CN106701730B - Alginate hydrogel microsphere carrier containing galactosyl chitosan molecule and its application - Google Patents

Abstract

The invention relates to an alginate-embedded hydrogel microsphere carrier containing galactosyl chitosan molecules inside and a preparation method thereof. The galactose group was grafted onto the chitosan molecule through amidation reaction, and the hepatocytes were cultured in the microcapsules formed by chitosan and alginic acid. Experiments have confirmed that the microcapsules prepared by galactosyl-grafted chitosan and alginic acid are used to culture rat primary hepatocytes, and the activity and function of hepatocytes are well maintained.

Description

Alginate hydrogel microsphere carrier containing galactosyl chitosan molecule and its application

technical field

The invention relates to an alginate hydrogel microcarrier, in particular to an alginate hydrogel microsphere carrier containing galactosyl chitosan molecules and its preparation and application.

Background technique

Biologically active substances are encapsulated in a selectively permeable membrane to form spherical microcapsules, which are called "biomicrocapsules" [Chang TMS.Hemoglobin corpuscles.Research report for honoursphysiology.Medical Library,McGill University,1957] . Since the 1990s, the use of biological microcapsules as the immune isolation and delivery tool of cells, the use of genetically recombined cells or the metabolites of primary cells to regulate the body's physiological functions, and the treatment of related diseases has become a research hotspot for biomedical workers [Basic D, Vacek I, Sun AM. Microencapsulation and transplantation of engineered cells: a new approach to somatic gene therapy. Art Cells Blood Subs Immob Biotechnol, 1996, 24(3): 219-255]. Alginic acid has become the main material of microencapsulation technology because of its excellent physical and chemical properties and biomedical properties. When the existing microencapsulation technology is used for culturing hepatocytes, due to the adherence-dependent growth characteristics of hepatocytes, the three-dimensional environment inside the microcapsules cannot rapidly adapt to the growth of hepatocytes. The galactose group is a specific ligand for the asialoglycoprotein receptor on the surface of hepatocytes, which can specifically recognize the asialoglycoprotein receptor on the surface of the hepatocyte. Therefore, the liver-targeted galactose was introduced into the microcapsule material. group, can induce and improve the adhesion and proliferation behavior of hepatocytes inside the microcapsules [Jun Yang, Galactosylated alginate as a scaffold for hepatocytes entrapment, Biomaterials, 2002, 23: 471-479]. The introduction of galactose groups inside the microcapsules can currently be done by blending or covalent modification on alginic acid. Macromolecular substances containing galactose groups [Seog-Jin Seo, Yun-Jaie Choi, Alginatemicrocapsules prepared with xyloglucan as a synthetic extracellular matrix for hepatocyte attachment, Biomaterials, 2005, 26:3607-3615] are mainly prepared by blending with alginic acid Microcapsules play a role, and the blended substances are prone to leakage, which leads to a decrease in the stability of such microcapsules; while the covalent modification of alginic acid mainly occurs at the carboxyl site of alginic acid, and the occupation of the carboxyl site makes the algae The degree of gelation of the acid is reduced when the microcapsules are formed by the gelation reaction, and the degree of galactosyl substitution is limited in order not to unduly affect the gel-forming properties of alginic acid. [Ivan Donati, Amedeo Vetere, Galactose-Substituted Alginate: Preliminary Characterization and Study of Gelling Properties, Biomacromolecules, 2003, 4:624-631]. Due to the limitation of the stability of the microcapsules and the degree of substitution of galactosyl groups, whether it is blending or covalently modifying alginic acid, the methods reported so far cannot introduce a higher concentration of galactose groups into the microcapsules.

Chitosan is a natural biological material with excellent biocompatibility. Practice has shown that microcapsules made of alginate and chitosan due to electrostatic interaction have a mild, simple preparation method and fast encapsulation speed. The microcapsules are spherical. Good degree of drug stability [Donati I, Holtan S, Morch YA, et al. New hypothes is on the role of alternating sequences in calcium-alginate gels, Biomacromolecules, 2005, 6(2): 1031-1040]. Therefore, significant progress has been made in the research of alginate/chitosan microcapsules in recent years. At present, there are three main methods for preparing alginate/chitosan microcapsules: alginate is dropped into the mixed solution of chitosan and divalent ions to form microcapsules [Thomas Chandy, Daniel L, Evaluation of Modified Alginate -Chitosan-PolyethyleneGlycol Microcapsules for Cell Encapsulation,Artificial Organs,1999]; Chitosan is dropped into alginate solution to form microcapsules [Sun-Hee Yu, Sung-Koo Kim, Encapsulation of rathepatocyte spheroids for the development of artificial liver, Biotechnology Techniques, 1999, 13:609–614; X.L.GUO, K.S.YANG, et al, Morphology and metabolism of Ba-alginate-encapsulated hepatocytes with galactosylated chitosan and poly(vinyl alcohol) as extracellular matrices, Journal of Biomaterials Science, Polymer Edition, 2014 , 6:551-565]; alginate is dropped into calcium solution to form embedded hydrogel microsphere carrier, and then the embedded hydrogel microsphere carrier is immersed in chitosan solution for further reaction to form a film. Microcapsules, i.e. AC microcapsules [Tasima Haque, In vitro study of alginate–chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure, Biotechnology Letters, 2005, 27: 317-322; Wujie Zhang, Shuting Zhao, A novel core – shellmicrocapsule for encapsulation and 3D culture of embryonic stem cells, Journal of Materials Chemistry B, 2013, 1: 1002-1009]. The first two methods have the following problems. First, due to the limitation of the instantaneous reaction speed of the two macromolecules, chitosan and alginic acid, the particle size of the formed microcapsules is about 1 mm or even larger. If the clusters grow too large, the central cells will be necrotic due to the limitation of mass transfer and oxygen transfer, which will affect the cell activity, and then affect the medical effect of the secreted products of the cells. Second, the instantaneous combination of macromolecules will form microcapsules with uneven and poor sphericity, which affects the Due to the mechanical strength of the microcapsules, the microcapsules are prone to damage when transplanted into the body and cause the body's inflammatory response; thirdly, whether alginic acid or chitosan is used as a gel bath, alginic acid cannot completely react with chitosan dissolved in calcium solution Or the chitosan cannot be completely combined with alginic acid, which leads to waste of raw materials. The third method is to drop alginic acid into a divalent salt solution to form an embedded hydrogel microsphere carrier with controllable size, good sphericity and mechanical strength. When the carrier is immersed in the chitosan solution and further reacts to form a film to form traditional AC microcapsules, the chitosan does not enter the inside of the microcapsule, which forms a core-shell microcapsule with alginic acid as the core and chitosan as the shell Structure, chitosan does not contact the cells inside the microcapsules, so that functional chitosan cannot play its due role.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention proposes an alginate-embedded hydrogel microsphere carrier containing galactosyl chitosan molecules inside, which is characterized in that: the alginate-embedded hydrogel microsphere carrier internally contains Galactosyl chitosan molecules, wherein electrostatic complexes are formed between galactosyl chitosan molecules and alginate molecules. That is, after the covalently modified galactosyl chitosan solution and the alginate solution are fully mixed evenly in a pH environment where neither of the two forms a precipitate, the mixed solution is formed into a jet under a high-voltage electrostatic field, and the pH partial acid is added dropwise. In the divalent salt gel bath of the chitosan, an embedded hydrogel microsphere carrier containing a galactose group is formed through the electrostatic complexation of chitosan and alginate.

Technical solutions

In the present invention, after the covalently modified galactosyl chitosan solution and the alginate solution are fully mixed evenly in an environment of pH 6.0-8.0, the mixed solution is formed into a jet under a high-voltage electrostatic field, and the solution is dropped into a pH of 5.0 In the divalent salt gel bath of -6.9, an embedded hydrogel microsphere carrier containing a galactose group inside is formed. The embedded hydrogel microsphere carrier can continue to react with polycations and complex on the surface of the microsphere carrier to form a membrane structure, which is called alginate/galactosyl chitosan-polycation microcapsules; Including any one or two or more of the following: polyamino acids (such as polylysine, polyornithine, polyarginine, polyhistidine, etc.), polyamines (such as polyethyleneimine, polyimide Methylguanidine, polyN vinyl caprolactam, carboxyl-propyl-acrylamide copolymer, DEAE-dextran, amino polyethylene glycol, etc.), chitosan, etc. The alginate/galactosyl chitosan-polycationic microcapsules can be immersed in an organometallic chelating agent solution to liquefy the alginate gel inside the microcapsules; the organometallic chelating agent solution involved in the liquefaction reaction is 40-70 mmol/ L of sodium citrate or 50-200mmol/L EDTA solution, the volume ratio of microcapsules to organometallic chelating agent solution is 1:1～1:40, react for 1-60 minutes, take out and wash with physiological saline, then obtain Microcapsules with internal liquid core.

Wherein, the galactosyl chitosan is produced from chitosan (Chitosan) and lactobionic acid (LA) through amidation reaction to generate galactosyl chitosan (GC), and its grafting degree is (per 100 chitosan) The percentage of sugar monomer grafted lactobionic acid molecules) 1%-99%, the reaction formula is as follows:

The specific preparation process of preparing galactosyl chitosan material by covalent modification of lactobionic acid and chitosan is as follows: TaekWoong Chung, Preparation of alginate/galactosylated chitosanscaffold for hepatocyte attachment, Biomaterials, 2002, 23: 2827-2834.

The alginate/galactosyl chitosan-polycationic microcapsule product prepared by the method is a spherical microcapsule with a particle size of 100-1000 microns; the thickness of the microcapsule film is 1-100 microns; the polycationic material in the microcapsule film includes : Chitosan with a degree of deacetylation of 60-98% and a molecular weight of 1kDa-800kDa (for example: 1kDa-5kDa; 10kDa-20kDa; 50kDa-100kDa; 100kDa-800kDa); α-polylysine, with a molecular weight of 2kDa - 500kDa (eg: 2kDa-10kDa; 70kDa-150kDa; 150kDa-300kDa; 300kDa-500kDa); ε-polylysine, with a molecular weight of 2kDa-500kDa (eg: 2kDa-50kDa; 50kDa-100kDa; 100kDa-350kDa; 350kDa) -500kDa); polyarginine, with a molecular weight of 1kDa-500kDa (eg: 1kDa-100kDa; 100kDa-350kDa; 350kDa-500kDa); polyornithine, with a molecular weight of 1kDa-500kDa (eg: 1kDa-50kDa; 50kDa-100kDa) ; 100kDa-350kDa; 350kDa-500kDa); polyhistidine with a molecular weight of 1kDa-500kDa (eg: 1kDa-50kDa; 50kDa-100kDa; 100kDa-350kDa; 350kDa-500kDa).

The alginate gel in the microcapsules is a divalent metal calcium, barium or zinc hydrogel, and the alginate (molecular weight 10KDa-10000kDa) solution is a sodium or potassium salt solution of alginic acid. The galactosyl chitosan solution is a solution of easily water-soluble galactosyl chitosan (molecular weight 1KDa-800KDa) dissolved with sodium salt or potassium salt.

The specific preparation steps of the product are:

1) A galactosyl modified chitosan material is prepared by amidation reaction of lactobionic acid and chitosan, wherein the graft ratio of chitosan grafted galactose group is 1%-99%;

2) Prepare a galactosyl chitosan solution with a concentration of 1-60g/L and an alginate solution with a concentration of 10-30g/L respectively, and adjust the pH environment of both to be between 6.0-8.0 to ensure that the two are sufficiently dissolve. Among them, the molecular weight of galactosyl chitosan is 1KDa-800KDa, the degree of deacetylation is 80-98%, and the molecular weight of alginic acid is 10KDa-10000kDa;

3) Mix the galactosyl chitosan solution described in 2) and the alginate solution in a volume ratio of 1:5-5:1, and stir at room temperature for 0.1-6h;

4) Use the mixed solution described in 3) to form droplets by the orifice extrusion method, the electrostatic droplet method, the emulsification method, the rotating disk method, etc., and drop them into a divalent salt gel bath with a pH of 5.0-6.9, and coagulate. After the gel is cured for 20-60 minutes, an embedded hydrogel microsphere carrier containing galactosyl chitosan molecules inside is prepared, which is called A microsphere.

5) The A microspheres in step 4) are immersed in the polycationic solution, the volume ratio of the A microspheres to the polycationic solution is 1:1-1:40, the reaction time is 1-60 minutes, and the reaction temperature is 0- At 37°C, an embedded hydrogel microsphere carrier loaded with galactose groups, namely alginate/galactosyl chitosan-polycationic microcapsules, was obtained.

The preparation method of the polycation solution is:

Chitosan is dissolved in acetic acid-sodium acetate buffer or 3-9g/L NaCl solution with a pH of 5.0-7.0, and the chitosan concentration is 0.1-15g/L;

Or, α-polylysine is dissolved in 3-9g/L NaCl solution, and the concentration of α-polylysine is 0.01-10g/L;

Or, ε-polylysine is dissolved in 3-9g/L NaCl solution, and the concentration of ε-polylysine is 0.01-10g/L;

Or, polyarginine is dissolved in 3-9g/L NaCl solution, and the polyarginine concentration is 0.01-10g/L;

Or, polyornithine is dissolved in 3-9g/L NaCl solution, and the concentration of polyornithine is 0.01-10g/L;

Or, polyhistidine is dissolved in 3-9g/L NaCl solution, and the concentration of polyornithine is 0.01-10g/L;

The microcapsules can be used for the embedding of living cells. After the galactosyl chitosan solution and the alginate solution are fully mixed, the mixed solution is mixed with the living cells to be embedded to prepare the microcapsules for embedding living cells. The content of microencapsulated cells was 10 ⁵ -2*10 ⁷ cells/mL, and the cell viability remained above 80%. The living cells are human or animal-derived isolated hepatocytes, stem cells, stem cells differentiated cells with hepatocyte function, hepatocyte lineage cells, transdifferentiated cells with hepatocyte function, endothelial cells, liver Kupffer cells , one or more of hepatic stellate cells, fibroblasts, and bone marrow mesenchymal stem cells.

The beneficial effects of the present invention

1. This method can ensure the formation of microcapsules with a controllable size and good sphericity, and avoids the phenomenon of central necrosis of the cell mass caused by the excessively large particle size of the microcapsules.

2. In the alginate/galactosyl chitosan-polycationic microcapsules prepared by this method, galactosyl chitosan can enter the interior of the microcapsules, which solves the problem that galactose in the traditional AC microcapsules can only be distributed in the microcapsules. The surface or even after entering the microcapsule will cause the microcapsule particle size to become larger and the sphericity to deteriorate.

3. Whether the alginate is dropped into the galactosyl chitosan solution or the galactosyl chitosan is dropped into the alginic acid solution, the galactose in the alginate/galactosyl chitosan hydrogel microsphere carrier is formed. In this method, alginic acid and galactosyl chitosan are pre-mixed under neutral conditions, so a larger concentration of galactose can be introduced into the microcapsule, which is beneficial to the galactose group inside the microcapsule. perform greater functions.

4. In this method, two important macromolecular raw materials, alginic acid and galactosyl chitosan, are directly mixed in advance to prepare microcapsules, which can save raw materials and control costs to the greatest extent.

5. The microcapsules prepared by this method maintain good sphericity and good mechanical strength, and can ensure the integrity of the microcapsules in the process of tissue cell transplantation and cell culture application.

6. Compared with the alginate/galactosyl chitosan microcapsules of the core-shell structure, the microcapsules prepared by this method can maintain the immune isolation performance while the permeability is not affected in any way. Cells get better mass and oxygen transfer in the microcapsules.

Detailed ways

The method for preparing alginate/galactosyl chitosan embedded hydrogel microsphere carrier is electrostatic drop method (reference: In Vivo Culture of Encapsulated Endostatin-Secreting Chinese Hamster Ovary Cells for Systemic Tumor Inhibition, Human Gene Therapy 2007, 18:474-481).

Example 1

1) Preparation of sodium alginate solution: 2.0 g of alginic acid was dissolved in 100 mL of physiological saline to prepare a 20 g/L sodium alginate solution, wherein the molecular weight of alginic acid was 350 kDa.

2) Preparation of galactosyl chitosan solution: dissolve galactosyl chitosan in physiological saline to prepare galactosyl chitosan with concentrations of 0, 15g/L, 20g/L, 30g/L, and pH 7.0 respectively. Glycan solution.

3) Preparation of gel bath solution: 11 g of anhydrous calcium chloride was dissolved in 1 L of deionized water.

4) Take 2 mL of sodium alginate solution, mix 2 mL of galactosyl chitosan solutions of different concentrations obtained in step 2), and stir at room temperature for 3 hours. At this time, the concentration of sodium alginate is 10 g/L, and the concentration of galactosyl chitosan is 10 g/L. 0, 7.5g/L, 10g/L, 15g/L in sequence, the calcium alginate/galactosyl chitosan embedded hydrogel microsphere carrier was prepared by electrostatic drop method.

5) Preparation of α-polylysine solution: α-polylysine was dissolved in physiological saline to prepare a 5 g/L α-polylysine solution. Among them, the molecular weight of α-polylysine is 30kDa.

6) Preparation of calcium alginate/galactosyl chitosan-α-polylysine microcapsules: Immerse the embedded hydrogel microsphere carrier prepared in step 4) into the α-polylysine prepared in step 5). In acid solution, the volume ratio of embedded hydrogel microspheres and α-polylysine solution is 1:10, react for 10 minutes, wash with normal saline, and prepare calcium alginate/galactosyl chitosan-α- Polylysine Microcapsules.

7) Put the calcium alginate/galactosyl chitosan-α-polylysine microcapsules obtained in step 6) into FITC-labeled BSA (bovine serum albumin) according to the volume ratio of microcapsules: BSA=1:20. ) solution, real-time observation under a laser confocal microscope, each group of microcapsules reached diffusion equilibrium within 2 hours, indicating that calcium alginate/galactosyl chitosan-α-polylysine microcapsules have good permeability .

8) The calcium alginate/galactosyl chitosan-α-polylysine microcapsules obtained in step 6) are placed in a conical flask together with agate balls and physiological saline, and under the conditions of 37 degrees and 170 r/min After oscillating ball milling for 24 hours, the integrity rate of the microcapsules in each group was above 90%, indicating that the calcium alginate/galactosyl chitosan-α-polylysine microcapsules had good mechanical strength.

Example 2

1) Preparation of sodium alginate solution: 3.0 g of alginic acid was dissolved in 100 mL of physiological saline to prepare a 30 g/L sodium alginate solution, wherein the molecular weight of alginic acid was 350 kDa.

2) Preparation of FITC-labeled galactosyl chitosan solution: The FITC-labeled galactosyl chitosan solution was dissolved in physiological saline to prepare a galactosyl chitosan solution with a concentration of 10 g/L and a pH of 7.0.

3) Preparation of gel bath solution: 11 g of anhydrous calcium chloride was dissolved in 1 L of deionized water.

4) Take 2 mL of sodium alginate solution, mix 2 mL of the galactosyl chitosan solution obtained in step 2), and stir at room temperature for 3 hours. At this time, the concentration of sodium alginate is 15 g/L, and the concentration of galactosyl chitosan is 5 g/L. L, Preparation of calcium alginate/galactosyl chitosan-embedded hydrogel microsphere carrier by electrostatic droplet method.

Example 3

1) Preparation of sodium alginate solution: 2.0 g of alginic acid was dissolved in 100 mL of physiological saline to prepare a 20 g/L sodium alginate solution, wherein the molecular weight of alginic acid was 350 kDa, and the solution was filtered and sterilized.

2) Preparation of galactosyl chitosan solution: Dissolve 0.4 g of galactosyl chitosan in 10 mL of physiological saline to prepare a 40 g/L chitosan solution, adjust the pH value to 7.2, and filter and sterilize.

3) Preparation of gel bath solution: dissolve 11 g of anhydrous calcium chloride in 1 L of deionized water, filter and sterilize.

4) Preparation of α-polylysine solution: α-polylysine was dissolved in physiological saline to prepare a 5g/L α-polylysine solution, which was filtered and sterilized. Among them, the molecular weight of α-polylysine is 30kDa.

5) Take 3 mL of sodium alginate solution, mix with 1 mL of galactosyl chitosan solution, and stir at room temperature for 1 h. Take 2 mL of the mixed solution, add 4*10 ⁶ rat primary hepatocytes, mix well, and use the electrostatic drop method to prepare calcium alginate/galactosyl chitosan mixed rat primary hepatocyte-embedded water. Gel microsphere carrier. Among them, the particle size of the embedded hydrogel microsphere carrier is about 350 μm.

6) Immerse the calcium alginate/galactosyl chitosan embedded hydrogel microsphere carrier embedded with primary rat hepatocytes into the polylysine solution prepared in step 4), and the embedded hydrogel The volume ratio of the gel microsphere carrier to the polylysine solution was 1:10, the reaction was performed for 10 minutes, and washed with physiological saline to prepare calcium alginate/galactosyl chitosan-α-polylysine microcapsules.

7) The primary rat hepatocytes in the microcapsules were cultured and the function of the hepatocytes was characterized. After one week, the microcapsules remained intact and the sphericity was good; The protein secretion amount was 1.29±0.33μg/10^6 cells, and the urea synthesis amount was 349±7μg/10^6 cells.

Comparative example

1) Preparation of sodium alginate solution: 2.0 g of alginic acid was dissolved in 100 mL of physiological saline to prepare a 20 g/L sodium alginate solution, wherein the molecular weight of alginic acid was 350 kDa, and the solution was filtered and sterilized.

2) Preparation of gel bath solution: dissolve 11 g of anhydrous calcium chloride in 1 L of deionized water, filter and sterilize.

3) Preparation of α-polylysine solution: α-polylysine was dissolved in physiological saline to prepare a 5g/L α-polylysine solution, and sterilized by filtration. Among them, the molecular weight of α-polylysine is 30kDa.

4) Take 3 mL of sodium alginate solution, mix with 1 mL of physiological saline solution, and stir at room temperature for 1 h. Take 2 mL of the mixed solution, add 4*10 ⁶ rat primary hepatocytes, and after mixing evenly, use the electrostatic drop method to prepare calcium alginate mixed rat primary hepatocyte-embedded hydrogel microsphere carrier. Among them, the particle size of the embedded hydrogel microsphere carrier is about 350 μm.

5) Immerse the calcium alginate embedded hydrogel microsphere carrier embedded with primary rat hepatocytes into the α-polylysine solution prepared in step 3), and the embedded hydrogel microsphere carrier and The volume ratio of the polylysine solution was 1:10, the reaction was performed for 10 minutes, and the solution was washed with physiological saline to prepare calcium alginate-α-polylysine microcapsules.

6) The primary rat hepatocytes in the microcapsules were cultured and the function of the hepatocytes was characterized. After one week, the microcapsules remained intact and the sphericity was good; The protein secretion amount was 0.46±0.05μg/10^6 cells, and the urea synthesis amount was 231±29μg/10^6 cells.

Claims (10)

1. The alginate-embedded hydrogel microsphere carrier containing galactose-based chitosan molecules is characterized in that: the alginate-embedded hydrogel microsphere carrier contains galactose-shell polysaccharide molecules inside, wherein electrostatic complexation is formed between the galactose-shell polysaccharide molecules and the alginate molecules;

the preparation method of the alginate-embedded hydrogel microsphere carrier containing galactose-based chitosan molecules comprises the following steps:

1) preparing a galactose-based chitosan material by covalent modification of lactobionic acid and chitosan, wherein the grafting rate of chitosan grafting galactose groups is 1% -99% (the percentage of lactobionic acid molecules grafted to each 100 chitosan monomers);

2) respectively preparing 1-60g/L galactose chitosan solution and 10-30g/L alginate solution, and adjusting pH of the two solutions to 6.0-8.0;

3) mixing the galactose chitosan solution prepared in the step 2) with the alginate solution according to the volume ratio of 1:5-5:1, and stirring at room temperature for 0.1-6 h;

4) forming liquid drops by using the mixed solution in the step 3) through an orifice extrusion method, an electrostatic liquid drop method, an emulsification method or a rotating disc method, dripping the liquid drops into a divalent salt gel bath with the pH value of 5.0-6.9, and curing the gel for 20-60 minutes to prepare the embedded hydrogel microsphere carrier containing galactose-based chitosan molecules.

2. The embedded hydrogel microsphere carrier of claim 1, wherein: the particle size of the microsphere carrier is 100-1000 microns.

3. The embedded hydrogel microsphere carrier of claim 1, wherein: the embedded hydrogel microspheres can continuously react with polycations to form a membrane structure, namely microcapsules, on the surfaces of the microspheres; wherein the polycation comprises any one or more than two of the following components: polyamino acids, polyamines and chitosan.

4. An embedded hydrogel microcapsule according to claim 3, wherein: the polyamino acids are polylysine, polyornithine, polyarginine and polyhistidine; the polyamine is selected from polyethyleneimine, polymethyleneguanidine, poly-N-vinylcaprolactam, carboxyl-propyl-acrylamide copolymer, DEAE-dextran, and amino polyethylene glycol.

5. The encapsulated hydrogel microcapsule according to claim 3, wherein said microcapsule is immersed in a solution of an organic metal chelating agent to liquefy alginate gel in the microcapsule, the solution of the organic metal chelating agent participating in the liquefaction reaction is 40-70mmol/L sodium citrate or 50-200mmol/L EDTA solution, the volume ratio of the microcapsule to the solution of the organic metal chelating agent is 1:1 ~ 1:40, the reaction is carried out for 1-60 minutes, and the microcapsule is taken out and washed with physiological saline to obtain a microcapsule with an inner liquid core.

6. The embedded hydrogel microsphere carrier of claim 1, wherein: the divalent salt gel bath is one or more than two of divalent metal calcium, barium or zinc ion solutions with the mass concentration of 3.3g/L-33 g/L.

7. The embedded hydrogel microsphere carrier of claim 1, wherein: the alginate solution in the hydrogel carrier preparation method comprises one or two of sodium alginate and potassium alginate which are necessarily contained, and one or more than two of calcium alginate, barium alginate or zinc alginate which are not necessarily contained, wherein the average molecular weight of the alginate is 10kDa-10000kDa, and the molar content of guluronic acid monomers in the alginate is 20-98 percent.

8. The embedded hydrogel microsphere carrier of claim 1, wherein: the chitosan is water-soluble chitosan, including low molecular weight chitosan with molecular weight of 1KDa-10KDa, water-soluble chitosan with molecular weight of 10KDa-800KDa, and chitosan grafted with hydrophilic group; the deacetylation degree of the chitosan is 60-98%, and the formed galactose chitosan keeps the water-soluble property.

9. The use of the encapsulated hydrogel microsphere carrier of claim 1, wherein: the microsphere carrier is mainly used for embedding living cells, and after the galactose-based chitosan solution and the alginate solution are fully mixed, the mixed solution is uniformly mixed with the living cells to be embedded.

10. The use of an embedded hydrogel microsphere carrier according to claim 9, wherein: the living cells are one or more than two of isolated liver cells, stem cell differentiated cells with liver cell functions, liver cell line cells, trans-differentiated cells with liver cell functions, endothelial cells, liver Kupffer cells, hepatic stellate cells, fibroblasts and bone marrow mesenchymal stem cells which are derived from human or animals.