阅读说明：本技术 一种表面接枝重组表皮生长因子纳米纤维的制备方法 (Preparation method of surface grafted recombinant epidermal growth factor nanofiber ) 是由 汤佳鹏 葛彦 操维芳 朱俐 于 2019-09-17 设计创作，主要内容包括：本发明公开了一种等离子体表面接枝重组表皮生长因子新型纳米纤维的制备方法,包括(1)将一定量O-羧甲基壳聚糖和聚氧化乙烯完全溶解于90％乙酸溶液,搅拌均匀,得到纺丝液；(2)采用纺丝液进行静电纺丝,得到O-羧甲基壳聚糖/聚氧化乙烯纳米纤维；(3)将O-羧甲基壳聚糖/聚氧化乙烯纳米纤维用DMEM培养基洗涤至中性,烘干之后经等离子处理器处理活化；(4)活化的纳米纤维浸泡在含有重组表皮生长因子EGF和CoCl<Sub>2</Sub>的DMEM培养基中进行负压闪爆,之后进行接枝反应,离心冻干。本发明制备的纳米纤维是一种能够诱导组织上皮损伤部的再生的医疗用材料,能够刺激多种细胞的增殖,适用于表皮细胞、内皮细胞。(The invention discloses a preparation method of novel plasma surface grafted recombinant epidermal growth factor nanofiber, which comprises the following steps(1) Completely dissolving a certain amount of O-carboxymethyl chitosan and polyoxyethylene in a 90% acetic acid solution, and uniformly stirring to obtain a spinning solution; (2) performing electrostatic spinning by using the spinning solution to obtain O-carboxymethyl chitosan/polyoxyethylene nano-fibers; (3) washing the O-carboxymethyl chitosan/polyethylene oxide nano-fiber to be neutral by using a DMEM culture medium, drying, and then treating and activating by using a plasma processor; (4) soaking the activated nanofiber in the solution containing recombinant epidermal growth factors EGF and CoCl 2 The DMEM medium is subjected to negative pressure flash explosion, then grafting reaction is carried out, and centrifugal freeze-drying is carried out. The nanofiber prepared by the invention is a medical material capable of inducing the regeneration of a tissue epithelial injury part, can stimulate the proliferation of various cells, and is suitable for epidermal cells and endothelial cells.)

1. A preparation method of surface grafting recombinant epidermal growth factor nano-fiber is characterized by comprising the following steps:

s1: completely dissolving a certain amount of O-carboxymethyl chitosan and polyoxyethylene in 90 v/v% acetic acid solution, and uniformly stirring to obtain spinning solution;

s2: performing electrostatic spinning by using the spinning solution to obtain O-carboxymethyl chitosan/polyoxyethylene nano-fibers;

s3: washing the O-carboxymethyl chitosan/polyethylene oxide nano-fiber to be neutral by using a DMEM culture medium, drying, and then treating and activating by using a plasma processor to obtain activated nano-fiber;

s4: soaking the activated nano-fiber obtained in the step S3 in a solution containing recombinant epidermal growth factor and CoCl₂And carrying out negative pressure flash explosion in the DMEM medium, then carrying out grafting reaction, and carrying out centrifugal freeze-drying to obtain the surface grafting recombinant epidermal growth factor nanofiber.

2. The method for preparing surface-grafted recombinant EGF nanofiber as claimed in claim 1, wherein the viscosity-average molecular weight of the O-carboxymethyl chitoglycan is 5.0 x 10⁵The degree of deacetylation is 80-85%, and the average molecular weight of the polyoxyethylene is 1.0 x 10⁶。

3. The method for preparing the surface-grafted recombinant epidermal growth factor nanofiber according to claim 1, wherein in the step S1, the total concentration of the O-carboxymethyl chitosan and the polyethylene oxide is 10-30 g/L, and the mass ratio of the O-carboxymethyl chitosan to the polyethylene oxide is 1: 1-4.

4. The method for preparing the surface-grafted recombinant epidermal growth factor nanofiber according to claim 1, wherein in the step S2, in the electrospinning process, the specification of an injector is 10ml, the specification of a needle head is a flat head, a 7-gauge needle is adopted, the voltage is 12-20 KV, the distance is 7-10 cm, the sample injection rate is 0.3-1.0 ml/h, and the temperature is 25-35 ℃.

5. The method for preparing the surface-grafted recombinant epidermal growth factor nanofiber according to claim 1, wherein in the step S3, the drying temperature is 37-45 ℃ and the drying time is 2-4 h.

6. The method of claim 1, wherein the plasma processor is configured to process the nanofibers in step S3 under the following conditions: the gas is nitrogen or oxygen, the treatment power is 250-300W, the pressure is 50-60 Pa, and the treatment time is 10-15 min.

7. The method of claim 1, wherein in step S4, the surface-grafted EGF nanofiber comprises EGF and CoCl₂The concentration of the recombinant epidermal growth factor in the DMEM medium is 40-80 mg/L, and the CoCl₂The concentration of (b) is 0.15-0.30 g/L.

8. The method of claim 1, wherein in step S4, the bath ratio of the grafting reaction is 1: 100-300, the soaking temperature is 0-4 ℃, and the soaking time is 12-24 h.

9. The method for preparing the surface-grafted recombinant epidermal growth factor nanofiber according to claim 1, wherein in step S4, the vacuum degree of the negative pressure flash explosion is 0.100-0.024 mBar, the freeze-drying temperature is-30 to-20 ℃, the vacuum degree is 0.100-0.024 mBar, and the freeze-drying time is 3-5 days.

Technical Field

The invention relates to the field of biomedical engineering, in particular to a preparation method of surface grafting recombinant epidermal growth factor nanofibers.

Background

Carboxymethyl chitosan and its derivatives have anti-infective effects in different degrees. The micromolecular chitosan has the functions of ammonium protonation, wherein the ammonium protonation and the cell membrane with negative charges of bacteria can adsorb and aggregate the bacteria, and simultaneously penetrate through cell walls to enter cells, disturb the metabolism and synthesis of the bacteria and have antibacterial effect. The chitosan with the relative molecular weight of 1500 has the strongest inhibition effect on escherichia coli, and the antibacterial effect is reduced along with the increase of the molecular weight. The chitosan has strong inhibiting effect on staphylococcus aureus, escherichia coli, yersinia enterocolitica, salmonella typhimurium and listeria monocytogenes. Carboxymethyl chitosan has been made into various medical dressings such as non-woven fabrics, cast films, coated gauzes and the like for clinical use, wherein the non-woven fabrics made of carboxymethyl chitosan and acetic acid have excellent air permeability and water permeability, are used for large-area burns and scalds, and have good anti-infection and wound healing promotion effects.

Human epidermal growth factor was first extracted from human urine in 1975. In the 90 s, gene recombination is carried out by adopting a bioengineering technology, namely, a production strain is selected, engineering bacteria fermentation is carried out, and then the recombinant human epidermal growth factor is prepared after purification and freeze-drying. The recombinant epidermal growth factor can promote cell mitosis and synthesis of sugar, protein, DNA and RNA, has the function of promoting division and proliferation of epithelial cells, and is closely related to the treatment of immunological dermatosis, wound tissue repair and operative vessel healing in clinical application. The recombinant human epidermal growth factor applied clinically can promote wound healing, has different healing accelerating effects on superficial II degree and deep II degree (especially the latter) chronic wounds, donor areas and the like of burns by matching with conventional burn wound treatment, and can shorten the hospitalization time of patients. At present, there are reports in literature that recombinant human epidermal growth factor is made into hydrogel, aqueous solution, common emulsion and other dosage forms, but because of the large molecular weight of the recombinant human epidermal growth factor, the stability of the recombinant human epidermal growth factor in these dosage forms is poor, and the dispersion matrix has no obvious absorption promoting effect, so that the transdermal effect of the preparation is poor, the absorption availability of the active ingredients is low, and especially, the operative vascular anastomosis has great difficulty.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing surface-grafted recombinant epidermal growth factor nanofibers, in which epidermal growth factors are grafted onto O-carboxymethyl chitosan nanofibers, and the obtained nanofibers can better promote the repair and healing of wound surfaces of corneal injury, burns, scalds, surgery, and the like.

In order to solve the technical problems, the invention provides a preparation method of surface grafting recombinant epidermal growth factor nano-fiber, which comprises the following steps:

s1: completely dissolving a certain amount of O-carboxymethyl chitosan and polyoxyethylene in 90 v/v% acetic acid solution, and uniformly stirring to obtain spinning solution;

s2: performing electrostatic spinning by using the spinning solution to obtain O-carboxymethyl chitosan/polyoxyethylene nano-fibers;

s3: washing the O-carboxymethyl chitosan/polyethylene oxide nano-fiber to be neutral by using a DMEM culture medium, drying, and then treating and activating by using a plasma processor to obtain activated nano-fiber;

s4: soaking the activated nano-fiber obtained in the step S3 in a solution containing recombinant epidermal growth factor and CoCl₂And carrying out negative pressure flash explosion in the DMEM medium, then carrying out grafting reaction, and carrying out centrifugal freeze-drying to obtain the surface grafting recombinant epidermal growth factor nanofiber.

Preferably, the viscosity average molecular weight of the O-carboxymethyl chitosan is 5.0 x 10⁵The degree of deacetylation is 80-85%, and the average molecular weight of the polyoxyethylene is 1.0 x 10⁶。

Preferably, in step S1, the total concentration of the O-carboxymethyl chitosan and the polyethylene oxide is 10-30 g/L, and the mass ratio of the O-carboxymethyl chitosan to the polyethylene oxide is 1: 1-4.

Preferably, in the step S2, in the electrostatic spinning process, the specification of the injector is 10ml, the specification of the needle head is a flat head, a No. 7 needle, the voltage is 12-20 KV, the distance is 7-10 cm, the sample injection speed is 0.3-1.0 ml/h, and the temperature is 25-35 ℃.

Preferably, in the step S3, the drying temperature is 37 to 45 ℃ and the drying time is 2 to 4 hours.

Preferably, in step S3, the plasma processor processing conditions are: the gas is nitrogen or oxygen, the treatment power is 250-300W, the pressure is 50-60 Pa, and the treatment time is 10-15 min.

Preferably, in step S4, the recombinant epidermal growth factor and CoCl are contained₂The concentration of the recombinant epidermal growth factor in the DMEM medium is 40-80 mg/L, and the CoCl₂The concentration of (b) is 0.15-0.30 g/L.

Preferably, in step S4, the soaking bath ratio of the grafting reaction is 1: 100-300, the soaking temperature is 0-4 ℃, and the soaking time is 12-24 hours.

Preferably, in step S4, the vacuum degree of the negative pressure flash explosion is 0.100 to 0.024mBar, the temperature of the freeze-drying is-30 to-20 ℃, the vacuum degree is 0.100 to 0.024mBar, and the freeze-drying time is 3 to 5 days.

Compared with the prior art, the invention has the following advantages and effects:

1) the O-carboxymethyl chitosan is adopted, and the high molecular compound has free carboxyl and amino, so that the water solubility and the biocompatibility are better than those of chitosan. The abundant carboxyl and amino can provide multiple anchoring sites for anchoring protein molecules, and the binding strength is enhanced.

2) Chelating Co with O-carboxymethyl chitosan²⁺The ions generate a chemical hypoxia environment at the cell repair position, induce the expression increase of hypoxia induction factor HIF in the cells, and induce the growth of epithelium and epidermis through the regulation and control of transcription factors.

Drawings

FIG. 1 shows the growth of rat lung epithelial cells treated in examples of the present invention and comparative examples after 24h culture;

FIG. 2 shows the cell proliferation rate of rat lung epithelial cells treated in examples of the present invention and comparative examples after 24 hours of culture.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow.

All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.

The technical scheme adopted by the invention is as follows:

a preparation method of novel nanofiber with surface grafted recombinant epidermal growth factor comprises the following steps:

s1: completely dissolving a certain amount of O-carboxymethyl chitosan and polyoxyethylene in 90 v/v% acetic acid solution, and uniformly stirring to obtain spinning solution;

s2: performing electrostatic spinning by using the spinning solution to obtain O-carboxymethyl chitosan/polyoxyethylene nano-fibers;

s3: washing the O-carboxymethyl chitosan/polyethylene oxide nano-fiber to be neutral by using a DMEM culture medium, drying, and then treating and activating by using a plasma processor;

s4: soaking the nanofiber obtained in the third step in a DMEM medium containing recombinant epidermal growth factor EGF for negative pressure flash explosion, and then carrying out grafting reaction;

s5: and (3) centrifugally freeze-drying the nano-fibers subjected to the grafting reaction.

In step S1, the O-carboxymethyl chitosan used has a viscosity average molecular weight of 5.0X 10⁵The degree of deacetylation is preferably 80 to 85%, more preferably 80%. The polyethylene oxide used has a specification of average molecular weight of 1.0X 10⁶. The total concentration of the O-carboxymethyl chitosan and the polyoxyethylene is preferably 10-30 g/L, more preferably 20g/L, and the O-carboxymethyl chitosan and the polyoxyethylene areThe mass ratio of the alkene is preferably 1: 1-4, and more preferably 1: 3.

In the step S2, in the electrostatic spinning process, the specification of the used injector is 10ml, and the specification of the needle head is a flat head 7-gauge needle; the electrostatic spinning condition is that the voltage is preferably 12-20 KV, more preferably 15KV, the distance is preferably 7-10 cm, more preferably 8cm, the sample injection rate is preferably 0.3-1.0 ml/h, more preferably 0.5ml/h, and the temperature is preferably 25-35 ℃, more preferably 30 ℃.

In step S3, the washing end point pH is 7, the drying temperature is preferably 37 to 45 ℃, more preferably 37 ℃, and the drying time is preferably 2 to 4 hours, more preferably 4 hours. The plasma treatment conditions were: the gas is preferably nitrogen or oxygen, more preferably oxygen, the treatment power is preferably 250-300W, more preferably 280W, the pressure is preferably 50-60 Pa, more preferably 55Pa, and the treatment time is preferably 10-15 min, more preferably 15 min.

In step S4, the concentration of the epidermal growth factor EGF is preferably 40-80 mg/L, more preferably 80mg/L, CoCl₂The concentration of (b) is preferably 0.15 to 0.30g/L, more preferably 0.20 g/L. The soaking bath ratio of the grafting reaction is preferably 1: 100-300, more preferably 1:200, the soaking temperature is preferably 0-4 ℃, more preferably 4 ℃, and the soaking time is preferably 12-24 hours, more preferably 24 hours. The vacuum degree of the negative pressure flash explosion is preferably 0.100-0.024 mBar, and more preferably 0.024 mBar.

In step S5, the temperature of lyophilization is preferably-30 to-20 ℃, more preferably-30 ℃, the vacuum degree is preferably 0.100 to 0.024mBar, more preferably 0.024mBar, and the lyophilization time is preferably 3 to 5 days, more preferably 4 days.

For further understanding of the present invention, the following examples are provided to illustrate the preparation method of the novel nanofiber with recombinant epidermal growth factor grafted on the surface, and the scope of the present invention is not limited by the following examples.