1. The protamine composite material is characterized by comprising protamine and a physical antibacterial material, wherein the protamine is coated outside the physical antibacterial material; the physical antibacterial material is one or two of graphene oxide or carbon nano tubes; the protamine is one or more of pure protamine, protamine sulfate, protamine hydrochloride or protamine fragments after enzyme digestion.

2. The protamine composite material of claim 1, wherein the outer side of the physical antimicrobial material is covalently bonded to the coated protamine.

3. The protamine composite of claim 1, wherein said protamine composite is in the form of a film.

4. The protamine composite material of claim 1, further comprising a gel forming agent cross-linked with the protamine coated outside the physical antibacterial material, the gel forming agent being one or more of sodium alginate, sodium alginate oxide, tannic acid, chitosan, cellulose, a polymer, gelatin, collagen or hyaluronic acid; the protamine composite material is in a gel shape.

5. A method of preparing the protamine composite material of claim 3, comprising the steps of:

s1, adding protamine into sterile water, and stirring until the protamine is completely dissolved to obtain a protamine solution;

s2, adding the physical antibacterial material into sterile water, and ultrasonically stirring until the physical antibacterial material is completely dissolved to obtain a physical antibacterial material solution;

s3, mixing the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2, and heating to obtain a composite suspension;

s4, performing suction filtration on the composite suspension prepared in the step S3, drying the film obtained by suction filtration, and separating the film from the basement membrane to obtain the membranous protamine composite material.

6. A method for preparing the protamine composite material of claim 4, comprising the steps of:

s1, adding protamine into sterile water, and stirring until the protamine is completely dissolved to obtain a protamine solution;

s2, adding the physical antibacterial material into sterile water, and ultrasonically stirring until the physical antibacterial material is completely dissolved to obtain a physical antibacterial material solution;

s3, mixing the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2, and heating to obtain a composite suspension;

s4, adding the composite suspension prepared in the step S3 into a gel forming agent, and pouring the gel forming agent into a gel mold, wherein the composite suspension forms a gelatinous protamine composite material through the crosslinking action of the gel forming agent; the mass fraction of the gel forming agent is 0.1-50%.

7. The method for preparing a protamine composite material according to claim 5 or 6, wherein the concentration of protamine in the protamine solution in the step S1 is 0.5g/L to 10 g/L.

8. The method of preparing a protamine composite material according to claim 5 or 6, wherein the concentration of the solution of the physical antibiotic material in the step S2 is 0.5g/L to 10 g/L.

9. The method for preparing a protamine composite material according to claim 5 or 6, wherein the step S3 is specifically that the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2 are mixed according to a volume ratio of 10: 1-1: 100, and then heated at 30-100 ℃ for 10-1000 min under a pH value of 1-14 to obtain a composite suspension.

10. Use of the protamine composite material of any one of claims 1 to 3 in the antibacterial field.

Background

Pathogenic microorganisms such as bacteria cause many hazards and impacts on human production and life. In particular, in recent years, global microbial disaster events caused by harmful bacteria frequently occur. Therefore, how to effectively control the infection and transmission of harmful microorganisms has become a key research topic of experts and scholars. Although antibiotics have strong bactericidal and bacteriostatic effects, drug resistance and environmental pollution caused by abuse of antibiotics become prominent problems and problems in the biomedical world nowadays. Therefore, research and development of novel, highly effective and safe antibacterial agents have become an urgent problem to be solved. The design of a composite material with multiple antibacterial activities is very important for the aspects of daily antibacterial requirements, treatment of infected wounds and the like.

Protamine is a protein with polycation property, contains more arginine, has stronger alkalinity and electropositivity, and has a molecular weight of about 4.2 KDa. The antibacterial peptide can act on a peptidoglycan part of a bacterial cell wall, has excellent antibacterial property and has important application prospect in the aspect of biomedical materials.

However, the existing protamine antibacterial method mainly uses protamine directly, which has the problems of large product dosage, high cost and the like, and protamine is easy to lose in the using process, thereby causing the antibacterial lasting effect of the product to be reduced. Therefore, how to develop an antibacterial material which is low in cost, simple in preparation method and capable of retaining an antibacterial component for a long time is one of the technical problems to be solved in the field.

Disclosure of Invention

The present invention is directed to overcoming at least one of the above-mentioned disadvantages of the prior art and providing a protamine composite material to improve the stability of protamine and reduce the loss of protamine during its use, which results in the decrease of antibacterial effect.

The technical scheme adopted by the invention is that the protamine composite material comprises protamine and a physical antibacterial material, wherein the protamine is coated outside the physical antibacterial material; the physical antibacterial material is one or two of graphene oxide or carbon nano tubes; the protamine is one or more of pure protamine, protamine sulfate, protamine hydrochloride or protamine fragments after enzyme digestion.

Further, the outer side of the physical antibacterial material is covalently bonded with the coated protamine.

Preferably, the protamine composite is in the form of a film.

Preferably, the protamine composite material further comprises a gel forming agent crosslinked with the protamine coated on the outer side of the physical antibacterial material, wherein the gel forming agent is one or more of sodium alginate, oxidized sodium alginate, tannic acid, chitosan, cellulose, polymer, gelatin, collagen or hyaluronic acid; the protamine composite material is in a gel shape.

Preferably, the crosslinking of said gel forming agent with said protamine comprises ionic crosslinking, covalent crosslinking, electrostatic crosslinking.

Preferably, the source of protamine includes, but is not limited to salmon, trout, herring and the like.

In the technical scheme, the protamine is coated on the outer side of the physical antibacterial material and is assembled through covalent, a large-size solid phase material is formed through compounding, the physical antibacterial material is not limited to graphene oxide or carbon nano tubes and can be reasonably replaced according to actual conditions, compared with common protamine, the protamine and the physical antibacterial material can be used as a reducing agent to generate a reduction reaction, so that a covalent effect is generated between the protamine and the physical antibacterial material, and a synergistic effect is formed, thereby enhancing the stability of the protamine and avoiding the protamine from losing in the using process, thereby increasing the continuous using time and the storage time of the protamine, meanwhile, when the protamine and the physical antibacterial material are compounded, besides the protamine can play an antibacterial effect on a wound surface when the protamine composite material is applied to wound surface treatment, the protamine can also generate degradation products such as an amino acid sequence rich in arginine on the wound surface, has healing promoting effect on wound surface.

The invention also provides a preparation method of the membrane-shaped protamine composite material, which comprises the following steps:

s1, adding protamine into sterile water, and stirring until the protamine is completely dissolved to obtain a protamine solution;

s2, adding the physical antibacterial material into sterile water, and ultrasonically stirring until the physical antibacterial material is completely dissolved to obtain a physical antibacterial material solution;

s3, mixing the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2, and heating to obtain a composite suspension;

s4, performing suction filtration on the composite suspension prepared in the step S3, drying the film obtained by suction filtration, and separating the film from the basement membrane to obtain the membranous protamine composite material.

Further, the concentration of protamine in the protamine solution in the step S1 is 0.5g/L to 10 g/L.

Further, the concentration of the physical antibacterial material solution in the step S2 is 0.5g/L to 10 g/L.

Further, the step S3 is specifically to mix the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2 in a volume ratio of 10:1 to 1:100, and heat the mixture at a pH of 1 to 14 and at a temperature of 30 to 100 ℃ for 10 to 1000min to obtain a composite suspension.

Further, the size of the membrane-shaped protamine composite material is controlled according to the size of the suction filter; the diameter of the membrane-shaped protamine composite material is 25-300 mm.

Preferably, the diameter of the membrane-shaped protamine composite is 50 mm.

Further, the thickness of the membrane-shaped protamine composite material is controlled by the using amount of the composite suspension for suction filtration; the thickness of the membrane-shaped protamine composite material is 10 nm-1 cm.

Preferably, the thickness of the membrane-shaped protamine composite is 2 μm.

The invention also provides a preparation method of the gelatinous protamine composite material, which comprises the following steps:

s1, adding protamine into sterile water, and stirring until the protamine is completely dissolved to obtain a protamine solution;

s2, adding the physical antibacterial material into sterile water, and ultrasonically stirring until the physical antibacterial material is completely dissolved to obtain a physical antibacterial material solution;

s3, mixing the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2, and heating to obtain a composite suspension;

s4, adding the composite suspension prepared in the step S3 into a gel forming agent, and pouring the gel forming agent into a gel mold, wherein the composite suspension forms a gelatinous protamine composite material through the crosslinking action of the gel forming agent; the mass fraction of the gel forming agent is 0.1-50%.

Further, the concentration of protamine in the protamine solution in the step S1 is 0.5g/L to 10 g/L.

Further, the concentration of the physical antibacterial material solution in the step S2 is 0.5g/L to 10 g/L.

Further, the steps S1 and S2 are performed at normal temperature and pressure.

Further, the step S3 is specifically to mix the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2 in a volume ratio of 10:1 to 1:100, and heat the mixture at a pH of 1 to 14 and at a temperature of 30 to 100 ℃ for 10 to 1000min to obtain a composite suspension.

Further, in step S3, the gel forming agent is sodium alginate or oxidized sodium alginate, and the sodium alginate or oxidized sodium alginate is added with calcium chloride solution as a cross-linking agent to generate a cross-linking effect so that the composite suspension forms a gel-like protamine composite material.

Further, in step S3, the gel-forming agent is tannic acid, and the tannic acid generates a cross-linking effect by adding iron ions as a cross-linking agent to enable the composite suspension to form a gelatinous protamine composite material.

Further, in step S3, the gel forming agent is chitosan, and the chitosan is cross-linked by adding polyaldehyde such as glutaraldehyde as a cross-linking agent to form the composite suspension into a gel-like protamine composite material.

Further, in step S3, the gel forming agent is cellulose, and the cellulose is cross-linked by adding an acidic solution as a cross-linking agent or freezing to form the composite suspension into a gelatinous protamine composite material.

Preferably, the mass fraction of the cross-linking agent is 0.1-10%.

Preferably, the gel forming agent and the cross-linking agent are mixed in a volume ratio of 20: 1-1: 10 to generate a cross-linking effect; or dripping the cross-linking agent into the gel mould to generate cross-linking action until the composite suspension forms the gelatinous protamine composite material.

Further, in step S3, the gel forming agent is a polymer, gelatin, collagen or hyaluronic acid, and the polymer, gelatin, collagen or hyaluronic acid forms a gelatinous protamine composite material through self-crosslinking.

Further, the gel mold is in any shape, such as round and long strip.

In the technical scheme, the protamine solution and the physical antibacterial material solution are mixed and heated to carry out reduction reaction, so that the outer side of the physical antibacterial material is covalently bonded and assembled with the coated protamine, and the reaction can be directly carried out with suction filtration or crosslinking without assembly to obtain the large-size solid-phase composite material, when the composite suspension is used for preparing the composite material, a suction filtration or gel crosslinking method can be selected according to actual requirements, and the size and the internal structure of the prepared composite material can be flexibly regulated and controlled by selecting different composite suspension liquid dosage, filter size or gel mold, thereby realizing different functions, when the antibacterial agent is used for the large-area open wound antibacterial, the large-size antibacterial film material can be prepared to be used as wound dressing, and when the large-size antibacterial film material is used for resisting bacteria of daily articles, the composite material can be attached to any solid surface for antibacterial modification.

The protamine composite material is applied to the antibacterial field.

Further, the protamine composite material is used for daily antibacterial and wound antibacterial treatment.

Compared with the prior art, the invention has the beneficial effects that:

(1) the protamine composite material is a large-size solid-phase material, and the stability of the protamine is enhanced by covalently bonding the protamine and a physical antibacterial material, so that the loss of the protamine during use is avoided, the antibacterial property is reduced, the continuous use time of the protamine is prolonged, the storage time of the protamine can be prolonged to more than 1 year, and the use cost of the protamine is reduced;

(2) the protamine composite material can be used as a reducing agent to carry out reduction reaction when the protamine and the physical antibacterial material are compounded, so that covalent interaction is generated between the protamine and the physical antibacterial material;

(3) the protamine composite material can control the size, structure and function according to actual needs, does not relate to the use of organic solvents, and has good biocompatibility;

(4) the protamine composite material provided by the invention compounds protamine with a biological antibacterial function and a physical antibacterial material to form a synergistic effect, so that the antibacterial effect of the protamine composite material can be improved, and meanwhile, a degradation product of the composite material can also generate a certain healing promoting effect, so that the composite material is more suitable for daily antibacterial and wound antibacterial treatment;

(5) the preparation method of the invention leads protamine and physical antibacterial material to carry out covalent bonding after reduction reaction by heating, and can directly prepare various sizes and structures without assembling after reaction, the synthesis steps are simple, and the large-size solid phase protamine composite material can be prepared.

Drawings

Fig. 1 is a scanning electron microscope image of the membrane-shaped protamine composite material prepared in example 1.

FIG. 2 is a photograph of a gel-like protamine composite prepared in example 2.

Fig. 3 is a scanning electron microscope image of the graphene oxide film prepared in example 3.

Fig. 4 is a graph showing data of an antibacterial experiment of the membrane-shaped protamine composite material prepared in example 1.

Detailed Description

In order to make the technical solutions of the present invention better understood, those skilled in the art will further describe the present invention with reference to the accompanying drawings.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

The embodiment provides a membrane-shaped protamine composite material, and the preparation method comprises the following steps:

s1, adding a certain mass of protamine into sterile water at normal temperature and normal pressure, stirring until the protamine is completely dissolved, and preparing a protamine solution with the concentration of 10 g/L;

s2, adding a certain mass of graphene oxide powder into sterile water at normal temperature and normal pressure, and ultrasonically stirring until the graphene oxide powder is completely dissolved to prepare 2g/L of physical antibacterial material solution;

s3, mixing the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2 according to the volume ratio of 1:10 at normal temperature and normal pressure, and heating the mixed solution at 60 ℃ for 200min under the condition that the pH value is 7 to obtain a composite suspension;

s4, performing suction filtration on the composite suspension prepared in the step S3, drying a film obtained by suction filtration, and separating the film from a basement membrane to obtain a film-shaped protamine composite material, wherein the diameter of the film-shaped protamine composite material is 50mm, and the thickness of the film-shaped protamine composite material is 2 microns.

Example 2

The embodiment provides a gelatinous protamine composite material, and the preparation method comprises the following steps:

s1, adding a certain mass of protamine into sterile water at normal temperature and normal pressure, stirring until the protamine is completely dissolved, and preparing a protamine solution with the concentration of 10 g/L;

s2, adding a certain mass of graphene oxide powder into sterile water at normal temperature and normal pressure, and ultrasonically stirring until the graphene oxide powder is completely dissolved to prepare 2g/L of physical antibacterial material solution;

s3, mixing the protamine solution prepared in the step S1 and the physical antibacterial material solution prepared in the step S2 according to the volume ratio of 1:10 at normal temperature and normal pressure, and heating the mixed solution at 60 ℃ for 200min under the condition that the pH value is 7 to obtain a composite suspension;

s4, mixing 10mL of the composite suspension prepared in the step S3 with 0.1g of sodium alginate, pouring the mixture into a round gel mold, adding 2mL of 4% calcium chloride solution, and enabling the composite suspension to form a gelatinous protamine composite material through the crosslinking action of the gel forming agent.

The photograph of the obtained gel-like protamine composite material is shown in FIG. 2.

Example 3

The embodiment provides a graphene oxide film, and a preparation method thereof comprises the following steps:

s1, adding a certain mass of graphene oxide powder into sterile water at normal temperature and normal pressure, and ultrasonically stirring until the graphene oxide powder is completely dissolved to prepare 2g/L of physical antibacterial material solution;

s2, performing suction filtration on the physical antibacterial material solution prepared in the step S1, drying the film obtained through suction filtration, and separating the film from the bottom film to obtain the graphene oxide film, wherein the diameter of the graphene oxide film is 50mm, and the thickness of the graphene oxide film is 2 microns.

In this example, the film-shaped protamine composite material of example 1 and the graphene oxide film prepared in this example were observed with a scanning electron microscope, and fig. 1 is a scanning electron microscope image of the film-shaped protamine composite material, and fig. 3 is a scanning electron microscope image of the graphene oxide film.

As can be seen from a comparison of fig. 1 and 3, the surface of the film-shaped protamine composite material of example 1 is rougher, the lamellar structure of graphene oxide becomes more blurred, and the thickness thereof is increased correspondingly, compared to the uncomplexed graphene oxide film, indicating that in the composite material, the protamine and the graphene oxide undergo covalent interaction. Meanwhile, the graphene oxide film prepared in the embodiment is brown, while the film-shaped protamine composite material prepared in the embodiment 1 is black, which can also show that protamine is successfully compounded on the surface of graphene oxide through covalent interaction.

Example 4

In this example, an antibacterial experiment was performed on the membrane-shaped protamine composite material prepared in example 1 to test its antibacterial effect. The antibacterial experiment specifically comprises the following steps:

s1 streaking the strain to be frozen and stored to a plate culture medium, and culturing at 37 ℃ for 24h to obtain activated strain;

s2 selecting single colony on the plate culture medium of S1, inoculating the single colony to 100ml of liquid culture medium, shaking-culturing overnight at 37 ℃ at a rotating speed of 200r/min to obtain bacterial liquid, washing the prepared bacterial liquid with PBS buffer solution, centrifuging to collect the bacterial, and diluting the bacterial with PBS buffer solution to obtain diluted bacterial liquid;

s3, dripping 1 mu L of the diluted bacteria prepared in the step S2 on the membrane-shaped protamine composite material prepared in the example 1, washing the membrane-shaped protamine composite material by PBS buffer solution after exposing for different time, and collecting washing liquid;

s4 pouring 20ml of plate culture medium into the sterilized plate, horizontally standing the plate until the culture medium is solidified, then inoculating 0.1ml of the washing liquid of the step S3 to the surface of the culture medium, uniformly coating the washing liquid, placing the washing liquid in an incubator, and culturing the washing liquid at 37 ℃ overnight;

s5 the medium cultured in step S4 is removed, and the number of colonies is counted and observed.

In the present example, three experimental groups were set, the exposure time in step S3 was 0min, 10min, and 30min, respectively, and the operation was consistent in the remaining steps. As shown in FIG. 4, it is understood from FIG. 4 that, when the diluted bacterial solution is not exposed on the surface of the membrane-like protamine composite material, the survival rate of the bacteria is 1, after the diluted bacterial liquid is exposed on the surface of the membranous protamine composite material for 10min, the survival rate of the bacteria is 1.5 percent, after the diluted bacterial liquid is exposed on the surface of the membranous protamine composite material for 30min, the survival rate of the bacteria is only 0.01 percent, which shows that compared with the protamine directly used, the prepared protamine composite material, the antibacterial effect is improved because the protamine and the physical antibacterial material graphene oxide are compounded to form a synergistic effect, the antibacterial effect is improved, and meanwhile, the covalent bonding between the protamine and the physical antibacterial material graphene oxide enhances the stability of the protamine, thereby ensuring that enough protamine can be preserved for a long time in the antibacterial process and further improving the antibacterial effect of the composite material.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.