A type of drug-resistant bone cement containing polypeptides

Abstract

The application discloses drug-resistant bacteria-resistant bone cement containing polypeptides and belongs to the technical field of medical materials. The novel bone cement obtained by adding polypeptides with drug-resistant bacteria resistance activity has good antibacterial property and biocompatibility, can effectively kill drug-resistant bacteria, and the drug-resistant bacteria are methicillin-resistant Staphylococcus aureus.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a bone cement containing polypeptides that resists drug-resistant bacteria. Background Technology

[0002] Bone cement is an important medical material in orthopedic treatment, widely used in surgeries such as bone defect repair and joint prosthesis placement. Among these, acrylic bone cement, especially polymethyl methacrylate (PMMA) bone cement, is gradually becoming the gold standard due to its superior mechanical properties and injectability. PMMA bone cement typically consists of a solid phase and a liquid phase. The solid phase includes PMMA powder, contrast agent, free radical initiator, and antibiotics, while the liquid phase often consists of methyl methacrylate (MMA), accelerator, and polymerization inhibitor. Because drug-resistant bacteria have developed resistance to antibiotics, adding antibiotics to PMMA is unlikely to have a bactericidal effect. Therefore, the development of novel bone cements capable of combating drug-resistant bacteria is an urgent clinical need. Summary of the Invention

[0003] To address the shortcomings of the existing technology, this invention provides a drug-resistant bone cement containing polypeptides. By adding polypeptides with anti-drug-resistant bacterial activity, this invention yields a novel bone cement exhibiting excellent antibacterial properties and biocompatibility.

[0004] Natural peptide LPRDA has poor stability. This invention modifies the N and C ends of the natural peptide (N-terminal acetylation, C-terminal amidation) to obtain LPRDA with only C-terminal amidation, LPRDA with only N-terminal acetylation, and LPRDA with both N-terminal acetylation and C-terminal amidation, in addition to the natural peptide.

[0005] The present invention relates to a peptide-containing anti-drug-resistant bone cement, comprising a solid phase agent and a liquid phase agent; wherein the solid phase agent comprises a peptide having anti-drug-resistant bacterial activity.

[0006] The polypeptide with anti-drug-resistant bacterial activity is selected from one or more of the following structural polypeptides:

[0007] .

[0008] Furthermore, the mass of the polypeptide with anti-drug-resistant bacterial activity is 0.5%-10% of the total mass of the solid agent, preferably 2%-5%.

[0009] The solid phase agent includes PMMA, dibenzoyl peroxide (BPO), barium sulfate, and the polypeptide with anti-drug-resistant bacteria activity.

[0010] The liquid phase agent includes MMA and N,N-dimethylaniline (DMPT).

[0011] The basic formulations and ratios of the solid and liquid phase agents can be followed according to conventional proportions. For example, in 1g of bone cement, the solid phase agent is 525 mg PMMA, 13 mg benzoyl peroxide (BPO), 100 mg barium sulfate, and polypeptides; the liquid phase agent is 379 mL MMA and 7.5 mL N,N-dimethyl-p-toluidine (DMPT).

[0012] The bone cement of this invention has good anti-drug-resistant bacterial activity and biocompatibility.

[0013] The drug-resistant bacteria is methicillin-resistant Staphylococcus aureus, abbreviated as MRSA.

[0014] The beneficial effects of this invention are reflected in:

[0015] (1) Compared with the current technology, the present invention adds a polypeptide LPRDA with both ends blocked, which avoids the use of antibiotics and can also effectively kill drug-resistant bacteria.

[0016] (2) The bone cement obtained by using novel antimicrobial peptides has good resistance to drug-resistant bacteria and good biocompatibility. Attached Figure Description

[0017] Figure 1 This is the mass spectrum of the polypeptide of the present invention.

[0018] Figure 2 This is a chromatogram of C-terminal amidation and N-terminal acetylation of LPRDA in an immersion experiment.

[0019] Figure 3 These are the mass spectra corresponding to the C-terminal amidation and N-terminal acetylation LPRDA chromatographic peaks in the immersion experiment.

[0020] Figure 4 The antibacterial rate corresponds to the C-terminal amidation and N-terminal acetylation LPRDA chromatographic peaks in the immersion experiment.

[0021] Figure 5 This is the result of the hemolysis rate test. Detailed Implementation

[0022] The technical solution of the present invention will now be described with reference to specific embodiments. The described embodiments are only some, not all, of the embodiments of the present invention.

[0023] Example 1: Synthesis of Polypeptides

[0024] Each polypeptide is artificially synthesized using the following method:

[0025] This invention employs solid-phase peptide synthesis (SPPS) to prepare peptides. First, 0.2 mmol of Rink amide resin is fully swollen in a v / v mixed solvent of N,N-dimethylformamide (DMF) / dichloromethane (DCM) to enhance its reactivity. Subsequently, a 20% piperidine / DMF (v / v) solution is added to remove the Fmoc protecting groups, exposing free amino sites on the resin. A stepwise condensation reaction is performed according to a standard Fmoc protection strategy: in each round, 4 equivalents of Fmoc-protected amino acid, 5 equivalents of ethyl 2-oxime (Oxyma), and 5 equivalents of N,N'-diisopropylcarbodiimide (DIC) are added as a coupling system, and the reaction is carried out in anhydrous DMF for 20 minutes. After each coupling, the resin was thoroughly washed with DMF and DCM (3 times each) to remove unreacted reagents and byproducts. This process was repeated until the target amino acid sequence was fully assembled. After polypeptide synthesis, a 10 mL cleavage system of trifluoroacetic acid / water / phenol / triisopropylsilane = 88 / 5 / 5 / 2 (v / v / v / v) was slowly added, and the reaction was carried out at room temperature for 3 hours to simultaneously deprotect the side chains and cleave the polypeptide from the resin.

[0026] After the reaction, the reaction solution was concentrated by purging with nitrogen, and the peptide was precipitated with cold diethyl ether. The crude product was collected by centrifugation to obtain the crude peptide. Subsequently, the crude product was dissolved in 10% acetonitrile / water solution and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The purified product was then freeze-dried to obtain the target peptide.

[0027] This method can be used to synthesize C-terminal amidated LPRDA.

[0028] The synthesis of C-terminal amidated and N-terminal acetylated LPRDA requires, after the synthesis of C-terminal amidated LPRDA and the completion of amino acid sequence assembly, the addition of acetic anhydride / NN-diisopropylethylamine (DIEA) / DMF = 1 / 1 / 8 (v / v / v) in 10 mL volume, followed by reaction for 10 minutes, and then cleavage.

[0029] To synthesize LPRDA, it is necessary to use Wang resin instead of the C-terminal amidation synthesis. After the resin swells, without removing Fmoc, the first amino acid is added, followed by the addition of 8 equivalents of Fmoc to protect the amino acid, 10 equivalents of Oxyma, 8 equivalents of DIC, and 1 equivalent of 4-(Dimethylamino)pyridine (DMAP). The reaction is carried out for 8 hours. After the first and third amino acids are added, the resin is blocked twice with acetic anhydride / DIEA / DMF = 1 / 1 / 8 (v / v / v), each time for 10 minutes (to block unreacted OH groups on the resin).

[0030] N-terminal acetylated LPRDA was synthesized. After the amino acid sequence was assembled based on the synthesized LPRDA, acetic anhydride / DIEA / DMF = 1 / 1 / 8 (v / v / v) was added in 10 mL, and the reaction was carried out for 10 minutes. Then, cleavage was performed.

[0031] Example 2: Investigation of antibacterial activity and serum stability

[0032] The antibacterial activity was evaluated as follows: Methicillin-resistant Staphylococcus aureus (MRSA) was revived and passaged to restore its activity. Bone cement samples were synthesized the day before the experiment and soaked in 3 mL of distilled water for 18 hours to remove unpolymerized PMMA monomers from the surface. On the day of the experiment, a concentration of (0.5 × 10⁻⁶) was prepared. 8 Bacterial suspensions of CFU / mL were prepared. Each bone cement sample was immersed in 1 mL of bacterial suspension and 1 mL of pure water / peptide stock solution and incubated in a CO2 incubator at 37°C for 6 hours. The samples were then removed and gently rinsed with 100 mL of distilled water to remove non-adherent bacteria. The samples were then placed in 5 mL of physiological saline and sonicated for 3 minutes to remove adhering bacteria. Subsequently, the bone cement samples were removed, and the remaining liquid was retained. 40 μL of this liquid was taken and diluted 100-fold, thoroughly mixed, and then 40 μL of the diluted bacterial solution was taken and evenly spread onto petri dishes. The petri dishes were incubated in a CO2 incubator at 37°C for 24 hours, and colony counts were performed to calculate the surface inhibition rate of bone cement containing different concentrations of peptides. Each bone cement sample was tested five times repeatedly, and the average value was taken.

[0033]

[0034] In the above formula for calculating the antibacterial rate, A represents the number of colonies on the blank group bone cement culture dish, while B represents the number of colonies on the polypeptide bone cement culture dish.

[0035]

[0036] Based on the antibacterial data, at the same concentration, the antibacterial rates of all four peptides, whether LPRDA or LPRDA modified at both ends of the peptides, were above 90%. This indicates that all four peptides are effective against bacteria at a concentration of 250 μg / mL.

[0037] Serum stability test: 200 µL of human serum was transferred to a 1.5 mL tube. 100 µg of peptide was dissolved in 100 µL of water and mixed with the serum, then incubated at 37 °C. At different time points (0 h, 1 h, 3 h), 100 μL of each sample was transferred to another 1.5 mL tube, and 300 μL of 90% acetonitrile aqueous solution (cooled after adding 1% formic acid) was added to precipitate plasma proteins. The tubes were cooled on ice for 45 min, and then centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was transferred to a third 1.5 mL tube, and acetonitrile was removed by nitrogen purging. Finally, 400 µL of H2O was added, and the tubes were centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was analyzed by RP-HPLC. The peak area at each time point was measured, and the residual peptide amount of each sample was determined by comparing it with the peak area at 0 h.

[0038]

[0039] In the above formula for calculating the dissociation rate, A represents the peak area at 0 hours, and B represents the peak area at different times. This is because, in the experiment, formic acid needs to be added at the 0-hour sampling point to terminate the reaction, but the mixing and stirring process lasts for more than ten seconds, during which the peptide may be completely degraded. Therefore, after chromatographic analysis of the 0-hour sample, there may be instances where the characteristic peaks of the raw material are not detected; this time point data is replaced with ---.

[0040]

[0041] The data in the table show that the natural peptide LPRDA has poor stability, almost completely decomposing after 3 hours. C-terminal blocking results in even worse stability (chromatographic analysis of the 0-hour sample immediately after mixing showed no characteristic peaks of the raw material, suggesting complete degradation of the peptide during mixing). N-terminal blocking LPRDA exhibits better stability, with a decomposition rate of less than 20% after 3 hours. N- and C-terminal blocking LPRDA demonstrates the best stability, with a decomposition rate of less than 10% after 3 hours. Since the antibacterial rates of the four peptides are similar, we selected N- and C-terminal blocking LPRDA for our subsequent research.

[0042] Example 3: Investigation of antibacterial properties and hemolysis rate

[0043] Bone cement contains a solid phase agent and a liquid phase agent, and its composition is shown in the table below:

[0044]

[0045] Preparation method of antibacterial bone cement: The solid phase agent and liquid phase agent are mixed according to the ratio in the table above, and formed into a cylinder with a height of 12 mm and a diameter of 6 mm in a mold. Sample 1 in the table is conventional PMMA bone cement, the amount of peptide added in Sample 2 is 2%, and the amount of peptide added in Sample 3 is 5%.

[0046] Immersion experiment: Peptides were added to bone cement at a solid phase concentration of 5%, followed by immersion in 1 mL of pure water. The water was changed every 24 hours. Samples were removed from the original EP tubes and transferred to new EP tubes containing fresh buffer. The samples from the first day were analyzed by high-performance liquid chromatography (HPLC). Specific HPLC parameters were as follows: a C18 column (4.6 × 250 mm) was used; mobile phase A consisted of an aqueous solution containing 0.1% trifluoroacetic acid (TFA), and mobile phase B was an acetonitrile solution containing 0.08% TFA. Linear gradient elution was used, increasing phase B from 10% to 90% within 30 minutes. The flow rate was set to 1.0 mL / min, and the detection wavelength was 254 nm. The resulting chromatographic solution was then subjected to mass spectrometry. Surface antibacterial rate experiments were performed on the samples obtained from the first day.

[0047] The polymerization process of bone cement is exothermic, and the successful release and maintenance of biological activity of incorporated peptides remains uncertain. Some peptides are difficult to release from bone cement. For example, nisin cannot be released from polymethyl methacrylate (PMMA) bone cement; relevant evidence can be found in patent CN119455109A. (The chromatogram is shown below.) Figure 3 and mass spectrum Figure 4 It was learned that C-terminal amidation and N-terminal acetylation of LPRDA peptides can be extracted from bone cement, and their antibacterial rate... Figure 5 It can be concluded that the released peptides retain their antibacterial properties.

[0048] Hemolysis rate determination: According to GB / T 16886 standard (State Administration for Market Regulation of China, 2022), bone cement extract was placed in a sterile centrifuge tube, and 200 μL of 2% rabbit erythrocyte suspension was added. Additionally, 200 μL of 2% rabbit erythrocyte suspension was added to 1.8 mL of physiological saline and pure water as negative and positive controls, respectively. After incubation at 37°C, 5% CO2 for 1 hour, centrifugation was performed at 3000 rpm for 5 minutes. The optical density (OD) of the supernatant was measured at 545 nm using a spectrophotometer. Each group of bone cement samples was tested three times. Figure 5 Calculate the hemolysis rate (HR) using the following formula:

[0049]

[0050] Among them, OD T1 It is the optical density (OD) of the experimental group solution. N1 The optical density (OD) of the negative control is... P The optical density is for the positive control. As shown in the table below, a hemolysis rate of less than 5% is considered to indicate that no hemolysis has occurred. This indicates that bone cement containing C-terminal amidation and N-terminal acetylation of LPRDA is similar to commercially available bone cement and does not cause hemolysis.

[0051] .

Claims

1. A bone cement containing polypeptides to resist drug-resistant bacteria, characterized in that: The drug-resistant bone cement comprises a solid phase agent and a liquid phase agent; The solid phase agent includes polypeptides with anti-drug-resistant bacteria activity; The polypeptide with anti-drug-resistant bacterial activity is selected from one or more of the following structural polypeptides: 。 2. The drug-resistant bone cement according to claim 1, characterized in that: The mass of the polypeptide with anti-drug-resistant bacterial activity is 0.5%-10% of the total mass of the solid agent.

3. The drug-resistant bone cement according to claim 1, characterized in that: The mass of the polypeptide with anti-drug-resistant bacterial activity is 2%-5% of the total mass of the solid agent.

4. The drug-resistant bone cement according to claim 1, 2 or 3, characterized in that: The structure of the polypeptide with anti-drug-resistant bacterial activity is shown below: 。

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