Use of ferrous ions in the manufacture of a product for the treatment of a bacterial infection

Abstract

This invention discloses the application of ferrous ions in the preparation of products for treating bacterial infections, belonging to the field of pharmaceutical technology. Antibacterial activity tests of ferrous ions against Staphylococcus aureus showed strong bactericidal effects, with a survival rate of less than 0.001% against Staphylococcus aureus and less than 0.01% against methicillin-resistant Staphylococcus aureus (MRSA). The use of ferrous ion-containing hydrogels in the treatment of keratitis and skin wound infections significantly reduced the risk of MRSA infection in the lungs and effectively prevented the proliferation and spread of microorganisms in the body, revealing that drugs containing ferrous compounds can be used to treat Staphylococcus aureus infections, including MRSA.

Description

[0001] This invention patent application is a divisional application of the invention patent application filed on April 25, 2022, with application number CN202210438990.1 and invention title "Application of ferrous ions in the preparation of products for treating bacterial infections". Technical Field

[0002] This invention relates to the field of pharmaceutical technology, specifically to the application of ferrous ions in the preparation of products for treating bacterial infections. Background Technology

[0003] Keratitis is a common eye disease and one of the leading causes of blindness in my country. Infections by bacteria, fungi, and viruses are among the most direct causes of keratitis. When the corneal epithelium is damaged, it is highly susceptible to infectious inflammation. In particular, improper use of contact lenses, contact with contaminated medications or water, etc., greatly increase the likelihood of developing infectious keratitis. It is noteworthy that, according to current authoritative clinical surveys, bacterial infectious keratitis caused by Staphylococcus aureus and Pseudomonas aeruginosa still has a high incidence rate, and the incidence is increasing year by year. Therefore, the prevention and treatment of this type of keratitis deserves attention.

[0004] Staphylococcus aureus is a major culprit in common corneal and epidermal infections. Currently, antibiotic treatment remains the standard strategy for treating Staphylococcus aureus infections. However, the improper use and overuse of antibiotics have led to increased antibiotic resistance in the human body, and the emergence of multidrug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA) poses a serious threat to human health. Therefore, there is an urgent need to find new antibacterial therapies to address this crisis.

[0005] Currently, the commonly used treatment strategies for Staphylococcus aureus-induced keratitis and epidermal tissue infections mainly consist of topical antibiotic eye drops, medical antibiotic hydrogels, and some essential topical steroid ointments, such as ofloxacin eye drops, vancomycin hydrogel, erythromycin hydrogel, and chlortetracycline ointment. Existing research indicates that while antibiotics possess highly effective bactericidal efficacy at low doses, they cannot completely eradicate bacteria. However, excessive use of these antibiotic-containing eye drops and ointments increases the risk of bacterial resistance and leads to drug dependence, forcing increased dosages and creating a vicious cycle. Repeated bacterial infections can lead to resistance to these antibiotic ointments, rendering them ineffective. Furthermore, this type of treatment cannot effectively prevent bacterial migration and colonization within the body, easily causing lesions in other tissues and organs. Therefore, there is an urgent need to find novel antibacterial materials to treat Staphylococcus aureus infections, especially methicillin-resistant Staphylococcus aureus (MRSA) infections. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of ferrous ions in the preparation of products for treating bacterial infections.

[0007] Application of ferrous ions in the preparation of products for treating bacterial infections.

[0008] Preferably, the bacterial infection is an ocular infection or an epidermal infection.

[0009] Preferably, the bacterial infection is caused by Staphylococcus aureus.

[0010] Preferably, the ferrous ions are derived from any one of ferrous sulfate, ferrous chloride, ferrous gluconate, and ferrous lactate.

[0011] Preferably, the hydrogel prepared from ferrous compounds, ascorbic acid, and compound A is used to treat the bacterial infection; wherein, compound A is selected from hyaluronic acid or sodium alginate.

[0012] Preferably, the concentration of the ferrous ion is ≥1 μM.

[0013] Preferably, the concentration of the ferrous ions is ≥16 μM.

[0014] More preferably, the concentration of the ferrous ion is 16-100 μM.

[0015] Preferably, the method for preparing the hydrogel includes the following steps:

[0016] S1. Dissolve the ferrous compound and ascorbic acid in water to prepare a mixed solution, and heat it to 45-55 °C. The concentration ratio of ascorbic acid to ferrous compound is 1:1.

[0017] S2. Add compound A during heating and stir until a stable colloid is formed to obtain the hydrogel. The amount of compound A added is 2% of the mass of the mixed solution.

[0018] Preferably, the concentration of the ferrous compound is 1 mM.

[0019] A hydrogel prepared by the above method.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1. The high bactericidal effect of the hydrogel provided by this invention against Staphylococcus aureus and MRSA indicates that ferrous compounds can serve as a potential drug to combat the current antibiotic crisis.

[0022] 2. This invention selects ferrous sulfate, a commonly used iron supplement, as a typical representative of ferrous compounds to study drug alternatives using ferrous ions as the core. A ferrous ion hydrogel was successfully prepared using hydrogel as a drug carrier. This invention utilizes ferrous sulfate as the core drug to replace antibiotics, and the prepared ferrous ion antibacterial hydrogel can effectively treat epidermal and ocular infections, significantly reduce the risk of MRSA infection in the lungs, and effectively and promptly prevent the reproduction and spread of microorganisms in the body.

[0023] 3. The hydrogel preparation method of the present invention is simple, but has strong material adaptability. It can be combined with various materials such as carrageenan, xanthan gum, low acyl colloids, hyaluronic acid, and carbomer to exert a strong bactericidal potential. Attached Figure Description

[0024] Figure 1 TEM image (A) of the hydrogel of Example 1, elemental distribution map (B) of the hydrogel of Example 1, SEM image (C) of the hyaluronic acid hydrogel without ferrous sulfate, SEM image (D) of the hydrogel of Example 1, rheological property map (E) of the hyaluronic acid hydrogel without ferrous sulfate, and rheological property map (F) of the hydrogel of Example 1.

[0025] Figure 2 SEM images (AB) of sodium alginate hydrogel without ferrous sulfate, SEM images (CD) of hydrogel in Example 2; TEM images (EF) of hydrogel in Example 2, elemental distribution map (G) of hydrogel in Example 2, rheological properties map (H) of sodium alginate hydrogel without ferrous sulfate, and rheological properties map (I) of hydrogel in Example 2.

[0026] Figure 3 The diagram shows the ferrous ion release rate of the hydrogel in Example 1 (A) and the ferrous ion release rate of the hydrogel in Example 2 (B).

[0027] Figure 4 The distribution diagram of the bactericidal activity of the hydrogel in Example 1 on PI and Syto 9 stained MRSA cells is shown in (A). The bactericidal activity effect diagram of the hydrogel in Example 1 is shown in (B). The distribution diagram of the bactericidal activity of the hydrogel in Example 2 on PI and Syto 9 stained MRSA cells is shown in (C). The bactericidal activity effect diagram of the hydrogel in Example 2 is shown in (D).

[0028] Figure 5 Schematic diagram of mouse survival rate in different treatment groups (A), schematic diagram of changes in inflammatory factor IL-1 in different treatment groups (B), schematic diagram of changes in inflammatory factor IL-6 in different treatment groups (C).

[0029] Figure 6The results of histological sections of mouse eyeballs and gross images of each group after treatment (A) and the results of histological sections of mouse lungs and gross images of each group after treatment (B);

[0030] Figure 7 A schematic diagram (A) showing the number of bacteria in the lungs of infected mice 3 days after hydrogel treatment, and a gross image of the wound healing in mice (B).

[0031] Figure 8 The diagram shows the antibacterial effect of 9 iron ions on Staphylococcus aureus (A), the antibacterial effect of ferrous sulfate on 6 pathogenic bacteria (B), and the antibacterial effect of ferrous sulfate on Staphylococcus aureus (C). Detailed Implementation

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0033] Example 1

[0034] A method for preparing a hydrogel includes the following steps:

[0035] S1. Dissolve ferrous sulfate and ascorbic acid in sterile ultrapure water to prepare a mixed solution, and heat it to 50 °C. The concentration of ferrous sulfate in the mixed solution is 1 mM, and the concentration ratio of ascorbic acid to ferrous sulfate is 1:1.

[0036] S2. Hyaluronic acid powder is added during the heating process and stirred until the hyaluronic acid powder dissolves and forms a stable colloid to obtain the hydrogel. The amount of hyaluronic acid added is 2% of the mass of the mixed solution.

[0037] The hydrogel prepared by the above method.

[0038] The hydrogel is used to treat bacterial keratitis.

[0039] Example 2

[0040] A method for preparing a hydrogel includes the following steps:

[0041] S1. Dissolve ferrous sulfate and ascorbic acid in sterile ultrapure water to prepare a mixed solution, and heat it to 50 °C. The concentration of ferrous sulfate in the mixed solution is 1 mM, and the concentration ratio of ascorbic acid to ferrous sulfate is 1:1.

[0042] S2. During the heating process, sodium alginate powder is added and stirred until the sodium alginate powder dissolves to form a stable colloid, thus obtaining the hydrogel. The amount of sodium alginate added is 2% of the mass of the mixed solution.

[0043] The hydrogel prepared by the above method.

[0044] The hydrogel is used to treat skin wound infections.

[0045] Example 3

[0046] The difference between Example 3 and Example 1 is that the ferrous compound is ferrous chloride.

[0047] Example 4

[0048] The difference between Example 4 and Example 1 is that the ferrous compound is ferrous gluconate.

[0049] Example 5

[0050] The difference between Example 5 and Example 1 is that the ferrous compound is 1 part ferrous lactate.

[0051] To verify the antibacterial activity of ferrous ions against Staphylococcus aureus, the following experiment was performed.

[0052] 1. Prepare aqueous solutions of five ferrous salts (ferrous sulfate, ferrous chloride, ferrous gluconate, ferrous lactate, and potassium ferrocyanide) and four ferric salts (ferric chloride, ferric citrate, ferric dextran, and potassium ferricyanide) to a final concentration of 100 μM. Use a 96-well plate method for 10... 8 The susceptibility of Staphylococcus aureus to nine iron salts was tested using CFU / mL iron. After 12 h of drug treatment, the total bacterial count was determined by drop plate assay to further investigate the antibacterial effect of iron ions on Staphylococcus aureus.

[0053] Result: Passed Figure 8 Results A showed that at the same concentration (100 μM), five different Fe... 2+ Salt has a much higher antibacterial effect against Staphylococcus aureus than iron. 3+ The presence of salt indicates that ferrous ions play a crucial role in the killing of Staphylococcus aureus. In particular, ferrous sulfate (FeSO4) can inhibit the growth of almost 100% of Staphylococcus aureus. This demonstrates the role of Fe... 2+ It can effectively inhibit the growth of Staphylococcus aureus.

[0054] 2. Prepare an aqueous solution of ferrous sulfate to a final concentration of 16 μM; and prepare 10 μL of each solution. 8 CFU / mL of Escherichia coli ( Escherichia coli Listeria monocytogenes ( Listeria monocytogenesVibrio parahaemolyticus ( Vibrio Parahemolyticus ), Enterobacter sakazakii ( Enterobacter sakazakii Staphylococcus aureus ( Staphylococcus aureus Six pathogenic bacteria were tested for ferrous sulfate susceptibility using a 96-well plate assay. After 12 h of drug treatment, the total bacterial count was determined by a drop plate test to further investigate the antibacterial effect of ferrous ions.

[0055] Results: Ferrous sulfate, with the best bactericidal effect, was further selected as the iron agent used in the study, and its drug susceptibility was tested against six different pathogenic bacteria. Figure 8 Results B showed that 16 μM ferrous sulfate exhibited strong bactericidal effects against all six pathogenic bacteria, indicating that ferrous ions possess broad-spectrum bactericidal efficacy and can effectively kill common pathogens that induce infection. Notably, ferrous sulfate showed significantly better inhibitory effects against Staphylococcus aureus than against other pathogens (Escherichia coli, Listeria monocytogenes, Vibrio parahaemolyticus, and Cronobacter sakazakii), with a survival rate of less than 0.001%. It is also noteworthy that ferrous sulfate demonstrated good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with a survival rate of less than 0.01% after treatment with 16 μM ferrous sulfate.

[0056] 3. Prepare aqueous solutions of ferrous sulfate to final concentrations of 1, 4, 16, and 64 μM. Prepare 100 μg / mL ampicillin and vancomycin as positive controls, and set up ultrapure water without ferrous sulfate as a blank control group. Prepare 10 8 CFU / mL Staphylococcus aureus ( Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) was detected. The susceptibility of ferrous sulfate to Staphylococcus aureus was tested using a 96-well plate assay at different concentrations. After 12 h of drug treatment, the total bacterial count was determined by a drop plate test to further investigate the minimum bactericidal concentration (MBC) of ferrous sulfate against MRSA.

[0057] Result: As Figure 8 Results C showed that Staphylococcus aureus treated with ferrous sulfate could not regrow after plating and reculturing, indicating that the bacteria were likely dead, and the degree of death was closely related to the concentration of ferrous sulfate. Ferrous sulfate has an MBC value of 16 μM for Staphylococcus aureus; at this concentration, the bactericidal rate can reach 99.99%, demonstrating excellent prospects for bactericidal applications.

[0058] To verify the performance of the hydrogel of the present invention, the following analysis was conducted:

[0059] Ampicillin and Vancomycin used in the experiment were purchased from Sigma-Aldrich.

[0060] I. Distribution Analysis of Ferrous Ions in Hydrogels

[0061] The hydrogels of Example 1 and Example 2 were placed in disposable petri dishes for 6 h and then frozen (-20 °C). Subsequently, they were placed in a freeze dryer for 48 h and then ground into powder with liquid nitrogen. Powder samples were then taken for testing.

[0062] (1) The effective elemental components in the hydrogel were analyzed by X-ray photoelectron spectroscopy (XPS);

[0063] (2) The phenotypic structure of the hydrogel was analyzed by field emission scanning electron microscopy (SEM);

[0064] (3) The rheological properties of the hydrogel were analyzed using a rotational rheometer: A 40 mm plate with a 1000 μm gap was used. Before testing, a thin film of silicone oil was applied around the hydrophobic valve to eliminate the effect of moisture evaporation. The equilibrium time was set to 7 minutes to allow the hydrogel to reach a stable state. The viscosity was tested at shear rates of 0.1–100 1 / s. Yield performance was tested in the range of 1–2000 Pa with an oscillation frequency of 1 Hz. The oscillatory strain behavior was within the strain range of 0.01% to 100% in the 1 Hz range. Creep and recovery tests were conducted within the linear viscoelastic range of 180 degrees and under a constant stress of 10 Pa.

[0065] Result: As Figure 1 and Figure 2 As shown, ferrous ions were successfully loaded into both hydrogel matrices and were uniformly distributed within the hydrogels. SEM images indicate that the ferrous ion-loaded hydrogels possess a heterogeneous surface and porous structure, which are beneficial for water retention and Fe2+ absorption. 2+ Diffusion and release from the hydrogel; the rheological properties comparison results of the two hydrogels show that the G' (elastic modulus) and G″ (loss modulus) of the ferrous ion-loaded hydrogel can recover to 100% within a few seconds at high shear rates, indicating that the FeSO4-loaded hydrogel has the potential flowability and resilience to facilitate drug diffusion.

[0066] The above results indicate that ferrous ion-loaded hydrogels have potential medical applications.

[0067] II. Analysis of Ferrous Ion Release Rate in Hydrogels

[0068] (1) 10 ml of water was injected into the hydrogel of Example 1 and the hydrogel of Example 2 respectively and then placed into dialysis bags (8000-14000 Da).

[0069] (2) Place 100 ml of ultrapure water into the dialysis bag and stir it for 12 h at 37 °C and 200 rpm using a magnetic stirrer.

[0070] (3) The concentration of ferrous ions was determined by colorimetric absorption spectroscopy of o-phenanthroline molecules. The measurement was performed every 30 min for a total of 12 h.

[0071] Results: The ferrous ion release experiment confirmed the results as follows. Figure 3 As shown, Fe in the hydrogel 2+ Both can be released from the hydrogel in a time-dependent manner, and the FeSO4 hydrogel has the characteristic of slow and long-term release of iron ions. Specifically, after 12 hours, the Fe in the hydrogel... 2+ The release rates were approximately 78.3% and 73.6%, respectively.

[0072] III. Analysis of the in vitro antibacterial activity of hydrogels

[0073] (1) The hydrogels of Example 1, Example 2, ferrous sulfate hyaluronic acid hydrogel, ascorbic acid hyaluronic acid hydrogel, Ampicillin hyaluronic acid hydrogel, Vancomycin hyaluronic acid hydrogel, ferrous sulfate sodium alginate hydrogel, ascorbic acid sodium alginate hydrogel, Ampicillin sodium alginate hydrogel and Vancomycin sodium alginate hydrogel were poured into sterile disposable petri dishes for later use;

[0074] (2) 10 8 Staphylococcus aureus cells at CFU / mL were washed twice with physiological saline and collected in 1 cm sterile filter paper.

[0075] (3) Place the filter paper with the cells collected facing up on each hydrogel, seal it, and incubate it in an incubator at 37°C.

[0076] (4) After culturing for 12 h, carefully remove the filter paper with sterile forceps, wipe off the excess hydrogel on the back of the filter paper with a sterile cotton swab, place it in a sterile EP tube, add 1 mL of sterile physiological saline to the tube, sonicate for 30 s, and collect the cells on the filter paper.

[0077] (6) The total bacterial count was determined by the drop plate method.

[0078] To determine the antibacterial mechanism of the FeSO4-loaded hydrogel, 100 μL of the hydrogel from Example 1 and 100 μL of the hydrogel from Example 2 were added to the center of a glass slide, and 5 μL of cleaned Staphylococcus aureus cells (10 μL) were added to the slide. 8 A PI (CFU / mL) solution was vertically added to the surface of a FeSO4 hydrogel and incubated at 37 °C for 3 h. Then, a PI solution (final concentration 1 μg / mL) was added to the FeSO4 hydrogel containing Staphylococcus aureus cells and incubated at 37 °C for 20 min. The antibacterial model of the FeSO4 hydrogel was determined using laser confocal scanning microscopy.

[0079] Results: Through simulated in vitro surface contact experiments between MRSA and ferrous ion hydrogel, it was found that both the hydrogels of Example 1 and Example 2 had good bactericidal effects. Figure 4 A shows the distribution of dead MRSA cells stained with PI and Syto 9 after hydrogel treatment in Example 1, as observed under a laser confocal microscope. The results indicate that the hydrogel containing ASC and FeSO4 exhibits significant bactericidal activity, with a bactericidal rate approaching 100%. Figure 4 As shown in B, treatment with a hydrogel containing only FeSO4 reduced CFU by only 3 lg, and the hydrogel containing only ASC had no bactericidal effect on MRSA. This indicates that the addition of ASC can make the hydrogel loaded with ferrous ions have the greatest bactericidal activity. Importantly, the hydrogel containing Ampicillin has a much lower ability to kill MRSA compared with the hydrogel in Example 1.

[0080] Figure 4 C shows the distribution of dead MRSA cells stained with PI and Syto 9 after hydrogel treatment in Example 2, as observed under a laser confocal microscope. The results indicate that the hydrogel also possesses significant bactericidal activity, and... Figure 4 As shown in D, the hydrogel containing vancomycin had a lower ability to kill MRSA than the hydrogel of Example 2. These results indicate that hydrogels containing both ferrous sulfate and ascorbic acid may be used for the treatment of MRSA infection.

[0081] IV. The following zoological experiments were conducted to further illustrate its effectiveness:

[0082] 1. Verification of the effect of Example 1

[0083] (1) Preparation of Staphylococcus aureus suspension

[0084] Overnight transfer of MRSA (from the Chinese Academy of Sciences) at a ratio of 1:200, grown to OD. 600 =0.3, collect 1×10 8 ~1×10 10CFU / mL, remove culture medium and resuspend in physiological saline for later use.

[0085] (2) Constructing a mouse model of Staphylococcus aureus keratitis

[0086] Mouse preparation: 40 male BALB / c mice, approximately 8 weeks old.

[0087] Model building:

[0088] a. Anesthesia: Each mouse was anesthetized by intramuscular injection of a 1:1 mixture of 0.125 mL ketamine hydrochloride (100 mg / mL): oxorazine (20 mg / mL). After about 3 to 5 minutes, mild signs of anesthesia appeared in the eye (loss of eyelid reflex and weak corneal reflex). One drop of 1% tetracaine hydrochloride was then instilled into the cornea.

[0089] b. Incision: Using a 25-30 gauge needle, make three parallel incisions, each 2 mm long, in the center of the corneal epithelium, without penetrating the surface stroma, to cause corneal damage in mice;

[0090] c. Inoculation: Place two drops of the prepared Staphylococcus aureus suspension (10 μL) into the mouse's eyeball. 8 (CFU / mL, 50 μL per drop, 100 μL total) Gently press the eyelid with your hand for 10 seconds to ensure full contact between the bacterial solution and the cornea, then gently restore the eyelid.

[0091] d. Identification: 24 / 48 h after inoculation (depending on the mouse's ocular infection status), observe the external eye with a flashlight. If there is vascular congestion, corneal opacity, and purulent accumulation in the eye, it can be considered that the model has been successfully established.

[0092] e. Grading: Divided into two grades: mild and severe. Mild: slight opaque discharge, partially or completely covering the pupil and anterior segment; Severe: dense opaque discharge, partially or completely covering the pupil or anterior segment, even corneal perforation.

[0093] (3) Animal grouping and administration

[0094] a. Grouping: After successfully establishing the mouse model of Staphylococcus aureus keratitis, the mice were divided into four groups of eight mice each, receiving drug treatment in each group.

[0095] Control group (8 mice): The eyes of mice were scratched, but no bacteria were inoculated. Sterile saline solution was dripped into their eyes, totaling 100 μL.

[0096] Model control group (8 mice): The constructed mouse model was treated with 100 μL of sterile saline in the eyes, followed by hyaluronic acid hydrogel treatment, for a total of 1 mL.

[0097] Treatment group 1 (8 mice): The constructed mouse model was treated with 100 μL of 1 mM ASC aqueous solution and 1 mM FeSO4 aqueous solution in its eyes, and then loaded with 1 mL of the hydrogel from Example 1.

[0098] Treatment group 2 (8 mice): The constructed mouse model was treated with 100 μL of 1 mM ASC aqueous solution in its eyes, and then treated with hyaluronic acid hydrogel loaded with ASC (1 mM), for a total of 1 mL;

[0099] Positive control group (8 mice): The constructed mouse model was treated with a total of 100 μL of 1 mg / mL ampicillin in its eyes, and then treated with hyaluronic acid hydrogel loaded with ampicillin (1 mg / mL), for a total of 1 mL.

[0100] b. Drug administration: Each group of mice was administered the drug twice a day according to the predefined group. Each time, the mice were first washed with saline containing the drug, followed by hydrogel drug application. The interval between the two applications was 10 minutes. The treatment lasted for 7 days.

[0101] c. Observation: Before administering medication each day during the experiment, each eye should be observed and recorded using a handheld flashlight. The condition of the mice's eyeballs should also be photographed and recorded daily.

[0102] d. Ocular colony counting: Gently swab the upper and lower eyelids with a sterile saline-moistened cotton ball, then immerse the cotton ball in 5 mL of sterile saline solution, mix well, extract 100 μL, perform serial dilution, drop the solution onto a plate, incubate, and count the colonies.

[0103] e. Blood marker testing: Detecting and identifying the types and quantities of bacteria in the blood, and testing related blood markers.

[0104] f. Lung colony count: Mice were sacrificed after 7 days of treatment, and their lungs were dissected and placed in 5 mL PBS. The lungs were sonicated and mixed. 100 μL of the supernatant was taken, serially diluted, dropped into a plate, cultured, and counted.

[0105] g. Ocular histopathological examination: Mice treated for 7 days were sacrificed, their eyeballs were removed, fixed in 10% formalin, embedded in paraffin, stained with hematoxylin and eosin (HE), and examined under a light microscope to observe histopathological changes.

[0106] Results: 48 hours post-infection, mice with MRSA-infected eyes were treated with the hydrogel of Example 1 for 7 days. The bactericidal activity of the self-assembled ferrous hydrogel was evaluated by analyzing its survival rate, cellular inflammatory factors, and pathological sections of the eyes and lungs. The results are as follows: Figure 5As shown in Figure A, compared with the 35% survival rate of mice in the control group hydrogel group, the survival rate of mice in the FeSO4+ASC hydrogel group was significantly improved, reaching 100%. Meanwhile, the serum cytokine IL-1 and IL-6 levels in the uninfected control group were 25 pg / mL and 35 pg / mL, respectively. The serum cytokine IL-1 and IL-6 levels in the blank hydrogel group were 30 pg / mL and 40 pg / mL, respectively. The IL-1 and IL-6 levels in the ASC hydrogel treatment group were slightly lower than those in the blank hydrogel treatment group. The serum IL-1 and IL-6 levels in the ampicillin hydrogel group and the FeSO4+ASC hydrogel group were significantly lower than those in the blank hydrogel group, with the most significant decrease in IL-6 and IL-1 observed in the FeSO4+ASC hydrogel group. This suggests that FeSO4+ASC hydrogel can reduce the inflammatory response during Staphylococcus aureus infection, and its effect is greater than that of ampicillin hydrogel. Figure 5 B, C). HE-stained microscopic images and histological analysis of mouse eyeballs and lungs after 7 days of treatment with different hydrogels showed that, regarding the mouse eyeballs, as... Figure 6 As shown in Figure A, mice in the blank hydrogel group had purulent and hemorrhagic discharge from their eyes, significant corneal edema, and a large number of neutrophils. In the ampicillin hydrogel group, the number of inflammatory cells in the cornea was significantly reduced, and the corneal thickness returned to normal; however, some corneal repair defects remained. In contrast, the self-assembled ferrous hydrogel group showed that the corneal tissue of mice had essentially returned to normal: corneal edema disappeared; there was no inflammatory response, and neovascularization was reduced. These results indicate that the hydrogel in Example 1 has a good therapeutic effect on Staphylococcus aureus infection of the cornea in mice. Figure 6 As shown in Figure B, compared with other treatment groups, mice treated with FeSO4+ASC hydrogel showed significant relief of lung damage. Their bronchial size was normal, alveolar structure was intact, and no inflammatory cells such as neutrophils and phagocytes were observed in the lungs, consistent with the uninfected group. These results indicate that the hydrogel in Example 1, in addition to its potential advantages in treating keratitis, can also effectively prevent the spread of corneal infection to the lungs.

[0107] 2. Verification of the effect of Example 2

[0108] (1) Preparation of Staphylococcus aureus suspension

[0109] Overnight transfer of MRSA (from the Chinese Academy of Sciences) at a ratio of 1:200, grown to OD. 600 =0.3, collect 1×10 8 ~1×10 10 CFU / mL, remove culture medium and resuspend in physiological saline for later use.

[0110] (2) Constructing a mouse model of Staphylococcus aureus infection of epidermal wounds

[0111] Mouse preparation: 30 male BALB / c mice, approximately 8 weeks old.

[0112] Model building:

[0113] a. Anesthesia: Each mouse was anesthetized by intramuscular injection of a 1:1 mixture of ketamine hydrochloride (100 mg / mL) and oxoperazine (20 mg / mL). The mice were observed for about 3 to 5 minutes to determine whether the anesthesia was successful by observing their respiratory status and heart rate (slowing respiratory rate and decreasing heart rate).

[0114] b. Back wound incision: Hair removal cream was applied to the backs of mice, the backs were scraped with saline gauze, and the backs were wiped with 75% ethanol. A full-thickness wound (10 mm × 10 mm) was created on the back skin of each mouse, extending deep into the subcutaneous tissue, and left open, resulting in the loss of skin damage on the mouse's back;

[0115] c. Inoculation: Apply two drops of the prepared Staphylococcus aureus suspension (10 μL) to the wound surface on the mouse's back. 8 (CFU / mL, 50 μL per drop, 100 μL total), and bandage the wound with sterile gauze;

[0116] d. Infection: Observe the abscess formation on the surface of the mouse wound 24 / 48 h after inoculation;

[0117] (3) Animal grouping and administration

[0118] a. Grouping: After successfully establishing the mouse model of Staphylococcus aureus infection with epidermal wounds, the mice were divided into 5 groups of 6 mice each, receiving drug treatment in each group.

[0119] Control group (6 mice): Mice had wounds on their backs, but were not inoculated with bacteria. Sterile saline solution was dripped into the wounds, totaling 100 μL.

[0120] Model control group (6 mice): The established mouse model was not treated with drugs. 100 μL of sterile physiological saline was dripped into the wound and then treated with sodium alginate hydrogel, for a total of 1 mL.

[0121] Treatment group 1 (6 mice): The constructed mouse model was treated with 100 μL of 1 mM ASC aqueous solution and 1 mM FeSO4 aqueous solution in the wound, and then treated with 1 mL of hydrogel from Example 2.

[0122] Treatment group 2 (6 mice): The established mouse model was treated with 100 μL of 1 mM ASC aqueous solution for wound treatment, followed by treatment with sodium alginate hydrogel loaded with ASC (1 mM), for a total of 1 mL;

[0123] Positive control group (6 mice): The established mouse model was treated with 100 μL of 1 mM vancomycin aqueous solution for wound treatment, followed by treatment with sodium alginate hydrogel loaded with vancomycin, for a total of 1 mL;

[0124] b. Drug administration: Each group of mice was administered the drug twice a day according to the predefined group. Each time, the mice were first washed with saline containing the drug, followed by hydrogel application of the drug. There was a 10-minute interval between the two treatments. The wounds were then bandaged with sterile gauze. The treatment lasted for 3 days.

[0125] c. Observation: Before administering the medication each day during the experiment, the wounds of each mouse should be observed and recorded using a handheld flashlight. The wound healing status of the mice should also be documented daily by taking photos.

[0126] d. Wound surface colony count: Gently apply a sterile saline-moistened cotton ball to the wound surface and place it into a 5 mL sterile saline solution. Mix well, take 100 μL, perform serial dilution, drop the solution onto a plate, incubate, and count the colonies.

[0127] e. Blood marker testing: Detecting and identifying the types and quantities of bacteria in the blood, and testing related blood markers.

[0128] f. Lung colony count: Mice were sacrificed after 7 days of treatment, and their lungs were dissected and placed in 5 mL PBS. The lungs were sonicated and mixed. 100 μL of the supernatant was taken, serially diluted, dropped into a plate, cultured, and counted.

[0129] Results: Staphylococcus aureus is a common human pathogen, with bacterial infections being the most frequent clinical manifestation. In particular, methicillin-resistant Staphylococcus aureus (MRSA) infections can cause purulent skin infections, seriously endangering life. Therefore, an important indicator for evaluating the potential application prospects of FeSO4 is the significant clearance capacity of MRSA cells from wounds and their ability to promote wound healing. Figure 7 As shown in Figure B, in the mouse epidermal infection model, the wound healing ability of the Example 2 hydrogel treatment group was superior to other groups on day 3, while the uninfected treatment group still had a small amount of pus in the wound. In the other five MRSA infection groups, the sodium alginate hydrogel treatment group containing only ASC and the vancomycin hydrogel treatment group failed to effectively alleviate wound healing, with severe purulent bleeding. Compared with the vancomycin hydrogel group, the Example 2 hydrogel was beneficial to wound healing; no purulent bleeding was observed in the wound, and the wound area was smaller than that on day 0. Dissection of the lungs of mice in each treatment group for Staphylococcus aureus detection revealed... Figure 7As shown in Figure A, FeSO4+ ASC hydrogel can eliminate at least 99.9% of MRSA cells, while the bactericidal effect of the other treatment groups is significantly weaker than that of ferrous hydrogel. FeSO4 hydrogel epidermal treatment has a potential preventive effect against MRSA cell infection to the lungs.

[0130] Therefore, it was confirmed that the FeSO4-containing hydrogel has potential antibacterial activity and can combat drug resistance of microorganisms in vivo.

[0131] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0132] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0133] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of a hydrogel for treating bacterial infections in the preparation of a medicament for treating bacterial infections caused by Staphylococcus aureus, characterized in that, The hydrogel was prepared using ferrous sulfate, ascorbic acid, and compound A as raw materials, wherein compound A is hyaluronic acid. The preparation method of the hydrogel includes the following steps: S1. Dissolve ferrous sulfate and ascorbic acid in sterile ultrapure water to prepare a mixed solution, and heat it to 50°C. The concentration of ferrous sulfate in the mixed solution is 1 mM, and the concentration ratio of ascorbic acid to ferrous sulfate is 1:

1. S2. Hyaluronic acid powder is added during the heating process and stirred until the hyaluronic acid powder dissolves and forms a stable colloid to obtain the hydrogel. The amount of hyaluronic acid added is 2% of the mass of the mixed solution. The bacterial infection is either an ocular infection or an epidermal infection.

2. The application according to claim 1, characterized in that, The bacterial infection was Staphylococcus aureus keratitis.

3. The application according to claim 1, characterized in that, The hydrogel has a bactericidal effect against Staphylococcus aureus.

4. The application according to claim 1, characterized in that, The hydrogel has a bactericidal effect against MRSA.

5. The application according to any one of claims 1 to 4, characterized in that, The hydrogel can effectively treat epidermal and ocular infections and significantly reduce the risk of MRSA infection in the lungs.

6. The application according to claim 1, characterized in that, The ferrous ions in the hydrogel were successfully loaded into the hydrogel matrix and uniformly distributed within the hydrogel. The ferrous ion-loaded hydrogel has a heterogeneous surface and porous structure, which is beneficial for water retention and also for Fe. 2+ Diffusion and release from the hydrogel.

7. The application according to claim 1, characterized in that, The ferrous ion-loaded hydrogels G′ and G″ in the hydrogel can recover to 100% within seconds under high shear rates, exhibiting fluidity and resilience that are beneficial for drug diffusion. Wherein, G′ is the elastic modulus and G″ is the loss modulus.

8. The application according to claim 1, characterized in that, Fe in the hydrogel 2+ It can be released from the hydrogel in a time-dependent manner, and the hydrogel has the characteristic of slow and long-term release of ferrous ions. After 12 h, Fe in the hydrogel 2+ The release rate was 78.3%.

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