阅读说明：本技术 用于周围神经损伤修复的磁力响应性外泌体及其制备方法 (Magnetic-responsive exosome for peripheral nerve injury repair and preparation method thereof ) 是由 刘鐘阳 唐佩福 张阳 张里程 李建涛 刘建恒 李明 崔翔 邓俊豪 于 2020-06-24 设计创作，主要内容包括：本发明属于神经修复技术领域,本发明公开了一种用于周围神经损伤修复的磁力响应性外泌体及制备方法,其中,制备方法包括如下步骤：用脂肪间充质干细胞内吞磁性纳米微粒,然后通过梯度超速离心法提取磁性生物材料得到所述的磁力响应型外泌体。本发明的外泌体具有生物相容性好的优点。(The invention belongs to the technical field of nerve repair, and discloses a magnetic-responsive exosome for repairing peripheral nerve injury and a preparation method thereof, wherein the preparation method comprises the following steps: and (2) endocytosing the magnetic nanoparticles by using adipose-derived mesenchymal stem cells, and then extracting the magnetic biomaterial by using a gradient ultracentrifugation method to obtain the magnetic response type exosome. The exosome has the advantage of good biocompatibility.)

1. A preparation method of a magnetic-force-responsive exosome for peripheral nerve injury repair is characterized by comprising the following steps: and (2) endocytosing the magnetic nanoparticles by using adipose-derived mesenchymal stem cells, and then extracting the magnetic biomaterial by using a gradient ultracentrifugation method to obtain the magnetic response type exosome.

2. The method for preparing a magnetically responsive exosome for peripheral nerve injury repair according to claim 1, comprising the steps of:

(1) primarily extracting and culturing adipose-derived mesenchymal stem cells;

(2) synthesizing magnetic nanoparticles: adding 0.01M sulfuric acid into iron sulfate heptahydrate, wherein the volume-mass ratio of the sulfuric acid to the iron sulfate heptahydrate is as follows: 48.7vol:1wt, fully mixing under nitrogen flow filling to prepare ferrous sulfate solution; mixing potassium nitrate and sodium hydroxide according to the mass ratio of 2.8:1 to obtain a mixture, and then dissolving the mixture in deionized water under the nitrogen state to obtain a mixture solution; adding the ferrous sulfate solution dropwise into the mixture solution under continuous stirring; after precipitation, heating to 85-95 ℃ under the condition of introducing nitrogen, and keeping for 24-36 hours to obtain black precipitate; cooling the black precipitate to room temperature by an ice bath method; separating the synthesized black product by magnetic decantation, and then washing with deionized water for several times to remove residual impurities to obtain the magnetic nano particles;

(3) Soaking the magnetic nano particles in poly-lysine solution for 20-24 hours to realize the surface modification of the magnetic nano particles;

(4) magnetically transfecting the surface-modified magnetic nanoparticles into adipose-derived mesenchymal stem cells according to the concentration of 10 mu g/mL, and extracting exosomes in supernate by adopting a gradient ultracentrifugation method after serum-free culture to obtain the magnetic-responsive exosomes for repairing peripheral nerve injury.

3. A magnetically-responsive exosome for peripheral nerve injury repair, prepared by the preparation method according to claim 1 or 2.

Technical Field

The invention relates to the technical field of nerve repair, in particular to a magnetic response exosome for repairing peripheral nerve injury and a preparation method thereof.

Background

The description of the background of the invention pertaining to the related art to which this invention pertains is given for the purpose of illustration and understanding only of the summary of the invention and is not to be construed as an admission that the applicant is explicitly or implicitly admitted to be prior art to the date of filing this application as first filed with this invention.

The peripheral and central nerve injuries have high incidence rate and serious harm, patients often have sensory and motor dysfunction, limb functional damage and even lifelong disability, and the treatment of the injuries is an unsolved worldwide problem. Reports have shown that about 1000-1500 million trauma patients are present worldwide per year, with 15-40% of these patients having peripheral and central nerve damage accounting for a very high percentage, with U.S. treatment costs as high as $ 150 million per year, and an average cost of about $ 47000 per person. About 2000 thousands of patients exist in China, the treatment cost is huge, and the heavy mental and economic burden is brought to the society and families. Therefore, the exploration of a new treatment strategy has important clinical significance and social value. Cell transplantation is an effective method for repairing nerve damage. Among them, Schwannscels (SCs) have various nerve regeneration promoting properties such as secretion of various neurotrophic factors, promotion of myelination, improvement of the nerve regeneration microenvironment and the like, and have been the focus of cell transplantation. However, after the SCs are subjected to in vitro primary separation culture, the transplantation activity and migration efficiency are reduced, and the neurotrophic factors cannot be efficiently and accurately released, the effective directional migration can be performed, and the regeneration microenvironment can not be improved in the nerve regeneration process, so that the further exertion of the nerve regeneration promoting function of the SCs is limited to a great extent. Therefore, how to realize better nerve injury repair effect by improving activity after SC transplantation is a hot point of domestic and foreign research.

Currently, methods for improving the activity of SCs cells include adding active factors, performing genetic modification, electrical stimulation, magnetic field stimulation, and the like. Wherein, the problem of explosive release often exists when the active factors are added, and the local concentration of the active factors is often higher than the physiological concentration, so that the side effect is difficult to avoid; genetically modified SCs present potential biosafety issues; electrical stimulation is difficult to realize under in-vivo non-invasive conditions, and the like. The magnetic field stimulation can overcome the problems, promote the secretion of neurotrophic factors, synthesize active proteins and improve the microenvironment for nerve regeneration. In addition, some current methods for creating a magnetic microenvironment to improve and regenerate by simply using Magnetic Nanoparticles (MNPs) have poor biocompatibility.

Disclosure of Invention

The embodiment of the invention aims to provide a magnetic-force-responsive exosome for repairing peripheral nerve injury and a preparation method thereof, and the magnetic-force-responsive exosome has the advantage of good biocompatibility.

In a first aspect, an embodiment of the present invention provides a method for preparing a magnetic-force-responsive exosome for repairing peripheral nerve injury, including the following steps: and (2) endocytosing the magnetic nanoparticles by using adipose-derived mesenchymal stem cells, and then extracting the magnetic biomaterial by using a gradient ultracentrifugation method to obtain the magnetic response type exosome.

Further, the method comprises the following steps:

(1) primarily extracting and culturing adipose-derived mesenchymal stem cells;

(2) synthesizing magnetic nanoparticles: adding 0.01M sulfuric acid into iron sulfate heptahydrate, wherein the volume-mass ratio of the sulfuric acid to the iron sulfate heptahydrate is as follows: 48.7vol:1wt, fully mixing under nitrogen flow filling to prepare ferrous sulfate solution; mixing potassium nitrate and sodium hydroxide according to the mass ratio of 2.8:1 to obtain a mixture, and then dissolving the mixture in deionized water under the nitrogen state to obtain a mixture solution; adding the ferrous sulfate solution dropwise into the mixture solution under continuous stirring; after precipitation, heating to 85-95 ℃ under the condition of introducing nitrogen, and keeping for 24-36 hours to obtain black precipitate; cooling the black precipitate to room temperature by an ice bath method; separating the synthesized black product by magnetic decantation, and then washing with deionized water for several times to remove residual impurities to obtain the magnetic nano particles;

(3) soaking the magnetic nano particles in poly-lysine solution for 20-24 hours to realize the surface modification of the magnetic nano particles;

(4) magnetically transfecting the surface-modified magnetic nanoparticles into adipose-derived mesenchymal stem cells according to the concentration of 10 mu g/mL, and extracting exosomes in supernate by adopting a gradient ultracentrifugation method after serum-free culture to obtain the magnetic-responsive exosomes for repairing peripheral nerve injury.

In a second aspect, the invention provides a magnetic-force-responsive exosome for peripheral nerve injury repair, which is prepared by the preparation method.

The embodiment of the invention has the following beneficial effects:

(1) the prepared magnetic exosome has the biological function of exosome and can exert biological activity in nerve regeneration;

(2) the prepared magnetic exosome can carry out targeted aggregation under the action of an external magnetic field, enrich in a target area, play a role and directionally guide regeneration of nerves.

Drawings

FIG. 1 is a TEM image of MNPs in the magnetically responsive exosomes for peripheral nerve injury repair and the preparation method thereof of the present invention;

FIG. 2 is TEM images of the exosomes secreted by ADSCs and the exosomes containing MNPs in the magnetic-responsive exosomes for peripheral nerve injury repair and the preparation method thereof of the present invention;

FIG. 3 is a diagram illustrating the practical effects of the application of an embodiment of the exosome in the magnetically responsive exosome for repairing peripheral nerve injury and the preparation method thereof of the present invention;

FIG. 4 is a diagram illustrating the practical effects of the application of another embodiment of the exosome in the magnetically responsive exosome for repairing peripheral nerve injury and the preparation method thereof of the present invention;

Fig. 5 is a diagram illustrating the practical effects of the application of another embodiment of the magnetic-responsive exosome for repairing peripheral nerve injury and the preparation method thereof according to the present invention.

Detailed Description

The present application is further described below with reference to examples.

In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art.

A preparation method of a magnetic-force-responsive exosome for peripheral nerve injury repair comprises the following steps: and (2) endocytosing the magnetic nanoparticles by using adipose-derived mesenchymal stem cells, and then extracting the magnetic biomaterial by using a gradient ultracentrifugation method to obtain the magnetic response type exosome.

Further, the method comprises the following steps:

(1) primarily extracting and culturing adipose-derived mesenchymal stem cells;

(2) synthesizing magnetic nanoparticles: adding 0.01M sulfuric acid into iron sulfate heptahydrate, wherein the volume-mass ratio of the sulfuric acid to the iron sulfate heptahydrate is as follows: 48.7vol:1wt, fully mixing under nitrogen flow filling to prepare ferrous sulfate solution; mixing potassium nitrate and sodium hydroxide according to the mass ratio of 2.8:1 to obtain a mixture, and then dissolving the mixture in deionized water under the nitrogen state to obtain a mixture solution; adding the ferrous sulfate solution dropwise into the mixture solution under continuous stirring; after precipitation, heating to 85-95 ℃ under the condition of introducing nitrogen, and keeping for 24-36 hours to obtain black precipitate; cooling the black precipitate to room temperature by an ice bath method; separating the synthesized black product by magnetic decantation, and then washing with deionized water for several times to remove residual impurities to obtain the magnetic nano particles;

(3) Soaking the magnetic nano particles in poly-lysine solution for 20-24 hours to realize the surface modification of the magnetic nano particles;

(4) magnetically transfecting the surface-modified magnetic nanoparticles into adipose-derived mesenchymal stem cells according to the concentration of 10 mu g/mL, and extracting exosomes in supernate by adopting a gradient ultracentrifugation method after serum-free culture to obtain the magnetic-responsive exosomes for repairing peripheral nerve injury.

A magnetic-force-responsive exosome for repairing peripheral nerve injury is prepared by the preparation method.

The preparation method comprises the following steps: MNPs are endocytosed by Adipose Derived Stem Cells (ADSCs), and then a novel magnetic biomaterial, namely 'magnetic responsive exosome (Mag-exo)', is extracted and prepared by a gradient ultracentrifugation method.

The method comprises the following specific steps:

(1) primarily extracting and culturing ADSCs, namely taking 2 SD rats of 3 weeks old, removing hairs at the inguinal position after cervical dislocation, taking adipose tissues in the inguinal region, placing the adipose tissues in a sterile PBS solution at 4 ℃, taking the second rat in the same method, carefully removing impurities such as blood vessels, fascia and the like in the adipose tissues, carefully cutting the adipose tissues by ophthalmic scissors, digesting and centrifuging, and then primarily culturing. Changing the liquid in a full amount every 2-3 days, carrying out passage when the cells are fused to 85% -90%, and substituting P3-P5 for the experiment;

(2) MNPs were synthesized. 15mL of 0.01M sulfuric acid was added to 0.308g of iron sulfate heptahydrate and mixed thoroughly under nitrogen flow to prepare a ferrous sulfate-sulfuric acid solution. 1.364g of potassium nitrate was mixed with 0.486g of sodium hydroxide, and the mixture was dissolved in deionized water under a nitrogen atmosphere. After the mixture was completely dissolved, the prepared ferrous sulfate solution was added dropwise to the above mixture with constant stirring. After precipitation, the temperature was heated to 90 ℃ for 24 hours under nitrogen. Finally, the obtained black product was cooled to room temperature by the ice bath method. The synthesized black product was isolated by magnetic decantation and then washed several times with deionized water to remove residual impurities.

(3) And soaking the MNPs in Polylysine (PLL) solution for 20 hours to realize surface modification of the MNPs so as to be beneficial to magnetic transfection.

(4) Performing magnetic transfection on MNPs (magnetic resonance proteins) into ADSCs (dependent Surveillance cells) according to the concentration of 10 mu g/mL, culturing, and extracting exosomes and exosomes in supernate by adopting a gradient ultracentrifugation method after serum-free culture, wherein the method comprises the following specific steps: and (3) extracting exosomes and exosomes in the supernatant by adopting a gradient ultracentrifugation technology in the serum removal culture, continuously culturing the ADSCs with the function of MNPs with the optimal concentration for 24 hours, washing cells by using a sterile 0.01MPBS solution, removing the unbound MNPs, and then adding a fresh serum-removal culture medium to culture for 72 hours. Cell supernatants were collected, 40mL per tube, and subjected to gradient centrifugation at 4 ℃ for 300g (10min to remove live cells), 2000g (15min to remove dead cells), 10000g (30min to remove cell debris, etc.), 120000g (120min to extract Mag-exo). And finally, carrying out magnetic separation and collecting Mag-exo. The ultrastructure was observed using TEM. And performing characterization detection.

(5) The magnetic exosome is injected into the damaged peripheral nerve region, so that the peripheral nerve regeneration is effectively promoted.

FIG. 1 shows the prepared magnetic nanoparticles, which are relatively uniform in size and uniform in particle size distribution when observed by TEM. The average particle size of the nanoparticles is 13.87 +/-6.44 nm.

FIG. 2 shows prepared exosomes and magnetic exosomes. TEM observations showed that the vesicles from both groups had a typical exosome-cup structure, with MNPs visible in Mag-exo.

Application Effect example 1

Magnetic exosomes were expressed as 1 × 10⁹The particles/mL concentration was injected into the sciatic nerve crush injury area, and nerve regeneration was observed by immunofluorescence staining 4 weeks after surgery, showing (FIG. 3) successful in-growth of regenerated nerve fibers.

Application Effect example 2

Magnetic exosomes were expressed as 1 × 10⁹The composites of particles/mL concentration and chitosan-collagen nerve conduit, the transplantation repairs the sciatic nerve 10mm defect, the nerve regeneration is observed by immunofluorescence staining after 8 weeks of operation, and the result shows (figure 4) that the regenerated nerve fiber successfully grows in.

Application Effect example 3

Magnetic exosomes were expressed as 1 × 10⁹The results of compounding particles/mL concentration with PCL nerve conduit, repairing sciatic nerve 10mm defect by transplantation, observing nerve regeneration by immunofluorescence staining after 8 weeks of operation show (figure 5) that regenerated nerve fiber successfully grows in.

It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.