Composite microneedle loaded with novel nano-enzyme Fe2N / CeO2-MnCO as well as preparation method and application of composite microneedle

Abstract

The invention discloses a novel nano-enzyme Fe2N / CeO2-MnCO loaded composite microneedle as well as a preparation method and application thereof, and the preparation method comprises the following steps: uniformly mixing Fe (NO3) 3.9 H2O and CeO2, and carrying out high-temperature calcination in an ammonia gas atmosphere to obtain a nano-enzyme Fe2N / CeO2; the preparation method comprises the following steps: respectively adding nano enzymes Fe2N / CeO2, MnCO and PVP into absolute ethyl alcohol, and uniformly mixing to respectively obtain a Fe2N / CeO2 ethanol solution, a MnCO ethanol solution and a PVP ethanol solution; the preparation method comprises the following steps: weighing PVP and HA, adding PVP and HA into deionized water together, and uniformly mixing to obtain a PVP-HA aqueous solution; uniformly mixing a Fe2N / CeO2 ethanol solution, a MnCO ethanol solution and a PVP ethanol solution to obtain a mixed solution, adding the mixed solution into a microneedle mold, and after centrifugation, filling the tip of the microneedle with the mixed solution; the PVP-HA aqueous solution is added into a microneedle mold, the microneedle mold containing a PVP-HA substrate and a microneedle tip is obtained after centrifugation, and the composite microneedle loaded with the novel nano-enzyme Fe2N / CeO2-MnCO is obtained after demolding. The composite microneedle loaded with the novel nano-enzyme Fe2N / CeO2-MnCO can be used for effectively realizing deep treatment of fungal skin infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composite microneedle loaded with a novel nanoenzyme Fe2N / CeO2-MnCO, a preparation method thereof and an application thereof. Background Art

[0002] Deep cutaneous fungal infection (DCFI) faces great challenges in treatment because it is difficult for drugs to reach the lesions. Fungal infections are characterized by high penetration depth and biofilm formation under the skin, which exacerbates the difficulty of drug treatment. Fungal skin infections are usually caused by pathogenic fungi such as Candida, Cryptococcus and Aspergillus, and are characterized by high incidence, strong infectivity, and easy recurrence or reinfection. At present, bactericidal drugs represented by fluconazole are the most typical first-line antibacterial drugs in clinical practice. However, with the widespread use of antibiotics, clinical treatment faces treatment difficulties such as low transdermal efficiency, poor antifungal effect, easy formation of drug resistance and fungal biofilm formation. Summary of the invention

[0003] The purpose of the present invention is to overcome the defects of existing antifungal drugs such as low transdermal efficiency, poor antibacterial effect, easy formation of drug resistance, etc., and to provide a safe and effective non-antibiotic method for treating deep skin fungal infections.

[0004] In order to achieve the above object, the present invention provides a method for preparing a composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO, the preparation method comprising at least the following steps:

[0005] Step 1, Fe(NO3)3·9H2O and CeO2 are mixed evenly, and calcined at high temperature in an ammonia atmosphere to obtain nanozyme Fe2N / CeO2;

[0006] Step 2, adding nanozyme Fe2N / CeO2, MnCO, and PVP into anhydrous ethanol respectively, and mixing them evenly to obtain Fe2N / CeO2 ethanol solution, MnCO ethanol solution, and PVP ethanol solution respectively;

[0007] Step 3, weigh PVP and HA, add them into deionized water, and mix them evenly to obtain a PVP-HA aqueous solution;

[0008] Step 4, the Fe2N / CeO2 ethanol solution, the MnCO ethanol solution, and the PVP ethanol solution are uniformly mixed to obtain a mixed solution, the mixed solution is added to the microneedle mold, and the mixed solution is centrifuged to fill the microneedle tip, and the mixed solution in the non-tip part of the microneedle mold is scraped off to obtain a microneedle tip containing Fe2N / CeO2 and MnCO;

[0009] Step 5, adding the PVP-HA aqueous solution into the microneedle mold, obtaining a microneedle mold containing a PVP-HA substrate and a microneedle tip after centrifugation, and obtaining the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO after demoulding.

[0010] Preferably, in step 1, the mass ratio of Fe(NO3)3·9H2O:CeO2 is (150-250):1.

[0011] Preferably, in step 1, the high temperature is 400° C. to 450° C., and the calcination time is 1 h to 2 h.

[0012] Preferably, in step 4, the concentration of the Fe2N / CeO2 ethanol solution is 20 mg / mL to 30 mg / mL, the concentration of the MnCO ethanol solution is 20 mg / mL to 30 mg / mL, and the concentration of the PVP ethanol solution is 300 mg / mL to 500 mg / mL.

[0013] Preferably, step 2 further comprises using an ultrasonic stripper to uniformly disperse the nanozymes Fe2N / CeO2 and MnCO in anhydrous ethanol, and the parameters of the ultrasonic stripper are: 650W, 30min, 25°C.

[0014] The present invention also provides a composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO prepared by the above preparation method.

[0015] Preferably, the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO comprises: a substrate containing PVP-HA, and a microneedle tip loaded with nanozyme Fe2N / CeO2 and MnCO located on the substrate.

[0016] Preferably, the tip height of the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO is 500 μm to 1500 μm, and the tip distance is 300 μm to 880 μm.

[0017] The present invention also provides an application of a composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO, wherein the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO is used to prepare a drug for treating skin fungal infections.

[0018] Preferably, the fungi include at least one or more of Candida albicans, Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least:

[0020] The invention uses Fe(NO3)3·9H2O and CeO2 as raw materials to prepare nanoenzyme Fe2N / CeO2, uses polyvinyl pyrrolidone and hyaluronic acid as microneedle substrate parts, loads nanoenzyme Fe2N / CeO2 and carbonyl manganese MnCO on the microneedle tip, and prepares a composite microneedle loaded with a novel nanoenzyme Fe2N / CeO2-MnCO by a template method. The composite microneedle loaded with the novel nanoenzyme Fe2N / CeO2-MnCO has good biocompatibility and mechanical properties, can effectively break through the stratum corneum barrier, and is conducive to deep drug administration, thereby realizing transdermal delivery and release of nanoenzyme Fe2N / CeO2 and MnCO; in the case of high concentration of hydrogen peroxide generated in a fungal infection environment, a large amount of ROS (mainly ·OH) and CO gas can be rapidly generated to achieve deep treatment of skin fungal infection, which not only solves the problems of low transdermal efficiency and poor treatment effect of existing clinical drugs for treating skin fungal infection, but also effectively inhibits the generation of drug resistance and the formation of fungal biofilm. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Electron microscopic images of CeO2 and nanozyme Fe2N / CeO2 prepared in Example 1 of the present invention;

[0022] A is the scanning electron microscope image of CeO2; B is the scanning electron microscope image of the nanozyme Fe2N / CeO2; C is the transmission electron microscope image of the nanozyme Fe2N / CeO2; D is the transmission electron microscope element distribution map of the nanozyme Fe2N / CeO2.

[0023] Figure 2 The mechanism of the reaction between Fe2N / CeO2 and MnCO2 in the evaluation nanozyme of the present invention;

[0024] A is the activity diagram of Fe2N / CeO2-type peroxidase; B is the production of ROS by hydrogen peroxide catalyzed by Fe2N / CeO2; C is the release of CO gas by MnCO under different conditions.

[0025] Figure 3 The structure diagram of the Fe2N / CeO2-MnCO composite microneedle of the present invention;

[0026] A is the actual picture and scanning electron microscope image of the Fe2N / CeO2-MnCO composite microneedle; B is a picture showing the recovery of the Fe2N / CeO2–MnCO composite microneedle after piercing the mouse skin.

[0027] Figure 4 To evaluate the biosafety of the Fe2N / CeO2-MnCO composite microneedles in vitro;

[0028] A shows the effect of Fe2N / CeO2-MnCO composite microneedle on L929 cell proliferation; B shows the hemolytic activity detection and result statistics of Fe2N / CeO2-MnCO composite microneedle.

[0029] Figure 5 Evaluation of the antibacterial effect of the composite microneedle based on Fe2N / CeO2-MnCO on Candida albicans;

[0030] A is a graph showing the antibacterial effect of Fe2N / CeO2-MnCO with increasing concentration; B is the CFU statistics; C is the destructive effect of different treatments on fungal biofilm, a graph showing live-dead fluorescence staining taken with a confocal microscope.

[0031] Figure 6 Evaluation of the therapeutic effect of the composite microneedle of Fe2N / CeO2-MnCO on the Candida albicans infection model in mice;

[0032] A is a graph showing bacterial masses after Fe2N / CeO2-MnCO composite microneedle treatment; B is a statistical comparison of the mass of bacterial masses of mice in different groups; C is a graph showing the antibacterial effect after Fe2N / CeO2-MnCO composite microneedle treatment. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0034] Microneedle drug delivery system is a transdermal drug delivery technology that can break through the stratum corneum barrier and is painless. Compared with traditional transdermal drug delivery strategies, it has the advantages of high transdermal efficiency, good patient compliance, and easy self-administration. In recent years, nanozymes, as a special nanomaterial with enzyme-mimicking activity, have shown considerable prospects in multifunctional biomedical applications due to their advantages such as simple synthesis, adjustable catalytic activity, high stability, low cost, and easy handling. They are promising alternatives to natural enzymes in various applications and have attracted widespread attention in the field of antibacterial drugs. Fe2N / CeO2 nanozymes have good superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)-like activities, can achieve effective antibacterial activity by generating reactive oxygen species, and can avoid drug resistance; in addition, the Fenton-like properties of CeO2 can also consume GSH in fungi and reduce fungal activity. MnCO is a CO molecular prodrug that can release CO gas under the stimulation of reactive oxygen species such as H2O2 and ·OH. As an effective bactericide, CO destroys the ATP supply of bacteria, targets the respiratory chain to enhance reactive oxygen species, and interferes with DNA synthesis and repair, showing good antibacterial potential against a variety of microorganisms. In addition, the degradation product of MnCO, Mn 2+And CO has been reported to activate the cyclic GMP-AMP interferon gene synthase stimulator (cGAS-STING) pathway from various aspects, thereby promoting the production of cyclic GMP-AMP (cGAMP) to enhancing cGAMP-STING binding affinity. STING plays an important role in the innate immune response triggered by DNA virus and bacterial infection, further synergistically enhancing the antibacterial effect.

[0035] The present invention provides a composite microneedle loaded with a novel nanoenzyme Fe2N / CeO2-MnCO. After pressing on the skin, the composite microneedle of Fe2N / CeO2-MnCO can dissolve into the dermis and release Fe2N / CeO2 and MnCO. Fe2N / CeO2 has strong POD-like and CAT-like activity, which can catalyze H2O2 to produce ·OH. At the same time, the prodrug MnCO can also release CO under H2O2 conditions, freely diffuse into the fungal cell membrane, and induce fungal death by inhibiting the fungal respiratory chain. Its metabolite Mn 2+ It activates the cGAS-STING pathway in different ways from CO, thereby achieving the goal of synergistically enhancing the antibacterial effect of chemotherapy (CDT), gas therapy and immunotherapy.

[0036] Note: The Fe2N / CeO2-MnCO mentioned in the present invention refers to the composite microneedle prepared into Fe2N / CeO2-MnCO, and the Fe2N / CeO2+MnCO mentioned in the present invention refers to the physical mixing of nanozyme Fe2N / CeO2 and MnCO, which is not prepared into a composite microneedle.

[0037] 1. Preparation of nanozyme Fe2N / CeO2:

[0038] (1) Weigh 2.0 g of Ce(NO3)3·6H2O and 1.0 g of polyvinyl pyrrolidone (PVP), completely dissolve them in 75 mL of ethylene glycol and 5 mL of deionized water using a magnetic stirrer, and mix and stir for 20 min.

[0039] (2) The mixed solution in (1) was transferred to a 100 ml reaction vessel lined with PTFE and placed in a forced air drying oven at 160°C for 10 h. The cooled sample was washed several times with H2O and ethanol by centrifugation, and then dried in a vacuum oven at 60°C.

[0040] (3) The dried sample prepared in step (2) was placed in a muffle furnace and calcined at 500°C for 2 h (heating rate of 10°C / min) to obtain a CeO2 sample.

[0041] (4) Accurately weigh 4.04 g (0.01 mol) of Fe(NO3)3·9H2O and 60 mg of CeO2 from step (3), put them into a mortar, and grind them thoroughly until mixed. Finally, the ground powder was calcined at 450°C for 2 h (heating rate of 5°C / min) under an ammonia atmosphere. Finally, the reaction product nanozyme Fe2N / CeO2 was obtained.

[0042] like Figure 1 As shown in A, the CeO2 prepared by the present invention is a spherical nanoparticle, which is approximately "bowl-shaped" with a relatively hollow middle part. This "bowl-shaped" structure can better combine with Fe2N to form the nanozyme Fe2N / CeO2 of the interface structure, and the structure is more stable.

[0043] like Figure 1 B. Figure 1 C. Figure 1 As shown in D, the morphology and composition of the prepared nanozyme Fe2N / CeO2 were characterized by scanning electron microscopy and transmission electron microscopy, indicating that the present invention successfully prepared the nanozyme Fe2N / CeO2.

[0044] 2. Nanozyme Fe2N / CeO2 and MnCO react to produce ROS and release CO gas:

[0045] (A) 1 mL NaAc buffer (pH 4.0) + 60 μg / mL Fe2N / CeO2 (aqueous solution) + 1 mM 3,3',5,5'-tetramethylbenzidine (TMB) + 8 mM 30% H2O2, react at room temperature in the dark for 10 minutes, and test the absorbance in the absorption wavelength range of 400 nm-800 nm.

[0046] (B) Hydroxyphenylfluorescein (HPF) was diluted with PBS to a final concentration of 200 μM. 100 μg / mL of Fe2N / CeO2 deionized water solution and 100 μg / mL of MnCO methanol solution were prepared respectively. The test group was divided into eight groups, namely control group, H2O2 group, MnCO group, Fe2N / CeO2 group, Fe2N / CeO2+MnCO group, MnCO+H2O2 group, Fe2N / CeO2+H2O2 group, and Fe2N / CeO2+MnCO+H2O2 group. 50 μL of HPF was added to each group, 50 μL of PBS was added to the control group, and 50 μL of single material was also added. The mixed materials were added at a ratio of 1:4 (Fe2N / CeO2:MnCO), and the H2O2 concentration was 100 μM. After mixing, the fluorescence intensity was measured using an enzyme marker (λex 490 nm; λem 515 nm).

[0047] (C) First, take PBS (ph7.4, 10mM) and deoxygenate it by nitrogen bubbling for 30 minutes, and set aside. Weigh 1.0836mg of hemoglobin (Hb) and dissolve it in PBS, and seal it immediately; then weigh 1.6mg of sodium dithionite (SDT) and dissolve it in the above solution, and seal it immediately; use the pre-deoxygenated PBS to prepare 500μg / mL of the material to be tested (400μg / mL of MnCO+100μg / mL of Fe2N / CeO2), take 200μL and quickly add it to the above solution, and seal it immediately; then quickly add 100μM H2O2 solution, and seal it immediately; finally, test it every 10min, for a total of 180min, and the test absorption wavelength range is 350nm-700nm.

[0048] like Figure 2 A. Figure 2 B. Figure 2 As shown in Figure 3C, the nanozyme Fe2N / CeO2 has efficient POD-like activity and catalyzes hydrogen peroxide to produce ROS, mainly ·OH; Fe2N / CeO2 can catalyze MnCO to release CO gas more quickly and in larger quantities.

[0049] 3. Preparation and non-invasiveness of Fe2N / CeO2-MnCO composite microneedles:

[0050] (1) Nanozymes Fe2N / CeO2 and MnCO were added to anhydrous ethanol respectively, and were evenly dispersed in the ethanol solution by an ultrasonic stripper (650 W, 30 min, 25 ° C). After mixing evenly, Fe2N / CeO2 ethanol solution and MnCO ethanol solution were obtained respectively. PVP was weighed and added to anhydrous ethanol, and mixed evenly at room temperature to obtain PVP ethanol solution.

[0051] (2) PVP and hyaluronic acid (HA) were weighed, added into deionized water, and mixed evenly at room temperature to obtain a PVP-HA aqueous solution.

[0052] (3) The Fe2N / CeO2 ethanol solution, MnCO ethanol solution and PVP ethanol solution are mixed uniformly to obtain a mixed solution, and the mixed solution is added to a microneedle mold. After centrifugation (4000 rpm, 5 min, room temperature), the mixed solution fills the microneedle tip, and the mixed solution in the non-tip part of the microneedle mold is scraped off to obtain a microneedle tip containing Fe2N / CeO2 and MnCO.

[0053] (4) The PVP-HA aqueous solution is added to the microneedle mold, and after centrifugation (4000 rpm, 5 min, room temperature), a microneedle mold containing a PVP-HA substrate and a microneedle tip is obtained, which is placed in a constant temperature and humidity chamber (40% humidity, 25°C), dried overnight, and demolded to obtain the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO.

[0054] The prepared composite microneedles loaded with the new nanozyme Fe2N / CeO2-MnCO were inserted into the mouse skin and then removed. The skin condition of the mouse was observed and photographed at different time points.

[0055] like Figure 3 A shows the prepared composite microneedle loaded with the new nanozyme Fe2N / CeO2-MnCO, as shown in Figure 3 As shown in B, after the composite microneedle loaded with the new nanozyme Fe2N / CeO2-MnCO was inserted into the mouse skin for 60 minutes, the skin could be completely restored to smoothness.

[0056] 4. Evaluation of the biosafety of Fe2N / CeO2-MnCO composite microneedles in vitro:

[0057] The cytotoxicity of Fe2N / CeO2-MnCO composite microneedles with different concentrations was evaluated by CCK-8 assay. 4 L929 cells were seeded in 96-well plates at 37°C for 24 h. They were then exposed to different cell culture media incubated with composite microneedles of Fe2N / CeO2-MnCO at different concentrations for 24 h. After 24 h, complete culture medium containing 10% CCK-8 was added to the cells at 37°C for 1 h, and then the absorbance at 450 nm was read using a hybrid multimode reader.

[0058] 1 mL of fresh BALB / c mouse blood was collected and centrifuged (3000 rpm) for 15 min to obtain red blood cells. After washing with PBS five times, the red blood cell suspension was diluted to 2% (v / v) with PBS and further treated with different groups, including positive group, negative group, blank microneedle MN group, MnCO MN group, Fe2N / CeO2 MN group, and Fe2N / CeO2-MnCO MN group, at 37°C for 3 h. Among them, 2% Triton X-100 was used as a positive control (+), and 20 μL of PBS was used as a negative control (-). After centrifugation at 3000 rpm for 15 min, the supernatant was aspirated and the absorbance of each group at 450 nm was measured using an enzyme reader. The hemolysis rate of these samples was calculated according to the following formula: Hemolysis rate (%) = (ODe-ODn) / (ODp-ODn)×100%. ODe represents the experimental group, ODn represents the PBS-treated group (negative group), and ODp represents the 2% Triton X-100 group (positive group).

[0059] Depend on Figure 4 A and Figure 4 It can be seen from B that the composite microneedle treatment of Fe2N / CeO2-MnCO has less toxicity to cells and does not affect the hemolysis rate of cells, and has good biosafety.

[0060] 5. Evaluation of the antibacterial effect of Fe2N / CeO2-MnCO composite microneedles on Candida albicans:

[0061] In order to evaluate the synergistic antibacterial ability of Fe2N / CeO2 and MnCO, 500 μL of Candida albicans suspension (1×10 6 CFU / mL) were added to 24-well plates, and further treated with different groups, including Fe2N / CeO2+MnCO (Fe2N / CeO2:MnCO=1:4) treatment at different dosing concentrations (250μg / mL, 325μg / mL, 500μg / mL, 625μg / mL, 750μg / mL, 1000μg / mL). After these treated plates were incubated at 37°C overnight, the Candida albicans suspension was diluted to 1:1000, and then 10μL of the diluted solution was spread on YPD agar medium (yeast agar medium), incubated at 37°C overnight, and then the CFU was calculated.

[0062] In order to evaluate the antifungal biofilm ability of Fe2N / CeO2-MnCO composite microneedles, 2 mL of Candida albicans suspension (1×10 6CFU / mL) was added to the confocal dish and left for two days. A milky white biofilm visible to the naked eye formed at the bottom of the dish. Different groups of treatments were further performed, including BlankMN, MnCO MN, Fe2N / CeO2 MN, and Fe2N / CeO2-MnCO MN. The needle tip was gently pressed on the biofilm until it dissolved. After leaving it overnight, it was stained with SYBR Green / PI dye and the 3D biofilm was observed and photographed under a confocal microscope.

[0063] Depend on Figure 5 A. Figure 5 B. Figure 5 It can be seen from the C that the antibacterial effect gradually increases with the increase of the dosage concentration. The antibacterial effect of Fe2N / CeO2 has a certain concentration dependence. The composite microneedle of Fe2N / CeO2-MnCO shows an obvious killing effect on fungal biofilm.

[0064] 6. Evaluation of the therapeutic effect of Fe2N / CeO2-MnCO composite microneedle on the Candida albicans infection model in mice:

[0065] First, a mouse model of subcutaneous infection with Candida albicans was established. Male BALB / c mice were depilated on their backs and subcutaneously inoculated with Candida albicans (1×10 6 CFU / mL) 100μL. The next day, the mice were randomly divided into 7 groups for treatment, as follows: (1) Model; (2) Positive; (3) Fe2N / CeO2+MnCO; (4) MN; (5) MnCO MN; (6) Fe2N / CeO2 MN; (7) Fe2N / CeO2-MnCO MN. If the day of modeling is regarded as day 0, microneedle anti-infection treatment will be performed on the 1st, 3rd, and 5th days thereafter. After 8 days of treatment, the mice were euthanized and their bacterial masses were dissected to obtain the size and quality of the mass. The bacterial mass was placed in 1mL PBS solution, cut into pieces and mixed, and 100μL was taken to coat the plate.

[0066] Depend on Figure 6 A. Figure 6 B and Figure 6 It can be seen from C that the Candida albicans infection in mice treated with Fe2N / CeO2-MnCO composite microneedles was weakened, the bacterial masses were reduced, and the mass was lightened, which had a significant effect on anti-subcutaneous fungal infection.

[0067] It can be understood that, in addition to Candida albicans, other fungi such as Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa can also achieve similar therapeutic effects using the Fe2N / CeO2-MnCO composite microneedle treatment of the present invention.

[0068] In summary, the present invention provides a method for preparing a composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO, comprising the following steps: step 1, evenly mixing Fe(NO3)3·9H2O and CeO2, and calcining at high temperature in an ammonia atmosphere to obtain the nanozyme Fe2N / CeO2; step 2, respectively adding the nanozyme Fe2N / CeO2, MnCO, and PVP into anhydrous ethanol, and mixing them evenly to obtain Fe2N / CeO2 ethanol solution, MnCO ethanol solution, and PVP ethanol solution, respectively; step 3, weighing PVP and HA, adding them together into deionized water, and mixing them evenly to obtain PVP. P-HA aqueous solution; step 4, the Fe2N / CeO2 ethanol solution, MnCO ethanol solution, and PVP ethanol solution are mixed evenly to obtain a mixed solution, the mixed solution is added to the microneedle mold, and the mixed solution is centrifuged to fill the microneedle tip, and the mixed solution of the non-tip part of the microneedle mold is scraped off to obtain a microneedle tip containing Fe2N / CeO2 and MnCO; step 5, the PVP-HA aqueous solution is added to the microneedle mold, and the microneedle mold containing the PVP-HA substrate and the microneedle tip is obtained after centrifugation, and the composite microneedle loaded with the new nanozyme Fe2N / CeO2-MnCO is obtained after demoulding. The composite microneedle loaded with the new nanozyme Fe2N / CeO2-MnCO of the present invention can effectively achieve deep treatment of skin fungal infections.

[0069] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing a composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO, characterized in that: The preparation method at least comprises the following steps: Step 1, Fe(NO3)3·9H2O and CeO2 are mixed evenly, and calcined at high temperature in an ammonia atmosphere to obtain nanozyme Fe2N / CeO2; Step 2, adding nanozyme Fe2N / CeO2, MnCO, and PVP into anhydrous ethanol respectively, and mixing them evenly to obtain Fe2N / CeO2 ethanol solution, MnCO ethanol solution, and PVP ethanol solution respectively; Step 3, weigh PVP and HA, add them into deionized water, mix them evenly, and obtain a PVP-HA aqueous solution; Step 4, the Fe2N / CeO2 ethanol solution, the MnCO ethanol solution, and the PVP ethanol solution are uniformly mixed to obtain a mixed solution, the mixed solution is added to the microneedle mold, and the mixed solution is centrifuged to fill the microneedle tip, and the mixed solution in the non-tip part of the microneedle mold is scraped off to obtain a microneedle tip containing Fe2N / CeO2 and MnCO; Step 5, adding the PVP-HA aqueous solution into the microneedle mold, obtaining a microneedle mold containing a PVP-HA substrate and a microneedle tip after centrifugation, and obtaining the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO after demoulding.

2. The preparation method according to claim 1, characterized in that In step 1, the mass ratio of Fe(NO3)3·9H2O:CeO2 is (150-250):

1.

3. The preparation method according to claim 1, characterized in that: In step 1, the high temperature is 400° C. to 450° C., and the calcination time is 1 h to 2 h.

4. The preparation method according to claim 1, characterized in that: In step 4, the concentration of the Fe2N / CeO2 ethanol solution is 20 mg / mL to 30 mg / mL, the concentration of the MnCO ethanol solution is 20 mg / mL to 30 mg / mL, and the concentration of the PVP ethanol solution is 300 mg / mL to 500 mg / mL.

5. The preparation method according to claim 1, characterized in that: Step 2 also includes using an ultrasonic stripper to evenly disperse the nanozymes Fe2N / CeO2 and MnCO in anhydrous ethanol, and the parameters of the ultrasonic stripper are: 650W, 30min, 25°C.

6. A composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO, characterized in that: The composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO is prepared by the preparation method described in any one of claims 1 to 5.

7. The composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO according to claim 6, characterized in that: The composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO comprises: a substrate containing PVP-HA, and a microneedle tip loaded with the nanozyme Fe2N / CeO2 and MnCO located on the substrate.

8. The composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO according to claim 6, characterized in that: The tip height of the composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO is 500 μm to 1500 μm, and the tip distance is 300 μm to 880 μm.

9. An application of a composite microneedle loaded with a novel nanozyme Fe2N / CeO2-MnCO as claimed in any one of claims 6 to 8, characterized in that: The composite microneedle loaded with the novel nanozyme Fe2N / CeO2-MnCO is used for preparing a drug for treating fungal skin infections.

10. The use according to claim 9, characterized in that The fungi include at least one or more of Candida albicans, Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa.

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