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

Abstract

The invention provides an amphiphilic ionic copolymer, a copolymer micelle drug delivery system and a preparation method thereof, and the amphiphilic ionic copolymer comprises a hydrophobic polymer formed by a hydrophobic monomer and a block copolymer formed by block copolymerization of a hydrophilic monomer; wherein the hydrophilic monomer has a structure as shown in a formula (A), the hydrophobic monomer has a structure as shown in a formula (B), R is independently selected from any one of C1-C6 alkyl groups, and adjacent R can be connected to form a ring. # imgabs0 #

Description

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

[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 drug delivery system for promoting the transmucosal delivery of hydrophobic drugs and a preparation method thereof.

[0002] Antibiotics account for a large proportion of clinical medications, which 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. At present, 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 limits the application of hydrophobic antibiotics to a certain extent. In order to obtain the expected therapeutic effect of the drug, it is usually necessary to administer the drug to the patient in a solubilized form. While increasing the dosage and prolonging the course of treatment, it faces 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 effect of antibiotics in the respiratory tract.

[0003] Among lung diseases, hospital-acquired pneumonia (HAP) secondary to Pseudomonas aeruginosa has a poor prognosis and a high mortality rate. It is estimated that the mortality rate of ventilator-associated pneumonia (VAP) secondary to Pseudomonas aeruginosa is as high as 32% to 42.8%. Pseudomonas aeruginosa colonizes in the bronchioles and alveoli, producing a large amount of extracellular polysaccharide matrix (EPS), which can occupy more than 90% of the total volume of Pseudomonas aeruginosa biofilm. It is mainly composed of polysaccharides, proteins and extracellular DNA. The dense bacterial biofilm composed of EPS forms a huge barrier that prevents antibiotics from penetrating. The formation of bacterial biofilm in the lungs is one of the reasons for the protracted and unhealed chronic pneumonia, which prevents neutrophils from phagocytosis, and the alkali resistance and heat resistance of bacterial biofilms further increase the difficulty of treatment. Therefore, the development of a copolymer that can carry drugs to the alveolar surface is of great significance in clinical research.

[0004]

[0005] In view of 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 technical solutions provided by the present disclosure are as follows:

[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 formula (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, a copolymer micelle drug delivery system is provided, 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 lapachone, 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 monomers are oxidized to obtain the hydrophilic monomers.

[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 to 80°C, and the reaction time is 8 to 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 to 80°C, and the reaction time is 8 to 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 min.

[0039] As a fourth aspect of the present disclosure, there is provided a use of the 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 solution, 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 of the present disclosure, a hydrophobic polymer formed by a hydrophobic monomer is used as a macromolecular chain transfer agent to form an amphiphilic copolymer with a hydrophilic monomer through block copolymerization. The surface charge is close to neutral, the particle size is small, the hydrophilic segment forms a zwitterionic surface outward, and the hydrophobic segment spontaneously forms a hydrophobic core. The biosafety is high, which provides ideal conditions for achieving the penetration and delivery of drugs by the amphiphilic ionic copolymer-drug self-assembly through the lung mucosal layer.

[0042] (2) In the embodiments of the present disclosure, due to the presence of the hydrophobic fragments, 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 inhaled 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 inhaled airway drug delivery by the copolymer micelle drug delivery system.

[0044] (4) In the embodiments of the present disclosure, 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 the biofilm penetration effect, and disassemble and release hydrophobic antibiotic drugs after breaking through the biofilm barrier, thereby achieving the efficient effect of antibiotics and exerting the synergistic antibacterial effect of the amphiphilic ionic copolymer and the antibacterial effect of the antibiotic.

[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 nuclear magnetic resonance hydrogen spectrum (1H NMR) of PEMA in Example 1 of the present disclosure;

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

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

[0052] FIG7 is a nuclear magnetic resonance hydrogen 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 pH 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 biofilm in Example 5 of the present disclosure;

[0063] FIG16 is a comparison diagram of 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 biofilm in Example 6 of the present disclosure;

[0065] FIG18 is a comparison diagram of 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 is 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 colony growth in mouse lung tissue in Example 8 of the present disclosure;

[0069] FIG22 is a statistical graph of 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 is the result of the hemolysis experiment of PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure;

[0072] FIG. 25 is an image of a hemolysis experiment of PPEMA-b-PPOEMA micelles in Example 9 of the present disclosure.

[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 combination with specific embodiments and with reference to the accompanying drawings.

[0074] At present, researchers have proposed some methods to use surfactants, polymers or carriers to increase the permeability of antibiotics in the mucus layer. Some literature reports that mucus permeable particles (MPP) can achieve mucus layer penetration. Their main characteristics are hydrophilicity, smaller than the gaps in the mucus network, and the surface charge is shielded and close to neutral, which can achieve the invisibility of the material in the mucus layer and avoid being captured by mucin and hydrophobic structure areas, thereby carrying hydrophobic drugs through the mucus layer smoothly. It is also 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, neutral charge is required to achieve mucus penetration, 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, which can freely pass through the mucin fiber network of the lung 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. It is also sensitive to pH changes, exposing the positive charge on the surface of the material under the acidic microenvironment of the biofilm, and can achieve the membrane-breaking bactericidal effect of cationic polymers and release drugs, and can exert the synergistic antibacterial effect of cationic polymers and antibiotic drugs to inhibit bacteriostasis, providing a feasible strategy for airway administration to treat bacterial biofilm infections in the lungs.

[0076] In order to achieve the above technical objectives, as a first aspect of the present disclosure, a 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 when pH = 6.8, it can trigger a response, and the degree of protonation increases sharply, which can increase the positive charge density and simulate the action mechanism 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-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, it can be 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 a hydrophilic monomer via a reversible addition-fragmentation chain transfer polymerization reaction. Preferably, the degree of polymerization of the hydrophilic segment to the hydrophobic segment in the formed polymer is 50:50.

[0087] As a second aspect of the present disclosure, a copolymer micelle drug delivery system is provided, 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 near-neutral nano-micelles in a neutral pH environment. The hydrophilic segments in the micelles form zwitterionic surfaces outward, and the hydrophobic segments spontaneously form a hydrophobic core. It can also be self-assembled with hydrophobic drugs to obtain a copolymer micelle drug delivery system, which provides ideal conditions for achieving drug penetration in 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, releases the hydrophobic drug, achieves the biomembrane penetration effect, and exerts the 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 lapachone, 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 1000Da, and molecules with a molecular weight below 1000Da 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 monomers are oxidized to obtain the hydrophilic monomers.

[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, wherein 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) using a compound as shown in formula (C) to carry out an esterification reaction with methacryloyl chloride to prepare a hydrophobic monomer, wherein the hydrophobic monomer has a structure as shown in formula (B);

[0105] (2) The hydrophobic monomer is oxidized in an ice bath to obtain a hydrophilic monomer, wherein the hydrophilic monomer has 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 acid dithiobenzoate (CPADB) as the chain transfer agent and azobisisobutyronitrile (AIBN) as the initiator, using methanol as the solvent to obtain a hydrophobic polymer. The hydrophobic polymer has a structure as 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 with 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. PBS is then 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 the embodiments of the present disclosure, the hydrophobic polymer formed by the polymerization of hydrophobic monomers is used as a macromolecular chain transfer agent, and after reversible addition-fragmentation chain transfer polymerization block copolymerization with hydrophilic monomers 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 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 the embodiments 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] Among them, the rotation speed of low-speed centrifugation is 2500-5000rpm, for example, it can be 2500rpm, 3000rpm, 3500rpm, 4000rpm, 5000rpm, etc.; the time of low-speed centrifugation is 5-10min, for example, it can be 5min, 6min, 8min, 10min, etc.

[0117] As a fourth aspect of the present disclosure, there is provided a use of the 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 solution and advantages of the present disclosure clearer and more specific, the technical solution and principle of the present disclosure are further explained below through specific embodiments combined with the accompanying drawings. It should be noted that the following specific embodiments are only used as examples, and the protection scope of the present disclosure is not limited thereto.

[0120] The test materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Specific techniques or conditions not specified in the examples are conventional methods and can be carried out 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 Bio-Tech Biotechnology Co., Ltd., and the mice used in the examples were purchased from Beijing Weitong Lihua 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, place in a 300mL round-bottom flask, add tetrahydrofuran and stir at a stirring speed of 400rpm, then weigh 10.4g of methacryloyl chloride and add dropwise to the flask. Heat the mixed solution to reflux at 80°C in tetrahydrofuran for more than 12 hours, collect the reaction solution and remove the precipitate by suction filtration, use a rotary evaporator to perform water pump rotary evaporation on the reaction solution at 35°C to remove the tetrahydrofuran solvent. Then perform vacuum distillation at 100°C to obtain 2-(piperidin-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] Weigh 1.0 g of 2-(piperidin-1-methyl)ethyl acrylate monomer (PEMA) and add it to 20 mL of anhydrous dichloromethane. Stir at 400 rpm under ice bath conditions to fully dissolve it. Then slowly add 1.295 g of 3-chloroperoxybenzoic acid to the flask. After the solution slowly warms up to room temperature, continue stirring for 2 hours. Use a rotary evaporator to perform water pump rotary evaporation on the reaction solution at 30°C to remove the dichloromethane solvent. Then, dissolve the product in water and extract it with ether three times in a separatory funnel. After freeze-drying the extracted product, a pale yellow jelly-like 2-piperidinyl methacrylate (POEMA) monomer is obtained, with a yield of about 70%.

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

[0129] 1972.8 mg of 2-(piperidin-1-methyl) ethyl acrylate monomer (PEMA), 111.75 mg of dithiobenzoic acid 4-cyanovaleric acid (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 by three freeze-pump-thaw cycles, and then placed in a 70°C preheated oil bath for 12 h to react. The reaction product was dialyzed in 500 mL of anhydrous methanol, and the dialysis bag had a molecular weight cutoff of 1000 Da (MW: 1000). The dialysis was repeated 3 times. The reaction solution was then subjected to water pump rotary evaporation at 30°C using a rotary evaporator to remove the solvent to obtain poly-2-piperidinyl methyl acrylate (PPEMA), which was a magenta viscous solid with a yield of about 70%.

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

[0131] Poly-2-piperidinyl ethyl methacrylate (PPEMA) was protonated with dilute hydrochloric acid, 810 mg of 2-piperidinyl ethyl methacrylate (POEMA), 390 mg of protonated PPEMA, and 2.8 mg of 4,4'-azobis(4-cyanovaleric acid) (ACVA) were weighed and dissolved in deionized water to obtain a mixture with a total volume of 2 mL. The mixture was degassed by 3 freeze-pump-thaw cycles, and then placed in a preheated oil bath at 70°C for 12 h. The reaction product was dialyzed in alkaline PBS buffer (MW: 1000) for 48 h to deprotonate, and dialyzed in methanol (MW: 1000) for 24 h to remove unpolymerized small molecules. The reaction solution was then subjected to water pump rotary evaporation at 38°C using a rotary evaporator to remove the methanol solvent, and then vacuum dried to obtain an amphiphilic ionic copolymer, which was a brown viscous solid poly-2-piperidinyl ethyl methacrylate-poly-2-oxidized piperidine ethyl methacrylate block copolymer, denoted as PPEMA-b-PPOEMA, with a yield of about 80%.

[0132] The prepared PEMA, POEMA, PPEMA and PPEMA-b-PPOEMA were tested by 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 rotary evaporation at 38°C to remove the methanol solvent so that the polymer formed a thin film skeleton. Then 10 mL of PBS buffer was 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] The PPEMA-b-PPOEMA polymer and AZM drug were dissolved in methanol, and the polymer was formed into a film after rotary evaporation. After ultrasonic hydration in PBS to form micelles, the unencapsulated free drugs were 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, and the scale bar in the figure is 400 nm.

[0140] As can be seen from Figure 8(A), in the PBS buffer solution at pH = 7.4, the particle size of the PPEMA-b-PPOEMA micelle is about 10-16 nm, and as can be seen from Figure 9(A), the particle size of the PPEMA-b-PPOEMA-AZM micelle is about 16-25.8 nm. As can be seen from Figures 8(B) and 9(B), in the PBS buffer solution at pH = 6.8, both the PPEMA-b-PPOEMA micelle and the PPEMA-b-PPOEMA-AZM micelle have disintegrated and no longer maintain a complete micelle 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 diagram 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 can be seen from Figures 10 and 12, the DLS results show that the number average particle size of PPEMA-b-PPOEMA micelles is about 25.60±4.61nm, and the surface potential is about -4.34±1.94mV; as can be seen from Figures 11 and 12, the number average particle size of PPEMA-b-PPOEMA-AZM micelles is about 47.18±2.06nm, and the surface potential is about -1.33±0.99mV. The difference in particle size between the dynamic light scattering (DLS) test and the transmission electron microscope (TEM) may be due to the swelling characteristics of the micelles in aqueous solution. After the pH of the micelle solution was adjusted to 6.8, the surface potentials of PPEMA-b-PPOEMA micelles and PPEMA-b-PPOEMA-AZM micelles were 9.92±3.10mV and 5.58±1.15mV, respectively.

[0144] Example 3

[0145] Preparation of amphiphilic ionic copolymer micelles loaded with Nile red (PPEMA-b-PPOEMA-Nilered): PPEMA-b-PPOEMA polymer and Nile red dye were dissolved in methanol, and the polymer was formed into a 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] 6-8w Balb / c mice were used to explore the direct penetration effect of the material through the lung mucus layer in vivo. The mice were divided into 2 groups and given free Nile red (Free Nilered) and PPEMA-b-PPOEMA-Nilered (containing an equal amount of Nile red) respectively. Free Nilered and PPEMA-b-PPOEMA-Nilered micelles were nebulized and given to mice. After 30 minutes of the experiment, the mice were anesthetized with sodium pentobarbital and fixed on the dissection table. The mouse lung tissue was dissected and peeled off to prepare frozen sections, and 4',6-diamidino-2-phenylindole (DAPI) was used to stain and locate the cell nuclei. The lung sections were observed using a laser scanning confocal microscope (CLSM).

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

[0148] As shown in Figures 13 and 14, the distribution of drugs in the bronchus and lungs of mice after airway administration was observed by CLSM. As shown in Figure 13, after atomization and inhalation of Free Nilered, the frozen section results showed that Free Nilered was mainly distributed in a ring along the airway and mostly accumulated on the airway surface, unable to penetrate into the alveoli. As shown in Figure 14, PPEMA-b-PPOEMA-Nilered micelles can deliver fluorescent drugs to deeper lung parenchyma. PPEMA-b-PPOEMA-Nilered is white, and the nucleus is gray after staining with 4',6-diamidino-2-phenylindole (DAPI). It is evenly distributed in the lungs, and no obvious accumulation of fluorescent drugs 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 Times, take 20 μL and apply it on the prepared MH bacterial agar plate, put it in a 37℃ constant temperature shaking incubator for overnight incubation, and count the colonies on the MH bacterial agar plate. The colony formation method was used to evaluate the killing activity of the material against biofilm. The experiment was repeated three times, and the average and standard deviation of the results were taken.

[0152] Example 5

[0153] The same test method as in Example 4 was used, except that the azithromycin (AZM) drug in Example 4 was replaced by PPEMA-b-PPOEMA micelles, PPEMA-b-PPOEMA-AZM micelles, and a control group (Control) in which an equal amount of sterile PBS buffer was added. The substance contents of the PPEMA-b-PPOEMA group and the AZM group were equivalent to the contents of the various substance components of the PPEMA-b-PPOEMA-AZM group.

[0154] The killing activity of each component on the biofilm of Pseudomonas aeruginosa was evaluated respectively. FIG15 shows the antibacterial effect of PPEMA-b-PPOEMA-AZM micelles on Pseudomonas aeruginosa biofilm in Example 5 of the present disclosure, and FIG16 shows the colony comparison diagram after the antibacterial effect test in Example 5 of the present disclosure. As shown in FIG15 and FIG16, all concentrations of AZM have no bactericidal effect. It is speculated that it 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-AZM 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 PPEMA-b-PPOEMA-AZM micelles to produce bactericidal effects. The bactericidal rate of PPEMA-b-PPOEMA micelles at a concentration of 200 μg / mL against Pseudomonas aeruginosa biofilm was 91.6%, while the bactericidal rate of PPEMA-b-PPOEMA-AZM micelles at a concentration of 200 μg / mL against Pseudomonas aeruginosa biofilm 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 off and diluted 10 with sterile PBS. 5 times, take 20 μL and apply it on the prepared MH bacterial agar plate, put it in a 37℃ constant temperature shaking incubator for overnight incubation, and count the colonies on the MH bacterial agar plate. The colony formation method was used to evaluate the inhibitory effect of copolymer micelles on Pseudomonas aeruginosa biofilm. The experiment was repeated three times, and the average and standard deviation of the results were taken.

[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 shows 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 biofilms, and the inhibitory effect of PPEMA-b-PPOEMA micelles on Pseudomonas aeruginosa biofilms may be affected by the bactericidal effect of sensitive acid reactions. In theory, PPEMA-b-PPOEMA micelles have no inhibitory effect on bacterial growth. When the concentration of AZM in PPEMA-b-PPOEMA-AZM micelles was increased to 200 μg / mL, the antibacterial effect of PPEMA-b-PPOEMA-AZM micelles on Pseudomonas aeruginosa biofilms could reach 97.7%, while the antibacterial effect of AZM was only 38.0%. AZM had a poor inhibitory effect on Pseudomonas aeruginosa biofilms, while PPEMA-b-PPOEMA-AZM micelles had an inhibitory effect on Pseudomonas aeruginosa biofilm bacteria, and the inhibitory effect accelerated with the increase of concentration. Hydrophilic cations are crucial for the permeability and reasonable distribution of biofilms. In PPEMA-b-PPOEMA-AZM micelles, AZM played an antibacterial role, while PPEMA-b-PPOEMA enhanced the permeability of biofilms and showed excellent antibacterial effects, which may be related to the fact that PPEMA-b-PPOEMA-AZM micelles increased the retention rate of drugs in biofilms after charge reversal.

[0158] Example 7

[0159] Pseudomonas aeruginosa was inoculated in a glass-bottomed culture dish, and a Pseudomonas aeruginosa biofilm was formed after 48 hours of culture. The upper culture medium was removed, and PPEMA-b-PPOEMA micelles, AZM, and PPEMA-b-PPOEMA-AZM micelles were added respectively 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 control, and the total system was 1mL. The upper culture medium was removed, washed twice with PBS, and propidium iodide (PI) PBS solution was added to the Pseudomonas aeruginosa biofilm and incubated for 20 minutes under dark conditions. The upper dye solution was removed, washed twice with PBS, and observed using a confocal laser microscope (CLSM) in Z-stack mode. The green fluorescent protein (GFP) excitation wavelength was 488nm, the emission wavelength was 510nm, the propidium iodide (PI) excitation wavelength was 535nm, and the emission wavelength was 617nm. The experimental results are shown in Figures 19 and 20.

[0160] Figure 19 is a confocal laser microscope (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 the scale is 25 μm. Imaris 9.0.1 software was then 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. The results of Figure 20 show that the volume of surviving bacteria in the Pseudomonas aeruginosa biofilm in the control group after incubation with PBS buffer solution is 82.03%, and AZM has no bactericidal effect on the Pseudomonas aeruginosa biofilm, and the volume of surviving bacteria is 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 is only 33.20% and 21.44%, respectively. The volume of surviving bacteria calculated by three-dimensional reconstruction is consistent with the trend of the number of surviving bacteria obtained in the plate count method biofilm sterilization experiment.

[0161] Example 8

[0162] Forty 6-8w Balb / c mice were randomly divided into four groups, with 10 mice in each group, to establish a mouse Pseudomonas aeruginosa chronic pneumonia infection model. After adaptive feeding for one week, logarithmic phase Pseudomonas aeruginosa were inoculated intranasally after anesthesia, with 20 μL per mouse, i.e., 1×10 7 CFU / mouse, and the 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 nebulization, and the groups were nebulized for 30 minutes. Among them, the AZM group and the PPEMA-b-PPOEMA-AZM micelle group both contained 10μg / mL of AZM. On the 11th day, the mice were killed, and the mouse lung tissues were taken. After grinding, the bacterial burden was evaluated by plating. According to 100mg / mL, PBS buffer solution was added for grinding, and 20μL×3 was taken for plating culture after dilution 10 times, and cultured 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. It can be seen from Figures 21 and 22 that the bacterial burden colony in the mouse lung tissue after atomization in the PPEMA-b-PPOEMA-AZM micelle group is the least, showing 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 adhered to the wall, the cells in the logarithmic growth phase were inoculated in a 96-well plate, with 100 μL per well containing about 8,000 RAW264.7 cells, and cultured in a 37°C cell culture incubator containing 5% CO2 for 24 hours. Then the prepared PPEMA-b-PPOEMA micelles were 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 original culture medium was replaced with 100 μL of serum-free culture medium containing 1 mg / mL MTT. After 4 hours, the culture 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 with an ELISA reader. The control group was cells incubated with complete culture medium. The cell survival rate 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 non-toxic at the cellular level.

[0168] The orbital venous plexus blood collection method was used to collect 0.5 mL of mouse venous blood in an anticoagulant tube, 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 h. 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 according to the absorbance of the supernatant. As shown in Figure 24, the hemolysis amount of PPEMA-b-PPOEMA micelles at a concentration of 1000μg / 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 implementations of the present disclosure described above do not constitute a limitation on the protection scope of the present disclosure. Any other corresponding changes and modifications made according to the technical concept of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Claims

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. 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. 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); 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. 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. 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. 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): 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. 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. 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 60-80°C, and the reaction time is 8-16h; 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. 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 60-80°C, and the reaction time is 8-16h; 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. 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. The preparation method according to claim 12, further comprising: The copolymer micelle drug delivery system is subjected to low-speed centrifugation 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 minutes. Use of the copolymer micelle drug delivery system as claimed in claim 5 or 6 in the preparation of a drug for treating bacterial biofilm infection in the lungs.

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