Preparation method of polygonatum kingianum exosome microcapsule

Abstract

The invention discloses a preparation method of rhizoma polygonati exosome microcapsules, and belongs to the technical field of biology, the rhizoma polygonati exosome microcapsules are prepared by four steps of crude extraction of rhizoma polygonati exosome, purification of the rhizoma polygonati exosome, preparation of rhizoma polygonati exosome freeze-dried powder and preparation of the rhizoma polygonati exosome microcapsules. The rhizoma polygonati exosome is wrapped in the tiny vesicles formed by the high polymer material, so that the external environment is effectively isolated, a protective barrier is provided for the exosome, the exosome is prevented from being degraded or inactivated in the external environment, and the stability of the exosome is improved; meanwhile, the polygonatum kingianum exosome can be prevented from being subjected to in-vivo enzymolysis and clearing through micro-capsule wrapping, the in-vivo residence time of the polygonatum kingianum exosome is prolonged, and the bioavailability is improved; the preparation process is simple, the operability is high, and the prepared exosome microcapsule has good application in the aspect of inhibiting gastric cancer cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically is a method for preparing polygonatum exosome microcapsules. Background Art

[0002] Polygonatum sibiricum is sweet, flat, non-toxic, and is a perennial herb. It is the dried rhizome of Polygonatum sibiricum, Polygonatum sibiricum, or Polygonatum sibiricum of the genus Polygonatum of the Liliaceae family. It has the effects of replenishing qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Modern pharmacological studies have shown that Polygonatum sibiricum has multiple pharmacological effects such as regulating immunity, anti-aging, anti-inflammatory, and lowering blood sugar, and has the same origin as medicine and food.

[0003] Exosomes are lipid bilayer vesicles with a diameter of 30 to 150 nm. They contain a variety of bioinformatics molecules such as nucleic acids, proteins, lipids and small molecules, and play an important role in the process of intercellular communication. The latest research shows that plants and plant-derived microorganisms can also secrete exosomes. These exosomes mediate the cross-border transmission of bioinformatics molecules between plants and microorganisms, thereby participating in the interaction process between plants and microorganisms. Plant exosomes have the advantages of low toxicity, low immunogenicity, high cellular uptake efficiency, high biocompatibility and stability. Plant exosomes themselves have a lipid bilayer structure, but when directly applied, they may be affected by environmental factors, resulting in a decrease in their stability.

[0004] Gastric cancer, as a common malignant tumor, has seriously threatened human life. Exosomes are special substances secreted by cells and help transmit biological information between cells. This information transmission effect is particularly significant in tumor cells. Exosomes can carry the genetic information of tumors and transmit cancer signals to surrounding cell tissues and even distant cell tissues. They play an important role in the occurrence mechanism of tumors and can participate in tumor growth, invasion, metastasis, drug resistance and other aspects. The mechanism of action of exosomes in gastric cancer has been confirmed to a certain extent. Exosomes can affect the signaling pathways related to gastric cancer through the function of information transmission, and play a certain guiding role in the treatment of gastric cancer. Summary of the invention

[0005] To solve the above problems, the present invention aims to provide a method for preparing exosome microcapsules of Polygonatum sibiricum.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] A method for preparing polygonatum exosome microcapsules comprises four steps: crude extraction of polygonatum exosomes, purification of polygonatum exosomes, preparation of polygonatum exosome freeze-dried powder and preparation of polygonatum exosome microcapsules.

[0008] The method for preparing the polygonatum exosome microcapsules specifically comprises the following steps:

[0009] 1) adding 100 parts by weight of washed and chopped fresh polygonatum to a mixture of 200 parts of glycerol and 10 mmol / L phosphate buffer, extracting the juice using a juicer to obtain polygonatum juice, filtering the obtained filtrate, centrifuging it at 8000 r / min at 4°C for 0.5 to 1 h, and collecting the supernatant;

[0010] 2) filtering the supernatant obtained in step 1) using a 0.45 μm filter membrane, concentrating the filtrate, adding the concentrated solution to polyethylene glycol 8000, stirring at 4° C. and 100 to 300 r / min for 12 to 24 h, centrifuging at 4° C. and 12000 r / min for 15 to 30 min, dissolving the obtained precipitate in 10 mmol / L phosphate buffer, centrifuging, and collecting the supernatant to obtain a crude extract of polygonatum exosomes;

[0011] 3) Purifying the crude extract of polygonatum sibiricum exosomes obtained in step 2) by using a chromatographic column to obtain a refined polygonatum sibiricum exosome solution;

[0012] 4) The refined polygonatum sibiricum exosome solution prepared in step 3) is configured into a polygonatum sibiricum exosome freeze-dried liquid, filtered through a 0.22 μm sterile filter membrane, and freeze-dried at -30 to -40°C and 10 to 80 Pa for 20 to 30 hours to obtain a polygonatum sibiricum exosome freeze-dried powder;

[0013] 5) Add 80% ethanol and 5% gum arabic aqueous solution to the freeze-dried powder of polygonatum sibiricum exosomes prepared in step 4), then add Tween-80, stir and mix evenly to obtain a mixed solution, drop the obtained mixed solution into 1% chitosan solution, stir evenly at a constant temperature to obtain a mixed emulsion, adjust the pH of the mixed emulsion to 3.8-4.2 with sodium hydroxide, add one times the amount of distilled water, cool to 30-35°C, stir in an ice water bath, adjust the pH of the system to 8-9 with a 20% formaldehyde aqueous solution, stir for 2-3 hours, pre-freeze for 12-15 hours, and dry for 20-24 hours to obtain polygonatum sibiricum exosome microcapsules.

[0014] The mass concentration of glycerol in the mixed solution of glycerol and 10 mmol / L phosphate buffer in step 1) is 10%.

[0015] The pressure of the filtrate concentration in step 2) is ≤0.2MPa.

[0016] The mass ratio of the concentrated solution to polyethylene glycol 8000 in step 2) is 8 to 10:1.

[0017] The mass ratio of the precipitate to the phosphate buffer in step 2) is 1:2-4.

[0018] The purification conditions of the chromatographic column in step 3) are: column: C18 liquid chromatography column; flow rate: 100 cm / h, and the sample loading amount is 5% of the column volume.

[0019] The polygonatum sibiricum exosome freeze-dried solution in step 4) is composed of the following raw materials, in parts by weight: 82 to 85 parts of refined polygonatum sibiricum exosome solution, 3 to 5 parts of polyethylene glycol 1500, 5 to 8 parts of glycerol, 6 to 8 parts of mannitol and 4 to 6 parts of phosphorylated modified trehalose.

[0020] The phosphorylated modified trehalose is prepared according to the following method:

[0021] In parts by weight, 6 to 8 parts of sodium tripolyphosphate and 1 part of sodium trimetaphosphate are added to 8 to 10 parts of distilled water, stirred and dissolved to obtain a composite phosphate solution, 5 to 8 parts of trehalose are added thereto, and after fully dissolved, the pH value of the system is adjusted to 9 with sodium hydroxide, the obtained reaction solution is placed in a 90°C constant temperature water bath oscillator, reacted for 7 to 10 hours, cooled to 25 to 30°C, added to 4 to 6 parts of ethanol with a mass concentration of 95%, allowed to stand for reaction for 12 to 24 hours, and then centrifuged at 4°C and 5000 r / min for 10 to 30 minutes, the obtained precipitate is dissolved with 2 to 4 parts of distilled water, and then precipitated with 2 to 4 parts of anhydrous ethanol for 2 to 3 times, centrifuged, and the obtained solid is dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0022] The preparation method of the 1% chitosan solution in step 5) is: adding chitosan to a 1% acetic acid solution, stirring and dissolving, and preparing a chitosan solution with a mass concentration of 1%.

[0023] The mass ratio of the polygonatum sibiricum exosome freeze-dried powder, 80% ethanol, 5% arabic gum aqueous solution, Tween-80, and 1% chitosan solution in step 5) is 7-10:70-80:6-8:2.1-2.5:1-3.

[0024] The present invention also includes the use of polygonatum exosome microcapsules in inhibiting gastric cancer cells.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The method for preparing polygonatum sibiricum exosome microcapsules of the present invention prepares polygonatum sibiricum exosome microcapsules through four steps of crude extraction of polygonatum sibiricum exosomes, purification of polygonatum sibiricum exosomes, preparation of polygonatum sibiricum exosome freeze-dried powder and preparation of polygonatum sibiricum exosome microcapsules. In the preparation process of polygonatum sibiricum exosome freeze-dried powder, phosphorylated modified trehalose is added to resist oxidative stress, form a stable glassy structure, prevent denaturation and inactivation during the freeze-drying process, enhance the antifreeze ability of exosomes, and thus reduce damage caused by freezing; not only can the freeze-drying and storage stability of exosomes be improved, but also the activity of exosomes under other physical operations can be improved, the integrity and dispersion of freeze-dried exosomes can be further improved, and the particle characteristics of exosomes can be protected.

[0027] The preparation method of the Polygonatum sibiricum exosome microcapsules of the present invention, the separation and purification of exosomes are prerequisites for studying the functions and mechanisms of exosomes. The present invention can encapsulate the Polygonatum sibiricum exosomes in tiny vesicles formed by polymer materials through microencapsulation technology, thereby effectively isolating the external environment and providing a protective barrier for the exosomes to prevent degradation or inactivation in the external environment, thereby improving their stability; at the same time, microencapsulation can also protect the Polygonatum sibiricum exosomes from enzymatic hydrolysis and clearance in the body, prolong their residence time in the body, and improve their bioavailability.

[0028] The preparation method of the polygonatum sibiricum exosome microcapsules of the present invention has a simple preparation process and strong operability, and the prepared exosome microcapsules have good application in inhibiting gastric cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a microscopic morphology of the refined polygonatum exosome solution prepared in Example 5 of the present invention at 100 nm;

[0030] Figure 2 This is a size distribution diagram of the refined polygonatum exosome solution prepared in Example 5 of the present invention;

[0031] Figure 3 This is a graph showing the inhibitory effect of the Polygonatum sibiricum exosome microcapsules prepared in Example 6 of the present invention on AGS cells. DETAILED DESCRIPTION

[0032] In order to better understand the technical solution of the present invention, the above content of the present invention is further described in detail below through specific implementation methods in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above content of the present invention belong to the scope of the present invention.

[0033] Example 1 Preparation of phosphorylated modified trehalose:

[0034] 0.6 kg of sodium tripolyphosphate and 0.1 kg of sodium trimetaphosphate were added to 0.8 kg of distilled water, stirred and dissolved to obtain a composite phosphate solution, and then 0.5 kg of trehalose was added thereto. After fully dissolved, the pH value of the system was adjusted to 9 with sodium hydroxide, and the obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator. After reacting for 7 hours, it was cooled to 25° C. and added to 0.4 kg of 95% ethanol by mass concentration. The reaction was allowed to stand for 12 hours, and then centrifuged at 4° C. and 5000 r / min for 10 minutes. The obtained precipitate was dissolved with 0.2 kg of distilled water, and then precipitated twice with 0.2 kg of anhydrous ethanol, centrifuged, and the obtained solid was dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0035] Example 2 Preparation of phosphorylated modified trehalose:

[0036] 0.7 kg of sodium tripolyphosphate and 0.1 kg of sodium trimetaphosphate were added to 0.9 kg of distilled water, stirred and dissolved to obtain a composite phosphate solution, and then 0.6 kg of trehalose was added thereto. After sufficient dissolution, the pH value of the system was adjusted to 9 with sodium hydroxide, and the obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator. After reacting for 8 hours, it was cooled to 26° C. and then added to 0.5 kg of 95% ethanol by mass concentration. The reaction was allowed to stand for 15 hours, and then centrifuged at 4° C. and 5000 r / min for 20 minutes. The obtained precipitate was dissolved with 0.3 kg of distilled water, and then precipitated with 0.3 kg of anhydrous ethanol for 3 times, centrifuged, and the obtained solid was dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0037] Example 3 Preparation of phosphorylation-modified trehalose:

[0038] 0.7 kg of sodium tripolyphosphate and 0.1 kg of sodium trimetaphosphate were added to 1 kg of distilled water, stirred and dissolved to obtain a composite phosphate solution, and then 0.7 kg of trehalose was added thereto. After fully dissolved, the pH value of the system was adjusted to 9 with sodium hydroxide, and the obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator. After reacting for 9 hours, it was cooled to 28° C. and added to 0.5 kg of 95% ethanol by mass concentration. The reaction was allowed to stand for 20 hours, and then centrifuged at 4° C. and 5000 r / min for 25 minutes. The obtained precipitate was dissolved with 0.4 kg of distilled water, and then precipitated with 0.4 kg of anhydrous ethanol for 3 times, centrifuged, and the obtained solid was dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0039] Example 4 Preparation of phosphorylation-modified trehalose:

[0040] 0.8 kg of sodium tripolyphosphate and 0.1 kg of sodium trimetaphosphate were added to 1 kg of distilled water, stirred and dissolved to obtain a composite phosphate solution, and then 0.8 kg of trehalose was added thereto. After fully dissolved, the pH value of the system was adjusted to 9 with sodium hydroxide, and the obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator. After reacting for 10 hours, it was cooled to 30° C. and added to 0.6 kg of 95% ethanol by mass concentration. The reaction was allowed to stand for 24 hours, and then centrifuged at 4° C. and 5000 r / min for 30 minutes. The obtained precipitate was dissolved with 0.4 kg of distilled water, and then precipitated with 0.4 kg of anhydrous ethanol for 3 times, centrifuged, and the obtained solid was dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0041] Example 5 Preparation of Polygonatum sibiricum Exosome Microcapsules:

[0042] 1) Add 1 kg of washed and chopped fresh polygonatum to a mixture of 2 kg of glycerol and 10 mmol / L phosphate buffer, wherein the mass concentration of glycerol in the mixture is 10%, squeeze the juice with a juicer to obtain polygonatum juice, filter, and centrifuge the filtrate at 8000 r / min at 4°C for 0.5 h to collect the supernatant;

[0043] 2) The obtained supernatant was filtered using a 0.45 μm filter membrane, and the obtained filtrate was concentrated at a pressure of ≤0.2 MPa. The obtained concentrate was added to polyethylene glycol 8000, wherein the mass ratio of the concentrate to polyethylene glycol 8000 was 8:1, and stirred at 4°C and 100 r / min for 12 h, and then centrifuged at 4°C and 12000 r / min for 15 min. The obtained precipitate was dissolved in 10 mmol / L phosphate buffer and centrifuged, wherein the mass ratio of the precipitate to the phosphate buffer was 1:2, and the supernatant was collected to obtain a crude extract of Polygonatum sibiricum exosomes;

[0044] 3) The obtained crude extract of polygonatum exosomes was purified by C18 liquid chromatography column, with a flow rate of 100 cm / h and a sample loading of 5% of the column volume; a refined polygonatum exosome solution was obtained; the obtained refined polygonatum exosome solution was observed by transmission electron microscopy for the morphology and size of polygonatum exosomes, and the results were as follows Figure 1 As shown by Figure 1 The results show that the exosomes prepared by the present invention present a typical "cup-and-disc" morphology wrapped by a lipid bilayer membrane, which is consistent with the morphology of exosomes. The particle size distribution of the refined Polygonatum sibiricum exosome solution was analyzed by nanoflow cytometry, and the results are as follows Figure 2 As shown by Figure 2 The results show that the diameter of the vesicles is about 100 nm, which is consistent with the morphological characteristics of plant exosomes. Figure 1 The results are consistent with the particle size obtained by transmission electron microscopy.

[0045] 4) 0.82 kg of refined polygonatum exosome solution, 0.03 kg of polyethylene glycol 1500, 0.05 kg of glycerol, 0.06 kg of mannitol and 0.04 kg of phosphorylated modified trehalose prepared in Example 1 were mixed evenly to prepare a polygonatum exosome freeze-dried liquid, filtered through a 0.22 μm sterile filter membrane, and freeze-dried at -30 ° C and 10 Pa for 20 h to obtain a polygonatum exosome freeze-dried powder;

[0046] 5) Add 7 kg of 80% ethanol and 0.6 kg of 5% gum arabic aqueous solution to 0.7 kg of polygonatum sibiricum exosome freeze-dried powder, and then add 0.21 kg of Tween-80, stir and mix evenly to obtain a mixed solution, and drop the obtained mixed solution into 0.1 kg of 1% chitosan solution, wherein the 1% chitosan solution is prepared by adding chitosan to 1% acetic acid solution and stirring to dissolve; stir evenly at a constant temperature to obtain a mixed emulsion, adjust the pH of the mixed emulsion to 3.8 with sodium hydroxide, add one times the amount of distilled water, cool to 30°C, stir in an ice water bath, adjust the pH of the system to 8 with a 20% formaldehyde aqueous solution, stir for 2 hours, pre-freeze for 12 hours, and dry for 20 hours to obtain polygonatum sibiricum exosome microcapsules.

[0047] Example 6 Preparation of Polygonatum sibiricum Exosome Microcapsules:

[0048] 1) Add 1 kg of washed and chopped fresh polygonatum to a mixture of 2 kg of glycerol and 10 mmol / L phosphate buffer, wherein the mass concentration of glycerol in the mixture is 10%, use a juicer to squeeze the juice to obtain polygonatum juice, filter, and centrifuge the filtrate at 8000 r / min at 4°C for 0.75 h to collect the supernatant;

[0049] 2) The obtained supernatant was filtered using a 0.45 μm filter membrane, and the obtained filtrate was concentrated at a pressure of ≤0.2 MPa. The obtained concentrate was added to polyethylene glycol 8000, wherein the mass ratio of the concentrate to polyethylene glycol 8000 was 9:1, and stirred at 4°C and 200 r / min for 15 h, and then centrifuged at 4°C and 12000 r / min for 20 min. The obtained precipitate was dissolved by adding 10 mmol / L phosphate buffer and centrifuged, wherein the mass ratio of the precipitate to the phosphate buffer was 1:3, and the supernatant was collected to obtain a crude extract of Polygonatum sibiricum exosomes;

[0050] 3) Purifying the obtained crude extract of polygonatum exosomes by using a C18 liquid chromatography column, with a flow rate of 100 cm / h and a sample loading amount of 5% of the column volume, to obtain a refined polygonatum exosome solution;

[0051] 4) 0.83 kg of refined polygonatum exosome solution, 0.04 kg of polyethylene glycol 1500, 0.06 kg of glycerol, 0.07 kg of mannitol and 0.05 kg of phosphorylated modified trehalose prepared in Example 2 were mixed evenly to prepare a polygonatum exosome freeze-dried liquid, filtered through a 0.22 μm sterile filter membrane, and freeze-dried at -35 ° C and 30 Pa for 25 h to obtain a polygonatum exosome freeze-dried powder;

[0052] 5) Add 7.5 kg of 80% ethanol and 0.7 kg of 5% gum arabic aqueous solution to 0.8 kg of polygonatum sibiricum exosome freeze-dried powder, and then add 0.23 kg of Tween-80, stir and mix evenly to obtain a mixed solution, and drop the obtained mixed solution into 0.2 kg of 1% chitosan solution, wherein the 1% chitosan solution is prepared by adding chitosan to 1% acetic acid solution, stirring and dissolving; stir evenly at a constant temperature to obtain a mixed emulsion, adjust the pH of the mixed emulsion to 4.0 with sodium hydroxide, add one times the amount of distilled water, cool to 32 ° C, stir in an ice water bath, adjust the pH of the system to 8.5 with a 20% formaldehyde aqueous solution, stir for 2.5 hours, pre-freeze for 13 hours, and dry for 22 hours to obtain polygonatum sibiricum exosome microcapsules.

[0053] Example 7 Preparation of Polygonatum sibiricum Exosome Microcapsules:

[0054] 1) Add 1 kg of washed and chopped fresh polygonatum to a mixture of 2 kg of glycerol and 10 mmol / L phosphate buffer, wherein the mass concentration of glycerol in the mixture is 10%, squeeze the juice with a juicer to obtain polygonatum juice, filter, and centrifuge the filtrate at 8000 r / min at 4°C for 0.9 h to collect the supernatant;

[0055] 2) The obtained supernatant was filtered using a 0.45 μm filter membrane, and the obtained filtrate was concentrated at a pressure of ≤0.2 MPa. The obtained concentrate was added to polyethylene glycol 8000, wherein the mass ratio of the concentrate to polyethylene glycol 8000 was 9:1, and stirred at 4°C and 250 r / min for 20 h, and then centrifuged at 4°C and 12000 r / min for 25 min. The obtained precipitate was dissolved in 10 mmol / L phosphate buffer and centrifuged, wherein the mass ratio of the precipitate to the phosphate buffer was 1:3, and the supernatant was collected to obtain a crude extract of Polygonatum sibiricum exosomes;

[0056] 3) Purifying the obtained crude extract of polygonatum exosomes by using a C18 liquid chromatography column, with a flow rate of 100 cm / h and a sample loading amount of 5% of the column volume, to obtain a refined polygonatum exosome solution;

[0057] 4) 0.84 kg of refined polygonatum exosome solution, 0.04 kg of polyethylene glycol 1500, 0.07 kg of glycerol, 0.07 kg of mannitol and 0.05 kg of phosphorylated modified trehalose prepared in Example 2 were mixed evenly to prepare a polygonatum exosome freeze-dried liquid, filtered through a 0.22 μm sterile filter membrane, and freeze-dried at -38 ° C and 50 Pa for 28 h to obtain a polygonatum exosome freeze-dried powder;

[0058] 5) Add 7.8 kg of 80% ethanol and 0.75 kg of 5% gum arabic aqueous solution to 0.9 kg of polygonatum sibiricum exosome freeze-dried powder, and then add 0.24 kg of Tween-80, stir and mix evenly to obtain a mixed solution, and drop the obtained mixed solution into 0.25 kg of 1% chitosan solution, wherein the 1% chitosan solution is prepared by adding chitosan to 1% acetic acid solution, stirring and dissolving; stir evenly at a constant temperature to obtain a mixed emulsion, adjust the pH of the mixed emulsion to 4.1 with sodium hydroxide, add one times the amount of distilled water, cool to 34 ° C, stir in an ice water bath, adjust the pH of the system to 8.7 with a 20% formaldehyde aqueous solution, stir for 2.7 hours, pre-freeze for 14 hours, and dry for 23 hours to obtain polygonatum sibiricum exosome microcapsules.

[0059] Example 8 Preparation of Polygonatum sibiricum Exosome Microcapsules:

[0060] 1) Add 1 kg of washed and chopped fresh polygonatum to a mixture of 2 kg of glycerol and 10 mmol / L phosphate buffer, wherein the mass concentration of glycerol in the mixture is 10%, squeeze the juice with a juicer to obtain polygonatum juice, filter, and centrifuge the filtrate at 8000 r / min at 4°C for 1 h to collect the supernatant;

[0061] 2) The obtained supernatant was filtered using a 0.45 μm filter membrane, and the obtained filtrate was concentrated at a pressure of ≤0.2 MPa. The obtained concentrate was added to polyethylene glycol 8000, wherein the mass ratio of the concentrate to polyethylene glycol 8000 was 10:1, and stirred at 4°C and 300 r / min for 24 h, and then centrifuged at 4°C and 12000 r / min for 30 min. The obtained precipitate was dissolved by adding 10 mmol / L phosphate buffer and centrifuged, wherein the mass ratio of the precipitate to the phosphate buffer was 1:4, and the supernatant was collected to obtain a crude extract of Polygonatum sibiricum exosomes;

[0062] 3) Purifying the obtained crude extract of polygonatum exosomes by using a C18 liquid chromatography column, with a flow rate of 100 cm / h and a sample loading amount of 5% of the column volume, to obtain a refined polygonatum exosome solution;

[0063] 4) 0.85 kg of refined polygonatum exosome solution, 0.05 kg of polyethylene glycol 1500, 0.08 kg of glycerol, 0.08 kg of mannitol and 0.06 kg of phosphorylated modified trehalose prepared in Example 3 were mixed evenly to prepare a polygonatum exosome freeze-dried liquid, filtered through a 0.22 μm sterile filter membrane, and freeze-dried at -40 ° C and 80 Pa for 30 h to obtain a polygonatum exosome freeze-dried powder;

[0064] 5) Add 8 kg of 80% ethanol and 0.8 kg of 5% gum arabic aqueous solution to 1 kg of polygonatum sibiricum exosome freeze-dried powder, and then add 0.25 kg of Tween-80, stir and mix evenly to obtain a mixed solution, and drop the obtained mixed solution into 0.3 kg of 1% chitosan solution, wherein the 1% chitosan solution is prepared by adding chitosan to 1% acetic acid solution and stirring to dissolve; stir evenly at a constant temperature to obtain a mixed emulsion, adjust the pH of the mixed emulsion to 4.2 with sodium hydroxide, add one times the amount of distilled water, cool to 35°C, stir in an ice water bath, adjust the pH of the system to 9 with a 20% formaldehyde aqueous solution, stir for 3 hours, pre-freeze for 15 hours, and dry for 24 hours to obtain polygonatum sibiricum exosome microcapsules.

[0065] Stability evaluation

[0066] The lyophilized powder of Polygonatum sibiricum exosomes and the Polygonatum sibiricum exosome microcapsules prepared in Example 6 were placed in a constant temperature incubator with light set to test the stability of Polygonatum sibiricum exosomes, wherein the temperature was set at 25°C ± 2°C and the relative humidity was 60% ± 5%. During the test, samples were taken at the 0th month and the 1st month for preliminary stability testing. The test items included: ① high performance liquid phase detection of purity; ② micro-spectrophotometer protein concentration; ③ pH value acidity and alkalinity changes. The test results are shown in Table 1. It can be seen from the results in Table 1 that the purity, concentration and pH of the Polygonatum sibiricum exosome microcapsules were less reduced than those of the lyophilized powder of Polygonatum sibiricum exosomes under the same conditions for one month, indicating that the Polygonatum sibiricum exosome microcapsules prepared by the present invention encapsulate the Polygonatum sibiricum exosomes in microvesicles formed by polymer materials, thereby effectively isolating the external environment and providing a protective barrier for the exosomes, so the stability is better.

[0067] Table 1 Stability test of Polygonatum sibiricum exosomes

[0068]

[0069] The Polygonatum sibiricum exosome microcapsules prepared in Example 6 of the present invention were dissolved in 0.01M PBS solution, and the concentrations of 160mg / mL, 240mg / mL, 320mg / mL, 400mg / mL, 480mg / mL, 640mg / mL, 800mg / mL and 960mg / mL were respectively prepared. AGS cells in the logarithmic growth phase were inoculated in a 96-well plate at a density of 4000 per well and cultured in a cell culture incubator for 12h. 10μL of the above-configured different concentrations of the drug solution were added to the experimental group, 6 replicates were set for each dosage group, and a blank control group was set at the same time. After adding the drug and culturing for 24 hours, take out the 96-well plate from the incubator, add 20 μL (5 mg / mL) of MTT solution to each well, continue to place it in the cell culture incubator for 4 hours, then aspirate and discard the liquid in the well, add 100 μL DMSO to each well, shake it in a constant temperature shaker for 10 minutes to fully dissolve the crystals, and finally use an ELISA reader to read the absorbance (A) value at 540nm and 655nm. Calculate the cell growth inhibition rate according to the following formula, and calculate the half inhibition concentration (IC50) using the Bliss method. Inhibition rate (%) = (A control-A experiment) / (A control-A blank), the results are as follows Figure 3 As shown by Figure 3 It can be seen that the Polygonatum sibiricum exosome microcapsules prepared by the present invention have an inhibitory effect on AGS cells at various concentrations, with an IC50 of 12 mg / mL, indicating that the Polygonatum sibiricum exosome microcapsules prepared by the present invention have good application in inhibiting gastric cancer cells.

[0070] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing polygonatum exosome microcapsules, characterized in that: The method comprises four steps: crude extraction of polygonatum sibiricum exosomes, purification of polygonatum sibiricum exosomes, preparation of polygonatum sibiricum exosome freeze-dried powder and preparation of polygonatum sibiricum exosome microcapsules.

2. The method for preparing the polygonatum exosome microcapsules according to claim 1, characterized in that: The specific steps include: 1) adding 100 parts by weight of washed and chopped fresh polygonatum to a mixture of 200 parts of glycerol and 10 mmol / L phosphate buffer, extracting the juice using a juicer to obtain polygonatum juice, filtering the obtained filtrate, centrifuging it at 8000 r / min at 4°C for 0.5 to 1 h, and collecting the supernatant; 2) filtering the supernatant obtained in step 1) using a 0.45 μm filter membrane, concentrating the filtrate, adding the concentrated solution to polyethylene glycol 8000, stirring at 4° C. and 100 to 300 r / min for 12 to 24 h, centrifuging at 4° C. and 12000 r / min for 15 to 30 min, dissolving the obtained precipitate in 10 mmol / L phosphate buffer, centrifuging, and collecting the supernatant to obtain a crude extract of polygonatum exosomes; 3) Purifying the crude extract of polygonatum sibiricum exosomes obtained in step 2) by using a chromatographic column to obtain a refined polygonatum sibiricum exosome solution; 4) The refined polygonatum sibiricum exosome solution prepared in step 3) is configured into a polygonatum sibiricum exosome freeze-dried liquid, filtered through a 0.22 μm sterile filter membrane, and freeze-dried at -30 to -40°C and 10 to 80 Pa for 20 to 30 hours to obtain a polygonatum sibiricum exosome freeze-dried powder; 5) Add 80% ethanol and 5% gum arabic aqueous solution to the freeze-dried powder of polygonatum sibiricum exosomes prepared in step 4), then add Tween-80, stir and mix evenly to obtain a mixed solution, drop the obtained mixed solution into 1% chitosan solution, stir evenly at a constant temperature to obtain a mixed emulsion, adjust the pH of the mixed emulsion to 3.8-4.2 with sodium hydroxide, add one times the amount of distilled water, cool to 30-35°C, stir in an ice water bath, adjust the pH of the system to 8-9 with a 20% formaldehyde aqueous solution, stir for 2-3 hours, pre-freeze for 12-15 hours, and dry for 20-24 hours to obtain polygonatum sibiricum exosome microcapsules.

3. The method for preparing the polygonatum exosome microcapsules according to claim 2, characterized in that: The mass concentration of glycerol in the mixed solution of glycerol and 10 mmol / L phosphate buffer in step 1) is 10%.

4. The method for preparing the polygonatum exosome microcapsules according to claim 2, characterized in that: The pressure of the filtrate concentration in step 2) is ≤0.2MPa; the mass ratio of the concentrate and polyethylene glycol 8000 in step 2) is 8-10:1; the mass ratio of the precipitate and phosphate buffer in step 2) is 1:2-4.

5. The method for preparing the polygonatum exosome microcapsules according to claim 2, characterized in that: The purification conditions of the chromatographic column in step 3) are: column: C18 liquid chromatography column; flow rate: 100 cm / h, and the sample loading amount is 5% of the column volume.

6. The method for preparing the polygonatum exosome microcapsules according to claim 2, characterized in that: The polygonatum sibiricum exosome freeze-dried solution in step 4) is composed of the following raw materials, in parts by weight: 82 to 85 parts of refined polygonatum sibiricum exosome solution, 3 to 5 parts of polyethylene glycol 1500, 5 to 8 parts of glycerol, 6 to 8 parts of mannitol and 4 to 6 parts of phosphorylated modified trehalose.

7. The method for preparing the polygonatum exosome microcapsules according to claim 6, characterized in that: The phosphorylated modified trehalose is prepared according to the following method: In parts by weight, 6 to 8 parts of sodium tripolyphosphate and 1 part of sodium trimetaphosphate are added to 8 to 10 parts of distilled water, stirred and dissolved to obtain a composite phosphate solution, 5 to 8 parts of trehalose are added thereto, and after fully dissolved, the pH value of the system is adjusted to 9 with sodium hydroxide, the obtained reaction solution is placed in a 90°C constant temperature water bath oscillator, reacted for 7 to 10 hours, cooled to 25 to 30°C, added to 4 to 6 parts of ethanol with a mass concentration of 95%, allowed to stand for reaction for 12 to 24 hours, and then centrifuged at 4°C and 5000 r / min for 10 to 30 minutes, the obtained precipitate is dissolved with 2 to 4 parts of distilled water, and then precipitated with 2 to 4 parts of anhydrous ethanol for 2 to 3 times, centrifuged, and the obtained solid is dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

8. The method for preparing the polygonatum exosome microcapsules according to claim 2, characterized in that: The preparation method of the 1% chitosan solution in step 5) is: adding chitosan to a 1% acetic acid solution, stirring and dissolving, and preparing a chitosan solution with a mass concentration of 1%.

9. The method for preparing the polygonatum exosome microcapsules according to claim 2, characterized in that: The mass ratio of the polygonatum sibiricum exosome freeze-dried powder, 80% ethanol, 5% arabic gum aqueous solution, Tween-80, and 1% chitosan solution in step 5) is 7-10:70-80:6-8:2.1-2.5:1-3.

10. Use of the polygonatum sibiricum exosome microcapsules according to claim 1 in inhibiting gastric cancer cells.

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