Glycyrrhizic acid hydrogel double-layer microneedle for treating malignant melanoma as well as preparation method and application of glycyrrhizic acid hydrogel double-layer microneedle

Abstract

The invention relates to the field of biological medicine, in particular to a glycyrrhizic acid hydrogel double-layer microneedle for treating malignant melanoma as well as a preparation method and application of the glycyrrhizic acid hydrogel double-layer microneedle. The microneedle (MNs) is an up-down double-layer microneedle composed of self-assembled glycyrrhizic acid (GA) hydrogel, the upper layer of the microneedle, namely a needle tip layer, is formed by wrapping triptolide (TP) and a photosensitizer chlorin e6 (Ce6) in the GA hydrogel, and the synergistic anti-tumor effect of chemotherapy and photodynamic therapy is achieved; the lower layer of the microneedle, namely the substrate layer, is a soluble microneedle containing GA hydrogel and auxiliary materials including hyaluronic acid and polyvinylpyrrolidone, the GA is released along with dissolution of the MNs, the anti-inflammatory effect is achieved, and potential damage of the MNs to the skin during transdermal penetration is reduced. According to the double-layer drug-loaded microneedle, the anti-cancer effect of a drug is remarkably improved, the double-layer drug-loaded microneedle has an anti-inflammatory effect on damaged skin, the double effects of cancer treatment and anti-inflammatory are achieved, and a new research direction is provided for treatment of melanoma.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a glycyrrhizic acid hydrogel bilayer microneedle for treating malignant melanoma, its preparation method, and its application. Background Technology

[0002] Malignant melanoma (MM), the most deadly type of skin cancer, is characterized by its high rate of metastasis and mortality. Conventional treatments often fail to deliver drugs accurately to the tumor site, resulting in low cure rates and rapid disease progression. Microneedles (MNs), a novel transdermal drug delivery technology, primarily function by penetrating the stratum corneum to create a micrometer-scale channel for drug delivery to the epidermis or dermis, allowing the drug to exert its effects directly under the skin. The MN needle tip possesses sufficient mechanical strength to pierce through the stratum corneum to the dermis, directly delivering the drug to the lesion, increasing drug distribution at the lesion site, minimizing drug diffusion to adjacent healthy tissues and organs, thus reducing side effects and prolonging patient survival. To effectively inhibit the progression and spread of MM, multi-mechanism therapy combinations are often employed, aiming to play a significant role in clinical practice.

[0003] Photodynamic therapy (PDT) utilizes photosensitizers to generate reactive oxygen species (ROS) under specific wavelengths of light, inducing apoptosis and death of tumor cells. The photosensitizer dihydroporphyrin e6 (Ce6) contains multiple conjugated double bonds in its molecular structure, enabling it to effectively absorb light of specific wavelengths, exhibiting strong absorption characteristics in the red light region (650-670 nm). Red light has good tissue penetration, making it suitable for treating lesions in deep tissues. Ce6 can selectively accumulate in tumor or diseased tissues, while having a weaker affinity for normal tissues, thus improving the precision of treatment. Under light irradiation, Ce6 can efficiently generate ROS, which damage cell membranes, proteins, and DNA through oxidation, thereby inducing apoptosis.

[0004] Triptolide (TP), a key active ingredient in the traditional Chinese medicine Tripterygium wilfordii, has exhibited broad-spectrum anti-tumor effects. However, its poor water solubility, strong toxic side effects, and rapid elimination from the body limit its clinical application. Glycyrrhizic acid (GA), the most abundant natural triterpenoid saponin extracted from licorice, is the main active ingredient of licorice. Studies have shown that GA has certain anti-inflammatory and detoxifying effects. Its anti-inflammatory mechanism mainly involves inhibiting antibody production, hindering the production of inflammatory cytokines and inflammatory mediators, and antagonizing the pro-inflammatory effects of inflammatory mediators. In addition, many reports in recent years have indicated that GA molecules can spontaneously form nanofibers or three-dimensional network structures in water through non-covalent interactions (such as hydrogen bonds, hydrophobic interactions, and π-π stacking), forming stable hydrogels. Therefore, GA can also be used as a drug delivery carrier. As a natural product, the metabolites produced after the degradation of GA hydrogels in vivo are harmless to the body, reducing the immune rejection or toxic reactions that may be caused by traditional synthetic materials. However, there are currently no reports of using a compound of the traditional Chinese medicine TP and GA for the treatment of malignant melanoma.

[0005] Chinese patent document CN118252787A discloses a controllable sequential release multilayered traditional Chinese medicine microneedle and its preparation method. Chinese patent document CN111568854A discloses a preparation method for a topical triptolide transdermal microneedle patch. However, no reports have been found regarding a glycyrrhizic acid hydrogel bilayer microneedle for treating malignant melanoma, its preparation method, and its application. Summary of the Invention

[0006] The purpose of this invention is to provide a glycyrrhizic acid hydrogel bilayer microneedle for the treatment of malignant melanoma, its preparation method and application. The microneedle can effectively inhibit tumor growth while reducing the inflammatory problems that the microneedle may cause to the skin during the transdermal process, while ensuring transdermal efficiency.

[0007] This invention utilizes glycyrrhizic acid as a carrier to encapsulate triptolide (TP), which can reduce TP toxicity, increase its solubility, and exert anti-inflammatory effects. This invention combines the traditional Chinese medicine triptolide (TP) with glycyrrhizic acid (GA). GA not only exerts its inherent "detoxification" and "anti-inflammatory" activities but also effectively encapsulates TP by forming a hydrogel carrier, thereby improving the solubility of poorly soluble drugs in water. It is a drug carrier with good biocompatibility and low toxicity. This invention uses microneedles as the primary treatment method and PDT as an adjunct therapy, loading TP and dihydroporphyrin e6 (Ce6) into a self-assembled GA hydrogel to exert a synergistic anti-malignant melanoma (MM) effect; simultaneously, it reduces TP toxicity and exerts the anti-inflammatory effect of GA itself.

[0008] In a first aspect, the present invention provides a glycyrrhizic acid hydrogel bilayer microneedle, comprising a drug-loaded needle tip layer and a hydrogel base layer, wherein the drug-loaded needle tip layer is made of glycyrrhizic acid (GA) hydrogel loaded with triptolide (TP) and photosensitizer dihydroporphyrin e6 (Ce6) and excipients, and the hydrogel base layer is made of glycyrrhizic acid (GA) hydrogel and excipients.

[0009] Furthermore, the drug-loaded needle tip layer is prepared from a drug-loaded solution, which is composed of a drug carrier, a drug, and excipients; the drug carrier is a self-assembled glycyrrhizic acid hydrogel, which contains the drugs TP and Ce6; the excipients are hyaluronic acid (HA) and polyvinylpyrrolidone (PVP K30).

[0010] The glycyrrhizic acid hydrogel has a glycyrrhizic acid concentration of 1-8 wt%; the drug loading solution has a TP to GA mass ratio of 1:100; and the drug loading solution has a dihydroporphyrin e6 (Ce6) concentration of 5 μM.

[0011] The hydrogel base layer is prepared from a base layer solution, which is composed of glycyrrhizic acid hydrogel and excipients, namely hyaluronic acid (HA) and polyvinylpyrrolidone (PVP K30).

[0012] Furthermore, the mass ratio of hyaluronic acid to polyvinylpyrrolidone in the excipients is 2:1. The mass percentage of hyaluronic acid in the bilayer microneedles is 5 wt%-30 wt%.

[0013] The self-assembling glycyrrhizic acid hydrogel comprises the following components: 1-8 wt% glycyrrhizic acid, purified water, and pH adjusters (HCl and NaOH), with a pH of 2-7. The preparation method includes the following steps: placing GA and an appropriate amount of purified water into a vial, and heating the vial in a heated magnetic stirrer (heating temperature 40℃-100℃) while stirring; adding a small amount of pH adjuster during the dissolution process; and storing the vial at 4℃ after all GA has dissolved.

[0014] A second aspect of the present invention provides a method for preparing the glycyrrhizic acid hydrogel bilayer microneedles as described above, which is obtained by repeatedly filling and applying negative pressure to the tip layer drug-loaded solution and the base layer solution, and finally drying them; specifically, it includes the following steps:

[0015] (A) Prepare the tip layer drug loading solution, namely the above-mentioned GA hydrogel solution containing TP and Ce6 and excipients HA and PVPK30; inject the tip layer drug loading solution into the polydimethylsiloxane (PDMS) mold and place it in a suitable temperature and vacuum environment for 15 min; add the drug loading solution again and place it in a suitable temperature and vacuum environment for 10 min; repeat the above steps once to ensure that the drug loading solution completely fills the tip of MNs; dry overnight at a suitable temperature.

[0016] (B) Prepare the base layer solution, namely blank GA hydrogel solution and excipients HA and PVP K30; fill the base layer solution into the dried MNs mold, dry it again overnight under suitable temperature conditions, and peel the microneedles off the mold along the edge to obtain a double-layer drug-loaded microneedle.

[0017] Furthermore, the suitable temperature is 25℃-55℃, and the vacuum environment conditions are -0.1 MPa to -0.8 MPa.

[0018] Furthermore, the preparation method of the needle tip layer drug-loaded solution is as follows: First, TP is sonicated with methanol until completely dissolved. During the dissolution of TP, Ce6 is added. Then, GA is poured into the methanol solution containing TP. The solution is then evaporated on a rotary evaporator to form a uniform film. After hydration with water for injection and heating to dissolve, the solution is refrigerated at 4°C to obtain a stable hydrogel.

[0019] The preparation method of the base layer solution is as follows: GA is placed in a round-bottom flask, purified water is added, and the solution is dissolved at 80°C.

[0020] The rotary evaporator has a rotation speed of 50 rpm to 300 rpm; a rotary evaporation temperature of 25℃ to 60℃; and a hydration heating temperature of 40℃ to 100℃.

[0021] A third aspect of the present invention provides the application of the glycyrrhizic acid hydrogel bilayer microneedles as described above in the preparation of a medicament for treating malignant melanoma.

[0022] The advantages of this invention are:

[0023] 1. This invention provides a method for preparing a self-assembled glycyrrhizic acid hydrogel and a double-layer drug-loaded microneedles for treating malignant melanoma. The method utilizes the detoxification and anti-inflammatory activity of GA and its ability to self-assemble into a hydrogel to make it a natural drug delivery carrier, thereby solving a series of problems caused by the poor water solubility, easy elimination in vivo, and high toxicity of TP, such as damage caused when TP is directly applied to the skin.

[0024] 2. The bilayer drug-loaded hydrogel microneedles prepared by the method of this invention have good mechanical strength, ensuring safe drug delivery. In addition, the drug loading of the microneedle patch can reach 15.62±0.07μg / mg, and after 15 days of storage, it remains stable at 15.51±0.17μg / mg, providing a basis for the long-term preservation of the microneedles. Solubility, skin healing, and hemolysis experiments show that the microneedles have good solubility in vivo, and there are no obvious adverse reactions and high safety after removal from MNs after a certain period of application. The cumulative transdermal release of the microneedles in vitro is more than 80%, proving that the drug can be effectively released from MNs, and the drug can be released more completely in the acidic environment of tumors.

[0025] 3. Through anti-cancer and anti-inflammatory experimental studies, this invention has found that the double-layered drug-loaded microneedles can effectively exert the anti-cancer effect of TP. At the same time, the MNs base can reduce oxidative stress and promote cell proliferation through GA, which has anti-inflammatory effects, thereby achieving anti-inflammatory effects on the skin. Attached Figure Description

[0026] Figure 1 This is a diagram showing the optimal ratio of TP to GA in Embodiment 1 of the present invention;

[0027] Figure 2 This is an electron micrograph of the GA hydrogel loaded with TP in Example 1 of the present invention;

[0028] Figure 3 The displacement percentage at 500 gf for different MNs matrix ratios (HA:PVP K30) in Example 2 of the present invention;

[0029] Figure 4 This is a morphological diagram of TP-GA@MNs in Embodiment 3 of the present invention;

[0030] Figure 5 This is a mechanical strength diagram of TP-GA@MNs in Embodiment 3 of the present invention;

[0031] Figure 6 This is a solubility diagram from Example 3 of the present invention;

[0032] Figure 7 This is a diagram of the healing process of nude mouse skin after microneedle pressure, as shown in Example 3 of the present invention.

[0033] Figure 8 This is the in vitro transdermal cumulative release curve in Example 3 of the present invention, n=3;

[0034] Figure 9 This is a cell invasion diagram from Example 4 of the present invention;

[0035] Figure 10 This is a diagram of intracellular reactive oxygen species in Example 4 of the present invention;

[0036] Figure 11 This is a diagram of the anti-inflammatory effect in Example 5 of the present invention, where A, B, and C are in vitro anti-inflammatory ELISA experimental diagrams, and D, E, and F are in vivo anti-inflammatory effect results diagrams;

[0037] Figure 12 This is a diagram illustrating the anticancer effect in Example 6 of the present invention;

[0038] Figure 13 This is a flowchart illustrating the preparation process of bilayer microneedles. Detailed Implementation

[0039] The specific embodiments provided by the present invention will be described in detail below with reference to the examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in this technical field.

[0040] Example 1: Self-assembled glycyrrhizic acid hydrogel

[0041] 1.1 Optimal Formulation for Self-Assembled Glycyrrhizic Acid Hydrogels

[0042] Previous studies have found that aqueous GA solutions can self-assemble into hydrogels under heating conditions, but the conditions and influencing factors for this formation remain unknown. Therefore, this invention first examines the effects of different influencing factors on the gelation of aqueous GA solutions, mainly including the following aspects:

[0043] (1) Concentration: Accurately weigh a fixed amount of GA, dissolve it in water, and prepare a stock solution of a certain concentration. Dilute the stock solution to prepare a series of surfactant solutions of different concentrations. Set the equilibrium temperature and place all solutions in a constant temperature water bath. Measure the surface tension of each concentration solution in sequence according to the surface tension meter instruction manual, record the surface tension values ​​at different concentrations, ensure that each concentration is measured at least three times, and calculate the average value. Plot the corresponding curve with GA concentration as the horizontal axis and surface tension as the vertical axis. The concentration value corresponding to the inflection point of the curve represents the critical micelle concentration (CMC). Using CMC as the lowest concentration reference, set up GA aqueous solutions of different concentrations and observe the gelation state.

[0044] (2) Different pH dispersion media: The difference in gelation behavior of GA was observed when the pH was set at 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 and 7.

[0045] (3) Heating temperature: Prepare a certain concentration of GA aqueous solution and heat it in a water bath at different temperatures (40℃, 50℃, 60℃, 70℃, 80℃, 90℃, 100℃). Stir slowly until the sample is completely dissolved and continue to heat in the water bath for a period of time to form a sol. Take it out and cool it at room temperature for a certain period of time to examine whether a gel can be formed at the heating temperature.

[0046] (4) Cooling method: The GA aqueous solution was heated in a water bath to form a sol. After being taken out, the effects of different cooling methods, including gradual cooling in a water bath, gradual cooling at room temperature, and sudden cooling at 4℃ or 0℃, on gel formation were investigated.

[0047] 1.2 The optimal ratio of glycyrrhizic acid to triptolide

[0048] The optimal ratio of the two drugs was evaluated using the CCK-8 assay. HaCaT cells in the logarithmic growth phase were digested, centrifuged, and resuspended in an appropriate amount of complete culture medium. Cells were counted and diluted to a density of 1 × 10⁻⁶. 5 Cells were seeded at 100 μL / mL into 96-well plates. The plates were incubated at 37°C with 5% CO2 for 24 h. After cell attachment, the supernatant was discarded and the cells were washed with PBS. 100 μL of different concentrations of TP and different TP:GA ratios (1:50, 1:100, 1:150) were added to the experimental groups, while 100 μL of DMEM was added to the control and blank groups. Each group had six replicates, and incubation was continued for 48 h under the same conditions. After incubation, the supernatant was discarded from the 96-well plates. Under light-protected conditions, 100 μL of 10% CCK-8 DMEM solution was added to each well. The plates were shaken for 60 s using a microplate reader to ensure uniform mixing. After standing at room temperature for 2 h, the absorbance of each well was measured at 450 nm using a microplate reader.

[0049] The results are as follows Figure 1 As shown, when TP was used alone, cell viability decreased significantly with increasing concentration, indicating that TP has a strong cytotoxic effect. When TP and GA were combined in different ratios (1:50, 1:100, 1:150), the cell viability was higher than when TP was used alone, indicating that GA can effectively reduce the cytotoxicity of TP. In particular, the cell viability was highest when the mass ratio of TP to GA was 1:100, indicating that the two drugs had the best effect at this ratio.

[0050] 1.3 Preparation of TP-loaded self-assembled GA hydrogels

[0051] TP was dissolved in methanol and placed in a round-bottom flask. Then, an appropriate amount of GA was added to the round-bottom flask. The round-bottom flask was attached to a rotary evaporator and rotary evaporated to form a uniform thin film. After hydration with a certain amount of water for injection, the solution was heated and stirred on a stirrer until it became a transparent solution. Then, it was placed directly in a 4°C environment and refrigerated for a period of time to obtain a stable TP-containing GA hydrogel.

[0052] 1.4 Electron microscopy of self-assembled glycyrrhizic acid hydrogel

[0053] A portion of the formed hydrogel sample was cut off with a blade and then placed in a temperature-controlled sample cup. After the sample cooled to -23°C, it was moved into the sample chamber for imaging.

[0054] Figure 2 It can be observed that the hydrogel exhibits a uniform porous structure, and these pores facilitate drug loading and release. The relatively uniform size and distribution of the pores indicate that TP is evenly distributed in the GA self-assembled hydrogel, without obvious aggregation. Furthermore, the surface of the hydrogel is smooth, without obvious cracks or collapses, confirming that the hydrogel generated by GA self-assembly possesses good structural properties.

[0055] Example 2: Preparation of a bilayer drug-loaded microneedle for treating melanoma

[0056] 2.1 Screening of optimal formulations for microneedle excipients

[0057] Common materials such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP K30), and hyaluronic acid (HA, 40-100KD, 10KD) were screened and prepared into 30% w / v sample solutions. These solutions were poured into microneedle molds and injection molding was performed at 30℃ and -0.5 MPa for 30 min. Air bubbles generated in the cavity were agitated using a pipette, and this process was repeated 2-3 times until no more air bubbles appeared in the grooves. The molds were then dried in an oven at 32℃ for 5 h. The microneedles were gently peeled off from the molds along the edges using tweezers to obtain the microneedle patches. The needle tip morphology was examined under a microscope. The selected materials were mixed and prepared into microneedle solutions at ratios of HA:PVP K30 of 1:1, 1:2, 1:3, 2:1, and 3:1, respectively, and then fabricated into microneedles. The prepared microneedles were pressed under a texture analyzer at 500 gf, and the degree of breakage of the microneedles was observed under a microscope.

[0058] This embodiment selected several matrix materials commonly used in the preparation of matrix elements (MNs), including PVA, PVP K30, and HA. Corresponding MNs samples were prepared while maintaining consistent material concentrations. The physical properties of the materials, such as hardness, viscosity, and moldability, were evaluated. Based on a comprehensive consideration of the experimental results, PVP K30, with its higher hardness, and HA 10KD, with its moderate viscosity and ease of molding, were selected as the preferred materials for subsequent MNs preparation.

[0059] Figure 3 To investigate the preparation of microneedles (MNs), different ratios of PVP K30 and HA 10KD were mixed, and a force of 500 gf was applied to the MNs samples to observe their fracture behavior. Since a 3:1 ratio of HA to PVP K30 failed to form microneedles under the same drying time conditions, this ratio was excluded. The lowest fracture rate, only 20% of the total, was observed when the ratio of HA to PVP K30 was 2:1. Therefore, a 2:1 ratio of HA to PVP K30 was ultimately determined as the standardized ratio for MN preparation.

[0060] 2.2 Preparation of drug-loaded solution at the needle tip and solution at the basement layer

[0061] The needle tip layer is a GA hydrogel solution containing TP and Ce6. The aforementioned TP-GA@Hydrogel is prepared by mixing 1 mg of TP with 100 mg of GA. First, 1 mg of TP is sonicated in methanol until completely dissolved. Then, 100 mg of GA is added to the methanol solution containing TP. The solution is then evaporated on a rotary evaporator to form a uniform thin film. After hydration with 5 mL of water for injection and heating to dissolve, the TP-GA@Hydrogel solution is obtained. Simultaneously, hyaluronic acid and polyvinylpyrrolidone are added to the TP-GA@Hydrogel solution in a 2:1 ratio and stirred until transparent and free of particles, thus obtaining the drug-loaded solution for the needle tip layer. Similarly, the difference between the TP-Ce6-GA@MNs solution and the TP-GA@Hydrogel solution is the introduction of Ce6. An appropriate amount of Ce6 (effective concentration 5 μM) needs to be added during the TP dissolution process. Subsequent steps are the same as for TP-GA@Hydrogel to obtain the microneedle tip layer solution.

[0062] The basal layer was a GA@Hydrogel solution. 100 mg of GA was weighed into a round-bottom flask, purified water was added, and the solution was dissolved at 80°C to obtain the GA@Hydrogel solution. Simultaneously, hyaluronic acid and polyvinylpyrrolidone were added to the GA@Hydrogel solution at a ratio of 2:1, and the mixture was stirred until clear and free of particles, thus obtaining the basal layer drug-loaded solution. The HA content accounted for 15 wt% of the total volume of the tip layer drug-loaded solution and the basal layer solution.

[0063] 2.3 Preparation of bilayer drug-loaded microneedles

[0064] The drug-loaded solution at the tip was injected into a polydimethylsiloxane (PDMS) mold and placed in a suitable temperature and vacuum environment for 15 min. The drug-loaded solution was added again and placed in a suitable temperature and vacuum environment for 10 min. The above steps were repeated once to ensure that the drug-loaded solution completely filled the tip of the MNs. The MNs were dried overnight at a suitable temperature. The base layer solution was then filled onto the dried MNs mold and dried overnight again at a suitable temperature. The microneedles were then peeled off from the mold along the edge to obtain a double-layered drug-loaded microneedle.

[0065] Example 3: Characterization of bilayer drug-loaded microneedles for the treatment of melanoma

[0066] 3.1 Morphology

[0067] The MNs sample is placed with the tip facing upwards and the substrate layer facing downwards, and fixed to the flat sample stage using carbon conductive tape. The electron microscope chamber door is opened, and the sample stage is placed inside the electron microscope chamber. Electron microscopy imaging is performed at 45° side, side, and top viewing angles, with magnifications of 300 μm, 500 μm, and 3 mm, respectively.

[0068] Figure 4 The morphology of MNs is shown in the images. MNs were imaged from above and the side at different magnifications (300 μm, 500 μm, and 3 mm) using a scanning electron microscope. The needle structure of MNs is a regular square pyramid, with a height of 900 μm, a base side length of 400 μm, and a needle spacing of 800 μm, and its morphology remains intact.

[0069] 3.2 Mechanical strength (mechanical properties, skin puncture, HE staining)

[0070] Mechanical properties: Measurements were performed using a texture analyzer TA.XT plusC. The microneedle was placed with the tip facing upwards and the basal layer downwards. A single test was conducted, and the stop point was set as the target location. A TA2 probe with a diameter of 2 mm was used, with a single indentation of 0.7 mm, a test speed of 0.02 mm / s, and a trigger force of 3 gf.

[0071] In vitro skin puncture experiment: Undamaged isolated pig skin samples were selected, and microneedles were applied with appropriate force to puncture the skin. After continuous pressure for 30 seconds, the microneedles were removed, and the puncture effect of the microneedles on the pig skin was observed.

[0072] H&E staining analysis: After the above puncture experiment was completed, the skin tissue samples after puncture were stained with paraformaldehyde solution, and the tissue sections were observed and analyzed under a microscope.

[0073] Figure 5The force-displacement diagrams show that MNs with a HA:PVP K30 ratio of 2:1 maintain structural stability under greater pressure and are less prone to deformation or breakage. MNs formed pinholes of uniform depth on pigskin. Microscopic observation of H&E-stained skin tissue sections clearly revealed the microneedle channels left by the MNs after puncture, indicating that MNs possess the ability to penetrate the skin. Furthermore, MNs exhibit good mechanical strength under this preparation process.

[0074] 3.3 In vitro dissolution

[0075] In vivo solubility assessment: Prepared blank MNs patches (n=3) were selected and pressed onto the backs of clean, wrinkle-free nude mice for 1 min, and then fixed to the backs of the mice with non-woven medical tape. The tape was removed at 15 min, 30 min and 45 min, and the solubility of the MNs patches was observed under a microscope. Figure 6 This indicates that MNs can be completely dissolved in nude mice.

[0076] 3.4 Skin healing

[0077] The prepared MNs were gently pressed onto the skin of nude mice and left in this position for approximately 5 minutes before being carefully removed. The recovery of the mouse skin was carefully observed and checked at time points of 0 seconds, 3 minutes, 6 minutes, and 10 minutes. To ensure accuracy, photos of the skin at each time point were taken using a mobile phone for subsequent analysis and research.

[0078] Figure 7 The results showed that regular pinholes appeared on the skin of nude mice after MNs treatment, and the skin recovered to its initial state by 10 minutes, with no bleeding observed. This indicates that MNs can penetrate animal skin without causing significant tissue damage.

[0079] 3.5 In vitro transdermal cumulative release

[0080] This experiment used the Franz diffusion cell method for determination. The effective diffusion area of ​​the diffusion cell was 1.77 cm², and PBS (pH 5.5 and pH 7.4) containing 0.5% Tween 80 and 10% ethanol was used as the diffusion medium. 10 mL of diffusion medium was added to the diffusion cell, and a piece of pigskin of appropriate size (2 cm × 2 cm) was placed at the upper end of the diffusion cell opening, ensuring the stratum corneum was facing upwards. The TP-Ce6-GA@MNs patch was fixed onto the pigskin, pressure was applied with the thumb, and finally sealed with sealing film. The mixture was stirred uniformly in a constant temperature water bath at 32℃ and 300 rpm. 2 mL samples of the transdermal receiving solution were taken at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h, and an equal volume of fresh medium was added to the diffusion cell. The obtained transdermal receiving solution was ultrasonically demulsified with methanol to ensure complete dissolution of TP. The transdermal permeation rate of TP under different pH conditions was determined by HPLC. The sample was filtered through a 0.45 μm filter membrane before injection. Based on the experimental results, a cumulative transdermal permeation rate curve of the drug was plotted.

[0081] Figure 8 The results showed that, under pH 5.5 conditions, after 24 hours of MN patch treatment, the cumulative transdermal drug release reached 82.66% ± 1.68%, while under a neutral environment at pH 7.4, this value was 71.91% ± 1.62. Under both conditions, the cumulative drug release exceeded 70%, indicating that the drug can dissolve and be effectively released from MNs. These experimental results demonstrate that drug release is more complete under an acidic environment at pH 5.5, thus confirming that an acidic environment promotes drug release.

[0082] Example 4: Investigation of the photodynamic properties of TP-Ce6-GA@MNs

[0083] 4.1 Cell Invasion

[0084] Pre-cooled DMEM was diluted 8 times with Matrigel gel, and 50 μL was added to each well of the Transwell chamber. The chamber was incubated at 37°C until the Matrigel gel solidified. A375 cells in logarithmic growth phase were then introduced at a rate of 2 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of 100 cells / well in 24-well plates. After cell attachment, the cells were treated according to the following groups: blank medium, GA, TP, TP-GA, TP-Ce6-GA (-), and TP-Ce6-GA (+). The TP concentration in all experimental groups was set at 100 ng / mL. The TP-Ce6-GA (+) group required intravenous infusion at 660 nm wavelength and 50 W / cm² after the infusion. 2Irradiate the cells with light at the specified intensity for 5 min, and incubate all treatment groups for a total of 4 h. Then, digestion, centrifugation, and resuspension in DMEM are performed. The treated cell suspension is seeded into the upper chamber of a Transwell incubator lined with Matrigel, while the lower chamber is incubated with DMEM medium containing 10% FBS as a chemokine. The Transwell chambers are incubated at 37°C with 5% CO2 for 24 h. Finally, uninvaded cells in the upper chamber are gently wiped away with a cotton swab, and cells that have invaded the lower chamber are fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The number of cells invading the lower chamber is counted under a microscope to assess the invasive ability of each group.

[0085] Invasion rate = ×100%

[0086] Figure 9 The image shows the results of the cell invasion experiment. The cell invasion rate of the TP-Ce6-GA (+) group after light irradiation was 31.47%, which was the lowest among the six groups. This indicates that the synergistic effect of TP and Ce6 achieved the strongest inhibitory effect on cell invasion.

[0087] 4.2 Detection of intracellular reactive oxygen species levels in A375 cells

[0088] With 6×10 per hole 5 A375 cells were seeded at a density of 100 cells / well in 6-well plates and incubated for 24 h. Subsequently, the cell supernatant was removed, and the cells were washed twice with PBS. Then, DMEM, TP, TP-Ce6-GA(-), and TP-Ce6-GA(+) were added, respectively, where (+) indicates 660 nm, 50 W / cm². 2 The light treatment lasted for 5 min. After 4 h of drug treatment, the supernatant was removed, and the cells were washed twice with PBS. Blank culture medium containing 5 μM H2DCFDA was added, and the cells were incubated at 37°C in the dark for another 20 min. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, and then washed three times with PBS. Finally, the slides were removed, dried, and inverted onto a glass slide containing 5 μL of mounting medium. The cells were photographed using a laser confocal microscope at an excitation wavelength of 488 nm and an emission wavelength of 525 nm (Ex / Em=488 / 525 nm), and the fluorescence intensity was statistically analyzed.

[0089] Figure 10The study observed that the green fluorescence of the TP group was not significant, while the two formulations loaded with Ce6 produced green fluorescence exceeding that of the TP group alone. This indicates that the introduction of Ce6 enhances the generation of ROS. We also noted that in the absence of 660nm light, there was no significant difference in the green fluorescence intensity between the Ce6 and TP groups; however, under illumination, the generation of ROS increased significantly. In summary, Ce6 significantly enhances intracellular ROS generation under laser irradiation, inducing oxidative stress in cells and ultimately leading to apoptosis. This is crucial for the therapeutic effect of photodynamic therapy.

[0090] Example 5: Evaluation of the anti-inflammatory effect of TP-Ce6-GA@MNs

[0091] 5.1 Effects of GA on the expression of inflammatory factors in skin cells

[0092] With 2.5×10 per hole 5 HaCaT cells in logarithmic growth phase were seeded into 24-well plates and incubated for 24 h. The adherent HaCaT cells were stimulated with 100 ng / ml TNF-α for 24 h, and the cell supernatant was collected. The levels of IFN-γ, IL-6, and IL-1β were measured according to the ELISA kit instructions. Next, 100 ng / ml TNF-α and the other drugs were added to the adherent HaCaT cells, with TNF-α serving as the control group. After 24 h of co-culture, the concentrations of IFN-γ, IL-6, and IL-1β in the treated cells were again measured using an ELISA kit.

[0093] 5.2 Investigation of anti-inflammatory effects in vivo

[0094] Skin tissue samples were excised from the microneedle administration site, i.e., the skin layer above the tumor. The samples were thoroughly rinsed with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood. Then, 0.1 g of tissue was taken from each sample and minced. The minced tissue was mixed with PBS at a 1:9 ratio and homogenized thoroughly in a tissue homogenizer (70 Hz, 300 s). To further lyse the tissue cells, the homogenate was subjected to sonication or repeated freeze-thaw cycles to ensure homogeneity. Finally, the homogenate was centrifuged at 5000×g for 10 min at 4°C, and the supernatant was collected. The levels of IFN-γ, IL-6, and IL-1β in the tissue samples were determined according to the standard operating procedure of the ELISA kit.

[0095] Figure 11 The image shows the results of an anti-inflammatory experiment, which verified that GA has a significant anti-inflammatory effect both in vitro and in vivo.

[0096] Example 6: Evaluation of the antitumor effect of TP-Ce6-GA@MNs

[0097] Thirty-six BALB / c nude mice were randomly selected and divided into 6 groups of 6 mice each. Each group received one patch of blank MNs, GA@MNs, TP@MNs, TP-GA@MNs, TP-Ce6-GA@MNs (-), or TP-Ce6-GA@MNs (+) on days 0, 3, 6, 9, 12, and 15, respectively. The patches were secured with adhesive bandages and further reinforced with medical-grade pressure-sensitive adhesive to prevent detachment. Specifically, in the TP-Ce6-GA@MNs (+) group, the mice were placed in an ether tank for general inhalation anesthesia 4 hours after administration. After anesthesia, the mice were removed and photodynamic laser irradiation (wavelength 660 nm, power density 50 mW / cm²) was applied to the microneedle administration site. 2 The treatment lasted for 10 minutes, with the light source approximately 2 cm away from the tumor site. The patch was removed 24 hours after administration, and the microneedle-pressed area was cleaned with povidone-iodine. The next administration was given 48 hours later. Administration was repeated every 3 days for a total of 6 administrations. During the experiment, the mice's condition was monitored daily, and the short diameter (a) and long diameter (b) of the tumors in each group of nude mice were measured before each administration. The results were calculated using the formula TV = a. 2 The tumor volume was calculated using the formula × b / 2. Two days after the last administration, mice were euthanized by cervical dislocation, and the tumor tissue was removed and weighed. The tumor inhibition rate was calculated using the formula TIR = (Wcontrol - Wtreat) / Wcontrol × 100%, the relative tumor volume was calculated using the formula RTV = Vt / V0 × 100%, and the relative tumor proliferation rate was calculated using the formula T / C = RTVtreat / RTVcontrol × 100%.

[0098] Figure 12 - A shows the solid tumor images of the control group and different microneedle formulation groups after drug treatment, visually reflecting the in vivo anti-tumor effects of each formulation group. On day 18, the mean tumor weights of the control group, GA@MNs group, TP@MNs group, TP-GA@MNs group, TP-Ce6-GA@MNs (-) group, and TP-Ce6-GA@MNs (+) group were 0.46 g, 0.40 g, 0.17 g, 0.16 g, 0.18 g, and 0.01 g, respectively. Figure 12 -B), the average tumor volume in each group increased from the initial 100 mm. 3 Up to 1090.79 mm 3 948.15 mm 3 379.71 mm 3373.01 mm 3 297.19 mm 3 90.81 mm 3 ( Figure 12 -C), the tumor inhibition rates of the groups were 16.11±8.34%, 61.38±14.19%, 61.33±9.71%, 65.75±10.85%, and 94.21±3.50%, respectively. Figure 12 -D). The results of this study indicate that the TP-Ce6-GA@MNs (+) group exhibited the best tumor-suppressing effect, significantly superior to the TP@MNs monotherapy group, the TP-GA@MNs group, and the TP-Ce6-GA@MNs (-) group without laser irradiation. This further confirms the significant synergistic anti-tumor effect of the cytotoxic drug TP combined with photodynamic therapy.

[0099] Figure 13 The fabrication process of the bilayer microneedles was demonstrated. First, the drug-loaded solution for the needle tip layer was injected into a polydimethylsiloxane (PDMS) mold and dried under vacuum for 15 min. The drug-loaded solution was then added to the PDMS a second time, and the mixture was dried under vacuum for another 10 min. This process was repeated once to ensure the mixture completely filled the tips of the microneedles (MNs). The mixture was then dried overnight at 32°C to prepare the needle tip layer of the MNs patch. Finally, a base layer solution containing a GA gel was injected into the dried PDMS mold, and the mixture was again dried overnight at 32°C to obtain the bilayer TP-Ce6-GA@MNs drug-loaded microneedle patch.

[0100] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A glycyrrhizic acid hydrogel bilayer microneedle, characterized in that, It includes a drug-loaded needle tip layer and a hydrogel base layer. The drug-loaded needle tip layer is made of glycyrrhizic acid hydrogel loaded with triptolide and dihydroporphyrin e6 and excipients. The hydrogel base layer is made of glycyrrhizic acid hydrogel and excipients. The excipients are hyaluronic acid (HA) and polyvinylpyrrolidone (PVP K30).

2. The glycyrrhizic acid hydrogel bilayer microneedles according to claim 1, characterized in that, The glycyrrhizic acid hydrogel has a glycyrrhizic acid concentration of 1-8 wt%; the drug loading solution has a triptolide to glycyrrhizic acid mass ratio of 1:100; and the drug loading solution has a dihydroporphyrin E6 concentration of 5 μM.

3. The glycyrrhizic acid hydrogel bilayer microneedles according to claim 1, characterized in that, The mass ratio of hyaluronic acid to polyvinylpyrrolidone in the excipients is 2:1; the mass ratio of hyaluronic acid is in the range of 5 wt%-30 wt%.

4. A method for preparing glycyrrhizic acid hydrogel bilayer microneedles as described in any one of claims 1-3, characterized in that, Includes the following steps: (A) Prepare a drug loading solution for the needle tip layer, namely a solution of glycyrrhizic acid hydrogel containing triptolide and dihydroporphyrin e6 and excipients HA and PVP K30; inject the drug loading solution into a polydimethylsiloxane mold and place it in a suitable temperature and vacuum environment for 15 min; add the drug loading solution again and place it in a suitable temperature and vacuum environment for 10 min; repeat the above steps once to ensure that the drug loading solution completely fills the tip of MNs; dry overnight at a suitable temperature. (B) Prepare a base layer solution, namely a solution containing glycyrrhizic acid hydrogel and excipients HA and PVP K30; fill the base layer solution onto the dried MNs mold, dry it again overnight at a suitable temperature, and then peel the microneedles off the mold along the edge to obtain a double-layer drug-loaded microneedle.

5. The preparation method according to claim 4, characterized in that, The suitable temperature is 25℃-55℃, and the vacuum environment conditions are -0.1 MPa to -0.8 MPa.

6. The preparation method according to claim 4, characterized in that, The preparation method of the needle tip drug-loaded solution is as follows: First, TP is sonicated with methanol until completely dissolved. During the dissolution of TP, Ce6 is added. Then, GA is poured into the methanol solution containing TP and Ce6. The solution is then evaporated into a uniform thin film using a rotary evaporator. After hydration and dissolution with water for injection, HA and PVP K30 are added to the above-dissolved solution at a ratio of 2:

1. The solution is stirred until it is transparent and free of particles, thus obtaining the needle tip drug-loaded solution.

7. The preparation method according to claim 4, characterized in that, The preparation method of the base layer solution is as follows: GA is dissolved in a round-bottom flask with purified water at 80°C. At the same time, HA and PVP K30 are added to the dissolved solution in a ratio of 2:

1. The mixture is stirred until it is transparent and free of particles to obtain the base layer solution.

8. The preparation method according to claim 6, characterized in that, The rotary evaporator has a rotation speed of 50 rpm to 300 rpm; a rotary evaporation temperature of 25℃ to 60℃; and a hydration heating temperature of 40℃ to 100℃.

9. The use of a glycyrrhizic acid hydrogel bilayer microneedle as described in any one of claims 1-3 in the preparation of a medicament for treating malignant melanoma.

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