CN112979968A

CN112979968A - Cartilage targeting zwitterionic polymer and preparation method and application thereof

Info

Publication number: CN112979968A
Application number: CN202110412807.6A
Authority: CN
Inventors: 谢婧; 李建树; 俞鹏; 孙辉; 任凯
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-04-16
Filing date: 2021-04-16
Publication date: 2021-06-18
Anticipated expiration: 2041-04-16
Also published as: CN112979968B

Abstract

The invention belongs to the field of high molecular functional materials, and particularly relates to a cartilage targeting zwitterionic polymer and a preparation method and application thereof, wherein a methacryloyl chloride modified N-hydroxysuccinimide monomer is synthesized for realizing efficient chemical grafting of cartilage targeting peptide; secondly, the cartilage targeting zwitterionic polymer is obtained through random copolymerization or RAFT polymerization. The polymer can be targeted to the cartilage surface, enhances the lubricating property of the articular cartilage, avoids abrasion in the movement process, and in addition, the antifouling property provided by the zwitterion unit can resist the damage of degrading enzymes to the cartilage matrix, thereby realizing hose protection. The cartilage targeting zwitterionic polymer has wide application prospect in bone tissue engineering.

Description

Cartilage targeting zwitterionic polymer and preparation method and application thereof

Technical Field

The invention belongs to the field of high molecular functional materials, and relates to a cartilage targeting zwitterionic polymer, a preparation method and application thereof, which can be used for enhancing the lubricating property of cartilage softening and degradation and weakening the damage of degradation enzymes to cartilage matrixes so as to protect articular cartilage, and has a wide biological application scene.

Background

The polymers currently used for cartilage protection are mainly zwitterionic polymers, anionic/cationic polyelectrolytes or zwitterionic modified natural polysaccharide materials, however, these materials can only be injected into the joint cavity as a lubrication supplement without robust interaction with cartilage tissue, and therefore, the polymers free in the joint cavity are easily lost or metabolized and do not have long-acting joint lubrication properties.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a cartilage targeting zwitterionic polymer and a preparation method and application thereof, and provides a general method for designing cartilage protection and osteoarthritis treatment materials by targeting a polymer on the surface of cartilage through targeting peptides on the basis of ensuring the excellent lubricating performance and anti-fouling performance of the zwitterionic polymer.

The specific technical scheme is as follows:

the preparation method of the cartilage targeting zwitterionic polymer realizes the chemical modification of the type II collagen binding polypeptide WYRRGRL by introducing an active ester monomer; obtained by random copolymerization or by chain transfer radical polymerization, namely RAFT.

The preparation method comprises the following steps:

(1) random copolymerization: dissolving a zwitterion monomer, an active ester monomer and an initiator in N, N-dimethylformamide according to a ratio, introducing nitrogen for 30 minutes, reacting for 24 hours at 70 ℃, precipitating products respectively by tetrahydrofuran, methanol and anhydrous ether, washing for multiple times, and drying in vacuum to obtain a product;

(2) RAFT polymerisation

The zwitterionic monomer, 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid and an initiator are dissolved in N, N-dimethylformamide, gas in a reaction system is removed by using 'three times freezing and three times pumping', and then nitrogen is introduced for 5 minutes. After reacting for 24 hours at the constant temperature of 70 ℃, precipitating a product by using an organic solvent, washing for 3 times, and then drying in vacuum to obtain a macromolecular initiator;

dissolving a macromolecular initiator, NHSMA and an initiator in N, N-dimethylformamide, removing gas in a system by adopting 'three-freezing and three-pumping', introducing nitrogen for 5 minutes, reacting at 70 ℃ for 24 hours, precipitating a product by using an organic solvent, washing for multiple times, and drying in vacuum to obtain a polymer;

(3) WYRRGRL polypeptide grafting

The polypeptide grafting process adopts 0.2M, pH ═ 8 phosphate buffer as solvent; firstly, preparing the polypeptide solution obtained in the step (1) and the polymer solution obtained in the step (2) with the concentrations of 10mg/mL and 0.2mg/mL respectively, mixing the two solutions in equal volumes, reacting at 37 ℃ for 24 hours, dialyzing for 3 days, and freeze-drying to obtain the final product, and storing at-20 ℃.

Wherein the zwitterionic monomer is MPC, SBMA or CBMA; obtaining products pMPC-pNHSMA, pSBMA-pNHSMA and pSBMA-pNHSMA.

The active ester monomer is NHSMA; the initiator is azobisisobutyronitrile.

The cartilage targeting zwitterionic polymer obtained by the invention is used for preparing raw materials of cartilage.

Preferably, the main synthetic process is as follows:

s1, preparing functional monomer HNSMA

In a 50mL round bottom flask, 1.173g of N-hydroxysuccinimide, 1.65mL of triethylamine, and 5mL of chromatographic grade dichloromethane were added; uniformly mixing 1mL of methacryloyl chloride and 5mL of chromatographic grade dichloromethane, dropwise adding the mixture into a round-bottom flask under the ice bath condition, controlling the dropwise adding speed to be 10 seconds per drop, and continuing to react for 12 hours after the reaction system is recovered to the room temperature.

S2, preparing functional polymer by random copolymerization

Dissolving a zwitterion monomer (MPC, SBMA or CBMA), an active ester monomer (NHSMA) and azobisisobutyronitrile (AIBN, initiator) into N, N-Dimethylformamide (DMF) according to a certain proportion, introducing nitrogen for 30 minutes, reacting for 24 hours at 70 ℃, precipitating products through tetrahydrofuran, methanol and anhydrous ether respectively, washing for multiple times, and drying in vacuum to obtain products pMPC-pNHSMA, pSBMA-pNHSMA and pSBMA-pNHSMA.

S3.RAFT polymerization for preparing functional polymer

18.0mmol of a zwitterionic monomer, 0.180mmol of 4-cyano-4- (phenylthioformylthio) pentanoic acid (CPAD) and 0.090mmol of AIBN were dissolved in 10mL of DMF, and after removing the gas from the reaction system by "three-freeze three-pump" operation, nitrogen gas was introduced for 5 minutes. After 24 hours of reaction at constant temperature (70 ℃), the product is precipitated by using a proper organic solvent, washed for 3 times and dried in vacuum to obtain the macromolecular initiator.

1.0g of macroinitiator, 0.6mmol of NHSMA and 0.02mol of AIBN are dissolved in 10mL of DMF, gas in the system is removed by 'three times freezing and three times pumping', nitrogen is introduced for 5 minutes, the mixture reacts for 24 hours at 70 ℃, a product is precipitated by using a proper organic solvent, and the product pMPC-b-pNHSMA is obtained after multiple washing and vacuum drying.

S4, chemically grafting cartilage targeting peptide

The polypeptide grafting procedure used 0.2M phosphate buffer (pH 7.2-7.4) as solvent. Firstly, preparing a polypeptide solution and a polymer solution (obtained in step 2 and step 3 respectively), wherein the concentrations of the polypeptide solution and the polymer solution are respectively 10mg/mL and 0.2mg/mL, mixing the two solutions in equal volumes, reacting at 37 ℃ for 24 hours, dialyzing for 3 days, and freeze-drying to obtain a final product, and storing at-20 ℃.

The "triple freeze triple pump" referred to above means the usual liquid nitrogen cooling-vacuum pumping-thawing cycle three times.

Compared with the prior art, the invention has the following beneficial technical effects:

1. the invention provides a preparation method of a cartilage target targeting zwitterionic polymer, which comprises the following specific synthesis design: the polymer of the zwitter-ion monomer and the active ester monomer is obtained through random copolymerization or RAFT active polymerization, and the high-efficiency reaction of the cartilage targeting peptide and the active ester unit is realized under the slightly alkaline environment. The polymerization and chemical grafting processes do not affect the "lubricating" and "anti-fouling" properties of the zwitterionic polymers themselves.

2. Experiments prove that the targeting peptide can be specifically combined in cartilage components, can realize the cartilage targeting performance of the polymer, and has wide application prospect.

3. The invention also provides a synthetic scheme of the cartilage targeting zwitterionic polymer, and the scheme has the advantages of simple preparation process, no need of special equipment, simple operation and preparation process, and easy realization of batch production and application and popularization.

Drawings

FIG. 1 shows the nuclear magnetic data of NHSMA in example 1;

FIG. 2 shows the UV spectrum (190-400nm) of the WYRRGRL polypeptide in example 1;

FIG. 3 is a fluorescence excitation-emission spectrum of the WYRRGRL polypeptide of example 1;

FIG. 4a is nuclear magnetic data of CBMA monomer of example 3;

FIG. 4b is mass spectral data of CBMA monomer of example 3;

FIG. 5 is an infrared spectrum of pMPC-pCOlBP in example 1;

FIG. 6 is an infrared spectrum of pSBMA-pColBP in example 2;

FIG. 7 shows nuclear magnetic data for pMPC-pNHSMA in example 1;

FIG. 8 shows nuclear magnetic data of pSBMA-pNHSMA in example 2;

FIG. 9 shows the results of the specific binding between the WYRRGRL polypeptide and the biological macromolecules in the cartilage component in example 7.

Detailed Description

The cartilage targeting zwitterionic polymers and the preparation methods thereof provided by the present invention are further illustrated by the following examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make certain insubstantial modifications and adaptations of the present invention based on the above disclosure and still fall within the scope of the present invention.

Example 1

In this embodiment, a method for preparing a cartilage-targeted zwitterionic polymer is provided, which includes the following steps:

(1) preparation of reactive ester monomer NHSMA

N-hydroxysuccinimide (NHS, 1.1736g, 12mmol) and triethylamine (1,650. mu.L, 12mmol) were dissolved in 5mL of chromatographic grade Dichloromethane (DCM) and stirred under ice bath conditions for 30 minutes while methacryloyl chloride (1mL, 11mmol) was dissolved in 5mL of chromatographic grade dichloromethane and added dropwise to the previously prepared solution at a rate of 1 drop for 10 seconds. After the solution was added dropwise, the reaction system was gradually returned to room temperature and the reaction was continued for 12 hours. After the reaction is finished, washing the reaction product for 3 times by using distilled water, wherein each time is 5 mL; then dried over anhydrous sodium sulfate, the resulting organic solution was concentrated by rotary evaporation, and precipitated in an ethyl acetate/n-hexane (1/3) mixed solution to give a white product. The reaction equation is shown as formula (I):

(2) random copolymerization synthesis of functionalized zwitterionic polymer pMPC-pNHSMA

2-Methacryloyloxyethyl Phosphorylcholine (MPC), NHSMA, Azobisisobutyronitrile (AIBN) were added to a 25mL reaction flask in the amounts shown in Table 1, 10mL of chromatographic grade N, N-Dimethylformamide (DMF) was added as a reaction solvent, and after introducing nitrogen gas into the reaction flask for 30 minutes, the reaction was carried out at 70 ℃ for 24 hours. After the reaction is finished, dropwise adding the reaction solution into precooled Tetrahydrofuran (THF) to precipitate a polymer, repeatedly washing for 3 times, and removing the organic solvent from the obtained polymer through vacuum drying to obtain the pMPC-pNHSMA. The reaction process is shown as the formula (II):

TABLE 1 pMPC-pNHSMA random copolymerization Material dosage

(3) Chemically grafted cartilage targeting peptide WYRRGRL

First a 0.2M PBS solution (pH 8) was prepared: weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, respectively dissolving in 100mL of water, mixing solutions with different volumes, adjusting pH to 8, adding 0.9g of sodium chloride, and finally fixing the volume to 100 mL. Then preparing a polypeptide solution with the solubility of 10 mg/mL; preparing a polymer solution with the concentration of 0.2mg/mL, and mixing the two solutions in equal volumes for reaction for 24 hours. Finally, dialysis was carried out for 3 days using a 2,000 molecular weight dialysis bag, and the polymer was obtained after freeze-drying and was designated pMPC-pCOLBP.

FIG. 1 shows NHSMA nuclear magnetic data of functional monomer of active ester; FIG. 2 shows the UV spectrum (190-400nm) of the WYRRGRL polypeptide; FIG. 3 is a WYRRGRL polypeptide fluorescence excitation-emission spectrum; FIG. 5 is an infrared spectrum of pMPC-pCOlBP; FIG. 7 shows nuclear magnetic data for pMPC-pNHSMA.

Example 2

(1) preparation of reactive ester monomer NHSMA

The procedure was as in example 1.

(2) Random copolymerization synthesis of functionalized zwitterionic polymer pSBMA-pNHSMA

[2- (methacryloyloxy) ethyl ] dimethyl- (3-sulfopropyl) ammonium hydroxide (SBMA), NHSMA, AIBN were added to a 25mL reaction flask in the amounts shown in Table 2, 10mL of chromatographic grade DMF was added as a reaction solvent, and after introducing nitrogen gas to the reaction flask for 30 minutes, the reaction was carried out at 70 ℃ for 24 hours. After the reaction is finished, dropwise adding the reaction solution into precooled methanol to precipitate a polymer, repeatedly washing for 3 times, and drying the obtained polymer in vacuum to remove the organic solvent to obtain the pSBMA-pNHSMA. The reaction is shown as formula (III):

TABLE 2 pSBMA-pNHSMA random copolymerization feed dosage

(3) Chemically grafted cartilage targeting peptide WYRRGRL

First a 0.2M PBS solution (pH 8) was prepared: weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, respectively dissolving in 100mL of water, mixing solutions with different volumes, adjusting pH to 8, adding 0.9g of sodium chloride, and finally fixing the volume to 100 mL. Then preparing a polypeptide solution with the solubility of 10 mg/mL; a polymer solution was prepared at a concentration of 0.2mg/mL, and the two solutions were mixed and reacted for 24 hours. Finally, dialysis was performed for 3 days using a 2,000 molecular weight dialysis bag, and the polymer was obtained after freeze-drying, and was designated as pSBMA-pCOLBP.

FIG. 6 is an infrared spectrum of pSBMA-pColBP; FIG. 8 shows nuclear magnetic data of pSBMA-pNHSMA.

Example 3

In this example, a preparation scheme of a cartilage targeting zwitterionic polymer is provided, which specifically comprises the following steps:

(1) preparation of reactive ester monomer NHSMA

The procedure was as in example 1.

(2) Preparation of the zwitterionic monomer [2- (methacryloyloxy) ethyl ] dimethyl- (3-carboxyethyl) ammonium hydroxide (CBMA)

Uniformly mixing 8.5mL of dimethylaminoethyl methacrylate (DMAEMA), 7mL of acrylic acid (AAc) and 0.01g of 4-methoxyphenol under an ice bath condition, and reacting for about 4 hours to thicken the solution; the reaction was continued for 24h after the addition of 5mL of acetone. After the reaction is finished, dropwise adding the reaction solution into 25mL of acetone to precipitate the product, repeatedly precipitating for 3 times, and performing vacuum drying to obtain the product CBMA.

(3) Random copolymerization synthesis of functionalized zwitterionic polymer pCBMA-pNHSMA

MPC, NHSMA and azobisisobutyronitrile were added to a 25mL reaction flask in the amounts shown in Table 3, followed by addition of 10mL of DMF as a reaction solvent, and after introducing nitrogen gas to the reaction flask for 30 minutes, the reaction was carried out at 70 ℃ for 24 hours. After the reaction is finished, dropwise adding the reaction solution into precooled anhydrous ether to precipitate a polymer, repeatedly washing for 3 times, and drying the obtained polymer in vacuum to remove the organic solvent to obtain pCBMA-pNHSMA. The reaction process is shown as the formula (V):

TABLE 3 pCBMA-pNHSMA random copolymerization Material amounts

(4) Chemically grafted cartilage targeting peptide WYRRGRL

First a 0.2M PBS solution (pH 8) was prepared: weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, respectively dissolving in 100mL of water, mixing solutions with different volumes, adjusting pH to 8, adding 0.9g of sodium chloride, and finally fixing the volume to 100 mL. Then preparing a polypeptide solution with the solubility of 10 mg/mL; a polymer solution was prepared at a concentration of 0.2mg/mL, and the two solutions were mixed and reacted for 24 hours. Finally, the mixture was dialyzed for 3 days using a 2,000 molecular weight dialysis bag and lyophilized to obtain a polymer designated pCBMA-pCOLBP.

FIG. 4a is CBMA monomer magnetic data and FIG. 4b is CBMA monomer mass spectral data.

Example 4

In this embodiment, a method for synthesizing a cartilage-targeted zwitterionic polymer is provided, which includes the following steps:

(1) preparation of reactive ester monomer NHSMA

The procedure was as in example 1.

(2) Preparation of macroinitiator by RAFT polymerization

18.0mmol of MPC, 0.180mmol of 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid (CPAD) and 0.090mmol of Azobisisobutyronitrile (AIBN) were dissolved in 10mL of DMF, and after the gas in the reaction system was purged by "triple freeze triple purge", nitrogen (N) was introduced₂) For 5 minutes. After 24 hours of reaction at constant temperature (70 ℃), products are precipitated by Tetrahydrofuran (THF), washed for three times and dried in vacuum to obtain the pMPC-CTA macroinitiator.

(3) Preparation of pMPC-b-pNHSMA by RAFT polymerization

Dissolving 1.0g of pMPC-CTA monomolecular initiator, 0.6mmol of NHSMA and 0.02mol of AIBN in 10mL of DMF, removing gas in the system by adopting 'three freezes and three pumps' operation, introducing N₂After 5 minutes, the reaction was carried out at 70 ℃ for 24 hours, the product was precipitated with THF, washed several times and dried under vacuum to give the product pMPC-co-pNHSMA.

(3) Chemically grafted cartilage targeting peptide WYRRGRL

First a 0.2M PBS solution (pH 8) was prepared: weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, respectively dissolving in 100mL of water, mixing solutions with different volumes, adjusting pH to 8, adding 0.9g of sodium chloride, and finally fixing the volume to 100 mL. Then preparing a polypeptide solution with the solubility of 2 mg/mL; a polymer solution was prepared at a concentration of 2mg/mL, and the two solutions were mixed and reacted for 24 hours. Finally, the mixture was dialyzed for 3 days using a dialysis bag of 2,000 molecular weight, and lyophilized to obtain a polymer designated pMPC-b-pCOLBP. The whole preparation process is shown as formula (VI):

example 5

(1) preparation of reactive ester monomer NHSMA

The procedure was as in example 1.

(2) Preparation of macroinitiator by RAFT polymerization

18.0mmol of SBMA, 0.180mmol of 4-cyano-4- (phenylthioformylthio) pentanoic acid (CPAD) and 0.090mmol of AIBN are dissolved in 10mL of DMF, the reaction system is degassed by "triple freezing and triple pumping", and then nitrogen (N) is introduced₂) For 5 minutes. After reacting for 24 hours at constant temperature (70 ℃), precipitating the product by Tetrahydrofuran (THF), washing for three times, and drying in vacuum to obtain pSBMA-CTA macroinitiator.

(3) Preparation of pSBMA-b-pNHSMA by RAFT polymerization

Dissolving 1.0g of pSBMA-CTA monomolecular initiator, 0.6mmol of NHSMA and 0.02mol of AIBN in 10mL of DMF, removing gas in the system by adopting 'three-freezing and three-pumping', introducing nitrogen for 5 minutes, reacting at 70 ℃ for 24 hours, precipitating a product by using methanol, washing for multiple times, and drying in vacuum to obtain a product pSBMA-b-pNHSMA.

(3) Chemically grafted cartilage targeting peptide WYRRGRL

First a 0.2M PBS solution (pH 8) was prepared: weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, respectively dissolving in 100mL of water, mixing solutions with different volumes, adjusting pH to 8, adding 0.9g of sodium chloride, and finally fixing the volume to 100 mL. Then preparing a polypeptide solution with the solubility of 10 mg/mL; a polymer solution was prepared at a concentration of 0.2mg/mL, and the two solutions were mixed and reacted for 24 hours. Finally, dialysis was performed for 3 days using a 2,000 molecular weight dialysis bag, and the polymer was obtained after freeze-drying, and was designated as pSBMA-b-pCOLBP. The whole preparation process is shown as a formula (VII):

example 6

(1) preparation of reactive ester monomer NHSMA

The procedure was as in example 1.

(2) Preparation of macroinitiator by RAFT polymerization

18.0mmol of CBMA monomer (prepared in accordance with example 3), 0.180mmol of 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid (CPAD) and 0.090mmol of Azobisisobutyronitrile (AIBN) were dissolved in 10mL of DMF, and after removing the gas from the reaction system by "triple freeze triple pump", nitrogen gas was introduced for 5 minutes. After 24 hours of reaction at constant temperature (70 ℃), the product is precipitated by using anhydrous ether, washed for three times and dried in vacuum to obtain the pCBMA-CTA macroinitiator.

(3) Preparation of pCBMA-b-pNHSMA by RAFT polymerization

1.0g of pCBMA-CTA monomolecular initiator, 0.6mmol of NHSMDissolving A and 0.02mol AIBN in 10mL DMF, removing gas in the system by adopting 'three-freezing three-pumping' operation, introducing N₂After 5 minutes, the reaction was carried out at 70 ℃ for 24 hours, and the product was precipitated with THF, washed several times and dried under vacuum to give the product pCBMA-b-pNHSMA.

(3) Chemically grafted cartilage targeting peptide WYRRGRL

First a 0.2M PBS solution (pH 8) was prepared: weighing sodium dihydrogen phosphate and disodium hydrogen phosphate, respectively dissolving in 100mL of water, mixing solutions with different volumes, adjusting pH to 8, adding 0.9g of sodium chloride, and finally fixing the volume to 100 mL. Then preparing a polypeptide solution with the solubility of 2 mg/mL; a polymer solution was prepared at a concentration of 2mg/mL, and the two solutions were mixed and reacted for 24 hours. Finally, the mixture was dialyzed for 3 days using a dialysis bag of 2,000 molecular weight, and lyophilized to obtain a polymer designated pMPC-b-pCOLBP. The whole preparation process is shown as a formula (VIII):

example 7

In this example, the ability of WYRGRL polypeptides to target type II collagen was tested by fluorescence spectrophotometer, which was not tested as follows:

(1) preparation of FITC-labeled WYRRGRL polypeptide (WYRRGRL-FITC)

First, 10mg of the polypeptide was dissolved in 5mL of phosphate buffer (PBS, pH 7.2 to 7.4), and then FITC was dissolved in 200 μ l dmso, and the solution was added dropwise to the polypeptide solution under ice-bath conditions, and reacted for 24 hours while being protected from light. After the reaction is finished, dialyzing (MWCO 1,000) for 3 days in the dark, freeze-drying and storing in an environment at-20 ℃.

(2) Fluorescence spectrophotometer testing

Firstly, preparing II type collagen, chondroitin sulfate and hyaluronic acid solutions with the concentration of 1mg/mL, and then co-culturing the II type collagen, the chondroitin sulfate and the hyaluronic acid solutions with the concentration of 1mg/mL FITC marked polypeptide solution, wherein the conditions are as follows: protected from light, water bath at 37 ℃ and 24 hours. Then dialyzed against light for 3 days using a dialysis bag having a molecular weight cut-off of 2,000. Finally, a fluorescence spectrophotometer is used for scanning the emission spectrum, the excitation wavelength is 488nm, and the scanning range is 500-600 nm.

FIG. 9 shows the results of specific binding between WYRRGRL polypeptide and the biological macromolecules in the cartilage component.

Example 8

In this example, the targeting properties of the polymer prepared were tested by a microcrystalline quartz balance (QCMD) and the specific operation was as follows:

first, sulfhydryl modified type II collagen (Col II-SH) is prepared: type II collagen was prepared as a 0.5% (w/v) aqueous solution and the pH was adjusted to 10 with NaOH (2M). Then dissolving 10 times of molar weight of gamma-thiobutyrolactone in the same volume of DMSO, and dropwise adding the gamma-thiobutyrolactone into the collagen solution, wherein the pH value is maintained at about 10 in the reaction process. After the reaction was complete, the pH was adjusted to 4.5 with HCl (5M), followed by dialysis for 3 days and lyophilization for use.

Before QCMD testing, the gold plaques were sonicated for 30min with toluene and isopropanol, respectively, then treated with ozone for 30min, then with toluene and isopropanol, N₂And (5) drying. In the test process, an HEPES buffer solution is used for stabilizing the running state of the instrument, and then the Col II-SH solution, the HEPES buffer solution, the block copolymer solution and the HEPES buffer solution are sequentially replaced. The test temperature was set at 25 ℃.

Example 9

In this example, the lubricating property of the material was tested by a universal material testing machine. The experimental sample is a sheet cut from bovine cartilage, and the sheet is tested after targeted modification of a block copolymer, wherein the test frequency is 1Hz, 3Hz and 5Hz respectively, and the test time is 40 min; the pressures were set to 1N, 2N and 5N, respectively. At the same time, the targeted lubrication performance of the polymer solutions of different concentrations was compared.

Claims

1. The preparation method of the cartilage targeting zwitterionic polymer is characterized in that the chemical modification of the type II collagen binding polypeptide WYRRGRL is realized by introducing an active ester monomer; obtained by random copolymerization or by chain transfer radical polymerization, namely RAFT.

2. The method for preparing the cartilage-targeting zwitterionic polymer according to claim 1, characterized in that the method comprises:

(2) RAFT polymerisation

(3) WYRRGRL polypeptide grafting

3. The method for preparing the cartilage-targeting zwitterionic polymer according to claim 2, wherein the RAFT polymerization step in step (2) is: the zwitterionic monomer, 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid and an initiator are dissolved in N, N-dimethylformamide, gas in a reaction system is removed by using 'three times freezing and three times pumping', and then nitrogen is introduced for 5 minutes. After reacting for 24 hours at the constant temperature of 70 ℃, precipitating a product by using an organic solvent, washing for 3 times, and then drying in vacuum to obtain a macromolecular initiator;

dissolving a macromolecular initiator, NHSMA and an initiator in N, N-dimethylformamide, removing gas in a system by adopting 'three freezing and three pumping', introducing nitrogen for 5 minutes, reacting at 70 ℃ for 24 hours, precipitating a product by using an organic solvent, washing for multiple times, and drying in vacuum to obtain the polymer.

4. The method for preparing a cartilage-targeting zwitterionic polymer according to claim 2 or 3, wherein the zwitterionic monomer is MPC, SBMA or CBMA; obtaining products pMPC-pNHSMA, pSBMA-pNHSMA and pSBMA-pNHSMA.

5. The method for preparing the cartilage-targeting zwitterionic polymer according to claim 2 or 3, wherein the active ester monomer is NHSMA; the initiator is azobisisobutyronitrile.

6. Cartilage-targeting zwitterionic polymers obtainable by the process according to any one of claims 1 to 5.

7. Use of a cartilage targeting zwitterionic polymer, characterised in that it is used for the preparation of a raw material for cartilage.