Ultrasonic contrast agent for targeting senescent cartilage cells

Abstract

The invention discloses an ultrasonic contrast agent for targeting senescent cartilage cells. The ultrasonic contrast agent is prepared from the following components in parts by weight: 80 to 120 parts of a hydroxyl-terminated lactic acid-glycolic acid copolymer, 80 to 120 parts of a polyethylene glycol-lignin conjugate, 8 to 12 parts of paclitaxel and 10 to 30 parts of a p16INK4a antibody. A hydroxyl-terminated lactic acid-glycolic acid copolymer and a polyethylene glycol-lignin conjugate are used as composite film-forming materials, a paclitaxel-coated polymer ultrasonic contrast agent is prepared by a double-emulsion solvent evaporation method, and the paclitaxel-coated polymer ultrasonic contrast agent is connected with a p16INK4a antibody by a carbodiimide method to prepare the paclitaxel-coated polymer ultrasonic contrast agent. And preparing the targeted ultrasonic contrast agent.

Description

Technical Field

[0001] The invention belongs to the technical field of ultrasound contrast imaging, and in particular relates to an ultrasound contrast agent targeting senescent chondrocytes. Background Art

[0002] Osteoarthritis (OA), also known as osteoarthritis, degenerative arthritis, and proliferative arthritis, is a chronic joint disease with unknown etiology characterized by degeneration of articular cartilage and secondary bone hyperplasia. It is prone to occur in the knee, hip, spine, and distal interphalangeal joints, which are subject to heavy loads, and clinically manifests as progressive joint pain.

[0003] Osteoarthritis is divided into primary osteoarthritis and secondary osteoarthritis. In recent years, more and more evidence has shown that the occurrence and development of OA is related to the increase in the number of senescent cells in joint tissues. Senescent cells and their senescence-related secretory phenotypes play an important role in cartilage degeneration and OA. Targeted elimination of senescent cells can prevent articular cartilage degeneration and OA progression.

[0004] The progression of osteoarthritis is closely related to senescent chondrocytes. Paying attention to the development of senescent chondrocytes is of great significance for delaying or treating osteoarthritis. However, there is currently no effective technical means to observe the development of senescent chondrocytes in vivo. Summary of the invention

[0005] The purpose of the present invention is to provide an ultrasound contrast agent targeting senescent chondrocytes.

[0006] An ultrasound contrast agent targeting senescent chondrocytes, the ultrasound contrast agent is made of the following components in parts by weight: 80-120 parts of hydroxyl-terminated lactic acid-glycolic acid copolymer, 80-120 parts of polyethylene glycol-lignin conjugate, 8-12 parts of paclitaxel, and p16 INK4a 10-30 copies of antibody.

[0007] The method for preparing the ultrasound contrast agent targeting senescent chondrocytes is carried out according to the following steps:

[0008] (1) taking a hydroxyl-terminated lactic acid-glycolic acid copolymer, a polyethylene glycol-lignin conjugate, and paclitaxel and fully dissolving them in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky white primary emulsion;

[0009] (2) adding a polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding an isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 2-4 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying them, and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel;

[0010] (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4a The antibody was incubated with shaking in an ice bath for 1-2 hours, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 24-48 hours, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes.

[0011] The preparation method of the polyethylene glycol-lignin conjugate is as follows: according to the weight ratio, 1-3 parts of alkali lignin are taken, dissolved in 100-200 parts of tetrahydrofuran, and 3-7 parts of polyethylene glycol are added; after sufficient dissolution, 2-5 parts of EDC and 2-3 parts of DMAP are added, and the mixture is stirred for reaction for 20-28 hours; after the reaction is completed, the reaction solution is dripped into anhydrous ether to precipitate; the precipitate is redissolved in 80-120 parts of pure water, dialyzed in pure water for 4-8 hours, unreacted reagents and organic solvents are removed, and freeze-dried to obtain the polyethylene glycol-lignin conjugate.

[0012] The coupling activator is EDC / NHS, the mass ratio of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) to NHS (N-hydroxysuccinimide) is 1:3, and the amount used is 10-40% of the mass of the hydroxyl-terminated lactic acid-hydroxyacetic acid copolymer.

[0013] The mass ratio of the hydroxyl-terminated lactic acid-glycolic acid copolymer, the polyethylene glycol-lignin conjugate, and paclitaxel is: (80-120): (80-120): (8-12).

[0014] The amount of dichloromethane added in step (1) is 2-8 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, the polyethylene glycol-lignin conjugate, and paclitaxel; the amount of double distilled water added is 2-5 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, the polyethylene glycol-lignin conjugate, and paclitaxel.

[0015] The volume of the polyvinyl alcohol solution added in step (2) is 15-30% of the volume of the primary emulsion; the volume of the isopropanol solution added is 15-30% of the volume of the secondary emulsion.

[0016] The beneficial effects of the present invention are as follows: the present invention adopts a hydroxyl-terminated lactic acid-glycolic acid copolymer and a polyethylene glycol-lignin conjugate as a composite film-forming material, prepares a polymer ultrasound contrast agent encapsulating paclitaxel by a double emulsion solvent volatilization method, and uses a carbodiimide method to bind the polymer ultrasound contrast agent encapsulating paclitaxel to p16 INK4a Antibodies are linked to prepare targeted ultrasound contrast agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of in vitro imaging experiments are shown in Figure 2.

[0018] In the figure, A is the experimental result of ultrasound contrast agent of Example 1; B is the experimental result of ultrasound contrast agent of Comparative Example 1; C is the experimental result of ultrasound contrast agent of Comparative Example 2. DETAILED DESCRIPTION

[0019] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0020] Example 1

[0021] An ultrasound contrast agent targeting senescent chondrocytes, the ultrasound contrast agent is made of the following components in parts by weight: 100 parts of hydroxyl-terminated lactic acid-glycolic acid copolymer, 100 parts of polyethylene glycol-lignin conjugate, 10 parts of paclitaxel, 16 INK4a 15 copies of antibodies.

[0022] The method for preparing the ultrasound contrast agent targeting senescent chondrocytes is carried out according to the following steps:

[0023] (1) fully dissolving the hydroxyl-terminated lactic acid-hydroxyacetic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky primary emulsion; the amount of the added dichloromethane is 6 times the total mass of the hydroxyl-terminated lactic acid-hydroxyacetic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel; the amount of the added double distilled water is 3 times the total mass of the hydroxyl-terminated lactic acid-hydroxyacetic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel;

[0024] (2) adding polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 3 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel; the volume of the polyvinyl alcohol solution added is 20% of the volume of the primary emulsion; the volume of the isopropanol solution added is 20% of the volume of the secondary emulsion;

[0025] (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4aThe antibody was incubated with shaking for 1.5 h under ice bath conditions, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 36 h, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes;

[0026] The coupling activator is EDC / NHS, the mass ratio of EDC to NHS is 1:3, and the amount used is 25% of the mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer;

[0027] The preparation method of the polyethylene glycol-lignin conjugate is as follows: according to the weight ratio, 2 parts of alkali lignin are taken, dissolved in 150 parts of tetrahydrofuran, and 5 parts of polyethylene glycol are added; after sufficient dissolution, 4 parts of EDC and 2 parts of DMAP are added, and the mixture is stirred for reaction for 24 hours; after the reaction is completed, the reaction solution is dropped into anhydrous ether to precipitate; the precipitate is redissolved in 100 parts of pure water, dialyzed in pure water for 6 hours, unreacted reagents and organic solvents are removed, and freeze-dried to obtain the polyethylene glycol-lignin conjugate.

[0028] Example 2

[0029] An ultrasound contrast agent targeting senescent chondrocytes, the ultrasound contrast agent is made of the following components in parts by weight: 80 parts of hydroxyl-terminated lactic acid-glycolic acid copolymer, 80 parts of polyethylene glycol-lignin conjugate, 8 parts of paclitaxel, and p16 INK4a Antibody 10 copies.

[0030] The method for preparing the ultrasound contrast agent targeting senescent chondrocytes is carried out according to the following steps:

[0031] (1) fully dissolving the hydroxyl-terminated lactic acid-glycolic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky primary emulsion; the amount of the added dichloromethane is 3 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel; the amount of the added double distilled water is 2 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel;

[0032] (2) adding polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 2 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel; the volume of the polyvinyl alcohol solution added is 15% of the volume of the primary emulsion; the volume of the isopropanol solution added is 15% of the volume of the secondary emulsion;

[0033] (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4a The antibody was incubated with shaking for 1 hour under ice bath conditions, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 24 hours, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes;

[0034] The coupling activator is EDC / NHS, the mass ratio of EDC to NHS is 1:3, and the amount used is 12% of the mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer;

[0035] The preparation method of the polyethylene glycol-lignin conjugate is as follows: according to the weight ratio, 1 part of alkali lignin is taken, dissolved in 100 parts of tetrahydrofuran, and 3 parts of polyethylene glycol are added; after sufficient dissolution, 2 parts of EDC and 2 parts of DMAP are added, and the mixture is stirred for reaction for 20 hours; after the reaction is completed, the reaction solution is dropped into anhydrous ether to precipitate; the precipitate is redissolved in 80 parts of pure water, dialyzed in pure water for 4 hours, unreacted reagents and organic solvents are removed, and freeze-dried to obtain the polyethylene glycol-lignin conjugate.

[0036] Example 3

[0037] An ultrasound contrast agent targeting senescent chondrocytes, the ultrasound contrast agent is made of the following components in parts by weight: 120 parts of hydroxyl-terminated lactic acid-glycolic acid copolymer, 120 parts of polyethylene glycol-lignin conjugate, 12 parts of paclitaxel, 16 INK4a 30 copies of antibody.

[0038] The method for preparing the ultrasound contrast agent targeting senescent chondrocytes is carried out according to the following steps:

[0039] (1) fully dissolving the hydroxyl-terminated lactic acid-glycolic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky primary emulsion; the amount of the added dichloromethane is 7 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel; the amount of the added double distilled water is 4 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, polyethylene glycol-lignin conjugate, and paclitaxel;

[0040] (2) adding polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 4 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel; the volume of the polyvinyl alcohol solution added is 30% of the volume of the primary emulsion; the volume of the isopropanol solution added is 30% of the volume of the secondary emulsion;

[0041] (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4a The antibody was shaken and incubated for 2 h under ice bath conditions, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 48 h, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes;

[0042] The coupling activator is EDC / NHS, the mass ratio of EDC to NHS is 1:3, and the amount used is 35% of the mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer;

[0043] The preparation method of the polyethylene glycol-lignin conjugate is as follows: according to the weight ratio, 3 parts of alkali lignin are taken, dissolved in 200 parts of tetrahydrofuran, and 7 parts of polyethylene glycol are added; after sufficient dissolution, 5 parts of EDC and 3 parts of DMAP are added, and the mixture is stirred for reaction for 28 hours; after the reaction is completed, the reaction solution is dropped into anhydrous ether to precipitate; the precipitate is redissolved in 120 parts of pure water, dialyzed in pure water for 8 hours, unreacted reagents and organic solvents are removed, and freeze-dried to obtain the polyethylene glycol-lignin conjugate.

[0044] Comparative Example 1

[0045] An ultrasound contrast agent targeting senescent chondrocytes, the ultrasound contrast agent is made of the following components in parts by weight: 200 parts of hydroxyl-terminated lactic acid-glycolic acid copolymer, 10 parts of paclitaxel, and p16 INK4a 15 copies of antibodies.

[0046] The method for preparing the ultrasound contrast agent targeting senescent chondrocytes is carried out according to the following steps:

[0047] (1) Fully dissolving a hydroxyl-terminated lactic acid-glycolic acid copolymer and paclitaxel in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky primary emulsion; the amount of the added dichloromethane is 6 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer and paclitaxel; the amount of the added double distilled water is 3 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer and paclitaxel;

[0048] (2) adding polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 3 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel; the volume of the polyvinyl alcohol solution added is 20% of the volume of the primary emulsion; the volume of the isopropanol solution added is 20% of the volume of the secondary emulsion;

[0049] (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4a The antibody was incubated with shaking for 1.5 h under ice bath conditions, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 36 h, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes;

[0050] The coupling activator is EDC / NHS, the mass ratio of EDC to NHS is 1:3, and the amount used is 12.5% ​​of the mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer;

[0051] Comparative Example 2

[0052] An ultrasound contrast agent targeting senescent chondrocytes, the ultrasound contrast agent is made of the following components in parts by weight: 200 parts of polyethylene glycol-lignin conjugate, 10 parts of paclitaxel, p16 INK4a 15 copies of antibodies.

[0053] The method for preparing the ultrasound contrast agent targeting senescent chondrocytes is carried out according to the following steps:

[0054] (1) taking polyethylene glycol-lignin conjugate and paclitaxel and fully dissolving them in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky white primary emulsion; the amount of the added dichloromethane is 6 times the total mass of the polyethylene glycol-lignin conjugate and paclitaxel; the amount of the added double distilled water is 3 times the total mass of the polyethylene glycol-lignin conjugate and paclitaxel;

[0055] (2) adding polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 3 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel; the volume of the polyvinyl alcohol solution added is 20% of the volume of the primary emulsion; the volume of the isopropanol solution added is 20% of the volume of the secondary emulsion;

[0056] (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4a The antibody was incubated with shaking for 1.5 h under ice bath conditions, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 36 h, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes;

[0057] The coupling activator is EDC / NHS, the mass ratio of EDC to NHS is 1:3, and the amount used is 12.5% ​​of the mass of the polyethylene glycol-lignin binder;

[0058] The preparation method of the polyethylene glycol-lignin conjugate is as follows: according to the weight ratio, 2 parts of alkali lignin are taken, dissolved in 150 parts of tetrahydrofuran, and 5 parts of polyethylene glycol are added; after sufficient dissolution, 4 parts of EDC and 2 parts of DMAP are added, and the mixture is stirred for reaction for 24 hours; after the reaction is completed, the reaction solution is dropped into anhydrous ether to precipitate; the precipitate is redissolved in 100 parts of pure water, dialyzed in pure water for 6 hours, unreacted reagents and organic solvents are removed, and freeze-dried to obtain the polyethylene glycol-lignin conjugate.

[0059] Experimental example:

[0060] The ultrasound contrast agents prepared in Example 1, Comparative Example 1 and Comparative Example 2 were used as experimental objects, and their ability to bind to cells was observed using a light microscope and a laser confocal microscope, their in vitro targeting effects were detected, and in vitro development experiments were performed (Example 1, Comparative Example 1, Comparative Example 2).

[0061] Results Figure 1 , Figure 1 A in the middle shows that the surrounding of senescent chondrocytes is targeted and identified by targeted contrast agents. The contrast agents have precise targeting properties. Figure 1 B and C show that the contrast agent has no obvious targeting effect on non-senescent chondrocytes. Paclitaxel, a drug encapsulated in the contrast agent microbubbles, can be irradiated with ultrasound. Under the cavitation effect of ultrasound, the microbubbles can be ruptured and the drug in the microbubbles can be released, thereby killing senescent chondrocytes and reducing the release of toxins from senescent chondrocytes. Senescent cells and their senescence-related secretory phenotypes play an important role in cartilage degeneration and OA. Targeted elimination of senescent cells can prevent articular cartilage degeneration and OA progression.

[0062] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An ultrasound contrast agent targeting senescent chondrocytes, characterized in that: The ultrasound contrast agent is made of the following components in parts by weight: 80-120 parts of hydroxyl-terminated lactic acid-glycolic acid copolymer, 80-120 parts of polyethylene glycol-lignin conjugate, 8-12 parts of paclitaxel, INK4a 10-30 copies of antibody.

2. The method for preparing the ultrasound contrast agent targeting senescent chondrocytes according to claim 1, characterized in that: Follow these steps: (1) taking a hydroxyl-terminated lactic acid-glycolic acid copolymer, a polyethylene glycol-lignin conjugate, and paclitaxel and fully dissolving them in dichloromethane as a continuous phase, then adding double distilled water as a dispersed phase, and sonicating with a sonicator to obtain a milky white primary emulsion; (2) adding a polyvinyl alcohol solution and performing secondary sonication to obtain a secondary emulsion; adding an isopropanol solution to the obtained secondary emulsion, magnetically stirring at room temperature for 2-4 hours, centrifuging in a high-speed centrifuge to obtain microspheres, collecting the microspheres and resuspending them in double distilled water, centrifuging and collecting the microspheres, freeze-drying them, and then filling them with fluorocarbon gas to obtain a polymer contrast agent encapsulating paclitaxel; (3) The polymer contrast agent encapsulating paclitaxel was dispersed and dissolved in MES buffer, and a coupling activator was added. The mixture was incubated on an oscillator at room temperature, and then washed with double distilled water and centrifuged to collect the surface-activated contrast agent. The surface-activated contrast agent was resuspended in MES buffer, and then p16 INK4a The antibody was incubated with shaking in an ice bath for 1-2 hours, then washed with double distilled water and centrifuged, the precipitate was collected, freeze-dried for 24-48 hours, and then filled with fluorocarbon gas to obtain an ultrasound contrast agent targeting senescent chondrocytes.

3. The method for preparing the ultrasound contrast agent targeting senescent chondrocytes according to claim 2, characterized in that: The preparation method of the polyethylene glycol-lignin conjugate is as follows: according to the weight ratio, 1-3 parts of alkali lignin are taken, dissolved in 100-200 parts of tetrahydrofuran, and 3-7 parts of polyethylene glycol are added; after sufficient dissolution, 2-5 parts of EDC and 2-3 parts of DMAP are added, and the mixture is stirred for reaction for 20-28 hours; after the reaction is completed, the reaction solution is dripped into anhydrous ether to precipitate; the precipitate is redissolved in 80-120 parts of pure water, dialyzed in pure water for 4-8 hours, unreacted reagents and organic solvents are removed, and freeze-dried to obtain the polyethylene glycol-lignin conjugate.

4. The method for preparing the ultrasound contrast agent targeting senescent chondrocytes according to claim 2, characterized in that: The coupling activator is EDC / NHS, the mass ratio of EDC to NHS is 1:3, and the dosage is 10-40% of the mass of the hydroxyl-terminated lactic acid-hydroxyacetic acid copolymer.

5. The method for preparing the ultrasound contrast agent targeting senescent chondrocytes according to claim 2, characterized in that: The mass ratio of the hydroxyl-terminated lactic acid-glycolic acid copolymer, the polyethylene glycol-lignin conjugate, and paclitaxel is: (80-120): (80-120): (8-12).

6. The method for preparing the ultrasound contrast agent targeting senescent chondrocytes according to claim 2, characterized in that: The amount of dichloromethane added in step (1) is 2-8 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, the polyethylene glycol-lignin conjugate, and paclitaxel; the amount of double distilled water added is 2-5 times the total mass of the hydroxyl-terminated lactic acid-glycolic acid copolymer, the polyethylene glycol-lignin conjugate, and paclitaxel.

7. The method for preparing the ultrasound contrast agent targeting senescent chondrocytes according to claim 2, characterized in that: The volume of the polyvinyl alcohol solution added in step (2) is 15-30% of the volume of the primary emulsion; the volume of the isopropanol solution added is 15-30% of the volume of the secondary emulsion.

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