CN106467613A - A kind of self-healing polyanion-chitin quarternary ammonium salt aquagel and its application - Google Patents

Abstract

The invention discloses a self-healing polyanion-chitosan quaternary ammonium hydrogel and its application in the field of medicine and biology. The preparation process of the hydrogel includes: preparing polyanion solution and chitosan quaternary ammonium salt solution respectively, after the two solutions are fully mixed, the two spontaneously aggregate under electrostatic interaction, a gelation reaction occurs, and the solid is collected The precipitate obtained polyanion-chitosan quaternary ammonium hydrogel. The raw materials for preparing the hydrogel are rich in sources and low in price. The hydrogel has good biocompatibility, has self-healing properties, and avoids factors such as organic solvents and acid-base environments during the formation process. It lays the foundation for the application of drugs and proteins, and has the advantages of injectability and high adhesion. When applied to tissue regeneration scaffolds, it has the advantages of mild preparation conditions, controllable shape and high water content, and is a good extracellular matrix material.

Description

A self-healing polyanion-chitosan quaternary ammonium hydrogel and its application

technical field

The invention relates to a polymer hydrogel, in particular to a self-healing polyanion-chitosan quaternary ammonium salt composite hydrogel and application thereof.

Background technique

Polymer hydrogel is a three-dimensional network structure polymer that can absorb and retain a large amount of water. The chemical cross-linking or physical cross-linking structure inside it can only swell but not dissolve. Polymer hydrogel has excellent viscoelasticity, high water content and water retention rate, and good biocompatibility. It can be applied in the fields of agriculture, food engineering and biomedicine. However, due to the high water content of polymer hydrogel, it will be destroyed due to low strength when subjected to tensile or compressive stress, which is difficult to meet the needs of practical applications.

Hydrogel materials with self-healing function are expected to solve the above problems and are suitable for long-term use of materials. Currently, there are mainly two approaches to prepare self-healing hydrogels, namely dynamic covalent crosslinking and noncovalent crosslinking. The chemical bonds involved in dynamic covalent crosslinking include acyl brown bonds, disulfide bonds, and Diels-Alder reactions, etc. These reactions cannot be carried out spontaneously, which limits their applications. In addition, the complicated chemical synthesis process is not only time-consuming and laborious, but the special chemical groups introduced in the whole process may induce toxic reactions. It is a challenge to prepare self-healing hydrogel materials with good biocompatibility using gentle methods. At present, there are very limited methods to prepare hydrogel materials with self-healing properties from natural polysaccharide materials, and the current reports still introduce aldehyde groups into the materials, which have potential cytotoxicity[Wei Zhao.Novel BiocompatiblePolysaccharide-Based Self-Healing Hydrogel , Advanced Functional Materials, 2015, 25(9): 1352-1359].

Chitosan quaternary ammonium salt is a positively charged chitosan derivative, which has the advantages of high charge density and good water solubility. Natural polyanionic materials and chitosan quaternary ammonium salts can form hydrogels through electrostatic interaction. The electrostatic interaction force is a non-covalent bond force, which can break and repair under suitable conditions. It is possible to prepare self-healing materials. Because all the polysaccharide materials of natural origin are used in the whole process, the large-scale synthesis technology of chitosan quaternary ammonium salt is also very perfect, so that the materials used are cheap and easy to obtain, and the reaction conditions of the gel preparation process are mild.

Due to the porosity of the hydrogel, the drug/protein can be released slowly, thereby realizing the slow and controlled release of the drug [Nathan P.Birch. -1843]. However, bulk gel materials are not easy to implant in the body, and the development of injectable materials has become a solution to this problem. At present, it is common to develop hydrogels with shear-thinning properties to achieve minimally invasive transplantation [Guvendiren, Murat .Shear-thinning hydrogels for biomedical applications, Soft Matter, 2012, 8(2):260-272]. In the field of tissue engineering, one of the important directions of simulating the cell microenvironment, the water content of hydrogel is close to that of human tissue, and the development of hydrogel with mild preparation conditions is a necessary condition to ensure cell activity. At present, calcium alginate is commonly used as the gel material, but once the hydrogel is prepared, its shape cannot be changed at will, which is not conducive to subsequent applications such as transplantation.

Contents of the invention

The purpose of the present invention is to provide a kind of hydrogel with self-healing property and its application in biomedical field, specifically a kind of self-healing polyanion-chitosan quaternary ammonium hydrogel, wherein, improve solution A higher ionic strength can increase the speed of the self-healing phenomenon and reduce the mechanical strength of the gel.

Technical scheme of the present invention is:

A self-healing polyanion-chitosan quaternary ammonium hydrogel, the preparation process includes: respectively preparing polyanion solution and chitosan quaternary ammonium salt solution, after the two solutions are fully mixed, the two are electrostatically interacted Spontaneous aggregation, gelation reaction occurs, and the solid precipitate is collected to obtain the polyanion-chitosan quaternary ammonium hydrogel.

The mass ratio of polyanion and chitosan quaternary ammonium salt in the hydrogel is 0.1:1-10:1, preferably the mass ratio is 0.5:1-2:1, and the water content of the hydrogel is between 50-99% Generally speaking, the higher the water content, the worse the mechanical strength. In practical applications, the best water content is 70-80%.

The hydrogel remains stable in the pH range of 4-14, and the hydrogel loses its self-healing function when the pH value is lower than 4. The hydrogel remains stable in the temperature range of 0-60°C, it will solidify below 0°C, and the solution may vaporize above 60°C.

The polyanion includes one or more of sodium alginate, sodium carboxymethyl cellulose, sodium hyaluronate, carboxymethyl chitosan and dextran sulfate.

After the hydrogel is randomly cut, the separated hydrogel has a self-healing function after being in contact again, forming a complete gel material, and the cracks generated by cutting disappear.

The molecular weight of the polyanion is 10kDa-2000kDa; the preferred molecular weight range is 100-1000kDa, the molecular weight of chitosan quaternary ammonium salt is 10kDa-2000kDa, the preferred molecular weight range is 10-100kDa, and the degree of substitution is 50%-100%, preferably within the range of degree of substitution 70%-100%.

The solvent of the polyanion solution prepared in the preparation process of the hydrogel is deionized water, or an aqueous solution with inorganic salt added, the solution concentration is 0.1-500g/L, the preferred concentration range is 1-100g/L, and the optimum concentration range is 1-10g/L; the solvent of the prepared quaternary ammonium salt solution is deionized water, or an aqueous solution with inorganic salts added, the solution concentration is 0.1-500g/L, the preferred concentration range is 1-100g/L, the optimal concentration range 1-10g/L, and the inorganic salt is one or more of sodium chloride, sodium nitrate, potassium chloride and potassium nitrate.

The solid precipitation collection method includes static precipitation, extrusion collection and centrifugation collection.

The centrifugation time for centrifugation collection is 1-120 minutes, and the centrifugation rate is 10-100000 rpm.

The invention provides the application of a self-healing polyanion-chitosan quaternary ammonium hydrogel in the field of biomedicine.

The hydrogel prepared by the invention is non-toxic, mild and self-healing, and can be used in biomedical fields such as drug sustained release, topical administration, and living cell culture, but not limited to.

In the aforementioned biomedical field, the hydrogel can be applied to, but not limited to, drug and protein localized delivery, and tissue regeneration scaffold. When the hydrogel is used for localized drug delivery, the drug is directly mixed with polyanion and chitosan quaternary ammonium salt, the final concentration of the drug is 1 μg/ml-1000 mg/ml, and the optimal concentration refers to the required dose of the drug for the human body.

When the hydrogel is applied to the tissue regeneration scaffold, the polyanion and the chitosan quaternary ammonium salt are mixed at 37°C to achieve the best charge neutralization, and the pH value is controlled between 6.4-7.8, and the pH= 7.4 best. Spread the prepared gel on a petri dish, and inoculate the surface with living cells. The reference density of inoculation is 1*10 ⁴ -2*10 ⁵ cells/cm ² , and the optimal inoculation density is 4*10 ⁴ cells/cm ² . The living cells are isolated hepatocytes derived from humans or animals, stem cells, stem cell differentiated cells with hepatocyte function, liver cell line cells, transdifferentiated cells with hepatocyte function, endothelial cells, hepatic Kupffer cells , one or more of hepatic stellate cells, fibroblasts, and bone marrow mesenchymal stem cells.

Beneficial effects of the present invention:

1. The composite gel of the present invention has a self-healing function, which can be performed spontaneously without external stimulation.

2. This compound prepared by polyanion and chitosan quaternary ammonium salt has ionic strength responsiveness, which is specifically manifested in increasing the ionic strength of the solution, which can increase the speed of self-healing phenomenon and reduce the mechanical strength of the gel. strength.

3. The polyanion material used in the present invention is derived from natural products. The chitosan quaternary ammonium salt is also modified and synthesized on the basis of the natural product chitin. The source of the material is abundant, the price is low, the biocompatibility is good, and it can heal itself In addition, organic solvents, acid-base environment and other factors are avoided during the formation of hydrogels, which lays the foundation for its application in the field of biomedicine. When used as a drug release carrier, the hydrogel material has the effect of sustained and controlled release. Because the gel has the effect of shear thinning, it can be used for injection, combined with the self-healing effect of the material, it can be administered at a fixed point through the injection method, and the material forms a block gel in the body. In addition, the hydrogel has strong adhesive properties, can adhere to the surface of various materials, and can be used as a dressing for drug administration. When applied to tissue engineering scaffolds, the shape of the hydrogel is easy to control, and the preparation conditions are mild. Combined with the high water content of the gel, it can simulate the cell microenvironment and promote cell proliferation and growth.

Description of drawings

Fig. 1 is the self-healing of sodium alginate-chitosan quaternary ammonium salt gel;

Fig. 2 is the 3ITT rheological experiment of carboxymethyl chitosan-chitosan quaternary ammonium salt gel;

Figure 3. Cell dead/live staining of the surface of the gel.

detailed description

For a better understanding of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto.

Example 1

(1) Prepare sodium alginate and chitosan quaternary ammonium salt solutions, the concentrations are respectively 20g/L and 40g/L, and the solvent is deionized water. Sodium alginate has a molecular weight of 430kDa and a G fragment content of 35%. The degree of substitution of chitosan quaternary ammonium salt is 85%, and the molecular weight is 78kDa.

(2) Add the chitosan quaternary ammonium salt solution into an equal volume of sodium alginate solution under shaking conditions, shake and mix well.

(3) Static precipitation for 2 hours to collect the product. The formed gels were stained with red basic fuchsin and blue methylene blue respectively, and the two cake-like gels with different colors were evenly divided into 4 parts, and rearranged according to the principle of staggered colors, so that the cut two pieces Gel close up. Thereafter, the gel state was observed at different times. The results are shown in Figure 1. The boundaries of the two gels close to each other began to blur after 1 hour of contact, and completely fused to form a complete gel after 7 hours.

Example 2

(1) Prepare carboxymethylcellulose sodium and chitosan quaternary ammonium salt solutions, the mass fraction is 20g/L, and the solvent is deionized water. The molecular weight of sodium carboxymethyl cellulose is 500kDa. The degree of substitution of chitosan quaternary ammonium salt is 85%, and the molecular weight is 78kDa.

(2) Add the chitosan quaternary ammonium salt solution into an equal volume of sodium alginate solution under shaking conditions, shake and mix well.

(3) Static sedimentation for 2 hours to collect the product and carry out rheological analysis, as shown in Figure 2, by increasing the strain, the structure of the gel is destroyed, and the structure of the gel is quickly restored after the stress is reduced, and the storage modulus returns to More than 65% of the original.

Example 3

(1) Prepare sodium alginate and chitosan quaternary ammonium salt solutions, the concentrations are respectively 20g/L and 40g/L, and the solvent is deionized water. Sodium alginate has a molecular weight of 430kDa and a G fragment content of 35%. The degree of substitution of chitosan quaternary ammonium salt is 85%, and the molecular weight is 78kDa.

(2) Add the chitosan quaternary ammonium salt solution into an equal volume of sodium alginate solution under shaking conditions, shake and mix well.

(3) Static precipitation for 2 hours to collect the product. The formed gel was mixed with water-soluble methylene blue and oil-soluble rhodamine B respectively, and the final drug concentration was 0.1 g/L.

(4) Collect 1 mL of the gel and place it in 10 mL of phosphate buffer (pH=7.4). At 25°C, the two drugs both exhibit fast and then slow release behavior. The drug release reaches 50% in 24 hours, and the drug is released after 7 days. The content can reach more than 90%, indicating that the hydrogel has a good sustained and controlled release effect.

Example 4

(1) Prepare sodium alginate and chitosan quaternary ammonium salt solutions, the concentrations are respectively 20g/L and 40g/L, and the solvent is deionized water. Sodium alginate has a molecular weight of 430kDa and a G fragment content of 35%. The degree of substitution of chitosan quaternary ammonium salt is 85%, and the molecular weight is 78kDa.

(2) Add the chitosan quaternary ammonium salt solution into an equal volume of sodium alginate solution under shaking conditions, shake and mix well.

(3) The product was collected by static precipitation for 2 hours, and 1 mL of the product was freeze-dried.

(4) The dried gel was placed in 10 mL of phosphate buffer (pH=7.4), and the gel absorbed water to reach equilibrium at 25°C for 6 hours, and the water content was about 75%.

(5) As shown in Figure 3, the sterilized gel was tiled in a small dish, and HepG2 cells were inoculated on the surface. After 24 hours of cultivation, the cells were attached to the surface of the gel, and the cell viability remained above 95% after dying and dying staining. .

(6) The cell function was characterized, and after one week, the cell activity remained at 80%, the albumin secretion was 0.46±0.05 μg/10 ⁶ cells, and the urea synthesis was 231±29 μg/10 ⁶ cells.

Comparative example 1

(1) Sodium alginate and chitosan solutions were prepared with concentrations of 20g/L and 40g/L respectively. Sodium alginate has a molecular weight of 430kDa and a G fragment content of 35%. The degree of deacetylation of chitosan is 85%, and the molecular weight is 78kDa. The solvent for preparing sodium alginate is water, and the solvent for preparing chitosan is 1% acetic acid.

(2) Add the chitosan solution into an equal volume of sodium alginate solution under shaking conditions, shake and mix well.

(3) Static precipitation for 2 hours to collect the product hydrogel.

(4) The sterilized gel was tiled in a small dish, and the surface was inoculated with mouse-derived L929 cells. After 24 hours of culture, the cells were attached to the surface of the gel, and the cell viability decreased to less than 20% after life and death staining. Due to the low solubility of chitosan in aqueous solution, the addition of acetic acid can increase the solubility of chitosan, while the overacid environment will lead to cell inactivation.

Claims (9)

1. a self-healing polyanion-chitosan quaternary ammonium hydrogel, is characterized in that: the preparation process of described hydrogel comprises: prepare polyanion solution and chitosan quaternary ammonium salt solution respectively, two kinds After the solutions are fully mixed, the two are spontaneously aggregated under the electrostatic interaction, and a gelation reaction occurs, and the solid precipitate is collected to obtain the polyanion-chitosan quaternary ammonium hydrogel. 2. self-healing polyanion-chitosan quaternary ammonium hydrogel according to claim 1, is characterized in that: described polyanion comprises sodium alginate, sodium carboxymethyl cellulose, sodium hyaluronate, One or more of carboxymethyl chitosan and dextran sulfate. 3. self-healing polyanion-chitosan quaternary ammonium hydrogel according to claim 1, is characterized in that: after this hydrogel is processed through arbitrary cutting, the separated hydrogel has self-healing after contacting again Function, form a complete gel material, and the cracks caused by cutting disappear. 4. self-healing polyanion-chitosan quaternary ammonium hydrogel according to claim 1, is characterized in that: the mass ratio of polyanion and chitosan quaternary ammonium salt in hydrogel is 0.1:1-10 : 1, the water content of the hydrogel is 50-99%; The molecular weight of the polyanion is 10kDa-2000kDa; the molecular weight of the chitosan quaternary ammonium salt is 10kDa-2000kDa, and the substitution degree is 50%-100%. 5. self-healing polyanion-chitosan quaternary ammonium hydrogel according to claim 1, is characterized in that: the solvent of the polyanion solution of preparation in the described hydrogel preparation process is deionized water, or adds The aqueous solution of inorganic salts, the solution concentration is 0.1-500g/L; the solvent of the prepared quaternary ammonium salt solution is deionized water, or the aqueous solution with inorganic salts added, the solution concentration is 0.1-500g/L, and the inorganic salt is chlorine One or more of sodium chloride, sodium nitrate, potassium chloride, and potassium nitrate. 6. The self-healing polyanion-chitosan quaternary ammonium hydrogel according to claim 1, characterized in that: the solid precipitation collection method comprises static precipitation, extrusion collection and centrifugal collection. 7. The self-healing polyanion-chitosan quaternary ammonium hydrogel according to claim 7, characterized in that: the centrifugation time collected by centrifugation is 1-120 minutes, and the centrifugation speed is 10-100000rpm. 8. The application of the self-healing polyanion-chitosan quaternary ammonium hydrogel according to any one of claims 1-7 in the field of biomedicine. 9. The application according to claim 8, characterized in that: when the hydrogel is applied to drug and protein location delivery, the drug/protein is directly mixed with polyanion and chitosan quaternary ammonium salt, and the final concentration of the drug/protein is 1-1000mg/ml, the optimal concentration refers to the dose required by the human body of the drug/protein; When the hydrogel is applied to the tissue regeneration scaffold, the polyanion and chitosan quaternary ammonium salt are mixed at 37°C to achieve the best charge neutralization, and the pH value is controlled between 6.4-7.8. A good gel is spread on a petri dish, and live cells are seeded on the surface, and the live cells are isolated liver cells derived from humans or animals, stem cells, cells with liver cell function differentiated from stem cells, cells of liver cell lines, transdifferentiated cells Cells with liver cell function, one or more of endothelial cells, hepatic Kupffer cells, hepatic stellate cells, fibroblasts, and bone marrow mesenchymal stem cells.