CN115999462A - A pH-responsive Mat@CS-Pro nanocapsule antifouling agent and its preparation method and application - Google Patents

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

A pH-responsive Mat@CS-Pro nanocapsule antifouling agent and its preparation method and application

technical field

The invention relates to an environmentally friendly pH-responsive 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 technique

Marine biofouling is a worldwide problem, which has a huge impact on our daily life, especially the economic loss caused by shipping and the safety risk of engineering equipment. When the material is immersed in seawater, fouling organisms in the ocean will adhere to the bottom of the ship, increasing navigation resistance, reducing ship speed, and consuming more fuel, seriously affecting continuous use. In addition, it will also accelerate metal corrosion and shorten the use of equipment. Lifespan even causes serious ecological damage such as biological invasion, which has a huge impact on the development of human oceans. Since the formation of microbial film is the basic condition for subsequent large-scale fouling organisms to adhere and fix, the research on the formation process of antibacterial and anti-biofouling in the process of converting marine antifouling into biofouling is an effective way to prevent and control organisms. Ideas and methods of defacement. In addition, because secretions such as acetic acid and lactic acid produced by bacterial metabolism can lower the pH value of the microenvironment to acidity, pH-responsive antifouling coatings have received more and more attention and developed rapidly in recent years. In addition, the biofilm formed on the surface of the material is also an important issue to be considered.

In the initial stage of the fouling process, bacteria and diatoms secrete mucus to form microbial mucous membranes on the surface of clean objects in the sea. In the developmental stage, the larvae of large fouling organisms begin to attach. The species and number of individuals continue to increase, and the volume and quality of the community continue to increase. The phenomenon is obvious. Some species with high individual density and rapid growth become the dominant species in the community; in the stable stage, species with long growth period and large individuals grow fully, crowding out or covering some attached medium and small species, complex and large species Community species formation. Therefore, the formation of biofilm creates conditions for subsequent large-scale fouling organisms to attach, which in turn leads to the formation of biofouling. Therefore, controlling the formation of microbial film is also a necessary condition for biofouling. However, the current research on antifouling agents at home and abroad mainly focuses on the improvement of antibacterial performance, but there are few reports on the introduction of anti-biofilm ability on the basis of improving the bactericidal effect.

Contents of the invention

In order to solve the above problems, the technical solution of the present invention proposes an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent and its preparation method, using natural organic matrine (matrine, Mat) as the core material, and using natural Polymer chitosan (chitosan, CS) was used as the wall material, and the microcapsules of CS encapsulated Mat were prepared by microemulsion polymerization, and D-Proline (D-Proline) was fixed on the capsules by stirring in a cold bath , and finally prepared a high-performance smart pH-responsive antibacterial/biofilm antifouling agent. The green and non-toxic Mat@CS capsule will improve the stability of the antimicrobial agent Mat, reduce the possibility of denaturation in an alkaline environment, and maximize the bactericidal efficiency of the microcapsules. Through the pH response of CS, Mat and D-Proline The release is more intelligent and sustainable. First, using CS as the water phase and Mat as the oil phase, O/W microcapsules were prepared by microemulsion method. The morphology and size of Mat@CS and Mat@CS-Pro microcapsules at different pH were analyzed by transmission electron microscopy (TEM). The characteristic functional groups of the microcapsules were analyzed by Fourier transform infrared spectroscopy (FTIR) and the components of the microcapsules were judged. The plate colony counting method and fluorescent live/dead bacteria detection were used to analyze the antibacterial performance and biofilm formation ability of the microcapsules before and after loading D-Proline, and the microcapsule sterilization at different pH was observed by the plate colony counting method. The microcapsules of D-Proline were characterized by drug release rate at different pH, and the pH-responsive long-term bactericidal mechanism of the microcapsules was comprehensively analyzed.

The invention prepares bactericidal microcapsules with pH response properties through microemulsion polymerization, and at the same time loads D-Proline to successfully synthesize antifouling capsules with pH response antibacterial and anti-biofilm properties. The Mat@CS-Pro microcapsule has excellent antibacterial, anti-biofilm and pH responsive properties, and has the functions of stable existence, intelligent control release, prolonging the service life of antibacterial agents and being environmentally friendly, and has a wide range of application prospects.

The purpose of the present invention is achieved through the following technical solutions:

According to the first aspect of the present invention, there is provided a method for preparing an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent, the preparation method comprising:

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

(2) dissolving natural polymer chitosan (chitosan, CS) in anhydrous acetic acid to prepare an aqueous phase;

(3) Mat@CS oil/water (O/W) microcapsules were prepared by the microemulsion method. After the water phase and oil phase were miscible, the water phase began to coat the oil phase, forming a Mat@CS oil with CS as the shell and Mat as the core. /water (O/W) microcapsules;

(4) Take the prepared Mat@CS oil/water (O/W) microcapsules and D-Proline to prepare an aqueous solution, stir in an ice-water bath, dialysis filter through a dialysis bag in a deionized aqueous solution, and then freeze Dry storage, thus obtaining the pH-responsive Mat@CS-Pro nanocapsule antifouling agent.

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

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

Further, the microemulsion method described in step (3) is: take the water phase and the oil phase respectively, slowly add the oil phase to the water phase to prepare uniformly distributed microspheres, and use the speed of 100-400rpm Stirring, the stirring time is more than 1-3h, dialysis filtration in PBS phosphate buffer solution through a dialysis bag, and then freeze-dried and stored to obtain the Mat@CS oil/water (O/W) microcapsules.

Further, the pH of the PBS phosphate buffer solution is 7.4.

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

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

According to the second aspect of the present invention, there is provided an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent, the pH-responsive Mat@CS-Pro nanocapsule antifouling agent adopts any of the above The preparation method is prepared.

Further, the pH-responsive Mat@CS-Pro nanocapsules are roughly spherical, and the particle diameter is between 200-500nm. The prepared capsules have intelligent pH response properties. Under marine (alkaline) conditions, CS can fully maintain the antibacterial activity of the internal Mat. release, to achieve the purpose of intelligent response antifouling.

According to the third aspect of the present invention, an application of an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent in marine environment decontamination is provided.

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

Beneficial effects of the present invention:

The present invention prepares O/W structure Mat@CS-Pro nano microcapsules by microemulsion method so that Mat can exist stably in seawater, reducing the possibility of oxidation reaction and saponification reaction between Mat and _O2 in the external environment. Mat@CS-Pro nanocapsules exhibited excellent antibacterial properties against S.aureus, P.aeruginosa and Escherichia coli (E.coli), and the capsules exhibited intelligent pH-responsive controlled-release bacteriostasis and inhibition of biofilm formation effect. In the marine environment, the secretions produced by bacterial metabolism will lead to a decrease in the local pH value. Due to the protonation process of the amino group in the CS of the Mat@CS-Pro capsule shell, electrostatic repulsion occurs between the CS molecules and the capsule expands to release Mat to kill the bacteria. , at the same time, D-proline is positively charged because it is less than the isoelectric point, and has the same charge as CS, so the two produce electrostatic mutual repulsion, which promotes the release of D-Proline and inhibits the formation of biofilm, showing the synergy of capsule sterilization Dual efficacy against biofilms. When the pH is high, the amino group is deprotonated, the capsule shrinks and Mat does not release, and Mat does not directly contact with the alkaline environment, which reduces the possibility that the lactam structure of Mat is directly exposed to the saponification reaction in the alkaline environment and causes denaturation. Mat exists stably for a long time. At the same time, because D-Proline is negatively charged like CS in alkaline environment, D-Proline can also release and inhibit the formation of biofilm in alkaline environment for antifouling.

In the present invention, D-Proline and Mat@CS are configured into an aqueous solution at a mass ratio of 1-5:1 (preferably 2:1), and stirred in an ice bath to fix D-Proline on the Mat@CS capsule, so that Mat@ CS is antibacterial and antifouling while adding D-amino acid anti-biofilm antifouling function. D-Proline is adsorbed and fixed to the surface of Mat@CS through electrostatic interaction. When the capsule expands under acidic conditions, Mat@CS and D-Proline are released simultaneously to realize It has the dual functions of capsule antibacterial and anti-biofilm, and shows good antifouling performance.

Description of drawings

Figure 1 is the TEM images of Mat@CS (part a, b) and Mat@CS-Pro (part c, d) nanocapsules at different pH values.

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

Figure 3 is the number of E.coli, S.aureus, P.aeruginosa (part a) and the counts of Mat, CS and Mat@CS nanocapsules in Luria-Bertani culture medium (LB) and 2216E medium for 18 hours The results correspond to the antimicrobial effect (part b).

Figure 4 shows the number of E.coli, S.aureus, and P.aeruginosa after Mat@CS nanocapsules, D-Proline and Mat@CS-Pro nanocapsules were applied to LB medium for 18 hours (part a), and the colony counts The antibacterial effect diagram corresponding to the results (part b).

Figure 5 is the SEM image of untreated E.coli (part a) and E.coli treated with Mat@CS-Pro nanocapsules (part a ₁ ); untreated S.aureus (part b) and Mat@CS-Pro - S. aureus treated with Pro nanocapsules (part b ₁ ); untreated P. aeruginosa (part c) and P. aeruginosa treated with Mat@CS-Pro nanocapsules (part c ₁ ).

Figure 6 is E.coli (part ac), S.aureus (part a ₁ -c ₁ ) and P.aeruginosa (part a ₂ -c ₂ ) with and without Mat@CS and Mat@CS-Pro nanocapsules The SEM image of the biofilm on the corresponding test piece after culturing in the culture medium of .

Figure 7 shows E.coli (part ac), S.aureus (part a ₁ -c ₁ ) and P.aeruginosa (part a ₂ -c ₂ ) in Mat@CS and Mat@CS-Pro nanocapsule conditions in Figure 6 Thickness graph of the biofilm formed after 3 days of incubation.

Figure 8 is the TEM image of Mat@CS nanocapsules at pH 5 (part a) and pH 8 (part b); Mat@CS-Pro nanocapsules at pH 5 (part c) and pH 8 (part d) TEM image below.

Figure 9 is the standard curve of Mat in PBS solution (part a); the concentration of Mat released from Mat@CS-Pro nanocapsules in PBS solutions of different pH for 10h (part b); Mat@CS-Pro nanocapsules Diameter after treatment in PBS solutions with different pH values (part c); after incubation for 4 h in LB and PBS solutions (volume ratio 1:1) containing Mat@CS-Pro nanocapsules (part d), three Optical density (O.D.) values of different types of bacterial strains.

Figure 10 is Mat@CS-Pro in pH 5, 6, 7 and 8 medium against E.coli (ad part), S.aureus (a ₁ -d ₁ part) and P.aeruginosa (a ₂ -d ₂ Part) Table colony plate photos.

Detailed ways:

The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and through specific embodiments.

The following examples are only exemplary, and can only be used to explain and illustrate the technical solution of the present invention, but cannot be construed as a limitation to the technical solution of the present invention.

The technical scheme of the present invention provides an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent and its preparation method, using natural organic matrine (matrine, Mat) as the core material, and using natural polymer shell Sugar (chitosan, CS) was used as the wall material, and the CS-encapsulated Mat microcapsules were prepared by microemulsion polymerization, and D-Proline (D-Proline) was fixed on the capsules by stirring in a cold bath, and finally prepared High-performance smart pH-responsive antimicrobial/biofilm antifouling agent.

Here, CS is a product obtained after the deacetylation reaction of chitin contained in crustaceans, and has good antibacterial properties. At the same time, as a polysaccharide, CS has rich biological and physical properties, including biodegradability, biocompatibility and non-toxicity. The microcapsules prepared with it as the shell have typical pH responsiveness. Under acidic conditions, a large number of amino groups carried in the CS side chain are positively charged, and the amino groups of CS are protonated. Due to electrostatic repulsion, the microcapsules expand and the pores open and expand; when the solution environment is alkaline, desorption occurs. Protonation causes the microcapsules to shrink and the pores to shrink or even close. Usually the marine environment is alkaline, and the secretions such as lactic acid produced by the metabolism of bacteria in the environment after colonization on the substrate will reduce the pH of the surrounding environment, promote the expansion of microcapsules, accelerate the release of drugs, and play a bactericidal effect; on the contrary, when the surrounding bacteria Rarely, the marine environment is alkaline, and the microcapsules shrink, so that the drug is wrapped by CS and stored, which greatly prolongs its service life. It can be seen that the microcapsules prepared by using CS can store and release drugs continuously, realize the delayed release of antibacterial agents and pH intelligent response to sterilization, improve the stability of drugs at the same time, and realize the maximum bactericidal effect of drugs. In addition, CS is also biodegradable, non-polluting to the environment, and non-toxic to organisms. It has achieved the goal of green environmental protection and has good application prospects in the fields of food safety, public health, and medical equipment.

And Mat is a kind of alkaloid that is made by extracting the dry roots, plants and fruits of Sophora flavescens through ethanol and other organic solvents. It has many pharmacological effects and effects, such as antibacterial, anti-inflammatory, anti-rheumatic, anti- Tumor, anti-allergy, anti-virus, anti-parasite, anti-arrhythmia, swelling and diuresis, immune and biological response regulation, etc. Mat is a plant-derived pesticide with specific and natural characteristics, and it only acts on specific organisms. It can be rapidly decomposed in nature, and the final products are water and carbon dioxide. Because a variety of chemical substances work together, it is not easy to cause harmful substances to develop drug 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 very green and environmentally friendly. However, due to the lactam structure in it, it is easy to undergo saponification reaction in alkaline environment and the structure will be destroyed. Therefore, increasing the stability of Mat and reducing its exposure to alkaline environment is to increase its antibacterial stability and long-term effect. The essential.

D-amino acids are produced as enantiomers of L-amino acids from racemates. Due to their degradability, nontoxicity and origin, they are widely used in drug synthesis, enzyme structure and function analysis, and fungicide applications. After years of experimental research, the applicant found that it is feasible to apply it to antifouling agents to achieve the purpose of antifouling by inhibiting the formation of biofilm.

Therefore, the present invention selects Mat as a natural antibacterial agent, prepares pH-responsive antibacterial microcapsules by coating CS, and then fixes D-Proline on the surface of the capsules to prepare environmentally friendly pH smart-responsive antibacterial/biofilm antifouling capsule. When the solution environment is acidic, the amino group on the capsule CS is protonated, and due to electrostatic repulsion, the Mat@CS microcapsule expands and the pores open and expand, and the antibacterial agent is released from the capsule to kill bacteria. At the same time, D-proline is less than the isoelectric point It is positively charged and has the same charge as CS, so the two produce electrostatic mutual repulsion, which promotes the release of D-Proline and inhibits the formation of biofilm. When the solution environment is alkaline, deprotonation occurs, causing the microcapsules to shrink, the pores to narrow and close, the antibacterial agent is not released, and Mat is blocked, reducing the lactam structure of Mat directly exposed to the alkaline environment. The possibility of denaturation caused by saponification makes Mat stable for long-term storage in the capsule, which improves the stability of Mat. In the environment, D-Proline can also be released to inhibit the formation of biofilm for antifouling. Usually, secretions such as lactic acid produced by bacterial metabolism will reduce the pH of the surrounding environment, promote the expansion of microcapsules, accelerate the release of Mat and D-Proline, and have a bactericidal effect; on the contrary, when there are few surrounding bacteria, the environment is alkaline. The microcapsules are shrunk so that Mat can be stored. It can be seen that Mat@CS-Pro microcapsules can exist stably in the marine environment and effectively store fungicide components. When bacteria are enriched and colonized, and the pH of the microenvironment decreases, Mat@CS-Pro microcapsules can effectively release Mat and D-proline to achieve the purpose of antifouling agent sterilization and synergistic inhibition of biofilm formation, while the added Mat, CS , D-Proline are biodegradable, and have no pollution to the environment and are non-toxic to organisms, achieving the goal of a green and environmentally friendly antifouling agent.

The preparation method comprises the following steps:

Mat was dissolved in absolute ethanol containing lecithin to prepare the oil phase, and CS was dissolved in anhydrous acetic acid to prepare the water phase. O/W structured microcapsules of Mat@CS were prepared by microemulsion method. After the water phase and oil phase are miscible, the water phase begins to coat the oil phase, forming a capsule structure with CS as the packaging material and Mat as the core material. Stir for about 2 hours at a speed of 100-400 rpm, dialyze through a dialysis bag, and then freeze-dry for storage. Dissolve 20-40 mg of D-Proline in 20 mL of aqueous solution, then add 10-20 mg of Mat@CS, and stir in an ice bath at 100-400 rpm for about 50 h. Free D-Proline was eliminated by dialysis in deionized aqueous solution. Mat@CS-Pro nanocapsules were collected after vacuum freezing at -50°C for about 40 h.

The amount of CS is 10 mg, and the volume of aqueous acetic acid (1%, v/v) is 10-30 mL. The microemulsion method is as follows: the Mat solution forms microdroplets through a liquid-liquid homogeneous nucleation process, and after blending with the CS solution, the Mat microdroplets are wrapped in the CS to form microcapsules. In this process, lecithin as an emulsifier participates in the liquid-liquid homogeneous nucleation process and realizes the microcapsule formation process. Finally, CS-coated Mat microcapsules were self-assembled according to the O/W mechanism of the microemulsion method. 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 coating structure of CS-coated Mat is stable. In alkaline environment, the CS coating material is deprotonated, the shrinkage channel of the capsule is tightly closed, and Mat does not release. Therefore, the alkaline seawater environment of Mat can be kept stable for a long period of time.

D-Proline is adsorbed to the surface of Mat@CS capsules through electrostatic interaction. While Mat@CS exerts antibacterial effect, D-Proline has the characteristics of anti-biofilm formation, realizing the dual effect of pH-responsive antibacterial and anti-biofilm marine defense dirty function.

The pH-responsive Mat@CS-Pro capsule has an intelligent pH-responsive controlled release function, which increases the stability of Mat in the marine environment. In the marine environment, the secretions produced by bacterial metabolism will lead to a decrease in the local pH value. Due to the protonation process of the amino group in the capsule shell CS, the capsule expands and releases Mat to kill the bacteria. Shrinking makes Mat sealed, reducing the possibility of saponification and denaturation of Mat due to direct exposure to alkaline environment due to the lactam structure, making Mat stable for long-term storage in the capsule, and improving the bactericidal stability of Mat.

Example 1

(1) Microcapsule solution with O/W structure was prepared by microemulsion method. Using a pipette, 32 mg of Mat was dissolved in 30 mg/mL absolute ethanol (400 μL) containing lecithin to prepare an oil phase. The aqueous phase was prepared by dissolving CS (10 mg) in 20 mL of aqueous acetic acid (1%, v/v). Slowly pour the oil phase into the water phase, and at room temperature, stir the mixed solution with a magnetic stirrer at a speed of 200 rpm for 2 hours until it becomes opalescent, and a microcapsule solution is obtained.

(2) Transfer the microcapsule solution to a dialysis bag, and soak in the dialysate (PBS phosphate buffer solution with a pH value of 7.4) for 12 hours to eliminate free Mat. The microcapsule solution was put into the freezer layer of the refrigerator to freeze for 2 hours, and after solidification, it was sent to a freeze dryer at -50° C. for 36 hours to obtain microcapsule powder.

(3) Dissolve 40 mg of D-Proline in 20 mL of aqueous solution, then add 20 mg of Mat@CS lyophilized powder into the solution, and stir at 200 rpm for 48 h in an ice bath. Free D-Proline was eliminated by dialysis in aqueous solution. Mat@CS-Pro nanocapsules were collected after vacuum freezing at -50°C for 36 h.

(4) The antibacterial properties of Mat@CS-Pro nanocapsules were analyzed by colony counting method. E.coli and S.aureus were chosen as representatives of Gram-negative and Gram-positive strains, and P.aeruginosa as a representative of marine bacteria. The initial concentration of each bacterium was approximately ~10 ⁸ CFU/mL. Inject 1 mL of E.coli or S.aureus suspension into 50 mL of LB culture medium, and shake and culture at 120 rpm for 18 hours in a constant temperature equipment at 37°C. Transfer 1mL of P.aeruginosa suspension to 50mL sterile 2216E medium, and shake and culture at 30°C for 18h. Thereafter, pipette 200 μL of bacterial suspension into 8 mL of LB or 2216E medium containing 2 mg/mL of different antibacterial agents (i.e. CS, Mat, D-Proline, Mat@CS nanocapsules, Mat@CS-Pro nanocapsules ). After incubating at 37°C or 30°C for 18 hours, take 20 μL of the diluted bacterial suspension and spread it on a solid medium plate, and cultivate overnight under corresponding temperature conditions. The antibacterial results were calculated according to the following formula:

In the formula, A is the number of colonies in the control group, and B is the number of colonies in the treatment group. In addition, the O.D. curve was detected at 600 nm by a UV spectrophotometer as a reference for evaluating the antibacterial properties of nanocapsules.

The O.D. value was measured at 600nm by a UV spectrophotometer, which provided a reference for evaluating the antibacterial properties of nanocapsules.

(5) The anti-adhesion performance of Mat@CS-Pro nanocapsules was evaluated by SEM. 316L stainless steel samples (1cm×1cm) were used to assist in evaluating the anti-adhesion properties of the prepared nanocapsules. The samples were polished with 1200-mesh silicon carbide paper, then ultrasonically cleaned with acetone and ethanol and dried with _N2 , and the metal samples were sterilized using ultraviolet light. Add 1 mL of E.coli or S.aureus suspension into 50 mL of sterile LB and shake at 120 rpm for 18 h at 37 °C. Add 1 mL of P. aeruginosa suspension into 50 mL of sterile 2216E medium and shake at 120 rpm for 18 h at 30 °C. Put each sample piece into a 24-well plate, and then inject 4 mL of LB medium (containing 2 mg/mL of Mat@CS or Mat@CS-Pro nanocapsules) and 100 μL of bacterial suspension. After 1 day and 3 days of static culture, the samples were removed and washed with deionized water to remove free-floating bacteria on the metal surface, and fixed with 2.5% (v/v) glutaraldehyde in phosphate buffered saline solution (PBS, pH 7.4) for 2 h . Subsequently, each sample was washed 3 times with PBS, 3 times with deionized water, and dehydrated for 15 minutes using 30%, 50%, 70%, 90% and 100% (v/v) graded ethanol. After the sample was sprayed with carbon, the bacterial film on the metal surface was observed by scanning electron microscopy.

Additionally, using Live/ The BacLight TM Bacterial Viability Kit further analyzed the anti-adhesion properties of Mat@CS-Pro nanocapsules. Each sample was immersed in different bacterial suspensions containing nanocapsules, and cultured for 1 day and 3 days, respectively. Each sample was then washed with deionized water to remove free bacteria on the metal surface, placed in a clean 24-well plate, and stained for 20 minutes in the dark.

(6) Put the Mat@CS-Pro nanocapsule suspension (2 mg/mL) into the dialysis bag and immerse in the PBS solution with pH 5, 6, 7 and 8 for 12 h. The dialysis solution was measured hourly by UV-Vis and the peak at 220 nm represents Mat. The size of Mat@CS-Pro nanocapsules treated in PBS solutions with different pH was detected by DLS.

(7) The colony plate method was used to evaluate the antibacterial performance of the pH response. Prepare a 2 mg/mL Mat@CS-Pro nanocapsule medium consisting of 4 mL of PBS solutions of different pH (pH 5, 6, 7, 8) and 4 mL of LB medium (or 2216E). Inoculate 200 μL of each bacterial suspension into 8 mL of culture medium and incubate at 37°C or 30°C for 4 hours. Spread 20 μL of the diluted bacterial suspension on the solid medium and incubate at the corresponding temperature for 18 h. The O.D. of each bacterial suspension after 4 h of incubation was also detected by UV-Vis at 600 nm as a reference for another method to evaluate the pH-responsive antibacterial properties of Mat@CS-Pro nanocapsules.

Embodiment 2 (effect embodiment)

Synthesis and performance testing of Mat@CS nanocapsules:

(1) Characterization of Mat@CS-Pro nanocapsules

The surface structure and morphology of the prepared nanocapsules before and after D-Proline immobilization were observed by transmission electron microscopy, as shown in Figure 1. It can be seen from Figure 1 that the Mat@CS nanocapsules prepared by the O/W microemulsion method have a uniform particle size, with an average particle size of about 250 nm. After immobilizing D-Proline, the capsules did not agglomerate, and the particle size increased slightly (parts a and c in Figure 1). However, the morphology of Mat@CS-Pro is different from that of Mat@CS nanocapsules, which shows a large number of small droplets inside the nanocapsules (part b in Fig. 1). According to the zeta potentials of CS, Mat and Mat@CS nanocapsules are 37.8 ± 1.1 mV, -16.9 ± 2.4 mV and 31.9 mV, respectively, it can be inferred that the droplets in the nanocapsules should be Mat, because the surface of free Mat droplets is negatively charged completely covered by cationic CS. In addition, the surface of Mat@CS nanocapsules is covered with irregularly shaped substances (part d in Fig. 1). The zeta potential distributions of D-Proline and Mat@CS-Pro nanocapsules were -7.7 ± 1.1 mV and -5.3 ± 0.3 mV, respectively, indicating that negatively charged D-Proline could be immobilized on the surface of Mat@CS nanocapsules. Therefore, D-Proline was immobilized on the surface of Mat@CS nanocapsules, and the diameter of Mat@CS-Pro nanocapsules was about 280 nm.

The FTIR of CS, Mat and the as-prepared Mat@CS nanocapsules are shown in part a of Fig. 2. For the CS line, the broad peaks located at 3500-3200 cm ^-1 belong to NH and OH stretching vibrations. The 1157cm ^-1 and 1091cm ^-1 peaks belong to COC stretching vibration. For the Mat spectrum, the peaks at 2935cm ^-1 and 2853cm ^-1 belong to CH stretching vibration in CH2, and the peak at 1635cm ^-1 belongs to C=O stretching vibration. The characteristics of the product spectrum of Mat@CS-Proline nanocapsules are similar to the CS spectrum. A broad peak at about 3500-3200cm ^-1 also appears on the Mat@CS spectrum in the figure, which belongs to the stretching vibration of NH and OH. In addition, a peak at 1157 cm ^-1 appears on Mat@CS, which belongs to the stretching vibration of COC. The 1085cm ^-1 peak (COC stretching vibration) in the Mat@CS spectrum corresponds to the 1019cm ^-1 peak in the CS spectrum, indicating that CS exists in the prepared capsules. At the same time, Mat related characteristic peaks can also be found in Mat@CS, such as the 1635cm ^-1 peak in the spectral line due to C=O stretching vibration is consistent with the pure Mat peak, and its intensity is weaker than that of the Mat spectral line . The 2935cm ^-1 and 2853cm ^-1 peaks in the Mat line due to the CH stretching vibration correspond to the 2930cm ^-1 and 2858cm ^-1 of Mat@CS respectively, and the 2875cm ^-1 peak on the CS caused by the CH stretching vibration covered by these two peaks. That is to say, Mat also exists in the prepared nanocapsules. In addition, a new peak located at ¹⁷³⁷ cm indicates the existence of H-bonding interactions between CS and Mat. Combined with the TEM results shown in Figure 1, it indicated that Mat@CS nanocapsules were successfully synthesized.

After the capsule was loaded with D-Proline, the product components were analyzed by FTIR, and the results are shown in part b of Figure 2. For the spectral line of D-Proline, the peaks at 3051cm ^-1 and 2979cm ^-1 are produced by the asymmetric stretching and symmetric stretching vibrations of CH bond respectively. The peaks located at 1600-1450 cm ^-1 in the D-Proline spectrum are related to the vibration of the skeleton ring, and there are strong peaks at similar positions on the spectrum of the synthesized Mat@CS-Pro nanocapsules. At the same time, the peaks at 848cm ^-1 and 791cm ^-1 caused by the rocking vibration of CH ₂ on the D-Proline line correspond to the peak positions at 856cm ^-1 and 773cm ^-1 of the Mat@CS-Pro line, respectively. Spectral lines Spectral lines In addition, the peaks at 1164cm ^-1 and 1085cm ^-1 of Mat@CS nanocapsules caused by COC vibration also appeared on Mat@CS-Pro spectral lines. Combined with the TEM results shown in Figure 1, it shows that D-proline was successfully immobilized on Mat@CS-Pro nanocapsules.

(2) Characterization of antibacterial properties of Mat@CS-Pro nanocapsules

Figure 3 is a graph of the colony count and antibacterial effect of CS, Mat and Mat@CS nanocapsules on E.coli, S.aureus and P.aeruginosa. In part a of Figure 3, the number of bacteria after the treatment of these three drugs was significantly lower than that of the blank control group (379CFU), indicating that CS, Mat and Mat@CS nanocapsules have significant antibacterial effects on these three strains. Part b of Figure 3 shows the antibacterial effects of the three drugs on E.coli, S.aureus and P.aeruginosa respectively, and the bactericidal rates on P.aeruginosa exceeded 87.71%, 77.90% and 73.39% respectively. Notably, the antibacterial effect of pure Mat and Mat@CS nanocapsules on Gram-positive bacteria was better than that on Gram-negative bacteria, and the antibacterial effect on P.aeruginosa was the worst. Specifically, the antibacterial rates of Mat and Mat@CS against P.aeruginosa were 78.02% and 89.16%, respectively, which were 15.06% and 5.39% lower than those of S.aureus, respectively. Compared with pure Mat, Mat@CS nanocapsules exhibited better antibacterial properties against E.coli, S.aureus, and P.aeruginosa. P. aeruginosa colonies were reduced by 8CFU, 6CFU and 39CFU, respectively. From the results in part b of Figure 3, the antibacterial rates of Mat@CS nanocapsules against the above three bacteria increased to 89.71%, 94.55% and 89.16%, respectively. Therefore, the antibacterial performance of Mat@CS nanocapsules was significantly improved after being coated with CS.

The colony counting method further showed the antibacterial effect of Mat@CS-Pro nanocapsules, and the results are shown in Figure 4. As shown in part b of Figure 4, the antibacterial effect of pure D-Proline is not satisfactory. The antibacterial rates of E.coli, S.aureus and P.aeruginosa were about 57.71%, 49.96% and 66.55%, respectively. However, after immobilizing D-Proline, the antibacterial rates of Mat@CS-Pro nanocapsules against E.coli and P.aeruginosa reached 92.85% and 96.28%, respectively. However, after the introduction of D-Proline, the antibacterial rate of Mat@CS-Pro nanocapsules against S.aureus decreased from 94.55% to 87.75%, which may be due to the poor antibacterial effect of the nanocapsule structure of D-Proline on S.aureus. Sincerely. After the surface of Mat@CS-Pro is coated with D-Proline, it may hinder the release of Mat, and the D-Proline on the surface cannot provide significant antibacterial effect, resulting in a slight decrease in the antibacterial performance of Mat@CS-Pro. Small. Overall, after the introduction of D-Proline, Mat@CS-Pro nanocapsules can almost maintain excellent antibacterial performance against Gram-positive bacteria, and even exhibit better antibacterial performance against Gram-negative bacteria.

After being sterilized by Mat@CS-Pro nanocapsules, the morphology of E.coli, S.aureus and P.aeruginosa was observed by scanning electron microscope. Compared with the control group, the cell membrane of the bacteria almost lost its complete structure, indicating that the nanocapsules caused irreversible damage to the bacterial cells. In addition, the cell surfaces of E.coli and P.aeruginosa were covered with a large amount of organic matter compared with the morphology of S.aureus, which demonstrated that Mat@CS-Pro nanocapsules were more effective on Gram-negative strains than on Gram-positive strains. influence is stronger. These results are consistent with the colony count results shown in Figure 4.

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

The effect of Mat@CS and Mat@CS-Pro nanocapsules on biofilm formation after 3 days was evaluated by SEM. For the control group, the bacteria attached to the metal surface were the most, and aggregation phenomenon occurred (part aa ₂ in Figure 6). After co-cultured with Mat@CS nanocapsules, the number of bacterial cells and aggregation on the surface of the substrate were slightly reduced (part bb ₂ in Figure 6). In the cc ₂ part in Figure 6, after introducing D-Proline into the Mat@CS nanocapsules, the number of bacteria and the aggregation phenomenon were further reduced. It can be seen from the figure that most of the bacterial cells are randomly scattered on the surface of the substrate in the form of single cells. And it can be seen that the bacterial morphology in the bacterial aggregation area is significantly different from that of the control group. When no nanocapsules were added to the medium, the bacteria had a complete cell morphology and the cell membrane had a clear edge. However, for the surface morphology of the substrate added with Mat@CS-Pro, especially the aggregation area, the bacterial morphology is similar to the a ₁ -c ₁ part in Figure 5, that is, the bacteria are aggregated (the cc ₂ part in Figure 6).

After 3 days of culture, the formed biofilm was evaluated by IPCM, and the results are shown in Figure 7. The green and red areas in the field of view represent live and dead bacteria, respectively. It can be seen from the figure that almost all of the control group are green, indicating that the 316L stainless steel sheet has no toxicity to these three bacteria, and the bacteria can survive on the surface of the substrate and form a complete and uniform biofilm. Among them, the biofilm thicknesses of E.coli, S.aureus, and P.aeruginosa were about 30 μm, 39 μm, and 48 μm, respectively (aa ₂ in Figure 6). After culturing with Mat@CS and Mat@CS-Pro nanocapsules, many red areas appeared in the visual field, indicating a large number of dead bacteria. In addition, after introducing D-Proline into Mat@CS nanocapsules, the proportion of green area was further reduced, indicating that the antibacterial effect of Mat@CS-Pro nanocapsules was better than that of Mat@CS nanocapsules, which was consistent with the results in Fig. 4. In the cc ₂ part in Figure 6, after 3 days of culture with Mat@CS-Pro nanocapsules, the biofilm thicknesses of E.coli, S.aureus and P.aeruginosa formed were about 20 μm, 10 μm and 28 μm, respectively. The biofilm dispersion ability of Mat@CS-Pro nanocapsules was significantly improved, and the biofilms of E.coli, S.aureus, and P.aeruginosa were reduced by about 33%, 74%, and 42%, respectively, compared with the control group. Therefore, on the basis of the existing antibacterial properties, the introduction of D-Proline helps to improve the biofilm dispersion properties of antifouling agents.

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

The morphology and structure of Mat@CS nanocapsules and Mat@CS-Pro nanocapsules treated in pH 5 and pH 8 PBS solutions were characterized by TEM. Under acidic conditions, the -NH ₂ group of CS was transformed into -NH ₃ ⁺ , resulting in positive charges and internal electrostatic repulsion in the system. Therefore, after soaking at pH 5, the Mat@CS nanocapsules swelled from 280 nm to around 430 nm (part a in Fig. 8). In the marine environment (pH 8), the Mat@CS nanocapsules shrunk to about 220 nm due to the deprotonation of the CS amino group, as shown in part b of Fig. 8. After immobilizing D-Proline, the pH-responsive properties of the nanocapsules still existed. In part c of Figure 8 and part d of Figure 8, the diameters of Mat@CS-Pro nanocapsules treated in alkaline and acidic PBS solutions were about 475 nm and 234 nm, respectively. The results show that the Mat@CS-Pro nanocapsules are pH-responsive, and the diameter of the nanocapsules can change with the change of environmental pH.

The pH-controlled release behavior and mechanism of Mat@CS-Pro nanocapsules were determined by UV-Vis and DLS analysis. The release concentration of Mat from Mat@CS-Pro nanocapsules calculated according to the standard curve (part a in Figure 9) is shown in part b in Figure 9. Under alkaline conditions, the release of Mat was maintained at a low level, from the initial 5.1ppm to 7.1ppm after 10h. At the same time, DLS results showed that the size of Mat@CS-Pro nanocapsules after soaking in PBS solution with pH 8 was the smallest, about 236±13nm. On the contrary, the release of Mat in the acidic solution continued to increase, and the concentration of Mat was about 28.5 ppm and 23.5 ppm after soaking in pH 5 and pH 6 solutions for 10 h, respectively. Compared with the initial state, the release concentration of Mat increased by 16.8ppm and 10.3ppm, respectively. In part c of Fig. 9, the diameters of nanocapsules treated in pH 5 and pH 6 solutions are 478 ± 18 nm and 396 ± 21 nm, respectively, which is consistent with the results in Fig. 8. Compared with the corresponding nanocapsules treated at pH 8, the diameters of the nanocapsules increased by about 242 nm and 160 nm, respectively. When the pH was 7, the release amount of Mat showed an upward trend within 10h, and the release amount was 7.6ppm after 1h, 9.4ppm after 5h, and further increased to 10.1ppm after 10h. The above results indicated that the pH-responsive properties of Mat@CS-Pro nanocapsules were not restricted after immobilizing D-Proline. As the pH of PBS solution increased, the release behavior of Mat and the size of nanocapsules decreased.

The effects of Mat@CS-Pro nanocapsules on the bacterial growth status of E.coli, S.aureus and P.aeruginosa in different pH media were evaluated by UV-Vis. The bacterial concentration increased with the increase of OD value. From part d of Figure 9, it can be seen that the OD values of the three bacterial strains increase with the increase of the pH value of the medium, indicating that the number of bacteria in the medium also increases with the increase of the pH value. Specifically, the OD values of E. coli were about 0.69±0.02, 0.70±0.02, 0.75±0.01 and 0.77±0.02 after cultured at pH 5, 6, 7 and 8, respectively. In parts a to d in Figure 10, the results of the colony plate photos are consistent with the OD data, that is, the number of bacterial colonies increases significantly with the increase of environmental pH. The change trend of OD value of S.aureus is similar to that of E.coli, which are 0.68±0.01, 0.69±0.01, 0.72±0.01, 0.72±0.01, which is consistent with the a ₁ -d ₁ part in Figure 10. As for the P.aeruginosa group, the OD value increased from 0.27±0.01 at pH 5 to 0.45±0.05 at pH 8. In part a ₂ -d ₂ in Figure 10, after culturing under pH 7 and 8 conditions, there are more colonies scattered on the solid medium, but after culturing under pH 6 and 7 conditions, there are fewer colonies on the solid medium . These results indicated that the antibacterial performance of Mat@CS-Pro decreased with the increase of environmental pH. Combining the results of parts b and c in Figure 9, it can be seen that the Mat@CS-Pro nanocapsules have better antibacterial properties in acidic environments due to the larger size of the nanocapsules and easy release of Mat in acidic environments.

In summary, the technical scheme of the present invention first dissolves matrine (Mat) in dehydrated alcohol containing lecithin to prepare an oil phase, dissolves chitosan (CS) in anhydrous acetic acid to prepare an aqueous phase, and prepares it by a microemulsion method Microcapsule solution with O/W structure, the capsules are distributed in a spherical shape. Through the encapsulation of CS, Mat, which is less stable in the alkaline conditions of the ocean, maintains bactericidal activity. Due to the protonation and deprotonation process of the amino groups in the capsule shell CS, the Mat@CS microcapsules swell or shrink, the pores open or close, and Mat is released from the capsules or stored without release. Compared with the alkaline environment, the prepared antibacterial capsules have better antibacterial properties in acidic solution. In acidic environment, the amino group on CS is protonated, and electrostatic mutual repulsion occurs between molecules, and the expansion pores of the microcapsules are opened to release the drug. Sterilize. The deprotonation of the amino group of CS under alkaline conditions prevents the release of Mat and increases the stability of the capsules in an alkaline environment. The isoelectric points of CS and D-Proline are similar. In acidic or alkaline environment, the zeta potentials of both are the same, and electrostatic mutual repulsion occurs between the molecules. This accelerates the release of D-Proline, making Mat@CS-Proline release D-Proline while releasing Mat antibacterial, thus realizing the nano-antifouling microcapsules with pH-corresponding functions of antibacterial and anti-biofilm dual functions. The material of the invention is environmentally friendly and has superior antibacterial effect, and provides ideas for exploring the research of intelligent pH-responsive antifouling agents in the marine field.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation, although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.

Claims (10)

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.

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