Mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibiosis as well as preparation method and application of mesoporous polydopamine drug-loaded microspheres

Abstract

The invention provides mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibiosis as well as a preparation method and application thereof, and belongs to the field of nano biological medicines. According to the invention, mesoporous polydopamine is used as a carrier, and hydrophilic modification and functional modification are carried out on the polydopamine, so that the biocompatibility and the loading performance of the mesoporous polydopamine are improved; a sound-sensitive agent and transition metal ions are loaded into mesoporous polydopamine, the concentration of GSH in a bacterial microenvironment is reduced through the reaction between the polydopamine and the GSH, and meanwhile oxygen is provided for the SDT process through the characteristic that the transition metal ions catalyze H2O2 to generate oxygen in the CDT process, so that the concentration of active oxygen in the microenvironment is increased, and the antibacterial effect of the SDT is improved; synergistic antibiosis of sonodynamic and chemical dynamic is realized; and the sound-sensitive agent and the transition metal ions are better released by utilizing cracking of the mesoporous polydopamine drug-loaded microspheres in the GSH consumption process, so that the antibacterial effect is further improved.

Description

Technical Field

[0001] The present invention relates to the field of nano-biomedicine, and in particular to mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties, and a preparation method and application thereof. Background Art

[0002] Treatments such as organ and prosthetic transplantation and tissue engineering have brought hope for the cure of many patients. However, there are still many clinical infections in clinical applications, among which bacterial infections are the most common. Bacterial infections can occur repeatedly alone or mixed with other pathogens, and the threat to human health cannot be underestimated. Therefore, the development of effective antibacterial materials or antibacterial methods to avoid bacterial infections in clinical practice is of great significance to modern medicine.

[0003] Antibacterial materials include organic antibacterial materials, sterile antibacterial materials, and organic-inorganic composite antibacterial materials. Organic antibacterial materials are characterized by rich variety, wide application range, obvious antibacterial effect, and relatively mature use technology. For example, antibiotics and quaternary ammonium ions belong to organic antibacterial materials, but long-term and high-dose use can easily lead to drug resistance, which limits their antibacterial effect. Inorganic antibacterial materials mainly use the direct destructive effect of transition metal ions, the penetration and photocatalytic properties of metal oxides, and the catalytic oxidation of nanoparticles to fight bacteria. They have the characteristics of stable antibacterial effect and good antibacterial performance. Whether it is the large-scale use of antibiotics, quaternary ammonium ions or metal ions, they are prone to drug resistance and have potential toxicity, which limits their application. Therefore, it is still necessary to develop more novel and effective antibacterial strategies to meet clinical needs. Organic-inorganic composite antibacterial materials are both biocompatible and have the characteristics of high porosity and large specific surface area. They can integrate multiple antibacterial methods and are currently an important research direction, such as Ag nanoparticles / chitosan composite antibacterial materials, zinc ion / tetradecyltributyl quaternary phosphonium salt composite antibacterial materials, etc. How to utilize the synergistic effect between different materials to achieve the technical effect of 1+1>2 is one of the key difficulties in the research and development process.

[0004] Sonodynamic therapy (SDT) is a non-invasive antibacterial therapy developed in recent years. It has shown great potential in the non-invasive treatment of deep bacterial infections. Its antibacterial principle is: ultrasound triggers sonosensitizers to convert oxygen into reactive oxygen species (ROS). High concentrations of reactive oxygen species can cause oxidative damage to bacteria and achieve antibacterial effects. However, there is always a high concentration of glutathione (GSH) in the microenvironment under bacterial infection. As an important member of the human immune system, GSH has antioxidant and integrated detoxification effects. It can react with ROS or H2O2 to generate oxidized glutathione (GSSG). The consumption of ROS and H2O2 aggravates the hypoxia of the bacterial microenvironment, which greatly affects the efficacy of SDT. Therefore, how to reduce the concentration of GSH during SDT and effectively improve the antibacterial effect of SDT is also an important technical problem that needs to be solved at present.

[0005] In view of this, it is necessary to design a preparation method and application of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties to solve the above problems. Summary of the invention

[0006] In view of the technical problems existing in the background technology, the present application provides a mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemodynamic synergistic antibacterial effect, as well as a preparation method and application thereof. Mesoporous polydopamine is used as a carrier, and the mesoporous polydopamine is hydrophilically modified and functionalized to improve the biocompatibility and loading performance of the mesoporous polydopamine. The sonodynamic and chemodynamic synergistic antibacterial effect is achieved by successively loading the sonosensitizer and transition metal ions into the mesoporous polydopamine, and the mesoporous polydopamine drug-loaded microspheres will undergo cleavage during the antibacterial process, so that the sonosensitizer and transition metal ions are fully released, thereby greatly improving the antibacterial effect.

[0007] In a first aspect, the present invention provides a method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties, comprising the following steps: S1, dissolving polyether F127 and dopamine hydrochloride in an ethanol-water solution, stirring vigorously until the solution is clear, then slowly dropping 1,3,5-trimethylbenzene under stirring, continuing stirring for 30-40 min, adding concentrated ammonia water dropwise under stirring, continuing stirring for 1-1.5 h, washing with ethanol and deionized water three times respectively, to obtain a mesoporous polydopamine nanoparticle dispersion; S2, adjusting the pH value of the mesoporous polydopamine nanoparticle dispersion of step S1 to 11-12 with an aqueous sodium hydroxide solution, adding a thiol-polyethylene glycol-carboxyl aqueous solution dropwise under stirring, and stirring for 2-3 hours to obtain a hydrophilically modified mesoporous polydopamine nanoparticle dispersion; S3, under stirring, adding the aqueous solution of the sonosensitizer to the hydrophilically modified mesoporous polydopamine nanoparticle dispersion of step S2, stirring in the dark for 8-12 h, then adding the aqueous solution containing transition metal ions, stirring for 1.5-3 h, and then centrifuging to obtain mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties; wherein the total mass of the sonosensitizer and the transition metal ions accounts for 1-5% of the mass of the mesoporous polydopamine drug-loaded microspheres, and the mass ratio of the sonosensitizer to the transition metal ions is 1:(0.5-1).

[0008] In some embodiments, in step S3, the sonosensitizer is one of protoporphyrin (PPIX), chlorophyll (Chl), cyanine (PC) or rose bengal (RhB); the sonosensitizer solution is prepared by dissolving the sonosensitizer in dimethyl sulfoxide at a concentration of (5-15) mg / mL, and then diluting it 10 times with deionized water; the transition metal in the transition metal ion is one of copper, iron, manganese, silver, cobalt, cadmium, nickel or molybdenum, and the concentration of the transition metal ion is (0.08-0.12) mol / L.

[0009] In some embodiments, in step S1, the mass ratio of the dopamine hydrochloride to the polyether F127 is 1:(1.5-2.5); the volume ratio of ethanol to water in the ethanol-water solution is 1:1; the volume ratio of the ethanol-water solution to the 1,3,5-trimethylbenzene is (45-55):1; the concentration of the dopamine hydrochloride in the ethanol-water solution is (3-7) mg / mL.

[0010] In some embodiments, in step S1, after adding concentrated aqueous ammonia, the pH value of the reaction system is 8-10.

[0011] In some embodiments, in step S2, the concentration of the sodium hydroxide aqueous solution is (0.1-0.2) mol / L, the concentration of the thiol-polyethylene glycol-carboxyl aqueous solution is (10-15) mg / mL, and the mass ratio of dopamine hydrochloride to thiol-polyethylene glycol-carboxyl is 1:(0.1-0.2).

[0012] In some embodiments, in step S3, the centrifugal speed is 11000-18000 rpm.

[0013] In the second aspect, the present application provides a mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties, which are prepared by any of the aforementioned preparation methods, wherein the mesoporous polydopamine drug-loaded microspheres use mesoporous polydopamine as a carrier to load sonosensitizers and transition metal ions; the sonosensitizer is one of protoporphyrin, chlorophyll, cyanine or rose bengal; the transition metal in the transition metal ions is one of copper, iron, manganese, silver, cobalt, cadmium, nickel or molybdenum.

[0014] In some embodiments, the average particle size of the mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties is 120-180 nm.

[0015] In the third aspect, the present application also provides an application of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial prepared by the preparation method described in any of the aforementioned schemes, or an application of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial in any of the aforementioned schemes, wherein the mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial are used to treat or prevent non-invasive treatment of deep bacterial infections, wherein the bacteria include Escherichia coli and Staphylococcus aureus.

[0016] The beneficial effects of this application are: The present application provides a mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemical dynamic synergistic antibacterial and its preparation method and application. Among them, mesoporous polydopamine is obtained by hydrophilic modification and structural modification of polydopamine, so as to improve biocompatibility and loading performance; and sonodynamic and chemical dynamic synergistic antibacterial is achieved by loading the sonosensitizer and transition metal ions into the mesoporous polydopamine in sequence.

[0017] In the process of sonodynamic and chemodynamic synergistic antibacterial, firstly, the sonosensitizer is excited to a high energy state under the action of ultrasound, and transfers its energy to the surrounding oxygen molecules to generate highly oxidative singlet oxygen and other reactive oxygen species (ROS). ROS can destroy the cell membrane of bacteria, oxidize proteins and enzymes to inactivate them, damage DNA and RNA, and ultimately lead to bacterial death, i.e., sonodynamic therapy (SDT); secondly, there is often overexpression of hydrogen peroxide (H2O2) at the site of bacterial infection, and transition metal ions catalyze the generation of H2O2 through the Fenton / Fenton-like reaction. Highly active hydroxyl radicals (•OH) cause oxidative damage to bacteria and produce oxygen, which is chemodynamic therapy (CDT); thirdly, there is overexpressed hydrogen peroxide in the bacterial infection site, which allows the CDT reaction to continue. The oxygen produced during the CDT process can be provided to SDT to avoid the formation of an oxygen-deficient environment in the bacterial infection site, thereby enhancing the antibacterial effect of SDT; fourthly, the degradation of polydopamine by GSH is used to achieve the consumption of GSH in the bacterial infection site, thereby reducing the reaction of GSH with ROS or H2O2 and increasing the lethality of reactive oxygen. At the same time, the process of consuming GSH is also accompanied by the cleavage of mesoporous polydopamine drug-loaded microspheres, which is conducive to the better release of sonosensitizers and transition metal ions by mesoporous polydopamine, further enhancing the antibacterial effect.

[0018] In addition, the modified mesoporous polydopamine nanoparticles provided in the present application have good biocompatibility, and their mesoporous structure enables the material to have a larger specific surface area, which can load more antibacterial agents, thereby achieving the purpose of increasing the concentration of active oxygen and enhancing the antibacterial effect.

[0019] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The surface morphology of the mesoporous polydopamine drug-loaded microspheres prepared in Example 1-2; wherein a is the surface morphology of MPDA-PEG@PPIX / Cu, and b is the surface morphology of MPDA-PEG@PC / Fe; Figure 2 The particle size variation diagram of the products prepared in Example 1 and Comparative Examples 1-3; Figure 3 The figure shows the consumption of glutathione and the change in particle size of the mesoporous polydopamine drug-loaded microspheres prepared in Example 1-2 under acidic conditions of pH=5.5; wherein, c is the absorbance curve of the residual glutathione in the solution after glutathione is treated with MPDA-PEG@PPIX / Cu for a certain period of time, d is the change in particle size after glutathione is treated with MPDA-PEG@PPIX / Cu for 6 h, e is the absorbance curve of the residual glutathione in the solution after glutathione is treated with MPDA-PEG@PC / Fe for a certain period of time, and f is the change in particle size after glutathione is treated with MPDA-PEG@PC / Fe for 6 h; Figure 4 This is a graph showing the absorbance variation of o-phenylenediamine at 410 nm for testing free radical and singlet oxygen concentrations in the antibacterial performance test of the products prepared by Examples 1-2 and Comparative Examples 3-6; Figure 5 This is a graph showing the change in fluorescence intensity at 525 nm of a green fluorescent probe (SOSG) for testing singlet oxygen concentration in the antibacterial performance test of the products prepared by Examples 1-2 and Comparative Examples 3-6; Figure 6 This is the antibacterial effect diagram obtained in Experimental Examples 1-8. DETAILED DESCRIPTION

[0022] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0024] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0025] In the description of the embodiments of the present application, the term "and / or" is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship. In the description of the embodiments of the present application, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0026] There is always a high concentration of glutathione (GSH) in the microenvironment under bacterial infection. As an important member of the human immune system, GSH has antioxidant and integrated detoxification effects. It can react with ROS or H2O2 to generate oxidized glutathione (GSSG). The consumption of ROS and H2O2 aggravates the hypoxia of the bacterial microenvironment, which greatly affects the efficacy of SDT. On the one hand, how to reduce the concentration of GSH during the SDT process and effectively improve the antibacterial effect of SDT is an important technical problem that needs to be solved urgently. On the other hand, how to use the synergistic effect between different materials to prepare antibacterial agents / antibacterial materials with better antibacterial properties is also one of the key difficulties in the research and development process.

[0027] The embodiment of the present application provides a mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemodynamic synergistic antibacterial properties, as well as a preparation method and application thereof. Mesoporous polydopamine is used as a carrier, and the mesoporous polydopamine is hydrophilically modified and functionalized to improve the biocompatibility and loading performance of the mesoporous polydopamine. Then, the sonosensitizer and transition metal ions are successively loaded into the mesoporous polydopamine to obtain mesoporous polydopamine drug-loaded microspheres having both sonodynamic and chemodynamic antibacterial functions.

[0028] The embodiment of the present application utilizes the reaction between polydopamine and GSH to reduce the concentration of GSH in the bacterial microenvironment, and at the same time utilizes the property of transition metal ions catalyzing H2O2 to produce oxygen in the CDT process to provide oxygen for the SDT process, so as to increase the concentration of active oxygen in the microenvironment, improve the antibacterial effect of SDT, and achieve the synergistic antibacterial effect of sonodynamics and chemical dynamics. The process of consuming GSH is also accompanied by the cleavage of mesoporous polydopamine drug-loaded microspheres, which is conducive to the better release of sonosensitizers and transition metal ions by mesoporous polydopamine, further improving the antibacterial effect.

[0029] In a first aspect, the present invention provides a method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties, comprising the following steps: S1, dissolving polyether F127 and dopamine hydrochloride in an ethanol-water solution, stirring vigorously until the solution is clear, then slowly dropping 1,3,5-trimethylbenzene under stirring, continuing stirring for 30-40 min, adding concentrated ammonia water dropwise under stirring, continuing stirring for 1-1.5 h, washing with ethanol and deionized water three times respectively, to obtain a mesoporous polydopamine nanoparticle dispersion.

[0030] In some embodiments, the mass ratio of dopamine hydrochloride to polyether F127 is 1:(1.5-2.5); the volume ratio of ethanol to water in the ethanol-water solution is 1:1; the volume ratio of ethanol-water solution to 1,3,5-trimethylbenzene is (45-55):1; the concentration of dopamine hydrochloride in the ethanol-water solution is (3-7) mg / mL.

[0031] In some embodiments, after adding concentrated aqueous ammonia, the pH value of the reaction system is 8-10.

[0032] In step S1, polyether F127 acts as a template and stabilizer, and can self-assemble into micelles, which form cavities or channels during the curing process of dopamine, and finally leave holes. The formation of these holes gives polydopamine a mesoporous structure, which provides a high specific surface area and porosity, and can also help regulate the polymerization reaction and avoid disordered aggregation of polydopamine particles, thereby obtaining a more uniform and controllable microstructure.

[0033] S2, adjusting the pH value of the mesoporous polydopamine nanoparticle dispersion of step S1 to 11-12 with a sodium hydroxide aqueous solution, and adding thiol-polyethylene glycol-carboxyl (SH-PEG 2000 -NH2) aqueous solution, and stirred for 2-3 h to obtain a hydrophilic modified mesoporous polydopamine nanoparticle dispersion.

[0034] In some embodiments, the concentration of the sodium hydroxide aqueous solution is (0.1-0.2) mol / L, SH-PEG 2000 The concentration of -NH2 is (10-15) mg / mL, dopamine hydrochloride and SH-PEG 2000 -NH2 mass ratio is 1:(0.1-0.2).

[0035] S3, under stirring, dropwise add the sonosensitizer aqueous solution to the hydrophilic modified mesoporous polydopamine nanoparticle dispersion of step S2, stir in the dark for 8-12 h, then dropwise add the aqueous solution containing transition metal ions, stir for 1.5-3 h, and then centrifuge to obtain mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial. The total mass of the sonosensitizer and the transition metal ions accounts for 1-5% of the mass of the mesoporous polydopamine drug-loaded microspheres, and the mass ratio of the sonosensitizer to the transition metal ions is 1: (0.5-1).

[0036] In some embodiments, the sonosensitizer is one of protoporphyrin, chlorophyll, cyanine or rose bengal, and the sonosensitizer solution is prepared by dissolving the sonosensitizer in dimethyl sulfoxide at a concentration of (5-15) mg / mL, and then diluting it 10 times with deionized water; the transition metal in the transition metal ion is one of copper, iron, manganese, silver, cobalt, cadmium, nickel or molybdenum, and the concentration of the transition metal ion is (0.08-0.12) mol / L.

[0037] In some embodiments, the centrifugation speed is 11000-18000 rpm.

[0038] In the process of sonodynamic and chemodynamic synergistic antibacterial, firstly, the sonosensitizer is excited to a high energy state under the action of ultrasound, and transfers its energy to the surrounding oxygen molecules, generating highly oxidative singlet oxygen and other reactive oxygen species (ROS). ROS can destroy the bacterial cell membrane, oxidize proteins and enzymes to inactivate them, damage DNA and RNA, and ultimately lead to bacterial death, i.e., sonodynamic antibacterial (SDT); secondly, there is often overexpression of hydrogen peroxide (H2O2) at the site of bacterial infection, and transition metal ions catalyze the generation of H2O2 through the Fenton / Fenton-like reaction. Highly active hydroxyl radicals (•OH) cause oxidative damage to bacteria and produce oxygen, which is chemical dynamic antibacterial (CDT); thirdly, there is overexpressed hydrogen peroxide in the bacterial infection site, which allows the CDT reaction to continue. The oxygen produced during the CDT process can be provided to SDT to avoid the formation of an oxygen-deficient environment in the bacterial infection site, thereby enhancing the antibacterial effect of SDT; fourthly, the degradation of polydopamine by GSH is used to achieve the consumption of GSH in the bacterial infection site, thereby reducing the reaction of GSH with ROS or H2O2 and increasing the lethality of reactive oxygen. At the same time, the process of consuming GSH is also accompanied by the cleavage of mesoporous polydopamine drug-loaded microspheres, which is conducive to the better release of sonosensitizers and transition metal ions by mesoporous polydopamine, further enhancing the antibacterial effect.

[0039] In the second aspect, the embodiment of the present application provides a mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemodynamic synergistic antibacterial, wherein the mesoporous polydopamine drug-loaded microsphere uses mesoporous polydopamine as a carrier to load sonosensitizer and transition metal ions, and the average particle size is 120-180 nm. Among them, the sonosensitizer is one of protoporphyrin, chlorophyll, cyanine or rose bengal; the transition metal in the transition metal ion is one of copper, iron, manganese, silver, cobalt, cadmium, nickel or molybdenum.

[0040] In a third aspect, the embodiments of the present application provide an application of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties, which can be used to treat or prevent non-invasive deep bacterial infections, including Escherichia coli and Staphylococcus aureus.

[0041] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0042] 1. Preparation method Example 1 Example 1 provides a method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial properties, comprising the following steps: S1, dissolve 0.2 g polyether F127 and 0.1 g dopamine hydrochloride in 20 mL ethanol-water solution (V 乙醇 :V 水 =1:1), stirred vigorously until clarified, then, 0.4 mL of 1,3,5-trimethylbenzene was slowly dropped under stirring, and stirring was continued for 30 min. Then, 1 mL of concentrated ammonia was added dropwise under stirring. After stirring for 1 h, the mixture was washed three times with ethanol and deionized water respectively to obtain a mesoporous polydopamine nanoparticle dispersion.

[0043] S2, adjusting the pH value of the mesoporous polydopamine nanoparticle dispersion of step S1 to 11-12 with 0.1 mol / L sodium hydroxide aqueous solution, adding 1 mL of thiol-polyethylene glycol-carboxyl aqueous solution (concentration of 10 mg / mL) dropwise under stirring, and stirring for 2-3 h to obtain a hydrophilically modified mesoporous polydopamine nanoparticle dispersion.

[0044] S3, dilute the dimethyl sulfoxide solution (10 mg / mL) of protoporphyrin (PPIX) to 1 mg / mL with deionized water, take 1 mL and add it dropwise to the hydrophilic modified mesoporous polydopamine nanoparticle dispersion in step S2 under stirring, stir for 10 h in the dark, then add 50 μL of Cu(NO3)2 aqueous solution (0.1 mol / L), stir for 2 h, and then centrifuge at 18000 r / min to obtain mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical synergistic antibacterial properties.

[0045] Example 1 also provides a mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemodynamic synergistic antibacterial properties, denoted as MPDA-PEG@PPIX / Cu.

[0046] Example 2 Example 2 provides a method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties. The difference between Example 2 and Example 1 is that the sonosensitizer protoporphyrin (PPIX) in step S3 is replaced by cyanine (PC), and Cu(NO3)2 is replaced by FeCl3, that is, the transition metal Cu 2+ Replace with Fe 3+ The rest of the contents are the same as those in Example 1 and will not be described again.

[0047] Example 2 also provides a mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemodynamic synergistic antibacterial properties, denoted as MPDA-PEG@PC / Fe.

[0048] Comparative Example 1 Comparative Example 1 provides a mesoporous polydopamine microsphere, denoted as MPDA. The preparation method thereof is different from that of Example 1 in that steps S2 and S3 are omitted, and the rest of the contents are the same as those of Example 1, which will not be described again.

[0049] Comparative Example 2 Comparative Example 2 provides a thiol-polyethylene glycol-carboxyl modified mesoporous polydopamine microspheres, denoted as MPDA-PEG. The preparation method thereof is different from that of Example 1 in that there is no step S3. The rest of the contents are the same as those of Example 1 and will not be repeated here.

[0050] Comparative Example 3 Comparative Example 3 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@PPIX. The preparation method thereof is different from that of Example 1 in that the mesoporous polydopamine drug-loaded microsphere is only loaded with the sonosensitizer PPIX, and no transition metal ion Cu is loaded. 2+ The rest of the contents are the same as those in Example 1 and will not be described again.

[0051] Comparative Example 4 Comparative Example 4 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@PC. The preparation method thereof is different from that of Example 2 in that the mesoporous polydopamine drug-loaded microsphere is only loaded with the sonosensitizer PC, and no transition metal ion Fe is loaded. 3+ The rest of the contents are the same as those in Example 1 and will not be described again.

[0052] Comparative Example 5 Comparative Example 5 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@Cu. The preparation method thereof is different from that of Example 1 in that the mesoporous polydopamine drug-loaded microsphere only loads the transition metal ion Cu. 2+ , without loading the sonosensitizer PPIX, and the rest of the contents are the same as those in Example 1 and will not be repeated here.

[0053] Comparative Example 6 Comparative Example 6 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@Fe. The difference between its preparation method and that of Example 1 is that the mesoporous polydopamine drug-loaded microsphere only loads the transition metal ion Fe. 3+ , without loading the sonosensitizer PC, the rest of the contents are the same as those in Example 1 and will not be repeated here.

[0054] Experimental Example 1 Experimental Example 1 provides an antibacterial performance test of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial, comprising the following steps: S1, take 0.0034g of the mesoporous polydopamine drug-loaded microspheres prepared in Example 1 and disperse them in ultrapure water to prepare an antibacterial agent with a concentration of 0.5 mg / mL, add the antibacterial agent to a concentration of 10 9 coli solution with a concentration of 100 CFU / mL to obtain a mixed solution, which was then cultured in a 37°C constant temperature shaker for 6 h.

[0055] S2, add 1 mM hydrogen peroxide solution to the mixed solution after shaking culture for 6 h in step S1, mix evenly, shake and culture in a constant temperature shaker at 37 °C for 5 h, and then ultrasonicate for 10 min at an ultrasonic intensity of 3 W / cm 2 Then, the mixture was placed in a 37°C constant temperature shaker for further shaking and incubation for 12 h. Finally, the solution after incubation was collected, and the antibacterial effect was detected by the plate coating method, and the relative activity (%) of the bacteria was calculated.

[0056] Experimental Example 2-8 The differences between Experimental Examples 2-8 and Experimental Example 1 are shown in the table below.

[0057] 2. Test Method (1) Surface morphology Scanning electron microscopy (SEM) was used to test the surface morphology of the product.

[0058] (2) Particle size test Dynamic light scattering (DLS) was used to test the hydrodynamic particle size of the product aqueous solution.

[0059] (3) GSH consumption performance test The consumption of glutathione by the test product under acidic conditions of pH=5.5 was specifically performed as follows: glutathione was dissolved in a pH=5.5 buffer to prepare a 1.2 mM glutathione solution, 5 mL of the solution was slowly added dropwise to 1 mL of a 3 mg / mL product aqueous solution, and after stirring for different times (0, 1, 2, 3, 4, 5, 6 h), 0.5 mL of the above mixed solution was slowly added dropwise to 0.5 mL of a 2 mM 5,5'-dithiobis(2-nitrobenzoic acid)-dimethyl sulfoxide solution, and after mixing, the UV spectrum was immediately measured using a UV spectrophotometer. .

[0060] (4) Antibacterial performance testa Using o-phenylenediamine as an indicator, the product was tested for its ability to generate active oxygen such as hydroxyl radicals and singlet oxygen under different conditions (ultrasound / hydrogen peroxide / (ultrasound+hydrogen peroxide)). The specific steps are as follows: Ultrasonic conditions: Add 0.3 mL of 0.1 M o-phenylenediamine aqueous solution and 0.3 mL of 1 mg / mL product aqueous solution to 2.4 mL of ultrapure water, mix well, and ultrasonicate for 10 min. Use an ultraviolet spectrophotometer to measure the ultraviolet spectrum of the system before and after ultrasound, and calculate the change in its absorbance at 410 nm.

[0061] Hydrogen peroxide conditions: Add 0.3 mL of 5 mM hydrogen peroxide solution, 0.1 M o-phenylenediamine solution, and 1 mg / mL product solution to 2.1 mL ultrapure water, mix well, and let stand for 5 h. Use a UV spectrophotometer to measure the UV spectrum of the system before and after standing, and calculate the change in its absorbance at 410 nm.

[0062] Ultrasonic + hydrogen peroxide conditions: Add 0.3 mL of 5 mM hydrogen peroxide solution, 0.1 M o-phenylenediamine solution, and 1 mg / mL product solution to 2.1 mL ultrapure water, mix well, let stand for 5 hours, and then ultrasonicate for 10 minutes. Use an ultraviolet spectrophotometer to measure the ultraviolet spectrum of the system before and after standing + ultrasonication, and calculate the change in its absorbance at 410 nm.

[0063] (5) Antibacterial performance test b Using green fluorescent probe (SOSG) as an indicator, the performance of the product in generating singlet oxygen under different conditions (ultrasound / hydrogen peroxide / (ultrasound+hydrogen peroxide)) was tested. The specific steps are as follows: Ultrasonic conditions: Add 10 μL of product solution (3.3 mg / mL) and 1 μL of green fluorescent probe (5 mM) to 322 μL of ultrapure water, mix well, and ultrasonicate for 10 min. Use a fluorescence spectrometer to record the fluorescence intensity value of the system at 525 nm before and after ultrasound, and calculate the fluorescence intensity change rate.

[0064] Hydrogen peroxide conditions: Add 17 μL hydrogen peroxide solution (10 mM), 10 μL product solution (3.3 mg / mL) and 1 μL green fluorescent probe (5 mM) to 305 μL ultrapure water, mix well, and let stand for 5 h. Use a fluorescence spectrometer to record the fluorescence intensity value of the system at 525 nm before and after standing, and calculate the fluorescence intensity change rate.

[0065] Ultrasound + hydrogen peroxide conditions: Add 17 μL hydrogen peroxide solution (10 mM), 10 μL product solution (3.3 mg / mL) and 1 μL green fluorescent probe (5 mM) to 305 μL ultrapure water, mix well, let stand for 5 h, and then ultrasonicate for 10 min. Use a fluorescence spectrometer to record the fluorescence intensity value of the system at 525 nm before and after standing + ultrasonication, and calculate the fluorescence intensity change rate.

[0066] III. Analysis of test results of various embodiments and comparative examples The surface morphology of the mesoporous polydopamine drug-loaded microspheres (MPDA-PEG@PPIX / Cu and MPDA-PEG@PC / Fe) prepared in Example 1-2 was tested according to the test method (1). Figure 1 As shown, a is the surface morphology of MPDA-PEG@PPIX / Cu, and b is the surface morphology of MPDA-PEG@PC / Fe. It can be seen that both are relatively regular spheres with many irregular holes on the surface for adsorbing sonosensitizers and transition metal ions. Such a structure enhances the functionality of the material in catalytic and biomedical applications, making it have higher reaction efficiency under ultrasonic action and Fenton-like reaction catalysis.

[0067] The particle sizes of the products (MPDA-PEG@PPIX / Cu, MPDA, MPDA-PEG, MPDA-PEG@PPIX) prepared in Example 1 and Comparative Examples 1-3 were tested according to test method (2). The test results are shown in Figure 2 As shown in the table below. It can be seen that after hydrophilic modification, the particle size of mesoporous polydopamine nanoparticles is significantly reduced, and the loading of PPIX and Cu 2+ This is because, after the hydrophilic modification, hydrophilic groups are introduced on the surface of the nanoparticles. These groups can form hydrogen bonds with water molecules, making their surface properties more hydrophilic, which helps to stabilize the suspension state of the nanoparticles and enable the nanoparticles to be better dispersed in aqueous media. The introduction of PPIX may have led to fine-tuning of the surface structure of the nanoparticles. This structural adjustment provides them with a higher structural compactness, which is manifested as a reduction in particle size in DLS measurements. Cu 2+ The introduction of further changes the charge density and distribution on the surface of the nanoparticles. Through electrostatic interaction and steric hindrance effect, the mutual repulsion between particles is enhanced, the aggregation tendency is reduced, and the dispersion is further improved by forming coordination bonds with functional groups such as carboxyl groups on the surface of the nanoparticles, so that the nanoparticles exhibit a smaller hydrated particle size in the solution.

[0068] According to test method (3), the mesoporous polydopamine drug-loaded microspheres (MPDA-PEG@PPIX / Cu and MPDA-PEG@PC / Fe) prepared in Example 1-2 were tested for glutathione consumption and particle size changes under acidic conditions (pH=5.5). Figure 3 As shown, c is the absorbance curve of residual glutathione in the solution after glutathione was treated with MPDA-PEG@PPIX / Cu for 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively; d is the particle size change diagram of MPDA-PEG@PPIX / Cu after glutathione was treated with MPDA-PEG@PPIX / Cu for 6 h; e is the absorbance curve of residual glutathione in the solution after glutathione was treated with MPDA-PEG@PC / Fe for 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively; f is the particle size change diagram of MPDA-PEG@PC / Fe after glutathione was treated with MPDA-PEG@PC / Fe for 6 h.

[0069] The smaller the absorbance at the absorption peak of 442 nm, the lower the concentration of glutathione in the solution. It can be seen that as the treatment time increases, the concentration of glutathione in the solution gradually decreases, especially after 6 h of treatment, indicating that the nanoparticles have the ability to consume glutathione. The functional groups of polydopamine can undergo redox reactions with glutathione to promote the degradation of glutathione, and this effect increases with time. As can be seen from Figures d and f, MPDA-PEG@PPIX / Cu or MPDA-PEG@PC / Fe are cleaved after reacting with glutathione, which is manifested as a decrease in the hydrated particle size. This is because MPDA as a matrix contains abundant phenolic and amine groups. These functional groups are easy to undergo redox reactions with the strong reducing agent glutathione, resulting in the reduction of MPDA. This reaction will cause the structure of MPDA to change at the molecular level, thereby affecting the stability and polymerization state of the overall drug-loaded microspheres, resulting in partial cleavage of the drug-loaded microsphere framework.

[0070] According to test method (4), the performance of the products prepared in Examples 1-2 and Comparative Examples 3-6 (MPDA-PEG@PPIX / Cu, MPDA-PEG@PC / Fe, MPDA-PEG@PPIX, MPDA-PEG@PC, MPDA-PEG@Cu and MPDA-PEG@Fe) in generating active oxygen such as hydroxyl radicals and singlet oxygen under different conditions was tested. The test results are shown in Figure 4 As shown in Table 2, the absorbance change value of the indicator o-phenylenediamine at 410 nm represents the change rate of the concentration of active oxygen such as hydroxyl radicals and singlet oxygen in the system.

[0071] Table 2. Absorbance changes of o-phenylenediamine at 410 nm in the system after treatment for a certain period of time See also Figure 4 As shown in Table 2, g is the absorbance change value at 410 nm of MPDA-PEG@PPIX / Cu, MPDA-PEG@PPIX and MPDA-PEG@Cu after being treated with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different time periods, and h is the absorbance change value at 410 nm of MPDA-PEG@PC / Fe, MPDA-PEG@PC and MPDA-PEG@Fe after being treated with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different time periods. It can be seen that when only ultrasonic treatment is used, the absorbance change values ​​of the sonosensitizer + metal ion system and the sonosensitizer system increase, and the values ​​are similar, both of which are much higher than the absorbance change value of the metal ion system; when only hydrogen peroxide treatment is used, the absorbance change values ​​of the sonosensitizer + metal ion system and the metal ion system increase, and the values ​​are similar, both of which are much higher than the absorbance change value of the sonosensitizer system; when ultrasound + hydrogen peroxide treatment is used, the absorbance change value of the sonosensitizer + metal ion system increases significantly. Although the absorbance change values ​​of the sonosensitizer system and the metal ion system also increase, the absorbance change value of the sonosensitizer + metal ion system is significantly higher than that of the other two systems.

[0072] The above analysis shows that the system containing sonosensitizer can effectively generate a large amount of reactive oxygen under ultrasonic excitation, while the system containing metal ions can generate hydroxyl radicals through (quasi) Fenton reaction in the presence of hydrogen peroxide. In addition, after the sonosensitizer + metal ion system was treated with ultrasound and hydrogen peroxide, under the effective excitation of the sonosensitizer and the synergistic catalysis of metal ions, the two jointly enhanced the generation efficiency of hydroxyl radicals and other reactive oxygen, making the sonosensitizer + metal ion system show the largest absorbance change value in the o-phenylenediamine test.

[0073] According to test method (5), the performance of the products prepared in Examples 1-2 and Comparative Examples 3-6 (MPDA-PEG@PPIX / Cu, MPDA-PEG@PC / Fe, MPDA-PEG@PPIX, MPDA-PEG@PC, MPDA-PEG@Cu and MPDA-PEG@Fe) in generating singlet oxygen under different conditions was tested. The test results are shown in Figure 5 As shown in the table below, the rate of change of the fluorescence intensity of the indicator SOSG at 525 nm represents the rate of change of the singlet oxygen concentration in the system.

[0074] Table 3. Change rate of fluorescence intensity of SOSG at 525 nm in the system after treatment for a certain period of time (%) See also Figure 5 As shown in Table 3, i is the fluorescence intensity change rate of MPDA-PEG@PPIX / Cu, MPDA-PEG@PPIX and MPDA-PEG@Cu at 525 nm after being treated with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different time periods, and j is the fluorescence intensity change rate of MPDA-PEG@PC / Fe, MPDA-PEG@PC and MPDA-PEG@Fe at 525 nm after being treated with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different time periods. It can be seen that after ultrasound treatment, the fluorescence intensity change rate of the sonosensitizer + metal ion system and the sonosensitizer system at 525 nm is much higher than that of the metal ion system, indicating that the system containing the sonosensitizer can effectively generate a large amount of singlet oxygen under ultrasound excitation, and then show a high fluorescence intensity change rate under the detection of the SOSG fluorescent probe.

[0075] See also Figure 6 As shown, it is the antibacterial effect diagram of Experimental Examples 1 to 8. It can be seen that after adding mesoporous polydopamine drug-loaded microspheres and hydrogen peroxide to the solution containing Escherichia coli / Staphylococcus aureus and performing ultrasonic treatment, the Escherichia coli / Staphylococcus aureus in the solution was almost completely killed, indicating that ultrasonic waves can cause the mesoporous polydopamine drug-loaded microspheres to produce a large amount of active oxygen, which has an excellent bactericidal effect and can cause bacterial death.

[0076] In summary, the present application provides a mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial effects of sonodynamics and chemodynamics, as well as a preparation method and application thereof. A sonosensitizer and transition metal ions are introduced into a mesoporous polydopamine matrix to obtain mesoporous polydopamine drug-loaded microspheres, so that the drug-loaded microspheres can not only remove the antioxidant glutathione, but also produce active oxygen such as hydroxyl free radicals and singlet oxygen through Fenton-like reactions and ultrasonic excitation, thereby achieving the antibacterial purpose. At the same time, the synergistic antibacterial effects of sonodynamics and chemodynamics and the cleavage of mesoporous polydopamine drug-loaded microspheres during the antibacterial process are utilized to greatly enhance the antibacterial effect.

[0077] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties, characterized in that: The steps include: S1, dissolving polyether F127 and dopamine hydrochloride in an ethanol-water solution, stirring vigorously until the solution is clear, then slowly dropping 1,3,5-trimethylbenzene under stirring, continuing stirring for 30-40 minutes, adding concentrated ammonia water dropwise under stirring, continuing stirring for 1-1.5 hours, washing with ethanol and deionized water three times respectively, to obtain a mesoporous polydopamine nanoparticle dispersion; S2, adjusting the pH value of the mesoporous polydopamine nanoparticle dispersion of step S1 to 11-12 with an aqueous sodium hydroxide solution, adding a thiol-polyethylene glycol-carboxyl aqueous solution dropwise under stirring, and stirring for 2-3 hours to obtain a hydrophilically modified mesoporous polydopamine nanoparticle dispersion; S3, under stirring, dropwise add the aqueous solution of the sonosensitizer to the hydrophilically modified mesoporous polydopamine nanoparticle dispersion of step S2, stir in the dark for 8-12 hours, then dropwise add the aqueous solution containing transition metal ions, stir for 1.5-3 hours, and then centrifuge to obtain mesoporous polydopamine drug-loaded microspheres with synergistic antibacterial properties based on sonodynamic and chemodynamic forces; wherein the total mass of the sonosensitizer and the transition metal ions accounts for 1-5% of the mass of the mesoporous polydopamine drug-loaded microspheres, and the mass ratio of the sonosensitizer to the transition metal ions is 1:(0.5-1).

2. The method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial according to claim 1, characterized in that: In step S3, the sonosensitizer is one of protoporphyrin, chlorophyll, cyanine or rose bengal; the sonosensitizer solution is prepared by dissolving the sonosensitizer in dimethyl sulfoxide at a concentration of (5-15) mg / mL, and then diluting it 10 times with deionized water; the transition metal in the transition metal ion is one of copper, iron, manganese, silver, cobalt, cadmium, nickel or molybdenum, and the concentration of the transition metal ion is (0.08-0.12) mol / L.

3. The method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial according to claim 1, characterized in that: In step S1, the mass ratio of the dopamine hydrochloride to the polyether F127 is 1:(1.5-2.5); the volume ratio of ethanol to water in the ethanol-water solution is 1:1; the volume ratio of the ethanol-water solution to the 1,3,5-trimethylbenzene is (45-55):1; and the concentration of the dopamine hydrochloride in the ethanol-water solution is (3-7) mg / mL.

4. The method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial according to claim 3, characterized in that: In step S1, after adding concentrated aqueous ammonia, the pH value of the reaction system is 8-10.

5. The method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial according to claim 1, characterized in that: In step S2, the concentration of the sodium hydroxide aqueous solution is (0.1-0.2) mol / L, the concentration of the thiol-polyethylene glycol-carboxyl aqueous solution is (10-15) mg / mL, and the mass ratio of dopamine hydrochloride to thiol-polyethylene glycol-carboxyl is 1:(0.1-0.2).

6. The method for preparing mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties according to claim 2, characterized in that: In step S3, the centrifugal speed is 11000-18000 rpm.

7. A mesoporous polydopamine drug-loaded microsphere based on sonodynamic and chemical dynamic synergistic antibacterial properties, characterized in that: The mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemodynamic synergistic antibacterial properties are prepared according to the preparation method described in any one of claims 1-6.

8. The mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial properties according to claim 7, characterized in that: The mesoporous polydopamine drug-loaded microspheres use mesoporous polydopamine as a carrier to load a sonosensitizer and transition metal ions; the sonosensitizer is one of protoporphyrin, chlorophyll, cyanine or rose bengal; the transition metal in the transition metal ions is one of copper, iron, manganese, silver, cobalt, cadmium, nickel or molybdenum.

9. The mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical dynamic synergistic antibacterial properties according to claim 8, characterized in that: The average particle size of the mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effect of sonodynamics and chemical dynamics is 120-180 nm.

10. The use of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical synergistic antibacterial prepared by the preparation method according to any one of claims 1 to 6 or the use of mesoporous polydopamine drug-loaded microspheres based on sonodynamic and chemical synergistic antibacterial prepared by the preparation method according to any one of claims 7 to 9, characterized in that: The mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effect of sonodynamics and chemodynamics are used for treating or preventing non-invasive deep bacterial infections, wherein the bacteria include Escherichia coli and Staphylococcus aureus.

Images