Zinc oxide nanoparticles / quaternary amine modified polystyrene nanospheres, and preparation method and application thereof

Abstract

The application provides a kind of zinc oxide nanoparticles / quaternary amine modified polystyrene nanospheres and its preparation method and application.The application prepares carboxyl functionalized PS / PVP nanospheres with high monodispersity by environmentally friendly water dispersion polymerization method;ZnONPs are successfully loaded after complexing zinc ions with the surface PVP pyrrolidone ring of PS / PVP nanospheres and reducing with reducing agent, and the surface of nanospheres is modified with quaternary amine groups by amidation and further quaternary amination of the surface carboxyl groups of PS / PVP nanospheres.The preparation method is simple, and the reaction condition is easy to realize.The material obtained by the method of the application is polystyrene nanosphere surface loaded with zinc oxide nanoparticles and grafted with quaternary amine groups;it is applied to antibacterial, and zinc oxide nanoparticles and quaternary amine groups play a synergistic effect, so that the material shows high antibacterial activity and can effectively inhibit the proliferation of escherichia coli and staphylococcus aureus.

Description

Technical Field

[0001] This invention relates to the field of antibacterial agents, specifically to a zinc oxide nanoparticle / quaternary ammonium modified polystyrene nanosphere, its preparation method, and its application. Background Technology

[0002] Bacterial proliferation has serious adverse effects in many fields, including food, construction, and medicine, especially endangering human health. With the improvement of living standards, people's awareness of antibacterial agents has gradually increased, and the research and application of antibacterial materials have received high attention in areas such as food safety, medical care, and industrial production. Due to the overuse of antibiotics in the past, bacterial resistance has become increasingly significant, which has become the biggest obstacle to combating the spread of infectious diseases and chemotherapy treatment. To address this problem, various nanomaterials with broad-spectrum antibacterial capabilities have been developed to combat drug-resistant bacteria, such as metal nanoparticles, metal oxide nanoparticles, and inorganic acid nanoparticles in inorganic antibacterial materials. These are widely used due to their non-polluting nature, strong antibacterial properties, and long-term effectiveness. Among them, zinc oxide nanoparticles (ZnONPs) are non-toxic, have good chemical stability, and are widely used in various fields such as antibacterial agents, catalysts, sensors, electrical and optical devices. Furthermore, they exhibit good biocompatibility with human cells, making them one of the most promising nanomaterials for antibacterial applications.

[0003] The size of ZnONPs is one of the key parameters affecting their antibacterial activity, but strong van der Waals forces and high surface energy cause them to aggregate, leading to a significant loss of antibacterial activity and high toxicity. To avoid these problems, various materials have been used to disperse ZnONPs, such as polymers like polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and polyethylene glycol (PEG), as well as solid carriers such as modified microspheres, zeolite networks, and glass fibers.

[0004] Polysaccharides, biguanides, and quaternary ammonium salts in organic antibacterial materials also play important roles in antibacterial applications. Among them, quaternary ammonium salt (QA) functionalized polymers are one of the most widely studied antibacterial materials. Their potential antibacterial mechanism is due to the adsorption of positively charged cationic groups on the negatively charged bacterial cell surface, disrupting the cell membrane and ultimately leading to cell death. An important class of polymer bactericides is based on quaternary ammonium (QA)-based polymers, where the QA group is a covalent or ionic bond attached to the polymer chain, and exerts antibacterial activity based on contact or release mechanisms. QA-based polymers have been applied in many fields such as medicine, hygiene, food, and agriculture due to their low toxicity, low cost, and broad-spectrum antibacterial activity.

[0005] In recent years, facing the emergence of bacterial resistance and numerous complex environmental factors, antimicrobial materials with single antimicrobial mechanisms are insufficient to address the problems of bacterial transmission and infection. Therefore, researching antimicrobial composite materials with synergistic effects of multiple mechanisms and exhibiting excellent antimicrobial activity is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides zinc oxide nanoparticle / quaternary ammonium modified polystyrene nanospheres, their preparation method, and applications. The preparation method of this invention is simple, and the reaction conditions are easy to achieve. The material obtained by this method is polystyrene nanospheres with zinc oxide nanoparticles loaded on the surface and simultaneously grafted with quaternary ammonium groups. In its antibacterial application, the zinc oxide nanoparticles and quaternary ammonium groups work synergistically, resulting in high antibacterial activity and effectively inhibiting the proliferation of *Escherichia coli* and *Staphylococcus aureus*.

[0007] This invention is achieved through the following technical solution:

[0008] A zinc oxide nanoparticle / quaternary ammonium modified polystyrene nanosphere, wherein the nanosphere is a polymer nanosphere on which zinc oxide nanoparticles are uniformly loaded, and the surface of the polymer nanosphere is also grafted with quaternary ammonium groups, the structural formula of which is as follows; the polymer nanosphere is a styrene-acrylic acid copolymer nanosphere coated with polyvinylpyrrolidone.

[0009]

[0010] According to a preferred embodiment of the present invention, the particle size of the zinc oxide nanoparticles / quaternary ammonium modified polystyrene nanospheres is 180-220 nm; the particle size of the zinc oxide nanoparticles is 5-15 nm.

[0011] The preparation method of the above zinc oxide nanoparticles / quaternary ammonium modified polystyrene nanospheres includes the following steps:

[0012] (1) Dissolve the initiator and polyvinylpyrrolidone (PVP) in deionized water, add styrene and acrylic monomer, disperse fully, then polymerize and dry to obtain polymer nanospheres (PS / PVP);

[0013] (2) The polymer nanospheres were fully dispersed in an anhydrous ethanol solution of Zn source, a reducing agent was added, and after reduction reaction, the polymer nanospheres loaded with zinc oxide (PS / PVP@ZnO) were obtained by centrifugation, washing and drying.

[0014] (3) The zinc oxide-loaded polymer nanospheres were fully dispersed in deionized water to obtain a dispersion. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added, and after activation, the dispersion was redispersed in deionized water after centrifugation and washing. N,N-diethyl-p-phenylenediamine was added, and after amidation, the dispersion was redispersed in anhydrous ethanol after centrifugation and washing. Bromoethane was added, and after quaternization, the dispersion was obtained by centrifugation, washing and drying to obtain zinc oxide nanoparticles / quaternary ammonium modified polystyrene nanospheres (quaternary ammonium PS / PVP@ZnO).

[0015] According to a preferred embodiment of the present invention, in step (1), the initiator is potassium persulfate (KPS); the mass of the initiator is 2-4% of the mass of styrene.

[0016] According to a preferred embodiment of the present invention, in step (1), the mass of polyvinylpyrrolidone (PVP) is 0.01-0.06% of the mass of deionized water.

[0017] According to a preferred embodiment of the present invention, in step (1), the mass ratio of styrene, acrylic acid and polyvinylpyrrolidone (PVP) is 3-4:1:0.02-0.11, preferably 3.6:1:0.072.

[0018] According to a preferred embodiment of the present invention, in step (1), the polymerization reaction conditions are as follows: under inert gas protection and stirring, the polymerization reaction is carried out at 70-90°C for 10-14 hours, followed by a polymerization reaction at 80-90°C for 0.1-1 hours. Preferably, the inert gas is nitrogen or argon.

[0019] According to a preferred embodiment of the present invention, in step (2), the Zn source is Zn(Ac)2·2H2O; the mass ratio of the Zn source to the polymer nanospheres is 2-3:1.

[0020] According to a preferred embodiment of the present invention, in step (2), the concentration of the ethanol solution of the Zn source is 20-40 mmol / L, preferably 30 mmol.

[0021] According to a preferred embodiment of the present invention, in step (2), after adding the Zn source, the mixture is stirred at 55-65°C for 0.5-2 hours to ensure thorough and uniform mixing, thereby ensuring that zinc ions are completely dispersed on the surface of the nanospheres.

[0022] According to a preferred embodiment of the present invention, in step (2), the reducing agent is a NaOH ethanol solution with a concentration of 50-100 mmol / L; the molar ratio of NaOH to Zn source is 2-3:1; and the reducing agent is added to the system dropwise.

[0023] According to a preferred embodiment of the present invention, in step (2), the reduction reaction temperature is 50-70°C, the reduction reaction time is 1-3 hours, and the reduction reaction is carried out under stirring conditions.

[0024] According to a preferred embodiment of the present invention, in step (3), the mass concentration of the dispersion is 0.005-0.1 g / mL.

[0025] According to a preferred embodiment of the present invention, in step (3), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxysuccinimide (NHS) is 0.5-1.5:0.5-1.5, preferably 1:1; the mass ratio of zinc oxide-supported polymer nanospheres to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) is 1:0.5-1, preferably 1:0.78.

[0026] According to a preferred embodiment of the present invention, in step (3), the activation reaction temperature is -5 to 5°C, the activation reaction time is 0.5 to 2 hours, and the activation reaction is carried out under stirring and light-proof conditions to activate the surface carboxyl groups.

[0027] According to a preferred embodiment of the present invention, in step (3), the mass ratio of the zinc oxide-loaded polymer nanospheres to the volume ratio of the deionized water used for redispersibility is 0.005-0.1 g / mL.

[0028] According to a preferred embodiment of the present invention, in step (3), the molar ratio of N-hydroxysuccinimide and N,N-diethyl-p-phenylenediamine is 0.5-1.5:0.5-1.5, preferably 1:1.

[0029] According to a preferred embodiment of the present invention, in step (3), the amidation reaction temperature is room temperature, the amidation reaction time is 10-15 h, and the amidation reaction is carried out under stirring and light-protected conditions.

[0030] According to a preferred embodiment of the present invention, in step (3), the mass ratio of the zinc oxide-loaded polymer nanospheres to the volume ratio of the anhydrous ethanol used for redispersibility is 0.005-0.1 g / mL.

[0031] According to a preferred embodiment of the present invention, in step (3), the molar ratio of N,N-diethyl-p-phenylenediamine and bromoethane is 1:4-6, preferably 1:5.

[0032] According to a preferred embodiment of the present invention, in step (3), the quaternization reaction temperature is 60-70°C, the quaternization reaction time is 10-15 h, and the quaternization reaction is carried out under stirring conditions.

[0033] The above-mentioned application of zinc oxide nanoparticles / quaternary ammonium modified polystyrene nanospheres in antibacterial applications.

[0034] According to a preferred embodiment of the present invention, the zinc oxide nanoparticles / quaternary ammonium modified polystyrene nanospheres are used as an antibacterial agent to inhibit Escherichia coli and / or Staphylococcus aureus.

[0035] Preparation and antibacterial mechanism of zinc oxide nanoparticles / quaternary ammonium modified polystyrene nanospheres in this invention:

[0036] This invention utilizes an environmentally friendly aqueous dispersion polymerization method to prepare carboxyl-functionalized PS / PVP nanospheres with high monodispersity properties, serving as an antibacterial agent carrier. ZnONPs are successfully loaded onto the PS / PVP nanospheres by utilizing the PVP pyrrolidone ring complexation of zinc ions on the surface of the nanospheres followed by reduction with a reducing agent. The surface carboxyl groups of the PS / PVP nanospheres are then amidated and further modified with quaternary ammoniation to impart quaternary ammonium groups to the nanospheres. The coexistence of ZnONPs and quaternary ammonium groups on the surface of the PS / PVP nanospheres prepares a composite antibacterial agent (quaternary ammonium PS / PVP@ZnO).

[0037] The antibacterial mechanism of the quaternary ammonium PS / PVP@ZnO composite antibacterial agent: The antibacterial activity of metal oxides depends on the total contact surface area, while the small-sized ZnONPs on the surface of the quaternary ammonium PS / PVP@ZnO composite nanospheres result in a large total contact surface area with bacteria. Therefore, the loading of ZnONPs gives the quaternary ammonium PS / PVP@ZnO antibacterial agent excellent antibacterial properties. It not only affects biofilm function by inhibiting bacterial acid production through the generation of zinc ions, but also induces bacterial oxidative stress by generating reactive oxygen species on the ZnONPs surface. This is the release-based bactericidal mechanism. In addition, the positively charged quaternary ammonium salt on the surface of the quaternary ammonium PS / PVP@ZnO composite nanospheres adsorbs negatively charged bacterial cells, and its hydrophobic groups insert into the lipid layer of bacterial cells, which can cause changes in cell membrane permeability, disrupt its structure, and cause leakage of intracellular substances. This is the contact-based bactericidal mechanism. The synergistic effect of multiple mechanisms gives the quaternary ammonium PS / PVP@ZnO of this invention excellent antibacterial effects.

[0038] The preparation method and conditions of zinc oxide nanoparticles / quaternary ammonium-modified polystyrene nanospheres of this invention work together as a whole to prepare ZnONPs and quaternary ammonium-modified polystyrene composite nanospheres with the structure and properties of this invention. The ratio of styrene to acrylic acid in the preparation of the polymer nanospheres of this invention needs to be appropriate; a small amount of acrylic acid will result in uneven particle size distribution of the polystyrene nanospheres, while an excessive amount of acrylic acid will cause severe adhesion of the polystyrene nanospheres. The amount of deionized water used in the water dispersion polymerization of this invention needs to be appropriate; as the amount of deionized water increases, the dispersion performance of the polystyrene nanospheres is improved. The ratio of PS / PVP nanospheres to Zn source, the order of ZnONPs modification and quaternization modification steps, and the proportions of raw materials such as EDC, NHS, N,N-diethyl-p-phenylenediamine, and bromoethane all have a certain influence on the properties of the obtained material. If these are not appropriate, the nanospheres with the specific structure and antibacterial properties of this invention will not be obtained.

[0039] The beneficial effects of this invention are as follows:

[0040] The polymer nanospheres (PS / PVP) obtained in this invention exhibit excellent particle size uniformity. The carboxyl groups on their surface prevent small-scale aggregation between particles through electrostatic repulsion, resulting in good dispersibility. Simultaneously, the carboxyl groups provide conditions for quaternization modification of the nanosphere surface. Furthermore, the polyvinylpyrrolidone rings present on the nanosphere surface provide loading sites for zinc oxide nanoparticles. The polystyrene nanospheres loaded with zinc oxide nanoparticles still maintain good dispersibility, and further quaternization modification yields composite nanospheres with highly efficient antibacterial properties.

[0041] The preparation method of this invention is simple, and the reaction conditions are easy to achieve. The zinc oxide nanoparticles / quaternary ammonium-modified polystyrene nanospheres of this invention are applied to antibacterial applications. The zinc oxide nanoparticles and quaternary ammonium groups work synergistically, resulting in high antibacterial activity and effectively inhibiting the proliferation of *Escherichia coli* and *Staphylococcus aureus*. Attached Figure Description

[0042] Figure 1 Infrared spectra (a) of PS / PVP and PS / PVP@ZnO prepared in Example 1, quaternary ammonium PS / PVP@ZnO prepared in Example 6, and quaternary ammonium PS / PVP and ZnO nanoparticles prepared in Comparative Example 1; XRD (b), Zeta potential (c), and particle size distribution (d) of PS / PVP@ZnO prepared in Example 1.

[0043] Figure 2 SEM image (a) and magnified image (b) of PS / PVP@ZnO prepared for Example 1;

[0044] Figure 3 TEM image of PS / PVP@ZnO prepared in Example 2;

[0045] Figure 4 TEM image of PS / PVP@ZnO prepared in Example 3;

[0046] Figure 5 TEM image of PS / PVP@ZnO prepared in Example 4;

[0047] Figure 6 Zeta potential (a) and particle size distribution (b) of the quaternary ammonium PS / PVP@ZnO prepared in Example 5; Zeta potential (c) and particle size distribution (d) of the quaternary ammonium PS / PVP prepared in Comparative Example 1.

[0048] Figure 7The antibacterial effects of PS / PVP / ZnO prepared in Example 1, quaternary ammonium PS / PVP / ZnO prepared in Example 5, and quaternary ammonium PS / PVP and ZnO nanoparticles prepared in Comparative Example 1 on Escherichia coli and Staphylococcus aureus are shown in Figure (a) and (b) of quaternary ammonium PS / PVP / ZnO prepared in Example 5 at different concentrations on Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0049] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0050] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example 1

[0052] The preparation method of PS / PVP@ZnO composite nanospheres is as follows:

[0053] (1) Weigh 0.1 g of KPS initiator and 2 wt% PVP stabilizer (mass fraction of styrene monomer) and dissolve them in 195 mL of deionized water. Mechanical stirring (500 rpm) was started at room temperature, and N2 was introduced to eliminate the influence of oxygen. After deoxygenation for 30 minutes, 3.6 g of St monomer and 1 g of AA monomer were added to the system, and stirring was continued for 20 minutes to completely disperse the monomers. After complete dispersion, the system temperature was raised to 80 °C, and the reaction was stirred under N2 atmosphere for 12 h to polymerize the monomers. After 12 h of polymerization, the system temperature was further raised to 85 °C, and the reaction was stirred for 30 minutes to ensure that all monomers participated in the polymerization reaction. After the reaction was completed, the system was cooled to room temperature to obtain a white PS / PVP nanoemulsion, which was then dried to obtain PS / PVP polymer nanospheres.

[0054] (2) 0.1 g of PS / PVP polymer nanospheres were ultrasonically dispersed in 40 mL of Zn(Ac)2·2H2O (30 mM) ethanol solution. The system was heated to 60 °C and stirred continuously for 1 h to ensure that zinc ions were completely dispersed on the surface of the nanospheres. To reduce the zinc ions, 40 mL of NaOH ethanol solution (75 mM) was slowly added dropwise to the system. After all the addition was completed, the system was allowed to continue reacting at 60 °C for 2 h. After the reaction was completed, the system was cooled to room temperature, centrifuged to obtain the product, washed several times with deionized water, and dried to obtain PS / PVP@ZnO composite nanospheres (PS / PVP / ZnO).

[0055] The infrared spectrum of the PS / PVP@ZnO nanospheres prepared in this embodiment is as follows: Figure 1 As shown in a, the spectrum appears at 3000 cm⁻¹ -1 The broad peak centered at 1720 cm⁻¹ originates from the stretching vibration of the OH bond in the carboxyl group of the AA monomer. Additionally, the C=O and CO bonds in the carboxyl group show a peak at 1720 cm⁻¹. -1 1180cm -1 A stretching vibration peak appears at 3060 cm⁻¹. The spectrum is at 3060 cm⁻¹. -1 The nearby absorption peak originates from the stretching vibration of the unsaturated CH bond in the benzene ring of the St monomer. Additionally, the 1600 cm⁻¹ peak... -1 1492cm -1 and 1452cm -1 The absorption peak at 755 cm⁻¹ originates from the vibration of the benzene ring skeleton; -1 and 698cm -1 The absorption peak at [location] is a characteristic absorption peak of a monosubstituted benzene ring. The infrared absorption spectrum of ZnONPs (Comparative Example 2) is in the range of 400-600 cm⁻¹. -1 The strong absorption peaks around 400-600 cm⁻¹ are attributed to the Zn-O bond vibrations in the ZnO crystal. The infrared absorption spectrum of PS / PVP@ZnO is similar to that of PS / PVP, but differs in the 400-600 cm⁻¹ range. -1 The appearance of a strong absorption peak proves that the loading of ZnONPs was successful.

[0056] XRD diffraction pattern of PS / PVP@ZnO ( Figure 1 (b) The spectrum shows a broad diffraction peak centered at 22°, which originates from the amorphous carbon framework that makes up the nanospheres. The spectrum also shows distinct diffraction peaks at 31.77°, 34.42°, 36.23°, 47.52°, 56.61°, 62.74°, and 68.76°, which correspond to the (100), (002), (101), (102), (110), (103), and (112) planes of hexagonal wurtzite ZnO crystals, further demonstrating the successful loading of ZnONPs.

[0057] The zeta potential of PS / PVP@ZnO is as follows: Figure 1 As shown in c, due to the presence of carboxyl groups on the surface of PS / PVP nanospheres, the PS / PVP@ZnO composite nanospheres as a whole still exhibit negative charge, which provides the conditions for preparing quaternary ammonium-modified carboxyl groups.

[0058] The particle size distribution of PS / PVP@ZnO is as follows: Figure 1 As shown in d, its particle size distribution is around 200nm, and it does not show an aggregated state.

[0059] SEM images of PS / PVP@ZnO composite nanospheres ( Figure 2a) This shows that the surface of the PS / PVP nanospheres is uniformly loaded with zinc oxide nanoparticles, thus avoiding their aggregation. A magnified SEM image ( Figure 2 b) The ZnONPs on the surface of PS / PVP nanospheres show good dispersibility and a particle size of about 10 nm.

[0060] Example 2

[0061] The preparation of PS / PVP@ZnO composite nanospheres is as shown in Example 1, except that the amount of PVP used in the preparation of PS / PVP polymer nanospheres is 1.4 wt% (the mass fraction of styrene monomer), and the other steps and conditions are the same as in Example 1.

[0062] TEM images of the PS / PVP@ZnO composite nanospheres prepared in this embodiment are shown below. Figure 3 As shown in the image, a small amount of PVP results in larger ZnONP particle sizes and aggregation on the surface of PS / PVP nanospheres.

[0063] Example 3

[0064] The preparation of PS / PVP@ZnO composite nanospheres is as shown in Example 1, except that the amount of PVP used in the preparation of PS / PVP polymer nanospheres is 2.8 wt% (as a percentage of the mass fraction of styrene monomer), and the other steps and conditions are the same as in Example 1.

[0065] TEM images of the PS / PVP@ZnO composite nanospheres prepared in this embodiment are shown below. Figure 4 As shown in the image, the presence of a large amount of PVP causes ZnONPs on the surface of PS / PVP nanospheres to aggregate at the loading points, which inevitably affects the antibacterial activity of ZnONPs.

[0066] Example 4

[0067] The preparation of PS / PVP@ZnO composite nanospheres is as shown in Example 1, except that the concentration of Zn(Ac)2·2H2O ethanol solution is 40mM and the concentration of NaOH ethanol solution is 100mM. Other steps and conditions are the same as in Example 1.

[0068] TEM images of the PS / PVP@ZnO composite nanospheres prepared in this embodiment are shown below. Figure 5 As shown in the image, the increased amount of Zn(Ac)2·2H2O will lead to excessive zinc oxide loading, resulting in the aggregation of zinc oxide nanoparticles, which has an adverse effect on antibacterial activity.

[0069] Example 5

[0070] The preparation steps of quaternary ammonium PS / PVP@ZnO composite nanospheres are as follows:

[0071] 0.4 g of the PS / PVP@ZnO composite nanospheres prepared in Example 1 were ultrasonically dispersed in 40 mL of deionized water and stirred in a cold bath at 0°C. EDC (2 mmol) and NHS (2 mmol) were added to the system, and the mixture was stirred in a dark environment at 0°C for 1 h to activate the surface carboxyl groups. The intermediate product was obtained by centrifugation, washed three times with deionized water, and then redispersed in 40 mL of deionized water. 2 mmol of N,N-diethyl-p-phenylenediamine was added to the system, and the mixture was stirred at room temperature in the dark for 12 h to carry out the amidation reaction. After the reaction, the mixture was centrifuged, washed with anhydrous ethanol, and the amidated PS / PVP@ZnO composite nanospheres were obtained. The amidated PS / PVP@ZnO composite nanospheres were redispersed in 40 mL of anhydrous ethanol, the system temperature was raised to 65°C, and 10 mmol of bromoethane was added. The mixture was stirred for 12 h. After the reaction, the system was cooled to room temperature, centrifuged, washed with anhydrous ethanol, and dried to obtain quaternary ammonium PS / PVP@ZnO nanospheres (quaternary ammonium PS / PVP / ZnO).

[0072] Zeta potential ( Figure 6 a) The quaternary ammonium PS / PVP@ZnO shows a positive charge, which proves the success of the quaternization reaction. Particle size distribution diagram ( Figure 6 b) shows that the particle size of the quaternary ammonium PS / PVP@ZnO nanospheres increases but still retains dispersibility.

[0073] Comparative Example 1

[0074] To determine the experimental sequence for zinc oxide loading and quaternization modification, quaternary ammonium PS / PVP was prepared by quaternization modification of unloaded zinc oxide nanospheres. The experimental procedures were as shown in Example 5, except that the PS / PVP@ZnO composite nanospheres were replaced with PS / PVP@ZnO composite nanospheres prepared in Example 1; other steps and conditions were the same as in Example 5.

[0075] Zeta potential ( Figure 6 c) shows that the quaternization modification experiment makes the PS / PVP nanospheres positively charged, but it also causes the nanospheres to aggregate. The particle size distribution diagram of the quaternary ammonium PS / PVP is shown. Figure 6 d) shows that its particle size is significantly increased and aggregation occurs. This is because the disappearance of electrostatic repulsion between nanospheres during the modification of carboxyl groups makes them unstable, which is not conducive to the uniform dispersion of ZnONP precursors on the surface of nanospheres.

[0076] Based on the above experimental results, we first selected to perform ZnONPs loading experiments on PS / PVP polymer nanospheres, and then modified the obtained PS / PVP@ZnO with quaternary ammonium to finally obtain quaternary ammonium PS / PVP@ZnO composite nanospheres as antibacterial agents.

[0077] Comparative Example 2

[0078] The preparation method of ZnONPs is as follows:

[0079] 40 mL of Zn(Ac)2·2H2O (30 mM) ethanol solution was heated to 60 °C and stirred continuously for 1 h. Then, 40 mL of NaOH ethanol solution (75 mM) was slowly added dropwise to the system. After all the addition was completed, the system was allowed to continue reacting at 60 °C for 2 h. After the reaction was completed, the system was cooled to room temperature, centrifuged to obtain the product, washed several times with deionized water, and dried to obtain white powder ZnONPs.

[0080] Experimental Example 1

[0081] Antibacterial properties investigation

[0082] The antibacterial properties of PS / PVP / ZnO prepared in Example 1, quaternary ammonium PS / PVP / ZnO prepared in Example 5, quaternary ammonium PS / PVP prepared in Comparative Example 1, and ZnONPs prepared in Comparative Example 2 as antibacterial agents against Gram-positive Staphylococcus aureus (ATCC 25923) and Gram-negative Escherichia coli (ATCC 25922).

[0083] The sample was ultrasonically dispersed in deionized water to prepare antibacterial agent dispersions at different concentrations (100, 150, 200, 250 μg / mL). Staphylococcus aureus or Escherichia coli were grown overnight in Luria-Bertani liquid medium (LB liquid medium) at 37°C. The resulting liquid culture was diluted with 2 mL of LB medium to a light density (OD600) of 0.2 at 600 nm. Then, 100 μL of antibacterial material was mixed with 400 μL of bacterial solution, and LB medium was added to a final volume of 2 mL. The mixture was then incubated at 37°C with shaking at 200 rpm for 24 hours. Bacterial viability was determined by measuring the light density at 600 nm.

[0084] The antibacterial effect of an antibacterial agent dispersion at a concentration of 100 μg / mL is as follows: Figure 7As shown in Figure a, for *Escherichia coli* and *Staphylococcus aureus*, the antibacterial effect of PS / PVP / ZnO is superior to that of ZnONPs. This is because the nanospheres increase the specific surface area of ​​ZnONPs, thereby enhancing their antibacterial activity. Quaternary ammonium PS / PVP nanospheres exhibit certain antibacterial effects, with their quaternary ammonium groups exerting their main antibacterial action through a contact antibacterial mechanism. Quaternary ammonium PS / PVP / ZnO reduced bacterial survival rates to a minimum of 33% and 24%, respectively, which strongly demonstrates the synergistic antibacterial effect between the quaternary ammonium groups and ZnONPs.

[0085] The antibacterial effects of quaternary ammonium PS / PVP / ZnO prepared in Example 5 at different concentrations are as follows: Figure 7 As shown in b, the antibacterial effect increases with increasing concentration; when the concentration is 250 μg / mL, the survival rate of Escherichia coli is only 3.1%, and the survival rate of Staphylococcus aureus is only 8.1%.

[0086] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of zinc oxide nanoparticles / quaternary amine modified polystyrene nanospheres in antibacterial applications, characterized in that, The nanosphere is a polymer nanosphere with zinc oxide nanoparticles uniformly loaded on the surface of the polymer nanosphere, and the surface of the polymer nanosphere is also grafted with quaternary amine groups, and the structure of the quaternary amine groups is as follows: the polymer nanosphere is a styrene-acrylic acid copolymer nanosphere coated with polyvinylpyrrolidone on the surface; ； The particle size of the zinc oxide nanoparticle / quaternary amine modified polystyrene nanosphere is 180-220 nm; the particle size of the zinc oxide nanoparticle is 5-15 nm; The preparation method of the zinc oxide nanoparticle / quaternary amine modified polystyrene nanosphere comprises the following steps: (1) The initiator, polyvinylpyrrolidone (PVP) is dissolved in deionized water, styrene and acrylic acid monomers are added, and then polymerization reaction is carried out, and the polymer nanosphere (PS / PVP) is obtained by drying; the mass ratio of styrene, acrylic acid and polyvinylpyrrolidone (PVP) is 3.6:1:0.072; (2) The polymer nanosphere is fully dispersed in the Zn source ethanol solution, a reducing agent is added, and then reduction reaction is carried out, and the zinc oxide loaded polymer nanosphere (PS / PVP@ZnO) is obtained by centrifugation, washing and drying; the Zn source is Zn(Ac)2·2H2O; the mass ratio of the Zn source and the polymer nanosphere is 2-3:1; (3) The zinc oxide loaded polymer nanosphere is fully dispersed in deionized water to obtain a dispersion liquid, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are added, and then activation reaction is carried out, and then the dispersion liquid is re-dispersed in deionized water after centrifugation and washing; N,N-diethyl-p-phenylenediamine is added, and then amidation reaction is carried out, and then the dispersion liquid is re-dispersed in anhydrous ethanol after centrifugation and washing; Bromine ethane is added, and then quaternary amination reaction is carried out, and then the zinc oxide nanoparticle / quaternary amine modified polystyrene nanosphere (quaternary amine PS / PVP@ZnO) is obtained by centrifugation, washing and drying.

2. Use according to claim 1, characterized in that, In step (1), one or more of the following conditions are included: i. The initiator is potassium persulfate (KPS); the mass of the initiator is 2-4% of the mass of styrene; ii. The mass of polyvinylpyrrolidone (PVP) is 0.01-0.06% of the mass of deionized water; iii. The polymerization reaction conditions are as follows: under the conditions of inert gas protection and stirring, the polymerization reaction is carried out at 70-90℃ for 10-14h, and then the polymerization reaction is carried out at 80-90℃ for 0.1-1h; the inert gas is nitrogen or argon.

3. Use according to claim 1, characterized in that, In step (2), one or more of the following conditions are included: i. The concentration of the Zn source ethanol solution is 20-40mmol / L; ii. After adding the Zn source, stirring is carried out at 55-65℃ for 0.5-2h to fully mix uniformly; iii. The reducing agent is NaOH ethanol solution with a concentration of 50-100mmol / L; the molar ratio of NaOH to Zn source is 2-3:1; the reducing agent is added into the system in a dropwise manner; iv. The reduction reaction temperature is 50-70℃, the reduction reaction time is 1-3h, and the reduction reaction is carried out under stirring conditions.

4. Use according to claim 1, characterized in that, In step (3), one or more of the following conditions are included: i. The mass concentration of the dispersion is 0.005-0.1 g / mL; ii. The molar ratio of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is 0.5-1.5:0.5-1.5; the mass ratio of zinc oxide loaded polymer nanospheres and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) is 1:0.5-1; iii. The activation reaction temperature is -5-5℃, the activation reaction time is 0.5-2h, and the activation reaction is carried out under stirring and light shielding conditions.

5. The use according to claim 1, characterized in that, In step (3), one or more of the following conditions are included: i. The mass of zinc oxide loaded polymer nanospheres and the volume of deionized water used for redispersion is 0.005-0.1 g / mL; ii. The molar ratio of N-hydroxysuccinimide and N,N-diethyl-p-phenylenediamine is 0.5-1.5:0.5-1.5; iii. The amidation reaction temperature is room temperature, the amidation reaction time is 10-15h, and the amidation reaction is carried out under stirring and light shielding conditions.

6. Use according to claim 1, characterized in that, In step (3), one or more of the following conditions are included: i. The mass of zinc oxide loaded polymer nanospheres and the volume of anhydrous ethanol used for redispersion is 0.005-0.1 g / mL; ii. The molar ratio of N,N-diethyl-p-phenylenediamine and bromoethane is 1:4-6; iii. The quaternary amination reaction temperature is 60-70℃, the quaternary amination reaction time is 10-15h, and the quaternary amination reaction is carried out under stirring conditions.

7. The use according to claim 1, characterized in that, The zinc oxide nanoparticles / quaternary amine modified polystyrene nanospheres are used as antibacterial agents to inhibit Escherichia coli or / and Staphylococcus aureus.

Images