CN113750294A

CN113750294A - Nerve repair stent loaded with multiple gene vector microspheres and preparation method thereof

Info

Publication number: CN113750294A
Application number: CN202111014868.3A
Authority: CN
Inventors: 黄忠兵; 王谕琳; 姜俊彦; 尹光福; 蒲曦鸣; 廖晓明
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-12-07
Anticipated expiration: 2041-08-31
Also published as: CN113750294B

Abstract

The invention relates to a nerve repair scaffold loaded with multiple gene vector microspheres and a preparation method thereof. Firstly, preparing a Polyethyleneimine (PEI) grafted Chitosan (CS) molecular chain by a carbodiimide method, and connecting RGD short peptide; forming gene carrier microspheres with positive charges by electrostatically adsorbing gene plasmids of nerve factors and electrostatically compressing; treating a degradable electrospun parallel membrane with negatively charged macromolecular chains; and finally, electrostatically adsorbing 2-6 gene carrier microspheres to the surface of the electrospun parallel membrane with negative charges by a layer-by-layer coating method, and curling to obtain the nerve regeneration scaffold for repairing the nerve gene therapy injury. The gene therapy nerve repair scaffold product obtained by the invention has good biocompatibility, in-vivo degradability and gene transfection performance, can be used for gene therapy of injury regeneration of spinal nerves and peripheral nerves, and has good potential clinical application value.

Description

Nerve repair stent loaded with multiple gene vector microspheres and preparation method thereof

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a nerve repair scaffold loaded with multiple gene vector microspheres and a preparation method thereof.

Background

Peripheral nerve damage causes sensory, motor and nutritional disorders in the area innervated by the nerve. Nerve repair by autologous or allogeneic nerve transplantation is difficult to accomplish due to limited sources, donor trauma, and other problems. Therefore, the selection of proper materials to repair the stent and the microsurgery technology are adopted, and the bridging and the sewing of the stent to repair the nerve and restore the nerve function become important means. In order to reduce the immunogen toxicity of the implant material to human bodies and avoid secondary operation wounds and the like, medical materials which can be degraded in vivo are selected. Research proves that Schwann cells can produce related nerve factors such as nerve growth factor NGF, transcription factor c-Jun, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF, short neurotrophic factor CNTF, neurotrophic factors NT-3, NT-4 and NT-5 and the like when nerves are damaged. Blood-derived macrophages that accumulate at nerve lesions also secrete interleukin 1 (IL-1), which IL-1 stimulates Schwann cells in their vicinity to secrete more nerve growth factor (Nature, 1987, 330(6149), 658 659). The gene plasmids of the factors have an important effect on treating peripheral nerve injury, and can stimulate Schwann cells to produce more nerve growth factors, thereby accelerating nerve cell regeneration and promoting nerve repair. The nerve gene of the coding growth factor is implanted through the repair bracket, slowly released and targeted to enter Schwann cells and macrophages, so that nerve cells at the damaged part can continuously and efficiently express more related growth factors through the transfection of the nerve gene, and a good microenvironment for nerve regeneration is created.

The cationic polymer Polyethyleneimine (PEI) has good proton sponge effect, so that PEI can have high electrostatic binding capacity with gene therapy pDNA (deoxyribonucleic acid), and is a gene carrier material with the highest potential; however, PEI is also cytotoxic and its toxicity increases as its molecular weight increases. The other cationic polymer, Chitosan (CS), has the characteristics of small cytotoxicity, good biocompatibility, no immunogenicity and the like, but the gene loading capacity of CS is very low. Therefore, by combining PEI with low molecular weight and CS with high molecular weight, the gene loading rate of PEI and CS can be improved, the cytotoxicity of PEI and CS can be greatly reduced, and good gene transfection efficiency of PEI and CS can be expected to be realized. The RGD short peptide as the modification consists of arginine, glycine and aspartic acid, exists in various extracellular matrixes, can be specifically combined with 11 integrins, can effectively promote the adhesion of cells to biological materials, and can help gene carrier microspheres to penetrate through membranes to enter cells, thereby further improving the gene transfection efficiency. Polylactic acid is a degradable high molecular material with no toxicity and good biocompatibility, but has low hydrophilicity and slow in-vivo degradation. The silk fibroin is natural high molecular protein directly extracted from silk, has good hydrophilicity, flexibility and tensile strength, can be rapidly degraded in a human body, and can be used as a protein or drug carrier material. The parallel silk membrane prepared by compounding the two materials and using an electrospinning method can be used as a carrier scaffold for tissue repair, and has the characteristics of good biocompatibility and high hydrophilicity, so that Schwann cells and neural axons can be guided to grow directionally along the direction of a silk axis.

At present, some progress has been made on nerve repair scaffolds, such as patent CN 107007882a, which discloses a porous scaffold for nerve repair, but fails to satisfy the growth condition of oriented spreading of nerve cells; patent CN201911143177.6 discloses a method for preparing a nerve repair scaffold, which can promote the repair of damaged nerves by electrical signals by endowing the scaffold with good electrical conductivity and forming a micro-current path with nerve tissues; the patent CN202020495157.7 discloses a nerve repair stent, which avoids the traction and secondary damage of the repair stent to the nerve tissue by the curve design of the two ends of the magnesium metal tube area. In genetic engineering, Guo Jia Song, Yangyun Hua, Zhu Si Pin and the like respectively report that gene modification of factors such as NT-3, cardiac muscle nutrient (CT-1), Fibroblast Growth Factor (FGF) and the like, Schwann cells and neural stem cells can promote the regeneration of injured tissues of spinal cord and brain (doctor academic thesis of Zhongshan university, 2003; doctor academic thesis of third military medical science, 2004; 2015 Zhejiang province bone science academic annual meeting); siniwei, Dougei et al reported that Schwann cells modified by a collagen-derived neurotrophic factor (GDNF) gene are compounded in a collagen-chitosan nerve scaffold or a PLGA nerve repair tube and bridged in an animal body to promote the repair of sciatic nerve injury (Chinese academy of medicine, 2013, 35, 655 and Beading 661; Master academic thesis of the university of Compound Dan, 2006); similarly, zhuangtian also reported that schwann cells modified by brain-derived neurotrophic factor BDNF gene, adhered to chitosan nerve scaffold, had good sciatic nerve repair effect (master's academic thesis of baoto medical college, university of inner mongol, 2010). These nerve repair scaffolds are either compounded with Schwann cells or neural stem cells transfected with a nerve factor to repair the sciatic nerve, or a nerve cell transfected with a nerve factor gene is directly injected into the lesion to repair the neural center; because the foreign body cells transfected by the gene are introduced, the problems of low biocompatibility, immunogenicity and the like still exist, the nerve repair capability of the gene is still not ideal, and the gene is difficult to apply to clinical repair treatment. Therefore, the development of a Schwann cell which has good clinical application value and can transfect the damaged part and load a plurality of gene carrier microspheres on the nerve repair bracket so as to promote the secretion of nerve factors by cells and the regeneration and repair of nerves is a current research focus.

Disclosure of Invention

In order to make up the technical defects of the existing nerve repair scaffold and improve the gene transfection efficiency and the continuous secretion of nerve factors at the damaged nerve, the patent provides a nerve repair scaffold capable of carrying out nerve gene therapy, and the nerve is guided to grow directionally by preparing a polylactic acid/silk fibroin composite parallel silk membrane; on the basis, the carrier microsphere carrying multiple nerve factor genes is adopted, so that the nerve repair scaffold with good biocompatibility and nerve gene treatment effect is developed.

In order to achieve the above object, in one aspect, the invention provides a nerve repair scaffold loaded with multiple gene vector microspheres, which is prepared by taking a polyethyleneimine grafted chitosan molecular chain, RGD peptide, gene plasmids of multiple nerve factors and a degradable electrospun parallel membrane as raw materials through a carbodiimide method, an electrostatic attraction-compression balling and a layer-by-layer coating technology; the gene carrier microspheres on the nerve repair scaffold loaded with the multiple gene carrier microspheres are formed by electrostatic adsorption and compression of the polyethylene imine grafted chitosan molecular chain modified by the RGD peptide and gene plasmids of multiple nerve factors respectively, and have surface electropositivity, cell targeting property and gene transfection property; the nerve repair stent body loaded with the multiple gene carrier microspheres is an electrospun parallel silk membrane, and has surface electronegativity and degradability; the nerve repair scaffold loaded with the multiple gene carrier microspheres is implanted at a nerve injury part through an operation, slowly released through the microspheres, penetrates through a membrane to enter nerve cells, and is used for transfecting the nerve cells to secrete corresponding nerve factors, so that the regeneration repair of the nerve injury is promoted.

On the other hand, the present invention provides the following technical solutions.

a) Preparation of CS-g-PEI: dissolving Chitosan (CS) in an acetic acid solution, followed by precipitation with NaOH solution; dissolving chitosan in dimethyl sulfoxide, mixing with a maleic anhydride solution, and heating to react for 8-12 h; precipitating the reactant with acetone to obtain maleated chitosan; dissolving the maleated chitosan in NaOH solution, adding PEI (200-20000 Da) aqueous solution, reacting for 6-36 h at 40-80 ℃, dialyzing in secondary water, and drying in vacuum to obtain PEI-g-CS; finally, the product was dissolved in PBS and the RGD peptide was attached to the amino group of PEI-g-CS by the carbodiimide method.

b) Preparing gene vector microspheres: preparing a PEI-g-CS-RGD aqueous solution with the concentration of 1-5 mg/mL, and then dropwise adding a nerve factor gene plasmid (pDNA) dispersion liquid until the solution has light microemulsion color, thus obtaining the carrier microsphere formed by electrostatic adsorption of gene plasmids and compression.

c) Preparation of an electrospun parallel membrane: dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to a certain proportion to obtain an electrostatic spinning solution; then, carrying out electrospinning in an electric field of 11-15 kV, and carrying out rotary collection by using a roller attached with metal foil paper to obtain parallel silk films attached to the metal foil paper; and soaking in absolute ethyl alcohol, and vacuum drying to obtain the electrospinning parallel membrane with enhanced mechanical strength.

d) Surface modification of the electrospun parallel membrane: soaking the silk membrane in a polylysine solution for 5-20 min, drying, and then soaking in a polyglutamic acid solution for 0.5-4 h, so that two layers of polyglutamic acid molecular chains with different charges and a large amount of negative charges are adsorbed on the surface of the electrospun parallel silk membrane.

e) Preparing a nerve repair scaffold loaded with gene vector microspheres: soaking the parallel silk film in a gene vector microsphere dispersion liquid for 0.5-3 h, and then soaking in a polyglutamic acid solution for 0.5-4 h; then soaking the microspheres in 1-8 mg/mL second gene vector microsphere dispersion for 0.5-3 h, and then soaking the microspheres in polyglutamic acid solution for 0.5-4 h; repeating the steps for 2-6 times, drying and then curling into a tube to obtain the nerve repair scaffold loaded with the multiple gene vector microspheres.

As a further scheme of the invention, in the step a), the chitosan is subjected to a process of dissolving in acetic acid and precipitating again, the molar ratio of the chitosan to the maleic anhydride is 2: 1-6, and the reaction is carried out for 8-12 h at 40-80 ℃; the molar ratio of the amino on the chitosan to the imino on the PEI is 2: 1-4, and the reaction is carried out for 6-36 h at the temperature of 40-80 ℃; the concentration of the CS-g-PEI is 1-3 mg/mL and 0.05-1.0 mg/mL respectively when the CS-g-PEI reacts with the RGD peptide.

As a further scheme of the invention, in the step b), in the balling process, the concentration of CS-g-PEI is 1-5 mg/mL, the concentration of gene plasmid pDNA suspension of each nerve factor is 0.01-1.0 mg/mL respectively, and the mass ratio of pDNA to the material is 1: 20-45.

As a further scheme of the invention, in the step b), the gene plasmid pDNA types of the nerve factors in the carrier microspheres comprise gene plasmids of nerve growth factor NGF, transcription factor c-Jun, interleukin 1 IL-1, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF, short form neurotrophic factor CNTF and neurotrophic factor III NT-3; one gene vector microsphere can be loaded in the nerve repair scaffold, and 2-6 gene plasmid vector microspheres can also be loaded in the nerve repair scaffold.

As a further scheme of the invention, in the step c), the mass ratio of polylactic acid to silk fibroin is 1-6: 1, the mass volume fraction of the polylactic acid and the silk fibroin after being dissolved in hexafluoroisopropanol is 6-15 wt%, the electric field intensity of electrospinning is 11-15 kV, the electrospinning time is 1.5-3 h, a silk film is collected on a roller attached with a metal foil, and the rotating speed is 400-2000 rpm; and soaking the parallel silk film attached to the metal foil in absolute ethyl alcohol for 5-30 min to improve the axial mechanical strength of the silk film.

As a further scheme of the invention, in the step d), the electrospun parallel filament membrane is firstly soaked in a polylysine solution with the concentration of 0.01-0.5 mg/mL for 5-20 min, and then soaked in a polyglutamic acid solution with the concentration of 1-8 mg/mL for 0.5-4 h, so that the outermost layer of the electrospun parallel filament membrane has a large amount of polyglutamic acid negative charges.

As a further scheme of the invention, in the step e), the two types of charge molecular chains are coated layer by layer, and the outermost layer of the electrospinning film is negative charge, the electrospinning film is firstly soaked in 1-8 mg/mL of gene carrier microsphere dispersion liquid for 0.5-3 h, and then soaked in 1-8 mg/mL of polyglutamic acid solution for 0.5-4 h; then soaking the microspheres in 1-8 mg/mL second gene vector microsphere dispersion liquid for 0.5-3 h; repeating the steps one to many times, drying and then curling into a tube to obtain the nerve repair scaffold loaded with the gene vector microspheres.

As a further scheme of the invention, in the step e), two kinds of charge molecular chains are coated layer by layer, the outermost layer is an electrospinning film with negative charges, and a plurality of gene carrier microspheres are loaded, wherein 2-3 kinds of interleukin 1 IL-1, transcription factor c-Jun and NGF are loaded, and then 0-3 kinds of any one of neurotrophic factor III NT-3, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF and short neurotrophic factor CNTF are loaded, so that the nerve repair scaffold with 2-6 kinds of carrier microspheres is combined.

As a further scheme of the invention, in the step e), 2-3 of interleukin 1 IL-1, transcription factor c-Jun and NGF are added into the gene vector microsphere dispersion liquid, and then the gene vector microspheres are mixed with 0-3 of any one of neurotrophic factor III NT-3, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF and short form neurotrophic factor CNTF to form 2-6 gene vector microspheres, wherein the mass ratio of the 2-6 gene vector microspheres is 1-4: 1, 1-4: 1-2: 1:1, and 1-4: 1-2: 1:1: 1.

Compared with the prior art, the invention has the beneficial effects that:

the electrospun membrane nerve repair scaffold loaded with the gene vector microspheres prepared by the invention has good biocompatibility, can well load nerve factor gene plasmids, and can be used for gene therapy of regeneration repair of long-segment nerve defects; the invention has simple preparation process, easily obtained material source and low cost, can be used as an ideal biological functional material and has good potential application value in the field of peripheral nerve repair and spinal nerve repair.

Drawings

FIG. 1 is a schematic diagram of the preparation process of gene vector microspheres.

FIG. 2 is a schematic diagram of the preparation process of the nerve repair scaffold loaded with multiple gene vector microspheres.

FIG. 3 is a transmission electron micrograph of a gene vector microsphere.

FIG. 4 is a photograph of gel electrophoresis of nucleic acids after gene vectors were combined with pDNA at various ratios.

FIG. 5 is a scanning electron micrograph of an electrospun parallel membrane loaded with gene vector microspheres.

FIG. 6 is a confocal laser microscopy photograph of Schwann cells grown on electrospun parallel-filamented neural restoration scaffolds loaded with gene vector microspheres.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Dissolving chitosan (CS, 6000 Da) in 1% acetic acid solution, precipitating with 0.1M NaOH solution, collecting, and washing; dissolving the obtained chitosan in dimethyl sulfoxide, adding the obtained chitosan into dimethyl sulfoxide containing maleic anhydride, and adjusting the molar ratio of amino on the chitosan to carboxyl on the maleic anhydride to be 2: 1; reacting at 60 ℃ for 8h, precipitating with acetone, and drying to obtain maleated chitosan; dissolving maleated chitosan in 0.1M NaOH solution, adding polyethyleneimine (PEI, 600 Da) aqueous solution, adjusting the mass ratio of chitosan to polyethyleneimine to be 2:1, stirring and reacting at 60 ℃ for 18h, then adjusting the pH value of the reaction solution to 7 by using hydrochloric acid, dialyzing for 3 days by using secondary water, and freeze-drying to obtain CS-g-PEI; dissolving the CS-g-PEI product in PBS to prepare a solution of 2mg/mL, and coupling RGD peptide by a carbodiimide method; preparing CS-g-PEI-RGD aqueous solution with the concentration of 2mg/mL, slowly dripping gene plasmid pDNA dispersion liquid of a nerve factor, and stopping dripping when the mass ratio of the pDNA to the material is 1:25, so as to obtain gene vector microspheres containing the pDNA; the method is used for preparing the microspheres containing NGF and IL-1 gene vectors respectively.

Dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to the mass ratio of 2:1 to prepare a spinning solution with the mass volume fraction of 7.5wt%, performing electrospinning for 1.5h in an electric field of 11kV, attaching the tinfoil paper to a collecting roller, adjusting the speed of the roller to be 500rpm, and collecting the parallel silk film on the tinfoil paper; soaking the silk film in absolute ethyl alcohol for 10min and drying at normal temperature to obtain a parallel silk film with improved strength; soaking the parallel silk film in 0.1 mg/mL polylysine solution for 5min, and then soaking in 1 mg/mL polyglutamic acid solution for 0.5h to make the surface of the silk film bring a large amount of negative charges; soaking the parallel silk membrane in 2mg/mL NGF gene vector microsphere dispersion liquid for 1h to obtain the parallel silk membrane loaded with NGF gene vector microspheres; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL IL-1 gene vector microsphere dispersion liquid for 1h to obtain a parallel silk membrane loaded with NGF and IL-1 gene vector microspheres; and finally, drying and curling into a tube to obtain the nerve repair scaffold loaded with the two gene vector microspheres.

Example 2

Dissolving chitosan (CS, 8000 Da) in 1% acetic acid solution, precipitating with 0.1M NaOH solution, collecting, and washing; dissolving the obtained chitosan in dimethyl sulfoxide, adding the obtained chitosan into dimethyl sulfoxide containing maleic anhydride, and adjusting the molar ratio of amino on the chitosan to carboxyl on the maleic anhydride to be 2: 1; reacting at 60 ℃ for 8h, precipitating with acetone, and drying to obtain maleated chitosan; dissolving maleated chitosan in 0.1M NaOH solution, adding polyethyleneimine (PEI, 400 Da) aqueous solution, adjusting the mass ratio of chitosan to polyethyleneimine to be 2:1, stirring and reacting at 60 ℃ for 18h, then adjusting the pH value of the reaction solution to 7 by using hydrochloric acid, dialyzing for 3 days by using secondary water, and freeze-drying to obtain CS-g-PEI; dissolving the CS-g-PEI product in PBS to prepare a solution of 2mg/mL, and coupling RGD peptide by a carbodiimide method; preparing CS-g-PEI-RGD aqueous solution with the concentration of 2mg/mL, slowly dripping gene plasmid pDNA dispersion liquid of a nerve factor, and stopping dripping when the mass ratio of the pDNA to the material is 1:25, so as to obtain gene vector microspheres containing the pDNA; the method is used for preparing the microspheres containing the NGF, the IL-1 and the c-Jun gene vectors respectively.

Dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to the mass ratio of 2:1 to prepare spinning solution with the mass volume fraction of 8.0wt%, performing electrospinning for 1.5h in a 10kV electric field, attaching the tin foil paper to a collecting roller, adjusting the speed of the roller to 700rpm, and collecting parallel silk films on the tin foil paper; soaking the silk film in absolute ethyl alcohol for 10min and drying at normal temperature to obtain a parallel silk film with improved strength; soaking the parallel silk film in 0.1 mg/mL polylysine solution for 5min, and then soaking in 1 mg/mL polyglutamic acid solution for 0.5h to make the surface of the silk film bring a large amount of negative charges; soaking the parallel silk membrane in 2mg/mL NGF gene vector microsphere dispersion liquid for 1h to obtain the parallel silk membrane loaded with NGF gene vector microspheres; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL IL-1 gene vector microsphere dispersion for 1 h; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL c-Jun gene vector microsphere dispersion liquid for 1h to obtain a parallel silk membrane loaded with NGF, IL-1 and c-Jun gene vector microspheres; and finally, drying and curling into a tube to obtain the nerve repair scaffold loaded with the three gene vector microspheres.

Example 3

Dissolving chitosan (CS, 7000 Da) in 1% acetic acid solution, precipitating with 0.1M NaOH solution, collecting, and washing; dissolving the obtained chitosan in dimethyl sulfoxide, adding the obtained chitosan into dimethyl sulfoxide containing maleic anhydride, and adjusting the molar ratio of amino on the chitosan to carboxyl on the maleic anhydride to be 2: 1; reacting at 60 ℃ for 8h, precipitating with acetone, and drying to obtain maleated chitosan; dissolving maleated chitosan into 0.1M NaOH solution, adding polyethyleneimine (PEI, 800 Da) aqueous solution, adjusting the mass ratio of chitosan to polyethyleneimine to be 2:1, stirring and reacting at 60 ℃ for 15h, then adjusting the pH value of the reaction solution to 7 by using hydrochloric acid, dialyzing for 3 days by using secondary water, and freeze-drying to obtain CS-g-PEI; dissolving the CS-g-PEI product in PBS to prepare a solution of 2mg/mL, and coupling RGD peptide by a carbodiimide method; preparing CS-g-PEI-RGD aqueous solution with the concentration of 2mg/mL, slowly dripping gene plasmid pDNA dispersion liquid of a nerve factor, and stopping dripping when the mass ratio of the pDNA to the material is 1:25, so as to obtain gene vector microspheres containing the pDNA; the method is used for preparing the microspheres containing four gene vectors of NGF, IL-1, c-Jun and NT-3 respectively.

Dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to the mass ratio of 2:1 to prepare spinning solution with the mass volume fraction of 8.0wt%, performing electrospinning for 1.5h in a 10kV electric field, attaching the tin foil paper to a collecting roller, adjusting the speed of the roller to 700rpm, and collecting parallel silk films on the tin foil paper; soaking the silk film in absolute ethyl alcohol for 10min and drying at normal temperature to obtain a parallel silk film with improved strength; soaking the parallel silk film in 0.1 mg/mL polylysine solution for 5min, and then soaking in 1 mg/mL polyglutamic acid solution for 0.5h to make the surface of the silk film bring a large amount of negative charges; soaking the parallel silk membrane in 2mg/mL NGF gene vector microsphere dispersion liquid for 1h to obtain the parallel silk membrane loaded with NGF gene vector microspheres; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL IL-1 gene vector microsphere dispersion for 1 h; then soaking the silk film in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk film in 2mg/mL c-Jun gene carrier microsphere dispersion liquid for 1h, then soaking the silk film in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk film in 2mg/mL NT-3 gene carrier microsphere dispersion liquid for 1h to obtain a parallel silk film loaded with four gene carrier microspheres of NGF, IL-1, c-Jun and NT-3; and finally, drying and curling into a tube to obtain the nerve repair scaffold loaded with the four gene vector microspheres.

Example 4

Dissolving chitosan (CS, 7000 Da) in 1% acetic acid solution, precipitating with 0.1M NaOH solution, collecting, and washing; dissolving the obtained chitosan in dimethyl sulfoxide, adding the obtained chitosan into dimethyl sulfoxide containing maleic anhydride, and adjusting the molar ratio of amino on the chitosan to carboxyl on the maleic anhydride to be 2: 1; reacting at 60 ℃ for 8h, precipitating with acetone, and drying to obtain maleated chitosan; dissolving maleated chitosan into 0.1M NaOH solution, adding polyethyleneimine (PEI, 800 Da) aqueous solution, adjusting the mass ratio of chitosan to polyethyleneimine to be 2:1, stirring and reacting at 60 ℃ for 18h, then adjusting the pH value of the reaction solution to 7 by using hydrochloric acid, dialyzing for 3 days by using secondary water, and freeze-drying to obtain CS-g-PEI; dissolving the CS-g-PEI product in PBS to prepare a solution of 2mg/mL, and coupling RGD peptide by a carbodiimide method; preparing CS-g-PEI-RGD aqueous solution with the concentration of 2mg/mL, slowly dripping gene plasmid pDNA dispersion liquid of a nerve factor, and stopping dripping when the mass ratio of the pDNA to the material is 1:25, so as to obtain gene vector microspheres containing the pDNA; the method is used for preparing five gene vector microspheres containing NGF, IL-1, c-Jun, NT-3 and BDNT respectively.

Dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to the mass ratio of 2:1 to prepare spinning solution with the mass volume fraction of 8.0wt%, performing electrospinning for 1.5h in a 10kV electric field, attaching the tin foil paper to a collecting roller, adjusting the speed of the roller to 700rpm, and collecting parallel silk films on the tin foil paper; soaking the silk film in absolute ethyl alcohol for 10min and drying at normal temperature to obtain a parallel silk film with improved strength; soaking the parallel silk film in 0.1 mg/mL polylysine solution for 5min, and then soaking in 1 mg/mL polyglutamic acid solution for 0.5h to make the surface of the silk film bring a large amount of negative charges; soaking the parallel silk membrane in 2mg/mL NGF gene vector microsphere dispersion liquid for 1h to obtain the parallel silk membrane loaded with NGF gene vector microspheres; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL IL-1 gene vector microsphere dispersion for 1 h; soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk membrane in 2mg/mL c-Jun gene carrier microsphere dispersion for 1h, then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk membrane in 2mg/mL NT-3 gene carrier microsphere dispersion for 1h, finally soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk membrane in 2mg/mL BDNF gene carrier microsphere dispersion for 1h, and obtaining the parallel silk membrane loaded with five gene carrier microspheres of NGF, IL-1, c-Jun, NT-3 and BDNT; and finally, drying and curling into a tube to obtain the nerve repair scaffold loaded with five gene vector microspheres.

Example 5

Dissolving chitosan (CS, 10000 Da) in 1% acetic acid solution, precipitating with 0.1M NaOH solution, collecting, and washing; dissolving the obtained chitosan in dimethyl sulfoxide, adding the obtained chitosan into dimethyl sulfoxide containing maleic anhydride, and adjusting the molar ratio of amino on the chitosan to carboxyl on the maleic anhydride to be 2: 1; reacting at 60 ℃ for 8h, precipitating with acetone, and drying to obtain maleated chitosan; dissolving maleated chitosan into 0.1M NaOH solution, adding polyethyleneimine (PEI, 800 Da) aqueous solution, adjusting the mass ratio of chitosan to polyethyleneimine to be 2:1, stirring and reacting at 60 ℃ for 18h, then adjusting the pH value of the reaction solution to 7 by using hydrochloric acid, dialyzing for 3 days by using secondary water, and freeze-drying to obtain CS-g-PEI; dissolving the CS-g-PEI product in PBS to prepare a solution of 2mg/mL, and coupling RGD peptide by a carbodiimide method; preparing CS-g-PEI-RGD aqueous solution with the concentration of 2mg/mL, slowly dripping gene plasmid pDNA dispersion liquid of a nerve factor, and stopping dripping when the mass ratio of the pDNA to the material is 1:25, so as to obtain gene vector microspheres containing the pDNA; the method is used for preparing six kinds of gene vector microspheres containing NGF, IL-1, c-Jun, NT-3, BDNF and GDNF respectively.

Dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to the mass ratio of 2:1 to prepare spinning solution with the mass volume fraction of 8.0wt%, performing electrospinning for 1.5h in a 10kV electric field, attaching the tin foil paper to a collecting roller, adjusting the speed of the roller to 700rpm, and collecting parallel silk films on the tin foil paper; soaking the silk film in absolute ethyl alcohol for 10min and drying at normal temperature to obtain a parallel silk film with improved strength; soaking the parallel silk film in 0.1 mg/mL polylysine solution for 5min, and then soaking in 1 mg/mL polyglutamic acid solution for 0.5h to make the surface of the silk film bring a large amount of negative charges; soaking the parallel silk membrane in 2mg/mL NGF gene vector microsphere dispersion liquid for 1h to obtain the parallel silk membrane loaded with NGF gene vector microspheres; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL IL-1 gene vector microsphere dispersion for 1 h; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, soaking the silk membrane in 2mg/mL c-Jun gene vector microsphere dispersion for 1h, then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk membrane in 2mg/mL NT-3 gene carrier microsphere dispersion liquid for 1h, then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk membrane in 2mg/mL BDNF gene carrier microsphere dispersion liquid for 1h, finally soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk membrane in 2mg/mL GDNF gene carrier microsphere dispersion liquid for 1h, and obtaining the parallel silk membrane loaded with six gene carrier microspheres of NGF, IL-1, c-Jun, NT-3, BDNF and GDNF; and finally, drying and curling into a tube to obtain the nerve repair scaffold loaded with six gene vector microspheres.

Example 6

Dissolving chitosan (CS, 10000 Da) in 1% acetic acid solution, precipitating with 0.1M NaOH solution, collecting, and washing; dissolving the obtained chitosan in dimethyl sulfoxide, adding the obtained chitosan into dimethyl sulfoxide containing maleic anhydride, and adjusting the molar ratio of amino on the chitosan to carboxyl on the maleic anhydride to be 2: 1; reacting at 60 ℃ for 8h, precipitating with acetone, and drying to obtain maleated chitosan; dissolving maleated chitosan into 0.1M NaOH solution, adding polyethyleneimine (PEI, 800 Da) aqueous solution, adjusting the mass ratio of chitosan to polyethyleneimine to be 2:1, stirring and reacting at 60 ℃ for 18h, then adjusting the pH value of the reaction solution to 7 by using hydrochloric acid, dialyzing for 3 days by using secondary water, and freeze-drying to obtain CS-g-PEI; dissolving the CS-g-PEI product in PBS to prepare a solution of 2mg/mL, and coupling RGD peptide by a carbodiimide method; preparing CS-g-PEI-RGD aqueous solution with the concentration of 2mg/mL, slowly dripping gene plasmid pDNA dispersion liquid of a nerve factor, and stopping dripping when the mass ratio of the pDNA to the material is 1:25, so as to obtain gene vector microspheres containing the pDNA; the method is used for preparing six kinds of gene vector microspheres containing NGF, IL-1, c-Jun, NT-3, BDNF and CNTF respectively.

Dissolving polylactic acid and silk fibroin in hexafluoroisopropanol according to the mass ratio of 2:1 to prepare spinning solution with the mass volume fraction of 8.0wt%, performing electrospinning for 1.5h in a 10kV electric field, attaching the tin foil paper to a collecting roller, adjusting the speed of the roller to 700rpm, and collecting parallel silk films on the tin foil paper; soaking the silk film in absolute ethyl alcohol for 10min and drying at normal temperature to obtain a parallel silk film with improved strength; soaking the parallel silk film in 0.1 mg/mL polylysine solution for 5min, and then soaking in 1 mg/mL polyglutamic acid solution for 0.5h to make the surface of the silk film bring a large amount of negative charges; soaking the parallel silk membrane in 2mg/mL NGF gene vector microsphere dispersion liquid for 1h to obtain the parallel silk membrane loaded with NGF gene vector microspheres; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, and then soaking the silk membrane in 2mg/mL IL-1 gene vector microsphere dispersion for 1 h; then soaking the silk membrane in 2mg/mL polyglutamic acid solution for 1h, soaking the silk membrane in 2mg/mL c-Jun gene vector microsphere dispersion for 1h, then soaking the silk film in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk film in 2mg/mL NT-3 gene carrier microsphere dispersion liquid for 1h, then soaking the silk film in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk film in 2mg/mL BDNF gene carrier microsphere dispersion liquid for 1h, finally soaking the silk film in 2mg/mL polyglutamic acid solution for 1h, then soaking the silk film in 2mg/mL CNTF gene carrier microsphere dispersion liquid for 1h, and obtaining the parallel silk film loaded with six gene carrier microspheres of NGF, IL-1, c-Jun, NT-3, BDNF and CNTF; and finally, drying and curling into a tube to obtain the nerve repair scaffold loaded with six gene vector microspheres.

FIG. 1 is a schematic diagram of the preparation process of gene vector microspheres; PEI and maleic acid maleated CS are reacted by a carbodiimide method to form PEI-g-CS, RGD peptide is connected to a PEI-g-CS chain by the carbodiimide method, and gene plasmid turbid liquid (pDNA) of various nerve factors is respectively and slowly dripped to obtain the carrier microsphere containing the corresponding nerve factor gene.

FIG. 2 is a schematic diagram of the preparation process of a nerve repair scaffold loaded with multiple gene vector microspheres; soaking the electro-spun parallel silk membrane collected on the metal foil paper in absolute ethyl alcohol, and drying to obtain the parallel silk membrane with improved strength; soaking the electrospun membrane in polyglutamic acid solution, wherein the surface layer of the electrospun membrane has a large amount of negative charges; soaking the electrospun parallel filament membrane by using gene carrier microsphere dispersion liquid with positive charges to load one gene carrier microsphere, and then soaking by using polyglutamic acid solution and second gene carrier microsphere dispersion liquid in sequence to load the second gene carrier microsphere; repeating the steps for a plurality of times, and then coiling the microspheres into tubes to obtain the nerve repair scaffold loaded with the gene vector microspheres.

FIG. 3 is a transmission electron micrograph of a gene vector microsphere; it can be clearly seen that the prepared gene vector fine particles have a spherical shape with a diameter of 50 to 200 nm.

FIG. 4 is a photograph of a nucleic acid gel electrophoresis of a gene vector in combination with pDNA at various ratios; when the mass ratio of CS-PEI to DNA was 2, a clear pDNA band could be observed in the gel; when the mass ratio of CS-PEI to DNA is 0.5 or 1, no band is generated in the gel, namely, pDNA is totally stayed in the spotting hole; these results show that CS-PEI can completely bind pDNA when the amount of CS-PEI added is 2 times or more of that of DNA, and electrostatically compress to form compact gene vector microspheres.

FIG. 5 is a scanning electron micrograph of an electrospun parallel filament membrane loaded with gene vector microspheres; the electrospinning filaments with the diameter of about 0.5-1.0 micron are mostly arranged in parallel, and white arrows indicate that a plurality of gene vector microspheres with the diameter of about 0.1 micron are adsorbed on the filament surfaces.

FIG. 6 is a confocal laser microscopy photograph of Schwann cells grown on electrospun parallel-filamented neural restoration scaffolds loaded with gene vector microspheres; grey arrows indicate fluorescently stained nuclei; white arrows indicate the cells, expressed corresponding proteins after successful transfection using microspheres loaded with a protein gene plasmid vector on the scaffold.

The present invention is not limited to the details of the above-described exemplary embodiments, and may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments are exemplary and non-limiting, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims

1. The nerve repair scaffold loaded with the multiple gene carrier microspheres is characterized by being prepared by taking a polyethyleneimine grafted chitosan molecular chain, arginine-glycine-aspartic acid RGD short peptide, multiple nerve factor gene plasmids and a degradable electrospun parallel membrane with negative charges as raw materials through carbodiimide reaction, electrostatic attraction-compression balling and dip-coating layer-by-layer coating technology; the gene carrier microspheres on the nerve repair scaffold loaded with the multiple gene carrier microspheres are polyethylene imine grafted chitosan molecular chains modified by RGD peptide and 2-6 nerve factor gene plasmids, and form 2-6 corresponding microspheres through electrostatic adsorption and compression, wherein the corresponding microspheres have a large amount of surface positive charge, cell targeting-cell penetrating peptide RGD and transfection nerve factor genes; the nerve repair scaffold loaded with the multiple gene carrier microspheres is a degradable electrospun parallel membrane with a negative charge macromolecular chain on the surface, and has a large amount of negative charges and degradability in vivo; the repairing bracket is combined with the nerve gene carrier microsphere through electrostatic adsorption; the repair scaffold loaded with the multiple nerve gene carrier microspheres is filled at a spinal nerve injury part through an operation, or is bridged and sutured at a peripheral nerve injury part through an operation, and then enters Schwann cells and the like at the injury part in vivo through microsphere diffusion, RGD peptide adhesion and membrane penetration, and nerve factor gene plasmids are released, so that Schwann cells and the like are transfected and corresponding nerve factor proteins are continuously secreted, and further, the proliferation of nerve cells, the extension of nerve axons, the regeneration and repair of injured nerves and the functional recovery of target muscles dominated by the nerve factor proteins are promoted.

2. The nerve repair scaffold loaded with multiple gene vector microspheres and the preparation method are characterized in that the preparation process comprises the following steps:

a) preparation of polyethyleneimine grafted chitosan (PEI-g-CS): dissolving chitosan (CS, molecular weight 2000-200000 Da) in acetic acid solution with concentration of 0.5-3.0 wt%, and precipitating and washing with 0.1M NaOH solution; dispersing the obtained chitosan in dimethyl sulfoxide, adding the chitosan into a dimethyl sulfoxide solution of maleic anhydride to enable the molar ratio of amino groups on the chitosan to carboxyl groups on the maleic anhydride to be 2: 1-6, and reacting for 8-12 hours at the temperature of 40-80 ℃; then, precipitating the product with acetone and drying to obtain maleated chitosan; dissolving the maleated chitosan in 0.01-1.0M NaOH solution, then adding polyethyleneimine (PEI, 200 plus 10000 Da) aqueous solution to enable the molar ratio of maleic acid carboxyl on the chitosan to imino on the PEI to be 2: 1-4, and reacting for 6-36 h at 40-80 ℃; then adjusting the pH value of the system to be neutral by using hydrochloric acid, dialyzing by using secondary water, and freeze-drying to obtain a PEI-g-CS product; finally, dissolving the product in PBS (pH value of 7.2) to prepare a solution of 1-3 mg/mL, and connecting the RGD peptide to a PEI-g-CS chain by using a carbodiimide technology to obtain an RGD-PEI-g-CS molecular chain product;

b) preparing PEI-g-CS gene carrier microspheres: respectively and slowly dripping gene plasmid (pDNA, the concentration of which is 0.01-1.0 mg/mL) turbid liquids of various nerve factors into a PEI-g-CS-RGD aqueous solution with the concentration of 1-5 mg/mL until slight opalescence appears in a transparent solution, namely that PEI-g-CS-RGD is adsorbed with pDNA through static electricity and is compressed to form carrier microspheres containing corresponding nerve factor genes; controlling the mass ratio of various pDNA to the material RGD-PEI-g-CS to be 1: 10-50;

c) preparing a reinforced electrospinning parallel filament membrane: mixing polylactic acid and silk fibroin according to the mass ratio of 1-8: 1, and dissolving the mixture in hexafluoroisopropanol to prepare an electrospinning solution with the mass volume fraction of 6-15 wt%; attaching the metal foil paper to a collecting roller, and adjusting the speed of the roller to 400-1500 rpm; electrospinning for 1.5-3 h in an electric field of 11-15 kV, collecting the electrospun fibers on metal foil paper, soaking in absolute ethyl alcohol for 5-30 min, and finally drying to obtain a reinforced electrospun parallel membrane;

d) surface modification of the electrospun parallel membrane: soaking the electrospun membrane in polylysine solution with the concentration of 0.01-0.5 mg/mL for 5-20 min, and then soaking in polyglutamic acid solution with the concentration of 1-8 mg/mL for 0.5-4 h, so that the surface of the electrospun parallel membrane is coated by two molecular chains with different charges layer by layer, and the outermost layer of the electrospun parallel membrane has a large amount of negative charges;

e) carrying gene vector microspheres on an electrospun parallel filament membrane: soaking the electrospun parallel filament membrane with the negative charges on the outer layer in 1-8 mg/mL gene carrier microsphere dispersion liquid for 0.5-3 h; then soaking the microspheres in a polyglutamic acid solution with the concentration of 1-8 mg/mL for 0.5-4 h, and soaking the microspheres in a second gene carrier microsphere dispersion liquid with the concentration of 1-8 mg/mL for 0.5-3 h; repeating the steps for a plurality of times to obtain a final product, namely the nerve repair scaffold loaded with the gene vector microspheres.

3. The nerve repair scaffold carrying multiple gene vector microspheres and the preparation method thereof as claimed in claim 2, wherein in the step a), the chitosan is subjected to dissolution, reprecipitation and precipitation, the molar ratio of amino groups on a molecular chain of the chitosan to carboxyl groups on maleic anhydride is 2: 1-6, and the two are subjected to reaction at 60 ℃ for 8-12 h; the molar ratio of the maleic acid carboxyl on the molecular chain of the chitosan to the imino group on the PEI is 2: 1-4, and the maleic acid carboxyl and the imino group on the PEI need to react for 6-36 h at the temperature of 40-80 ℃; the concentration of the CS-g-PEI is 1-3 mg/mL and 0.05-1.0 mg/mL respectively when the CS-g-PEI reacts with the RGD peptide.

4. The nerve repair scaffold loaded with multiple gene carrier microspheres and the preparation method of the nerve repair scaffold, as claimed in claim 2, are characterized in that in step b), the concentration of CS-g-PEI is 1-5 mg/mL, the concentration of gene plasmid pDNA suspension of each nerve factor is 0.01-1.0 mg/mL, and the mass ratio of each pDNA to CS-g-PEI is 1: 20-45.

5. The nerve repair scaffold loaded with multiple gene carrier microspheres and the preparation method thereof as claimed in claim 2, wherein in step b), the gene plasmid pDNA types of the cell factors in the carrier microspheres comprise gene plasmids of nerve growth factor NGF, transcription factor c-Jun, interleukin 1 IL-1, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF, short form neurotrophic factor CNTF, and neurotrophic factor III NT-3; one gene vector microsphere can be loaded in the nerve repair scaffold, and 2-6 gene plasmid vector microspheres can also be loaded in the nerve repair scaffold.

6. The method for preparing the gene vector microsphere-loaded nerve repair scaffold as claimed in claim 2, wherein in step c), the mass ratio of polylactic acid to silk fibroin is 1-6: 1, the mass volume fraction of polylactic acid and silk fibroin dissolved in hexafluoroisopropanol is 6-15 wt%, the electric field intensity during electrospinning is 11-15 kV, and the electrospinning time is 1.5-3 h; collecting the silk film on a roller attached with metal foil, wherein the rotating speed of the roller is 400-2000 rpm; and soaking the parallel silk film attached to the metal foil in absolute ethyl alcohol for 5-30 min.

7. The nerve repair scaffold carrying multiple gene vector microspheres and the preparation method of the nerve repair scaffold carrying multiple gene vector microspheres of claim 2 are characterized in that in the step d), the electrospun parallel silk membrane is firstly soaked in a polylysine solution with the concentration of 0.01-0.5 mg/mL for 5-20 min, and then soaked in a polyglutamic acid solution with the concentration of 1-8 mg/mL for 0.5-4 h, so that the outermost layer of the electrospun parallel silk membrane carries a large amount of negative charges.

8. The nerve repair scaffold loaded with multiple gene carrier microspheres and the preparation method thereof as claimed in claim 2, wherein in step e), two kinds of charged molecular chains are coated layer by layer, and the outermost layer is an electrospun membrane with negative charges, which is firstly soaked in 1-8 mg/mL of a gene carrier microsphere dispersion liquid for 0.5-3 h; then soaking the microspheres in a polyglutamic acid solution with the concentration of 1-8 mg/mL for 0.5-4 h, and soaking the microspheres in a second gene carrier microsphere dispersion liquid with the concentration of 1-8 mg/mL for 0.5-3 h; repeating the steps for a plurality of times to obtain a final product, namely the nerve repair scaffold loaded with the gene vector microspheres.

9. The nerve repair scaffold loaded with multiple gene carrier microspheres and the preparation method thereof as claimed in claims 2 and 5, wherein in step e), the outermost layer is an electrospun parallel membrane with negative charges, and the loaded multiple gene carrier microspheres are: 2-3 of interleukin 1 IL-1, transcription factor c-Jun and NGF, namely 0-3 of any of neurotrophic factor III NT-3, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF and short neurotrophic factor CNTF, so that the composition is a nerve repair scaffold loaded with 2-6 carrier microspheres.

10. The nerve repair scaffold carrying gene vector microspheres and the preparation method of the nerve repair scaffold carrying gene vector microspheres according to claims 5 and 9 are characterized in that in the step e), 2-3 of interleukin 1 IL-1, transcription factor c-Jun and NGF are added into a gene vector microsphere dispersion liquid, and then the mass ratio of 2-6 gene vector microspheres to any 0-3 of neurotrophic factor III NT-3, brain-derived neurotrophic factor BDNF, glial-derived neurotrophic factor GDNF and short form neurotrophic factor CNTF is 1-4: 1, 1-4: 1-2: 1:1 and 1-4: 1-2: 1:1: 1.