Selenium-containing nanoparticles, their preparation methods, and their application in drugs for treating inflammatory mucosal diseases

Abstract

This application relates to the field of nanomedicine technology, and in particular to a selenium-containing nanoparticle, its preparation method, and its application in drugs for treating inflammatory mucosal diseases. The selenium-containing nanoparticle has a core comprising zero-valent selenium or ferrous selenide, and a shell comprising at least one of polyserotonin, dopamine, and sodium alginate; the two are bonded together by C-Se covalent bonds to form a stable organic-inorganic nanocomposite. This selenium-containing nanoparticle overcomes the bottlenecks of traditional selenium nanoparticles, such as easy aggregation and poor retention, and achieves synergistic enhancement of antioxidant, anti-inflammatory, and antibacterial functions. When applied to the treatment of inflammatory mucosal diseases, it can effectively treat inflammatory mucosal diseases in complex environments, such as oral mucositis, based on the "seed-soil" synergistic treatment concept, by synergistically regulating the triple mechanism of "epithelial cells (seeds)," "immune microenvironment (soil)," and "local flora."

Description

[0001] This application claims priority to Chinese Patent Application No. 2025120311711, filed on December 30, 2025, entitled "Selenium-containing nanoparticles and their preparation method and their application in a drug for treating inflammatory mucosal diseases", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of nanomedicine technology, and in particular to a selenium-containing nanoparticle, its preparation method, and its application in drugs for treating inflammatory mucosal diseases. Background Technology

[0003] Oral mucositis (OM) is a common inflammatory condition with complex causes, including radiotherapy, chemotherapy, infection, medications, and trauma. Its incidence remains high in the elderly and immunocompromised individuals, severely hindering the treatment of underlying diseases and significantly reducing patients' quality of life. The core pathological contradiction of OM lies in the vicious cycle formed by the persistent destruction of the mucosal epithelial barrier and the uncontrolled local inflammatory response, clinically manifesting as mucosal redness, swelling, erosion, or ulceration. These symptoms not only cause persistent and severe pain, leading to swallowing difficulties and nutritional intake problems, but also greatly increase the risk of secondary or systemic infections, making it a challenging clinical problem.

[0004] Despite the emergence of various OM treatment strategies in recent years, most have failed to break free from the traditional "single-target, linear intervention" model, making it difficult to reverse the disease progression at a systemic level. Specifically: in terms of antioxidant therapy, while N-acetylcysteine ​​and superoxide dismutase mimics can scavenge some reactive oxygen species, they cannot effectively prevent the inactivation of the key antioxidant enzyme GPX4 and its mediated ferroptosis pathway; in the field of immunomodulation, while glucocorticoids such as dexamethasone or tumor necrosis factor-α signaling blockers can suppress inflammatory responses in the short term, they inhibit the repair function of the immune system and may even increase the risk of secondary infections; in terms of anti-ferroptosis, although specific inhibitors (such as Ferrostatin-1) can directly block the ferroptosis process by scavenging lipid free radicals, the effectiveness of such single-step interventions is often weakened by continuous oxidative stress and immune imbalance. Overall, existing strategies treat the complex, multi-dimensional pathological network of OM as isolated links, neglecting the self-reinforcing closed loop formed between "oxidative stress—ferroptosis—immune imbalance," thus leading to a clinical dilemma of "treating the symptoms but not the root cause." Therefore, developing an integrated synergistic treatment system that can regulate the "seed-soil" process and simultaneously achieve ferroptosis blocking, oxidative damage clearance, and immune microenvironment remodeling has become an urgent need and an inevitable direction for breaking through the current predicament of OM treatment.

[0005] The trace element selenium (Se) has attracted our attention due to its remarkable ability to inhibit ferroptosis and resist oxidative stress. Selenium is an essential cofactor for the glutathione peroxidase (GPX) family, especially GPX4 (a key negative regulator of ferroptosis in cells). Therefore, selenium not only plays a fundamental role in maintaining cellular redox homeostasis, but also plays an irreplaceable physiological role in directly inhibiting ferroptosis and regulating immune inflammatory responses. However, traditional selenium supplements (such as sodium selenite) suffer from low bioavailability, narrow therapeutic window, and systemic toxicity risks. Even improved nano-selenium (SeNPs) still face bottlenecks such as easy aggregation and inactivation in the physiological environment, short retention time on the oral mucosa, and limited function, making it difficult to synergistically regulate multiple pathological networks and meet the multidimensional treatment needs of ophthalmic fibrosis (OM). Summary of the Invention

[0006] Therefore, it is necessary to provide selenium-containing nanoparticles that can remain in the lesion environment such as the oral cavity for a longer period of time and have a good therapeutic effect on inflammation; and further, to provide a method for preparing the selenium-containing nanoparticles, as well as the application of the above-mentioned selenium-containing nanoparticles in the preparation of drugs for the prevention and / or treatment of inflammatory mucosal diseases.

[0007] First, this application provides a selenium-containing nanoparticle, comprising a core composed of a selenium-containing material and a biomimetic adhesive polymer shell coating the surface of the core, wherein the selenium-containing material comprises at least one of zero-valent selenium and ferrous selenide; and the biomimetic adhesive polymer comprises at least one of polyserotonin, dopamine, and sodium alginate.

[0008] It is understandable that the aforementioned zero-valent selenium is also elemental selenium.

[0009] Preferably, the selenium-containing substance is bonded to the biomimetic adhesive polymer via C-Se covalent bonds. This C-Se covalent bonding enhances the material's structural stability, enabling it to resist saliva erosion and achieve effective retention at the ulcer site.

[0010] Preferably, the nanoparticles are spherical.

[0011] Preferably, the particle size of the nanoparticle core is 10~20nm.

[0012] Preferably, the hydrated particle size of the nanoparticles is 80-110 nm. Controlling the particle size of the nanoparticles can give them excellent colloidal stability, mucosal permeability, and dispersibility.

[0013] Preferably, the zeta potential of the nanoparticles is from -10mV to 0mV.

[0014] Nanoparticles with the above-mentioned particle size and potential value can maintain better nanoparticle mucosal retention, colloidal stability and biological activity.

[0015] Preferably, based on the total mass of the nanoparticles as 100%, the carbon content in the nanoparticles is 30%~40%, and the selenium content is 20%~25%.

[0016] Based on a general inventive concept, this application also provides a method for preparing selenium-containing nanoparticles, comprising the following steps:

[0017] The monomer of the biomimetic adhesive polymer, sodium selenite, and ferric ion salt were dissolved in water and stirred for 30-40 minutes. The pH was adjusted to 7.5-9.5, and a hydrothermal reaction was carried out at 60-100℃ for 2-6 hours. After the reaction was completed, the mixture was centrifuged and freeze-dried to obtain the selenium-containing nanoparticles. The molar ratio of the monomer of the biomimetic adhesive polymer to sodium selenite was (2-3):1.

[0018] Preferably, the stirring reaction rate is 60~100 r / min.

[0019] Preferably, the centrifugation speed is 8000~16000 r / min and the centrifugation time is 10~15 min.

[0020] Preferably, the freeze-drying temperature is -60°C to -50°C, and the freeze-drying time is 24 to 36 hours.

[0021] Preferably, the monomer of the biomimetic adhesive polymer is serotonin; in the hydrothermal reaction, the initial concentration of serotonin is 0.5~1 mmol / L, and the initial concentration of sodium selenite is 0.2~0.5 mmol / L.

[0022] Based on a general inventive concept, this application also provides the use of the above-described selenium-containing nanoparticles in the preparation of medicaments for the prevention and / or treatment of inflammatory mucosal diseases.

[0023] Preferably, the inflammatory mucosal disease includes at least one of oral mucositis, esophagitis, gastritis, and ulcerative colitis.

[0024] Preferably, the drug comprises the above-mentioned selenium-containing nanoparticles and pharmaceutically acceptable excipients.

[0025] Preferably, the selenium-containing nanoparticles have a mass content of 20% to 40% based on the total mass of the drug (100%).

[0026] Preferably, pharmaceutically acceptable excipients include, but are not limited to, fillers, flavoring agents, and binders.

[0027] Preferably, pharmaceutically acceptable excipients include maltodextrin, steviol glycosides, and hydroxypropyl methylcellulose.

[0028] Preferably, the dosage form of the drug is a gel, spray, mouthwash, or patch.

[0029] The selenium-containing nanoparticles provided in this application have a core comprising zero-valent selenium or ferrous selenide, and a shell comprising at least one of polyserotonin, dopamine, and sodium alginate; the two are bound together by C-Se covalent bonds to form a stable organic-inorganic nanocomposite. When applied to the treatment of inflammatory mucosal diseases, they can exert a therapeutic effect based on the "seed-soil" synergistic treatment concept, synergistically regulating the triple mechanism of "epithelial cells (seeds)," "immune microenvironment (soil)," and "local flora."

[0030] On the one hand, the "seed" protection mechanism efficiently delivers active selenium through nanoparticles, upregulating GPX4 expression in mucosal epithelial cells of lesions such as the oral cavity, clearing lipid peroxides, thereby specifically inhibiting ferroptosis and directly protecting and promoting epithelial cell repair. On the other hand, the "soil" remodeling mechanism utilizes the broad-spectrum free radical scavenging ability of biomimetic adhesive polymer shells such as polyserotonin (PST) and the biological functions of selenium to efficiently remove reactive oxygen species / nitrogen species and polarize macrophages from a pro-inflammatory M1 phenotype to an anti-inflammatory / repair M2 phenotype, thereby significantly improving the immune microenvironment. In addition, these selenium-containing nanoparticles also integrate the inherent broad-spectrum antibacterial activity of selenium, working synergistically with PST to effectively inhibit the growth of common pathogenic bacteria on ulcer surfaces, creating a clean microenvironment for tissue repair, thus forming a new paradigm of synergistic treatment of "antibacterial-anti-inflammatory-antiferroptosis". Furthermore, thanks to the biomimetic adhesive properties of the shell such as PST, the nanoparticles can achieve long-term local retention in the ulcer area for up to 120 hours, ensuring that the antibacterial and biological functions of selenium can be continuously exerted, effectively overcoming the problem of drug loss caused by the complex oral environment.

[0031] Compared with the prior art, this application has the following advantages:

[0032] 1. For the first time, we systematically applied the "seed-soil" synergistic theory to the treatment of oral mucositis. By simultaneously intervening in three core pathological processes—"epithelial cell ferroptosis," "immune microenvironment imbalance," and "local microbial infection"—using a single nano-formulation, we broke the vicious cycle of "oxidative stress—ferroptosis—inflammation—infection." In terms of material design, we innovatively utilized serotonin (PST), which acts as a reducing agent, stabilizer, and functional component, to construct structurally stable core-shell nanoparticles through a one-step synthesis method. This design not only overcomes the bottlenecks of traditional selenium nanoparticles, such as easy aggregation and poor retention, but also achieves synergistic enhancement of antioxidant, anti-inflammatory, and antibacterial functions.

[0033] 2. Regarding the preparation method, (1) a one-step synthesis method is adopted, which does not require complex post-processing, and the yield is increased by 26% compared with the traditional method. It is also green and has no chemical residues. The process is simple and efficient. (2) The selenium-containing nanoparticles formed under the above specific conditions can enable selenium-containing substances to be combined with biomimetic adhesive polymers through C-Se covalent bonds. Compared with the weak and unstable non-covalent or coordinate bond method in traditional selenium modification (physical adsorption / encapsulation, coordination bond / metal-ligand interaction), the application creatively adopts and realizes the strong chemical bonding strategy of C-Se covalent bonds. This not only achieves the ultra-stable residence of the nanoplatform in the harsh oral environment in terms of physics, but also achieves the precise anchoring and controllable release of active ingredients in terms of chemistry, and at the same time, it promotes the synergistic and enhanced therapeutic effect in terms of function. The selenium-containing nanoparticles formed by the above method solve the pain points of the instability and easy leakage of traditional nano-selenium through strong chemical bonding, and achieve synergistic and efficient treatment of complex mucositis through functional integration, which has a clear transformation advantage.

[0034] 3. These selenium-containing nanoparticles also demonstrate outstanding advantages in comprehensive performance and clinical translational potential. In terms of delivery performance, their biomimetic adhesion properties achieve long-term retention in oral ulcers for up to 120 hours, five times more efficient than traditional nano-selenium, ensuring sustained antibacterial and repair effects. In terms of biological efficacy, this formulation significantly enhances the scavenging ability of various free radicals and increases epithelial cell proliferation by 50%, achieving a migration healing rate as high as 53.9%. Specifically, in vitro antibacterial experiments confirmed that PST-SeNPs exhibit significant inhibitory effects on common oral bacterial strains (such as Staphylococcus aureus and Candida albicans), with antibacterial performance superior to traditional nano-selenium. Animal experiments confirmed that its ulcer healing speed (basic healing within 5 days) and tissue repair quality are significantly better than clinical control drugs, and it can effectively control the ulcer microbiota, reducing the risk of secondary infection. Further mechanistic studies show that this formulation can precisely regulate GPX4 / ACSL4 expression bidirectionally at the molecular level, and transcriptomics confirmed that it can systematically reverse multi-pathway gene disorders related to oral mucositis, with effects far exceeding those of single components. Furthermore, the system's biosafety assessment has confirmed that it has no significant toxic side effects on normal mucosa and flora at effective doses, giving it excellent prospects for clinical translation.

[0035] When this product contains selenium nanoparticles for the preparation of corresponding drugs, it can be flexibly formulated into various dosage forms such as mouthwash, spray, gel or lozenge. Among them, the mouthwash dosage form is convenient for patients to use and can achieve broad drug coverage, showing good clinical application prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0037] Figure 1 Figures show the synthesis, characterization, and antioxidant evaluation of PST-SeNPs. (A) Schematic diagram of one-step hydrothermal synthesis of PST-SeNPs. (B) Representative photographs of color changes during the synthesis process. (C) Transmission electron microscopy images showing uniform spherical PST-SeNP morphology. (D) UV-Vis spectra of PST and PST-SeNPs. (E~I) XPS analysis of PST-SeNPs: (E) Full spectrum; (F) High-resolution XPS C1s plot; (G) High-resolution XPS N1s plot; (H) High-resolution XPS O1s plot; (I) High-resolution XPS Se3d plot. (JM) PST-SeNPs versus ABTS. + ·、·O2 - Quantitative analysis of the activity of PST-SeNPs in scavenging ·OH and ·NO free radicals. (NO) is represented by an ESR spectrum showing the activity of PST-SeNPs in scavenging ·OH and ·O2. - Graph showing the relationship between free radical scavenging effect and concentration.

[0038] Figure 2 The figures show the particle size distribution and potential diagrams of PST and PST-SeNPs, where (A) is the particle size distribution curve and (B) is the potential diagram.

[0039] Figure 3 To evaluate the effects of different concentrations of PST-SeNPs on free ABTS + · and · O 2- The image shows the effect of the cleanup, where (A) represents the effect on the free ABTS. + The effect diagram of the removal of · is shown in (B), which represents the effect of removing ·O. 2- The result of the cleanup is shown in the image.

[0040] Figure 4 This is a comparison of the particle size, synthesis efficiency, stability, antioxidant activity, and oral adhesion of PST-SeNPs and traditional GSH-SeNPs in Example 2. (A) is a schematic diagram showing the differences in composition and function between GSH-SeNPs and PST-SeNPs; (B) is a dynamic light scattering analysis diagram of the particle size distribution of GSH-SeNPs and PST-SeNPs; (C) is a comparison diagram of the product yields of GSH-SeNPs and PST-SeNPs; and (D) is the IC50 of free radical scavenging by GSH-SeNPs and PST-SeNPs. 50Value Comparison Bar Chart (ABTS) + O2 - (E) Concentration-dependent O2 scavenging activity curve; (F) Representative fluorescence images of free FITC, GSH-SeNPs / FITC, and PST-SeNPs / FITC on mucus-coated slides; (G) Quantitative analysis of mucosal adhesion efficiency; (H) Representative in vivo fluorescence images of the retention behavior of PBS, GSH-SeNPs / Cy5.5, and PST-SeNPs / Cy5.5 in the mouse oral cavity at different time points; (I) Quantitative fluorescence intensity analysis of (H); (J) Fluorescence colocalization images showing the distribution of Cy5.5-labeled SeNPs (red), CK5 (green), and cell nuclei (blue) after 8 h of drug administration; (K) Quantitative analysis of the colocalization results (J).

[0041] Figure 5 This is a representative TEM image of the GSH-SeNPs prepared in Example 2.

[0042] Figure 6 The diagrams show the scavenging activity of GSH-SeNPs and PST-SeNPs at various concentration gradients in Example 2 against free radicals such as ABTS, •OH, and •NO; where (A) is the scavenging activity against ABTS free radicals, (B) is the scavenging activity against •OH free radicals, and (C) is the scavenging activity against •NO free radicals.

[0043] Figure 7 The particle size variation curves of GSH-SeNPs and PST-SeNPs in Example 2 at different storage times are shown.

[0044] Figure 8The graph shows the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SeNPs, PST-SeNPs, PST, CS-SeNPs, and CS against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli in Example 3. (A) is a schematic diagram of the antibacterial evaluation of PST-SeNPs; (B) shows the bacterial turbidity of Staphylococcus aureus treated with conventional SeNPs, PST-SeNPs, free PST polymer, chitosan-stabilized selenium nanoparticles CS-SeNPs, and free chitosan CS at pH 7.4 using the bacterial turbidity method; (C) shows the bacterial turbidity of Staphylococcus aureus treated with SeNPs, PST-SeNPs, PST, CS-SeNPs, or CS at pH 5.5 using the bacterial turbidity method. (Aureus) bacterial turbidity; (D) bacterial turbidity of E. coli after treatment with SeNPs, PST-SeNPs, PST, CS-SeNPs or CS in a pH 7.4 environment (simulating normal physiological environment); (E) bacterial turbidity of E. coli after treatment with SeNPs, PST-SeNPs, PST, CS-SeNPs or CS in a pH 5.5 environment (simulating the lesion microenvironment after oral infection) using bacterial turbidity method.

[0045] Figure 9 The cell viability graphs of SeNPs, PST, and PST-SeNPs against HOK and HUVECs in Example 4 are shown; where (A) is the cell viability graph against HOK and (B) is the cell viability graph against HUVECs.

[0046] Figure 10 The image shows the results of PST-SeNPs promoting oral keratinocyte proliferation, migration, and angiogenesis in Example 4; where (A) is a schematic diagram of PST-SeNPs cell evaluation; (B) is the OD at different time points after treatment with control, SeNPs, PST, and PST-SeNPs groups as shown by CCK-8 analysis. 450 (C) is a representative image of a cell colony formation assay comparing the proliferation effects of different drug treatments; (D) is a quantitative analysis of the number of cell colonies in (C); (E) is a wound healing assay showing the migration-promoting activities of SeNPs, PST, and PST-SeNPs, with live cells stained with Calcein-AM; (F) is the quantitative wound healing rate derived from (E); (G) is the result of a blood vessel formation assay evaluating the angiogenesis activity of different drug treatments; (H~K) are quantitative statistical analyses of the number of tubes, loops, nodes, and total tube length in (G).

[0047] Figure 11 The image shows the results of PST-SeNPs protecting oral keratinocytes by upregulating GPX4 and inhibiting ferroptosis in Example 5. (A) shows representative immunofluorescence images of GPX4 (green) and cell nuclei (blue) after different treatments; (B) shows the quantitative GPX4 fluorescence intensity in (A); (C-D) show the relative expression levels of GPX4 and ACSL4 mRNA detected by RT-PCR; (E-F) show the intracellular GSH / GSSG ratio and MDA content measured by a biochemical kit; (G) shows a representative fluorescence image of lipid peroxidation detected using the BODIPY 581 / 591 C11 probe, displaying oxidation (green) and reduction (red) states; (H-I) shows flow cytometry analysis and quantitative lipid oxidation levels; (J) shows a representative intracellular ROS fluorescence image; (K-L) shows the results of flow cytometry analysis and quantitative cellular ROS levels; (M) is a schematic diagram of the molecular mechanism by which PST-SeNPs regulate ferroptosis in HOK cells through the GPX4 signaling pathway.

[0048] Figure 12 The image shows the cell viability of RAW cells measured by CCK8 in Example 6, specifically GSH-SeNPs, PST, and PST-SeNPs.

[0049] Figure 13 This is a graph showing the results of PST-SeNPs scavenging multiple free radicals and inducing macrophage polarization from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype in Example 6; where (A) shows the effects of different groups of nanoparticles on RONS, H2O2, OH / ONOO. - •, O2 - Representative fluorescence images of various free radicals such as RONS, H2O2, OH / ONOO, etc. (scale bar = 200 μm); (BF) are fluorescence images of RONS, H2O2, OH / ONOO, etc., obtained by flow cytometry. - •, O2 - • Quantitative analysis of free radicals such as NO•; (G) is a heatmap showing the mRNA expression of classical inflammatory factors IL-6, TNF-α and IL-10, M1 phenotypic markers iNOS and IL-1β, and M2 phenotypic markers Arg-1 and CD206, etc., detected by RT-PCR; (H) is a representative fluorescence colocalization image (scale bar = 25 μm) of M1 macrophages (CD86, green), M2 macrophages (CD206, red) and cell nuclei (DAPI, blue) after different group interventions; (I~J) is the quantitative statistical analysis of CD86 and CD206 in (H); (K~L) is the expression of CD86 and CD206 detected by flow cytometry; (M~N) is the quantitative analysis of the flow cytometry results in (K~L); (O) is a schematic diagram showing the molecular regulatory mechanism of PST-SeNPs on RAW264.7 cells.

[0050] Figure 14 The following diagram shows the results of PST-SeNPs in reducing oral mucosal inflammation and promoting tissue repair in vivo in Example 7. Among them, (A) is a schematic diagram of the animal treatment plan; (B) is a representative macroscopic image of the oral mucosa at different time points after treatment; (C) is the clinical score of oral mucosal damage in each group; (D) is the quantitative analysis of ulcer area changes during treatment; (E) is the food consumption curve of mice during treatment; (F) is the weight change of mice during treatment; (G) is a representative H&E staining image of oral mucosal tissue (scale bar = 100 μm); (H) is a representative Masson trichrome staining image of oral mucosal tissue (scale bar = 100 μm); (I~J) are quantitative analysis diagrams of epithelial thickness and re-epithelialization rate in (E).

[0051] Figure 15 This is a molecular network diagram of PST-SeNPs' systemic reversal of oral mucositis, revealed by RNA-Seq analysis in Example 8. (A) is principal component analysis (PCA) of the transcriptome profiles of the Sham, PBS, and PST-SeNPs groups; (B) is a Venn diagram showing differentially expressed genes (DEGs) among the groups; (C) is a KEGG pathway enrichment analysis of DEGs, highlighting signaling pathways related to antioxidation, immunity, and tissue repair; (D) is a gene ontology (GO) enrichment analysis, categorized by biological processes (BP), cellular components (CC), and molecular functions (MF); (E) shows a heatmap of gene expression levels related to selenium metabolism and ferroptosis; (F) shows a heatmap of genes related to oxidative stress regulation; and (G) shows a heatmap of gene expression related to mucosal repair.

[0052] Figure 16 This is the result of PST-SeNPs exerting a therapeutic effect by inhibiting epithelial ferroptosis and regulating macrophage polarization in Example 9; where (A) are representative immunofluorescence images of GPX4 (red) and cell nuclei (blue) in the oral mucosa tissue of each treatment group; (B) are immunohistochemical staining images of ACSL4 expression in different treatment groups; (C) are representative triple immunofluorescence images of macrophages: F4 / 80 (magenta), CD86 (red, M1 marker), CD206 (green, M2 marker), and cell nuclei (DAPI, blue). (A) (D) Quantitative analysis of GPX4 fluorescence intensity in (A); (E) Quantitative analysis of ACSL4 positive region in (B); (F~G) Quantitative analysis of CD86+ and CD206+ macrophages in (C); (H~I) Representative immunohistochemical images of IL-6 and TNF-α; (J~K) Quantitative analysis of IL-6 and TNF-α positive staining in (H and I); (L) Schematic diagram of PST-SeNPs inhibiting iron deposition in oral mucosa through the GPX4 pathway, thereby maintaining tissue integrity.

[0053] Figure 17 The following diagrams show the expression of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β in mouse serum as determined by ELISA in the examples. Among them, (A) shows the expression of IL-6 pro-inflammatory factor in mouse serum; (B) shows the expression of TNF-α pro-inflammatory factor in mouse serum; and (C) shows the expression of IL-1β pro-inflammatory factor in mouse serum.

[0054] Figure 18 This image shows the effect of PST-SeNPs in accelerating mucosal repair by coordinating epithelial proliferation, angiogenesis, and immune regulation in Example 10. (A) is a representative immunofluorescence image of cytokeratin 5 (CK5, green) and cell nucleus (DAPI, blue) in oral mucosal tissue after treatment (scale bar = 50 μm); (B) is a double immunofluorescence staining of the angiogenesis marker CD31 (green), the cytoskeletal protein α-SMA (red), and the cell nucleus (DAPI, blue) showing vascular structures (scale bar = 50 μm); (C) is a quantitative analysis of CK5 fluorescence intensity. Analysis; (D~E) Quantitative analysis of CD31+ endothelial cells and α-SMA+ pericytes; (F) Representative fluorescence images of CD11b+ inflammatory cells (red) and nuclei (blue) (scale bar = 50 μm); (G) Quantitative analysis of CD11b+ cell infiltration; (H~I) Representative immunohistochemical staining images of TGF-β and VEGF (scale bar = 100 μm); (J~K) Quantitative analysis of TGF-β and VEGF positive areas; (L) Schematic diagram of the multidimensional regeneration program triggered by PST-SeNPs in oral mucosa tissue.

[0055] Figure 19 The graphs show the changes in blood biochemical indicators alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and urea (BUN) after different group treatments in Example 11; where (A) to (D) are the changes in ALT, AST, CRE, and BUN, respectively.

[0056] Figure 20 This chart shows the changes in blood routine indicators (white blood cell count, neutrophil percentage, lymphocyte percentage, monocyte percentage, red blood cell count, hemoglobin concentration, hematocrit, and platelet count) after different group treatments in Example 11. In the chart, (A) through (H) show the changes in the blood routine indicators WBC, Neu%, Lym%, Mon%, RBC, HGB, HCT, and PLT, respectively.

[0057] Figure 21These are representative H&E staining images of the major organs—heart, liver, spleen, lung, and kidney—after different group treatments in Example 11. Detailed Implementation

[0058] The embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0059] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0060] Those skilled in the art will understand that the order in which the steps are written in the various embodiments or examples does not imply a strict execution order and does not limit the implementation process in any way. The detailed execution order of each step should be determined by its function and possible internal logic. Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, but sequentially is preferred.

[0061] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0062] Example 1: Preparation, characterization and antioxidant performance evaluation of PST-SeNPs

[0063] 1. Preparation and Basic Characterization

[0064] PST-SeNPs (e.g., prepared by a one-step hydrothermal synthesis process) were prepared. Figure 1(As shown in A). The specific steps are as follows: Dissolve 10 mg of serotonin hydrochloride in 10 mL of deionized water, then add 1 mL of 0.1 M Na₂SeO₃ solution and 0.3 mL of 0.1 M FeCl₃ solution sequentially. Stir at 100 r / min for 30 minutes, then add 1 mL of 0.1 M NaOH solution dropwise until the pH reaches 8.0, and start the polymerization. Transfer the mixture to a hydrothermal reactor and react at 70 °C for 2 hours. Observe that the solution changes from colorless to dark brownish-red (as shown in A). Figure 1 (As shown in B), indicating the formation of zero-valent selenium nanoparticles. The reaction solution was purified by dialysis and freeze-dried to obtain reddish-brown PST-SeNPs powder. The hydrated particle size of the PST-SeNPs was determined using dynamic light scattering (DLS). The hydrated particle size was 93.5 ± 2.6 nm. Figure 2 A), the Zeta potential is -4.7 ± 1.6 mV ( Figure 2 B, where n=3 indicates that the number of test samples is 3), indicates that the particles have good colloidal stability. Transmission electron microscopy (TEM) shows ( Figure 1 C) It is a uniform sphere with a core particle size of approximately 15±5 nm.

[0065] 2. Structural and chemical composition analysis

[0066] The chemical structure and surface properties of the product were systematically characterized using ultraviolet-visible absorption spectroscopy (UV-vis) and X-ray photoelectron spectroscopy (XPS). Its structure is shown in the UV-vis spectrum (…). Figure 1 D) shows that PST-SeNPs exhibit a broad absorption band similar to PST in the 270–320 nm range, indicating that its surface successfully retains the functional group structure of PST. XPS full-spectrum analysis ( Figure 1 E~I) confirmed that the material is composed of four elements: C (36.8%), N, O, and Se (21.35%). The significant C=O and CO peaks in the high-resolution C1s spectrum indicate that the material surface is rich in carboxyl and phenolic hydroxyl groups, which is consistent with its electronegativity and potential adhesive properties. The crucial Se3d spectrum exhibits characteristic peaks at 56.5 eV and 51.8 eV, confirming the presence of zero-valent selenium (Se). 0 The ) was successfully formed and exists stably in the structure in the form of C-Se covalent bonds.

[0067] 3. Evaluation of in vitro antioxidant properties

[0068] The antioxidant potential of PST-SeNPs was evaluated using a variety of free radical scavenging experimental systems. The results showed that ( Figure 1 J~M), which is related to ABTS + ·、·O2 - The scavenging activity of various free radicals such as ·OH and ·NO is concentration-dependent. Figure 3Meanwhile, this material is effective against O2. - Various pathology-related free radicals, such as ·OH and ·NO, also exhibited highly efficient dose-dependent scavenging capabilities. Figure 1 J~M). To further verify its direct antioxidant mechanism, electron spin resonance (ESR) technology was used for detection, and the results clearly showed ( Figure 1 N、 Figure 1 PST-SeNPs can effectively quench ·OH and ·O2 in a concentration-dependent manner. - Free radicals. The above data collectively demonstrate that PST-SeNPs possess a powerful and broad-spectrum free radical scavenging ability, providing a solid basis for its application in the antioxidant treatment of oral mucositis.

[0069] Example 2: Comprehensive optimization of PST-SeNPs performance and multi-dimensional comparison with traditional SeNPs

[0070] To systematically evaluate the comprehensive performance advantages of PST-SeNPs, we conducted a multi-dimensional comparative analysis with SeNPs prepared by the classic glutathione (GSH) reduction method. Figure 4 The results show a comparison between PST-SeNPs and traditional SeNPs in terms of synthesis efficiency, stability, antioxidant activity, and oral adhesion. Specifically, (A) is a schematic diagram illustrating the differences in composition and function between GSH-SeNPs and PST-SeNPs; (B) is a dynamic light scattering analysis diagram of the particle size distribution of GSH-SeNPs and PST-SeNPs; (C) is a comparison diagram of the product yields of GSH-SeNPs and PST-SeNPs; and (D) is the IC50 of free radical scavenging by GSH-SeNPs and PST-SeNPs. 50 Value Comparison Bar Chart (ABTS) + O2 - (E) Concentration-dependent O2 scavenging activity curves; (F) Representative fluorescence images of free FITC, GSH-SeNPs / FITC, and PST-SeNPs / FITC on mucus-coated slides; (G) Quantitative analysis of mucosal adhesion efficiency; (H) Representative in vivo fluorescence images of the retention behavior of PBS, GSH-SeNPs / Cy5.5, and PST-SeNPs / Cy5.5 in the mouse oral cavity at different time points; (I) Quantitative fluorescence intensity analysis of (H); (J) Fluorescence colocalization images showing the distribution of Cy5.5-labeled SeNPs (red), CK5 (green), and cell nuclei (blue) after 8 H administration; (K) Quantitative analysis of the colocalization results (J). *ns: P>0.05, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.001. Figure 5Representative TEM images of GSH-SeNPs are shown (scale bar: 100 nm). Figure 6 A series of concentration gradients of GSH-SeNPs and PST-SeNPs were demonstrated for their effects on ABTS. + ·, O2 - Comparison of free radical scavenging activities such as •, •OH and •NO (n=3). Figure 7 The particle size variation curves of GSH-SeNPs and PST-SeNPs at different storage times are shown (n=3). Detailed analysis is as follows:

[0071] 1. Physicochemical properties and antioxidant performance

[0072] from Figure 4 B and Figure 4 As can be seen from C, in terms of physicochemical properties, PST-SeNPs exhibit smaller hydrated particle size (93.5±2.6 nm vs. 203±3 nm) and higher synthesis yield (an increase of approximately 26%). Transmission electron microscopy results show that PST-SeNPs are uniformly dispersed. Figure 1 C), while GSH-SeNPs showed significant aggregation (C). Figure 5 PST-SeNPs exhibit superior colloidal stability. Figure 7 ).

[0073] from Figure 4 As can be seen from D, in terms of antioxidant function, PST-SeNPs have an effect on ABTS. + ·、·O 2- The scavenging ability of various free radicals, such as ·OH, is significantly better than that of GSH-SeNPs, exhibiting a lower IC50 value. 50 The performance advantage primarily stems from the abundance of phenolic hydroxyl groups in the PST shell, which provide ample reaction sites for free radical scavenging. Although slightly inferior to GSH-SeNPs in ·NO scavenging, possibly due to differences in scavenging mechanisms, overall, PST modification significantly enhances the broad-spectrum antioxidant capacity of SeNPs. Concentration gradient experiments further confirmed this. Figure 6 Under the same Se concentration conditions, PST-SeNPs generally exhibit higher free radical scavenging efficiency. Furthermore, from... Figure 7 It can be seen that the particle size of PST-SeNPs remained stable after 8 days of storage at room temperature, which is significantly better than that of GSH-SeNPs, which are prone to aggregation, and shows superior potential for practical applications.

[0074] 2. Mucosal adhesion and retention behavior in vivo

[0075] from Figure 4From F to 4G, it can be seen that in the in vitro adhesion experiment simulating the oral mucosa environment, PST-SeNPs exhibited a strong and uniform fluorescence signal, and its adhesion strength was about 67% higher than that of GSH-SeNPs. This superior performance is attributed to the multiple non-covalent bonds formed between the catechol groups in the PST molecule and the mucin.

[0076] Small animal live imaging experiments further confirmed ( Figure 4 (H~I) PST-SeNPs exhibit a significant retention advantage in the oral cavity. Following local administration, the fluorescence signal of GSH-SeNPs decays to background levels within 24 hours, while the signal of PST-SeNPs can be detected up to 120 hours, representing a 4-fold increase in retention time. Figure 4 H). Tissue section colocalization analysis showed that 8 hours after drug administration, PST-SeNPs (red) exhibited high colocalization with oral epithelial cell keratin CK5 (green) and cell nuclei (blue). Figure 4 J) indicates that it can specifically accumulate and remain in the oral mucosal epithelium for a long time. Simultaneously, dispersed nanoparticle signals were also observed in the lamina propria of the mucosa. Figure 4 The presence of J indicates that it possesses a certain degree of tissue penetration. In contrast, the weak and diffuse distribution of GSH-SeNPs signals further highlights the unique advantages of PST modification in enhancing mucosal targeting and retention.

[0077] Therefore, PST-SeNPs are significantly superior to traditional SeNPs in terms of synthesis efficiency, storage stability, antioxidant properties and oral adhesion. Its dual characteristics of "macroscopic long-term retention" and "microscopic tissue enrichment" lay a solid foundation for the local continuous treatment of oral mucositis.

[0078] Example 3: Evaluation of the in vitro antibacterial properties of PST-SeNPs

[0079] To evaluate the antibacterial potential of PST-SeNPs, we selected Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) as representative pathogens. Under simulated normal physiological environment (pH 7.4) and the common slightly acidic lesion environment after oral infection (pH 5.5), we systematically compared the antibacterial activity of PST-SeNPs with traditional SeNPs, free PST polymers, chitosan-stabilized selenium nanoparticles (CS-SeNPs), and free chitosan (CS). Figure 8The results of PST-SeNPs' antibacterial effect against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli are shown. (A) is a schematic diagram of the antibacterial evaluation of PST-SeNPs; (B) shows the bacterial turbidity of Staphylococcus aureus treated with traditional SeNPs, PST-SeNPs, free PST polymer, chitosan-stabilized selenium nanoparticles (CS-SeNPs), and free chitosan (CS) at pH 7.4 using a bacterial turbidity method; (C) shows the bacterial turbidity of Staphylococcus aureus treated with SeNPs, PST-SeNPs, PST, CS-SeNPs, or CS at pH 5.5 using a bacterial turbidity method; (D) shows the bacterial turbidity of Escherichia coli treated with SeNPs, PST-SeNPs, PST, CS-SeNPs, or CS at pH 7.4 (simulating normal physiological conditions) using a bacterial turbidity method. (E) represents the bacterial turbidity of *Escherichia coli* after treatment with SeNPs, PST-SeNPs, PST, CS-SeNPs, or CS in a pH 5.5 environment (simulating the lesion microenvironment after oral infection). Table 1 shows the MIC and MBC values ​​of *Staphylococcus aureus* and *Escherichia coli* at two pH values ​​(pH 7.4 and pH 5.5) after treatment with SeNPs, PST-SeNPs, PST, CS-SeNPs, or CS. Data are expressed as mean ± SD (n=3 per group). Statistical significance was analyzed using one-way ANOVA and Tukey post-hoc test. *ns: P>0.05, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.001. Specific analysis is as follows:

[0080] 1. Antibacterial activity analysis

[0081] like Figure 8 As shown in BE, the bacterial turbidity method clearly demonstrates that all selenium-containing nanomaterials exhibited a certain degree of bacterial growth inhibition at the tested concentrations. Among them, PST-SeNPs showed significant antibacterial advantages against both types of bacteria under both pH conditions, with its culture medium exhibiting significantly lower turbidity than the other groups. It is noteworthy that pH environment has a decisive influence on antibacterial efficacy: under slightly acidic conditions at pH 5.5, the antibacterial activity of all tested materials (including PST-SeNPs) was significantly stronger than under neutral conditions at pH 7.4. Figure 8 C, E vs. Figure 8 B,D).

[0082] This phenomenon is further confirmed by the quantitative minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) data in Table 1. The data show that the MIC / MBC values ​​of PST-SeNPs against S. aureus and E. coli are 2-4 times lower at pH 5.5 than at pH 7.4, indicating that their antibacterial efficacy is greatly enhanced in acidic environments. This pH dependence may be related to the following mechanisms: (1) the acidic environment may promote the ionization of selenium on the surface of SeNPs or the efficiency of reactive oxygen species (ROS) generation; (2) the slightly acidic conditions themselves may have put some pressure on bacterial metabolism, making them more sensitive to oxidative stress; (3) the surface charge or conformation of the PST polymer shell may change when the pH value changes, enhancing the interaction with the bacterial cell wall.

[0083] Table 1. MIC and MBC of different intervention groups against Escherichia coli and Staphylococcus aureus at pH 7.4 and pH 5.5.

[0084]

[0085] Furthermore, the data consistently showed that PST-SeNPs exhibited superior antibacterial activity against Gram-positive bacteria (S. aureus) compared to Gram-negative bacteria (E. coli). This may stem from the fundamental differences in their cell wall structures: the thicker peptidoglycan layer of Gram-positive bacteria may be more readily interacting with nanomaterials, while the lipopolysaccharide layer in the outer membrane of Gram-negative bacteria constitutes an additional permeation barrier.

[0086] In summary, this study confirms that PST-SeNPs possess broad-spectrum and highly efficient antibacterial properties, with activity significantly superior to that of pure SeNPs or PST polymers, indicating a synergistic antibacterial effect between PST modification and the selenium core. Crucially, its antibacterial activity is significantly enhanced in the slightly acidic environment simulating infectious lesions. This characteristic is clinically significant because oral mucosal ulcers often present a locally acidic environment due to inflammation and bacterial metabolism. This implies that PST-SeNPs can achieve a "smart response," maximizing its antibacterial efficacy at the lesion site where bactericidal action is most needed, thus providing a new nanotechnology solution for the precise and efficient control of secondary infections of oral mucositis (OM). Future work will further explore its specific antibacterial molecular mechanisms, such as ROS generation, cell membrane disruption, and biofilm inhibition capabilities.

[0087] Example 4: Study on the in vitro cell proliferation, migration and angiogenesis activity of PST-SeNPs

[0088] To systematically evaluate the promoting effect of PST-SeNPs on oral mucosal healing, this embodiment used human primary oral mucosal epithelial cells (HOK) and human umbilical vein endothelial cells (HUVEC) as models to simulate epithelial regeneration and angiogenesis processes, respectively, and compared them with GSH-SeNPs and free PST. Figure 9 The cell viability of HOK and HUVECs was demonstrated by CCK8 assays of SeNPs, PST, and PST-SeNPs (n=3). Figure 10 The results of PST-SeNPs promoting oral keratinocyte proliferation, migration, and angiogenesis are shown. (A) is a schematic diagram of PST-SeNPs cell evaluation; (B) is a CCK-8 analysis showing the OD at different time points after treatment with control, SeNPs, PST, or PST-SeNPs groups. 450 (C) is a representative image of the colony formation assay comparing the proliferation effects of different drug treatments; (D) is a quantitative analysis of the number of colonies in (C); (E) is a wound healing assay showing the migration-promoting activities of SeNPs, PST, and PST-SeNPs, with live cells stained with Calcein-AM; (F) is the quantitative wound healing rate derived from (E); (G) is the result of a blood vessel formation assay evaluating the angiogenesis activity of different drug treatments, shown by Calcein-AM staining; (HK) are the results of quantitative analysis of tube number, loop number, branch point number, and total tube length, respectively. Data are expressed as mean ± SD (n=3 per group). Statistical significance was analyzed using one-way ANOVA and Tukey post-hoc test. *ns: P>0.05, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.001. Specific analysis is as follows:

[0089] 1. Cell safety evaluation

[0090] The cytotoxicity of PST-SeNPs against HOK and HUVECs was assessed using the CCK-8 assay. Results showed ( Figure 9 When the Se concentration was not higher than 320 ng / mL, the survival rate of both cell types remained above 80%, indicating that PST-SeNPs had good biocompatibility within this concentration range, providing a safety basis for subsequent functional experiments.

[0091] 2. Promotes epithelial cell proliferation and migration.

[0092] CCK-8 experiments showed that PST-SeNPs exhibited the best HOK proliferation-promoting ability at all time points. On day 5 of culture, its OD... 450The value reached 2.24±0.15, a 50% increase compared to the control group, and significantly better than the GSH-SeNPs group (14.3% increase) and the PST group (31.3% increase). Figure 10 B). Plate colony formation experiments further confirmed that the number of colonies formed in the PST-SeNPs group was (1.84±0.10)×10⁻⁶. 3 The number of cells / well was significantly higher than that in the control groups ( ). Figure 10 C Figure 10 D). Scratch test results showed that after 24 hours of culture, the healing rate of the PST-SeNPs group reached 53.9±3.5%, significantly higher than that of the control group (17.5±6.5%), the GSH-SeNPs group (38.8±3.6%), and the PST group (31.1±3.8%). Figure 10 E, Figure 10 F). This superior migration-promoting ability may stem from the synergistic effect of PST's cell adhesion properties and Se's oxidative stress regulation function.

[0093] 3. Angiogenesis-promoting ability

[0094] The effect of PST-SeNPs on the tube-forming ability of HUVECs was evaluated using an in vitro angiogenesis assay with matrix gel. The results showed that ( Figure 10 G~ Figure 10 PST-SeNPs significantly outperformed the control groups in all evaluation metrics (including the number of tubules, ring structure, number of branching points, and total tubular length). PST-SeNPs could induce the formation of tightly connected and morphologically complete tubular networks within 6 hours, while the control groups could only form fragmented structures.

[0095] This embodiment demonstrates that PST-SeNPs exhibit significantly superior biological activity compared to single-component or conventional SeNPs in promoting epithelial cell proliferation and migration, as well as enhancing angiogenesis. This synergistic enhancement effect stems from the complementary and integrated molecular functions of PST and Se, providing solid experimental evidence for its application as a highly effective oral mucosal repair agent.

[0096] Example 5: PST-SeNPs inhibit ferroptosis and maintain cellular redox homeostasis by regulating the GPX4 pathway.

[0097] To elucidate the molecular mechanism of PST-SeNPs in the treatment of oral mucositis, this embodiment systematically evaluated its core regulatory role in the key pathway of ferroptosis using an H2O2-induced HOK cell oxidative damage model. Figure 11This study demonstrates the results of PST-SeNPs protecting oral keratinocytes by upregulating GPX4 and inhibiting ferroptosis. (A) shows representative immunofluorescence images of GPX4 (green) and cell nuclei (blue) after different treatments (scale bar = 25 μm); (B) shows the quantitative analysis of GPX4 fluorescence intensity in (A); (CD) shows the relative expression levels of GPX4 and ACSL4 mRNA detected by RT-PCR; (EF) shows the intracellular GSH / GSSG ratio and MDA content determined by a biochemical kit; (G) shows representative fluorescence images of lipid peroxidation detected using the BODIPY 581 / 591 C11 probe, displaying oxidized (green) and reduced (red) states; (HI) shows flow cytometry analysis and quantitative analysis of lipid oxidation levels; (J) shows representative intracellular ROS fluorescence images (scale bar = 200 μm); (KL) shows flow cytometry analysis and quantitative analysis of cellular ROS levels; and (M) is a schematic diagram illustrating the molecular mechanism by which PST-SeNPs regulate ferroptosis in HOK cells through the GPX4 signaling pathway. Data are expressed as mean ± SD (n=3 per group). Statistical significance was analyzed using one-way ANOVA and Tukey's post-hoc test. *ns: P>0.05, P<0.05, **P<0.01, ***, P<0.005, ****, P<0.001. The detailed analysis follows:

[0098] 1. Activation effect on the GPX4 pathway

[0099] Immunofluorescence and qPCR analysis results showed that ( Figure 11 A, Figure 11 B Figure 11 (C) PST-SeNPs significantly upregulated GPX4 expression. Its upregulation at the protein level (3.2-fold compared to the control group) and at the mRNA level (approximately 2-fold compared to the control group) were significantly superior to those of GSH-SeNPs and the free PST treatment group. This result confirms that PST-SeNPs can effectively activate this key negative regulatory pathway of ferroptosis.

[0100] 2. Two-way regulation of key nodes in ferrodeogenesis

[0101] The relative expression level of ACSL4 mRNA was detected by RT-PCR. Figure 11 D): In addition to effectively upregulating GPX4, PST-SeNPs also significantly inhibited the expression of ACSL4, a positive regulator of ferroptosis. This bidirectional regulatory mode of "enhancing defense (GPX4↑) and weakening attack (ACSL4↓)" synergistically blocked ferroptosis in key pathways.

[0102] 3. Improvement of oxidative damage indicators

[0103] Biochemical reagent kits were used to measure the intracellular GSH / GSSG ratio and MDA content, and it was found that ( Figure 11 E, Figure 11 F): Regarding oxidative stress indicators, PST-SeNPs treatment significantly reversed H2O2-induced oxidative damage, reducing MDA content by 61% (to 0.52±0.11 μM / mg protein) and restoring the GSH / GSSG ratio from 3.1±0.4 to 6.8±0.9, demonstrating superior improvement compared to individual components. This synergistic protective mechanism relies on the functional complementarity of PST and Se: Se directly scavenges lipid peroxides by enhancing GPX4 activity, while PST replenishes intracellular antioxidant reserves by promoting GSH synthase expression.

[0104] 4. Lipid peroxidation inhibition and ROS scavenging ability

[0105] Detection using the BODIPY 581 / 591 C11 probe revealed that the oxidized fluorescence intensity of the PST-SeNPs-treated group was only 15.3% of that of the control group. Figure 11 G, Figure 11 H, Figure 11 I) confirmed that it can effectively inhibit the lipid peroxidation process. Meanwhile, fluorescence imaging and flow cytometry analysis showed that PST-SeNPs exhibited the strongest ROS scavenging ability ( Figure 11 J、 Figure 11 K, Figure 11 L) can effectively inhibit H2O2-induced ROS accumulation to near physiological levels.

[0106] This embodiment demonstrates that PST-SeNPs effectively block the ferroptosis pathway at the molecular level through multiple mechanisms, including synergistic upregulation of GPX4, inhibition of ACSL4, reduction of lipid peroxidation levels, and scavenging of ROS, providing a solid mechanistic basis for its application in the treatment of oral mucositis.

[0107] Example 6: PST-SeNPs exert a dual anti-inflammatory effect through free radical scavenging and macrophage phenotype regulation.

[0108] To elucidate the anti-inflammatory mechanism of PST-SeNPs at the level of immune regulation, this study systematically evaluated its regulatory effects on free radical scavenging and macrophage polarization using LPS-induced RAW 264.7 macrophages as a model. Specific results are as follows: Figures 12-13 As shown, where, Figure 12 The cell viability of RAW264.7 cells (n=3) was demonstrated by CCK8 assay using GSH-SeNPs, PST, and PST-SeNPs. Figure 13The results show that PST-SeNPs scavenge multiple free radicals and induce macrophage polarization from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype. In this study, (A) shows the effects of different groups of nanoparticles on RONS, H2O2, and OH / ONOO. - •,O2 - Representative fluorescence images of various free radicals such as RONS, H2O2, OH / ONOO, etc. (scale bar = 200 μm); (BF) represents fluorescence images of RONS, H2O2, OH / ONOO, etc., obtained by flow cytometry. - •,O2 - • Quantitative analysis of free radicals such as NO•; (G) is a heatmap showing the mRNA expression of classical inflammatory factors IL-6, TNF-α, and IL-10, M1 phenotypic markers iNOS and IL-1β, and M2 phenotypic markers Arg-1 and CD206, etc., detected by RT-PCR; (H) is a representative fluorescence colocalization image (scale bar = 25 μm) of M1 macrophages (CD86, green), M2 macrophages (CD206, red), and cell nuclei (DAPI, blue) after different group interventions; (I~J) are the quantitative statistical analysis of CD86 and CD206 in (H); (K~L) are the expression of CD86 and CD206 detected by flow cytometry; (MN) is the quantitative analysis of the flow cytometry results in (K~L). (O) is a schematic diagram showing the molecular regulatory mechanism of PST-SeNPs on RAW264.7 cells. Data are expressed as mean ± SD (n=3 per group). Statistical significance was analyzed using one-way ANOVA and Tukey's post-hoc test. *ns: P>0.05, P<0.05, **P<0.01, ***, P<0.005, ****, P<0.001. The detailed analysis follows:

[0109] 1. Cell safety and free radical scavenging ability

[0110] CCK-8 assays confirmed that PST-SeNPs had no significant cytotoxicity at selenium concentrations ≤320 ng / mL. Figure 12 Based on this, fluorescence probe detection revealed that PST-SeNPs are effective against various reactive oxygen species (including H2O2, O2). - 、·OH、ONOO - Both (and NO) showed highly efficient scavenging ability. Figure 13 A, Figure 13 B Figure 13 C Figure 13 D、 Figure 13 E, Figure 13The overall clearance rate of PST (F) was approximately 70%, significantly better than that of GSH-SeNPs (approximately 50%) and free PST (approximately 30%). This broad-spectrum clearance capability stems from the synergistic mechanism between PST and Se: PST achieves rapid exogenous clearance through phenolic hydroxyl groups, while Se completes endogenous regulation by activating the antioxidant enzyme system.

[0111] 2. Macrophage phenotypic regulation

[0112] qPCR analysis showed that PST-SeNPs significantly downregulated M1 marker genes such as TNF-α, IL-6, and iNOS, while upregulating M2 marker genes such as Arg-1, CD206, and IL-10. Figure 13 G). Immunofluorescence staining further confirmed ( Figure 13 H, Figure 13 I, Figure 13 J), PST-SeNPs treatment significantly increased CD206 + The proportion of M2 macrophages was reduced, while CD86 was also decreased. + M1 type cells. Quantitative analysis by flow cytometry showed that ( Figure 13 K, Figure 13 L, Figure 13 M, Figure 13 PST-SeNPs reduced the proportion of M1 cells by 70% and increased the proportion of M2 cells by 59%.

[0113] This embodiment demonstrates that PST-SeNPs synergistically scavenge free radicals through "exogenous scavenging-endogenous regulation" and effectively guide macrophages to transform from a pro-inflammatory M1 phenotype to an anti-inflammatory / repair M2 phenotype, thereby achieving effective remodeling of the immune microenvironment in oral mucosal inflammation. Figure 13 O).

[0114] Example 7: In vivo efficacy evaluation of PST-SeNPs in treating oral mucositis

[0115] To verify the in vivo therapeutic effect of PST-SeNPs, this embodiment successfully established a mouse oral mucositis model by stimulation with 5-fluorouracil combined with acetic acid. The modeling animals were randomly divided into six groups: sham-operated group, PBS control group, GSH-SeNPs group, PST group, PST-SeNPs group, and chlorhexidine mouthwash group. The models were systematically evaluated from three dimensions: macroscopic healing, functional indicators, and histopathology. Figure 14This study demonstrates the efficacy of PST-SeNPs in reducing oral mucosal inflammation and promoting tissue repair in vivo. (A) is a schematic diagram of the animal treatment regimen; (B) are representative macroscopic images of the oral mucosa at different time points after treatment; (C) are clinical scores of oral mucosal damage in each group; (D) is a quantitative analysis of ulcer area changes during treatment; (E) is the food consumption curve of mice during treatment; (F) is the weight change of mice during treatment; (G) are representative H&E staining images of oral mucosal tissue (scale bar = 100 μm); (H) are representative Masson's trichrome staining images of oral mucosal tissue (scale bar = 100 μm); and (IJ) is a quantitative analysis of epithelial thickness and re-epithelialization rate in (E). Data are expressed as mean ± SD (n = 5 mice per group). Statistical significance was analyzed using one-way ANOVA and Tukey's post-hoc test. *ns: P>0.05, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.001. The following is a detailed analysis:

[0116] 1. Macroscopic healing evaluation

[0117] from Figure 14 B~ Figure 14 As shown in Figure D, on day 1 after model establishment, all experimental groups developed typical ulcer lesions. The PST-SeNPs group exhibited the fastest healing speed: the ulcer area was smallest on day 4, and it was almost completely healed on day 5, with the mucosal morphology approaching normal levels. Its healing process was significantly better than that of the chlorhexidine group (which required complete healing on day 6). The results of damage scoring and quantitative analysis of ulcer area consistently showed that the PST-SeNPs group had the best improvement effect.

[0118] 2. Recovery of nutritional and metabolic functions

[0119] By monitoring food intake ( Figure 14 E) and weight change ( Figure 14 F) Assess functional recovery. The PBS control group showed persistent decreased food intake and weight loss; the SeNPs and PST groups showed limited improvement; while the PST-SeNPs group experienced rapid recovery of food intake and a simultaneous significant increase in weight from day 4 of administration. These results indicate that PST-SeNPs not only promotes structural healing but also effectively improves nutritional intake function.

[0120] 3. Histopathological analysis

[0121] H&E staining showed ( Figure 14 G, Figure 14 I, Figure 14J), the PST-SeNPs group showed the most complete tissue repair morphology: continuous and intact epithelium, high reepithelialization rate, abundant granulation tissue, and minimal inflammatory cell infiltration. Masson staining further confirmed ( Figure 14 The PST-SeNPs group (H) showed the most significant collagen deposition, with tightly and regularly arranged fibers, indicating its excellent tissue remodeling ability.

[0122] This embodiment demonstrates that PST-SeNPs showed significantly better therapeutic effects than the control groups in promoting structural healing, functional recovery, and tissue maturation of oral mucositis, providing solid in vivo experimental evidence for its clinical application.

[0123] Example 8: In vivo efficacy evaluation of PST-SeNPs in treating oral mucositis

[0124] Based on transcriptomics analysis, this embodiment reveals the multi-pathway synergistic mechanism by which PST-SeNPs promote oral mucosal repair through KEGG and GO enrichment analysis systems. Figure 15 This study presents a molecular network diagram revealing the systemic reversal of oral mucositis by PST-SeNPs through RNA-Seq analysis. Specifically: (A) Principal component analysis (PCA) of the transcriptome profiles of the Sham, PBS, and PST-SeNPs groups; (B) Venn diagram showing differentially expressed genes (DEGs) among the groups; (C) KEGG pathway enrichment analysis of DEGs, highlighting signaling pathways related to antioxidation, immunity, and tissue repair; (D) Gene Ontology (GO) enrichment analysis, categorized by biological processes (BP), cellular components (CC), and molecular functions (MF); (E) a heatmap showing the expression levels of genes related to selenium metabolism and ferroptosis; (F) a heatmap showing genes related to oxidative stress regulation; and (G) a heatmap showing the expression of genes related to mucosal repair. (n=3 per group). Detailed analysis follows:

[0125] 1. Pathway enrichment analysis

[0126] KEGG analysis showed that ( Figure 15 C) PST-SeNPs are significantly enriched in three major pathways: tissue repair-related pathways (PI3K-Akt, focal adhesion, and ECM-receptor interactions), antioxidant and iron metabolism pathways (ferroptosis, glutathione metabolism, and mineral uptake), and immune regulation pathways (TNF, Toll-like receptors, and JAK-STAT signaling pathways). This multi-pathway synergistic mechanism provides a systematic molecular basis for its promotion of mucosal repair.

[0127] 2. Functional Analysis

[0128] GO analysis revealed its mechanism of action from three dimensions. Figure 15D): At the biological process (BP) level, it is significantly enriched in iron homeostasis, glutathione metabolism, and reactive oxygen species metabolism; at the cellular composition (CC) level, it mainly acts on key cellular sites such as the mitochondrial matrix, peroxisomes, and extracellular matrix; at the molecular function (MF) level, it significantly enhances antioxidant activity, oxidoreductase activity, and cytokine binding. The results of these three levels of analysis together construct a multi-level regulatory network of "molecule-cell-tissue".

[0129] 3. Regulation of key genes

[0130] Heatmap analysis shows ( Figure 15 PST-SeNPs (E~15G) can systematically regulate three core gene classes: significantly upregulating the selenoprotein family (GPX1-4, SELENOP, TXNRD1-3, etc.) and antioxidant / repair factors (HMOX1, SOD, MUC1, VEGFA, etc.), while effectively inhibiting the expression of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α). This coordinated gene expression pattern confirms its ability to correct molecular disorders of disease at the systemic level.

[0131] This embodiment demonstrates through multi-omics analysis that PST-SeNPs can reverse the molecular pathological state of oral mucositis at the systemic level by synergistically regulating three major functional modules: anti-oxidation, anti-inflammation, and tissue repair, providing in-depth mechanistic evidence for its clinical application.

[0132] Example 9: PST-SeNPs synergistically inhibit mucosal damage in vivo through ferroptosis inhibition and immune reprogramming.

[0133] To validate the therapeutic mechanism of PST-SeNPs at the tissue level, this embodiment conducted a systematic molecular and protein-level analysis of mouse oral mucosa tissue. Figure 16This study demonstrates that PST-SeNPs exert their therapeutic effects by inhibiting epithelial ferroptosis and regulating macrophage polarization. (A) shows representative immunofluorescence images of GPX4 (red) and cell nuclei (blue) in the oral mucosa tissue of each treatment group; (B) shows immunohistochemical staining images of ACSL4 expression in different treatment groups; and (C) shows representative triple immunofluorescence images of macrophages: F4 / 80 (magenta), CD86 (red, M1 marker), CD206 (green, M2 marker), and cell nuclei (DAPI, blue). (D) Quantitative analysis of GPX4 fluorescence intensity in (A); (E) Quantitative analysis of ACSL4-positive areas in (B); (FG) Quantitative analysis of CD86+ and CD206+ macrophages in (C); (HI) Representative immunohistochemical staining of IL-6 and TNF-α; (JK) Quantitative analysis of IL-6 and TNF-α positive staining in (H and I); (L) Schematic diagram of PST-SeNPs inhibiting ferroptosis in oral mucosa through the GPX4 pathway, thereby maintaining tissue integrity. Data are expressed as mean ± SD (n=5 per group). One-way ANOVA and Tukey post-hoc test were used for statistical significance analysis. *ns: P>0.05, P<0.05,**, P<0.01,***, P<0.005,****, P<0.001. Detailed analysis is as follows:

[0134] 1. Validation of the regulation of the ferroptosis pathway

[0135] Immunofluorescence ( Figure 16 A, Figure 16 D) and immunohistochemical analysis ( Figure 16 B Figure 16 E) showed that PST-SeNPs significantly restored and enhanced the expression of GPX4 protein in mucosal epithelial cells, with better results than the control groups. Simultaneously, this formulation effectively inhibited the expression of ACSL4, a key driver of ferroptosis. The results indicate that PST-SeNPs blocked the ferroptosis pathway through a synergistic effect of "upregulating GPX4, inhibiting lipid peroxidation, and downregulating ACSL4".

[0136] 2. Role in immune microenvironment remodeling

[0137] Observed by triple immunofluorescence staining ( Figure 16 C Figure 16 F~ Figure 16 G), F4 / 80 in the PST-SeNPs treatment group + CD206 + The proportion of M2 macrophages increased significantly, while the proportion of F4 / 80 macrophages increased significantly. + CD86 + M1 type cells decreased accordingly. Immunohistochemistry ( Figure 16H~ Figure 16 K) and ELISA analysis ( Figure 17 Further evidence confirms that PST-SeNPs can significantly reduce the expression and secretion of pro-inflammatory factors such as IL-6, TNF-α, and IL-1β.

[0138] This embodiment demonstrates that PST-SeNPs construct a self-reinforcing positive feedback loop through a dual mechanism of synergistically inhibiting epithelial cell ferroptosis and promoting macrophage M2 polarization: ferroptosis inhibition reduces inflammation triggering, while immune microenvironment remodeling further promotes epithelial repair. This bidirectional interactive mechanism is the key to its superior efficacy compared to single-component formulations and traditional drugs.

[0139] Example 10: PST-SeNPs reconstruct mucosal tissue structure and accelerate repair in vivo through a multidimensional mechanism.

[0140] To systematically evaluate the pro-repair effect of PST-SeNPs on oral mucosal tissue, this embodiment comprehensively analyzes its repair mechanism from three core aspects: epithelial regeneration, angiogenesis, and immune regulation. Figure 18 The results of PST-SeNPs accelerating mucosal repair by coordinating epithelial proliferation, angiogenesis, and immune regulation are shown. (A) is a representative immunofluorescence image of cytokeratin 5 (CK5, green) and cell nucleus (DAPI, blue) in oral mucosal tissue after treatment (scale bar = 50 μm); (B) is a dual immunofluorescence staining of the angiogenesis marker CD31 (green), the cytoskeletal protein α-SMA (red), and the cell nucleus (DAPI, blue) showing vascular structures (scale bar = 50 μm); (C) is a quantitative analysis of CK5 fluorescence intensity. (DE) Quantitative analysis of CD31+ endothelial cells and α-SMA+ pericytes; (F) Representative fluorescence images of CD11b+ inflammatory cells (red) and nuclei (blue) (scale bar = 50 μm); (G) Quantitative analysis of CD11b+ cell infiltration; (HI) Representative immunohistochemical staining images of TGF-β and VEGF (scale bar = 100 μm); (JK) Quantitative analysis of TGF-β and VEGF positive areas; (L) Schematic diagram of the multidimensional regeneration process triggered by PST-SeNPs in oral mucosa tissue. Data are expressed as mean ± SD (n = 5 per group). One-way ANOVA and Tukey post-hoc test were used for statistical significance analysis. *ns: P>0.05, P<0.05, **, P<0.01, ***, P<0.005, ****, P<0.001. Specific analysis is as follows:

[0141] 1. Promotes epithelial regeneration

[0142] Epithelial regeneration was assessed using immunofluorescence staining for cytokeratin 5 (CK5). Results showed ( Figure 18 A, Figure 18 (C) The PST-SeNPs treatment group formed a thick, continuous, and orderly arranged CK5-positive epithelial layer, with significantly better structural integrity and fluorescence intensity than the control groups, approaching the level of the sham-operated group. This indicates that PST-SeNPs can effectively promote the proliferation and differentiation of basal epithelial cells and accelerate the re-epithelialization process.

[0143] 2. Angiogenesis Induction

[0144] From the CD31 and α-SMA dual immunofluorescence staining experiment, it can be seen that ( Figure 18 B Figure 18 D、 Figure 18 E): In the PST-SeNPs treatment group, not only was the number of newly formed microvessels significantly increased, but the lumen of CD31-positive endothelial cells was also tightly wrapped by α-SMA-positive pericytes, forming structurally complete functional vascular units. This mature vascular network provides sufficient nutritional support for tissue repair and reflects moderate extracellular matrix remodeling.

[0145] 3. Remodeling of the immune microenvironment

[0146] CD11b immunofluorescence assay showed that ( Figure 18 In the F~18G group, inflammatory cell infiltration was significantly reduced, and most residual cells were in a resting state. Simultaneously, immunohistochemical results showed ( Figure 18 In this group (H~18K), the expression of two key repair factors, TGF-β and VEGF, showed the most significant upregulation, indicating that they effectively activated the repair signaling pathway while controlling inflammation.

[0147] This embodiment demonstrates that PST-SeNPs drive the complete repair of oral mucosal tissue in multiple dimensions by synergistically promoting epithelial barrier reconstruction, functional angiogenesis, inflammation reduction, and activation of repair signals (see schematic diagram of the multidimensional regeneration process triggered by PST-SeNPs). Figure 16 As shown in L), this demonstrates its comprehensive advantages as a treatment for oral mucositis.

[0148] Example 11: Biosafety assessment of PST-SeNPs

[0149] To systematically evaluate the in vivo safety of PST-SeNPs, this embodiment comprehensively evaluated the mice in each group through hematological tests and histopathological analysis after the 5-day treatment cycle. Figures 19-21 The results of the in vivo safety evaluation were presented; among them, Figure 19 A~D represent the changes in blood biochemical indicators ALT, AST, CRE and BUN after different treatment groups; Figure 20Figures A through H show the changes in blood routine indicators (WBC, Neu%, Lym%, Mon%, RBC, HGB, HCT, and PLT) after different treatment groups; Figure 22 shows representative H&E staining images (Scar bar = 100 μm, n = 4) of major organs: heart, liver, spleen, lungs, and kidneys. Detailed analysis follows:

[0150] 1. Blood biochemical index analysis

[0151] Blood biochemistry test results showed ( Figure 19 (A~D) In ​​all treatment groups, the liver function indicators (ALT, AST) and kidney function indicators (BUN, Cre) of mice remained within the normal physiological range, and no drug-related abnormal changes were observed.

[0152] 2. Blood routine parameter testing

[0153] Blood routine analysis showed that ( Figure 20 All indicators (A~H), including white blood cell count, erythrocyte parameters, and platelets, were within the normal reference range, confirming that PST-SeNPs had no significant effect on hematopoietic function.

[0154] 3. Histopathological evaluation

[0155] H&E staining of major organs (heart, liver, spleen, lungs, kidneys) showed ( Figure 21 In the PST-SeNPs treatment group, all examined organs maintained intact tissue structure, and no pathological changes such as inflammatory cell infiltration, cell degeneration or necrosis were observed. Their morphological characteristics were basically consistent with those of the normal control group.

[0156] Under the dosage and dosing cycle set in this embodiment, PST-SeNPs did not cause significant systemic toxicity or organ-specific damage, demonstrating excellent biocompatibility and in vivo safety, providing important experimental evidence for its clinical translation.

[0157] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A selenium-containing nanoparticle, characterized in that, The device includes a core made of selenium-containing material and a biomimetic adhesive polymer shell covering the surface of the core, wherein the selenium-containing material includes at least one of zero-valent selenium and ferrous selenide; and the biomimetic adhesive polymer includes at least one of polyserotonin, dopamine, and sodium alginate.

2. The selenium-containing nanoparticles according to claim 1, characterized in that, The selenium-containing substance is bonded to the biomimetic adhesive polymer via C-Se covalent bonds.

3. The selenium-containing nanoparticles according to claim 1, characterized in that, The nanoparticles satisfy at least one of the following conditions: (1) The nanoparticles are spherical; (2) The particle size of the nanoparticle core is 10~20nm; (3) The hydrated particle size of the nanoparticles is 80~110nm; (4) The zeta potential of the nanoparticles is -10mV to 0mV.

4. The method for preparing selenium-containing nanoparticles according to any one of claims 1 to 3, characterized in that, Includes the following steps: The monomers of the biomimetic adhesive polymer, sodium selenite, and ferric ion salt were dissolved in water, stirred for 30-40 minutes, and the pH was adjusted to 7.5-9.

5. The hydrothermal reaction was carried out at 60-100℃ for 2-6 hours. After the reaction was completed, the mixture was centrifuged and freeze-dried to obtain the selenium-containing nanoparticles. The molar ratio of the monomer of the biomimetic adhesive polymer to sodium selenite is (2~3):

1.

5. The method according to claim 6, characterized in that, The monomer of the biomimetic adhesive polymer is serotonin; in the hydrothermal reaction, the initial concentration of serotonin is 0.5~1 mmol / L, and the initial concentration of sodium selenite is 0.2~0.5 mmol / L.

6. The use of selenium-containing nanoparticles as described in any one of claims 1 to 3 in the preparation of medicaments for the prevention and / or treatment of inflammatory mucosal diseases.

7. The application as described in claim 6, characterized in that, The inflammatory mucosal diseases include at least one of oral mucositis, esophagitis, gastritis, and ulcerative colitis.

8. The application as described in claim 6, characterized in that, The drug comprises selenium-containing nanoparticles as described in any one of claims 1 to 3 and pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, Based on the total mass of the drug (100%), the mass content of the selenium-containing nanoparticles is 20% to 40%.

10. The application according to claim 9, characterized in that, The dosage form of the drug is a gel, spray, mouthwash, or patch.

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