Mechanical property controllable polymer microneedle and preparation method thereof
By controlling the degree of crosslinking of polymer microneedles, microneedles with different mechanical properties were prepared, which solved the problem of insufficient adaptability caused by fixed mechanical properties in the existing technology, and realized dynamic control and optimization effects in drug delivery and skin wound healing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIHANG UNIV
- Filing Date
- 2025-04-16
- Publication Date
- 2026-06-16
AI Technical Summary
Existing polymer microneedles are difficult to dynamically regulate mechanical properties to adapt to the needs of different physiological environments while maintaining biological functions, which affects their effectiveness in applications such as drug delivery and skin wound healing.
By adjusting the proportion of crosslinking agents to change the degree of crosslinking of polymer microneedles, their mechanical properties can be controlled. Microneedles with high elastic modulus and high tensile strength can be prepared for early wound healing, while microneedles with low elastic modulus and low tensile strength can be prepared for later healing. Combinations of polymer materials such as collagen, gelatin, and chitosan with crosslinking agents such as genipin and glutaraldehyde are used.
It achieves dynamic control of mechanical properties without affecting the structure and composition of microneedles, adapts to changes in the physiological environment, promotes early and rapid wound healing and reduces scar formation, and is suitable for drug delivery and skin wound healing.
Smart Images
Figure CN120241584B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of polymer microneedle preparation, specifically to a polymer microneedle with tunable mechanical properties and its preparation method. Background Technology
[0002] Microneedles, a relatively new material form, consist of needles with dimensions on the micrometer scale. This miniaturized array of needles, with its minimally invasive and painless characteristics, offers revolutionary solutions for drug delivery, disease diagnosis, and treatment. In drug delivery, microneedles can penetrate the outermost layer of the skin, delivering drugs specifically to the epidermis or dermis, thereby improving drug bioavailability and reducing systemic side effects. This is particularly suitable for insulin injections, vaccinations, and topical drug treatments in diabetic patients. In 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. Furthermore, microneedles can be used in tissue engineering and regenerative medicine, promoting wound healing and tissue repair through mechanical stimulation of skin regeneration and collagen synthesis.
[0003] Polymer microneedles are arrays of tiny needles made from biodegradable or biocompatible polymer materials, widely used in drug delivery, cosmetic skincare, and biomedicine. Their core advantage lies in the safety of the materials used; commonly used polymers include polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), and hyaluronic acid (HA). These materials have excellent biocompatibility, gradually degrading within the body and avoiding the need for secondary surgical removal.
[0004] Different polymer materials possess varying biological functions (e.g., anti-inflammatory effects, coagulation properties, cell migration promotion, and cell proliferation promotion) and mechanical properties (e.g., strength, plasticity, and elasticity). In clinical applications, different scenarios demand not only the biological functions of polymer microneedles but also their mechanical strength. For example, drug delivery requires not only rapid degradation but also high strength to achieve rapid skin penetration and drug release. Furthermore, within the same application scenario, changes in the physiological environment over time can alter the mechanical performance requirements of microneedles. For instance, high-stiffness microneedles promote wound healing in the early stages, but in later stages, they can promote scar formation, hindering wound repair. Existing polymer microneedles often neglect the importance of mechanical properties, resulting in microneedles with fixed mechanical properties. This makes it difficult to dynamically adjust mechanical properties to adapt to dynamically changing physiological environments while maintaining the original biological functions. The mechanical properties of hydrogels can be regulated by the degree of crosslinking. Therefore, microneedles with tunable mechanical properties can be prepared by simply controlling the degree of crosslinking of the hydrogel used to prepare the microneedles. Summary of the Invention
[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a polymer microneedle with controllable 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, methacrylamide gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, and polylactic-co-glycolic acid copolymer; and crosslinking agents, including one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, and CaCl2. By changing the proportion of crosslinking agent, the degree of crosslinking of the polymer microneedle is controlled, thereby changing the mechanical properties of the polymer microneedle. The invention also discloses the application of polymer microneedles with different mechanical properties in skin wound healing, which has good application prospects.
[0006] To achieve the above technical effects, the following technical solution is adopted:
[0007] A polymeric microneedle with tunable mechanical properties comprises the following components:
[0008] Polymer materials and crosslinking agents;
[0009] The polymeric materials include one or more of the following: collagen, gelatin, methacrylamide gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, and polylactic acid-glycolic acid copolymer.
[0010] The crosslinking agent includes one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, and CaCl2.
[0011] A method for preparing polymer microneedles with tunable mechanical properties is as follows:
[0012] The polymer material solution and the crosslinking agent solution are thoroughly mixed in a molar ratio, added to a microneedle mold, and then subjected to low-speed centrifugation or vacuum treatment in a vacuum dryer. After crosslinking, the mixture becomes a hydrogel. After drying, the polymer microneedles are removed from the mold to obtain the prepared polymer microneedles.
[0013] Furthermore, the molar ratio of the crosslinking agent solution to the polymer material solution is 1:50 to 1:3000.
[0014] Furthermore, the low-speed centrifugation or vacuum dryer vacuum treatment temperature is 4°C.
[0015] Furthermore, the crosslinking conditions are 4℃~26℃ and 40~80%RH.
[0016] Furthermore, the crosslinking time is 24 to 48 hours.
[0017] Furthermore, the drying conditions are 4℃~26℃, and the drying time is 24h.
[0018] A method for applying polymer microneedles with tunable mechanical properties to the healing of skin wounds, the specific application method is as follows:
[0019] In the early stages of skin wound healing, high-elasticity and high-tensile-strength polymer microneedles, i.e., highly cross-linked polymer microneedles, are selected to improve the mechanical environment of the wound and promote the wound closure speed. In the later stages of skin wound healing, low-elasticity and low-tensile-strength polymer microneedles, i.e., low-cross-linked polymer microneedles, are selected to reduce the proliferation of fibroblasts, the differentiation of myofibroblasts, and the overexpression of collagen during the wound repair process, thus achieving rapid repair and scarless repair.
[0020] The beneficial effects of this invention are as follows:
[0021] This invention discloses a polymer microneedle with tunable 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, methacrylamide gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, and polylactic-co-glycolic acid copolymer; and crosslinking agents, including one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, and CaCl2. The crosslinking degree of the polymer microneedle is controlled by changing the proportion of the crosslinking agent, thereby changing the mechanical properties of the polymer microneedle. The invention also discloses the application of polymer microneedles with different mechanical properties in skin wound healing, which has good application prospects.
[0022] (1) The mechanical properties of the polymer microneedles of the present invention can be controlled, which realizes the control of the mechanical properties of the microneedles without affecting the structure and composition of the microneedles.
[0023] (2) The mechanical properties of the polymer microneedles of the present invention can be adjusted to adapt to the needs of dynamically changing physiological environment by dynamically adjusting the mechanical properties while maintaining the original biological function of the microneedles, thereby improving the effect of microneedles in different biomedical application scenarios (such as skin repair, vaccine delivery, etc.).
[0024] (3) The microneedle preparation method of the present invention is simple, has a wide range of applications, low production cost, and is easy to mass-produce. Attached Figure Description
[0025] Figure 1 This is the chemical reaction formula for the crosslinking of CMCS and genipin in the embodiments of the present invention;
[0026] Figure 2 This is a flowchart illustrating the preparation process of CMCS microneedles with different degrees of crosslinking in this invention.
[0027] Figure 3 This is a schematic diagram of the tensile strength test specimen of the microneedle material in an embodiment of the present invention;
[0028] Figure 4 These are macroscopic morphology images of CMCS microneedles with different degrees of crosslinking in the embodiments of the present invention;
[0029] Figure 5 This is a morphological image of the MN400 microneedles observed under a stereomicroscope in an embodiment of the present invention.
[0030] Figure 6 This is a morphological image of the MN100 microneedles observed under a stereomicroscope in an embodiment of the present invention.
[0031] Figure 7 This is a diagram showing the morphology and height of the MN400 microneedle under a stereomicroscope in an embodiment of the present invention.
[0032] Figure 8 This is a diagram showing the morphology and height of the MN100 microneedle under a stereomicroscope in an embodiment of the present invention.
[0033] Figure 9 This is a comparison diagram of the needle height of CMCS microneedles with different degrees of crosslinking in the embodiments of the present invention;
[0034] Figure 10 This is a comparison diagram of the elastic modulus of dried microneedle materials with different degrees of crosslinking in the embodiments of the present invention;
[0035] Figure 11 This is a comparison diagram of the elastic modulus of microneedle materials swollen with different degrees of crosslinking in the embodiments of the present invention;
[0036] Figure 12 These are specimen images of tensile strength of microneedle materials with different degrees of crosslinking in the embodiments of the present invention;
[0037] Figure 13 This is a comparison diagram of the tensile strength of microneedle materials with different degrees of crosslinking in the embodiments of the present invention;
[0038] Figure 14 This is a comparison of force-displacement curves of single microneedles with different degrees of crosslinking in the embodiments of the present invention;
[0039] Figure 15This is a diagram showing the staining results of live and dead cells of fibroblasts implanted on microbeads with different mechanical properties at a scale bar of 100 μm in an embodiment of the present invention.
[0040] Figure 16 The diagram shows the proliferation of fibroblasts seeded on microbeads with different mechanical properties in the embodiments of the present invention after 1, 3 and 5 days of culture in MNs or 48-well plates.
[0041] Figure 17 The figures show microneedles with different mechanical properties promoting wound healing in rats in embodiments of the present invention; wherein, Figure 17 a is a schematic diagram of an animal experiment; Figure 17 b shows the macroscopic wound morphology of rat wounds on days 3 and 7 after the application of microneedles with different mechanical properties; Figure 17 c represents the statistical results of rat wound size on days 3 and 7; Figure 17 d represents the H&E staining results of rat wound tissue on day 3; Figure 17 e shows the H&E staining results of rat wound tissue on day 7; the red triangles indicate the fibroblast aggregation areas; n=5, *P < 0.05, **P < 0.01 and ***P < 0.001;
[0042] Figure 18 The figures shown are of different mechanical properties of microneedles used in the present invention to repair fibrosis in rat wounds; wherein, Figure 18 a, Figure 18 b shows the Masson staining and Sirius red staining results of rat wound tissue on day 7. Figure 18 In b, orange-red represents type I collagen, and yellow-green represents type III collagen. Figure 18 c. Figure 18 d represents the α-SMA immunohistochemical staining image and statistical results of mouse wound tissue d7; n = 5, *P < 0.05, **P < 0.01 and ***P < 0.001. Detailed Implementation
[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0044] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
[0045] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.
[0046] Example 1:
[0047] Microneedle preparation:
[0048] 1. Preparation of carboxymethyl chitosan (CMCS) solutions with different degrees of crosslinking
[0049] Genipin can react with the amino groups of CMCS to achieve network cross-linking, as shown in the chemical reaction formula. Figure 1 As shown. Genipin powder was added to ultrapure water and evenly dispersed using a magnetic stirrer to prepare a 1% w / v genipin aqueous solution. CMCS powder was added to ultrapure water and completely dissolved using a magnetic stirrer to prepare a 7.5% w / v CMCS aqueous solution. The 7.5% w / v CMCS aqueous solution was then placed in an ultrasonic cleaner to remove air bubbles using ultrasound.
[0050] A 1% w / v genipin aqueous solution and a 7.5% w / v CMCS aqueous solution were mixed at genipin to CMCS molar ratios of 1:100 and 1:400, respectively. The mixtures were allowed to stand at room temperature for 24 hours to allow for complete cross-linking, resulting in CMCS solutions with different degrees of cross-linking.
[0051] 2. Preparation of CMCS microneedles with different degrees of crosslinking
[0052] according to Figure 2 The procedure shown is for preparing microneedles. Microneedle molds containing CMCS solutions with different degrees of crosslinking were placed in a vacuum desiccator and evacuated to a vacuum level to ensure complete solution filling and air bubbles were removed. The molds were then placed in a high-humidity environment for 24 hours for crosslinking, followed by natural air drying at room temperature. After air drying, the molds were demolded to obtain microneedles with different degrees of crosslinking. In subsequent experiments, MN100 and MN400 were used to refer to the 1:100 and 1:400 ratio samples and microneedles, respectively.
[0053] 3. Preparation of elastic modulus samples of dried microneedle materials
[0054] Solutions of genipin and CMCS at molar ratios of 1:100 and 1:400 were prepared, poured into cylindrical molds, and after cross-linking for 24 hours, the samples were demolded and air-dried 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.
[0055] 4. Preparation of tensile strength test specimens for microneedle materials
[0056] Prepare solutions of genipin and CMCS at molar ratios of 1:100 and 1:400, respectively. Pour these solutions onto a glass slide covered with a PDMS mold (10 cm long and 2 cm wide), ensuring a solution thickness of 5 mm. Place the slide in a high-humidity vacuum desiccator and allow it to crosslink for 24 hours, then remove and air-dry. Peel off the dried material strips with a blade. Cut the strips into shapes using a laser cutter. Figure 3 The dumbbell shape shown is 12 mm wide at both ends, 70 mm long in total, and 25 mm long and 5 mm wide in the middle. Five samples were prepared for each group as parallel samples.
[0057] 5. Preparation of single microneedle samples
[0058] CMCS microneedles with different degrees of crosslinking were prepared. The microneedles were cut into individual pieces with a scalpel, ensuring that the morphology of the needle body was not damaged and that the needle body base was flat during the process.
[0059] Representation method:
[0060] 1. Determination of elastic modulus using a mechanical testing machine
[0061] A dry material sample was placed on the stage of a universal testing machine. An unconfined compression test was conducted using a 1 kN force sensor. The sample was mechanically loaded at a loading rate of 0.5 mm / min, and the stress-strain curve was recorded until the sample structure failed. The elastic modulus of the dry material was calculated using data from the strain range of 0-10%.
[0062] The swollen material specimen was placed on the stage of a universal testing machine. An unconfined compression test was conducted using a 50 N force sensor. The specimen was mechanically loaded at a loading rate of 1 mm / min, and the stress-strain curve was recorded until the specimen structure failed. The elastic modulus of the swollen material was calculated using the stress-strain curve before structural failure.
[0063] 2. Tensile strength is determined using a mechanical testing machine.
[0064] The material specimen to be tested is clamped in a universal testing machine, and a uniaxial tensile test is performed using a 100 N force sensor. A mechanical loading rate of 0.5 mm / min is applied to the specimen, and the force-displacement curve is recorded until the specimen fails. The tensile strength of the material is calculated based on the maximum force and the original cross-section.
[0065] 3. The maximum force on a single microneedle is determined using a mechanical testing machine.
[0066] The single microneedle specimen to be tested was placed on the stage of a universal testing machine. An unconfined compression test was conducted using a 50 N force sensor. The specimen was mechanically loaded at a loading rate of 0.03 mm / min, and the force-displacement curve was recorded until the specimen failed. The maximum force experienced by the microneedle was analyzed.
[0067] Experimental results:
[0068] 1. Microneedle morphology analysis observed using a stereomicroscope
[0069] MN100 and MN400 microneedles were prepared as follows Figure 4 As shown, the two exhibit different shades of blue due to their different degrees of crosslinking; MN100 is dark blue, while MN400 is light blue.
[0070] Observe the top view and 45° oblique morphology of the microneedles under a stereomicroscope, such as... Figure 5 and Figure 6 As shown, both sets of cross-linked solutions can be used to prepare microneedles, and the microneedles are arranged in a regular matrix with intact morphology, no bubbles, and no bending.
[0071] By observing the side view of the microneedles under a stereomicroscope, compare the differences in needle height among CMCS microneedles with different degrees of cross-linking. For example... Figure 7 and Figure 8 As shown, the two sets of microneedles have similar needle heights and both have good morphology.
[0072] The height of the needles was statistically analyzed using the built-in software of the stereomicroscope. Three samples were taken from each group of needles, and the height of 10 needles was recorded for each sample. Figure 9 The results show that there is no significant difference in needle height between the two groups of needles.
[0073] 2. Analysis of elastic modulus determined by mechanical testing machine
[0074] After preparing genipin-crosslinked CMCS with different degrees of crosslinking, it is necessary to investigate whether the degree of crosslinking affects the various mechanical properties of the material. First, the elastic modulus of the material is analyzed. After compression tests on MN100 and MN400 samples, the elastic modulus of the dried microneedles is obtained by analyzing the strain range of 0-10%. Figure 10 As 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. A t-test shows that there is a significant difference between the two groups.
[0075] Since the prepared microneedles are hydrogel microneedles, which exhibit swelling properties, it is necessary to analyze the mechanical properties of the material after swelling. After inserting the dried microneedle material into a 2% agarose block immersed in PBS buffer for 24 hours, simulating a skin environment, the microneedle material showed significant swelling. Data obtained from compression tests on the swollen microneedle material were analyzed, and the results are as follows: Figure 11 As shown, the elastic modulus of the swollen microneedle material decreased significantly, with MN100 after swelling being (8.78 ± 6.16) × 10⁻⁶. -2 MPa, while MN400 is (2.73 ± 0.73) × 10 -3 The t-test showed a significant difference between the two values (MPa). The elastic modulus of human soft tissue is (2.70 ± 0.70) × 10⁻⁶ MPa. -2 Therefore, after swelling, the elastic modulus of MN100 is higher than that of human soft tissue, while the elastic modulus of MN400 is lower than that of human soft tissue.
[0076] In summary, the elastic modulus of CMCS microneedles can be altered by changing the degree of crosslinking in the CMCS solution, and the degree of crosslinking is positively correlated with the elastic modulus. Furthermore, regardless of whether the material is dry or swollen, the elastic modulus of CMCS materials with different degrees of crosslinking exhibits significant differences.
[0077] 3. Tensile strength analysis determined by mechanical testing machine
[0078] Solutions of genipin and CMCS at molar ratios of 1:100 and 1:400 were prepared as follows: Figure 12 The dumbbell-shaped specimen shown.
[0079] The tensile strength of a material can be obtained through a tensile test using a mechanical testing machine. Figure 13 As shown, the tensile strength of MN100 is 37.40 ± 1.26 MPa, and the tensile strength of MN400 is 26.57 ± 2.14 MPa. The t-test showed a significant difference between the two groups.
[0080] In summary, the tensile strength of MN100 is significantly higher than that of MN400. Therefore, when microneedles made from materials with different degrees of crosslinking are inserted into wounds, the difference in tensile strength of the microneedle substrate may affect the wound closure effect.
[0081] 4. Analysis of the maximum force on a single microneedle
[0082] When a single microneedle is placed on a stage and pressure is applied to it, the force-displacement curve of the microneedle can be observed as follows: Figure 14As shown, MN400 exhibited significant failure at 5 N, while MN100 did not show significant failure up to 10 N. Both sets of microneedles were able to successfully penetrate the skin under the maximum force. However, due to the inconsistent elastic modulus of the two sets of microneedles, the slopes of the force-displacement curves were significantly different.
[0083] 5. CMCS microneedles with different mechanical properties regulate fibroblasts
[0084] Microneedles were soaked in DMEM high-glucose medium (containing 10% fetal bovine serum) for 12 hours, and single-well microneedles of the appropriate size were cut and placed at the bottom of the 48-well plate. 1×10-1 microneedles were then inoculated into each well. 5 NIH 3T3 cells were seeded on microneedles or in well plates, and 1 ml of DMEM high-glucose medium (containing 10% fetal bovine serum) was added. The cells were cultured at 37°C and 5% CO2 for 24 h. Cytotoxicity was evaluated using acridine orange / ethidium bromide (AO / EB) staining. In cell proliferation assays, 4000 NIH 3T3 cells were seeded on microneedles or in well plates, and 1 ml of DMEM high-glucose medium (containing 10% fetal bovine serum) was added. The cells were cultured at 37°C and 5% CO2. CCK8 reagent was added on days 1, 3, and 5, and the absorbance at 450 nm was measured after 2 h of incubation.
[0085] The toxicity of microbes with different mechanical properties to fibroblasts was observed by AO / EB live / dead cell staining, such as... Figure 15 As shown, both MN100 and MN400 exhibit good biocompatibility (red represents dead cells, green represents live cells). However, cells on MN100 aggregated and were denser than those in 48-well plates, while cell density on MN400 was lower than that in 48-well plates, indicating inconsistent effects on cell proliferation. Further monitoring using CCK8 to observe the effect of the mechanical properties of MN cultured for 5 days on fibroblast proliferation revealed that the proliferation rate of fibroblasts cultured on MN100 was significantly higher than that in 48-well plates, while the proliferation rate of fibroblasts cultured on MN400 was significantly lower than that in 48-well plates. Figure 16 As shown, the mechanical properties of microneedles have different effects on fibroblast proliferation. Microneedles with high elastic modulus upregulate fibroblast proliferation, while microneedles with low elastic modulus downregulate fibroblast proliferation.
[0086] 6. Effects of CMCS micro-applications with different mechanical properties on wound repair in rats
[0087] Eight-week-old male SD rats were used in the experiment. Two fusiform wounds, 3 cm long and 1 cm wide, were made along the back of each SD rat, equidistant from the midline and perpendicular to the line of minimum tension (i.e., along the spine). The two wounds were spaced 2.5 cm apart to ensure no influence between different groups on the wounds. The fusiform wounds on the backs of the SD rats were divided into four groups: Control, Suture, MN100, and MN400. Each group was further divided into a 3-day sampling group and a 7-day sampling group. Each group had four replicates, requiring a total of 32 fusiform wounds, or 16 SD rats.
[0088] This experiment has been reviewed and approved by the Biological and Medical Ethics Committee of Beijing University of Aeronautics and Astronautics, approval number: BM20220026.
[0089] To ensure that the location of the experimental wounds on the upper and lower backs of the SD rats would not affect the experimental results, the experimental groups were designed to have two wounds on both the upper and lower backs of the SD rats in each group. The specific groupings are shown in Table 1 below.
[0090] Table 1 Animal Experiment Grouping
[0091]
[0092] Representation method:
[0093] (1) Photographing the appearance of animal wounds
[0094] The wound morphology was photographed after the wound was created in 0D and during the 3D and 7D sampling.
[0095] (2) ImageJ analysis of wound area change rate
[0096] The wound morphology of SD rats photographed in section 4.3.3.1 was processed using ImageJ software, and the wound area was selected as the Region of Interest (ROI). When selecting the ROI, ensure that the edge of the ROI is close to the edge of the wound or the edge of the scar tissue after wound healing. The healed scar area was also included in the wound area calculation. The wound area change rate for each group was obtained by dividing the wound area at the time of sampling by the wound area at day 0.
[0097] (3) Optical microscope images of H&E stained, Masson stained and α-SMA stained sections.
[0098] H&E-stained and Masson-stained sections were photographed under bright-field and bottom-lit microscopes. Exposure time and contrast were adjusted during photography to ensure consistent color tone in each image.
[0099] (4) Photographs of Sirius red stained sections using a polarized light microscope
[0100] Rotate the polarizer to make it 90 degrees from the analyzer, and adjust the aperture to make the overall image darker, showing only the yellow-green and orange-red of collagen.
[0101] (5) Statistical analysis
[0102] Each experiment had four parallel samples. Data were analyzed using SPSS software, and a two-sample t-test was used for analysis. A p-value < 0.05 was considered statistically significant. ns indicates no difference, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.
[0103] Experimental procedure:
[0104] (1) Animal surgery
[0105] After anesthetizing and removing the hair from rats, the skin surface on the rats' backs was wiped with alcohol and iodine. Two 3 cm long and 1 cm wide fusiform full-thickness wounds were created on the rat skin, with a 2.5 cm gap between the wounds to avoid mutual interference. The following treatments were applied to the four animal groups:
[0106] 1) Control: Apply a layer of 3M-Tegaderm membrane directly to the wound.
[0107] 2) Suture: Close the wound with absorbable sutures. Apply a 3M-Tegaderm membrane after suturing.
[0108] 3) MN100: After pulling the skin on both sides of the wound closed, insert microneedles. Insert 3 MN100 needles into each wound to ensure that the microneedles cover the entire wound. Then apply a layer of 3M-Tegaderm membrane.
[0109] 4) MN400: After pulling the skin on both sides of the wound closed, insert microneedles. Insert 3 MN400 needles into each wound to ensure that the microneedles cover the entire wound. Then apply a layer of 3M-Tegaderm membrane.
[0110] Rats were housed individually after surgery. Their physical condition and feeding status were observed daily to prevent infection, and bedding was changed and food and water replenished as needed.
[0111] (2) Animal-based materials
[0112] Samples were taken from rats at the sampling point. A 3 × 1 cm rectangular piece of skin tissue was cut from the initial wound area, paraffin sections were prepared, and H&E staining, Masson staining, Sirius red staining, and α-SMA staining were performed on the sections.
[0113] Experimental results:
[0114] like Figure 17As shown, Figure 17 In group b, compared to the untreated open wound control group, the Surture group, MN100 group, and MN400 group (as controls) all closed by day 3. However, the Surture group showed significant scab formation and redness, and the closure effect of MN400 was not as good as that of MN100. Due to the anti-inflammatory ability of CMCS in MN and the ability of hydrogel microneedles to absorb exudate, the surface inflammation of the wound treated with MN was significantly improved. By day 7, the Control group still had not closed and showed significant inflammation, while the Surture group had closed, but the sutures had not been absorbed. The wound repair effect using MN was the best.
[0115] The wound dimensions of each group were quantitatively analyzed using ImageJ, such as... Figure 17 c, In 3d, the Summary group is 1.31 × 10⁻⁶. -1 The MN100 group is 3.50 × 10 -2 MN400 is 5.42 × 10 -2 This indicates that the use of microneedles can indeed accelerate wound closure, and faster than commonly used surgical sutures. Furthermore, microneedles with high elastic modulus and high tensile strength close faster than those with low elastic modulus and low tensile strength. At 7 days, the Surture group showed a closure rate of 5.72 × 10⁻⁶. -2 The MN100 group has a concentration of 1.71 × 10⁻⁶. -2 MN400 is 1.64 × 10 -2 The microneedling group still showed better wound closure than the Suture group, but the difference between the two groups was no longer significant. This indicates that microneedling does promote wound closure, and the higher elastic modulus and tensile strength in the early stages of wound healing lead to faster wound closure. However, the mechanical properties of the microneedles have no significant impact on the closure speed in the later stages.
[0116] Observe the H&E-stained sections of the repaired wound skin, such as... Figure 17 d and Figure 17 As shown in Figure 3, at 3 days, the wound tissue in the Control group consisted of granulation tissue with inflammation and a large number of fibroblasts. In the Suture group, compared to the MN group, there were obvious gaps and blood crusts at the skin sutures on both sides, indicating that the wound was not completely closed. Furthermore, the Suture group had fewer fibroblasts at the wound site, while the MN group showed clearly fibroblasts filling the gaps between the two wounds, connecting the skin on both sides, and the MN100 group had more fibroblasts than the MN400 group. This indicates that microneedles accelerated fibroblast migration and inhibited the inflammatory response in the early stages of wound healing. The number of fibroblasts was consistent with the results of in vitro cell culture, suggesting that the high-elasticity modulus microneedles' promoting effect on fibroblast proliferation also led to a faster closure rate.
[0117] In the tissues at 7 days, the newly formed tissue in the Control group was higher than the surrounding normal skin, and contained a large number of non-apoptotic fibroblasts and inflammatory cells. In the Suture group, although the wound closed, the newly formed tissue was lower than the surrounding normal skin height. In the MN100 group, the wound closed, but the newly formed tissue was also lower than the surrounding normal skin height, and contained a large number of fibroblasts. Although the wound in the MN400 group closed more slowly than in the MN100 group, the newly formed tissue was at the same height as the normal skin, and the number of fibroblasts in the tissue was closest to that of normal tissue, indicating the best repair effect.
[0118] like Figure 18 As shown, the fibrosis status of MN-repaired wounds was further evaluated by characterizing the collagen content and types in the newly formed wound tissue after 7 days, as well as the myofibroblasts associated with scar formation. Figure 18 Figure a shows the collagen distribution in the newly formed tissue after wound repair. It can be seen that the collagen in the control group is lower and more disorganized. In contrast, the collagen in the MNs patch group and the suture group is more aligned along the wound surface. The type I collagen / type III collagen ratio can be used to further evaluate the fibrosis status of the repaired tissue. Figure 18 b. Compared to the MN400 and Suture groups, the Control and MN100 groups showed a higher proportion of orange-red type I collagen in their tissues, indicating that fibrosis occurred in both untreated wounds and wounds treated with MN100, while no fibrosis was observed in wounds treated with sutures or MN400. Furthermore, an increased proportion of type I collagen was observed in the undamaged areas of the skin on both sides of the MN100 group. Figure 18 The immunohistochemical staining results of group c show that myofibroblasts are present in the newly formed tissue of all three groups. However, α-SMA in the MN100 group is also distributed in the subepidermal layer of the skin, which may be related to the mechanism of action of microneedles. Figure 18 The quantitative statistical results of d show that the content of myofibroblasts in wound tissue using MN100 is significantly higher than that using MN400 and sutures, indicating that microneedles with a higher elastic modulus than the skin will increase the differentiation of myofibroblasts during wound repair in the later stage, thus leading to more severe fibrosis.
[0119] In summary, while MN100 resulted in faster wound closure in the early stages of wound healing, its excessive proliferation of fibroblasts, increased differentiation of myofibroblasts, and elevated expression of type I collagen in the later stages led to fibrosis of newly formed wound tissue and even surrounding uninjured skin. In contrast, MN400 achieved even better repair results than wounds treated with sutures, exhibiting collagen content and distribution closer to normal tissue, thus achieving scarless repair.
[0120] In summary, the specific application methods of polymer microneedles in skin wound healing should be as follows:
[0121] In the early stages of skin wound healing, high-elasticity and high-tensile-strength polymer microneedles, i.e., highly cross-linked polymer microneedles, are selected to improve the mechanical environment of the wound and promote the wound closure speed. In the later stages of skin wound healing, low-elasticity and low-tensile-strength polymer microneedles, i.e., low-cross-linked polymer microneedles, are selected to reduce the proliferation of fibroblasts, the differentiation of myofibroblasts, and the overexpression of collagen during the wound repair process, thus achieving rapid repair and scarless repair.
[0122] In summary, this invention discloses a polymer microneedle with tunable 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, methacrylamide gelatin, chitosan, carboxymethyl chitosan, polyethyleneimine, cellulose and its derivatives, polyvinyl alcohol, hyaluronic acid, sodium alginate, polyacrylic acid, and polylactic-co-glycolic acid copolymer; and crosslinking agents, including one of genipin, glutaraldehyde, photoinitiator, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, NaOH, and CaCl2. By changing the proportion of crosslinking agent, the degree of crosslinking of the polymer microneedle is controlled, thereby changing the mechanical properties of the polymer microneedle. Furthermore, the application of polymer microneedles with different mechanical properties in skin wound healing is disclosed, showing good application prospects.
[0123] Therefore, those skilled in the art will recognize that although embodiments of the present invention have been shown and described in detail herein, many other variations or modifications conforming to the principles of the present invention can be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the invention. Therefore, the scope of the present invention should be understood and recognized as covering all such other variations or modifications.
Claims
1. A skin wound healing product, characterized in that, The polymer microneedle includes high and low cross-linking degrees, and is composed of the following components: a polymer material and a cross-linking agent; the polymer material is carboxymethyl chitosan; the cross-linking agent is genipin; a preparation method of the polymer microneedle is as follows: the polymer material solution and the cross-linking agent solution are mixed at a molar ratio, and then are added into a microneedle mold, and are subjected to low-speed centrifugation or vacuum treatment of a vacuum dryer, so as to become a hydrogel after cross-linking, and the prepared polymer microneedle is obtained after drying and taking out from the mold; in the polymer microneedle with high cross-linking degree, the molar ratio of genipin to carboxymethyl chitosan is 1:100; and in the polymer microneedle with low cross-linking degree, the molar ratio of genipin to carboxymethyl chitosan is 1:
400.
2. A skin wound healing product as claimed in claim 1 characterised in that, The temperature of the low-speed centrifugation or the vacuum treatment of the vacuum dryer is 4℃.
3. A skin wound healing product as claimed in claim 1, characterised in that The cross-linking condition is 4℃-26℃ and 40-80 %RH humidity.
4. A skin wound healing product as claimed in claim 1, characterised in that The cross-linking time is 24-48 hours.
5. A skin wound healing product as claimed in claim 1, characterised in that The drying condition is 4℃-26℃, and the drying time is 24 hours.