Alginate-tempo conjugated polymer, its preparation method and application

Abstract

This invention relates to the field of biomedical materials, disclosing an alginate-TEMPO coupled polymer, its preparation method, and its applications. The polymer is prepared by amidation of alginate and 4-amino-TEMPO, followed by crosslinking with calcium chloride to form a hydrogel. This hydrogel possesses excellent reactive oxygen species (ROS) scavenging properties and biocompatibility, promoting cell migration and regulating macrophage polarization, thus enabling its use in skin wound repair. Based on the synergistic effect between its components, the hydrogel provided by this invention exhibits significant effects in scavenging ROS and promoting tissue regeneration, offering a promising material for clinical skin wound repair.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to an alginate-TEMPO coupling polymer, its preparation method, and its applications. Background Technology

[0002] The repair process of skin wounds (such as trauma, surgical incisions, burns, and chronic ulcers) typically involves stages such as hemostasis, inflammatory response, proliferation, and remodeling. The homeostasis of the wound microenvironment is crucial for the healing process. However, under conditions such as infection, ischemia, diabetes, and persistent inflammation, the wound often exists in an abnormal microenvironment, leading to prolonged repair cycles or even the formation of chronic, refractory wounds, posing significant challenges to clinical treatment.

[0003] Existing research and clinical observations indicate that reactive oxygen species (ROS) play a dual role in wound repair: appropriate amounts of ROS participate in antibacterial defense and cell signal regulation, while excessive ROS production and continuous accumulation trigger oxidative stress, leading to cell membrane lipid peroxidation, protein and nucleic acid damage, inhibiting the migration and proliferation of fibroblasts and keratinocytes, and further amplifying the inflammatory response, thereby affecting key processes such as re-epithelialization, collagen deposition, and angiogenesis (Advanced Materials, 2026, 38(3): 12719-12755). Therefore, reducing excessive ROS in the wound microenvironment and alleviating oxidative stress are considered important strategies for promoting rapid and high-quality wound healing.

[0004] To address the problem of excessive ROS, existing technologies include using small-molecule antioxidants, free radical scavengers, and anti-inflammatory drugs to improve the wound microenvironment. However, small-molecule antioxidants often suffer from short retention times at the wound site, are easily diluted or rapidly cleared, and require repeated administration. Furthermore, some free radical scavengers, being small molecules, may pose irritant or potential biosafety risks, limiting their feasibility for long-term or high-dose application at the wound site. In addition, relying solely on drugs to clear ROS often fails to simultaneously address the needs of moist healing, exudate management, and tissue regeneration support.

[0005] Hydrogels, due to their high water content, softness, ability to create a moist healing environment, and certain barrier protection, have become an important material system for wound dressings and local drug delivery carriers (ACS Nano, 2021, 15(8): 12687-12722). Among them, alginate (Alg) materials are widely available, have mild gelation conditions, good biocompatibility, and can achieve rapid ionic cross-linking to form gels through divalent ions, thus having a wide range of applications in wound dressings. However, traditional alginate hydrogels mainly focus on moisturizing, liquid absorption, and physical barrier functions, and have limited ability to regulate excessive ROS and persistent inflammation in the wound microenvironment, making it difficult to fundamentally improve the problem of delayed healing caused by oxidative stress.

[0006] Therefore, there is an urgent need for a functional hydrogel material that can maintain the good biological properties and gelling advantages of alginate hydrogels, further endow the material with stable and effective free radical / reactive oxygen species scavenging capabilities, and synergistically regulate the inflammatory microenvironment of wounds, promote cell migration and tissue regeneration, so as to better meet the needs of clinical wound repair.

[0007] 4-Amino-2,2,6,6-Tetramethylpiperidine-1-oxo radical (TEMPO) is a stable nitroxide radical with excellent free radical scavenging ability and antioxidant properties, effectively scavenging various reactive oxygen species (Biomaterials, 2018, 177: 98-112). It has potential application value in inhibiting oxidative stress and regulating the inflammatory microenvironment. However, as a small molecule free radical scavenger, TEMPO is easily diffused or cleared rapidly in the body fluid environment in its free state, making it difficult to maintain an effective concentration at the wound site. Furthermore, it may exhibit certain cytotoxicity at higher doses, thus limiting its direct application in biomedical materials. To improve its stability and reduce potential biotoxicity, current research typically involves covalently immobilizing TEMPO onto a polymeric material framework (Acta Biomaterialia, 2022, 152: 171-185), thereby maintaining its free radical scavenging activity while reducing the irritation and safety risks associated with its small molecule form. Therefore, grafting TEMPO, which has free radical scavenging capabilities, onto natural polymers with good biocompatibility and gelling properties to construct biomedical hydrogel systems that combine gelling ability and antioxidant function has significant research significance and application prospects. Summary of the Invention

[0008] To address the limitations of existing technologies such as the limited ability of alginate hydrogels to regulate oxidative stress in wounds, the short retention time of small molecule free radical scavengers in the wound area, and their potential irritation, the present invention aims to provide an alginate-based functional hydrogel material with stable reactive oxygen species scavenging capabilities. This material effectively regulates the level of excessive reactive oxygen species in the wound microenvironment while maintaining the good biocompatibility and gelling properties of alginate, thereby promoting rapid repair and tissue regeneration of skin wounds.

[0009] To achieve the above objectives, the present invention provides an alginate-TEMPO coupling polymer, which is formed by covalently grafting amino-containing TEMPO or its derivatives onto the molecular chain of alginate or its salt.

[0010] Furthermore, the present invention provides an alginate-TEMPO coupling polymer hydrogel, wherein the hydrogel is formed by ionic crosslinking of the alginate-TEMPO coupling polymer in the presence of divalent ions, preferably by crosslinking in a calcium chloride solution to form a three-dimensional network structure.

[0011] In some embodiments of the present invention, the molar ratio of mannuronic acid (M) to guluronic acid (G) in the alginate is 1:2, and the average molecular weight is 140-150 kDa; the TEMPO grafting rate is 1%-20%.

[0012] The present invention also provides a method for preparing the alginate-TEMPO coupling polymer, comprising: activating the carboxyl group of alginate or its salt in an aqueous system; adding amino-containing TEMPO or its derivative to carry out a coupling reaction to form an amide bond; and purifying the product to obtain the alginate-TEMPO coupling polymer.

[0013] The present invention also provides a method for preparing the hydrogel, which involves dissolving the alginate-TEMPO coupling polymer in an aqueous system, and then adding a divalent ion solution for ionic crosslinking to form a hydrogel, preferably by adding 0.5% (w / v) calcium chloride solution for crosslinking to form a hydrogel.

[0014] The present invention also provides the application of the alginate-TEMPO coupling polymer or the hydrogel in the preparation of materials for skin wound repair.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention covalently grafts stable free radical TEMPO onto the alginate molecular chain, so that the material retains the good biocompatibility and ionic gelation properties of alginate while giving it a stable and continuous reactive oxygen scavenging ability, thereby effectively alleviating the oxidative stress microenvironment of the wound. (2) The alginate-TEMPO hydrogel described in this invention can form a moist three-dimensional network structure in the local wound area, which can not only maintain a suitable healing environment and absorb exudate, but also promote cell migration, induce macrophages to polarize to the M2 phenotype, and further promote tissue regeneration processes such as re-epithelialization, collagen deposition and angiogenesis. (3) The present invention fixes the small molecule free radical scavenger in the polymer hydrogel network, which significantly improves the retention and stability of the free radical scavenging component in the local wound and reduces the irritation risk that small molecules may cause, thereby improving the safety and effectiveness of the material in wound repair. (4) The hydrogel of the present invention has mild preparation conditions, can be injected or gelled in situ, has good biocompatibility and application feasibility, and is suitable for the repair of various types of skin wounds. (5) By regulating the degree of grafting of TEMPO onto the alginate molecular chain, this invention can achieve synergistic regulation of free radical scavenging ability and material biological properties, so that the resulting hydrogel can maintain good gelling properties and biocompatibility while having adjustable reactive oxygen scavenging intensity and duration of action, thus being more conducive to the personalized repair needs of different types of wounds. Attached Figure Description

[0016] To more clearly illustrate the technical solution of the present invention, the present invention will be further described below with reference to the accompanying drawings.

[0017] In the figure: Alg represents alginate or sodium alginate; 4-amino-TEMPO represents 4-amino-substituted TEMPO; Alg-TEMPO represents alginate-TEMPO coupling polymer; Alg-T1 represents alginate-TEMPO coupling polymer with a grafting rate of approximately 3%; Alg-T2 represents alginate-TEMPO coupling polymer with a grafting rate of approximately 8%.

[0018] Figure 1 This diagram illustrates the preparation process of alginate-TEMPO coupled hydrogels and their role in wound repair.

[0019] Figure 2 Figure 1 shows the structural characterization and free radical scavenging performance results of the alginate-TEMPO coupling polymer.

[0020] Figure 3 Images show the appearance of sodium alginate solutions of different concentrations.

[0021] Figure 4 The images show the appearance of sodium alginate solutions of different concentrations under different pH conditions.

[0022] Figure 5 The images show the gelation state of a 0.5% sodium alginate solution mixed with calcium chloride of different concentrations.

[0023] Figure 6 This is a diagram showing the crosslinking and gelation state of the alginate-TEMPO coupling polymer under different concentrations of calcium chloride.

[0024] Figure 7 Figures show the appearance and rheological properties of the alginate-TEMPO hydrogel.

[0025] Figure 8 The figure shows the experimental results of the effects of alginate-TEMPO coupled polymer on cell migration and macrophage polarization.

[0026] Figure 9 The figure shows the experimental results of the effect of alginate-TEMPO coupling polymer on intracellular reactive oxygen species levels.

[0027] Figure 10 This image shows the results of an in vivo wound repair experiment using alginate-TEMPO hydrogel.

[0028] Figure 11 Histological and immunohistochemical results of alginate-TEMPO hydrogel promoting wound tissue regeneration.

[0029] Figure 12 This figure shows the results of the in vitro and in vivo biocompatibility evaluation of alginate-TEMPO hydrogel.

[0030] Figure 13 The figure shows the results of the in vitro blood compatibility test of the alginate-TEMPO coupling polymer. Detailed Implementation

[0031] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Various modifications, equivalent substitutions, or variations made to this invention by those skilled in the art without departing from the spirit and substance of this invention should fall within the scope of protection of this invention.

[0032] Example 1: Preparation of Alginic Acid-TEMPO Coupling Polymer

[0033] Raw materials and reagents: High-G sodium alginate was selected, with a molar ratio of mannuronic acid (M) to guluronic acid (G) of 1:2 and a weight-average molecular weight of 140-150 kDa; 4-amino-TEMPO was selected; EDCI and NHS were selected as carboxyl activation / coupling reagents; the buffer was MES buffer (50 mM, pH 6.0).

[0034] The preparation steps are as follows: (1) Dissolve sodium alginate in MES buffer to obtain an alginate solution with a mass-volume concentration of 0.5% (w / v); (2) Add EDCI and NHS to the alginic acid solution to make the molar ratio of EDCI and NHS to the carboxyl group of alginic acid 1:1 (equimolar ratio relative to the carboxyl group of alginic acid), and react at room temperature for 1 h to activate the carboxyl group. (3) After adjusting the pH of the reaction system to 7.6, add 4-amino-TEMPO, so that the molar ratio of 4-amino-TEMPO to the carboxyl group of alginate is 10% or 30% respectively, and stir gently at room temperature overnight. (4) After the reaction is complete, anhydrous ethanol is used to repeatedly precipitate and wash to remove unreacted small molecules and byproducts, and then an 80% (v / v) ethanol aqueous solution is used for final washing. (5) The purified product was freeze-dried to obtain alginate-TEMPO coupling polymer.

[0035] The product with a feed molar ratio of 10% is designated as Alg-T1, and the product with a feed molar ratio of 30% is designated as Alg-T2.

[0036] Example 2: Structural characterization and grafting rate determination of coupling polymers

[0037] The product obtained in Example 1 was characterized by ¹H-NMR and UV-Vis absorption spectroscopy. ¹H-NMR results showed that the coupled product retained the characteristic signal of alginate while exhibiting a characteristic peak attributed to the TEMPO group; the UV-Vis absorption spectrum showed a characteristic absorption band of TEMPO at approximately 250 nm, indicating that 4-amino-TEMPO had been successfully covalently coupled to the alginate backbone.

[0038] Based on the results of UV-Vis absorption measurements, the TEMPO grafting rates of Alg-T1 and Alg-T2 are approximately 3% and 8%, respectively.

[0039] Example 3: Preparation of Alginic Acid-TEMPO Hydrogel

[0040] The Alg-T1 or Alg-T2 obtained in Example 1 was dissolved in double-distilled water to prepare a solution with a mass-volume concentration of 0.5% (w / v). A calcium chloride solution with a mass-volume concentration of 0.5% (w / v) was added to the above solution, and the mixture was allowed to stand at room temperature for 2 hours to crosslink, forming a stable ionicly crosslinked hydrogel, thus obtaining the Alg-T1 hydrogel or Alg-T2 hydrogel. In some comparative embodiments, a calcium chloride solution with a mass-volume concentration of 1% (w / v) can also be used for ionic crosslinking to compare the effects of different crosslinking ion concentrations on the gelation state and rheological properties of the hydrogel.

[0041] In this embodiment, the hydrogel has good gelation stability and operability, making it suitable for local application to the wound surface.

[0042] Example 4: Observation of the dissolution and gelation state of alginic acid and the alginic acid-TEMPO system under different conditions

[0043] Sodium alginate with different mass-volume concentrations (0.5%–5%) was dissolved in water, and the changes in the appearance and fluidity of the solutions were observed. The results are as follows: Figure 4 As shown in the figure. 0.5% and 1% sodium alginate were dissolved in aqueous systems with different pH conditions, and their dissolution states and appearance differences were observed. The results are as follows. Figure 5 As shown in the figure. A 0.5% sodium alginate solution was mixed with calcium chloride solutions of different concentrations, and their gel-forming state and inverted stability were observed. The results are as follows. Figure 6 As shown in the figure. The alginate-TEMPO coupling polymer solution was mixed with 0.5% and 1% calcium chloride solutions, respectively, and the appearance of the crosslinked gel was observed. The results are as follows. Figure 7 As shown.

[0044] Example 5: Rheological properties and shear-thinning characteristics of hydrogels

[0045] The viscosity-shear rate relationship of the hydrogel samples before and after crosslinking was determined using a rheometer. The results showed that Alg-T1 and Alg-T2 hydrogels had high viscosity at low shear rates, and the viscosity decreased significantly with increasing shear rate, exhibiting obvious shear-thinning behavior, which is beneficial for spreading and uniformly coating on the skin surface.

[0046] Example 6 Evaluation of in vitro free radical / reactive oxygen species scavenging capacity

[0047] The coupling polymer obtained in Example 1 was tested using in vitro evaluation methods related to free radical scavenging. The results showed that the alginate-TEMPO coupling polymer had significant free radical / reactive oxygen species scavenging ability; and the scavenging ability was further enhanced under conditions of higher grafting rate.

[0048] Example 7 Cell compatibility evaluation

[0049] RAW 264.7 macrophages and HUVEC cells were selected as in vitro evaluation models. Cells were seeded in 96-well plates and cultured overnight. Then, different concentrations of alginate, Alg-T1, or Alg-T2 solutions were added for 24 h, and cell viability was detected using the CCK-8 assay. The results showed that the conjugated polymer exhibited good cell compatibility over a wide concentration range.

[0050] Example 8 Evaluation of cell migration promotion ability

[0051] The effect of scratch assay on cell migration was evaluated. HUVEC cells were seeded in 24-well plates and cultured to confluence. Scratches were made with a pipette tip, and cell debris was removed by washing with PBS. Then, medium containing 1% FBS (negative control), alginate (1 mg / mL), Alg-T1 (1 mg / mL), Alg-T2 (1 mg / mL), and 10% FBS (positive control) were added, respectively. Images were taken at specified time points, and the migration area was calculated using image processing software. The results showed that the Alg-T conjugate treatment group exhibited significantly enhanced cell migration ability, comparable to the alginate group, indicating that TEMPO grafting does not weaken the migration-promoting biological function of alginate.

[0052] Example 9 Evaluation of Macrophage Polarization

[0053] RAW 264.7 cells were seeded in 24-well plates and cultured overnight. Then, they were treated for 24 h with culture medium (control group), alginate (1 mg / mL), Alg-T1 (1 mg / mL), or Alg-T2 (1 mg / mL), respectively. Cells were collected and analyzed by flow cytometry using CD80 and CD206 labeled with antibodies. The results showed that Alg-T treatment promoted the transformation of macrophage phenotype towards the M2-related phenotype, which helps improve the inflammatory microenvironment of wounds.

[0054] Example 10 Evaluation of intracellular ROS inhibition under oxidative stress conditions

[0055] After RAW 264.7 cells were seeded and cultured overnight, they were treated with culture medium (control group), alginate (1 mg / mL), Alg-T1 (1 mg / mL), or Alg-T2 (1 mg / mL) for 24 h, respectively. An oxidative stress model was then established by stimulation with 100 μM H2O2 for 2 h. Cells were then stained with DCFH-DA (10 μM) in the dark for 30 min, and the fluorescence intensity was quantified by fluorescence microscopy and / or microplate reader. The results showed that Alg-T treatment effectively reduced intracellular ROS levels under oxidative stress conditions.

[0056] Example 11 Evaluation of in vitro hemolysis and isotonic compatibility

[0057] The blood compatibility of the samples was evaluated by observing erythrocyte morphology and measuring hemolysis rate. A 1% erythrocyte suspension was co-incubated with alginate, Alg-T1, and Alg-T2 (final concentration 1 mg / mL), and changes in erythrocyte morphology were observed. The hemolysis rate was calculated by measuring the absorbance of the supernatant at 540 nm. The results showed that the coupled polymer had good blood compatibility.

[0058] Application Example 1: Validation of the effect on full-thickness skin wound repair in mice

[0059] A full-thickness skin defect model was established using 6-week-old female BALB / c mice. After anesthesia, a 10 mm diameter full-thickness skin defect was created on the back. Animals were randomly assigned to four groups: a saline control group, alginate group, free 4-amino-TEMPO group, dexamethasone gel control group, Alg-T1 hydrogel group, and Alg-T2 hydrogel group. Each group received topical application / treatment once daily, and wound changes were recorded daily using photographic software. Wound area and closure rate were calculated.

[0060] The results showed that the Alg-T hydrogel group could significantly accelerate wound closure. Specifically, the local application of Alg-T hydrogel achieved a wound closure rate of approximately 98% on day 12, which was significantly better than the control group.

[0061] Wound tissue samples were collected at predetermined time points for histological and immunohistochemical analysis, including HE staining, Masson staining, and CD31 immunostaining. The results showed that Alg-T hydrogel promoted re-epithelialization, collagen deposition, and angiogenesis, while reducing inflammatory cell infiltration, suggesting a combined effect of promoting tissue regeneration and regulating the inflammatory microenvironment.

[0062] Application Example 2: In vivo biosafety evaluation

[0063] Blood samples were collected from animals at the experimental endpoint for routine blood tests, and major organs were weighed to calculate organ indices. Pathological examinations were also performed on relevant tissues. The results indicate that Alg-T hydrogel exhibits good biocompatibility and safety when used in vivo.

[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Various modifications, equivalent substitutions, or improvements made by those skilled in the art without departing from the spirit and substance of the present invention should be included within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims. Industrial applicability

[0065] The alginate-TEMPO coupling polymer and its hydrogel provided by this invention have good biocompatibility, stable reactive oxygen species scavenging ability and excellent wound repair promotion effect. The preparation method is simple, the conditions are mild, and it can be mass-produced. It is suitable for preparing wound repair dressings or related medical materials and has good industrial application prospects.

Claims

1. An alginate-TEMPO coupling polymer, characterized in that, The alginate-TEMPO coupling polymer is prepared by an amidation reaction of alginate and 4-amino-TEMPO. The alginate is in the form of alginate and its common sodium, potassium, calcium, magnesium, etc. The molar ratio (M / G) of mannuronic acid (M) to guluronic acid (G) in the molecular chain is in the range of 0.25 to 2.5, preferably 0.4 to 0.6, and the average molecular weight is 100 to 200 kDa, preferably 140 to 150 kDa. The molar proportion of TEMPO structural units in the alginate-TEMPO coupling polymer is 0.01 to 0.20 of the total molar amount of mannuronic acid (M) and guluronic acid (G) in the alginate molecular chain.

2. The method for preparing the alginate-TEMPO coupling polymer according to claim 1, characterized in that, Alginic acid and 4-amino-TEMPO are prepared by an amidation reaction, including the following steps: Step 1: In a buffer solution, the carboxyl group of alginate is activated using a carboxyl activating agent. Step 2: Add 4-amino-TEMPO to the activated alginate, and purify the reaction product to obtain the alginate-TEMPO coupling polymer.

3. The coupling polymer according to claim 1 or 2, characterized in that, The carboxyl activating agent is one or a combination of dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 4-N,N-dimethylpyridine (DMAP), 1-hydroxybenzotriazole (HoBt), and N-hydroxysuccinimide (NHS); the buffer solution is a buffer solution with pH 6.0 to 8.

5.

4. An alginate-TEMPO coupled polymer hydrogel, characterized in that, The hydrogel is formed by crosslinking the alginate-TEMPO coupling polymer according to any one of claims 1 to 3 with a calcium chloride solution.

5. The method for preparing the hydrogel according to claim 4, characterized in that, Formed by crosslinking alginate-TEMPO coupling polymer with ions, including the following steps: Step 1: Dissolve the alginate-TEMPO coupling polymer in an aqueous system; Step 2: Add calcium chloride solution to form a hydrogel through ionic cross-linking.

6. The hydrogel according to claim 4 or 5, characterized in that, The aqueous system is water, physiological saline, or a buffer solution with a pH of 6.0–8.5; the concentration of the alginate-TEMPO coupling polymer in the aqueous system is 0.1%–5% (w / v), preferably 1%–2%; further, the concentration of the calcium chloride solution is 0.1%–5% (w / v), preferably 0.5%–1% (w / v).

7. The use of the hydrogel according to any one of claims 4 to 6 in the preparation of materials for skin wound repair.

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