Double-drug-loading soluble microneedle patch with function of improving hypertrophic scar and preparation method and application of double-drug-loading soluble microneedle patch

Abstract

The invention discloses a double-drug-loading soluble microneedle patch with a hypertrophic scar improving function as well as a preparation method and application of the double-drug-loading soluble microneedle patch, and belongs to the technical field of medical cosmetology. The microneedle patch comprises a substrate and a needle body arranged on the surface of the substrate, wherein the needle body is mainly prepared from a cyclodextrin inclusion compound, colchicine and a microneedle framework material; the cyclodextrin inclusion compound is prepared from an insoluble drug shikonin and beta-cyclodextrin, and the microneedle framework material is prepared from sodium hyaluronate and polyvinylpyrrolidone. The double-drug-loading soluble microneedle patch has good mechanical strength and can accurately penetrate through scar skin to form a drug delivery channel. Compared with a traditional transdermal patch, the soluble microneedle has the advantages that the transdermal permeation efficiency of the medicine is greatly improved, and compared with traditional injection administration, the soluble microneedle reduces long-term irritation to the skin, reduces the risk of adverse reactions such as infection and bleeding and has a very good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical aesthetics technology, and specifically relates to a dual-drug-loaded soluble microneedle patch with the function of improving hypertrophic scars, its preparation method and application. Background Technology

[0002] Hypertrophic scars (HS) are raised, swollen inflammatory lesions primarily caused by excessive proliferation of fibroblasts and disordered collagen deposition following skin injury, severely impacting patients' appearance and overall well-being. Typically, MMP-1 (matrix metalloproteinase-1) and TIMP (inhibitors of matrix metalloproteinases) participate in the formation of hypertrophic scars by regulating collagen metabolism; an imbalance between these two is one of the core mechanisms of excessive scar fibrosis. Furthermore, in hypertrophic scars, transforming growth factor-β (TGF-β) expression is enhanced, stimulating fibroblast migration, proliferation, and extracellular matrix (ECM) synthesis while inhibiting collagenase production. Among the three different isoforms of TGF-β, TGF-β1 plays a crucial role in scar hyperplasia, exhibiting overexpression and inducing collagen synthesis and accumulation. Meanwhile, TGF-β1 is a key upstream signaling molecule that induces α-SMA expression, and α-SMA is a core marker protein for myofibroblast activation under the influence of TGF-β1. α-SMA can drive myofibroblast activation and play a central role in the contraction, collagen deposition, and abnormal signaling pathways of hypertrophic scars. Therefore, its expression level can serve as a biomarker for scar pathological assessment.

[0003] Traditional treatments for hypertrophic scars include surgical excision, corticosteroid therapy, 5-fluorouracil therapy, laser therapy, and pressure therapy. However, these traditional methods also have many drawbacks. Surgical procedures are highly invasive, have long recovery times, and carry risks such as infection and bleeding; the scar may regrow after surgery, requiring further treatment. Corticosteroid injections are painful and may cause side effects such as pigmentation and local tissue collapse; long-term use of corticosteroids may lead to systemic side effects such as osteoporosis and hypertension. 5-fluorouracil injections are also painful and may cause side effects such as local tissue necrosis and pigmentation; long-term use may have adverse effects on other tissues. Laser therapy may cause a series of adverse reactions, such as edematous erythema and crusting, delayed erythema, pigmentation, and even infection and allergic reactions. Pressure therapy has a relatively low effectiveness rate, only working in 60%–85% of patients. Furthermore, pressure therapy requires specific parameters, such as pressure exceeding the capillary pressure of the skin, but not excessively high, otherwise it may lead to numbness or blistering of the limbs. Prolonged wearing of high-pressure clothing may affect the growth and development of young patients. Therefore, the goal of drug treatment for scars is to establish a convenient, safe, and repeatedly applicable method of drug administration that can easily penetrate scar tissue, achieve effective local drug concentrations, and be repeated. Summary of the Invention

[0004] To overcome the aforementioned shortcomings and deficiencies in existing scar treatment processes, the present invention aims to provide a dual-drug-loaded soluble microneedle patch with the function of improving hypertrophic scars, its preparation method, and its application. On one hand, the dual-drug-loaded soluble microneedle patch of the present invention acts on scar tissue through a biaxial synergistic mechanism of "inhibition of fibrosis signaling (TGF-β1 / α-SMA) - remodeling of collagen metabolism (MMP1 / TIMP)," exhibiting excellent anti-scarring effects. On the other hand, the mechanical strength of the microneedle patch is sufficient to penetrate the skin, forming a drug delivery channel. Compared with traditional transdermal patches, this soluble microneedle significantly improves the transdermal penetration efficiency of drugs; compared with traditional injection administration, this soluble microneedle reduces long-term skin irritation and lowers the risk of adverse reactions such as infection and bleeding.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a dual-drug-loaded soluble microneedle patch with the function of improving hypertrophic scars, comprising the following steps: (1) Shikonin ethanol solution was added to β-cyclodextrin aqueous solution at a molar ratio of 2:1 to 1:10, stirred to carry out inclusion reaction, ethanol was evaporated, and freeze-dried to obtain shikonin inclusion complex; (2) Add hyaluronic acid and polyvinylpyrrolidone to water in a mass ratio of 1:1 to 1:20, stir to dissolve, let stand to remove bubbles, and obtain a matrix material solution; (3) Add the shikonin inclusion complex and colchicine obtained in step (1) to the matrix material solution obtained in step (2), stir evenly, and obtain the needle solution; (4) Under vacuum conditions, the needle solution obtained in step (3) is filled into the microneedle mold so that the needle solution completely fills the needle tip part and the backing part of the mold, solidifies, and demolds to obtain a microneedle patch.

[0006] Based on the above technical solution, further, in step (1), the molar ratio of shikonin and β-cyclodextrin is 1:1 to 1:5.

[0007] Based on the above technical solution, further, the concentration of the shikonin ethanol solution in step (1) is 0.1~0.5 mol / L; the concentration of the β-cyclodextrin aqueous solution is 0.2~1 mol / L.

[0008] Based on the above technical solution, further, the β-cyclodextrin in step (1) is at least one of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and sulfopropyl ether-β-cyclodextrin.

[0009] Based on the above technical solution, in step (1), after adding the shikonin ethanol solution to the β-cyclodextrin aqueous solution, an additional 0.5 to 2 times the total volume of the mixed solution is added to water.

[0010] Based on the above technical solution, further, in step (1), the inclusion reaction temperature is 40~70℃ and the inclusion reaction time is 0.5~4h.

[0011] Based on the above technical solution, further, in step (2), the mass ratio of hyaluronic acid and polyvinylpyrrolidone is 1:2 to 1:6, the molecular weight of hyaluronic acid is 10KDa to 1000KDa, and the polyvinylpyrrolidone is at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90.

[0012] Based on the above technical solution, further, the mass percentage of hyaluronic acid in the matrix material solution in step (2) is 1~5%.

[0013] Based on the above technical solution, further, the mass percentage of shikonin in the needle solution in step (3) is 0.005~0.1%, and the mass percentage of colchicine is 0.05~0.3%.

[0014] Based on the above technical solution, further, the vacuum pressure in step (4) is -0.07 MPa to -0.09 MPa; the curing temperature is room temperature, and the curing time is 4 to 12 hours.

[0015] Secondly, the present invention provides a dual-drug-loaded soluble microneedle patch prepared by the above preparation method.

[0016] Thirdly, the present invention provides the application of the above-mentioned dual-drug-loaded soluble microneedle patch in the preparation of a drug for treating and improving hypertrophic scars.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention prepares a poorly soluble drug, shikonin, into an inclusion complex with a specific type of cyclodextrin in a certain ratio. This complex is then combined with a specific type of microneedle matrix material in a certain ratio to prepare soluble microneedles with good mechanical properties and good transdermal absorption. The preparation of the drug into a cyclodextrin inclusion complex overcomes the problems of low drug loading and poor transdermal absorption caused by the low solubility of the poorly soluble anti-scarring drug shikonin. By selecting a suitable type of cyclodextrin and combining it with a specific type of microneedle matrix material, the prepared microneedles possess good mechanical strength while effectively improving the drug loading and transdermal delivery efficiency, thereby enhancing the bioavailability of the drug.

[0018] 2. The soluble microneedles containing poorly soluble anti-scarring drugs of the present invention can form a large number of micron-sized mechanical channels on the skin surface, through which the drug can penetrate into the skin; after the microneedles are inserted into the skin, the needle tip dissolves, and the loaded anti-scarring drug penetrates into the skin through the mechanical channels. This can deliver a large amount of drug transdermally in a short time, be absorbed into the blood by capillaries to exert its effect, and greatly improve the transdermal penetration efficiency of the drug.

[0019] 3. The present invention relates to a dual-drug-loaded soluble microneedle patch for improving hypertrophic scars, which combines shikonin and colchicine. It addresses different mechanisms of scar formation: shikonin disrupts the positive feedback loop of "TGF-β1 → α-SMA → myofibroblast activation → collagen deposition" in hypertrophic scars by doubly inhibiting TGF-β1 signaling and α-SMA expression; colchicine regulates collagen metabolism through a dual mechanism: on the one hand, it upregulates MMP-1 expression and activity, enhancing collagen degradation; on the other hand, it inhibits TIMP-1 synthesis and function, relieving the inhibition of MMP. This "bidirectional regulation" reshapes the imbalanced MMP1 / TIMP1 axis in hypertrophic scars. The combined use of shikonin and colchicine, through a dual-axis synergistic mechanism of "inhibiting fibrosis signals (TGF-β1 / α-SMA) - reshaping collagen metabolism (MMP1 / TIMP1)," demonstrates superior efficacy compared to single-drug therapy in the treatment of hypertrophic scars. Its core advantages lie in: 1) multi-target complementary regulation, simultaneously inhibiting collagen synthesis and promoting degradation; 2) cross-blocking of signaling pathways, reducing resistance to single targets; and 3) dose-synergistic effect, reducing single-drug toxicity. Furthermore, biodegradable natural polymers such as hyaluronic acid inhibit fibroblast proliferation, and abnormal fibroblast proliferation is the main mechanism of pathological scar progression. Therefore, using these raw material components as the matrix material for microneedles to carry scar repair drugs can exert a combined therapeutic effect. Simultaneously, the aforementioned matrix is ​​a biodegradable material with good biosafety, can be digested and absorbed by tissues, and is harmless to the human body.

[0020] 4. The preparation conditions of the dual-drug-loaded microneedle patch of the present invention are simple and the process is easy to control, which is conducive to the large-scale production of microneedles. Attached Figure Description

[0021] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0022] Figure 1 The graph shows the inclusion rate of the shikonin inclusion complexes prepared under different conditions in Example 1. In the graph, A represents the effect of the molar ratio of hydroxypropyl-β-cyclodextrin and shikonin on the inclusion rate, B represents the effect of inclusion time on the inclusion rate, C represents the effect of inclusion temperature on the inclusion rate, and D represents the effect of water addition on the inclusion rate.

[0023] Figure 2 Microscopic image (A) and scanning electron microscopy image (B) of the dual-drug-loaded soluble microneedles prepared in Example 2.

[0024] Figure 3 This is a confocal microscope image of the effective depth of transdermal transmission of the dual-drug-loaded soluble microneedles in Example 4.

[0025] Figure 4The image shows the results of the in vitro transdermal experiments of shikonin colchicine microneedles, shikonin inclusion complex colchicine microneedles, and shikonin in PBS buffer in Example 5.

[0026] Figure 5 This image shows the in vitro transdermal test results of shikonin and colchicine in the colchicine microneedles containing the shikonin inclusion complex in Example 5.

[0027] Figure 6 This is a Western blot diagram showing the protein expression bands of TGF-β1, α-SMA, MMP1, and TIMP in each experimental group as shown in Example 6.

[0028] Figure 7 The bar chart shows the relative protein expression levels of TGF-β1 (A), α-SMA (B), MMP1 (C), and TIMP (D) in each experimental group as detected by Western blot in Example 6.

[0029] Figure 8 This is a comparison of the rabbit ear hypertrophic scar models before and after treatment in each group of experiments in Example 7.

[0030] Figure 9 This is a comparison of the results of rabbit ear hypertrophic scars before (A) and after (B) treatment in the colchicine microneedling treatment group of the shikonin inclusion complex in Example 7. Detailed Implementation

[0031] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0032] Example 1 Single-factor investigation experiment of shikonin inclusion complex (1) Preparation of Shikonin Inclusion Complex: First, 5 mg of shikonin (SHI) was dissolved in 50 mL of ethanol. Then, 46.24 mg of hydroxypropyl-β-cyclodextrin (HP-β-CD) was dissolved in 50 mL of distilled water and heated to a certain temperature. The SHI ethanol solution was slowly added to the HP-β-CD solution according to a certain molar ratio. Then, a certain amount of water was added. The mixture was kept at a constant temperature and magnetically stirred for a period of time to carry out the inclusion reaction. After the inclusion reaction was completed, the inclusion complex solution was left to stand overnight to evaporate the ethanol. The mixture was filtered twice through a 0.45 μm filter membrane and freeze-dried to obtain the shikonin inclusion complex.

[0033] (2) Single-factor investigation experiment of inclusion complex A. Molar ratio: Prepare SHI / HP-β-CD inclusion solutions according to the above process. Transfer 2.88 ml of SHI ethanol solution (0.001 mmol / 2.88 ml). The molar ratio of HP-β-CD to SHI is 1:2, 1:1, 2:1, 3:1, and 5:1, respectively. Other parameters are set as follows: inclusion time is 2 h, inclusion temperature is 60℃, and an additional 10 ml of water is added. Each experiment is repeated 3 times. The concentration of SHI is determined by ultraviolet spectrophotometry, the inclusion rate is calculated, and the effect of the feed ratio on the inclusion rate is investigated.

[0034] B. Inclusion Time: The SHI / HP-β-CD inclusion solution was prepared according to the above process. 2.88 ml of SHI ethanol solution (0.001 mmol / 2.88 ml) was transferred, and the inclusion times were set to 0.5 h, 1 h, 2 h, 3 h, and 4 h, respectively. Other parameters were set as follows: inclusion temperature 60℃, HP-β-CD to SHI molar ratio 3:1, and 10 ml of additional water added. Each experiment was repeated three times. The SHI concentration was determined by UV spectrophotometry, and the inclusion rate was calculated to investigate the effect of inclusion time on the inclusion rate.

[0035] C. Inclusion Temperature: The SHI / HP-β-CD inclusion solution was prepared according to the above process. 2.88 ml of SHI ethanol solution (0.001 mmol / 2.88 ml) was transferred. The inclusion temperatures were set at 30℃, 40℃, 50℃, 60℃, and 70℃, respectively. Other parameters were set as follows: inclusion time 2 h, HP-β-CD to SHI molar ratio 3:1, and 10 ml of additional water added. Each experiment was repeated three times. The SHI concentration was determined by UV spectrophotometry, and the inclusion rate was calculated to investigate the effect of temperature on the inclusion rate.

[0036] D. Additional Water Addition: The SHI / HP-β-CD inclusion solution was prepared according to the above process. 2.88 ml of SHI ethanol solution (0.001 mmol / 2.88 ml) was transferred. Additional water addition was set at 5 ml, 7.5 ml, 10 ml, and 12.5 ml. Other parameters were set as follows: inclusion time 2 h, inclusion temperature 60℃, and HP-β-CD to SHI molar ratio 3:1. Each experiment was repeated three times. The SHI concentration was determined by UV spectrophotometry, and the inclusion rate was calculated to investigate the effect of additional water addition on the inclusion rate.

[0037] Example 2 This embodiment provides a method for preparing a dual-drug-loaded soluble microneedle patch with the function of improving hypertrophic scars, including the following steps: (1) Preparation of shikonin inclusion complex: Dissolve 5 mg of shikonin in 50 ml of ethanol for later use (SHI solution), and then take 2.88 ml of SHI solution (0.001 mmol / 2.88 ml) for each subsequent experiment.

[0038] Add 46.24 mg of hydroxypropyl-β-cyclodextrin to pure water and bring the volume to 50 ml. Then take 5 ml of HP-β-CD solution (0.003 mmol / 5 ml) for each subsequent experiment.

[0039] Transfer 5 ml of HP-β-CD solution to a reagent bottle, add 2.88 ml of SHI solution at 60℃, with a molar ratio of shikonin to hydroxypropyl-β-cyclodextrin of 1:3, and add 10 ml of pure water. Stir magnetically at constant temperature for 2 h to carry out the inclusion reaction. Let stand overnight at room temperature to evaporate the ethanol solvent, filter twice through a 0.45 μm filter membrane, and freeze-dry to obtain the shikonin inclusion complex for later use. (2) Preparation of matrix material: Weigh 60 mg of hyaluronic acid with a molecular weight of 20 KDa~40 KDa and 240 mg of polyvinylpyrrolidone K30 and dissolve them in 2 ml of pure water. Stir to dissolve and let stand for 1 h to remove bubbles to obtain blank microneedle matrix material solution. (3) Preparation of needle solution: The above-obtained shikonin inclusion complex was added to the obtained matrix material solution, and 2 mg of colchicine was added. The mixture was stirred and dissolved to obtain the needle solution. (4) Preparation of blank microneedle patches Take 200 μL of the blank microneedle matrix material solution prepared according to the above method and inject it into the polydimethylsiloxane microneedle mold. Then, place the mold in a vacuum desiccator and maintain a negative pressure of -0.085 MPa for 5 minutes. The negative pressure drives the solution to completely fill the micron-scale needle tip cavity of the mold. To completely eliminate any air bubbles that may be trapped during the molding process, after releasing the vacuum, systematically remove air bubbles from the mold surface using a micropipette and replenish with an equal volume of solution. This step is repeated twice. After filling, allow the mold to dry at room temperature and ambient humidity for 8 hours. Once the matrix material has completely solidified, demold using tweezers to obtain the blank microneedle patch (the microneedle patch includes the microneedle portion and the backing portion).

[0040] (5) Preparation of drug-loaded microneedle patches For dual-drug-loaded microneedles, the needle solution prepared in step (3) is magnetically stirred until all components are completely dissolved to form a uniform drug-loaded matrix mixture solution. The subsequent mold filling, vacuum treatment, bubble removal and room temperature curing processes are consistent with the preparation process of blank microneedles.

[0041] For the shikonin-colchicine drug microneedles (control), shikonin was not included in the preparation process; only an equal amount of shikonin was added, and the other steps were the same as described above.

[0042] Microscopic and scanning electron microscope images of the dual-drug-loaded soluble microneedles prepared in this embodiment are shown below. Figure 2 As shown.

[0043] Example 3 Determination of needle tip utilization rate of dual-drug-loaded soluble microneedle patches First, the drug content of the whole microneedle was determined. The whole microneedle was dissolved in water, and the absorbance of shikonin and colchicine was measured at 515 nm and 243 nm using a UV spectrophotometer. The content was calculated and recorded as D_whole_needle (μg). The tips of different types of microneedles were carefully scraped off with a blade, and the remaining backing was dissolved in water to determine the drug content of the backing, recorded as D_backing (μg). Finally, the drug utilization rate of the microneedle tip was calculated according to the following formula: Needle tip drug utilization rate % = ×100% The results are shown in Table 1-2.

[0044] Table 1. Shikonin content of the dual-drug-loaded soluble microneedle patch in Example 1

[0045] Table 2 Colchicine content of the dual-drug-loaded soluble microneedle patch in Example 1

[0046] The results showed that the utilization rate of shikonin at the tip of the microneedle, specifically the hydroxypropyl shikonin inclusion complex-colchicine dual-loaded drug, was higher. β Cyclodextrin showed good solubilizing effect on shikonin; the colchicine utilization rate of the two dual-drug-loaded microneedles was comparable.

[0047] Example 4 In vitro effective transdermal depth experiment of the dual drug-loaded soluble microneedle patch prepared in Example 2 Since pig skin has a structure similar to human skin, microneedle patches loaded with rhodamine 123 (Rh123) were inserted into pig skin (with shikonin replaced by rhodamine 123, and the other preparation process was exactly the same as in Example 2) to evaluate transdermal delivery capability. To observe the distribution and penetration of the drug in the local skin, fluorescence microscopy images were taken at different depths perpendicular to the skin surface using a confocal laser scanning microscope (CLSM).

[0048] Prepare a piece of fresh pigskin, remove surface hair, wash, place on a glass slide, and dry. Insert a soluble microneedle loaded with Rhodamine 123 vertically into the pigskin, press for 5 minutes, and then remove it. A regularly arranged matrix of holes appears on the pigskin surface. After the microneedles dissolve, Rhodamine 123 diffuses within the tissue. Observation using a laser confocal microscope reveals the formation of green hollow circles around the holes. This experimental result demonstrates that the drug-loaded tip of the microneedle can penetrate the skin and dissolve, delivering the loaded drug into the tissue. After determining the xy-plane with the highest and lowest fluorescence intensities in CLSM, fluorescence images were obtained from the xy-plane, with a scan interval of 20 μm along the z-axis to show the diffusion of the dye in the skin.

[0049] Experimental results are as follows Figure 3 As shown, the effective transdermal depth of microneedles can reach 300 µm, achieving effective tissue penetration.

[0050] Example 5 This embodiment uses a modified Franz diffusion cell to conduct microneedle in vitro drug transdermal experiments. (1) In vitro transdermal experiments were conducted using the shikonin-colchicine microneedles (SHI-COL-MN) prepared in Example 2, the shikonin inclusion complex colchicine microneedles (HP-β-CD(SHI)-COL-MN) prepared in Example 2, and PBS buffer (pH 7.4) containing an equal amount of shikonin to determine the transdermal efficiency of different forms of shikonin. A modified Franz apparatus (receiving cell volume of 3 mL, effective contact area of ​​the receiving cell of 0.28 cm²) was used. 2 To conduct experiments.

[0051] The colchicine microneedles, the shikonin inclusion complex prepared in Example 2, were pressed onto isolated mouse skin (stratum corneum) with a force of 50 N and held for 30 s. The skin with the microneedles was then fixed between the two halves of the diffusion chamber, with the stratum corneum facing the supply chamber. Pre-warmed (37±0.5℃) pH 7.4 PBS solution was added to the receiving chamber. The chamber was then placed in a constant temperature shaking incubator at (37±0.5℃). At different time points, 1 mL of the receiving solution was collected, and the same volume of pre-warmed (37℃) pH 7.4 PBS solution was immediately added. The absorbance of the receiving solution was measured at 515 nm and 243 nm using a UV spectrophotometer, and the transdermal dissolution curve of the drug was plotted.

[0052] The in vitro transdermal drug delivery process of the shikonin-colchicine microneedles is the same as that of the shikonin inclusion complex colchicine microneedles prepared in Example 2 above.

[0053] The in vitro transdermal drug transdermal assay for shikonin with PBS buffer was performed as follows: Isolated mouse skin was fixed between the two halves of a diffusion cell, with the stratum corneum facing the donor cell. Shikonin-PBS buffer was added to the donor cell, and pre-warmed (37±0.5℃) pH 7.4 PBS solution was added to the receiver cell. The cell was then placed in a constant-temperature shaking incubator at (37±0.5℃). At different time points, 1 mL of the receiver solution was collected, and the same volume of pre-warmed (37℃) pH 7.4 PBS solution was immediately added. The absorbance of the receiver solutions for shikonin and colchicine was measured at 515 nm and 243 nm using a UV spectrophotometer, and transdermal dissolution curves were plotted.

[0054] The results are as follows Figure 4 As shown, the cumulative permeability Qt (μg / cm²) of skin per unit area at each time point is calculated according to the following formula. 2 ), where Ci is the COL content (μg / mL) measured at the i-th time point, V is the volume of the receiving cell (approximately 3 mL), and S is the surface area of ​​the drug delivery cell (0.28 cm). 2 Plot the Qt-t curve for in vitro percutaneous infiltration, and perform linear fitting on the straight line segment. The resulting slope is the steady-state infiltration rate Jst [μg / (cm²)]. 2 The release behavior of transdermal drug delivery is generally considered to be passive diffusion, which can be represented by Fix's diffusion law, and Jst is the diffusion rate after the drug diffusion has stabilized.

[0055]

[0056] The measurement results were plotted as a Qt-t curve, and the transdermal release parameters of shikonin were calculated. The specific results are shown in Table 3.

[0057] Table 3. Transdermal release parameters of shikonin

[0058] The results above indicate that the transdermal efficiency of the colchicine microneedles containing shikonin is significantly higher than that of shikonin-colchicine microneedles and shikonin buffer solution. Preparing shikonin into a cyclodextrin inclusion complex significantly improves the solubility of this poorly soluble drug. This cyclodextrin inclusion complex overcomes the problems of low drug loading and poor transdermal absorption caused by the low solubility of the poorly soluble anti-scarring drug shikonin. Hydroxypropyl-β-cyclodextrin, through its unique cage-like structure, encapsulates the shikonin drug molecule, resulting in high dispersibility. Furthermore, the hydrophilicity of the external polyhydroxyl groups of cyclodextrin provides excellent wettability, thus achieving a solubilizing effect on poorly soluble drugs. This improves the solubility and dissolution rate, thereby enhancing the transdermal efficiency of shikonin. Cyclodextrin inclusion complexes can also alter the lipid barrier at the drug absorption site, thereby enhancing drug absorption. In vitro transdermal experiments showed that the initial transdermal rate and cumulative transdermal volume of inclusion complex microneedles were higher than those of ordinary microneedles or drug suspensions, and the time delay was shorter.

[0059] (2) Simultaneously, an in vitro transdermal experiment was conducted on the shikonin inclusion complex and colchicine dual-drug microneedles prepared in Example 2 to determine the in vitro transdermal properties of shikonin and colchicine in the dual-drug microneedles. The specific operation followed the method described above. The measurement results were plotted as a Qt-t curve, and the transdermal release parameters were calculated. Specific results are shown in Table 4 and... Figure 5 As shown.

[0060] Table 4. Transdermal release parameters of shikonin and colchicine

[0061] The results above show that in the in vitro transdermal drug release experiment of colchicine-containing microneedles with shikonin inclusion complex, the cumulative unit permeability of both SHI and COL increased over time, indicating that both drugs can be released transdermally through microneedles. At the same time point, the cumulative unit permeability of SHI was higher than that of COL. SHI exists in the form of a cyclodextrin inclusion complex, which may have altered its physicochemical properties (such as solubility and dispersibility), making it easier to release from microneedles and penetrate the skin, thus exhibiting a faster and greater transdermal release effect than COL.

[0062] Example 6 In this embodiment, the expression levels of TGF-β1, α-SMA, MMP1, and TIMP proteins were detected by Western blot. The experiment was divided into a Control group and a TGF-β1 stimulation group, and SHI MN, COL MN, and SHI-COL MN were prepared according to the preparation method in Example 2. SHI MN (containing only SHI), COL MN (containing only COL), and SHI-COL MN (containing both SHI inclusion complex and COL) were prepared in DMEM medium with effective ingredient concentrations of 2.4 μg / ml SHI, 14.4 μg / ml COL, and 2.4 μg / ml SHI + 14.4 μg / ml COL, respectively, for later use.

[0063] Human skin fibroblasts (HSF) were used as cell samples and treated as follows: Control group: conventional culture in DMEM medium containing 15% fetal bovine serum for 6 h; TGF-β1 stimulation group: culture in DMEM medium containing 15% fetal bovine serum and 10 ng / mL recombinant human TGF-β1 for 6 h; SHI group: culture in medium containing 15% fetal bovine serum and 2.4 μg / mL SHI for 6 h; COL group: culture in medium containing 15% fetal bovine serum and 14.4 μg / mL COL for 6 h; SHI-COL group: culture in medium containing 15% fetal bovine serum, 2.4 μg / mL SHI and 14.4 μg / mL COL for 6 h. Each group was set up with three biological replicates.

[0064] The specific procedure for detecting the expression levels of TGF-β1, α-SMA, MMP1, and TIMP proteins using Western blot is as follows: Main reagents: Protein extraction reagent: RIPA lysis buffer; Protein quantification reagent: BCA protein quantification kit; Electrophoresis reagent: 10% SDS-PAGE precast gel, Tris-glycine electrophoresis buffer; Transfer reagent: 0.45 μm PVDF membrane, transfer buffer; Blocking buffer: 5% skim milk powder in TBST solution (containing 0.1% Tween-20); Primary antibodies: anti-TGF-β1 monoclonal antibody, anti-α-SMA monoclonal antibody, anti-MMP1 monoclonal antibody, anti-β-actin monoclonal antibody; Secondary antibody: HRP-labeled goat anti-rabbit IgG; Chromogenic reagent: ECL ultrasensitive luminescence kit.

[0065] Protein sample preparation: After cell culture in each group was completed, the culture medium was discarded, and the cells were washed three times with pre-cooled PBS buffer (5 min each time, 4℃). 200 μL of RIPA lysis buffer was added to each well, and the cells were incubated on ice for 30 min (vortexed once every 10 min). The cells were centrifuged at 12,000 rpm for 15 min at 4℃, and the supernatant was transferred to a new EP tube. The protein concentration was determined by the BCA method, and the protein concentration of each group was adjusted to 2 μg / μL with lysis buffer. 5× loading buffer was added at a 4:1 volume ratio, and the cells were denatured by boiling at 100℃ for 10 min and stored at -80℃ for later use.

[0066] SDS-PAGE electrophoresis: Remove the pre-made gel and install it in the electrophoresis tank. Add Tris-glycine electrophoresis buffer to submerge the sample wells. Load 20 μg of protein sample (10 μL) into each well. Add 5 μL of protein marker to the left sample well. Set the electrophoresis parameters as follows: 80 V constant voltage electrophoresis for 30 min (stack gel stage), 120 V constant voltage electrophoresis for 90 min (separating gel stage), until the bromophenol blue indicator migrates to 1 cm from the bottom of the gel.

[0067] Transfer operation: The PVDF membrane was activated by soaking in methanol for 10 seconds. It was then assembled with the gel and filter paper in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge" (avoiding air bubbles). The transfer buffer was pre-cooled to 4°C. The transfer conditions were set as follows: 200mA constant current and transfer time of 90 min. After the transfer, the membrane was washed three times with TBST buffer (5 min each time at room temperature).

[0068] Blocking and antibody incubation: Completely immerse the PVDF membrane in 5% skim milk powder TBST solution and block it on a shaker (60 rpm) at room temperature for 2 h; discard the blocking solution and wash three times with TBST (5 min each time); cut the membrane according to the molecular weight of the target protein, add the corresponding primary antibody dilution solution (1% BSA TBST), and incubate overnight on a shaker (40 rpm) at 4°C; recover the primary antibody the next day and wash four times with TBST (10 min each time); add HRP-labeled secondary antibody dilution solution and incubate on a shaker (60 rpm) at room temperature for 1 h; wash four times with TBST (10 min each time).

[0069] Color rendering and imaging: ECL luminescent solution A and solution B were mixed in a 1:1 volume ratio and uniformly dropped onto the membrane surface; the ChemiDoc MP imaging system was used for exposure, automatically adjusting the exposure time according to the band brightness to acquire and save clear band images. The results are as follows: Figure 6 .

[0070] ImageJ software was used to quantitatively analyze the gray values ​​of the bands. β-actin was used as the internal reference protein. The ratio of the gray value of each target protein band to the gray value of the corresponding internal reference band was calculated as the relative protein expression level (normalized with the expression level of the control group as 1.00). The results are as follows: Figure 7 See Table 5.

[0071] Table 5. Relative expression levels of target proteins in HSF cells of each group

[0072] TGF-β1 stimulation can significantly alter the expression pattern of hypertrophic scar-related proteins in HSF cells, namely upregulating the expression of TGF-β1, α-SMA, and TIMP while downregulating MMP1 expression. SHI and COL, alone or in combination, can reverse this effect. The combination of SHI and COL has a better regulatory effect on protein expression, indicating that SHI and COL have a synergistic effect in improving the expression of hypertrophic scar-related proteins. Example 7 Establishment and treatment of a rabbit ear hypertrophic scar model Experimental Methods: Japanese white rabbits, weighing 3-3.5 kg, were used. No other medications were administered prior to the experiment. Anesthesia was achieved via intravenous injection of 20% urethane. After anesthesia, the rabbits were fixed in a supine position on the operating table. The ears were disinfected with povidone-iodine and then deiodized with 75% ethanol. A circular full-thickness skin defect, measuring 6 mm × 6 mm, was created on the ventral side of each ear, avoiding visible blood vessels. The entire skin layer was removed, and the perichondrium of the ear was scraped clean with a scalpel to expose the cartilage surface. Four wounds were created per ear, spaced at least 1 cm apart. Hemostasis was achieved by applying pressure with cotton balls, and the wounds were disinfected and left exposed to heal naturally. Seven days post-injury, the animals were anesthetized again intravenously. The ears were disinfected with povidone-iodine, and newly formed scabs were removed with a scalpel and sterile forceps. The wounds were then disinfected with benzalkonium chloride solution and left to heal spontaneously. Twenty-one days after the full-thickness skin excision, hypertrophic scars formed on each wound. All rabbits were randomly divided into 5 groups (n=3): hypertrophic scar group (HS), drug-free microneedling group, colchicine microneedling treatment group (COL MNs), shikonin microneedling treatment group (SHI MNs), and shikonin inclusion complex-colchicine dual-load microneedling treatment group (SHI-COL MNs). Treatment was administered on days 21, 28, 35, and 42 post-surgery, once weekly for 4 consecutive weeks. In the shikonin inclusion complex-colchicine dual-load microneedling treatment group, one SHI-COL MN tablet was used per hypertrophic scar site, containing a total of 0.023 mg of SHI and 0.18 mg of COL. In the colchicine microneedling treatment group, one COL MN tablet was used per hypertrophic scar site, containing 0.18 mg of COL. In the shikonin microneedling treatment group, one SHI MN tablet was used per hypertrophic scar site, containing 0.023 mg of SHI. The HS group received no treatment. Day 21 after surgery is considered before administration, day 28 (one week after the first administration) is considered the first treatment, day 35 (one week after the second administration) is considered the second treatment, day 42 (one week after the third administration) is considered the third treatment, and day 49 (one week after the fourth administration) is considered the fourth treatment.

[0073] Treatment results as follows Figure 8-9 ( Figure 9 A represents the result before SHI-COL MN treatment. Figure 9(B represents the area after SHI-COL MN treatment). After a period of treatment, the rabbit ear scar area treated with microneedles (SHI-COL MNs) in the dual-drug-loaded group showed significant improvement in appearance compared to other control groups: the overall color of the scar was significantly lighter, changing from the typical dark red and purplish-red of the control group to light pink or close to the skin color of normal skin, with uniform color and no obvious pigmentation; the scar thickness was significantly thinner, the degree of elevation was greatly reduced, and some areas were almost flat, with a significant reduction in the height difference with the surrounding normal skin; the texture was significantly softer, without the hardness and toughness of the control group, and the elasticity was enhanced, allowing for slight deformation with rabbit ear movement; the scar edge contour became blurred and softer, no longer as clearly defined and raised as the control group, with a natural transition to the surrounding normal skin tissue, improved surface smoothness, no obvious rough granular feel or epidermal hyperplasia protrusion, and the overall appearance was closer to the normal skin morphology.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a dual-drug-loaded soluble microneedle patch with the function of improving hypertrophic scars, characterized in that, Includes the following steps: (1) Shikonin ethanol solution was added to β-cyclodextrin aqueous solution at a molar ratio of 2:1 to 1:10, stirred to carry out inclusion reaction, ethanol was evaporated, and freeze-dried to obtain shikonin inclusion complex; (2) Add hyaluronic acid and polyvinylpyrrolidone to water in a mass ratio of 1:1 to 1:20, stir to dissolve, let stand to remove bubbles, and obtain a matrix material solution; (3) Add the shikonin inclusion complex and colchicine obtained in step (1) to the matrix material solution obtained in step (2), stir evenly, and obtain the needle solution; (4) Under vacuum conditions, the needle solution obtained in step (3) is filled into the microneedle mold so that the needle solution completely fills the needle tip part and the backing part of the mold, solidifies, and demolds to obtain a microneedle patch.

2. The preparation method according to claim 1, characterized in that, In step (1), the molar ratio of shikonin and β-cyclodextrin is 1:1 to 1:5; the concentration of the shikonin ethanol solution is 0.1 to 0.5 mol / L; and the concentration of the β-cyclodextrin aqueous solution is 0.2 to 1 mol / L.

3. The preparation method according to claim 1, characterized in that, The β-cyclodextrin mentioned in step (1) is at least one of hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and sulfopropyl ether-β-cyclodextrin.

4. The preparation method according to claim 1, characterized in that, In step (1), after adding the shikonin ethanol solution to the β-cyclodextrin aqueous solution, an additional 0.5 to 2 times the total volume of the mixed solution is added to water.

5. The preparation method according to claim 1, characterized in that, In step (1), the inclusion reaction temperature is 40~70℃ and the inclusion reaction time is 0.5~4h.

6. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of hyaluronic acid to polyvinylpyrrolidone is 1:2 to 1:6, the molecular weight of hyaluronic acid is 10 kDa to 1000 kDa, and the polyvinylpyrrolidone is at least one of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K90; the mass percentage of hyaluronic acid in the matrix material solution is 1 to 5%.

7. The preparation method according to claim 1, characterized in that, The mass percentage of shikonin in the needle solution mentioned in step (3) is 0.005~0.1%, and the mass percentage of colchicine is 0.05~0.3%.

8. The preparation method according to claim 1, characterized in that, The vacuum pressure mentioned in step (4) is -0.07 MPa to -0.09 MPa; the curing temperature is room temperature, and the curing time is 4 to 12 hours.

9. The drug-loaded soluble microneedle patch prepared by the preparation method according to any one of claims 1-8.

10. The use of the dual-drug-loaded soluble microneedle patch according to claim 9 in the preparation of a medicament for treating and improving hypertrophic scars.

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