CN115999462A

CN115999462A - PH response type Mat@CS-Pro nanocapsule anti-fouling agent and preparation method and application thereof

Info

Publication number: CN115999462A
Application number: CN202210636699.5A
Authority: CN
Inventors: 郝湘平; 张达威; 闫卫路
Original assignee: University of Science and Technology Beijing USTB
Current assignee: University of Science and Technology Beijing USTB
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2023-04-25

Abstract

The invention discloses a pH response Mat@CS-Pro nanocapsule anti-fouling agent, and a preparation method and application thereof, belonging to the technical field of marine antibacterial anti-fouling and nanocomposite materials, and comprising the following steps: dissolving matrine in absolute ethanol containing lecithin to prepare an oil phase; dissolving chitosan in anhydrous acetic acid to prepare a water phase; preparing Mat@CS oil/water microcapsules by a microemulsion method; and preparing the prepared Mat@CS oil/water microcapsule and D-Proline into an aqueous solution, stirring in an ice-water bath, dialyzing and filtering in a deionized water solution through a dialysis bag, freeze-drying and preserving to obtain the pH response Mat@CS-Pro nanocapsule anti-fouling agent. The material provided by the invention is environment-friendly and has excellent antibacterial effect, and a thought is provided for exploring the research of intelligent pH response type antifouling agents in the ocean field.

Description

PH response type Mat@CS-Pro nanocapsule anti-fouling agent and preparation method and application thereof

Technical Field

The invention relates to an environment-friendly pH response Mat@CS-Pro antibacterial synergistic anti-biofilm antifouling capsule and a preparation method thereof, belonging to the technical field of marine antibacterial antifouling and nanocomposite materials.

Background

Marine biofouling is a worldwide problem that has a tremendous impact on our daily lives, especially the economic loss of shipping and the safety risk of engineering equipment. When the material is immersed in seawater, fouling organisms in the ocean can be attached to the bottom of a ship, so that the sailing resistance is increased, the ship speed is reduced, the fuel consumption is reduced, the sustainable use is seriously affected, in addition, the metal corrosion can be accelerated, the service life of instruments and equipment is shortened, even serious ecological damages such as biological invasion are caused, and the development of the human ocean is greatly affected. As the formation of the microbial film is a basic condition for the adhesion and fixation of the subsequent large-scale fouling organisms, the research on the antibacterial and anti-biofilm formation process in the process of converting marine antifouling into the early stage of biofouling is an effective thinking and method for preventing and controlling the biofouling. In addition, since secretions such as acetic acid and lactic acid generated by bacterial metabolism can lower the pH of the microenvironment to be acidic, pH-responsive antifouling paints have received increasing attention and have rapidly developed in recent years. In addition, biofilm formation on the surface of materials is also an important issue to be considered.

The initial stage of the fouling process is that bacteria and diatom secrete mucus form microbial mucosa on the surface of a clean object in the sea, the development stage is that larvae of large fouling organisms begin to adhere, the types and the number of individuals are continuously increased, the volume and the quality of communities are continuously increased, succession phenomena are obvious, some individuals have high density, and the types which grow rapidly become dominant species of communities; the stable phase is to grow long, large species of individuals fully, and to crowd or cover some attached medium and small species, complex and large-quality community species are formed. Biofilm formation thus creates conditions for subsequent attachment of large fouling organisms, which in turn leads to the formation of biofouling. Control of microbial film formation is also a requirement for biofouling. However, the research on the antifouling agent at home and abroad is mainly focused on the improvement of antibacterial property, and on the basis of improving the bactericidal effect, the related research on the capability of inhibiting the biological membrane is introduced, but has been reported.

Disclosure of Invention

In order to solve the problems, the technical scheme of the invention provides an environment-friendly pH response Mat@CS-Pro nanocapsule anti-fouling agent and a preparation method thereof, wherein natural organic matrine (Mat) is used as a core material, natural polymer Chitosan (CS) is used as a wall material, a micro-emulsion polymerization method is used for preparing a CS encapsulated Mat microcapsule, and D-Proline (D-Proline) is fixed on the capsule by a cold bath stirring method, so that the intelligent pH response antibacterial/biomembrane anti-fouling agent with high performance is finally prepared. The green nontoxic Mat@CS capsule can improve the stability of the antibacterial agent Mat, reduce the possibility of denaturation in an alkaline environment, maximize the sterilization efficiency of the microcapsule, and enable the release of Mat and D-Proline to be more intelligent and sustainable through the pH response effect of CS. Firstly, CS is used as a water phase, mat is used as an oil phase, and a microemulsion method is used for preparing the O/W structure microcapsule. The morphology and size of Mat@CS and Mat@CS-Pro microcapsules at different pH values were analyzed by Transmission Electron Microscopy (TEM). The characteristic functional groups of the microcapsules were analyzed by fourier infrared spectroscopy (FTIR) and the microcapsule components were judged. The antibacterial performance and biofilm formation inhibition capacity of the microcapsules before and after loading the D-Proline are analyzed by adopting a plate colony counting method and fluorescence live/dead bacteria detection, the sterilization conditions of the microcapsules under different pH values are observed by adopting the plate colony counting method, the drug release rate characterization under different pH values is carried out on the microcapsules loaded with the D-Proline, and the pH response long-acting sterilization mechanism of the microcapsules is comprehensively analyzed.

The invention prepares the sterilizing microcapsule with pH response property by a microemulsion polymerization method, and simultaneously loads D-Proline to successfully synthesize the anti-fouling capsule with pH response antibacterial and anti-biofilm properties. The Mat@CS-Pro microcapsule has excellent antibacterial, anti-biofilm and pH response properties, has the functions of stable existence, intelligent controlled release, prolonged service life of an antibacterial agent, environmental friendliness and the like, and has a wide application prospect.

The invention aims at realizing the following technical scheme:

according to a first aspect of the present invention, there is provided a method for preparing an environmentally friendly pH-responsive mat@cs-Pro nanocapsule stain-resist agent, the method comprising:

(1) Dissolving natural organic matrine (Mat) in absolute ethanol containing lecithin to prepare oil phase;

(2) Dissolving natural polymer Chitosan (CS) in anhydrous acetic acid to prepare a water phase;

(3) Preparing Mat@CS oil/water (O/W) microcapsules by a microemulsion method, wherein after the water phase and the oil phase are mixed, the water phase starts to cover the oil phase, so as to form Mat@CS oil/water (O/W) microcapsules with a CS shell and a Mat core;

(4) And preparing the prepared Mat@CS oil/water (O/W) microcapsule and D-Proline into an aqueous solution, stirring in an ice-water bath, dialyzing and filtering in a deionized water solution through a dialysis bag, and freeze-drying and preserving to obtain the pH response Mat@CS-Pro nanocapsule anti-fouling agent.

Further, the Mat amount in the step (1) is 10-40 mg, the concentration of the absolute ethyl alcohol containing lecithin is 20-40mg/mL, preferably 30mg/mL, and the volume is 200-500 mu L.

Further, the CS content in the step (2) is 5-15mg, preferably 10mg, and the volume of the aqueous solution (1%, v/v) of acetic acid is 10-30mL, preferably 20mL.

Further, the microemulsion method in the step (3) comprises the following steps: and respectively taking the water phase and the oil phase, slowly adding the oil phase into the water phase to prepare microspheres with uniform distribution, stirring at a rotating speed of 100-400 rpm for more than 1-3 hours, dialyzing and filtering in a PBS (phosphate buffer) solution by a dialysis bag, and freeze-drying and preserving to obtain the Mat@CS oil/water (O/W) microcapsule.

Further, the PBS phosphate buffer solution pH was 7.4.

Further, the water phase is glacial acetic acid, CS and deionized water, and the oil phase is lecithin, absolute ethyl alcohol and Mat.

Further, the mass of the Mat@CS capsule in the step (4) is 20-40mg, the mass of the D-Proline is 10-20 mg, and the mass ratio of the Mat@CS oil/water (O/W) microcapsule to the D-Proline is 1-5:1, preferably 2:1; the deionized water solution is added to have a volume of 10-30mL, preferably 20mL, and is stirred in an ice-water bath at a stirring speed of 100-400 rpm for 40-50 h, preferably 48h.

According to a second aspect of the present invention, there is provided an environmentally friendly pH-responsive mat@cs-Pro nanocapsule stain-proofing agent prepared by the preparation method according to any one of the above aspects.

Further, the pH response Mat@CS-Pro nanocapsule is approximately spherical, and the particle size is between 200 and 500 nm. The prepared capsule has intelligent pH response performance, under the ocean (alkaline) condition, CS can fully maintain the antibacterial activity of internal Mat, and after the surrounding acidification is caused by mass propagation of microorganisms, the microcapsule structure is changed to release Mat and D-Proline, so that the purpose of intelligent response and pollution prevention is realized.

According to a third aspect of the invention, there is provided the use of an environmentally friendly pH responsive Mat@CS-Pro nanocapsule stain blocker in decontamination of marine environments.

Further, the pH value range of the marine environment to which the pH response Mat@CS-Pro nanocapsule antifouling agent is applied is 8.0-8.3.

The invention has the beneficial effects that:

the invention prepares the Mat@CS-Pro nano microcapsule with the O/W structure by a microemulsion method to ensure that the Mat stably exists in the sea water, and reduces the O in the Mat and the external environment ₂ Oxidation and saponification may occur. Mat@CS-Pro nano microcapsules show excellent antibacterial performance on S.aureus, P.aeroginosa and escherichia coli (E.coli), and the capsules show that the Mat@CS-Pro nano microcapsules have intelligent pH response controlled release antibacterial and biofilm formation inhibiting effects. In marine environment, secretion generated by bacterial metabolism can lead to local pH value reduction, and due to the protonation process of amino groups in the CS of the Mat@CS-Pro capsule shell, electrostatic repulsion action occurs among CS molecules to enable the capsule to expand and release Mat to kill bacteria, meanwhile, D-Pro line is positively charged due to being smaller than isoelectric points and is charged with CS in the same type, so that the two substances generate electrostatic repulsion action, the release of D-Pro line is promoted, the formation of a biological film is further inhibited, and the dual effects of capsule sterilization and synergistic anti-biological film are shown. And when the pH is higher, the amino is deprotonated, the capsule is reduced, the Mat is not released, the Mat is not in direct contact with an alkaline environment, the possibility of denaturation caused by saponification reaction of the lactam structure of the Mat when directly exposed to the alkaline environment is reduced, and the Mat is stably present for a long time. At the same time D-Proline is in the alkaline ringThe charges in the environment are negative as CS, so D-Proline can also release and inhibit the formation of biological film to prevent fouling in alkaline environment.

According to the invention, D-Proline and Mat@CS are prepared into aqueous solution according to the mass ratio of 1-5:1 (preferably 2:1), and the aqueous solution is stirred in an ice bath to fix the D-Proline on a Mat@CS capsule, so that the antibacterial and antifouling functions of the Mat@CS are increased, the anti-biofilm antifouling functions of the D-amino acid are simultaneously increased, the D-Proline is adsorbed and fixed on the surface of the Mat@CS through electrostatic effect, and the Mat@CS and the D-Proline are simultaneously released when the capsule expands under an acidic condition, thereby realizing the dual functions of the antibacterial and the anti-biofilm functions of the capsule and showing good antifouling performance.

Drawings

FIG. 1 is a TEM image of Mat@CS (parts a, b) and Mat@CS-Pro (parts c, d) nanocapsules at different pH values.

FIG. 2 is a schematic illustration of FTIR spectra according to an embodiment of the present invention, wherein part a in FIG. 2 is the FTIR spectra of Mat@CS nanocapsules, mat and CS, and part b is the FTIR spectra of Mat@CS-Pro nanocapsules, mat@CS nanocapsules and D-Proline.

FIG. 3 shows the number of E.coli, S.aureus, P.aeroginosa (part a) after 18h of Mat, CS and Mat@CS nanocapsules in Luria-Bertani broth (LB) and 2216E medium, and the antibacterial effect (part b) corresponding to each counting result.

FIG. 4 is a graph showing the number of E.coli, S.aureus, P.aeroginosa (part a) after Mat@CS nanocapsules, D-Proline and Mat@CS-Pro nanocapsules are applied to LB medium for 18h, respectively, and the antibacterial effect corresponding to the counting result of each colony (part b).

FIG. 5 is untreated E.coli (part a) and Mat@CS-Pro nanocapsules treated E.coli (a) ₁ SEM images of part); untreated S.aureus (part b) and Mat@CS-Pro nanocapsules treated S.aureus (b) ₁ A portion); untreated P.aerocinosa (part c) and Mat@CS-Pro nanocapsules treated P.aerocinosa (c) ₁ Part(s).

FIG. 6 is E.coli (part a-c), S.aureus (a) ₁ -c ₁ Part) and P.aeroginosa (a) ₂ -c ₂ Part) with and without Mat@CS and Mat@CS-Pro NaSEM image of biofilm on the corresponding test strip after incubation in medium of rice capsules.

FIG. 7 is the E.coli (part a-c), S.aureus (a) of FIG. 6 ₁ -c ₁ Part) and P.aeroginosa (a) ₂ -c ₂ Part) thickness map of biofilm formed after 3 days of incubation under Mat@CS and Mat@CS-Pro nanocapsules.

FIG. 8 is a TEM image of Mat@CS nanocapsules at pH5 (part a) and pH 8 (part b); TEM image of Mat@CS-Pro nanocapsules at pH5 (part c) and pH 8 (part d).

FIG. 9 is a standard curve of Mat in PBS solution (part a); concentration of Mat released from Mat@CS-Pro nanocapsules in PBS solution of different pH for 10h (part b); diameter (part c) of Mat@CS-Pro nanocapsules after treatment in PBS solutions with different pH values; optical density (o.d.) values for three different types of bacterial strains after 4h (part d) incubation in LB and PBS solutions (volume ratio 1:1) at different pH containing mat@cs-Pro nanocapsules.

FIG. 10 is Mat@CS-Pro against E.coli (a-d fraction), S.aureus (a) in pH5, 6, 7 and 8 medium ₁ -d ₁ Part) and P.aeroginosa (a) ₂ -d ₂ Part) of the surface colony plate photograph.

The specific embodiment is as follows:

the technical scheme of the invention is further described below by means of specific embodiments in combination with the accompanying drawings.

The following examples are merely illustrative and are only intended to illustrate and describe the technical solutions of the present invention, and are not to be construed as limiting the technical solutions of the present invention.

The technical scheme of the invention provides an environment-friendly pH response Mat@CS-Pro nano-capsule anti-fouling agent and a preparation method thereof, wherein natural organic matrine (Mat) is used as a core material, natural high polymer Chitosan (CS) is used as a wall material, a micro-capsule of CS encapsulation Mat is prepared by a microemulsion polymerization method, and D-Proline (D-Proline) is fixed on the capsule by a cold bath stirring method, so that the intelligent pH response antibacterial/biomembrane anti-fouling agent with high performance is finally prepared.

Here, CS is a product obtained by deacetylation of chitin contained in crustaceans, and has excellent antibacterial properties. Meanwhile, CS as a polysaccharide has rich biological and physical properties including biodegradability, biocompatibility, nontoxicity and the like. Microcapsules prepared from them as shells have typical pH responsiveness. Under the acidic condition, a large number of amino groups carried in a CS side chain are positively charged, the amino groups of CS are protonated, and the microcapsule expands due to the electrostatic repulsion effect, so that a pore canal is opened and enlarged; when the solution environment is alkaline, deprotonation occurs, resulting in shrinkage of the microcapsules, shrinking or even closing of the channels. Normally, the marine environment is alkaline, and secretions such as lactic acid and the like generated by metabolism of bacteria in the environment after matrix fixation can reduce the pH of the surrounding environment, promote microcapsule expansion, accelerate drug release and play a role in sterilization; on the contrary, when the surrounding bacteria are few, the marine environment is alkaline, and the microcapsule is contracted, so that the medicine is wrapped by CS and stored, and the service life of the medicine is greatly prolonged. Therefore, the microcapsule prepared by the CS can store and sustainably release the medicine, so that the delayed release of the antibacterial agent and the intelligent pH response sterilization are realized, the stability of the medicine is improved, and the maximum sterilization effect of the medicine is realized. In addition, CS can be biodegraded, has no pollution to the environment, is nontoxic to organisms, achieves the aim of green environmental protection, and has good application prospects in the fields of food safety, public health, medical appliances and the like.

Mat is an alkaloid prepared by extracting dried root, plant and fruit of Sophora flavescens ait of Leguminosae with organic solvent such as ethanol, and has various pharmacological effects and effects such as antibacterial, antiinflammatory, antirheumatic, antitumor, antiallergic, antiviral, antiparasitic, antiarrhythmic, repercussive, diuretic, immunity and biological response regulating effects. Mat is a botanical pesticide, has the characteristics of specificity and naturality, only plays a role on specific organisms, can be rapidly decomposed in nature, and is finally water and carbon dioxide. Because of the combined action of a plurality of chemical substances, the pesticide is not easy to cause the harmful substances to generate pesticide resistance, and can be used for a long time. Therefore, mat is obviously different from general high-toxicity and high-residue chemical pesticides, and is quite green and environment-friendly. However, since the lactam structure is contained in the Mat, saponification reaction is easy to occur in an alkaline environment to damage the structure, so that the Mat stability is increased, and the Mat is reduced in exposure to the alkaline environment, which is a key for increasing the antibacterial stability and long-acting property of the Mat.

D-amino acids are produced as enantiomers of L-amino acids from racemates. Because of their degradability, non-toxicity and origin, they are widely used in pharmaceutical synthesis, enzymatic structural and functional analysis, and in bactericidal applications. The applicant has found that the application of the anti-fouling agent to the anti-fouling agent can achieve the anti-fouling purpose by inhibiting the formation of a biological film through many years of experimental researches.

Therefore, mat is selected as a natural antibacterial agent, and after the pH response antibacterial microcapsule is prepared by coating CS, D-Proline is fixed on the surface of the capsule, so that the environment-friendly antibacterial/biological film antifouling capsule with intelligent pH response is prepared. When the environment of the solution is acidic, amino groups on the capsule CS are protonated, and due to electrostatic repulsion, mat@CS microcapsules expand to open and expand pore channels, so that the antibacterial agent releases and kills bacteria from the capsule, and meanwhile, D-pro line is positively charged due to being smaller than an isoelectric point and is charged with the same kind as CS, so that electrostatic repulsion is generated between the two microcapsules, and the release of the D-pro line is promoted to inhibit the formation of a biological film. When the solution environment is alkaline, deprotonation occurs, so that the microcapsule is contracted, the pore canal is contracted and closed, the antibacterial agent is not released, mat is sealed, the possibility of denaturation caused by saponification reaction when the lactam structure of Mat is directly exposed to the alkaline environment is reduced, the Mat is stably stored in the capsule for a long time, the stability of the Mat is improved, meanwhile, D-Proline is negatively charged as CS (sodium dodecyl sulfate) due to the fact that the charges in the alkaline environment are the same, and D-Proline can also be released to inhibit the formation of a biological film in the alkaline environment for antifouling. In general, the pH of the surrounding environment is reduced due to secretion such as lactic acid generated by bacterial metabolism, so that the expansion of the microcapsules is promoted, the release of Mat and D-Proline is accelerated, and the sterilization effect is achieved; conversely, when there are few surrounding bacteria, the environment is alkaline, causing the microcapsules to shrink, and thus Mat can be stored. Thus, mat@CS-Pro microcapsules can exist stably in a marine environment and effectively store bactericide components. When bacteria are enriched and planted and the pH of the microenvironment is reduced, mat@CS-Pro microcapsules can effectively release Mat and D-Pro, the aim of sterilizing and synergistically inhibiting the formation of a biological film by an anti-fouling agent is fulfilled, and the added Mat, CS and D-Pro can be biodegraded, so that the method has no pollution to the environment and no toxicity to organisms, and the aim of a green environment-friendly anti-fouling agent is fulfilled.

The preparation method comprises the following steps:

mat is dissolved in absolute ethyl alcohol containing lecithin to prepare an oil phase, and CS is dissolved in absolute acetic acid to prepare a water phase. The O/W structure microcapsule of Mat@CS is prepared by a microemulsion method. After the water phase and the oil phase are mixed, the water phase starts to coat the oil phase, a capsule structure with a packing material CS and a core material Mat is formed, the mixture is stirred for about 2 hours under the condition of rotating speed of 100-400 rpm, dialyzed by a dialysis bag, and freeze-dried and preserved. 20-40mg of D-Proline is dissolved in 20mL of aqueous solution, then 10-20 mg of Mat@CS is added and stirred in an ice bath at 100-400 rpm for about 50h. Free D-Proline was dialyzed against deionized water solution. Mat@CS-Pro nanocapsules were collected after vacuum freezing at-50℃for about 40 h.

The CS content was 10mg, and the volume of the aqueous acetic acid solution (1%, v/v) was 10 to 30mL. The microemulsion method comprises the following steps: the Mat solution forms micro-droplets through a liquid-liquid homogeneous nucleation process, and the Mat micro-droplets are wrapped in CS to form microcapsules after being blended with CS solution. In this process, lecithin as an emulsifier participates in the liquid-liquid homogeneous nucleation process, and a microcapsule formation process is achieved. Finally, self-assembling according to a microemulsion O/W mechanism to form the CS coated Mat microcapsule. The tension of the oil/water interface decreases under the action of the surfactant and even produces a transient negative interfacial tension, so the system will spontaneously expand the interface until the interfacial tension returns to zero or a slightly positive value to form a microemulsion.

The CS coating Mat has stable coating structure, the CS coating material is deprotonated in an alkaline environment, the shrinkage channel of the capsule is tightly closed, and the Mat is not released. Therefore, the Mat alkaline seawater environment can be kept stable for a long time.

The D-Proline is adsorbed on the surface of the Mat@CS capsule through electrostatic action, and plays an antibacterial role in Mat@CS, and meanwhile, the D-Proline plays a role in resisting the formation of a biological film, so that the marine antifouling function of pH response antibacterial synergistic antibacterial film is realized.

The pH response type Mat@CS-Pro capsule has an intelligent pH response controlled release effect, and the stability of the Mat in a marine environment is improved. In marine environment, secretion generated by bacterial metabolism can lead to local pH value reduction, and due to the protonation process of amino in the capsule shell CS, the capsule is expanded to release Mat to kill bacteria, and when the pH value is higher, the amino is deprotonated, the capsule is reduced to seal the Mat, so that the possibility of saponification denaturation of the Mat caused by direct exposure of the Mat to an alkaline environment due to a lactam structure is reduced, the Mat is stably stored in the capsule for a long time, and the sterilization stability of the Mat is improved.

Example 1

(1) The O/W structure microcapsule solution is prepared by a microemulsion method. An oil phase was prepared by dissolving 32mg of Mat in 30mg/mL absolute ethanol (400. Mu.L) containing lecithin using a pipette. CS (10 mg) was dissolved in 20mL of an aqueous acetic acid solution (1%, v/v) to prepare an aqueous phase. Slowly pouring the oil phase into the water phase, and stirring the mixed solution for 2 hours at room temperature by a magnetic stirrer at a rotating speed of 200rpm until the mixed solution is opalescent, thereby obtaining a microcapsule solution.

(2) The microcapsule solution was transferred to a dialysis bag and immersed in a dialysis solution (PBS phosphate buffer solution at pH 7.4) for 12 hours to eliminate free Mat. And (3) putting the microcapsule solution into a refrigerator freezing layer to freeze for 2 hours, and after solidification, putting into a freeze dryer to freeze-dry for 36 hours at the temperature of minus 50 ℃ to obtain microcapsule powder.

(3) 40mg of D-Proline was dissolved in 20mL of aqueous solution, then 20mg of Mat@CS lyophilized powder was added to the solution, and stirred in an ice bath at 200rpm for 48h. Free D-pro line was eliminated by dialysis in aqueous solution. Mat@CS-Pro nanocapsules were collected after 36h vacuum freezing at-50 ℃.

(4) And analyzing the antibacterial property of the Mat@CS-Pro nanocapsule by adopting a colony counting method. E.coli and s.aureus were chosen as representative of gram-negative and gram-positive strains, p.aeromonas as representative of marine bacteria. The initial concentration of each bacteria was about-10 ⁸ CFU/mL. 1mL of E.coli or S.aureus suspension was poured into 50mL of LB medium, and shake-cultured at 120rpm for 18 hours in a constant temperature apparatus at 37 ℃. 1mL of P.aeThe ruginosa suspension was transferred to 50mL of sterile 2216E medium and cultured with shaking at 30℃for 18h. Thereafter, 200. Mu.L of the bacterial suspension was removed to 8mL LB or 2216E medium containing 2mg/mL of different antibacterial agents (i.e., CS, mat, D-Proline, mat@CS nanocapsules, mat@CS-Pro nanocapsules). After 18h incubation at 37℃or 30℃20. Mu.L of the diluted bacterial suspension was spread on a solid culture substrate and incubated overnight at the corresponding temperature. The bacteriostasis result is calculated according to the following formula:

wherein A is the colony count of the control group, and B is the colony count of the treatment group. In addition, the o.d. curve was detected at 600nm by an ultraviolet spectrophotometer as a reference for evaluating the antibacterial performance of nanocapsules.

The O.D. value of the nano capsule is measured at 600nm by an ultraviolet spectrophotometer, so that a reference is provided for evaluating the antibacterial property of the nano capsule.

(5) The anti-adhesion properties of Mat@CS-Pro nanocapsules were evaluated by SEM. A316L stainless steel coupon (1 cm. Times.1 cm) was used to aid in evaluating the anti-adhesion properties of the prepared nanocapsules. Polishing the sample with 1200 mesh silicon carbide paper, ultrasonic cleaning, washing with acetone and ethanol, and washing with N ₂ Blow-drying, and performing aseptic treatment on the metal sample by using ultraviolet rays. 1mL of E.coli or S.aureus suspension was added to 50mL of sterile LB and shaken at 120rpm for 18h at 37 ℃. 1mL of the P.aerocosa suspension was added to 50mL of sterile 2216E medium and shaken at 120rpm for 18h at 30 ℃. Each coupon was placed in a 24-well plate and then injected with 4mL of LB medium (containing 2mg/mL Mat@CS or Mat@CS-Pro nanocapsules) and 100. Mu.L of bacterial suspension. After 1 and 3 days of stationary culture, samples were removed and washed with deionized water to remove free-floating bacteria on the metal surface, and the solution (PBS, pH 7.4) was fixed with 2.5% (v/v) glutaraldehyde phosphate buffer for 2h. Subsequently, each sample was washed 3 times with PBS, 3 times with deionized water, and dehydrated using 30%, 50%, 70%, 90% and 100% (v/v) gradient ethanol for 15 minutes. After the sample is sprayed with carbon, a bacterial film on the metal surface is observed through a scanning electron microscope.

Furthermore, live ∈

The BacLight (TM) bacteria viability kit further analyzes the anti-adhesion performance of Mat@CS-Pro nanocapsules. Each sample was immersed in a different nanocapsule-containing bacterial suspension and incubated for 1 day and 3 days, respectively. Each sample was then washed with deionized water to remove free bacteria from the metal surface, placed in a clean 24-well plate, and stained in the dark for 20 minutes.

(6) Mat@CS-Pro nanocapsule suspension (2 mg/mL) was placed in a dialysis bag and immersed in PBS solutions at pH5, 6, 7 and 8, respectively, for 12h. Dialysis solutions were measured by UV-Vis per hour, with the peak at 220nm representing Mat. The size of Mat@CS-Pro nanocapsules after treatment in PBS solutions of different pH was detected by DLS.

(7) Colony plating was used to evaluate the antibacterial properties of the pH response. 2mg/mL Mat@CS-Pro nanocapsule medium consisting of 4mL PBS solutions of different pH (

pH

5, 6, 7, 8) and 4mL LB medium (or 2216E) was prepared. 200. Mu.L of each bacterial suspension was inoculated into 8mL of a medium and cultured at 37℃or 30℃for 4 hours. mu.L of the diluted bacterial suspension was spread on a solid medium and cultured at the corresponding temperature for 18 hours. The o.d. of each bacterial suspension after 4h incubation was also tested by UV-Vis at 600nm as a reference for another method of assessing the pH response antibacterial properties of mat@cs-Pro nanocapsules.

Example 2 (Effect example)

Synthesizing Mat@CS nano microcapsules and testing performance:

(1) Characterization of Mat@CS-Pro nanocapsules

The structure and morphology of the nanocapsule surface prepared before and after D-line fixation were observed by transmission electron microscopy as shown in fig. 1. As can be seen from FIG. 1, the Mat@CS nanocapsules prepared by the O/W microemulsion method have uniform particle sizes and have an average particle size of about 250nm. After the immobilization of D-Proline, the capsules did not agglomerate and the particle size slightly increased (parts a, c in FIG. 1). However, the morphology of Mat@CS-Pro is different from Mat@CS nanocapsules, which show a large number of small droplets inside the nanocapsules (part b in FIG. 1). From the Zeta potentials of the CS, mat and Mat@CS nanocapsules being 37.8+ -1.1 mV, -16.9+ -2.4 mV and 31.9mV, respectively, it can be inferred that the droplets in the nanocapsules should be Mat, since the surface negative charge of the free Mat droplets is completely covered by the cation CS. In addition, the surface of the Mat@CS nanocapsules is covered with irregularly shaped substances (part d in FIG. 1). Zeta potential distributions of the D-Proline and Mat@CS-Pro nanocapsules are-7.7+ -1.1 mV and-5.3+ -0.3 mV respectively, which indicates that the negatively charged D-Proline can be fixed on the surface of the Mat@CS nanocapsules. Thus, the D-Proline was immobilized on the surface of Mat@CS nanocapsules, which had a diameter of about 280nm.

FTIR of CS, mat and mat@cs nanocapsules prepared are shown in part a of fig. 2. For CS lines, lie between 3500 and 3200cm ^-1 The broad peaks of (2) belong to N-H and OH stretching vibrations. 1157cm ^-1 And 1091cm ^-1 The peak belongs to C-O-C stretching vibration. 2935cm for Mat line ^-1 And 2853cm ^-1 Peak belongs to C-H stretching vibration in CH2, 1635cm ^-1 The peak belongs to c=o stretching vibration. The characteristics of the Mat@CS-Proline nanocapsule product spectral line are similar to those of the CS spectral line. Also shown in the figure are Mat@CS spectral lines of about 3500-3200cm ^-1 Is a stretching vibration of N-H and O-H. Furthermore, 1157cm appear on Mat@CS ^-1 It belongs to the C-O-C stretching vibration. Whereas 1085cm in line Mat@CS ^-1 The peak (C-O-C stretching vibration) corresponds to 1019cm on CS spectrum ^-1 Peaks, indicating the presence of CS in the prepared capsules. Meanwhile, related characteristic peaks of Mat can be found in Mat@CS, such as 1635cm appearing in spectral line due to C=O stretching vibration ^-1 The peak is consistent with the peak position of the pure Mat, and the intensity of the peak is weaker than that of the Mat spectral line. 2935cm of Mat spectrum line generated by C-H stretching vibration ^-1 And 2853cm ^-1 The peak positions respectively correspond to 2930cm of Mat@CS ^-1 And 2858cm ^-1 On CS, 2875cm caused by C-H stretching vibration ^-1 The peaks are then covered by these two peaks. That is, mat is also present in the prepared nanocapsules. Furthermore, at 1737cm ^-1 The new peak of (2) indicates that there is an H bond between CS and Mat. Combining the TEM results shown in FIG. 1, it was shown that Mat@CS nanocapsules were successfully synthesized.

Capsule load D-PrFTIR analysis of the product components after oline was performed and the results are shown in part b of fig. 2. For the spectral line of D-Proline, 3051cm ^-1 And 2979cm ^-1 The peaks of (2) are generated by asymmetric stretching and symmetric stretching vibration of the C-H bond, respectively. In the D-Proline spectrum at 1600-1450 cm ^-1 The peaks of the spectral lines are related to backbone ring vibration, while strong peaks appear at similar positions on the synthesized Mat@CS-Pro nanocapsule spectral lines. At the same time, on the D-Proline spectrum line, by CH ₂ 848cm caused by rocking vibration of (C) ^-1 And 791cm ^-1 The peaks at which correspond to 856cm of Mat@CS-Pro spectral lines, respectively ^-1 And 773cm ^-1 Peak position. Spectral lines furthermore, 1164cm of Mat@CS nanocapsules caused by C-O-C vibrations ^-1 And 1085cm ^-1 The peak at this point also appears on the Mat@CS-Pro spectral line. In combination with the TEM results shown in FIG. 1, it was shown that D-Pro line was successfully immobilized on Mat@CS-Pro nanocapsules.

(2) Antibacterial property characterization of Mat@CS-Pro nanocapsules

FIG. 3 is a graph of colony counts and bacteriostatic effects of CS, mat, and Mat@CS nanocapsules on E.coli, S.aureus, and P.aeromonas. In part a of fig. 3, the bacterial numbers after the three drug treatments were significantly reduced compared to the blank (379 CFU), indicating that CS, mat and mat@cs nanocapsules had significant antibacterial effects on all three strains. Part b of fig. 3 shows the bacteriostatic effect of the three drugs on e.coli, s.aureus and p.aeroginosa, respectively, with the bactericidal rate on p.aeroginosa exceeding 87.71%, 77.90% and 73.39%, respectively. It is notable that the antibacterial effect of pure Mat and Mat@CS nanocapsules on gram-positive bacteria is better than that of gram-negative bacteria, and the antibacterial effect on P.aeroginosa is the worst. Specifically, the bacteriostasis rates of Mat and Mat@CS to P.aeroginosa are 78.02% and 89.16%, respectively, which are reduced by 15.06% and 5.39% compared with the corresponding bacteriostasis rates of S.aureus. Mat@CS nanocapsules exhibit better antibacterial properties to E.coli, S.aureus and P.aeromonas compared to pure Mat. Aerucinosa colonies decreased by 8CFU, 6CFU and 39CFU, respectively. From the results of the part b in fig. 3, the antibacterial rates of the Mat@CS nanocapsules on the three bacteria are respectively improved to 89.71%, 94.55% and 89.16%. Therefore, after CS coating, the antibacterial property of the Mat@CS nanocapsule is obviously improved.

The colony counting method further shows the antibacterial effect of Mat@CS-Pro nanocapsules, and the result is shown in FIG. 4. As shown in part b of FIG. 4, the antimicrobial effect of pure D-Proline is not ideal. The bacteriostasis rates of E.coli, S.aureus and P.aeroginosa were approximately 57.71%, 49.96% and 66.55%, respectively. However, after fixation of D-Proline, the antibacterial rates of Mat@CS-Pro nanocapsules on E.coli and P.aeroginosa reached 92.85% and 96.28%, respectively. However, after the introduction of D-Proline, the antibacterial rate of Mat@CS-Pro nanocapsules on S.aureus was reduced from 94.55% to 87.75%, probably due to the poor antibacterial property of the nanocapsule structure of D-Proline on S.aureus. After the surface of Mat@CS-Pro is coated with D-Proline, the release of Mat is possibly hindered, and the D-Proline on the surface cannot provide a remarkable antibacterial effect, so that the antibacterial performance of Mat@CS-Pro is slightly reduced. In general, after the D-Proline is introduced, mat@CS-Pro nanocapsules can almost maintain excellent antibacterial performance on gram-positive bacteria and even exert more excellent antibacterial performance on gram-negative bacteria.

After the Mat@CS-Pro nanocapsule sterilization treatment, the morphology of E.coli, S.aureus and P.aeromonas was observed by a scanning electron microscope. The membrane of the bacteria almost lost the intact structure compared to the control group, indicating that the nanocapsules caused irreversible damage to the bacterial cells. Furthermore, the cell surfaces of E.coli and P.aeroginosa were covered with a large amount of organics compared to the S.aureus morphology, demonstrating that Mat@CS-Pro nanocapsules have a stronger effect on gram negative strains than gram positive strains. These results are consistent with the colony count results shown in FIG. 4.

(3) Biofilm dispersibility of Mat@CS-Pro nanocapsules

The effect on biofilm formation was assessed by SEM after 3 days of Mat@CS and Mat@CS-Pro nanocapsules. For the control group, the metal surface had the most bacteria attached, and aggregation occurred (a-a in FIG. 6 ₂ Part(s). After incubation with Mat@CS nanocapsules, the bacterial cell count and aggregation on the substrate surface were slightly reduced (b-b in FIG. 6) ₂ Part(s). C-c in FIG. 6 ₂ Part of theAfter D-Proline is introduced into Mat@CS nanocapsules, the number of bacteria and aggregation phenomenon are further reduced. As can be seen, most bacterial cells are randomly dispersed on the substrate surface in single cell form. Moreover, it can be seen that the bacterial morphology of the bacterial aggregation area is significantly different from that of the control group, and that the bacteria have a complete cell morphology and the cell membrane has a clear edge when no nanocapsules are added to the medium. However, for the substrate surface morphology, especially the aggregation zone, with Mat@CS-Pro addition, the bacterial morphology is similar to that of FIG. 5 a ₁ -c ₁ Partially similar, i.e. the bacteria agglomerate (c-c in FIG. 6) ₂ Part(s).

After 3 days of culture, the formed biofilm was evaluated with IPCM and the results are shown in FIG. 7. The green and red areas of the field represent live and dead bacteria, respectively. From the graph, almost all of the control groups are green, which indicates that the 316L stainless steel sheet does not show toxicity to all three bacteria, and the bacteria can survive on the surface of the substrate and form a complete and uniform biological film. Wherein E.coli, S.aureus and P.aeromonas biofilms were approximately 30 μm, 39 μm and 48 μm thick, respectively (a-a in FIG. 6) ₂ Part(s). Many red areas appeared in the field of view after incubation with Mat@CS and Mat@CS-Pro nanocapsules, indicating the presence of a large number of dead bacteria. In addition, after D-Pro line was introduced into the mat@cs nanocapsules, the green area ratio was further reduced, indicating that the antibacterial effect of the mat@cs-Pro nanocapsules was superior to that of the mat@cs nanocapsules, which is consistent with the results in fig. 4. C-c in FIG. 6 ₂ In part, E.coli, S.aureus and P.aerobacking formed after 3 days incubation with Mat@CS-Pro nanocapsules had biofilm thicknesses of about 20 μm,10 μm and 28 μm, respectively. The dispersing ability of the biological film of Mat@CS-Pro nanocapsule is obviously improved, and compared with a control group, E.coli, S.aureus and P.aeromonas biological films are respectively reduced by about 33%, 74% and 42%. Therefore, on the basis of the existing antibacterial performance, the introduction of D-Proline is helpful for improving the biological film dispersion performance of the antifouling agent.

(4) pH controlled release and antibacterial Properties of Mat@CS-Pro nanocapsules

Morphology and structure of Mat@CS nanocapsules and Mat@CS-Pro nanocapsules treated in PBS solutions at pH5 and pH 8 were characterized by TEM. At the position ofCS-NH under acidic conditions ₂ Conversion of groups to-NH ₃ ⁺ Resulting in positive charges and internal electrostatic repulsive forces in the system. Thus, after soaking at pH5, mat@CS nanocapsules swell from 280nm to about 430nm (part a in FIG. 8). In the marine environment (pH 8), the mat@cs nanocapsules shrink to about 220nm due to deprotonation of the CS amino groups, as shown in part b of fig. 8. After immobilization of D-pro line, the pH response characteristics of the nanocapsules remain. In FIG. 8, part c and part d of FIG. 8, the Mat@CS-Pro nanocapsules after treatment in alkaline and acidic PBS solutions had diameters of about 475nm and 234nm, respectively. The results show that Mat@CS-Pro nanocapsules have pH responsiveness, and the diameter of the nanocapsules can be changed along with the change of environmental pH.

The pH controlled release behavior and mechanism of Mat@CS-Pro nanocapsules was determined by UV-Vis and DLS analysis. The release concentration of Mat from Mat@CS-Pro nanocapsules calculated from the standard curve (part a in FIG. 9) is shown in part b in FIG. 9. Under alkaline conditions, the amount of Mat released was maintained at a low level, from the initial 5.1ppm to 7.1ppm after 10 hours. Meanwhile, DLS results show that the size of Mat@CS-Pro nanocapsules after being soaked in PBS solution with pH of 8 is minimum, and the size is about 236+/-13 nm. In contrast, the release of Mat in acidic solutions continues to increase, and after 10h of immersion in pH5 and pH 6 solutions, the Mat concentrations were about 28.5ppm and 23.5ppm, respectively. The release concentration of Mat was increased by 16.8ppm and 10.3ppm, respectively, compared to the initial state. In FIG. 9, part c, the nanocapsules after treatment in pH5 and pH 6 solutions had diameters of 478.+ -.18 nm and 396.+ -.21 nm, respectively, which are consistent with the results of FIG. 8. The nanocapsules were increased in diameter by about 242nm and 160nm, respectively, compared to the corresponding nanocapsules treated at pH 8. At pH 7, the release amount of Mat increased in 10 hours, 7.6ppm after 1 hour, 9.4ppm after 5 hours, and further increased to 10.1ppm after 10 hours. The above results indicate that the pH response characteristics of Mat@CS-Pro nanocapsules after immobilization of D-Proline are not limited. As the pH of the PBS solution increases, the release behavior of the Mat and the size of the nanocapsules decrease.

Evaluation of Mat@CS-Pro nanocapsules by UV-Vis for E.coli, S.aureus and P.aerogin in different pH mediaInfluence of bacterial growth status of osa. The bacterial concentration increases with increasing o.d. value. From part d of fig. 9 it can be seen that the o.d. values of the three bacterial strains increase with increasing pH of the medium, indicating that the number of bacteria in the medium also increases with increasing pH. Specifically, after incubation at pH5, 6, 7 and 8, the E.coli has O.D. values of about 0.69.+ -. 0.02, 0.70.+ -. 0.02, 0.75.+ -. 0.01 and 0.77.+ -. 0.02, respectively. In parts a to d of fig. 10, the results of colony plating are consistent with o.d. data, i.e. the number of bacterial colonies increases significantly with increasing environmental pH. The trend of the O.D. value of S.aureus is similar to E.coli, and is 0.68+ -0.01, 0.69+ -0.01, 0.72+ -0.01 in sequence, which is the same as that of a in FIG. 10 ₁ -d ₁ Partially identical. As for the p.aerocosa group, the o.d. value increased from 0.27±0.01 at pH5 to 0.45±0.05 at pH 8. In FIG. 10 a ₂ -d ₂ In part, after incubation at

pH

7 and 8, there were more colonies scattered on the solid medium, but after incubation at

pH

6 and 7, there were fewer colonies on the solid medium. These results indicate that the antibacterial properties of Mat@CS-Pro decrease with increasing environmental pH. As can be seen from the results of combining parts b and c in fig. 9, the mat@cs-Pro nanocapsules have better antibacterial properties in an acidic environment, since the nanocapsules are larger in size and easily release Mat in an acidic environment.

In summary, the technical scheme of the invention is that matrine (Mat) is dissolved in absolute ethyl alcohol containing lecithin to prepare an oil phase, chitosan (CS) is dissolved in absolute acetic acid to prepare a water phase, and a microemulsion method is used for preparing microcapsule solution with an O/W structure, wherein the capsules are spherically distributed. Through CS encapsulation, mat with poor stability under the ocean alkaline condition can maintain the bactericidal activity. Due to the protonation and deprotonation processes of the amino groups in the capsule shell CS, the mat@cs microcapsules expand or contract, the pore channels open or close, and the Mat is released from the capsules or stored without release. Compared with an alkaline environment, the prepared antibacterial capsule has more excellent antibacterial performance in an acidic solution, amino groups on CS in the acidic environment are protonated, electrostatic mutual exclusion is generated among molecules, and an expansion pore canal of the microcapsule is opened to release medicines for sterilization. The deprotonation of CS amino groups under alkaline conditions can prevent Mat release and increase the stability of the capsule in alkaline environment. CS and D-Proline have isoelectric points similar to each other, and under acidic or alkaline environment, zeta potential is the same, and electrostatic mutual exclusion is generated between molecules. The release of the D-Proline is accelerated, so that the Mat@CS-Proline releases the D-Proline while releasing the Mat antibacterial, thereby realizing the nano anti-fouling microcapsule with the antibacterial and anti-biofilm dual functions of the corresponding pH function. The material provided by the invention is environment-friendly and has excellent antibacterial effect, and a thought is provided for exploring the research of intelligent pH response type antifouling agents in the ocean field.

It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, and the scope of the claims of the present invention should be covered.

Claims

1. The preparation method of the pH response type Mat@CS-Pro nanocapsule anti-fouling agent is characterized by comprising the following steps of:

(1) Dissolving matrine in absolute ethanol containing lecithin to prepare an oil phase;

(2) Dissolving chitosan in anhydrous acetic acid to prepare a water phase;

(3) Preparing Mat@CS oil/water microcapsules by a microemulsion method, wherein after water phase and oil phase are mixed, the water phase starts to cover the oil phase, so as to form Mat@CS oil/water microcapsules with chitosan as shells and matrine as cores;

(4) And preparing the prepared Mat@CS oil/water microcapsule and D-Proline into an aqueous solution, stirring in an ice-water bath, dialyzing and filtering in a deionized water solution through a dialysis bag, and freeze-drying and preserving to obtain the pH response Mat@CS-Pro nanocapsule anti-fouling agent.

2. The preparation method according to claim 1, wherein the matrine amount in the step (1) is 10-40 mg, the concentration of absolute ethanol containing lecithin is 20-40mg/mL, and the volume is 200-500 μl.

3. The method according to claim 1, wherein the chitosan in the step (2) is 5-15mg, and the volume ratio of the aqueous acetic acid solution is 1%, and the volume is 10-30mL.

4. The method according to claim 1, wherein the microemulsion method in step (3) is: and (3) respectively taking the water phase and the oil phase, slowly adding the oil phase into the water phase to prepare microspheres with uniform distribution, stirring at a rotating speed of 100-400 rpm for more than 1-3 hours, dialyzing and filtering in a phosphate buffer solution through a dialysis bag, and freeze-drying and preserving to obtain the Mat@CS water/oil microcapsule.

5. The method according to claim 4, wherein the phosphate buffer solution has a pH of 7.4.

6. The preparation method of claim 4, wherein the water phase is glacial acetic acid, chitosan and deionized water, and the oil phase is lecithin, absolute ethyl alcohol and matrine.

7. The preparation method according to claim 1, wherein the mass of the Mat@CS oil/water microcapsule in the step (4) is 20-40mg, and the mass of the D-Proline is 10-20 mg; the mass ratio of the Mat@CS oil/water microcapsule to the D-Proline is 1-5:1.

8. The method according to claim 1, wherein the deionized water solution in the step (4) has a volume of 10-30mL, and is stirred in an ice-water bath at a stirring speed of 100-400 rpm for 40-50 hours.

9. A pH-responsive mat@cs-Pro nanocapsule stain-proofing agent prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a pH-responsive mat@cs-Pro nanocapsule stain-resist agent according to claim 9 in decontamination of marine environments, said pH-responsive mat@cs-Pro nanocapsule stain-resist agent being applied to a marine environment having a pH range of 8.0 to 8.3.