Nanometer assembly and preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to a nano assembly as well as a preparation method and application thereof. The nano assembly is formed by assembling the following three components: an amphiphilic block polymer, an antibiotic and a sound-sensitive agent, wherein the antibiotic is a hydrophobic antibiotic, the sound-sensitive agent is a porphyrin sound-sensitive agent, the structural formula of the amphiphilic block polymer is as follows: # imgabs0 #, m is an integer from 40 to 120, and n is an integer from 50 to 100. The nano assembly has excellent sonodynamic performance, antibacterial performance, pH responsiveness and nuclear magnetic resonance imaging capability, and has wide application prospects in treatment of bacterial and fungal infection, bacterial biofilm infection and nuclear magnetic resonance imaging.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a nano-assembly and its preparation method and application. Background Art

[0002] A biofilm is a film-like structure formed by a polymeric matrix secreted extracellularly by microorganisms adhering to the surface of a medium to enclose themselves. When bacteria form a biofilm, the bacteria inside the film are not easily cleared by the host immune system due to the shielding effect of the biofilm, reducing the antibacterial activity of antibiotics and leading to a significant increase in bacterial drug resistance. Therefore, it is of great significance to develop a treatment strategy that can effectively destroy the biofilm structure, enhance the antibacterial activity of antibiotics, and reduce bacterial drug resistance.

[0003] In recent years, the rapid development of nanotechnology has provided new ideas for the treatment of bacterial biofilm infections. Many nanomaterials have been developed to destroy the biofilm structure and enhance the penetration of antibiotics inside the biofilm to improve the antibacterial activity of antibiotics. However, existing nanomaterials still have problems with insufficient antibacterial performance, limiting their applications.

[0004] Sonodynamic therapy (SDT) is a new non-invasive treatment method based on the synergistic effect of a sonosensitizer and ultrasound. Its principle is to specifically generate reactive oxygen species (ROS) at the tumor site under the action of ultrasound and the sonosensitizer, thereby clearing rapidly dividing tumor cells. Compared with photodynamic therapy (PDT), SDT has deeper tissue penetration ability and higher safety, and has been widely used in the field of tumor treatment. Research shows that ROS, as a strong oxidant, has a wide range of bactericidal abilities, can effectively eliminate bacteria, spores, viruses, fungi (such as molds), also has a significant killing effect on protozoa and their oocysts, and can destroy bacterial toxins and hepatitis B surface antigen, etc.

[0005] Therefore, combining sonodynamic therapy (SDT) with nanotechnology to develop a nanomaterial with excellent sonodynamic performance and antibacterial performance is an urgent problem to be solved. Summary of the Invention

[0006] The present invention provides a nano-assembly, which has excellent sonodynamic performance, antibacterial performance, pH responsiveness, and nuclear magnetic resonance imaging ability.

[0007] The present invention also provides a preparation method of the nano-assembly. Through this method, the above-mentioned nano-assembly with excellent sonodynamic performance, antibacterial performance, pH responsiveness, and nuclear magnetic resonance imaging ability can be prepared.

[0008] The present invention also provides an application of the above-mentioned nano-assembly or a nano-assembly prepared by the preparation method of the above-mentioned nano-assembly in the preparation of products for treating bacterial and fungal infections or in the preparation of products for treating biofilm infections. Through the research of the inventor, it is shown that: when the nano-assembly is used in the antibacterial activity tests of Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and molds resistant to levofloxacin, under the action of ultrasonic waves, it releases a photosensitizer and an antibiotic, and kills bacteria through the sonodynamic performance (releasing ROS) of the photosensitizer and the antibacterial performance of the antibiotic, showing strong antibacterial activity. After killing Klebsiella pneumoniae resistant to levofloxacin, the survival rate of Klebsiella pneumoniae resistant to levofloxacin is 8%, after killing Escherichia coli, the survival rate of Escherichia coli is 9%, after killing Staphylococcus aureus, the survival rate of Staphylococcus aureus is 9%, and after killing molds, the survival rate of molds is 11%. This shows that the nano-assembly can effectively reduce bacterial drug resistance, kill bacteria and fungi, and has excellent antibacterial activity against both bacteria and fungi. Therefore, it can be used in the preparation of products for treating bacterial and / or fungal infections; when the nano-assembly is used in the bacterial biofilm inhibition test, under the action of ultrasonic waves, it can effectively remove the bacterial biofilm, destroy the structure of the bacterial biofilm, release a photosensitizer and an antibiotic, and kill bacteria through the sonodynamic performance (releasing ROS) of the photosensitizer and the antibacterial performance of the antibiotic. Therefore, it can be used in the preparation of products for treating bacterial biofilm infections.

[0009] The present invention also provides an application of the above-mentioned nano-assembly or a nano-assembly prepared by the preparation method of the above-mentioned nano-assembly in the preparation of a magnetic resonance imaging contrast agent. Through the research of the inventor, it is shown that: when the nano-assembly is used in the magnetic resonance imaging test, it can respond to the acidic microenvironment, significantly improve the signal-to-noise ratio (SNR) of T1-weighted magnetic resonance imaging, and achieve signal enhancement. Therefore, it can be used in the preparation of a magnetic resonance imaging contrast agent.

[0010] The first aspect of the present invention provides a nano-assembly, which is assembled from the following three components:

[0011] An amphiphilic block polymer, an antibiotic, a photosensitizer; wherein, the structural formula of the amphiphilic block polymer is shown in Formula I, the antibiotic is a hydrophobic antibiotic, and the photosensitizer is a porphyrin-based photosensitizer;

[0012] Formula I

[0013] In Formula I, m is an integer from 40 to 120, and n is an integer from 50 to 100.

[0014] For the above-mentioned nano-assembly, the particle size of the nano-assembly is 50 - 200 nm.

[0015] The nano-assembly as described above, in the nano-assembly, the mass ratio of the amphiphilic block polymer, the antibiotic and the photosensitizer is (10-20):(1-3):(1-5).

[0016] The nano-assembly as described above, the hydrophobic antibiotics include at least one of levofloxacin, tetracycline, chloramphenicol, ciprofloxacin, azithromycin, doxycycline, linezolid, norfloxacin;

[0017] And / or, the porphyrin photosensitizer includes manganese(II) 5,10,15,20-tetraphenyl-21H,23H-porphyrin.

[0018] The second aspect of the present invention provides a preparation method of the nano-assembly as described above, including the following steps:

[0019] Dissolve the amphiphilic block polymer, the antibiotic and the photosensitizer in an organic solvent respectively to prepare an amphiphilic block polymer mother liquor, an antibiotic mother liquor and a photosensitizer mother liquor;

[0020] Mix the amphiphilic block polymer mother liquor, the antibiotic mother liquor and the photosensitizer mother liquor with PBS solution, and then carry out stirring treatment and dialysis treatment to obtain the nano-assembly.

[0021] The preparation method of the nano-assembly as described above, the concentration of the amphiphilic block polymer in the amphiphilic block polymer mother liquor is 14-18 mg / mL, the concentration of the antibiotic in the antibiotic mother liquor is 1.5-3 mg / mL, and the concentration of the photosensitizer in the photosensitizer mother liquor is 1.5-3 mg / mL;

[0022] The volume ratio of the amphiphilic block polymer mother liquor, the antibiotic mother liquor and the photosensitizer mother liquor is (1-2):1:1.

[0023] The preparation method of the nano-assembly as described above, in the stirring treatment, the rotation speed is 1000-1500 rpm / min, the temperature is 20-30 °C, and the time is 5-10 min;

[0024] And / or, in the dialysis treatment, the cut-off molecular weight of the dialysis bag used is 3000-20000 Da, and the dialysis time is 8-12 h;

[0025] And / or, the organic solvent includes dimethyl sulfoxide.

[0026] The preparation method of the nano-assembly as described above, the amphiphilic block polymer is obtained by a preparation method including the following process:

[0027] Dissolve 2-(azepan-1-yl)ethyl methacrylate, a macromolecular chain transfer agent PEG-CTA, and an initiator in dimethyl sulfoxide to obtain a mixed solution;

[0028] Carry out a polymerization reaction on the mixed solution at 50 - 80 °C for 6 - 12 h to obtain the amphiphilic block polymer;

[0029] Wherein the initiator includes azobisisobutyronitrile;

[0030] The mass ratio of the 2-(azepan-1-yl)ethyl methacrylate, the macromolecular chain transfer agent PEG-CTA, and the initiator is (50 - 150):1:(0.1 - 0.3).

[0031] The third aspect of the present invention provides an application of the described nano-assembly or a nano-assembly prepared by the preparation method of the described nano-assembly in the preparation of products for treating bacterial and fungal infections or in the preparation of products for treating bacterial biofilm infections.

[0032] The fourth aspect of the present invention provides an application of the described nano-assembly or a nano-assembly prepared by the preparation method of the described nano-assembly in the preparation of magnetic resonance imaging contrast agents.

[0033] The solution of the present invention has at least the following effects:

[0034] The nano-assembly provided by the present invention has excellent sonodynamic performance and pH responsiveness. It generates reactive oxygen species (ROS) under the action of ultrasonic waves, and it can respond to the acidic microenvironment and generate more ROS under the action of ultrasound; this nano-assembly has excellent sonodynamic performance and antibacterial performance. Under the action of ultrasonic waves, it can effectively remove bacterial biofilms, destroy the structure of bacterial biofilms, release photosensitizers and antibiotics, and kill bacteria through the sonodynamic performance (releasing ROS) of the photosensitizer and the antibacterial performance of the antibiotic, and can be used to prepare products for treating bacterial biofilm infections; and under the action of ultrasonic waves, this nano-assembly releases photosensitizers and antibiotics, and through the sonodynamic performance (releasing ROS) of the photosensitizer and the antibacterial performance of the antibiotic, it can effectively kill Klebsiella pneumoniae resistant to levofloxacin, Escherichia coli, Staphylococcus aureus, and molds, showing strong antibacterial activity, indicating that this nano-assembly can effectively reduce bacterial drug resistance and has excellent antibacterial activity against both bacteria and fungi. Therefore, it can be used to prepare products for treating bacterial and fungal infections; this nano-assembly has excellent pH-responsive nuclear magnetic resonance imaging ability. It can respond to the acidic microenvironment, significantly improve the signal-to-noise ratio (SNR) of T1-weighted magnetic resonance imaging, and achieve signal enhancement. Therefore, it can be used to prepare magnetic resonance imaging contrast agents and has broad application prospects in magnetic resonance imaging. Description of the Drawings

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0036] Figure 1 1H NMR spectrum of the amphiphilic block polymer in Example 1 of the present invention;

[0037] Figure 2 UV absorption spectrum of the nano-assembly in Example 1 of the present invention;

[0038] Figure 3 Particle size distribution diagram and transmission electron microscope (TEM) image of the nano-assembly in Example 1 of the present invention, where Figure 3 a is the particle size distribution diagram of the nano-assembly in Example 1, Figure 3 b is the TEM image of the nano-assembly in Example 1;

[0039] Figure 4 ROS release curves of LEV+MnTCPP@PC7A solutions with different pH values before and after ultrasonic treatment, where Figure 4 a is the ROS release curve of the LEV+MnTCPP@PC7A solution with pH = 6.0 before and after ultrasonic treatment, Figure 4 b is the ROS release curve of the LEV+MnTCPP@PC7A solution with pH = 7.4 before and after ultrasonic treatment;

[0040] Figure 5 Antibacterial activity test, where Figure 5 a is the antibacterial activity of the nano-assembly against Klebsiella pneumoniae resistant to levofloxacin, Figure 5 b is the antibacterial activity of the nano-assembly against Escherichia coli, Figure 5 c is the antibacterial activity of the nano-assembly against Staphylococcus aureus, Figure 5 d is the antibacterial activity of the nano-assembly against molds;

[0041] Figure 6 Crystal violet staining result diagram in the biofilm inhibition test;

[0042] Figure 7 SEM test result diagram in the biofilm inhibition test;

[0043] Figure 8 Magnetic resonance imaging test; where Figure 8a is the result of processing the scanned images of nano-assembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at different pH values using Sante DICOM software. Figure 8 b is the signal-to-noise ratio (SNR) of magnetic resonance of nano-assembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at different pH values. Detailed implementation manners

[0044] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention. For the embodiments not specifying specific technologies or conditions, the technologies or conditions described in the literature in the field or according to the product specifications shall be followed. The reagents or instruments not indicating the manufacturer can be conventional products obtained through commercial purchase.

[0045] It should be noted that "and / or" in the specification indicates at least one of the connected objects. The character " / " generally indicates an "or" relationship between the associated objects before and after.

[0046] In the present invention, the sonodynamic performance refers to the ability of a sonosensitizer to generate reactive oxygen species (ROS) under the action of ultrasonic waves.

[0047] The first aspect of the present invention provides a nano-assembly, which is assembled from the following three components:

[0048] An amphiphilic block polymer, an antibiotic, and a sonosensitizer; wherein, the structural formula of the amphiphilic block polymer is as shown in Formula I, the antibiotic is a hydrophobic antibiotic, and the sonosensitizer is a porphyrin-based sonosensitizer;

[0049] Formula I

[0050] In Formula I, m is an integer from 40 to 120, and n is an integer from 50 to 100.

[0051] The nanoassemblies of the present invention are assembled from amphiphilic block polymers, antibiotics, and sonosensitizers, and the nanoassemblies have excellent sonodynamic performance, antibacterial performance, pH responsiveness, and nuclear magnetic resonance imaging capabilities. Research by the inventors has shown that: (1) The nanoassemblies can generate reactive oxygen species (ROS) under the action of ultrasonic waves, and they can respond to acidic microenvironments and generate more ROS under the action of ultrasound. Therefore, they have excellent sonodynamic performance and pH responsiveness; (2) The nanoassemblies can effectively remove bacterial biofilms and destroy the structure of bacterial biofilms under the action of ultrasonic waves, releasing sonosensitizers and antibiotics. Bacteria are killed through the sonodynamic performance (releasing ROS) of the sonosensitizers and the antibacterial performance of the antibiotics. Moreover, under the action of ultrasonic waves, the nanoassemblies can also effectively kill Klebsiella pneumoniae resistant to levofloxacin, Escherichia coli, Staphylococcus aureus, and molds, showing strong antibacterial activity. This shows that the nanoassemblies can effectively reduce bacterial drug resistance and have excellent antibacterial activity against both bacteria and fungi. Therefore, they have excellent sonodynamic performance and antibacterial performance; (3) The nanoassemblies can respond to acidic microenvironments, significantly improving the signal-to-noise ratio (SNR) of T1-weighted magnetic resonance imaging and achieving signal enhancement. Therefore, they have pH responsiveness and nuclear magnetic resonance imaging capabilities.

[0052] In a specific embodiment, the particle size of the above nanoassemblies is 50-200 nm. When the particle size of the nanoassemblies is within the above range, the nanoassemblies can effectively enter the biofilm and remove bacteria.

[0053] In a specific embodiment, in the above nanoassemblies, the mass ratio of the amphiphilic block polymer, antibiotic, and sonosensitizer is (10-20):(1-3):(1-5).

[0054] When the mass ratio of the amphiphilic block polymer, antibiotic, and sonosensitizer in the nanoassemblies is within the above range, the amphiphilic block polymer, antibiotic, and sonosensitizer are better matched, thereby obtaining nanoassemblies with excellent sonodynamic performance, antibacterial performance, pH responsiveness, and nuclear magnetic resonance imaging capabilities.

[0055] In a specific embodiment, the above hydrophobic antibiotics include at least one of levofloxacin, tetracycline, chloramphenicol, ciprofloxacin, azithromycin, doxycycline, linezolid, and norfloxacin.

[0056] In a specific embodiment, the above porphyrin-based sonosensitizer includes manganese(II) 5,10,15,20-tetraphenyl-21H,23H-porphyrin.

[0057] The second aspect of the present invention provides a preparation method of the above nanoassemblies, including the following steps:

[0058] Dissolve the amphiphilic block polymer, antibiotic, and sonosensitizer in organic solvents respectively to prepare an amphiphilic block polymer mother liquor, an antibiotic mother liquor, and a sonosensitizer mother liquor.

[0059] Mix the amphiphilic block polymer mother liquor, the antibiotic mother liquor, the sonosensitizer mother liquor with PBS solution, and then carry out stirring treatment and dialysis treatment to obtain the nano-assembly.

[0060] In a specific embodiment, the concentration of the amphiphilic block polymer in the above amphiphilic block polymer mother liquor is 14 - 18 mg / mL. For example, the concentration of the amphiphilic block polymer in the amphiphilic block polymer mother liquor is 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, etc.; the concentration of the antibiotic in the above antibiotic mother liquor is 1.5 - 3 mg / mL. For example, the concentration of the antibiotic in the antibiotic mother liquor is 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, etc.; the concentration of the sonosensitizer in the above sonosensitizer mother liquor is 1.5 - 3 mg / mL. For example, the concentration of the sonosensitizer in the sonosensitizer mother liquor is 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, etc.

[0061] When the concentration of the amphiphilic block polymer in the amphiphilic block polymer mother liquor, the concentration of the antibiotic in the antibiotic mother liquor, and the concentration of the sonosensitizer in the sonosensitizer mother liquor are each within the above ranges, the finally prepared nano-assembly can effectively kill bacteria.

[0062] In a specific embodiment, the volume ratio of the above amphiphilic block polymer mother liquor, the above antibiotic mother liquor, and the above sonosensitizer mother liquor is (1 - 2):1:1.

[0063] In a specific embodiment, in the above stirring treatment, the rotation speed is 1000 - 1500 rpm / min, the temperature is 20 - 30 °C, and the time is 5 - 10 min.

[0064] When in the stirring treatment, the parameters of the rotation speed, temperature, and time are each within the above ranges, the amphiphilic block polymer, the antibiotic solution, and the sonosensitizer solution can be fully assembled with each other, so as to prepare a nano-assembly with excellent sonodynamic performance, antibacterial performance, pH responsiveness, and nuclear magnetic resonance imaging ability.

[0065] Exemplarily, in the above stirring treatment, the rotation speed can be any one of 1000 rpm / min, 1100 rpm / min, 1200 rpm / min, 1300 rpm / min, 1400 rpm / min, 1500 rpm / min or the range composed of any two of them;

[0066] A range composed of any one or any two of 20 °C, 22 °C, 24 °C, 26 °C, 28 °C, and 30 °C;

[0067] A range composed of any one or any two of 5 min, 6 min, 7 min, 8 min, 9 min, and 10 min.

[0068] In a specific embodiment, in the above dialysis treatment, the cut-off molecular weight of the dialysis bag used is 3000 - 20000 Da, and the dialysis time is 8 - 12 h.

[0069] When the parameters of the pore size of the dialysis bag and the dialysis time in the dialysis treatment are each within the above ranges, the loss of the loaded drugs (antibiotics and photosensitizers) is reduced and the organic solvent is removed to the greatest extent.

[0070] In a specific embodiment, the above organic solvent includes dimethyl sulfoxide.

[0071] In a specific embodiment, the above amphiphilic block copolymer is obtained by a preparation method including the following process:

[0072] Dissolve 2-(azepan-1-yl)ethyl methacrylate, a macromolecular chain transfer agent PEG-CTA, and an initiator in dimethyl sulfoxide to obtain a mixed solution;

[0073] Carry out a polymerization reaction on the mixed solution at 50 - 80 °C for 6 - 12 h to obtain the amphiphilic block copolymer.

[0074] The present invention can prepare an amphiphilic block copolymer through the above preparation method. Assembling the amphiphilic block copolymer with antibiotics and photosensitizers can prepare a nano-assembly with excellent sonodynamic performance, antibacterial performance, pH responsiveness, and nuclear magnetic resonance imaging ability.

[0075] The present invention does not particularly limit the specific preparation method of the above macromolecular chain transfer agent PEG-CTA, and it can be prepared according to the methods well-known to those skilled in the art. In some embodiments, the preparation method of the macromolecular chain transfer agent PEG-CTA is as follows:

[0076] Dissolve 2.5 mmol of methoxypolyethylene glycol in 50 mL of anhydrous tetrahydrofuran to obtain a methoxypolyethylene glycol solution;

[0077] Dissolve 5 mmol of 4-cyano-4-(((ethylthio)thioxomethyl)thio)pentanoic acid, 5 mmol of dicyclohexylcarbodiimide, and 0.5 mmol of 4-dimethylaminopyridine in 40 mL of anhydrous tetrahydrofuran to obtain a mixture solution;

[0078] The mixture solution is mixed with the polyethylene glycol monomethyl ether solution, and then subjected to an esterification reaction under stirring at 25 °C for 72 h to obtain a reaction product;

[0079] The reaction product is successively subjected to first filtration, vacuum distillation, precipitation, second filtration and vacuum drying to obtain the macromolecular chain transfer agent PEG-CTA.

[0080] In a specific embodiment, the initiator includes azobisisobutyronitrile.

[0081] In a specific embodiment, the mass ratio of the above-mentioned 2-(azepan-1-yl)ethyl methacrylate, the above-mentioned macromolecular chain transfer agent PEG-CTA and the above-mentioned initiator is (50-150):1:(0.1-0.3).

[0082] The third aspect of the present invention provides an application of the above-mentioned nano-assembly or a nano-assembly prepared by the preparation method of the above-mentioned nano-assembly in the preparation of products for treating bacterial and fungal infections or in the preparation of products for treating bacterial biofilm infections. The inventors' research shows that: under the action of ultrasonic waves, the nano-assembly can effectively reduce bacterial drug resistance and has excellent antibacterial activity against both bacteria and molds. Therefore, it can be used to prepare products for treating bacterial and / or fungal infections; under the action of ultrasonic waves, the nano-assembly can effectively remove bacterial biofilms, destroy the structure of bacterial biofilms, release photosensitizers and antibiotics, and kill bacteria through the sonodynamic performance (release of ROS) of the photosensitizer and the antibacterial performance of the antibiotics. Therefore, it can be used to prepare products for treating bacterial biofilm infections.

[0083] The fourth aspect of the present invention provides an application of the above-mentioned nano-assembly or a nano-assembly prepared by the preparation method of the above-mentioned nano-assembly in the preparation of magnetic resonance imaging contrast agents. The inventors' research shows that: when the nano-assembly is used in magnetic resonance imaging experiments, it can respond to the acidic microenvironment, significantly improve the magnetic resonance signal-to-noise ratio (SNR) of T1-weighted magnetic resonance imaging, and achieve signal enhancement. Therefore, it can be used to prepare magnetic resonance imaging contrast agents.

[0084] Hereinafter, the present invention will be further introduced through specific examples.

[0085] Example 1

[0086] This example provides a preparation method of a nano-assembly, including the following steps:

[0087] (1) Preparation of the macromolecular chain transfer agent PEG-CTA:

[0088] Dissolve polyethylene glycol monomethyl ether (2.5 mmol, 12.5 g) in 50 mL of anhydrous tetrahydrofuran to obtain a polyethylene glycol monomethyl ether solution;

[0089] Dissolve 4-cyano-4-(((ethylthio)thiocarbonyl)thio)pentanoic acid (5 mmol, 1.38 g), dicyclohexylcarbodiimide (5 mmol, 1.03 g) and 4-dimethylaminopyridine (0.5 mmol, 0.06 g) in 40 mL of anhydrous tetrahydrofuran to obtain a mixed solution;

[0090] Mix the mixed solution with a polyethylene glycol monomethyl ether solution and carry out an esterification reaction at 25 °C with stirring for 72 h to obtain a reaction product;

[0091] Carry out first filtration, vacuum distillation, precipitation, second filtration and vacuum drying on the reaction product in sequence to obtain a macromolecular chain transfer agent PEG-CTA (abbreviation: PEG-CTA); The chemical structural formula of PEG-CTA is as follows:

[0092] .

[0093] (2) Preparation of amphiphilic block polymer:

[0094] Dissolve 552 mg of 2-(azepan-1-yl)ethyl methacrylate, 207 mg of PEG-CTA and 2 mg of azobisisobutyronitrile (AIBN) in 500 μL of dimethyl sulfoxide, and carry out three freeze-thaw cycles to obtain a mixed solution;

[0095] Carry out a polymerization reaction on the mixed solution at 70 °C for 8 h to obtain a polymerized product; Add 2 mL of tetrahydrofuran to the polymerized product for dilution to obtain a diluted product; Carry out dialysis and freeze-drying treatments on the diluted product in sequence to obtain an amphiphilic block polymer (denoted as PC7A); The chemical structural formula of the amphiphilic block polymer is as follows:

[0096] , where m is 113 and n is 57.

[0097] (3) Preparation of nano-assembly:

[0098] Dissolve the amphiphilic block polymer (PC7A), levofloxacin (LEV) and manganese(II) 5,10,15,20-tetraphenyl-21H,23H-porphyrin in dimethyl sulfoxide (DMSO) respectively to prepare a PC7A mother liquor with a concentration of 16 mg / mL, a LEV mother liquor with a concentration of 1.8 mg / mL and a MnTCPP mother liquor with a concentration of 2 mg / mL;

[0099] Mix 0.3 mL of the PC7A mother liquor, 0.2 mL of the LEV mother liquor and 0.2 mL of the MnTCPP mother liquor to obtain a mixed solution;

[0100] Place the mixture solution in an assembled bottle containing 7 mL of phosphate buffer solution (PBS solution), and carry out stirring treatment for 5 min under the conditions of a rotation speed of 1500 rpm / min and a temperature of 20 °C to obtain the product after stirring treatment;

[0101] Use a dialysis bag with a pore size of 3000 Da to perform dialysis treatment on the product after stirring treatment for 12 h to obtain a nano-assembly (denoted as LEV+MnTCPP@PC7A).

[0102] Performance test:

[0103] 1. 1H NMR test

[0104] Perform 1H NMR test on the amphiphilic block polymer in Example 1 of the present invention, and the results are as Figure 1 shown; Figure 1 is the 1H NMR spectrum of the amphiphilic block polymer in Example 1 of the present invention.

[0105] It can be seen from Figure 1 that the amphiphilic block polymer with the structure shown in Formula I was successfully prepared.

[0106] 2. UV absorption spectrum test

[0107] Perform UV absorption spectrum test on the nano-assembly in Example 1, and the results are as Figure 2 shown; Figure 2 is the UV absorption spectrum of the nano-assembly in Example 1.

[0108] It can be seen from Figure 2 that the nano-assembly in Example 1 has two obvious absorption peaks at wavelengths of 330 nm and 470 nm. Among them, the absorption peak at 330 nm is the characteristic absorption spectrum peak of LEV, and the absorption peak at 470 nm is the characteristic absorption spectrum peak of MnTCPP.

[0109] 3. Particle size and morphology test

[0110] Perform particle size and morphology tests on the nano-assembly in Example 1 of the present invention respectively, and the results are as Figure 3 shown in a-b of; Figure 3 is the particle size distribution diagram and transmission electron microscope (TEM) diagram of the nano-assembly in Example 1 of the present invention, where Figure 3 a of is the particle size distribution diagram of the nano-assembly in Example 1, Figure 3 b of is the TEM diagram of the nano-assembly in Example 1.

[0111] It can be seen from Figure 3 a of that the particle size of the nano-assembly in Example 1 is 164 nm.

[0112] It can be seen fromFigure 3 As can be seen from b of Example 1, the nanoassemblies are circular particles.

[0113] 4. pH Responsiveness and Sonodynamic Performance Tests

[0114] The sonodynamic performance of the nanoassemblies in Example 1 of the present invention was tested. The specific method is as follows:

[0115] The nanoassemblies (LEV+MnTCPP@PC7A) in Example 1 were configured into a nanoassembly (LEV+MnTCPP@PC7A) solution with pH = 6.0 using phosphate buffer solution (PBS solution). The LEV+MnTCPP@PC7A solution with pH = 6.0 was used as experimental group A, and the phosphate buffer solution with pH = 6.0 was used as control group A;

[0116] The nanoassemblies (LEV+MnTCPP@PC7A) in Example 1 were configured into a LEV+MnTCPP@PC7A solution with pH = 7.4 using PBS solution. The LEV+MnTCPP@PC7A solution with pH = 7.4 was used as experimental group B, and the phosphate buffer solution with pH = 7.4 was used as control group B;

[0117] Under the action of 1.5 W ultrasonic wave, experimental group A, control group A, experimental group B and control group B were ultrasonically treated for 2 min respectively. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to measure the release amount of reactive oxygen species (ROS) in each group. During the ultrasonic treatment process, an excitation wavelength of 488 nm was used to record the change of fluorescence intensity in each group. The results are as Figure 4 shown in a and b; Figure 4 is the ROS release curve of LEV+MnTCPP@PC7A solutions with different pH values before and after ultrasonic treatment, where Figure 4 a is the ROS release curve of the LEV+MnTCPP@PC7A solution with pH = 6.0 before and after ultrasonic treatment, Figure 4 b is the ROS release curve of the LEV+MnTCPP@PC7A solution with pH = 7.4 before and after ultrasonic treatment.

[0118] From Figure 4As can be seen from a-b, under the action of 1.5 W ultrasonic waves, the release amounts of reactive oxygen species (ROS) in LEV+MnTCPP@PC7A solutions with different pH values are different; compared with the LEV+MnTCPP@PC7A solution with pH = 7.4, the LEV+MnTCPP@PC7A solution with pH = 6.0 generates more ROS with the prolongation of ultrasonic treatment time. The above results illustrate that the nano-assembly provided by the embodiment of the present invention can respond to an acidic microenvironment, generate more ROS under the action of ultrasonic waves, and has excellent sonodynamic performance and pH responsiveness.

[0119] 5. Antibacterial Activity Test

[0120] The antibacterial performance of the nano-assembly in Example 1 of the present invention was tested, and the specific method is as follows:

[0121] (1) Bacterial culture:

[0122] Klebsiella pneumoniae resistant to levofloxacin, Escherichia coli, and Staphylococcus aureus were respectively inoculated into fresh tryptone soy broth (TSB) medium and incubated at 37°C for 15 h to obtain bacterial suspensions; then 50 μL of the bacterial suspension was added to 5 mL of fresh TSB medium and incubated at 37°C until the bacteria reached the logarithmic growth phase. The bacteria were collected by centrifugation and washed twice with sterile phosphate buffer solution (PBS), and centrifugation was performed after each washing (10,000 rpm, for 5 min, with the temperature controlled at 4°C). After two washings, the bacterial concentration was adjusted to 1.5×10 6 CFU / mL to obtain Klebsiella pneumoniae resistant to levofloxacin suspension, Escherichia coli suspension, and Staphylococcus aureus suspension respectively.

[0123] (2) Fungal (mold) culture:

[0124] The mold was inoculated into yeast extract peptone dextrose medium (YPD), placed in a shaker at 30°C and 200 rpm / min, and cultured for 15 h. The mold cells were collected by centrifugation and diluted with sterile PBS to 2×10 5 CFU / mL to obtain a mold suspension.

[0125] (3) Antibacterial performance test of the nano-assembly:

[0126] 1. Antibacterial activity of the nano-assembly against levofloxacin-resistant Klebsiella pneumoniae: ① Experimental group without sonication (-US): Add 100 μL of the assembly to a 96-well plate, then add 50 μL of the levofloxacin-resistant Klebsiella pneumoniae suspension to each well, and incubate at 37 °C for 20 min to obtain the incubated levofloxacin-resistant Klebsiella pneumoniae suspension; continue to incubate the incubated levofloxacin-resistant Klebsiella pneumoniae suspension at 37 °C for 30 min, then dilute the bacterial suspension 100-fold with sterile PBS to obtain the diluted levofloxacin-resistant Klebsiella pneumoniae suspension; place 20 μL of the diluted levofloxacin-resistant Klebsiella pneumoniae suspension on a tryptic soy agar plate and incubate at 37 °C for 15 h to obtain the levofloxacin-resistant Klebsiella pneumoniae suspension treated with the nano-assembly (LEV+MnTCPP@PC7A);

[0127] Control group without sonication (-US): The procedure is basically the same as that of the experimental group without sonication (-US), except that 100 μL of the nano-assembly is replaced with 100 μL of phosphate buffer solution (PBS), so finally the levofloxacin-resistant Klebsiella pneumoniae suspension treated with PBS (PBS) is obtained;

[0128] ② Experimental group with sonication (+US): Add 100 μL of the nano-assembly to a 96-well plate, then add 50 μL of the levofloxacin-resistant Klebsiella pneumoniae suspension to each well, and incubate at 37 °C for 20 min to obtain the incubated levofloxacin-resistant Klebsiella pneumoniae suspension; sonicate the incubated levofloxacin-resistant Klebsiella pneumoniae suspension under ultrasonic waves at 1.5 W for 2 min, then continue to incubate at 37 °C for 30 min, and then dilute the bacterial suspension 100-fold with sterile PBS to obtain the diluted levofloxacin-resistant Klebsiella pneumoniae suspension; place 20 μL of the diluted levofloxacin-resistant Klebsiella pneumoniae suspension on a tryptic soy agar plate and incubate at 37 °C for 15 h to obtain the levofloxacin-resistant Klebsiella pneumoniae suspension treated with the nano-assembly (LEV+MnTCPP@PC7A);

[0129] Control group with sonication (+US): The procedure is basically the same as that of the experimental group with sonication (+US), except that 100 μL of the nano-assembly is replaced with 100 μL of phosphate buffer solution (PBS), so finally the levofloxacin-resistant Klebsiella pneumoniae suspension treated with PBS (PBS) is obtained;

[0130] Bacterial viability tests were performed on Klebsiella pneumoniae suspensions resistant to levofloxacin (LEV+MnTCPP@PC7A) treated with nanoassemblies in the experimental group without sonication (-US), Klebsiella pneumoniae suspensions resistant to levofloxacin (PBS) treated with PBS in the control group without sonication (-US), Klebsiella pneumoniae suspensions resistant to levofloxacin (LEV+MnTCPP@PC7A) treated with nanoassemblies in the experimental group with sonication (+US), and Klebsiella pneumoniae suspensions resistant to levofloxacin (PBS) treated with PBS in the control group with sonication (+US). The results are as Figure 5 shown in a of

[0131] 2. Antibacterial activity of nanoassemblies against Escherichia coli: ① Experimental group without sonication (-US): The procedure was basically the same as that of the experimental group without sonication (-US) in the antibacterial activity of nanoassemblies against Klebsiella pneumoniae resistant to levofloxacin, except that the Klebsiella pneumoniae suspension resistant to levofloxacin was replaced with an Escherichia coli suspension. Thus, an Escherichia coli suspension treated with nanoassemblies (LEV+MnTCPP@PC7A) was finally obtained;

[0132] Control group without sonication (-US): The procedure was basically the same as that of the experimental group without sonication (-US), except that 100 μL of nanoassemblies was replaced with 100 μL of phosphate buffer solution (PBS). Thus, an Escherichia coli suspension treated with PBS (PBS) was finally obtained;

[0133] ② Experimental group with sonication (+US): The procedure was basically the same as that of the experimental group with sonication (+US) in the antibacterial activity of nanoassemblies against Klebsiella pneumoniae resistant to levofloxacin, except that the Klebsiella pneumoniae suspension resistant to levofloxacin was replaced with an Escherichia coli suspension. Thus, an Escherichia coli suspension treated with nanoassemblies (LEV+MnTCPP@PC7A) was finally obtained;

[0134] Control group with sonication (+US): The procedure was basically the same as that of the experimental group with sonication (+US), except that 100 μL of nanoassemblies was replaced with 100 μL of phosphate buffer solution (PBS). Thus, an Escherichia coli suspension treated with PBS (PBS) was finally obtained;

[0135] Bacterial viability tests were performed on Escherichia coli suspensions treated with nanoassemblies (LEV+MnTCPP@PC7A) in the experimental group without sonication (-US), Escherichia coli suspensions treated with PBS in the control group without sonication (-US), Escherichia coli suspensions treated with nanoassemblies (LEV+MnTCPP@PC7A) in the experimental group with sonication (+US), and Escherichia coli suspensions treated with PBS in the control group with sonication (+US). The results are shown in Figure 5 as shown in b of

[0136] 3. Antibacterial activity of nanoassemblies against Staphylococcus aureus: ① Experimental group without sonication (-US): The procedure was basically the same as that of the experimental group without sonication (-US) in the antibacterial activity of nanoassemblies against levofloxacin-resistant Klebsiella pneumoniae, except that the levofloxacin-resistant Klebsiella pneumoniae suspension was replaced with a Staphylococcus aureus suspension. Thus, a Staphylococcus aureus suspension treated with nanoassemblies (LEV+MnTCPP@PC7A) was finally obtained;

[0137] Control group without sonication (-US): The procedure was basically the same as that of the experimental group without sonication (-US), except that 100 μL of nanoassemblies was replaced with 100 μL of phosphate buffer solution (PBS). Thus, a Staphylococcus aureus suspension treated with PBS (PBS) was finally obtained;

[0138] ② Experimental group with sonication (+US): The procedure was basically the same as that of the experimental group with sonication (+US) in the antibacterial activity of nanoassemblies against levofloxacin-resistant Klebsiella pneumoniae, except that the levofloxacin-resistant Klebsiella pneumoniae suspension was replaced with a Staphylococcus aureus suspension. Thus, a Staphylococcus aureus suspension treated with nanoassemblies (LEV+MnTCPP@PC7A) was finally obtained;

[0139] Control group with sonication (+US): The procedure was basically the same as that of the experimental group with sonication (+US), except that 100 μL of nanoassemblies was replaced with 100 μL of phosphate buffer solution (PBS). Thus, a Staphylococcus aureus suspension treated with PBS (PBS) was finally obtained;

[0140] Bacterial viability tests were performed on Staphylococcus aureus suspensions treated with nanoassemblies (LEV+MnTCPP@PC7A) in the experimental group without sonication (-US), Staphylococcus aureus suspensions treated with PBS in the control group without sonication (-US), Staphylococcus aureus suspensions treated with nanoassemblies (LEV+MnTCPP@PC7A) in the experimental group with sonication (+US), and Staphylococcus aureus suspensions treated with PBS in the control group with sonication (+US). The results are shown in Figure 5 as shown in c of

[0141] 4. Antifungal activity of nanoassemblies: ① Experimental group without sonication (-US): The procedure was basically the same as that of the experimental group without sonication (-US) in the antifungal activity of nanoassemblies against levofloxacin-resistant Klebsiella pneumoniae, except that the levofloxacin-resistant Klebsiella pneumoniae suspension was replaced with a mold suspension and the tryptone soy agar plate was replaced with a yeast extract peptone dextrose agar plate. Thus, a mold suspension treated with nanoassemblies (LEV+MnTCPP@PC7A) was finally obtained;

[0142] Control group without sonication (-US): The procedure was basically the same as that of the experimental group without sonication (-US), except that 100 μL of nanoassemblies was replaced with 100 μL of phosphate buffer solution (PBS). Thus, a mold suspension treated with PBS (PBS) was finally obtained;

[0143] ② Experimental group with sonication (+US): The procedure was basically the same as that of the experimental group with sonication (+US) in the antifungal activity of nanoassemblies against levofloxacin-resistant Klebsiella pneumoniae, except that the levofloxacin-resistant Klebsiella pneumoniae suspension was replaced with a mold suspension and the tryptone soy agar plate was replaced with a yeast extract peptone dextrose agar plate. Thus, a mold suspension treated with nanoassemblies (LEV+MnTCPP@PC7A) was finally obtained;

[0144] Control group with sonication (+US): The procedure was basically the same as that of the experimental group with sonication (+US), except that 100 μL of nanoassemblies was replaced with 100 μL of phosphate buffer solution (PBS). Thus, a mold suspension treated with PBS (PBS) was finally obtained;

[0145] Bacterial viability tests were performed on the mold suspensions treated with the nanoassemblies (LEV+MnTCPP@PC7A) in the experimental group without sonication (-US), the PBS-treated mold suspensions (PBS) in the control group without sonication (-US), the nanoassembly-treated mold suspensions (LEV+MnTCPP@PC7A) in the experimental group with sonication (+US), and the PBS-treated mold suspensions (PBS) in the control group with sonication (+US), respectively. The results are as Figure 5 shown in d of

[0146] Figure 5 This is an antibacterial activity test, in which Figure 5 a of Figure 5 shows the antibacterial activity of the nanoassemblies against Klebsiella pneumoniae resistant to levofloxacin, Figure 5 b of Figure 5 shows the antibacterial activity of the nanoassemblies against Escherichia coli,

[0147] From Figure 5 a of

[0148] it can be seen that when sonication is not performed, the survival rate of Klebsiella pneumoniae resistant to levofloxacin is 77% after being sterilized by the nanoassemblies; while when sonication is performed, the survival rate of Klebsiella pneumoniae resistant to levofloxacin is 8% after being sterilized by the nanoassemblies. Figure 5 From

[0149] it can be seen that when sonication is not performed, the survival rate of Escherichia coli is 82% after being sterilized by the nanoassemblies; while when sonication is performed, the survival rate of Escherichia coli is 9% after being sterilized by the nanoassemblies. Figure 5 From

[0150] it can be seen that when sonication is not performed, the survival rate of Staphylococcus aureus is 84% after being sterilized by the nanoassemblies; while when sonication is performed, the survival rate of Staphylococcus aureus is 9% after being sterilized by the nanoassemblies. Figure 5 From

[0151] The above results demonstrate that the nanoassemblies provided by the present invention can effectively reduce bacterial drug resistance, kill bacteria and fungi under ultrasonic action, and have excellent antibacterial activity against both bacteria and fungi, indicating that the nanoassemblies provided by the present invention have excellent antibacterial properties.

[0152] 5. Biofilm inhibition test

[0153] The biofilm inhibition test was carried out on the nanoassemblies in Example 1 of the present invention, and the method is as follows:

[0154] (1) Biofilm culture: Add 500 μL of tryptic soy broth (TSB) medium and 100 μL of Klebsiella pneumoniae bacterial suspension (10 8 CFU / mL) to each well of a 24-well plate, incubate in an incubator at 37 °C for 24 h. Subsequently, replace the old TSB medium with fresh TSB medium and continue to incubate for 24 h to obtain the cultured biofilm.

[0155] (2) Removal effect of nanoassemblies on biofilm: ① Experimental group without ultrasonic treatment (-US): Mix the cultured biofilm with 100 μL of nanoassemblies (nanoassemblies prepared according to the preparation method of Example 1), then incubate at 37 °C for 1 h, then continue to incubate at 37 °C for 30 min, wash twice with PBS, add 0.5 mL of crystal violet staining solution and incubate for 20 min, wash twice with PBS again, and finally add 500 μL of absolute ethanol to obtain the biofilm suspension treated with nanoassemblies (LEV+MnTCPP@PC7A);

[0156] Control group without ultrasonic treatment (-US): The steps are basically the same as those of the experimental group without ultrasonic treatment (-US), except that 100 μL of nanoassemblies are replaced with 100 μL of phosphate buffer solution (PBS). Therefore, the biofilm suspension treated with PBS (PBS) is finally obtained;

[0157] ② Experimental group with ultrasonic treatment (+US): Mix the cultured biofilm with 100 μL of nanoassemblies (nanoassemblies prepared according to the preparation method of Example 1), then incubate at 37 °C for 1 h to obtain the incubated biofilm; After ultrasonic treatment of the incubated biofilm under ultrasonic action of 1.5 W for 2 min, continue to incubate at 37 °C for 30 min, wash twice with PBS, add 0.5 mL of crystal violet staining solution and incubate for 20 min, wash twice with PBS again, and finally add 500 μL of absolute ethanol to obtain the biofilm suspension treated with nanoassemblies (LEV+MnTCPP@PC7A);

[0158] Control group treated with sonication (+US): The procedure was basically the same as that of the experimental group treated with sonication (+US), except that 100 μL of the nano-assembly was replaced with 100 μL of phosphate buffer solution (PBS), and thus a PBS-treated biofilm suspension (PBS) was finally obtained.

[0159] Photographs were taken of the nano-assembly-treated biofilm suspension (LEV+MnTCPP@PC7A) in the experimental group without sonication (-US), the PBS-treated biofilm suspension (PBS) in the control group without sonication (-US), the nano-assembly-treated biofilm suspension (LEV+MnTCPP@PC7A) in the experimental group with sonication (+US), and the PBS-treated biofilm suspension (PBS) in the control group with sonication (+US). The results are as Figure 6 shown; Figure 6 This is the crystal violet staining result diagram in the biofilm inhibition test.

[0160] It can be seen from Figure 6 that the nano-assembly provided in the embodiments of the present invention can effectively remove bacterial biofilms and destroy the structure of bacterial biofilms under the action of ultrasonic waves. This is because under the action of ultrasonic waves, the nano-assembly releases MnTCPP and LEV, and kills bacteria through the sonodynamic performance (releasing ROS) of MnTCPP and the antibacterial performance of LEV, thereby effectively removing bacterial biofilms and destroying the structure of bacterial biofilms.

[0161] The morphological observations were respectively carried out on the nano-assembly-treated biofilm suspension (LEV+MnTCPP@PC7A) in the experimental group without sonication (-US), the PBS-treated biofilm suspension (PBS) in the control group without sonication (-US), the nano-assembly-treated biofilm suspension (LEV+MnTCPP@PC7A) in the experimental group with sonication (+US), and the PBS-treated biofilm suspension (PBS) in the control group with sonication (+US) by using a scanning electron microscope (SEM). The specific method is as follows:

[0162] Each group was washed three times with phosphate buffer solution (PBS) to remove excess culture medium and metabolites. After washing, each group was placed in an environment at 4 °C and fixed overnight with 1 mL of paraformaldehyde solution (concentration 2.5 wt%) to ensure the structural stability of the biofilm in each group. After fixation, to further facilitate SEM observation, serial dehydration treatment with gradient ethanol solutions was performed on the biofilms of each group. This process included ethanol solutions with different mass fractions (30%, 50%, 70%, 80%, 90%, 95% and 100%), and each mass fraction of ethanol solution was used to dehydrate the biofilms of each group; finally, the dehydrated biofilms of each group were placed under vacuum for overnight drying, and the dried biofilms of each group were tested and observed using a scanning electron microscope (SEM). The results are as Figure 7 shown; Figure 7 It is the SEM test result diagram in the biofilm inhibition test.

[0163] Figure 7 The results further illustrate that the nanoassemblies provided in the embodiments of the present invention can effectively remove bacterial biofilms and destroy the structure of bacterial biofilms under the action of ultrasonic waves.

[0164] 6. Magnetic resonance imaging test

[0165] The nanoassemblies (prepared according to the preparation method of the nanoassemblies in Example 1) were respectively configured into nanoassembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at pH = 6.0 using PBS solution, where the concentration refers to the concentration of MnTCPP in the nanoassembly solution;

[0166] The nanoassemblies (prepared according to the preparation method of the nanoassemblies in Example 1) were respectively configured into nanoassembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at pH = 7.4 using PBS solution, where the concentration refers to the concentration of MnTCPP in the nanoassembly solution;

[0167] T1-weighted sequence scanning experiments were respectively performed on nanoassembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at pH = 6.0 and nanoassembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at pH = 7.4 on a Philips 3.0T magnetic resonance imaging system to obtain scanned images, and then the scanned images were processed using Sante DICOM software ( Figure 8a), the magnetic resonance signal-to-noise ratio (SNR) of nano-assembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at pH = 6.0 or pH = 7.4 was calculated and recorded ( Figure 8 b).

[0168] Figure 8 For magnetic resonance imaging experiments; among them Figure 8 a is the result of processing the scanned images of nano-assembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at different pH values using Sante DICOM software, Figure 8 b is the magnetic resonance signal-to-noise ratio (SNR) of nano-assembly solutions with different concentrations (0 mM, 0.4 mM, 0.2 mM, 0.1 mM, 0.05 mM) at different pH values.

[0169] From Figure 8 a and b, it can be seen that as the concentration of MnTCPP in the nano-assembly solution increases, the SNR shows a certain trend of change. Specifically, when the concentration of MnTCPP in the nano-assembly solution is low, the SNR value is relatively low, indicating a weak signal intensity; while as the concentration of MnTCPP in the nano-assembly solution increases, the SNR value gradually increases, indicating an enhanced signal intensity. And from Figure 8 a and b, it can be seen that when the concentration of MnTCPP in the nano-assembly solution is the same, the SNR values of nano-assembly solutions with different pH values are different, and the SNR value of the nano-assembly solution at pH = 6.0 is higher than that of the nano-assembly solution at pH = 7.4. The above results illustrate that the nano-assembly provided in the embodiments of the present invention can respond to an acidic microenvironment, significantly improve the SNR of T1-weighted magnetic resonance imaging, achieve signal enhancement, has the ability of nuclear magnetic resonance imaging, and can be used as a T1-enhanced magnetic resonance imaging contrast agent.

[0170] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nano-assembly, characterized in that, The nano-assembly is assembled from the following three components: An amphiphilic block polymer, an antibiotic, and a sonosensitizer; wherein, the structural formula of the amphiphilic block polymer is shown in Formula I, the antibiotic is a hydrophobic antibiotic, and the sonosensitizer is a porphyrin-based sonosensitizer; Formula Ⅰ In Formula I, m is an integer from 40 to 120, and n is an integer from 50 to 100.

2. The nano-assembly according to claim 1, wherein, The particle size of the nano-assembly is 50 - 200 nm.

3. The nano-assembly according to claim 1, wherein In the nano-assembly, the mass ratio of the amphiphilic block polymer, the antibiotic, and the sonosensitizer is (10 - 20):(1 - 3):(1 - 5).

4. The nano-assembly according to claim 1, wherein The hydrophobic antibiotics include at least one of levofloxacin, tetracycline, chloramphenicol, ciprofloxacin, azithromycin, doxycycline, linezolid, and norfloxacin; And / or, the porphyrin-based sonosensitizer includes manganese(II) 5,10,15,20-tetraphenyl-21H,23H-porphyrin.

5. A method for preparing the nano-assembly according to any one of claims 1-4, characterized in that, It includes the following steps: Dissolve the amphiphilic block polymer, the antibiotic, and the sonosensitizer in an organic solvent respectively to prepare an amphiphilic block polymer mother liquor, an antibiotic mother liquor, and a sonosensitizer mother liquor; Mix the amphiphilic block polymer mother liquor, the antibiotic mother liquor, the sonosensitizer mother liquor with PBS solution, and then carry out stirring treatment and dialysis treatment to obtain the nano-assembly.

6. The preparation method of the nano-assembly according to claim 5, wherein, The concentration of the amphiphilic block polymer in the amphiphilic block polymer mother liquor is 14 - 18 mg / mL, the concentration of the antibiotic in the antibiotic mother liquor is 1.5 - 3 mg / mL, and the concentration of the sonosensitizer in the sonosensitizer mother liquor is 1.5 - 3 mg / mL; The volume ratio of the amphiphilic block polymer mother liquor, the antibiotic mother liquor, and the sonosensitizer mother liquor is (1 - 2):1:

1.

7. The preparation method of the nano-assembly according to claim 5, characterized in that, In the stirring treatment, the rotation speed is 1000 - 1500 rpm / min, the temperature is 20 - 30 °C, and the time is 5 - 10 min; And / or, in the dialysis treatment, the cut-off molecular weight of the dialysis bag used is 3000 - 20000 Da, and the dialysis time is 8 - 12 h; And / or, the organic solvent includes dimethyl sulfoxide.

8. The preparation method of the nano-assembly according to claim 5, wherein, The amphiphilic block polymer is obtained by a preparation method including the following process: Dissolve 2-(azepan-1-yl)ethyl methacrylate, a macromolecular chain transfer agent PEG-CTA, and an initiator in dimethyl sulfoxide to obtain a mixed solution; Carry out a polymerization reaction on the mixed solution at 50 - 80 °C for 6 - 12 h to obtain the amphiphilic block polymer; Wherein the initiator includes azobisisobutyronitrile; The mass ratio of 2-(azepan-1-yl)ethyl methacrylate, the macromolecular chain transfer agent PEG-CTA, and the initiator is (50 - 150):1:(0.1 - 0.3).

9. Use of the nano-assembly according to any one of claims 1 - 4 or the nano-assembly prepared by the preparation method of the nano-assembly according to any one of claims 5 - 8 in the preparation of products for treating bacterial and fungal infections or in the preparation of products for treating bacterial biofilm infections.

10. Use of the nano-assembly according to any one of claims 1-4 or the nano-assembly prepared by the preparation method of the nano-assembly according to any one of claims 5-8 in the preparation of a magnetic resonance imaging contrast agent.

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