PH-responsive pocket molecule functionalized antibacterial implant as well as preparation method and application thereof

Abstract

The invention discloses a pH response type pocket molecule functionalized antibacterial implant as well as a preparation method and application thereof. The preparation method comprises the steps of alkali heat treatment of the titanium-based implant, silane coupling agent surface modification, pocket molecule grafting and antibacterial agent loading. The pocket molecules are modified on the surface of the implant through a'thiol-ene 'click reaction, so that the implant is endowed with dual functions of pH-responsive antibiosis and tissue repair promotion. On one hand, the pocket molecules serve as an antibacterial agent carrier and can release an antibacterial agent in an acidic environment at an infected part, so that bacteria are effectively killed and biofilm formation is inhibited; and on the other hand, the cholic acid compounds in the pocket molecules endow the implant with excellent biological activity, so that the prepared implant has excellent stability, pH responsiveness, antibacterial property and biocompatibility, can avoid high cytotoxicity caused by burst release of an antibacterial agent, is suitable for orthopedic implant infection prevention and treatment, and has wide application prospects. The important clinical application value is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a pH-responsive pocket molecule-functionalized antibacterial implant (Ti-S-MP-C+) and its preparation method and application. Background Art

[0002] Bioactive biomaterials, as structural and functional replacement implants, play an important role in the field of bone tissue repair. Titanium and its alloys, etc., have been widely used in bone tissue repair and regeneration implant materials, such as artificial joints, bone screws, and dental implants, due to their excellent mechanical properties, good corrosion resistance, and excellent biocompatibility. However, due to the lack of antibacterial properties of titanium-based materials themselves, various factors during the implantation process can lead to bacterial infection, colonization, and biofilm formation in the bone. The infected bacteria adhere to the surface of the bone implant to form a biofilm. The biofilm becomes a barrier that is difficult for antibiotics and other drugs to penetrate, complicating the eradication of bacteria, promoting bacterial growth and reproduction, and ultimately leading to implant failure. It is reported that the infection rate of global orthopedic surgeries is about 2-5%, and the infection rate of primary knee and hip replacement surgeries is about 1.5-2.5%. Among all cases of implant failure, about 20% of implant failures are directly related to bacterial infection of the implant, and bacterial infection has become one of the main reasons for implant failure. Once implant infection occurs, clinical treatment mainly relies on antibiotic treatment and surgical debridement. However, bacterial drug resistance greatly reduces the therapeutic effect of antibiotics, and surgical debridement prolongs the healing time of patients, bringing pain and economic pressure to patients.

[0003] Constructing an antibacterial coating on the surface of the implant is an effective method to prevent infection during the entire implantation process. Currently, a variety of antibacterial implants have been developed, such as antibiotic, metal ion coating, antimicrobial peptide, or antimicrobial polymer (quaternary ammonium salt, etc.) coatings. However, these antibacterial implants still have inherent limitations: 1) Long-term exposure of human tissues and cells to bactericides may cause adverse effects; 2) Uncontrolled release of antibacterial molecules cannot maintain long-term therapeutic effects, and excessive accumulation of drugs will cause serious side effects on healthy cells and tissues; 3) The antibacterial implant has a single function. In view of the high incidence of implant bacterial infection and the limitations of traditional antibacterial implants, developing a bio-adaptable implant that can exhibit different functions such as antibacterial and promoting tissue repair around the implant under microenvironment stimulation has become an urgent clinical need and is of great significance in preventing and treating implant infections. Summary of the Invention

[0004] In order to overcome the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide a method for preparing a pH-responsive pocket molecule-functionalized antibacterial implant. The present invention uses a titanium-based implant commonly used in orthopedic implants as the base material, and modifies the pH-responsive pocket molecule on the surface of the titanium-based implant by using the "thiol-ene" click reaction, endowing it with anti-infection function and biological activity. The present invention uses the pH-responsive pocket molecule as an antibacterial agent carrier, and the prepared functionalized antibacterial implant has pH-responsive antibacterial properties and the function of promoting the repair of tissues around the implant.

[0005] The second object of the present invention is to provide a pH-responsive pocket molecule-functionalized antibacterial implant prepared by the above preparation method.

[0006] The third object of the present invention is to provide an application of the pH-responsive pocket molecule-functionalized antibacterial implant.

[0007] The primary object of the present invention is achieved by the following technical solutions:

[0008] A method for preparing a pH-responsive pocket molecule-functionalized antibacterial implant, comprising the following steps:

[0009] (1) Subject the pretreated titanium-based implant (Ti) to alkali heat treatment;

[0010] (2) Dissolve the thiol-containing silane coupling agent in an organic solvent to prepare a silane coupling agent / organic solvent;

[0011] (3) Immerse the titanium-based implant after alkali heat treatment in step (1) in the silane coupling agent / organic solvent, take it out after soaking, cure at high temperature, wash, and dry with nitrogen to obtain a surface-silanized titanium-based implant (Ti-S);

[0012] (4) Dissolve the pH-responsive pocket molecule and the initiator in deionized water to prepare a mixed solution, add it to the surface of the surface-silanized titanium-based implant (Ti-S) in step (3), and carry out a reaction under ultraviolet light irradiation to obtain a pocket molecule-grafted titanium-based implant (Ti-S-MP);

[0013] (5) Immerse the pocket molecule-grafted titanium-based implant (Ti-S-MP) in step (4) in an acidic buffer solution containing an antibacterial agent, then rinse with a neutral buffer solution, and dry with nitrogen to obtain a drug-loaded antibacterial implant (Ti-S-MP-C+), that is, a pH-responsive pocket molecule-functionalized antibacterial implant.

[0014] Furthermore, the alkali solution used in the alkali heat treatment in step (1) is an aqueous NaOH solution, and the concentration of the alkali solution is 4-8 mol / L, preferably 5 mol / L.

[0015] Further, the alkali heat treatment conditions in step (1) are as follows: temperature 50 - 80°C, time 18 - 30 h, preferably temperature 60°C, time 24 h.

[0016] Further, the pretreatment in step (1) is specifically as follows: The titanium-based implant is ultrasonically cleaned with deionized water and absolute ethanol in sequence for 10 - 20 min, and then dried with nitrogen.

[0017] Further, the organic solvent in step (2) is an ethanol solution with a volume fraction of 95%, the silane coupling agent is one of 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane or 3-mercaptopropylmethyldimethoxysilane, and the concentration of the silane coupling agent / organic solvent is 0.5 - 2.0 mg / mL. Preferably, the silane coupling agent is 3-mercaptopropyltrimethoxysilane and the concentration is 1.0 mg / mL.

[0018] Further, the soaking treatment conditions in step (3) are as follows: room temperature, time 8 - 24 h, preferably 12 h; the temperature for high-temperature curing is 90 - 120°C, and the time for high-temperature curing is 0.5 - 2 h. Preferably, the temperature for high-temperature curing is 100°C and the time for high-temperature curing is 1 h.

[0019] Further, in step (4), the structural formula of the pH-responsive pocket molecule is Formula I:

[0020]

[0021] Among them, R1, R2 are -H or -OH, n represents the degree of polymerization of histidine units, and n is an integer between 5 and 15. Preferably, R1 = -OH, R2 = -OH, and n = 7.

[0022] The preparation method of the pH-responsive pocket molecule described in the present invention refers to the invention patent "A pH-responsive pocket molecule and its preparation method and application" submitted by the research group of the present inventor on the same day.

[0023] Further, in step (4), the surface-silanized titanium-based implant (Ti-S) is rinsed twice with deionized water before grafting the pocket molecule to remove the silane coupling agent not grafted on the surface, and then dried with nitrogen.

[0024] Further, in the mixed solution in step (4), the concentration of the pH-responsive pocket molecule is 50 - 200 μmol / L, the concentration of the initiator is 0.5 - 2.0 mg / mL, and the ultraviolet light irradiation reaction time is 10 - 90 min. Preferably, the concentration of the pocket molecule is 100 μmol / L, the concentration of the initiator (I2959) is 1.0 mg / mL, and the ultraviolet light irradiation reaction time is 60 min.

[0025] Further, the initiator described in step (4) is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959).

[0026] Further, the pocket molecule-functionalized implant Ti-S-MP described in step (5) is rinsed twice with deionized water and dried with nitrogen before drug loading.

[0027] Further, the antibacterial agent described in step (5) is one of triclosan, chlorhexidine, metronidazole or tinidazole, and the concentration of the antibacterial agent is 50 - 500 μg / mL. Preferably, the antibacterial agent is a chlorhexidine solution with a concentration of 100 μg / mL.

[0028] Further, the acidic buffer solution described in step (5) is PBS with a pH of 5.0 - 6.0, preferably PBS with a pH of 6.0; the neutral buffer solution is PBS with a pH of 7.4.

[0029] The second object of the present invention is achieved by the following technical solutions:

[0030] A pH-responsive pocket molecule-functionalized antibacterial implant is prepared by the above preparation method.

[0031] The third object of the present invention is achieved by the following technical solutions:

[0032] An application of a pH-responsive pocket molecule-functionalized implant in the preparation of biomedical materials.

[0033] The present invention has the following advantages and beneficial effects compared with the prior art:

[0034] (1) The pH-responsive pocket molecule-functionalized antibacterial implant prepared by the present invention has excellent stability and can adapt to the entire healing period after the orthopedic implant is implanted.

[0035] (2) The pH-responsive pocket molecule-functionalized antibacterial implant prepared by the present invention has pH responsiveness and can be endowed with excellent antibacterial and anti-biofilm formation activities by loading different antibacterial agents. The implant can release antibacterial agents to kill bacteria in an acidic environment.

[0036] (3) The surface of the pH-responsive pocket molecule-functionalized antibacterial implant prepared by the present invention contains highly bioactive cholic acid compounds, enabling it to have functions such as promoting bone integration.

[0037] (4) The surface of the pH-responsive pocket molecule-functionalized antibacterial implant prepared by the present invention has excellent biocompatibility. The pocket molecules modified on the surface of the titanium-based implant can control the release of antibacterial agents and avoid the sudden release of antibacterial agents causing excessive cytotoxicity. Brief Description of the Drawings

[0038] Figure 1 The surface N element peak characterized by XPS after the pocket molecule grafted titanium-based implant (Ti-S-MP) prepared in Example 1 was continuously incubated in a simulated oral environment for 90 days;

[0039] Figure 2 The survival rate results of Staphylococcus aureus cultured on the titanium-based implant (Ti) and pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in Example 1;

[0040] Figure 3 The (a) crystal violet staining map and (b) quantitative analysis results of biofilms after Staphylococcus aureus was cultured on the titanium-based implant (Ti), pocket molecule grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in Example 1 for 48 h;

[0041] Figure 4 The results of cytocompatibility tests on the surfaces of the titanium-based implant (Ti), pocket molecule grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in Example 1;

[0042] Figure 5 The results of in vitro osteogenic performance tests on the surfaces of the titanium-based implant (Ti), pocket molecule grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in Example 1. Detailed implementation manners

[0043] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0044] The pH-responsive pocket molecule used in this embodiment has the following structural formula:

[0045] Among them, R1 = -OH, R2 = -OH, n = 7.

[0046] The specific preparation steps of the pH-responsive pocket molecule used in this embodiment are as follows:

[0047] (1) Weigh 1 g of Rink Amide-MBHA Resin with a substitution degree of 0.38 mmol / g, put it into the reaction tube of the peptide synthesizer, and add 15 mL of anhydrous DCM. After shaking for 30 min, remove the DCM;

[0048] (2) Add 15 mL of 20% piperidine / DMF solution to the reaction tube, wash for 10 min, and then remove the solvent;

[0049] (3) Dissolve 706.5 mg of Fmoc-His(Trt)-OH, 432.3 mg of HBTU, and 491.2 mg of DIEA in 15 mL of anhydrous DMF, add them to the above reactor, and after vibrating the reaction for 60 min, remove the solvent;

[0050] (4) Continue to add 15 mL of a 20% piperidine / DMF solution to the reaction tube, oscillate the reaction for 15 min, and then remove the solvent;

[0051] (5) Add DMF, methanol, and DMF in sequence for two washes, with the solvent dosage being 15 mL each time;

[0052] (6) Repeat the operation steps (3) to (5) six more times to form a polypeptide containing 7 amino acid units on the resin;

[0053] (7) Add DMF, DCM, and methanol in sequence to the reactor for three washes, with the solvent dosage being 15 mL each time. After the washing is completed, drain the solvent;

[0054] (8) Prepare a cleavage solution containing 95% TFA, 2% water, 2% EDT, and 1% TIS in 10 mL, add it to the reactor, and after cleavage for 180 min, collect the cleavage solution;

[0055] (9) Blow dry the cleavage solution with nitrogen, add ether to precipitate, wash it with ether multiple times, centrifuge to remove the supernatant, and then volatilize it at room temperature to obtain polyhistidine polypeptide;

[0056] (10) Weigh 10 g of cholanic acid, dissolve it in 30 mL of anhydrous DMF, add 3.95 g of K2CO3 and 3.488 g of benzyl bromide respectively, and reflux at 50 °C for 20 h; after the reaction is completed, filter to remove inorganic salts, disperse the organic phase in a 5 wt% NaHCO3 solution to form a white precipitate; filter and collect the precipitate, wash it with deionized water multiple times, and vacuum dry it overnight to obtain Compound I;

[0057] (11) Dissolve 1.5 g of Compound I and 285.5 mg of anhydrous pyridine in 10 mL of anhydrous THF, and stir and mix them in a nitrogen atmosphere and an ice bath for 30 min; weigh 725 mg of p-nitrophenyl chloroformate, dissolve it in 5 mL of anhydrous THF, and slowly add this solution dropwise to the above mixed solution containing Compound I using a constant pressure dropping funnel; after the dropping is completed, react at room temperature for 5 h, and separate and purify the mixture by column chromatography to obtain Compound II, with the eluent being a mixed solvent of petroleum ether:ethyl acetate = 3:1;

[0058] (12) Weigh 1.5 g of Compound II, dissolve it in 15 mL of anhydrous DMF, add 91.75 mg of tris(2-amino)ethylamine, and react at room temperature for 5 h. After the reaction, Compound III is obtained by separation and purification using column chromatography, and the eluent is a mixed solvent of petroleum ether:ethyl acetate = 4:1;

[0059] (13) Weigh 100 mg of Compound III, dissolve it in 1 mL of anhydrous DMF, add 703.23 mg of 3-bromopropene, and reflux at 60 °C for 24 h; after the reaction, the organic solution is dispersed in diethyl ether, the precipitate is collected, washed with clean diethyl ether, and dried under vacuum overnight to obtain Compound IV;

[0060] (14) Weigh 112.2 mg of Compound IV, dissolve it in a mixed solution of 3 mL of anhydrous THF and 1 mL of deionized water, add 8.8 mg of LiOH to the above system, stir to dissolve, after 4 h, rotary evaporate to remove THF, adjust the pH of the solution to 2 with hydrochloric acid, a white precipitate appears, filter to collect the precipitate, wash it with deionized water multiple times, and dry under vacuum overnight to obtain Compound V;

[0061] (15) Weigh 38.15 mg of Compound V, 43.76 mg of HATU, 15.55 mg of HOBt, and 14.13 mg of DMAP, dissolve them in 2 mL of anhydrous DMF, and stir and activate at room temperature for 2 h; after dissolving 100 mg of polyhistidine polypeptide in 1 mL of anhydrous DMF, add it to the above solution, and stir and react at room temperature for 24 h; after the reaction, the organic solution is dispersed in diethyl ether, the precipitate is collected, washed with clean diethyl ether, and dried under vacuum overnight to obtain the pH-responsive pocket molecule.

[0062] Example 1

[0063] The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant described in this example includes the following steps:

[0064] (1) Ultrasonically clean the titanium-based implant with anhydrous ethanol and deionized water in sequence for 20 min, dry it with nitrogen, and then place it in a 5 mol / L NaOH aqueous solution for alkali heat treatment at 60 °C for 24 h;

[0065] (2) Dissolve the thiol-containing 3-mercaptopropyltrimethoxysilane coupling agent in a 95% ethanol solution by volume to prepare a 3-mercaptopropyltrimethoxysilane coupling agent / ethanol solution with a concentration of 1 mg / mL;

[0066] (3) Place the titanium-based implant after alkali heat treatment in step (1) in a well plate, add 3-mercaptopropyltrimethoxysilane coupling agent / ethanol solution to each well to submerge the implant, soak it at room temperature for 12 h, then take out the implant and cure it in an oven at 100 °C for 1 h;

[0067] (4) Wash the implant solidified in step (3) twice with deionized water to remove the silane coupling agent not grafted on the surface, and dry it with nitrogen to obtain a surface-silanized titanium-based implant (Ti-S).

[0068] (5) Dissolve the pH-responsive pocket molecule and initiator (I2959) in deionized water, and add them to the surface of the surface-silanized titanium-based implant (Ti-S) so that the final concentration of the pocket molecule is 100 μM and the final concentration of the initiator (I2959) is 1.0 mg / mL, and initiate for 60 min under ultraviolet light irradiation.

[0069] (6) Wash the sample in step (5) twice with deionized water to remove the pocket molecules not grafted on the surface, and dry it with nitrogen to obtain a pocket molecule-grafted titanium-based implant (Ti-S-MP).

[0070] (7) Immerse the pocket molecule-grafted titanium-based implant (Ti-S-MP) in a chlorhexidine solution with a pH of 6.0 containing 100 μg / mL, then transfer it to a PBS buffer with a pH of 7.4, and dry it with nitrogen to obtain a drug-loaded antibacterial implant (Ti-S-MP-C+), that is, a pH-responsive pocket molecule-functionalized antibacterial implant.

[0071] The grafting stability of the pocket molecule-grafted titanium-based implant (Ti-S-MP) prepared in this example was detected. The pocket molecule-grafted titanium-based implant (Ti-S-MP) was immersed in simulated body fluid and continuously incubated on a shaker at 37 °C and 100 rpm for 90 days. Samples were taken out and dried every 10 days, and the N element distribution on the surface of Ti-S-MP was detected by XPS. The results are shown in Figure 1 。 From Figure 1 It can be seen that the elemental peak intensity (N 1s) of the surface-grafted pocket molecule remains basically unchanged, indicating that the content of the surface-grafted pocket molecule remains basically unchanged. The pocket molecule-grafted titanium-based implant (Ti-S-MP) has excellent stability in a simulated human environment and can adapt to the entire cycle of bone implant healing after implantation.

[0072] The pH-responsive antibacterial property of the pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in this example was detected, and Staphylococcus aureus (S. aureus ATCC6538) was selected for the experiment. All consumables used in the bacterial experiments were sterilized by high-temperature and high-pressure treatment to prevent the influence of bacterial contamination on the experimental results. A small amount of Staphylococcus aureus colonies were picked up with an inoculation needle and added to sterile nutrient broth, and incubated on a shaker at 37 °C and 220 rpm for 12 h, and then diluted to 1×10 with PBS with pH values of 7.4, 6.0, and 5.0, respectively 6Bacterial suspension with a concentration of CFU / mL. Place the Ti-S-MP-C+ sample prepared in this example in a well plate, drop 20 μL of bacterial suspensions with different pH values onto the surface of each sample, and incubate in a 37 °C mold incubator for 2 h. Add PBS with a pH of 7.4 to each sample well to dilute the bacteria to a concentration of 1×10 4 CFU / mL. Take 10 μL and spread it on the surface of an agar plate. After the agar plate is placed in a 37 °C mold incubator for 16 h, count the colonies and calculate their bacterial survival rate. The antibacterial activity of the implant prepared in this example is as Figure 2 shown. The pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) does not release chlorhexidine to sterilize in an environment with a pH of 7.4, while the antibacterial rates increase from 16.83% to 95.64% and 96.80% respectively in environments with pH values of 6.0 and 5.0, indicating that the pH-responsive pocket molecule-functionalized surface can respond to the acidic environment of bacterial infection and release more chlorhexidine to sterilize.

[0073] The anti-biofilm formation of the pocket molecule-grafted titanium-based implant prepared in this example was detected, and Staphylococcus aureus (S. aureus ATCC6538) was selected for the experiment. Dilute the bacteria to 10 8 CFU / mL with TSB broth, pipette 10 μL of the bacterial solution and drop it onto the surface of the sample, then add 990 μL of TSB broth, and incubate in a 37 °C mold incubator for 48 h. Every 24 h, pipette 500 μL of the broth in the sample and replenish 500 μL of fresh TSB broth. After culturing for 48 h, discard the culture medium, and gently wash the surface of the sample twice with sterile PBS to remove the adsorbed surface bacteria. Add 300 μL of crystal violet staining solution to each sample, stain in the dark for 30 min, and then gently wash the surface twice with PBS. Observe the formation of biofilms under bright field microscopy. To quantify the formation of biofilms, transfer the samples to a new 24-well plate, add 300 μL of absolute ethanol to each well to dissolve the biofilm and crystal violet complex on the surface, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 590 nm to quantitatively analyze the biofilms on the surface of the samples. The anti-biofilm formation activities of the titanium-based implant (Ti), pocket molecule-grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized antibacterial implant (Ti-S-MP-C+) prepared in this example are as Figure 3 shown. As can be seen from Figure 3 a, a thick and firm biofilm has formed on the surfaces of the Ti and Ti-S-MP groups and is stained purple by crystal violet, while the pocket molecule-functionalized surface-loaded chlorhexidine Ti-S-MP-C+ group can respond to the local acidic environment of biofilm growth and release chlorhexidine to resist biofilm formation. Figure 3The quantitative results showed that Ti-S-MP-C+ could resist 87.36% of biofilm formation.

[0074] The cytocompatibility of the pocket molecule-grafted titanium-based implant prepared in this example was detected, and human mesenchymal stem cells (hMSCs) were selected for the experiment. The implant was placed in a 24-well plate and sterilized overnight by ultraviolet light. The hMSCs in the culture flask were digested with trypsin, centrifuged and resuspended, counted and diluted to an appropriate concentration, and then inoculated on the surface of the samples in the well plate at an inoculation density of 2×10 4 cells / well. The well plate was incubated in an incubator at 37°C and 5% CO2. On the 1st, 3rd, and 5th days of culture, the CCK-8 kit was used to detect the survival rate of hMSCs cells on the surface of the implant. The cytocompatibility results of the titanium-based implant (Ti), pocket molecule-grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized antibacterial implant (Ti-S-MP-C+) prepared in this example are as Figure 4 shown. On the 5th day, the cell viabilities of the Ti-S-MP and Ti-S-MP-C+ groups were 0.96 times and 0.87 times that of pure Ti, respectively. After the pH-responsive pocket molecule-functionalized surface was loaded with chlorhexidine, it could control the release of chlorhexidine molecules, and the cell viability of the Ti-S-MP-C+ group decreased slightly, avoiding the high cytotoxicity caused by the burst release of chlorhexidine.

[0075] The osteogenic ALP activity of the pocket molecule-grafted titanium-based implant prepared in this example was detected, and human mesenchymal stem cells (hMSCs) were selected for the experiment. The implant was placed in a 24-well plate and sterilized overnight by ultraviolet light. The hMSCs in the culture flask were digested with trypsin, centrifuged and resuspended, counted and diluted to an appropriate concentration, and then inoculated on the surface of the samples in the well plate at an inoculation density of 5×10 4 cells / well. The well plate was incubated in an incubator at 37°C and 5% CO2. After 1 day of culture, the culture medium was aspirated and osteogenic induction medium was added. After continuing to culture for 7 days or 14 days, the implant was transferred to a new 24-well plate and Western and IP cell lysate was added. The lysate was collected in a centrifuge tube and centrifuged at 12,000 rpm / min for 3 min, and the ALP activity of the cell supernatant was detected using an ALP detection kit. The cytocompatibility results of the titanium-based implant (Ti), pocket molecule-grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized antibacterial implant (Ti-S-MP-C+) prepared in this example are as Figure 5As shown, on day 7, the ALP activity of cells grown on Ti-S-MP and Ti-S-MP-C+ surfaces was 1.18 and 0.93 times higher than that on the blank Ti surface, respectively; on day 14, the ALP activity of cells grown on these surfaces was 1.14 and 0.89 times higher than that on the blank Ti surface, respectively. Ti-S-MP further enhanced ALP activity, facilitating the differentiation of stem cells into osteoblasts. However, after loading with chlorhexidine, the ALP activity of the Ti-S-MP-C+ group decreased slightly due to the cytotoxicity of chlorhexidine, but the stem cells still showed a strong tendency to differentiate into osteoblasts.

[0076] Example 2

[0077] (1) The titanium-based implant was ultrasonically cleaned with anhydrous ethanol and deionized water for 20 min, dried with nitrogen, and then placed in a 6 mol / L NaOH aqueous solution for alkaline heat treatment at 60°C for 24 h.

[0078] (2) dissolving a thiol-containing 3-mercaptopropyltrimethoxysilane coupling agent in a 95% by volume ethanol solution to prepare a 2 mg / mL 3-mercaptopropyltrimethoxysilane coupling agent / ethanol solution;

[0079] (3) The titanium-based implant subjected to the alkali heat treatment in step (1) was placed in a well plate, and a 3-mercaptopropyltrimethoxysilane coupling agent / ethanol solution was added to each well to cover the implant. After soaking at room temperature for 10 hours, the implant was removed and placed in a 100° C. oven for curing for 1 hour;

[0080] (4) The implant cured in step (3) was washed twice with deionized water to remove the silane coupling agent not grafted on the surface, and dried with nitrogen to obtain a surface silanized titanium-based implant (Ti-S);

[0081] (5) The pH-responsive pocket molecule and initiator (I2959) were dissolved in deionized water and added to the surface of the titanium-silanized implant (Ti-S) to a final concentration of 150 μM for the pocket molecule and 1.0 mg / mL for the initiator (I2959). The initiation was carried out under ultraviolet light for 60 min.

[0082] (6) The sample of step (5) was washed twice with deionized water to remove the pocket molecules not grafted on the surface, and dried with nitrogen to obtain a pocket molecule grafted titanium-based implant (Ti-S-MP);

[0083] (7) The pocket molecule grafted titanium-based implant (Ti-S-MP) was immersed in a chlorhexidine solution containing 100 μg / mL at pH 6.0, then transferred to a PBS buffer solution at pH 7.4, and blown dry with nitrogen to obtain a drug-loaded antibacterial implant (Ti-S-MP-C+), namely, a pH-responsive pocket molecule functionalized antibacterial implant.

[0084] The antibacterial rates of the pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in this example were increased from 15.23% in a pH 7.4 environment to 96.13% and 95.40% in pH 6.0 and 5.0 environments, respectively, and Ti-S-MP-C+ was able to resist 89.53% of biofilm formation. The in vitro osteogenic activities of the titanium-based implant (Ti), the pocket molecule-grafted titanium-based implant (Ti-S-MP), and the pH-responsive pocket molecule-functionalized antibacterial implant (Ti-S-MP-C+) prepared in this example were detected. The results showed that: on the 7th day, the ALP activities of the cells on the surfaces of Ti-S-MP and Ti-S-MP-C+ were 1.23 and 0.95 times that of the blank Ti surface, respectively; on the 14th day, the ALP activities of the above-mentioned surface cells were 1.19 and 0.90 times that of the blank Ti surface, respectively.

[0085] Example 3

[0086] (1) The titanium-based implant was ultrasonically cleaned with absolute ethanol and deionized water for 20 min in sequence, dried with nitrogen, and then subjected to alkali heat treatment in a 6 mol / L NaOH aqueous solution at 60 °C for 20 h;

[0087] (2) The mercapto group-containing 3-mercaptopropyltrimethoxysilane coupling agent was dissolved in an ethanol solution with a volume fraction of 95% to prepare a 3-mercaptopropyltrimethoxysilane coupling agent / ethanol solution with a concentration of 1 mg / mL;

[0088] (3) The titanium-based implant subjected to alkali heat treatment in step (1) was placed in a well plate, and the 3-mercaptopropyltrimethoxysilane coupling agent / ethanol solution was added to each well to submerge the implant. After soaking at room temperature for 16 h, the implant was taken out and placed in an oven at 100 °C for curing for 1 h;

[0089] (4) The implant cured in step (3) was washed twice with deionized water to remove the silane coupling agent not grafted on the surface, and dried with nitrogen to obtain a surface-silanized titanium-based implant (Ti-S);

[0090] (5) The pH-responsive pocket molecule and the initiator (I2959) were dissolved in deionized water and added to the surface of the surface-silanized titanium-based implant (Ti-S) to make the final concentration of the pocket molecule 100 μM and the final concentration of the initiator (I2959) 2.0 mg / mL, and initiated under ultraviolet light for 30 min;

[0091] (6) The sample in step (5) was washed twice with deionized water to remove the pocket molecule not grafted on the surface, and dried with nitrogen to obtain a pocket molecule-grafted titanium-based implant (Ti-S-MP);

[0092] (7) Immerse the pocket molecule-grafted titanium-based implant (Ti-S-MP) in a chlorhexidine solution with a pH of 6.0 containing 100 μg / mL, then transfer it to a PBS buffer with a pH of 7.4, and dry it with nitrogen to obtain a drug-loaded antibacterial implant (Ti-S-MP-C+), that is, a pH-responsive pocket molecule-functionalized antibacterial implant.

[0093] The antibacterial rates of the pH-responsive pocket molecule-functionalized implant (Ti-S-MP-C+) prepared in this example at pH 6.0 and 5.0 environments increased from 13.69% in the pH 7.4 environment to 95.78% and 97.30% respectively, and Ti-S-MP-C+ was able to resist 87.51% of biofilm formation. The in vitro osteogenic activities of the titanium-based implant (Ti), pocket molecule-grafted titanium-based implant (Ti-S-MP), and pH-responsive pocket molecule-functionalized antibacterial implant (Ti-S-MP-C+) prepared in this example were detected. The results showed that: at day 7, the ALP activities of the cells on the surfaces of Ti-S-MP and Ti-S-MP-C+ were 1.30 and 0.87 times that of the blank Ti surface respectively; at day 14, the ALP activities of the above-mentioned surface cells were 1.21 and 0.93 times that of the blank Ti surface respectively.

[0094] 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 preparation method of a pH-responsive pocket molecule-functionalized antibacterial implant, characterized in that It includes the following steps: (1) Subject the pretreated titanium-based implant to alkali heat treatment; (2) Dissolve the thiol-containing silane coupling agent in an organic solvent to prepare a silane coupling agent / organic solvent solution; (3) Immerse the titanium-based implant after alkali heat treatment in step (1) in the silane coupling agent / organic solvent solution, take it out after the immersion, cure it at a high temperature, wash it, and dry it with nitrogen to obtain a surface-silanized titanium-based implant; (4) Dissolve the pH-responsive pocket molecule and the initiator in deionized water to prepare a mixed solution, add it to the surface of the surface-silanized titanium-based implant in step (3), and carry out a reaction under ultraviolet light irradiation to obtain a pocket molecule-grafted titanium-based implant; (5) Immerse the pocket molecule-grafted titanium-based implant in step (4) in an acidic buffer solution containing an antibacterial agent, then rinse it with a neutral buffer solution, and dry it with nitrogen to obtain a drug-loaded antibacterial implant, that is, a pH-responsive pocket molecule-functionalized antibacterial implant.

2. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that, The alkali solution used in the alkali heat treatment in step (1) is an aqueous NaOH solution.

3. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that The pretreatment in step (1) is specifically: ultrasonically clean the titanium-based implant with deionized water and absolute ethanol in sequence for 10 - 20 min, and then dry it with nitrogen.

4. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that, The organic solvent in step (2) is an ethanol solution with a volume fraction of 95%, the silane coupling agent is one of 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, or 3-mercaptopropylmethyldimethoxysilane, and the concentration of the silane coupling agent / organic solvent is 0.5 - 2.0 mg / mL.

5. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that, The conditions for the immersion treatment in step (3) are: room temperature, time 8 - 24 h, preferably 12 h; the temperature for high-temperature curing is 90 - 120 °C, and the time for high-temperature curing is 0.5 - 2 h.

6. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that, In step (4), the structural formula of the pH-responsive pocket molecule is formula I: Among them, R1 and R2 are -H or -OH, n represents the polymerization degree of the histidine unit, and n is an integer between 5 and 15.

7. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that, In the mixed solution in step (4), the concentration of the pH-responsive pocket molecule is 50 - 200 μmol / L, the concentration of the initiator is 0.5 - 2.0 mg / mL, and the reaction time under ultraviolet light irradiation is 10 - 90 min.

8. The preparation method of the pH-responsive pocket molecule-functionalized antibacterial implant according to claim 1, characterized in that, The antibacterial agent in step (5) is one of triclosan, chlorhexidine, metronidazole, or tinidazole, and the concentration of the antibacterial agent is 50 - 500 μg / mL.

9. A pH-responsive pocket molecule-functionalized antibacterial implant, characterized in that, Prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the pH-responsive pocket molecule-functionalized implant according to claim 9 in the preparation of biomedical materials.

Images