A preparation method of polygonatum exosome microcapsules

Abstract

The present invention discloses a method for preparing polygonatum exosome microcapsules, belonging to the field of biotechnology. The polygonatum exosome microcapsules are prepared through four steps: crude extraction of polygonatum exosomes, purification of polygonatum exosomes, preparation of polygonatum exosome freeze-dried powder and preparation of polygonatum exosome microcapsules. The polygonatum exosomes are encapsulated in tiny vesicles formed by polymer materials, 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 exosomes from enzymatic hydrolysis and clearance in the body, prolong their residence time in the body, and improve their bioavailability. The preparation process of the present invention is simple and highly operable. The prepared exosome microcapsules have good application in inhibiting gastric cancer cells.

Description

Technical Field

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

[0002] Polygonatum sibiricum (Polygonatum sibiricum) is a sweet, neutral, and non-toxic perennial herb. It is the dried rhizome of Polygonatum sibiricum, Polygonatum sibiricum, or Polygonatum multiflorum, all of the Liliaceae family. It has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys. Modern pharmacological research has shown that Polygonatum sibiricum has multiple pharmacological effects, including immune regulation, anti-aging, anti-inflammatory, and blood sugar-lowering, demonstrating its medicinal and edible properties.

[0003] Exosomes are lipid bilayer vesicles with a diameter of 30 to 150 nm. They contain a variety of bioinformatics molecules, including nucleic acids, proteins, lipids, and small molecules, and play an important role in intercellular communication. Recent studies have shown that plants and plant-derived microorganisms can also secrete exosomes. These exosomes mediate the cross-domain transmission of bioinformatics molecules between plants and microorganisms, thereby participating in the interaction between plants and microorganisms. Plant exosomes have the advantages of low toxicity, low immunogenicity, high cellular uptake efficiency, high biocompatibility, and high stability. Plant exosomes themselves have a lipid bilayer structure, but direct application may be affected by environmental factors, resulting in reduced stability.

[0004] Gastric cancer, a common malignancy, poses a serious threat to human life. Exosomes are specialized substances secreted by cells that facilitate the transfer of biological information between cells. This information transfer is particularly pronounced in tumor cells. Exosomes can carry genetic information from the tumor and transmit cancer signals to surrounding cells and tissues, even to distant tissues. They play a crucial role in tumorigenesis, participating in multiple stages of tumor growth, invasion, metastasis, and drug resistance. The mechanism of action of exosomes in gastric cancer has been confirmed to some extent. Through their information transfer function, exosomes can influence gastric cancer-related signaling pathways, providing guidance for gastric cancer treatment. Summary of the Invention

[0005] In order to solve the above problems, the purpose of the present invention is to provide a method for preparing Polygonatum sibiricum exosome microcapsules.

[0006] To achieve the above-mentioned purpose, 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 preparation method of the polygonatum exosome microcapsules specifically comprises the following steps:

[0009] 1) Add 100 parts by weight of washed and chopped fresh polygonatum to a mixture of 200 parts of glycerol and 10 mmol / L phosphate buffer, extract the juice using a juicer to obtain polygonatum juice, filter the resulting filtrate, and centrifuge at 8000 rpm at 4°C for 0.5-1 h to collect 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 100-300 r / min for 12-24 hours, and then centrifuging at 4°C and 12000 r / min for 15-30 minutes. The resulting precipitate was dissolved in 10 mmol / L phosphate buffer, centrifuged, and the supernatant was collected to obtain a crude extract of Polygonatum sibiricum exosomes;

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

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

[0013] 5) To the Polygonatum sibiricum exosome freeze-dried powder prepared in step 4), 80% ethanol and 5% gum arabic aqueous solution were added, and Tween-80 was added and stirred to obtain a mixed solution. The obtained mixed solution was dropped into a 1% chitosan solution and stirred at a constant temperature to obtain a mixed emulsion. The pH of the mixed emulsion was adjusted to 3.8-4.2 using sodium hydroxide, and then distilled water was added thereto, cooled to 30-35 ° C, stirred in an ice water bath, and the pH of the system was adjusted to 8-9 with a 20% formaldehyde aqueous solution. After stirring for 2-3 hours, the mixture was pre-frozen for 12-15 hours and dried 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 ≤0.2 MPa.

[0016] The mass ratio of the concentrated solution and 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 as follows: 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: 82-85 parts of refined polygonatum sibiricum exosome solution, 3-5 parts of polyethylene glycol 1500, 5-8 parts of glycerol, 6-8 parts of mannitol and 4-6 parts of phosphorylated modified trehalose.

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

[0021] The method comprises the following steps: adding 6 to 8 parts of sodium tripolyphosphate and 1 part of sodium trimetaphosphate to 8 to 10 parts of distilled water, stirring and dissolving the mixture to obtain a composite phosphate solution, adding 5 to 8 parts of trehalose to the solution, fully dissolving the mixture, adjusting the pH value of the system to 9 with sodium hydroxide, placing the obtained reaction solution in a 90°C constant temperature water bath oscillator, reacting for 7 to 10 hours, cooling the solution to 25 to 30°C, adding 4 to 6 parts of 95% ethanol, allowing the solution to react for 12 to 24 hours, and centrifuging the solution at 4°C and 5000 r / min for 10 to 30 minutes. The obtained precipitate is dissolved in 2 to 4 parts of distilled water, precipitated 2 to 3 times with 2 to 4 parts of anhydrous ethanol, and centrifuged. 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 as follows: chitosan is added to a 1% acetic acid solution, and stirred to dissolve, thereby preparing a chitosan solution with a mass concentration of 1%.

[0023] The mass ratio of the polygonatum exosome freeze-dried powder, 80% ethanol, 5% aqueous gum arabic 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 preparation method of the polygonatum exosome microcapsules of the present invention comprises four steps of crude extraction of polygonatum exosomes, purification of polygonatum exosomes, preparation of polygonatum exosome freeze-dried powder and preparation of polygonatum exosome microcapsules to obtain polygonatum exosome microcapsules. During the preparation of the polygonatum 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 the exosomes, and thus reduce damage caused by freezing. The method can not only improve the freeze-drying and storage stability of the exosomes, but also improve the activity of the exosomes under other physical operations, further improve the integrity and dispersion of the freeze-dried exosomes, and protect the particle characteristics of the exosomes.

[0027] The preparation method of the Polygonatum sibiricum exosome microcapsules of the present invention and the isolation and purification of exosomes are prerequisites for studying the functions and mechanisms of exosomes. The present invention uses microencapsulation technology to encapsulate Polygonatum sibiricum exosomes in tiny vesicles formed by polymer materials, 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 Polygonatum sibiricum exosomes from enzymatic hydrolysis and clearance in the body, prolonging their residence time in the body and improving their bioavailability.

[0028] The preparation method of the polygonatum 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 sibiricum 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 sibiricum exosome solution prepared in Example 5 of the present invention;

[0031] Figure 3 This is a diagram 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 solutions of the present invention, the following is a further detailed description of the above content of the present invention through specific implementation methods in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within 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 sufficient dissolution, the pH value of the system was adjusted to 9 with sodium hydroxide. The obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator, reacted for 7 hours, cooled to 25° C., added to 0.4 kg of 95% ethanol, allowed to react 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. 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. The obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator, reacted for 8 hours, cooled to 26° C., added to 0.5 kg of 95% ethanol, allowed to react 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 three times. The obtained solid was dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0037] Example 3 Preparation of phosphorylated 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 sufficient dissolution, the pH value of the system was adjusted to 9 with sodium hydroxide. The obtained reaction solution was placed in a 90° C. constant temperature water bath oscillator, reacted for 9 hours, cooled to 28° C., added to 0.5 kg of 95% ethanol, allowed to react 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 three times. The obtained solid was dialyzed and freeze-dried to obtain phosphorylated modified trehalose.

[0039] Example 4 Preparation of phosphorylated 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 sufficient dissolution, the pH value of the system was adjusted to 9 with sodium hydroxide. The obtained reaction solution was placed in a 90°C constant temperature water bath oscillator, reacted for 10 hours, cooled to 30°C, added to 0.6 kg of 95% ethanol, and allowed to react for 24 hours. 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 three 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 cleaned 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 extract the polygonatum juice, filter the juice, and centrifuge the filtrate at 8000 rpm 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 hours, and then centrifuged at 4°C and 12000 r / min for 15 minutes. 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 at a flow rate of 100 cm / h and a sample loading of 5% of the column volume to obtain a refined polygonatum exosome solution; the obtained refined polygonatum exosome solution was observed using a transmission electron microscope to observe the morphology and size of the polygonatum exosomes. The results are 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 using a nanoflow cytometry instrument, and the results are as follows Figure 2 As shown by Figure 2 The results show that the diameter of the vesicles is about 100nm, 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 sibiricum 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 sibiricum 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 sibiricum exosome freeze-dried powder;

[0046] 5) 0.7 kg of Polygonatum sibiricum exosome freeze-dried powder was added with 7 kg of 80% ethanol and 0.6 kg of 5% gum arabic aqueous solution, and then 0.21 kg of Tween-80 was added and stirred to obtain a mixed solution, which was added dropwise to 0.1 kg of 1% chitosan solution, wherein the 1% chitosan solution was prepared by adding chitosan to 1% acetic acid solution and stirring to dissolve; stirring at a constant temperature to obtain a mixed emulsion, adjusting the pH of the mixed emulsion to 3.8 with sodium hydroxide, and then adding one times the amount of distilled water to the mixture, cooling to 30 ° C, stirring in an ice water bath, and adjusting the pH of the system to 8 with a 20% formaldehyde aqueous solution. After stirring 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 cleaned 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 extract the polygonatum juice, filter it, and centrifuge the filtrate at 8000 rpm 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 hours, and then centrifuged at 4°C and 12000 r / min for 20 minutes. 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;

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

[0051] 4) 0.83 kg of refined Polygonatum sibiricum 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 sibiricum 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 sibiricum exosome freeze-dried powder;

[0052] 5) 0.8 kg of Polygonatum sibiricum exosome freeze-dried powder was added with 7.5 kg of 80% ethanol and 0.7 kg of 5% gum arabic aqueous solution, and then 0.23 kg of Tween-80 was added and stirred to obtain a mixed solution, which was added dropwise to 0.2 kg of 1% chitosan solution, wherein the 1% chitosan solution was prepared by adding chitosan to 1% acetic acid solution and stirring to dissolve; the mixture was stirred at a constant temperature to obtain a mixed emulsion, and the pH of the mixed emulsion was adjusted to 4.0 with sodium hydroxide, and then one times the amount of distilled water was added thereto, cooled to 32 ° C, stirred in an ice water bath, and the pH of the system was adjusted to 8.5 with a 20% formaldehyde aqueous solution. After stirring for 2.5 h, it was pre-frozen for 13 h and dried for 22 h to obtain Polygonatum sibiricum exosome microcapsules.

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

[0054] 1) Add 1 kg of cleaned 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 extract the polygonatum juice, filter the filtrate, and centrifuge the filtrate at 8000 rpm at 4°C for 0.9 h, collecting 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 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 Polygonatum sibiricum exosome extract using a C18 liquid chromatography column at a flow rate of 100 cm / h and a sample loading amount of 5% of the column volume to obtain a refined Polygonatum sibiricum exosome solution;

[0057] 4) 0.84 kg of refined Polygonatum sibiricum 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 sibiricum 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 sibiricum exosome freeze-dried powder;

[0058] 5) 0.9 kg of Polygonatum sibiricum exosome freeze-dried powder was added with 7.8 kg of 80% ethanol and 0.75 kg of 5% gum arabic aqueous solution, and then 0.24 kg of Tween-80 was added and stirred to obtain a mixed solution, which was added dropwise to 0.25 kg of 1% chitosan solution, wherein the 1% chitosan solution was prepared by adding chitosan to 1% acetic acid solution and stirring to dissolve; the mixture was stirred at a constant temperature to obtain a mixed emulsion, and the pH of the mixed emulsion was adjusted to 4.1 with sodium hydroxide, and then one times the amount of distilled water was added thereto, cooled to 34 ° C, stirred in an ice water bath, and the pH of the system was adjusted to 8.7 with a 20% formaldehyde aqueous solution. After stirring for 2.7 h, it was pre-frozen for 14 h and dried for 23 h to obtain Polygonatum sibiricum exosome microcapsules.

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

[0060] 1) Add 1 kg of cleaned 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 extract the polygonatum juice, filter it, and centrifuge the filtrate at 8000 rpm at 4°C for 1 hour 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 hours, and then centrifuged at 4°C and 12000 r / min for 30 minutes. The obtained precipitate was dissolved in 10 mmol / L phosphate buffer and centrifuged, wherein the mass ratio of the precipitate to 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 Polygonatum sibiricum exosome extract using a C18 liquid chromatography column at a flow rate of 100 cm / h and a sample loading amount of 5% of the column volume to obtain a refined Polygonatum sibiricum exosome solution;

[0063] 4) 0.85 kg of refined Polygonatum sibiricum 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 sibiricum 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 sibiricum exosome freeze-dried powder;

[0064] 5) 1 kg of Polygonatum sibiricum exosome freeze-dried powder was added with 8 kg of 80% ethanol and 0.8 kg of 5% gum arabic aqueous solution, and then 0.25 kg of Tween-80 was added and stirred to obtain a mixed solution, which was added dropwise to 0.3 kg of 1% chitosan solution, wherein the 1% chitosan solution was prepared by adding chitosan to 1% acetic acid solution and stirring to dissolve; the mixture was stirred at a constant temperature to obtain a mixed emulsion, and the pH of the mixed emulsion was adjusted to 4.2 with sodium hydroxide, and then one times the amount of distilled water was added thereto, cooled to 35 ° C, stirred in an ice water bath, and the pH of the system was adjusted to 9 with a 20% formaldehyde aqueous solution. After stirring for 3 hours, it was pre-frozen for 15 hours and dried for 24 hours to obtain Polygonatum sibiricum exosome microcapsules.

[0065] Stability evaluation

[0066] The stability of the Polygonatum sibiricum exosome lyophilized powder and Polygonatum sibiricum exosome microcapsules prepared in Example 6 was tested in a constant-temperature incubator set to 25°C ± 2°C and a relative humidity of 60% ± 5% under illumination. Samples were collected at month 0 and month 1 for preliminary stability testing. The testing criteria included: ① purity by HPLC; ② protein concentration by microspectrophotometry; and ③ pH changes. The results are shown in Table 1. As shown in Table 1, after one month of storage under the same conditions, the purity, concentration, and pH of the Polygonatum sibiricum exosome microcapsules decreased less than those of the Polygonatum sibiricum exosome lyophilized powder. This suggests that the Polygonatum sibiricum exosome microcapsules prepared in the present invention encapsulate the Polygonatum sibiricum exosomes within microvesicles formed from a polymer material, effectively isolating them from the external environment and providing a protective barrier for the exosomes, resulting in improved stability.

[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 cells per well and cultured in a cell culture incubator for 12h. 10μL of the above-prepared solution of different concentrations was added to the experimental group, 6 replicates were set for each dose group, and a blank control group was set at the same time. After adding the drug and culturing for 24 hours, remove the 96-well plate from the incubator, add 20 μL (5 mg / mL) MTT solution to each well, continue to place it in the cell culture incubator and culture for 4 hours, then aspirate and discard the liquid in the well, add 100 μL DMSO to each well, place it in a constant temperature shaker and shake for 10 minutes to fully dissolve the crystals, and finally use a microplate 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-maximal inhibitory 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 sibiricum exosome microcapsules, characterized by: The method includes four steps: crude extraction of polygonatum exosomes, purification of polygonatum exosomes, preparation of polygonatum exosome freeze-dried powder and preparation of polygonatum exosome microcapsules; The specific steps include: 1) Add 100 parts by weight of washed and chopped fresh polygonatum to a mixture of 200 parts of glycerol and 10 mmol / L phosphate buffer. Use a juicer to extract the polygonatum juice. Filter the resulting filtrate and centrifuge it at 8000 rpm at 4°C for 0.5-1 h. Collect the supernatant. 2) Filtering the supernatant obtained in step 1) using a 0.45 μm filter membrane, concentrating the resulting filtrate, adding the resulting concentrate to polyethylene glycol 8000, stirring at 4°C and 100-300 rpm for 12-24 hours, and then centrifuging at 4°C and 12,000 rpm for 15-30 minutes. Dissolving the resulting precipitate in 10 mmol / L phosphate buffer, centrifuging, and collecting the supernatant to obtain a crude Polygonatum sibiricum exosome extract; 3) Purifying the crude exosome extract of polygonatum sibiricum obtained in step 2) using a chromatographic column to obtain a refined exosome solution of polygonatum sibiricum; 4) preparing the refined polygonatum exosome solution prepared in step 3) into a polygonatum exosome freeze-dried liquid, filtering the solution through a 0.22 μm sterile filter membrane, and freeze-drying the solution at -30 to -40°C and 10 to 80 Pa for 20 to 30 hours to obtain a polygonatum exosome freeze-dried powder; 5) adding 80% ethanol and 5% aqueous solution of gum arabic to the freeze-dried powder of polygonatum exosomes prepared in step 4), and then adding Tween-80, stirring and mixing uniformly to obtain a mixed solution, dropping the obtained mixed solution into a 1% chitosan solution, stirring uniformly at a constant temperature to obtain a mixed emulsion, adjusting the pH of the mixed emulsion to 3.8-4.2 with sodium hydroxide, and then adding one times the amount of distilled water to the mixed emulsion, cooling to 30-35°C, stirring in an ice water bath, and adjusting the pH of the system to 8-9 with a 20% aqueous formaldehyde solution. After stirring for 2-3 hours, pre-freeze for 12-15 hours, and dry for 20-24 hours to obtain polygonatum exosome microcapsules; The polygonatum sibiricum exosome freeze-dried solution in step 4) is composed of the following raw materials, in parts by weight: 82-85 parts of refined polygonatum sibiricum exosome solution, 3-5 parts of polyethylene glycol 1500, 5-8 parts of glycerol, 6-8 parts of mannitol, and 4-6 parts of phosphorylated modified trehalose; 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 sufficient dissolution, 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 with 4 to 6 parts of 95% ethanol, 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.

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

3. The method for preparing the polygonatum exosome microcapsules according to claim 1, wherein: The pressure of the filtrate concentration in step 2) is ≤0.2 MPa; 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.

4. The method for preparing the polygonatum exosome microcapsules according to claim 1, wherein: The chromatographic column purification conditions in step 3) are as follows: column: C18 liquid chromatography column; flow rate: 100 cm / h, and the sample loading amount is 5% of the column volume.

5. The method for preparing the polygonatum exosome microcapsules according to claim 1, wherein: The preparation method of the 1% chitosan solution in step 5) is as follows: chitosan is added to a 1% acetic acid solution, and stirred to dissolve, thereby preparing a chitosan solution with a mass concentration of 1%.

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

7. Use of the polygonatum exosome microcapsules prepared by the method for preparing polygonatum exosome microcapsules according to claim 1 in preparing drugs for inhibiting gastric cancer cells.

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