Microneedle preparation for co-delivery of photothermal nanolaser and elemane and preparation method and application thereof

Abstract

The application provides a microneedle preparation for jointly delivering photothermal nanolaser and elemene and a preparation method and application thereof. The application first prepares a novel photothermal nanolaser Au@MoS2 through a two-step aqueous phase synthesis strategy. Benefiting from the synergistic effect of the hybrid material, the nanolaser exhibits significantly enhanced near-infrared (NIR) photothermal effect, peroxidase (POD) activity and glutathione peroxidase (GSHOx) activity, can effectively remodel the tumor microenvironment and destroy the oxidation-reduction homeostasis. Further, the application successfully prepares a novel soluble microneedle preparation through a template method by taking the photothermal nanolaser, beta-ELE and hyaluronic acid as raw materials. The novel composite microneedle of the application is in a sharp quadrangular pyramid shape, and can effectively inhibit the growth of melanoma through transdermal delivery of the photothermal nanolaser and beta-ELE and has no significant systemic toxicity, thereby providing an innovative strategy for non-invasive, efficient and safe combined treatment of melanoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a microneedle formulation that co-delivers photothermal nanoenzymes and elemene, its preparation method, and its application. Background Technology

[0002] Cutaneous melanoma (CM) is the deadliest form of skin cancer, characterized by high invasiveness and the potential for distant metastasis. Current treatments for melanoma include surgical excision, chemotherapy, and immunotherapy. However, the clinical efficacy of surgery and chemotherapy for this disease is generally poor. Surgical treatment is primarily suitable for early-stage melanoma, but its effectiveness is limited for advanced or metastatic melanoma, with a high recurrence rate. Traditional chemotherapy drugs (such as dacarbazine) have low efficacy against melanoma, are prone to drug resistance, and are accompanied by severe systemic side effects. Existing clinical treatments have failed to significantly improve the overall survival rate of melanoma patients; therefore, the development of highly effective and safe new therapies for melanoma is urgently needed.

[0003] In recent years, microneedles (MNs), as a percutaneous drug delivery tool, have shown promise as an ideal option for local treatment of melanoma due to their advantages such as being painless, minimally invasive, and allowing for adjustable delivery doses. Soluble polymer MN systems can cross the skin barrier above melanoma, penetrate the stratum corneum, and create microchannels. As the polymer dissolves, therapeutic molecules are deposited directly at the tumor site, achieving targeted delivery and release of local drugs without the need for systemic circulation. To date, many soluble polymer MN systems have been integrated with drug molecules, nanomaterials, photothermal agents, and photosensitizers for the treatment of melanoma. Although these soluble MN systems have shown high delivery efficiency, the therapeutic dose delivered to the tumor site remains insufficient due to the limited patch area. Furthermore, the effects of many therapeutic molecules are diminished in the complex and metabolically abnormal tumor microenvironment (TME) of tumor cells. Extensive evidence suggests that cancer is a metabolically abnormal disease with an aberrant redox balance in the TME, characterized by high levels of reactive oxygen species (ROS) and overexpression of the antioxidant glutathione (GSH). These properties promote tumor cell metabolism and proliferation, ultimately severely weakening the antitumor efficacy of therapeutic agents. To address these issues, it is necessary to integrate ROS generation and GSH consumption to reshape the redox state of the TME, thereby enhancing the therapeutic effect of melanoma.

[0004] Nanozymes are a class of nanomaterials with catalytic activities similar to those of natural enzymes, capable of modulating tumor microenvironment (TME) in various ways. For example, nanozymes with peroxidase-like (POD) activity can induce the conversion of endogenous H2O2 in the TME into highly toxic hydroxyl radicals (·OH), catalyzing a Fenton-like reaction to achieve oxidative damage to tumor cells, thereby inhibiting tumor growth. Furthermore, nanozymes with glutathione oxidase (GSHOx)-like activity can consume overexpressed GSH in the TME, disrupting the antioxidant defense system, leading to GPx-4 inactivation and lipid peroxide (LPO) accumulation, thereby enhancing the killing effect on tumor cells and can be combined with other therapies to further inhibit tumor progression. Therefore, the rational design and construction of high-performance photothermal nanozyme microneedles plays a crucial role in enhancing the efficacy of combined tumor treatments. Generally, the design of nanozymes should consider multiple aspects, such as simple preparation processes, diverse catalytic activities, high catalytic efficiency, and multifunctionality, in order to maximize therapeutic effects for clinical application.

[0005] Currently, some studies have attempted combination therapies to improve efficacy. However, traditional injection or oral administration methods struggle to achieve targeted co-delivery of nanozymes and hydrophobic drugs to tumors, and carry a high risk of systemic toxicity. Furthermore, most existing microneedle systems use nanozymes or drugs alone, lacking mechanisms for systemic regulation of the tumor microenvironment (TME), thus limiting their anti-tumor effects. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a microneedle formulation that co-delivers photothermal nanozymes and elemene, along with its preparation method and application, to achieve enhanced transdermal therapy combining nanocatalysis, photothermal therapy, and traditional Chinese medicine.

[0007] In a first aspect, the present invention provides a method for preparing a microneedle formulation that co-delivers photothermal nanozyme and elemene, comprising the following steps:

[0008] Molybdenum disulfide (MoS2) solution was mixed with polyvinylpyrrolidone (PVP), and chloroauric acid (HAuCl4) solution was added. The reaction was carried out under stirring to obtain photothermal nanoenzyme Au@MoS2.

[0009] The photothermal nanozyme Au@MoS2, β-elemene, and hyaluronic acid (HA) solution were mixed, placed in a microneedle mold, centrifuged, and dried to obtain the microneedle formulation.

[0010] Preferably, the mass ratio of molybdenum disulfide (MoS2) to chloroauric acid (HAuCl4) is (0.5-5):1.

[0011] Preferably, the molybdenum disulfide (MoS2) solution is obtained by dissolving MoS2 powder in an ethanol solution, followed by ultrasonication in an ice bath and centrifugation. Further, the ultrasonication time is 4-6 hours, and the centrifugation speed is 6000-6500 rpm.

[0012] Preferably, the stirring time is 0.5-1 hour.

[0013] Preferably, the mass ratio of photothermal nanoenzyme Au@MoS2 to β-elemene is 1:(1-5).

[0014] Preferably, the concentration of the hyaluronic acid solution is 5%-10% (w / v).

[0015] Preferably, the drying time is 6-8 hours and the drying temperature is 40-45℃.

[0016] Preferably, the centrifugation time is 15-20 min and the centrifugation speed is 3500-4000 rpm.

[0017] Secondly, the present invention provides a microneedle formulation for co-delivering photothermal nanozymes and elemene, which is prepared by the above method.

[0018] Thirdly, the present invention provides the application of the microneedle formulation that co-delivers photothermal nanozyme and elemene in the preparation of a drug for treating malignant skin melanoma.

[0019] Preferably, the microneedle formulation that co-delivers photothermal nanoenzymes and elemene synergistically responds to glutathione and hydrogen peroxide.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention utilizes soluble MNs to simultaneously deliver a novel photothermal nanozyme (Au-modified MoS2, Au@MoS2) and the traditional Chinese medicine molecule β-elemene (β-ELE) transdermally. First, two-dimensional (2D) MoS2 nanosheets are obtained from MoS2 powder using a classical liquid-phase exfoliation method. Subsequently, Au nanoparticles are grown in situ on the MoS2 nanosheets using the intrinsic self-reducing ability of MoS2 to form the Au@MoS2 photothermal nanozyme without the need for additional reducing agents.

[0022] High-density Au nanoparticle doping significantly enhances the NIR photothermal conversion and nanocatalytic efficiency of MoS2, thereby maximizing its potential for combined photothermal / catalytic therapy. In vitro experiments show that photothermal nanozymes can efficiently catalyze the decomposition of H2O2 to generate ·OH, while continuously consuming overexpressed GSH through a GSHox-like catalytic reaction. This dual effect significantly remodels the tumor microenzyme (TME) and further enhances the tumor cell killing effect under the combined action of β-ELE and NIR photothermal therapy. Photothermal nanozymes and β-ELE are integrated into biocompatible and water-soluble hyaluronic acid (HA)MNs for local delivery to melanoma lesions. In vivo experiments demonstrate that the prepared MNs can effectively achieve combined treatment of melanoma, with the highest tumor suppression efficiency and negligible side effects.

[0023] In summary, this invention provides valuable insights into the rational design of nanozymes and offers a novel and efficient combined strategy for enhancing melanoma treatment in a clinical setting. Attached Figure Description

[0024] Figure 1 The image shows the characterization of Au@MoS2, where A: TEM image of Au@MoS2; B: high-resolution TEM image of Au@MoS2; C: HAADF-STEM image of Au@MoS2; D: EDX mapping image of Au@MoS2: S (green), Mo (blue), and Au (red), with a scale bar of 100 nm.

[0025] Figure 2 A: Particle size of MoS2 and Au@MoS2; B: Zeta potential of MoS2 and Au@MoS2; C: UV-vis-NIR spectra of Au nanoparticles, MoS2 and Au@MoS2; D: Raman spectra of MoS2 and Au@MoS2.

[0026] Figure 3 A: Schematic diagram of the TMB color development principle; B: UV-vis-NIR absorption spectra of TMB oxidation reaction catalyzed by Au nanoparticles, MoS2 and Au@MoS2; C: Absorbance curves at 650 nm at different times of TMB oxidation reaction catalyzed by Au nanoparticles, MoS2 and Au@MoS2.

[0027] Figure 4 A: Schematic diagram of DTNB color development principle; B: Absorbance curve at 412 nm of DTNB oxidation reaction catalyzed by MoS2 and Au@MoS2 as a function of time.

[0028] Figure 5The image shows the photothermal properties of Au@MoS2, where A: thermal images of water, MoS2, and Au@MoS2 after 5 min of irradiation with an 808 nm near-infrared laser; B: photothermal heating curves of water, MoS2, and Au@MoS2 after 5 min of irradiation with an 808 nm near-infrared laser.

[0029] Figure 6 The figures show the photothermal conversion curves and time constants of MoS2 and Au@MoS2, where A: photothermal conversion temperature curve of MoS2, B: time constant of MoS2, C: photothermal conversion temperature curve of Au@MoS2, and D: time constant of Au@MoS2.

[0030] Figure 7 The values ​​represent the survival rates of B16F10 cells under different conditions, where A: different concentrations of HAMNs; B: different concentrations of β-ELE; C: different concentrations of Au@MoS2 and Au@MoS2+β-ELE without laser irradiation; D: different concentrations of Au@MoS2 and Au@MoS2+β-ELE with laser irradiation.

[0031] Figure 8 The images show the double staining results of B16F10 cells in dead / live states, where A: double staining images of B16F10 cells in dead / live states with / without laser irradiation; B: quantitative fluorescence results of B16F10 cells in dead / live states without laser irradiation; and C: quantitative fluorescence results of B16F10 cells in dead / live states under laser irradiation.

[0032] Figure 9 A: Preparation process of Au@MoS2+β-ELE MNs; B: Stereomicroscopic image of HAMNs; C: Stereomicroscopic image of Au@MoS2+β-ELEMNs; D: SEM image of Au@MoS2+β-ELE MNs.

[0033] Figure 10 The diagram shows the performance characterization of the microneedle formulation, where A represents the mechanical strength of HAMNs and Au@MoS2+β-ELE MNs, and B represents the photothermal properties of HAMNs and Au@MoS2+β-ELE MNs.

[0034] Figure 11 The images show the skin insertion results of the microneedle formulation, where A: H&E staining of Au@MoS2+β-ELE MNs after insertion into pigskin; B: in vitro skin insertion capability of Au@MoS2+β-ELE MNs.

[0035] Figure 12 Tumor growth in each group of mice during treatment.

[0036] Figure 13Anatomical images of tumors in mice in each group after treatment.

[0037] Figure 14 Images of H&E staining of major organs in mice of each group 12 days after drug administration. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] As mentioned above, the present invention provides a method for preparing a microneedle formulation that co-delivers photothermal nanozyme and elemene, comprising the following steps:

[0040] Step (1): Dissolve molybdenum disulfide (MoS2) powder in an ethanol solution and sonicate in an ice bath for 4-6 hours. Then centrifuge at 6000-6500 rpm to obtain a molybdenum disulfide (MoS2) solution. Add polyvinylpyrrolidone (PVP) stabilizer and mix and stir. During stirring, add chloroauric acid (HAuCl4) solution and continue stirring for 0.5-1 hours to obtain photothermal nanoenzyme Au@MoS2. The mass ratio of molybdenum disulfide (MoS2) to chloroauric acid (HAuCl4) is (0.5-5):1, for example, it can be 5:1, 4:1, 3:1, 2:1, 1:1, 1:2 or other ratios within this range. It can be selected according to actual needs and is not limited here.

[0041] Step (2): Mix the photothermal nanoenzyme Au@MoS2 and β-elemene at a mass ratio of 1:(1-5), add 5%-10% (w / v) hyaluronic acid (HA) solution, place in a microneedle mold, centrifuge at 3500-4000 rpm for 15-20 min, and dry at 40-45℃ for 6-8 h to obtain the microneedle formulation. The mass ratio of the photothermal nanoenzyme Au@MoS2 and β-elemene can be 1:1, 1:2, 1:3, 1:4, 1:5 or other ratios within this range, which can be selected according to actual needs and are not limited here.

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0043] 1. Preparation and characterization of Au@MoS2

[0044] Weigh 2g of MoS2 powder and dissolve it in 50mL of 45% ethanol solution. After sonicating in an ice bath for 4 hours, centrifuge at 6000rpm and collect the supernatant for storage. Mix 10mL of the sonicated MoS2 solution with 100μl of PVP solution and stir. During stirring, add 1mL of HAuCl4 solution and continue stirring for 30min to obtain Au@MoS2 solution. Then wash with deionized water 2-3 times and store at 4℃ for later use.

[0045] The morphology, particle size, and zeta potential of Au@MoS2 nanosheets were characterized using instruments such as DLS, TEM, AFM, XPS, and UV-vis-NIR. MoS2 and Au@MoS2 solutions were dropped onto copper grids, dried, and then the nanosheets were characterized using TEM to determine their morphology and size. High-resolution TEM was then used to analyze the structure and elemental distribution of Au@MoS2. MoS2 and Au@MoS2 solutions were injected into quartz cuvettes and potential cells, respectively, and their particle size and zeta potential were measured using DLS at 25°C. Au NPs, MoS2, and Au@MoS2 solutions were injected into quartz cuvettes, and the absorption spectra of the three materials in the 300nm-800nm ​​range were measured using a UV-vis-NIR spectrophotometer. MoS2 and Au@MoS2 solutions were dropped onto silicon wafers, dried, and then the nanosheets were characterized using AFM to determine their thickness. After centrifuging the Au@MoS2 solution to remove the supernatant, the solution was freeze-dried into powder and then characterized using XPS to determine the elemental composition and chemical bonding properties of the material. The Au@MoS2 solution was also freeze-dried into powder and then characterized using Raman spectroscopy to determine the molecular structure of the material based on characteristic vibrational peaks.

[0046] 2. Detection of the properties of Au@MoS2 nanozymes

[0047] Au@MoS2 was tested for its POD-like and GSHOx properties using UV-Vis-NIR spectrophotometers and other instruments with 3,3',5,5'-tetramethylbenzidine (TMB) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as colorimetric reagents.

[0048] 3. Detection of photothermal properties of Au@MoS2

[0049] To investigate the in vitro photothermal heating of different materials, water, MoS2, and Au@MoS2 were placed in 48-well plates and heated using an 808nm near-infrared laser at 1.5W·cm². -2 Irradiation was performed, and infrared thermal images were taken at 0, 1, 2, 3, 4, and 5 minutes using an infrared thermal imager, and the temperature changes at different time points were recorded.

[0050] To investigate the photothermal conversion efficiency of MoS2 and Au@MoS2, aqueous solutions of MoS2 and Au@MoS2 were exposed to 1.5 W·cm⁻¹. -2 Near-infrared laser light (808nm) was applied for 5 minutes, followed by natural cooling to room temperature. Temperature changes at different time points were recorded, and the photothermal conversion efficiency of MoS2 and Au@MoS2 was calculated. Photothermal conversion efficiency (η) refers to the material's ability to convert light energy into heat energy, and is calculated using the following formula:

[0051]

[0052] h is the heat transfer coefficient (W / cm·K); S is the surface area of ​​the container (cm²). 2 );T max The equilibrium temperature (K) at which the temperature reaches a steady state; T a A represents ambient temperature; I represents laser power; A represents ambient temperature; 808 Q is the absorbance of the material at 808 nm. w Q represents the energy produced by water under laser irradiation. w The calculation formula is as follows:

[0053] Q w =hS(T maxw -T a (6)

[0054] T maxw This represents the highest equilibrium temperature of water; Formula 7 is used to calculate hS:

[0055]

[0056] τ s Represents a typical time constant; m w and c w These are the mass of the solvent and its specific heat capacity, respectively.

[0057] 4. Cell Culture

[0058] Mouse melanoma B16F10 cells were seeded in cell culture dishes, and 10 mL of DMEM-F12 high-glucose medium containing 1% streptomycin, penicillin, and 10% (v / v) fetal bovine serum was added. The dishes were then incubated at 37°C with 5% CO2. When the cell density reached 80%–90%, the cells were passaged.

[0059] 5. Cytotoxicity

[0060] The toxicity of HAMN to B16F10 cells was investigated using the MTT assay. Cell suspensions of 50,000 cells / mL were prepared and seeded into 96-well plates, which were then incubated overnight. The following day, B16F10 cells were co-incubated with HAMN at concentrations ranging from 0 to 500 μg / mL for 24 hours. Then, 100 μl of MTT solution was added to each well using a multipipeline, and the plates were incubated at 37°C for 4 hours. After incubation, the supernatant was discarded, and 200 μl of dimethyl sulfoxide (DMSO) was added to each well using a multipipeline. The plates were then placed in a microplate reader and shaken (medium speed) for 5 minutes. The absorbance of each well was measured at 570 nm, and cell viability was calculated.

[0061] The MTT assay was used to investigate the toxicity of different treatment groups to B16F10 cells. B16F10 cells were seeded at a density of 50,000 cells per well in 96-well plates and incubated for 24 hours. Then, the following groups were treated with the appropriate drugs: Control group, Control+NIR group, β-ELE group, β-ELE+NIR group, Au@MoS2 group, Au@MoS2+NIR group, Au@MoS2+β-ELE group, and Au@MoS2+β-ELE+NIR group (material-to-drug ratio was 1:1). Generally, the second column of the 96-well plate was the control group, with only 25 μl of cell culture medium added. Columns 3-11 each contained 25 μl of the drug. Groups requiring NIR treatment were treated with an 808 nm near-infrared laser (1 W / cm²) after drug administration. 2 Cell suspension was irradiated for 5 min per well, and then the 96-well plate was incubated for 24 h. Cell viability was detected using the MTT assay.

[0062] 6. Double staining of live and dead cells

[0063] Live and dead cells in different treatment groups were measured using the Calcein-AM / PI live / dead cell double staining kit. B16F10 cells were seeded at a density of 50,000 cells per well in 96-well plates and incubated for 24 h. Then, the cells were administered according to the experimental groups: Control group, Control+NIR group, β-ELE group, β-ELE+NIR group, Au@MoS2 group, Au@MoS2+NIR group, Au@MoS2+β-ELE group, and Au@MoS2+β-ELE+NIR group.

[0064] After laser irradiation, cells were cultured for another 24 hours. After incubation, the liquid in the wells was aspirated, and the cells were thoroughly washed 2-3 times with 1×Assay Buffer to remove residual esterase activity. Then, 100 μl of staining working solution was added to 200 μl of cell suspension, mixed well, and incubated at 37°C for 15 min. The dye was then aspirated, and fluorescence was observed using an inverted fluorescence microscope.

[0065] 7. Preparation and Characterization of Novel MN Formulations

[0066] Au@MoS2 and β-ELE were mixed with 5% HA at a mass ratio of 1:1 (both at a concentration of 50 μg / mL), and the mixture was placed in a PDMS mold containing microneedles. After centrifugation at 3500 rpm for 15 min, the mixture was placed in a 40℃ oven and dried for 6 h. The resulting product was then stored in a desiccator. The morphology of Au@MoS2+β-ELE MN was characterized using stereomicroscopy and scanning electron microscopy.

[0067] To investigate whether Au@MoS2+β-ELE MN can successfully penetrate the skin, a universal testing machine was used to test the mechanical properties of Au@MoS2+β-ELE MN.

[0068] To investigate the photothermal properties of Au@MoS2+β-ELE MN, an 808nm near-infrared laser and an infrared thermal imager were used to record the temperature changes of Au@MoS2+β-ELE MN.

[0069] To investigate the solubility of Au@MoS2+β-ELE MN, the tip of Au@MoS2+β-ELE MN was placed in a PBS solution at pH 6.0, and the dissolution of the MN tip was observed and recorded using a stereomicroscope.

[0070] To investigate the in vitro skin insertion ability of Au@MoS2+β-ELE MN, MN was inserted into porcine skin, and changes on the skin surface were observed and recorded at 0 min, 10 min, 20 min, and 30 min. Subsequently, the in vitro skin insertion ability of Au@MoS2+β-ELE MN was further determined by hematoxylin and eosin (H&E) staining.

[0071] To investigate the biocompatibility of Au@MoS2+β-ELE MN, the MTT assay was used to detect the cytotoxicity of Au@MoS2+β-ELE MN against human immortalized epidermal cells (Hacat cells) and mouse embryonic fibroblast cell line (NIH3T3 cells). Cytotoxicity was assessed after incubating Au@MoS2+β-ELE MN at concentrations ranging from 0 to 500 μg / mL with both types of normal cells for 24 hours.

[0072] 8. Establishment of a mouse model of melanoma

[0073] Using mouse melanoma cells B16F10 as a model cell, cells in the logarithmic growth phase were digested with trypsin, collected by centrifugation, and diluted with culture medium to a concentration of 1×10⁻⁶. 6A cell suspension of 100 μl / mL was prepared and kept on ice until needed. 100 μl of the cell suspension was subcutaneously injected into the dorsal side of the right upper limb of mice to establish a tumor-bearing mouse model. Tumor growth was monitored every two days after modeling was completed.

[0074] 9. In vivo anti-tumor experiments

[0075] Using B16F10 tumor-bearing mice as a model animal, the in vivo antitumor effect of Au@MoS2+β-ELE MNs was studied. Tumors with a volume of 100 mm² were used. 3 B16F10 tumor-bearing mice were randomly divided into 6 groups of 8 mice each: HAMNs group, HAMNs+NIR group, Au@MoS2 MNs group, Au@MoS2 MNs+NIR group, Au@MoS2+β-ELE MNs group, and Au@MoS2+β-ELE MNs+NIR group. Drug was administered via microneedle, with a second dose given five days after the first dose, for a total of two doses.

[0076] Starting from the first day of drug administration, the body weight and tumor volume of tumor-bearing mice were measured every two days. The formula for calculating tumor volume is shown below:

[0077] V = L × W 2 / 2

[0078] V: Tumor volume, L: Tumor length, W: Tumor width

[0079] Twelve days after drug administration, all mice were sacrificed. One representative mouse from each group was photographed at the tumor site. After cervical dislocation and sacrifice, tumor tissue was collected from each group. The tumor tissue was immediately washed with PBS, fixed overnight with 10 volumes of 4% paraformaldehyde, embedded in paraffin, sectioned, and mounted on glass slides for H&E staining. The sections were then scanned using an automated slide scanning imaging system to observe tumor necrosis. Additionally, the tumor tissue underwent TUNEL, Ki-67, GPX-4, and LPO staining analysis.

[0080] 10. In vivo biosafety assessment

[0081] Starting from the first day of drug administration, mice in each group were weighed and their weights recorded every two days. After treatment, vital organs (heart, liver, spleen, lungs, and kidneys) of mice in each group were collected, washed once with PBS, fixed overnight with 4% paraformaldehyde, embedded in paraffin, sectioned, stained with H&E, and the extent of organ damage was observed using an automated section scanning imaging system.

[0082] Results analysis and discussion:

[0083] 1. Synthesis and Characterization of Materials

[0084] This invention uses MoS2 powder and HAuCl4 as raw materials, and PVP as a stabilizer, to prepare Au@MoS2 nanosheets via a self-reduction reaction. During the reaction, gold seeds first form and disperse at the defects and edges of the MoS2 nanosheets, and then Au nanoparticles can further grow along the gold seeds. Figure 1 As shown in Figure A, the two-dimensional MoS2 nanosheets are deposited with uniform and high-density Au nanoparticles, confirming the formation of a phase-agnostic nanostructure. In the high-resolution transmission electron microscopy (HRTEM) image, two lattice spacings of 0.2 nm and 0.27 nm were observed, corresponding to the (200) and (100) crystal planes of Au and MoS2, respectively. Figure 1 In addition, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images (B). Figure 1 C) and element mapping image ( Figure 1 D) in the figure demonstrates the distribution of S, Mo and Au elements in the non-uniform composite material.

[0085] The particle sizes of MoS2 and Au@MoS2 were measured by dynamic light scattering (DLS) and were 220 nm and 180 nm, respectively. Figure 2 As shown in Figure A), the size of Au@MoS2 is slightly smaller than that of MoS2 nanosheets, which may be due to the in-situ reduction depletion of some molybdenum disulfide. Additionally, the Zeta potentials of Au@MoS2 and MoS2 are approximately -20 mV. Figure 2 The B in the figure indicates that both nanomaterials are relatively stable in aqueous solution.

[0086] In addition, the UV-vis-NIR spectrum of Au nanoparticles ( Figure 2 C) shows that it has an absorption peak at 520 nm. MoS2 produces representative absorption peaks at wavelengths of approximately 400, 620, and 650 nm. Au@MoS2 shows a new absorption peak corresponding to Au around 550 nm, indicating the successful synthesis of Au@MoS2. These results demonstrate that Au nanoparticles have been effectively deposited on the MoS2 surface, and the characteristic surface plasmon resonance peaks of Au contribute to the observed spectral shift.

[0087] Raman spectroscopy Figure 2 D) shows that both MoS2 and Au@MoS2 nanosheets produced two characteristic vibrational peaks, and Au@MoS2's E 2g and A 1g The peak shifted to the left by about 4cm -1 This may be due to the lattice strain caused by the change in the curvature of the MoS2 shell due to Au doping, indicating the generation of Au@MoS2 heterostructure, which further proves the successful synthesis of Au@MoS2 nanosheets.

[0088] 2. Dual catalytic activity of nanozymes

[0089] Nanozymes with peroxidase-like functions can induce the decomposition of overexpressed H2O2 in endogenous tumor microenzymes (TMEs) to produce highly toxic ·OH, thereby leading to tumor cell necrosis or apoptosis. This type of ROS-mediated nanozyme catalysis has important clinical value for tumor therapy. Therefore, the peroxidase-like activity of Au@MoS2 in an acidic environment (pH = 3.5) was first tested. Its peroxidase-like properties were investigated using the 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric method. Figure 3 As shown in Figure A, POD catalyzes the decomposition of H₂O₂ to generate ·OH, which oxidizes TMB to produce the blue oxide product oxTMB, with a characteristic absorption wavelength of 650 nm. Au nanoparticles, MoS₂, and Au@MoS₂ were introduced into a mixture of H₂O₂ and TMB, respectively, and the reaction was continued for 5 min at a normal physiological temperature of 37 °C. The results are as follows. Figure 3 As shown in Figure B, all three materials exhibit characteristic absorption peaks at 370 nm and 650 nm, indicating successful catalytic oxidation of TMB. The resulting color is blue, with Au@MoS2 showing the highest and deepest peak, indicating that all three materials possess peroxidase mimicry activity, with Au@MoS2 exhibiting the best mimicry activity. Furthermore, the change in absorbance at 650 nm over time was investigated, revealing that Au@MoS2 exhibits the fastest reaction rate and the best mimicry activity. Figure 3 (C in the text). In addition, the absorbance values ​​of all three materials tended to stabilize after 10 minutes, reaching reaction equilibrium.

[0090] Typically, the generated ·OH groups are largely consumed by the excess GSH in the TME. Therefore, further investigation is needed to determine whether Au@MoS2 can consume the excess GSH. The reaction product of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and GSH (TNB) exhibits a typical absorption peak at 412 nm. Therefore, the content of residual GSH can be monitored by observing the change in the TNB absorption peak over reaction time. Figure 4 A in the example. Figure 4 As time progresses, GSH is continuously consumed, and the absorbance at 412 nm gradually decreases, indicating that Au@MoS2 and MoS2 possess glutathione peroxidase mimicry activity.

[0091] 3. Photothermal activity

[0092] The photothermal properties of Au@MoS2 were investigated under 808 nm near-infrared laser irradiation. First, the photothermal properties of different materials were studied. For example... Figure 5As shown, during NIR laser irradiation, the temperature of the aqueous solution can increase by only 5℃, while the temperatures in MoS2 and Au@MoS2 solutions increase from 22℃ to 42℃ and 53℃, respectively. This indicates that both MoS2 and Au@MoS2 have photothermal properties, and Au@MoS2 has better photothermal properties than MoS2.

[0093] like Figure 6 As shown, for MoS2, the temperature change over time during the cooling phase was calculated to be τ = 135 s, and the photothermal conversion efficiency η was 39.3%. The correlation coefficient between the photothermal conversion curve and the first-order function fitting curve was 0.99473 (>0.99), indicating a good fit. Similarly, for Au@MoS2, the temperature change over time during the cooling phase was calculated to be τ = 146 s, and the photothermal conversion efficiency η was 51.2%. The correlation coefficient between the photothermal conversion curve and the first-order function fitting curve was 0.99701 (>0.99), indicating a good fit. All these results indicate that Au@MoS2 has good photothermal performance and can be used for synergistic therapy.

[0094] 4. The combined antitumor effect of photothermal nanozymes and ELE

[0095] The cytotoxicity of microneedle precursor HA to melanoma cells (B16F10 cells) was assessed using the MTT assay. After incubation with HAMNs at concentrations ranging from 0 to 500 μg / mL for 24 hours, the cytotoxicity was... Figure 7 As shown in Figure A, the survival rate of B16F10 cells remained above 85% with increasing HAMN concentration, indicating that HAMN has low toxicity to B16F10 cells and good biocompatibility.

[0096] To evaluate the anti-melanoma effect of Au@MoS2+β-ELE, B16F10 cells were co-incubated with Au@MoS2, Au@MoS2+β-ELE, or β-ELE, and then subjected to 808 nm near-infrared laser (1 W·cm⁻¹). -2 Irradiate for 5 minutes. (As shown) Figure 7 The β-ELE group showed lower cytotoxicity against B16F10 cells at lower concentrations, while its cytotoxicity was significantly enhanced at higher concentrations. Under laser irradiation, the cell viability of the β-ELE group was not significantly different from that of the unirradiated group, indicating that β-ELE has no photothermal effect.

[0097] like Figure 7As shown in C and D, Au@MoS2 and Au@MoS2+β-ELE were cytotoxic to B16F10 cells and significantly inhibited the proliferation of B16F10 cells in a concentration-dependent manner. Moreover, the cytotoxicity of the Au@MoS2+β-ELE group was significantly higher than that of the Au@MoS2 group at the same concentration. In addition, the viability of B16F10 cells in the laser irradiation group was further decreased, indicating that both Au@MoS2 and Au@MoS2+β-ELE had photothermal effects and could synergistically inhibit the growth of tumor cells.

[0098] To more clearly confirm cell death, Calcein-AM and PI were respectively applied to stain live cells (green) and dead cells (red). As Figure 8 shown in A, the experimental results showed that the Au@MoS2+β-ELE group exhibited the brightest red fluorescence, indicating the largest number of dead cells, suggesting that Au@MoS2+β-ELE had the best cytotoxic effect on cells. In addition, under the condition of photothermal stimulation, more dead cells were observed in the Au@MoS2 and Au@MoS2+β-ELE groups, indicating that photothermal stimulation could enhance the cytotoxicity of the materials to B16F10 cells, which was consistent with the above MTT results.

[0099] In addition, the quantitative results of the fluorescence intensity of dead cells / live cells ( Figure 8 shown in B and C) also showed that the fluorescence intensity of dead B16F10 cells was Control < β-ELE < Au@MoS2 < Au@MoS2+β-ELE, and the fluorescence intensity of live cells was opposite, and the fluorescence intensity of dead cells was higher under photothermal stimulation. These results indicated that the synergistic therapeutic effect of Au@MoS2 nanozyme, β-ELE, and photothermal was significantly improved.

[0100] 5. Preparation and Characterization of MNs Co-Loaded with Photothermal Nanozyme and ELE

[0101] Based on the significant nanozyme catalytic and photothermal properties of Au@MoS2 in vitro and the anti-tumor properties of β-ELE, Au@MoS2 and β-ELE were further integrated into a hyaluronic acid (HA) matrix to develop a dissolvable microneedle patch specifically for melanoma treatment. HA with good biocompatibility and water solubility was used as the matrix of MNs. As Figure 9 shown in A, Au@MoS2+β-ELE MNs were fabricated by a stepwise casting method. First, the PDMS microneedle mold was filled with a mixture of HA and Au@MoS2+β-ELE by centrifugation, then dried at 40 °C for 6 h, and finally, the Au@MoS2+β-ELE MNs were obtained by slowly separating them from the mold by clamping the edge area of the microneedles with tweezers. As Figure 9As shown in BD, the neatly arranged Au@MoS2+β-ELE MNs have a complete pyramid shape, with a needle tip height of 750 μm and an array size of 10 × 10 mm. 2 .

[0102] Meanwhile, the mechanical strength of the microneedles was tested using a universal testing machine, and the experimental results ( Figure 10 A) indicates that the mechanical strength of Au@MoS2+β-ELE MNs is greater than that of blank HA microneedles. The pressure range of Au@MoS2+β-ELE MNs during deformation is much greater than the minimum force (0.045N) required to penetrate the stratum corneum, proving that the mechanical strength of Au@MoS2+β-ELE MNs is sufficient to penetrate the skin and subcutaneous tissue. Furthermore, the photothermal properties of Au@MoS2+β-ELE MNs were studied using an 808nm near-infrared laser. The results are as follows... Figure 10 As shown in Figure B, after 5 minutes of laser irradiation, the temperature of Au@MoS2+β-ELE MNs reached 63℃, while the temperature of HA MNs under the same conditions was only slightly higher than room temperature. This indicates that the heating rate of Au@MoS2+β-ELE MNs is faster than that of HA MNs, proving that Au@MoS2+β-ELE MNs has good photothermal properties.

[0103] To investigate the solubility of Au@MoS2+β-ELE MNs, the tips of Au@MoS2+β-ELE MNs were placed in a PBS solution at pH 6.0. The tips of the microneedles dissolved completely within 20 seconds. Figure 11 The A in the figure indicates that Au@MoS2+β-ELE MNs have good solubility. Subsequently, the in vitro skin insertion ability of Au@MoS2 MNs was evaluated by puncturing pig skin. MNs were inserted into pig skin, and changes on the pig skin surface were observed at 0 min, 10 min, 20 min, and 30 min. Figure 11 (C) Micropores were observed on the surface of pigskin after MN insertion, demonstrating that MN can successfully penetrate the skin and has good mechanical strength. At 30 minutes, the micropores almost disappeared, indicating that the in vitro healing time of MN in pigskin is 30 minutes. Simultaneously, hematoxylin and eosin (H&E) staining revealed that Au@MoS2+β-ELE MNs can pierce the stratum corneum to form microchannels (C). Figure 11 (B) further confirms that Au@MoS2+β-ELE MNs can effectively penetrate the skin barrier for drug delivery.

[0104] 6. In vivo antitumor effect of co-loaded photothermal nanozymes and ELE's MNs

[0105] To investigate whether Au@MoS2+β-ELE MNs can effectively inhibit tumor growth, B16F10 tumor-bearing mice (a subcutaneous melanoma-bearing mouse model) were used as animal models to study its in vivo anti-tumor effect. Tumor-bearing mice were randomly divided into 6 groups of 8 mice each: HAMNs group, HAMNs+NIR group, Au@MoS2MNs group, Au@MoS2MNs+NIR group, Au@MoS2+β-ELE MNs group, and Au@MoS2+β-ELE MNs+NIR group. The drug was administered via microneedle. A second dose was given five days after the first dose, for a total of two doses. Tumor volume and body weight were recorded every two days.

[0106] Figure 12 The study showed changes in tumor volume in mice across different treatment groups during the treatment observation period. Results indicated that the HA MNs and HA MNs+NIR groups had no significant tumor-inhibiting effect; the Au@MoS2 MNs, Au@MoS2 MN+NIR, and Au@MoS2+β-ELE MNs groups showed moderate tumor inhibition; the Au@MoS2+β-ELE MNs+NIR group exhibited the most significant and effective tumor inhibition, almost completely eliminating the tumor. These results demonstrate that laser and chemotherapy alone cannot produce effective tumor inhibition. Effective tumor ablation is only achieved when Au@MoS2+β-ELE is released from the microneedle formulation and undergoes effective photothermal conversion under 808nm near-infrared laser irradiation. Laser irradiation promotes the release of Au@MoS2+β-ELE, which reacts with excessive GSH and H2O2 in the tumor microenvironment, producing toxic ·OH. •OH directly damages tumor tissue and also causes damage to intracellular mitochondria, promoting oxidative stress. Simultaneously, the synergistic effect of photothermal stimulation further enhances the tumor-suppressive effect. From Figure 13 This allows for a more direct observation of the tumor status in different treatment groups of tumor-bearing mice after the treatment observation period. The results show that the tumor in the Au@MoS2+β-ELE MNs+NIR group was almost completely eliminated, demonstrating the best tumor inhibition effect. This result is consistent with the tumor volume change curves mentioned above.

[0107] Furthermore, observation of H&E staining of major organs in mice showed no significant morphological differences in the major organs (heart, liver, spleen, lung, and kidney) between the drug-treated group and the HAMN group, further indicating that Au@MoS2+β-ELEMN is an effective and safe nano-formulation for treating tumors. Figure 14 ).

[0108] The above description is merely an embodiment of the present invention and should not be construed as limiting the scope of the present invention. For those skilled in the art, modifications and innovations can be made to the claims and drawings, or the invention can be applied to other technical fields. However, it should be noted that all modifications to the present invention should be included within the scope of patent protection of the present invention.

Claims

1. A method for the preparation of a microneedle formulation for the co-delivery of photothermal nanolaser and elemane, characterized by, The preparation method includes the following steps: Molybdenum disulfide (MoS2) solution was mixed with polyvinylpyrrolidone (PVP), and chloroauric acid (HAuCl4) solution was added. The reaction was carried out under stirring to obtain photothermal nanoenzyme Au@MoS2. The photothermal nanozyme Au@MoS2, β-elemene, and hyaluronic acid (HA) solution were mixed, placed in a microneedle mold, centrifuged, and dried to obtain the microneedle formulation; wherein the drying time was 6-8 hours and the drying temperature was 40-45℃.

2. The production method according to claim 1, characterized by, The mass ratio of molybdenum disulfide (MoS2) to chloroauric acid (HAuCl4) is (0.5-5):

1.

3. The preparation method according to claim 1, characterized in that, The molybdenum disulfide (MoS2) solution was obtained by dissolving MoS2 powder in an ethanol solution, followed by ultrasonication in an ice bath and centrifugation; wherein the ultrasonication time was 4-6 hours and the centrifugation speed was 6000-6500 rpm.

4. The production method according to claim 1, characterized by, The stirring time is 0.5-1 hour.

5. The preparation method according to claim 1, characterized in that, The mass ratio of photothermal nanozyme Au@MoS2 to β-elemene is 1:(1-5).

6. The preparation method according to claim 1, characterized in that, The mass percentage concentration of the hyaluronic acid solution is 5%-10%.

7. A microneedle formulation for co-delivering photothermal nanozymes and elemene, characterized in that, It is prepared by the method described in any one of claims 1-6.

8. The use of the microneedle formulation for co-delivering photothermal nanozyme and elemene as described in claim 7 in the preparation of a therapeutic drug for malignant skin melanoma.

9. The application according to claim 8, characterized in that, The microneedle formulation that co-delivers photothermal nanozymes and elemene synergistically responds to glutathione and hydrogen peroxide.

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