Antibacterial composition, antibacterial nano-emulsion, antibacterial preparation and application of antibacterial composition, antibacterial nano-emulsion and antibacterial preparation

Abstract

The invention belongs to the technical field of preparation of antibacterial preparations, and particularly relates to an antibacterial composition, an antibacterial nano-emulsion, an antibacterial preparation and application of the antibacterial composition, the antibacterial nano-emulsion and the antibacterial preparation. Through verification, the benzalkonium bromide and the chlorhexidine acetate can achieve the effect of synergistically resisting methicillin-resistant staphylococcus aureus (MRSA) by inhibiting or removing a biological membrane (cell membrane), and particularly, the nano-emulsion prepared from the benzalkonium bromide and the chlorhexidine acetate has an excellent MRSA resisting effect; the minimum inhibitory concentration (MIC) of the prepared nanoemulsion on MRSA bacteria is 1.5625 [mu] g / mL, and the minimum bactericidal concentration (MBC) of the prepared nanoemulsion on MRSA bacteria is 6.25 [mu] g / mL; in addition, the prepared nanoemulsion is low in toxicity and excellent in stability. Moreover, the antibacterial preparation disclosed by the invention is simple in preparation process, stable in quality and suitable for large-scale production, can be widely applied to infection of multiple drug-resistant bacteria of skin wounds, and has a good market application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial preparation technology, specifically relating to an antibacterial composition, an antibacterial nanoemulsion, an antibacterial preparation, and its uses. Background Technology

[0002] Methicillin-resistant Staphylococcus aureus (MRSA) has become a significant threat to global healthcare due to its biofilm-forming ability and multidrug resistance. Statistics show that in 2019, MRSA directly caused over 100,000 deaths due to drug resistance. While traditional antibiotics (such as daptomycin and linezolid) are the mainstay of treatment, their permeability is significantly weakened by biofilm barriers, forcing clinicians to adopt high-dose strategies, leading to increased toxicity and accelerated evolution of drug resistance. Therefore, there is an urgent need to develop innovative strategies that combine high antibacterial efficacy, anti-biofilm activity, and improved safety.

[0003] Current antibacterial strategies largely focus on single-drug or antibiotic combinations. For example, while non-antibiotic disinfectants such as chlorhexidine acetate (CHX) possess broad-spectrum antibacterial activity, their single-drug action struggles to penetrate mature biofilms, and high concentrations can easily induce cytotoxicity. On the other hand, quaternary ammonium surfactants (such as benzalkonium bromide) can enhance bactericidal effects by disrupting cell membranes, but their high toxicity severely limits their clinical application. While existing combination therapies can partially improve antibacterial efficiency, over-reliance on antibiotic combinations accelerates the spread of resistance, and systematic research on the synergistic mechanisms and toxicity control of non-antibiotic drugs remains lacking.

[0004] Existing technologies have not fully explored the potential of combined use of non-antibiotic antimicrobial agents, especially how to optimize drug compatibility to synergistically exert different mechanisms of action (such as metabolic interference and membrane disruption) to enhance anti-biofilm activity while reducing cytotoxicity. Summary of the Invention

[0005] This invention verifies that benzalkonium bromide and chlorhexidine acetate can synergistically inhibit or remove biofilms to combat methicillin-resistant Staphylococcus aureus (MRSA). In particular, the nanoemulsion prepared from benzalkonium bromide and chlorhexidine acetate has excellent anti-MRSA effects, with a minimum inhibitory concentration (MIC) of 1.5625 μg / mL and a minimum bactericidal concentration (MBC) of 6.25 μg / mL against MRSA bacteria. Furthermore, the nanoemulsion exhibits low toxicity and excellent stability.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides an antibacterial composition comprising A and B, wherein A is benzalkonium bromide or its nanoemulsion, and B is chlorhexidine acetate or its nanoemulsion.

[0008] Preferably, in the above antibacterial composition, the mass ratio of A to B is (1-9):(1-9).

[0009] More preferably, in the above antibacterial composition, the mass ratio of A to B is 4:6.

[0010] Another aspect of the present invention provides an antibacterial nanoemulsion comprising benzalkonium bromide and chlorhexidine acetate.

[0011] Preferably, in the above-mentioned antibacterial nanoemulsion, the mass ratio of benzalkonium bromide to chlorhexidine acetate is (1-9):(1-9).

[0012] Preferably, the above-mentioned antibacterial nanoemulsion further includes a surfactant, a co-surfactant, and an oil phase.

[0013] More preferably, the above-mentioned antibacterial nanoemulsion satisfies one or more of the following conditions:

[0014] (i) The surfactant is one or more of the following: castor oil polyoxyethylene 35 ether, Tween-80, castor oil polyoxyethylene ether, polyoxyethylene 40 hydrogenated castor oil or lecithin.

[0015] (ii) The co-surfactant is one or more combinations of propylene glycol, ethanol, glycerol or polyethylene glycol 400;

[0016] (iii) The oil phase is one or more of isopropyl myristate, soybean oil, ethyl oleate or medium-chain triglycerides;

[0017] (iv) The mass ratio of surfactant, co-surfactant and oil phase is (6-8):(1-3):1.

[0018] In another aspect, the present invention provides an antibacterial preparation comprising the antibacterial composition of the present invention or the antibacterial nanoemulsion of the present invention.

[0019] In another aspect, this invention provides the use of the antibacterial composition, antibacterial nanoemulsion, or antibacterial preparation of this invention, the use including:

[0020] (a) Use of antimicrobial compositions, antimicrobial nanoemulsions, or antimicrobial agents against methicillin-resistant Staphylococcus aureus; or

[0021] (b) Use of an antimicrobial composition, antimicrobial nanoemulsion, or antimicrobial preparation in the preparation of a medicament for the prevention of wound infection by methicillin-resistant Staphylococcus aureus; or

[0022] (c) Use of antimicrobial compositions or antimicrobial nanoemulsions or antimicrobial preparations in the preparation of medicaments for wound healing of wounds infected with methicillin-resistant Staphylococcus aureus.

[0023] Preferably, in the above-described uses, the antibacterial composition, antibacterial nanoemulsion, or antibacterial preparation has one or more of the following functions:

[0024] (a) It has the function of inhibiting the growth of methicillin-resistant Staphylococcus aureus biofilm;

[0025] (b) It has the function of clearing biofilms of methicillin-resistant Staphylococcus aureus;

[0026] (c) It has the function of disrupting the cell wall and cell membrane integrity of methicillin-resistant Staphylococcus aureus;

[0027] (d) It has the function of reducing the hydrophobicity of the cell membrane of methicillin-resistant Staphylococcus aureus.

[0028] The beneficial effects of this invention include:

[0029] (1) In this invention, benzalkonium bromide (BZL) and chlorhexidine acetate (CHX) have synergistic antibacterial effects (especially methicillin-resistant Staphylococcus aureus), and can achieve antibacterial effects by inhibiting or clearing biofilms. The antibacterial effect of the combination of the two is significantly better than that of the single component.

[0030] (2) In this invention, the antibacterial effect of the nanoemulsion ((BZL+CHX)NE) prepared from benzalkonium bromide and chlorhexidine acetate is superior to that of the aqueous solution of benzalkonium bromide and chlorhexidine acetate (BZL+CHX) and superior to the antibacterial effect of the mixture after benzalkonium bromide and chlorhexidine acetate are each prepared as a nanoemulsion (BZL+(CHX)NE or (BZL)+CHXNE). Verification revealed that the minimum inhibitory concentration (MIC) of the nanoemulsion prepared from benzalkonium bromide and chlorhexidine acetate against MRSA bacteria was 1.5625 μg / mL, and the minimum bactericidal concentration (MBC) was 6.25 μg / mL; however, the MIC and MBC of the aqueous solution composed of benzalkonium bromide and chlorhexidine acetate against MRSA were 3.125 μg / mL and 12.5 μg / mL, respectively. That is, the MIC and MBC of (BZL+CHX)NE are both twice lower than those of BZL+CHX.

[0031] (3) The nanoemulsion ((BZL+CHX)NE) prepared by benzalkonium bromide and chlorhexidine acetate in this invention has good stability. After being stored at room temperature for 3 years, the particle size, PDI and zeta potential did not change significantly.

[0032] (4) The nanoemulsion ((BZL+CHX)NE) prepared by benzalkonium bromide and chlorhexidine acetate in this invention has lower cytotoxicity than the aqueous solution (BZL+CHX) of benzalkonium bromide and chlorhexidine acetate.

[0033] (5) In this invention, the combined use of benzalkonium bromide and chlorine acetate can promote wound healing of methicillin-resistant Staphylococcus aureus, and the healing speed of nanoemulsion ((BZL+CHX)NE) is better than that of BZL+CHX. Attached Figure Description

[0034] Figure 1a TEM image of nanoemulsion (BZL+CHX)NE;

[0035] Figure 1b AFM plot of nanoemulsion (BZL+CHX)NE;

[0036] Figure 1c The particle size distribution of the nanoemulsion (BZL+CHX)NE;

[0037] Figure 1d The zeta potential of the nanoemulsion (BZL+CHX)NE;

[0038] Figure 2a Differential scanning calorimetry analysis of nanoemulsion (BZL+CHX)NE;

[0039] Figure 2b Thermogravimetric analysis of nanoemulsion (BZL+CHX)NE;

[0040] Figure 2c The FTIR characteristics of the nanoemulsion (BZL+CHX)NE;

[0041] Figure 3a The antibacterial effects of nanoemulsions prepared with different mass ratios of BZL and CHX;

[0042] Figure 3b The antibacterial effects of antibacterial agents prepared by different combinations of BZL and CHX;

[0043] Figure 4a The particle size of nanoemulsion (BZL+CHX)NE after being stored for different times;

[0044] Figure 4b PDI of nanoemulsion (BZL+CHX)NE after different storage times;

[0045] Figure 4c The zeta potential of nanoemulsion (BZL+CHX)NE after being placed for different times;

[0046] Figure 5 The cytotoxicity test results of the nanoemulsion (BZL+CHX)NE;

[0047] Figure 6aThe MIC and MBC values ​​of the nanoemulsion (BZL+CHX)NE are shown.

[0048] Figure 6b The bacterial colony status after treatment with nanoemulsion (BZL+CHX)NE;

[0049] Figure 6c The colony count of bacteria after treatment with nanoemulsion (BZL+CHX)NE;

[0050] Figure 7a Bacterial colony counts were performed on the wounds of trauma model mice in each treatment group;

[0051] Figure 7b The ratio of wound healing area in each treatment group of trauma model mice;

[0052] Figure 7c Photographs of the appearance of wounds in the trauma model mice of each treatment group;

[0053] Figure 7d Histological analysis of skin samples from wound sites in trauma model mice of each treatment group;

[0054] Figure 8a SEM image of the MRSA biofilm in the control group during the biofilm inhibition experiment;

[0055] Figure 8b SEM images of the MRSA biofilm in the BZL+CHX group during the biofilm inhibition experiment;

[0056] Figure 8c SEM images of MRSA biofilm in the (BZL+CHX)NE group during biofilm inhibition experiments;

[0057] Figure 8d Quantitative analysis of MRSA biofilm in different treatment groups during biofilm inhibition experiments.

[0058] Figure 8e The image shows a CLSM image of the MRSA biofilm in the control group during the biofilm inhibition experiment.

[0059] Figure 8f CLSM image of MRSA biofilm in the BZL+CHX group during biofilm inhibition experiment;

[0060] Figure 8g CLSM image of MRSA biofilm in the (BZL+CHX)NE group during biofilm inhibition experiment;

[0061] Figure 8h The ratio of dead to alive bacteria in different treatment groups during the biofilm inhibition experiment;

[0062] Figure 9aSEM image of the MRSA biofilm in the control group during the biofilm clearance experiment;

[0063] Figure 9b SEM images of MRSA biofilm in the BZL+CHX group during biofilm clearance experiments;

[0064] Figure 9c SEM images of MRSA biofilm in the (BZL+CHX)NE group during biofilm clearance experiments;

[0065] Figure 9d Quantitative analysis of MRSA biofilm in different treatment groups during biofilm clearance experiments;

[0066] Figure 9e The image shows a CLSM image of the MRSA biofilm in the control group during the biofilm clearance experiment.

[0067] Figure 9f CLSM image of MRSA biofilm in the BZL+CHX group during biofilm clearance experiment;

[0068] Figure 9g CLSM image of MRSA biofilm in the (BZL+CHX)NE group during biofilm clearance experiment;

[0069] Figure 9h The ratio of dead to alive bacteria in different treatment groups during the biofilm removal experiment;

[0070] Figure 10 TEM images of MRSA biofilms after different treatments in a cell damage study experiment; (a) is the original MRSA biofilm; (b) is the MRSA biofilm after (BZL+CHX)NE treatment; (c) is the MRSA biofilm after BZL+CHX treatment.

[0071] Figure 11 The surface hydrophobicity index of MRSA cell membranes after different treatments in a cell damage study experiment. Detailed Implementation

[0072] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0073] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0074] In one aspect, the present invention provides an antibacterial composition comprising A and B, wherein A is benzalkonium bromide or its nanoemulsion, and B is chlorhexidine acetate or its nanoemulsion.

[0075] It should be noted that the present invention has verified that benzalkonium bromide and chlorhexidine acetate have a synergistic antibacterial effect. The combined antibacterial effect of benzalkonium bromide and chlorhexidine acetate is significantly better than that of single antibacterial agents, especially against methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, it should be noted that the antibacterial composition may contain other antibacterial active ingredients besides benzalkonium bromide and chlorhexidine acetate, which are known in the art.

[0076] It should also be noted that CHX is a widely used disinfectant in dermatology and root canal treatments, exhibiting broad-spectrum antibacterial activity. In its nanoparticle form (CNE), CHX possesses enhanced antibacterial efficacy and potent anti-biofilm activity. Furthermore, CNE demonstrated adequate safety in a mouse model of MRSA-infected burn wounds and exhibited stronger antibacterial activity than CHX. Benzalkonium bromide (BZL) is a lipophilic quaternary ammonium cationic surfactant. BZL can penetrate the phospholipid bilayer of cell membranes, leading to increased membrane permeability, altered ionic resistance, and even cell membrane disruption. These properties endow BZL with bactericidal activity, and it has been used as a skin disinfectant and eye drop preservative. However, its strong cytotoxicity limits its application.

[0077] In some specific examples, the mass ratio of A to B in the above antibacterial composition is (1-9):(1-9).

[0078] It should be noted that when benzalkonium bromide and chlorhexidine acetate are used in combination, the mass ratio of the two can be selected as (1-9):(1-9). For example, the mass ratio of benzalkonium bromide to chlorhexidine acetate can be 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1; among these, the preferred mass ratio is 4:6, as the combined antibacterial effect of this mass ratio is superior to other mass ratios.

[0079] In some specific examples, the nanoemulsion in the above-mentioned antibacterial composition also includes surfactants, co-surfactants, and an oil phase.

[0080] It should be noted that the method for preparing benzalkonium bromide or chlorhexidine acetate into nanoemulsions in this invention is well known in the art. It is generally prepared by mixing surfactants, co-surfactants, and an oil phase with benzalkonium bromide and / or chlorhexidine acetate.

[0081] In some specific examples, the nanoemulsion in the above antibacterial compositions satisfies one or more of the following conditions:

[0082] (i) The surfactant is one or more of the following: castor oil polyoxyethylene 35 ether, Tween-80, castor oil polyoxyethylene ether, polyoxyethylene 40 hydrogenated castor oil or lecithin.

[0083] (ii) The co-surfactant is one or more combinations of propylene glycol, ethanol, glycerol or polyethylene glycol 400;

[0084] (iii) The oil phase is one or more of isopropyl myristate, soybean oil, ethyl oleate or medium-chain triglycerides;

[0085] (iv) The mass ratio of surfactant, co-surfactant and oil phase is (6-8):(1-3):1.

[0086] It should be noted that the surfactant, co-surfactant, and oil phase in this invention are all well-known in the art, including but not limited to castor oil polyoxyethylene 35 ether, propylene glycol, and isopropyl myristate listed above; in addition, the mass ratio of surfactant, co-surfactant, and oil phase can be (6-8):(1-3):1, for example 7:1:1, 7:2:1, 7:3:1, 6:2:1, or 8:2:1, etc. Furthermore, the above-mentioned nanoemulsion can simultaneously satisfy conditions (i) to (iv), or it can satisfy only one of the above conditions, preferably simultaneously satisfying the above conditions.

[0087] This invention also provides an antibacterial nanoemulsion comprising benzalkonium bromide and chlorhexidine acetate.

[0088] It should be noted that, as mentioned above, although benzalkonium bromide and chlorhexidine acetate can be used in combination via aqueous solution or by mixing them after forming nanoemulsions of one or both benzalkonium bromide and chlorhexidine acetate, the effect is not as good as preparing a single nanoemulsion of benzalkonium bromide and chlorhexidine acetate. In this invention, it was found that the minimum inhibitory concentration (MIC) of the nanoemulsion ((BZL+CHX)NE) prepared from benzalkonium bromide and chlorhexidine acetate as a single system against MRSA bacteria was 1.5625 μg / mL, and the minimum bactericidal concentration (MBC) was 6.25 μg / mL; however, the MIC and MBC of the aqueous solution composed of benzalkonium bromide and chlorhexidine acetate against MRSA were 3.125 μg / mL and 12.5 μg / mL, respectively. That is, the MIC and MBC of (BZL+CHX)NE are both twice lower than those of BZL+CHX. This indicates that the nanoemulsion exhibits a stronger antibacterial effect than the aqueous solution.

[0089] In some specific examples, the mass ratio of benzalkonium bromide to chlorhexidine acetate in the above antibacterial composition can be (1-9):(1-9).

[0090] It should be noted that in the same system of nanoemulsions, the mass ratio of benzalkonium bromide to chlorhexidine acetate can also be (1-9):(1-9). For example, the mass ratio of benzalkonium bromide to chlorhexidine acetate can be 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:1. Among these, the preferred mass ratio is 4:6, as the combined antibacterial effect of this mass ratio is better than that of other mass ratios.

[0091] In some specific examples, the aforementioned antibacterial nanoemulsions also include surfactants, co-surfactants, and an oil phase.

[0092] It should be noted that the method for preparing benzalkonium bromide and chlorhexidine acetate into a nanoemulsion in this invention is well known in the art. It is generally prepared by mixing a surfactant, a co-surfactant, an oil phase, and benzalkonium bromide and chlorhexidine acetate.

[0093] In some specific examples, the above-mentioned antibacterial nanoemulsions meet one or more of the following conditions:

[0094] (i) The surfactant is one or more of the following: castor oil polyoxyethylene 35 ether, Tween-80, castor oil polyoxyethylene ether, polyoxyethylene 40 hydrogenated castor oil or lecithin.

[0095] (ii) The co-surfactant is one or more combinations of propylene glycol, ethanol, glycerol or polyethylene glycol 400;

[0096] (iii) The oil phase is one or more of isopropyl myristate, soybean oil, ethyl oleate or medium-chain triglycerides;

[0097] (iv) The mass ratio of surfactant, co-surfactant and oil phase is (6-8):(1-3):1.

[0098] It should be noted that the surfactant, co-surfactant, and oil phase in this invention are all well-known in the art, including but not limited to castor oil polyoxyethylene 35 ether, propylene glycol, and isopropyl myristate listed above; in addition, the mass ratio of surfactant, co-surfactant, and oil phase can be (6-8):(1-3):1, for example 7:1:1, 7:2:1, 7:3:1, 6:2:1, or 8:2:1, etc. Furthermore, the above-mentioned nanoemulsion can simultaneously satisfy conditions (i) to (iv), or it can satisfy only one of the above conditions, preferably simultaneously satisfying the above conditions.

[0099] The present invention also provides an antibacterial preparation comprising the above-described antibacterial composition or the above-described antibacterial nanoemulsion.

[0100] It should be noted that the antibacterial composition and antibacterial nanoemulsion in this invention can be used directly as an antibacterial agent, or they can be prepared into different formulations for different clinical needs by adding other excipients. Furthermore, it should be understood that the antibacterial preparation can be a drug in different dosage forms, or it can be other preparations used for in vitro bactericidal action.

[0101] This invention also provides a use of the antibacterial composition, antibacterial nanoemulsion, or antibacterial preparation of this invention, the uses including:

[0102] (a) Use of antimicrobial compositions, antimicrobial nanoemulsions, or antimicrobial agents against methicillin-resistant Staphylococcus aureus; or

[0103] (b) Use of an antimicrobial composition, antimicrobial nanoemulsion, or antimicrobial preparation in the preparation of a medicament for the prevention of wound infection by methicillin-resistant Staphylococcus aureus; or

[0104] (c) Use of antimicrobial compositions or antimicrobial nanoemulsions or antimicrobial preparations in the preparation of medicaments for wound healing of wounds infected with methicillin-resistant Staphylococcus aureus.

[0105] It should be understood that, of the above uses, use (a) mainly involves applying the antibacterial composition, antibacterial nanoemulsion, or antibacterial preparation in in vitro sterilization; use (b) includes, but is not limited to, using the antibacterial composition, antibacterial nanoemulsion, or antibacterial preparation of the present invention for sterilization treatment before and after surgery; and use (c) includes, but is not limited to, using the antibacterial composition, antibacterial nanoemulsion, or antibacterial preparation of the present invention for wound cleaning treatment after wound infection with methicillin-resistant Staphylococcus aureus.

[0106] In some specific examples, in the above-described uses, the antimicrobial composition, antimicrobial nanoemulsion, or antimicrobial preparation has one or more of the following functions:

[0107] (a) It has the function of inhibiting the formation of biofilms by methicillin-resistant Staphylococcus aureus;

[0108] (b) It has the function of clearing biofilms of methicillin-resistant Staphylococcus aureus;

[0109] (c) It has the function of disrupting the cell wall and cell membrane integrity of methicillin-resistant Staphylococcus aureus;

[0110] (d) It has the function of reducing the hydrophobicity of the cell membrane of methicillin-resistant Staphylococcus aureus.

[0111] It should be noted that the bacterial composition, antibacterial nanoemulsion, or antibacterial preparation in this invention achieves its antibacterial purpose by inhibiting the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms, clearing MRSA biofilms, damaging MRSA cell walls and cell membranes, and reducing the hydrophobicity of MRSA cell membranes.

[0112] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0113] In the following examples, BZL (benzalofop-methyl) (5%) was purchased from Chongqing Yusheng Pharmaceutical Co., Ltd. (Chongqing, China); CHX (chlorhexidine acetate) was purchased from Jinzhou Jiutai Pharmaceutical Co., Ltd. (Liaoning, China); isopropyl myristate (6%, IPM) was purchased from Croda (Gull, UK); propylene glycol (PG) was purchased from Chongqing Wanda Biotechnology Co., Ltd. (Chongqing, China); EL35 was purchased from (supplier information omitted here); Mueller-Hinton agar (MH(A)), Mueller-Hinton broth (MH(B)) and tryptone soybean broth (TSB) were all purchased from AOBOX (Beijing, China);

[0114] In the following examples, 96-well and 24-well cell culture plates were purchased from Labselect (Beijing, China); MTT was purchased from Sigma Chemical Company (St. Louis, Missouri, USA); fetal bovine serum (FBS), DMEM medium, 0.25% trypsin, and penicillin-streptomycin were all purchased from Gibico (California, USA); the NCTC clone 929 (L929) cell line was provided by the American Type Culture Collection (ATCC) (Manassas, Virginia, USA); coverslips and the LIVE / DEAD 7021 bacterial viability kit were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Rochester, USA); and the potassium assay kit was purchased from Nanjing Jiancheng Biotechnology Institute (C001-3, Nanjing, China).

[0115] In the following examples, Staphylococcus aureus strain MRSA252 was purchased from ATCC (Manassas, Virginia, USA); the same method was used to prepare bacterial suspensions in all experiments, with the following steps: bacteria (e.g., MRSA252) were inoculated onto MH(A) and cultured overnight at 37°C; then, a single MRSA252 colony was picked and suspended in MH(B), and cultured at 37°C and 220 rpm for 12–16 hours; then, 200 μL of the suspension was added to 20 mL of fresh MH(B); after incubation under the same conditions for 4 hours, the suspension was collected and washed twice with physiological saline; then, the bacteria were resuspended, and their absorbance (OD) at 600 nm was adjusted to 1.0 (1 × 10⁻⁶). 9 (CFU / mL); finally, the suspension was diluted 1000 times in MH(B) to obtain a final concentration of approximately 1×10⁻⁶ CFU / mL. 6 A CFU / mL suspension was used for all experiments; OD was measured by a UV spectrophotometer (Ultrospec 8000, GE Healthcare Bio-Science AB).

[0116] I. Preparation of Antibacterial Agents

[0117] Example 1

[0118] (1) Weigh 7 mg EL35 (castor oil polyoxyethylene 35 ether), 2 mg PG (propylene glycol) and 1 mg IPM (isopropyl myristate) respectively and place them in a 50 mL container and mix thoroughly to obtain a mixture;

[0119] (2) Slowly add BZL, CHX and water (the mass ratio of the mixture of BZL and CHX to water is w / w = 1:60) to the mixture and stir the mixture clockwise to form nanoemulsion (BZL+CHX)NE (the total mass concentration of the active ingredient (BZL+CHX) is 10 mg / mL, the same below); wherein, in the mixture of BZL and CHX, the mass ratio of BZL to CHX is 1:9.

[0120] Examples 2 to 9

[0121] The difference between Examples 2 to 9 and Example 1 is that in step (2), the mass ratio of BZL to CHX in the mixture of BZL and CHX is different from that in Example 1, but the rest is the same as in Example 1; in Examples 2 to 9, the mass ratio of BZL to CHX is 2:8 (Example 2), 3:7 (Example 3), 4:6 (Example 4), 5:5 (Example 5), 6:4 (Example 6), 7:3 (Example 7), 8:2 (Example 8) and 9:1 (Example 9).

[0122] Example 10

[0123] Preparation of BZL+CHX: Weigh BZL and CHX and dissolve them in pure water so that the mass ratio of the mixture of BZL and CHX to water is w / w = 1:60. Vortex thoroughly to mix, which is the aqueous solution of BZL+CHX (i.e., BZL+CHX); wherein the mass ratio of BZL to CHX is 4:6.

[0124] Example 11

[0125] (1) Weigh 7 mg EL35 (castor oil polyoxyethylene 35 ether), 2 mg PG (propylene glycol) and 1 mg IPM (isopropyl myristate) respectively and place them in a 50 mL container and mix thoroughly to obtain a mixture;

[0126] (2) BZL and water (mass ratio of BZL to water w / w = 1:60) were slowly added dropwise to the mixture, and the mixture was stirred clockwise to prepare (BZL)NE (10 mg / mL);

[0127] (3) Prepare (BZL)NE+CHX by mixing (BZL)NE with CHX solution (in which the mass ratio of CHX to water is w / w = 1:60).

[0128] Example 12

[0129] (1) Weigh 7 mg EL35 (castor oil polyoxyethylene 35 ether), 2 mg PG (propylene glycol) and 1 mg IPM (isopropyl myristate) respectively and place them in a 50 mL container and mix thoroughly to obtain a mixture;

[0130] (2) CHX and water (mass ratio of CHX to water w / w = 1:60) were slowly added dropwise to the mixture, and the mixture was stirred clockwise to prepare (CHX)NE (10 mg / mL);

[0131] (3) BZL+(CHX)NE was prepared by mixing (CHX)NE with BZL solution (in which the mass ratio of BZL to water is w / w = 1:60).

[0132] Example 13

[0133] Weigh out BZL and CHX and dissolve them in pure water so that the mass ratio of the BZL and CHX mixture (BZL and CHX mass ratio of 4:6) to the water mass ratio w / w = 1:60. Vortex thoroughly to mix and set aside. Then weigh out 7 mg of EL35 (castor oil polyoxyethylene 35 ether), 2 mg of PG (propylene glycol) and 1 mg of IPM (isopropyl myristate) and place them in a 50 mL container and mix thoroughly to obtain a mixture. Mix this mixture with the BZL+CHX aqueous solution to obtain BZL+CHX+BNE.

[0134] Comparative Example 1

[0135] (1) Weigh 7 mg EL35 (castor oil polyoxyethylene 35 ether), 2 mg PG (propylene glycol) and 1 mg IPM (isopropyl myristate) respectively and place them in a 50 mL container and mix thoroughly to obtain a mixture;

[0136] (2) CHX and water were slowly added dropwise to the mixture (the mass ratio of CHX to water was w / w = 1:60), and the mixture was stirred clockwise to form a nanoemulsion (CHX)NE (the final mass concentration of CHX was 10 mg / mL).

[0137] Comparative Example 2

[0138] (1) Weigh 7 mg EL35 (castor oil polyoxyethylene 35 ether), 2 mg PG (propylene glycol) and 1 mg IPM (isopropyl myristate) respectively and place them in a 50 mL container and mix thoroughly to obtain a mixture;

[0139] (2) Slowly add BZL and water (BZL to water mass concentration w / w = 1:60) to the mixture and stir the mixture clockwise to form nanoemulsion (BZL)NE (final mass concentration of BZL 10 mg / mL).

[0140] II. Characterization of Antibacterial Agents

[0141] (I) Characterization and physical state of nanoemulsions

[0142] Characterization of (BZL+CHX)NE

[0143] The gross morphology of the nanoemulsion (BZL+CHX)NE prepared in Example 1 was observed using a transmission electron microscope (TEM) (Tecnai 10, Philips). Specifically, the nanoemulsion was diluted with pure water to a ratio of 1:200 for detection; after absorption on a copper grid for 2 hours, it was stained with 2% phosphotungstic acid for 30 minutes; the naturally dried sample was then observed using TEM. The results are as follows: Figure 1a As shown, the results indicate that the dark droplets of (BZL+CHX)NE are distributed in a bright environment. BZL and CHX are encapsulated in nanoemulsions to form particles, most of which are between 1 and 200 nanometers in size. No droplet aggregation was observed.

[0144] In addition, the ultrastructure of the nanoemulsion (BZL+CHX)NE prepared in Example 1 was observed using atomic force microscopy (AFM) (IPC-208B, Chongqing University). The results are as follows: Figure 1b As shown, the results indicate that most droplets are spherical and well dispersed.

[0145] Furthermore, according to the manufacturer's instructions, the particle size and zeta potential of the nanoemulsion (BZL+CHX)NE prepared in Example 1 were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd, UK). The particle size characteristics are as follows... Figure 1c As shown, the results indicate that (BZL+CHX)NE exhibits excellent properties, including a small particle size of 15.05±0.139 nm and a PDI value <0.3 (0.139±0.021); the zeta potential is as follows. Figure 1d As shown, the results indicate that the average zeta potential of (BZL+CHX)NE is 19.4±1.217mV.

[0146] The above results demonstrate that the nanoemulsifiers loaded with BZL and CHX possess excellent physicochemical properties.

[0147] II. Physical State of (BZL+CHX)NE

[0148] Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were performed on the nanoemulsion (BZL+CHX)NE prepared in Example 1 and the BZL+CHX prepared in Example 10, respectively, using a TA Instruments Q600 system (Newcastle, USA) heated from room temperature to 200°C at a heating rate of 10°C / min under a nitrogen atmosphere. The results of differential scanning calorimetry (DSC) are as follows: Figure 2a As shown, the results indicate that BZL+CHX and (BZL+CHX)NE reached significant peaks at 85.61℃ and 86.37℃, respectively, suggesting that the encapsulation of the drug in the novel formulation led to a peak shift in the TG curve, with the peak value of the nanoemulsion being much lower than that of the aqueous solution. Thermogravimetric analysis (TGA) results are shown below. Figure 2b As shown, the results indicate that the weight loss patterns of BZL+CHX and (BZL+CHX)NE are similar, both starting at around 24°C. The maximum weight loss ratios of BZL+CHX and (BZL+CHX)NE are 87.63% and 91.84%, respectively.

[0149] In addition, a Lambda 950 spectrometer (Perkin Elmer, Boston, USA) was used at 400 to 4000 cm⁻¹. -1 Within the spectral range at 4cm -1 The Fourier transform infrared (FTIR) spectra of the nanoemulsion (BZL+CHX)NE prepared in Example 1 and the BZL+CHX prepared in Example 10 were measured by 64 scans at a resolution. The results are as follows: Figure 2c As shown in the figure, the results indicate that there are four characteristic peaks at 1120 cm⁻¹, 1640 cm⁻¹, 2110 cm⁻¹, and 3440 cm⁻¹, which correspond to the XY stretching vibration, double bond stretching vibration, CH stretching vibration, and OH stretching vibration, respectively.

[0150] The above results indicate that BZL and CHX compounds have been successfully encapsulated in a self-nanoemulsion system and possess good physicochemical properties.

[0151] III. Antibacterial Effects of Different Antibacterial Agents

[0152] The antibacterial effects of the nanoemulsions prepared in Examples 1 to 9, and Comparative Examples 1 and 2 were tested. Specifically, MRSA bacterial suspensions cultured overnight were taken, and the absorbance value at 595 nm was adjusted to 1. The suspensions were then diluted 1000 times with fresh TSB medium. Each analyte was then diluted to 50 μg / mL with the diluted bacterial suspension, and 200 μL / well was added to a 96-well plate for three replicates. For each replicate, 100 μL / well of the bacterial suspension was added to a 96-well plate. 100 μL of the bacterial suspension was then taken from each well containing 25 μg / mL using a pipette. μL was added to the next concentration well, and then each analyte was serially diluted 2-fold to the 7th concentration using this dilution method. The resulting concentrations were 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL, 0.7825 μg / mL, and 0.39125 μg / mL, respectively. The 96-well plate was incubated overnight at 37°C. Bacterial growth at each concentration was observed visually, and the absorbance (OD) at 595 nm was measured. 595 ), OD 595 A value less than 0.1 indicates no bacterial growth, as shown in the results. Figure 3a As shown, the results indicate that when the mass ratio of BZL to CHX is 4:6 (Example 4), the drug concentration is lowest when there is no bacterial growth, meaning that this formulation has the best antibacterial effect.

[0153] In addition, the antibacterial effects of the antibacterial agents prepared in Examples 1, 10 to 13 were tested (measured at a wavelength of 595 nm (OD)). 595 The optical density of the sample is as follows: Figure 3b As shown in the figure, the results indicate that the nanoemulsifier (BZL+CHX)NE prepared from BZL and CHX exhibits better antibacterial effects compared to other antibacterial agents.

[0154] IV. Stability and Cytotoxicity Testing of Nanoemulsions

[0155] (I) Stability

[0156] The nanoemulsion (BZL+CHX)NE prepared in Example 1 was diluted to 1:200 with deionized water to evaluate its stability. Specifically, the size, PDI, and zeta potential of (BZL+CHX)NE were measured after 0, 1, and 2 years (0 years being the freshly prepared nanoemulsion) at well-ventilated conditions, humidity of 50%–60%, and room temperature (25°C).

[0157] Save the results for different years as follows Figure 4a , 4b As shown in Figure 4c, the results indicate that the size, PDI, and zeta potential of (BZL+CHX)NE did not differ significantly over three years, indicating good stability.

[0158] (II) Cytotoxicity Test

[0159] In the following experiments, the mass ratio of BZL to CHX in BZL+CHX and (BZL+CHX)NE is 4:6.

[0160] (1) The L929 cell line was cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution at 37°C and 5% CO2; L929 cells were seeded into 96-well plates (1×10⁶ cells per well). 4 Add fresh complete DMEM medium to each sample and incubate for 24 hours.

[0161] (2) Dilute BZL+CHX with a total concentration of 10 mg / mL and (BZL+CHX)NE with a total concentration of 10 mg / mL with complete DMEM medium to 100 μg / mL, and then serially dilute to the 10th concentration in a 2-fold gradient. After discarding the cell culture medium, add 100 μL of each of the above-diluted drug concentrations to each well, with 3 replicates for each concentration, and incubate at 37°C and 5% CO2 for 24 hours.

[0162] (3) After treatment, add 20 μL of 5 mg / mL LMTT dye solution to the cells and incubate for 4 hours;

[0163] (4) After incubation, discard the MTT solution and add 150 μL of dimethyl sulfoxide to each well;

[0164] (5) OD at 490 nm was detected using a microplate reader (Thermo Fisher Scientific, USA); cell viability was calculated as follows: viability = (OD sample / OD control) × 100%, where the OD sample and OD control were from cells treated with the drug and untreated cells, respectively.

[0165] Test results as follows Figure 5 As shown, the results indicate that (BZL+CHX)NE exhibits lower cytotoxicity than BZL+CHX. The safe concentrations (survival >90%) for BZL+CHX and (BZL+CHX)NE in L929 cells are ≤3.125 μg / mL and 6.25 μg / mL, respectively. Therefore, (BZL+CHX)NE is stable and exhibits low cytotoxicity to L929 cells.

[0166] V. In vitro antibacterial activity detection

[0167] In the following tests, the mass ratio of BZL to CHX in BZL+CHX and (BZL+CHX)NE is 4:6.

[0168] (I) MIC and MBC Measurement

[0169] The minimum inhibitory concentration (MIC) was determined using the broth microdilution method, specifically including:

[0170] (1) Different concentrations (final concentration of active ingredient) of BZL+CHX and (BZL+CHX)NE were added to a suspension containing MRSA252 bacteria (1×10⁻⁶). 6 In a 96-well microtiter plate (CFU / mL), the final concentrations ranged from 100 μg / mL to 0.78125 μg / mL. After incubation at 37°C for 24 hours, the OD600 was detected using a microplate reader.

[0171] (2) The microtiter plate used for MIC (the lowest concentration that inhibits MRSA growth (OD600 equals blank) is the minimum bactericidal concentration) is used to determine the minimum bactericidal concentration (MBC). Specifically, 5 μL of the suspension without bacterial growth after incubation in step (1) is added to the MH(A) plate and incubated overnight at 37°C. The lowest concentration without bacterial growth is the MBC.

[0172] Test results as follows Figure 6aAs shown, the results indicate that the minimum inhibitory concentration (MIC) of (BZL+CHX)NE against MRSA252 is 1.5625 μg / mL. Figure 6b As shown, the minimum bactericidal concentration (MBC) of (BZL+CHX)NE against MRSA252 was 6.25 μg / mL; however, the MIC and MBC of the aqueous solution against MRSA252 were 3.125 μg / mL and 12.5 μg / mL, respectively. It can be seen that the MIC and MBC of (BZL+CHX)NE were both twice as low as those of BZL+CHX. These results indicate that the nanoemulsion exhibits a stronger antibacterial effect than the aqueous solution.

[0173] (II) Time-Killing Kinetics

[0174] To assess the minimum time required for (BZL+CHX)NE to kill all bacterial cells, time-kinetic kinetics were performed; specifically including:

[0175] (1) Dilute (BZL+CHX)NE to 12.5 μg / mL with MH(B) medium, then take 200 μL of the diluted solution and 200 μL of the prepared MRSA suspension (1×10⁻⁶). 6 The mixture (CFU / mL) was mixed and incubated at 37°C and 220 rpm. After incubation for 0, 1, 2, 4, 8, 16 and 24 hours, 10 μL of the mixture was diluted to a series of multiples, and 5 μL of each concentration gradient dilution was added to MH(A) plates for incubation to identify surviving bacteria.

[0176] (2) After incubating on a plate for 24 hours, count the bacterial colonies and calculate the bacterial concentration in the original mixture based on the dilution factor.

[0177] Bacterial colony count as follows Figure 6c As shown, the results indicate that at the same time point, the number of bacteria in the system treated with (BZL+CHX)NE was less than that in the system treated with BZL+CHX. At a concentration of 6.25 μg / mL, (BZL+CHX)NE killed approximately 90% of MRSA252 within 30 min and all bacteria within 4 hours; however, at the same concentration, BZL+CHX required 1 hour to kill 90% of MRSA252. Therefore, (BZL+CHX)NE exhibits more effective and faster bactericidal activity than BZL+CHX.

[0178] VI. In vivo antibacterial activity test

[0179] In the following tests, the mass ratio of BZL to CHX in BZL+CHX and (BZL+CHX)NE is 4:6.

[0180] (I) Grouping

[0181] A trauma model was established using female BALB / c mice (6-8 weeks old, weighing 18±2g, Beijing Huafukang Biotechnology). The process included anesthetizing the mice with an air isoflurane anesthesia machine, preparing the skin, and performing a 1cm×1cm wound surgery on the back of each mouse to obtain the trauma model mice.

[0182] Mice with trauma models were randomly divided into four groups of five mice each: a negative control group, an MRSA 252 control group, a 5 mg / mL (BZL+CHX) NE group, and a 5 mg / mL BZL+CHX group. Except for the negative control group, the other three groups of mice were inoculated at the wound site with 50 μL of the prepared MRSA suspension (1×10⁻⁶). 7 CFU / mL); 24 hours after inoculation, except for the negative control group, the other groups of mice were treated with 50μL 5mg / mL BZL+CHX, 50μL 5mg / mL (BZL+CHX)NE and 50μL physiological saline solution, respectively, twice a day, morning and evening, with an interval of 8 hours.

[0183] (ii) Bacterial colony count

[0184] On days 0, 3, 5, 7 and 14 after drug administration, the wound of each mouse was scraped ten times with a sterile cotton swab; then, the cotton swab was immersed in 1 mL of sterile PBS solution for 5 minutes to obtain a suspension; the suspension was diluted to a series of multiples, and 5 μL of each dilution was added to MH(A) plates and incubated at 37°C for 24 hours, and the bacterial colonies in the wound were counted.

[0185] The results are as follows Figure 7a As shown, the results indicated that, compared to the same dose of BZL+CHX, the bacterial count in mice treated with 5 mg / mL (BZL+CHX)NE decreased much faster; on the first day after treatment, the bacterial count in the 5 mg / mL (BZL+CHX)NE-treated group decreased by 90%, while the 5 mg / mL BZL+CHX-treated group only saw a 40% reduction; by day 14, the bacterial count in the 5 mg / mL (BZL+CHX)NE-treated group had dropped to zero. These results suggest that (BZL+CHX)NE has stronger bactericidal activity against MRSA infection in skin wounds.

[0186] (III) Wound Healing Assessment

[0187] Wound area was measured on days 0, 3, 5, 7, and 14 of the initial medication treatment, and the relative wound area ratio was calculated: Relative wound area ratio (%) = Wound area on specified days / Wound area on day 0 × 100%. The results are shown below. Figure 7bAs shown, the results indicated that the wound healing rate in mice treated with (BZL+CHX)NE was similar to that in mice not infected with MRSA, but significantly faster than that in mice treated with BZL+CHX (P<0.05).

[0188] In addition, photographs of the overall appearance of the wound were taken on days 0, 3, 5, 7, and 14 after the start of medication, and the results were as follows: Figure 7c As shown in the figure, the results indicate that the wound healing rate of mice treated with (BZL+CHX)NE was significantly faster than that of mice treated with BZL+CHX.

[0189] In addition, skin samples were taken from the wound site on day 14 of the start of medication and fixed with 4% formaldehyde; the skin samples were embedded in paraffin and sectioned; histological analysis was performed by hematoxylin and eosin (H&E) staining. Results are as follows: Figure 7d As shown, the results indicated that uninfected MRSA skin wounds were largely intact (normal skin consists of three layers: epidermis, dermis, and subcutaneous layer), with few inflammatory cells found beneath the dermis. In contrast, skin wounds treated with BZL+CHX were similar to the MRSA-infected control group, exhibiting discontinuous skin with a large number of inflammatory cells distributed beneath the dermis (indicated by red arrows). However, skin wounds treated with (BZL+CHX)NE resembled uninfected MRSA skin wounds, with very few inflammatory cells found beneath the dermis. These results demonstrate that (BZL+CHX)NE has a more effective bactericidal activity against MRSA infection in skin wounds.

[0190] VII. Anti-biofilm activity test

[0191] In the following tests, the mass ratio of BZL to CHX in BZL+CHX and (BZL+CHX)NE is 4:6.

[0192] In the following tests, the SEM observation method was as follows: the biofilm on the coverslip was washed three times with 0.1M PBS and fixed with 2.5% glutaraldehyde; then, it was dehydrated with a series of ethanol (10%, 30%, 50%, 70%, 90%, and 100%) and isobutanol (10%, 30%, 50%, 70%, 90%, and 100%); subsequently, the sample was treated with a sputter coater and treated with hexamethyldisilazane for 10 minutes, followed by a gold coating; finally, the sample was observed using a scanning electron microscope (SEM) (AMRAY1000 B, Amray Inc., Bedford, Massachusetts, USA).

[0193] In the following tests, the CLSM observation method included: labeling live and dead cells with SYTO-9 dye (green) and propidium iodide (PI) (red), respectively; staining the prepared biofilm with 6 μM SYTO-9 dye and 30 μM PI at room temperature for 15 minutes; then observing the biofilm using a confocal laser scanning microscope (CLSM 780, Zeiss, Oberkochen, Germany); generating the maximum projection of all image stacks using ZEN soft (Zeiss) and quantifying the red (dead) and green (live) regions in the biofilm; and calculating the percentage of live cells and the dead / live ratio.

[0194] In the following tests, the biofilm was quantified as follows: the biofilm was washed twice with 0.1M PBS, fixed with methanol for 15 minutes, and then stained with 1 mg / mL crystal violet for 15 minutes; after washing again, the biofilm was dissolved with 1 mL of 30% (v / v) acetic acid; the OD at 570 nm was detected using a microplate reader (Bio-Rad Laboratories Inc.).

[0195] (I) Biomembrane Inhibition Effect Test

[0196] 1.8 mL of MRSA 252 bacterial suspension (prepared according to the above method) (1×10 7 (CFU / mL) was added to 0.2 mL of (BZL+CHX)NE at 2 times the MIC and 0.2 mL of BZL+CHX at 2 times the MIC in 24-well plates (the plates were in TSB medium containing 0.5% (w / v) glucose and 2% (w / v) sodium chloride) with coverslips for SEM and CLSM observation, and incubated at 37°C for 24 hours. A control group was also set up with only TSB medium containing 0.5% (w / v) glucose and 2% (w / v) sodium chloride without any antibacterial agents.

[0197] 1) SEM observation and biofilm quantification

[0198] After incubation, the integrity, surface morphology, and ultrastructural changes of the MRSA biofilm in each well were observed using scanning electron microscopy. The results showed that the biofilm images of the control group showed dense biofilms, with cells maintaining their original spherical shape and smooth, intact surfaces. Figure 8a The biofilm in the BZL+CHX group showed almost no change, and the cells maintained their original morphology. Figure 8b However, MRSA treated with (BZL+CHX)NE formed a thin biofilm with numerous visible pores on the cell membrane, making the cell capsule indistinct. Furthermore, aggregated and ruptured cells were also observed. Figure 8c ).

[0199] In addition, after incubation, the MRSA cell membranes formed in each well were quantitatively analyzed, and the results were as follows: Figure 8d As shown in the results, the biofilm formed by MRSA treated with (BZL+CHX)NE was thinner than that formed by BZL+CHX, indicating that the nanoemulsion had a stronger inhibitory effect on biofilm formation.

[0200] (ii) CLSM observation

[0201] After incubation, the MRSA biofilm in each well was observed using a fluorescence microscope (live bacteria were stained green, and dead bacteria were stained red). The results showed that the biofilm image of the control group was mainly covered by green fluorescence. Figure 8e The same applies to the BZL+CHX group. Figure 8f However, images of the (BZL+CHX)NE group show more red fluorescence. Figure 8g In addition, the proportion of live bacteria and the dead / live ratio were calculated based on the average fluorescence intensity of all groups, and the results are as follows: Figure 8h As shown, the results are consistent with the images, indicating that (BZL+CHX)NE has a stronger inhibitory effect on biofilm formation.

[0202] (II) Biofilm removal efficiency test

[0203] Add 2 mL of MRSA252 bacterial suspension (1×10⁻⁶) 7 CFU / mL was added to a 24-well plate (the plate was infused with TSB medium containing 0.5% (w / v) glucose and 2% (w / v) sodium chloride) (with coverslips for SEM and CLSM observation), and incubated at 37°C for 24 hours. After incubation, the supernatant was removed, and the biofilm was treated with 0.2 mL of 2×MIC (BZL+CHX)NE and 0.2 mL of 2×MIC BZL+CHX for 8 hours, respectively. In addition, a control group was set up with bacterial culture under the same conditions but without the drug.

[0204] 1) SEM observation and biofilm quantification

[0205] After treatment, the integrity, surface morphology, and ultrastructural changes of the MRSA biofilm in each well plate were observed using scanning electron microscopy. The results showed that the biofilm treated with BZL+CHX (… Figure 9b ) and control group ( Figure 9a Similar to the first type, the first type exhibits a dense and thick biofilm, with cells maintaining their original spherical shape and a smooth, intact surface; however, the biofilm treated with (BZL+CHX)NE is sparse and incomplete, with damaged cells and many visible pores on the membrane. Figure 9c ).

[0206] In addition, after the treatment, the MRSA cell membranes formed in each well were quantitatively analyzed, and the results are as follows: Figure 9d As shown, the results indicate that the biofilm formed by MRSA treated with (BZL+CHX)NE is thinner than that formed by MRSA treated with BZL+CHX.

[0207] The above results indicate that (BZL+CHX)NE can effectively remove biofilms compared to aqueous solutions.

[0208] (ii) CLSM observation

[0209] After treatment, the MRSA biofilm in each well was observed using a fluorescence microscope (live bacteria were stained green, and dead bacteria were stained red). The results showed that the MRSA control group ( Figure 9e ) and BZL+CHX treated biofilms ( Figure 9f The green fluorescence was significantly greater than the red fluorescence; while the (BZL+CHX)NE group ( Figure 9g The group that showed higher red fluorescence than the other groups also showed higher average fluorescence intensity. Figure 9h This also indicates that (BZL+CHX)NE induced a greater number of dead bacteria than BZL+CHX. These results confirm that (BZL+CHX)NE has a stronger scavenging effect on MRSA biofilms.

[0210] (III) Cell damage research

[0211] 1) Observing ultrastructure using TEM

[0212] Add 1 mL of MRSA252 bacterial suspension (1×10⁻⁶) 7 The samples were collected by centrifugation at 5000 rpm (CFU / mL) and treated with 1 mL of (BZL+CHX)NE and BZL+CHX at 2 times MIC for 1 hour, respectively. After treatment, the samples were washed twice with 0.1 M PBS solution and fixed with 2.5% (v / v) glutaraldehyde, and then treated with 1% OsO4 (w / v) at room temperature for 1 hour. Subsequently, the samples were washed twice and dehydrated with a series of ethanol solutions (50%, 70%, 80%, 90%, 95%, and 100%) in ascending order of concentration. The samples were then embedded in epoxy resin and cut into ultrathin sections using an ultramicrotome (Ultracut E, SuperNova, Reichert-JungOptische Werke, Austria), and stained with alkaline lead citrate and uranium acetate. Finally, the samples were observed using a transmission electron microscope (TEM) (PHILIPS TECNAI-10, Netherlands).

[0213] The results are as follows Figure 10 As shown, the results indicate that the original MRSA cell membrane morphology is as follows: Figure 10As shown in Figure a, the cells are nearly spherical, with plump cytoplasm, intact cell membranes, and smooth surfaces; while MRSA treated with BZL+CHX ( Figure 10 c) Similar to the control group, no significant changes were observed; conversely, (BZL+CHX)NE significantly damaged the MRSA cell membrane, exhibiting many obvious changes (indicated by red arrows). Figure 10 (b) These processes include vacuolar formation, cell membrane damage, and intracellular efflux. Therefore, (BZL+CHX)NE exhibits significantly higher bactericidal activity against MRSA than its aqueous solution.

[0214] II) Determination of hydrophobicity

[0215] To confirm the effect of the novel emulsion on the maturation and aggregation stages of MRSA biofilms, cell surface hydrophobicity (CSH) was further measured, specifically by adding 2 mL of MRSA252 bacterial suspension (1×10⁻⁶). 8 CFU / mL was added to 24-well microplates (the plates were infused with TSB medium containing 0.5% (w / v) glucose and 2% (w / v) sodium chloride), and the plates were treated with a series of concentrations of (BZL+CHX)NE and BZL+CHX for 16 hours. The OD600 value was then measured and recorded as A0. Next, 0.4 mL of xylene was added, and the samples were incubated at room temperature for 1 hour, and the OD600 value was measured and recorded as A. The surface hydrophobicity index was calculated as: A% = (A0 - A) / A0 × 100%.

[0216] Surface hydrophobicity indices of different groups, such as Figure 11 As shown, the results indicate that (BZL+CHX)NE significantly reduced the surface hydrophobicity index at multiple concentrations, suggesting that the nanoemulsion caused damage to the cell wall and membrane. Furthermore, (BZL+CHX)NE caused more significant damage to the MRSA cell membrane than BZL+CHX.

[0217] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antibacterial composition, characterized in that, It consists of A and B, where A is benzalkonium bromide or its nanoemulsion, and B is chlorhexidine acetate or its nanoemulsion.

2. The antibacterial composition according to claim 1, characterized in that, The mass ratio of A to B is (1-9):(1-9).

3. The antibacterial composition according to claim 2, characterized in that, The mass ratio of A to B is 4:

6.

4. An antibacterial nanoemulsion, characterized in that, This includes benzalkonium bromide and chlorhexidine acetate.

5. The antibacterial nanoemulsion according to claim 4, characterized in that, The mass ratio of benzalkonium bromide to chlorhexidine acetate is (1-9):(1-9).

6. The antibacterial nanoemulsion according to claim 4 or 5, characterized in that, Antibacterial nanoemulsions also include surfactants, co-surfactants, and an oil phase.

7. The antibacterial nanoemulsion according to claim 6, characterized in that, Antibacterial nanoemulsions meet one or more of the following conditions: (i) The surfactant is one or more of the following: castor oil polyoxyethylene 35 ether, Tween-80, castor oil polyoxyethylene ether, polyoxyethylene 40 hydrogenated castor oil or lecithin. (ii) The co-surfactant is one or more combinations of propylene glycol, ethanol, glycerol or polyethylene glycol 400; (iii) The oil phase is one or more of isopropyl myristate, soybean oil, ethyl oleate or medium-chain triglycerides; (iv) The mass ratio of surfactant, co-surfactant and oil phase is (6-8):(1-3):

1.

8. An antibacterial agent, characterized in that, This includes the antibacterial composition according to any one of claims 1 to 3 or the antibacterial nanoemulsion according to any one of claims 4 to 7.

9. Use of the antimicrobial composition according to any one of claims 1 to 3, or the antimicrobial nanoemulsion according to any one of claims 4 to 7, or the antimicrobial agent according to claim 9, wherein the use includes: (a) Use of antimicrobial compositions, antimicrobial nanoemulsions, or antimicrobial agents against methicillin-resistant Staphylococcus aureus; or (b) Use of an antimicrobial composition, antimicrobial nanoemulsion, or antimicrobial preparation in the preparation of a medicament for the prevention of wound infection by methicillin-resistant Staphylococcus aureus; or (c) Use of antimicrobial compositions or antimicrobial nanoemulsions or antimicrobial preparations in the preparation of medicaments for wound healing of wounds infected with methicillin-resistant Staphylococcus aureus.

10. The use according to claim 9, characterized in that, Antimicrobial compositions, antimicrobial nanoemulsions, or antimicrobial agents have one or more of the following functions: (a) It has the function of inhibiting the growth of methicillin-resistant Staphylococcus aureus biofilm; (b) It has the function of clearing biofilms of methicillin-resistant Staphylococcus aureus; (c) It has the function of disrupting the cell wall and cell membrane integrity of methicillin-resistant Staphylococcus aureus; (d) It has the function of reducing the hydrophobicity of the cell membrane of methicillin-resistant Staphylococcus aureus.

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