Preparation method and application of tremella polysaccharide intelligent hydrogel for inhibiting ferroptosis

Abstract

The invention provides a preparation method and application of tremella polysaccharide intelligent hydrogel capable of inhibiting ferroptosis, and relates to the technical field of biological material preparation, and the tremella polysaccharide intelligent hydrogel capable of inhibiting ferroptosis is prepared from methacrylated tremella polysaccharide TrepMA, oxidized tremella polysaccharide O-Trep and ketal thiol diamine TK-NH2. The tremella polysaccharide intelligent hydrogel prepared in the invention presents a three-dimensional network structure, has good mechanical properties, and can load growth factors or active molecules to accelerate bone regeneration. In addition, the tremella polysaccharide intelligent hydrogel can inhibit ferroptosis and promote healing of bone defects accompanied by osteoporosis. The tremella polysaccharide intelligent hydrogel prepared by the invention shows various biological activities, not only provides a certain supporting effect for bone regeneration, but also has the effects of inhibiting ferroptosis, improving local osteoporosis, resisting oxidation / inflammation, inducing osteogenic differentiation and the like through the excellent biocompatibility, biological activity and drug controlled release effect of the tremella polysaccharide intelligent hydrogel; and bone tissue regeneration can be effectively promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterial preparation, and in particular to a method for preparing a Tremella polysaccharide intelligent hydrogel for inhibiting ferroptosis and an application thereof. Background Art

[0002] In recent years, the incidence of bone injuries has continued to rise, which has driven the growth of demand for bone repair. The bone repair process mainly includes three intersecting stages: inflammation, bone formation, and bone remodeling. At present, the "gold standard" for clinical treatment of bone defects is still based on autologous / allogeneic bone transplantation or artificial bone transplantation. Due to factors such as donor restrictions, immune rejection and infection, scar healing, delayed healing, and even poor bone connection are inevitable. In addition, autologous bone transplantation has limited sources and may induce immune responses, which brings great trouble to clinical results. With the development of biomaterials, the design of new tissue engineering scaffolds with biological activity and function based on natural materials has gradually become a research hotspot. Among them, hydrogel, as a promising material, can effectively promote the exchange of nutrients due to its porous three-dimensional network structure similar to the extracellular matrix. At the same time, it has certain mechanical properties and can provide filling and support for bone defects. However, given the high incidence of osteoporosis in the aging population, the healing of bone defects accompanied by osteoporosis has gradually become a clinical problem. Bone loss caused by osteoporosis not only reduces the biomechanical properties of bone tissue, increases the incidence of fractures, but also reduces the potential for bone regeneration, leading to further slowing of healing. Currently, the development of new materials that can improve bone defects associated with osteoporosis remains to be studied.

[0003] Studies have shown that osteoporosis is accompanied by an imbalance in human iron metabolism. A large amount of iron cannot be metabolized and accumulates in the bones, causing iron overload in osteoblasts. Osteoblasts are prone to ferroptosis under iron overload conditions. Iron overload can significantly inhibit the differentiation ability of osteoblasts and weaken their functions, thereby causing an imbalance in bone metabolism in the body and ultimately leading to osteoporosis. In addition, ferroptosis can further aggravate bone formation disorders by regulating key signaling pathways in osteoblasts. Osteoclasts are responsible for the bone resorption process and degrade bone matrix by secreting acidic substances and enzymes. Under iron overload conditions, osteoclast activity is enhanced and bone resorption is aggravated, resulting in a rate of bone loss exceeding the rate of new bone formation, thereby accelerating the development of osteoporosis. Osteoporosis is a metabolic bone disease caused by an imbalance in bone homeostasis, which is mainly regulated by osteoblasts and osteoclasts. Due to the inhibition of osteogenic differentiation, overactive osteoclasts and impaired angiogenesis, the repair of osteoporotic bone defects becomes more difficult, and a single treatment strategy is difficult to achieve a good repair effect. Therefore, the development of bioactive materials and treatment strategies based on the concept of multiple repairs is of great significance for this type of disease. Summary of the invention

[0004] The object of the present invention is to solve the problem in the prior art that due to the inhibition of osteogenic differentiation, the over-activity of osteoclasts and the impairment of angiogenesis ability, the repair of osteoporotic bone defects becomes more difficult, and a single treatment strategy is difficult to achieve a good repair effect.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] An intelligent tremella polysaccharide hydrogel with ferroptosis inhibition, which is prepared by using methacrylated tremella polysaccharide TrepMA, oxidized tremella polysaccharide O-Trep and ketothiol diamine TK-NH2.

[0007] Preferably, the TrepMA is obtained by reacting a tremella polysaccharide raw material with methacrylic anhydride, and the O-Trep is synthesized based on the oxidation of tremella polysaccharide by sodium periodate.

[0008] Preferably, the preparation method of the intelligent tremella polysaccharide hydrogel is as follows:

[0009] S1: Synthesis of TrepMA

[0010] Dissolve tremella polysaccharide in deionized water to prepare a tremella polysaccharide solution. Subsequently, add methacrylic anhydride to the tremella polysaccharide solution to form a mixed solution, stir the mixed solution and adjust the pH of the mixed solution to 8.0. Then, place the mixed solution in an ice bath for reaction. After the reaction is completed, use a dialysis bag to dialyze the mixed solution in deionized water to remove unreacted methacrylic anhydride. After dialysis is completed, freeze-dry to obtain solid TrepMA;

[0011] S2: Synthesis of O-Trep:

[0012] Prepare a tremella polysaccharide solution, then add sodium periodate to the tremella polysaccharide solution to form a mixed solution, stir the mixed solution, and then place the mixed solution at room temperature for reaction. Use a dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove sodium periodate. After dialysis is completed, freeze-dry to obtain solid O-Trep;

[0013] S3: Preparation of hydrogel precursor solution:

[0014] Dissolve the TrepMA synthesized in S1 and the O-Trep synthesized in S2 in deionized water, then add a photoinitiator, TK-NH2 and an active molecule, and ultrasonically vibrate for 5 minutes to fully mix. The resulting mixed solution is the hydrogel precursor solution. The subsequent operation must be carried out within 1 hour after the preparation of the precursor solution is completed;

[0015] S4: Preparation of DTrep

[0016] The hydrogel precursor solution obtained in S3 is filled into a syringe, and the solution is injected into a mold or a bone defect site. After irradiating the solution with ultraviolet light at a wavelength of 405 nm for 1 - 60 seconds, a cured DTrep composite hydrogel is obtained.

[0017] Preferably, the molecular weight of the tremella polysaccharide selected in S1 and S2 is above 3000 Da, and the tremella polysaccharide is dissolved in deionized water with a water bath heating at 50 °C to promote dissolution. The concentration of the tremella polysaccharide solution prepared in S1 and S2 is 0.5% - 6% wt.

[0018] Preferably, the concentration of methacrylic anhydride in the mixed solution in S1 is 1% - 20% wt, the concentration of sodium periodate in the mixed solution in S2 is 0.5 - 3 mg / mL, and the stirring rate of the mixed solution in S1 and S2 is 300 rpm.

[0019] Preferably, a 1 M sodium hydroxide solution is used in S1 to adjust the pH of the mixed solution.

[0020] Preferably, the added concentration of TrepMA in S3 is 3% - 8% wt, and the mass ratio of TrepMA to O - Trep is 1:1 - 1:3.

[0021] Preferably, the photoinitiator in S3 is lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate, and its concentration is 1% - 5% wt. The concentration of TK - NH2 is 0.05 - 0.2 mg / mL. The active molecule is at least one of EGF, FGF, PDGF, TGF - β, VEGF, BMP - 2, BMP - 4, BMP - 7, OGP, and its content in the mixed solution is 0.01 - 0.5 μg / mL.

[0022] The present application also provides the application of the tremella polysaccharide intelligent hydrogel with ferroptosis inhibition in the preparation of products for repairing bone defects. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition is the above - mentioned tremella polysaccharide intelligent hydrogel.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] In this application, Tremella polysaccharide derived from natural plants was ingeniously selected. First, by virtue of the hydroxyl groups of Tremella polysaccharide, it reacted with methacrylic anhydride to introduce carbon-carbon double bonds, synthesizing TrepMA. Additionally, aldehyde-functionalized Tremella polysaccharide O-Trep was prepared based on the oxidation of sodium periodate. With the assistance of a photoinitiator and under the excitation of blue light or ultraviolet light, the carbon-carbon double bonds combined with each other to form a hydrogel from the precursor solution. The amino groups at both ends in TK-NH2 reacted with the aldehyde groups in O-Trep, playing a role in secondary cross-linking. By adding growth factors or active molecules to the precursor solution in the hydrogel, it played a role in drug loading, ultimately forming an intelligent Tremella polysaccharide hydrogel. Compared with other processes, the process is simple, the components are controllable, the antioxidant property of Tremella polysaccharide can be retained, and the TK in the hydrogel can intelligently respond to high concentrations of ROS in the microenvironment, accelerating the release of active molecules in an environment with high concentrations of ROS, playing a role in accelerating the healing of severely damaged areas.

[0025] The intelligent Tremella polysaccharide hydrogel prepared in this invention exhibits various biological activities. It not only provides a certain supporting role for bone regeneration, but also can, through its excellent biocompatibility, biological activity, and drug controlled-release effect, inhibit ferroptosis, improve local osteoporosis, have antioxidant / anti-inflammatory effects, induce osteogenic differentiation, etc., and can effectively promote the regeneration of bone tissue. Description of the Drawings

[0026] Figure 1 It is the equations and schematic diagrams involved in the preparation process of the intelligent Tremella polysaccharide hydrogel of this invention.

[0027] Figure 2 They are the characterization data and physical photos of an intelligent Tremella polysaccharide hydrogel with a ferroptosis inhibitory effect in Example 1 of this invention. (A) is the 1H-NMR spectra of Tremella polysaccharide (TP), O-Trep, and TrepMA. The characteristic peaks marked with blue frames show that the aldehyde groups in O-Trep and the methacryloyl groups in TrepMA were successfully modified; (B) is the infrared spectra of Tremella polysaccharide (TP), O-Trep, and TrepMA, which also confirms that O-Trep and TrepMA were successfully modified; (C) is the physical photo of the OGP@DTrep hydrogel in Example 1 of this invention. The precursor solution solidified into a hydrogel after ultraviolet irradiation; (D) are the physical photos and scanning electron microscope photos of the OGP@DTrep hydrogel, TrepMA, and DTerp hydrogels in Example 1 of this invention, showing that OGP@DTrep presents a three-dimensional network structure with clearly visible pores; (E) is the rheological property test of the OGP@DTrep hydrogel in Example 1 of this invention, confirming its hydrogel characteristics; (F) is the creep rate test of the OGP@DTrep hydrogel in Example 1 of this invention.

[0028] Figure 3This is the test of the OGP@DTrep hydrogel chelating iron ions in Example 1 of the present invention. (A) is the molecular docking simulation diagram of tremella polysaccharide molecules chelating iron ions. It can be seen that the binding energy between tremella polysaccharide molecules and iron ions reaches -11.74 kcal / mol, showing excellent binding ability; (B) is the photo of the hydrogel adsorbing iron ions. The hydrogel is immersed in the ferric chloride solution, which can clarify the solution in a short time, that is, chelate the free iron ions in the solution; (C) is the Mapping elemental analysis of the hydrogel immersed in the ferric chloride solution and (D) energy spectrum analysis. It can be seen that iron ions are successfully adsorbed in the hydrogel. Therefore, the above results show that the hydrogel proposed by the present invention has excellent ability to chelate iron ions.

[0029] Figure 4 This is the biocompatibility of the OGP@DTrep hydrogel and its role in promoting osteoblast differentiation in Example 1 of the present invention. (A) Fluorescent staining of osteogenic differentiation-related protein OPN of osteoblast MC3T3 seeded on the OGP@DTrep hydrogel, showing that the hydrogel increases the level of OPN in cells; (B) Expression of osteogenic differentiation-related genes of MC3T3 cells seeded on the OGP@DTrep hydrogel, showing high expression of osteogenic differentiation-related genes OCN, OPN, Runx2 and COL-1 in cells; (C) is the alkaline phosphatase (ALP) and alizarin red (ARS) staining of MC3T3 cells seeded on the OGP@DTrep hydrogel; (D) is the quantitative analysis of ARS staining; (E) is the quantitative analysis of ALP staining. The above staining data suggest that the hydrogel promotes the mineralization deposition of MC3T3 cells, indicating that the hydrogel can promote the secretion of bone matrix by cells; (F) is the tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts co-cultured with the hydrogel, suggesting that the hydrogel inhibits the activity of osteoclasts; (G) Western Blot analysis of the expression of osteogenic differentiation-related proteins of MC3T3 cells seeded on the OGP@DTrep hydrogel and (H) its quantitative data further illustrate that the OGP@DTrep hydrogel in Example 1 of the present invention can promote the osteogenic differentiation of osteoblasts.

[0030] Figure 5Effect of OGP@DTrep hydrogel on ferroptosis of osteoblasts in Example 1 of the present invention. After inoculating MC3T3 cells with OGP@DTrep hydrogel, the ferroptosis pathway of the cells was activated by stimulating with the ferroptosis activator ammonium iron(III) citrate (AIC). (A) shows the fluorescence photographs of intracellular ROS labeled with the ROS fluorescent probe DCFH-DA after AIC stimulation, and (B-C) flow cytometry and its quantitative data analysis. The results show that OGP@DTrep hydrogel significantly reduces the ROS level in the cells; (D) shows the fluorescence images of lipid peroxides in the cells detected by the C11 BODIPY fluorescent probe; (E) shows the fluorescence images of intracellular iron ion levels analyzed by FerroGreen staining; (F) shows the fluorescence staining of the ferroptosis-related antioxidant enzyme GPX4 in the cells cultured in each group of hydrogels; (G-H) Western Blot bands and their quantitative analysis verify the effect of OGP@DTrep hydrogel on the expression of ferroptosis-related proteins in osteoblasts; (I) Transmission electron microscope (TEM) images show the morphology and structure of the mitochondria of MC3T3 cells after stimulation. The above results show that the hydrogel reduces the ROS level, lipid peroxide level and iron ion concentration in MC3T3 cells after AIC stimulation. In particular, the hydrogel inhibits the activation of the ferroptosis signaling pathway and protects the structure and morphology of cell mitochondria. Therefore, it is fully proved that OGP@DTrep hydrogel has a good inhibitory effect on ferroptosis of osteoblasts. (*P<0.05, **P<0.01 or ***P<0.001).

[0031] Figure 6 Repair effect of OGP@DTrep hydrogel on bone defects with osteoporosis in rats in Example 1 of the present invention. An osteoporosis animal model was constructed in rats by ovariectomy, and a bone defect with a diameter of 2 mm was further created in the femoral condyle, thus constructing a bone defect model with osteoporosis in rats. Subsequently, the OGP@DTrep hydrogel was used to repair the bone defect site. After 4 weeks and 8 weeks of repairing the femoral condyle defect with the hydrogel, specimens were taken for histopathological staining analysis. (A-B) show the hematoxylin and eosin (H&E), Masson's and toluidine blue (T.B.) staining images of the pathological sections; (C-D) MicroCT images show the quantitative analysis of the relevant bone mass at the defect site after 4 weeks and 8 weeks of repairing the femoral condyle defect with the hydrogel. Subsequently, bone mass analysis was performed based on the MicroCT images, including (E) bone connectivity density (Conn.D), (F) trabecular bone number (Tb.N), (G) trabecular bone spacing (Tb.Sp) and (H) total volume ratio (Tb.BV / TV). The above results show that the hydrogel accelerates the healing of bone defects with osteoporosis in rats and the regeneration of trabecular bone. Detailed implementation mode

[0032] The present invention will be further described in detail below in conjunction with specific embodiments.

[0033] An intelligent Tremella polysaccharide hydrogel with ferroptosis inhibition is prepared using methacrylated Tremella polysaccharide (TrepMA), oxidized Tremella polysaccharide (O-Trep), and ketoketal diamine (TK-NH2).

[0034] Among them, the TrepMA is obtained by reacting a Tremella polysaccharide raw material with methacrylic anhydride, and the O-Trep is synthesized based on the oxidation of Tremella polysaccharide with sodium periodate.

[0035] In addition, the present application also provides a preparation method for an intelligent Tremella polysaccharide hydrogel with ferroptosis inhibition. Please refer to Figure 1 , in the figure, TP: Tremella polysaccharide, NaIO4: sodium periodate, MA: methacrylic anhydride, OGP: growth factor or active molecule loaded in the hydrogel. Taking OGP as an example here, the specific steps of the preparation method are as follows:

[0036] S1: Synthesis of methacrylated Tremella polysaccharide (TrepMA)

[0037] Dissolve Tremella polysaccharide in deionized water, heat it in a water bath at 50 °C to promote dissolution, and make a Tremella polysaccharide solution with a concentration of 0.5%-6% wt. Subsequently, add a certain amount of methacrylic anhydride to the Tremella polysaccharide solution to make a mixed solution. The concentration of methacrylic anhydride in the mixed solution is 1%-20% wt. At this time, the mixed solution is stirred at a speed of 300 rpm, and the pH of the mixed solution is adjusted to 8.0 with a 1 M sodium hydroxide solution. Subsequently, the mixed solution reacts in an ice bath for 24 hours. After the reaction is completed, use a dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove unreacted methacrylic anhydride. In one embodiment, the molecular weight cut-off of the dialysis bag is 500-20000 Da. After dialysis is completed, freeze-dry to obtain solid TrepMA;

[0038] S2: Synthesis of oxidized Tremella polysaccharide (O-Trep):

[0039] Dissolve Tremella polysaccharide in deionized water, heat it in a water bath at 50 °C to promote dissolution, and make a Tremella polysaccharide solution with a concentration of 0.5%-6% wt. Subsequently, add a certain amount of sodium periodate to the Tremella polysaccharide solution to make a mixed solution. The concentration of sodium periodate in the mixed solution is 0.5-3 mg / mL. At this time, the mixed solution is stirred at a speed of 300 rpm, and then the mixed solution reacts at room temperature for 24 hours. After the reaction is completed, use a dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove sodium periodate. In one embodiment, the molecular weight cut-off of the dialysis bag is 500-20000 Da. After dialysis is completed, freeze-dry to obtain solid O-Trep;

[0040] The molecular weight of the tremella polysaccharide selected in S1 and S2 is above 3000 Da.

[0041] S3: Preparation of hydrogel precursor solution:

[0042] Dissolve TrepMA synthesized in S1 and O-Trep synthesized in S2 in deionized water according to a certain ratio and concentration. In one embodiment, the concentration of TrepMA is 3%-8% wt, and the mass ratio of TrepMA to O-Trep is 1:1 - 1:3. Further add a certain amount of photoinitiator, ketothioketal diamine (TK-NH2) and active molecules. Among them, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and the concentration is 1%-5% wt. The concentration of TK-NH2 is 0.05 - 0.2 mg / mL. The active molecule is at least one of EGF, FGF, PDGF, TGF-β, VEGF, BMP-2, BMP-4, BMP-7, OGP, and its content in the mixed solution is 0.01 - 0.5 μg / mL. Ultrasonic oscillation for 5 minutes to fully mix. The obtained mixed solution is the hydrogel precursor solution, and subsequent operations must be carried out within 1 hour after the preparation of the precursor solution;

[0043] S4: Preparation of tremella polysaccharide intelligent hydrogel (DTrep):

[0044] Load the hydrogel precursor solution obtained in S3 into a syringe, inject the solution into a mold or a bone defect site, and irradiate the solution with ultraviolet light of 405 nm wavelength for 1 - 60 s to obtain the cured DTrep composite hydrogel.

[0045] Based on the above preparation method, the present application also provides the application of the tremella polysaccharide intelligent hydrogel with ferroptosis inhibition in the preparation of products for repairing bone defects.

[0046] The tremella polysaccharide intelligent hydrogel provided by the present application can inhibit ferroptosis in the following ways and promote the healing of bone defects accompanied by osteoporosis:

[0047] (1) The ketothioketal group in DTrep can react quickly with reactive oxygen species (ROS), playing the roles of intelligent response, scavenging ROS, anti-inflammatory and controlling the release of loaded drugs. The released drugs can promote the osteogenic differentiation of osteoblasts;

[0048] (2) The carboxyl groups rich in tremella polysaccharide in DTrep can form an efficient chelation with iron ions. Thus, by reducing the iron ion concentration in the microenvironment, the activation of the cell ferroptosis-related signaling pathway can be inhibited;

[0049] (3) DTrep can activate the Nrf2 signaling pathway in osteoblasts, activate the endogenous antioxidant effect in osteoblasts, and inhibit osteoblast apoptosis.

[0050] The above content will be elaborated below in combination with specific examples.

[0051] Experimental materials and sources:

[0052]

[0053] Example 1:

[0054] (1) Synthesis of Tremella polysaccharide modified with methacrylic anhydride (TrepMA): Dissolve 3 g of Tremella polysaccharide (molecular weight 50 kDa) in 100 mL of deionized water, heat it in a water bath at 50 °C until fully dissolved to prepare a 3% wt Tremella polysaccharide solution. Then add 5 g of methacrylic anhydride to the Tremella polysaccharide solution to form a mixed solution. At this time, the mixed solution is stirred at a speed of 300 rpm, and the pH of the mixed solution is adjusted to 8.0 with a 1 M sodium hydroxide solution. Then the mixed solution reacts in an ice bath for 24 hours. After the reaction is completed, the mixed solution is dialyzed against deionized water using a 5000 Da dialysis bag for 3 days to remove the unreacted methacrylic anhydride, and then freeze-dried to obtain solid TrepMA;

[0055] (2) Synthesis of oxidized Tremella polysaccharide (O-Trep): Dissolve 3 g of Tremella polysaccharide (molecular weight 50 kDa) in 100 mL of deionized water, heat it in a water bath at 50 °C until fully dissolved to prepare a 3% wt Tremella polysaccharide solution. Then add sodium periodate to the Tremella polysaccharide solution so that the concentration of sodium periodate in the mixed solution is 1 mg / mL. At this time, the mixed solution is stirred at a speed of 300 rpm, and then the mixed solution reacts at room temperature for 24 hours. After the reaction is completed, the mixed solution is dialyzed against deionized water using a dialysis bag for 3 days to remove sodium periodate, and then freeze-dried to obtain solid O-Trep;

[0056] (3) Preparation of hydrogel precursor solution: Dissolve the TrepMA synthesized in step (1) and the O-Trep synthesized in step (2) in 10 mL of deionized water according to a mass ratio of 1:2. The concentration of TrepMA is 3% wt. Further add the photoinitiator LAP, ketoketal thiol (TK-NH2), and osteogenic polypeptide OGP, with concentrations of 0.02% wt, 0.1 mg / mL, and 0.1 μg / mL respectively, and ultrasonically vibrate for 5 minutes to fully mix. The resulting mixed solution is the hydrogel precursor solution. The subsequent operations are carried out within 1 hour after the preparation of the precursor solution;

[0057] (4) Preparation of Tremella polysaccharide intelligent hydrogel: The hydrogel precursor solution obtained in step (3) was loaded into a syringe, and the solution was injected into a mold or the bone defect site. After being irradiated with ultraviolet light at a wavelength of 405 nm for 30 seconds, the solution was solidified to obtain the Tremella polysaccharide intelligent hydrogel, which was named OGP@DTrep.

[0058] Example 2

[0059] (1) Synthesis of methacrylic anhydride-modified Tremella polysaccharide (TrepMA): 5 g of Tremella polysaccharide (molecular weight 50 kDa) was dissolved in 100 mL of deionized water and heated in a water bath at 50 °C until fully dissolved to prepare a Tremella polysaccharide solution. Subsequently, 6 g of methacrylic anhydride was added to the Tremella polysaccharide solution to form a mixed solution. At this time, the mixed solution was stirred at a speed of 300 rpm, and the pH of the mixed solution was adjusted to 8.0 with a 1 M sodium hydroxide solution. Subsequently, the mixed solution was reacted in an ice bath for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water using a 5000 Da dialysis bag for 3 days to remove the unreacted methacrylic anhydride, and then freeze-dried to obtain solid TrepMA;

[0060] (2) Synthesis of oxidized Tremella polysaccharide (O-Trep): 5 g of Tremella polysaccharide (molecular weight 50 kDa) was dissolved in 100 mL of deionized water and heated in a water bath at 50 °C until fully dissolved to prepare a Tremella polysaccharide solution. Subsequently, sodium periodate was added to the Tremella polysaccharide solution to make the concentration of sodium periodate in the mixed solution 1.5 mg / mL. At this time, the mixed solution was stirred at a speed of 300 rpm, and then the mixed solution was reacted at room temperature for 24 hours. After the reaction was completed, the mixed solution was dialyzed in deionized water using a dialysis bag for 3 days to remove sodium periodate, and then freeze-dried to obtain solid O-Trep;

[0061] (3) Preparation of hydrogel precursor solution: The TrepMA synthesized in step (1) and the O-Trep synthesized in step (2) were dissolved in 10 mL of deionized water according to a mass ratio of 1:1. The concentration of TrepMA was 5% wt. Further, a photoinitiator LAP, ketoketal thiol (TK-NH2), and growth factor VEGF were added, with concentrations of 0.02% wt, 0.1 mg / mL, and 0.1 μg / mL, respectively. The mixture was sonicated for 5 minutes to mix well, and the resulting mixed solution was the hydrogel precursor solution. The subsequent operations were carried out within 1 hour after the preparation of the precursor solution;

[0062] (4) Preparation of Tremella polysaccharide intelligent hydrogel: The hydrogel precursor solution obtained in step (3) was loaded into a syringe, and the solution was injected into a mold or the bone defect site. After being irradiated with ultraviolet light at a wavelength of 405 nm for 30 seconds, the solution was solidified to obtain the Tremella polysaccharide intelligent hydrogel loaded with VEGF.

[0063] Example 3

[0064] (1) Synthesis of Tremella polysaccharide modified with methacrylic anhydride (TrepMA): Dissolve 3 g of Tremella polysaccharide (molecular weight 50 kDa) in 100 mL of deionized water, heat it in a water bath at 50 °C until fully dissolved to prepare a Tremella polysaccharide solution with a concentration of 3% wt. Subsequently, add 5 g of methacrylic anhydride to the Tremella polysaccharide solution to form a mixed solution. At this time, the mixed solution is stirred at a speed of 300 rpm, and the pH of the mixed solution is adjusted to 8.0 with a 1 M sodium hydroxide solution. Then, the mixed solution reacts in an ice bath for 24 hours. After the reaction is completed, use a 5000 Da dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove the unreacted methacrylic anhydride. After dialysis is completed, freeze-dry to obtain solid TrepMA;

[0065] (2) Synthesis of oxidized Tremella polysaccharide (O-Trep): Dissolve 3 g of Tremella polysaccharide (molecular weight 50 kDa) in 100 mL of deionized water, heat it in a water bath at 50 °C until fully dissolved to prepare a Tremella polysaccharide solution with a concentration of 3%. Subsequently, add sodium periodate to the Tremella polysaccharide solution so that the concentration of sodium periodate in the mixed solution is 1 mg / mL. At this time, the mixed solution is stirred at a speed of 300 rpm, and then the mixed solution reacts at room temperature for 24 hours. After the reaction is completed, use a dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove sodium periodate. After dialysis is completed, freeze-dry to obtain solid O-Trep;

[0066] (3) Preparation of hydrogel precursor solution: Dissolve the TrepMA synthesized in step (1) and the O-Trep synthesized in step (2) in 10 mL of deionized water according to a mass ratio of 1:2. The concentration of TrepMA is 3% wt. Further add photoinitiator LAP, ketoketal thiol (TK-NH2), and growth factor BMP-2, with concentrations of 0.02% wt, 0.1 mg / ml, and 0.1 μg / ml respectively. Ultrasonically vibrate for 5 minutes to fully mix. The resulting mixed solution is the hydrogel precursor solution. Perform subsequent operations within 1 hour after the preparation of the precursor solution is completed;

[0067] (4) Preparation of Tremella polysaccharide intelligent hydrogel: Load the hydrogel precursor solution obtained in step (3) into a syringe, inject the solution into a mold or bone defect site, and after irradiating with ultraviolet light with a wavelength of 405 nm for 30 seconds, the solution solidifies to obtain a Tremella polysaccharide intelligent hydrogel loaded with BMP-2.

[0068] Verification experiment 1:

[0069] In this application, nuclear magnetic resonance spectroscopy (1H-NMR), infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), rheology, and creep were used to characterize the material properties of Tremella polysaccharide derivatives. The results are as Figure 2As shown, the 1H-NMR spectrum of Trep-MA showed characteristic peaks of methacryloyl groups at 5.1 and 5.6 ppm ( Figure 2 A). The 1H-NMR spectrum of O-Trep showed characteristic peaks of aldehyde groups at 5.2 and 5.6 ppm. As Figure 2 shown in B, FT-IR spectral analysis confirmed the successful synthesis of these two components. Compared with unmodified tremella polysaccharide (TP), the characteristic peak of Trep-MA spectrum gradually increased at 1735 cm -1 , representing the C=O group in the methacryloyl structure. In the FT-IR spectrum of O-Trep, the intensity of the characteristic peak at 1735 cm -1 representing aldehyde groups (C=O) also increased. In addition, all samples showed typical tremella polysaccharide characteristic peaks at 1636, 1422, 3431, 2927, and 1074 cm -1 , corresponding to O-H stretching vibration, C-H stretching vibration, carbonyl C-O stretching vibration in uronic acid, C-H bending vibration, and C-O-C stretching vibration, respectively. Therefore, the above results indicated the successful preparation of the monomers constituting the tremella polysaccharide-based OGP@DTrep double-network hydrogel. Subsequently, three hydrogels were constructed: Trep-MA (without O-Trep and TK), DTrep hydrogel, and the OGP@DTrep hydrogel constructed in Example 1. As Figure 2 shown in D, DTrep also exhibited excellent formability and injectability in a humid environment and could be cured into a hydrogel under ultraviolet light induction. Figure 2 The SEM images in D showed that the OGP@DTrep hydrogels constructed in Example 1 all presented a porous microstructure. This typical hydrogel structural feature was beneficial to cell migration and tissue ingrowth. In addition, the results of rheological experiments showed ( Figure 2 E) that each group of hydrogels had a high storage modulus (G') and a low loss modulus (G"); that is, the hydrogels exhibited solid-like viscoelasticity, indicating that OGP@DTrep was successfully transformed into a gel state. Rheological data also showed that due to its double-network structure, the elastic moduli of OGP@DTrep and DTrep were greater than that of Trep-MA. Subsequently, the stability of various hydrogels was evaluated by swelling ratio tests. As Figure 2 shown in F, OGP@DTrep reached a stable state within 6 hours in a liquid environment and showed a low swelling ratio. However, within 48 hours, the swelling ratio of Trep-MA further increased, while the creep of OGP@DTrep changed little, indicating that the OGP@DTrep hydrogel had better stability. Maintaining the stability of the hydrogel structure helped to maintain the shape and integrity during bone regeneration, while minimizing compression on surrounding tissues or separation from the target organ.

[0070] Verification experiment 2

[0071] The tremella polysaccharide-based intelligent hydrogel can block the ferroptosis process of osteoblasts by reducing the concentration of free iron ions in the microenvironment. This application verified the chelating ability of the above-mentioned tremella polysaccharide-based hydrogel and iron ions. As Figure 3 shown in A, molecular docking simulations were performed on the coordination of TP molecules and Fe 3+ ions. The results showed that the binding energy of Fe 3+ ions to TP was -11.47 kcal / mol, indicating that Fe 3+ could efficiently bind to the tremella polysaccharide molecular chain. Interaction analysis showed that the iron ions formed salt bridge interactions with the carboxyl groups in TP and hydrogen bond interactions with the hydroxyl groups in TP. The distance of the hydrogen bond was 2.8 Å. Subsequently, the ability of the hydrogel to adsorb Fe 3+ ions was tested in this application. As Figure 3 shown in B, after being soaked in the FeCl3 solution for only 1 minute, Trep-MA quickly turned yellow due to the adsorption of a large amount of iron ions, while the original yellow supernatant solution turned colorless. Subsequently, the iron content in the central part of the hydrogel was verified in this application. As Figure 3 shown in C&D, the results of Mapping elemental analysis and EDS energy spectrum analysis showed that after Trep-MA was soaked in the FeCl3 solution, a large amount of iron elements were enriched in the gel network in a very short time (1 minute). Thus, it can be seen that the hydrogel prepared based on TP shows excellent chelating ability for iron ions, which means that it may be able to block ferroptosis by regulating the concentration of iron ions in the microenvironment.

[0072] Verification experiment 3:

[0073] This application further evaluated the effect of the OGP@DTrep hydrogel constructed in Example 1 on osteoblast differentiation in the ferroptosis state. The hydrogel was made into a disc-shaped sample with a diameter of 1.5 cm and a height of 3 mm, placed at the bottom of a 24-well plate, and then osteoblast MC3T3 cells were seeded into the 24-well plate at a density of 2×10 5 and cultured in DMEM medium for subsequent experiments. Figure 4 The immunofluorescence staining images in A showed that OGP@DTrep significantly increased the level of osteopontin (OPN) in osteoblast MC3T3 cells. As Figure 4 shown in B, the RT-PCR results further supported this finding. After co-culturing MC3T3 cells with OGP@DTrep, the osteogenic differentiation-related genes Runx2, OCN, OPN, and COL-1 were significantly highly expressed. As Figure 4As shown by the results of alizarin red staining (ARS) and alkaline phosphatase (ALP) staining in C, MC3T3 cells co-cultured with OGP@DTrep showed the highest levels of alkaline phosphatase and mineral deposition, and quantitative analysis confirmed this ( Figure 4 D&E). It indicates that OGP@DTrep can stimulate osteoblasts to secrete ECM to form new bone. In addition, Western Blot (WB) data showed that OGP@DTrep increased the protein levels of wnt7b, Runx2, and OPN related to bone regeneration ( Figure 4 G&H). In addition, as Figure 4 shown in F, the results of tartrate-resistant acid phosphatase (TRAP) staining showed that OGP@DTrep could inhibit osteoclast activity. The above data indicate that OGP@DTrep helps to restore the osteoblast / osteoclast balance in the pathological microenvironment of osteoporosis.

[0074] Subsequently, in this application, MC3T3 cells were stimulated to activate the ferroptosis pathway to verify the regulatory effect of the OGP@DTrep hydrogel constructed in Example 1 on the ferroptosis pathway of osteoblast MC3T3. Ammonium ferric citrate (AIC) was used as a ferroptosis activator for cells to stimulate the ferroptosis pathway of MC3T3 cells. As Figure 5 shown in A, after stimulation with AIC, compared with the blank group, MC3T3 cells co-cultured with the OGP@DTrep hydrogel showed lower levels of green fluorescence, which represents a decrease in the level of intracellular reactive oxygen species (ROS). As Figure 5 shown in B&C, the results of flow cytometry and related quantitative analysis showed that OGP@DTrep had the greatest ability to scavenge ROS, indicating that the hydrogel could remove excessive ROS in cells. In addition, the accumulation of lipid peroxides and iron ions is an important manifestation of cell ferroptosis. Subsequently, the C11 BODIPY fluorescent probe was used in this application to verify the level of intracellular lipid peroxides. As Figure 5 shown in D, OGP@DTrep showed the lowest level of lipid peroxides. In addition, according to the results of FerroGreen staining, the level of iron ion enrichment in MC3T3 cells co-cultured with OGP@DTrep was also the lowest. The WB bands of ferroptosis-related proteins and their quantitative statistical data are as Figure 5 shown in G&H. The OGP@DTrep hydrogel significantly promoted the high expression of FTH1, GSTP1, GPX4, and Nrf2 proteins in MC3T3. In addition, the expressions of ACSL4 and Cox-2 were significantly decreased. Therefore, it shows that the ferroptosis pathway activated by AIC stimulation in cells was blocked. Figure 5Figure I shows that the OGP@DTrep hydrogel protected the structural integrity of cellular mitochondria, and both the outer cell membrane and inner cristae showed relatively intact morphology. In addition, the hydrogel activated the intracellular Nrf2 / Gpx4 pathway, indicating enhanced intracellular endogenous antioxidant capacity.

[0075] Verification experiment 4:

[0076] This application further evaluated the effect of the OGP@DTrep hydrogel constructed in Example 1 on implanting into the bone defect site in a rat osteoporosis model to promote new bone regeneration. Animal modeling: All animal experimental procedures were approved ethically and scientifically by the Animal Care and Use Committee of Nantong University (approval number: S20240617-003). All experimental procedures complied with the ARRIVE guidelines and were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 and related guidelines. A rat osteoporosis bone defect model was established using female SD rats aged 12 weeks and weighing 300 - 400 g. Bilateral ovariectomy was performed on the female rats for the experiment. After 15 days of postoperative observation, the estrogen level began to decline. One month later, the estrogen level dropped to the lowest level. After 4 to 8 weeks, an osteoporosis model was established. Then, bone defect surgeries were performed on the ovariectomized group and the sham operation control group. A scalpel was used to cut through the subcutaneous area of the lateral condyle of the rat femur to expose the bone surface. Then, a bone defect model with a diameter of 2.5 mm and a depth of 3 mm under the lateral condyle was created. The hydrogel precursor solution was injected to fill the defect and cured by ultraviolet crosslinking. Subsequently, the joint capsule and skin incision were sutured. The rats were allowed to move freely in the cage. The animals were randomly assigned to each group.

[0077] The results showed that at 4 weeks postoperatively, the H&E Masson and toluidine blue (TB) staining results showed ( Figure 6 A&B), OGP@DTrep significantly promoted the ingrowth of osteoid tissue into the defect site, and a certain amount of trabecular bone was formed. The defect sites in the other groups still showed a certain degree of bone defect and less trabecular bone formation. At 8 weeks postoperatively, the blank OVX group produced a small amount of trabecular bone, but there was still a large cavity, while the OGP@DTrep group showed the highest level of trabecular bone formation. As Figure 6 shown in C-H, the MicroCT results showed that untreated osteoporotic rats (OVX group) showed fewer trabeculae, poor bone structure, and low bone content, and their pathological characteristics were similar to those of osteoporosis models in previous studies. The defect site in the OGP@DTrep group had the most trabecular bone. Bone mineral density (BMD) and total volume ratio (Tb.BV / TV) can reflect bone quality and strength. Trabecular number (Tb.N) and trabecular separation (Tb.Sp) are the main indicators for evaluating the spatial morphology and structure of trabeculae. Figure 6The statistical results of E-H showed that the BMD, Tb.N, Tb.Sp, and BV / TV indexes of the OGP@DTrep group were significantly greater than those of other groups, indicating that OGP@DTrep significantly promoted new bone regeneration and improved bone strength. Generally speaking, after the defect site of the rat model was repaired with OGP@DTrep, bone regeneration and trabecular bone formation were significantly promoted.

[0078] In this application, tremella polysaccharide from natural plant sources was selected. First, with the help of the hydroxyl groups of tremella polysaccharide, it was reacted with methacrylic anhydride to introduce carbon-carbon double bonds, and TrepMA was synthesized. In addition, aldehyde group-modified tremella polysaccharide O-Trep was prepared based on the oxidation of sodium periodate. Under the assistance of a photoinitiator and under the excitation of blue light or ultraviolet light, the carbon-carbon double bonds combined with each other to form a hydrogel in the precursor solution. The amino groups at both ends in TK-NH2 reacted with the aldehyde groups in O-Trep, playing a role in secondary cross-linking. By adding growth factors or active molecules to the precursor solution in the hydrogel, it played a role in drug loading, and finally a tremella polysaccharide intelligent hydrogel was formed. Compared with other processes, the process is simple, the components are controllable, the antioxidant property of tremella polysaccharide can be retained, and the TK in the hydrogel can intelligently respond to high concentrations of ROS in the microenvironment, accelerating the release of active molecules in a high-concentration ROS environment and playing a role in accelerating the healing of severely damaged areas.

[0079] The tremella polysaccharide intelligent hydrogel prepared in this invention exhibits various biological activities. It not only provides a certain supporting role for bone regeneration, but also can inhibit ferroptosis, improve local osteoporosis, have antioxidant / anti-inflammatory effects, induce osteogenic differentiation, etc. through its excellent biocompatibility, biological activity, and drug controlled-release effect, and can effectively promote the regeneration of bone tissue.

Claims

1. A Tremella polysaccharide intelligent hydrogel with ferroptosis inhibition, characterized in that: It is prepared by using methacrylated Tremella polysaccharide TrepMA, oxidized Tremella polysaccharide O-Trep and ketothiol diamine TK-NH2.

2. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 1, characterized in that: The TrepMA is obtained by reacting a Tremella polysaccharide raw material with methacrylic anhydride, and the O-Trep is synthesized based on sodium periodate oxidation of Tremella polysaccharide.

3. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 2, characterized in that: The preparation method of the Tremella polysaccharide intelligent hydrogel is as follows: S1: Synthesis of TrepMA Dissolve Tremella polysaccharide in deionized water to prepare a Tremella polysaccharide solution. Subsequently, add methacrylic anhydride to the Tremella polysaccharide solution to form a mixed solution. Stir the mixed solution and adjust the pH of the mixed solution to 8.

0. Then, place the mixed solution in an ice bath for reaction. After the reaction is completed, use a dialysis bag to dialyze the mixed solution in deionized water to remove unreacted methacrylic anhydride. After dialysis is completed, freeze-dry to obtain solid TrepMA; S2: Synthesis of O-Trep: Prepare a Tremella polysaccharide solution, then add sodium periodate to the Tremella polysaccharide solution to form a mixed solution. Stir the mixed solution, and then place the mixed solution at room temperature for reaction. Use a dialysis bag to dialyze the mixed solution in deionized water for 3 days to remove sodium periodate. After dialysis is completed, freeze-dry to obtain solid O-Trep; S3: Preparation of hydrogel precursor solution: Dissolve the TrepMA synthesized in S1 and the O-Trep synthesized in S2 in deionized water, then add a photoinitiator, TK-NH2 and an active molecule, and ultrasonically vibrate for 5 minutes to fully mix. The resulting mixed solution is the hydrogel precursor solution. Subsequent operations must be carried out within 1 hour after the preparation of the precursor solution; S4: Preparation of DTrep Load the hydrogel precursor solution obtained in S3 into a syringe, inject the solution into a mold or a bone defect site, and irradiate the solution with ultraviolet light of 405 nm wavelength for 1 - 60 seconds to obtain a cured DTrep composite hydrogel.

4. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 3, wherein: The molecular weight of the Tremella polysaccharide selected in S1 and S2 is above 3000 Da, and the dissolution of Tremella polysaccharide in deionized water is promoted by heating in a water bath at 50 °C. The concentration of the Tremella polysaccharide solution prepared in S1 and S2 is 0.5% - 6% wt.

5. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 3, characterized in that: The concentration of methacrylic anhydride in the mixed solution in S1 is 1% - 20% wt, the concentration of sodium periodate in the mixed solution in S2 is 0.5 - 3 mg / mL, and the stirring rate of the mixed solution in S1 and S2 is 300 rpm.

6. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 3, characterized in that: The pH of the mixed solution in S1 is adjusted using a 1 M sodium hydroxide solution.

7. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 3, characterized in that: The addition concentration of TrepMA in S3 is 3% - 8% wt, and the mass ratio of TrepMA to O-Trep is 1:1 - 1:

3.

8. The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition according to claim 3, characterized in that: The photoinitiator in S3 is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and the concentration is 1%-5% wt. The concentration of TK-NH2 is 0.05-0.2 mg / mL. The active molecule is at least one of EGF, FGF, PDGF, TGF-β, VEGF, BMP-2, BMP-4, BMP-7, OGP, and its content in the mixed solution is 0.01-0.5 μg / mL.

9. Application of Tremella polysaccharide intelligent hydrogel with ferroptosis inhibition in the preparation of products for repairing bone defects, characterized in that: The tremella polysaccharide intelligent hydrogel with ferroptosis inhibition is the tremella polysaccharide intelligent hydrogel according to any one of claims 1-8.

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