Antibacterial and antioxidant dynamic nanogel and preparation method and application thereof

Abstract

The application discloses an antibacterial and antioxidative dynamic nanogel as well as a preparation method and application thereof. L-arginine is chemically modified by using methacrylic anhydride to obtain an antibacterial monomer M-Arg; M-Arg powder, N-isopropyl acrylamide, N,N'-methylene bisacrylamide, cetyltrimethylammonium bromide and 2,2'-azobis (2-amidinopropane) dihydrochloride are dissolved in deionized water to perform precipitation polymerization, dialysis, concentration and pH value adjustment, and thus an antibacterial nanogel is obtained; dopamine is grafted into the antibacterial nanogel through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide activation, dialysis, concentration and pH value adjustment, and thus the antibacterial and antioxidative dynamic nanogel is obtained; the antibacterial and antioxidative dynamic nanogel can exert good antibacterial and antioxidative effects under different temperatures and different acidic environments, and can be used for the treatment of infected wounds.

Description

Technical Field

[0001] This invention belongs to the field of polymer technology, specifically relating to an antibacterial and antioxidant dynamic nanogel, its preparation method, and its application. Background Technology

[0002] Tissue damage is often accompanied by bacterial infection, severely hindering wound healing and potentially affecting the normal functioning of internal organs, posing a significant threat to public health. Hydrogels are three-dimensional polymer networks formed by physical or chemical cross-linking, widely used in medical devices, wound dressings, and drug delivery systems. Due to their unique extracellular matrix-like properties and tunable physicochemical properties, they have become a hot topic in antimicrobial materials research. Based on their mechanism of action, antimicrobial hydrogels can be broadly classified into three types: (i) hydrogels containing antimicrobial agents; (ii) hydrogels with inherent antimicrobial capabilities; and (iii) stimulus-responsive antimicrobial hydrogels.

[0003] By introducing antibacterial agents, such as silver ions, metal oxide nanoparticles, and antibacterial drugs, into hydrogels, small molecules with antibacterial activity can directly act on the cellular structure or metabolic activities 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 antibacterial agents are often difficult to control precisely, which may lead to problems such as burst release or insufficient release, thus affecting the durability and stability of the antibacterial effect. In addition, the cytotoxicity and long-term retention of metal-based biomaterials in vivo remain to be solved. Choosing raw materials with antibacterial properties, such as antimicrobial peptides, to manufacture inherently antimicrobial hydrogels is an effective approach (Su, Y.; Andrabi, SM; Shahriar, SMS; Wong, SL; Wang, G.; Xie, J., Journal of Controlled Release 2023, 356, 131–141.). Inherently antimicrobial hydrogel dressings do not require the introduction of additional antimicrobial agents; they primarily rely on the mechanism of functional groups contacting the bacterial surface for antimicrobial action, such as influencing the structure of the bacterial cell membrane to cause the leakage of cellular contents. On the one hand, it can effectively reduce bacterial resistance and provide long-lasting antimicrobial effects. On the other hand, inherently antimicrobial hydrogels exhibit good cell compatibility. However, most currently studied antimicrobial hydrogels are macroscopic bulk hydrogels, which often exhibit relatively poor antimicrobial effects due to insufficient contact with bacteria. Furthermore, natural antimicrobial components typically require extraction, separation, and purification before gelation, a cumbersome process with limited yield. The preparation technology of synthetic antimicrobial substances such as antimicrobial peptides is complex and expensive, which greatly limits their large-scale application and commercialization. In recent years, researchers have discovered that hydrogels have high antimicrobial 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 species to kill bacteria (Badran, Z.; Rahman, B.; DeBonfils, P.; Nun, P.; Coeffard, V.; Verron, E., Drug Discovery Today 2023, 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] Furthermore, increased local reactive oxygen species at the site of wound infection often lead to inflammatory responses, severely hindering wound healing and exacerbating patient suffering. Ordinary antibacterial hydrogels lack inherent antioxidant capacity; therefore, integrating antibacterial and antioxidant properties into hydrogels to achieve synergistic effects could potentially improve tissue repair outcomes.

[0005] Developing polymeric nanogel sprays with both antibacterial and antioxidant capabilities can help overcome the limitations of traditional antibacterial hydrogels. Polymeric nanogels not only possess the basic properties of hydrogels but also have a large specific surface area, allowing for more thorough contact with the affected area, and exhibit better biocompatibility than inorganic metal particles. By screening suitable monomers and cross-linking agents, nanogels with intrinsic antibacterial properties can be constructed. Intrinsically antibacterial nanogels can anchor to tissue surfaces through bridging interactions to achieve long-lasting antibacterial effects, and their large specific surface area facilitates sufficient contact with bacteria at the affected area, thereby improving antibacterial efficiency. Further simple chemical modification of the nanogels, introducing antioxidant groups, can achieve a dual synergistic effect of antibacterial and antioxidant properties. Currently, the methods for synthesizing nanogels are very mature, but most research focuses on using nanogels as drug carriers for drug release; reports on nanogels with intrinsic antibacterial and antioxidant functions are scarce.

[0006] To address the current problems of complex functional construction, poor antibacterial effect, short antibacterial duration, lack of antioxidant capacity, and biotoxicity in antibacterial hydrogels, a nanogel spray with simple preparation process, long-lasting antibacterial and antioxidant properties, and easy application can be developed. Its inhibitory effect on bacterial growth and reproduction, its ability to scavenge reactive oxygen species, and the establishment of a general model for nanogel antibacterial agents will lay the foundation for their development and provide new ideas for the application of antibacterial materials in the biomedical field. Summary of the Invention

[0007] To overcome the shortcomings and drawbacks of existing technologies, the primary objective of this invention is to provide a method for preparing antibacterial dynamic nanogels. This invention modifies arginine (Arg) containing guanidine cationic groups with methacrylic anhydride (MA) to prepare an antibacterial functional monomer (M-Arg). Using the thermosensitive monomer N-isopropylacrylamide (NIPAM) and the antibacterial guanidine-functionalized zwitterion (M-Arg) as monomers, and employing N,N'-methylenebisacrylamide (MBA) as a crosslinking agent and hexadecyltrimethylammonium bromide (CTAB) as a surfactant, a high-molecular-weight dynamic nanogel antibacterial agent is obtained through aqueous precipitation polymerization initiated by 2,2'-azobisisobutylammonium dihydrochloride (V-50).

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

[0009] A third objective of this invention is to provide a method for preparing dynamic antibacterial and antioxidant nanogels. This invention further incorporates dopamine (DA), which possesses antioxidant properties, into the nanogel antibacterial agent through coupling 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Compared with traditional antibacterial hydrogels, this nanogel spray has advantages such as simple preparation, convenient application, high antibacterial efficiency, and antioxidant properties. This nanogel spray is temperature- and pH-sensitive, allowing its particle size to be controlled by temperature. It can respond promptly to environmental pH and regulate antibacterial activity, thus broadening the application range of nanogels.

[0010] The fourth objective of this invention is to provide an antibacterial and antioxidant dynamic nanogel obtained by the above preparation method.

[0011] The fifth objective of this invention is to provide applications of the aforementioned dynamic nanogels.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] A method for preparing an antibacterial dynamic nanogel includes the following steps:

[0014] (1) Dissolve L-arginine (Arg) in a mixed solvent of water, dioxane and triethylamine (TEA) to obtain an Arg solution;

[0015] (2) Add methacrylic anhydride (MA) dropwise to the Arg solution described in step (1) and stir until homogeneous to obtain a mixed reaction solution;

[0016] (3) Stir the reaction solution of the mixture described in step (2), recrystallize the reaction solution with acetone, and dry the precipitate under vacuum to obtain white M-Arg powder;

[0017] (4) Dissolve the M-Arg powder described in step (3) with N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (MBA), hexadecyltrimethylammonium bromide (CTAB), and 2,2'-azobisisobutylammonium dihydrochloride (V-50) in water, stir, and dialyze to obtain a nanogel dispersion;

[0018] (5) Adjust the concentration and pH value of the nanogel dispersion in step (4) to obtain the antibacterial dynamic nanogel.

[0019] Furthermore, all water mentioned in this invention is deionized water.

[0020] Further, the concentration of L-arginine in the Arg solution in step (1) is 6±1 g / 100 mL; the proportions of each component in the mixed solvent are as follows: deionized water 60±5 v / v%, dioxane 26±3 v / v%, triethylamine 14±2 v / v.

[0021] Furthermore, the ratio of L-arginine, water, dioxane, and triethylamine in step (1) is 2 g: 20 mL: 8.5 mL: 4.5 mL.

[0022] Further, the concentration of methacrylic anhydride in the mixture reaction solution described in step (2) is 8.4 ± 1 v / v.

[0023] 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.

[0024] Furthermore, the stirring conditions described in step (2) are: speed 200-300 rpm, time 10-20 min.

[0025] Furthermore, the stirring conditions described in step (3) are: speed 200-300 rpm, time overnight (8-18h).

[0026] Furthermore, the amount of acetone used in step (3) is 3 to 5 times the volume of the mixture reaction solution.

[0027] Further, in step (4), the concentration of M-Arg in the nanogel solution is 0.1–0.5 g / 100 mL, preferably 0.5 g / mL; the concentration of N-isopropylacrylamide is 1–5 g / 100 mL, preferably 2.5 g / 100 mL; the concentration of N,N'-methylenebisacrylamide is 0.01–0.05 g / 100 mL, preferably 0.025 g / 100 mL; the concentration of 2,2'-azobisisobutylammonium dihydrochloride is 0.01–0.1 g / 100 mL, preferably 0.05 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.25–0.5 g / 100 mL, preferably 0.3 g / 100 mL.

[0028] Furthermore, the stirring conditions described in step (4) are: speed 200-300 rpm, time 7-9 h, and temperature 65-75℃.

[0029] Furthermore, the molecular weight cutoff of the dialysis bag used in step (4) is 8k to 14kDa.

[0030] Further, the concentration of the nanogel solution in step (5) is 1 to 5 g / 100 mL, preferably 3 to 5 g / 100 mL.

[0031] Furthermore, the pH value of the nanogel solution in step (5) is 2 to 6.

[0032] Furthermore, the nanogel solution in step (5) is adjusted using ultrapure water.

[0033] An antibacterial dynamic nanogel was obtained by the above preparation method.

[0034] A method for preparing antibacterial and antioxidant dynamic nanogels, comprising all the steps in the above-described method for preparing antibacterial dynamic nanogels and the following steps:

[0035] (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the antibacterial dynamic nanogel, and stir under a nitrogen atmosphere to obtain a mixture;

[0036] (7) Inject dopamine (DA) solution into the mixture in step (6), stir, dialyze, and obtain an antibacterial and antioxidant nanogel dispersion;

[0037] (8) Adjust the concentration and pH value of the nanogel dispersion in step (7) to obtain the antibacterial and antioxidant dynamic nanogel.

[0038] Further, in the mixture described 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.

[0039] Further, the concentration of the dopamine solution in step (7) is 0.1 to 0.2 g / mL, preferably 0.11 g / mL.

[0040] Further, the amount of dopamine solution used in step (7) is calculated as antibacterial dynamic nanogel: dopamine solution = 40-50 mL: 1-3 mL, preferably 45 mL: 2 mL.

[0041] 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.

[0042] Furthermore, the stirring conditions described in step (6) are: speed 200-300 rpm, time 0.5-2 h.

[0043] Furthermore, the stirring conditions described in step (7) are: speed 200-300 rpm, time 20-30 h, and temperature 20-30℃.

[0044] Furthermore, the molecular weight cutoff of the dialysis bag used in step (7) is 8k to 14kDa.

[0045] Furthermore, the pH value of the nanogel dispersion in step (8) is 2 to 6.

[0046] Further, the concentration of the nanogel dispersion in step (8) is 1 to 5 g / 100 mL, preferably 3 to 5 g / 100 mL.

[0047] An antibacterial and antioxidant dynamic nanogel was obtained by the above preparation method.

[0048] The above-mentioned antibacterial and antioxidant dynamic nanogels, or antibacterial and antioxidant dynamic nanogels, are used in the preparation of drugs that promote wound healing and tissue regeneration.

[0049] In this invention, NIPAM is the core of nanogel formation, M-Arg provides antibacterial properties, and dopamine provides antioxidant properties. The nanogel is easy to spray and has a large specific surface area, overcoming the problems of poor antibacterial effect of bulk hydrogels and poor biocompatibility of inorganic metal particle antibacterial agents. The polymeric nanogel spray prepared by this invention is pH-sensitive and temperature-sensitive. The nanogel of this invention gradually decreases in particle size as the temperature increases (25–60°C) and increases in particle size as the temperature decreases. The nanogel of this invention can achieve reversible particle size reduction after repeated treatment at different temperatures. The nanogel of this invention is positively charged in the pH range of 2–6, exhibiting good inhibitory effects on bacterial activity. The dynamic nanogel is used to kill or inhibit bacterial growth and reproduction at wound infection sites and to remove reactive oxygen species from affected areas.

[0050] The present invention has the following advantages and effects compared with the prior art:

[0051] (1) The present invention uses precipitation polymerization to synthesize dynamic polymer nanogel antibacterial agent, which is simple to operate, easy to synthesize, and convenient to spray.

[0052] (2) The present invention overcomes the problem of poor antibacterial effect caused by insufficient contact of large hydrogels.

[0053] (3) The present invention overcomes the problem of poor biocompatibility of inorganic metal particle antibacterial agents.

[0054] (4) The polymer nanogel antibacterial agent prepared by the present invention exhibits positive charge under acidic pH values ​​and can be applied in bacterial microenvironments with different acidity.

[0055] (5) The nanogel prepared by the present invention is temperature sensitive and has excellent antibacterial effect at different temperatures.

[0056] (6) The nanogel spray prepared by the present invention has both antibacterial and antioxidant effects, which is beneficial to further promote wound healing. Attached Figure Description

[0057] Figure 1 This is a transmission electron microscope image of the nanogel in Example 1;

[0058] Figure 2 This is a graph showing the change in nanogel particle size with temperature in Example 2;

[0059] Figure 3 This is a photograph showing the changes in the nanogel during repeated heating and cooling in Example 3.

[0060] Figure 4 This is a potential diagram of the nanogel at different pH values ​​in Example 4;

[0061] Figure 5 The graph shows the results of the antibacterial rate determination of the nanogel against Staphylococcus aureus in Example 5 and Comparative Example 1.

[0062] Figure 6 The graph shows the scavenging rate of ABTS free radicals by the nanogel in Example 6, Comparative Example 2, Comparative Example 3, and Comparative Example 4.

[0063] Figure 7 This is a graph showing the cell activity effect of mouse fibroblasts co-cultured with nanogel extract for 3 days in Example 7. Detailed Implementation

[0064] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. However, the implementation of the present invention is not limited thereto. The advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are merely exemplary in nature and do not constitute any limitation on the scope of the present invention.

[0065] The following description of the sources of raw materials used in the examples is as follows: L-arginine, methacrylic anhydride, N-isopropylacrylamide, N,N'-methylenebisacrylamide, hexadecyltrimethylammonium bromide, 2,2'-azobisisobutylammonium dihydrochloride, triethylamine, dioxane, acetone, dopamine hydrochloride, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were all purchased from Aladdin.

[0066] Example 1

[0067] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0068] (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;

[0069] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0070] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0071] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0072] (5) Stir 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;

[0073] (6) The nanogel solution obtained in step (5) is 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%.

[0074] 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. The results are as follows. Figure 1 As shown in the figure, the nanogels appear as spherical particles with relatively uniform size under electron microscopy.

[0075] Example 2

[0076] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0077] (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;

[0078] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0079] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0080] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0081] (5) Stir 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;

[0082] (6) The nanogel solution obtained in step (5) is 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%.

[0083] The nanogel solution was diluted with ultrapure water to 0.1 w / v%, and the change in nanogel particle size with temperature was characterized. The results are as follows: Figure 2 As shown in the figure, it can be seen that within the temperature range of 25℃ to 60℃, the particle size of the nanogel decreases as the temperature increases.

[0084] Example 3

[0085] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0086] (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;

[0087] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0088] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0089] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0090] (5) Stir 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;

[0091] (6) The nanogel solution obtained in step (5) is 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%.

[0092] The nanogel solution was diluted to 0.1 w / v% with ultrapure water, and the reversibility of the change in nanogel particle size with temperature was characterized by repeated heating and cooling. The results are as follows: Figure 3 As shown in the figure, it can be seen that the nanogel particle size is reversible with repeated heating and cooling. The heated nanogel solution (60℃) turns milky white, indicating that the nanogel particle size decreases, while the cooled nanogel solution (25℃) becomes clear and transparent, indicating that the hydrophilic nanogel particle size increases.

[0093] Example 4

[0094] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0095] (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;

[0096] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0097] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0098] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0099] (5) Stir 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;

[0100] (6) The nanogel solution obtained in step (5) is 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%.

[0101] First, the pH of the nanogel solution was adjusted to pH=2.0, pH=4.0, and pH=6.0. 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 as follows: Figure 4 As shown in the figure, the nanogels are positively charged within the pH range of 2-6, and the positive charge of the nanogels increases with increasing acidity.

[0102] Example 5

[0103] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0104] (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;

[0105] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0106] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0107] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0108] (5) Stir 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;

[0109] (6) The nanogel solution obtained in step (5) is 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%.

[0110] The 3 w / v nanogel was concentrated to 5 w / v, and then 200 μL of the 3 w / v and 5 w / v nanogel solutions were respectively mixed with 800 μL of a 10% concentration. 6 A mixture of CFU / mL Staphylococcus aureus was prepared, with 200 μL of PBS solution mixed with the bacterial culture as a control. All mixtures were incubated at 37°C for 2 h. The bacterial cultures were then diluted to 10⁻⁶. 4 After CFU / mL concentration, 10 μL of each sample was evenly spread onto an agar plate and incubated at 37°C for 12 h. The agar plates were then removed, and colonies were counted for each group. The antibacterial rate was calculated using the following formula: (where N is the antibacterial rate). x N represents the number of bacterial colonies in the nanogel group. c Colony count for control group: Antibacterial rate (%) = N x / N c ×100%.

[0111] Figure 5 The bar charts for the antibacterial rates of nanogels at different concentrations in Example 5 and Comparative Example 1 show that the nanogels have good antibacterial properties, and the higher the concentration, the stronger the antibacterial effect.

[0112] Example 6

[0113] A method for preparing an antibacterial and antioxidant nanogel spray includes the following steps:

[0114] (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;

[0115] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0116] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0117] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0118] (5) Stir 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;

[0119] (6) The nanogel solution obtained in step (5) is 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%.

[0120] (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 mixture;

[0121] (8) Inject 2 mL of 0.11 g / mL dopamine solution into the mixture in (7), stir at 250 rpm for 24 h at 25℃ to obtain an antibacterial and antioxidant nanogel dispersion;

[0122] (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. The solution was concentrated to obtain an antibacterial and antioxidant nanogel solution with a concentration of 3 w / v%, which was named NGDA.

[0123] 200 μL of the 3 w / v NGDA nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azono-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS). An ABTS solution without NGDA was used as a control group. Each mixture was incubated at 37°C in a shaker for 30 min. 100 μL of each mixture was then taken to measure the absorbance at 734 nm to calculate the free radical scavenging rate. Scavenging rate (%) = (1-A x / A c ) × 100%, A x A represents the absorbance measured in the experimental group. c This represents the absorbance measured in the control group under the same conditions.

[0124] Figure 6 The bar chart shows the scavenging rate of ABTS free radicals by the nanogels in Examples 6, 2, 3, and 4. It can be seen that the nanogel with added dopamine (NGDA) exhibits superior antioxidant properties compared to the nanogel without added dopamine (NG). Within a certain range, the higher the NGDA concentration, the stronger the antioxidant effect. When the concentration reaches 3 w / v%, the free radical scavenging ability basically reaches equilibrium, and further increasing the NGDA concentration does not significantly improve the free radical scavenging effect.

[0125] Example 7

[0126] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0127] (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;

[0128] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0129] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0130] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0131] (5) Stir 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;

[0132] (6) The nanogel solution obtained in step (5) is 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%.

[0133] (7) Dilute the nanogel solution obtained in step (6) with culture medium to obtain a culture medium containing 1 w / v%% nanogel, and co-culture it with mouse fibroblasts. Figure 7 This image shows the cell viability of mouse fibroblasts co-cultured with nanogel for 3 days in Example 7. It can be seen that no significant cell death was observed after 3 days of co-culture with nanogel, indicating that the nanogel has good biocompatibility.

[0134] Comparative Example 1

[0135] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0136] (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;

[0137] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0138] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0139] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0140] (5) Stir 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;

[0141] (6) Transfer the nanogel solution obtained in step (5) to a dialysis bag with a molecular weight cutoff of 8k-14kDa and dialyze for 3 days. Dilute the nanogel solution to obtain a nanogel solution with a concentration of 1 w / v%.

[0142] Take 200 μL of 1 w / v% nanogel solution and 800 μL of 10% nanogel solution. 6 A mixture of CFU / mL Staphylococcus aureus was prepared and incubated at 37°C for 2 hours. The bacterial culture was then diluted to 10⁻⁶. 4 After CFU / mL concentration, 10 μL of each nanogel was evenly spread onto an agar plate and incubated at 37°C for 12 h. The agar plates were then removed, colonies were counted, and the antibacterial rate was calculated. The antibacterial rate of the 1 w / v% concentration nanogel prepared in this comparative example is as follows: Figure 5 .

[0143] Comparative Example 2

[0144] A method for preparing a dynamic nanogel antibacterial agent includes the following steps:

[0145] (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;

[0146] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0147] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0148] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0149] (5) Stir 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;

[0150] (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.

[0151] 200 μL of the 3 w / v% NG nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azono-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS). The mixture was incubated in a shaker at 37°C for 30 min, and then 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 ABTS nanogel prepared in this comparative example is as follows: Figure 6 .

[0152] Comparative Example 3

[0153] A method for preparing an antibacterial and antioxidant nanogel spray includes the following steps:

[0154] (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;

[0155] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0156] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0157] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0158] (5) Stir 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;

[0159] (6) The nanogel solution obtained in step (5) is 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%.

[0160] (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 mixture;

[0161] (8) Inject 2 mL of 0.11 g / mL dopamine solution into the mixture in (7), stir at 250 rpm for 24 h at 25℃ to obtain an antibacterial and antioxidant nanogel dispersion;

[0162] (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. The solution was diluted to obtain an antibacterial and antioxidant nanogel spray with a concentration of 1 w / v%, which was named NGDA.

[0163] 200 μL of the 1 w / v% NGDA nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azono-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS). The mixture was incubated in a shaker at 37°C for 30 min, and then 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 ABTS nanogel prepared in this comparative example is as follows: Figure 6 .

[0164] Comparative Example 4

[0165] A method for preparing an antibacterial and antioxidant nanogel spray includes the following steps:

[0166] (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;

[0167] (2) Add 3 mL of methacrylic anhydride dropwise to the Arg solution in step (1) under ice bath and stir until homogeneous. The stirring speed is 200 rpm to obtain the reaction solution of the mixture.

[0168] (3) Stir the reaction solution of the mixture in step (2) overnight, recrystallize the reaction solution with 150 mL of acetone and dry the precipitate under vacuum to obtain white M-Arg powder;

[0169] (4) Prepare a solution containing 2.5 w / v% N-isopropylacrylamide (NIPAM), 0.5 w / v% M-Arg powder from 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;

[0170] (5) Stir 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;

[0171] (6) The nanogel solution obtained in step (5) is 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%.

[0172] (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 mixture;

[0173] (8) Inject 2 mL of 0.11 g / mL dopamine solution into the mixture in (7), stir at 250 rpm for 24 h at 25℃ to obtain an antibacterial and antioxidant nanogel dispersion;

[0174] (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. The concentration was then concentrated to obtain an antibacterial and antioxidant nanogel spray with a concentration of 5 w / v%, which was named NGDA.

[0175] 200 μL of the 5 w / v% NGDA nanogel solution prepared in this example was mixed with 400 μL of 140 mM 2,2-azono-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS). The mixture was incubated in a shaker at 37°C for 30 min. 100 μL of the mixture was then taken to measure the absorbance at 734 nm to calculate the free radical scavenging rate. The free radical scavenging rate of the ABTS nanogel prepared in this comparative example is as follows: Figure 6 .

[0176] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial and antioxidant dynamic nanogel, characterized in that: Includes the following steps: (1) Dissolve L-arginine in a mixed solvent of water, dioxane and triethylamine to obtain an Arg solution; (2) Add methacrylic anhydride dropwise to the Arg solution described in step (1) and stir until homogeneous to obtain a mixed reaction solution; (3) Stir the reaction solution of the mixture described in step (2), recrystallize the reaction solution with acetone, and dry the precipitate under vacuum to obtain white M-Arg powder; (4) Dissolve the M-Arg powder described in step (3) with N-isopropylacrylamide, N,N'-methylenebisacrylamide, hexadecyltrimethylammonium bromide and 2,2'-azobisisobutylammonium dihydrochloride in water, stir, and dialyze to obtain a nanogel dispersion; (5) Adjust the concentration and pH value of the nanogel dispersion in step (4) to obtain antibacterial dynamic nanogel; the concentration of the nanogel dispersion is 3-5 g / 100 mL. (6) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the antibacterial dynamic nanogel described in step (5), and stir under a nitrogen atmosphere to obtain a mixture; (7) Inject dopamine solution into the mixture in step (6), stir, dialyze, and obtain antibacterial and antioxidant nanogel dispersion; (8) Adjust the concentration and pH value of the antibacterial and antioxidant nanogel dispersion in step (7) to obtain the antibacterial and antioxidant dynamic nanogel; the concentration of the antibacterial and antioxidant nanogel dispersion is 3-5 g / 100 mL.

2. The method for preparing the antibacterial and antioxidant 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 each component in the mixed solvent are as follows: deionized water 60±5 v / v%, dioxane 26±3 v / v%, triethylamine 14±2 v / v%. The concentration of methacrylic anhydride in the reaction mixture described in step (2) is 8.4 ± 1 v / v% In step (4), the concentration of M-Arg in the nanogel dispersion 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'-azobisisobutylammonium dihydrochloride is 0.01–0.1 g / 100 mL; and the concentration of hexadecyltrimethylammonium bromide is 0.25–0.5 g / 100 mL.

3. The method for preparing the antibacterial and antioxidant 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; In step (4), the concentrations of M-Arg in the nanogel dispersion are: 0.5 g / 100 mL; N-isopropylacrylamide: 2.5 g / 100 mL; N,N'-methylenebisacrylamide: 0.025 g / 100 mL; 2,2'-azobisisobutylammonium dihydrochloride: 0.05 g / 100 mL; and hexadecyltrimethylammonium bromide: 0.3 g / 100 mL. The pH value of the nanogel dispersion in step (5) is 2 to 6.

4. The method for preparing the antibacterial and antioxidant dynamic nanogel according to any one of claims 1 to 3, characterized in that: The stirring conditions described in step (2) are: speed 200-300 rpm, time 10-20 min; The stirring conditions described in step (3) are: speed 200-300 rpm, time 8-18h; The amount of acetone used in step (3) is 3 to 5 times the volume of the mixture reaction solution; The stirring conditions described in step (4) are: speed 200-300 rpm, time 7-9 h, and temperature 65-75℃; The dialysis bag used in step (4) has a molecular weight cutoff of 8k to 14kDa.

5. The method for preparing the antibacterial and antioxidant dynamic nanogel according to claim 1, characterized in that: In the mixture described in step (6), the concentration of 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 dopamine solution used in step (7) is calculated as antibacterial dynamic nanogel: dopamine solution = 40-50 mL: 1-3 mL.

6. The method for preparing the antibacterial and antioxidant dynamic nanogel according to claim 1, characterized in that: In the mixture described in step (6), the concentration of the antibacterial dynamic nanogel is 3 g / 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 described in step (6) are: speed 200-300 rpm, time 0.5-2 h; The stirring conditions described in step (7) are: speed 200-300 rpm, time 20-30 h, and temperature 20-30℃; The molecular weight cutoff of the dialysis bag used in step (7) is 8k to 14kDa; The pH value of the nanogel dispersion in step (8) is 2 to 6.

7. A dynamic nanogel with antibacterial and antioxidant properties, characterized in that: It is obtained by the preparation method described in any one of claims 1 to 6.

8. The use of the antibacterial and antioxidant dynamic nanogel as described in claim 7 in the preparation of drugs that promote wound healing and tissue regeneration.

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