Anaerobic-activated ultrasound-responsive antibacterial osteogenic coating, implant, and preparation method thereof

Abstract

The present invention relates to the technical field of implant materials, and more specifically, to an ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation, an implant, and a preparation method thereof. The ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation comprises a base material, Cu-TCPP, and antibiotics and probiotic vesicles carried on the Cu-TCPP via pDA. The antibacterial osteogenic coating is activated by ultrasound and can effectively generate antibacterial active substances under hypoxic conditions, and combined with osteogenic factors, it can not only effectively eliminate biofilm-related infections, but also promote bone tissue regeneration at the implant site, thereby improving the overall clinical effect of the implant.

Description

Technical Field

[0001] The present invention relates to the technical field of implant materials, and in particular to an ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation, an implant and a preparation method thereof. Background Art

[0002] Implant-associated infections (IAIs) are common and serious complications in clinical practice, especially in orthopedic prosthesis implantation surgery. These infections are usually caused by bacteria forming biofilms on the surface of implants. The formation of biofilms not only hinders the penetration of antibiotics, but also protects bacteria from attacks by the immune system. Due to the hypoxic microenvironment within the biofilm, the therapeutic effect of traditional antibiotics is often significantly limited. Especially in the deep areas of the biofilm, the metabolic activity of bacteria is low, which further reduces the effectiveness of antimicrobial treatment. Therefore, existing treatment methods have obvious limitations in preventing and controlling implant-associated infections.

[0003] Furthermore, implant-related infections not only lead to surgical failure but can also trigger chronic inflammation, which in turn affects bone healing and regeneration. In these cases, simple antimicrobial therapy often fails to meet clinical needs, as increased antibiotic resistance and the re-formation of biofilms lead to recurrent infections. Therefore, there is an urgent need for innovative coating technologies that can effectively function under hypoxic conditions and promote osteogenesis to improve the clinical efficacy of prostheses.

[0004] In recent years, sonodynamic therapy (SDT) has gradually attracted attention as a non-antibiotic antibacterial strategy. SDT activates sonosensitizers through ultrasound to produce reactive oxygen species (ROS), which can kill bacteria to a certain extent. However, the antibacterial effect of SDT may be limited due to the hypoxic environment within the biofilm. In addition, current SDT strategies mostly focus on antibacterial functions, and there is little research on how to simultaneously promote bone tissue regeneration, which limits its widespread application in orthopedic implants.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide an ultrasound-responsive antibacterial osteogenic coating and implant based on anaerobic activation, as well as methods for preparing the same. The present invention provides an antibacterial osteogenic coating that, through ultrasound activation, effectively generates antimicrobial active substances under hypoxic conditions. Combined with osteogenic factors, the coating effectively eliminates biofilm-related infections and promotes bone regeneration at the implant site, thereby improving the overall clinical efficacy of the implant.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation, which comprises a base material Cu-TCPP and antibiotics and probiotic vesicles loaded on the Cu-TCPP via pDA.

[0009] In an optional embodiment, the probiotic vesicles include any one of Lactobacillus animalis vesicles, Lactobacillus rhamnosus vesicles and Lactococcus lactis vesicles.

[0010] In an optional embodiment, the antibiotic is a nitroazole antibiotic, preferably metronidazole.

[0011] In a second aspect, the present invention provides an implant comprising a substrate and the ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation as described in the aforementioned embodiment, wherein the antibacterial osteogenic coating is disposed on the surface of the substrate.

[0012] In an optional embodiment, the surface of the substrate is loaded with pDA, and Cu-TCPP, antibiotics and probiotic vesicles are all loaded on the substrate via pDA.

[0013] In an optional embodiment, the substrate is a PEKK scaffold.

[0014] In a third aspect, the present invention provides a method for preparing the implant described in the aforementioned embodiment, comprising: immersing a substrate in a DA buffer solution so that the surface of the substrate is loaded with pDA, and then immersing the substrate in an antibiotic solution, a probiotic vesicle solution, and a Cu-TCPP solution.

[0015] In an optional embodiment, the substrate is stirred in a Tris-HCl solution containing 2.5-3.5 mg / ml DA for 20-24 hours; then, it is immersed in a 0.8-1.2 mg / ml antibiotic solution for 20 minutes; then immersed in a 1.5-2.5 mg / mL probiotic vesicle solution for 20-24 hours, and then immersed in a 0.8-1.2 mg / ml Cu-TCPP solution for 1-1.5 hours.

[0016] In an optional embodiment, the substrate is formed by 3D printing technology.

[0017] In an optional embodiment, the preparation steps of Cu-TCPP are as follows: TCPP and divalent copper ions are mixed and reacted at 60-80°C.

[0018] The present invention has the following beneficial effects: the embodiments of the present invention can enable Cu-TCPP, antibiotics and probiotic vesicles to interact with each other through pDA to form a coating, which can be activated by ultrasound and effectively generate antimicrobial active substances under hypoxic conditions and promote the expression of osteogenic factors. It can not only effectively eliminate biofilm-related infections, but also promote bone tissue regeneration at the implant site, thereby improving the overall clinical effect of the implant. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 This is a representation diagram of the implant provided in Example 1 of the present invention;

[0021] Figure 2 The experimental result diagram provided for Experimental Example 1 of the present invention;

[0022] Figure 3 The experimental result diagram provided for Experimental Example 2 of the present invention;

[0023] Figure 4 This is the experimental result diagram provided for Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0025] In a first aspect, the present invention provides an ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation. The coating is based on Cu-TCPP, and antibiotics and probiotic vesicles are carried on the Cu-TCPP through the adhesion effect of pDA.

[0026] The coating provided by the embodiments of the present invention can effectively solve the following problems:

[0027] (1) In the prior art, the formation of bacterial biofilms on the surface of prostheses is the main reason why prosthesis-related infections are difficult to treat. Especially in an oxygen-deficient microenvironment, bacteria in the biofilms have reduced metabolic activity and increased resistance to antibiotics, which greatly reduces the effectiveness of traditional antibacterial treatments.

[0028] Therefore, the above-mentioned antibacterial osteogenic coating provided in the embodiment of the present invention can be activated by ultrasound under hypoxic conditions to generate highly effective antibacterial active substances, such as reactive oxygen species (ROS), thereby effectively eliminating drug-resistant bacteria in the biofilm, and then breaking through the limitations of the biofilm protective layer, thereby improving the effect of antibacterial treatment in the hypoxic microenvironment.

[0029] (2) Break through the limitations of the biofilm protective layer and improve the effectiveness of antibacterial treatment in hypoxic microenvironments.

[0030] The antibacterial osteogenic coating provided in the embodiment of the present invention enhances copper-induced bacterial death (copper death mechanism) through the dopamine (pDA) electron transfer mechanism, thereby effectively removing residual bacteria in the biofilm. Specifically, under ultrasonic activation, the coating uses dopamine as an electron donor to reduce Cu(II) ions to Cu(I) ions with stronger antibacterial activity. Cu(I) induces copper-like death in bacteria by interfering with the tricarboxylic acid cycle (TCA cycle) and other key metabolic pathways of bacteria, thereby achieving a complete killing of bacteria deep in the biofilm. At the same time, the antibacterial osteogenic coating provided in the embodiment of the present invention has the characteristics of long-term release. The electron transfer effect of dopamine enables Cu(I) to maintain high-efficiency antibacterial activity for a long time, prevent the re-formation of biofilm, and significantly reduce the risk of recurrence of infection. In short, the antibacterial osteogenic coating provided in the embodiment of the present invention enhances copper-induced bacterial death through the endogenous dopamine electron transfer mechanism and controllable reduction, providing a continuous and long-term antibacterial strategy, effectively solving the problem of high recurrence rate of prosthetic surface infection, and improving the clinical safety and service life of the prosthesis.

[0031] (3) The inflammatory response triggered by infection can cause macrophages to polarize toward the M1 type, thereby enhancing osteoclast activity, leading to tissue damage and inhibiting bone tissue regeneration.

[0032] The antibacterial osteogenic coating provided by the embodiment of the present invention can effectively induce macrophage polarization from M1 type to M2 type through the regulatory effect of probiotic vesicles, thereby weakening the inflammatory response, reducing osteoclast activity, and promoting tissue repair. In addition, the vesicles can promote the expression of cellular bone morphogenetic proteins (BMPs), alkaline phosphatase (ALP) and other factors that promote osteogenesis, contribute to the proliferation and differentiation of osteoblasts, and significantly accelerate the formation of new bone. It can be seen that the embodiment of the present invention aims to provide a comprehensive and effective solution to the problem of tissue damage caused by infection through the precise regulation of the immune response by probiotic vesicles, combined with the regenerative function of osteogenic factors, to ensure the long-term stability and functionality of the prosthesis.

[0033] In summary, the embodiments of the present invention enhance the antibacterial activity of copper-induced bacterial death (Cu(I)) through the electrons provided by controlled reduction, while Cu(I) provides a carrier for electron transfer during the controlled reduction process. This establishes an intrinsic link between immunomodulation and the promotion of osteogenic factor release in the coating, achieving the dual effects of antibacterial and bone regeneration. The coating provided by the embodiments of the present invention simultaneously addresses the challenges of infection and bone damage.

[0034] Furthermore, the probiotic vesicles include any one of Lactobacillus animalis vesicles, Lactobacillus rhamnosus vesicles and Lactococcus lactis vesicles. The antibiotic is a nitroazole antibiotic, for example including but not limited to metronidazole.

[0035] In a second aspect, the present invention provides an implant comprising a substrate and the anaerobic-activated, ultrasound-responsive, antibacterial osteogenic coating described in the aforementioned embodiments, disposed on the surface of the substrate. Specifically, the substrate is loaded with Cu-TCPP as a base material, an anaerobic-activated antibiotic, and probiotic vesicles that modulate macrophage differentiation through the adhesion of pDA.

[0036] The matrix may be a PEKK scaffold. It is understood that a scaffold formed of other polymer materials may also be used.

[0037] In a third aspect, the present invention provides a method for preparing the implant described in the aforementioned embodiment, comprising: immersing a substrate in a DA buffer solution so that the surface of the substrate is loaded with pDA, and then immersing the substrate in an antibiotic solution, a probiotic vesicle solution, and a Cu-TCPP solution.

[0038] Specifically, to synthesize Cu-TCPP: TCPP and divalent copper ions were mixed and reacted at 60-80°C. Specifically, CuSO4·5H2O was dissolved in DMF to obtain a uniform copper ion solution. Subsequently, TCPP was dissolved in DMF and slowly added to the copper sulfate solution under stirring. The mixed solution was reacted at 60-80°C and ultrasonically treated to promote the reaction. After the reaction was completed, the product was washed with ethanol and centrifuged, and then freeze-dried. Through the above steps, Cu-TCPP powder was successfully prepared.

[0039] The substrate was prepared using 3D printing technology based on fused deposition modeling.

[0040] The substrate was stirred in a Tris-HCl solution containing 2.5-3.5 mg / ml DA for 20-24 hours; then, it was immersed in a 0.8-1.2 mg / ml antibiotic solution for 20 minutes; then immersed in a 1.5-2.5 mg / mL probiotic vesicle solution for 20-24 hours, and then immersed in a 0.8-1.2 mg / ml Cu-TCPP solution for 1-1.5 hours.

[0041] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0042] Example 1

[0043] An embodiment of the present invention provides a method for preparing an implant (hereinafter abbreviated as PO-Cu™), comprising:

[0044] Synthesis of Cu-TCPP: CuSO4·5H2O (12.5 mg, 0.05 mmol) was dissolved in 8 mL of DMF to obtain a uniform copper ion solution. Subsequently, TCPP (4.0 mg, 0.0075 mmol) was dissolved in 2 mL of DMF and slowly added to the copper sulfate solution under stirring. The mixed solution was reacted at 80°C for 2 hours and ultrasonically treated for 10 minutes to promote the reaction. After the reaction was completed, the product was washed with ethanol and centrifuged at 8000 rpm for 10 minutes, and then freeze-dried. Through the above steps, Cu-TCPP powder was successfully prepared.

[0045] Fabrication of the PEKK scaffold: The PEKK scaffold was produced using fused deposition modeling 3D printing technology. The structural design was completed using Materialise 3-matic software, with a scaffold diameter of 3 mm and a height of 4 mm. Medical-grade PEKK filament was extruded into the 3D printer's deposition chamber, where the scaffold was fabricated layer by layer according to the predetermined shape at a temperature of 200°C.

[0046] PEKK scaffolds were stirred in a Tris-HCl solution (10 mM, Sigma, USA) containing 3 mg / ml DA (Sigma, USA) for 24 hours to produce PEKK@pDA(Pp). The Pp scaffolds were then immersed in a 1 mg / mL metronidazole (MNZ) solution for 20 minutes and air-dried at room temperature to form an MNZ coating. The scaffolds were then immersed in a 2 mg / mL OMV solution for 24 hours and air-dried at room temperature. Finally, the scaffolds were immersed in a 1 mg / mL Cu-TCPP solution for 1 hour, then gently rinsed and air-dried to form implants containing an anaerobic-activated, ultrasound-responsive, antibacterial, osteogenic coating.

[0047] Characterization

[0048] The implant of Example 1 was characterized using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The morphology and elemental composition of the implant of Example 1 were characterized. The results are shown in Figure 1 .according to Figure 1It can be seen that SEM and TEM showed that the coating surface successfully loaded Cu-TCPP with a thin layer structure, and XPS showed that the coating contained elements such as Cu, N, and O, which proved the successful loading of OMVs and MNZ.

[0049] Experimental Example 1

[0050] Copper (I) release performance test

[0051] Methods: The PO-CuTM scaffold provided in Example 1 was placed in PBS at pH 5.5 and pH 7.4 to conduct a Cu ion release experiment. Samples were taken at different time points (1 day, 3 days, 5 days, and 7 days), and the Cu ion concentration in the solution was measured by inductively coupled plasma mass spectrometry (ICP-MS). Ultrasound (US) was also used during the test. The ultrasound conditions included: 1.0 W / cm 2 , 10 minutes.

[0052] Meanwhile, X-ray photoelectron spectroscopy (XPS) was used to analyze the surface of the PO-CuTM scaffold before and after ultrasonic treatment.

[0053] Results see Figure 2 ,according to Figure 2 Under ultrasound (US) at pH 5.5, the Cu release concentration on day 7 was 313 ng / mL; under US at pH 5.5, the Cu release concentration on day 7 was 230 ng / mL. Before US treatment, the proportion of Cu(I) was 9% and the proportion of Cu(II) was 91%. After US treatment, the proportion of Cu(I) increased to 28% and the proportion of Cu(II) decreased to 72%. This change was achieved through electron transfer via polydopamine (pDA), indicating that US treatment effectively promoted the reduction of Cu(II) to Cu(I), enhancing the antibacterial activity of the scaffold.

[0054] Comparative Example 1: The PEKK@pDA(Pp) formed in Example 1 was directly immersed in a 2 mg / mL OMVs solution and left to soak at room temperature for 24 hours, followed by gentle rinsing and air-drying to form an implant containing a Pp-OMVs coating.

[0055] Comparative Example 2: The PEKK@pDA(Pp) formed in Example 1 was directly immersed in a 1 mg / mL Cu-TCPP solution for 1 hour, then gently rinsed and air-dried to form an implant containing a Pp-Cu-TCPP coating.

[0056] Experimental Example 2

[0057] Antibacterial testing

[0058] The effects of PO-CuTM of Example 1 on Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) with and without US irradiation were evaluated using a spread plate method. Implants were exposed to a bacterial suspension (2×10 7 CFU / mL) for the specified time and subjected to 1W / cm 2 The cells were irradiated with US for 10 minutes or left untreated. The cells were cultured on agar plates at 37°C for 18 hours and the colony forming units (CFU) were quantified.

[0059] Meanwhile, the above operation was also performed on the implant containing only Pp of Example 1, the implant containing Pp-OMVs coating of Comparative Example 1, and the implant containing Pp-Cu-TCPP coating of Comparative Example 2.

[0060] Results see Figure 3 ,according to Figure 3 It can be seen that the antibacterial effect of the coating formed by only dopamine, dopamine and Cu-TCPP, or dopamine and Lactococcus lactis vesicles is poor, while the antibacterial osteogenic coating based on anaerobic activation and ultrasound response provided by the embodiment of the present invention has excellent antibacterial effect.

[0061] Experimental Example 3

[0062] Osteogenesis performance test

[0063] To assess the osteogenic capacity of PO-CuTM using ALP and ARS, MC3T3-E1 cells were seeded into each well of a 12-well plate. When cell confluence reached 70%, the α-MEM medium was replaced with osteogenic induction medium containing 10 mM β-glycerophosphate, 50 μg / mL ascorbic acid (Sigma), 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate (Sigma), and 10 nM dexamethasone (Sigma) to promote osteogenic commitment of the MC3T3-E1 osteoblasts and scaffold material. The osteoinduction medium was refreshed along with the material every 3 days. On days 7 and 14, ALP activity was assessed using a 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolium (BCIP / NBT) ALP colorimetric kit (Beyotime, China). To assess the calcified extracellular matrix, cells were fixed and treated with ARS dye (Beyotime, China), and ARS assays were performed on days 14 and 21. Briefly, cells were fixed and treated with ARS dye (Beyotime, China) and then, after removing excess dye, images were captured using a scanner.

[0064] Meanwhile, the above operation was also performed on the implant containing only Pp of Example 1, the implant containing Pp-OMVs coating of Comparative Example 1, and the implant containing Pp-Cu-TCPP coating of Comparative Example 2.

[0065] Results see Figure 4 The implant provided by the embodiment of the present invention has excellent osteogenesis ability. The results showed that the Pp-OMVs and PO-CuTM groups increased calcium and phosphate deposition and matrix mineralization. In particular, the calcium and phosphate deposition and matrix mineralization of PO-CuTM provided by the embodiment of the present invention increased significantly, and the calcium and phosphate deposition and matrix mineralization increased significantly relative to Pp-OMVs. This not only verified the promoting effect of Lactococcus lactis vesicles on osteogenesis and the good osteogenesis induction effect of PO-CuTM, but also demonstrated that the implant provided by the embodiment of the present invention can promote osteogenesis.

[0066] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation, characterized in that: The method comprises a basic material Cu-TCPP and antibiotic and probiotic vesicles loaded on the Cu-TCPP via pDA; The preparation steps of the antibacterial osteogenic coating include: immersing a substrate in a DA buffer solution to load pDA on the surface of the substrate, and then immersing the substrate in an antibiotic solution, a probiotic vesicle solution and a Cu-TCPP solution.

2. The ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation according to claim 1, characterized in that: The probiotic vesicles include any one of Lactobacillus animalis vesicles, Lactobacillus rhamnosus vesicles and Lactococcus lactis vesicles.

3. The ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation according to claim 1, characterized in that: The antibiotic is a nitroazole antibiotic.

4. The ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation according to claim 1, characterized in that: The antibiotic is metronidazole.

5. The ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation according to any one of claims 1 to 4, characterized in that: The substrate was stirred in a Tris-HCl solution containing 2.5-3.5 mg / mL DA for 20-24 hours; then, it was immersed in a 0.8-1.2 mg / mL antibiotic solution for 20 minutes; then immersed in a 1.5-2.5 mg / mL probiotic vesicle solution for 20-24 hours, and then immersed in a 0.8-1.2 mg / mL Cu-TCPP solution for 1-1.5 hours.

6. The ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation according to any one of claims 1 to 4, characterized in that: The preparation steps of the Cu-TCPP are as follows: TCPP and divalent copper ions are mixed and reacted at 60-80°C.

7. An implant, characterized in that: The invention comprises a substrate and the ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation according to claim 1, wherein the antibacterial osteogenic coating is arranged on the surface of the substrate.

8. The implant according to claim 7, characterized in that The surface of the substrate is loaded with pDA, and Cu-TCPP, antibiotics and probiotic vesicles are all loaded on the substrate through pDA.

9. The implant according to claim 7, characterized in that The matrix is ​​a PEKK bracket.

10. The implant according to claim 7, characterized in that The matrix is ​​formed by 3D printing technology.

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