Piezoelectric MOF (Metal Organic Framework) sound-sensitive agent with ultrasonic response ROS / CO synergistic antibacterial function as well as preparation method and application of piezoelectric MOF sound-sensitive agent

Abstract

The invention discloses a preparation method of a piezoelectric MOF (Metal Organic Framework) sound-sensitive agent with an ultrasonic response ROS / CO synergistic antibacterial function. The preparation method comprises the following steps: synthesizing a catechol modified ruthenium carbonyl carbon monoxide donor through a coupling reaction of 2, 4-dihydroxyl-1-phenylalanine and carbonyl ruthenium chloride; the preparation method comprises the following steps: mixing a carnosine aqueous solution and a metal ion aqueous solution in a solvent, and self-assembling the carnosine aqueous solution and the metal ion aqueous solution by a hydrothermal method to obtain a piezoelectric metal organic framework (PMOF); and modifying the carbon monoxide donor on the surface of the PMOF through a complexation reaction between metal ions on the surface of the PMOF and phenolic hydroxyl groups of the carbon monoxide donor to obtain the piezoelectric MOF sound-sensitive agent with ultrasonic response ROS / CO synergistic antibiosis. The invention also discloses the piezoelectric MOF sonosensitizer with ultrasonic response ROS / CO synergistic antibacterial effect obtained by the preparation method and application of the piezoelectric MOF sonosensitizer in preparation of sonosensitizer hydrophobic drugs and / or antibacterial agents. The sonosensitizer provided by the invention has excellent biocompatibility, can controllably start release of active oxygen and carbon monoxide through ultrasound to achieve synergistic antibiosis, enhances the antibacterial ability, and has excellent biological membrane penetrating ability.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and nanomaterials, and particularly relates to a piezoelectric MOF sonosensitizer capable of ultrasonically responding to ROS / CO for synergistic antibacterial effects, and a preparation method and application thereof. Background Art

[0002] Due to the increase in antibiotic-resistant bacteria and the difficulty in removing bacterial biofilms, traditional antibacterial therapy faces major challenges (S. Sharma, J. Mohler, SD Mahajan, et al. Microorganisms 11, 1614 (2023).). To address these issues, there are now a variety of methods to combat drug-resistant bacterial infections, including sonodynamic therapy (SDT). Sonodynamic therapy uses ultrasound as an excitation source to trigger sonosensitizers to convert acoustic energy into chemical energy of reactive oxygen species (ROS), with high therapeutic accuracy and deep tissue penetration. Traditional sonosensitizers in sonodynamic therapy include porphyrin-based single-atom catalysts and precious metal-doped titanium dioxide particles. However, the efficiency of traditional sonosensitizers in generating reactive oxygen is limited by the electron-hole (e-hole) reaction. - -h + ) limits rapid recombination (R. Zhao, H. Zhu, L. Feng, Y. Zhu, B. Liu, C. Yu, S. Gai, P. Yang, Small 19, e2301349 (2023).). Metal-organic frameworks (MOFs) exhibit excellent ROS generation capabilities due to their high specific surface area and adjustable porous structure. However, the biological toxicity of most sonosensitizers affects their application prospects as clinical sonosensitizers.

[0003] For example, a Chinese patent with publication number CN118717975A discloses a sonodynamic nanoparticle, its preparation method and application. The core of the sonodynamic nanoparticle is β-NaGdF4: 18% Yb, 2% Er@NaYF4@NaNdF4 nanoparticle, and the shell is mesoporous silica and CeO2 adsorbed in the shell. It can also produce a photothermal effect under 808nm laser irradiation to enhance the catalase activity of cerium oxide and increase the production of reactive oxygen. However, the components of the sonodynamic particles are toxic to a certain extent and cannot be completely degraded in the body. And due to the limitations of the laser generating equipment, it is impossible to achieve a non-invasive and passive treatment approach like medical ultrasound equipment.

[0004] Carbon monoxide (CO) gas therapy is also becoming a promising antibacterial method. CO is a gaseous transmitter with antibacterial effects. It can inhibit the oxidative phosphorylation process of the bacterial respiratory chain by binding to heme proteins in bacterial cells, inhibit the normal energy metabolism of bacteria, and induce bacterial death (D. Wu, AR Mehdipour, F. Finke, et al. Nat Chem Biol 19, 992–1003 (2023).). In addition, CO can also affect the intracellular ion balance by binding to metal elements, trigger free radical reactions, and damage macromolecules such as DNA. Based on these advances, antibacterial agents involving CO have attracted the attention of many researchers.

[0005] For example, a Chinese patent with publication number CN116510071A discloses a method for preparing a CO antibacterial dressing with a pH indicator. The invention indicates the wound infection through the color change of the phenol red indicator loaded by the composite aerogel, controls the temperature of the near-infrared (NIR) light source on / off, adjusts the release of CO, and realizes the on-demand activation of the CO antibacterial effect, thereby achieving an efficient and controllable wound treatment effect. However, the invention did not conduct biofilm antibacterial tests, and the antibacterial effect of relying solely on CO is limited, especially it is difficult to remove the biofilm formed by drug-resistant bacteria.

[0006] L-carnosine (β-alanyl-L-histidine) is an endogenous antioxidant that is present in large quantities in the heart, skeletal muscle fibers and the brain. L-carnosine can also chelate divalent zinc cations and is widely used in zinc supplementation and treatment of gastric ulcers (K. Sakae, H. Yanagisawa, Biol. Trace Elem. Res. 158 (3), 280-288 (2014).). Therefore, it is of great research significance to design an antibacterial sonosensitizer that has a simple and efficient preparation method, good biocompatibility, high bactericidal efficiency and high biomembrane penetration by using chemical components naturally present in the human body. Summary of the invention

[0007] The purpose of the present invention is to provide a piezoelectric MOF sonosensitizer with ultrasonic response to ROS / CO synergistic antibacterial properties and a preparation method and application thereof. The sonosensitizer provided by the present invention has excellent biocompatibility, can controllably initiate the release of reactive oxygen and carbon monoxide through ultrasound to achieve synergistic antibacterial properties, enhance antibacterial ability, and has excellent biomembrane penetration.

[0008] The present invention provides the following technical solutions:

[0009] A method for preparing a piezoelectric MOF sonosensitizer with ultrasonic response to ROS / CO synergistic antibacterial properties, the preparation method comprising the following steps:

[0010] (1) The catechol-modified ruthenium carbonyl carbon monoxide donor was synthesized by coupling reaction of 2,4-dihydroxy-l-phenylalanine (L-DOPA) with carbonyl ruthenium chloride;

[0011] (2) mixing an aqueous solution of carnosine and an aqueous solution of metal ions in a solvent, and using a hydrothermal method to self-assemble the two to obtain a piezoelectric metal organic framework (PMOF);

[0012] (3) Through the complexation reaction between the metal ions on the PMOF surface and the phenolic hydroxyl groups of the carbon monoxide donor, the carbon monoxide donor was modified on its surface to obtain a piezoelectric MOF sonosensitizer with ultrasonic responsive ROS / CO synergistic antibacterial effect.

[0013] The preparation method provided by the present invention comprises three steps: firstly, a catechol-modified ruthenium carbonyl carbon monoxide donor is obtained by coupling reaction of 2,4-dihydroxy-L-phenylalanine L-DOPA and carbonyl ruthenium chloride; then, a piezoelectric metal organic framework PMOF is synthesized by self-assembly of carnosine and metal ions (such as zinc ions) by a hydrothermal method; finally, the catechol-modified ruthenium carbonyl carbon monoxide donor is complexed with metal ions (such as zinc ions) on the surface of PMOF to modify the carbon monoxide donor, thereby giving the sonosensitizer the ability to release carbon monoxide under ultrasound triggering.

[0014] In the present invention, L-carnosine, which exists naturally in large quantities in the heart, skeletal muscle fibers and brain, is used as the skeleton of the metal organic framework, giving the piezoelectric MOF with ultrasound-responsive ROS / CO synergistic antibacterial excellent biocompatibility and degradation performance, and has potential ROS production ability, and can be used as a sonosensitizer; then by introducing a carbon monoxide donor, the piezoelectric metal organic framework (PMOF) is added with the function of releasing carbon monoxide and destroying the bacterial respiratory chain on the basis of its active oxygen sterilization function, thereby enhancing its antibacterial performance and reducing the dosage of the sonosensitizer.

[0015] In step (1), carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine L-DOPA are coupled in the presence of an alkaline catalyst, and the molar ratio of carbonyl ruthenium chloride, L-DOPA and the alkaline catalyst is 1-5:1-10:1-10.

[0016] Preferably, the alkaline catalyst is sodium methoxide.

[0017] Furthermore, the step (1) is specifically as follows:

[0018] Carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine (L-DOPA) are dissolved in anhydrous methanol at a molar ratio of 1-5:1-10. After three freeze-pump-thaw cycles, the solution is deoxygenated, and then a basic catalyst sodium methoxide solution (1-10 times the molar equivalent of carbonyl ruthenium chloride) is added; the reaction solution is stirred in the dark at room temperature for 18 hours, the methanol is evaporated under reduced pressure, and it is dissolved again with tetrahydrofuran; the filtrate is collected, the tetrahydrofuran is evaporated under reduced pressure, and it is further precipitated in hexane to obtain a carbon monoxide donor.

[0019] In step (1), the carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine (L-DOPA) react as follows:

[0020]

[0021] In step (2), L-carnosine is used as carnosine, zinc ion is used as metal ion, and the molar ratio of L-carnosine to zinc oxide is 1-10:1-10; in step (2), the solvent used is selected from acetonitrile, N,N-dimethylformamide, acetone or ethanol.

[0022] In step (2), the volume ratio of L-carnosine aqueous solution, zinc oxide aqueous solution, solvent and water is 1-10:1-10:2-20:1-10, the concentration of L-carnosine aqueous solution is 80-160mM, and the concentration of zinc oxide aqueous solution is 80-160mM solvent.

[0023] In step (2), the reaction temperature of the hydrothermal method is 80-120° C., and the reaction time is 4-12 hours.

[0024] In step (3), in the complexation reaction between PMOF and carbon monoxide donor, the mass ratio of PMOF to carbon monoxide donor is 1-10:1-10.

[0025] The present invention also provides a piezoelectric MOF sonosensitizer with ultrasonic response to ROS / CO synergistic antibacterial properties obtained by the above preparation method.

[0026] Furthermore, the average particle size of the sonosensitizer is 1255 nm.

[0027] Furthermore, the medical ultrasonic parameters used for the ultrasonic response trigger are: power 0.5-3W·cm -2 , frequency 0.5-3MHz, duty cycle 10-100%.

[0028] The present invention also provides an application of the above-mentioned ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer in the preparation of sonosensitizer hydrophobic drugs and / or antibacterial agents (antibacterial drugs for treating drug-resistant bacteria).

[0029] When used, the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF) obtained in step (3) is dispersed in water, or mixed with other antibacterial agents, and heated with a power of 0.5-3 W·cm -2 Medical ultrasound with a frequency of 0.5-3MHz and a duty cycle of 10-100% triggers its ultrasonic response, exerts its antibacterial effect, and implements ultrasound-triggered active oxygen / carbon monoxide synergistic antibacterial sonodynamic therapy.

[0030] Compared with the prior art, the technical effects of the present invention are:

[0031] (1) For the first time, the reactive oxygen species / carbon monoxide (ROS / CO) synergistic antibacterial sonodynamic therapy was proposed. The release of reactive oxygen species and carbon monoxide can be controlled by ultrasound. The combination of the two achieves a strong antibacterial effect. It is also beneficial to reduce the dose of sonosensitizers and lower the ultrasound intensity to avoid tissue damage.

[0032] (2) The main components of CO-PMOF in the present invention, L-carnosine and zinc ions, are both present in large quantities in the human body. The hemolysis test, cytotoxicity test and in vivo degradation test of the modified PMOF all show that it has excellent biocompatibility and will not cause toxicity to the organism after degradation.

[0033] (3) The preparation method provided by the present invention is simple and fast, avoids complicated manufacturing process, and can be widely promoted.

[0034] (4) The CO-PMOF in the present invention can effectively inhibit the colonies of drug-resistant bacteria (91.5%) and remove the biofilm of drug-resistant bacteria (98.5%) through the synergistic antibacterial effect of ROS / CO.

[0035] (5) The CO-PMOF in the present invention can effectively destroy the biofilm formed by drug-resistant bacteria through the synergistic antibacterial effect of ROS / CO, thus overcoming the problem that the biofilm of drug-resistant bacteria is difficult to remove.

[0036] (6) The present invention is based on a medical ultrasonic generator commonly used in clinical practice, which can be used efficiently and controllably, providing a more convenient and practical solution for clinical implementation, and will become a new treatment approach for wounds infected with drug-resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The mechanism of ultrasound-responsive ROS / CO synergistic antibacterial sonodynamic therapy of piezoelectric MOF.

[0038] Figure 2 These are the nuclear magnetic resonance spectra, infrared spectra and ultraviolet spectra of the carbon monoxide donors in Example 1, Example 2 and Example 3.

[0039] Figure 3SEM and TEM images of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) and piezoelectric metal-organic framework (PMOF) in Example 1.

[0040] Figure 4 The results of using dynamic light scattering to characterize the particle size of piezoelectric MOF (CO-PMOF) and piezoelectric metal-organic framework (PMOF) with ultrasonic response to ROS / CO synergistic antibacterial properties in Example 1.

[0041] Figure 5 This is the infrared spectrum of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1, Example 2, and Example 3.

[0042] Figure 6 This is the X-ray photoelectron spectrum of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1.

[0043] Figure 7 The piezoelectric properties of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1 were measured using a piezoelectric response force microscope (PFM).

[0044] Figure 8 These are the electron paramagnetic resonance spectra of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) with DMPO and TEMP adducts under ultrasound in Example 1, Example 2, and Example 3.

[0045] Fig. 9 The rate at which the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1 releases carbon monoxide under ultrasound.

[0046] Fig.10 These are the antibacterial test results of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1.

[0047] Fig.11 This is a confocal laser scanning microscopy image of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1, which shows the effect of destroying the biofilm of drug-resistant bacteria. DETAILED DESCRIPTION

[0048] The technical solution of the present invention is further described below in conjunction with specific embodiments, but these embodiments are not intended to limit the present invention.

[0049] The ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer provided by the present invention and the preparation method thereof include the following steps:

[0050] (1) A catechol-modified ruthenium carbonyl carbon monoxide donor is obtained by reacting 2,4-dihydroxy-L-phenylalanine (L-DOPA) with carbonyl ruthenium chloride: carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine (L-DOPA) are dissolved in anhydrous methanol at a molar ratio of 1-10:1-10. After three freeze-pump-thaw cycles, the solution is deoxygenated, and then sodium methoxide solution (1-10 times the molar equivalent of carbonyl ruthenium chloride) is added; the reaction solution is stirred in the dark at room temperature for 18 hours, the methanol is evaporated under reduced pressure, and it is dissolved again with tetrahydrofuran; the filtrate is collected, the tetrahydrofuran is evaporated under reduced pressure, and it is further precipitated in hexane to obtain a carbon monoxide donor.

[0051] (2) L-carnosine aqueous solution and metal ion aqueous solution are mixed in a solvent, and a piezoelectric metal organic framework (PMOF) is synthesized by a hydrothermal method: a certain amount of L-carnosine powder and zinc oxide are dissolved in water respectively, and the L-carnosine solution, zinc oxide solution, solvent and water are mixed in a ratio of 1-10:1-10:2-20:1-10 and placed in a 20 mL scintillation vial. Then the mixture is reacted at 100°C for 4-12 hours; after the reaction, the formed crystals are washed with ethanol and deionized water in large quantities to remove the residual reactants, and then dried at 60°C for 12 hours to obtain a carnosine-zinc piezoelectric metal organic framework.

[0052] (3) Through the complexation between the metal ions contained in PMOF and the phenolic hydroxyl groups, the carbon monoxide donor was modified on the surface of PMOF to obtain a piezoelectric MOF with ultrasonic response to ROS / CO synergistic antibacterial: the ethanol dispersion of carnosine-zinc piezoelectric metal organic framework particles was dropped into deionized water with a stirring speed of 1000 r / min; then, the carbon monoxide donor was dissolved in deionized water, the pH was adjusted to 8.5, and then the above dispersion was added and stirred for 24 hours for self-polymerization reaction. The product was collected by centrifugation at a speed of 10000 r / min for 10 minutes, and then washed with deionized water to obtain a piezoelectric MOF sonosensitizer (CO-PMOF) with ultrasonic response to ROS / CO synergistic antibacterial.

[0053] (4) The ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF) obtained in step (3) was dispersed in water to a concentration of 10 μM and heated at a power of 1.5 W cm -2 , medical ultrasound with a frequency of 1 MHz and a duty cycle of 50% was used to trigger its ultrasonic response to ROS / CO, and its antibacterial properties were tested.

[0054] Example 1

[0055] Taking zinc ion as metal ion, acetonitrile and water as solvent, L-carnosine: zinc oxide molar ratio of 1:1, 80mM L-carnosine aqueous solution: 160mM zinc oxide aqueous solution: acetonitrile: water volume ratio of 2:1:4:1, and carbon monoxide donor 2,4-dihydroxy-L-phenylalanine (L-DOPA) and carbonyl ruthenium chloride as an example, the preparation of ultrasonic responsive ROS / CO synergistic antibacterial piezoelectric MOF is described.

[0056] (1) Carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine (L-DOPA) are dissolved in anhydrous methanol at a molar ratio of 1:2. After three freeze-pump-thaw cycles, the solution is deoxygenated and then sodium methoxide solution (4 times the molar equivalent of carbonyl ruthenium chloride) is added. The reaction solution is stirred in the dark at room temperature for 18 hours, the methanol is evaporated under reduced pressure, and it is dissolved again in tetrahydrofuran. The filtrate is collected, the tetrahydrofuran is evaporated under reduced pressure, and further precipitated in hexane to obtain a carbon monoxide donor.

[0057] (2) A certain amount of carnosine powder was dissolved in water to a concentration of 80 mM. 80 mM carnosine stock solution (2 mL) was placed in a 20 mL volumetric scintillation vial with 160 mM zinc oxide (1 mL), 4 mL acetonitrile and 2 mL deionized water. The mixture was then ultrasonically mixed and then reacted at 100°C for 4 hours. After the reaction, the formed crystals were washed with ethanol and deionized water to remove the residual reactants, and then dried at 60°C for 12 hours to obtain a carnosine-zinc piezoelectric metal organic framework.

[0058] (3) 200 μL of 25 mg / mL ethanol dispersion of carnosine-zinc piezoelectric metal organic framework particles was dropped into 10 mL of deionized water, and the stirring speed was 1000 r / min. Subsequently, 6 mg of carbon monoxide donor was dissolved in 5 mL of deionized water, the pH was adjusted to 8.5, and then the above dispersion was added and stirred for 24 hours for self-polymerization reaction. The product was collected by centrifugation at 10000 r / min for 10 min, and then washed with deionized water to obtain an ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF).

[0059] (4) The ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF) obtained in step (3) was dispersed in water to a concentration of 10 μM and heated at a power of 1.5 W cm -2 , frequency of 1 MHz, and duty cycle of 50% medical ultrasonic wave were used to trigger its ultrasonic response and test its antibacterial property.

[0060] Example 2

[0061] Taking zinc ion as metal ion, acetone as solvent, L-carnosine: zinc oxide molar ratio of 1:1, 80mM L-carnosine aqueous solution: 80mM zinc oxide aqueous solution: acetone volume ratio of 2:2:4, and carbon monoxide donor 2,4-dihydroxy-L-phenylalanine (L-DOPA) and carbonyl ruthenium chloride as an example, the preparation of ultrasonic responsive ROS / CO synergistic antibacterial piezoelectric MOF is described.

[0062] (1) Carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine (L-DOPA) are dissolved in anhydrous methanol at a molar ratio of 1:2. After three freeze-pump-thaw cycles, the solution is deoxygenated and then sodium methoxide solution (4 times the molar equivalent of carbonyl ruthenium chloride) is added. The reaction solution is stirred in the dark at room temperature for 18 hours, the methanol is evaporated under reduced pressure, and it is dissolved again in tetrahydrofuran. The filtrate is collected, the tetrahydrofuran is evaporated under reduced pressure, and further precipitated in hexane to obtain a carbon monoxide donor.

[0063] (2) A certain amount of carnosine powder was dissolved in water to a concentration of 80 mM. 80 mM carnosine stock solution (2 mL) was placed in a 20 mL scintillation vial with 80 mM zinc oxide (2 mL) and 4 mL acetone. The mixture was then ultrasonically mixed and then reacted at 100°C for 4 hours. After the reaction, the formed crystals were washed with ethanol and deionized water to remove the residual reactants, and then dried at 60°C for 12 hours to obtain a carnosine-zinc piezoelectric metal organic framework.

[0064] (3) 200 μL of 25 mg / mL ethanol dispersion of carnosine-zinc piezoelectric metal organic framework particles was dropped into 10 mL of deionized water, and the stirring speed was 1000 r / min. Subsequently, 6 mg of carbon monoxide donor was dissolved in 5 mL of deionized water, the pH was adjusted to 8.5, and then the above dispersion was added and stirred for 24 hours for self-polymerization reaction. The product was collected by centrifugation at 10000 r / min for 10 min, and then washed with deionized water to obtain an ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF).

[0065] (4) The ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF) obtained in step (3) was dispersed in water to a concentration of 10 μM and heated at a power of 1.5 W cm -2 , 1MHz frequency, and 50% duty cycle medical ultrasound was used to trigger its ultrasonic response to test its antibacterial properties.

[0066] Example 3

[0067] Zinc ions were used as metal ions, ethanol and water were used as solvents, the molar ratio of L-carnosine: zinc oxide was 1:1, the volume ratio of 80mM L-carnosine aqueous solution: 160mM zinc oxide aqueous solution: ethanol: water was 2:1:6:1, and the carbon monoxide donor was 2,4-dihydroxy-L-phenylalanine (L-DOPA) and carbonyl ruthenium chloride. The preparation of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF was described.

[0068] (1) Carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine (L-DOPA) are dissolved in anhydrous methanol at a molar ratio of 1:2. After three freeze-pump-thaw cycles, the solution is deoxygenated and then sodium methoxide solution (4 times the molar equivalent of carbonyl ruthenium chloride) is added. The reaction solution is stirred in the dark at room temperature for 18 hours, the methanol is evaporated under reduced pressure, and it is dissolved again in tetrahydrofuran. The filtrate is collected, the tetrahydrofuran is evaporated under reduced pressure, and further precipitated in hexane to obtain a carbon monoxide donor.

[0069] (2) A certain amount of carnosine powder was dissolved in water to a concentration of 80 mM. 80 mM carnosine stock solution (2 mL) was placed in a 20 mL scintillation vial with 160 mM zinc oxide (1 mL), 6 mL ethanol and 2 mL deionized water. The mixture was then ultrasonically mixed and then reacted at 100°C for 6 hours. After the reaction, the formed crystals were washed with ethanol and deionized water to remove the residual reactants, and then dried at 60°C for 12 hours to obtain a carnosine-zinc piezoelectric metal organic framework.

[0070] (3) 200 μL of 25 mg / mL ethanol dispersion of carnosine-zinc piezoelectric metal organic framework particles was dropped into 10 mL of deionized water, and the stirring speed was 1000 r / min. Subsequently, 6 mg of carbon monoxide donor was dissolved in 5 mL of deionized water, the pH was adjusted to 8.5, and then the above dispersion was added and stirred for 24 hours for self-polymerization reaction. The product was collected by centrifugation at 10000 r / min for 10 min, and then washed with deionized water to obtain an ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF).

[0071] (4) The ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer (CO-PMOF) obtained in step (3) was dispersed in water to a concentration of 10 μM and heated at a power of 1.5 W cm -2 , 1MHz frequency, and 50% duty cycle medical ultrasound was used to trigger its ultrasonic response to test its antibacterial properties.

[0072] like Figure 1 Shown is the mechanism of ultrasound-responsive ROS / CO synergistic antibacterial sonodynamic therapy of piezoelectric MOF reactive oxygen species / carbon monoxide synergistic antibacterial.

[0073] like Figure 2 As shown, the nuclear magnetic resonance spectrum, infrared spectrum and ultraviolet spectrum of the carbon monoxide donor in Example 1, Example 2 and Example 3. 1 The H NMR spectrum shows that the catechol proton characteristic peak of 2,4-dihydroxy-L-phenylalanine (L-DOPA) is present in the 5-10 ppm spectral region of DO-DOPA. The infrared spectrum of carbon monoxide donor has a peak of 2300-1800 cm -1 The characteristic peaks of carbonyl groups are shown in the spectral region. The characteristic peaks of the carbon monoxide donor UV spectrum at 260nm and 285nm show the chloride ions in carbonyl ruthenium chloride and the benzene rings in 2,4-dihydroxy-L-phenylalanine (L-DOPA).

[0074] like Figure 3 As shown, the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) and piezoelectric metal organic framework (PMOF) in Example 1 were observed under a scanning electron microscope and a transmission electron microscope, showing a modified layer of carbon monoxide donor on the surface of the particles, with an average thickness of 287±90 nm.

[0075] like Figure 4 As shown, the particle size of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1 was characterized by dynamic light scattering, and the average diameter of CO-PMOF was 1255 nm.

[0076] like Figure 5 As shown, the infrared spectra of the piezoelectric MOF (CO-PMOF) with ultrasonic response to ROS / CO synergistic antibacterial in Example 1, Example 2, and Example 3 are 1800-2300 cm -1 The carbonyl characteristic peaks indicated that the carbon monoxide donor was successfully modified onto the PMOF.

[0077] like Figure 6 As shown, in the X-ray photoelectron spectrum of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1, in addition to the four strong peaks of O1s, N 1s, C 1s and Zn 2p, new peaks of Ru 3d (284.93 eV and 281.43 eV) and Ru 3p (476.03 eV and 462.78 eV) were found, further confirming the chemical structure of the obtained CO-PMOF.

[0078] like Figure 7As shown, the piezoelectric force microscope (PFM) was used to measure the piezoelectricity of the ultrasonic response ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in the embodiment. Under different excitation voltages, the PFM vertical response showed a resonance peak of 294kHz, indicating the presence of electric dipoles in the CO-PMOF crystal. Under the reversal of the DC bias field, the phase angle changes by 180°, forming a complete butterfly circuit, which further verifies that CO-PMOF has ferroelectricity. These evidences verify the piezoelectric properties of CO-PMOF.

[0079] like Figure 8 As shown, the ultrasonic response ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1, Example 2, and Example 3 was triggered by medical ultrasound for 10 minutes (power 1.5 W·cm -2 , frequency 1 MHz, duty cycle 50%), a representative intensity ratio of 1:1:1 (singlet oxygen 1 The electron paramagnetic spectroscopy signals of CO-PMOF (O2) and 1:2:2:1 (hydroxyl radical OH) indicate that CO-PMOF simultaneously produces 1 There are two active oxygen species: O2 and ·OH.

[0080] like Fig. 9 As shown, the carbon monoxide release capacity of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1 was detected by a carbon monoxide gas detector in a closed container under ultrasound triggering. -2 , frequency 1MHz, duty cycle 50%), the carbon monoxide release increased to 10.05μmol·mg -1 The second round of ultrasound investigated the reproducibility of ultrasound-triggered carbon monoxide release behavior, and the final total carbon monoxide concentration reached 17.41 μmol·mg -1 .

[0081] like Fig.10As shown, the antibacterial ability of the ultrasonic response ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) in Example 1 was tested by colony coating experiments of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E.coli) and live bacteria / dead bacteria staining experiments. The bacterial activity of each group can be judged by the number of colonies after different treatments (PBS buffer, ultrasound-triggered PBS buffer, ultrasound-triggered piezoelectric metal organic framework, ultrasound-triggered ultrasound-responsive ROS / CO synergistic antibacterial piezoelectric MOF) coated on LB medium at 37°C for 24 hours, and the live bacteria / dead bacteria staining results under confocal laser scanning microscopy (green fluorescence for live bacteria, red fluorescence for dead bacteria). The bacterial activity of the group treated with the ultrasonic response ROS / CO synergistic antibacterial piezoelectric MOF (CO-PMOF) was the lowest. In the coating experiment, the MRSA activity of this group was only 8.5%.

[0082] like Fig.11 As shown, the methicillin-resistant Staphylococcus aureus (MRSA) biofilm was cultured in nutrient broth at 37°C for 48 hours. After 60 minutes of different treatments (PBS buffer, ultrasound-triggered PBS buffer, ultrasound-triggered ultrasound-responsive ROS / CO synergistic antibacterial piezoelectric MOF), the ultrasound time was 10 minutes (power 1.5 W·cm -2 , frequency 1MHz, duty cycle 50%). The biofilm was stained with SYTO9 dye and PI dye for live bacteria / dead bacteria, and observed with confocal laser scanning microscopy (CLSM). Green is live bacteria, red is dead bacteria. The biofilm thickness was measured by crystal violet staining. The biofilm thickness of the ultrasound group was 91.5% of that of the PBS group. The thickness of the remaining MRSA biofilm in the CO-PMOF group was only 11.5% of that in the PBS group.

[0083] Biocompatibility verification: Whole blood from healthy SD rats was collected using EDTA-K2 anticoagulant tubes. The diluted blood was incubated with a series of concentrations (1-1000 μg·mL-1) of CO-PMOF NPs for 24 hours. The absorbance value of the supernatant at 540 nm was measured using an enzyme marker to quantitatively determine the hemolysis rate. Normal saline and deionized water were used as negative and positive control groups, respectively. Subsequently, L929 cells were selected as representative cells to verify the cell compatibility of CO-PMOF. Finally, the CO-PMOF dispersion was used to treat the wound surface of mice, and the contents of zinc (Zn) and ruthenium (Ru) in the main organs of mice were determined by inductively coupled plasma (ICP) proton emission spectrometry after 7 and 18 days, respectively. At 1000 μg·mL -1The hemolysis rate under high concentration of CO-PMOF is only 2.3%, and the cell viability in the cytotoxicity test reaches 95%. In the in vivo experiment of the sonosensitizer of the present invention, the ICP results show that the zinc and ruthenium elements contained in the sonosensitizer will not accumulate in major organs and cause biological toxicity.

[0084] The present invention is described above by way of example. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by those skilled in the art without inventive effort falls within the protection scope of the present invention.

Claims

1. A method for preparing a piezoelectric MOF sonosensitizer with ultrasonic response to ROS / CO synergistic antibacterial properties, characterized in that: The preparation method comprises the following steps: (1) The catechol-modified ruthenium carbonyl carbon monoxide donor was synthesized by the coupling reaction of 2,4-dihydroxy-l-phenylalanine L-DOPA and carbonyl ruthenium chloride; (2) mixing an aqueous solution of carnosine and an aqueous solution of metal ions in a solvent, and using a hydrothermal method to self-assemble the two to obtain a piezoelectric metal organic framework (PMOF); (3) Through the complexation reaction between the metal ions on the PMOF surface and the phenolic hydroxyl groups of the carbon monoxide donor, the carbon monoxide donor was modified on its surface to obtain a piezoelectric MOF sonosensitizer with ultrasonic responsive ROS / CO synergistic antibacterial effect.

2. The method for preparing the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 1, characterized in that: In step (1), carbonyl ruthenium chloride and 2,4-dihydroxy-L-phenylalanine L-DOPA are coupled in the presence of an alkaline catalyst, and the molar ratio of carbonyl ruthenium chloride, L-DOPA and the alkaline catalyst is 1-5:1-10:1-10.

3. The method for preparing the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 1, characterized in that: In step (2), L-carnosine and zinc ions are used as metal ions, the molar ratio of L-carnosine to zinc oxide is 1-10:1-10, and the solvent used is selected from acetonitrile, N,N-dimethylformamide, acetone or ethanol.

4. The method for preparing the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 3, characterized in that: In step (2), the volume ratio of the L-carnosine aqueous solution, the zinc oxide aqueous solution, the solvent and water is 1-10:1-10:2-20:1-10, the concentration of the L-carnosine aqueous solution is 80-160 mM, and the concentration of the zinc oxide aqueous solution is 80-160 mM.

5. The method for preparing the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 1, characterized in that: In step (2), the reaction temperature of the hydrothermal method is 80-120° C., and the reaction time is 4-12 hours.

6. The method for preparing the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 1, characterized in that: In step (3), in the complexation reaction between PMOF and carbon monoxide donor, the mass ratio of PMOF to carbon monoxide donor is 1-10:1-10.

7. A piezoelectric MOF sonosensitizer with ultrasonic response to ROS / CO synergistic antibacterial properties obtained by the preparation method according to any one of claims 1 to 6.

8. The ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 7, characterized in that: The medical ultrasonic parameters used for the ultrasonic response trigger are: power 0.5-3W·cm -2 , frequency 0.5-3MHz, duty cycle 10-100%.

9. Use of the ultrasonically responsive ROS / CO synergistic antibacterial piezoelectric MOF sonosensitizer according to claim 7 in the preparation of sonosensitizer hydrophobic drugs and / or antibacterial agents.

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