Amphiphilic ionic copolymer, copolymer micellar drug delivery system, and preparation method thereof

Abstract

The present disclosure provides an amphiphilic ionic copolymer, a copolymer micelle drug delivery system, and a preparation method thereof. The amphiphilic ionic copolymer comprises: a block copolymer formed by block copolymerization of a hydrophobic polymer formed by a hydrophobic monomer and a hydrophilic monomer; and the hydrophilic monomer has a structure as shown in formula (A), and the hydrophobic monomer has a structure as shown in formula (B), wherein R is independently selected from any one of the C1-C6 alkyl groups, and adjacent R groups can be connected to form a ring. FIG. 1: AA%%%Formula (A)

Description

Amphiphilic ionic copolymer, copolymer micelle drug delivery system and preparation method thereof Technical Field

[0001] The present disclosure belongs to the field of biomaterial technology and relates to an amphiphilic ionic copolymer, a copolymer micelle drug delivery system and a preparation method thereof, and more specifically, to an amphiphilic ionic copolymer-promoting drug delivery system across the mucosa and a preparation method thereof. Background Art

[0002] Antibiotics account for a large proportion of clinical medications and can effectively reduce a series of complications caused by bacterial infections and reduce mortality. The invention of antibiotics is hailed as one of the greatest achievements in the history of science. Currently, the main routes of administration of antibiotics in clinical practice are injection and oral administration. However, hydrophobic antibiotics have poor solubility in water and low bioavailability, which to a certain extent limits their application. In order to obtain the expected therapeutic effect of the drug, it is usually necessary to administer the drug in a solubilized form to the patient. While increasing the dosage and extending the course of treatment, there are the disadvantages of increased biological resistance and increased toxic side effects. In response to the problems of hydrophobic drugs, nanotechnology provides new possibilities for antibiotic delivery. Inhalation drug delivery systems can be used to improve the delivery of antibiotics in the respiratory tract.

[0003] Among lung diseases, hospital-acquired pneumonia (HAP) secondary to Pseudomonas aeruginosa carries a poor prognosis and high mortality. Ventilator-associated pneumonia (VAP) secondary to P. aeruginosa has an estimated mortality rate of 32% to 42.8%. P. aeruginosa colonizes the bronchioles and alveoli, producing a large exopolysaccharide matrix (EPS), which can account for over 90% of the total volume of the P. aeruginosa biofilm. Composed primarily of polysaccharides, proteins, and extracellular DNA, the dense bacterial biofilm forms a massive barrier, impermeable to antibiotics. The formation of bacterial biofilms in the lungs is one of the causes of chronic pneumonia, hindering neutrophil phagocytosis. Furthermore, the alkali- and heat-resistant properties of bacterial biofilms further complicate treatment. Therefore, the development of copolymers capable of carrying drugs to the alveolar surface is of great clinical significance.

[0004] Summary of the Invention

[0005] In response to the above technical problems, the present disclosure provides an amphiphilic ionic copolymer, a copolymer micelle drug delivery system and a preparation method thereof, in order to at least partially solve the above-mentioned technical problems.

[0006] In order to solve the above technical problems, the present disclosure provides the following technical solutions:

[0007] As a first aspect of the present disclosure, there is provided an amphiphilic ionic copolymer comprising:

[0008] A block copolymer formed by block copolymerization of a hydrophobic polymer formed by a hydrophobic monomer and a hydrophilic monomer;

[0009] Wherein, the hydrophilic monomer has a structure as shown in formula (A):

[0010] The hydrophobic monomer has a structure as shown in formula (B):

[0011] Wherein, each R is independently selected from any one of C1 to C6 alkyl groups, and adjacent R groups may be connected to form a ring.

[0012] In one embodiment, R is independently selected from a C1-C6 straight-chain alkane, and the ring formed by connecting adjacent Rs can be a five-membered ring, a six-membered ring, or a seven-membered ring.

[0013] In one embodiment, the hydrophilic monomer includes any one of the structures shown in formulas (A1) to (A4);

[0014] The hydrophobic monomer includes any one of the structures shown in formulas (B1) to (B4);

[0015] In one embodiment, the degree of polymerization of the hydrophilic monomer and the hydrophobic monomer in the block copolymer is 40-60:40-60.

[0016] As a second aspect of the present disclosure, there is provided a copolymer micelle drug delivery system comprising:

[0017] hydrophobic drugs;

[0018] The amphiphilic ionic copolymer self-assembles with the hydrophobic drug to form a copolymer micelle drug delivery system.

[0019] In one embodiment, the hydrophobic drug is any one of lapacone, doxorubicin, camptothecin, 17-AAG, mitomycin C, or indocyanine green.

[0020] As a third aspect of the present disclosure, a method for preparing an amphiphilic ionic copolymer is provided, comprising:

[0021] subjecting a hydrophobic monomer to a first reversible addition-fragmentation chain transfer polymerization reaction to obtain a hydrophobic polymer, wherein the hydrophobic monomer has a structure as shown in formula (B);

[0022] The hydrophobic polymer is then protonated and used as a second chain transfer agent to undergo a second reversible addition-fragmentation chain transfer polymerization reaction with the hydrophilic monomer to obtain an amphiphilic ionic copolymer, wherein the hydrophilic monomer has a structure as shown in formula (A):

[0023] In one embodiment, the method for preparing the amphiphilic ionic copolymer further comprises:

[0024] The amphiphilic ionic copolymer obtained by the reaction is dialyzed and deprotonated in an alkaline PBS buffer solution, and then dialyzed in a methanol solution and vacuum dried to obtain a purified amphiphilic ionic copolymer.

[0025] In one embodiment, the method for preparing the amphiphilic ionic copolymer further comprises:

[0026] The compound represented by formula (C) is subjected to an esterification reaction with methacryloyl chloride to prepare a hydrophobic monomer;

[0027] The hydrophobic monomer is oxidized to obtain the hydrophilic monomer.

[0028] In one embodiment, in the first reversible addition-fragmentation chain transfer polymerization reaction, the first solvent is methanol, the first chain transfer agent is 4-cyanovaleric dithiobenzoate (CPADB), and the first initiator is azobisisobutyronitrile (AIBN);

[0029] The temperature of the first reversible addition-fragmentation chain transfer polymerization reaction is 60-80°C, and the reaction time is 8-16 hours;

[0030] The molar concentration ratio of the hydrophobic monomer, the first chain transfer agent and the first initiator is 50n:n:1, wherein n is 3-5.

[0031] In one embodiment, in the second reversible addition-fragmentation chain transfer polymerization reaction, the second solvent is water, and the second initiator is 4,4'-azobis(4-cyanovaleric acid) (ACVA);

[0032] The temperature of the second reversible addition-fragmentation chain transfer polymerization reaction is 60-80°C, and the reaction time is 8-16 hours;

[0033] The molar concentration ratio of the hydrophilic monomer, the second chain transfer agent and the second initiator is 50n:n:1, wherein n is 3-5.

[0034] As a third aspect of the present disclosure, a method for preparing a copolymer micelle drug delivery system is provided, comprising:

[0035] The amphiphilic ionic copolymer and the hydrophobic drug are self-assembled by thin film hydration method to obtain a copolymer micelle drug delivery system.

[0036] In one embodiment, the method for preparing the copolymer micelle drug delivery system further comprises:

[0037] The copolymer micellar drug delivery system was subjected to low-speed centrifugation to remove unencapsulated drugs;

[0038] The rotation speed of the low-speed centrifugation is 2500-5000 rpm, and the time of the low-speed centrifugation is 5-10 minutes.

[0039] As a fourth aspect of the present disclosure, there is provided a use of the above-mentioned copolymer micelle drug delivery system in the preparation of a drug for treating bacterial biofilm infection in the lungs.

[0040] Based on the above technical solutions, the amphiphilic ionic copolymer, copolymer micelle drug delivery system and preparation method thereof provided by the present disclosure have at least one of the following beneficial effects:

[0041] (1) In the embodiments disclosed herein, a hydrophobic polymer formed from a hydrophobic monomer is used as a macromolecular chain transfer agent to form an amphiphilic copolymer through block copolymerization with a hydrophilic monomer. The surface charge is close to neutral, the particle size is small, the hydrophilic segments form a zwitterionic surface outward, and the hydrophobic segments spontaneously form a hydrophobic core, which has high biosafety and provides ideal conditions for achieving the penetration and delivery of drugs through the lung mucosa by the amphiphilic ionic copolymer-drug self-assembly.

[0042] (2) In the embodiments of the present disclosure, due to the presence of hydrophobic segments, the amphiphilic ionic copolymer is pH responsive and can trigger a response at pH = 6.8. The degree of protonation increases sharply, increasing the positive charge density, which can destroy bacterial biofilms and negatively charged bacterial membranes, achieving a bactericidal effect.

[0043] (3) In the embodiments of the present disclosure, hydrophobic drugs and amphiphilic ionic copolymers are self-assembled to form copolymer micelle drug delivery systems for inhalation drug delivery. The systems can freely pass through the mucin fiber network in the lung mucus layer and carry the drugs to the alveolar surface, thus overcoming the delivery difficulties of inhaled drugs caused by mucus layer penetration and bacterial biofilm infection in the lungs, and facilitating direct inhalation airway drug delivery by the copolymer micelle drug delivery system.

[0044] (4) In the embodiments disclosed herein, the copolymer micelle drug delivery system can not only carry drugs through the airway mucus layer, but also expose the positive charge on the surface of the material in the specific acidic pH environment of the bacterial biofilm, thereby achieving a biofilm penetration effect, breaking through the biofilm barrier and disassembling to release hydrophobic antibiotic drugs, thereby achieving a highly efficient antibiotic effect and exerting the synergistic antibacterial effect of the amphiphilic ionic copolymer killing bacteria and the antibiotic inhibiting bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a schematic diagram of synthesizing an amphiphilic ionic copolymer in an embodiment of the present disclosure;

[0046] FIG2 is a schematic diagram of the self-assembly and acid-responsive disintegration release of PPEMA-b-PPOEMA-AZM micelles in an embodiment of the present disclosure;

[0047] FIG3(A) is a schematic diagram of the structure of mouse alveoli in an embodiment of the present disclosure;

[0048] FIG3(B) is a schematic diagram of the action of PPEMA-b-PPOEMA-AZM micelles in an embodiment of the present disclosure;

[0049] FIG4 is a hydrogen nuclear magnetic resonance spectrum ( 1H NMR) of PEMA in Example 1 of the present disclosure;

[0050] FIG5 is a hydrogen nuclear magnetic resonance spectrum ( 1H NMR) of POEMA in Example 1 of the present disclosure;

[0051] FIG6 is a hydrogen nuclear magnetic resonance spectrum (1H NMR) of PPEMA in Example 1 of the present disclosure;

[0052] FIG7 is a hydrogen nuclear magnetic resonance spectrum ( 1H NMR) of PPEMA-b-PPOEMA in Example 1 of the present disclosure;

[0053] FIG8(A) is a transmission electron microscopy (TEM) image of PPEMA-b-PPOEMA micelles at a pH of 7.4 in Example 2 of the present disclosure;

[0054] FIG8(B) is a transmission electron microscopy (TEM) image of PPEMA-b-PPOEMA micelles at a pH of 6.8 in Example 2 of the present disclosure;

[0055] FIG9(A) is a transmission electron microscopy (TEM) image of PPEMA-b-PPOEMA-AZM micelles at a pH of 7.4 in Example 2 of the present disclosure;

[0056] FIG9(B) is a transmission electron microscopy (TEM) image of PPEMA-b-PPOEMA-AZM micelles at a pH of 6.8 in Example 2 of the present disclosure;

[0057] FIG10 is a size distribution diagram of PPEMA-b-PPOEMA micelles in Example 2 of the present disclosure;

[0058] FIG11 is a size distribution diagram of PPEMA-b-PPOEMA-AZM micelles in Example 2 of the present disclosure;

[0059] FIG12 is a surface potential diagram of PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles in Example 2 of the present disclosure;

[0060] FIG13 is a confocal laser scanning microscope (CLSM) image of the bronchus and lung of mice after administration of free Nile red (Free Nilered) in the airway in Example 3 of the present disclosure;

[0061] FIG14 is a confocal laser scanning microscope (CLSM) image of the bronchus and lung of mice after airway administration of PPEMA-b-PPOEMA-Nilered in Example 3 of the present disclosure;

[0062] FIG15 shows the antibacterial effect of PPEMA-b-PPOEMA-AZM micelles on Pseudomonas aeruginosa biofilms in Example 5 of the present disclosure;

[0063] FIG16 is a comparison diagram of bacterial colonies after antibacterial effect testing in Example 5 of the present disclosure;

[0064] FIG17 shows the antibacterial effect of PPEMA-b-PPOEMA-AZM micelles on Pseudomonas aeruginosa biofilms in Example 6 of the present disclosure;

[0065] FIG18 is a comparison diagram of bacterial colonies after antibacterial effect testing in Example 6 of the present disclosure;

[0066] FIG19 is a confocal laser microscopy (CLSM) image of the incubated Pseudomonas aeruginosa biofilm in Example 7 of the present disclosure;

[0067] FIG20 shows the volume of surviving bacteria in the Pseudomonas aeruginosa biofilm after three-dimensional reconstruction in Example 7 of the present disclosure;

[0068] FIG21 is an image of bacterial burden and colony growth in mouse lung tissue in Example 8 of the present disclosure;

[0069] FIG22 is a statistical graph showing bacterial burden and colony growth in lung tissue of mice after treatment in Example 8 of the present disclosure;

[0070] FIG23 shows the cell viability after incubation with PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure;

[0071] FIG24 shows the results of a hemolysis experiment of PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure;

[0072] FIG25 is an image of a hemolysis experiment of PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure. DETAILED DESCRIPTION

[0073] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0074] Currently, researchers have proposed methods to increase the permeability of antibiotics in the mucus layer using surfactants, polymers, or carriers. Literature has reported that mucus-permeable particles (MPPs) can achieve mucus penetration. Their main characteristics are hydrophilicity, smaller than the pores of the mucus network, and a shielded, nearly neutral surface charge. This allows the material to be invisible in the mucus layer, avoiding capture by mucin and hydrophobic structural regions, thereby carrying hydrophobic drugs through the mucus layer. It has also been reported that hydrophilic cations have excellent biomembrane penetration ability, which may be related to the capture-release phenomenon of the extracellular polysaccharide matrix (EPS). However, achieving mucus penetration requires a neutral charge, and the biomembrane penetration ability provided by hydrophilic cations is inconsistent with the characteristics of mucus penetration.

[0075] In view of the technical problems existing in the related art, the present disclosure provides an amphiphilic ionic copolymer, a copolymer micelle drug delivery system and a preparation method thereof. An amphiphilic ionic copolymer and a copolymer micelle drug delivery system with a small particle size and a surface charge close to neutral are synthesized by block copolymerization. They can freely pass through the mucin fiber network of the pulmonary mucus layer, carry drugs to the alveolar surface, promote the cross-mucus penetration of hydrophobic drugs, and improve the penetration and retention of the loaded drugs. They are also sensitive to pH changes and expose the positive charge on the surface of the material in the acidic microenvironment of the biofilm, which can achieve the membrane-breaking bactericidal effect of cationic polymers and release drugs. They can exert the synergistic antibacterial effect of cationic polymers and the antibacterial effect of antibiotics, providing a feasible strategy for the treatment of bacterial biofilm infections in the lungs through airway administration.

[0076] In order to achieve the above technical objectives, as a first aspect of the present disclosure, an amphiphilic ionic copolymer is provided, comprising:

[0077] A block copolymer formed by block copolymerization of a hydrophobic polymer formed by a hydrophobic monomer and a hydrophilic monomer;

[0078] Wherein, the hydrophilic monomer has a structure as shown in formula (A):

[0079] The hydrophobic monomer has a structure as shown in formula (B):

[0080] Wherein, each R is independently selected from any one of C1 to C6 alkyl groups, and adjacent R groups may be connected to form a ring.

[0081] According to the embodiments of the present disclosure, the hydrophobic monomer structure in the amphiphilic ionic copolymer is pH-responsive, especially at pH = 6.8, it can trigger a response, the degree of protonation increases sharply, and the positive charge density can be increased to simulate the mechanism of action of cationic antimicrobial peptides. It was found that it can destroy bacterial biofilms and negatively charged bacterial membranes, thereby achieving a bactericidal effect.

[0082] According to an embodiment of the present disclosure, R is independently selected from a C1 to C6 straight-chain alkane, and the ring formed by connecting adjacent Rs can be a five-membered ring, a six-membered ring, or a seven-membered ring.

[0083] According to an embodiment of the present disclosure, the hydrophilic monomer includes any one of the structures shown in formulas (A1) to (A4);

[0084] The hydrophobic monomer includes any one of the structures shown in formulas (B1) to (B4);

[0085] According to an embodiment of the present disclosure, the degree of polymerization of the hydrophilic monomer and the hydrophobic monomer in the block copolymer is 40-60:40-60, for example, 40:60, 45:55, 50:50, 55:45, 60:40, etc.

[0086] According to the embodiments of the present disclosure, the amphiphilic ionic copolymer can be formed by forming a hydrophobic polymer from a hydrophobic monomer, and then using the hydrophobic polymer as a macromolecular chain transfer agent to block copolymerize with the hydrophilic monomer through a reversible addition-fragmentation chain transfer polymerization reaction. Preferably, the degree of polymerization of the hydrophilic segment and the hydrophobic segment in the formed polymer is 50:50.

[0087] As a second aspect of the present disclosure, there is provided a copolymer micelle drug delivery system comprising:

[0088] hydrophobic drugs;

[0089] The amphiphilic ionic copolymer self-assembles with the hydrophobic drug to form a copolymer micelle drug delivery system.

[0090] According to the embodiments of the present disclosure, the copolymer micelle drug delivery system can promote the penetration of hydrophobic drugs across the mucus layer. The amphiphilic ionic copolymer can self-assemble into smaller, nearly neutral nano-micelles in a neutral pH environment. The hydrophilic segments in the micelles form a zwitterionic surface outward, and the hydrophobic segments spontaneously form a hydrophobic core. It can also be self-assembled together with hydrophobic drugs to obtain a copolymer micelle drug delivery system, which provides ideal conditions for achieving drug penetration into the pulmonary mucus layer. The hydrophobic drug is pre-loaded in the self-assembled micelles, and the disassembly of the copolymer micelles at an ideal pH exposes the positive charge on the surface of the material, releasing the hydrophobic drug, achieving a biomembrane penetration effect, and exerting a synergistic effect of the bactericidal effect of the cationic polymer and the antibacterial effect of the hydrophobic drug.

[0091] According to an embodiment of the present disclosure, the hydrophobic drug is any one of lapacone, doxorubicin, camptothecin, 17-AAG, mitomycin C, or indocyanine green.

[0092] As a third aspect of the present disclosure, a method for preparing an amphiphilic ionic copolymer is provided, comprising:

[0093] subjecting a hydrophobic monomer to a first reversible addition-fragmentation chain transfer polymerization reaction to obtain a hydrophobic polymer, wherein the hydrophobic monomer has a structure as shown in formula (B);

[0094] The hydrophobic polymer is then protonated and used as a second chain transfer agent to undergo a second reversible addition-fragmentation chain transfer polymerization reaction with the hydrophilic monomer to obtain an amphiphilic ionic copolymer, wherein the hydrophilic monomer has a structure as shown in formula (A):

[0095] According to an embodiment of the present disclosure, the method for preparing the amphiphilic ionic copolymer further includes:

[0096] The amphiphilic ionic copolymer obtained by the reaction is dialyzed and deprotonated in an alkaline PBS buffer solution, and then dialyzed in a methanol solution and vacuum dried to obtain a purified amphiphilic ionic copolymer.

[0097] According to an embodiment of the present disclosure, unpolymerized small molecule substances are removed by dialyzing in a methanol solution, wherein the molecular weight cutoff MW of the dialysis bag may be 1000 Da, and molecules with a molecular weight below 1000 Da are separated and removed.

[0098] According to an embodiment of the present disclosure, the method for preparing the amphiphilic ionic copolymer further includes:

[0099] The compound represented by formula (C) is subjected to an esterification reaction with methacryloyl chloride to prepare a hydrophobic monomer;

[0100] The hydrophobic monomer is oxidized to obtain the hydrophilic monomer.

[0101] According to an embodiment of the present disclosure, in the first reversible addition-fragmentation chain transfer polymerization reaction of polymerizing a hydrophobic monomer, the first solvent is methanol, the first chain transfer agent is 4-cyanovaleric dithiobenzoate (CPADB), and the first initiator is azobisisobutyronitrile (AIBN); the temperature of the first reversible addition-fragmentation chain transfer polymerization reaction is 60-80°C, for example, it can be 60°C, 65°C, 70°C, 75°C, 80°C, etc., and the reaction time is 8-16h, for example, it can be 8h, 10h, 12h, 14h, 16h, etc.; when the degree of polymerization is 50, the molar concentration ratio of the hydrophobic monomer, the first chain transfer agent, and the first initiator is 50n:n:1, where n is 3-5, for example, n can be 3, 3.5, 4, 4.5, 5, etc. When n is 3, the molar concentration ratio of the hydrophobic monomer, the first chain transfer agent, and the first initiator is 150:3:1; when n is 4, the molar concentration ratio of the hydrophobic monomer, the first chain transfer agent, and the first initiator is 200:4:1; when n is 5, the molar concentration ratio of the hydrophobic monomer, the first chain transfer agent, and the first initiator is 250:5:1.

[0102] According to an embodiment of the present disclosure, in the second reversible addition-fragmentation chain transfer polymerization reaction of polymerizing a hydrophilic monomer, the second solvent is water, and the second initiator is 4,4'-azobis(4-cyanovaleric acid) (ACVA); the temperature of the second reversible addition-fragmentation chain transfer polymerization reaction is 60-80°C, for example, it can be 60°C, 65°C, 70°C, 75°C, 80°C, etc., and the reaction time is 8-16h, for example, it can be 8h, 10h, 12h, 14h, 16h, etc.; when the degree of polymerization is 50, the molar concentration ratio of the hydrophilic monomer, the second chain transfer agent, and the second initiator is 50n:n:1, where n is 3-5, for example, n can be 3, 3.5, 4, 4.5, 5, etc. When n is 3, the molar concentration ratio of the hydrophilic monomer, the second chain transfer agent, and the second initiator is 150:3:1; when n is 4, the molar concentration ratio of the hydrophilic monomer, the second chain transfer agent, and the second initiator is 200:4:1; when n is 5, the molar concentration ratio of the hydrophilic monomer, the second chain transfer agent, and the second initiator is 250:5:1.

[0103] FIG1 is a schematic diagram of synthesizing an amphiphilic ionic copolymer in an embodiment of the present disclosure. The preparation method is briefly described below in conjunction with FIG1 :

[0104] (1) esterifying a compound represented by formula (C) with methacryloyl chloride to prepare a hydrophobic monomer having a structure represented by formula (B);

[0105] (2) The hydrophobic monomer is oxidized in an ice bath to obtain a hydrophilic monomer having a structure as shown in formula (A):

[0106] (3) The hydrophobic monomer is subjected to a reversible addition-fragmentation chain transfer polymerization reaction at 70° C. with 4-cyanovaleric dithiobenzoate (CPADB) as a chain transfer agent and azobisisobutyronitrile (AIBN) as an initiator, using methanol as a solvent to obtain a hydrophobic polymer having a structure shown in formula (D):

[0107] (4) The hydrophobic polymer is then protonated to obtain a protonated hydrophobic polymer, and the protonated hydrophobic polymer is used as a chain transfer agent, 4,4'-azobis(4-cyanovaleric acid) (ACVA) is selected as an initiator, and water is used as a solvent to cause the hydrophilic monomer to undergo a reversible addition-fragmentation chain transfer polymerization reaction to obtain an amphiphilic ionic copolymer. The amphiphilic ionic copolymer has a structure as shown in formula (E):

[0108] (5) The amphiphilic ionic copolymer is dialyzed and deprotonated in an alkaline PBS buffer solution, and then dialyzed in a methanol solution and vacuum dried to obtain a purified amphiphilic ionic copolymer.

[0109] According to an embodiment of the present disclosure, 2-(piperidin-1-methyl)ethyl acrylate (PEMA) monomer is prepared by esterification reaction of acyl chloride, and PEMA is then oxidized to prepare 2-piperidinyl methacrylate (POEMA) monomer; using dithiobenzoic acid 4-cyanovaleric acid (CPADB) as a chain transfer agent, PEMA is subjected to reversible addition-fragmentation chain transfer polymerization block copolymerization to prepare poly-2-piperidinyl methacrylate (PPEMA); PPEMA is protonated using dilute hydrochloric acid to increase the positive charge density of the polymer, and the protonated PPEMA is used as a macromolecular chain transfer agent to prepare a block copolymer of poly-2-piperidinyl methacrylate-poly-2-piperidinyl methacrylate (PPEMA-b-PPOEMA), which is an amphiphilic ionic copolymer, and the degree of polymerization of the hydrophilic segment and the hydrophobic segment in the amphiphilic ionic copolymer is 50:50.

[0110] According to actual needs, PPEMA-b-PPOEMA micelles can be prepared by thin film hydration method.

[0111] As a third aspect of the present disclosure, a method for preparing a copolymer micelle drug delivery system is provided, comprising:

[0112] The amphiphilic ionic copolymer and the hydrophobic drug are self-assembled by a thin film hydration method. The amphiphilic polymer and the hydrophobic drug are dissolved in an organic solvent. The amphiphilic ionic copolymer has good solubility in the organic solvent. A polymer film skeleton is formed by vacuum rotary evaporation. Subsequently, PBS is added for ultrasonic hydration. During this process, the amphiphilic polymer and the hydrophobic drug self-assemble into micelles to obtain a copolymer micelle drug delivery system.

[0113] According to an embodiment of the present disclosure, a hydrophobic polymer formed by polymerization of a hydrophobic monomer is used as a macromolecular chain transfer agent. After copolymerization with a hydrophilic monomer through reversible addition-fragmentation chain transfer polymerization to form an amphiphilic ionic copolymer PPEMA-b-PPOEMA, the amphiphilic ionic copolymer (PPEMA-b-PPOEMA) and the hydrophobic antibiotic drug azithromycin (AZM) are self-assembled by a thin film hydration method to form drug-loaded micelles PPEMA-b-PPOEMA-AZM, which is a copolymer micelle drug delivery system. Figure 2 is a schematic diagram of the self-assembly and acid-responsive disintegration release of PPEMA-b-PPOEMA-AZM micelles in an embodiment of the present disclosure. As shown in Figure 2, after PPEMA-b-PPOEMA and AZM self-assemble to form PPEMA-b-PPOEMA-AZM micelles, they are sensitive to pH and can be disassembled and released under acidic conditions of pH = 6.8.

[0114] According to an embodiment of the present disclosure, the method for preparing the copolymer micelle drug delivery system further includes:

[0115] The copolymer micellar drug delivery system was subjected to low-speed centrifugation to remove unencapsulated drugs;

[0116] The rotation speed of the low-speed centrifugation is 2500-5000 rpm, for example, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 5000 rpm, etc.; the time of the low-speed centrifugation is 5-10 min, for example, 5 min, 6 min, 8 min, 10 min, etc.

[0117] As a fourth aspect of the present disclosure, there is provided a use of the above-mentioned copolymer micelle drug delivery system in the preparation of a drug for treating bacterial biofilm infection in the lungs.

[0118] According to the embodiments of the present disclosure, the above-mentioned copolymer micelle drug delivery system can be used for the preparation of drugs for treating Pseudomonas aeruginosa biofilm pneumonia caused by bacterial infection. Figure 3(A) is a schematic diagram of the structure of mouse alveoli in the embodiments of the present disclosure, and Figure 3(B) is a schematic diagram of the action of PPEMA-b-PPOEMA-AZM micelles in the embodiments of the present disclosure. As shown in Figure 3(B), PPEMA-b-PPOEMA-AZM micelles can penetrate into the lung mucus layer, exert a pH-responsive cationic bactericidal effect and release AZM for synergistic antibacterial effect, while free AZM is difficult to exert an antibacterial effect through the dual barriers of the mucus layer and the biofilm.

[0119] In order to make the purpose, technical solutions and advantages of the present disclosure more clear and explicit, the technical solutions and principles of the present disclosure are further explained below through specific embodiments in combination with the accompanying drawings. It should be noted that the following specific embodiments are only for illustration and the scope of protection of the present disclosure is not limited thereto.

[0120] Unless otherwise specified, the experimental materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the examples, these are conventional methods and can be performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0121] The reagents used in the examples were purchased from Sinopharm Chemical Reagent Co., Ltd. and Shanghai Beyotime Biotechnology Co., Ltd., and the mice used in the examples were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0122] Example 1

[0123] A method for preparing an amphiphilic ionic copolymer comprises:

[0124] (1) Preparation of hydrophobic monomer: 2-(piperidin-1-methyl)ethyl acrylate (PEMA)

[0125] Weigh 12.9g of N-(2-hydroxyethyl)piperidine, 10.1g of triethylamine, and 0.13g of phloroglucinol into a 300mL round-bottom flask. Add tetrahydrofuran and stir at 400rpm. Then weigh 10.4g of methacryloyl chloride and add it dropwise to the flask. The mixed solution is heated to reflux at 80°C in tetrahydrofuran for more than 12 hours. The reaction solution is collected and filtered to remove the precipitate. The reaction solution is then subjected to water pump rotary evaporation at 35°C using a rotary evaporator to remove the tetrahydrofuran solvent. Then, vacuum distillation is performed at 100°C to obtain 2-(piperidinyl-1-methyl)ethyl acrylate monomer (PEMA), which is a colorless liquid at room temperature with a yield of 40%.

[0126] (2) Preparation of hydrophilic monomer: 2-piperidinyl methacrylate (POEMA)

[0127] 1.0 g of 2-(piperidinyl-1-methyl)ethyl acrylate (PEMA) monomer was weighed and added to 20 mL of anhydrous dichloromethane. Stirring was carried out at 400 rpm in an ice bath to fully dissolve the mixture. 1.295 g of 3-chloroperoxybenzoic acid was then slowly added to the flask. After the solution slowly warmed to room temperature, stirring was continued for 2 hours. The reaction solution was subjected to rotary evaporation at 30°C using a water pump to remove the dichloromethane solvent. The product was then dissolved in water and extracted three times with diethyl ether in a separatory funnel. The extracted product was lyophilized to obtain 2-piperidinyl-1-methylethyl acrylate (POEMA) monomer as a pale yellow jelly with a yield of approximately 70%.

[0128] (3) Preparation of hydrophobic polymer: poly-2-piperidinyl ethyl methacrylate (PPEMA)

[0129] 1972.8 mg of 2-(piperidinyl-1-methyl)ethyl acrylate (PEMA), 111.75 mg of 4-cyanovaleric dithiobenzoate (CPADB), and 16.42 mg of azobisisobutyronitrile (AIBN) were dissolved in 2 mL of anhydrous dichloromethane to obtain a mixed solution. The mixed solution was degassed through three freeze-pump-thaw cycles and then placed in a preheated oil bath at 70°C for 12 hours. The reaction product was dialyzed against 500 mL of anhydrous methanol using a dialysis bag with a molecular weight cutoff of 1000 Da (MW: 1000) and the dialysis was repeated three times. The reaction solution was then subjected to rotary evaporation at 30°C to remove the solvent, yielding poly-2-piperidinyl methyl acrylate (PPEMA) as a magenta viscous solid in approximately 70% yield.

[0130] (4) Preparation of amphiphilic ionic copolymer: poly-2-piperidinyl ethyl methacrylate-poly-2-piperidinyl oxyethyl methacrylate block copolymer (PPEMA-b-PPOEMA)

[0131] Poly-2-piperidinylethyl methacrylate (PPEMA) was protonated with dilute hydrochloric acid. 810 mg of 2-piperidinylethyl methacrylate (POEMA), 390 mg of protonated PPEMA, and 2.8 mg of 4,4'-azobis(4-cyanovaleric acid) (ACVA) were dissolved in deionized water to give a total volume of 2 mL. The mixture was degassed using three freeze-pump-thaw cycles and then reacted in a preheated oil bath at 70°C for 12 hours. The reaction product was dialyzed against alkaline PBS buffer (MW: 1000) for 48 hours to deprotonate and then dialyzed against methanol (MW: 1000) for 24 hours to remove unpolymerized small molecules. The reaction solution was then subjected to water pump rotary evaporation using a rotary evaporator at 38°C to remove the methanol solvent, and then vacuum dried to obtain an amphiphilic ionic copolymer, which was a brown sticky solid poly-2-piperidinyl ethyl methacrylate-poly-2-oxidized piperidine ethyl methacrylate block copolymer, denoted as PPEMA-b-PPOEMA, with a yield of approximately 80%.

[0132] The prepared PEMA, POEMA, PPEMA, and PPEMA-b-PPOEMA were tested using nuclear magnetic resonance spectroscopy (NMR) with hydrogen nuclei as the measurement object. As shown in Figures 4 to 7, the nuclear magnetic resonance hydrogen spectra (1H NMR) of PEMA, POEMA, PPEMA, and PPEMA-b-PPOEMA were obtained, respectively.

[0133] Example 2

[0134] (1) Preparation of PPEMA-b-PPOEMA micelles:

[0135] 10 mg of PPEMA-b-PPOEMA polymer was dissolved in 10 mL of methanol. PPEMA-b-PPOEMA polymer has good solubility in methanol. A rotary evaporator was used to perform water pump evaporation at 38°C to remove the methanol solvent, so that the polymer formed a thin film skeleton. 10 mL of PBS buffer was then added and ultrasonic hydration was performed to form stable PPEMA-b-PPOEMA micelles.

[0136] (2) Preparation of copolymer micelle drug delivery system: amphiphilic ionic copolymer micelles loaded with azithromycin (AZM) (PPEMA-b-PPOEMA-AZM)

[0137] PPEMA-b-PPOEMA polymer and AZM drug were dissolved in methanol, and the polymer was formed into a thin film after rotary evaporation. After ultrasonic hydration in PBS to form micelles, the unencapsulated free drug was removed by low-speed centrifugation at 3000 rpm for 5 minutes to obtain amphiphilic ionic copolymer micelles loaded with AZM, which were recorded as PPEMA-b-PPOEMA-AZM.

[0138] PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles were drop-coated on a copper mesh. After the copper mesh was placed in an oven to dry, the morphological characteristics of the PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles were observed under a transmission electron microscope.

[0139] Figure 8(A) is a transmission electron microscope (TEM) image of PPEMA-b-PPOEMA micelles at a pH of 7.4 in Example 2 of the present disclosure, and Figure 8(B) is a transmission electron microscope (TEM) image of PPEMA-b-PPOEMA micelles at a pH of 6.8 in Example 2 of the present disclosure; Figure 9(A) is a transmission electron microscope (TEM) image of PPEMA-b-PPOEMA-AZM micelles at a pH of 7.4 in Example 2 of the present disclosure, and Figure 9(B) is a transmission electron microscope (TEM) image of PPEMA-b-PPOEMA-AZM micelles at a pH of 6.8 in Example 2 of the present disclosure. The scale bar in the figure is 400 nm.

[0140] As can be seen from Figure 8(A), in a PBS buffer solution at pH 7.4, the particle size of the PPEMA-b-PPOEMA micelles is approximately 10-16 nm, and as can be seen from Figure 9(A), the particle size of the PPEMA-b-PPOEMA-AZM micelles is approximately 16-25.8 nm. As can be seen from Figures 8(B) and 9(B), in a PBS buffer solution at pH 6.8, both the PPEMA-b-PPOEMA micelles and the PPEMA-b-PPOEMA-AZM micelles have disintegrated and no longer maintain a complete micellar morphology.

[0141] Dynamic light scattering (DLS) was used to detect PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles, and the particle size distribution and potential diagram of the micelles were obtained.

[0142] Figure 10 is a size distribution diagram of PPEMA-b-PPOEMA micelles in Example 2 of the present disclosure, and Figure 11 is a size distribution diagram of PPEMA-b-PPOEMA-AZM micelles in Example 2 of the present disclosure; Figure 12 is a surface potential diagram of PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles in Example 2 of the present disclosure.

[0143] As shown in Figures 10 and 12, DLS results show that the number average particle size of PPEMA-b-PPOEMA micelles is approximately 25.60±4.61 nm and the surface potential is approximately -4.34±1.94 mV. As shown in Figures 11 and 12, the number average particle size of PPEMA-b-PPOEMA-AZM micelles is approximately 47.18±2.06 nm and the surface potential is approximately -1.33±0.99 mV. The difference in particle size between dynamic light scattering (DLS) and transmission electron microscopy (TEM) is likely due to the swelling properties of the micelles in aqueous solution. After adjusting the pH of the micelle solution to 6.8, the surface potentials of PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles were 9.92±3.10 mV and 5.58±1.15 mV, respectively.

[0144] Example 3

[0145] Preparation of Nile red loaded amphiphilic ionic copolymer micelles (PPEMA-b-PPOEMA-Nilered): PPEMA-b-PPOEMA polymer and Nile red dye were dissolved in methanol, and the polymer was formed into a thin film after rotary evaporation. After ultrasonic hydration in PBS to form micelles, the unencapsulated dye was removed by low-speed centrifugation at 3000 rpm for 5 min to obtain amphiphilic ionic copolymer micelles loaded with Nile red dye, which were recorded as PPEMA-b-PPOEMA-Nilered.

[0146] 6-8 week old Balb / c mice were used to investigate the direct penetration of the material through the pulmonary mucus layer in vivo. The mice were divided into two groups and administered free Nile Red (Free Nilered) or PPEMA-b-PPOEMA-Nilered (containing an equal amount of Nile Red). Free Nilered and PPEMA-b-PPOEMA-Nilered micelles were administered to the mice via aerosolization. After 30 minutes of the experiment, the mice were anesthetized with sodium pentobarbital and fixed on a dissecting table. The lung tissue was dissected and dissected for frozen sections. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and the lung sections were observed using confocal laser scanning microscopy (CLSM).

[0147] Figure 13 is a confocal laser scanning microscope (CLSM) image of the bronchi and lungs of mice after Free Nilered was administered through the airways in Example 3 of the present disclosure, and Figure 14 is a confocal laser scanning microscope (CLSM) image of the bronchi and lungs of mice after PPEMA-b-PPOEMA-Nilered was administered through the airways in Example 3 of the present disclosure.

[0148] As shown in Figures 13 and 14, CLSM was used to observe the drug distribution in the bronchi and lungs of mice after airway administration. As shown in Figure 13, after aerosol inhalation of Free Nilered, frozen sections showed that Free Nilered was mainly distributed in a ring shape along the airways, mostly accumulated on the airway surface, and unable to penetrate into the alveoli. As shown in Figure 14, PPEMA-b-PPOEMA-Nilered micelles can deliver fluorescent drugs deeper into the lung parenchyma. PPEMA-b-PPOEMA-Nilered is white, and the cell nuclei are gray after staining with 4',6-diamidino-2-phenylindole (DAPI). It is relatively evenly distributed in the lungs, and no obvious fluorescent drug accumulation is observed around the small airways.

[0149] Example 4

[0150] 100 μL of Pseudomonas aeruginosa bacterial liquid and 300 μL of sterile Mueller-Hinton (MH) medium were placed in a 24-well plate and cultured in a 37°C constant temperature shaking incubator for 24 hours. The upper bacterial liquid and medium were removed, and the lower biofilm was retained. Fresh sterile MH medium was replaced and cultured for another 24 hours to obtain a Pseudomonas aeruginosa biofilm.

[0151] 200 μL of azithromycin (AZM) diluted with PBS buffer solution was added to the Pseudomonas aeruginosa biofilm, and the 24-well plate was placed in a 37°C constant temperature shaking incubator for 3 h. After the incubation, the biofilm was blown away and diluted with sterile PBS buffer solution for 10 5 20 μL of the solution was then spotted onto a pre-prepared MH agar plate. The plate was then incubated overnight in a 37°C constant temperature shaking incubator. The colonies on the MH agar plate were counted. The biofilm-killing activity of the material was assessed using the colony formation assay. The experiment was repeated three times, and the average and standard deviation of the results were calculated.

[0152] Example 5

[0153] The same testing method as in Example 4 was used, except that the azithromycin (AZM) drug in Example 4 was replaced with PPEMA-b-PPOEMA micelles, PPEMA-b-PPOEMA-AZM micelles, and a control group (Control) containing an equal amount of sterile PBS buffer. The substance contents in the PPEMA-b-PPOEMA and AZM groups were comparable to the contents of the respective substance components in the PPEMA-b-PPOEMA-AZM group.

[0154] The killing activity of each component on the biofilm of Pseudomonas aeruginosa was evaluated separately. Figure 15 shows the antibacterial effect of PPEMA-b-PPOEMA-AZM micelles on Pseudomonas aeruginosa biofilm in Example 5 of the present disclosure, and Figure 16 is a colony comparison diagram after the antibacterial effect test in Example 5 of the present disclosure. As shown in Figures 15 and 16, all concentrations of AZM have no bactericidal effect. It is speculated that this may be due to the barrier effect of the biofilm, which prevents the antibiotic from penetrating. As the concentration of PPEMA-b-PPOEMA micelles increases, PPEMA-b-PPOEMA micelles show a more excellent bactericidal effect. The bactericidal effect of PPEMA-b-PPOEMA-AZM micelles is the best. PPEMA-b-PPOEMA micelles are used as a pH-responsive cationic bactericide and are the main component of the bactericidal effect of PPEMA-b-PPOEMA-AZM micelles. The bactericidal rate of PPEMA-b-PPOEMA micelles at a concentration of 200 μg / mL against Pseudomonas aeruginosa biofilms was 91.6%, while the bactericidal rate of PPEMA-b-PPOEMA-AZM micelles at a concentration of 200 μg / mL against Pseudomonas aeruginosa biofilms was 94.9%.

[0155] Example 6

[0156] 200 μL of azithromycin (AZM), PPEMA-b-PPOEMA micelles, PPEMA-b-PPOEMA-AZM micelles diluted with MH medium, and a control group with an equal amount of sterile MH medium were added to the 24-well plate, and the 24-well plate was placed in a 37°C constant temperature shaking incubator for 3 h. After the incubation, the biofilm was blown away and diluted 10% with sterile PBS. 5 The solution was diluted 20 μL and spotted onto a pre-prepared MH agar plate. The plate was then incubated overnight in a 37°C constant temperature shaking incubator. The colonies on the MH agar plate were counted. The colony formation assay was used to evaluate the inhibitory effect of the copolymer micelles on Pseudomonas aeruginosa biofilms. The experiment was repeated three times, and the average and standard deviation of the results were calculated.

[0157] Figure 17 shows the antibacterial effect of PPEMA-b-PPOEMA-AZM micelles on Pseudomonas aeruginosa biofilm in Example 6 of the present disclosure, and Figure 18 is a colony comparison diagram after the antibacterial effect test in Example 6 of the present disclosure. It can be seen from Figures 17 and 18 that different concentrations of PPEMA-b-PPOEMA micelles do not statistically significantly reduce the growth of biofilm. The inhibitory effect of PPEMA-b-PPOEMA micelles on Pseudomonas aeruginosa biofilm may be affected by the bactericidal effect of sensitive acid reaction. In theory, PPEMA-b-PPOEMA micelles have no inhibitory effect on bacterial growth. When the AZM concentration in the PPEMA-b-PPOEMA-AZM micelles was increased to 200 μg / mL, the PPEMA-b-PPOEMA-AZM micelles exhibited an antibacterial effect of 97.7% against P. aeruginosa biofilms, while the antibacterial effect of AZM was only 38.0%. AZM exhibited a poor inhibitory effect against P. aeruginosa biofilms, while the PPEMA-b-PPOEMA-AZM micelles exhibited an inhibitory effect against P. aeruginosa biofilm bacteria, with the inhibitory effect accelerating with increasing concentration. Hydrophilic cations are crucial for the permeability and proper distribution of biofilms. In the PPEMA-b-PPOEMA-AZM micelles, AZM primarily exerted the antibacterial effect, while PPEMA-b-PPOEMA enhanced the permeability of the biofilm, demonstrating excellent antibacterial activity. This may be due to the enhanced drug retention within the biofilm after charge reversal in the PPEMA-b-PPOEMA-AZM micelles.

[0158] Example 7

[0159] Pseudomonas aeruginosa was inoculated into a glass-bottomed culture dish and cultured for 48 hours to form a Pseudomonas aeruginosa biofilm. The upper culture medium was removed, and PPEMA-b-PPOEMA micelles, AZM, and PPEMA-b-PPOEMA-AZM micelles were added and incubated in a 37°C incubator for 3 hours. The control group was added with an equal amount of sterile PBS buffer solution for comparison. The total system was 1 mL. The upper culture medium was removed, the dish was washed twice with PBS, and propidium iodide (PI) PBS solution was added to the Pseudomonas aeruginosa biofilm and incubated in the dark for 20 minutes. The upper dye solution was removed, the dish was washed twice with PBS, and the sample was observed using a confocal laser microscope (CLSM) in Z-stack mode. The excitation wavelength of green fluorescent protein (GFP) was 488 nm, the emission wavelength was 510 nm, and the excitation wavelength of propidium iodide (PI) was 535 nm, and the emission wavelength was 617 nm. The experimental results are shown in Figures 19 and 20.

[0160] Figure 19 is a confocal laser microscopy (CLSM) image of the incubation of Pseudomonas aeruginosa biofilm in Example 7 of the present disclosure, with 3 different fields of view randomly selected for each group, and a scale of 25 μm. Subsequently, Imaris 9.0.1 software was used for three-dimensional reconstruction analysis, and Figure 20 is the volume of surviving bacteria in the Pseudomonas aeruginosa biofilm after three-dimensional reconstruction in Example 7 of the present disclosure. As shown in the results of Figure 20, the volume of surviving bacteria in the Pseudomonas aeruginosa biofilm in the control group after incubation with PBS buffer solution was 82.03%, and AZM had no bactericidal effect on the Pseudomonas aeruginosa biofilm, and the volume of surviving bacteria was close to that of the PBS group, reaching 81.86%. The volume of surviving bacteria in the biofilm incubated with PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles was only 33.20% and 21.44% respectively. The volume of surviving bacteria calculated by three-dimensional reconstruction was consistent with the trend of the number of surviving bacteria obtained in the biofilm sterilization experiment using the plate count method.

[0161] Example 8

[0162] Forty 6-8 week old Balb / c mice were randomly divided into four groups, with 10 mice in each group. A mouse model of chronic pneumonia caused by Pseudomonas aeruginosa was established. After adaptive feeding for one week, the mice were anesthetized and inoculated with logarithmic-phase Pseudomonas aeruginosa intranasally at a rate of 20 μL per mouse, i.e., 1×10 7 CFU / mouse, and a chronic pneumonia model of Pseudomonas aeruginosa was established after 7 days. On days 8-10, mice were given PBS buffer solution (Control), PPEMA-b-PPOEMA micelles, AZM, and PPEMA-b-PPOEMA-AZM micelles for 30 minutes each. Both the AZM group and the PPEMA-b-PPOEMA-AZM micelle group contained 10 μg / mL of AZM. On day 11, mice were sacrificed, and lung tissues were obtained and ground and plated to assess bacterial burden. The tissues were ground at 100 mg / mL with PBS buffer solution, diluted 10-fold, and 20 μL × 3 were plated for culture and incubated for 24 hours.

[0163] Figure 21 is an image of bacterial burden colony growth in mouse lung tissue in Example 8 of the present disclosure, and Figure 22 is a statistical graph of bacterial burden colony growth in mouse lung tissue after treatment in Example 8 of the present disclosure. As can be seen from Figures 21 and 22, the bacterial burden colony in the mouse lung tissue of the PPEMA-b-PPOEMA-AZM micelle group after aerosolization is the lowest, demonstrating the good bactericidal effect of PPEMA-b-PPOEMA-AZM micelles.

[0164] Example 9

[0165] The cytotoxicity of PPEMA-b-PPOEMA micelles was tested by the MTT method. RAW264.7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After the cells attached to the wall, the cells in the logarithmic growth phase were seeded in 96-well plates. Each well contained approximately 8,000 RAW264.7 cells in 100 μL and cultured in a 37°C cell culture incubator containing 5% CO2 for 24 hours. The prepared PPEMA-b-PPOEMA micelles were then diluted to a certain concentration with serum-containing RPMI 1640 medium, and 100 μL was taken to replace the original medium. The control group used 100 μL of complete medium. After 24 hours of incubation, the culture medium was replaced with 100 μL of serum-free medium containing 1 mg / mL MTT. After 4 hours, the medium was discarded and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were incubated in a 37°C cell culture incubator for 10 minutes to completely dissolve the purple solid. The absorbance at 488 nm was measured using a microplate reader. The control group consisted of cells incubated in complete medium. Cell viability was calculated according to the following formula:

[0166] Cell viability (%) = [(OD sample -OD blank )] / [(OD control control-OD blank )]×100%

[0167] Figure 23 shows the cell survival rate after incubation with PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure. As shown in Figure 23, the experimental results indicate that the cell survival rate of PPEMA-b-PPOEMA micelles is almost 100% at 25 to 200 μg / mL, indicating that PPEMA-b-PPOEMA micelles are not toxic at the cellular level.

[0168] 0.5 mL of mouse venous blood was collected in an anticoagulant tube using the orbital venous plexus blood collection method, and the lower layer of blood cells was collected by centrifugation for 15 min (3000 rpm, 4 ° C). 1 mL of PPEMA-b-PPOEMA micelles, 1 mL of deionized water, and 1 mL of PBS were mixed with 20 μL of blood cells and incubated in a 37 ° C constant temperature shaking incubator for 4 hours. After centrifugation for 15 min (3000 rpm, 4 ° C), the supernatant was retained and 100 μL was added to a 96-well cell culture plate. The absorbance was measured at 576 nm. The experiment was repeated three times, and the results were averaged and standard deviations were taken. The hemolysis rate was calculated according to the following formula:

[0169] Hemolysis rate (%) = [(OD sample -OD PBS )] / [(OD ddH2O-OD PBS )]×100%

[0170] Figure 24 is the result of the hemolysis experiment of PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure, and Figure 25 is the image of the hemolysis experiment of PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure. As can be seen from Figure 25, after the mouse anticoagulated blood was co-incubated with PPEMA-b-PPOEMA micelles, there was no hemolysis visible to the naked eye. The hemolysis rate after incubation of venous blood with different concentrations of drugs was calculated based on the absorbance of the supernatant. As shown in Figure 24, the hemolysis amount of PPEMA-b-PPOEMA micelles at a concentration of 1000 μg / mL is still less than 5%. It can be considered that at this concentration, PPEMA-b-PPOEMA micelles are not hemolytic, which suggests that PPEMA-b-PPOEMA micelles have good biocompatibility.

[0171] The specific embodiments of the present disclosure described above do not limit the scope of protection of the present disclosure. Any other corresponding changes and modifications made based on the technical concept of the present disclosure should be included in the scope of protection of the claims of the present disclosure.

Claims

1. An amphiphilic ionic copolymer comprising: A block copolymer formed by block copolymerization of a hydrophobic polymer formed by a hydrophobic monomer and a hydrophilic monomer; Wherein, the hydrophilic monomer has a structure as shown in formula (A): The hydrophobic monomer has a structure as shown in formula (B): Wherein, each R is independently selected from any one of C1 to C6 alkyl groups, and adjacent R groups may be connected to form a ring.

2. The amphiphilic ionic copolymer according to claim 1, wherein R is independently selected from C1-C6 straight-chain alkanes, and the ring formed by connecting adjacent Rs can be a five-membered ring, a six-membered ring or a seven-membered ring.

3. The amphiphilic ionic copolymer according to claim 1 or 2, wherein The hydrophilic monomer includes any one of the structures shown in formulas (A1) to (A4); The hydrophobic monomer includes any one of the structures shown in formulas (B1) to (B4); 4. The amphiphilic ionic copolymer according to claim 1, wherein The polymerization degrees of the hydrophilic monomer and the hydrophobic monomer in the block copolymer are 40-60:40-60.

5. A copolymer micellar drug delivery system, comprising: Hydrophobic drugs; The amphiphilic ionic copolymer according to any one of claims 1 to 4, self-assembles with the hydrophobic drug to form a copolymer micelle drug delivery system.

6. The copolymer micellar drug delivery system according to claim 5, wherein: The hydrophobic drug is any one of lapachone, doxorubicin, camptothecin, 17-AAG, mitomycin C or indocyanine green.

7. A method for preparing the amphiphilic ionic copolymer according to any one of claims 1 to 4, comprising: Subjecting a hydrophobic monomer to a first reversible addition-fragmentation chain transfer polymerization reaction to obtain a hydrophobic polymer, wherein the hydrophobic monomer has a structure as shown in formula (B); The hydrophobic polymer is then protonated and used as a second chain transfer agent to undergo a second reversible addition-fragmentation chain transfer polymerization reaction with a hydrophilic monomer to obtain the amphiphilic ionic copolymer, wherein the hydrophilic monomer has a structure as shown in formula (A):

8. The preparation method according to claim 7, further comprising: The amphiphilic ionic copolymer obtained by the reaction is dialyzed and deprotonated in an alkaline PBS buffer solution, and then dialyzed in a methanol solution and vacuum dried to obtain a purified amphiphilic ionic copolymer.

9. The preparation method according to claim 7, further comprising: The compound represented by formula (C) is subjected to an esterification reaction with methacryloyl chloride to prepare the hydrophobic monomer; The hydrophobic monomer is oxidized to obtain the hydrophilic monomer.

10. The preparation method according to claim 7, wherein: In the first reversible addition-fragmentation chain transfer polymerization reaction, the first solvent is methanol, the first chain transfer agent is 4-cyanovaleric dithiobenzoate (CPADB), and the first initiator is azobisisobutyronitrile (AIBN); The temperature of the first reversible addition-fragmentation chain transfer polymerization reaction is 60 to 80° C., and the reaction time is 8 to 16 hours; The molar concentration ratio of the hydrophobic monomer, the first chain transfer agent and the first initiator is 50n:n:1, wherein n is 3-5.

11. The preparation method according to claim 7, wherein: In the second reversible addition-fragmentation chain transfer polymerization reaction, the second solvent is water, and the second initiator is 4,4'-azobis(4-cyanovaleric acid) (ACVA); The temperature of the second reversible addition-fragmentation chain transfer polymerization reaction is 60 to 80° C., and the reaction time is 8 to 16 hours; The molar concentration ratio of the hydrophilic monomer, the second chain transfer agent and the second initiator is 50n:n:1, wherein n is 3-5.

12. A method for preparing a copolymer micelle drug delivery system, comprising: The amphiphilic ionic copolymer according to any one of claims 1 to 4 and a hydrophobic drug are self-assembled by a thin film hydration method to obtain a copolymer micelle drug delivery system.

13. The preparation method according to claim 12, further comprising: performing low-speed centrifugation on the copolymer micelle drug delivery system to remove unencapsulated drugs; Wherein, the rotation speed of the low-speed centrifugation is 2500-5000 rpm, and the time of the low-speed centrifugation is 5-10 min.

14. Use of the copolymer micelle drug delivery system according to claim 5 or 6 in the preparation of a drug for treating bacterial biofilm infection in the lungs.

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