Micro-needle preparation for co-delivering photo-thermal nano-enzyme and elemene as well as preparation method and application of micro-needle preparation

Abstract

The invention provides a microneedle preparation for co-delivering photo-thermal nano-enzyme and elemene as well as a preparation method and application of the microneedle preparation. The preparation method comprises the following steps: firstly, preparing a novel photo-thermal nano enzyme Au (at) MoS2 through a two-step water phase synthesis strategy; benefited from the synergistic effect of the hybrid material, the nano-enzyme shows significantly enhanced near-infrared (NIR) photothermal effect, peroxidase-like (POD) activity and glutathione-like peroxidase (GSHOx) activity, and can effectively remodel the tumor microenvironment and destroy the redox steady state. Furthermore, photo-thermal nano enzyme, beta-ELE and hyaluronic acid are used as raw materials, and a novel soluble microneedle preparation is successfully prepared through a template method. The novel composite microneedle is in a sharp rectangular pyramid shape, growth of melanoma can be effectively inhibited through percutaneous delivery of photo-thermal nano-enzyme and beta-ELE, remarkable systemic toxicity is avoided, and an innovative strategy is provided for non-invasive, efficient and safe melanoma combined treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a microneedle preparation for co-delivering photothermal nanozyme and elemene, a preparation method thereof, and an application thereof. Background Art

[0002] Malignant cutaneous melanoma (CM) is the most lethal skin cancer, with high invasiveness and the potential for distant metastasis. At present, the main treatment methods for melanoma include surgical resection, chemotherapy, and immunotherapy, etc. However, the clinical effects of surgery or chemotherapy for this disease are poor. Surgical treatment is mainly applicable to early melanoma, but for advanced or metastatic melanoma, the treatment effect is limited, and the postoperative recurrence rate is relatively high; the efficacy of traditional chemotherapy drugs (such as dacarbazine) for melanoma is low, and it is easy to produce drug resistance, and at the same time, it is accompanied by relatively serious systemic side effects. Existing clinical treatments have not significantly improved the overall survival rate of melanoma patients. Therefore, it is urgent to develop new, efficient, and safe treatments for melanoma.

[0003] In recent years, microneedles (MNs) as a transdermal drug delivery tool have the advantages of being painless, minimally invasive, and having adjustable delivery doses, and are expected to become an ideal choice for local treatment of melanoma. The soluble polymer MNs system can cross the skin barrier above CM, penetrate the stratum corneum, and create microchannels. As the polymer dissolves, the therapeutic molecules are directly deposited at the tumor site, realizing targeted delivery and release of local drugs without passing through the body circulation. So far, many soluble polymer MNs systems have been integrated with drug molecules, nanomaterials, photothermal agents, and photosensitizers for the treatment of CM. Although these soluble MNs systems show high delivery efficiency, due to the limited area of the MNs patch, the therapeutic dose delivered to the tumor site is still insufficient. In addition, the effects of many therapeutic molecules are weakened in the complex and metabolically abnormal tumor microenvironment (TME) of tumor cells. A large amount of evidence shows that tumors are diseases with abnormal metabolism, and the redox balance in the TME is abnormal, manifested as high levels of reactive oxygen species (ROS) and overexpression of the antioxidant glutathione (GSH). These characteristics promote tumor cell metabolism and proliferation, and ultimately severely weaken the anti-tumor efficiency of therapeutic agents. To solve the above problems, it is necessary to integrate ROS generation and GSH consumption to reshape the redox state of the TME, thereby enhancing the treatment effect of melanoma.

[0004] Nanozymes are a class of nanomaterials with catalytic activities similar to natural enzymes and can regulate the TME in various ways. For example, nanozymes with peroxidase (POD)-like activity can induce the conversion of endogenous H2O2 in the TME into highly toxic hydroxyl radicals (·OH), and achieve oxidative damage to tumor cells by catalyzing the Fenton-like reaction, thereby inhibiting tumor growth. In addition, nanozymes with glutathione oxidase (GSHOx)-like activity can consume the overexpressed GSH in the TME, disrupt the antioxidant defense system, lead to the inactivation of GPx-4 and the accumulation of lipid peroxides (LPO), 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 play an important role in enhancing the combined tumor therapy effect. Generally speaking, when designing nanozymes, multiple aspects should be considered, such as a simple preparation process, diverse catalytic activities, high catalytic efficiency, and multifunctionality, so as to maximize the therapeutic effect for clinical application.

[0005] At present, some studies have attempted combination therapies to improve the curative effect. However, it is difficult to achieve the tumor-targeted co-delivery of nanozymes and hydrophobic drugs by traditional injection or oral administration, and the risk of systemic toxicity is high. In addition, most existing microneedle systems use nanozymes or drugs alone, lacking systematic regulation means for the TME, resulting in limited anti-tumor effects. Summary of the Invention

[0006] The object of the present invention is to provide a microneedle preparation for co-delivering photothermal nanozymes and elemene, and its preparation method and application, aiming to achieve enhanced nano-catalytic / photothermal / traditional Chinese medicine combined transdermal therapy in view of the deficiencies of the prior art.

[0007] In the first aspect, the present invention provides a preparation method of a microneedle preparation for co-delivering photothermal nanozymes and elemene, comprising the following steps:

[0008] Mix a molybdenum disulfide MoS2 solution with polyvinylpyrrolidone PVP, add a chloroauric acid HAuCl4 solution, and react under stirring to obtain a photothermal nanozyme Au@MoS2;

[0009] Mix the photothermal nanozyme Au@MoS2, β-elemene, and a hyaluronic acid HA solution, place them in a microneedle mold for centrifugation, and obtain the microneedle preparation after drying.

[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, sonicating in an ice bath, and then centrifuging. Further, the sonication time is 4-6 h, and the centrifugation speed is 6000-6500 rpm.

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

[0013] Preferably, the mass ratio of the photothermal nanozyme 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 h and the drying temperature is 40 - 45 °C.

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

[0017] In a second aspect, the present invention provides a microneedle preparation for co - delivering a photothermal nanozyme and elemene, which is prepared by the above - mentioned method.

[0018] In a third aspect, the present invention provides the application of the above - mentioned microneedle preparation for co - delivering a photothermal nanozyme and elemene in the preparation of a therapeutic drug for malignant cutaneous melanoma.

[0019] Preferably, the microneedle preparation for co - delivering a photothermal nanozyme 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] The present invention uses soluble MNs to simultaneously transdermally deliver a novel photothermal nanozyme (Au - modified MoS2, Au@MoS2) and the traditional Chinese medicine molecule β - elemene (β - ELE). First, two - dimensional (2D) MoS2 nanosheets are obtained from MoS2 powder by the classical liquid - phase exfoliation method. Subsequently, Au nanoparticles are in - situ grown on the MoS2 nanosheets using the intrinsic self - reduction ability of MoS2 to form the Au@MoS2 photothermal nanozyme without adding an additional reducing agent.

[0022] Among them, the doping of high-density Au nanoparticles significantly enhances the NIR photothermal conversion and nano-catalytic efficiency of MoS2, thus maximizing its combined photothermal / catalytic therapy potential. In vitro experiments show that the photothermal nanozyme can efficiently catalyze the decomposition of H2O2 to generate ·OH, and at the same time continuously consume overexpressed GSH through the GSHOx-like catalytic reaction. This dual effect significantly remodels the TME and further enhances the killing effect of tumor cells under the combined action of β-ELE and NIR photothermal therapy. The photothermal nanozyme 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 the combined treatment of melanoma, with the highest tumor suppression efficiency and negligible side effects.

[0023] In summary, the present invention provides valuable insights for the rational design of nanozymes and also provides a novel and efficient combined strategy for enhancing melanoma treatment in a clinical setting. Description of the Drawings

[0024] Figure 1 It is a characterization diagram 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 images corresponding to S (green), Mo (blue), and Au (red) of Au@MoS2, and the scale bar is 100 nm.

[0025] Figure 2 Among them, A: Particle sizes of MoS2 and Au@MoS2; B: Zeta potentials 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 Among them, A: Schematic diagram of the TMB color development principle; B: UV-vis-NIR absorption spectra of Au nanoparticles, MoS2, and Au@MoS2 catalyzing the oxidation reaction of TMB; C: Absorbance value curves at 650 nm at different times of Au nanoparticles, MoS2, and Au@MoS2 catalyzing the oxidation reaction of TMB.

[0027] Figure 4 Among them, A: Schematic diagram of the DTNB color development principle; B: Absorbance value curves at 412 nm of MoS2 and Au@MoS2 catalyzing the oxidation reaction of DTNB over time.

[0028] Figure 5Photothermal property results of Au@MoS2, where A: Thermograms of water, MoS2, and Au@MoS2 under 808 nm near-infrared laser irradiation for 5 min; B: Photothermal heating curves of water, MoS2, and Au@MoS2 under 808 nm near-infrared laser irradiation for 5 min.

[0029] Figure 6 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; D: Time constant of Au@MoS2.

[0030] Figure 7 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 Dead / live double staining of B16F10 cells, where A: Dead / live double staining images of B16F10 cells with / without laser irradiation; B: Fluorescence quantitative results of dead / live double staining of B16F10 cells without laser irradiation; C: Fluorescence quantitative results of dead / live double staining of B16F10 cells with laser irradiation.

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

[0033] Figure 10 Performance characterization diagrams of microneedle preparations, where A: Mechanical strength of HAMNs and Au@MoS2 + β-ELE MNs; B: Photothermal properties of HAMNs and Au@MoS2 + β-ELE MNs.

[0034] Figure 11 Skin insertion results of microneedle preparations, where A: H&E staining of Au@MoS2 + β-ELE MNs after insertion into porcine skin; B: In vitro skin insertion ability of Au@MoS2 + β-ELE MNs.

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

[0036] Figure 13Anatomical diagrams of the tumor masses of mice in each group after the treatment.

[0037] Figure 14 H&E staining pictures of the main organs of mice in each group 12 days after drug administration. Specific implementation manners

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

[0039] As described above, the present invention provides a preparation method of a microneedle preparation for co-delivering a photothermal nanozyme and elemene, comprising the following steps:

[0040] Step (1): Dissolve molybdenum disulfide (MoS2) powder in an ethanol solution and ultrasonicate in an ice bath for 4 - 6 h, then centrifuge at a rotation speed of 6000 - 6500 rpm to obtain a molybdenum disulfide (MoS2) solution; add a stabilizer polyvinylpyrrolidone (PVP) and mix and stir, during the stirring process, add chloroauric acid (HAuCl4) solution, and continue stirring for 0.5 - 1 h to react to obtain a photothermal nanozyme Au@MoS2, wherein 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, which can be selected according to actual needs and are not limited herein.

[0041] Step (2): Mix the photothermal nanozyme Au@MoS2 and β-elemene according to a mass ratio of 1:(1 - 5), add a hyaluronic acid (HA) solution with a concentration of 5% - 10% (w / v), place it in a microneedle mold and centrifuge at a rotation speed of 3500 - 4000 rpm for 15 - 20 min, and dry at 40 - 45 °C for 6 - 8 h to obtain the microneedle preparation, wherein the mass ratio of the photothermal nanozyme Au@MoS2 to 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 herein.

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present 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 2 g of MoS2 powder and dissolve it in 50 mL of 45% ethanol solution. After ultrasonic treatment in an ice bath for 4 h, centrifuge at 6000 rpm and save the supernatant. Mix 10 mL of the ultrasonicated and centrifuged MoS2 solution with 100 μL of PVP solution and stir. During stirring, add 1 mL of HAuCl4 solution and continue stirring for 30 min to obtain the Au@MoS2 solution. Then wash it with deionized water 2 - 3 times and store it in a refrigerator at 4 °C for later use.

[0045] Characterize the morphology, particle size, Zeta potential, etc. of the Au@MoS2 nanosheets using instruments such as DLS, TEM, AFM, XPS, UV-vis-NIR. Drop the MoS2 and Au@MoS2 solutions onto copper grids respectively, dry them, and use TEM to characterize the nanosheets to determine their morphology and size. Then analyze the structure and element distribution of Au@MoS2 by high-resolution TEM. Inject the MoS2 and Au@MoS2 solutions into quartz cuvettes and potentiometric cells respectively, and measure their particle size and Zeta potential at 25 °C using DLS. Inject the Au NPs, MoS2, and Au@MoS2 solutions into quartz cuvettes respectively, and use a UV-vis-NIR spectrophotometer to measure the absorption spectra of the three materials in the range of 300 nm - 800 nm. Drop the MoS2 and Au@MoS2 solutions onto silicon wafers respectively, dry them, and use AFM to characterize the nanosheets to determine their thickness. Centrifuge the Au@MoS2 solution to remove the supernatant, then use a freeze dryer to turn it into powder and use XPS to characterize Au@MoS2 to determine the element composition and chemical bonding properties in the material. Centrifuge the Au@MoS2 solution to remove the supernatant, then use a freeze dryer to turn it into powder and use Raman spectroscopy to characterize Au@MoS2, and determine the molecular structure of the material according to the characteristic vibration peaks.

[0046] 2. Detection of nanozyme properties of Au@MoS2

[0047] Use 3,3',5,5'-tetramethylbenzidine (TMB) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as chromogenic agents for Au@MoS2 respectively, and use instruments such as a UV-vis-NIR spectrophotometer to detect the POD-like and GSHOx properties of Au@MoS2.

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

[0049] To study the in vitro photothermal temperature rise of different materials, place water, MoS2, and Au@MoS2 in 48-well plates respectively, and irradiate them with an 808 nm near-infrared laser at 1.5 W·cm -2 Irradiate, and use an infrared thermal imager to take infrared thermal images at 0, 1, 2, 3, 4, and 5 min and record the temperature changes at different time points.

[0050] To study the photothermal conversion efficiency of MoS2 and Au@MoS2, the aqueous solutions of MoS2 and Au@MoS2 were respectively exposed to a near-infrared laser (808 nm) of 1.5 W·cm -2 for 5 min, and then naturally cooled to room temperature. The temperature changes at different time points were recorded and the photothermal conversion efficiencies of MoS2 and Au@MoS2 were calculated. The photothermal conversion efficiency (η) refers to the ability of the material 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 (cm 2 ); T max is the equilibrium temperature (K) when the temperature reaches a steady state; T a is the ambient temperature; I represents the laser power; A 808 is the absorbance of the material at 808 nm. Q w represents the energy generated by water under laser irradiation, and the calculation formula of Q w is as follows:

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

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

[0055]

[0056] τ s represents the typical time constant; m w and c w are the mass and specific heat capacity of the solvent 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 and penicillin and 10% (v / v) fetal bovine serum was added, and then placed in a constant temperature incubator at 37 °C with a CO2 concentration of 5% for culture. When the cell density reached 80% - 90%, subculture could be carried out.

[0059] 5. Cytotoxicity

[0060] The cytotoxicity of HAMN against B16F10 cells was investigated by MTT colorimetric assay. First, the cell suspension was prepared at a density of 50,000 cells / mL and inoculated into 96-well plates, then cultured overnight in an incubator. The next day, HAMN at a concentration range of (0 - 500 μg / mL) was co-incubated with B16F10 cells for 24 hours. After that, 100 μl of MTT solution was added to each well using a multi-channel pipette and incubated in a 37°C incubator for 4 hours. After incubation, the culture supernatant in the wells was aspirated, and 200 μl of dimethyl sulfoxide (DMSO) was added to each well using a multi-channel pipette. The plate was placed in a microplate reader and shaken (medium speed) for 5 minutes. The absorbance at 570 nm was selected to measure the optical absorbance of each well and calculate the cell survival rate.

[0061] The cytotoxicity of different treatment groups against B16F10 cells was investigated by MTT colorimetric assay. B16F10 cells were inoculated onto 96-well plates at a density of 50,000 cells per well and incubated for 24 hours. Then, according to the experimental grouping, drugs were administered: Control group, Control + NIR group, β-ELE group, β-ELE + NIR group, Au@MoS2 group, Au@MoS2 + NIR group, Au@MoS2 + β-ELE group, Au@MoS2 + β-ELE + NIR group (where the ratio of material to drug was 1:1). Generally, the second column in the 96-well plate was the control group, and only 25 μl of cell culture medium with the same volume was added. In each well of the 3rd - 11th columns, 25 μl of the drug was added. For the groups that required NIR, after adding the drug, the cell suspension was irradiated with an 808 nm near-infrared laser (1 W / cm 2 ) for 5 minutes per well. After that, the 96-well plate was placed in an incubator and cultured for 24 hours. The cell survival rate was detected by the MTT method.

[0062] 6. Dual staining of live / dead cells

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

[0064] After laser irradiation, the cells were cultured for another 24 hours. After the incubation ended, the liquid in the wells was aspirated, and the cells were washed 2 - 3 times with 1×Assay Buffer to fully remove the residual esterase activity. Subsequently, 100 μl of the staining working solution was added to 200 μl of the cell suspension, mixed well, and incubated at 37°C for 15 minutes. The dye was aspirated, and the 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), loaded into the PDMS mold of the microneedles, centrifuged at 3500 rpm for 15 min, then placed in an oven at 40 °C. After 6 h, they were taken out and stored in a desiccator. The morphology of Au@MoS2+β-ELE MN was characterized by instruments such as a stereomicroscope and a scanning electron microscope.

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

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

[0069] To study 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 a stereomicroscope was used to observe and record the dissolution of the MN tip.

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

[0071] To study the biocompatibility of Au@MoS2+β-ELE MN, the MTT method 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). The cytotoxicity was detected after incubating Au@MoS2+β-ELE MN with two normal cell lines at a concentration range of 0 - 500 μg / mL for 24 hours.

[0072] 8. Establishment of Melanoma Mouse Model

[0073] Using mouse melanoma cell B16F10 as the model cell, the cells in the logarithmic growth phase were digested with trypsin, collected by centrifugation, and then diluted with the culture medium to 1×10 6A cell suspension of cells / mL was placed on ice for later use. 100 μL of the cell suspension was inoculated subcutaneously into the back of the right upper limb of mice to construct a tumor-bearing mouse model. After the modeling was completed, the growth of the tumors in the mice was monitored every 2 days.

[0074] 9. In vivo anti-tumor experiment

[0075] Using B16F10 tumor-bearing mice as model animals, the in vivo anti-tumor effect of Au@MoS2+β-ELE MNs was studied. B16F10 tumor-bearing mice with a tumor volume of 100 mm 3 were randomly divided into 6 groups, with 8 mice in each group: the HAMNs group, the HAMNs+NIR group, the Au@MoS2 MNs group, the Au@MoS2 MNs+NIR group, the Au@MoS2+β-ELE MNs group, and the Au@MoS2+β-ELE MNs+NIR group. Micro-needle administration was used. After the first administration, the drug was given again after 5 days, and a total of 2 administrations were given.

[0076] From the first day of drug administration, the body weight and tumor volume of the tumor-bearing mice were measured every 2 days. The formula for calculating the tumor volume is as follows:

[0077] V = L×W 2 / 2

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

[0079] All the mice were sacrificed 12 days after drug administration. One representative mouse from each group was taken, and its tumor site was photographed. After cervical dislocation and sacrifice, the tumor tissues of the mice in each group were collected. The tumor tissues were immediately rinsed with PBS, then fixed overnight with 10 times the volume of 4% paraformaldehyde, embedded in paraffin, sectioned and attached to glass slides for H&E staining, and the sections were scanned with a fully automatic section scanning imaging instrument to observe the necrosis of the tumors. In addition, the tumor tissues also needed to be analyzed by TUNEL, Ki-67, GPX-4, and LPO staining.

[0080] 10. In vivo biosafety evaluation

[0081] From the first day of drug administration, the mice in each group were weighed and recorded every two days. After the treatment was completed, the important organs (heart, liver, spleen, lung, kidney) of the mice in each group were collected, rinsed once with PBS, fixed overnight with 4% paraformaldehyde by volume fraction, embedded in paraffin, sectioned and stained with H&E, and the damage of the organs was observed with a fully automatic section scanning imaging instrument.

[0082] Result analysis and discussion:

[0083] 1. Synthesis and characterization of materials

[0084] Using MoS2 powder and HAuCl4 as raw materials and PVP as a stabilizer, Au@MoS2 nanosheets were prepared through a self-reduction reaction. During the reaction, gold seeds can first form and disperse at the defects and edges of MoS2 nanosheets, and then Au nanoparticles can further grow along the gold seeds. As Figure 1 shown in A of Figure 1 , the two-dimensional MoS2 nanosheets are evenly deposited with a high density of Au nanoparticles, confirming the formation of a heterophase 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 B of Figure 1 ). In addition, the high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image (

[0085] C of Figure 2 ) and the elemental mapping image ( Figure 2 D of

[0086] ) prove the distribution of S, Mo, and Au elements in the inhomogeneous composite material. Figure 2

[0087] The particle sizes of MoS2 and Au@MoS2 were measured by dynamic light scattering (DLS) to be 220 nm and 180 nm, respectively ( Figure 2 A of ), indicating that the size of Au@MoS2 is slightly smaller than that of MoS2 nanosheets, which may be due to the in-situ reduction depleting part of the molybdenum disulfide. In addition, the Zeta potential of Au@MoS2 and MoS2 is about -20 mV ( 2g ), 1g -1 indicating that both of these two nanomaterials are relatively stable in aqueous solution. ​

[0088] 2. Dual Catalytic Activity of Nanozymes

[0089] Nanozymes with mimetic peroxidase function can induce the decomposition of overexpressed H2O2 in the endogenous TME to generate highly toxic ·OH, leading to necrosis or apoptosis of tumor cells. This ROS-mediated nanozyme catalysis has important clinical value for tumor therapy. Therefore, the mimetic POD activity of Au@MoS2 in an acidic environment (pH = 3.5) was first tested. The 3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric method was used to study its peroxidase-like properties. As shown in A of Figure 3 , POD catalyzes the decomposition of H2O2 to generate ·OH, which oxidizes TMB to produce a blue oxidation product oxTMB, and its characteristic absorption wavelength is 650 nm. After introducing Au nanoparticles, MoS2, and Au@MoS2 into the mixture of H2O2 and TMB, the reaction continued at 37 °C, the normal physiological temperature, for 5 min. The results are shown in B of Figure 3 . All three materials have characteristic absorption peaks at 370 nm and 650 nm, indicating that TMB was successfully catalytically oxidized, and the color after the reaction is blue. Moreover, the peak of Au@MoS2 is the highest and the color is the deepest, indicating that all three materials have peroxidase mimetic activity and Au@MoS2 has the best mimetic activity. At the same time, the change in the absorption values of the three materials at 650 nm over time was also studied. The results show that the reaction rate of Au@MoS2 is the fastest and the mimetic activity is the best ( Figure 3 C). In addition, the absorption values of the three materials tend to be stable after 10 min, reaching the reaction equilibrium.

[0090] Generally, the generated ·OH will be basically depleted by the excessive GSH in the TME. Therefore, it was further explored whether Au@MoS2 could consume excessive GSH. The reaction product (TNB) of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and GSH has a typical absorption peak at 412 nm. Therefore, the content of residual GSH can be monitored by the change in the absorption peak of TNB over the reaction time ( Figure 4 A). As shown in B of Figure 4 , as time increases, GSH is continuously consumed, and the absorbance at 412 nm gradually decreases, indicating that Au@MoS2 and MoS2 have glutathione peroxidase mimetic activity.

[0091] 3. Photothermal Activity

[0092] The photothermal properties of Au@MoS2 were studied under 808 nm near-infrared laser irradiation. First, the photothermal performance of different materials was studied. As shown in Figure 5As shown, during NIR laser irradiation, the temperature of the aqueous solution can only increase by 5 °C, while the temperatures in the MoS2 and Au@MoS2 solutions increase from 22 °C to 42 °C and 53 °C respectively, indicating that both MoS2 and Au@MoS2 have photothermal properties, and the photothermal property of Au@MoS2 is better than that of MoS2.

[0093] As Figure 6 shown, the τ calculated using the temperature change with time during the cooling stage of MoS2 is 135 s, and the photothermal conversion efficiency η is 39.3%. The correlation coefficient between the photothermal conversion curve and the fitting curve of the first-order function is 0.99473 (>0.99), indicating a good fitting effect; similarly, the τ calculated using the temperature change with time during the cooling stage of Au@MoS2 is 146 s, and the photothermal conversion efficiency η is 51.2%. The correlation coefficient between the photothermal conversion curve and the fitting curve of the first-order function is 0.99701 (>0.99), indicating a good fitting effect. All the above results show that Au@MoS2 has good photothermal properties and can be used for synergistic therapy.

[0094] 4. Anti-tumor effect of the combination of photothermal nanozyme and ELE

[0095] The MTT method was used to detect the cytotoxicity of the micro-needle raw material HA on melanoma cells (B16F10 cells). After incubating HAMNs with B16F10 cells at a concentration range of (0 - 500 μg / mL) for 24 hours, as Figure 7 shown in A, as the concentration of HAMN increases, the survival rate of B16F10 cells is above 85%, indicating that HAMN has low cytotoxicity to B16F10 cells and good biocompatibility.

[0096] To evaluate the anti-melanoma effect of Au@MoS2 + β-ELE, B16F10 cells were incubated with Au@MoS2, Au@MoS2 + β-ELE, or β-ELE, and then irradiated with 808 nm near-infrared laser (1 W·cm -2 ) for 5 minutes. As Figure 7 shown in B, the β-ELE group has little killing effect on B16F10 cells at lower concentrations, while its killing effect on cells is significantly enhanced at higher concentrations. Under laser irradiation, there is no significant difference in the cell survival rate between the β-ELE group and the non-irradiated laser group, indicating that β-ELE has no photothermal effect.

[0097] As 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 cell killing ability 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 reduced, 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 used to stain live cells (green) and dead cells (red), respectively. As Figure 8 shown in A, the experimental results showed that the Au@MoS2+β-ELE group exhibited the brightest red fluorescence, indicating that it had the most dead cells, suggesting that Au@MoS2+β-ELE had the best killing effect on cells. In addition, under the condition of photothermal stimulation, there were more dead cells in the Au@MoS2 and Au@MoS2+β-ELE groups, indicating that photothermal stimulation could enhance the killing ability of the materials on 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 the 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, 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 B - D in [reference], the neatly arranged Au@MoS2+β - ELE MNs have a complete pyramid shape, with a tip height of 750 μm and an array size of 10×10 mm 2 .

[0102] Meanwhile, the mechanical strength of the microneedles was detected by a universal testing machine. The experimental results ( Figure 10 A in [reference]) show 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 larger than the minimum force (0.045 N) 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 tissues. In addition, the photothermal properties of Au@MoS2+β - ELE MNs were studied by 808 - nm near - infrared laser. The results are as shown in Figure 10 B in [reference]. When irradiated with laser for 5 min, the temperature of Au@MoS2+β - ELE MNs reaches as high as 63 °C, while the temperature of HA MNs under the same conditions is only slightly higher than room temperature, indicating that the heating rate of Au@MoS2+β - ELE MNs is faster than that of HA MNs, proving that Au@MoS2+β - ELE MNs have good photothermal properties.

[0103] To study the solubility of Au@MoS2+β - ELE MNs, the tips of Au@MoS2+β - ELE MNs were placed in PBS solution with pH 6.0, and the tips of the microneedles could be completely dissolved within 20 s ( Figure 11 A in [reference]), indicating that Au@MoS2+β - ELE MNs have good solubility. Subsequently, the in - vitro skin insertion ability of Au@MoS2 MNs was evaluated by piercing porcine skin. The MNs were inserted into porcine skin, and the surface changes of porcine skin were observed at 0 min, 10 min, 20 min, and 30 min respectively ( Figure 11 C in [reference]). Microchannels on the surface of porcine skin could be observed after inserting the MNs, proving that the MNs can successfully penetrate the skin and have good mechanical strength. At 30 min, the microchannels almost disappeared, indicating that the in - vitro healing time of MNs in porcine skin is 30 min. Meanwhile, hematoxylin and eosin (H&E) staining determination reveals that Au@MoS2+β - ELE MNs can pierce the stratum corneum to form microchannels ( Figure 11 B in [reference]), further confirming that Au@MoS2+β - ELE MNs can effectively penetrate the skin barrier for drug delivery.

[0104] 6. Antitumor effect in vivo of MNs co - loaded with photothermal nanozyme and ELE

[0105] To investigate whether Au@MoS2+β-ELE MNs can effectively inhibit tumor growth, B16F10 tumor-bearing mice (subcutaneous tumor-bearing model of mouse melanoma) were used as model animals to study their in vivo anti-tumor effects. The tumor-bearing mice were randomly divided into 6 groups, with 8 mice in each group: HAMNs group, HAMNs+NIR group, Au@MoS2MNs group, Au@MoS2 MNs+NIR group, Au@MoS2+β-ELE MNs group, Au@MoS2+β-ELE MNs+NIR group. Micro-needle administration was used. After the first administration, the drug was given again after a five-day interval, and the drug was administered a total of 2 times. The tumor volume and body weight of the mice were recorded every two days.

[0106] Figure 12 shows the changes in the tumor volume of mice in different treatment groups during the treatment observation period. The results show that the HA MNs group and the HA MNs+NIR group did not have obvious tumor inhibitory effects; the Au@MoS2 MNs group, the Au@MoS2 MN+NIR group, and the Au@MoS2+β-ELE MNs group had moderate tumor inhibitory effects; the Au@MoS2+β-ELE MNs+NIR group showed the most significant and effective tumor inhibitory effect, almost completely eliminating the tumor. The above results indicate that single laser and chemotherapy cannot produce effective tumor inhibitory effects. When Au@MoS2+β-ELE in the micro-needle preparation is released and undergoes effective photothermal conversion under 808 nm near-infrared laser irradiation, effective tumor thermal ablation can be achieved for the purpose of treatment. Laser irradiation promotes the release of Au@MoS2+β-ELE and reacts with excessive GSH and H2O2 in the tumor microenvironment to produce toxic ·OH. On the one hand, ·OH directly damages tumor tissues, and on the other hand, it causes damage to intracellular mitochondria, promotes oxidative stress, and at the same time the photothermal synergistic effect further enhances the tumor inhibitory effect. From Figure 13 it can be more intuitively seen the tumor conditions of tumor-bearing mice in different treatment groups after the end of the treatment observation period. The results show that the tumor in the Au@MoS2+β-ELE MNs+NIR group was almost completely eliminated, showing the best tumor inhibitory effect, which is consistent with the above tumor volume change curve.

[0107] In addition, by observing the H&E staining of the main organs of the mice, it can be seen that there are no obvious morphological differences in the main organs (heart, liver, spleen, lung, kidney) of the mice in the drug administration group compared with the HAMN group, further indicating that Au@MoS2+β-ELE MN is an effective and safe nano-formulation for treating tumors ( Figure 14 ).

[0108] The above are only embodiments of the present invention and should not be construed as limiting the scope of the present invention. For those skilled in the art, the claims and the content of the drawings can be modified, innovated or applied to other technical fields. However, it should be noted that all modifications to the present invention should be included within the scope of the patent protection of the present invention.

Claims

1. A preparation method of a microneedle preparation for co - delivering photothermal nanozyme and elemene, characterized in that, The preparation method comprises the following steps: Mix a molybdenum disulfide MoS2 solution with polyvinylpyrrolidone PVP, add a chloroauric acid HAuCl4 solution, and react under stirring to obtain a photothermal nanozyme Au@MoS2; Mix the photothermal nanozyme Au@MoS2, β-elemene, and a hyaluronic acid HA solution, place them in a microneedle mold for centrifugation, and obtain the microneedle preparation after drying.

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

1.

3. The preparation method according to claim 1, wherein, The molybdenum disulfide MoS2 solution is obtained by dissolving MoS2 powder in an ethanol solution, performing ice bath ultrasound, and then centrifuging; wherein, the ultrasound time is 4 - 6 h, and the centrifugation speed is 6000 - 6500 rpm.

4. The preparation method according to claim 1, characterized in that, The stirring time is 0.5 - 1 h.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the 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. The preparation method according to claim 1, characterized in that, The drying time is 6 - 8 h, and the drying temperature is 40 - 45 °C.

8. A microneedle preparation for co - delivering photothermal nanozyme and elemene, characterized in that, Prepared by the method according to any one of claims 1 - 7.

9. Use of the microneedle preparation for co-delivering a photothermal nanozyme and elemene in the preparation of a therapeutic drug for malignant cutaneous melanoma as claimed in claim 8.

10. The application according to claim 9, wherein The microneedle preparation for co-delivering a photothermal nanozyme and elemene synergistically responds to glutathione and hydrogen peroxide.

Images