Double-anchoring enzyme response polypeptide as well as preparation method and application thereof

Abstract

The invention discloses a double-anchoring enzyme response polypeptide as well as a preparation method and application thereof, and the preparation method comprises the following steps: introducing a gelatinase response sequence GPLGV between an antibacterial peptide HHC36 and a tissue repair promoting peptide, and carrying out double-end azido modification to obtain the double-anchoring enzyme response polypeptide. The peptide for promoting tissue repair can be cell adhesion peptide, osteogenic peptide, neurogenic peptide, angiogenic peptide and other functional polypeptides. The polypeptide is double-anchored on the surface of the titanium sheet through double-end azide modification. The activity of the polypeptide is closely related to the degree of freedom of the polypeptide, two ends of the polypeptide are fixed on the surface by azide groups in a non-infection environment, cell membranes cannot be destroyed through model antibacterial mechanisms such as annular holes, and at the moment, osteogenic activity is expressed. In a bacterial infection environment, gelatinase secreted by bacteria can enable antibacterial polypeptide to be unilaterally released to obtain chain segment freedom, and the antibacterial property is improved, so that antibacterial / osteogenesis functional switching of bacterial gelatinase responsiveness is realized, and a functionalized titanium-based implant surface is constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of antibacterial materials on the surface of titanium metal, and particularly relates to a dual-anchored enzyme-responsive polypeptide, a preparation method thereof, and an application thereof. Background Art

[0002] Titanium-based implants are widely used in bone and dental clinics, and the problems of implant failure and revision caused by infection and aseptic loosening need to be solved urgently. At present, the main research direction is to design a surface with osteogenic and antibacterial responsive switching, which can exhibit tissue repair and osteogenic properties in a non-infected environment and antibacterial properties in an infected environment. According to statistics, the infection of bone implants is mainly caused by open fractures or intraoperative infections, mainly occurring in the early stage of implantation. Since the infection has a latent period and the symptoms in the early stage of infection are not very obvious, this allows bacteria to obtain a window period for rapid growth and reproduction in the body. After the formation of a biofilm, antibiotics cannot penetrate the biofilm, resulting in drug resistance and increasing the treatment difficulty. Therefore, an endogenously responsive antibacterial implant is more suitable for clinical use, which can kill bacteria in the early stage of infection, without introducing exogenous equipment, and can achieve sterilization in a timely and efficient manner, which is beneficial to subsequent osteogenic repair. Among them, pH and enzymes belong to the range of internal environmental homeostasis and are not easily affected by external factors. Different bacteria secrete different enzymes. More than 74% of the pathogenic bacteria of implants secrete gelatinase, which is an important factor for microenvironment responsiveness. In addition, the high-molecular coatings corresponding to hyaluronidase and lipase are prone to peeling problems, and coagulase-negative bacteria are an important cause of late infection. Therefore, gelatinase is a more suitable responsive factor.

[0003] As a kind of antibacterial molecule with good biocompatibility, antibacterial peptides are not easy to produce drug resistance and their metabolic pathways are clear. At present, a large number of antibacterial peptides have been used in research. Among them, vancomycin, as a glycopeptide antibiotic, has been commercialized and is commonly used for the treatment of drug-resistant bacteria after the failure of β-lactam antibiotic treatment. In addition, antibacterial peptides are also an important direction for future antibacterial drug research. Therefore, using antibacterial peptides as antibacterial components for surface modification of bone implants can achieve good antibacterial performance. The antibacterial mechanism of antibacterial peptides is related to their hydrophilicity-hydrophobicity and secondary structure. When antibacterial peptides are grafted onto the surface, their antibacterial performance is related to the surface conformation. Therefore, the expression of antibacterial properties can be controlled by changing the surface conformation of the polypeptide.

[0004] In previous studies on combining gelatinase-responsive fragments GPLGV and antimicrobial peptides to achieve responsive antibacterial effects, unilateral grafting was mainly used to construct sequences such as "antimicrobial peptide-responsive enzymatic cleavage fragment" or "antimicrobial peptide-responsive enzymatic cleavage fragment-tissue repair promoting polypeptide" grafted onto the surface. When infection occurs, the enzyme-responsive fragment is enzymatically cleaved, and the antimicrobial peptide becomes free, increasing its antibacterial activity. Or after grafting this polypeptide onto the surface, the antimicrobial peptide is exposed and exhibits antibacterial properties. After infection, in the case of the above two surfaces, on the first type of "antimicrobial peptide-responsive enzymatic cleavage fragment" surface, the antimicrobial peptide is completely free in the body fluid after the first infection, only exerting a single antibacterial effect; on the second type of "antimicrobial peptide-responsive enzymatic cleavage fragment-tissue repair promoting polypeptide" surface, the tissue repair promoting peptide detaches, which is not conducive to tissue repair after infection. In existing studies, the modification of titanium metal surfaces with polypeptides is mainly achieved through chemical grafting by single-terminal specific group modification or physical adsorption under porous structures. There is no study on double-terminal group modification of polypeptides to achieve controllable design of the polypeptide surface conformation while retaining the functional polypeptide on the other side after enzymatic cleavage. Summary of the Invention

[0005] To overcome the deficiencies and drawbacks of the prior art, the primary object of the present invention is to provide a method for preparing a double-anchored enzyme-responsive polypeptide. This preparation method involves introducing a gelatinase-responsive sequence GPLGV (Gly-Pro-Leu-Gly-Val) between an antimicrobial peptide HHC36 (Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg) and a tissue repair promoting peptide, and after double-terminal azide modification, obtaining the double-anchored enzyme-responsive polypeptide. The tissue repair promoting peptide can be other functional polypeptides such as cell adhesion peptides, osteogenic peptides, neurogenic peptides, and angiogenic peptides.

[0006] The second object of the present invention is to provide a double-anchored enzyme-responsive polypeptide prepared by the above preparation method. After grafting this double-anchored enzyme-responsive polypeptide onto the titanium metal surface, it can exhibit excellent antibacterial properties and biocompatibility, with a specifically designed polypeptide sequence.

[0007] The third object of the present invention is to provide an application of the double-anchored enzyme-responsive polypeptide, specifically designed as a modular gelatinase-responsive antibacterial implant surface design.

[0008] The primary object of the present invention is achieved through the following technical solutions:

[0009] A method for preparing a double-anchored enzyme-responsive polypeptide, the double-anchored enzyme-responsive polypeptide is obtained by solid-phase synthesis, and the synthesis sequence is from the carboxyl terminus to the amino terminus. Specifically, it includes the following steps:

[0010] (1) Attachment of the first amino acid: The 2-chlorotrityl chloride resin is soaked in DMF for swelling, and DIEA and the Fmoc-protected carboxyl-terminal amino acid are added. If the carboxyl-terminal amino acid is a non-basic amino acid, then Fmoc-Lys or Fmoc-Arg is added;

[0011] (2) Synthesis of the polypeptide segment: The 20 wt% piperidine DMF solution is used to deprotect the first amino acid attached in step (1), and Kaiser test is performed. If the Kaiser test is positive, the adjacent Fmoc-amino acid, HBTU and DIEA are added for condensation; if the Kaiser test is negative, the corresponding amino acids are added in the order of C→N and step (2) is repeated until the sequence is completed;

[0012] (3) Azide modification: N3-OEG4-CH2CH2COOH, HBTU and DIEA are added for reaction, and finally DMF is added for dissolution;

[0013] (4) The polypeptide powder is obtained after cleavage, purification and lyophilization.

[0014] Preferably, in step (1), the molar volume ratio of the 2-chlorotrityl chloride resin to DMF (N,N-dimethylformamide) is 1 mmol: 5 - 35 ml.

[0015] Preferably, in step (1), the molar volume ratio of the 2-chlorotrityl chloride resin to DMF (N,N-dimethylformamide) is 1 mmol: 15 ml.

[0016] Preferably, in step (1), the molar ratio of DIEA to the 2-chlorotrityl chloride resin is 2 - 6:1; the molar ratio of the Fmoc-protected carboxyl-terminal amino acid to the 2-chlorotrityl chloride resin is 3 - 18:1.

[0017] Preferably, in step (1), the molar ratio of DIEA to the 2-chlorotrityl chloride resin is 3:1; the molar ratio of the Fmoc-protected carboxyl-terminal amino acid to the 2-chlorotrityl chloride resin is 10:1.

[0018] Preferably, in step (2), if the Kaiser test is positive, the molar ratio of the Fmoc-amino acid, HBTU, DIEA to the 2-chlorotrityl chloride resin in step (1) is 2 - 6:1 - 5:3 - 27:1.

[0019] Preferably, in step (2), if the Kaiser test is positive, the molar ratio of the Fmoc-amino acid, HBTU, DIEA to the 2-chlorotrityl chloride resin in step (1) is 3:3:10:1.

[0020] Preferably, the molar ratio of N3-OEG4-CH2CH2COOH, HBTU, DIEA to 2-chlorotrityl chloride resin in step (3) is 2-8:4-8:10-40:1.

[0021] Preferably, the molar ratio of N3-OEG4-CH2CH2COOH, HBTU, DIEA to 2-chlorotrityl chloride resin in step (3) is 6:6:20:1.

[0022] The design of a dual-anchored enzyme-responsive polypeptide provided by the present invention is analyzed as follows:

[0023] (1) During the polypeptide synthesis process, the HHC36-GPLGV-RGDK sequence

[0024] (Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg-Gly-Pro-Leu-Gly-Val-Arg-Gly-Asp-Lys) is prepared by a polypeptide semi-automatic synthesizer;

[0025] (2) The α-NH2 at the amino terminus of the antimicrobial peptide reacts with N3-OEG4-CH2CH2COOH to introduce an azide group;

[0026] (3) If the carboxyl terminus is an acidic amino acid, a basic amino acid such as lysine Lys or arginine Arg needs to be added to introduce an amino group for grafting -OEG4-N3;

[0027] (4) The dual-anchored enzyme-responsive polypeptide sequence is (N3-OEG4)-Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg-Gly-Pro-Leu-Gly-Val-Arg-Gly-Asp-Lys-(OEG4-N3).

[0028] The second object of the present invention is achieved by the following technical solutions:

[0029] A dual-anchored enzyme-responsive polypeptide prepared by the above preparation method.

[0030] Preferably, the end-group modification group of the dual-anchored enzyme-responsive polypeptide is an azide group, and the sequence is -OEG4-N3, and the specific structure is N3-OEG4-antimicrobial peptide-enzyme-responsive sequence-tissue repair promoting peptide-OEG4-N3.

[0031] Preferably, the tissue repair promoting peptide in N3-OEG4-antimicrobial peptide-enzyme-responsive sequence-tissue repair promoting peptide-OEG4-N3 is a cell adhesion peptide, an osteogenic peptide, a neurogenic peptide, and an angiogenic peptide.

[0032] The third object of the present invention is achieved by the following technical solutions:

[0033] Application of a dual-anchored enzyme-responsive polypeptide in the preparation of an antibacterial bone implant.

[0034] The present invention has the following advantages and beneficial effects compared with the prior art:

[0035] The present invention realizes the controllable construction of the surface polypeptide conformation through dual-terminal group modification, and realizes the switching of antibacterial / osteogenic responsiveness. To promote osteogenic repair after infection and avoid the detachment of tissue repair-promoting peptides after enzymatic hydrolysis and the toxicity caused by the long-term exposure of antibacterial peptides, the present invention dual-anchors the polypeptide on the titanium sheet surface through dual-terminal azide modification. The activity of the polypeptide is closely related to its degree of freedom. In a non-infected environment, both ends of the polypeptide are fixed on the surface by azide groups and cannot damage the cell membrane through the antibacterial mechanism of circular pores and other models. At this time, osteogenic activity is expressed. In a bacterial infection environment, the gelatinase secreted by bacteria can cause unilateral release of the antibacterial polypeptide to obtain the degree of freedom of the chain segment, and the antibacterial performance is improved, thereby realizing the bacteria gelatinase-responsive antibacterial / osteogenic functional switching and constructing a functionalized titanium-based implant surface.

[0036] Compared with the prior art, the present invention realizes the controllable regulation of the polypeptide conformation relatively simply through dual-terminal grafting and enzymatic hydrolysis of polypeptide fragments. The surface before enzymatic hydrolysis can promote osteogenic differentiation. When bacteria secrete gelatinase, the surface can switch to an antibacterial conformation. For gelatinase-positive bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa, when the grafting concentration reaches more than 50 μM, the antibacterial rate of the surface reaches more than 95%, and the formation of surface biofilms can be inhibited, effectively realizing the surface antibacterial performance. In addition, compared with the enzyme-responsive surface with unilateral grafting, the biocompatibility of the surface with dual-terminal grafting retains the cell adhesion-promoting peptide after enzymatic hydrolysis, effectively improving the osteogenic effect after infection. Description of the Drawings

[0037] Figure 1 Schematic diagram of the grafting of a polypeptide surface with dual-terminal azide modification;

[0038] Figure 2 Structural formula of the dual-anchored enzyme-responsive polypeptide molecule 1 in Example 1;

[0039] Figure 3 Structural formula of the dual-anchored enzyme-responsive polypeptide molecule 2 in Example 2;

[0040] Figure 4 Structural formula of the dual-anchored enzyme-responsive polypeptide molecule 3 in Example 3;

[0041] Figure 5 Structural formula of the dual-anchored enzyme-responsive polypeptide molecule 4 in Example 4. Detailed Embodiments

[0042] The present invention will be further described in detail below in conjunction with specific embodiments, but the implementation manners of the present invention are not limited thereto. The materials used in the examples of the present invention can all be obtained by commercial purchase.

[0043] Example 1

[0044] This example provides a preparation method of a dual-anchored enzyme-responsive polypeptide. The polypeptide is obtained by solid-phase synthesis, and the synthesis sequence is from the carboxyl terminus to the amino terminus. Among them, the antimicrobial peptide is HHC36, and the bioactive peptide is RGD. Since the carboxyl terminus is aspartic acid Asp, lysine Lys needs to be introduced. This dual-anchored enzyme-responsive polypeptide 1 is (N3-OEG4)-Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg-Gly-Pro-Leu-Gly-Val-Arg-Gly-Asp-Lys-(OEG4-N3).

[0045] The preparation method of the dual-anchored enzyme-responsive polypeptide in this example includes the following steps:

[0046] (1) Swelling of 2-chlorotrityl chloride resin: Add 2-chlorotrityl chloride resin to the reaction tube. Add 15 ml of DMF (N,N-dimethylformamide) per 1 mmol of 2-chlorotrityl chloride resin. After swelling, remove the solvent.

[0047] (2) Connecting the carboxyl terminus: Add 3-fold molar amount of Fmoc-Lys (9-fluorenylmethoxycarbonyl-protected lysine) and 10-fold molar amount of DIEA (N,N-diisopropylethylamine) of 2-chlorotrityl chloride resin and react for 30 min. After dissolving with DMF, soak in methanol for 30 min for blocking.

[0048] (3) Deprotection: Remove the solvent. Add 15 ml of 20 wt% piperidine DMF solution per 2 mmol of 2-chlorotrityl chloride resin and soak for 10 min and repeat once. Remove the solvent.

[0049] (4) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min, and observe the appearance of blue as positive, which proves grafting. Wash it twice with DMF, methanol, and DMF in turn.

[0050] (5) Condensation: In the direction from the carboxyl terminus to the amino terminus, add ortho-Fmoc-amino acid with a molar amount 3 times that of 2-chlorotrityl chloride resin, 3-fold molar amount of HBTU, and 10-fold molar amount of DIEA and react for 30 min. Wash it three times with DMF.

[0051] (6) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min, and observe that it turns colorless, indicating that the reaction is complete;

[0052] (7) Amino-terminal azide modification: Repeat steps (3)-(6), where in step (5), synthesize the middle polypeptide sequence in the order of Lys-Asp-Gly-Arg-Val-Gly-Leu-Pro-Gly-Arg-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Lys, remove the N-terminal Fmoc protecting group and the K-side chain Dde protecting group, add 6-fold molar amount of N3-OEG4-CH2CH2COOH, 6-fold molar amount of HBTU, and 20-fold molar amount of DIEA relative to the 2-chlorotrityl chloride resin, react for 30 min, finally dissolve in DMF to obtain the resin synthesized by solid phase synthesis, and obtain the polypeptide powder after cleavage, purification, and lyophilization, namely the dual-anchored enzyme-responsive polypeptide.

[0053] Modify the surface of the titanium-based material with the dual-anchored enzyme-responsive polypeptide prepared in this example. The specific steps are as follows:

[0054] (1) Place the titanium metal in 5 M sodium hydroxide solution and react at 60 °C for 24 h. After the reaction, add water and ultrasonically clean;

[0055] (2) Prepare a 0.5 mg / mL DBCO-PEG 3.4k -silane coupling agent solution with a volume fraction of 95% ethanol;

[0056] (3) Add 1 mL of the silane coupling agent per square centimeter of the titanium sheet, place it at room temperature for reaction for 24 h, and then place it in an oven at 100 °C for curing;

[0057] (4) Add the 50 μM polypeptide solution to the surface of the titanium metal prepared in step (3) and react;

[0058] (5) Wash the sample in step (4) thoroughly with deionized water and dry it with nitrogen to obtain the surface of the titanium-based material modified with the dual-anchored enzyme-responsive polypeptide.

[0059] The titanium-based material modified with the dual-anchored enzyme-responsive polypeptide can kill 98.68% of Staphylococcus aureus and 99.84% of Pseudomonas aeruginosa. The surface of the modified titanium-based material can promote the proliferation of mouse mesenchymal stem cells, and the CCK-8 value after cell proliferation is 1.08 times that of the pure titanium group. Compared with the enzyme-responsive polypeptide grafted at one end, the CCK-8 corresponding OD value of the dual-anchored group is 1.26 times that of it.

[0060] Example 2

[0061] This example provides a method for preparing a dual-anchored enzyme-responsive polypeptide. The polypeptide is obtained by solid-phase synthesis, and the synthesis sequence is from the carboxyl terminus to the amino terminus. Among them, the antimicrobial peptide is HHC36, the bioactive peptide is YGFGG, and the dual-anchored enzyme-responsive polypeptide 2 is (N3-OEG4)-Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg-Gly-Pro-Leu-Gly-Val-Tyr-Gly-Phe-Gly-Gly-Lys-(OEG4-N3).

[0062] The method for preparing the dual-anchored enzyme-responsive polypeptide in this example includes the following steps:

[0063] (1) Swelling of 2-chlorotrityl chloride resin: Add 2-chlorotrityl chloride resin to a reaction tube. For every 1 mmol of 2-chlorotrityl chloride resin, add 15 ml of DMF (N,N-dimethylformamide). After swelling, remove the solvent.

[0064] (2) Coupling of the carboxyl terminus: Add 3-fold molar amount of Fmoc-Lys (9-fluorenylmethoxycarbonyl-protected lysine) and 10-fold molar amount of DIEA (N,N-diisopropylethylamine) based on 2-chlorotrityl chloride resin and react for 30 min. After dissolving with DMF, soak in methanol for 30 min for blocking.

[0065] (3) Deprotection: Remove the solvent. For every 1 mmol of 2-chlorotrityl chloride resin, add 20 ml of 20 wt% piperidine DMF solution and soak for 10 min, and repeat once. Then remove the solvent.

[0066] (4) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min, and observe the appearance of blue as positive, indicating grafting. Then wash it twice with DMF, methanol, and DMF in sequence.

[0067] (5) Condensation: In the direction from the carboxyl terminus to the amino terminus, add ortho-Fmoc-amino acid with a molar amount 3 times that of 2-chlorotrityl chloride resin, 3-fold molar amount of HBTU, and 9-fold molar amount of DIEA and react for 30 min. Wash it three times with DMF.

[0068] (6) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min, and observe colorless, indicating that the reaction is complete.

[0069] (7) Amino-terminal azide modification: Repeat steps (3)-(6), where in step (5), the intermediate polypeptide sequence is synthesized in the order of Lys-Gly-Gly-Phe-Gly-Tyr-Val-Gly-Leu-Pro-Gly-Arg-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Lys. Remove the N-terminal Fmoc protecting group and the K-side chain Dde protecting group. Add 5-fold molar amount of N3-OEG4-CH2CH2COOH, 5-fold molar amount of HBTU, and 15-fold molar amount of DIEA to 2-chlorotrityl chloride resin and react for 30 min. Finally, dissolve in DMF to obtain the resin synthesized by solid phase method. After cleavage, purification, and lyophilization, the polypeptide powder, namely the dual-anchored enzyme-responsive polypeptide, is obtained.

[0070] Modify the surface of the titanium-based material with the dual-anchored enzyme-responsive polypeptide prepared in this example. The specific steps are as follows:

[0071] (1) Place the titanium metal in a 5 M sodium hydroxide solution and react at 60 °C for 24 h. After the reaction, add water and ultrasonically clean.

[0072] (2) Prepare a 0.5 mg / mL DBCO-PEG 3.4k -silane coupling agent solution with 95% ethanol by mass fraction;

[0073] (3) Add 1 mL of the silane coupling agent per square centimeter of the titanium sheet, place it at room temperature for reaction for 24 h, and then place it in an oven at 100 °C for curing;

[0074] (4) Add the 100 μM polypeptide solution to the surface of the titanium metal prepared in step (3) and carry out the reaction;

[0075] (5) Thoroughly wash the sample in step (4) with deionized water and dry it with nitrogen to obtain the surface of the modified titanium-based material.

[0076] The material modified with the dual-anchored enzyme-responsive polypeptide can kill 96.12% of Staphylococcus aureus and 97.43% of Pseudomonas aeruginosa. The surface of the modified material can promote the differentiation of mouse mesenchymal stem cells. The alizarin red quantification result after 14 days of osteogenic induction is 1.52 times that of the pure titanium group.

[0077] Example 3

[0078] This example provides a method for preparing a dual-anchored enzyme-responsive polypeptide. The polypeptide is obtained by solid-phase synthesis, and the synthesis sequence is from the carboxyl terminus to the amino terminus. Among them, the antimicrobial peptide is LWKKIYRKWKRW, the bioactive peptide is YGFGG, and the dual-anchored enzyme-responsive polypeptide 3 is (N3-OEG4)-Leu-Trp-Lys-Lys-Ile-Tyr-Arg-Lys-Trp-Lys-Arg-Trp-Gly-Pro-Leu-Gly-Val-Tyr-Gly-Phe-Gly-Gly-Lys-(OEG4-N3).

[0079] The method for preparing the dual-anchored enzyme-responsive polypeptide in this example includes the following steps:

[0080] (1) Swelling of 2-chlorotrityl chloride resin: Add 2-chlorotrityl chloride resin to a reaction tube. Add 20 ml of DMF (N,N-dimethylformamide) per 1 mmol of 2-chlorotrityl chloride resin. After swelling, remove the solvent.

[0081] (2) Attachment of the carboxyl terminus: Add 3-fold molar amount of Fmoc-Lys (9-fluorenylmethoxycarbonyl-protected lysine) and 10-fold molar amount of DIEA (N,N-diisopropylethylamine) based on 2-chlorotrityl chloride resin and react for 30 min. After dissolving in DMF, soak in methanol for 30 min for blocking.

[0082] (3) Deprotection: Remove the solvent. Add 20 ml of 20 wt% piperidine DMF solution per 1 mmol of 2-chlorotrityl chloride resin, soak for 10 min and repeat once, then remove the solvent.

[0083] (4) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min. If blue appears, it is positive, indicating grafting. Wash it twice with DMF, methanol, and DMF in sequence.

[0084] (5) Condensation: In the direction from the carboxyl terminus to the amino terminus, add ortho-Fmoc-amino acid with a molar amount 3 times that of 2-chlorotrityl chloride resin, 3-fold molar amount of HBTU, and 10-fold molar amount of DIEA and react for 30 min. Wash it three times with DMF.

[0085] (6) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min. If it is colorless, the reaction is complete.

[0086] (7) Amino-terminal azide modification: Repeat steps (3)-(6), where in step (5), the intermediate polypeptide sequence is synthesized in the order of Lys-Gly-Gly-Phe-Gly-Tyr-Val-Gly-Leu-Pro-Gly-Trp-Arg-Lys-Trp-Lys-Arg-Tyr-Ile-Lys-Lys-Trp-Leu. Remove the N-terminal Fmoc protecting group and the K-side chain Dde protecting group. Add 4-fold molar amount of N3-OEG4-CH2CH2COOH, 4-fold molar amount of HBTU, and 24-fold molar amount of DIEA to the 2-chlorotrityl chloride resin and react for 30 min. Finally, dissolve the resin obtained by solid-phase synthesis in DMF, and obtain the polypeptide powder after cleavage, purification, and lyophilization, namely the dual-anchored enzyme-responsive polypeptide.

[0087] Modify the surface of the titanium-based material with the dual-anchored enzyme-responsive polypeptide prepared in this example. The specific steps are as follows:

[0088] (1) Place the titanium metal in a 5 M sodium hydroxide solution and react at 60 °C for 24 h. After the reaction, add water and ultrasonically clean.

[0089] (2) Prepare a 0.5 mg / mL DBCO-PEG 3.4k -silane coupling agent solution with 95% ethanol by mass fraction;

[0090] (3) Add 1 mL of the silane coupling agent per square centimeter of the titanium sheet, place it at room temperature for reaction for 24 h, and then place it in an oven at 100 °C for curing;

[0091] (4) Add the 50 μM polypeptide solution to the surface of the titanium metal prepared in step (3) and carry out the reaction;

[0092] (5) Thoroughly wash the sample in step (4) with deionized water and dry it with nitrogen to obtain the modified titanium-based material surface.

[0093] The material modified with the dual-anchored enzyme-responsive polypeptide can kill 94.39% of Staphylococcus aureus and 94.27% of Pseudomonas aeruginosa. The surface of the modified material can promote the differentiation of mouse mesenchymal stem cells, and the quantitative result of alizarin red after 14 days of osteogenic induction is 1.22 times that of the pure titanium group.

[0094] Example 4

[0095] The present invention provides a method for preparing a dual-anchored enzyme-responsive polypeptide, which is obtained by solid-phase synthesis in the order from the carboxyl terminus to the amino terminus. Among them, the antimicrobial peptide is KFKWWRMLI, the bioactive peptide is DGEA, and the dual-anchored enzyme-responsive polypeptide 4 is (N3-OEG4)-Lys-Phe-Lys-Trp-Trp-Arg-Met-Leu-Ile-Gly-Pro-Leu-Gly-Val-Asp-Gly-Glu-Ala-Lys-(OEG4-N3).

[0096] The method for preparing the dual-anchored enzyme-responsive polypeptide in this example includes the following steps:

[0097] (1) Swelling of 2-chlorotrityl chloride resin: Put 2-chlorotrityl chloride resin into a reaction tube. Add 20 ml of DMF (N,N-dimethylformamide) per 1 mmol of 2-chlorotrityl chloride resin. After swelling, remove the solvent.

[0098] (2) Connecting the carboxyl terminus: Add Fmoc-Lys (lysine protected by 9-fluorenylmethoxycarbonyl) in a molar amount 3 times that of 2-chlorotrityl chloride resin and DIEA (N,N-diisopropylethylamine) in a molar amount 10 times that of 2-chlorotrityl chloride resin and react for 30 min. After dissolving in DMF, soak in methanol for 30 min for blocking.

[0099] (3) Deprotection: Remove the solvent. Add 20 ml of 20 wt% piperidine DMF solution per 1 mmol of 2-chlorotrityl chloride resin, soak for 10 min and repeat once. Then remove the solvent.

[0100] (4) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min, and observe the appearance of blue, which indicates a positive result and proves grafting. Then wash it twice with DMF, methanol, and DMF in sequence.

[0101] (5) Condensation: In the direction from the carboxyl terminus to the amino terminus, add ortho-Fmoc-amino acid in a molar amount 3 times that of 2-chlorotrityl chloride resin, HBTU in a molar amount 3 times, and DIEA in a molar amount 10 times and react for 30 min. Then wash it three times with DMF.

[0102] (6) Detection: Take 0.2 g of 2-chlorotrityl chloride resin, wash it three times with ethanol, add one drop of Kaiser kit solution each, heat at 105 °C for 5 min, and observe colorless, indicating that the reaction is complete.

[0103] (7) Amino-terminal azide modification: Repeat steps (3)-(6), where in step (5), the intermediate polypeptide sequence is synthesized in the order of Lys-Ala-Glu-Gly-Asp-Val-Gly-Leu-Pro-Gly-Ile-Leu-Met-Arg-Trp-Trp-Lys-Phe-Lys. Remove the N-terminal Fmoc protecting group and the K-side chain Dde protecting group. Add 7-fold molar amount of N3-OEG4-CH2CH2COOH, 7-fold molar amount of HBTU, and 25-fold molar amount of DIEA to the 2-chlorotrityl chloride resin and react for 30 min. Finally, dissolve the resin obtained by solid-phase synthesis with DMF, and obtain the polypeptide powder after cleavage, purification, and lyophilization, namely the dual-anchored enzyme-responsive polypeptide.

[0104] Modify the surface of the titanium-based material with the dual-anchored enzyme-responsive polypeptide prepared in this example. The specific steps are as follows:

[0105] (1) Place the titanium metal in a 5 M sodium hydroxide solution and react at 60 °C for 24 h. After the reaction, add water and ultrasonically clean.

[0106] (2) Prepare a 0.5 mg / mL DBCO-PEG 3.4k -silane coupling agent solution with 95% ethanol by mass fraction;

[0107] (3) Add 1 mL of the silane coupling agent per square centimeter of the titanium sheet, place it at room temperature for reaction for 24 h, and then place it in an oven at 100 °C for curing;

[0108] (4) Add the 150 μM polypeptide solution to the surface of the titanium metal prepared in step (3) and carry out the reaction;

[0109] (5) Thoroughly wash the sample in step (4) with deionized water and dry it with nitrogen to obtain the modified titanium-based material surface.

[0110] The material modified with the dual-anchored enzyme-responsive polypeptide can kill 93.22% of Staphylococcus aureus and 97.26% of Pseudomonas aeruginosa. The modified material surface can promote the proliferation of mouse mesenchymal stem cells, and the CCK-8 value after cell proliferation is 1.12 times that of the pure titanium group.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing a double-anchored enzyme-responsive polypeptide, wherein the double-anchored enzyme-responsive polypeptide is obtained by solid phase synthesis, and the synthesis order is from the carboxyl end to the amino end, characterized in that: The specific steps include: (1) Connecting the first amino acid: 2-Chlorotrityl chloride resin was soaked in DMF to swell, and DIEA and Fmoc-protected carboxyl terminal amino acid were added. If the carboxyl terminal amino acid was a non-basic amino acid, Fmoc-Lys or Fmoc-Arg was added; (2) Synthesizing peptide segments: Deprotect the first amino acid in step (1) with 20 wt% piperidine DMF solution, and perform Kaiser detection. If the Kaiser detection is positive, add ortho-Fmoc-amino acid, HBTU and DIEA for condensation; if the Kaiser detection is negative, repeat step (2) in the order of C→N to add the corresponding amino acid until the sequence is completed; (3) Azide modification: add N3-OEG4-CH2CH2COOH, HBTU and DIEA to react, and finally add DMF to dissolve; (4) After cutting, purification and freeze-drying, the polypeptide powder is obtained.

2. The method for preparing the double-anchored enzyme-responsive polypeptide according to claim 1, characterized in that: In step (1), the molar volume ratio of 2-chlorotrityl chloride resin and DMF is 1 mmol:5-35 ml.

3. The method for preparing the double-anchored enzyme-responsive polypeptide according to claim 1, characterized in that: In step (1), the molar ratio of DIEA to 2-chlorotrityl chloride resin is 2-6:1; and the molar ratio of the Fmoc-protected carboxyl terminal amino acid to 2-chlorotrityl chloride resin is 3-18:

1.

4. The method for preparing a double-anchored enzyme-responsive polypeptide according to claim 1, characterized in that: If the Kaiser test is positive in step (2), the molar ratio of the Fmoc-amino acid, HBTU, DIEA and the 2-chlorotrityl chloride resin in step (1) is 2-6:1-5:3-27:

1.

5. The method for preparing the double-anchored enzyme-responsive polypeptide according to claim 1, characterized in that: The molar ratio of N3-OEG4-CH2CH2COOH, HBTU, DIEA in step (3) to 2-chlorotrityl chloride resin in step (1) is 2-8:4-8:10-40:

1.

6. A double anchoring enzyme responsive polypeptide, characterized in that: The method is prepared according to any one of claims 1 to 5.

7. The double anchoring enzyme responsive polypeptide according to claim 6, characterized in that: The terminal modification group of the double-anchored enzyme-responsive polypeptide is an azido group, the sequence is -OEG4-N3, and the specific structure is N3-OEG4-antimicrobial peptide-enzyme-responsive sequence-tissue repair-promoting peptide-OEG4-N3.

8. The double anchoring enzyme responsive polypeptide according to claim 7, characterized in that: The tissue repair promoting peptides in the N3-OEG4-antimicrobial peptide-enzyme response sequence-tissue repair promoting peptide-OEG4-N3 are cell adhesion peptides, osteogenic peptides, neurogenic peptides and angiogenic peptides.

9. Use of the double-anchored enzyme-responsive polypeptide according to any one of claims 6 to 8 in the preparation of an antibacterial bone implant.

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