Dopamine-modified alginate polysaccharide and polysaccharide derivatives, hydrogels prepared therefrom, and applications thereof

Abstract

The application discloses a dopamine modified seaweed polysaccharide and a polysaccharide derivative, a hydrogel and application thereof, and belongs to the technical field of seaweed polysaccharide derivatives. The double dynamic bond synergistic hydrogel is made through in-situ cross-linking reaction of a bifunctional polysaccharide derivative and a metal-polyphenol complex cross-linking agent, wherein the bifunctional polysaccharide derivative is obtained by sequentially grafting catechol compounds and phenylboronic acid compounds on seaweed polysaccharide, and the hydrogel has excellent injectability, rapid self-healing, intelligent response to a microenvironment, and multiple biological activities such as anti-inflammatory and repair promotion. The application further discloses a preparation method, and the method is mild and controllable. The hydrogel can be widely applied to the fields of microenvironment response type drug controlled release carriers, tissue engineering scaffolds and functional wound dressings, and has great potential in improving microenvironment disorder and has popularization value.

Description

Technical Field

[0001] This invention belongs to the field of seaweed polysaccharide derivative technology, specifically relating to a dopamine-modified seaweed polysaccharide and its prepared polysaccharide derivatives, hydrogels, and applications. Background Technology

[0002] Injectable hydrogels have attracted much attention in the biomedical field due to their minimally invasive implantation and perfect fit to irregular defects. An ideal injectable hydrogel needs to possess excellent shear-thinning / self-healing rheological properties, intelligent responsiveness to complex lesion microenvironments, and bioactivity that promotes healing. However, current technologies typically only address one or two of these aspects, making it difficult to achieve efficient integration of multiple functions.

[0003] Currently, hydrogels based on dynamic covalent bonds (such as phenylboronic acid ester bonds and imine bonds) have achieved responses to biomarkers such as pH and glucose, but their mechanical strength is often limited and they lack active tissue integration and repair capabilities. On the other hand, hydrogels based on non-covalent bonds (such as metal coordination and hydrogen bonds), such as tannic acid-metal ion networks, although possessing self-healing and high toughness, lack the ability to precisely respond to specific biochemical signals.

[0004] Seaweed polysaccharides (such as Ulva lactuca polysaccharide and Ulva prolifera polysaccharide) are a class of natural anionic polysaccharides derived from marine algae. Rich in carboxyl and sulfate groups, they possess good biocompatibility, degradability, and certain bioactivities (such as anti-inflammatory and antioxidant properties). However, they are difficult to form stable, injectable, and intelligently responsive gels on their own. Functional modification of polysaccharides is an effective strategy to improve their performance. Dopamine modification can introduce adhesion and coordination sites, while phenylboronic acid modification can introduce responsive crosslinking sites. However, existing technologies typically only modify polysaccharides by a single type, or although multiple modifications are performed, they fail to synergistically construct integrated networks with a clear "structure-function" relationship with suitable crosslinking agents. In particular, the construction of an "integrated" injectable hydrogel platform that combines excellent mechanical properties, intelligent responsiveness, and multiple bioactivities has not yet been reported. Summary of the Invention

[0005] To address the shortcomings of the aforementioned technologies, the present invention aims to provide a dopamine-modified seaweed polysaccharide, its prepared polysaccharide derivatives, hydrogels, and applications. Based on a seaweed polysaccharide with specific dual modifications, the present invention combines it with a functional crosslinking agent to form a hydrogel with a synergistic dynamic network of both borate ester bonds and metal-polyphenol coordination bonds. The invention also describes its applications in microenvironment-responsive drug release, tissue repair, and regenerative medicine. The prepared dopamine-modified seaweed polysaccharide hydrogel and the dual-dynamic-bond synergistic hydrogel exhibit good injectability, intelligent responsiveness, and excellent biocompatibility, and can be widely used in medical adhesives, wound dressings, drug delivery, and tissue engineering.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] This invention provides a dopamine-modified seaweed polysaccharide, the preparation method of which specifically includes the following steps:

[0008] S1: Weigh out seaweed polysaccharides, prepare seaweed polysaccharide solutions, and continuously introduce nitrogen gas to remove oxygen;

[0009] S2: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 4-dimethylaminopyridine are added sequentially to the seaweed polysaccharide solution for catalytic activation to obtain a mixed solution;

[0010] S3: Under nitrogen protection, a light-protected reagent is slowly added to the mixed solution using a micro-injection pump to carry out a light-protected reaction, thereby obtaining a dopamine-modified seaweed polysaccharide solution.

[0011] S4: The dopamine-modified seaweed polysaccharide solution is dialyzed and dried to obtain dopamine-modified seaweed polysaccharide.

[0012] Furthermore, in step S1, the solvent in the seaweed polysaccharide solution is morpholine ethanesulfonic acid buffer, the molar concentration of morpholine ethanesulfonic acid is 10-50 mM, and the pH is 5.5-7.0; the seaweed polysaccharide is selected from at least one of Ulva polysaccharide, Ulva polysaccharide, and sodium alginate, and the mass-volume concentration of the seaweed polysaccharide is 0.005-0.03 g / mL.

[0013] Furthermore, in step S2, the mass ratio of seaweed polysaccharide: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride: N-hydroxysuccinimide: 4-dimethylaminopyridine in the mixed solution is 1:2~6:2~6:0.1~3; the catalytic reaction is carried out at a temperature of 25~35℃ for 1~5 h.

[0014] Furthermore, the light-shielding reagent in step S3 is a nitrogen-saturated solution containing ascorbic acid and catechol compounds. The molar concentration of ascorbic acid is 1-5 mM, and the mass-volume concentration of the catechol compounds is 0.005-0.05 g / mL, based on the volume of the light-shielding reagent. The catechol compounds are levodopa and / or dopamine hydrochloride.

[0015] Furthermore, in step S3, a light-protected reagent is added to the activated mixed solution using a micro-injection pump, wherein the flow rate of the micro-injection pump is 2 to 5 mL / h.

[0016] This invention also provides a dopamine-modified seaweed polysaccharide hydrogel prepared from the aforementioned dopamine-modified seaweed polysaccharide, wherein the preparation method of the dopamine-modified seaweed polysaccharide hydrogel includes the following steps:

[0017] S1: Dissolve the dopamine-modified seaweed polysaccharide in PBS solution to obtain a dispersion of dopamine-modified seaweed polysaccharide;

[0018] S2: Dissolve the protein in ultrapure water to obtain a protein solution;

[0019] S3: Add the protein solution to the dispersion, stir evenly, and incubate at a temperature of 50~90 ℃ for 0.5~3 h to obtain a highly adhesive dopamine-modified seaweed polysaccharide hydrogel.

[0020] Furthermore, in step S3, the volume ratio of the dispersion to the protein solution is 1:1 to 4, and the mass-volume concentration of the dopamine-modified seaweed polysaccharide is 0.01 to 0.05 g / mL, and the mass-volume concentration of the protein solution is 0.05 to 0.5 g / mL, based on the volume of the dispersion.

[0021] Furthermore, the proteins include animal proteins and / or plant proteins containing amino acids.

[0022] Furthermore, the pH range of the solution during incubation in step S3 is 4.5 to 11.

[0023] Furthermore, the pH range of the PBS solution in step S1 is 4.5 to 7.4.

[0024] This invention also provides a polysaccharide derivative prepared from the dopamine-modified seaweed polysaccharide, wherein the preparation method of the polysaccharide derivative includes the following steps:

[0025] (1) The dopamine-modified seaweed polysaccharide was prepared into a dopamine-modified seaweed polysaccharide solution, and a phenylboronic acid compound was slowly added to the dopamine-modified seaweed polysaccharide solution to carry out a secondary grafting reaction to obtain a secondary grafting solution.

[0026] (2) The secondary grafting solution is dialyzed and dried to obtain a polysaccharide derivative based on dual modification.

[0027] Furthermore, in step (1), the phenylboronic acid compound is 3-aminophenylboronic acid and / or 4-carboxyphenylboronic acid, and the mass-volume concentration of the phenylboronic acid compound is 0.003 ~ 0.05 g / mL based on the volume of the secondary grafting solution, and the mass-volume concentration of the dopamine-modified seaweed polysaccharide is 0.005 ~ 0.03 g / mL.

[0028] This invention also provides an injectable double dynamic bond synergistic hydrogel prepared from the aforementioned polysaccharide derivative, wherein the preparation method of the double dynamic bond synergistic hydrogel includes the following steps:

[0029] (1) Dissolve the polysaccharide derivative in PBS solution to obtain a polysaccharide derivative dispersion;

[0030] (2) Dissolve the polyphenol compound and the metal salt in PBS solution to obtain a metal-polyphenol complex solution;

[0031] (3) After stirring the polysaccharide derivative dispersion and the metal-polyphenol complex solution evenly, an in-situ crosslinking reaction is carried out to obtain an injectable double dynamic bond synergistic hydrogel.

[0032] Furthermore, the pH range of the PBS solution in step (1) is 4.5 to 7.4, and the mass-volume concentration of the polysaccharide derivative is 0.01 to 0.1 g / mL, based on the volume of the polysaccharide derivative dispersion.

[0033] Furthermore, the pH range of the PBS solution in step (2) is 4.5 to 7.4.

[0034] Furthermore, the volume ratio of the polysaccharide derivative dispersion to the metal-polyphenol complex solution is 5~15:1.

[0035] Furthermore, the polyphenolic compound in step (2) is selected from any one of tannic acid, catechin, and proanthocyanidins, and the molar concentration of the polyphenolic compound is 5-200 mM based on the volume of the metal-polyphenol complex solution; the metal ion in the metal salt is selected from Zn. 2+ Fe 2+ Ca 2+ Or Al 3+ The mass-volume concentration of the metal salt is 0.01 ~ 0.1 g / mL, based on the volume of the metal-polyphenol complex solution.

[0036] The present invention also provides the application of the dopamine-modified seaweed polysaccharide, the dopamine-modified seaweed polysaccharide hydrogel, the polysaccharide derivative, or the double dynamic bond synergistic hydrogel in the preparation of biomedical materials.

[0037] The injectable dual dynamic bond synergistic hydrogel provided by the present invention is prepared by bifunctionalized polysaccharide derivatives: the bifunctionalized polysaccharide derivatives are prepared by sequentially grafting catechol compounds and phenylboronic acid compounds onto seaweed polysaccharides via covalent bonds, and then using a metal-polyphenol complex solution as a crosslinking agent to undergo an in-situ crosslinking reaction with the polysaccharide derivatives to form an injectable dual dynamic bond synergistic hydrogel.

[0038] The metal-polyphenol complex solution contains polyphenol compounds and metal ions; the in-situ crosslinking reaction forms at least a borate ester dynamic covalent bond and a metal-polyphenol coordination dynamic non-covalent bond. The borate ester bond is formed by the reaction of the phenylboronic acid group in the bifunctionalized polysaccharide derivative with the catechol group in the bifunctionalized polysaccharide derivative and / or the ortho-hydroxyl group on the polyphenol compound in the metal-polyphenol complexing crosslinking agent. The metal-polyphenol coordination bond is formed by the coordination of the metal ion in the metal-polyphenol complexing crosslinking agent with the polyphenol compound in the metal-polyphenol complexing crosslinking agent and the catechol group in the bifunctionalized polysaccharide derivative.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The present invention also provides a hydrogel prepared from dopamine-modified seaweed polysaccharide, which has an adhesion strength of 15~70 kPa, significantly higher than that of unmodified seaweed polysaccharide hydrogel. Due to its non-toxicity and high adhesion, the hydrogel can be used in biomedical fields such as oral delivery, wound repair and tissue engineering scaffolds.

[0041] 2. This invention provides a method for preparing bifunctional polysaccharide derivatives and injectable hydrogels. The extraction method is green and efficient, without the use of strong acid or strong base reagents, which is of reference value for the large-scale production of related products in the future. In addition, the hydrogels prepared by the dual-modified seaweed polysaccharides can maintain stability in the injection state, and have non-toxicity and high biocompatibility, which can be used in biomedical fields such as oral delivery, wound repair and tissue engineering scaffolds.

[0042] 3. This invention utilizes the covalent interaction between catechol groups and phenylboronic acid groups under mild conditions, thereby constructing a dual dynamic network of "boronic acid ester bonds - metal coordination bonds" with a metal-polyphenol complexing crosslinking agent. These two dynamic networks synergistically construct the three-dimensional structure of the hydrogel, thus endowing the material with intelligent responsiveness and high mechanical strength. Experiments show that dual modification of seaweed polysaccharides can significantly improve the tissue adhesion and mechanical properties of the hydrogel. Therefore, this invention introduces two functional groups, catechol and phenylboronic acid, into the seaweed polysaccharide backbone, and then constructs a dual dynamic network of "boronic acid ester bonds - metal coordination bonds" with a metal-polyphenol complexing crosslinking agent. This not only improves the stability of its network structure but also enhances its responsiveness in the pathological microenvironment, providing an effective approach for developing novel high-performance hydrogels. Attached Figure Description

[0043] Figure 1 Images show the adhesion of dopamine-modified polysaccharide-modified hydrogels to various material surfaces.

[0044] Figure 2 The figure shows the adhesion test results of the dopamine-modified polysaccharide high-adhesion hydrogel.

[0045] Figure 3 The viscosity of the high-adhesion hydrogel of dopamine-modified polysaccharide;

[0046] Figure 4 A schematic diagram of the cross-linked network structure of a dual dynamic bond synergistic injectable hydrogel;

[0047] Figure 5 The appearance of the injectable hydrogel with dual dynamic bond synergy is shown in the left image, which is APDA-1G and the right image is APDA-2G.

[0048] Figure 6 The rheological behavior of the double dynamic bond synergistic injectable hydrogel;

[0049] Figure 7 The viscosity of the injectable hydrogel with dual dynamic bond synergy;

[0050] Figure 8 The free radical scavenging rate of the double dynamic bond synergistic injectable hydrogel;

[0051] Figure 9 To enhance the biocompatibility of injectable hydrogels with dual dynamic bonds. Detailed Implementation

[0052] To better illustrate the objectives, technical solutions, and advantages of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0053] Unless otherwise specified in the embodiments of the present invention, the conditions shall be performed in accordance with conventional conditions or conditions recommended by the manufacturer.

[0054] In the embodiments of this invention, reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0055] Example 1: Preparation of the bifunctionalized polysaccharide derivative of the present invention

[0056] 1. Weigh 0.2 g of morpholine ethanesulfonic acid and mix it with 89.3 mL of ultrapure water to prepare morpholine ethanesulfonic acid buffer. Adjust the pH of the buffer to 7.0. Add 0.5 g of Ulva polysaccharide to prepare a Ulva polysaccharide solution containing 0.005 g / mL of Ulva polysaccharide. Continuously purge with high-purity nitrogen gas to remove oxygen for 20 min.

[0057] 2. Add 2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1 g of N-hydroxysuccinimide (NHS), and 0.1 g of 4-dimethylaminopyridine (DMAP) sequentially to the *Ulva prolifera* polysaccharide solution, and activate the mixture by mixing and reacting at 25 °C for 5 h to obtain the activated mixed solution.

[0058] 3. Under a nitrogen protective atmosphere, a light-protected reaction is carried out using a light-protected reagent. The light-protected reagent is 10 mL of nitrogen-saturated solution containing 17.6 mg of ascorbic acid (i.e., the molar concentration of ascorbic acid is 1 mM) and 500 mg of dopamine hydrochloride. The light-protected reagent is slowly added to the activated mixed solution using a micro-injection pump at a flow rate of 2 mL / h. The reaction is carried out at 20 °C in the dark for 8 h to obtain an intermediate solution, namely, a dopamine-modified polysaccharide solution.

[0059] 4. Transfer the intermediate solution to a dialysis bag and dialyze it in ultrapure water until no small molecule impurities are present in the solution. Then freeze-dry it to obtain dopamine-modified polysaccharide (UPDA-1).

[0060] 5. Slowly add 4-carboxyphenylboronic acid to the intermediate solution described in step 3, and react at 25 °C in the dark for 3 h to obtain a secondary grafting solution. The mass-volume concentration of the 4-carboxyphenylboronic acid is 0.003 g / mL and the mass-volume concentration of the dopamine-modified polysaccharide is 0.005 g / mL, based on the volume of the secondary grafting solution.

[0061] 6. Transfer the secondary grafting solution to a dialysis bag and place it in ultrapure water for dialysis. Replace the dialysis water periodically until there are no small molecule impurities in the solution to obtain the purified product. Freeze-dry the purified product to obtain the bifunctional polysaccharide derivative (APDA-1).

[0062] Example 2: Preparation of the bifunctionalized polysaccharide derivative of the present invention

[0063] 1. Weigh 0.3 g of morpholine ethanesulfonic acid and mix it with 88.7 mL of ultrapure water to prepare morpholine ethanesulfonic acid buffer. Adjust the pH of the buffer to 5.5. Add 1 g of Ulva polysaccharide to prepare a Ulva polysaccharide solution containing 0.01 g / mL of Ulva polysaccharide. Continuously purge with high-purity nitrogen gas to remove oxygen for 30 min.

[0064] 2. Add 3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 2 g of N-hydroxysuccinimide (NHS), and 0.3 g of 4-dimethylaminopyridine (DMAP) to the Ulva polysaccharide solution in sequence, and activate the mixture by mixing and reacting at 30 °C for 3 h to obtain the activated mixed solution.

[0065] 3. Under a nitrogen protective atmosphere, a light-protected reaction is carried out using a light-protected reagent. The light-protected reagent is 10 mL of nitrogen-saturated solution containing 35.2 mg of ascorbic acid (i.e., the molar concentration of ascorbic acid is 2 mM) and 1 g of dopamine hydrochloride. The light-protected reagent is slowly added to the activated mixed solution using a micro-injection pump at a flow rate of 3 mL / h. The reaction is carried out at 25 °C for 12 h in the dark to obtain an intermediate solution, namely, a dopamine-modified polysaccharide solution.

[0066] 4. Transfer the intermediate solution to a dialysis bag and dialyze it in ultrapure water until no small molecule impurities are present in the solution. Then freeze-dry it to obtain dopamine-modified polysaccharide (UPDA-2).

[0067] 5. Slowly add 4-carboxyphenylboronic acid to the intermediate solution described in step 3, and react at 30 °C in the dark for 4 h to obtain a secondary grafting solution. The mass-volume concentration of the 4-carboxyphenylboronic acid is 0.02 g / mL and the mass-volume concentration of the dopamine-modified polysaccharide is 0.01 g / mL, based on the volume of the secondary grafting solution.

[0068] 6. Transfer the secondary grafting solution to a dialysis bag and place it in ultrapure water for dialysis. Replace the dialysis water periodically until there are no small molecule impurities in the solution to obtain the purified product. Freeze-dry the purified product to obtain the bifunctional polysaccharide derivative (APDA-2).

[0069] Example 3: Preparation of the bifunctionalized polysaccharide derivative of the present invention

[0070] 1. Weigh 0.35 g of morpholine ethanesulfonic acid and mix it with 87.65 mL of ultrapure water to prepare morpholine ethanesulfonic acid buffer. Adjust the pH of the buffer to 6.5 and add 2 g of sodium alginate to prepare a sodium alginate solution containing 0.02 g / mL of sodium alginate. Continuously purge with high-purity nitrogen gas to remove oxygen for 40 min.

[0071] 2. Add 4 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 6 g of N-hydroxysuccinimide (NHS), and 0.4 g of 4-dimethylaminopyridine (DMAP) sequentially to the sodium alginate solution, and activate the mixture by mixing and reacting at 30 °C for 2 h to obtain the activated mixed solution.

[0072] 3. Under a nitrogen protective atmosphere, a light-protected reaction is carried out using a light-protected reagent. The light-protected reagent is 10 mL of nitrogen-saturated solution containing 52.8 mg of ascorbic acid (i.e., the molar concentration of ascorbic acid is 3 mM) and 1.5 g of levodopa. The light-protected reagent is slowly added to the activated mixed solution using a micro-injection pump at a flow rate of 4 mL / h. The reaction is carried out at 30 ℃ in the dark for 10 h to obtain an intermediate solution, namely, a dopamine-modified polysaccharide solution.

[0073] 4. Transfer the intermediate solution to a dialysis bag and dialyze it in ultrapure water until there are no small molecule impurities in the solution. Then freeze-dry it to obtain dopamine-modified polysaccharide (UPDA-3).

[0074] 5. Slowly add 3-aminophenylboronic acid to the intermediate solution described in step 3, and react at 20 °C in the dark for 3 h to obtain a secondary grafting solution. The mass-volume concentration of the 3-aminophenylboronic acid is 0.03 g / mL and the mass-volume concentration of the dopamine-modified polysaccharide is 0.02 g / mL, based on the volume of the secondary grafting solution.

[0075] 6. Transfer the secondary grafting solution to a dialysis bag and place it in ultrapure water for dialysis. Replace the dialysis water periodically until there are no small molecule impurities in the solution to obtain the purified product. Freeze-dry the purified product to obtain the bifunctional polysaccharide derivative (APDA-3).

[0076] Example 4: Preparation of the bifunctionalized polysaccharide derivative of the present invention

[0077] 1. Weigh 0.4 g of morpholine ethanesulfonic acid and mix it with 86.6 mL of ultrapure water to prepare morpholine ethanesulfonic acid buffer. Adjust the pH of the buffer to 6.0. Add 3 g of Ulva polysaccharide to prepare a Ulva polysaccharide solution containing 0.03 g / mL of Ulva polysaccharide. Continuously purge with high-purity nitrogen gas to remove oxygen for 30 min.

[0078] 2. Add 12 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 9 g of N-hydroxysuccinimide (NHS), and 0.5 g of 4-dimethylaminopyridine (DMAP) sequentially to the Ulva polysaccharide solution, and activate the mixture by mixing and reacting at 35 °C for 1 h to obtain the activated mixed solution.

[0079] 3. Under a nitrogen protective atmosphere, a light-protected reaction is carried out using a light-protected reagent. The light-protected reagent is 10 mL of nitrogen-saturated solution containing 70.4 mg of ascorbic acid (i.e., the molar concentration of ascorbic acid is 4 mM) and 2 g of levodopa. The light-protected reagent is slowly added to the activated mixed solution using a micro-injection pump at a flow rate of 5 mL / h. The reaction is carried out at 35 °C for 4 h in the dark to obtain an intermediate solution, i.e., a modified polysaccharide solution.

[0080] 4. Transfer the intermediate solution to a dialysis bag and dialyze it in ultrapure water until there are no small molecule impurities in the solution. Then freeze-dry it to obtain dopamine-modified polysaccharide (UPDA-4).

[0081] 5. Slowly add 3-aminophenylboronic acid to the intermediate solution described in step 3, and react at 25 °C in the dark for 4 h to obtain a secondary grafting solution. The mass-volume concentration of the 3-aminophenylboronic acid is 0.05 g / mL and the mass-volume concentration of the dopamine-modified polysaccharide is 0.03 g / mL, based on the volume of the secondary grafting solution.

[0082] 6. Transfer the secondary grafting solution to a dialysis bag and place it in ultrapure water for dialysis. Replace the dialysis water periodically until there are no small molecule impurities in the solution to obtain the purified product. Freeze-dry the purified product to obtain the bifunctional polysaccharide derivative (APDA-4).

[0083] Example 5: Preparation of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0084] 1. Weigh 0.5 g of dopamine-modified polysaccharide UPDA-1 and dissolve it in 49.5 mL of PBS solution at pH 4.5. The mass-volume concentration of the prepared dopamine-modified polysaccharide dispersion is 0.01 g / mL.

[0085] 2. Dissolve 2.5 g of gelatin in 47.5 mL of ultrapure water to prepare a gelatin solution with a mass-volume concentration of 0.05 g / mL.

[0086] 3. Mix 25 mL of dopamine-modified polysaccharide dispersion and 25 mL of gelatin solution evenly, hydrate at 4 °C, adjust the pH of the mixed solution to 4.5, and incubate at 50 °C for 3 h to obtain dopamine-modified polysaccharide high-adhesion hydrogel (UPDA-1G).

[0087] Example 6: Preparation of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0088] 1. Weigh 1 g of dopamine-modified polysaccharide UPDA-2 and dissolve it in 49 mL of PBS solution at pH 5.5. The mass-volume concentration of the prepared dopamine-modified polysaccharide dispersion is 0.02 g / mL.

[0089] 2. Dissolve 5 g of whey protein isolate in 45 mL of ultrapure water to prepare a whey protein isolate solution with a mass-volume concentration of 0.1 g / mL.

[0090] 3. Mix 25 mL of dopamine-modified polysaccharide dispersion and 50 mL of whey protein isolate solution evenly, hydrate at 4 °C, adjust the pH of the mixed solution to 7.0, and incubate at 70 °C for 2 h to obtain dopamine-modified polysaccharide high-adhesion hydrogel (UPDA-2G).

[0091] Example 7: Preparation of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0092] 1. Weigh 1.5 g of dopamine-modified polysaccharide UPDA-3 and dissolve it in 48.5 mL of PBS solution at pH 6.4. The mass-volume concentration of the prepared dopamine-modified polysaccharide dispersion is 0.03 g / mL.

[0093] 2. Dissolve 6 g of soy protein isolate in 44 mL of ultrapure water to prepare a soy protein isolate solution with a mass-volume concentration of 0.12 g / mL.

[0094] 3. Mix 25 mL of dopamine-modified polysaccharide dispersion and 75 mL of soy protein isolate solution evenly, hydrate at 4 ℃, adjust the pH of the mixed solution to 9.0, and incubate at 80 ℃ for 1 h to obtain dopamine-modified polysaccharide high adhesion hydrogel (UPDA-3G).

[0095] Example 8: Preparation of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0096] 1. Weigh 2 g of dopamine-modified polysaccharide UPDA-4 and dissolve it in 48 mL of PBS solution at pH 7.4. The mass-volume concentration of the prepared dopamine-modified polysaccharide dispersion is 0.04 g / mL.

[0097] 2. Dissolve 4 g of soy protein isolate in 46 mL of ultrapure water to prepare a soy protein isolate solution with a mass-volume concentration of 0.08 g / mL.

[0098] 3. Mix 25 mL of dopamine-modified polysaccharide dispersion and 100 mL of soy protein isolate solution evenly, hydrate at 4 °C, adjust the pH of the mixed solution to 11.0, and incubate at 90 °C for 0.5 h to obtain dopamine-modified polysaccharide high-adhesion hydrogel (UPDA-4G).

[0099] Example 9: Preparation of injectable dual-dynamic bond synergistic hydrogel of dual-modified polysaccharide of the present invention

[0100] 1. Weigh 0.5 g of the bifunctionalized polysaccharide derivative APDA-1 and dissolve it in 50 mL of PBS solution at pH 4.5 to obtain a polysaccharide derivative dispersion. The mass-volume concentration of APDA-1 in the polysaccharide derivative dispersion is 0.01 g / mL.

[0101] 2. Dissolve 0.06 g of proanthocyanidins and 0.10 g of ferric chloride in 10 mL of PBS solution at pH 4.5 to obtain a metal-polyphenol complex solution;

[0102] 3. The polysaccharide derivative dispersion and the metal-polyphenol complex solution are mixed and stirred at a volume ratio of 5:1 to carry out an in-situ cross-linking reaction, thereby obtaining an injectable double dynamic bond synergistic hydrogel (APDA-1G).

[0103] Example 10: Preparation of an injectable dual-dynamic bond synergistic hydrogel of the present invention with dual-modified polysaccharide

[0104] 1. Weigh 3 g of the bifunctionalized polysaccharide derivative APDA-2 and dissolve it in 42 mL of PBS solution at pH 5.5 to obtain a polysaccharide derivative dispersion. The mass-volume concentration of APDA-2 in the polysaccharide derivative dispersion is 0.07 g / mL.

[0105] 2. Dissolve 0.06 g of tannic acid and 0.2 g of zinc chloride in 4.84 mL of PBS solution at pH 5.5 to obtain a metal-polyphenol complex solution;

[0106] 3. The polysaccharide derivative dispersion and the metal-polyphenol complex solution are mixed and stirred at a volume ratio of 9:1 to carry out an in-situ cross-linking reaction, thereby obtaining an injectable double dynamic bond synergistic hydrogel (APDA-2G).

[0107] Example 11: Preparation of injectable dual-dynamic bond synergistic hydrogel of dual-modified polysaccharide of the present invention

[0108] 1. Weigh 4.5 g of the bifunctionalized polysaccharide derivative APDA-3 and dissolve it in 45 mL of PBS solution at pH 6.4 to obtain a polysaccharide derivative dispersion. The mass-volume concentration of APDA-3 in the polysaccharide derivative dispersion is 0.1 g / mL.

[0109] 2. Dissolve 0.15 g of catechin and 0.25 g of calcium chloride in 3 mL of PBS solution at pH 6.4 to obtain a metal-polyphenol complex solution;

[0110] 3. The polysaccharide derivative dispersion and the metal-polyphenol complex solution are mixed and stirred at a volume ratio of 15:1 to carry out an in-situ cross-linking reaction, thereby obtaining an injectable double dynamic bond synergistic hydrogel (APDA-3G).

[0111] Example 12: Preparation of injectable dual-dynamic bond synergistic hydrogel of dual-modified polysaccharide of the present invention

[0112] 1. Weigh 2.2 g of the bifunctionalized polysaccharide derivative APDA-4 and dissolve it in 55 mL of PBS solution at pH 7.0 to obtain a polysaccharide derivative dispersion. The mass-volume concentration of APDA-4 in the polysaccharide derivative dispersion is 0.04 g / mL.

[0113] 2. Dissolve 0.075 g of tannic acid and 0.3 g of aluminum chloride in 5 mL of PBS solution at pH 7.0 to obtain a metal-polyphenol complex solution;

[0114] 3. The polysaccharide derivative dispersion and the metal-polyphenol complex solution are mixed and stirred at a volume ratio of 11:1 to carry out an in-situ cross-linking reaction, thereby obtaining an injectable double dynamic bond synergistic hydrogel (APDA-4G).

[0115] Example 13: Verification of the adhesiveness of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0116] The hydrogels used in this embodiment are UPDA-1G prepared in Example 5, UPDA-2G prepared in Example 6, UPDA-3G prepared in Example 7, and UPDA-4G prepared in Example 8.

[0117] The formed hydrogel samples were adhered to and photographed on the surfaces of various materials (glass, latex gloves, polypropylene). The results are shown in the figure. Figure 1 .

[0118] like Figure 1 As shown, all dopamine-modified polysaccharide hydrogels can stably adhere to the surfaces of various materials.

[0119] Example 14: Adhesion test of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0120] The hydrogels used in this embodiment are UPDA-1G prepared in Example 5, UPDA-2G prepared in Example 6, UPDA-3G prepared in Example 7, and UPDA-4G prepared in Example 8.

[0121] The prepared hydrogel was applied to the surface of pigskin and cured by ultraviolet light. The pigskin was then held in place, and a tensile testing machine was used to apply tension to the hydrogel on the surface of the pigskin at a speed of 25 mm / min until it was separated.

[0122] The results are as follows Figure 2 As shown, the hydrogels prepared according to Examples 5, 6, 7 and 8 have excellent adhesion strength. The overall adhesion strength of the prepared hydrogels is 15-70 KPa, among which the adhesion strength of UPDA-4G hydrogel is 60-70 KPa, which is the best.

[0123] Example 15: Viscosity determination of the high-adhesion dopamine-modified polysaccharide hydrogel of the present invention

[0124] The hydrogels used in this embodiment are UPDA-1G prepared in Example 5, UPDA-2G prepared in Example 6, UPDA-3G prepared in Example 7, and UPDA-4G prepared in Example 8.

[0125] The viscosity of the hydrogel was determined at room temperature using a controlled stress rheometer (AR2000EX). The sample was placed on a 20 mm stainless steel parallel plate, and steady-state shear measurements were performed in the range of 0.1–100 s⁻¹. -1 The viscosity variation with frequency was recorded within the shear frequency range.

[0126] The results are as follows Figure 3 As shown, the hydrogels prepared according to Examples 5, 6, 7, and 8 exhibited a gradual decrease in viscosity with increasing shear rate, demonstrating significant shear thinning, indicating that all UPDA hydrogels were pseudoplastic fluids. Among them, UPDA-4G exhibited the strongest network structure (initial viscosity > 10). 7 (mPa·s). Across the entire frequency range, the viscosity curves of UPDA-4G and UPDA-2G were significantly higher than those of UPDA-1G and UPDA-3G. This indicates that the hydrogel prepared by UPDA-4G possesses the densest or strongest three-dimensional network structure, effectively resisting deformation under low shear. Secondly, UPDA-2G exhibits a moderate strength structure, indicating that the integrity and strength of its network structure are superior to UPDA-1G and UPDA-3G. UPDA-1G has the lowest viscosity among the four samples, suggesting that its internal crosslinking density or the interaction between polymer chains is relatively weak, making its network structure more susceptible to shear failure.

[0127] Example 16: Appearance of the injectable double dynamic bond synergistic hydrogel of the dual-modified polysaccharide of the present invention

[0128] The hydrogels used in this embodiment are APDA-1G prepared in Example 9 and APDA-2G prepared in Example 10.

[0129] A schematic diagram of the cross-linked network structure of the dual dynamic bond synergistic hydrogel is shown below. Figure 4 As shown. The formed hydrogel sample was injected into PBS buffer at pH 6.4, and the results are shown in the figure. Figure 5 .

[0130] The results are as follows Figure 5 As shown, the injectable double dynamic bond synergistic hydrogels prepared based on dual-modified polysaccharides can all be injected and stabilized in PBS buffer solution.

[0131] Example 17: Rheological behavior of the injectable double dynamic bond synergistic hydrogel of the dual-modified polysaccharide of the present invention

[0132] The hydrogels used in this embodiment are APDA-1G prepared in Example 9, APDA-2G prepared in Example 10, APDA-3G prepared in Example 11, and APDA-4G prepared in Example 12.

[0133] A series of rheological properties of hydrogels were determined at room temperature using a controllable stress rheometer (AR2000EX). The samples were placed on a 20 mm stainless steel parallel plate and the frequency was swept by angular frequency changes from 1 to 100 rad / s.

[0134] The results are as follows Figure 6 As shown, the storage modulus (G') of the hydrogels prepared according to Examples 9, 10, 11, and 12 is greater than the loss modulus (G''), indicating that the hydrogels prepared in this invention have greater elastic characteristics than viscous characteristics and possess rheological properties of solid gels. APDA-4G hydrogel exhibits a higher storage modulus (651.78 Pa) during structural failure, followed by APDA-2G and APDA-3G, while APDA-1G has the weakest rheological properties (G' = 198.61 Pa). Therefore, the injectable double dynamic bond synergistic hydrogel prepared in this invention has excellent rheological behavior and broad development prospects in the fields of food, agriculture, tissue engineering, biopharmaceuticals, and biomaterials.

[0135] Example 18: Viscosity determination of the injectable double dynamic bond synergistic hydrogel of the dual-modified polysaccharide of the present invention

[0136] The hydrogels used in this embodiment are APDA-1G prepared in Example 9, APDA-2G prepared in Example 10, APDA-3G prepared in Example 11, and APDA-4G prepared in Example 12.

[0137] The viscosity of the hydrogel was determined at room temperature using a controlled stress rheometer (AR2000EX). The sample was placed on a 20 mm stainless steel parallel plate, and steady-state shear measurements were performed in the range of 0.1–100 s⁻¹. -1 The viscosity variation with frequency was recorded within the shear frequency range.

[0138] The results are as follows Figure 7 As shown, the hydrogels prepared according to Examples 9, 10, 11, and 12 exhibited a gradual decrease in gel viscosity with increasing shear rate, demonstrating significant shear thinning, indicating that the APDA hydrogels were all pseudoplastic fluids. Figure 3 In comparison, the hydrogel of the dual-modified polysaccharide showed a significant increase in viscosity compared to the dopamine-modified polysaccharide hydrogel, with the initial viscosity increasing from 10...7 mPa·s rose to 10 8 The results showed that the additional group modifications introduced more crosslinking sites into the system, significantly enhancing the interactions between molecular chains and thus constructing a more robust three-dimensional network. The APDA-4 hydrogel exhibited the highest viscosity across the entire shear rate range. This indicates that the internal network structure of the hydrogel in this formulation is the most dense or robust, possessing the strongest resistance to deformation under external forces and exhibiting the best network integrity.

[0139] Example 19: Free radical scavenging rate of the injectable double-dynamic bond synergistic hydrogel of the dual-modified polysaccharide of the present invention

[0140] The hydrogels used in this embodiment are APDA-1G prepared in Example 9, APDA-2G prepared in Example 10, APDA-3G prepared in Example 11, and APDA-4G prepared in Example 12.

[0141] Weigh 0.1 g of hydrogel into a centrifuge tube containing 2 mL of PBS. Incubate at 37°C and 100 rpm in the dark for 24 hours. Add 100 μL of the supernatant and 0.2 mM DPPH solution to each well of a 96-well plate. After addition, wrap the plate with aluminum foil and allow it to stand in the dark for 30 minutes. Use ascorbic acid as a positive control. Measure the absorbance at 517 nm using a microplate reader and calculate the DPPH radical scavenging rate.

[0142] The results are as follows Figure 8 As shown, the hydrogels prepared according to Examples 9, 10, 11, and 12 exhibited excellent free radical scavenging rates, ranging from 66.43% to 85.34% overall. The APDA-2G hydrogel showed the best free radical scavenging rate at 85.34%. This indicates that the injectable, dual-dynamic-bond synergistic hydrogel with dual-modified polysaccharides can effectively neutralize excess free radicals under inflammatory conditions and reduce oxidative stress damage.

[0143] Example 20: Biocompatibility of the injectable double dynamic bond synergistic hydrogel of the dual-modified polysaccharide of the present invention

[0144] The hydrogels used in this embodiment are APDA-1G prepared in Example 9, APDA-2G prepared in Example 10, APDA-3G prepared in Example 11, and APDA-4G prepared in Example 12.

[0145] The biocompatibility of four hydrogels in RAW 264.7 cells was evaluated using the CCK-8 assay. Hydrogel samples of different concentrations were immersed in DMEM medium containing 10% fetal bovine serum and incubated at 37 °C for 24 hours to obtain extracts at concentrations of 0.625, 1.25, 2.5, and 5 mg / mL. RAW 264.7 cells were seeded in 96-well plates and cultured for 24 hours. The original medium was then removed, and 100 μL of each concentration of hydrogel extract was added. 10 μL of CCK-8 reagent was added to the cells after 24 hours of culture, and incubation continued for another 2 hours. Using the culture medium alone as a background control, absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated.

[0146] The results are as follows Figure 9 As shown, the cell viability of the hydrogels prepared according to Examples 9, 10, 11, and 12 was above 75% at different concentrations. With decreasing hydrogel concentration, cell viability in all groups showed an increasing trend, indicating that high-concentration hydrogel extracts have a slight inhibitory effect on cell metabolism, while low concentrations showed better material compatibility, exhibiting good dose-dependency overall. These results demonstrate that cells can survive and proliferate at each hydrogel concentration, implying that the injectable double dynamic bond synergistic hydrogel described in this invention has good biocompatibility. Among them, APDA-2G exhibited the most superior cell compatibility results.

[0147] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A polysaccharide derivative prepared from dopamine-modified seaweed polysaccharide, characterized in that, The preparation method of the polysaccharide derivative includes the following steps: S1: Weigh out seaweed polysaccharides, prepare seaweed polysaccharide solutions, and continuously introduce nitrogen gas to remove oxygen; The solvent in the seaweed polysaccharide solution is morpholine ethanesulfonic acid buffer, wherein the molar concentration of morpholine ethanesulfonic acid is 10-50 mM and the pH is 5.5-7.0; the seaweed polysaccharide is selected from Ulva polysaccharide and / or Ulva prolifera polysaccharide, and the mass-volume concentration of the seaweed polysaccharide is 0.005-0.03 g / mL; S2: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 4-dimethylaminopyridine are added sequentially to the seaweed polysaccharide solution, and the mixture is activated by catalysis to obtain a mixed solution; The mass ratio of seaweed polysaccharide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and 4-dimethylaminopyridine in the mixed solution is 1:2~6:2~6:0.1~3; the catalytic reaction is carried out at a temperature of 25~35℃ for 1~5 h. S3: Under nitrogen protection, a light-protected reagent is slowly added to the mixed solution using a micro-injection pump to carry out a light-protected reaction, thereby obtaining a dopamine-modified seaweed polysaccharide solution. The light-shielding reagent is a nitrogen-saturated solution containing ascorbic acid and catechol compounds, wherein the molar concentration of ascorbic acid is 1-5 mM, and the mass-volume concentration of the catechol compounds is 0.005-0.05 g / mL; the catechol compounds are levodopa and / or dopamine hydrochloride. S4: The dopamine-modified seaweed polysaccharide solution is dialyzed and dried to obtain dopamine-modified seaweed polysaccharide; S5: The dopamine-modified seaweed polysaccharide is prepared into a dopamine-modified seaweed polysaccharide solution. A phenylboronic acid compound is slowly added to the dopamine-modified seaweed polysaccharide solution to carry out a secondary grafting reaction, obtaining a secondary grafting solution. The phenylboronic acid compound is 3-aminophenylboronic acid and / or 4-carboxyphenylboronic acid. Based on the volume of the secondary grafting solution, the mass-volume concentration of the phenylboronic acid compound is 0.003 ~ 0.05 g / mL, and the mass-volume concentration of the dopamine-modified seaweed polysaccharide is 0.005 ~ 0.03 g / mL. S6: The secondary grafting solution is dialyzed and dried to obtain a polysaccharide derivative based on dual modification.

2. The double dynamic bond synergistic hydrogel prepared from the polysaccharide derivative according to claim 1, characterized in that, The preparation method of the dual dynamic bond synergistic hydrogel includes the following steps: (1) Dissolve the polysaccharide derivative in PBS solution to obtain a polysaccharide derivative dispersion; (2) Dissolve the polyphenol compound and the metal salt in PBS solution to obtain a metal-polyphenol complex solution; (3) After stirring the polysaccharide derivative dispersion and the metal-polyphenol complex solution evenly, an in-situ crosslinking reaction is carried out to obtain a double dynamic bond synergistic hydrogel; The polyphenol compound is selected from any one of tannic acid, catechin and procyanidin, and the molar concentration of the polyphenol compound is 5-200 mM in volume of the metal-polyphenol complex solution; the metal ion in the metal salt is selected from Zn 2+ , Fe 2 + , Ca 2+ or Al 3+ , and the mass-volume concentration of the metal salt is 0.01-0.1 g / mL in volume of the metal-polyphenol complex solution.

3. The application of the polysaccharide derivative of claim 1 or the double dynamic bond synergistic hydrogel of claim 2 in the preparation of biomedical materials.

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