Polypeptide-silver-manganese co-assembled nano-enzyme antibacterial coating for ureteral stent and preparation method of polypeptide-silver-manganese co-assembled nano-enzyme antibacterial coating

Abstract

The invention discloses a polypeptide-silver-manganese co-assembled nano-enzyme antibacterial coating for a ureteral stent and a preparation method of the polypeptide-silver-manganese co-assembled nano-enzyme antibacterial coating, and belongs to the technical field of preparation of ureteral stent coatings. The preparation method comprises the following steps: sequentially immersing a polyurethane ureteral stent into a hydrogen peroxide-sulfuric acid mixed solution, a gamma-aminopropyltriethoxysilane solution, a polypeptide-EDC-NHS mixed solution, a silver-containing solution and a manganese-containing solution, and respectively carrying out activation, amino modification, polypeptide coupling, surface silver plating and surface manganese plating; the preparation of the ureteral stent surface polypeptide-silver-manganese co-assembled nano-enzyme antibacterial coating is completed. According to the invention, the high selectivity and specificity of biomolecules are utilized to prepare the coating, and the preparation can be carried out under mild conditions. The prepared novel ureteral stent realizes stent surface functionalization, can reduce stent surface protein, bacterial adhesion and biofilm formation, reduces the incidence rate of complications such as urinary tract infection and the like, improves the curative effect of a patient and improves the prognosis of the patient.

Description

Technical Field

[0001] The present invention belongs to the technical field of ureteral stent coating preparation, and more specifically relates to a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating for a ureteral stent and a preparation method thereof. Background Art

[0002] The ureters are a pair of long and thin tubes that connect the kidneys and bladder in the human body, with an average diameter of 0.5-0.7 cm. Ureteral obstruction is easily caused by trauma, congenital malformations, tumors, stones, etc. Ureteral stents are implanted to reconstruct or maintain ureteral patency, and are usually placed in situ in the patient's body for several days to months. Various stent-related complications are very likely to occur during the retention period, especially the probability of urinary tract infection, which increases at a rate of 3-10% per day. In addition, as the retention time increases, the frequency and amplitude of ureteral peristalsis decrease, which will further lead to retrograde bacterial infection and even sepsis, which is life-threatening.

[0003] With the increasing use of ureteral stents, the effective prevention and treatment of ureteral stent-related urinary tract infections is a major challenge currently faced. It is currently believed that the occurrence of these complications is closely related to the adhesion of bacteria on the surface of the ureteral stent and the formation of biofilms. The ureteral stents currently used are mostly made of polyurethane (PU) materials, which have good flexibility, elasticity and biocompatibility. However, their hydrophobic surface reduces the antibacterial properties and easily leads to protein adsorption and increased bacterial adhesion in the urine environment, forming biofilms. The use of antibiotics is a conventional treatment for implant-related urinary tract infections. However, bacteria that form biofilms have a certain resistance to antibiotics. In most cases, antibiotic treatment alone is not enough to eradicate biofilm infections. In recent years, in order to reduce the occurrence of bacterial infections and implant-related complications, surface modification of ureteral stent materials has become an ideal solution.

[0004] In recent years, nanotechnology has been used to solve the problem of biological contamination of medical devices including ureteral stents. However, most of the current research on modifying the surface of ureteral stents through nanotechnology focuses on enhancing the bactericidal effect of ureteral stents. However, there is a gap in research on how to reduce the adhesion and biofilm formation of proteins and bacteria on the surface of the stent, solve the impact of protein and bacterial biofilm formation on the stent from the root, reduce the incidence of complications such as urinary tract infections, improve patient efficacy and improve patient prognosis. In addition, the existing technology for surface modification of ureteral stents through nanotechnology usually requires harsh reaction conditions. Therefore, it is of great significance to develop a ureteral stent surface modification method with mild modification conditions and which can solve the impact of protein and bacterial biofilm formation on the stent from the root. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating for ureteral stents and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art, solve the influence of the formation of protein and bacterial biofilm on the stent from the root, reduce the incidence of complications such as urinary tract infection, improve patient efficacy and improve patient prognosis, and at the same time, the conditions required for the modification process are mild, which can reduce energy consumption.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is to provide a method for preparing a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating, comprising the following steps:

[0008] The substrate material is sequentially immersed in a hydrogen peroxide-sulfuric acid mixture, a γ-aminopropyltriethoxysilane solution, a polypeptide-EDC-NHS mixture, a silver-containing solution, and a manganese-containing solution for activation, amino modification, polypeptide coupling, surface silver plating, and surface manganese plating, respectively, to complete the preparation of the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating;

[0009] The hydrogen peroxide-sulfuric acid mixed liquid is a mixed liquid consisting of hydrogen peroxide and sulfuric acid;

[0010] The polypeptide-EDC-NHS mixed solution is a mixed solution composed of polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0011] Preferably, the base material is a ureteral stent.

[0012] Preferably, the volume ratio of hydrogen peroxide to sulfuric acid in the hydrogen peroxide-sulfuric acid mixture is 1-5:5-9; the mass fraction of sulfuric acid is 98±0.3%; and the activation time is 0.5-5h.

[0013] Preferably, the solvent of the γ-aminopropyltriethoxysilane solution includes ethanol; the volume ratio of γ-aminopropyltriethoxysilane to the solvent in the γ-aminopropyltriethoxysilane solution is 1:9; and the amino modification time is 6 to 8 hours.

[0014] Preferably, the polypeptide includes one or more of a cationic polypeptide, a defensin polypeptide and variants thereof; the cationic polypeptide includes one or more of polypeptide (RGD-NNN-KWKWKKA), Poly-Arginine, LL-37, Magainin 2, CecropinA and Tachyplesin I; the defensin polypeptide includes one or more of human β-defensin, Bacitracin, Indolicidin and Histatin 5.

[0015] Preferably, the preparation step of the polypeptide-EDC-NHS mixed solution comprises: mixing the polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, N-hydroxysuccinimide solution and water to obtain the polypeptide-EDC-NHS mixed solution;

[0016] The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution is 1 to 30 mmol / L;

[0017] The dosage ratio of the polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, N-hydroxysuccinimide solution and water is 0.001-0.005 g:0.5-2 mL:0.5-2 mL:0.5-2 mL, further preferably 0.002-0.003 g:0.8-1.2 mL:0.8-1.2 mL:0.8-1.2 mL, more preferably 0.002-0.003 g:1 mL:1 mL:1 mL.

[0018] Preferably, the polypeptide coupling time is 3 hours.

[0019] Preferably, the silver in the silver-containing solution is added in the form of silver salt; the silver salt includes one or more of silver nitrate, silver chloride, silver sulfide, silver ammonia solution, silver carbonate and silver-silver nitrate complex; the concentration of the silver-containing solution is 20 mmol / mL; and the surface silver plating time is 48 hours.

[0020] Preferably, the manganese in the manganese-containing solution is added in the form of manganese salt; the manganese salt includes one or more of potassium permanganate, manganese chloride, manganese sulfate, manganese carbonate, manganate, manganese oxide and manganese nitrate; the concentration of the manganese-containing solution is 10 to 200 mmol / L; the surface manganese plating includes: first reacting at 0±5°C for 1 to 4 hours, and then reacting under ultraviolet light irradiation and 0±5°C for 2 to 12 hours.

[0021] Furthermore, the polyurethane ureteral stent may be pre-treated before the above treatment; the pre-treatment may include the steps of cleaning and drying.

[0022] The formation of biofilm on the surface of ureteral stents involves complex interactions among host biological tissues, implanted stents, and bacteria. Therefore, the ideal antibacterial ureteral stent surface should have two characteristics: one is to resist the adsorption of host proteins to prevent the initial attachment of bacteria, and the other is to repel bacterial adhesion and kill attached bacteria. Due to the small size of nanoparticles and their potential ability to penetrate the biofilm layer of implants, the application of nanoparticles in preventing biofilm-mediated infection has attracted much attention. Nanoparticles bind to bacterial cell walls and cause membrane damage through direct interaction or the generation of free radicals. Mammalian cells can phagocytize nanoparticles and then degrade these particles through lysosomal fusion, thereby reducing toxicity and free radical damage. Nanoparticles include metal nanoparticles, metal oxide nanoparticles, and organic nanomaterials. Therefore, the prior art has developed a variety of solutions for surface modification of ureteral stents through nanotechnology. However, the research on this solution in the prior art only stays at the level of the bactericidal effect of nanoparticles.

[0023] Compared with the ureteral stent surface coating in the prior art which can only exhibit a bactericidal effect, the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating formed on the surface of the ureteral stent in the present invention can not only repel bacterial adhesion and kill attached bacteria, but also counteract the adsorption of host proteins to prevent the initial attachment of bacteria.

[0024] The present invention constructs a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating on the surface of the ureteral stent. The specific construction mechanism is as follows: first, the surface of the ureteral stent is hydroxylated by treating with hydrogen peroxide and sulfuric acid, and then the polypeptide is modified on the surface of the ureteral stent by amino modification using γ-aminopropyltriethoxysilane as a coupling agent, and the silver ions and manganese ions in the silver-containing solution and the manganese-containing solution are reduced using the polypeptide as a reducing agent to generate nanosilver (AgNPs) and nanomanganese (MnNPs) loaded on the surface of the ureteral stent. In addition, the surface of the ureteral stent modified by hydroxylation, amino and polypeptide contains a large number of active oxygen-containing groups. , such as hydroxyl and carboxyl groups, can provide abundant binding sites, improve the shortcomings of metal nanoparticles such as easy aggregation and uneven particle size; in addition, the hydroxyl groups on the surface of the hydroxylated ureteral stent can undergo condensation reaction with the amine groups in γ-aminopropyltriethoxysilane to form chemical bonds, further enhancing the amino modification effect; the carboxyl groups contained in the intermediate generated by the reaction of polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide react with the amino groups contained in the amino-modified ureteral stent to form a stable covalent amide bond (peptide bond), further enhancing the polypeptide modification effect.

[0025] The present invention uses polypeptides to reduce metals, which reduces the use of toxic chemicals and is a green chemical method. At the same time, polypeptides also have the ability to self-assemble into complex structures, which can further assemble metal nanoparticles to form highly ordered nanostructures, further improving the bactericidal effect.

[0026] On the one hand, the metal nanoparticles increase the roughness of the stent surface to form an anti-adhesion surface, thereby inhibiting bacterial adhesion and biofilm formation on the stent surface; on the other hand, the metal ions released by the coating and the metal nanoparticles on the coating surface can bind to the DNA of bacterial cells, to the enzymes that control respiration and other key cell functions of bacterial cells, or to receptors on the cell membrane of bacterial cells with chemical functions, thereby affecting the bacterial cell membrane potential and integrity and destroying bacterial cells. In addition, the coating surface can release reactive oxygen species, which can kill bacteria and destroy biofilms. The metal ions released by the coating, the metal nanoparticles on the coating surface, and the reactive oxygen species released by the coating can exert an efficient bactericidal effect without producing drug resistance, thereby achieving the effect of sterilization and destruction of biofilms, reducing the incidence of complications such as urinary tract infections after ureteral stent placement, and improving patient efficacy and prognosis.

[0027] The second technical solution of the present invention is to provide a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating prepared by the above preparation method.

[0028] The third technical solution of the present invention: provides the application of the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating in the modified ureteral stent.

[0029] The fourth technical solution of the present invention is to provide a method for reducing the adhesion of proteins and bacteria on the surface of a ureteral stent and the formation of biofilm, comprising the following steps: treating the ureteral stent using the above-mentioned preparation method.

[0030] The present invention discloses the following technical effects:

[0031] 1. The synthesis of the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating on the surface of the ureteral stent described in the present invention utilizes the high selectivity and specificity of biological molecules and can be carried out under mild conditions. It is an environmentally friendly antibacterial material synthesis strategy.

[0032] 2. The novel ureteral stent prepared by the method described in the present invention realizes the functionalization of the stent surface, can reduce the adhesion of proteins and bacteria on the stent surface, biofilm formation, reduce the incidence of complications such as urinary tract infection, improve patient efficacy and improve patient prognosis, and has clinical application value.

[0033] 3. The bactericidal test, TMB test and anti-adhesion test showed that the new ureteral stent prepared by the method of the present invention achieved synergistic antibacterial and anti-protein adhesion activities. The new ureteral stent can provide a simple, safe and effective prevention and treatment approach for implant-related urinary tract infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a scan of the ureteral stent with the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating obtained in Example 1;

[0035] Figure 2 The full X-ray photoelectron spectroscopy (XPS) spectra of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D;

[0036] Figure 3 The fine spectra of C1s, N1s and O1s of sample A, sample B, sample C and sample D, where A is sample A, B is sample B, C is sample C, and D is sample D;

[0037] Figure 4 The fine spectra of Ag3d of samples C and D and the fine spectra of Mn2p of sample D;

[0038] Figure 5 3D morphology images of different ureteral stent surfaces displayed by atomic force microscopy (AFM), where A is sample A, B is sample B, C is sample C, and D is sample D;

[0039] Figure 6 The antibacterial effect diagram of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D;

[0040] Figure 7 The sterilization effect diagram of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D;

[0041] Figure 8 The UV-visible absorption spectra of Ox-TMB of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D;

[0042] Fig. 9 is the amount of protein adhesion on the surface of different ureteral stents, where a is the OD value of the polyurethane ureteral stent containing the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating under incubation with different concentrations of BSA, and b is the OD value of different ureteral stents under incubation with 4 mg / mL BSA;

[0043] Fig.10Surface scanning electron micrographs of different ureteral stents after incubation in Escherichia coli solution for 24 hours, where A is sample A, B is sample B, C is sample C, and D is sample D. DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] The raw materials used in the examples, comparative examples and performance tests of the present invention are described below:

[0050] The polyurethane ureteral stent (PU) used was a double J-type polyurethane ureteral stent (F6) purchased from Zhangjiagang Shagong Medical Equipment Technology Development Co., Ltd.

[0051] The peptide used (RGD-NNN-KWKWKKA) was purchased from Nanjing Peptide Valley Biotechnology Co., Ltd.

[0052] The BCA protein concentration determination kit (BCA Protein Assay Kit) used was purchased from Beyotime Biotechnology Co., Ltd.

[0053] TMB colorimetric reagent and bovine serum albumin (BSA) were purchased from Sigma-Aldrich.

[0054] Escherichia coli (ATCC 25922) was purchased from Thermo Fisher Scientific.

[0055] Other raw materials are commercially available unless otherwise specified.

[0056] The room temperature involved in the present invention is 25±5°C unless otherwise specified.

[0057] Example 1

[0058] This embodiment provides a method for preparing a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating for a ureteral stent, the steps of which are as follows:

[0059] (1) Surface pretreatment of ureteral stents: Double J-shaped polyurethane ureteral stents (F6) were cut into 1 cm pieces, immersed in anhydrous ethanol, and ultrasonicated in a 40 kHz ultrasonic cleaner for 5 min. The stents were taken out and placed in ultrapure water and ultrasonicated again in a 40 kHz ultrasonic cleaner for 5 min. The above operation was repeated three times and then the stents were taken out and the liquid on the surface of the stents was completely blown dry with nitrogen.

[0060] (2) Activate the polyurethane ureteral stent: Add hydrogen peroxide (H 2 O 2 ) Slowly add 98% concentrated sulfuric acid (H 2 SO 4 ) (volume ratio of 3:7), and then put the pretreated ureteral stent in to ensure that the stent is completely immersed in the solution, stir at room temperature for 1 hour, then take out and rinse with pure water, blow dry with nitrogen, and repeat the above operation 3 times.

[0061] (3) Surface modification of amino groups: γ-aminopropyltriethoxysilane was diluted in an ethanol solution at a volume ratio of 1:9. During this process, the three ethoxy groups of γ-aminopropyltriethoxysilane were gradually hydrolyzed to form hydroxide ions and three ethanol molecules. The activated ureteral stent was placed in the solution and completely immersed in it. It was stirred at room temperature for 7 hours to allow the hydroxyl groups on the surface of the activated ureteral stent to undergo a condensation reaction with the amine groups in γ-aminopropyltriethoxysilane to form a chemical bond. The ureteral stent was removed and rinsed with pure water three times.

[0062] (4) Peptide coupling: 6 mmol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride aqueous solution and N-hydroxysuccinimide aqueous solution were prepared as carboxyl activation reagents, 1 mL of each solution was mixed, 0.002 g of peptide (RGD-NNN-KWKWKKA) was added, and 1 mL of pure water was added. After stirring evenly, the amino-modified ureteral stent was placed in the stent to couple the modified amino group on the stent with the carboxyl group of the peptide to form a peptide bond. After 3 hours, the stent was removed and rinsed with pure water to obtain a peptide-modified ureteral stent, which was recorded as sample B.

[0063] (5) Surface silver plating: Add 20 mmol / mL AgNO to the peptide-modified ureteral stent. 3 The mixture was completely immersed in the solution and stirred at room temperature for 48 h to obtain a polypeptide-Ag modified ureteral stent, which was recorded as sample C.

[0064] (6) Surface manganese plating: The polypeptide-Ag modified ureteral stent was added to a 20 mmol / L potassium permanganate solution and stirred in an ice bath for 2 h. The mixed solution was then irradiated with ultraviolet light for 6 h while being kept in an ice bath and stirred. A ureteral stent containing a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating was obtained, which was recorded as sample D.

[0065] Performance Verification:

[0066] 1. Characterization analysis:

[0067] (1) Observe sample D using a scanning electron microscope (SEM) to obtain a surface scan of sample D. The results are as follows: Figure 1 shown.

[0068] Figure 1 This is a scan of the ureteral stent with the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating obtained in Example 1.

[0069] Depend on Figure 1 It can be seen that on the surface of the ureteral stent with the peptide-silver-manganese co-assembled nanozyme antibacterial coating, the C, O, and N elements are evenly distributed on the stent surface without obvious accumulation, providing a basis for subsequent silver and manganese plating. The distribution of Ag and Mn elements is also relatively uniform, indicating that silver and manganese have been successfully grafted on the stent surface, further improving the bactericidal effect.

[0070] (2) The untreated double J-type polyurethane ureteral stent (F6) was recorded as sample A. The chemical elements and chemical states of samples A, B, C, and D were determined by X-ray photoelectron spectroscopy (XPS). The results are shown in the following table. Figure 2 , 3 and 4.

[0071] Figure 2The full X-ray photoelectron spectroscopy (XPS) spectra of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D; Figure 3 The detailed spectra of C1s, N1s, and O1s of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D; Figure 4 These are the fine spectra of Ag3d and Mn2p of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D.

[0072] Figure 2 The full XPS spectra of different ureteral stent surfaces were given, and the binding energy was corrected with C1s = 284.8 eV as the benchmark. The peptide-silver-manganese co-assembled nanozyme antibacterial coating showed a characteristic peak of Ag3d at a binding energy of 368.2 eV, and a characteristic peak of Mn2p at a binding energy of 645.0 eV.

[0073] Figure 3 The fine spectra of C1s, N1s and O1s of samples A, B, C and D are given. The peptide-modified ureteral stent (sample B) has peaks corresponding to carbonyl (C=O), (CO / CN) and (CC) at binding energies of 288.9, 286.4 and 284.8 eV. Compared with the untreated double J-type polyurethane ureteral stent (F6) (sample A), the C=O content increased significantly, indicating that the modified amino groups on the stent were coupled with the carboxyl groups of the peptide to form a peptide bond. The peaks of C=O and CO / CN of the peptide-Ag modified ureteral stent (sample C) and the ureteral stent containing the peptide-silver-manganese co-assembled nanozyme antibacterial coating (sample D) shifted to low binding energies, indicating that there was a strong interaction between AgNPs, MnNPs and the C=O and CO groups on the peptide-modified ureteral stent (sample B). In the N1s fine spectrum, in the comparison between the peptide-modified ureteral stent (sample B) and the untreated double J-type polyurethane ureteral stent (F6) (sample A), it can be clearly seen that the peak at the binding energy of 400 eV shifted toward the low binding energy direction by about 0.5 eV. This is due to the coupling reaction between the amino group and the peptide, which once again shows that γ-aminopropyltriethoxysilane successfully modified the amino group on the stent surface.

[0074] The signal of the ureteral stent (sample D) containing the peptide-silver-manganese co-assembled nanozyme antibacterial coating in the O1s fine spectrum is quite different from that of the other three ureteral stents. This is because the addition of manganese oxides results in the presence of multiple oxide signals in the O1s fine spectrum of sample D, of which the peak of 530.2eV is attributed to the signal of AgO, and the peak of 529.4eV is attributed to the signal of manganese oxide. This shows that the Ag and Mn elements are successfully modified on the surface of the ureteral stent.

[0075] Combined with the full spectrum ( Figure 2 ) and Ag3d fine spectrum ( Figure 4 ), it can be seen that the peak with a binding energy of 368eV is attributed to Ag, and obvious energy loss peaks appear at 371eV and 376eV, which proves the existence of Ag on the surface of the support. However, not all Ag elements exist in the form of Ag. Combined with the O1s fine spectrum ( Figure 3 ), the peak at 367.6 eV is attributed to the signal of AgO. From the Mn2p fine spectrum ( Figure 4 ) It can be seen that the peak at 641eV belongs to trivalent manganese, and there is MnO at 642eV~646eV. 2 The multiple split peaks of Mn element on the surface of the scaffold are shown in Figure 2. 2 O 3 and MnO 2 For the main.

[0076] (3) The untreated double J-shaped polyurethane ureteral stent (F6) was recorded as sample A. The surfaces of sample A, sample B, sample C, and sample D were examined by atomic force microscopy (AFM) and their roughness was observed. The results are shown in Figure 2. Figure 5 shown.

[0077] Figure 5 3D morphology images of different ureteral stent surfaces displayed by atomic force microscopy (AFM), where A is sample A, B is sample B, C is sample C, and D is sample D.

[0078] Depend on Figure 5 It can be seen that after modifying the surface of the ureteral stent with peptide, peptide-Ag, and peptide-silver-manganese co-assembled nanozyme antibacterial coating, the surface morphology of the stent was significantly changed, the roughness was significantly increased, and the adsorption of host proteins could be counteracted to prevent the initial attachment of bacteria and repel bacterial adhesion.

[0079] 2. The untreated double J-type polyurethane ureteral stent (F6) is recorded as sample A. The sterilization test is performed on samples A, B, C and D:

[0080] Test method: Dilute the Escherichia coli solution to 1.5×10 8 CFU / ml, 100 μL was evenly dropped into LB agar medium (commercially available), and evenly spread with a coating rod until the bacterial solution was completely absorbed. Two groups of 8 ureteral stents were sterilized and placed in their respective LB agar medium (commercially available), and incubated in a 37°C constant temperature incubator for 48h and 72h respectively. The results are shown in Figure 6 shown.

[0081] Figure 6The antibacterial effect diagram of different ureteral stents, where A is sample A, B is sample B, C is sample C, and D is sample D.

[0082] Ureteral stents with antibacterial activity can inhibit the growth of Escherichia coli and form transparent zones. Figure 6 It can be seen that a significant antibacterial effect was observed on the ureteral stent (sample D) containing the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating, and the antibacterial area showed a clearly visible circle. This shows that the ureteral stent effectively inhibited the growth of Escherichia coli and preliminarily verified its antibacterial properties. It can be seen that the coating has potential application prospects in preventing ureteral stent-related urinary tract infections.

[0083] To further evaluate the bactericidal effect of the scaffold sample, the scaffold was co-cultured with the Escherichia coli solution resuspended in sterile saline to observe the colonies formed on the LB culture medium, as follows:

[0084] After resuspending Escherichia coli with sterile saline, the concentration of the bacterial solution was adjusted to 1.5×10 8 CFU / ml, immerse each ureteral stent in a centrifuge tube containing 1mL of diluted bacterial suspension, and after culturing for 12h, take 100μL of bacterial suspension and evenly spread it on LB medium, and observe the growth of colonies after cultivation. Figure 7 shown.

[0085] Figure 7 The sterilization effect diagrams of different ureteral stents, among which A is sample A, B is sample B, C is sample C, and D is sample D.

[0086] Depend on Figure 7 It can be seen that the bactericidal effect of the ureteral stent (sample D) containing the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating is significantly better than that of samples A, B and C.

[0087] 3. The untreated double J-type polyurethane ureteral stent (F6) was recorded as sample A. TMB test was performed on samples A, B, C and D:

[0088] Since ureteral stents are placed in H 2 O 2 In the presence of hydroxyl radical, it reacts with TMB to catalyze the production of ·OH to oxidize TMB, forming a blue oxidized TMB (Ox-TMB) product. The absorbance of Ox-TMB at a wavelength of 650nm is proportional to the concentration of Ox-TMB. By detecting the absorbance at 650nm, H 2 O 2 →·OH conversion, i.e., the POD enzyme catalytic ability of the nanoparticles.

[0089] Test method: UV-visible absorption spectroscopy was used to detect the production of Ox-TMB in sample A, sample B, sample C and sample D in acidic PBS buffer (pH=6.5). Figure 8 shown.

[0090] Figure 8 The UV-visible absorption spectra of different ureteral stents Ox-TMB, where A is sample A, B is sample B, C is sample C, and D is sample D.

[0091] Depend on Figure 8 It can be seen that the ureteral stent (sample D) containing the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating showed the characteristic absorbance peak of Ox-TMB at 650nm, indicating that the ureteral stent has peroxidase-like activity. In addition, there is no obvious absorbance in sample A, indicating that no oxidation reaction occurs and there is no peroxidase-like activity. Peroxidase-like activity refers to a substance with catalytic activity similar to peroxidase, which can help remove peroxides in the body, maintain the redox balance of cells, and reduce the damage to cells caused by oxidative stress.

[0092] 4. The untreated double J-type polyurethane ureteral stent (F6) was recorded as sample A. The anti-adhesion test was performed on samples A, B, C and D:

[0093] When a ureteral stent is placed in the body, the adsorption of proteins on the stent surface damages the surface properties, which promotes further bacterial adhesion and biofilm formation. The inherent hydrophobic and electrostatic interactions between the ureteral stent surface and proteins make improving the hydrophilicity of the surface one of the effective ways to achieve the antibacterial function of the ureteral stent.

[0094] The amount of surface protein adhered to samples A, B, C and D after incubation with bovine serum albumin was determined by BCA analysis.

[0095] Bovine serum albumin (BSA, PI = 4.7) was used as a model protein. The anti-protein adsorption performance of the coated ureteral stent surface was evaluated by BCA analysis. The amount of adhered albumin was evaluated by measuring the absorbance of the eluate at 562nm with an ELISA reader. Different concentrations of BSA solutions (1mg / ml, 2mg / ml, 4mg / ml) were prepared with sterile PBS solution. The sterilized 1cm D samples were placed in different concentrations of BSA solutions and incubated in a 37℃ constant temperature incubator for 1h. The free albumin that was not adhered to the surface of the ureteral stent was removed by washing with PBS buffer (pH = 7.4) and ultrapure water, and the excess water was absorbed by filter paper. Then, the adhered albumin was eluted with 0.5mL SDS (1% w / v) solution, and the ureteral stents incubated with different BSA concentrations were placed in a 37℃ constant temperature incubator and incubated for 2h. Take 20uL of the eluate in each well of a 96-well plate, add 200uL of BCA working solution, place in a 37°C constant temperature incubator for 30 minutes, and then use an ELISA reader to measure the absorbance at 562nm to obtain the optimal concentration of albumin adhesion and make a calibration curve. Repeat the above test three times under the same conditions at different times. The results are as follows Fig. 9 As shown in a in .

[0096] Samples A, B, C, and D were immersed in 1 mL of BSA solution (4 mg / mL) respectively, and then cultured in a 37°C constant temperature incubator for 1 hour. The above process was repeated to obtain the absorbance at 562 nm. The above test was repeated three times under the same conditions at different times. The results are shown in Figure 2. Fig. 9 As shown in b.

[0097] GraphPad Prism (GraphPad Software Inc, Version 9.0.0) was used for statistical analysis and drawing of graphs. One-way ANOVA was used to compare differences among groups. The levels of statistical significance are as follows: (****) indicates p<0.0001, (***) indicates p<0.001, (**) indicates p<0.01, and (*) indicates p<0.05.

[0098] Fig. 9 is the amount of protein adhesion on the surface of different ureteral stents, where a is the OD value of the polyurethane ureteral stent containing the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating incubated with different concentrations of BSA, and b is the OD value of different ureteral stents incubated with 4 mg / mL BSA. Fig. 9 In the table, * indicates P < 0.05, **** indicates P < 0.0001; in addition, A is A sample, B is B sample, C is C sample, and D is D sample.

[0099] Depend on Fig. 9It can be seen that compared with samples A, B and C, the ureteral stent (sample D) containing the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating has the best anti-protein adhesion performance. This is because the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating has a higher hydrophilicity.

[0100] It is currently believed that the adhesion of bacteria to ureteral stents is affected by two forces. On the one hand, the coated ureteral stent has a superhydrophilic surface, and similar to the protein adhesion process, the bacterial adhesion site is also affected by the isolation effect of the hydration layer. On the other hand, the coated ureteral stent has a contact bactericidal effect on bacteria. After the bacteria are inactivated, the contents flow out, and the subsequent adhesion mechanism of bacterial fragments is consistent with the protein adhesion mechanism. Based on the above research on the protein adhesion behavior and bactericidal behavior of ureteral stents, the ability of different ureteral stents to resist bacterial adhesion in eutrophic bacterial suspensions was further verified, as follows:

[0101] Antibacterial adhesion test The modified surface and biofilm morphology of the coated ureteral stents were observed under a scanning electron microscope. Four different ureteral stents were placed in a sterile 24-well plate and 1 mL of 1.5×10 8 CFU / mL of Escherichia coli liquid was incubated in a 37°C constant temperature incubator for 24 hours. The bacterial liquid in the well was aspirated, and the surface of the ureteral stent was gently washed three times with sterile PBS solution to remove non-adherent Escherichia coli. After fixation, dehydration, and gold spraying, scanning electron microscopy (SEM) imaging was performed to observe the number and morphology of bacteria attached to the surface of the ureteral stent. The results are as follows Fig.10 shown.

[0102] Fig.10 Surface scanning electron micrographs of different ureteral stents after incubation in Escherichia coli solution for 24 hours, where A is sample A, B is sample B, C is sample C, and D is sample D.

[0103] Depend on Fig.10 It can be seen that compared with sample A, the amount of E. coli adhesion on the surface of samples B, C and D is significantly reduced, especially in sample D, where the amount of E. coli adhesion is more significantly reduced. This is because the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating produces reactive oxygen species (ROS), which can damage cell components such as bacterial cell membranes, proteins, and DNA, resulting in the inhibition of bacterial growth or direct death without affecting surrounding human tissues. This shows that the new coated ureteral stent prepared by the present invention has excellent anti-bacterial adhesion effect and has broad medical application prospects.

[0104] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0105] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a polypeptide-silver-manganese co-assembled nanozyme antibacterial coating, characterized in that: The steps include: The substrate material is sequentially immersed in a hydrogen peroxide-sulfuric acid mixture, a γ-aminopropyltriethoxysilane solution, a polypeptide-EDC-NHS mixture, a silver-containing solution, and a manganese-containing solution for activation, amino modification, polypeptide coupling, surface silver plating, and surface manganese plating, respectively, to complete the preparation of the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating; The hydrogen peroxide-sulfuric acid mixed liquid is a mixed liquid consisting of hydrogen peroxide and sulfuric acid; The polypeptide-EDC-NHS mixed solution is a mixed solution composed of polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

2. The preparation method according to claim 1, characterized in that: The volume ratio of hydrogen peroxide to sulfuric acid in the hydrogen peroxide-sulfuric acid mixture is 1-5:5-9; the mass fraction of the sulfuric acid is 98±0.3%; and / or the activation time is 0.5-5h.

3. The preparation method according to claim 1, characterized in that: The solvent of the γ-aminopropyltriethoxysilane solution includes ethanol; and / or the volume ratio of γ-aminopropyltriethoxysilane to the solvent in the γ-aminopropyltriethoxysilane solution is 1:9; and / or the time of the amino modification is 6 to 8 hours.

4. The preparation method according to claim 1, characterized in that: The preparation step of the polypeptide-EDC-NHS mixed solution includes: mixing the polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, N-hydroxysuccinimide solution and water to obtain the polypeptide-EDC-NHS mixed solution; The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide solution is 1 to 30 mmol / L; The dosage ratio of the polypeptide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution, N-hydroxysuccinimide solution and water is 0.001-0.005 g: 0.5-2 mL: 0.5-2 mL: 0.5-2 mL.

5. The preparation method according to claim 1, characterized in that: The polypeptide coupling time is 3h.

6. The preparation method according to claim 1, characterized in that: The silver in the silver-containing solution is added in the form of silver salt; the silver salt includes one or more of silver nitrate, silver chloride, silver sulfide, silver ammonia solution, silver carbonate and silver-silver nitrate complex; and / or the concentration of the silver-containing solution is 20 mmol / mL; and / or the surface silver plating time is 48 hours.

7. The preparation method according to claim 1, characterized in that: The manganese in the manganese-containing solution is added in the form of manganese salt; the manganese salt includes one or more of potassium permanganate, manganese chloride, manganese sulfate, manganese carbonate, manganate, manganese oxide and manganese nitrate; and / or, the concentration of the manganese-containing solution is 10 to 200 mmol / L; and / or, the surface manganese plating includes: first reacting at 0±5°C for 1 to 4 hours, and then reacting under ultraviolet light irradiation at 0±5°C for 2 to 12 hours.

8. The polypeptide-silver-manganese co-assembled nanozyme antibacterial coating prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the polypeptide-silver-manganese co-assembled nanozyme antibacterial coating according to claim 8 in a modified ureteral stent.

10. A method for reducing the adhesion of proteins and bacteria on the surface of a ureteral stent and the formation of biofilm, characterized in that: The method comprises the following steps: treating the ureteral stent by using the preparation method according to any one of claims 1 to 7.

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