Ultrasonic response type antibacterial osteogenesis coating based on anaerobic activation, implant and preparation method thereof

Abstract

The invention relates to the technical field of implant materials, in particular to an ultrasonic response type antibacterial osteogenesis coating based on anaerobic activation, an implant and a preparation method of the implant. The ultrasonic response type antibacterial osteogenesis coating based on anaerobic activation comprises a base material Cu-TCPP, an antibiotic and probiotic vesicles, wherein the antibiotic and the probiotic vesicles are carried on the Cu-TCPP through pDA. The antibacterial osteogenic coating can effectively generate antibacterial active substances through ultrasonic activation under an anoxic condition, and is combined with osteogenic factors, so that biological membrane related infection can be effectively eliminated, bone tissue regeneration of an implanted part can be promoted, and the overall clinical effect of an implant is improved.

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 one of the 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 presence of anoxic microenvironments in biofilms, the therapeutic effects of traditional antibiotics are often significantly limited. Especially in the deep areas of biofilms, the metabolic activity of bacteria is low, which further reduces the effectiveness of antimicrobial therapy. Therefore, existing treatments have obvious limitations in preventing and controlling implant-associated infections.

[0003] In addition, implant-related infection can not only lead to surgical failure, but also cause chronic inflammation, which in turn affects the healing and regeneration of bone tissue. In these cases, antimicrobial therapy alone often cannot meet clinical needs, as the increase in antibiotic resistance and the re-formation of biofilms lead to repeated infections. Therefore, there is an urgent need for an innovative coating technology that can function effectively under hypoxic conditions and has the function of promoting osteogenesis to improve the clinical application 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 inside the biofilm. In addition, current SDT strategies are mostly focused on antibacterial functions, and there are few studies on how to promote bone tissue regeneration at the same time, which limits its wide application in orthopedic implants.

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

[0006] The purpose of the present invention is to provide an ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation, an implant and a preparation method thereof. The embodiment of the present invention provides an antibacterial osteogenic coating that can effectively generate antibacterial active substances under hypoxic conditions through ultrasound activation, and combined with osteogenic factors, it can effectively eliminate biofilm-related infections and promote bone tissue regeneration at the implant site, thereby improving the overall clinical effect 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 nidazole 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 buffer solution of DA so that the surface of the substrate is loaded with pDA, and then immersing it 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 embodiment 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 effectively generate antibacterial active substances under hypoxic conditions through ultrasonic activation and promote the expression of osteogenic factors, which can effectively eliminate biofilm-related infections and 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 drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 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 a diagram of experimental results provided for Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all 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, wherein 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 biofilm on the surface of prostheses is the main reason why prosthesis-related infections are difficult to treat. Especially in a hypoxic microenvironment, the bacteria in the biofilm have reduced metabolic activity and increased resistance to antibiotics, which greatly reduces the effectiveness of traditional antibacterial treatment.

[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 removing 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 a 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 thorough 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, and the electron transfer effect of dopamine enables Cu(I) to maintain high-efficiency antibacterial activity for a long time, prevent the reformation 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 endogenous dopamine electron transfer mechanism and controllable reduction, providing a continuous and long-term antibacterial strategy, effectively solving the high recurrence rate of prosthesis 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 probiotic vesicles on the immune response, combined with the regeneration 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 the controllable reduction, and Cu(I) provides a carrier for electron transfer in the controllable reduction process. The intrinsic connection between immunomodulation and promotion of osteogenic factor release in the coating is established, achieving the dual effects of antibacterial and bone regeneration. The coating provided by the embodiments of the present invention solves the problems of infection and bone damage at the same time.

[0034] Further, the probiotic vesicles include any one of Lactobacillus animalis vesicles, Lactobacillus rhamnosus vesicles and Lactococcus lactis vesicles. The antibiotic is a nidazole 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 ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation as described in the above embodiment, wherein the antibacterial osteogenic coating is disposed on the surface of the substrate. Specifically, through the adhesion of pDA, Cu-TCPP as a base material, an anaerobic-activated antibiotic, and a probiotic vesicle that regulates the differentiation ability of macrophages are co-loaded on the substrate.

[0036] The matrix may be a PEKK bracket, and it is understandable that a bracket 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 buffer solution of DA so that the surface of the substrate is loaded with pDA, and then immersing it in an antibiotic solution, a probiotic vesicle solution and a Cu-TCPP solution.

[0038] Specifically, synthesis of 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 a 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 using 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 in conjunction with the embodiments.

[0042] Example 1

[0043] The present invention provides a method for preparing an implant (hereinafter referred to as PO-CuTM), comprising:

[0044] Synthesis of Cu-TCPP: Dissolve CuSO4·5H2O (12.5 mg, 0.05 mmol) 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] Preparation of PEKK stents: The PEKK stents were prepared using 3D printing technology using fused deposition modeling. The structural design was completed using Materialise3-matic software, and the applied stent had a diameter of 3 mm and a height of 4 mm. Medical-grade PEKK filament was extruded into the deposition box of the 3D printer, and the stent was prepared layer by layer according to a predetermined shape at a temperature of 200°C.

[0046] The PEKK scaffold was stirred in a Tris-HCl solution (10Mm, sigma, USA) containing 3mg / ml DA (sigma, USA) for 24h to obtain PEKK@pDA (Pp). The Pp scaffold was immersed in a 1mg / mL metronidazole (MNZ) solution for 20 minutes and then dried at room temperature to form an MNZ coating. It was then immersed in a 2mg / mL OMVs solution for 24 hours and then dried at room temperature. Finally, the scaffold was immersed in a 1mg / mL Cu-TCPP solution for 1 hour, then gently rinsed and air-dried to form an implant containing an ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation.

[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 show that the coating surface successfully loads Cu-TCPP with a thin layer structure, and XPS shows that the coating contains elements such as Cu, N, and O, which proves the successful loading of OMVs and MNZ.

[0049] Experimental Example 1

[0050] Copper (I) release performance test

[0051] Method: The PO-CuTM scaffold provided in Example 1 was placed in PBS at pH 5.5 and pH 7.4 to perform 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 detected by inductively coupled plasma mass spectrometry (ICP-MS). Ultrasound (US) can also be used during the test, and the conditions for ultrasound include: 1.0 W / cm 2 , 10 minutes.

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

[0053] Results Figure 2 ,according to Figure 2 It can be seen that under pH 5.5 and ultrasound (US), the release concentration of Cu on the 7th day was 313 ng / mL; under pH 5.5 and ultrasound, the release concentration of Cu on the 7th day was 230 ng / mL. Before ultrasound treatment, the proportion of Cu(I) was 9% and the proportion of Cu(II) was 91%; after ultrasound treatment, the proportion of Cu(I) increased to 28% and the proportion of Cu(II) decreased to 72%. This change was achieved through pDA (polydopamine) electron transfer, indicating that ultrasound treatment can effectively promote the reduction of Cu(II) to Cu(I) and enhance 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, left to soak at room temperature for 24 hours, then gently rinsed and air-dried 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] Antimicrobial 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 bacterial suspensions (2×10 7 CFU / mL) for a specified time and 1W / cm 2 The cells were irradiated with US for 10 min or left untreated. The cells were cultured on agar plates at 37°C for 18 h 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 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] Detection of PO-CuTM osteogenic capacity with ALP and ARS MC3T3-E1 cells were seeded into each well of a 12-well plate. When the 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 the osteogenic commitment of MC3T3-E1 osteoblasts and scaffold materials. The osteoinduction medium was renewed every 3 days together with the materials. On days 7 and 14, the activity of ALP was evaluated using a 5-bromo-4-chloro-3-indoleyl phosphate / nitro blue tetrazolium (BCIP / NBT) ALP colorimetric kit (Beyotime, China). To evaluate the calcified extracellular matrix, the 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). 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 Figure 4 , the implant provided by the embodiment of the present invention has a good osteogenic ability. The results showed that the calcium-phosphorus deposition and matrix mineralization of the Pp-OMVs and PO-CuTM groups increased, especially the calcium-phosphorus deposition and matrix mineralization of the PO-CuTM provided by the embodiment of the present invention increased significantly, and the calcium-phosphorus deposition and matrix mineralization of the Pp-OMVs increased significantly, which not only verified the promoting effect of Lactococcus lactis vesicles on osteogenesis and the good osteogenic induction effect of PO-CuTM, but also showed that the implant provided by the embodiment of the present invention can promote osteogenesis.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An ultrasound-responsive antibacterial osteogenic coating based on anaerobic activation, characterized in that: The invention comprises a basic material Cu-TCPP and antibiotic and probiotic vesicles loaded on the Cu-TCPP via pDA.

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 nidazole antibiotic, preferably metronidazole.

4. An implant, characterized in that: It 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.

5. The implant according to claim 4, 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.

6. The implant according to claim 4, characterized in that The matrix is ​​a PEKK bracket.

7. A method for preparing the implant according to claim 4, characterized in that: include: The substrate was immersed in a DA buffer solution to load pDA on the surface of the substrate, and then immersed in an antibiotic solution, a probiotic vesicle solution and a Cu-TCPP solution.

8. The preparation method according to claim 7, 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.

9. The preparation method according to claim 7, characterized in that: The substrate is formed by 3D printing technology.

10. The preparation method according to claim 7, 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.

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