Polymer microneedle with adjustable mechanical property and preparation method thereof

Abstract

The invention discloses a polymer microneedle with adjustable and controllable mechanical properties and a preparation method of the polymer microneedle. Main components of the polymer microneedle comprise the following substances: a polymer material comprising one or more of collagen, gelatin, methacrylated gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and derivatives thereof, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid and a polylactic acid-glycolic acid copolymer, and a cross-linking agent, comprising one of genipin, glutaraldehyde, a photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide, NaOH and CaCl2; the cross-linking degree of the polymer microneedle is regulated and controlled by changing the proportion of the cross-linking agent, so that the mechanical property of the polymer microneedle is changed, the application of the polymer microneedle with different mechanical properties in skin wound healing is disclosed, and the polymer microneedle has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of preparation of polymer microneedles, and particularly to a polymer microneedle with adjustable mechanical properties and a preparation method thereof. Background Art

[0002] Microneedles are a newly emerging form of material in recent years, with needle tips on the micron scale. This miniaturized needle array provides a revolutionary solution for drug delivery, disease diagnosis and treatment with its minimally invasive and painless characteristics. In terms of drug delivery, microneedles can penetrate the outermost layer of the skin and deliver drugs directly to the epidermis or dermis, thereby improving the bioavailability of drugs and reducing systemic side effects. It is particularly suitable for insulin injection, vaccination and local drug treatment for diabetic patients. In the field of disease diagnosis, microneedles can painlessly collect interstitial fluid or trace amounts of blood for real-time monitoring of blood glucose levels and detection of biomarkers, facilitating personalized medicine and early diagnosis. In addition, microneedles can also be used in tissue engineering and regenerative medicine, and can promote wound healing and tissue repair by mechanically stimulating skin regeneration and collagen synthesis.

[0003] Polymer microneedles are a type of micro-needle array made of biodegradable or biocompatible polymer materials, and are widely used in the fields of drug delivery, beauty care and biomedicine. Their core advantage lies in the safety of the materials. Commonly used polymer materials include polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), hyaluronic acid (HA), etc. These materials have good biocompatibility and can be gradually degraded in the body, avoiding the need for secondary surgical removal.

[0004] Different polymer materials have different biological functions (such as anti-inflammatory effects, blood coagulation properties, promoting cell migration and promoting cell proliferation, etc.) and also have different mechanical properties (such as strength, plasticity and elasticity, etc.). In the process of clinical application, different application scenarios have requirements not only for the biological functions of polymer microneedles, but also for the mechanical strength of polymer microneedles. For example, for drug delivery, it is required that the microneedles not only have fast degradation properties but also have high strength to achieve rapid skin puncture and drug release. In addition, in the same application scenario, with the passage of time, changes in the physiological environment will also bring changes in the requirements for the mechanical properties of microneedles. For example, high-stiffness microneedles can promote wound healing in the early stage of wound repair, while in the late stage, high-stiffness microneedles will promote scar formation of the wound, which is not conducive to wound repair. Existing polymer microneedles often neglect the importance of mechanical properties, and the prepared microneedles have fixed mechanical properties, and it is difficult to dynamically adjust the mechanical properties to meet the needs of the dynamically changing physiological environment while maintaining the original biological functions. The mechanical properties of hydrogels can be regulated by the degree of crosslinking. Therefore, microneedles with adjustable mechanical properties can be prepared by only regulating the degree of crosslinking of the hydrogels used to prepare the microneedles. Summary of the Invention

[0005] The object of the present invention is to overcome the disadvantages of the prior art and provide a polymer microneedle with adjustable mechanical properties and a preparation method thereof; the main components of the polymer microneedle include the following substances: a polymer material, including one or more of collagen, gelatin, methacrylated gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, poly(lactic-co-glycolic acid), and a cross-linking agent, including one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, CaCl2; by changing the proportion of the cross-linking agent, the cross-linking degree of the polymer microneedle is regulated, thereby changing the mechanical properties of the polymer microneedle, and the application of polymer microneedles with different mechanical properties in skin wound healing is disclosed, which has good application prospects.

[0006] To achieve the above technical effects, the following technical solutions are adopted: A polymer microneedle with adjustable mechanical properties, comprising the following components: Polymer material and cross-linking agent; The polymer material includes one or more of collagen, gelatin, methacrylated gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, poly(lactic-co-glycolic acid); The cross-linking agent includes one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, CaCl2.

[0007] A preparation method of a polymer microneedle with adjustable mechanical properties is as follows: Fully mix the polymer material solution and the cross-linking agent solution according to the molar ratio, add it to the microneedle mold, and perform low-speed centrifugation or vacuum treatment with a vacuum dryer. After cross-linking, it becomes a hydrogel, and after drying, it is taken out from the mold to obtain the prepared polymer microneedle.

[0008] Further, the molar ratio of the cross-linking agent solution to the polymer material solution is 1:50 to 1:3000.

[0009] Further, the temperature of the low-speed centrifugation or vacuum treatment with a vacuum dryer is 4°C.

[0010] Further, the cross-linking conditions are in an environment of 4°C to 26°C and a humidity of 40 to 80% RH.

[0011] Further, the cross-linking time is 24 to 48 hours.

[0012] Furthermore, the drying conditions are 4°C to 26°C, and the drying time is 24 hours.

[0013] An application method of a polymer microneedle with adjustable mechanical properties, which is applied to the healing of skin wounds. The specific application method is as follows: In the early stage of skin wound healing, polymer microneedles with high elastic modulus and high tensile strength, that is, polymer microneedles with high cross-linking degree, are selected to improve the mechanical environment of the wound and promote the wound closure speed. In the late stage of skin wound healing, polymer microneedles with low elastic modulus and low tensile strength, that is, polymer microneedles with low cross-linking degree, are selected to reduce the proliferation of fibroblasts, the differentiation of myofibroblasts and the overexpression of collagen during the wound repair process, achieving fast repair speed and scarless repair.

[0014] The beneficial effects of the present invention are as follows: The present invention discloses a polymer microneedle with adjustable mechanical properties and its preparation method. The main components of the polymer microneedle include the following substances: polymer materials, including one or more of collagen, gelatin, methacrylated gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, poly(lactic-co-glycolic acid), and cross-linking agents, including one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, CaCl2; the cross-linking degree of the polymer microneedle is regulated by changing the proportion of the cross-linking agent, thereby changing the mechanical properties of the polymer microneedle, and the application of polymer microneedles with different mechanical properties in skin wound healing is disclosed, having good application prospects.

[0015] (1) The polymer microneedle with adjustable mechanical properties of the present invention realizes the regulation of the mechanical properties of the microneedle without affecting the structure and composition of the microneedle.

[0016] (2) The polymer microneedle with adjustable mechanical properties of the present invention realizes the adaptation to the needs of the dynamically changing physiological environment by dynamically regulating the mechanical properties while maintaining the original biological functions of the microneedle, improving the effect of the microneedle in different biomedical application scenarios (such as skin repair, vaccine delivery, etc.).

[0017] (3) The microneedle preparation method of the present invention is simple, has a wide application range, low production cost, and is easy to mass-produce. Description of the Drawings

[0018] Figure 1 It is the chemical reaction formula of the cross-linking of CMCS and genipin in the embodiment of the present invention; Figure 2Flow chart for the preparation of CMCS microneedles with different crosslinking degrees in the embodiments of the present invention; Figure 3 Schematic diagram of the tensile strength specimen of the microneedle material in the embodiments of the present invention; Figure 4 Macromorphology diagrams of CMCS microneedles with different crosslinking degrees in the embodiments of the present invention; Figure 5 Morphology diagram of MN400 microneedles observed under a stereomicroscope in the embodiments of the present invention; Figure 6 Morphology diagram of MN100 microneedles observed under a stereomicroscope in the embodiments of the present invention; Figure 7 Needle body morphology and height diagram of MN400 microneedles observed under a stereomicroscope in the embodiments of the present invention; Figure 8 Needle body morphology and height diagram of MN100 microneedles observed under a stereomicroscope in the embodiments of the present invention; Figure 9 Needle body height comparison diagram of CMCS microneedles with different crosslinking degrees in the embodiments of the present invention; Figure 10 Elastic modulus comparison diagram of dry microneedle materials with different crosslinking degrees in the embodiments of the present invention; Figure 11 Elastic modulus comparison diagram of swollen microneedle materials with different crosslinking degrees in the embodiments of the present invention; Figure 12 Tensile strength specimen diagrams of microneedle materials with different crosslinking degrees in the embodiments of the present invention; Figure 13 Tensile strength comparison diagram of microneedle materials with different crosslinking degrees in the embodiments of the present invention; Figure 14 Force-displacement curve comparison diagram of single microneedles with different crosslinking degrees in the embodiments of the present invention; Figure 15 Live-dead cell staining results diagram of microneedles with different mechanical properties and a scale of 100 μm for the fibroblasts planted on them in the embodiments of the present invention; Figure 16 Proliferation diagrams of fibroblasts planted on microneedles with different mechanical properties after 1, 3, and 5 days of culture on MNs or in 48-well plates in the embodiments of the present invention; Figure 17 Diagram of microneedles with different mechanical properties promoting the wound healing of rats in the embodiments of the present invention; among them, Figure 17 a is a schematic diagram of the animal experiment; Figure 17 b is the macroscopic wound morphology of the rat wounds at d3 and d7 under the application of microneedles with different mechanical properties; Figure 17 c is the statistical result of the wound size of the rat wounds at d3 and d7; Figure 17d shows the H&E staining results of the rat wound tissue on day 3; Figure 17 e shows the H&E staining results of the rat wound tissue on day 7; the area pointed by the red triangle is the fibroblast aggregation area; n = 5, *P < 0.05, **P < 0.01 and ***P < 0.001; Figure 18 This is the diagram showing the fibrosis condition of the rat wounds repaired by microneedles with different mechanical properties in the embodiment of the present invention; wherein, Figure 18 a, Figure 18 b respectively show the Masson staining and Sirius red staining results of the rat wound tissue on day 7 ( Figure 18 in b, the orange - red color is type I collagen, and the yellow - green color is type III collagen); Figure 18 c, Figure 18 d shows the α - SMA immunohistochemical staining image and the statistical result diagram of the mouse wound tissue on day 7; n = 5, *P < 0.05, **P < 0.01 and ***P < 0.001. Detailed implementation mode

[0019] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific implementation modes and are not intended to limit the exemplary implementation modes of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof.

[0022] Example 1: Microneedle preparation: 1. Preparation of carboxymethyl chitosan (CMCS) solutions with different cross - linking degrees Genipin can react with the amino groups of CMCS to achieve network cross - linking. The chemical reaction formula is as Figure 1As shown, genipin powder was added to ultrapure water and evenly dispersed in the ultrapure water using a magnetic stirrer to prepare a 1% w / v genipin aqueous solution. CMCS powder was added to ultrapure water and mixed evenly with a magnetic stirrer to completely dissolve it in the ultrapure water, preparing a 7.5% w / v CMCS aqueous solution. The 7.5% w / v CMCS aqueous solution was placed in an ultrasonic cleaner to remove air bubbles by ultrasonic waves.

[0023] The 1% w / v genipin aqueous solution and the 7.5% w / v CMCS aqueous solution were mixed at a molar ratio of genipin to CMCS of 1:100 and 1:400, respectively. The mixed solution was allowed to stand at room temperature for 24 h to fully crosslink, obtaining CMCS solutions with different degrees of crosslinking.

[0024] 2. Preparation of CMCS microneedles with different degrees of crosslinking According to Figure 2 the shown process, microneedles were prepared. The microneedle molds added with CMCS solutions with different degrees of crosslinking were placed in a vacuum dryer and evacuated with a vacuum pump to vacuum, so that the solution completely filled the mold and the air bubbles in the mold were discharged. After crosslinking in a high-humidity environment for 24 h, it was allowed to air-dry naturally at room temperature. After air-drying, the microneedles with different degrees of crosslinking were demolded. In subsequent experiments, MN100 and MN400 were used to represent the specimens and microneedles with a ratio of 1:100 and 1:400 in the two groups, respectively.

[0025] 3. Preparation of elastic modulus specimens of dried microneedle materials Solutions with a molar ratio of genipin to CMCS of 1:100 and 1:400 were prepared, poured into a cylindrical mold, demolded after crosslinking for 24 h, and air-dried naturally to obtain cylindrical specimens with a diameter of 6 mm and a height of 7 mm. Five samples were prepared for each group as parallel samples.

[0026] 4. Preparation of tensile strength specimens of microneedle materials Solutions with a molar ratio of genipin to CMCS of 1:100 and 1:400 were prepared and poured onto a glass slide covered with a PDMS mold with a length of 10 cm and a width of 2 cm, ensuring that the thickness of the solution was 5 mm. It was placed in a vacuum dryer with a high environmental humidity, taken out and air-dried after crosslinking for 24 h. The air-dried material strip was peeled off with a blade. It was cut into a dumbbell shape as Figure 3 shown, with a width of 12 mm at both ends, a total length of 70 mm, a middle section length of 25 mm, and a width of 5 mm. Five samples were prepared for each group as parallel samples.

[0027] 5. Preparation of single microneedle samples CMCS microneedles with different degrees of crosslinking were prepared, and the microneedles were cut into single and independent ones with a scalpel, ensuring that the morphology of the needle body was not damaged and the base of the needle body was flat during the process.

[0028] Characterization method: 1. Measuring the elastic modulus with a mechanical testing machine Place the dried material specimen on the loading platform of a universal mechanical testing machine. Select a 1 kN mechanical sensor to conduct a compression test under unconfined conditions. Apply mechanical loading to the specimen at a loading speed of 0.5 mm / min, and record the stress-strain curve until the specimen structure fails. Select the data in the strain range of 0 - 10% to calculate the elastic modulus of the dried material.

[0029] Place the swollen material specimen on the loading platform of a universal mechanical testing machine. Select a 50 N mechanical sensor to conduct a compression test under unconfined conditions. Apply mechanical loading to the specimen at a loading speed of 1 mm / min, and record the stress-strain curve until the specimen structure fails. Select the stress-strain curve before the structural strength failure to calculate the elastic modulus of the swollen material.

[0030] 2. Measuring the tensile strength with a mechanical testing machine Clamp the material specimen to be tested on a universal mechanical testing machine. Select a 100 N mechanical sensor to conduct a unidirectional tensile test. Apply mechanical loading to the specimen at a loading speed of 0.5 mm / min, and record the force-displacement curve until the specimen fails. Calculate the tensile strength of the material based on the maximum force and the original cross-section.

[0031] 3. Measuring the maximum force on a single microneedle with a mechanical testing machine Place the single microneedle specimen to be tested on the loading platform of a universal mechanical testing machine. Select a 50 N mechanical sensor to conduct a compression test under unconfined conditions. Apply mechanical loading to the specimen at a loading speed of 0.03 mm / min, record the force-displacement curve until the specimen fails. Analyze the maximum force on the microneedle.

[0032] Experimental results: 1. Analyzing the microneedle morphology by stereomicroscope The prepared MN100 and MN400 microneedles are as Figure 4 shown. Due to different degrees of crosslinking, both show blue with different shades. Among them, MN100 is dark blue and MN400 is light blue.

[0033] Observe the top view and 45° oblique view of the microneedles under a stereomicroscope, as Figure 5 and Figure 6 shown. The solutions with two degrees of crosslinking can both be prepared into microneedles, and the microneedles are arranged in a regular matrix, with the needle body morphology intact, no bubbles and no bending.

[0034] Observe the side view of the microneedles under a stereomicroscope and compare whether there are differences in the needle body height of CMCS microneedles with different degrees of crosslinking. As Figure 7 and Figure 8As shown, the needle bodies of the two groups of microneedles have similar heights and good morphologies.

[0035] The software built into the stereomicroscope was used to statistically analyze the needle body height. For each group of needles, 3 samples were taken, and the heights of 10 needles were statistically analyzed for each sample, obtaining Figure 9 results that show no significant difference in the needle body heights between the two groups of needles.

[0036] 2. Analysis of Elastic Modulus Determined by Mechanical Testing Machine After preparing genipin-crosslinked CMCS with different degrees of crosslinking, it is necessary to explore whether the degree of crosslinking will affect the mechanical properties of the material. First, the elastic modulus of the material is analyzed. After performing compression tests on MN100 and MN400 specimens, the elastic moduli of the dry microneedles were obtained by analyzing the data in the strain range of 0 - 10% as Figure 10 shown. The elastic modulus of MN100 is 499.45 ± 61.74 MPa, and the elastic modulus of MN400 is 255.36 ± 77.55 MPa. The elastic modulus of MN100 is approximately twice that of MN400. Through t-test, it can be obtained that the two groups have significant differences.

[0037] Since the prepared microneedles are hydrogel microneedles with the property of swelling, it is necessary to analyze the mechanical properties of the material after swelling. After inserting the dry microneedle material into a 2% agarose block soaked in PBS buffer solution simulating the skin environment for 24 hours, obvious swelling of the microneedle material occurred. By analyzing the data obtained from the compression test of the swollen microneedle material, the results are as Figure 11 shown. The elastic modulus of the swollen microneedle material decreased significantly. After swelling, MN100 is (8.78 ± 6.16) × 10 -2 MPa, while MN400 is (2.73 ± 0.73) × 10 -3 MPa. Through t-test, it can be obtained that there are significant differences between the two. Since the elastic modulus of human soft tissue is (2.70 ± 0.70) × 10 -2 Mpa, therefore, the elastic modulus of MN100 after swelling is higher than that of human soft tissue, while the elastic modulus of MN400 is lower than that of human soft tissue.

[0038] In summary, by changing the degree of crosslinking of the CMCS solution, the elastic modulus of the CMCS microneedle material can be changed, and the degree of crosslinking is positively correlated with the elastic modulus. In addition, whether it is the dry material or the swollen material, the elastic moduli of CMCS materials with different degrees of crosslinking have significant differences.

[0039] 3. Analysis of Tensile Strength Determined by Mechanical Testing Machine Solutions with a genipin to CMCS molar ratio of 1:100 and 1:400 were prepared as Figure 12The dumbbell-shaped specimen shown

[0040] The tensile strength of the material can be obtained through the tensile test of a mechanical testing machine as Figure 13 shown. The tensile strength of MN100 is 37.40 ± 1.26 MPa, and the tensile strength of MN400 is 26.57 ± 2.14 MPa. Through t-test, there are significant differences between the two groups

[0041] In summary, the tensile strength of MN100 is significantly higher than that of MN400. Therefore, when microneedles prepared from materials with different crosslinking degrees are inserted into the wound, due to the differences in the tensile strength of the microneedle substrate, the wound closure effect may be affected

[0042] 4. Analysis of the maximum force on a single microneedle Place a single microneedle on the stage and apply pressure to the microneedle. It can be seen that the force-displacement curve of the microneedle is as Figure 14 shown. MN400 showed obvious failure at 5 N, while MN100 did not show obvious failure until 10 N. The maximum forces on the two groups of microneedles can ensure their smooth insertion into the skin. However, due to the inconsistent elastic moduli of the two groups of microneedles, the slopes of the force-displacement curves are significantly different

[0043] 5. CMCS microneedles with different mechanical properties regulate fibroblasts Soak the microneedles in high-glucose DMEM medium (containing 10% fetal bovine serum) for 12 h, and cut out microneedles with the size of a single well of a 48-well plate and spread them on the bottom of the well. Inoculate 1×10 5 NIH 3T3 cells on the microneedles or in the wells, and add 1 ml of high-glucose DMEM medium (containing 10% fetal bovine serum) and culture at 37 °C and 5% CO2 for 24 h. Acridine orange / ethidium bromide (AO / EB) staining is used to evaluate the cytotoxicity. In the cell proliferation experiment, 4000 NIH 3T3 cells are inoculated on the microneedles or in the wells, add 1 ml of high-glucose DMEM medium (containing 10% fetal bovine serum) and culture at 37 °C and 5% CO2. Add cck8 reagent on days 1, 3, and 5 respectively, and measure the absorbance at 450 nm after incubation for 2 h

[0044] The toxicity of microneedles with different mechanical properties to fibroblasts was observed by AO / EB live-dead cell staining, as Figure 15As shown, both MN100 and MN400 have good biocompatibility (red represents dead cells and green represents live cells). However, the cells on MN100 are aggregated and have a higher density than those cultured in a 48-well plate, while the cell density on MN400 is lower than that cultured in a 48-well plate, indicating that their effects on cell proliferation are inconsistent. By further specifically monitoring the effect of the mechanical properties of MNs on fibroblast proliferation after 5 days of culture using cck8, it was found that the proliferation rate of fibroblasts cultured on MN100 was significantly higher than that of fibroblasts cultured in a 48-well plate, while the rate of fibroblasts cultured on MN400 was significantly lower than that of fibroblasts cultured in a 48-well plate, as Figure 16 shown. Therefore, the mechanical properties of microneedles have different effects on fibroblast proliferation. Microneedles with a high elastic modulus upregulate fibroblast proliferation, while microneedles with a low elastic modulus downregulate fibroblast proliferation.

[0045] 6. Effects of CMCS microneedles with different mechanical properties on wound repair in rats Male SD rats at 8 weeks old were selected for the experiment. Two full-thickness skin wounds in the shape of a spindle with a length of 3 cm and a width of 1 cm were made on the back of each SD rat. The wounds were equidistant from the midline and perpendicular to the minimum tension line (i.e., along the spinal column direction). The distance between the two wounds was 2.5 cm to ensure that there was no influence on the wounds between different groups. The spindle-shaped wounds on the back of SD rats were divided into four groups: blank group (Control), suture group (Suture), MN100, and MN400. Each group was further divided into a 3-day sampling group and a 7-day sampling group. Each group had 4 parallel samples, and a total of 32 spindle-shaped wounds were required, that is, a total of 16 SD rats were needed.

[0046] This experiment has obtained the review and approval of the Bioethics Committee of Beihang University, approval number: BM20220026.

[0047] To ensure that making the experimental wounds on the upper and lower backs of SD rats has no influence on the experimental results, the experimental groups were arranged to ensure that there were 2 wounds on both the upper and lower backs of SD rats in each group. The specific grouping was carried out according to Table 1 below.

[0048] Table 1 Animal experiment grouping

[0049] Characterization methods: (1) Photograph the morphology of animal wounds The morphology of the wounds was photographed respectively after making the wounds at 0 d and when sampling at 3 d and 7 d.

[0050] (2) Analyze the wound area change rate using ImageJ Use ImageJ software to process the wound morphology of SD rats photographed in 4.3.3.1, and frame the wound area as the ROI. When selecting, ensure that the edge of the ROI area is closely attached to the edge of the wound or the edge of the scar tissue after wound healing. The scar area after healing is also included in the wound area. Divide the wound area at the time of material collection by the wound area at 0 d to obtain the wound area change rate of each group.

[0051] (3)Take pictures of H&E staining, Masson staining sections, and α-SMA staining sections with an optical microscope Take pictures of H&E staining and Masson staining sections under the bright field of the microscope and with lighting from below. Adjust the exposure time and contrast during shooting to ensure that the tone of each photo is consistent.

[0052] (4)Take pictures of Sirius red staining sections with a polarized light microscope Rotate and adjust the polarizer so that the polarizer and analyzer are at a 90-degree angle. Adjust the aperture size to make the overall image screen darker, and only the yellow-green and orange-red colors of collagen appear.

[0053] (5)Statistical analysis There are four parallel samples in each group of experiments. The data is analyzed using SPSS software, and the data is analyzed using a two-sample t-test. When p < 0.05, there is a significant difference between the data. ns indicates no difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0054] Experimental procedure: (1)Animal surgery After anesthetizing the rats, depilate them, and wipe the skin surface of the rats' backs with alcohol and iodophor. Make 2 full-thickness injury wounds on the rats' skin, each with a length of 3 cm and a width of 1 cm, and the wounds are spaced 2.5 cm apart to avoid mutual influence of the wounds. The following treatment methods are taken for the four animal groups respectively: 1) Control: Directly apply a layer of 3M-Tegaderm film to the wound.

[0055] 2) Suture: Close the wound with absorbable suture. Apply a layer of 3M-Tegaderm film after suture.

[0056] 3) MN100: Pull the skin on both sides of the wound to close it and then insert microneedles. Insert 3 MN100 into each wound to ensure that the microneedles cover the entire wound, and then apply a layer of 3M-Tegaderm film.

[0057] 4) MN400: Pull the skin on both sides of the wound to close it and then insert microneedles. Insert 3 MN400 into each wound to ensure that the microneedles cover the entire wound, and then apply a layer of 3M-Tegaderm film.

[0058] After the operation, the rats were housed individually in cages. The physical condition and feeding status of the rats were observed daily to avoid infection, and the bedding was changed in a timely manner to replenish food and water.

[0059] (2)Animal sampling At the sampling point, the rats were sampled. A rectangular skin tissue with a width of 3 × 1 cm was cut from the initial wound area, paraffin sections were prepared, and H&E staining, Masson staining sections, Sirius red staining, and α-SMA staining sections were completed.

[0060] Experimental results: As Figure 17 shown, as Figure 17 in b, compared with the open wound Control group without treatment, the Suture group, MN100 group, and MN400 group used as controls had all closed on the 3rd day. However, obvious scabs and swelling appeared in the Suture group, and the closing effect of MN400 was also inferior to that of MN100. Due to the anti-inflammatory ability of CMCS in MN and the ability of hydrogel microneedles to absorb exudative tissue fluid, the surface inflammation of the wounds treated with MN was also significantly improved. By the 7th day, the Control group had not closed yet and obvious inflammation occurred. Although the Suture group had closed, the suture had not been absorbed. The wound repair effect of using MN was the best.

[0061] The wound sizes of each group were quantitatively analyzed using ImageJ. As Figure 17 shown in c, at 3d, the area of the Suture group was 1.31× 10 -1 , the area of the MN100 group was 3.50 × 10 -2 , and the area of MN400 was 5.42 × 10 -2 , indicating that the use of microneedles can indeed accelerate wound closure, and faster than the sutures commonly used in surgery. In addition, microneedles with high elastic modulus and high tensile strength had a faster closing speed than those with low elastic modulus and low tensile strength. At 7d, the area of the Suture group was 5.72 × 10 -2 , the area of the MN100 group was 1.71 × 10 -2 , and the area of MN400 was 1.64 × 10 -2 . The closing effect of the microneedle group on the wound was still better than that of the Suture group, but the difference between the two microneedle groups was no longer obvious. It shows that the use of microneedles can indeed promote wound closure, and higher elastic modulus and tensile strength can bring a faster wound closing speed in the early stage of wound repair, but the mechanical properties of microneedles have no obvious effect on the closing speed in the later stage.

[0062] The H&E stained sections of the repaired wound skin were observed. As Figure 17 shown in d and Figure 17e. It can be seen that the wound tissue in the Control group at 3D is granulation tissue, and there is inflammation in the wound, accompanied by a large number of fibroblasts. There are obvious gaps and scabs at the seam of the two sides of the skin in the Suture group compared with the MN group, indicating that the wound is not completely closed. In addition, the number of fibroblasts at the wound in the Suture group is small, while in the MN group, fibroblasts can be clearly seen filling the gap between the two sides of the wound and connecting the two sides of the skin, and the number of fibroblasts in the MN100 group is more than that in the MN400 group. This shows that microneedles accelerate the migration of fibroblasts and inhibit the inflammatory response in the early stage of wound healing. And the number of fibroblasts is consistent with the results of in vitro cell culture, indicating that the promotion of fibroblast proliferation by the high elastic modulus microneedles also brings a faster closing rate.

[0063] In the tissue at 7D, it can be seen that the newly formed tissue at the wound in the Control group is higher than the normal skin, and a large number of non-apoptotic fibroblasts and inflammatory cells appear. Although the wound in the Suture group is closed, the newly formed tissue is lower than the height of the surrounding normal skin. The wound in the MN100 group is closed, but the newly formed tissue is also lower than the height of the surrounding normal skin, and there are a large number of fibroblasts. Although the closing rate of the wound in the MN400 group is slower than that in the MN100 group, the newly formed tissue is consistent with the height of the normal skin, and the number of fibroblasts in the tissue is closest to the normal tissue, showing the best repair effect.

[0064] As Figure 18 shown, by characterizing the collagen content and types in the newly formed tissue at the wound after 7D and the myofibroblasts related to scar formation, the fibrosis situation of the wound repaired by MN was further evaluated. Figure 18 As shown in a, the collagen distribution in the newly formed tissue at the wound after repair can be seen. The collagen in the wound surface of the control group is lower and arranged disorderly. In contrast, the collagen in the MNs patch group and the suture group is more arranged along the wound surface. The fibrosis situation of the repaired tissue can be further evaluated by the ratio of type I collagen / type III collagen. As Figure 18 shown in b, compared with the MN400 group and the Suture group, the proportion of type I collagen showing orange-red color in the tissues of the Control group and the MN100 group is larger, indicating that fibrosis has occurred in the untreated wound and the wound treated with MN100, while the wounds treated with suture and MN400 have no fibrosis. In addition, an increase in the proportion of type I collagen was also observed in the undamaged areas of the two sides of the skin in the MN100 group. From Figure 18 the immunohistochemical staining result diagram in c, it can be seen that myofibroblasts exist in the newly formed tissues of all three groups, but α-SMA in the MN100 group is also distributed under the epidermis of the skin, which may be related to the way of microneedles piercing the skin. According to Figure 18From the quantitative statistical results of d, it can be seen that the content of myofibroblasts in the wound tissue using MN100 is significantly higher than that using MN400 and suture, indicating that microneedles with a higher skin elastic modulus will increase the differentiation of myofibroblasts during the later stage of wound repair, thus bringing about more serious fibrosis.

[0065] In summary, although MN100 brought a faster wound closure speed in the early stage of wound repair, it led to fibrosis of the newly formed tissue in the wound and even the uninjured skin around it due to the excessive proliferation of fibroblasts, increased differentiation of myofibroblasts, and increased expression of type I collagen in the later stage of wound repair. The final repair effect of MN400 is even better than that of the wound using suture, with a collagen content and arrangement closer to normal tissue, achieving scarless repair.

[0066] Generally speaking, the specific application method of the polymer microneedle in skin wound healing should be as follows: In the early stage of skin wound healing, select polymer microneedles with a high elastic modulus and high tensile strength, that is, polymer microneedles with a high degree of crosslinking, to improve the wound mechanical environment and promote the wound closure speed. In the later stage of skin wound healing, select polymer microneedles with a low elastic modulus and low tensile strength, that is, polymer microneedles with a low degree of crosslinking, to reduce the proliferation of fibroblasts, the differentiation of myofibroblasts, and the overexpression of collagen during the wound repair process, achieving a fast repair speed and scarless repair.

[0067] In summary, the present invention discloses a polymer microneedle with adjustable mechanical properties and its preparation method; the main components of the polymer microneedle include the following substances: a polymer material, including one or more of collagen, gelatin, methacrylated gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, poly(lactic-co-glycolic acid), and a crosslinking agent, including one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, CaCl2; by changing the proportion of the crosslinking agent, the degree of crosslinking of the polymer microneedle is regulated, thereby changing the mechanical properties of the polymer microneedle, and the application of polymer microneedles with different mechanical properties in skin wound healing is disclosed, having good application prospects.

[0068] At this point, those skilled in the art recognize that although the embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or deduced from the content disclosed in the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and determined to cover all these other variations or modifications.

Claims

1. A polymer microneedle with adjustable mechanical properties, characterized in that, The polymer microneedles are composed of the following components: Polymer materials and crosslinking agents; The polymer materials include one or more of collagen, gelatin, methacrylated gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, poly(lactic-co-glycolic acid); The crosslinking agents include one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, CaCl2.

2. The preparation method of a polymer microneedle with adjustable mechanical properties as described in claim 1, characterized in that, The preparation method is as follows: Fully mix the polymer material solution and the crosslinking agent solution according to the molar ratio, add it to the microneedle mold, and perform low-speed centrifugation or vacuum treatment in a vacuum dryer. After crosslinking, it becomes a hydrogel, and after drying, it can be taken out of the mold to obtain the prepared polymer microneedles.

3. The preparation method of a polymer microneedle with adjustable mechanical properties as described in claim 2, characterized in that, The molar ratio of the crosslinking agent solution to the polymer material solution is 1:50 to 1:3000.

4. The preparation method of a polymer microneedle with adjustable mechanical properties as described in claim 2, characterized in that, The temperature for low-speed centrifugation or vacuum treatment in a vacuum dryer is 4°C.

5. The preparation method of a polymer microneedle with adjustable mechanical properties as described in claim 2, characterized in that, The crosslinking conditions are in an environment of 4°C to 26°C and a humidity of 40 to 80 %RH.

6. The preparation method of a polymer microneedle with adjustable mechanical properties as described in claim 2, characterized in that, The crosslinking time is 24 to 48 hours.

7. The preparation method of a polymer microneedle with adjustable mechanical properties as described in claim 2, characterized in that, The drying conditions are 4°C to 26°C, and the drying time is 24h.

8. The application method of a polymer microneedle with adjustable mechanical properties as described in claim 1, characterized in that, The polymer microneedles are applied to the healing of skin wounds. The specific application method is as follows: In the early stage of skin wound healing, polymer microneedles with high elastic modulus and high tensile strength, that is, polymer microneedles with high crosslinking degree, are selected to improve the wound mechanical environment and promote the wound closure speed. In the late stage of skin wound healing, polymer microneedles with low elastic modulus and low tensile strength, that is, polymer microneedles with low crosslinking degree, are selected to reduce the proliferation of fibroblasts, the differentiation of myofibroblasts and the overexpression of collagen during the wound repair process, achieving fast repair speed and scarless repair.

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