CN108904890B - Dynamic electrostatic deposition compound natural material bionic porous microcarrier and preparation method thereof - Google Patents
Dynamic electrostatic deposition compound natural material bionic porous microcarrier and preparation method thereof Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
- A61L27/34—Macromolecular materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
Abstract
The invention discloses a dynamic electrostatic deposition compound natural material bionic porous microcarrier and a preparation method thereof, relates to the technical field of biomedical materials or biological composite materials, and discloses a natural material bionic porous microcarrier and a preparation method thereof, wherein the natural material bionic porous microcarrier can be used in the fields of human soft and hard tissue culture, tissue engineering micro tissue construction, human tissue repair, cell amplification and drug release. The natural materials comprise chitosan, nano-cellulose, animal protein, plant protein, polyamino acid, polypeptide and the like. The nano-cellulose is processed by deflocculation, the protein is processed by dissolution, centrifugation and low-temperature precipitation, and the bionic porous microcarrier is prepared by a dynamic electrostatic deposition method. The invention has the beneficial effects that: the bionic porous microcarrier is prepared by simulating the components and the structure of the extracellular matrix and carrying out in-situ dynamic electrostatic deposition, so that the bionic porous microcarrier has good biocompatibility and mechanical property and the capability of promoting cell activity. In addition, no cross-linking agent is used in the process of preparing the bionic porous microcarrier, so that cytotoxicity caused by residual cross-linking agent is avoided.
Description
Technical Field
The invention relates to the technical field of biomedical materials or biological composite materials, in particular to a natural material bionic porous microcarrier and a preparation method thereof.
Background
Loss or dysfunction of tissues and organs is one of the major risks facing human health and is the leading cause of human disease and death. With the development of tissue engineering technology, tissue engineering is becoming an effective and feasible means for repairing tissues or organs. For the tissue defect with irregular shape, the traditional block tissue engineering scaffold has the defect of difficult fusion with the tissue defect. Aiming at the problem, the tissue engineering microcarrier is produced by inoculating seed cells into the microcarrier, culturing the seed cells in a bioreactor to obtain a micro-tissue with a large amount of cells adhered and proliferated in the microcarrier, and accurately attaching the micro-tissue to an injured part through injection, so that the tissue shape has controllability and the pain of a patient can be reduced. The ideal microcarrier should have good biocompatibility, porosity, degradability, good mechanical properties, etc. Researchers have been working on developing and modifying various tissue engineering microcarrier materials to better assist in the repair of tissues or organs.
The chitosan has the advantages of good biocompatibility and degradability, no toxicity of products degraded by enzymes in human bodies, easiness in forming spherical porous structures by freeze-drying acid solutions, and the like, and is widely applied to research of tissue engineering microcarriers. However, when chitosan is singly used as a tissue engineering microcarrier material, the defects of insufficient mechanical strength, difficulty in simulating a microenvironment required by cell growth, lack of cell recognition sites, low cell activity promoting capacity (such as osteoblasts, nerve cells and the like) and the like exist. The method for carrying out composite modification on chitosan by adopting other natural materials is widely concerned by students, and in the existing research, when the chitosan or chitosan composite other materials are adopted to prepare the tissue engineering microcarrier, the microenvironment required by cell growth cannot be well simulated, the mechanical property is poor, and partial cell activity cannot be promoted. In addition, a cross-linking agent is commonly used in the prior research, and the residual cross-linking agent generates cytotoxicity and cannot be used for tissue engineering. The ideal tissue engineering microcarrier has good biocompatibility and excellent mechanical property, and can simulate the microenvironment required by cell growth, promote cell activity and the like. How to endow the microcarrier with excellent mechanical property and simultaneously ensure that the microcarrier can well simulate the microenvironment required by cell growth, which is a problem that is not solved at home and abroad.
Aiming at the current situation, the invention designs and prepares the natural material bionic tissue engineering porous microcarrier which has good biocompatibility and excellent mechanical property and can well simulate the microenvironment required by cell growth according to the components and the structure of the extracellular matrix.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a natural material bionic tissue engineering porous microcarrier and a preparation method thereof.
The nano cellulose (BS) has a nano fiber network structure similar to that of an extracellular matrix on a microstructure, the fiber diameter is close to that of collagen fibers in the extracellular matrix, the mechanical strength is good, and the nano cellulose has unique advantages in structure bionic design; animal protein (such as collagen, gelatin and the like), plant protein (such as soybean protein, corn protein and the like), polyamino acid and polypeptide can be used for simulating protein components in extracellular matrix, providing cell active sites and promoting cell activity, and the protein, polyamino acid and polypeptide (HS-P) subjected to physical separation and ionization treatment can improve solubility on the basis of maintaining the type and quantity of active functional groups, and is favorable for forming polyelectrolyte with other materials through electrostatic adsorption; chitosan (CS) is similar in chemical structure to glycosaminoglycans in the extracellular matrix. The three components are compounded to prepare the microcarrier, so that double bionics on the structure and the components are achieved, a microenvironment required by cell growth is well simulated, a certain mechanical strength is achieved, and cell activity is promoted.
The invention relates to a dynamic electrostatic deposition compound natural material bionic porous microcarrier, which comprises a porous microcarrier and a protein component coated on the surface of the porous microcarrier;
the bionic porous microcarrier is prepared by compounding nano-cellulose and chitosan, and the protein component is subjected to in-situ dynamic electrostatic deposition.
Further, the mass ratio of chitosan to nano-cellulose in the porous microcarrier is 1: 1-5: 1.
Further, the protein component comprises one or more of animal protein, plant protein, polyamino acid or polypeptide.
Furthermore, the bionic porous microcarrier simulates the structure and the components of the bionic extracellular matrix through the components of nano cellulose, chitosan and protein.
Further, the particle size range of the bionic porous microcarrier is 50-1000 μm; the aperture range is 10-100 mu m; the porosity is more than 80%; the mechanical strength is 50 kPa to 300 kPa.
The invention also aims to provide a preparation method of the bionic porous microcarrier, which comprises the following steps:
step one, deflocculating nano-cellulose:
washing the nano cellulose membrane with deionized water; soaking the nano cellulose membrane in NaOH solution, and boiling for 1-10 h in a high-temperature water bath; washing with deionized water for many times until the solution is neutral to obtain a pure nano cellulose membrane; and (3) deflocculating the obtained pure nano cellulose membrane under the action of high-speed shearing, and centrifuging at a high speed to obtain a lower-layer precipitate for later use.
Step two, preparing the BC/CS porous microcarrier:
fully mixing the BC slurry and the CS solution according to the mass ratio of the solute to the mixed solution of 1: 1-5: 1, stirring at the rotating speed of less than 150 r/min, and uniformly stirring the mixed solution; adding 2-10% of volume fraction emulsifier into the mixed solution, increasing the rotating speed, stirring the mixed solution at the rotating speed of 300-1000 r/min, and stabilizing the particle size and the pore diameter of the microcarrier by the nano-cellulose with the nano-fiber structure in the stirring process; placing the stirred liquid in a low-temperature environment of minus 70 ℃ to minus 150 ℃, and quickly curing and separating the phase; cleaning the microspheres with a low-temperature organic solvent to obtain solid microspheres; and (3) drying the microspheres in a low-temperature negative-pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa to obtain the BC/CS porous microcarrier.
Step three, obtaining a high-solubility protein component:
dispersing the protein component in the solution to dissolve to obtain high-solubility protein for later use;
step four, preparing the HS-P/BC/CS bionic porous microcarrier by a dynamic electrostatic deposition method:
preparing the high-solubility protein obtained in the third step into a solution with the concentration of 0.5-5%, adjusting the pH value to 6-10, adding the BC/CS microcarrier obtained in the second step into the solution, controlling the stirring speed to be 20-200 r/min, and stirring for 1-24 h to generate controllable dynamic electrostatic adsorption between HS-P and chitosan in the solution, so as to form the shell-core fiber structure porous microspheres through self-assembly, placing the microspheres subjected to solid-liquid separation in a low-temperature negative pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa for solvent separation, and finally obtaining the HS-P/BC/CS porous microcarrier.
Further, the protein component is divided into a protein component with higher solubility and a protein component with lower solubility;
the protein component with higher solubility is directly dispersed in the solution to be dissolved for later use;
and the protein component with low solubility is extracted by physical separation, wherein the physical separation comprises high-speed centrifugation, low-temperature precipitation and freeze drying, and the protein with high solubility is finally obtained.
The bionic porous microcarrier prepared by the method is applied to human soft and hard tissue culture, tissue engineering micro tissue construction, human tissue repair, cell amplification or drug release.
Compared with the prior art, the invention has the beneficial effects that:
1. the polyelectrolyte microspheres are formed by compounding the nano-cellulose and the chitosan, and the nano-cellulose with a nano-fiber structure plays a role in stabilizing the particle size and the pore diameter of the porous microcarrier;
2. no cross-linking agent is introduced in the process of synthesizing the porous microcarrier by dynamic electrostatic deposition, so that the synthesis step is simplified, and the toxicity caused by introducing the cross-linking agent is eliminated;
3. the bionic porous microcarrier is prepared by simulating the components and the structure of the extracellular matrix and selecting a natural degradable material, so that the bionic porous microcarrier has good biocompatibility and mechanical property, and can provide a microenvironment similar to the extracellular matrix for the in-vitro proliferation and differentiation of cells.
Drawings
FIG. 1 shows a macroscopic view of a porous microcarrier according to an embodiment of the invention.
FIGS. 2 and 3 are scanning electron micrographs of a porous microcarrier according to an embodiment of the present invention.
Detailed Description
The invention is further illustrated in the following description with reference to the specific figures. It should be understood that the examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various equivalent modifications of the present invention can be made by those skilled in the art after reading the teaching of the present invention, and the modifications also fall within the scope of the claims of the present application.
As shown in the figure, the bionic porous microcarrier of the dynamic electrostatic deposition compound natural material comprises a porous microcarrier and a protein component coated on the surface of the porous microcarrier;
the bionic porous microcarrier is prepared by compounding nano-cellulose and chitosan, and the protein component is subjected to in-situ dynamic electrostatic deposition.
Further, the mass ratio of chitosan to nano-cellulose in the porous microcarrier is 1: 1-5: 1.
Further, the protein component comprises one or more of animal protein, plant protein, polyamino acid or polypeptide.
Furthermore, the bionic porous microcarrier simulates the structure and the components of the bionic extracellular matrix through the components of nano cellulose, chitosan and protein.
Further, the particle size range of the bionic porous microcarrier is 50-1000 μm; the aperture range is 10-100 mu m; the porosity is more than 80%; the mechanical strength is 50 kPa to 300 kPa.
The invention also aims to provide a preparation method of the bionic porous microcarrier, which comprises the following steps:
step one, deflocculation of nano-cellulose
Washing the nano cellulose membrane with deionized water; soaking the nano cellulose membrane in NaOH solution, and boiling for 1-10 h in a high-temperature water bath; washing with deionized water for many times until the solution is neutral to obtain a pure nano cellulose membrane; and (3) deflocculating the obtained pure nano cellulose membrane under the action of high-speed shearing, and centrifuging at a high speed to obtain a lower-layer precipitate for later use.
Step two, preparing the BC/CS porous microcarrier
Fully mixing the BC slurry and the CS solution according to the mass ratio of the solute to the mixed solution of 1: 1-5: 1, stirring at the rotating speed of less than 150 r/min, and uniformly stirring the mixed solution; adding 2-10% of volume fraction emulsifier into the mixed solution, increasing the rotating speed, stirring the mixed solution at the rotating speed of 300-1000 r/min, and stabilizing the particle size and the pore diameter of the microcarrier by the nano-cellulose with the nano-fiber structure in the stirring process; placing the stirred liquid in a low-temperature environment of minus 70 ℃ to minus 150 ℃, and quickly curing and separating the phase; cleaning the microspheres with a low-temperature organic solvent to obtain solid microspheres; and (3) drying the microspheres in a low-temperature negative-pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa to obtain the BC/CS porous microcarrier.
Step three, obtaining a high-solubility protein component:
dispersing the protein component in the solution to dissolve to obtain high-solubility protein for later use;
step four, preparing the HS-P/BC/CS bionic porous microcarrier by a dynamic electrostatic deposition method
Preparing the high-solubility protein obtained in the third step into a solution with the concentration of 0.5-5%, adjusting the pH value to 6-10, adding the BC/CS microcarrier obtained in the second step into the solution, controlling the stirring speed to be 20-200 r/min, and stirring for 1-24 h to generate controllable dynamic electrostatic adsorption between HS-P and chitosan in the solution, so as to form the shell-core fiber structure porous microspheres through self-assembly, placing the microspheres subjected to solid-liquid separation in a low-temperature negative pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa for solvent separation, and finally obtaining the HS-P/BC/CS porous microcarrier.
Further, the protein component is divided into a protein component with higher solubility and a protein component with lower solubility;
the protein component with higher solubility is directly dispersed in the solution to be dissolved for later use;
and the protein component with low solubility is extracted by physical separation, wherein the physical separation comprises high-speed centrifugation, low-temperature precipitation and freeze drying, and the protein with high solubility is finally obtained.
The bionic porous microcarrier prepared by the method is applied to human soft and hard tissue culture, tissue engineering micro tissue construction, human tissue repair, cell amplification or drug release.
Example (b):
step one, deflocculation of nano-cellulose
Washing a nano cellulose (BC) membrane with clear water for multiple times to remove impurities on the surface of the membrane, soaking the membrane in 0.01mol/L NaOH solution, boiling in a water bath at 100 ℃ for 2 hours, and removing thalli in a liquid membrane; washing with distilled water for many times until the solution is neutral to obtain a pure BC membrane; the pure nano cellulose membrane is deflocculated under the action of high-speed shearing, and the sediment at the lower layer is centrifuged at high speed for standby.
Step two, preparing the BC/CS porous microcarrier
Weighing a certain mass of BC in a 2% (V/V) acetic acid solution to prepare BC suspension slurry with the mass concentration of 2% (W/V); weighing a certain mass of CS and dissolving the CS in 2 percent (V/V) acetic acid solution to prepare CS solution with the mass concentration of 2 percent (W/V). Fully mixing the CS acetic acid solution and the BC acetic acid suspension slurry according to the volume ratio of 1: 1; adding 2ml of emulsifier into 100ml of mixed solution, increasing the rotating speed and stirring the mixed solution at the rotating speed of 500 r/min, wherein the nano-cellulose with the nano-fiber structure can stabilize the particle size and the pore diameter of the microcarrier in the stirring process; placing the liquid formed after stirring for 1h in a low-temperature environment below-150 ℃, and quickly curing and separating the phases; cleaning with low-temperature petroleum ether to obtain solid microspheres; and (3) drying the microspheres in a low-temperature negative-pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa to obtain the BC/CS porous microcarrier.
Step three, obtaining the high-solubility soybean protein
Dissolving low-temperature defatted soybean powder in deionized water, adjusting pH to weak alkalinity with NaOH (2 mol/L), centrifuging at 5000 r/min for 30 min, collecting supernatant, adding a small amount of sulfite until the Solution is turbid, adjusting pH with HCl (2 mol/L) until the Solution is clear, storing at low temperature for 24h to separate out a large amount of Protein, centrifuging at High speed, collecting lower layer precipitate, storing in a low-temperature environment for freezing, and drying at a temperature lower than-20 deg.C and a pressure lower than 100Pa to obtain High-solubility soybean Protein (HS-SP).
Step four, preparing the HS-SP/BC/CS composite bionic porous microcarrier by a dynamic electrostatic deposition method
Preparing the high-solubility soybean protein obtained in the third step into a solution with the concentration of 2%, adjusting the pH to be =7, adding the BC/CS microcarrier obtained in the second step into the solution, stirring the mixture for 4 hours at the rotating speed of 60r/min, wherein in the stirring process, HS-P in the solution and chitosan generate electrostatic attraction to realize dynamic electrostatic adsorption, so that the HS-P is deposited on the surface of the chitosan to form a shell core fiber structure, and placing the microspheres subjected to solid-liquid separation in a low-temperature negative pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa for drying to finally obtain the HS-P/BC/CS porous microcarrier.
Compared with the prior art, the invention has the beneficial effects that:
1. the polyelectrolyte microspheres are formed by compounding the nano-cellulose and the chitosan, and the nano-cellulose with a nano-fiber structure plays a role in stabilizing the particle size and the pore diameter of the porous microcarrier;
2. no cross-linking agent is introduced in the process of synthesizing the porous microcarrier by dynamic electrostatic deposition, so that the synthesis step is simplified, and the toxicity caused by introducing the cross-linking agent is eliminated;
3. the bionic porous microcarrier is prepared by simulating the components and the structure of the extracellular matrix and selecting a natural degradable material, so that the bionic porous microcarrier has good biocompatibility and mechanical property, and can provide a microenvironment similar to the extracellular matrix for the in-vitro proliferation and differentiation of cells.
4. The materials are non-animal-derived natural materials, so that the risk of carrying animal viruses is avoided, and the use is safe. This is an added benefit in this embodiment.
Examples
Step one, deflocculating nano-cellulose:
washing a nano cellulose (BC) membrane with clear water for multiple times to remove impurities on the surface of the membrane, soaking the membrane in 0.02mol/L NaOH solution, boiling in a water bath at 100 ℃ for 3 hours, and removing thalli in a liquid membrane; washing with distilled water for many times until the solution is neutral to obtain a pure BC membrane; the pure nano cellulose membrane is deflocculated under the action of high-speed shearing, and the sediment at the lower layer is centrifuged at high speed for standby.
Step two, preparing the BC/CS porous microcarrier:
weighing a certain mass of BC in a 2% (V/V) acetic acid solution to prepare BC suspension slurry with the mass concentration of 2% (W/V); weighing a certain mass of CS and dissolving the CS in 2 percent (V/V) acetic acid solution to prepare CS solution with the mass concentration of 2 percent (W/V). Fully mixing the CS acetic acid solution and the BC acetic acid suspension slurry according to the volume ratio of 2: 1; adding 2ml of emulsifier into 100ml of mixed solution, increasing the rotating speed and stirring the mixed solution at the rotating speed of 600 r/min, wherein in the stirring process, the nano-cellulose with a nano-fiber structure can stabilize the particle size and the pore diameter of the microcarrier; placing the liquid formed after stirring for 1h in a low-temperature environment below-100 ℃, and quickly curing and separating the phases; cleaning with low-temperature petroleum ether to obtain solid microspheres; and (3) drying the microspheres in a low-temperature negative-pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa to obtain the BC/CS porous microcarrier.
Step three, preparing the Col/BC/CS bionic porous microcarrier by a dynamic electrostatic deposition method:
preparing a 2% (W/V) collagen (Col) solution, adjusting the pH =8, adding the BC/CS microcarrier obtained in the step two into the solution, stirring for 4h at the rotating speed of 100r/min, wherein in the stirring process, electrostatic attraction is generated between the Col in the solution and chitosan to realize dynamic electrostatic adsorption, so that the Col is deposited on the surface of the chitosan to form a shell core fiber structure, and drying the microspheres subjected to solid-liquid separation in a low-temperature negative pressure environment of lower than-20 ℃ and lower than 100Pa to finally obtain the Col/BC/CS porous microcarrier.
Compared with the prior art, the invention has the beneficial effects that:
1. the polyelectrolyte microspheres are formed by compounding the nano-cellulose and the chitosan, and the nano-cellulose with a nano-fiber structure plays a role in stabilizing the particle size and the pore diameter of the porous microcarrier;
2. no cross-linking agent is introduced in the process of synthesizing the porous microcarrier by dynamic electrostatic deposition, so that the synthesis step is simplified, and the toxicity caused by introducing the cross-linking agent is eliminated;
3. the bionic porous microcarrier is prepared by simulating the components and the structure of the extracellular matrix and selecting a natural degradable material, so that the bionic porous microcarrier has good biocompatibility and mechanical property, and can provide a microenvironment similar to the extracellular matrix for the in-vitro proliferation and differentiation of cells.
Claims (3)
1. A dynamic electrostatic deposition compound natural material bionic porous microcarrier is characterized in that the bionic porous microcarrier comprises a porous microcarrier and a protein component coated on the surface of the porous microcarrier;
the bionic porous microcarrier is prepared by compounding nano-cellulose and chitosan, and the protein component is subjected to in-situ dynamic electrostatic deposition;
the mass ratio of chitosan to nano cellulose in the porous microcarrier is 1: 1-5: 1;
the particle size range of the bionic porous microcarrier is 50-1000 μm; the aperture range is 10-100 mu m; the porosity is more than 80%; the mechanical strength is 50 kPa-300 kPa;
the protein component comprises one or more of animal protein, plant protein, polyamino acid or polypeptide;
the bionic porous microcarrier simulates the structure and components of a bionic extracellular matrix through components of nano cellulose, chitosan and protein.
2. The preparation method of the bionic porous microcarrier according to claim 1, characterized by comprising the following steps:
step one, deflocculation of nano-cellulose
Washing the nano cellulose membrane with deionized water; soaking the nano cellulose membrane in NaOH solution, and boiling for 1-10 h in a high-temperature water bath; washing with deionized water for many times until the solution is neutral to obtain a pure nano cellulose membrane; and (3) deflocculating the obtained pure nano cellulose membrane under the action of high-speed shearing, and centrifuging at a high speed to obtain a lower-layer precipitate for later use.
Step two, preparing the nano-cellulose/chitosan porous microcarrier
Stirring the chitosan solution and the nano cellulose pulp at a solute mass ratio of 1: 1-5: 1 at a rotating speed of less than 150 r/min, and uniformly stirring the mixed solution; adding 2-10% of volume fraction emulsifier into the mixed solution, increasing the rotating speed, stirring the mixed solution at the rotating speed of 300-1000 r/min, and stabilizing the particle size and the pore diameter of the microcarrier by the nano-cellulose with the nano-fiber structure in the stirring process; placing the stirred liquid in a low-temperature environment of minus 70 ℃ to minus 150 ℃, and quickly curing and separating the phase; cleaning the microspheres with a low-temperature organic solvent to obtain solid microspheres; and (3) drying the microspheres in a low-temperature negative pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa to obtain the nano-cellulose/chitosan porous microcarrier.
Step three, obtaining a high-solubility protein component:
dispersing the protein component in the solution to dissolve to obtain high-solubility protein for later use;
step four, preparing the high-solubility protein/nano-cellulose/chitosan bionic porous microcarrier by a dynamic electrostatic deposition method
Preparing the high-solubility protein obtained in the third step into a solution with the concentration of 0.5-5%, adjusting the pH value to 6-10, adding the nano-cellulose/chitosan microcarrier obtained in the second step into the solution, controlling the stirring speed to be 20-200 r/min, and stirring for 1-24 h to generate controllable dynamic electrostatic adsorption between the high-solubility protein and chitosan in the solution, so that the porous microspheres with the shell-core fiber structure are formed by self-assembly, placing the microspheres subjected to solid-liquid separation in a low-temperature negative pressure environment at the temperature of lower than-20 ℃ and lower than 100Pa for solvent separation, and finally obtaining the high-solubility protein/nano-cellulose/chitosan porous microcarrier.
3. The method of claim 2, wherein the protein component is divided into a more soluble protein component and a less soluble protein component;
the protein component with higher solubility is directly dispersed in the solution to be dissolved for later use;
and the protein component with low solubility is extracted by physical separation, wherein the physical separation comprises high-speed centrifugation, low-temperature precipitation and freeze drying, and the protein with high solubility is finally obtained.
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