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CN-122005487-A - Preparation method of ROS response microsphere encapsulated mitochondrion targeted nocardione loaded Mxene multifunctional composite material

CN122005487ACN 122005487 ACN122005487 ACN 122005487ACN-122005487-A

Abstract

The invention discloses a preparation method of a ROS-responsive microsphere encapsulated mitochondria-targeted nocarbazone-loaded Mxene multifunctional composite material, which relates to the technical field of biological medicines, and is characterized in that carboxylated Mxene nanosheets are prepared, dissolved in dimethyl sulfoxide to obtain a product MXene-COOH@NK, then dissolved in 2-morpholinoethanesulfonic acid MES solution to obtain a product MXene-COOH@NK-Man, and the ROS-responsive microsphere encapsulated mitochondria-targeted Mxene-loaded nocarbazone is prepared on the basis. The ROS response microsphere prepared by the invention encapsulates mitochondrially-targeted Mxene loaded nocardianone, MXene has near infrared light heat conversion performance to support light-controlled drug release and local mild hyperthermia, and MXene-COOH@NK can be directly enriched with chondrocyte mitochondria after being modified by mannosamine, so that the expression of inflammatory factors is further inhibited, boric acid ester bonds are contained in the microsphere, the microsphere is broken under the condition of high ROS environment of an OA focus, the fixed point of the drug nocardianone is realized, the release is carried out according to needs, and the systemic exposure is reduced.

Inventors

  • LI JINYU
  • MU XIAOHONG
  • HAN SONGJIE
  • Fan Fangyang

Assignees

  • 北京中医药大学东直门医院

Dates

Publication Date
20260512
Application Date
20260306

Claims (10)

  1. 1. The preparation method of the ROS response microsphere encapsulated mitochondria targeted nocardinone loaded Mxene multifunctional composite material is characterized by comprising the following steps of: s1, preparing carboxylated Mxene nano-sheets; S1-1, dissolving lithium fluoride in a proper amount of hydrochloric acid solution, stirring for 10-15 min at 30-35 ℃, then adding MAX phase powder, raising the temperature to 42-48 ℃, and continuously stirring for 18-30 h at constant temperature to obtain a suspension; S1-2, transferring the suspension obtained in the step S1-1 into a centrifuge tube, and washing with deionized water for several times in a differential centrifugation mode until the pH value of the supernatant is 6.5-7 to obtain a precipitate I; S1-3, dissolving the precipitate I obtained in the step S1-2 in deionized water, placing in ice water bath for ultrasonic stripping, stripping the multilayer material into single-layer or less-layer Mxene, centrifuging, washing the obtained upper-layer suspension, and freeze-drying to obtain a product MXene; s1-4, taking the MXene nano-sheet obtained in the step S1-3, adding an NaOH aqueous solution into the MXene nano-sheet, and heating to 75-80 ℃; Magnetically stirring for 32-48 h in a nitrogen environment, dialyzing to pH=6.8-7.4, and freeze-drying to obtain Mxene-COOH; s2, dissolving the product Mxene-COOH obtained in the step S1 in dimethyl sulfoxide, after ultrasonic dispersion, adding nootkatone, incubating for 12-16 hours by a constant-temperature shaking table, washing the dimethyl sulfoxide with deionized water, and freeze-drying to obtain a product MXene-COOH@NK; S3, dissolving the MXene-COOH@NK obtained in the step S2 in 2-morpholinoethanesulfonic acid MES solution, sequentially adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, activating for 15-30 min at room temperature, adjusting the pH to 7.8-8.5 by using borax-boric acid buffer solution, adding D-mannosamine hydrochloride Man-NH 2 & HCl, continuing to react for 12-18 h, washing with deionized water, and freeze-drying to obtain a product MXene-COOH@NK-Man; S4, preparing the ROS response microsphere encapsulated mitochondria targeted Mxene loaded nocardinone; S4-1, dissolving MXene-COOH@NK-Man obtained in the S3 in PBS, then adding methacrylic acylated gelatin GelMA and 3-acrylamidophenylboronic acid 3-AAPBA, and stirring in a dark place at 35-55 ℃ until the mixture is completely dissolved to obtain a uniform water phase II; S4-2, mixing liquid paraffin and Span-80 according to a volume ratio of 15:1-25:1, and stirring for 5-30 min at room temperature to obtain a uniform oil phase III; S4-3, dropwise adding the uniform water phase II obtained in the S4-1 into the uniform oil phase III, emulsifying for 5-15 min, then irradiating for 1-10 min under an ultraviolet light source of 5-50 mW/cm <2>, and 365nm, centrifuging for 3-10 min at 2000-4000 rpm, washing for 1-5 times with absolute ethyl alcohol, and freeze-drying to obtain a product HMs@MXene-COOH@NK-Man; S4-4, dispersing the microspheres obtained in the S4-3 in a hyaluronic acid solution activated by 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, reacting for 6-18 hours at 4-37 ℃, and then dialyzing and freeze-drying to obtain a target product which is named as HA-HMs@MXene-COOH@NK-Man.
  2. 2. The method for preparing the ROS-responsive microsphere encapsulated mitochondrially-targeted nocardinone loaded Mxene multifunctional composite material according to claim 1, wherein in the step S1-1, the molar ratio of MAX phase powder to lithium fluoride to hydrochloric acid is 1:15:47.
  3. 3. The preparation method of the ROS-responsive microsphere encapsulated mitochondria-targeted nocardinone loaded Mxene multifunctional composite material is characterized in that in the step S1-2, the centrifugal speed is 3000-11000 rpm, and the centrifugal time is 5-15 min.
  4. 4. The preparation method of the ROS-responsive microsphere encapsulated mitochondria-targeted nocardinone loaded Mxene multifunctional composite material is characterized in that in the step S1-3, the whole ultrasonic process is carried out in an ice bath, the ultrasonic temperature is lower than 15 ℃, the centrifugal speed is 8000-10000 rpm, and the composite material is frozen and vacuumized and dried at-67 ℃.
  5. 5. The preparation method of the ROS-responsive microsphere-encapsulated mitochondria-targeted nocardianone-loaded Mxene multifunctional composite material is characterized in that in the step 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 cut-off MWCO=3 kDa of a dialysis bag.
  6. 6. The method for preparing the ROS-responsive microsphere-encapsulated mitochondrially-targeted nocardiane-loaded Mxene multifunctional composite material according to claim 1, wherein in the step S2, the molar ratio of nocardiane, mxene-COOH and dimethyl sulfoxide is 0.87:1:268.
  7. 7. The preparation method of the ROS-responsive microsphere-encapsulated mitochondria-targeted nocardinone-loaded Mxene multifunctional composite material is characterized in that in the step S3, the concentration of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC, N-hydroxysuccinimide NHS and D-mannosamine hydrochloride in a mixed solution is 0.020-0.030 mol/L, 0.045-0.060 mol/L and 0.0085-0.0098 mol/L respectively.
  8. 8. The preparation method of the ROS-responsive microsphere encapsulated mitochondria-targeted nocarone-loaded Mxene multifunctional composite material is characterized in that in the step S4-1, the concentration of MXene-COOH@NK-Man, methacryloylated gelatin GelMA and 3-acrylamidophenylboronic acid is 1-5%, 5-15% w/v and 3-7% w/v respectively.
  9. 9. The preparation method of the ROS-responsive microsphere encapsulated mitochondria-targeted nocardinone-loaded Mxene multifunctional composite material is characterized in that in the step S4-4, the concentration of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC, N-hydroxysuccinimide NHS and hyaluronic acid in a mixed solution is 0.030-0.060 mol/L, 0.045-0.060 mol/L and 0.01-0.03 mol/L respectively.
  10. 10. An ROS-responsive microsphere encapsulated mitochondrially targeted nocardione loaded to Mxene multifunctional composite prepared by the method of any one of claims 1-9.

Description

Preparation method of ROS response microsphere encapsulated mitochondrion targeted nocardione loaded Mxene multifunctional composite material Technical Field The invention relates to the technical field of biological medicine, in particular to a preparation method of a ROS response microsphere encapsulated mitochondria targeted nocardione loaded Mxene multifunctional composite material. Background Osteoarthritis (Osteoarthritis, OA) is a common degenerative joint disease characterized by progressive destruction of articular cartilage, synovial inflammation and subchondral bone sclerosis. At present, clinical treatment of OA mainly aims at relieving pain and improving functions, such as nonsteroidal anti-inflammatory drugs, sodium hyaluronate injection into joint cavities and the like, but the methods cannot reverse cartilage injury, and have obvious side effects after long-term use. Recent studies have found that the pathological course of OA is closely related to the excessive accumulation of Reactive Oxygen Species (ROS) within the joint cavity. Excessive ROS can induce mitochondrial dysfunction, exacerbate inflammatory responses (e.g., promote the release of inflammatory factors such as IL-1 β, TNF- α, etc.), and lead to chondrocyte apoptosis and degradation of extracellular matrix (e.g., type II collagen and proteoglycans). Thus, targeting mitochondria and scavenging excess ROS, while inhibiting inflammation, is a new strategy for treating OA. Nocarpus ketone is a natural sesquiterpene compound and has excellent anti-inflammatory and antioxidant activities, and researches show that the nocarpus ketone can effectively protect chondrocytes. However, nocardinone is poorly water-soluble, metabolized rapidly in vivo, lacks targeting, and limits its therapeutic effect. Mxene is an emerging two-dimensional nanomaterial with a large specific surface area, which can be used to efficiently load hydrophobic drugs (e.g., nocardiane), and has excellent photothermal conversion properties, which can generate local heat under near infrared light irradiation, further enhancing anti-inflammatory effects and promoting drug release. The hyaluronic acid modified ROS response microsphere improves the lubricating performance, and the phenylboronic acid ester bond is introduced to release the medicament on demand. Therefore, a preparation method of the ROS response microsphere encapsulated mitochondria targeted nocardinone loaded to Mxene multifunctional composite material is provided. Disclosure of Invention In order to solve the problems of poor water solubility, fast in vivo metabolism and lack of targeting of nocardione in the prior art, the treatment effect of nocardione is limited. The invention provides a preparation method of a ROS response microsphere encapsulated mitochondria targeted nocardinone loaded Mxene multifunctional composite material, which adopts the following technical scheme: 1. the preparation method of the ROS response microsphere encapsulated mitochondria targeted nocardinone loaded Mxene multifunctional composite material comprises the following steps: s1, preparing carboxylated Mxene nano-sheets; S1-1, dissolving lithium fluoride in a proper amount of hydrochloric acid solution, stirring for 10-15 min at 30-35 ℃, then adding MAX phase powder, raising the temperature to 42-48 ℃, and continuously stirring for 18-30 h at constant temperature to obtain a suspension; S1-2, transferring the suspension obtained in the step S1-1 into a centrifuge tube, and washing with deionized water for several times in a differential centrifugation mode until the pH value of the supernatant is 6.5-7 to obtain a precipitate I; S1-3, dissolving the precipitate I obtained in the step S1-2 in deionized water, placing in ice water bath for ultrasonic stripping, stripping the multilayer material into single-layer or less-layer Mxene, centrifuging, washing the obtained upper-layer suspension, and freeze-drying to obtain a product MXene; s1-4, taking the MXene nano-sheet obtained in the step S1-3, adding an NaOH aqueous solution into the MXene nano-sheet, and heating to 75-80 ℃; Magnetically stirring for 32-48 h in a nitrogen environment, dialyzing to pH=6.8-7.4, and freeze-drying to obtain Mxene-COOH; s2, dissolving the product Mxene-COOH obtained in the step S1 in dimethyl sulfoxide, after ultrasonic dispersion, adding nootkatone, incubating for 12-16 hours by a constant-temperature shaking table, washing the dimethyl sulfoxide with deionized water, and freeze-drying to obtain a product MXene-COOH@NK; S3, dissolving the MXene-COOH@NK obtained in the step S2 in 2-morpholinoethanesulfonic acid MES solution, sequentially adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS, activating for 15-30 min at room temperature, adjusting the pH to 7.8-8.5 by using borax-boric acid buffer solution, adding D-mannosamine hydrochloride Man-NH 2 & HCl, continuing to react for 12-18 h, washing wi