An injectable hydrogel, a method for preparing the same, and use thereof

Abstract

The application discloses an injectable hydrogel, a preparation method thereof and application thereof. The hydrogel is a chemical-physical double network crosslinked hydrogel prepared by using a blending solution of magnesium acrylate, carboxymethyl chitosan and phytic acid as main components. Covalent crosslinking is derived from a magnesium acrylate network constructed by a free radical polymerization reaction. Non-covalent crosslinking is derived from hydrogen bonds and electrostatic interactions among components in the interpenetrating network, and a carboxymethyl chitosan chain entanglement network gradually induced in the application process in a physiological environment. In the preparation process, the pH value of the reaction system is controlled by controlling the concentration of phytic acid, magnesium ions are chelated, and then the structural strength of the hydrogel is controlled, so that the injectable effect is achieved. Meanwhile, the phytic acid can chelate proteins, and the adhesion to tissues is enhanced. The preparation process is simple, easy to control and suitable for large-scale industrial production, and all raw materials are easy to obtain and low in price. The hydrogel has high adhesion efficiency and wet adhesion strength to tissues.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of an injectable hydrogel suitable for continuous adhesion in a physiological environment and its application, and belongs to the technical field of medical adhesive preparations. BACKGROUND

[0002] As a kind of adhesive material with soft matter characteristics, hydrogel adhesive plays a key role in a wide range of wet environment applications, including underwater industrial repair and mechanical movement, tissue adhesion and sealing in physiological environment, etc. Compared with the adhesive showing viscoelastic liquid behavior, hydrogel adhesive can provide better modulus matching and transmission performance with the substrate. Most traditional tough hydrogels show poor adhesion due to the lack of high molecular interfacial anchoring between the substrate surface or the destruction of interfacial interaction by hydration layer. Especially when the hydrogel adhesive is directly applied in a wet environment, or after long-term application, such disadvantages are particularly obvious. In order to solve the above problems, the following two effects are needed to work together. On the one hand, the low hydration swelling of the hydrogel matrix should be ensured, so that it is difficult to be destroyed by hydration, thereby showing long-term stable mechanical properties. On the other hand, when directly applied in a wet environment, the interfacial hydration layer must be removed through the polymer network of the hydrogel adhesive or the surface interfacial micropatterning strategy, while covalent bonding is established at the adhesive interface.

[0003] Due to the rich functional carboxyl groups that can serve as multiple binding sites, acrylic acid magnesium with extremely strong adhesion has been widely used in industrial adhesives. It is extremely attractive to choose acrylic acid magnesium as the main component of the hydrogel. However, the underwater long-term adhesion effect of polyacrylic acid magnesium (PAMg) single network hydrogel is not ideal. Therefore, a physical network with non-covalent cross-linking is sought to construct a physical-chemical double network gel with the theory of "sacrificial bond" in combination with polyacrylic acid magnesium, as a kind of double enhanced polymer network material, which provides the possibility to maintain and even enhance the mechanical properties of the adhesive material applied in a wet environment for a long time. Chitosan (CS) is the only alkaline polysaccharide discovered so far with biocompatibility and biodegradability. After simple treatment in a monovalent anion salt solution, a chain entanglement network can be generated. The positively charged N-glucosamine units in the CS chain are attracted to the excess Cl-, thereby reducing the electrostatic repulsion between the N-glucosamine units, and further leading to the entanglement of the CS chain and the volume shrinkage of the system. SUMMARY

[0004] Invention purposes: In view of the defects that the adhesion ability of the existing medical adhesive is greatly reduced in long-term wet environment, the purpose of the present application is to provide an injectable hydrogel, a preparation method thereof and an application thereof, a chemical-physical cross-linked double network hydrogel is prepared by using a blended solution of magnesium acrylate, carboxymethyl chitosan (CMCS) and phytic acid (PA) as main components. The covalent cross-linking is derived from the polyacrylic acid network constructed by free radical polymerization, the non-covalent cross-linking is derived from the hydrogen bond and electrostatic interaction between the components in the interpenetrating network, and the carboxymethyl chitosan chain entanglement network gradually induced by salting-out effect, shielded electrostatic repulsion and enhanced hydrophobic interchain interaction in the application process in physiological environment. In addition, the strong chelating ability of phytic acid contained in the gel can form more stable electrovalent bond with protein. The synergistic effect of the dissipation matrix not easily affected by swelling and the multiple bonded adhesion interface can realize the long-term stable adhesion ability of the hydrogel adhesive in the physiological environment.

[0005] Technical scheme: In order to achieve the above purpose, the present application adopts the following technical scheme:

[0006] An injectable hydrogel is a chemical-physical cross-linked double network hydrogel constructed by a blended solution of magnesium acrylate, carboxymethyl chitosan and phytic acid as main components, wherein the covalent cross-linking is derived from the polyacrylic acid network constructed by free radical polymerization, the non-covalent cross-linking is derived from the hydrogen bond and electrostatic interaction between the components in the interpenetrating network, and the carboxymethyl chitosan chain entanglement network gradually induced by salting-out effect, shielded electrostatic repulsion and enhanced hydrophobic interchain interaction in the application process in physiological environment. Phytic acid forms more stable electrovalent bond with protein by virtue of strong chelating ability. The synergistic effect of the dissipation matrix not easily affected by swelling and the multiple bonded adhesion interface can realize the long-term stable adhesion ability of the hydrogel adhesive in the physiological environment.

[0007] The present application provides a preparation method of an injectable hydrogel, comprising the following steps:

[0008] Step one: disperse magnesium hydroxide powder in deionized water, then add acrylic acid solution to prepare 10-50 wt% magnesium acrylate solution.

[0009] Step two: dissolve carboxymethyl chitosan powder and prepared crosslinking agent solution in the aqueous solution of step one, fully stir and uniformly configure into carboxymethyl chitosan-crosslinking agent-magnesium acrylate aqueous solution.

[0010] The degree of substitution of carboxymethyl chitosan is greater than or equal to 80%, and the concentration is 1.0-5.0 wt%; the crosslinking agent is N, N'-methylene bisacrylamide (MBAm), the solution concentration is 20 mg / mL, and the volume ratio of crosslinking agent solution to acrylic acid monomer is 0-1:8 and greater than 0.

[0011] Step 3: Add 0~50 wt% phytic acid solution (greater than 0) dropwise to the solution prepared in Step 2 above, stir for 10~40 min to prepare the precursor solution of hydrogel. The mechanical strength of the hydrogel can be controlled by adjusting the amount of phytic acid added.

[0012] Step 4: Heat the precursor solution of the gel described in Step 3 in a water bath, add the freshly prepared initiator solution (ammonium persulfate as the initiator) dropwise, raise the temperature to 50~70℃, and then continue stirring for 10~40 minutes to disperse the components in the solution evenly, and wait for the polymerization to be completed.

[0013] The resulting injectable hydrogel, used as a rapid hemostatic material, is applied by directly adhering the hydrogel to the surface of the bleeding tissue, thus obtaining a hydrogel-tissue adhesion interface that can function continuously in a physiological environment. In one embodiment, the bleeding is liver bleeding, and the hydrogel is directly adhered to the surface of the bleeding tissue.

[0014] The principle of this invention is as follows: To overcome the barrier of hydration disruption, actively and effectively utilize the saline solution in the physiological environment to improve its solubility and increase the injectability of the gel, the inventors replaced the hydroxyl groups on chitosan with carboxyl groups to obtain carboxymethyl chitosan. Carboxymethyl chitosan has the advantages of better procoagulant properties and reduced scar formation. Constructing a magnesium polyacrylate-carboxymethyl chitosan hybrid hydrogel provides significant advantages for the wet adhesion of the hydrogel. To further improve the adhesion strength of the dual-network hydrogel matrix, phytic acid (PA) was introduced. Phytic acid consists of six identical phosphate groups on the inositol ring. PA contains a large amount of electronegative oxygen, has strong electronegativity, and readily reacts with -NH2 on tissues to form stable valence bonds, preventing degradation; phytic acid has strong chelating properties and readily reacts with Mg... 2+ Ca 2+ divalent cations and Fe 3+ Phytic acid chelates with Mg to form more stable coordination bonds. Therefore, by adjusting the phytic acid content, the pH of the solution can be controlled, thus controlling its interaction with Mg in the solution. 2+ The coordination formation of magnesium phytate controls its structural strength. Furthermore, it readily chelates Fe. 3+ The presence of proteins further promotes hemostasis and enhances adhesion properties. In summary, the synergistic effect of a dissipative matrix unaffected by swelling and a multi-bonded adhesion interface enables the hydrogel to achieve injectability and long-term stable adhesion in physiological environments. Beneficial effects

[0015] 1. The hydrogel matrix prepared by this invention can achieve rapid, high-strength, and repeatable adhesion to different surfaces (including engineered solids and tissues) in dry and physiological environments.

[0016] 2. The introduction of phytic acid at the adhesive interface can significantly improve the adhesive ability of the hydrogel adhesive in a wet environment (underwater). The combination of the dissipation matrix which is not easily affected by swelling and the multiple bonded adhesive interface can achieve long-term stable adhesive ability of the hydrogel adhesive in a physiological environment.

[0017] 3. The synergistic effect of phytic acid and carboxymethyl chitosan in the hydrogel matrix prepared by the application can improve the structural strength and mechanical properties of the product, so that it can adapt to more application environments.

[0018] 4. The hydrogel matrix prepared by the application achieves high burst pressure on various tissue surfaces in air and simulated physiological environment. In addition, it has a significant and rapid hemostatic effect on the rat in vitro liver bleeding model.

[0019] 5. The hydrogel prepared by the application has raw materials that are easy to obtain. It can be prepared by in-situ polymerization of the precursor solution and the initiator. The preparation process is simple and rapid, easy to operate, control and large-scale industrial production, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The adhesive tensile strength of the hydrogel matrix prepared in the embodiment of the application in a dry environment; the adhesive tensile strength can be up to 132.8kPa; wherein (a) the effect of different crosslinker concentrations on the adhesive tensile properties of the product in Example 1; (b) the effect of carboxymethyl chitosan on the adhesive tensile properties of the product in Example 2; (c) the effect of phytic acid concentration on the mechanical properties of the product in Example 3;

[0021] Figure 2 Rheological test of the hydrogel prepared in samples 1, 2, 3, 4, 6, 7, 8 in Examples 1, 2, and 3 of the application;

[0022] Figure 3 The burst pressure test of the hydrogel matrix prepared in Example 1 of the application (sample 2) after adhesion on different tissue surfaces: skin;

[0023] Figure 4 The in vitro hemostatic effect of the hydrogel matrix prepared according to Example 1 of the application (sample 3, sample 5, sample 7) on the SD rat liver bleeding model. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the application more clear and obvious, the application will be further described in detail below in combination with specific examples and with reference to the drawings.

[0025] Example 1: Effect of crosslinker concentration on the mechanical properties of the product:

[0026] (1) 0.2 g N, N'-methylenebisacrylamide was dissolved in 10 mL deionized water to prepare a crosslinker solution; 2 g ammonium persulfate was dissolved in 10 mL deionized water to prepare an initiator solution.

[0027] (2) 0.9 g magnesium hydroxide was dispersed in 7.5 mL deionized water; 2 mL acrylic acid was gradually added to obtain a magnesium acrylate solution.

[0028] (3) 0, 100 μL of the crosslinker solution prepared in step (1) was added to the magnesium acrylate solution prepared in step (2) together with 0.2 g carboxymethyl chitosan, and the mixture was stirred uniformly.

[0029] (4) 1.5 mL of a phytic acid solution with a concentration of 50 wt% was added dropwise to the solution prepared in step (3), and the mixture was stirred in a 50°C water bath for 30 min; then 500 μL of the initiator solution prepared in step (1) was added dropwise, and the water bath was heated to 65°C and the stirring was continued until the crosslinking was completed (to obtain sample 1, 2).

[0030] The adhesion ability of the hydrogel matrix was evaluated according to ASTM F2258 tensile test, from Figure 1 a, it can be seen that the crosslinker concentration significantly affects the crosslinking density of the hydrogel matrix, and sample 2 with 100 μL of crosslinker solution has a higher crosslinking density and thus a higher adhesion tensile strength. From Figure 2 a, it can be seen that sample 2 with a higher crosslinker concentration has a higher storage modulus G'. Moreover, affected by the crosslinker concentration, the storage modulus G' of sample 1 changes more rapidly with the angular frequency ω at low frequency (0-1 rad / s), while at high angular frequency, the material exhibits a stronger elastic behavior. The increase of the crosslinker concentration increases the rigidity of the whole system, so the storage modulus G' of sample 2 increases at medium-high frequency, and the change rate tends to be flat at this time, because the elasticity of the crosslinking network dominates the response of the system.

[0031] Example 2: Effect of carboxymethyl chitosan on the mechanical properties of the product: (1) 0.2 g N, N'-methylenebisacrylamide was dissolved in 10 mL deionized water to prepare a crosslinker solution; 2 g ammonium persulfate was dissolved in 10 mL deionized water to prepare an initiator solution (2) 0.9 g magnesium hydroxide was dispersed in 7.5 mL deionized water; 2 mL of acrylic acid was added dropwise to obtain a magnesium acrylate solution.

[0032] (3) 100 μL of the crosslinker solution prepared in step (1) was added to the magnesium acrylate solution prepared in step (2) together with 0 g and 0.2 g carboxymethyl chitosan, respectively, and the mixture was stirred uniformly.

[0033] (4) Add 1.5 mL of 50 wt.% phytic acid solution dropwise to the solution of step (2) and stir in a 50 °C water bath for 30 min, then add 500 μL of initiator solution of step (1) dropwise, raise the temperature of the water bath to 65 °C and continue to heat and stir until it solidifies and crosslinks (obtain samples 3 and 4).

[0034] The adhesion ability of the hydrogel matrix was evaluated according to ASTM F2258 tensile test from Figure 1 It can be seen from b that sample 4 with the addition of carboxymethyl chitosan has better adhesion tensile strength. From Figure 2 It can be seen from b that sample 3 has a higher storage modulus G' at low angular frequency ω. For the hydrogel without carboxymethyl chitosan, it may exhibit a more stable physical crosslinking network in this region, or the interaction between its molecular chains is relatively tight, so the elastic response is stronger and the storage modulus is higher. In the high frequency region, sample 4 exhibits a higher storage modulus because it contains carboxymethyl chitosan, has more crosslinking points and higher crosslinking density.

[0035] Example 3: Effect of phytic acid concentration on the mechanical properties of the product: (1) Dissolve 0.2 g of N, N'-methylenebisacrylamide in 10 mL of deionized water to prepare a crosslinking agent solution; dissolve 2 g of ammonium persulfate in 10 mL of deionized water to prepare an initiator solution.

[0036] (2) Disperse 0.9 g of magnesium hydroxide in 7.5 mL of deionized water; gradually add 2 mL of acrylic acid to obtain a magnesium acrylate solution.

[0037] (3) Add 100 μL of the crosslinking agent solution prepared in step (1) to the magnesium acrylate solution prepared in step (2), and stir well.

[0038] (4) Add 0, 1.0 mL, 1.5, and 2 mL of 50 wt.% phytic acid solution dropwise to the solution of step (2) respectively, and stir in a 50 °C water bath for 30 min, then add 500 μL of initiator solution of step (1) dropwise, raise the temperature of the water bath to 65 °C and continue to heat and stir until it solidifies and crosslinks (obtain samples 5, 6, 7, and 8).

[0039] The adhesion ability of the hydrogel matrix was evaluated according to ASTM F2258 tensile test from Figure 1 It can be seen from c that as the concentration of phytic acid increases, the adhesion tensile strength of the sample decreases. From Figure 2 c, it can be seen that the concentration of phytic acid significantly affects the crosslinking density, and samples with lower phytic acid concentration have higher storage modulus, showing higher structural strength.Figure 2 It can be seen from c that the storage modulus of the sample gradually decreases with the increase of the phytic acid concentration.

[0040] Among them, samples 2, 4, and 7 are samples of the same component. Example 4

[0041] By building an in-vitro burst pressure experimental device, the burst pressure of the hydrogel prepared in Example 1 (sample 2) after adhering to the surface of different tissues was measured. The hydrogel prepared in Example 1 (diameter 8 mm, thickness 2-3 mm) was directly sealed at the gap with a diameter of 2 mm on the tissue surface, and the device was immediately pressurized. The pressure peak value displayed when the hydrogel adhesion failed or cohesion failed was recorded, which was the burst pressure value. In a dry environment, the burst pressure of sample 2 on the skin was 0.8 psi. The high burst pressure exhibited by the hydrogel matrix prepared by the present application expands its application potential in the field of medical sealants or occluders. Example 5

[0042] By building a liver injury bleeding model of SD rats, the in-vitro hemostatic effect of the hydrogel prepared by the present application was evaluated. Here, the D rats (260-270 g) were fixed on the operation board after anesthesia, and the operation board was kept at an angle of 30° with the horizontal plane. The liver was exposed through the abdominal incision, and the excess serous fluid around the liver was carefully removed with sterile gauze. The pre-weighed qualitative filter paper was placed at the bottom of the liver, and a large enough area of sealing film was placed at the bottom of the filter paper to prevent the influence of tissue serous fluid on the weight of the filter paper. Subsequently, a pair of scissors was used to induce liver bleeding, and sample 3, sample 5, and sample 7 hydrogels were immediately injected or adhered to the bleeding site in situ, and the timing was started. The group without any treatment was used as a blank group. Pictures were taken at 30s, 60s, 90s, and 120s, respectively. Finally, the recording was stopped at 120s, and the cumulative blood loss was recorded by weighing the filter paper. The figure shows the bleeding situation of a 1cm×1cm cross-shaped incision on the surface of the rat liver at 30s, 60s, 90s, and 120s, respectively. The bleeding amount at 120s is 0.0788g for the blank group, 0.0551g for sample 3, 0.0153g for sample 5, and 0.0249g for sample 7. The bleeding amount from large to small is blank group>sample 3>sample 7>sample 5, among which it can be seen that the hemostatic effect of sample 5 is the best, followed by sample 7, and sample 3 is the worst. Although sample 5 without phytic acid has better hemostatic effect, the adhesion effect in a wet environment is poor when phytic acid is not added. Sample 7 with phytic acid has better wet adhesion effect and better hemostatic ability. Therefore, after balancing hemostasis and adhesion, the comprehensive effect of sample 7 with phytic acid is the best.

[0043] The rapid hemostatic ability of the hydrogel obtained from sample 7 can be attributed to the wound closure in wet environment and the ability of carboxymethyl chitosan to promote hemostasis and the synergistic effect of the poly(magnesium acrylate) based hydrogel to seal the bleeding site. The carboxyl groups on the surface of the hydrogel and the phosphate in phytic acid form non-covalent molecular bonding with the tissue surface, including hydrogen bonds, mutual electrostatic interaction, coordination bonds, so that the hydrogel can quickly adhere to the bleeding site, seal the wound and promote hemostasis. At the same time, carboxymethyl chitosan can promote the adhesion and aggregation of platelets, so that red blood cells can quickly aggregate, thereby promoting rapid hemostasis.

[0044] It can be seen that whether carboxymethyl chitosan is contained in the hydrogel matrix greatly affects the hemostatic speed of the sample. However, in sample 5 containing only carboxymethyl chitosan, the adhesion effect of the tissue and the sample is not obvious in a wet environment. It is speculated that this may be because the bond energy of hydrogen bonds and electrostatic interactions is very low, and the polarity of water in blood is very strong, which can break the bond, resulting in the inability to firmly adhere in a wet environment. The adhesion effect is greatly improved in sample 7 containing phytic acid, which can firmly adhere to the bleeding wound. Compared with the control group containing carboxymethyl chitosan, only sample 3 containing magnesium acrylate and phytic acid has the highest adhesion effect, which may be because the strong chelation of phytic acid and some amino groups on carboxymethyl chitosan reduces the phosphate groups, thereby reducing the adhesion strength. However, in sample 7 containing carboxymethyl chitosan and phytic acid, the hemostatic effect of carboxymethyl chitosan can promote blood coagulation in the wound, so the total bleeding amount is less. At the same time, the addition of phytic acid in the sample can enhance its flowability, making the application range of the hydrogel wider.

Claims

1. An injectable hydrogel, characterized in that, This is a chemically-physically cross-linked dual-network hydrogel constructed from a blend solution of magnesium acrylate, carboxymethyl chitosan, and phytic acid as the main components. The covalent cross-links originate from the polyacrylic acid network constructed by free radical polymerization, while the non-covalent cross-links originate from hydrogen bonds and electrostatic interactions between the components in the interpenetrating network, as well as the carboxymethyl chitosan chain entanglement network gradually induced during application in physiological environments due to salting-out effect, shielding electrostatic repulsion, and enhanced hydrophobic chain interactions. Phytic acid forms more stable valence bonds with proteins through its strong chelating ability. Together with a dissipative matrix that is not easily affected by swelling and a multi-bonded adhesion interface, the hydrogel achieves long-term stable adhesion in physiological environments. The method for preparing the injectable hydrogel includes the following steps: Step 1: Disperse magnesium hydroxide powder in deionized water, add acrylic acid solution, and react to prepare an aqueous solution of magnesium acrylate; Step 2: Add carboxymethyl chitosan powder and freshly prepared crosslinking agent solution to the aqueous solution of magnesium acrylate obtained in Step 1, stir thoroughly to prepare a carboxymethyl chitosan-crosslinking agent-magnesium acrylate aqueous solution; Step 3: Add a phytic acid solution with a concentration of 0~50 wt% (greater than 0) dropwise to the solution prepared in Step 2, and stir for 10~40 min to obtain the precursor solution of the hydrogel; the mechanical strength of the hydrogel can be controlled by adjusting the amount of phytic acid added. Step 4: Heat the precursor solution of the hydrogel described in Step 3 in a water bath, add the freshly prepared initiator solution dropwise, raise the temperature, and continue stirring until polymerization is complete to obtain the hydrogel. The adhesion strength of the hydrogel can be controlled by adjusting the content of magnesium acrylate, carboxymethyl chitosan, and crosslinking agent.

2. The injectable hydrogel according to claim 1, characterized in that, In step two, the concentration of the magnesium acrylate solution is 10-50 wt%; the degree of substitution of carboxymethyl chitosan is ≥80%, and the concentration is 1.0-5.0 wt%; the crosslinking agent is N,N′-methylenebisacrylamide, the solution concentration is 20 mg / mL, and the volume ratio of the crosslinking agent solution to the acrylic monomer solution is 0-1:8 and greater than 0.

3. The injectable hydrogel according to claim 1, characterized in that, In step four, the hydrogel precursor solution is heated to 50-70°C for 10-40 minutes; the initiator is ammonium persulfate.

4. The use of the injectable hydrogel of claim 1 in the preparation of a rapid hemostatic material.

5. The application of the injectable hydrogel of claim 1 in the preparation of tissue adhesion materials under physiological conditions.

6. The application according to claim 5, characterized in that, It involves directly adhering the hydrogel to the surface of the bleeding tissue.

7. The application according to claim 6, characterized in that, This involves directly adhering hydrogels to the tissue surface to obtain a hydrogel-tissue adhesion interface that can function continuously in the physiological environment.

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