Plant-derived exosome as well as preparation method and application thereof

Abstract

Disclosed a plant-derived exosome as well as a preparation method and an application thereof in preparation of drugs or scaffolds for animal tissue regeneration therapy. The preparation method includes: soaking and infiltrating any part of a natural plant with a 2-(N-morpholine) ethanesulfonic acid buffer solution; removing a supernatant; collecting a wet treated sample; refrigerating, centrifuging and extracting the sample to obtain apoplastic fluid, wherein the soaking and infiltrating method is as follows: vacuum supply is performed within 6-24 h after soaking for 2-5 times, vacuum supply time is independently 5-15 s each time, and interval time between two adjacent times of vacuum supply is independently 10 s-1 min; and centrifuging the apoplastic fluid at an ultra-high speed to obtain the plant-derived exosome, wherein ultra-high speed centrifugation conditions are as follows: centrifugal force is not lower than 100000 g, centrifugation time is 1-7 h, and a temperature is 0-4° C.

Claims

1. A method for preparing a Sophora japonica-derived exosome, comprising: a) soaking a Sophora japonica sample with a 2-(N-morpholino)ethanesulfonic acid buffer solution, and performing vacuum supply 2-5 times with 5-15 seconds each time within 6-24 hours to produce a first supernatant, wherein the vacuum supply is performed every 10 seconds-1 minute; b) removing the first supernatant obtained in step a) to collect a wet treated sample; c) centrifuging the wet treated sample under a refrigerating condition to extract an apoplastic fluid from the wet treated sample; d) performing centrifugal fragment removal treatment comprising: performing centrifugation on the apoplastic fluid obtained in step c) for 15 minutes-1 hour at a centrifugal force of 2500-3500 g and a centrifugation temperature of 0-4° C., followed by another centrifugation for 15 minutes-1 hour at a centrifugal force of 9000-11000 g and a centrifugation temperature of 0-4° C. to produce a second supernatant; and e) centrifuging the second supernatant obtained in step d) for 1-7 hours at a centrifugal force of not lower than 100000 g and a centrifugation temperature of 0-4° C. to obtain the Sophora japonica-derived exosome.

2. The method of claim 1, wherein the 2-(N-morpholino)ethanesulfonic acid buffer solution comprises 2-(N-morpholino)ethanesulfonic acid, NaCl, CaCl.sub.2.Math.H.sub.2O and water; and a pH of the 2-(N-morpholino)ethanesulfonic acid buffer solution is 5.5-6.5.

3. The method of claim 1, wherein in step c), a centrifugal force is 600-800 g; and a centrifugation time is 15-25 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a transmission electron microscope morphology observation photo of a Sophora japonica exosome prepared in embodiments;

(2) FIG. 2 is a particle size analysis result diagram of a Sophora japonica exosome prepared in embodiments by a particle tracking analyzer, wherein in the figure, a horizontal coordinate represents the particle size, and a vertical coordinate represents a number of particles per ml; and

(3) FIG. 3 is a staining result diagram of an intra-tissue peroxidation product of a Sophora japonica exosome prepared in embodiments in combination with biological scaffold material transplantation and scaffold material transplantation alone for treatment of spinal cord injury of rats, wherein in the figure, scale of the upper figure is 1 mm, and the scale of two lower figures is 100 μm.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

(4) The present invention will be further described below in combination with drawings and specific embodiments. It should be understood that, these embodiments are merely used for describing the present invention, rather than limiting the scope of the present invention. Operating methods without specific indicated conditions in embodiments below are generally conducted in accordance with general conditions or conditions suggested by manufacturers. 1. A preparation method of a plant-derived exosome:

(5) 4.26 g of 2-(N-morpholine) ethanesulfonic acid, 5.85 g of NaCl and 0.27 g of CaCl.sub.2.Math.H.sub.2O were dissolved into ultrapure water; a pH value of the solution was regulated to 6.0 with 5M of NaOH solution; a 2-(N-morpholine) ethanesulfonic acid buffer solution was prepared.

(6) Dried Sophora japonica was soaked in the above buffer solution for 12-18 h and subjected to vacuum supply for 10 s after soaking for preset time; interval time was 30 s each time; and vacuum supply was performed for totally 3 times.

(7) The supernatant was removed; a wet treated Sophora japonica sample was collected and filled in an outer barrel of a syringe; the syringe barrel was placed in a 50 mL of centrifuge tube; and the sample was refrigerated, centrifuged and extracted so as to obtain apoplastic fluid, wherein a centrifugal force was 700 g, and centrifugation time was 20 min.

(8) The obtained apoplastic fluid was collected and subjected to centrifugal treatment at 4° C. so as to remove fragments; centrifugation was performed twice at a centrifugal force of 3000 g for 20 min; then centrifugation was performed once at a centrifugal force of 10000 g for 30 min; and finally the supernatant was collected.

(9) The supernatant was centrifuged at a centrifugal force of 100000 g for 3 h at a temperature of 4° C.; the supernatant was removed; and a precipitate was collected, thereby obtaining the plant-derived exosome.

(10) A transmission electron microscope photo of the extracted plant-derived exosome is shown in FIG. 1. It shows that, the prepared plant-derived exosome has a particle size of about 100 nm and is of a typical cup-like exosome vesicle structure.

(11) A detection result of the extracted plant exosome by a particle tracking analyzer is shown in FIG. 2. It shows that, the prepared plant-derived exosome has a main particle size distribution of about 150 nm and is in line with the definition of the particle size of the exosome. 2. Preparation method of a dopamine enveloped hyaluronic acid (HA) hydrogel scaffold:

(12) Aldehyde groups and amino were respectively grafted on molecular chains of HA; and Schiff base condensation was conducted, thereby obtaining the hydrogel scaffold.

(13) 500 mg of HA having a molecular weight of 2.3 MDa was weighed and dissolved into 150 mL of ultrapure water; 134 mg of NaIO.sub.4 was weighed and dissolved into 13.4 mL of water; an aqueous solution of HA was added dropwise in dark conditions; the solution was stirred and reacted for 2 d; 600 μL of ethylene glycol was added; the solution was continuously stirred for 1 h; and the solution was freeze-dried within 3 d after dialysis so as to obtain aldehyde-modified HA.

(14) 270 mg of HA having a molecular weight of 1.3 MDa was dissolved into 150 mL of ultrapure water; 4.644 g of adipic acid dihydrazide (ADH) was added and uniformly stirred; 0.96 g of 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride and 0.675 g of 1-hydroxybenzotriazole were dissolved into 10 mL of a mixed solution of dimethyl sulfoxide and water according to a ratio of 1:1; the mixed solution was dropwise added into the HA solution; a pH value was maintained at 6.8; the solution was stirred for 4 h; and the reaction was terminated when the pH value was regulated to 7, and the solution was freeze-dried within 3 d after dialysis so as to obtain amino-modified HA.

(15) The amino-modified HA was dissolved into a PBS buffer solution so as to prepare a 12 mg/mL of solution; the aldehyde-modified HA was dissolved into the PBS buffer solution so as to prepare a 20 mg/mL of solution; the 12 mg/mL of solution and the 20 mg/mL of solution were mixed in an isovolumetric manner and then stood so as to form gel.

(16) The hydrogel was freeze-dried and then swelled in a Tris buffer solution having a pH of 8.5 so as to form a 1 mg/mL of dopamine Tris solution; the swelling hydrogel was stirred in the solution overnight in dark conditions, thereby obtaining the dopamine enveloped hydrogel. 3. Animal experiment: (1) Establishment of spinal cord injury (SCI) injury:

(17) Female SD rats having the weight of 220-250 g were selected and subjected to SCI model establishment surgery.

(18) The rats were anesthetized with 1% of pentobarbital sodium; hair on the back of the anesthetized rats was shaved off; T9-T10 sections of the spines were found; by taking the T9-T10 sections as the center, upper and lower parts of the spines were cut open by 2 cm by an operating knife so as to expose the spinal backs; and muscles on two sides of the spines on the T9-T10 sections were isolated.

(19) The spines with free muscular tissues were cut open so as to expose the spines; spinal cord tissues were clipped; fractures of about 4 mm were made; through inspection of microscope forceps, it was ensured that the spines were fully isolated; bleeding was stopped; residual tissues were wiped; the hydrogel was transplanted to the spinal injury fractures; 20 μL of PBS suspension (P-ES group) of the prepared Sophora japonica exosome was injected by a micropipettor; a hydrogel scaffold was transplanted after the spines were cut off in a blank group without injecting the exosome; and the wound was stitched and daubed with iodine tincture.

(20) The rats were killed within 7 days after modeling; and spinal cord tissues of the rats were taken and subjected to immumohistochemical staining inspection with a peroxidation product, i.e., 4-hydroxynonenal (4-HNE).

(21) Partial regions of a lesion span of P-ES group and blank group in a figure located on the upper part of FIG. 3 were respectively intercepted and magnified so as to obtain two smaller figures located on the lower part of FIG. 3. As shown in FIG. 3, antioxidation of the plant-derived exosome is relatively obvious. Through treatment of the plant-derived exosome, the amount of a peroxidation injury product, i.e., the 4-HNE, in the tissues is decreased significantly.

(22) In addition, it should be understood that, after reading the above descriptions of the present invention, those skilled in the art may make various changes or modifications to the present invention. These equivalent forms shall be included in the scope defined by claims in the present application.