A metal polyphenol network coupled functional block peptide coating and a preparation method thereof, a functional implant material

Abstract

The application provides a metal polyphenol network coupling functional block peptide coating and a preparation method thereof, and a functional implant material, and belongs to the technical field of biomedical engineering.The intermediate layer of the metal polyphenol network is constructed, the phenolic hydroxyl groups in the polyphenol have high affinity and adhesion, can not only provide extensive substrate adhesion capacity, but also can be assembled with the polybasic amino acid units of the functional block peptide, so that the functional block peptide is firmly fixed on the surface of the implant, and the high biological activity of the polypeptide is maintained.The stable and soft metal polyphenol network surface modification strategy is adopted, the functional block peptide in which the polybasic amino acid is reasonably connected with the functional polypeptide is designed, and these functional peptide segments can provide the implant with a wide range of antibacterial, anti-protein adsorption, cell adhesion promotion, bone repair, angiogenesis or immune regulation and other biological functions, so that the implant is endowed with the designed biological activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a metal polyphenol network-coupled functional block peptide coating and its preparation method, as well as a functional implant material. Background Technology

[0002] Various bioengineering materials, primarily titanium and its alloys, are widely used in bioimplants due to their excellent mechanical properties, corrosion resistance, and biocompatibility. However, the poor bioinertness of the implant surface can easily lead to implantation failure under pathological conditions in vivo. Surface modification of implants to enhance their biocompatibility, anti-inflammatory, antioxidant, osteogenic and osteoclast-inhibiting, antibacterial, and angiogenic functions is a promising strategy.

[0003] Researchers have developed various functional implant coatings using calcium phosphates (hydroxyapatite and calcium hydrogen phosphate) and bioactive molecules (collagen, bone morphogenetic proteins, DNA, growth factors, and peptides). Among these, bioactive peptides have attracted considerable attention due to their excellent biocompatibility, stability, broad functional selectivity, and high degree of design flexibility, making them ideal materials for implant surface modification. However, due to insufficient implant surface activity, bioactive molecules struggle to effectively bind to the implant, potentially leading to the loss of the implant's bioactivity. Summary of the Invention

[0004] In view of this, the present invention aims to provide a metal polyphenol network-coupled functional block peptide coating, its preparation method, and a functional implant material. The metal polyphenol network-coupled functional block peptide coating provided by the present invention can effectively modify implants and improve the biological function of the implant surface.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing a metal polyphenol network-coupled functional block peptide coating, comprising the following steps:

[0007] A functional block peptide is provided; the structure of the functional block peptide includes a polybasic amino acid at the amino terminus and a functional polymer at the carboxyl terminus;

[0008] The implant was placed in a mixture of plant polyphenol solution and metal ion solution, and a first oscillation was performed. After standing, Tris-HCl solution was added to the obtained first oscillation mixture, and a second oscillation was performed to obtain an implant with a metal polyphenol network intermediate layer on the surface.

[0009] The implant with the surface-coated metal polyphenol network intermediate layer is placed in the functional block peptide solution to perform a self-assembly reaction, thereby obtaining a metal polyphenol network coupled functional block peptide coating on the implant surface.

[0010] Preferably, the polybasic amino acid includes one or more of polylysine, polyarginine, and polyhistidine; the degree of polymerization of the polybasic amino acid is 3 to 24.

[0011] The functional polymer comprises functional peptides and / or functional polymer segments; the functional peptides comprise one or more of RGD, OGP, and CAMEL0, and the functional polymer segments comprise polyethylene glycol.

[0012] Preferably, the plant polyphenols include one or more of tannic acid, proanthocyanidins, catechins, and epigallocatechin gallate.

[0013] The metal ions include Mg 2+ Fe 2+ Fe 3+ Co 2+ Al 3+ Cu 2+ Ti 4+ Ag + V 4+ and Mo 3+ One or more of them.

[0014] Preferably, the metal ions are provided by a metal ion source;

[0015] When the plant polyphenol is proanthocyanidin, the mass ratio of the plant polyphenol to the metal ion source is 1-2:3-6.

[0016] When the plant polyphenol is catechin, the mass ratio of the plant polyphenol to the metal ion source is 1-4:3-12;

[0017] When the plant polyphenol is epigallocatechin gallate, the mass ratio of the plant polyphenol to the metal ion source is 1-4:3-12.

[0018] When the plant polyphenol is tannic acid, the mass ratio of the plant polyphenol to the metal ion source is 1-4:3-12.

[0019] Preferably, the first oscillation mixing is a vortex oscillation, and the first oscillation mixing rate is 500-1000 rpm, and the time is 10-60 s;

[0020] The settling time is 0–30 min;

[0021] The second oscillation mixing is a vortex oscillation, and the speed of the second oscillation mixing is 500-1000 rpm, and the time is 10-60 s.

[0022] Preferably, after the second oscillation mixing, the process further includes repeating the first oscillation mixing-resting-second oscillation mixing process, wherein the number of repetitions is ≥5 times.

[0023] Preferably, the solvent for the functional block peptide solution is a Tris-HCl solution with a pH of 7-9 and a concentration of 10 mM; the concentration of the functional block peptide is 0.5-5 mg / mL.

[0024] Preferably, the self-assembly reaction takes 12 to 48 hours.

[0025] The present invention provides a metal polyphenol network coupled functional block peptide coating prepared by the above preparation method, comprising a metal polyphenol network intermediate layer located on the surface of the implant and a functional block peptide coupled to the metal polyphenol network intermediate layer.

[0026] The present invention provides a functional implant material, comprising an implant substrate and a metal polyphenol network coupled with functional peptide coating covering the surface of the implant substrate.

[0027] This invention provides a method for preparing a metal-polyphenol network-coupled functional block peptide coating. The method involves placing an implant in a mixture of plant polyphenol solution and metal ion solution, performing a first shaking and mixing, and then, after standing, adding Tris-HCl solution to the resulting first shaking and mixing system, followed by a second shaking and mixing to obtain an implant with a metal-polyphenol network intermediate layer. The implant with the metal-polyphenol network intermediate layer is then placed in the functional block peptide solution for a self-assembly reaction to obtain the metal-polyphenol network-coupled functional block peptide coating. This invention, by constructing an intermediate layer of a metal-polyphenol network, leverages the high affinity and adhesion of the phenolic hydroxyl groups in the polyphenols, providing broad substrate adhesion capabilities and enabling assembly with the polybasic amino acid units of the functional block peptides. This firmly fixes the functional block peptides to the implant surface, maintaining the high bioactivity of the peptides. This invention employs a stable and gentle metal polyphenol network surface modification strategy. By designing functional block peptides that rationally link polybasic amino acids with functional polypeptides, these functional peptides can provide implants with a variety of biological functions, such as broad-spectrum antibacterial, anti-protein adsorption, cell adhesion promotion, bone repair, angiogenesis, or immune regulation, thus endowing implants with designable bioactivity.

[0028] The functional block peptide structures in the metal-polyphenol network-coupled functional peptide coating constructed in this invention can be easily designed according to actual applications, enabling multifunctionalization of the implant surface. Specifically, the basic amino acid can be selected from polyarginine, polyhistidine, or polylysine, providing various supramolecular interaction forces; the functional peptides can be selected from antimicrobial peptides, antifouling peptides, cell adhesion peptides, osteogenic peptides, angiogenic peptides, or immunomodulatory peptides, achieving broad-spectrum antibacterial, anti-protein adsorption, cell adhesion promotion, bone repair, angiogenesis, immunomodulation, and other biological functions. Furthermore, the metal-polyphenol network-coupled functional peptide coating of this invention has strong versatility for implant substrates and can be used for surface functionalization of various biomaterials, exhibiting good surface bioactivity modification effects on metallic substrates such as titanium, aluminum, magnesium, and steel, as well as non-metallic substrates such as ceramics, plastics, quartz, and glass.

[0029] Meanwhile, the coating preparation method provided by this invention is simple to operate and easy to achieve industrial mass production. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the preparation process of the metal polyphenol network coupled functional block peptide coating of the present invention;

[0031] Figure 2 The thickness diagrams show the different coating surfaces of the silicon wafers prepared in Examples 1-4;

[0032] Figure 3 The diagram shows the water contact angles of different coating surfaces of the titanium plates prepared in Examples 1-4.

[0033] Figure 4 Optical photographs of the PC / Fe-MPN and PC / Fe@K6-(linker-RGD)3 coatings used in Example 1 on various substrates;

[0034] Figure 5 SEM and AFM images of the silicon wafers used in Examples 1-2, and the obtained PC / Fe-MPN, PC / Fe@K6-(linker-RGD)3, and PC / Fe@K6-linker-(YGFGG)2;

[0035] Figure 6 SEM images of Ti used in Example 1, and the resulting PC / Fe-MPN and PC / Fe@K6-(linker-RGD)3 surface cell growth and adhesion;

[0036] Figure 7 The images show ALP and Alizarin Red staining of glass slides and osteoblasts on the surface of PC / Fe-MPN and PC / Fe@K6-linker-(YGFGG)2 cells from Example 2.

[0037] Figure 8 SEM images of the Ti screws obtained in Example 2 with and without PC / Fe@K6-linker-(YGFGG)2 coating;

[0038] Figure 9 The graph shows the BSA adsorption resistance per unit area of ​​PC / Fe-MPN and PC / Fe@K6-Cys(PEG2000-MAL) surfaces measured by the QCM-D chip used in Example 3.

[0039] Figure 10 Photographs of Ti used in Example 4 and the resulting PC / Fe-MPN and PC / Fe@K6-linker-KWKLFKKIGAVLKVL-NH2 surfaces showing bactericidal effects against Gram-negative E. coli and Gram-positive S. aureus. Detailed Implementation

[0040] This invention provides a method for preparing a metal polyphenol network-coupled functional block peptide coating, comprising the following steps:

[0041] A functional block peptide is provided; the structure of the functional block peptide includes a polybasic amino acid at the amino terminus, a functional polymer at the carboxyl terminus, and a linker connecting the polybasic amino acid and the functional polymer;

[0042] The implant was placed in a mixture of plant polyphenol solution and metal ion solution, and a first oscillation was performed. After standing, Tris-HCl solution was added to the obtained first oscillation mixture, and a second oscillation was performed to obtain an implant with a metal polyphenol network intermediate layer on the surface.

[0043] The implant with the surface-coated metal polyphenol network intermediate layer is placed in the functional block peptide solution to perform a self-assembly reaction, thereby obtaining a metal polyphenol network coupled functional block peptide coating on the implant surface.

[0044] This invention first provides a functional block peptide. In this invention, the structure of the functional block peptide comprises a polybasic amino acid at the amino terminus and a functional polymer at the carboxyl terminus. Preferably, the polybasic amino acid comprises polylysine. n K n Polyarginine n R n Polyhistidine n H n One or more of the following: the degree of polymerization n of the polybasic amino acid is preferably 3 to 24, more preferably 6 to 20.

[0045] In this invention, the functional polymer preferably comprises functional peptides and / or functional polymer chains; the functional peptides include one or more of RGD, OGP and CAMEL0, and the functional polymer chains include polyethylene glycol, preferably PEG2000.

[0046] In this invention, the connection method between the polybasic amino acid and the functional polymer is preferably one of peptide bond connection, disulfide bond connection and click chemical connection.

[0047] In this invention, the polybasic amino acids and functional polymers are preferably linked by a linker to maximize the bioactivity of the block peptide and maintain a good conformation; in this invention, the linker is preferably 6-aminohexanoic acid (ACP) or maleimide.

[0048] As a specific embodiment of the present invention, the functional block peptide is preferably one of K6-(linker-RGD)3, K6-linker-(YGFGG)2, K6-Cys(PEG2000-MAL), and K6-acp-KWKLFKKIGAVLKVL-NH2.

[0049] In this invention, (linker-RGD)3 in K6-(linker-RGD)3 represents the linker-RGD structure repeated three times. In this invention, RGD in K6-(linker-RGD)3 consists of arginine, glycine, and aspartic acid, with the sequence RGD.

[0050] In this invention, (YGFGG)2 in K6-linker-(YGFGG)2 represents YGFGG repeated twice. In this invention, the sequence of (YGFGG)2 in K6-linker-(YGFGG)2 is as shown in SEQ ID NO.1, specifically YGFGGYGFGG.

[0051] In this invention, Cys(PEG2000-MAL) in K6-Cys(PEG2000-MAL) indicates that PEG2000 is modified onto the polypeptide by binding the thiol group of Cys to MAL, where MAL is maleimide.

[0052] In this invention, the sequence in K6-acp-KWKLFKKIGAVLKVL-NH2 is as shown in SEQ ID NO.2, specifically KWKLFKKIGAVLKVL.

[0053] This invention does not impose any special requirements on the synthesis method of the functional block peptides; conventional peptide synthesis methods in the art, such as solid-phase long peptide synthesis technology, can be used. In the embodiments of this invention, the functional block peptides are synthesized by a synthetic company, specifically Nanjing Peptide Valley Biotechnology Co., Ltd.

[0054] This invention involves placing the implant in a mixture of plant polyphenol solution and metal ion solution, performing a first shaking mixture, and then, after standing, adding Tris-HCl solution to the resulting first shaking mixture, followed by a second shaking mixture to obtain an implant with a surface coated with a metal polyphenol network intermediate layer. In this invention, the implant is preferably a medical implant. This invention does not impose special requirements on the shape or size of the implant; implants of any shape and size are suitable for the metal polyphenol network-coupled functional block peptide coating modification using the method described in this invention.

[0055] In this invention, the implant material is preferably a metal substrate or a non-metal substrate; the metal substrate is preferably one or more of titanium, aluminum, magnesium, steel, titanium alloy, aluminum alloy and magnesium alloy; the non-metal substrate is preferably one or more of ceramic, plastic, quartz and glass.

[0056] In this invention, prior to the first oscillation mixing, the implant is preferably pretreated. In this invention, the pretreatment preferably includes the following steps:

[0057] The implant is ultrasonically cleaned and dried to remove surface oil and dirt.

[0058] In this invention, the cleaning solution used for ultrasonic cleaning is preferably acetone, ethanol, and water in that order. This invention does not specify a particular method for ultrasonic cleaning. In this invention, the drying is preferably achieved by baking.

[0059] For implants with an oxide layer on their surface, the present invention preferably involves polishing the implant to remove the oxide layer. The present invention does not specify the particular method of polishing.

[0060] In this invention, the plant polyphenols include one or more of tannic acid, proanthocyanidins, catechins, and epigallocatechin gallate.

[0061] In this invention, the metal ions include Mg. 2+ Fe 2+ Fe 3+ Co 2+ Al 3+ Cu 2+ Ti 4+ Ag + V 4+ and Mo 3+One or more of the following. In this invention, the metal ion source providing the metal ions is preferably one or more of soluble metal chloride salts, soluble metal nitrates, and soluble metal sulfates.

[0062] In this invention, when the plant polyphenol is proanthocyanidins, the mass ratio of the plant polyphenol to the metal ion source is preferably 1-2:3-6, more preferably 1-2:4-5. That is, in parts by mass, the amount of proanthocyanidins added is 1-2 parts, and the amount of the metal ion source added is 3-6 parts, preferably 4-5 parts.

[0063] In this invention, when the plant polyphenol is catechin, the mass ratio of the plant polyphenol to the metal ion source is preferably 1-4:3-12, more preferably 2-3:5-10. That is, in parts by mass, the amount of catechin added is 1-4 parts, preferably 2-3 parts; the amount of the metal ion source added is 3-12 parts, preferably 5-10 parts.

[0064] In this invention, when the plant polyphenol is epigallocatechin gallate, the mass ratio of the plant polyphenol to the metal ion source is preferably 1-4:3-12, more preferably 2-3:5-10. That is, in parts by mass, the amount of epigallocatechin gallate added is 1-4 parts, preferably 2-3 parts; the amount of the metal ion source added is 3-12 parts, preferably 5-10 parts.

[0065] In this invention, when the plant polyphenol is tannic acid, the mass ratio of the plant polyphenol to the metal ion source is preferably 1-4:3-12, more preferably 2-3:5-10. That is, in parts by mass, the amount of tannic acid added is 1-4 parts, preferably 2-3 parts; the amount of the metal ion source added is 3-12 parts, preferably 5-10 parts.

[0066] In this invention, the preferred solutions for the plant polyphenols are water, a 5-20 mM sodium acetate solution with a pH of 4-5.5, a 5-20 mM Bis-tris-HCl solution with a pH of 6-7, or a 5-20 mM Tris-HCl solution with a pH of 7-9; the concentration of the polyphenol solution is the soluble concentration in the corresponding buffer solution, including 0.5 mM to 5 mM. To obtain a more stable and reproducible coating, proanthocyanidins and catechins are preferably dissolved using a 10 mM sodium acetate buffer solution with a pH of 4.5, and tannic acid and epigallocatechin gallate are preferably dissolved using a 10 mM sodium acetate buffer solution with a pH of 5.5.

[0067] In this invention, the preferred solution for the metal ions is water, a sodium acetate solution with a concentration of 5–20 mM and a pH of 4–5.5, a Bis-tris-HCl solution with a concentration of 5–20 mM and a pH of 6–7, or a Tris-HCl solution with a concentration of 5–20 mM and a pH of 7–9. An aqueous solution is preferred for obtaining a more stable and repeatable coating.

[0068] In this invention, it is preferable to first prepare a plant polyphenol solution and a metal ion solution separately, and then mix the plant polyphenol solution and the metal ion solution according to the above-mentioned weight parts to make the solution volume for immersing the implant appropriate; in this invention, the plant polyphenol solution and the metal ion solution are preferably mixed in equal volumes. In this invention, when the plant polyphenol is proanthocyanidin and the proanthocyanidin solvent is a 10mM sodium acetate buffer solution with pH=4.5, the concentration of the proanthocyanidin solution is preferably 0.25-0.5mM; the concentration of metal ions in the metal ion solution is 0.75-3mM.

[0069] In this invention, when the plant polyphenol is catechin and the catechin solvent is a 10mM sodium acetate buffer solution with pH=4.5, the concentration of the catechin solution is 0.25-1mM, and the concentration of the metal ion in the metal ion solution is 0.75-12mM.

[0070] In this invention, when the plant polyphenol is epigallocatechin gallate and the epigallocatechin gallate solvent is a 10mM sodium acetate buffer solution with pH=5.5, the concentration of the epigallocatechin gallate solution is 0.25-1mM, and the concentration of the metal ion in the metal ion solution is 0.75-12mM.

[0071] In this invention, when the plant polyphenol is tannic acid, the tannic acid solvent is pH=5.5, and the 10mM sodium acetate buffer solution is used, the concentration of the tannic acid solution is 0.25-1mM, and the concentration of the metal ions in the metal ion solution is 0.75-12mM.

[0072] In this invention, the first oscillation mixing is preferably vortex oscillation, and the speed of the first oscillation mixing is preferably 500-1000 rpm, more preferably 600-800 rpm; the time is preferably 10-60 s, more preferably 30 s. In this invention, the settling time is preferably 0-30 min, preferably 10 min. This invention, through the first oscillation and settling, assembles a metal polyphenol coating formed from plant polyphenols and metal ions onto the surface of the implant.

[0073] In this invention, the pH value of the Tris-HCl solution is preferably 7-9, and the concentration is preferably 0.2-0.5M. In this invention, the volume ratio of the mixture of the Tris-HCl solution, the plant polyphenol solution, and the metal ion solution is preferably 1:1.

[0074] In this invention, the second oscillation mixing is a vortex oscillation, and the preferred oscillation mixing rate is 500-1000 rpm, more preferably 600-800 rpm; the preferred time is 10-60 s, more preferably 30 s. To improve experimental repeatability and ensure sufficient reaction and complete assembly, it is preferable to let the mixture stand for 10 minutes after the first oscillation mixing, and then add a 0.3 M Tris-HCl solution with pH=8.

[0075] In this invention, the first oscillation mixing, the settling, and the second oscillation mixing are preferably carried out at room temperature.

[0076] To ensure that the metal polyphenol coating has a thickness suitable for peptide grafting, after the second oscillation mixing, the present invention further includes repeating the process of first oscillation mixing-standing-second oscillation mixing, the number of repetitions being ≥5 times.

[0077] After the second oscillation mixing is completed, the implant is preferably removed and dried. In this invention, the drying is preferably performed by blowing with a gentle airflow.

[0078] After obtaining the metal polyphenol network coating, the present invention places the implant with the metal polyphenol network intermediate layer on the surface into the functional block peptide solution to perform a self-assembly reaction, thereby obtaining a metal polyphenol network coupled with a functional block peptide coating on the implant surface. In the present invention, the solvent of the functional block peptide solution is a Tris-HCl solution with a pH value preferably of 7-9 and a concentration preferably of 10 mM; the concentration of the functional block peptide is preferably 0.5-5 mg / mL, more preferably 1 mg / mL.

[0079] In this invention, the self-assembly reaction is preferably carried out in a shaker; the shaking rate is preferably 100-200 rpm. In this invention, the temperature of the self-assembly reaction is preferably room temperature, and the time is preferably 12-48 h, more preferably 24 h.

[0080] As a specific embodiment of the present invention, to ensure the reproducibility and stability of the self-assembly reaction, K6-(linker-RGD)3 is preferably reacted in Tris-HCl at pH 7 and 10 mM for 24 hours; K6-linker-(YGFGG)2 is preferably reacted in Tris-HCl at pH 8 and 10 mM for 24 hours; and K6-Cys(PEG2000-MAL) and K6-linker-KWKLFKKIGAVLKVL-NH2 are preferably reacted in Tris-HCl at pH 9 and 10 mM for 24 hours.

[0081] Following the self-assembly reaction, the implant is removed and sequentially rinsed with deionized water and dried. In this invention, the drying is preferably performed using a gentle airflow.

[0082] In this invention, the preparation process of the metal polyphenol network-coupled functional block peptide coating is shown in the schematic diagram below. Figure 1 As shown.

[0083] This invention provides a metal-polyphenol network-coupled functional block peptide coating prepared by the above-described method, comprising a metal-polyphenol network intermediate layer located on the surface of an implant and functional block peptides coupled to the metal-polyphenol network intermediate layer. In this invention, the thickness of the metal-polyphenol network-coupled functional block peptide coating is preferably 10–100 nm.

[0084] This invention provides a functional implant material, comprising an implant substrate and a metal polyphenol network-coupled functional peptide coating covering the surface of the implant substrate. In this invention, the implant substrate is preferably made of a metal substrate or a non-metal substrate; the metal substrate is preferably one or more of titanium, aluminum, magnesium, steel, titanium alloy, aluminum alloy, and magnesium alloy; the non-metal substrate is preferably one or more of ceramic, plastic, quartz, and glass.

[0085] The following detailed description, in conjunction with embodiments, illustrates a metal polyphenol network-coupled functional block peptide coating and its preparation method, as well as a functional implant material provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0086] In the following examples, K6-(linker-RGD)3, K6-linker-(YGFGG)2, K6-Cys(PEG2000-MAL), and K6-acp-KWKLFKKIGAVLKVL-NH2 were synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd., using solid-phase long peptide synthesis technology.

[0087] Example 1

[0088] A method for preparing a metal polyphenol network-coupled functional block peptide coating (this coating is a surface cell adhesion-promoting coating) includes the following steps:

[0089] 1) Place a titanium plate, glass plate, or silicon wafer (2cm in size) 2 The surface should be ultrasonically cleaned sequentially with acetone, ethanol, and water to remove oil and dirt, then dried for later use. Metals need to be polished to remove the surface oxide layer.

[0090] 2) Place the cleaned implant in a 5 mL centrifuge tube, add 1 mL of 0.5 mM proanthocyanidin solution (pH 4.5, 10 mM sodium acetate buffer) and 1 mL of 1.5 mM Fe(NO3)3 aqueous solution, then vortex rapidly at 500–1000 rpm for 30 seconds and let stand for 10 minutes. Add Tris-HCl solution at pH 8, vortex rapidly at 500–1000 rpm for 30 seconds to stabilize the metal polyphenol network, then rinse and dry with a gentle airflow. Repeat the above steps 5 times to obtain a titanium plate, glass slide, or silicon wafer with an assembled (PC / Fe)5-MPNs nanofilm.

[0091] 3) Prepare a 1 mg / mL Tris-HCl solution of K6-(linker-RGD)3 (pH = 7, 10 mM). Immerse a titanium plate, glass slide, or silicon wafer with the (PC / Fe)5-MPNs nanofilm interlayer assembled in 500 μL of the above solution and react at room temperature with shaking at 200 rpm for 24 hours. Then, remove the sample, rinse with deionized water for 1 minute, and dry with a gentle airflow to obtain a titanium plate, glass slide, or silicon wafer coated with a metal polyphenol network-coupled functional block peptide coating, designated as (PC / Fe)5@K6-RGD sample. Store at 4℃ in a cool, dry place.

[0092] Example 2

[0093] A method for preparing a metal polyphenol network-coupled functional block peptide coating (this coating is a surface osteogenic coating) differs from Example 1 in that:

[0094] 3) Prepare a 1 mg / mL Tris-HCl solution of K6-linker-(YGFGG)2 (pH = 8, 10 mM). Immerse a titanium plate, glass slide, or silicon wafer with the (PC / Fe)5-MPNs nanofilm interlayer assembled in 500 μL of the above solution and react at room temperature with shaking at 200 rpm for 24 hours. Then, remove the sample, rinse with deionized water for 1 minute, and dry with a gentle airflow at room temperature to obtain a titanium plate, glass slide, or silicon wafer coated with a metal polyphenol network-coupled functional block peptide coating, designated as (PC / Fe)5@K6-OGP sample. Store at 4℃ in a cool, dry place.

[0095] Example 3

[0096] A method for preparing a metal polyphenol network-coupled functional block peptide coating (this coating is a surface anti-protein adsorption coating) differs from Example 1 in that:

[0097] 3) Prepare a 1 mg / mL Tris-HCl solution of K6-Cys (PEG2000-MAL) (pH = 9, 10 mM). Immerse the titanium plate, silicon wafer, or QCM-D chip with the (PC / Fe)5-MPNs nanofilm interlayer in 500 μL of the above solution and react at room temperature with shaking at 200 rpm for 24 hours. Then, remove the sample, rinse with deionized water for 1 minute, and dry with a gentle airflow at room temperature to obtain the titanium plate, silicon wafer, or QCM-D chip coated with a metal polyphenol network-coupled functional block peptide coating, designated as the (PC / Fe)5@K6-PEG sample. Store at 4℃ in a dry, refrigerated environment.

[0098] Example 4

[0099] A method for preparing a metal polyphenol network-coupled functional block peptide coating (this coating is a surface antibacterial coating) differs from Example 1 in that:

[0100] 3) Prepare a 1 mg / mL Tris-HCl solution of K6-linker-KWKLFKKIGAVLKVL-NH2 (pH = 9, 10 mM). Immerse a titanium plate, glass plate, or silicon wafer with the (PC / Fe)5-MPNs nanofilm interlayer assembled in 500 μL of the above solution and react at room temperature with shaking at 200 rpm for 24 hours. Then, remove the sample, rinse with deionized water for 1 minute, and dry with a gentle airflow at room temperature to obtain a titanium plate, glass plate, quartz plate, or silicon wafer coated with a metal polyphenol network-coupled functional block peptide coating, designated as (PC / Fe)5@K6-CAMEL0 sample. Store at 4℃ in a cool, dry place.

[0101] Structural characterization

[0102] (1) The thickness of the coating on the silicon wafers obtained in Examples 1-4 was measured using an elliptic polarization spectrometer. The thickness of the coatings on the silicon wafers prepared in Examples 1-4 is shown in the figure. Figure 2 As shown, by Figure 2 It can be seen that after coupling functional block peptides onto the metal polyphenol network, the coating thickness increases significantly, indicating that the functional block peptides were successfully deposited onto the surface of the metal polyphenol network.

[0103] (2) The change process of the contact angle of the coating on the silicon wafer surface obtained in Examples 1-4 was characterized using a contact angle measuring instrument. The water contact angle (WCA) of the titanium plates with different coating surfaces prepared in Examples 1-4 is shown in the figure. Figure 3 As shown, by Figure 3 It can be seen that the titanium plate has a large surface contact angle, exhibiting obvious hydrophobicity. When the titanium plate surface is deposited with a metal polyphenol network and coupled with functional block peptides, the surface water contact angle of the material becomes significantly smaller, showing more hydrophilic properties, which is beneficial to increasing the biocompatibility of the implant material.

[0104] (3) Photographs of the coating on various substrates were taken using a camera. Optical photographs of the PC / Fe@K6-(linker-RGD)3 coating used in Example 1 on various substrates are shown below. Figure 4 As shown, by Figure 4 As can be seen, after the PC / Fe@K6-(linker-RGD)3 coating is deposited on the substrate surface, the surface color turns dark brown, indicating the successful deposition of the PC / Fe@K6-(linker-RGD)3 coating. In addition, the PC / Fe@K6-(linker-RGD)3 coating can be successfully coated on the surfaces of substrates such as glass sheets, silicon wafers, calcium fluoride sheets, polystyrene sheets, aluminum sheets, and ceramic sheets, indicating that the coating has substrate universality.

[0105] (4) The morphological structure of the coated silicon wafer surface was characterized using SEM and AFM. The silicon wafers used in Examples 1 and 2, and the SEM and AFM images of the obtained PC / Fe-MPN, PC / Fe@K6-(linker-RGD)3, and PC / Fe@K6-linker-(YGFGG)2 are shown below. Figure 5 As shown, by Figure 5 It can be seen that, compared with the metal polyphenol network coating PC / Fe-MPN, the surface-coupled functional block peptide coatings PC / Fe@K6-(linker-RGD)3 and PC / Fe@K6-linker-(YGFGG)2 have more and larger particles on their surface, and have higher roughness and thickness.

[0106] Performance testing

[0107] (1) To verify the effect of the multifunctional coating prepared in this invention, the (PC / Fe)5@K6-RGD coating on the surface of the Ti plate prepared in Example 1 was subjected to a cell compatibility test. The specific experiment is as follows:

[0108] A. Culture NIH / 3T3 cells in DMEM complete medium containing 10% FBS and 1% penicillin antibiotics, then transfer 2cm... 2 Ti-(PC / Fe)5@K6-RGD sample and 1×10 4Cells were co-incubated. They were cultured for 3 days in a humid environment of 37°C and 5% CO2, then fixed overnight with 2.5% glutaraldehyde at 4°C, followed by fixation with 1% osmium tetroxide, dehydration with a gradient ethanol solution, drying using a supercritical desiccator, and gold sputtering before SEM observation. SEM images of the Ti used in Example 1, and the cell growth and adhesion on the obtained PC / Fe-MPN and PC / Fe@K6-(linker-RGD)3 surfaces are shown below. Figure 6 As shown, NIH / 3T3 cells exhibit a spindle-shaped morphology and are in a state of good adhesion, with a higher cell count in the coated group than in the bare titanium group. This demonstrates the good cell compatibility of the coating, which is beneficial for cell adhesion and proliferation.

[0109] (2) To verify the effect of the multifunctional coating prepared in this invention, the (PC / Fe)5@K6-OGP coating on the surface of the glass slide prepared in Example 2 was tested for osteogenic ability by ALP and Alizarin Red staining. The specific experiments are as follows:

[0110] A. 1×10 4 MC3T3-E1 cells were seeded at a depth of 2 cm. 2 The surface of glass slides coated with (PC / Fe)5@K6-OGP was examined. The slides were cultured in osteogenic induction medium for 7 and 21 days, respectively, followed by fixation with 4% paraformaldehyde. BCIP / NBT staining was used to reflect ALP activity, and Alizarin Red S staining (2%, pH 4.2) was used to reflect late-stage osteogenic calcification nodules. ALP staining and Alizarin Red staining images of the glass slides obtained in Example 2 and osteoblasts on the surfaces of PC / Fe-MPN and PC / Fe@K6-linker-(YGFGG)2 are shown below. Figure 7 As shown, the (PC / Fe)5@K6-OGP coating showed higher ALP and Alizarin Red staining than the glass slide surface, demonstrating the coating's ability to promote bone differentiation.

[0111] To verify the effectiveness of the multifunctional coating prepared in this invention, a Ti screw coating test was conducted on the (PC / Fe)5@K6-OGP coating prepared in Example 2. The specific experiment is as follows:

[0112] B. Ti screws coated and uncoated with (PC / Fe)5@K6-OGP coating were fixed to the SEM stage with conductive adhesive, sputter-coated with gold for 90 seconds, and the coating condition of the titanium screw surface was observed in the SEM instrument. SEM images of the Ti screws obtained in Example 2 with and without PC / Fe@K6-linker-(YGFGG)2 coating are shown below. Figure 8 As shown, the (PC / Fe)5@K6-OGP coating can be uniformly applied to the surface of the titanium screw.

[0113] (3) To verify the effect of the multifunctional coating prepared in this invention, the reactive anti-protein adsorption capacity of the (PC / Fe)5@K6-PEG coating on the surface of the QCM-D chip prepared in Example 3 was tested. The specific experiment is as follows:

[0114] A. A QCM-D chip coated with (PC / Fe)5@K6-PEG was placed in the reaction cell of the QCM-D instrument. A 1 mg / ml BSA aqueous solution was introduced into the reaction cell, and the adsorption kinetics were recorded using the auxiliary software. The adsorption mass per unit area of ​​BSA protein was also recorded using the auxiliary software. The adsorption mass per unit area of ​​PC / Fe-MPN and PC / Fe@K6-Cys(PEG2000-MAL) surfaces measured by the QCM-D chip used in Example 3 is shown in the figure below. Figure 9 As shown, after the BSA solution was introduced, obvious protein adsorption was observed on the (PC / Fe)5-MPN surface, while no obvious adsorption was observed on the (PC / Fe)5@K6-PEG coating, which proves that the (PC / Fe)5@K6-PEG coating has good anti-protein adhesion ability.

[0115] (4) To verify the effect of the multifunctional coating prepared in this invention, the bactericidal performance of the (PC / Fe)5@K6-CAMEL0 coating on the surface of the Ti plate prepared in Example 4 was tested. The specific experiment is as follows:

[0116] A. All prepared samples were sterilized beforehand. Single colonies of Gram-negative E. coli and Gram-positive S. aureus were selected from the bacterial culture plates and incubated in LB medium with shaking for several hours until the bacterial population reached mid-log phase (optical density at 600 nm reached 0.5). Then, 20 μL of 1×10⁻⁶ samples were added. 7 CFU mL -1 The bacterial suspension was dropped onto the sample surface prepared in Example 4 and incubated for 1 hour in a bacterial incubator at 37°C. Subsequently, 980 μL of sterile PBS was added to the wells, and bacteria attached to each sample were separated by sonication. Then, 20 μL of bacterial suspension from each well containing the sample was dispersed onto a solid LB agar plate using a spreader and incubated overnight at 37°C for photographing. The bactericidal smear images of the Ti used in Example 4 and the obtained PC / Fe-MPN and PC / Fe@K6-linker-KWKLFKKIGAVLKVL-NH2 surfaces against Gram-negative E. coli and Gram-positive S. aureus are shown below. Figure 10 As shown, the number of colonies on the plate of the (PC / Fe)5@K6-CAMEL0 coating group was less than that of the (PC / Fe)5-MPN group, which proves the good bactericidal ability of the coating.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a metal polyphenol network-coupled functional block peptide coating, comprising the following steps: A functional block peptide is provided; the structure of the functional block peptide includes a polybasic amino acid at the amino terminus and a functional polymer at the carboxyl terminus; The implant was placed in a mixture of plant polyphenol solution and metal ion solution, and a first oscillation was performed. After standing, Tris-HCl solution was added to the obtained first oscillation mixture, and a second oscillation was performed to obtain an implant with a metal polyphenol network intermediate layer on the surface. The implant with the surface-coated metal polyphenol network intermediate layer was placed in the functional block peptide solution to carry out a self-assembly reaction, thereby obtaining a metal polyphenol network coupled functional block peptide coating on the surface of the implant. The polybasic amino acid includes one or more of polylysine, polyarginine, and polyhistidine; the degree of polymerization of the polybasic amino acid is 3 to 24. The functional polymer comprises functional peptides and / or functional polymer chains; the functional peptides comprise one or more of RGD, OGP, and CAMEL0, and the functional polymer chains comprise polyethylene glycol. The linking method between the polybasic amino acid and the functional polymer is a linker linking, wherein the linker is 6-aminohexanoic acid or maleimide. The metal ions include Mg 2+ Fe 2+ Fe 3+ Co 2+ Al 3+ Cu 2+ Ti 4+ Ag + V 4+ and Mo 3+ One or more of them; The concentration of the functional peptide in the functional block peptide solution is 0.5~5 mg / mL.

2. The preparation method according to claim 1, characterized in that, The plant polyphenols include one or more of tannic acid, proanthocyanidins, catechins, and epigallocatechin gallate.

3. The preparation method according to claim 2, characterized in that, The metal ions are provided by a metal ion source; When the plant polyphenol is proanthocyanidin, the mass ratio of the plant polyphenol to the metal ion source is 1~2:3~6; When the plant polyphenol is catechin, the mass ratio of the plant polyphenol to the metal ion source is 1~4:3~12; When the plant polyphenol is epigallocatechin gallate, the mass ratio of the plant polyphenol to the metal ion source is 1~4:3~12; When the plant polyphenol is tannic acid, the mass ratio of the plant polyphenol to the metal ion source is 1~4:3~12.

4. The preparation method according to claim 1, characterized in that, The first oscillation mixing is a vortex oscillation, and the speed of the first oscillation mixing is 500~1000rpm, and the time is 10~60s; The settling time is 10-30 minutes; The second oscillation mixing is a vortex oscillation, and the speed of the second oscillation mixing is 500~1000rpm, and the time is 10~60s.

5. The preparation method according to claim 1 or 4, characterized in that, After the second oscillation mixing, the process further includes repeating the first oscillation mixing-resting-second oscillation mixing process, and the number of repetitions is ≥5 times.

6. The preparation method according to claim 1, characterized in that, The solvent for the functional block peptide solution is a Tris-HCl solution with a pH of 7-9 and a concentration of 10 mM.

7. The preparation method according to claim 1 or 6, characterized in that, The self-assembly reaction takes 12 to 48 hours.

8. The metal polyphenol network coupled functional block peptide coating prepared by the preparation method according to any one of claims 1 to 7 includes a metal polyphenol network intermediate layer located on the surface of the implant and a functional block peptide coupled to the metal polyphenol network intermediate layer.

9. A functional implant material comprising an implant substrate and a metal polyphenol network coupled functional peptide coating of claim 8 covering the surface of the implant substrate.

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