Preparation method and application of a double-network sprayable hydrogel for repairing radioactive skin damage

Abstract

This invention discloses a method for preparing and applying a dual-network sprayable hydrogel for the repair of radiation-induced skin damage. The hydrogel introduces aldehyde groups into Polygonatum odoratum polysaccharide via sodium periodate oxidation, causing it to chemically crosslink with the amino groups of quaternized chitosan to form a first-layer network. Simultaneously, sodium alginate is introduced into the system, and calcium ions are used to crosslink and construct a second-layer physical crosslinking network. This invention achieves instant gelation upon injection under physiological conditions, effectively avoiding the operational complexity caused by pre-mixed crosslinking agents and optimizing gel performance. The resulting hydrogel exhibits significantly improved mechanical and adhesive properties. In vitro cell experiments and in vivo animal experiments show that this sprayable hydrogel can effectively promote cell proliferation and migration, effectively maintain macrophage mitochondrial dysfunction caused by ionizing radiation, and possesses good antioxidant and anti-inflammatory activities, demonstrating broad application prospects in the repair and treatment of radiation dermatitis.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a sprayable gel with a double cross-linked network, its preparation method, and its application in the repair of radiation-induced skin damage. Background Technology

[0002] Radiation therapy is a key treatment for cancer, with over 50% of cancer patients requiring it. Although the efficacy of radiation therapy has been proven, unnecessary radiation exposure to adjacent healthy tissues remains a significant challenge in clinical practice. Radiation-induced skin damage is one of the most common side effects of radiation therapy, with an incidence rate as high as 95%. Early symptoms include mild erythema, dry, scaly skin, and itching. As the radiation dose accumulates, the damage gradually worsens, potentially leading to moist desquamation, oozing, and even hemorrhagic crusting. In severe cases, it can cause skin ulceration and necrosis, placing a heavy financial burden on cancer patients and impacting the progress of clinical treatment. Therefore, exploring new therapeutic targets and developing novel treatment strategies for radiation-induced skin damage are of great importance.

[0003] In the pathological process of radiation-induced skin injury, macrophage dysfunction and energy metabolism imbalance are key factors hindering tissue repair. Ionizing radiation induces a burst of reactive oxygen species (ROS) in mitochondria, disrupting mitochondrial integrity, weakening oxidative phosphorylation capacity, and inhibiting the production of adenosine triphosphate (ATP) within macrophages. As a core organelle of cellular energy metabolism, mitochondrial dysfunction further reduces the oxidative phosphorylation efficiency of macrophages and decreases ATP synthesis, thereby disrupting the energy supply required for immune response and tissue repair. Simultaneously, metabolic dysregulation drives macrophages to polarize towards a pro-inflammatory phenotype, exacerbating local inflammation and inhibiting their transition to a repair state, ultimately hindering skin epithelialization and tissue regeneration. Although antioxidant therapy can partially alleviate the above-mentioned damage, a precise strategy that can simultaneously clear mitochondrial ROS, restore mitochondrial bioenergy metabolism, and reprogram macrophage immune metabolism is still lacking. Therefore, this invention develops a hydrogel based on Polygonatum polysaccharide, which can reshape macrophage mitochondrial function and rebuild immune metabolic balance, and is of great significance for the treatment of radiation dermatitis.

[0004] To date, treatments for radiation-induced skin injuries primarily include topical medications, biomaterial dressings, cell therapy, and surgery. However, the poor chemical stability of small-molecule drugs, their susceptibility to inactivation during long-term storage, limited ability to scavenge free radicals, and significant side effects severely hinder their bioavailability and practicality. Meanwhile, growth factor and stem cell therapies are hampered by high costs, a lack of long-term safety data, and insufficient large-scale clinical trials, making large-scale adoption difficult. Currently, biomaterial treatments for radiation-induced skin injuries have garnered widespread attention; however, existing traditional gels are limited by poor adhesion, inability to form gels in situ, and insufficient mechanical strength, making them unsuitable for treating radiation-induced skin injuries. Therefore, designing a gel dressing that can form gels in situ is the best option to overcome the shortcomings of existing gels. Sodium alginate cross-linked with calcium ions is a common method for in-situ gel formation, often involving dual-network cross-linking with other gels to address the poor tissue integration and insufficient toughness of single-gel networks.

[0005] Polygonatum polysaccharides are a class of natural compounds and one of the main active ingredients of Polygonatum, exhibiting good biocompatibility. Modern pharmacology shows that they can protect cells from oxidative damage by scavenging free radicals. Furthermore, they can effectively reduce inflammation by inhibiting the secretion of pro-inflammatory factors, while also possessing immunomodulatory, mitochondrial protective, and anti-radiation effects, making them an effective candidate material for repairing radiation-induced skin damage. However, the low solubility of pure Polygonatum polysaccharides makes it difficult to meet the practical application requirements for repairing radiation-induced skin damage. Therefore, developing a hydrogel with enhanced bioactivity and optimized engineering properties would have significant clinical application value.

[0006] In this study, the invention innovatively employs sodium periodate oxidation to aldehyde-modify Polygonatum polysaccharides, improving their water solubility while simultaneously generating a Polygonatum polysaccharide hydrogel through a Schiff base reaction between the aldehyde groups of the oxidized Polygonatum polysaccharides and the amino groups on chitosan. To further enhance the mechanical and adhesive properties of the hydrogel, sodium alginate solution was added to the first network system of the Polygonatum polysaccharide hydrogel, and a double-network hydrogel was prepared through chelation crosslinking of sodium alginate with calcium ions. This material not only effectively removes excess reactive oxygen species within macrophages and restores mitochondrial membrane potential and ultrastructural integrity, but also promotes mitochondrial biosynthesis by activating the AMPK signaling pathway while inhibiting the NF-κB inflammatory cascade, thereby reprogramming immune metabolic function. Systematic validation from molecular, cellular, and animal models demonstrates that the PCA hydrogel has pleiotropic effects in alleviating oxidative stress, correcting energy metabolism disorders, and promoting tissue regeneration. This work not only provides a novel treatment strategy with clinical translational potential for radiation dermatitis but also offers new insights into the design of immunomodulatory materials based on natural polysaccharides. Summary of the Invention

[0007] The purpose of this invention is to repair radiation-induced skin damage. One of the objectives of this invention is to provide a sprayable dual-network hydrogel (PCA) and its preparation method. Compared with previously reported hydrogel dressings, it has the following advantages: (1) Aldehyde-modified Polygonatum polysaccharide has better solubility and bioavailability; (2) Aldehyde-modified Polygonatum polysaccharide and chitosan form a chemical bond to realize the preparation of Polygonatum polysaccharide hydrogel, while enhancing the mechanical properties and adhesion properties of the material; (3) Calcium ion-crosslinked sodium alginate not only helps maintain the moist environment of the wound, but the resulting dual-network portable sprayable gel also has a good effect on the repair of radiation-induced skin damage in mice.

[0008] The technical solution of the present invention: A dual-network sprayable hydrogel for repairing radiation-induced skin damage comprises cross-linked oxidized polygonatum polysaccharide (OPSP), quaternized chitosan (CS), sodium alginate (SA), and CaCl2; wherein the mass ratio of oxidized polygonatum polysaccharide (OPSP), quaternized chitosan, sodium alginate (SA), and CaCl2 is 20:25-75:1.5-12.5:1.11-11.1. Preferably, the mass ratio of Polygonatum polysaccharide OPSP, quaternized chitosan CS, sodium alginate SA and CaCl2 is 20 : 25-75 : 3.75-6.25 : 1.11-5.55.

[0009] The pore size distribution of the dual-network sprayable hydrogel is 65-75 μm; preferably 72.33 μm.

[0010] The FT-IR spectrum of the dual-network sprayable hydrogel was observed at 1662 cm⁻¹. -1 1481 cm -1 1641cm -1 and 1599cm -1 A characteristic peak exists at this location.

[0011] The pore size distribution of the dual-network sprayable hydrogel is 65-75 μm; preferably 72.33 μm.

[0012] The FT-IR spectrum of the dual-network sprayable hydrogel was observed at 1662 cm⁻¹. -1 1481 cm -1 1641 cm -1 and 1599 cm -1 A characteristic peak exists at this location.

[0013] The above-mentioned method for preparing a dual-network sprayable hydrogel includes the following steps: 1) Mix the aqueous solution of oxidized Polygonatum polysaccharide with quaternized chitosan evenly to obtain a cross-linked gel of oxidized Polygonatum polysaccharide and quaternized chitosan; The mass ratio of the oxidized Polygonatum polysaccharide to the quaternized chitosan is 20:75; 2) The cross-linked PC gel obtained in step 1) is mixed with sodium alginate solution at a volume ratio of 10:1~5 to obtain oxidized Polygonatum polysaccharide, quaternized chitosan and sodium alginate gel (SA-PC gel). 3) Spray 0.1-5 M CaCl2 solution onto the surface of the SA-PC gel to obtain a double-network sprayable hydrogel (PCA gel).

[0014] The oxidized Polygonatum polysaccharide OPSP was prepared by the following method: Under light-protected conditions, 20 mL of 0.1–5 mol / L NaIO4 solution was added dropwise to 500 mL of Polygonatum polysaccharide (PSP) aqueous solution, and the mixture was stirred at 4 °C for 2 h. Then, 5 mL of ethylene glycol was added, and the reaction was quenched by stirring for 30 min. The reaction solution was then dialyzed for 3 days and freeze-dried to obtain OPSP. The concentration of Polygonatum polysaccharide is 20~30 mg / mL.

[0015] The mass ratio of sodium periodate to Polygonatum polysaccharide is 0.3~3:5, preferably 0.3~2:5; The concentration of the sodium periodate aqueous solution is 0.1~1 mol / L, preferably 0.03 g / mL.

[0016] The concentration of the Polygonatum polysaccharide aqueous solution was 20 mg / mL.

[0017] The concentration of the sodium alginate solution is 10~30 mg / mL, preferably 15~25 mg / mL; The concentration of quaternized chitosan is 0.01~0.1 g / mL, preferably 0.025~0.075 g / mL.

[0018] The concentration of the CaCl2 solution is 0.1~1 mol / L, preferably 0.1~0.5 mol / L, and more preferably 0.1~0.2 mol / L.

[0019] The volume ratio of the cross-linked gel to sodium alginate is 10:0.25; The amount of CaCl2 solution used during spraying is 50-200 μL. Preferably, 100 μL of 0.1M CaCl2 solution is sprayed onto the surface of 1 mL of SA-PC gel. The above-described dual-network sprayable hydrogel, or the dual-network sprayable hydrogel obtained according to the above preparation method, is used in the preparation of gel wound dressings; the wound dressing is used to apply to the surface of wounds on the body surface or internal cavity.

[0020] A biocompatible dressing comprising the above-described dual-network sprayable hydrogel, or a dual-network sprayable hydrogel prepared according to the above-described preparation method.

[0021] The beneficial effects of the above-described technical solution of the present invention are as follows: 1) Aldehyde-modified Polygonatum polysaccharide undergoes a Schiff base reaction with quaternized chitosan. Compared with the simple electrostatic interaction between the two, this not only increases the solubility of Polygonatum polysaccharide but also improves the crosslinking degree of the hydrogel.

[0022] 2) Using Ca 2+ Cross-linked sodium alginate not only synergistically enhances the anti-inflammatory effects of the hydrogel but also improves its mechanical and adhesive properties. The multi-layered cross-linked network structure ensures the stability of the hydrogel's structure and mechanical properties during swelling tests. The dual-network hydrogel described in this invention integrates mechanical protection, durable adhesion, and antioxidant and anti-inflammatory functions, providing a breakthrough treatment strategy for radiation dermatitis.

[0023] 3) The sprayable hydrogel of this invention achieves in-situ dual-network cross-linking under physiological conditions through a physicochemical synergistic cross-linking mechanism, without the need for exogenous chemical cross-linking agents. The breakthrough of this invention lies in overcoming technical bottlenecks such as premixed cross-linking agents and uncontrollable gelation time. It enables rapid treatment in animal models of radiation dermatitis and significantly improves wound healing rates, providing a revolutionary solution for special scenarios such as emergency trauma and wartime rescue.

[0024] 4) In vitro cell experiments have confirmed that the sprayable hydrogel provided by this invention is non-cytotoxic, exhibits good cell proliferation and spreading effects, and promotes cell migration. The hydrogel provided by this invention can promote the growth of epidermal cells after ionizing radiation damage in vitro and protect the energy metabolism and mitochondrial morphology of macrophages damaged by ionizing radiation.

[0025] 5) The present invention further confirms through a mouse model of localized radiation dermatitis that the dual-network hydrogel provided by the present invention adheres very well to the skin surface. It can not only act as a physical barrier to protect skin wounds from secondary infection, but also the antioxidant and anti-inflammatory properties of the hydrogel can promote the restoration of local skin microenvironment homeostasis, accelerate the formation of new matrix and collagen deposition.

[0026] The hydrogel of this invention introduces aldehyde groups into Polygonatum polysaccharide via sodium periodate oxidation, causing it to chemically crosslink with the amino groups of quaternized chitosan to form a first-layer network. Simultaneously, sodium alginate is introduced into the system, and calcium ions are used to crosslink and construct a second-layer physical crosslinking network. This invention achieves instant gel formation upon injection under physiological conditions, effectively avoiding the operational complexity caused by premixed crosslinking agents and optimizing gel performance. The mechanical and adhesive properties of the prepared hydrogel are significantly improved. In vitro cell experiments and in vivo animal experiments show that this sprayable hydrogel can effectively promote cell proliferation and migration, effectively maintain macrophage mitochondrial dysfunction caused by ionizing radiation, and has good antioxidant and anti-inflammatory activities, demonstrating broad application prospects in the repair and treatment of radiation dermatitis. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the present invention, the accompanying drawings used in the description of the implementation examples will be briefly introduced below.

[0028] Figure 1 This section describes the preparation and characterization of oxidized Polygonatum polysaccharide OPSP. (a) shows the FT-IR spectra of PSP and OPSP; (b) shows the UV-Vis spectra of PSP and OPSP; and (c) shows the UV-Vis spectra of PSP and OPSP. 1 (d) is the HPLC chromatogram of the mixed monosaccharide standard and PSP and OPSP polysaccharides. Figure 2 This section describes the preparation and characterization of PC gels. (a) shows SEM images and histograms of pore size distribution of PC gels prepared with different concentrations of OPSP (1%, 1.5%, 2%, w / v); (be) shows the rheological properties of PC gels with different CS dosages: (b) gelation time, (c) viscosity, (d) shear resistance, and (e) self-healing test of PC gels in the strain range of 100%–500%. Figure 3 This section describes the preparation and characterization of PCA gels. (a) shows SEM images and histograms of pore size distribution of PCA gels with PC to SA volume ratios of 1:0.25, 1:0.5, and 1:0.75; (b) shows the viscoelasticity of the PCA gel (G' and G''); (c) shows the self-healing ability of the PCA gel within a strain range of 100%–500%; (d) shows the FT-IR spectra of SA, CS, PC gels, and PCA gel; and (e) demonstrates the adhesion properties of the PCA gel on fresh pigskin (bending, torsion, and compression).

[0029] Figure 4This is a biocompatibility analysis of PCA gels; (a) shows the CCK-8 cell viability detection results after 72 h of co-culturing HaCaT cells with different gel extracts; (c) live and dead cell staining fluorescence images of HaCaT cells treated with 40-fold diluted gel extracts for 1, 2, and 3 days, scale bar: 100 μm; (b) is the quantitative analysis of cell viability corresponding to (c); (d) cytoskeleton staining images of HaCaT cells treated with different gel extracts for 24 h, scale bar: 50 μm; (e) the hemolysis rate detection results of erythrocytes treated with PCA gel extracts at different dilutions (the inset shows the corresponding hemolysis images).

[0030] Figure 5 This is an evaluation of the radiation protection capability of PCA hydrogel for cells. (a) Representative images of cell clone formation after different treatments; (b) Representative images of cell scratch healing after different treatments; (e) Representative images of cell migration in the Transwell experiment.

[0031] Figure 6 This is an evaluation of the ROS scavenging effect of PCA hydrogel on cells. (a) Representative images of cell viability and mortality staining after different treatments and (b) quantitative analysis of viability, scale bar: 200 μm; (c) Representative fluorescence images of intracellular ROS detected by DCFH-DA staining and (d) quantitative analysis of fluorescence intensity.

[0032] Figure 7 The diagram shows the protective effect of PCA hydrogel against radiation-induced skin injury. Among them, (a) is a schematic diagram of the establishment and treatment process of the radiation-induced skin injury model; (b) photographs of wound healing in mice on day 7 and day 14 after treatment with different hydrogels; (c) quantitative analysis of wound closure rate; and (df) representative images of H&E staining, Masson trichrome staining and Sirius red staining of skin tissue sections on day 7 and day 14 after different treatments.

[0033] Figure 8 The differential gene expression analysis of radiation-induced skin injury after PCA gel intervention included: (a) gene correlation analysis among samples from the control group, IR group, and PCA hydrogel group; (b) cluster heatmap of differentially expressed genes between the PCA hydrogel group and the IR group; (cf) GSEA enrichment analysis of differentially expressed genes between the IR group and the PCA gel group; (c) GSEA enrichment analysis of TCA cycle, (d) oxidative phosphorylation, (e) NF-κB signaling pathway, and (f) AMPK signaling pathway.

[0034] Figure 9This study evaluates the reduction of oxidative stress in irradiated macrophages by PCA hydrogel. Among them, (a) is a representative image of ROS detected by DCFH-DA staining and (b) is the corresponding quantitative analysis of fluorescence intensity; (c) is the flow cytometry analysis of ROS levels in macrophages after different treatments; (d) is the detection map of ATP expression levels in macrophages after treatment in each group; and (e) is the representative TEM image of the ultrastructure of mitochondria in macrophages after different treatments.

[0035] Figure 10 The results are an evaluation of PCA hydrogel's ability to improve mitochondrial dysfunction in irradiated macrophages; (a) is a representative image of functional mitochondria (MitoTracker Red / MitoTracker Green ratio) and (b) quantitative analysis; (a) is a representative image of JC-1 staining and (c) corresponding quantitative analysis of fluorescence intensity; (a) is a representative image of mitochondrial reactive oxygen species detected by MitoSOX™ Red staining and (d) corresponding quantitative analysis of fluorescence intensity. Detailed Implementation

[0036] The following examples further illustrate the dual-network hydrogel, its preparation method, and its applications provided by the present invention. It should be noted that the following examples are only for further illustration and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the invention to implement it are still within the scope of protection of the present invention. The implementation conditions not specifically specified in the examples are generally those used in conventional experiments.

[0037] Materials and reagents: RPMI 1640 medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0038] DMEM high-glucose medium was purchased from Hyclone.

[0039] New Zealand fetal bovine serum was purchased from Hyclone (Cytiva).

[0040] The penicillin-streptomycin solution (100× bispecific antibody) was purchased from BaiSha Biotechnology Co., Ltd.

[0041] Polygonatum polysaccharide (PSP, item number S27804) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0042] NaIO (item number S104091) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0043] Quaternized chitosan (item number 850124-100g) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0044] Sodium alginate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0045] Trifluoroacetic acid (TFA) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0046] The chloroform was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0047] 1-Phenylene-3-methyl-5-pyrazolone (PMP) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0048] D2O (item number DLM-4-10X0.55) was purchased from CAMBRIDGE ISOTOPE.

[0049] The CCK-8 kit was purchased from Beyotime Biotechnology Co., Ltd.

[0050] The Calcein / PI cell viability and cytotoxicity assay kit was purchased from Beyotime Biotechnology Co., Ltd.

[0051] Crystal violet staining solution was purchased from Beyotime Biotechnology Co., Ltd.

[0052] Hoechst 33342 live cell staining solution was purchased from Beyotime Biotechnology Co., Ltd.

[0053] Sirius red staining kit was purchased from Beyotime Biotechnology Co., Ltd.

[0054] The phalloidin was purchased from Beyotime Biotechnology Co., Ltd.

[0055] The JC-1 detection kit was purchased from Beyotime Biotechnology Co., Ltd.

[0056] MitoSOX™ Red mitochondrial superoxide indicator was purchased from Beyotime Biotechnology Co., Ltd.

[0057] MitoTracker Red was purchased from Beyotime Biotechnology Co., Ltd.

[0058] MitoTracker Green was purchased from Beyotime Biotechnology Co., Ltd.

[0059] The Annexin V-FITC / PI apoptosis detection kit was purchased from Yisheng Biotechnology Co., Ltd.

[0060] The reactive oxygen species (ROS) assay kit (DCFH-DA) was purchased from Solarbio Science & Technology Co., Ltd.

[0061] Masson trichrome staining kit was purchased from Solarbio Science & Technology Co., Ltd.

[0062] Bovine serum albumin (BSA) was purchased from Sigma-Aldrich.

[0063] TRIzol was purchased from Invitrogen (Thermo Fisher Scientific).

[0064] The PrimeScript RT Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0065] The ChamQ SYBR Color qPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0066] The mouse IL-6 enzyme-linked immunosorbent assay kit was purchased from CUSABIO, USA.

[0067] The mouse TNF-α enzyme-linked immunosorbent assay kit was purchased from CUSABIO, USA.

[0068] The mouse IL-1β enzyme-linked immunosorbent assay kit was purchased from CUSABIO, USA.

[0069] The 3422 Transwell chamber (24-well plate) was purchased from Corning.

[0070] Isoflurane (product number R510) was purchased from RWD Life Sciences Co., Ltd.

[0071] Hematoxylin-eosin (HE) staining solution was purchased from Aifang Biotechnology Co., Ltd.

[0072] HaCaT cells were purchased from Beijing Beina Chuanglian Biotechnology Research Institute.

[0073] Macrophages were extracted from peritoneal macrophages of C57 mice.

[0074] BALB / c mice were purchased from the Animal Experiment Center of Army Medical University.

[0075] C57 mice were purchased from the Animal Experiment Center of Army Medical University.

[0076] Unless otherwise specified, all reagents used in this embodiment are conventional analytical grade products.

[0077] Instruments and equipment:

[0078] Example 1: Preparation and structural characterization of oxidized Polygonatum polysaccharide (OPSP) Methods: OPSP was prepared by sodium periodate oxidation. 10 g of Polygonatum polysaccharide (PSP) was dissolved in an appropriate amount of deionized water, sonicated for 30 min until completely dissolved, and the solution was brought to a final volume of 500 mL to prepare a 20 mg / mL PSP solution. 0.6 g of NaIO4 (sodium periodate) was dissolved in 20 mL of deionized water and added dropwise to the PSP solution under light-protected conditions. The mixture was stirred at 4°C for 2 h. 5 mL of ethylene glycol was added, and the reaction was quenched by stirring for another 30 min. The reaction solution was dialyzed for 3 days and then freeze-dried to obtain OPSP.

[0079] The sample structure was analyzed using FTIR spectroscopy: the dry powder sample was placed directly in the crystal window for measurement, with wavenumbers ranging from 400 to 4000 cm⁻¹. -1 The samples were scanned 32 times. The UV absorption spectra of the samples were measured using a UV-Vis spectrophotometer. OPSP and PSP were each prepared into 0.5 mg / mL solutions with deionized water, and scanned within the wavelength range of 200-400 nm. NMR spectroscopy was used to collect the data. 1 ¹H-NMR spectrum. 6 mg of OPSP (with PSP as a control) was dissolved in 0.5 mL of D₂O and scanned 16 times at room temperature, with a spectral width of 12 ppm. Monosaccharide composition was determined using pre-column derivatization-HPLC with PMP: 15 mg of PSP or OPSP was hydrolyzed in 2 M trifluoroacetic acid at 110 °C for 2 h. The hydrolysate was dried over methanol-N₂ to remove TFA. The residue was dissolved in NaOH and reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70 °C for 2 h. After chloroform extraction, the sample was analyzed using a C18 column (30 °C, 245 nm). The mobile phase was sodium phosphate buffer (pH 6.7)-acetonitrile, and the flow rate was 1 mL / min.

[0080] Results: Using FTIR, UV-Vis, 1 The structure of OPSP was characterized by 1H-NMR and PMP pre-column derivatization-HPLC. FT-IR spectroscopy ( Figure 1 As shown in a), compared to the PSP, the OPSP has a larger screen size at 3360cm. -1 The stretching vibration of OH is weakened at 1192 cm. -1 and 817cm -1 The disappearance of the CO stretching vibration peak indicates that the hydroxyl groups have been consumed and the polysaccharide side chains have undergone oxidative breakage; 2934 cm⁻¹ -1 CH peak and 1740cm -1 The C=O peak is retained at 1617 cm⁻¹. -1 With 1410cm -1 The characteristic peak of glucuronic acid is present in both. UV-Vis spectroscopy ( Figure 1As shown in b), neither PSP nor OPSP showed significant absorption in the 200-400 nm range, indicating that the content of protein and nucleic acid impurities was extremely low. 1 H-NMR spectrum ( Figure 1 As shown in c), the rhamnose (Rha) methyl proton signal at 0.97 ppm in the PSP disappeared in the OPSP, indicating that the 1→2 or 1→4 linked Rha units were oxidized and broken; the acetyl methyl signal disappeared at 1.73 ppm and 1.90 ppm, attributed to acetyl hydrolysis caused by a basic side reaction; the signal in the OPSP was significantly enhanced at 3.45 ppm, due to the enrichment of methylene protons in the glyceraldehyde structure generated by the cleavage of the ortho-trihydroxy group, indicating the presence of 1→4 / 1→6 linked glycosidic units; the anomeric proton signal at 5.2 ppm and the sugar ring proton signal at 3.9-4.0 ppm remained stable in the OPSP, confirming that the β-glucan backbone linked by 1→3 bonds remained intact. Monosaccharide composition analysis ( Figure 1 Figure d) shows that glucose (Glc) is the main monosaccharide in both PSP and OPSP, with molar percentages of 82.96% and 70.17%, respectively. Furthermore, changes in the proportions of mannose (Man, 2.86%→2.92%), galacturonic acid (GalUA, 2.92%→7.64%), galactose (Gal, 4.73%→10.29%), and arabinose (Ara, 3.09%→5.55%) were observed between the two. In summary, OPSP retains the main structure of PSP, with oxidation primarily occurring in the side chains, while the main chain β-glucan backbone remains intact.

[0081] Example 2: Preparation and structural characterization of PC hydrogel Methods: To prepare hydrogels crosslinked with oxidized Polygonatum polysaccharide and quaternized chitosan, different masses of OPSP were dissolved in 1 mL of deionized water to prepare homogeneous OPSP solutions with mass concentrations of 1%, 1.5%, and 2%. Then, 75 mg of quaternized chitosan was added to each solution and mixed by high-speed vortexing (350 rpm / min-400 rpm / min) in a vortex mixer to form PC gels. After successfully preparing PC (1%), PC (1.5%), and PC (2%) containing different concentrations of OPSP, the optimal pore size distribution of the gels was confirmed by scanning electron microscopy. After testing, 2% (w / v) OPSP was selected as the optimal concentration for gel preparation. Different masses of quaternized chitosan (0.05 g, 0.075 g, 0.1 g) were added to 1 mL of OPSP solution containing 2% (w / v) and PC gels were prepared under the same vortex conditions. After testing the gelation time, viscosity, maximum shear force and self-healing properties with a rheometer, it was finally determined that adding 75 mg of quaternized chitosan to 1 mL of OPSP solution was the optimal ratio for preparing PC gels.

[0082] Results: PC hydrogels were prepared via a Schiff base reaction between the amino groups of quaternized chitosan (CS) and the aldehyde groups of OPSP. First, the amount of CS was fixed, and the OPSP concentrations (1%, 1.5%, 2%, w / v) were screened. SEM images ( Figure 2 As shown in Figure a), the gel prepared with 2% OPSP has a uniform pore structure and a small pore size distribution (69.27 μm), therefore 2% OPSP was chosen for subsequent experiments. Further rheological performance evaluation determined the amount of CS required. The results showed that adding 75 mg of CS per milliliter of 2% OPSP solution resulted in a gel with ideal gelation time, suitable viscosity, and shear resistance. Figure 2 (middle bd). Furthermore, although the PC gel prepared under these conditions does not possess self-healing ability, it exhibits excellent toughness within the 100%-500% strain range ( Figure 2 (e).

[0083] Example 3: Preparation and structural characterization of a dual-network sprayable hydrogel (PCA) Methods: Based on the successful preparation of PC gel, different volumes (0.1, 0.25, 0.5 mL) of sodium alginate (SA) solution (15 mg / mL) were added to 1 mL of PC gel system (0.075 g of quaternized chitosan added to 2% PSP). After thorough mixing in a vortex mixer (350 rpm / min-400 rpm / min), approximately 100 μL of 0.1 MCaCl2 solution was sprayed onto the gel surface to obtain a double-network sprayable hydrogel. The optimal amount of SA added to the PC gel was then confirmed using SEM. After freeze-drying, the microstructure of the hydrogel was observed using scanning electron microscopy at an accelerating voltage of 5 kV. The freeze-dried PC, PCA, and SA hydrogels were cut into small pieces, sputter-coated with gold for 15 s, and observed. The pore size distribution was quantitatively analyzed using ImageJ software. Simultaneously, the rheological properties of the hydrogel were tested using a Haake MARS rheometer. The hydrogel was placed between parallel plates with a diameter of 10 mm and a gap of 1 mm, and measurements were performed at 25°C. Elastic modulus (G') and viscous modulus (G'') were measured over a 60-s time scan at 1 Hz to assess gelation time. G' and G'' were measured over a 0-100% strain scan at 1 Hz to assess gel viscosity. The critical point of the hydrogel was tested by strain amplitude scanning. The self-healing ability of the hydrogel was assessed by alternating scans at 1% and 500% strain (switching every 20 s). The adhesion properties of the PCA hydrogel were tested using fresh pigskin of different shapes. The in vitro swelling characteristics of the hydrogel were determined using the weight difference method. The initial mass (W0) of the fresh hydrogel was weighed and immersed in PBS buffer at 37°C. The hydrogel was removed at different time points, the surface moisture was absorbed with filter paper, and the mass (Wt) was weighed. This process was repeated until the gel mass stabilized.

[0084] Results: To further improve the performance of PC gel, a sodium alginate (SA) and calcium ion gel system was introduced to prepare PCA hydrogel. When the volume ratio of PC to SA was 1:0.25, the SEM image of the PCA gel showed that its pore size distribution was uniform (72.33 μm), similar to that of the PC gel. Figure 3 (a) Rheological tests show that the PCA gel has suitable viscoelasticity (G'>G'') Figure 3 (b), and successfully achieved self-healing capability within a strain range of 100%-500% ( Figure 3 (c) Therefore, a PC to SA volume ratio of 1:0.25 was chosen for subsequent experiments. FT-IR spectroscopy confirmed the successful construction of the hydrogel network: the characteristic peak of CS was located at 1662 cm⁻¹. -1 (C=O stretching vibration) and 1481cm -1 (CH bending vibration) Figure 3(d) PC gel at 1641cm -1 A new peak appeared at [a certain point], attributed to the C=N stretching vibration, confirming the occurrence of the Schiff base reaction; a peak at 1599 cm⁻¹ was further observed in the PCA gel. -1 The characteristic peak at the [location] corresponds to the asymmetric stretching vibration of the carboxylate group (-COO-) in SA, confirming the successful introduction of SA into the network structure. This gel exhibits excellent extensibility on fresh pigskin and maintains strong adhesion under bending, torsion, and compression conditions. Figure 3 (e) This strong adhesion ensures that the gel adheres closely to the wound, helping to maintain a moist environment and prevent bacterial invasion.

[0085] Example 4 Biocompatibility analysis of PCA gel Methods: The toxicity of the hydrogel to HaCaT cells was detected using the CCK-8 assay. Unless otherwise specified, HaCaT cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (New Zealand fetal bovine serum) and 1% penicillin-streptomycin solution (100X penicillin-streptomycin solution). 200 μL of the hydrogel sample was immersed in 30 mL of serum-free RPMI 1640 medium and incubated at 4 ℃ for 12 h to obtain the extract. HaCaT cells were then cultured at 5 × 10⁻⁶ cells / mL. 3 HaCaT cells were seeded at a density of 1 cell / well in 96-well plates and cultured overnight. Then, 100 μL of filtered, sterile gel extraction medium was added, and the cells were cultured for 1, 2, and 3 days. After incubation for 2 h, 10 μL of CCK-8 working solution was added, and the absorbance at 450 nm was measured to calculate cell viability. Cell viability was assessed using the Live / Dead staining method (Calcein / PI cell viability and cytotoxicity assay kit): HaCaT cells were seeded in 24-well plates, and after culturing for 1, 2, and 3 days with 500 μL of gel extraction medium, they were stained with serum-free medium containing 2 μM calcein-AM and 4 μM PI for 30 min. Cell viability was observed under a fluorescence microscope. The cytoskeleton was observed using rhodamine-labeled phalloidin staining: HaCaT cells were co-cultured with gel extract for 24 h, fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, blocked with 2% BSA (bovine serum albumin), and incubated overnight at 4°C with rhodamine-labeled phalloidin. The nuclei were stained with Hoechst (Hoechst 33342 live cell staining solution) and observed under a confocal microscope. Blood compatibility was evaluated using a hemolysis test: Mouse erythrocytes were incubated at 37°C for 2 h with physiological saline (positive control), deionized water (negative control), and different volumes of PCA gel extract, respectively. The supernatant was centrifuged, and the absorbance at 540 nm was measured to calculate the hemolysis rate.

[0086] Results: Excellent biocompatibility is a key prerequisite for the clinical application of hydrogel wound dressings. To assess the biosafety of the hydrogel, cell viability was first examined after co-culturing cells with different concentrations of gel extract for 72 h. The results showed that the CCK-8 value of the 40-fold diluted gel extract group was comparable to that of normal cells after 72 h of culture. Figure 4 (a) Therefore, this concentration was chosen for subsequent cell experiments. Under these co-culture conditions, the cell viability remained above 90% within 72 h. Figure 4 (b, c), and the cells still maintained their characteristic morphology after 24 h of culture ( Figure 4 (d). Furthermore, the hemocompatibility of the PCA hydrogel was assessed by co-incubating erythrocytes with serially diluted PCA gel extract. The results showed that the PCA gel extract, when mixed with an equal volume of erythrocyte suspension, exhibited excellent erythrocyte protection, with a hemolysis rate of less than 3% (d). Figure 4 (e). In summary, this study successfully prepared a PCA hydrogel with good biocompatibility.

[0087] Example 5: Evaluation of the in vitro radiation damage protection effect of PCA gel Methods: The effect of hydrogels on cell proliferation was assessed using a colony formation assay. HaCaT cells were seeded at a density of 400 cells / well in 6-well plates and cultured overnight. Afterward, they were treated with fresh RPMI 1640 medium containing SA, PC, or PCA gel extracts, or PBS (control) for 24 h, followed by irradiation with 4 Gy X-rays. After 5 days of further culture, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and the colony count and viability were calculated. Cell migration ability was assessed using a scratch assay. HaCaT cells were seeded at a density of 3 × 10⁶ cells / well. 5 HaCaT cells were seeded at a density of 3 × 3 × 10⁶ cells / well in 6-well plates. After treatment with different gel extraction solutions for 24 h to form a monolayer, the cells were irradiated with 30 Gy X-rays. Cross-shaped wounds were made on the monolayer cells using a sterile 200 μL pipette tip. After washing with PBS to remove exfoliated cells, the cells were cultured again. Images were taken at 0, 18, and 30 h, and the wound area and healing rate were calculated using ImageJ software. Transwell assays (3422 Transwell chambers, 24-well plates) were used to further evaluate cell migration ability. HaCaT cells were treated with different gel extraction solutions for 24 h and then seeded at a density of 3 × 3 × 10⁶ cells / well. 4HaCaT cells were seeded at a density of 1 cell / well in the upper layer of Transwell chambers of 24-well plates (containing serum-free medium). The lower layer was treated with 800 μL of RPMI 1640 medium containing the appropriate gel extraction solution. After 15 h of migration, the chambers were removed, fixed with 4% paraformaldehyde for 30 min, stained with crystal violet, and the number of migrated cells was counted and the migration rate was calculated. Apoptosis was assessed using the Annexin V-FITC / PI double staining method (V-FITC / PI apoptosis detection kit). HaCaT cells were seeded in 24-well plates, treated with different gel extraction solutions for 24 h, and then irradiated with 30 Gy X-rays. After 48 h of culture, cells were collected by trypsin digestion with EDTA-free enzyme, washed with PBS, and then incubated with 100 μL binding buffer, 5 μL Annexin V-FITC, and 10 μL PI staining solution at room temperature in the dark for 15 min. The proportion of apoptotic cells was detected by flow cytometry. Cell viability after radiation was further assessed using Live / Dead staining (Calcein / PI cell viability and cytotoxicity detection kit). Cell treatment and irradiation conditions were the same as above. Live and dead cells were stained 48 hours after irradiation, observed under a fluorescence microscope, and analyzed using ImageJ software. Intracellular ROS levels were detected using the DCFH-DA fluorescent probe (Reactive Oxygen Spectroscopy ROS Assay Kit (DCFH-DA)). HaCaT cells were irradiated at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured overnight. The culture medium was then replaced with gel extract medium and cultured for another 24 h. Subsequently, the cells were irradiated with 30 Gy X-rays (dose rate 1 Gy / min). 24 h after irradiation, serum-free RPMI 1640 medium containing 1 μL / mL DCFH-DA was added, and the cells were incubated at 37°C for 30 min. After washing with PBS, ROS fluorescence intensity was observed using a confocal laser scanning microscope, and ROS levels were quantitatively detected using flow cytometry (FITC channel). 10,000 cells were collected from each sample for analysis.

[0088] Results: The radiation protection effect of PCA hydrogel was evaluated using a clonal formation assay. Figure 5 (a). The results showed that, compared with the SA treatment group, PC and PCA hydrogels significantly restored the clonogenic ability of radiation-damaged cells. Cell scratch assays and Transwell assays were used to assess cell migration ability after 30 Gy X-ray irradiation. The results showed that, compared with the control group, the PC and PCA treatment groups effectively promoted cell migration within 30 h. Figure 5 (b); Transwell experiments further confirmed that both methods can significantly restore the migration ability of radiation-damaged cells. Figure 5 (c) Cell viability assays showed that PC and PCA gels effectively protected cells from radiation-induced cytotoxicity, with cell viability in the treated groups remaining above 80%. Figure 6 (a, b). The above results indicate that PCA hydrogel has multiple radiation protection effects, including enhancing colony formation ability, promoting cell migration, and inhibiting apoptosis.

[0089] Excessive production of reactive oxygen species (ROS) is not only a major factor in ionizing radiation-induced cell damage, but also a key reason why the healing process of radiation-induced skin injuries is more complex than that of other wounds. Based on DPPH and ABTS measurements, the ability of PSP-based hydrogels to scavenge radiation-induced intracellular ROS accumulation was further evaluated. Intracellular ROS levels were quantitatively detected using the DCFH-DA fluorescent probe. Results showed that, compared with the PC and PCA treatment groups, cells in the SA treatment group exhibited higher fluorescence intensity after X-ray irradiation (…). Figure 6 (c, d) indicates that the ROS level is elevated. PC and PCA hydrogels significantly reduce radiation-induced ROS production and effectively protect cells from ROS-mediated oxidative damage.

[0090] Example 6: Evaluation of the in vivo radiation damage protection effect of PCA gel Methods: A radiation-induced skin injury model was established using male BALB / c mice (7-8 weeks old, 20-22 g) to evaluate the therapeutic effect of hydrogel. After isoflurane inhalation anesthesia, the hair on the back was shaved, and a 2 cm × 2 cm irradiation area was marked. Mice were fixed on foam boards and irradiated with 40 Gy X-rays at a dose rate of 2 Gy / min. On day 7 post-irradiation, mice were randomly divided into 5 groups: control group, SA group, PC group, PCA group, and Kangfuxin liquid positive control group. Local treatment was administered to the wound every other day for 14 days. The wounds were observed and photographed at specific time points, and the wound closure rate of each group was quantitatively analyzed using ImageJ software, and normalized to the wound size on day 7 post-irradiation. Skin tissue samples from the irradiated area were collected on days 7 and 14 after the start of treatment, fixed or cryopreserved for subsequent analysis. Skin tissue samples from the irradiated area were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, and then prepared into 2.5 μm thick paraffin sections. Histological evaluation was performed using hematoxylin-eosin (H&E) staining (Hematoxylin-eosin (HE) staining solution); collagen deposition was observed using Masson's trichrome staining kit; and collagen typing was performed using Sirius red staining kit. Stained sections were imaged using a slide scanner, and quantitative analysis was performed using ImageJ software.

[0091] Results: After confirming the good radiation protection effect of PSP-based hydrogels in vitro, this invention further evaluated the therapeutic effect of PCA gels in a Balb / c mouse radiation-induced skin injury model. Mice were treated with hydrogels one week after receiving 40 Gy X-ray irradiation, with topical administration every other day. Wound skin tissue was collected on days 7 and 14 post-treatment for further analysis. Figure 7 (a) A commercially available Kangfuxin liquid treatment group was used as a positive control. Results showed that both the PC and PCA treatment groups exhibited significant wound closure on day 7 post-treatment, with healing rates exceeding 50%. Figure 7 The levels of B and C were significantly higher than those in the control group and the SA treatment group. Simultaneously, H&E staining showed that granulation tissue formation and re-epithelialization were essentially complete in the PC and PCA groups by day 7 post-treatment. Figure 7 (d). Furthermore, the PC and PCA hydrogel treatment groups showed significant collagen deposition, with a distribution pattern more closely resembling that of normal skin after treatment. Figure 7 (e). Sirius red staining showed significant type III collagen deposition in the PCA treatment group ( Figure 7 The results (f) indicate that it has an excellent ability to promote normal tissue remodeling and regeneration after treatment. Notably, H&E staining suggests that the PCA group formed a thinner epithelial layer and more new skin appendages compared to the PC group. This observation may be attributed to the structural modification of the PSP-based hydrogel by sodium alginate, making it more conducive to promoting functional skin repair.

[0092] Example 7 Transcriptome Sequencing and Analysis Methods: Samples for transcriptome sequencing were obtained from dorsal skin wound tissue of mice in the normal control group, irradiation group, and PCA hydrogel treatment group. Wound tissue was collected using sterile surgical instruments, rinsed with pre-cooled PBS, gently blotted dry with sterile filter paper, immediately flash-frozen in liquid nitrogen, and transported on dry ice to Shanghai Meiji Biotechnology Co., Ltd. for total RNA extraction, library construction, and eukaryotic transcriptome sequencing. After sequencing, differentially expressed genes were screened by statistical analysis of expression levels. Subsequently, GSEA functional enrichment analysis was performed to determine changes in cellular components, molecular functions, biological processes, and signaling pathways. This study focused on analyzing differentially expressed genes and their pathway enrichment related to inflammation regulation, mitochondrial biogenesis, and metabolic function.

[0093] Results: Based on the protective effect of PCA hydrogel against ionizing radiation damage confirmed by in vitro and in vivo experiments, this invention further employed transcriptomics technology to analyze mouse skin tissue treated with PCA hydrogel for 14 days to elucidate the potential mechanism of action of PCA gel. Skin samples from the normal skin group, IR group, and PCA group were collected for RNA sequencing. PCA correlation analysis showed a high degree of consistency in gene expression among samples within each group (…). Figure 8 (a). Meanwhile, the heatmap showed that the difference in gene expression between the normal skin group and the PCA group was smaller than that between the IR group ( Figure 8 (b) indicates that PCA treatment induced significant transcriptomic changes compared to IR irradiation. Furthermore, gene set enrichment analysis was used to assess activated and repressed signaling pathways ( Figure 8 (cf). The results showed that oxidative phosphorylation and the tricarboxylic acid cycle pathway were significantly enriched in the PCA group. In addition, compared with the IR group, the PCA treatment group significantly activated the AMPK signaling pathway and inhibited the NF-κB signaling pathway.

[0094] Example 8: PCA hydrogel protects macrophages from radiation damage by restoring mitochondrial homeostasis. Methods: Hydrogel toxicity was assessed using live / dead staining: Macrophages were seeded in 24-well plates and co-cultured with DMEM high-glucose medium containing gel extract for different time periods, followed by staining and observation. The culture medium used for culturing macrophages in this invention was DMEM high-glucose medium containing 10% fetal bovine serum (New Zealand fetal bovine serum) and 1% penicillin-streptomycin solution (100X penicillin-streptomycin solution). Anti-inflammatory effect evaluation: Macrophages were treated with different gel extracts for 24 h and then irradiated with 4 Gy X-rays. After another 24 h of culture, they were washed twice with pre-cooled PBS buffer and lysed with TRIzol. Total RNA was extracted and reverse transcribed into cDNA using PrimerScript RT Mix. Gene expression quantification was performed using ChamQ SYBR Color qPCR Master Mix on a Bio-Rad real-time quantitative PCR system. Primer sequences are shown in Table S2. Actin was used as an internal reference gene, and gene expression levels were calculated using the 2-ΔΔCt method. Simultaneously, cell culture supernatant was collected, and the secretion level of inflammatory factors was detected using enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. Intracellular ROS scavenging capacity was evaluated: the ability of cells to scavenge ROS was assessed using the DCFH-DA fluorescent probe. Macrophages were stained with DCFH-DA and then observed using fluorescence microscopy and flow cytometry. The specific experimental methods were consistent with the aforementioned HaCaT cell experiments, but ROS detection was performed 12 h after 4 Gy irradiation. Mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) levels were detected: mitochondrial membrane potential and ROS levels were assessed using the JC-1 assay kit (Mitochondrial Membrane Potential Assay Kit (JC-1)) and the MitoSOX™ Red mitochondrial superoxide indicator, respectively. Macrophages were seeded in confocal culture dishes, treated with different gel extraction solutions, irradiated with 4 Gy, and cultured for 24 h. After washing with PBS, JC-1 working solution (diluted 1:400 in serum-free medium) was added, and the cells were incubated at 37°C for 20 min. Discard the staining solution, wash twice with PBS, and stain the nuclei with Hoechst 33342 for 5 min. JC-1 monomers showed green fluorescence, and JC-1 aggregates showed red fluorescence. For mitochondrial reactive oxygen species detection, cells were incubated with MitoSOX™ Red (1:3000 dilution) at 37°C for 20 min. Fluorescence images were acquired using a confocal laser scanning microscope, and quantitative analysis was performed using ImageJ software. Mitochondrial ultrastructure observation: The ultrastructure of mitochondria was observed using a transmission electron microscope. Macrophages were treated with different gel extraction solutions, and cells were collected 24 h after irradiation. They were fixed overnight at 4°C with 2.5% glutaraldehyde. TEM detection was performed using eTest Technology Co., Ltd., following the steps: after washing with PBS, fixation with 1% osmium tetroxide at room temperature in the dark for 2 h.After washing twice with deionized water, the sections were dehydrated with graded ethanol, permeated with acetone, and embedded in epoxy resin. Ultrathin sections of 50-80 nm were prepared using an ultramicrotome and stained with uranium acetate and lead citrate. Mitochondrial morphology was observed using transmission electron microscopy.

[0095] Results: An in vitro model of macrophage X-ray radiation injury was established to evaluate the radioprotective effect and related mechanisms of PCA gel. The intracellular ROS scavenging capacity of PCA hydrogel was assessed using the fluorescent probe DCFH-DA. Immunofluorescence staining ( Figure 9 (a, b) and flow cytometry analysis ( Figure 9 Both (c) and (d) indicate that the PCA treatment group significantly reduced ROS levels in macrophages after irradiation, demonstrating its protective effect against radiation-induced oxidative damage. Simultaneously, this invention observed that PCA hydrogel can protect macrophages from radiation-induced ATP depletion (…). Figure 9 (d). Studies have shown that mitochondria are the energy factories of cells, maintaining cellular function by producing ATP. However, dysfunctional mitochondria produce excessive amounts of ROS, exacerbating damage and creating a vicious cycle. Given that PCA hydrogel can scavenge ROS and maintain ATP levels, this invention hypothesizes that it can protect macrophages from radiation-induced mitochondrial oxidative stress and dysfunction. Furthermore, transmission electron microscopy was used to assess changes in macrophage mitochondrial structure after irradiation. The results showed that radiation caused severe damage to the ultrastructure of mitochondria (d). Figure 9 The main manifestations of mitochondrial dysfunction (MDMA) were membrane rupture, cristae loss, and swelling. In contrast, the PC and PCA treatment groups maintained double-membrane integrity and cristae morphology, and the number of mitochondria increased, indicating their ability to protect mitochondrial structure. To assess whether mitochondrial dysfunction occurred after ionizing radiation, this invention used MitoTracker Red (a functional mitochondrial marker) and MitoTracker Green (a total mitochondrial marker) for immunofluorescence staining. The results showed that radiation significantly reduced the fluorescence ratio of MitoTracker Red to MitoTracker Green (MRF). Figure 10 (a, b) indicates functional mitochondrial depletion after irradiation. In contrast, the PC and PCA treatment groups maintained this ratio, indicating that they can alleviate mitochondrial membrane potential depletion and maintain functional integrity, thereby maintaining cellular energy and redox homeostasis under irradiation conditions. Consistent with these results, JC-1 staining confirmed that the PCA treatment group maintained a higher red / green fluorescence ratio than other groups ( Figure 10(a, c) indicates the stability of the mitochondrial membrane potential. Given that a stable mitochondrial membrane potential is crucial for maintaining normal mitochondrial function, and its disruption leads to electron transport chain leakage and increased ROS, this invention further examined the level of mitochondrial reactive oxygen species (ROS). MitoSOX Red staining confirmed that PCA treatment effectively removed excess mitochondrial ROS after irradiation. Figure 10 (a, d). This result indicates that PCA gel can break the vicious cycle of ROS and mitochondrial damage, providing direct evidence of redox levels for mitochondrial protection.

[0096] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A dual-network sprayable hydrogel for repairing radiation-induced skin damage, characterized in that: It contains cross-linked oxidized Polygonatum polysaccharide, quaternized chitosan, sodium alginate and CaCl2; wherein the mass ratio of oxidized Polygonatum polysaccharide, quaternized chitosan, sodium alginate and CaCl2 is: 20:25-75:1.5-12.5:1.11-11.1; Preferably, the mass ratio of Polygonatum polysaccharide, quaternized chitosan, sodium alginate and CaCl2 is 20 : 25-75 : 3.75-6.25 : 1.11-5.

55.

2. The dual-network sprayable hydrogel as described in claim 1, characterized in that: The pore size distribution of the dual-network sprayable hydrogel is 65-75 μm; preferably 72.33 μm.

3. The dual-network sprayable hydrogel as described in claim 1 or 2, characterized in that: The FT-IR spectrum of the dual-network sprayable hydrogel was observed at 1662 cm⁻¹. -1 1481 cm -1 1641 cm -1 and 1599cm -1 A characteristic peak exists at this location.

4. The method for preparing a dual-network sprayable hydrogel as described in any one of claims 1-3, characterized in that, Includes the following steps: 1) Mix the aqueous solution of oxidized Polygonatum polysaccharide with quaternized chitosan evenly to obtain a cross-linked gel of oxidized Polygonatum polysaccharide and quaternized chitosan; The mass ratio of the oxidized Polygonatum polysaccharide to the quaternized chitosan is 20:75; 2) The cross-linked PC gel obtained in step 1) is mixed with sodium alginate solution at a volume ratio of 10:1~5 to obtain oxidized Polygonatum polysaccharide, quaternized chitosan and sodium alginate gel (SA-PC gel). 3) Spray 0.1-5 M CaCl2 solution onto the surface of the SA-PC gel to obtain a double-network sprayable hydrogel (PCA gel).

5. The preparation method according to claim 4, characterized in that, The oxidized Polygonatum polysaccharide was prepared using the following method: Under light-protected conditions, 20 mL of NaIO4 solution with a concentration of 0.1~5 mol / L was added dropwise to 500 mL of Polygonatum polysaccharide aqueous solution, and the mixture was stirred at 4 ℃ for 2 h. Then, 5 mL of ethylene glycol was added, and the reaction was quenched by stirring for 30 min. The reaction solution was then dialyzed for 3 days and freeze-dried to obtain oxidized Polygonatum polysaccharide. The concentration of Polygonatum polysaccharide is 20~30 mg / mL.

6. The preparation method according to claim 5, characterized in that, The mass ratio of sodium periodate to Polygonatum polysaccharide is 0.3~3:5, preferably 0.3~2:5; The concentration of the sodium periodate aqueous solution is 0.1~1 mol / L, preferably 0.03 g / mL; The concentration of the Polygonatum polysaccharide aqueous solution was 20 mg / mL.

7. The preparation method according to claim 4, characterized in that, The concentration of the sodium alginate solution is 10~30 mg / mL, preferably 15~25 mg / mL; The concentration of quaternized chitosan is 0.01~0.1 g / mL, preferably 0.025~0.075 g / mL; The concentration of the CaCl2 solution is 0.1~1 mol / L, preferably 0.1~0.5 mol / L, and more preferably 0.1~0.2 mol / L.

8. The preparation method according to claim 4, characterized in that, The volume ratio of the cross-linked gel to sodium alginate is 10:0.25; The amount of CaCl2 solution used during spraying is 50-200 μL. Preferably, 100 μL of 0.1M CaCl2 solution is sprayed onto the surface of 1 mL of SA-PC gel.

9. The use of the dual-network sprayable hydrogel as described in any one of claims 1-3, or the dual-network sprayable hydrogel obtained by the preparation method according to any one of claims 4-8, in the preparation of gel wound dressings; the wound dressings are used to be applied to the surface of wounds on the body surface or the surface of wounds in internal cavities.

10. A biocompatible dressing comprising a dual-network sprayable hydrogel as described in any one of claims 1-3, or a dual-network sprayable hydrogel prepared by the preparation method as described in any one of claims 4-8.

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