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

Abstract

The present invention discloses a pH-responsive Mat@CS-Pro nanocapsule antifouling agent, its preparation method, and application, belonging to the technical field of marine antibacterial antifouling and nanocomposite materials. The invention comprises the following steps: dissolving matrine in anhydrous ethanol containing lecithin to prepare an oil phase; dissolving chitosan in anhydrous acetic acid to prepare an aqueous phase; preparing Mat@CS oil / water microcapsules by a microemulsion method; and preparing an aqueous solution of the prepared Mat@CS oil / water microcapsules and D-Proline. The solution is stirred in an ice-water bath, filtered through a dialysis bag in a deionized water solution, and freeze-dried to obtain the pH-responsive Mat@CS-Pro nanocapsule antifouling agent. The material disclosed is environmentally friendly and has excellent antibacterial properties, providing insights into the exploration of intelligent pH-responsive antifouling agents in the marine field.

Description

Technical Field

[0001] The present 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 and antifouling and nanocomposite materials. Background Art

[0002] Marine biofouling is a global problem that significantly impacts our daily lives, particularly through economic losses to shipping and safety risks to engineering equipment. When materials are immersed in seawater, marine biofouling organisms adhere to the bottom of ships, increasing resistance, reducing speed, and increasing fuel consumption, severely impacting their continued use. Furthermore, they accelerate metal corrosion, shorten the lifespan of equipment, and even cause serious ecological damage, such as bioinvasion, significantly impacting human marine development. Because the formation of microbial biofilms is essential for the subsequent attachment and fixation of large-scale fouling organisms, transforming marine antifouling efforts into research on antibacterial and biofilm-resistant processes during the early stages of biofouling is an effective approach and method for biofouling prevention. Furthermore, because secretions such as acetic and lactic acids produced by bacterial metabolism can lower the pH of the microenvironment to acidic levels, pH-responsive antifouling coatings have garnered increasing attention and rapid development in recent years. Furthermore, the formation of biofilms on material surfaces is also a critical consideration.

[0003] In the initial stage of the fouling process, bacteria and diatoms secrete mucus to form a microbial film on the surface of clean marine objects. In the development stage, the larvae of large fouling organisms begin to attach, the number of species and individuals continues to increase, and the community volume and mass continue to grow. Succession is obvious, and some species with high individual density and rapid growth become the dominant species in the community. In the stable stage, species with long growth periods and large individuals fully grow, crowding out or covering some of the attached medium and small species, forming a complex and large-scale community. Therefore, biofilm formation creates conditions for the subsequent attachment of large fouling organisms, which in turn leads to the formation of biofouling. Therefore, controlling the formation of microbial films is also a necessary condition for biofouling. However, current research on antifouling agents at home and abroad focuses mainly on improving antimicrobial properties, while research on introducing biofilm inhibition capabilities in addition to improving bactericidal effects has rarely been reported. Summary of the Invention

[0004] To address the above issues, the present invention proposes an environmentally friendly, pH-responsive Mat@CS-Pro nanocapsule antifoulant and its preparation method. Using natural organic matrine (Mat) as the core material and the natural polymer chitosan (CS) as the wall material, Mat-encapsulated microcapsules are prepared via microemulsion polymerization. D-proline (D-Proline) is then immobilized onto the capsules using a cold bath stirring method, ultimately creating a high-performance, intelligent, pH-responsive antibacterial / biofilm antifoulant. The green, non-toxic Mat@CS capsules improve the stability of the antibacterial Mat and reduce its potential for denaturation in alkaline environments, maximizing the microcapsule's bactericidal efficiency. The pH-responsive nature of CS allows for a more intelligent and sustainable release of Mat and D-Proline. First, an O / W microcapsule structure is prepared using a microemulsion method with CS as the aqueous phase and Mat as the oil phase. The morphology and size of the Mat@CS and Mat@CS-Pro microcapsules at different pH values ​​are analyzed using transmission electron microscopy (TEM). Fourier transform infrared spectroscopy (FTIR) was used to analyze the characteristic functional groups of the microcapsules and identify their composition. Plate colony counts and fluorescent live / dead bacteria assays were used to analyze the antibacterial properties and biofilm inhibition of the microcapsules before and after loading with D-Proline. Plate colony counts were also used to observe the bactericidal activity of the microcapsules at different pH values. The drug release rate of the D-Proline-loaded microcapsules at different pH values ​​was characterized, and the pH-responsive, long-lasting bactericidal mechanism of the microcapsules was comprehensively analyzed.

[0005] This study uses microemulsion polymerization to prepare pH-responsive bactericidal microcapsules. D-Proline is then loaded into the microemulsion to create antifouling capsules with pH-responsive antibacterial and anti-biofilm properties. These Mat@CS-Pro microcapsules exhibit excellent antibacterial, anti-biofilm, and pH-responsive properties. They offer a wide range of applications, including stable existence, intelligent controlled release, extended antimicrobial life, and environmental friendliness.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] According to a first aspect of the present invention, a method for preparing an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent is provided, the preparation method comprising:

[0008] (1) Dissolving natural organic matrine (Mat) in anhydrous ethanol containing lecithin to prepare an oil phase;

[0009] (2) dissolving natural polymer chitosan (CS) in anhydrous acetic acid to prepare an aqueous phase;

[0010] (3) Mat@CS oil / water (O / W) microcapsules were prepared by the microemulsion method. After the water phase and the oil phase were miscible, the water phase began to cover the oil phase, forming Mat@CS oil / water (O / W) microcapsules with a CS shell and a Mat core;

[0011] (4) The prepared Mat@CS oil / water (O / W) microcapsules were mixed with D-Proline to prepare an aqueous solution, stirred in an ice-water bath, dialyzed and filtered through a dialysis bag in a deionized water solution, and then freeze-dried for storage, thereby obtaining the pH-responsive Mat@CS-Pro nanocapsule antifouling agent.

[0012] Furthermore, the amount of Mat in step (1) is 10-40 mg, the concentration of the lecithin-containing anhydrous ethanol is 20-40 mg / mL, preferably 30 mg / mL, and the volume is 200-500 μL.

[0013] Furthermore, 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.

[0014] Furthermore, the microemulsion method described in step (3) is as follows: taking the aqueous phase and the oil phase separately, slowly adding the oil phase to the aqueous phase to prepare uniformly distributed microspheres, stirring at a speed of 100 to 400 rpm for more than 1 to 3 hours, dialysis filtering through a dialysis bag in a PBS phosphate buffer solution, and then freeze-drying and storing to obtain the Mat@CS oil / water (O / W) microcapsules.

[0015] Furthermore, the pH of the PBS phosphate buffer solution is 7.4.

[0016] Furthermore, the aqueous phase comprises glacial acetic acid, CS, and deionized water, and the oil phase comprises lecithin, anhydrous ethanol, and Mat.

[0017] Furthermore, the mass of the 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 10-30 mL, preferably 20 mL, and stirring is carried out in an ice-water bath at a stirring speed of 100-400 rpm and a stirring time of 40-50 h, preferably 48 h.

[0018] According to a second aspect of the present invention, an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent is provided. The pH-responsive Mat@CS-Pro nanocapsule antifouling agent is prepared by the preparation method according to any of the above aspects.

[0019] Furthermore, the pH-responsive Mat@CS-Pro nanocapsules are roughly spherical, with a particle size between 200 and 500 nm. These capsules exhibit intelligent pH-responsive properties. Under alkaline marine conditions, the CS can fully maintain the antibacterial activity of the Mat within. However, when microbial proliferation causes acidification of the surrounding area, the microcapsule structure changes, releasing the Mat and D-Proline, achieving intelligent responsive antifouling properties.

[0020] According to a third aspect of the present invention, there is provided an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent for use in marine environment decontamination.

[0021] Furthermore, the pH value range of the marine environment in which the pH-responsive Mat@CS-Pro nanocapsule antifouling agent is applied is 8.0 to 8.3.

[0022] Beneficial effects of the present invention:

[0023] The present invention prepares O / W structured Mat@CS-Pro nano-microcapsules by a microemulsion method to allow Mat to exist stably in seawater, reducing the possibility of oxidation reaction and saponification reaction between Mat and O2 in the external environment. Mat@CS-Pro nano-microcapsules show excellent antibacterial properties against S.aureus, P.aeruginosa and Escherichia coli (E.coli). The capsules demonstrate intelligent pH-responsive controlled-release antibacterial and biofilm formation inhibition. In the marine environment, secretions produced by bacterial metabolism can cause the local pH value to decrease. Due to the protonation process of the amino group in the Mat@CS-Pro capsule shell CS, electrostatic repulsion occurs between CS molecules, causing the capsule to swell and release Mat to kill 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 repulsion, which promotes the release of D-proline and inhibits the formation of biofilm, showing the dual efficacy of capsule sterilization and synergistic anti-biofilm. At higher pH levels, the amino group deprotonates, the capsule shrinks, and the Mat is not released. This prevents the Mat from direct contact with the alkaline environment, reducing the possibility of saponification and denaturation of the Mat's lactam structure. This ensures the long-term stability of the Mat. Furthermore, because D-Proline carries a negative charge in alkaline environments, similar to CS, it can also be released in alkaline environments to inhibit biofilm formation and prevent fouling.

[0024] The present invention prepares an aqueous solution of D-Proline and Mat@CS at a mass ratio of 1-5:1 (preferably 2:1), and stirs the solution in an ice bath to fix the D-Proline on the Mat@CS capsule. This not only improves the antibacterial and antifouling properties of the Mat@CS but also adds the anti-biofilm and antifouling functions of D-amino acids. The D-Proline is adsorbed and fixed to the surface of the Mat@CS through electrostatic action. When the capsule expands under acidic conditions, the Mat@CS and D-Proline are simultaneously released, thereby achieving the dual antibacterial and anti-biofilm functions of the capsule and exhibiting excellent antifouling performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 TEM images of Mat@CS (parts a and b) and Mat@CS-Pro (parts c and d) nanocapsules at different pH values.

[0026] Figure 2 is a schematic diagram of an FTIR spectrum according to an embodiment of the present invention, wherein: Figure 2 Part a 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.

[0027] Figure 3 The numbers of E. coli, S. aureus, and P. aeruginosa of Mat, CS, and Mat@CS nanocapsules in Luria-Bertani broth (LB) and 2216E medium after 18 h of treatment (part a), and the antibacterial effects corresponding to the counting results (part b).

[0028] Figure 4 The numbers of E. coli, S. aureus, and P. aeruginosa after 18 hours of treatment with Mat@CS nanocapsules, D-Proline, and Mat@CS-Pro nanocapsules, respectively, are shown (part a), as well as the antibacterial effect diagram corresponding to the colony count results (part b).

[0029] Figure 5 SEM images of untreated E. coli (part a) and E. coli treated with Mat@CS-Pro nanocapsules (part a1); untreated S. aureus (part b) and S. aureus treated with Mat@CS-Pro nanocapsules (part b1); untreated P. aeruginosa (part c) and P. aeruginosa treated with Mat@CS-Pro nanocapsules (part c1).

[0030] Figure 6These are the SEM images of the biofilms on the corresponding test pieces after E. coli (ac parts), S. aureus (a1-c1 parts) and P. aeruginosa (a2-c2 parts) were cultured in culture medium with and without the addition of Mat@CS and Mat@CS-Pro nanocapsules.

[0031] Figure 7 yes Figure 6 Thickness of biofilms formed by E. coli (parts ac), S. aureus (parts a1-c1), and P. aeruginosa (parts a2-c2) after culturing in the presence of Mat@CS and Mat@CS-Pro nanocapsules for 3 days.

[0032] Figure 8 TEM images of Mat@CS nanocapsules at pH 5 (part a) and pH 8 (part b); TEM images of Mat@CS-Pro nanocapsules at pH 5 (part c) and pH 8 (part d).

[0033] Figure 9 Figure 3 is the standard curve of Mat in PBS solution (part a); the concentration of Mat released from Mat@CS-Pro nanocapsules in PBS solutions with different pH values ​​for 10 h (part b); the diameter of Mat@CS-Pro nanocapsules after treatment in PBS solutions with different pH values ​​(part c); the optical density (OD) values ​​of three different types of bacterial strains after culture in LB and PBS solutions with different pH values ​​(volume ratio 1:1) containing Mat@CS-Pro nanocapsules for 4 h (part d).

[0034] Figure 10 These are photos of colony plates showing Mat@CS-Pro against E. coli (fractions ad), S. aureus (fractions a1-d1) and P. aeruginosa (fractions a2-d2) in pH 5, 6, 7 and 8 culture media. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments.

[0036] The following embodiments are merely exemplary and can only be used to explain and illustrate the technical solutions of the present invention, and should not be construed as limiting the technical solutions of the present invention.

[0037] The technical solution of the present invention provides an environmentally friendly pH-responsive Mat@CS-Pro nanocapsule antifouling agent and a preparation method thereof. Natural organic matrine (Mat) is used as the core material, and natural polymer chitosan (CS) is used as the wall material. CS-encapsulated Mat microcapsules are prepared by microemulsion polymerization, and D-proline (D-Proline) is fixed to the capsules by cold bath stirring, ultimately preparing a high-performance intelligent pH-responsive antibacterial / biofilm antifouling agent.

[0038] CS, a product derived from the deacetylation of chitin, a substance found in crustaceans, exhibits excellent antibacterial properties. Furthermore, as a polysaccharide, CS possesses a wealth of biological and physical properties, including biodegradability, biocompatibility, and nontoxicity. Microcapsules prepared using it as a shell exhibit typical pH responsiveness. Under acidic conditions, the numerous amino groups in the CS side chains carry a positive charge, causing them to protonate. Due to electrostatic repulsion, the microcapsules expand, opening and expanding their pores. In alkaline environments, deprotonation occurs, causing the microcapsules to shrink, with the pores shrinking or even closing. The marine environment is typically alkaline, and the secretions of bacteria that colonize the substrate, such as lactic acid, produced by their metabolism, lower the pH of the surrounding environment, promoting microcapsule expansion and accelerating drug release, resulting in a bactericidal effect. Conversely, when the surrounding bacteria are scarce, the marine environment becomes alkaline, causing the microcapsules to shrink, trapping the drug within the CS and storing it, significantly extending its lifespan. These results demonstrate that microcapsules prepared using CS can store and sustainably release drugs, achieving delayed release of antimicrobial agents and pH-responsive sterilization, while also improving drug stability and maximizing their bactericidal effect. Furthermore, CS is biodegradable, environmentally friendly, and non-toxic to organisms, achieving environmentally friendly applications. This promising technology has promising applications in areas such as food safety, public health, and medical devices.

[0039] Mat is an alkaloid extracted from the dried roots, plants, and fruits of the leguminous plant Sophora flavescens, using organic solvents such as ethanol. It possesses a wide range of pharmacological effects and efficacies, including antibacterial, anti-inflammatory, anti-rheumatic, anti-tumor, anti-allergic, antiviral, antiparasitic, anti-arrhythmic, anti-swelling and diuretic, as well as immune and biological response modulatory properties. Mat is a plant-derived pesticide characterized by its specificity and natural properties. It only affects specific organisms and rapidly decomposes in nature, ultimately producing water and carbon dioxide. The combined action of multiple chemical substances makes it less likely to induce resistance in pests and allows for long-term use. Therefore, Mat is distinct from conventional highly toxic and residual chemical pesticides and is highly environmentally friendly. However, due to its lactam structure, it is susceptible to saponification in alkaline environments, causing its structure to break down. Therefore, increasing Mat's stability and minimizing its exposure to alkaline environments are key to enhancing its antimicrobial stability and long-term efficacy.

[0040] D-amino acids, as enantiomers of L-amino acids, are produced from racemates. Due to their biodegradability, non-toxicity, and availability, they are widely used in drug synthesis, enzyme structure and function analysis, and biocide applications. After years of experimental research, the applicant has discovered that their application in antifouling agents can achieve antifouling effects by inhibiting biofilm formation.

[0041] Therefore, the present invention uses Mat as a natural antimicrobial agent. By coating CS with Mat, pH-responsive antibacterial microcapsules are prepared. D-Proline is then immobilized on the capsule surface to create environmentally friendly, pH-responsive antimicrobial / biofilm-resistant antifouling capsules. When the solution environment is acidic, the amino groups on the CS capsules become protonated. Due to electrostatic repulsion, the Mat@CS microcapsules swell, opening and expanding the pores. The antimicrobial agent is released from the capsules to kill bacteria. Simultaneously, D-Proline, positively charged because its charge is below its isoelectric point, shares the same charge with CS. This electrostatic repulsion promotes D-Proline release and inhibits biofilm formation. When the solution environment is alkaline, deprotonation occurs, causing the microcapsules to shrink, the pores to shrink and close, and the antimicrobial agent to be prevented from being released. The Mat is enclosed, reducing the possibility of saponification and denaturation of the Mat's lactam structure from direct exposure to the alkaline environment. This allows for long-term stable storage of the Mat within the capsule, enhancing its stability. Furthermore, because D-Proline carries a negative charge in alkaline environments, similar to CS, it can also be released in alkaline environments to inhibit biofilm formation and provide antifouling protection. Normally, secretions such as lactic acid produced by bacterial metabolism lower the pH of the surrounding environment, promoting microcapsule expansion and accelerating the release of Mat and D-Proline, thus achieving a bactericidal effect. Conversely, when the surrounding bacteria are sparse, the alkaline environment causes the microcapsules to shrink, allowing the Mat to be stored. This demonstrates that Mat@CS-Pro microcapsules can be stable in marine environments and effectively store the bactericidal ingredients. When bacteria are enriched and colonized, causing the pH of the microenvironment to decrease, Mat@CS-Pro microcapsules can effectively release Mat and D-proline, achieving the purpose of antifouling agent sterilization and synergistic inhibition of biofilm formation. The added Mat, CS, and D-Proline are all biodegradable, and are non-polluting to the environment and non-toxic to organisms, achieving the goal of green and environmentally friendly antifouling agent.

[0042] The preparation method comprises the following steps:

[0043] Mat was dissolved in anhydrous ethanol containing lecithin to prepare the oil phase, and CS was dissolved in anhydrous acetic acid to prepare the aqueous phase. Mat@CS O / W microcapsules were prepared by a microemulsion method. After the aqueous and oil phases became miscible, the aqueous phase began to coat the oil phase, forming a capsule structure with CS as the coating and Mat as the core. The capsules were stirred at 100-400 rpm for approximately 2 hours, dialyzed through a dialysis bag, and then freeze-dried for storage. 20-40 mg of D-Proline was dissolved in 20 mL of aqueous solution, followed by the addition of 10-20 mg of Mat@CS. The mixture was stirred in an ice bath at 100-400 rpm for approximately 50 hours. Free D-Proline was dialyzed against deionized water. The Mat@CS-Pro nanocapsules were frozen at -50°C in a vacuum freezer for approximately 40 hours and then collected.

[0044] The amount of CS is 10 mg, and the volume of acetic acid aqueous solution (1%, v / v) is 10-30 mL. The microemulsion method involves forming microdroplets from the Mat solution through a liquid-liquid homogeneous nucleation process. After blending with the CS solution, the Mat droplets are encapsulated within the CS to form microcapsules. During this process, lecithin, an emulsifier, participates in the liquid-liquid homogeneous nucleation process and facilitates microcapsule formation. Ultimately, CS-coated Mat microcapsules are self-assembled according to the microemulsion O / W mechanism. The surfactant reduces the tension at the oil / water interface, even generating a transient negative interfacial tension. Consequently, the system spontaneously expands the interface until the interfacial tension returns to zero or a small positive value, forming a microemulsion.

[0045] The CS-coated Mat has a stable coating structure. In an alkaline environment, the CS packaging material deprotonates, the capsule shrinks and the channels close, preventing the release of Mat. Therefore, the Mat can remain stable in the alkaline seawater environment for a long time.

[0046] D-Proline is adsorbed onto the surface of Mat@CS capsules through electrostatic action. While Mat@CS exerts its antibacterial effect, D-Proline also has the property of resisting biofilm formation, realizing the marine antifouling function of pH-responsive antibacterial and synergistic anti-biofilm dual effects.

[0047] The pH-responsive Mat@CS-Pro capsules feature intelligent pH-responsive controlled release, enhancing the stability of the Mat in marine environments. In marine environments, secretions produced by bacterial metabolism can cause a local decrease in pH. The protonation of amino groups in the capsule shell (CS) causes the capsule to expand, releasing the Mat to kill bacteria. At higher pH levels, the amino groups deprotonate, causing the capsule to shrink and seal the Mat. This reduces the potential for saponification and denaturation of the Mat due to its lactam structure, which could be directly exposed to alkaline environments. This allows for long-term stable storage of the Mat within the capsule, enhancing its bactericidal stability.

[0048] Example 1

[0049] (1) Prepare an O / W microcapsule solution using a microemulsion method. Use a pipette to dissolve 32 mg of Mat in 30 mg / mL lecithin-containing anhydrous ethanol (400 μL) to prepare an oil phase. Dissolve CS (10 mg) in 20 mL of 1% aqueous acetic acid solution (v / v) to prepare an aqueous phase. Slowly pour the oil phase into the aqueous phase and stir the mixed solution at room temperature with a magnetic stirrer at 200 rpm for 2 h until opalescence occurs, thereby obtaining a microcapsule solution.

[0050] (2) The microcapsule solution was transferred to a dialysis bag and immersed in a dialysate (PBS phosphate buffer solution with a pH value of 7.4) for 12 hours to eliminate free Mat. The microcapsule solution was placed in the freezer of a refrigerator for 2 hours. After solidification, it was sent to a freeze dryer and freeze-dried at -50°C for 36 hours to obtain microcapsule powder.

[0051] (3) 40 mg of D-Proline was dissolved in 20 mL of aqueous solution. 20 mg of Mat@CS lyophilized powder was then added to the solution and stirred at 200 rpm in an ice bath for 48 h. Free D-Proline was eliminated by dialysis against aqueous solution. Mat@CS-Pro nanocapsules were collected after freezing at -50°C in a vacuum freezer for 36 h.

[0052] (4) The antibacterial properties of Mat@CS-Pro nanocapsules were analyzed by colony counting method. E. coli and S. aureus were selected as representatives of Gram-negative and Gram-positive strains, and P. aeruginosa was selected 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 culture at 120 rpm in a constant temperature device at 37°C for 18 hours. Transfer 1 mL of P. aeruginosa suspension to 50 mL of sterile 2216E culture medium and culture at 30°C for 18 hours. Thereafter, transfer 200 μL of bacterial suspension to 8 mL of LB or 2216E culture medium containing 2 mg / mL of different antibacterial agents (i.e., CS, Mat, D-Proline, Mat@CS nanocapsules, Mat@CS-Pro nanocapsules). After culturing at 37°C or 30°C for 18 hours, take 20 μL of the diluted bacterial suspension and apply it to a solid culture medium plate and culture overnight under the corresponding temperature conditions. The antibacterial results are calculated according to the following formula:

[0053]

[0054] Where A is the number of colonies in the control group and B is the number of colonies in the treatment group. In addition, the OD curve was detected at 600 nm by UV spectrophotometer as a reference for evaluating the antibacterial properties of the nanocapsules.

[0055] The OD value was measured at 600 nm using an ultraviolet spectrophotometer to provide a reference for evaluating the antibacterial properties of the nanocapsules.

[0056] (5) The anti-adhesion properties of Mat@CS-Pro nanocapsules were evaluated by SEM. 316L stainless steel specimens (1 cm × 1 cm) were used to assist in evaluating the anti-adhesion properties of the prepared nanocapsules. The specimens were polished with 1200 mesh silicon carbide paper, then ultrasonically cleaned, cleaned with acetone and ethanol, and blown dry with N2. The metal specimens were sterilized using ultraviolet light. 1 mL of E. coli or S. aureus suspension was added to 50 mL of sterile LB and shaken at 120 rpm for 18 h at 37 °C. 1 mL of P. aeruginosa suspension was added to 50 mL of sterile 2216E culture medium and shaken at 120 rpm for 18 h at 30 °C. Each specimen was placed in a 24-well plate, and then injected with 4 mL of LB culture medium (containing 2 mg / mL of Mat@CS or Mat@CS-Pro nanocapsules) and 100 μL of bacterial suspension. After 1 and 3 days of static incubation, the samples were removed and washed with deionized water to remove free-floating bacteria on the metal surface. They were then fixed with 2.5% (v / v) glutaraldehyde in phosphate-buffered saline (PBS, pH 7.4) for 2 hours. Subsequently, each sample was washed three times with PBS and three times with deionized water, and dehydrated using a gradient of 30%, 50%, 70%, 90%, and 100% (v / v) ethanol for 15 minutes. After carbon spraying, the bacterial membranes on the metal surface were observed using a scanning electron microscope.

[0057] In addition, using Live / The anti-adhesion properties of Mat@CS-Pro nanocapsules were further analyzed using the BacLight™ Bacterial Viability Kit. Each sample was immersed in a different bacterial suspension containing nanocapsules and cultured for one and three days. 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.

[0058] (6) A suspension of Mat@CS-Pro nanocapsules (2 mg / mL) was placed in a dialysis bag and immersed in PBS solutions of pH 5, 6, 7, and 8 for 12 h. The dialyzed solution was measured every hour by UV-Vis spectroscopy, and the peak at 220 nm represented Mat. The size of the Mat@CS-Pro nanocapsules after treatment in PBS solutions of different pH values ​​was determined by DLS.

[0059] (7) The colony plate method was used to evaluate the pH-responsive antibacterial properties. A 2 mg / mL Mat@CS-Pro nanocapsule culture medium consisting of 4 mL of PBS solution with different pH values ​​(pH 5, 6, 7, 8) and 4 mL of LB medium (or 2216E) was prepared. 200 μL of each bacterial suspension was inoculated into 8 mL of culture medium and cultured at 37°C or 30°C for 4 h. 20 μL of the diluted bacterial suspension was plated on a solid culture medium and cultured at the corresponding temperature for 18 h. The OD of each bacterial suspension after 4 h of culture 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.

[0060] Example 2 (Effect Example)

[0061] Synthesis and performance testing of Mat@CS nanocapsules:

[0062] (1) Characterization of Mat@CS-Pro nanocapsules

[0063] The surface structure and morphology of the prepared nanocapsules before and after D-Proline fixation were observed by transmission electron microscopy. Figure 1 As shown. Figure 1 It can be seen that the Mat@CS nanocapsules prepared by the O / W microemulsion method have uniform particle size, with an average particle size of about 250nm. After fixing D-Proline, the capsules did not agglomerate, and the particle size increased slightly ( Figure 1 However, the morphology of Mat@CS-Pro is different from that of Mat@CS nanocapsules, which show a large number of small droplets inside the nanocapsules ( Figure 1 Part b). According to the Zeta potential of CS, Mat, and Mat@CS nanocapsules of 37.8±1.1mV, -16.9±2.4mV, and 31.9mV, respectively, it can be inferred that the droplets in the nanocapsules should be Mat, because the surface negative charge of the free Mat droplets is completely covered by the cationic CS. In addition, the surface of the Mat@CS nanocapsules is covered with irregularly shaped substances ( Figure 1 (Part d) The zeta potentials 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 can be immobilized on the surface of Mat@CS nanocapsules. Therefore, D-Proline is immobilized on the surface of Mat@CS nanocapsules, which have a diameter of approximately 280 nm.

[0064] FTIR of CS, Mat and prepared Mat@CS nanocapsules Figure 2As shown in part a. For the CS line, it is located at 3500-3200cm -1 The broad peak at 1157 cm is attributed to the stretching vibration of NH and OH. -1 and 1091cm -1 The peak belongs to COC stretching vibration. For Mat spectrum, 2935cm -1 and 2853cm -1 The peak belongs to the CH stretching vibration of CH2, 1635cm -1 The peak belongs to C=O stretching vibration. The characteristics of the spectrum of Mat@CS-Proline nanocapsule product are similar to the CS spectrum. The spectrum of Mat@CS also appears around 3500-3200 cm -1 The broad peak at 1157 cm appears on Mat@CS, which belongs to the stretching vibration of NH and OH. -1 The peak of 1085cm in the spectrum of Mat@CS belongs to the stretching vibration of COC. -1 The peak (COC stretching vibration) corresponds to 1019 cm on the CS spectrum. -1 The peak indicates the presence of CS in the prepared capsules. At the same time, the relevant characteristic peaks of Mat can also be found in Mat@CS, such as the 1635cm peak in the spectrum due to C=O stretching vibration. -1 The peak is consistent with the pure Mat peak, and its intensity is weaker than the Mat spectrum. The 2935cm peak in the Mat spectrum is caused by CH stretching vibration. -1 and 2853cm -1 The peak positions correspond to 2930cm of Mat@CS. -1 and 2858cm -1 and 2875cm on CS caused by CH stretching vibration -1 The peak is covered by these two peaks. That is to say, Mat also exists in the prepared nanocapsules. In addition, the peak at 1737cm -1 The new peaks indicate the existence of H-bonding between CS and Mat. Figure 1 The TEM results shown indicate that the Mat@CS nanocapsules were successfully synthesized.

[0065] After the capsules were loaded with D-Proline, the product components were analyzed by FTIR. The results were as follows: Figure 2 As shown in part b. For the spectrum line of D-Proline, 3051cm -1 and 2979cm -1 The peaks are generated by the asymmetric stretching and symmetric stretching vibrations of the CH bond. In the D-Proline spectrum, the peaks are located at 1600~1450cm -1The peak of the spectrum is related to the vibration of the skeleton ring, and a strong peak also appears at a similar position on the spectrum of the synthesized Mat@CS-Pro nanocapsules. At the same time, the 848cm peak on the D-Proline spectrum is caused by the rocking vibration of CH2. -1 and 791cm -1 The peaks at 856cm correspond to the Mat@CS-Pro spectrum line. -1 and 773cm -1 In addition, the 1164cm peak of Mat@CS nanocapsules is caused by COC vibration. -1 and 1085cm -1 The peak at also appears on the Mat@CS-Pro spectrum. Figure 1 The TEM results shown indicate that D-proline was successfully immobilized on the Mat@CS-Pro nanocapsules.

[0066] (2) Characterization of the antibacterial properties of Mat@CS-Pro nanocapsules

[0067] Figure 3 The colony counts and antibacterial effects of CS, Mat and Mat@CS nanocapsules on E.coli, S.aureus and P.aeruginosa are shown in the figure. Figure 3 In part a, the number of bacteria after treatment with these three drugs was significantly lower than that of the blank control group (379 CFU), indicating that CS, Mat and Mat@CS nanocapsules have significant antibacterial effects on these three strains. Figure 3 Part b shows the antibacterial effects of the three drugs on E. coli, S. aureus and P. aeruginosa, respectively, among which the bactericidal rates against P. aeruginosa exceeded 87.71%, 77.90% and 73.39%, respectively. It is worth noting that pure Mat and Mat@CS nanocapsules have better antibacterial effects on Gram-positive bacteria than Gram-negative bacteria, and the worst antibacterial effect on P. aeruginosa. 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 the corresponding bactericidal rates of S. aureus. Compared with pure Mat, Mat@CS nanocapsules showed better antibacterial properties against E. coli, S. aureus and P. aeruginosa. The colonies of P. aeruginosa were reduced by 8 CFU, 6 CFU and 39 CFU, respectively. From Figure 3The results in part b show that 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 properties of Mat@CS nanocapsules were significantly improved after being coated with CS.

[0068] The colony counting method further demonstrated the antibacterial effect of Mat@CS-Pro nanocapsules. Figure 4 As shown. Figure 4 As shown in part (b), the antibacterial effect of pure D-Proline is not ideal. The inhibition rates against E. coli, S. aureus, and P. aeruginosa are approximately 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 inhibition rate of Mat@CS-Pro nanocapsules against S. aureus decreased from 94.55% to 87.75%, likely due to the poor antibacterial properties of D-Proline's nanocapsule structure against S. aureus. Coating D-Proline on the surface of Mat@CS-Pro may hinder the release of Mat, and the surface-localized D-Proline cannot provide significant antibacterial activity, resulting in a slight reduction in the antibacterial performance of Mat@CS-Pro. Overall, after the introduction of D-Proline, Mat@CS-Pro nanocapsules can almost maintain excellent antibacterial performance against Gram-positive bacteria and even exert better antibacterial performance against Gram-negative bacteria.

[0069] After the sterilization treatment with Mat@CS-Pro nanocapsules, the morphology of E. coli, S. aureus, and P. aeruginosa was observed using a scanning electron microscope. Compared with the control group, the bacterial cell membrane almost lost its complete structure, indicating that the nanocapsules caused irreversible damage to the bacterial cells. In addition, compared with the morphology of S. aureus, the cell surface of E. coli and P. aeruginosa was covered with a large amount of organic matter, which proves that the effect of Mat@CS-Pro nanocapsules on Gram-negative strains is stronger than that on Gram-positive strains. These results are consistent with Figure 4 The colony count results shown are consistent.

[0070] (3) Biofilm dispersibility of Mat@CS-Pro nanocapsules

[0071] The effects of Mat@CS and Mat@CS-Pro nanocapsules on biofilm formation after 3 days of action were evaluated by SEM. In the control group, the most bacteria adhered to the metal surface and aggregation occurred ( Figure 6 After incubation with Mat@CS nanocapsules, the number of bacterial cells and aggregation on the substrate surface were slightly reduced ( Figure 6 b-b2 in the Figure 6 In the c-c2 part, after the introduction of D-Proline into the Mat@CS nanocapsules, the number of bacteria and the aggregation phenomenon were further reduced. As can be seen from the figure, most of the bacterial cells are randomly dispersed on the surface of the substrate in the form of single cells. It can also be seen that the bacterial morphology in the bacterial aggregation area is significantly different from that in the control group. When the nanocapsules are not added to the culture medium, the bacteria have a complete cell morphology and the cell membrane has a clear edge. However, for the surface morphology of the substrate with the addition of Mat@CS-Pro, especially the aggregation area, the bacterial morphology is different from that in the control group. Figure 5 The a1-c1 parts are similar, that is, the bacteria are clustered together ( Figure 6 (section c-c2 in the figure).

[0072] After 3 days of culture, the formed biofilm was evaluated using IPCM. Figure 7 As shown. The green and red areas in the field of view represent live bacteria and dead bacteria, respectively. As can be seen from the figure, the control group is almost entirely 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 thickness of the E. coli, S. aureus and P. aeruginosa biofilms are approximately 30μm, 39μm and 48μm, respectively. Figure 6 (a-a2 in the figure). After incubation with Mat@CS and Mat@CS-Pro nanocapsules, many red areas appeared in the visual field, indicating the presence of a large number of dead bacteria. In addition, after the introduction of D-Proline into Mat@CS nanocapsules, the proportion of green areas further decreased, indicating that the antibacterial effect of Mat@CS-Pro nanocapsules was better than that of Mat@CS nanocapsules, which is consistent with the Figure 4 The results are consistent with those in Figure 6 In the c-c2 section, after three days of incubation with Mat@CS-Pro nanocapsules, the biofilms formed were approximately 20 μm thick for E. coli, 10 μm thick for S. aureus, and 28 μm thick for P. aeruginosa. The Mat@CS-Pro nanocapsules significantly improved their biofilm dispersibility, reducing E. coli, S. aureus, and P. aeruginosa biofilms by approximately 33%, 74%, and 42%, respectively, compared to the control group. Therefore, the introduction of D-Proline, in addition to its existing antimicrobial properties, helps enhance the biofilm dispersibility of the antifouling agent.

[0073] (4) pH-controlled release and antibacterial properties of Mat@CS-Pro nanocapsules

[0074] The morphology and structure of Mat@CS nanocapsules and Mat@CS-Pro nanocapsules treated in PBS solutions at pH 5 and pH 8 were characterized by TEM. Under acidic conditions, the -NH2 group of CS is converted into -NH3 + , resulting in positive charge and internal electrostatic repulsion in the system. Therefore, after immersion at pH 5, the Mat@CS nanocapsules expanded from 280 nm to about 430 nm ( Figure 8 In the marine environment (pH 8), the Mat@CS nanocapsules shrink to about 220 nm due to the deprotonation of the CS amino group, as shown in Figure 2. Figure 8 As shown in part b of Figure 2, after D-Proline was fixed, the pH response characteristics of the nanocapsules still existed. Figure 8 In part c of Figure 8 and part d of Figure 8, the diameters of the Mat@CS-Pro nanocapsules after treatment in alkaline and acidic PBS solutions were approximately 475 nm and 234 nm, respectively. These results indicate that the Mat@CS-Pro nanocapsules are pH-responsive, and their diameters can change with changes in the ambient pH.

[0075] The pH controlled release behavior and mechanism of Mat@CS-Pro nanocapsules were determined by UV-Vis and DLS analysis. Figure 9 The release concentration of Mat from Mat@CS-Pro nanocapsules calculated in part a) is as follows Figure 9 As shown in part b. 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 was the smallest after immersion in PBS solution with a pH of 8, which was about 236±13nm. In contrast, the release of Mat in acidic solution continued to increase. After immersion in pH 5 and pH 6 solutions for 10h, the concentrations of Mat were approximately 28.5ppm and 23.5ppm, respectively. Compared with the initial state, the release concentrations of Mat increased by 16.8ppm and 10.3ppm, respectively. Figure 9 In the middle part c, the diameters of the nanocapsules treated in pH 5 and pH 6 solutions were 478 ± 18 nm and 396 ± 21 nm, respectively, which is consistent with the Figure 8The results are consistent with those of the previous study. Compared with the corresponding nanocapsules treated at pH 8, the diameters of the nanocapsules increased by approximately 242 nm and 160 nm, respectively. At pH 7, the release of Mat within 10 h showed an upward trend, with the release amount reaching 7.6 ppm after 1 h, 9.4 ppm after 5 h, and further increasing to 10.1 ppm after 10 h. The above results indicate that the pH response characteristics of Mat@CS-Pro nanocapsules are not restricted after immobilization of D-Proline. As the pH of the PBS solution increases, the release behavior of Mat and the size of the nanocapsules decrease.

[0076] The effect of Mat@CS-Pro nanocapsules on the bacterial growth of E. coli, S. aureus and P. aeruginosa in different pH media was evaluated by UV-Vis. The bacterial concentration increased with the increase of OD value. Figure 9 In the middle d part, it can be seen that the OD values ​​of the three bacterial strains increase with the increase of the pH value of the culture medium, indicating that the number of bacteria in the culture medium increases with the increase of pH value. Specifically, after culture under pH 5, 6, 7 and 8, the OD values ​​of E. coli were approximately 0.69±0.02, 0.70±0.02, 0.75±0.01 and 0.77±0.02, respectively. Figure 10 From a to d in the figure, 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 OD value change trend of S. aureus is similar to that of E. coli, which is 0.68±0.01, 0.69±0.01, 0.72±0.01, and 0.72±0.01, respectively. This result is consistent with 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. Figure 10 In parts a2-d2, after incubation at pH 7 and 8, more colonies were scattered on the solid culture medium, but after incubation at pH 6 and 7, fewer colonies were found on the solid culture medium. These results indicate that the antibacterial activity of Mat@CS-Pro decreases with increasing environmental pH. Figure 9 The results in parts b and c show that Mat@CS-Pro nanocapsules have better antibacterial properties in acidic environments because the nanocapsules are larger in size and easier to release Mat in acidic environments.

[0077] In summary, the technical solution of the present invention first dissolves matrine (Mat) in anhydrous ethanol containing lecithin to prepare an oil phase, dissolves chitosan (CS) in anhydrous acetic acid to prepare an aqueous phase, and prepares an O / W structured microcapsule solution by a microemulsion method, with the capsules being distributed in a spherical shape. By encapsulating CS, Mat, which has poor stability under alkaline conditions such as the ocean, maintains its bactericidal activity. Due to the protonation and deprotonation process of the amino group in the capsule shell CS, the Mat@CS microcapsules expand or contract, the pores open or close, and Mat is released from the capsule or stored without being released. Compared with alkaline environments, the prepared antibacterial capsules have more outstanding antibacterial properties in acidic solutions. In acidic environments, the amino groups on CS are protonated, and electrostatic repulsion occurs between molecules. The microcapsules expand and the pores open to release drugs for sterilization. The deprotonation of the amino groups of CS under alkaline conditions can prevent the release of Mat and increase the stability of the capsule in an alkaline environment. CS and D-Proline have similar isoelectric points. In acidic or alkaline environments, their zeta potentials are identical, resulting in electrostatic repulsion between the molecules. This accelerates the release of D-Proline, allowing Mat@CS-Proline to release both the antibacterial Mat and the D-Proline simultaneously, thus creating a nano-antifouling microcapsule with dual antibacterial and anti-biofilm properties tailored to pH. This environmentally friendly material exhibits superior antibacterial properties, providing insights into the development of intelligent pH-responsive antifouling agents for the marine industry.

[0078] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a pH-responsive Mat@CS-Pro nanocapsule antifouling agent, characterized in that: The preparation method comprises: (1) Dissolving matrine in anhydrous ethanol containing lecithin to prepare an oil phase; (2) dissolving chitosan in a 1% by volume aqueous acetic acid solution to prepare an aqueous phase, wherein the volume of the aqueous acetic acid solution is 10-30 mL; (3) Mat@CS oil / water microcapsules were prepared by the microemulsion method. After the water phase and the oil phase were mixed, the water phase began to cover the oil phase, forming Mat@CS oil / water microcapsules with a chitosan shell and matrine as the core; (4) The prepared Mat@CS oil / water microcapsules were mixed with D-Proline to prepare an aqueous solution, stirred in an ice-water bath, dialyzed and filtered through a dialysis bag in a deionized aqueous solution, and then freeze-dried for storage, thereby obtaining the pH-responsive Mat@CS-Pro nanocapsule antifouling agent.

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

3. The preparation method according to claim 1, characterized in that The amount of chitosan in step (2) is 5-15 mg.

4. The preparation method according to claim 1, characterized in that The microemulsion method in step (3) is as follows: taking the aqueous phase and the oil phase respectively, slowly adding the oil phase into the aqueous phase to prepare uniformly distributed microspheres, stirring at a speed of 100-400 rpm for more than 1-3 h, dialysis and filtration through a dialysis bag in a phosphate buffer solution, and then freeze-drying and storage to obtain the Mat@CS water / oil microcapsules.

5. The preparation method according to claim 4, characterized in that The pH value of the phosphate buffer solution is 7.

4.

6. The preparation method according to claim 4, characterized in that The aqueous phase comprises glacial acetic acid, chitosan and deionized water, and the oil phase comprises lecithin, anhydrous ethanol and matrine.

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

1.

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

9. A pH-responsive Mat@CS-Pro nanocapsule antifouling agent, wherein the pH-responsive Mat@CS-Pro nanocapsule antifouling agent is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the pH-responsive Mat@CS-Pro nanocapsule antifouling agent according to claim 9 in marine environment decontamination, wherein the pH value of the marine environment in which the pH-responsive Mat@CS-Pro nanocapsule antifouling agent is used is in the range of 8.0 to 8.3.

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