Soluble microneedle patch based on laminarin and its preparation method

Abstract

This invention belongs to the field of pharmaceutical formulation and transdermal drug delivery technology, specifically relating to a drug-excipient soluble microneedle patch based on laminarin and its preparation method. The drug-excipient soluble microneedle patch uses laminarin as the supporting material for the microneedle structure and a pharmacologically active therapeutic agent. The molecular weight of the laminarin is 5-30 kDa. The drug-excipient soluble microneedle patch also includes an active pharmaceutical ingredient; the mass ratio of the active pharmaceutical ingredient to the laminarin is 1:(1-50). The active pharmaceutical ingredient includes one or more of cardiovascular therapeutic drugs, antitumor drugs, or immunomodulatory drugs. The drug-excipient soluble microneedle patch provided by this invention uses laminarin as the matrix material, exhibiting good biocompatibility, biodegradability, film-forming ability, and natural pharmacological activity, enabling it not only to serve as a microneedle supporting material but also to exert a synergistic therapeutic effect with the loaded drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation and transdermal drug delivery technology, specifically relating to a drug-excipient integrated soluble microneedle patch based on laminarin and its preparation method. Background Technology

[0002] Microneedling is an innovative drug delivery method that uses an array of micron-sized needles to pierce the outermost layer of the skin, the stratum corneum, creating microchannels to facilitate transdermal drug absorption. This technology combines the high bioavailability of subcutaneous injections with the painless, convenient, and safe benefits of transdermal patches. Among the various types of microneedles, dissolvable microneedles are highly favored due to their "disposable, non-sharp medical waste" characteristics. These microneedles are typically composed of water-soluble polymers (such as hyaluronic acid, polyvinylpyrrolidone, sodium alginate, etc.), loaded with drug, and inserted into the skin. The needle rapidly dissolves in the tissue fluid, releasing the drug.

[0003] Currently, the selection of matrix materials for soluble microneedles is mostly based on their mechanical strength, solubility, and biocompatibility. These materials are usually considered inert excipients in formulations, and their main function is to construct the microneedle structure and deliver drugs; they do not possess therapeutic activity themselves. This means that all therapeutic effects must depend entirely on the loaded drug, and the function of the formulation is relatively singular.

[0004] The patent entitled "A Hematoporphyrin Microneedle Formulation and Its Preparation Method" (publication number: CN118717640A) discloses a hematoporphyrin microneedle formulation that uses laminarin polysaccharide. However, it only adds a small amount of laminarin polysaccharide as one of the components to increase the solid content and improve puncture resistance. Moreover, this added component can be replaced by other sugars.

[0005] Therefore, there is a significant gap in the existing technology: can a natural polysaccharide with specific biological activity (such as kelp polysaccharide) be defined from the traditional "inert excipient" as an active matrix material with synergistic therapeutic function? Developing such a microneedle platform can not only achieve efficient drug delivery, but its matrix material itself can also contribute additional therapeutic benefits, realizing "drug-excipient synergistic therapy", thereby greatly enhancing the overall efficacy and application potential of the formulation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a drug-excipient integrated soluble microneedle patch based on kelp polysaccharide and its preparation method; the aim is to overcome the above-mentioned defects of existing soluble microneedle technology.

[0007] This invention is achieved through the following technical solution: A soluble microneedle patch based on laminarin polysaccharide, wherein the soluble microneedle patch uses laminarin polysaccharide as a supporting material for the microneedle structure and a pharmacologically active therapeutic agent.

[0008] Furthermore, the molecular weight of the kelp polysaccharide is 5-30 kDa, preferably 20-25 kDa.

[0009] Furthermore, the drug-excipient soluble microneedle patch also includes active pharmaceutical ingredients; The mass ratio of the active pharmaceutical ingredient to laminarin is 1:(1-50); preferably 1:(2.5-20).

[0010] Furthermore, the active pharmaceutical ingredient includes one or more of cardiovascular therapeutic drugs, antitumor drugs, or immunomodulatory drugs; preferably, the active pharmaceutical ingredient is cycloviniferin D.

[0011] Furthermore, the needle height of the drug-excipient integrated soluble microneedle patch is 200-900 μm, preferably 300-600 μm; the needle width is 200-600 μm, preferably 300-600 μm; and the needle tip distance is 150-900 µm, preferably 200-500 μm. The drug-excipient soluble microneedle patch dissolves in the interstitial fluid of the skin in less than 30 minutes.

[0012] A method for preparing a drug-excipient soluble microneedle patch based on laminarin polysaccharide, the method comprising the following steps: Laminaria polysaccharide was dissolved in deionized water, stirred until completely dissolved, and centrifuged to remove air bubbles, thus obtaining an aqueous solution of laminarin polysaccharide. The laminarin polysaccharide self-assembled to form a three-dimensional hydrogen bond network structure. Based on the three-dimensional hydrogen bond network structure, it is not necessary to add chemical cross-linking agents to the microneedle patch.

[0013] The kelp polysaccharide aqueous solution is applied to the surface of the microneedle mold, and vacuum decompression treatment is performed to fully fill the micropores of the mold with the kelp polysaccharide aqueous solution. After drying, the mold is peeled off to obtain the drug-excipient integrated soluble microneedle patch.

[0014] Furthermore, the concentration of laminarin in the laminarin aqueous solution is 5-10% (w / t).

[0015] Furthermore, the conditions for the vacuum decompression treatment are: vacuum suction time of 8 to 30 minutes; and drying temperature of 20 to 45°C.

[0016] Furthermore, when the prepared drug-excipient soluble microneedle patch also includes an active pharmaceutical ingredient, the preparation method is as follows: The active pharmaceutical ingredient and laminarin polysaccharide were dissolved in deionized water. After complete dissolution, the mixture was stirred evenly and centrifuged to remove air bubbles, thus obtaining an aqueous solution of laminarin polysaccharide / active pharmaceutical ingredient. The kelp polysaccharide / active drug aqueous solution is applied to the surface of the microneedle mold, and vacuum decompression treatment is performed to fully fill the micropores of the mold with the kelp polysaccharide / active drug aqueous solution. After drying, the mold is peeled off to obtain the drug-excipient integrated soluble microneedle patch. The active pharmaceutical ingredient includes one or more of cardiovascular therapeutic drugs, antitumor drugs, or immunomodulatory drugs; the mass ratio of the active pharmaceutical ingredient to laminarin is 1:(1-50).

[0017] The application of a drug-excipient soluble microneedle patch, wherein the drug-excipient soluble microneedle patch is used in the preparation of drugs for the treatment of cardiovascular diseases or immune-related diseases.

[0018] Laminarin is a natural, water-soluble polysaccharide derived from kelp. With a low molecular weight, it exhibits good biocompatibility, biodegradability, and film-forming ability, making it a highly promising candidate material for microneedle matrices. Preliminary research in this invention further demonstrates that laminarin can self-assemble into a dense three-dimensional hydrogen-bonded network in aqueous solution, thus enabling the preparation of mechanically strong, self-supporting, soluble microneedles without the addition of any chemical cross-linking agents or other auxiliary materials. In existing technologies, polysaccharide materials in microneedle formulations are primarily used as traditional excipients for structural support and drug delivery, while their inherent biological activities have not been fully recognized and utilized. This invention shows that laminarin possesses various pharmacological activities, including excellent anticoagulant, antioxidant, antitumor, and immunomodulatory effects. This characteristic allows laminarin to transcend the limitations of traditional excipients, exerting synergistic therapeutic functions while loading drugs, significantly enhancing the overall efficacy and application prospects of the formulation.

[0019] Beneficial technical effects of the present invention: This invention successfully applies kelp polysaccharide to the field of microneedles. This invention not only proves that kelp polysaccharide can form a microneedle body with good puncture properties on its own, but its key lies in achieving the integration of "drug and adjuvant". In addition to serving as a carrier, kelp polysaccharide has its own anti-myocardial ischemia, antioxidant and immunomodulatory activities, and can synergistically enhance the therapeutic effect with loaded drugs.

[0020] The preparation process of the drug-excipient integrated soluble microneedle patch provided by this invention is simple and green, requiring no chemical cross-linking agents, thus improving the biosafety of the product; The drug-excipient integrated soluble microneedle patch provided by this invention has excellent puncture and dissolution properties, which can effectively promote transdermal drug absorption and achieve synergistic delivery of drugs and excipients. The drug-excipient soluble microneedle patch provided by this invention pioneers the application of laminarin in the field of microneedles, giving full play to its high biocompatibility, wide availability, and synergistic therapeutic properties, and providing a new, efficient, and safe drug delivery strategy for the treatment of cardiovascular, anti-tumor, and other diseases.

[0021] The method for preparing the drug-excipient integrated soluble microneedle patch provided by the present invention adopts vacuum casting, which successfully achieves one-time molding to produce a microneedle structure with a high aspect ratio (needle height 200-900 μm, needle spacing 150-900 μm). Attached Figure Description

[0022] Figure 1 : Schematic diagram of the preparation method of kelp polysaccharide microneedles in this embodiment of the invention; Figure 2 : Microscopic images of 5% kelp polysaccharide microneedles in the embodiments of the present invention; Figure 3 : Microscopic images of 8% kelp polysaccharide microneedles in the embodiments of the present invention; Figure 4 : Microscopic images of 10% kelp polysaccharide microneedles in the embodiments of the present invention; Figure 5 : Microscopic images of kelp polysaccharide microneedles in the embodiments of the present invention (showing the tip height); Figure 6 SEM images of kelp polysaccharide microneedles in the embodiments of the present invention (showing the integrity of the needle tip); Figure 7 The puncture results of kelp polysaccharide microneedles in the embodiments of the present invention; Figure 8 HE staining results of skin after puncture with kelp polysaccharide microneedles in this embodiment of the invention; Figure 9 The mechanical property test results of kelp polysaccharide microneedles in the embodiments of the present invention; Figure 10 The in vitro dissolution results of kelp polysaccharide microneedles in the embodiments of the present invention; Figure 11 The skin irritation results of the kelp polysaccharide microneedles in the embodiments of the present invention; Figure 12 The results of erythrocyte hemolysis using kelp polysaccharide microneedles in the embodiments of the present invention; Figure 13 The histopathological examination results of the main organs of the kelp polysaccharide microneedles in the embodiments of the present invention; Figure 14 The longitudinal body weight monitoring results of rats using kelp polysaccharide microneedles in this embodiment of the invention; Figure 15 The results of the CCK8 cytotoxicity test of kelp polysaccharide microneedles in L929 and HaCaT cells in the embodiments of the present invention; Figure 16 The results of L929 and HaCaT cell viability staining of kelp polysaccharide microneedles in the embodiments of the present invention; Figure 17The in vitro antitumor activity verification results of kelp polysaccharide in the embodiments of the present invention; Figure 18 The following are the efficacy verification results of kelp polysaccharide microneedles in treating mouse melanoma in the embodiments of the present invention. a is a photo of the tumors of nude mice in each treatment group, and b is the tumor volume of mice in each treatment group during the drug administration period. Figure 19 : These are the stereomicroscopic morphology (a) and puncture properties (b) of the microneedles loaded with kelp polysaccharide in the embodiments of the present invention; Figure 20 Efficacy validation of synergistic in vitro treatment of IHD with kelp polysaccharide and CVB-D. a) Effect of CVB-D and LAM on the in vitro viability of H9c2 cells under hypoxia model. b) LDH leakage in H9c2 cells treated with CVB-D and LAM under hypoxia conditions. c) CK content in H9c2 cells treated with CVB-D and LAM under hypoxia conditions. d) Hoechst 33258 staining results of H9c2 cells treated with CVB-D and LAM under hypoxia conditions. Figure 21 Results of ventricular wall motion ultrasound examination in rats one month after treatment; Figure 22 Example 5: Detection of biomarkers of myocardial injury in rats with myocardial ischemia treated with CVB-D@LAM DMNs microneedling: a is the CK-MB content in rat serum, b is the cTnT content in rat serum, c is the MDA content in rat serum, and d is the SOD content in rat serum. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Conversely, this invention encompasses any substitutions, modifications, equivalent methods, and solutions made within the spirit and scope of the invention as defined in the claims. Furthermore, to provide a better understanding of the invention, certain specific details are described in detail below. However, those skilled in the art will fully understand the invention even without these detailed descriptions.

[0025] In existing technologies, the matrix material of microneedles is typically used only as an inert excipient, with a single function and no therapeutic benefit; all efficacy depends entirely on the loaded drug. This invention provides a drug-excipient integrated soluble microneedle patch using laminarin as the supporting material for the microneedle structure and a pharmacologically active therapeutic agent. The drug-excipient integrated soluble microneedle patch provided by this invention exhibits good biocompatibility, biodegradability, film-forming ability, and natural pharmacological activity, enabling it not only to serve as a microneedle support material but also to exert a synergistic therapeutic effect with the loaded drug, significantly enhancing the application efficacy of formulations in cardiovascular diseases, anti-tumor treatments, and other fields. Furthermore, the preparation method provided by this invention is simple, green, and safe, resulting in microneedles with high mechanical strength and good solubility, achieving integrated synergistic delivery of the drug and excipient. Specific embodiments are as follows: Example 1: A method for preparing a soluble microneedle patch based on laminarin polysaccharide, comprising pharmacologically active therapeutic agents and laminarin polysaccharide as the supporting material for the microneedle structure, wherein the soluble microneedle patch is an integrated laminarin polysaccharide needle, such as... Figure 1 As shown, it includes the following steps: Laminaria polysaccharide was dissolved in deionized water to prepare an aqueous solution of laminarin; the concentration of laminarin in the aqueous solution was 5 wt%. Specifically, in this embodiment, 0.25 g of laminarin was accurately weighed into a 5 mL centrifuge tube, 4.75 mL of ultrapure water was added to completely dissolve it, the mixture was stirred evenly, centrifuged to remove air bubbles, and a homogeneous matrix solution was obtained. The homogeneous matrix solution was added to the microneedle mold using a pipette, 80 μL per well, and vacuum aspirated for 30 min. Afterward, the solution was dried at room temperature. The mold was then peeled off to obtain a drug-excipient soluble microneedle patch (i.e., laminarin microneedles). The microneedle morphology is as follows. Figure 2 As shown, the prepared microneedles have complete tips and good morphology.

[0026] Example 2: This example provides a drug-excipient integrated soluble microneedle patch based on laminarin, with a preparation method basically the same as that provided in Example 1, except that the concentration of laminarin in the aqueous solution is 8 wt%. Specifically, in this example, 0.4 g of laminarin was accurately weighed into a 5 mL centrifuge tube, and 4.6 mL of ultrapure water was added to completely dissolve it. After stirring evenly and centrifuging to remove air bubbles, an aqueous solution of laminarin was obtained. The aqueous solution of laminarin was added to the microneedle mold using a pipette, with 80 μL added to each well, and vacuum aspiration was performed for 30 min. After completion, the solution was dried at room temperature, and the mold was peeled off to obtain the drug-excipient integrated soluble microneedle patch (i.e., laminarin microneedles).

[0027] The morphology of the laminarin microneedles prepared in this embodiment is as follows: Figure 3 As shown, the prepared microneedles have complete tips and good morphology.

[0028] Example 3: This example provides a drug-excipient integrated soluble microneedle patch based on laminarin, with a preparation method basically the same as that provided in Example 1, except that the concentration of laminarin in the aqueous solution is 10wt%. Specifically, in this example, 0.5g of laminarin is accurately weighed into a 5mL centrifuge tube, 4.6mL of ultrapure water is added to completely dissolve it, and the mixture is stirred evenly, centrifuged to remove air bubbles, and an aqueous solution of laminarin is obtained. The aqueous solution of laminarin is added to the microneedle mold using a pipette, 80μL per well, and vacuum aspirated for 30min. After completion, the solution is dried at room temperature, and the mold is peeled off to obtain the drug-excipient integrated soluble microneedle patch (i.e., laminarin microneedles).

[0029] The morphology of the laminarin microneedles prepared in this embodiment is as follows: Figure 4 As shown, the prepared microneedles have complete tips and good morphology.

[0030] Performance characterization of kelp polysaccharide microneedle patches: (1) Morphology and needle height characterization of kelp polysaccharide microneedles: Kelp polysaccharide microneedles were prepared according to Example 1, and then their morphology was tested using a stereomicroscope and a scanning electron microscope. The results are as follows: Figure 5 and 6 As shown, the prepared microneedles have complete tips and good morphology.

[0031] (2) Characterization of the puncture performance of kelp polysaccharide microneedle patches: Kelp polysaccharide microneedles were prepared according to Example 1, and then the puncture performance of the microneedles was tested. Using ex vivo pig skin, the microneedles were attached to a hydrogel backing, and then an inserter was used to assist in insertion. After 20 seconds, the patches were removed, observed, and photographed. The puncture results are as follows: Figure 7 As shown, a clear array of micropores is visible on the pigskin, indicating that the prepared microneedles have good puncture performance. Furthermore, the punctured pigskin was fixed with tissue fixative and stained with hematoxylin and eosin (HE) to observe the microneedle insertion performance. The insertion results are as follows: Figure 8 As shown, the microneedles have good puncture performance.

[0032] (3) Characterization of the mechanical properties of kelp polysaccharide microneedle patches: Kelp polysaccharide microneedles were prepared according to Example 1, and their compressive mechanical properties were evaluated using a TA-XT texture analyzer in compression mode. Specifically, the kelp polysaccharide microneedle samples were placed on a platform with the tips facing upwards. An axial force was applied at a constant speed of 0.5 mm / s using a 10 mm diameter stainless steel flat-end cylindrical probe. The preload was set to 0.05 N, and data acquisition continued until the sensor displacement reached 0.49 mm (corresponding to 70% deformation of the microneedle) to ensure complete characterization of the stress-strain behavior. The mechanical test results are as follows: Figure 9 As shown, the microneedles possess good mechanical strength.

[0033] (4) Characterization of in vitro dissolution properties of kelp polysaccharide microneedle patches: Kelp polysaccharide microneedles were prepared according to Example 1. Then, using excised porcine skin, a medical tape backing was applied to the dorsal side of the kelp polysaccharide microneedle patch to ensure stable adhesion to the skin matrix. Using a microneedle applicator (20 N / cm²), the DMN was applied to the porcine skin, and the height of residual microneedles was dynamically monitored using an inverted stereomicroscope (ICX41, SOPTOP, China). Results are as follows: Figure 10 As shown, the rapid in vitro dissolution properties of kelp polysaccharides are demonstrated.

[0034] Biocompatibility validation of kelp polysaccharide microneedle patches: (1) Skin irritation verification of kelp polysaccharide microneedle patches: Kelp polysaccharide microneedles were prepared according to Example 1, and the skin irritation of LAM-DMNs was evaluated using New Zealand white rabbits. Before use, back hair was removed using an electric shaver and depilatory cream, followed by a 24-hour adaptation period. Subsequently, HA-DMNs and LAM-DMNs were applied to the prepared skin sites and secured with medical-grade adhesive tape backing. After maintaining the patches for 8 hours, all devices were carefully removed. During the observation period, local skin reactions, including erythema and edema, and subsequent recovery, were systematically recorded using digital photography at 0, 24, and 48 hours after removal. Skin irritation results are as follows: Figure 11 As shown, the kelp polysaccharide microneedles do not cause skin irritation.

[0035] (2) Verification of erythrocyte compatibility of kelp polysaccharide microneedle patches: Kelp polysaccharide microneedles were prepared according to Example 1. Fresh rabbit blood (1 mL) was then mixed with 4 mL of Tris buffer and centrifuged at 1000 × g for 10 minutes. The supernatant was discarded, and the erythrocyte (RBC) globules were washed twice with Tris buffer until the supernatant became clear. The washed erythrocytes were resuspended in fresh Tris buffer to a concentration of 5% (v / v) and stored for subsequent analysis. One device was dissolved in 1 mL of physiological saline buffer to obtain a 100% stock solution. A series of dilutions (50% and 25% v / v) were then generated using the same saline buffer. Positive and negative controls were established using 0.1% Triton X-100 added to Tris buffer (positive control) and fresh physiological saline (negative control), respectively. Add equal aliquots (20 μL) of red blood cell suspension to each centrifuge tube, with each tube containing 1 mL of test solution (100%, 50%, and 25% microneedle concentrations, positive control, and negative control). Incubate at 37°C with shaking (200 rpm) for 1 hour. After centrifugation at 10000×g for 10 minutes, photograph the supernatant and visually assess hemolysis. Quantify the absorbance of each sample at 570 nm using a microplate reader and calculate the hemolysis rate according to the following formula.

[0036] Hemolysis rate (%) = (P - P)(P - P) × 100% In the formula, H, Hn, and Hp are the absorbance values ​​measured in the microneedle sample group, negative control group, and positive control group, respectively. Hemolysis results are as follows: Figure 12 As shown, the kelp polysaccharide microneedles do not cause hemolysis. (3) Kelp polysaccharide microneedles were prepared according to Example 1, and then the systemic toxicity was studied by repeated patch application to mice for 28 days, and the histopathological examination of major organs was also performed. Figure 13 ) and longitudinal weight monitoring ( Figure 14 The result is as follows: Figure 13 and 14 As shown, no obvious adverse reactions were observed.

[0037] (4) Prepare kelp polysaccharide microneedles according to Example 1, dissolve them thoroughly in sterile culture medium, and then filter them through a sterile 0.22µm filter membrane for later use. L929 fibroblasts and HaCaT keratinocytes were then cultured at a concentration of 1×10⁻⁶. 4 Cells were seeded at 96-well plates at their specified density and treated for 24 hours with medium containing laminarin microneedle extract (25%, 50%, 100%). CCK-8 assay showed that both cell types exhibited ≥90% viability (negative control was set at 100%). Simultaneous AO / EB fluorescence staining showed that the treated cells exhibited a uniform bright green viable cell morphology (excitation / emission: 490 / 530 nm), consistent with the negative control, while the 5% DMSO positive control group showed significant cell death (orange-red fluorescence aggregation). These results confirm that laminarin microneedles are non-cytotoxic at the tested concentrations. Relevant cell viability data and fluorescence images are shown below. Figure 15 and Figure 16 The in vitro biosafety was further verified by two methods: (1) CCK-8 assay showed that the cell viability of L929 fibroblasts and HaCaT keratinocytes was maintained at >90% ( Figure 15 ), and (2) AO / EB fluorescence imaging showed that the cells were bright green and evenly distributed, compared with the negative control, while the number of cells in the positive control group was significantly reduced ( Figure 16 This confirms that laminarin microneedles are non-cytotoxic and possess good biocompatibility. In summary, these findings establish laminarin microneedles as a biosafe platform for continuous transdermal therapy.

[0038] To investigate the in vitro antitumor activity of laminarin, the CCK-8 assay was used to verify the in vitro antitumor activity of laminarin in mouse melanoma B16-F10 cells. The specific steps were as follows: First, cells were seeded into plates. The tumor cell suspension was counted using a hemocytometer. Based on the counting results, the cell density was adjusted to 80,000 cells / mL, and 100 μL was seeded into 96-well plates. 100 μL of PBS was added to the outer wells to prevent evaporation. The plates were incubated at 37°C and 5% CO2 for 24 hours. At the same time, the cells were passaged and counted. The cell suspension was diluted 10-fold, counted, and the concentration was adjusted to 50,000–80,000 cells / mL. The cells were seeded into 96-well plates at a density of 5,000–8,000 cells per well and cultured for another 24 hours. Subsequently, laminarin sample solutions were prepared: 32.0 mg of laminarin was accurately weighed and added to 5.0 mL of RPMI 1640 medium to prepare a stock solution of 6400 μg / mL. This stock solution was then sequentially diluted with the medium to obtain a series of concentration solutions of 100, 200, 400, 800, 1200, 1600, 3200, and 6400 μg / mL. Different concentration sample solutions were added to 96-well plates after culture, and after a certain period of incubation, the cell proliferation inhibition rate was detected using the CCK-8 assay, and the IC50 was calculated. 50 The value is used to evaluate its antitumor activity.

[0039] result Figure 17 This indicates that laminarin has anti-tumor activity, suggesting that laminarin can synergistically work with drugs that have anti-tumor effects in treating melanoma.

[0040] To investigate the therapeutic effect of kelp polysaccharide microneedle patches on mouse melanoma, a mouse melanoma animal model was established for pharmacological evaluation. The kelp polysaccharide microneedle patches were prepared in the same manner as in Example 1, and the experimental protocol is as follows: Animal model: A mouse orthotopic melanoma model was established by subcutaneous injection of B16-F10 cells.

[0041] Grouping Design and Microneedle Administration: Fifteen successfully modeled mice were randomly divided into three groups: kelp polysaccharide microneedle administration group, positive drug group, and model group, with five mice in each group. Twenty-four hours prior to the experiment, abdominal hair was removed from the rats using a hair removal device and cream, and the rats were returned to their cages for normal feeding. Drug administration began the following day. Microneedle administration was performed according to the pre-set groupings. A hydrogel backing was used to fix the microneedles to the skin of the hair-removed area on the rat's abdomen, and the needles were inserted using a needle inserter (20 N / cm²). 2 Press the microneedle for 20 seconds, then use medical tape to secure the microneedle patch to prevent rats from biting each other. Administer once daily for 12 consecutive days.

[0042] After successful modeling, the tumor volume of the mice was measured and recorded with calipers before each day's administration, until the tumors in the model group mice grew to nearly 1500 mm. 3 Mice were sacrificed at the designated time, and the size of the tumor was recorded before sacrifice to observe the tumor-inhibiting effect of kelp polysaccharide microbes.

[0043] The results are as follows Figure 18 As shown in the results, continuous administration of laminarin significantly inhibited tumor growth in mice. This is consistent with the conclusions reported in numerous studies that laminarin exerts its anti-tumor effects through multiple mechanisms, including activating immune responses and inducing tumor cell apoptosis.

[0044] In summary, laminarin can effectively inhibit tumor growth, providing important preclinical data to support its potential as an anti-tumor drug or adjuvant therapy. Further research can delve deeper into its precise molecular mechanism of action and consider combining it with existing chemotherapy drugs to achieve synergistic effects and reduce toxicity.

[0045] Example 5: To investigate the synergistic therapeutic effect of laminarin on myocardial ischemia while simultaneously loading drugs, the synergistic protective effect of a combination of cycloviniferin D and laminarin in a myocardial cell ischemia model was evaluated. A myocardial hypoxia model of H9c2 cardiomyocytes was established using the AnaeroPack® anaerobic culture system, and the experimental protocol is as follows: Cell model: H9c2 cardiomyocyte myocardial hypoxia model; Grouped administration: control group (noroxic), hypoxia group, CVB-D alone group, laminarin alone group, and CVB-D + laminarin combination group.

[0046] Cell viability was detected using the CCK-8 assay. Figure 19 The results showed that the cell viability in the combined treatment group was 93.7±8.5%, significantly higher than that in the hypoxia model group (58.3±5.6%) (p<0.01), and significantly higher than that in the CVB-D monotherapy group (72.4±6.2%) and the laminarin monotherapy group (79.1±7.3%) (p<0.05). Figure 19 As shown in Figures b and c, the detection of myocardial injury markers indicated that the release of LDH and CK in the combined medication group was reduced by 42.0% and 38.2%, respectively, compared to the model group (p<0.01). Figure 19 As shown in the data, Hoechst 33258 staining revealed that the proportion of apoptotic cells in the combined drug group (9.2±1.1%) was close to that in the normal control group (5.3±0.8%), and significantly lower than that in each single drug group.

[0047] The results showed that CVB-D and laminarin had a synergistic cardioprotective effect at a ratio of 200 μg / mL + 1600 μg / mL, indicating that laminarin can synergistically treat myocardial ischemia with CVB-D.

[0048] Example 5: This example provides a method for preparing a soluble microneedle patch loaded with kelp polysaccharide (cycloviscose D), which is basically the same as the preparation method provided in Example 1, except that the kelp polysaccharide aqueous solution is mixed with the traditional Chinese medicine monomer cycloviscose D. Specifically, in this example, 0.25 g of kelp polysaccharide and 0.2 g of cycloviscose D are accurately weighed and thoroughly ground in a mortar, then transferred to a 5 mL centrifuge tube, 4.55 mL of ultrapure water is added to completely dissolve the mixture, and the mixture is stirred evenly and centrifuged to remove air bubbles, thereby obtaining a cycloviscose D-kelp polysaccharide aqueous solution. The cycloviscose D-kelp polysaccharide aqueous solution is added to the microneedle mold using a pipette, with 80 μL added to each well, and vacuum aspirated for 30 min. After completion, the solution is dried at room temperature, and the mold is peeled off after drying to obtain the soluble microneedle patch (i.e., cycloviscose D-kelp polysaccharide microneedles).

[0049] The morphology of the cycloviscosity-containing Buxus microneedles D-lamb polysaccharide prepared in this embodiment is as follows: Figure 20 As shown in Figure a, the prepared microneedles have complete tips and good morphology.

[0050] The puncture performance of the microneedles was then tested. Using excised pig skin, the microneedles were attached to a hydrogel backing, and then an inserter was used to assist insertion. After 20 seconds, the skin was removed, observed, and photographed. The puncture results are as follows: Figure 20 As shown in Figure b, a clear array of micropores is visible on the pig skin, indicating that the prepared microneedles have good puncture properties. The results demonstrate that the addition of small drug molecules to kelp polysaccharides resulted in good needle-forming and puncture properties.

[0051] To investigate the synergistic therapeutic effect of kelp polysaccharide microneedle patches on myocardial ischemia, a myocardial ischemia model was established using SD rats for pharmacological evaluation. The preparation method of the kelp polysaccharide microneedle patches was the same as in Example 5, and the experimental protocol is as follows: Animal model: Myocardial ischemia model was established by ligation of the left anterior descending coronary artery in SD rats; Grouped drug administration: Sham surgery group: open chest suturing only; Model group (MI): ischemic treatment; Microneedle group (CVB-D@LAM DMNs): daily application of CVB-D microneedles (1 patch); Oral group: CVB-D solution administered by gavage; Blank control group (LAM-DMNs): drug-free laminarin microneedles; Detection indicators: Cardiac function: echocardiography; Myocardial injury: serum CK-MB, cTnT, MDA, and SOD.

[0052] Experimental results are as follows Figure 21 As shown, after 4 weeks of continuous administration of CVB-D@LAM microneedle patches, the left ventricular end-diastolic diameter (LVIDd) and left ventricular end-systolic diameter in rats in the microneedle group were significantly improved compared with the model group, and the efficacy of the CVB-D microneedle administration group was comparable to that of the gavage administration group. The kelp polysaccharide microneedle group also showed a synergistic therapeutic effect. The CVB-D@LAM microneedle group showed significant improvement in cardiac function, specifically an increase in left ventricular ejection fraction (EF), a decrease in left ventricular systolic pressure (LVSP), and positive changes in parameters such as left ventricular fractional shortening (FS%), stroke volume (SV), cardiac output (CO), and left ventricular end-diastolic volume (LVEDV). The blank microneedle group also showed improvement in the above indicators, suggesting that it may synergistically promote myocardial repair through mechanical tissue remodeling. Furthermore, the serum levels of myocardial injury biomarkers (including CK-MB, cTnT, MDA, and SOD) in the microneedle administration group were significantly lower than those in the model group. Figure 22 The effect was superior to that of the gavage control group. The blank microneedle group also showed improvement in various indicators, further confirming its potential for synergistic treatment of myocardial ischemia with CVB-D. These results indicate that CVB-D@LAM microneedles can serve as a synergistic drug delivery and mechanical tissue remodeling platform, promoting myocardial repair and providing a new strategy for the long-term treatment of chronic diseases such as myocardial ischemia. In summary, this study suggests that laminarin can synergistically enhance the efficacy of CVB-D in treating myocardial ischemia in rats, highlighting the comprehensive advantages of microneedle drug delivery systems in myocardial tissue repair.

[0053] Compared with the prior art, the present invention has the following outstanding advantages: The preparation process is simplified and product safety is improved: Utilizing the property of laminarin polysaccharide to form a three-dimensional hydrogen-bonded network through self-assembly in aqueous solution, self-supporting microneedles with excellent mechanical strength can be prepared without the addition of any chemical cross-linking agents. This preparation method is mild, simple, and reproducible, avoiding the biosafety risks that may be caused by chemical cross-linking agents, and meeting the requirements of green pharmaceuticals.

[0054] This innovative model of "drug-excipient synergistic therapy" has been achieved: kelp polysaccharide has been redefined from a traditional inert excipient as an active matrix material with active therapeutic functions. While fulfilling its functions as a microneedle structural support and drug carrier, it can also synergistically enhance the overall therapeutic effect by interacting with the loaded drug through its multiple pharmacological activities, including antioxidant and immunomodulatory effects.

[0055] This invention expands the application prospects of microneedle formulations: It not only provides a specific active microneedle product, but also pioneers a new microneedle design paradigm of "active matrix material + therapeutic drug", providing a universal technical platform for the development of various transdermal drug delivery systems with synergistic therapeutic functions, with broad application potential.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A soluble microneedle patch based on laminarin polysaccharide, characterized in that, The drug-excipient soluble microneedle patch uses laminarin as a supporting material for the microneedle structure and also as a pharmacologically active therapeutic agent; the molecular weight of the laminarin is 5-30 kDa.

2. A method for preparing a drug-excipient soluble microneedle patch based on laminarin polysaccharide, used to prepare the drug-excipient soluble microneedle patch according to claim 1, characterized in that, The preparation method includes the following steps: Laminaria polysaccharides were dissolved in deionized water. After complete dissolution and thorough stirring, the mixture was centrifuged to remove air bubbles, yielding an aqueous solution of laminarin. The laminarin self-assembled to form a three-dimensional hydrogen-bonded network structure. The concentration of laminarin in the aqueous solution was 5-10 wt%. The kelp polysaccharide aqueous solution is applied to the surface of the microneedle mold, and vacuum decompression treatment is performed to fully fill the micropores of the mold with the kelp polysaccharide aqueous solution. After drying, the mold is peeled off to obtain the drug-excipient integrated soluble microneedle patch.

3. The preparation method of the drug-excipient integrated soluble microneedle patch based on laminarin according to claim 2, characterized in that, The conditions for the vacuum decompression treatment are: vacuum suction time of 8 to 30 minutes; and drying temperature of 25 ± 0.2°C.

4. The application of the soluble microneedle patch based on laminarin polysaccharide according to claim 1, characterized in that, The application of the drug-excipient integrated soluble microneedle patch in the preparation of drugs for the treatment of melanoma.

5. A soluble microneedle patch based on laminarin polysaccharide, characterized in that, The drug-excipient integrated soluble microneedle patch uses laminarin as a supporting material for the microneedle structure and also as a pharmacologically active therapeutic agent; the molecular weight of the laminarin is 5-30 kDa; the drug-excipient integrated soluble microneedle patch also includes cycloviniferin D; The mass ratio of the cyclovinifera star D to kelp polysaccharide is 1:(2.5-20).

6. The soluble microneedle patch based on laminarin polysaccharide according to claim 5, characterized in that, The drug-excipient integrated soluble microneedle patch has a needle height of 200-900μm, a needle width of 200-600μm, and a needle tip distance of 150-900µm. The drug-excipient soluble microneedle patch dissolves in the interstitial fluid of the skin in less than 30 minutes.

7. A method for preparing a drug-excipient soluble microneedle patch based on laminarin polysaccharide, used to prepare the drug-excipient soluble microneedle patch according to claim 5 or 6, characterized in that, The preparation method includes the following steps: Cyclofibrin D and laminarin were dissolved in deionized water. After complete dissolution, the mixture was stirred evenly and centrifuged to remove air bubbles, thus obtaining an aqueous solution of laminarin and cyclofibrin D. The laminarin and cycloviscose D aqueous solution were applied to the surface of the microneedle mold, and vacuum decompression treatment was performed to allow the laminarin and cycloviscose D aqueous solution to fully fill the micropores of the mold. After drying, the mold was peeled off to obtain the drug-excipient integrated soluble microneedle patch. The mass ratio of the cyclovinifera star D to kelp polysaccharide is 1:(2.5-20).

8. The application of the soluble microneedle patch based on laminarin polysaccharide according to claim 5 or 6, characterized in that, The application of the drug-excipient integrated soluble microneedle patch in the preparation of drugs for treating myocardial ischemia.

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