Antibacterial and antioxidant dynamic nanogel as well as preparation method and application thereof

Abstract

The invention discloses antibacterial and antioxidant dynamic nanogel as well as a preparation method and application thereof. The preparation method comprises the following steps: chemically modifying L-arginine with methacrylic anhydride to obtain an antibacterial monomer M-Arg; dissolving the M-Arg powder, N-isopropylacrylamide, N, N '-methylene bisacrylamide, hexadecyl trimethyl ammonium bromide and 2, 2'-azobis (isobutylamidine) dihydrochloride in deionized water, carrying out precipitation polymerization, carrying out dialysis, and adjusting the concentration and the pH value of the solution to obtain a nanogel antibacterial agent; the preparation method comprises the following steps: grafting dopamine into a nanogel antibacterial agent through activation of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, dialyzing, and adjusting the concentration and pH value of a solution to obtain the antibacterial and antioxidant dynamic nanogel. Good antibacterial and antioxidant effects can be achieved at different temperatures and in different acidic environments, and the antibacterial wound dressing can be used for treating infected wounds.

Description

Technical Field

[0001] The invention belongs to the field of polymer technology, and specifically relates to an antibacterial and antioxidant dynamic nanogel and a preparation method and application thereof. Background Art

[0002] Tissue damage is often accompanied by bacterial infection, which seriously hinders wound healing and may even affect the normal function of internal organs, posing a huge threat to public health. Hydrogel is a three-dimensional polymer network formed by physical or chemical cross-linking, which is widely used in medical devices, wound dressings and drug delivery systems. Due to its unique extracellular matrix-like properties and adjustable physicochemical properties, it has become one of the hot topics in antibacterial material research. Antibacterial hydrogels can be roughly divided into three types according to their mechanism of action: (i) hydrogels containing antibacterial agents; (ii) hydrogels with inherent antibacterial ability; and (iii) stimuli-responsive antibacterial hydrogels.

[0003] By introducing antimicrobial agents such as silver ions, metal oxide nanoparticles, antimicrobial drugs and other small molecules with antimicrobial activity into hydrogels, they can directly act on the cell structure or metabolic activity of bacteria, leading to bacterial death or growth inhibition. For example, silver ions can bind to bacterial DNA, blocking its replication process, and can also affect bacterial protein synthesis (Fan, Z.; Liu, B.; Wang, J.; Zhang, S.; Lin, Q.; Gong, P.; Ma, L.; Yang, S., Advanced Functional Materials 2014, 24(25): 3933–3943.). However, the release rate and amount of antimicrobial agents are often difficult to accurately control, which may lead to problems such as sudden release or insufficient release, thereby affecting the durability and stability of the antimicrobial effect. In addition, the cytotoxicity and long-term retention of metal-based biomaterials in the body remain to be solved. Selecting raw materials with antibacterial properties, such as antimicrobial peptides, to make intrinsic antibacterial hydrogels is an effective way (Su, Y.; Andrabi, SM; Shahriar, SMS; Wong, SL; Wang, G.; Xie, J., Journal of Controlled Release 2023, 356, 131–141.). Intrinsic antibacterial hydrogel dressings do not require the introduction of additional antibacterial agents, and mainly rely on the antibacterial mechanism of functional groups contacting the bacterial surface, such as affecting the structure of bacterial cell membranes to cause cell content outflow. On the one hand, it can effectively reduce bacterial resistance and have a long-lasting antibacterial effect. On the other hand, intrinsic antibacterial hydrogels show good cell compatibility. However, most of the antibacterial hydrogels currently studied are macroscopic bulk hydrogels, which often show relatively poor antibacterial effects due to insufficient contact with bacteria. At the same time, natural antibacterial components usually need to be extracted, separated and purified before gelation, which is cumbersome and has limited yield. The preparation technology of synthetic antibacterial substances such as antimicrobial peptides is complex and expensive, which greatly limits their large-scale application and commercialization. In recent years, researchers have found that hydrogels have high antibacterial activity under external stimuli such as light, heat, and electricity, including introducing photothermal agents into hydrogels to increase the temperature of the affected area and using photodynamic agents to release reactive oxygen to kill bacteria (Badran, Z.; Rahman, B.; DeBonfils, P.; Nun, P.; Coeffard, V.; Verron, E., Drug Discovery Today2023, 28(4), 103493.). However, external stimuli usually require specific conditions, such as the wavelength and intensity of light, which limits their application in deep tissues. The stability and biocompatibility of photosensitizers also need further research and improvement.

[0004] In addition, the increase of local reactive oxygen species at the wound infection site usually leads to an inflammatory response, which seriously hinders wound repair and aggravates the patient's pain. Ordinary antibacterial hydrogels lack intrinsic antioxidant capacity. If antibacterial and antioxidant properties can be integrated into hydrogels to achieve synergistic effects, it is expected to improve the tissue repair effect.

[0005] The development of polymer nanogel sprays with both antibacterial and antioxidant capabilities will help to break through the limitations of traditional antibacterial hydrogels. Polymer nanogels not only have the basic properties of hydrogels, but also have a large specific surface area, which allows them to have more adequate contact with the affected area, and their biocompatibility is also better than that of inorganic metal particles. Nanogels with intrinsic antibacterial properties can be constructed by screening suitable monomers and crosslinkers. Intrinsically antibacterial nanogels can be anchored on the tissue surface through bridging to achieve long-term antibacterial effects. The large specific surface area is conducive to their full contact with the bacteria in the affected area, thereby improving the antibacterial efficiency. Further simple chemical modification of the nanogel and the introduction of antioxidant groups can achieve dual synergistic effects of antibacterial and antioxidant effects. At present, the method of synthesizing nanogels is very mature, but most of the research is related to the use of nanogels as drug carriers for drug release. There are few reports on nanogels with intrinsic antibacterial and antioxidant functions.

[0006] In order to solve the problems of complex functional construction of current antibacterial hydrogels, poor antibacterial effect, short antibacterial time, lack of antioxidant capacity, and biological toxicity, a nanogel spray with a simple preparation process, long-lasting antibacterial and antioxidant properties, and easy application can be developed to explore its inhibitory effect on bacterial growth and reproduction and its ability to scavenge reactive oxygen species, establish a general model of nanogel antibacterial agents, lay the foundation for the development of nanogel antibacterial agents, and provide new ideas for the application of antibacterial materials in the biomedical field. Summary of the invention

[0007] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a method for preparing an antibacterial dynamic nanogel. In the present invention, arginine (Arg) containing a guanidine cationic group is modified by methacrylic anhydride (MA) to prepare an antibacterial functional monomer (M-Arg). The thermosensitive monomer N-isopropylacrylamide (NIPAM) and the guanidine functionalized zwitterion (M-Arg) with antibacterial function are used as monomers, N,N'-methylenebisacrylamide (MBA) is used as a crosslinker, and hexadecyltrimethylammonium bromide (CTAB) is used as a surfactant. The high molecular dynamic nanogel antibacterial agent is obtained by aqueous phase precipitation polymerization initiated by 2,2'-azobisisobutylamidine dihydrochloride (V-50).

[0008] The second object of the present invention is to provide an antibacterial dynamic nanogel obtained by the above preparation method.

[0009] The third object of the present invention is to provide a method for preparing antibacterial and antioxidant dynamic nanogels. The present invention further introduces dopamine (DA) with antioxidant properties into the nanogel antibacterial agent by coupling 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Compared with traditional antibacterial hydrogels, the nanogel spray has the advantages of simple preparation, convenient application, high antibacterial efficiency, and antioxidant. The nanogel spray is temperature-sensitive and pH-sensitive, and its particle size can be regulated by temperature. It can respond to the pH of the environment in a timely manner and regulate the antibacterial property, which broadens the application range of the nanogel.

[0010] A fourth object of the present invention is to provide antibacterial and antioxidant dynamic nanogel obtained by the above preparation method.

[0011] A fifth object of the present invention is to provide an application of the dynamic nanogel.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing an antibacterial dynamic nanogel comprises the following steps: (1) dissolving L-arginine (Arg) in a mixed solvent of water, dioxane and triethylamine (TEA) to obtain an Arg solution; (2) adding methacrylic anhydride (MA) dropwise to the Arg solution in step (1) and stirring evenly to obtain a mixture reaction solution; (3) stirring the reaction mixture in step (2), recrystallizing the reaction mixture with acetone, and vacuum drying the precipitate to obtain a white M-Arg powder; (4) dissolving the M-Arg powder described in step (3) with N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (MBA), hexadecyltrimethylammonium bromide (CTAB), and 2,2'-azobisisobutylamidine dihydrochloride (V-50) in water, stirring, and dialyzing to obtain a nanogel dispersion; (5) Adjusting the concentration and pH value of the nanogel dispersion in step (4) to obtain the antibacterial dynamic nanogel.

[0013] Furthermore, the water described in the present invention is all deionized water.

[0014] Furthermore, in step (1), the concentration of L-arginine in the Arg solution is 6±1 g / 100 mL; the proportions of the components in the mixed solvent are: deionized water 60±5 v / v%, dioxane 26±3 v / v%, and triethylamine 14±2 v / v%.

[0015] Furthermore, in step (1), the ratio of L-arginine, water, dioxane and triethylamine is 2 g: 20 mL: 8.5 mL: 4.5 mL.

[0016] Furthermore, the concentration of methacrylic anhydride in the mixture reaction solution in step (2) is 8.4±1 v / v%.

[0017] Furthermore, based on the amount of L-arginine used in step (1) being 2 g, the amount of methacrylic anhydride added in step (2) is 3 mL.

[0018] Furthermore, the stirring conditions in step (2) are: speed 200-300 rpm, time 10-20 min.

[0019] Furthermore, the stirring conditions in step (3) are: a speed of 200 to 300 rpm, and a time of overnight (8 to 18 hours).

[0020] Furthermore, the amount of acetone used in step (3) is 3 to 5 times the volume of the mixed reaction liquid.

[0021] Furthermore, in the nanogel solution in step (4), the concentration of M-Arg is 0.1 to 0.5 g / 100 mL, preferably 0.5 g / mL; the concentration of N-isopropylacrylamide is 1 to 5 g / 100 mL, preferably 2.5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.01 to 0.05 g / 100 mL, preferably 0.025 g / 100 mL; the concentration of 2,2'-azobisisobutylamidine dihydrochloride is 0.01 to 0.1 g / 100 mL, preferably 0.05 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.25 to 0.5 g / 100 mL, preferably 0.3 g / 100 mL.

[0022] Furthermore, the stirring conditions in step (4) are: speed 200-300 rpm, time 7-9 h, temperature 65-75°C.

[0023] Furthermore, the molecular weight cut-off of the dialysis bag used for dialysis in step (4) is 8k to 14kDa.

[0024] Furthermore, the concentration of the nanogel solution in step (5) is 1 to 5 g / 100 mL, preferably 3 to 5 g / 100 mL.

[0025] Furthermore, the pH value of the nanogel solution in step (5) is 2-6.

[0026] Furthermore, the adjustment of the nanogel solution in step (5) is achieved by using ultrapure water.

[0027] An antibacterial dynamic nanogel is obtained by the preparation method.

[0028] A method for preparing an antibacterial and antioxidant dynamic nanogel, comprising all the steps in the above-mentioned method for preparing an antibacterial dynamic nanogel and the following steps: (6) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the antibacterial dynamic nanogel, stirring under a nitrogen atmosphere to obtain a mixed solution; (7) injecting dopamine (DA) solution into the mixed solution in step (6), stirring, and dialyzing to obtain an antibacterial and antioxidant nanogel dispersion; (8) Adjusting the concentration and pH value of the nanogel dispersion in step (7) to obtain the antibacterial and antioxidant dynamic nanogel.

[0029] Furthermore, in the mixed solution in step (6), the concentration of the antibacterial dynamic nanogel is 3-5 g / 100 mL, preferably 3 mg / 100 mL, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.5±0.2 g / 100 mL, and the concentration of N-hydroxysuccinimide is 1±0.2 g / 100 mL.

[0030] Furthermore, the concentration of the dopamine solution in step (7) is 0.1-0.2 g / mL, preferably 0.11 g / mL.

[0031] Furthermore, the amount of the dopamine solution in step (7) is calculated as follows: antibacterial dynamic nanogel: dopamine solution = 40-50 mL: 1-3 mL, preferably 45 mL: 2 mL.

[0032] Furthermore, the ratio of the antibacterial dynamic nanogel, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and dopamine solution in steps (6) and (7) is 45 mL: 0.6 g: 0.36 g: 2 mL.

[0033] Furthermore, the stirring conditions in step (6) are: speed 200-300 rpm, time 0.5-2 h.

[0034] Furthermore, the stirring conditions in step (7) are: speed 200-300 rpm, time 20-30 h, temperature 20-30°C.

[0035] Furthermore, the molecular weight cut-off of the dialysis bag used for dialysis in step (7) is 8k to 14kDa.

[0036] Furthermore, the pH value of the nanogel dispersion in step (8) is 2-6.

[0037] Furthermore, the concentration of the nanogel dispersion in step (8) is 1 to 5 g / 100 mL, preferably 3 to 5 g / 100 mL.

[0038] An antibacterial and antioxidant dynamic nanogel is obtained by the preparation method.

[0039] The above antibacterial and antioxidant dynamic nanogel, or the use of the antibacterial and antioxidant dynamic nanogel in the preparation of drugs for promoting wound healing and tissue regeneration.

[0040] In the present invention, NIPAM is the core of nanogel formation, M-Arg provides antibacterial properties, and dopamine provides antioxidant properties. Nanogel spraying is simple and convenient, with a large specific surface area, which overcomes the problems of poor antibacterial effect of bulk hydrogels with inherent antibacterial ability and poor biocompatibility of inorganic metal particle antibacterial agents. The polymer nanogel spray prepared by the present invention has pH sensitivity and temperature sensitivity. The particle size of the nanogel of the present invention gradually decreases with the increase of temperature at 25 to 60°C; and increases with the decrease of temperature. The particle size of the nanogel of the present invention can be reversible when repeatedly treated at different temperatures. The nanogel of the present invention is positively charged in the pH range of 2 to 6 and has a good inhibitory effect on bacterial activity. The dynamic nanogel is used to kill or inhibit the growth and reproduction of bacteria in wound infection sites and remove active oxygen in the affected area.

[0041] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention adopts precipitation polymerization to synthesize dynamic polymer nanogel antibacterial agent, which is simple to operate, convenient to synthesize, and easy to spray and apply.

[0042] (2) The present invention overcomes the problem of poor antibacterial effect caused by insufficient contact of large hydrogel blocks.

[0043] (3) The present invention overcomes the problem of poor biocompatibility of inorganic metal particle antibacterial agents.

[0044] (4) The polymer nanogel antibacterial agent prepared by the present invention is positively charged at acidic pH values ​​and can be used in bacterial microenvironments of different acidities.

[0045] (5) The nanogel prepared by the present invention is temperature sensitive and has excellent antibacterial effect at different temperatures.

[0046] (6) The nanogel spray prepared by the present invention has both antibacterial and antioxidant effects, which is beneficial to further promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a transmission electron microscopy image of the nanogel in Example 1; Figure 2 The graph showing the change of nanogel particle size with temperature in Example 2; Figure 3 This is a physical picture of the nanogel in Example 3 after repeated heating and cooling; Figure 4 is the potential diagram of the nanogel at different pH in Example 4; Figure 5 The graph is a result of measuring the antibacterial rate of the nanogel against Staphylococcus aureus in Example 5 and Comparative Example 1; Figure 6 The figure is a result of measuring the scavenging rate of ABTS free radicals by the nanogel in Example 6, Comparative Example 2, Comparative Example 3 and Comparative Example 4; Figure 7 This is a diagram showing the cell activity effect of mouse fibroblasts co-cultured with the nanogel extract for 3 days in Example 7. DETAILED DESCRIPTION

[0048] The present invention will be further described in detail below in conjunction with specific embodiments and drawings, but the embodiments of the present invention are not limited thereto. The advantages and features of the present invention will become clearer as the description proceeds, but the embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention.

[0049] Sources of raw materials used in the following examples are as follows: L-arginine, methacrylic anhydride, N-isopropylacrylamide, N,N'-methylenebisacrylamide, hexadecyltrimethylammonium bromide, 2,2'-azobisisobutylamidine dihydrochloride, triethylamine, dioxane, acetone, dopamine hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were all purchased from Aladdin.

[0050] Example 1 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0051] The nanogel solution was diluted with ultrapure water to 0.05 w / v% and the morphology of the nanogel was observed under a transmission electron microscope. Figure 1 As shown in Figure 2, it can be seen that the nanogels are spherical particles with relatively uniform size under the electron microscope.

[0052] Example 2 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0053] The nanogel solution was diluted to 0.1 w / v% with ultrapure water to characterize the change of nanogel particle size with temperature. Figure 2 It can be seen that within the range of 25℃~60℃, as the temperature increases, the particle size of the nanogel decreases.

[0054] Example 3 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0055] The nanogel solution was diluted to 0.1 w / v% with ultrapure water, and the temperature was repeatedly raised and lowered to characterize the reversibility of the change of nanogel particle size with temperature. Figure 3 As shown. It can be seen that with repeated heating and cooling, the nanogel particle size is reversible. The heated nanogel solution (60°C) becomes milky white, indicating that the nanogel particle size is reduced, and the cooled nanogel solution (25°C) becomes clear and transparent, indicating that the hydrophilic particle size of the nanogel is enlarged.

[0056] Example 4 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0057] First, the pH of the nanogel solution was adjusted to pH = 2.0, pH = 4.0, and pH = 6.0, and then the nanogel solution was diluted to 0.1 w / v% with a solution of the same pH value to characterize the potential of the nanogel at different pH values. The results are shown in Figure 4 As shown. It can be seen that in the pH range of 2-6, the nanogels are all positively charged, and as the acidity increases, the positive charge of the nanogels increases.

[0058] Example 5 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0059] The 3 w / v% nanogel was concentrated to 5 w / v%, and then 200 μL of 3 w / v% and 5 w / v% nanogel solutions were mixed with 800 μL of 10 6 CFU / mL of Staphylococcus aureus, and 200 μL of PBS solution was mixed with the above bacterial solution as the control group. The mixed solution of each group was placed in a 37℃ incubator for 2 h. 4 After the test, 10 μL of each was evenly spread on the agar plate and placed in a 37°C incubator for 12 h. The agar plate was taken out to count the colonies in each group and the antibacterial rate was calculated as follows, where N x is the colony number of nanogel group, N c is the colony count of the control group: Antibacterial rate (%) = N x / N c ×100%.

[0060] Figure 5 This is a bar graph of the antibacterial rate of nanogels of different concentrations in Example 5 and Comparative Example 1. It can be seen that the nanogel has good antibacterial properties, and the higher the concentration, the stronger the antibacterial property.

[0061] Example 6 A method for preparing an antibacterial and antioxidant nanogel spray comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0062] (7) Take 45 mL of the nanogel antibacterial agent in step (6), add 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and stir at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution; (8) Inject 2 mL of 0.11 g / mL dopamine solution into the mixed solution in (7), and stir at 250 rpm for 24 h at 25 °C to obtain an antibacterial and antioxidant nanogel dispersion; (9) The nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain an antibacterial and antioxidant nanogel solution with a concentration of 3 w / v%, named NGDA.

[0063] 200 μL of the 3 w / v% NGDA nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and the ABTS solution without NGDA was used as the control group. After incubating the mixed solution in a shaker at 37°C for 30 minutes, 100 μL was taken to measure the absorbance at 734 nm to calculate the free radical scavenging rate. Scavenging rate (%) = (1-A x / A c ) × 100%, A xrepresents the absorbance measured in the experimental group, A c It represents the absorbance measured by the control group under the same conditions.

[0064] Figure 6 The bar graphs are of the scavenging rates of the nanogels for ABTS free radicals in Example 6, Comparative Example 2, Comparative Example 3, and Comparative Example 4. It can be seen that the nanogel (NGDA) after adding dopamine has excellent antioxidant properties compared with the nanogel (NG) without adding dopamine. Within a certain range, the higher the NGDA concentration, the stronger the antioxidant property. When it reaches 3 w / v%, the free radical scavenging ability basically reaches a balance, and further increasing the NGDA concentration has no obvious improvement on the free radical scavenging effect.

[0065] Example 7 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0066] (7) The nanogel solution obtained in step (6) was diluted with culture medium to obtain a culture medium containing 1 w / v% nanogel, and the culture medium was co-cultured with mouse fibroblasts. Figure 7This is a diagram showing the cell activity effect of mouse fibroblasts co-cultured with nanogels for 3 days in Example 7. It can be seen that there are no obvious dead cells after mouse fibroblasts co-cultured with nanogels for 3 days, indicating that the nanogels have good biocompatibility.

[0067] Comparative Example 1 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and the nanogel solution was diluted to obtain a nanogel solution with a concentration of 1 w / v%.

[0068] Take 200 μL of 1 w / v% nanogel solution and 800 μL of 10 6 CFU / mL of Staphylococcus aureus, and incubate the mixture in a 37°C incubator for 2 h. 4 After the 10 μL of each was evenly spread on the agar plate, it was placed in a 37°C incubator for 12 h. The agar plate was taken out to count the colonies and calculate the antibacterial rate. The antibacterial rate of the 1 w / v% concentration of the nanogel prepared in this comparative example is as follows: Figure 5 .

[0069] Comparative Example 2 A method for preparing a dynamic nanogel antibacterial agent comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days. The nanogel solution was concentrated to obtain a nanogel solution with a concentration of 3 w / v%, which was named NG.

[0070] 200 μL of the 3 w / v% NG nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and the mixture was incubated on a shaker at 37°C for 30 min. 100 μL was taken to measure the absorbance at 734 nm to calculate the free radical scavenging rate. The free radical scavenging rate of the nanogel ABTS prepared in this comparative example is shown in FIG. Figure 6 .

[0071] Comparative Example 3 A method for preparing an antibacterial and antioxidant nanogel spray comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0072] (7) Take 45 mL of the nanogel antibacterial agent in step (6), add 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and stir at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution; (8) Inject 2 mL of 0.11 g / mL dopamine solution into the mixed solution in (7), and stir at 250 rpm for 24 h at 25 °C to obtain an antibacterial and antioxidant nanogel dispersion; (9) The nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and diluted to obtain an antibacterial and antioxidant nanogel spray with a concentration of 1 w / v%, named NGDA.

[0073] 200 μL of the 1 w / v% NGDA nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and the mixture was incubated on a shaker at 37°C for 30 min. 100 μL was taken to measure the absorbance at 734 nm to calculate the free radical scavenging rate. The free radical scavenging rate of the nanogel ABTS prepared in this comparative example is shown in FIG. Figure 6 .

[0074] Comparative Example 4 A method for preparing an antibacterial and antioxidant nanogel spray comprises the following steps: (1) Dissolve 2 g of L-arginine (Arg) in a mixed solvent of 20 mL of deionized water, 8.5 mL of dioxane, and 4.5 mL of triethylamine (TEA) to obtain an Arg solution; (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under an ice bath and stir evenly at a stirring speed of 200 rpm to obtain a mixed reaction solution; (3) The reaction mixture in step (2) was stirred overnight, the reaction mixture was recrystallized with 150 mL of acetone, and the precipitate was vacuum dried to obtain a white M-Arg powder; (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder prepared in step (3), 0.025 w / v% N,N'-methylenebisacrylamide (MBA), 0.3 w / v% hexadecyltrimethylammonium bromide (CTAB), and 0.05 w / v% 2,2'-azobisisobutylamidine dihydrochloride (V-50) using 20 mL of deionized water; (5) stirring the solution in step (4) in a water bath at 70° C. for 8 h at a stirring speed of 250 rpm to obtain a nanogel solution; (6) The nanogel solution obtained in step (5) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days to obtain a nanogel solution with a concentration of 3 w / v%.

[0075] (7) Take 45 mL of the nanogel antibacterial agent in step (6), add 0.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.36 g of N-hydroxysuccinimide (NHS), and stir at 250 rpm for 1 h under a nitrogen atmosphere to obtain a mixed solution; (8) Inject 2 mL of 0.11 g / mL dopamine solution into the mixed solution in (7), and stir at 250 rpm for 24 h at 25 °C to obtain an antibacterial and antioxidant nanogel dispersion; (9) The nanogel dispersion in step (8) was transferred to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyzed for 3 days, and concentrated to obtain an antibacterial and antioxidant nanogel spray with a concentration of 5 w / v%, named NGDA.

[0076] 200 μL of the 5 w / v% NGDA nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and the mixture was incubated on a shaker at 37°C for 30 min. 100 μL was taken to measure the absorbance at 734 nm to calculate the free radical scavenging rate. The free radical scavenging rate of the nanogel ABTS prepared in this comparative example is shown in FIG. Figure 6 .

[0077] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial dynamic nanogel, characterized in that: The steps include: (1) dissolving L-arginine in a mixed solvent of water, dioxane and triethylamine to obtain an Arg solution; (2) adding methacrylic anhydride dropwise to the Arg solution in step (1) and stirring evenly to obtain a mixture reaction liquid; (3) stirring the reaction mixture in step (2), recrystallizing the reaction mixture with acetone, and vacuum drying the precipitate to obtain a white M-Arg powder; (4) dissolving the M-Arg powder described in step (3) with N-isopropylacrylamide, N,N'-methylenebisacrylamide, hexadecyltrimethylammonium bromide, and 2,2'-azobisisobutylamidine dihydrochloride in water, stirring, and dialyzing to obtain a nanogel dispersion; (5) Adjusting the concentration and pH value of the nanogel dispersion in step (4) to obtain the antibacterial dynamic nanogel.

2. The method for preparing the antibacterial dynamic nanogel according to claim 1, characterized in that: The concentration of L-arginine in the Arg solution in step (1) is 6±1 g / 100 mL; the proportions of the components in the mixed solvent are: deionized water 60±5 v / v%, dioxane 26±3 v / v%, triethylamine 14±2 v / v; The concentration of methacrylic anhydride in the mixed reaction solution in step (2) is 8.4±1 v / v%; In the nanogel solution in step (4), the concentration of M-Arg is 0.1-0.5 g / 100 mL; the concentration of N-isopropylacrylamide is 1-5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.01-0.05 g / 100 mL; the concentration of 2,2'-azobisisobutylamidine dihydrochloride is 0.01-0.1 g / 100 mL; the concentration of hexadecyltrimethylammonium bromide is 0.25-0.5 g / 100 mL; The concentration of the nanogel solution in step (5) is 1 to 5 g / 100 mL.

3. The method for preparing the antibacterial dynamic nanogel according to claim 2, characterized in that: The ratio of L-arginine, water, dioxane and triethylamine in step (1) is 2 g: 20 mL: 8.5 mL: 4.5 mL; Based on the amount of L-arginine used in step (1) being 2 g, the amount of methacrylic anhydride added in step (2) is 3 mL; The concentration of M-Arg in the nanogel solution in step (4) is 0.5 g / 100 mL; the concentration of N-isopropylacrylamide is 2.5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.025 g / 100 mL; the concentration of 2,2'-azobisisobutylamidine dihydrochloride is 0.05 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.3 g / 100 mL. The concentration of the nanogel solution in step (5) is 3 to 5 g / 100 mL; The pH value of the nanogel solution in step (5) is 2-6.

4. The method for preparing the antibacterial dynamic nanogel according to any one of claims 1 to 3, characterized in that: The stirring conditions in step (2) are: speed 200-300 rpm, time 10-20 min; The stirring conditions in step (3) are: speed 200-300 rpm, time 8-18 hours; The amount of acetone used in step (3) is 3 to 5 times the volume of the mixed reaction liquid; The stirring conditions in step (4) are: speed 200-300 rpm, time 7-9 h, temperature 65-75°C; The molecular weight cut-off of the dialysis bag used in the dialysis in step (4) is 8k to 14kDa.

5. An antibacterial dynamic nanogel, characterized in that: The preparation method according to any one of claims 1 to 4 is obtained.

6. A method for preparing an antibacterial and antioxidant dynamic nanogel, characterized in that: The method comprises all the steps in the method for preparing the antibacterial dynamic nanogel as described in any one of claims 1 to 4 and the following steps: (6) adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the antibacterial dynamic nanogel, stirring under a nitrogen atmosphere to obtain a mixed solution; (7) injecting dopamine solution into the mixed solution in step (6), stirring, and dialyzing to obtain an antibacterial and antioxidant nanogel dispersion; (8) Adjusting the concentration and pH value of the nanogel dispersion in step (7) to obtain the antibacterial and antioxidant dynamic nanogel.

7. The method for preparing the antibacterial and antioxidant dynamic nanogel according to claim 6, characterized in that: In the mixed solution described in step (6), the concentration of the antibacterial dynamic nanogel is 3-5 g / 100 mL; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1.5±0.2 g / 100 mL; The concentration of the dopamine solution in step (7) is 0.1-0.2 g / mL; The amount of the dopamine solution in step (7) is calculated as follows: antibacterial dynamic nanogel: dopamine solution = 40-50 mL: 1-3 mL; The concentration of the nanogel dispersion in step (8) is 1 to 5 g / 100 mL.

8. The method for preparing the antibacterial and antioxidant dynamic nanogel according to claim 7, characterized in that: In the mixed solution described in step (6), the concentration of the antibacterial dynamic nanogel is 3 mg / 100 mL; The concentration of the dopamine solution in step (7) is 0.11 g / mL; The ratio of the antibacterial dynamic nanogel, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and dopamine solution is 45 mL: 0.6 g: 0.36 g: 2 mL; The stirring conditions in step (6) are: speed 200-300 rpm, time 0.5-2 h; The stirring conditions in step (7) are: speed 200-300 rpm, time 20-30 h, temperature 20-30°C; The molecular weight cut-off of the dialysis bag used in the dialysis in step (7) is 8k to 14kDa; The pH value of the nanogel dispersion in step (8) is 2 to 6; The concentration of the nanogel dispersion in step (8) is 3 to 5 g / 100 mL.

9. An antibacterial and antioxidant dynamic nanogel, characterized in that: The method is obtained by the preparation method described in any one of claims 6 to 8.

10. Use of the antibacterial and antioxidant dynamic nanogel described in claim 5 or the antibacterial and antioxidant dynamic nanogel described in claim 9 in the preparation of drugs for promoting wound healing and tissue regeneration.

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