CN114774348A - Kiwi extracellular vesicles and application thereof in drug carriers - Google Patents
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Abstract
The invention discloses kiwi extracellular vesicles and application thereof in a drug carrier, wherein the preparation method comprises the following steps: selecting fresh kiwi fruits, peeling and extruding to obtain kiwi fruit juice, carrying out differential centrifugation to remove plant fibers and cell debris precipitates, passing the obtained supernatant through a 0.45-micron filter membrane, carrying out ultra-high speed centrifugation on the filtrate to obtain low-purity kiwi fruit extracellular vesicle precipitates, dissolving the low-purity kiwi fruit extracellular vesicle precipitates in PBS, carrying out centrifugation by a sucrose concentration gradient centrifugation method, collecting a solution with 30% -45% of strips, washing the solution with PBS, and carrying out ultra-high speed centrifugation to obtain high-purity kiwi fruit extracellular vesicles. The kiwi extracellular vesicles separated from kiwi are used as a drug carrier, and are helpful for assisting the drug to escape from the capture of an immune system, and the circulation time of the drug in vivo is prolonged. Animal experiments show that the compound can cross the intestinal barrier to show the capacity of liver accumulation, realize effective encapsulation and reduce the release of the medicine in the gastrointestinal tract.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to kiwi extracellular vesicles and application thereof in a medicine carrier.
Background
For a long time, many drugs have been limited in clinical application due to problems of poor solubility, stability and large side effects, and methods such as chemical modification and liposome loading have been used to improve the bioavailability of the drugs, however, these modifications still face the problems of poor targeting and easy clearance by the immune system. Therefore, it is very important to develop a drug carrier with good biocompatibility and high targeting.
In recent years, research on extracellular vesicles has received much attention. Based on the lipid bilayer structure of the extracellular vesicles, the loading of hydrophilic drugs and hydrophobic drugs can be realized, and good biological stability and biocompatibility are shown in a body. In addition, the specific protein on the surface of the extracellular vesicle of the mammal is helpful for realizing tumor targeting and immune escape, and has been used for loading various medicaments to enhance the therapeutic effect of the medicaments. Unfortunately, mammalian extracellular vesicles for drug delivery still face several obstacles such as intolerance to the gastrointestinal environment, low yields, and the presence of immunogenicity.
In the prior art, plant cells are found to secrete EV similar to extracellular vesicles of mammals and have good drug encapsulation capacity and targeting function. The kiwi fruits are called as kiwi fruits, fruit gold ores and VC king, and have a long history of food and drug application, which means that kiwi fruit extracellular vesicles (KEVs) have considerable safety. The kiwi fruit not only contains rich nutrient components, but also has the functions of resisting cancer, oxidation, inflammation and the like, researches show that the extracellular vesicles derived from plants inherit part of the biological activity of the source plants, and related reports on the kiwi fruit extracellular vesicles serving as drug carriers are not seen at present.
Disclosure of Invention
In view of the defects in the prior art, the first purpose of the invention is to provide a preparation method of kiwi fruit extracellular vesicles.
The second purpose of the invention is to provide a kiwi fruit extracellular vesicle prepared by the preparation method of the kiwi fruit extracellular vesicle.
The third purpose of the invention is to provide the application of the kiwi fruit extracellular vesicles in a drug carrier.
In order to realize the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of kiwi extracellular vesicles, which comprises the following steps:
(1) selecting fresh kiwi fruits, peeling and extruding to obtain kiwi fruit juice;
(2) centrifuging the kiwi fruit juice obtained in the step (1) for several times at a differential speed from low to high to remove plant fibers and cell debris precipitates to obtain supernatant;
(3) filtering the supernatant obtained in the step (2) with a 0.45-micron filter membrane, centrifuging the filtrate at a high speed, and retaining the precipitate to obtain low-purity kiwi extracellular vesicle precipitate;
(4) and (5) dissolving the low-purity kiwi fruit extracellular vesicle precipitate obtained in the step (4) in PBS, centrifuging by a sucrose concentration gradient centrifugation method, collecting a solution with 30% -45% of bands, washing by PBS, and centrifuging at ultrahigh speed to obtain the high-purity kiwi fruit extracellular vesicle.
Preferably, in the step (1), the kiwi fruits are peeled and extruded and then pass through 200-mesh filter cloth.
Preferably, in step (2), the differential centrifugation process is: 300g, 10min × 2; 1000g, 10min × 2; 3000g, 10min × 2; 10000g, 30min x 2; 20000g, 30min × 2.
Preferably, in step (3), the ultra-high speed centrifugation is 120000g for 2 hours.
Preferably, in step (4), the sucrose concentration gradient in the sucrose concentration gradient centrifugation method is 8%, 15%, 30%, 45% and 60%, respectively.
The invention also provides application of the kiwi fruit extracellular vesicles in a drug carrier.
The invention also provides application of the kiwi fruit extracellular vesicles serving as a drug carrier to entrap an anti-cancer drug.
Preferably, the anticancer drug is sorafenib, wherein the mass ratio of the kiwi fruit extracellular vesicles to the sorafenib is 1-20: 1, more preferably 10: 1.
compared with the prior art, the invention has the beneficial effects that:
according to the invention, the kiwi fruit extracellular vesicles are separated from kiwi fruits and can play a role in cooperative treatment when being used as a drug carrier, and the coating of the kiwi fruit extracellular vesicles helps to assist the drug to escape from the capture of an immune system, so that the circulation time of the drug in a body is prolonged. In addition, compared to mammalian vesicles and other pharmaceutical carriers, extracellular vesicles derived from kiwi fruit are readily available and simple to prepare. Animal experiments show that the mouse can penetrate through the intestinal barrier to show the capacity of liver accumulation after the mouse takes the extracellular vesicles orally, and when the kiwi fruit extracellular vesicles are used as a drug carrier to carry drugs, the kiwi fruit extracellular vesicles can effectively carry out encapsulation and simultaneously reduce the release of the drugs in the gastrointestinal tract, so that the kiwi fruit extracellular vesicles have great potential to become oral drug carriers.
Drawings
Fig. 1 is a process flow diagram of the preparation process of the kiwi extracellular vesicles in the example.
FIG. 2 is the distribution of kiwi fruit outer vesicles in mice in the examples after oral administration.
Fig. 3 shows the encapsulation efficiency of the kiwi fruit extracellular vesicles on sorafenib drugs in the example.
Fig. 4 shows the release of sorafenib in the extracellular vesicles of kiwi fruits in simulated gastrointestinal fluids in the examples.
FIG. 5 is the in vitro inhibition of HepG2 by sorafenib entrapped in the extracellular vesicles of Actinidia chinensis in the examples.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without inventive step, are within the scope of protection of the invention.
Example 1
Separation and purification of kiwi extracellular vesicles
Peeling fresh fructus Actinidiae chinensis, squeezing to obtain fructus Actinidiae chinensis juice, and filtering with 200 filter cloth; passing through 300g, 10min × 2; 1000g, 10min × 2; 3000g, 10min × 2; 10000g, 30min x 2; 20000g, 30min × 2 centrifuging to remove plant fiber, cell debris, etc.; filtering the supernatant with 0.45 μm filter membrane; centrifuging the filtrate by a CP70-ME ultracentrifuge for 120000g for 2h to obtain low-purity kiwi fruit outer vesicle sediment, dissolving the sediment in PBS, performing sucrose gradient centrifugation (8%, 15%, 0%, 45%, 60%) to obtain 30% -45% solution, washing the solution by PBS, and ultracentrifuging for 2h to remove sucrose solution to obtain kiwi fruit outer vesicle.
Secondly, the kiwi fruit extracellular vesicles are distributed in the body of a mouse after being orally taken
The fluorescent dye DiR is used for marking the kiwi fruit extracellular vesicles, and after oral administration, the mouse viscera is taken for in vitro tissue imaging, and the result is shown in figure 2, the kiwi fruit extracellular vesicles show strong liver accumulation in 12h and 24h, the spleen also has partial fluorescence, the gastrointestinal fluorescence is strong in 6h, and the gastrointestinal fluorescence is weakened after 12h, so that the kiwi fruit extracellular vesicles have good liver targeting capability.
Example 2
Kiwi extracellular vesicles coated with sorafenib as anticancer drug
The kiwi fruit extracellular vesicle-sorafenib mixed solution with different mass ratios is subjected to ultrasonic treatment for 10min multiplied by 2, the structure of the extracellular vesicle is recovered after standing and incubating for 1h at room temperature, the non-encapsulated sorafenib is removed by an ultrafiltration centrifugal tube with the molecular weight of 30kDa, the kiwi fruit extracellular vesicle-sorafenib mixed solution is subjected to precipitation for 120000g and 2h, the precipitate is dissolved in PBS, the encapsulation amount of the sorafenib is determined by an HPLC method, an Agilent C18 column (250mm multiplied by 4.6mm and 5 mu m) has the mobile phase of 20mmol/L ammonium acetate-acetonitrile (28: 72), the detection wavelength is 266nm, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the sample introduction amount is 20 mu L.
The results are shown in FIG. 3, and are shown at 1: the kiwifruit extracellular vesicles can achieve better encapsulation of sorafenib at the ratio of 10, the encapsulation rate is about 78%, and the drug loading rate at the ratio is 6.5%.
Second, medicine release of kiwi fruit extracellular vesicle loaded with sorafenib
The drug release condition of the kiwi fruit extracellular vesicle-sorafenib in simulated gastric juice and intestinal fluid is inspected by a dialysis bag method, 1mL of kiwi fruit extracellular vesicle-sorafenib solution is placed in a dialysis bag with a molecular weight of 3500Da, is respectively immersed in 50mL of simulated gastric juice and intestinal fluid, is stirred at 37 ℃ and 100rpm, 1mL of gastric juice or intestinal fluid is respectively taken at 5, 10, 30, 60, 90, 120, 180 and 240min (1 mL of gastric juice and intestinal fluid is simultaneously added), and the content of sorafenib in the gastric juice or intestinal fluid is measured according to the previous HPLC method.
The result is shown in fig. 4, which shows that under the entrapment of the kiwi fruit extracellular vesicles, sorafenib only leaks by less than 30% after incubation for 2h in simulated gastric fluid and intestinal fluid, and shows that the entrapment of the kiwi fruit extracellular vesicles helps to improve the leakage condition of sorafenib drugs in gastrointestinal tracts.
Third, the in vitro anti-tumor capacity of the kiwi fruit extracellular vesicle loaded with sorafenib
HepG2 liver cancer cells at 1X 10 per ml5The concentration of individual cells was added to a 96-well plate at 100. mu.L per well for 12h, Sorafenib (SFB) and kiwifruit extracellular vesicle-loaded sorafenib (KEVs-SFB) at different concentrations were added to the 96-well plate and incubated for 48h, 20. mu.L, 5mg/mL MTT solution and 180. mu.L media were added per well, incubated for 4h, the culture broth was removed, DMSO was added at 150. mu.L per well, and the absorbance value was measured with an microplate reader at 490 nm.
The results are shown in fig. 5, and show that the encapsulation of sorafenib by the kiwi extracellular vesicles does not reduce the cancer inhibition efficiency of sorafenib, and even improves the capability of sorafenib in inhibiting HepG2 cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A preparation method of kiwi extracellular vesicles is characterized by comprising the following steps:
(1) selecting fresh kiwi fruits, peeling and extruding to obtain kiwi fruit juice;
(2) centrifuging the kiwi fruit juice obtained in the step (1) for several times at a differential speed from low to high to remove plant fibers and cell debris precipitates to obtain supernatant;
(3) filtering the supernatant obtained in the step (2) with a 0.45-micron filter membrane, centrifuging the filtrate at a high speed, and retaining the precipitate to obtain low-purity kiwi extracellular vesicle precipitate;
(4) and (5) dissolving the low-purity kiwi fruit extracellular vesicle precipitate obtained in the step (4) in PBS, centrifuging by a sucrose concentration gradient centrifugation method, collecting a solution with 30% -45% of bands, washing by PBS, and centrifuging at ultrahigh speed to obtain the high-purity kiwi fruit extracellular vesicle.
2. The method for preparing the kiwi extracellular vesicles according to claim 1, wherein in step (1), kiwi fruits are peeled and squeezed, and then are filtered through a 200-mesh filter cloth.
3. The method for preparing the kiwi extracellular vesicles according to claim 1, wherein in the step (2), the differential centrifugation process comprises: 300g, 10min × 2; 1000g, 10min × 2; 3000g, 10min × 2; 10000g, 30min x 2; 20000g, 30min × 2.
4. The method for preparing the kiwi extracellular vesicles according to claim 1, wherein the ultra-high speed centrifugation in step (3) is 120000g for 2 hours.
5. The method for preparing the kiwi extracellular vesicles of claim 1, wherein in step (4), the sucrose concentration gradient in the sucrose concentration gradient centrifugation method is 8%, 15%, 30%, 45%, and 60%, respectively.
6. A kiwi extracellular vesicle prepared by the method for preparing a kiwi extracellular vesicle according to any one of claims 1 to 5.
7. Use of the kiwi extracellular vesicle of claim 6 in a pharmaceutical carrier.
8. The use of claim 7, wherein said kiwi extracellular vesicles are used as a drug carrier to encapsulate an anticancer drug.
9. The use of claim 8, wherein the anticancer drug is sorafenib, and wherein the mass ratio of the kiwifruit extracellular vesicles to sorafenib is 1-20: 1.
10. the use according to claim 9, wherein the mass ratio of kiwifruit extracellular vesicles to sorafenib is 10: 1.
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| CN114854667A (en) * | 2022-06-14 | 2022-08-05 | 江苏省中医药研究院(江苏省中西医结合医院) | Kiwi-derived plant nano vesicle and application thereof |
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| CN112501112A (en) * | 2020-12-22 | 2021-03-16 | 北京恩泽康泰生物科技有限公司 | Separation and enrichment method for rapidly extracting tissue extracellular vesicles |
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|---|---|---|---|---|
| CN114854667A (en) * | 2022-06-14 | 2022-08-05 | 江苏省中医药研究院(江苏省中西医结合医院) | Kiwi-derived plant nano vesicle and application thereof |
| CN114854667B (en) * | 2022-06-14 | 2023-08-22 | 江苏省中医药研究院(江苏省中西医结合医院) | Plant nano vesicle from kiwi fruits and application thereof |
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| CN114774348B (en) | 2023-11-03 |
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