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 consists of the following components: 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. Moreover, 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 application of the invention patent application has an application date of August 14, 2023, an application number of 202311020808.1, and an invention title of "A Copper-containing Hydrogel and Its Application in Inhibiting Bacterial Infection". Technical Field

[0002] The present invention relates to the field of pharmaceutical 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 penicillin came out 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 being applied clinically 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 MRSA colony, 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 bacteria in the colony are sensitive to methicillin. A large number of sensitive bacteria are killed within a few hours after using antibiotics, but a small number of resistant strains grow slowly and then multiply rapidly after several hours. Since its discovery, MRSA infections have almost spread 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, its 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 an 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 infection, including the application of the copper-containing hydrogel in the preparation of a product for treating bacterial infection 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 kills Staphylococcus aureus, its survival rate is less than 0.001%.

[0020] Preferably, when the copper-containing hydrogel kills 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 using a copper ion compound, sodium alginate, and auxiliary solvent A as raw materials. The antibacterial activity test of copper ions against Staphylococcus aureus shows a strong bactericidal effect, having a good killing effect on it, and being able to keep the skin moist, non-irritating, and not easily generating drug resistance. It reveals 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, has uniform administration, and 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-containing 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; (C) is a schematic diagram of the bactericidal effect of copper citrate; (D) is a schematic diagram of the bactericidal effect of cuprous iodide; (E) is a schematic diagram of the bactericidal effect of sodium 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 a 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 following further describes the present invention in detail with specific embodiments, which is an explanation rather than a limitation of the present invention.

[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 accompanying 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 efforts 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 selected as water, to make 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 selected as water, to make 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 selected as water, to form 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: Sterile fill the semi-finished copper-containing hydrogel to obtain a finished copper-containing hydrogel.

[0049] The application of the described 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 selected as water, to form 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: Sterile fill the semi-finished copper-containing hydrogel to obtain a finished copper-containing hydrogel.

[0055] The application of the described 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 selected as dimethyl sulfoxide, to form 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: Sterile fill the semi-finished copper-containing hydrogel to obtain a finished copper-containing hydrogel.

[0061] The application of the described 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 measured by gradient drop plate experiment to explore the antibacterial 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, almost 100% of the growth of Staphylococcus aureus could be inhibited.

[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 measured 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, 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 antibacterial 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 loaded with PI probes and a control group 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 results of fluorescence microscopy 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 antibacterial 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 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 shows that Example 1 (A) and Example 2 (B) have good antibacterial effects, and the antibacterial rates are 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 effect:

[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 for standby.

[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 anesthesia of the mouse is successful;

[0089] b. Dorsal 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 deep into the subcutaneous tissue and keep it open, causing 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 dorsal wound of the mouse, and bandage the wound with a sterile gauze;

[0091] d. Infection: Observe the abscess condition on the surface of the mouse wound 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 treat it with 1 mL of the hydrogel loaded in Example 1;

[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. Each time, the wound was first cleaned with physiological saline containing the drug, and then the hydrogel was applied. The interval between the two times was 10 minutes, 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. Especially methicillin-resistant Staphylococcus aureus infection can cause suppurative skin infections and seriously endanger life. Therefore, an important index for evaluating the potential application prospect of CuSO4 is the huge ability of MRSA cells to be cleared 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 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 or blood on its wound surface, and the wound area was smaller than the wound area 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 only for the purpose of describing specific embodiments 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 as shown in the accompanying drawings of the specification and as described above; 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 substantial technology of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of a copper sulfate hydrogel in the preparation of a product for treating bacterial infections caused by Staphylococcus aureus, characterized in that, including, the copper sulfate hydrogel is prepared from copper sulfate, sodium alginate and an auxiliary solvent; the auxiliary solvent is water; the concentration of copper sulfate in the copper sulfate hydrogel is 16 μM; when the MBC value of copper sulfate against the standard strain of Staphylococcus aureus is 16 μM, its bactericidal rate reaches 99.99%; when the copper sulfate hydrogel sterilizes Staphylococcus aureus, its survival rate is less than 0.001%; when the copper sulfate concentration reaches 1 / 4 MBC, the morphology of Staphylococcus aureus becomes smaller and wrinkles appear on the surface; when the concentration reaches MBC, membrane perforation occurs in Staphylococcus aureus, indicating that the introduction of copper sulfate destroys the morphology of Staphylococcus aureus, and the degree of destruction intensifies with the increase of copper sulfate concentration; the copper sulfate can induce the death of the standard strain of Staphylococcus aureus to varying degrees, and the degree of death shows concentration dependence.

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