CN101445971A - Method for preparing bionic extracellular matrix silk fibroin/chitosan composite nanometer fibre - Google Patents

Abstract

The invention relates to a preparation method of bionic extracellular matrix silk fibroin/ _chitosan composite nanofibers, comprising: (1) boiling de-chrysalis cocoons in _Na2CO3 solution for three times, washing them, and drying them at 45°C , dissolving it in a ternary solvent, hydrolysis, dialysis, and freeze-drying to obtain loose porous solid silk fibroin; (2) dissolving the above silk fibroin in hexafluoroisopropanol to obtain a silk fibroin solution; ( 3) dissolving chitosan in a mixed solvent of HFIP and TFA to obtain a chitosan solution; (4) mixing the silk fibroin solution and the chitosan solution and stirring evenly to obtain a blended electrospinning solution; (5) ) Electrospinning the mixed solution to obtain silk fibroin/chitosan composite nanofibers. The silk fibroin/chitosan composite fiber of the invention has excellent mechanical properties and degradation behavior, and provides the best bionic physiological environment for cell growth and tissue regeneration; the preparation method is simple and easy to realize industrial production.

Description

Preparation method of biomimetic extracellular matrix silk fibroin/chitosan composite nanofibers

technical field

The invention belongs to the field of preparation of composite nanofibers, in particular to a method for preparing bionic extracellular matrix silk fibroin/chitosan composite nanofibers.

Background technique

The development of tissue engineering and regenerative medicine provides an effective treatment for rebuilding or repairing human diseased tissues and organs. Degradable scaffolds provide the necessary space and support for cell growth and tissue regeneration, and provide effective channels for signal transduction, which is one of the key factors that determine the success of tissue engineering.

Silk fibroin is degummed from silkworms. It is a natural protein with excellent properties such as good biocompatibility, biodegradability, good air permeability, moisture permeability, and non-immunogenicity. It is widely used in the repair and regeneration of skin, bone, cartilage, tendon, nerve conduit, blood vessel and other tissues. Park et al[1] prepared silk fibroin nanofiber membranes by electrospinning with formic acid as a solvent, which can promote the adhesion, proliferation and differentiation of mouse preosteoblasts (MC3T3-E1), induce the production of osteocalcin, and promote bone tissue repair and regeneration, Chinese patent CN1844509A discloses "a preparation method of silk fibroin porous material", which uses water as a solvent to electrospin nanofibers with good biocompatibility and high porosity, but a single silk fibroin nanofiber Brittle and poor flexibility reduce its practical value, and as a tissue engineering scaffold material, the best design should be biomimetic extracellular matrix in terms of structure and composition. The natural extracellular matrix is composed of proteins and polysaccharides with a nanofiber network structure [2]. Adding a single natural component cannot completely simulate the composition of the extracellular matrix, so it cannot achieve functional bionics.

Chitosan is the second largest natural polysaccharide after cellulose in the world. It has good biocompatibility and biodegradability, and has good antibacterial, antithrombotic and non-immunogenic properties. Chitosan and its derivatives, spongy porous scaffolds prepared by blends with other polymers, injectable hydrogels and microsphere scaffolds, and fibrous scaffolds have been used as a variety of different tissues, including bone, liver, etc. , nerve tissue, blood vessels, cartilage tissue and skin and other tissue engineering fields.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing bionic extracellular matrix silk fibroin/chitosan composite nanofibers. The silk fibroin/chitosan composite fibers of the present invention have excellent mechanical properties and degradation behavior, especially The flexibility has been greatly improved; the composite fiber can well simulate the composition and structure of human extracellular matrix, provide the best bionic physiological environment for cell growth and tissue regeneration, and is an ideal tissue engineering scaffold; the preparation The method is simple and easy, the raw material resources are abundant, and the industrialized production can be easily realized.

A kind of preparation method of biomimetic extracellular matrix silk fibroin/chitosan composite nanofiber of the present invention, comprises

(1) Extraction of silk fibroin: Boil the silkworm cocoons without chrysalis in an aqueous solution of 0.5% Na ₂ CO ₃ at 100° C. for 30 minutes each time, wash them thoroughly with distilled water, and dry them at 45° C. The finished silk fibers were dissolved in a ternary solvent with a bath ratio of 1:10, hydrolyzed at a constant temperature of 70°C for 1 hour, then dialyzed with distilled water for 72 hours, then placed in -80°C for 12 hours, and then freeze-dried at -58°C to obtain a loose silk fiber Porous solid silk fibroin;

(2) Preparation of silk fibroin solution: dissolve the above silk fibroin in hexafluoroisopropanol, stir at room temperature until completely dissolved, and obtain a silk fibroin solution with a concentration of 4-10% (g/ml);

(3) Prepare chitosan solution: dissolve chitosan in a mixed solvent of hexafluoroisopropanol (HFIP) and trifluoroacetic acid (TFA), stir until transparent, and obtain a concentration of 4-8% (g/ml ) chitosan solution;

(4) Prepare a mixed solution of silk fibroin and chitosan: mix the silk fibroin solution and the chitosan solution at a volume ratio of 0.15-2.4:1, and stir evenly to obtain a blended electrospinning solution;

(5) Electrospin the silk fibroin and chitosan mixed solution, adjust the electrospinning process parameters, control the voltage to 12-30 kV, the electric field distance is 60-200 mm, and the spinning rate is 0.5-1.5 ml / hour to obtain silk fibroin/chitosan composite nanofibers.

The ternary solvent in the step (1) is a mixture of CaCl ₂ , C ₂ H ₅ OH and H ₂ O, wherein the molar ratio of CaCl ₂ , C ₂ H ₅ OH and H ₂ O is 1:2:8 ;

The mixed solvent of hexafluoroisopropanol HFIP and trifluoroacetic acid TFA in described step (3), wherein the volume ratio of hexafluoroisopropanol HFIP and trifluoroacetic acid TFA is 9:1;

The silk fibroin in the step (4) accounts for 10%-80% of the total mass fraction of the solute, and chitosan accounts for 20%-90% of the total mass fraction of the solute;

Described solute is silk fibroin and chitosan;

The diameter of the silk fibroin/chitosan composite nanofiber increases gradually with the increase of the concentration of the spinning solution, and decreases gradually with the increase of the chitosan content.

The silk fibroin/chitosan composite fiber of the invention can be applied to the research of tissue engineering in the field of biomedicine.

Beneficial effect

(1) The mechanical properties and degradation behavior of the silk fibroin/chitosan composite fiber of the present invention are excellent, especially the flexibility has been greatly improved;

(2) The composite fiber can well simulate the composition and structure of human extracellular matrix, provide the best bionic physiological environment for cell growth and tissue regeneration, and is an ideal tissue engineering scaffold;

(3) The preparation method is simple and easy, the raw material resources are abundant, and industrial production is easy to realize.

Description of drawings

Fig. 1 is the scanning electron micrograph of silk fibroin/chitosan blending electrospun nanofiber;

Fig. 2 is a photograph of silk fibroin/chitosan blended electrospun nanofiber membrane.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

First add 100 grams of silkworm cocoons to 1 liter of 0.5% Na ₂ CO ₃ aqueous solution, boil for 30 minutes, repeat the treatment three times, wash them thoroughly with distilled water, put them in a drying oven at 45°C and dry them to obtain degummed silk fiber. A ternary solvent was prepared with a molar ratio of CaCl ₂ :C ₂ H ₅ OH:H ₂ O=1:2:8, and the silk fiber was hydrolyzed in a water bath at 70°C for 1 hour at a constant temperature with a bath ratio of 1:10. Obtain the completely dissolved brown-yellow silk fiber hydrolysis solution. Put the hydrolyzed solution into a dialysis bag, dialyze with distilled water for 72 hours, put the dialyzed silk fiber hydrolyzed solution into -80°C for 12 hours, and then freeze-dry it at -58°C until dry to obtain a white, odorless, loose porous solid silk fibroin.

Example 2

Dissolve 1.0 gram of silk fibroin in 10 milliliters of hexafluoroisopropanol, stir until fully dissolved to obtain a silk fibroin solution with a concentration of 10% (g/ml); 0.6 gram of chitosan is dissolved in 10 milliliters of In the hexafluoroisopropanol and the trifluoroacetic acid mixed solvent that ratio 90:10 mixes, stir until dissolving completely, obtain the chitosan solution that concentration is 6% (gram/milliliter); Two kinds of solutions are with volume ratio 12:5, The mass ratio of solute is mixed at 4:1, and the mixed solution is subjected to electrospinning, spinning conditions: voltage, 20 kV; electric field distance, 130 mm; spinning rate, 80 ml/hour, to obtain an average fiber diameter of 305 nanometers Composite nanofiber membrane.

Example 3

Take by weighing 1.0 grams of silk fibroin prepared in Example 1, dissolve in 10 milliliters of hexafluoroisopropanol, stir until completely dissolved, and obtain a silk fibroin solution with a concentration of 10% (g/ml); Chitosan was dissolved in 10 milliliters of hexafluoroisopropanol and trifluoroacetic acid mixed solvent with a volume ratio of 90:10, stirred until fully dissolved to obtain a chitosan solution with a concentration of 6% (g/ml); the two The two solutions were mixed at a volume ratio of 3:5 and a solute mass ratio of 1:1, and the mixed solution was subjected to electrospinning, spinning conditions: voltage, 20 kV; electric field distance, 130 mm; spinning rate, 80 ml/hour , to obtain a composite nanofibrous membrane with an average fiber diameter of 202 nm.

Example 4

Take by weighing 1.0 grams of silk fibroin prepared in Example 1, dissolve in 10 milliliters of hexafluoroisopropanol, stir until completely dissolved, and obtain a silk fibroin solution with a concentration of 10% (g/ml); Chitosan was dissolved in 10 milliliters of hexafluoroisopropanol and trifluoroacetic acid mixed solvent with a volume ratio of 90:10, stirred until fully dissolved to obtain a chitosan solution with a concentration of 6% (g/ml); the two The two solutions are mixed at a volume ratio of 3:20 and a solute mass ratio of 1:4, and the mixed solution is electrospun, and the spinning conditions are: voltage, 20 kV; electric field distance, 130 mm; spinning rate, 80 ml/hour , to obtain a composite nanofibrous membrane with an average fiber diameter of 148 nm.

Example 5

Take by weighing 0.8 grams of silk fibroin prepared in Example 1, dissolve in 10 milliliters of hexafluoroisopropanol, stir until completely dissolved, and obtain a silk fibroin solution with a concentration of 8% (g/ml); Chitosan was dissolved in 10 milliliters of hexafluoroisopropanol and trifluoroacetic acid mixed solvent mixed with a volume ratio of 90:10, stirred until completely dissolving, obtaining a chitosan solution with a concentration of 8% (g/ml); The two solutions are mixed at a volume ratio of 1:1 and a solute mass ratio of 1:1, and the mixed solution is subjected to electrospinning, spinning conditions: voltage, 20 kV; electric field distance, 130 mm; spinning rate, 80 ml/hour , to obtain a composite nanofibrous membrane with an average fiber diameter of 102 nm.

Claims (7)

1. A preparation method of biomimetic extracellular matrix silk fibroin/chitosan composite nanofiber, comprising (1) Extraction of silk fibroin: Boil the silkworm cocoons without chrysalis in an aqueous solution of 0.5% Na ₂ CO ₃ at 100° C. for 30 minutes each time, wash them thoroughly with distilled water, and dry them at 45° C. The finished silk fibers were dissolved in a ternary solvent with a bath ratio of 1:10, hydrolyzed at a constant temperature of 70°C for 1 hour, then dialyzed with distilled water for 72 hours, then placed in -80°C for 12 hours, and then freeze-dried at -58°C to obtain a loose silk fiber Porous solid silk fibroin; (2) Preparation of silk fibroin solution: dissolve the above silk fibroin in hexafluoroisopropanol, stir at room temperature until completely dissolved, and obtain a silk fibroin solution with a concentration of 4-10% g/ml; (3) Prepare chitosan solution: dissolve chitosan in a mixed solvent of hexafluoroisopropanol HFIP and trifluoroacetic acid TFA, stir until transparent, and obtain a chitosan solution with a concentration of 4-8% g/ml ; (4) Prepare a mixed solution of silk fibroin and chitosan: mix the silk fibroin solution and the chitosan solution at a volume ratio of 0.15-2.4:1, and stir evenly to obtain a blended electrospinning solution; (5) Electrospin the silk fibroin and chitosan mixed solution, adjust the electrospinning process parameters, control the voltage to 12-30 kV, the electric field distance is 60-200 mm, and the spinning rate is 0.5-1.5 ml / hour to obtain silk fibroin/chitosan composite nanofibers. 2. A method for preparing biomimetic extracellular matrix silk fibroin/chitosan composite nanofibers according to claim 1, characterized in that: the ternary solvent in the step (1) is CaCl ₂ , C ₂ A mixture of H ₅ OH and H ₂ O, wherein the molar ratio of CaCl ₂ , C ₂ H ₅ OH and H ₂ O is 1:2:8. 3. the preparation method of a kind of biomimetic extracellular matrix silk fibroin/chitosan composite nanofiber according to claim 1 is characterized in that: hexafluoroisopropanol HFIP and trifluoroisopropanol in described step (3) A mixed solvent of acetic acid TFA, wherein the volume ratio of hexafluoroisopropanol HFIP and trifluoroacetic acid TFA is 9:1. 4. the preparation method of a kind of biomimetic extracellular matrix silk fibroin/chitosan composite nanofiber according to claim 1, is characterized in that: the silk fibroin in the described step (4) accounts for 10% of the total solute mass fraction 10%-80%, chitosan accounts for 20%-90% of the total mass fraction of the solute. 5 . The method for preparing a biomimetic extracellular matrix silk fibroin/chitosan composite nanofiber according to claim 4 , wherein the solute is silk fibroin and chitosan. 6. the preparation method of a kind of biomimetic extracellular matrix silk fibroin/chitosan composite nanofiber according to claim 1, is characterized in that: the diameter of described silk fibroin/chitosan composite nanofiber varies with spinning The concentration of silk solution increased gradually, and gradually decreased with the increase of chitosan content. 7. A method for preparing biomimetic extracellular matrix silk fibroin/chitosan composite nanofibers according to claim 1, characterized in that: said silk fibroin/chitosan composite fibers can be applied to biomedicine Research in the field of tissue engineering.

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