Application of antioxidant hydrogel based on iron steady-state regulation and control

Abstract

The invention relates to the technical field of biological medicine, in particular to application of antioxidant hydrogel based on iron steady state regulation and control. The iron steady state regulation-based antioxidant hydrogel comprises a Schiff base bond-C = N-structure and a catechol structure, the application comprises the step of preparing a biomedical material by taking the iron steady state regulation-based antioxidant hydrogel as a bioactive component and / or a drug delivery system, wherein the biomedical material is suitable for iron overload and / or oxidative stress related diseases. Cell experiment results show that the hydrogel can effectively reduce radiation-induced intracellular iron ion and ROS accumulation, and is beneficial to maintaining balance of iron ions and ROS in a body; animal experiment results show that the hydrogel can significantly promote wound repair; after treatment for 4 weeks, the total iron content and GSH content of the skin tissue of the irradiated part are recovered to be close to the normal tissue level, main organs of the mouse are not subjected to pathological injury, and blood routine examination is within a normal clinical reference range.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an antioxidant hydrogel based on iron homeostasis regulation. Background Technology

[0002] As the body's largest barrier organ, the skin is susceptible to damage from exogenous stimuli (such as X-ray radiotherapy) and endogenous metabolic abnormalities (such as diabetes). X-ray radiotherapy is a core treatment for malignant tumors, but most patients experience radiation-induced skin damage after radiotherapy, including acute damage (erythema, blisters) or chronic damage (fibrosis, ulcers), severely affecting radiotherapy adherence. X-ray damage to the skin is divided into two categories: direct (DNA strand breaks) and indirect (ionization of water molecules to generate reactive oxygen species), with indirect damage being the dominant type. Diabetic patients often experience skin complications due to long-term high blood sugar causing skin microcirculation disorders and weakened immunity, with diabetic foot ulcers significantly increasing the risk of amputation.

[0003] Recent studies have found that both diseases share a core pathological chain of "iron overload-oxidative stress-glutathione (GSH) depletion": X-rays induce the generation of reactive oxygen species (ROS) through ionization, which, combined with iron overload, amplifies oxidative stress through the Fenton reaction; the high-glucose environment of diabetes disrupts iron metabolism homeostasis, promotes iron accumulation and exacerbates ROS generation, ultimately leading to GSH depletion, collapse of the antioxidant system, and resulting in skin barrier damage, inflammation, and impaired tissue repair.

[0004] Iron is an essential trace element for skin cell proliferation, differentiation, and enzymatic reactions. Iron homeostasis refers to the balance of iron absorption, transport, storage, and utilization within cells and tissues; ROS balance refers to the dynamic stability of ROS generation and clearance in the body. Iron homeostasis and ROS balance are key links in maintaining skin physiological function and damage repair. They regulate each other, and their imbalance is not only an important pathological cause of skin damage but also a core target for intervention in skin injury diseases.

[0005] Hydrogels are three-dimensional network structures formed by the physical or chemical cross-linking of hydrophilic polymer chains. They can swell and lock in large amounts of water as a dispersion medium while maintaining structural integrity and remaining insoluble. Their porous structure can load drugs and achieve controlled release, reducing systemic side effects, making them particularly suitable for local drug delivery (such as the treatment of diabetic foot ulcers or radiation-induced skin damage).

[0006] Iron overload-related diseases are a challenge in clinical treatment. Multi-target intervention of hydrogels to ferroptosis pathways and microenvironment remodeling may be a solution for iron overload-related diseases. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the object of this invention is to provide an application of an iron-based, stable-state regulated antioxidant hydrogel.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The iron-based antioxidant hydrogel comprises a Schiff base bond -C=N- structure and a catechol structure, as shown below: ; The applications include the preparation of biomedical materials using the iron homeostasis-regulated antioxidant hydrogel as a bioactive component and / or drug delivery system, suitable for iron overload and / or oxidative stress-related diseases.

[0009] Furthermore, the iron-based antioxidant hydrogel is used to capture iron ions to reduce the abnormal accumulation of iron ions in the body.

[0010] Furthermore, the iron-based antioxidant hydrogel is used to remove excess reactive oxygen species in the body to maintain the balance of reactive oxygen species in the body.

[0011] Furthermore, the iron-based antioxidant hydrogel is used to increase the content of glutathione in the body in order to maintain the balance of glutathione in the body.

[0012] Furthermore, the biomedical material includes pharmaceutically acceptable excipients, excipients, or carriers.

[0013] Furthermore, the biomedical material is an oral biomedical material or a topical biomedical material.

[0014] Furthermore, the externally used biomedical biomaterial is an external dressing.

[0015] Furthermore, the iron overload / related diseases include skin lesions.

[0016] Furthermore, the skin damage includes radiation-induced skin damage and diabetes-related skin damage.

[0017] Furthermore, the iron-based antioxidant hydrogel can promote wound healing.

[0018] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides an application of an iron-based antioxidant hydrogel, which comprises a Schiff base bond -C=N- structure and a catechol structure, the catechol structure being shown below: Applications include the preparation of biomedical materials using the aforementioned iron-based antioxidant hydrogel as a bioactive component and / or drug delivery system, suitable for iron overload and / or oxidative stress-related diseases.

[0019] I. Cellular experimental results show that: Iron homeostasis-regulated antioxidant hydrogels can effectively reduce radiation-induced intracellular iron ion and ROS accumulation, which is beneficial for maintaining the balance of iron ions and ROS in the body.

[0020] II. The hemolysis test results showed that no hemolysis occurred in the red blood cell suspension with iron homeostasis-regulated antioxidant hydrogel, which suggests that it has good biocompatibility.

[0021] III. Animal experimental results show that: (I) Wound healing effect: Based on the iron homeostasis-regulated antioxidant hydrogel, the wound repair was significantly promoted. After 3 to 4 weeks of treatment, the skin of the injured area in mice basically returned to normal. (II) Iron ion regulation ability: After 4 weeks of treatment, the total iron content of the skin tissue at the irradiation site returned to the normal level. This result indicates that the iron homeostasis-regulating antioxidant hydrogel can efficiently chelate abnormally accumulated iron ions at the wound site. (III) Antioxidant function: After 4 weeks of treatment, the GSH content of the skin tissue at the irradiation site recovered to a level close to that of normal tissue. This result indicates that the iron homeostasis-regulated antioxidant hydrogel can effectively alleviate radiation-induced GSH depletion. (iv) Organ safety: After 4 weeks of treatment, H&E staining (hematoxylin-eosin staining method) showed no pathological damage to the major organs of mice in the iron homeostasis-regulated antioxidant hydrogel treatment group. (V) System safety assessment: After 4 weeks of treatment, the blood routine of mice was within the normal clinical reference range.

[0022] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0023] Figure 1 The nuclear magnetic resonance hydrogen spectra of HA, AHA, and DAHA provided in Example 1 of this invention are... 1 H NMR spectrum.

[0024] Figure 2 This is a diagram showing the results of intracellular Ferro Orange fluorescence staining provided in Experimental Example 1 of this invention.

[0025] Figure 3 This is a diagram showing the results of intracellular DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence staining provided in Experimental Example 2 of this invention.

[0026] Figure 4 This is a diagram showing the supernatant in the hemolysis experiment provided in Experiment Example 3 of this invention.

[0027] Figure 5 This is a bar chart of the hemolysis experiment results provided in Experiment Example 3 of this invention.

[0028] Figure 6 This is a diagram illustrating the therapeutic effects of different substances on mice provided in Experiment Example 4 of this invention.

[0029] Figure 7 This is a line graph showing the effects of different substances on the RTOG grading of mice after treatment, as provided in Experimental Example 4 of this invention.

[0030] Figure 8 This is a graph showing the results of iron content detection in the skin tissue of mice after 28 days of treatment with different substances provided in Experimental Example 4 of this invention.

[0031] Figure 9 This is a graph showing the detection results of GSH content in the skin tissue of mice after 28 days of treatment with different substances provided in Experimental Example 4 of this invention.

[0032] Figure 10 This is an H&E staining image of the major organs of mice after 28 days of treatment with different substances provided in Experiment Example 4 of this invention.

[0033] Figure 11 This is a bar chart showing the results of blood routine tests on mice treated with different substances for 28 days, as provided in Experimental Example 4 of this invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0035] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0036] Example 1 The preparation process of iron-steady-state regulated oxidative hydrogels is as follows: I. Preparation of Aldehyde-Tylated Hyaluronic Acid (AHA): The process is as follows: Sodium hyaluronate (HA) (5 kDa, 1 g) was dissolved in 100 mL of water, and then sodium periodate aqueous solution (0.5 M, 5 mL) was added. The reaction was carried out under stirring at 250 rpm in the dark for 2 h. Ethylene glycol (1 mL) was added to terminate the reaction. The mixture was then dialyzed with water for 3 days and freeze-dried to obtain AHA.

[0037] II. Preparation of catecholated hyaluronic acid (DAHA), the process is as follows: Dissolve 800 mg of AHA in water (10 mL), add 400 mg of dopamine (DA), react under nitrogen protection and in the dark for 8 h, then dialyze with deoxygenated deionized water for 1 day, and freeze dry to obtain DAHA.

[0038] The determination of HA, AHA, and DAHA was carried out using deuterated solvents. 1 H NMR spectrum, such as Figure 1 As shown, HA was successfully oxidized to obtain AHA, and AHA was successfully linked with DA to obtain DAHA.

[0039] III. Preparation of different types of hydrogel samples, the process is as follows: Aqueous solutions of AHA, carboxymethyl chitosan (CMCS), and DAHA were prepared respectively, the process is as follows: Dissolve 6g of AHA in 100ml of water and stir until homogeneous to obtain an AHA aqueous solution with a concentration of 60mg / mL. Dissolve CMCS (6g) in water (100ml) and stir until homogeneous to obtain a CMCS aqueous solution with a concentration of 60mg / mL; Dissolve 6g of DAHA in 100ml of water and stir until homogeneous to obtain an aqueous solution of DAHA with a concentration of 60mg / mL.

[0040] The preparation processes for HACS and DACS hydrogels are as follows: Mix 10 ml of AHA aqueous solution and 10 ml of CMCS aqueous solution evenly, and let stand until gelation occurs to obtain HACS hydrogel.

[0041] After mixing 50 ml of DAHA aqueous solution and 50 ml of CMCS aqueous solution evenly, the mixture was allowed to stand until it gelled, resulting in an iron-based antioxidant hydrogel, denoted as DACS hydrogel.

[0042] Experiment 1: Investigation of the iron-capturing ability of antioxidant hydrogels based on iron homeostasis regulation.

[0043] Five different groups were set up: PBS, PBS+IR, DFO+IR, HACS+IR and DACS+IR; The PBS group served as the blank control group, and its process was the same as that of the other groups except that it was not exposed to X-rays. PBS and DFO were obtained through commercial purchase; HACS and DACS hydrogels were prepared according to the method in Example 1; PBS is phosphate buffer; DFO stands for deferoxamine, and the working solution of DFO is a solution of DFO dissolved in PBS (concentration of 20 μmol / L).

[0044] IR stands for ionizing radiation irradiation. In this experimental example, the ionizing radiation irradiation method is X-ray irradiation.

[0045] The ability of the hydrogel to capture iron ions was investigated using Ferro Orange fluorescence staining, as follows: 3T3 cells were stored at a density of 5 × 10⁻⁶. 4 Cells were seeded per well on a culture plate and incubated in complete culture medium for 24 h. The medium was then discarded, and the cells were washed three times with PBS. Subsequently, 100 μL of different treatments (PBS, DFO working solution, HACS, DACS) were added, and the cells were co-cultured for another 12 h. The treatment solutions were then discarded, and the cells were washed with PBS. Cells were stained with FerroOrange probes and then exposed to X-ray irradiation (dose rate 1 Gy / min, duration 6 min). The staining results were observed under a laser confocal microscope. Figure 2 As shown, the results indicate that the iron homeostasis-regulated antioxidant hydrogel (DACS) can capture iron ions and reduce the accumulation of iron ions in cells.

[0046] Experiment 2: Investigation of the ROS scavenging ability of antioxidant hydrogels based on iron homeostasis regulation.

[0047] Five groups were set up, and their grouping was the same as in Experiment 1 except that DFO was replaced with SOD in Experiment 1. SOD is superoxide dismutase. The working solution of SOD is a solution of SOD dissolved in PBS (concentration of 50 μg / ml). The ability of the hydrogel to scavenge ROS was investigated using DCFH-DA fluorescence staining, as follows: 3T3 cells were stored at a density of 5 × 10⁻⁶. 4Cells were seeded per well on a culture plate and incubated in complete culture medium for 24 h. The medium was then discarded, and the cells were washed three times with PBS. Subsequently, 100 μL of different treatment substances (PBS, SOD working solution, HACS, DACS) were added, and the cells were co-cultured for another 12 h. The treatment solutions were then discarded, and the cells were washed with PBS. Cells were stained with the DCFH-DA probe and then exposed to X-ray irradiation (dose rate 1 Gy / min, duration 6 min). The staining results were observed under a laser confocal microscope. Figure 3 As shown, the results indicate that DACS hydrogel can alleviate radiation-induced intracellular ROS accumulation.

[0048] Example 3: In vitro biosafety study of antioxidant hydrogels based on iron homeostasis regulation.

[0049] Five different groups were set up: PBS (negative control group), DA, HACS, DACS and Triton X-100 (positive control group); Among them, PBS served as the negative control group, and Triton X-100 served as the positive control group; Triton X-100 working solution: PBS solution with a concentration of 0.1 wt% Triton X-100; The in vitro biocompatibility of the hydrogel was investigated using a hemolysis test, as follows: 2% red blood cell suspension (1 mL) was incubated with 100 μL of PBS, DA, HACS, DACS, and Triton X-100, respectively, at 37 °C for 4 h. After centrifugation at 3000 rpm for 3 min, the supernatant was photographed. Figure 4 As shown; the absorbance of the supernatant at 570 nm was measured, and the hemolysis rate of each group was calculated using the following formula: The calculation results are shown in a bar chart, such as... Figure 5 As shown; from Figure 4 and Figure 5 As can be seen, the iron homeostasis-regulated antioxidant hydrogel (DACS) exhibits almost no hemolysis and good in vitro biocompatibility.

[0050] Experiment 4: Evaluation of the efficacy and safety of iron-based antioxidant hydrogels in an animal model of skin injury.

[0051] Five different groups were set up: PBS, hydrogel wound dressing (3M Tegaderm, denoted as 3M), SOD, HACS and DACS. The animal experiment process is as follows: A mouse model of radiation-induced skin injury was established by subjecting the right leg of a mouse to a single local irradiation of 40 Gy X-rays (2 Gy / min, 20 min) for 10 days. After successfully establishing the animal model of injury, PBS, 3M, SOD, HACS, and DACS were applied to the skin injury sites on the right legs of mice according to the corresponding groups. Treatment continued for 28 days, with daily changes. The wound condition of the mice was observed and photographed. Results Figure 6 As shown in the figure, the appearance of the feet of mice in each group was similar at the initial drug administration. From day 21 to day 28 of treatment, the skin of mice in the group with iron homeostasis-regulated antioxidant hydrogel (DACS) basically recovered to normal. After 28 days of treatment, the skin of mice in other groups still had obvious damage marks. The RTOG skin scoring system was used to grade the degree of skin damage in mice. The RTOG skin scoring system is the acute radiation-induced skin injury grading system of the Radiation Therapy Oncology Group (RTOG), a commonly used tool in clinical practice for assessing acute skin reactions after radiation. The RTOG skin scoring system is shown in the table below: According to the above criteria, mouse skin was scored and graded, and a line graph was plotted. The results are as follows: Figure 7 As shown, the results indicate that mice treated with iron homeostasis-regulated antioxidant hydrogel (DACS) recovered from skin damage faster and had a more significant therapeutic effect.

[0052] Twenty-eight days after treatment, the total iron and glutathione (GSH) levels in the skin tissue of the irradiated areas of mice were measured. The results are as follows: Figure 8 and Figure 9 As shown, the ratios of total iron and GSH content in the samples were calculated to those in normal tissues, and the ratios were close to 1. This result indicates that iron homeostasis-regulated antioxidant hydrogels (DACS) are beneficial for maintaining the balance of iron and GSH in the body.

[0053] The in vivo biocompatibility of the hydrogel was assessed using H&E staining of major organs. Major organs (heart, liver, spleen, lung, and kidney) were collected from mice 28 days after treatment. After formalin fixation for 48 h, tissue dehydration, paraffin embedding, sectioning, and routine H&E staining were performed. The results are as follows: Figure 10 As shown, the results indicate that the iron homeostasis-regulated antioxidant hydrogel (DACS) does not cause damage to major organs and has good in vivo biocompatibility. The in vivo biocompatibility of the hydrogel was further investigated using a complete blood count (CBC) test. After hydrogel treatment, orbital blood samples were collected from mice for a CBC test, and the results were as follows: Figure 11As shown, the results indicate that the iron homeostasis-regulated antioxidant hydrogel (DACS) has no hematological side effects on mice.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of an antioxidant hydrogel based on iron homeostasis regulation, characterized in that, The iron-based antioxidant hydrogel comprises a Schiff base bond -C=N- structure and a catechol structure, as shown below: ； The applications include the preparation of biomedical materials using the iron homeostasis-regulated antioxidant hydrogel as a bioactive component and / or drug delivery system, suitable for iron overload and / or oxidative stress-related diseases.

2. The application of the iron-based antioxidant hydrogel as described in claim 1, characterized in that, The iron-based antioxidant hydrogel is used to capture iron ions to reduce the abnormal accumulation of iron ions in the body.

3. The application of the iron-based antioxidant hydrogel as described in claim 1, characterized in that, The iron-based antioxidant hydrogel is used to remove excess reactive oxygen species in the body to maintain the balance of reactive oxygen species in the body.

4. The application of the iron-based antioxidant hydrogel as described in claim 1, characterized in that, The iron-based antioxidant hydrogel is used to increase the content of glutathione in the body in order to maintain the balance of glutathione in the body.

5. The application of the iron-based antioxidant hydrogel as described in claim 1, characterized in that, The biomedical materials include pharmaceutically acceptable excipients, excipients, or carriers.

6. The application of the iron-based antioxidant hydrogel as described in claim 5, characterized in that, The biomedical material is either an oral biomedical material or a topical biomedical material.

7. The application of the iron-based antioxidant hydrogel as described in claim 6, characterized in that, The externally used biomedical biomaterial is a topical dressing.

8. The application of the iron-based antioxidant hydrogel as described in claim 7, characterized in that, The iron overload / related diseases include skin lesions.

9. The application of the iron-based antioxidant hydrogel as described in claim 8, characterized in that, The skin injury includes any one of radiation-induced skin injury, diabetes-related skin injury, and burn injury.

10. The application of the iron-based antioxidant hydrogel as described in claim 8 or 9, characterized in that, The iron-based antioxidant hydrogel can promote wound healing.

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