Preparation method and application of a two-step microneedle for regulating reactive oxygen species homeostasis

Abstract

The present application relates to the technical field of biological materials, and particularly relates to a preparation method of a two-stage microneedle for regulating reactive oxygen species homeostasis and application thereof.The present application combines two complementary materials of Fe-MOF for oxidative antibiosis and CeO2 for ROS removal to construct a two-stage microneedle for regulating reactive oxygen species, which can not only realize local delivery of drugs, but also effectively treat periodontitis through antibiosis, ROS balance and bone promotion.The present application utilizes Fe-MOF and CeO2 nanomaterials to synergistically regulate ROS level, Fe-MOF catalyzes Fenton-like reaction to generate ROS in the periodontal pocket microenvironment, realizes efficient, rapid and broad-spectrum antibiosis, and compared with traditional antibiotics, can reduce the risk of drug resistance, while CeO2 has excellent antioxidant enzyme simulation activity, removes excess ROS and reduces inflammatory response, and the innovative combination of the two materials solves the core contradiction between efficient antibiosis demand and oxidative stress damage in the treatment of periodontitis.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a method for preparing bi-stage microneedles that regulate reactive oxygen species homeostasis and their applications. Background Technology

[0002] Periodontitis is a chronic inflammatory disease caused by dental plaque biofilm, characterized by periodontal tissue destruction and alveolar bone resorption. Reactive oxygen species (ROS) can damage the cell membranes, proteins, DNA, and other key structures of microorganisms through oxidation, disrupting their physiological functions and leading to their death. Therefore, generating excessive ROS at pathological sites to achieve ideal antibacterial effects has become a common strategy for treating many diseases. ROS plays a crucial role in the treatment of periodontitis, but current research focuses on increasing ROS production to enhance antibacterial effects, neglecting the dual nature of ROS function: excessive ROS can cause oxidative stress, further damaging host tissue structures. Studies have confirmed that the inflammatory response in periodontitis is closely related to increased local and systemic oxidative stress and impaired antioxidant capacity. Traditional treatments such as mechanical debridement and antibiotic therapy have limitations, including difficulty in reaching deep into the gingival sulcus, significant side effects from systemic administration, and the tendency to develop drug resistance. Therefore, developing novel treatment strategies that can not only deliver drugs locally but also effectively combat bacteria, regulate ROS balance, reduce inflammation, and promote tissue regeneration is of great significance.

[0003] The role of regulating reactive oxygen species in the treatment of inflammatory diseases has attracted widespread attention. Metal-organic frameworks (MOFs) are a class of porous coordination polymers formed by the self-assembly of metal ions or metal clusters with organic ligands through coordination bonds. Iron-based metal-organic frameworks (Fe-MOFs) specifically refer to MOF materials with iron ions as metal nodes. Due to their good biocompatibility, diverse coordination geometries, and variable redox states, they can achieve highly efficient antibacterial effects and have shown great potential in biocatalysis, antibacterial therapy, and drug sustained release. CeO2 has a unique face-centered cubic fluorite lattice structure and contains Ce... 3+ and Ce 4+ It exhibits two valence states and can rapidly switch between them, thus acting as both an oxidant and a reducing agent, demonstrating mimicry activities of superoxide dismutase (SOD) and catalase (CAT). In CeO2 and CeO 2-xDuring the alternating transformation process, the loss of oxygen atoms or the change in electronic configuration in the crystal lattice creates oxygen vacancies in the crystal structure, providing binding sites for the catalytic decomposition of ROS and thus clearing excess ROS. However, there are no reports on strategies for effectively combining these two complementary materials to construct a treatment for periodontitis that not only delivers the substance locally but also effectively fights bacteria, regulates ROS balance, reduces inflammation, and promotes tissue regeneration. Summary of the Invention

[0004] Based on the above background, this invention develops a method for preparing a two-stage microneedle that regulates reactive oxygen species homeostasis and its application. This invention combines two complementary materials, Fe-MOF with oxidative antibacterial properties and CeO2 with ROS scavenging properties, to construct a two-stage microneedle that regulates reactive oxygen species. This not only enables local drug delivery but also provides effective treatment for periodontitis through antibacterial activity, ROS balancing, and bone-promoting effects.

[0005] The technical solution of the present invention: A method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis includes the following steps: Preparation of S1 upper layer liquid, lower layer liquid and backing liquid Preparation of the upper layer liquid: Lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid was dissolved in PBS to prepare a LAP solution with a concentration of 5 mg / ml. Fe-MOF was dissolved in LAP solution to prepare Fe-MOF / LAP stock solution; Take Fe-MOF / LAP stock solution and add LAP solution and SilMA (methacrylamide silk fibroin) to prepare the upper layer solution. The concentration of Fe-MOF in the upper layer solution is 80-500 μg / ml. Preparation of the lower layer liquid: Dissolve CeO2 nanozyme in 5 mg / ml LAP solution to prepare CeO2 / LAP stock solution; The CeO2 / LAP stock solution was taken and LAP solution and SilMA were added to prepare the lower layer solution, wherein the concentration of CeO2 nanozyme in the lower layer solution was 80-500 μg / ml; Preparation of backing liquid: A gel prepolymer solution was prepared using the gel matrix as a backing solution. S2 adds the upper liquid to the mold, draws the absolute pressure in the sealed cavity containing the mold to and maintains it between 5 kPa and 20 kPa, vacuums the mold, centrifuges the mold to pour the solution into the mold, then heats and concentrates it, gradually increasing the concentration to improve the mechanical strength of the microneedles, and finally cures it with ultraviolet light at 365 nm, 6 W, and 1 min. S3 adds the lower layer liquid into the mold, performs vacuum treatment, concentrates the liquid, and then performs UV curing. S4 adds backing liquid into the mold, performs vacuum treatment, and then dries the mold to prepare the bi-dimensional microneedles e-MOF / CeO2@DL-MN.

[0006] Furthermore, the preparation of the upper layer liquid using Fe-MOF in step S1 includes the following steps: 1) At room temperature, 1,4,5,8-naphthalenetetracarboxylic anhydride, 4-amino-1,2,4-triazole were mixed with a solvent, stirred until homogeneous, and then heated under reflux. 2) After the reaction is complete, a precipitant is added to the reaction product to obtain a solid-liquid mixture; 3) After solid-liquid separation, a solid product is obtained, which is then dried to obtain the DBPT ligand; 4) After dissolving DBPT ligand and FeCl3 in an organic solvent containing tetrafluoroboric acid or its salts, the mixture is heated to react. The reaction product is then filtered, and the filtrate is taken as Fe-MOF.

[0007] Furthermore, the preparation of the lower layer liquid with CeO2 nanozyme in step S1 includes the following steps: ① Take Ce(NO3)3·6H2O and oleylamine and add them to a non-coordinated high-boiling-point solvent. After stirring at room temperature, a reaction solution is obtained. ② The reaction solution is reacted in an inert atmosphere at 80-125℃ for a period of time, then the temperature is raised to 220-260℃ and maintained for a period of time to obtain the reaction product. ③ After adding a separating agent to the reaction product, a solid-liquid mixture is obtained; ④ After solid-liquid separation of the solid-liquid mixture, a solid intermediate product is obtained; ⑤ CeO2 nanozymes can be prepared by sintering the solid intermediate product at 350-420℃.

[0008] Furthermore, the drying temperature in step S4 is 35-40℃.

[0009] Further, the preparation of the backing liquid in step S1: Polyvinyl alcohol and polyvinylpyrrolidone are added to deionized water and heated until the solution becomes transparent. The mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is (6-12):1.

[0010] Furthermore, the concentration of methacrylamide silk fibroin in the upper and lower liquids is 145-150 mg / ml.

[0011] Furthermore, in steps S2 and S3, the concentration ratio of Fe-MOF and CeO2 nanozymes in the upper and lower liquids added to the mold is 1:1.

[0012] Based on the same inventive concept, this invention also provides a bi-stage microneedle prepared by the aforementioned method for preparing a bi-stage microneedle for regulating reactive oxygen species homeostasis.

[0013] Based on the same inventive concept, the present invention also provides the application of the aforementioned bi-stage microneedles in the preparation of periodontitis treatment agents.

[0014] The beneficial effects achieved by this invention are as follows: 1) This invention innovatively combines nanomaterials to achieve ROS balance: Fe-MOF and CeO2 nanomaterials synergistically regulate ROS levels. Fe-MOF catalyzes a Fenton-like reaction in the periodontal pocket microenvironment to generate ROS, achieving highly efficient, rapid, and broad-spectrum antibacterial activity. Compared to traditional antibiotics, it reduces the risk of drug resistance. Simultaneously, CeO2, with its excellent antioxidant enzyme mimicry activity, scavenge excess ROS and alleviate inflammatory responses. This innovative combination of two materials resolves the core contradiction in periodontitis treatment: the need for highly efficient antibacterial activity versus oxidative stress damage. 2) Using a dual-stage microneedle as a local delivery system, the loaded material is delivered directly to the core area of ​​infection and inflammation through painless and minimally invasive penetration of the gingival tissue, so as to achieve efficient treatment and avoid the side effects of systemic administration and the disadvantages of insufficient local drug concentration caused by gingival crevicular fluid flushing.

[0015] 3) This dual-layer microneedle system integrates three key therapeutic functions: antibacterial, ROS balancing, and bone-promoting, avoiding the complexity and potential interactions associated with the combined use of multiple drugs. It provides a novel, non-surgical, minimally invasive, long-lasting local treatment option for periodontitis that promotes tissue regeneration, potentially becoming a powerful supplement or alternative to traditional mechanical debridement and antibiotic therapy. Attached Figure Description

[0016] Figure 1 These are microscopic morphology characterization images of the two materials prepared in this invention.

[0017] Figure 2 The image shows the morphology and structure of the hydrogel bi-dimensional microneedles according to an embodiment of the present invention.

[0018] Figure 3 For different concentrations of CeO2 in this embodiment of the invention, A: SOD enzyme activity, B: CAT enzyme activity, and C: total antioxidant capacity.

[0019] Figure 4 This is the UV absorption spectrum of the ability of different concentrations of Fe-MOF and CeO2 to balance ROS, as determined by ABDA in this invention.

[0020] Figure 5 The results of detecting intracellular ROS levels in BMSC cells of each group using the DCFH-DA fluorescent probe described in this invention.

[0021] Figure 6 This image shows the antibacterial effect of the micro-target described in this invention on S. aurenus and E. coli.

[0022] Figure 7 The results of osteogenic-associated alizarin red S and alkaline phosphatase staining of the microneedles described in this invention are shown. Detailed Implementation

[0023] The preparation method and application of the bi-stage microneedles for regulating reactive oxygen species homeostasis according to the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; unless otherwise specified, the experimental materials used in the following embodiments are all purchased from commercial channels.

[0025] Example 1: A method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis, comprising the following steps: Preparation of S1 upper layer liquid, lower layer liquid and backing liquid Preparation of the upper layer liquid: Weigh 500 μg of Fe-MOF and dissolve it in 1 ml of LAP solution with a concentration of 5 mg / ml (prepared with PBS as solvent), then disperse it by sonication to obtain Fe-MOF / LAP stock solution.

[0026] Take 100 μl of Fe-MOF / LAP stock solution and add it to 400 μl of LAP solution (concentration of 5 mg / ml) and 75 mg of SilMA (methacrylamide silk fibroin) to form the upper layer of Fe-MOF loaded with 100 μg / ml. Preparation of the lower layer liquid: Weigh 500 μg of CeO2 and dissolve it in 1 ml of LAP solution with a concentration of 5 mg / ml (prepared with PBS as solvent), and disperse it by sonication to obtain CeO2 / LAP stock solution; Take 100 μl of CeO2 / LAP stock solution, add 400 μl of LAP solution (concentration of 5 mg / ml) and 75 mg of SilMA respectively to form a lower layer liquid loaded with 100 μg / ml CeO2; Preparation of backing liquid: Add 180 mg PVA (polyvinyl alcohol) and 20 mg PVP (polyvinylpyrrolidone) to 1 ml of deionized water, and heat in a water bath at 100°C until the solution is clear and transparent to form the backing solution.

[0027] In the next steps S2 to S4, the mold is placed in a sealed cavity, and the absolute pressure in the sealed cavity is drawn down and maintained between 5 kPa and 20 kPa.

[0028] S2 Add 100 μl of supernatant to a PDMS mold (16.9×16.9 mm in size, 2 mm in groove depth, and 15-20 µm conical needle tip), evacuate for 2 min, remove and gently remove bubbles with a pipette tip, evacuate again for 2 min, centrifuge the PDMS mold in a 50 ml centrifuge tube at 4000 r / min for 4 min, concentrate at 37 °C for 1.5 h, and then cure under UV light for 1 min (365 nm, 6 W). S3 Add 100 μl of the lower layer liquid into the PDMS mold, vacuum for 2 min, concentrate at 37°C for 1.5 h, and cure under UV light (365 nm, 6 w). S4 Add all the backing liquid into the mold, vacuum for 2 minutes, dry at 37°C for 17 hours to form the mold, and the bi-stage microneedles Fe-MOF / CeO2@DL-MN can be prepared. The concentration ratio of Fe-MOF to CeO2 is 1:1. The preparation of Fe-MOF is shown below: 1) At room temperature, 2 mmol NTCDA (1,4,5,8-naphthalenetetracarboxylic anhydride), 4.2 mmol C2H4N4 (4-amino-1,2,4-triazole) and 30 ml DMF (N,N-dimethylformamide) were mixed, stirred evenly, and refluxed at 150 °C for 8 h.

[0029] 2) After the solution cools, add 30 ml of water and 20 ml of methanol, stir, and a brownish-red solid precipitate is obtained.

[0030] 3) After filtering the solid, washing it with methanol, and drying it at 37°C, the DBPT ligand can be obtained.

[0031] 4) Add 15 mg DBPT ligand and 10 mg FeCl3 to a mixed solution containing 3 ml DMF and 200 μl tetrafluoroboric acid, and sonicate the mixture to ensure uniform dispersion and dissolution.

[0032] 5) Then stir overnight in an oil bath at 120°C, cool to room temperature, and centrifuge to separate the solid.

[0033] 6) Wash three times alternately with ethanol and deionized water, and dry at 37°C.

[0034] The preparation of CeO2 nanozymes is as follows: ①Ce(NO3)3·6H2O (1.0 mmol) and oleylamine (3.0 mmol) were added to 4 g of octadecene (ODE) and stirred continuously at room temperature for 1 hour.

[0035] ② Heat the above mixture in argon to 120°C, maintain for 2 hours, and then maintain at 260°C for 1.5 hours.

[0036] ③ Cool to room temperature, then add 10 ml of cyclohexane and 40 ml of ethanol to the mixture.

[0037] ④ Centrifuge at 11000 r / min for 20 minutes, repeat three times.

[0038] ⑤ Collect the precipitate by centrifugation.

[0039] ⑥ Sinter at 400℃ for 8 hours in a high-temperature sintering furnace.

[0040] ⑦ The above precipitate was dried in an oven at 37°C to obtain CeO2 nanozyme.

[0041] (1) Preparation of Fe-MOF@DL-MN Weigh 500 μg Fe-MOF and dissolve it in 1 ml of 0.5% LAP. Disperse the solution by sonication. Take 100 μl of the solution, add 400 μl of LAP, and add 75 mg of SilMA to form the supernatant loaded with 100 μg / ml Fe-MOF.

[0042] Weigh 75 mg of SilMA and dissolve it in 500 μl of 0.5% LAP to form the lower layer.

[0043] Add 180 mg PVA (polyvinyl alcohol) and 20 mg PVP (polyvinylpyrrolidone) to 1 ml of deionized water, and heat in a water bath at 100°C until the solution is clear and transparent to form the backing solution.

[0044] Add 100 μl of the supernatant to the PDMS mold, vacuum for 2 min, remove and gently remove bubbles with a pipette tip, then vacuum again for 2 min.

[0045] Centrifuge the PDMS mold in a 50ml centrifuge tube at 4000r / min for 4min.

[0046] Concentrate at 37℃ for 1.5 hours, then cure under UV light for 1 minute.

[0047] Add the lower layer liquid, vacuum for 2 minutes, concentrate at 37°C for 1.5 hours, and then cure under UV light.

[0048] Add backing liquid to the PDMS mold and vacuum for 2 minutes.

[0049] After drying at 37℃ for 17 hours, it is molded to form Fe-MOF @DL-MN.

[0050] (2) Preparation of CeO2@DL-MN Weigh 75 mg of SilMA and dissolve it in 500 μl of 0.5% LAP to form the supernatant.

[0051] Weigh 500 μg CeO2 and dissolve it in 1 ml of 0.5% LAP. Disperse the solution by sonication. Take 100 μl of the solution and add 400 μl of LAP. Add 75 mg of SilMA to form the lower layer solution loaded with 100 μg / ml CeO2. Add 180 mg PVA (polyvinyl alcohol) and 20 mg PVP (polyvinylpyrrolidone) to 1 ml of deionized water, and heat in a water bath at 100°C until the solution is clear and transparent to form the backing solution.

[0052] (3) Pure SilMA DL-MN Weigh 75 mg of SilMA and dissolve it in 500 μl of 0.5% LAP to form the upper and lower layers. Add 100 μl of the supernatant to the PDMS mold, vacuum for 2 min, remove and gently remove bubbles with a pipette tip, then vacuum again for 2 min.

[0053] Centrifuge the PDMS mold in a 50ml centrifuge tube at 4000r / min for 4min. Concentrate at 37℃ for 1.5h and cure under UV light for 1min.

[0054] Add the lower layer liquid, vacuum for 2 minutes, concentrate at 37°C for 1.5 hours, and then cure under UV light.

[0055] Add backing liquid to the PDMS mold and vacuum for 2 minutes.

[0056] After drying at 37°C for 17 hours, it is molded to form pure SilMA DL-MN.

[0057] Relevant characterization experiments: (1) The morphology of CeO2 nanozymes, Fe-MOF and their bi-dimensional microneedles was characterized, and the results are shown in the figure. Figure 1 and Figure 2 .

[0058] Figure 1 These are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of CeO2 nanozymes. As can be seen from the images, the CeO2 particle size is approximately 20 nm. Figure 1 (C) is a SEM image of Fe-MOF, showing its regular polyhedral morphology. Test conditions: Observation was performed using a scanning electron microscope. The sample was platinum-sprayed, with a thickness of approximately 3.5 mm. The accelerating voltage was 10 kV, the working distance was 10 mm, and the magnification was ×15000. Figure 2 This is a morphology and structural characterization diagram of the hydrogel bi-stage microneedles described in this invention. Figure 2(A) is an optical micrograph of the microneedles (taken through an external lens), showing the macroscopic array structure of the conical microneedles; Figure 2 (B) is a SEM image of the microneedle, clearly showing its sharp tip and smooth conical surface. The microneedle is approximately 700 μm high, the substrate diameter is approximately 280 μm, and the tip-to-tip distance is approximately 650 μm. Figure 2 (C) is a laser scanning confocal microscope image of the microneedle. In the image, red fluorescence and green fluorescence mark the upper and lower layers of the microneedle, respectively. The boundary between the two is clear and there is no mixing between the layers, which verifies the two-level structure of the microneedle.

[0059] (2) Study on the ability of Fe-MOF / CeO2 to balance ROS and screening of ratios ① Detecting the antioxidant capacity of CeO2 nanozymes a. Total SOD activity assay kit (WST-8 method) for detecting SOD activity in CeO2. S1. Prepare CeO2 at concentrations of 100 μg / ml, 200 μg / ml, and 500 μg / ml using PBS.

[0060] S2. Prepare WST-8 / enzyme working solution: Take 3775 μl of SOD detection buffer, add 200 μl of WST-8 and 25 μl of enzyme solution.

[0061] S3. Prepare the startup solution: Add 273 μl of SOD buffer to 7 μl of startup solution.

[0062] S4. Take a 96-well plate and set up sample wells and various blank control wells.

[0063] Add 20 μl of the sample to be tested, 160 μl of WST-8 / enzyme working solution, and 20 μl of starter solution to the sample well in sequence; Add 20 μl of SOD detection buffer, 160 μl of WST-8 / enzyme working solution, and 20 μl of starter solution to well 1 of the blank control; add 40 μl of SOD detection buffer and 160 μl of WST-8 / enzyme working solution to well 2 of the blank control. Add 20 μl of the test sample, 20 μl of SOD detection buffer, and 160 μl of WST-8 / enzyme working solution to each of the three wells in the blank control.

[0064] S5, incubate at 37℃ for 30 min, measure absorbance at 450 nm, and calculate the inhibition percentage.

[0065] b. Detection of CAT enzyme activity in CeO2 using a catalase assay kit. S1. Prepare CeO2 at concentrations of 100 μg / ml, 200 μg / ml, and 500 μg / ml using PBS.

[0066] S2. Prepare 5mM H2O2 solution: Take 10μl H2O2 and add it to 1990μl catalase detection buffer; Prepare 250mM H2O2 solution: Take 50μl H2O2 and add it to 150μl catalase detection buffer.

[0067] S3. Take an appropriate amount of peroxidase and prepare a colorimetric working solution by mixing peroxidase and chromogenic substrate at a ratio of 1:1000.

[0068] S4. Take 0, 12.5, 25, 50, and 75 μl of 5 mM hydrogen peroxide solution into 1.5 ml centrifuge tubes, add catalase detection buffer to 100 μl, mix well, and prepare hydrogen peroxide standard solutions.

[0069] S5. Set up sample wells and blank control wells in a 96-well plate. Add 40 μl of catalase detection buffer and 10 μl of 250 mM hydrogen peroxide solution to the blank control wells.

[0070] S6. Add 40 μl of sample and 10 μl of 250 mM hydrogen peroxide to the sample well, mix well, react at 25 °C for 5 min, then add 450 μl of hydrogen peroxide reaction stop solution, invert and mix well, take 10 μl of the solution and add 40 μl of catalase detection buffer, mix well, and then take 10 μl of the solution and add it to a 96-well plate.

[0071] Add 4 μl each of S7 and hydrogen peroxide standard solution to a 96-well plate. Simultaneously add 200 μl of colorimetric working solution to the sample wells and blank control wells. Incubate at 25°C for 15 min, then measure A. 520 .

[0072] S8. Calculate the number of micromoles of residual hydrogen peroxide in the sample based on the standard curve to obtain the hydrogen peroxide consumption rate.

[0073] c. Total antioxidant capacity assay kit (FRAP method) for detecting the total antioxidant capacity of CeO2. S1. Prepare CeO2 at concentrations of 100 μg / ml, 200 μg / ml, and 500 μg / ml using PBS.

[0074] S2. Preparation of working solution: Mix 3000 μl of TPTZ diluent with 300 μl of TPTZ solution, add 300 μl of detection buffer, and incubate at 37°C.

[0075] S3. Preparation for standard curve determination: Take 27.8 mg FeSO4·7H2O, add deionized water to a final volume of 1 ml to obtain a 100 mM FeSO4 solution, and dilute to 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM.

[0076] S4. Set up sample wells, blank wells, and standard curve wells in a 96-well plate. Add 180 μl of FRAP working solution to each test well, 5 μl of sample to each sample well, and 5 μl of PBS to each blank well.

[0077] After incubating at 37℃ for 5 minutes, A was measured. 593 The total antioxidant capacity of the sample was calculated using the corresponding concentration of the FeSO4 standard solution.

[0078] d. Detect ROS levels The balancing ability of Fe-MOF and CeO2 on ROS was determined using ABDA (9,10-anthrayl-bis(methylene)dimalonic acid).

[0079] S1. Prepare Fe-MOF and CeO2 at concentrations of 100 μg / ml, 200 μg / ml, and 500 μg / ml using PBS.

[0080] S2. Take 9 centrifuge tubes and divide them into three groups: Fe-MOF, CeO2, and Fe-MOF / CeO2. Add 1 ml of each concentration to the centrifuge tubes of the Fe-MOF and CeO2 groups. For the Fe-MOF / CeO2 groups, first add 100 μg / ml of Fe-MOF, and then add 100 μg / ml, 200 μg / ml, and 500 μg / ml of CeO2 respectively after 24 h.

[0081] After 24 hours, 2 mg of ABDA was dissolved in 1 ml of DMSO. 1 ml of each sample was taken and 20 μl of ABDA was added. The waveform at 400 nm was detected using a UV spectrophotometer.

[0082] e. Use the DCFH-DA fluorescent probe to detect the balancing ability of Fe-MOF and CeO2 on ROS.

[0083] S1. Weigh 1 mg of Fe-MOF and CeO2, autoclave, and then prepare and dilute with α-MEM medium to prepare Fe-MOF culture medium containing 100 μg / ml and CeO2 culture medium containing 100 μg / ml, 200 μg / ml, and 500 μg / ml. Periodontal ligament stem cells were cultured in S2 and 24-well plates. After adhesion, the culture medium was discarded, and the cells were washed with PBS. 100 μg / ml Fe-MOF culture medium was added to Fe-MOF wells; 100 μg / ml CeO2 culture medium was added to CeO2 wells; and 100 μg / ml Fe-MOF culture medium was added to Fe-MOF / CeO2 wells.

[0084] After S3 and 24h, the solution in the Fe-MOF / CeO2 wells was discarded, and the wells were rinsed with PBS. CeO2 culture medium at concentrations of 100 μg / ml, 200 μg / ml, and 500 μg / ml was added, respectively. After 24 hours, add Rosup to the positive control well at a ratio of 1:1000 and incubate for 30 minutes.

[0085] S5 and DCFH-DA were diluted 1:1000 with serum-free medium, the liquid in the wells was discarded, and 400 μl was added to each well. The mixture was then incubated at 37°C for 40 min.

[0086] S6. Discard the liquid, wash three times with α-MEM medium, and observe under a confocal microscope.

[0087] The results are attached. Figure 3 To be continued Figure 5 .

[0088] Figure 3 The figure shows the SOD enzyme activity, CAT enzyme activity, and total antioxidant capacity of CeO2 at different concentrations as described in this invention. As can be seen from the figure, the SOD enzyme activity and total antioxidant capacity of 100 μg / ml CeO2 were significantly higher than those of the other concentration groups, while the CAT enzyme activity test results showed no statistically significant difference among the three concentrations.

[0089] Figure 4 The following are the UV absorption spectra of the ability of different concentrations of Fe-MOF and CeO2 to balance ROS as described in this invention: Figure 4 (A) shows the absorption spectra of CeO2 treatment groups of 100 μg / ml, 200 μg / ml, and 500 μg / ml, reflecting their antioxidant capacity. The results show that the 100 μg / ml group has the strongest antioxidant capacity. Figure 4 (B) shows the absorption spectra of Fe-MOF treatment groups at concentrations of 100 μg / ml, 200 μg / ml, and 500 μg / ml, indicating that their ROS generation capacity increases with increasing concentration. Figure 4 (C) shows the absorption spectra of 100 μg / ml Fe-MOF and CeO2 composite treatment groups with different concentrations. CeO2 was added 24 h after Fe-MOF was added, and its synergistic regulatory effect was detected after 24 h. The results show that when the ratio of 100 μg / ml Fe-MOF to 100 μg / ml CeO2 is 1:1, ROS can be controlled at a low level.

[0090] Figure 5 The results of detecting intracellular ROS levels in each group of cells using the DCFH-DA fluorescent probe described in this invention.

[0091] Figure 5 (A) shows the fluorescence microscopy images of each group. Green fluorescence indicates the ROS level. The results show that the fluorescence intensity of the Fe-MOF to CeO2 concentration ratio of 1:1 is weaker than that of other ratio groups. Figure 5(B) is the corresponding quantitative fluorescence intensity statistical chart, showing that the 1:1 concentration ratio exhibits the best ROS scavenging ability, with its ROS level being only about 1 / 7 of that of the Fe-MOF single group. Antibacterial performance evaluation.

[0092] (3) Evaluation of antibacterial properties S1. 30 μL of bacterial suspensions of *Escherichia coli* and *Staphylococcus aureus* were mixed with 270 μL of LB liquid medium, and then blended with DL-MN, Fe-MOF@DL-MN, CeO2@DL-MN, and Fe-MOF / CeO2@DL-MN microneedles. The Fe-MOF and CeO2 loading concentrations were both 100 μg / ml. The mixtures were incubated at 37℃ for 3 hours. The control group consisted of blank wells.

[0093] S2. Inoculate 100 μL of the diluted bacterial solution onto an agar plate, incubate at 37°C for 12 hours, and observe and calculate the antibacterial rate. The results showed that Fe-MOF@DL-MN had good antibacterial activity, with an antibacterial rate of 80.88 ± 3.48% against S. aurens and 90.09 ± 0.99% against E. coli. When Fe-MOF and CeO2 were loaded at a concentration ratio of 1:1, the antibacterial performance of Fe-MOF / CeO2@DL-MN was even more outstanding, with an antibacterial rate of 96.83 ± 0.72% against S. aurens and 93.20 ± 1.03% against E. coli.

[0094] (4) Evaluation of osteogenic performance a. Staining with BCIP / NBT alkaline phosphatase colorimetric kit S1. Prepare DL-MN, Fe-MOF@DL-MN, CeO2@DL-MN, and Fe-MOF / CeO2@DL-MN, and sterilize with ultraviolet light for 24 hours.

[0095] S2. Place each group of microneedles in a 24-well plate, add α-MEM medium, and extract at 37°C for 24 hours. Centrifuge the extract at 10,000 r / min for 10 minutes, collect the supernatant, filter through a 0.22 μm filter membrane, add 10% FBS, and prepare the complete working solution.

[0096] S3. Periodontal ligament stem cells were seeded into 24-well plates and cultured for 24 hours. After cell adhesion, the original culture medium was removed, the cells were washed with PBS, and complete working solution was added. The medium was changed every 3 days.

[0097] S4. After 7 days, remove the working solution from the hole, wash with deionized water 3 times, add 4% paraformaldehyde for fixation for 10 minutes, remove the fixative, and wash with deionized water 3 times.

[0098] S5. Take 33 μl of BCIP (5-bromo-4-chloro-3-indolyl-phosphate) solution and add it to 66 μl of NBT (nitroblue tetrazolium) solution. Mix the solution with 10 ml of alkaline phosphatase colorimetric buffer to prepare the colorimetric working solution.

[0099] S6. Add 600 μl of colorimetric working solution to each well, incubate at room temperature in the dark for 30 min, remove the staining solution, wash 3 times with deionized water, and observe under a microscope.

[0100] b. Alizarin Red S staining S1. Prepare DL-MN, Fe-MOF@DL-MN, CeO2@DL-MN, and Fe-MOF / CeO2@DL-MN, and sterilize with ultraviolet light for 24 hours.

[0101] S2. Place each group of microneedles in a 24-well plate, add α-MEM medium, and extract at 37°C for 24 hours. Centrifuge the extract at 10,000 r / min for 10 minutes, collect the supernatant, filter through a 0.22 μm filter membrane, add 10% FBS, and prepare the complete working solution.

[0102] S3. Periodontal ligament stem cells were seeded into 24-well plates and cultured for 24 hours. After cell adhesion, the original culture medium was removed, the cells were washed with PBS, and complete working solution was added. The medium was changed every 3 days.

[0103] S4. After 14 days, remove the working solution from the well, wash with deionized water 3 times, add 95% ethanol for 10 minutes to fix, remove the fixative, and wash with deionized water 3 times.

[0104] S5. Add 600 μl of Alizarin Red S staining solution to each well, incubate at 37°C in the dark for 30 min, remove the staining solution, wash 3 times with deionized water, and observe under a microscope.

[0105] The results showed that both staining methods in the Fe-MOF / CeO2@DL-MN group were significantly deeper than those in the control group and the single-material group, suggesting that the two materials have the potential to synergistically promote osteogenic formation.

[0106] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis, characterized in that, Includes the following steps: Preparation of S1 upper layer liquid, lower layer liquid and backing liquid Preparation of the upper layer liquid: Fe-MOF was dissolved in LAP solution to prepare Fe-MOF / LAP stock solution; Take Fe-MOF / LAP stock solution and add LAP solution and methacrylamide silk fibroin to prepare the upper layer solution, wherein the concentration of Fe-MOF in the upper layer solution is 80-500 μg / ml; Preparation of the lower layer liquid: CeO2 nanozyme was dissolved in LAP solution to prepare CeO2 / LAP stock solution; Take CeO2 / LAP stock solution and add LAP solution and methacrylamide silk fibroin to prepare the lower layer solution. The concentration of CeO2 nanozyme in the lower layer solution is 80-500 μg / ml. Preparation of backing liquid: A gel prepolymer solution was prepared using the gel matrix as a backing solution. S2 adds the upper liquid to the mold, vacuums it, centrifuges the mold, heats the upper liquid to concentrate it, and finally cures it with ultraviolet light. S3 adds the lower layer liquid into the mold, then vacuums it, heats and concentrates the lower layer liquid, and then performs ultraviolet curing. S4 adds backing liquid into the mold, vacuums it, heats and dries it to form the double-stage microneedles Fe-MOF / CeO2@DL-MN.

2. The method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis according to claim 1, characterized in that, The preparation of the upper layer liquid using Fe-MOF in step S100 includes the following steps: 1) At room temperature, 1,4,5,8-naphthalenetetracarboxylic anhydride, 4-amino-1,2,4-triazole were mixed with a solvent, stirred until homogeneous, and then heated under reflux. 2) After the reaction is complete, a precipitant is added to the reaction product to obtain a solid-liquid mixture; 3) After solid-liquid separation, a solid product is obtained, which is then dried to obtain the DBPT ligand; 4) After dissolving DBPT ligand and FeCl3 in an organic solvent containing tetrafluoroboric acid or its salts, the mixture is heated to react. The reaction product is then filtered, and the filtrate is taken as Fe-MOF.

3. The method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis according to claim 1, characterized in that, The preparation of the lower layer liquid using CeO2 nanozyme in step S100 includes the following steps: ① Take Ce(NO3)3·6H2O and oleylamine and add them to a non-coordinated high-boiling-point solvent. After stirring at room temperature, a reaction solution is obtained. ② The reaction solution is reacted in an inert atmosphere at 80-125℃ for a period of time, then the temperature is raised to 220-260℃ and maintained for a period of time to obtain the reaction product. ③ After adding a separating agent to the reaction product, a solid-liquid mixture is obtained; ④ After solid-liquid separation of the solid-liquid mixture, a solid intermediate product is obtained; ⑤ CeO2 nanozymes can be prepared by sintering the solid intermediate product at 350-420℃.

4. The method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis according to claim 1, characterized in that, The drying temperature in step S4 is 35-40℃.

5. The method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis according to claim 1, characterized in that, Preparation of the backing liquid in step S1: Polyvinyl alcohol and polyvinylpyrrolidone are added to deionized water and heated until the solution becomes transparent. The mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is (6-12):

1.

6. The method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis according to claim 1, characterized in that, The concentration of methacrylamide silk fibroin in the upper and lower liquids in step S1 is 145-150 mg / ml.

7. The method for preparing a two-stage microneedle for regulating reactive oxygen species homeostasis according to claim 1, characterized in that, In steps S2 and S3, the concentration ratio of Fe-MOF and CeO2 nanozymes in the upper and lower liquids added to the mold is 1:

1.

8. The bi-stage microneedles prepared by the method for preparing bi-stage microneedles for regulating reactive oxygen species homeostasis as described in any one of claims 1 to 7.

9. The use of the bi-stage microneedles according to claim 8 in the preparation of periodontitis treatment agents.

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