A soluble microneedle for treating hypertrophic scars and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses soluble microneedles for treating hyperplastic scars, and a preparation method and application thereof. The hyaluronic acid is modified to obtain sulfonated hyaluronic acid, which is blended with natural penetration enhancer dextro-borneol having anti-inflammatory activity, rotary evaporated and freeze-dried to obtain a sulfonated hyaluronic acid-dextro-borneol solid dispersion, and then the solid dispersion is loaded into the microneedles to obtain the soluble microneedles having the function of treating hyperplastic scars. The method improves the bioavailability and biocompatibility of dextro-borneol, and the dextro-borneol is loaded into the microneedles, so that the anti-inflammatory and migration inhibition of fibroblasts of the solid dispersion can be maximized, and the microneedles and dextro-borneol can penetrate the dense scar tissue to achieve the purpose of deep drug delivery under the double penetration of the microneedles and dextro-borneol. The application provides a safe and efficient novel transdermal drug delivery system for treating hyperplastic scars, and has a wide clinical application prospect and market value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a soluble microneedle for treating hypertrophic scars, its preparation method, and its application. Background Technology

[0002] Hypertrophic scars (HS), also known as raised scars, are fibrotic skin tissue lesions resulting from surgical trauma or burns and the pathological healing of wounds after injury. Globally, over 100 million people have scars due to surgery or trauma, with hypertrophic scars accounting for 15% of these cases. Clinically, they present as irregular raised scars, often accompanied by itching and pain. They are characterized by three major challenges: high incidence, complex pathogenesis, and difficulty in treatment. Inhibiting hypertrophic scar formation remains a hot research topic.

[0003] Scar formation occurs in three phases: the inflammatory phase, the proliferative phase, and the remodeling phase. During the inflammatory phase, inflammatory cells migrate to the wound and release inflammatory factors, which directly or indirectly lead to the proliferation of fibroblasts (HSF). During the proliferative phase, the release of large amounts of transforming growth factor-β (TGF-β) causes excessive proliferation and migration of fibroblasts, further promoting the deposition of large amounts of collagen-based extracellular matrix (ECM). During the remodeling phase, the excessively accumulated ECM is difficult for the body to absorb or remodel in time, and ECM interacts with HSF, resulting in fibrotic lesions. Therefore, the histological characteristics of hypertrophic scars are inflammatory cell infiltration and the large deposition of fibroblasts and extracellular matrix. Therefore, reducing the content of inflammatory factors and regulating the excessive proliferation and migration of fibroblasts are crucial steps in inhibiting scar hyperplasia.

[0004] Currently, there are three commonly used treatment methods in clinical practice: oral administration, mainly using anti-inflammatory drugs, only relieves symptoms and suffers from the first-pass effect in the gastrointestinal tract; topical administration, although it can regulate collagen arrangement and reduce pigmentation, suffers from low drug penetration due to the density of scar tissue; and intralesional injection of corticosteroids, a combination of drug and device therapy, has been the most commonly used and effective method since 1960. It can penetrate dense scar tissue to reach the dermis and release the drug, but long-term corticosteroid injections can lead to pain, skin atrophy, and pigmentation, and require multiple injections by doctors, resulting in high costs and negative emotions for patients. Therefore, there is an urgent clinical need for safe and effective drugs and drug delivery systems for treating hypertrophic scars. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing soluble microneedles for treating hypertrophic scars.

[0006] Another object of the present invention is to provide soluble microneedles for treating hypertrophic scars obtained by the above preparation method.

[0007] Another object of the present invention is to provide the application of the above-mentioned soluble microneedles for treating hypertrophic scars.

[0008] The objective of this invention is achieved through the following technical solution: a method for preparing soluble microneedles for treating hypertrophic scars, comprising the following steps:

[0009] S1. Preparation of sulfonated hyaluronic acid (SHA): First, hyaluronic acid is mixed with ammonium salt solution to obtain modified fat-soluble hyaluronic acid. Then, sulfonating reagent is added under inert gas and ice bath conditions to react. Next, water is added to terminate the reaction. Then, alkaline solution is added to adjust the pH value. Organic solvent is added to precipitate the product. Solid-liquid separation is performed. The obtained solid is dissolved in water and dialyzed to obtain SHA.

[0010] S2. Preparation of sulfonated hyaluronic acid-dextrin solid dispersion (SHA-NB-SD): Dextrin (NB) and SHA prepared in step S1 are mixed evenly in an organic solvent, the organic solvent is removed by rotary evaporation, and the solid dispersion is obtained by freeze drying.

[0011] S3. Preparation of needle layer solution: Mix the needle excipient with water evenly to obtain solution A; mix the solid dispersion obtained in step S2 with water evenly to obtain solution B; mix solution A and solution B to obtain needle layer solution; wherein, the amount of each component is as follows according to the mass (g) to volume (mL) ratio: needle excipient 5-15%, solid dispersion 5-15%, water as the balance; preferably as follows: needle excipient 10%, solid dispersion 10%, water as the balance;

[0012] S4. Preparation of backing layer solution: Mix soluble polymer and water evenly to obtain solution C; then mix evenly with needle excipient material; wherein, the amount of each component is as follows by mass (g) and volume (mL): soluble polymer 3-7%, needle excipient material 8-12%, water as balance; preferably as follows: soluble polymer 5%, needle excipient material 10%, water as balance;

[0013] S5. Preparation of microneedles:

[0014] A. Add the needle body layer solution prepared in step S3 into the microneedle negative mold to fill the microneedle negative mold, scrape off the excess liquid, and dry and solidify to form the microneedle body layer;

[0015] B. Add the backing layer solution prepared in step S4 to the microneedle body layer in step A to connect the needle body layer with the backing layer. Dry the entire microneedle to obtain soluble microneedles for preventing hypertrophic scars.

[0016] The molecular weight of the hyaluronic acid mentioned in step S1 is preferably 5000-7000 Da; more preferably 6000 Da.

[0017] The ammonium salt mentioned in step S1 is selected from tetrabutylammonium hydroxide.

[0018] The inert gas mentioned in step S1 is selected from nitrogen.

[0019] The sulfonating agent mentioned in step S1 is selected from sulfur trioxide-pyridine.

[0020] The reaction time in step S1 is preferably 1 to 2 hours; more preferably 1 hour.

[0021] The alkaline solution mentioned in step S1 is selected from sodium hydroxide solution; preferably, it is a sodium hydroxide solution with a concentration of 4-6% w / v; more preferably, it is a sodium hydroxide solution with a concentration of 5% w / v.

[0022] The pH value mentioned in step S1 is preferably 8 to 8.5; more preferably 8.5.

[0023] The organic solvent mentioned in step S1 is selected from anhydrous ethanol.

[0024] The preferred number of precipitation cycles in step S1 is 3.

[0025] The preferred method for solid-liquid separation in step S1 is centrifugation.

[0026] The preferred centrifugation conditions are centrifugation at 6000-10000 r / min for 5-15 min; more preferably, centrifugation at 8000 r / min for 10 min.

[0027] The preferred specification of the dialysis bag used in step S1 is 3000-3500 Da.

[0028] The dialysis time in step S1 is preferably 24 to 48 hours; more preferably 36 hours.

[0029] In step S2, the SHA and the NB are preferably mixed in a mass ratio of 1:1 to 1.1; more preferably in a mass ratio of 1:1.

[0030] The organic solvent mentioned in step S2 is preferably an ethanol solution; more preferably a 40-60% v / v ethanol solution; and most preferably a 50% v / v ethanol solution.

[0031] The preferred conditions for rotary evaporation in step S2 are a temperature of 30–50°C and a rotation speed of 100–150 r / min; more preferably, a temperature of 40°C and a rotation speed of 100 r / min.

[0032] The needle shaping material mentioned in step S3 is preferably at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90, more preferably polyvinylpyrrolidone K90.

[0033] The needle-shaped material solution and the solid dispersion solution mentioned in step S3 are mixed at a volume ratio of 0.5 to 1.5:1; preferably at a volume ratio of 1:1.

[0034] The soluble polymer mentioned in step S4 is preferably polyvinyl alcohol, more preferably polyvinyl alcohol 1788.

[0035] The soluble polymer and water are mixed evenly in step S4, preferably prepared by the following steps: under heating conditions, the soluble polymer is dissolved in water to obtain an aqueous solution of the soluble polymer.

[0036] The heating temperature is preferably 80-90°C; more preferably 85°C.

[0037] The needle-shaped material solution and the soluble polymer solution mentioned in step S4 are mixed at a volume ratio of 0.5 to 1.5:1; preferably at a volume ratio of 1:1.

[0038] The preferred method for filling the microneedle negative mold in step S5A is vacuum treatment; more preferably, vacuuming for 15 to 20 minutes in an environment with a vacuum degree of -0.09 to -0.1 MPa.

[0039] The drying conditions described in step S5A are preferably drying at 25-40°C until cured; more preferably drying at 25-40°C for 4-5 hours.

[0040] The preferred method for connecting the needle body layer and the backing layer in step S5B is vacuum treatment; more preferably, vacuuming for 15 to 20 minutes in an environment with a vacuum degree of -0.09 to -0.1 MPa.

[0041] The drying conditions described in step S5B are preferably drying at 25-40°C until cured; more preferably drying at 25-40°C for 18-24 hours.

[0042] A soluble microneedle for treating hypertrophic scars is obtained by the above preparation method.

[0043] The above-mentioned soluble microneedles for treating hypertrophic scars are used in the preparation of drugs for treating hypertrophic scars.

[0044] The drug is used to inhibit the expression of inflammatory factors and to suppress the excessive proliferation and migration of fibroblasts.

[0045] Compared with existing technologies, the microneedles prepared in this invention treat the formation of hypertrophic scars from multiple dimensions, and have the following advantages and effects:

[0046] (1) Molecular Engineering: From "Inert Carriers" to "Smart Active Drugs"

[0047] A. Functionalized HA modification: By sulfonation, HA is endowed with strong negative charge, anti-inflammatory targeting and HSF excessive migration effects, which upgrades it from a traditional moisturizing material to a dual-function drug of "anti-inflammatory and anti-fibrotic" by inhibiting the activation of TGF-β / NF-κB / MAPK signaling pathway.

[0048] B. Enhanced efficacy of natural drugs: The anti-inflammatory and fibroblast migration-inhibiting effects of SHA combined with the permeation-promoting and anti-inflammatory effects of NB create a therapeutic effect of "1+1>2". At the same time, it realizes the design concept of "carrier as drug". Chemical modification endows the material with active therapeutic function and reduces the need for exogenous drug addition.

[0049] (2) Security breakthrough

[0050] Solid dispersion technology: SHA and NB are prepared as a solid dispersion, solving the industry problems of poor water solubility and high toxicity of NB, and significantly improving its bioavailability. Zebrafish experiments confirmed that SHA-NB-SD is practically non-toxic, superior to single-component dextrorotatory borneol. This is because the introduction of negatively charged sulfonic acid groups can highly bind to receptors on the surface of immune cells and effectively activate the immune response, enhancing the immune reaction; at the same time, the sulfonic acid groups have high hydrophilicity, which can enhance the interaction between the material and biomolecules, thereby improving the biocompatibility of the material.

[0051] (3) Delivery System: From “Single Penetration” to “Spatiotemporal Dynamic Collaboration”

[0052] A. Optimized Penetration: Microneedles directly penetrate dense scar tissue, changing SHA's previous method of topical drug delivery and overcoming the bottleneck of low penetration rate in traditional transdermal drug delivery.

[0053] B. Dual Permeation Enhancement Design: The physical penetration of microneedles and the chemical penetration of NB combine to achieve deep drug delivery "from point to surface".

[0054] (4) Treatment strategy: From "single-target therapy" to "closed-loop regulation of the entire pathology process"

[0055] A. Inflammatory factors: The negative charge of SHA can bind to the positively charged pro-inflammatory factors IL-1β, IL-6, and TNF-α, eliminating inflammatory factors; while NB has anti-inflammatory activity; when the two are prepared as a solid dispersion, they can synergistically exert anti-inflammatory effects. The results of the zebrafish neutrophil migration inhibition experiment also show that, at the same concentration, the solid dispersion has a better anti-inflammatory effect.

[0056] B. Inhibition of fibroblast activity and migration: SHA-NB-SD can inhibit the activation of NF-κB and MAPK signaling pathways and reduce HSF activation through significant anti-inflammatory activity; TGF-β is a key factor for HSF activation and proliferation, and SHA can compete with fibronectin for the release site of TGF-β, LTBP protein, resulting in reduced TGF-β release, thereby reducing HSF migration and activation.

[0057] C. Microneedle mechanical regulation: Microneedles penetrate the skin, causing local stimulation, disrupting the collagen arrangement, and causing rearrangement; at the same time, microneedles can penetrate dense scar tissue to achieve deep drug delivery.

[0058] (5) Industrialization and clinical translation potential

[0059] A. Standardized process: The invention has a clear preparation process, from raw material modification to the preparation of solid dispersions and then to microneedle molding, which is suitable for large-scale production.

[0060] B. High patient compliance: Microneedling is painless and does not require multiple injections, overcoming the side effects such as atrophy and pain caused by corticosteroid therapy.

[0061] C. Core Breakthrough: Innovation across the entire chain, from molecular design to delivery systems to treatment strategies.

[0062] (6) Sulfonated hyaluronic acid-dextrin-based soluble microneedles have significant advantages in the treatment of hypertrophic scars, achieving a quadruple breakthrough of "molecular design-safety breakthrough-delivery synergy-pathological closed loop", providing the first transdermal treatment solution for hypertrophic scars that is highly efficient, safe and convenient. Attached Figure Description

[0063] Figure 1 This is the Fourier transform infrared spectrum of the sulfonated hyaluronic acid prepared in Example 1.

[0064] Figure 2 This is a potential diagram of the sulfonated hyaluronic acid prepared in Example 1.

[0065] Figure 3 This is a comparison diagram of the dissolution states of dextrorotatory borneol (left) and the sulfonated hyaluronic acid-dextrorotatory borneol solid dispersion prepared in Example 2 (right).

[0066] Figure 4 This is a schematic diagram of the structure of a soluble microneedle prepared in Example 3 for treating hypertrophic scars.

[0067] Figure 5 This is a graph showing the test results of different samples on zebrafish embryo toxicity.

[0068] Figure 6This is a graph showing the detection results of the therapeutic effects of different samples on CuSO4-induced inflammation in zebrafish.

[0069] Figure 7 This is a graph showing the detection results of different samples on the inhibition effect of HSF migration.

[0070] Figure 8 This is a graph showing the test results of the therapeutic effect of a soluble microparticle prepared in Example 3 for treating hypertrophic scars on the formation of hypertrophic scars in rabbit ears. Detailed Implementation

[0071] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0072] Unless otherwise specified, all reagents used in the examples are commercially available.

[0073] Example 1

[0074] The preparation method of sulfonated hyaluronic acid, with specific steps as follows:

[0075] (1) Hydrophobic modification of hyaluronic acid (HA)

[0076] Weigh 3g of IR120 hydrogen-type cation exchange resin and place it in a 250mL Erlenmeyer flask. First, soak it in anhydrous ethanol for 2 hours, then wash it with deionized water until neutral. Next, soak it in 5% w / v sodium hydroxide solution for 2 hours, then wash it with deionized water until neutral. Finally, soak it in 5% w / v hydrochloric acid solution for 2 hours, then wash it with deionized water until neutral. Set aside for use.

[0077] Weigh 1.0 g HA (MW 6 kDa), dissolve it completely in 100 mL of deionized water, then add the H-type cation exchange resin IR120 prepared according to the above steps, stir at room temperature for 3 h, filter, take the filtrate and adjust the pH to 7.0 with 25% w / v tetrabutylammonium hydroxide solution, stir at room temperature for 2 h, freeze dry to obtain the hydrophobic modified product of HA (HA-TBA).

[0078] (2) Preparation of sulfonated hyaluronic acid (SHA)

[0079] Weigh 0.3 g of HA-TBA into a 200 mL beaker, add 60 mL of DMF (N,N-dimethylformamide), stir until dissolved, and purge with nitrogen for 20 min to obtain an HA-TBA-DMF solution. Weigh 0.9 g of pyridine sulfur trioxide and dissolve it in 20 mL of DMF, stirring until homogeneous to prepare an esterification reagent. Add the esterification reagent to the HA-TBA-DMF solution under ice bath conditions, react for 1 h under a nitrogen atmosphere, and then add an equal volume of water to terminate the reaction. Adjust the pH to 7-8.5 with 5% w / v sodium hydroxide solution. Add anhydrous ethanol to precipitate three times. Centrifuge, discard the supernatant, and dry the precipitate in a vacuum drying oven. Then add an appropriate amount of deionized water to dissolve the precipitate, transfer it to a 3 kDa dialysis bag, and dialyze it with running water. After dialyzing in deionized water for 1.5 days, freeze-dry the dialysate in the dialysis bag, and store it in a sealed container at 4 °C to obtain sulfonated hyaluronic acid (SHA).

[0080] SHA was observed using Fourier transform infrared spectroscopy. Figure 1 It can be seen that at 1276cm -1 An absorption peak for the stretching vibration of the S=O double bond appears at 826 cm⁻¹, and at 826 cm⁻¹... -1 The appearance of a COS stretching vibration absorption peak at the point confirmed the sulfonation process. Zeta potential was also determined. Figure 2 It can be seen from the Zeta potential that the introduction of SO into HA... 3- The group increases the density of negative charge, with the Zeta potential of HA decreasing from -29.47±7.8 mV to -49.70±2.9 mV of SHA. The decrease in the SHA potential indicates that sulfonation was successful.

[0081] Example 2

[0082] The preparation method of sulfonated hyaluronic acid-dextral borneol solid dispersion (SHA-NB-SD) is as follows:

[0083] Weigh out equal amounts of SHA and dextrorotatory borneol (NB), mix them together in a 50% v / v ethanol solution, stir well, and rotary evaporate at 40℃ and 100 r / min until no more ethanol drips down. Pour out the solution from the rotary evaporation flask and freeze dry to obtain SHA-NB-SD.

[0084] The state of NB precipitation after rotary evaporation of 50% ethanol solution was observed with the naked eye and recorded with a camera. Figure 3 It can be seen that when NB is dissolved in a 50% ethanol solution, and the ethanol is removed by rotary evaporation, NB precipitates out (part of the precipitated NB is as follows). Figure 3(As indicated by the arrow in the image); SHA and NB were dissolved together in a 50% ethanol solution, and the ethanol was removed by rotary evaporation. NB did not precipitate, resulting in a clear and transparent solution. This demonstrates that SHA can physically blend with NB via hydrogen bonds or van der Waals forces to form a solid dispersion, thereby improving the bioavailability of NB.

[0085] Example 3

[0086] The preparation method of sulfonated hyaluronic acid-dextrin-based soluble microneedles (SHA-NB-SD@DMNs) is as follows:

[0087] (1) Preparation of needle layer solution: Mix 20% w / v SHA-NB-SD (solvent is deionized water) and 20% w / v polyvinylpyrrolidone PVP K90 solution (solvent is deionized water) at a volume ratio of 1:1 to obtain the needle layer solution.

[0088] (2) Preparation of backing layer solution: Polyvinyl alcohol (PVA) 1788 was dissolved in deionized water under heating (85°C) to obtain a 10% w / v PVA 1788 solution. A 20% w / v PVP K90 solution and a 10% w / v PVA 1788 solution were mixed at a volume ratio of 1:1 to prepare the backing layer solution.

[0089] (3) Preparation of SHA-NB-SD@DMNs: Microneedles were prepared using a two-step method. Specifically, 150 μL of the needle layer solution was precisely pipetted into the mold groove, and bubbles were scraped off to ensure even spreading. The microneedle mold was transferred to a vacuum drying oven for 20 min to degas, ensuring the needle solution filled the micropores within the mold. This process was repeated twice. The mold was then dried at 25°C for 5 h. Next, 200 μL of the backing layer solution was added to the dried portion, and the solution was spread evenly. The microneedle mold was then transferred to a vacuum drying oven for 20 min to degas, and this process was repeated twice. The molds were dried under the same conditions for 24 h. After complete drying, the microneedles were demolded to obtain SHA-NB-SD@DMNs, which were then stored in a dry, light-protected place. Sulfonylrhodamine B (SRB) of the same concentration was used as a mimic drug to replace SHA-NB-SD, with the same operating steps as described above, for subsequent investigation of drug distribution patterns.

[0090] (4) Results: Observation using an inverted fluorescence microscope revealed that... Figure 4As shown in the left image, the bright-field soluble microneedle (DMN) array consists of conical needles arranged in a sharp pattern. The needle tip height is approximately 550 μm, the base diameter is 200 μm, and the tip-to-tip distance is 600 μm. Due to volume shrinkage during vacuum drying, these dimensions are slightly smaller than those of an anion exchange membrane. Furthermore, fluorescence observation reveals that the simulated drug sulfonylrhodamine B (SRB) is uniformly distributed along the microneedle axis, indirectly demonstrating the uniform distribution of the drug along the microneedle axis (e.g., ...). Figure 4 (As shown in the right figure).

[0091] Example 4

[0092] The inhibitory effect of sulfonated hyaluronic acid-dextral borneol solid dispersion on CuSO4-induced inflammation in a zebrafish model is studied as follows:

[0093] Following the preparation methods of Examples 1 and 2, SHA-NB-SD was obtained and subjected to anti-inflammatory experiments in zebrafish.

[0094] (1) Toxicity test of zebrafish

[0095] Normal zebrafish embryos (Huante Laboratory Technology (Hangzhou) Co., Ltd.) at 6–8 hpf were randomly selected using a plastic dropper under a digital microscope. A 6-well plate was used as the experimental medium, with 15–20 embryos added to each well. The culture solution was then carefully aspirated with a pipette, and 3–4 mL of pre-prepared sample solution was added to each well. The plate was then covered, wrapped in aluminum foil, and transferred to a 28±0.5℃ incubator in the dark for 96 hpf. Embryo mortality was recorded. Three parallel groups were set up for each concentration, along with a blank control group or a solution control group. Sample solutions were prepared directly with water for HA, SHA, and SHA-NB-SD; NB was prepared by first preparing a 100 mg / mL stock solution with DMSO and then diluting it with water.

[0096] Depend on Figure 5It can be seen that zebrafish are essentially unable to survive at a concentration of 500 μg / mL for HA and NB; however, at a concentration of 1000 μg / mL for SHA and SHA-NB-SD, the survival rates of zebrafish are 93.3% and 91.09%, respectively. According to the group standard "Acute Toxicity Testing of Zebrafish Embryos," the toxicity of SHA and SHA-NB-SD to zebrafish embryos is preliminarily determined to be practically non-toxic. Therefore, compared with HA and NB, SHA and SHA-NB-SD exhibit better biocompatibility in zebrafish embryos, not only improving the bioavailability of NB but also reducing its toxicity. This is because the introduction of negatively charged sulfonic acid groups can highly bind to receptors on the surface of immune cells and effectively activate the immune response, enhancing the immune reaction; simultaneously, the sulfonic acid groups have high hydrophilicity, which can enhance the interaction between the material and biomolecules, thereby improving the biocompatibility of the material.

[0097] (2) Therapeutic effects of SHA, NB, and SHA-NB-SD on CuSO4-induced inflammation in zebrafish

[0098] Normally developing zebrafish embryos at 3 days post-fertilization (dpf) were selected. A 6-well plate was used as the experimental medium, with 15 zebrafish added to each well. The culture medium was carefully aspirated using a pipette, and 3 mL of distilled water was added to each well. Then, except for the control group, 3 μL of CuSO4 solution was added to each well to induce inflammation, resulting in a final CuSO4 concentration of 10 μmol / L. The plate was then covered with a lid, wrapped in aluminum foil, and incubated at 28±0.5℃ in the dark for 30–40 min. The solution in the wells was then aspirated. 3 mL of distilled water was added to the model group, and 3 mL of sample solution was added to each sample group (SHA, NB, and SHA-NB-SD, respectively). Both groups were incubated for 4–5 hpf under the same conditions. The solution in the sample groups was then aspirated, and 3 mL of distilled water was added. Each concentration was set up in triplicate. Ten zebrafish were randomly selected, fixed on a glass slide with 5% methylcellulose, and observed and photographed under an inverted fluorescence microscope. The number of neutrophils was recorded using ImageJ image analysis software.

[0099] Table 1 and Figure 6 The results showed that the number of neutrophils in the neurothalamic cell region decreased with increasing sample concentration. SHA and SHA-NB-SD exhibited more significant anti-inflammatory effects, which may be due to the introduction of sulfonic acid groups, allowing the negative charge to adsorb more inflammatory factors and achieve a better anti-inflammatory effect. Furthermore, at a concentration of 0.5 mg / mL, all zebrafish died, further demonstrating that SHA-NB-SD had a superior anti-inflammatory effect compared to SHA.

[0100] Table 1. Number of neutrophils produced in a CuSO4-induced zebrafish inflammation model by different samples (X±SD)

[0101]

[0102] Note: The concentrations of SHA-NB-SD in the table are based on SHA equivalent concentrations.

[0103] Example 5

[0104] Investigation on the inhibitory effect of sulfonated hyaluronic acid-dextral borneol solid dispersion on HSF migration

[0105] In the cell scratch assay, mouse fibroblast L929 cells were subjected to a 2×10⁻⁶ saturation rate. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated at 37°C with 5% CO2 for 12 hours until a monolayer of cells formed in the wells. Subsequently, using a sterile 200 μL pipette tip and a ruler, a uniform vertical scratch was made in the center of the cell monolayer. The cells were washed three times with PBS to remove any detached cells from the scratch, ensuring the purity of the control group and the accuracy of the experimental results. HA (100 μg / mL), SHA (100 μg / mL), NB (100 μg / mL, prepared using a 20 mg / mL stock solution in DMSO), and SHA-NB-SD (100 μg / mL SHA equivalent concentration, already converted to SHA equivalent concentration) were prepared using low-serum MEM medium containing 2% v / v FBS. 2 mL of the pre-prepared culture medium was added to each 6-well plate, and 2 mL of low-serum MEM medium containing 2% v / v FBS was added to the control group. Cell culture plates were placed in an incubator at 37°C and 5% CO2. Cell migration was observed by photographing the cells using an inverted fluorescence microscope at 0 hours and 48 hours. Finally, the images were analyzed using ImageJ software to calculate the spacing of the scratches, record the changes in scratch width at different time points, and employ appropriate statistical methods for data analysis to determine the effects of different treatments on cell migration.

[0106] From Table 2 and Figure 7 As can be seen, SHA-NB-SD significantly slowed down the closure speed of scratches, indicating that SHA-NB-SD had the highest inhibition rate of HSF migration, followed by SHA. This may be because SHA inhibited the release of TGF-β, which can stimulate the release of large amounts of HSF, thus enabling SHA to effectively inhibit HSF proliferation and migration. At the same time, SHA-NB-SD has a synergistic anti-inflammatory effect, possibly by inhibiting the expression of inflammatory factors such as IL-1 and TNF-α, thereby preventing the activation of NF-κB and MAPK signaling pathways, and thus inhibiting fibroblast proliferation and migration.

[0107] Table 2. Scratch healing rate (X±SD) of different samples after 48 hours of treatment on HSF cells.

[0108] Group Scratch healing rate (%) Control 66.30±1.56 HA 67.36±5.57 NB 59.06±3.05 SHA 58.40±3.38 SHA-NB-SD <![CDATA[39.45±5.94 *** ]]>

[0109] Example 6

[0110] The therapeutic effect of this invention on hypertrophic scars is further illustrated by the elimination of hypertrophic scars on the ears of New Zealand rabbits. The specific steps are as follows:

[0111] (1) Experimental materials

[0112] 1) Laboratory animals

[0113] All SPF-grade New Zealand rabbits (male, 2kg) used in this application were provided by the Experimental Animal Center of South China University of Technology. The breeding conditions were: separate cages, normal diet, and acclimatization feeding for 7 days before the experiment.

[0114] 2) Test drug

[0115] Treatment group 1 was given SHA-NB-SD@DMNs; treatment group 2 was given SHA@DMNs obtained according to the method of Example 3 (i.e., in step (1) of Example 3, the SHA-NB-SD solution was replaced with SHA solution of the same concentration, and the solvent of SHA solution was water); treatment group 3 was given NB@DMNs obtained according to the method of Example 3 (i.e., in step (1) of Example 3, the SHA-NB-SD solution was replaced with NB solution of the same concentration, and the solvent of NB solution was 50% ethanol); treatment group 4 was given Drug-free@DMNs obtained according to the method of Example 3 (i.e., in step (1) of Example 3, the SHA-NB-SD solution was replaced with deionized water).

[0116] (2) Experimental methods

[0117] 1) Establishment of a rabbit ear hypertrophic scar model

[0118] New Zealand rabbits were housed individually under standard conditions for one week. Prior to the experiment, they were fasted and deprived of water for 8 hours, then anesthetized with sodium pentobarbital. After satisfactory anesthesia, the rabbits were fixed on a specialized experimental table, and four wounds (1×1cm) were made along the long axis of the mid-ventral side of the rabbit's ear, with a distance >1cm between wounds. The wounds were then cleaned with 0.1% benzalkonium chloride. After 7 days of recovery, newly formed scabs were removed, exposing the wounds again. A hypertrophic scar model was successfully established after 21 days.

[0119] 2) Grouping and microneedle therapy

[0120] Thirty New Zealand rabbits that successfully underwent modeling were selected and divided into a control group and a treatment group, with six rabbits in each group. The control group received no drug treatment, while treatment groups 1-4 received different types of microneedling treatment. The microneedles were inserted into the skin, pressed for 1 minute, and then fixed with medical breathable tape for 10 minutes. After the microneedles were fully dissolved, they were removed, thus completing the microneedling drug administration treatment. The drug was administered once a week for a total of 3 times. On the 21st day of drug administration, scar tissue was collected and H&E stained sections were prepared. The H&E stained sections were observed under a microscope and photographed. The vertical height from the highest point of the scar protrusion to the normal skin layer was measured using ImageJ software and recorded as A; the vertical distance from the normal skin around the scar to the surface of the ear cartilage was recorded as B. The scar hyperplasia index (SEI) was calculated according to formula (1).

[0121] Calculation formula:

[0122] 3) Experimental Results

[0123] From Table 3 and Figure 8 It can be seen that the scar hyperplasia index of the treatment group was significantly reduced compared with the control group, and SHA-NB-SD@DMNs had the best effect. This may be due to the synergistic effect of the drug in moderate anti-inflammatory and inhibiting excessive proliferation and migration of HSF. At the same time, the microneedles can penetrate the stratum corneum to deliver drugs to the dermis. The anti-inflammatory drug NB loaded in them also acts as a natural penetration enhancer, enabling the drug to be released "from point to surface". The synergy of the two plays a dual role in promoting penetration and more effectively treating hypertrophic scars.

[0124] Table 3 Comparison of the effects of different sample groups on the scar hyperplasia index of rabbit ears (X±SD)

[0125] Group Scar hyperplasia index control group 4.29±0.18 Treatment group 1 <![CDATA[0.69±0.06 **** ]]> Treatment group 2 <![CDATA[1.45±0.04 **** ]]> Treatment group 3 <![CDATA[2.01±0.03 **** ]]> Treatment group 4 <![CDATA[2.82±0.02 **** ]]>

[0126] The above description is merely a preferred embodiment of the present invention, but the implementation of the present invention is not limited to the above examples. For those skilled in the art, appropriate modifications can be made to the embodiments of the present invention without departing from the spirit of the invention. However, these modifications should not be considered as limitations on the scope of the present invention. Therefore, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent alternatives and fall within the protection scope of the present invention.

Claims

1. A method for preparing soluble microneedles for treating hypertrophic scars, characterized in that... Includes the following steps: S1. Preparation of sulfonated hyaluronic acid: First, hyaluronic acid is mixed with ammonium salt solution to obtain modified fat-soluble hyaluronic acid. Then, sulfonating reagent is added under inert gas and ice bath conditions to react. Next, water is added to terminate the reaction. Then, alkaline solution is added to adjust the pH value. Organic solvent is added to precipitate the solid and separate the solid and liquid. The obtained solid is dissolved in water and dialyzed to obtain SHA. S2. Preparation of sulfonated hyaluronic acid-dextrin solid dispersion: Dextrin and SHA prepared in step S1 are mixed evenly in an organic solvent, the organic solvent is removed by rotary evaporation, and the solid dispersion is obtained by freeze drying. S3. Preparation of needle layer solution: Mix the needle excipient with water evenly to obtain solution A; mix the solid dispersion obtained in step S2 with water evenly to obtain solution B; mix solution A and solution B to obtain needle layer solution; wherein, the amount of each component is as follows according to the mass-volume ratio: needle excipient 5-15%, solid dispersion 5-15%, water is the balance; S4. Preparation of backing layer solution: Mix soluble polymer and water evenly to obtain solution C; then mix it evenly with needle excipient material solution; wherein, the amount of each component is as follows according to the mass-volume ratio: soluble polymer 3-7%, needle excipient material 8-12%, water is the balance; S5. Preparation of microneedles: A. Add the needle body layer solution prepared in step S3 into the microneedle negative mold to fill the microneedle negative mold, scrape off the excess liquid, and dry and solidify to form the microneedle body layer; B. Add the backing layer solution prepared in step S4 to the microneedle body layer in step A to connect the needle body layer with the backing layer. Dry the entire microneedle to obtain soluble microneedles for treating hypertrophic scars.

2. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 1, characterized in that: The hyaluronic acid mentioned in step S1 has a molecular weight of 5000-7000 Da; The ammonium salt mentioned in step S1 is tetrabutylammonium hydroxide; The sulfonating agent mentioned in step S1 is sulfur trioxide-pyridine; The needle shaping material mentioned in step S3 is at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90; The soluble polymer mentioned in step S4 is polyvinyl alcohol.

3. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 1, characterized in that: The inert gas mentioned in step S1 is nitrogen; The alkaline solution mentioned in step S1 is selected from sodium hydroxide solution; The organic solvent mentioned in step S1 is anhydrous ethanol; The organic solvent mentioned in step S2 is an ethanol solution.

4. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 1, characterized in that: The pH value mentioned in step S1 is 8 to 8.5; The SHA and the dextrorotatory borneol mentioned in step S2 are mixed in a mass ratio of 1:1 to 1.1; In step S3, solution A and solution B are mixed in a volume ratio of 0.5 to 1.5:

1. The needle-shaped material solution and the solution C mentioned in step S4 are mixed in a volume ratio of 0.5 to 1.5:

1.

5. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 1, characterized in that: The reaction time described in step S1 is 1 to 2 hours; The dialysis bag specifications mentioned in step S1 are 3000-3500 Da; The dialysis time described in step S1 is 24–48 hours; The conditions for rotary evaporation in step S2 are a temperature of 30–50°C and a rotation speed of 100–150 r / min.

6. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 1, characterized in that: The precipitation process described in step S1 is repeated three times. The solid-liquid separation method described in step S1 is centrifugation; The soluble polymer and water mentioned in step S4 are prepared by the following steps: under heating conditions, the soluble polymer is dissolved in water to obtain an aqueous solution of the soluble polymer; The method of filling the microneedle negative mold as described in step S5A is vacuum treatment; The drying conditions described in step S5A are drying at 25–40 °C until cured; The method of connecting the needle body layer and the backing layer in step S5B is vacuum treatment; The drying conditions described in step S5B are drying at 25–40 °C until cured.

7. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 6, characterized in that: The centrifugation conditions are 6000-10000 r / min for 5-15 min; The vacuum treatment conditions are as follows: vacuuming for 15 to 20 minutes in an environment with a vacuum degree of -0.09 to -0.1 MPa; The drying conditions described in step S5A are drying at a temperature of 25–40 °C for 4–5 h; The drying conditions described in step S5B are 25–40 °C for 18–24 h.

8. A soluble microneedle for treating hypertrophic scars, characterized in that: It is obtained by the preparation method according to any one of claims 1 to 7.

9. The use of the soluble microneedles for treating hypertrophic scars as described in claim 8 in the preparation of a medicament for treating hypertrophic scars.

10. The application of the soluble microneedles for treating hypertrophic scars according to claim 9 in the preparation of a drug for treating hypertrophic scars, characterized in that: The drug is used to inhibit the expression of inflammatory factors and to suppress the excessive proliferation and migration of fibroblasts.

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