Injectable self-repairing polysaccharide-based hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable self-repairing polysaccharide-based hydrogel as well as a preparation method and application thereof. The injectable polysaccharide-based hydrogel is formed by carrying out reversible covalent polymerization on phenylboronic acid modified open-loop hyaluronic acid and polyvinyl alcohol; the hydrogel has a uniform porous network structure, and the pore diameter of the hydrogel is within 1 mu m. After the hyaluronic acid is subjected to ring opening treatment by an oxidizing agent, more aminophenylboronic acid can be grafted on aldehyde groups and carboxyl groups, so that the grafting rate is increased, and the hyaluronic acid and rich hydroxyl groups of polyvinyl alcohol can form more boric acid ester bonds, so that gelatinization can be quicker; the borate bond is a reversible covalent bond, so that the hydrogel has the characteristic of self-repairing. The two polymers forming the hydrogel are high in biosafety, and the formed hydrogel has excellent injectable performance and self-repairing capability, can be directly suitable for irregular wounds in an injection mode, and is suitable for wound closure in an emergency.

Description

TECHNICAL FIELD

[0001] The present application relates to an injectable self-healing polysaccharide-based hydrogel, a preparation method and application thereof, and belongs to the field of biomedical polymer materials. BACKGROUND

[0002] In clinical practice, acute bleeding and wound infection are the main clinical factors leading to patient death after tissue injury such as accidental trauma and surgical incision. Although suture and staple suture are the most commonly used wound closure methods in current clinical treatment, they are time-consuming and technically demanding, and are not suitable for many emergency situations, such as sudden natural disasters and wars. In recent years, bioadhesives have become a research hotspot in the field of wound repair and are widely used in wound closure, intraoperative hemostasis, anastomotic fistula prevention, and wound healing in clinical scenarios. However, due to inherent defects such as insufficient tissue adhesion, potential toxic ingredients, and poor controllability of biodegradation, commercially available bioadhesives still cannot meet the actual needs of clinical practice. For example, fibrin glue, although approved by the US FDA for the closure and hemostasis of various wounds, has a low tissue adhesion strength (5-10 kPa) and a risk of blood-borne virus transmission. Cyanacrylate adhesives have a high adhesion strength and are approved for use in skin wound closure, but the release of toxic cyanacrylate monomers and polymer degradation products greatly limits their application in in vivo clinical environments.

[0003] Hyaluronic acid, as a natural polymer material, has excellent biocompatibility and abundant functional group modification sites. Polyvinyl alcohol, as a synthetic polymer that has been widely used and has excellent biocompatibility. However, there is no report on the construction of an injectable dynamic hydrogel system using polyvinyl alcohol and benzene boronic acid-modified hyaluronic acid cross-linked through reversible boronate ester bonds. SUMMARY

[0004] Based on the existing technical problems, the purpose of the present application is to provide an injectable self-healing polysaccharide-based hydrogel, a preparation method and application thereof. Hyaluronic acid is used as the main raw material, which is modified by ring opening and benzene boronic acid to obtain benzene boronic acid-modified ring-opening hyaluronic acid. It is mixed with an equal volume of polyvinyl alcohol solution to construct a dynamic covalent network using the boronate ester bonds formed between the benzene boronic acid and the hydroxyl groups on the polyvinyl alcohol molecular chain, to prepare an injectable self-healing hydrogel suitable for wound closure in emergency situations.

[0005] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions:

[0006] The injectable self-repairing polysaccharide-based hydrogel is prepared by using benzene boronic acid modified open ring hyaluronic acid and polyvinyl alcohol as raw materials, the benzene boronic acid on the molecule of the former and the rich hydroxyl of the latter form reversible covalent bond-boric acid ester bond, and then form the injectable and self-healing hydrogel. The hydrogel has a uniform porous network structure, and the pore size is within 1 μm.

[0007] The volume ratio of the benzene boronic acid modified open ring hyaluronic acid to the polyvinyl alcohol is 1:1.

[0008] The concentration of the benzene boronic acid modified open ring hyaluronic acid is 2wt%-5wt%, and the concentration of the polyvinyl alcohol is 5wt%.

[0009] The molecular weight of the hyaluronic acid is 400,000, and the oxidation degree of the hyaluronic acid after the open ring treatment is 35%-45%.

[0010] The weight average molecular weight of the polyvinyl alcohol is 8.9×10 4 -9.8×10 4 .

[0011] The aldehyde group hyaluronic acid formed after the open ring treatment of the hyaluronic acid can graft more aminobenzene boronic acid on the aldehyde group and the carboxyl group, form the benzene boronic acid modified open ring hyaluronic acid with better solubility and more effective functional groups. And the grafting rate is improved, so that it can form more boric acid ester bonds with the rich hydroxyl of the polyvinyl alcohol, thereby gelling faster; the benzene boronic acid of the benzene boronic acid modified open ring hyaluronic acid generates boric acid ester bond with the hydroxyl of the polyvinyl alcohol, forming a dynamic covalent network.

[0012] The preparation method of the above-mentioned injectable self-repairing polysaccharide-based hydrogel, the reaction formula is as follows:

[0013]

[0014] Specifically includes the following steps:

[0015] Step one: preparation of phenylboronic acid modified open ring hyaluronic acid ([1] Jeon EJ, Han SY, et al. pH-Universal Catechol-Amine Chemistry for Versatile Hyaluronic Acid Bioadhesives. Small. 2022; 18(41): e2202729. doi: 10.1002 / smll.202202729; [2] Lei L, Cong R, Ni Y, et al. Dual-Functional Injectable Hydrogel for Osteoarthritis Treatments. Adv Healthc Mater. 2024; 13(5): e2302551. doi: 10.1002 / adhm.202302551):

[0016] (1) Oxidize hyaluronic acid (HA) with sodium periodate (NaIO4) to obtain open ring hyaluronic acid. Specifically, the concentration of hyaluronic acid is 2.5wt%, and the concentration of sodium periodate is 0.5mol / L. React for 2h in the dark, and add ethylene glycol to terminate the reaction.

[0017] (2) React the open ring hyaluronic acid with 3-aminophenylboronic acid (3A-PBA) to obtain phenylboronic acid modified open ring hyaluronic acid; dissolve the phenylboronic acid modified open ring hyaluronic acid in ultrapure water to obtain a phenylboronic acid modified open ring hyaluronic acid aqueous solution. Specifically, dissolve the open ring hyaluronic acid in 2-morpholinoethanesulfonic acid buffer, add 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (DMTMM) to activate the carboxyl group of the open ring hyaluronic acid for 30min, then add 3-aminophenylboronic acid and react at room temperature for 24h, dialyze and freeze-dry.

[0018] Step two: disperse polyvinyl alcohol in ultrapure water, stir and heat to 80℃ to dissolve, to obtain a polyvinyl alcohol aqueous solution with a concentration of 5wt%. Prepare a phenylboronic acid modified open ring hyaluronic acid aqueous solution with a concentration of 2-5wt%.

[0019] Step three: mix the phenylboronic acid modified open ring hyaluronic acid aqueous solution with the polyvinyl alcohol aqueous solution obtained in step two in equal volume to form dynamic covalent boronic ester bonds, to obtain an injectable self-healing polysaccharide-based hydrogel.

[0020] The injectable self-healing polysaccharide-based hydrogel is used for preparing a material for direct injection into an open wound, can be directly injected into an irregular wound, is used for wound closure in emergency situations, has high biological safety and is degradable, and can replace sutures to achieve wound closure.

[0021] Beneficial effects: compared with the prior art, the present application has the following characteristics:

[0022] (1) The hydrogel of the present application has simple preparation process, short preparation period, and raw materials are easy to obtain, and can realize large-scale industrial production.

[0023] (2) By modifying hyaluronic acid, benzene boronic acid modified open ring hyaluronic acid is prepared. After the ring opening of hyaluronic acid, the side chain of hyaluronic acid adds aldehyde functional group, so that after the addition of 3-aminobenzene boronic acid, benzene boronic acid can be grafted on the carboxyl and aldehyde functional group of open ring hyaluronic acid, and the grafting rate of benzene boronic acid is improved.

[0024] (3) The reversible borate ester bond is formed between benzene boronic acid and hydroxyl group, and a crosslinking network based on dynamic covalent bond is constructed, so that it has good injectability and self-repairing ability, which is beneficial to fill and close irregular shape and deep wound, and is suitable for different wound defects. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The morphology diagram of the hydrogel prepared in example 1 of the present application (figures a-d);

[0026] Figure 2 The process diagram of the hydrogel prepared in example 1 of the present application (figure a), the self-repairing experiment diagram (figure b), the shear thinning property test result diagram (figure c) and the self-healing scanning test result diagram (figure d);

[0027] Figure 3 The wound closure effect diagram of the hydrogel prepared in example 1 of the present application in the full defect model of the back skin of SD rats; (a) experimental process diagram, (b) different experimental groups after operation Different days of wound healing diagram. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application is further described in detail below in combination with specific examples and with reference to the drawings.

[0029] Example 1:

[0030] The preparation method of the injectable self-repairing polysaccharide-based hydrogel is as follows:

[0031] Step 1: Preparation of phenylboronic acid-modified open-ring hyaluronic acid ([1 Jeon EJ, Han SY, et al. pH-Universal Catechol-Amine Chemistry for Versatile Hyaluronic Acid Bioadhesives. Small. 2022;18(41):e2202729.doi:10.1002 / smll.202202729;[2] Lei L, Cong R, Ni Y, et al. Dual-Functional Injectable Hydrogel for Osteoarthritis Treatments. Adv Healthc Mater. 2024;13(5):e2302551.doi:10.1002 / adhm.202302551):

[0032] (1) Dissolve 3.0 g of hyaluronic acid (Mn = 400 kDa, Ron Chemical, purchased from Shanghai Yi'en Chemical Technology Co., Ltd.) in 120 mL of ultrapure water, and heat and stir to form an aqueous solution of hyaluronic acid. Add 30 mL of 0.5 M NaIO4, react in the dark for 2 h, and then add 10 mL of ethylene glycol to terminate the reaction. Dialyze the mixed solution for 3 days, and then freeze-dry to obtain ring-opening hyaluronic acid (OHA).

[0033] (2) 1.0 g of ring-opening hyaluronic acid was dissolved in 200 mL of 0.1 M 2-morpholine ethanesulfonic acid solution to obtain a ring-opening hyaluronic acid solution. 1.5 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) was added to activate the carboxyl group for 30 min; 0.7 g of 3-aminophenylboronic acid (3A-PBA) was added, and the reaction was carried out at room temperature for 24 h. The resulting solution was dialyzed for 3 days and then lyophilized to obtain phenylboronic acid-modified ring-opening hyaluronic acid (OHA-PBA). The phenylboronic acid-modified ring-opening hyaluronic acid was dissolved in ultrapure water at a concentration of 2–5 wt%.

[0034] Step 2: Place polyvinyl alcohol in ultrapure water and heat to dissolve, to obtain a 5wt% polyvinyl alcohol (PVA) aqueous solution.

[0035] Step 3: Mix equal volumes of phenylboronic acid-modified open-ring hyaluronic acid aqueous solution and polyvinyl alcohol aqueous solution at a volume ratio of 1:1 to obtain injectable self-healing polysaccharide hydrogel.

[0036] Figure 1SEM images in a-d show that the four hydrogels (corresponding to sample 1, sample 2, sample 3 and sample 4 respectively) prepared by mixing equal volume of 2wt%, 3wt%, 4wt% and 5wt% of the aqueous solution of benzene boronic acid modified open hyaluronic acid prepared in example 1 and 5wt% of the aqueous solution of polyvinyl alcohol all have uniform porous network structure with pore diameter within 1 μm. With the increase of the concentration of benzene boronic acid modified open hyaluronic acid from 2wt% to 5wt%, the pore size of the hydrogels gradually decreases, which indicates that the cross-linked network structure of the hydrogels becomes more and more compact with the increase of the concentration of benzene boronic acid modified open hyaluronic acid. The above results demonstrate that the concentration of benzene boronic acid modified open hyaluronic acid can be adjusted to meet the practical application requirements in different scenarios.

[0037] Example 2:

[0038] 1 mL of 3wt% benzene boronic acid modified open hyaluronic acid aqueous solution prepared in example 1 and 1 mL of 5wt% polyvinyl alcohol aqueous solution (Mw: 89000-98000, Rohn, purchased from Shanghai Yinn Chemical Technology Co., Ltd.) were separately placed in two syringes (2.5 mL) connected by a luer adapter, and pushed back and forth until mixed into a uniform injectable hydrogel (sample 3). The rheological properties of the hydrogel were tested using a rotational rheometer.

[0039] Figure 2 a is a schematic diagram of the preparation of an injectable self-healing hydrogel, and the aqueous solution of benzene boronic acid modified open hyaluronic acid (OHA-PBA) and the aqueous solution of polyvinyl alcohol (PVA) are mixed in equal volume, and pushed back and forth until mixed uniformly to form an injectable hydrogel (OHA-PBA / PVA).

[0040] After the hydrogel is dyed with two different dyes, a mold is used to make the hydrogel with a symmetrical shape Figure 2 I), III) in b, cut the two hydrogels of different colors (II, IV) and splice the two parts of different colors together, as shown in Figure 2 b, the two parts cut finally can be spliced into a whole by self-healing Figure 2 V) in b, which shows that the hydrogel has self-repairing ability. From Figure 2 c, it can be seen that the hydrogel sample has shear thinning properties, so the hydrogel has injectable properties. Under constant oscillation rate, the hydrogel was subjected to 1% and 300% strain alternating scanning, and the results are shown in Figure 2 d, which shows that the hydrogel has good self-repairing ability. The rheological results show that the hydrogel has excellent extrusion-injection-in-situ plasticity, and thus has the potential to be applied to irregular wounds.

[0041] Example 3:

[0042] The hydrogel prepared in Example 2 (sample 3) was evaluated for wound closure using a rat dorsal skin defect model Figure 3 a) After anesthesia, SD rats (6-8 weeks, male, ~250 g, N=4) were cut with surgical scissors to make a 2 cm long incision on the back of the rats. The hydrogel was injected at the wound site, and the skin on both sides of the wound was adhered together. The wound was photographed on the day of surgery and on the 4th, 7th, 10th, and 14th day after surgery Figure 3 b) The control group was not treated, the suture group was treated with sutures, and the experimental group was only treated with the injectable self-healing hydrogel. The results showed that the hydrogel group showed similar closure effect as the suture, and compared with the suture group, the hydrogel avoided the tissue damage caused by suture puncture, therefore, the injectable self-healing hydrogel has the potential to replace sutures to achieve wound closure.

[0043] The above merely provides a specific implementation of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. An injectable self-healing polysaccharide-based hydrogel, characterized in that, The benzene boronic acid modified open ring hyaluronic acid and polyvinyl alcohol are used as raw materials, the benzene boronic acid grafted on the former and the abundant hydroxyl of the latter form reversible covalent bond-boric acid ester bond, and then form injectable and self-healing hydrogel; the hydrogel has uniform porous network structure, and the pore size is within 1 μm.

2. A process for the preparation of an injectable self-healing polysaccharide-based hydrogel according to claim 1, characterized in that, The reaction formula is as follows: 。 3. A process for the preparation of an injectable self-healing polysaccharide-based hydrogel according to claim 1, characterized in that, The method comprises the following steps: Step one: preparation of benzene boronic acid modified open ring hyaluronic acid (1) hyaluronic acid is oxidized by sodium periodate to obtain open ring hyaluronic acid; (2) the open ring hyaluronic acid is reacted with 3-aminobenzene boronic acid to obtain benzene boronic acid modified open ring hyaluronic acid; (3) a benzene boronic acid modified open ring hyaluronic acid aqueous solution with a concentration of 2-5 wt% is prepared and reserved; Step two: polyvinyl alcohol is dispersed in ultrapure water, stirred and heated to dissolve to obtain a 5 wt% polyvinyl alcohol aqueous solution; Step three: the benzene boronic acid modified open ring hyaluronic acid aqueous solution and the polyvinyl alcohol aqueous solution are mixed, the benzene boronic acid grafted on the former and the abundant hydroxyl of the polyvinyl alcohol form dynamic reversible covalent bond-boric acid ester bond, and then an injectable self-repairing polysaccharide-based hydrogel is obtained.

4. The process for the preparation of injectable self-healing polysaccharide-based hydrogels according to claim 3, characterized in that, In step three, the volume ratio of the benzene boronic acid modified open ring hyaluronic acid aqueous solution to the polyvinyl alcohol aqueous solution is 1:

1.

5. The method of claim 3, wherein the injectable self-healing polysaccharide-based hydrogel is prepared by, In step one, the molecular weight of hyaluronic acid is 400,000, and the oxidation degree of hyaluronic acid after oxidative ring opening is 35-45%; the weight average molecular weight of polyvinyl alcohol is 8.9x10 4 ~9.8x10 4 .

6. Use of the injectable self-repairing polysaccharide-based hydrogel obtained in claim 1 for preparing a material directly injected into an open wound.

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