A medical titanium metal surface slow-release antibacterial peptide coating and a preparation method and application thereof

Abstract

The application discloses a medical titanium metal surface slow-release antibacterial peptide coating and a preparation method and application thereof, relates to the technical field of biomedical materials, and comprises the following steps: after medical titanium is activated, silane polyethylene glycol active ester, azide-active oxygen response connecting arm N3-TK-PEG-NH2 and DBCO modified antibacterial peptide DBCO-GL13K are sequentially grafted; and an active oxygen response slow-release antibacterial coating is constructed on the surface of the medical titanium through freeze drying; the medical titanium metal surface slow-release antibacterial peptide coating and the preparation method and application thereof connect the active oxygen response bond TK and the antibacterial peptide GL13K on the surface of the medical titanium metal; the TK bond is oxidized and broken by active oxygen to release the antibacterial peptide GL13K, drug controlled release and precise release are achieved, excessive active oxygen is consumed, the local microenvironment homeostasis is maintained, and tissue healing and regeneration in an inflammatory state are promoted.

Description

Technical Field

[0001] This invention relates to biomedical materials technology, specifically to a medical titanium metal surface sustained-release antimicrobial peptide coating, its preparation method, and its application. Background Technology

[0002] Implantable medical devices refer to devices that are inserted into the human body, either wholly or partially, through surgical procedures and are expected to remain in place for at least 30 days. Examples include titanium and titanium alloy bone fixation devices in the orthopedic field (such as bone plates, bone screws, intramedullary nails, spinal fixation systems and their accessories) and titanium-based dental implants in the field of dental restoration.

[0003] One of the core challenges facing the clinical application of such devices is implant-related infection. This infection stems from the introduction of pathogenic microorganisms (such as Staphylococcus aureus and Staphylococcus epidermidis) during the implantation process. These microorganisms readily adhere to and aggregate on the implant surface, secreting extracellular matrix to form a biofilm with a complex three-dimensional structure. This biofilm poses a significant obstacle to traditional antibacterial methods: on the one hand, its physical barrier effect significantly reduces the permeability and effectiveness of antibiotics; on the other hand, it can facilitate the escape of pathogens from the host's immune system (such as macrophages and neutrophils) from recognition and clearance, leading to chronic, latent, and recurrent infections. Once an infection occurs, a second surgery is often required to replace or remove the implant, not only increasing the patient's suffering, medical risks, and economic burden, but also resulting in a huge drain on public medical resources. Summary of the Invention

[0004] The purpose of this invention is to provide a sustained-release antimicrobial peptide coating for medical titanium metal surfaces, its preparation method, and its application, in order to solve the problem that existing implantable medical devices are prone to causing biofilm-related infections.

[0005] To achieve the above objectives, the present invention provides a method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface, comprising the following steps:

[0006] S1. After cleaning and drying the pure titanium / titanium alloy implant, acid etching and alkaline etching are performed, followed by nitrogen drying to obtain eTi; Silance-PEG-NHS is dissolved in dimethyl sulfoxide (DMSO) to obtain mixed solution a;

[0007] S2. React the mixed solution a with eTi to obtain eTi-NHS after the reaction is complete;

[0008] S3. Dissolve N3-TK-PEG-NH2 in deionized water to obtain mixed solution b; soak eTi-NHS in mixed solution b to react, and after the reaction is complete, eTi-TK-N3 is obtained.

[0009] S4. Dissolve DBCO-GL13K in deionized water to obtain mixed solution c; soak eTi-TK-N3 in mixed solution c for 1 hour, clean and dry after soaking, then pre-freeze at -80℃ for 4 hours, freeze-dry for 4 hours to obtain eTi-TK-GL13K, that is, generate a sustained-release antimicrobial peptide coating on the surface of medical titanium metal.

[0010] Furthermore, the preparation method of N3-TK-PEG-NH2 in S3 includes the following steps:

[0011] A1. The diamino ketone thioglycolate copolymer (NH2-TK-NH2) was dissolved in N,N-dimethylformamide (DMF) and subjected to a condensation reaction with azidoacetic acid in the presence of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) to specifically modify one of its amino groups. After the reaction was completed, the intermediate N3-TK-NH2 was obtained by concentration, column chromatography purification and drying.

[0012] A2. BOC-polyethylene glycol carboxyl group (Boc-NH-PEG) 2000 -COOH) and N3-TK-NH2 were condensed in DMF under the catalysis of PyBOP and DIPEA. The reaction solution was concentrated, dissolved in dichloromethane, washed and purified with water. The resulting product was treated with trifluoroacetic acid, and finally obtained by precipitation with diethyl ether, centrifugation and drying to obtain the target product N3-TK-PEG-NH2.

[0013] Furthermore, the structural formula of DBCO-GL13K in S4 is as follows:

[0014] .

[0015] Furthermore, the preparation method of DBCO-GL13K in S4 includes the following steps:

[0016] B1. Using the Fmoc solid-phase synthesis method, dichloropolymer resin was used as the solid-phase support. The amino acids corresponding to the sequence Gly-Gly-Gly-Lys-Ile-Ile-Lys-Leu-Lys-Ala-Ser-Leu-Lys-Leu-Leu were sequentially linked from the C-terminus to the N-terminus. Each cycle included amino acid condensation, Fmoc protecting group removal, and washing steps, and the reaction efficiency was monitored by the ninhydrin method. After all peptide chains were assembled, the target peptide was cleaved from the resin using a mixed lysis buffer containing trifluoroacetic acid, water, and triisopropylsilane. The cleaved peptide was then precipitated with cold diethyl ether, centrifuged, and vacuum dried to obtain the crude product GGGKIIKLKASLKLL-NH2.

[0017] B2. GGGKIIKLKASLKLL-NH2 was dissolved in DMF and reacted with diphenylcyclooctyne-carboxylic acid (DBCO-COOH) in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and DIPEA. After the reaction was completed, the product was separated and purified by reversed-phase high-performance liquid chromatography. The main peak fraction containing the target product was collected and freeze-dried to obtain the final product DBCO-GL13K.

[0018] Furthermore, the acids used for etching in S1 are HCl and H2SO4.

[0019] Furthermore, the alkali used for alkaline etching in S1 is NaOH.

[0020] Furthermore, the reaction time in S2 is 1 hour.

[0021] Furthermore, the reaction time in S3 is 1 hour.

[0022] Another aspect of the present invention provides a medical titanium metal surface sustained-release antimicrobial peptide coating, which is prepared by the aforementioned preparation method.

[0023] Another aspect of the present invention provides the application of a sustained-release antimicrobial peptide coating on the surface of medical titanium metal in the preparation of orthopedic titanium-based implants and dental titanium-based implants.

[0024] Compared with existing technologies, the present invention provides a sustained-release antimicrobial peptide coating for medical titanium metal surfaces, its preparation method, and its application. By linking reactive oxygen species (ROS) responsive bonds TK and antimicrobial peptide GL13K to the surface of medical titanium metal, the antimicrobial peptide GL13K can inhibit the formation of bacterial biofilms in implants and reduce the risk of inflammation. Once inflammation occurs, the TK bonds are oxidized and broken by ROS to release the antimicrobial peptide GL13K, achieving effective controlled and precise drug release. At the same time, it consumes excess ROS, maintains local microenvironment homeostasis, and promotes tissue healing and regeneration under inflammatory conditions. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0026] Figure 1 The proton NMR spectrum of N3-TK-PEG-NH2 provided in the embodiments of the present invention;

[0027] Figure 2The proton NMR spectrum of DBCO-GL13K provided in this embodiment of the invention;

[0028] Figure 3 Physical property characterization of eTi, eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K provided in embodiments of the present invention;

[0029] Figure 4 XPS energy spectrum of eTi-TK-GL13K provided in an embodiment of the present invention;

[0030] Figure 5 Scanning electron microscope images of eTi, eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K provided in embodiments of the present invention;

[0031] Figure 6 AFM height maps of eTi, eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K provided in embodiments of the present invention;

[0032] Figure 7 Biocompatibility of eTi-TK-GL13K provided in the embodiments of the present invention;

[0033] Figure 8 The graph shows the relative fluorescence intensity of fluorescein released by eTi-Flu and eTi-TK-Flu in different concentrations of H2O2 as a function of time, according to embodiments of the present invention.

[0034] Figure 9 The in vitro antioxidant effects of eTi, eTi-GL13K, and eTi-TK-GL13K provided in the embodiments of the present invention are shown in the figure.

[0035] Figure 10 CFU analysis of bacteria on the surface of eTi, eTi-GL13K, and eTi-TK-GL13K provided in the embodiments of the present invention;

[0036] Figure 11 Scanning electron microscope images of bacterial morphology on the surfaces of eTi, eTi-GL13K, and eTi-TK-GL13K provided in embodiments of the present invention;

[0037] Figure 12 Immunofluorescence staining of RAW264.7 cell polarization markers on the surface of eTi, eTi-GL13K, and eTi-TK-GL13K under LPS stimulation, as provided in embodiments of the present invention;

[0038] Figure 13The relative expression levels of inflammation-related genes in RAW264.7 cells stimulated with LPS on the surfaces of eTi, eTi-GL13K, and eTi-TK-GL13K, as provided in the embodiments of the present invention;

[0039] Figure 14 CFU analysis of the in vivo antibacterial effects of eTi, eTi-GL13K, and eTi-TK-GL13K provided in embodiments of the present invention;

[0040] Figure 15 H&E staining results of in vivo chronic inflammation of eTi, eTi-GL13K, and eTi-TK-GL13K provided in embodiments of the present invention;

[0041] Figure 16 The image shows the HO-1 immunohistochemical staining results of eTi, eTi-GL13K, and eTi-TK-GL13K in vivo for chronic inflammation provided in the embodiments of the present invention. Detailed Implementation

[0042] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0043] Example 1:

[0044] Please see Figure 1 , Figure 2 and Figure 3 A, a method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface, includes the following steps:

[0045] S1. After cleaning and drying the pure titanium / titanium alloy implant, acid etching and alkaline etching are performed, followed by nitrogen drying to obtain eTi; Silance-PEG-NHS is dissolved in DMSO to obtain mixed solution a;

[0046] Pure titanium / titanium alloy implants were sequentially cleaned with acetone, anhydrous ethanol, and deionized water, dried at 40°C, and then acid-etched with a mixture of 5.8 mol / L HCl and 8.96 mol / L H2SO4. The implants were then immersed in a water bath at 60°C for 30 minutes, and this process was repeated three times for 10 minutes each time. Next, alkaline etching was performed using 5 mol / L NaOH, followed by immersion in a water bath at 60°C for 24 hours. This process was repeated three times for 10 minutes each time, and the implants were dried under nitrogen to obtain eTi. 1 mM Silance-PEG-NHS was dissolved in DMSO to obtain mixed solution a.

[0047] S2. React the mixed solution a with eTi to obtain eTi-NHS after the reaction is complete;

[0048] The mixed solution a was reacted with eTi at room temperature for 1 hour, and the mixture was shaken at 50 rpm. After washing with DMSO 3 times and deionized water 3-5 times, and dried with nitrogen, eTi-NHS was obtained.

[0049] S3. Dissolve N3-TK-PEG-NH2 in deionized water to obtain mixed solution b; soak eTi-NHS in mixed solution b to react, and after the reaction is complete, eTi-TK-N3 is obtained.

[0050] Synthesis of N3-TK-PEG-NH2:

[0051] Weigh 500 mg of NH2-TK-NH2 and dissolve it in 3 mL of DMF. Add 1.05 eq. of azidoacetic acid, 1.2 eq. of PyBOP reagent, and 3.0 eq. of DIPEA and dissolve completely. React at room temperature for 0.5 hours. After concentration under reduced pressure, purify by column chromatography and dry under vacuum to obtain N3-TK-NH2.

[0052] Weigh 1g of Boc-NH-PEG 2000 -COOH was dissolved in 3 mL of DMF, and N3-TK-NH2 (1.05 eq.), PyBOP reagent (1.2 eq.), and DIPEA (3.0 eq.) were added and dissolved completely. The reaction was carried out at room temperature for 0.5 hours. After concentration under reduced pressure, dichloromethane was added to redissolve the solution, and the mixture was washed three times with water. The reaction solution was concentrated under reduced pressure and redissolved in 6 mL of dichloromethane. 1 mL of trifluoroacetic acid was added, and the reaction was carried out in an ice bath for 30 min. The reaction solution was concentrated under reduced pressure and the precipitate was poured into a large amount of ice-cold diethyl ether. The product was collected by centrifugation and dried under vacuum to obtain N3-TK-PEG-NH2.

[0053] The structural formula of N3-TK-PEG-NH2 is as follows:

[0054] .

[0055] Please see Figure 11H NMR spectrum of N3-TK-PEG-NH2 NMR (7.26 ppm (h / h') shows a proton hydrogen signal peak on the amide at 7.26 ppm in the low field region; two sets of signal peaks at 4.24 ppm (g) are considered to be two sets of methylene proton hydrogen signal peaks adjacent to the carbonyl group, with the chemical shift shifting to the lower field due to the influence of electron-withdrawing groups; the multiplet at 3.66 ppm (a) is a stacked multiple methylene proton hydrogen signal peak on the PEF group, and the nearby 3.52 ppm (c) is a methylene proton hydrogen signal peak adjacent to oxygen; the multiplet at 3.16 ppm (b) is considered to be a proton hydrogen at the α-position of the amino group, which splits into multiplets due to the interaction of the amino and methylene groups; there is a stacked multiplet at 3.11 ppm (e / e'), considered to be multiple sets of methylene proton hydrogen signal peaks adjacent to sulfur; the signal peak at 2.11 ppm (f) is considered to be a proton hydrogen at the terminal amino group; finally, the signal peak at 1.42 ppm (d) in the high field region is the elution position of the proton hydrogen signal peaks of the two methyl groups.

[0056] Dissolve 1 mM N3-TK-PEG-NH2 in deionized water, soak at room temperature for 1 hour, and wash three times with deionized water to obtain eTi-TK-N3.

[0057] S4. Dissolve DBCO-GL13K in deionized water to obtain mixed solution c; soak eTi-TK-N3 in mixed solution c for 1 hour, clean and dry after soaking, then pre-freeze at -80℃ for 4 hours, freeze-dry for 4 hours to obtain eTi-TK-GL13K, that is, generate a sustained-release antimicrobial peptide coating on the surface of medical titanium metal.

[0058] Synthesis of DBCO-GL13K:

[0059] The specific structure of DBCO-GL13K is DBCO-GGGKIIKLKASLKLL-NH2. It is synthesized using the Fmoc solid-phase method with dichlororesin as the solid-phase support. Amino acids are sequentially linked from the C-terminus to the N-terminus. The amino acid sequence is Gly-Gly-Gly-Lys-Ile-Ile-Lys-Leu-Lys-Ala-Ser-Leu-Lys-Leu-Leu, abbreviated as GGGKIIKLKASLKLL, as shown in SEQ ID NO.1 of the amino acid sequence listing.

[0060] (1) Synthesis of Fmoc-Leu-NH2:

[0061] Dichloromethane resin (1.0 eq.) was placed in a peptide synthesis reactor and soaked in excess dichloromethane (DCM). After washing the resin with DMF, it was rotary evaporated and dried under vacuum to obtain NH2- resin. N-(9-fluorenylmethoxycarbonyl)-L-leucine (Fmoc-Leu-OH) (3.0 eq.), the first amino acid at the C-terminus, was added to the reactor, followed by HATU (3.0 eq.), DCM, and DIPEA (6.0 eq.). After thorough mixing, NH2- resin was added, and the reaction was carried out at room temperature for 2 hours. After the reaction was complete, the reaction system was washed multiple times with DMF and dried to obtain Fmoc-Leu-NH2. A 20% (v / v) piperidine solution was added to Fmoc-Leu-NH2 to remove the Fmoc protecting group. After the reaction was complete, the reaction system was washed multiple times with DMF, dried, and the deprotection efficiency was assessed using the ninhydrin method to ensure complete deprotection.

[0062] (2) Linking of the remaining amino acids:

[0063] Connecting the second amino acid at the C-terminus: Add Fmoc-Leu-OH (the second amino acid at the C-terminus) (3.0 eq.), 1-hydroxybenzotriazole (HOBT) (3.0 eq.), and diisopropylcarbodiimide (DIC) (3.0 eq.) to the above reactor, stir evenly, and react at room temperature for 1 hour. After the reaction is complete, the reaction efficiency is detected by the ninhydrin method to ensure that the reaction is complete.

[0064] Deprotection: Wash the reaction system thoroughly with DMF multiple times, dry it, and then add a 20% piperidine solution (by volume) to the reactor. Stir at room temperature for 5-10 minutes to remove the Fmoc protecting group. After the reaction is complete, wash the system thoroughly with DMF multiple times and dry it. Use the ninhydrin method to detect the deprotection efficiency to ensure complete deprotection.

[0065] Subsequent amino acid ligation: Following the above steps for ligating and deprotecting amino acids, the remaining amino acids were ligated sequentially. After each amino acid was ligated, a deprotection operation was performed. The reaction efficiency and deprotection efficiency were detected using the ninhydrin method to ensure that each step of the reaction was complete. After all amino acids were ligated, peptide chain resin was obtained.

[0066] (3) Peptide chain cleavage and purification:

[0067] Peptide chain cleavage: The ligated peptide chains were added to an excess of cleavage buffer containing trifluoroacetic acid (TFA), H2O, and triisopropylsilane (TIS). The mixture was stirred at room temperature for 2 hours to cleave the resin, releasing the peptide chains and yielding a lysate. The lysate was then added dropwise to an excess of 4°C diethyl ether, centrifuged, and the precipitate was collected and dried under vacuum to obtain the target peptide fragment GGGKIIKLKASLKLL-NH2.

[0068] Peptide chain purification: The obtained peptide GGGKIIKLKASLKLL-NH2 was dissolved in DMF, and DBCO-COOH (1.5 eq.), HATU (1.5 eq.) and DIPEA (3.0 eq.) were added. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the product was separated and purified by reversed-phase high-performance liquid chromatography. The main peak fraction containing the target product was collected and freeze-dried to obtain the final product DBCO-GL13K.

[0069] The structural formula of DBCO-GL13K is as follows:

[0070] .

[0071] Please see Figure 2 The 1H NMR of DBCO-GL13K showed multiple signal peaks at 7.46 ppm (A) in the aromatic region, due to the asymmetry of the benzo[a]azine heterocyclic structure in the compound's structural formula and the stacking of multiple signal peaks. Two sets of multiplets with similar chemical environments were observed at 3.76 ppm and 4.33 ppm (E), considered to be the ortho and meta proton signal peaks of the butylamine side chain. The multiplet at 2.89 ppm (B) was considered to be the proton hydrogen signal peak of the methylene group adjacent to the carbonyl group, with the chemical shift towards a higher field due to the influence of electron-withdrawing groups. Finally, the signal peaks at 1.31 ppm (D) and 0.80 ppm (C) in the high-field region were identified as aliphatic proton hydrogens, representing the proton hydrogen signal peaks of the methyl and methylene groups in different chemical environments, respectively.

[0072] 1 mM DBCO-GL13K was first dissolved in deionized water at a ratio of 1:100, vortexed to ensure complete dissolution, then diluted 10 times with deionized water, soaked at room temperature for 1 hour, washed 3 times with deionized water, pre-dried under nitrogen, then pre-frozen at -80℃ for 4 hours, freeze-dried for 4 hours, and dried and stored at -20℃ to obtain eTi-TK-GL13K, which is a sustained-release antimicrobial peptide coating generated on the surface of medical titanium metal.

[0073] Please see Figure 3 Fourier transform infrared spectroscopy (FTIR) analysis of A in the image shows that at 3230 cm⁻¹... -1 The broad peak at 3350 cm⁻¹ is attributed to the stretching vibration of the hydroxyl group (-OH), indicating that the eTi sample contains free hydroxyl groups and has high reactivity. After eTi is coupled with Silance-PEG-NHS, eTi-NHS exhibits a peak at 3350 cm⁻¹. -1 The absorption peak corresponds to the NH stretching vibration of the amide group (-NH-), and a peak at 1622 cm⁻¹ also appears. -1The sharp peak of the asymmetric stretching vibration is consistent with the stretching vibration of the carbonyl group (C=O) in the amide and ester groups. After eTi-NHS is connected to N3-TK-PEG-NH2, the hydrogen bonding of eTi-TK-N3 is significantly enhanced, as evidenced by a red shift of the amide group (-NH-) to 3267 cm⁻¹. -1 At the same time, 1430cm -1 The stretching vibration of CN. After eTi-NHS is connected to DBCO-GL13K, eTi-TK-GL13K exhibits stretching vibration at 1622 cm⁻¹. -1 The absorption peak (asymmetric stretching vibration of C=O) at 1535 cm⁻¹ becomes significantly sharper and higher, indicating that the vibrational response of the C=O group is more concentrated and enhanced. -1 The NH and CH bending vibrations at the point originate from the 1,2,3-triazole ring structure formed by the reaction of the DBCO group and the azide group. These characteristic absorption peaks indicate the presence of functional groups such as hydroxyl, amide, and carbonyl groups in the sample, which is highly consistent with the characteristic chemical structure of eTi-TK-GL13K.

[0074] Comparative Example 1:

[0075] This comparative example provides a technical solution based on Example 1: preparation of eTi-GL13K without ROS-responsive bonds:

[0076] To compare the effect with that of sustained-release antimicrobial peptide coatings on medical titanium metal surfaces, eTi-GL13K without ROS-responsive bonds was synthesized.

[0077] The synthesis process of eTi-GL13K differs from that in Example 1 in that the N3-TK-PEG-NH2 in S3 is dissolved in deionized water and replaced with 11-azido-3,6,9-trioxaundecan-1-amine dissolved in deionized water, thereby preparing eTi-GL13K without ROS-responsive bonds.

[0078] Comparative Example 2:

[0079] This comparative example provides a technical solution based on Example 1: eTi-Flu and eTi-TK-Flu are prepared by replacing the antimicrobial peptide GL13K with fluorescein Flu.

[0080] Preparation method of eTi-Flu: The synthesis process of eTi-Flu differs from that in Example 1 in that: N3-TK-PEG-NH2 in S3 is dissolved in deionized water and then 11-azido-3,6,9-trioxaundecan-1-amine is dissolved in deionized water and then reacted with eTi-NHS to obtain eTi-N3; DBCO-GL13K in S4 is replaced with DBCO-fluorescein (DBCO-Flu), and eTi-N3 reacts with DBCO-Flu to obtain eTi-Flu.

[0081] Preparation method of eTi-TK-Flu: The synthesis process of eTi-TK-Flu differs from that in Example 1 in that DBCO-GL13K in S4 is replaced with DBCO-fluorescein (DBCO-Flu), and eTi-TK-Flu is finally prepared.

[0082] Example 2:

[0083] Please see Figure 3 B and Figures 4 to 6 This embodiment provides a technical solution based on Embodiment 1: Characterization experiments of eTi-TK-GL13K:

[0084] 1. Water contact angle test:

[0085] The water contact angles of eTi, eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K samples were measured using a contact angle meter. The procedure was as follows: the samples were dried with nitrogen and placed on the test stage of the contact angle meter. The meter was turned on and preheated. Water was dispensed using a micro-syringe, and a droplet was squeezed close to the sample surface. Images were acquired through the optical system, and the contact angles were analyzed. All data are expressed as mean ± standard deviation. Levene's test was used to determine homogeneity of variance, and one-way ANOVA and Dunnett's T3 post-hoc test were used to determine statistical differences between groups. The criterion was set at α = 0.05, and p < 0.05 was considered statistically significant. .

[0086] Please see Figure 3 In Figure B, the water contact angle shows that the water contact angle of eTi is less than 20°, indicating that the activated hydroxyl groups make eTi hydrophilic. The water contact angles of eTi-NHS and eTi-TK-N3 increase slightly but remain below 40°. The addition of DBCO-GL13K significantly increases the water contact angle to over 120°, making it a superhydrophobic structure. The hydrophobic surface GL13K peptide is rich in cationic and hydrophobic amino acids, including Ala, Ile, and Leu. Superhydrophobic materials can prevent bacterial adhesion and protect against hydrolysis and degradation in high-pressure, high-temperature water vapor environments, which is beneficial for pre-application sterilization.

[0087] 2. X-ray photoelectron spectroscopy (XPS) characterization experiment:

[0088] The microfocusing X-ray photoelectron spectrometer attaches the eTi-TK-GL13K sample to the sample stage with conductive tape, introduces it into the vacuum chamber, selects a region, scans the sample surface, records the photoelectron signals of different elements, calibrates the original photoelectron signals, and analyzes the chemical state of the elements through peak fitting.

[0089] Please see Figure 4 XPS spectroscopy revealed that all surface element characteristic peaks, including Ti 2p, C 1s, N 1s, O 1s, and S 2p, were identified. Among them, the characteristic element of the TK bond, the S 2p peak, appeared at 163.2 eV, which is a CS bond. This position is clearly distinguishable from oxidized sulfur (>164 eV) or sulfonic acid / sulfone (>168 eV), ruling out excessive oxidation.

[0090] 3. Scanning electron microscopy characterization experiment:

[0091] The eTi, eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K samples were characterized using a scanning electron microscope (SEM). First, the samples were dried with nitrogen gas. The dried samples were then attached to the sample stage with conductive tape, mounted on the SEM sample holder, placed in the SEM, and the observation parameters were adjusted. Images of each sample were then captured.

[0092] Please see Figure 5 Scanning electron microscopy revealed a porous honeycomb structure on the eTi surface with pore sizes of 100-200 nm. This indicates that the titanium sheet developed nanostructure textures after alkaline etching, increasing the surface area and providing ample anchoring sites for subsequent physical adsorption and chemical bonding of molecules. The stepwise coupling of eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K resulted in the pores being filled layer by layer. In the eTi-NHS stage, the pore size decreased to 50-100 nm; in the eTi-TK-N3 stage, only 20-40 nm micropores remained; finally, a continuous organic coating with a coverage rate of >90% was formed on the eTi-TK-GL13K surface, confirming that the drug / peptide layer uniformly coated the entire titanium sheet without aggregation or island-like distribution.

[0093] 4. Atomic Force Microscopy (AFM) Characterization Experiment:

[0094] The eTi, eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K samples were characterized using AFM. Probes were mounted on a high-speed imaging atomic force microscope (AFM), and the samples were placed sequentially on the sample stage. The probe holders were inserted into their respective positions and adjusted. The laser position was adjusted, and the following settings were established: Scan Size 10 μm, Points & Lines 256, Scan Rate 1.22 Hz, Setpoint 608.12 mV, Drive Amplitude 5.34 mV. Scanning was then initiated to acquire data.

[0095] Please see Figure 6AFM height maps showed that the surface of eTi after alkaline etching exhibited a three-dimensional nanoporous structure with an RMS roughness (Rq) of approximately 1.0 μm and a depth of 20-50 nm, forming a honeycomb texture. After sequential modification, the surface undulations of eTi-NHS, eTi-TK-N3, and eTi-TK-GL13K gradually decreased: Rq decreased slightly in the eTi-NHS stage, and further decreased to 0.75 μm in the eTi-TK-N3 stage; the surface of the final product eTi-TK-GL13K reached 0.5-0.75 μm, confirming that the organic coating had uniformly covered the original nanopores, which is consistent with the SEM results.

[0096] Example 3:

[0097] Please see Figure 7 This embodiment provides a technical solution based on Embodiment 1: Biocompatibility testing of eTi-TK-GL13K:

[0098] Biocompatibility of mouse mononuclear macrophage leukemia cells (RAW264.7) cells ( Figure 7 A) and mouse embryonic osteoblasts (MC3T3-E1) cells ( Figure 7 In step B), the biocompatibility of eTi, eTi-GL13K and eTi-TK-GL13K samples was tested.

[0099] Different samples were irradiated with UV light on both sides for 20 min each, and then placed in 96-well plates. Healthy RAW 264.7 cells were collected by gently blowing them away, followed by centrifugation and resuspending. MC3T3-E1 cells were washed with PBS buffer, digested with 0.25% trypsin, and the cell suspension was collected by centrifugation and resuspending. After dilution to appropriate concentrations, cell counting was performed, with RAW 264.7 cells at 5 × 10⁶ cells per well. 3 MC3T3-E1 cells were seeded onto the sample at a rate of 2 × 10⁶ cells per well. 3 Cells were seeded onto the sample in each well, and CCK-8 working solution was added. A cell-free control group was included. Absorbance at 450 nm was measured using a multi-mode microplate reader. During measurement, the cell solution was transferred to the blank wells without sample, and then transferred back to the corresponding sample wells after measurement. Cell proliferation of RAW264.7 cells was measured on days 1 and 3, and on days 5 and 7. All data are expressed as mean ± standard deviation. Levene's test was used to determine homogeneity of variance, and one-way ANOVA and Dunnett's T3 post-hoc test were used to determine statistical differences between groups. The criterion for statistical significance was set at α = 0.05, and p < 0.05 was considered statistically significant. .

[0100] After 3 days of RAW264.7 cell culture and 7 days of MC3T3-E1 cell culture, there were no significant differences in the proliferation and metabolic activities of eTi, eTi-GL13K, and eTi-TK-GL13K compared with the Control group. eTi-TK-GL13K showed a tendency to promote the proliferation of MC3T3-E1 cells. This indicates that the introduction of ROS-responsive bonds and the antimicrobial peptide GL13K did not produce significant cytotoxicity, and the coating did not have a negative impact on cell adhesion.

[0101] Example 4:

[0102] Please see Figure 8 This embodiment provides a technical solution based on Embodiment 1 and Comparative Example 2: ROS responsive release effect detection of eTi-TK-GL13K:

[0103] Using fluorescein to replace the antimicrobial peptide GL13K, the change in fluorescence intensity in solution was measured to represent the relative release of GL13K. eTi was soaked in 1 mM DBCO-Flu as a control for fluorescein self-adhesion on titanium sheets. eTi, eTi-Flu, and eTi-TK-Flu were soaked in 0 mM, 0.01 mM, 0.1 mM, and 1.0 mM H2O2, respectively, and the fluorescence intensity of the leachate was measured for 10 consecutive days (excitation wavelength 480 nm, emission wavelength 525 nm), and the change in fluorescence intensity was calculated.

[0104] Please see Figure 8 eTi-Flu slowly released fluorescein in different concentrations of H2O2, but there was no significant difference between the concentrations, indicating that coatings without ROS-responsive TK bonds could not responsively release the target drug. However, the fluorescein release of eTi-TK-Flu in 0 mM H2O2 was similar to that of eTi-Flu. The fluorescence intensity increased in 0.01, 0.1, and 1.0 mM H2O2 concentrations, and the rate of increase accelerated with increasing H2O2 concentration. eTi-TK-Flu immersed in 0.01 mM H2O2 took approximately 7 days to reach a plateau, the 0.1 mM H2O2 group took 5 days, and the 1.0 mM H2O2 group reached a plateau in 3 days. This indicates that the release rate of the target drug by the ROS-responsive TK bonds increases with the concentration of environmental ROS, enabling controlled release of the target drug.

[0105] Example 5:

[0106] Please see Figure 9 This embodiment provides a technical solution based on Embodiment 1: the in vitro antioxidant effect of eTi-TK-GL13K:

[0107] To verify the in vitro antioxidant effect of eTi-TK-GL13K, its scavenging effect on extracellular and intracellular ROS was examined. The extracellular ROS scavenging effect was assessed using the total antioxidant capacity assay kit (ABTS rapid method). eTi, eTi-GL13K, and eTi-TK-GL13K were incubated with ABTS working solution and peroxidase working solution at 37°C, respectively, and the absorbance in the 400-1000 nm range was scanned. Furthermore, RAW264.7 cells cultured in different groups of samples (eTi, eTi-GL13K, and eTi-TK-GL13K) were stimulated with 0, 0.1, 1.0, and 10.0 μg / mL lipopolysaccharide (LPS). After 12 hours of LPS stimulation, intracellular ROS were observed using DCFH-DA staining and an inverted fluorescence microscope. The fluorescence area was calculated to evaluate the intracellular ROS scavenging effect.

[0108] Please see Figure 9 The absorption spectrum of A shows absorption peaks of ABTS+ after ABTS oxidation at 414 nm and 734 nm. eTi and eTi-GL13K have absorbance exceeding 0.5 at 414 nm and exceeding 0.2 at 734 nm, while eTi-TK-GL13K has absorbance less than 0.2 at 414 nm and less than 0.1 at 734 nm, indicating that eTi-TK-GL13K can effectively scavenge ROS from the extracellular environment.

[0109] Please see Figure 9 The relative fluorescence intensity plots of RAW264.7 cells stimulated with different concentrations of LPS (B) show that intracellular ROS in RAW264.7 cells stimulated on eTi gradually increases with increasing LPS concentration. Although the fluorescence intensity of the eTi-GL13K group increased, it was generally lower than that of eTi because GL13K has an immunomodulatory effect, which can inhibit ROS generation and enhance its clearance to a certain extent. However, eTi-TK-GL13K has the presence of the ROS-responsive bond TK. TK has high reactivity to ROS and is cleaved by excess ROS oxidation. In this process, a portion of ROS is directly consumed, thereby temporarily downregulating the intracellular oxidative load. At the same time, the thiol intermediate generated by the cleavage can further undergo secondary redox reactions with hydrogen peroxide, forming sulfonic acid or disulfide bonds, and continue to clear free radicals.

[0110] Example 6:

[0111] Please see Figure 10 and Figure 11 This embodiment provides a technical solution based on Embodiment 1: In vitro antibacterial effect detection of eTi-TK-GL13K:

[0112] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the main pathogens causing implant infections. The in vitro antibacterial effect of eTi-TK-GL13K was assessed using methicillin-resistant Staphylococcus aureus (MRSA) and E. coli. eTi, eTi-GL13K, and eTi-TK-GL13K were co-cultured with MRSA and E. coli for 6 hours, respectively. Colony-forming unit (CFU) counts were used to analyze bacterial adhesion on titanium sheet samples with different coatings. Scanning electron microscopy was performed on the titanium sheet samples to observe morphological changes in bacteria on the sample surface, thus evaluating the in vitro antibacterial effect of eTi-TK-GL13K. All data are expressed as mean ± standard deviation. Levene's test was used to determine homogeneity of variance, and one-way ANOVA and Dunnett's T3 post-hoc test were employed to determine statistical differences between groups. The criterion was set at α = 0.05, and p < 0.05 was considered statistically significant. .

[0113] Please see Figure 10 The results show that eTi-TK-GL13K has good bactericidal effects on both MRSA and E. coli, reducing the number of MRSA by more than 80% and the number of E. coli by more than 90% compared to eTi.

[0114] Please see Figure 11 Scanning electron microscopy images showed that MRSA and E. coli on the surface of eTi-GL13K and eTi-TK-GL13K had varying degrees of rupture and cell membrane shrinkage. The cells lost their original plump shape and the surface was "raisin-like", indicating loss of cell integrity, decrease in turgor pressure and resulting in lethal structural collapse.

[0115] In the two sets of experiments above, the antibacterial effect of eTi-TK-GL13K was similar to that of eTi-GL13K, indicating that the introduction of GL13K is the reason why eTi-TK-GL13K has excellent antibacterial ability.

[0116] Example 7:

[0117] Please see Figure 12 and Figure 13 This embodiment provides a technical solution based on Embodiment 1 and Comparative Example 1: the in vitro anti-inflammatory effect of eTi-TK-GL13K:

[0118] RAW264.7 cells were cultured on different samples (eTi, eTi-GL13K, and eTi-TK-GL13K) and stimulated with 1.0 μg / mL LPS for 12 hours. Immunofluorescence staining: Primary antibodies against CD86 (a surface marker for M1 macrophages) and CD206 (a surface marker for M2 macrophages) were added, along with secondary antibodies labeled with different fluorescein. Finally, DAPI was added to label the nuclei, and observation was performed using a confocal microscope. qRT-PCR: RNA was extracted from cells in each group using TRIzol, and reverse transcription was performed using a one-step qRT-PCR reaction. Fluorescence signals were collected, and the melting curve showed a single peak. -ΔΔCt Relative expressions were calculated. All data are expressed as mean ± standard deviation. Levene's test was used to determine homogeneity of variance, and one-way ANOVA and Dunnett's T3 post-hoc test were used to determine statistical differences between groups. The criterion was set at α = 0.05, and p < 0.05 was considered statistically significant. .

[0119] Please see Figure 12 In Figure A, immunofluorescence staining showed that the green fluorescence (CD86) of cells in the eTi-TK-GL13K group was significantly reduced compared to eTi; please refer to [link / reference needed]. Figure 12 The significant increase in B, red fluorescence (CD206) indicates that RAW264.7 cells showed decreased polarization towards M1 macrophages and increased polarization towards M2 macrophages.

[0120] Please see Figure 13 qRT-PCR results showed that compared with the eTi group, the relative expression levels of IL-6 and IL-1β in the eTi-TK-GL13K group were significantly reduced, while the relative expression levels of IL-10 and CD206 were significantly increased. This was corroborated by the immunofluorescence staining results, indicating that eTi-TK-GL13K promotes the transition of chronic inflammation to the healing stage by reshaping macrophage polarization.

[0121] Example 8:

[0122] Please see Figures 14 to 16 This embodiment provides a technical solution based on Embodiment 1 and Comparative Example 1: in vivo application effect detection of eTi-TK-GL13K:

[0123] Male ICR mice (7 weeks old) were used for experiments after one week of acclimatization. Subcutaneous sacs were created on both sides of the mouse's back, and 10 μL of a 1×10⁻⁶ concentration was injected. 5Inflammation was induced by CFU / mL MRSA bacterial suspension, and different groups of samples (eTi, eTi-GL13K, and eTi-TK-GL13K) were then introduced. The blank control group only underwent surgical preparation of cysts and was denoted as "No bacteria". Mice were sacrificed on days 5 and 7 post-surgery, and samples were collected for CFU analysis, H&E staining, and HO-1 immunohistochemical staining of surrounding tissues to evaluate the in vivo antibacterial effect and therapeutic effect on chronic inflammation of eTi-TK-GL13K. All data are expressed as mean ± standard deviation. Levene's test was used to determine homogeneity of variance, and one-way ANOVA and Dunnett's T3 post-hoc test were used to determine statistical differences between groups. The statistical threshold was set at α = 0.05, and P < 0.05 was considered statistically significant. .

[0124] Please see Figure 14 CFU analysis showed that the number of bacteria adhering to the surface of eTi-TK-GL13K was reduced by 10 times compared to eTi, indicating that it still has a good killing effect on drug-resistant strains and inhibits the formation of bacterial biofilms when applied in vivo.

[0125] Please see Figure 15 H&E staining showed that the area of ​​inflammatory cell infiltration in the tissue sections surrounding the eTi-TK-GL13K group was significantly reduced, suggesting that the drug effectively inhibited the local immune-inflammatory response and that the drug remodeled the immune microenvironment: inhibiting chronic inflammatory cell infiltration, promoting macrophage M2 polarization, accelerating the inflammation-repair switching, and achieving chronic inflammation healing and tissue regeneration.

[0126] Please see Figure 16 Immunohistochemical staining for HO-1 showed an increase in HO-1-positive cells in the eTi-TK-GL13K group, indicating that the antioxidant drug successfully activated the Nrf2 / HO-1 pathway and exerted its cell-protective and tissue-repairing effects through a three-tiered mechanism of "upregulating antioxidant enzymes - directly consuming ROS - inhibiting inflammation." These in vivo experimental results demonstrate that eTi-TK-GL13K can inhibit implantation surface formation, accelerate inflammation healing and regeneration, and exert multiple antibacterial, anti-inflammatory, and antioxidant effects under implantation infection conditions.

[0127] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a sustained-release antimicrobial peptide coating on the surface of medical titanium metal, characterized in that, Includes the following steps: S1. After cleaning and drying the pure titanium / titanium alloy implant, acid etching and alkaline etching are performed, followed by nitrogen drying to obtain eTi; silane polyethylene glycol active ester is dissolved in dimethyl sulfoxide to obtain mixed solution a; S2. React the mixed solution a with eTi to obtain eTi-NHS after the reaction is complete; S3. Dissolve N3-TK-PEG-NH2 in deionized water to obtain mixed solution b; soak eTi-NHS in mixed solution b to react, and after the reaction is complete, eTi-TK-N3 is obtained. The preparation method of N3-TK-PEG-NH2 includes the following steps: A1. The diamino ketone thioglycol copolymer was dissolved in N,N-dimethylformamide and condensed with azidoacetic acid in the presence of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate and N,N-diisopropylethylamine to specifically modify one of the amino groups. After the reaction was completed, the intermediate N3-TK-NH2 was obtained by concentration, column chromatography purification and drying. A2. BOC-polyethylene glycol carboxyl groups and N3-TK-NH2 were condensed in N,N-dimethylformamide under the catalysis of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate and N,N-diisopropylethylamine. The reaction solution was concentrated, dissolved in dichloromethane, washed and purified with water. The product was treated with trifluoroacetic acid, and finally obtained by ether precipitation, centrifugation and drying to obtain the target product N3-TK-PEG-NH2. S4. Dissolve DBCO-GL13K in deionized water to obtain mixed solution c; soak eTi-TK-N3 in mixed solution c for 1 hour, clean and dry after soaking, then pre-freeze at -80℃ for 4 hours, freeze-dry for 4 hours to obtain eTi-TK-GL13K, that is, generate a sustained-release antimicrobial peptide coating on the surface of medical titanium metal. The preparation method of the DBCO-GL13K includes the following steps: B1. Using the Fmoc solid-phase synthesis method, dichloropolymer resin was used as the solid-phase support. The amino acids corresponding to the sequence Gly-Gly-Gly-Lys-Ile-Ile-Lys-Leu-Lys-Ala-Ser-Leu-Lys-Leu-Leu were sequentially linked from the C-terminus to the N-terminus. Each cycle included amino acid condensation, Fmoc protecting group removal, and washing steps, and the reaction efficiency was monitored by the ninhydrin method. After all peptide chains were assembled, the target peptide was cleaved from the resin using a mixed lysis buffer containing trifluoroacetic acid, water, and triisopropylsilane. The cleaved peptide was then precipitated with cold diethyl ether, centrifuged, and vacuum dried to obtain the crude product GGGKIIKLKASLKLL-NH2. B2. GGGKIIKLKASLKLL-NH2 was dissolved in N,N-dimethylformamide and reacted with diphenylcyclooctyne-carboxylic acid in the presence of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine. After the reaction was completed, the product was separated and purified by reversed-phase high-performance liquid chromatography. The main peak fraction containing the target product was collected and freeze-dried to obtain the final product DBCO-GL13K.

2. The method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface according to claim 1, characterized in that, The structural formula of DBCO-GL13K in S4 is as follows: 。 3. The method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface according to claim 1, characterized in that, The acids used for etching in S1 are HCl and H2SO4.

4. The method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface according to claim 1, characterized in that, The alkali used in the alkaline etching of S1 is NaOH.

5. The method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface according to claim 1, characterized in that, The reaction time in S2 is 1 hour.

6. The method for preparing a sustained-release antimicrobial peptide coating on a medical titanium metal surface according to claim 1, characterized in that, The reaction time in S3 is 1 hour.

7. A medical-grade titanium metal surface sustained-release antibacterial peptide coating, characterized in that, It is prepared by the preparation method described in any one of claims 1-6.

8. The application of the sustained-release antimicrobial peptide coating on the surface of medical titanium metal as described in claim 7 in the preparation of orthopedic titanium-based implants and dental titanium-based implants.

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