Preparation method of quaternary ammonium salt antibacterial fiber, and preparation method and application thereof

Abstract

The present invention relates to the technical field of antibacterial material preparation, and particularly relates to a preparation method and application of quaternary ammonium salt antibacterial fibers. The present invention provides a preparation method of quaternary ammonium salt antibacterial fibers, which comprises the following steps: (1) mixing methanol and epibromohydrin to obtain solvent 1; mixing quaternary ammonium salt and methanol to obtain solvent 2; (2) dropwise adding the solvent 1 into the solvent 2 for a first reaction, removing methanol to obtain a product, and recrystallizing the product and then drying it under vacuum to obtain a quaternary ammonium salt intermediate; (3) mixing the quaternary ammonium salt intermediate, dextran, NaOH and water for a second reaction, adjusting the pH, followed by dialysis and freeze-drying to obtain the quaternary ammonium salt antibacterial fibers. The present invention connects QAS to the Dextran molecular backbone to form a novel QAS polymer, improving the blood compatibility, biocompatibility and antibacterial property of the antibacterial polymer, which is an effective way to develop novel antibacterial materials.

Description

Technical Field

[0001] The present invention relates to the technical field of antibacterial material preparation, and particularly relates to a preparation method and application of quaternary ammonium salt antibacterial fibers. Background Art

[0002] Potential sources of bacterial infection can spread through droplets, contact, or blood, leading to bacterial infections and even death. Currently, antibiotics are mainly used clinically to treat bacterial infections. With the widespread use of antibiotics, the problem of bacterial drug resistance has become increasingly prominent. Long-term use of multiple antibiotics can lead to multiple drug-resistant infections and may result in high mortality. To prevent multiple drug-resistant infections, the development of new and effective non-antibiotic bacteriostatic agents has become inevitable.

[0003] Staphylococcus aureus (referred to as S. aureus), is a common human pathogen. S. aureus can invade the underlying layer or blood of the body, causing inflammation. The resistance of S. aureus to antibiotics makes treatment more complex and becomes one of the most difficult problems in current clinical practice.

[0004] The problem of drug resistance is becoming increasingly serious. When a drug is infected by bacteria, it will develop into a new biofilm. A biofilm is a microbial community composed of an extracellular matrix produced by bacteria. Existing experiments have shown that to kill the bacteria in the biofilm, more than 10 million times the amount of antibacterial agent is required. The enhancement of these drug resistances is due to the limitation of the biofilm or the repulsive force of the biofilm, resulting in a very low penetration rate of most drugs, making the cells inside directly exposed to the antibiotics. In addition, due to the slow growth and low metabolic activity of the cells in the body, this will also inhibit and weaken the effect of antibiotics, especially drugs that inhibit bacterial activity. The biofilm formed by S. aureus not only has good adaptability to the surrounding environmental conditions, but also has the ability to evade the body's specific and non-specific immunity, which will increase the durability and reversibility of the implant, causing great difficulties in clinical treatment.

[0005] In recent years, antibacterial materials have been developed to address bacterial infections and drug resistance issues. These materials mainly include metal-organic frameworks (MOFs), transition metals, organic materials, carbon-based materials, and noble metal materials, etc. MOF-based antibacterial materials have excellent antibacterial capabilities, but their biocompatibility still needs to be addressed. Transition metal materials have advantages such as good physiological stability and semiconduction, but their preparation process is complex and their biosafety still needs to be improved. Organic materials can be flexibly processed and have a certain degree of biocompatibility, but their synthesis process is complex. Although carbon-based materials have advantages such as adjustable parameters, good thermal stability, and low cost, they have potential toxicity and poor antibacterial performance. Therefore, developing antibacterial materials with high biocompatibility, high biosafety, and strong antibacterial ability is an important topic in antibacterial research.

[0006] Quaternary ammonium salts are a type of cationic surfactant with good bactericidal properties. All four hydrogen atoms in the ammonium ion of quaternary ammonium salts are replaced by hydrocarbon groups, which can adsorb negatively charged groups in the bacterial cell wall, such as teichoic acid and lipoteichoic acid, and damage the cell wall. The non-polar action of the hydrophobic long chain of quaternary ammonium salts can penetrate the membrane of the bacterial cell wall and lyse the bacterial cell wall. By destroying the bacterial cell wall, the bacterial contents leak out, thus killing the bacteria. Bacteria use sugars or chemical synthesis to produce dextran, which has a large number of hydroxyl groups and is easily mixed with drugs and proteins through non-covalent or covalent interactions. Due to its good physicochemical properties and minimal cytotoxicity to cells, dextran has become the most promising macromolecular carrier for a series of drugs. Dextran is a type of branched dextran that can improve blood compatibility and has various effects on blood coagulation homeostasis, including inhibition of platelet activation and inhibition of red blood cell formation.

[0007] Although quaternary ammonium salt polymers (QAS) have high antibacterial efficiency, proteins and red blood cells in damaged tissues are prone to bind to the QAS moiety, and the blood compatibility and biocompatibility of QAS-based antibacterial materials are still insufficient. Therefore, improving blood compatibility and biocompatibility is a difficult problem in the research and development of QAS antibacterial materials. Summary of the Invention

[0008] The purpose of the present invention is to provide a silver-plated polyamide fiber, its preparation method, and its application. In the present invention, QAS is connected to the Dextran molecular backbone to form a novel QAS polymer, which improves the blood compatibility, biocompatibility, and antibacterial properties of antibacterial polymers, and is an effective way to develop novel antibacterial materials.

[0009] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0010] The present invention provides a preparation method of quaternary ammonium salt antibacterial fiber, which comprises the following steps:

[0011] (1) Mix methanol and epibromohydrin to obtain solvent 1; mix quaternary ammonium salt and methanol to obtain solvent 2;

[0012] (2) Dropwise add the solvent 1 into the solvent 2 for a primary reaction, remove methanol to obtain a product, recrystallize the product and then vacuum dry it to obtain a quaternary ammonium salt intermediate;

[0013] (3) Mix the quaternary ammonium salt intermediate, dextran, NaOH and water for a secondary reaction, adjust the pH, then dialyze and freeze-dry to obtain the quaternary ammonium salt antibacterial fiber.

[0014] Preferably, the quaternary ammonium salt in step (2) is one of N,N-dimethyldodecylamine, N,N-dimethyltetradecylamine, N,N-dimethylhexadecylamine and N,N-dimethyloctadecylamine.

[0015] Preferably, the volume-mass ratio of methanol to epibromohydrin in step (1) is 4-6 ml: 6.5-7.5 g; the mass-volume ratio of the quaternary ammonium salt to methanol is 8-10 g: 4-6 ml.

[0016] Preferably, the primary reaction in step (2) is carried out under the protection of nitrogen; the temperature of the primary reaction is 40-50 °C; the time of the primary reaction is 1.5-2.5 h.

[0017] Preferably, the number of recrystallizations in step (2) is 4-6 times; the temperature of the vacuum drying is 35-45 °C, and the time is 20-28 h.

[0018] Preferably, the mass-volume ratio of the quaternary ammonium salt intermediate, dextran, NaOH and water in step (3) is 5-9 g: 3-5 g: 3-5 g: 90-110 ml.

[0019] Preferably, the temperature of the secondary reaction in step (3) is 45-55 °C, and the time is 2-4 h.

[0020] Preferably, the pH is adjusted to 6.8-7.2 in step (3); the dialysis time is 2-4 d.

[0021] The present invention also provides the quaternary ammonium salt antibacterial fiber prepared by the above-mentioned preparation method.

[0022] The present invention also provides the application of the quaternary ammonium salt antibacterial fiber prepared by the above-mentioned preparation method or the quaternary ammonium salt antibacterial fiber in the preparation of antibacterial or wound surface healing promoting drugs or protective articles.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Quaternary ammonium salt polymers are a type of cationic antibacterial agent that can penetrate into bacteria, disrupt electrolytes, and damage cell membranes, thereby playing a bactericidal role. The cationic quaternary ammonium groups in the QAS structure can be fixed or anchored on the polymer chain. Research has shown that the antibacterial properties of QAS are related to its structure. As the alkyl chain length on the nitrogen atom in the quaternary ammonium salt increases, the antibacterial effect of QAS enhances. When the chain length is 12 - 18 carbon atoms, the antibacterial effect is optimal.

[0025] In this invention, quaternary ammonium salt intermediates (QASI) with different carbon chain lengths were first synthesized, and then QASI was grafted onto dextran to construct novel quaternary ammonium salt antibacterial fibers - quaternary ammonium salt dextran fibers (QASAF). They are: QASAF-C12, QASAF-C14, QASAF-C16, and QASAF-C18. Then, their structures were characterized by nuclear magnetic resonance hydrogen spectroscopy, and their biocompatibility and biosafety were evaluated through hemolysis and cytotoxicity. Finally, through antibacterial growth curves, plate coating experiments, and MIC value determination, the antibacterial properties of QASAF against Staphylococcus aureus (S. aureus) and Methicillin-resistant Staphylococcus aureus (MRSA) were studied, and the role of QASAF in promoting wound healing in infected mice was evaluated through a mouse wound model. Brief Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0027] Figure 1 For the synthesis of quaternary ammonium salt dextran fibers and their in vitro and in vivo mechanisms.

[0028] Figure 2 For the synthesis route of quaternary ammonium salt intermediate compounds.

[0029] Figure 3 For the synthesis route of quaternary ammonium salt antibacterial fibers with different carbon chains.

[0030] Figure 41H NMR of (A) QASI-C12, (B) QASI-C14, (C) QASI-C16, and (D) QASI-C18.

[0031] Figure 5 1H NMR of (A) QASAF-C12, (B) QASAF-C14, (C) QASAF-C16, and (D) QASAF-C18.

[0032] Figure 6 Hemolysis rates of QASAF-C12, QASAF-C14, QASAF-C16, and QASAF-C18.

[0033] Figure 7 Relative cell viability of Hep G2 incubated with media treated with different concentrations of QASAF.

[0034] Figure 8 Cell morphology and survival status of QASAF and Hep G2 at 75 μg / mL.

[0035] Figure 9 Bacterial survival rate after co-culturing QASAF and Staphylococcus aureus for 10 h.

[0036] Figure 10 Growth kinetic curves of co-culturing QASAF-C18 and Staphylococcus aureus at different concentrations.

[0037] Figure 11 Co-culturing QASAF-C18 and Staphylococcus aureus at different concentrations. (A) Dilution plating observation and (B) quantitative analysis.

[0038] Figure 12 Mouse subcutaneous infection model. (A) Timeline and (B) injection sites of Staphylococcus aureus and QASAF-C18.

[0039] Figure 13 Tissue bacterial dilution plating observation and quantitative analysis.

[0040] Figure 14 Tissue sections of heart, liver, spleen, lung, and kidney (scale bar = 100 μm).

[0041] Figure 15 Wound healing of mice treated with QASAF-C18 (scale bar = 1 cm).

[0042] Figure 16 Wound healing ratio of mice treated with QASAF-C18 (n = 3). (**P < 0.01).

[0043] Figure 17 Wound healing area of mice treated with QASAF-C18.

[0044] Figure 18 H&E staining and Masson staining of mouse skin tissue sections in the wound healing model.

[0045] Figure 19 Bacterial survival rate after co - culturing QASAF and MRSA for 10 h.

[0046] Figure 20 Growth kinetic curve of co - culturing QASAF - C12 and MRSA.

[0047] Figure 21 (A) Dilution coating observation and (B) quantitative analysis of co - culturing QASAF - C12 with MRSA.

[0048] Figure 22 For subcutaneous injection of MRSA in mice, QASAF - C12 site; (B) Dilution coating observation of bacteria in QASAF - C12 tissue; (C) Quantitative analysis.

[0049] Figure 23 Tissue sections of mouse heart, liver, spleen, lung and kidney (scale bar = 100 μm).

[0050] Figure 24 Wound healing of mice treated with QASAF - C12 (scale bar = 1 cm).

[0051] Figure 25 Wound healing ratio of mice treated with QASAF - C12 (n = 3). (**P < 0.01).

[0052] Figure 26 Wound healing area of mice with wound infection treated with QASAF - C12.

[0053] Figure 27 H&E and Masson staining of tissue sections of the epidermis of mice with wound infection treated with QASAF - C12 (scale bar = 500 μm). Detailed implementation manner

[0054] The present invention provides a preparation method of quaternary ammonium salt antibacterial fiber, which comprises the following steps:

[0055] (1) Mix methanol and epibromohydrin to obtain solvent 1; mix quaternary ammonium salt and methanol to obtain solvent 2;

[0056] (2) Dropwise add the solvent 1 into the solvent 2 for a one - time reaction, remove methanol to obtain a product, and recrystallize the product and then vacuum - dry it to obtain a quaternary ammonium salt intermediate;

[0057] (3) Mix the quaternary ammonium salt intermediate, dextran, NaOH, and water for a secondary reaction. After adjusting the pH, dialyze and freeze-dry to obtain the quaternary ammonium salt antibacterial fiber.

[0058] In the present invention, the quaternary ammonium salt described in step (2) is one of N,N-dimethyldodecylamine, N,N-dimethyltetradecylamine, N,N-dimethylhexadecylamine, and N,N-dimethyloctadecylamine; preferably N,N-dimethyldodecylamine.

[0059] In the present invention, the volume-mass ratio of methanol to epibromohydrin in step (1) is 4 - 6 ml: 6.5 - 7.5 g; preferably 5 ml: 6.7 - 7.3 g; more preferably 5 ml: 6.9 - 7.1 g; most preferably 5 ml: 7 g.

[0060] In the present invention, the mass-volume ratio of the quaternary ammonium salt to methanol in step (1) is 8 - 10 g: 4 - 6 ml; preferably 9 g: 4 - 6 ml; more preferably 9 g: 5 ml.

[0061] In the present invention, the primary reaction in step (2) is carried out under the protection of nitrogen.

[0062] In the present invention, the temperature of the primary reaction in step (1) is 40 - 50 °C; preferably 42 - 48 °C; more preferably 44 - 46 °C; most preferably 45 °C.

[0063] In the present invention, the time of the primary reaction in step (2) is 1.5 - 2.5 h; preferably 2 h.

[0064] In the present invention, the number of recrystallizations in step (2) is 4 - 6 times; preferably 5 times.

[0065] In the present invention, the temperature of the vacuum drying in step (1) is 35 - 45 °C; preferably 37 - 43 °C; more preferably 39 - 41 °C; most preferably 40 °C.

[0066] In the present invention, the time of the vacuum drying in step (1) is 20 - 28 h; preferably 21 - 27 h; more preferably 22 - 26 h; most preferably 24 h.

[0067] In the present invention, the mass-volume ratio of the quaternary ammonium salt intermediate, dextran, NaOH, and water in step (3) is 5 - 9 g: 3 - 5 g: 3 - 5 g: 90 - 110 ml; preferably 6 - 8 g: 4 g: 4 g: 94 - 106 ml; more preferably 7 g: 4 g: 4 g: 98 - 102 ml; most preferably 7 g: 4 g: 4 g: 100 ml.

[0068] In the present invention, the temperature of the secondary reaction in step (3) is 45 to 55 °C; preferably 47 to 53 °C; more preferably 49 to 51 °C; and even more preferably 50 °C.

[0069] In the present invention, the time of the secondary reaction in step (3) is 2 to 4 h; preferably 3 h.

[0070] In the present invention, the pH is adjusted to 6.8 to 7.2 in step (3); preferably 6.9 to 7.1; more preferably 7.0.

[0071] In the present invention, the dialysis time in step (2) is 2 to 4 d; preferably 3 d.

[0072] The present invention also provides a quaternary ammonium salt antibacterial fiber prepared by the described preparation method.

[0073] The present invention also provides the application of the quaternary ammonium salt antibacterial fiber prepared by the described preparation method or the described quaternary ammonium salt antibacterial fiber in the preparation of antibacterial or wound healing promoting drugs or protective articles.

[0074] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0075] Example 1

[0076] Preparation and Characterization of 1QASAF

[0077] 1.1 Materials and Methods

[0078] 1.1.1 Experimental Materials

[0079] (1) Experimental Animals

[0080] Kunming mice were purchased from the Experimental Animal Center of the Medical College and met the SPF standard. All experimental animals were carried out in the standardized laboratory of the Animal Experiment Center of Guilin Medical College and strictly followed the "3R" experimental animal ethical guidelines and norms. The animal ethics number was: GLMC202103275.

[0081] (2) Experimental Cells

[0082] Hep G2 cells were cryopreserved and stored in the Laboratory of Biochemistry and Molecular Biology of Guilin Medical College.

[0083] (3) Experimental Reagents

[0084] N,N-dimethyldodecylamine, N,N-dimethyltetradecylamine, N,N-dimethylhexadecylamine, N,N-dimethyloctadecylamine, methanol epibromohydrin, dextran, deuterated dimethyl sulfoxide (DMSO-d6), heavy water (D2O), anticoagulant were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; ethyl acetate, sodium hydroxide, glacial acetic acid, ethanol, potassium bromide, normal saline were purchased from Sinopharm Reagent Co., Ltd.; penicillin, streptomycin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beijing Solarbio Science & Technology Co., Ltd.; high-glucose DMEM medium, fetal bovine serum were purchased from Gibco Company of the United States; trypsin was purchased from Hyclone Company of the United States; 2.5% glutaraldehyde was purchased from Beijing Huanyu Kechuang Biotechnology Development Co., Ltd.; CCK-8 kit was purchased from Shanghai Dongren Technology Co., Ltd.

[0085] (4) Experimental instruments

[0086] Multiskan FC microplate reader was purchased from Jiangsu Scientific Instruments Co., Ltd. (51119000); freeze dryer LC-10N-50A was purchased from Shanghai Lichen Bangxi Instrument Technology Co., Ltd.; nuclear magnetic resonance spectrometer

[0087] DRX 400MHz was purchased from Bruker Company of Germany; Zeta potential analyzer Zetasizer Nano ZS was purchased from Malvern Company of the United Kingdom; field emission scanning electron microscope FE-SEMS4800 was purchased from Hitachi Company of Japan; cell incubator ThermoScientific TM 370 was purchased from Thermo Company of the United States.

[0088] 1.1.2 Experimental methods

[0089] (1) Synthesis of quaternary ammonium salt intermediates with different carbon chains

[0090] To synthesize the quaternary ammonium salt intermediate QASI with a straight-chain alkane carbon chain length of C12, N,N-dimethyldodecylamine and epibromohydrin were used as raw materials, and the synthesis route is as Figure 2 shown.

[0091] Specifically: Add N,N-dimethyldodecylamine (9.8085 g) and methanol (anhydrous methanol) (5 mL) into a 100 mL two-necked round-bottom flask respectively. Then, dropwise add the mixed solution of methanol (5 mL) and epibromohydrin (7.2012 g) (purity 98%) into the above two-necked round-bottom flask, and continuously react at 45 °C for 2 h under nitrogen protection. After the reaction is completed, remove methanol under reduced pressure by a rotary evaporator. Recrystallize the obtained product 5 times with ethyl acetate (analytical pure, 99.5%), with 200 mL of ethyl acetate used each time, and vacuum dry at 40 °C for 24 h to obtain the product, named QASI-C12.Figure 2 Synthesis routes of each quaternary ammonium salt intermediate compound.

[0092] For the synthesis of quaternary ammonium salt intermediates with linear alkane carbon chain lengths of C14, C16, and C18 respectively, N,N-dimethyltetradecylamine, N,N-dimethylhexadecylamine, N,N-dimethyloctadecylamine, and epibromohydrin are selected as raw materials, and the synthesis method is the same as above.

[0093] The synthesized quaternary ammonium salt intermediate compounds are named QASI-C14, QASI-C16, and QASI-C18 respectively, and each compound is characterized by 1H NMR.

[0094] (2) Synthesis of quaternary ammonium salt antibacterial fibers with different carbon chains

[0095] The synthesis routes of different QASAFs are as Figure 3 shown. Specifically, 4.0 g of dextran is dissolved in 400 mL of distilled water, 7.0 g of C12 quaternary ammonium salt intermediate (QASI-C12) and 4.0 g of NaOH (purity 98%) are added, and the reaction is carried out continuously at 50 °C for 3 h. After the reaction is completed, it is cooled to room temperature, neutralized to neutral with glacial acetic acid (purity 99.8%), transferred to a 3.5 kd dialysis bag and dialyzed with pure water for three days, and finally freeze-dried to obtain C12 quaternary ammonium salt antibacterial fiber, named QASAF-C12.

[0096] The method for synthesizing quaternary ammonium salt antibacterial fibers with linear alkane carbon chain lengths of C14, C16, and C18 respectively is the same as above. The obtained quaternary ammonium salt antibacterial fiber compounds are named QASAF-C14, QASAF-C16, and QASAF-C18 respectively, and each compound is characterized by 1H NMR. The results are as Figure 4 shown.

[0097] (3) Nuclear magnetic resonance spectroscopy (NMR) analysis of QASI and QASAF

[0098] Approximately 20 mg of solid-phase samples of different QASI or QASAF are respectively placed in a clean NMR tube, and 0.5 mL of DMSO-d6 (deuterated dimethyl sulfoxide) solvent is added to each with a pipette for dissolution. Then, at room temperature, the 1H NMR of each compound is measured using a 400 MHz Bruker AVANCE III HD MRI nuclear magnetic resonance spectrometer. 1 1H NMR). The results are as Figure 5 shown.

[0099] Results: By comparing with the nuclear magnetic resonance hydrogen spectra of the raw material epibromohydrin and each raw material (N,N-dimethyldodecylamine, N,N-dimethyltetradecylamine, N,N-dimethylhexadecylamine, and N,N-dimethyloctadecylamine), it is found that each tertiary amine reaction raw material has been successfully linked to the molecular framework of propylene oxide through nucleophilic substitution reaction with epibromohydrin, becoming quaternary ammonium salt intermediates with different carbon chain lengths.

[0100] The experimental results of the nuclear magnetic resonance hydrogen spectra of the raw material dextran and each quaternary ammonium salt intermediate raw material (QASI-C12, QASI-C14, QASI-C16, and QASI-C18) are as Figure 5 shown. Each QASAF compound has the characteristic hydrogen proton signal peaks of the corresponding quaternary ammonium salt intermediate raw material and dextran, indicating that each quaternary ammonium salt intermediate compound has been successfully linked to the molecular framework of the dextran macromolecule through reaction with dextran, becoming quaternary ammonium salt antibacterial fibers with different carbon chain lengths.

[0101] (4) Zeta potential determination of QASI and QASAF

[0102] The Zeta potentials of QASI and QASAF were measured using a Malvern laser particle size analyzer (JEN3690). Each quaternary ammonium salt intermediate compound and each quaternary ammonium salt antibacterial fiber were prepared into aqueous solutions with a concentration of 1.0 mg / mL using ultrapure water. After ultrasonic treatment at a power of 120 W and a frequency of 40 KHz for 30 min, the Zeta potentials of each compound were measured using a Malvern laser particle size analyzer (JEN3690).

[0103] Through the analysis of the chemical structures of dextran, each quaternary ammonium salt intermediate compound (QASI-C12, QASI-C14, QASI-C16, and QASI-C18), and each quaternary ammonium salt antibacterial fiber (QASAF-C12, QASAF-C14, QASAF-C16, and QASAF-C18) in the present invention, it can be known that the dextran macromolecule is a macromolecular polymer formed by the connection of glucose basic units through glycosidic bonds, and the molecule is basically uncharged or slightly negatively charged, while a large number of positively charged ammonium ions are contained in the molecules of each quaternary ammonium salt intermediate compound and each quaternary ammonium salt antibacterial fiber, so they are all positively charged.

[0104] The Zeta potentials of dextran, QASI-C12, and QASAF-C12 were measured using a Zeta potential particle size analyzer, and the experimental results are shown in Table 1.

[0105] Table 1 Zeta potentials (mv) of DEX, QASI-C12, and QASAF-C12

[0106]

[0107] As can be seen from Table 1, the dextran polymer solution is weakly negatively charged, with a Zeta potential ξ = -1.46 mV, while the Zeta of QASI-C12 and QASAF-C12 is approximately +25 mV. The experimental results further indicate that the positively charged QASI-C12 has been successfully connected to the dextran polymer backbone, forming a positively charged quaternary ammonium salt dextran antibacterial fiber.

[0108] (5) Hemolysis experiment of QASAF

[0109] Collect 1.0 mL of fresh mouse blood and place it in an EP tube (2 mL) containing a small amount of anticoagulant. Centrifuge (at a speed of 1000 rpm for 10 min), discard the supernatant, and wash it 3 times with PBS solution containing normal saline. Then resuspend it with PBS solution containing normal saline to obtain a red blood cell suspension. Take 500 μL of the red blood cell suspension and mix it with an equal volume of QASAF solutions with different carbon chain lengths at concentrations of 5, 25, 50, 75, 100, and 125 μg / mL. Use pure water as the positive control group (PC) and PBS solution containing normal saline as the negative control group (NC). After incubating at 37 °C for 60 min, centrifuge (at a speed of 1000 rpm for 10 min). Take the supernatant and place it in a 96-well plate, and measure the absorbance value of each sample at 590 nm. Calculate the relative hemolysis rate according to the formula "Sun X, Li L, Zhang H, et al. Near-Infrared Light-Regulated Drug-Food Homologous Bioactive Molecules and Photothermal Collaborative Precise Antibacterial Therapy Nanoplatform with Controlled Release Property[J]. Advanced Healthcare Materials, 2021, 10(16): 2100546.":

[0110] Hemolysis%=(X - Y) / (Z - Y)×100%

[0111] In the formula: X is the absorbance value of the experimental group, Y is the absorbance value of the negative control group, and Z is the absorbance value of the positive control group.

[0112] (6) Cell culture

[0113] Human hepatocellular carcinoma cells (Hep G2) are derived from the School of Intelligent Medicine and Biotechnology, Guilin Medical University. The cell culture medium used is DMEM medium containing streptomycin (100 μg / mL), penicillin (100 U / mL), and fetal bovine serum (10%).

[0114] The specific operations are as follows: The Hep G2 cells are cultured in a constant temperature cell incubator at 37°C with 5% carbon dioxide. The culture medium is changed every other day, and subsequent experimental operations such as cell passage, seeding, and cryopreservation are carried out after the cell adherent density reaches 80%.

[0115] (7) Cytotoxicity experiment of QASAF

[0116] The cytotoxicity of different QASAFs was determined by the thiazolyl blue tetrazolium bromide (MTT method) "Shu H, Chun-Xuan W, Yang K, et al. Preparation of rapid expansion alginate / silica fiber composite scaffold and application of rapid hemostatic function[J]. J Mater Eng, 2019, 47:124-9.".

[0117] The specific operations are as follows: Hep G2 cells in the logarithmic growth phase are seeded in a 96-well plate at a density of 5×10 4 cells / well. After culturing in the cell incubator for 24 h, the solution in the original wells is replaced with DMEM complete medium containing 25, 50, 75, 100, 125 μg / mL QASAF, and the cells are cultured in the incubator for another 24 h. Next, 10 μL of MTT (5 mg / mL) solution is added to each well. After culturing in the incubator for 4 h, the solution in each well of the 96-well plate is removed, and 150 μL of DMSO is added to each well. After shaking for 10 min, the absorbance at 490 nm is measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the relative cell survival rate is calculated.

[0118] To further verify the biosafety of the synthesized QASAF, the hemolytic properties of 4 QASAFs at different concentrations on mouse red blood cells were determined, and the experimental results are as Figure 6 shown. The results show that when the concentration of QASAF is relatively high, the hemolysis rates of the 4 QASAFs are still <5%, while within the effective antibacterial concentration range, the hemolysis rates of the 4 QASAFs are relatively low, indicating that the 4 QASAFs have slight hemolytic properties only at relatively high concentrations and have good biosafety.

[0119] Whether the biocompatibility meets the requirements is one of the necessary conditions for judging the qualification of wound healing materials. To further evaluate the biocompatibility of the synthesized QASAF, Hep G2 cells were then selected as the cell model, and the inhibitory activity of 4 kinds of QASAF at different concentrations on Hep G2 cells was measured by the MTT method. The experimental results are as Figure 7 shown.

[0120] The results showed that when the concentrations of the 4 kinds of QASAF were in the range of 25 - 125 μg / mL, the survival rate of Hep G2 cells was greater than 85% (>70%, no cytotoxicity

[53] ). The experimental results indicated that the synthesized QASAF had very low cytotoxicity, that is, QASAF had good biocompatibility.

[0121] The present invention then took electron microscope photos of the cell state of HepG2 cells treated with 4 kinds of QASAF with a concentration of 125 μg / mL. The experimental results are as Figure 8 shown. The results showed that compared with the control group, there were no obvious differences in the morphology and growth state of Hep G2 cells in each group, further indicating that QASAF had very low toxicity to cells and had good biocompatibility.

[0122] When the hemolysis of a biomaterial is less than 5%, it indicates that the biosafety of the biomaterial is relatively high. Since the surfaces of human and mammalian cells are all negatively charged, cationic quaternary ammonium salt compounds with a positive charge can cause varying degrees of damage and killing effects on anionic biological structures. Therefore, cationic quaternary ammonium salt antibacterial materials have a slight hemolytic effect.

[0123] The present invention investigated the hemolytic effect of four different QASAF on red blood cells through hemolysis. The results showed that the hemolysis rates of the four QASAF with a concentration of 125 μg / mL on red blood cells were between 2 - 5%, with a slight hemolytic effect. The cytotoxic side effects of the four QASAF on cells were explored through MTT experiments, and the biosafety of QASAF was further investigated. The results showed that after Hep G2 cells were treated with the four QASAF at different concentrations, the cell survival rate was greater than 85% (as Figure 7 shown), and there were no obvious differences in the morphology and growth state of the cells in each group compared with the control group (as Figure 8 shown), indicating that the cytotoxic side effects of the four QASAF on cells were very low and had good biosafety.

[0124] Experimental Example 2 Study on the antibacterial performance and wound repair of QASAF against Staphylococcus aureus

[0125] 1.2 Materials and methods

[0126] 1.2.1 Experimental materials

[0127] (1) Experimental bacterial strains

[0128] Staphylococcus aureus (S. aureus, ATCC 25923) was purchased from the Culture Collection Center of Guangdong Institute of Microbiology.

[0129] (2) Experimental animals

[0130] The same experimental animals as in Experimental Example 1.

[0131] (3) Experimental reagents

[0132] Neutral resin, 1% hydrochloric acid ethanol, and Masson stain were purchased from Beijing Solarbio Science & Technology Co., Ltd. Xylene and ammonia water were purchased from Xilong Scientific Co., Ltd.; hematoxylin and eosin were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.

[0133] (4) Experimental instruments

[0134] An 8-mm small animal skin puncher was purchased from Shanghai Yuyan Scientific Instruments Co., Ltd.

[0135] 1.2.2 Experimental methods

[0136] (1) Bacterial culture

[0137] Bacterial culture: In a UV-sterilized laminar flow hood, dip a bacterial inoculation loop into the S. aureus (S. aureus, ATCC 25923) bacterial solution, then use the inoculation loop to streak on an LB agar plate, and place it in a 37°C constant temperature incubator for 13 h to obtain single bacterial colonies. Use a bacterial inoculation loop to pick a single S. aureus colony and disperse it in 5 mL of LB broth liquid medium, and incubate it overnight in a 37°C constant temperature shaker (rotation speed of 180 rpm). The next day, take 100 μL of the bacterial culture solution and add it to 7 mL of fresh LB broth liquid medium, and continue to culture it in a 37°C constant temperature shaker (rotation speed of 180 rpm) for about 2.5 h. Use a UV spectrophotometer to measure the optical density (OD) value of the bacterial solution at 600 nm OD 600 , and dilute it with physiological saline to make the OD of the bacterial solution 600 = 0.5 (when OD 600 = 0.5, the number of colonies ≈ 1×10 8 CFU / mL), centrifuge at 7000 rpm for 5 min, discard the supernatant, resuspend the bacterial pellet with physiological saline and centrifuge and wash it 2 times, and finally resuspend it with physiological saline to obtain a bacterial suspension with a colony count of 1×10 8 CFU / mL for standby.

[0138] (2) Determination of the antibacterial growth curve

[0139] Add 80 μL of normal saline to each well of a 96-well plate, then add 10 μL of QASAF-C12, QASAF-C14, QASAF-C16 or QASAF-C18 samples with different concentration gradients respectively. After thorough mixing, add 10 μL of OD 600 = 0.5 bacterial suspension. After mixing evenly, incubate in a constant temperature shaker at 37 °C (rotation speed 180 rpm) for 10 min. Add 10 μL of 10×LB broth liquid medium and measure its OD 600 .

[0140] (3) Determination of Minimal Inhibitory Concentration (MIC)

[0141] Dilute the bacterial suspension with OD 600 = 0.5 ten-fold with normal saline. Add 80 μL of normal saline to each well of a 96-well plate, then add 10 μL of QASAF-C12, QASAF-C14, QASAF-C16 or QASAF-C18 samples with different concentration gradients respectively. After thorough mixing, add 10 μL of the diluted bacterial suspension respectively. After mixing evenly, incubate in a constant temperature shaker at 37 °C (rotation speed 180 rpm) for 10 min. Add 10 μL of 10×LB broth liquid medium and continue to incubate in a constant temperature shaker at 37 °C (rotation speed 180 rpm) for 18 h. Add 10 μL of CCK-8 solution to each well, shake in an enzyme-labeled instrument for 10 min and measure its absorbance at 450 nm, and judge the inhibitory ability of different concentrations of different antibacterial fibers on bacteria according to the absorbance.

[0142] (4) Plate coating experiment

[0143] Add 400 μL of normal saline to EP tubes respectively, then add 50 μL of QASAF-C12, QASAF-C14, QASAF-C16 or QASAF-C18 samples with different concentration gradients respectively. After thorough mixing, add 50 μL of OD 600 = 0.5 bacterial suspension. After mixing evenly, incubate in a constant temperature shaker at 37 °C (rotation speed 180 rpm) for 10 min. Pipette 50 μL from each tube and drop it on an LB agar plate culture dish, spread evenly with a glass spreader, then place the plate upside down in a constant temperature incubator at 37 °C and incubate statically for 18 h, take pictures and count the number of colonies.

[0144] Results: The in vitro antibacterial properties of 4 kinds of QASAF against Staphylococcus aureus were studied by the antibacterial growth curve method and the plate coating method. As Figure 9As shown, the antibacterial growth curve indicates that all four QASAFs have strong bactericidal ability against Staphylococcus aureus, and their antibacterial ability is positively correlated with the dose. Among them, QASAF-C18 has the best antibacterial performance against Staphylococcus aureus. The antibacterial performance of QASAF against Staphylococcus aureus is in the order of: QASAF-C18 > QASAF-C16 > QASAF-C14 > QASAF-C12, suggesting that among the four quaternary ammonium salt antibacterial fibers with different carbon chain lengths, the longer the straight-chain alkane carbon chain length, the better its antibacterial performance against common Staphylococcus aureus.

[0145] In addition, QASAF-C18 at a concentration of 25 μg / mL can achieve 100% sterilization, while other QASAF-C16, QASAF-C14, and QASAF-C12 can achieve 100% sterilization only when the concentrations are 50 μg / mL, 75 μg / mL, and 150 μg / mL, respectively. Therefore, it is concluded that QASAF-C18 has the best antibacterial performance against Staphylococcus aureus ( Figure 9 ).

[0146] The present invention then used a CCK-8 kit to measure the minimum inhibitory concentration (MIC) of four different concentrations of QASAF against Staphylococcus aureus. The experimental results are shown in Table 2. The MIC values of QASAF-C12, QASAF-C14, QASAF-C16, and QASAF-C18 (all prepared from Experimental Example 1) are in the order of: 150 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL. This result is consistent with the result obtained by the above antibacterial growth curve method, further verifying that QASAF-C18 has the best antibacterial performance against Staphylococcus aureus.

[0147] Table 2 MIC values of four QASAFs against Staphylococcus aureus (μg / mL)

[0148]

[0149]

[0150] On this basis, the relationship between the optical density value OD at 600 nm and time when different concentrations of QASAF-C18 act on Staphylococcus aureus was further measured, and the growth inhibition curve of QASAF-C18 against Staphylococcus aureus was obtained. The results are as 600 shown. Figure 10

[0151] After culturing for a period of time, the OD 600 values of the samples treated with QASAF-C18 at a concentration < 25 μg / mL increased continuously with the extension of time. After 24 h, the OD 600 ​The value decreased with the prolongation of time, which was caused by the depletion of nutrients and the death of some Staphylococcus aureus. And QASAF-C18 with a concentration of 25 μg / mL completely inhibited the growth of Staphylococcus aureus within 48 hours, and the optical density value OD of the bacterial solution at 600 nm 600 was all 0.

[0152] Furthermore, the antibacterial properties of QASAF-C18 at different concentrations against Staphylococcus aureus were studied by the plate coating method, and the experimental results are as Figure 11 shown. Figure 11 (A) and Figure 11 (B) As can be seen from the results, compared with the control group, as the concentration of QASAF-C18 increased from 10 μg / mL to 50 μg / mL, the number of bacterial colonies in the LB agar petri dish decreased significantly. When the concentration of QASAF-C18 was 25 μg / mL and 50 μg / mL, no colonies were formed in the petri dish.

[0153] (5) Establishment of a mouse epidermal infection model and in vivo antibacterial property experiment

[0154] ① Female Kunming mice (weighing 25 - 30 g) were anesthetized by intraperitoneal injection of 0.94% sodium pentobarbital and depilated with depilatory cream;

[0155] ② After subcutaneous injection of 100 μL of a bacterial solution with OD 600 = 1.0 for 2 days, the infected mice were randomly grouped;

[0156] ③ On the 3rd and 4th days after subcutaneous injection of bacteria for infection, the control group was subcutaneously injected with 100 μL of PBS at the infection site, and the experimental group was subcutaneously injected with 100 μL of QASAF-C18 at the infection site.

[0157] ④ All the mice were sacrificed on the 5th day, and the hearts, livers, spleens, lungs, kidneys and subcutaneously infected tissues of each mouse were collected.

[0158] ⑤ The hearts, livers, spleens, lungs and kidneys of each mouse collected were immersed in 4% paraformaldehyde solution for fixation, sent to the pathology department of the affiliated hospital of the medical college for pathological sections, and the sections were stained with hematoxylin (H) and eosin (E) to make H&E stained pathological sections and photographed.

[0159] ⑥ After weighing the collected subcutaneous infected tissues, add normal saline and 56 steel beads with a diameter of about 1 mm according to 10 mg / mL (tissue weight / normal saline volume), and use a tissue crusher for homogenization treatment (frequency: 50 Hz, time interval: 10 s, homogenization treatment time: 20 min). After homogenization, add different volumes of normal saline to dilute 0, 10, 100, 1000, and 10,000 times respectively. Then, take 50 μL of the homogenized tissue dilution and evenly coat it on an LB agar plate culture dish. Invert the plate and place it in a constant temperature incubator at 37 °C for static incubation for 18 h, take pictures and count the number of colonies.

[0160] In this invention, a mouse model of Staphylococcus aureus subcutaneous infection was used as the research object to evaluate the in vivo antibacterial performance of QASAF-C18. Figure 12 (A) is the timeline for establishing the mouse model of subcutaneous infection, Figure 12 (B) Sites of subcutaneous injection of Staphylococcus aureus and QASAF-C18 in mice. From the mouse photos, it can be seen that the subcutaneous infection site of the control group mice is significantly more severe than that of the QASAF-C18 treatment group. In the subcutaneous infection site of the control mice, obvious pale white purulent tissues can be seen, while the subcutaneous infection site of the mice treated with QASAF-C18 by in situ subcutaneous injection at the infection site has improved significantly.

[0161] Next, in this invention, the subcutaneous infection sites of the two groups of mice on the 5th day were collected and homogenized. Then, the obtained homogenate was serially diluted and cultured on plates. The treatment effect was evaluated by comparing the number of bacterial colonies in the infection sites of the two groups of mice, and the results are as Figure 13 shown. The results show that the number of bacterial colonies in different concentration gradients of the QASAF-C18 treatment group mice is significantly less than that of the control group, revealing that QASAF-C18 still has excellent antibacterial effects in vivo.

[0162] On the 5th day, the two groups of mice were dissected, and the main organ tissues such as the heart, liver, spleen, lung, and kidney of the two groups of mice were collected. Pathological sections were made to further study the damage to the health of mice after QASAF-C18 was subcutaneously injected into the mice. As Figure 14 shown, through histological examination and analysis of the tissue sections, it can be seen that the pathological sections of the main organs such as the heart, liver, spleen, lung, and kidney in the QASAF-C18 group all maintained normal tissue structures, without any obvious organ damage or inflammatory damage, indicating that QASAF-C18 did not cause damage to the mice after being subcutaneously injected into the body. This result further shows that QASAF-C18 has good biocompatibility.

[0163] (6) Mouse wound healing experiment

[0164] ① Female Kunming mice (weighing 25 - 30 g) were anesthetized by intraperitoneal injection of 0.94% sodium pentobarbital, their hair was removed with depilatory cream, and a wound surface with a diameter of about 8 mm was obtained by removing the skin at the depilated area using a sharp stainless - steel punch (8 mm in diameter) and stainless - steel scissors. 100 μL of a Staphylococcus aureus suspension with a concentration of 1×10 8 CFU / mL was slowly dropped onto the wound surface to infect the wound, and the mice were randomly grouped.

[0165] ② In the experimental group, QASAF - C18 was dropped onto the wound surface of the mice to treat the wound on days 0, 1, and 2 after wound infection. In the control group, PBS was dropped onto the wound surface of the mice to treat the wound on days 0, 1, and 2 after wound infection.

[0166] ③ Mice in each group were photographed on days 0, 3, 6, 9, and 14. The initial total wound area (A0) of each mouse and the total wound area (A d ) on days 0, 3, 6, 9, and 14 were accurately measured using Image J. The wound healing rate (P, %) was calculated according to the following formula:

[0167] P = (A0 - A d ) / A0 × 100%

[0168] ④ After photographing on days 0, 3, 6, 9, and 14, some mice in each group were sacrificed, the wound tissues were collected, immersed in 4% paraformaldehyde solution for fixation, made into pathological sections, and made into H&E - stained pathological sections and Masson - stained pathological sections and photographed.

[0169] (7) Tissue paraffin sections

[0170] Hearts, livers, spleens, lungs, kidneys of the skin - infected mouse models on day 5 and skin tissues of the full - thickness skin defect models on days 9 and 14 on the backs of mice were taken. The tissue samples were fixed in PBS solution containing 4% formaldehyde at 4°C for 24 h. Subsequently, the tissues were transferred to 70% ethanol and stored at 4°C at low temperature. The tissues were successively added to 70% ethanol for 2 h, 80% ethanol for 2 h, 90% ethanol for 2 h, 95% ethanol and absolute ethanol for 2 h each for tissue gradient dehydration.

[0171] Next, the dehydrated tissues were placed in xylene I (analytical pure, 99%) and xylene II (analytical pure, 99%) for 10 min each. The tissues were successively soaked in paraffin I (melting point 48 - 50°C) melted in a 65°C incubator for 30 min, paraffin II (melting point 56 - 58°C) for 30 min, and paraffin III (melting point 56 - 58°C) for 30 min for tissue impregnation with paraffin. Finally, the tissues were embedded with a tissue embedding machine, and successively sliced (section thickness 3 - 4 μm), spread, and pasted; they were placed in an oven at 65°C and baked overnight.

[0172] (8) HE staining of paraffin tissue sections

[0173] Place the baked glass slides in xylene I for 40 min and xylene II for 40 min for dewaxing. After dewaxing, the sections are immersed in absolute ethanol, 95% ethanol, 80% ethanol, and 70% ethanol for 10 min each, followed by washing twice with pure water. Next, soak the tissue sections in hematoxylin stain for 5 min and rinse twice with running water. Put the tissue sections into 1% hydrochloric acid ethanol and shake quickly for 30 s, then wash twice with pure water. Carry out blueing by quickly shaking in weak ammonia water for 30 s. Observe the hematoxylin staining effect under the microscope, then soak in eosin stain for 5 min and wash twice with pure water. Gradient dehydration is carried out by successively placing the stained sections in 70% ethanol, 80% ethanol, 90% ethanol, absolute ethanol I, and absolute ethanol II for 10 min each. Subsequently, place the tissue sections in xylene I and xylene II for 15 min each for clearing. Finally, seal the slides with neutral resin, observe the slides with an inverted microscope, and collect data.

[0174] (9) Masson staining of paraffin tissue sections

[0175] Soak the tissue sections in xylene I for 40 min and xylene II for 40 min. Then soak the sections in Masson A solution overnight and rinse with pure water. Successively place the sections in the dye solution prepared by mixing Masson B solution and Masson C solution in equal proportion, immerse for 1 min and then wash with pure water. Next, put the tissue sections into 1% hydrochloric acid ethanol and shake quickly for 30 s, then wash twice with pure water. Immerse the sections in Masson D solution and stain for 6 min, then rinse with pure water. Stain with Masson E solution for 1 min. Do not wash with water here. After slightly drying, soak it in Masson F solution for 2 - 30 s. Subsequently, rinse with 1% glacial acetic acid and dehydrate with absolute ethanol. Place the sections in xylene solution for 5 min and then seal with neutral resin. Finally, observe and collect data on the stained samples with an inverted microscope.

[0176] (10) Statistical methods

[0177] All experiments were independently repeated three times. The data were expressed in the form of mean ± standard deviation. Statistical comparisons were performed using t - tests. *P < 0.05 and **P < 0.01 were considered to be statistically significant.

[0178] The wound - healing effect of QASAF - C18 on infected wound surfaces was studied by applying bacteria to the wound surfaces of mice. Figure 15The experimental results show that, compared with the control group, QASAF-C18 has an obvious effect of promoting wound healing. For wounds of the same size, the wound surface treated with QASAF-C18 had scabbed on the 3rd day after treatment, and the wound area was significantly smaller than that of the control group. The ratio of the healing area of the infected wound surface of each group of mice at different time points to the initial infected wound surface area is as Figure 16 shown. In the QASAF-C18 treatment group, the wound surface of the mice was basically healed on the 14th day, and its wound surface area was only 1.41% of the initial wound surface area, and the healing rate reached 98.59%; while in the control group, there were still large scabbed tissue on the wound surface on the 14th day, and its wound surface area was still 11.83% of the initial wound surface area ( Figure 16 ). Thus, it can be seen that QASAF-C18 has an obvious effect of promoting the healing of wound surfaces.

[0179] Figure 15 During the healing process of the infected wound surface of each group of mice in Figure 16 , the healing trajectory of the wound surface and the intuitive statistical situation of the wound surface area size are as

[0180] shown. The results show that the wound surface area of the mice treated with QASAF-C18 at different time points is significantly smaller than that of the control group. QASAF-C18 can promote the healing of the infected wound surface because QASAF-C18 can kill the bacteria at the infected wound surface in time, avoiding the difficulty of wound healing caused by the inflammatory reaction caused by infection. Therefore, QASAF-C18 can promote the healing of the infected wound surface. Figure 18 Next, the effect of QASAF-C18 on promoting the healing of infected wound surfaces was further evaluated by pathological sections of the wound tissue. The tissue at the wound surface on the 14th day was stained with H&E and Masson, and made into H&E staining pathological sections and Masson staining pathological sections, as

[0181] shown. The inflammatory reaction and inflammatory cells generated during the wound healing process can be evaluated by H&E staining, and the collagen generated during the wound healing process can be evaluated by Masson staining. During the wound healing process, both groups produced more inflammatory cells, but the number of inflammatory cells in the QASAF-C18 treatment group was less than that in the control group. This is because the QASAF-C18 treatment group can kill the bacteria on the wound surface in time, avoiding the inflammatory reaction, so the number of its inflammatory cells is less than that of the control group.

[0182] The in vitro and in vivo research results of the present invention are consistent with the above research. The antibacterial abilities of the four different QASAFs are QASAF-C18 > QASAF-C16 > QASAF-C14 > QASAF-C12. The longer the length of the straight-chain alkane carbon chain, the better the antibacterial performance against Staphylococcus aureus (as Figure 9 shown). Among them, QASAF-C18 has the best antibacterial performance against Staphylococcus aureus.

[0183] Bacteria are mainly composed of components such as cell walls, cytoplasmic membranes, cytoplasm, and nucleoids. According to the differences in the structure and chemical composition of bacterial cell walls, bacteria are divided into two major categories: Gram-positive and Gram-negative bacteria. Staphylococcus aureus belongs to Gram-positive bacteria. The cell wall of Gram-positive bacteria is relatively thick, and its composition is relatively simple. The outermost layer of the cell wall is composed of dozens of layers of peptidoglycan, accounting for about 90% of the cell wall, with 75% of the subunits cross-linked, and the network is tight and firm. The teichoic acid in the inner layer accounts for about 10%. QAC is a cationic surfactant that can adsorb negatively charged groups in the bacterial cell wall and damage the cell wall

[26] . The non-polar action of the hydrophobic long chain penetrates and dissolves the bacterial cell wall membrane, causing the leakage of bacterial contents by damaging the bacterial wall, thereby killing the bacteria. Among the four QASAFs with different carbon chain lengths in the present invention, QASAF-C18 has the strongest antibacterial ability. This may be due to its hydrophobic straight-chain alkane, which can better penetrate and dissolve the bacterial cell wall membrane and kill the bacteria.

[0184] The results of the agar plate counting experiment show (as Figure 11 shown) that when QASAF-C18 is at 25 μg / mL, there are no colonies formed in the petri dish; when QASAF-C12 is at 100 μg / mL, there are no colonies formed in the petri dish. This shows that QASAF-C18 has good antibacterial performance against Staphylococcus aureus, and QASAF-C12 has good antibacterial performance against MRSA. The MIC value of QASAF-C18 against Staphylococcus aureus measured in the experiment of the present invention is 25 μg / mL, and this value is lower than the above literature. In short, MIC further proves the high antibacterial performance of QASAF-C18 against Staphylococcus aureus.

[0185] The wound healing process is generally divided into an exudative phase, a proliferative phase, and a scar phase. This complex process involves inflammation, cell proliferation and cell migration, the formation of new capillaries, the recruitment of fibroblasts, and the deposition of collagen, etc.

[54] Once the wound is infected with bacteria during the healing process, an inflammatory reaction will occur, prolonging the wound healing time. Timely killing of the bacteria on the wound surface and preventing wound infection are beneficial to wound healing and repair. The more collagen deposited during the wound healing process, the more it can promote wound healing, the faster the wound heals, and the better the healing effect.

[0186] The in vivo research results of the present invention show (asFigure 15 , Figure 16 , Figure 17 As shown in Figure 17 , the QASAF-C18 group could well promote wound healing. QASAF-C18 could promptly remove the bacteria on the infected wound surface, thus promoting the wound healing. The wound healing rate reached 98.86% on the 14th day. Therefore, QASAF-C18 had a significant effect on promoting the healing of the wound surface infected by Staphylococcus aureus in mice.

[0187] The results showed that among the 4 kinds of QASAF with different carbon chain lengths, QASAF-C18 had the best antibacterial performance against Staphylococcus aureus, and its MIC was 25 μg / mL. Explanation: The antibacterial effect of QASAF on common Staphylococcus aureus was related to the length of the alkane carbon chain. The longer the length of the straight-chain alkane carbon chain, the better its antibacterial performance against common Staphylococcus aureus.

[0188] Experimental Example 3 Antibacterial Performance and Wound Repair Study of QASAF against MRSA

[0189] 1.3 Materials and Methods

[0190] 1.3.1 Experimental Materials

[0191] (1) Experimental Bacteria

[0192] The methicillin-resistant Staphylococcus aureus (MRSA, ATCC 29213) used in this experiment was from the Culture Collection Center of Guangdong Institute of Microbiology.

[0193] (2) Experimental Animals

[0194] Kunming mice were purchased from the Experimental Animal Center of the Medical College and met the SPF-level standard. All the experimental animals were carried out in the standardized laboratory of the Animal Experiment Center of our school and strictly followed the "3R" experimental animal ethical guidelines and norms. The animal ethics number was: GLMC202103275.

[0195] (3) Experimental Reagents

[0196] 0.94% sodium pentobarbital was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; LB agar and LB broth were purchased from Beijing Land Bridge; 1% hydrochloric acid ethanol was purchased from Beijing Solarbio Science & Technology Co., Ltd. 1.3.2 Experimental Methods

[0197] The experimental methods adopted were the same as those of each experiment in Experimental Example 2 and would not be elaborated here. The strains selected for each experiment of the present invention were multi-drug resistant MRSA.

[0198] 1.4 Results and Discussion

[0199] 1.4.1 In Vitro Antibacterial Performance of QASAF against MRSA

[0200] In this invention, the in vitro antibacterial properties of 4 kinds of QASAF against MRSA were studied by using the antibacterial growth curve method, the plate coating method and the MIC value determination experiment. The results obtained by the antibacterial growth curve method are as Figure 19 and Figure 20 shown.

[0201] The results show that not all QASAF have strong bactericidal ability against MRSA. Among them, only QASAF-C12 has the best antibacterial performance against MRSA. The antibacterial properties of the 4 kinds of QASAF against MRSA are in the order of: QASAF-C12 > QASAF-C14 > QASAF-C16 > QASAF-C18 (all from the preparation in Experimental Example 1), indicating that among the quaternary ammonium salt antibacterial fibers with 4 different carbon chain lengths, the shorter the length of the straight-chain alkane carbon chain, the better its antibacterial performance against MRSA.

[0202] Table 3 MIC values (μg / mL) of four kinds of QASAF against MRSA

[0203]

[0204] In addition, the experimental results reveal that QASAF-C12 at a concentration of 100 μg / mL can achieve 100% sterilization, while at the same concentration, QASAF-C14, QASAF-C16 and QASAF-C18 have no significant effect on inhibiting MRSA. Therefore, QASAF-C12 has the best antibacterial performance against MRSA.

[0205] In this invention, the antibacterial properties of QASAF-C12 against MRSA were explored through the plate coating experiment. Figure 21 As shown in (A), as the concentration increased from 25 μg / mL to 125 μg / mL, the survival rate of bacteria decreased significantly. And when the concentration increased to 100 μg / mL, there was almost no MRSA in the plate. Figure 21 (B) Quantitative determination and analysis show that the antibacterial performance of the QASAF-C12 group is significantly improved compared with the group without QASAF-C12. The survival rate of bacteria in the plate co-cultured with MRSA in the QASAF-C12 group was almost 0% at concentrations of 100 μg / mL and 125 μg / mL. This result further shows that QASAF-C12 has good antibacterial performance at a concentration of 100 μg / mL.

[0206] 1.4.2 In vivo antibacterial properties of QASAF

[0207] In this invention, a mouse model of subcutaneous infection with MRSA was constructed to evaluate the in vivo antibacterial properties of QASAF-C12. The experimental results are as Figure 22As shown in (A), the subcutaneous infection site of the control group mice was significantly more severe than that of the QASAF-C12 treatment group, with pale white purulent tissue, while the subcutaneous infection site of the mice treated by in-situ subcutaneous injection of QASAF-C12 at the infection site improved significantly.

[0208] Next, the subcutaneous infection sites of the two groups of mice on the 5th day were collected and homogenized, and the obtained homogenates were serially diluted and plated for culture. The bacterial colony counts at the infection sites of the two groups of mice were compared to evaluate the treatment effect, and the results are as Figure 22 shown in (B) and (C). The results showed that the bacterial colony counts at different concentration gradients of the QASAF-C12 treatment group mice were significantly less than those of the control group. This result indicates that QASAF-C12 has excellent antibacterial effects in vivo.

[0209] After the 5th day of the experiment, the two groups of mice were dissected, and the main organ tissues such as the heart, liver, spleen, lungs, and kidneys of the mice were collected for pathological section observation. The organ damage results of the mice after QASAF-C12 was subcutaneously injected into the mice were obtained. As Figure 23 shown, compared with the control group, there were no obvious organ damages or inflammatory lesions in the pathological sections of the main organs such as the heart, liver, spleen, lungs, and kidneys of the group treated with QASAF-C12. This result suggests that: after QASAF-C12 was subcutaneously injected into the body, it did not cause damage to the mice. Therefore, QASAF-C12 has good biocompatibility.

[0210] 1.4.3 Promoting wound healing performance of QASAF-C12

[0211] By coating bacteria on the wound surfaces of mice, the promoting wound healing effect of QASAF-C12 on infected wound surfaces was evaluated. The ratio of the healing area of the infected wound surfaces at different time points to the initial infected wound surface area is as Figure 24 shown. Compared with the control group, QASAF-C12 has an obvious effect of promoting the healing of wound surfaces. On the 3rd day after treatment with QASAF-C12, the wounds with the same area had scabbed, and the area was significantly smaller than that of the control group. The wound surfaces of the QASAF-C12 treatment group mice were basically healed on the 14th day, and the wound surface area was only 1.93% of the initial wound surface area; there were still large scabbed tissues on the wound surfaces of the control group on the 14th day, and the wound surface area was still 6.00% of the initial wound surface area. Thus, it can be seen that QASAF-C12 has an obvious effect of promoting the healing of wound surfaces.

[0212] Figure 25 It is the intuitive statistical situation of the wound healing trajectory and the wound surface area size during the healing process of the infected wound surface. Figure 26It was shown that the wound surface area of mice treated with QASAF-C12 at different time points was significantly smaller than that of the control group. The reason is that QASAF-C12 promptly kills the bacteria at the wound surface of the infected wound, preventing the wound surface from being difficult to heal due to the inflammatory reaction caused by infection. Therefore, it is not difficult to conclude that QASAF-C12 can promote the healing of infected wound surfaces.

[0213] Finally, the tissue at the wound surface on the 14th day was subjected to H&E staining and Masson staining to prepare H&E staining pathological sections and Masson staining pathological sections to evaluate the wound healing-promoting effect of QASAF-C12 on infected wounds. The H&E staining and Masson staining pathological sections are as Figure 27 shown. The results suggest that a large number of inflammatory cells were generated in both the control group and the QASAF-C12 treatment group during the wound healing process; however, the number of inflammatory cells in the QASAF-C12 treatment group was less than that in the control group, indicating that QASAF-C12 can promptly kill the bacteria at the wound surface, thereby avoiding the inflammatory reaction. The results of Masson staining suggest that more collagen was generated in the wound surface of the QASAF-C12 treatment group than in the control group, further suggesting the wound healing-promoting effect of QASAF-C12 on infected wound surfaces.

[0214] The research of the present invention shows that when the concentration of QASAF-C12 is 100 μg / mL, no colonies appear on the agar plate (as Figure 21 shown), indicating that QASAF-C12 has excellent antibacterial properties against MRSA at 100 μg / mL.

[0215] The present invention studied the effect of QASAF-C12 on promoting the wound healing of MRSA-infected mice. On the 14th day, the wound healing rate reached 98.86%; HE staining showed that there were fewer inflammatory cells in the QASAF-C12 treatment group; the results of Masson staining suggest that more collagen was generated in the wound surface of the QASAF-C12 treatment group than in the control group. These results further suggest that QASAF-C12 has a wound healing-promoting effect on MRSA-infected wound surfaces.

[0216] The present invention studied the antibacterial effects of QASAF-C12, QASAF-C14, QASAF-C16, and QASAF-C18 against MRSA. The results suggest that among the 4 QASAFs with different carbon chain lengths, QASAF-C12 has the best antibacterial properties against MRSA, and its MIC is 100 μg / mL. This chapter also evaluated the in vivo antibacterial properties of QASAF-C12 against MRSA and its ability to promote the healing of infected wound surfaces. The results suggest that QASAF-C12 can be used to treat subcutaneous MRSA infection diseases, has good biosafety, and has a significant wound healing-promoting effect on the infected wound surfaces of mice.

[0217] Quaternary ammonium salt polymers have good antibacterial properties, good biocompatibility, and are easy to be functionalized and modified, etc., becoming a hot spot in antibacterial research. The present invention aims to develop a new type of quaternary ammonium salt antibacterial material with good biocompatibility and biosafety, and having dual functions of antibacterial infection and promoting wound healing. Quaternary ammonium salt intermediates QASI with different carbon chain lengths were synthesized, and QASI was grafted onto Dextran to construct new quaternary ammonium salt antibacterial fibers: QASAF-C12, QASAF-C14, QASAF-C16, and QASAF-C18. Then, its structure was characterized by nuclear magnetic resonance hydrogen spectrum, and its biocompatibility and biosafety were evaluated by hemolysis and cytotoxicity. Next, the antibacterial properties of QASAF against Staphylococcus aureus and MRSA were studied by antibacterial growth curve, plate coating experiment, and MIC value determination. Finally, the effect of QASAF on promoting wound healing in infected mice was evaluated through a mouse wound model. The following conclusions were drawn from the present invention:

[0218] First of all, QASAF has good biocompatibility and biosafety. In the concentration range of 25 - 125 μg / mL, it has low cytotoxicity (relative cell survival rate is greater than 85%), and has very slight hemolysis (hemolysis rate is all < 5%).

[0219] Secondly, the results of antibacterial growth curve determination, plate coating experiment, and MIC value determination show that the antibacterial properties of QASAF against Staphylococcus aureus are in the order of: QASAF-C18 > QASAF-C16 > QASAF-C14 > QASAF-C12. Among the 4 quaternary ammonium salt antibacterial fibers with different carbon chain lengths, the longer the length of the straight-chain alkane carbon chain, the better the antibacterial property against Staphylococcus aureus. QASAF-C18 has the best antibacterial property against Staphylococcus aureus, and the MIC value is 25 μg / mL. QASAF-C12 has the best antibacterial property against MRSA, and the MIC value is 100 μg / mL.

[0220] Finally, QASAF-C18 and QASAF-C12 respectively have a significant effect on promoting the healing of wounds infected with Staphylococcus aureus and MRSA in mice. The healing rate of QASAF-C18 reached 98.59% on the 14th day of the Staphylococcus aureus-infected wound surface. The healing rate of QASAF-C12 reached 98.86% on the 14th day of the MRSA-infected wound surface.

[0221] In summary, QASAF may be a potential wound dressing, having dual functions of antibacterial infection and promoting wound healing.

[0222] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of quaternary ammonium salt antibacterial fiber in preparing antibacterial or wound healing promoting drugs or protective articles, wherein the bacteria are Staphylococcus aureus and methicillin-resistant Staphylococcus aureus; The preparation method of the quaternary ammonium salt antibacterial fiber comprises the following steps: (1) Mix methanol and epibromohydrin to obtain solvent 1; mix quaternary ammonium salt and methanol to obtain solvent 2; (2) Dropwise add the solvent 1 into the solvent 2 for a first reaction, remove methanol to obtain a product, recrystallize the product and then vacuum dry to obtain a quaternary ammonium salt intermediate; (3) Mix the quaternary ammonium salt intermediate, dextran, NaOH and water for a second reaction, adjust the pH, then dialyze and freeze-dry to obtain the quaternary ammonium salt antibacterial fiber; The quaternary ammonium salt in step (2) is N,N-dimethyldodecylamine or N,N-dimethyloctadecylamine; The volume-mass ratio of methanol to epibromohydrin in step (1) is 4-6 ml: 6.5-7.5 g; the mass-volume ratio of the quaternary ammonium salt to methanol is 8-10 g: 4-6 ml; The first reaction in step (2) is carried out under the protection of nitrogen; the temperature of the first reaction is 40-50 °C; the time of the first reaction is 1.5-2.5 h; The mass-volume ratio of the quaternary ammonium salt intermediate, dextran, NaOH and water in step (3) is 5-9 g: 3-5 g: 3-5 g: 90-110 ml; The temperature of the second reaction in step (3) is 45-55 °C, and the time is 2-4 h.

2. The application according to claim 1, wherein The number of recrystallizations in step (2) is 4-6 times; the temperature of the vacuum drying is 35-45 °C, and the time is 20-28 h.

3. The application according to claim 1, wherein In step (3), the pH is adjusted to 6.8-7.2; the dialysis time is 2-4 d.

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