomodulatory characterization result graph of MN-MgH 2 ; among them, a is the SEM image of the polarization of Raw264.7 cells after being treated with MN-PLGA extract, MN-MgH 2 extract, and MgH 2 solution for 24 h. b is the determination of the reactive oxygen species content of Raw264.7 cells after being treated with different materials and without different materials for 24 h. c is the flow cytometry analysis result graph of the ROS fluorescence staining of Raw264.7 cells under different treatments. d is the statistical result graph of ROS generation in each treatment group. e is the PCR detection statistical graph of the expression levels of IL-6, IL-1β, iNOS, and Arg-1 after incubation for 24 h; in each statistical graph, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0019] Figure 3 is the in-vitro cytotoxicity, cell regeneration, and angiogenesis effect result graph of MN-MgH 2 ; among them, a is the statistical graph of the viability of fibroblasts co-cultured with MN-PLGA, MN-MgH 2 extract, or normal cell culture medium for 72 h. b is the statistical graph of the viability of fibroblasts co-cultured with different concentrations of MgH 2 for 72 h. c is the confocal image of migrating HUVECs after incubation under different conditions for 24 h. d is the statistical graph of the change in the cell migration ability of HUVECs after 24 h when treated with MN-PLGA extract, MN-MgH 2 extract, MgH 2 solution (1 μg / mL), or normal cell culture medium. e is the statistical graph of the cell migration ability when treated with different materials (MN-PLGA extract, MN-MgH 2 extract, and MgH 2Detection chart of the difference in the migration ability of fibroblasts after co - culture with a solution or normal cell culture medium (control group). f is the quantitative statistical chart of the gap closure rate between different groups. g is the image of HUVECs co - cultured with different materials or normal cell culture medium for 4 h. h is the quantitative statistics chart of blood vessel nodes and tube length in each group. In each statistical chart, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0020] Figure 4 is MN - MgH 2 In - vivo wound healing efficacy test. Among them, a is the schematic diagram of the in - vivo treatment plan. b is the representative photographic images of diabetic injury mice treated or untreated with MgH 2 or MN - MgH 2 at 0, 3, 5, 7, 10, 12, and 14 days. c is the quantification of the wound healing rate in different groups. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0021] Figure 5 is the result chart of ROS staining and staining quantification of each group of mice on the 3rd and 7th days. Among them, a is the representative images of ROS staining of the control group, MgH 2 group, and MN - MgH 2 group of mice on the 3rd and 7th days. b is the statistical chart of the ROS intensity measured in each group on the 3rd and 7th days. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0022] Figure 6 is the result chart of the change in the cell morphology of MN - MgH 2 in the in - vivo study of immunomodulatory effects. Among them, a is the representative double - stained images of CD68 and iNOS of the control group, MgH 2 group, and MN - MgH 2 group of mice on the 3rd, 7th, and 14th days after treatment. b is the quantitative statistical chart of the intensity of CD68 + iNOS + cells on the 3rd, 7th, and 14th days. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0023] Figure 7 is the result chart of the change in the cell morphology of MN - MgH 2 in the in - vivo study of immunomodulatory effects. Among them, a is the representative double - stained images of CD68 and iNOS of the control group, MgH 2 group, and MN - MgH 2Representative images of double staining of CD68 and CD206 in the mouse groups after 3, 7, and 14 days of treatment. b is the quantitative statistical chart of the intensity of CD68+CD206+ cells on days 3, 7, and 14. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0024] Figure 8 Histological section staining for tissue regeneration and collagen fiber repair after treatment. H&E and Masson's trichrome staining images of each treatment group on day 14. The white line indicates fat cavitation;

[0025] Figure 9 For MN-MgH 2 In vivo verification results of the effects on cell proliferation and angiogenesis. Among them, a is the CD31 immunofluorescence staining image, b is the ki67 immunofluorescence staining image, c is the statistical chart of capillary density in the skin tissues of each group, d is the statistical chart of cell proliferation rate in the epidermal skin tissues of each group, and e is the statistical chart of cell proliferation rate in the dermal skin tissues of each group. In each statistical chart, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed implementation mode

[0026] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and not to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solution of the invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention, and it should be covered by the scope of the claims of the present invention.

[0027] The specific test methods used in the following examples of the present invention are described in detail as follows:

[0028] 1) Experimental materials

[0029] Materials and cell culture: PLGA was purchased from Sigma-Aldrich. MgH was prepared by the Hydrogen Science Center of Shanghai Jiao Tong University. 2 . The micro-needle patch (MN) mold was made of polydimethylsiloxane (PDMS) and purchased from Micropoint Technology Co., Ltd. in Singapore. Each MN had a 10×10 array, and the array size was 200 μm×200 μm×500 μm (width×length×height). High-glucose DMEM cell culture medium and fetal bovine serum were purchased from Gibco in the UK. Fibroblasts, HUVECs, and Raw264.7 cells were cultured in a complete medium (high-glucose DMEM cell culture medium containing 10% fetal bovine serum) in an incubator (37 °C, 5% CO 2 ).

[0030] 2) Preparation of MN-PLGA and MN-MgH 2 Preparation

[0031] For a 10% PLGA solution, 100 mg of PLGA was dissolved in 1 mL of 1,4-dioxane and stirred for 30 minutes. 10 mg of MgH 2 was evenly distributed into the above PLGA solution to obtain a PLGA-MgH 2 solution. The MgH 2 suspension was added to the microneedle mold and centrifuged at 3000 rpm for 5 min to ensure that the solution completely filled the tip of the microneedle mold. Each microneedle mold contained 300 μL of PLGA-MgH 2 solution, and then it was placed in a dryer at 40 °C. After 12 hours, the microneedles reached a certain hardness and were demolded to obtain microneedle patches containing MgH 2 (MN-MgH 2 ).

[0032] Microneedles (MN-PLGA) made of pure PLGA solution were prepared using the same process as described above.

[0033] 3) In vitro release of Mg 2 and H 2 from MgH 2+ and MN-MgH 2 To test the in vitro release profiles of MgH

[0034] powder and MN-MgH2, 3 mg of MgH 2 powder or MN-MgH 2 was placed at the bottom of a test tube, and then 15 mL of simulated body fluid (pH = 7.0) was added. A hydrogen electrode was used to detect the release of hydrogen. MgH 2 and MN-MgH 2 released Mg 2 in pure water (pH = 7.0) and simulated body fluid (pH = 7.0). Every 3 mg of MgH 2+ and 1 piece of MN-MgH 2 were immersed in 15 mL of simulated body fluid and then placed in a dialysis bag. Samples were collected at different time points (30 minutes, 1 hour, 6 hours, 1 day, 2 days, 4 days) for ICP detection. 2

[0035] 4) CCK-8 assay

[0036] Used to detect the cytotoxicity of PLGA and MgH 2 . Each MN-PLGA or MN-MgH 2 ​The extract was obtained by soaking in 5 ml of PBS for 3 days. Fibroblasts were seeded into 96-well plates at a density of 3000 cells per well and divided into a control group, an MN-PLGA group, and an MN-MgH 2 group. After starving the fibroblasts with 100 μL of high-glucose DMEM for 12 hours, different groups of fibroblasts were mixed with 90 μL of complete medium and 10 μL of PBS (control group), MN-PLGA, or MN-MgH 2 extract and co-cultured for 3 days. On the third day, complete medium containing 10% CCK8 solution was added to each well to interact with the live cells. After incubation in the incubator for 2 hours, the 96-well plates were analyzed using a microplate reader (SpectraMAX iD3, Molecular Devices.LLC., USA). In determining the optimal concentration of MgH 2 for promoting cell growth, fibroblasts were divided into different groups, starved with high-glucose DMEM for 12 hours, and then co-cultured with different concentrations of MgH 2 solution (0, 0.5, 1, 2, 3, 4, 5 μg / mL) for 3 days. The OD values were measured on the last day using the same method as above.

[0037] 5) Double staining of live and dead cells

[0038] HUVECs were divided into three groups and seeded into confocal dishes at a density of 40×10 4 cells per dish. Different groups were co-cultured with MN-PLGA, MN-MgH 2 or only with cell culture medium (control group) for 1 day or 3 days. At the designated time points, the medium was removed, and 300 μL of Calcein-AM / PI assay (the dye reagent was prepared according to the manufacturer's instructions) was added to each dish. After incubation at 37 °C and 5% CO 2 for 15 minutes, the cells in different groups were photographed using a confocal microscope (Leica SP5, Leica Camera AG, Germany), and representative images were recorded.

[0039] 6) Tube formation assay

[0040] First, according to the manufacturer's protocol, 50 μL of Matrigel was added to each well of a 96-well plate. HUVECs were dispersed in DMEM and seeded into the 96-well plate at a density of 2×10 4 cells per well. HUVECs were divided into 3 groups and incubated with normal cell culture medium (control group), MgH 2 solution, or MN-MgH 2 extract for 4 hours, and representative images of each group were taken.

[0041] 7) In vitro migration and wound healing analysis

[0042] Place 750 μL of complete medium in each well of a 24-well plate. Suspend 2 × 10 4 HUVECs in 200 μL of high-glucose DMEM, and then seed them into one well of a 24-well Transwell plate. The 24 wells of the Transwell plate have different treatment factors according to different groups, including interaction with 100 μL of MN-PLGA, MN-MgH 2 , MgH 2 solution (1 μg / mL) or normal cell culture medium. After co-culturing for 24 hours, the cells are first fixed with paraformaldehyde for 15 minutes and then stained with Hoechst. After wiping the cells in the migration wells, the migrated cells are photographed using a confocal microscope. The fibroblasts are divided into four groups, scratched in the center of each well, and co-cultured with MN-PLGA, MN-MgH 2 extracts, or MgH 2 solution (1 μg / mL) or normal cell culture medium. After co-culturing for 0, 6, 12, and 20 hours, the cell migration pattern is recorded under an optical microscope.

[0043] 8) In vitro macrophage polarization analysis

[0044] Seed 5 × 10 4 Raw264.7 cells into one well of a 24-well plate containing a sterile silicon wafer for SEM analysis. After co-culturing with MN-PLGA, MN-MgH 2 extracts and MgH 2 solution extracts or normal cell culture medium for 24 h, take the silicon wafer with Raw264.7 cells from the plate, fix it with 2.5% glutaraldehyde for 30 min, and dehydrate it according to the alcohol concentration gradient (30% for 5 min, 50% for 5 min, 70% for 10 min, 80% for 10 min, 95% for 15 min, 100% for 15 min). Subsequently, SEM pictures are taken. For RT-PCR analysis, co-culture Raw264.7 cells with the above different materials for 24 h. Then, completely isolate the RNA, use GAPDH as an internal reference, and quantitatively detect the RNA expression of IL-6, IL-1, iNOS, and Arg-1.

[0045] 9) In vitro flow analysis of ROS production

[0046] First, seed Raw264.7 cells into a 6-well plate containing 2 μg of LPS per well for flow analysis, divide them into 4 groups, and co-culture with 1 mL of MN-PLGA, MN-MgH 2 extracts and MgH 2Co-cultured with a solution or normal cell culture medium (2 mL of normal cell culture medium, control group). After 24 hours of co-culture, Raw264.7 cells were stained using a ROS detection kit according to the instructions. The stained cells were first photographed with a fluorescence microscope and then collected and analyzed by flow cytometry.

[0047] 10) In vivo establishment of diabetic wound model and MgH 2 powder and MN-MgH 2 Wound healing efficacy test

[0048] A chronic wound model was established using diabetic (db / db) mice (at a certain week age). After anesthetizing and shaving the mice, a circular wound with a diameter of 6 mm was created on the back of the mice. Subsequently, 12 mice were randomly divided into 3 groups (control group, MN-MgH 2 group, MgH 2 powder group). The control group was not given any treatment. Each mouse in the MN-MgH 2 group was treated with one MN-MgH 2 patch until it fell off naturally. Each mouse in the MgH 2 powder group was given 3 mg of MgH 2 powder. Each mouse was housed in a separate cage with sufficient food and water. Wound images were taken at 0, 3, 5, 7, 10, 12, and 14 days after treatment, respectively.

[0049] 11) In vivo wound healing, ROS reduction, and macrophage polarization analysis

[0050] On the 14th day after treatment, the mice were sacrificed with an overdose of 4% chloral hydrate. First, the wound skin was removed and immersed in 4% paraformaldehyde for 24 hours. After embedding the samples in wax blocks, sections with a thickness of 5 μm were prepared for subsequent staining. Hematoxylin-eosin staining (H&E), Masson's trichrome staining, and immunofluorescence staining with anti-CD31 (1:500) antibody and anti-Ki67 (1:500) antibody were used to detect the wound healing process. Antibodies against ROS were used to detect the reduction rate of ROS in different groups. Antibodies against CD68, CD206, and iNOS were used in this part to detect macrophage polarization.

[0051] Example 1 MN-MgH 2 Characterization

[0052] The microneedles containing only PLGA were named MN-PLGA ( Figure 1 a). ICP-MS was used to detect the purity of MgH 2 powder, demonstrating that the Mg element content reached 99.9%. SEM images showed that MgH 2 had a relatively uniform spherical morphology with an average diameter of 8.1 μm ( Figure 1 b). XRD was used to further verify MgH2 has a high crystallinity, with a small proportion of Mg monomers ( Figure 1 c). In addition, the chemical stability of MgH 2 was characterized by thermogravimetry (TG) and differential scanning calorimetry (DSC). Both TG and DSC results indicated that MgH 2 remained stable above 403 °C ( Figure 1 d), which ensured that the preparation process (40 °C) of MN-MgH 2 would not change the properties of MgH 2 .

[0053] Each MN-MgH 2 patch has a 10×10 array ( Figure 1 e). The shape of each needle is a rectangular cone with a size of 200 μm×200 μm×500 μm (W×L×H) ( Figure 1 f).

[0054] After successfully preparing MN-MgH 2 , the in-situ and in-vivo release profiles of Mg and H 2 from MgH 2 and MN-MgH 2 were investigated ( Figure 1 g). MgH 2 and MN-MgH 2 were immersed in simulated body fluid. First, the release of Mg 2 from MgH 2 and MN-MgH 2+ was tested in simulated body fluid ( Figure 1 h). Over time, both MgH 2 and MN-MgH 2 released Mg 2+ , but the amount of Mg 2 produced by MN-MgH 2+ was 1.46 times that of MgH 2 . This result was consistent with our previous hypothesis that the acid degradation products of PLGA would promote the release of Mg 2+ . Next, the H 2 released from MgH 2 and MN-MgH 2 in simulated body fluid was measured using a hydrogen electrode ( Figure 1 i). The release curve of MN-MgH 2 showed that H 2 was continuously released after several days (>30 h), while the release curve of H 2 from MgH 2 powder was much faster, reaching the release peak at 0.15 h. Thus, it was verified that incorporating MgH2 Loaded into the MN based on hydrogel, Mg 2+ release amount increased significantly, H 2 release time extended significantly, MN-MgH 2 has significant sustained release of H 2 advantage.

[0055] Example 2 MN-MgH 2 In vitro immunomodulation

[0056] MN-MgH 2 Verification of reducing ROS generation in vitro

[0057] First, lipopolysaccharide (LPS) was added to Raw264.7 cells in each well to induce excessive production of ROS. The cells were seeded in 24-well plates and co-cultured with MN-PLGA extract, MN-MgH 2 extract, MgH 2 solution or normal cell culture medium (control group). After 24 h of co-culture, Raw264.7 cells were stained by ROS staining method and imaged under a fluorescence microscope. Representative images of each group showed that MgH 2 or MN-MgH 2 application significantly reduced the production of ROS ( Figure 2 a). To quantify the reduction of ROS generation, the stained Raw264.7 cells were collected for flow cytometry analysis ( Figure 2 b). Flow cytometry results showed ( Figure 2 c) that MgH 2 and MN-MgH 2 reduced the production of reactive oxygen species to 70.77% and 50.24% respectively, achieving a decrease in the production of ROS and confirming their ability to reduce the level of reactive oxygen species.

[0058] MN-MgH 2 Characterization of in vitro induced M2 polarization

[0059] After verifying the advantage of MN-MgH 2 to reduce ROS through H 2 treatment, its function of inducing macrophage phenotype change by releasing Mg 2+ was further verified. Compared with the control (untreated) or MN-PLGA extract-treated Raw264.7 cells, SEM images showed that MgH 2 or MN-MgH 2 extract-treated cells had morphological changes, indicating that Mg 2+ induced macrophage polarization ( Figure 2 d). For more detailed research, we used MN-MgH 2Extract or MgH 2 RT-PCR experiments were performed on Raw264.7 cells treated with the solution (1 μg / mL) for 24 h. Untreated cells were used as the control group. RT-PCR was used to detect the expression of M1 macrophage biomarkers (IL-6, IL-1, and iNOS) and M2 macrophage biomarker (Arg-1) at the mRNA level ( Figure 2 e). The results showed that the expressions of IL-6, IL-1, and iNOS in the MgH 2 group and the MN-MgH 2 group were significantly decreased statistically, indicating that M1 polarization was inhibited after treatment. However, the expression of Arg-1 in the MgH 2 and MN-MgH 2 groups increased significantly, indicating that M2 repolarization increased simultaneously after treatment. In summary, MN-MgH 2 promoted the polarization of pro-inflammatory M1 macrophages into pro-healing M2 macrophages.

[0060] Example 3 MN-MgH 2 Can promote cell proliferation and migration in vitro

[0061] The CCK-8 method was used to study the effects of MgH 2 and MN-MgH 2 on cell viability and proliferation. After co-culturing for 72 h, there was no significant difference in the viability of fibroblasts between the MN-PLGA group and the control group, while the cell viability in the MN-MgH 2 group increased significantly statistically, indicating that MN-PLGA had no obvious cytotoxic effect on fibroblasts, while MN-MgH 2 would further promote the proliferation of fibroblasts ( Figure 3 a). At the same time, it was proved that the optimal concentration of MgH 2 to promote fibroblast proliferation was 1 μg / mL ( Figure 3 b). Therefore, unless otherwise stated, 1 μg / mL MgH 2 solution was used in subsequent experiments. In addition, live and dead staining experiments also confirmed this result and obtained similar conclusions.

[0062] Then, the effect of MN-MgH 2 on cell migration ability was studied. The nuclei of migrating HUVECs were stained with Hoechst ( Figure 3 c). Quantitative analysis found that the migration of HUVECs increased significantly after treatment with MgH 2 solution or MN-MgH 2 extract ( Figure 3 d). In addition, the cell migration ability of fibroblasts was also evaluated by an in vitro wound healing scratch assay. MgH was used2 Solution or MN-MgH 2 Observation of the extract promoting wound healing( Figure 3 e). The quantitative analysis results showed that MgH 2 Solution group and MN-MgH 2 The gap closure rates of the extract groups were 1.28 times and 1.94 times higher than those of the control group, respectively( Figure 3 f). In addition, a tube formation experiment was also carried out. HUVECs were incubated with normal cell culture medium (control group), MgH 2 Solution or MN-MgH 2 Extract for 4 h( Figure 3 g). The nodes and tube lengths of different groups were quantified. MN-MgH 2 Treatment increased the nodes and tube lengths by 3.54 times and 2.22 times, respectively( Figure 3 h).

[0063] Therefore, the ability of MN-MgH 2 to promote cell proliferation and migration was verified in vitro, and thus it is expected to promote angiogenesis and tissue regeneration in vivo.

[0064] Example 4 MN-MgH 2 Promoting wound healing in vivo

[0065] The wound healing effect of MN-MgH 2 in diabetic skin injury mice was further evaluated. As Figure 4 shown in a, the basic method was to create a 6-mm circular wound on the back of diabetic (db / db) mice to establish a mouse model. After the operation, the mice were randomly divided into three groups and were given PBS (control group), MgH 2 Powder or MN-MgH 2 . Record the morphological changes of MN-MgH 2 migrating over time after insertion into the skin. The results are as Figure 4 shown in b. As H 2 gas was released, the MN-MgH 2 patch began to turn white and then more and more small bubbles appeared. At the same time, the wounds of mice in different treatment groups were photographed on day 0, 3, 5, 7, 10, 12, and 14. On day 10, the damaged area of the mice in the MN-MgH 2 group had healed significantly, while the other groups had not. The wound percentages at different times were quantified. As Figure 4 shown in c, compared with the control group and the MgH 2 powder group, the MN-MgH 2 group showed a significant reduction in wound area on day 14.

[0066] Example 5 MN-MgH2 Reducing ROS generation in vivo

[0067] Detecting MgH by ROS staining 2 Reductive effect on ROS in vivo. Skin specimens of diabetic mice in each group were taken on the 3rd and 7th days respectively, stained with ROS and representative images were taken ( Figure 5 a). The quantitative results on the 3rd day showed that compared with the control group, the ROS generation in the MgH 2 and MN-MgH 2 groups decreased by 1.65-fold and 1.95-fold respectively. At the same time, on the 7th day, after treatment with MgH 2 and MN-MgH 2 the reactive oxygen production still decreased by 1.32-fold and 1.64-fold respectively ( Figure 5 b). Thus, it can be seen that treatment with MN-MgH 2 can significantly reduce the production of ROS, thereby promoting wound healing.

[0068] Example 6 MN-MgH 2 Inducing M2 polarization in vivo

[0069] To verify the role of MN-MgH 2 in M2 polarization, in vivo immunofluorescence staining of macrophage markers (CD68, red), M1 macrophage marker (iNOS, green) and M2 macrophage marker (CD206, green) was performed on tissues of different groups. Normally, M1 macrophages are the main macrophage phenotype from the 1st to 3rd days after injury, while M2 macrophages usually reach their peak on the 7th day after injury. Representative images and fluorescence intensity quantification diagrams of CD68 / iNOS double staining on the 3rd day showed ( Figure 6 a), compared with the control group, treatment with MgH 2 powder and MN-MgH 2 reduced M1 polarization by 1.90-fold and 2.63-fold respectively ( Figure 6 b). On the 7th day, the number of M1 macrophages in the MN-MgH 2 group decreased by 2.03-fold compared with the control group, while the MgH 2 group only decreased by 1.28-fold ( Figure 6 b). On the 14th day, MN-MgH 2 still reduced M1 polarization by 1.69-fold, while there was no significant difference between the MgH 2 group and the control group ( Figure 6 b).

[0070] CD68 / CD206 double staining analysis showed that MgH 2 and MN-MgH 2On the 3rd day after treatment, the repolarization of M2 increased by 2.37 times and 3.50 times respectively ( Figure 7 a, 7b). On the 7th day, compared with the control group, the repolarization of M2 in the MgH 2 group and the MN-MgH 2 group increased by 1.44 times and 1.77 times respectively ( Figure 7 b). On the 14th day, compared with the control group, the repolarization continued to increase by 1.32 times and 1.54 times after treatment with MgH 2 and MN-MgH 2 respectively ( Figure 7 b). Compared with the MgH 2 powder group, the repolarization of M2 in the MN-MgH 2 group increased by 1.23 times and 1.16 times on the 7th day and the 14th day respectively, indicating that the release of Mg 2 by MN-MgH 2+ was enhanced.

[0071] Thus, it was verified that the application of MgH 2 and MN-MgH 2 reduced the M1 concentration in vivo and promoted the repolarization of M2. The therapeutic effect of MN-MgH 2 was better than that of MgH 2 .

[0072] Example 7 MN-MgH 2 can promote tissue regeneration, collagen fiber repair and angiogenesis in vivo

[0073] H&E staining was used to observe granulation tissue formation and measure the epithelial formation process at the tissue level ( Figure 8 , the first column). The results showed that the epithelial tissue was completely formed in the MN-MgH 2 group 10 days after treatment. In contrast, there were still multiple adipose cavities in the epithelial tissues of the control group and the MgH 2 powder group, indicating incomplete healing. In addition, Masson's trichrome staining was used to detect and distinguish collagen fibers and muscle fibers in animal tissues by staining collagen fibers blue (or green), muscle fibers red, and cell nuclei blue ( Figure 8 , the second column). The MN-MgH 2 group had the best collagen deposition and directional arrangement in the tissue, and showed better wound healing and tissue remodeling effects.

[0074] Next, anti-CD31 and anti-ki67 immunofluorescence staining were used to evaluate cell proliferation and capillary formation in vivo after different treatments ( Figure 9 a, 9b). Quantitative analysis of regenerated capillaries demonstrated that the capillary density in the MN-MgH 2 group increased by 2.48 times compared with the control group (Figure 9 c), compared with the MgH 2 group, it increased by 1.48 times.

[0075] At the same time, the proliferating cells in the epidermis and dermis were quantified separately. Although both MgH 2 and MN-MgH 2 treatments could promote the proliferation of epidermal cells ( Figure 9 d), but the proliferation in the dermis of the MN-MgH 2 group was 5.2 times that of the control group and 1.5 times that of the MgH 2 group ( Figure 9 e). In summary, capillary formation and cell proliferation were mainly observed on the tissue surface in the control group and the MgH 2 group, while due to the advantages of microneedle drug delivery, more regenerated capillaries and cells could be formed in the dermis tissue layer in the MN-MgH 2 group.

Claims

1. Use of a microneedle patch in the preparation of a medical device product for promoting the healing of diabetic wounds, reducing the production of reactive oxygen species, promoting cell proliferation and migration, or enhancing angiogenesis. Characterized in that The described microneedle patch includes a support layer and microneedles. One end of the microneedles is connected to the support layer. The needle body of the microneedles is made of PLGA, and the tip contains MgH 2 particles with an average diameter of 8.1 μm.

2. A microneedle patch as described in claim 1. Characterized in that The described MgH 2 has a purity of 98% to 99.9%.

3. A microneedle patch as described in claim 1. Characterized in that Each microneedle on it has a rectangular pyramid shape with specifications of width × length × height being 200μm × 200μm × 500μm.

4. A microneedle patch as described in claim 1. Characterized in that It is a 10×10 microneedle array.

5. A microneedle patch as described in claim 1. Characterized in that It is composed of several units, and each unit is a 10×10 microneedle array.

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