PLGA / Zn-KNN piezoelectric antibacterial scaffold material as well as preparation method and application thereof

Abstract

The invention discloses an ultrasonic-driven PLGA / Zn-KNN piezoelectric antibacterial scaffold material as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. The PLGA / Zn-KNN piezoelectric antibacterial scaffold material provided by the invention is prepared by combining zinc oxide (ZnO) into potassium sodium niobate (KNN), and is compounded with polylactic acid-glycolic acid copolymer (PLGA) to realize multi-combination antibiosis for bone infection. Moreover, the physical and chemical properties of the PLGA / Zn-KNN piezoelectric antibacterial scaffold material are analyzed, and bacteria, cell and animal experiments are carried out to characterize the antibacterial and infection treatment capabilities of the piezoelectric scaffold. According to the invention, ZnO particles are embedded into a KNN solid solution matrix, so that the piezoelectric property of the PLGA / Zn-KNN stent is enhanced. In addition, the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the invention releases zinc ions, and ultrasonic-driven electrical stimulation generates a remarkable antibacterial effect through direct and immunoregulation antibacterial ways.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and specifically relates to an ultrasonically driven PLGA / Zn-KNN piezoelectric antibacterial stent material and a preparation method and application thereof. Background Art

[0002] Bone infection is becoming a global economic and health challenge, which can lead to destruction of bone and surrounding soft tissue, implant failure, and even systemic inflammatory response. Antibiotic administration is the main clinical method for infection treatment. However, long-term use can lead to intestinal microbiota disturbance, bacterial evolution, gastrointestinal discomfort, and abnormal liver and kidney function. In addition, once bacterial biofilms are formed in the host, antibiotic resistance and chronic inflammation will occur, which is very difficult to cure. Therefore, new biomaterials and antimicrobial strategies for alternative antimicrobial therapies are urgently needed.

[0003] Physical stimulation is a promising antibacterial therapy that destroys the structure of microorganisms by mechanical stretching or interfering with metabolism. It can not only reduce the drug resistance of microorganisms, but also kill resistant bacteria. Piezoelectric materials are biofriendly and show good piezoelectric effect. They can form built-in electric fields and surface potentials under external mechanical stress stimulation. Moreover, researchers have found that exogenous ultrasound can promote the piezoelectric effect. Therefore, ultrasound-driven piezoelectric materials have become a new antibacterial method that integrates electrical and acoustic dynamic processing.

[0004] At present, the most widely available piezoelectric materials mainly include piezoelectric crystals, piezoelectric ceramics and organic piezoelectric materials. Zhu et al. designed piezoelectric barium titanate with antibacterial drugs and self-assembled MOF, which showed synergistic antibacterial efficacy by inducing ROS production through sonodynamic treatment and drug release (Dynamically evolving piezoelectric nanocompositesfor antibacterial and repair-promoting applications in infectedwoundhealing.Acta Biomater.2023;157:566-577.). Zhang et al. prepared sulfur-doped graphdiyne nanosheets, which enhanced peroxidase-like activity by piezoelectric effect for bacterial disinfection (Piezoelectric enhancedperoxidase-like activity of metal-free sulfur doped graphdiyne nanosheets forefficientwater pollutant degradation and bacterial disinfection.Nano Today,2022(43-):43.). However, piezoelectric materials in the prior art still have many problems, such as non-biodegradation or toxicity. In addition, electrical stimulation also has many problems, including high energy consumption, low sterilization efficiency and safety issues. Potassium sodium niobate (K 0.5 Na 0.5 NbO3, KNN) possesses piezoelectric properties, biocompatibility, and biodegradability, but it lacks antibacterial properties. Therefore, the preparation of KNN with antibacterial efficacy is an interesting solution for non-antibacterial treatments.

[0005] Many metal-based nanomaterials and metal ions, such as silver ions (Ag + ), magnesium ion (Mg 2+ ), copper ions (Cu 2+ ) and zinc ions (Zn 2+ ), which exhibits outstanding antibacterial effects by inducing bacterial cell membrane rupture, oxidative stress, and intracellular biomolecule rupture. However, these effects are dose-dependent. Excessive free ions can cause cytotoxicity and even cause serious damage to organisms. In addition, the application problems of piezoelectric materials also include low electrocatalytic efficiency, which limits their application in the antibacterial field.

[0006] Based on the above reasons, this application is filed. Summary of the invention

[0007] Based on the above reasons, in view of the problems or defects existing in the prior art, the purpose of the present invention is to provide a PLGA / Zn-KNN piezoelectric antibacterial scaffold material and its preparation method and application, so as to solve or at least partially solve the above technical defects existing in the prior art: the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the present invention has direct and immunomodulatory antibacterial activity, and can be driven by ultrasound.

[0008] In order to achieve one of the above purposes of the present invention, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a PLGA / Zn-KNN piezoelectric antibacterial stent material, the method specifically comprising the following steps:

[0010] (1) Synthesis of Zn-KNN composite particles

[0011] The KNN particles and ZnO nanoparticles are sequentially added to the dispersion medium according to the ratio, ball-milled and mixed, dried and then calcined at 800-900° C. for 10-20 min; after the calcination, the obtained product is cooled to room temperature to obtain the Zn-KNN composite particles;

[0012] (2) Preparation of PLGA / Zn-KNN piezoelectric antibacterial scaffold material

[0013] The Zn-KNN composite particles in step (1) are added to a Tris-HCl aqueous solution and uniformly dispersed to obtain a Zn-KNN dispersion; dopamine hydrochloride (DA-HCl) is then added to the Zn-KNN dispersion according to a ratio, and the mixture is stirred for reaction for 10-15 hours. The obtained reaction product is washed and vacuum dried to obtain polydopamine (PDA)-modified Zn-KNN;

[0014] The PDA-modified Zn-KNN is uniformly dispersed in hexafluoroisopropanol, PLGA is added according to a ratio, and stirred evenly. The resulting mixed solution is poured into a polytetrafluoroethylene mold and dried to obtain the PLGA / Zn-KNN piezoelectric antibacterial stent material.

[0015] Furthermore, in the above technical solution, the particle size of the ZnO nanoparticles in step (1) is 10-50 nm.

[0016] Furthermore, in the above technical solution, the mass ratio of the KNN particles to the ZnO nanoparticles in step (1) is 20:1.

[0017] Furthermore, in the above technical solution, the dispersion medium in step (1) is an alcohol solvent, and the alcohol solvent can be any one of anhydrous ethanol, isopropanol or polyethylene glycol.

[0018] Furthermore, in the above technical solution, the amount of the dispersion medium in step (1) is not specifically limited, as long as the KNN particles and the ZnO nanoparticles can be completely and evenly dispersed. For example, the total mass of the KNN particles and the ZnO nanoparticles and the volume ratio of the dispersion medium is (1-10) mg: (5-10) mL.

[0019] Furthermore, in the above technical solution, the ball milling time in step (1) is 20-30 hours.

[0020] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the calcination temperature is 850° C. and the calcination time is 15 minutes.

[0021] Furthermore, in the above technical solution, the KNN particles in step (1) are prepared by the following method, the steps are as follows:

[0022] (a) Potassium chloride, potassium carbonate and niobium pentoxide (Nb2O5) are mixed according to a ratio, ball-milled and calcined at 950-1050°C for 2-4 hours; after calcination, the obtained product is cooled to room temperature, soaked in dilute acid, washed and dried in sequence to obtain a KNN particle precursor;

[0023] (b) The KNN particle precursor is mixed with sodium carbonate, sodium chloride, potassium chloride and potassium carbonate according to a ratio, ball-milled to obtain a uniform mixture, and then calcined at 800-900° C. for 5-15 min to obtain the KNN particles.

[0024] Furthermore, in the above technical solution, the mass ratio of potassium chloride, potassium carbonate and niobium pentoxide in step (a) is 11:1:4.

[0025] Specifically, in the above technical solution, the purpose of soaking with dilute acid in step (b) is to remove the reaction raw materials that have not participated in the reaction.

[0026] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the calcination temperature in step (a) is 1000° C. and the calcination time is 3 hours.

[0027] Furthermore, in the above technical solution, in step (b), the mass ratio of the KNN particle precursor, sodium carbonate, sodium chloride, potassium chloride and potassium carbonate is 100:3:11:1:11.

[0028] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the calcination temperature in step (b) is 850° C. and the calcination time is 10 min.

[0029] Furthermore, in the above technical solution, the concentration of the Tris-HCl aqueous solution in step (2) is 0.5-2 mM, and the pH value is 8.5-9.0.

[0030] Furthermore, in the above technical solution, the mass ratio of dopamine hydrochloride to Zn-KNN composite particles in step (2) is 1:10.

[0031] Furthermore, in the above technical solution, in a preferred embodiment of the present invention, the stirring reaction time in step (2) is 12 hours.

[0032] Furthermore, in the above technical scheme, the amount of the PDA-modified Zn-KNN and hexafluoroisopropanol used in step (2) may not be specifically limited, as long as the PDA-modified Zn-KNN can be evenly dispersed. For example, in a preferred embodiment of the present invention, the amount ratio of the PDA-modified Zn-KNN to hexafluoroisopropanol is 0.5 g:10 mL.

[0033] Furthermore, in the above technical solution, in step (2), the mass ratio of the PDA-modified Zn-KNN to PLGA is 1:2.

[0034] Furthermore, in the above technical solution, the molecular weight of the PLGA in step (2) is between 10k and 150k.

[0035] The second object of the present invention is to provide a PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the above method, wherein the scaffold material is biodegradable and has direct and immunomodulatory antibacterial activity against bone infection under controllable ultrasonic drive.

[0036] The third object of the present invention is to provide the use of the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the above method in the preparation of antibacterial drugs for preventing and treating bone infection.

[0037] The present invention also provides an antibacterial drug for preventing and treating bone infection, comprising the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the above method.

[0038] The mechanism involved in the present invention is as follows:

[0039] The invention introduces ZnO dopant into KNN-based piezoelectric ceramics to enhance the lattice distortion of BO6 octahedron, thereby enhancing the piezoelectric properties, including enhancing the piezoelectric coefficient (kp), piezoelectric charge constant (d33) and Curie temperature (Tc). Therefore, adjusting the phase transition temperature of KNN piezoelectric ceramics by adding ZnO may be an effective way to obtain antibacterial and piezoelectric properties.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The PLGA / Zn-KNN piezoelectric antibacterial scaffold material provided by the present invention is prepared by combining zinc oxide (ZnO) into potassium sodium niobate (KNN), and compounded with polylactic acid-glycolic acid copolymer (PLGA) to achieve multi-combination antibacterial for bone infection. In addition, the present invention analyzes the physicochemical properties of the PLGA / Zn-KNN piezoelectric antibacterial scaffold material, and conducts bacteria, cell and animal experiments to characterize the antibacterial and infection treatment capabilities of the piezoelectric scaffold. The present invention also enhances the piezoelectric properties of the PLGA / Zn-KNN scaffold by embedding ZnO particles into the KNN solid solution matrix. In addition, the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the present invention releases zinc ions, and ultrasound-driven electrical stimulation produces significant antibacterial effects through direct and immunomodulatory antibacterial pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 Ultrasound-driven PLGA / Zn-KNN piezoelectric antibacterial scaffold material promotes bone infection: (A) Synthesis and manufacturing process of PLGA / Zn-KNN piezoelectric antibacterial scaffold material in Example 1; (B) Direct antibacterial effect through ion release and electricity generation; (C) Indirect antibacterial effect through regulation of M1 macrophages; (D) Ultrasound-driven PLGA / Zn-KNN piezoelectric antibacterial scaffold material treats bone infection in SD rats.

[0044] Figure 2 Characterization of KNN and Zn-KNN composite particles prepared in Example 1: (A) Scanning electron microscope image of KNN particles; (B) Scanning electron microscope image of Zn-KNN composite particles; (C) EDS elemental map of Zn-KNN composite particles; (D) HRTEM image of KNN particles; (E) HRTEM image of Zn-KNN composite particles; (F) Electron diffraction spots of KNN and Zn-KNN composite particles; (G) XRD diagram of KNN and Zn-KNN composite particles.

[0045] Figure 3Characterization of the composite scaffold materials prepared in Example 1 and Comparative Example 1, respectively: (AB) SEM images and EDS elemental maps of PLGA / KNN and PLGA / Zn-KNN scaffolds; (C) FTIR of PLGA / KNN and PLGA / Zn-KNN scaffolds; (D) ion concentration in the salt solution after immersion of PLGA / KNN and PLGA / Zn-KNN scaffolds for different times; (EF) open circuit voltage generated by PLGA / KNN scaffold under ultrasonic drive (frequency 1 MHz, pulse width 1 ms, pulse interval 0.5 s); (GH) open circuit voltage generated by PLGA / Zn-KNN scaffold under ultrasonic drive (frequency 1 MHz, pulse width 1 ms, pulse interval 0.5 s); (I) piezoelectric constants of PLGA / KNN and PLGA / Zn-KNN scaffolds in simulated environments with different shear force directions and crystal polarization directions.

[0046] Figure 4 : (A) The open circuit voltage generated by the PLGA / KNN scaffold prepared in Comparative Example 1 under ultrasonic drive (frequency 1 MHz, pulse width 1 ms, pulse interval 0.5 s) after immersion in PBS for 14 days; (B) The open circuit voltage generated by the PLGA / Zn-KNN scaffold prepared in Example 1 under ultrasonic drive (frequency 1 MHz, pulse width 1 ms, pulse interval 0.5 s) after immersion in PBS for 14 days.

[0047] Figure 5 The cell compatibility and osteogenic properties of the composite scaffold materials prepared in Example 1 and Comparative Example 1, respectively: (A) F-actin and nuclear staining were performed to detect the effects of PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) on the function of BMSCs; (B) Live-dead cell staining of BMSCs incubated with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) for 24 hours; (C) ALP staining was performed to evaluate the effects of PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) on the osteogenic differentiation of BMSCs; (D) Relative gene expression of osteoclast marker genes OPN, OCN and Col-1 in BMSCs incubated with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) for 14 days; (E) Cell proliferation assay results of bone marrow mesenchymal stem cells treated with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+).

[0048] Figure 6Direct antibacterial effects of the composite scaffold materials prepared in Example 1 and Comparative Example 1 under ultrasound. (AB) Live colonies on standard agar plates after E. coli and MRSA were treated with various scaffolds and ultrasound (+); (CD) Bacterial colony counts of E. coli and MRSA; (E) OD values ​​of bacteria treated with various scaffolds and ultrasound (+); (F) Live / dead staining analysis of bacteria treated with various scaffolds and ultrasound (+).

[0049] Figure 7 Indirect antibacterial effect of the composite scaffold materials prepared in Example 1 and Comparative Example 1 under ultrasound. (A) Macrophages treated with scaffold and ultrasound (+) can capture and consume more CFDA-labeled MRSA (B) TEM image of bacterial phagocytosis by macrophages treated with scaffold and ultrasound (+); (C) Immunofluorescence staining image of CD86 and TNF-α after 2 days of treatment with PLGA / Zn-KNN scaffold and ultrasound (+).

[0050] Figure 8 Anti-infection effects of the composite scaffold materials prepared in Example 1 and Comparative Example 1 respectively in vivo: (A) Reconstructed micro-CT images of femoral condyle infection in SD rats treated with various scaffolds for 2 months; (B) Quantitative analysis of bone healing area; (C) Quantitative analysis of scaffold degradation rate; (D) Quantitative analysis of bone volume / total volume (BV / TV).

[0051] Fig. 9 Histopathology Gram stain of bone tissue.

[0052] Fig.10 In vivo histological analysis of bone infection after treatment of the composite scaffold materials prepared in Example 1 and Comparative Example 1, respectively: (A) H&E staining of histopathological bone tissue; (B) TRAP staining of histopathological bone tissue treated with various piezoelectric scaffolds and ultrasound; (C) Immunofluorescence staining of osteocalcin (OCN, red) and osteocalcin (OCN, green) proteins in different scaffold groups. DETAILED DESCRIPTION

[0053] The present invention develops a biodegradable PLGA / Zn-KNN composite material, which can be used as a biodegradable antibacterial tissue scaffold and a controllable electrical stimulator at the same time. The incorporation of ZnO into KNN greatly enhances the piezoelectric properties of Zn-KNN particles, showing inherent solubility and degradation behavior. In addition, the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the present invention can release zinc ions, and can also directly and immunomodulate antibacterial through ultrasound-driven electrical stimulation, produce significant antibacterial effects, and provide guidance for the immuno-antibacterial treatment of clinical infections.

[0054] The present invention is further described in detail below through implementation cases. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following implementation cases.

[0055] According to the information contained in this application, various changes can be easily made to the precise description of the present invention for those skilled in the art.It should be understood that the scope of the present invention is not limited to defined processes, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention.

[0056] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in this application to express the amount, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained based on the reported significant figures and by conventional rounding methods.

[0057] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0058] The ZnO nanoparticles used in the following examples of the present invention were purchased from Aladdin Reagent Company and had a particle size of 30 nm.

[0059] The polylactic acid-glycolic acid copolymer (PLGA) (intrinsic viscosity = 0.6 dL / g, polylactic acid:glycolic acid = 75:25, mol / mol) used in the following examples of the present invention was purchased from Changchun Shengboma Biomaterial Co., Ltd.

[0060] Example 1

[0061] A method for preparing a PLGA / Zn-KNN piezoelectric antibacterial stent material in this embodiment specifically comprises the following steps:

[0062] (1) Synthesis of KNN particles by solid phase reaction

[0063] Potassium chloride, potassium carbonate and niobium pentoxide were mixed in a mass ratio of 11:1:4 by ball milling, and then calcined at 1000°C in a high-temperature resistant crucible for 3 hours. After the calcination, the mixture was cooled to room temperature, and the resulting product was immersed in a 2M nitric acid solution for 2 hours, washed with deionized water, and dried in a forced air drying oven to obtain a KNN particle precursor.

[0064] The KNN particle precursor was mixed with sodium carbonate, sodium chloride, potassium chloride and potassium carbonate in a mass ratio of 100:3:11:1:11, ball milled, and calcined in a crucible at 850°C for 10 min to completely convert into KNN particles. Finally, the KNN particles were collected by washing and drying in a forced air drying oven.

[0065] (2) Synthesis of Zn-KNN composite particles

[0066] The KNN particles prepared in step (1) and ZnO nanoparticles (30 nm, Aladdin) were ball-milled in alcohol at a mass ratio of 20:1 for 24 hours. After drying, the resulting mixture was sintered at 850° C. in a covered alumina crucible for 15 minutes and then cooled to room temperature to obtain Zn-KNN composite particles.

[0067] (3) Preparation of PLGA / Zn-KNN piezoelectric antibacterial scaffold material

[0068] (i) 1 g of the Zn-KNN composite particles described in step (2) was dispersed in 100 mL of 1 mM Tris-HCl aqueous solution (pH 8.6), followed by ultrasonic treatment (50 kHz) for 60 minutes and uniform stirring for 2 hours. Then, 0.1 g of dopamine hydrochloride (DA-HCl) was transferred to the above mixed solution and stirred for 12 hours. Finally, the obtained reaction product was washed and dried in a vacuum oven to obtain PDA-modified Zn-KNN.

[0069] (ii) 0.5 g of the PDA-modified Zn-KNN prepared in step (i) was dispersed in 10 mL of hexafluoroisopropanol by ultrasound (50 kHz) for 60 minutes and stirred for 2 hours, and 1 g of PLGA powder was added to the solution and stirred for 2 hours to form a uniform solution. Subsequently, the resulting mixture was cast into a polytetrafluoroethylene mold and dried at room temperature to obtain the PLGA / Zn-KNN piezoelectric antibacterial scaffold material.

[0070] Comparative Example 1

[0071] The preparation method of a PLGA / KNN scaffold material in this comparative example is basically the same as that in Example 1, except that this comparative example does not contain step (2), and other processes and parameters are the same.

[0072] (I) Structural characterization

[0073] The present invention characterized the size and morphology of the KNN and Zn-KNN composite particles prepared in Example 1 by SEM ( Figure 2 A and 2B). The crystal length of KNN particles is about 1 to 20 μm, and the crystal length of Zn-KNN composite particles is about 1 to 10 μm. Detailed element distribution is provided ( Figure 2C). K, Na, Nb, and Zn elements occupy the same region, while the distribution of some Zn is isolated (indicated by white arrows). The isolated Zn was further analyzed by HRTEM micrographs and selected area diffraction patterns. The (002) interplanar spacing of KNN particles is about 0.3948nm ( Figure 2 D). Under the same synthesis conditions, the (002) interplanar spacing of Zn-KNN composite particles is about 0.4120 μm ( Figure 2 E). The isolated Zn is adsorbed on the grain boundary surface of Zn-KNN composite particles in the form of nano-ZnO ( Figure 2 E). The electron diffraction patterns show that KNN and Zn-KNN particles have the same crystal structure ( Figure 2 F) XRD further confirmed this result. Figure 2 G shows the XRD pattern of KNN particles. The diffraction peaks of KNN particles (PDF#77-0038) are assigned to (002), (022), (400), (204), (422) and (440). The diffraction peaks of ZnO particles (PDF#76-0704) are assigned to (100), (002), (101), (102) and (103). The above results confirm the composite structure of KNN and ZnO. It can be expected that ZnO exists in two forms. Some zinc is randomly distributed in the KNN matrix, which increases its interplanar spacing. Another part of ZnO is adsorbed on the grain boundaries of KNN, acting as a sintering aid.

[0074] (II) Ion release performance test of composite scaffold materials:

[0075] The PLGA / KNN scaffold material prepared in Comparative Example 1 and the PLGA / Zn-KNN scaffold material prepared in Example 1 were immersed in 9 mg / L sodium chloride solution, respectively. The mixed solution was centrifuged (10000 rpm) for 5 minutes to collect the supernatant, which was soaked again with a fresh 9 mg / L NaCl solution to dilute it at 37°C. Supernatant samples were collected every 3 days for 2 weeks. Then, the Zn in the supernatant was measured using an inductively coupled plasma-optical emission spectrometer (ICP-AES, Optima 4300DV). 2+ and Nb 5+ concentration.

[0076] (III) Piezoelectric performance test:

[0077] ViennaAb Initio Simulations: The piezoelectric properties of ZnO / KNbO3 solid vacuum heterojunctions and solid KNbO3 were compared using the ViennaAb Initio Simulation Package (VASP). In order to accurately obtain and compare the piezoelectric properties of PLGA / KNN and PLGA / Zn-KNN scaffolds, it is necessary to calculate their piezoelectric constants dij and piezoelectric tensors eij, paying attention to the elastic constants cij. First, the geometric structures of ZnO / KNbO3 vacuum heterojunctions and solid KNbO3 were optimized using VASP to ensure the accuracy of the calculations. During the optimization process, the lattice parameters and atomic positions should be considered to be adjusted until the energy and force converge. The stress-strain relationship was calculated to obtain the elastic constant matrix of the material. The piezoelectric tensor was obtained by applying a small strain and calculating the polarization of the system using the built-in function of VASP. By combining the elastic constant and the piezoelectric tensor, the piezoelectric constant can be obtained by the following relationship:

[0078] dij=eij·sij

[0079] Among them, sij is the elastic compliance matrix (i.e., the inverse matrix of cij). The piezoelectric properties of ZnO / KNbO3 solid vacuum heterojunction and KNbO3 solid are comprehensively compared, and their advantages and differences in practical applications are clarified.

[0080] like Figure 3 As shown in A, the piezoelectric scaffold was fabricated by doping PDA-modified KNN or Zn-KNN composite particles into PLGA. The KNN or Zn-KNN composite particles distributed in the piezoelectric scaffold were also confirmed by the elemental mapping of K, Na, Nb, and Zn in SEM characterization ( Figure 3 B) FTIR spectrum of the piezoelectric support is shown in Figure 3 C. 1164cm -1 (COC stretch), 1721cm -1 (C=O stretching), 2867cm -1 (CH2 stretching) and 2946cm -1 The band at (CH3 stretching) can be attributed to the characteristic peak of PLGA. Figure 3 D shows the Zn released in the supernatant collected from days 1 to 12. 2+ and Nb 5+ The concentration of Zn 2+ and Nb 5+ The release was relatively small in the first 6 days, reaching 0.4 and 0.8 mg / L respectively. 2+ The concentration of Nb was between 0.4 and 0.6 mg / L. 5+ The concentration of Zn is between 0.8 and 1.0 ± 0.01 mg / L. 2+ and Nb 5+The sustained release of PLGA / KNN clearly indicates the dissolution of KNN and Zn-KNN in saline solution. To measure the electrical output of the piezoelectric scaffold under ultrasonic actuation, the ultrasonic driving signal (frequency 1 MHz) was set to a pulse width of 1 ms and a pulse interval of 0.5 s. The open circuit voltage generated by the PLGA / KNN scaffold under ultrasonic actuation was 20 mV ( Figure 3 E-3F). The open circuit voltage of the PLGA / Zn-KNN scaffold under ultrasound actuation was 40 mV ( Figure 3 G-3H). Therefore, the addition of ZnO to KNN can greatly improve the piezoelectric properties, which is compared by using VASP (Table 1-Table 2). In the simulated environment with different shear force directions and crystal polarization directions, the piezoelectric constant of PLGA / Zn-KNN scaffolds is higher than that of PLGA / KNN ( Figure 3 I), and this piezoelectric constant was still maintained after being immersed in PBS for 14 days.

[0081] Table 1 Piezoelectric tensor and piezoelectric constant of PLGA / KNN scaffold prepared in Comparative Example 1

[0082]

[0083] Table 2 Piezoelectric tensor and piezoelectric constant of PLGA / Zn-KNN scaffold prepared in Example 1

[0084]

[0085] (IV) Cytocompatibility and osteogenic properties testing of composite scaffold materials:

[0086] Cell viability analysis: Rat bone marrow mesenchymal stem cells were obtained from male Wistar rats. The cells were dispersed, centrifuged (1000 rpm, 10 minutes) and resuspended in α-MEM containing 20% ​​FBS and inoculated into 25 cm 2 The cells were cultured in plastic flasks and incubated at 37°C in a humidified atmosphere with 5% CO2 for 24 h before the first culture medium was replaced. Then, the culture medium was replaced every 3 days and the cells were used after 1 week. BMSCs from passages 2 to 5 were used for all experiments. The cultured BMSCs were then plated at 100,000 cells / cm 2The cells were seeded at a density of 10% in α-MEM supplemented with 10% fetal bovine serum, 1% penicillin and streptomycin, and the culture medium was changed every 3 days. A cell proliferation assay was performed to evaluate the viability and survival of bone marrow mesenchymal stem cells (BMSCs) under ultrasound exposure in PLGA / KNN scaffolds, PLGA / Zn-KNN scaffolds and PLGA / Zn-KNN scaffolds treated with ultrasound (the driving signal frequency of ultrasound was 1 MHz, set to a pulse width of 1 ms and a pulse interval of 0.5 s). Specifically, BMSCs were seeded on sterile scaffolds in 96-well plates, respectively. The culture medium was used as a control group. After 1, 3 and 5 days of culture, the cell viability was studied using the CCK-8 assay according to the kit instructions to evaluate the effects of ultrasound and PLGA / Zn-KNN scaffolds on cell proliferation. BMSCs treated with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) for 3 days were stained with TRITC alkaloids (5 μg / mL) and DAPI (5 μg / mL) to display the cytoskeleton and nucleus, respectively. The cell status of BMSCs on day 1 was evaluated by live-dead cell staining using calcein-acetoxymethyl ester (calcein-AM) / propidium iodide (PI) double staining. The cells were observed by fluorescence microscopy (Olympus IX71, Japan).

[0087] (V) In vitro osteogenic performance test

[0088] The present invention performed CCK-8 analysis on the PLGA / KNN scaffold material prepared in Comparative Example 1 and the PLGA / Zn-KNN scaffold material prepared in Example 1, respectively, to explore the effects of PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) (the driving signal frequency of ultrasound was 1 MHz, set to a pulse width of 1 ms and a pulse interval of 0.5 s) on the proliferation of bone marrow mesenchymal stem cells (BMSCs). The data showed that PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) had no effect on the activity of BMSCs. BMSCs treated with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) for 3 days were stained with TRITC alkaloids and DAPI to show the cytoskeleton and cell nucleus, respectively. The results are as follows Figure 5 As shown in A, it is shown that after 2 days of treatment with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+), BMSCs spread smoothly and maintained a normal shape. Live / dead staining of BMSCs co-cultured with PLGA / KNN and PLGA / Zn-KNN scaffolds and ultrasound (+) group grew to the entire area, and no number of dead cells was found ( Figure 5 B). Then, early markers of osteogenic differentiation were assessed by alkaline phosphatase (ALP) staining. After adding PLGA / KNN or PLGA / Zn-KNN scaffolds and ultrasound (+), Figure 5 C. Meanwhile, the relative gene expression of osteoclast marker genes OPN, OCN, and Col-1 in BMSCs incubated with PLGA / KNN or PLGA / Zn-KNN scaffolds and ultrasound (+) for 14 days showed significantly upregulated expression ( Figure 5 D), which means that the scaffold and ultrasound (+) effect are beneficial to the osteogenic differentiation of BMSCs.

[0089] (VI) Direct antibacterial effect of releasing zinc ions and generating electricity

[0090] Ultrasonication of bacterial strains with scaffolds: For the ultrasonication experiments, Escherichia coli (E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) were grown overnight at 37°C with shaking at 180 rpm in LB medium supplemented with 10 g / L peptone, 7 g / L beef extract, and 5 g / L NaCl. Subsequently, E. coli and MRSA were washed in PBS and centrifuged for 5 min, and then diluted with PBS to 1 to 5 × 10 based on the optical density value. 7 The density of CFU / mL was calculated. 200 μL of E. coli and MRSA suspensions were mixed with ZnO (0.14 mg / mL, equal to 9 mg PLGA / Zn-KNN), PLGA / KNN (9 mg / mL), and PLGA / Zn-KNN (9 mg / mL) scaffolds and then diluted to a final volume of 4 mL with PBS in a sterile microcentrifuge tube. The groups included the control group, PLGA / KNN scaffolds, PLGA / Zn-KNN scaffolds, and PLGA / Zn-KNN scaffolds, which were exposed to ultrasound at 37°C for 25 min (frequency: 100 kHz, pulse period: 0.5 s). After ultrasound treatment, the number of surviving bacteria was determined by the colony counting method. Briefly, each sample was serially diluted with PBS to a final volume of 10 7 Then, 200 μL of the diluted E. coli and MRSA suspensions were applied to the surface of LB agar plates. After incubation at 37°C for 24 h, the colonies of E. coli and MRSA were counted in CFU / mL. The number of surviving bacteria was determined by measuring the absorbance at 600 nm using a microplate reader.

[0091] Bacterial live / dead staining analysis: AO / EB staining kit is used to qualitatively determine the integrity of E. coli and MRSA bacterial cell membranes. 5-8×10 7Fresh PBS bacterial suspensions of E. coli and MRSA at CFU / mL were mixed with PLGA / KNN (9 mg / mL) and PLGA / KNN scaffolds (9 mg / mL) and then diluted to 4 mL with PBS in sterile microcentrifuge tubes. The mixtures in different microcentrifuge tubes were then exposed to ultrasound (frequency: 100 kHz, pulse period: 0.5 s) at 37°C for 25 min. The control group was not exposed to ultrasound and incubated at 37°C for 25 min. After different treatments, 1 μM AO and 2 μM EB were added and the mixtures were incubated in the dark for 10 min. Then, the excess AO and EB dyes were washed with PBS. Finally, E. coli and MRSA bacteria were observed using a fluorescence microscope (Olympus IX71) at an excitation wavelength of 488 nm.

[0092] The inactivation effects of the piezoelectric stents with and without ultrasound treatment on E. coli and MRSA were compared with the control without any stent or ultrasound treatment ( Figure 6 A and 6B). CFU assays are used to assess bacterial inactivation to determine the number of viable cells remaining after treatment. Plate counts are shown on Figure 6 C and 6D. There was no significant difference in the number of colonies between the PLGA / KNN group and the control group. The number of colonies in the PLGA / Zn-KNN group was significantly lower than that in the control group and the PLGA / KNN treatment group. The number of colonies in the PLGA / Zn-KNN synergistic ultrasound treatment group was significantly lower than that in the PLGA / Zn-KNN single treatment group. Pure PLGA / KNN has no antibacterial ability, but ultrasonic treatment makes it have antibacterial properties. After the introduction of nano zinc oxide, PLGA / Zn-KNN has antibacterial ability, and ultrasonic treatment further enhances the antibacterial ability of PLGA / Zn-KNN. Figure 6 E shows that after PLGA / Zn-KNN combined with ultrasonic treatment, the OD values ​​of Escherichia coli and MRSA decreased sharply, which is consistent with the previous data. Figure 6 F shows that E. coli and MRSA in the control group and PLGA / KNN group emitted green fluorescence, indicating that all bacteria were living cells. After treatment with zinc oxide and PLGA / Zn-KNN, a small number of bacteria were dyed red, indicating that the bactericidal effect of PLGA / Zn-KNN had changed. After PLGA / Zn-KNN combined with ultrasonic treatment, the proportion of red-dyed bacteria increased significantly, indicating that PLGA / Zn-KNN combined with ultrasonic treatment significantly improved the bactericidal effect against E. coli and methicillin-resistant Staphylococcus aureus.

[0093] (VII) Indirect antibacterial effect of regulating macrophage phagocytic function

[0094] Morphological macrophage phagocytosis assay: Mouse mononuclear macrophages (RAW264.7) were purchased from China Center for Type Culture Collection (Shanghai, China). The complete medium for RAW264.7 cells included 500 mL high glucose medium (Gibco), 10 mL fetal bovine serum (Gibco), and 5 mL penicillin / streptomycin solution (Gibco). During cell culture, the medium was replaced every 2 days. The complete medium for RAW264.7 cells included 500 mL high glucose medium (Gibco), 10 mL fetal bovine serum (Gibco), and 5 mL penicillin / streptomycin solution (Gibco). During cell culture, the medium was replaced every 2 days. The fused RAW264.7 cells were co-cultured on PLGA / KNN and PLGA / Zn-KNN scaffolds with or without ultrasonic treatment for 2 days. Then, CFDA-labeled Escherichia coli and MRSA were added to the wells and co-cultured with RAW264.7 cells for 1 hour. Finally, the phagocytosis results were observed and recorded using a fluorescence microscope. For TEM, the medium was replaced with gradient acetone after dehydration, and the cells were immersed in embedding medium overnight. After cross-linking polymerization at 37°C, 45°C, and 60°C for 12 h, the samples were cut into thin slices with a thickness of about 80 nm using an ultramicrotome (Leica emuc7). For visualization, uranyl acetate and lead dye solutions were used for double staining. Finally, TEM images were taken on a Hitachi H-7000FA with an accelerating voltage. For visualization at 2500x and 10,000x magnification.

[0095] Immunofluorescence staining: RAW264.7 cells were cultured on the piezoelectric support for 2 days and then fixed with 4% paraformaldehyde for 30 minutes at 37°C. The fixed RAW264.7 cells were permeabilized with 0.1% Triton-X-100 at 25°C for 5 minutes and blocked with 1% BSA / PBS at 25°C for 30 minutes. Fixed RAW264.7 cells were washed with PBS and incubated with primary TNF-α (1:800; 60291-1-LG; Proteintech) and CD86 (1:800; 13395-1-AP; Proteintech) after washing three times with PBS. Fixed RAW264.7 cells were then incubated with Cy3-conjugated affinity-purified goat anti-rabbit IgG (H+L) secondary antibody (sa 00009-2; Proteintech) and fluorescein (FITC)-conjugated affinity-purified goat anti-rabbit IgG (H+1) secondary antibody (sa 00003-2; Proteintech). After washing three times with PBS, fixed RAW264.7 cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and recorded using a laser scanning confocal microscope (Olympus FV4000, Japan).

[0096] Fluorescence staining was used to observe the phagocytosis of MRSA by macrophages. Figure 7A shows that the engulfed MRSA cells were stained green, while the nuclei of macrophages were stained blue. Obviously, the PLGA / Zn-KNN scaffolds in the ultrasound (+) group (the driving signal frequency of the ultrasound was 1 MHz, set to a pulse width of 1 ms and a pulse interval of 0.5 s) showed more MRSA phagocytosis in macrophages than the other groups. The fluorescence intensity increased in the order of control, PLGA / KNN, PLGA / KNN+, PLGA / Zn-KNN, and PLGA / Zn-KNN+ ( Figure 7 A), which indicates that most bacteria were phagocytosed by macrophages cultured on PLGA / Zn-KNN+. Therefore, macrophages cultured on PLGA / Zn-KNN+ scaffolds showed the greatest ability to phagocytize MRSA. Transmission electron microscopy further detected the phagocytosis of MRSA by macrophages. Figure 7 B illustrates that the PLGA / Zn-KNN scaffolds in the ultrasound (+) group showed more phagocytosed MRSA in macrophages than the other groups. Among the other groups, the PLGA / Zn-KNN+ scaffolds showed the highest phagocytosis rate. Immunofluorescence staining was performed to mark polarized macrophages (M1: TNF-α positive, CD86 positive). Figure 7 As shown in C, PLGA / Zn-KNN scaffold combined with ultrasound treatment (PLGA / Zn-KNN+) exerted the highest proportion of M1 polarized macrophages and inflammatory cytokines (TNF-α), indicating that ultrasound-mediated electrical stimulation promoted the activation of M1 polarized macrophages and the secretion of cytokines, which may enhance the phagocytosis of bacteria by macrophages.

[0097] (VIII) In vivo therapeutic effect of bone infection

[0098] The PLGA / Zn-KNN scaffolds showed excellent antibacterial properties after in vitro ultrasound treatment, which prompted the inventors to explore the potential in vivo effects. In order to further study the degradation process of the scaffolds and the formation of new bone in vivo, Figure 8 A shows a reconstructed 3D image of the left femur at 2 months. The PLGA / Zn-KNN+ group had only a small amount of new bone formation at the defect site. Obvious osteolysis and severe trabecular disorder indicated that a typical bone infection was successfully established, but there was still a significant unrepaired gap at the defect site. In contrast, the other groups showed milder symptoms of osteomyelitis and new bone formation, confirming the importance of pore structure in bone regeneration. In addition, rats treated with PLGA / Zn-KNN+ had a larger bone healing area ( Figure 8 B) and the degradation rate of the scaffold ( Figure 8 C). Figure 8 Quantitative analysis in D confirmed that the BV / TV ratio of the PLGA / Zn-KNN+ group was significantly higher than that of the other groups. In conclusion, the use of PLGA / Zn-KNN scaffolds and their acoustic-electric effect improved the microenvironment of osteomyelitis.

[0099] Gram staining is used to observe bacteria in tissues, where dark (purple) is Gram-positive bacteria and red is Gram-negative bacteria. Fig. 9 As shown, in the control group and PLGA / KNN group, the defect site was filled only with a large amount of dark purple area. However, the PLGA / Zn-KNN group showed a small amount of dark purple area, while the PLGA / Zn-KNN+ group did not show any dark purple area. The main reason for this phenomenon is that ZnO in PLGA / Zn-KNN has certain antibacterial properties. It is worth noting that ultrasonic treatment even further stimulated the discharge of PLGA / Zn-KNN and enhanced its antibacterial properties. H&E and Masson staining were used to evaluate the effects of PLGA / Zn-KNN scaffolds and ultrasonic exposure on bone regeneration in the lateral femoral condyle area. As Fig.10 As shown in A, after 2 months, the PLGA / Zn-KNN+ group showed more newly formed bone, while the other groups had limited formed bone and fibrous tissue around the implants. TRAP is a specific osteoclast enzyme, and TRAP staining was used to indicate the number and distribution of osteoclasts. Fig.10 As shown in B, osteoclasts were stained dark. The combination of PLGA / Zn-KNN scaffold and ultrasound (+) significantly reduced the dark color, indicating that osteoclast activity in new bone tissue was inhibited in the PLGA / Zn-KNN+ group. Immunofluorescence staining of osteocalcin (OCN, red) and osteopontin (OPN, green) in different scaffold groups is shown in Fig.10 As shown in C. The fluorescence intensity of OCN and OPN in the PLGA / Zn-KNN+ group was significantly enhanced compared with other groups, providing the required regenerative microenvironment for bone infection.

Claims

1. A method for preparing a PLGA / Zn-KNN piezoelectric antibacterial stent material, characterized in that: The method specifically comprises the following steps: (1) Synthesis of Zn-KNN composite particles The KNN particles and ZnO nanoparticles are sequentially added to the dispersion medium according to the ratio, ball-milled and mixed, dried and then calcined at 800-900° C. for 10-20 min; after the calcination, the obtained product is cooled to room temperature to obtain the Zn-KNN composite particles; (2) Preparation of PLGA / Zn-KNN piezoelectric antibacterial scaffold material The Zn-KNN composite particles in step (1) are added to a Tris-HCl aqueous solution and uniformly dispersed to obtain a Zn-KNN dispersion; dopamine hydrochloride (DA-HCl) is then added to the Zn-KNN dispersion according to a ratio, and the mixture is stirred for reaction for 10-15 hours. The obtained reaction product is washed and vacuum dried to obtain polydopamine (PDA)-modified Zn-KNN; The PDA-modified Zn-KNN is uniformly dispersed in hexafluoroisopropanol, PLGA is added according to a ratio, and stirred evenly. The resulting mixed solution is poured into a polytetrafluoroethylene mold and dried to obtain the PLGA / Zn-KNN piezoelectric antibacterial stent material.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the KNN particles to the ZnO nanoparticles in step (1) is 20:

1.

3. The preparation method according to claim 1 or 2, characterized in that: The KNN particles in step (1) are prepared by the following method: (a) Potassium chloride, potassium carbonate and niobium pentoxide (Nb2O5) are mixed according to a ratio, ball-milled and calcined at 950-1050°C for 2-4 hours; after calcination, the obtained product is cooled to room temperature, soaked in dilute acid, washed and dried in sequence to obtain a KNN particle precursor; (b) The KNN particle precursor is mixed with sodium carbonate, sodium chloride, potassium chloride and potassium carbonate according to a ratio, ball-milled to obtain a uniform mixture, and then calcined at 800-900° C. for 5-15 min to obtain the KNN particles.

4. The preparation method according to claim 3, characterized in that: The mass ratio of potassium chloride, potassium carbonate and niobium pentoxide in step (a) is 11:1:

4.

5. The preparation method according to claim 3, characterized in that: In step (b), the mass ratio of the KNN particle precursor, sodium carbonate, sodium chloride, potassium chloride and potassium carbonate is 100:3:11:1:

11.

6. The preparation method according to claim 1, characterized in that: The mass ratio of dopamine hydrochloride to Zn-KNN composite particles in step (2) is 1:

10.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the PDA-modified Zn-KNN to PLGA in step (2) is 1:

2.

8. The PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the preparation method according to any one of claims 1 to 7 in preparing antibacterial drugs for preventing and treating bone infection.

10. An antibacterial drug for preventing and treating bone infection, characterized in that: The invention comprises a PLGA / Zn-KNN piezoelectric antibacterial scaffold material prepared by the preparation method according to any one of claims 1 to 7.

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