Methacrylic acid 3, 4-dichloro-5-hydroxy-5H-furan-2-ketone ester modified antibacterial bone cement

Abstract

The invention belongs to the field of biomedical materials, and discloses an antibacterial bone cement modified by methacrylic acid 3, 4-dichloro-5-hydroxy-5H-furan-2-ketone ester and a preparation method of the antibacterial bone cement modified by methacrylic acid 3, 4-dichloro-5-hydroxy-5H-furan-2-ketone ester. According to the invention, 3, 4-dichloro-5-hydroxy-5H-furan-2-ketone (DHF) is covalently connected with methacrylic acid (MAA) to prepare a novel monomer, namely methacrylic acid 3, 4-dichloro-5-hydroxy-5H-furan-2-ketone ester (DHF-MAA), which is an oily liquid easily dissolved in methyl methacrylate (MMA). The obtained DHF-MAA is used for liquid phase modification of bone cement, and the DHF-MAA modified antibacterial bone cement is finally prepared and is novel NLBC. The DHF-MAA modified bone cement is excellent in antibacterial activity, mechanical strength and biocompatibility, and the excellent performance of the DHF-MAA modified bone cement shows that the DHF-MAA modified bone cement has a wide prospect in clinical application.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to an antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate. Background Art

[0002] In orthopedic surgery, polymethyl methacrylate (PMMA) bone cement is a commonly used biomedical material and is widely used in surgical procedures such as joint replacement and vertebroplasty. However, its insufficient antibacterial activity has always been a concern. Staphylococcus aureus is the most common pathogen in orthopedics, which may cause postoperative infection, delayed healing and even surgical failure, seriously affecting the patient's prognosis and quality of life. To meet this severe challenge, the traditional improvement method is to mix antibiotics into bone cement to make antibiotic-loaded bone cement (ALBC). However, this method has problems such as explosive release of antibiotics and bacterial resistance, which limits its further use in clinical applications. In this context, the research and development of new antibacterial bone cement has become one of the hot topics in the field of orthopedics.

[0003] Non-leaching bone cement (NLBC) combines antibacterial substances with the PMMA skeleton by covalent bonding or physical adsorption, so that the antibacterial substances are not released in the body, but sterilized by contact, thus avoiding the above problems. At present, the mainstream NLBC mainly includes two categories: quaternary ammonium compounds and heterocyclic compounds. Among them, as a type of heterocyclic compound, furan derivatives have been widely studied due to their good antibacterial activity. However, there is currently no NLBC with excellent antibacterial activity, mechanical strength and biocompatibility. It is usually necessary to compromise in other aspects to give priority to a certain performance, which limits the further research and application of NLBC. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a new antibacterial bone cement that performs well in all aspects. The present invention covalently links 3,4-dichloro-5-hydroxy-5H-furan-2-one (DHF) and methacrylic acid (MAA) to prepare a new monomer, namely 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate (DHF-MAA), which is an oily liquid that is easily soluble in methyl methacrylate (MMA). The obtained DHF-MAA is used for liquid phase modification of bone cement, and finally a DHF-MAA-modified antibacterial bone cement is prepared, which is a new type of NLBC. The bone cement modified by DHF-MAA has excellent antibacterial activity, mechanical strength and biocompatibility, and its excellent performance shows that it has broad prospects in clinical applications.

[0005] To achieve the purpose of the present invention, the antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate of the present invention is obtained by adding DHF-MAA into bone cement.

[0006] Furthermore, in some embodiments of the present invention, the bone cement is polymethyl methacrylate (PMMA) bone cement.

[0007] Furthermore, in some embodiments of the present invention, the polymethyl methacrylate bone cement comprises a solid phase and a liquid phase, and the solid-liquid ratio is 1.5-2.5:1.

[0008] Furthermore, in some embodiments of the present invention, the concentration of DHF-MAA in the bone cement is greater than or equal to 2.5%, for example, greater than or equal to 5%, and for example, greater than or equal to 7.5%, preferably greater than or equal to 9%, and more preferably greater than or equal to 10%, wherein the concentration of DHF-MAA in the bone cement is calculated according to the mass ratio of DHF-MAA in the liquid phase of the bone cement.

[0009] Further, in some embodiments of the present invention, the preparation method of DHF-MMA is:

[0010] (1) Add DHF and MAA to dichloromethane, then add EDCI and DMAP, and stir at room temperature;

[0011] (2) After the reaction of step (1) is completed, dichloromethane is removed, the remaining product is dissolved in ethyl acetate, extracted, the organic layer is dried, the solvent is removed, and then the crude product is purified to obtain the final product DHF-MAA.

[0012] Furthermore, in some embodiments of the present invention, in step (1) of the method for preparing DHF-MMA, the volume molar ratio of dichloromethane, DHF and MAA is 40-60 ml: 25-35 mmol: 30-40 mmol.

[0013] Furthermore, in some embodiments of the present invention, in step (1) of the method for preparing DHF-MMA, the molar ratio of MAA, EDCI and DMAP is 5-7:5-7:1.

[0014] Furthermore, in some embodiments of the present invention, the removal of dichloromethane in step (2) of the method for preparing DHF-MMA is carried out using a rotary evaporator at 35-45°C.

[0015] Furthermore, in some embodiments of the present invention, the extraction in step (2) of the method for preparing DHF-MMA is carried out using hydrochloric acid and sodium carbonate solution.

[0016] Furthermore, in some embodiments of the present invention, in step (2) of the method for preparing DHF-MMA, the organic layer is dried with anhydrous sodium sulfate.

[0017] Furthermore, in some embodiments of the present invention, the removal of the solvent in step (2) of the method for preparing DHF-MMA is carried out using a rotary evaporator at 50°C.

[0018] Furthermore, in some embodiments of the present invention, in step (2) of the method for preparing DHF-MMA, the crude product is purified by silica gel column chromatography.

[0019] Furthermore, in some embodiments of the present invention, the specific steps of adding DHF-MAA to bone cement are as follows: adding DHF-MAA to the liquid phase of bone cement, shaking it thoroughly, mixing it with the solid phase of bone cement and stirring it evenly, then pouring the mixture into a mold, and after curing, demolding it to obtain a bone cement sample.

[0020] The DHF-MMA modified bone cement sample prepared by the present invention was tested for mechanical properties and antibacterial activity, and an leaching solution was prepared for biocompatibility evaluation. The compressive strength of the bone cement meets the ISO5833 standard, the antibacterial activity is strong, the antibacterial activity of the modified bone cement containing 10% DHF-MMA is 100%, and the biocompatibility is good without hemolytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the synthetic route of DHF-MMA prepared by the present invention;

[0022] Figure 2 is a hydrogen nuclear magnetic resonance spectrum of the final product DHF-MAA synthesized in Example 1 of the present invention;

[0023] Figure 3 is the analysis result of the prepared sample using Fourier transform infrared spectrometer (FT-IR) in Example 2 of the present invention;

[0024] Figure 4 is the compressive strength test result of the bone cement sample in Example 3 of the present invention;

[0025] Figure 5 is the antibacterial activity test result of the bone cement sample in Example 4 of the present invention;

[0026] Figure 6 This is the hemolysis rate measurement result of the bone cement sample obtained in Example 5 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. It should be understood that the following description is only used to explain the present invention and is not intended to limit the present invention.

[0028] When amount, concentration or other value or parameter is expressed as range, preferred range or a series of upper preferred value and lower preferred value limit range, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when disclosing range "1 to 5", described range should be interpreted as including range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc. When numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0029] Singular forms include plural references unless the context clearly indicates otherwise. "Optional" or "either" means that the subsequently described event or incident may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0030] The indefinite articles "a" and "an" before the elements or components of the present invention have no limitation on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0031] In addition, the descriptions of the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" described below mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0032] The polymethyl methacrylate (PMMA) bone cement involved in the embodiments of the present invention was purchased from Heraeus Medical AG of Germany, and includes a solid phase and a liquid phase with a solid-liquid ratio of 2:1.

[0033] Example 1

[0034] The synthesis method of DHF-MMA is as follows:

[0035] DHF (30 mmol, 1.0 eq) and MAA (36 mmol, 1.2 eq) were added to 50 ml of dichloromethane. Then, EDCI (36 mmol, 1.2 eq) and DMAP (6 mmol, 0.2 eq) were added and stirred at room temperature for 3 hours to ensure that the reaction was complete. After the reaction was completed, dichloromethane was removed at 40 ° C using a rotary evaporator. The remaining product was dissolved in 350 ml of ethyl acetate, extracted with 1M hydrochloric acid and 5% sodium carbonate solution, and the organic layer was dried with 200 g of anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator at 50 ° C. The crude product was purified by silica gel column chromatography to obtain the final product DHF-MAA (4.0 g, 16.9 mmol). 1 HNMR (400MHz, CDCl3) δ7.02-6.94(m,1H),6.28(p,J=0.9Hz,1H),5.88-5.78(m,1H),2.01(ddd,J=2.6,1.6,1.0Hz,3H).

[0036] The preparation method of DHF-MMA modified bone cement is as follows: Table 1 below shows the bone cement formulas of different concentrations. First, DHF-MAA is added to the liquid phase of bone cement (the concentration of DHF-MAA is calculated according to the mass ratio in the liquid phase). After fully shaking, mix with the solid phase of bone cement and stir evenly. Then pour the mixture into a mold. After curing, demolding obtains a cylindrical bone cement sample with a diameter of (6.0±0.1) mm and a height of (12.0±0.1) mm. These samples were tested for mechanical properties and antibacterial activity, and an leaching solution was prepared for biocompatibility evaluation. The polymethyl methacrylate (PMMA) bone cement involved in the present invention was purchased from Heraeus Medical AG, Germany, including a solid phase and a liquid phase, with a solid-liquid ratio of 2:1.

[0037] Table 1 Bone cement formulas of different concentrations

[0038]

[0039] Example 2

[0040] The bone cement powder prepared according to the formula in Table 1 was thoroughly mixed with potassium bromide at a ratio of about 1:50, ground and pressed into tablets. The prepared samples were analyzed using Fourier transform infrared spectrometer (FT-IR) (see Appendix Figure 3 ).

[0041] Example 3

[0042] Mechanical strength test is as follows: The bone cement samples (5 per group) prepared according to the formula in Table 1 were polished with 1000 mesh sandpaper until the top and bottom surfaces were parallel. The samples were incubated at 37°C and 100% humidity for 24 hours. At room temperature, the deformation-load curves of the bone cement samples were drawn using a material testing machine (MTS809, Bose, USA) at a loading rate of 20 mm / min. The compressive strength and elastic modulus of each bone cement sample were calculated from these curves. All operations were carried out in accordance with the requirements and standards of ISO 5833.

[0043] Example 4

[0044] The antibacterial activity of DHF-MAA modified bone cement was evaluated as follows: Staphylococcus aureus (ACTT25923) was revived and passaged to restore its activity. Bone cement samples were synthesized one day before the experiment and soaked in 3 mL of distilled water for 18 h to remove unpolymerized DHF-MAA monomers on the surface. On the day of the experiment, a concentration of (0.5 × 10 8 ) CFU / mL of bacterial suspension. Each bone cement sample was soaked in 1 ml of bacterial suspension and incubated in a CO2 incubator at 37°C for 6 hours. The sample was then removed and gently rinsed with 100 ml of distilled water to remove non-adherent bacteria. The sample was placed in 5 ml of normal saline and ultrasonically treated for 3 minutes to remove adherent bacteria. Subsequently, the bone cement sample was removed, and the remaining liquid was retained, 40 microliters was drawn from it and diluted 100 times, and after thorough mixing, 40 microliters was drawn from the diluted bacterial solution and evenly spread on the culture dish. The culture dish was incubated in a CO2 incubator at 37°C for one day, and the colony count was performed to calculate the surface inhibition rate of bone cement containing different concentrations of DHF-MAA. Each concentration of bone cement was tested five times.

[0045]

[0046] In the above antibacterial rate calculation formula, A represents the number of colonies on the culture dish of the control group bone cement, and B represents the number of colonies on the culture dish of the different concentrations of DHF-MAA bone cement groups. Figure 5 .

[0047] Example 5

[0048] The hemolysis rate was determined as follows:

[0049] 1. To prepare a 2% rabbit erythrocyte suspension, collect arterial blood from male New Zealand rabbits and centrifuge it in a test tube at 1000 rpm for 15 minutes to remove plasma and other fluids. Resuspend the precipitated erythrocytes with saline and centrifuge again to remove the supernatant to obtain higher purity. Finally, add saline to prepare a 2% rabbit erythrocyte suspension and store it at 4°C.

[0050] 2. Hemolysis rate test, first soak the commercially available Heraeus bone cement and the bone cement modified with DHF-MAA in Example 1 of the present invention in 3 ml of normal saline for 1 hour to prepare an extract. Then, each extract was mixed with a 2% rabbit red blood cell suspension at a ratio of 9:1 and incubated in an incubator at 37°C for 1 hour. Finally, the mixture was centrifuged at 3000 rpm for 5 minutes, and the optical density (OD) of the supernatant was measured using a spectrophotometer. At the same time, the OD values ​​of the negative control group (normal saline) and the positive control group (distilled water) were measured at the same ratio to calculate the hemolysis rate of each group of bone cement extracts.

[0051]

[0052] In the above hemolysis rate calculation formula, OD T Represents the OD value of the experimental group, OD N represents the OD value of the negative control group (normal saline), OD P Represents the OD value of the positive control group (distilled water). See the attached test results. Figure 6 .

[0053] Those skilled in the art will easily understand that the above descriptions are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate, characterized in that: The antibacterial bone cement modified by 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate is obtained by adding DHF-MAA into bone cement.

2. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 1, characterized in that: The bone cement is polymethyl methacrylate bone cement.

3. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 2, characterized in that: The polymethyl methacrylate bone cement comprises a solid phase and a liquid phase, and the solid-liquid ratio is 1.5-2.5:

1.

4. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 1, characterized in that: The concentration of DHF-MAA in the bone cement is greater than or equal to 2.5%, for example greater than or equal to 5%, for example greater than or equal to 7.5%, preferably greater than or equal to 9%, more preferably greater than or equal to 10%, wherein the concentration of DHF-MAA in the bone cement is calculated according to the mass ratio of DHF-MAA in the liquid phase of the bone cement.

5. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 1, characterized in that: The preparation method of the DHF-MMA is: (1) Add DHF and MAA to dichloromethane, then add EDCI and DMAP, and stir at room temperature; (2) After the reaction of step (1) is completed, dichloromethane is removed, the remaining product is dissolved in ethyl acetate, extracted, the organic layer is dried, the solvent is removed, and then the crude product is purified to obtain the final product DHF-MAA.

6. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 5, characterized in that: In step (1) of the preparation method of DHF-MMA, the volume molar ratio of dichloromethane, DHF and MAA is 40-60 ml: 25-35 mmol: 30-40 mmol.

7. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 5, characterized in that: In step (1) of the preparation method of DHF-MMA, the molar ratio of MAA, EDCI and DMAP is 5-7:5-7:

1.

8. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 5, characterized in that: In the step (2) of the preparation method of DHF-MMA, the removal of dichloromethane is carried out using a rotary evaporator at 35-45°C; preferably, in the step (2) of the preparation method of DHF-MMA, the extraction is carried out using hydrochloric acid and sodium carbonate solution; preferably, in the step (2) of the preparation method of DHF-MMA, the organic layer is dried with anhydrous sodium sulfate; preferably, in the step (2) of the preparation method of DHF-MMA, the removal of the solvent is carried out using a rotary evaporator at 50°C.

9. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 5, characterized in that: In step (2) of the preparation method of DHF-MMA, the crude product is purified by silica gel column chromatography.

10. The antibacterial bone cement modified with 3,4-dichloro-5-hydroxy-5H-furan-2-one methacrylate according to claim 1, characterized in that: The specific steps of adding DHF-MAA to bone cement are as follows: adding DHF-MAA to the liquid phase of bone cement, shaking it thoroughly, mixing it with the solid phase of bone cement and stirring it evenly, then pouring the mixture into a mold, and after solidification, demolding it to obtain a bone cement sample.

Images