Mesoporous polydopamine-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics, their preparation method and application

Abstract

This application provides a mesoporous polydopamine drug-loaded microsphere based on synergistic antibacterial action of acoustic-dynamic and chemical-dynamic processes, its preparation method, and its application, belonging to the field of nanobiomedicine. This application uses mesoporous polydopamine as a carrier, improving its biocompatibility and loading capacity through hydrophilic modification and functionalization. By loading acoustic sensitizers and transition metal ions into the mesoporous polydopamine, the reaction between polydopamine and GSH reduces the GSH concentration in the bacterial microenvironment. Simultaneously, the characteristic of transition metal ions catalyzing the production of oxygen from H2O2 during CDT provides oxygen for SDT, thereby increasing the concentration of reactive oxygen species in the microenvironment and enhancing the antibacterial effect of SDT, achieving synergistic antibacterial action of acoustic-dynamic and chemical-dynamic processes. Furthermore, the lysis of the mesoporous polydopamine drug-loaded microspheres accompanying the GSH consumption process better releases the acoustic sensitizers and transition metal ions, further enhancing the antibacterial effect.

Description

Technical Field

[0001] This invention relates to the field of nanobiomedicine, specifically to a mesoporous polydopamine drug-loaded microsphere based on the synergistic antibacterial effects of acoustic and chemical dynamics, its preparation method, and its application. Background Technology

[0002] Organ transplants, prosthesis transplants, and tissue engineering have brought hope for cures to many patients. However, clinical infections remain a significant issue, with bacterial infections being the most common. Bacterial infections can occur alone or repeatedly, or they can co-infect with other pathogens, posing a considerable threat to human health. Therefore, developing effective antimicrobial materials or methods to prevent bacterial infections in clinical settings is of paramount importance to modern medicine.

[0003] Antimicrobial materials include organic antimicrobial materials, sterile antimicrobial materials, and organic-inorganic composite antimicrobial materials. Organic antimicrobial materials are characterized by their rich variety, wide range of applications, significant antimicrobial effects, and relatively mature application technologies. Antibiotics and quaternary ammonium ions are examples of organic antimicrobial materials. However, prolonged and high-dose use can easily lead to drug resistance, limiting their antimicrobial efficacy. Inorganic antimicrobial materials mainly utilize the direct destructive effect of transition metal ions, the penetration effect and photocatalytic properties of metal oxides, and the catalytic oxidation effect of nanoparticles for antimicrobial activity. They are characterized by stable antimicrobial effects and good antimicrobial performance. Regardless of the large-scale use of antibiotics, quaternary ammonium ions, or metal ions, drug resistance is easily generated, and they possess potential toxicity, limiting their application. Therefore, there is still a need to develop more novel and effective antimicrobial strategies to meet clinical needs. Organic-inorganic composite antibacterial materials possess both biocompatibility and the characteristics of high porosity and large specific surface area, and can integrate multiple antibacterial mechanisms, making them an important research direction. Examples include Ag nanoparticle / chitosan composite antibacterial materials and zinc ion / tetradecyltributyl quaternary phosphonium salt composite antibacterial materials. How to utilize the synergistic effect between different materials to achieve a technical effect of 1+1>2 is one of the key challenges in the research and development process.

[0004] Sonodynamic therapy (SDT) is a non-invasive antibacterial therapy that has developed in recent years and has shown great potential in the non-invasive treatment of deep bacterial infections. Its antibacterial mechanism is that ultrasound triggers a sonosensitive agent to convert oxygen into reactive oxygen species (ROS). High concentrations of ROS can cause oxidative damage to bacteria, achieving an antibacterial effect. However, the microenvironment under bacterial infection always contains high concentrations of glutathione (GSH). As an important member of the human immune system, GSH has antioxidant and detoxification functions and can react with ROS or H2O2 to generate oxidized glutathione (GSSG). The consumption of ROS and H2O2 exacerbates the hypoxia in the bacterial microenvironment, greatly affecting the efficacy of SDT. Therefore, how to reduce the concentration of GSH during SDT and effectively improve its antibacterial effect is a crucial technical challenge that needs to be addressed.

[0005] In view of this, it is necessary to design a method for preparing and applying mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effect of acoustic and chemical dynamics in order to solve the above problems. Summary of the Invention

[0006] In view of the technical problems existing in the background art, this application provides a mesoporous polydopamine drug-loaded microsphere based on the synergistic antibacterial effect of acoustic and chemical dynamics, its preparation method and application. Using mesoporous polydopamine as a carrier, the biocompatibility and loading performance of mesoporous polydopamine are improved by hydrophilic modification and functionalization. By loading acoustic sensitizer and transition metal ions sequentially into mesoporous polydopamine, the synergistic antibacterial effect of acoustic and chemical dynamics is achieved. Moreover, the mesoporous polydopamine drug-loaded microspheres will cleave during the antibacterial process, so that the acoustic sensitizer and transition metal ions are fully released, which greatly improves the antibacterial effect.

[0007] In a first aspect, embodiments of this application provide a method for preparing mesoporous polydopamine-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics, comprising the following steps:

[0008] S1, dissolve polyether F127 and dopamine hydrochloride in an ethanol-water solution and stir vigorously until clear. Then, slowly add 1,3,5-trimethylbenzene dropwise while stirring. Continue stirring for 30-40 min, then add concentrated ammonia dropwise while stirring. Continue stirring for 1-1.5 h, then wash three times with ethanol and deionized water respectively to obtain a mesoporous polydopamine nanoparticle dispersion.

[0009] S2, adjust the pH of the mesoporous polydopamine nanoparticle dispersion from step S1 to 11-12 with sodium hydroxide aqueous solution, add mercapto-polyethylene glycol-carboxyl aqueous solution dropwise under stirring, and stir for 2-3 hours to obtain hydrophilic modified mesoporous polydopamine nanoparticle dispersion.

[0010] S3, under stirring, the aqueous solution of the acoustic sensitizer is added dropwise to the hydrophilic modified mesoporous polydopamine nanoparticle dispersion of step S2, and stirred in the dark for 8-12 hours. Then, an aqueous solution containing transition metal ions is added dropwise, and the mixture is stirred for 1.5-3 hours. After centrifugation, mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial action of acoustic and chemical kinetics are obtained. The total mass of the acoustic sensitizer 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 acoustic sensitizer to the transition metal ions is 1:(0.5-1).

[0011] In some embodiments, in step S3, the sound sensitizer is one of protoporphyrin (PPIX), chlorophyll (Chl), cyanin (PC), or rose red (RhB); the sound sensitizer solution is prepared by dissolving the sound sensitizer in dimethyl sulfoxide at a concentration of (5-15) mg / mL, and then diluting it 10 times with deionized water; 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.

[0012] In some embodiments, in step S1, 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 the ethanol-water solution to 1,3,5-trimethylbenzene is (45-55):1; and the concentration of dopamine hydrochloride in the ethanol-water solution is (3-7) mg / mL.

[0013] In some embodiments, after adding concentrated ammonia in step S1, the pH value of the reaction system is 8-10.

[0014] 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 mercapto-polyethylene glycol-carboxyl aqueous solution is (10-15) mg / mL, and the mass ratio of dopamine hydrochloride to mercapto-polyethylene glycol-carboxyl is 1:(0.1-0.2).

[0015] In some embodiments, in step S3, the centrifugation speed is 11000-18000 rpm.

[0016] Secondly, this application provides a mesoporous polydopamine drug-loaded microsphere based on the synergistic antibacterial effect of acoustic and chemical dynamics, which is prepared by any of the aforementioned preparation methods. The mesoporous polydopamine drug-loaded microsphere uses mesoporous polydopamine as a carrier to load a acoustic sensitizer and transition metal ions. The acoustic sensitizer is one of protoporphyrin, chlorophyll, anthocyanin, or rose red. The transition metal ion is one of copper, iron, manganese, silver, cobalt, cadmium, nickel, or molybdenum.

[0017] In some embodiments, the average particle size of the mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics is 120-180 nm.

[0018] Thirdly, this application also provides an application of mesoporous polydopamine drug-loaded microspheres prepared by any of the preparation methods described in any of the foregoing schemes, based on the synergistic antibacterial effects of acoustic dynamics and chemodynamics, or an application of mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effects of acoustic dynamics and chemodynamics in any of the foregoing schemes. The mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effects of acoustic dynamics and chemodynamics are used for the prevention or non-invasive treatment of deep bacterial infections, wherein the bacteria include Escherichia coli and Staphylococcus aureus.

[0019] The beneficial effects of this application are:

[0020] This application provides a mesoporous polydopamine drug-loaded microsphere based on synergistic antibacterial action of acoustic and chemical kinetics, its preparation method, and its application. Specifically, mesoporous polydopamine is obtained by hydrophilic modification and structural alteration of polydopamine, thereby improving biocompatibility and loading capacity. Synergistic antibacterial action of acoustic and chemical kinetics is achieved by sequentially loading a sonosensitive agent and transition metal ions into the mesoporous polydopamine.

[0021] In the synergistic antibacterial process of sonodynamics and chemodynamics, firstly, the sonosensitive agent is excited to a high-energy state under the action of ultrasound, and transfers its energy to the surrounding oxygen molecules, generating highly oxidizing reactive oxygen species (ROS), such as singlet oxygen. ROS can destroy the bacterial cell membrane, oxidize proteins and enzymes, inactivating them, damage DNA and RNA, and ultimately lead to bacterial death, i.e., sonodynamic therapy (SDT); secondly, the bacterial infection site often has overexpressed hydrogen peroxide (H2O2), and transition metal ions catalyze the generation of H2O2 through Fenton / Fenton-like reactions. Highly reactive hydroxyl radicals (·OH) cause oxidative damage to bacteria and simultaneously generate oxygen, i.e., chemokinetic therapy (CDT). Thirdly, the presence of overexpressed hydrogen peroxide at the bacterial infection site allows the CDT reaction to continue. The oxygen generated during CDT can be supplied to spontaneously activated hydroxyl radicals (SDT), preventing the formation of a hypoxic environment at the bacterial infection site and thus enhancing the antibacterial effect of SDT. Fourthly, the degradation of polydopamine by glucosamine (GSH) depletes GSH at the bacterial infection site, reducing the reaction of GSH with ROS or H2O2 and increasing the lethality of reactive oxygen species. Simultaneously, the consumption of GSH is accompanied by the lysis of mesoporous polydopamine-loaded microspheres, a phenomenon that facilitates better release of sonosensitive agents and transition metal ions from the mesoporous polydopamine, further enhancing the antibacterial effect.

[0022] Furthermore, the modified mesoporous polydopamine nanoparticles provided in this application have good biocompatibility. Their mesoporous structure gives the material a larger specific surface area, which can load more antibacterial reagents, thereby increasing the concentration of reactive oxygen species and enhancing the antibacterial effect.

[0023] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0025] Figure 1 The images show the surface morphology of the mesoporous polydopamine drug-loaded microspheres prepared in Examples 1-2; where a is the surface morphology of MPDA-PEG@PPIX / Cu and b is the surface morphology of MPDA-PEG@PC / Fe.

[0026] Figure 2 Particle size variation graphs of the products prepared in Example 1 and Comparative Examples 1-3;

[0027] Figure 3 The graphs show the consumption of glutathione and particle size changes of the mesoporous polydopamine drug-loaded microspheres prepared in Examples 1-2 under acidic conditions of pH 5.5; where c is the absorbance curve of residual glutathione in the solution after treating glutathione with MPDA-PEG@PPIX / Cu for a certain time, d is the particle size change graph after treating glutathione with MPDA-PEG@PPIX / Cu for 6 h, e is the absorbance curve of residual glutathione in the solution after treating glutathione with MPDA-PEG@PC / Fe for a certain time, and f is the particle size change graph after treating glutathione with MPDA-PEG@PC / Fe for 6 h.

[0028] Figure 4 The graph shows the absorbance changes of o-phenylenediamine at 410 nm during the antibacterial performance test of the products prepared by Examples 1-2 and Comparative Examples 3-6, which tested the concentration of free radicals and singlet oxygen.

[0029] Figure 5 The graph shows the fluorescence intensity change at 525 nm of the green fluorescent probe (SOSG) used to test the singlet oxygen concentration in the antibacterial performance test of the products prepared by Examples 1-2 and Comparative Examples 3-6.

[0030] Figure 6 The image shows the antibacterial effect obtained in Experiment 1-8. Detailed Implementation

[0031] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0033] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0034] In the description of the embodiments of this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0035] High concentrations of glutathione (GSH) are always present in the bacterial microenvironment. As an important member of the human immune system, GSH has antioxidant and detoxification functions. It can react with ROS or H2O2 to generate oxidized glutathione (GSSG). The consumption of ROS and H2O2 exacerbates the hypoxia in the bacterial microenvironment, greatly affecting the efficacy of glutathione toxicology (SDT). On the one hand, how to reduce the concentration of GSH during SDT and effectively improve its antibacterial effect is a critical technical challenge that urgently needs to be solved. On the other hand, how to utilize the synergistic effects between different materials to prepare antibacterial agents / materials with better antibacterial properties is also one of the key difficulties in the research and development process.

[0036] This application provides a mesoporous polydopamine drug-loaded microsphere based on synergistic antibacterial activity of acoustic and chemical dynamics, its preparation method, and its application. Using mesoporous polydopamine as a carrier, the biocompatibility and loading capacity of mesoporous polydopamine are improved by hydrophilic modification and functionalization. Then, acoustic sensitizers and transition metal ions are loaded into mesoporous polydopamine sequentially to obtain mesoporous polydopamine drug-loaded microspheres with both acoustic and chemical dynamic antibacterial functions.

[0037] This application utilizes the reaction between polydopamine and GSH to reduce the GSH concentration in the bacterial microenvironment. Simultaneously, it leverages the characteristic of transition metal ions catalyzing the production of oxygen from H₂O₂ during the CDT process to provide oxygen for the SDT process, thereby increasing the concentration of reactive oxygen species in the microenvironment and enhancing the antibacterial effect of SDT, achieving synergistic antibacterial action through sonodynamic and chemodynamic processes. The consumption of GSH is accompanied by the lysis of mesoporous polydopamine-loaded microspheres, a phenomenon that facilitates better release of the sonosensitive agent and transition metal ions from the mesoporous polydopamine, further enhancing the antibacterial effect.

[0038] In a first aspect, embodiments of this application provide a method for preparing mesoporous polydopamine-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics, comprising the following steps:

[0039] S1, dissolve polyether F127 and dopamine hydrochloride in an ethanol-water solution and stir vigorously until clear. Then, slowly add 1,3,5-trimethylbenzene dropwise while stirring. Continue stirring for 30-40 min, then add concentrated ammonia dropwise while stirring. Continue stirring for 1-1.5 h, then wash three times with ethanol and deionized water respectively to obtain a mesoporous polydopamine nanoparticle dispersion.

[0040] 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 the ethanol-water solution to 1,3,5-trimethylbenzene is (45-55):1; and the concentration of dopamine hydrochloride in the ethanol-water solution is (3-7) mg / mL.

[0041] In some embodiments, after adding concentrated ammonia, the pH value of the reaction system is 8-10.

[0042] In step S1, polyether F127 acts as a template agent and stabilizer. It can self-assemble into microcells, which form cavities or channels during the dopamine curing process, ultimately leaving pores. The formation of these pores gives polydopamine a mesoporous structure, which provides high specific surface area and porosity. It also helps to regulate the polymerization reaction, avoid the disordered aggregation of polydopamine particles, and thus obtain a more uniform and controllable microstructure.

[0043] S2, adjust the pH of the mesoporous polydopamine nanoparticle dispersion from step S1 to 11-12 using sodium hydroxide aqueous solution, and add mercapto-polyethylene glycol-carboxyl (SH-PEG) dropwise under stirring. 2000 A hydrophilic modified mesoporous polydopamine nanoparticle dispersion was obtained by stirring an aqueous solution of -NH2 for 2-3 hours.

[0044] 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 was (10-15) mg / mL, and dopamine hydrochloride was reacted with SH-PEG. 2000 The mass ratio of -NH2 is 1:(0.1-0.2).

[0045] S3, under stirring, the aqueous solution of the acoustic sensitizer is added dropwise to the hydrophilic modified mesoporous polydopamine nanoparticle dispersion from step S2. The mixture is stirred in the dark for 8-12 hours. Then, an aqueous solution containing transition metal ions is added dropwise, and the mixture is stirred for 1.5-3 hours. The mixture is then centrifuged to obtain mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical kinetics. The total mass of the acoustic sensitizer and transition metal ions accounts for 1-5% of the mass of the mesoporous polydopamine drug-loaded microspheres, and the mass ratio of the acoustic sensitizer to the transition metal ions is 1:(0.5-1).

[0046] In some embodiments, the sound sensitizer is one of protoporphyrin, chlorophyll, cyanin, or rose red. The sound sensitizer solution is prepared by dissolving the sound sensitizer in dimethyl sulfoxide at a concentration of (5-15) mg / mL, and then diluting it 10 times with deionized water. 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.

[0047] In some embodiments, the centrifugation speed is 11,000-18,000 rpm.

[0048] In the synergistic antibacterial process of sonodynamics and chemodynamics, firstly, the sonosensitive agent is excited to a high-energy state under the action of ultrasound, and transfers its energy to the surrounding oxygen molecules, generating highly oxidizing reactive oxygen species (ROS), such as singlet oxygen. ROS can destroy the bacterial cell membrane, oxidize proteins and enzymes, inactivating them, damage DNA and RNA, and ultimately lead to bacterial death, i.e., sonodynamic antibacterial (SDT); secondly, the bacterial infection site often has overexpressed hydrogen peroxide (H2O2), and transition metal ions catalyze the generation of H2O2 through Fenton / Fenton-like reactions. Highly reactive hydroxyl radicals (·OH) cause oxidative damage to bacteria and simultaneously generate oxygen, i.e., chemodynamic antibacterial action (CDT). Thirdly, the presence of overexpressed hydrogen peroxide at the bacterial infection site allows the CDT reaction to continue. The oxygen generated during CDT can be supplied to SDT, preventing the formation of a hypoxic environment at the bacterial infection site and thus enhancing the antibacterial effect of SDT. Fourthly, the degradation of polydopamine by GSH consumes GSH at the bacterial infection site, reducing the reaction of GSH with ROS or H2O2 and increasing the lethality of reactive oxygen species. Simultaneously, the consumption of GSH is accompanied by the lysis of mesoporous polydopamine-loaded microspheres, a phenomenon that facilitates better release of sonosensitive agents and transition metal ions from the mesoporous polydopamine, further enhancing the antibacterial effect.

[0049] Secondly, embodiments of this application provide mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics. These microspheres use mesoporous polydopamine as a carrier to load a acoustic sensitizer and transition metal ions, with an average particle size of 120-180 nm. The acoustic sensitizer is one of protoporphyrin, chlorophyll, anthocyanin, or rose red; the transition metal ion is one of copper, iron, manganese, silver, cobalt, cadmium, nickel, or molybdenum.

[0050] Thirdly, embodiments of this application provide an application of mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial action of acoustic and chemical dynamics, which can be used for the prevention or non-invasive treatment of deep bacterial infections, including Escherichia coli and Staphylococcus aureus.

[0051] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0052] I. Preparation Method

[0053] Example 1

[0054] Example 1 provides a method for preparing mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics, comprising the following steps:

[0055] S1, Dissolve 0.2 g of polyether F127 and 0.1 g of dopamine hydrochloride in 20 mL of ethanol-water solution (V 乙醇 V 水 In a mixture of 1:1, the mixture was stirred vigorously until clear. Then, 0.4 mL of 1,3,5-trimethylbenzene was slowly added dropwise while stirring. After stirring for 30 min, 1 mL of concentrated ammonia was added dropwise while 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.

[0056] S2, adjust the pH of the mesoporous polydopamine nanoparticle dispersion from step S1 to 11-12 with 0.1 mol / L sodium hydroxide aqueous solution, add 1 mL of mercapto-polyethylene glycol-carboxyl aqueous solution (concentration 10 mg / mL) dropwise while stirring, and obtain the hydrophilic modified mesoporous polydopamine nanoparticle dispersion after stirring for 2-3 h.

[0057] S3, dilute the dimethyl sulfoxide solution of protoporphyrin (PPIX) (10 mg / mL) 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 in the dark for 10 h, then add 50 μL 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 the synergistic antibacterial effect of acoustic and chemical kinetics.

[0058] Example 1 also provides a mesoporous polydopamine drug-loaded microsphere based on the synergistic antibacterial effect of acoustic and chemical dynamics, denoted as MPDA-PEG@PPIX / Cu.

[0059] Example 2

[0060] Example 2 provides a method for preparing mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics. The difference between Example 2 and Example 1 is that the acoustic sensitizer protoporphyrin (PPIX) in step S3 is replaced with cyanine (PC), and Cu(NO3)2 is replaced with FeCl3, i.e., the transition metal Cu... 2+ Replace with Fe 3+ The rest of the content is the same as in Example 1, and will not be repeated here.

[0061] Example 2 also provides a mesoporous polydopamine drug-loaded microsphere based on the synergistic antibacterial effect of acoustic and chemical dynamics, denoted as MPDA-PEG@PC / Fe.

[0062] Comparative Example 1

[0063] Comparative Example 1 provides a mesoporous polydopamine microsphere, denoted as MPDA. Its preparation method differs from that of Example 1 in that steps S2 and S3 are omitted. The rest of the content is the same as that of Example 1 and will not be repeated here.

[0064] Comparative Example 2

[0065] Comparative Example 2 provides a thiol-polyethylene glycol-carboxyl modified mesoporous polydopamine microsphere, denoted as MPDA-PEG. Its preparation method differs from that of Example 1 in that step S3 is omitted. The rest of the content is the same as that of Example 1, and will not be repeated here.

[0066] Comparative Example 3

[0067] Comparative Example 3 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@PPIX. The preparation method differs from Example 1 in that this mesoporous polydopamine drug-loaded microsphere only loads the sonosensitive agent PPIX and does not load the transition metal ion Cu. 2+ The rest of the content is the same as in Example 1, and will not be repeated here.

[0068] Comparative Example 4

[0069] Comparative Example 4 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@PC. The preparation method differs from Example 2 in that this mesoporous polydopamine drug-loaded microsphere only loads the sonosensitive agent PC and does not load the transition metal ion Fe. 3+ The rest of the content is the same as in Example 1, and will not be repeated here.

[0070] Comparative Example 5

[0071] Comparative Example 5 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@Cu. The preparation method differs from Example 1 in that this mesoporous polydopamine drug-loaded microsphere is loaded only with the transition metal ion Cu. 2+ This one does not contain the sound-sensitive agent PPIX, and the rest is the same as in Example 1, so it will not be repeated here.

[0072] Comparative Example 6

[0073] Comparative Example 6 provides a modified mesoporous polydopamine drug-loaded microsphere, denoted as MPDA-PEG@Fe. The preparation method differs from Example 1 in that this mesoporous polydopamine drug-loaded microsphere is loaded only with transition metal ions Fe. 3+ This one does not contain the sound-sensitive agent PC, and the rest is the same as in Example 1, so it will not be repeated here.

[0074] Experimental Example 1

[0075] Example 1 provides a method for testing the antibacterial properties of mesoporous polydopamine-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics, including the following steps:

[0076] S1, 0.0034 g of the mesoporous polydopamine drug-loaded microspheres prepared in Example 1 were dispersed in ultrapure water to prepare an antibacterial reagent with a concentration of 0.5 mg / mL. The antibacterial reagent was added to a concentration of 10... 9 The mixture was prepared by adding CFU / mL of Escherichia coli solution and then incubating it in a shaker at 37°C for 6 hours.

[0077] S2, Add 1mM hydrogen peroxide aqueous solution to the mixed solution after shaking culture for 6h in step S1, mix evenly, and shake culture in a constant temperature shaker at 37℃ for 5h, followed by ultrasonic treatment for 10min at an ultrasonic intensity of 3W / cm. 2 Then, the solution was placed in a constant temperature shaker at 37°C and incubated for 12 hours. Finally, the solution after incubation was collected, and the antibacterial effect was detected by plate coating method, and the relative activity (%) of bacteria was calculated.

[0078] Experimental Example 2-8

[0079] The differences between Experiment 2-8 and Experiment 1 are shown in the table below.

[0080]

[0081] II. Testing Methods

[0082] (1) Surface morphology

[0083] The surface morphology of the product was tested using a scanning electron microscope (SEM).

[0084] (2) Particle size test

[0085] The hydrodynamic particle size of the product's aqueous solution was measured using a dynamic light scattering (DLS) instrument.

[0086] (3) GSH power consumption performance test

[0087] The test investigated the product's consumption of glutathione under acidic conditions (pH 5.5). The specific steps were as follows: Glutathione was dissolved in a pH 5.5 buffer solution to prepare a 1.2 mM glutathione solution. 5 mL of this solution was slowly added dropwise to 1 mL of a 3 mg / mL aqueous solution of the product. After stirring for different times (0, 1, 2, 3, 4, 5, 6 h), 0.5 mL of the mixture was taken, and 0.5 mL of a 2 mM 5,5'-dithiobis(2-nitrobenzoic acid)-dimethyl sulfoxide solution was slowly added dropwise. After mixing, the UV spectrum was immediately measured using a UV spectrophotometer.

[0088] (4) Antibacterial performance test a

[0089] Using o-phenylenediamine as an indicator, the product's ability to generate reactive oxygen species such as hydroxyl radicals and singlet oxygen under different conditions (ultrasound / hydrogen peroxide / (ultrasound + hydrogen peroxide)) was tested. The specific steps were as follows:

[0090] 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 thoroughly, and sonicate for 10 min. Measure the UV spectrum of the system before and after ultrasonication using a UV spectrophotometer, and calculate the change in absorbance at 410 nm.

[0091] Hydrogen peroxide conditions: Add 0.3 mL each of a 5 mM hydrogen peroxide aqueous solution, a 0.1 M o-phenylenediamine aqueous solution, and a 1 mg / mL product aqueous solution to 2.1 mL of ultrapure water, mix thoroughly, and let stand for 5 h. Measure the UV spectrum of this system before and after standing using a UV spectrophotometer, and calculate the change in absorbance at 410 nm.

[0092] Ultrasonic treatment with hydrogen peroxide: Add 0.3 mL each of a 5 mM hydrogen peroxide aqueous solution, a 0.1 M o-phenylenediamine aqueous solution, and a 1 mg / mL product aqueous solution to 2.1 mL of ultrapure water. Mix thoroughly, let stand for 5 h, and then sonicate for 10 min. Measure the UV spectrum of the system before and after standing and sonication using a UV spectrophotometer, and calculate the change in absorbance at 410 nm.

[0093] (5) Antibacterial performance test b

[0094] Using a 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:

[0095] 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 thoroughly, and sonicate for 10 min. Record the fluorescence intensity of the system at 525 nm before and after sonication using a fluorescence spectrometer, and calculate the rate of change in fluorescence intensity.

[0096] Hydrogen peroxide conditions: Add 17 μL of hydrogen peroxide solution (10 mM), 10 μL of product solution (3.3 mg / mL), and 1 μL of green fluorescent probe (5 mM) to 305 μL of ultrapure water, mix well, and let stand for 5 h. Record the fluorescence intensity of the system at 525 nm before and after standing using a fluorescence spectrometer, and calculate the rate of change of fluorescence intensity.

[0097] Ultrasonic treatment with hydrogen peroxide: Add 17 μL of hydrogen peroxide solution (10 mM), 10 μL of product solution (3.3 mg / mL), and 1 μL of green fluorescent probe (5 mM) to 305 μL of ultrapure water, mix thoroughly, let stand for 5 h, and then sonicate for 10 min. Record the fluorescence intensity of the system at 525 nm before and after standing and sonication using a fluorescence spectrometer, and calculate the rate of change in fluorescence intensity.

[0098] III. Analysis of Test Results for Each Embodiment and Comparative Example

[0099] The surface morphology of the mesoporous polydopamine drug-loaded microspheres (MPDA-PEG@PPIX / Cu and MPDA-PEG@PC / Fe) prepared in Examples 1-2 was tested according to test method (1). Please refer to [link to test method]. 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 pores on the surface, which are used to adsorb sound-sensing agents and transition metal ions. This structure enhances the functionality of the material in catalysis and biomedical applications, making it more efficient in ultrasonic catalysis and Fenton-like reaction catalysis.

[0100] The particle size of the products (MPDA-PEG@PPIX / Cu, MPDA, MPDA-PEG, MPDA-PEG@PPIX) prepared in Example 1 and Comparative Examples 1-3 was tested according to test method (2). The test results are shown in [reference]. Figure 2 As shown in the table below, the hydrophilic modification significantly reduced the particle size of the mesoporous polydopamine nanoparticles, and the loading of PPIX and Cu... 2+ The size further decreases. This is because hydrophilic modification introduces hydrophilic groups onto the nanoparticle surface. These groups can form hydrogen bonds with water molecules, making the surface more hydrophilic. This helps stabilize the suspension of the nanoparticles, allowing them to disperse better in aqueous media. The introduction of PPIX may have led to a fine-tuning of the nanoparticle surface structure. This structural adjustment provides greater structural compactness, resulting in a smaller particle size in DLS measurements. 2+ The introduction of [a specific substance] further alters the charge density and distribution on the surface of nanoparticles. Through electrostatic interactions and steric hindrance, it enhances the mutual repulsion between particles, reduces the tendency to aggregate, and further improves dispersibility by forming coordination bonds with functional groups such as carboxyl groups on the surface of nanoparticles, resulting in smaller hydrated particle sizes in the solution.

[0101] Example 1 MPDA-PEG@PPIX / Cu 341 Comparative Example 1 MPDA 1106 Comparative Example 2 MPDA-PEG 458 Comparative Example 3 MPDA-PEG@PPIX 396

[0102] The consumption of glutathione and particle size changes of the mesoporous polydopamine-loaded microspheres (MPDA-PEG@PPIX / Cu and MPDA-PEG@PC / Fe) prepared in Examples 1-2 were tested according to test method (3) under acidic conditions (pH=5.5). Please refer to [link to test method (3)]. Figure 3 As shown, c is the absorbance curve of residual glutathione in the solution after treatment with MPDA-PEG@PPIX / Cu for 0h, 1h, 2h, 3h, 4h, 5h, and 6h, respectively; d is the particle size change of MPDA-PEG@PPIX / Cu after treatment with MPDA-PEG@PPIX / Cu for 6h; e is the absorbance curve of residual glutathione in the solution after treatment with MPDA-PEG@PC / Fe for 0h, 1h, 2h, 3h, 4h, 5h, and 6h, respectively; and f is the particle size change of MPDA-PEG@PC / Fe after treatment with MPDA-PEG@PC / Fe for 6h.

[0103] The lower the absorbance at the absorption peak of 442 nm, the lower the concentration of glutathione in the solution. It can be seen that the concentration of glutathione in the solution gradually decreases with prolonged treatment time, especially after 6 hours, indicating that the nanospheres have the ability to consume glutathione. The functional groups of polydopamine can undergo redox reactions with glutathione, thereby promoting its degradation, and this effect increases with time. Figures d and f show that MPDA-PEG@PPIX / Cu or MPDA-PEG@PC / Fe both undergo cleavage after reacting with glutathione, resulting in a smaller hydrated particle size. This is because MPDA, as the matrix, contains abundant phenolic and amino groups. These functional groups readily undergo redox reactions with the strong reducing agent glutathione, leading to the reduction of MPDA. This reaction alters the structure of MPDA at the molecular level, affecting the overall stability and polymerization state of the drug-loaded microspheres, resulting in partial cleavage of the microsphere framework.

[0104] The performance of the products (MPDA-PEG@PPIX / Cu, MPDA-PEG@PC / Fe, MPDA-PEG@PPIX, MPDA-PEG@PC, MPDA-PEG@Cu and MPDA-PEG@Fe) prepared in Examples 1-2 and Comparative Examples 3-6 in generating reactive oxygen species such as hydroxyl radicals and singlet oxygen was tested under different conditions according to test method (4). The test results are shown in […]. Figure 4 As shown in Table 2, the change in absorbance of the indicator o-phenylenediamine at 410 nm represents the rate of change in the concentration of reactive oxygen species such as hydroxyl radicals and singlet oxygen in the system.

[0105] Table 2. Changes in absorbance of o-phenylenediamine at 410 nm after treatment for a certain period of time.

[0106]

[0107] Please see Figure 4 As shown in Table 2, g represents the change in absorbance at 410 nm of the systems MPDA-PEG@PPIX / Cu, MPDA-PEG@PPIX, and MPDA-PEG@Cu after treatment with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different times, respectively; h represents the change in absorbance at 410 nm of the systems MPDA-PEG@PC / Fe, MPDA-PEG@PC, and MPDA-PEG@Fe after treatment with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different times, respectively. As can be seen, when only ultrasonic treatment is used, the absorbance changes of the acoustic sensitizer + metal ion system and the acoustic sensitizer system increase, and the values ​​are similar, both much higher than the absorbance change of the metal ion system. When only hydrogen peroxide treatment is used, the absorbance changes of the acoustic sensitizer + metal ion system and the metal ion system increase, and the values ​​are similar, both much higher than the absorbance change of the acoustic sensitizer system. When ultrasonic + hydrogen peroxide treatment is used, the absorbance change of the acoustic sensitizer + metal ion system increases significantly. Although the absorbance changes of the acoustic sensitizer system and the metal ion system also increase, the absorbance change of the acoustic sensitizer + metal ion system is significantly higher than that of the other two systems.

[0108] The above analysis demonstrates that systems containing acoustic sensitizers can effectively generate large amounts of reactive oxygen species under ultrasonic excitation, while systems containing metal ions can generate hydroxyl radicals through a (Fenton-like) reaction in the presence of hydrogen peroxide. Furthermore, after treatment with both ultrasound and hydrogen peroxide, the acoustic sensitizer + metal ion system, under the effective excitation of the acoustic sensitizer and the synergistic catalytic effect of the metal ions, jointly enhances the generation efficiency of hydroxyl radicals and other reactive oxygen species, resulting in the acoustic sensitizer + metal ion system exhibiting the largest absorbance change value in the o-phenylenediamine test.

[0109] The performance of the products (MPDA-PEG@PPIX / Cu, MPDA-PEG@PC / Fe, MPDA-PEG@PPIX, MPDA-PEG@PC, MPDA-PEG@Cu and MPDA-PEG@Fe) prepared in Examples 1-2 and Comparative Examples 3-6 in generating singlet oxygen under different conditions was tested according to test method (5). The test results are shown in [Figure 5]. Figure 5 As shown in the table below, the rate of change in fluorescence intensity of the indicator SOSG at 525 nm represents the rate of change in the concentration of singlet oxygen in the system.

[0110] Table 3. Change rate (%) of SOSG fluorescence intensity at 525 nm in the system after treatment for a certain period of time

[0111]

[0112] Please see Figure 5 As shown in Table 3, i represents the fluorescence intensity change rate at 525 nm of the MPDA-PEG@PPIX / Cu, MPDA-PEG@PPIX, and MPDA-PEG@Cu systems after treatment with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different times, respectively; j represents the fluorescence intensity change rate at 525 nm of the MPDA-PEG@PC / Fe, MPDA-PEG@PC, and MPDA-PEG@Fe systems after treatment with ultrasound, hydrogen peroxide, and ultrasound + hydrogen peroxide for different times, respectively. It can be seen that after ultrasound treatment, the fluorescence intensity change rate at 525 nm of the sonication agent + metal ion system and the sonication agent system is much higher than that of the metal ion system. This indicates that the system containing the sonication agent can effectively generate a large amount of singlet oxygen under ultrasonic excitation, thus exhibiting a high fluorescence intensity change rate under SOSG fluorescent probe detection.

[0113] Please see Figure 6 The image shows the antibacterial effect of Experiments 1-8. It can be seen that after adding mesoporous polydopamine-loaded microspheres and hydrogen peroxide to a solution containing *E. coli* / *Staphylococcus aureus*, and then subjecting it to ultrasonic treatment, the *E. coli* / *Staphylococcus aureus* in the solution were almost completely killed. This indicates that ultrasound can cause the mesoporous polydopamine-loaded microspheres to generate a large amount of reactive oxygen species. Reactive oxygen species have excellent bactericidal effects and can lead to bacterial death.

[0114] In summary, this application provides a mesoporous polydopamine drug-loaded microsphere based on the synergistic antibacterial effect of acoustic and chemical dynamics, its preparation method, and its application. The mesoporous polydopamine drug-loaded microsphere is obtained by introducing a acoustic sensitizer and transition metal ions onto a mesoporous polydopamine matrix. This allows the drug-loaded microsphere to not only scavenge the antioxidant glutathione but also generate reactive oxygen species such as hydroxyl radicals and singlet oxygen through Fenton-like reactions and ultrasonic excitation, achieving the antibacterial purpose. Furthermore, the synergistic antibacterial effect of acoustic and chemical dynamics, along with the cleavage of the mesoporous polydopamine drug-loaded microsphere during the antibacterial process, significantly enhances the antibacterial effect.

[0115] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for preparing mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics, characterized in that, Includes the following steps: S1, dissolve polyether F127 and dopamine hydrochloride in an ethanol-water solution and stir vigorously until clear. Then, slowly add 1,3,5-trimethylbenzene dropwise while stirring. Continue stirring for 30-40 min, then add concentrated ammonia dropwise while stirring. Continue stirring for 1-1.5 h, then wash three times with ethanol and deionized water respectively to obtain a mesoporous polydopamine nanoparticle dispersion. S2, adjust the pH of the mesoporous polydopamine nanoparticle dispersion from step S1 to 11-12 with sodium hydroxide aqueous solution, add mercapto-polyethylene glycol-carboxyl aqueous solution dropwise under stirring, and obtain hydrophilic modified mesoporous polydopamine nanoparticle dispersion after stirring for 2-3 h. S3, under stirring, the aqueous solution of the acoustic sensitizer is added dropwise to the hydrophilic modified mesoporous polydopamine nanoparticle dispersion of step S2, and stirred in the dark for 8-12 h. Then, an aqueous solution containing transition metal ions is added dropwise, and the mixture is stirred for 1.5-3 h. After centrifugation, mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics are obtained. The total mass of the acoustic sensitizer 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 acoustic sensitizer to the transition metal ions is 1:(0.5-1). In step S1, 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 the ethanol-water solution to 1,3,5-trimethylbenzene is (45-55):1; and the concentration of dopamine hydrochloride in the ethanol-water solution is (3-7) mg / mL. The polydopamine in the mesoporous polydopamine drug-loaded microspheres will undergo a degradation reaction with GSH. During the consumption of GSH, the mesoporous polydopamine drug-loaded microspheres will also be pyrolyzed, which is conducive to the better release of the sonosensitive agent and transition metal ions by the mesoporous polydopamine. The mesoporous polydopamine drug-loaded microspheres lyse under the action of glutathione in the bacterial infection microenvironment, releasing the sonosensitive agent and the transition metal ions; the transition metal ions catalyze hydrogen peroxide to generate hydroxyl radicals and oxygen, and the oxygen supplies the sonosensitive agent to generate reactive oxygen species under the action of ultrasound, thereby achieving synergistic antibacterial action of sonodynamics and chemodynamics. The transition metal in the transition metal ion is one of copper, iron, manganese, cobalt, and nickel.

2. The method for preparing mesoporous polydopamine-loaded microspheres based on the synergistic antibacterial effects of acoustic and chemical dynamics according to claim 1, characterized in that, In step S3, the sound sensitizer is one of protoporphyrin, chlorophyll, cyanin, or rose red; the sound sensitizer solution is prepared by dissolving the sound sensitizer in dimethyl sulfoxide at a concentration of (5-15) mg / mL, and then diluting it 10 times with deionized water; 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 the synergistic antibacterial effects of acoustic and chemical dynamics according to claim 1, characterized in that, In step S1, after adding concentrated ammonia, the pH of the reaction system is 8-10.

4. The method for preparing mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effect of acoustic and chemical dynamics 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 mercapto-polyethylene glycol-carboxyl aqueous solution is (10-15) mg / mL, and the mass ratio of dopamine hydrochloride to mercapto-polyethylene glycol-carboxyl is 1:(0.1-0.2).

5. The method for preparing mesoporous polydopamine-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics according to claim 2, characterized in that, In step S3, the centrifugation speed is 11000-18000 rpm.

6. A mesoporous polydopamine-loaded microsphere based on synergistic antibacterial action of acoustic and chemical dynamics, characterized in that, The mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial action of acoustic and chemical dynamics are prepared according to any one of claims 1-5.

7. The mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics according to claim 6, characterized in that, The mesoporous polydopamine drug-loaded microspheres use mesoporous polydopamine as a carrier to load a sonosensitive agent and transition metal ions; the sonosensitive agent is one of protoporphyrin, chlorophyll, anthocyanin or rose red.

8. The mesoporous polydopamine drug-loaded microspheres based on synergistic antibacterial action of acoustic and chemical dynamics according to claim 7, characterized in that, The average particle size of the mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial action of acoustic and chemical dynamics is 120-180 nm.

9. The application of the mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effect of acoustic and chemodynamic processes prepared by the preparation method according to any one of claims 1-5, or the application of the mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial effect of acoustic and chemodynamic processes according to any one of claims 6-8, characterized in that, The application of the mesoporous polydopamine drug-loaded microspheres based on the synergistic antibacterial action of acoustic and chemical kinetics in the preparation of drugs for the prevention or non-invasive treatment of deep bacterial infections, wherein the bacteria are Escherichia coli and / or Staphylococcus aureus.

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