Preparation method of copper ion mediated agglomerated microdroplets with double antibacterial effects

Abstract

The invention discloses a preparation method of copper ion-mediated condensed micro-droplets with double antibacterial effects, and relates to a preparation method of condensed micro-droplets with double antibacterial effects. The invention provides a preparation method of copper ion-mediated condensed micro-droplets with double antibacterial effects, which comprises the following steps: on the basis of a membrane-free condensed body, firstly constructing a phospholipid membrane to protect the condensed body from being damaged, and then adding functional phospholipid and copper ions to obtain a condensed body with a copper ion catalysis effect on the surface, so that a drug does not need to be loaded; in the presence of a substrate, the reaction is performed to generate active molecules NO and hydroxyl radicals to kill bacteria. The preparation method is mild in condition, simple to operate and efficient; the agglomerated microdroplet is composed of a biocompatible material (polysaccharide), can be spontaneously formed in a buffer solution, and is easy to degrade. The agglomerated microdroplet has better environmental friendliness and biosafety, and can more comprehensively destroy the defense mechanism of bacteria and reduce the generation of bacterial drug resistance.

Description

Technical Field

[0001] The invention relates to a method for preparing condensed microdroplets with dual antibacterial effects. Background Art

[0002] In recent years, foodborne diseases caused by pathogenic Escherichia coli have frequently broken out, posing a serious threat to human health. Although antibiotics are effective in killing Escherichia coli, the emergence of drug-resistant bacteria (i.e., "superbugs") with the abuse of antibiotics has become a major challenge to global public health. Pathogenic bacteria usually adhere to host tissues and cause lesions by specifically recognizing sugar molecules on the surface of host cells through lectins, thereby infecting host tissues and causing lesions. Therefore, in-depth research on the interaction between pathogens and host cell surface receptors not only helps to reveal the pathogenic mechanism of infectious diseases, but also provides new strategies and ideas for the specific detection of pathogens and the treatment of infectious diseases.

[0003] Traditional antibiotic development primarily targets specific bacterial targets, such as the cell wall, protein synthesis, or DNA replication. However, due to the rapid evolution and adaptability of bacteria, the limitations of this strategy are becoming increasingly apparent. In recent years, researchers have begun exploring novel antimicrobial strategies based on bacterial-host interactions. For example, these strategies inhibit infection by interfering with bacterial adhesion, invasion, or virulence factors, rather than directly killing the bacteria. This approach not only reduces the selective pressure of antibiotics but also has the potential to delay the development of drug resistance.

[0004] The increasing prevalence of bacterial drug resistance is forcing researchers to seek novel antimicrobial strategies. Membraneless aggregates, as emerging biomolecular assemblies, offer new insights into bacterial virulence regulation, stress responses, and drug resistance mechanisms. By targeting membraneless aggregates or mimicking their functions, novel antimicrobial materials and treatments can be developed, offering innovative solutions to address the threat of drug-resistant bacteria. This research direction not only holds significant scientific significance but also offers broad application prospects.

[0005] Although membraneless aggregates can be used as ideal drug carriers, they rely on weak interactions such as hydrophobic and electrostatic interactions to maintain their structure. They are prone to disintegration in the complex physiological environment of high ionic strength and enzymatic degradation, leading to premature leakage of antimicrobial components. The lack of a physical barrier makes it difficult to control the drug release rate, leading to a "burst effect" where excessive drug is released in a short period of time and an inability to maintain an effective antimicrobial concentration. The self-assembly process of membraneless aggregates may repel hydrophobic or charged antimicrobial molecules, resulting in low encapsulation efficiency (typically <30%). Summary of the Invention

[0006] In order to address the deficiencies of the existing prior art, the present invention proposes a method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets. The present invention is based on membraneless coacervates. A phospholipid membrane is first constructed to protect the coacervate from damage. Then, functional phospholipids and copper ions are added to obtain a coacervate with copper ion catalysis on the surface. No drug loading is required. In the presence of a substrate, a reaction occurs to produce active molecules NO and hydroxyl free radicals to kill bacteria.

[0007] The method for preparing copper ion-mediated dual antibacterial efficacy coacervate microdroplets of the present invention is carried out according to the following steps:

[0008] Step 1: Preparation of membrane-free microdroplet solution

[0009] (1) Synthesis of carboxylated dextran: succinic anhydride was mixed with dextran, dimethyl sulfoxide (DMSO) was added, and the mixture was heated to 55-60°C in an oil bath and stirred to completely dissolve the succinic anhydride and dextran. 4-dimethylaminopyridine (DMAP) was then added and stirred at 55-60°C for 11-12 hours. The reaction solution was diluted with deionized water and dialyzed. Finally, the mixture was filtered and freeze-dried to obtain carboxylated dextran.

[0010] The mass ratio of the succinic anhydride, dextran and 4-dimethylaminopyridine is 185:100:5;

[0011] The ratio of the mass of the succinic anhydride to the volume of dimethyl sulfoxide is 185 mg:15 mL;

[0012] (2) Preparation of diethylaminoethyl dextran solution: Dissolve diethylaminoethyl dextran in phosphate buffer solution to prepare a 9-10 mg / mL diethylaminoethyl dextran solution;

[0013] (3) Preparation of carboxylated dextran solution: dissolve carboxylated dextran in phosphate buffer solution to prepare a 9-10 mg / mL carboxylated dextran solution;

[0014] (4) adding the diethylaminoethyl dextran solution to the carboxylated dextran solution and shaking it using a vortex shaker to obtain a film-free microdroplet solution;

[0015] The volume ratio of the diethylaminoethyl dextran solution to the carboxylated dextran solution is 7:3-4;

[0016] Step 2: Preparation of membrane condensation solution

[0017] (1) Preparation of dioleoylphosphatidylcholine (DOPC) solution: Dissolve dioleoylphosphatidylcholine in anhydrous ethanol to obtain a 18-20 mg / mL dioleoylphosphatidylcholine solution;

[0018] (2) adding the dioleoylphosphatidylcholine solution to the membrane-free microdroplet solution prepared in step 2, and allowing the solution to stand at room temperature to obtain a membrane-containing condensation solution;

[0019] The volume ratio of the dioleoylphosphatidylcholine solution to the membraneless microdroplet solution is 6-8:100;

[0020] Step 3. Preparation of functionalized coagulation suspension

[0021] (1) Preparation of functional phospholipid solution: Dissolve the functional phospholipid in anhydrous ethanol solution to prepare a 18-20 mg / mL functional phospholipid solution;

[0022] (2) adding the functional phospholipid solution to the membrane-forming coacervate solution obtained in step 2, shaking the solution using a vortex shaker, and then allowing the solution to stand to obtain a functionalized microdroplet suspension;

[0023] The volume ratio of the functional phospholipid solution to the membrane-forming coacervate solution is 4-6:100;

[0024] Step 4. Preparation of copper ion-mediated coacervation droplets

[0025] The functionalized microdroplet suspension obtained in step 3 is added to the CuCl2 solution, shaken using a vortex shaker, and then allowed to stand to obtain a copper ion-mediated dual antibacterial efficacy condensed microdroplet solution;

[0026] The concentration of the CuCl2 solution is 1.5-2 mg / mL;

[0027] The volume ratio of the CuCl2 solution to the functionalized microdroplet suspension is 3-4:100.

[0028] The beneficial effects of the present invention compared to the prior art are:

[0029] 1. The present invention constructs a primitive cell model based on the membrane-less unstable condensed droplet structure. By self-assembling a phospholipid membrane on the surface of the condensed droplets, stable condensed droplets with a membrane structure are successfully obtained. The introduction of the phospholipid membrane not only significantly improves the stability of the condensed droplets, but also can wrap macromolecules such as glucose oxidase inside the condensed droplets, so that glucose oxidase and the like can stably carry out enzyme-catalyzed reactions inside the droplets, thereby giving the condensed droplets the function of a local reactor. In addition, by modifying the surface of the phospholipid membrane with functionalized phospholipids, the functionality of the condensed droplets is further enhanced. The functionalized phospholipid has an orthohydroxy structure, which can efficiently chelate copper ions on the surface of the condensed droplets through the coordination effect of the orthohydroxyl groups with metal ions; under the catalysis of copper ions, the addition of glucose can initiate a cascade reaction to generate hydroxyl radicals with strong oxidizing properties. The synergistic effect of hydroxyl radicals and nitric oxide can effectively inhibit or kill bacteria, showing significant antibacterial effects. This design not only realizes the structural transformation of the condensed droplets from membrane-free to membrane-containing, but also endows the condensed droplets with multiple catalytic capabilities and antibacterial functions through functional modification, providing innovative ideas for the development of new bionic antibacterial materials.

[0030] 2. The present invention discloses a method and approach for functionalized condensed droplets to simulate cells killing bacteria. The present invention constructs a functionalized membrane-enclosed condensate with copper ions on the surface. The copper ions act as enzyme proteins on the membrane surface, which can catalyze GSNO to produce NO, and can also cooperate with the glucose oxidase inside the condensate to catalyze glucose to produce hydroxyl radicals; this simulates the inducible nitric oxide synthase (iNOS) in macrophages being activated under the stimulation of inflammatory signals (such as IFN-γ, LPS), catalyzing L-arginine and oxygen to produce NO and citrulline. And the NADPH oxidase on the macrophage membrane converts oxygen (O2) into superoxide anions (O2 - ), which is then converted into hydrogen peroxide (H2O2) by superoxide dismutase (SOD). 2+ Under catalysis (Fenton-like reaction), H2O2 is further decomposed into highly reactive ·OH.

[0031] 3. The coacervate droplets of the present invention are artificial cell models constructed based on aggregate camouflage. The preparation method is mild, simple, and highly efficient. Composed of biocompatible materials (polysaccharides), the coacervate droplets spontaneously form in buffer solutions and are easily degradable. Compared to traditional chemical fungicides, the coacervate droplets are more environmentally friendly and biosafe.

[0032] 4. The condensed droplets of the present invention are formed by liquid-liquid phase separation, which can highly concentrate specific molecules (such as glucose and GSNO, the precursor molecules for producing ·OH and NO) inside or on the surface of the droplets. This local high concentration effect can significantly enhance the generation efficiency of ·OH and NO, and form a high-concentration bactericidal environment around the droplets, thereby killing bacteria more effectively. Hydroxyl radicals (·OH) and nitric oxide (NO) have different bactericidal mechanisms. ·OH destroys the cell structure by oxidizing bacterial proteins, lipids and DNA, while NO inhibits bacterial growth by interfering with bacterial metabolism and signaling pathways. Condensed droplets can simultaneously produce these two active molecules, exerting a synergistic bactericidal effect and improving the antibacterial effect. Through synergistic effects, condensed droplets can more comprehensively destroy the bacterial defense mechanism and reduce the development of bacterial resistance. It can also simulate the bactericidal mechanism of the natural immune system (when the human immune system responds to bacterial infections, it produces ·OH and NO through immune cells such as macrophages to kill pathogens). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is an optical microscope image of the membraneless condensed droplets obtained in Example 1;

[0034] Figure 2 This is an optical microscope image of the membrane-coated droplets obtained in Example 1;

[0035] Figure 3 This is an optical microscope image of the functionalized coacervate droplets obtained in Example 1;

[0036] Figure 4 This is an optical microscope image of the copper ion-mediated condensed droplets obtained in Example 1;

[0037] Figure 5 This is a laser confocal microscope image of membrane-coated droplets;

[0038] Figure 6 This is a 3D laser confocal microscopy image of the membrane-coated droplets obtained in Example 1;

[0039] Figure 7 This is a picture of the inhibition zone of Escherichia coli mediated by copper ion-mediated coacervate droplets;

[0040] Figure 8 This is a picture of the inhibition zone of copper ion-mediated coacervate droplets against Staphylococcus aureus;

[0041] Figure 9 This is a scanning electron microscope image of a membrane-free condensed droplet;

[0042] Figure 10 This is a scanning electron microscope image of a membrane-coated droplet;

[0043] Figure 11This is a scanning electron microscope image of functionalized coacervate droplets;

[0044] Figure 12 This is a scanning electron microscopy image of copper ion-mediated condensed droplets;

[0045] Figure 13 This is a diagram showing the adhesion and killing effects of copper ion-mediated coacervate droplets on bacteria under confocal microscopy. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is not limited to the specific implementation methods listed below, but also includes any reasonable combination of the specific implementation methods.

[0047] Specific embodiment 1: The preparation method of copper ion-mediated dual antibacterial effect coacervate droplets of this embodiment is carried out according to the following steps:

[0048] Step 1: Preparation of membrane-free microdroplet solution

[0049] (1) Synthesis of carboxylated dextran: succinic anhydride was mixed with dextran, dimethyl sulfoxide (DMSO) was added, and the mixture was heated to 55-60°C in an oil bath and stirred to completely dissolve the succinic anhydride and dextran. 4-dimethylaminopyridine (DMAP) was then added and stirred at 55-60°C for 11-12 hours. The reaction solution was diluted with deionized water and dialyzed. Finally, the mixture was filtered and freeze-dried to obtain carboxylated dextran.

[0050] The mass ratio of the succinic anhydride, dextran and 4-dimethylaminopyridine is 185:100:5;

[0051] The ratio of the mass of the succinic anhydride to the volume of dimethyl sulfoxide is 185 mg:15 mL;

[0052] (2) Preparation of diethylaminoethyl dextran solution: Dissolve diethylaminoethyl dextran in phosphate buffer solution to prepare a 9-10 mg / mL diethylaminoethyl dextran solution;

[0053] (3) Preparation of carboxylated dextran solution: dissolve carboxylated dextran in phosphate buffer solution to prepare a 9-10 mg / mL carboxylated dextran solution;

[0054] (4) adding the diethylaminoethyl dextran solution to the carboxylated dextran solution and shaking it using a vortex shaker to obtain a film-free microdroplet solution;

[0055] The volume ratio of the diethylaminoethyl dextran solution to the carboxylated dextran solution is 7:3-4;

[0056] Step 2: Preparation of membrane condensation solution

[0057] (1) Preparation of dioleoylphosphatidylcholine (DOPC) solution: Dissolve dioleoylphosphatidylcholine in anhydrous ethanol to obtain a 18-20 mg / mL dioleoylphosphatidylcholine solution;

[0058] (2) adding the dioleoylphosphatidylcholine solution to the membrane-free microdroplet solution prepared in step 2, and allowing the solution to stand at room temperature to obtain a membrane-containing condensation solution;

[0059] The volume ratio of the dioleoylphosphatidylcholine solution to the membraneless microdroplet solution is 6-8:100;

[0060] Step 3. Preparation of functionalized coagulation suspension

[0061] (1) Preparation of functional phospholipid solution: Dissolve the functional phospholipid in anhydrous ethanol solution to prepare a 18-20 mg / mL functional phospholipid solution;

[0062] (2) adding the functional phospholipid solution to the membrane-forming coacervate solution obtained in step 2, shaking the solution using a vortex shaker, and then allowing the solution to stand to obtain a functionalized microdroplet suspension;

[0063] The volume ratio of the functional phospholipid solution to the membrane-forming coacervate solution is 4-6:100;

[0064] Step 4. Preparation of copper ion-mediated coacervation droplets

[0065] The functionalized microdroplet suspension obtained in step 3 is added to the CuCl2 solution, shaken using a vortex shaker, and then allowed to stand to obtain a copper ion-mediated dual antibacterial efficacy condensed microdroplet solution;

[0066] The concentration of the CuCl2 solution is 1.5-2 mg / mL;

[0067] The volume ratio of the CuCl2 solution to the functionalized microdroplet suspension is 3-4:100.

[0068] This embodiment has the following beneficial effects:

[0069] 1. This embodiment is based on the unstable membrane-free condensed droplet structure to construct a primitive cell model. By self-assembling a phospholipid membrane on the surface of the condensed droplets, a stable condensed droplet with a membrane structure was successfully obtained. The introduction of the phospholipid membrane not only significantly improves the stability of the condensed droplets, but also can wrap macromolecules such as glucose oxidase inside the condensed droplets, so that glucose oxidase and the like can stably carry out enzyme-catalyzed reactions inside the droplets, thereby giving the condensed droplets the function of a local reactor. In addition, by modifying the surface of the phospholipid membrane with functionalized phospholipids, the functionality of the condensed droplets is further enhanced. The functionalized phospholipid has an orthohydroxy structure, which can efficiently chelate copper ions on the surface of the condensed droplets through the coordination effect of orthohydroxyl groups with metal ions; under the catalysis of copper ions, the addition of glucose can initiate a cascade reaction to generate hydroxyl radicals with strong oxidizing properties. The synergistic effect of hydroxyl radicals and nitric oxide can effectively inhibit or kill bacteria, showing significant antibacterial effects. This design not only realizes the structural transformation of the condensed droplets from membrane-free to membrane-containing, but also endows the condensed droplets with multiple catalytic capabilities and antibacterial functions through functional modification, providing innovative ideas for the development of new bionic antibacterial materials.

[0070] 2. This embodiment discloses a method and approach for functionalized condensed droplets to simulate cells killing bacteria. The surface of the functionalized membrane-enclosed condensate constructed in this embodiment has copper ions. The copper ions act as enzyme proteins on the membrane surface, which can catalyze GSNO to produce NO, and can also cooperate with the glucose oxidase inside the condensate to catalyze glucose to produce hydroxyl radicals; this simulates the inducible nitric oxide synthase (iNOS) in macrophages being activated under the stimulation of inflammatory signals (such as IFN-γ, LPS), catalyzing L-arginine and oxygen to produce NO and citrulline. And the NADPH oxidase on the macrophage membrane converts oxygen (O2) into superoxide anions (O2 - ), which is then converted into hydrogen peroxide (H2O2) by superoxide dismutase (SOD). 2 + Under catalysis (Fenton-like reaction), H2O2 is further decomposed into highly reactive ·OH.

[0071] 3. The coacervate droplets of this embodiment are artificial cell models constructed based on aggregate camouflage. The preparation method is mild, simple, and efficient. Composed of biocompatible materials (polysaccharides), the coacervate droplets can spontaneously form in buffer solutions and are easily degradable. Compared with traditional chemical fungicides, the coacervate droplets are more environmentally friendly and biosafe.

[0072] 4. In this embodiment, the condensed droplets are formed by liquid-liquid phase separation, which can highly concentrate specific molecules (such as glucose and GSNO, the precursor molecules for producing ·OH and NO) inside or on the surface of the droplets. This local high concentration effect can significantly enhance the generation efficiency of ·OH and NO, and form a high-concentration bactericidal environment around the droplets, thereby killing bacteria more effectively. Hydroxyl radicals (·OH) and nitric oxide (NO) have different bactericidal mechanisms. ·OH destroys the cell structure by oxidizing bacterial proteins, lipids and DNA, while NO inhibits the growth of bacteria by interfering with their metabolism and signaling pathways. Condensed droplets can produce these two active molecules at the same time, exerting a synergistic bactericidal effect and improving the antibacterial effect. Through synergistic action, condensed droplets can more comprehensively destroy the bacterial defense mechanism and reduce the development of bacterial resistance. It can also simulate the bactericidal mechanism of the natural immune system (when the human immune system responds to bacterial infections, it produces ·OH and NO through immune cells such as macrophages to kill pathogens).

[0073] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that: in step 1 (1), the dialysis uses a dialysis bag with a molecular weight cut-off of 8000-14000, and the dialysis is carried out for three days.

[0074] Specific embodiment three: This embodiment differs from specific embodiment one or two in that: the pH of the phosphate buffer solution in step one (2) is 7.5 and the concentration is 0.01 mol / L.

[0075] Specific embodiment 4: This embodiment differs from any one of specific embodiments 1 to 3 in that: the pH of the phosphate buffer solution in step 1 (3) is 7.5 and the concentration is 0.01 mol / L.

[0076] Specific embodiment 5: The difference between this embodiment and any one of specific embodiments 1 to 4 is that: Step 1 (4) uses a vortex shaker to shake for 20 to 30 seconds.

[0077] Specific embodiment 6: This embodiment differs from any one of specific embodiments 1 to 5 in that: Step 2 (2) is to stand at room temperature for 2-3 hours.

[0078] Specific embodiment seven: This embodiment differs from any one of specific embodiments one to six in that: the functional phospholipid in step three (1) is DSPE-PEG-DA.

[0079] Specific embodiment eight: This embodiment differs from any one of specific embodiments one to seven in that: in step three (2), the mixture is shaken for 30 seconds using a vortex shaker and then allowed to stand for 1 hour.

[0080] Specific embodiment 9: This embodiment differs from any one of specific embodiments 1 to 8 in that: in step 4, the mixture is shaken for 30 seconds using a vortex shaker and then allowed to stand for 1 hour.

[0081] Specific embodiment ten: This embodiment differs from any one of specific embodiments one to nine in that: in step one (1), dialysis is performed for three days.

[0082] Example 1

[0083] The preparation method of copper ion-mediated dual antibacterial efficacy coacervate microdroplets of this embodiment is carried out according to the following steps:

[0084] Step 1: Preparation of membrane-free microdroplet solution

[0085] (1) Synthesis of carboxylated dextran: Succinic anhydride was mixed with dextran, dimethyl sulfoxide (DMSO) was added, and the mixture was heated to 60°C in an oil bath and stirred to completely dissolve the succinic anhydride and dextran. 4-dimethylaminopyridine (DMAP) was then added and stirred at 60°C for 12 h. The reaction solution was diluted with deionized water and dialyzed. Finally, the mixture was filtered and freeze-dried to obtain carboxylated dextran.

[0086] The mass ratio of the succinic anhydride, dextran and 4-dimethylaminopyridine is 185:100:5;

[0087] The ratio of the mass of the succinic anhydride to the volume of dimethyl sulfoxide is 185 mg:15 mL;

[0088] The dialysis was performed using a dialysis bag with a molecular weight cut-off of 8K-14K for three days;

[0089] (2) Preparation of DEAE-dextran solution: Dissolve 10 mg of DEAE-dextran in 1 mL of phosphate buffer solution to prepare a 10 mg / mL DEAE-dextran solution.

[0090] The pH of the phosphate buffer solution is 7.5 and the concentration is 0.01 mol / L;

[0091] (3) Preparation of carboxylated dextran solution: Dissolve 10 mg of carboxylated dextran in 1 mL of phosphate buffer solution to prepare a 10 mg / mL carboxylated dextran solution;

[0092] The pH of the phosphate buffer solution is 7.5 and the concentration is 0.01 mol / L;

[0093] (4) Add 70 μL of diethylaminoethyl dextran solution to 30 μL of carboxylated dextran solution and vortex for 20–30 s to obtain a film-free microdroplet solution; Figure 1 This is an optical microscope image of the membrane-free coagulated droplets obtained in Example 1. It can be seen that the surface morphology of the agglomerates is smooth and the size distribution is uniform; Figure 9 The scanning electron microscope image of the membrane-free condensed droplets obtained in Example 1 shows that the surface is rough and wrinkled. This is because the external environment destroys the condensates without the protection of the membrane.

[0094] Step 2: Preparation of membrane condensation solution

[0095] (1) Preparation of dioleoylphosphatidylcholine (DOPC) solution: Dissolve 20 mg of DOPC in 1 mL of anhydrous ethanol to obtain a 20 mg / mL DOPC solution;

[0096] (2) Add 8 μL of dioleoylphosphatidylcholine solution to 100 μL of the membrane-free microdroplet solution prepared in step 2, and let it stand at room temperature for 3 h to obtain a membrane-containing condensation solution; Figure 2 This is an optical microscope image of the film-coated droplet obtained in Example 1. It can be seen that the surface is still smooth; Figure 5 This is a laser confocal microscope image of membrane-coated droplets; Figure 5 Figure a is a laser confocal microscopy image of the membrane-coated microdroplets obtained in Example 1, in which DEAE-dextran is labeled with green fluorescent FITC; Figure b is a laser confocal microscopy image of the membrane-coated microdroplets obtained in Example 1, in which the phospholipid membrane is labeled with red fluorescent Dil; Figure 6 This is a 3D laser confocal microscopy image of the membrane-containing condensed droplets obtained in Example 1, in which DEAE-dextran was labeled with green fluorescent FITC and the phospholipid membrane was labeled with red fluorescent Dil. It can be seen from the figure that the phospholipid membrane is relatively densely distributed on the surface of the condensed droplets, the outer circle of the condensed droplets shows a uniform red color, and the interior of the condensed droplets shows a uniform green fluorescence. Figure 10 The scanning electron microscope image of the membrane-coated droplets obtained in Example 1 shows that the surface is smooth and wrinkle-free. This is because the phospholipid membrane protects the droplets from damage by the external environment.

[0097] Step 3. Preparation of functionalized coagulation suspension

[0098] (1) Preparation of functional phospholipid solution: Dissolve 20 mg of functional phospholipid in 1 mL of anhydrous ethanol to prepare a 20 mg / mL functional phospholipid solution;

[0099] The functional phospholipid is DSPE-PEG-DA;

[0100] (2) Add 6 μL of the functional phospholipid solution to 100 μL of the membrane-forming coacervate solution obtained in step 2, vortex for 30 seconds, and then let it stand for 1 hour to obtain a functionalized microdroplet suspension; Figure 3 This is an optical microscope picture of the functionalized coacervate droplets obtained in Example 1. It can be seen that the surface is still smooth and evenly dispersed. Figure 11 The scanning electron microscopy image of the functionalized coacervate droplets obtained in Example 1 shows that the surface is smooth and wrinkle-free. The addition of functional phospholipids does not destroy the integrity of the phospholipid membrane. Therefore, the protection of the phospholipid membrane can prevent the coacervate from being damaged by the external environment.

[0101] Step 4. Preparation of copper ion-mediated coacervation droplets

[0102] Add 4 μL of the functionalized microdroplet suspension obtained in step 3 to 100 μL of the CuCl2 solution, shake it for 30 seconds using a vortex shaker, and then let it stand for 1 hour to obtain a copper ion-mediated dual antibacterial efficacy condensed microdroplet solution; the concentration of the CuCl2 solution is 2 mg / mL. Figure 4 This is an optical microscope picture of the copper ion-mediated coacervate droplets obtained in Example 1. It can be seen that the coacervates are aggregated together. Figure 12 The scanning electron microscopy image of the copper ion-mediated coacervate droplets obtained in Example 1 shows that the coacervate droplets aggregate together, but do not fuse together due to the protection of the phospholipid membrane.

[0103] Test of the inhibitory or killing effect of copper ion-mediated coacervation droplets on bacteria:

[0104] 1. Culture of Escherichia coli and Staphylococcus aureus:

[0105] (1) The strain was placed in LB liquid medium and cultured in a shaker at 37°C and 150 rpm until the exponential growth phase;

[0106] (2) The cultured E. coli was washed with 0.01-0.05 mol / L phosphate buffer solution, the bacteria were collected by centrifugation, and redispersed in sterile phosphate buffer solution to obtain 10 8 ~10 9 CFU / mL bacterial solution; sterile phosphate buffer solution pH 7.4, concentration 0.01mol / L;

[0107] (3) The collected bacteria were labeled with 0.1 mg / mL DAPI solution (4',6-diamidino-2-phenylindole) to make the bacteria emit blue fluorescence under laser confocal microscopy;

[0108] The two bacterial solutions of Escherichia coli and Staphylococcus aureus were mixed with the dual antibacterial efficacy condensed microdroplet solution at a volume ratio of 1:10 to 100;

[0109] 2. Observe the inhibitory or killing effect of copper ion-mediated condensed droplet dual antibacterial efficacy factors on bacteria through solid culture medium inhibition zone experiment.

[0110] Inoculate the two cultured bacteria onto a solid medium in a sterile environment. Use a ball bearing to evenly distribute the bacteria across the solid medium. Place antibacterial cotton in the center of the solid medium. Add equal amounts of copper ion-mediated coacervation droplets onto the cotton. Perform three separate experiments: Group A (glucose only), Group B (GSNO (S-nitrosoglutathione) only), and Group C (glucose and GSNO simultaneously). Culture the cells in an incubator at 37°C and 150 rpm for 24 hours. Observe and photograph the cells. Figure 7 This is a picture of the inhibition zone of Escherichia coli mediated by copper ion-mediated coacervate droplets; Figure 7 Figure a shows the inhibition zone of E. coli produced by copper ion-mediated condensed droplets producing only ·OH, Figure b shows the inhibition zone of E. coli produced by copper ion-mediated condensed droplets producing only NO, and Figure c shows the inhibition zone of E. coli produced by copper ion-mediated condensed droplets producing both ·OH and NO. Figure 7 There are three groups, A, B and C, for Escherichia coli. It can be seen that the antibacterial effect of group A and group B on Escherichia coli is not very obvious because they only have a single antibacterial factor, while group C with dual antibacterial efficacy has a significantly improved antibacterial effect on Escherichia coli. Figure 8 Figure a shows the inhibition zone of S. aureus produced by copper ion-mediated condensed microdroplets only producing ·OH; Figure b shows the inhibition zone of S. aureus produced by copper ion-mediated condensed microdroplets only producing NO; Figure c shows the inhibition zone of S. aureus produced by copper ion-mediated condensed microdroplets simultaneously producing ·OH and NO; Figure 8 These are three groups, A, B, and C, for Staphylococcus aureus. It can be seen that the antibacterial effects of only a single antibacterial factor in group A and group B on Staphylococcus aureus are not very obvious, while the antibacterial effect of group C, which has dual antibacterial efficacy, on Staphylococcus aureus is significantly improved; this is because hydroxyl radicals (·OH) and nitric oxide (NO) have different bactericidal mechanisms. ·OH destroys the cell structure by oxidizing bacterial proteins, lipids, and DNA, while NO inhibits bacterial growth by interfering with bacterial metabolism and signaling pathways. Coacervated droplets can produce these two active molecules at the same time, exerting a synergistic bactericidal effect and improving the antibacterial effect. Through synergistic effects, coacervated droplets can more comprehensively destroy the bacterial defense mechanism and reduce the development of bacterial resistance.

[0111] 3. Use laser confocal microscopy to observe the location of copper ion-mediated condensed droplets and bacteria, as well as their inhibitory or killing effects. Take a 5μm suspension of Escherichia coli labeled with DAPI solution and add it to the copper ion-mediated condensed droplet solution. Let the bacteria and copper ion-mediated condensed droplets fully incubate for 30 minutes, then add 0.2μL of freshly prepared propidium iodide (PI) stain, stir at low speed (150r / min), and stain the solution in the dark for 30 minutes. The life and death of the bacteria are observed by laser confocal microscopy. Among them, the concentration of propidium iodide is 1mg / mL. The incubated copper ion-mediated condensed droplets are dropped on a small dish, and then glucose and GSNO are added. Figure 13 This is a diagram showing the adhesion and killing effects of copper ion-mediated coacervate droplets on bacteria under confocal microscopy. Figure 13 Figure a shows DAPI-labeled Escherichia coli. It can be seen that Escherichia coli is adsorbed on the surface of the copper ion-mediated condensation droplet and does not enter the interior of the condensation droplet. Figure 13 Figure b shows dead E. coli labeled with PI. It can be seen that almost all the E. coli on the surface of the copper ion-mediated condensation droplets were killed, with a killing rate of up to 95%; Figure 13 Figure c shows an image of FITC-labeled condensed droplets. Laser confocal microscopy revealed that copper-mediated condensed droplets can adsorb bacteria to the surface. Then, in the presence of copper ions, the addition of glucose and GSNO triggers a cascade reaction on the surface of the copper-mediated condensed droplets to produce ·OH and NO, which inhibit and kill bacteria.

Claims

1. A method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets, characterized by: The preparation method of copper ion-mediated dual antibacterial effect coacervate microdroplets is carried out according to the following steps: Step 1: Preparation of membrane-free microdroplet solution (1) Synthesis of carboxylated dextran: succinic anhydride was mixed with dextran, dimethyl sulfoxide was added, and the mixture was heated to 55-60°C in an oil bath and stirred to completely dissolve the succinic anhydride and dextran. 4-dimethylaminopyridine was then added and stirred at 55-60°C for 11-12 hours. The reaction solution was diluted with deionized water and dialyzed. Finally, the mixture was filtered and freeze-dried to obtain carboxylated dextran. The mass ratio of the succinic anhydride, dextran and 4-dimethylaminopyridine is 185:100:5; The ratio of the mass of the succinic anhydride to the volume of dimethyl sulfoxide is 185 mg:15 mL; (2) Preparation of diethylaminoethyl dextran solution: Dissolve diethylaminoethyl dextran in phosphate buffer solution to prepare a 9-10 mg / mL diethylaminoethyl dextran solution; (3) Preparation of carboxylated dextran solution: dissolve carboxylated dextran in phosphate buffer solution to prepare a 9-10 mg / mL carboxylated dextran solution; (4) adding the diethylaminoethyl dextran solution to the carboxylated dextran solution and shaking it using a vortex shaker to obtain a film-free microdroplet solution; The volume ratio of the diethylaminoethyl dextran solution to the carboxylated dextran solution is 7:3-4; Step 2: Preparation of membrane condensation solution (1) Preparation of dioleoylphosphatidylcholine solution: dissolve dioleoylphosphatidylcholine in anhydrous ethanol to obtain 18-20 mg / mL dioleoylphosphatidylcholine solution; (2) adding the dioleoylphosphatidylcholine solution to the membrane-free microdroplet solution prepared in step 2, and allowing the solution to stand at room temperature to obtain a membrane-containing condensation solution; The volume ratio of the dioleoylphosphatidylcholine solution to the membraneless microdroplet solution is 6-8:100; Step 3. Preparation of functionalized coagulation suspension (1) Preparation of functional phospholipid solution: Dissolve the functional phospholipid in anhydrous ethanol solution to prepare a 18-20 mg / mL functional phospholipid solution; (2) adding the functional phospholipid solution to the membrane-forming coacervate solution obtained in step 2, shaking the solution using a vortex shaker, and then allowing the solution to stand to obtain a functionalized microdroplet suspension; The volume ratio of the functional phospholipid solution to the membrane-forming coacervate solution is 4-6:100; Step 4. Preparation of copper ion-mediated coacervation droplets The functionalized microdroplet suspension obtained in step 3 is added to the CuCl2 solution, shaken using a vortex shaker, and then allowed to stand to obtain a copper ion-mediated dual antibacterial efficacy condensed microdroplet solution; The concentration of the CuCl2 solution is 1.5-2 mg / mL; The volume ratio of the CuCl2 solution to the functionalized microdroplet suspension is 3-4:

100.

2. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: The dialysis in step 1 (1) uses a dialysis bag with a molecular weight cut-off of 8000-14000, and the dialysis is carried out for three days.

3. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: The pH of the phosphate buffer solution in step 1 (2) is 7.5 and the concentration is 0.01 mol / L.

4. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: The pH of the phosphate buffer solution in step 1 (3) is 7.5 and the concentration is 0.01 mol / L.

5. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: Step 1 (4) Oscillate using a vortex shaker for 20 to 30 seconds.

6. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: Step 2 (2) Let stand at room temperature for 2-3 hours.

7. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: The functional phospholipid in step 3 (1) is DSPE-PEG-DA.

8. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: As described in step 4, the mixture was shaken on a vortex shaker for 30 seconds and then allowed to stand for 1 hour.

9. The method for preparing copper ion-mediated dual antibacterial efficacy coacervate droplets according to claim 1, characterized in that: In step 1 (1), dialysis was performed for three days.

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