CN110975000A

CN110975000A - Preparation and application of antibacterial modified exosome burn wound healing promotion biological dressing

Info

Publication number: CN110975000A
Application number: CN201911165878.XA
Authority: CN
Inventors: 刘海峰; 钱智勇; 樊瑜波; 白雅婷
Original assignee: Beihang University
Current assignee: Beihang University
Priority date: 2019-11-25
Filing date: 2019-11-25
Publication date: 2020-04-10

Abstract

Preparation and application of an antibacterial modified exosome burn wound healing promotion biological dressing. The raw dressing is a novel artificial modified exosome prepared by taking exosomes from cell sources as carriers of an antibacterial agent and combining a broad-spectrum antibacterial agent. And loading the modified exosome into the asymmetric chitosan porous moisturizing dressing. The weight ratio of chitosan, water-absorbing molecules, exosomes and antibacterial agents in the dressing is as follows: (40-60), (20-40), (5-20), (1-10). The dressing has broad-spectrum antibacterial effect, and is effective in promoting blood vessel repair, nerve repair and wound healing. Meanwhile, the dressing has moisture retention, air permeability, barrier property, high strength and easy uncovering property, and is a novel antibacterial composite dressing with a broad-spectrum antibacterial effect. The dressing is prepared by freeze-drying, and has the advantages of simple preparation process, low cost, remarkable treatment effect, good safety, obvious medical value and industrialization potential.

Description

Preparation and application of antibacterial modified exosome burn wound healing promotion biological dressing

Technical Field

The invention relates to an antibacterial modified exosome burn wound healing promotion biological dressing, and in particular relates to a water-absorbing antibacterial healing promotion hydrogel antibacterial dressing for an infectious wound surface.

Background

The skin is the largest organ of the human body, can not only protect the body from being damaged and isolate pathogens to play a role in barrier protection, but also plays an important role in preventing excessive water loss of the body. Trauma often impairs normal skin tissue structure and physiological function. Infection caused by the wound in the treatment process is always a main problem in the clinical treatment process. Pseudomonas aeruginosa (p. aeruginosa, a typical gram-negative bacterium) is a relatively broad range of microorganisms in nature and is one of the major pathogenic bacteria of nosocomial infections. Infection of the wound due to invasion of the wound by pseudomonas aeruginosa inhibits repair of epithelial tissue, resulting in persistent infection and delayed wound healing. Delayed wound healing can lead to the risk of gangrene, amputation and even death. In addition, the treatment of large areas of infectious wounds generally requires the use of antibiotics in high frequency and high doses, which treatment results in the emergence of clinically resistant strains. Therefore, improving the performance of the traditional dressing has important significance for effectively treating the infected wound.

Good wound dressings are particularly important for the effective treatment of infected wounds. An ideal wound dressing should have good moisture retention, electrolyte balance, hemostasis, analgesia, good antibacterial properties, and wound healing promotion. Wound dressings can be broadly divided into traditional dressings, synthetic dressings, and biological dressings. Traditional dressings such as sterile gauze and the like cannot keep the wound surface wet for a long time, and the dressings soaked by body fluid are easy to pass pathogens to cause secondary infection. In addition, dressing fiber is easy to fall off to cause foreign body reaction, and the dressing fiber is adhered to the wound surface to cause new epidermal injury when being uncovered, so that the healing of the wound surface is influenced, and the pain of a patient is caused. The synthetic dressing is made of high molecular materials, such as polyurethane, polyvinyl alcohol and the like, and can be made into films, sponges, hydrogel and the like, and most of the materials have a good barrier effect but have no obvious promotion effect on wound tissue regeneration. The biological dressing mainly comprises xenogenic skin, amniotic membrane, animal peritoneum, collagen membrane, chitin dressing and the like, wherein the xenogenic skin has limited source, and the xenogenic skin has serious immunological rejection reaction with animal amniotic membrane and peritoneal membrane materials. And the collagen, silk fibroin and chitin materials show better application potential due to mature processing technology, rich modification means and easy large-scale production. Although some wound dressings are clinically applied, for example, commercially available dressings such as Syvek-Patch, Chitopack C, Tegasorb, HemCon Bandage, and KytoCel are clinically applied, but the antibacterial effect is limited, and the treatment effect on infectious wounds is not good.

Under physiological conditions, wound healing involves many complex processes such as hemostasis, inflammation, new tissue formation and remodeling of skin appendages. An ideal wound dressing should provide good antimicrobial effects, promote wound healing, retain moisture, maintain electrolyte balance, and stop bleeding. Nano-silver is a broad-spectrum antimicrobial metal particle that has been used clinically for many years. However, the nano-silver application is still limited for the following reasons: (1) it has no wound healing promoting effect; (2) it can pass through the surface of biological mucosa and enter the animal's organs.

Exosomes are produced by cell fusion, endocytosis, or receptor cell-specific phagocytosis. They provide a variety of bioactive molecules, including membrane receptors, proteins, mRNA, and microRNA, among others. Research in recent years has shown that many exosomes can promote cell proliferation, angiogenesis and re-epithelialization and thus skin repair and regeneration. Researchers found that exosomes in human cord blood could promote angiogenesis and fibroblast proliferation to promote skin wound healing. In addition, the human urinary stem cell-derived exosome can accelerate the healing of the diabetic wound. The exosome can be used as a stem cell paracrine factor to play a biological effect, and a new way is provided for further realizing cell-free treatment. However, exosomes have limited utility in the treatment of infectious wounds due to their lack of antibacterial function.

We have proved in previous studies that silk fibroin egg can be used as nano-silver slow release material, and the nano-silver slow release material is slowly released in a medium. The invention mixes the silver nano-particle solution and the exosome solution, and utilizes ultrasonic waves to guide the nano-silver particles into the exosome, thereby preparing the exosome with the broad-spectrum antibacterial function. The exosome with the broad-spectrum antibacterial function is loaded into the asymmetric moisturizing dressing, and a novel broad-spectrum antibacterial moisturizing dressing (CTS-SF/SA/Ag-Exo dressing) is prepared by secondary freeze-drying. The CTS-SF/SA/Ag-Exo dressing has a broad-spectrum antibacterial function, and can effectively kill Candida albicans, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. In a pseudomonas aeruginosa infected mouse full-thickness skin defect model, the wound healing is promoted, and the effects of blood vessel regeneration and nerve regeneration are obvious. The invention is believed to provide new opportunities and treatment strategies for the treatment of clinically infectious refractory wounds.

Disclosure of Invention

Aiming at the defects of the existing clinical burn wound dressing products, the invention loads the nano-silver modified exosome compound into the asymmetric moisturizing dressing to prepare the antibacterial modified exosome burn wound healing-promoting biological dressing. The dressing has broad-spectrum antibacterial property, and has wound healing promoting effects, such as water absorption, moisture keeping, air permeability, barrier property, hemostatic effect, and easy uncovering property.

In order to achieve the purpose, the invention comprises the following technical scheme:

an antibacterial modified exosome burn wound healing promotion biological dressing is prepared by taking exosome as an antibacterial agent slow-release carrier and loading the antibacterial agent modified exosome on an asymmetric chitosan water-absorbing moisturizing dressing. The mass ratio of chitosan, water absorption molecules, exosomes, antibacterial agents and plasticizers in the antibacterial modified exosome burn wound healing promotion biological dressing is 1: (0.2-1): (0.1-2): (0.5-2): (1-5); wherein the content of the first and second substances,

the chitosan is one or a mixture of acid-soluble chitosan, water-soluble chitosan and anhydride modified chitosan derivatives;

the water-absorbing polymer is silk fibroin, gelatin, collagen, polyethylene glycol, polyacrylamide, polymethyl methacrylate and/or polyvinyl alcohol;

the antibacterial agent is one or more of nano-metal antibacterial materials selected from nano-gold, nano-silver or nano-zinc, the nano-nonmetal antibacterial materials are one or more selected from nano-ferroferric oxide, nano-zinc oxide or nano-titanium dioxide, and the quaternary ammonium salt antibacterial materials are one or more selected from chitosan quaternary ammonium salt and guanidine salt antibacterial materials;

the antibacterial agent slow-release carrier is one or more of exosomes selected from stem cell-derived exosomes, platelet-derived exosomes or blood-derived exosomes;

the asymmetric modified alkane derivative is selected from one or more of fatty acid anhydride, fatty acid and polyester.

The antibacterial modified exosome burn wound healing-promoting biological dressing as described above, preferably,

the chitosan is one or more of chitosan with high deacetylation degree, methacrylic anhydride modified methacrylic anhydride terminated chitosan, acrylic anhydride modified acrylic anhydride terminated chitosan, maleic anhydride modified maleic anhydride terminated chitosan and itaconic anhydride modified itaconic anhydride terminated chitosan;

the acrylic anhydride compound is methacrylic anhydride, acrylic anhydride, maleic anhydride or itaconic anhydride.

the nano antibacterial material is selected from one or more of nano gold, nano silver or nano zinc, the nano non-metallic material is selected from nano ferroferric oxide, nano zinc oxide or nano titanium dioxide, and the quaternary ammonium salt antibacterial material is selected from one or more of chitosan quaternary ammonium salt and guanidine salt antibacterial material.

The antibacterial modified exosome burn wound healing promoting biological dressing as described above, preferably, the dressing further comprises a plasticizer, an emulsifier and/or an antioxidant.

On the other hand, the invention provides a preparation method of the antibiotic function modified exosome burn wound healing promotion biological dressing, which comprises the following steps:

(1) the stem cell culture medium was collected and centrifuged to remove cell debris and dead cells from the supernatant. The centrifuged supernatant was collected and filtered through a 0.22 μm filter. Centrifuging the filtered culture supernatant and collecting the ultrafiltered liquid;

(2) and (3) enabling the ultrafiltration liquid collected in the step (1) to pass through an qEV-exosome separation column, and continuously collecting an exosome solution. Placing the collected exosome solution into an ultrafiltration centrifugal tube (30KD) again for centrifugation, concentrating the collected exosome solution, and storing at-80 ℃ for later use;

(3) adding 1mL of isopropanol into 5% silver nitrate solution, magnetically stirring for 10min, adding 2g of glucose, and magnetically stirring for 2h at 80 ℃ to obtain nano-silver solution;

(4) mixing 800 mu L of the exosome solution separated in the step (2) with 200 mu L of the nano-silver solution (diluted by 32 times) prepared in the step (3), and performing ultrasonic treatment for 1min to obtain a nano-silver modified exosome colloidal solution;

(5) adding 20mL of 4% silk fibroin solution into 1mL of glycerol, mechanically stirring for 10 minutes, adding 10mL of 2% chitosan solution into the silk fibroin solution, mechanically stirring for 30 minutes, pouring into a container with the size of 100 multiplied by 150mm, standing at 4 ℃ for 1 hour, standing at-20 ℃ for 4 hours, standing at-70 ℃ for 6 hours, and freeze-drying by a freeze-dryer to obtain CTS-SF sponge for later use;

(6) fully absorbing deionized water by using CTS-SF sponge, standing at-20 ℃ for 4h, uniformly pouring 8mL of stearic acid solution (40mmol/L of ethanol and DCC as a dehydrating agent) on the smooth surface of the CTS-SF sponge, continuously placing in a freezing chamber of a refrigerator for 2 hours, and washing the smooth surface of the CTS-SF dressing by using absolute ethanol at 20 ℃ for 3 times to obtain CTS-SF/SA sponge for later use;

(7) and (3) adding 800 mu L of the colloidal solution prepared in the step (4) into the CTS-SF/SA sponge with the size of 20 x 20mm prepared in the step (6), standing at 4 ℃ for 30min, standing at-20 ℃ for 2h, standing at-70 ℃ for 6h, and freeze-drying by a freeze-dryer to obtain the antibacterial healing-promoting modified exosome burn wound biological dressing (CTS-SF/SA/Ag-Exo dressing).

The preparation method as described above, preferably, the specific operation of the step (1) is as follows:

the stem cell culture medium was collected, and the collected cell culture supernatant was centrifuged at 2000g at 4 ℃ to remove cell debris and dead cells from the supernatant. The centrifuged supernatant was collected and filtered through a 0.22 μm filter. Placing the filtered culture supernatant in an ultrafiltration centrifuge tube (30KD), centrifuging at 4 deg.C and 5000g for 20min, and collecting the ultrafiltered liquid solution;

the preparation method as described above, preferably, the specific operation of the step (2) is as follows:

and (3) enabling the collected ultrafiltration liquid in the step (1) to pass through an qEV-exosome separation column, and placing a 15ml centrifuge tube below the column for collecting separated exosomes. Before the exosomes were extracted, the column was washed once with 10ml of PBS, 0.5ml of the ultrafiltered cell culture supernatant was added, 3ml of waste liquid was discarded, and 2ml of exosome solution was collected, at which time the column was carefully supplemented with PBS to prevent drying. qEV were washed again with 10ml PBS and the above procedure was repeated to collect exosomes continuously. Placing the collected exosome in an ultrafiltration centrifugal tube (30KD) again, centrifuging for 20min at 4 ℃ and 5000g, concentrating the collected exosome, and storing at-80 ℃ for later use;

in still another aspect, the invention provides an antibacterial function modified exosome burn wound healing promotion biological dressing, which is prepared by adopting the method.

In another aspect, the invention provides a preparation method of the antibacterial modified exosome burn wound healing-promoting biological dressing, which is mainly used for treating large-area burns, large-area wounds, deep burns, large-area infected wounds, drug-resistant strain infected wounds and large-area drug-resistant strain infected wounds.

The broad-spectrum antibacterial agent is preferably a quaternary ammonium salt antibacterial agent, a nano metal antibacterial agent or a nano nonmetal antibacterial agent.

The chitosan is preferably one or more of methacrylic anhydride-terminated chitosan modified by methacrylic anhydride, acrylic anhydride-modified acrylic anhydride-terminated chitosan, maleic anhydride-modified maleic anhydride-terminated chitosan and itaconic anhydride-modified itaconic anhydride-terminated chitosan.

The plasticizer used in the present invention is preferably glycerin, propylene glycol or sorbitol.

The polyethylene glycol used in the invention is preferably one or more of PEG-400, PEG-600, PEG-1500, PEG-4000, PEG-6000 and PEG-20000.

The antibacterial modified exosome burn wound healing promotion biological dressing is an asymmetric moisturizing dressing as a carrier of an antibacterial healing promotion material, exosomes modified by a spectral antibacterial agent are combined to serve as slow-release medicines, and macromolecules or polymers with flexible structures and water absorption capacity serve as moisturizing and water absorbing agents. The dressing has good mechanical strength due to the rigid molecular junction of the chitosan, and macromolecules or polymers with flexible structures and water absorption capacity are doped in the chitosan, so that the dressing has good flexibility and is comfortable to contact with the skin. The loaded antibacterial agent modified exosome not only has a broad-spectrum antibacterial function, but also has a good effect of promoting wound healing.

The antibacterial agent slow release carrier disclosed by the invention is of a phospholipid bimolecular membrane structure, and bioactive molecules including bioactive proteins, DNA, RNA and the like are arranged inside the antibacterial agent slow release carrier. The bioactive molecules have good wound repair effect, vascular repair effect, nerve repair effect and the like. The carrier loaded with the antibacterial agent is dissolved when meeting the phospholipase secreted by bacteria, and the antibacterial agent and bioactive molecules are released, so that the dressing has double functions of antibiosis and healing promotion. The beneficial effects of the invention are as follows:

(1) the water-absorbing macromolecules or polymers in a proper proportion in the dressing have certain water-absorbing and water-locking functions. The water absorption rate is 10-15 times of the self weight, and the water can be absorbed instantly when meeting water. When the dressing is contacted with body fluid, the material swells to form gel, so that the wound surface can be effectively isolated from the outside, and the dressing has good air permeability. The water-absorbing macromolecules in the dressing are rich in carboxyl or hydroxyl, and are combined with water molecules through hydrogen bonds, so that the dressing has the intramolecular water-locking characteristic, and absorbed water cannot be squeezed out through external force. The water absorption function is helpful for wound hemostasis, and when the water absorption function is contacted with blood, the hemostatic components such as platelet, thrombin, fibrin and the like in the blood are highly concentrated, so that the formation of blood clots is accelerated and strengthened. Meanwhile, the dressing can absorb redundant exudates and reduce the breeding of bacteria. The moisture-locking function of the dressing enables the contact surface to keep certain humidity, thereby being beneficial to accelerating the formation of epithelial tissues, relieving pain, decomposing necrotic tissues and slowly releasing the antibacterial agent.

(2) The antibacterial agent slow-release carrier in the dressing can play the roles of slow-release antibacterial agent and broad-spectrum antibacterial for a long time.

(3) The dressing can be tightly attached to the wound surface, seals the wound surface, effectively kills bacteria in the environment, prevents harmful particles from contacting the wound surface, has good air permeability, and cannot be adhered to the wound surface tissue.

(5) The antibacterial modified exosome burn wound healing-promoting biological dressing can be used for treatment of large-area burn, large-area wound, deep burn wound, large-area infection wound, drug-resistant strain infection wound, large-area drug-resistant strain infection wound and the like.

(6) The antibacterial modified exosome burn wound healing-promoting biological dressing is prepared by a freeze-drying method, a separation and purification process is not needed in the middle, the cost is saved, the quality control is convenient, and the large-scale production is facilitated.

Drawings

FIG. 1 is an electron microscope photograph of the biological dressing for promoting healing of burn and wound prepared by the exosome with modified antibacterial function in example 1.

FIG. 2 the porosity, swelling ratio, moisture retention property and protein slow release property of the antibiotic function modified exosome burn wound healing-promoting biological dressing prepared in the embodiment 1 to 4.

FIG. 3 is an in vitro broad-spectrum antibacterial experiment of the modified exosome burn wound healing promoting biological dressing prepared in example 1.

Fig. 4 the antimicrobial healing-promoting biological dressing prepared in example 1 was used for the treatment of infectious wounds.

FIG. 5 is a process flow diagram of the synthesis of the antibiotic modified exosome burn wound healing promoting biological dressing.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 preparation of antibacterial modified exosome burn wound healing-promoting biological dressing sample 1 (the synthetic process flow is shown in figure 5)

(1) The stem cell culture medium was collected, and the collected cell culture supernatant was centrifuged at 2000g at 4 ℃ to remove cell debris and dead cells from the supernatant. The centrifuged supernatant was collected and filtered through a 0.22 μm filter. Placing the filtered culture supernatant in an ultrafiltration centrifuge tube (30KD), centrifuging at 4 deg.C and 5000g for 20min, and collecting the ultrafiltered liquid;

(2) and (3) enabling the collected ultrafiltration liquid in the step (1) to pass through an qEV-exosome separation column, and placing a 15ml centrifuge tube below the column for collecting separated exosomes. Before the exosomes were extracted, the column was washed once with 10ml of PBS, 0.5ml of the ultrafiltered cell culture supernatant was added, 3ml of waste liquid was discarded, and 2ml of exosome solution was collected, at which time the column was carefully supplemented with PBS to prevent drying. qEV were washed again with 10ml PBS and the above procedure was repeated to collect the exosome solutions continuously. Placing the collected exosome solution into an ultrafiltration centrifugal tube (30KD) again, centrifuging for 20min at 4 ℃ and 5000g, concentrating the collected exosome solution, and storing at-80 ℃ for later use;

(7) and (3) adding 800 mu L of the colloidal solution prepared in the step (4) into the CTS-SF/SA sponge with the size of 20 x 20mm prepared in the step (6), standing at 4 ℃ for 30min, standing at-20 ℃ for 2h, standing at-70 ℃ for 6h, and freeze-drying by a freeze-dryer to obtain the antibacterial modified exosome burn wound healing-promoting biological dressing (CTS-SF/SA/Ag-Exo dressing). Packaging, cutting, and sterilizing by cobalt 60 irradiation.

Example 2 preparation of antibacterial modified exosome burn wound healing promoting biological dressing sample 2

(2) and (3) enabling the collected ultrafiltration liquid in the step (1) to pass through an qEV-exosome separation column, and placing a 15ml centrifuge tube below the column for collecting separated exosomes. Before the exosomes were extracted, the column was washed once with 10ml of PBS, 0.5ml of the ultrafiltered cell culture supernatant was added, 3ml of waste liquid was discarded, and 2ml of exosomes began to be collected, at which time the column was carefully supplemented with PBS to prevent drying. qEV were washed again with 10ml PBS and the above procedure was repeated to collect the exosome solutions continuously. Placing the collected exosome solution into an ultrafiltration centrifugal tube (30KD) again, centrifuging for 20min at 4 ℃ and 5000g, concentrating the collected exosome solution, and storing at-80 ℃ for later use;

(3) mixing 800 mu L of the exosome solution separated in the step (2) with 200 mu L of 0.156% nano zinc oxide, and carrying out ultrasonic treatment for 1min to obtain a nano zinc oxide modified exosome colloidal solution;

(4) adding 20mL of 4% polyvinyl alcohol solution into 1mL of glycerol, mechanically stirring for 10 minutes, adding 10mL of 2% chitosan solution into the polyvinyl alcohol solution, mechanically stirring for 30 minutes, pouring into a container with the size of 100 multiplied by 150mm, standing at 4 ℃ for 1 hour, standing at-20 ℃ for 4 hours, standing at-70 ℃ for 6 hours, and freeze-drying by a freeze-dryer to obtain CTS-PVA sponge for later use;

(5) fully absorbing deionized water by using CTS-PVA sponge, standing at-20 ℃ for 4h, uniformly pouring 8mL of stearic acid solution (40mmol/L ethanol, DCC serving as a dehydrating agent) on the smooth surface of the CTS-PVA sponge, continuously placing in a freezing chamber of a refrigerator for 2h, and washing the smooth surface of the CTS-PVA dressing by using absolute ethanol at 20 ℃ for 3 times to obtain CTS-PVA/SA sponge for later use;

(6) and (3) adding 800 mu L of the colloidal solution prepared in the step (3) into the CTS-PVA/SA sponge with the size of 20 multiplied by 20mm prepared in the step (5), standing for 30min at the temperature of 4 ℃, standing for 2h at the temperature of-20 ℃, standing for 6h at the temperature of-70 ℃, and freeze-drying by a freeze-drying machine to obtain the antibacterial modified exosome burn wound healing-promoting biological dressing (CTS-PVA/SA/Ag-Exo dressing). Packaging, cutting, and sterilizing by cobalt 60 irradiation.

Example 3 preparation of antibacterial modified exosome burn wound healing promoting biological dressing sample 3

(3) mixing 800 mu L of the exosome solution separated in the step (2) with 200 mu L of 0.156% polyhexamethylene biguanide hydrochloride, and carrying out ultrasonic treatment for 1min to obtain a colloid solution of the polyhexamethylene biguanide hydrochloride modified exosome;

(4) adding 20mL of 4% polyethylene glycol-6000 solution into 1mL of glycerol, mechanically stirring for 10 minutes, adding 10mL of 2% chitosan solution into PEG-6k solution, mechanically stirring for 30 minutes, pouring into a container with the size of 100 multiplied by 150mm, standing at 4 ℃ for 1 hour, standing at-20 ℃ for 4 hours, standing at-70 ℃ for 6 hours, and freeze-drying by a freeze-dryer to obtain CTS-PEG-6k sponge for later use;

(5) fully absorbing deionized water by using CTS-PEG-6k sponge, standing at the temperature of-20 ℃ for 4h, uniformly pouring 8mL of stearic acid solution (40mmol/L ethanol and DCC as a dehydrating agent) on the smooth surface of the CTS-PEG-6k sponge, continuously placing in a freezing chamber of a refrigerator for 2h, and washing the smooth surface of the CTS-PEG-6k dressing by using absolute ethanol at the temperature of 20 ℃ for 3 times to obtain the CTS-PEG-6k/SA sponge for later use;

(6) and (3) adding 800 mu L of the colloidal solution prepared in the step (3) into the CTS-PEG-6k/SA sponge with the size of 20 multiplied by 20mm prepared in the step (5), standing at 4 ℃ for 30min, standing at-20 ℃ for 2h, standing at-70 ℃ for 6h, and freeze-drying by a freeze-drying machine to obtain the antibacterial modified exosome burn wound healing-promoting biological dressing (CTS-PEG-6k/SA/Ag-Exo dressing). Packaging, cutting, and sterilizing by cobalt 60 irradiation.

Example 4 preparation of antibacterial modified exosome burn wound healing promoting biological dressing sample 4

(3) mixing 800 mu L of the exosome solution separated in the step (2) with 200 mu L of 0.156% polyhexamethylene guanidine hydrochloride, and performing ultrasonic treatment for 1min to obtain polyhexamethylene guanidine hydrochloride modified exosome colloidal solution;

(4) adding 20mL of 4% silk fibroin solution into 1mL of glycerol, mechanically stirring for 10 minutes, adding 10mL of 2% chitosan solution into the silk fibroin solution, mechanically stirring for 30 minutes, pouring into a container with the size of 100 multiplied by 150mm, standing at 4 ℃ for 1 hour, standing at-20 ℃ for 4 hours, standing at-70 ℃ for 6 hours, and freeze-drying by a freeze-dryer to obtain CTS-SF sponge for later use;

(5) fully absorbing deionized water by using CTS-SF sponge, standing at-20 ℃ for 4h, uniformly pouring 8mL of stearic acid solution (40mmol/L of ethanol and DCC as a dehydrating agent) on the smooth surface of the CTS-SF sponge, continuously placing in a freezing chamber of a refrigerator for 2 hours, and washing the smooth surface of the CTS-SF dressing by using absolute ethanol at 20 ℃ for 3 times to obtain CTS-SF/SA sponge for later use;

(6) and (3) adding 800 mu L of the colloidal solution prepared in the step (3) into the CTS-SF/SA sponge with the size of 20 x 20mm prepared in the step (5), standing at 4 ℃ for 30min, standing at-20 ℃ for 2h, standing at-70 ℃ for 6h, and freeze-drying by a freeze-dryer to obtain the antibacterial modified exosome burn wound healing-promoting biological dressing (CTS-SF/SA/Ag-Exo dressing). Packaging, cutting, and sterilizing by cobalt 60 irradiation.

Example 5 physical and structural characterization

The antibacterial modified exosome burn wound healing promotion biological dressing obtained in the examples 1-4 is of a porous dressing-like structure, the appearance of the dressing is characterized by selecting the dressing 1 (as shown in figure 1), the porosity is 40% -80%, the pore size is 100 μm-1mm, and the dressing is of a mutually-penetrated pore structure (as shown in figure 1). After contacting with body fluid, the pore structure of the porous material quickly sucks the body fluid into the pores and quickly gelatinizes, so that on one hand, the pore wall thickens and gelatinizes after absorbing water, the tube cavity becomes narrow, and the viscoelasticity of the tube wall is increased; on the other hand, the quick gelling dressing begins to slowly release the nano-silver modified exosome, and plays roles in broad-spectrum antibiosis and healing promotion.

The antibacterial modified exosome burn wound healing promotion biological dressing samples prepared in examples 1-4 were subjected to gross structural observation, and microstructure observation was performed by scanning electron microscopy. As represented by sample 1, as shown in fig. 1, the samples were all in the form of soft porous dressing, elastic, crimpable, and free of odor. And (5) displaying the interconnected porous channel structure under an electron microscope.

Example 6 physical Property characterization

The swelling performance of the antibacterial modified exosome burn wound healing-promoting biological dressing obtained in examples 1-4 is tested (shown in fig. 2A). 0.100g of the sample obtained in examples 1-4 is accurately weighed, soaked in deionized water (pH 7.0), physiological saline, phosphate buffer, α -MEM culture medium and blood serum, completely swells after absorbing water at 37 ℃, absorbs surface water, and weighs the mass of the sample after absorbing the liquid, and the water absorption rate of the sample is calculated.

TABLE1 Water absorption Capacity of different media

Results as shown in table1, samples 1-4 exhibited similar water absorption, and the swelling ratios in different media (shown in fig. 2A) were as follows, taking example 1 to prepare a dressing: the swelling ratio in water is between 12 and 15; the swelling ratio in the saline is 10-12; the swelling ratio in the phosphate buffer solution is between 8 and 9; swelling multiplying power in the cell culture solution is 6-7; the swelling ratio in serum is between 5 and 6.

The antibacterial modified exosome burn wound healing promoting biological dressing obtained in the examples 1 to 4 is tested for moisturizing performance, the moisturizing performance is similar, the dressing prepared in the example 1 is taken as an example to illustrate the moisturizing performance of the dressing (shown in fig. 2B), the moisturizing performance is superior to that of a control group, and the moisturizing time is over 13 hours.

The porosity detection of the antibacterial modified exosome burn wound healing promoting biological dressing obtained in examples 1-4 is close, and the dressing porosity is illustrated by taking the dressing prepared in example 1 as an example (shown in fig. 2C). The porosity distribution of the control group and the experimental group is 70-85%.

The antibacterial modified exosome burn wound healing promoting biological dressing obtained in the examples 1-4 has the same protein slow release function. The slow release function of the dressing is illustrated by preparing the dressing in example 1 (shown in fig. 2D). 0.100g of the sample in example 1 was accurately weighed, immersed in physiological saline at 37 ℃, and then subjected to physiological saline for 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h and 72h, respectively, and then the release of the protein in the antibacterial dressing was detected by using the BCA total protein kit, and a sustained-release curve was plotted (shown in FIG. 2D). The results show that the slow release time of the protein of the sample prepared in example 1 is longer than that of the control group, and the sustained and effective antibiosis and wound healing promotion can be ensured when the dressing is in contact with the wound.

Example 7 in vitro broad-spectrum antibacterial Performance test

The antibacterial activity of the antibacterial modified exosome burn wound healing promoting biological dressings prepared in examples 1-4 was tested by the bacteriostatic loop method. The dressing was evaluated for antimicrobial activity using staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, candida albicans. 70. mu.L of bacterial suspension (1X 10)⁸CFU/mL) was spread on an LB agar plate, sterile gauze, a commercially available antibacterial dressing, and the water-absorbing burn wound antibacterial dressings prepared in examples 1 to 4 were placed on the surface of the agar, and after incubation at 37 ℃ for 12 hours, the diameter of the antibacterial ring was measured. Table 2 shows the killing effect of 6 dressings on 6 strains (candida albicans, escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, staphylococcus aureus-resistant strains and pseudomonas aeruginosa-resistant strains). The dressing has an antibacterial effect superior to that of a commercially available nano-silver dressing by analyzing the size of the antibacterial zone.

TABLE 2 evaluation of the antibacterial Effect of six strains

Note: non-antimicrobial "-"; the antibacterial activity is indicated by "+", wherein the diameter of the zone of inhibition is greater than 3mm is indicated by "+".

Example 8 in vivo assessment of infectious wound healing Effect

The biological and medical ethics committee of the university of beijing aerospace approved in vivo animal experiments. 100 BALB/c mice, male, each weighing about 18g + -2 g, 7-10 weeks old. Randomly dividing into 5 groups (20 mice per group), intraperitoneally injecting pentobarbital sodium (20mg/kg) to anesthetize mice, removing skin hair, making phi 1cm full-thickness skin injury on the back of each mouse, and dripping 1 × 10 concentration on wound surface⁸Each wound surface of the CFU pseudomonas aeruginosa bacterial liquid is 100 mul, and an infected wound surface is formed. The wound surface is tightly covered by the antibacterial dressing 1-4, the commercially available nano-silver dressing and the sterile gauze respectively. Changes were made 2 times per week.

The wound healing effect of the antibacterial dressing is evaluated according to the repairing condition of the infected wound of a BALB/c mouse. FIG. 4 shows wound healing for CTS-SF/SA/Ag-Exo, CTS-SF/Ag/SA, Acosin, gauze-Exo and gauze prepared in example 1 of the present invention on

days

3, 7 and 12. The wounds treated with 5 dressings all had scabbing to varying degrees and the wounds started to contract. The wound surface repaired by the antibacterial dressing is more obvious in shrinkage and the treatment effect is optimal. The results of the 8 samples are shown in Table 3, and the wound treated by the antibacterial dressing group of the invention reaches 53.24 +/-2.12% on the 3 rd day after the wound, and the commercial nano silver group and gauze group reach 21.84 +/-1.21% and 32.08 +/-2.23%. The wound surface edge of the antibacterial dressing group is not inflamed, the commercially available nano silver group has obvious inflamed wound surface edge, and the gauze group still can see part of infected exudates. Through observation of wound surfaces of mice on the 7 th day after wound, the healing rate of the antibacterial dressing 1-4 groups is found to reach 73.34 +/-2.32-78.54 +/-1.33% on average. The gauze-Ag group dressing has serious adhesion with the wound surface, and the red and swollen phenomenon of the edge of the wound surface is most obvious. The granulation of the gauze group was evident and a partial inflammatory exudate remained. On the 12 th day after operation, the optimal healing rate of the antibacterial dressing 1-4 groups can reach 98.32 +/-1.74%, the healing rates of the commercially available nano-silver group and the gauze group are 83.42 +/-2.14% and 63.24 +/-1.32%, and the healing rates of the three groups have obvious statistical difference (Table1 p is less than 0.01), and the results show that the antibacterial dressing group is optimal, and the commercially available nano-silver group is inferior, and the gauze group is poor.

TABLE 3 wound healing Rate of infectious wound

Claims

1. An antibacterial modified exosome burn wound healing promotion biological dressing is characterized in that a novel modified exosome is generated by combining an exosome from a cell source with a broad-spectrum antibacterial agent, and the modified exosome is loaded into an asymmetric chitosan porous moisturizing dressing. The chitosan is used as the skeleton of the dressing, and the water-absorbing polymer is used as water-absorbing and flexible molecules, so that the asymmetric dressing has good water-absorbing and flexibility. The weight ratio of the chitosan, the water-absorbing polymer, the exosome and the antibacterial agent in the dressing is as follows: (40-60), (20-40), (5-20), (1-10).

Wherein the chitosan is one or more of acid-soluble chitosan, water-soluble chitosan and acid anhydride modified chitosan derivatives;

the water-absorbing polymer is one or more of silk fibroin, gelatin, collagen, polyethylene glycol, sodium polyacrylate, polyacrylamide, polymethyl methacrylate and/or polyvinyl alcohol;

the exosome is an exosome derived from cells, and comprises a stem cell source, a fibroblast source, a platelet source and the like;

the broad-spectrum antibacterial agent is one or more of nano antibacterial agents selected from nano metal particles and nano nonmetal particles, quaternary ammonium salt antibacterial agents and guanidine salt antibacterial agents.

2. The healing-promoting exosome according to claim 1, wherein the exosome is derived from stem cells, from fibroblasts, from platelets, or the like.

3. The antibacterial healing-promoting modified exosome burn wound biological dressing according to claim 1 or 2, wherein the modified exosome is an antibacterial agent modified exosome, and the burn wound biological dressing is modified by stearic acid, palmitic acid and one or more of stearic anhydride and palmitic anhydride.

4. The antibiotic modified exosome burn wound biological dressing according to claim 1 or 2, characterized in that the nano metal antibiotic material is selected from one or more of nano gold, nano silver or nano zinc, the nano nonmetal antibiotic material is selected from one or more of nano ferroferric oxide, nano zinc oxide or nano titanium dioxide, and the quaternary ammonium salt antibiotic material is selected from one or more of chitosan quaternary ammonium salt and guanidine salt antibiotic material.

5. The antimicrobial modified exosome burn wound biological dressing of any one of claims 1-4, further comprising a plasticizer, an emulsifier and/or an antioxidant.

6. A preparation method of an antibacterial modified exosome burn wound healing-promoting biological dressing is characterized by comprising the following steps:

I. mixing chitosan, water-absorbing molecules, an antibacterial agent modified exosome and a plasticizer, then emulsifying by mechanical stirring, pouring into a grinding tool, and freeze-drying by a freeze dryer to obtain porous sponge;

II, the antibacterial healing-promoting modified exosome burn wound biological dressing obtained in the step I is prepared from chitosan, water absorbing molecules, exosomes, an antibacterial agent and a plasticizer in a mass ratio of 1: (0.2-1): (0.1-2): (0.5-2): (1-5).

7. The antibacterial healing-promoting modified exosome burn wound biological dressing of any one of claims 1-6, for use in treating a large area burn wound, a deep burn wound, a large area infected wound, a drug-resistant strain infected wound, a large area drug-resistant strain infected wound.