阅读说明：本技术 氨基酸基聚酯氨静电纺纳米纤维组织工程皮肤支架的制备方法 (Preparation method of amino acid-based polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold ) 是由 吴德群 李梦娜 仇威王 王倩 韩华 李发学 王学利 俞建勇 于 2019-09-02 设计创作，主要内容包括：本发明公开了一种氨基酸基聚酯氨静电纺纳米纤维组织工程皮肤支架的制备方法,其特征在于,合成氨基酸基二胺、对二硝基苯活性酯；将氨基酸基二胺与对二硝基苯活性酯两种单体进行溶液缩聚,得到氨基酸基聚酯氨聚合物；将氨基酸基聚酯氨聚合物进行静电纺得到氨基酸基聚酯氨静电纺纳米纤维组织工程皮肤支架。本发明不仅具有稳定的形态,均匀的直径分布,还具备优良的生物相容性,且方法简单易行,绿色环保,可大规模生产。在伤口抗菌、伤口修复等生物医用领域具有潜在的应用。(the invention discloses a preparation method of amino acid base polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold, which is characterized in that amino acid base diamine and p-dinitrobenzene active ester are synthesized; carrying out solution polycondensation on two monomers, namely amino acid-based diamine and p-dinitrobenzene active ester to obtain an amino acid-based polyester ammonia polymer; and (3) carrying out electrostatic spinning on the amino acid-based polyester ammonia polymer to obtain the amino acid-based polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold. The invention has stable shape, uniform diameter distribution and excellent biocompatibility, and the method is simple and easy to implement, green and environment-friendly and can be used for large-scale production. Has potential application in the biomedical fields of wound antibiosis, wound repair and the like.)

1. A preparation method of an amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold is characterized by comprising the following steps:

Step 1): synthesizing amino acid-based diamine, and obtaining the amino acid-based diamine through esterification reaction of tryptophan and diol;

Step 2): reacting diacyl chloride with p-nitrophenol to obtain p-dinitrobenzene active ester;

Step 3): carrying out solution polycondensation on two monomers, namely amino acid-based diamine and p-dinitrobenzene active ester to obtain an amino acid-based polyester ammonia polymer;

Step 4): and (3) carrying out electrostatic spinning on the amino acid-based polyester ammonia polymer to obtain the amino acid-based polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold.

2. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold as claimed in claim 1, wherein the step 1) is specifically as follows: directly mixing L-tryptophan, diol and p-toluenesulfonic acid, and placing the mixture in a container filled with toluene; then heating the solid-liquid reaction mixture to 70-130 ℃, and keeping refluxing for 16-24 hours; cooling the reaction mixture to room temperature, removing toluene, repeatedly dissolving and precipitating in deionized water for three times to purify the product, filtering and collecting white substances, drying in vacuum, sealing and storing.

3. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold as claimed in claim 2, wherein the diol is any one of chain aliphatic diols with 3-8 carbon atoms; the feeding molar ratio of the glycol to the L-tryptophan to the p-toluenesulfonic acid is 1: (2-4): (4-6); the volume of toluene is 10-30 times the volume of the solid.

4. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold as claimed in claim 1, wherein the step 2) is specifically as follows: dissolving p-nitrophenol in an organic solvent, adding a catalyst, dropwise adding diacyl chloride, reacting overnight under the action of mechanical stirring, filtering, drying to obtain a needle-shaped off-white solid, sealing, and storing in a drying dish.

5. The method for preparing the amino acid based polyesteramide electrospun nanofiber tissue engineering skin scaffold of claim 4, wherein the organic solvent is acetone, DMF, DMAC, THF and CHCl₃Any one or more of them; the catalyst is alkaline catalyst, and DMAP, DIEA and ET are adopted₃Any one or more of N and pyridine; the diacid chloride is fatty diacid chloride and comprises any one of succinyl chloride, glutaryl chloride, adipoyl chloride and sebacoyl chloride; the temperature of the reaction is-90 to-30 ℃.

6. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold as claimed in claim 1, wherein the step 3) is specifically as follows: dissolving amino acid-based diamine and p-dinitrobenzene active ester in an organic solution, adding a catalyst, carrying out polymerization reaction, precipitating in ethyl acetate, filtering, purifying a polymer by a Soxhlet extraction method to obtain a white solid, and sealing in a drying dish for storage.

7. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold as claimed in claim 6, wherein the organic solution is any one or more of DMF, DMAC, DMSO and THF; the temperature of the polymerization reaction is 60-100 ℃, and the reaction time is 6-24 hours.

8. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold as claimed in claim 1, wherein the step 4) is specifically as follows: dissolving a polymer in an organic solvent to prepare spinning solution, obtaining a nanofiber membrane by using an electrostatic spinning technology, drying the membrane in a ventilated place, cutting the nanofiber membrane according to the shape of a wound, and preparing the amino acid based polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold.

9. The method for preparing the amino acid based polyesterammonia electrospun nanofiber tissue engineering skin scaffold according to claim 8, wherein the mass concentration of the polyesterammonia in the spinning solution is 5-35%; the organic solvent is any one of DMF, DMSO, TFA and HFIP.

10. The method for preparing the amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold according to claim 8, wherein the technological parameters of the electrospinning are as follows: the temperature is 25-45 ℃, the relative humidity is 15-45%, the injection speed is 0.5-3 mL/h, and the receiving distance is 10-25 cm; the diameter of the fiber in the prepared nanofiber membrane is 500-1000 nm.

Technical Field

The invention relates to a preparation method of an amino acid based polyester ammonia electrospun nanofiber tissue engineering skin scaffold, belonging to the technical field of biomedical materials.

Background

In the biomedical field, due to the increasing demands and higher requirements of sutures, surgical implants, controlled drug release preparations, tissue engineering scaffolds and the like, the development of biodegradable polymers is an increasingly important research field. Polyesteramine is a high molecular compound with good mechanical properties and excellent biocompatibility, has the characteristics of both polyester and polyamide, and is receiving most attention.

There are two classes of polyesteramines, one derived from non-amino acids, such as aliphatic diamines. Another class is derived from amino acids such as L-phenylalanine, L-leucine and L-lysine. The amino acid-based polyester has amido bonds and ester bonds in the molecular chain, so that the polymer has the characteristics of polyurethane and protein, namely, the surface erosion biodegradation catalyzed by enzyme combines the mechanical, physical and biological compatibility into a single entity. Polyurethane polyurethanes based on alpha-amino acids are a new generation of synthetic materials, with versatility, exhibiting good processability, excellent mechanical properties, also exhibiting good biocompatibility and low inflammatory response, and potentially enhancing cell-substance interactions. As a family of potential biodegradable polymers, amino acid-based polyester ammonia contains natural amino acid fragments in molecular chains, can be biodegraded, is susceptible to hydrolysis and enzymatic degradation of the hydrolysis, has low toxicity sources in degraded products, and can be absorbed by human bodies through proteolytic enzymes. It is also worth mentioning that the hard and soft segments in their structures can be designed and changed and the amide bond/ester bond ratio can be adjusted, and the degradation period can be adjusted and controlled to adapt to the healing of different wounds.

It is well known that the native extracellular matrix (ECM) is an extremely complex fluid environment, consisting of fibrogenic proteins (such as collagen and elastic fibers) and non-fibrogenic proteins (proteoglycans, glycosaminoglycans and soluble proteoglycans), which are the microenvironments upon which cells provide survival. Electrospinning has been widely used for tissue engineering, mainly because their microstructures can easily modulate extracellular matrix (ECM) -like structures through a simple electrospinning process. The nanofiber membrane has high surface area-to-volume ratio and good water vapor transmission rate, and the pore structure of the nanofiber membrane can stimulate cell adhesion, migration and proliferation. During the electrostatic spinning process, many substances (such as epidermal growth factor, antibacterial agent and the like) can be easily doped in the fiber, so that the wound healing efficiency and the biological activity are improved.

Disclosure of Invention

the technical problem to be solved by the invention is as follows: provides a preparation method of an amino acid-based polyester ammonia electrospun nanofiber tissue engineering skin scaffold.

In order to solve the technical problem, the invention provides a preparation method of an amino acid-based polyester ammonia electrospun nanofiber tissue engineering skin scaffold, which is characterized by comprising the following steps of:

Step 1): synthesizing amino acid-based diamine, and obtaining the amino acid-based diamine through esterification reaction of tryptophan and diol;

Step 2): reacting diacyl chloride with p-nitrophenol to obtain p-dinitrobenzene active ester;

Step 3): carrying out solution polycondensation on two monomers, namely amino acid-based diamine and p-dinitrobenzene active ester to obtain an amino acid-based polyester ammonia polymer;

Step 4): and (3) carrying out electrostatic spinning on the amino acid-based polyester ammonia polymer to obtain the amino acid-based polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold. The nanofiber scaffold not only has stable shape and uniform diameter distribution, but also has excellent biocompatibility, and the method is simple and easy to implement, green and environment-friendly, and can be used for large-scale production.

preferably, the step 1) is specifically: directly mixing L-tryptophan, diol and p-toluenesulfonic acid, and placing the mixture in a container filled with toluene; then heating the solid-liquid reaction mixture to 70-130 ℃, and keeping refluxing for 16-24 hours; cooling the reaction mixture to room temperature, removing toluene, repeatedly dissolving and precipitating in deionized water for three times to purify the product, filtering and collecting white substances, drying in vacuum, sealing and storing.

More preferably, the diol is any one of chain aliphatic diols having 3 to 8 carbon atoms; the feeding molar ratio of the glycol to the L-tryptophan to the p-toluenesulfonic acid is 1: (2-4): (4-6); the volume of toluene is 10-30 times the volume of the solid.

Preferably, the step 2) is specifically: dissolving p-nitrophenol in an organic solvent, adding a catalyst, dropwise adding diacyl chloride, reacting overnight under the action of mechanical stirring, filtering, drying to obtain a needle-shaped off-white solid, sealing, and storing in a drying dish.

More preferably, the organic solvent is acetone, DMF, DMAC, THF and CHCl₃Any one or more of them; the catalyst is alkaline catalyst, and DMAP, DIEA and ET are adopted₃Any one or more of N and pyridine; the diacid chloride is fatty diacid chloride and comprises any one of succinyl chloride, glutaryl chloride, adipoyl chloride and sebacoyl chloride; the temperature of the reaction is-90 to-30 ℃.

Preferably, the step 3) is specifically: dissolving amino acid-based diamine and p-dinitrobenzene active ester in an organic solution, adding a catalyst, carrying out polymerization reaction, precipitating in ethyl acetate, filtering, purifying a polymer by a Soxhlet extraction method to obtain a white solid, and sealing in a drying dish for storage. The degradation sites and the degradation rate of a plurality of enzymes such as ester bonds, amido bonds and the like are matched with the wound healing cycle by changing the types, the concentrations and the degradation time of the enzymes.

More preferably, the organic solution is any one or more of DMF, DMAC, DMSO and THF; the temperature of the polymerization reaction is 60-100 ℃, and the reaction time is 6-24 hours.

Preferably, the step 4) is specifically: dissolving a polymer in an organic solvent to prepare spinning solution, obtaining a nanofiber membrane by using an electrostatic spinning technology, drying the membrane in a ventilated place, cutting the nanofiber membrane according to the shape of a wound, and preparing the amino acid based polyester ammonia electrostatic spinning nanofiber tissue engineering skin scaffold.

More preferably, the mass concentration of the polyester ammonia in the spinning solution is 5-35%; the organic solvent is any one of DMF, DMSO, TFA and HFIP.

more preferably, the electrostatic spinning process parameters are as follows: the temperature is 25-45 ℃, the relative humidity is 15-45%, the injection speed is 0.5-3 mL/h, and the receiving distance is 10-25 cm; the diameter of the fiber in the prepared nanofiber membrane is 500-1000 nm.

the amino acid-based polyurethane nanofiber scaffold is prepared by an electrostatic spinning technology, the amino acid-based polyurethane is a polyurethane polymer prepared by solution polycondensation of two monomers, and amino acid and active ester fragments in the polymer are nontoxic and harmless to human bodies and the environment after enzymatic degradation, and have good biocompatibility.

the nanofiber membrane scaffold prepared by electrostatic spinning not only has stable form and uniform diameter distribution, but also has excellent biocompatibility, and the method is simple and easy to implement, green and environment-friendly, and can be produced in a large scale. Has potential application in the biomedical fields of wound antibiosis, wound repair and the like.

Compared with the prior polyester ammonia material, the invention has the beneficial effects that:

Aliphatic polyesters are the most widely studied family of biodegradable polymers, and although several polymers of aliphatic polyesters based on glycolic acid, L-lactic acid, epsilon-caprolactone and copolymers thereof have been used as biodegradable biomaterials they still lack certain optimum properties. For example, since most of these polyesters are synthesized from a single synthesis, the degradation rate monomer of aliphatic polyesters cannot be easily adjusted. Furthermore, the acidic degradation by-products of these polyesters have been shown to be toxic to certain cells, limiting their use as functional tissue engineering scaffolds.

(1) The amino acid-based polyurethane material prepared by the invention has a large number of amido bonds and ester bonds, and can be degraded by a plurality of enzymes, and the degradation sites and the degradation rate of the ester bonds, the amido bonds and the like can be matched with the period of wound repair by changing the types of the enzymes, the concentration of the enzymes and the degradation time.

(2) The amino acid-based polyurethane prepared by the invention endows the polymer with the characteristics of polyurethane and protein (a large number of amido bonds), namely, the surface erosion biodegradation catalyzed by enzyme combines the mechanical, physical and biological compatibility into a single entity. Polyurethane polyurethanes based on alpha-amino acids are a new generation of synthetic materials, with versatility, exhibiting good processability, excellent mechanical properties, also exhibiting good biocompatibility and low inflammatory response, and potentially enhancing cell-substance interactions.

(3) The amino acid-based polyurethane nano material prepared by the invention can be used as a scaffold material, can well simulate a natural cytoplasm matrix, has a high surface area-to-volume ratio and a good water vapor transmission rate, and can stimulate cell adhesion, migration and proliferation due to the pore structure. During the electrostatic spinning process, many substances (such as epidermal growth factor, antibacterial agent and the like) can be easily doped in the fiber, so that the wound healing efficiency and the biological activity are improved.

(4) The amino acid based polyester ammonia nanofiber scaffold prepared by the invention is prepared by electrostatic spinning, and has the advantages of simple process, high production efficiency, environmental protection and safety. Can well simulate natural cytoplasm matrix, and has high surface area-to-volume ratio, good water vapor permeability, and pore structure capable of stimulating cell adhesion, migration and proliferation. During the electrostatic spinning process, many substances (such as epidermal growth factor, antibacterial agent and the like) can be easily doped in the fiber, so that the wound healing efficiency and the biological activity are improved.

Drawings

FIG. 1 is a schematic diagram of the synthesis of amino acid based diamines; wherein n is 1-3 and represents the number of carbon atoms in diol;

FIG. 2 is a schematic diagram of the synthesis of amino acid based polyesteramines; wherein the content of the first and second substances,m is 1-3 and represents the number of carbon atoms in diol; k is 2-8 and represents the number of carbon atoms in acyl chloride;

FIG. 3 is a scanning electron microscope image of amino acid based polyurethane nanofiber membrane.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.