A kind of polypeptide hydrogel, its preparation method and application

Abstract

The invention discloses a preparing method of polypeptide hydrogel. The method includes dissolving polypeptide into a dispersing medium to form a polypeptide solution, and then the polypeptide hydrogel can be formed after several minutes. The polypeptide is formed by n repeated xKEALKEy amino acid sequences, wherein the n is an integer from 2 to 10, the x is a hydrophobic amino acid, the y is a hydrophilic amino acid, the K is lysine, the E is glutamic acid, the A is alanine and the L is leucine. The polypeptide hydrogel and applications thereof are also disclosed by the invention. The preparing method is simple and convenient in operation, components of the polypeptide hydrogel are pure and controllable and the main folding manner of the polypeptide used in the method is an alpha spiral manner, thus avoiding possible safety problems fundamentally. The polypeptide hydrogel prepared by the method can maintain a gel state in a pH range of 5.8-7.6, and is liquid when a pH value is out of the range.

Description

technical field

[0001] The invention belongs to the field of material preparation, and relates to a polypeptide hydrogel, its preparation method and its application in a bionic scaffold material for three-dimensional culture of stem cells, a bionic scaffold material for promoting the repair of defective tissue, and a carrier for controlling the release of growth factors. Background technique

[0002] Hydrogel is a kind of polymer with a three-dimensional network structure that has hydrophilic groups and can be swelled by water but insoluble in water. It can absorb a large amount of water and swell significantly in water, and can continue to maintain its original structure without being dissolved after significant swelling. It can perceive small changes in external stimuli, such as temperature, pH value, ionic strength, electric field, magnetic field, etc., and can respond sensitively to stimuli, usually through swelling or shrinking of the volume. This characteristic of hyd...

Examples

Embodiment 1

[0039] First synthesize a biologically active high-purity polypeptide (powder, its amino acid sequence is xKEALKEyxKEALKEyxKEALKEY, K is lysine, E is glutamic acid, A is alanine, L is leucine, x is glutamic acid, y is leucine).

[0040] The polypeptide can be synthesized by any one of the following two methods:

[0041] i Synthesize by chemical method, that is, use a peptide synthesizer to synthesize a specific sequence of peptides, which can be used after purification;

[0042] ii Through biological production, that is, introducing related genes into Escherichia coli or CHO cells to produce polypeptides with specific sequences, isolating high-yielding cell lines, conducting expanded culture, and obtaining polypeptides after purification.

[0043] The synthesized polypeptide was made into a solution with a concentration of 3mg / mL with deionized water, and then a small amount of alpha-MEM medium solution was added dropwise. After standing for several minutes at 37°C and pH 7, ...

Embodiment 2

[0046] First synthesize a biologically active high-purity polypeptide (powder, its amino acid sequence is xKEALKEYyxKEALKEYyxKEALKEYy, K is lysine, E is glutamic acid, A is alanine, L is leucine, x is histidine , y is valine). Wherein, the synthesis method of the polypeptide is the same as that in Example 1.

[0047] The polypeptide is made into a solution with a concentration of 0.5mg / mL with deionized water, and the mixed solution is injected into the irregularly shaped tissue defect area with a sterile syringe, and in situ self-assembles to form a hydrogel under the induction of interstitial fluid (such as blood) , as a scaffold material to promote the repair of tissue defects.

Embodiment 3

[0049] First synthesize a biologically active high-purity polypeptide (powder, its amino acid sequence is xKEALKEYyxKEALKEYyxKEALKEYy, K is lysine, E is glutamic acid, A is alanine, L is leucine, x is glutamine , y is tyrosine). Wherein, the synthesis method of the polypeptide is the same as that in Example 1.

[0050] Make the polypeptide material into a solution with a concentration of 10mg / mL with deionized water, and then add a certain amount of FITC fluorescently labeled EGF dropwise to make the final concentration 1-10mg / mL, at 37°C and pH 7 After standing for a few minutes, the polypeptide hydrogel gradually formed. image 3 The release situation of 1mg / mLEGF and 10mg / mLEGF in the polypeptide hydrogel is shown (wherein, the curve located at the top shows the release situation of 10mg / mLEGF in the polypeptide hydrogel, and the curve located at the bottom shows the release situation of 1mg / mLEGF The release of mLEGF in the polypeptide hydrogel); Figure 4 On the 21st d...

technical field

[0001] The invention belongs to the field of material preparation, and relates to a polypeptide hydrogel, its preparation method and its application in a bionic scaffold material for three-dimensional culture of stem cells, a bionic scaffold material for promoting the repair of defective tissue, and a carrier for controlling the release of growth factors. Background technique

[0002] Hydrogel is a kind of polymer with a three-dimensional network structure that has hydrophilic groups and can be swelled by water but insoluble in water. It can absorb a large amount of water and swell significantly in water, and can continue to maintain its original structure without being dissolved after significant swelling. It can perceive small changes in external stimuli, such as temperature, pH value, ionic strength, electric field, magnetic field, etc., and can respond sensitively to stimuli, usually through swelling or shrinking of the volume. This characteristic of hyd...

Examples

Embodiment 1

[0039] First synthesize a biologically active high-purity polypeptide (powder, its amino acid sequence is xKEALKEyxKEALKEyxKEALKEY, K is lysine, E is glutamic acid, A is alanine, L is leucine, x is glutamic acid, y is leucine).

[0040] The polypeptide can be synthesized by any one of the following two methods:

[0041] i Synthesize by chemical method, that is, use a peptide synthesizer to synthesize a specific sequence of peptides, which can be used after purification;

[0042] ii Through biological production, that is, introducing related genes into Escherichia coli or CHO cells to produce polypeptides with specific sequences, isolating high-yielding cell lines, conducting expanded culture, and obtaining polypeptides after purification.

[0043] The synthesized polypeptide was made into a solution with a concentration of 3mg / mL with deionized water, and then a small amount of alpha-MEM medium solution was added dropwise. After standing for several minutes at 37°C and pH 7, ...

Embodiment 2

[0046] First synthesize a biologically active high-purity polypeptide (powder, its amino acid sequence is xKEALKEYyxKEALKEYyxKEALKEYy, K is lysine, E is glutamic acid, A is alanine, L is leucine, x is histidine , y is valine). Wherein, the synthesis method of the polypeptide is the same as that in Example 1.

[0047] The polypeptide is made into a solution with a concentration of 0.5mg / mL with deionized water, and the mixed solution is injected into the irregularly shaped tissue defect area with a sterile syringe, and in situ self-assembles to form a hydrogel under the induction of interstitial fluid (such as blood) , as a scaffold material to promote the repair of tissue defects.

Embodiment 3

[0049] First synthesize a biologically active high-purity polypeptide (powder, its amino acid sequence is xKEALKEYyxKEALKEYyxKEALKEYy, K is lysine, E is glutamic acid, A is alanine, L is leucine, x is glutamine , y is tyrosine). Wherein, the synthesis method of the polypeptide is the same as that in Example 1.

[0050] Make the polypeptide material into a solution with a concentration of 10mg / mL with deionized water, and then add a certain amount of FITC fluorescently labeled EGF dropwise to make the final concentration 1-10mg / mL, at 37°C and pH 7 After standing for a few minutes, the polypeptide hydrogel gradually formed. image 3 The release situation of 1mg / mLEGF and 10mg / mLEGF in the polypeptide hydrogel is shown (wherein, the curve located at the top shows the release situation of 10mg / mLEGF in the polypeptide hydrogel, and the curve located at the bottom shows the release situation of 1mg / mLEGF The release of mLEGF in the polypeptide hydrogel); Figure 4 On the 21st d...

Images