CN103993424B - Preparing method of polyurethane-keratin composite nano fiber film - Google Patents

Abstract

The invention relates to a preparation method of a polyurethane-keratin composite nanofiber film, comprising: dissolving polyurethane in a solvent, stirring and dissolving to obtain a polyurethane solution; dissolving keratin in a solvent, stirring and dissolving to obtain a keratin solution; Polyurethane solution and keratin solution are mixed at a mass ratio of 95:5-5:95, stirred and mixed to obtain polyurethane-keratin spinning solution, and then electrospun, and aluminum foil or cloth is used to receive nano-filaments to obtain the product. The polyurethane-keratin composite nanofiber membrane prepared by the invention not only maintains good biological activity of keratin, but also has good toughness and mechanical strength of polyurethane, and is expected to be applied in fields such as medical dressings.

Description

A kind of preparation method of polyurethane-keratin composite nanofiber film

technical field

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

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 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. Medical grade polyurethane is a commercial synthetic polymer material with good flexibility, high mechanical strength, breathable and waterproof, etc. It has been widely used in wound care, cardiology, plastic surgery, vascular and other fields. However, the polyurethane material itself has no biological activity and lacks cell recognition signal sites, which is not conducive to the adhesion and growth of tissue cells.

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 medical dressings, tissue engineering scaffolds and other fields.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing a polyurethane-keratin composite nanofiber membrane. The polyurethane-keratin composite nanofiber membrane prepared by the present invention not only maintains the good biological activity of keratin, but also has the good biological activity of polyurethane. The toughness and mechanical strength are expected to be applied in medical dressings and other fields.

A kind of preparation method of polyurethane-keratin composite nanofiber film of the present invention comprises:

(1) Polyurethane is dissolved in a solvent, stirred and dissolved to obtain a polyurethane solution;

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

(3) Mix polyurethane solution and keratin solution at a mass ratio of 95:5-5:95, stir and mix to obtain polyurethane-keratin spinning solution, then perform electrospinning, and receive nanowires with aluminum foil or cloth , to obtain polyurethane-keratin composite nanofiber membrane.

In described step (1), polyurethane is Tecothane ^™ , Techphilic ^TM , Corthane ^TM , Chronoflex AR, Chronoflex CL, One or more of Elast Eon, Cardiomat, and Avcothane.

The concentration of the polyurethane 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.

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

The solvent of the step (1), (2) is hexafluoroisopropanol, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, dichloromethane, di One or more of ethyl chloride, chloroform, tetrahydrofuran.

The solvent is preferably hexafluoroisopropanol.

In the step (3), other reagents that can be dissolved in the mixed solution system are added to the polyurethane-keratin spinning solution: drugs, inorganic antibacterial agents or organic antibacterial agents, etc.

Electrospinning process parameters in the step (3) are: voltage 13-35kV, receiving distance 8-22cm, spinning rate 0.5-1.5ml/h, spinneret inner diameter 0.7-0.9mm, spinning temperature 20 -30°C, spinning humidity 45-65%.

The polyurethane-keratin composite nanofiber membrane is cross-linked with a cross-linking agent to improve its stability and mechanical strength, and the cross-linking agent is glutaraldehyde, genipin, carbodiimide, divinyl sulfone , formaldehyde, glyoxal or one or more of them.

Beneficial effect

(1) The composite nanofiber membrane prepared by the present invention has a three-dimensional network structure, and the fiber diameter is 50-800nm, which can be used in fields such as wound care;

(2) The polyurethane-keratin composite nanofiber membrane prepared by the present invention not only maintains the good biological activity of keratin, but also possesses the good toughness and mechanical strength of polyurethane, and is expected to be applied in fields such as medical dressings.

detailed description

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, then dialyzed for 36 hours with a dialysis bag with a molecular weight cut-off of 40,000, and 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 polyurethane-keratin composite nanofiber membrane: 0.15 g of polyurethane was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a polyurethane solution with a concentration of 15 mg/ml. 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. Mix the above polyurethane and keratin solution according to the mass ratio of 50:50, and magnetically stir until the mixture is uniform. The polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 15kV, receiving distance 12cm, spinning rate 0.5ml/h, spinneret inner diameter 0.7mm, spinning temperature 25°C, spinning Humidity 50%. The aluminum foil is used as a receiver to receive the nanowires generated by the jet, and a polyurethane-keratin composite nanofiber membrane is 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, then dialyzed with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours, and 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 polyurethane-keratin composite nanofiber membrane: 0.2 g of polyurethane was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a polyurethane solution with a concentration of 20 mg/ml. 0.15 g of solid keratin was dissolved in hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a keratin solution with a concentration of 15 mg/ml. Mix the above-mentioned polyurethane and keratin solution according to the mass ratio of 20:80, and magnetically stir until the mixture is uniform. The polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 13kV, receiving distance 10cm, spinning rate 0.8ml/h, spinneret inner diameter 0.7mm, spinning temperature 30°C, spinning Humidity 65%. The aluminum foil is used as a receiver to receive the nanowires generated by the jet, and a polyurethane-keratin composite nanofiber membrane is 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 liquid 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, then dialyzed with a dialysis bag with a molecular weight cut-off of 8,000 for 36 hours, and 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 polyurethane-keratin composite nanofiber membrane: 0.05 g of polyurethane was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a polyurethane solution with a concentration of 5 mg/ml. 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. Mix the above-mentioned polyurethane and keratin solution according to the mass ratio of 95:5, and magnetically stir until the mixture is uniform. The polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 13kV, receiving distance 8cm, spinning rate 1.5ml/h, spinneret inner diameter 0.9mm, spinning temperature 20°C, spinning Humidity 45%. The aluminum foil is used as a receiver to receive the nanowires generated by the jet, and a polyurethane-keratin composite nanofiber membrane is 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 for 36 hours with a dialysis bag with a molecular weight cut-off of 8,000. 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 polyurethane-keratin composite nanofiber membrane: 0.15 g of polyurethane was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a polyurethane solution with a concentration of 15 mg/ml. 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. Mix the above-mentioned polyurethane and keratin solution according to the mass ratio of 5:95, and magnetically stir until the mixture is uniform. The polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 35kV, receiving distance 22cm, spinning rate 1.5ml/h, spinneret inner diameter 0.9mm, spinning temperature 20°C, spinning Humidity 55%. The cloth is used as a receiver to receive the nanofilaments generated by the jet, and a polyurethane-keratin composite nanofiber membrane is 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 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 treatment, and then put it in a freeze dryer to dry to obtain dry carboxymethyl angle protein powder.

Preparation of polyurethane-keratin composite nanofiber membrane: 0.15 g of polyurethane was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a polyurethane solution with a concentration of 15 mg/ml. 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. Mix the above-mentioned polyurethane and keratin solution according to the mass ratio of 5:95, and magnetically stir until the mixture is uniform. The polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 15kV, receiving distance 12cm, spinning rate 1.0ml/h, spinneret inner diameter 0.7mm, spinning temperature 15°C, spinning Humidity 55%. The aluminum foil is used as a receiver to receive the nanowires generated by the jet, and a polyurethane-keratin composite nanofiber membrane is 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. The above-mentioned human hair was added into a mixed solution of 200ml of water and 5g of sodium metabisulfite, the pH was adjusted to about 10.0 with NaOH, and then reacted 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 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 treatment, and then put it in a freeze dryer to dry to obtain dry carboxymethyl angle protein powder.

Preparation of polyurethane-keratin composite nanofiber membrane: 0.15 g of polyurethane was dissolved in 10 ml of hexafluoroisopropanol, and magnetically stirred at room temperature until completely dissolved to obtain a polyurethane solution with a concentration of 15 mg/ml. 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. Mix the above polyurethane and keratin solution according to the mass ratio of 50:50, and magnetically stir until the mixture is uniform. The polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 15kV, receiving distance 12cm, spinning rate 1.2ml/h, spinneret inner diameter 0.7mm, spinning temperature 15°C, spinning Humidity 55%. The aluminum foil is used as a receiver to receive the nanowires generated by the jet, and a polyurethane-keratin composite nanofiber membrane is obtained.

Example 7

According to the preparation method described in Example 1, the polyurethane/keratin composite nanofiber membrane prepared by mixing Techphilic ^TM polyurethane material with wool keratin was determined by analysis, and the breaking strength of the composite nanofiber membrane in a dry state was 4.5MPa , The elongation at break is 75%, and the water absorption is 900%. Using rat fibroblast L929 and vascular endothelial cells as cell models, the adhesion morphology of cells on the material was observed by scanning electron microscopy, and the proliferation behavior of cells was evaluated by MTT method. The results showed that fibroblasts and endothelial cells were Polyurethane/keratin composite nanofiber membranes showed good adhesion morphology and proliferation behavior, which were significantly different from those of single polyurethane nanofiber membranes. According to the preparation method described in Example 1, the membrane material prepared from a single keratin is highly brittle and easily broken in a dry state.

Claims (1)

1. A preparation method of polyurethane-keratin composite nanofiber membrane, comprising: Use wool as raw material to extract keratin by reduction 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 dry it in air; add the above wool to 100ml, In 1mol/l mercaptoethanol, use NaOH to adjust the pH to about 10.0, and then react at 40°C for 12 hours; after the reaction, collect the reaction solution and record it as filtrate 1; at the same time, filter and collect the wool solid remaining in the reaction, and continue to use 100ml , 0.1mol/l Tris lye was treated at 40°C for 2 hours, then filtered to remove solids, and the filtrate was collected and recorded as filtrate 2; the filtrates 1 and 2 were combined, centrifuged at 6000rpm for 5 minutes, and then filtered with a molecular weight cut-off of 40,000 The dialysis bag was dialyzed for 36 hours, and finally the dialysate was added to liquid nitrogen for quick-freezing treatment, and then placed in a freeze dryer to dry to obtain dry keratin powder; Preparation of polyurethane-keratin composite nanofiber membrane: Dissolve 0.15 g of polyurethane in 10 ml of hexafluoroisopropanol, stir magnetically at room temperature until completely dissolved, and obtain a polyurethane solution with a concentration of 15 mg/ml; dissolve 0.2 g of keratin The protein solid was dissolved in 10ml of hexafluoroisopropanol, and magnetically stirred at room temperature until it was completely dissolved to obtain a keratin solution with a concentration of 20mg/ml; the above polyurethane and keratin solution were mixed according to the mass ratio of 50:50, magnetically Stir until uniformly mixed; the polyurethane/keratin solution obtained above is subjected to electrospinning, the electrospinning parameters are voltage 15kV, receiving distance 12cm, spinning rate 0.5ml/h, spinneret inner diameter 0.7mm, spinning temperature 25°C, spinning humidity 50%; aluminum foil was used as a receiver to receive the nanofilaments produced by jetting, and a polyurethane-keratin composite nanofiber membrane was obtained.