Hyaluronic Acid-Based Zwitterionic Polymer Brush, Preparation Method Thereof, and Use Thereof

Abstract

Disclosed are a hyaluronic acid-based zwitterionic polymer brush, a preparation method thereof, and the use thereof. The structure of the zwitterionic polymer brush is represented by formula (I), wherein n is an integer from 60-150, x is an integer from 589-686, and y is an integer from 125-230. The above-mentioned zwitterionic polymer brush can delay the pathological progress of osteoarthritis, promote cartilage regeneration, and even treat osteoarthritis and improve conditions such as the generation of abraded fragments of implants such as joints and related diseases triggered thereby, and can be combined with cartilage repairing materials to improve the frictional mechanical properties thereof for achieving better repair results. Moreover, the above-mentioned zwitterionic polymer brush can selectively bind to proteins in cartilage and has an excellent lubricating effect. The present invention has the advantages of simple method, convenient operation, easy purification, high yield, and the like.

##STR00001##

Claims

1. A zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine, comprising the following structure: ##STR00003## wherein n is an integer from 60-150, x is an integer from 589-686, and y is an integer from 125-230.

2. A preparation method for the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 1, comprising the following steps: (1) reacting hyaluronic acid and adipic acid dihydrazide to obtain an amino-modified hyaluronic acid; (2) subjecting 2 methyacryloxyloxyethyl phosphorylcholine to reversible addition-fragmentation chain transfer radical polymerization to obtain poly(2-methacryloyloxyethyl phosphorylcholine) carrying a carboxyl group at the terminal under the action of an initiator and a chain transfer agent, by taking 2-methacryloyloxyethyl phosphorylcholine as a polymerization monomer; and (3) mixing the above-mentioned amino-modified hyaluronic acid and poly(2-methacryloyloxyethyl phosphorylcholine) carrying a carboxyl group at the terminal with activators, and reacting same in an aqueous solution system to obtain the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine.

3. The preparation method for the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 2, wherein: the reaction in step (1) is carried out under the action of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride; the pH of the reaction system is 4.7-4.85; and the pH value of the reaction system in step (3) is 5-7.

4. The preparation method for the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 2, wherein: the mass ratio of the hyaluronic acid to adipic acid dihydrazide in step (1) is 1:4 to 1:18; the amounts of the amino-modified hyaluronic acid and the poly(2-methacryloyloxyethyl phosphorylcholine) carrying a carboxyl group at the terminal used in step (3) satisfy the following condition: the molar ratio of the amino group to the carboxyl group is 1:(1.1-2).

5. The preparation method for the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 2, wherein: the chain transfer agent in step (2) is 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid; the initiator in step (2) is 4,4′-azobis(4-cyanovaleric acid), and the activators in step (3) are 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

6. The preparation method for the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 5, wherein: the molar ratio of the chain transfer agent to 2-methacryloyloxyethyl phosphorylcholine is 1:(100-200); the molar ratio of the initiator to the chain transfer agent is 1:(2-10); and the mass ratio of the activators EDC to NHS used therein is 1:1 to 1:2.

7. The preparation method for the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 2, wherein: the temperature of the polymerization in step (2) is 60° C.-70° C., and the polymerization time is 6-24 h; the solvent used in the polymerization in step (2) is one or more of ethanol, water, and methanol; the temperature of the reaction in step (3) is 25° C.-35° C., and the reaction time is 18-36 h; the temperature of the reaction in step (1) is 25° C.-35° C.

8. A biomedical product comprising the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine according to claim 1.

9. The biomedical product according to claim 8, wherein: the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine is especially used for preparing medicines for treating osteoarthritis, and for preparing a lubricant for improving the lubricating and frictional properties of implanted prostheses, and can be combined with cartilage repairing materials to improve the frictional mechanical properties thereof.

10. The biomedical product according to claim 8, wherein: the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine is especially used as a biomimetic lubricant or a modified material for improving the frictional property of the cartilage repairing materials.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a schematic diagram of the synthetic route of the zwitterionic polymer brush, in which FIG. 1-(1) is a schematic diagram of the reaction of adipic acid dihydrazide modified hyaluronic acid; FIG. 1-(2) is a polymerization reaction diagram of 2-methacryloyloxyethyl phosphorylcholine; and FIG. 1-(3) is a preparation reaction diagram of the zwitterionic polymer brush.

[0034] FIG. 2 shows the NMR spectrum 2-(1) and the infrared spectrum 2-(2) of the zwitterionic polymer brush obtained in Embodiment 1, wherein in the figures, HPM is the zwitterionic polymer brush, PMPC is the poly(2-methacryloyloxyethyl phosphorylcholine) carrying a carboxyl group at the terminal, and HA-ADH is the amino-modified hyaluronic acid.

[0035] FIG. 3 shows the lubricating effect of the zwitterionic polymer brush obtained in Embodiment 1, wherein HPM is the zwitterionic polymer brush, and HA is hyaluronic acid.

[0036] FIG. 4 shows a diagram of the effect of the zwitterionic polymer brush obtained in Embodiment 1 on slowing osteoarthritis (scale: 500 μm).

DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with the embodiments and accompanying drawings, but this does not limit the implementation of the present invention. The reagents in the following embodiments are all commercially available.

[0038] The schematic diagram of the synthetic route of the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine of the present invention is as shown in FIG. 1, in which FIG. 1-(1) is a schematic diagram of the reaction of adipic acid dihydrazide modified hyaluronic acid; FIG. 1-(2) is a schematic diagram of the polymerization reaction of 2-methacryloyloxyethyl phosphorylcholine; and FIG. 1-(3) is a schematic diagram of the preparation reaction of the zwitterionic polymer brush.

Embodiment 1

[0039] Preparation of a Zwitterionic Polymer Brush Based on Hyaluronic Acid and 2-Methacryloyloxyethyl Phosphorylcholine:

[0040] (1) dissolving 100 mg of hyaluronic acid with a molecular weight of 1-1.5 million in 20 mL of deionized water, and stirring same for dissolution, so as to obtain a hyaluronic acid solution; mixing 1.736 g of adipic acid dihydrazide with the hyaluronic acid solution, the mass ratio of the adipic acid dihydrazide to hyaluronic acid being 17.36:1; adjusting the pH value of the solution to 4.75 with 1 M hydrochloric acid, and adding 0.191 g of 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC) in a solid form, to further adjust the pH value of the solution to 4.75; carrying out a reaction at 30° C. until the pH value does not change; and dialyzing and lyophilizing same to obtain the amino-modified hyaluronic acid;

[0041] (2) dissolving 10 g of 2-methacryloyloxyethyl phosphorylcholine (MPC) in 30 mL of anhydrous methanol, and weighing 37.5 mg of 4,4′-azobis(4-cyanovaleric acid) (ACVA) as the initiator, and 75 mg of 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CTP) as the chain transfer agent, adding the initiator and the chain transfer agent to the solution of MPC in anhydrous methanol, stirring same for dissolution, then sealing same, and introducing nitrogen for oxygen removal in an ice bath for 40 minutes;

[0042] (3) placing the deoxygenated MPC solution in an oil bath preheated to 60° C. to react for 12 h, quenching the reaction via liquid nitrogen, and after returning to room temperature, precipitating same with 1 L of acetone, pouring out the supernatant, and then drying same under a vacuum at 30° C., to obtain 7.981 g of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) carrying a carboxyl group at the terminal; and

[0043] (4) taking 100 mg of the product in step (1) and 2 g of the product in step (3) and dissolving same in deionized water, adjusting the pH value of the solution to 5.5, adding 60 mg of EDC/NHS (a molar ratio of 1:1), then carrying out a reaction at 30° C., pH=5.5 for 24 h, the molar ratio of the amino group to the carboxyl group in the amino-modified hyaluronic acid and PMPC carrying a carboxyl group at the terminal being 1:1.5, after the completion of the reaction, dialyzing and lyophilizing same to obtain 154.1 mg of the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine.

[0044] FIG. 2 shows the NMR spectrum 2-(1) and the infrared spectrum 2-(2) of the zwitterionic polymer brush obtained in Embodiment 1.

Embodiment 2

[0045] Preparation of an Anionic Polymer Brush Based on Hyaluronic Acid and 2-Acrylamido-2-Methylpropanesulfonic Acid:

[0046] (1) dissolving 100 mg of hyaluronic acid with a molecular weight of 1-1.5 million in 20 mL of deionized water, and stirring same for dissolution, so as to obtain a hyaluronic acid solution; mixing 435.4 mg of adipic acid dihydrazide with the hyaluronic acid solution, the mass ratio of the adipic acid dihydrazide to hyaluronic acid being 4.35:1; adjusting the pH value of the solution to 4.8 with 1 M hydrochloric acid, and adding 30 mg of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) in a solid form, to further adjust the pH value of the solution to 4.8; carrying out a reaction at 30° C. until the pH value does not change; and dialyzing and lyophilizing same to obtain the amino-modified hyaluronic acid;

[0047] (2) dissolving 2 g of 2-methacryloyloxyethyl phosphorylcholine (MPC) in 6 mL of anhydrous methanol, and weighing 7.5 mg of 4,4′-azobis(4-cyanovaleric acid) (ACVA) as the initiator, and 15 mg of 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CTP) as the chain transfer agent, adding the initiator and the chain transfer agent to the solution of MPC in anhydrous methanol, stirring same for dissolution, then sealing same, and introducing nitrogen for oxygen removal in an ice bath for 30 minutes;

[0048] (3) placing the deoxygenated MPC solution in an oil bath preheated to 60° C. to react for 12 h, quenching the reaction via liquid nitrogen, and after returning to room temperature, precipitating same with 300 mL of acetone, pouring out the supernatant, and then drying same under a vacuum at 30° C., to obtain 1.535 g of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) carrying a carboxyl group at the terminal; and

[0049] (4) taking 50 mg of the product in step (1) and 1 g of the product in step (3) and dissolving same in deionized water, adjusting the pH value of the solution to 5.5, adding 60 mg of EDC/NHS (a molar ratio of 1:1), then carrying out a reaction at 30° C., pH=5.5 for 24 h, the molar ratio of the amino group to the carboxyl group in the amino-modified hyaluronic acid and PMPC carrying a carboxyl group at the terminal being 1:1.5, after the completion of the reaction, dialyzing and lyophilizing same to obtain 83.5 mg of the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine.

Embodiment 3

[0050] Preparation of an Anionic Polymer Brush Based on Hyaluronic Acid and 2-Acrylamido-2-Methylpropanesulfonic Acid:

[0051] (1) dissolving 100 mg of hyaluronic acid with a molecular weight of 1-1.5 million in 20 mL of deionized water, and stirring same for dissolution, so as to obtain a hyaluronic acid solution; mixing 868 mg of adipic acid dihydrazide with the hyaluronic acid solution, the mass ratio of the adipic acid dihydrazide to hyaluronic acid being 8.68:1; adjusting the pH value of the solution to 4.8 with 1 M hydrochloric acid, and adding 60 mg of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) in a solid form, to further adjust the pH value of the solution to 4.8; carrying out a reaction at 30° C. until the pH value does not change; and dialyzing and lyophilizing same to obtain the amino-modified hyaluronic acid;

[0052] (2) dissolving 5 g of 2-methacryloyloxyethyl phosphorylcholine (MPC) in 15 mL of anhydrous methanol, and weighing 19 mg of 4,4′-azobis(4-cyanovaleric acid) (ACVA) as the initiator, and 38 mg of 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTP) as the chain transfer agent, adding the initiator and the chain transfer agent to the solution of MPC in anhydrous methanol, stirring same for dissolution, then sealing same, and introducing nitrogen for oxygen removal in an ice bath for 30 minutes;

[0053] (3) placing the deoxygenated MPC solution in an oil bath preheated to 60° C. to react for 12 h, quenching the reaction via liquid nitrogen, and after returning to room temperature, precipitating same with 600 mL of acetone, pouring out the supernatant, and then drying same under a vacuum at 30° C., to obtain 3.643 g of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) carrying a carboxyl group at the terminal; and

[0054] (4) taking 150 mg of the product in step (1) and 3 g of the product in step (3) and dissolving same in deionized water, adjusting the pH value of the solution to 5.5, adding 60 mg of EDC/NHS (a molar ratio of 1:1), then carrying out a reaction at 30° C., pH=5.5 for 24 h, the molar ratio of the amino group to the carboxyl group in the amino-modified hyaluronic acid and PMPC carrying a carboxyl group at the terminal being 1:1.5, after the completion of the reaction, dialyzing and lyophilizing same to obtain 201 mg of the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine.

Embodiment 4

[0055] Testing of the lubricating property of the polymer brush prepared in Embodiment 1 using a Bruker frictional abrasion testing machine. The specific method is as follows:

[0056] (1) formulating a solution of polymer brush in PBS with a concentration of 0.2 mg/mL, and a solution of hyaluronic acid in PBS with a concentration of 1 mg/mL; and

[0057] (2) taking fresh pig joints, removing the articular cartilage (avoiding any damage to the cartilage), cutting the cartilage into a size of about 1.5 cm×1 cm, at the same time using a trephine to take a cartilage nail with a diameter of 6 mm, and treating same with 0.5% pancreatin for 3 h, then washing same with serum to imitate cartilage with osteoarthritic lesions, selecting a cartilage sheet with a flat surface and fixing same on a pathological grade glass slide, and then fixing the pathological grade glass slide with immobilized cartilage to a petri dish with a diameter of 10 mm, the cartilage nails being fixed by the built-in fixture of the frictional abrasion testing machine. Subsequently, 0.2 mg/mL of the solution of polymer brush in PBS, 1 mg/mL of the solution of hyaluronic acid in PBS and a natural bovine synovial fluid are added to measure the friction coefficient under different lubrication media, and the measurement is repeated 3 times. The results are summarized in FIG. 3.

[0058] It can be seen from FIG. 3 that compared with hyaluronic acid and natural bovine synovial fluid, the zwitterionic polymer brush based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine has a better lubricating effect, and the friction coefficient significantly decreases.

Embodiment 5

[0059] Evaluation of the effect of the polymer brush prepared in Embodiment 1 on delaying the progress of osteoarthritis using a rat osteoarthritis model. The specific method is as follows:

[0060] taking 24 SPF grade SD rats, which are male and 4 months old; after passing the quarantine inspection, anesthetizing all animals with zoletil (40 mg/kg) and xylazine (10 mg/kg), opening the rat's right knee joint capsule and cutting the anterior cruciate ligament with a scalpel; randomly dividing the rats for which a model was successfully established and which survived into 4 groups of A, B, C, and D, with 6 rats per group; performing intra-articular injection into the animals' right knee at day 14, day 21, day 35 and day 42 of rearing after the operation, wherein the rats in group A were not treated and used as a blank control group, the rats in group B were injected with 100 μL of PBS into the articular cavity, the rats in group C were injected with 100 μL of hyaluronic acid solution into the articular cavity, and the rats in group D were injected with 100 μL of a solution of the zwitterionic polymer brush (HPM) based on hyaluronic acid and 2-methacryloyloxyethyl phosphorylcholine into the articular cavity.

Testing:

[0061] (1) observing and recording the general clinical conditions of the animals every day, and measuring the animals' body weight once a week;

[0062] (2) on day 49 after the operation, sacrificing the rats and taking out their articular cartilage, taking the articular cartilage from the left untreated joint of all the animals, and taking the left articular cartilage of 3 animals as the reference groups;

[0063] (3) decalcifying and dehydrating the obtained rat joints, cutting out 15 μm×15 μm cartilage samples, embedding same in paraffin, and then staining same with toluidine blue and safranin O; and staining type II collagen with an anti-col 2A1 monoclonal antibody.

[0064] The results are summarized in FIG. 4.

[0065] It can be seen from FIG. 4 that the articular cartilage is obviously thinned during rearing after the anterior cruciate ligament of the SD rats is removed, the surface of the cartilage is rough, uneven, and discontinuous, and local cartilage detaches, showing obvious symptoms of osteoarthritis, and indicating that the osteoarthritis model is successfully established, while the surface of the articular cartilage of healthy rats is flat and smooth. Compared with the hyaluronic acid and PBS treatment groups, the roughness and unevenness of the cartilage surface in the HPM treatment group are significantly improved, and the OARSI score is also significantly reduced, indicating that under the action of HPM, abrasion and degradation of cartilage are effectively improved, which thus effectively slows the pathological progress of osteoarthritis.

Embodiment 6

[0066] Investigation of the interaction of the polymer brush (HPM) prepared in Embodiment 1 with synovial fluid and proteins in cartilage using a VP-ITC isothermal calorimetric titrator (MICROCAL, USA). The specific method is as follows:

[0067] (1) formulating 0.2 mg/mL of a solution of HPM in PBS and 0.2 mg/mL of an acetic acid solution (the concentration of acetic acid being 5 mg/mL), 7 mg/mL and 11 mg/mL of solutions of albumin and γ-globulin in PBS, 0.5 mg/mL of a type II collagen solution (5 mg/mL of acetic acid as a solvent), and 0.5 mg/mL of a fibronectin solution (PBS as a solvent);

[0068] (2) titrating the blank curves of the solvent PBS and 5 mg/mL of acetic acid using a VP-ITC isothermal calorimetric titrator, as the experimental backgrounds;

[0069] (3) titrating the interaction curves of HPM and various proteins, separately, and obtaining the binding constants of the interaction between HPM and various proteins after subtracting the experimental background corresponding to the previous step.

[0070] The results are summarized in Table 1. As shown in Table 1, the binding constants of HPM with albumin, globulin, fibronectin and type II collagen vary greatly, wherein the binding constants with type II collagen and fibronectin have an order of magnitude difference from the binding constants with albumin and globulin, showing a selective interaction with the proteins, such that HPM can selectively bind to the proteins in cartilage, and the way by which same binds to the cartilage is naturally harmless. While hyaluronic acid does not show such selectivity. (HA is hyaluronic acid)

TABLE-US-00001 TABLE 1 Binding constants of zwitterionic polymer brush with major proteins in synovial fluid and cartilage Fibronectin Type II collagen γ-globulin Albumin HA 0.072 1.17 3220 9040 HPM 8.15 × 10.sup.6 4.46 × 10.sup.6 265 70.3

[0071] The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention should all be equivalent replacement methods and should all be included in the scope of protection of the present invention.