CN104005179A - Method for preparing polycaprolactone-keratin composite nanometer fiber pipe - Google Patents

Abstract

The invention relates to a preparation method of a polycaprolactone-keratin composite nanofiber tube, comprising: dissolving polycaprolactone in a solvent, stirring and dissolving to obtain a polycaprolactone solution; dissolving keratin in a solvent, Stir and dissolve to obtain a keratin solution; mix the polycaprolactone solution and the keratin solution at a mass ratio of 95:5-5:95, stir and mix to obtain a spinning solution, and then carry out electrospinning and spin in a cylindrical shape The collection roller was used as a receiver to obtain polycaprolactone-keratin composite nanofiber tubes. The tubular material obtained by the invention not only maintains good biological activity of keratin, but also has good toughness and mechanical strength of polycaprolactone, and has the advantages of adjustable inner diameter and simple preparation method, and is expected to be used in artificial blood vessels and other fields.

Description

A kind of preparation method of polycaprolactone-keratin composite nanofiber tube

technical field

The invention belongs to the field of preparation of nanofiber tubes, in particular to a preparation method of polycaprolactone-keratin composite nanofiber tubes.

Background technique

Keratin is an insoluble fibrous animal protein, a structural protein of ectodermal cells, widely present in animal skin and skin appendages, such as hair, hooves, shells, claws, horns, scales, etc. A large number of studies in recent years have shown that keratin is a high-quality biomedical material with good biocompatibility and not rejected by the body's immune system, and has broad application prospects. Most prominently, the amino acid sequence of keratin derived from wool and other sources was found to contain the same Arg-Gly-Asp (RGD) tripeptide sequence. This tripeptide sequence is recognized as an effective binding site for cell binding in the extracellular matrix, and has the function of promoting cell adsorption. Therefore, a large number of basic research and animal experiment research on keratin-based biomaterials have been carried out at home and abroad, and have been widely used in wound dressings [Wound Repair and Regeneration, 2012, 20: 236-242], artificial bone [Journal of Bioactive and Compatible Polymers, 2013,28:141-153] and nerve repair [Biomaterials34 (2013) 5907-5914] have achieved good results, and some products have been used clinically.

However, existing studies have shown that due to the low molecular weight of keratin, the membrane material made of a single keratin material is usually brittle and has low mechanical strength, which limits its application. Therefore, at present, most keratin-based biomaterials often use keratin and natural polymers or artificial polymers, which can maintain and improve the poor mechanical properties of single keratin, such as silk fibroin [Biomacromolecules, 2008 ,9,1299–1305], PVA[Advances in Materials Science and Engineering,2014,Article ID163678], PLGA[Journal of Bioactive and Compatible Polymers,2013,28:141-153], PLLA[Biomed.Mater.2013,8:1 -9] etc. Polycaprolactone (PCL) is a commercial medical polymer material with good biocompatibility and degradability. It has been approved by the US Food and Drug Administration and is widely used in drug carriers, tissue engineering materials, etc. biomedical field. However, the polycaprolactone material itself has no biological activity and lacks cell recognition signal sites, which is not conducive to the adhesion and growth of tissue cells such as endothelial cells.

Accordingly, the present invention proposes to compound keratin and polycaprolactone, and prepare nanofibrous tubular materials by electrospinning, in order to improve the low mechanical properties of single keratin fiber materials. Electrospinning technology refers to the use of a high-voltage electric field environment to form a charged jet flow from the polymer spinning solution. The jet flow is elongated under the action of the electric field, the solvent is volatilized, and finally nanofibers of a certain shape are formed on the receiving device. In the past ten years, this technology has become one of the effective ways to prepare nanofiber materials. The obtained nanofibers have high porosity and controllable shape, and are widely used in the field of membrane and tubular tissue engineering materials.

Contents of the invention

The technical problem to be solved by the present invention is to provide a preparation method of polycaprolactone-keratin composite nanofiber tube. Toughness and mechanical strength, as well as the advantages of adjustable inner diameter and simple preparation method, are expected to be used in artificial blood vessels and other fields.

A kind of preparation method of polycaprolactone-keratin composite nanofiber tube of the present invention comprises:

(1) dissolving polycaprolactone in a solvent, stirring and dissolving to obtain a polycaprolactone solution;

(2) dissolving keratin in a solvent, stirring and dissolving to obtain a keratin solution;

(3) Mix polycaprolactone solution and keratin solution at a mass ratio of 95:5-5:95, stir and mix to obtain spinning solution (polycaprolactone/keratin mixed solution), and then perform electrospinning Filament, and the cylindrical collection roller as a receiver, to obtain polycaprolactone-keratin composite nanofiber tubes.

In the described step (1), the solvent is hexafluoroisopropanol, formic acid, acetic acid, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, dichloromethane, di One or more of ethyl chloride, chloroform, dimethyl sulfoxide, and tetrahydrofuran. Hexafluoroisopropanol is preferred.

The concentration of the polycaprolactone solution in the step (1) is 5-20 mg/ml.

The molecular weight of keratin in the step (2) is 3-300kDa.

The keratin can be keratin extracted and prepared by various methods reported so far, including reduction method, oxidation method and hydrolysis method, etc., and can also be a derivative of the above-mentioned keratin, such as carboxymethyl keratin.

The keratin can be extracted from human or animal hairs such as human hair, wool, poultry feathers, cow hair, or other reported animal bodies.

In the step (2), the solvent is hexafluoroisopropanol, formic acid, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dichloromethane, di One or more of ethyl chloride, chloroform, dimethyl sulfoxide, and tetrahydrofuran. Hexafluoroisopropanol is preferred.

The concentration of the keratin solution in the step (2) is 10-20 mg/ml.

The mass percent concentration of the spinning solution in the step (3) is 5-20%.

The polycaprolactone/keratin mixed solution in the step (3) can be added with other substances that are conducive to improving and improving its application performance, including drugs, inorganic or organic antibacterial agents, anticoagulants such as heparin, and the like.

The electrospinning process parameters in the step (3) are a voltage of 13-35kV, a receiving distance of 8-22cm, a spinning rate of 0.3-1.5ml/h, a collection roller rotation rate of 60-130rpm, and an inner diameter of a spinneret hole of 0.7- 0.9mm, spinning temperature 20-30℃, spinning humidity 45-65%.

The diameter of the cylindrical collection roller in the step (3) can be 0.6-2 cm, or be adjusted according to the inner diameter of the required tubular material; the axis of the collection roller is perpendicular to the opening direction of the spinning nozzle and rotates at a constant speed , the speed is 60-130rpm.

Beneficial effect

The tubular material obtained by the invention not only maintains good biological activity of keratin, but also has good toughness and mechanical strength of polycaprolactone, and has the advantages of adjustable inner diameter, good biocompatibility, degradability, and simple preparation method. , is expected to be used in artificial blood vessels and other fields.

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

The keratin was extracted from wool by reduction method. The specific method was: weigh 5g of wool, use petroleum ether as solvent to remove the oil on the surface of wool by Soxhlet extraction, then wash the wool with ethanol and air-dry it. Add the above-mentioned wool into 100ml, 1mol/l mercaptoethanol, adjust the pH to about 10.0 with NaOH, and then react at 40°C for 12h. After the reaction was finished, the reaction solution was collected (referred to as filtrate 1), and the wool solid remaining in the reaction was collected by filtration, and continued to be treated with 100ml, 0.1mol/l Tris lye at 40°C for 2h, and then filtered to remove the solid. The filtrate was collected (designated as filtrate 2). .The filtrates 1 and 2 were combined, centrifuged at 6000rpm for 5 minutes, and then dialyzed with a dialysis bag with a molecular weight cut-off of 40,000 for 36 hours. Finally, the dialysate was added to liquid nitrogen for quick freezing, and then dried in a freeze dryer to obtain dry keratin powder .

Preparation of polycaprolactone-keratin composite nanofiber membrane: dissolve polycaprolactone with a mass of 0.15 g in 10 ml hexafluoroisopropanol, and stir magnetically at room temperature until completely dissolved to obtain a polycaprolactone with a concentration of 15 mg/ml. caprolactone solution. 0.2 g of solid keratin was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a keratin solution with a concentration of 20 mg/ml. The above polycaprolactone and keratin solution were mixed according to the mass ratio of 50:50, the total concentration of the spinning solution was 15%, and magnetically stirred until uniformly mixed. The polycaprolactone/keratin solution obtained above was subjected to electrospinning, the electrospinning parameters were voltage 15kV, receiving distance 12cm, spinning rate 0.5ml/h, spinneret inner diameter 0.7mm, spinning temperature 25°C , Spinning humidity 50%. A cylindrical collection roller with a diameter of 1.0 cm was used as a receiver to receive the nanofilaments generated by the jet. The rotation speed of the collection roller was 100 rpm. After 4 hours of collection, the polycaprolactone-keratin composite nanofiber tubular material was obtained.

Example 2

Using human hair as raw material to extract keratin by reduction method, the specific method is: weigh 5g of human hair, use petroleum ether as solvent to remove human hair surface oil by Soxhlet extraction, then wash human hair with ethanol and air-dry. Add the above-mentioned human hair into a mixed solution of 200ml of water and 5g of sodium metabisulfite, adjust the pH to about 10.0 with NaOH, and then react at 60°C for 5h. After the reaction, collect the reaction solution (referred to as filtrate 1), and collect the remaining human hair solid by filtration at the same time, continue to treat it with 100ml, 0.1mol/l Tris lye at 40°C for 2h, and then filter to remove the solid , collect the filtrate (referred to as filtrate 2). .The filtrates 1 and 2 were combined, centrifuged at 6000rpm for 5 minutes, and then dialyzed with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours. Finally, the dialysate was added to liquid nitrogen for quick freezing, and then dried in a freeze dryer to obtain dry keratin powder .

Preparation of polycaprolactone-keratin composite nanofiber membrane: Dissolve polycaprolactone with a mass of 0.2 g in 10 ml formic acid, stir magnetically at room temperature until completely dissolved, and obtain polycaprolactone with a concentration of 20 mg/ml solution. 0.15 g of solid keratin was dissolved in formic acid, and magnetically stirred at room temperature until completely dissolved to obtain a keratin solution with a concentration of 15 mg/ml. The above polycaprolactone and keratin solution were mixed according to the mass ratio of 20:80, the total concentration of the spinning solution was 10%, and magnetically stirred until uniformly mixed. The polycaprolactone/keratin solution obtained above was subjected to electrospinning, the electrospinning parameters were voltage 13kV, receiving distance 10cm, spinning rate 0.8ml/h, spinneret inner diameter 0.7mm, spinning temperature 30°C , Spinning humidity 65%. A cylindrical collection roller with a diameter of 0.6 cm was used as a receiver to receive the nanofilaments produced by the jet. The rotation speed of the collection roller was 60 rpm. After 4 hours of collection, the polycaprolactone-keratin composite nanofiber tubular material was obtained.

Example 3

The keratin is extracted from wool by oxidation method. The specific method is: weigh 5g of wool, use petroleum ether as solvent to remove the oil on the surface of wool by Soxhlet extraction, then wash the wool with ethanol and air-dry it. 100ml of deionized water and 5ml of 30% hydrogen peroxide were added to the wool, and then reacted at 100°C for 2h. After the reaction was finished, the reaction solution was collected (referred to as filtrate 1), and the wool solid remaining in the reaction was collected by filtration, and continued to be treated with 100ml, 0.1mol/l Tris lye at 40°C for 2h, and then filtered to remove the solid. The filtrate was collected (designated as filtrate 2). .The filtrates 1 and 2 were combined, centrifuged at 6000rpm for 5 minutes, and then dialyzed with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours. Finally, the dialysate was added to liquid nitrogen for quick freezing, and then dried in a freeze dryer to obtain dry horn protein powder.

Preparation of polycaprolactone-keratin composite nanofiber membrane: Dissolve polycaprolactone with a mass of 0.05 g in 10 ml hexafluoroisopropanol, stir magnetically at room temperature until completely dissolved, and obtain a polycaprolactone with a concentration of 5 mg/ml Polycaprolactone solution. 0.1 g of solid keratin was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a keratin solution with a concentration of 10 mg/ml. The above polycaprolactone and keratin solution were mixed according to the mass ratio of 95:5, the total concentration of the spinning solution was 5%, and magnetically stirred until uniformly mixed. The polycaprolactone/keratin solution obtained above was subjected to electrospinning, the electrospinning parameters were voltage 13kV, receiving distance 8cm, spinning rate 1.5ml/h, spinneret inner diameter 0.9mm, spinning temperature 20°C , Spinning humidity 45%. A cylindrical collection roller with a diameter of 1.5 cm was used as a receiver to receive the nanofilaments generated by the jet. The rotation speed of the collection roller was 110 rpm. After 3 hours of collection, the polycaprolactone-keratin composite nanofiber tubular material was obtained.

Example 4

The keratin is extracted from wool by oxidation method. The specific method is: weigh 5g of wool, use petroleum ether as solvent to remove the oil on the surface of wool by Soxhlet extraction, then wash the wool with ethanol and air-dry it. Add 100 ml of 4% peroxyacetic acid to the above wool, and then react at 60° C. for 5 h. After the reaction was finished, the reaction solution was collected (referred to as filtrate 1), and the wool solid remaining in the reaction was collected by filtration, and continued to be treated with 100ml, 0.1mol/l Tris lye at 40°C for 2h, and then filtered to remove the solid. The filtrate was collected (designated as filtrate 2). .The filtrates 1 and 2 were combined, centrifuged at 6000rpm for 5 minutes, and then dialyzed with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours. Finally, the dialysate was added to liquid nitrogen for quick freezing, and then dried in a freeze dryer to obtain dry keratin powder .

Preparation of polycaprolactone-keratin composite nanofiber membrane: Dissolve polycaprolactone with a mass of 0.15 g in 10 ml formic acid, stir magnetically at room temperature until completely dissolved, and obtain polycaprolactone with a concentration of 15 mg/ml solution. 0.2 g of solid keratin was dissolved in 10 ml of formic acid, and magnetically stirred at room temperature until completely dissolved to obtain a keratin solution with a concentration of 20 mg/ml. The above polycaprolactone and keratin solution were mixed according to the mass ratio of 5:95, the total concentration of the spinning solution was 20%, and magnetically stirred until uniformly mixed. The polycaprolactone/keratin solution obtained above was subjected to electrospinning, the electrospinning parameters were voltage 35kV, receiving distance 22cm, spinning rate 1.5ml/h, spinneret inner diameter 0.9mm, spinning temperature 20°C , Spinning humidity 55%. A cylindrical collection roller with a diameter of 1.0 cm was used as a receiver to receive the nanofilaments generated by the jet. The rotation speed of the collection roller was 130 rpm. After 3 hours of collection, the polycaprolactone-keratin composite nanofiber tubular material was obtained.

Example 5

The wool is used as raw material to extract keratin by reduction method, and the keratin is derivatized and modified. The specific method is as follows: take 5 g of wool, use petroleum ether as a solvent to remove the oil on the surface of the wool by Soxhlet extraction, then wash the wool with ethanol and air-dry it. Add the above-mentioned wool into 100ml, 1mol/l mercaptoethanol, adjust the pH to about 10.0 with NaOH, and then react at 40°C for 12h. Then, the pH of the above reaction solution was adjusted to 7.5 with acetic acid, 100 ml of 0.33 mol/l NaBrO3 solution was added thereto, and stirring was continued for 24 h at room temperature. After the reaction, remove the solid matter by filtration, collect the filtrate and dialyze it with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours, and finally add the dialysate to liquid nitrogen for quick freezing, and then dry it in a freeze dryer to obtain dry carboxymethyl keratin powder .

Preparation of polycaprolactone-keratin composite nanofiber membrane: Dissolve polycaprolactone with a mass of 0.15 g in 10 ml hexafluoroisopropanol, and stir magnetically at room temperature until it is completely dissolved to obtain a polycaprolactone with a concentration of 15 mg/ml. Polycaprolactone solution. 0.1 g of solid carboxymethyl keratin was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a carboxymethyl keratin solution with a concentration of 10 mg/ml. The above polycaprolactone and keratin solution were mixed according to the mass ratio of 5:95, and magnetically stirred until the mixture was uniform. The polycaprolactone/keratin solution obtained above is electrospun, the total concentration of the spinning solution is 15%, the electrospinning parameters are a voltage of 15kV, a receiving distance of 12cm, a spinning rate of 1.0ml/h, and a spinneret hole The inner diameter is 0.7mm, the spinning temperature is 15°C, and the spinning humidity is 55%. A cylindrical collection roller with a diameter of 1.0 cm was used as a receiver to receive the nanofilaments generated by the jet. The rotation speed of the collection roller was 100 rpm. After 4 hours of collection, the polycaprolactone-keratin composite nanofiber tubular material was obtained.

Example 6

Human hair is used as raw material to extract keratin by reduction method, and the keratin is derivatized and modified. The specific method is as follows: 5g of human hair is weighed, oil on the surface of the human hair is removed by Soxhlet extraction with petroleum ether, then the human hair is washed with ethanol and air-dried. Add the above-mentioned human hair into a mixed solution of 200ml of water and 5g of sodium metabisulfite, adjust the pH to about 10.0 with NaOH, and then react at 60°C for 5h. Then, the pH of the above reaction solution was adjusted to 8.5 with acetic acid, 8.0 g of iodoacetic acid was added thereto, and stirring was continued for 6 h at room temperature. After the reaction, remove the solid matter by filtration, collect the filtrate and dialyze it with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours, and finally add the dialysate to liquid nitrogen for quick freezing, and then dry it in a freeze dryer to obtain dry carboxymethyl keratin powder .

Preparation of polycaprolactone-keratin composite nanofiber membrane: dissolve polycaprolactone with a mass of 0.15 g in 10 ml hexafluoroisopropanol, and stir magnetically at room temperature until completely dissolved to obtain a polycaprolactone with a concentration of 15 mg/ml. caprolactone solution. 0.2 g of carboxymethyl keratin solid was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a carboxymethyl keratin solution with a concentration of 20 mg/ml. The above polycaprolactone and keratin solution were mixed according to the mass ratio of 50:50, the total concentration of the spinning solution was 15%, and magnetically stirred until uniformly mixed. The polycaprolactone/keratin solution obtained above was subjected to electrospinning, the electrospinning parameters were voltage 15kV, receiving distance 12cm, spinning rate 1.2ml/h, spinneret inner diameter 0.7mm, spinning temperature 15°C , Spinning humidity 55%. A cylindrical collection roller with a diameter of 1.0 cm was used as a receiver to receive the nanofilaments generated by the jet. The rotation speed of the collection roller was 100 rpm. After 4 hours of collection, the polycaprolactone-keratin composite nanofiber tubular material was obtained.

Example 7

Prepare the polycaprolactone-keratin composite nanofiber tubular material according to the preparation method described in Example 1. After analysis, the composite nanofiber material has a breaking strength of 1.35MPa in a dry state and an elongation at break of 60%. , Porosity 91%. Taking vascular endothelial cells as the cell model, the adhesion morphology of cells on the material was observed by scanning electron microscope, and the proliferation behavior of cells was evaluated by MTT method. The results showed that endothelial cells showed good adhesion morphology on the composite nanofiber material. And proliferation behavior, compared with single polycaprolactone nanofibrous material, there are significant differences. According to the preparation method described in Example 1, the nanofibrous material prepared from a single keratin is highly brittle and easily broken in a dry state.

Claims (9)

1. a preparation method for polycaprolactone-keratin composite nano fiber pipe, comprising:

(1) polycaprolactone is dissolved in solvent, stirring and dissolving, obtains polycaprolactone solution;

(2) keratin is dissolved in solvent, stirring and dissolving, obtains keratin solution;

(3) by polycaprolactone solution and keratin solution in mass ratio for 95:5-5:95 mixes, stir and evenly mix, obtain spinning solution, then carry out electrostatic spinning, and taking columniform collecting drum as receiver, obtain polycaprolactone-keratin composite nano fiber pipe.

2. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (1), (2), solvent is hexafluoroisopropanol, formic acid, acetic acid, N, one or more in dinethylformamide, DMA, 1-METHYLPYRROLIDONE, carrene, dichloroethanes, chloroform, dimethyl sulfoxide (DMSO), oxolane.

3. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 2, is characterized in that:

Described solvent is hexafluoroisopropanol.

4. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (1), polycaprolactone solution concentration is 5-20mg/ml.

5. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (2), keratic molecular weight is 3-300kDa.

6. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (2), keratin solution concentration is 10-20mg/ml.

7. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (3), the mass percentage concentration of spinning solution is 5-20%.

8. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (3), electrostatic spinning process parameter is voltage 13-35kV, receiving range 8-22cm, and spinning speed is 0.3-1.5ml/h, collecting drum slewing rate 60-130rpm, spinneret orifice internal diameter is 0.7-0.9mm, spinning temperature 20-30 DEG C, spinning humidity 45-65%.

9. the preparation method of a kind of polycaprolactone-keratin composite nano fiber pipe according to claim 1, is characterized in that:

In described step (3), columniform collecting drum diameter dimension is 0.6-2 centimetre; The axle of collecting drum is perpendicular to spinning-nozzle opening direction, and does uniform rotation.