Anionic surfactant and polyarginine co-assembled antibacterial agent
By co-assembling the anionic surfactant with polyarginine to form an antibacterial agent with a specific structure, the problem of insufficient antibacterial activity of anionic surfactant is solved, and efficient bactericidal and biocompatibility is achieved for bacteria.
Patent Information
- Application Number
- CN202311360122.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-19
- Publication Date
- 2025-04-22
AI Technical Summary
In the prior art, anionic surfactants lack antibacterial activity or have low antibacterial activity, making it difficult to significantly improve their antibacterial properties while maintaining biocompatibility.
By co-assembling the anionic surfactant with polyarginine, an antibacterial agent with a layered structure or micelle structure is formed, and its assembly structure is regulated to improve antibacterial activity.
The efficient antibacterial activity of anionic surfactants is achieved, especially for E. coli and Staphylococcus aureus, while maintaining biocompatibility for mammalian cells.
Smart Images
Figure CN119857108A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of antibacterial agents, and in particular relates to an antibacterial agent based on the co-assembly of an anionic surfactant and polyarginine. Background Art
[0002] Pathogenic microorganisms such as bacteria and viruses pose a serious threat to human health and life, and antibiotics are the main means for people to fight against pathogenic microorganisms. However, traditional antibiotics often cause microbial resistance, thus losing their therapeutic effects. Surfactants are a class of amphiphilic molecules with surface activity and strong self-assembly ability. Among them, cationic surfactants have become a class of antibacterial agents that have attracted much attention due to their broad-spectrum and efficient antibacterial activity, and they can also effectively overcome the problem of drug resistance. Cationic surfactants mainly bind to the bacterial surface through electrostatic binding of the head group, and then insert into the bacterial membrane through the hydrophobic effect of the alkyl chain, thereby changing the permeability and osmotic pressure of the membrane, causing the leakage of bacterial contents, and ultimately leading to bacterial death. Although cationic surfactants have high antibacterial activity, their strong positive charge also leads to their high cytotoxicity, which is limited in practical applications. Compared with cationic surfactants, anionic surfactants have better biocompatibility and are also widely used surfactants in production and life, but they do not have antibacterial activity or have low antibacterial activity. It is of great significance to significantly improve the antibacterial activity of anionic surfactants and maintain their biocompatibility, which will help expand the application field of anionic surfactants. However, how to endow anionic surfactants with efficient antibacterial activity is a challenging research topic, and no relevant reports have been seen so far. Summary of the invention
[0003] In view of the defects and shortcomings of the prior art, the present invention constructs an antibacterial agent based on the co-assembly of polyarginine and anionic surfactant. The antibacterial agent has the advantages of simple preparation, safety, non-toxicity, and high antibacterial efficiency.
[0004] The invention provides an antibacterial agent, which is an aggregate formed by co-assembly of an anionic surfactant and polyarginine.
[0005] According to an embodiment of the present invention, the antibacterial agent has a lamellar structure, a micellar structure (eg, a worm-like micellar structure), and preferably a lamellar structure.
[0006] According to an embodiment of the present invention, the molecular formula of polyarginine is (Arg) n , where n is selected from any natural number between 2 and 20.
[0007] According to an embodiment of the present invention, n is selected from any natural number between 4 and 16, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16.
[0008] According to an embodiment of the present invention, polyarginine (Arg) n It is poly-arginine (Arg)8.
[0009] According to an embodiment of the present invention, the anionic surfactant is selected from one, two or more of a carboxylate type anionic surfactant, a sulfonate type anionic surfactant, a sulfate type anionic surfactant, and a phosphate type anionic surfactant.
[0010] According to an embodiment of the present invention, the anionic surfactant is selected from one, two or more of sodium dodecyl sulfate, sodium α-olefin sulfonate, lauryl amide propyl betaine, sodium dodecyl fatty alcohol ether sulfate, and sodium lauryl sulfate.
[0011] According to an embodiment of the present invention, the charge ratio of the anionic surfactant to polyarginine is 1:1-20, preferably 1:2-10, such as 1:4, 1:5, 1:6, 1:7, 1:8.
[0012] According to an embodiment of the present invention, the molar ratio of the anionic surfactant to polyarginine is 1-20:1, preferably 1-10:1, for example 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1.
[0013] The present invention also provides a method for preparing the above antibacterial agent, and the method for preparing the antibacterial agent is as follows:
[0014] The antibacterial agent is obtained by mixing the anionic surfactant solution and the polyarginine solution.
[0015] According to an embodiment of the present invention, the anionic surfactant solution and / or the polyarginine solution is an aqueous solution.
[0016] According to an embodiment of the present invention, the concentration of the anionic surfactant solution is 1 μM-1000 μM, preferably 10 μM-500 μM, more preferably 50 μM-300 μM, such as 100 μM, 150 μM.
[0017] According to an embodiment of the present invention, the concentration of the poly-arginine solution is 5 μM-500 μM, preferably 10 μM-300 μM, such as 100 μM, 50 μM, 25 μM.
[0018] According to an embodiment of the present invention, the pH of the poly-arginine solution is 6.0-8.0, for example, 7.0.
[0019] According to an embodiment of the present invention, the mixing condition is room temperature.
[0020] According to an embodiment of the present invention, the volume ratio of the anionic surfactant solution and the polyarginine solution when mixed is 0.1-10:1, preferably 5-0.5:1, such as 3:1, 1.5:1, 1:1.
[0021] According to an embodiment of the present invention, the charge ratio of the anionic surfactant to polyarginine is 1:1-20, preferably 1:2-10, such as 1:4, 1:5, 1:6, 1:7, 1:8.
[0022] According to an embodiment of the present invention, the molar ratio of the anionic surfactant to polyarginine is 1-20:1, preferably 1-10:1, for example 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1.
[0023] The present invention also provides the use of the antibacterial agent in preparing medicines for inhibiting bacterial growth or killing bacteria.
[0024] According to an embodiment of the present invention, the bacteria are Gram-negative bacteria or Gram-positive bacteria; for example, Escherichia coli, Staphylococcus aureus.
[0025] The present invention also provides the use of the above antibacterial agent in the preparation of a medicament for promoting wound healing. According to an embodiment of the present invention, the wound is a wound infected by bacteria.
[0026] According to an embodiment of the present invention, the bacteria are Gram-negative bacteria or Gram-positive bacteria; for example, Escherichia coli, Staphylococcus aureus.
[0027] Beneficial Effects
[0028] The present invention provides a safe and efficient antibacterial agent. The antibacterial agent provided by the present invention is composed of polyarginine (Arg) n The aggregates formed by co-assembly with anionic surfactants have a simple preparation method. The antibacterial agent has high safety and is non-toxic to mammalian cells; and has significant antibacterial activity against Escherichia coli and Staphylococcus aureus.
[0029] The present invention can adjust the number of arginine in polyarginine (e.g., 4, 8, 12 or 16) and the charge ratio between polyarginine and anionic surfactant to regulate the assembly structure of the antibacterial agent; and the antibacterial agent has the strongest antibacterial activity when forming a layered structure. At the same time, in the in vivo mouse experiment, the antibacterial agent also exhibits efficient antibacterial activity and can promote wound healing. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Cryo-TEM image of antibacterial agent 2, which is a layered structure formed by (Arg)8 and anionic surfactant SDS.
[0031] Figure 2 The cytotoxicity results of antimicrobial agent 2, which is a layered structure formed by (Arg)8 and anionic surfactant SDS.
[0032] Figure 3 This is a diagram showing the killing effect of antibacterial agent 2 formed by (Arg)8 and anionic surfactant SDS on Escherichia coli and Staphylococcus aureus.
[0033] Figure 4 It is a statistical chart of the sterilization efficiency after treatment of each embodiment and comparative example.
[0034] Figure 5 The result of the antibacterial agent 4, which is a layered structure formed by (Arg)8 and the anionic surfactant SDS, promoting wound healing. DETAILED DESCRIPTION
[0035] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.
[0036] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
[0037] In the following examples and comparative examples, the bactericidal efficiency is obtained by the following method:
[0038] The bactericidal efficiency was evaluated by the traditional plate counting method, that is, the bactericidal efficiency of the antimicrobial agent against Escherichia coli and Staphylococcus aureus before and after treatment was evaluated by the reduction rate of colony forming units (CFU). The prepared antimicrobial agent was mixed with the bacterial solution and co-cultured at 37°C for 2 hours. Finally, 100 μL of the diluted Escherichia coli and Staphylococcus aureus suspension was spread on the TSA agar plate. After culturing at 37°C for 16 to 18 hours, the number of colonies was recorded, and the percentage of CFU reduction, i.e., the bactericidal efficiency, was calculated according to the following formula.
[0039]
[0040] Where c0 is the number of colonies in the blank group, and c is the number of colonies in the experimental group.
[0041] Example 1
[0042] A 150 μM SDS aqueous solution and a 100 μM (Arg) 8 aqueous solution with a pH of 7 were prepared respectively, and the two aqueous solutions were mixed at a volume ratio of 3:1 at room temperature to obtain an antibacterial agent 1 having a worm-like micelle structure. Fresh slant cultures of Escherichia coli and Staphylococcus aureus were washed with PBS to prepare 10 9 CFU / mL bacterial suspension. Take 1mL of the prepared antimicrobial agent 1 and place it in two sterile test tubes, take 10μL of the above two bacterial suspensions and add them to two sterile test tubes, and take 10μL of the above two bacterial suspensions and add them to two 1mL sterile water test tubes as controls. After a period of action, make appropriate dilutions, take 100μL of each, inoculate it in agar culture medium by pouring method, culture at 37℃ for 16-20h, count the live bacterial colonies, and repeat the experiment three times. The bactericidal efficiency of antimicrobial agent 1 against Escherichia coli is 78.9%, and the bactericidal efficiency against Staphylococcus aureus is 57.5%.
[0043] Example 2
[0044] A 100 μM SDS aqueous solution and a 50 μM (Arg) 8 aqueous solution with a pH of 7 were prepared respectively, and the two aqueous solutions were mixed at a volume ratio of 3:2 at room temperature to obtain a layered structure ( Figure 1 ) to obtain antibacterial agent 2. Fresh slant cultures of Escherichia coli and Staphylococcus aureus were washed with PBS to prepare 10 9 CFU / mL bacterial suspension. Take 1mL of the prepared antimicrobial agent 2 and place it in two sterile test tubes, take 10μL of the above two bacterial suspensions and add them to two sterile test tubes, and at the same time take 10μL of the above two bacterial suspensions and add them to two 1mL sterile water test tubes as controls. After a period of action, make appropriate dilutions, take 100μL of each, inoculate it into agar culture medium by pouring method, culture at 37℃ for 16-20h, count the live bacterial colonies, and repeat the experiment three times. The bactericidal efficiency of antimicrobial agent 2 against Escherichia coli is 100%, and the bactericidal efficiency against Staphylococcus aureus is 100% (such as Figure 3 shown).
[0045] Example 3
[0046] A 150 μM SDS aqueous solution and a 25 μM (Arg) 8 aqueous solution with a pH of 7 were prepared respectively, and the two aqueous solutions were mixed at a volume ratio of 1:1 at room temperature to obtain an antibacterial agent 3 having a worm-like micelle structure. Fresh slant cultures of Escherichia coli and Staphylococcus aureus were washed with PBS to prepare 10 9CFU / mL bacterial suspension. Take 1mL of the prepared antimicrobial agent 3 and place it in two sterile test tubes, take 10μL of the above two bacterial suspensions and add them to two sterile test tubes, and take 10μL of the above two bacterial suspensions and add them to two 1mL sterile water test tubes as controls. After a period of action, make appropriate dilutions, take 100μL of each, inoculate it in agar culture medium by pouring method, culture at 37℃ for 16-20h, count the live bacterial colonies, and repeat the experiment three times. The bactericidal efficiency of antimicrobial agent 3 against Escherichia coli is 77.1%, and the bactericidal efficiency against Staphylococcus aureus is 55.9%.
[0047] Example 4
[0048] The antimicrobial agent 2 in Example 2 was prepared into different concentrations (6.25 / 12.5 / 25 / 50 / 100 μM), and its toxicity to human vascular endothelial HUVEC cells and human renal epithelial 293T cells was tested by MTT method. 3 The cells were inoculated into a 96-well culture plate at a density of 100 μL / well. After 12 hours, 100 μL (Arg) 8 / SDS solution containing DMEM medium was added to each well. Then, the cells were grown at 37°C and in a humidified atmosphere containing 5% CO2 for 24 hours until they adhered to the wall. After incubation at 37°C for 24 hours, the supernatant was removed, MTT (0.5 mg / mL culture medium, 100 μL / well) was added to the wells, incubated at 37°C for 4 hours, the supernatant was removed, and 100 μL dimethyl sulfoxide (DMSO) was added to each well. After shaking the plate for 5 minutes, the absorbance value at 570 nm was read with an enzyme reader to calculate the cell viability.
[0049] The results are as follows Figure 2 As shown, under the experimental concentration conditions, the (Arg)8 / SDS antimicrobial agent exhibited good biocompatibility to HUVEC cells and 293T cells, and even had the effect of promoting cell growth under higher concentration conditions.
[0050] Example 5
[0051] A 1 mM SDS aqueous solution and a 200 μM (Arg) 8 aqueous solution with a pH of 7 were prepared respectively, and the two aqueous solutions were mixed at a volume ratio of 1:1 at room temperature to obtain an antibacterial agent 4 having a layered structure. The antibacterial agent 4 was used in a mouse wound healing experiment.
[0052] On the first day, wounds with a diameter of 8 cm were created on the backs of all mice and Staphylococcus aureus (10 5 CFU / mL) for wound infection. After a period of infection, the control group was treated with PBS buffer and the experimental group was treated with antimicrobial agent 4 for two consecutive days. Wound photos were recorded on the 1st, 4th, 7th, 10th and 12th days. The results showed that antimicrobial agent 4 had a significant effect on promoting wound healing ( Figure 5).
[0053] Comparative Example 1
[0054] Prepare 100 μM SDS aqueous solution, wash the fresh slant culture of Escherichia coli and Staphylococcus aureus with PBS, and prepare 10 9 CFU / mL bacterial suspension. Take 1mL of the prepared SDS aqueous solution and place it in two sterile test tubes, take 10μL of the above two bacterial suspensions and add them to two sterile test tubes, and take 10μL of the above two bacterial suspensions and add them to two 1mL sterile water test tubes as controls. After a period of action, make appropriate dilutions, take 100μL of each, inoculate it in agar culture medium by pouring method, culture at 37℃ for 16-20h, count the live bacterial colonies, and repeat the experiment three times. The bactericidal efficiency of SDS against Escherichia coli is 4%, and the bactericidal efficiency against Staphylococcus aureus is 5%.
[0055] Comparative Example 2
[0056] Prepare a 50 μM (Arg) 8 aqueous solution at pH 7, wash the fresh slant cultures of Escherichia coli and Staphylococcus aureus with PBS, and prepare 10 9 CFU / mL bacterial suspension. Take 1mL of the prepared (Arg)8 aqueous solution and place it in two sterile test tubes, take 10μL of the above two bacterial suspensions and add them to two sterile test tubes, and take 10μL of the above two bacterial suspensions and add them to two 1mL sterile water test tubes as controls. After a period of action, make appropriate dilutions, take 100μL of each, inoculate it in agar culture medium by pouring method, culture at 37℃ for 16-20h, count the live bacterial colonies, and repeat the experiment three times. The bactericidal efficiency of (Arg)8 against Escherichia coli is 6%, and the bactericidal efficiency against Staphylococcus aureus is 8%.
[0057] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. An antibacterial agent, which is an aggregate formed by co-assembly of an anionic surfactant and polyarginine.
2. The antibacterial agent according to claim 1, characterized in that The antibacterial agent has a lamellar structure or a micellar structure (e.g., a worm-like micellar structure), preferably a lamellar structure; And / or, the molecular formula of the polyarginine is (Arg) n , wherein n is selected from any natural number between 2 and 20; preferably, n is selected from any natural number between 4 and 16; further preferably, polyarginine (Arg) n is polyarginine (Arg)8; And / or, the anionic surfactant is selected from one, two or more of carboxylate anionic surfactants, sulfonate anionic surfactants, sulfate ester salt anionic surfactants, and phosphate ester salt anionic surfactants; preferably, the anionic surfactant is selected from one, two or more of sodium dodecyl sulfate, sodium α-olefin sulfonate, lauryl amide propyl betaine, sodium dodecyl fatty alcohol ether sulfate, and sodium lauryl sulfate.
3. The antibacterial agent according to claim 1 or 2, characterized in that According to an embodiment of the present invention, the charge ratio of the anionic surfactant to polyarginine is 1:1-20, preferably 1:2-10, such as 1:4, 1:5, 1:6, 1:7, 1:
8. According to an embodiment of the present invention, the molar ratio of the anionic surfactant to polyarginine is 1-20:1, preferably 1-10:1, for example 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:
1.
4. The method for preparing the antibacterial agent according to any one of claims 1 to 3, wherein the method for preparing the antibacterial agent is as follows: The antibacterial agent is obtained by mixing the anionic surfactant solution and the polyarginine solution.
5. The preparation method according to claim 4, characterized in that: The anionic surfactant solution and / or polyarginine solution is an aqueous solution; and / or, the concentration of the anionic surfactant solution is 1 μM-1000 μM, preferably 10 μM-500 μM, more preferably 50 μM-300 μM, for example 100 μM, 150 μM; and / or, the concentration of the polyarginine solution is 5 μM-500 μM, preferably 10 μM-300 μM, such as 100 μM, 50 μM, 25 μM; And / or, the pH of the polyarginine solution is 6.0-8.0, for example, 7.0; and / or, the mixing condition is room temperature; and / or, the volume ratio of the anionic surfactant solution and the polyarginine solution when mixed is 0.1-10:1, preferably 5-0.5:1, such as 3:1, 1.5:1, 1:1; and / or, the charge ratio of the anionic surfactant to polyarginine is 1:1-20, preferably 1:2-10, for example 1:4, 1:5, 1:6, 1:7, 1:8; And / or, the molar ratio of the anionic surfactant to polyarginine is 1-20:1, preferably 1-10:1, for example 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:
1.
6. Use of the antibacterial agent according to any one of claims 1 to 3 in the preparation of a drug for inhibiting bacterial growth or killing bacteria.
7. The use according to claim 6, characterized in that: The bacteria are Gram-negative bacteria or Gram-positive bacteria; for example, Escherichia coli and Staphylococcus aureus.
8. Use of the antibacterial agent according to any one of claims 1 to 3 in the preparation of a medicament for promoting wound healing.
9. The use according to claim 8, characterized in that: The wound is a wound infected by bacteria; And / or, the bacteria are Gram-negative bacteria or Gram-positive bacteria; for example, Escherichia coli, Staphylococcus aureus.