Zingiberone-loaded self-assembled nano-drug, soluble microneedle patch, and preparation method and application of zingiberone-loaded self-assembled nano-drug

Abstract

The invention belongs to the technical field of biological medicine, and provides a zingiberone-loaded self-assembled nano-drug, a soluble microneedle patch as well as a preparation method and application thereof, and the zingiberone-loaded self-assembled nano-drug comprises zingiberone, indocyanine green and a polymer mPEG-PLA in a mass ratio of (1.5-5): (0.35-1.4): (10-20). The invention also provides a preparation method of the zingiberone-loaded self-assembled nano-drug. The invention further provides a soluble microneedle patch, the soluble microneedle patch comprises the zingiberone-carrying self-assembled nano-drug, and the soluble microneedle patch comprises a patch substrate and a needle head arranged on the surface of the patch substrate; the needle head is prepared from a ball-carrying zingerone self-assembled nano-drug and a soluble matrix material. The zingiberone-loaded self-assembled nano-drug provided by the invention has an anti-tumor effect, can be used for treating melanoma, and improves the solubility and bioavailability of zingiberone.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a self-assembled nanomedicine loaded with gingerone, a soluble microneedle patch, its preparation method and application. Background Technology

[0002] Zermombone (ZER) is isolated from ginger volatile oil. Existing studies have shown that zermombone can significantly reduce melanin accumulation in mouse B16F10 melanocyte-producing cells stimulated by melanocyte-stimulating hormone (-MSH). Although zermombone has excellent anti-cancer properties, its therapeutic application is hampered by poor water solubility, resulting in poor subsequent absorption, bioavailability, and delivery to targeted tissues and organs. Currently, research on improving the solubility and bioavailability of zermombone is very limited.

[0003] Melanoma is one of the most aggressive and treatment-resistant skin cancers, originating from the malignant transformation of nerves and melanocytes in the skin. It accounts for approximately 1% of skin cancer cases but is the leading cause of death from skin cancer. While surgical excision is currently the most common treatment for melanoma, it has drawbacks such as limited surgical scope, difficulty in completely inspecting excision margins, high risk of local recurrence, and poor patient compliance due to the invasive nature of the procedure. Other treatments for melanoma include photothermal therapy (PTT), which has emerged as a potential anti-tumor treatment. Near-infrared dye indocyanine green (ICG) can be used for photothermal therapy to inhibit tumor growth or induce tumor cell apoptosis; however, ICG has poor photostability and is oxygen-dependent.

[0004] Methoxy polyethylene glycol-polylactic acid (mPEG-PLA) is a biodegradable and biocompatible amphiphilic polymer with promising applications in drug delivery and gene delivery systems.

[0005] Transdermal microneedle drug delivery, as an emerging drug delivery system, can increase drug concentration and reduce adverse reactions during treatment. Drug delivery through the skin increases drug distribution in deeper tumor areas and reduces leakage. Furthermore, it offers advantages such as simplicity, safety, and effectiveness for mild pain. Nanomedicines are revolutionizing traditional cancer chemotherapy by improving drug pharmacokinetics and optimizing the biodistribution of therapeutic drugs. Dissolvable nanoparticles (DMNs) are the most widely studied type of nanoparticle in recent years. They are created by encapsulating drugs in water-soluble substrates (such as hyaluronic acid), which then dissolve or polymerize. Injecting dissolved nanoparticles into the skin, followed by drug release, makes it easier to achieve higher drug loading rates. Moreover, soluble microneedles can achieve uniform three-dimensional channel distribution, improving intratumoral drug accumulation and reducing systemic side effects.

[0006] For example, Chinese patent CN118267343A discloses a method for preparing photothermal-photodynamic synergistic antibacterial freeze-dried microneedles. It uses hyaluronic acid to encapsulate ZIF-8 nanoparticles loaded with indocyanine green as a metal-organic framework nanocarrier, which improves the efficiency of ICG photodynamic therapy and at the same time endows it with acidic pH response characteristics, thereby enhancing the drug's responsiveness in the bacterial microenvironment.

[0007] Therefore, researching safe and efficient composite nanoparticles loaded with gingerone to broaden their application prospects in the pharmaceutical field is of great significance. Summary of the Invention

[0008] This invention proposes a self-assembled nanomedicine loaded with gingerone, a soluble microneedle patch, its preparation method, and its application. The nanomedicine has anti-tumor effects and can be used to treat melanoma. It improves the bioavailability of gingerone and has high safety, enabling efficient, non-invasive, and painless treatment.

[0009] The technical solution of this invention is implemented as follows: Technical Topic 1 A self-assembled nanomedicine loaded with shogazone comprises shogazone (ZER), indocyanine green (ICG), and polymer mPEG-PLA in a mass ratio of 1.5-5:0.35-1.4:10-20.

[0010] Preferably, the mass ratio of gingerone (ZER), indocyanine green (ICG), and the polymer is 1.5-5:0.7:10-20.

[0011] Preferably, the mass ratio of gingerone (ZER), indocyanine green (ICG), and the polymer is 1.5-4:0.7:20.

[0012] Preferably, the mass ratio of gingerone (ZER), indocyanine green (ICG), and polymer mPEG-PLA is 4:0.7:20.

[0013] Preferably, the polymer mPEG-PLA is mPEG. 2000 -PLA 2000 .

[0014] Technical Theme Two This invention also provides a method for preparing the above-mentioned spheroidized gingerone self-assembled nanomedicine, comprising the following steps: Gingerol, polymer mPEG-PLA and organic solvent were mixed and stirred. Indocyanine green aqueous solution was added, and the mixture was subjected to initial ultrasonic treatment at room temperature in the dark to obtain a mixed solution. The organic solvent in the mixed solution was removed by rotary evaporation, then hydrated with ultrapure water, sonicated again, and filtered to obtain the self-assembled nanomedicine loaded with gingerone.

[0015] Preferably, the concentration of the indocyanine green aqueous solution is 1-10 mM.

[0016] Preferably, the concentration of the indocyanine green aqueous solution is 10 mM.

[0017] Preferably, the organic solvent is acetonitrile.

[0018] Preferably, the ratio of the organic solvent, gingerone, and ultrapure water is 2 mL: 1.5-5 mg: 4-10 mL.

[0019] Preferably, the ratio of the organic solvent, gingerone, and ultrapure water is 2 mL: 1.5-4 mg: 4-10 mL.

[0020] Preferably, the specific steps of the rotary evaporation include: rotary evaporation in a vacuum rotary evaporator for 18-22 minutes under reduced pressure, at a temperature of 40°C.

[0021] Preferably, the power of the initial ultrasonic treatment is 150-250W, the frequency is 35-45KHz, and the time is 5-10min.

[0022] Preferably, the power of the second ultrasonic treatment is 150-250W, the frequency is 35-45KHz, and the time is 5-10min.

[0023] Preferably, the filtration is performed using a 0.22μm filter membrane.

[0024] Technical Theme 3 The present invention also provides a soluble microneedle patch comprising the above-mentioned self-assembled spheroidized gingerone nanomedicine.

[0025] The soluble microneedle patch includes a patch substrate and a needle disposed on the surface of the patch substrate; the needle is prepared from a self-assembled nanomedicine loaded with gingerone and a soluble matrix material.

[0026] The soluble matrix material is polyvinylpyrrolidone and / or polyvinyl alcohol.

[0027] Preferably, the material of the patch substrate is hyaluronic acid.

[0028] Preferably, the material of the patch substrate is a hyaluronic acid aqueous solution with a mass concentration of 15% to 30%.

[0029] Preferably, the molecular weight of the hyaluronic acid is 40,000 to 100,000 Daltons.

[0030] Preferably, the hyaluronic acid has a molecular weight of 50,000 Daltons.

[0031] Preferably, the ratio of gingerone-loaded self-assembled nanomedicine, polyvinylpyrrolidone, and polyvinyl alcohol in the needle is 0.8-1.2 mL: 250 μg: 100 μg.

[0032] Preferably, the polyvinylpyrrolidone is PVP-K30.

[0033] Preferably, the soluble microneedle patch has a needle height of 500-700 μm, a needle tip spacing of 300-500 μm, and a needle base (i.e., the diameter of the base of each needle in a pyramid shape) diameter of 150-300 μm.

[0034] Preferably, the soluble microneedle patch contains 70-90 μg of gingerone self-assembled nanomedicine per patch, with 15*15 needles per patch.

[0035] Technical Theme 4 This invention also provides a method for preparing a soluble microneedle patch, comprising the following steps: Gingerol self-assembled nanomedicine was mixed with a soluble matrix material to obtain a needle tip mixture solution; hyaluronic acid was pre-swelled in water overnight to form a base solution. The needle tip mixture solution is poured into a mold, and the mold cavity is filled by vacuum method. Base liquid is added, then dried and demolded to obtain soluble microneedles.

[0036] The vacuum method includes the following steps: degassing in a vacuum oven (pressure 0.8 MPa) at room temperature for 15 minutes, gently scraping the surface of the mold to remove excess air bubbles, repeating the above steps 2-3 times, and scraping off excess solution.

[0037] Preferably, the drying temperature is 20℃~40℃, the air humidity is 45%~60%, and the drying time is 20-26h.

[0038] Technology Theme 5 The present invention provides the application of the above-mentioned spheroidized gingerone self-assembled nanomedicine in the preparation of drugs for anti-tumor or treatment of melanoma.

[0039] The beneficial effects of this invention are as follows: 1. This invention prepares a nanomedicine from gingerone, indocyanine green, and polymer mPEG-PLA. Gingerone and indocyanine green in this nanomedicine have synergistic anti-tumor effects and can be used to treat melanoma. This improves the bioavailability of gingerone. At the same time, this nanomedicine improves the stability and solubility of gingerone, effectively solving the problem of low bioavailability caused by the low water solubility of gingerone.

[0040] 2. The preparation method of the self-assembled spheroidized gingerone nanomedicine provided by the present invention has the advantages of being simple and easy to implement, and requiring no special equipment.

[0041] 3. The soluble microneedle patch provided by this invention is made by mixing self-assembled nanomedicine loaded with gingerone with polyvinylpyrrolidone and polyvinyl alcohol. Polyvinylpyrrolidone and polyvinyl alcohol endow the soluble microneedles with the necessary mechanical strength and toughness. The soluble microneedles can be used for transdermal drug delivery, which can break through the stratum corneum skin barrier and deliver drugs in a highly efficient, simple and minimally invasive manner. At the same time, the soluble microneedle patch prepared by this invention has an anti-tumor effect, and the drug targeting of the microneedles enables transdermal delivery of poorly soluble drugs, improving the skin penetration rate of gingerone. It has application value in the field of biomedical technology. Attached Figure Description

[0042] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0043] Figure 1 This is a zeta potential distribution diagram of the self-assembled gingerone nanomedicine loaded with spheres prepared in Example 2 of the present invention.

[0044] Figure 2 This is a particle size distribution diagram of the spheroidized gingerone self-assembled nanomedicine prepared in Example 2 of the present invention.

[0045] Figure 3 This is a transmission electron microscope image of the self-assembled gingerone nanomedicine loaded with spheres prepared in Example 2 of the present invention.

[0046] Figure 4 This is a scanning electron microscope image of the microneedle patch of the self-assembled gingerone nanomedicine loaded with spheres prepared in Example 4 of the present invention.

[0047] Figure 5 This is a cell fluorescence microscope image used in the in vitro antitumor efficacy assay of this invention.

[0048] Figure 6This is a microscopic image of the orange fluorescence intensity inside the mitochondria during the in vitro antitumor efficacy assay of this invention.

[0049] Figure 7 Images of tumors after treatment in different groups during the in vivo melanoma inhibition trial of this invention.

[0050] Figure 8 The weight of the animal at the end of the in vivo melanoma inhibition trial of this invention.

[0051] Figure 9 This represents the tumor volume of the corresponding cohort at the end of the in vivo melanoma inhibition trial of this invention.

[0052] Figure 10 This refers to the tumor weight of the corresponding cohort at the end of the in vivo melanoma inhibition trial of this invention.

[0053] Figure 11 These are immunofluorescence staining images of melan-A, ki-67, and F4 / 80 in tumor tissues from various groups in the in vivo melanoma inhibition assay of this invention.

[0054] Figure 12 This is a SOX-10 staining image of tumor tissues from each group in the in vivo melanoma inhibition experiment of this invention.

[0055] Figure 5 and Figure 6 In the text, ICG-NMs, ICG-NMs+NIR, ZER-NMs, ZER-ICG-NMs, and ZER-ICG-NMs+NIR represent indocyanine green nanomedicine treatment, indocyanine green nanomedicine treatment + laser treatment, succinyl ketone nanomedicine treatment, nanomedicine treatment prepared in Example 2 (succinyl ketone and indocyanine green nanomedicine treatment), and nanomedicine treatment prepared in Example 2 + laser treatment (succinyl ketone and indocyanine green nanomedicine + laser treatment), respectively.

[0056] Figure 7-10 In the figure, AG represents: (A) no treatment (blank control); (B) ICG@DMNs; (C) ICG@DMNs+NIR; (D) free-ZER@DMNs; (E) ZER@DMNs; (F) ZER-ICG@DMNs; (G) ZER-ICG@DMNs+NIR.

[0057] Figure 11-12In this context, ICG@DMNs, ICG@DMNs+NIR, free-ZER@DMNs, ZER@DMNs, ZER-ICG@DMNs, and ZER-ICG@DMNs+NIR represent ICG@DMNs microneedle patch, ICG@DMNs microneedle patch + 808nm laser irradiation for 5 minutes, free-ZER@DMNs microneedle patch, ZER@DMNs microneedle patch, ZER-ICG@DMNs microneedle patch (microneedle patch prepared in Example 4), and ZER-ICG@DMNs microneedle patch + 808nm laser irradiation for 5 minutes (microneedle patch prepared in Example 4 + 808nm laser), respectively. Detailed Implementation

[0058] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0060] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, clearly defined in the relevant application fields. Those skilled in the art can understand the conventional process steps based on these names and apply the corresponding equipment, implementing the process under conventional conditions or conditions recommended by the manufacturer.

[0061] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0062] The raw material mPEG used in this invention 2000 -PLA 2000 Purchased from Guangzhou Tanshtech, China, product number 80010401-2K-2K, mPEG 2000 -PLA2000 This indicates a methoxylated polyethylene glycol-polylactic acid block copolymer.

[0063] Polyvinylpyrrolidone (PVP-K30) was purchased from Aladdin Reagents (P274371-100g).

[0064] Polyvinyl alcohol was purchased from Aladdin Reagent, type 1788, with a degree of alcoholysis of 87.0-89.0% (mol / mol).

[0065] Example 1 A method for preparing a self-assembled nanomedicine loaded with gingerone includes the following steps: S1. Prepare an aqueous solution of indocyanine green with a concentration of 10 mM; S2, 1.5mg of gingerone granules and 20mg of polymer mPEG 2000 -PLA 2000 The particles were mixed with 2 mL of acetonitrile and stirred thoroughly. The indocyanine green aqueous solution prepared in step S1 was then added. The mixture was protected from light and subjected to initial sonication at room temperature for 5 min (ultrasonic power 250 W, frequency 35 kHz) to obtain a mixed solution containing gingerone, indocyanine green, and the polymer mPEG. 2000 -PLA 2000 The mass ratio is 1.5:0.7:20; S3. The mixed solution was rotary evaporated under reduced pressure at 40°C for 20 minutes using a vacuum rotary evaporator (EYELA N-1100, speed 6, with the connected vacuum pump rotating at 2800 rpm). 10 mL of ultrapure water was added for hydration, and then the solution was sonicated again for 5 minutes (ultrasonic power 250 W, frequency 35 kHz). After filtration through a 0.22 μm filter membrane, the self-assembled spheroidized gingerone nanomedicine was obtained.

[0066] Example 2 A method for preparing a self-assembled nanomedicine loaded with gingerone includes the following steps: S1. Prepare an aqueous solution of indocyanine green with a concentration of 10 mM; S2, Add 4mg of gingerone granules and 20mg of polymer mPEG. 2000 -PLA 2000 The particles were mixed with 2 mL of acetonitrile and stirred thoroughly. The indocyanine green (ICG) aqueous solution prepared in step S1 was then added. The mixture was protected from light and subjected to initial sonication at room temperature for 5 min (ultrasonic power 200 W, frequency 40 kHz) to obtain a mixed solution containing gingerone, indocyanine green, and the polymer mPEG. 2000 -PLA 2000 The mass ratio is 4:0.7:20; S3. The mixed solution was rotary evaporated under reduced pressure at 40°C for 20 minutes using a vacuum rotary evaporator (EYELA N-1100, speed 6, connected vacuum pump speed 2800 rpm). 10 mL of ultrapure water was added for hydration, and then the solution was sonicated again for 5 minutes (ultrasonic power 200 W, frequency 40 kHz). After filtration through a 0.22 μm filter membrane, the self-assembled spheroidized gingerone nanomedicine was obtained at room temperature.

[0067] Example 3 A method for preparing a self-assembled nanomedicine loaded with gingerone includes the following steps: S1. Prepare an aqueous solution of indocyanine green with a concentration of 1 mM; S2, Add 3mg of gingerone granules and 20mg of polymer mPEG. 2000 -PLA 2000 The particles were mixed with 2 mL of acetonitrile and stirred thoroughly. The indocyanine green aqueous solution prepared in step S1 was then added. The mixture was protected from light and subjected to initial sonication at room temperature for 10 min (ultrasonic power 150 W, frequency 45 kHz) to obtain a mixed solution containing gingerone, indocyanine green, and the polymer mPEG. 2000 -PLA 2000 The mass ratio is 3:0.7:20; S3. The mixed solution was rotary evaporated under reduced pressure at 40°C for 22 minutes using a vacuum rotary evaporator (EYELA N-1100, speed 6, connected vacuum pump speed 2800 rpm). 10 mL of ultrapure water was added for hydration, and then the solution was sonicated again for 10 min (ultrasonic power 150 W, frequency 45 kHz). After filtration through a 0.22 μm filter membrane, the self-assembled spheroidized gingerone nanomedicine was obtained at room temperature.

[0068] Example 4 A method for preparing a soluble microneedle patch includes the following steps: S1. Dissolve and mix 250 μg of polyvinylpyrrolidone and 100 μg of polyvinyl alcohol in 1 mL of self-assembled nanomedicine with spheroidized gingerone (prepared in Example 2) to obtain a needle tip mixed solution; pre-swell hyaluronic acid in water overnight to prepare an aqueous solution with a mass concentration of 20% to form a base solution; wherein, the molecular weight of hyaluronic acid is 50,000; S2. The needle tip mixture from S1 is poured into a PDMS mold, and the mold cavity is filled by vacuum filling. Specifically, the mold is degassed in a vacuum oven (pressure 0.8 MPa) at room temperature for 15 minutes. Excess air bubbles are removed by gently scraping the mold surface. This step is repeated 3 times, and excess solution is scraped off. Then, 1 mL of base solution is added, and the mold is dried at room temperature for 24 hours. The mold is then demolded to obtain soluble microneedles. The humidity of the drying air is 50%. The content of nanomedicine in each soluble microneedle patch can be adjusted to 70~90μg / patch as needed (80.5 ±0.17μg / patch in this example). Each patch contains 15*15 needles. The height of the prepared needles, the spacing between the needle tips, and the diameter of the needle base (i.e., the diameter of the pyramid-shaped base of each needle) can be adjusted as needed. For example, the needle height is 500-700μm, the spacing between the needle tips is 300-500μm, and the diameter of the needle base (i.e., the diameter of the pyramid-shaped base of each needle) is 150-300μm.

[0069] Example 5 A method for preparing a soluble microneedle patch includes the following steps: S1. Dissolve and mix 250 μg of polyvinylpyrrolidone and 100 μg of polyvinyl alcohol in 1 mL of self-assembled nanomedicine with spheroidized gingerone (prepared in Example 2) to obtain a needle tip mixed solution; pre-swell hyaluronic acid in water overnight to prepare a hyaluronic acid aqueous solution with a mass concentration of 30% to form a base solution; wherein, the molecular weight of hyaluronic acid is 40,000. S2. Cast the needle tip mixture from S1 into a PDMS mold and fill the mold cavity using a vacuum method. Specifically, degas the mold in a vacuum oven (pressure 0.8 MPa) for 15 minutes at room temperature, gently scrape the mold surface to remove excess air bubbles, and repeat the above steps 3 times, scraping off excess solution. Then add 1 mL of base liquid, and dry at room temperature for 24 hours. Demold to obtain soluble microneedles. The humidity of the drying air is 50%. The content of nanomedicine in each soluble microneedle patch can be adjusted to 70~90 μg / patch as needed. Each patch contains 15*15 needles. The height of the prepared needles, the spacing between the needle tips, and the diameter of the needle base (i.e., the diameter of the pyramid-shaped base of each needle) can be adjusted as needed. For example, the needle height is 500-700 μm, the spacing between the needle tips is 300-500 μm, and the diameter of the needle base (i.e., the diameter of the pyramid-shaped base of each needle) is 150-300 μm.

[0070] Example 6 A method for preparing a soluble microneedle patch includes the following steps: S1. Dissolve and mix 250 μg of polyvinylpyrrolidone and 100 μg of polyvinyl alcohol in 1 mL of gingerone-loaded self-assembled nanomedicine (prepared in Example 2) to obtain a needle tip mixture solution; pre-swell hyaluronic acid in water overnight to prepare a 15% (w / w) hyaluronic acid aqueous solution to form a base solution; wherein the molecular weight of hyaluronic acid is 100,000 and the concentration of hyaluronic acid is 15%; S2. The needle tip mixture from S1 is poured into a PDMS mold, and the mold cavity is filled using a vacuum method. Specifically, the mold is degassed for 15 minutes at room temperature using a vacuum oven (0.8 MPa pressure, YB-I vacuum constant temperature drying oven, purchased from Tianjin Xinzhou Technology Co., Ltd.). Excess air bubbles are removed by gently scraping the mold surface. This step is repeated twice, and excess solution is scraped off. Then, 1 mL of base solution is added, and the mold is dried at room temperature for 24 hours. The mold is then demolded to obtain soluble microneedles. The humidity of the drying air is 50%. The content of nanomedicine in each soluble microneedle patch can be adjusted to 70~90μg / patch as needed. Each patch contains 15*15 needles. The height of the prepared needles, the spacing between the needle tips, and the diameter of the needle base (i.e., the diameter of the pyramid-shaped base of each needle) can be adjusted as needed. For example, the needle height is 500-700μm, the spacing between the needle tips is 300-500μm, and the diameter of the needle base (i.e., the diameter of the pyramid-shaped base of each needle) is 150-300μm.

[0071] Test case 1. Zeta potential detection The specific steps are as follows: S1. Take an appropriate amount of the spheroidized gingerone self-assembled nanomedicine prepared in Examples 1-3; S2. Turn on the instrument and preheat for 30 minutes until the light source stabilizes. Pour the prepared spheroidized gingerone self-assembled nanomedicine into the Malvern potential sample cell. The solution volume should reach the metal plate of the sample cell but not overflow, ensuring the sample is correctly inserted into the sample cell. Test results show that the zeta potentials measured for the spheroidized gingerone self-assembled nanomedicines prepared in Examples 1-3 are all negative.

[0072] from Figure 1 As shown, the self-assembled nanomedicine of gingerone prepared in Example 2 has negative zeta potentials, indicating good stability of the colloid, which is crucial for ensuring the dispersion integrity of the micelles in subsequent biological experiments.

[0073] 2. Particle size distribution determination Specific procedures: The particle size distribution of Examples 1-3 was determined using a Nano-ZS90 nanoparticle size analyzer (Malvern Panalytical, Malvern City, UK), and the results are shown in Table 1 below. The particle size distribution of the nanomedicine prepared in Example 2 is also shown below. Figure 2 .

[0074] Table 1 3. Morphological characteristics The self-assembled gingerone nanomedicines prepared in Examples 1-3 were examined by transmission electron microscopy. The results showed that the nanomicelle particles were approximately spherical with an average diameter of about 250 nm, which is in good agreement with the hydrodynamic diameter measured by Malvern. Figure 3 As shown, the spherical gingerone self-assembled nanomedicine prepared in Example 2 has a roughly spherical shape.

[0075] 4. Detection of soluble microneedle patches The soluble microneedle patches prepared in Examples 4-6 were examined using a scanning electron microscope. The results showed that the microneedle tips of the soluble microneedle patches were neatly and regularly arranged under the scanning electron microscope, with no defects in the tips, thus proving that structurally complete soluble microneedle patches were successfully prepared. Figure 4 The image shown is a scanning electron microscope image of the soluble microneedle patch prepared in Example 4.

[0076] 5. In vitro antitumor efficacy assay Measurement Experiment 1 (1) Experimental materials B16F10 cells (mouse melanoma cells). Cell source (Wuhan Service Biotechnology Co., Ltd. (Wuhan, China)).

[0077] Culture conditions: Cultured in RPMI 1640 medium containing 10% inactivated fetal bovine serum, 1% penicillin and 1% streptomycin at 37°C under 5% CO2.

[0078] Number of generations: 3.

[0079] (2) Experimental grouping A blank control group (without any treatment) and an experimental group (treated with different drugs and treatments) were set up. The experimental group was divided into five groups: ICG-NMs, ICG-NMs+NIR, ZER-NMs, ZER-ICG-NMs, and ZER-ICG-NMs+NIR, representing indocyanine green nanomedicine treatment, indocyanine green nanomedicine treatment + laser treatment, succinyl ketone nanomedicine treatment, nanomedicine treatment prepared in Example 2 (succinyl ketone and indocyanine green nanomedicine treatment), and nanomedicine treatment prepared in Example 2 + laser treatment (succinyl ketone and indocyanine green nanomedicine + laser treatment).

[0080] The preparation methods for indocyanine green nanomedicines (ICG-NMs) include: S1. Prepare a 10 mM indocyanine green (ICG) aqueous solution; S2, Add 20mg of polymer mPEG 2000 -PLA 2000 The particles were mixed with 2 mL of acetonitrile and stirred thoroughly. The indocyanine green (ICG) aqueous solution prepared in step S1 was added. The mixture was protected from light and subjected to initial sonication at room temperature for 5 min (ultrasonic power 200 W, frequency 40 kHz) to obtain a mixed solution. The mass ratio of indocyanine green (ICG) to polymer was 0.7:20. S3. The mixed solution was rotary evaporated under reduced pressure at 40°C for 20 minutes using a vacuum rotary evaporator (EYELA N-1100, speed 6, connected vacuum pump speed 2800 rpm). 10 mL of ultrapure water was added for hydration, and then the solution was sonicated again for 5 minutes (ultrasonic power 200 W, frequency 40 kHz). After filtration through a 0.22 μm filter membrane, indocyanine green nanomedicine (ICG-NMs) was obtained at room temperature.

[0081] The preparation methods of ZER-NMs (zingerone nanomedicines) include: S1, Mix 4mg of gingerone granules and 20mg of polymer mPEG. 2000 -PLA 2000 The particles were mixed with 2 mL of acetonitrile, stirred thoroughly, protected from light, and subjected to initial sonication at room temperature for 5 min (ultrasonic power 200 W, frequency 40 kHz) to obtain a mixed solution. S2. The mixed solution was rotary evaporated under reduced pressure at 40°C for 20 minutes using a vacuum rotary evaporator (EYELA N-1100, speed 6, connected vacuum pump speed 2800 rpm). 10 mL of ultrapure water was added for hydration, and then the solution was sonicated again for 5 minutes (ultrasonic power 200 W, frequency 40 kHz). After filtration through a 0.22 μm filter membrane, the gingerone nanoparticles (ZER-NMs) were obtained at room temperature.

[0082] Storage conditions for each nanomedicine: Store at 4 degrees Celsius away from light. (3) Experimental methods Cellular experiments: First, B16F10 cells were seeded into 24-well plates, 1*10 cells per well. 5 After culturing for 24 hours, B16F10 cells were treated with different nanomedicines, ICG-NMs, ZER-NMs, and ZER-ICG-NMs, for 2 hours each. Specifically, ZER-ICG-NMs were diluted with serum-free medium to a ZER concentration of 10 μg / mL, and the ICG concentration was calculated and recorded. 1 mL of this treatment solution was added to each well for co-incubation with the cells. Similarly, ZER-NMs were diluted with serum-free medium to a ZER concentration of 10 μg / mL, and 1 mL of this treatment solution was added to each well for co-incubation with the cells. ICG-NMs were diluted with serum-free medium to an ICG concentration equivalent to that in the ZER-ICG-NMs treatment solution, and 1 mL of this treatment solution was added to each well for co-incubation with the cells. (The amounts of ICG and ZER used in the ICG-NMs and ZER-NMs treatments are the same as those in the ZER-ICG-NMs treatment.) The amounts of each medium were the same; serum-free medium refers to RPMI 1640 medium containing 1% penicillin and 1% streptomycin. The medium was irradiated with an 808nm laser at 2 W / cm² for 5 min (the laser was held vertically against the well plate). Staining was performed using a live-dead cell staining kit (Beyotime Biotechnology (Shanghai, China)). Images were taken using an inverted fluorescence microscope. Results are as follows: Figure 5 The results showed that a small number of cells died in the ICG-NMs group (red indicates dead cells), while the number of dead cells increased in the ICG-NMs+NIR group; the proportion of dead cells was similar in the ZER-NMs and ZER-ICG-NMs groups. The ZER-ICG-NMs group treated with laser showed the lowest cell viability, indicating that the nanomedicine prepared in this invention has cytotoxic effects. Therefore, it is demonstrated that ZER and ICG can exert a synergistic anti-tumor effect under 808nm laser light.

[0083] Measurement Experiment 2 To further confirm this invention, mitochondrial membrane potential was measured. First, B16F10 cells were seeded into 24-well plates, 1*10 cells per well. 5After 24 hours of cultivation (culture conditions: RPMI 1640 medium containing 10% inactivated fetal bovine serum, 1% penicillin, and 1% streptomycin, cultured at 37°C under 5% CO2), B16F10 cells were incubated for 2 hours with different nanoparticles of ICG-NMs, ZER-NMs, and ZER-ICG-NMs (specifically, the prepared ZER-ICG-NMs were diluted with serum-free medium to prepare a treatment solution with a ZER concentration of 10 μg / mL, and the ICG concentration in the treatment solution was calculated and recorded at this time). For the ZER-NMs treatment, 1 mL of treatment solution was added to each well and incubated with the cells. Similarly, ZER-NMs were diluted with serum-free medium to a ZER concentration of 10 μg / mL, and 1 mL of treatment solution was added to each well and incubated with the cells. ICG-NMs were diluted with serum-free medium to an ICG concentration equivalent to that in the ZER-ICG-NMs treatment solution, and 1 mL of treatment solution was added to each well and incubated with the cells. The amounts of ICG and ZER used for the ICG-NMS and ZER-NMs treatments are the same as those used for the ZER-ICG-NMs treatment. In drug treatment, the amounts of ICG and ZER were the same. Serum-free medium referred to RPMI 1640 medium containing 1% penicillin and 1% streptomycin. Cells were irradiated with an 808nm laser at 2 W / cm² for 5 min, then stained with 500nM TMRE (tetramethylrhodamine ethyl ester dye, Beyotime Biotechnology (Shanghai, China) dye), incubated at 37℃ for 20 min, and the nuclei were stained with Hoechst 33342. The staining was then observed using an inverted fluorescence microscope. Under normal conditions, mitochondria contain a large number of negative charges. TMRE, acting as a cationic probe, accumulates in mitochondria and emits bright orange fluorescence. During apoptosis, the mitochondrial membrane potential is lost, TMRE is released into the cytoplasm, and the intensity of orange fluorescence in the mitochondria decreases significantly. To confirm the effects of different nanomedicines under laser irradiation or without irradiation, TMRE staining was used. Figure 6 The results showed that the intensity of orange-red fluorescence in mitochondria was significantly reduced, and the fluorescence intensity of the ZER-ICG-NMs+NIR group was the weakest, with the most melanocytes undergoing apoptosis. This indicates that the combined effect of ZER and ICG (nanomedicine prepared in Example 2) was strongest under 808nm laser irradiation.

[0084] 6. In vivo melanoma inhibition test Based on the ability of ZER-ICG-NMs nanoparticles to effectively kill B16F10 tumors in vitro, this invention further investigates the anti-melanoma effect of ZER-ICG@DMNs microneedle patches in vivo.

[0085] Experiment 1 (1) Experimental materials B16F10 tumor-bearing mice.

[0086] Method for establishing animal models: 3*10 5 A tumor-bearing mouse model was established by injecting B16F10 cells into the right ventral dorsal region of female Balb / c nude mice.

[0087] Animal species: Balb / c nude mouse.

[0088] Gender: Female.

[0089] Age: 5 weeks old.

[0090] Weight: Approximately 18-20g.

[0091] Husbandry conditions: room temperature, rat house.

[0092] (2) Experimental grouping When the tumor volume reaches approximately 60-80 mm 3 Research on anti-tumor efficacy began at that time.

[0093] Five mice were used in each group and were given the following treatments: (A) no treatment (i.e., blank control, Control); (B) ICG@DMNs microneedle patch (i.e., ICG@DMNs); (C) ICG@DMNs microneedle patch + 808nm laser irradiation at 2 W / cm² for 5 minutes (i.e., ICG@DMNs+NIR); (D) free-ZER@DMNs microneedle patch (i.e., free-ZER@DMNs); (E) ZER@DMNs microneedle patch (i.e., ZER@DMNs); (F) ZER-ICG@DMNs microneedle patch (microneedle patch prepared in Example 4, ZER-ICG@DMNs); (G) ZER-ICG@DMNs microneedle patch + 808nm laser irradiation for 5 minutes (microneedle patch prepared in Example 4 + 808nm laser, i.e., ZER-ICG@DMNs+NIR).

[0094] The preparation method of the ICG@DMNs microneedle patch is the same as in Example 4, namely: S1. Dissolve and mix 250 μg of polyvinylpyrrolidone and 100 μg of polyvinyl alcohol with indocyanine green nanomedicine ICG-NMs (indocyanine green nanomedicine ICG-NMs prepared for in vitro antitumor efficacy assay, the volume of which is such that the ICG content in each patch is the same as the ICG content in the ZER-ICG@DMNs patch) to obtain a needle tip mixture solution; pre-swell hyaluronic acid in water overnight to prepare a 20% (w / w) hyaluronic acid aqueous solution to form the base solution; wherein, the molecular weight of hyaluronic acid is 50,000; S2. The needle tip mixture from S1 is poured into a PDMS mold, and the mold cavity is filled by vacuum filling. Specifically, the mold is degassed in a vacuum oven (pressure 0.8 MPa) at room temperature for 15 minutes. Excess air bubbles are removed by gently scraping the mold surface. This step is repeated 3 times, and excess solution is scraped off. Then, 1 mL of base solution is added, and the mold is dried at room temperature for 24 hours. The mold is then demolded to obtain ICG@DMNs microneedle patches. The humidity of the drying air is 50%, and each patch contains 15*15 needles.

[0095] The preparation method of ZER@DMNs microneedle patches is the same as that of ICG@DMNs microneedle patches, the only difference being that ICG-NMs are replaced with ZER-NMs. Specifically, the following steps are included: S1. Dissolve and mix 250 μg of polyvinylpyrrolidone and 100 μg of polyvinyl alcohol with ZER-NMs (ZER-NMs, a nanomedicine prepared for in vitro antitumor efficacy assay, with a volume ratio such that the ZER content in each patch is the same as the ZER content in the ZER-ICG@DMNs patch) to obtain a needle tip mixture solution; pre-swell hyaluronic acid in water overnight to prepare a 20% (w / w) hyaluronic acid aqueous solution to form the base solution; wherein, the molecular weight of hyaluronic acid is 50,000; S2. Cast the needle tip mixture from S1 into a PDMS mold and fill the mold cavity using a vacuum method. Specifically, degas the mold in a vacuum oven (0.8 MPa pressure) for 15 minutes at room temperature, gently scrape the mold surface to remove excess air bubbles, and repeat the above steps 3 times, scraping off excess solution. Then add 1 mL of base solution, and dry at room temperature for 24 hours. Demold to obtain soluble microneedles. The humidity of the drying air is 50%, and each piece contains 15*15 needles.

[0096] The preparation method of free-ZER@DMNs microneedle patches is the same as that of ZER@DMNs microneedle patches. The only difference is that ZER-NMs is replaced with ZER, the amount of ZER used is the same as that in ZER-NMs, and an equal amount of ZER is directly dissolved in acetonitrile. The remaining steps are the same.

[0097] (3) Experimental methods Microneedle patches were applied to the surface of the tumor for 5 minutes, followed by laser irradiation for another 5 minutes; Dosage: one microneedle patch; Dosage frequency: once daily; Tumor volume measurement: every 2 days; Weight monitoring: every 2 days.

[0098] Data Analysis: All results were expressed as mean ± standard deviation (SD). One-way ANOVA was performed using SPSS 22.0 software for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001). Statistical analysis was performed using Prism 5.01 GraphPad software.

[0099] Mice were euthanized after 9 days of continuous treatment, and tumor tissue and organs from the tumor sites of each group were collected. Figure 7-10 , Figure 7 Digital images showing tumors after different treatment groups; Figure 8 This indicates the animal's weight at the end of treatment; Figure 9 Indicates the tumor volume of the corresponding queue at the end; Figure 10 The figures indicate the tumor weight of the corresponding cohort at the end of the treatment. In the figure, AG represents: (A) untreated (blank control); (B) ICG@DMNs; (C) ICG@DMNs+NIR; (D) free-ZER@DMNs; (E) ZER@DMNs; (F) ZER-ICG@DMNs; (G) ZER-ICG@DMNs+NIR. It can be seen that the combined treatment group (patch prepared in Example 4 + laser treatment, ZER-ICG@DMNs+NIR) had the smallest tumor volume and the lowest tumor weight, verifying the inhibitory effect of ZER-ICG@DMNs patch combined with laser irradiation on melanoma. In mice treated with ICG@DMNs + NIR and ZER@DMNs, significant anti-tumor effects were observed, with tumor inhibition rates of 26.75% and 60.35%, respectively, indicating that both photothermal and chemotherapy are helpful in tumor suppression. Notably, the combination therapy group using ZER-ICG@DMNs + NIR showed the most significant therapeutic benefit, achieving a significant tumor inhibition rate of 99.35%, and complete tumor ablation was observed in 3 mice.

[0100] Experiment 2 Tumor tissues from different treatments in Experiment 1 were collected and subjected to immunofluorescence staining. In the ZER@DMNs group, ZER-ICG@DMNs group, ZER-ICG@DMNs+NIR group, and free-ZER@DMNs group (free ZER@DMNs group), melan-A and F4 / 80 expression were downregulated (e.g., Figure 11 (Images showing immunofluorescence staining of tumor tissues for melan-A, Ki-67, and F4 / 80 in each group), corresponding to tumor volume and weight results. Figure 11In the diagram, green represents melan-A; red represents F4 / 80; and pink represents Ki-67. The ZER-ICG@DMNs+NIR group showed the highest apoptosis level, with cell proliferation and apoptosis occurring in tandem. These results indicate that photothermal therapy is a promising treatment for skin cancer. Ki67 staining further confirmed its inhibitory effect on tumor growth by observing tumor cell proliferation. In conclusion, these results demonstrate that the drug-based photothermal therapy composed of ZER-ICG@DMNs microneedle patches has a good inhibitory effect on tumor growth and exhibits no significant toxicity. Immunohistochemical staining was used to observe the expression of SOX-10 in mouse tumor tissues after different treatment groups. Figure 12 SOX-10 staining images of tumor tissues in each group were shown. The results showed that SOX-10 positive expression was weakest in tumor tissues treated with ZER-ICG@DMNs under 808nm laser irradiation.

[0101] In summary, the self-assembled nanomedicine loaded with gingerone of the present invention comprises gingerone, indocyanine green and polymer mPEG-PLA. In this nanomedicine, gingerone and indocyanine green have synergistic anti-tumor effects and can be used to treat melanoma, thereby improving the bioavailability of gingerone.

[0102] The preparation method of the spherical gingerone self-assembled nanomedicine of the present invention has the advantages of being simple and easy to implement, and forms a stable spherical structure, thus successfully realizing the preparation of the spherical gingerone self-assembled nanomedicine.

[0103] The soluble microneedle patch of the present invention imparts the necessary mechanical strength and toughness to the soluble microneedles through hyaluronic acid. The soluble microneedles can be used for transdermal drug delivery, which can break through the skin barrier of the stratum corneum and deliver drugs in a highly efficient, simple and minimally invasive manner. The prepared soluble microneedle patch has anti-tumor effects and can achieve transdermal drug delivery of poorly soluble drugs through the drug targeting of microneedles, thereby improving the skin penetration rate of gingerol. It has promotional application value in the field of biomedical technology.

[0104] The above are merely preferred embodiments of the present invention and are 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 self-assembled nanomedicine loaded with gingerone, characterized in that, It includes gingerone, indocyanine green, and polymer mPEG-PLA in a mass ratio of 1.5-5:0.35-1.4:10-20.

2. The self-assembled spheroidized gingerone nanomedicine according to claim 1, characterized in that, The mass ratio of gingerone, indocyanine green, and polymer mPEG-PLA is 1.5-5:0.7:10-20.

3. The self-assembled spheroidized gingerone nanomedicine according to claim 1, characterized in that, The mass ratio of gingerone, indocyanine green, and polymer mPEG-PLA is 4:0.7:20; The polymer mPEG-PLA is mPEG. 2000 -PLA 2000 .

4. The method for preparing spheroidized gingerone self-assembled nanomedicine according to any one of claims 1-3, characterized in that, Includes the following steps: Gingerol, polymer mPEG-PLA and organic solvent were mixed and stirred. Indocyanine green aqueous solution was added, and the mixture was subjected to initial ultrasonic treatment at room temperature in the dark to obtain a mixed solution. The organic solvent in the mixed solution was removed by rotary evaporation, then hydrated with ultrapure water, sonicated again, and filtered to obtain the self-assembled nanomedicine loaded with gingerone.

5. The method for preparing a self-assembled spheroidized gingerone nanomedicine according to claim 4, characterized in that, The concentration of the indocyanine green aqueous solution is 1-10 mM; The ratio of the organic solvent, gingerone, and ultrapure water used is 2 mL: 1.5-5 mg: 4-10 mL; The power of the initial ultrasonic treatment is 150-250w, the frequency is 35-45KHz, and the time is 5-10min; The power of the second ultrasonic treatment is 150-250w, the frequency is 35-45KHz, and the time is 5-10min; The filtration process uses a 0.22μm filter membrane.

6. A soluble microneedle patch, characterized in that, Including the spheroidized gingerone self-assembled nanomedicine according to any one of claims 1-3.

7. A soluble microneedle patch according to claim 6, characterized in that, The soluble microneedle patch includes a patch substrate and a needle disposed on the surface of the patch substrate; the needle is prepared from a self-assembled nanomedicine loaded with gingerone and a soluble matrix material; The soluble matrix material is polyvinylpyrrolidone and / or polyvinyl alcohol; The substrate material of the patch is hyaluronic acid.

8. A soluble microneedle patch according to claim 7, characterized in that, The soluble microneedle patch meets one or more of the following conditions: 1) The substrate material of the patch is a hyaluronic acid aqueous solution with a mass concentration of 15% to 30%; the molecular weight of the hyaluronic acid is 40,000 to 100,000 Daltons; 2) The ratio of gingerone-loaded self-assembled nanomedicine, polyvinylpyrrolidone, and polyvinyl alcohol in the needle is 0.8-1.2 mL: 250 μg: 100 μg; 3) Polyvinylpyrrolidone is PVP-K30; 4) The height of the soluble microneedle patch is 500-700μm, the distance between the needle tips is 300-500μm, and the diameter of the needle base is 150-300μm. 5) The content of the soluble microneedle patch containing spheroidized gingerone self-assembled nanomedicine is 70~90μg / patch, and each patch contains 15*15 needles.

9. A method for preparing a soluble microneedle patch as described in any one of claims 6-8, characterized in that, Includes the following steps: Gingerol self-assembled nanomedicine was mixed with a soluble matrix material to obtain a needle tip mixture solution; hyaluronic acid was pre-swelled in water overnight to form a base solution. The needle tip mixture solution is poured into a mold, and the mold cavity is filled by vacuum method. Base liquid is added, then dried and demolded to obtain soluble microneedles.

10. The use of a spheroidized gingerone self-assembled nanomedicine as described in any one of claims 1-3 in the preparation of a drug for anti-tumor or treatment of melanoma.

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