阅读说明：本技术 一种纤维材料及其制备方法和应用 (fiber material and preparation method and application thereof ) 是由 于谦 郑琰君 张燕霞 周洋 于 2019-07-24 设计创作，主要内容包括：本发明涉及一种纤维材料,其包括纤维核层和包覆在所述的纤维核层外的纤维壳层,所述的纤维核层包括外源分子和聚乙烯亚胺；所述的纤维壳层包括金纳米棒和生物可降解材料。本发明的纤维材料实现了外源分子的释放与高效递送的双重目的,且外源分子的负载方式简单,传递效率高,释放前不易被降解失效,在组织工程等生物医用领域具有潜在的应用价值。(The invention relates to a fiber material, which comprises a fiber core layer and a fiber shell layer coated outside the fiber core layer, wherein the fiber core layer comprises exogenous molecules and polyethyleneimine; the fiber shell layer comprises gold nanorods and biodegradable materials. The fiber material provided by the invention realizes dual purposes of release and efficient delivery of exogenous molecules, has a simple exogenous molecule loading mode and high transfer efficiency, is not easily degraded and ineffective before release, and has potential application value in biomedical fields such as tissue engineering and the like.)

1. A fibrous material characterized by: the fiber core layer comprises exogenous molecules and polyethyleneimine; the fiber shell layer comprises gold nanorods and biodegradable materials.

2. The fibrous material according to claim 1, characterized in that: the exogenous molecule comprises one or more of plasmid DNA, nucleic acid, saccharide, protein and medicine.

3. The fibrous material according to claim 2, characterized in that: the N/P molar ratio of the polyethyleneimine to the plasmid DNA in the fiber core layer is 15-40.

4. The fibrous material according to claim 1, characterized in that: the polyethyleneimine is low molecular weight polyethyleneimine; the biodegradable material is one or more of hyaluronic acid, gelatin, alginate, fibroin, chitosan, polyhydroxy butyrate valerate, polyhydroxy butyrate caproate, polycaprolactone, polyglycolide, polylactide, polylactic acid and a copolymer of polylactic acid and caprolactone or glycolide.

5. The fibrous material according to claim 4, characterized in that: the biodegradable material is a mixture of gelatin and levorotatory polylactic acid.

6. the fibrous material according to claim 1, characterized in that: the mass ratio of the gold nanorods to the biodegradable material is 1: 150-200.

7. The fibrous material according to claim 1, characterized in that: the fiber material is 0.27W/cm in near infrared light²～3.9W/cm²When the fiber material is irradiated for 10-60 seconds under the illumination intensity, the temperature of the fiber material is increased, and the exogenous molecules can be released from the fiber material.

8. A process for the preparation of a fibrous material according to any of claims 1 to 7, characterized in that: the fiber material is prepared by coaxial electrostatic spinning.

9. Use of a fibrous material according to any of claims 1 to 7 in medical products or devices such as haemostatic materials, wound dressings, tissue engineering scaffold materials, drug release films, wound dressings, drug delivery materials and the like.

10. A method for in vitro cell delivery of a fibrous material according to any of claims 1 to 7, wherein: the method comprises the following steps:

(1) seeding cells onto said fibrous material;

(2) Adding serum-free cell culture medium, and applying near infrared light at 0.27W/cm²～3.9W/cm²The illumination intensity of the light source is irradiated for 10 to 60 seconds;

(3) And 3-6 h after the irradiation is finished, replacing the serum-free cell culture medium with a serum cell culture medium, and continuously culturing to obtain the cells containing the exogenous molecules.

Technical Field

The invention particularly relates to a fiber material and a preparation method and application thereof.

Background

Gene transfer is an important link in gene therapy and tissue engineering. Among these, surface-mediated gene delivery has shown greater advantage. Different from the traditional gene transfer mode, the research loads gene molecules on the surface or the body of a biological material, and when the material is contacted with cells in vitro or implanted into an intervention body, the genes on the interface of the surface of the material are endocytosed by the cells so as to realize gene transfer. Compared with other traditional delivery methods, the surface-mediated gene delivery has the irreplaceable advantages of high transfection efficiency, stable vector, long-acting gene release and the like.

In recent researches, functional genes with regulation and control functions are embedded or loaded on the surface of biological materials and devices, and the functional genes are transfected to surrounding tissues after the materials are implanted into the body, so that on one hand, adverse reactions such as inflammation and complications are hopeful to be inhibited, and on the other hand, the biological activity for further treating diseases at the parts can be given to the materials according to needs. However, for the gene transfer mediated by the surface of the biomaterial, the gene therapy effect cannot be completely guaranteed only by solving the problem of loading and protecting the gene on the material, and the gene molecules loaded in or on the material must be released from the material in a proper manner so as to effectively transfect the tissue and achieve the purpose of gene therapy.

The electrostatic spinning nanofiber attracts great attention as a carrier of surface-mediated gene transfer, mainly because the electrostatic spinning nanofiber has high specific surface area and high porosity, and the electrostatic spinning nanofiber can simulate an interconnected porous geometric structure of an extracellular matrix (ECM) topological structure, so that the adhesion and growth of cells are facilitated. Some current studies bind DNA to electrospun microfibers. The synthetic polymer/DNA composite scaffold was initially fabricated by mixed electrode, after which numerous studies have shown that loading naked DNA or gene-vector complexes presents a number of problems, such as the difficulty of controlling the loading of the gene on the material, the susceptibility of the vector to inactivation; the gene is easy to be attacked by enzyme before being released, the release behavior of the gene in an in vivo environment is uncontrollable, the problems of too fast or too slow release and the like are easy to occur, and the release and transfection of the gene have no selectivity; the efficiency of gene transfection between cells and tissues is still low.

In the design of surface-mediated gene transfer materials, the gene transfer capability of the materials is improved by taking the property of the implant intervention substrate material as a starting point, and the cell state of the cells during transfection and the behavior of various cells have decisive influence on the transfection efficiency. The cell membrane is the most critical barrier in gene transfection, and thus the state of the cell membrane is one of the most important cell states for gene transfection among various cell states. At present, methods for disturbing cell membranes and increasing membrane permeability by means of electroporation, ultrasound, or the like have been used to promote gene transfection. However, these measures are poorly controllable and tend to cause cell death.

The patent name: a method for preparing a cell loaded with exogenous molecules by adopting a photo-induced perforation mode, a base material for preparing the cell and the cell (patent number: CN 105420278A), wherein the base material is obtained by settling gold nanoparticle aggregates in culture holes of a culture plate, exogenous molecules are added into a serum-free culture medium, and the transfection of the exogenous molecules into the cell is realized by utilizing laser irradiation. The base material in the mode has no universality and has certain limitation in practical application.

The patent name: a macromolecular system modified by a nucleic acid aptamer is spun into a fiber membrane for controlled release through electrostatic spinning (patent number: CN 103705438A), and DNA double chains are respectively grafted to linear polyacrylamide macromolecular polymers and added with guest molecules to form a loading system for wrapping the guest molecules. And further spinning the loading system into a fiber membrane by an electrostatic spinning technology, and decomposing the loading system by externally adding target molecules with strong binding capacity to compete and bind the aptamers to release the guest molecules. The loading and releasing processes of this approach are complex, limiting their practical application.

disclosure of Invention

The invention aims to solve the technical problem of providing a fiber material with a photothermal effect, a preparation method and application thereof, wherein the fiber material can realize the controllable release of exogenous molecules by utilizing the photothermal effect while ensuring the protection of the loaded exogenous molecules; meanwhile, the photothermal effect of the material can change the state of cell membranes of cells adhered to the fiber material, promote exogenous molecules to enter the cells and improve the transfer efficiency.

In order to solve the technical problems, the invention adopts the following technical scheme:

The invention provides a fiber material, which comprises a fiber core layer and a fiber shell layer coated outside the fiber core layer, wherein the fiber core layer comprises exogenous molecules and polyethyleneimine; the fiber shell layer comprises gold nanorods and biodegradable materials.

the exogenous molecule in the invention comprises one or more of plasmid DNA (pDNA), nucleic acid, saccharide, protein and medicine.

The fiber material is a surface-mediated transfer material, and compared with other traditional transfer modes, the fiber material has the irreplaceable advantages of high transfer efficiency, stable carrier, long-acting release of exogenous molecules and the like.

Based on the advantages of photothermal transfection, we developed a fiber material with a core-shell structure loaded with an exogenous molecule having a photothermal effect, and in order to protect the exogenous molecule, particularly pDNA, we used Polyethyleneimine (PEI) to electrostatically condense with a negatively charged DNA molecule to protect the pDNA and improve the transfection efficiency. In the aspect of releasing exogenous molecules, the invention utilizes the photothermal effect to increase the permeability of the fiber material, so that the exogenous molecules are released from the fiber material, and the irradiation intensity and the illumination time can be regulated and controlled according to requirements to adapt to the requirements of different cell systems for transmission and improve the transmission efficiency of cell systems difficult to transmit. Meanwhile, the permeability of a cell membrane can be enhanced simultaneously under the irradiation of near infrared light, so that released exogenous molecules can enter cells to successfully transfer the exogenous molecules to the cells, and the cells can obtain certain functions.

In addition, the gold nanorods, as a commonly used functional nano material, have good photo-thermal effect. Under the irradiation of near infrared light, the gold nanorods can effectively convert absorbed light into heat, and meanwhile, light can penetrate to the maximum extent. Therefore, the invention adopts the gold nanorods as the photo-thermal conversion medium.

the fiber material can be prepared by a coaxial electrostatic spinning technology to obtain the fiber with a core-shell structure. Compared with other gene vectors, the fiber material prepared by electrostatic spinning has the advantages of strong functionality, high porosity, similar extracellular matrix structure and the like. In addition, the fiber material in the invention can change the material composition and properties to realize different functions.

Preferably, the N/P molar ratio of the polyethyleneimine to the plasmid DNA in the fiber core layer is 15-40. The N/P molar ratio is preferably 15 to 40, more preferably 30, because transfection efficiency is low when N/P is low, and cytotoxicity is caused when N/P is high.

preferably, the polyethyleneimine is low molecular weight polyethyleneimine (LPEI). The Mw of the low molecular weight polyethyleneimine (LPEI) used in the present invention is 0.2 to 3 kDA. Further preferably 2 kDA.

In the invention, the biodegradable material is one or more of hyaluronic acid, gelatin, alginate, fibroin, chitosan, polyhydroxybutyrate valerate, polyhydroxybutyrate caproate, polycaprolactone, polyglycolide, polylactide, polylactic acid, and a copolymer of polylactic acid and caprolactone or glycolide.

Preferably, the biodegradable material is a mixture of gelatin and L-polylactic acid, the gelatin and the L-polylactic acid are used in a composite manner, so that cell adhesion is enhanced, and the L-polylactic acid can enhance the electrospinning property of the gelatin. More preferably, the mass ratio of the gelatin to the L-polylactic acid is 1: 10-20, and more preferably 1: 12-16.

Preferably, the mass ratio of the gold nanorods to the biodegradable material is 1: 150 to 200 parts.

The gold nanorods can be obtained commercially or prepared by a seed growth method, and the seed growth method can be obtained by referring to reports in the prior art.

The fiber material of the invention has the near infrared light of 0.27W/cm²～3.9W/cm²When the fiber material is irradiated for 10-60 seconds under the illumination intensity, the exogenous molecules can be released from the fiber material, and the temperature of the fiber material is increased. The control of the illumination intensity and the illumination time in the invention is important, if the illumination intensity is too high or the illumination time is too long, the fiber material can be deformed and burnt, if the intensity is too low or the illumination time is too short, the exogenous molecules can not be effectively released from the fiber material, and the temperature rise of the fiber material is limited, so that the permeability of cell membranes can not be well improved, and the improvement of the transmission efficiency is not facilitated.

In a second aspect of the invention, a method for preparing the fiber material is provided, wherein the fiber material is prepared by coaxial electrospinning.

Specifically, a core layer spinning solution and a shell layer spinning solution are prepared respectively, and then the core layer spinning solution and the shell layer spinning solution are spun through a coaxial electrostatic spinning device to obtain the composite material.

The preparation method of the core layer spinning solution comprises the following steps: mixing exogenous molecule with the water solution of polyethyleneimine.

When the exogenous molecule is pDNA, the preparation method comprises the following steps: culturing glycerol strain of pDNA, extracting and purifying plasmid, and mixing polyethyleneimine solution and the extracted and purified plasmid to obtain the product, wherein the solvent of the polyethyleneimine solution is ultrapure water.

The preparation method of the shell spinning solution comprises the following steps: adding the biodegradable material and the gold nanorods into a solvent, and stirring to obtain the nano gold/.

The specific method of coaxial electrostatic spinning comprises the following steps: and injecting the core layer spinning solution and the shell layer spinning solution into a needle head communicated with a positive electrode and a negative electrode according to the volume ratio of 1: 2-3 for spinning, wherein the inner diameter of the needle head is 0.4-0.8 mm, the voltage of the positive electrode is 12-16 KV, and the voltage of the negative electrode is-2-6 KV. A schematic of coaxial electrospinning is shown in fig. 1.

The third aspect of the invention provides an application of the fiber material in medical products or medical devices, and the fiber material can be made into medical products or medical devices such as hemostatic materials, wound coating materials, tissue engineering scaffold materials, drug release films, wound dressings, drug delivery materials and the like, and is used for treating required diseases. For example, by varying the composition of the fibrous material and electrospinning a factor having hemostatic function, it can be applied to wound treatment of massive bleeding. The nuclear layer is made of a fiber material which is prepared by pDNA (pFGF) loaded with and encoding fibroblast growth factors, so that the proliferation and migration of fibroblasts can be efficiently promoted, the purpose of promoting wound healing is achieved, and the fiber material has potential application value in the biomedical fields of tissue engineering and the like. In addition, the modification of pDNA supported in the nuclear layer, for example, the replacement with a plasmid encoding a growth factor such as VEGF (vascular endothelial growth factor), IGF-1 (insulin-like growth factor 1), or the like, can be applied to the fields of vascular endothelialization and osteoblast proliferation and differentiation, or the replacement of pDNA with a protein, a drug, or the like, for example, to the fields of drug release.

The fourth aspect of the present invention provides a method for in vitro cell delivery of the fiber material, comprising the following steps:

(1) Seeding cells onto said fibrous material;

(2) Adding serum-free cell culture medium, and applying near infrared light at 0.27W/cm²～3.9W/cm²the illumination intensity of the light source is irradiated for 10 to 60 seconds;

(3) And 3-6 h after the irradiation is finished, replacing the serum-free cell culture medium with a serum cell culture medium, and continuously culturing to obtain the cells containing the plasmid DNA.

due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:

the fiber material provided by the invention realizes dual purposes of release and efficient delivery of exogenous molecules, has a simple exogenous molecule loading mode and high transfer efficiency, is not easily degraded and ineffective before release, and has potential application value in biomedical fields such as tissue engineering and the like.

Drawings

FIG. 1 is a schematic illustration of a coaxial electrospinning process;

FIG. 2 is a graph showing the results of the measurement of CCK-8 cell activity and the results of transfection efficiency of the coaxial electrospun fibers at different N/P ratios in example 1;

FIG. 3 is SEM and TEM images of the fibrous material prepared in example 2;

FIG. 4 is a graph showing the photothermal effect measurement results of the electrospun fiber of example 2;

FIG. 5 is a graph of the results of flow cytometry characterization of transfection efficiency for example 3;

FIG. 6 is a graph showing the results of example 3 on the proliferation effect of NIH-3T3 cells;

FIG. 7 is the result of cell migration of example 3, wherein FIG. 7(a) is the result of characterizing cell migration using Transwell; FIG. 7(b) is a graph showing the results of a scratch test for cell migration.

Detailed Description

in order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used are not indicated by the manufacturer and are commercially available.

Based on the advantages of surface-assisted gene transfection of photothermal method and the advantages of electrospun fiber, a related study is conducted below with the aim of promoting proliferation and migration of fibroblasts, the core layer of the electrospun fiber is pDNA (pFGF) loaded with growth factors encoding fibroblasts, gelatin/poly-L-lactic acid (PLLA) with good biocompatibility and biodegradability is used as the outer layer of the spun fiber, and a certain amount of Gold Nanorods (GNR) are doped in the outer layer, so as to provide excellent photothermal effect of the spun fiber. Finally, the high-porosity photo-thermal controllable electrostatic spinning fiber membrane is obtained, under the irradiation of near infrared light with certain intensity, pGF of a nuclear layer can be released from spinning fibers to transfect fibroblasts on the surface nearby in situ, and meanwhile, the irradiated near infrared light can perforate on the surface of a cell membrane to complete the delivery of the pGF to the cells, so that the proliferation and migration of the fibroblasts are accelerated, and the purpose of promoting wound healing is achieved.