Bone and joint biological super-lubricating copolymer and preparation method thereof

Abstract

The present invention relates to the technical field of biomedical materials, and specifically provides a bone and joint biological super-lubricating copolymer and a preparation method thereof. Dopamine is modified with methacrylic anhydride to prepare dopamine methacrylamide, and then a positively charged poly (2-ethyl-2-oxazoline) with good hydrophilicity and hydration lubrication performance and dopamine methacrylamide with adhesion performance are subjected to free radical polymerization to form a super-lubricating copolymer. The positively charged super-lubricating copolymer has good cell affinity, can promote the attachment, growth and differentiation of chondrocytes, and is beneficial to tissue repair and regeneration. The positively charged super-lubricating copolymer has high hydrophilicity, excellent hydration ability and lubricating properties, can adhere to the joint surface, absorb surrounding water molecules to form a hydration layer, effectively reduce surface friction and wear, and the unique adhesion properties of the copolymer and the phenol structure can effectively scavenge free radicals to achieve an anti-inflammatory effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular provides a bone and joint biological super-lubricating copolymer and a preparation method thereof. Background Art

[0002] Osteoarthritis is a common, chronic joint disease that can cause pain, stiffness, and limited joint function. Osteoarthritis often leads to reduced or disrupted joint lubrication, accelerating the wear of articular cartilage. Lubricating fluid in normal joints reduces friction and wear on joint surfaces, protecting articular cartilage from damage. However, in patients with osteoarthritis, the quality and quantity of lubricating fluid may be impaired, leading to increased friction across the joints and exacerbating arthritis symptoms. Therefore, maintaining good joint lubrication can help slow the progression of osteoarthritis and alleviate symptoms.

[0003] Research on biolubricants has garnered significant attention in the field of bone and joint health. Currently, improving lubrication between cartilages in osteoarthritis is primarily achieved by injecting exogenous lubricants, such as hyaluronic acid and chitosan, into the joint cavity. However, this approach requires frequent injections, which can further damage the articular cartilage and exacerbate the disease.

[0004] Therefore, in view of the limitations of current traditional lubricants in the fields of artificial joints and cartilage repair, it is crucial to develop materials with biocompatibility and excellent lubrication properties. Summary of the Invention

[0005] The present invention aims to address the problems of existing traditional lubricants and provide a biological super-lubricating copolymer and its preparation method. The bone and joint biological super-lubricating copolymer can be directly modified on pharmaceutical microspheres and has the characteristics of strong adhesion and excellent hydration and lubrication properties.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The bone and joint biological super-lubricating copolymer material comprises dopamine methacrylamide and a positively charged poly (2-ethyl-2-oxazoline) copolymer; the structural formula of the super-lubricating copolymer is shown in the following formula I:

[0008]

[0009] In formula I: x=8~9, y=6~7.

[0010] The preparation process of the bone and joint biological super lubricating copolymer is as follows:

[0011]

[0012] In formula II: MeOTs is methyl p-toluenesulfonate, and MA is methacrylic anhydride.

[0013] The preparation method of dopamine methacrylamide comprises: mixing sodium tetraborate and sodium hydroxide solution under nitrogen protection, rapidly adding dopamine hydrochloride at room temperature, and simultaneously dropping a mixed solution of methacrylic anhydride and tetrahydrofuran to carry out a grafting reaction to obtain modified dopamine methacrylamide;

[0014] The mass ratio of dopamine hydrochloride to sodium tetraborate is 1:(2-5), the volume ratio of methacrylic anhydride to tetrahydrofuran is 1:(2-8), the grafting reaction time is 12-24 hours, the reaction temperature is 20-30° C., and the pH needs to be controlled at 2-3.

[0015] The preparation method of the positively charged poly (2-ethyl-2-oxazoline) copolymer is as follows: 2-ethyl-2-oxazoline is mixed with an acetonitrile solution, and methyl p-toluenesulfonate and N-(3-dimethylaminopropyl) methacrylamide are added to carry out a grafting reaction to obtain the positively charged poly (2-ethyl-2-oxazoline).

[0016] The mass ratio of 2-ethyl-2-oxazoline to methyl p-toluenesulfonate is (5-15):1, the molar ratio of methyl p-toluenesulfonate to N-(3-dimethylaminopropyl)methacrylamide is 1:(1-20), the grafting reaction time is 12-24 hours, and the reaction temperature is 60-80°C.

[0017] The poly (2-ethyl-2-oxazoline) of the present invention has a positive charge, N + The introduction of ions gives the polymer a positive charge, which offers advantages in the treatment of osteoarthritis, including: ① Anti-inflammatory effect: Positively charged polymer materials can adsorb and neutralize negatively charged inflammatory mediators, thereby reducing the inflammatory response and helping to alleviate pain and discomfort in osteoarthritis patients. ② Removal of degradation substances: Positively charged polymers can adsorb and remove degradation products and metabolites within the joint, helping to maintain a clean and stable environment within the joint. ③ Promotion of tissue growth and repair: Positively charged polymers have good cell affinity and can promote the attachment, growth, and differentiation of bone and cartilage cells, facilitating tissue repair and regeneration. Furthermore, this positively charged polymer is highly hydrophilic, possessing excellent hydration capacity and lubricating properties. It can adhere to the surface of the joint, absorb surrounding water molecules to form a hydration layer, and effectively reduce surface friction and wear. When copolymerized with dopamine methacrylamide, dopamine's unique adhesion properties and phenol structure can effectively scavenge free radicals, achieving an anti-inflammatory effect.

[0018] The modified dopamine methacrylamide and positively charged poly (2-ethyl-2-oxazoline) are mixed with N, N-dimethylformamide solvent and subjected to free radical polymerization to obtain a biological super-lubricating copolymer.

[0019] The mass ratio of dopamine methacrylamide to positively charged poly (2-ethyl-2-oxazoline) is 1: (1-10), the free radical polymerization reaction time is 12-48 hours, and the reaction temperature is 60-80°C.

[0020] After the first grafting reaction is completed, the resulting product is extracted, precipitated, and vacuum-dried to obtain the final product; after the second grafting reaction is completed, the resulting product is precipitated and vacuum-dried to obtain the final product; after the third free radical polymerization reaction is completed, the resulting product is dialyzed and freeze-dried in sequence.

[0021] The present invention also provides a method for preparing a bone and joint biological super-lubricating copolymer solution, wherein a copolymer obtained by polymerizing modified dopamine methacrylamide and positively charged poly (2-ethyl-2-oxazoline) is dissolved in the good solvent to obtain the bone and joint biological super-lubricating copolymer solution.

[0022] The preferred good solvent is Tris-HCl buffer. The concentration of the bone and joint biological super-lubricating copolymer in the good solvent is 1-10 mg / mL.

[0023] The present invention also provides the use of a bone and joint biological super-lubricating copolymer and a copolymer solution in modifying new dosage form microspheres for treating osteoarthritis.

[0024] Another aspect of the present invention:

[0025] The carrier microspheres of the bone and joint biological super-lubricating copolymer material are prepared using a microfluidic device. The preparation method includes the following steps:

[0026] (1) dissolving methacrylated gelatin in phosphate buffer to form solution A as the dispersed phase;

[0027] (2) Liquid paraffin is solution B, serving as the continuous phase;

[0028] (3) Based on the microfluidic platform, the flow rates of the continuous phase and the dispersed phase were set, and methacryloylated gelatin (GelMA) was formed into microspheres under UV cross-linking.

[0029] (4) After washing away the excess oil phase on the surface of the microspheres with anhydrous ethanol and PBS, the microspheres were added to the biological super-lubricating copolymer solution and incubated at 37°C on a shaker for 24 h to successfully modify the copolymer on the surface of the microspheres to form lubricating microspheres.

[0030] The beneficial effects of the present invention compared to the prior art are:

[0031] 1. Dopamine, a key derivative of dihydroxyphenylalanine, inherits the adhesive properties of mussels while also demonstrating potential benefits in bone and joint health. Dopamine methacrylamide (DMA), a dopamine derivative, can be combined with other biomedical materials to play a role in cartilage repair and alleviate conditions like arthritis, offering new solutions for bone and joint health.

[0032] 2. Poly(2-ethyl-2-oxazoline) (POX) is a highly hydrophilic polymer with excellent hydration and lubrication properties. In bone joints, it functions through its hydration-lubrication mechanism. When POX adheres to the joint surface, it absorbs surrounding water molecules to form a hydration layer, effectively reducing surface friction and wear, thereby providing a lubricating effect. Its lubricating properties can reduce friction between bones and cartilage, alleviating the pain and discomfort caused by arthritis.

[0033] 3. The positively charged biological superlubricating copolymer material provided by the present invention can adsorb and neutralize negatively charged inflammatory mediators, has good cell affinity, can promote the growth and differentiation of chondrocytes, thereby reducing inflammatory responses, and its lubricating and adhesive properties effectively reduce the friction of the lubricating microspheres in the joint cavity, helping to alleviate the discomfort of osteoarthritis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The DMA obtained in Example 5 1 H NMR spectrum;

[0035] Figure 2 The POX obtained in Example 5 1 H NMR spectrum;

[0036] Figure 3 The DMA-POX obtained in Example 5 1 H NMR spectrum;

[0037] Figure 4 This is the Fourier transform infrared spectrum of DMA obtained in Example 5;

[0038] Figure 5 This is the Fourier transform infrared spectrum of POX obtained in Example 5;

[0039] Figure 6 This is the Fourier transform infrared spectrum of DMA-POX obtained in Example 5;

[0040] Figure 7 FTIR spectra of blank microspheres and lubricated microspheres obtained in Example 6;

[0041] Figure 8Surface charge test results of DMA-POX, GelMA, and GelMA@DMA-POX;

[0042] Figure 9 The friction coefficient test results of PBS, GelMA, and GelMA@DMA-POX;

[0043] Figure 10 The histological therapeutic effects of PBS, GelMA, and GelMA@DMA-POX on rats with osteoarthritis;

[0044] Figure 11 This is the imaging detection effect of PBS, GelMA, and GelMA@DMA-POX on rats with osteoarthritis. DETAILED DESCRIPTION

[0045] The present invention provides a bone and joint biological super-lubricating copolymer, the structural formula of which is shown in Formula I below:

[0046]

[0047] In the present invention, x represents the graft ratio of dopamine methacrylamide, and y represents the graft ratio of positively charged poly (2-ethyl-2-oxazoline).

[0048] In the present invention, x is preferably 8-9, and y is preferably 6-7.

[0049] In the present invention, the preparation method of the copolymer of dopamine methacrylamide and positively charged poly (2-ethyl-2-oxazoline) as a biological superlubricating material comprises the following steps:

[0050] The present invention mixes sodium tetraborate and sodium hydroxide solution under a nitrogen atmosphere, quickly adds dopamine hydrochloride at room temperature, and simultaneously drops a mixed solution of methacrylic acid and tetrahydrofuran to carry out a first grafting reaction to obtain dopamine methacrylamide (denoted as DMA). The mass ratio of sodium hydroxide to sodium tetraborate is 1:(2-5), more preferably 1:(2-3), the amount of dopamine hydrochloride added is 2-10g, more preferably 4-6g, the volume ratio of methacrylic anhydride to tetrahydrofuran is 1:(2-8), more preferably 1:(4-6), the temperature of the first grafting reaction is preferably 20-30°C, more preferably 25-28°C, the time of the first grafting reaction is preferably 12-24h, more preferably 18-24h, and the pH at the end of the first grafting reaction needs to be controlled at 2-3, more preferably 2-2.5.

[0051] After the first grafting reaction is completed, the resulting product is extracted, precipitated, and vacuum dried to obtain the final product, dopamine methacrylamide. In the present invention, the product obtained from the first grafting reaction is preferably extracted with ethyl acetate, dried using anhydrous magnesium sulfate, and precipitated using petroleum ether. The vacuum drying time is preferably 12 to 36 hours.

[0052] The present invention mixes 2-ethyl-2-oxazoline with an acetonitrile solution, adds methyl p-toluenesulfonate and N-(3-dimethylaminopropyl)methacrylamide to carry out a second-step grafting reaction to obtain positively charged poly(2-ethyl-2-oxazoline). The mass ratio of the 2-ethyl-2-oxazoline to the methyl p-toluenesulfonate is (5-15):1, more preferably (8-12):1, and more preferably 10:1; the volume ratio of the 2-ethyl-2-oxazoline to the anhydrous acetonitrile solvent is 1:1, the molar ratio of the methyl p-toluenesulfonate to the N-(3-dimethylaminopropyl)methacrylamide is 1:(1-20), more preferably 1:(4-6), the reaction temperature of the second-step grafting reaction is preferably 60-80°C, more preferably 70-80°C, and the reaction time of the second-step grafting reaction is preferably 12-24 hours, more preferably 18-24 hours.

[0053] After the second grafting reaction is completed, the resulting product is precipitated and vacuum dried to obtain the final product, namely, positively charged poly(2-ethyl-2-oxazoline). The product obtained from the second grafting reaction is preferably precipitated with cold anhydrous ether, and the product is preferably vacuum dried. The vacuum drying time is preferably 12 to 36 hours.

[0054] After obtaining dopamine methacrylamide and positively charged poly(2-ethyl-2-oxazoline), the modified dopamine methacrylamide and positively charged poly(2-ethyl-2-oxazoline) are dissolved in an N,N-dimethylformamide solvent. The preferred mass ratio of dopamine methacrylamide to positively charged poly(2-ethyl-2-oxazoline) is 1:(1-10), and more preferably 1:(2-4). The amount ratio of the initiator azobisisobutyronitrile to the solvent N,N-dimethylformamide is preferably 1 mg:(1-5) mL, and more preferably 1 mg:(1-3) mL. The temperature of the third step free radical polymerization reaction is preferably 60-80° C., and more preferably 70-80° C. The time of the third step free radical polymerization reaction is preferably 12-48 hours, and more preferably 24-48 hours. The third step free radical polymerization reaction is preferably carried out under nitrogen protection.

[0055] After the third step of free radical polymerization reaction, the obtained lubricating copolymer (denoted as DMA-POX) is preferably dialyzed and freeze-dried in sequence; the dialysis solvent is preferably deionized water, the molecular weight cutoff of the dialysis bag is preferably 2000-8000 kDa, the dialysis time is preferably 2-5 days, more preferably 3 days; the freeze-drying temperature is preferably -70--80°C, and the freeze-drying time is preferably 12-36 hours.

[0056] The present invention also provides a method for preparing the bone and joint super-lubricating copolymer solution described in the above scheme, comprising the following steps: dissolving the copolymer obtained from dopamine methacrylamide and positively charged poly (2-ethyl-2-oxazoline) in the good solvent to obtain a biological super-lubricating solution. The present invention has no specific requirements for the dissolution conditions of the copolymer, and it can be stirred at room temperature until it is completely dissolved.

[0057] In the present invention, the good solvent is preferably Tris-HCl buffer, the pH value of the Tris-HCl buffer is preferably 8-9, and the concentration of the copolymer in the bone and joint super-lubricating copolymer solution is preferably (0.1-10) mg / mL, more preferably (1-8) mg / mL, and further preferably 4 mg / mL.

[0058] The present invention also provides the use of the above-mentioned super-lubricating copolymer solution in preparing new dosage form microspheres for treating osteoarthritis. The method comprises the following steps:

[0059] The bone and joint biological super-lubricating copolymer material is dissolved in the good solvent to obtain a super-lubricating solution, and gelatin microspheres are prepared based on microfluidic technology. Preferably, the microspheres are added to the super-lubricating solution, and the amount ratio of the microspheres to the super-lubricating solution is preferably (4-10) mg / mL to obtain super-lubricating microspheres. The bone and joint biological super-lubricating copolymer solution and super-lubricating microspheres provided by the present invention have good biocompatibility, strong interfacial adhesion and excellent water lubrication properties, and have excellent application prospects in the treatment of osteoarthritis.

[0060] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.

[0061] Example 1

[0062] Step 1: Under an inert gas atmosphere, add 4g of sodium hydroxide, 10g of sodium tetraborate and 100mL of deionized water to a 250mL three-necked flask to fully dissolve them. At room temperature, quickly add 3g of dopamine hydrochloride, and at the same time, drop a mixed solution of 5mL of methacrylic anhydride and 25mL of tetrahydrofuran into the round-bottom flask, and then adjust the pH value of the solution to 8.5 with sodium hydroxide. At the same time, monitor the pH value of the solution every 2h and control it at 8.5. After reacting at room temperature for 12h, adjust the pH of the reaction mixture to 2 with hydrochloric acid. After the reaction is complete, extract with ethyl acetate, dry with anhydrous magnesium sulfate, precipitate with petroleum ether, and vacuum dry the product to finally obtain a white solid powder, i.e., dopamine methacrylamide DMA.

[0063] Step 2: Under an inert gas atmosphere, 5 g of 2-ethyl-2-oxazoline, 1 g of methyl p-toluenesulfonate and 40 mL of anhydrous acetonitrile were added to the reaction bottle, placed in an 80 ° C oil bath, stirred for 12 hours, and then cooled to room temperature. 3.5 g of N-(3-dimethylaminopropyl) methacrylamide was added dropwise via a syringe and stirred for 12 hours at room temperature until the reaction was completed. The excess solvent was removed by rotary evaporation, and the remaining product was precipitated with anhydrous ether and dried in vacuo to obtain the final product, i.e., positively charged poly (2-ethyl-2-oxazoline) POX.

[0064] Step 3: Under an inert atmosphere, dissolve 0.5 g of DMA and 2.0 g of POX monomer in 10 mL of N,N-dimethylformamide (DMF). Quickly add 6 mg of azobisisobutyronitrile (AIBN) as an initiator. Stir at 70°C for 24 hours to allow the reaction to occur, observing the viscosity change. After the reaction, dialyze against deionized water for 3 days and freeze-dry for 1 day to obtain the final product, DMA-POX.

[0065] Example 2

[0066] Step 1: Under an inert gas atmosphere, add 4g of sodium hydroxide, 10g of sodium tetraborate and 100mL of deionized water to a 250mL three-necked flask to fully dissolve them. At room temperature, quickly add 3g of dopamine hydrochloride, and at the same time, drop a mixed solution of 5mL of methacrylic anhydride and 25mL of tetrahydrofuran into the round-bottom flask, and then adjust the pH value of the solution to 8.5 with sodium hydroxide. At the same time, monitor the pH value of the solution every 2h and control it at 8.5. After reacting at room temperature for 12h, adjust the pH of the reaction mixture to 2 with hydrochloric acid. After the reaction is complete, extract with ethyl acetate, dry with anhydrous magnesium sulfate, precipitate with petroleum ether, and vacuum dry the product to finally obtain a white solid powder, i.e., dopamine methacrylamide DMA.

[0067] Step 2: Under an inert gas atmosphere, 5 g of 2-ethyl-2-oxazoline, 1 g of methyl p-toluenesulfonate and 40 mL of anhydrous acetonitrile were added to the reaction bottle, placed in an 80 ° C oil bath, stirred for 12 hours, and then cooled to room temperature. 4.5 g of N-(3-dimethylaminopropyl) methacrylamide was added dropwise via a syringe and stirred at room temperature for 12 hours. The reaction was completed, and the excess solvent was removed by rotary evaporation. The remaining product was precipitated with anhydrous ether and dried in vacuo to obtain the final product, i.e., positively charged poly (2-ethyl-2-oxazoline) POX.

[0068] Step 3: Under an inert atmosphere, dissolve 0.5 g of DMA and 2.0 g of POX monomer in 10 mL of N,N-dimethylformamide (DMF). Quickly add 6 mg of azobisisobutyronitrile (AIBN) as an initiator. Stir at 70°C for 24 hours to allow the reaction to occur, observing the viscosity change. After the reaction, dialyze against deionized water for 3 days and freeze-dry for 1 day to obtain the final product, DMA-POX.

[0069] Example 3

[0070] Step 1: Under an inert gas atmosphere, add 4g of sodium hydroxide, 10g of sodium tetraborate and 100mL of deionized water to a 250mL three-necked flask to fully dissolve them. At room temperature, quickly add 3g of dopamine hydrochloride, and at the same time, drop a mixed solution of 5mL of methacrylic anhydride and 25mL of tetrahydrofuran into the round-bottom flask, and then adjust the pH value of the solution to 8.5 with sodium hydroxide. At the same time, monitor the pH value of the solution every 2h and control it at 8.5. After reacting at room temperature for 12h, adjust the pH of the reaction mixture to 2 with hydrochloric acid. After the reaction is complete, extract with ethyl acetate, dry with anhydrous magnesium sulfate, precipitate with petroleum ether, and vacuum dry the product to finally obtain a white solid powder, i.e., dopamine methacrylamide DMA.

[0071] Step 2: Under an inert gas atmosphere, 5 g of 2-ethyl-2-oxazoline, 1 g of methyl p-toluenesulfonate and 40 mL of anhydrous acetonitrile were added to the reaction bottle, placed in an 80 ° C oil bath, stirred for 12 hours, and then cooled to room temperature. 5.5 g of N-(3-dimethylaminopropyl) methacrylamide was added dropwise via a syringe and stirred at room temperature for 12 hours. The reaction was completed, and the excess solvent was removed by rotary evaporation. The remaining product was precipitated with anhydrous ether and dried in vacuo to obtain the final product, i.e., positively charged poly (2-ethyl-2-oxazoline) POX.

[0072] Step 3: Under an inert atmosphere, dissolve 0.1 g of DMA and 0.4 g of POX monomer in 10 mL of N,N-dimethylformamide (DMF). Quickly add 6 mg of azobisisobutyronitrile (AIBN) as an initiator. Stir at 70°C for 24 hours to allow the reaction to occur, observing the viscosity change. After the reaction, dialyze against deionized water for 3 days and freeze-dry for 1 day to obtain the final product, DMA-POX.

[0073] Example 4

[0074] Step 1: Under an inert gas atmosphere, add 4g of sodium hydroxide, 10g of sodium tetraborate and 100mL of deionized water to a 250mL three-necked flask to fully dissolve them. At room temperature, quickly add 3g of dopamine hydrochloride, and at the same time, drop a mixed solution of 5mL of methacrylic anhydride and 25mL of tetrahydrofuran into the round-bottom flask, and then adjust the pH value of the solution to 8.5 with sodium hydroxide. At the same time, monitor the pH value of the solution every 2h and control it at 8.5. After reacting at room temperature for 12h, adjust the pH of the reaction mixture to 2 with hydrochloric acid. After the reaction is complete, extract with ethyl acetate, dry with anhydrous magnesium sulfate, precipitate with petroleum ether, and vacuum dry the product to finally obtain a white solid powder, i.e., dopamine methacrylamide DMA.

[0075] Step 2: Under an inert gas atmosphere, 5 g of 2-ethyl-2-oxazoline, 1 g of methyl p-toluenesulfonate and 40 mL of anhydrous acetonitrile were added to the reaction bottle, placed in an 80 ° C oil bath, stirred for 12 hours, and then cooled to room temperature. 5.5 g of N-(3-dimethylaminopropyl) methacrylamide was added dropwise via a syringe and stirred at room temperature for 12 hours. The reaction was completed, and the excess solvent was removed by rotary evaporation. The remaining product was precipitated with anhydrous ether and dried in vacuo to obtain the final product, i.e., positively charged poly (2-ethyl-2-oxazoline) POX.

[0076] Step 3: Under an inert atmosphere, dissolve 0.3 g of DMA and 1.2 g of POX monomer in 10 mL of N,N-dimethylformamide (DMF). Quickly add 6 mg of azobisisobutyronitrile (AIBN) as an initiator. Stir at 70°C for 24 hours to allow the reaction to occur, observing the viscosity change. After the reaction, dialyze against deionized water for 3 days and freeze-dry for 1 day to obtain the final product, DMA-POX.

[0077] Example 5

[0078] Step 1: Under an inert gas atmosphere, add 4g of sodium hydroxide, 10g of sodium tetraborate and 100mL of deionized water to a 250mL three-necked flask to fully dissolve them. At room temperature, quickly add 3g of dopamine hydrochloride, and at the same time, drop a mixed solution of 5mL of methacrylic anhydride and 25mL of tetrahydrofuran into the round-bottom flask, and then adjust the pH value of the solution to 8.5 with sodium hydroxide. At the same time, monitor the pH value of the solution every 2h and control it at 8.5. After reacting at room temperature for 12h, adjust the pH of the reaction mixture to 2 with hydrochloric acid. After the reaction is complete, extract with ethyl acetate, dry with anhydrous magnesium sulfate, precipitate with petroleum ether, and vacuum dry the product to finally obtain a white solid powder, i.e., dopamine methacrylamide DMA.

[0079] Step 2: Under an inert gas atmosphere, 5 g of 2-ethyl-2-oxazoline, 1 g of methyl p-toluenesulfonate and 40 mL of anhydrous acetonitrile were added to the reaction bottle, placed in an 80 ° C oil bath, stirred for 12 hours, and then cooled to room temperature. 5.5 g of N-(3-dimethylaminopropyl) methacrylamide was added dropwise via a syringe and stirred at room temperature for 12 hours. The reaction was completed, and the excess solvent was removed by rotary evaporation. The remaining product was precipitated with anhydrous ether and dried in vacuo to obtain the final product, i.e., positively charged poly (2-ethyl-2-oxazoline) POX.

[0080] Step 3: Under an inert atmosphere, dissolve 0.5 g of DMA and 2.0 g of POX monomer in 10 mL of N,N-dimethylformamide (DMF). Quickly add 6 mg of azobisisobutyronitrile (AIBN) as an initiator. Stir at 70°C for 24 hours to allow the reaction to occur, observing the viscosity change. After the reaction, dialyze against deionized water for 3 days and freeze-dry for 1 day to obtain the final product, DMA-POX.

[0081] Figure 1 The DMA obtained in Example 5 1 H NMR spectroscopy, Figure 2 The POX obtained in Example 5 1 H NMR spectroscopy, Figure 3 The DMA-POX obtained in Example 5 1 H NMR spectroscopy, Figure 4 This is the Fourier infrared spectrum of DMA obtained in Example 5, Figure 5 This is the Fourier infrared spectrum of POX obtained in Example 5, Figure 6 This is the Fourier transform infrared spectrum of DMA-POX obtained in Example 5. Comparing the infrared spectrum of the super lubricating copolymer DMA-POX with the two reactants, we can see that DMA-POX has characteristic peaks of amide groups such as OH, C=O, NH and CN, as well as the peak at 3110 cm -1 The stretching vibration peak of Ar-H at 1370 cm-1 According to the above figure, it can be determined that the obtained product has the target structure.

[0082] Table 1 is a summary of the performance characterization results of the copolymers obtained in Examples 1-5

[0083]

[0084] Example 6

[0085] Step 1: Prepare a dual-channel microfluidic injection pump. The external phase is the continuous phase, and 5mL of liquid paraffin is added. The dispersed phase is a 10wt% methacrylated gelatin solution and a LAP photoinitiator with a mass of 0.5wt% of the methacrylated gelatin. Place syringes containing 5mL of aqueous solution and 5mL of oily solution horizontally on the microinjection pump, and connect the needles to the inlet of the chip using a capillary. Set the push speed to 1μL / s for the aqueous channel and 5μL / s for the oily channel. Start the device. Under the shear force of the continuous phase, the dispersed phase solution will be sheared into dispersed droplets. The droplets collected at the outlet pipe will polymerize into microspheres after being exposed to UV light (365nm) for 20 seconds. The collected microspheres are washed three times with anhydrous ethanol and PBS to obtain gelatin microspheres (blank microspheres, i.e., GelMA microspheres).

[0086] Step 2: The DMA-MPC copolymer (4 mg / mL) prepared in Example 5 was dispersed in Tris-HCl buffer (pH = 8.5, 1 M), and then 16 mg of gelatin microspheres were added to the above solution (4 mL). The mixture was shaken for 24 h in the dark and rinsed with deionized water to obtain GelMA@DMA-POX lubricating microspheres. The surface charge of the microspheres was tested and Fourier infrared spectroscopy was used to characterize the successful modification of the lubricating copolymer onto the microsphere surface.

[0087] The surface charge test results of blank microspheres, super lubricating copolymers and lubricating microspheres are as follows Figure 7 As shown, according to Figure 7 The results show that the blank microspheres are negatively charged, and the superlubricating copolymer is positively charged due to the presence of positively charged poly (2-ethyl-2-oxazoline). The superlubricating microspheres are successfully modified with the copolymer and are positively charged. Figure 8 The Fourier transform infrared spectrum results show that the FT-IR comparison of blank microspheres and lubricated microspheres is 2923, 1640, 1548, 1100 cm -1 The peaks corresponding to the CH stretching vibration, C=O stretching vibration, NH vibration and CN stretching vibration on the amide group are shown in Figure 2. It can be clearly seen that the amide peak intensity of the lubricated microspheres is significantly higher than that of the blank microspheres, and the peak at 3200 cm -1 The OH stretching vibration peak is strong, which verifies that the superlubricating copolymer is successfully modified onto the surface of the microspheres.

[0088] Example 7

[0089] This embodiment is an evaluation of the tribological properties of a biological super-lubricating copolymer, and its preparation steps are as follows:

[0090] Friction coefficient tests were performed using phosphate buffered saline (PBS), blank microspheres (10 mg / mL), and the superlubricating microspheres (10 mg / mL) prepared in Example 6 as the test samples. Tribological tests were performed using a universal materials testing instrument in reciprocating mode, with PBS as the control group. Each test was repeated three times to ensure data accuracy.

[0091] The friction coefficient test results are as follows Figure 9 As shown, according to Figure 9 The results show that compared with the PBS group, the friction coefficients of GelMA microspheres and DMA-POX-modified GelMA microspheres are reduced. Among them, the microspheres modified with DMA-POX have better lubrication properties, and the friction coefficient is reduced to about 0.018, which is 40% lower than that of the PBS group and 33% lower than that of the GelMA group.

[0092] Example 8

[0093] This example is a rat animal osteoarthritis treatment verification

[0094] Six-week-old male Sprague-Dawley rats were anesthetized by intraperitoneal injection and immobilized in the supine position. The knee joint hair was shaved, and the skin around the knee joint was disinfected with povidone-iodine solution. A small incision was made on the medial side of the knee joint under aseptic conditions. The subcutaneous tissue and muscle layers were dissected layer by layer to expose the joint cavity. The anterior cruciate ligament was transected and the medial meniscus was removed. The joint cavity was then closed and sutured layer by layer. In the sham-operated group, only the muscle layer was incised to expose the joint cavity; no other procedures were performed. Postoperatively, 50,000 to 100,000 units of the antibiotic penicillin sodium were injected daily for three days to prevent wound infection. Four weeks after surgery, the rats were randomly divided into four groups (n=6 each): sham-operated group (Sham), model group (PBS), GelMA-treated group, and GelMA@DMA-POX-treated group. Every two weeks, 150 μL of PBS was injected into the joint cavity of the model group rats; 150 μL of blank GelMA microspheres and GelMA@DMA-POX lubricating microspheres described in Example 6 were injected into the lubricant-treated group. The treatment course lasted eight weeks, and after completion of treatment, the knee joints were removed for radiographic and histological evaluation.

[0095] Figure 10 The therapeutic effects of PBS, GelMA blank microspheres and GelMA@DMA-POX lubricated microspheres on osteoarthritis rats. Figure 10The results showed that in the normal group, the surface of the articular cartilage was smooth and intact; in the modeling group, the synovial membrane proliferated and infiltrated into the joint cavity, the cartilage surface was severely defective, the subchondral bone was damaged, the chondrocytes and ECM were destroyed, and the staining was reduced. There was no significant difference between the GeMA group and the PBS group. In contrast, the cartilage destruction in the GelMA@DMA-POX lubricating microsphere group was improved to varying degrees, and the depth of cartilage erosion was significantly reduced. According to the results of safranin staining, the glycosaminoglycan deposition was analyzed, showing that the GelMA@DMA-POX lubricating microsphere group had the highest glycosaminoglycan (GAG) content, and the progression of osteoarthritis was inhibited.

[0096] Figure 11 Micro-CT test results of PBS, GelMA blank microspheres and GelMA@DMA-POX lubricated microspheres on osteoarthritis rats. Figure 11 The results showed that compared with the smooth bone joints of the normal group, the modeling group had the most severe osteoarthritis symptoms, the most osteophytes were formed and the volume was the largest, and the subchondral bone was severely damaged and had bone defects. There was no significant difference between the blank microsphere GelMA group and the modeling group. In the microsphere treatment group with lubricating copolymer, the volume of osteophytes was significantly reduced, the subchondral bone defects were reduced, and it had the best inhibitory effect on osteoarthritis.

[0097] The results of the above examples show that the present invention, through molecular structure design, selects dopamine modified with methacrylic anhydride, which has adhesive properties, and poly(2-ethyl-2-oxazoline), which has excellent hydration and lubrication properties, to synthesize a super-lubricating copolymer material, effectively forming a lubricating layer, reducing friction and wear on the bone joint surface, and thus providing a lubricating effect. The super-lubricating copolymer modified with gelatin microspheres has lubricating properties that can reduce friction between bones and cartilage, alleviating pain and discomfort caused by arthritis. The DMA-POX lubricating copolymer provided by the present invention is a new type of biolubricating material with broad application prospects in the treatment of osteoarthritis.

[0098] The foregoing description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications should be considered within the scope of protection of the present invention. Therefore, the scope of protection of this patent application shall be determined by the appended claims, and the specification may be used to interpret the claims.

Claims

1. A bone and joint biological super lubricating copolymer, characterized in that: The copolymer is prepared by free radical polymerization of dopamine methacrylamide with adhesiveness and poly (2-ethyl-2-oxazoline) with positive charge. The structural formula of the biological super-lubricating copolymer is shown in Formula I: In formula I: x=8~9, y=6~7.

2. The bone and joint biological super lubricating copolymer according to claim 1, characterized in that: The preparation method of the dopamine methacrylamide comprises the following steps: mixing sodium tetraborate and sodium hydroxide solution under nitrogen protection, adding dopamine hydrochloride at room temperature, and simultaneously dripping a mixed solution of methacrylic anhydride and tetrahydrofuran to carry out a grafting reaction to obtain the dopamine methacrylamide.

3. The bone and joint biological super lubricating copolymer according to claim 2, characterized in that: The mass ratio of dopamine hydrochloride to sodium tetraborate is 1:2-5, the volume ratio of methacrylic anhydride to tetrahydrofuran is 1:2-8, the grafting reaction temperature is 20-30° C., the reaction time is 12-24 hours, and the pH needs to be controlled at 2-3.

4. The bone and joint biological super lubricating copolymer according to claim 1, characterized in that: The preparation method of the positively charged poly(2-ethyl-2-oxazoline) comprises: mixing 2-ethyl-2-oxazoline with an acetonitrile solution, adding methyl p-toluenesulfonate and N-(3-dimethylaminopropyl)methacrylamide to carry out a grafting reaction to obtain the positively charged poly(2-ethyl-2-oxazoline).

5. The bone and joint biological super lubricating copolymer according to claim 4, characterized in that: The mass ratio of the 2-ethyl-2-oxazoline to methyl p-toluenesulfonate is 5 to 15:1, and the molar ratio of the methyl p-toluenesulfonate to N-(3-dimethylaminopropyl) methacrylamide is 1:1 to 20; the temperature of the grafting reaction is 60 to 80° C., and the reaction time is 12 to 24 hours.

6. A method for preparing a bone and joint biological super-lubricating copolymer according to claim 1, characterized in that: The preparation method comprises the following steps: mixing dopamine methacrylamide and positively charged poly (2-ethyl-2-oxazoline) with an N,N-dimethylformamide solvent, and performing free radical polymerization to obtain a biological super-lubricating copolymer.

7. The method for preparing the bone and joint biological super lubricating copolymer according to claim 6, characterized in that: The mass ratio of the dopamine methacrylamide to the positively charged poly (2-ethyl-2-oxazoline) is 1:1-10, the reaction temperature of the free radical polymerization reaction is 60-80° C., and the reaction time is 12-48 hours.

8. A bone and joint biological super lubricating copolymer solution, characterized in that: The copolymer solution is prepared by dissolving the copolymer as claimed in claim 1 in a good solvent to obtain a bone and joint biological super-lubricating copolymer solution.

9. The bone and joint biological super-lubricating copolymer solution according to claim 8, characterized in that: The good solvent is Tris-HCl buffer; the concentration of the bone and joint biological super-lubricating copolymer in the solvent is 1-10 mg / mL.

10. The bone and joint biological super-lubricating copolymer or the bone and joint biological super-lubricating copolymer solution as claimed in claim 1 or 8 is used for preparing pharmaceutical microspheres for treating osteoarthritis.

Images