Preparation method of injectable calcium alginate hydrogel based on novel polysaccharide hybrid calcium source

Abstract

The invention discloses a preparation method of injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source. An injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that under the action of weak acid (such as glucolactone, tartaric acid and the like) or polyphenol (such as tannic acid, gallic acid, tea polyphenol and the like), a polysaccharide (such as sodium alginate, carboxymethyl chitosan and hyaluronic acid) hybrid calcium phosphate solution with good organic-inorganic interface compatibility can be prepared into the injectable calcium alginate hydrogel based on the novel polysaccharide hybrid calcium source. The injectable calcium alginate hydrogel with good performance is prepared by releasing calcium ions and cross-linking sodium alginate, and the method is simple, convenient and effective, and safe in process.

Description

Technical Field

[0001] The present invention belongs to the field of injectable hydrogel biomaterials; specifically, it relates to a biocompatible injectable calcium alginate hydrogel and a preparation method thereof. Background Art

[0002] Sodium alginate, as a natural anionic polysaccharide, is one of the popular materials in biomedical engineering due to its good biocompatibility, biodegradability, non-toxicity, and non-immunogenicity. Cations such as Ca 2+ , Ba 2+ , Cu 2+ , Sr 2+ , Fe 3+ etc. can directly form ionic bonds with the carboxyl groups on alginate, thus constructing calcium alginate hydrogels. Currently, using calcium carbonate, hydroxyapatite, bioactive glass, etc. as calcium sources, calcium ions are slowly released under the action of gluconolactone to control the crosslinking rate of sodium alginate and calcium alginate, thereby designing injectable calcium alginate hydrogels. This is a major direction for optimizing the performance of hydrogels and expanding their application prospects, showing great promise in biomedical fields such as drug or cell delivery, cosmetic beauty, etc. Currently, the design of injectable calcium alginate hydrogels mostly focuses on the chemical modification of sodium alginate, viscosity, adjustment of the G / M ratio, and introduction of other metal ions into inorganic calcium salts. These design strategies do not pay attention to the serious interfacial compatibility problem between inorganic phases such as calcium carbonate, hydroxyapatite, and bioactive glass as calcium sources and the organic component alginate. These calcium sources are difficult to be stable in the alginate solution for a long time and are prone to sedimentation problems, which will affect the uniformity of the calcium alginate hydrogel structure and thus its mechanical properties, restricting its application in the biomedical field. In addition, most studies only use gluconolactone to mediate the formation of calcium alginate hydrogels, but ignore the development of functional injectable calcium alginate hydrogels by introducing weak acids with biological activity and functionality.

[0003] The patent application with the publication number CN201710233147.9 discloses an injectable hydrogel for myocardial repair and its preparation and application. This patent injects the sodium alginate solution in a liquid form into the myocardial injury site, and then the bioactive glass therein gradually releases calcium ions under the action of gluconolactone to crosslink sodium alginate to form a calcium alginate hydrogel, preventing ventricular dilation. At the same time, the solidified hydrogel has a porous structure, facilitating cell ingrowth; various ions generated by the decomposition of bioactive glass can promote angiogenesis and myocardial cell repair by promoting cells to secrete key growth factors.

[0004] The patent application with the publication number CN201910379468.9 discloses a sustained-release material system for tissue repair and its preparation method. This active substance sustained-release material system consists of three parts: an outer layer, a middle layer, and an inner layer. The outer layer is a sodium alginate / bioactive glass / glucono-δ-lactone composite injectable hydrogel layer, the middle layer is a sodium alginate hydrogel microsphere layer encapsulating repair factors, and the inner layer is a poly(lactic-co-glycolic acid) microsphere layer encapsulating drugs. The present invention also discloses the preparation method of this active substance sustained-release material system. The active substance sustained-release material system of the present invention can orderly release the repair factors and drugs encapsulated inside the material according to different stages of tissue regeneration during wound repair, providing the corresponding active substances required for tissue regeneration at different stages, and can better exert the combined effect of repair factors.

[0005] The above-mentioned preparation methods of injectable calcium alginate hydrogels mainly involve adding inorganic calcium salts and bioactive glass to the sodium alginate organic system, and releasing calcium ions in bioactive glass with the participation of weak acid glucono-δ-lactone to crosslink sodium alginate to obtain injectable calcium alginate hydrogels. However, all inorganic calcium salts, including bioactive glass, whether at the nanoscale or microscale, have problems such as interfacial barriers and dispersibility with the organic substance sodium alginate, which will affect the mechanical properties of injectable calcium alginate hydrogels and thus limit their application in the field of biomedical engineering. Even using ultrasonic mixing cannot ensure the stable dispersion of inorganic calcium salts in the alginate solution. Means such as surface graft modification of inorganic calcium salts not only bring inconvenience in operation but also pose a risk of introducing harmful substances. The patent application with the publication number CN104958766A discloses a sodium alginate-hydroxyapatite hybrid nanoparticle and its preparation method. This method synthesizes sodium alginate-hydroxyapatite hybrid nanoparticles by adding calcium salts and phosphates to an aqueous sodium alginate solution. This organic-inorganic hybrid system can well solve the problems of interfacial compatibility and dispersibility between inorganic and organic substances and will not introduce other components. Secondly, the commonly prepared injectable calcium alginate hydrogels by releasing calcium ions in inorganic calcium salts with weak acid glucono-δ-lactone to crosslink sodium alginate are often only injected into the damaged site as tissue scaffolds or used as carriers to load functional components for treatment, which greatly limits the application of injectable calcium alginate hydrogels. Polyphenols show weak acidity due to the presence of phenolic hydroxyl groups and have many effects such as anti-inflammatory, antioxidant, and photoprotection, which can endow injectable calcium alginate hydrogels with functionality. Fruit acids are widely used in the field of medical beauty, and the injectable calcium alginate hydrogels prepared by introducing fruit acids also have broad application prospects. Summary of the Invention

[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source and a preparation method thereof. The hybrid calcium phosphate solution is stable and uniformly mixed in the sodium alginate organic system. After adding a weak acid or polyphenol solution, calcium ions are slowly and controllably released to crosslink sodium alginate, obtaining an injectable calcium alginate hydrogel with a uniform structure, controllable gel time and mechanical properties. At the same time, the introduction of the polyphenol system endows the injectable calcium alginate hydrogel with anti-inflammatory, antioxidant and other properties.

[0007] The present invention has the following beneficial effects: 1. The hybrid calcium phosphate solution is directly synthesized in an organic system, and the concentrated hybrid calcium phosphate solution has good dispersion stability in the organic system, overcoming the problems of poor dispersion and interface compatibility of the inorganic phase in the organic system existing in the current injectable calcium alginate hydrogels using hydroxyapatite (calcium phosphate), calcium carbonate or bioactive glass - weak acid as calcium sources; 2. The injectability of the calcium alginate hydrogel of the present invention is realized by the slow release of calcium ions from the sodium alginate hybrid calcium phosphate. Compared with other methods of improving interface adaptability by surface grafting and other means, it avoids the risk of easy contamination of external primers and the potential harmful substances brought; 3. The weak acids such as glucono - δ - lactone or fruit acid used in the calcium alginate hydrogel of the present invention have natural advantages in the application of the medical beauty industry such as chemical peeling; 4. The calcium alginate hydrogel of the present invention has good pore structure, mechanical properties and injectability, and has good application prospects in the biomedical engineering fields such as cell delivery and tissue repair; 5. The present invention first introduces a polyphenol system to release calcium ions in the hybrid calcium phosphate for crosslinking sodium alginate, and prepares an injectable calcium alginate hydrogel with anti - inflammatory and antioxidant functions, which can be used to treat diseases such as acute and chronic gastritis and wound repair.

[0008] The object of the present invention is to optimize the deficiencies of the existing preparation methods of injectable calcium alginate hydrogels, optimize the mechanical properties of injectable calcium alginate hydrogels and broaden the application of injectable calcium alginate hydrogels in the field of biomedical engineering, and provide a new preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source.

[0009] In order to achieve the above object, the technical solution of the present invention is: a preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source, which is characterized by comprising the following steps: (1) At room temperature, sodium alginate was dissolved in pure water to obtain a sodium alginate solution with a mass concentration of 0.1% - 1.0%. Under stirring, an aqueous calcium salt solution and an aqueous phosphate solution were gradually and dropwise dispersed into the above sodium alginate solution to form a reaction system. Then, the pH of the reaction system was adjusted to 12 - 14, and after stopping stirring, it was allowed to stand and age for 7 days. The reaction solution was dialyzed for 3 - 7 days using a dialysis bag (MWCO = 8000 - 14000 kDa), and finally, it was concentrated by rotary evaporation to obtain a sodium alginate hybrid calcium phosphate solution; (2) Sodium alginate was dissolved in the hybrid calcium phosphate solution to obtain a sodium alginate - hybrid calcium phosphate mixture, which was then mixed with a weak acid or polyphenol solution. The hybrid calcium phosphate reacted with the weak acid or polyphenol to release calcium ions to cross - link sodium alginate, obtaining an injectable calcium alginate hydrogel.

[0010] A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the molecular weight of the sodium alginate used for preparing sodium alginate hybrid calcium phosphate is 30 kDa - 190 kDa; A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the calcium salt solution is an aqueous solution of Ca(NO3)2•4H2O or an aqueous solution of CaCl2, and the phosphate solution is one of Na2HPO4•12H2O, NaH2PO4•2H2O, (NH4)2HPO4•2H2O, K2HPO4•3H2O, KH2PO4, NH4H2PO4; A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the concentrations of the aqueous calcium salt solution and the aqueous phosphate solution dispersed and added are 1 mol / L and 0.6 mol / L respectively, and the Ca / P ratio of the aqueous calcium salt solution and the aqueous phosphate solution dispersed and added is (1.0 - 2.0):1; A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the volumes of the aqueous calcium salt solution and the aqueous phosphate solution dispersed and added to the sodium alginate solution are 1 / 10 - 1 / 20 of the aqueous sodium alginate solution; A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the size of the hybrid calcium phosphate is 40 - 200 nanometers; A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the concentration of sodium alginate in the sodium alginate - hybrid calcium phosphate mixture is 0.5% - 6%, the G / M ratio of sodium alginate is 0.67 - 2.3, the calcium ion concentration of the hybrid calcium phosphate solution is 5 - 20 µmol / L, and the concentration of the weak acid or polyphenol is 0.1% - 10%, and the volume ratio of the alginate - calcium phosphate mixture to the weak acid or polyphenol is adjustable; A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source is characterized in that the injectable calcium alginate hydrogel prepared by this method. Description of the Drawings

[0011] Figure 1 It is a scanning electron micrograph of the injectable calcium alginate hydrogel prepared in Example 1 magnified 100 times. The scale bar in the figure is 200 microns.

[0012] Figure 2 It is a scanning electron micrograph of the sodium alginate hybrid calcium phosphate prepared in Example 1 magnified 100,000 times. The scale bar in the figure is 200 nanometers.

[0013] Figure 3 It is a photograph of the injectable calcium alginate hydrogel prepared in Example 3.

[0014] Figure 4 It is the effect diagram and the photograph of the residual hydrogel of the injectable calcium alginate hydrogel containing polyphenols prepared in Example 4 in the treatment of acute gastritis in C57 mice. Detailed implementation mode

[0015] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with examples, but the content of the present invention is not limited to the following examples.

[0016] Application Example 1: (1) Dissolve 0.8 g of sodium alginate in 100 mL of pure water. While continuously stirring, gradually add 5 mL of calcium chloride (CaCl2) solution (1 mol / L), and then continue to add 5 mL of sodium dihydrogen phosphate (NaH2PO4) solution (0.6 mol / L). After stirring evenly, add sodium hydroxide (NaOH) solution (2 mol / L) to adjust the pH of the solution to 12. Let it stand for 7 days and then dialyze for three days using a dialysis bag (MWCO = 14000). Subsequently, use a rotary evaporator concentrator to concentrate the obtained hybrid calcium phosphate solution until the calcium ion (Ca2+) concentration in the solution is 7 µmol / L; (2) Dissolve 0.03 g of sodium alginate in 1 mL of the above hybrid calcium phosphate solution to obtain a sodium alginate / hybrid calcium phosphate mixture with a sodium alginate concentration of 3%; (3) Dissolve 0.06 g of glucono - δ - lactone (GDL) in 1 mL of pure water to obtain a GDL solution with a concentration of 6%; (4) Mix the two solutions in a volume ratio of 1:1 to prepare an injectable calcium alginate hydrogel. The microstructure of the freeze - dried hydrogel is as shown in Figure 1 shown, and the microscopic morphology of the hybrid calcium phosphate is as shown in Figure 2 shown.

[0017] Application Example 2: (1) Adjust the concentration of the calcium salt in the hybrid calcium phosphate solution (10 µmol / L) and the concentration of sodium alginate dissolved in the hybrid calcium phosphate solution (1% - 5%) respectively; (2) After adjusting the concentration of GDL to (2% - 10%), the two solutions were mixed at a volume ratio of 1:1 to obtain an injectable calcium alginate hydrogel with different gel times and mechanical properties.

[0018] Application Example 3: (1) 0.02 g of sodium alginate with different G / M values of (0.67 - 2.3) was dissolved in 1 mL of hybrid calcium phosphate (7 μmol / L) solution to obtain a mixed solution with a sodium alginate concentration of 2%; (2) 0.06 g of GDL was dissolved in 1 mL of pure water to obtain a GDL solution with a concentration of 6%; (3) The two solutions were mixed at a volume ratio of 1:1 to obtain an injectable calcium alginate hydrogel with different G / M ratios. The digital photo of the calcium alginate hydrogel with a G / M ratio of 1:1 is shown as Figure 3 follows.

[0019] Application Example 4: (1) 0.02 g of sodium alginate with a G / M ratio of 1:1 was dissolved in a hybrid calcium phosphate solution (7 μmol / L) to obtain a mixed solution with a sodium alginate concentration of 2%; (2) 0.05 g of tannic acid or 0.005 g of gallic acid was dissolved in 1 mL of pure water to obtain a tannic acid solution with a concentration of 5% or a gallic acid solution with a concentration of 0.5%; (3) The polyphenol solution and the sodium alginate mixed solution were mixed at a volume ratio of 1:1 to obtain an injectable calcium alginate hydrogel containing the functional ingredient polyphenol. This hydrogel has good anti - inflammatory and antioxidant effects and has good effects in the treatment of acute gastritis. In the treatment of the acute gastritis model of C57 mice, this injectable calcium alginate hydrogel has good treatment effects. The gastric tissue photos of the mice are shown as Figure 4 follows.

[0020] In the present invention, injectable calcium alginate hydrogels with different properties are obtained by adjusting the concentrations of sodium alginate, hybrid calcium phosphate, weak acid, and the G / M ratio of sodium alginate. At the same time, functional injectable calcium alginate hydrogels can be prepared by introducing functional weak - acid substances to replace weak acids such as glucono - δ - lactone. Therefore, both the upper and lower limit values and the interval values of the process parameters of the present invention can achieve the present invention, so no further examples are given one by one.

Claims

1. A preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source, characterized in that The method includes the following steps: (1) At room temperature, sodium alginate is dissolved in pure water to prepare a sodium alginate solution with a mass concentration of 0.1% - 1.0%. Under stirring, an aqueous calcium salt solution and an aqueous phosphate solution are gradually and dropwise dispersed into the above sodium alginate solution to form a reaction system. Then, the pH of the reaction system is adjusted to 12 - 14 and stirring is stopped. After standing and aging for 7 days, the reaction solution is dialyzed with a dialysis bag for 3 - 7 days, and then rotary evaporation is used for concentration to obtain a sodium alginate hybrid calcium phosphate solution; (2) Sodium alginate is dissolved in the hybrid calcium phosphate solution to obtain a sodium alginate - hybrid calcium phosphate mixture, which is then mixed with a weak acid or polyphenol solution. The hybrid calcium phosphate reacts with the weak acid or polyphenol to release calcium ions to crosslink sodium alginate, obtaining an injectable sodium alginate calcium hydrogel.

2. The preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source according to claim 1, characterized in that The molecular weight of the sodium alginate used for preparing the sodium alginate hybrid calcium phosphate is 30 kDa - 190 kDa.

3. The preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source according to claim 1, wherein The calcium salt solution is an aqueous solution of Ca(NO3)2•4H2O or an aqueous solution of CaCl2, and the phosphate solution is one of Na2HPO4•12H2O, NaH2PO4•2H2O, (NH4)2HPO4•2H2O, K2HPO4•3H2O, KH2PO4, NH4H2PO4.

4. The preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source according to claim 1, wherein The concentrations of the aqueous calcium salt solution and the aqueous phosphate solution added by dispersion are 1 mol / L and 0.6 mol / L respectively, and the Ca / P ratio of the aqueous calcium salt solution and the aqueous phosphate solution added by dispersion is (1.0 - 2.0):

1.

5. The preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source according to claim 1, wherein The volumes of the aqueous calcium salt solution and the aqueous phosphate solution added by dispersion into the sodium alginate solution are 1 / 10 - 1 / 20 of the aqueous sodium alginate solution.

6. The preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source according to claim 1, wherein The size of the hybrid calcium phosphate is 40 - 200 nanometers.

7. The preparation method of an injectable calcium alginate hydrogel based on a novel polysaccharide hybrid calcium source according to claim 1, characterized in that The concentration of sodium alginate in the sodium alginate mixture is 0.5% - 6%, the G / M ratio of sodium alginate is 0.67 - 2.3, the calcium ion concentration of the hybrid calcium phosphate solution is 5 - 20 µmol / L, and the concentration of the weak acid or polyphenol is 0.1% - 10%. The volume ratio of the sodium alginate - hybrid calcium phosphate mixture to the weak acid or polyphenol is adjustable.

8. An injectable sodium alginate calcium hydrogel prepared by the method according to any one of claims 1 - 7.

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