Method for obtaining extracellular vesicles of rhodiola rosea and application of extracellular vesicles

Abstract

The invention provides a rhodiola rosea extracellular vesicle obtaining method and application. The rhodiola rosea extracellular vesicle obtaining method comprises rhodiola rosea crude extract preparation, rhodiola rosea extracellular vesicle crude extraction, rhodiola rosea extracellular vesicle fine extraction and rhodiola rosea extracellular vesicle surface modification. According to the method for obtaining the extracellular vesicles provided by the invention, the extracellular vesicles of the rhodiola rosea can be obtained, and the extracellular vesicles of the rhodiola rosea have good biocompatibility, not only can effectively protect the drug activity, but also have a potential synergistic interaction effect and have a relatively good anti-cancer effect.

Description

Technical Field

[0001] The present invention relates to the technical field of extracellular vesicles, and in particular to a method for obtaining Rhodiola rosea extracellular vesicles and an application thereof. Background Art

[0002] Extracellular vesicles are phospholipid bilayer vesicles secreted by cells, encapsulating a variety of functional nucleic acids, proteins and other components, and have similar physiological functions to their source cells. As one of the effective methods of drug delivery carriers, plant extracellular vesicles have the advantages of small size and strong tissue penetration, and show significant advantages in biocompatibility, stability, in vivo distribution, and extended half-life. At the same time, in terms of safety, plant-derived vesicles do not contain any zoonotic or human pathogens, and have a low immune risk in the body. Therefore, plant extracellular vesicles have great potential in disease treatment and drug delivery.

[0003] Currently, most studies on plant extracellular vesicles are from fruits and vegetables, such as ginger, grapes, grapefruit, and broccoli. Only a few studies have been conducted with medicinal plant extracellular vesicles. Therefore, it is necessary to further explore the application of medicinal plant extracellular vesicles. Summary of the invention

[0004] In a first aspect, a method for obtaining Rhodiola rosea extracellular vesicles is provided, comprising the following steps:

[0005] S10: Preparation of crude Rhodiola rosea extract, crushing fresh Rhodiola rosea into a homogenate, adding PBS buffer, filtering and centrifuging, collecting the supernatant to form a crude Rhodiola rosea extract;

[0006] S20: crude extraction of extracellular vesicles of Rhodiola rosea, centrifugation and concentration treatment, centrifugation treatment, impurity removal of the crude extract of Rhodiola rosea, collecting the supernatant, adding PBS buffer, and filtering with a filter membrane for sterilization to obtain the crude extract of extracellular vesicles of Rhodiola rosea;

[0007] S30: Extraction of extracellular vesicles from Rhodiola rosea. The crude extract of extracellular vesicles from Rhodiola rosea was centrifuged and the precipitate was resuspended and incubated with paraformaldehyde and dithiothreitol in PBS at 4 °C. The supernatant was then centrifuged and collected to remove impurities, debris and microvesicles in the vesicle formation buffer. The supernatant was processed through a centrifugal filter to obtain extracellular vesicles from Rhodiola rosea.

[0008] S40: Surface modification of Rhodiola rosea extracellular vesicles by inserting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipids between the lipid bilayers of the extracellular vesicle membrane through hydrophobic interactions to impart targeting ability.

[0009] The method for obtaining the extracellular vesicle provided by the present invention can obtain the extracellular vesicle of Rhodiola rosea. On the one hand, the extracellular vesicle of Rhodiola rosea has good biocompatibility. The extracellular vesicle of Rhodiola rosea is derived from natural Rhodiola rosea plant cells. Its composition and structure have certain compatibility with the environment of the organism itself. Compared with some synthetic drug delivery carriers, it is less likely to be recognized as a foreign body by the immune system, thereby reducing the risk of inducing an immune response, such as reducing the probability of occurrence of inflammatory reactions, allergic reactions, etc., making the drug delivery process safer and more stable. On the other hand, the extracellular vesicle of Rhodiola rosea can effectively protect the activity of the drug. The extracellular vesicle of Rhodiola rosea can physically wrap the drug inside it. In the whole process of drug production, transportation, entering the human body and reaching the tumor site, this wrapping effect can prevent the drug from prematurely contacting with adverse factors in the external environment (such as gastric acid, certain enzymes in the blood, etc.) and causing chemical reactions to cause loss of activity, ensuring that the drug reaches the cancer cells in an effective active state and maintaining the therapeutic efficacy of the drug. Finally, Rhodiola rosea extracellular vesicles also have potential synergistic effects. Rhodiola rosea itself contains a variety of ingredients with anti-cancer activity, such as salidroside, tyrosol, etc. When Rhodiola rosea extracellular vesicles are used as drug delivery carriers, these components of Rhodiola rosea itself may synergize with the delivered anti-cancer drugs at the tumor site. Rhodiola rosea components may enhance the killing effect of the delivered drugs on cancer cells, and the delivered drugs may further enhance the anti-cancer activity of Rhodiola rosea components. The two work together to provide more powerful firepower for cancer treatment.

[0010] Preferably, in step S10, the homogenate is filtered, and the filtrate is centrifuged at 3°C ​​to 5°C, 8000g / min to 12000g / min for 20min to 30min, the supernatant is collected, and the crude Rhodiola rosea extract is obtained and stored at -25°C to -15°C.

[0011] Preferably, in step S10, the homogenate is filtered, and the filtrate is centrifuged at 4°C, 10000g / min for 25min, the supernatant is collected, and the crude Rhodiola rosea extract is obtained and stored at -20°C.

[0012] Preferably, in step S20, the crude extract of Rhodiola rosea is centrifuged and concentrated at a low temperature of 3°C to 5°C and 2300g / min to 2700g / min, and then the concentrate is centrifuged at 3°C ​​to 5°C and 8000g / min to 12000g / min for 8min-12min. After removing impurities, the supernatant is collected and centrifuged at 3°C ​​to 5°C and 48000g / min to 52000g / min for 80min-100min to obtain the Rhodiola rosea extracellular vesicle precipitate, and PBS buffer is added, and after sufficient blowing, it is filtered and sterilized with a filter membrane to obtain the crude extract of Rhodiola rosea extracellular vesicles.

[0013] Preferably, in step S20, the crude extract of Rhodiola rosea is centrifuged and concentrated at a low temperature of 4°C and 2500g / min, and then the concentrate is centrifuged at 4°C and 10000g / min for 10min. After removing impurities, the supernatant is collected and centrifuged at 4°C and 50000g / min for 90min to obtain the Rhodiola rosea extracellular vesicle precipitate, and PBS buffer is added, and after being fully blown, it is filtered and sterilized with a 0.22um filter membrane to obtain the crude extract of Rhodiola rosea extracellular vesicles.

[0014] Preferably, in step S30, the crude extract of Rhodiola rosea is centrifuged at 200g-240g for 4min-6min, and then the precipitate is resuspended and incubated with 1% paraformaldehyde and 3mM dithiothreitol in PBS at 35°C-39°C and 4.5%-5.5% CO2 for 2.5h-3.5h; then, the supernatant is collected by centrifugation at 200g-240g for 4.5min-5.5min at room temperature, and then the supernatant is collected by centrifugation at 9000g-10000g for 13min-17min at room temperature, and the supernatant containing plant extracellular vesicles initially screened out is concentrated at room temperature for 12min-18min at 3300g-3700g using a 100kDa centrifugal filter, and rinsed with PBS, and repeated 1-3 times with an equal amount of PBS to obtain Rhodiola rosea extracellular vesicles.

[0015] Preferably, in step S30, the crude extract of Rhodiola rosea is centrifuged at 220g for 56min, and then the precipitate is resuspended and incubated with 1% paraformaldehyde and 3mM dithiothreitol in PBS at 37°C and 5% CO2 for 3h; then, the supernatant is collected by centrifugation at 220g for 5min at room temperature, and then the supernatant is collected by centrifugation at 9500g for 15min at room temperature, and the supernatant containing plant extracellular vesicles initially screened out is concentrated at 3500g for 15min at room temperature using a 100kDa centrifugal filter, and rinsed with PBS, and repeated 1 to 3 times with an equal amount of PBS to obtain Rhodiola rosea extracellular vesicles.

[0016] In a second aspect, a Rhodiola rosea extracellular vesicle is provided, which is obtained by the method described in the first aspect.

[0017] In a third aspect, an application of Rhodiola rosea extracellular vesicles is provided, wherein the Rhodiola rosea extracellular vesicles are obtained by the method described in the first aspect as a drug delivery carrier.

[0018] In a fourth aspect, an application of Rhodiola rosea extracellular vesicles is provided, wherein the Rhodiola rosea extracellular vesicles obtained by the method described in the first aspect are used as anticancer drug delivery carriers.

[0019] In an embodiment of the present invention, a method for obtaining Rhodiola rosea extracellular vesicles and an application thereof are provided. The obtained Rhodiola rosea extracellular vesicles and the application thereof have the following beneficial effects:

[0020] 1. Rhodiola rosea extracellular vesicles have good biocompatibility: Rhodiola rosea extracellular vesicles are derived from natural Rhodiola rosea plant cells. Their composition and structure are compatible with the organism's own environment. Compared with some synthetic drug delivery carriers, they are less likely to be recognized as foreign matter by the immune system, thereby reducing the risk of triggering immune responses, such as reducing the probability of inflammatory reactions and allergic reactions, making the drug delivery process safer and smoother;

[0021] 2. Rhodiola rosea extracellular vesicles can effectively protect drug activity: Rhodiola rosea extracellular vesicles can physically encapsulate drugs inside them. During the entire process from drug production and transportation to entering the human body and reaching the tumor site, this encapsulation effect can prevent the drug from prematurely contacting with adverse factors in the external environment (such as gastric acid, certain enzymes in the blood, etc.) and causing chemical reactions that lead to loss of activity, ensuring that the drug reaches cancer cells in an effective active state and maintaining the therapeutic efficacy of the drug;

[0022] 3. Rhodiola rosea extracellular vesicles also have potential synergistic effects: Rhodiola rosea itself contains a variety of ingredients with anti-cancer activity, such as salidroside, tyrosol, etc. When Rhodiola rosea extracellular vesicles are used as drug delivery carriers, these components of Rhodiola rosea itself may synergize with the delivered anti-cancer drugs at the tumor site. Rhodiola rosea components may enhance the killing effect of the delivered drugs on cancer cells, and the delivered drugs may further enhance the anti-cancer activity of Rhodiola rosea components. The two work together to provide more powerful firepower for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] in:

[0025] Figure 1 is a flow chart of the steps for obtaining and validating Rhodiola rosea extracellular vesicles;

[0026] Figure 2 This is a schematic diagram of the morphology of Rhodiola rosea extracellular vesicles observed by transmission electron microscopy;

[0027] Figure 3 This is a schematic diagram of the DLS detection of the size of Rhodiola rosea extracellular vesicles;

[0028] Figure 4 This is a schematic diagram of the Zeta potential measurement of the surface potential of Rhodiola rosea extracellular vesicles;

[0029] Figure 5It is a schematic diagram of tumor photos, volume and weight in in vivo validation;

[0030] Figure 6 This is a schematic diagram of TUNEL staining to detect apoptosis of tumor tissue cells in in vivo validation;

[0031] Figure 7 This is a schematic diagram of CCK8 detection of cell viability in in vitro validation;

[0032] Figure 8 This is a schematic diagram of the in vitro validation of the cellular uptake of Rhodiola rosea extracellular vesicles detected by confocal microscopy;

[0033] Fig. 9 This is a schematic diagram of Transwell's detection of cell migration ability in in vitro validation;

[0034] Fig.10 This is a schematic diagram of Transwell's detection of cell invasion ability in in vitro validation; DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings, and "multiple" generally includes at least two.

[0037] As used herein, the words "if" and "if" may be interpreted as "at the time of" or "when" or "in response to determining" or "in response to detecting", depending on the context. Similarly, the phrases "if it is determined" or "if (stated condition or event) is detected" may be interpreted as "when it is determined" or "in response to determining" or "when detecting (stated condition or event)" or "in response to detecting (stated condition or event)", depending on the context.

[0038] Extracellular vesicles are phospholipid bilayer vesicles secreted by cells, which encapsulate a variety of functional nucleic acids, proteins and other components. They have similar physiological functions to their source cells. EVs are divided into several subtypes according to different cells and characteristics, such as microvesicles, exosomes, exosomes, and exosomes.

[0039] At present, it is believed that almost all living cells, including plant cells, secrete EVs. Compared with mammalian EVs, the study of plant EVs is still in its infancy. Plant exosome vesicles or vesicle-like nanoparticles have been successfully isolated from many plant species, including ginseng, ginger, astragalus, wolfberry, broccoli, bitter melon, oats, grapes, grapefruit, lemon, blueberry, orange, sunflower seeds and Arabidopsis.

[0040] As one of the effective methods of drug delivery carriers, plant extracellular vesicles have the advantages of small size and strong tissue penetration, and can maintain good physical and chemical stability at different pH and temperatures. In addition, plant extracellular vesicles have the morphology and characteristics of nanocarriers, and show significant advantages in biocompatibility, stability, in vivo distribution, and extended half-life. In terms of safety, plant-derived vesicles do not contain any zoonotic or human pathogens, and the risk of in vivo immunity is low.

[0041] Rhodiola rosea belongs to the genus Rhodiola of the Rhodiola family. It is a top-level drug for strengthening and nourishing deficiency. Modern research has found that Rhodiola rosea has multiple pharmacological effects such as anti-fatigue, anti-oxidation, anti-aging, anti-inflammatory, and anti-tumor. Salidroside, as the main active ingredient of Rhodiola rosea, has a concentration-dependent mechanism to exert anti-tumor effects by inhibiting tumor cell proliferation, inhibiting cell migration, promoting cell apoptosis, inducing autophagy, inhibiting angiogenesis and infiltration and metastasis, and enhancing immunity. For example, salidroside induces autophagy, apoptosis, infiltration and metastasis of liver cancer cells through the PI 3K / Akt / mTOR signaling pathway; salidroside inhibits the proliferation and migration of prostate cancer through the PI 3K / AKT pathway; salidroside regulates the tumor microenvironment of non-small cell lung cancer through the Hsp70 / Stub1 / Foxp3 pathway in Tregs; salidroside reduces neuronal ferroptosis by activating the Nrf2 / HO1 signaling pathway in Aβ.

[0042] In order to develop a safe and effective drug delivery system, the present invention develops a method for preparing Rhodiola rosea extracellular vesicles, and verifies its anti-tumor effect in vivo and in vitro.

[0043] After introducing the basic principles of the present invention, various non-limiting embodiments of the present invention are specifically introduced below. It should be noted that the embodiments provided by the present invention are only shown to facilitate understanding of the spirit and principles of the present invention, and the embodiments of the present invention are not subject to any limitation in this respect. On the contrary, the embodiments of the present invention can be applied to any applicable system.

[0044] Embodiment 1:

[0045] A method for obtaining Rhodiola rosea extracellular vesicles, comprising:

[0046] S10: Preparation of crude Rhodiola rosea extract, crushing fresh Rhodiola rosea into a homogenate, adding PBS buffer, filtering and centrifuging, collecting the supernatant to form a crude Rhodiola rosea extract;

[0047] In this step, the homogenate is filtered, and the filtrate is centrifuged at 3°C ​​to 5°C, 8000g / min to 12000g / min for 20min to 30min, the supernatant is collected, and the crude Rhodiola rosea extract is obtained and stored at -25°C to -15°C.

[0048] Preferably, the homogenate is filtered, and the filtrate is centrifuged at 4°C, 10000g / min for 25min, the supernatant is collected, and the crude Rhodiola rosea extract is obtained and stored at -20°C.

[0049] S20: crude extraction of extracellular vesicles of Rhodiola rosea, centrifugation and concentration treatment, centrifugation treatment, impurity removal of the crude extract of Rhodiola rosea, collecting the supernatant, adding PBS buffer, and filtering with a filter membrane for sterilization to obtain the crude extract of extracellular vesicles of Rhodiola rosea;

[0050] In this step, the crude extract of Rhodiola rosea is concentrated by centrifugation at a low temperature of 3°C to 5°C and 2300g / min to 2700g / min, and then the concentrate is centrifuged at 3°C ​​to 5°C and 8000g / min to 12000g / min for 8min-12min. After removing impurities, the supernatant is collected and centrifuged at 3°C ​​to 5°C and 48000g / min to 52000g / min for 80min-100min to obtain the Rhodiola rosea extracellular vesicle precipitate, and PBS buffer is added. After being fully blown, it is filtered and sterilized with a filter membrane to obtain the crude extract of Rhodiola rosea extracellular vesicles.

[0051] Preferably, the crude extract of Rhodiola rosea is centrifuged and concentrated at a low temperature of 4°C and 2500g / min, and then the concentrate is centrifuged at 4°C and 10000g / min for 10 min. After removing impurities, the supernatant is collected and centrifuged at 4°C and 50000g / min for 90 min to obtain the Rhodiola rosea extracellular vesicle precipitate, and PBS buffer is added. After being fully blown, it is filtered and sterilized with a 0.22um filter membrane to obtain the crude extract of Rhodiola rosea extracellular vesicles.

[0052] S30: Extraction of extracellular vesicles from Rhodiola rosea. The crude extract of extracellular vesicles from Rhodiola rosea was centrifuged and the precipitate was resuspended and incubated with paraformaldehyde and dithiothreitol in PBS at 4 °C. The supernatant was then centrifuged and collected to remove impurities, debris and microvesicles in the vesicle formation buffer. The supernatant was processed through a centrifugal filter to obtain extracellular vesicles from Rhodiola rosea.

[0053] In this step, the crude extract of Rhodiola rosea is centrifuged at 200g-240g for 4min-6min, and then the precipitate is resuspended and incubated with 1% paraformaldehyde and 3mM dithiothreitol in PBS at 35°C-39°C and 4.5%-5.5% CO2 for 2.5h-3.5h; then, the supernatant is collected by centrifugation at 200g-240g for 4.5min-5.5min at room temperature, and then the supernatant is collected by centrifugation at 9000g-10000g for 13min-17min at room temperature, and the supernatant containing plant extracellular vesicles initially screened out is concentrated at 3300g-3700g for 12min-18min at room temperature using a 100kDa centrifugal filter, and rinsed with PBS, and repeated 1-3 times with an equal amount of PBS to obtain Rhodiola rosea extracellular vesicles.

[0054] Preferably, the crude extract of Rhodiola rosea is centrifuged at 220g for 56min, and then the precipitate is resuspended and incubated with 1% paraformaldehyde and 3mM dithiothreitol in PBS at 37°C and 5% CO2 for 3h; subsequently, the supernatant is collected by centrifugation at 220g for 5min at room temperature, and then the supernatant is collected by centrifugation at 9500g for 15min at room temperature, and the supernatant containing plant extracellular vesicles initially screened out is concentrated using a 100kDa centrifugal filter at 3500g for 15min at room temperature, and rinsed with PBS, and repeated 1 to 3 times with an equal amount of PBS to obtain Rhodiola rosea extracellular vesicles.

[0055] S40: Surface modification of Rhodiola rosea extracellular vesicles by inserting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipids between the lipid bilayers of the extracellular vesicle membrane through hydrophobic interactions to impart targeting ability.

[0056] S40: Transmission electron microscopy was used to observe the morphology of Rhodiola rosea extracellular vesicles. Figure 2 ;

[0057] S50: DLS was used to detect the size of Rhodiola rosea extracellular vesicles. Figure 3 ;

[0058] S60: Zeta potential was used to measure the surface potential of Rhodiola rosea extracellular vesicles, such as Figure 4 ;

[0059] Based on the above embodiment, the steps are also included.

[0060] S70: In vivo verification of the anti-tumor effect of Rhodiola rosea extracellular vesicles, specifically:

[0061] A nude mouse model of liver cancer was established and Rhodiola rosea extracellular vesicles were intraperitoneally injected. After 4 weeks, samples were collected and photographed and weighed. Figure 5 ;

[0062] TUNEL staining was performed on tumor tissues to detect tumor cell apoptosis. Figure 6 .

[0063] Based on the above embodiment, the steps are also included.

[0064] S80: In vitro verification that Rhodiola rosea extracellular vesicles inhibit tumor cell proliferation, specifically:

[0065] Human hepatoma cells (HepG2) and human thyroid cancer cells (8505C) were cultured and treated with different concentrations of Rhodiola rosea extracellular vesicles. Cell viability was detected by CCK8. Figure 7 ;

[0066] The extracellular vesicles of Rhodiola rosea were labeled with DiO green fluorescent dye, the cell nuclei were stained with Hoechst, and the cellular uptake of the extracellular vesicles of Rhodiola rosea was detected using confocal microscopy. Figure 8 ;

[0067] Transwell was used to detect cell invasion and migration abilities, respectively. Fig. 9 and Fig.10 .

[0068] Through in vivo and in vitro verification, it can be shown that the Rhodiola rosea extracellular vesicles obtained by the method of the present invention have significant anti-tumor effects, which are specifically manifested as follows:

[0069] 1. Rhodiola rosea extracellular vesicles have good biocompatibility. Rhodiola rosea extracellular vesicles are derived from natural Rhodiola rosea plant cells. Their composition and structure are compatible with the organism's own environment. Compared with some synthetic drug delivery carriers, it is less likely to be recognized as a foreign body by the immune system, thereby reducing the risk of triggering an immune response, such as reducing the probability of inflammatory reactions and allergic reactions, making the drug delivery process safer and smoother;

[0070] 2. Rhodiola rosea extracellular vesicles can effectively protect the activity of drugs. Rhodiola rosea extracellular vesicles can physically wrap drugs inside them. During the entire process from drug production and transportation to entering the human body and reaching the tumor site, this wrapping effect can prevent the drug from prematurely contacting with adverse factors in the external environment (such as gastric acid, certain enzymes in the blood, etc.) and causing chemical reactions that lead to loss of activity, ensuring that the drug reaches cancer cells in an effective active state and maintaining the therapeutic efficacy of the drug;

[0071] 3. Rhodiola rosea extracellular vesicles also have potential synergistic effects. Rhodiola rosea itself contains a variety of ingredients with anti-cancer activity, such as salidroside, tyrosol, etc. When Rhodiola rosea extracellular vesicles are used as drug delivery carriers, these components of Rhodiola rosea itself may synergize with the delivered anti-cancer drugs at the tumor site. Rhodiola rosea components may enhance the killing effect of the delivered drugs on cancer cells, and the delivered drugs may further enhance the anti-cancer activity of Rhodiola rosea components. The two work together to provide more powerful firepower for cancer treatment.

[0072] Embodiment 2:

[0073] The Rhodiola rosea extracellular vesicles obtained by the method provided in Example 1 have good biocompatibility, can not only effectively protect the drug activity, but also have potential synergistic effects. In comparison, traditional nanomaterials have the following shortcomings:

[0074] 1. Immune response: Many nanomaterials may be recognized as foreign bodies by the immune system after entering the human body, thereby triggering an immune response, which will not only affect the effect of drug delivery, but may also cause additional burden on the patient's body.

[0075] 2. Cytotoxicity: Some nanomaterials themselves may be cytotoxic, causing damage to normal cells, affecting the normal tissues around the target cells, and reducing the safety and effectiveness of treatment.

[0076] 3. Difficulty in degradation: Some nanomaterials are difficult to degrade in the body and may remain in the body for a long time. Long-term residual nanomaterials may continue to have potential effects on the body, such as causing chronic inflammation and affecting organ function.

[0077] 4. High preparation cost: The preparation of nanomaterials usually requires relatively complex processes and special equipment, which makes its preparation cost relatively high. From the selection of raw materials, energy consumption in the preparation process, equipment maintenance and other aspects, a large amount of money needs to be invested.

[0078] Embodiment three:

[0079] Provided is an application of Rhodiola rosea extracellular vesicles, wherein the Rhodiola rosea extracellular vesicles are obtained by the method described in Example 1 as a drug delivery carrier.

[0080] Embodiment 4:

[0081] Provided is an application of Rhodiola rosea extracellular vesicles, wherein the Rhodiola rosea extracellular vesicles are obtained by the method described in Example 1 and used as an anticancer drug delivery carrier.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for obtaining Rhodiola rosea extracellular vesicles, characterized in that: include: S10: Preparation of crude Rhodiola rosea extract, crushing fresh Rhodiola rosea into a homogenate, adding PBS buffer, filtering and centrifuging, collecting the supernatant to form a crude Rhodiola rosea extract; S20: crude extraction of extracellular vesicles of Rhodiola rosea, centrifugation and concentration treatment, centrifugation treatment, impurity removal of the crude extract of Rhodiola rosea, collecting the supernatant, adding PBS buffer, and filtering with a filter membrane for sterilization to obtain the crude extract of extracellular vesicles of Rhodiola rosea; S30: Extraction of extracellular vesicles from Rhodiola rosea. The crude extract of extracellular vesicles from Rhodiola rosea was centrifuged and the precipitate was resuspended and incubated with paraformaldehyde and dithiothreitol in PBS at 4 °C. The supernatant was then centrifuged and collected to remove impurities, debris and microvesicles in the vesicle formation buffer. The supernatant was processed through a centrifugal filter to obtain extracellular vesicles from Rhodiola rosea. S40: Surface modification of Rhodiola rosea extracellular vesicles by inserting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipids between the lipid bilayers of the extracellular vesicle membrane through hydrophobic interactions to impart targeting ability.

2. The method for obtaining Rhodiola rosea extracellular vesicles according to claim 1, characterized in that: In the step S10, the homogenate is filtered, and the filtrate is centrifuged at 3°C ​​to 5°C, 8000g / min to 12000g / min for 20min to 30min, and the supernatant is collected to obtain the crude Rhodiola rosea extract and store it at -25°C to -15°C.

3. The method for obtaining Rhodiola rosea extracellular vesicles according to claim 2, characterized in that: In the step S10, the homogenate is filtered, and the filtrate is centrifuged at 4°C, 10000g / min for 25min, and the supernatant is collected to obtain the crude Rhodiola rosea extract and stored at -20°C.

4. The method for obtaining Rhodiola rosea extracellular vesicles according to claim 1, characterized in that: In the step S20, the crude extract of Rhodiola rosea is centrifuged and concentrated at a low temperature of 3°C to 5°C and 2300g / min to 2700g / min, and then the concentrate is centrifuged at 3°C ​​to 5°C and 8000g / min to 12000g / min for 8min-12min. After removing impurities, the supernatant is collected and centrifuged at 3°C ​​to 5°C and 48000g / min to 52000g / min for 80min-100min to obtain the Rhodiola rosea extracellular vesicle precipitate, and PBS buffer is added, and after being fully blown, it is filtered and sterilized with a filter membrane to obtain the crude extract of Rhodiola rosea extracellular vesicles.

5. The method for obtaining Rhodiola rosea extracellular vesicles according to claim 4, characterized in that: In the step S20, the crude extract of Rhodiola rosea is concentrated by centrifugation at a low temperature of 4°C and 2500g / min, and then the concentrate is centrifuged at 4°C and 10000g / min for 10min. After removing impurities, the supernatant is collected and centrifuged at 4°C and 50000g / min for 90min to obtain the Rhodiola rosea extracellular vesicle precipitate, and PBS buffer is added. After being fully blown, it is filtered and sterilized with a 0.22um filter membrane to obtain the crude extract of Rhodiola rosea extracellular vesicles.

6. The method for obtaining Rhodiola rosea extracellular vesicles according to claim 1, characterized in that: In the step S30, the crude extract of Rhodiola rosea is centrifuged at 200g-240g for 4min-6min, and then the precipitate is resuspended and incubated with 1% paraformaldehyde and 3mM dithiothreitol in PBS at 35°C-39°C and 4.5%-5.5% CO2 for 2.5h-3.5h; then, the supernatant is collected by centrifugation at 200g-240g for 4.5min-5.5min at room temperature, and then the supernatant is collected by centrifugation at 9000g-10000g for 13min-17min at room temperature, and the supernatant containing plant extracellular vesicles initially screened out is concentrated at 3300g-3700g for 12min-18min at room temperature using a 100kDa centrifugal filter, and rinsed with PBS, and repeated 1-3 times with an equal amount of PBS to obtain Rhodiola rosea extracellular vesicles.

7. The method for obtaining Rhodiola rosea extracellular vesicles according to claim 1, characterized in that: In the step S30, the crude extract of Rhodiola rosea is centrifuged at 220 g for 56 min, and then the precipitate is resuspended and incubated with 1% paraformaldehyde and 3 mM dithiothreitol in PBS at 37° C. and 5% CO 2 for 3 h; Subsequently, the supernatant was collected by centrifugation at 220g for 5 min at room temperature, and then centrifuged at 9500g for 15 min at room temperature and the supernatant was collected. The supernatant containing plant extracellular vesicles initially screened out was concentrated using a 100 kDa centrifugal filter at 3500g for 15 min at room temperature and rinsed with PBS. This was repeated 1 to 3 times with an equal amount of PBS to obtain Rhodiola rosea extracellular vesicles.

8. A Rhodiola rosea extracellular vesicle, characterized in that The method according to any one of claims 1 to 7 is used to obtain the 9. An application of Rhodiola rosea extracellular vesicles, characterized in that: The Rhodiola rosea extracellular vesicles are obtained by the method described in any one of claims 1 to 7 as a drug delivery carrier.

10. An application of Rhodiola rosea extracellular vesicles, characterized in that: The method described in any one of claims 1 to 7 is used to obtain Rhodiola rosea extracellular vesicles as an anticancer drug delivery carrier.

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