A copper-containing hydrogel and its application in inhibiting bacterial infection

Abstract

The present invention discloses a copper-containing hydrogel and its application in inhibiting bacterial infection. The copper-containing hydrogel is composed of the following: a copper ion compound, sodium alginate, and an auxiliary solvent. The antibacterial active ingredient is the copper ion compound. Through the antibacterial activity test of Staphylococcus aureus, it is determined that the copper ion compound has a strong bactericidal effect. When killing Staphylococcus aureus, its survival rate is less than 0.001%, and when killing methicillin-resistant Staphylococcus aureus (MRSA), its survival rate is less than 0.01%. Using a mouse model of Staphylococcus aureus-infected epidermal wound, we compared the copper-containing hydrogel with commonly used drugs erythromycin ointment and mupirocin ointment on the market and found that the copper-containing hydrogel has the strongest healing ability and wound surface clearance ability, and the copper ion compound does not belong to antibiotics and is not prone to drug resistance. Therefore, the copper-containing hydrogel has great application potential in wound infection.

Description

[0001] This divisional patent application is for the invention patent application with the application date of August 14, 2023, application number CN 202311020808.1, and invention title "A Copper-containing Hydrogel and Its Application in Inhibiting Bacterial Infection". Technical Field

[0002] The present invention relates to the field of medical technology and discloses a copper-containing hydrogel and its application in inhibiting bacterial infection. Background Art

[0003] Staphylococcus aureus belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It is a common foodborne pathogenic microorganism and one of the important pathogenic bacteria causing bacterial food poisoning and hospital clinical infections, seriously threatening food safety and human health. Staphylococcus aureus is a clinically common bacterium with relatively strong toxicity. Since the advent of penicillin in the 1940s, infectious diseases caused by Staphylococcus aureus have been relatively well controlled. However, with the widespread use of penicillin, some Staphylococcus aureus produce penicillinase, which can hydrolyze the β-lactam ring, showing resistance to penicillin. Scientists developed a new semi-synthetic penicillin that can resist penicillinase, namely methicillin. After its application in clinical practice in 1959, it effectively controlled the infection of Staphylococcus aureus strains producing enzymes. However, Jevons in the UK first discovered methicillin-resistant Staphylococcus aureus (MRSA). In the colonies of MRSA, there are two subpopulations of bacteria, sensitive and resistant. That is, only a small part of the bacteria in a strain of MRSA, about 10 -4 ~10 -7 , are highly resistant to methicillin and can still survive under the condition of 50 μg / ml methicillin. Most of the bacteria in the colony are sensitive to methicillin. A large number of sensitive bacteria are killed within a few hours after the use of antibiotics, but a small number of resistant strains grow slowly and then proliferate rapidly after several hours. Since its discovery, MRSA infections have spread almost all over the world. Methicillin-resistant Staphylococcus aureus is a clinically common bacterium with relatively strong toxicity and has become one of the important pathogenic bacteria in hospital and community infections. At present, antibiotics are mainly used for the prevention and treatment of Staphylococcus aureus at home and abroad. However, due to the abuse of antibiotics, their drug resistance has increased and super-resistant bacteria have emerged. Therefore, there is an urgent need to develop new antibacterial agents and explore new antibacterial products. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides the application of a copper-containing hydrogel in inhibiting bacterial infection. It has a good killing effect, can keep the skin moist, non-irritating, and is not prone to drug resistance, revealing that copper ion compounds can be used as drugs to treat Staphylococcus aureus infections including MRSA.

[0005] The present invention is realized through the following technical solutions:

[0006] A copper-containing hydrogel, comprising

[0007] The copper-containing hydrogel is prepared from a copper ion compound, sodium alginate, and an auxiliary solvent.

[0008] Preferably, the copper ion compound is any one of copper sulfate, copper gluconate, cuprous iodide, copper citrate, and copper chlorophyllin sodium.

[0009] Preferably, the concentration of copper ions in the copper-containing hydrogel is 16 - 256 μM.

[0010] Preferably, the auxiliary solvent is water or dimethyl sulfoxide.

[0011] Preferably, the specific preparation process of the copper-containing hydrogel is as follows:

[0012] S1: Dissolve the copper ion compound in the auxiliary solvent to form a mixed solution A;

[0013] S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished copper-containing hydrogel;

[0014] S3: Filter the semi-finished copper-containing hydrogel and fill it aseptically to obtain the copper-containing hydrogel.

[0015] Preferably, the addition amount of sodium alginate is 2% of the mass of the mixed solution A.

[0016] Preferably, the semi-finished copper-containing hydrogel in S3 is filtered with an organic filter membrane.

[0017] An application of a copper-containing hydrogel in inhibiting bacterial infections, including the application of the copper-containing hydrogel in the preparation of a product for treating bacterial infections caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0018] Preferably, the bacterial infection is an epidermal infection, and the bacterial infection is an epidermal wound infection caused by Staphylococcus aureus or methicillin-resistant Staphylococcus aureus.

[0019] Preferably, when the copper-containing hydrogel sterilizes Staphylococcus aureus, its survival rate is less than 0.001%.

[0020] Preferably, when the copper-containing hydrogel sterilizes methicillin-resistant Staphylococcus aureus, its survival rate is less than 0.01%.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] The present invention discloses the application of a copper-containing hydrogel in inhibiting bacterial infections. A copper-containing gel is prepared from a copper ion compound, sodium alginate, and auxiliary solvent A. The antibacterial activity test of copper ions against Staphylococcus aureus shows a strong bactericidal effect, with good killing effect on it, and it can keep the skin moist, non-irritating, and not easily develop drug resistance, revealing that the copper ion compound can be used as a drug to treat Staphylococcus aureus infections including MRSA. The preparation method of the copper-containing hydrogel agent of the present invention is simple, and it is administered in the form of a gel agent, which is convenient to use, evenly administered, and has a rapid onset. At the same time, the moisturizer sodium alginate is added, which is beneficial to the moisturizing and repair of the skin.

[0023] Furthermore, the present invention provides that the copper-containing hydrogel agent has a high bactericidal effect on both standard strains and drug-resistant strains of Staphylococcus aureus. When killing Staphylococcus aureus, its survival rate is less than 0.001%, and when killing methicillin-resistant Staphylococcus aureus (MRSA), its survival rate is less than 0.01%. Using the copper ion gel to treat bacterial infections such as Staphylococcus aureus indicates that copper ions can provide a new strategy for solving the current antibiotic crisis. Brief Description of the Drawings

[0024] Figure 1 It is a schematic diagram of the antibacterial results of five copper ion compounds against Staphylococcus aureus. (A) is a schematic diagram of the bactericidal effect of copper sulfate; (B) is a schematic diagram of the bactericidal effect of copper gluconate (B); (C) is a schematic diagram of the bactericidal effect of copper citrate; (D) is a schematic diagram of the bactericidal effect of cuprous iodide (D); (E) is a schematic diagram of the bactericidal effect of copper chlorophyllin.

[0025] Figure 2 It is a schematic diagram of the antibacterial results of copper sulfate in the examples. (A) is a schematic diagram of the antibacterial results of copper sulfate against Staphylococcus aureus ATCC6538; (B) is a schematic diagram of the antibacterial results of copper sulfate against methicillin-resistant Staphylococcus aureus.

[0026] Figure 3 It is a schematic diagram of the effect diagram of Staphylococcus aureus after being treated with copper sulfate in the examples. (A) is an inverted fluorescence microscope image of Staphylococcus aureus ATCC 6538 after being treated with copper sulfate; (B) is a flow cytometry image of Staphylococcus aureus ATCC6538 after being treated with copper sulfate;

[0027] Figure 4 It is a schematic diagram of the field emission scanning electron microscope (SEM) of Staphylococcus aureus after being treated with copper sulfate.

[0028] Figure 5 It is a schematic diagram of the bactericidal effects of Example 1 and Example 2.

[0029] Figure 6Gross view of wound healing in mice after 5 days of hydrogel treatment. Detailed implementation mode

[0030] The present invention will be further described in detail below in conjunction with specific embodiments, which are explanations of the present invention rather than limitations.

[0031] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0032] Example 1

[0033] A copper-containing hydrogel, comprising the following steps:

[0034] S1: Dissolve copper sulfate in an auxiliary solvent, which can be water, to form a mixed solution A.

[0035] S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished copper-containing hydrogel. The addition amount of sodium alginate is 2% of the mass of the mixed solution.

[0036] S3: Sterilely fill the semi-finished copper-containing hydrogel to make a finished copper-containing hydrogel.

[0037] The application of the copper-containing hydrogel in inhibiting bacterial infection.

[0038] Example 2

[0039] A copper-containing hydrogel, comprising the following steps:

[0040] S1: Dissolve copper gluconate in an auxiliary solvent, which can be water, to form a mixed solution A.

[0041] S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished copper-containing hydrogel. The addition amount of sodium alginate is 2% of the mass of the mixed solution.

[0042] S3: Sterilely fill the semi-finished copper-containing hydrogel to make a finished copper-containing hydrogel.

[0043] The application of the copper-containing hydrogel in inhibiting bacterial infection.

[0044] Example 3

[0045] A copper-containing hydrogel, comprising the following steps:

[0046] S1: Dissolve cuprous iodide in an auxiliary solvent, which can be water, to make a mixed solution A.

[0047] S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished copper-containing hydrogel. The addition amount of sodium alginate is 2% of the mass of the mixed solution.

[0048] S3: Sterilely fill the semi-finished copper-containing hydrogel to make a finished copper-containing hydrogel.

[0049] The application of the copper-containing hydrogel in inhibiting bacterial infection.

[0050] Example 4

[0051] A copper-containing hydrogel, comprising the following steps:

[0052] S1: Dissolve copper citrate in an auxiliary solvent, which can be water, to make a mixed solution A.

[0053] S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished copper-containing hydrogel. The addition amount of sodium alginate is 2% of the mass of the mixed solution A.

[0054] S3: Sterilely fill the semi-finished copper-containing hydrogel to make a finished copper-containing hydrogel.

[0055] The application of the copper-containing hydrogel in inhibiting bacterial infection.

[0056] Example 5

[0057] A copper-containing hydrogel, comprising the following steps:

[0058] S1: Dissolve sodium copper chlorophyllin in an auxiliary solvent, which can be dimethyl sulfoxide, to make a mixed solution A.

[0059] S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished copper-containing hydrogel. The addition amount of sodium alginate is 2% of the mass of the mixed solution.

[0060] S3: Sterilely fill the semi-finished copper-containing hydrogel to make a finished copper-containing hydrogel.

[0061] The application of the copper-containing hydrogel in inhibiting bacterial infection.

[0062] Example 6

[0063] The difference between Example 6 and Example 1 is that the concentration of copper ions is 4 μM;

[0064] Example 7

[0065] Example 7 is different from Example 1 in that the concentration of copper ions is 16 μM;

[0066] Example 8

[0067] Example 8 is different from Example 1 in that the concentration of copper ions is 64 μM;

[0068] Example 9

[0069] Example 9 is different from Example 1 in that the concentration of copper ions is 256 μM;

[0070] To verify the antibacterial activity of copper ions against Staphylococcus aureus, the following experiments were conducted:

[0071] 1. Five copper ion compounds were separately prepared, including: copper sulfate, copper gluconate, cuprous iodide, copper citrate, and sodium copper chlorophyllin. After 3 h of drug treatment, 10 8 CFU / mL of Staphylococcus aureus were prepared, and the total number of bacterial colonies was determined by gradient drop plate experiment to explore the bacteriostatic effect of copper ions on Staphylococcus aureus.

[0072] Results: Through Figure 1 Results A - E showed that when the concentration of copper salts was 256 μM or less, the bactericidal rate of the five copper salts against Staphylococcus aureus reached over 99.9%, indicating that copper ions play an important role in the process of killing Staphylococcus aureus. In particular, when the concentrations of copper sulfate and copper gluconate were 16 μM, they could inhibit the growth of almost 100% of Staphylococcus aureus.

[0073] 2. Copper sulfate solutions with better bactericidal effects were prepared at concentrations of 4 μM, 16 μM, 64 μM, and 256 μM respectively. 10 8 CFU / mL of Staphylococcus aureus and methicillin - resistant Staphylococcus aureus (MRSA) were prepared. The total number of bacterial colonies was determined by gradient drop plate experiment to explore the minimum bactericidal concentration (MBC) of copper ions against Staphylococcus aureus standard strain and MASA.

[0074] Results: As Figure 2 the results showed that copper sulfate could induce Figure 2 A Staphylococcus aureus standard strain (ATCC 6538) and Figure 2 B methicillin - resistant Staphylococcus aureus (MRSA) to die to varying degrees, and the degree of death showed concentration - dependence. From the bactericidal results, it can be known that the MBC value of copper sulfate against Staphylococcus aureus standard strain (ATCC 6538) was 16 μM, and at this concentration, the bactericidal rate could reach 99.99%; the MBC value of copper sulfate against methicillin - resistant Staphylococcus aureus (MRSA) was 64 μM.

[0075] Therefore, copper ion compounds have good prospects for bactericidal applications and can combat the drug resistance of methicillin-resistant Staphylococcus aureus.

[0076] 3. To further exclude the possibility that copper ions induce Staphylococcus aureus to enter the viable but non-culturable (VBNC) state, a copper sulfate treatment group and a control group loaded with PI probes were prepared and observed by inverted fluorescence microscopy and flow cytometry.

[0077] Results: As Figure 3 shown in B, compared with the control group, the copper sulfate group showed a significant right shift and presented a dose-dependence. Figure 3 The fluorescence microscopy results in A were consistent with the flow cytometry results. The above results further excluded the possibility that copper ions cause Staphylococcus aureus to enter the VBNC state and verified its bactericidal effect.

[0078] 4. Analyze the phenotypic structure of Staphylococcus aureus after treatment with copper sulfate by field emission scanning electron microscopy (SEM)

[0079] Results: After treatment with 4 μM and 16 μM copper sulfate for 3 h, the morphology of Staphylococcus aureus was as Figure 4 shown. When the copper sulfate concentration reached 1 / 4 MBC, the bacteria became significantly smaller and wrinkles appeared on the surface. When the concentration reached MBC, obvious membrane perforation phenomena occurred in the bacteria. This indicates that the introduction of copper sulfate will damage the morphology of Staphylococcus aureus, and the degree of damage intensifies with the increase of copper sulfate concentration, while the basic morphological structure is the basis for the normal function of cells.

[0080] 5. To verify the performance of the copper-containing hydrogel of the present invention, antibacterial activity analysis was specifically carried out on Example 1 and Example 2: Prepare Staphylococcus aureus at 10 8 CFU / mL, and take the gel solutions of Example 1 and Example 2 for treatment respectively. Use the gradient drop plate experiment to measure the total number of bacterial colonies and explore the antibacterial effects of Example 1 and Example 2 on Staphylococcus aureus respectively.

[0081] Results: Figure 5 It was shown that Example 1 (A) and Example 2 (B) had good bactericidal effects, and the bactericidal rates were both above 99.9%, showing good prospects for antibacterial applications.

[0082] 6. To verify the performance of the copper-containing hydrogel of the present invention, the following zoological tests were specifically carried out on Example 9 to further illustrate its effects:

[0083] (1) Preparation of Staphylococcus aureus suspension

[0084] MRSA (from the Chinese Academy of Sciences) overnight bacteria were subcultured (1:200) until the OD600 = 0.3, collect 1×10 8 ~1×10 10 CFU / mL, resuspend it in physiological saline after removing the culture medium and set it aside for use.

[0085] (2) Establish an animal model of Staphylococcus aureus-infected epidermal wound in mice

[0086] Mouse preparation: 30 KM mice, about 7 weeks old, male.

[0087] Model establishment:

[0088] a. Anesthesia: Inject 0.003 mL / kg chloral hydrate intraperitoneally into each mouse for anesthesia. Observe the respiratory state and heart beating frequency of the mouse for about 3 - 5 minutes (the respiratory rhythm slows down and the heart beating frequency decreases) to judge whether the mouse is successfully anesthetized;

[0089] b. Back wound incision: Depilate the back of the mouse with depilatory cream, scrape the back with a saline gauze, and wipe the back of the mouse with 75% ethanol. Create a full-thickness wound (10 mm × 10 mm) on the back skin of each mouse until the deep subcutaneous tissue, and keep it open to cause skin injury and loss on the back of the mouse;

[0090] c. Bacteria inoculation: Drop two drops of the prepared Staphylococcus aureus suspension (10 8 CFU / mL, 50 μL per drop, a total of 100 μL) onto the surface of the back wound of the mouse, and bandage the wound with a sterile gauze;

[0091] d. Infection: Observe the abscess situation on the wound surface of the mouse 48 h after the inoculation treatment;

[0092] (3) Animal grouping and drug administration

[0093] a. Grouping: After the animal model of Staphylococcus aureus-infected epidermal wound in mice is successfully established, divide it into 6 groups with 6 mice in each group for drug administration treatment, namely:

[0094] Blank group (6 mice): There are invasive wounds on the back of the mouse, but no bacteria are inoculated. Drop 100 μL of sterile physiological saline onto the wound;

[0095] Model group (6 mice): The established mouse model, without drug treatment, drop 100 μL of sterile physiological saline onto the wound;

[0096] Erythromycin ointment treatment group (6 mice): The established mouse model, smear 100 μL of erythromycin ointment on the wound for treatment, and then perform the treatment with the hydrogel loaded in Example 1, a total of 1 mL;

[0097] Mupirocin Ointment Treatment Group (6 mice): For the established mouse model, the wound was treated with 100 μL of mupirocin ointment.

[0098] Sodium Alginate Hydrogel Group (6 mice): For the established mouse model, the wound was treated with 100 μL of sodium alginate hydrogel.

[0099] Copper-Containing Sodium Alginate Hydrogel Group (6 mice): For the established mouse model, the wound was treated with 100 μL of 256 μM copper-containing sodium alginate hydrogel.

[0100] b. Drug Administration: The mice in each group were administered drugs according to the divided groups, twice a day. First, the wound was cleaned with physiological saline containing the drug, and then the hydrogel was applied. The interval between the two times was 10 min, and then the wound was bandaged with a sterile gauze again. The treatment lasted for 5 days.

[0101] c. Observation: Before drug administration every day during the experiment, the wounds of each mouse were observed and recorded. And the wound healing of the mice was photographed and recorded every day.

[0102] Results: Staphylococcus aureus is a common human pathogen, and bacterial infections are the most common clinically. In particular, methicillin-resistant Staphylococcus aureus infections can cause suppurative skin infections and seriously endanger life. Therefore, an important indicator for evaluating the potential application prospect of CuSO4 is the great ability to remove MRSA cells from the wound surface and the ability to promote wound healing. As Figure 6 shown, in the mouse epidermal infection model, it was observed that the wound healing ability of the hydrogel treatment group in Example 9 on the 5th day was better than that of other groups. There was still a small amount of pus in the wound surface of the non-infected treatment group. Among the other 5 MRSA-infected groups, compared with the sodium alginate hydrogel treatment group and the ointment group, the copper-containing hydrogel in Example 9 was beneficial to wound healing. There was no pus and blood in its wound surface, and the wound area was smaller than that on the 0th day. Therefore, the copper-containing hydrogel has broad application prospects in the treatment of wound infections.

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of this invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0104] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any ordinary technician in the industry can smoothly implement the present invention according to the illustrations in the specification and the above description. However, any minor changes, modifications, and equivalent variations made by those skilled in the art within the scope of the technical solution of the present invention by using the technical content disclosed above are equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of the hydrogel prepared based on copper gluconate in the preparation of products for treating bacterial infections, including The hydrogel prepared based on copper gluconate is prepared from copper gluconate, sodium alginate and an auxiliary solvent; the auxiliary solvent is water; The specific process for preparing the hydrogel based on copper gluconate is as follows: S1: Dissolve copper gluconate in the auxiliary solvent to form a mixed solution A; S2: Add sodium alginate to the mixed solution A and stir evenly to obtain a semi-finished product of copper gluconate hydrogel; S3: Sterile fill the semi-finished product of copper gluconate hydrogel to obtain copper gluconate hydrogel; The bacterial infection is caused by Staphylococcus aureus; The addition amount of the sodium alginate is 2% of the mass of the mixed solution A; The bactericidal rate of copper gluconate against Staphylococcus aureus reaches more than 99.9%; When the concentration of copper gluconate is 256 μM and it is used to kill Staphylococcus aureus, its survival rate is less than 0.001%.

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