Hydrogel of selenium-containing polymer compound, preparation method therefor, and use thereof

Abstract

The present invention belongs to the technical field of biomedical engineering, and relates to a hydrogel of a selenium-containing polymer compound, a preparation method therefor, and the use thereof. The hydrogel of a selenium-containing polymer compound is formed by means of dispersing the selenium-containing polymer compound in an aqueous solution of a hydrogel carrier material, and the selenium-containing polymer compound is prepared from raw materials containing R1-R2-Se-R3-R4, a compound of formula (I), a diisocyanate, and mPEG-R5 by means of a reaction. The hydrogel of the selenium-containing polymer compound can be used to further improve the therapeutic effect on skin wounds, especially diabetic skin wounds, and can achieve a better preventive and / or therapeutic effect on oral inflammation, especially peri-implantitis and periodontitis.

Description

A hydrogel containing a selenium polymer compound and its preparation method and use Technical Field

[0001] The invention belongs to the technical field of biomedical engineering and relates to a hydrogel containing a selenium polymer compound and a preparation method and application thereof. Background Art

[0002] The complex microenvironment of diabetic wounds, including high blood sugar, high reactive oxygen species (ROS) and low pH, significantly increases the risk of infection, hinders angiogenesis and delays the healing process. Traditional wound dressings, such as gauze bandages, can stop bleeding, absorb exudate and prevent infection, but they are difficult to promote healing. Hydrogels have become an important research direction for new wet wound dressings due to their excellent biocompatibility, moisture retention and transparency. However, although existing hydrogel dressings can absorb exudate and keep wounds moist, they are difficult to adapt to the unique challenges of diabetic wounds (see Chinese patent application 202111464355.2).

[0003] In order to overcome the limitations of traditional hydrogel dressings in the treatment of diabetic wounds, smart responsive hydrogels have been developed that can precisely respond to the specific environment of diabetic wounds (such as high blood sugar, high reactive oxygen species (ROS) and low pH) or external stimuli (such as temperature, light, and magnetism). However, although these smart hydrogels have achieved precise regulation in drug delivery, avoiding the overuse of antibiotics and thus reducing the risk of drug resistance, most of them are currently still mainly dependent on antibiotics as their core therapeutic ingredient in design (see Chinese patent application CN202111405340.9). The complexity of diabetic wounds goes far beyond bacterial infection, and also includes oxidative stress, slowed cell migration, angiogenesis disorders and other pathophysiological processes. Smart hydrogels that rely solely on antibiotics may not be able to fully meet these challenges.

[0004] Therefore, when developing new smart responsive hydrogels, it is necessary to explore more non-antibiotic active ingredients, such as growth factors, antioxidants, immunomodulators, etc., to achieve a more comprehensive, multi-target treatment strategy.

[0005] Selenium is an essential trace element for the human body. At low doses, it can act as an antioxidant, supporting cell survival and growth, reducing inflammation, and having certain antibacterial effects. Currently, research on selenium-containing polymers focuses on targeted tumor therapy. For example, the patent application "A targeted reduction-sensitive polymer micelle, preparation method, and application" (Application Number: CN202211516175.9) uses high-dose levels of selenium to kill tumor cells. However, the optimal concentration of low-dose selenium in inflammatory environments remains undetermined, and its effect on osteogenesis is also unclear.

[0006] Furthermore, periimplantitis and periodontitis, as common inflammatory diseases in the oral cavity, present complex and variable pathological environments, including high microbial loads, localized high oxidative stress (high levels of reactive oxygen species (ROS)), a low pH environment, and the massive release of inflammatory mediators. The interaction of these unfavorable factors not only increases the risk of infection and leads to rapid inflammation progression, but also severely hinders angiogenesis and osseointegration, leading to destruction of soft and hard tissues around implants and delayed healing, seriously threatening the long-term stability of implants and the oral health of patients.

[0007] Although traditional treatments such as local cleaning and antibiotics can temporarily relieve symptoms, it is difficult to fundamentally solve the deep-seated problems of peri-implantitis and periodontitis. Currently, most dressings used for the prevention and treatment of peri-implantitis and periodontitis on the market are single dosage forms, such as antibacterial coatings (for example, see Chinese patent application CN202111169202.5), gels or rinsing solutions (for example, see Chinese patent application CN202180033519.8). Although they can inhibit bacterial growth or reduce inflammation to a certain extent, they are often difficult to fully cover the complex pathophysiological processes of the oral inflammatory environment.

[0008] Therefore, it is particularly important to develop a new therapy that can accurately respond to the challenges of the microenvironment around the dentition and implants and promote tissue regeneration. Summary of the Invention

[0009] The primary purpose of the present invention is to provide a hydrogel containing a selenium polymer compound to further enhance the therapeutic effect of skin wounds, especially diabetic skin wounds; and to achieve better prevention and / or treatment effects on oral inflammation, especially peri-implantitis and periodontitis.

[0010] To achieve this object, in a basic embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein the hydrogel containing the selenium polymer compound is formed by dispersing the selenium polymer compound in an aqueous solution of a hydrogel carrier material, and the selenium polymer compound is prepared by reacting raw materials comprising R1-R2-Se-R3-R4, a compound of formula (I), a diisocyanate, and mPEG-R5.

[0011] in:

[0012] R1 and R4 are independently selected from hydroxyl or amino;

[0013] R2 and R3 are independently selected from C 2-20 alkyl;

[0014] R5 is selected from hydroxyl or amino;

[0015] R6 and R7 are independently selected from C 1-5 alkyl.

[0016] In a preferred embodiment, the present invention provides a hydrogel of a selenium-containing polymer compound, wherein the average molecular weight of the selenium-containing polymer compound is 10,000-40,000.

[0017] In a preferred embodiment, the present invention provides a hydrogel of a selenium-containing polymer compound, wherein R1 and R4 are the same, and / or R2 and R3 are the same, and / or R6 and R7 are the same.

[0018] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein R2 and R3 are independently selected from C 3-12 alkyl.

[0019] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein the diisocyanate is selected from 2,4-toluene diisocyanate, hexamethylene diisocyanate, diphenylmethane diisocyanate or isophorone diisocyanate.

[0020] In a preferred embodiment, the present invention provides a hydrogel of a selenium-containing polymer compound, wherein the selenium-containing polymer compound is prepared by reacting raw materials comprising R1-R2-Se-R3-R4, 1,4-di(2-hydroxyethyl)piperazine, diisocyanate, and polyethylene glycol monomethyl ether.

[0021] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein the R1-R2-Se-R3-R4 is H2N-(CH2)3-Se-(CH2)3-NH2 or HO-(CH2) 11 -Se-(CH2) 11 -OH.

[0022] In a preferred embodiment, the present invention provides a hydrogel of a selenium-containing polymer compound, wherein the structure of the selenium-containing polymer compound is as shown in the following formula (II) or formula (III):

[0023] wherein m and n are independently selected from natural numbers of 1-250, and x and y are independently selected from natural numbers of 1-100.

[0024] In a preferred embodiment, the present invention provides a hydrogel of a selenium-containing polymer compound, wherein the preparation method of the selenium-containing polymer compound comprises: mixing R1-R2-Se-R3-R4, a compound of formula (I), a diisocyanate, mPEG-R5, and a catalyst in an organic solvent for reaction, and recrystallizing and washing the reaction product.

[0025] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein:

[0026] The organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate and n-butyl acetate;

[0027] The molar ratio of R1-R2-Se-R3-R4, the compound of formula (I), the diisocyanate, and mPEG-R5 is x:1:(1.1x+1.1):(0.2x+0.2), wherein x is 0.1 to 10;

[0028] The catalyst is dibutyltin dilaurate.

[0029] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein the reaction temperature is 30-100° C. and the reaction time is 1-48 hours.

[0030] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein the recrystallization is performed using ether, and the washing is performed using ethanol with a volume percentage concentration of 5-50%.

[0031] In a preferred embodiment, the present invention provides a hydrogel containing a selenium polymer compound, wherein the hydrogel carrier material is selected from one or more of methacrylated gelatin, methacrylated chitosan, methacrylated sodium alginate, methacrylated chondroitin sulfate, hydroxybutyl chitosan, sodium alginate, hyaluronic acid, chondroitin sulfate, polyethylene glycol diacrylate, and polyvinyl alcohol.

[0032] In a preferred embodiment, the present invention provides a hydrogel containing a selenium-containing polymer compound, wherein the concentration of the selenium-containing polymer compound in the hydrogel is 0.2-5 μg / mL (preferably 0.5-2 μg / mL); the concentration of the hydrogel carrier material in the hydrogel is 3-10 wt%.

[0033] The second object of the present invention is to provide a method for preparing the hydrogel of the selenium-containing polymer compound as described above, so as to better prepare the hydrogel of the selenium-containing polymer compound as described above.

[0034] To achieve this object, in a basic embodiment, the present invention provides a method for preparing a hydrogel of the selenium-containing polymer compound as described above, the preparation method comprising: dissolving the selenium-containing polymer compound as described above in an organic solvent, then adding it to an aqueous solution of a hydrogel carrier material, dispersing to obtain an injectable hydrogel, and heating to obtain a solidified hydrogel.

[0035] In a preferred embodiment, the present invention provides a method for preparing a hydrogel containing a selenium polymer compound as described above, wherein:

[0036] The organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide;

[0037] The concentration of the selenium-containing polymer compound after being dissolved in an organic solvent is 1-10 mg / mL.

[0038] In a preferred embodiment, the present invention provides a method for preparing a hydrogel containing a selenium polymer compound as described above, wherein:

[0039] The selenium-containing polymer compound is dissolved in an organic solvent and then added to an aqueous solution of a hydrogel carrier material, and the temperature of the injectable hydrogel obtained after dispersion is 2-10°C;

[0040] The heating is to 35-40°C.

[0041] The third object of the present invention is to provide the use of the hydrogel of the selenium-containing polymer compound as described above for preparing skin wound dressings, or for preparing a medicament for preventing and / or treating oral inflammation, so that after the hydrogel of the selenium-containing polymer compound as described above is used as a skin wound dressing, the therapeutic effect of skin wounds, especially diabetic skin wounds, can be further improved; or it can have a better preventive and / or therapeutic effect on oral inflammation, especially peri-implantitis and periodontitis.

[0042] To achieve this object, in a basic embodiment, the present invention provides the use of the hydrogel of the selenium-containing polymer compound as described above for preparing a skin wound dressing, or for preparing a medicament for preventing and / or treating oral inflammation.

[0043] In a preferred embodiment, the present invention provides the use of the hydrogel containing selenium polymer compound as described above for preparing skin wound dressing, or for preparing a medicament for preventing and / or treating oral inflammation, wherein the skin wound is a diabetic skin wound.

[0044] In a preferred embodiment, the present invention provides the use of the hydrogel of the selenium-containing polymer compound as described above for preparing a skin wound dressing, or for preparing a medicament for preventing and / or treating oral inflammation, wherein the oral inflammation is peri-implantitis and / or periodontitis.

[0045] The beneficial effects of the present invention are that the hydrogel containing the selenium polymer compound of the present invention can further enhance the therapeutic effect of skin wounds, especially diabetic skin wounds; and can achieve better prevention and / or treatment effects on oral inflammation, especially peri-implantitis and periodontitis.

[0046] The beneficial effects of the present invention are specifically embodied in:

[0047] (1) The hydrogel of the selenium-containing polymer compound of the present invention is thermosensitive and injectable. The selenium element it contains, as an essential trace element for the human body, has significant antioxidant function, so that it will not produce antibiotic resistance, and is expected to provide a more efficient and safe healing solution for the high ROS environment of diabetic wounds. The hydrogel of the selenium-containing polymer compound of the present invention is thermosensitive, gels at body temperature, and can stably and efficiently release selenium in a relatively short period of time, which meets the soft tissue healing time window. The hydrogel of the selenium-containing polymer compound of the present invention has the effect of promoting adhesion, extension and migration of human skin fibroblasts. The hydrogel of the selenium-containing polymer compound of the present invention can exert anti-inflammatory and antioxidant effects, relieve inflammation of skin area tissue (inhibit inflammatory gene expression, promote the expression of TGFβ), and promote wound healing. In a diabetic environment, the hydrogel of the selenium-containing polymer compound of the present invention can efficiently promote the healing of diabetic skin wounds.

[0048] (2) The hydrogel of the selenium-containing polymer compound of the present invention can promote the adhesion, extension and migration of human gingival fibroblasts, inhibit the expression of inflammatory genes, and achieve comprehensive prevention and treatment of peri-implantitis and periodontitis. The hydrogel dosage form is applied to the surface of the implant after inflammation occurs or in the "periodontal pocket" around the implant. The hydrogel not only has good biocompatibility and moisture retention, but can also maintain a local moist environment, promote cell migration and angiogenesis, and the selenium element therein can also be continuously released in the microenvironment around the implant, through the synergistic effects of multiple pathways such as anti-oxidation, anti-inflammatory, and promotion of tissue repair, thereby comprehensively improving the pathological state of peri-implantitis and periodontitis, accelerating the healing process, and playing a role in treating peri-implantitis and periodontitis. Therefore, the present invention can meet different needs and is suitable for the prevention and treatment of peri-implantitis and periodontitis. In summary, the present invention demonstrates significant advantages in the prevention and treatment of peri-implantitis and periodontitis. By precisely responding to the complex pathophysiological processes around implants, it can not only relieve inflammation and promote soft tissue regeneration, but also effectively avoid excessive use of antibiotics and drug resistance problems, thereby providing a safer, more efficient and comprehensive solution for the treatment of peri-implantitis and periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 shows the characterization results of the selenium-containing small molecule prepared in Example 1, wherein Figure 1a shows the structure of the selenium-containing small molecule; Figure 1b shows the H NMR spectrum; Figure 1c shows the C NMR spectrum; Figure 1d shows the characterization results of the Se NMR spectrum; Figure 1e shows the experimental results of electrospray ionization mass spectrometry; and Figure 1f shows the peak shape of the theoretical simulation of the electrospray ionization mass spectrometry. Predicted molecular weight [M+Na] + :445.26, experimentally determined molecular weight [M+Na] + :445.25.

[0050] FIG2 is the nuclear magnetic resonance hydrogen spectrum detection result of the selenium-containing polymer compound prepared in Example 1, wherein SePU1 is a 25% Se selenium-containing polymer compound, SePU2 is a 50% Se selenium-containing polymer compound, and SePU3 is a 75% Se selenium-containing polymer compound.

[0051] FIG3 is the gel permeation chromatography test results of the selenium-containing polymer compound prepared in Example 1, wherein SePU1 is a 25% Se selenium-containing polymer compound, SePU2 is a 50% Se selenium-containing polymer compound, and SePU3 is a 75% Se selenium-containing polymer compound.

[0052] Figure 4 is a diagram showing the characterization results of the hydrogel containing a selenium polymer compound in Example 3, wherein Figure a is a diagram showing the temperature response results of the hydrogel containing a selenium polymer compound; and Figure b is a diagram showing the release ratio of selenium in the simulated body fluid of the hydrogel containing a selenium polymer compound.

[0053] FIG5 is a diagram showing the cell compatibility (cytotoxicity) test results of the selenium-containing polymer hydrogel of Example 4.

[0054] Figure 6 shows the results of in vitro fibroblast extension and migration detection of the hydrogel of selenium-containing polymer compounds in Example 5, wherein a is a cytoskeleton fluorescence staining image of human skin fibroblasts (HSFs) by hydrogels of selenium-containing polymer compounds at different concentrations; b is a quantitative analysis image of the average cell area of ​​cytoskeleton fluorescence staining; c is a horizontal migration scratch image of HSFs by hydrogels of selenium-containing polymer compounds at different concentrations; and d is a quantitative image of migration width.

[0055] Figure 7 shows the results of the in vitro regulation test of the adhesion and inflammation-related genes by the hydrogel of the selenium-containing polymer compound in Example 6, wherein a is the result of qPCR detection of the expression of adhesion-related genes IGTA1 and IGTB1 in the hydrogel of selenium-containing polymer compound of different concentrations; b is the result of qPCR detection of the expression of inflammation-related genes TNFα and TGFβ.

[0056] FIG8 is a graph showing the results of an experiment on promoting skin healing in diabetic mice using the selenium-containing polymer hydrogel of Example 7, wherein a is a graph showing the results of skin healing in diabetic mice; and b is a graph showing the quantitative analysis results of the skin healing rate.

[0057] Figure 9 shows the Micro-CT scan and bone quantitative analysis results of the selenium-containing polymer hydrogel in Example 8 for preventing and treating periodontitis in vivo, wherein a is the Micro-CT image and three-dimensional modeling image of the maxillary bone for preventing and treating periodontitis by the selenium-containing polymer hydrogel; b is the quantitative analysis result of the enamel-cementum junction and the alveolar ridge top difference (ECJ-ABC); and c is the bone quantitative analysis result. DETAILED DESCRIPTION

[0058] The specific embodiments of the present invention are further described below through examples.

[0059] Example 1: Preparation and characterization of selenium-containing polymer compounds

[0060] 1.0 g of selenium powder (0.0114 mol) and 2.0 g of sodium borohydride (0.0528 mol) were added to a 250 mL round-bottom flask. 30 mL of water was slowly added dropwise in an ice-water bath. After the reaction gradually subsided and gas production ceased, the flask was sealed with a rubber stopper fitted with a balloon to obtain a colorless, transparent solution. This solution was then brought to room temperature and used for further processing. 5.7 g of 11-bromo-1-undecanol (0.0227 mol) was dissolved in 40 mL of tetrahydrofuran to obtain a solution. The prepared 11-bromo-1-undecanol solution was added to the colorless, transparent solution using a syringe and syringe with stirring. The reaction was heated at 50°C for 6 h. After the reaction was completed, the reaction apparatus was cooled to room temperature. Dichloromethane and deionized water were added to the remaining mixture, and the organic phase was separated and collected. The aqueous phase was washed with dichloromethane until colorless, and the organic phase solutions were combined. Anhydrous sodium sulfate was added for drying, and the anhydrous sodium sulfate was removed by filtration. Most of the dichloromethane was removed by rotary evaporation to obtain a concentrated solution, which was then added dropwise into vigorously stirred petroleum ether for recrystallization. Filtering afforded a pale yellowish-white solid product. The molecular structure of the resulting selenium-containing small molecule is shown in Figure 1a. The H NMR, C NMR, and Se NMR spectra are shown in Figures 1b, 1c, and 1d, respectively. The electrospray ionization mass spectrometry experimental results and the peak shapes simulated by electrospray ionization mass spectrometry are shown in Figures 1e and 1f, respectively.

[0061] Results of H NMR spectrum of selenium-containing small molecules: 1 H NMR(400MHz,Chloroform-d)δ3.63(dt,J1=J2=6.1Hz,4H,-CH2-OH),2.54(t,J=7.5Hz,4H,-Se-CH2-),1.70-1.60(p, J=6.8Hz,4H,-CH2CH2-OH),1.56(p,J=6.8Hz,4H,-Se-CH2CH2-),1.42-1.22(m,28H,-Se-CH2CH2(CH2)7CH2CH2-OH).

[0062] Results of C-NMR spectrometry of selenium-containing small molecules: 13 C NMR(101MHz,Chloroform-d)δ63.22(-CH2-OH),32.93(-CH2CH2-OH),30.80(-CH2CH2CH2-OH),30.11(-CH2CH2CH2CH2-OH),29 .71-29.55(-Se-CH2CH2CH2-(CH2)4-CH2CH2CH2CH2-OH),29.29(-Se-CH2CH2CH2-),25.86(-Se-CH2CH2-),24.13(-Se-CH2-).

[0063] Results of NMR selenium spectrum test of selenium-containing small molecules: 77 Se NMR(76MHz,Chloroform-d)δ154.90.

[0064] 1.0 mmol of 1,4-bis(2-hydroxyethyl)piperazine and 1.0 mmol of the selenium-containing small molecule HO-(CH2) prepared above were added to 8 mL of tetrahydrofuran. 11 -Se-(CH2) 11 -OH, and 383.2 mg of 2,4-diisocyanatotoluene (2,4-TDI, 2.2 mmol) (see Table 1 below for specific proportions and amounts), along with 20 μL of dibutyltin dilaurate, were added. The reaction vessel was sealed with a rubber stopper, and nitrogen was purged through the system for 5 minutes to remove air from the system. The reaction was then heated at 50°C overnight. Subsequently, 0.8 g of dried polyethylene glycol monomethyl ether (weight-average molecular weight = 2000, 0.4 mmol) was weighed and dissolved in 5 mL of tetrahydrofuran. This was added to the reaction system via syringe and needle, and the reaction was continued at 50°C for 24 hours. After the reaction, most of the tetrahydrofuran was removed by rotary evaporation to obtain a concentrated solution. The solution was then added dropwise into vigorously stirred icy ether for recrystallization. The product was collected by filtration to obtain an egg yolk-white viscous solid, washed five times with a 10% ethanol solution, and lyophilized to remove moisture. The structure of the purified polymer is shown in Formula (III). It was characterized by proton nuclear magnetic resonance spectroscopy and gel permeation chromatography (Figures 2 and 3, respectively).

[0065] The results of H-NMR detection of selenium-containing polymer compounds, taking SePU2 (50% Se) as an example: 1 H NMR(400MHz,Chloroform-d)δ4.33(b,-NH2COO-CH2CH2-N),4.11(b,-NH2COO-CH2(CH2)9CH2-Se-),3.64(b,-OCH2CH2-of mPEG),3.40(b,CH3-OCH2CH2-of mPEG),2.86(b,-NH2COO-CH2CH2-N)2.54(b,-CH2-Se-CH2-),2.18(b,-N(CH2CH2)2N-),1.64( b,-NH2COO-CH2CH2(CH2)7CH2CH2-Se-),1.30-1.25(b,-NH2COO-CH2CH2(CH2)7CH2CH2-Se-).

[0066] Table 1

[0067] Example 2: Preparation of a hydrogel solution containing a selenium polymer compound

[0068] (1) Preparation of hydroxybutyl chitosan (HBC)

[0069] Weigh 1g of chitosan powder and disperse it in a 50% w / w NaOH solution for alkalization. Stir thoroughly at room temperature for 48 hours, then filter to remove excess alkali. Filter the alkalized chitosan through nylon cloth, washing the NaOH with MilliQ water during filtration to remove excess water. Place the filtered chitosan in a glass bottle, add 20mL of a 1:1 v / v isopropanol / water solution, and stir at room temperature to completely disperse the chitosan in the isopropanol / water system. Slowly add an equal volume of 1,2-butylene oxide dropwise to the glass bottle, and place the bottle in a heated magnetic stirrer at 55°C for thorough reaction. After the reaction is complete, cool to room temperature and adjust the pH to neutral by adding HCl solution. Place the neutral solution in a dialysis bag and dialyze against distilled water for 3 days; the solution will become increasingly transparent during dialysis. After dialysis, the dialyzed liquid was taken out, filtered through nylon cloth to remove insoluble impurities, divided into 50 mL centrifuge tubes, placed in a -20°C refrigerator and frozen overnight, and freeze-dried for about 2-3 days to obtain freeze-dried HBC.

[0070] (2) Preparation of hydrogel solution containing selenium polymer compound

[0071] The selenium-containing polymer compound prepared in Example 1 (25% SePU, hereinafter referred to as SePU) was weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain a 10 mg / mL SePU solution. The 10 mg / mL SePU solution was then diluted with DMSO to five different concentrations of SePU: 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, and 5.0 mg / mL, as needed. The lyophilized HBC prepared above was weighed and dissolved in sterile PBS under ice bath conditions to obtain a 5 wt% HBC solution. The five SePU solutions were then mixed with the 5 wt% HBC solution on ice at a volume ratio of 1:1000. After mixing, the five hydrogel solutions of the selenium-containing polymer compound were obtained, with final SePU concentrations of 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, and 5.0 μg / mL, respectively.

[0072] Example 3: Characterization of Selenium-Containing Polymer Hydrogels

[0073] (1) Characterization of temperature response of hydrogels containing selenium polymer compounds

[0074] The hydrogel solution (2.0 μg / mL) of the selenium-containing polymer compound prepared in Example 2 was placed at 4°C and 37°C to evaluate its temperature-responsive sol-gel transformation process. The hydrogel solution's state at different temperatures was captured by photographing the transition from a flowable liquid to a non-flowable gel. The results are shown in Figure 4a. As shown in Figure 4a, the hydrogel exhibits fluidity at 4°C, but rapidly solidifies into a gel at 37°C, losing its fluidity.

[0075] (2) Release of selenium from hydrogels containing selenium polymers in simulated body fluids

[0076] The hydrogel solution (2.0 μg / mL) of the selenium-containing polymer compound prepared in Example 2 was solidified at 37°C and then immersed in a simulated body fluid with a pH of 7.4 (Coollebo, China, Product No.: SL6710). Samples were taken at different immersion time points from 0 to 48 hours, and the concentration of released selenium was detected by ultraviolet spectrophotometry (detection wavelength of 231 nm), and the selenium release rate was calculated (NanoDrop8000 ultramicro UV spectrophotometer (ThermoFisher, USA) was used to construct a standard curve, and the concentration of selenium in each sample was detected to calculate the release rate). The results are shown in Figure 4b, indicating that selenium can be released to reach a plateau within 48 hours, and 70% of the selenium-containing polymer compound in the hydrogel can be released.

[0077] Example 4: Cytocompatibility testing of hydrogels containing selenium polymer compounds

[0078] The cytocompatibility of the selenium-containing polymer hydrogels was evaluated using a cell proliferation and toxicity assay kit (Solaibao, China). Human skin fibroblasts (HSFs) were seeded in 96-well plates and treated with different concentrations of selenium-containing polymer hydrogels in cell proliferation medium (DMEM, 10% FBS, 1% penicillin-streptomycin) to obtain extracts (hydrogel:proliferation medium volume ratio = 1:10, immersion for 24 hours). Cell proliferation in the extracts of the selenium-containing polymer hydrogels at different concentrations was assessed using a cell proliferation and toxicity assay kit (CCK-8) on days 0, 1, 3, 5, and 7 of culture. After incubation of HSFs with CCK-8 for 1 hour, the absorbance at 405 nm was measured using an enzyme-linked immunosorbent assay (ELISA) to analyze cell proliferation and cytotoxicity. The results are shown in Figure 5. As can be seen from Figure 5, the hydrogels of selenium-containing polymer compounds at all concentrations had no obvious cytotoxicity, among which the concentrations of 0.2 μg / mL and 0.5 μg / mL had no significant effect on cell proliferation compared with the control (5wt% HBC without selenium-containing polymer compounds); while the concentrations of 1.0 μg / mL, 2.0 μg / mL and 5.0 μg / mL could promote cell proliferation.

[0079] Example 5: Selenium-containing polymer hydrogel promotes fibroblast expansion and migration in vitro

[0080] Cytoskeleton fluorescence staining was used to evaluate the effect of selenium-containing polymer hydrogels on fibroblast extension. Specifically, 4',6-diamidino-2-phenylindole (DAPI) was used to label the cell nucleus, and phalloidin was used to label the cytoskeleton. HSFs were photographed using a TE2000-U inverted fluorescence microscope (Nikon, Japan) coupled with F3.0 digital photography system software (NIS-Elements, Japan). The fluorescence color of the cell nucleus was blue, and the fluorescence color of the cytoskeleton was green. The results are shown in Figure 6a. As shown in Figure 6a, the cell extension of the hydrogel containing a selenium-containing polymer at a concentration of 0.2 μg / mL was worse than that of the control, while the cell extension of the hydrogel containing a selenium-containing polymer at concentrations of 1.0 μg / mL, 2.0 μg / mL, and 5.0 μg / mL was better. The average cell area was quantitatively analyzed using ImageJ, and the results are shown in Figure 6b. As can be seen from Figure 6b, the hydrogels containing selenium-containing polymers at concentrations of 1.0 μg / mL, 2.0 μg / mL and 5.0 μg / mL showed statistical differences compared with the control (5 wt% HBC without the addition of selenium-containing polymers), indicating that the hydrogels containing selenium-containing polymers can promote the expansion of human skin fibroblasts (HSFs) in vitro.

[0081] A cell scratch test was used to evaluate the effect of selenium-containing polymer hydrogels on the horizontal migration of fibroblasts. The specific method was as follows: HSFs were evenly seeded into a 6-well plate. When the cell confluence reached 100%, a 200μL plastic pipette tip was used to scratch the bottom of the well plate perpendicularly, forming gaps of uniform width between the cells. A TE2000-U inverted fluorescence microscope (Nikon, Japan) equipped with F3.0 digital photography system software (NIS-Elements, Japan) was used to photograph the HSFs in bright field at 0 and 24 hours after the scratch. The results after 24 hours of scratching are shown in Figure 6c. As shown in Figure 6c, except for the hydrogel with a concentration of 0.2μg / mL of selenium-containing polymer, the hydrogels with other concentrations of selenium-containing polymer all showed more significant migration than the control (5wt% HBC without selenium-containing polymer), among which the hydrogel with a concentration of 2.0μg / mL of selenium-containing polymer had the fastest migration rate. ImageJ was used to quantify the average scratch width and calculate the migration rate, as shown in Figure 6d. As shown in Figure 6d, the selenium-containing polymer compounds at concentrations of 2.0 μg / mL and 5.0 μg / mL were statistically different from the control, suggesting that the selenium-containing polymer compounds can promote the horizontal migration of HSFs in vitro.

[0082] Example 6: In vitro regulation of adhesion and inflammation-related genes by hydrogels containing selenium polymers

[0083] Real-time fluorescence quantitative PCR (qPCR) was used to evaluate the effects of selenium-containing polymer hydrogels on the expression of genes related to adhesion and inflammation in HSFs. The expression levels of adhesion- and inflammation-related genes were measured, respectively. The results, shown in Figures 7a and 7b, show that the 2.0 μg / mL selenium-containing polymer hydrogel showed statistically significant differences compared to the control (5wt% HSFs without selenium-containing polymers), with the most significant effect. The expression levels of adhesion-related genes ITGA1 and ITGB1 were significantly upregulated, the expression level of the pro-inflammatory gene TNFα was downregulated, and the expression level of the anti-inflammatory gene TGFβ was significantly upregulated, indicating that the selenium-containing polymer hydrogel can promote cell adhesion and inhibit cellular inflammation in vitro.

[0084] Example 7: Experiment on the effect of hydrogel containing selenium polymer compound on promoting skin healing in diabetic mice

[0085] A 10mm diameter full-thickness skin defect model was created using 6-8 week old male db / db C57BLKS / J mice. The skin defect was located in the central back, at the natural protrusion of the spine. The mice were divided into blank, HBC, and HBC + SePU (SePU / HBC) groups, with six mice in each group. Based on in vitro results, the optimal SePU concentration in the selenium-containing polymer hydrogel for the HBC + SePU group in in vivo experiments was 2.0 μg / mL. 100 μL of the hydrogel, pre-cooled at 4°C, was evenly applied to the full-thickness skin defect using a syringe. Due to body temperature, the hydrogel solution rapidly cross-linked through a sol-gel reaction to form a hydrogel. Skin wounds were photographed on days 0, 3, 7, 10, and 14 after surgery (Figure 8a). As shown in Figure 8a, the group treated with the selenium-containing polymer hydrogel (HBC + SePU) healed faster than the other two groups. Wound area changes were analyzed using ImageJ, and the healing rate was calculated (Figure 8b). As shown in Figure 8b, there was a statistical difference between the HBC+SePU group and the other two groups, suggesting that the hydrogel containing selenium polymer compounds can promote skin wound healing in diabetic mice.

[0086] Example 8: In vivo study of the prevention and treatment of periodontitis using a hydrogel containing a selenium polymer compound

[0087] Male SD rats aged 6-8 weeks were used and divided into 5 groups: healthy group (Blank), periodontitis model group (Control), prevention group (Prevent), treatment group (Treatment) and positive control group (Periocline), with 3 rats in each group. A periodontitis model was prepared by ligating the maxillary first molars bilaterally with orthodontic ligatures (0.2 mm in diameter) and injecting 200 μL of 1 mg / mL LPS solution (once a week for 4 weeks) to create the model. At the same time as the modeling, 10 minutes after LPS injection, 500 μL of 1.0 μg / mL selenium-containing polymer hydrogel was used for the prevention of periodontitis, so that the hydrogel could evenly cover the periodontal tissue (once a week until sampling, prevention group). After modeling, the ligature and surrounding plaque were removed. A 500 μL solution of a 1.0 μg / mL selenium-containing polymer hydrogel was applied for periodontitis treatment (once weekly until sampling). A positive control group (500 μL, once weekly) was also administered minocycline hydrochloride ointment (Periocline), a clinically used antibiotic for periodontitis treatment. This allowed the hydrogel and Periocline ointment to evenly cover the periodontal tissue. Two weeks after treatment, samples were collected for micro-CT analysis. As shown in Figure 9a, micro-CT scans revealed decreased alveolar bone density in the periodontitis modeling group compared to the healthy group, indicating successful periodontitis modeling. The prevention group showed a significant preventive effect compared to the periodontitis modeling group, with alveolar bone density only slightly lower than that of the healthy group. A comparison of the treatment group with the positive control group revealed no significant difference in alveolar bone density between the selenium-containing polymer hydrogel and the periodontitis drug Periocline, demonstrating the positive therapeutic effect of the selenium-containing polymer hydrogel. As shown in Figure 9b, 3D modeling visually demonstrates the distance between the enamel-cemental junction (ECJ) and the alveolar bone crest (ABC) (ECJ-ABC). Consistent with the results of Micro-CT, the prevention group was able to prevent the progression of periodontitis, with the ECJ-ABC being significantly smaller than that of the periodontitis modeling group, the treatment group, and the positive control group. There was no significant difference in efficacy between the treatment group and the positive control group. As shown in Figure 9c, bone quantitative analysis (BV / TV) results showed that the prevention group effectively alleviated bone loss, while there was no significant difference between the treatment group and the positive control group, indicating that the selenium-containing polymer hydrogel can prevent and treat periodontitis in vivo.

[0088] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these changes and variations. The above embodiments or implementation methods are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described implementation methods should be regarded as illustrative and not restrictive in any respect. The scope of the present invention should be described by the appended claims, and any changes that are equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A hydrogel of a selenium-containing polymer compound, characterized in that: The hydrogel of the selenium-containing polymer compound is formed by dispersing the selenium-containing polymer compound in an aqueous solution of a hydrogel carrier material. The selenium-containing polymer compound is prepared by reacting raw materials including R1-R2-Se-R3-R4, a compound of formula (I), a diisocyanate, and mPEG-R5. Wherein: R1 and R4 are each independently selected from a hydroxyl group or an amino group; R2 and R3 are each independently selected from C 2-20 alkyl; R5 is selected from a hydroxyl group or an amino group; R6 and R7 are each independently selected from C 1-5 alkyl groups.

2. The hydrogel of the selenium-containing polymer compound according to claim 1, characterized in that: R1 and R4 are the same, and / or R2 and R3 are the same, and / or R6 and R7 are the same.

3. The hydrogel of the selenium-containing high molecular compound according to claim 1, characterized in that: The diisocyanate is selected from tolylene 2,4-diisocyanate, hexamethylene diisocyanate, diphenylmethane diisocyanate or isophorone diisocyanate.

4. The hydrogel of the selenium-containing polymer compound according to claim 1, characterized in that: The structure of the selenium-containing polymer compound is as shown in the following formula (II) or formula (III). Wherein m and n are each independently selected from natural numbers from 1 to 250, and x and y are each independently selected from natural numbers from 1 to 100.

5. The hydrogel of the selenium-containing polymer compound according to any one of claims 1-4, characterized in that, The method for preparing the selenium-containing polymer compound comprises: reacting R1-R2-Se-R3-R4, the compound of formula (I), a diisocyanate, mPEG-R5, and a catalyst in an organic solvent, and subjecting the reaction product to recrystallization and washing.

6. The hydrogel of the selenium-containing polymer compound according to claim 5, characterized in that: The organic solvent is selected from one or more of tetrahydrofuran, N,N-dimethylformamide, dichloromethane, chloroform, 1,2-dichloroethane, ethyl acetate and n-butyl acetate; The molar ratio of R1-R2-Se-R3-R4, the compound of formula (I), the diisocyanate, and mPEG-R5 is x:1:(1.1x + 1.1):(0.2x + 0.2), where x is 0.1 to 10; The catalyst is dibutyltin dilaurate.

7. The hydrogel of the selenium-containing polymer compound according to claim 5, characterized in that: The reaction temperature is 30-100 °C, and the reaction time is 1-48 hours.

8. The hydrogel of the selenium-containing high molecular compound according to claim 1, characterized in that: The hydrogel carrier material is selected from one or more of methacrylated gelatin, methacrylated chitosan, methacrylated sodium alginate, methacrylated chondroitin sulfate, hydroxybutyl chitosan, sodium alginate, hyaluronic acid, chondroitin sulfate, polyethylene glycol diacrylate, and polyvinyl alcohol.

9. The hydrogel of the selenium-containing high molecular compound according to claim 1, characterized in that: The concentration of the selenium-containing polymer compound in the hydrogel is 0.2-5 μg / mL; the concentration of the hydrogel carrier material in the hydrogel is 3-10 wt%.

10. A method for preparing a hydrogel of a selenium-containing high molecular compound according to any one of claims 1-9, characterized in that, The preparation method comprises: dissolving the selenium-containing polymer compound in an organic solvent, and then adding it to an aqueous solution of the hydrogel carrier material, and dispersing to obtain an injectable hydrogel, and heating to obtain a cured hydrogel.

11. The preparation method according to claim 10, characterized in that: The organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide; The concentration of the selenium-containing polymer compound dissolved in the organic solvent is 1-10 mg / mL.

12. The preparation method according to claim 10, characterized in that: The temperature for dissolving the selenium-containing polymer compound in the organic solvent and then adding it to the aqueous solution of the hydrogel carrier material and dispersing to obtain an injectable hydrogel is 2-10 °C; The heating is heating to 35-40 °C.

13. Use of the hydrogel of the selenium-containing polymer compound according to any one of claims 1-9 for preparing a skin wound dressing, or for preparing an agent for preventing and / or treating oral inflammation.

14. The use according to claim 13, characterized in that: The skin wound is a diabetic skin wound.

15. The use according to claim 13, wherein: The oral inflammation is peri-implantitis and / or periodontitis.

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