Soluble microneedle for treating hyperplastic scars as well as preparation method and application of soluble microneedle

Abstract

The invention belongs to the technical field of biomedicine, and discloses a soluble microneedle for treating hyperplastic scars as well as a preparation method and application of the soluble microneedle. The preparation method comprises the following steps: modifying hyaluronic acid to obtain sulfonated hyaluronic acid, blending the sulfonated hyaluronic acid with a natural penetration enhancer d-borneol with anti-inflammatory activity, carrying out rotary evaporation and freeze-drying to obtain a sulfonated hyaluronic acid-d-borneol solid dispersion, and loading the solid dispersion into a microneedle to obtain the soluble microneedle with the hyperplastic scar treatment function. The preparation method is simple and easy to operate, and large-scale production can be realized. According to the preparation method, the sulfonated hyaluronic acid and the d-borneol are effectively combined, the bioavailability and biocompatibility of the d-borneol are improved, and the d-borneol is loaded into the microneedle, so that the anti-inflammatory and fibroblast migration inhibition capability of the solid dispersion is maximized, and meanwhile, the anti-inflammatory and fibroblast migration inhibition capability of the solid dispersion can be further improved under the dual permeation promotion of the microneedle and the d-borneol. And the aim of deeply delivering the medicine is fulfilled by penetrating dense scar tissues. The problems that in a traditional scar treatment method, medicine permeability is poor, and the treatment effect is limited are solved, a safe and efficient novel transdermal drug delivery system is provided for treating hyperplastic scars, and wide clinical application prospects and market value are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a soluble microneedle for treating hypertrophic scars, and a preparation method and application thereof. Background Art

[0002] Hypertrophic scars (HS), also known as raised scars, occur secondary to surgical trauma and burns and are fibrotic lesions of skin tissue caused by pathological wound healing. Over 100 million people worldwide suffer scars from surgery or trauma, of which 15% suffer from hypertrophic scars. Clinically, these scars present as irregular, raised areas often accompanied by itching and pain. They are associated with a high incidence, complex pathogenesis, and significant treatment challenges. Inhibiting the formation of hypertrophic scars remains a research hotspot.

[0003] Scar formation is divided into three phases: inflammatory, proliferative, and remodeling. During the inflammatory phase, inflammatory cells migrate to the wound surface 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 (TGF-β) causes excessive fibroblast proliferation and migration, which in turn promotes the deposition of a large amount of extracellular matrix (ECM), mainly collagen. During the remodeling phase, the excessive accumulation of ECM is difficult to be absorbed or remodeled by the body in a timely manner, and ECM and HSF form a linking effect, resulting in fibrotic lesions. Therefore, the tissue characteristics of hypertrophic scars are inflammatory cell infiltration and the deposition of large amounts of fibroblasts and extracellular matrix. Therefore, how to reduce the content of inflammatory factors and regulate the excessive proliferation and migration of fibroblasts are important links in inhibiting scar hyperplasia.

[0004] There are currently three common treatment methods used clinically. Oral administration is mainly based on anti-inflammatory drugs, which only relieve symptoms and have a first-pass gastrointestinal effect. Although topical administration can regulate collagen arrangement and reduce pigmentation, the traditional transdermal method has a low drug penetration rate due to the density of scar tissue. Intralesional injection of corticosteroids in combined drug-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 drugs. However, long-term injection of corticosteroids can cause pain, skin atrophy, and pigmentation, and requires doctors to inject multiple times, which is expensive. This treatment method causes negative emotions in patients. Therefore, there is an urgent need for safe and efficient drugs and drug-enhanced delivery systems for the treatment of hypertrophic scars. Summary of the Invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to 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 an application of the above-mentioned soluble microneedles for treating hypertrophic scars.

[0008] The purpose of the present invention is achieved by 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 an ammonium salt solution to obtain a modified fat-soluble hyaluronic acid, then a sulfonating reagent is added to react under inert gas and an ice bath, followed by addition of water to terminate the reaction, then an alkaline solution is added to adjust the pH, an organic solvent is added for precipitation, and solid-liquid separation is performed. The resulting solid is dissolved in water and then dialyzed to obtain SHA;

[0010] S2. Preparation of sulfonated hyaluronic acid-dextrorotatory borneol solid dispersion (SHA-NB-SD): D-borneol (NB) and SHA prepared in step S1 were uniformly mixed in an organic solvent, the organic solvent was removed by rotary evaporation, and the solid dispersion was obtained by freeze-drying;

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

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

[0013] S5. Preparation of microneedles:

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

[0015] B. Add the backing layer solution prepared in step S4 to the microneedle body layer of step A to connect the body layer to the backing layer, and dry the entire microneedle to obtain soluble microneedles for preventing hypertrophic scars.

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

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

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

[0019] The sulfonating agent described 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 in step S1 is selected from sodium hydroxide solution; preferably, the concentration is 4-6% w / v sodium hydroxide solution; more preferably, the concentration is 5% w / v sodium hydroxide solution.

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

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

[0024] The number of precipitations in step S1 is preferably 3 times.

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

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

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

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

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

[0030] The organic solvent 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 rotary evaporation conditions in step S2 are preferably 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 body excipient material described in step S3 is preferably at least one of polyvinyl pyrrolidone K30 and polyvinyl pyrrolidone K90, more preferably polyvinyl pyrrolidone K90.

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

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

[0035] The uniform mixing of the soluble polymer and water in step S4 is preferably prepared by the following steps: dissolving the soluble polymer in water under heating conditions to obtain a soluble polymer aqueous solution.

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

[0037] The needle body excipient solution and the soluble polymer solution in step S4 are mixed in a volume ratio of 0.5 to 1.5:1, preferably in a volume ratio of 1:1.

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

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

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

[0041] The drying conditions in step S5B are preferably drying at 25-40° C. until solidified; 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] Application of the above-mentioned soluble microneedles for treating hypertrophic scars in the preparation of drugs for treating hypertrophic scars.

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

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

[0046] (1) Molecular Engineering: From “Inert Carrier” to “Smart Active Drug”

[0047] A. Functionalized HA modification: Sulfonation is used to give HA strong negative charge, anti-inflammatory targeting and the ability to inhibit excessive migration of HSF, thereby upgrading it from a traditional moisturizing material to an "anti-inflammatory-fibrosis inhibition" dual-functional drug by inhibiting the activation of the TGF-β / NF-κB / MAPK signaling pathway.

[0048] B. Natural drug synergy: SHA's anti-inflammatory and fibroblast migration inhibition effects combined with NB's penetration-promoting and anti-inflammatory effects create a "1+1>2" therapeutic effect, while also realizing the "carrier as drug" design concept. Chemical modification gives the material active therapeutic functions, reducing the addition of exogenous drugs.

[0049] (2) Security breakthrough

[0050] Solid Dispersion Technology: SHA and NB are prepared as a solid dispersion, resolving the industry challenges of NB's poor water solubility and high toxicity, significantly improving its bioavailability. Zebrafish experiments confirm that SHA-NB-SD is virtually non-toxic, outperforming single-dextrorotatory borneol. This is due to the introduction of negatively charged sulfonic acid groups, which strongly bind to receptors on the surface of immune cells and effectively activate and enhance immune responses. Furthermore, the sulfonic acid groups are highly hydrophilic, enhancing interactions between the material and biomolecules, thereby improving its biocompatibility.

[0051] (3) Delivery system: from “single penetration” to “dynamic coordination in time and space”

[0052] A. Penetration optimization: Microneedles directly penetrate dense scar tissue, changing the traditional SHA drug delivery method of smearing and breaking through the bottleneck of low penetration rate of traditional transdermal drug delivery.

[0053] B. Double penetration-enhancing design: The physical penetration of microneedles and the chemical penetration of NB are combined 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; NB has anti-inflammatory activity; the two prepared as a solid dispersion can synergistically exert anti-inflammatory effects. The results of the zebrafish neutrophil migration inhibition experiment also showed that at equivalent concentrations, 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 through significant anti-inflammatory activity, thereby reducing HSF activation. TGF-β is a key factor in HSF activation and proliferation. At the same time, SHA can compete with fibronectin for the TGF-β release site LTBP protein, resulting in reduced TGF-β release, thereby reducing HSF migration and activation.

[0057] C. Microneedle mechanical regulation: When microneedles penetrate the skin, they can cause local irritation, disrupt the order of collagen, and cause rearrangement; at the same time, microneedles can penetrate dense scar tissue to achieve the purpose of deep drug delivery.

[0058] (5) Industrialization and clinical transformation potential

[0059] A. Process standardization: The invention has a clear preparation process, which progresses step by step from raw material modification to the preparation of solid dispersion and then to microneedle molding, making it suitable for large-scale production.

[0060] B. High patient compliance: Microneedles are painless and do not require multiple injections, overcoming the side effects of corticosteroid therapy such as atrophy and pain.

[0061] C. Core breakthrough: innovation in the entire chain from molecular design to delivery system to treatment strategy.

[0062] (6) Sulfonated hyaluronic acid-D-borneol-based soluble microneedles have significant advantages in the treatment of hypertrophic scars, achieving a quadruple breakthrough in "molecular design-safety breakthrough-delivery synergy-pathological closed loop", providing the first transdermal treatment solution for hypertrophic scars that is both efficient, safe and convenient. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0064] Figure 2 3 is the potential diagram of the sulfonated hyaluronic acid prepared in Example 1.

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

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

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

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

[0069] Figure 7 This is a graph showing the test results of investigating the inhibitory effects of different samples on HSF migration ability.

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

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

[0072] Unless otherwise specified, all reagents used in the examples can be purchased from the market.

[0073] Example 1

[0074] The preparation method of sulfonated hyaluronic acid comprises the following specific steps:

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

[0076] Weigh 3 g of IR120 hydrogen form cation exchange resin and place it in a 250 mL conical flask. First soak it in anhydrous ethanol for 2 h, wash it with deionized water until it is neutral, then soak it in 5% w / v sodium hydroxide solution for 2 h, wash it with deionized water until it is neutral, then soak it in 5% w / v hydrochloric acid solution for 2 h, wash it with deionized water until it is neutral, and set aside.

[0077] 1.0 g of HA (MW 6 kDa) was weighed and fully dissolved in 100 mL of deionized water. Then, H-type cation exchange resin IR120 treated according to the above steps was added and stirred at room temperature for 3 h. The mixture was filtered and the pH of the filtrate was adjusted to 7.0 with 25% w / v tetrabutylammonium hydroxide solution. The mixture was stirred at room temperature for 2 h and freeze-dried to obtain the HA hydrophobically modified product (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) and stir until dissolved, and exhaust with nitrogen for 20 minutes to obtain a HA-TBA-DMF solution; weigh 0.9 g of pyridine sulfur trioxide and dissolve it in 20 mL of DMF and stir evenly to prepare an esterification reagent; add the esterification reagent to the HA-TBA-DMF solution under ice bath conditions, react for 1 hour under a nitrogen atmosphere, and then add an equal volume of water to terminate the reaction; adjust the pH value to 7-8.5 with 5% w / v sodium hydroxide solution; add anhydrous ethanol for precipitation three times; centrifuge, remove the supernatant, and place the precipitate in a vacuum drying oven to dry; then add an appropriate amount of deionized water to dissolve, transfer to a 3 kDa dialysis bag for flowing water dialysis; after dialysis in deionized water for 1.5 days, the dialysate in the dialysis bag is freeze-dried and sealed and stored at 4°C to obtain sulfonated hyaluronic acid SHA.

[0080] The SHA was observed by Fourier transform infrared spectroscopy. Figure 1 It can be seen that at 1276cm -1 The stretching vibration absorption peak of S=O double bond appears at 826cm -1 The stretching vibration absorption peak of COS appeared at , which confirmed the sulfonation effect. At the same time, the Zeta Potential was also determined. Figure 2 It can be seen from the Zeta potential that the introduction of SO into HA 3- The groups increased the density of negative charges, where the Zeta potential of HA decreased from -29.47 ± 7.8 mV to -49.70 ± 2.9 mV of SHA, and the decrease in SHA potential indicated that the sulfonation was successful.

[0081] Example 2

[0082] The preparation method of sulfonated hyaluronic acid-D-borneol solid dispersion (SHA-NB-SD) comprises the following steps:

[0083] Weigh equal amounts of SHA and borneol (NB), mix them in a 50% v / v ethanol solution, stir evenly, and rotary evaporate at 40°C and 100 rpm until no more ethanol is dripped. Then stop the rotary evaporation, pour out the solution in the rotary evaporation bottle, and lyophilize to obtain SHA-NB-SD.

[0084] The precipitation of NB after rotary evaporation of 50% ethanol solution was recorded by naked eye and camera. Figure 3 It can be seen that when NB is dissolved in 50% ethanol solution and the ethanol is removed by rotary evaporation, NB is precipitated (part of the precipitated NB is as shown in FIG. Figure 3SHA and NB were dissolved in 50% ethanol and the ethanol was removed by rotary evaporation. No NB precipitated, resulting in a clear, transparent solution. This demonstrates that SHA can physically blend with NB through hydrogen bonding or van der Waals forces to form a solid dispersion, enhancing the bioavailability of NB.

[0085] Example 3

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

[0087] (1) Preparation of needle body layer solution: 20% w / v SHA-NB-SD (solvent is deionized water) and 20% w / v polyvinylpyrrolidone PVP K90 solution (solvent is deionized water) are mixed in a volume ratio of 1:1 to prepare the needle body 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 polyvinyl alcohol (PVA) 1788 solution. A 20% w / v PVP K90 solution and a 10% w / v polyvinyl alcohol (PVA) 1788 solution were mixed in a volume ratio of 1:1 to obtain a backing layer solution.

[0089] (3) Preparation of SHA-NB-SD@DMNs: A two-step method was used to prepare microneedles. The specific method was to precisely pipette 150 μL of the needle body layer solution into the groove of the mold and scrape it to spread it evenly. The microneedle mold was transferred to a vacuum drying oven for degassing for 20 minutes, so that the needle body solution filled the micropores in the microneedle mold, and repeated twice. It was dried at room temperature of 25°C for 5 hours. Then, 200 μL of the backing layer solution was added to the dried whole part to spread it evenly. The microneedle mold was transferred to a vacuum drying oven for degassing for 20 minutes, repeated twice, and dried under the same conditions for 24 hours. After complete drying, SHA-NB-SD@DMNs were demolded and stored in a dry and dark place. The same concentration of sulforhodamine B (SRB) was used as a simulated drug to replace SHA-NB-SD. The operation steps were the same as above and used for subsequent drug distribution law investigation.

[0090] (4) Results: Through inverted fluorescence microscopy, Figure 4As can be seen from the left picture in the figure, the soluble microneedle (DMNs) array under bright field is composed of conical needles with sharp needles. The tip height of the microneedle is about 550μm, the bottom diameter is 200μm, and the tip distance is 600μm. Since the volume of DMNs shrinks during vacuum drying, the size is slightly smaller than that of the microneedle negative film. In addition, it can be seen from the observation under fluorescence that the simulated drug sulfonylrhodamine B (SRB) is evenly distributed in the microneedle needle shaft, which indirectly proves the uniform distribution of the drug in the microneedle needle shaft (such as Figure 4 (as shown in the right figure in the figure).

[0091] Example 4

[0092] The inhibitory effect of sulfonated hyaluronic acid-D-borneol solid dispersion on the CuSO4-induced zebrafish inflammation model was studied as follows:

[0093] SHA-NB-SD was obtained by referring to the preparation methods of Examples 1 and 2, and anti-inflammatory experiments were performed on zebrafish.

[0094] (1) Zebrafish toxicity test

[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. Using a 6-well plate as the experimental carrier, 15-20 embryos were added to each well. The culture solution was then carefully aspirated with a pipette, and 3-4 mL of the pre-prepared sample solution was added to each well. The plate was covered with a lid and wrapped with aluminum foil. The cells were transferred to a 28±0.5°C incubator and cultured in the dark for 96 hpf. Embryonic 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 for HA, SHA, and SHA-NB-SD were prepared directly with water; NB was prepared by first preparing a 100 mg / mL stock solution in DMSO and then diluting it with water.

[0096] Depend on Figure 5As can be seen, at a concentration of 500 μg / mL for HA and NB, zebrafish survival was essentially nonexistent. At a concentration of 1000 μg / mL for SHA and SHA-NB-SD, the survival rates were 93.3% and 91.09%, respectively. Based on the group standard for acute toxicity testing of zebrafish embryos, SHA and SHA-NB-SD were preliminarily determined to be practically nontoxic to zebrafish embryos. This indicates that SHA and SHA-NB-SD exhibited superior biocompatibility compared to HA and NB in zebrafish embryos, increasing NB bioavailability and reducing toxicity. This is due to the introduction of negatively charged sulfonic acid groups, which bind strongly to receptors on the surface of immune cells and effectively activate and enhance immune responses. Furthermore, the sulfonic acid groups are highly hydrophilic, which enhances interactions between the material and biomolecules, thereby improving its biocompatibility.

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

[0098] Normally developed zebrafish embryos at 3 dpf were selected and used as experimental carriers in 6-well plates, with 15 zebrafish placed in each well. The culture medium was carefully aspirated with a pipette, and 3 mL of distilled water was added to each well. Subsequently, 3 μL of CuSO₄ solution was added to each well, except for the blank control group, to establish an inflammation model. The final concentration of the CuSO₄ solution was 10 μmol / L. The plates were covered and wrapped with aluminum foil. The plates were transferred to a 28±0.5°C incubator and incubated in the dark for 30–40 min. After that, the solution was aspirated. The model group was treated with 3 mL of distilled water, and the sample groups (SHA, NB, and SHA-NB-SD) were each treated with 3 mL of sample solution. The cells were incubated under the same conditions for 4–5 hpf. The sample group solution was then aspirated and replaced with 3 mL of distilled water. Three replicates were run for each concentration. Ten zebrafish were randomly selected, mounted on glass slides with 5% methylcellulose, and observed and photographed under an inverted fluorescence microscope. Image J software was used to analyze and record the number of neutrophils.

[0099] Table 1 and Figure 6 Results showed that the number of neutrophils in the neuromast cell region decreased with increasing sample concentration. SHA and SHA-NB-SD showed a more pronounced anti-inflammatory effect, likely due to the introduction of sulfonic acid groups. The negative charge allows for the adsorption of more inflammatory factors, resulting in a better anti-inflammatory effect. Furthermore, at a concentration of 0.5 mg / mL, all zebrafish died, further demonstrating that SHA-NB-SD exhibits superior anti-inflammatory effects compared to SHA.

[0100] Table 1 The number of neutrophils produced by different samples in the CuSO4-induced zebrafish inflammation model (X±SD)

[0101]

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

[0103] Example 5

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

[0105] In the cell scratch test, mouse fibroblast L929 cells were cultured at a rate of 2 × 10 5 Cells were seeded at a density of 10 cells / well in a 6-well plate and incubated in a 37°C, 5% CO2 incubator for 12 hours until the cells formed a confluent monolayer within the wells. Subsequently, a sterile 200 μL pipette tip was used to make a uniform vertical scratch in the center of the cell monolayer with the aid of a ruler. The cells were then washed three times with PBS to remove cells that had fallen off the scratch and ensure 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 stock solution at a concentration of 20 mg / mL in DMSO), and SHA-NB-SD (calculated at a SHA equivalent concentration of 100 μg / mL, which has been converted to a SHA equivalent concentration) were prepared in low-serum MEM medium containing 2% v / v FBS. 2 mL of the prepared culture medium was pipetted into the 6-well plate, and 2 mL of low-serum MEM medium containing 2% v / v FBS was added to the blank group. The cell plates were placed in a 37°C, 5% CO2 incubator and photographed using an inverted fluorescence microscope after 0 and 48 hours to observe cell migration. Finally, the photographs were analyzed using ImageJ software to calculate the spacing between scratches and record changes in scratch width at different time points. Appropriate statistical methods were used for data analysis to determine the effects of different treatments on cell migration.

[0106] From Table 2 and Figure 7 As can be seen in the figure, SHA-NB-SD significantly slows the closure of scratches, indicating that SHA-NB-SD has the highest inhibition rate on HSF migration, followed by SHA. This may be due to SHA's inhibitory effect on the release of TGF-β, which stimulates the release of large amounts of HSF, leading to SHA's effective inhibition of HSF proliferation and migration. SHA-NB-SD also has a synergistic anti-inflammatory effect, possibly inhibiting the expression of inflammatory factors such as IL-1 and TNF-α, thereby preventing the activation of NF-κB and MAPK signaling pathways, thereby inhibiting the proliferation and migration of fibroblasts.

[0107] Table 2 Scratch healing rate of different samples on HSF cells for 48 h (X±SD)

[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 effect of the present invention on treating 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) Experimental animals

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

[0114] 2) Test drug

[0115] Treatment group 1 was administered with SHA-NB-SD@DMNs; treatment group 2 was administered with 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 the SHA solution was water); treatment group 3 was administered with 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 the NB solution was 50% ethanol); treatment group 4 was administered with 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 rabbit ear hypertrophic scar model

[0118] New Zealand rabbits were housed in single cages under standard conditions for one week. They were deprived of food and water for 8 hours before the experiment and then anesthetized with sodium pentobarbital. Once anesthesia was satisfactory, the rabbits were secured on a dedicated laboratory table. Four wounds (1 x 1 cm) were created along the long axis of the mid-ventral part of the rabbit ear, with a distance of >1 cm between the wounds. The wounds were then cleaned with 0.1% benzalkonium chloride. After 7 days of recovery, the newly formed scabs were removed and the wounds were re-exposed. After 21 days, a hypertrophic scar model was successfully established.

[0119] 2) Grouping and microneedle treatment

[0120] Thirty New Zealand rabbits with successful modeling were selected and divided into a control group and a treatment group, with 6 rabbits in each group. The control group did not receive any drug treatment, while treatment groups 1-4 received different types of microneedle 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 to complete the microneedle drug administration treatment. The drug was administered once a week for a total of 3 times. On the 21st day of administration, the scar tissue was sampled and H&E stained and sliced. The H&E stained slices 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 Image J software, 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 of the present application was significantly reduced compared with that of 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 inhibition of excessive proliferation and migration of HSF; at the same time, the microneedles can penetrate the stratum corneum to reach the dermis to deliver drugs, and the loaded anti-inflammatory drug NB also acts as a natural penetration enhancer, allowing the drug to be released "from point to surface". The synergy of the two has a dual penetration-promoting effect, which is more effective in treating hypertrophic scars.

[0124] Table 3 Comparison of the effects of different sample groups on rabbit ear scar hyperplasia index (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 content is only a preferred embodiment of the present invention, but the embodiments of the present invention are not limited to the above examples. For those skilled in the art, without violating the spirit of the present invention, the embodiments of the present invention can be appropriately modified. However, these modifications should not be regarded as limiting the scope of the rights 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 regarded as equivalent alternatives and fall within the scope of protection of the present invention.

Claims

1. A method for preparing soluble microneedles for treating hypertrophic scars, characterized in that The steps include: S1. Preparation of sulfonated hyaluronic acid: first, hyaluronic acid is mixed with an ammonium salt solution to obtain modified fat-soluble hyaluronic acid, then a sulfonating reagent is added to react under inert gas and an ice bath, followed by addition of water to terminate the reaction, and then an alkaline solution is added to adjust the pH value, and an organic solvent is added for precipitation, followed by solid-liquid separation, and the obtained solid is dissolved in water and dialyzed to obtain SHA; S2. Preparation of sulfonated hyaluronic acid-D-borneol solid dispersion: D-borneol and SHA prepared in step S1 were uniformly mixed in an organic solvent, the organic solvent was removed by rotary evaporation, and the solid dispersion was obtained by freeze-drying; S3. Prepare a needle layer solution: uniformly mix the needle excipient and water to obtain solution A; uniformly mix the solid dispersion obtained in step S2 with water to obtain solution B; and mix solution A and solution B to obtain a needle layer solution; wherein the amounts of each component are calculated by mass volume ratio as follows: 5-15% needle excipient, 5-15% solid dispersion, and the balance water; S4. Prepare a backing layer solution: Mix a soluble polymer and water to obtain a solution C; then mix the solution C with the needle-forming material; wherein the amounts of the components are calculated by mass to volume as follows: 3-7% soluble polymer, 8-12% needle-forming material, and the balance water; S5. Preparation of microneedles: A. Add the needle body layer solution prepared in step S3 to the microneedle negative mold to fill the microneedle negative mold, scrape off excess liquid, and dry and solidify to form a microneedle body layer; B. Add the backing layer solution prepared in step S4 to the microneedle body layer of step A to connect the body layer to the backing layer, and dry the entire microneedle to obtain soluble microneedles for preventing hypertrophic scars.

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

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

4. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 1, wherein: The pH value described in step S1 is 8 to 8.5; The SHA and NB described in step S2 are mixed at a mass ratio of 1:1 to 1.1; The needle body excipient solution and the solid dispersion solution described in step S3 are mixed in a volume ratio of 0.5 to 1.5:1; The needle body excipient solution and the soluble polymer solution 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, wherein: The reaction time in step S1 is 1 to 2 hours; The specification of the dialysis bag in the dialysis described in step S1 is 3000-3500Da; The dialysis time in step S1 is 24 to 48 hours; The conditions for the 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, wherein: The number of precipitations in step S1 is 3 times; The solid-liquid separation method in step S1 is centrifugation; The soluble polymer and water are uniformly mixed in step S4 and prepared by the following steps: dissolving the soluble polymer in water under heating conditions to obtain a soluble polymer aqueous solution; The method of filling the microneedle negative mold in step S5A is vacuum treatment; The drying condition in step S5A is drying at 25-40° C. until solidified; The method for connecting the needle body layer and the backing layer in step S5B is vacuum treatment; The drying condition in step S5B is 25-40° C. until solidified.

7. The method for preparing soluble microneedles for treating hypertrophic scars according to claim 6, characterized in that: The centrifugal conditions are 6000-10000 r / min for 5-15 minutes; The vacuum treatment condition is to evacuate the environment at a vacuum degree of -0.09 to -0.1 MPa for 15 to 20 minutes; The drying conditions in step S5A are drying at a temperature of 25-40° C. for 4-5 hours; The drying conditions in step S5B are 25-40° C. for 18-24 hours.

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

9. Use of the soluble microneedle for treating hypertrophic scars according to claim 8 in the preparation of a drug for treating hypertrophic scars.

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

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