Active oxygen response hydrogel and application thereof in bacteriostasis and repair promotion

Abstract

The application discloses an active oxygen response type bacteriostatic and repair promoting hydrogel and a preparation method and application thereof, the hydrogel is formed by disulfide bond cross-linked hydrogel in response to active oxygen free radicals in the vagina of vaginitis patients through thiol modified hyaluronic acid (HASH), and can enhance in-vivo retention by thiol and vaginal tissue interaction. Cationic surfactant such as didecyldimethylammonium chloride (DDAC) as the bacteriostatic component of the hydrogel, efficiently killing pathogenic bacteria while having good biocompatibility, and is not prone to cause bacterial drug resistance. As a natural immune regulator, beta-glucan regulates the vaginal microenvironment, promotes the polarization of macrophages into anti-inflammatory M2 type and promotes cell migration, etc., to promote the repair of the vaginal immune barrier, regulate the vaginal flora, and reduce the probability of vaginitis recurrence. The HASH hydrogel provides an efficient, convenient and highly transformative potential strategy to provide more choices for the treatment of vaginitis.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and medical devices, specifically to a method for preparing and applying a reactive oxygen species-responsive antibacterial and repair-promoting hydrogel, belonging to the field of antibacterial and repair-promoting hydrogels. Background Technology

[0002] Vaginitis is a common gynecological disease among women of all ages. Common symptoms include abnormal vaginal discharge, itching, and burning. The most common causes are poor hygiene, prolonged use of antibiotics, and weakened immunity leading to bacterial invasion. Common pathogens include bacteria, trichomonas, and Candida albicans. Patients with vaginitis have an abnormal vaginal environment, a damaged immune barrier, and are at risk of transmission. Oral or vaginal antibiotics are the primary clinical treatment for vaginitis; common medications include metronidazole gel and clindamycin. However, frequent use of antibiotics can lead to antibiotic resistance, and simply using antibiotic solutions cannot repair the damaged immune barrier, making recurrent vaginitis a common problem during treatment.

[0003] Hydrogels are widely used in the treatment of vaginitis due to their good biocompatibility, sustained-release effect, and wound protection. However, existing hydrogels face the following challenges in vaginitis treatment: 1) They easily induce bacterial resistance; 2) The adhesion between hydrogels and the vagina is insufficient, especially since the moist environment and constantly renewing vaginal mucus make hydrogels prone to dissolution, structural damage, and premature expulsion from the body; 3) Current vaginitis treatments focus primarily on antibacterial activity and fail to repair the damaged vaginal immune barrier. To address these pain points and challenges, there is an urgent need to develop new antibacterial hydrogel systems. Summary of the Invention

[0004] The purpose of this invention is to disclose a reactive oxygen species-responsive antibacterial and repair-promoting hydrogel, comprising a hydrogel material with lubricating, moisturizing, biocompatible and tissue-adhesive properties, a broad-spectrum, highly effective antibacterial component with strong selective pathogen killing ability, a repair agent that promotes the repair of the vaginal immune barrier and regulates the vaginal flora, and its application in the treatment of vaginitis, especially vaginitis caused by trichomoniasis, Candida albicans and bacterial infections.

[0005] The present invention adopts the following technical solution.

[0006] A reactive oxygen species (ROS) responsive hydrogel includes an ROS-responsive hydrogel material; the hydrogel material includes a polymeric framework material modified with tissue adhesion groups.

[0007] An active oxygen-responsive antibacterial hydrogel includes an active oxygen-responsive hydrogel material and an antibacterial agent loaded thereon; the hydrogel material includes a polymeric framework material modified with tissue adhesion groups.

[0008] A reactive oxygen species-responsive pro-repair hydrogel includes a reactive oxygen species-responsive hydrogel material and an immunomodulator loaded thereon; the hydrogel material includes a polymeric framework material modified with tissue adhesion groups.

[0009] A reactive oxygen species-responsive antibacterial and repair-promoting hydrogel includes a reactive oxygen species-responsive hydrogel material and an antibacterial agent and an immunomodulator loaded thereon; the hydrogel material includes a polymeric framework material modified with tissue adhesion groups.

[0010] In this invention, the polymeric framework material includes hyaluronic acid, gelatin, chitosan, cyclodextrin, collagen, sodium alginate, dextran, or other polysaccharides; the tissue adhesion groups include one or more of thiol, N-hydroxysuccinimide (NHS), catechol, amino, carboxyl, aldehyde, acrylate, and tannic acid; the modification ratio of the tissue adhesion groups is 10-70%; the hydrogel has lubricating, tissue adhesion, antibacterial, and repair-promoting functions.

[0011] In this invention, the concentration of the immunomodulator is 0.001-100 mg / mL; the concentration of the antibacterial agent is 0.001-10 mmol / mL. Preferably, the concentration of the immunomodulator is 0.5-50 mg / mL; the concentration of the antibacterial agent is 0.01-1 mmol / mL. More preferably, the concentration of the immunomodulator is 5-20 mg / mL; the concentration of the antibacterial agent is 0.02-0.5 mmol / mL.

[0012] In this invention, the immunomodulator is β-glucan; β-glucan includes one or more of the following: yeast and fungi, cereals, algae, and bacterial β-glucan.

[0013] In this invention, the antibacterial agent is a cationic surfactant; the cationic surfactant includes one or more of the following: dimethyldisdecylammonium chloride (DDAC), dimethyldisoctylammonium chloride (DODAC), benzalkonium chloride (n=12, DDBAC), benzalkonium chloride (n=14, BAC), benzalkonium chloride (n=16, HDBAC), trimethylhexadecylammonium chloride (CTAC), trimethylhexadecylammonium bromide (CTAB), trimethyldodecylammonium chloride (DTAC), and trimethyldodecylammonium bromide (DTAB).

[0014] This invention discloses a method for preparing the above-mentioned reactive oxygen species responsive hydrogel, which includes the following steps: modifying tissue adhesion groups on a polymer backbone material to obtain a reactive oxygen species responsive hydrogel.

[0015] This invention discloses a method for preparing the above-mentioned reactive oxygen species responsive antibacterial hydrogel, which includes the following steps: mixing the reactive oxygen species responsive hydrogel with an antibacterial agent to obtain the reactive oxygen species responsive antibacterial hydrogel.

[0016] This invention discloses a method for preparing the above-mentioned reactive oxygen species-responsive repair-promoting hydrogel, which includes the following steps: mixing the reactive oxygen species-responsive hydrogel with an immunomodulator to obtain the reactive oxygen species-responsive repair-promoting hydrogel.

[0017] This invention discloses a method for preparing the above-mentioned reactive oxygen species-responsive antibacterial and repair-promoting hydrogel, which includes the following steps: mixing the reactive oxygen species-responsive hydrogel with an antibacterial agent and an immunomodulator to obtain the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel.

[0018] The hydrogel disclosed in this invention is preferably an active oxygen-responsive antibacterial and repair-promoting hydrogel. Specifically, the components can be combined in the following ways: HASH + surfactant, HASH + surfactant + immunomodulator, HASH + immunomodulator, and HASH.

[0019] This invention discloses the application of the above-mentioned hydrogel in the preparation of drugs.

[0020] This invention discloses a drug whose active ingredient includes the above-mentioned hydrogel.

[0021] In this invention, the drug is an anti-inflammatory drug and / or a tissue repair drug; further, the drug is a vaginal anti-inflammatory drug and / or a vaginal tissue repair drug.

[0022] Preferably, the inflammation includes vaginitis caused by trichomoniasis, Candida albicans, or bacterial infections.

[0023] In this invention, a reactive oxygen species (ROS) responsive antibacterial and repair-promoting hydrogel reacts to form a hydrogel in the presence of ROS in patients with vaginitis, and enhances adhesion by interacting with tissues. The hydrogel, remaining in the vagina, protects the damaged vaginal barrier, scavenges ROS to alleviate inflammation, and slowly releases antibacterial and repair agents. The antibacterial agent inhibits bacterial growth through broad-spectrum bactericidal action, treats bacterial infections, and is less likely to induce bacterial resistance. The repair agent promotes macrophage polarization and vaginal tissue repair. The HASH, antibacterial agent, and repair agent work synergistically to regulate the vaginal immune barrier, treat vaginitis, and reduce its recurrence.

[0024] The positive and progressive effects of this invention are as follows: The modified hyaluronic acid used in this invention can self-crosslink to form a hydrogel without additional initiation, and can crosslink with thiol groups on vaginal tissue to enhance the in vivo retention of the hydrogel, exhibiting a better retention effect compared to traditional hydrogels.

[0025] The hydrogel uses the cationic surfactant DDAC as an antibacterial agent, which is highly effective in inhibiting bacteria, is not prone to inducing bacterial resistance, and causes little damage to cells within the application concentration range; moreover, the surfactant is stable, easy to transport, has a well-established preparation technology, and is low in cost.

[0026] The hydrogel uses beta-glucan, an immunomodulator that can promote the repair of the vaginal immune barrier.

[0027] Hydrogels can regulate vaginal flora and promote the improvement of the vaginal immune microenvironment. Attached Figure Description

[0028] Figure 1 This diagram illustrates the synthetic pathway and gelation mechanism of a reactive oxygen species-responsive gel.

[0029] Figure 2 This is the hydrogen NMR spectrum of HASH.

[0030] Figure 3 The hydrogel precursor solution forms a hydrogel in response to reactive oxygen species.

[0031] Figure 4 For subsequent experiments on hydrogels, A is a scanning electron microscope image of the hydrogel after freeze-drying; B is the relationship between the gelation time of the reactive oxygen species (ROS) responsive hydrogel precursor solution and hyaluronic acid solution and the H2O2 concentration; C is the storage modulus and loss modulus values ​​of the ROS responsive hydrogel precursor solution and hyaluronic acid solution mixed with 50 μM H2O2 solution in a frequency scanning test at 1% strain.

[0032] Figure 5 For the hydrogel animal experiments, A represents the retention effect of reactive oxygen species (ROS) responsive hydrogel, hyaluronic acid, and Cy5.5 in a mouse model of vaginitis within 48 hours; B represents the retention effect of the hydrogel in the vagina after 48 hours in the mouse model of vaginitis; C represents the distribution of the hydrogel, hyaluronic acid, and Cy5.5 in major organs after 48 hours in the mouse model of vaginitis (V for vagina, Lv for liver, Lu for lung, H for heart, S for spleen, and K for kidney); D represents the fluorescence signal value of the hydrogel, hyaluronic acid, and Cy5.5 in the mouse model of vaginitis within 48 hours; E represents the fluorescence signal value of the hydrogel in the vagina after 48 hours in the mouse model of vaginitis; and F represents the fluorescence signal value of the hydrogel in major organs after 48 hours in the mouse model of vaginitis.

[0033] Figure 6To represent the antibacterial effect of hydrogels, A represents the in vivo inhibitory effect of various hydrogels on Candida albicans, a common pathogen causing vaginitis; B represents the in vivo inhibitory effect of various hydrogels on multiple pathogenic bacteria causing vaginitis (Candida albicans, Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus); C represents the in vivo inhibition rate of various hydrogels on Candida albicans, a common pathogen causing vaginitis; and D represents the in vivo inhibition rate of various hydrogels on multiple pathogenic bacteria causing vaginitis (Candida albicans, Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus).

[0034] Figure 7 To illustrate the in vivo antibacterial effect of the hydrogel, A shows the improvement in vulvar redness and swelling in mice during the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel; B shows the change in the area of ​​vulvar redness and swelling in mice during the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel; and C shows the change in mouse body weight during the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel.

[0035] Figure 8 To illustrate the in vivo repair effect of the hydrogel, A represents the number of bacteria in the vaginal irrigation fluid of mice during the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel; B represents the survival rate of bacteria in the vaginal irrigation fluid of mice during the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel; C represents the polarization of macrophages in the vaginal tissue of mice after the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel; and D represents the ratio of macrophages polarized to M2 and M1 types in the vaginal tissue of mice after the treatment of vaginitis with the reactive oxygen species-responsive antibacterial and repair-promoting hydrogel.

[0036] Figure 9 To assess the repair effect of the hydrogel, the following images are presented: A) PAS staining of vaginal tissue sections from mice with a vaginitis model treated with the reactive oxygen species (ROS) responsive antibacterial and repair-promoting hydrogel; B) HE staining of vaginal tissue sections from mice with a vaginitis model treated with the ROS responsive antibacterial and repair-promoting hydrogel; and C) iNOS staining of vaginal tissue sections from mice with a vaginitis model treated with the ROS responsive antibacterial and repair-promoting hydrogel.

[0037] Figure 10To illustrate the effect of hydrogels on the vaginal flora, A represents the Shannon index of the vaginal flora in mice treated with reactive oxygen species-responsive antibacterial and repair-promoting hydrogels for vaginitis; B represents the Simpson index of the vaginal flora in mice treated with reactive oxygen species-responsive antibacterial and repair-promoting hydrogels for vaginitis; and C represents the Pielou index of the species evenness of the vaginal flora in mice treated with reactive oxygen species-responsive antibacterial and repair-promoting hydrogels for vaginitis. D represents the relative abundance of Firmicutes in the vaginal flora of mice after treatment with reactive oxygen species-responsive antibacterial and repair-promoting hydrogel for vaginitis; E represents the relative abundance of Proteobacteria in the vaginal flora of mice after treatment with reactive oxygen species-responsive antibacterial and repair-promoting hydrogel for vaginitis; F represents the relative abundance of Enterococcus in the Lactobacillales order of the vaginal flora of mice after treatment with reactive oxygen species-responsive antibacterial and repair-promoting hydrogel for vaginitis; G represents the PCoA analysis diagram of the vaginal flora of mice after treatment with reactive oxygen species-responsive antibacterial and repair-promoting hydrogel for vaginitis; H represents the NMDS analysis diagram of the vaginal flora of mice after treatment with reactive oxygen species-responsive antibacterial and repair-promoting hydrogel for vaginitis.

[0038] Figure 11 The relative abundance of each species at the phylum level after treatment of a mouse model of vaginitis with a reactive oxygen species-responsive antibacterial and repair-promoting hydrogel.

[0039] Figure 12 This is a graph showing the relative abundance of species at the genus level in mice with a reactive oxygen species-responsive antibacterial and repair-promoting hydrogel for treating vaginitis.

[0040] Figure 13 The in vitro antibacterial effects of two antibacterial hydrogels were compared. Detailed Implementation

[0041] The polymeric framework material of the hydrogel of this invention includes one or more of the following: Hyaluronic acid, with a molecular weight of 10-2000 kDa, preferably 80-150 kDa; Collagen, with a molecular weight of 50-300 kDa, preferably 200-300 kDa; Chitosan, with a molecular weight of 10-300 kDa, preferably 100-300 kDa; Sodium alginate, with a molecular weight of 50-500 kDa, preferably 200-500 kDa; Gelatin, preferably with a molecular weight of 50-100 kDa; Cyclodextrin, preferably with a molecular weight of 5-50 kDa; Dextran, preferably with a molecular weight of 10-200 kDa.

[0042] This invention modifies the side chains of polymers with tissue adhesion groups, including thiol, NHS, catechol, amino, carboxyl, aldehyde, acrylate, and tannic acid; preferably, thiol-modified hyaluronic acid, denoted as HASH, to improve tissue adhesion and increase the vaginal retention capacity of hydrogel materials.

[0043] This invention improves the antibacterial effect and biosafety of the hydrogel by adding a cationic surfactant as an antibacterial material, including one or more of dimethyldisdecylammonium chloride (DDAC), dimethyldisoctylammonium chloride (DODAC), benzalkonium chloride (n=12, DDBAC), benzalkonium chloride (n=14, BAC), benzalkonium chloride (n=16, HDBAC), trimethylhexadecylammonium chloride (CTAC), trimethylhexadecylammonium bromide (CTAB), trimethyldodecylammonium chloride (DTAC), and trimethyldodecylammonium bromide (DTAB), preferably dimethyldisdecylammonium chloride (DDAC), and reduces the risk of bacterial resistance.

[0044] In this invention, β-glucan is preferably used as a repair agent for hydrogels, and its sources include one or more of yeast and fungi, cereals, algae, and bacteria; β-glucan derived from yeast is preferred as a repair-promoting material for hydrogels, thereby enhancing the hydrogel's effect on promoting vaginal barrier repair and flora regulation.

[0045] The present invention specifically discloses the following technical solutions.

[0046] A reactive oxygen species responsive hydrogel, comprising HASH.

[0047] A reactive oxygen species-responsive antibacterial and repair-promoting hydrogel comprising HASH and an antibacterial agent.

[0048] A reactive oxygen species-responsive repair-promoting hydrogel includes HASH and a repair agent.

[0049] A reactive oxygen species-responsive antibacterial and repair-promoting hydrogel, comprising HASH, an antibacterial agent, and a repair agent.

[0050] This invention discloses a method for preparing the above-mentioned reactive oxygen species-responsive antibacterial and repair-promoting hydrogel, comprising the following steps: using HASH, antibacterial agent, and repair agent as raw materials to prepare a reactive oxygen species-responsive antibacterial and repair-promoting hydrogel.

[0051] This invention's reactive oxygen species-responsive antibacterial and repair-promoting hydrogel can effectively inhibit the growth of vaginal pathogens, such as Candida albicans, Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus. It can also promote the repair of the vaginal immune barrier and the regulation of vaginal flora through its repairing agent.

[0052] In this invention, the reactive oxygen species responsive hydrogel material is thiol-modified hyaluronic acid (HASH), and the solvent is water. The grafting rate of HASH is 10-80%, preferably 40-70%, and more preferably 55-65%. The concentration of HASH is 5-20 mg / mL, preferably 10-15 mg / mL.

[0053] In this invention, the application concentration of the antibacterial agent is 0.001-1 mmol / mL, and the preferred concentration is 0.02-0.5 mmol / mL.

[0054] In this invention, the application concentration of the repair agent is 0.001-100 mg / mL, and the preferred concentration is 5-20 mg / mL.

[0055] This invention discloses the application of the above-mentioned reactive oxygen species-responsive antibacterial and repair-promoting hydrogel in the treatment of vaginitis. The vaginitis referred to is vaginitis caused by Candida albicans infection or vaginitis caused by mixed pathogens, particularly its application in vaginitis caused by mixed pathogens.

[0056] This invention adds β-glucan, a repair-promoting ingredient, to the surfactant without inducing bacterial resistance. This allows for the simultaneous inhibition of bacteria and repair of the damaged antibacterial barrier. In particular, the hydrogel component is a reactive oxygen species (ROS) responsive component (HASH), which enhances drug retention and maximizes efficacy. The β-glucan in this invention effectively repairs the damaged vaginal barrier and, as a natural immunomodulator, can also regulate the vaginal flora to some extent. Furthermore, the antibacterial ingredient in this invention is abundant, inexpensive, and stable. Compared to existing hydrogels loaded with lactobacilli, this hydrogel is easier to store and more readily adaptable for practical application. In summary, no research has yet explored the application of repair-promoting therapy in the treatment of vaginitis. This invention is the first to introduce an immunomodulator that can repair the damaged vaginal barrier while exerting antibacterial activity, thus curing vaginitis.

[0057] The raw materials and bacteria used in this invention are existing products, and all that is required is that they meet the modeling requirements; unless otherwise specified, the molecular weights are number-average molecular weights; the specific preparation operations and performance tests are conventional techniques. Animal experiments comply with the relevant requirements of Soochow University, and statistical analysis is standard.

[0058] Example 1 Figure 1The synthesis pathway and gelation mechanism of reactive oxygen species-responsive gel are shown in the diagram. Sodium morpholine ethanesulfonate (MES-Na, 1.974 g) and hyaluronic acid (HA, 400 mg) were dissolved in 80 mL of deionized water. After stirring until the hyaluronic acid was completely dissolved, the pH was adjusted to 5.5 using hydrochloric acid and H2O2 solution. While stirring, 2,2'-dithiodimethyldiethylamine (CSA, 641.17 mg), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 782.45 mg), and N-hydroxysuccinimide (NHS, 270 mg) were added sequentially. The reaction was carried out under nitrogen protection for 3 h. The reaction product was dialyzed against deionized water for 3 days, with the water changed every 24 h. After dialyzing, 1 g of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added to the product, and the reaction was stirred and carried out under nitrogen protection for 3 h. The reaction product was dialyzed against deionized water at pH 3.5 for 3 days, with nitrogen protection and dialyzed every 24 h. The water is changed once every hour, and the dialysate is freeze-dried to obtain the target product HASH.

[0059] The obtained product was dissolved in deuterium oxide (D2O). After complete dissolution, the insoluble matter was removed by filtration. The nuclear magnetic resonance hydrogen spectrum of the product was obtained by nuclear magnetic resonance spectroscopy to confirm the synthesis of the product. Figure 2 The image shows the 1H NMR spectrum of HASH. Compared to the 1H NMR spectrum of hyaluronic acid, the product exhibits characteristic peaks at δ 2.63 ppm and δ 2.85 ppm. These characteristic peaks are attributed to the hydrogen atoms on the methylene group linked to the thiol group, and the modification rate can be calculated by integrating these characteristic peaks. The grafting rate is calculated using the following formula:

[0060] In Example 1 of this invention, the standard peak selected is the peak at δ 1.9 ppm, which is attributed to the methyl group on hyaluronic acid.

[0061] The hyaluronic acid in Example 1 of this invention has a molecular weight of 80-150 kDa.

[0062] Example 2 This invention provides an active oxygen-responsive hydrogel, an active oxygen-responsive antibacterial hydrogel, an active oxygen-responsive repair-promoting hydrogel, and an active oxygen-responsive antibacterial and repair-promoting hydrogel, achieved through the following technical solutions: A homogeneous aqueous solution I was obtained by dispersing thiol-modified hyaluronic acid (HASH) in PBS and then sonicating it; an aqueous solution II was obtained by dispersing an antibacterial agent in PBS and then sonicating it; and an aqueous solution III was obtained by dispersing a repair agent in PBS and then sonicating it.

[0063] The product obtained by uniformly mixing aqueous solution I with PBS is a reactive oxygen species responsive hydrogel, with a volume ratio of aqueous solution I to PBS of 8:2.

[0064] A reactive oxygen species-responsive antibacterial hydrogel was prepared by uniformly mixing aqueous solution I with PBS and aqueous solution II. The volume ratio of aqueous solution I to PBS and aqueous solution II was 8:1:1.

[0065] A reactive oxygen species-responsive pro-repair hydrogel was prepared by mixing aqueous solution I with PBS and aqueous solution III, with a mixing volume ratio of 8:1:1.

[0066] A reactive oxygen species-responsive antibacterial and repair-promoting hydrogel was prepared by mixing aqueous solution I with aqueous solution II and aqueous solution III. The mixing volume ratio of aqueous solution I to aqueous solution II and aqueous solution III was 8:1:1.

[0067] The grafting rate of the above-mentioned HASH was 60%, and the concentration of aqueous solution I was 12.5 mg / mL.

[0068] In the above aqueous solution II, the antibacterial agent is dimethyldisdecylammonium chloride (DDAC) with a concentration of 0.1 mmol / mL.

[0069] The repair agent in the above aqueous solution III is β-glucan, which is derived from yeast (product number G304913-1g, CAS9012-72-0) and has a concentration of 10 mg / mL.

[0070] Example 3 The reactive oxygen species responsive hydrogel of the present invention forms a hydrogel by reacting with reactive oxygen species and enhances tissue adhesion through the interaction of thiol groups with tissues.

[0071] 1. This experiment is used to demonstrate the ability of reactive oxygen species-responsive hydrogels to form hydrogels in response to reactive oxygen species.

[0072] Add 0.05 mL of hydrogen peroxide solution with a concentration of 10-5000 μmol / mL to 0.5 mL of reactive oxygen species responsive hydrogel. Start timing when adding the solution. The standard for gel formation is that the hydrogel does not flow down when the gel is inverted and gently shaken. Record the gel formation time of the hydrogel.

[0073] The formation principle of reactive oxygen species responsive hydrogels is as follows: Figure 1 The thiol group reacts with reactive oxygen species (ROS) to form disulfide bonds, which then link together the hyaluronic acid backbone to form a water-containing three-dimensional network structure and a hydrogel.

[0074] Referring to Example 2, aqueous solution I was mixed with PBS to obtain a reactive oxygen species responsive hydrogel precursor solution, with a volume ratio of aqueous solution I to PBS of 8:2; hyaluronic acid was mixed with PBS to obtain a hyaluronic acid solution, with a volume ratio of hyaluronic acid to PBS of 8:2, as a control.

[0075] Figure 3 The precursor solution for reactive oxygen species-responsive hydrogels forms a hydrogel in response to reactive oxygen species, and does not drip when inverted and gently shaken. Figure 4 Image A is a scanning electron microscope image of the freeze-dried hydrogel; Figure 4 In Figure B, the relationship between the gelation time of reactive oxygen species-responsive hydrogel precursor solution and hyaluronic acid solution and H2O2 concentration is shown. Figure 4 In the figure, C represents the storage modulus and loss modulus values ​​of a reactive oxygen species-responsive hydrogel precursor solution, a hyaluronic acid solution, and a 50 μM H2O2 solution, obtained by mixing these components under a frequency scan at 1% strain.

[0076] Depend on Figure 3 , Figure 4 A, Figure 4 B indicates that HASH exhibits the characteristic of forming hydrogels in response to ROS; after hydrogel formation, it is not horizontal when tilted and does not flow when inverted. From... Figure 4 As can be seen from C, the formed product exhibits the rheological characteristics of a hydrogel, namely, the elastic modulus is greater than the viscous modulus (G' > G), which further verifies the formation of the hydrogel.

[0077] In treating vaginitis, adhesion is a crucial consideration. Hydrogels with low adhesion are easily washed away by constantly changing vaginal mucus, preventing them from fully exerting their therapeutic effect and leading to their expulsion. The HASH technology used in this invention contains thiol groups, enabling it to interact with vaginal tissue and adhere to the vagina. It also releases a sustained-release drug to fully utilize the loaded medication.

[0078] 2. This experiment is used to illustrate the in vivo retention capacity of reactive oxygen species-responsive antibacterial and repair-promoting hydrogels.

[0079] Sample preparation: According to Example 1, EDCI, NHS, and 100 μL of 0.1 mg / ml Cy5.5-NH2 were added, and the reaction was carried out for 3 h. The reaction product was dialyzed and dried to obtain HA-Cy5.5 (hyaluronic acid without thiol groups but with Cy5.5).

[0080] According to Example 1, after adding NHS, EDCI, and 2,2'-dithiodimethyldiethylamine (CSA), 100 μL of 0.1 mg / ml Cy5.5-NH2 was added while stirring. The subsequent steps remained unchanged, and the reaction product was dialyzed and dried to obtain HASH-Cy5.5.

[0081] The entire process of preparing HASH-Cy5.5 and HA-Cy5.5 was conducted in the dark.

[0082] Test methods Before the experiment, nine 8-week-old Kunming mice were divided into three groups of three: HASH, HA, and Cy5.5. The mice were treated with HASH-Cy5.5, HA-Cy5.5, and NH2-Cy5.5, respectively. Tryptic soy broth (TS medium) was prepared before the experiment. Gardnerella vaginalis culture medium was prepared with 10% defibrinated rabbit blood added.

[0083] One week prior to the experiment, mice were anesthetized every other day using a small animal anesthesia device and injected with 0.1 mL of estradiol benzoate injection (2 mg / mL) for six consecutive days. Two days before the experiment, four tubes of 10 mL TS medium were taken, and 100 μL of Candida albicans, Gardnerella vaginalis, Escherichia coli, and Staphylococcus aureus bacterial suspensions were added and incubated overnight at 37 ℃ and 150 rpm. On the last day, 20 μL of Candida albicans suspension (diluted to a final concentration of 10) was inoculated. 9 (CFU / mL), and HASH-Cy5.5, HA-Cy5.5, and Cy5.5 were dissolved; on the morning of the seventh day, mice were anesthetized using a small animal anesthesia device, and 50 μL of HASH-Cy5.5, HA-Cy5.5, and NH2-Cy5.5 were injected into each group using a syringe. Imaging was performed using a small animal in vivo imaging device, and the time point was recorded as 0 h; mice were anesthetized and controlled using a small animal in vivo imaging device at 3 h, 6 h, 12 h, 24 h, and 48 h; another 9 mice were treated in the same way, and 48 h after drug injection, they were euthanized by CO2 and dissected, and the heart, liver, spleen, lungs, kidneys, and vagina were collected for fluorescence imaging.

[0084] Figure 5 In the study, groups A, B, and C showed that the HASH group still exhibited strong fluorescence signals after 48 h, while the fluorescence signals of the HA and Cy5.5 groups were almost completely cleared after 6 h. This demonstrates that HASH has a good vaginal retention effect, and can form a hydrogel in the vagina of mice with vaginitis to protect injured tissue and release the loaded drug, thus exerting its effect.

[0085] Figure 5 In the images, D, E, and F show fluorescence signals mainly concentrated in the vagina, indicating that HASH is primarily retained in the vaginal area, ensuring the hydrogel functions effectively.

[0086] Example 4 Antibacterial efficacy is an important evaluation indicator in the treatment of vaginitis. Common clinical drugs mainly use antibiotics to exert antibacterial effects, but the overuse of antibiotics can easily lead to bacterial resistance, resulting in recurrent vaginitis. Surfactants exert their effects through different pathways than antibiotics and are less likely to induce bacterial resistance. Antibacterial experiments were conducted to demonstrate the in vivo antibacterial effects of reactive oxygen species-responsive antibacterial and repair-promoting hydrogels.

[0087] 1. This test is used to illustrate the inhibitory effect of the hydrogel of the present invention on Candida albicans, a common pathogen causing vaginitis, in vivo.

[0088] Sample preparation: The reactive oxygen species responsive hydrogel and reactive oxygen species responsive antibacterial hydrogel from Example 2, as well as hyaluronic acid and antibacterial agent, were used as experimental materials.

[0089] Strain selection: Candida albicans (Ca), a common pathogenic bacterium in vaginitis.

[0090] Test method: The antibacterial properties of the hydrogel were measured using the agar plate coating method. Twenty-four mice were randomly divided into six groups, with a density of 10... 6 Five groups (20 μL / mouse) were injected with CFU / mL bacterial suspension, labeled as the vaginitis control group (Control), the hydrogel-only treatment group (HASH), the surfactant treatment group (DDAC), the hyaluronic acid treatment group (HA+DDAC), and the hydrogel treatment group (HASH+DDAC, HD). The group that did not receive bacterial suspension was designated as the health group. Two hours after bacterial suspension injection, the corresponding drugs were administered (20 μL / mouse, vaginal injection). One day after treatment, the mice were sacrificed, and 250 μL of PBS vaginal irrigation solution was serially diluted and plated. The plates were incubated overnight at 37°C, and colonies were counted.

[0091] Depend on Figure 6 As shown in A and C, the HASH+DDAC hydrogel treatment group had the lowest bacterial survival rate, indicating that HASH+DDAC, by treating and releasing antibacterial agents in the vagina, fully exerts its antibacterial effect and has the potential to effectively kill bacteria in the vagina of mice and thus treat vaginitis.

[0092] 2. This test is used to illustrate the inhibitory effect of the hydrogel of the present invention on a variety of common pathogens causing vaginitis in vivo.

[0093] Sample preparation: The reactive oxygen species responsive hydrogel and reactive oxygen species responsive antibacterial hydrogel from Example 2, as well as hyaluronic acid and antibacterial agent, were used as experimental materials.

[0094] Strain selection: The pathogenic bacteria for vaginitis are Candida albicans (Ca), Gardnerella vaginalis (Gv), Staphylococcus aureus (Sa), and Escherichia coli (Ec).

[0095] Test method: The antibacterial properties of the hydrogel were measured using the agar plate coating method. Twenty-four mice were randomly divided into six groups, with a density of 10... 6 A mixed bacterial solution of CFU / mL was injected into five groups (20 μL / mouse), and labeled as the vaginitis control group (Control), the hydrogel-only treatment group (HASH), the surfactant treatment group (DDAC), the hyaluronic acid treatment group (HA+DDAC), and the hydrogel treatment group (HASH+DDAC). The group that did not receive the bacterial solution was the healthy group (Health). Two hours after the bacterial solution injection, the corresponding drugs (20 μL / mouse) were administered. One day after treatment, the mice were sacrificed, and 250 μL of PBS vaginal irrigation solution was serially diluted and plated. The plates were incubated overnight at 37°C, and the colonies were counted.

[0096] Depend on Figure 6 As shown in B and D, the HASH+DDAC hydrogel treatment group had the lowest bacterial survival rate, proving that the DDAC-loaded HASH hydrogel can effectively inhibit a variety of pathogens in mice, thereby reducing vaginal infection and promoting the repair of vaginitis.

[0097] Example 5 This experiment was conducted to demonstrate the therapeutic effect of the hydrogel of the present invention on mice with vaginitis.

[0098] Thirty mice were divided into five groups of six each: Health (healthy group), Control (positive control group), DG glucan (DDAC + β-glucan solution treatment group), HD (HASH + DDAC treatment group), and HDG (HASH + DDAC + β-glucan). Except for the Health group, the other four groups received 0.1 mL of 2 mg / mL estradiol benzoate solution every other day for six days prior to the experiment. The other four groups received a 10 mg / mL solution of estradiol benzoate solution one day prior to the experiment. 9 A mixed bacterial solution of CFU / mL (containing the pathogenic species Candida albicans (Ca), Gardnerella vaginalis (Gv), Staphylococcus aureus (Sa), and Escherichia coli (Ec)) was administered once daily according to the respective groups (20 μL / mouse). Vulvar photographs and body weight were recorded. Mice were sacrificed on day seven, and vaginal irrigation fluid and vaginal tissue were collected. Data such as vaginal redness and swelling, and bacterial colony count were analyzed.

[0099] like Figure 7As shown in A and 8A, the vulvar redness and swelling in mice treated with the hydrogel of this invention rapidly decreased and returned to normal levels. The HD group was superior to the Control group, indicating that the antibacterial agent and the repair agent can exert a good therapeutic effect. Compared with the HD and DG groups, the HDG group had the most significant therapeutic effect, indicating that the strong retention effect of HASH in vivo can fully exert the effects of the antibacterial agent and the repair agent, and the repair agent can effectively promote the recovery of vaginitis. Figure 8 As shown in Figure B, the HDG-treated mice exhibited a similar weight change trend to the healthy group, indicating that their weight change returned to normal after treatment and they were relatively healthy; Figure 7 As shown in B and 8D, the bacterial count in the vaginal irrigation fluid of the HDG group mice returned to normal levels; Figure 7 As shown in C and 8C, the number of M2 macrophages in the vaginal tissue of HDG group mice was reduced, and inflammation was suppressed.

[0100] As shown in 9A, the HDG group exhibited the fewest hyphae, demonstrating that the pathogens in the vagina of the HDG group mice were basically clear; Figure 9 As shown in B, the HDG group exhibited less epithelial tissue damage and showed similar epithelial integrity and inflammatory cell infiltration as the healthy group; Figure 9 As shown in Figure C, the HDG group exhibited the lowest levels of reactive oxygen species (ROS). Vaginal tissue sections demonstrated the powerful therapeutic effects of HDG, including pathogen elimination, promotion of vaginal tissue repair, and scavenging of ROS.

[0101] like Figure 10 As shown in A, B, and C, the vaginal flora in the HDG group recovered to a healthy level in terms of diversity and uniformity after treatment. Figure 11 , 10 D and 10E demonstrate that after HDG treatment, the vaginal flora in mice recovered to a level similar to that of the healthy group at the phylum level, particularly with an increase in the abundance of the probiotic Firmicutes phylum and a decrease in the abundance of the pathogenic Proteobacteria phylum. Figure 12 , 10 F demonstrated that after HDG treatment, the vaginal flora in mice recovered to a level similar to that of the healthy group at the genus level, especially with an increase in the abundance of Enterococcus genus in the Lactobacillus order. Figure 10 G and H demonstrate that after HDG treatment, the vaginal flora of mice was most similar to that of the healthy group, and the difference from that of the control group was most significant.

[0102] Example 6 Different concentrations of surfactant solutions were prepared using PBS, and then antibacterial experiments were conducted. Commonly used bacteria included *Candida albicans* (Ca), *Gardnerella vaginalis* (Gv), *Staphylococcus aureus* (Sa), and *Escherichia coli* (Ec). After 24 hours of inhibition, the half-maximal concentration (IC50) of the bacteria was calculated (μM), as follows:

[0103] In the table: Dimethyldisdecylammonium chloride (DDAC), Dimethyldisoctylammonium chloride (DODAC), Benzalkonium chloride (n=12, DDBAC), Dioctadecyldimethylammonium chloride (DODMAC), Trimethylhexadecylammonium chloride (CTAC), Trimethylhexadecylammonium bromide (CTAB), Trimethyldodecylammonium chloride (DTAC), Trimethyldodecylammonium bromide (DTAB), Benzalkonium chloride (n=16, HDBAC).

[0104] Referring to Example 2, antibacterial hydrogels containing HDBAC reactive oxygen species (40 μM) and DDAC reactive oxygen species (20 μM) were prepared. In vitro antibacterial experiments were then conducted using common bacteria such as Candida albicans (Ca), Gardnerella vaginalis (Gv), Staphylococcus aureus (Sa), and Escherichia coli (Ec). After 24 hours of inhibition, bacterial survival rates were calculated. (See [reference needed]). Figure 13 The in vitro antibacterial concentration of HASH hydrogels containing DDAC or HDBAC was tested, and it was found that only DDAC could completely inhibit the proliferation of four pathogens in the hydrogel.

[0105] Existing literature mainly focuses on antibacterial therapy or probiotic supplementation. This invention addresses the challenges of vaginal barrier repair and, compared to existing research, offers readily available, low-cost, and easy-to-store raw materials. Specifically, this invention discloses a reactive oxygen species-responsive antibacterial and repair-promoting hydrogel. It effectively kills pathogens in patients with vaginitis using antibacterial agents and promotes the repair of the immune barrier through immunomodulators. HASH, by modifying thiol groups on HA, becomes responsive to reactive oxygen species, enabling the formation of hydrogels and enhancing tissue adhesion through disulfide bonds generated by oxidation, ensuring the full effectiveness of the loaded antibacterial agents and immunomodulators. The hydrogel effectively inhibits bacteria through DDAC with minimal impact on normal cells, selectively clearing pathogens without easily inducing drug resistance. Through the multiple regulatory effects of the immunomodulator β-glucan, it promotes vaginal immune barrier repair and flora restoration, treating vaginitis at its root and preventing recurrence.

Claims

1. A reactive oxygen species responsive hydrogel, characterized in that, The invention includes reactive oxygen species (ROS) responsive hydrogel materials; said ROS responsive hydrogel materials include polymeric framework materials modified with tissue adhesion groups.

2. A reactive oxygen species-responsive antibacterial hydrogel, characterized in that, The invention includes reactive oxygen species (ROS) responsive hydrogel materials and their loaded antibacterial agents; the ROS responsive hydrogel materials include polymeric framework materials modified with tissue adhesion groups.

3. A reactive oxygen species-responsive, repair-promoting hydrogel, characterized in that, The invention includes reactive oxygen species (ROS) responsive hydrogel materials and their loaded immunomodulators; the ROS responsive hydrogel materials include polymeric framework materials modified with tissue adhesion groups.

4. A reactive oxygen species-responsive antibacterial and repair-promoting hydrogel, characterized in that, The invention includes reactive oxygen species (ROS) responsive hydrogel materials and their loaded antibacterial agents and immunomodulators; the ROS responsive hydrogel materials include polymeric framework materials modified with tissue adhesion groups.

5. The hydrogel according to claim 1, 2, 3 or 4, characterized in that, The polymeric framework material includes hyaluronic acid, gelatin, chitosan, cyclodextrin, collagen, sodium alginate, dextran, or other polysaccharides; the tissue adhesion groups include one or more of the following: thiol, N-hydroxysuccinimide, catechol, amino, carboxyl, aldehyde, acrylate, and tannic acid groups; the modification ratio of the tissue adhesion groups is 10-70%.

6. The hydrogel according to claim 2, 3, or 4, characterized in that, The immunomodulator is β-glucan; the antibacterial agent is a cationic surfactant; the concentration of the immunomodulator is 0.001-100 mg / mL; the concentration of the antibacterial agent is 0.001-1 mmol / mL.

7. The hydrogel according to claim 6, characterized in that, β-glucan includes one or more of β-glucan derived from yeast and fungi, cereals, algae, and bacteria; cationic surfactants include one or more of dimethyldisdecylammonium chloride, dimethyldisoctylammonium chloride, benzalkonium chloride, benzalkonium chloride, benzalkonium chloride, trimethylhexadecylammonium chloride, trimethylhexadecylammonium bromide, trimethyldodecylammonium chloride, and trimethyldodecylammonium bromide.

8. The method for preparing the hydrogel according to claim 1, 2, 3 or 4, characterized in that, By modifying tissue adhesion groups in a polymer framework material, a reactive oxygen species (ROS) responsive hydrogel is obtained; by mixing the ROS responsive hydrogel with an antibacterial agent, an ROS responsive antibacterial hydrogel is obtained; by mixing the ROS responsive hydrogel with an immunomodulator, an ROS responsive repair-promoting hydrogel is obtained; and by mixing the ROS responsive hydrogel with both an antibacterial agent and an immunomodulator, an ROS responsive antibacterial and repair-promoting hydrogel is obtained.

9. The use of the hydrogel according to claim 1, 2, 3 or 4 in the preparation of a drug.

10. A drug, characterized in that, The active ingredient includes the hydrogel described in claim 1, 2, 3 or 4.

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