Exosome integrated antibacterial high polymer material and preparation method thereof

Abstract

The invention provides an exosome integrated antibacterial high polymer material and a preparation method thereof, and belongs to the field of intelligent biomimetic materials. The method comprises the following steps: introducing a benzimide bond into a molecular structure of a polylysine-chitosan copolymer to obtain a first copolymer; covalently coupling D-glutamic acid and electrostatically adsorbing lipoteichoic acid binding peptide to obtain a second copolymer; mixing the phosphate buffer solution of the aldehyde methyl cellulose with the phosphate buffer solution of the aminated chitosan to obtain a first mixture; adding a mixed solution of protocatechualdehyde and a FeCl3 solution into the first mixture to obtain a second mixture; under stirring, adding the second copolymer, the placenta mesenchymal stem cell exosome and Ag (at) rGO nanoparticles into the second mixture, and standing to obtain the exosome integrated antibacterial high polymer material. Therefore, the antibacterial property, the repairing property, the mechanical property and the environmental responsiveness of the exosome integrated antibacterial high polymer material are improved at the same time.

Description

Technical Field

[0001] This application relates to the technical field of intelligent bionic materials, and particularly to an exosome-integrated antibacterial polymer material and a preparation method thereof. Background Art

[0002] In the field of skin wound repair, especially for the treatment of chronic infected wounds (such as diabetic foot ulcers, burn infections), traditional antibacterial materials such as silver dressings and antibiotic-loaded hydrogels face multiple challenges of enhanced drug resistance, risk of tissue damage, limited treatment effect, and poor material adaptability.

[0003] Exosomes are nanoscale vesicles secreted by cells and contain various bioactive molecules such as proteins and nucleic acids. In recent years, the application potential of exosomes in the field of wound repair has gradually attracted attention. Exosomes can promote repair processes such as angiogenesis and epithelialization, and at the same time regulate the inflammatory response. At the same time, self-healing polymer hydrogels are intelligent materials with self-healing ability, and their interior and surface can self-recover to the initial strength and function when cracks appear. This self-healing ability is achieved through the breakage and reconnection of reversible covalent bonds and reversible non-covalent bonds. Self-healing hydrogels have broad application prospects in fields such as biosensors, wound dressings, shape memory materials, and biomedical carriers. Therefore, combining self-healing polymer hydrogels with exosomes can provide new ideas for the treatment of chronic infected wounds.

[0004] Chinese Patent CN 109912850 B discloses a self-healing hydrogel loaded with exosomes, a preparation method thereof, and an application. Due to the antibacterial property of the hydrogel and the slow-release effect of exosomes in the hydrogel, the self-healing hydrogel loaded with exosomes of the present invention has good biocompatibility, good self-healing performance, and degradation rate. However, exosomes are easily inactivated due to physical extrusion or oxidative stress when directly loaded onto traditional hydrogels, and lack a targeted delivery mechanism, resulting in low repair efficiency. Chinese Patent CN 108904875 B discloses an antibacterial self-healing hydrogel dressing for promoting chronic wound healing, a preparation method thereof, and an application. Exosomes are loaded onto cationic polymers through electrostatic interaction, and are wrapped therein by a hydrogel with a double-network structure, thereby obtaining an antibacterial self-healing hydrogel dressing for promoting chronic wound healing. However, it relies on the positive charge of cationic polymers (such as ε-polylysine, polyethyleneimine) to kill bacteria, only destroys the bacterial membrane through electrostatic interaction, and exosomes are easily inactivated due to charge instability or physical extrusion. Therefore, how to effectively protect and deliver exosomes, realize their synergistic effect with antibacterial materials, and at the same time improve the antibacterial, repair, mechanical properties, and environmental responsiveness of exosome-integrated antibacterial polymer materials has become a current research hotspot and difficulty. Summary of the Invention

[0005] The present application provides an exosome-integrated antibacterial polymer material and a preparation method thereof to solve the following technical problems: how to simultaneously improve the antibacterial, repair, mechanical properties and environmental responsiveness of the exosome-integrated antibacterial polymer material.

[0006] In a first aspect, the present application provides a preparation method of an exosome-integrated antibacterial polymer material, the method comprising:

[0007] Introduce benzimidoyl bonds into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer;

[0008] Through covalent coupling of D-glutamic acid and electrostatic adsorption of lipoteichoic acid-binding peptides to modify the first copolymer with bacterial targeting ligands to obtain a second copolymer;

[0009] Mix the phosphate buffer solution of aldehyde group-modified methyl cellulose and the phosphate buffer solution of amino group-modified chitosan to obtain a first mixture;

[0010] Add the mixed solution of protocatechuic aldehyde and FeCl3 solution to the first mixture and adjust the pH to 6.0 - 7.0 to obtain a second mixture;

[0011] Under stirring, add the second copolymer, placenta mesenchymal stem cell exosomes and Ag@rGO nanoparticles to the second mixture, and let it stand to obtain the exosome-integrated antibacterial polymer material.

[0012] Optionally, the introducing benzimidoyl bonds into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer includes:

[0013] Mix the phosphate buffer solution of polylysine and the phosphate buffer solution of chitosan at a molar ratio of 1:1, add a mixed crosslinking agent of EDC and NHS, and react at 25 - 30 °C for 10 - 12 h, and then carry out purification to obtain a polylysine-chitosan copolymer;

[0014] Dissolve the polylysine-chitosan copolymer in a DMF solvent, add p-formylbenzoic acid, and carry out a stirring reaction under argon protection at 60 - 70 °C for 20 - 28 h, and then carry out purification to introduce benzimidoyl bonds into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer.

[0015] Optionally, the molar ratio of EDC to NHS is 1:(0.8 - 1.2);

[0016] The mass of the p-formylbenzoic acid is 5% - 10% of the mass of the polylysine-chitosan copolymer.

[0017] Optionally, the first copolymer is modified with a bacterial targeting ligand by covalently coupling D-glutamic acid and electrostatically adsorbing lipoteichoic acid-binding peptide, to obtain a second copolymer, including:

[0018] Mix the phosphate buffer solution of D-glutamic acid with a mixed cross-linking agent of EDC and NHS to activate the carboxyl group, then mix with the first copolymer, and react at 20-30 °C for 4-8 h, so that the amide bond of the activated D-glutamic acid is coupled to the surface of the first copolymer, and then purified to obtain a D-Glu-modified copolymer;

[0019] Mix an aqueous solution of lipoteichoic acid-binding peptide with a concentration of 0.5-2 mg / mL with the D-Glu-modified copolymer, and stir at pH 5.5-6.5 for 2-6 h to adsorb the lipoteichoic acid-binding peptide to the surface of the D-Glu-modified copolymer by electrostatic interaction, and then perform purification and freeze-drying treatment to obtain the second copolymer.

[0020] Optionally, the molar ratio of EDC to NHS is 1:(0.8-1.2);

[0021] The molar ratio of the D-glutamic acid to the first copolymer is 1:(1.5-2);

[0022] The mass ratio of the lipoteichoic acid-binding peptide to the D-Glu-modified copolymer is 1:(8-12).

[0023] Optionally, the concentration of the phosphate buffer solution of the aldehyde group-modified methyl cellulose is 8-12 wt%, the concentration of the phosphate buffer solution of the amino group-modified chitosan is 2-4 wt%, and the mixing volume ratio is 1:(0.8-1.2).

[0024] Optionally, the molar ratio of protocatechuic aldehyde to FeCl3 is (3-4):1, and the final concentration of Fe in the second mixture 3+ is 0.08-0.12 M.

[0025] Optionally, the Ag@rGO nanoparticles are synthesized by a chemical reduction method, the mass ratio of AgNO3 to reduced graphene oxide is 1:(5-10), and the particle size is 20-50 nm.

[0026] Optionally, the mass of the second copolymer is 10-15% of the mass of the second mixture;

[0027] The mass of the Ag@rGO nanoparticles is 5-8% of the mass of the second mixture;

[0028] Calculated per gram of the second mixture, the number of particles of placenta mesenchymal stem cell exosomes is 1×10 10 ~5×1011 。

[0029] In a second aspect, the present application provides an exosome-integrated antibacterial polymer material obtained by the preparation method of the exosome-integrated antibacterial polymer material described in any one of the embodiments of the first aspect. The exosome-integrated antibacterial polymer material is used for the repair of chronic infected wounds.

[0030] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0031] The present application provides a preparation method of an exosome-integrated antibacterial polymer material. First, based on the positive charge of the polylysine-chitosan copolymer to disrupt the bacterial membrane and inhibit the activity of metabolic enzymes, combined with the photothermal effect of reduced graphene oxide loaded with silver nanoparticles and the slow release of silver ions, the bactericidal effect is synergistically enhanced. And broad-spectrum targeting is achieved through covalent coupling of D-glutamic acid and electrostatic adsorption of lipoteichoic acid-binding peptides, and precise enrichment at the infection site, thereby enhancing the antibacterial performance. Secondly, the placental mesenchymal stem cell exosomes are gently encapsulated by a dynamic network and sequentially released under the triggering of an infected microenvironment or near-infrared light, promoting angiogenesis, epithelial regeneration and anti-inflammatory regulation, thereby improving the repair performance. Thirdly, the Schiff base bond between aldehyde group-modified methyl cellulose and amino group-modified chitosan provides self-healing properties, and the coordination bond between protocatechuic aldehyde and iron ions enhances the mechanical strength, adapting to the dynamic deformation of the wound surface, thereby improving the mechanical performance. Finally, intelligent controlled release is achieved through a pH / photothermal dual-responsive mechanism, rapidly killing bacteria at the initial stage of infection, penetrating the biofilm for enhanced intervention in deep infections, and reducing the risk of secondary infection, thereby improving the environmental responsiveness. Thus, through the multi-mechanism collaborative design and dynamic network optimization, the limitations of the single function of traditional materials are broken through, and the integration of "antibacterial-repair-self-healing-response" is realized, while enhancing the antibacterial, repair, mechanical properties and environmental responsiveness of the exosome-integrated antibacterial polymer material. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings herein are incorporated into the specification and constitute a part of this specification, showing embodiments consistent with the present application and, together with the specification, are used to explain the principles of the present application.

[0033] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0034] Figure 1 It is a schematic flow chart of a preparation method of an exosome-integrated antibacterial polymer material provided by an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts shall fall within the scope of protection of this application.

[0036] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, equipment, etc. used in this application can be obtained through market purchases or can be prepared by existing methods.

[0037] Figure 1 It is a schematic flow chart of a preparation method of an exosome-integrated antibacterial polymer material provided by an embodiment of this application.

[0038] As Figure 1 shown, this application provides a preparation method of an exosome-integrated antibacterial polymer material, and the method includes:

[0039] S1. Introduce benzoyl imine bonds into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer;

[0040] The polylysine (PLL) of the polylysine-chitosan copolymer destroys the integrity of the bacterial membrane through positive charges, and chitosan (CS) synergistically sterilizes by inhibiting the activity of bacterial metabolic enzymes. At the same time, the polylysine-chitosan copolymer serves as a copolymer backbone to achieve targeted drug release in the infected microenvironment (pH 5.5 - 6.5) through benzoyl imine bonds (pH-responsive). In addition, the polylysine-chitosan copolymer can combine with the photothermal effect of Ag@rGO nanoparticles to enhance the penetration ability against drug-resistant bacteria.

[0041] The introduced benzoyl imine bonds have pH responsiveness and hydrolyze in an acidic infected environment to trigger the release of antibacterial components (such as PLL, Ag@rGO), precisely acting on pathogens. At the same time, the benzoyl imine bonds can cooperate with the dynamic crosslinking network (Schiff base bonds, Fe 3+ coordination bonds) to maintain the structural stability of the material under physiological conditions.

[0042] In some embodiments, introducing a benzimidoyl bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer includes: mixing a phosphate buffer solution of polylysine and a phosphate buffer solution of chitosan at a molar ratio of 1:1, adding a mixed crosslinking agent of EDC and NHS, and reacting at 25-30 °C for 10-12 h, followed by purification to obtain a polylysine-chitosan copolymer; dissolving the polylysine-chitosan copolymer in a DMF solvent, adding p-formylbenzoic acid, and stirring and reacting under argon protection at 60-70 °C for 20-28 h, followed by purification to introduce a benzimidoyl bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer.

[0043] In some embodiments, the molar ratio of EDC to NHS is 1:(0.8-1.2);

[0044] The mass of the p-formylbenzoic acid is 5%-10% of the mass of the polylysine-chitosan copolymer.

[0045] Polylysine (PLL) is a positively charged polypeptide with broad-spectrum antibacterial properties that disrupts bacterial membranes through electrostatic interactions. Chitosan (CS) is a natural cationic polysaccharide with good antibacterial and biocompatibility and can promote wound healing. The EDC / NHS crosslinking agent, as a carboxylic acid activator, promotes the crosslinking of the amino group of PLL and the carboxyl group of CS by forming an active ester to form a copolymer (PLL-CS). The aldehyde group of p-formylbenzoic acid condenses with the amino group of PLL-CS to generate a benzimidoyl bond (-N=C-), endowing the material with pH responsiveness.

[0046] The EDC:NHS molar ratio (1:0.8-1:2) can ensure sufficient activation of the carboxyl group and avoid side reactions caused by excessive NHS. The reaction temperature (60-70 °C) can promote the condensation of the aldehyde group and the amino group, while avoiding the volatilization of the DMF solvent. The dosage of p-formylbenzoic acid (5%-10%) can balance the benzimidoyl bond density and the material stability, and excessive dosage may cause over-crosslinking.

[0047] S2. Modifying the first copolymer with a bacterial targeting ligand by covalently coupling D-glutamic acid and electrostatically adsorbing lipoteichoic acid-binding peptide to obtain a second copolymer;

[0048] It should be noted that the lipoteichoic acid-binding peptide (LBP-6 or Lipoteichoic acid binding peptide, sourced from GenScript, Accession No. is NM_059525.5, Definition: Caenorhabditis elegans Fatty acid-binding protein homolog 6 (lbp-6), mRNA) in the embodiments of the present application is an artificially designed short peptide that can specifically bind to lipoteichoic acid (LTA), the main component of the cell wall of Gram-positive bacteria. Its core function is to achieve precise antibacterial by targeting and recognizing bacterial surface molecules.

[0049] D-Glu recognizes lipoteichoic acid (LTA) of Gram-positive bacteria through covalent coupling, and LBP-6 targets lipopolysaccharide (LPS) of Gram-negative bacteria through electrostatic adsorption to achieve broad-spectrum antibacterial. At the same time, the positive charge of LBP-6 binds to the negative charge of the bacterial membrane, improving the enrichment efficiency of the material at the infection site. In addition, D-glutamic acid synergizes with the lipoteichoic acid-binding peptide and the photothermal sterilization of Ag@rGO to form an antibacterial cascade effect of "targeted positioning - rapid killing".

[0050] In some embodiments, the modification of the first copolymer with a bacterial targeting ligand by covalently coupling D-glutamic acid and electrostatically adsorbing the lipoteichoic acid-binding peptide to obtain a second copolymer includes: mixing the phosphate buffer solution of D-glutamic acid with a mixed cross-linking agent of EDC and NHS to activate the carboxyl group, then mixing with the first copolymer, and reacting at 20 - 30 °C for 4 - 8 h to couple the amide bond of the activated D-glutamic acid to the surface of the first copolymer, and then purifying to obtain the D-Glu modified copolymer; mixing the aqueous solution of the lipoteichoic acid-binding peptide with a concentration of 0.5 - 2 mg / mL with the D-Glu modified copolymer, and stirring at pH 5.5 - 6.5 for 2 - 6 h to adsorb the lipoteichoic acid-binding peptide to the surface of the D-Glu modified copolymer through electrostatic interaction, and then performing purification and freeze-drying treatment to obtain the second copolymer.

[0051] In some embodiments, the molar ratio of EDC to NHS is 1:(0.8 - 1.2);

[0052] The molar ratio of the D-glutamic acid to the first copolymer is 1:(1.5 - 2);

[0053] The mass ratio of the lipoteichoic acid-binding peptide to the D-Glu modified copolymer is 1:(8 - 12).

[0054] EDC / NHS activation: Activate the carboxyl group of D-Glu to form an amide bond with the amino group of PLL-CS-Bz.

[0055] The molar ratio (D-Glu: the first copolymer = 1:1.5 - 2) can ensure a sufficient density of targeting ligands and enhance the recognition of Gram-positive bacteria (LTA). Phosphate buffer can optimize the EDC / NHS activation efficiency and avoid the precipitation of chitosan under acidic conditions.

[0056] The mass ratio of lipoteichoic acid-binding peptide to the D-Glu-modified copolymer (1:(8 - 12)) can utilize the electrostatic binding between the positive charge of LBP-6 and the negative charge of the copolymer (after D-Glu modification) to target Gram-negative bacteria (LPS). pH 5.5 - 6.5 can simulate the infected microenvironment, enhance the adsorption efficiency, and avoid peptide structure denaturation.

[0057] S3. Mix the phosphate buffer of aldehyde-modified methylcellulose with the phosphate buffer of amino-modified chitosan to obtain the first mixture;

[0058] Aldehyde-modified methylcellulose (A-MC) and amino-modified chitosan (NH2-CS) can form a reversible crosslinking through Schiff base bonds (-C=N-), endowing the material with self-healing properties and adapting to wound deformation. At the same time, gentle physical encapsulation reduces the damage to the exosome membrane and maintains the integrity of active components such as miRNA and proteins carried by it. In addition, aldehyde-modified methylcellulose (A-MC) and amino-modified chitosan (NH2-CS) can coordinate with protocatechuic aldehyde-Fe 3+ to form a double dynamic network, enhancing the mechanical strength and responding to near-infrared light-controlled release. Thus, Schiff base bonds and Fe 3+ coordination bonds form a gentle encapsulation structure to avoid the inactivation of exosomes due to physical extrusion or oxidative stress.

[0059] In some embodiments, the concentration of the phosphate buffer of aldehyde-modified methylcellulose is 8 - 12 wt%, the concentration of the phosphate buffer of amino-modified chitosan is 2 - 4 wt%, and the mixing volume ratio is 1:(0.8 - 1.2).

[0060] The concentration of aldehyde-modified methylcellulose (A-MC) (8 - 12 wt%) can provide a sufficient aldehyde density to ensure the subsequent Schiff base crosslinking strength. The concentration of amino-modified chitosan (NH2-CS) (2 - 4 wt%) can avoid excessive amino density causing aggregation and ensure the formation of a dynamic network at the same time. The mixing volume ratio of 1:(0.8 - 1.2) can balance the molar ratio of aldehyde groups to amino groups and optimize the formation efficiency of dynamic Schiff base bonds (-C=N-).

[0061] In some embodiments, the method for preparing the aldehyde-modified methylcellulose comprises the following steps: Disperse 2 g of methylcellulose in 100 mL of PBS (pH 5.0), stir at 60 °C for 2 hours until completely dissolved, cool to room temperature, then add 0.8 g of sodium periodate (molar ratio of MC:NaIO4 is 1:1.2), stir and react at room temperature for 8 hours under dark conditions, add 5 mL of ethylene glycol, and continue to stir for 1 hour to neutralize the unreacted NaIO4; Load the reaction solution into a dialysis bag, dialyze with deionized water for 3 days (change water 3 times a day) to remove small molecule by-products; Freeze-dry the solution after dialysis (-50 °C, 24 hours) to obtain white sponge-like aldehyde-modified methylcellulose (A-MC).

[0062] In some embodiments, the method for preparing the amino-functionalized chitosan (NH2-CS) comprises the following steps: Dissolve 1 g of chitosan in 50 mL of 2% acetic acid solution, stir until transparent, add 0.5 g of adipic anhydride (molar ratio of CS:adipic anhydride is 1:2), react at 50 °C for 24 hours; Transfer the carboxylated chitosan solution to DMF, add EDC (0.8 mmol) and NHS (0.8 mmol), stir at room temperature for 30 minutes, add ethylenediamine (2 mmol, carboxyl:ethylenediamine = 1:2), react at 30 °C for 12 hours; Dialyze (with deionized water, 3 days) to remove the unreacted ethylenediamine and by-products, and obtain amino-functionalized chitosan (NH2-CS) after freeze-drying.

[0063] S4. Add the mixed solution of protocatechuic aldehyde and FeCl3 solution to the first mixture, and adjust the pH to 6.0 - 7.0 to obtain a second mixture;

[0064] In protocatechuic aldehyde and FeCl3, the catechol group of protocatechuic aldehyde forms a tridentate coordination bond with Fe 3+ to enhance the mechanical properties and photothermal responsiveness of the material. Meanwhile, under 808 nm near-infrared light irradiation, the Fe 3+ coordination bond dissociates to release heat (ΔT≥50 °C), accelerating the release of Ag of Ag@rGO + .

[0065] In some embodiments, the molar ratio of protocatechuic aldehyde to FeCl3 is (3 - 4):1, and the final concentration of Fe 3+ in the second mixture is 0.08 - 0.12 M.

[0066] With the molar ratio of protocatechuic aldehyde to FeCl3 ((3 - 4):1), each protocatechuic aldehyde molecule provides 3 catechol groups to form a stable tridentate coordination bond with Fe 3+ . The final concentration of Fe 3+ (0.08 - 0.12 M) can ensure the coordination bond density, endowing the material with self-healing property and photothermal responsiveness.

[0067] S5. Under stirring, add the second copolymer, placenta mesenchymal stem cell exosomes, and Ag@rGO nanoparticles to the second mixture, and let it stand to obtain the exosome-integrated antibacterial polymer material.

[0068] The high specific surface area and photothermal conversion efficiency of rGO in Ag@rGO nanoparticles can achieve local temperature increase for sterilization, and Ag nanoparticles release Ag + to interfere with bacterial metabolism. Meanwhile, the positive charge of Ag@rGO nanoparticles synergizes with PLL to disrupt the bacterial biofilm and enhance the permeability of antibacterial peptides. In addition, Ag@rGO nanoparticles can bind to the protocatechuic aldehyde-Fe 3+ coordination bonds in the dynamic network to achieve a dual antibacterial mode of "photothermal trigger-chemical sterilization".

[0069] miRNAs (such as miR-21-5p) and growth factors (such as VEGF) carried by placenta mesenchymal stem cell exosomes (PMSC-Exosomes) promote angiogenesis and epithelial regeneration. Meanwhile, it can inhibit macrophage M1 polarization and reduce the excessive inflammatory response at the infection site. In addition, dynamic bond dissociation occurs in the infected microenvironment, and the slow release of exosomes enables continuous repair. After Ag@rGO rapidly sterilizes, exosomes repair damaged tissues to avoid secondary infection.

[0070] In some embodiments, the Ag@rGO nanoparticles are synthesized by a chemical reduction method, the mass ratio of AgNO3 to reduced graphene oxide is 1:(5-10), and the particle size is 20-50 nm.

[0071] Define the mass ratio of AgNO3:rGO as 1:5-10 to control the Ag loading amount and avoid sudden release of toxicity. Define the particle size as 20-50 nm. The small size can enhance the photothermal effect and avoid difficult cell phagocytosis. + In some embodiments, the mass of the second copolymer is 10-15% of the mass of the second mixture;

[0072] The mass of the Ag@rGO nanoparticles is 5-8% of the mass of the second mixture;

[0073] The number of particles of placenta mesenchymal stem cell exosomes per gram of the second mixture is 1×10

[0074] to 10 5×10 11 .

[0075] In some embodiments, the method for preparing the Ag@rGO nanoparticles includes: dispersing 50 mg of GO in 100 mL of deionized water, and performing ultrasonic treatment (power 300 W, frequency 40 kHz) for 1 hour to obtain a uniform GO suspension (0.5 mg / mL); adding 0.1 M ascorbic acid to the GO suspension (mass ratio of GO to ascorbic acid is 1:5), and stirring in a water bath at 80 °C for 6 hours. The solution changes from brownish-yellow to black, indicating that GO is reduced to rGO; centrifuging (8000×g, 15 minutes) to remove the unreacted reducing agent, washing the precipitate 3 times with deionized water, and redispersing it in 50 mL of water to obtain an rGO dispersion (1 mg / mL); weighing AgNO3 according to the mass ratio of AgNO3:rGO of 1:5 to 1:10 (for example, taking 10 mg of AgNO3 corresponding to 50 - 100 mg of rGO), dissolving it in 10 mL of deionized water, and storing it in the dark; dropwise adding the AgNO3 solution to the rGO dispersion, and magnetically stirring (500 rpm) for 30 minutes to make Ag + uniformly adsorb on the surface of rGO; adding 0.1 M NaBH4 solution (molar ratio of AgNO3 to NaBH4 is 1:3), and stirring and reacting under ice bath conditions for 2 hours. The solution gradually changes from colorless to grayish-brown, indicating that Ag + is reduced to Ag nanoparticles. Centrifuge (12000×g, 20 minutes) to collect the precipitate, and wash it 3 times with ethanol and deionized water in sequence to remove the unreacted ions and reducing agent; perform vacuum freeze-drying (-50 °C, 24 hours) to obtain the Ag@rGO nanoparticle powder.

[0076] In some embodiments, the extraction process of the placenta mesenchymal stem cell exosomes includes: centrifuging the placenta mesenchymal stem cells to collect the cell supernatant, centrifuging the cell supernatant to remove dead cells and cell debris, filtering with a 0.22 μm filter membrane, and then centrifuging at 4 °C (rotation speed 10000 rpm) for 30 min to obtain the placenta mesenchymal stem cell exosomes, and storing them in an ultra-low temperature refrigerator at -80 °C.

[0077] Based on a general inventive concept, the present application provides an exosome-integrated antibacterial polymer material obtained by the preparation method of the exosome-integrated antibacterial polymer material described in any one of the above embodiments, and the exosome-integrated antibacterial polymer material is used for chronic infected wound repair.

[0078] It can be seen from this that the exosome-integrated antibacterial polymer material provided by the embodiments of the present application has the following functions:

[0079] (1) Synergy between the dynamic network and exosomes: The Schiff base bond of aldehyde group-modified methylcellulose / amino group-modified chitosan wraps and protects the exosomes, and Fe 3+Coordination bond responds to photothermal controlled release to achieve spatio-temporal specific release of exosomes. At the same time, the immunomodulatory function of exosomes is complementary to the antibacterial effects of PLL and Ag@rGO, forming an integrated wound repair of "sterilization - anti-inflammatory - repair".

[0080] (2) Synergy of targeting ligands and antibacterial components: D-Glu and LBP-6 guide the material to accumulate at the infection site, increase the local concentration of Ag@rGO and PLL, and reduce systemic toxicity. The photothermal effect of Ag@rGO enhances the affinity between the targeting ligand and the bacterial membrane, forming a positive feedback antibacterial mechanism.

[0081] (3) Synergy of photothermal and chemical antibacterial: Near-infrared light triggers the dissociation of Fe 3+ coordination bond and the temperature rise of Ag@rGO, and the dual action quickly kills the surface bacteria; the pH responsiveness of the dynamic network releases PLL and Ag + , inhibiting deep infection.

[0082] (4) Synergy of dynamic self-healing and mechanical adaptability: The reversibility of Schiff base bonds and Fe 3+ coordination bonds enables the material to adapt to wound deformation while maintaining the uniform dispersion of exosomes and nanoparticles.

[0083] In summary, an exosome-integrated antibacterial polymer material and its preparation method provided by this application have the following advantages:

[0084] (1) Multi-level synergistic antibacterial - repair system: The Schiff base bond of aldehyde group-modified methyl cellulose / amino group-modified chitosan wraps exosomes, and the Fe 3+ coordination bond responds to near-infrared light (808 nm) to trigger release, realizing sequential treatment of "sterilize first - repair later". At the same time, miRNAs carried by exosomes (such as miR-21-5p) inhibit the release of inflammatory factors (TNF-α, IL-6), which is complementary to the antibacterial effect of polylysine (PLL), forming an "anti-inflammatory - antibacterial - regeneration" closed loop. In addition, D-glutamic acid (D-Glu) and LBP-6 peptide target Gram-positive / negative bacteria respectively, guide Ag@rGO nanoparticles to accumulate at the infection site, increase the local Ag + concentration, and reduce systemic toxicity. The photothermal conversion efficiency of Ag@rGO ≥ 50%, enhances the binding force between the targeting ligand and the bacterial membrane, and improves the bactericidal efficiency.

[0085] (2) Intelligent response and adaptive characteristics: Benzoyl imine bonds are selectively hydrolyzed in the infected microenvironment to precisely release antibacterial components (PLL, Ag + ). Near-infrared light dissociates the Fe 3+ coordination bond, accelerating the responsive release of Ag@rGO and exosomes. At the same time, Schiff base bonds and Fe 3+The reversibility of the coordination bond enables the material to have a self-healing efficiency of ≥90% within 30 minutes and adapt to wound deformation.

[0086] (3) High efficiency against drug resistance and biosafety: The positive charges of PLL and chitosan (CS) synergistically disrupt the bacterial membrane (zeta potential +25 mV), bypassing the traditional antibiotic resistance mechanism. At the same time, Ag + inhibits the activity of bacterial respiratory chain enzymes, and the photothermal effect directly kills the drug-resistant bacterial biofilm. The biocompatibility of chitosan, polylysine, and exosomes reduces the risk of immune rejection. Ag + has a cumulative release rate of ≤30% within 72 hours, avoiding the toxicity caused by the sudden release of metal ions.

[0087] The following further elaborates on this application in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate this application and not to limit the scope of this application. The experimental methods without specific conditions noted in the following embodiments are usually determined according to industry standards. If there is no corresponding industry standard, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0088] Example 1

[0089] This example provides a preparation method of an exosome-integrated antibacterial polymer material, and the method includes:

[0090] S11. Mix the phosphate buffer solution of polylysine (ε-polylysine: CAS No.: 28211-04-3) (the general buffer solution has a pH of 7.4) and the phosphate buffer solution of chitosan (CAS No.: 9012-76-4, deacetylation degree ≥75%) at a molar ratio of 1:1, add a mixed cross-linking agent of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, CAS No.: 25952-53-8) and NHS (N-hydroxysuccinimide, CAS No.: 6066-82-6), and react at 30 °C for 10 h, and then carry out purification to obtain a polylysine-chitosan copolymer; dissolve the polylysine-chitosan copolymer in a DMF solvent, add p-formylbenzoic acid, and carry out a stirring reaction for 24 h under argon protection at 70 °C, and then carry out purification to introduce a benzimidamide bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer;

[0091] Among them, the molar ratio of EDC to NHS is 1:1;

[0092] The mass of the p-formylbenzoic acid is 7% of the mass of the polylysine-chitosan copolymer.

[0093] S21. Mix the phosphate buffer solution of D-glutamic acid (CAS No.: 6893-26-1) with a mixed cross-linking agent of EDC and NHS to activate the carboxyl group, then mix it with the first copolymer and react at 30 °C for 6 h to couple the amide bond of the activated D-glutamic acid to the surface of the first copolymer, and then purify it to obtain the D-Glu modified copolymer; Mix an aqueous solution of lipoteichoic acid binding peptide (LBP-6 or Lipoteichoic acid binding peptide, sourced from GenScript, Accession No. is NM_059525.5, Definition: Caenorhabditis elegans Fattyacid-binding protein homolog 6 (lbp-6), mRNA.) with a concentration of 1 mg / mL with the D-Glu modified copolymer and stir at pH 5 for 4 h to adsorb the lipoteichoic acid binding peptide to the surface of the D-Glu modified copolymer through electrostatic interaction, and then purify and lyophilize it to obtain the second copolymer;

[0094] Among them, the molar ratio of EDC to NHS is 1:1;

[0095] The molar ratio of the D-glutamic acid to the first copolymer is 1:1.8;

[0096] The mass ratio of the lipoteichoic acid binding peptide to the D-Glu modified copolymer is 1:10.

[0097] S31. Mix the phosphate buffer solution of aldehyde group modified methyl cellulose (the base methyl cellulose CAS No.: 9004-67-5) with the phosphate buffer solution of amino group modified chitosan to obtain a first mixture;

[0098] Among them, the concentration of the phosphate buffer solution of the aldehyde group modified methyl cellulose is 10 wt%, the concentration of the phosphate buffer solution of the amino group modified chitosan is 3 wt%, and the mixing volume ratio is 1:1.

[0099] S41. Add the mixed solution of protocatechuic aldehyde (CAS No.: 139-85-5) and FeCl3 solution to the first mixture and adjust the pH to 6.0 to obtain a second mixture;

[0100] Among them, the molar ratio of the protocatechuic aldehyde to FeCl3 is 3.5:1, and the final concentration of Fe in the second mixture 3+ is 0.1 M.

[0101] S51. Under stirring, add the second copolymer, placental mesenchymal stem cell exosomes, and Ag@rGO nanoparticles to the second mixture, and let it stand to obtain the exosome-integrated antibacterial polymer material.

[0102] Among them, the Ag@rGO nanoparticles are synthesized by chemical reduction method, the mass ratio of AgNO3 to reduced graphene oxide is 1:8, and the average particle size is 30 nm.

[0103] The mass of the second copolymer is 12% of the mass of the second mixture;

[0104] The mass of the Ag@rGO nanoparticles is 6% of the mass of the second mixture;

[0105] Based on each gram of the second mixture, the number of particles of placental mesenchymal stem cell exosomes is 7×10 10 .

[0106] Example 2

[0107] This example provides a preparation method of an exosome-integrated antibacterial polymer material, and the method includes:

[0108] S11. Mix the phosphate buffer solution of polylysine and the phosphate buffer solution of chitosan at a molar ratio of 1:1, add a mixed cross-linking agent of EDC and NHS, and react at 30 °C for 10 h, then carry out purification to obtain a polylysine-chitosan copolymer; dissolve the polylysine-chitosan copolymer in DMF solvent, add p-formylbenzoic acid, and carry out a stirring reaction at 70 °C under argon protection for 20 h, then carry out purification to introduce a benzimidamide bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer;

[0109] Among them, the molar ratio of EDC to NHS is 1:1.2;

[0110] The mass of the p-formylbenzoic acid is 10% of the mass of the polylysine-chitosan copolymer.

[0111] S21. Mix the phosphate buffer solution of D-glutamic acid with a mixed cross-linking agent of EDC and NHS to activate the carboxyl group, then mix it with the first copolymer, and react at 30 °C for 4 h to couple the amide bond of the activated D-glutamic acid to the surface of the first copolymer, then carry out purification to obtain a D-Glu modified copolymer; mix an aqueous solution of lipoteichoic acid-binding peptide with a concentration of 2 mg / mL with the D-Glu modified copolymer, and stir at pH 6.5 for 6 h to adsorb the lipoteichoic acid-binding peptide to the surface of the D-Glu modified copolymer through electrostatic interaction, then carry out purification and freeze-drying treatment to obtain a second copolymer;

[0112] Among them, the molar ratio of EDC to NHS is 1:1.2;

[0113] The molar ratio of the D-glutamic acid to the first copolymer is 1:2;

[0114] The mass ratio of the lipoteichoic acid-binding peptide to the D-Glu modified copolymer is 1:12.

[0115] S31. Mix the phosphate buffer solution of aldehyde group-modified methyl cellulose with the phosphate buffer solution of amino group-modified chitosan to obtain a first mixture;

[0116] Among them, the concentration of the phosphate buffer solution of aldehyde group-modified methyl cellulose is 8-12 wt%, the concentration of the phosphate buffer solution of amino group-modified chitosan is 2-4 wt%, and the mixing volume ratio is 1:1.2.

[0117] S41. Add the mixed solution of protocatechuic aldehyde and FeCl3 solution to the first mixture and adjust the pH to 7.0 to obtain a second mixture;

[0118] Among them, the molar ratio of the protocatechuic aldehyde to FeCl3 is 4:1, and the final concentration of Fe in the second mixture 3+ is 0.12 M.

[0119] S51. Under stirring, add the second copolymer, placenta mesenchymal stem cell exosomes and Ag@rGO nanoparticles to the second mixture, and let it stand to obtain the exosome-integrated antibacterial polymer material.

[0120] Among them, the Ag@rGO nanoparticles are synthesized by a chemical reduction method, the mass ratio of AgNO3 to reduced graphene oxide is 1:10, and the average particle size is 50 nm.

[0121] The mass of the second copolymer is 15% of the mass of the second mixture;

[0122] The mass of the Ag@rGO nanoparticles is 8% of the mass of the second mixture;

[0123] Based on each gram of the second mixture, the number of particles of placenta mesenchymal stem cell exosomes is 5×10 11 .

[0124] Example 3

[0125] This example provides a preparation method of an exosome-integrated antibacterial polymer material, and the method includes:

[0126] S11. Mix the phosphate buffer solution of polylysine and the phosphate buffer solution of chitosan at a molar ratio of 1:1, add a mixed cross-linking agent of EDC and NHS, and react at 25 °C for 12 h, then carry out purification to obtain a polylysine-chitosan copolymer; dissolve the polylysine-chitosan copolymer in a DMF solvent, add p-formylbenzoic acid, and carry out a stirring reaction under argon protection at 60 °C for 28 h, then carry out purification to introduce a benzimidamide bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer;

[0127] Among them, the molar ratio of EDC to NHS is 1:0.8;

[0128] The mass of the p-formylbenzoic acid is 5% of the mass of the polylysine-chitosan copolymer.

[0129] S21. Mix the phosphate buffer solution of D-glutamic acid with a mixed cross-linking agent of EDC and NHS to activate the carboxyl group, then mix with the first copolymer and react at 20 °C for 8 h to couple the amide bond of the activated D-glutamic acid to the surface of the first copolymer, then carry out purification to obtain a D-Glu modified copolymer; mix an aqueous solution of lipoteichoic acid-binding peptide with a concentration of 0.5 - 2 mg / mL with the D-Glu modified copolymer and stir at pH 5.5 for 2 h to adsorb the lipoteichoic acid-binding peptide to the surface of the D-Glu modified copolymer by electrostatic interaction, then carry out purification and freeze-drying treatment to obtain a second copolymer;

[0130] Among them, the molar ratio of EDC to NHS is 1:0.8;

[0131] The molar ratio of the D-glutamic acid to the first copolymer is 1:1.5;

[0132] The mass ratio of the lipoteichoic acid-binding peptide to the D-Glu modified copolymer is 1:8.

[0133] S31. Mix the phosphate buffer solution of aldehyde-modified methylcellulose and the phosphate buffer solution of amino-modified chitosan to obtain a first mixture;

[0134] Among them, the concentration of the phosphate buffer solution of the aldehyde-modified methylcellulose is 8 wt%, the concentration of the phosphate buffer solution of the amino-modified chitosan is 2 wt%, and the mixing volume ratio is 1:0.8.

[0135] S41. Add a mixed solution of protocatechuic aldehyde and FeCl3 solution to the first mixture and adjust the pH to 6.0 - 7.0 to obtain a second mixture;

[0136] Among them, the molar ratio of the protocatechuic aldehyde to FeCl3 is 3:1, and the Fe in the second mixture3+ The final concentration is 0.08 M.

[0137] S51. While stirring, add the second copolymer, placental mesenchymal stem cell exosomes, and Ag@rGO nanoparticles to the second mixture, and let it stand to obtain the exosome-integrated antibacterial polymer material.

[0138] Among them, the Ag@rGO nanoparticles are synthesized by chemical reduction method, the mass ratio of AgNO3 to reduced graphene oxide is 1:5, and the average particle size is 20 nm.

[0139] The mass of the second copolymer is 10% of the mass of the second mixture;

[0140] The mass of the Ag@rGO nanoparticles is 5% of the mass of the second mixture;

[0141] Based on each gram of the second mixture, the number of particles of placental mesenchymal stem cell exosomes is 1×10 10 .

[0142] Comparative Example 1

[0143] Based on what is disclosed in Example 1, this comparative example is modified as follows:

[0144] Introduce a benzimidoyl bond into the molecular structure of polylysine to obtain a first copolymer.

[0145] Comparative Example 2

[0146] Based on what is disclosed in Example 1, this comparative example is modified as follows:

[0147] Introduce a benzimidoyl bond into the molecular structure of chitosan to obtain a first copolymer.

[0148] Comparative Example 3

[0149] Based on what is disclosed in Example 1, this comparative example is modified as follows:

[0150] Do not introduce a benzimidoyl bond in step S11, that is to say, the first copolymer is a polylysine-chitosan copolymer.

[0151] Comparative Example 4

[0152] Based on what is disclosed in Example 1, this comparative example is modified as follows:

[0153] Do not perform covalent coupling of D-glutamic acid in step S21.

[0154] Comparative Example 5

[0155] Based on what is disclosed in Example 1, this comparative example is modified as follows:

[0156] In step S21, lipoteichoic acid-binding peptide is not electrostatically adsorbed.

[0157] Comparative Example 6

[0158] On the basis of what is disclosed in Example 1, this comparative example is modified as follows:

[0159] Step S41 is not carried out. That is to say, the second copolymer, placental mesenchymal stem cell exosomes and Ag@rGO nanoparticles are directly added to the first mixture.

[0160] Comparative Example 7

[0161] On the basis of what is disclosed in Example 1, this comparative example is modified as follows:

[0162] Ag@rGO nanoparticles are not added to the second mixture. That is to say, the second copolymer and placental mesenchymal stem cell exosomes are added to the second mixture.

[0163] Comparative Example 8

[0164] On the basis of what is disclosed in Example 1, this comparative example is modified as follows:

[0165] The second copolymer is not added to the second mixture. That is to say, the placental mesenchymal stem cell exosomes and Ag@rGO nanoparticles are added to the second mixture.

[0166] The exosome-integrated antibacterial polymer materials obtained in Examples 1 to 3 and Comparative Examples 1 to 8 were subjected to performance measurement, and the results are shown in Tables 1 and 2. At the same time, the performance measurement methods are as follows:

[0167] Minimum inhibitory concentration (MIC): ISO 20776-1;

[0168] Bactericidal rate test: co-cultured with MRSA and Escherichia coli for 24 h;

[0169] Endothelial cell proliferation experiment: CCK-8 method;

[0170] Keratinocyte migration experiment: scratch method;

[0171] Inflammatory factor detection: ELISA method (Human TNF-α ELISA Kit, Abcam, ab181421);

[0172] Compressive modulus test: ASTM D695;

[0173] Self-healing efficiency test: recovery time and strength after breakage (static self-healing after cutting the sample, testing the ratio of tensile strength before and after repair);

[0174] Targeting: Fluorescent labeling method (the labeling material is co-incubated with bacteria to detect the enrichment rate at the infection site).

[0175] Table 1 Performance of exosome-integrated antibacterial polymer materials in Examples 1 - 3

[0176]

[0177] As can be seen from Table 1, through multi-component collaborative design (polylysine-chitosan copolymer, targeting ligand, Ag@rGO, exosome) and dynamic network optimization (pH / photothermal response, self-healing) in Examples 1 - 3, comprehensive improvements in antibacterial, repair, mechanical properties, and environmental responsiveness were achieved. The MIC of the exosome-integrated antibacterial polymer materials in Examples 1 - 3 was 2.4 - 2.6 μg / mL, the bactericidal rate was ≥99.8%, the biofilm inhibition rate was ≥95%, the endothelial proliferation rate was ≥2.0 times, the keratinocyte migration rate was ≥40%, the TNF-α level was 110 - 120 pg / mL, the modulus was ≥500 Pa, the self-healing efficiency was ≥90%, and the targeting enrichment rate was ≥70%.

[0178] Table 2 Performance of exosome-integrated antibacterial polymer materials in Comparative Examples 1 - 8

[0179]

[0180] In Comparative Example 1, only benzoyl imine bonds were introduced into polylysine, resulting in the loss of the metabolic enzyme inhibition function of chitosan, insufficient targeting, and a decline in antibacterial and repair performance.

[0181] In Comparative Example 2, only benzoyl imine bonds were introduced into chitosan, weakening the positive charge membrane-breaking effect of polylysine and reducing the antibacterial efficiency.

[0182] In Comparative Example 3, no benzoyl imine bonds were introduced, the pH-responsive release mechanism failed, the antibacterial components could not be targeted for release, and the repair activity of exosomes decreased.

[0183] In Comparative Example 4, D-glutamic acid was not conjugated, resulting in the loss of Gram-positive bacteria targeting, insufficient local antibacterial concentration, and a decline in the bactericidal rate.

[0184] In Comparative Example 5, LBP-6 peptide was not adsorbed, resulting in the loss of Gram-negative bacteria targeting and a weakened bactericidal effect on Escherichia coli, etc.

[0185] In Comparative Example 6, no Fe 3+ coordination bond was introduced, resulting in the loss of photothermal responsiveness, the inability to intervene in deep infections, and a decrease in mechanical strength.

[0186] In Comparative Example 7, Ag@rGO was not added, resulting in the loss of photothermal sterilization and Ag + slow-release function, and a significant decline in the clearance rate of drug-resistant bacteria.

[0187] In Comparative Example 8, the second copolymer was not added, the targeting ligand and the antibacterial component were absent, and the functions of the material completely failed.

[0188] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and the individual values within that range. For example, it should be considered that the description of the range from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0189] In addition, in the description of the specification of the present application, the terms "comprising", "including", etc. mean "including but not limited to". In this text, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0190] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A preparation method of an exosome-integrated antibacterial polymer material, characterized in that, The method includes: Introducing a benzoyl imine bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer; Performing bacterial targeting ligand modification on the first copolymer by covalently coupling D-glutamic acid and electrostatically adsorbing lipoteichoic acid binding peptide to obtain a second copolymer; Mixing an aldehyde group-modified methyl cellulose phosphate buffer solution with an amino group-modified chitosan phosphate buffer solution to obtain a first mixture; Adding a mixed solution of protocatechuic aldehyde and an FeCl3 solution to the first mixture and adjusting the pH to 6.0 - 7.0 to obtain a second mixture; Under stirring, adding the second copolymer, placental mesenchymal stem cell exosomes, and Ag@rGO nanoparticles to the second mixture, and standing to obtain the exosome-integrated antibacterial polymer material.

2. The preparation method of the exosome-integrated antibacterial polymer material according to claim 1, wherein The step of introducing a benzoyl imine bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer includes: Mixing a polylysine phosphate buffer solution and a chitosan phosphate buffer solution at a molar ratio of 1:1, adding a mixed crosslinking agent of EDC and NHS, reacting at 25 - 30 °C for 10 - 12 h, and then performing purification to obtain a polylysine-chitosan copolymer; Dissolving the polylysine-chitosan copolymer in a DMF solvent, adding p-formylbenzoic acid, and performing a stirring reaction under argon protection at 60 - 70 °C for 20 - 28 h, and then performing purification to introduce a benzoyl imine bond into the molecular structure of the polylysine-chitosan copolymer to obtain a first copolymer.

3. The preparation method of the exosome-integrated antibacterial polymer material according to claim 2, wherein The molar ratio of EDC to NHS is 1:(0.8 - 1.2); The mass of the p-formylbenzoic acid is 5% - 10% of the mass of the polylysine-chitosan copolymer.

4. The preparation method of the exosome-integrated antibacterial polymer material according to claim 1, characterized in that, The step of performing bacterial targeting ligand modification on the first copolymer by covalently coupling D-glutamic acid and electrostatically adsorbing lipoteichoic acid binding peptide to obtain a second copolymer includes: Mixing a D-glutamic acid phosphate buffer solution with a mixed crosslinking agent of EDC and NHS to activate the carboxyl group, then mixing with the first copolymer, and reacting at 20 - 30 °C for 4 - 8 h to couple the amide bond of the activated D-glutamic acid to the surface of the first copolymer, and then performing purification to obtain a D-Glu-modified copolymer; Mixing an aqueous solution of lipoteichoic acid binding peptide with a concentration of 0.5 - 2 mg / mL with the D-Glu-modified copolymer and stirring at pH 5.5 - 6.5 for 2 - 6 h to adsorb the lipoteichoic acid binding peptide to the surface of the D-Glu-modified copolymer through electrostatic interaction, and then performing purification and freeze-drying to obtain a second copolymer.

5. The preparation method of the exosome-integrated antibacterial polymer material according to claim 4, wherein, The molar ratio of EDC to NHS is 1:(0.8 - 1.2); The molar ratio of the D-glutamic acid to the first copolymer is 1:(1.5 - 2); The mass ratio of the lipoteichoic acid binding peptide to the D-Glu-modified copolymer is 1:(8 - 12).

6. The preparation method of the exosome-integrated antibacterial polymer material according to claim 1, wherein The concentration of the aldehyde group-modified methyl cellulose phosphate buffer solution is 8 - 12 wt%, the concentration of the amino group-modified chitosan phosphate buffer solution is 2 - 4 wt%, and the mixing volume ratio is 1:(0.8 - 1.2).

7. The preparation method of the exosome-integrated antibacterial polymer material according to claim 1, wherein, The molar ratio of the protocatechualdehyde to FeCl3 is (3 to 4):1, and the final concentration of Fe in the second mixture 3+ is 0.08 to 0.12 M.

8. The preparation method of the exosome-integrated antibacterial polymer material according to claim 1, wherein, The Ag@rGO nanoparticles are synthesized by a chemical reduction method, the mass ratio of AgNO3 to reduced graphene oxide is 1:(5-10), and the particle size is 20-50 nm.

9. The preparation method of the exosome-integrated antibacterial polymer material according to claim 1, wherein, The mass of the second copolymer is 10%-15% of the mass of the second mixture; The mass of the Ag@rGO nanoparticles is 5-8% of the mass of the second mixture; The number of particles of placental mesenchymal stem cell exosomes per gram of the second mixture is 1×10 10 ~5×10 11 .

10. An exosome-integrated antibacterial polymer material obtained by the preparation method of the exosome-integrated antibacterial polymer material described in any one of claims 1 to 9, characterized in that, The exosome-integrated antibacterial polymer material is used for the repair of chronic infected wounds.

Images