CN115245168A - A kind of long-acting antibacterial agent suitable for various substrates, preparation method and application thereof - Google Patents

Abstract

The invention discloses a long-acting antibacterial agent suitable for various substrates and a preparation method and application thereof. The long-acting antibacterial agent is a water-insoluble aggregate formed by co-assembly of a polyphenol compound and a quaternary ammonium salt surfactant under non-covalent interaction. The long-acting antibacterial agent of the invention can effectively enhance the long-acting antibacterial effect of the quaternary ammonium salt surfactant, improve the recyclability of its use, and has a simple preparation method. The antibacterial agent can form a stable antibacterial film on different types of substrates, and the antibacterial film can remain resistant to gram-negative and gram-positive bacteria after being recycled for 20 times and immersed in water for 30 days Efficient and broad-spectrum antibacterial properties with low toxicity to normal cells and good selectivity.

Description

A kind of long-acting antibacterial agent suitable for various substrates, preparation method and application thereof

technical field

The invention belongs to the technical field of antibacterial, and in particular relates to a long-acting antibacterial agent suitable for various substrates and a preparation method and application thereof.

Background technique

So far, a variety of quaternary ammonium salt-type surfactants with different structures have been designed as antibacterial agents. Quaternary ammonium salt surfactants can assemble to form aggregates, which can increase their local concentration and cationic charge density, thus exhibiting excellent antibacterial activity. Unlike traditional antibiotics, the morphology of bacteria is basically maintained after inhibiting or killing bacteria. After quaternary ammonium salt surfactants come into contact with bacteria, firstly, the positively charged head group is bound to the surface of bacteria through electrostatic interaction. Then, the surfactant hydrophobic chain inserts into the bacterial membrane through the hydrophobic interaction with the bacterial membrane, which ruptures the bacterial membrane and leads to the death of the bacteria, so it can effectively inhibit the generation of bacterial drug resistance.

However, due to the good water solubility of quaternary ammonium salt surfactants, they are not resistant to washing in actual production and life, resulting in poor antibacterial stability and long-term effect. Usually after one use, most of the quaternary ammonium salt surfactant is wasted with the discharge of production and domestic sewage. However, the high recovery cost limits the recycling of quaternary ammonium salt surfactants, resulting in a large accumulation of quaternary ammonium salt surfactants in the environment, posing a greater threat to the ecological environment. Therefore, it is urgent to develop new high-efficiency and long-acting antibacterial agents, prolong the sterilization time of quaternary ammonium salt surfactants and improve their recyclability.

SUMMARY OF THE INVENTION

In order to improve the above-mentioned technical problems, the present invention provides an aggregate, which is formed by co-assembly of a polyphenolic compound and a quaternary ammonium salt surfactant through non-covalent interaction;

The quaternary ammonium salt type surfactant is selected from at least one of the structural compounds shown in formula I and the structural compounds shown in formula II,

wherein, R ₁ , R ₁ ′, R ₁ ″, R ₂ , R ₂ ′ and R ₂ ″ are the same or different, and are independently selected from unsubstituted or by one, two or more C _6-14 aryl groups Substituted C _1-8 alkyl;

R ₃ , R ₃ ′ and R ₃ ″ are the same or different, and are independently selected from C _1-18 alkyl;

n is selected from an integer between 2 and 8;

X ^- is selected from Br ^- , F ^- , Cl ^- , I ^- , SO ₄ ^2- , HCOO ^- or HSO ₄ ^- .

According to an embodiment of the present invention, the non-covalent interaction may be at least one of electrostatic interaction, hydrophobic effect, hydrogen bonding, van der Waals force and π-π conjugation effect.

According to an embodiment of the present invention, the polyphenolic compound may be selected from gallic acid, tannic acid, epigallocatechin gallate, anthocyanin, catechin, quercetin, ellagic acid, arbutin , at least one of protocatechuic acid, tea polyphenols and chlorogenic acid.

According to an embodiment of the present invention, the R ₁ , R ₁ ′, R ₁ ″, R ₂ , R ₂ ′ and R ₂ ″ are the same or different, and independently of each other are selected from unsubstituted or by one, two or more phenyl substituted C _1-4 alkyl; R ₃ , R ₃ ' and R ₃ " are the same or different, independently selected from C _1-12 alkyl, n is selected from an integer between 2 and 6; X ^- is selected from Br ^- , F ^- , Cl ^- , I ^- , SO ₄ ^2- , HCOO ^- or HSO ₄ ^- .

According to an embodiment of the present invention, the R ₁ , R ₁ ′, R ₁ ″, R ₂ , R ₂ ′ and R ₂ ″ are the same or different, and independently of each other are selected from unsubstituted or by one, two or more phenyl substituted C _1-4 alkyl; R ₃ , R ₃ ' and R ₃ " are the same or different, independently selected from C _1-12 alkyl, n is selected from an integer between 2 and 6; X ^- is selected from Br ^- , F ^- , Cl ^- , I ^- , SO ₄ ^2- , HCOO ^- or HSO ₄ ^- .

According to an embodiment of the present invention, the quaternary ammonium salt surfactant may be selected from gemini-type quaternary ammonium salt surfactants, such as trimethylene-1,3-bis-dodecyldimethylammonium bromide [ C ₁₂ H ₂₅ N(CH ₃ ) ₂ (CH ₂ ) ₃ (CH ₃ ) ₂ NC ₁₂ H ₂₅ Br ₂ , 12-3-12(Br) ₂ ], [C ₁₂ H ₂₅ N(CH ₃ ) ₂ ( CH ₂ ) ₆ (CH ₃ ) ₂ NC ₁₂ H ₂₅ Br ₂ (ie, 12-6-12(Br) ₂ ); single-headed double-tailed quaternary ammonium salt surfactants, such as diddecyldimethyl bromide ammonium chloride (DDAB); or an oligomeric quaternary ammonium salt surfactant with a degree of polymerization of 3 to 6, such as an oligomeric quaternary ammonium salt surfactant with a degree of polymerization of 3 12-3-12-3-12 (Br ) ₃ , 12-6-12-6-12(Br) ₃ , its molecular formula is shown in formula III:

The present invention further provides the preparation method of the above-mentioned aggregate, comprising the following steps:

(1) after the quaternary ammonium salt surfactant is dissolved in water, the pH is adjusted to obtain the quaternary ammonium salt surfactant aqueous solution;

(2) adjusting the pH after dissolving the polyphenolic compound in water to obtain an aqueous solution of the polyphenolic compound;

(3) mixing the quaternary ammonium salt surfactant aqueous solution and the polyphenolic compound aqueous solution to obtain the aggregate;

The quaternary ammonium salt surfactants and polyphenolic compounds are as defined above.

According to an embodiment of the present invention, in step (1), the pH value of the quaternary ammonium salt surfactant aqueous solution can be 6-8, exemplarily being 6, 7, and 8;

According to an embodiment of the present invention, in step (1), the concentration of the aqueous quaternary ammonium salt surfactant solution may be 0.1-15 mM, for example, 0.1-5 mM, exemplarily 0.1 mM, 0.2 mM, 0.5 mM.

According to an embodiment of the present invention, in step (2), the pH value of the polyphenolic compound aqueous solution may be 4-11, such as 4-9, exemplarily 4, 5, 6, 7, 8, and 9;

According to an embodiment of the present invention, in step (2), the concentration of the polyphenolic compound aqueous solution may be 1-10 mM, such as 1-5 mM, exemplarily 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM.

According to an embodiment of the present invention, in step (3), the volume ratio of the aqueous solution of the quaternary ammonium salt surfactant and the aqueous solution of the polyphenolic compound may be (0.1-10):(0.1-10), for example ( 1-8):(1-8), exemplarily 1:1, 1:2, 1:3, 1:5, 5:1, 3:1, 2:1;

According to an embodiment of the present invention, in step (3), the mixing temperature may be 0-60° C., for example, 20-40° C., exemplarily 25° C.; the mixing time may be 10 seconds-5 minutes , for example 0.5-3 minutes, exemplarily 1 minute.

According to an embodiment of the present invention, step (3) further includes a post-processing step, and the post-processing may be to perform centrifugation, washing and drying on the aggregates in sequence to obtain dried aggregates.

The present invention also provides the application of the above aggregates in the field of antibacterial, such as for antibacterial agents, preferably long-acting antibacterial agents.

The present invention also provides an antibacterial agent comprising the aggregate. Preferably, the antibacterial agent is a long-acting antibacterial agent.

The present invention also provides an antibacterial film comprising the aggregate. Preferably, the antibacterial agent is a long-acting antibacterial film.

The present invention also provides a method for preparing an antibacterial film, which comprises dissolving the aggregate in a solvent, spraying or coating it on the surface of a substrate, and obtaining the antibacterial film on the surface of the substrate after drying.

According to an embodiment of the present invention, the solvent may be an organic solvent or a mixed solvent of an organic solvent and water; for example, the solvent may be an aqueous solution containing 75% ethanol, ethanol, methanol, chloroform, dichloromethane, n-hexane , at least one of ethyl acetate and ether, preferably 75% ethanol in water.

According to an embodiment of the present invention, the concentration of the aggregates dissolved in the solvent may be 1-1000 μg/mL, such as 10-800 μg/mL, exemplarily 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL mL, 500 μg/mL, 600 μg/mL.

According to embodiments of the present invention, the substrate may be plastic, rubber, stainless steel, glass, silica, silicon, mica, metal alloy or cotton; for example, the plastic may be a polypropylene material.

According to an embodiment of the present invention, the amount of the aggregates sprayed or coated on the surface of the substrate may be 2-20 μg/cm ² , such as 2-10 μg/cm ² , exemplarily 4 μg/cm ² , 6 μg/cm 2 ^2. 8 μg/cm ² .

According to an embodiment of the present invention, the drying may be natural volatilization; the drying time may be 20 seconds to 30 minutes, such as 1 to 20 minutes, and exemplarily 5 minutes.

The present invention also provides the use of the aggregates, antibacterial agents or antibacterial films in inhibiting or inactivating bacteria or fungi.

According to an embodiment of the present invention, the bacteria may be Gram-negative bacteria or Gram-positive bacteria;

For example, the Gram-negative bacteria can be Escherichia coli;

For example, the gram-positive bacteria can be Staphylococcus aureus, lactic acid bacteria or streptococcus;

For example, the fungus may be Candida albicans, mold or yeast.

beneficial effect

The invention provides a long-acting antibacterial aggregate, an antibacterial agent/film and a preparation method and application thereof. The antibacterial agent is composed of polyphenolic compounds and quaternary ammonium salt surfactants (especially gemini quaternary ammonium salt surfactants) under non-covalent interactions, such as electrostatic interactions, hydrophobic effects, hydrogen bonding, van der Waals forces Water-insoluble aggregates formed by co-assembly driven by interaction and/or π-π conjugation effect. The aggregates can be effectively deposited on a variety of substrates, effectively enhancing their long-term antibacterial properties and improving their recyclability. The preparation method of the aggregate is simple, the time required is short, and a stable antibacterial film can be formed on different types of substrates. The high-efficiency and broad-spectrum antibacterial properties of Lanseria-positive bacteria and fungi (see Figure 5), while having low toxicity to normal cells and good selectivity.

Description of drawings

Fig. 1 is the particle size distribution diagram of the aggregate of embodiment 1;

Fig. 2 is the electron microscope image of the surface morphology of the aggregate of Example 1 adsorbed on the substrate;

Fig. 3 is the cycle sterilization test result of the substrate coated with the antibacterial agent of embodiment 9 to Escherichia coli;

Fig. 4 is the contact angle result of the water of embodiment 10 on the substrate not coated with antibacterial agent and the substrate coated with antibacterial agent;

Figure 5 is the result of the stability of the antibacterial agent of Example 10 coated on the substrate;

6 is a graph showing the relationship between the coating amount of the antibacterial agent of Example 11 on the substrate on the killing effect of bacteria and fungi;

Fig. 7 is the change diagram of the surface topography of the bacteria and fungi of Example 12 before and after the action with the antibacterial agent-coated substrate;

8 is a graph showing the relationship between the cytotoxicity of the antibacterial agent of Example 13 and its coating amount.

Definition and Explanation of Terms

The term "C _1-18 alkyl" is understood to preferably denote a linear or branched saturated monovalent hydrocarbon radical having 1 to 18 carbon atoms. For example, " _C1-8 alkyl" refers to straight and branched chain alkyl groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl , 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc. or their isomers.

The term "C _6-14 aryl" is to be understood as preferably denoting a monovalent aromatic or partially aromatic monocyclic, bicyclic or Tricyclic hydrocarbon rings ("C _6-14 aryl"), especially rings with 6 carbon atoms ("C ₆ aryl"), such as phenyl; or biphenyl, or those with 9 carbon atoms a ring ("C ₉ aryl") such as indanyl or indenyl, or a ring having 10 carbon atoms ("C ₁₀ aryl") such as tetrahydronaphthyl, dihydronaphthyl or naphthyl, Either a ring with 13 carbon atoms (" _C13 aryl"), such as fluorenyl, or a ring with 14 carbon atoms (" _C14 aryl"), such as anthracenyl. When the _C6-20 aryl group is substituted, it may be monosubstituted or polysubstituted. Also, the substitution site is not limited, for example, it may be ortho-, para- or meta-substitution.

In the present invention, the "long-acting antibacterial" means that the antibacterial agent/film can be recycled at least 10 times (preferably 20 times) and/or soaked in water for at least 15 days (preferably 30 days), and still can maintain a high antibacterial rate and Broad-spectrum antibacterial properties, the antibacterial rate remains above 99%.

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the intended protection scope of the present invention.

Unless otherwise stated, the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods.

"mM" stands for concentration mmol/L.

Example 1

Prepare 3 mM gallic acid aqueous solution with pH 4 and 0.5 mM 12-6-12(Br) ₂ aqueous solution with pH 7, and mix the above two aqueous solutions in a volume ratio of 1:1 at room temperature to form a water-insoluble After the aggregate, the supernatant was discarded by centrifugation, the aggregate was washed three times with water three times, and the powder was obtained by vacuum drying. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.2 mg/mL. The above transparent solution was sprayed on the polypropylene sheet at an amount of 20 μg/cm ² , and after about 10 minutes of natural evaporation of the solvent, an antibacterial film was obtained on the polypropylene surface.

The above aggregates were dissolved in 75% ethanol to prepare a solution with a concentration of 0.5 mg/mL. The results of dynamic light scattering showed that the average Z-average diameter of the aggregates formed by the co-assembly of the system was 220 nm, and the particle size distribution of the aggregates is shown in Figure 1. .

After coating the 75% ethanol solution of the above-mentioned aggregates with a concentration of 0.5 mg/mL on the substrate, the electron microscope image of the surface morphology is shown in Figure 2, indicating that the aggregates can be adsorbed on the surface of the substrate to form a layer composed of a large number of spherical aggregates. Antimicrobial membranes formed by tight junctions.

Example 2

Prepare 2.5mM gallic acid aqueous solution with pH 4 and 0.5mM 12-6-12-6-12(Br) ₃ aqueous solution with pH 7, and mix the above two aqueous solutions in a volume ratio of 3:1 at room temperature, After water-insoluble aggregates were formed, the supernatant was discarded by centrifugation, and the aggregates were washed three times with water and dried in vacuum to obtain powder. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.2 mg/mL. The above transparent solution is sprayed on the cotton cloth, and after about 10 minutes of natural volatilization of the solvent, an antibacterial film is obtained on the surface of the cotton cloth.

Example 3

Prepare 2.5mM gallic acid aqueous solution with pH 4 and 0.1mM 12-3-12(Br) ₂ aqueous solution with pH 7, and mix the above two aqueous solutions in a volume ratio of 5:1 at room temperature to form a water-insoluble solution After the aggregates were obtained, the supernatant was discarded by centrifugation, the aggregates were washed three times with water three times, and the powder was obtained by vacuum drying. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.5 mg/mL. The above transparent solution was sprayed on the rubber, and after about 10 minutes of natural volatilization of the solvent, an antibacterial film was obtained on the surface of the rubber.

Example 4

Prepare 1.5 mM gallic acid aqueous solution with pH 4 and 0.1 mM 12-3-12-3-12(Br) ₂ aqueous solution with pH 7, and mix the above two aqueous solutions in a volume ratio of 2:1 at room temperature, After water-insoluble aggregates were formed, the supernatant was discarded by centrifugation, and the aggregates were washed three times with water and dried in vacuum to obtain powder. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.2 mg/mL. The above transparent solution is sprayed on the silica, and after about 10 minutes of natural volatilization of the solvent, an antibacterial film is obtained on the surface of the silica.

Example 5

Prepare 1 mM tannic acid aqueous solution with pH 9 and 0.1 mM 12-3-12(Br) ₂ aqueous solution with pH 7, and mix the above two aqueous solutions in a volume ratio of 1:3 at room temperature to form a water-insoluble solution After the aggregates were obtained, the supernatant was discarded by centrifugation, the aggregates were washed three times with water three times, and the powder was obtained by vacuum drying. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.5 mg/mL. The above transparent solution was sprayed on the stainless steel sheet, and after about 10 minutes of natural volatilization of the solvent, an antibacterial film was obtained on the surface of the stainless steel sheet.

Example 6

Prepare 1 mM tannic acid aqueous solution with pH 9 and 0.2 mM 12-6-12-6-12(Br) ₃ aqueous solution with pH 7, and mix the above two aqueous solutions in a volume ratio of 1:2 at room temperature. After water-insoluble aggregates were formed, the supernatant was discarded by centrifugation, and the aggregates were washed three times with water and dried in vacuum to obtain powder. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.5 mg/mL. The above transparent solution is sprayed on the mica sheet, and after about 10 minutes of natural volatilization of the solvent, an antibacterial film is obtained on the mica surface.

Example 7

Prepare 2.5mM aqueous solution of epigallocatechin gallate at pH 9 and 0.5mM aqueous solution of 12-6-12(Br) ₂ at pH 7, and mix the above two in a volume ratio of 1:1 at room temperature Aqueous solution, after forming water-insoluble aggregates, the supernatant was discarded by centrifugation, the aggregates were washed three times with water, and the powder was obtained by vacuum drying. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.2 mg/mL. The above transparent solution is sprayed on the glass sheet, and after about 10 minutes of natural volatilization of the solvent, an antibacterial film is obtained on the glass surface.

Example 8

Prepare 2mM aqueous solution of epigallocatechin gallate at pH 9 and 0.1mM aqueous solution of 12-3-12-3-12(Br) ₂ at pH 7 and mix at room temperature in a volume ratio of 1:3 After the above two aqueous solutions formed water-insoluble aggregates, the supernatant was discarded by centrifugation, the aggregates were washed three times with water three times, and the powder was obtained by vacuum drying. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.5 mg/mL. The above transparent solution is sprayed on the silicon wafer, and after about 10 minutes of natural evaporation of the solvent, an antibacterial film is obtained on the surface of the silicon wafer.

Comparative Example 1

Prepare a 3mM, pH 9 gallic acid aqueous solution and a 0.1mM pH 7 DTAB (dodecyl trimethyl ammonium bromide) aqueous solution, and mix the above two aqueous solutions in a volume ratio of 5:1 at room temperature to form After the water-insoluble gallic acid-monoquaternary ammonium salt surfactant complex was obtained, the supernatant was discarded by centrifugation, the aggregates were washed three times with water three times, and the powder was obtained by vacuum drying. Take the dry powder and dissolve it in 75% ethanol to prepare a clear solution with a concentration of 0.5 mg/mL. The above transparent solution was sprayed on the silicon wafer, and after about 10 minutes of natural evaporation of the solvent, a film coated with a gallic acid-monoquaternary ammonium salt surfactant complex was obtained on the surface of the silicon wafer.

Comparative Example 2

An ethanol solution containing 0.5 mg/mL 12-3-12(Br) ₂ with a pH of 5.8 was prepared, and the above solution was sprayed on the silicon wafer. Layers of quaternary ammonium surfactant films.

Example 9 Long-acting antibacterial properties of antibacterial agents

Experimental group: take 25 μL of Escherichia coli suspension (OD=0.2) and respectively react with the PBS solution of the substrate (2cm×2cm) coated with 0.5mg/mL antibacterial agent obtained in Examples 1-8 at 37°C for 30min, The antibacterial activity of the above-mentioned antibacterial films against Escherichia coli at the first use was evaluated by the plate method. Then use three times of water to clean the substrate after the action of Escherichia coli under the rotating speed of 350rpm, and vacuum dry the substrate after cleaning for 30min. Continue to investigate the recycling of the antibacterial film formed by the antibacterial agent on the substrate according to the above experimental conditions after multiple washings. antibacterial activity.

Control group 1: also take 25 μL of E. coli suspension (OD=0.2) and the PBS solution of the substrate (2cm×2cm) coated with 0.5mg/mL of the complex in Comparative Example 1 at 37°C for 30min, pass through the plate method to evaluate the antibacterial activity of the complex against E. coli when it was first used. Subsequently, the substrate after washing with Escherichia coli was washed three times with water at a rotating speed of 350 rpm. After cleaning for 30 min, the substrate was vacuum-dried. Continue to investigate the antibacterial activity of the composite after repeated washings according to the above experimental conditions.

Control group 2: Take 25 μL of Escherichia coli suspension (OD=0.2) and the PBS solution whose surface is only coated with 0.5 mg/mL of gemini quaternary ammonium salt surfactant in Comparative Example 2 (2cm×2cm) at 37 The antibacterial activity of the gemini quaternary ammonium salt surfactant against Escherichia coli was evaluated by the plate method at ℃ for 30min. Subsequently, the substrate after washing with Escherichia coli was washed with three times of water at a rotating speed of 350 rpm. After cleaning for 30 min, the substrate was vacuum-dried. Continue to investigate the antibacterial effect of the cyclic use of the gemini quaternary ammonium salt surfactant after multiple washings according to the above experimental conditions. active.

The antibacterial activity results of the above-mentioned experimental group and control group are shown in FIG. 3 .

It can be seen from Figure 3 that under the same external conditions of the control experiment, the experimental group coated with antibacterial agent on the substrate can still show 99.9% antibacterial activity against Escherichia coli after 10 cycles of use; The control group 1 covered with gallic acid-single-chain quaternary ammonium salt surfactant complex had a great decrease in antibacterial activity after the second cycle of use, and the antibacterial activity dropped to ~51.3%, and after the sixth cycle of use The antibacterial activity against Escherichia coli was completely lost; and the antibacterial activity of the control group 2, which was coated with a gemini quaternary ammonium salt surfactant on the substrate, was greatly reduced after the second cycle of use, and the antibacterial activity dropped to ~ 43.5%, completely lost the antibacterial activity against Escherichia coli after the 5th cycle.

Example 10 Stability of the antibacterial film formed by the antibacterial agent coating on the substrate

Water was dropped three times on the substrates without the antibacterial agent and on the substrates coated with the antibacterial agent prepared in Example 2 at 0.5 mg/mL, respectively, and the size of the contact angle of water on the above two substrates was tested. As shown in Figure 4, the contact angle of water molecules on the substrate without antibacterial agent was 62.2°±0.9°, while the contact angle of water molecules on the substrate coated with antibacterial agent was improved to 88.2°±0.6°, indicating aggregation It adsorbs on the substrate surface and improves the hydrophobicity of the substrate surface.

The substrate coated with the antibacterial agent was further immersed in water three times for one month, and then the contact angle of water molecules on the substrate was tested and the long-acting antibacterial activity of the antibacterial agent was evaluated, as shown in FIG. 5 .

It can be seen from Figure 5 that the water molecule contact angle of the substrate coated with the antibacterial agent after immersion in water for three times for one month is 83.6°±0.2°, which still shows a higher contact angle than that of the substrate without the aggregate coating, which proves that The antibacterial film formed by the antibacterial agent on the substrate has better stability. And the antibacterial film can still maintain >99% antibacterial activity against Escherichia coli after soaking for one month.

Example 11 The relationship between the coating amount of antibacterial agent and the killing effect of bacteria and fungi

25 μL of Escherichia coli suspension (OD=0.2), Staphylococcus aureus suspension (OD=0.2) and Candida albicans suspension (OD=0.4) were respectively coated with the antibacterial agent prepared in Example 2 with different qualities on the above surfaces. The substrate was exposed to PBS solution at 37 °C for 30 min, and the effect of the coating amount of antibacterial agent on the killing effect of three bacteria and fungi was evaluated by the plate method, as shown in Figure 6.

It can be seen from Figure 6 that with the increase of the quality of the antibacterial agent coated on the substrate, its antibacterial activity is also gradually enhanced. The antibacterial agent can achieve 99.9% antibacterial activity against Staphylococcus aureus when the coating amount of the substrate is 0.26μg/cm ² ; the antibacterial agent can achieve 99.9% antibacterial activity against Escherichia coli when the coating amount of the substrate is 0.41 μg/cm ² . % antibacterial activity; the antibacterial agent can reach 99.9% antibacterial activity against Candida albicans when the coating amount of the substrate is 2.04 μg/cm ² .

Example 12 Morphology changes of bacteria and fungi before and after the action with antibacterial agents

The bacterial liquid before and after the action with the antibacterial agent prepared in Example 2 was centrifuged at 7100 rpm for 5 min, the supernatant was removed, and then the bacterial cells were resuspended in sterilized water. Take 5 μL of bacterial droplets on a clean silicon wafer, and immediately immerse the silicon wafer in a 0.1% glutaraldehyde aqueous solution after air-drying in an ultra-clean bench, and fix it in a 4°C refrigerator overnight. The samples were washed twice with sterilized water, and then dehydrated with 50%, 70%, 90% and 100% ethanol gradients for 6 min each time. After the samples were naturally dried, they were vacuum-dried for 2 h and then sprayed with gold. The surface morphology changes of Escherichia coli, Staphylococcus aureus and Candida albicans membranes before and after the action of the antibacterial agent were directly observed by SEM, as shown in FIG. 7 .

As can be seen from Figure 7, before contacting with the substrate coated with the antibacterial agent, the scanning electron microscope image of the control group showed that the cells of Escherichia coli, Staphylococcus aureus and Candida albicans were intact and the edges were clearly visible. On contact with substrates coated with aggregated antimicrobial agents, bacterial and fungal membranes collapsed, ruptured, and internal cytoplasmic leakage led to bacterial and fungal death.

Example 13 Cytotoxicity of antibacterial agents

Experimental group: The antibacterial agents prepared in Example 2 of different quality were coated on a 96-well cell culture plate, and 6 parallel wells were made in each group. Subsequently, HeLa cells in logarithmic growth phase were counted in a cytometer, and seeded in the above-mentioned 96-well cell culture plate treated with antibacterial agents at a cell density of 8×10 ³ cells/well and a final volume of 100 μL in each well. Then put into 5% CO ₂ , 37 ℃ constant temperature incubator for 24 hours.

Control group: Hela cells in logarithmic growth phase were counted in a cytometer, and seeded in a 96-well cell culture plate without any treatment at a cell density of 8×10 ³ cells/well, with a final volume of 100 μL per well. Then put into 5% CO ₂ , 37 ℃ constant temperature incubator for 24 hours to make it adhere to the wall.

After removing the medium of the above experimental group and control group, add 100 μL of 0.5 mg/mL MTT to each well, continue to culture at 37°C for 4 h, remove the supernatant, add 100 μL of DMSO to each well, put the 96-well plate into the enzyme In the standard instrument, shake for 5 minutes to fully dissolve the blue-purple formazan particles, and measure the absorbance at 520 nm. The cell viability was calculated according to the following formula: Cell viability(%)=A/A ₀ ×100, where A ₀ was the absorbance value of the control group, and A was the absorbance value of the experimental group. The relationship between cell viability and antibacterial agent coating amount is shown in Figure 8.

It is known from FIG. 6 that when the content of the antibacterial agent coated on the substrate reaches 2.04 μg/cm ² , it has 99.9% antibacterial activity against Escherichia coli, Staphylococcus aureus and Candida albicans. However, it can be seen from Figure 8 that when the content of the antibacterial agent coated on the substrate is as high as 3.02 μg/cm ² , there is still no obvious cytotoxicity to HeLa cells. This may be because the negative charge on the surface of bacteria and fungi is stronger than that of mammalian cells, and the electrostatic interaction between antibacterial agents and bacteria is stronger than that between them and HeLa cells, so the above antibacterial agents show excellent antibacterial activity without obvious cytotoxicity .

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. an aggregate is characterized in that, the aggregate is formed by co-assembly of polyphenolic compound and quaternary ammonium salt surfactant through non-covalent interaction; The quaternary ammonium salt type surfactant is selected from at least one of the structural compounds shown in formula I and the structural compounds shown in formula II,

wherein, R ₁ , R ₁ ′, R ₁ ″, R ₂ , R ₂ ′ and R ₂ ″ are the same or different, and are independently selected from unsubstituted or by one, two or more C _6-14 aryl groups Substituted C _1-8 alkyl; R ₃ , R ₃ ' and R ₃ ″ are the same or different and independently selected from C _1-18 alkyl, n is selected from an integer between 2 and 8; X ^- is selected from Br ^- , F ^- , Cl ^- , I ^- , SO ₄ ^2- , HCOO ^- or HSO ₄ ^- . 2. The aggregate according to claim 1, wherein the polyphenolic compound is selected from the group consisting of gallic acid, tannic acid, epigallocatechin gallate, anthocyanin, catechin, quercetin , at least one of ellagic acid, arbutin, protocatechuic acid, tea polyphenols and chlorogenic acid; Preferably, the non-covalent interaction is at least one of electrostatic interaction, hydrophobic effect, hydrogen bonding effect, van der Waals force effect and π-π conjugation effect. 3. The aggregate according to claim 1 or 2, wherein the R ₁ , R ₁ ', R ₁ ", R ₂ , R ₂ ' and R ₂ " are the same or different, and are independently selected from each other C _1-4 alkyl unsubstituted or substituted by one, two or more phenyl groups; R ₃ , R ₃ ' and R ₃ " are the same or different, independently selected from C _1-12 alkyl, n is selected from an integer between 2 and 6; X ^- is selected from Br ^- , F ^- , Cl ^- , I ^- , SO ₄ ^2- , HCOO ^- or HSO ₄ ^- . 4. The aggregate according to any one of claims 1-3, wherein the quaternary ammonium salt surfactant can be selected from gemini-type quaternary ammonium salt surfactants, such as trimethylene-1,3 -Bis-dodecyldimethylammonium bromide [C ₁₂ H ₂₅ N(CH ₃ ) ₂ (CH ₂ ) ₃ (CH ₃ ) ₂ NC ₁₂ H ₂₅ Br ₂ , 12-3-12(Br) ₂ ], [C ₁₂ H ₂₅ N(CH ₃ ) ₂ (CH ₂ ) ₆ (CH ₃ ) ₂ NC ₁₂ H ₂₅ Br ₂ (ie 12-6-12(Br) ₂ ); single-head double-tail quaternary ammonium salt Surfactants, such as diddecyldimethylammonium bromide (DDAB); oligomeric quaternary ammonium salt surfactants with a degree of polymerization of 3 to 6, such as oligomeric quaternary ammonium salts with a degree of polymerization of 3 Active agents 12-3-12-3-12(Br) ₃ , 12-6-12-6-12(Br) ₃ , whose molecular formula is shown in formula III,

5. the preparation method of the aggregate described in any one of claim 1-4, is characterized in that, described preparation method comprises the steps: (1) after the quaternary ammonium salt surfactant is dissolved in water, the pH is adjusted to obtain the quaternary ammonium salt surfactant aqueous solution; (2) adjusting the pH after dissolving the polyphenolic compound in water to obtain an aqueous solution of the polyphenolic compound; (3) mixing the quaternary ammonium salt surfactant aqueous solution and the polyphenolic compound aqueous solution to obtain the aggregate; Wherein, the quaternary ammonium salt surfactant and polyphenolic compound have the definition as described in any one of claims 1-4; Preferably, in step (1), the pH value of the quaternary ammonium salt surfactant aqueous solution is 6-8; Preferably, in step (1), the concentration of the quaternary ammonium salt surfactant aqueous solution is 0.1-15mM, for example, 0.1-5mM; Preferably, in step (2), the pH value of the polyphenolic compound aqueous solution is 4-11, for example, 4-9; Preferably, in step (2), the concentration of the polyphenolic compound aqueous solution is 1-10 mM, for example, 1-5 mM; Preferably, in step (3), the volume ratio of the quaternary ammonium salt surfactant aqueous solution and the polyphenolic compound aqueous solution is (0.1-10): (0.1-10), such as (1-8): (1-8); Preferably, in step (3), the mixing temperature is 0-60° C., for example, 20-40° C.; the mixing time is 10 seconds-5 minutes, such as 0.5-3 minutes; Preferably, step (3) further includes a post-processing step, wherein the post-processing is to sequentially perform centrifugation, washing and drying on the aggregates to obtain dried aggregates. 6. The application of the aggregate described in any one of claims 1 to 4 in the field of antibacterial, for example in the preparation of an antibacterial agent, preferably in the preparation of a long-acting antibacterial agent. 7. An antibacterial agent, characterized in that the antibacterial agent comprises the aggregate of any one of claims 1-4; preferably, the antibacterial agent is a long-acting antibacterial agent. 8. An antibacterial film, characterized in that the antibacterial film comprises the aggregate of any one of claims 1-4; preferably, the antibacterial agent is a long-acting antibacterial film. 9. A preparation method of an antibacterial film, characterized in that the preparation method comprises dissolving the aggregate described in any one of claims 1-4 in a solvent, spraying or coating on the surface of the substrate, and drying on the surface of the substrate to obtain the antibacterial film; Preferably, the solvent is an organic solvent or a mixed solvent of an organic solvent and water; the organic solvent is an aqueous solution containing 75% ethanol, ethanol, methanol, chloroform, dichloromethane, n-hexane, ethyl acetate and ether At least one, preferably an aqueous solution of 75% ethanol; Preferably, the concentration of the antibacterial aggregate after being dissolved in the solvent is 1-1000 μg/mL, for example, 10-800 μg/mL; Preferably, the substrate is plastic, rubber, stainless steel, glass, silica, silicon, mica, metal alloy or cotton; the plastic is polypropylene material; Preferably, the amount of the antibacterial aggregates sprayed or coated on the surface of the substrate is 2-20 μg/cm ² , for example, 2-10 μg/cm ² ; Preferably, the drying is natural volatilization; the drying time is 20 seconds to 30 minutes, for example, 1 to 20 minutes. 10. The application of the aggregate described in any one of claims 1-4, the antibacterial agent of claim 7 or the antibacterial film of claim 8 in inhibiting or inactivating bacteria or fungi; Preferably, the bacteria are Gram-negative bacteria or Gram-positive bacteria; Preferably, the Gram-negative bacteria is Escherichia coli; Preferably, the gram-positive bacteria are Staphylococcus aureus, lactic acid bacteria or streptococcus; Preferably, the fungus is Candida albicans, mold or yeast.

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