Preparation method of ROS (reactive oxygen species) response microsphere encapsulated mitochondrial targeted nootkatone loaded to Mxene multifunctional composite material

Abstract

The invention discloses a preparation method of an ROS response microsphere encapsulated mitochondrial targeted nootkatone loaded Mxene multifunctional composite material, and relates to the technical field of biomedicine.The preparation method comprises the steps that carboxylated Mxene nanosheets are prepared and dissolved in dimethyl sulfoxide to obtain a product MXene-COOH-NK, then the product MXene-COOH-NK is dissolved in a 2-morpholine ethanesulfonic acid MES solution to obtain a product MXene-COOH-NK-Man, and the MXene-COOH-NK-Man is loaded on the Mxene multifunctional composite material to obtain the ROS response microsphere encapsulated mitochondrial targeted nootkatone loaded Mxene multifunctional composite material. On the basis, the ROS response microsphere encapsulated mitochondrial targeted Mxene loaded nootkatone is prepared. MXene has near-infrared photothermal conversion performance and supports light controlled drug release and local mild thermal therapy, MXene-COOH at NK can directly enrich chondrocyte mitochondria after being modified by mannosamine, expression of inflammatory factors is further inhibited, boric acid ester bonds are contained in the microspheres, and the MXene-COOH at NK can be used for preparing a drug for treating chondrocyte mitochondria, so that the MXene-COOH at NK can be used for preparing a drug for treating chondrocyte mitochondria. The nootkatone is broken under the high ROS environment condition of an OA focus, fixed-point and on-demand release of the medicine nootkatone is achieved, and whole body exposure is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing a multifunctional composite material of mitochondrial-targeting nocaketone loaded onto ROS-responsive microspheres. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative joint disease characterized by progressive destruction of articular cartilage, synovitis, and subchondral bone sclerosis. Currently, clinical treatment for OA primarily aims to relieve pain and improve function, such as with nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular injections of sodium hyaluronate. However, these methods cannot reverse cartilage damage, and long-term use can lead to significant side effects.

[0003] Recent studies have found that the pathological progression of osteoarthritis (OA) is closely related to the excessive accumulation of reactive oxygen species (ROS) in the joint cavity. Excessive ROS can induce mitochondrial dysfunction, exacerbate inflammatory responses (such as promoting the release of inflammatory factors like IL-1β and TNF-α), and lead to chondrocyte apoptosis and degradation of the extracellular matrix (such as type II collagen and proteoglycans). Therefore, targeting mitochondria and clearing excess ROS while simultaneously inhibiting inflammation has become a novel strategy for treating OA.

[0004] Nocaketone is a natural sesquiterpene compound with excellent anti-inflammatory and antioxidant activities, and studies have shown that it can effectively protect chondrocytes. However, its poor water solubility, rapid in vivo metabolism, and lack of targeting limit its therapeutic effects. Mxene is an emerging two-dimensional nanomaterial with a large specific surface area, which can be used to efficiently load hydrophobic drugs (such as nocaketone). It also possesses excellent photothermal conversion properties, generating local heat under near-infrared light irradiation, further enhancing the anti-inflammatory effect and promoting drug release. Hyaluronic acid-modified ROS-responsive microspheres improve lubrication properties, and the introduction of phenylboronic acid ester bonds enables on-demand drug release. Therefore, a method for preparing a multifunctional composite material of ROS-responsive microspheres encapsulating mitochondrial-targeted nocaketone loaded onto Mxene is proposed. Summary of the Invention

[0005] In order to address the problems of poor water solubility, rapid metabolism in vivo, and lack of targeting in the aforementioned background technologies, which limit the therapeutic effect of nocaketone.

[0006] This invention provides a method for preparing a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto Mxene and encapsulated in ROS-responsive microspheres, using the following technical solution: 1. A method for preparing a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto ROS-responsive microspheres, comprising the following steps: S1. Preparation of carboxylated Mxene nanosheets; S1-1. Dissolve lithium fluoride in an appropriate amount of hydrochloric acid solution, stir at 30~35℃ for 10~15min, then add MAX phase powder, raise the temperature to 42~48℃, and stir continuously at a constant temperature for 18~30h to obtain a suspension. S1-2. Transfer the suspension obtained in step S1-1 to a centrifuge tube and wash it several times with deionized water by differential centrifugation until the pH of the supernatant is 6.5-7 to obtain precipitate I. S1-3. Dissolve the precipitate I obtained in step S1-2 in deionized water, place it in an ice-water bath for ultrasonic exfoliation, and peel the multilayer material into a single layer or few layers of Mxene. Centrifuge and wash the upper suspension to freeze-dry the product MXene. S1-4. Take the MXene nanosheets obtained in step S1-3, add NaOH aqueous solution to them, and heat to 75~80℃; The mixture was magnetically stirred in a nitrogen atmosphere for 32–48 h, then dialyzed to pH 6.8–7.4, and freeze-dried to obtain Mxene-COOH. S2. Dissolve the product Mxene-COOH obtained in step S1 in dimethyl sulfoxide, disperse it by ultrasonication, then add nocaketone, incubate in a constant temperature shaker for 12-16 h, wash the dimethyl sulfoxide with deionized water and freeze dry to obtain the product MXene–COOH@NK. S3. Dissolve MXene–COOH@NK obtained in step S2 in 2-morpholinoethanesulfonic acid (MES) solution, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) sequentially, activate at room temperature for 15–30 min, adjust the pH to 7.8–8.5 with borax-boric acid buffer, add D-mannosamine hydrochloride (Man–NH2·HCl), continue the reaction for 12–18 h, wash with deionized water and freeze dry to obtain the product MXene–COOH@NK–Man; S4. Preparation of ROS-responsive microspheres encapsulating mitochondrial-targeted Mxene-loaded nocaketone; S4-1. Dissolve the MXene–COOH@NK–Man obtained in S3 in PBS, then add methacrylamide gelatin (GelMA) and 3-acrylamidophenylboronic acid (3-AAPBA), and stir in the dark at 35~55℃ until completely dissolved to obtain a homogeneous aqueous phase II. S4-2. Mix liquid paraffin with Span-80 at a volume ratio of 15:1 to 25:1 and stir at room temperature for 5 to 30 minutes to obtain a homogeneous oil phase III. S4-3. The homogeneous aqueous phase II obtained in S4-1 is added dropwise to the homogeneous oil phase III, emulsified for 5-15 min, then irradiated under a UV light source of 5-50 mW / cm2 and 365 nm for 1-10 min, then centrifuged at 2000-4000 rpm for 3-10 min, washed 1-5 times with anhydrous ethanol, and freeze-dried to obtain the product HMs@MXene–COOH@NK–Man. S4-4. The microspheres obtained in S4-3 were dispersed in a hyaluronic acid solution activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The reaction was carried out at 4-37°C for 6-18 hours. The product was then dialyzed and freeze-dried to obtain the target product, named HA-HMs@MXene–COOH@NK–Man.

[0007] Preferably, in step S1-1, the molar ratio of MAX phase powder, lithium fluoride and hydrochloric acid is 1:15:47.

[0008] Preferably, in steps S1-2, the centrifugation speed is 3000~11000 rpm and the centrifugation time is 5~15 min.

[0009] Preferably, in steps S1-3, the ultrasonic process is carried out entirely in an ice bath, the ultrasonic temperature is below 15°C, the centrifugation rate is 8000~10000 rpm, and the process is freeze-dried at -67°C under vacuum.

[0010] Preferably, in steps S1-4, the concentration of Mxene is 0.2~0.28 w / v, the molar concentration of NaOH is 1~5 mol / L, and the molecular weight cutoff of the dialysis bag is MWCO=3kDa.

[0011] Preferably, in step S2, the molar ratio of nocaketone, Mxene-COOH, and dimethyl sulfoxide is 0.87:1:268.

[0012] Preferably, in step S3, the concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC, N-hydroxysuccinimide NHS, and D-mannosamine hydrochloride in the mixed solution are 0.020~0.030 mol / L, 0.045~0.060 mol / L, and 0.0085~0.0098 mol / L, respectively.

[0013] Preferably, in step S4-1, the concentrations of MXene–COOH@NK–Man, methacrylamide gelatin (GelMA), and 3-acrylamidophenylboronic acid (3-AAPBA) are 1~5%, 5~15% w / v, and 3~7% w / v, respectively.

[0014] Preferably, in step S4-4, the concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and hyaluronic acid in the mixed solution are 0.030~0.060 mol / L, 0.045~0.060 mol / L, and 0.01~0.03 mol / L, respectively.

[0015] The present invention provides a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto ROS-responsive microspheres prepared by the above preparation method.

[0016] In summary, the present invention has the following beneficial effects: 1. This invention uses ROS-responsive element 3-acrylamidophenylboronic acid 3-AAPBA, methacrylamide gelatin, paraffin and Span 80 to encapsulate MXene–COOH@NK–Man loaded with hydrophobic drugs in a suitable shell as microspheres, thereby supporting the hydrophilic formulation in the composite material; 2. MXene has near-infrared photothermal conversion properties that support photocontrolled drug release and local mild thermotherapy. At the same time, after MXene–COOH@NK is modified with mannosamine, it can be directly enriched in chondrocyte mitochondria and further inhibit the expression of inflammatory factors. 3. The microspheres contain borate ester bonds, which break under the high ROS environment of OA lesions, enabling the targeted and on-demand release of the drug nocaketone and reducing systemic exposure. Attached Figure Description

[0017] Figure 1 This is the XRD pattern of MXene from the present invention; Figure 2 This is an XPS plot of MXene from the present invention; Figure 3 This is a temperature rise curve of MXene according to the present invention; Figure 4 This is a thermal stability diagram of MXene from the present invention; Figure 5 The TEM of MXene–COOH@NK–Man in this invention; Figure 6 This is the HRTEM and elemental distribution diagram of MXene–COOH@NK–Ma of the present invention; Figure 7 This invention relates to HA-HMs@MXene–COOH@NK–ManSEM; Figure 8 HA-HMs@MXene–COOH@NK–Man SEM elemental distribution diagram of this invention; Figure 9 This is an in vitro release diagram of HA-HMs@MXene–COOH@NK–Man of the present invention; Figure 10 The friction coefficient diagrams for HMs@MXene–COOH@NK–Man and HA-HMs@MXene–COOH@NK–Man of this invention are shown. Figure 11 This is a particle size distribution diagram of the different groups of materials in this invention; Figure 12 This is a potential distribution diagram of different grouped materials of the present invention; Figure 13 Infrared images of different grouping materials of the present invention. Detailed Implementation

[0018] The following is in conjunction with the appendix Figures 1-13 The present invention will be described in further detail below.

[0019] One embodiment of the present invention provides a method for preparing a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto ROS-responsive microspheres, comprising the following steps: S1. Preparation of carboxylated Mxene nanosheets; S1-1. Dissolve lithium fluoride in an appropriate amount of hydrochloric acid solution and stir at 30~35℃ for 10~15min. Then add MAX phase powder. The molar ratio of MAX phase powder, lithium fluoride and hydrochloric acid is 1:15:47. Raise the temperature to 42~48℃ and stir continuously at a constant temperature for 18~30h to obtain a suspension. The entire reaction is carried out in a fume hood. S1-2. Transfer the suspension obtained in step S1-1 to a centrifuge tube and wash it several times with deionized water by differential centrifugation at a speed of 3000~11000 rpm for 5~15 min until the pH of the supernatant is 6.5~7, and obtain precipitate I. S1-3. Dissolve the precipitate I obtained in step S1-2 in deionized water and place it in an ice-water bath for ultrasonic exfoliation. The ultrasonic process is carried out in an ice bath with an ultrasonic temperature below 15°C. The multilayer material is exfoliated into a single layer or a few layers of Mxene. The upper suspension obtained by centrifugation and washing is freeze-dried to obtain the product MXene. The centrifugation speed is 8000~10000 rpm, and the product is frozen and vacuum-dried at -67°C. S1-4. Take the MXene nanosheets obtained in step S1-3, with an MXene concentration of 0.2~0.28 w / v, add NaOH aqueous solution with a NaOH molar concentration of 1~5 mol / L, and heat to 75~80℃. The mixture was magnetically stirred in a nitrogen atmosphere for 32–48 h, then dialyzed to pH 6.8–7.4. The molecular weight cutoff (MWCO) of the dialysis bag was 3 kDa. The mixture was then freeze-dried to obtain Mxene-COOH. S2. Dissolve the product Mxene-COOH obtained in step S1 in dimethyl sulfoxide, disperse it by ultrasonication, then add nocaketone, incubate in a constant temperature shaker for 12-16 h, wash the dimethyl sulfoxide with deionized water and freeze dry to obtain the product MXene–COOH@NK. The molar ratio of nocaketone, Mxene-COOH and dimethyl sulfoxide is 0.87:1:268. S3. Dissolve MXene–COOH@NK obtained in step S2 in 2-morpholinoethanesulfonic acid (MES) solution, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) sequentially. Activate at room temperature for 15–30 min, adjust the pH to 7.8–8.5 with borax-boric acid buffer, add D-mannosamine hydrochloride (Man–NH2·HCl), and continue the reaction for 12–18 h. Wash with deionized water and freeze-dry to obtain the product MXene–COOH@NK–Man. The concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and D-mannosamine hydrochloride in the mixed solution are 0.020–0.030 mol / L, 0.045–0.060 mol / L, and 0.0085–0.0098 mol / L, respectively. S4. Preparation of ROS-responsive microspheres encapsulating mitochondrial-targeted Mxene-loaded nocaketone; S4-1. Dissolve MXene–COOH@NK–Man obtained in S3 in PBS, then add methacrylamide gelatin (GelMA) and 3-acrylamidophenylboronic acid (3-AAPBA), and stir in the dark at 35–55°C until completely dissolved to obtain a homogeneous aqueous phase II. The concentrations of MXene–COOH@NK–Man, methacrylamide gelatin (GelMA), and 3-acrylamidophenylboronic acid (3-AAPBA) are 1–5%, 5–15% w / v, and 3–7% w / v, respectively. S4-2. Mix liquid paraffin with Span-80 at a volume ratio of 15:1 to 25:1 and stir at room temperature for 5 to 30 minutes to obtain a homogeneous oil phase III. S4-3. The homogeneous aqueous phase II obtained in S4-1 is added dropwise to the homogeneous oil phase III, emulsified for 5-15 min, then irradiated under a UV light source of 5-50 mW / cm2 and 365 nm for 1-10 min, then centrifuged at 2000-4000 rpm for 3-10 min, washed 1-5 times with anhydrous ethanol, and freeze-dried to obtain the product HMs@MXene–COOH@NK–Man. S4-4. The microspheres obtained in S4-3 were dispersed in a hyaluronic acid solution activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The reaction was carried out at 4–37 °C for 6–18 h, followed by dialyzing and freeze-drying to obtain the target product, named HA-HMs@MXene–COOH@NK–Man. The concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and hyaluronic acid in the mixed solution were 0.030–0.060 mol / L, 0.045–0.060 mol / L, and 0.01–0.03 mol / L, respectively.

[0020] XRD testing of MXene was performed using a Bruker D8 Advance X-ray diffractometer, employing a grazing incidence method to analyze the phase composition and crystal structure of the material surface. The crystal structure of MXene was analyzed using XRD. Figure 1 This indicates that the diffraction peaks around 2θ=6° correspond to the (002) crystal plane and the interlayer spacing of Mxene, and the diffraction peaks around 2θ=39° correspond to the (104) crystal plane disappear, confirming that the Al layer was successfully etched away from the MAX phase precursor and a regular layered structure was formed.

[0021] XPS testing uses X-ray photoelectron spectroscopy to analyze the elemental composition and chemical state of material surfaces. Figure 2 This indicates that a wide scan clearly revealed strong characteristic peaks for carbon (C), oxygen (O), and titanium (Ti), as well as a weak nitrogen (N) signal. Peak fitting of the C 1s spectrum revealed C-Ti and C-Ti-T... x Peaks were observed in the O 1s spectrum for Ti-O (~282.8 eV), CO (~286.2 eV), and C=O (~288.5 eV); peaks were observed in the O 1s spectrum for Ti-O (~530.0 eV), Ti-OH (~531.5 eV), and H2O (~533.0 eV); peaks were observed in the Ti 2p spectrum for Ti-C (~455.0 eV 22p). 3 / 2 The peaks demonstrate the characteristic C-Ti bonds of the material Mxene, while the surface is rich in oxygen-containing functional groups.

[0022] Mxene's heating performance and thermal stability were tested using an 808nm laser to examine the material's photothermal properties. Figure 3 The results showed that the PBS group did not experience any temperature increase within 600 seconds of laser irradiation, and different concentrations of Mxene (25ug / mL, 50ug / mL, 100ug / mL, 150ug / mL) were positively correlated with the temperature increase. Figure 4This indicates that the temperature rise of Mxene is highly repeatable: it exhibits remarkably similar trends from second 0 to second 6000. Each rise and fall curve follows a trend of rapid to slow temperature increase, reaching a peak, and then rapid to slow temperature decrease.

[0023] The morphology of MXene–COOH@NK–Man was characterized using a Tecnai G2 F20 transmission electron microscope. Figure 5 It can be seen that the Mxene nanosheets loaded with nocaketone modified mannosamine exhibit an irregular, curved-edge sheet structure with a single sheet size of about 140 nm.

[0024] Validation was performed using HRTEM and element-mapping distribution. Figure 6 The synthesized MXene–COOH@NK–Man exhibits a thin, wrinkled two-dimensional lamellar structure. The densely and uniformly distributed red dots represent Ti, the main metallic element in the MXene framework. Their highly uniform distribution perfectly matches the morphology of the sample, indicating that the two-dimensional lamellar structure of MXene remains intact, without significant segregation or loss of Ti, thus demonstrating the stability of the material matrix. Due to the copper mesh and carbon deposition, C is present throughout the lamellar structure; the green dots represent C. Furthermore, due to the surface modification with mannosamine, small amounts of N and O are also present; the blue dots represent N, and the pink dots represent O. Figure 6 The diagram consists of 6 figures, which, from left to right and top to bottom, represent: the two-dimensional layered structure of MXene–COOH@NK–Man; the overall elemental distribution; the C elemental distribution; the N elemental distribution; the O elemental distribution; and the Ti elemental distribution.

[0025] SEM and elemental distribution analysis were performed using a JSM-IT210 field emission scanning microscope. HMs@MXene–COOH@NK–Man powder was dispersed in anhydrous ethanol and ultrasonically dispersed. 10 μL of the dispersion was dropped onto a single-crystal Si wafer, dried, and then sputtered with gold.

[0026] SEM images are provided by Figure 7 and Figure 8 The results show that HA-HMs@MXene–COOH@NK–Man exhibits a typical spherical structure, with individual spheres having a particle size of approximately 10 μm. Elemental distribution indicates that it mainly contains C, N, O, and Ti elements, confirming that MXene–COOH@NK–Man is successfully encapsulated within the microspheres.

[0027] The HA-HMs@MXene–COOH@NK–Man prepared in S4-4 were placed in solutions containing 10 μL H₂O₂ and 100 μL H₂O₂, respectively, and the drug release capacity of HA-HMs@MXene–COOH@NK–Man at different time points was determined. Dialysis bags were placed in centrifuge tubes containing 9 mL of H₂O₂ solution corresponding to different concentrations, and samples were taken at 37℃ at different time points. Equal volumes of buffer were added, and the absorbance of the drug was measured using a UV spectrophotometer. The cumulative release rate was calculated based on the nocaketone standard curve.

[0028] In vitro release results by Figure 9 The results indicate that the drug release rate in the 10uMH2O2 environment is slow. During the 10-day observation period, there was no explosive release or plateau. On the contrary, the high-concentration H2O2 environment caused the drug to exhibit rapid release characteristics. By day 6, the release rate had reached over 80%, and a clear plateau appeared, with the release rate stabilizing between 80-90%. This suggests that the microspheres possess both the ability to prolong drug release and the ability to respond to ROS.

[0029] The coefficient of friction was tested using the horizontal slider method (slider mass 200g, speed 100mm / min). Figure 10 The comparison of the friction coefficient curves of the prepared HMs@MXene-COOH / NK–Man and HA–HMs@MXene-COOH / NK–Man composite microspheres shows that the friction coefficient μs of HA–HMs@MXene-COOH / NK–Man is 0.22, which is about 16% lower than that of HMs@MXene-COOH / NK–Man (μs=0.26). This indicates that the hyaluronic acid surface layer improves the lubrication effect of the microspheres.

[0030] The functional groups of the different groups of materials were characterized using Fourier transform infrared spectroscopy. 1 mg of the lyophilized material powder was ground with 150 mg of potassium bromide, then pressed into thin sheets using a tablet press, and subjected to infrared detection.

[0031] The particle size and potential of different molecular materials were characterized using a particle size analyzer. Figure 11 The results showed that Mxene-COOH, MXene-COOH / NK, MXene-COOH / NK–Man, HMs@MXene-COOH / NK–Man, and HA-HMs@MXene-COOH / NK–Man (each molecular material in Figure 10 The particle sizes (corresponding sequentially from top to bottom and left to right) are approximately 168 nm, 170 nm, 177 nm, 10.9 μm and 11.4 μm, respectively. Figure 12The results showed that Mxene-COOH, MXene-COOH / NK, MXene-COOH / NK–Man, HMs@MXene-COOH / NK–Man, and HA-HMs@MXene-COOH / NK–Man (each molecular material in Figure 11 The Zeta values ​​(corresponding from left to right in the bar chart) are approximately -27.3 mV, -33.3 mV, -20.6 mV, -8.3 mV, and -10.5 mV, respectively. These results are consistent with SEM and TEM electron microscopy, and the Zeta values ​​also corroborate the successful preparation of the material.

[0032] Depend on Figure 13 Infrared analysis revealed that Mxenen nanosheets have a density of 500–600 cm⁻¹ -1 Vibrational absorption peaks of metal-carbon / oxygen bonds Ti-C and Ti-O appeared at 1500 cm⁻¹; in Mxene-COOH nanosheets, the peak value was 1500 cm⁻¹. -1 The presence of C=O bond stretching vibrations at this point confirms successful carboxylation modification; MXene-COOH / NK–Man exhibits stretching vibrations at 1650 cm⁻¹. -1 An absorption peak appeared at (amide I band, C=O), which was caused by the amidation reaction of Mxene-COOH with mannosamine; compared with microspheres without HA modification, HA-HMs@MXene-COOH / NK–Man showed an absorption peak at 1650 cm⁻¹. -1 and 1050cm -1 The amide peak and COC absorption peak of HA were observed, which proved the successful modification of HA on the surface of microspheres.

Claims

1. A method for preparing a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto ROS-responsive microspheres, characterized in that, Includes the following steps: S1. Preparation of carboxylated Mxene nanosheets; S1-1. Dissolve lithium fluoride in an appropriate amount of hydrochloric acid solution, stir at 30~35℃ for 10~15min, then add MAX phase powder, raise the temperature to 42~48℃, and stir continuously at a constant temperature for 18~30h to obtain a suspension. S1-2. Transfer the suspension obtained in step S1-1 to a centrifuge tube and wash it several times with deionized water by differential centrifugation until the pH of the supernatant is 6.5-7 to obtain precipitate I. S1-3. Dissolve the precipitate I obtained in step S1-2 in deionized water, place it in an ice-water bath for ultrasonic exfoliation, and peel the multilayer material into a single layer or few layers of Mxene. Centrifuge and wash the upper suspension to freeze-dry the product MXene. S1-4. Take the MXene nanosheets obtained in step S1-3, add NaOH aqueous solution to them, and heat to 75~80℃; The mixture was magnetically stirred in a nitrogen atmosphere for 32–48 h, then dialyzed to pH 6.8–7.4, and freeze-dried to obtain Mxene-COOH. S2. Dissolve the product Mxene-COOH obtained in step S1 in dimethyl sulfoxide, disperse it by ultrasonication, then add nocaketone, incubate in a constant temperature shaker for 12-16 h, wash the dimethyl sulfoxide with deionized water and freeze dry to obtain the product MXene–COOH@NK. S3. Dissolve MXene–COOH@NK obtained in step S2 in 2-morpholinoethanesulfonic acid (MES) solution, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) sequentially, activate at room temperature for 15–30 min, adjust the pH to 7.8–8.5 with borax-boric acid buffer, add D-mannosamine hydrochloride (Man–NH2·HCl), continue the reaction for 12–18 h, wash with deionized water and freeze dry to obtain the product MXene–COOH@NK–Man; S4. Preparation of ROS-responsive microspheres encapsulating mitochondrial-targeted Mxene-loaded nocaketone; S4-1. Dissolve the MXene–COOH@NK–Man obtained in S3 in PBS, then add methacrylamide gelatin (GelMA) and 3-acrylamidophenylboronic acid (3-AAPBA), and stir in the dark at 35~55℃ until completely dissolved to obtain a homogeneous aqueous phase II. S4-2. Mix liquid paraffin with Span-80 at a volume ratio of 15:1 to 25:1 and stir at room temperature for 5 to 30 minutes to obtain a homogeneous oil phase III. S4-3. The homogeneous aqueous phase II obtained in S4-1 is added dropwise to the homogeneous oil phase III, emulsified for 5-15 min, then irradiated under a UV light source of 5-50 mW / cm2 and 365 nm for 1-10 min, then centrifuged at 2000-4000 rpm for 3-10 min, washed 1-5 times with anhydrous ethanol, and freeze-dried to obtain the product HMs@MXene–COOH@NK–Man. S4-4. The microspheres obtained in S4-3 were dispersed in a hyaluronic acid solution activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The reaction was carried out at 4-37°C for 6-18 hours. The product was then dialyzed and freeze-dried to obtain the target product, named HA-HMs@MXene–COOH@NK–Man.

2. The method for preparing a ROS-responsive microsphere-encapsulated mitochondrial-targeted nocacolone loaded onto Mxene multifunctional composite material according to claim 1, characterized in that: In step S1-1, the molar ratio of MAX phase powder, lithium fluoride, and hydrochloric acid is 1:15:

47.

3. The method for preparing a multifunctional composite material of mitochondrial-targeted nocacolone loaded onto ROS-responsive microspheres according to claim 1, characterized in that: In steps S1-2, the centrifugation speed is 3000~11000 rpm and the centrifugation time is 5~15 min.

4. The method for preparing a multifunctional composite material of ROS-responsive microspheres encapsulating mitochondrial-targeted nocacolone loaded onto Mxene according to claim 1, characterized in that: In steps S1-3, the entire ultrasonic process is carried out in an ice bath with an ultrasonic temperature below 15°C, a centrifugation rate of 8000~10000 rpm, and freeze-drying at -67°C under vacuum.

5. The method for preparing a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto ROS-responsive microspheres according to claim 1, characterized in that: In steps S1-4, the concentration of Mxene is 0.2~0.28 w / v, the molar concentration of NaOH is 1~5 mol / L, and the molecular weight cutoff of the dialysis bag is MWCO=3kDa.

6. The method for preparing a multifunctional composite material of mitochondrial-targeted nocacolone loaded onto ROS-responsive microspheres according to claim 1, characterized in that: In step S2, the molar ratio of nocaketone, Mxene-COOH, and dimethyl sulfoxide is 0.87:1:

268.

7. The method for preparing a multifunctional composite material of mitochondrial-targeted nocacolone loaded onto ROS-responsive microspheres according to claim 1, characterized in that: In step S3, the concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and D-mannosamine hydrochloride in the mixed solution are 0.020~0.030 mol / L, 0.045~0.060 mol / L, and 0.0085~0.0098 mol / L, respectively.

8. The method for preparing a multifunctional composite material of mitochondrial-targeted nocacolone loaded onto ROS-responsive microspheres according to claim 1, characterized in that: In step S4-1, the concentrations of MXene–COOH@NK–Man, methacrylamide gelatin (GelMA), and 3-acrylamidophenylboronic acid (3-AAPBA) are 1–5%, 5–15% w / v, and 3–7% w / v, respectively.

9. The method for preparing a multifunctional composite material of mitochondrial-targeted nocaketone loaded onto ROS-responsive microspheres according to claim 1, characterized in that: In step S4-4, the concentrations of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and hyaluronic acid in the mixed solution are 0.030~0.060 mol / L, 0.045~0.060 mol / L, and 0.01~0.03 mol / L, respectively.

10. A multifunctional composite material containing mitochondrial-targeted nocaketone encapsulated in ROS-responsive microspheres prepared by any one of claims 1-9.

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