Photo-thermal soluble microneedle patch for promoting skin wound healing and preparation method

Abstract

The invention relates to a photo-thermal soluble microneedle patch for promoting skin wound healing and a preparation method. The soluble photo-thermal microneedle patch is composed of a needle tip layer hyaluronic acid and a backing layer PVP K60 as matrixes, the composition comprises chlorogenic acid, prussian blue and asiaticoside, the chlorogenic acid, the prussian blue and the hyaluronic acid are blended to prepare the needle tip layer, and the asiaticoside and the PVP K60 are blended to prepare the backing layer. The photo-thermal microneedle patch disclosed by the invention has the effect of remarkably promoting healing of skin wounds, and can be used for inhibiting bacteria, destroying bacterial biofilms and promoting synthesis of collagen. The soluble photo-thermal microneedle patch under the concentration of hyaluronic acid and PVP K60 has enough mechanical strength to penetrate into the skin, shows high biocompatibility in wound treatment, also has a good healing promoting effect, not only can promote cell proliferation, but also can promote synthesis of collagen and generation of blood vessels, and has a good application prospect. Certain clinical popularization and application values are realized.

Description

Technical Field

[0001] This invention belongs to the field of preparation technology for promoting skin repair, and particularly relates to a photothermal soluble microneedle patch for promoting skin wound healing and its preparation method. Background Technology

[0002] Chronic wounds are wounds that fail to heal properly and persist for more than 4-6 weeks without achieving anatomical and functional integrity through a systematic repair process. Their healing is difficult due to complex pathological microenvironments, such as persistent inflammation, oxidative stress, formation of drug-resistant bacterial biofilms, and impaired angiogenesis.

[0003] With the growing crisis of antibiotic resistance, it is estimated that nearly 10 million people will die from chronic wound diseases by 2050. Therefore, it is essential to actively explore new antibacterial formulations to replace traditional antibiotics and promote chronic wound healing.

[0004] Hyaluronic acid exhibits excellent biocompatibility, making it suitable for various medical and cosmetic applications, particularly for long-term topical application and wound healing. PVP K60 is also a biocompatible material; although synthetic, it has wide applications in many drug delivery systems, especially for short-term treatments. PVP K60 is non-irritating to the skin and suitable for use in wound dressings. Both materials demonstrate good biocompatibility in the treatment of chronic wounds, drug delivery, and other biomedical applications.

[0005] Microneedle systems have become a research hotspot in the treatment of chronic wounds in recent years, offering a precise, efficient, low-side-effect, and long-lasting drug delivery method for treating chronic bacterial infections. Microneedle systems can effectively deliver various types of drugs to treat chronic wounds, particularly suitable for antimicrobial peptides, growth factors, nanomedicines, and anti-inflammatory drugs requiring efficient, precise, and localized delivery. The high efficiency and strong targeting characteristics of microneedles make them a promising treatment option for chronic bacterial infections.

[0006] Compared to traditional dosage forms such as hydrogels, the advantages of microneedles are mainly reflected in targeted drug delivery, improved bioavailability, reduced systemic toxicity, and enhanced patient compliance. Therefore, microneedle technology has greater potential and application prospects in the treatment of chronic trauma. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a photothermal soluble microneedle patch for promoting skin wound healing and its preparation method.

[0008] One object of the present invention is to provide a photothermal soluble microneedle patch for disrupting biomembrane barriers and promoting wound healing.

[0009] One object of the present invention is to provide a photothermal soluble microneedle patch loaded with chlorogenic acid, which can exert antibacterial and anti-inflammatory effects to kill Staphylococcus aureus in infected wounds and promote wound healing.

[0010] One object of the present invention is to provide a photothermal soluble microneedle patch loaded with Prussian blue (PB) nanomaterials, as a MOF material, having cyano-linked Fe... 2+ with Fe 3+ Its structure, coupled with good biocompatibility and safety, and its excellent photothermal conversion capability, make it play an important role in delivery and photothermal therapy. It is used to exert photothermal bactericidal effects, which is beneficial for promoting wound healing.

[0011] One objective of this invention is to provide a photothermal soluble microneedle patch, the tip layer of which is loaded with chlorogenic acid and Prussian blue. The two can work together to exert a photothermal antibacterial effect, which can not only kill bacteria that infect wounds, but also destroy the bacterial biofilm formed by them, thereby promoting wound healing.

[0012] One objective of this invention is to provide a photothermal soluble microneedle patch loaded with asiaticoside, which can exert its effects through mechanisms such as anti-inflammatory, antioxidant, collagen synthesis promotion, and epithelialization, thereby promoting wound healing.

[0013] The technical solution of this invention is as follows: A photothermal soluble microneedle patch is provided, which contains a sterile water mixture containing chlorogenic acid (0-4 mg / mL, preferably 2 mg / mL for better effect) and Prussian blue (20-100 μg / mL, preferably 60 μg / mL considering cytotoxicity and previous work of our research group). This mixture is then added to hyaluronic acid powder with a final concentration of 15 mg / mL. The patch is incubated overnight in a shaker at 37-40°C, undergoing hygroscopic, swelling, and dispersion stages to obtain a needle tip gel. The gel is then ultrasonically dissolved in a mixture of 0.5% DMSO and sterile water to a concentration of 300 μg / mL (300 μg / mL is preferred considering both solubility and effect between 100 and 300 μg / mL). A solution of asiaticoside (μg / mL) was prepared and placed in a shaker at 37-40°C overnight with the needle tip layer to obtain a backing matrix. A composition that promotes wound healing was obtained. The prepared needle tip layer and backing matrix were filled into a PDMS mold, and the photothermal soluble microneedle patch was obtained after vacuum negative pressure, centrifugation and drying.

[0014] The photothermal soluble microneedle patch has a solid conical structure with a needle length of 710 μm, a bottom diameter of 280 μm, a needle tip distance of 600 μm between two adjacent conical needles, an array of 15*15 needles, an area containing needles of approximately 8.7 * 8.7 mm, and a patch size of 12*12 mm.

[0015] The aforementioned photothermal soluble microneedle patch, with its hyaluronic acid matrix, exhibits excellent biocompatibility, making it suitable for various medical and cosmetic applications, particularly for long-term local application and wound healing. PVP K60 is also a biocompatible material; although synthetic, it has wide applications in many drug delivery systems, especially for short-term treatments. PVP K60 is non-irritating to the skin and suitable for use in wound dressings. Both materials demonstrate good biocompatibility in the treatment of chronic wounds, drug delivery, and other biomedical applications.

[0016] The present invention also provides a method for using the aforementioned composition, comprising the following steps: 1) Preparation of the tip layer: First, centrifuge the tip layer matrix containing chlorogenic acid and Prussian blue to remove air bubbles (0~4℃, 3000~5000 rpm, 5~10min). After centrifugation, use a syringe to draw about 100 μL and add it to the PDMS mold for even spreading. Then, centrifuge under vacuum (0.08 MPa, 90~120s) at the following conditions: horizontal centrifugation speed 4000 rpm, time 15~20min, temperature 0~4℃, 2~3 times. After centrifugation, remove the excess matrix to obtain the tip layer. 2) Preparation of backing layer: First, centrifuge the backing layer matrix containing asiaticoside to remove air bubbles (0~4℃, 3000~5000rpm, 10min). After centrifugation, use a syringe to draw about 200 μL and add it to the backing groove of the PDMS mold to obtain the backing layer. 3) Place the obtained microneedle patch in an oven to dry at a temperature of 37~38℃ for 24~36h. After demolding, the photothermal soluble microneedle patch is obtained.

[0017] This invention analyzes the efficacy of each component in the above-mentioned photothermal soluble microneedle patch that promotes wound healing, and it has the following advantages: This invention has been demonstrated through literature review and extensive experiments: 1) Chlorogenic acid, as the main active ingredient of honeysuckle, has broad-spectrum antibacterial, antiviral, antioxidant and anti-malignant tumor effects. Therefore, chlorogenic acid has certain development prospects as an antibacterial ingredient for healing infected wounds. However, using it alone for antibacterial purposes may have the disadvantages of large dosage and certain toxicity to cells.

[0018] 2) As a MOF material, PB has been shown in existing literature to possess certain anti-inflammatory effects. It contains cyano-linked Fe... 2 + with Fe 3+ Its structure, coupled with good biocompatibility and safety, and its excellent photothermal conversion capability, make it play an important role in delivery and photothermal antibacterial therapy. Therefore, it can be used to exert photothermal bactericidal effects. Combined with chlorogenic acid under near-infrared light, it can not only significantly enhance the bactericidal effect and scavenge ROS, but also reduce the amount of chlorogenic acid required and improve biocompatibility.

[0019] 3) Asiaticoside plays a multifaceted role in promoting the healing of infected wounds. It not only accelerates healing through mechanisms such as anti-inflammation, anti-oxidation, and promotion of collagen synthesis and epithelialization, but also protects the wound from further infection. Therefore, Asiaticoside has significant advantages in the treatment of chronic infected wounds, especially in repair and healing.

[0020] 4) The hyaluronic acid (15 mg / mL, 40-100 KDa) and PVP K60 (400 mg / mL) used in the true system have sufficient mechanical strength to penetrate the skin for the treatment of wound infection, and both have good biocompatibility.

[0021] Experiments have demonstrated that the photothermal microneedle patch of this invention significantly promotes skin wound healing, exerting antibacterial effects, disrupting bacterial biofilms, and promoting collagen synthesis. It exhibits high biocompatibility in wound treatment while also demonstrating excellent healing-promoting effects, thus possessing significant clinical application value. Attached Figure Description

[0022] Figure 1 Taking bright-field photos of photothermal soluble microneedle patches with a mobile phone; Figure 2 Images of photothermal soluble microneedle patches under field emission scanning electron microscopy (SEM); Figure 3 Figure 1 shows the experimental results of mechanical strength testing of photothermal soluble microneedle patches; Figure 4 Figure showing the experimental results of photothermal stability testing of photothermal soluble microneedle patches; Figure 5 Figure 1 shows the results of an in vitro antibacterial plate coating experiment for each component of the photothermal soluble microneedle patch. Figure 6 Figure 1 shows the SEM results of the in vitro antibacterial activity of each component of the photothermal soluble microneedle patch. Figure 7 Figure 1 shows the results of the anti-Staphylococcus aureus live / dead staining experiment of each component of the photothermal soluble microneedle patch; Figure 8 Figure 1 shows the results of the crystal violet staining experiment on the anti-biofilm properties of the various components of the photothermal soluble microneedle patch. Figure 9 Figure 1 shows the results of the anti-biofilm liveness and deadness staining experiment of each component of the photothermal soluble microneedle patch; Figure 10 Figure 1 shows the cytotoxicity test results of each component of the photothermal soluble microneedle patch. Figure 10 In the diagram, 'a' represents the toxicity of PB to L929 cells. Figure 10 b in the diagram is a schematic representation of the toxicity of CGA to L929 cells; Figure 10 In the diagram, 'c' represents the toxicity of AS to L929 cells. Figure 10 The diagram below, d, illustrates the toxicity of PB to HaCat cells. Figure 10 The 'e' in the diagram represents the toxicity of CGA to HaCat cells; Figure 10 f in the figure is a schematic diagram of the toxicity of AS to HaCat cells; Figure 11 The figure shows the experimental results of the effect of asiaticoside on the migration of HaCat cells in vitro. Figure 12 The graph shows the changes in body weight of mice in each group from the time of modeling to the end of treatment. Figure 13 A graph showing the temperature changes during photothermal therapy in mice in the photothermal therapy group; Figure 14 This is a graph showing the changes in wounds of mice in each group during treatment. Figure 15 A quantitative graph showing the changes in wounds in each group of experimental mice during the treatment period; Figure 16 Figure 1 shows the results of a plate application experiment on wound infection status of mice in each group before and after treatment. Figure 17 The results of routine blood tests for each group of mice are shown in the figure. Figure 18 The images show representative HE and Masson staining results at the wound sites of mice in each group. Figure 19 HE staining results of the heart, liver, spleen, lungs and kidneys of mice in each group; Figure 20 The images show the fluorescence immunohistochemical staining of Ki67, CD31, collagen-I, and collagen-III at the wound sites of mice in each group. Figure 21 Immunohistochemical images of IL-10 at the wound sites of mice in each group. Detailed Implementation

[0023] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this disclosure.

[0024] The following detailed description, in conjunction with specific embodiments, provides a further detailed explanation of the composition and preparation method of a photothermal soluble microneedle patch for promoting skin wound healing according to the present invention.

[0025] Chemical reagents and materials All raw materials used in this invention are commercially available common raw materials. Asiaticoside, chlorogenic acid, and hyaluronic acid (molecular weight 40-100 kDa) were purchased from Shanghai Maclean Biotechnology Co., Ltd.; PVP K60 was purchased from Beijing Coolplay Technology Co., Ltd.; PDMS molds (array: 15*15, needle length 710 μm, bottom diameter 280 μm) were purchased from Taizhou Microchip Pharmaceutical Technology Co., Ltd.; L929 and HaCaT cells were purchased from Wuhan Zishan Biotechnology Co., Ltd.; and high-glucose DMEM culture medium, penicillin-streptomycin mixed antibiotic solution, and fetal bovine serum (FBS) were purchased from Gibco, UK.

[0026] Example 1: A method for preparing a photothermal soluble microneedle patch to promote skin wound healing Preparation of the needle tip matrix: Prepare a sterile water mixture containing chlorogenic acid (2 mg / mL) and Prussian blue (60 μg / mL), then add it to hyaluronic acid powder with a final concentration of 15 mg / mL (40-100 KDa), and incubate overnight in a shaker at 37°C. After the hygroscopic, swelling and dispersion stages, the needle tip gel is obtained.

[0027] Preparation of the backing matrix: Dissolve the 300 μg / mL asiaticoside solution in a mixture of 0.5% DMSO and sterile water by sonication, and place it in a shaker at 37°C overnight with the needle tip layer to obtain the backing matrix; Preparation of the tip layer: First, centrifuge the tip layer matrix containing chlorogenic acid and Prussian blue to remove air bubbles (4℃, 3000~5000 rpm, 10min). After centrifugation, use a syringe to draw about 100 μL and add it to the PDMS mold for even spreading. Then, centrifuge under vacuum (0.08 MPa, 90~120s) at the following conditions: horizontal centrifugation speed 4000 rpm, time 15~20 min, temperature 4℃, 2~3 times. After centrifugation, remove excess matrix to obtain the tip layer. Preparation of the backing layer: First, centrifuge the backing layer matrix containing asiaticoside to remove air bubbles (4℃, 3000~5000 rpm, 10min). After centrifugation, use a syringe to draw about 200 μL and add it to the backing groove of the PDMS mold to obtain the backing layer. The obtained microneedle patch was placed in an oven to dry at a temperature of 37-38°C for 24-36 hours. After demolding, the photothermal soluble microneedle patch was obtained.

[0028] Example 2 Characterization of photothermal soluble microneedle patches Its morphological characterization was observed using a mobile phone and a field emission scanning electron microscope (SEM, Hitachi, Japan). Characterization results are as follows: Figure 1 and Figure 2 As shown, the results indicate that the microneedles have a complete needle structure and uniform needle size.

[0029] Example 3 Mechanical strength testing of photothermal soluble microneedle patches The mechanical strength of the microneedle patch was tested using a universal mechanical analyzer. According to relevant literature (Ning, Xiaoyu, et al. "A double-layered microneedle platform fabricated through frozen spray-coating." Advanced healthcare materials 9.10 (2020): 2000147.), the force required for a single microneedle to penetrate human skin is 0.058 N. The results are as follows... Figure 3 As shown, the needle tip layer is pure hyaluronic acid. The needle tip layer contains Prussian blue and the needle tip layer contains both Prussian blue and chlorogenic acid. The force corresponding to the hyaluronic acid microneedles at 400 μm is greater than the force reported in the literature, and there is no breakage point, indicating that they have sufficient mechanical strength to penetrate human skin.

[0030] Example 4: Photothermal stability testing of photothermally soluble microneedle patches The final microneedle drug delivery group (MN group) was placed on a near-infrared excitation stage and subjected to 1.5 W / cm² excitation at a wavelength of 808 nm. 2 The light source was irradiated with high power for 3 minutes, during which the temperature rise was recorded every 20 seconds using thermal imaging. After 3 minutes, the light source was turned off, and the temperature drop was recorded every 20 seconds for the same period. After 5 cycles, the photothermal soluble microneedle patch exhibited good photothermal stability. The experimental results are as follows: Figure 4 As shown.

[0031] Example 5: In vitro antibacterial plate coating experiment verification of each component Group setup: Seven groups were set up, namely: control group (needle tip layer and backing layer contained pure hyaluronic acid and PVP K60, named: CK); needle tip layer containing Prussian blue (PB) (named: PM); needle tip layer containing PB and chlorogenic acid (CGA) (named: PCM); needle tip layer containing PB and CGA, and backing layer containing asiaticoside (AS) (named: MN); and the other three groups were PM + NIR, PCM + NIR, and MN + NIR (NIR: near-infrared light wavelength of 808 nm, power of 1.5 W / cm). 2 The irradiation time is 10 minutes.

[0032] Experimental procedure: *Escherichia coli* and *Staphylococcus aureus* were cultured in LB and NA media respectively at 37°C in a shaker. After centrifugation at 5000 rpm for 10 minutes, both bacteria were resuspended in sterile PBS. The OD values ​​of the two bacteria were then analyzed using a UV spectrophotometer. 600 Adjust to approximately 0.6 and dilute 10,000 times for later use. Take 14 sterile 1.5 mL centrifuge tubes and divide them into two groups, corresponding to the seven groups set in Example 4. Perform experiments on each group, with a final volume of 500 μL in the centrifuge tubes. Add 100 μL of diluted Staphylococcus aureus and Escherichia coli to each of the two groups' centrifuge tubes, followed by 400 μL of sterile PBS. For groups requiring NIR, use near-infrared light at a wavelength of 808 nm and a power of 1.5 W / cm². 2 The irradiation time was 10 minutes. Microneedle patches were added to each group, and all groups were incubated together at 37°C for 24 hours. After 24 hours, 50 μL of each group was taken and plated on LB agar plates (each sample was repeated three times). After plating, all groups were inverted and incubated at 37°C for 24 hours, and colony growth was recorded by photography. The experimental results are as follows: Figure 5 SEM results showed that the photothermal soluble microneedle patch administration groups (MN+NIR group and PCM+NIR group) had a better inhibitory effect on the growth of Staphylococcus aureus and Escherichia coli compared with other groups.

[0033] Example 6: In vitro antibacterial field emission scanning electron microscopy experiment to verify the antibacterial properties of each component. The experimental groups were the same as in Example 5, with the addition of a positive control group (70% ethanol group, using 400 μL of 70% ethanol instead of sterile PBS). The experimental procedures up to incubation were the same as in Example 4. After incubation, the bacterial samples from each group were washed with PBS by centrifugation (6000 rpm, 10 min, 2-3 times), and then fixed overnight at 4°C with 2.5% glutaraldehyde. The next day, the samples were washed 2-3 times with PBS, dehydrated for 15 min with a gradually increasing ethanol concentration gradient (30%, 50%, 80%, 90%, 95%), and finally treated with a gradient of tert-butanol (30%, 50%, 100%) for 15 min before being dried in a vacuum dryer. After drying, the samples were adhered to a sample stage containing conductive adhesive for gold sputtering and observation. The experimental results are as follows: Figure 6 As shown, the photothermal soluble microneedle patch drug delivery groups (MN+NIR group and PCM+NIR group) caused greater damage to Staphylococcus aureus and Escherichia coli compared to other groups.

[0034] Example 7: In vitro anti-Staphylococcus aureus live / dead staining (NucGreen / EthD-III) experiment verification of each component The experimental groups were the same as in Example 6, and the experimental procedures up to incubation were the same as in Example 4. The specific staining steps are as follows: (1) Centrifuge each bacterial sample (5000 rpm, 5 min) and wash with sterile PBS 2-3 times.

[0035] (2) After centrifugation, remove the supernatant and add 1 mL of 0.9% NaCl solution to resuspend the bacteria.

[0036] (4) Centrifuge again and resuspend the bacteria in 1 mL of 0.9% NaCl solution.

[0037] (5) Preparation of staining working solution: Take 1 μL of NucGreen and 2 μL of EthD-III, mix them thoroughly, and then add 8 μL of 0.9% NaCl solution and mix well.

[0038] (6) Add 10 μL of staining working solution to each group. Mix thoroughly and incubate at room temperature in the dark for 15 min.

[0039] (7) After staining, 10 μL of bacterial suspension was dropped onto a clean glass slide and photographed using laser confocal microscopy under dark conditions. The experimental results are as follows: Figure 7 As shown in the results of the live-dead staining experiment, the photothermal soluble microneedle patch administration groups (MN+NIR group and PCM+NIR group) showed stronger bactericidal ability against Staphylococcus aureus compared with other groups.

[0040] Example 8: In vitro verification of the anti-crystal violet effect of each component on biofilms The experimental groups were the same as in Example 6. Staphylococcus aureus was cultured in NA medium on a shaker at 37°C, and then OD was adjusted... 600 100 μL of Staphylococcus aureus at 0.6 g / L and 400 μL of TSB medium were used to form a biofilm in a 24-well plate for 48 h. A suitable amount of 1% glucose solution was added to promote biofilm formation. The culture conditions were a 37°C incubator for 48 h, during which TSB medium and 1% glucose solution could be added as needed. After biofilm formation, subsequent anti-biofilm experiments were performed, following the same drug administration procedure as in Example 4. After drug administration, crystal violet staining was performed, with the staining procedure as follows: (1) After incubation, remove the 24-well plate, discard the culture medium and wash twice with PBS.

[0041] (2) Add 200 μL of 4% paraformaldehyde to each well and fix for 20 min.

[0042] (4) Remove paraformaldehyde and carefully add 300 μL PBS to each well to wash away any residual fixative.

[0043] (5) Add 200 μL of 0.1% crystal violet solution to each well and let it stand at room temperature for 30 min to stain.

[0044] (6) Discard the crystal violet, wash three times with PBS, and air dry.

[0045] (7) Take photos to record the process after drying.

[0046] Experimental results are as follows Figure 8 As shown, the photothermal soluble microneedle patch drug delivery groups (MN+NIR group and PCM+NIR group) showed stronger destructive ability against bacterial biofilms compared to other groups.

[0047] Example 9: Verification of the in vitro anti-Staphylococcus aureus biofilm live / dead staining experiment of each component The experimental groups and procedures were the same as in Example 8, except that the culture vessel for film formation was replaced with a laser confocal microplate. After drug administration, the sample was carefully washed twice with sterile PBS, and then 1 mL of sterile PBS was added for staining as in Example 6 (steps 5-6). After staining, photographs were taken and recorded under laser confocal microscopy. The experimental results are as follows: Figure 9 As shown, the photothermal soluble microneedle patch drug delivery groups (MN+NIR group and PCM+NIR group) showed stronger destructive and bactericidal abilities against bacterial biofilms compared to other groups.

[0048] Example 10: In vitro MTT cytotoxicity assay of each component The cytotoxicity of chlorogenic acid, Prussian blue, and asiaticoside was verified using L929 and HaCat cells, respectively. The experimental steps are as follows: Cells frozen in liquid nitrogen were rapidly thawed in a 37°C water bath. After thawing, they were transferred to sterile 15 mL centrifuge tubes and centrifuged (1500 rpm, 5 min). After centrifugation, the cryopreservation solution was discarded, and the cells were gently resuspended in 1 mL of complete culture medium (10% fetal bovine serum, 1% penicillin-dextrin). The cells were then transferred to T25 cell culture flasks containing culture medium and cultured in a cell culture incubator (37°C, 5% CO2). When the cells reached 80-90% confluence, they were passaged. After 2-3 passages and when the cells were stable, cell counting was performed, and the cells were seeded into 96-well plates. 100 μL of low-serum (1% FBS, 1% penicillin-dextrin) culture medium containing approximately 6000 cells was added to each well and incubated overnight. On the second day, the culture medium was discarded, and 100 μL of complete culture medium containing different drug concentrations was added to each of the three parallel wells in each group. After culturing for 24 h, 10 μL of MTT (5 mg / mL) reagent was added to each well, and the cells were incubated in a cell culture incubator at 37℃ for 3.5–4 h. All culture medium and MTT were then discarded, and the cells were carefully washed 1–2 times with PBS. 100 μL of DMSO solution was added to each well, and the readings were taken at 570 nm using a microplate reader. The experimental results are as follows: Figure 10 As shown, Figure 10 In the diagram, 'a' represents the toxicity of PB to L929 cells. Figure 10 In the diagram, b represents the toxicity of CGA to L929 cells. Figure 10 In the diagram, 'c' represents the toxicity of AS to L929 cells. Figure 10 The diagram below, labeled 'd', illustrates the toxicity of PB to HaCat cells. Figure 10 The 'e' in the diagram represents the toxicity of CGA to HaCat cells. Figure 10 f in the figure is a schematic diagram of the toxicity of AS to HaCat cells. The experimental results show that each component in the photothermal soluble microneedle patch has good biocompatibility with L929 and HaCat cells, and asiaticoside and chlorogenic acid also have a certain effect on promoting cell proliferation.

[0049] Example 11 Verification of asiaticoside HaCat cell migration assay HaCat cells were cultured as in Example 10. After the cells stabilized, cell counting was performed, and the cells were seeded in 24-well plates. 500 μL of approximately 8*10 cells were added to each well. 4 Incubate the cells in complete culture medium overnight. The next day, use a white pipette tip to scratch the cells, and then carefully wash with PBS 1-2 times to remove any loose cells before administering the drug.

[0050] The dosing groups are set as follows: Blank group: 500 μL of culture medium containing 1% FBS and 1% antibiotics; Positive control group: 500 μL of culture medium containing 3% FBS and 1% antibiotics; Test sample groups: 500 μL of asiaticoside solutions of different concentrations (100, 300 μg / mL) containing 1% FBS complete culture medium.

[0051] Cell migration was recorded by photographing at 0 h, 24 h, and 48 h after drug administration, and the migration rate was calculated. The experimental results are as follows: Figure 11 As shown, a 300 μg / mL solution of asiaticoside significantly promoted the migration of HaCat.

[0052] Example 12: Pharmacodynamic Experiment of Photothermal Soluble Microneedle Patch Sixty-four healthy male Balb / c mice, aged 6-8 weeks and weighing 18-22g, were selected as subjects for this experiment. After being housed under P2-level experimental conditions for one week, the 64 mice were randomly divided into 8 groups as follows: CK group: The needle tip layer and backing layer are made of pure hyaluronic acid and PVP K60; 3M group: Purchased from the market as a positive control group; PM group: Prussian blue (PB) group in the apical layer; PCM group: Needle tip layer containing PB and chlorogenic acid (CGA); MN group: The needle tip layer contains PB and CGA, and the backing layer contains asiaticoside (AS) group; PM + NIR group: Prussian blue (PB) group in the tip layer; PCM + NIR group: Needle tip layer containing PB and chlorogenic acid (CGA); MN + NIR group: The needle tip layer contains PB and CGA, and the backing layer contains asiaticoside (AS) group; (NIR: Near-infrared light wavelength of 808 nm, power of 1.5 W / cm²) 2 The irradiation time is 10 minutes.

[0053] Animal experimental processing steps: (1) The day before the wound was created, the back of the mouse was treated with a shaver and hair removal cream, and the remaining hair was removed with hair removal cream. The weight was recorded (every 3 days until the end of the experiment).

[0054] (2) On the second day, an incision was made on the back of all mice using a 10 mm diameter mouse skin punch and surgical scissors with aphrodisiac (dosage: 20 μL / g). The size of the incision was recorded by photograph (every 3 days, depending on the situation when the incision is almost healed). 50 μL of OD was applied to the incision. 600 An infection model was created using 0.6% Staphylococcus aureus bacterial suspension, and samples were taken the next day for plate smearing to observe the infection status.

[0055] (3) Each group of mice was treated with corresponding 3M patches and microneedle patches. The PM + NIR, PCM + NIR and MN + NIR groups were treated with near-infrared light at a wavelength of 808 nm and a power of 1.5 W / cm². 2 Irradiate for 10 minutes, and record the photothermal temperature every 90 seconds.

[0056] (4) When the wounds of a group of mice that received the drug were almost completely healed, samples were taken from all the mice's wounds, and the recovery was observed by smearing the samples on a plate. The size of all the mice's wounds was also recorded.

[0057] (5) The next day, the mice were dissected, blood was taken from the eyeballs for routine blood tests, and skin from the internal organs and wounds was fixed with 4% paraformaldehyde for HE and Masson staining analysis.

[0058] Experimental results are as follows Figure 12-19 As shown. Figure 12 The results showed that the weight of the mice decreased after modeling due to infection and anesthetic, but after treatment with microneedle patches, their weight gradually increased to normal. Figure 13 The results showed that the treatment temperature in the photothermal group mice stabilized at approximately 46°C after 6 minutes. Excessively high temperatures could cause burns to the wound. Literature studies (Gu, Wanrong, et al. "Bilayer hydrogel microneedles with mild photothermal effect promote infectious skin regeneration." Journal of Materials Chemistry B 13.25 (2025): 7366-7380.) indicate that when the photothermal temperature rises to approximately or above 50°C, it may cause irreversible damage to normal tissues, hindering collagen deposition and angiogenesis, and even affecting healing. Therefore, this photothermal soluble microneedle patch offers a gentler treatment for the skin. Figure 14 and Figure 15 The results showed that both the MN group and the MN + NIR group in the mouse group contained asiaticoside, which had a better wound healing effect under the same time conditions. Figure 16 The results showed that the wound infection status of mice was significantly improved after treatment in the PCM + NIR group and the MN + NIR group. Figure 17 The blood routine test results showed that all components in the microneedle patch had good biocompatibility; Figure 18 HE test results showed that after treatment in the MN + NIR group, the mouse wounds showed more complete epithelialization and skin regeneration compared to the positive control 3M group; Masson test results showed that after treatment in the MN + NIR group, the mouse wounds showed more collagen deposition. Figure 19 HE staining results of the heart, liver, spleen, lungs and kidneys of mice in each group showed that each component of the microneedle patch had good biocompatibility with mice.

[0059] Example 13: Immunohistochemical results of Ki67, CD31, Col-I, and Col-III at the wound sites of mice in each group. Experimental results are as follows Figure 20 As shown in the figure. Ki67 fluorescence immunohistochemical results indicated that mice treated with MN + NIR showed increased cell proliferation at the wound site; CD31 fluorescence immunohistochemical results indicated that mice treated with MN + NIR showed better angiogenesis at the wound site; Col-I and Col-III fluorescence immunohistochemical results indicated that mice treated with MN + NIR showed increased collagen deposition at the wound site, with type III collagen deposition significantly higher than type I collagen, indicating an inhibitory effect on scar formation at the wound site.

[0060] Example 14 Immunohistochemical results of IL-10 at the wound site in each group of mice Experimental results are as follows Figure 21 As shown in the figure, the immunohistochemical results of the control group mice showed higher expression of IL-10. This indicates that the inflammation at the wound site was more severe in the control group mice, while the MN + NIR group mice showed lower expression of IL-10 at the wound site. This suggests that the photothermal soluble microneedle patch has a good anti-inflammatory effect.

Claims

1. A photothermal soluble microneedle patch for promoting skin wound healing, characterized in that, Includes the needle tip layer and the backing layer supporting the needle tip layer; The needle tip layer includes hyaluronic acid, chlorogenic acid, and Prussian blue; The backing layer includes PVP K60 and asiaticoside.

2. The photothermal soluble microneedle patch for promoting skin wound healing according to claim 1, characterized in that, The needle tip layer contains 15 mg / mL hyaluronic acid as a matrix, and 0-4 mg / mL chlorogenic acid and 20-100 μg / mL Prussian blue.

3. The photothermal soluble microneedle patch according to claim 1, characterized in that, The backing layer contains 100-300 μg / mL of asiaticoside, with 400 mg / mL of PVP K60 as the matrix.

4. The photothermal soluble microneedle patch according to claim 1, characterized in that, The needle tip layer contains 15 mg / mL hyaluronic acid as a matrix, 2 mg / mL chlorogenic acid, and 60 μg / mL Prussian blue; the backing layer contains 400 mg / mL PVP K60 as a matrix, and 300 μg / mL asiaticoside.

5. The photothermal soluble microneedle patch according to claim 1, characterized in that, The photothermal soluble microneedle patch has a solid conical structure with a needle length of 710 μm, a bottom diameter of 280 μm, a needle tip distance of 600 μm between two adjacent conical needles, an array of 15*15 needles, an area containing needles of approximately 8.7 * 8.7 mm, and a patch size of 12*12 mm.

6. A method for preparing the photothermal soluble microneedle patch according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Preparation of the tip layer: Centrifuge the tip layer matrix containing chlorogenic acid and Prussian blue to remove air bubbles at a temperature of 0~4℃, a centrifugation speed of 3000~5000 rpm, and a time of 5~10 min; after centrifugation, use a syringe to draw 100 μL and add it to the PDMS mold to spread evenly. After vacuum negative pressure and centrifugation, remove the excess matrix to obtain the tip layer; (2) Preparation of backing layer: Centrifuge the backing layer matrix containing asiaticoside to remove air bubbles at a temperature of 0~4℃, a centrifugation speed of 3000~5000 rpm, and a time of 10min; after centrifugation, use a syringe to draw about 200 μL and add it to the backing groove of the PDMS mold to obtain the backing layer. (3) The obtained needle tip layer and backing layer are placed in an oven to dry, and after demolding, the photothermal soluble microneedle patch is obtained.

7. The preparation method according to claim 6, characterized in that, The preparation of the needle tip matrix includes: first, preparing a sterile water mixture containing 2 mg / mL chlorogenic acid and 60 μg / mL Prussian blue, adding it to hyaluronic acid powder with a final concentration of 15 mg / mL, and incubating it overnight in a shaker at 37~40℃ through the hygroscopic, swelling and dispersion stages to obtain the needle tip gel. The preparation of the backing layer matrix includes: ultrasonically dissolving a 300 μg / mL asiaticoside solution in a mixed solution of 0.5% DMSO and sterile water, and then placing it in a shaker at 37-40°C overnight with the needle tip layer to obtain the backing layer matrix.

8. The preparation method according to claim 6, characterized in that, In step (1), the vacuum negative pressure condition is 0.08MPa, 90~120s; the centrifugation conditions are horizontal centrifugation speed of 4000 rpm, time of 15~20 min, temperature of 0~4℃, and number of times of centrifugation is 2~3.

9. The preparation method according to claim 6, characterized in that, In step (3), the drying temperature is 37~38℃ and the drying time is 24~36 h.

10. The use of the photothermal soluble microneedle patch according to any one of claims 1 to 5 in the preparation of any one of wound healing medical devices and wound antibacterial medical devices.

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